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.

In vitro immunotoxicology of quantum dots and comparison with dissolved cadmium and tellurium.

The increasing use of products derived from nanotechnology has raised concerns about their potential toxicity, especially at the immunocompetence level in organisms. This study compared the immunotoxicity of cadmium sulfate/cadmium telluride (CdS/Cd-Te) mixture quantum dots (QDs) and their dissolved components, cadmium chloride (CdCl2 )/sodium telluride (NaTeO3 ) salts, and a CdCl2 /NaTeO3 mixture on four animal models commonly used in risk assessment studies: one bivalve (Mytilus edulis), one fish (Oncorhynchus mykiss), and two mammals (mice and humans). Our results of viability and phagocytosis biomarkers revealed that QDs were more toxic than dissolved metals for blue mussels. For other species, dissolved metals (Cd, Te, and Cd-Te mixture) were more toxic than the nanoparticles (NPs). The most sensitive species toward QDs, according to innate immune cells, was humans (inhibitory concentration [IC50 ] = 217 µg/mL). However, for adaptative immunity, lymphoblastic transformation in mice was decreased for small QD concentrations (EC50 = 4 µg/mL), and was more sensitive than other model species tested. Discriminant function analysis revealed that blue mussel hemocytes were able to discriminate the toxicity of QDs, Cd, Te, and Cd-Te mixture (Partial Wilk's λ = 0.021 and p < 0.0001). For rainbow trout and human cells, the immunotoxic effects of QDs were similar to those obtained with the dissolved fraction of Cd and Te mixture. For mice, the toxicity of QDs markedly differed from those observed with Cd, Te, and dissolved Cd-Te mixture. The results also suggest that aquatic species responded more differently than vertebrates to these compounds. The results lead to the recommendation that mussels and mice were most able to discriminate the effects of Cd-based NPs from the effects of dissolved Cd and Te at the immunocompetence level.

Transcriptomic and cellular response to bacterial challenge (pathogenic Vibrio parahaemolyticus) in farmed juvenile Haliotis rufescens fed with or without probiotic diet.

The abalone production in Chile has increased considerably in recent years with no sign of tapering off. Open and semi-closed circuits in the marine water zones in the north and south of Chile are the preferred areas of culture. Coastal ecosystems are subjected to a wide variety of contaminants that generate stress that affects populations via their impacts to individuals at both physiological and genetic levels. This work investigated the genomic and cellular response of post-weaning juvenile Haliotis rufescens abalone under hatchery conditions, fed with probiotic diets, and subsequently challenged with Vibrio parahaemolyticus. The expression patterns of 16 selected genes associated with different metabolic pathways were analyzed using Real-Time PCR. Gene expression was then compared to immunological response parameters in the abalone and quantification of V. parahaemolyticus during the experimental period. Both transcriptomic and immunological analyses indicated significant alteration of physiological processes in H. rufescens correlated to exposure to the pathogenic bacteria, as well as to probiotic nutrition.

Does the environmental history of mussels have an effect on the physiological response to additional stress under experimental conditions?

Expected effects on marine biota of the ongoing elevation of water temperature and high latitudes is of major concern when considering the reliability of coastal ecosystem production. To compare the capacity of coastal organisms to cope with a temperature increase depending on their environmental history, responses of adult blue mussels (Mytilus spp.) taken from two sites differentially exposed to chemical pollution were investigated during an experimental exposure to a thermal stress. Immune parameters were notably altered by extreme warming and transcriptional changes for a broad selection of genes were associated to the temperature increase following a two-step response pattern. Site-specific responses suggested an influence of environmental history and support the possibility of a genetic basis in the physiological response. However no meaningful difference was detected between the response of hybrids and M galloprovincialis. This study brings new information about the capacity of mussels to cope with the ongoing elevation of water temperature in these coastal ecosystems.

In vivo effects of the soluble fraction of light cycle oil on immune functions in the European sea bass, Dicentrarchus labrax (Linné).

Hydrocarbons are major contaminants that may affect biota at various trophic levels in estuaries and coastal ecosystems. The effects of accidental pollution by light cycle oil (LCO), a refined product of heavy fuel oil, on bioaccumulation, depuration processes and immune-related parameters in the European sea bass, Dicentrarchus labrax, were investigated in the laboratory after 7 days of exposure and a 2-week recovery period. Exposure of fish to the soluble fraction of LCO (1600ngL(-1)) for 7 days led to the bioaccumulation of some polycyclic aromatic hydrocarbons (PAHs) in muscles: naphthalene, acenaphthene, fluorene, phenanthrene and anthracene. After 7 days of recovery period, half-elimination of naphthalene was reported in fish muscles due to facilitated diffusive loss by the epithelium and a faster elimination rate proven by the presence of a high level of naphthalene biliary metabolites. The other bioaccumulated molecules displayed a slower depuration rate due to their elimination by the formation of hydrophobic metabolites excreted through bile or urine. Three days after the beginning of the recovery period, each contaminated fish showed severe external lesions (tissue necrosis, suppurative exudates, haemorrhagic area). The hypothesis of a possible link with inflammatory phenomenon was supported by (i) an inversion of the leucocyte sub-population percentage, (ii) a significant up-expression in the spleen of the tumour necrosis factor alpha gene, (iii) a significant increase in ACH(50). Moreover, the lack of C3 gene regulation in the spleen suggested a non-renewal of this component. The reduction of phagocytic activity and lysozyme concentration reflected immune suppression. Finally, LCO toxicity in this fish was clearly demonstrated to be related to inflammatory reaction and immune depletion.

