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.

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.

Effects of aluminium and bacterial lipopolysaccharide on oxidative stress and immune parameters in roach, Rutilus rutilus L.

Aluminium is used in diverse anthropogenic processes at the origin of pollution events in aquatic ecosystems. In the Champagne region (France), high concentrations of aluminium (Al) are detected due to vine-growing practices. In fish, little is known about the possible immune-related effects at relevant environmental concentrations. The present study analyzes the simultaneous effects of aluminium and bacterial lipopolysaccharide (LPS), alone and in combination, on toxicological biomarkers in the freshwater fish species Rutilus rutilus. For this purpose, roach treated or not with LPS were exposed to environmental concentrations of aluminium (100 µg/L) under laboratory-controlled conditions for 2, 7, 14 and 21 days. After each exposure time, we assessed hepatic lipoperoxidation, catalase activity, glutathione reductase activity and total glutathione content. We also analyzed cellular components related to the LPS-induced inflammatory response in possible target tissues, i.e. head kidney and spleen. Our results revealed a significant prooxidant effect in the liver cells and head kidney leukocytes of roach exposed to 100 µg of Al/L for 2 days. In liver, we observed more lipoperoxidation products and lower endogenous antioxidant activity levels such as glutathione reductase activity and total glutathione content. These prooxidant effects were associated with a higher oxidative burst in head kidney leukocytes, and they were all the more important in fish stimulated by LPS injection. These findings demonstrate that environmental concentrations of Al induce oxidative and immunotoxic effects in fish and are associated to an immunomodulatory process related to the inflammatory response.

Immunodetection of proteins from grapes and yeast in a white wine.

The objective of this study was to analyze the origin of proteins of a Chardonnay wine. Three various polyclonal antibodies raised against must, yeast, and bacteria proteins were produced. For microorganisms, only the secreted macromolecules were used. To this end, yeast and bacteria were cultured in a model medium under conditions close to those of winemaking. Results obtained using these specific antibodies indicate that most of the wine proteins came from grapes and many of them were glycoproteins. Some proteins of this Chardonnay wine came from the yeast; they were released during the alcoholic fermentation and consisted of high molecular weight mannoproteins. In contrast, no bacteria proteins were detected in this Chardonnay wine.

Wheat gluten used as a clarifying agent of red wines.

Bovine spongiform encephalopathy caused a situation of crisis leading the public and winemakers to lose their confidence in the use of gelatin as a fining agent and to reject animal proteins in general. Therefore, we started the search for a substitute for gelatin and egg protein by comparing gluten with these fining treatments currently used. This study concerned the fining of a Burgundy red wine (Rully, Controlled Appellation). For 6 g/hL, enzymatically hydrolyzed glutens (EHG) gave better efficiencies than deamidated glutens. The efficiency of the egg proteins treatment was situated between those of the hydrolyzed glutens and deamidated glutens. For 12 and 18 g/hL, turbidities of the wine treated by five glutens were 67 to 86% less than that of the control wine. Better results were obtained with egg proteins for short kinetics particularly. Wine fining with gluten was always better than gelatin treatments. The differences between the five glutens became very small when the dose incorporated in the wine increased. The volumes of lees generated by fining with gluten are situated between the values obtained with egg proteins and gelatin. After fining, immunodetection with gluten polyclonal antibodies failed to detect residual deamidated gluten.

Detection and quantification of lysozyme in champagne wines.

We describe here techniques to detect and quantify lysozyme in Pinot noir and Chardonnay Champagne wines. Using a dot-blot technique, lysozyme antibodies were able to recognize their antigens even when the concentration of lysozyme in wine was 75 mg/L. SDS-PAGE was the second technique used. After Coomassie Brilliant Blue (CBB) staining or antibody immunostaining was performed, the wine originating from the lysozyme-treated must gave only one band corresponding to the lysozyme. It is then possible to precisely determine the concentration of lysozyme in a must or a wine by densitometric measurement of this band. The control wine gave no band with the CBB staining, such as with the immunostaining. The quantification of lysozyme with HPLC is another useable technique because the lysozyme elution time is largely superior to that of all of the wine compounds. In wines, losses of lysozyme were higher when the enzyme was added at one time to the must (-34% for the Pinot noir and -37% for the Chardonnay) than when lysozyme is added in 2-fold both in the must and in the wine (around -26% for the two wines). The lowest diminution is observed when lysozyme was added to the wine only (-18%) in comparison to the addition to the must at 300 mg/L (-43%).