First evidence of the induction of domoic acid production in Pseudo-nitzschia australis by the copepod Temora longicornis from the French coast.

Diatoms of the genus Pseudo-nitzschia are widespread in marine waters. Some of them can produce the toxin domoic acid (DA) which can be responsible for amnesic shellfish poisoning (ASP) when transferred into the food web. These ASP events are of major concern, due to their ecological and socio-economic repercussions, particularly on the shellfish industry. Many studies have focused on the influence of abiotic factors on DA induction, less on the role of biotic interactions. Recently, the presence of predators has been shown to increase DA production in several Pseudo-nitzschia species, in particular in Arctic areas. In order to investigate the relationship between Pseudo-nitzschia species and grazers from the French coast, exposures between one strain of three species (P. australis, P. pungens, P. fraudulenta) and the copepod Temora longicornis were conducted for 5 days. Cellular and dissolved DA content were enhanced by 1,203 % and 1,556 % respectively after the 5-days exposure of P.australis whereas no DA induction was observed in P. pungens and P. fraudulenta. T. longicornis consumed all three Pseudo-nitzschia species. The copepod survival was not related to DA content. This study is an essential first step to better understanding the interactions between planktonic species from the French coast and highlights the potential key role of copepods in the Pseudo-nitzschia bloom events in the temperate ecosystems.

The copepod Eurytemora affinis as a relevant species to assess estuarine sediment toxicity: Effects on gene expression and swimming behavior.

Compared to freshwater ecosystems, the health status of estuarine waters remains little studied despite their importance for many species. They represent a zone of interest for Human settlements that make them the final sink of pollution in both the water column and sediment. Once in sediments, pollutants could represent a threat to benthic as well as pelagic estuarine species through resuspension events. In the Seine estuary, the copepod Eurytemora affinis has been previously presented as a relevant species to assess resuspended sediment contamination through fitness-related effects at the individual level. The aim of the present study was to use E. affinis copepods to assess estuarine sediment-derived elutriates toxicity at environmental concentrations of particles using a molecular (i.e. transcriptomics) and a behavioral approach. Two sites along the Seine estuary were sampled. The analysis of sediments reveals that both sites have the same granulometric composition and close contamination profiles with the detection of PCBs, PAHs and pyrethroid insecticides. The transcriptomic analysis reveals that exposure to elutriates from both sites triggers the dysregulation of genes involved in biological function as defense response, immunity, ecdysone pathway or neurotoxicity with 66% and 36% of shared genes at the highest concentration for Tancarville and Fatouville. This analysis also reveals a higher count of dysregulated genes in the Fatouville site compared to the Tancarville (271 vs 148) despite their close contamination profile. These results emphasize the molecular approach sensitivity to assess environmental matrix toxicity with E. affinis. The analysis of the swimming behavior of E. affinis did not highlight significant effects after elutriate exposure. However, our strategy to assess E. affinis swimming behavior allows the discrimination of basal swimming behavior i.e. dark/light velocity changes and strong thigmotaxis behavior. Thus, it represents a promising standardized tool to assess copepods swimming behavior in ecotoxicological studies.

Organ-oriented proteogenomics functional atlas of three aquatic invertebrate sentinel species.

Proteogenomic methodologies have enabled the identification of protein sequences in wild species without annotated genomes, shedding light on molecular mechanisms affected by pollution. However, proteomic resources for sentinel species are limited, and organ-level investigations are necessary to expand our understanding of their molecular biology. This study presents proteomic resources obtained from proteogenomic analyses of key organs (hepatopancreas, gills, hemolymph) from three established aquatic sentinel invertebrate species of interest in ecotoxicological/ecological research and environmental monitoring: Gammarus fossarum, Dreissena polymorpha, and Palaemon serratus. Proteogenomic analyses identified thousands of proteins for each species, with over 90% of them being annotated to putative function. Functional analysis validated the relevance of the proteomic atlases by revealing similarities in functional annotation of catalogues of proteins across analogous organs in the three species, while deep contrasts between functional profiles are delimited across different organs in the same organism. These organ-level proteomic atlases are crucial for future research on these sentinel animals, aiding in the evaluation of aquatic environmental risks and providing a valuable resource for ecotoxicological studies.

Decoupled responses of the copepod Eurytemora affinis transcriptome and its microbiota to dissolved copper exposure.

