Advancing environmental monitoring across the water continuum combining biomarker analysis in multiple sentinel species: A case study in the Seine-Normandie Basin (France).

Nowadays, biomarkers are recognized as valuable tools to complement chemical and ecological assessments in biomonitoring programs. They provide insights into the effects of contaminant exposures on individuals and establish connections between environmental pressure and biological response at higher levels. In the last decade, strong improvements in the design of experimental protocols and the result interpretation facilitated the use of biomarker across wide geographical areas, including aquatic continua. Notably, the statistical establishment of reference values and thresholds enabled the discrimination of contamination effects in environmental conditions, allowed interspecies comparisons, and eliminated the need of a reference site. The aim of this work was to study freshwater-estuarine-coastal water continua by applying biomarker measurements in multi-species caged organisms. During two campaigns, eight sentinel species, encompassing fish, mollusks, and crustaceans, were deployed to cover 25 sites from rivers to the sea. As much as possible, a common methodology was employed for biomarker measurements (DNA damage and phagocytosis efficiency) and data interpretation based on guidelines established using reference values and induction/inhibition thresholds (establishment of three effect levels). The methodology was successfully implemented and allowed us to assess the environmental quality. Employing multiple species per site enhances confidence in observed trends. The results highlight the feasibility of integrating biomarker-based environmental monitoring programs across a continuum scale. Biomarker results align with Water Framework Directive indicators in cases of poor site quality. Additionally, when discrepancies arise between chemical and ecological statuses, biomarker findings offer a comprehensive perspective to elucidate the disparities. Presented as a pilot project, this work contributes to gain insights into current biomonitoring needs, providing new questions and perspectives.

Comparative immune responses of blue mussel and zebra mussel haemocytes to simultaneous chemical and bacterial exposure.

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.

Modulation of haemocyte motility by chemical and biological stresses in Mytilus edulis and Dreissena polymorpha.

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.

Metal Contamination and Biomarkers in Cerastoderma glaucum: A Multi-level Approach.

In this study, we focused on evaluating the responses of the cockle, Cerastoderma glaucum to in situ exposures to metals at three sites in the Gulf of Gabes in the coastal zone of Tunisia differing in levels of metal contamination. Firstly, we examined the general physiological state of the organisms. Secondly, we evaluated the bioaccumulation of several metals (Cd, Cu, Zn, Ni) in the cockles. Thirdly, we focused on evaluating histologically changes in gametogenesis and sexual maturity of the organisms. Finally, we determined the expression of seven key genes encoding enzymes or proteins involved in responses to different types of environmental stressors. Results showed a decrease in the general physiological status of the cockles, including a reduced condition index, sex ratios skewed to females (70% and 80% females in the intermediate and the contaminated site, respectively) and greater mortalities in tests under anoxic conditions (i.e., stress on stress test) in cockles collected from the most contaminated site (LT50 = 2.88 days) compared to the cockles from the intermediate site (LT50 = 5 days) and the less contaminated site (LT50 = 6 days). Results for metal bioaccumulation showed that the levels of Cd, Cu, Zn and Ni in cockles were consistent with the contaminant gradient, with the highest levels in cockles from the most contaminated site (1.04; 4.92; 52.76 and 13.81 µg/g dw, respectively), followed by those from the intermediate site (0.34; 2.94; 36.94; 17.40 µg/g dw, respectively) and then the less contaminated site (0.065; 1.27; 21.62 and 5.40 µg/g dw, respectively). Results from the gametogenesis and maturity index showed few differences in the reproductive cycle of cockles collected from the three study sites. There were different patterns of gene expression that were divided into three groups in terms of responses: (1) expression of genes involved in metal detoxification, ATP Binding Cassette Subfamily B Member 1 (ABCB1) and metallothionein MT) and genes for superoxide dismutases (i.e., Mn SOD and CuZn SOD), which did not show any difference in their levels of expression; (2) heat shock protein 70 (HSP70) gene expression, which decreased in cockles according to the pollution gradient, and (3) expression of catalase (CAT) and cytochrome oxidase subunit 1 (COI) genes was threefold and 1000-fold higher in cockles from intermediate and most contaminated sites compared to the less contaminated site. Therefore, changes in overall physiological condition, sex ratios and expression of HSP70, CAT and COI genes may be appropriate biomarkers for in situ studies of the impacts of metals in cockles. However, these biomarkers should be coupled to proteomics studies.

Integrative biomarker response - Threshold (IBR-T): Refinement of IBRv2 to consider the reference and threshold values of biomarkers.

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 < r2 < 0.998) with the percentage of biomarkers significantly modulated than the IBRv2 (0.002 < r2 < 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.

Blue mussel (Mytilus edulis)-A bioindicator of marine water contamination by protozoa: Laboratory and in situ approaches.

