Julie Letendre
email: julie.letendre@univ-lehavre.fr

Bastien Chouquet

Hélène Manduzio

Matthieu Marin

Florence Bultelle

François Leboulenger

Fabrice Durand

Tidal height influences the levels of enzymatic antioxidant defences in

Keywords: Mytilus edulis, air exposure, antioxidant defences, intertidal, biomarkers

Introduction

Production of reactive oxygen species (ROS) occurs naturally during cellular aerobic respiration mechanisms as a result of partial reduction of oxygen to water, but also during metabolic processing of certain xenobiotics [START_REF] Livingstone | Oxyradical production as a pollution-mediated mechanism of toxicity in the common mussel, Mytilus edulis L., and other molluscs[END_REF]. Even if cells possess diverse mechanisms to limit the deleterious effects of ROS on biomolecules (oxidation of proteins, lipids, DNA), the generation of oxyradicals can overwhelm antioxidant defence capacities and lead to oxidative stress [START_REF] Kappus | Toxic drug effects associated with oxygen metabolism: redox cycling and lipid peroxidation[END_REF][START_REF] Sies | Oxidative stress: from basic research to clinical application[END_REF][START_REF] Winston | Prooxidant and antioxidant mechanisms in aquatic organisms[END_REF][START_REF] Manduzio | The point about oxidative stress in molluscs[END_REF]. For any organism such stress is an important and frequent consequence of exposure to contaminants, that is why measurements of oxidative damage and antioxidant responses are commonly used as biomarkers in ecotoxicological studies. In particular, measurement of antioxidant enzymatic activities is a means to diagnose the adverse effects of environmental pollution on organisms such as the blue mussel Mytilus edulis. This filter-feeding and sessile species is widely distributed and known for its ability to accumulate chemical compounds and its high tolerance to diverse environmental stress [START_REF] Kennedy | Desiccation, higher temperatures and upper intertidal limits of three species of sea mussels (Mollusca: Bivalvia) in New Zealand[END_REF][START_REF] Widdows | Physiological and biochemical responses of bivalve molluscs to exposure to air[END_REF]. With this species, the main antioxidant enzymatic activities described are: cupper/zinc superoxide dismutase (Cu/Zn-SOD), catalase (CAT), glutathione-peroxidase (GPx, selenium-dependant or independent), glutathione reductase (GRd) and glutathione transferase (GST) [START_REF] Ramos-Martinez | Purification and properties of glutathione reductase from hepatopancreas of Mytilus edulis L[END_REF][START_REF] Livingstone | Oxyradical production as a pollution-mediated mechanism of toxicity in the common mussel, Mytilus edulis L., and other molluscs[END_REF][START_REF] Fitzpatrick | Separation of multiple forms of glutathione Stransferase from the blue mussel, Mytilus edulis[END_REF].

Although seasonal variations of antioxidants are well-described in different mussel models [START_REF] Power | Seasonal variation in the antioxidant defence systems of gill and digestive gland of the blue mussel, Mytilus edulis[END_REF][START_REF] Regoli | Seasonal variations of susceptibility to oxidative stress in Adamussium colbecki, a key bioindicator species for the Antarctic marine environment[END_REF][START_REF] Manduzio | Seasonal variations in antioxidant defences in blue mussels Mytilus edulis collected from a polluted area: major contributions in gills of an inducible isoform of Cu/Zn-superoxide dismutase and of glutathione S-transferase[END_REF][START_REF] Bocchetti | Seasonal variability of oxidative biomarkers, lysosomal parameters, metallothioneins and peroxisomal enzymes in the Mediterranean mussel Mytilus galloprovincialis from Adriatic Sea[END_REF], the variability regarding the natural parameters is only partially assessed, which could explain some conflicting results that were obtained in the past [START_REF] Manduzio | The point about oxidative stress in molluscs[END_REF]. Thus, for intertidal populations of blue mussels which are subjected to wide fluctuations in different abiotic factors (oxygenation, temperature, hygrometry, etc), tidal status represents a supplementary aspect to be taken into account when studying antioxidant capacities.

Different laboratory experiments have investigated the specific effects of changes in temperature or aerial exposure on antioxidants in intertidal organisms [START_REF] Abele | Temperature-dependence of mitochondrial function and production of reactive oxygen species in the intertidal mud clam Mya arenaria[END_REF][START_REF] Almeida | Oxidative stress in digestive gland and gill of the brown mussel (Perna perna) exposed to air and re-submersed[END_REF][START_REF] Almeida | Effects of aerial exposure on antioxidant defenses in the brown mussel Perna perna[END_REF][START_REF] De Oliveira | Effects of environmental anoxia and different periods of reoxygenation on oxidative balance in gills of the estuarine crab Chasmagnathus granulata[END_REF]. However, the acclimation aspects that the chronicity of tidal cycles could induce in the field are not revealed in these cases. Blue mussels can be spatially distributed from high shore, where communities are exposed to air for >50% of the time, to the lowest levels of the shore where the period of water deprivation is short or non-existent. As the length and frequency of air exposure is likely to influence the intensity of stress, the vertical position of mussels on the shore should have some effect on the responses of antioxidant activities. This study aims at determining the possible differences in activities of enzymatic antioxidant defences in the gills and digestive gland of M. edulis after 2h of emersion according to its spatial distribution on the shore within a clean site. This represents a preliminary work for the investigation of the influence of tidal condition on enzymatic antioxidants in the blue mussel.

Materials and methods

In situ sampling

Mussels were collected on the intertidal rocky-shore of Yport (Seine-Maritime, France). On this shore, the mussel-bed is developed from the upper shore (5.80 m) to the lower shore (1.8 m). Two sampling areas were chosen according to the respective amount of time they were exposed to air (emersion). Twice-daily mussels from high-shore (HS) experienced airexposure of up to 6 hours, whereas mussels from low-shore (LS) experienced at the most two periods of 2 -2.5 hours of air-exposure a few days a month. HS (40-45 mm) and LS mussels (45-50 mm) were collected after 2 hours of air exposure, so that both groups would be exposed to the same emersion period. Gills and digestive glands were dissected out on site immediately after collection, placed in 2 ml sample tubes and directly deep-frozen in liquid nitrogen. In the laboratory, tissues were transferred and kept at -80°C until analysis. Sampling was performed in October 2004 and repeated in November 2005.

Preparation of tissue extracts

The gills and digestive glands were homogenised on ice in 10 mM Tris, 10 mM EDTA, 5 mM 2-β mercaptoethanol and 1%Triton X100, pH = 7.5, (1:3 and 1:10 volume respectively) using an electric homogeniser (Ultraturax). Homogenates were treated using ultrasonics at 20 kHz (gills, 2 x 10s periods, digestive gland 2 x 20s periods) and centrifuged for 15 min at 10 000 g and 4°C. Supernatants were kept on ice and used immediately for enzymatic assays. Protein concentration of supernatants was measured according to the method of Bradford [START_REF] Bradford | A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding[END_REF], using bovine serum albumin as a standard.

Measurements of enzymatic activities

Catalase (CAT, EC 1.15.1.1)

CAT activity was measured by the adapted method of Claiborne (1985) according to [START_REF] Livingstone | Oxyradical production as a pollution-mediated mechanism of toxicity in the common mussel, Mytilus edulis L., and other molluscs[END_REF]. H 2 O 2 disappearance was recorded at 240 nm. Activity was expressed as micromoles of H 2 O 2 per minute per milligram of protein, or U.mg -1 .

Superoxide dismutase. (SOD, EC 1.15.1.1)

Isoelectric focusing on high resolution gels (pH 4.0 -5.0) was performed in a Multiphor II apparatus (Amersham Pharmacia Biotech, France). For each extract, a volume corresponding to 50µg of total protein content was loaded on the gel. Staining of SOD activity was performed immediately after the completion of electrophoresis, using the photochemical method of [START_REF] Beauchamp | Superoxide dismutase: improved assays and an assay applicable to acrylamide gels[END_REF]. Gels were first soaked in 2.5 mM nitrobluetetrazolium (NBT) for 30 min in darkness under gentle agitation. They were then incubated in a solution of 1M phosphate buffer (pH 7.8) containing 0.04 mM riboflavine and 0.5% TEMED for 30 min in the dark and under gentle agitation. Detection of SOD isoforms and associated activity was carried out by soaking gels in 10 mM phosphate buffer (pH 7.8) under light conditions. In mussel extracts, using IEF, three isoforms of SOD with acidic isoelectric points were detected: 4.7 for the major band, SOD-1, 4.6 for SOD-2 and 4.55 for the minor one, named SOD-3.

Gels were scanned and subsequently analysed using the ImageMaster TotalLab software from Amersham Pharmacia Biotech (France). The activity of each SOD band was determined from the pixel intensity of its volume (Fig. 1). The total activity of SOD was compared to a standard of Cu/Zn-SOD of bovine erythrocyte and expressed in U.mg -1 prot.

The contribution of each isoform was expressed as percentage of the total activity.

Glutathione peroxidase (GPx, EC 1.11.1.9)

Total GPx activity was measured using the Bioxytech GPx-340TM kit (OxysInternational Inc.) adapted for mussel extracts by [START_REF] Manduzio | Etude des modifications d' expression protéique sous l' effet d' un stress environnemental chez deux bivalves estuariens, la moule zébrée (Dreissena polymorpha) et la moule bleue (Mytilus edulis) : suivi de marqueurs de défense cellulaire et approche protéomique[END_REF]. Briefly, the enzymatic assay was based on a coupled enzyme system, where the GSSG (glutathione oxidized form) formed in the GPx reaction with a tert-butyl substrate was converted to the reduced form GSH by GRd [START_REF] Lawrence | Glutathione peroxidase activity in selenium-deficient rat liver[END_REF]. The consumption of NADPH was monitored at 340 nm and directly proportional to the GPx activity in the sample. Activities were calculated with the extinction coefficient ε = 6220 M -1 .cm -1 . Results were expressed as nanomoles of NADPH per minute per milligram of protein, or mU.mg -1 prot.

