Andrea Luna-Acosta

Hélène Thomas-Guyon

Myriam Amari

Eric Rosenfeld

Paco Bustamante

Ingrid Fruitier-Arnaudin

Differential tissue distribution and specificity of phenoloxidases from the Pacific oyster Crassostrea gigas

Keywords: bivalve, phenoloxidase, laccase, catecholase, zymography

Phenoloxidases (POs) play a key role in melanin production, are involved in invertebrate immune mechanisms, and have been detected in different bivalves. Recently, we identified catecholase-and laccase-like PO activities in plasma and haemocyte lysate supernatant (HLS) of the Pacific oyster Crassostrea gigas. To go further in our investigations, the aims of this study were (i) to determine the tissue distribution of PO activities in C. gigas, and (ii) to identify and characterise the different sub-classes of POs (i.e. tyrosinase, catecholase and/or laccase) involved in these oxido-reductase activities. With dopamine and p-phenylenediamine (PPD) but not with L-tyrosine used as substrates, PO-activities were detected by spectrophotometry in the gills, digestive gland, mantle, and muscle. These results suggest the presence of catecholase and laccase but not of tyrosinase activities in oyster tissues. The highest activity was recovered in the digestive gland. PO-like activities were all inhibited by 1-phenyl-2-thiourea (PTU) and by the specific laccase inhibitor, cethyltrimethylammonium bromide (CTAB). With dopamine as substrate, the catecholase inhibitor 4-hexylresorcinol (4-HR) only inhibited PO in the muscle. SDS-PAGE zymographic assays with dopamine and PPD elicited a unique ~40 kDa protein band in the muscle. In the other tissues, laccase-like activities could be related to ~10 kDa and/or ~200 kDa protein bands. The ~10 kDa protein band was also detected in plasma and HLS, confirming the presence of a laccase in the later compartments, and probably in most of the tissues of C.gigas. This is the first time to our knowledge that a ~10 kDa protein band is associated to a laccase-like activity in a mollusc species, contributing to the characterisation of phenoloxidase activities in marine bivalves.

Introduction

Phenoloxidases (POs,EC 1.14.18.1) are a class of copper proteins widely distributed in bacteria, fungi, plants and animals [START_REF] Cerenius | The proPO-system: pros and cons for its role in invertebrate immunity[END_REF]. They play a key role in melanin production and are implicated in immune defence mechanisms in invertebrates. This class of enzymes include tyrosinases (EC 1.14.18.1), catecholases (EC 1.10.3.1) and laccases (EC 1.10.3.2), all capable of o-diphenol oxidation. However, among these three enzymes, only tyrosinases can hydroxylate monophenols (e.g. L-tyrosine), and only laccases can oxidise pdiphenols and aromatic compounds containing amine groups (e.g. p-phenylenediamine, PPD) [START_REF] Thurston | The structure and function of fungal laccases[END_REF][START_REF] Solomon | Multicopper oxidases and oxygenases[END_REF]. In addition to that, a panel of inhibitors exert different actions on these three types of enzymes: while 1-phenyl-2-thiourea (PTU) inhibits the three types of PO activities (Williamson 1997, Jordan and[START_REF] Jordan | Characterization of phenoloxidase from Crassostrea virginica hemocytes and the effect of Perkinsus marinus on phenoloxidase activity in the hemolymph of Crassostrea virginica and Geukensia demissa[END_REF], 4-hexylresorcinol (4-HR) inhibits tyrosinase and catecholase but not laccase activities [START_REF] Dawley | Differentiation of tyrosinase and laccase using 4hexylresorcinol, a tyrosinase inhibitor[END_REF]Flurkey 1993, Zavarzina and[START_REF] Zavarzina | Laccase and tyrosinase activities in lichens[END_REF] and cethyltrimethylammonium bromide (CTAB) specifically inhibits laccase activity [START_REF] Walker | The selective inhibition of ortho-and para-diphenol oxidases[END_REF]. Recently, we conducted a study to identify PO activities present in the haemolymph of the Pacific oyster Crassostrea gigas (Luna-Acosta et al. 2010a). By using different PO substrates, such as L-tyrosine, L-3,4dihydroxyphenylalanine (L-DOPA), dopamine or PPD, and different PO inhibitors, such as PTU, 4-HR and CTAB, results suggested the presence of both catecholase-and laccase-like activities in the plasma, and the presence of a laccase-like activity in the haemocyte lysate supernatant (HLS, Luna-Acosta et al. 2010a). Our interest in C. gigas comes from the fact that this organism dominates over all other molluscs with respect to global distribution and aquaculture production, but suffers from massive summer mortality each year [START_REF] Cheney | Summer mortality of Pacific oysters Crassostrea gigas (Thunberg): Initial findings on multiple environmental stressors in Puget Sound, Washington[END_REF]. Summer mortality of C. gigas has been suggested to be the result of a complex interaction between the host, pathogens and environmental factors [START_REF] Cheney | Summer mortality of Pacific oysters Crassostrea gigas (Thunberg): Initial findings on multiple environmental stressors in Puget Sound, Washington[END_REF].

Importantly, studies in C. gigas have shown that PO activities, usually detected by using the o-diphenol substrates L-DOPA or dopamine, can be modulated by environmental factors, such as the presence of heavy metals or hydrocarbons (Gagnaire et al. 2004a[START_REF] Bado-Nilles | Effects of 16 pure hydrocarbons and two oils on haemocyte and haemolymphatic parameters in the Pacific oyster, Crassostrea gigas (Thunberg)[END_REF], Luna-Acosta et al. 2010b). In addition to that, a gene coding for a laccase in the haemocytes from C. gigas was modulated in the presence of hydrocarbons [START_REF] Bado-Nilles | In vivo effects of LCO soluble fraction on immune-related functions and gene transcription in the Pacific oyster, Crassostrea gigas (Thunberg)[END_REF]. To the best of our knowledge, studies on POs in C. gigas have only been carried out in the haemolymphatic compartment. However, POs may be present in other body tissues in bivalves, e.g. in the prismatic shell layer [START_REF] Nagai | Tyrosinase localization in mollusc shells[END_REF] or in the byssus gland [START_REF] Hellio | Phenoloxidase (E.C. 1.14.18.1) from Mytilus edulis byssus gland: purification, partial characterization and application for screening products with potential antifouling activities[END_REF]. A better characterisation and localisation of POs in C. gigas is needed to expand our knowledge on the immune defence mechanisms in this organism and therefore to a better understanding of the potential causes of summer mortality events.

In this general context, our goal was to determine the distribution and the nature of PO activities (tyrosinase, catecholase, and laccase) in different oyster body compartments, namely gills, digestive gland, mantle, muscle, plasma and HLS. PO activities were determined by spectrophotometry using different PO substrates (L-tyrosine, dopamine and PPD) and PO inhibitors (PTU, 4-HR, CTAB). Electrophoretic techniques using polyacrylamide gels are useful to detect PO enzymes and their associated molecular weights in crude extracts without the necessity of enzyme purification [START_REF] Cardenas | Cresolase, catecholase and laccase activities in haemocytes of the red swamp crayfish[END_REF][START_REF] Decker | SDS-induced phenoloxidase activity of hemocyanins from Limulus polyphemus, Eurypelma californicum, and Cancer magister[END_REF][START_REF] Dicko | Zymography of monophenolase and o-diphenolase activities of polyphenol oxidase[END_REF][START_REF] Perdomo-Morales | Phenoloxidase activity in the hemolymph of the spiny lobster Panulirus argus[END_REF]). Hence, SDS-PAGE zymographic assays were carried out on crude and partially purified samples from the different oyster compartments.

Differences between tissues, in terms of PO-like activity and molecular weight characteristics, are discussed.

Materials and methods

Chemicals and materials

L-tyrosine, dopamine, p-phenylenediamine (PPD), 1-phenyl-2-thiourea (PTU), 4hexylresorcinol (4-HR), cethyltrimethylammonium bromide (CTAB), 3-methyl-2benzothiazolinone hydrazone (MBTH), trizma hydrochloride (Tris HCl), sodium chloride (NaCl), ammonium sulfate (AS), sodium dodecyl sulfate (SDS), trizma base, glycine, N,N,N',N'-tetramethylethylenediamine (TEMED), ammonium persulfate, glacial acetic acid, Coomassie brilliant blue, bovine serum albumin (BSA), copper sulfate and bicinchoninic acid were obtained from Sigma-Aldrich (France). Magnesium chloride (MgCl 2 ) and calcium chloride (CaCl 2 ) were obtained from Acros organics (France). Acrylamide/Bis acrylamide 30% was obtained from Bio-Rad.

Oysters

Three years old Pacific oysters, Crassostrea gigas (n= 30; mean ± SD; weight: 75.5 ± 8.7 g; length: 9 ± 3 cm) were purchased during October-November 2008 from shellfish farms in Aytré Bay (Charente Maritime, France), on the French Atlantic coast, and were processed immediately after their arrival in the laboratory.

Collection of oyster tissues

After opening the oyster shells by cutting off the adductor muscle, a quantity (0.5-1 ml) of haemolymph was withdrawn directly from the pericardial cavity with a 1-ml syringe equipped with a needle (0.9 x 25 mm), and the haemolymph from 10 oysters was pooled to reduce inter-individual variation [START_REF] Gagnaire | In vitro and in vivo effects of mercury on haemocytes of Pacific oyster, Crassostrea gigas (Thunberg): development of techniques evaluating estuarine pollution[END_REF]). Haemolymph samples were centrifuged (260 g, 10 min, 4°C) to separate the cellular fraction (i.e. haemocytes) from plasma, as described previously [START_REF] Hellio | Demonstration of a true phenoloxidase activity and activation of a ProPO cascade in Pacific oyster, Crassostrea gigas (Thunberg) in vitro[END_REF]. Gills, digestive gland, mantle and muscle were removed from oysters and pooled. Three replicates from 10 oysters were prepared per tissue.

Haemocytes, gills, digestive gland, mantle and muscle were homogenized at 4°C in Tris buffer (0.1 M Tris HCl, 0.45 M NaCl, 26 mM MgCl 2 and 10 mM CaCl 2 ) adjusted to pH 7.