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.

Evaluation of real-time qPCR-based methods to detect the DNA of the three protozoan parasites Cryptosporidium parvum, Giardia duodenalis and Toxoplasma gondii in the tissue and hemolymph of blue mussels (M. edulis).

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.

First evidence of SARS-CoV-2 genome detection in zebra mussel (Dreissena polymorpha).

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.

Subcellular Distribution of Dietary Methyl-Mercury in Gammarus fossarum and Its Impact on the Amphipod Proteome.

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.

Toxic effects of a mixture of five pharmaceutical drugs assessed using Fontinalis antipyretica Hedw.

The potential health risks associated with the pharmaceuticals released into the environment through effluents from sewage treatment plants have become a major cause for concern. Owing to the lack of effective indicators, monitoring the concentration of these pollutants in the aquatic environment is challenging. The aim of this study was to assess the toxicity of a mixture of five pharmaceutical drugs (paracetamol, carbamazepine, diclofenac, irbesartan, and naproxen) using the aquatic moss Fontinalis antipyretica as a bioindicator and bioaccumulator. We examined the effects of the drug mixture on the cellular antioxidant system, chlorophyll content, and morphological traits of F. antipyretica. The plant was exposed for 5 months to three concentrations of the mixture, including the environmental concentration (MX1), and 10- (MX10) and 100-times (MX100) the environmental concentration. The results showed that only carbamazepine and irbesartan were accumulated by the species. The bioconcentration level increased with exposure time, with the maximum uptake at the 4th month of exposure. The increase in bioaccumulation with exposure time was more evident in plants exposed to MX100. Analysis of the activity of antioxidant enzymes showed that superoxide dismutase (SOD, EC 1.15.1.1.) and catalase (EC 1.11.1.6.) were highly sensitive to the drug mixture. The activity of the enzymes was significantly higher in plants exposed to MX100; however, the activity of guaiacol peroxidase (GPX, EC 1.11.1.7.) was not significantly affected. Plants exposed to MX10 and MX100 had significantly lower total chlorophyll content and chlorophyll a/b ratio compared with those of plants in the control group; however, photosynthetic activity was restored after 5 months of exposure. The morphological characteristics of F. antipyretica were less sensitive to the treatment conditions.

Water quality of the Meuse watershed: Assessment using a multi-biomarker approach with caged three-spined stickleback (Gasterosteus aculeatus L.).

The use of a multi-biomarker approach with three-spined sticklebacks (Gasterosteus aculeatus) through an active biomonitoring strategy appears to be a promising tool in water quality assessment. The present work proposes to assess the efficiency of these tools in the discrimination of some sites in a large scale on the Meuse basin in Europe. The study was part of an EU program which aims to assess water quality in the Meuse across the French-Belgian border. Sticklebacks were caged 21 days upstream and downstream from the wastewater treatment plants (WWTPs) of Namur (Belgium), Charleville-Mézières (France), Bouillon (Belgium) and Avesnes-sur-Helpe (France). First, the state of a variety of physiological functions was assessed using a battery of biomarkers that represented innate immunity (leucocyte mortality and distribution, phagocytosis activity, respiratory burst), antioxidant system (GPx, CAT, SOD and total GSH content), oxidative damages to the membrane lipids (TBARS), biotransformation enzymes (EROD, GST), synaptic transmission (AChE) and reproduction system (spiggin and vitellogenin concentration). The impacts of the effluents were first analysed for each biomarker using a mixed model ANOVA followed by post-hoc analyses. Secondly, the global river contamination was assessed using a principal component analysis (PCA) followed by a hierarchical agglomerative clustering (HAC). The results highlighted a small number of effects of WWTP effluents on the physiological parameters in caged sticklebacks. Despite a significant effect of the "localisation" factor (upstream/downstream) in the mixed ANOVA for several biomarkers, post-hoc analyses revealed few differences between upstream and downstream of the WWTPs. Only a significant decrease of innate immune responses was observed downstream from the WWTPs of Avesnes-sur-Helpe and Namur. Other biomarker responses were not impacted by WWTP effluents. However, the multivariate analyses (PCA and HAC) of the biomarker responses helped to clearly discriminate the different study sites from the reference but also amongst themselves. Thus, a reduction of general condition (condition index and HSI) was observed in all groups of caged sticklebacks, associated with a weaker AChE activity in comparison with the reference population. A strong oxidative stress was highlighted in fish caged in the Meuse river at Charleville-Mézières whereas sticklebacks caged in the Meuse river at Namur exhibited weaker innate immune responses than others. Conversely, sticklebacks caged in the Helpe-Majeure river at Avesnes-sur-Helpe exhibited higher immune responses. Furthermore, weak defence capacities were recorded in fish caged in the Semois river at Bouillon. This experiment was the first to propose an active biomonitoring approach using three-spined stickleback to assess such varied environments. Low mortality and encouraging results in site discrimination support the use of this tool to assess the quality of a large number of water bodies.

