Protein expression profiles in Bathymodiolus azoricus exposed to cadmium.

Proteomic changes in the "gill-bacteria complex" of the hydrothermal vent mussel B. azoricus exposed to cadmium in pressurized chambers ((Incubateurs Pressurises pour l'Observation en Culture d'Animaux Marins Profonds - IPOCAMP) were analyzed and compared with the non-exposed control group. 2-D Fluorescence Difference Gel Electrophoresis (2D-DIGE) showed that less than 1.5% of the proteome of mussels and symbiotic bacteria were affected by a short-term (24 h) Cd exposure. Twelve proteins of the more abundant differentially expressed proteins of which six were up-regulated and six were down-regulated were excised, digested and identified by mass spectrometry. The identified proteins included structural proteins (actin/actin like proteins), metabolic proteins (calreticulin/calnexin, peptidyl-prolyl cis-trans isomerase, aminotransferase class-III, electron transfer flavoprotein, proteasome, alpha-subunit and carbonic anhydrase) and stress response proteins (chaperone protein htpG, selenium-binding protein and glutathione transferases). All differently expressed proteins are tightly connected to Cd exposure and are affected by oxidative stress. It was also demonstrated that B. azoricus was well adapted to Cd contamination therefore B. azoricus from hydrothermal vent areas may be considered a good bioindicator.

Trace metal concentrations in post-hatching cuttlefish Sepia officinalis and consequences of dissolved zinc exposure.

In this study, we investigated the changes of 13 trace metal and metalloid concentrations (i.e. Ag, As, Cd, Co, Cr, Cu, Fe, Mn, Ni, Pb, Se, V, Zn) and their subcellular fractionation in juvenile cuttlefish Sepia officinalis reared in controlled conditions between hatching and 2 months post-hatching. In parallel, metallothionein concentrations were determined. Our results highlighted contrasting changes of studied metals. Indeed, As and Fe concentrations measured in hatchlings suggested a maternal transfer of these elements in cuttlefish. The non-essential elements Ag and Cd presented the highest accumulation during our study, correlated with the digestive gland maturation. During the 6 first weeks of study, soluble fractions of most of essential trace metals (i.e. Co, Cr, Cu, Fe, Se, Zn) slowly increased consistently with the progressive needs of cuttlefish metabolism during this period. In order to determine for the first time in a cephalopod how metal concentrations and their subcellular distributions are impacted when the animals are trace metal-exposed, we studied previously described parameters in juveniles exposed to dissolved Zn at environmental (i.e. 50 µg l(-1)) and sublethal (i.e. 200 µg l(-1)) levels. Moreover, oxidative stress (i.e. glutathione S-transferase (GST), superoxide dismutase (SOD) and catalase activities, and lipid peroxidation (LPO)) was assessed in digestive gland and gills after 1 and 2 months exposures. Our results highlighted no or low ability of this stage of life to regulate dissolved Zn accumulation during the studied period, consistently with high sensitivity of this organism. Notably, Zn exposures caused a concentration-dependent Mn depletion in juvenile cuttlefish, and an increase of soluble fraction of Ag, Cd, Cu without accumulation modifications, suggesting substitution of these elements (i.e. Mn, Ag, Cd, Cu) by Zn. In parallel, metallothionein concentrations decreased in individuals most exposed to Zn. Finally, no perturbations in oxidative stress management were detected in gills, whereas modifications of GST, SOD and catalase activity levels were recorded in digestive gland, resulting in an increase of LPO content after a 6-week exposure to low Zn concentration. Altogether, these perturbations are consistent with previously described high sensitivity of juvenile cuttlefish towards Zn. Our results underlined the need to study deeply contamination impact on this animal at this stage of life.

Evidence of species-specific detoxification processes for trace elements in shorebirds.