Haemocytes were lysed using a Thomas-Potter homogenizer (IKA-Labortechnik, clearance 0.13-0.18mm) at 200 rpm for 1 min. Gills, digestive gland, mantle and muscle were homogenized, as described previously (Luna-Acosta et al. 2010b), using an Ultra Turrax (T25 basic, IKA-WERKE) at 19 000 rpm for 1 min followed by twelve up and down strokes of Thomas-Potter homogenizer at 200 rpm for 1 min (IKA-Labortechnik RW 20.n, size 0.13-0.18mm, BB). All homogenized samples were centrifuged at 10 000 g for 10 min at 4°C. The resulting haemocyte lysate supernatant (HLS) and tissue supernatants were collected for enzymatic studies.

Aliquots (100 µl) of plasma, HLS and tissue samples were stored at -80°C. Each aliquot was used only once per microplate for spectrophotometric analysis, or per gel running for zymographic studies.

Partial purification

A previous analysis, by using different concentrations of saturated ammonium sulfate (0, 30, 40, 60, 70, 80, 100%), revealed that precipitation with 60% saturated ammonium sulfate (60P-SAS) was the best condition for protein concentration to detect PO-like activity for oyster tissues, i.e. the gills, digestive gland, mantle and muscle (data not shown), and was in agreement with other studies [START_REF] Cong | Purification and characterization of phenoloxidase from clam Ruditapes philippinarum[END_REF][START_REF] Liu | Purification and characterization of phenoloxidase from crab Charybdis japonica[END_REF]. Therefore, proteins of collected supernatants from oyster tissues were brought to 60% saturation concentration by addition of solid ammonium sulfate at 4°C, and allowed to stand overnight. The resulting precipitate was collected by centrifugation (15 500 g for 10 min), dissolved in a small volume of Tris buffer , and dialysed at 4°C against distilled water for 12h and twice against Tris buffer for 8h. Crude plasma samples were concentrated with Centricon-5 centrifugal concentration units (AmiconTM).

Phenoloxidase assays

Phenoloxidase-like (PO-like) activity was measured spectrophotometrically by recording the formation of o-quinones, as described previously (Luna-Acosta et al. 2010a). PO assays were conducted in 96-well microplates (Nunc, France). Dopamine or p-phenylenediamine (PPD) were used as substrates, at final concentrations of 100 mM and 50 mM, respectively. Dopamine (100 mM) was prepared just before being used in Tris buffer. At 25°C, 10 µl of sample was incubated with 80 µl of dopamine and 50 µl of Tris buffer. Several control wells were systematically used: 'buffer control' containing only buffer, 'sample control' containing only sample and buffer, and 'non-enzymatic control' containing only substrate and buffer, always in a final volume reaction of 140µl. Immediately after dopamine addition, PO-like activity was monitored during 4h by using a VersaMax™ microplate reader (Molecular Devices) and by following the increase of absorbance at 490 nm. Because of solubility constraints, the protocol was slightly modified in the case of PPD: the sample was incubated with 7 µl of PPD (50 mM diluted in methanol) and 123 µl of buffer (no effect of methanol was observed on the enzymatic reactions). PO-like activity was monitored during 2h at 420 nm. For all conditions, the experiments were performed with three pooled oyster samples.

Each pool was tested in triplicate wells and average rates were calculated by dividing the sum of replicate measurements from the three oyster pools, by the number of measurements, i.e. 9

(3 replicate measurements x 3 oyster pools).

For enzymatic oxidation, the results were systematically corrected for non-enzymatic autoxidation of the substrate and were expressed in specific activity (SA), i.e. in international units (IU) per mg of protein. One IU is defined as the amount of enzyme that catalyzes the appearance of 1 µmole of product per min [START_REF] Fenoll | Unification for the expression of the monophenolase and diphenolase activities of tyrosinase[END_REF] under the above conditions using molar extinction coefficient of dopamine and PPD reactions products of 3 300 M -1 cm -1 [START_REF] Waite | Calculating extinction coefficients for enzymatically produced o-quinones[END_REF]) and 43 160 M -1 cm -1 [START_REF] Eggert | The lignolytic system of the white rot fungus Pycnoporus cinnabarinus: Purification and characterization of the laccase[END_REF][START_REF] Paranjpe | Characterization of tyrosinase and accompanying laccase from Amorphophallus campanulatus[END_REF], respectively.

Phenoloxidase inhibition assays

Working solutions of inhibitors were prepared just before being used in Tris buffer. PO inhibition assays were performed by preincubating 10 µl of the specific PO inhibitor PTU (5 mM, final concentration), the specific tyrosinase and catecholase inhibitor 4-HR (1 mM, final concentration), or the specific laccase inhibitor CTAB (1 mM, final concentration), with 10 µl of sample for 20 minutes. Then, PO assay was carried out with dopamine (100 mM, prepared in Tris buffer) or PPD (50 mM, prepared in methanol). Appropriate controls were used as described before. Experiments were performed with three pooled oyster samples.

Each pool was tested in triplicate wells and average rates were calculated.

Protein assays

Protein concentrations were determined by the slightly modified Lowry method, as described previously [START_REF] Smith | Measurement of a protein using bicinchoninic acid[END_REF], using bovin serum albumin as standard.

Gel electrophoresis and zymography

To associate PO enzyme activities with individual proteins, and estimate the molecular weights of the enzymes, SDS-PAGE and 1 D-zymography were used. Aliquots of the different oyster tissues (equivalent to 76 µg of proteins for gills, 76 µg for digestive gland, 57 µg for mantle, 40 µg for muscle, 47 µg for plasma and 1.55 µg for HLS) were mixed with sample buffer (65 mM Tris HCl pH 6.8, 25% glycerol, 2% SDS, 0.01% Bromophenol blue).

Samples were then applied to 7% SDS-PAGE gels or 15% SDS-PAGE gels in non reducing conditions (i.e. without boiling samples after the addition of sample buffer) and with an upper gel of 4% using a Mini-PROTEAN III Cell (Bio-Rad). Electrophoresis was carried out according to the method of [START_REF] Laemmli | Cleavage of structural proteins during the assembly of the head of bacteriophage T4[END_REF] at 110V for 2h45. Two gels containing the same samples were run and processed in parallel. For each tissue, samples previously brought to 0, 30, 40 or 60% saturation concentration by addition of solid ammonium sulfate at 4°C were runned per gel. After electrophoresis, SDS-PAGE gels were washed 2 x 10 min in distilled water and 2 x 10 min in Tris buffer.

The first SDS-PAGE gel was stained with a solution containing 100 mM L-tyrosine and 5 mM MBTH (to detect tyrosinase activity), 100 mM dopamine and 5 mM MBTH (to detect catecholase activity), or 100 mM PPD (to detect laccase activity). MBTH was used, according to the method of [START_REF] Dicko | Zymography of monophenolase and o-diphenolase activities of polyphenol oxidase[END_REF], to trap o-quinone products originating from the oxidation of phenolic compounds by phenoloxidases. All substrates were dissolved in Tris buffer. The gels were developed for 1 h, at 25°C and then rinsed with distilled water several times, dried at room temperature and photographed.

The second SDS-PAGE gels were immediately washed with distilled water and stained with Coomassie brilliant blue R-250 for visualizing total proteins. The molecular weight of PO activity bands were estimated with pre-stained molecular weight markers (Broad Range Markers, Tebu Bio, France) that were run together with samples (data not shown).

In order to test the specificity of the zymographic assay, a purified laccase from Trametes versicolor (20 µg) and a purified superoxide dismutase (SOD) from bovine erythrocytes (20 µg) were included in the activity gels.

Statistical analysis

All values are reported as mean ± standard deviation (SD). Statistical analysis was carried out with SYSTAT 11.0. Values were tested for normality (Shapiro test) and homogeneity of variances (Bartlett test). In some cases, logarithmic transformations (Log 10 ) were used to meet the underlying assumptions of normality and homogeneity of variances. For normal values, one-way nested ANOVA tests were used followed by a Tukey post-hoc test. For non normal values, Kruskal-Wallis tests were applied, followed by Dunn's multiple comparisons test [START_REF] Zar | Biostatistical analysis[END_REF]. The statistical significance was designed as being at the level of p<0.05.

Results

Spectrophotometric studies

Different PO substrates (L-tyrosine, dopamine and PPD), the common PO inhibitor, PTU, the tyrosinase and catecholase inhibitor, 4-HR, and the laccase inhibitor, CTAB, were used.

When L-tyrosine was used as substrate, no PO-like activity was detected in any of the tissues that were tested, i.e. gills, digestive gland, mantle, and muscle (data not shown). When dopamine and PPD were used as substrates, PO-like activity was detected in all oyster tissues (Fig. 1). PO-like activity was inhibited by PTU. The inhibition was total in muscle with dopamine as substrate (Fig. 1g), and in digestive gland (Fig. 1d), mantle (Fig. 1f), and muscle (Fig. 1h) with PPD as substrate. PO-like activity was insensitive to 4-HR except in the muscle with dopamine as substrate (Fig. 1g). By contrast, PO-like activity was fully (or almost fully) inhibited by the laccase inhibitor CTAB (1 mM) in all the oyster tissues with both dopamine and PPD as substrates (Fig. 1).

Since fresh weight differs between the different analyzed tissues (i.e. the gills, digestive gland, mantle and muscle), tissue distribution of PO-like activity was also examined in terms of recovery of enzymatic activity (Table 1). With dopamine as substrate, the highest total POlike activity was recovered in the digestive gland, followed by the gills, mantle and muscle (Table 1). With PPD as substrate, the total PO-like activity was considerably higher in the digestive gland compared to the other compartments.

SDS-PAGE zymographic assays

When gels were stained with L-tyrosine, no bands were observed, and this, for all the oyster tissues tested, i.e. the gills, digestive gland, mantle, muscle, plasma and HLS (data not shown). However, PO-like activity was detected in all oyster tissues that were analyzed by SDS-PAGE zymographs, with both dopamine and PPD as substrates.

PO substrates such as dopamine or PPD can oxidise non-enzymatically. This leads to different intermediary products of the melanization cascade such as quinone radicals. Unspecific reactions between quinone radicals issued from non-enzymatically oxidation reactions and radical species that could be produced by SOD-like proteins are thus likely to take place in zymographic studies [START_REF] Eibl | Zinc-metallothionein: a potential mediator of antioxidant defence mechanisms in response to dopamine-induced stress[END_REF]). However, PO-like activity was detected in the presence of laccase from T. versicolor but not in the presence of SOD from bovine erythrocytes (data not shown), confirming that our zymographic conditions were well adapted to discriminate between true PO-like activities and other enzymatic activities involving radical species.

