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The fate and effects of CuO nanoparticles (CuO NPs) were examined in endobenthic species (Scrobicularia plana , Hediste diversicolor), under environmentally realistic conditions in outdoor mesocosms (exposure to Cu at 10 μg L(-1) in particulate (CuO NPs) or soluble salt (CuNO(3)) forms) for 21 days. Labile Cu was determined in water and sediment by using diffusive gradient in thin films. No labile Cu being detected from CuO NPs; the observed effects in invertebrates exposed to CuO NPs were mainly attributed to the toxicity of nanoparticulate rather than dissolved Cu toxicity. Bioaccumulation of CuO NPs was observed in both species. Biomarkers were examined at different levels of biological organization: biochemical markers of defense and damage, biomarkers of genotoxicity (comet assay), and behavioral biomarkers (feeding and burrowing). Behavioral biomarkers, antioxidant defenses (catalase, glutathion S-transferase, metallothionein), and genotoxicity are the most sensitive tools to highlight the effect of soluble or nanoparticulate metal forms. Concerning other biomarkers of defense (superoxide dismutase, lactate dehydrogenase, laccase) and damage (thiobarbituric acid reactive substances, acetylcholinesterase, acid phosphatase), no significant effects were detected. This experiment shows the suitability of mesocosms for studying the environmental effects of nanoparticles.

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Phenoloxidases (POs) are a family of enzymes including tyrosinases, catecholases and laccases, which play an important role in immune defence mechanisms in various invertebrates. The aim of this study was to thoroughly identify the PO-like activity present in the hemolymph of the Pacific oyster Crassostrea gigas, by using different substrates (i.e. dopamine and p-phenylenediamine, PPD) and different PO inhibitors. In order to go deeper in this analysis, we considered separately plasma and hemocyte lysate supernatant (HLS). In crude plasma, oxygraphic assays confirmed the presence of true oxidase activities. Moreover, the involvement of peroxidase(s) was excluded. In contrast to other molluscs, no tyrosinase-like activity was detected. With dopamine as substrate, PO-like activity was inhibited by the PO inhibitors tropolone, phenylthiourea (PTU), salicylhydroxamic acid and diethyldithio-carbamic acid, by a specific inhibitor of tyrosinases and catecholases, i.e. 4-hexylresorcinol (4-HR), and by a specific inhibitor of laccases, i.e. cetyltrimethylammonium bromide (CTAB). With PPD as substrate, PO-like activity was inhibited by PTU and CTAB. In precipitated protein fractions from plasma, and with dopamine and PPD as substrates, PTU and 4-HR, and PTU and CTAB inhibited PO-like activity, respectively. In precipitated protein fractions from hemocyte lysate supernatant, PTU and CTAB inhibited PO-like activity, independently of the substrate. Taken together, these results suggest the presence of both catecholase- and laccase-like activities in plasma, and the presence of a laccase-like activity in HLS. To the best of our knowledge, this is the first time that a laccase-like activity is identified in a mollusc by using specific substrates and inhibitors for laccase, opening new perspectives for studying the implication of this enzyme in immune defence mechanisms of molluscs of high economic value such as C. gigas.

In the context of massive summer mortality events of the Pacific oyster Crassostrea gigas, the aim of this study was to investigate the early effects on genes, enzymes and haemocyte parameters implicated in immune defence mechanisms in C. gigas oysters exposed to a potentially hostile environment, i.e. to an herbicide alone or within a mixture. Following 2 h of exposure to the herbicide diuron at 1 μg L(-1), the repression of different genes implicated in immune defence mechanisms in the haemocytes and the inhibition of enzyme activities, such as laccase-type phenoloxidase (PO) in the plasma, were observed. The inhibition of superoxide dismutase (SOD) activity in the plasma was also observed after 6 and 24 h of exposure. In the mixture with the herbicides diuron and isoproturon, and the pharmaceutical ibuprofen, catecholase-type PO activity in the plasma and the percentage of phagocytosis in the haemocytes were reduced after 6 h of exposure. Our results showed that early effects on molecular, biochemical and cellular parameters can be detected in the presence of diuron alone or within a mixture, giving an insight of its potential effect in situations that can be found in natural environments, i.e. relatively high concentrations for short periods of time.

