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The occurrence of iodinated contrast agents (ICAs) in the aquatic environment is relatively well documented, showing that these compounds can be found at several µg/L in natural waters, and up to hundreds of µg/L in waste water treatment plants inlets. Nevertheless, only few studies address their potential impacts and fate in aquatic organisms mainly because these compounds are considered non-toxic due to their intrinsic properties. However, as aquatic organisms are continuously exposed to these compounds, they could nonetheless induce some adverse effects on aquatic populations like filter feeder organisms. To verify this, we exposed model organisms, Dreissena polymorpha mollusks, to 100 µg/L of an ICA, diatrizoic acid (DTZ), to determine the potential biological effects caused by this compound using a non-targeted metabolomic approach based on liquid chromatography coupled to high resolution mass spectrometry. Metabolic profiles showed a slight effect of DTZ, with some metabolome variations linked to exposure. Indeed, to avoid any misinterpretation of DTZ effects, we also studied the natural evolution of the metabolome over time in unexposed mussels, showing that control mussels exhibited metabolomic changes over the exposure period. During DTZ exposure, we showed that the carnitine shuttle pathway of fatty acids and pyrimidine metabolisms were impacted, leading to dysregulation of mussels' energy metabolism. Thus, this study demonstrates for the first time that compounds considered non-toxic like ICAs can have an impact on aquatic organisms such as bivalves by slightly modulating their metabolome., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Iodinated contrast media (ICMs) used in X-ray imaging for medical diagnostics are released into wastewater and then encountered in river water at concentrations ranging from several dozen to hundreds of µg/L, and even thousands of µg/L in hospital effluents. ICMs are considered as emerging pollutants as their occurrence and impact on ecosystems and the environment are poorly documented. Even if they are considered inert for humans, aquatic organisms are continuously exposed to ICMs, and their potential deleterious effects are therefore questioned as we have recently demonstrated that they enter into organisms such as the zebra mussels. To answer this question, Dreissena polymorpha were exposed to two ICMs of different osmolality, diatrizoic acid and iohexol, at an environmental concentration (100 µg/L) for 21 days before a depuration phase of 4 days. The occurrence, fate, and impact of both ICMs in these organisms were studied using a metallomic approach. Thus, iodine as well as endogenous copper and zinc were quantified and analyzed in cytosolic extracts of digestive glands, gills, and gonads of mussels by size exclusion chromatography coupled to ICP MS. This work shows that a subcellular fractionation is necessary to distinguish variations in total element content. The cytosolic iodoprotein chromatographic pattern was consistent for the three organs and confirmed the presence of ICMs in cytosols. Additionally, this exploratory work tends to show a weak biological effect of ICMs with a substantial variation of the profile of Cu-binding proteins mostly in the gill cytosol and to a lesser extent, in the digestive gland cytosol., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Biomonitoring appears to be a key approach to assess chemical or microbiological contaminations. The freshwater mussel, Dreissena polymorpha (D. polymorpha), is a suitable tool already used to monitor chemical and, more recently, microbiological pollution. In the present study, we used this sentinel species to monitor viral contamination of fecal origin over a wide geographical distribution. An active approach was implemented based on caging of calibrated and pathogen-free organisms with the same exposure conditions, allowing spatio-temporal comparisons between different water bodies. In addition, different types of sites were selected to investigate the range of environmental concentrations that D. polymorpha are able to translate. Different viral genome targets were measured: norovirus genogroup I and II (NoV GI and GII) and F-specific RNA bacteriophages belonging to the genogroup -I and -II (FRNAPH-I and -II). Total infectious FRNAPH were also monitored. D. polymorpha was able to translate a wide range of concentrations for all the viral targets studied, meaning that this sentinel species can be used for both low and highly anthropised sites. Moreover, D. polymorpha caging proved effective in achieving gradients of viral contamination of fecal origin pressure and to highlight the contribution of tributaries to the main rivers. D. polymorpha provided spatial and temporal variations of the viral contamination. It allowed to highlight the prevalence of the FRNAPH-I and -II genogroups according to the caging site. FRNAPH-II was found to be dominant in urban areas and FRNAPH-I in rural areas. This strategy uses the caging of the sentinel species D. polymorpha on selected sites with standardised analysis methods has proven to be a promising tool for characterizing viral contamination at both large and very fine scales., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Ltd.)