With dopamine as substrate, the activity detected for the gills and the mantle corresponded to one upper band with an estimated molecular weight of ~200 kDa and to a lower band with an estimated molecular weight of ~10 kDa (Fig. 2a). In both tissues, most of the PO activity appeared in the higher molecular mass band. For the digestive gland the activity corresponded to an upper band of ~200 kDa and for the plasma and the HLS to one lower band with an estimated molecular weight of ~10 kDa (Fig. 2a). Similarly, an upper band with a molecular weight of ~200 kDa was observed in the presence of PPD for the gills and the mantle, but not for the digestive gland (Fig. 2b), and a lower band with a molecular weight of ~10 kDa was detected in the presence of PPD for the gills, digestive gland, mantle, crude plasma and crude HLS (Fig. 2b). Again, most activity in samples from the gills and the mantle was evident in higher molecular mass bands. The bands of ~10 kDa are not likely to be an artefact since they stained differentially with dopamine and PPD, depending on the tissue that was analyzed, e.g. stained with PPD but not with dopamine in the digestive gland. Contrary to the other tissues, the activity detected for the muscle, with dopamine and PPD as substrates, corresponded to a band with an estimated molecular weight of ~40 kDa.

Discussion

POs are of widespread occurrence in bacteria, fungi, plants, invertebrates and vertebrates [START_REF] Sanchez-Ferrer | Tyrosinase: a comprehensive review of its mechanism[END_REF][START_REF] Cerenius | The proPO-system: pros and cons for its role in invertebrate immunity[END_REF]. Despite the importance of the reactions and the functional roles associated to POs, the enzymes belonging to the PO class have not been thoroughly characterised in molluscs and especially in bivalve species. In the present study, spectrophotometric analyses were conducted to identify PO-like activity in different tissues from C. gigas. L-tyrosine, dopamine and PPD were used as substrates and different PO inhibitors were tested. When L-tyrosine was used as substrate, no PO-like activity was detected in any of the tissues that were tested, i.e. the gills, digestive gland, mantle, and muscle. Similar results were recently obtained with plasma and HLS (Luna-Acosta et al. 2010a). These data suggest the total absence of tyrosinase-like activity in C. gigas. Inhibition assays in the present study were thus only conducted with dopamine and PPD as substrates.

The choice on PO specific inhibitors was based on a previous study carried out on the PO inhibitors described in the literature (Luna-Acosta et al. 2010a). Indeed, PO substrates can chemically oxidise in the absence of PO (autoxidation) and many PO inhibitors described in the literature such as reducing agents (e.g. 2-mercaptoethanol or sodium azide) can inhibit autoxidation reactions (Luna-Acosta et al. 2010a). Such inhibitors are likely to react with the substrate and/or the quinone intermediates derived from the autoxidation reaction. Therefore, this type of inhibitors should be avoided for identifying PO activity. Among PO inhibitors described in different species, PTU was chosen because it has been described as a common inhibitor of all POs [START_REF] Arias | Kraft pulp biobleaching and mediated oxidation of a nonphenolic substrate by laccase from Streptomyces cyaneus CECT 3335[END_REF][START_REF] Zufelato | Phenoloxidase activity in Apis mellifera honey bee pupae, and ecdysteroid-dependent expression of the prophenoloxidase mRNA[END_REF]), 4-HR as a tyrosinase and a catecholase but not a laccase specific inhibitor [START_REF] Dawley | Differentiation of tyrosinase and laccase using 4hexylresorcinol, a tyrosinase inhibitor[END_REF]Flurkey 1993, Zavarzina and[START_REF] Zavarzina | Laccase and tyrosinase activities in lichens[END_REF] and CTAB as a laccase but not a tyrosinase or a catecholase specific inhibitor [START_REF] Walker | The selective inhibition of ortho-and para-diphenol oxidases[END_REF]McCallion 1980, Martinez-Alvarez et al. 2008). Moreover, no inhibitory effect was observed with these chemicals in dopamine or PPD autoxidation reactions (Luna-Acosta et al. 2010a).

In the present study, PO-like activity in the muscle was completely inhibited in the presence of PTU (5 mM) and 4-HR (1 mM) with dopamine as substrate, and in the presence of PTU (5 mM) and CTAB (1 mM) with PPD as substrate (Fig. 1), suggesting the presence of catecholase and laccase in this tissue. These results are in agreement with those obtained previously with plasma (Luna-Acosta et al. 2010a). However, in the previous study, no inhibition was exerted by CTAB with plasma and with dopamine as substrate (Luna-Acosta et Interestingly, PO-like activities in all other tissues were partially inhibited in the presence of PTU and completely inhibited in the presence of CTAB with dopamine and PPD as substrates, suggesting the presence of laccase activity in different tissues from C. gigas (Fig. 1). These results are in agreement with those obtained previously with HLS (Luna-Acosta et al. 2010a).

In zymographic studies, catecholase-and/or laccase-but not tyrosinase-like activities were detected in the gills, digestive gland, mantle, muscle, plasma and HLS (Fig. 2). This coincides with properties of the Asian swimming crab Charybdis japonica [START_REF] Liu | Purification and characterization of phenoloxidase from crab Charybdis japonica[END_REF]) and the eastern oyster C. virginica [START_REF] Jordan | Characterization of phenoloxidase from Crassostrea virginica hemocytes and the effect of Perkinsus marinus on phenoloxidase activity in the hemolymph of Crassostrea virginica and Geukensia demissa[END_REF], and differs from tyrosinase-type POs from other invertebrates, such as the vinegar fly Drosophila melanogaster [START_REF] Asada | Activation of prophenoloxidase with 2-propanol and other organic compounds in Drosophila melanogaster[END_REF], the bloodfluke planorb Biomphalaria glabrata [START_REF] Bai | Isolation and characterization of phenoloxidase from egg masses of the gastropod mollusc, Biomphalaria glabrata[END_REF], the Manila clam Ruditapes philippinarum [START_REF] Cong | Purification and characterization of phenoloxidase from clam Ruditapes philippinarum[END_REF]) and the Sydney rock oyster Saccostrea glomerata [START_REF] Aladaileh | Characterization of phenoloxidase activity in Sidney rock oysters (Saccostrea glomerata)[END_REF]).

In the present study, when dopamine or PPD were used as substrates, PO-positive bands of ~10, 40 or 200 kDa were detected in C. gigas depending on the tissue. These tissue-dependent differences in molecular weights may be due to (i) the activation state of POs or (ii) the existence of polymeric forms of the enzyme. Indeed, the molecular weights of POs vary depending on the activation state, animal tissue and animal species that are studied (Table 2).

In molluscs, the molecular weights of POs estimated by exclusion chromatography or SDS-PAGE electrophoresis are in the range of 35 to 381 kDa, and in invertebrates, POs occur as monomers, dimers, tetramers or pentamers [START_REF] Jaenicke | Tyrosinases from crustaceans form hexamers[END_REF]. Thus, differences in molecular weights in the present study may be explained by the existence of polymeric forms of the enzyme. Generally, the molecular weight of monomeric forms is about 40 to 45 kDa, and generally each subunit possesses two copper atoms [START_REF] Prota | Occurence and properties of tyrosinase in ejected ink of cephalopods[END_REF]). In the present study, a PO-positive band of ~40 kDa was detected in the muscle of C. gigas, suggesting that a monomeric form of a laccase sensitive to inhibition by 4-HR, or a catecholase sensitive to inhibition by CTAB could be present in the muscle of this species. In the other analyzed tissues, PO-positive bands of ~10 or ~200 kDa were detected, and in the haemolymphatic compartments, PO-positive bands of ~10 kDa were detected. Results of the upper band of ~200 kDa are in agreement with the large range of molecular weights of PO reported for molluscs, i.e. from 35 to 381 kDa (Table 2). However, to our knowledge, this is the first time that a PO-positive band of ~10 kDa is reported in a mollusc species. A PO-positive band of ~10 kDa was detected in a non-mollusc aquatic invertebrate, the red swamp crayfish Procambarus clarkii [START_REF] Cardenas | Cresolase, catecholase and laccase activities in haemocytes of the red swamp crayfish[END_REF], suggesting that C. gigas possesses PO with characteristics (i.e. PO-like activity and molecular weight) comparable to that of arthropods.

Overall, in the present study, differences between tissues were observed in terms of (i) substrate affinity (ii) effect of PO inhibitors, and (iii) number and molecular weight of the bands detected by zymography. The data presented here suggest that zymography can be a useful way of characterising PO-like activities present in C. gigas. Interestingly, results of the present study revealed that numerous differences exist between the tissues and the haemolymphatic compartment in C. gigas, both in terms of PO-like activities and in terms of proteins that may be responsible for these activities.

  al. 2010a), while, in the present study, an inhibitory effect by CTAB was observed in the muscle and with dopamine as substrate. Since 4-HR and CTAB are specific catecholase and laccase inhibitors (van Doorn and Vaslier 2002), respectively, results in the muscle suggest the presence of a laccase sensitive to inhibition by 4-HR, or a catecholase sensitive to inhibition by CTAB.

  Our results indicate that at least three oligomeric forms of POs coexist in the Pacific oyster. This comparative study gives first evidences of structure-function relationships of tissue POs in C. gigas, contributing to the understanding of tissue-specific heterogeneity of PO activities in this marine organism. As POs are involved in immune response, tests based on modifications in oligomeric forms and functions of this class of enzymes, and more particularly of laccase, could be used as a probe to measure health conditions in this economically important species.

Figure 1 .Figure 2 .

 12 Figure 1. Inhibition of phenoloxidase-like activity in precipitated protein fractions from 498

Table 1 . Total phenoloxidase-like activity (Mean ± standard deviation, µmol.min -1 .mg prot -1 .g fresh weight, n=3) from different oyster body tissues with dopamine and PPD as substrates. Body tissue Total phenoloxidase-like activity

 1

		Dopamine	PPD
	Gills	1.8 ± 0.3	0.2 ± 0.0
	Digestive gland	3.0 ± 0.4	50.4 ± 4.3
	Mantle	0.9 ± 0.1	0.2 ± 0.0
	Muscle	0.5 ± 0.2	0.3 ± 0.1

Acknowledgements: This study was supported by a PhD grant from the Conseil Général of the Charente-Maritime for A. Luna-Acosta. The Conseil Régional de Poitou-Charentes is acknowledged for financial support through the research project 'POLERON' (Modifications chimiques de polluants organiques dans le bassin de Marennes-Oléron, toxicité des produits de dégradation sur l'huître creuse). This study has also been supported financially by the CPER (Contrat de Projet Etat-Région).