A new protocol for the simultaneous flow cytometric analysis of cytotoxicity and immunotoxicity on zebra mussel (Dreissena polymorpha) hemocytes.

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.

Cellular and molecular complementary immune stress markers for the model species Dreissena polymorpha.

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.

From shotgun to targeted proteomics: rapid Scout-MRM assay development for monitoring potential immunomarkers in Dreissena polymorpha.

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.

Comparative evaluation of loop-mediated isothermal amplification (LAMP) vs qPCR for detection of Toxoplasma gondii oocysts DNA in mussels.

The apicomplexan parasite Toxoplasma gondii can infect humans and cause toxoplasmosis. T. gondii has been highly prioritized among the foodborne parasites regarding its global impact on public health. Human infection can occur through multiple routes, including the ingestion of raw or undercooked food contaminated with T. gondii oocysts, such as fresh produce and bivalves. As filter-feeders, bivalves can accumulate and concentrate contaminants, including protozoan (oo)cysts. Although detection of T. gondii in different bivalves by molecular techniques (PCR and qPCR) has been achieved, routine application is currently limited by lack of sensitivity or equipment costs. Here, we describe the assessment of a loop-mediated isothermal amplification (LAMP)-based assay to detect T. gondii oocysts in spiked mussels. Detection limit was down to 5 oocysts/g in tissue and 5 oocyst/ml in hemolymph, and, under the experimental conditions tested, LAMP was found to provide a promising alternative to qPCR.

Effects of a chronic exposure to different water temperatures and/or to an environmental cadmium concentration on the reproduction of the threespine stickleback (Gasterosteus aculeatus).

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).

Impact of confinement and food access restriction on the three-spined stickleback (Gasterosteus aculeatus, L.) during caging: a multi-biomarker approach.

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.

Procedures for leukocytes isolation from lymphoid tissues and consequences on immune endpoints used to evaluate fish immune status: A case study on roach (Rutilus rutilus).

The effects of two protocols (density gradient versus hypotonic lysis) used for leukocyte isolation from three major lymphoid tissue of fish (head-kidney, spleen and blood) were examined on some cell functional activities (tissue leucocytes distributions, phagocytosis, basal and burst oxidative activities) classically used to estimate the fish immune status. Experiments were conducted on roach (Rutilus rutilus), a cyprinid fish model often studied in different eco-physiological contexts (aquaculture, ecotoxicology …). All of immune endpoints were assessed either immediately after cell isolation or after a 12 h of incubation in order to observe if a post-isolation incubation may influence the leukocytes activities. Compared to the density gradient, hypotonic lysis is associated with granulocytes enrichments of cell suspensions. This is particularly true for leukocyte suspensions isolated from head kidney where granulocytes are naturally abundant. However, important variabilities in leukocyte distributions were observed in head kidney and spleen cells samples obtained by the use of hypotonic lysis for two incubation conditions used (no incubation or 12 h of incubation at 4 °C). The density gradient protocol leads to a transitory increase in basal ROS production in spleen lymphocytes and macrophages The blood leukocytes isolated by this same method exhibit high basal oxidative activities after 12 h of incubation at 4 °C and for the three leukocyte types (lymphocytes, monocytes and granulocytes). The hypotonic lysis is associated with an increase in PMA-induced ROS production especially in head kidney leukocytes. The increases in cell oxidative activities are consistent with increases in granulocyte proportions observed in leukocyte suspensions obtained by hypotonic lysis. Finally, the two protocols have no effect on leukocyte mortality and phagocytic activity. Within limits of our experimental conditions, the spleen is the organ whose leukocyte oxidative activities (stimulated or not) are only slightly influenced by the methods used for leukocyte isolation. This is also the case for the anterior kidney, but for this tissue, it is necessary to incubate the isolated cells for 12 h at 4 °C before functional analyses. Each of the two methodologies used has advantages and disadvantages. The hypotonic lysis allows to isolate a greater variety of leukocytes types whereas the density gradient used ensures a better stability of cells distributions over time. However, for the same fish species and for the same tissue, the method used to isolate leukocytes influences results and must be taken into consideration during acquired data analysis for evaluation of fish immune status.

Differential sensitivity to cadmium of immunomarkers measured in hemocyte subpopulations of zebra mussel Dreissena polymorpha.

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 EC50 of 3.71±0.53×10-4M for phagocytic activity and 2.79±0.19×10-4M 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.

Functional features of hemocyte subpopulations of the invasive mollusk species Dreissena polymorpha.

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.