Elise David

Transcriptomic response of Argopecten purpuratus post-larvae to copper exposure under experimental conditions.

Few studies have described the molecular response of mollusk larvae to heavy metal exposure. We investigated the response of Chilean scallop Argopecten purpuratus post-larvae to copper exposure under experimental conditions. Post-larvae were maintained with and without copper stress in tanks containing netlon collectors with biofilms that were formed by the bacterium Halomonas sp. and the diatom Amphora sp., known to increase larval settlement. We focused on the analysis of the differential expression patterns of genes associated with copper response. A suppression subtractive hybridization method was used to identify copper-specific up- and down-regulated genes in the post-larvae following 4 days and 8 days exposure to 2.5 and 10 microg/l Cu(+2). This method revealed 145 different sequences corresponding to 10 major physiological functions. The expression of 15 potentially regulated genes was analyzed by real-time PCR in post-larvae at different sampling times during the copper stress. The genes chosen were alpha tubulin, elongation factor 1-alpha (EF1A), tributylin binding protein type 1 (TBT), NADH dehydrogenase subunit 2 (NADH2), cavortin, glutathione peroxidase (GPx), ferritin, heat shock protein 70 (HSP70), Pam highwire rpm1 (Phr1), insulin-like growth factor 1 (IGF1), cellulase, and ribosomal proteins: L18, L44, S3a and S15. This study contributes to the characterization of potential genetic markers that could be used in future environmental monitoring and to explore new mechanisms of stress tolerance in marine mollusk species, especially in early stages of development.

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.

Variations in gene expression levels in four European zebra mussel, Dreissena polymorpha, populations in relation to metal bioaccumulation: A field study.

The present study was performed to validate the suitability of using gene expression in zebra mussels, Dreissena polymorpha, for biomonitoring of freshwater environment. Mussels were collected in four French rivers (Meuse, Moselle, Oise and Vilaine) in spring and autumn. Relative gene expression of 9 candidate genes involved in cellular metabolic activities (Cytochrome-c-oxidase - cox, and ATP synthase - atp), detoxification process (Metallothionein - mt and Glutathion-S-Transferase - gst), oxidative stress (Catalase - cat, Superoxyde Dismutase - sod and Glutathion peroxidase - gpx) and digestive functions (Amylase - amy and Cellulase - ghf) were measured in digestive gland. Metal bioaccumulation in tissues and morphometric parameters were also analyzed to interpret molecular responses. All our results are consistent with different physiological reactions to environmental condition between zebra mussel populations. In spring, the levels of mt, sod, gpx, cat, atp, amy and ghf relative expression were significantly higher in mussels with the lowest metal bioaccumulation (the Meuse) compared to at least one of the other sites. In autumn, this higher expression levels in Meuse River were still observed for gpx, cat, atp and amy. This study has also pointed out different sources of variability in gene expression (individual size, season, trophic resources and origin of mussels) which are inevitable in natural fluctuant environment. This underlines the importance to take them into account in field study to propose a correct interpretation of biomarker responses.

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.

FOOD DEPRIVATION AND MODULATION OF HEMOCYTE ACTIVITY IN THE ZEBRA MUSSEL (DREISSENA POLYMORPHA)

ABSTRACT Bivalve hemocyte responses are modulated by numerous biotic and abiotic environmental factors. This study investigates how nutritional stress canmodulate two hemocyte functions classically studied in bivalves, i.e., phagocytic and oxidative activities. For this purpose, we exposed zebra mussels (Dreissena polymorpha) to three nutritional conditions, for 42 days: (1) one groupwas fedwith 1×106 algal cells permussel and per day, (2) a second groupwas fed with 10%of the previous food intake (1×105 cells per mussel and per day), and (3) the third one was deprived of food. Hemocyte responses were assessed by flow cytometry every week for 42 days. Food deprivation was associated with a significant decrease in granulocyte size. Phagocytic activity increased in mussels exposed to the three diets, but it was more pronounced in mussels partially deprived of food (10% food intake). Mussels exposed to stressful nutritional conditions (10% and 0%) displayed significantly decreased oxidative activity from 14 days of exposure, whereas those fed on a normal diet displayed stable intracellular oxidative activity throughout the experiment. This study shows that nutritional conditions affect hemocyte morphometry and activity. Zebra mussel sensitivity to the nutritional conditions seemed low, even with total food deprivation for 42 days. It is necessary to estimate the physiological consequences of these food conditions, especially on the energetic status of this bivalve organism but also on its ability to resist to infection.

Differentiation of sympatric zebra and quagga mussels in ecotoxicological studies: A comparison of morphometric data, gene expression, and body metal concentrations

The zebra mussel is among the best studied freshwater molluscs in ecotoxicology, but information on the quagga mussel is lacking. Considering its potential spread, we selected a river in France in which zebra and quagga mussels coexisted, and then we used genetic markers to differentiate the two species and compared morphological parameters. cDNA sequencing assays of ten genes already used in zebra mussels were performed on quagga mussels to obtain functional specific primers. Then we analyzed the expression of genes involved in cellular metabolic activities (Cytochrome-c-oxidase - cox, and ATP synthase - atp), detoxification processes (Glutathione-S-Transferase - gst), oxidative stress (Catalase - cat), and digestive functions (Amylase - amy) on the two species. Whereas morphometric analysis underlined similarities in shape between the two species, relative gene expression profiles and metal concentrations evidenced strong differences. Quagga mussels notably presented half as high concentrations in Cd and Pb, two particularly toxic elements, as zebra mussels. These results imply that i) particular attention should be paid to properly distinguish the two species considering their similar external appearance, and ii) zebra mussels cannot be replaced by quagga mussels in ecotoxicological studies without preliminary investigations on biomarker response patterns. To our knowledge, this study is the first to have undertaken such an approach in gene expression analysis in quagga mussels, and more generally to have compared such biomarker responses of zebra and quagga mussels in the field.

Mussel as a Tool to Define Continental Watershed Quality

Bivalves appear as relevant sentinel species in aquatic ecotoxicology and water quality assessment. This is particularly true in marine ecosystems. In fact, several biomonitoring frameworks in the world used mollusks since several decades on the base of contaminant accumulation (Mussel Watch, ROCCH) and/or biological responses called biomarker (OSPAR) measurements. In freshwater systems, zebra and quagga mussels could represent alternative sentinels, which could be seen as the counterparts of mussel marine species. This chapter presents original studies and projects underlying the interest of these freshwater mussels for water quality monitoring based on contaminant accumulation and biomarker development measurements. These sentinel species could be used as a tool for chemical/biological monitoring of biota under the European water framework directive and for the development of effect-based monitoring tools.