The detection all pathogenic enteric viruses in water is expensive, time-consuming, and limited by numerous technical difficulties. Consequently, using reliable indicators such as F-specific RNA phages (FRNAPH) can be well adapted to assess the risk of viral contamination of fecal origin in surface waters. However, the variability of results inherent to the water matrix makes it difficult to use them routinely and to interpret viral risk. Spatial and temporal variability of surface waters can lead to underestimate this risk, in particular in the case of low loading. The use of bivalve mollusks as accumulating systems appears as a promising alternative, as recently highlighted with the freshwater mussel Dreissena polymorpha, but its capacity to accumulate and depurate FRNAPH needs to be better understood and described. The purpose of this study is to characterise the kinetics of accumulation and elimination of infectious FRNAPH by D. polymorpha in laboratory conditions, formalised by a toxico-kinetic (TK) mechanistic model. Accumulation and depuration experiments were performed at a laboratory scale to determine the relationship between the concentration of infectious FRNAPH in water and the concentration accumulated by D. polymorpha. The mussels accumulated infectious FRNAPH (3-5.4 × 10 4  PFU/g) in a fast and concentration-dependent way in only 48 h, as already recently demonstrated. The second exposure demonstrated that the kinetics of infectious FRNAPH depuration by D. polymorpha was independent to the exposure dose, with a T 90 (time required to depurate 90 % of the accumulated concentration) of approximately 6 days. These results highlight the capacities of D. polymorpha to detect and reflect the viral pollution in an integrative way and over time, which is not possible with point water sampling. Different TK models were fitted based on the concentrations measured in the digestive tissues (DT) of D. polymorpha. The model has been developed to formalise the kinetics of phage accumulation in mussels tissues through the simultaneous estimation of accumulation and depuration rates. This model showed that accumulation depended on the exposure concentration, while depuration did not. Standardized D. polymorpha could be easily transplanted to the environment to predict viral concentrations using the TK model defined in the present study to predict the level of contamination of bodies of water on the basis of the level of phages accumulated by the organisms. It will be also provide a better understanding of the dynamics of the virus in continental waters at different time and spatial scales, and thereby contribute to the protection of freshwater resources., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The zebra mussel Dreissena polymorpha (Pallas, 1771) is an invasive species and a valuable bioindicator in the inland waters. Nevertheless, the biochemical reasons for the unique competitiveness of zebra mussels are not clear. This study aimed to compare the native and invasive populations of D. polymorpha in their ability to withstand the same multiple environmental challenges (i.e. chemical: microplastics and caffeine; physical: temperatures). The specimens from the invasive population in west Ukraine (Tn) and native population at lower streams of river Dnipro (south Ukraine, Kh) were sampled in the August of 2021 y. Molluscs from both populations were treated simultaneously with microplastics (MP, 1 mg L -1 , pore size 2 μm); caffeine (Caf, 20.0 μg L -1 ) at 18 °C, elevated temperature (25 °C) and MP and Caf combinations at 18 °C and 25 °C for 14 days. Untreated molluscs exposed at 18 °C represented control groups. A set of the 20 markers of oxidative stress, biotransformation, detoxification and apoptosis were assayed in the total soft tissues. From the two controls, Kh-group indicated lower stressful impact. However, both populations increased caspase-3 and GST activities and lysosomal instability in most exposures, and cholinesterase and phenoloxidase activities under the heating and combine exposures, indicating the remarkable properties to respond to new challenges and synergistic effect of mixtures. Inter-population differences were related to the metallothionein, cathepsin D, cytochrome P540 and oxidative stress responses that reflect population-dependent adverse outcome pathways. The discriminant analysis separated two populations with a substantially lesser magnitude of responses in the invasive population as a sign of higher resistance., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

The Integrated Biomarker Response (IBR) is one of the most used index in biomonitoring, especially the IBRv2 integrating a reference condition. However, some limitations remain for its routine and large-scale use. The IBRv2 is proportional to the total number of biomarkers, is dependent on the nature of biomarkers and considers all biomarkers modulations, even small and biologically non-significant. In addition, IBRv2 relies on reference values but the references are often different between each study, making it difficult to compare results between studies and/or campaigns. To overcome these limitations, the present work proposed a new index called IBR-T ("Integrated Biomarker Response - Threshold") which considers the threshold values of biomarkers by limiting the calculation of the IBR value to biomarkers with significant modulations. The IBRv2 and the IBR-T were calculated and compared on four datasets from active biomonitoring campaigns using Dreissena polymorpha, a bivalve widely used in freshwater biomonitoring studies. The comparison between indices has demonstrated that the IBR-T presents a better correlation (0.907 < r 2  < 0.998) with the percentage of biomarkers significantly modulated than the IBRv2 (0.002 < r 2  < 0.759). The IBRv2 could not be equal to 0 (0.915 < intercept <1.694) because the value was dependent on the total number of biomarkers, whereas the IBR-T reached 0 when no biomarker was significantly modulated, which appears more biologically relevant. The final ranking of sites was different between the two index and the IBR-T ranking tends to be more ecologically relevant that the IBRv2 ranking. This IBR-T have shown an undeniable interest for biomonitoring and could be used by environmental managers to simplify the interpretation of large datasets, directly interpret the contamination status of the site, use it to decision-making, and finally to easily communicate the results of biomonitoring studies to the general public., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper, (Copyright © 2023. Published by Elsevier Ltd.)