Andrea Luna-Acosta

R Kanan

Stéphane Le Floch

Valérie Huet

Philippe Pineau

Paco Bustamante

Hélène Thomas-Guyon

Le Floch

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

Keywords: chemical dispersion, oil spill, bivalve, defence mechanisms, tissue-dependent response

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

The biological and economical consequences of numerous accidental oil spills that have occurred during the last 40 years are concrete proof of the need to develop reliable oil spill countermeasures. Large oil spills (> 100 tonnes) represent only 6% of the total introduction of oil into the environment (NCR, 2003). Indeed, most oil spills are relatively small (~1 m 3 ). They occur close to the shore and oil slicks hit shorelines relatively quickly. As a result of the Torrey Canyon incident, where large amounts of toxic dispersants were used and caused severe long-term biological impacts, research efforts have focused on the mechanical clean-up and containment of floating oil. However, these types of techniques in special areas, such as estuaries and closed bays, can also cause additional harm to oil-impacted zones [START_REF] Marchand | Marketing of solid waste management services in Tingloy, the Philippines[END_REF]. During the same period, chemical dispersants evolved from first generation products, effective but highly toxic, to third generation products, with low toxicity and high biodegradability. Chemical dispersants are complex mixtures, primarily containing surfactants and solvents, which reduce the interfacial tension at the oil-water interface, and therefore facilitate the downward mixing of oil into the water [START_REF] Canevari | Development of the 'next generation' chemical dispersants[END_REF][START_REF] Li | The relationship between oil droplet size and upper ocean turbulence[END_REF]. In this way, oil can be dispersed to concentrations below toxicity threshold limits [START_REF] Lessard | The significance of oil spill dispersants[END_REF][START_REF] Page | Behavior of a chemically-dispersed oil and a whole oil on a near-shore environment[END_REF], become more accessible to hydrocarbon-degrading bacteria [START_REF] Venosa | Biodegradation of crude oil contaminating marine shorelines and freshwater wetlands[END_REF], and have a lower impact on shorelines.

Moreover, third generation dispersants are considered non-toxic and biodegradable. Unfortunately, little is known about the behavior and effects of dispersed oil in the nearshore environment (ASTM Committee, 1984). Indeed, because of the higher exposure of organisms to petroleum compounds that may be triggered by the use of chemical dispersants in zones with low water-column depth, e.g. coastal areas and estuaries, most countries that allow the use of dispersants have strict rules: minimum water depth (normally 20 m), minimum current speed (normally 1 meter per second), and minimum distance from the shore or from offshore islands (normally 2 km) [START_REF] Ramachandran | Oil dispersant increases PAH uptake by fish exposed to crude oil[END_REF]. Recently, a Net Environmental Benefit Analysis (NEBA) carried out by [START_REF] Baca | Net environmental benefit analysis (NEBA) of dispersed oil on near shore tropical ecosystems derived from the 20 years 'tropics' field study[END_REF] on a 20-year field study, revealed the lack of long-term environmental impact of dispersed oil in nearshore tropical areas.

However, dispersants are a wide-ranging group of chemicals with varying chemical properties and related toxicities. Therefore, many questions remain unanswered about the possible direct or indirect effects of their use in other nearshore zones, in response to oil spills, where human activities are heavily concentrated, such as in the case of the Transocean Deepwater Horizon oil rig explosion.

Coasts and estuaries are considered to be sensitive areas since they provide habitats for a large number of organisms, constitute nursery grounds for juveniles of several commercially important species, and bear very high productivity. Sessile and filter-feeder organisms inhabiting these zones, such as the Pacific oyster Crassostrea gigas (Thunberg, 1753), are constantly in contact with various chemical molecules.

Environmental stress from pollutants is likely to be an important determining factor in weakening defence mechanisms in these organisms and therefore promoting the occurrence or increase in diseases, particularly at early life stages, i.e. larvae and juveniles [START_REF] Perdue | Some relationships between gametogenic cycle and summer mortality phenomenon in the Pacific oyster (Crassostrea gigas) in Washington State[END_REF][START_REF] Lacoste | Stress and stress-induced neuroendocrine changes increase the susceptibility of juvenile oysters (Crassostrea gigas) to Vibrio splendidus[END_REF]. Among immune defence mechanisms in bivalves, antioxidant enzyme activities, e.g. superoxide dismutase (SOD), catalase, glutathione peroxidase (GPx), and enzyme activities involved in humoral innate defences, e.g. phenoloxidase (PO) and lysozyme, have been shown to be modulated by the presence of several types of pollutants [START_REF] Verlecar | Biochemical markers of oxidative stress in Perna viridis exposed to mercury and temperature[END_REF][START_REF] Bado-Nilles | Immune effects of HFO on European sea bass, Dicentrarchus labrax, and Pacific oyster, Crassostrea gigas[END_REF][START_REF] Stabili | Effect of zinc on lysozyme-like activity of the seastar Marthasterias glacialis (Echinodermata, Asteroidea) mucus[END_REF]. In this respect, these responses have been shown to be tissue-dependent [START_REF] Cheung | Relationships between tissue concentrations of polycyclic aromatic hydrocarbons and antioxidative responses of marine mussels, Perna viridis[END_REF]Luna-Acosta et al., 2010a).

In this general context, the aim of this study was to experimentally assess 1) the bioaccumulation and 2) the effects of chemically-dispersed oil on immune defence and/or detoxification mechanisms, i.e. SOD, catalase, GPx, catecholase-and laccase-type PO, and lysozyme activities of Pacific oyster C. gigas juveniles. For this purpose, enzyme activities were determined in different tissues, i.e. gills, digestive gland, mantle, and in the haemocytes and plasma, or acellular fraction, of the haemolymph. To this end, comparisons were made between oysters exposed to oil subjected to chemical dispersion (CD) or mechanical dispersion (MD), to the water soluble fraction of the oil (WSF) and to the dispersant alone (D).

Material and methods

Chemicals

Oil • A Brut Arabian Light crude oil (BAL 110) was used for this study. The crude oil was topped at 110°C to remove the most volatile components, in order to simulate the natural weathering of the oil after its release at sea (evaporation of most volatile components), before it reaches coastal zones. BAL 110 possesses the following physico-chemical characteristics, similar to the oil spilled by the Amoco Cadiz in 1978: density of 0.860 at 20°C, viscosity of 60 mPa s at 15°C, 12% polar compounds, 34% aromatic hydrocarbons and 54% saturated hydrocarbons. Dispersant • The chemical dispersant used in this study was selected following an evaluation carried out by the Centre of Documentation, Research and Experimentation on Accidental Water Pollution (Cedre), which defines it as 1) effective for use in the marine environment, 2) non-toxic at the concentration recommended by the manufacturer Total Fluides (i.e. 5% v/v) and (3) biodegradable. Its physicochemical characteristics were not available for reasons of confidentiality.

Biological material

Pacific oyster C. gigas juveniles (3-4 cm in height, less than 1 year old) were purchased from the hatchery France Naissain, located in Bouin (France). The oysters were acclimatised in the laboratory at 15 ± 1°C for two weeks before starting the experiments. They were fed daily with an algal diet (5 x 10 4 cell ml -1 ) composed of Heteroskeletonema sp. (Bacillariophyceae) purchased from the hatchery SATMAR (Normandie, France).

Experimental design

The experiment, consisting of an exposure period followed by a depuration period, was carried out three times to provide analysis replicates.

Experimental system • The experimental system consisted of 300 L static water tanks. Because of the hydrophobic character of the oil, a funnel (at the surface of each tank) was connected to a submersed Johnson L450 water pump (at the bottom of the tank), in order to maintain the mixture of oil and dispersant as a homogenous solution. Preliminary tests confirmed that total petroleum hydrocarbon concentrations in the water column were depth-independent, suggesting that small petroleum droplets were homogeneously dispersed in the water column (data not shown). The oxygen saturation of water in each tank was maintained at around 96% by a compressor that injected air via an air stone. The experimental system was placed in a thermoregulated greenhouse, in order to maintain the temperature of the exposure studies at 15 ± 1 °C. Exposure • One experimental system was used per condition, making a total of five experimental systems.

Thirty oysters were transferred into each experimental system. The different exposure conditions are summarized in Table 1. The exposure period lasted 2 days. The seawater used for this study (pH: 7.95 ± 0.05, salinity: 35.2 ± 0.1 p.s.u.) was provided by Oceanopolis, Brest (France). This seawater was treated by UV-light and filtrated at 0.45 µm before use. In the first tank, the control exposure condition was set up with clean seawater. For the amount of BAL selected, the aim of this study was to obtain a concentration of dispersed oil in the same rank than those reported in situ, following an oil spill, such as reported by [START_REF] Lunel | The Braer oil spill: oil fate governed by dispersion, International oil spill conference[END_REF] following the Braer oil spill. Thus, in the second tank, the chemical dispersion (CD) condition was set up by pouring 20 g of BAL 110 and the volume of dispersant recommended by the manufacturer, i.e. 1.2 g of dispersant, into the funnel of the experimental system. In the third tank, the mechanical dispersion (MD) condition was set up by pouring 20 g of BAL 110 into the funnel. In the fourth tank, the toxicity of molecules that naturally dissolve in seawater was tested, by exposing oysters to a water-soluble fraction (WSF) of BAL 110. To obtain the WSF, a plastic circle was set on the surface of the seawater in the experimental system. The BAL 110 (20 g) was then poured into the plastic circle, in order to contain the oil slick at the surface, without mixing. The oysters were therefore only exposed to the soluble fraction of the oil, i.e. free of particles of bulk material, and, contrary to the wateraccommodated fraction (WAF), free of soluble and volatile compounds that can naturally evaporate [START_REF] Anderson | Characteristics of dispersions and watersoluble extracts of crude and refined oils and their toxicity to estuarine crustaceans and fish[END_REF][START_REF] Singer | Standardization of the preparation and quantitation of water-accommodated fractions of petroleum for toxicity testing[END_REF]. In the fifth tank, an internal control for the CD condition was set up by pouring 1.2 g of chemical dispersant into the funnel (i.e., dispersant condition, D). All experimental systems contained a funnel connected to a submersed water pump. All exposure media were added to the tanks 13h before adding the oysters, the time needed to obtain a relatively stable oil concentration in the water column. The oysters were not fed during the exposure period. Depuration • Ten oysters per treatment condition were placed in a decontamination tank, located in the thermoregulated greenhouse (T = 15 ± 1°C) and containing clean seawater, for a recovery period of 15 days. The oysters were fed daily with an algal diet (5 x 10 4 cell ml -1 ) composed of Heteroskeletonema sp.