Glutathione reductase (GRd, EC 1.6.4.2)

GRd activity was determined using the Bioxytech GR-340TM kit (OxysInternational Inc.) according to the method of Carlberg and Mannervik (1985), by measuring the oxidation rate of NADPH to NAD + by GRd in the presence of oxidized glutathione. One GRd unit was defined as the reduction of one micromole of GSSG per minute per milligram of protein at pH 7.6 and 25°C [START_REF] Manduzio | Seasonal variations in antioxidant defences in blue mussels Mytilus edulis collected from a polluted area: major contributions in gills of an inducible isoform of Cu/Zn-superoxide dismutase and of glutathione S-transferase[END_REF], and activity was expressed as mU.mg -1 prot.

Glutathione transferase (GST, EC 2.5.1.18)

GST activity was measured according to the method of [START_REF] Habig | Glutathione-S-transferases. The first enzymatic step in mercapturic acid formation[END_REF], using CDNB (1chloro-2,4-dinitrobenzene) and GSH (glutathione reduced form) as substrates. Absorbance was read at 340 nm, and activities were expressed as nanomoles of conjugated product formed per minute per milligram of protein, or mU.mg -1 prot.

Statistical analysis

Values were expressed as mean values ± SEM of ten independent individual values. Statistically significant differences between samples were determined with SigmaStat (SPPS, Chicago, USA) using the Student's t test. P<0.05 was accepted as significant.

Results

HS mussels exhibited higher levels of antioxidant activities than LS mussels for both tissues and almost every enzyme. Varying levels were sometimes observed between 2004 and 2005 for a single station.

In gills, equivalent levels of catalase activity were measured for the two years (HS: 99.0±7.9 and 103.6±4.2 U CAT.mg -1 ; LS: 62.4±4.7 and 68.6±5.4 U CAT.mg -1 ); in the digestive gland, the difference in CAT levels between HS and LS mussels occuring in 2004 disappeared in 2005 (fig. 2a,2d). Concerning Cu/Zn SOD, the total activity in gills was also higher in HS mussels than in LS especially in November 2005 (fig. 2b,2e); no significant differences were observed in the relative contribution of isoforms between the two sampling points and between the two stations (around 47% SOD-1, 30% SOD-2 , 23% SOD-3). In the digestive gland, HS mussels exhibited a 50% higher level of total Cu/Zn SOD activity in 2004 but not in 2005. The contribution of the three isoforms showed a similar pattern to that observed in gills, and in October 2004 SOD-1 presented a 25% higher contribution to the total activity in HS mussels than in LS (Figs. 2c,2f). Levels of GPx and GRd activities observed for HS mussels were 35% to 132% higher than those observed in LS mussels whatever the tissue (Figs. 3a,3b,3d,3e). GST activity is the one which exhibited the least significant differences between HS and LS mussels, especially in gills (no difference in 2004; Fig. 3c); activity was 37% and 50% higher in the digestive gland of HS mussels than in LS respectively in 2004 and 2005 (Fig. 3f).

Discussion

We found significantly greater antioxidant enzyme activities in mussels living at the upper part of the shore than mussels inhabiting the lower part of the shore. Anthropogenic and natural factors influencing antioxidant defence capacities are contamination of the environment, aging, feeding level and variations in abiotic parameters [START_REF] Livingstone | Oxyradical production as a pollution-mediated mechanism of toxicity in the common mussel, Mytilus edulis L., and other molluscs[END_REF][START_REF] Viarengo | Seasonal variations in the antioxidant defence systems and lipid peroxidation of the digestive gland of mussels[END_REF]. i) The two sampling stations were located on the same shore, which is used as reference site in biomonitoring studies. Very low levels of organic contaminants in water is have been reported, and the body burdens of these contaminants are the same for HS and LS mussels (data not shown). Thus, the levels of contaminants in mussels' environment can not be implicated in the differences of antioxidant activities measured between high and low shore mussels. ii) Estimation of age was performed by counting the annuli on mussel shell surfaces and no significant differences appeared between HS and LS sampled mussels [START_REF] Haskin | Age determination in molluscs[END_REF]. Hence, a difference in aging between HS mussels and LS mussels could not be involved in the variability observed in antioxidant activities. iii) Mussels inhabiting low shore have a much longer feeding time than those living on the upper part of the shore, but diverse studies suggest that mussels which are submitted to regular and prolonged aerial exposure partially compensate for the loss of feeding time due to this situation through metabolic, physiological and morphological adjustments [START_REF] Widdows | Physiological responses of Mytilus edulis and Cardium edule to aerial exposure[END_REF][START_REF] Franz | Allometry of shell and body weight in relation to shore level in the intertidal bivalve Geukensia demissa (Bivalvia: Mytilidae)[END_REF][START_REF] Charles | Digestive physiology of the ribbed mussel Geukensia demissa (Dillwyn) held at different tidal heights[END_REF]. Accordingly, access to food seems not to be different between HS and LS mussels in terms of molecular antioxidants provided by the food. However, the enhanced metabolic rate and subsequent ROS production in HS mussels at re-submersion could generate a difference in the oxidative insult between the two groups.

Air-exposure implies the isolation from the surrounding environment and consequent decrease in partial pressure of O 2 during low tide. Subsequently, there is a 90% decrease in the initial metabolic level, followed by a switch to anaerobic pathways [START_REF] De Zwaan | Molluscs[END_REF].

When recovery occurs, partial pressure of oxygen in tissues rises suddenly and a 2-or 3-fold increase in the respiratory rate is observed during the first hour [START_REF] Widdows | Physiological responses of Mytilus edulis and Cardium edule to aerial exposure[END_REF]. This situation, which would be comparable to an ischemia-reperfusion event, is generally associated with ROS overproduction and oxidative injuries [START_REF] Ferreira | Oxidative stress and ischaemia-reperfusion injury in the heart[END_REF][START_REF] Abele | Formation of reactive species and induction of antioxidant defence systems in polar and temperate marine invertebrates and fish[END_REF][START_REF] De Oliveira | Effects of environmental anoxia and different periods of reoxygenation on oxidative balance in gills of the estuarine crab Chasmagnathus granulata[END_REF]. The length of air-exposure recurrently endured by HS mussels is likely to lead to a complete tissue anoxia at the end of emersion, adding to accumulation of reducing equivalents; the metabolic burst and drastic increase in oxygen tension at recovery must induce a stronger rise in free radical production than in LS mussel tissues. In an ischemia-reperfusion experiment, it was demonstrated that prolongation of the hypoxic phase increases oxidative damage to molecules and concentration of hydroperoxides during recovery [START_REF] Venditti | Effects of myocardial ischemia and reperfusion on mitochondrial function and susceptibility to oxidative stress[END_REF]. In addition, the intertidal condition induces variations in other abiotic parameters such as temperature, which are known to influence the redox balance [START_REF] Prakash | Superoxide Anion Radical Generation as a Temperature Stress Response in the Gills of Freshwater Catfish Heteropneustes fossilis: Role in Mucus Exudation Under Elevated Temperature[END_REF][START_REF] Martínez-Álvarez | Physiological changes of sturgeon Acipenser naccarii caused by increasing environmental salinity[END_REF][START_REF] Khessiba | Effects of some environmental parameters on catalase activity measured in the mussel (Mytilus galloprovincialis) exposed to lindane[END_REF][START_REF] Heise | Oxidative stress during stressful heat exposure and recovery in the North Sea eelpout Zoarces viviparus L[END_REF]. Fluctuations in the diverse environmental and physiological parameters linked to tidal oscillations are more pronounced for HS mussels than for LS, moreover the cyclic repetition of these conditions probably intensifies the stress endured by HS mussels. Hence, the elevated levels of antioxidant enzymatic activities observed in HS mussels in comparison to those measured in LS mussels may be related to the more demanding conditions imposed by their high location on the shore. Such observations are corroborated by a study on two close species of limpets inhabiting respectively high and low parts of the shore, and where the authors suggest that HS species exhibit high and effective antioxidant defences allowing the limitation of oxidative damage [START_REF] Malanga | Oxidative stress in limpets exposed to different environmental conditions in the Beagle Channel[END_REF]. Here, the plasticity of antioxidant capacities evolved by mussels may allow those inhabiting high shore to acclimate to their more extreme habitat, through enhancing their defences in order to minimize the likely oxidative insults due to this fluctuating environment. Measurement of oxidative damages could help to verify this hypothesis.