Chemical contamination is a common threat to biota thriving in estuarine and coastal ecosystems. Of particular importance is that trace metals tend to accumulate and exert deleterious effects on small invertebrates such as zooplankton, which are essential trophic links between phytoplankton and higher-level consumers in aquatic food webs. Beyond the direct effects of the contamination, we hypothesized that metal exposure could also affect the zooplankton microbiota, which in turn might further impair host fitness. To assess this assumption, copepods (Eurytemora affinis) were sampled in the oligo-mesohaline zone of the Seine estuary and exposed to dissolved copper (25 µg.L-1) over a 72-hour time period. The copepod response to copper treatment was assessed by determining transcriptomic changes in E. affinis and the alteration of its microbiota. Unexpectedly, very few genes were differentially expressed in the copper-treated copepods compared to the controls for both male and female samples, while a clear dichotomy between sex was highlighted with 80% of the genes showing sex-biased expression. In contrast, copper increased the taxonomic diversity of the microbiota and resulted in substantial compositional changes at both the phyla and genus levels. Phylogenetic reconstruction of the microbiota further suggested that copper mitigated the phylogenetic relatedness of taxa at the basal tree structure of the phylogeny, whereas it strengthened it at the terminal branches. Increased terminal phylogenetic clustering in the copper-treated copepods coincided with higher proportions of bacterial genera previously identified as copper resistant (e.g., Pseudomonas, Acinetobacter, Alkanindiges, Colwellia) and a higher relative abundance of the copAox gene encoding a periplasmic inducible multi-copper oxidase. The enrichment in micro-organisms likely to perform copper sequestration and/or enzymatic transformation processes, underlines the need to consider the microbial component during evaluation of the vulnerability of zooplankton to metallic stress.

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

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

Effects of Chemical Compounds on the Activity of the N-acetyl-ß-D-Glucosaminidase of the Marine Prawn, Palaemon serratus: Screening In Vitro.

N-acetyl-ß-D-glucosaminidase (NAGase) is important for crustaceans because the enzyme activity is necessary for the molting process. The present study aimed to assess the sensitivity of Palaemon serratus NAGase activity to a set of compounds of diverse chemical families in the context of in vitro exposures. Compounds representing different chemical families were selected according to their abundance, impact in the environment, and relevance as disruptors of the molting process. In a first step, four solvents (dimethylsulfoxide [DMSO], methanol, acetone, and ethanol) were tested to determine their suitability to dissolve hydrophobic compounds without affecting NAGase activity. Exclusively, ethanol had no effect on enzyme activity and on the integrity of the proteins present in the enzyme extract. The 18 other compounds were tested and four of these compounds, pentoxifylline, fenoxycarb, dithiocarbamate, and RH5849, showed a specific alteration on the activity of NAGase, without affecting the protein content. However, cadmium, zinc, and glyphosate showed a nonspecific alteration, affecting both the enzyme activity and the proteins, whereas ibuprofen exclusively altered the protein content. Finally, 10 of the 22 tested compounds (including DMSO, acetone, and methanol) showed a direct alteration of NAGase activity. Environ Toxicol Chem 2023;42:846-858. © 2023 SETAC.

Susceptibility of the Non-Targeted Crustacean Eurytemora affinis to the Endocrine Disruptor Tebufenozide: A Transcriptomic Approach.

Copepods are zooplanktonic crustaceans ubiquitously widespread in aquatic systems. Although they are not the target, copepods are exposed to a wide variety of pollutants such as insect growth regulators (IGRs). The aim of this study was to investigate the molecular response of a non-targeted organism, the copepod Eurytemora affinis, to an IGR. Adult males and females were exposed to two sub-lethal concentrations of tebufenozide (TEB). Our results indicate a sex-specific response with a higher sensitivity in males, potentially due to a differential activation of stress response pathways. In both sexes, exposure to TEB triggered similar pathways to those found in targeted species by modulating the transcription of early and late ecdysone responsive genes. Among them were genes involved in cuticle metabolism, muscle contraction, neurotransmission, and gametogenesis, whose mis-regulation could lead to moult, locomotor, and reproductive impairments. Furthermore, genes involved in epigenetic processes were found in both sexes, which highlights the potential impact of exposure to TEB on future generations. This work allows identification of (i) potential biomarkers of ecdysone agonists and (ii) further assessment of putative physiological responses to characterize the effects of TEB at higher biological levels. The present study reinforces the suitability of using E. affinis as an ecotoxicological model.