Correction to: From legacy pollutants to emerging contaminants: recent inputs from the 2018 conference of EcoBIM network.

The original publication of this paper contains a mistake.

Immune effects of HFO on European sea bass, Dicentrarchus labrax, and Pacific oyster, Crassostrea gigas.

The European sea bass, Dicentrarchus labrax, and the Pacific oyster, Crassostrea gigas, were exposed to a soluble fraction of heavy fuel oil for 5 and 9 days, respectively. The organisms were then transferred to non-contaminated seawater for 1 month. The bioaccumulation and elimination of PAHs in contaminated tissues were dissimilar between species. In fish, acenaphthene and naphthalene were detected and naphthalene was still detectable 30 days after the beginning of the recovery period. In oysters, on the other hand, pyrene and phenanthrene were bioaccumulated and 14 days after exposure no more PAHs were detected. Concerning innate immune parameters, the increase of haemolytic activity of the alternative complement pathway in fish and the reduction of phenoloxidase activity in oysters endured, respectively, 1 and 2 weeks in contaminated organisms. This indicates that these two enzymatic cascades could be quite useful for monitoring pollution by oil.

Biomarkers as tools for monitoring within the Water Framework Directive context: concept, opinions and advancement of expertise.

The Water 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 chemicals makes it possible to properly discriminate the role of toxicants within 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.

Flow cytometric measurement of the clearance rate in the blue mussel Mytilus edulis and the development of a new individual exposure system for aquatic immunotoxicological studies.

Animals in poor health condition are not relevant biological models. The current study focused on the use of the clearance rate of Mytilus edulis to assess the gross physiological condition of individuals maintained in stressful experimental conditions. This approach was developed in a new, highly controlled experimental exposure device designed to investigate individual responses in aquatic ecotoxicological studies. Both clearance rate values and immune parameters analysis indicated that the health condition of mussels kept in 50ml tubes for 24h or 48h was not altered compared to controls, while most parameters were depressed after 72h. Moreover, this study confirms the relevance of flow cytometric for the measurement of clearance rate compared to techniques utilizing microscopy. Current results prompted us to perform further 24h chemical exposure using this "in tubo" device.

Multiple experimental approaches of immunotoxic effects of mercury chloride in the blue mussel, Mytilus edulis, through in vivo, in tubo and in vitro exposures.

Biological impairments due to mercury discharge into the environment are now an issue of global concern. From the three forms of mercury found in aquatic ecosystems, the immunotoxic effects of mercury chloride were examined in the model animal, the blue mussel. In order to investigate the toxic potency of this chemical, three exposure regimes were carried out: chronic exposure of groups of individuals, a new protocol "in tubo" designed for sub-acute exposures of individuals, and acute exposures of target cells. Chronic exposure revealed significant immunotoxic effects after 7 days at 10(-6)M, while acute exposures showed significant inhibition of phagocytosis at 10(-4)M and 10(-3)M. In sub-acute exposures both circulating haemocytes and haemocyte mortality increased at 10(-4)M and 10(-3)M while phagocytosis and the clearance rate drew hormetic toxic effects on healthy individuals. These results suggest the use of the "in tubo" design for bivalve toxicological individual studies.

Field biomonitoring using the zebra mussel Dreissena polymorpha and the quagga mussel Dreissena bugensis following immunotoxic reponses. Is there a need to separate the two species?