The greatest spatial variability among the enzymes measured was for GPX, GRd and catalase activities, which play a role in the destruction of peroxides. In general, antioxidant activities are clearly higher in gills than in the digestive gland except for CAT and GPX whose activities are of the same order for both tissues, and therefore consistent with previous reports [START_REF] Power | Seasonal variation in the antioxidant defence systems of gill and digestive gland of the blue mussel, Mytilus edulis[END_REF][START_REF] Manduzio | Seasonal variations in antioxidant defences in blue mussels Mytilus edulis collected from a polluted area: major contributions in gills of an inducible isoform of Cu/Zn-superoxide dismutase and of glutathione S-transferase[END_REF]. This difference could be related to the position of gill tissue at the interface between external and internal medium, thus being a likely site of major oxidative stress. Absolute levels sometimes vary between October 2004 and November 2005, possibly because of a difference in temperature between the two sampling points, but the HS/LS pattern is generally confirmed. Autumn corresponds to a rest period and also to the onset of gametogenesis, which can be influenced by environmental factors [START_REF] Sprung | Reproduction and fecundity of the mussel Mytilus edulis at Helgoland (North Sea)[END_REF]. This implies a spatial difference in the distribution of energy between gamete building and stress response [START_REF] Petes | Effects of environmental stress on intertidal mussel reproduction[END_REF], with potential adjustments of antioxidant defences. The HS/LS pattern certainly varies with the time of year and thus emphasizes that temporal variations need to be assessed, particularly since seasonal changes in antioxidant activities have already been reported in Mytilus sp. [START_REF] Viarengo | Seasonal variations in the antioxidant defence systems and lipid peroxidation of the digestive gland of mussels[END_REF][START_REF] Power | Seasonal variation in the antioxidant defence systems of gill and digestive gland of the blue mussel, Mytilus edulis[END_REF][START_REF] Manduzio | Seasonal variations in antioxidant defences in blue mussels Mytilus edulis collected from a polluted area: major contributions in gills of an inducible isoform of Cu/Zn-superoxide dismutase and of glutathione S-transferase[END_REF][START_REF] Borkovic | The activity of antioxidant defence enzymes in the mussel Mytilus galloprovincialis from the Adriatic Sea[END_REF][START_REF] Bocchetti | Seasonal variability of oxidative biomarkers, lysosomal parameters, metallothioneins and peroxisomal enzymes in the Mediterranean mussel Mytilus galloprovincialis from Adriatic Sea[END_REF]. We already notice changes in the HS/LS difference and in the SOD isoforms' contribution to the total activity between the present work and a previous study, which deals with the tidal variations of Cu/Zn-SOD in tissues of mussels sampled in summer [START_REF] Letendre | Differential pattern of Cu/Zn superoxide dismutase isoforms in relation to tidal spatiotemporal changes in the blue mussel Mytilus edulis[END_REF]. In addition, this study shows that SOD activity is affected by the tidal cycle in a tissue-specific manner.

The antioxidant response to the alternation of emersion-submersion could be different according to the vertical location of organisms and the effect of tidal cycle on the HS/LS pattern could itself depend on the season. Future studies are therefore investigating the variations of antioxidant activities exhibited by HS and LS organisms during a whole tidal cycle for different periods of the year.

Conclusion

This study shows that in a clean environmental context, antioxidant enzymatic activities are higher for mussels living in the upper intertidal zone, suggesting an ability to adjust their antioxidant defences in response to the stressful situation induced by living in this part of the habitat. These data suggest the importance of the vertical position of animals on the shore in the response of enzymatic antioxidant defences, which is of great interest for sampling in monitoring studies. Nonetheless, further investigations concerning tidal and seasonal variations have to be performed to provide a complete picture of the impact of intertidal condition on antioxidant enzymatic activities.

Fig. 1: Densitograms of gels after SOD staining. SOD activity was revealed by the NBT method after IEF on pH gradient 4.0-5.0 gels as described in the Materials and Methods. Fig. 2: Activities of catalase and Cu-Zn SOD and relative contribution of the SOD isoforms to the total activity in gills (A, B, C) and digestive gland (D, E, F) of mussels sampled at high shore (HS) and low shore (LS) after 2h of emersion in October 2004 and November 2005. For each sample, total SOD activity was the sum of the activity of all revealed SOD isoforms. Pie charts represent the percentage of contribution of each SOD isoform to the total activity. Data are expressed as mean ± S.E.M. of 10 inter-individual measurements. Differences of values between LS and HS mussels were considered as significant from p<0.05 (*), p<0.01 (**), p<0.001 (***).

Fig. 3 :

 3 Fig. 3: Activities of GPx, GRd and GST in gills (A,B,C) and digestive gland (C,D,E) of mussels sampled at high shore (HS) and low shore (LS) after 2h of emersion in October 2004 and November 2005. All values are means of 10 inter-individual measurements. Data are expressed as mean ± S.E.M. of 10 inter-individual measurements. Differences of values between LS and HS mussels were considered as significant from p<0.05 (*), p<0.01 (**), p<0.001 (***).

  Figure1

Acknowledgements

This work was supported by a grant from French Ministry for Research and Technology and funded by the scientific program EcotDyn (CNRS-INSUE). The collaboration of Dawn Hallidy for the English revision of the manuscript is greatly appreciated.


Isabel Caçador

Miguel Caetano
email: mcaetano@ipimar.pt

Bernardo Duarte

Carlos Vale

Stock and losses of trace metals from salt marsh plants

Keywords: halophytes, metals, detritus, salt-marshes, metal-containing litter

Pools of Zn, Cu, Cd and Co in leaf, stem and root tissues of Sarcocornia fruticosa, S. perennis, Halimione portulacoides and Spartina maritima were analysed for a Tagus estuary (Portugal) salt marsh. Pools of Cu and Cd in the salt marsh were higher in spring/summer indicating a net uptake of these metals during the growing season. Standing stocks of Zn, Cu, Cd and Co in leaf and stem biomass of S. fruticosa, S. perennis and H. portulacoides showed a strong seasonal variation, with higher values recorded in autumn. The metal-containing leaves and stems that shed in the autumn become metal-containing detritus. The amount of this material washed out from the total marsh area (200 ha) was estimated as 68 kg of Zn, 8.2 kg of Cu, 13 kg of Co and 0.35 kg of Cd. The high tidal amplitude, a branched system of channels and semidiurnal tidal cycle greatly favour the export of the organic detritus to adjoining marsh area.

Introduction

Salt marsh plants are widely recognized to influence the transfer of metals between the aquatic compartments [START_REF] Rozema | Accumulation of heavy metals in estuarine salt marsh sediment and uptake of heavy metals by salt marsh halophytes In[END_REF][START_REF] Crowder | Acidification, metals and macrophytes[END_REF]. These metals may be immobilized and stored in belowground biomass, sediment or in aboveground tissues [START_REF] Jacob | Conflicting processes in the wetland plant rhizosphere: Metal retention or mobilization?[END_REF][START_REF] Weis | Metal uptake, transport and release by wetland plants: implications for phytoremediation and restoration[END_REF]. Several plant species have the remarkable ability to concentrate metals in their tissues and are therefore being used for phytoremediation proposes [START_REF] Meagher | Phytoremediation of toxic elemental and organic pollutants[END_REF][START_REF] Williams | Phytoremediation in wetland ecosystems: Progress, problems, and potential[END_REF]. Plants included in that category have been widely studied and their capacity to accumulate metals in roots is well documented [START_REF] Almeida | Influence of the sea rush Juncus maritimus on metal concentration and speciation in estuarine sediment colonized by the plant[END_REF][START_REF] Cundy | Heavy metal distribution and accumulation in two Spartina sp.-dominated macrotidal salt marshes from the Seine estuary (France) and the Medway estuary (UK)[END_REF][START_REF] Deng | Lead and zinc accumulation and tolerance in populations of six wetland plants[END_REF].

Since most salt marsh plants show low translocation of metals to their aboveground tissues, they have been considered as a long-term sink [START_REF] Mcfarlane | Accumulation and distribution of heavy metals in grey mangrove, Avicennia marina (Forsk.) Vierh.: biological indication potential[END_REF][START_REF] Fitzgerald | Copper and lead concentrations in salt marsh plants on the Suir Estuary, Ireland[END_REF]. Nevertheless, some studies on natural marsh plants have shown that a small portion of the accumulated metals may be released back to the aquatic environment through salt glands in living leaves, according to the physiological and morphological properties of plants [START_REF] Burke | Release of metals by the leaves of the salt marsh grasses Spartina alterniflora and Phragmites australis[END_REF][START_REF] Weis | Release into the environment of metals by two vascular salt marsh plants[END_REF]. Furthermore, during the seasonal life cycle of salt marsh plants aboveground plant portions die and fall to the sediment becoming organic-rich detritus [START_REF] Giblin | Uptake and Losses of Heavy Metals in Sewage Sludge by a New England Salt Marsh[END_REF][START_REF] Valiela | Decomposition in salt marsh ecosystems: the phases and major factors affecting disappearance of aboveground organic matter[END_REF][START_REF] Weis | Metal uptake, transport and release by wetland plants: implications for phytoremediation and restoration[END_REF]. This detritus can be retained and/or exported out from the marsh according to the flooding regime, elevation gradient, marsh topography and wind [START_REF] Halupa | Effects of tidally mediated litter moisture contents on decomposition of Spartina alterniflora and S. patens[END_REF]Neckless and Neill, 2004). Flooding and wind increase the rate of mass loss from aboveground litter [START_REF] Dame | The flux of floating macrodetritus in the North Inlet estuarine ecosystem[END_REF][START_REF] Bouchard | Fate of plant detritus in a European salt marsh dominated by Atriplex portulacoides (L.) Aellen[END_REF][START_REF] Bouchard | Primary production and macro-detritus dynamics in a European salt marsh: carbon and nitrogen budgets[END_REF]. Otherwise, detritus retention increases with the elevation gradient from low marsh (frequently inundated) to higher marsh area [START_REF] Mitsch | Wetlands[END_REF]. The branched system of channels across that gradient also promotes the transfer of litter between marsh zones and the export to adjacent areas [START_REF] Caçador | Carbon storage in Tagus salt marsh sediments[END_REF]. A number of studies have focused on in situ plant litter decay using nylon mesh litter bags (e.g. [START_REF] Valiela | Decomposition in salt marsh ecosystems: the phases and major factors affecting disappearance of aboveground organic matter[END_REF][START_REF] Halupa | Effects of tidally mediated litter moisture contents on decomposition of Spartina alterniflora and S. patens[END_REF]. The decomposition of litter is strongly regulated by climate conditions, litter quality, sediment properties, bacterial and fungal populations [START_REF] Valiela | Decomposition in salt marsh ecosystems: the phases and major factors affecting disappearance of aboveground organic matter[END_REF][START_REF] Graça | Grazing rates of organic matter and living fung biomass of decaying Spartina alterniflora by three species of salt-marsh invertebrates[END_REF]. Since metals in aboveground plant parts are likely to persist when tissues die and fall off, the leaching or retention of metals in decaying litter over time has been extensively investigated [START_REF] Banus | Export of lead from salt marshes[END_REF][START_REF] Dorgelo | Effects of diet and heavy metals on growth rate and fertility in deposit-feeding snail Potamopyrgus jenkinsi (Smith) (Gastropoda: Hydrobiidae)[END_REF][START_REF] Zawislanski | Accumulation of selenium and trace metals on plant litter in a tidal marsh[END_REF][START_REF] Laing | Metal accumulation in intertidal litter through decomposing leaf blades, sheaths and stems of Phragmites australis[END_REF]. Otherwise, metal-containing detritus can also be transported to adjacent marsh areas. The large majority of salt marsh ecosystems exported energy-rich substances (reduced nitrogen compounds, dissolved and particulate organic matter) to deeper waters [START_REF] Mitsch | Wetlands[END_REF][START_REF] Valiela | Salt marshes: biological controls of food webs in a diminishing environment[END_REF]. [START_REF] Banus | Export of lead from salt marshes[END_REF] highlight that substantial amounts of Pb associated with S. alterniflora litter may be removed by tides from the marsh surfaces. However, where metal-containing fertilizers were supplied to the marsh a minor export of Pb, Zn and Cd associated with litter was obtained [START_REF] Banus | Lead, zinc and cadmium budgets in experimentally enriched salt marsh ecosystems[END_REF]. Consequently, there is little agreement on the transfer of trace metals to marine environment across the detritus generated by salt marsh plants. The fate of detritus is crucial to the understanding of the metals budgets at the estuarine ecosystem level.