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

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

Controversial use of vitellogenin as a biomarker of endocrine disruption in crustaceans: New adverse pieces of evidence in the copepod Eurytemora affinis.

In recent years, the interest in the use of vitellogenin (VTG) as a biomarker of endocrine disruption in fish has led to VTG being considered as a potential tool in invertebrates. Among aquatic invertebrate models in ecotoxicology, the copepods are considered as reference species in marine, estuarine and freshwater ecosystems. In this context, we identified a VTG cDNA in Eurytemora affinis. The Ea-VTG2 cDNA is 5416bp in length with an open reading frame (ORF) of 5310bp that encodes a putative protein of 1769 amino acids residues. Phylogenetic analysis confirmed the hypothesis of a VTG duplication event before the emergence of the copepod species. The analysis of the Ea-VTG2 expression by qPCR in males and females according to their reproductive stages allowed transcript basal levels to be determined. The expression pattern revealed a gradual increase of transcript levels during maturation in females. Important inter-sex differences were observed with a VTG level in males ranging from about 1900- to 6800-fold lower than in females depending on their stage. Moreover, the protein was only detected in ovigerous females. The inducibility of Ea-VTG2 by chemicals was studied in males exposed to either a model of endocrine disruptor in vertebrates i.e. 4-nonylphenol (4-NP) or a crustacean hormone i.e. Methyl Farnesoate (MF), and in males sampled from a multi-contaminated estuary. No induction was highlighted. The VTG should not be considered as an appropriate biomarker in E. affinis as previously suggested for other crustaceans.

Bioaccumulation, distribution and elimination of chlordecone in the giant freshwater prawn Macrobrachium rosenbergii: Field and laboratory studies.

Chlordecone is a persistent organochlorine pesticide that has been widely used in Guadeloupe (French West Indies) to control the banana weevil Cosmopolites sordidus from 1972 to 1993. A few years after its introduction, widespread contamination of soils, rivers, wild animals and aquatic organisms was reported. Although high chlordecone concentrations have been reported in several crustacean species, its uptake, internal distribution, and elimination in aquatic species have never been described. This study aimed at investigating the accumulation and tissue distribution of chlordecone in the giant freshwater prawn Macrobrachium rosenbergii, using both laboratory (30 days exposure) and field (8 months exposure) approaches. In addition, depuration in chlordecone-free water was studied. Results showed that chlordecone bioconcentration in prawns was dose-dependent and time-dependent. Moreover, females appeared to be less contaminated than males after 5 and 7 months of exposure, probably due to successive spawning leading in the elimination of chlordecone through the eggs. Chlordecone distribution in tissues of exposed prawns showed that cephalothorax organs, mainly represented by the hepatopancreas, was the most contaminated. Results also showed that chlordecone was accumulated in cuticle, up to levels of 40% of the chlordecone body burden, which could be considered as a depuration mechanism since chlordecone is eliminated with the exuviae during successive moults. Finally, this study underlined the similarity of results obtained in laboratory and field approaches, which highlights their complementarities in the chlordecone behaviour understanding in M. rosenbergii.

Proteomic response of Macrobrachium rosenbergii hepatopancreas exposed to chlordecone: Identification of endocrine disruption biomarkers?

The present work is the first study investigating the impacts of chlordecone, an organochlorine insecticide, on the proteome of the decapod crustacean Macrobrachium rosenbergii, by gel-free proteomic analysis. The hepatopancreas protein expression variations were analysed in organisms exposed to three environmental relevant concentrations of chlordecone (i.e. 0.2, 2 and 20µg/L). Results revealed that 62 proteins were significantly up- or down-regulated in exposed prawns compared to controls. Most of these proteins are involved in important physiological processes such as ion transport, defense mechanisms and immune system, cytoskeleton dynamics, or protein synthesis and degradation. Moreover, it appears that 6% of the deregulated protein are involved in the endocrine system and in the hormonal control of reproduction or development processes of M. rosenbergii (e.g. vitellogenin, farnesoic acid o-methyltransferase). These results indicate that chlordecone is potentially an endocrine disruptor compound for decapods, as already observed in vertebrates. These protein modifications could lead to disruptions of M. rosenbergii growth and reproduction, and therefore of the fitness population on the long-term. Besides, these disrupted proteins could be suggested as biomarkers of exposure for endocrine disruptions in invertebrates. However, further investigations are needed to complete understanding of action mechanisms of chlordecone on proteome and endocrine system of crustaceans.