This study investigated sub-lethal effects and detoxification processes activated in free-ranging Red Knots (RKs) (Calidris canutus) from the Pertuis Charentais on the Atlantic coast of France, and compared the results with previous data obtained on another shorebird species, the Black-tailed Godwit (Limosa limosa). The concentrations of 13 trace elements (Ag, As, Cd, Co, Cr, Cu, Fe, Hg, Mn, Ni, Pb, Se, Zn) were assessed in the liver, kidneys, muscle and feathers. Stable isotope analyses of carbon and nitrogen were carried out to determine whether differences in diet explained variations in elemental uptake. The mRNA expression of relevant genes (cytochrome c oxidase 1, acetyl-CoA carboxylase, Cu/Zn and Mn superoxide dismutase, catalase, metallothionein, malic enzyme), antioxidant enzyme activities (catalase, glutathione peroxidase (GPx), superoxide dismutase), and metallothionein (MT) levels were investigated to shed light on trace element detoxification and toxic effects. Although Red Knots were characterized by elevated As and Se concentrations which were potentially toxic, most elements were usually below toxicity threshold levels. The results strongly suggested a dietary specialization of Red Knots, with individuals feeding on higher trophic status prey experiencing higher As, Hg and Se burdens. Red Knots and Godwits also showed discrepancies in elemental accumulation and detoxification processes. Higher As and Se concentrations in Red Knots enhanced catalase gene expression and enzyme activity, while Godwits had higher Ag, Cu, Fe and Zn levels and showed higher MT production and GPx activity. The results strongly suggest that detoxification pathways are essentially trace element- and species-specific.

Insight on trace element detoxification in the Black-tailed Godwit (Limosa limosa) through genetic, enzymatic and metallothionein analyses.

Trace element concentrations (Ag, As, Cd, Co, Cr, Cu, Fe, Hg, Mn, Ni, Pb, Se, Zn) were investigated in the liver, kidneys, muscle and feathers of 31 black-tailed godwits (Limosa limosa) accidentally killed during catches by mist net in the Pertuis Charentais, Atlantic coast of France. Analyses of carbon and nitrogen stable isotope ratios were carried out in liver, muscle and feathers in order to elucidate dietary patterns and to determine whether differences in diet explained the variation in elemental uptake. This study also aimed to have a preliminary assessment of sub-lethal effects triggered by trace elements through the investigation of gene expressions by quantitative real-time PCR, antioxidant enzyme activities (catalase, superoxide dismutase, glutathione peroxidase), and metallothionein (MT) levels. The results showed that Cr and Ni concentrations in tissues of adults were lower than in juveniles in part because adults may have eliminated these trace elements through moulting. Except for Cd and Ni, trace element concentrations were negatively correlated to the body mass of godwits. Ag, As, Hg and Se concentrations were positively linked with the trophic position of birds. The diet could be considered as a fundamental route of exposure for these elements demonstrating therefore the qualitative linkage between dietary habits of godwits and their contaminant concentrations. Our results strongly suggest that even though trace element concentrations were mostly below toxicity threshold level, the elevated concentrations of As, Ag, Cd, Cu, Fe and Se may however trigger sub-lethal effects. Trace elements appear to enhance expression of genes involved in oxidative stress defence, which indicates the production of reactive oxygen species. Moreover, birds with the highest concentrations appeared to have an increased mitochondrial metabolism suggesting that the fight against trace element toxicity requires additional energetic needs notably to produce detoxification mechanisms such as metallothioneins.

The influence of nutritional conditions on metal uptake by the mixotrophic dual symbiosis harboring vent mussel Bathymodiolus azoricus.

The vent mussel Bathymodiolus azoricus, host thioautotrophic and methanotrophic bacteria, in their gills and complementary, is able to digest suspended organic matter. But the involvement of nutritional status in metal uptake and storage remains unclear. The influence of B. azoricus physiological condition on its response to the exposure of a mixture of metals in solution is addressed. Mussels from the Menez Gwen field were exposed to 50 µgL(-1) Cd, plus 25 µgL(-1) Cu and 100 µgL(-1) Zn for 24 days. Four conditions were tested: (i) mussels harboring both bacteria but not feed, (ii) harboring only methanotrophic bacteria, (iii) without bacteria but fed during exposure and (iv) without bacteria during starvation. Unexposed mussels under the same conditions were used as controls. Eventual seasonal variations were assessed. Metal levels were quantified in subcellular fractions in gills and digestive gland. Metallothionein levels and condition indices were also quantified. Gill sections were used for fluorescence in situ hybridization (FISH) to assess the temporal distribution of symbiotic associations. Starvation damages metal homeostasis mechanisms and increase the intracellular Zn and MT levels function. There is a clear metallic competition for soluble and insoluble intracellular ligands at each condition. Seasonal variations were observed at metal uptake and storage.

(210)Po and (210)Pb in the tissues of the deep-sea hydrothermal vent mussel Bathymodiolus azoricus from the Menez Gwen field (Mid-Atlantic Ridge).