The aim of this work was to assess the budget of selected metals (Zn, Cu, Cd and Co) exported from a moderately contaminated salt marsh during a one-year growing season.

While Zn and Cu are essential metals, and no metabolic functions are known for Cd, less evidence exists on the beneficial effects of Co. The partition of Zn, Cu, Cd and Co concentrations between aboveground plant parts and belowground biomass are also studied. We focused our study on the most common plants of south European salt marshes: Sarcocornia fruticosa, Sarcocornia perennis, Halimione portulacoides and Spartina maritima. The standing stock of metals was estimated for plants as a whole and also for the individual plant parts.

Materials and methods

Study area

The Tagus estuary is one of the largest on the Atlantic coast of Europe and subjected to high human pressure with ca. 4 million people living around it. Around 40% of the estuary is composed of intertidal mudflats and its southern and eastern shores contain extensive areas of salt marshes colonised mainly by Sarcocornia fruticosa (Caryophyllalles, Chenopodiaceae), Sarcocornia perennis (Caryophyllalles, Chenopodiaceae), Halimione portulacoides (Caryophyllalles, Chenopodiaceae) and Spartina maritima (Poales, Poaceae). One of the marshes (Rosário) located in the southern shoreline of the Tagus estuary (Figure 1) covers an area of 200 ha [START_REF] Crespo | Cartografia do habitat potencial de Passeriformes no Estuário do Tejo por processamento digital de imagem[END_REF] and is characterized by a typical zonation with homogeneous stands of S. maritima as a pioneer species, colonizing bare mud in the lower marsh area. Across the elevation (20-50 cm) transect pure stands of H. portulacoides follow S. maritima, while S. fruticosa and S. perennis are found in the upper salt marsh. This marsh is fully inundated twice a day by tidal action (2-4m of tidal amplitude) through a highly branched system of channels that cross the elevation transect. These channels have 0.5 to 1.5 m depth promoting the inundation of the higher marsh even at low amplitude tides. During the ebb tide water is drained into the channels due to the water table difference. The Rosário marsh is located in the proximity of a heavy industrialised area that includes several chemical plants. During the past four decades these industries discharged effluents enriched in several contaminates directly into the estuary.

According to [START_REF] Vale | Temporal variations of particulate metals in the Tagus river estuary[END_REF] both the semi-diurnal tidal inundation and the proximity to these pollution sources contribute to metal inputs to the Rosário salt marsh. Pore water depth profiles obtained from intertidal sediments will give erroneous information of the metals budgets introduced into the marsh due to post-depositional early diagenetic reactions (Shaw et al., 1990) associated with tidal flushing of pore water solutes (Caetano et al., 1995). Thus, in this particular situation it is inaccurate to estimate the amount of metals flushed to the estuary and establish the pools of metals introduced/retained in the marsh. However, available data on depth profiles of Zn, Cu, Cd and Pb in non-vegetated sediments showed that concentrations in the upper 10 cm layers are up two 10 times higher than pre-industrial levels [START_REF] Caçador | Effects of plants on the accumulation of Zn, Pb, Cu and Cd in sediments of the Tagus estuary salt marshes[END_REF][START_REF] Zenk | Heavy metal detoxification in higher plants -a review[END_REF][START_REF] Caetano | Mobility of Pb in salt marshes recorded by total content and stable isotopic signature[END_REF].

Plant material and sampling

Pure stands of S. fruticosa, S. perennis, H. portulacoides and S. maritima were sampled bimonthly at Rosário salt marsh at low tide across a growing season, from October 2001 to June 2002. The aboveground biomass was assessed for each species by clipping out five squares of 0.3x0.3 m, and storing in plastic bags. In the laboratory plant parts were washed with Milli-Q water (18.2 MΩ.cm) to remove dust and sediment, and separated into photosynthetic and non-photosynthetic tissues. Sarcocornia fruticosa and S. perennis do not have a true shoot system with leaves and stems. From these plants the swollen photosynthetic stems (referred to as "leaves" hereafter) were separated from the dry perennial shoots (referred to as "stems" hereafter). The similar procedure was followed for S. maritima where the tillers were divided in leaves and in nonphotosynthetic tissues (referred to as "stems" hereafter). Otherwise, the shoot system of H. portulacoides allows separating leaves from stems. For all species, no flowering plants were sampled. In each 0.3x0.3 m square all the shedding parts of salt marsh plants (hereafter referred as detritus) found on sediment surface were collected with tweezers and transported to the laboratory in plastic bags. This detritus material corresponds to the dead plant portions retained within the salt marsh. Samples were washed with Milli-Q water and oven dried at 60 ºC until constant weight. Five sediment cores were taken at each study site using a core tube, 7 cm in diameter and 100 cm long.

Only the upper layer in each core (0-25 cm) was used since it contains the majority of belowground components [START_REF] Gross | Relationship between aboveground and belowground biomass of Spartina alterniflora (smooth cordgrass)[END_REF]. The belowground biomass of this layer was sorted out from the cores and carefully separated from the sediment under a flux of Milli-Q water using a sieve with 212 µm mesh size to remove any adhering particulate matter. Plant parts were oven dried at 60 ºC and powdered in a grinding ball mill (Glen CrestomMM2000) [START_REF] Gross | Relationship between aboveground and belowground biomass of Spartina alterniflora (smooth cordgrass)[END_REF]. Sediment samples were oven dried at 60 ºC until constant weight, cleaned of roots with tweezers, passed through a 0.25 mm mesh, homogenised and ground with an agate mortar [START_REF] Caçador | Biomass of Spartina maritima, Halimione portulacoides and Arthrocnemum fruticosum in Tagus estuary salt marshes. Halophyte uses in different climates[END_REF].

Analytical Methods

Redox potential (Eh) and pH of the sediment between the roots were measured in situ in all sampling periods except October 2001. Five replicate measurements were obtained between 10 and 15-cm depth. The pH was measured using a combined glass electrode with one Ag/AgCl reference electrode while for Eh measurements a platinum electrode with a calomel reference electrode was used. Values of redox potential were corrected for the reference potential of H 2 . Pore water was extracted by centrifugation (3000 rpm, 10 min) to measure salinity using a refractometer (Atago, S/Mill-E). Particle size (sand, silt, or clay) in each sediment sample was determined by the pipette method [START_REF] Gee | Particle size analysis[END_REF]. The organic matter content of the sediment was determined in dried samples by loss of ignition (LOI) at 600 º C for 2 hours. Sediment samples ( 100 mg) were digested with 10 mL of HNO 3 /HCl (3:1 v/v) at 130 ºC. This procedure was repeated twice, as described by [START_REF] Otte | Heavy metals and arsenic in vegetation of salt marshes and floodplains[END_REF]. Concentrations of Zn were determined by flame atomic absorption spectrometry (FAAS) with an air-acetylene flame while levels of Cu, Co and Cd were determined by graphite furnace atomic absorption spectrometry (GFAAS). Plant samples and detritus ( 200 mg) were digested with 10 mL of HNO 3 /HClO 4 (7:1 v/v) at 130 ºC according to the method described by [START_REF] Otte | Heavy metals and arsenic in vegetation of salt marshes and floodplains[END_REF].

Levels of Zn, Cu, Co and Cd were determined by GFAAS and detection limits were 0.01, 0.10, 0.05 and 0.05 µg g -1 , respectively, for all the analytical procedures used.

International certified reference materials of sewage sludge (CRM 145 and CRM 146) and plant material (BCR 62-Olea europaea) were used to ensure accuracy of our procedure. For all metals investigated, obtained values were consistently within the ranges of certified values (p<0.05).

The area occupied by each plant species in Rosário salt marsh was estimated on the basis of patches from aerial photographs scaling 1:2000. Halimione portulacoides colonized 132 ha, the equivalent to 66 % of the total salt marsh area (200 ha), S. maritima and S. fruticosa occupied 37.6 and 28.2 ha, respectively and S. perennis was found only in small patches within the marsh.