Individual and mixture acute toxicity of model pesticides chlordecone and pyriproxyfen in the estuarine copepod Eurytemora affinis.

Due to the increase in the use of phytosanitary products during the last few decades, the importance to study the effect of pesticide mixtures has been established. In this study, we investigated the acute toxicity of two model insecticides, chlordecone (CLD) and pyriproxyfen (PXF), alone and in mixtures, in the estuarine copepod Eurytemora affinis. After 48 h of exposure, the relative LC50 were 73.24 and 131.61 µg/L for PXF and CLD, respectively. The lower concentration tested (10 µg/L) did not affect the mortality of E. affinis whatever the considered chemical compound. To understand the interaction between compounds in mixture, the results were fitted to the concentration addition, Vølund, and Hewlett models. The best fit was obtained with the Hewlett model, suggesting a synergistic effect of the mixture.

Vitellogenin and vitellogenin receptor gene expression and 20-hydroxyecdysone concentration in Macrobrachium rosenbergii exposed to chlordecone.

Chlordecone is a persistent organochlorine pesticide widely used in Guadeloupe (French West Indies) to control the banana weevil Cosmopolites sordidus. Although it was previously highlighted that chlordecone may affect the reproduction and growth of vertebrate species, little information is available on the chlordecone effects in invertebrates. The present study investigated the effects of chlordecone on a hormone and a protein having key roles in reproduction and growth of the decapod crustacean Macrobrachium rosenbergii, by measuring the 20-hydroxyecdysone concentration, vitellogenin, and vitellogenin receptor gene expression, as well as the bioconcentration of chlordecone in exposed prawns. First, the results revealed that chlordecone was accumulated in M. rosenbergii. Then, it was found that Vg and VgR gene expression were increased in male and female M. rosenbergii exposed to chlordecone for 90 and 240 days, while the 20-hydroxyecdysone concentrations were decreased. This work suggests that chlordecone accumulates in prawn tissues and could affect key molecules involved in the reproduction and the growth of the invertebrate M. rosenbergii. However, many questions remain unresolved regarding the impacts of chlordecone on growth and reproduction and the signaling pathways responsible for these effects, as well as the potential role of confounding factors present in in situ studies.

Transcriptome analysis of the copepod Eurytemora affinis upon exposure to endocrine disruptor pesticides: Focus on reproduction and development.

Copepods-which include freshwater and marine species-represent the most abundant group of aquatic invertebrates. Among them, the calanoid copepod Eurytemora affinis is widely represented in the northern hemisphere estuaries and has become a species of interest in ecotoxicology. Like other non-target organisms, E. affinis may be exposed to a wide range of chemicals such as endocrine disruptors (EDs). This study investigated the gene expression variation in E. affinis after exposure to ED pesticides-chosen as model EDs-in order to (i) improve the knowledge on their effects in crustaceans, and (ii) highlight relevant transcripts for further development of potential biomarkers of ED exposure/effect. The study focused on the reproduction function in response to ED. Copepods were exposed to sublethal concentrations of pyriproxyfen (PXF) and chlordecone (CLD) separately. After 48h, males and females (400 individuals each) were sorted for RNA extraction. Their transcriptome was pyrosequenced using the Illumina(®) technology. Contigs were blasted and functionally annotated using Blast2GO(®). The differential expression analysis between ED- and acetone-exposed organisms was performed according to sexes and contaminants. Half of the 19,721 contigs provided by pyrosequencing were annotated, mostly (80%) from arthropod sequences. Overall, 2,566 different genes were differentially expressed after ED exposures in comparison with controls. As many genes were differentially expressed after PXF exposure as after CLD exposure. In contrast, more genes were differentially expressed in males than in females after both exposures. Ninety-seven genes overlapped in all conditions. Finally, 31 transcripts involved in reproduction, growth and development, and changed in both chemical exposures were selected as potential candidates for future development of biomarkers.

