David Costantini
email: david.costantini@mnhn.fr

Gábor Á Czirják

Paco Bustamante

Sara Bumrungsri

Christian C Voigt

Impacts of land use on an insectivorous tropical bat: the importance of mercury, physio-immunology and trophic position

Keywords: Agriculture, Antioxidants, Deforestation, Oxidative stress, Rice, Stable isotopes

Deforestation, agricultural intensification, and habitat homogenization are critical threats to biodiversity in Southeast Asia. Limited information is available on the trophic and physiological responses of tropical animals to these environmental changes. The wrinklelipped free-tailed bat Chaerephon plicatus is a cave roosting species that is experiencing population declines across Southeast Asia, where landscapes have been drastically modified.

In our study site in central Thailand, we tested the hypothesis that wrinkle-lipped free-tailed bats living in landscapes that contrast in heterogeneity and land-use differed in mercury contamination, trophic position and physio-immunological status. Bats from less heterogeneous landscapes (dominated by rice crops, absence of large forest patches) occupied a lower trophic position than conspecifics from more heterogeneous landscapes (including large forest patches). Additionally, bats from these habitats had lower concentrations of mercury in erythrocytes, lower body mass, higher antioxidant superoxide dismutase (SOD), lower antioxidant glutathione peroxidase (GPx) and lower values of the GPx/SOD ratio than bats from more heterogeneous landscapes. Individual bat mercury concentrations were positively correlated with body mass and two immune markers (lysozyme and immunoglobulin) but were negatively correlated with plasma non-enzymatic antioxidant capacity. Our results suggest various links between landscape heterogeneity, mercury accumulation/exposure, and health status of wildlife in Southeast Asian countries.

Introduction

Conversion of forests to agricultural areas has been a major way through which forests have vanished from tropical regions between 1990 and 2015 (FAO, 2015). The Food and Agriculture Organization estimate that South and Southeast Asia experienced the highest loss of canopy cover (over 50 million ha) from 2000 to 2012 and the highest loss in carbon stock from 2010 to 2015 compared to other regions (FAO, 2015). In many tropical regions, deforestation and conversion of natural lands into agricultural lands, soil erosion, and gold mining have also increased exposure of wildlife and humans to mercury contamination [START_REF] Stubner | Sulfur oxidation in rice field soil: activity, enumeration, isolation and characterization of thiosulfate-oxidizing bacteria[END_REF][START_REF] Horvat | Total mercury, methylmercury and selenium in mercury polluted areas in the province Guizhou, China[END_REF][START_REF] Meng | Distribution patterns of inorganic mercury and methylmercury in tissues of rice (Oryza sativa L.) plants and possible bioaccumulation pathways[END_REF][START_REF] Meng | The process of methylmercury accumulation in rice (Oryza sativa L.)[END_REF][START_REF] Krisnayanti | Assessment of environmental mercury discharge at a four-year old artisanal gold mining area on Lombok Island, Indonesia[END_REF][START_REF] Tang | Mercury in rice (Oryza sativa L.) and rice-paddy soils under long-term fertilizer and organic amendment[END_REF]. For example, several studies in Asia have found that rice paddy soil hosts bacteria that can methylate inorganic mercury, which facilitates the bioaccumulation of mercury along food chains (e.g., [START_REF] Stubner | Sulfur oxidation in rice field soil: activity, enumeration, isolation and characterization of thiosulfate-oxidizing bacteria[END_REF][START_REF] Meng | Distribution patterns of inorganic mercury and methylmercury in tissues of rice (Oryza sativa L.) plants and possible bioaccumulation pathways[END_REF][START_REF] Meng | The process of methylmercury accumulation in rice (Oryza sativa L.)[END_REF]. Mercury contamination is a priority topic for the United Nations (Minamata Convention) because it represents a significant threat to biodiversity, ecosystems, and human health [START_REF] Eagles-Smith | Modulators of mercury risk to wildlife and humans in the context of rapid global change[END_REF]Whitney and Cristol, 2018). Mercury is neurotoxic and can impair several functions in organisms, including reproduction and immunity (Whitney and Cristol, 2018).