Data analysis

Statistical analysis was performed using a one-way analysis of variance to detect differences between metal concentrations, plant type and dates. The same test was used to look for differences in the sediment characteristics. One-way ANOVA and Newman-Keuls multiple comparison tests were used to detect differences between standing stocks of metals. To obtain homogeneity of variance, data were log-transformed [START_REF] Sokal | Redox chemistry in the root zone of a salt marsh sediment in the Tagus estuary, Portugal[END_REF].

Estimation of aboveground plant detritus

The difference between the maximum and the minimum metal standing stocks for leaves and stems over the annual period of study, allows the estimation of aboveground net metal production of detritus (NMePD; mol m -2 ) for each plant species [START_REF] Caçador | Carbon storage in Tagus salt marsh sediments[END_REF]:

NMePD i = (max Me A-stk -min Me A-stk ) i ( 1
)
where i is the plant species and Me A-stk (mol m -2 ) is the aboveground metal standing stock for that species. By using the surface area colonized by each plant species in the Rosário marsh, the amount of metal in aboveground plant detritus produced in the total marsh area (Q Me ; kg) may be estimated by:

Q Me = NMePD i A i ( 2
)
where i is the plant species and A is the area colonized by each plant species (m 2 ).

Results and Discussion

Sediment characteristics

Sediments from the upper layer (0-25cm) were formed mainly of silt (60±0.4 %) and clays (38±0.4 %) in all plots studied and at all sampling times. This suggests that these plants grow in sediment with similar grain size and retained similar suspended matter [START_REF] Salgueiro | Short-term sedimentation in Tagus estuary, Portugal: the influence of salt marsh plants[END_REF]. Pore water salinity varied within the salt marsh depending on vegetation species. Pore waters from sediments colonised by high marsh plants (S. fruticosa and S. perennis) contained more salts (salinity = 42 ± 1.0 and 41 ± 1.7) than those from the lower marsh (H. portulacoides: salinity = 36 ± 0.5 and S.

maritima: salinity = 31 ± 0.2). This difference was observed all over the seasonal survey, although salinity was lower in spring. A seasonal variation of pH was also observed, with lower values in the winter (Table 1). In a previous study, [START_REF] Sokal | Redox chemistry in the root zone of a salt marsh sediment in the Tagus estuary, Portugal[END_REF] showed that the release of oxygen by roots was lower in winter, changing the state of sediment to reduced conditions. Therefore, the reactions involving the Fe 2+ /Fe 3+ , Mn 2+ /Mn 4+ and S 2-/SO 4 2-pairs prevail in the rhizosphere during the winter. As oxygen reduces in the rizosphere the decrease of pH appears to be related to the oxidation of iron sulphide compounds by Fe(III) organic complexes [START_REF] Luther | Dissolved organic Fe(III) and Fe(II) complexes in salt marsh porewaters[END_REF]. Since no statistically significant differences of pH were found between plant species, early diagenetic reactions had similar consequences on sediment acidity and metal availability. The redox potential (Table 1) varied irregularly between plants and throughout the year with no seasonal pattern. These results are consistent with previous work done by [START_REF] Caçador | Accumulation of Zn, Pb, Cu and Ni in sediments between roots of the Tagus estuary salt marshes, Portugal[END_REF] in sediments coming from areas colonised by the four plant species included in our study. Lack of seasonal variation in the metal concentrations may be attributed to higher non-reactive fraction of these metals in the solids [START_REF] Caçador | Accumulation of Zn, Pb, Cu and Ni in sediments between roots of the Tagus estuary salt marshes, Portugal[END_REF]. However, it is possible that variations within the sediment layer may have occurred, but this must have been on a scale of a few centimetres only and could not be resolved by our sampling methods.

Plant biomass

From October 2001 to February 2002, the plant biomass overall was larger in plots of S.

fruticosa, while areas with S. maritima had the lowest biomass values recorded from all the plant species included in this study (Table 3). In April and June total biomass was not statistically different for all the studied species. Root biomass may account for up to 80 % (d.w.) of the total biomass. An exception was recorded in October for S. fruticosa, when the aboveground biomass accounted for 50 % of the total biomass. Presumably because dead and live roots are not easily discerned, rather small seasonal variation were observed in the biomass of roots, although higher values were found in the summer for S. maritima and in spring for the other species. Results from aboveground biomass showed statistically significant differences between plant species (p<0.05) and also between leaf and stem biomass (p<0.05). Although both Sarcocornia species had similar morphology, leaf and stem biomasses of S. fruticosa were up to one order of magnitude higher than those calculated for S. perennis. The same difference was also found by comparing aboveground plant parts biomass of S. fruticosa with those of S.

maritima and H. portulacoides (Table 3). Furthermore, a clear seasonal variation was observed for leaf and stem biomass in all four species. Higher biomass values were observed in October, followed by a logarithmic decrease. However, the decline of biomass was more sharp for S. fruticosa (leaf: from 3516±341 to 50±19 g m -2 and stem: from 1919±195 to 110±29 g m -2 ) than for S. perennis (leaf: from 167±39 to 37±5 g m -2

and stem: from 425±40 to 43±17 g m -2 ) and H. portulacoides (leaf: from 212±57 to 46±3 g m -2 and stem: from 733±122 to 180±49 g m -2 ). Leaf biomass in the latter species was most reduced in December after some senescent leaves had fallen from the standing stems. A different pattern was observed for S. maritima since aboveground biomass was lower in December but increased afterwards. Differences in the phenology of plant species attest to specific patterns of carbon allocation to the aboveground tissues, with S. perennis, S. fruticosa, and H. portulacoides promoting early leaf production when compared to S. maritima [START_REF] Windham | Uptake and distribution of metals in two dominant salt marsh macrophytes, Spartina alterniflora (cordgrass) and Phragmites australis (common reed)[END_REF].

Metal concentrations in plant parts

Concentrations of Zn, Cu, Co and Cd in plant tissues of S. fruticosa, S. perennis, H. portulacoides and S. maritima are presented in Table 4. Seasonal and tissue allocation patterns of Zn, Cu, and Cd differed from Co in the four plant species studied under field conditions. For all metals except Co, the concentrations in roots were, in general, up to one order of magnitude higher than levels found in the aboveground tissues. The partitioning indicates that for S. fruticosa, S. perennis, H. portulacoides and S. maritima the dominant uptake of metals was via the root system with only small amounts being translocated to the aerial plant parts [START_REF] Fitzgerald | Copper and lead concentrations in salt marsh plants on the Suir Estuary, Ireland[END_REF]MacFarlane et al., 2003;[START_REF] Weis | Metal uptake, transport and release by wetland plants: implications for phytoremediation and restoration[END_REF]. Metal concentrations in leaves showed no statistically significant differences when compared to equivalent levels in stems within the same plant or [START_REF] Zenk | Heavy metal detoxification in higher plants -a review[END_REF]. However, Co does not activate the phytochelatin synthase [START_REF] Oven | Increase of free cystein and citric acid in plant cells exposed to cobalt ions[END_REF] suggesting that this metal may induce other detoxification mechanisms or play a physiological role similar to that described for algae. The coenzyme cobalamin has cobalt as component and is essential for Euglena gracilis growth, being located in several subcellular compartments, including the tilakoids of the chloroplasts [START_REF] Marschner | Mineral Nutrition in Higher Plants[END_REF].

Metal stocks in plants

Our data of metal concentrations in plant parts combined with biomass allow us to compare the standing stocks of given elements in whole plant tissues and subsets, or to compare between plant species and for different seasons. Considering the standing stocks in the whole plant structure for all species in our study (Figure 2), metal pools were generally lower in S. maritima as a consequence of comparatively smaller biomass values. Standing stocks of Cu and Cd were higher for all plants in spring/summer (p<0.05) indicating a net uptake of these metals in the growing season. When standing stocks of metals were analysed in leaves and stems two different patterns were observed (Figure 3). In the cases of S. perennis, H. portulacoides and S. maritima, stems contained comparatively higher aboveground standing stocks of Zn, Cu, Co and Cd (p<0.05) while for S. fruticosa leaves emerge as the major contributor (p<0.05). These different patterns observed for standing stocks are explained by the higher aboveground biomass of S. fruticosa (Table 3). There were also differences in the seasonality of aboveground metal standing stocks. While S. perennis, S. fruticosa and H.

portulacoides showed a strong seasonal variation with higher values occurring in the autumn (p<0.05; Figure 3), the standing stocks of metals in S. maritima were higher in February (p<0.05; Figure 3). The early leaf production of S. maritima seems to be coupled to an increased transfer of some metals into the aboveground tissues.

Belowground standing stocks in all plant species showed small irregular variability throughout the seasonal survey (not shown), but values were considerably higher than aboveground. This result is consistent with other investigations elsewhere showing difficulty in observing well-defined seasonal patterns for metal concentrations in roots [START_REF] Gleason | Seasonal and environmental variation in Mn, Fe, Cu and Zn content of Spartina alterniflora[END_REF][START_REF] Heller | Seasonal study of speciation of mercury (II) and monomethyl mercury in Spartina alterniflora from Great Bay Estuary, NH[END_REF][START_REF] Windham | Uptake and distribution of metals in two dominant salt marsh macrophytes, Spartina alterniflora (cordgrass) and Phragmites australis (common reed)[END_REF].

Metal containing detritus

Metals in aboveground tissues are likely to remain when these tissues die and turn into detritus. The seasonal variation of the metal standing stocks in S. perennis, S. fruticosa and H. portulacoides was mainly associated with aboveground biomass production.