(Bacillariophyceae).

Sampling procedure

Pooled gills, digestive glands, mantles, haemocyte fraction and plasma of ten oysters were used for each replicate sample, and three replicates were prepared per treatment. After opening the oyster shells by cutting off the adductor muscle, approx. 0.3-0.5 ml of haemolymph was withdrawn from the pericardial cavity using a 1-ml syringe equipped with a needle (0.9 x 25 mm). Haemolymph samples were centrifuged at 260 x g for 10 min at 4°C in order to separate the cellular (haemocytes) fraction from the plasma. The gills, digestive gland and mantle were removed from the soft tissues and homogenized at 4°C in 0.1 M Tris HCl buffer pH 7.0 (0.45 M NaCl, 26 mM MgCl 2 , 10 mM CaCl 2 ; 0.5 ml of buffer.g -1 of fresh weight for the gills and the mantle, and 1 ml.g -1 of fresh weight for the digestive gland), using an Ultra Turrax (T25 basic, IKA-WERKE) and a Thomas-Potter homogenizer (IKA-Labortechnik RW 20.n, size 0.13-0.18 mm, BB). The homogenates were centrifuged at 10 000 x g for 10 min at 4°C. The resulting supernatant was collected for enzymatic studies.

Biochemical analysis

Superoxide dismutase assay • SOD was determined as described previously (Luna-Acosta et al., 2010a) based on competition of SOD with iodonitrotetrazolium (INT) for dismutation of superoxide anion (O 2 -).

In the presence of O 2 -, INT is reduced into a red formazan dye that can be measured at 505 nm at 25°C (Kit Ransod SD 125, Randox, France). One unit of SOD is defined as the amount of enzyme that promotes a 50% decrease in the rate of INT reduction.

Glutathione peroxidase assay • GPx activity was determined as described previously (Luna-Acosta et al., 2010a). In the presence of glutathione reductase and substrates (i.e. reduced glutathione and cumene hydroperoxide), the decrease of absorbance at 340 nm was proportional to the reduction of the oxidised glutathione by NADPH, H + (Kit Ransel RS 504, Randox, France). One unit of GPx oxidises 1 µmol of NADPH (ε NADPH = 6.22 mM -1 cm -1 ) per minute.

Catalase assay • Catalase activity was determined according to the method of [START_REF] Fossati | Use of 3,5-dichloro-2-hydroxybenzenesulfonic acid/4aminophenazone chromogenic system in direct enzymic assay of uric acid in serum and urine[END_REF]. This method is based on the measurement of the hydrogen peroxide substrate remaining after the action of catalase. First, catalase converts hydrogen peroxide into water and oxygen and then this enzymatic reaction is stopped with sodium azide. An aliquot of the reaction mix was then assayed for the amount of hydrogen peroxide remaining using a colorimetric method (Kit Catalase CAT-100, Sigma).

Catecholase-type and laccase-type phenoloxidase assay • Catecholase-and laccase-type phenoloxidase (PO) activities were determined according to the method described previously (Luna-Acosta et al., 2010b). For PO assays, 100 mM of dopamine or 50 mM of PPD were used as substrates and the increase of absorbance at 490 and 420 nm was monitored for 4 and 2h for catecholase-and laccase-type PO activity, respectively. Nonenzymatic oxidation by the substrate was monitored in wells without oyster sample and subtracted from oxidation of the substrate with oyster sample. One unit of catecholase-and laccase-type PO activities corresponds to the amount of enzyme that catalyzes the production of 1 µmole of product (ε=3300 M -1 cm -1 and 43 160 M -1 cm -1 , respectively), per minute.

Lysozyme assay • Lysozyme was determined as described previously (Luna-Acosta et al., 2010a). The lysozyme assay was done in triplicate for each sample and compared to hen egg white lysozyme standards (2.5-20 µg ml -1 ), in the presence of Micrococcus lysodeikticus (Sigma-Aldrich, France). One unit of lysozyme corresponds to the amount of enzyme that diminishes absorbance at 450 nm by 0.001 per minute at pH 7.0, at 25°C. Protein assay • All activities were expressed in relation to the protein concentration measured according to the Lowry method with slight modifications, using bicinchoninic acid and copper sulphate 4% [START_REF] Smith | Measurement of protein using bicinchoninic acid[END_REF]. Serum albumin was used as protein standard (Sigma-Aldrich, France).

All enzyme activities were measured in the gills, digestive gland, mantle, plasma and HLS, except for lysozyme that was not measured in the HLS, making a total of 29 enzymatic analyses.

Chemical analysis in oysters

The levels of polycyclic aromatic hydrocarbons (PAHs) in oysters were determined with a Varian Saturn 2100 T GC-MS device, using the procedure of [START_REF] Baumard | Analytical procedure for the analysis of PAHs in biological tissues by gas chromatography coupled to mass spectrometry: application to mussels[END_REF] with some modifications. Three pools of five oysters were analysed per treatment. Prior to extraction, each oyster sample was homogenized using an Ultra Turrax (Janke and Kunkel, IKA ® -Labortechnik). 150 µl of perdeuterated internal standards (CUS-7249, Ultra Scientific, Analytical solutions) were added to 3 g of homogenized oyster samples and the mixtures obtained were digested for 4 h under reflux in 50 ml of an ethanolic solution of potassium hydroxide (2 M, Fisher Chemicals). After cooling, settling and addition of 20 ml of demineralised water, the digest was extracted in a 250 ml funnel twice with 20 ml of pentane (Carlo Erba Reactifs, SDS). The extract was evaporated with a TurboVap 500 concentrator (Zyman, Hopkinton, MA, USA, at 880 mbar and 50 °C) to obtain 1 ml of concentrated extract. The purification of the extract was performed by transfer to a silica column (5 g of silica). Hydrocarbons were eluted with 50 ml of pentane: dichloromethane (80:20, v:v, SDS) and concentrated to 200 µl by means of a TurboVap 500 concentrator (Zyman, 880 mbar, 50°C). Aromatic compounds were analysed by GC-MS, with a detection limit of 0.005 µg g -1 of dry weight, and PAHs were quantified relative to the perdeuterated internal standards introduced at the beginning of the sample preparation [START_REF] Roy | On-site determination of polynuclear aromatic hydrocarbons in seawater by stir bar sorptive extraction (SBSE) and thermal desorption GC-MS[END_REF]. Five perdeuterated standard compounds, i.e. Naphthalene d8, Biphenyl d10, Phenanthrene d10, Chrysene d12, and Benzo[a]pyrene d12, representative of all the PAHs analysed, were used as internal standards. A total of 20 parent PAHs and 25 alkylated compounds were quantified. The PAH recovery was > 60% and the relative standard deviation (RSD, i.e. (standard error / mean x 100) was < 15%.

Statistical analysis

All values are reported as mean ± standard deviation (SD). Statistical analysis was carried out with STATISTICA 7.0. Values were tested for normality (Shapiro test) and homogeneity of variances (Bartlett test). In some cases, logarithmic transformations (Log 10 ) were used to meet the underlying assumptions of normality and homogeneity of variances. Two-way nested MANOVA were used to analyse results, with treatment and period as fixed factors, and pool as a random factor. The period factor corresponds to the exposure period and the depuration period. Pool was nested within each combination of treatment and time [START_REF] Zar | Biostatistical analysis[END_REF]. When the null hypothesis (H 0 : no difference between treatments or within treatment at different time intervals) was rejected, significant differences were tested using Tukey's HSD test. For non normal values, i.e. to compare oysters' PAH content between different treatments, a Kruskal-Wallis test was used, followed by a Dunn's multiple comparisons test. Statistical significance was determined as being at the level of p < 0.05. The relationships between body burdens for the different PAH categories (HMW PAHs, LMW PAHs, parent PAHs and alkylated PAHs) and the responses of the enzyme activities were analysed using principal component analysis (PCA) and redundancy analysis (RDA) [START_REF] Leps | Multivariate analysis of ecological data: course material[END_REF]. Separate analyses were performed for the different organs. Data for enzyme activities were centered and standardized before analysis. PCA and RDA were performed using CANOCO for Windows® software package, Version 4.5 (Center for Biometry, Wageningen, The Netherlands). For RDA analyses, the significance of the relationship between enzyme activities and PAH body burdens were tested using a global Monte Carlo permutation test.

Results

Chemical analyses

After 2 days of exposure, PAHs had been efficiently bioaccumulated in the soft tissues of C. gigas. The total PAH content in oysters (∑ PAHs) was equal to 7 ± 1, 530 ± 75, 198 ± 22, 56 ± 50 and 8 ± 3 µg g -1 dry weight for the control, the chemically dispersed oil (CD), the mechanically dispersed oil (MD), the water soluble fraction (WSF) and the dispersant (D) treatments, respectively. Significant differences were found between the control and the CD or the MD, but not with the WSF conditions (F 4,15 = 12.83, p = 0.01). This lack of significant difference could be explained by the high variation in total PAH levels in the WSF condition. Importantly, the total PAH content in oysters in the CD condition was almost 3 times higher than in the MD condition.

After 15 days of depuration, the ∑ PAHs was equal to 3 ± 0, 15 ± 6, 14 ± 6, 2 ± 0 and 2 ± 2 µg g -1 dry weight in control, CD, MD, WSF and D treatments, respectively. Even if the PAH content in oysters had decreased by 97 and 93% in CD and MD conditions, respectively, it remained significantly higher than the control condition (F 4,15 = 10.27, p = 0.03).

Independently of time and treatment, light PAHs (≤ 3 rings) and their alkylated homologues accounted for at least 90% of the total PAHs in oyster tissues (Fig. 1). After 2 days of exposure to the CD condition, alkylated naphthalenes (NaF), alkylated dibenzothiophenes (DBT) and alkylated fluorenes (Fl) accounted for most of the PAH content in oyster soft tissues with 55 ± 6, 19 ± 1, 18 ± 8% for alkylated NaF, alkylated DBT and alkylated Fl, respectively (Fig. 1). In the MD condition, alkylated DBT and alkylated Fl accounted for most of the PAH content in the oysters' soft tissues, representing 38 ± 2 and 40 ± 2% of the total PAHs, respectively. Alkylated DBT (22 ± 0%) and alkylated Fl (53 ± 0%) also accounted for a large proportion of the PAH content in the oysters' soft tissues in the WSF condition. For the D condition, alkylated DBT (45 ± 6%) and anthracene (30 ± 7%) accounted for most of the PAH content in oyster soft tissues.