Healthy ecosystems provide various services of economic value to humans. Bats are renowned for the services they provide to silviculture and agriculture (reviewed in Kunz et al., 2011;[START_REF] Ghanem | Increasing awareness of ecosystem services provided by bats[END_REF]. For example, many bats are predators of pest insects in natural forests [START_REF] Kalka | Bats limit arthropods and herbivory in a tropical forest[END_REF][START_REF] Böhm | Top-down control of herbivory by birds and bats in the canopy of temperate broad-leaved oaks (Quercus robur)[END_REF] and agricultural habitats (Williams-Guillen et al., 2008;[START_REF] Maas | Bats and birds increase crop yield in tropical agroforestry landscapes[END_REF]. Bats may enhance crop production and economic benefits through a top-down reduction of herbivorous insects (Williams-Guillen et al., 2008;[START_REF] Boyles | Economic importance of bats in agriculture[END_REF][START_REF] Karp | Cascading effects of insectivorous birds and bats in tropical coffee plantations[END_REF]. However, bats are also one of the most threatened taxa [START_REF] Voigt | Bats in the Anthropocene: conservation of bats in a changing world[END_REF]IUCN, 2018). While bat species diversity is higher in tropical regions, where land-use changes and mercury contamination are widespread, little work has been done to understand the effects of such environmental changes on tropical species as compared to other regions [START_REF] Struebig | Conservation value of forest fragments to Palaeotropical bats[END_REF][START_REF] Struebig | Conservation importance of limestone karst outcrops for Palaeotropical bats in a fragmented landscape[END_REF][START_REF] Syaripuddin | Mercury accumulation in bats near hydroelectric reservoirs in Peninsular Malaysia[END_REF]Becker et al., 2017Becker et al., , 2018a;;[START_REF] Kumar | Presence of artisanal gold mining predicts mercury bioaccumulation in five genera of bats (Chiroptera)[END_REF]. It is therefore important to identify potential threats for bats if we are to develop evidence-based landscape planning to limit anthropogenic impacts on bats and other wildlife.

In the last decade, there has been growing awareness that immunological and physiological techniques may allow conservation practitioners and wildlife managers to assess and, predict the impact of environmental changes on wildlife [START_REF] Beaulieu | Biomarkers of oxidative status: missing tools in conservation physiology[END_REF][START_REF] Seltmann | Habitat disturbance results in chronic stress and impaired health status in forest-dwelling paleotropical bats[END_REF][START_REF] Madliger | The conservation physiology toolbox: status and opportunities[END_REF]Becker et al., 2019). Immune markers are one important component of this toolbox because the immune system provides protection against parasites and pathogens [START_REF] Martin | An introduction to ecological immunology[END_REF]. Oxidative status markers are also important because the generation of molecular oxidative damage and antioxidant defenses can affect important fitness-related traits [START_REF] Beaulieu | Biomarkers of oxidative status: missing tools in conservation physiology[END_REF]. Both immunological and oxidative status markers provide complementary information about the impact of anthropogenic challenges on wildlife [START_REF] Beaulieu | Biomarkers of oxidative status: missing tools in conservation physiology[END_REF]Whitney and Cristol, 2018).

Changes in land-use also affect the amount and diversity of prey available for bats [START_REF] Treitler | The effect of local land use and loss of forests on bats and nocturnal insects[END_REF]. Measuring stable isotope ratios of nitrogen and carbon in tissues or body products (e.g., blood or fur, respectively) can provide information on what animals have consumed, in which habitat they foraged, and their trophic position (DeNiro andEpstein, 1978, 1981;[START_REF] Voigt | Host preference of the common vampire bat (Desmodus rotundus; Chiroptera) assessed by stable isotopes[END_REF]. This technique is based on the evidence that the isotope composition of an animal reflects that of its diet within a temporal window that depends on the isotopic incorporation rate of the analyzed tissue or body product (DeNiro andEpstein, 1978, 1981;[START_REF] Voigt | Host preference of the common vampire bat (Desmodus rotundus; Chiroptera) assessed by stable isotopes[END_REF].