Therefore the aboveground plant detritus -QMe may be estimated for a growing season using equations 1 and 2. Results show that plants in our study area (200 ha) generate organic detritus containing 96 kg of Zn, 13 kg of Cu, 18 kg of Co and 0.51 kg of Cd during one growing season. However, the pool of organic material derived from aerial plant parts greatly exceeds the litter pool found in the sediment surface. The detritus on the sediment surface follows a seasonal pattern with higher values at the end of the growing season (S. fruticosa -1020 g m -2 ; S. perennis -212 g m -2 ; H.

portulacoides -134 g m -2 ; S. maritima -45 g m -2 ) and a decrease in winter (S. fruticosa -146 g m -2 ; S. perennis -25 g m -2 ; H. portulacoides -62 g m -2 ; S. maritima -21 g m - 2 ). This is in line with the findings of [START_REF] Caçador | Carbon storage in Tagus salt marsh sediments[END_REF] where more than 86% of the produced carbon from aboveground plant parts was exported from two different salt marshes in the Tagus estuary. The hydrological conditions of Rosário salt marsh (branched system of channels and semidiurnal tidal cycle) greatly favour the transport of organic detritus to adjoining marsh areas. The litter production may be estimated by different methods [START_REF] Bouchard | Primary production and macro-detritus dynamics in a European salt marsh: carbon and nitrogen budgets[END_REF], although all have limitations (Hopksion et al., 1978). Thus, we chose the simple method, viz. the difference between maximum and minimum litter pools [START_REF] Caçador | Carbon storage in Tagus salt marsh sediments[END_REF]. By comparing the aboveground biomass production (Table 3) with the litter pool we calculate that only 5% (S. maritima), 21 % (H. portulacoides) 17 % (S. perennis) and 46 % (S. fruticosa) of the detritus produced by aboveground plant parts remain at the site. By apply these proportions to the pool of the metal-containing detritus (Q Me ) we found that most of the metal containing detritus was flushed out from the marsh. During one growing season 68 kg of Zn, 8.2 kg of Cu, 13 kg of Co and 0.35 kg of Cd was exported to areas adjoining the Rosário salt marsh. This metal containing detritus may act as a source for these metals before being buried in the sediments since they are easily degraded by decomposers and weathering [START_REF] Weis | Metal uptake, transport and release by wetland plants: implications for phytoremediation and restoration[END_REF][START_REF] Laing | Metal accumulation in intertidal litter through decomposing leaf blades, sheaths and stems of Phragmites australis[END_REF].

Conclusions

There is an important export of trace metals associated with marsh detritus to the estuary, even considering that all plant species included in this study have much higher belowground than aboveground biomass. The export route involves uptake of metals from sediment by roots, translocation to the aerial parts of plants, and return to the soil after senescence and fall of aerial plant parts. The major fraction of the metal-containing detritus is not retained within the marsh. Therefore, when assessing remediation techniques in a contaminated marsh we must take into account the balance between retention and export of metals from the marsh, the biology of the plant species involved and the marsh ecology.

Figure Captions

  at several salt marshes in the Tagus estuary, showing that pH and Eh variations have no statistical consistency. No seasonal differences were found also for organic matter content. However, sediment colonized by S. maritima contained less organic matter (13±1.0 %) than the sediments from areas colonised by the other plant species (S. fruticosa = 21±1.4 %; S. perennis = 20±1.6 %; and H. portulacoides = 20±1.5 %). Mean concentrations of Zn, Cu, Co and Cd found in sediment between roots of the four plant species included in this study are shown in Table 2. Levels of Cu, Co and Cd were relatively constant during the sampling period as can be inferred by the low standard deviations. Although an enhanced variability was found for Zn concentrations, the standard deviation never exceed 10 % of the mean value. Moreover, no statistically significant differences were found among the metal (Zn, Cu, Co and Cd) concentrations

  between plant species. The similarity of metal accumulation in aboveground plant parts of S. maritima and of both Sarcocornia species may result from the undistinguished shoot system of these plants. Moreover, we may suggest that photosynthetic tissues play a minor role on the storage of metals in aboveground plant parts. No seasonal pattern was observed for the concentration of essential elements, Zn and Cu in the aboveground parts of the four species. However, concentrations of Cd in leaf and stem biomass of the same plant species showed a distinct seasonal variation, with increased accumulation in February. The accumulation of Co in the tissues of the studied plants showed a distinctive pattern, quite different from that observed for the remaining elements. Up to 50 % of the total metal pool was retained in the aboveground parts. While for Zn, Cu and Cd the roots act as a barrier for upward translocation this retention was not observed for Co. Detoxification of those metals by plants are associated with the phytochelatins in plant cells

Figure 1 -

 1 Figure 1 -Location of the Tagus estuary and Rosário salt marsh.

Figure 2 -

 2 Figure 2 -Whole plant standing stock of each metal. Values presented as mean±SD.

Figure 3 -

 3 Figure 3 -Standing stocks of Zn, Cu Co and Cd for aboveground tissues of S. fruticosa, S. perennis, H. portulacoides and S. maritima. Values presented as mean±SD.Dissimilar letters denote significant differences (p<0.05).

  Figure3

Table 3 -

 3 Average (n=5) dry weight biomass (g m -2 ) and standard deviations for total and individual plant tissues of S. fruticosa, S. perennis, H. portulacoides, and S.

	maritima.
			Leaf	Stem	Root	Total
			biomass	biomass	biomass	biomass
					(g m -2 )
	Sarcocornia	Oct.	3516±341	1919±195	4366±225	9010±1753
	fruticosa	Dec.	630±151	704±215	4574±176	6023±277
		Feb.	50±19	212±67	7176±366	7287±219
		Apr.	67±20	123±50	5354±466	5172±96
		Jun.	82±16	110±29	4252±163	4444±178
	Sarcocornia	Oct.	167±39	425±40	3900±329	4594±194
	perennis	Dec.	59±18	148±56	4172±429	4179±209
		Feb.	37±5	85±2	4550±206	4725±195
		Apr.	43±6	43±17	4860±94	4946±103
		Jun.	64±12	82±17	4090 146	4287±105
	Halimione	Oct.	211±57	733±122	4128±111	5072±264
	portulacoides	Dec.	65±16	320±50	3576±283	4183±159
		Feb.	56±11	189±51	4476±276	4623±110
		Apr.	46±3	180±49	4416±75	4642±61
		Jun.	60±16	188±31	4386±227	4643±15
	Spartina	Oct.	154±56	254±62	2556±596	2998±162
	maritima	Dec.	61±16	148±29	1510±62	1751±21
		Feb.	127±30	363±77	3678±492	4360±353
		Apr.	104±18	271±53	3386±620	3730±127
		Jun.	79±20	181±80	4268±360	4379±191

Table 3 Table 4 -

 34 Metal concentrations for individual plant tissues of S. fruticosa, S. perennis, H. portulacoides, and S. maritima.

	Table 4

Acknowledgments

This study was supported by POCTI/PNAT/BIA/15035/99 and Programa Operacional Ciência, Tecnologia, Inovação do Quadro Comunitário de Apoio III. The authors are grateful to Ivan Valiela for the helpful comments.

ACCEPTED MANUSCRIPT


MANGROVE SWAMPS Mélina C Z Laurent
email: melina.laurent@univ-ag.fr

Olivier Gros

Jean-Pierre Brulport

Françoise Gaill

Nadine Le Bris

SUNKEN WOOD HABITAT FOR THIOTROPHIC SYMBIOSIS IN

Keywords: wood falls, in situ measurements, sulfur-oxidizing symbiosis, microeukaryotes, ciliates, mangrove swamp

Large organic falls to the benthic environment, such as dead wood or whale bones, harbour organisms relying on sulfide-oxidizing symbionts. Nothing is known however concerning sulfide enrichment at the wood surface and its relation to wood colonization by sulfideoxidizing symbiotic organisms.

In this study we combined in situ hydrogen sulfide and pH measurements on sunken wood, with associated fauna microscopy analyses in a tropical mangrove swamp. This shallow environment is known to harbour thiotrophic symbioses and is also abundantly supplied with sunken wood. A significant sulfide enrichment at the wood surface was revealed. A 72 hour sequence of measurements emphasized the wide fluctuation of sulfide levels (0.1 to >100 µM) over time with both a tidal influence and rapid fluctuations. Protozoans observed on the wood surface were similar to Zoothamnium niveum and to Vorticellids.

Our SEM observations revealed their association with ectosymbiotic bacteria, which are likely to be sulfide-oxidizers. These results support the idea that sunken wood surfaces constitute an environment suitable for sulfide-oxidizing symbioses.

INTRODUCTION

Thiotrophic symbioses have long been associated with particular geological environments in the deep-ocean, such as hydrothermal vents or methane seeps. In these environments productive communities of invertebrates have been shown to rely on chemoautotrophic carbon fixation by microbes [START_REF] Childress | The biology of hydrothermal vent animals: physiology, biochemistry, and autotrophic symbioses[END_REF]. Among them, several species of bivalves and siboglinid tubeworms harbor endosymbiotic sulfideoxidizing bacteria [START_REF] Cavanaugh | Symbioses of chemoautotrophic bacteria and marine invertebrates from hydrothermal vents and reducing sediments[END_REF][START_REF] Felbeck | Carbon release from purified chemoautotrophic bacterial symbionts of the hydrothermal vent tubeworm Riftia pachyptila[END_REF][START_REF] Duperron | Dual symbiosis in a Bathymodiolus sp. mussel from a methane seep on the Gabon Continental Margin (Southeast Atlantic): 16S rRNA phylogeny and distribution of the symbionts in gills[END_REF].

