Recent findings on phenoloxidases in bivalves.

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.

Persistent organic pollutants in a marine bivalve on the Marennes-Oléron Bay and the Gironde Estuary (French Atlantic Coast) - part 2: potential biological effects.

Contaminant effects on defence responses of ecologically and economically important organisms, such as the Pacific oyster Crassostrea gigas, are likely to influence their ability to resist infectious diseases, particularly at the young stages. The aim of this study was to explore the potential relationships between organic contaminants accumulated in the soft tissues of juvenile oysters, defence responses and physiological condition. Oysters were transplanted during summer and winter periods in different sites in the Marennes-Oléron Bay, the first area of oyster production in France, and in the Gironde Estuary, the biggest estuary in Occidental Europe. Amongst the battery of biochemical and physiological biomarkers applied in the present work [superoxide dismutase (SOD), catalase, glutathione peroxidase (GPx), malondyaldehyde (MDA), catecholase, laccase and lysozyme in gills, digestive glands, mantle and haemolymph, glycogen, proteins and lipids in the digestive gland and the condition index at the whole-organism level], MDA and lysozyme in the digestive gland and SOD, GPx and laccase in plasma contributed in order to significantly discriminate the sites in which oysters bioaccumulated different levels of heavy polycyclic aromatic hydrocarbons (HPAHs), polychlorobiphenyls (PCBs), polybromodiphenylethers (PBDEs), dichlorodiphenyltrichloroethanes (DDTs) and lindane. These results strengthen the hypothesis that it is possible to differentiate sites depending on their contamination levels and biological effects by carrying out studies with transplanted juvenile oysters. In addition, correlations were found between antioxidant and immune-defence responses, and PAH and DDT body burdens in the first area of oyster production in France (the Marennes-Oléron Bay) and where considerable oyster mortalities have been reported. This result suggests that the presence of organic chemical contaminants in the Marennes-Oléron Bay may influence defence responses in juveniles of C. gigas, and, therefore, could influence their ability to resist infectious diseases.

Persistent organic pollutants in a marine bivalve on the Marennes-Oléron Bay and the Gironde Estuary (French Atlantic Coast) - part 1: bioaccumulation.

The aim of this study was to determine 1) the relevance of using the Pacific oyster Crassostrea gigas as a sentinel organism, at a juvenile stage, for polycyclic aromatic hydrocarbon (PAH) and persistent organic pollutant (polychlorobiphenyl, PCB, polybromodimethylether, PBDE, and organochlorine pesticide, OCP) contamination, 2) the potential levels of chemical organic contamination in the Marennes-Oléron Bay, and their potential sources and 3) the potential influence of physiological or environmental factors on contaminant body burdens in oysters. To this end, juvenile oysters purchased from an oyster hatchery were transplanted to a reference site, in Bouin, and to different transplantation sites in the Marennes-Oléron Bay, the first oyster production area in France, and in the Gironde Estuary, the biggest estuary in Occidental Europe. Transplantations were done during summer and winter. Whole oyster soft tissues from each site were analysed for PAHs, PCBs, PBDEs and OCPs. Results obtained with a transplantation period of 3months suggest that C. gigas, at the juvenile stage, is a relevant sentinel organism for short-term assessment of contamination for these contaminants. In addition, no significant effects of physiological factors on contaminant body burdens were observed. Principal component analysis revealed two distinct groups of contaminants (PAHs and OPCs, and PCBs and PBDEs) and three groups of sites: 1) the reference site, 2) Les Palles (LP) and Boyard (BOY) in winter and 3) all the other sites. The group of LP and BOY was clearly defined by the levels of PAHs and OCPs, suggesting higher levels of contamination of these chemical compounds on these sites, potentially due to local contamination sources. In addition, no relevant effects of physiological or environmental factors on contaminant body burdens were observed. Results suggest also a predominance of contaminants related to agricultural activities along the Marennes-Oléron Bay, and therefore, further studies on the presence of pesticides in this region should be considered.

Detection of early effects of a single herbicide (diuron) and a mix of herbicides and pharmaceuticals (diuron, isoproturon, ibuprofen) on immunological parameters of Pacific oyster (Crassostrea gigas) spat.

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.

Bioaccumulation and metabolisation of (14)C-pyrene by the Pacific oyster Crassostrea gigas exposed via seawater.

The first objective of this study was to determine the bioaccumulation kinetics of pyrene in the soft tissues of Crassostrea gigas (mantle, muscle, gills, digestive gland, and the remaining soft tissues). As bivalves can biotransform hydrocarbons in more polar compounds (metabolites) that are more easily excreted, the second objective was to investigate the oyster capacity to metabolize pyrene into its metabolite, the 1-hydroxypyrene. To these ends, oysters were exposed 24 h to waterborne (14)C-pyrene then placed in depuration conditions for 15 d. Oysters efficiently bioaccumulated pyrene in their soft tissues and equilibrium was reached within the exposure time. The metabolite1-hydroxypyrene was also detected in oyster tissues but represented only 4-14% of the parent pyrene. At the end of the exposure period, the gills and the mantle showed the highest pyrene proportion of total soft tissue content, i.e. 47% and 26%, respectively. After 15 d of depuration, the mantle contained 32% and 30% of the remaining pyrene and 1-hydroxypyrene, respectively. As C. gigas did not display a high capacity for metabolizing pyrene, it can be considered as a good bioindicator species to survey and monitor pyrene contamination in the coastal marine environment.