After 15 days of depuration, alkylated DBT were the predominant PAH compounds in oyster tissues of the CD, MD, WSF and D conditions, representing 56 ± 19%, 48 ± 7%, 40 ± 5% and 50 ± 16% of the total PAHs, respectively (Fig. 1).

Enzymatic analysis

Overall, no significant differences in enzymatic activities were observed between the control conditions from both exposure and depuration periods, whatever the tissue (Figs. 234567). In contrast, all enzyme activities were affected by both treatment and period, independently of the considered tissue, except for catecholase and laccase activities in the mantle, which were only affected by the treatment (Figs. 5 and6).

No significant effect was observed for treatment for lysozyme activity in the gills and plasma (data not shown).

After 2 days of exposure for the CD condition, and relative to the control condition, catalase (Fig. 3) and GPx (Fig. 4) activities were completely inhibited and laccase activity (Fig. 6) was 1.4-fold higher in the gills. SOD (Fig. 2) and GPx (Fig. 4) activities were 30% and 50% lower in the digestive gland, respectively. Catalase activity (Fig. 3) was 75% lower in the mantle. Interestingly, results from the HLS and the plasma differed. While no significant effect was observed in the HLS, SOD activity (Fig. 2) was 2-fold higher and catalase (Fig. 3) and laccase (Fig. 6) activities were completely inhibited in the plasma.

For the MD condition, SOD (Fig. 2) and laccase (Fig. 6) activities were 2.5-fold and 1.4-fold higher in the gills, respectively. GPx activity (Fig. 4) was 1.5-fold higher in the mantle. While SOD (Fig. 2), GPx (Fig. 4), catecholase (Fig. 5) and laccase (Fig. 6) activities were 2-4-fold higher in the HLS, catalase activity (Fig. 3) was completely inhibited in the plasma. For the WSF condition, SOD (Fig. 2) and laccase (Fig. 6) activities were 2.5-and 1.7-fold higher in the gills, respectively. SOD (Fig. 2) and catecholase (Fig. 5) activities were 4-and 2-fold higher in the HLS, respectively. It is important to notice that the dispersant alone (D condition), which is considered as a harmless product, also modulated different enzyme activities in C. gigas: laccase activity (Fig. 6) was 1.4-fold higher in the gills; SOD (Fig. 2) and lysozyme (Fig. 7) activities (Fig. 2) were 70 and 55% lower in the digestive gland, respectively, and SOD (Fig. 2), catecholase (Fig. 5) and laccase (Fig. 6) activities were 2.5-to 3-fold higher in the HLS.

After 15 days of depuration, some activities returned to control levels, depending on the tissue and the treatment. In some tissues and for some activities, effects were observed only at this period, e.g. SOD activity (Fig. 2) was 1.3-fold higher in the mantle for the MD condition; catalase activity (Fig. 3) was 2to 2.5-fold higher in the digestive gland for the CD, WSF and D conditions; SOD activity (Fig. 2) was 1.3-fold higher for the MD condition; lysozyme activity (Fig. 7) was 1.4-to 1.6-fold higher for the CD, WSF and D conditions in the mantle; catecholase activity (Fig. 5) was inhibited by 20 to 30% in the plasma for the CD, MD and WSF conditions, and 1.4-fold higher in the D condition.

In addition to these results, a significant treatment, but not period, effect was observed in the mantle for catecholase (Fig. 5) and laccase (Fig. 6) activities. In this tissue, catecholase activity was inhibited by 20% for the CD and MD conditions and laccase activity was 1.2-fold higher for the CD and the D conditions.

Relationships between PAH contents in oyster soft tissues and enzyme activities

At the end of the exposure period, significant correlations were observed between different enzyme activities and PAH contents in oyster soft tissues and responses varied according to the tissue that was analysed. The results of PCA and RDA are presented in Fig. 8 and Table 2, respectively. RDA indicated PAH body burdens as the significant variable explaining 49%, 43%, 43%, 39%, 44% of total variation in enzyme activities in the gills, digestive gland, mantle, plasma and HLS, respectively. Enzyme activities in the gills, plasma and mantle were significantly correlated to parent PAHs. Enzyme activities in the plasma were also significantly correlated to LMW PAHs. Four out of six enzyme activities analysed in the gills and plasma were negatively correlated to body burdens of the various PAH categories: catalase, GPx, lysozyme, and catecholase or laccase, in the gills and plasma, respectively (Fig. 8, Table 2). SOD, GPx, catecholase and lysozyme in the mantle were positively correlated to body burdens of the various PAH categories (Fig. 8,Table 2). Four out of five enzyme activities analysed in the HLS, i.e. SOD, GPx, catecholase and laccase, were negatively correlated to body burdens of the various PAH categories, but were positively correlated to parent PAHs (Fig. 8, Table 2). Enzyme activities in the digestive gland were significantly correlated to HMW PAHs. Five out of six enzyme activities analysed in the digestive gland were positively correlated to body burdens of the various PAH categories: SOD, catalase, catecholase, laccase and lysozyme (Fig. 8, Table 2).

Discussion

PAH bioaccumulation and depuration in oyster tissues

The aim of this study was to assess 1) the bioaccumulation and 2) the effects of chemically dispersed hydrocarbons on a species inhabiting coastal and estuarine zones, the Pacific oyster C. gigas. This marine bivalve is a good indicator of the presence and bioavailability of oil in the water column and benthic sediments. In this species, bioconcentration factors (BCF) of petroleum hydrocarbons range from 10 to 50 000 [START_REF] Michel | Oil uptake and depuration in oysters after use of dispersants in shallow water in El Salvador[END_REF], so even low levels of exposure are likely to be detectable in oyster tissues. PAHs, with high octanol-water partition coefficients (log K ow > 3.5), are readily taken up by organisms [START_REF] Meador | Bioacccumulation of PAHs in marine invertebrates[END_REF]. Thus, evaluating PAH bioaccumulation plays an important part in assessing the risk that chemical dispersants are likely to pose to marine organisms inhabiting coasts and estuaries, especially carcinogenic, mutagenic or teratogenic PAHs. Because no PAH measurements were carried out in seawater, we were not able to calculate BCF, but PAH body burden analysis was conducted in oyster soft tissues. Results showed that, independently of the treatment (i.e. CD, MD, WSF or D), heavy PAHs were poorly accumulated in oyster tissues (0-3% of the total PAH content), while light PAHs and mainly their alkylated homologues, which are generally more toxic than the parent compounds, were present in large proportions in all the treatment conditions. Indeed, PAHs with low molecular weight (≤ 178.2 g mol -1 ) such as NaF, DBT and Fl are more likely to be bioaccumulated due to high water solubility and higher bioavailability for organisms [START_REF] Neff | Bioaccumulation in marine organisms: effect of contaminants from oil well produced water[END_REF].

A high heterogeneity in PAH body burden was observed for the WSF treatment. The WSF corresponds to hydrocarbon molecules that are liable to naturally dissolve, meaning that the solution can be considered as homogeneous. Therefore, the heterogeneity of results for the WSF treatment suggests that 1) organisms depurate at different rates and/or that 2) organisms accumulate at different rates [START_REF] Neff | Bioaccumulation in marine organisms: effect of contaminants from oil well produced water[END_REF].

Interestingly, for the same amount of dispersed oil with (CD condition) or without (MD condition) a chemical dispersant, and for the same exposure time, i.e. 2 days, oysters bioaccumulated approximately 2.7 times more PAHs in the presence of the chemical dispersant, showing that the dispersant increased the bioavailability of PAHs and thus their uptake rate in C. gigas. The bioaccumulated PAH compounds were completely different between both CD and MD conditions. In the CD condition, 55% of the total PAH content was composed of high alkylated NaF. These compounds were poorly bioaccumulated in the MD and WSF conditions (Fig. 1), whereas alkylated Fl and alkylated DBT represented between 70 and 80% of the total PAH content (Fig. 1). Our results for the CD condition but not for the MD condition are in agreement with a recent study carried out on the Iceland scallop Chlamys islandica where C3-NaF accounted for a large proportion of the PAHs that were accumulated in the tissues after 15 days of exposure to mechanically dispersed oil [START_REF] Hannam | Immune function in the Arctic Scallop, Chlamys islandica, following dispersed oil exposure[END_REF]. In the present study, the exposure experiment was carried out for only 2 days, and, since uptake rate constants of PAHs in bivalves generally increase with molecular weight or molecular complexity [START_REF] Mcleese | Comparative accumulation of PAHs in four marine invertebrates[END_REF], it cannot be excluded that a longer exposure period would result in a higher alkylated NaF uptake in oyster tissues. Nonetheless, our results clearly demonstrated that the presence of the chemical dispersant increased the bioaccumulation rate of alkylated NaF in oyster soft tissues. However, the processes that could alter 1) the bioconcentration and/or 2) the type of components accumulated, when oil is chemically dispersed, have been poorly described in the scientific literature. A possible contributing factor for bioaccumulation of some components is that dispersing spilled oil converts the oil from a surface slick to a plume of small oil droplets dispersed in the water column. These oil droplets might be more easily ingested by filter-feeding organisms, such as oysters [START_REF] Baussant | Bioaccumulation of polycyclic aromatic compounds: 2. Modeling bioaccumulation in marine organisms chronically exposed to dispersed oil[END_REF]. Additionally, particles trapped on the oysters' gills are directed into the gut and later incorporated into endocytic vacuoles in the digestive gland. Oil particles retained in intracellular vacuoles can also be assimilated to the tissue lipids [START_REF] Baussant | Bioaccumulation of polycyclic aromatic compounds: 2. Modeling bioaccumulation in marine organisms chronically exposed to dispersed oil[END_REF]. Thus, in the CD treatment, a significant amount of the larger PAHs bound to particles could be retained and accumulated in intracellular vacuoles or in tissue lipids during the exposure period.

After 15 days of depuration, more than 90% of the PAH burden had been depurated from oysters' soft tissues. Even if PAH contents in CD and MD conditions were still significantly higher than the control condition, this result clearly shows that oysters can eliminate high quantities of PAHs very rapidly.