More recently, organisms relying on sulfide-oxidizing symbionts have been described in association with organic falls in the shallow and deep-sea, most of them being related to whale falls [START_REF] Deming | Evidence for the microbial basis of a chemoautotrophic invertebrate community at a whale fall on the deep seafloor: Bone-colonizing bacteria and invertebrate endosymbionts[END_REF]Smith and Baco 2003;[START_REF] Fujiwara | Three-year investigations into sperm whale-fall ecosystems in Japan[END_REF][START_REF] Lorion | Several deep-sea mussels and their associated symbionts are able to live both on wood and whale falls[END_REF]. Symbiotic associations were notably described for the mussel Idasola washingtonia, which lives attached to the bones, and the burrowing clam Vesicomya c.f. gigas found in the sediments surrounding whale skeletons [START_REF] Deming | Evidence for the microbial basis of a chemoautotrophic invertebrate community at a whale fall on the deep seafloor: Bone-colonizing bacteria and invertebrate endosymbionts[END_REF]. From fluorescence in Situ hybridization analyses and transmission electron microscopy (TEM) observations, symbiotic associations were recently described for several mytilid species attached to the wood pieces (Gros and Gaill 2007;Gros et al. 2007). While extracellularly located, comparative analyses of 16S rDNA and adenosine-5'-phosphosulfate (APS) reductase gene sequences from these "epibionts" indicate that they are closely related to sulfideoxidizing gill-endosymbionts of other Bathymodiolinidae [START_REF] Duperron | Sulphur-oxidizing extracellular bacteria in the gills of Mytilidae associated with wood falls[END_REF] providing the first molecular evidence for the occurrence of thiotrophic symbiosis in direct association with sunken wood.

Mangrove swamp is a shallow seawater environment known to offer a variety of habitats for thiotrophic symbioses. High organic carbon influx from the mangrove forest result in almost millimolar sulfide enrichment in the sediment, through anaerobic degradation processes. Some invertebrates like the lucinid bivalves Lucina pectinata and/or Anodontia alba living in these sediments were shown to harbour intracellular sulfide-oxidizing symbionts [START_REF] Durand | Phylogenetic characterization of sulfur-oxidizing bacterial endosymbionts in three tropical Lucinidae by 16S rDNA sequence analysis[END_REF][START_REF] Frenkiel | Gill structure in Lucina pectinata (Bivalvia: Lucinidae) with reference to hemoglobin in bivalves with symbiotic sulphur-oxidizing bacteria[END_REF][START_REF] Gros | Interspecific infection of aposymbiotic juveniles of Codakia orbicularis by various tropical lucinid gill-endosymbionts[END_REF].

Moreover some ciliate colonies sampled from the mangrove swamp have also been shown to develop from artificial sulfide sources in the laboratory (Vopel et al. 2001), and were later confirmed to harbour sulfide-oxidizing epibionts [START_REF] Rinke | Candidatus thiobios zoothamnicoli," an ectosymbiotic bacterium covering the giant marine ciliate Zoothamnium niveum[END_REF]. Sulfide gradients characterized in situ in the environment of symbiotic ciliates confirmed their close association with sulfide "minivents" sustained by the degrading vegetable materials (Vopel et al. 2005). These ciliates have been found on disturbed bacterial mats at the peat surface, as well as on decaying leaves and rootlets from the mangrove peat (Bauer-Nebelsick et al. 1996a;1996b;Ott and Bright 2004;Ott et al. 2004;[START_REF] Rinke | Candidatus thiobios zoothamnicoli," an ectosymbiotic bacterium covering the giant marine ciliate Zoothamnium niveum[END_REF]), but have not been described associated with dead wood.

To our knowledge, no study has been specifically dedicated to dead woodassociated habitats in mangrove swamps. Nevertheless, naturally sunken dead wood is abundant in mangrove swamps. These might constitute substantial habitats for organisms relying on thiotrophic symbiosis in this environment. Furthermore, it was anticipated that their study could provide some clues to address the relationship between sulfide enrichment and the colonization by thiotrophic organisms.

The questions we addressed in this study were twofold: (1) what characterizes these environments in terms of sulfide exposure? (2) does sunken wood provide a suitable habitat for thiotrophic symbioses in a mangrove swamp? Our aim was to characterize biological and chemical habitat conditions at the interface of sunken wood and seawater.

By deploying autonomous sulfide sensors on pieces of naturally sunken wood, we were able to directly assess (in situ) the daily variability of sulfide concentration at the surface of the wood. The associated organisms were subsequently observed and analyzed using scanning electron microscopy (SEM).

MATERIALS AND METHODS

Sampling

For chemical measurements pieces of sunken wood from a few centimeters to 1 meter in length with a diameter up to 15 cm were analyzed. Such wood was located in < 1 meter depth in the mangrove swamp in Guadeloupe (16°N, 61.5°W) in the Caribbean area.

These pieces of wood, as well as the other collected vegetable material (leaves and seeds), all derive from the dicotyledon species Rhizophora mangle (Linnaeus, 1753). A series of these samples were transferred to the lab in order to analyze the associated microfauna using SEM.

Collected samples were observed with a stereomicroscope before preparation for SEM observations in order to detect possible symbioses among the organisms associated with these items of vegetable debris. Underwater pictures were taken using a Nikon Coolpix 5600 camera inside a DiCAPac bag.

Sulfide concentration measurements

Autonomous probes were used to measure in situ hydrogen sulfide concentration and pH at the surface of sunken wood pieces (Fig. 1) and at the interface of mangrove swamp water and sediment. The sulfide measuring system composed of a custom-made Ag/Ag 2 S sulfide electrode (0.8mm diameter, 2 mm length) was combined with a 2 mm diameter pH glass electrode, both being connected to submersible potentiometric autonomous data loggers (NKE, France). These sensors have been used in a variety of deep-sea chemosynthetic habitats (Le [START_REF] Bris | Autonomous potentiometric sensor for in situ sulfide monitoring in marine sulfidic media[END_REF]. The pH measuring system is similar to the one described by Le [START_REF] Bris | A new deep-sea probe for in situ pH measurement in the environment of hydrothermal vent biological communities[END_REF]. A series of successive short-term measurements were recorded on the surface of the wood, in the sediment and the overlying water with tightly attached sulfide and pH electrodes. Following these snapshot measurements, chemical conditions were continuously monitored over three days on a piece of wood about 1 meter long and 10 cm wide (Fig. 1). One sulfide electrode was attached to the wood, in order to maintain its sensing part in close contact with the surface of the wood (Fig. 2). The sensing tip of the electrode was precisely positioned within a white patch of a few millimeters diameter on the wood surface that was suspected to indicate the presence of a ciliate colony. The 2 mm diameter pH electrode was positioned a few centimeters apart from the sulfide electrode on another white patch (Fig. 1, 2). Our reason for decoupling the electrodes was to overcome a too large physical disturbance that would have resulted in both electrodes positioned in the same patch. For comparison, a second sulfide electrode was positioned in the water a few centimeters from the wood surface (Fig. 1, 2). The manipulation was performed very carefully avoiding displacing the wood in order to preserve as much as possible the natural hydrodynamic conditions. The electrodes were calibrated in the laboratory before deployment. The Nernstian sensitivity of the silver sulfide electrode to sulfide ions offers the possibility to detect free sulfide over a wide range of concentrations. Quantitative assessments of total free sulfide concentration were done using the H 2 S acidity constant of Rickard and Luther III (2007) and the pH value simultaneously recorded with the glass electrode. It was assumed that the pH variation is similar on both white patches, which might not be rigorously the case. As the pH is needed to calculate the sulfide concentration from raw data, the results should be regarded as first order estimates rather than high accuracy measurements. Nevertheless, the continuous data provide access to the temporal variability of the medium, which is of greater relevance to the addressed questions than would be a limited series of highly accurate discrete measurements.

Scanning Electron Microscopy observations

Samples were fixed 2 hours at 4°C in 2% glutaraldehyde fixative solution in caccodylate buffer (900 mOsm, pH 7.2). They were dehydrated in an ascending series of acetone, critical point dried in CO 2 , and sputter-coated with gold before viewed with a Hitachi S 2500 at 20 kV.

RESULTS

Sulfide concentrations

The mangrove sediments were confirmed to be highly enriched in sulfide while no trace of sulfide was detected in the overlying water (Table 1). Concentrations of hydrogen sulfide up to almost 1mM were detected in the mangrove sediment (data not shown), consistent with the measurements of [START_REF] Ott | The ecology of a novel symbiosis between a marine peritrich ciliate and chemoautotrophic bacteria[END_REF] in the sediment of the mangrove swamp of the Twin Cays (Belize). Sulfide variation with depth in the sediment appeared to be very steep in the first centimeters. Microscale sediment profiling could not be achieved with our sensors and was out of the scope of this study.

Short-term measurements within white patches on the surface of sunken wood exposed to seawater also revealed local sulfide enrichments, up to at least 114 µM, while no sulfide was detected in the surrounding water (Table 1). The 72 hour sequence, additionally, showed that the sulfide levels and pH values on the surface of a naturally sunken wood were very variable over time. Large fluctuations in the levels of sulfide were observed, with concentration changes that can exceed 100 µM within 1 hour or less (Fig. 7a). In addition, a slower cycle was observed showing successive stages with low sulfide maxima (< 50 µM to undetectable) and high sulfide maxima (>100 µM) (Fig. 7a). This periodicity was demonstrated by both the sulfide (Fig. 7a) and the pH (Fig. 7b) electrode responses, even though the two measurements were completely independent, indicating that this behavior was not a measurement artifact. A few centimeters from the wood surface, the second sulfide sensor did not record any detectable sulfide in water. Our

Organisms analyzed

All the wood samples analyzed were colonized by a very diversified fauna, composed of organisms belonging to Annelida, Arthropoda, Ciliophora, Chordata, Cnidaria, Kamptozoa, Mollusca, Nematoda, Plathelminthes, and Urochordata with different abundances. Among these organisms, the colonial ciliate Zoothamnium niveum (Hemprich & Ehrenberg, 1831), which was attached to the surface of the wood, constituted the white patches noticed during in situ sampling and chemical measurements. SEM observations revealed the presence of numerous bacteria with 2 morphotypes covering the whole body of the organisms similar to previous studies (Fig. 5) (Bauer-Nebelsick et al. 1996a;1996b;[START_REF] Rinke | Candidatus thiobios zoothamnicoli," an ectosymbiotic bacterium covering the giant marine ciliate Zoothamnium niveum[END_REF].