The zebra mussel, Dreissena polymorpha constitutes an extensively used sentinel species for biomonitoring in European and North American freshwater systems. However, this invasive species is gradually replaced in freshwater ecosystem by Dreissena bugensis, a closely related dreissenid species that shares common morphological characteristics but possess some physiological differences. However, few are known about differences on more integrated physiological processes that are generally used as biomarkers in biological monitoring studies. Declining of zebra mussel populations raises the question of the sustainability of using one or both species indifferently to maintain the quality of environmental pollution monitoring data. In our study, we performed a field comparative study measuring immune-related markers and bioaccumulation of PCBs, PAHs and PBDEs in sympatrically occurring mussel populations from three sites of the St. Lawrence River. For tested organisms, species were identified using RFLP analysis. Measurement of bioaccumulated organic compounds indicated a higher accumulation of PCBs and PBDEs in D. bugensis soft tissues compared to D. polymorpha while no differences were noticed for PAHs. Results of hemocytic parameters highlighted that differences of hemocyte distributions were associated to modulations of phagocytic activities. Moreover, marked differences occurred in measurement of hemocytic oxidative activity, indicating divergences between the two species for ROS regulation strategies. This physiological characteristic may deeply influence species responses facing environmental or pollution related stress and induce bias if the two species are not differentiated in further biomarker or bioaccumulation measurement-based studies.

Effect of Temperature on Immunocompetence of the Blue Mussel (Mytilus Edulis).

The blue mussel is a filter-feeding bivalve commonly used in ecotoxicological monitoring as a sentinel species. Due to climate change and the increase of temperature expected in marine environment, it is important to anticipate potential impacts on this species. The aim of this study was to investigate the immunocompetence of blue mussels acclimated to different temperatures and on the effects of increasing temperatures (5, 10 and 20°C). Different indices and gonad maturation stages were also determined throughout the experiments. Cell viability, phagocytosis, serum lysozyme activity and cyclooxygenase (COX) activity were evaluated as immune parameters. The cellular immunity was also evaluated after hemocytes exposure to various cadmium concentrations in vitro. The results obtained demonstrate modulation of hemocyte viability and the ability of these cells to phagocytize in absence of contaminants. After the exposure to cadmium, hemocytes showed greater viability at 5°C while maintaining a higher phagocytic competence. In addition, the lysozyme activity stayed stable at all tested temperatures, contrary to that of COX, which increased when the mussels were maintained at 20°C. The evaluation of indices demonstrated no reduction of general conditions during all the experiment despite the increase of temperature and the reduction of the digestive gland weight. Moreover, the lack of food does not affect gonad maturation and the spawning process.

Active and passive biomonitoring suggest metabolic adaptation in blue mussels (Mytilus spp.) chronically exposed to a moderate contamination in Brest harbor (France).

Brest harbor (Bay of Brest, Brittany, France) has a severe past of anthropogenic chemical contamination, but inputs tended to decrease, indicating a reassessment of its ecotoxicological status should be carried out. Here, native and caged mussels (Mytilus spp.) were used in combination to evaluate biological effects of chronic chemical contamination in Brest harbor. Polycyclic aromatic hydrocarbon (PAH) contamination was measured in mussel tissues as a proxy of harbor and urban pollution. Biochemical biomarkers of xenobiotic biotransformation, antioxidant defenses, generation of reducing equivalents, energy metabolism and oxidative damage were studied in both gills and digestive glands of native and caged mussels. In particular, activities of glutathione-S-transferase (GST), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), NADP-dependent isocitrate dehydrogenase (IDP), pyruvate kinase (PK) and phosphoenolpyruvate carboxykinase (PEPCK) were measured and lipid peroxidation was assessed by malondialdehyde (MDA) quantification. In addition, a condition index was calculated to assess the overall health of the mussels. Moderate PAH contamination was detected in digestive glands of both native and caged individuals from the exposed site. Modulations of biomarkers were detected in digestive glands of native harbor mussels indicating the presence of a chemical pressure. In particular, results suggested increased biotransformation (GST), antioxidant defenses (CAT), NADPH generation (IDP) and gluconeogenesis (PEPCK), which could represent a coordinated response against chemically-induced cellular stress. Lipid peroxidation assessment and condition index indicated an absence of acute stress in the same mussels suggesting metabolic changes could, at least partially, offset the negative effects of contamination. In caged mussels, only GR was found modulated compared to non-exposed mussels but significant differences in oxidative stress and energy-related biomarkers were observed compared to native harbor mussels. Overall, these results suggested mussels chronically exposed to contamination have set up metabolic adaptation, which may contribute to their survival in the moderately contaminated harbor of Brest. Whether these adaptive traits result from phenotypic plasticity or genetic adaptation needs to be further investigated.

Proteomic analysis of the European flounder Platichthys flesus response to experimental PAH-PCB contamination.

Platichthys flesus is often used as a sentinel species to monitor the estuarine water quality. In this study, we carried out an experimental contamination of fish using a PAHs/PCBs mixture, which was designed to mimic the concentrations found in the Seine estuary (C1) and 10 times these concentrations (C2). We used a proteomic approach to understand the molecular mechanisms implied in the response of P. flesus to these xenobiotics. We showed that 54 proteins were differentially accumulated in one or several conditions, which 34 displayed accumulation factors higher than two. 18 of these proteins were identified by MALDI TOF-TOF mass spectrometry. The results indicated the deregulation of oxidative stress- and glutathione metabolism-(GST, GPx) proteins as well as of several proteins belonging to the betaine demethylation pathway and the methionine cycle (BHMT, SHMT, SAHH), suggesting a role for these different pathways in the P. flesus response to chemical contamination.