Mussels are constantly exposed to various pollutants in the environment, which can impair their immune defences against microbes and thus threaten their survival. In this study, we expand the insight into a key parameter of immune response in two mussel species by exploring the impact of exposure to pollutants or bacteria or simultaneous chemical and biological exposure on haemocyte motility. Basal haemocyte velocity in primary culture was high and increasing over time in Mytilus edulis (mean cell speed of 2.32 μm/min ± 1.57) whereas Dreissena polymorpha showed a constant and rather low cell motility with time (mean cell speed of 0.59 μm/min ± 0.1). In the presence of bacteria, the motility of haemocytes was instantly enhanced and slowed down after 90 min for M. edulis. In contrast, in vitro exposure of haemocytes to chemicals, either Bisphenol A, oestradiol, copper, or caffeine, induced an inhibition of cell motility in both mussel species. Finally, the cellular activation observed during bacterial challenges was inhibited by simultaneous exposure to bacteria and pollutants. Overall, our results indicate that chemical contaminants can alter haemocyte migration in mussels which can weaken their response to pathogens and therefore increase their susceptibility to infectious diseases., Competing Interests: Declaration of competing interest None., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Biomonitoring at the scale of the aquatic continuum and based on biomarkers, requires various representative species and a knowledge of their sensitivity to contaminants. Mussel immunomarkers are established tools for evaluating immunotoxic stress, but little is known about the consequences of an immune activation by local microorganisms on their response to pollution. This study aims to compare the sensitivity of cellular immunomarkers in two mussel species from different environments, the marine mussel Mytilus edulis (blue mussel) and the freshwater mussel Dreissena polymorpha (zebra mussel), to chemical stressors combined with bacterial challenge. Haemocytes were exposed ex vivo to the contaminants (bisphenol A, caffeine, copper chloride, oestradiol, ionomycin) for 4 h. The chemical exposures were coupled with simultaneous bacterial challenges (Vibrio splendidus and Pseudomonas fluorescens) to trigger activation of the immune response. Cellular mortality, phagocytosis efficiency and phagocytosis avidity were then measured by flow cytometry. The two mussel species had different basal levels since D. polymorpha showed higher cell mortality than M. edulis (23.9 ± 11% and 5.5 ± 3% dead cells respectively), and lower phagocytosis efficiency (52.6 ± 12% and 62.2 ± 9%), but similar phagocytosis avidity (17.4 ± 5 and 13.4 ± 4 internalised beads). Both bacterial strains led to an increase in cellular mortality (+8.4% dead cells in D. polymorpha, +4.9% in M. edulis), as well an activation of phagocytosis (+9.2% of efficient cells in D. polymorpha, +6.2% efficient cells and +3 internalised beads per cell in M. edulis). All chemicals triggered an increase in haemocyte mortality and/or phagocytotic modulations, except for bisphenol A. The two species differed in the amplitude of their response. The addition of a bacterial challenge significantly altered cell responses to chemicals with synergetic and antagonistic variations compared to a single exposure, depending on the compound used and the mussel species. This work highlights the species-specific sensitivity of mussel immunomarkers to contaminants, with or without bacterial challenge, and the necessity of considering the presence of in natura non-pathogenic microorganisms for future in situ applications of immunomarkers., Competing Interests: Declaration of competing interest None., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

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 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., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

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 1 H-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 1 H-NMR metabolomics can be an efficient approach to assess the health status of zebra mussels in the biomonitoring of aquatic environments., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

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 mussel Dreissena 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. Graphical abstract.

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

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., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

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® 33342, the membrane impermeant dye propidium iodide and yellow-green fluorescent microspheres allowed the simultaneous analysis of cytotoxicity and phagocytosis 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., Competing Interests: Competing interest statement The authors have no competing interests to declare., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

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.

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., (Copyright © 2019 Australian Society for Parasitology. Published by Elsevier Ltd. All rights reserved.)

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., (© 2018 Society for Applied Microbiology and John Wiley & Sons Ltd.)

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).