The hydrothermal deep-sea vent fauna is naturally exposed to a highly specific environment enriched in potentially toxic species such as sulfides, metals and natural radionuclides due to the convective seawater circulation inside the oceanic crust and its interaction with basaltic or ultramafic host rocks. However, data on radionuclides in biota from such environment are very limited. An investigation was carried out on tissue partitioning of (210)Po and (210)Pb, two natural radionuclides within the (238)U decay chain, in Bathymodiolus azoricus specimens from the Mid-Atlantic Ridge (Menez Gwen field). These two elements showed different distributions with high (210)Pb levels in gills and high (210)Po levels in both gills and especially in the remaining parts of the body tissue (including the digestive gland). Various factors that may explain such partitioning are discussed. However, (210)Po levels encountered in B. azoricus were not exceptionally high, leading to weighted internal dose rate in the range 3 to 4 µGy h⁻¹. These levels are slightly higher than levels characterizing coastal mussels (~1 µGy h⁻¹).

Sub-lethal effects of cadmium on the antioxidant defence system of the hydrothermal vent mussel Bathymodiolus azoricus.

The mussel Bathymodiolus azoricus is one of the most abundant species in the Mid-Atlantic Ridge hydrothermal vents and is continually exposed to the high-temperature venting fluids containing high metal concentrations and enriched in sulphides and methane, which constitute a potential toxic environment for marine species. The aim of this study was to assess the effects of a sub-lethal Cd concentration on the antioxidant defence system of this mussel. B. azoricus were collected at Menez Gwen vent site (37 degrees 51'N, 32 degrees 31'W) and exposed to Cd (50 microg l(-1)) during 24 days, followed by a depuration period of six days. A battery of stress related biomarkers including antioxidant enzymes (superoxide dismutase-SOD, catalase-CAT; glutathione peroxidases-GPx), metallothioneins (MT), lipid peroxidation (LPO) and total oxyradical scavenging capacity (TOSC) were measured in the gills and mantle of B. azoricus. Cd was accumulated linearly during the exposure period in both tissues and no significant elimination occurred after the 6 days of depuration. Antioxidant enzymes activities were significantly higher in the gills. Cyt-SOD, T-GPx and Se-GPx were induced during the experiment but this was also observed in control organisms. Mit-SOD and CAT activities remained relatively unchanged. MT levels increased linearly in the gills of exposed mussels in the first 18 days of exposure. No significant differences were observed between LPO levels of control and exposed mussels. TOSC levels remained unchanged in control and exposed mussels. This suggests that although Cd is being accumulated in the tissues of exposed mussels, MT defence system is enough to detoxify the effect of Cd accumulated in the tissues. Furthermore, other factors besides the presence of Cd are influencing the antioxidant defence system in B. azoricus.

Responses of wild small mammals to a pollution gradient: host factors influence metal and metallothionein levels.

We investigated how host factors (species, age, gender) modulated Cd, Pb, Zn, and Cu concentrations, metallothionein levels (MTs) and their relationships in 7 sympatric small mammal species along a pollution gradient. Cd concentrations in liver and kidneys increased with age in all species. Age effect on other metals and MTs differs among species. Gender did not influence metal and MT levels except in the bank vole. Three patterns linking internal metal concentrations and MTs were observed along the gradient: a low metal accumulation with a (i) high (wood mouse) or (ii) low (bank vole) level of MTs accompanied by a slight or no increase of MTs with Cd accumulation; (iii) an elevated metal accumulation with a sharp increase of MTs (common and pygmy shrews). In risk assessment and biomonitoring perspectives, we conclude that measurements of MTs and metals might be associated because they cannot be interpreted properly when considered separately.

Spatial variation of metal bioaccumulation in the hydrothermal vent mussel Bathymodiolus azoricus.

The variability of the bioaccumulation of metals (Ag, Cd, Cu, Fe, Mn and Zn) was extensively studied in the mussel Bathymodiolus azoricus from five hydrothermal vent sites inside three main vent fields of increasing depth along the Mid-Atlantic Ridge: Menez Gwen, Lucky Strike and Rainbow. Metal bioaccumulation varied greatly between vent fields and even between sites inside a vent field with B. azoricus showing a great capacity to accumulate metals. The bioaccumulation of these metals also varied significantly among tissues. The main target was the gills where metals were mainly associated with soluble compounds whereas in the digestive gland they were mainly associated with insoluble compounds. Storage of metals under insoluble forms in B. azoricus seems to be a major pathway for the detoxification of both essential and non-essential metals. Mussels from the studied fields can be discriminated following their metallic load but the segregation relies partially on the composition of the metal-enriched fluids.