In this study, we tested the hypothesis that bats living in landscapes in Thailand that contrast in extension of forest patches and in the type of cultivated crops would differ in their trophic position, mercury contamination, and physio-immunological status. In particular, we used stable nitrogen and carbon isotope ratios in erythrocytes as a proxy for the trophic position and foraging behaviour of bats (Ruadreo et al., 2019). We measured mercury concentrations in erythrocytes because, conversely to other matrices (e.g., fur), they integrate mercury accumulated over the most immediate past, which is the reference period for the stable isotope ratios and physio-immunological parameters. Moreover, most mercury in blood is located in the erythrocytes (e.g., Kershaw et al., 1980;[START_REF] Magos | The absorption, distribution and excretion of methyl mercury[END_REF][START_REF] Berglund | Interindividual variations of human mercury exposure biomarkers: a cross-sectional assessment[END_REF]. We also measured the following physio-immunological parameters to assess bat health: body mass, four immunological markers, and four oxidative status markers. As study species, we selected the wrinkle-lipped free-tailed bat, Chaerephon plicatus (see supplementary material for a description of the species) because it forages above agricultural lands, it provides significant ecosystem services to the rice industry in Thailand and is experiencing population declines across Southeast Asia (Leelapaibul et al., 2005;[START_REF] Srilopan | The wrinkle-lipped free-tailed bat (Chaerephon plicatus Buchannan, 1800) feeds mainly on brown planthoppers in rice fields of Central Thailand[END_REF]Ruadreo et al. 2019). To this end, we selected four cave roosts that were located in landscapes with different land-use intensity within a radius of 25 km (assumed foraging area of the study species, [START_REF] Utthammachai | Foraging habitat use by acoustic monitoring of Tadarida plicata (Buchannan, 1800) in an agricultural landscape, Ratchaburi province[END_REF][START_REF] Wanger | Bat pest control contributes to food security in Thailand[END_REF]): more heterogeneous landscapes (mosaic of land uses, including large forest patches) and less heterogenous landscapes (dominated by farmlands, particularly rice crops, with only a few forest patches).

We predicted that bats living in the less heterogenous landscapes would have a different trophic niche, higher erythrocyte concentrations of mercury, lower body mass, lower immune function, higher oxidative damage, and contrasting antioxidant defenses. We also predicted that bats with higher mercury concentrations would show lower immune function and higher oxidative stress.

Material and Methods

Study area and sampling

Sampling was carried out at four caves located in two habitat types (Table 1 andFig Upon capture (Table 1), bats were placed in individual holding bags. A blood sample was collected from the antebrachial vein using heparinised microvettes (Sarstedt, Nümbrecht, Germany) as outlined in Weise et al. (2017) within 30 minutes from capture (the order of bleeding was not significantly correlated with any physiological or immunological trait). Bats were released as soon as possible after bleeding. Blood samples were maintained cool until the end of the trapping session. Tubes were spun for five minutes, plasma was separated from the erythrocytes, and both were stored on dry ice while in the field and at -80°C at the laboratory. From each bat, we identified sex and recorded body mass using a handheld spring balance (Pesola, Switzerland). All individuals were identified as adults because they were at least one year old according to the closure of the epiphyseal gap.

Laboratory analyses

Stable carbon isotope and nitrogen isotopes

Details regarding sample preparation and analysis are given in Ruadreo et al. (2019). Briefly, we measured stable carbon isotope ratios in dried samples using an elemental analyzer (Flash EA 1112, Thermo Scientific, Bremen, Germany) connected in sequence via a ConFlo to a Delta V Advantage isotope ratio mass spectrometer (both ThermoScientific, Bremen Germany). Values are reported in δ 13 C and δ 15 N notation as parts per mille (‰) deviation from the international standard V-PDB for carbon and atmospheric nitrogen for nitrogen. For both elements, analytical precision was always better than 0.1‰ (one standard deviation) for repeated measurements of laboratory protein standards.