Genotoxic and immunotoxic potential effects of selected psychotropic drugs and antibiotics on blue mussel (Mytilus edulis) hemocytes.

The potential toxicity of pharmaceuticals towards aquatic invertebrates is still poorly understood and sometimes controversial. This study aims to document the in vitro genotoxicity and immunotoxicity of psychotropic drugs and antibiotics on Mytilus edulis. Mussel hemocytes were exposed to fluoxetine, paroxetine, venlafaxine, carbamazepine, sulfamethoxazole, trimethoprim and erythromycin, at concentrations ranging from µg/L to mg/L. Paroxetine at 1.5 µg/L led to DNA damage while the same concentration of venlafaxine caused immunomodulation. Fluoxetine exposure resulted in genotoxicity, immunotoxicity and cytotoxicity. In the case of antibiotics, trimethoprim was genotoxic at 200 µg/L and immunotoxic at 20 mg/L whereas erythromycin elicited same detrimental effects at higher concentrations. DNA metabolism seems to be a highly sensitive target for psychotropic drugs and antibiotics. Furthermore, these compounds affect the immune system of bivalves, with varying intensity. This attests the relevance of these endpoints to assess the toxic mode of action of pharmaceuticals in the aquatic environment.

Comparative analysis of hemocyte properties from Mytilus edulis desolationis and Aulacomya ater in the Kerguelen Islands.

The capability of bivalve molluscs to respond to environmental stresses largely depends upon their cellular immunity. Accordingly, shift in habitat conditions following thermal stress or exposure to pollutants may harm sensitive species differently, thereby modulating the biodiversity of a given ecosystem by favoring stress-tolerant species. Here, we have compared the sensitivity of hemocytes from Mytilus edulis desolationis (M. edulis desolationis) and Aulacomya ater (A. ater) to acute thermal stress and exposure to cadmium. The two subantarctic species are commonly found in the same habitat in the isolated Kerguelen archipelago. Our results showed that the phagocytic activity and viability of hemocytes from both species were equally sensitive to increasing concentrations of cadmium. However, although in vitro exposure to cadmium induced apoptosis in hemocytes of M. edulis desolationis and A. ater, flow cytometric analyses showed that the apoptotic profile of both species differed greatly when using Annexin V and YO-PRO-1 as apoptotic markers. We also found that the total hemocyte counts decreased strongly in A. ater but not in M. edulis desolationis following an acute thermal stress. Taken together, these results showed that stress responses differed significantly in hemocytes from both species. This suggests that the co-existence of both species may be at risk following exposure to pollutants and/or changes in temperature.

Molecular identification and expression of heat shock cognate 70 (hsc70) and heat shock protein 70 (hsp70) genes in the Pacific oyster Crassostrea gigas.

The 70-kDa heat shock protein (Hsp) family is composed of both environmentally inducible (Hsp) and constitutively expressed (Hsc) family members. We sequenced 2 genes encoding an Hsp70 and an Hsc70 in the Pacific oyster Crassostrea gigas. The Cghsc70 gene contained introns, whereas the Cghsp70 gene did not. Moreover, the corresponding amino acid sequences of the 2 genes presented all the characteristic motifs of the Hsp70 family. We also investigated the expression of Hsp70 in tissues of oysters experimentally exposed to metal. A recombinant Hsc72 was used as an antigen to produce a polyclonal antibody to quantify soluble Hsp70 by enzyme-linked immunosorbent assay in protein samples extracted from oysters. Our results showed that metals (copper and cadmium) induced a decrease in cytosolic Hsp70 level in gills and digestive gland of oysters experimentally exposed to metal. These data suggest that metals may inhibit stress protein synthesis.

In situ hybridisation for flow cytometry: a molecular method for monitoring stress-gene expression in hemolymph cells of oysters.

In the present study, we developed a molecular method for flow cytometry to detect the effects of environmental factors on the stress-response in immunocompetent cells of the European flat oyster, Ostrea edulis. Stress-generating conditions were applied to individuals acclimated in the laboratory to environmental salinity and temperature. Oligonucleotidic probes were applied to quantify the expression of HSP/C70 or metallothionein genes. After a heat-stress, a response was reflected in hemocytes by an increased amount of HSP/C70 mRNA and quantitative changes in HSP/C70 protein expression. The technique of in situ hybridisation described here also allowed to quantify the expression of metallothionein mRNA in oysters exposed to a heavy metal-exposure. The detection of stress protein markers by using such quantitative methods could be applied in contamination studies in oysters and other bivalves where monitoring the status of hemolymph cells is required.