Effects of chlordecone on 20-hydroxyecdysone concentration and chitobiase activity in a decapod crustacean, Macrobrachium rosenbergii.

Chlordecone (CLD) is an organochlorine insecticide abundant in aquatic environment of the French West Indies. However, few studies have investigated its impact on freshwater invertebrates. Whereas CLD is suspected of inducing endocrine disruption, this work aimed to study the effects of environmentally relevant concentrations of CLD on the 20-hydroxyecdysone (20-HE) hormone concentration and on the chitobiase activity, both having key roles in the molting process of crustaceans. In addition, the bioaccumulation of CLD was measured in the muscle tissue of Macrobrachium rosenbergii to underline potential dose-response relationship. The results have shown that CLD was bioaccumulated in exposed organisms according to a trend to a dose-response relationship. Moreover, it was observed that CLD decreased the 20-HE concentration in exposed prawns when compared to control, whatever the duration of exposure, as well as it inhibited the chitobiase activity after 30days of exposure. The present study indicates that CLD could interfere with molting process of M. rosenbergii by disturbing the 20-HE concentration and the activity of chitobiase, suggesting consequences at the long term on the shrimp development. This study also confirmed that CLD could be an endocrine disruptor in decapod crustaceans, as it was already observed in vertebrates.

Differential protein expression in the estuarine copepod Eurytemora affinis after diuron and alkylphenol exposures.

Proteomics was used in the calanoid copepod Eurytemora affinis for screening of protein expression modifications induced by organic contaminants. The copepods were exposed in a continuous flow-through system for 86 h to environmentally relevant concentrations of contaminants representative of the pollution in the Seine Estuary (Haute-Normandie, France; diuron, 500 ng L(-1) ; alkylphenol mixture, 1000 ng L(-1) ). Proteome analysis of whole-body copepod extracts by 2-dimensional gel electrophoresis revealed that the contaminants induced modifications in protein expression, with the highest quantitative variations occurring after diuron exposure. Specifically, 88 and 41 proteins were differentially expressed after diuron and alkylphenol treatments, respectively. After mass spectrometry analysis, 51 (diuron exposure) and 15 (alkylphenol exposure) proteins were identified. The identified proteins were potentially related to energy metabolism, cell growth, nervous signal conductivity, excitotoxicity, oxidative stress response, and antioxidant defense. The data suggest a massive general disturbance of physiological functions of E. affinis after diuron exposure, whereas alkylphenols induced an alteration of a few targeted physiological functions. The protein expression signatures identified after contaminant exposure deserve further investigation in terms of the development of novel potential biomarkers for water quality assessment. Environ Toxicol Chem 2016;35:1860-1871. © 2015 SETAC.

Toxicity of sediment-bound pollutants in the Seine estuary, France, using a Eurytemora affinis larval bioassay.

Coastal urbanisation exposes surrounding estuarine environments to urban-related contaminants such as polycyclic aromatic hydrocarbons (PAHs), polychlorobiphenyls (PCBs) and pesticide mixtures. Hydrophobic contaminants can adsorb on estuarine sediments. They can subsequently be released on a massive scale in the aquatic environment due to artificial or natural phenomena (e.g. dredging, tides), thereby threatening living organisms. The contamination of sediment is a significant ecological issue in the Seine estuary, France. However, few relevant methods have been developed to assess sediment toxicity and its ecological impacts in a cost-effective way. In this context, we aimed to assess the toxicity of natural sediments from the Seine estuary on the development of the calanoid copepod Eurytemora affinis using a previously developed larval bioassay. This assay involves direct exposure of nauplii to elutriates of sediments for six days. Sediments were collected along the Seine estuary from six polluted sites and one reference site. Pollutants in this estuary included PAHs, PCBs and OCPs (organochlorine pesticides). Nauplius survival was significantly more affected by exposure to all contaminated sediment elutriates, than by exposure to sediment from Yville-sur-Seine (the reference site), whereas nauplius growth was significantly reduced after exposure to contaminated sediment elutriates from four of the six contaminated sites. We identified two distinct site clusters, one including both the sand-rich and the least polluted sediments (Oissel, Quillebeuf-sur-Seine, Caudebec-en-Caux) and the other including both the clay- and silt-rich, and the most polluted sediments (La Bouille, Poses, Pont de Normandie). As expected, survival was significantly more impacted after exposure to elutriates from the second cluster than from the first. This work enables (i) assessment of the toxicity of natural sediments in the Seine estuary and (ii) validation of the larval bioassay previously developed using sorbed sediment with model molecules.