Antioxidant biochemical responses to long-term copper exposure in Bathymodiolus azoricus from Menez-Gwen hydrothermal vent.

Copper (Cu) is essential to various physiological processes in marine organisms. However, at high concentrations this redox-active transition metal may enhance the formation of reactive oxygen species (ROS) and subsequently initiate oxidative damage. High concentrations of Cu may increase oxidative damage to lipids, proteins and DNA. Bathymodiolus azoricus is a Mytilid bivalve very common in hydrothermal environments near the Azores Triple Junction continuously exposed to high metal concentrations, including Cu, emanating from the vent fluids. The knowledge of antioxidant defence system and other stress related biomarkers in these organisms is still scarce. The aim of this work was to study the effect of Cu (25 microg l(-1); 24 days exposure; 6 days depuration) on the antioxidant stress biomarkers in the gills and mantle of B. azoricus. The expression of stress related biomarkers was tissue-dependent and results suggest that other factors than metal exposure may influence stress biomarkers, since little variation in antioxidant enzymes activities, MT concentrations, LPO and total oxyradical scavenging capacity (TOSC) occurred in both control and Cu-exposed mussels. Moreover, there is a general tendency for these parameters to increase with time, in both control and Cu-exposed mussels, suggesting that reactive oxygen species (ROS) formation is not metal dependent, and may be related with poor physiological conditions of the animals after long periods in adverse conditions compared to those in hydrothermal environments.

Mercury in seabird feathers: insight on dietary habits and evidence for exposure levels in the western Indian Ocean.

Breast feathers were used to estimate mercury levels in six marine birds nesting in the tropical western Indian Ocean, i.e. Sooty Tern (Sterna fuscata), Brown Noddy (Anous stolidus), Lesser Noddy (Anous tenuirostris), Audubon Shearwater (Puffinus lherminieri bailloni), Barau's Petrel (Pterodroma baraui) and the White-tailed Tropicbird (Phaethon lepturus). Juveniles consistently showed lower plumage mercury than adults. The lowest mean level was noted in juvenile Sooty Terns from the Glorioso Archipelago (0.05 microg g(-1)). The highest levels were obtained for adult Barau's Petrels from Reunion Island (0.96 microg g(-1)). An inter-site analysis of Sooty Tern showed higher mercury levels in birds nesting on Juan de Nova Island. Levels were low in comparison with values reported in the plumage of seabirds worldwide. The potential impacts of the size, the type (fish/cephalopod) and the origin (epi-/meso-pelagic) of prey on mercury intake in birds are discussed. Although the diet composition of individuals within a species appeared to be quite variable, combining results on mercury levels with common knowledge of each species allowed additional information on their dietary and foraging habits to be unraveled.

Bioaccumulation of trace elements in pelagic fish from the Western Indian Ocean.

Trace elements were analyzed in fish of commercial interest to determine their importance in marine systems of the Western Indian Ocean and their bioaccumulation patterns. The results are equivalent or lower than levels reported in ichthyofauna worldwide. Certain values of muscular Cd, Hg, Pb and Zn were, however, above thresholds for human consumption. Levels varied among tissues, species and fish length, but were seldom influenced by the nutritional condition of the fish, its gender and its reproductive status. Correlations between hepatic Hg and Se levels in Swordfish (r2=0.747) and Yellowfin Tunas (r2=0.226), and among metallothionein linking metals imply the existence of detoxification processes in these species. Level differences between fish from the Mozambique Channel and Reunion Island reflect differences of diets rather than differences of elemental availability in both environments.

Metal influence on metallothionein synthesis in the hydrothermal vent mussel Bathymodiolus thermophilus.

The present study reports on the metallothionein expression in the hydrothermal vent mussel Bathymodiolus thermophilus. Metallothioneins (MT) are proteins involved in intracellular metal regulation and conserved throughout the animal kingdom. The hydrothermal vent environment presents peculiarities (high levels of sulfides and metals, low pH, anoxia) that may have driven associated species to develop original evolutionary ways to face these extreme living conditions. Mussels were exposed to different metal solutions at the atmospheric pressure. The MT mRNA levels and MT contents were measured in gills and mantles of each exposed mussel. The intracellular metal distribution was estimated in fractions obtained after the centrifugation of tissue homogenates. A few of the tested metals (Ag, Cu, Cd, Hg and Zn) were able to significantly induce MT mRNA levels. Silver was the only one that produced a significant increase of the MT protein level in both mantle and gills. The gills always presented higher MT protein levels than the mantle did, while their MT mRNA levels were similar. Our data show that MT mRNA and MT protein levels do not follow a clear relationship in the gills and mantle of B. thermophilus and we assume that a posttranscriptional control occurs in these mussels.