Responses of juvenile sea bass, Dicentrarchus labrax, exposed to acute concentrations of crude oil, as assessed by molecular and physiological biomarkers.

In the present study, juvenile sea bass were exposed for 48 and 96 h to an Arabian light crude oil and their responses were assessed at the molecular and physiological levels. The aim of the study was therefore to assess (i) the short term effects of crude oil exposure by the measurement of several molecular biomarkers, (ii) the consequences of this short term exposure on fish health by using growth and condition indices measured after a decontamination period of 28 and 26 d in seawater. Hydrocarbon petroleum concentrations was monitored during the 96 h experiments and an increase of PAH concentrations were found in fish following both exposure times. An 7-ethoxyresorufin-O-deethylase (EROD) induction was observed after 48 h of exposure, while a significant decrease in the sea bass specific growth rate in length and for the RNA:DNA ratio was observed 28 d after that exposure ceased. The EROD induction doubled after the 96 h exposure, and a significant increase in GST activities was observed. A significant decrease in the specific growth rates, the otolith recent growth, the RNA:DNA ratio and the Fulton's K condition index were then observed in sea bass 26 d after the 96 h exposure to mechanically dispersed crude oil compared to the control. The present study shows that growth and condition indices can prove useful in assessing fish health status following an oil spill. Their complementary analysis with sensitive molecular biomarkers as EROD could improve the determination of oil spill impact on fish populations.

Enhanced immunological and detoxification responses in Pacific oysters, Crassostrea gigas, exposed to chemically dispersed oil.

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.

Phenoloxidase activation in the embryo of the common cuttlefish Sepia officinalis and responses to the Ag and Cu exposure.

The prophenoloxidase (proPO) system catalyzing the melanin production is considered as implicated in the innate immune system in invertebrates. The phenoloxidase (PO)-like activity was detected in the cuttlefish embryo sampled at the end of the organogenesis and few hours before hatching. Various modulators of the PO activity were used to assess the triggering of the proPO activating system. The results demonstrated the evidence of a true PO activity in the cuttlefish embryo. However, SDS and LPS granted contrasting effects on the PO-like activity between the developmental stages suggesting a progressive maturation of the proPO system from the embryonic to the juvenile stages. In eggs exposed to dissolved trace metals all along the embryonic development, Ag (1.2 microg L(-1)) inhibited the PO-like activity in the cuttlefish embryo except at hatching time, suggesting the synthesis of a new "juvenile" form of the PO enzyme. In similar conditions as for Ag, Cu (230 microg L(-1)) stimulated and then inhibited the PO-like activity according to a progressive metal accumulation within the egg and suggesting the occurrence of a threshold, above which the toxicity of the essential metal reduced the PO activity.

Effects of two oils and 16 pure polycyclic aromatic hydrocarbons on plasmatic immune parameters in the European sea bass, Dicentrarchus labrax (Linné).

The in vitro effects of polycyclic aromatic hydrocarbons (PAHs) on two plasmatic immune parameters, lysozyme concentration and haemolytic alternative complement activity, of the European sea bass, Dicentrarchus labrax, were tested using field (10(-7) and 10(-9) mg mL(-1)) and high concentrations (10(-3) and 10(-5) mg mL(-1)) observed during oil spills. Peripheral blood from 105 fish was collected, centrifuged at 1200 g, for 10 min, at 4 degrees C and three plasma pools, each of 35 fish, were constituted. Two oils (heavy fuel oil and light cycle oil) and 16 pure PAHs, selected on the basis of the American Environmental Protection Agency list (US EPA), were tested in vitro on the two humoral immune parameters. Only three pure PAHs (anthracene, chrysene and dibenz[a,h]anthracene) modulated lysozyme concentration. Acenaphthene, acenaphthylene, anthracene, benzo[a]anthracene, benzo[a]pyrene, benzo[b]fluoranthene, benzo[k]fluoranthene, pyrene and light cycle oil modified the haemolytic alternative complement activity after 4h of incubation. This study investigates the direct effects of several PAHs on fish humoral immune functions and describes the haemolytic complement activity of fish as suitable biomarkers of oil pollution.

Pollutant effects on Pacific oyster, Crassostrea gigas (Thunberg), hemocytes: screening of 23 molecules using flow cytometry.