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

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

The multi-xenobiotic resistance (MXR) efflux activity in hemocytes of Mytilus edulis is mediated by an ATP binding cassette transporter of class C (ABCC) principally inducible in eosinophilic granulocytes.

In marine and estuarine species, immunotoxic and/or immunomodulatory mechanisms are the crossroad of interactions between xenobiotics, microorganisms and physicochemical variations of the environment. In mussels, immunity relies exclusively on innate responses carried out by cells collectively called hemocytes and found in the open hemolymphatic circulatory system of these organisms. However, hemocytes do not form a homogenous population of immune cells since distinct subtypes of mussel blood cells can be distinguished by cytochemistry, flow cytometry or cell motility analysis. Previous studies have also shown that these cells are able to efflux xenobiotics by means of ATP binding cassette (ABC) transporter activities conferring a multixenobiotic resistance (MXR) phenotype. ABC transporters corresponding to vertebrate class B/P-glycoprotein (P-gp) and to class C/multidrug resistance related protein (MRP) are characterized in Mytilidae. Herein, we have investigated the relative contributions of ABCB- and ABCC-mediated efflux within the different hemocyte subpopulations of Mytilus edulis mussels, collected from areas differentially impacted by chemical contaminants in Normandy (France). RT-PCR analyses provide evidence for the presence of ABCB and ABCC transporters transcripts in hemocytes. Immunodetection of ABCB/P-gp with the monoclonal antibody UIC2 in living hemocytes revealed that expression was restricted to granular structures of spread cells. Efflux transporter activities, with calcein-AM as fluorescent probe, were measured by combining flow cytometry to accurate Coulter cell size measurements in order to get a cell-volume normalized fluorescence concentration. In these conditions, basal fluorescence levels were higher in hemocytes originating from Yport (control site) than in cells collected from the harbor of Le Havre, where mussels are more exposed to with persistent pollutants. By using specific ABCB/P-gp (verapamil, PSC833, zosuquidar) and ABCC/MRP (MK571) blockers, we show that MXR activity is only carried out by MRP-type transporters in M. edulis hemocytes. In addition, cell-type-gated flow cytometry and calculation of the MXR activity factor indicate that ABCC-efflux activity is higher and more inducible in eosinophilic granulocytes than in other hemocyte subtypes. We conclude that, in the hemocytes of M. edulis, MXR phenotype is mediated by an ABCC/MRP-type transporter activity principally supported by eosinophilic granulocytes. A role for ABC transporters in hemocyte migration is discussed.

Development of a larval bioassay using the calanoid copepod, Eurytemora affinis to assess the toxicity of sediment-bound pollutants.

Hydrophobic pollutants, in particular sediment-sorbed organic compounds, are widespread in the aquatic environment and could represent a threat to living organisms. Estuarine species, which live in turbulent ecosystems, are particularly exposed to this mode of contamination. For precise evaluation of the toxicity of hydrophobic contaminants desorbed from particles, a new larval assay using nauplii of the estuarine calanoid copepod Eurytemora affinis was developed. It consists of the direct exposure of copepods during naupliar development to elutriates of an unpolluted sediment spiked with different model contaminants. This bioassay measures the toxicity of the bioavailable fraction of particle-sorbed pollutants on the naupliar stage of copepods. Mortality and growth (non-invasive endpoints) in nauplii were analysed after six days of exposure. This approach was validated using six pollutants with different modes of action: benzo[a]pyrene (BaP), dimethylbenzo[a]anthracene (DMBA), phenanthrene (PHE), polychlorinated biphenyls (PCB 126, PCB 153) and 4-nonylphenol (4-NP). All these compounds induced a dose-dependent increase in toxic effects. Lethal effects only occurred at the highest tested concentrations: 58,541 and 6092 ng g(-1) dry weight sediment (dws), for PHE and DMBA, respectively. Sublethal effects (growth inhibition) were observed at lower concentrations for all tested compounds except PCB 153, from 8, 142, 297, 6092 and 8453 ng g(-1) dws for PCB 126, BaP, PHE, DMBA and 4-NP, respectively.