Mercury content in commercial pelagic fish and its risk assessment in the Western Indian Ocean.

As top predators of pelagic food webs, large fish naturally bioaccumulate mercury (Hg). Determining Hg burdens in commercialized fish is essential considering the concern about effects of contaminants on human health and the legal thresholds that are therefore set for local consumption and/or exportation. Total Hg levels were measured in the muscular tissue of 183 fish of five commercially important species from the tropical zone of the Western Indian Ocean. All individuals were measured and sexed in order to study the impregnation of Hg with size and sex within each species. Values of Hg found in this part of the Indian Ocean were comparable to Hg in muscular tissue of the same species studied in other areas. The highest Hg levels were noted in Swordfish (Xiphias gladius) caught in waters surrounding Reunion Island (3.97+/-2.67 microg g(-1) dry weight). Following the Swordfish, in decreasing order of Hg content, were the Yellowfin Tuna (Thunnus albacares) and the Skipjack (Katsuwonus pelamis), then the Common Dolphinfish (Coryphaena hippurus) and the Wahoo (Acanthocybium solandri). In the North of the Mozambique Channel, Swordfish had higher Hg levels than Yellowfin Tunas, and Dolphinfish exhibited intermediate Hg levels. The size of a fish was a determining factor of its Hg burden, as was the species. Differences in size-normalized Hg levels were observed between the two study zones for Swordfish and Common Dolphinfish. Sex, in contrast, did not influence Hg levels suggesting that females and males have similar feeding habits. The muscular Hg levels presented here suggest that consumers of fish originating from the Western Indian Ocean should limit themselves to one Swordfish based meal per week, or one fish meal a day if they choose to eat tuna or Common Dolphinfish.

Effect of pressure on pulse radiolysis reduction of proteins.

Pulse radiolysis experiments were performed on proteins under pressure. Whereas many spectroscopic techniques have shown protein modifications at different pressure ranges, the present measurements performed using the water radiolysis allowed to generate radical species and to study the mechanisms implied in their reactions with proteins. This work gives the first results obtained on the effects of pressure on the rate constants of the proteins reduction by the hydrated electron at pressures up to 100 MPa. The reaction with the hydrated electron was investigated on two classes of protein: the horse myoglobin and the mussel metallothioneins. We have successively studied the influence of the pH value of metmyoglobin solutions (pH 6, 7 and 8) and the influence of the metals nature (Zn,Cu,Cd) bound to metallothioneins. For both protein, whatever the experimental conditions, the pressure does not influence the value of the reduction rate constant in the investigated range (0.1-100 MPa).

Do organisms living around hydrothermal vent sites contain specific metallothioneins? The case of the genus Bathymodiolus (Bivalvia, Mytilidae).

The unusual characteristics of the hydrothermal vent environment (high pressure and metal concentrations, low pH, etc.) leads us to wonder how species living in this particular biotope have adjusted to these severe living conditions. To investigate the consequences of high metal concentrations, filter-feeding organisms are commonly used in ecotoxicological studies. Metallothioneins (MTs) are proteins conserved throughout the animal kingdom and involved in intracellular metal regulation. Therefore, we tried here to find out whether the metallothioneins of hydrothermal bivalves are different from those of coastal bivalves. The characterization of DNA sequences coding MTs from some of the most common hydrothermal bivalves, belonging to the genus Bathymodiolus (Mytilidae) was performed by reverse transcription polymerase chain reactions (RT-PCRs). The complementary DNAs (cDNAs) of MT-10 and MT-20 isoforms were obtained for the Atlantic and Pacific hydrothermal mussels (Bathymodiolus azoricus and Bathymodiolus thermophilus). The MT-10 transcripts were 222 nucleotides long and the MT-20 transcripts, 207 nucleotides. The polymorphism of the MT cDNAs in these two hydrothermal species is discussed. The comparison between metallothionein cDNA sequences of the Mytilus and the Bathymodiolus genera shows strong homologies among metallothioneins of coastal and hydrothermal mussels.