Interestingly, no differences in the total PAH content between CD and MD were found for the depuration period. This result is in agreement with a previous study carried out on mussels [START_REF] Gilfillan | Effects of test spills of chemically dispersed and nondispersed oil on the activity of aspartate amino-transferase and glucose-6-phosphate dehydrogenase in two intertidal bivalves, Mya arenaria and Mytilus edulis[END_REF]. Among PAHs, alkylated DBTs were the most persistent PAHs in all treatments (Fig. 1). This result is in agreement with [START_REF] Berthou | The occurrence of hydrocarbons and histopathological abnormalities in oysters for seven years following the wreck of the Amoco Cadiz in Brittany (France)[END_REF] who report that DBT persist in oyster tissues for at least one year.

As in the present study, C3-DBT was among the most persistent PAHs [START_REF] Berthou | The occurrence of hydrocarbons and histopathological abnormalities in oysters for seven years following the wreck of the Amoco Cadiz in Brittany (France)[END_REF], raising questions about its potential toxic effects. Further studies are therefore needed in order to assess the longterm toxicity of this compound.

Effect on enzyme activities

Disease emergence and organism survival are determined partly by the condition of the immune system.

Therefore, the measurement of defence mechanisms can provide important early warning signals of the sub-lethal effects of exposure to contaminants and the susceptibility of animals to infectious diseases [START_REF] Hannam | Immune function in the Arctic Scallop, Chlamys islandica, following dispersed oil exposure[END_REF].

Bivalve molluscs possess two types of innate responses: 1) cellular, i.e. phagocytosis and encapsulation; and 2) humoral, e.g. PO and lysozyme enzyme activities [START_REF] Tryphonas | Investigative Immunotoxicology[END_REF]. During phagocytosis, reactive oxygen species (ROS), such as the superoxide anion (O 2 -) and hydrogen peroxide (H 2 O 2 ), play an important role in protection against pathogens. However, ROS can also interact with hosts' biological macromolecules which can result in enzyme inactivation, lipid peroxidation, DNA damage or cell death [START_REF] Cazenave | Attenuating effects of natural organic matter on microcystin toxicity in zebra fish (Danio rerio) embryos -benefits and costs of microcystin detoxication[END_REF]. The extent to which oxyradical generation produces biological damage depends on the effectiveness of antioxidant defences, such as SOD, which participates in the transformation of O 2 into H 2 O 2 and H 2 O, and of catalase and GPx, which participate in the transformation of H 2 O 2 into O 2 and H 2 O [START_REF] Neumann | Antimicrobial mechanisms of fish phagocytes and their role in host defense[END_REF].

Environmental contaminants, such as PAHs, can lead to the formation of ROS and enhance oxidative stress in aquatic organisms [START_REF] Winston | Oxidants and antioxidants in aquatic animals[END_REF]. Results from the present study showed a positive correlation between PAH contents and SOD activities. Such a relationship has already been reported for different bivalve species exposed to hydrocarbons [START_REF] Sole | The use of biomarkers for assessing the effects of organic pollution in mussels[END_REF][START_REF] Orbea | Antioxidant enzymes and peroxisome proliferation in relation to contaminant body burdens of PAHs and PCBs in bivalve molluscs, crabs and fish from the Urdaibai and Plentzia estuaries (Bay of Biscay)[END_REF][START_REF] Richardson | Antioxidant responses to polycyclic aromatic hydrocarbons and organochlorine pesticides in green-lipped mussels (Perna viridis): Do mussels "integrate" biomarker responses?[END_REF] and suggests that hydrocarbons induce oxidative stress by producing ROS such as O 2 -. However, in the present study, catalase and GPx activities were generally negatively correlated with different PAH body burdens. This may be due to the inhibition of enzyme synthesis by PAHs or to enzyme inactivation caused by high tissue contaminant concentrations [START_REF] Borg | Cytotoxicity from coupled redox cycling of autoxidizing xenobiotics and metals[END_REF]. Moreover, since GPx and catalase catalyze the transformation of H 2 O 2 into H 2 O, they may act on common substrates, and thus competition may exist for the same group of substrates [START_REF] Kappus | Lipid peroxidation: mechanisms, analysis, enzymology and biological relevance[END_REF]. This may explain the positive correlations of catalase activity and negative correlations of GPx activity with PAH content for the same tissues in the present study (Table 2).

Among enzymes involved in humoral immune defences in bivalves, POs are the key enzymes of melanization, participating in the entrapment of foreign material in a melanin capsule or in the direct killing of microbes by the toxic quinone intermediates produced during the melanin production cascade [START_REF] Söderhäll | Role of the prophenoloxidase-activating system in invertebrate immunity[END_REF]. Tyrosinases, catecholases, and laccases belong to the family of POs.

While tyrosinase possesses monophenolase (hydroxylation of monophenols) and catecholase (oxidation of diphenols) activities, catecholase only possesses catecholase activities and laccase possesses both catecholase and laccase activities (oxidation of o-, p-and m-diphenols and other non phenolic substrates such as diamines). In a recent study, two types of PO activity, catecholase and laccase, were detected in C. gigas (Luna-Acosta et al., 2010b). In the present study, catecholase activities were generally inhibited in the presence of hydrocarbons, except for HLS catecholase activity. These results are in agreement with other studies on C. gigas where exposure to hydrocarbons induced an inhibitory effect in PO activities [START_REF] Bado-Nilles | Immune effects of HFO on European sea bass, Dicentrarchus labrax, and Pacific oyster, Crassostrea gigas[END_REF]. Moreover, when using PPD as a substrate for PO assays in this study, laccase activities were generally stimulated in the presence of hydrocarbons, except for laccase activity in plasma.

These results are in agreement with previous studies where the exposure of C. gigas to a light cycle oil (LCO) soluble fraction induced an increase in the mRNA expression of a laccase (multicopper oxidase) gene in haemocytes after 7 days of exposure [START_REF] Bado-Nilles | In vivo effects of LCO soluble fraction on immune-related functions and gene transcription in the Pacific oyster, Crassostrea gigas (Thunberg)[END_REF]. Since laccases can catalyze oxidation of aromatics, such as PAHs, by an indirect mechanism involving the participation of an oxidative mediator [START_REF] Dodor | Oxidation of anthracene and benzo[a]pyrene by immobilized laccase from Trametes versicolor[END_REF], an increase in laccase gene transcription in C. gigas may enable protection against bioaccumulated PAHs. Laccase could therefore be a potential defence biomarker candidate in ecotoxicological studies.

Lysozyme is one of the most important bacteriolytic agents against several species of Gram-positive and Gram-negative bacteria, and has been recorded for various bivalve species [START_REF] Mchenery | Distribution of lysozyme-like activity in 30 bivalve species[END_REF].

During phagocytosis, the release of lysosomal enzymes, such as lysozymes, participates in the inactivation of invading pathogens. In the present study, inhibition of lysozyme activity was observed in the digestive gland in the D condition. Previous studies have shown inhibition of lysozyme activity or lysozyme gene expression in organisms exposed to hydrocarbons [START_REF] Boutet | Response of the Pacific oyster Crassostrea gigas to hydrocarbon contamination under experimental conditions[END_REF][START_REF] Gopalakrishnan | Immunomodulation in the marine gastropod Haliotis diversicolor exposed to benzo(a)pyrene[END_REF]. However, in this study no lysozyme inhibition was observed in the presence of hydrocarbons, except in the digestive gland for the D condition. Activation of mantle lysozyme activity was observed during the depuration period. Although generally pollutant exposure has shown to inhibit lysozyme activity in bivalves (for review, see [START_REF] Giron-Perez | Relationships between innate immunity in bivalve molluscs and environmental pollution[END_REF], some studies have shown an induction of lysozyme activity (e.g., [START_REF] Oliver | Greater hemocyte bactericidal activity in oysters (Crassostrea virginica) from a relatively contaminated site in Pensacola Bay[END_REF][START_REF] Ordas | Assessment of in vivo effects of the Prestige fuel oil spill on the Mediterranean mussel immune system[END_REF][START_REF] Hannam | Immune function in the Arctic Scallop, Chlamys islandica, following dispersed oil exposure[END_REF]. However, to our knowledge, no studies have shown the modulation of lysozyme activity during the depuration period. The activation of this enzyme activity suggests a potential immunostimulation during the depuration period. Uptake of xenobiotics from seawater is generally accomplished by filtration over the gill structure, although diffusion through the integument may also contribute to tissue concentrations [START_REF] Landrum | Role of respiration in the accumulation of organic xenobiotics by the amphipod, Pontoporeia hoyi[END_REF]. As the organism directs seawater over the gill surface to extract oxygen, hydrophobic contaminants are very efficiently taken up because of the large surface area and lipid-rich membranes.

Moreover, bivalves possess an open circulatory system and are thus continually exposed to fluctuations of environmental factors including contaminants [START_REF] Cheng | Invertebrate blood cells[END_REF]. As a general trend, enzyme activities in the plasma and gills were generally inhibited, especially in the CD condition (Figs. 234567), suggesting that compartments that are the more exposed to the marine environment, such as the gills and plasma, are likely to be more affected by the presence of hydrocarbons, in comparison to tissues less exposed to the marine environment, such as the digestive gland, mantle and HLS [START_REF] Cheng | Invertebrate blood cells[END_REF][START_REF] Frouin | Physiological effects of polycyclic aromatic hydrocarbons on soft-shell clam Mya arenaria[END_REF].

Enzyme activities in the digestive gland were less modulated than in the other tissues but were strongly correlated with PAH contents in this tissue, highlighting its role in xenobiotic detoxification (Chafai-El [START_REF] Alaoui | Etude de quelques aspects de la contamination métallique chez certains mollusques et poissons marins : Etude environnementale (côte atlantique marocaine) et approche expérimentale[END_REF][START_REF] Chu | Relationship between PCB accumulation and reproductive output in conditioned oysters Crassostrea virginica fed a contaminated algal diet[END_REF].