SEM observations of a vorticellid ciliate regularly recorded nearby Zoothamnium niveum colonies on the wood samples, revealed the presence of a bacterial coat covering the whole zooid and stalk surface (Fig. 6). The rod-shaped bacteria were organized in one single layer on the surface of the organism.

DISCUSSION

Wood as a suitable substrate to sustain thiotrophic symbioses

Although our measurements did not achieve the sub-millimetric resolution of the microelectrode used for the study of rootlets, they confirmed that sulfide is similarly enriched at the surface of large pieces of sunken wood within the mangrove. Direct degradation of wood material by microbes is likely to be the source for this sulfide production, as the presence of a Teredinid burrow or a local accumulation of particulate organic matter could be excluded. The mechanism that sustains sulfide production from wood material still has to be detailed, but it is likely that microbial degradation of the wood generates anoxic niches suitable for SRB activity as in organically enriched sediments. The substantial pH decrease observed at the surface of the wood (7.3 to 7.6) compared to mangrove swamp water (8.0) is also consistent with this assumption.

While Z. niveum and the vorticellid ciliates were already known in the mangrove environment or on small pieces of decomposing organic substrate (Bauer-Nebelsick et al. 1996a;1996b;Ott and Bright 2004;Ott et al. 2004), this study is the first to report their presence on less easily degraded wood material. The sulfide enrichments measured on the white patches of the sunken wood are comparable with those determined in the laboratory by Vopel et al. (2001) on decomposing rootlets from peat pieces where Z. niveum developed (110 and 360µM H 2 S) and in situ in the water contained in decomposing rootlet tubes (up to 739 µM H 2 S) (Vopel et al. 2005).

While the symbiotic association of Z. niveum with its sulfide-oxidizing symbionts has been well described (Bauer-Nebelsick 1996 a and b;[START_REF] Ott | The ecology of a novel symbiosis between a marine peritrich ciliate and chemoautotrophic bacteria[END_REF]Ott and Bright 2004;Ott et al. 2004;[START_REF] Rinke | Candidatus thiobios zoothamnicoli," an ectosymbiotic bacterium covering the giant marine ciliate Zoothamnium niveum[END_REF]2007), there was no phylogenetic identification of the vorticellid ciliate and its symbionts. In previous studies similar vorticellid ciliates, identified as Vorticella sp., were able to develop on artificial sulfide sources (Vopel et al. 2001) and were observed close to Z. niveum colonies in the natural environment (Ott and Bright, 2004). The vorticellid ciliate sampled in our study was identified as Pseudovorticella sp. based on phylogenetic analyses (Muller, personal communication).

The presence of elemental sulphur, suggested by its white colour in incident light is confirmed by Raman spectrometry [START_REF] Maurin | Preliminary assumption of sulphur-oxidizing bacterial symbiosis by Raman microspectrometry: an application on mangrove swamp meiofauna of Guadeloupe[END_REF].

Regular observations of natural sunken wood pieces showed that Z. niveum colonies persisted for several weeks on the surface of a single piece of wood, while their life span is estimated at 7 days (Ott and Bright 2004), suggesting that these colonies find long-lasting favourable conditions, implying that there is long-term sulfide availability.

Sulfide variability in wood microhabitats and potential importance for chemolithoautotrophic growth of symbiotic organisms

The fact that the ciliates are concentrated on small patches suggests that suitable conditions only occur on some particular features of the wood surface, presumably where specific topographic features prevent the medium being totally flushed with oxygenated seawater. Another important outcome of this study lies in the fact that at a single point the sulfide concentration fluctuates over time. Sharp transitions commonly exceeding 100 µM H 2 S in amplitude over a period of < 1 hour and a marked periodicity of sulfide concentration maxima were observed over longer time periods. This study showed that periodicity was a characteristic feature of the sunken wood habitat and reflected a tidal influence, and this is the first time that this has been recorded in such a microhabitat. The lowest sulfide maxima were recorded during low tide and high values corresponded with high tide. A likely explanation for this fluctuation is the tidal variability of current intensity in the area (Bouchereau, personal communication). As shown by Vopel et al. (2005) on shorter timescales, higher velocities may induce a reduction of the thickness of the boundary layer at the surface of the wood and result in the replacement of the sulfidic layer with an oxygenated layer. If we assume that the pH increase at low tide is indicating the advection of mangrove water in the ciliate microenvironment, the reduction of sulfide levels should be related to an increase in the oxygen concentration. The fact that pH never goes up to the mangrove water value, even when sulfide is not detectable, indicates that the chemical microenvironment remains permanently under the influence of wood degradation processes and is thus likely to remain hypoxic.

The tidal modulation of oxygen and sulfide exerts significant constraints on the availability of chemical substrates for chemolithoautotrophic growth, with periods of low electron donor availability. Conversely, the rapid and sharp fluctuations can be considered to create optimal conditions for growth of epibionts, providing them with both sulfide and oxygen. The concomitant pH increase suggests that oxygenated seawater is periodically supplied to the microhabitat. These pulsations could be hydrodynamically-driven fluctuations under the influence of local flow speed, as described in Vopel et al. (2005).

CONCLUSION

Fluctuating chemical conditions characterizing microhabitats on the surface of the wood are likely to be a prerequisite for the settlement of sulfide-oxidizing symbioses.

Unlike peat rootlets, decaying leaves or disturbed peat surfaces, the production of sulfide from the large piece of wood is potentially sustained over much longer period. This would have important consequences on the capacity of thiotrophic organisms to colonize sunken wood after its transport to the benthic habitat. From the analysis of woodassociated fauna diversity, [START_REF] Distel | Do mussels take wooden steps to deep-sea vents?[END_REF] reported close relationships with vent and seep species and suggested that these habitats might have served as stepping stones, at evolutionary scale, for the colonization of deep-sea sulfidic habitats. This idea is consistent with the fact that sulfide production can be supported by bacteria during the wood degradation process. The potential for long-term evolution of colonization at the surface of the wood needs to be further studied, particularly monitoring sulfide levels and colonization over the course of the wood degradation.

ACKNOWLEDGMENTS

This study was carried out with PhD salary grants from "Région de la Guadeloupe", "Fond Social Européen", and ANR "Deep Oases" to M.L.. The development of autonomous sulfide and pH sensors was supported by IFREMER, UMR7138 fundings and the DIWOOD European research group. In situ probes were partly funded by a special grant from "Ministère de l'Outre Mer, (ref n°06GUA 1). We thank also Yoshihiro Fujiwara for the realisation of the underwater pictures. Zoothamnium niveum feather-shaped colonies (Fig. 3) and Vorticella sp. (Fig. 4) appear white-colored in incident light. The little zooid (curved arrow) of Vorticella sp. is on a long stalk (straight arrow).

FIGURE CAPTIONS

Figures 56: SEM views of the protozoan ciliates collected on wood samples. The retracted coiled central stalk (arrow) of the feather-shaped colonial ciliate (Zoothamnium niveum) with numerous zooids. Both, zooids and stalk, are covered by bacteria (Fig. 5).

The solitary vorticellid ciliate is composed of a single inverted bell-shaped zooid (asterisk) borne by a long spring-shaped stalk when contracted (straight arrow) (Fig. 6).

Both, zooids and stalks, are also covered by bacteria of about 1.5µm long (insert). Only the adhesive disk and the more basal part of the central stalk are devoid of bacteria (curved arrow).

  measurement of sulfide therefore illustrates the environmental conditions at the surface of the wood. Variations of 0.3 to 0.4 pH units in amplitude were observed over the 72 hour period. Both pH and sulfide records correlated well with the water height at Pointe-à-Pitre, suggesting a tidal influence on the local chemical conditions at the wood surface (Figs. 7 a and b).

Figure 1 :

 1 Figure 1: In situ measurement set-up during the 3 day autonomous deployment. The pH

Figure 2 :

 2 Figure 2: Close up view of the sensing tips sulfide (curved arrow) and pH (straight arrow)

Figures 3- 4 :

 4 Figures 3-4: Colonies of ciliates are restricted to specific areas of wood surface.

Figure 7 :

 7 Figure 7: Variation of the total H 2 S concentration (a) and pH (b) over 72 hours on the surface of a sunken piece of wood, together with predicted water height at Pointe-à-

c cm m

1µ µm m

oligonucleotides and tyramide signal amplification. Applied and Environmental Microbiology 63, 3268-3273. Sekar, R., Fuchs, B.M., Amann, R., Pernthaler, J., 2004. Flow sorting of marine bacterioplankton after fluorescence in situ hybridization. Applied and Environmental Microbiology 70, 6210-6219. Smith, C.R., Baco, A.R., 2003. Ecology of whale falls at the deep-sea floor. Oceanography and Marine Biology 41, 311-354. Vopel, K., Po hn, M., Sorgo, A., Ott, J., 2001. Ciliate-generated advective seawater transport supplies chemoautotrophic ectosymbionts. Marine Ecology Progress Series 210, 93-99. Vopel, K., Thistle, D., Ott, J., Bright, M., Røy, H., 2005. Wave-induced H2S flux sustains a chemoautotrophic symbiosis. Limnology and Oceanography 50, 128-133.

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