The environmental neurotoxin β- N -Methylamino-l-alanine (BMAA) has been pointed out to be involved in human neurodegenerative diseases. This molecule is known to be bioaccumulated by bivalves. However, little data about its toxic effects on freshwater mussels is available, particularly on the hemolymphatic compartment and its hemocyte cells involved in various physiological processes such as immune defenses, digestion and excretion, tissue repair, and shell production. Here we exposed Dreissena polymorpha to dissolved BMAA, at the environmental concentration of 7.5 µg of /mussel/3 days, during 21 days followed by 14 days of depuration in clear water, with the objective of assessing the BMAA presence in the hemolymphatic compartment, as well as the impact of the hemocyte cells in terms of potential cytotoxicity, immunotoxicity, and genotoxiciy. Data showed that hemocytes were in contact with BMAA. The presence of BMAA in hemolymph did not induce significant effect on hemocytes phagocytosis activity. However, significant DNA damage on hemocytes occurred during the first week (days 3 and 8) of BMAA exposure, followed by an increase of hemocyte mortality after 2 weeks of exposure. Those effects might be an indirect consequence of the BMAA-induced oxidative stress in cells. However, DNA strand breaks and mortality did not persist during the entire exposure, despite the BMAA persistence in the hemolymph, suggesting potential induction of some DNA-repair mechanisms., Competing Interests: The authors declare no conflict of interest.

Increasing discharge of industrial wastes into the environment results in pollution transfer towards hydrosystems. These activities release heavy metals such as cadmium, known as persistent pollutant that is accumulated by molluscs and exercise immunotoxicological effects. Among molluscs, the zebra mussel, Dreissena polymorpha constitutes a suitable support for freshwater ecotoxicological studies. In molluscs, homeostasis maintain is ensured in part by hemocytes that are composed of several cell populations involved in multiple physiological processes such as cell-mediated immune response or metal metabolism. Thus, hemocytes constitute a target of concern to study adverse effects of heavy metals. The objectives of this work were to determine whether immune-related endpoints assessed were of different sensitivity to cadmium and whether hemocyte functionalities were differentially affected depending on hemocyte subpopulation considered. Hemocytes were exposed ex vivo to concentrations of cadmium ranging from 10 -6 M to 10 -3 M for 21h prior flow cytometric analysis of cellular markers. Measured parameters (viability, phagocytosis, oxidative activity, lysosomal content) decreased in a dose-dependent manner with sensitivity differences depending on endpoint and cell type considered. Our results indicated that phagocytosis related endpoints were the most sensitive studied mechanisms to cadmium compared to other markers with EC 50 of 3.71±0.53×10 -4 M for phagocytic activity and 2.79±0.19×10 -4 M considering mean number of beads per phagocytic cell. Lysosomal content of granulocytes was less affected compared to other cell types, indicating lower sensitivity to cadmium. This suggests that granulocyte population is greatly involved in metal metabolism. Mitochondrial activity was reduced only in blast-like hemocytes that are considered to be cell precursors. Impairment of these cell functionalities may potentially compromise functions ensured by differentiated cells. We concluded that analysis of hemocyte activities should be performed at sub-population scale for more accurate results in ecotoxicological studies., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Dreissena polymorpha is a mussel species that invaded many lotic and lentic inland waters in Western Europe and North America. Its positive or negative interactions with biotic and abiotic components of ecosystems are numerous, making this bivalve the subject of numerous studies in ecology, ecophysiology and ecotoxicology. In these contexts, the functional characterization of the zebra mussel hemocytes is of particular interest, as hemocytes are central cells involved in vital functions (immunity, growth, reproduction) of molluscan physiology. Dreissena polymorpha circulating hemocytes populations were characterized by a combination of structural and functional analysis. Assessments were performed during two contrasted physiological periods for mussels (gametogenesis and spawning). Three hemocyte types were identified as hyalinocytes and blast-like cells for agranular hemocytes and one granulocyte population. Flow cytometry analysis of hemocytes functionalities indicated that blast-like cells had low oxidative and mitochondrial activities and low lysosomal content. Hyalinocytes and granulocytes are fully equipped to perform innate immune response. Hyalinocytes exhibit higher oxidative activity than granulocytes. Such observation is not common since numerous studies show that granulocytes are usually cells that have the highest cellular activities. This result demonstrates the significant functional variability of hemocyte subpopulations. Moreover, our findings reveal that spawning period of Dreissena polymorpha was associated with an increase of hyalinocyte percentage in relation to low levels of biological activities in hemocytes. This reduction in hemocyte activity would reflect the important physiological changes associated with the spawning period of this invasive species known for its high reproductive potential., (Copyright © 2016 Elsevier Ltd. All rights reserved.)