Enzyme activities in the mantle and haemocytes, which are known to play an important role in defence mechanisms in oysters [START_REF] Cheng | Invertebrate blood cells[END_REF], were generally activated in comparison to the control. Among tissues that were analysed, haemolymph can be considered as a key tissue because this fluid irrigates the whole body and, therefore, it can distribute contaminants and/or their metabolites throughout the organism [START_REF] Cheng | Invertebrate blood cells[END_REF]. Interestingly, enzyme activities differed notably from the plasma and HLS of the haemolymph. These differences could be due to alterations in the membrane integrity by PAHs and/or their metabolites, as suggested by in vitro and in vivo investigations in the blue mussel Mytilus edulis [START_REF] Grundy | Phagocytic reduction and effects on lysosomal membranes by polycylic aromatic hydrocarbons, in haemocytes of Mytilus edulis[END_REF]. The extent of membrane alterations could be dependent on the physical (e.g. linear versus angular or branched configuration of isomers) and/or chemical (e.g. low molecular weight with high solubility versus high molecular weight with low solubility) properties of the compounds. In addition, modulation of enzyme activities in the plasma can be attributed to 1) normal mechanisms, such as secretion or 2) pathological features, such as cell lysis. Indeed, PAHs can cause cytolysis in haemocytes [START_REF] Mccormick-Ray | Hemocytes of Mytilus edulis affected by Prudhoe Bay crude oil emulsion[END_REF], due to 1) a depletion or stimulation of metabolites or coenzymes, 2) an inhibition or stimulation of enzymes and other specific proteins, 3) an activation of a xenobiotic to a more toxic molecular species, or 4) membrane disturbances (for review, see [START_REF] Moore | Cellular responses to pollutants[END_REF]. Cytolysis may lead to 1) an increase in haemocyte number indicating compensation for cell lysis and/or 2) the release of cell contents in the plasma and consequently, a significant increase in plasma enzyme activities.

Alternatively, direct induction or repression by PAHs of humoral factors, such as lysozyme [START_REF] Luna-Gonzalez | Ontogenic variations of hydrolytic enzymes in the Pacific oyster Crassostrea gigas[END_REF] and pro-phenoloxidase [START_REF] Söderhäll | Role of the prophenoloxidase-activating system in invertebrate immunity[END_REF], may contribute to the modulation of enzyme activities found in the plasma.

Effect of chemical dispersion (CD) and dispersant (D) conditions

Increased activity of an enzyme involved in defence mechanisms can be interpreted as a response of the organism to protect itself against a non-self molecule, e.g. xenobiotics. Persistent or excessive activation can lead to overstimulation of the immune system, which can be detrimental to the organism. Inhibition of the enzyme activity can be interpreted as saturation of the system because of the presence of a xenobiotic, overpowering of antioxidant enzymes by oxygen radicals, or an immunodeficiency resulting in direct or an indirect inhibition of mechanisms that modulate this enzyme activity [START_REF] Huggett | Biomarkers: biochemical, physiological and histological makers of anthropogenic stress[END_REF].

In both cases, changes in defencive enzymes may affect the survival of the organisms when challenged with infectious pathogens [START_REF] Thiagarajan | Immunomodulation the marine green mussel Perna viridis exposed to sub-lethal concentrations of Cu and Hg[END_REF].

Twenty nine enzymatic analyses were carried out in this study. As a general trend and relative to the control, CD modulated a higher number of enzyme activities than D, following the exposure period.

Indeed, an equal or greater effect was observed in 13/18 (i.e. 13 out of 18) enzyme activities modulated by CD and/or D: 5/5, 2/3, 3/3, 3/3 and 0/4 enzyme activities modulated by CD and/or D in the gills, digestive gland, mantle, plasma and HLS, respectively (Figs. 234567). When comparing CD and MD conditions, following the exposure period, CD modulated a higher number of enzyme activities than MD, relative to the control condition, with an equal or greater effect observed in 13/19 activities modulated by CD and/or MD: 4/5, 2/2, 3/4, 4/5 and 0/4 enzyme activities modulated by CD and/or MD in the gills, digestive gland, mantle, plasma and HLS, respectively (Figs. 23456). Moreover, CD exerted an equal or greater effect in a higher number of enzyme activities in the gills and plasma in comparison to other tissues, i.e. 4/5 enzyme activities modulated by CD and/or MD (Figs. 23456) , suggesting that the gills and plasma are sensitive compartments in C. gigas. Moreover, some enzyme activities that were modulated by the CD treatment following the exposure period were also modulated following the depuration period, e.g. SOD activity in the gills, mantle and plasma (Fig. 2), or laccase activity in the plasma (Fig. 6), suggesting that CD may exert long-term effects. Importantly, D also modulated enzyme activities, especially at the end of the depuration period, e.g. SOD activity in the plasma and HLS (Fig. 2), catalase activity in the digestive gland (Fig. 3), GPx activity in the plasma (Fig. 4), catecholase activity in the plasma and HLS (Fig. 5), laccase activity in the gills and digestive gland (Fig. 6) and lysozyme activity in the mantle (Fig. 7), suggesting that the dispersant used in the present study could also induce long-term effects, i.e. following 15 days of depuration. Our results are thus in agreement with previous studies that have shown the effects of other third generation dispersants on biological functions in marine invertebrates [START_REF] Gilfillan | Effects of test spills of chemically dispersed and nondispersed oil on the activity of aspartate amino-transferase and glucose-6-phosphate dehydrogenase in two intertidal bivalves, Mya arenaria and Mytilus edulis[END_REF][START_REF] Shafir | Short-and long-term toxicity of crude oil and oil dispersants to two representative coral species[END_REF].

Differences between oysters from the control and the treatment conditions resided only in the presence of oil and/or dispersants in the water column (i.e. experimental devices and acclimatization conditions were identical in the control and the treatment conditions). Additionally, the D treatment (i.e. in the absence of oil) induced effects in enzyme activities such as laccase in the gills, SOD, catalase and lysozyme in the digestive gland, and SOD in the plasma, following the exposure or the depuration periods. Therefore, significant differences in biological responses between the control and the different treatments could not be considered as specific to contamination by hydrocarbons, but rather as indicators of unspecific stress in oysters, induced by chemicals (oil and/or dispersants) that have entered the organism from the water column. It is important to notice that transient effects were observed for different enzyme activities and thus longer periods of exposure should be studied. Moreover, not all the enzyme activities returned to control levels after the 15-day depuration period and thus longer depuration periods could give better insight into long-term effects.

Conclusions

• Responses of enzyme activities involved in immune and detoxification mechanisms in juveniles of the Pacific oyster C. gigas were highly variable depending on the treatment, the time and the tissue that was studied, highlighting the importance of carrying out studies in different tissues and with multiple enzymes involved in crucial biological responses.

• Enzyme activities were generally inhibited in the gills and plasma, while they were generally activated in the mantle and haemocytes, suggesting that the gills and the plasma are sensitive compartments in C. gigas and that the mantle and the haemocytes are likely to play an important role in protection against PAHs.

• Among the various parameters and during the exposure period, enzyme activities in the digestive gland, mantle and haemocytes were generally positively correlated with PAH body burdens.

Enzyme activities in the gills and plasma were generally negatively correlated with PAH body burdens, suggesting potential suppressive effects of pollutants in immune and/or detoxification mechanisms through the inhibition of enzyme activities involved in these biological responses.

• Finally, the presence of dispersants or of chemically dispersed oil modulates different biological responses in C. gigas. These results raise questions as to the potential effects of chemically dispersed oil in nearshore areas on immune and/or detoxification responses for this estuarine species, such as in the case of the Deepwater Horizon oil spill, in which a high percentage of oil was dispersed in the water column, with a large number of unknowns on the long-term impact of the oil-dispersant association. No data a The arrows between parenthesis show the trend associated with body burden of parent PAHs

Figure 1 .

 1 Figure 1. Proportion (%, mean ± SD, n=3) of the main PAHs contained in oyster tissues after 2 days of exposure to chemical dispersion (CD), mechanical dispersion (MD), water soluble fraction (WSF) and dispersant (D) treatments, followed by 15 days of depuration. PAHs: polycyclic aromatic hydrocarbons; NaF: naphthalene; Ac: acenaphtylene; Fl: fluorene; Fen: phenanthrene; Ant: anthracene; DBT: dibenzothiophene.

Figure 2 .Figure 3 .Figure 4 .Figure 5 .Figure 6 .Figure 7 .Figure 8 .

 2345678 Figure 2. Superoxide dismutase activity in the gills, digestive gland, mantle, plasma and haemocyte lysate supernatant (HLS) in C. gigas after 2 days of exposure to chemical dispersion (CD), mechanical dispersion (MD), water soluble fraction (WSF) and dispersant (D) treatments, followed by 15 days of depuration. Data are expressed as mean ± SD U.mg prot -1 , n=9 (i.e. 3 sample replicates from 3 experiment replicates). For clarity of results, only significant differences between control and treatment and for a given time (after 2 d of exposure or after 15 d of depuration) are shown; * p<0.05, ** p<0.01, *** p<0.001.

Table 1 .

 1 Experimental conditions used in the study. CD: chemical dispersion, MD: mechanical dispersion, WSF: water soluble fraction and D: dispersant. BAL 110: Brut Arabian Light crude oil topped at 110°C.Quantity of the product added to the water column (mg.l -1 ) # In order to have a stable concentration of the products in the water column, the products were added 13 hours before introducing animals in the tanks

	BAL 110	Dispersant

Table 2 Relationships between enzyme activities in tissues and haemolymphatic compartments and body burdens of the various PAH categories measured in the whole organism.

 2

		Gills	Digestive gland	Mantle	Plasma	HLS
	Significance of enzyme activities-PAH body burden relationship (RDA; Monte Carlo test, p-value)
		0.002	0.009	0.001	0.021	0.025
	Amount of total variation in enzyme activities explained by PAH body burdens (%)
		48.6	42.5	43.3	38.7	43.5
	Correlation with RDA 1st axis
	HMW PAHs	0.778	0.822	0.382	0.673	0.183
	LMW PAHs	0.631	0.756	0.620	0.776	0.369
	Parent PAHs	0.871	0.618	0.873	0.623	-0.216
	Alkylated PAHs	0.630	0.758	0.380	0.774	0.373
	Trend with increasing body burden of the various PAH categories, i.e. HMW, LMW, parent and alkylated PAHs
	SOD					( ) a
	Catalase					( )
	GPx					( )
	Catecholase					( )
	Laccase					( )
	Lysozyme

Acknowledgements: This study was supported by a PhD grant for A. Luna-Acosta from the Conseil Général de la Charente-Maritime. The Programme Ecotechnologies et Développement Durable (PRECODD) of the Agence Nationale de la Recherche (ANR) and especially M. Girin and G. Le Lann are acknowledged for financial support for the project 'DISCOBIOL' ("Dispersants et technique de lutte en milieux côtiers : effets biologiques et apports à la réglementation"), managed by F. X. Merlin. The authors also acknowledge Total Fluides for providing chemicals. Special thanks go to M. Menguy and M. Pommier for their help and assistance during the study and during the experimental procedures. The authors thank also S.

Ferguson (Alba Traduction, Quimper, France) for her revision of the English language. Finally, the authors specially acknowledge Dr. T. Caquet and Dr. P-G. Sauriau for their help and assistance for statistical analysis.