The shellfish industry is an important economic activity in France, occurring mostly in estuarine zones subject to pollution due to anthropogenic activities. The harmful effects of pollutants on species inhabiting these estuarine zones are not well known. Among marine species, bivalve mollusks--particularly Pacific oyster, Crassostrea gigas--may serve a model of interest. The species is sedentary and filter-feeding, which favors bioaccumulation of pollutants in their tissues. Oysters may be suitable for studies on disturbance by pollutants of physiological activities, among which defense mechanisms are poorly documented in bivalves. In this study, effects of pollutants on hemocyte functions were monitored in Pacific oyster, C. gigas. Hemocytes were exposed in vitro to selected pollutants. The strategy for investigating the effects of pollutants on hemocyte functions is based on several biomarkers, which is more relevant than that of published papers based on single-endpoint experiments. Pollutants belonging to the most important groups of xenobiotics (PAHs, PCBs, and pesticides) were selected and their effect on hemocyte activities was analyzed using flow cytometry. Twenty-three pollutants were tested and eight of them showed significant modulation of hemocyte activities. PAHs and PCB 77 induced a decrease of hemocyte activity after an incubation periods of 4 and 24 h at 200 micro mol/L. Three pesticides (2,4D, paraoxon, and chlorothalonil) modulated hemocyte activities. A mixture of eight pesticides also decreased phagocytotic activity. This study is one of the first to investigate the effects of so many pollutants on hemocyte functions at the same time and therefore allows a real comparison of different pollutant effects.

A flow cytometric approach to study intracellular-free Ca2+ in Crassostrea gigas haemocytes.

Bivalve haemocytes are essential in defence mechanisms including phagocytosis. They also produce molecules including hydrolytic enzymes and antimicrobial peptides that contribute to pathogen destruction. Although haemocyte activities have been extensively studied, relatively little is known about the intracellular signalling pathways that are evoked during haemocyte activation and especially the role of calcium. Flow cytometry has been used for the first time to define the effect of cell incubation in haemolymph and artificial sea water (ASW) on Pacific oyster, Crassostrea gigas, haemocytes. Cell viability, enzymatic activities (esterases and aminopeptidases), phagocytosis and granulocyte percentage were analysed. Viability and some activities were different in haemolymph and ASW. Cytoplasmic-free calcium in circulating haemocytes was then investigated by flow cytometry in both media using a calcium probe (Fluo-3/AM). To explore calcium homeostasis, different calcium modulators were tested. The calcium chelator Bapta/AM (10 microM) reduced significantly the percentage of Fluo-3-positive cells in ASW. In addition, ryanodine (5 microM) induced a significant enhancement of the percentage of Fluo-3 positive cells in haemolymph and in ASW. Flow cytometry may be used to study calcium movements in C. gigas haemocytes, but several haemocyte incubation media need to be tested in order to confirm results. The objective of the study should be considered before selecting a particular experimental medium.

In vitro effects of cadmium and mercury on Pacific oyster, Crassostrea gigas (Thunberg), haemocytes.

In the past decades, shellfish culture has developed in a significant way around the world. However, culture areas are often subject to recurring anthropic pollution. The recrudescent presence of industrial wastes is a source of heavy metals and results in pollutant transfer towards the aquatic environment in estuarine areas. Because of their mode of life, bivalves, including mussels and oysters, are suggested as ideal indicator organisms. The development of techniques allowing the analysis of the effects of pollutants on bivalve biology may lead to the monitoring of pollutant transfer in estuarine areas. In this context, the effects of cadmium and mercury on defence mechanisms were analysed in Pacific oysters, Crassostrea gigas. Pollutant effects were tested in vitro on oyster haemocytes. Cell viability and enzymatic activities (esterase, peroxidase, aminopeptidase, phagocytosis activities) were monitored by flow cytometry. Enzymatic phenoloxidase-like activity was also evaluated by spectrophotometry. High pollutant concentrations were used in order to detect the acute effect and to approach real pollutant concentrations existing in animal tissues. Cadmium induced no effect on oyster haemocytes under the tested conditions. On the contrary, mercury caused a significant haemocyte mortality after a 24 h in vitro incubation. Aminopeptidase positive cell percentage was enhanced by the pollutant, and phenoloxidase-like activity was inhibited. These in vitro results show that mercury may be expected to have an impact on bivalve immune functions in contaminated areas.

Study of atrazine effects on Pacific oyster, Crassostrea gigas, haemocytes.

Shellfish farming is an important economic activity around the world. This activity often takes place in areas subjected to various recurring pollutions. The recrudescent use of herbicides in agriculture including atrazine implies pollutant transfer towards aquatic environment in estuarine areas. Harmful effects of such substances on animals in marine environment, particularly on cultured bivalves, are poorly documented. Bivalve molluscs such as mussels and oysters have been postulated as ideal indicator organisms because of their way of life. They filter large volumes of seawater and may therefore accumulate and concentrate contaminants within their tissues. Moreover, development of techniques allowing effect analysis of such compounds on bivalve biology may lead to the development of diagnosis tools adapted to analyze pollutant transfer towards estuarine areas. In this context, influence of atrazine on defence mechanisms was analyzed in Pacific oysters, Crassostrea gigas. Atrazine was tested in vitro and in vivo on oyster haemocytes, and its effects were analyzed by flow cytometry. Haemocyte viability, cell cycle and cellular activities were monitored. Atrazine induced no significant effect in oyster under tested conditions except for peroxidase activity.