The production of melanin is a complex process involving biochemical cascades, such as the pro-phenoloxidase (proPO) system, and enzymes, such as phenoloxidases (POs). Different studies have shown a strong correlation between the decrease in PO activities and the occurrence of diseases in bivalve invertebrates, leading to mortalities in the host. Results of these studies suggest that POs could play a fundamental role in defense mechanisms in bivalves. This article reviews the fundamental knowledge on the proPO system in bivalves and the methods used to assess PO activities. Finally, this is the first report on the major findings of laboratory and field studies that indicate that a type of PO in bivalves, the laccase enzyme, is inducible and involved in the 1) immune 2) antioxidant and 3) detoxification roles in bivalves, and might be an ecological potential biomarker of environmental stress.

Phenoloxidases (POs) are a group of copper proteins including tyrosinase, catecholase and laccase. In several insects and crustaceans, antibacterial substances are produced through the PO cascade, participating in the direct killing of invading microorganisms. However, although POs are widely recognised as an integral part of the invertebrate immune defence system, experimental evidence is lacking that these properties are conserved in molluscs, and more particularly in the Pacific oyster Crassostrea gigas. In the present study, Vibrio splendidus LGP32 and Vibrio aestuarianus 02/041 growths were affected, after being treated with C. gigas haemocyte lysate supernatant (HLS), and either a common substrate of POs, l-3,4-dihydroxyphenylalanine (L-DOPA), to detect catecholase-type PO activity, or a specific substrate of laccase, p-phenylenediamine (PPD), to detect laccase-type PO activity. Interestingly, a higher bacterial growth inhibition was observed in the presence of PPD than in the presence of L-DOPA. These effects were suppressed when the specific PO inhibitor, phenylthiourea (PTU), was added to the medium. Results of the present study suggest, for the first time in a mollusc species, that antibacterial activities of HLS from C. gigas potentially involve POs, and more particularly laccase catalysed reactions.

The aim of this study was to evaluate the effects of chemically dispersed oil on an economically and ecologically important species inhabiting coasts and estuaries, the Pacific oyster Crassostrea gigas. Studies were carried out with juveniles, known to generally be more sensitive to environmental stress than adults. A set of enzyme activities involved in immune defence mechanisms and detoxification processes, i.e. superoxide dismutase (SOD), catalase, glutathione peroxidase (GPx), catecholase-type phenoloxidase (PO), laccase-type PO and lysozyme were analysed in different oyster tissues, i.e. the gills, digestive gland and mantle, and in the plasma and the haemoycte lysate supernatant (HLS) of the haemolymph. Results indicated that total PAH body burdens were 2.7 times higher in the presence than in the absence of the chemical dispersant. After 2 days of exposure to chemically dispersed oil, alkylated naphthalenes accounted for 55% of the total PAH body burden, whereas alkylated fluorenes and alkylated dibenzothiophenes accounted for 80% when the chemical dispersant was absent. Importantly, a higher number of enzyme activities were modified when oil was chemically dispersed, especially in the plasma and gills. Moreover, independently of the presence or absence of chemical dispersant, oil exposure generally inhibited enzyme activities in the gills and plasma, while they were generally activated in the mantle and haemocytes. These results suggest that the gills and plasma constitute sensitive compartments in C. gigas, and that the mantle and haemocytes may play an important role in protection against xenobiotics. Among the six enzyme activities that were analysed in these body compartments, five were modulated in the chemical dispersion (CD) treatment while only half of the enzyme activities were modulated in the mechanical dispersion treatment. Furthermore, CD treatment effects were often observed following exposure, but also during depuration periods. These results suggest that immune and/or detoxification responses are likely to be affected when dispersants are used to treat oil spills in shallow waters.