Mercury

The total concentration of mercury was quantified in freeze-dried erythrocytes using an atomic absorption spectrophotometer (Advanced Mercury Analyser-254, Altec) on dried tissue aliquots (ranging from 0.5 to 3 mg) as described by [START_REF] Chouvelon | Hg concentrations and related risk assessment in coral reef crustaceans, molluscs and fish from New Caledonia[END_REF]. Prior work on bats has shown that the total concentration of mercury in blood is highly and positively correlated with the highly toxic methylated form [START_REF] Karouna-Renier | Assessment of mitochondrial DNA damage in little brown bats (Myotis lucifugus) collected near a mercury-contaminated river[END_REF]. Total mercury in erythrocytes is also a proxy measure of methylmercury in humans (e.g., [START_REF] Skerfving | Mercury in women exposed to methylmercury through fish consumption, and in their newborn babies and breast milk[END_REF][START_REF] Sakamoto | Declining risk of methylmercury exposure to infants during lactation[END_REF]. The analytical quality (i.e., accuracy and reproducibility) of the mercury measurements was assessed by the analyses of blanks and certified reference material (CRM) TORT-2 Lobster Hepatopancreas (NRC, Canada; certified mercury concentration: 0.27 ± 0.06 µg/g dw). The CRM were analyzed at the beginning and at the end of the analytical cycle, and by running controls for every 10 samples [START_REF] Bustamante | Metal and metalloid concentrations in the giant squid Architeuthis dux from Iberian waters[END_REF]. Mass of the CRM was adjusted to represent an amount of mercury similar to that in bat samples. Our measured values for the CRM were 0.262 ± 0.013 µg/g dw (n = 12) showing a recovery of 97%. Blanks were analyzed at the beginning of each set of samples and the quantification limit of the method was 0.05 ng. Data for mercury concentrations are presented as ng/g relative to the dry weight (dw).

Immunological markers

We selected four immune markers used in eco-immunological studies [START_REF] Demas | Beyond phytohaemagglutinin: assessing vertebrate immune function across ecological contexts[END_REF], characterizing both constitutive (bacterial killing ability [BKA], lysozyme) and induced innate (haptoglobin) and adaptive (immunoglobulin G [IgG]) humoral immune responses [START_REF] Heinrich | Cheetahs have a stronger constitutive innate immunity than leopards[END_REF]. All these assays require small blood volumes and have been validated for bats (Schneeberger et al., 2013a(Schneeberger et al., , 2014a;;Becker et al., 2017Becker et al., , 2018a;;Ruoss et al., 2019).

We report a detailed description of assays in the supplementary material.

Markers of oxidative status

We selected four markers used in ecological studies of mammalian species (e.g., [START_REF] Costantini | Interplay between plasma oxidative status, cortisol and coping styles in wild alpine marmots, Marmota marmota[END_REF][START_REF] Costantini | Socioecological and environmental predictors of physiological stress markers in a threatened feline species[END_REF]Schneeberger et al., 2013bSchneeberger et al., , 2014b) ) characterizing biomolecules that have been oxidized by free radicals (reactive oxygen metabolites), antioxidant protection from non-enzymatic antioxidants (e.g., those derived from diet), detoxification of cells from accumulation of hydrogen peroxide and organic hydroperoxides (activity of the enzyme glutathione peroxidase), and protection against the strong pro-oxidant action of the free radical superoxide generated by cells (activity of superoxide dismutase). We report a detailed description of assays in the supplementary material.

Statistical analyses

We used general linear models to compare isotopic signature, mercury, body mass, immunological markers and oxidative status markers between bats from more and less heterogeneous environments. We also compared the GPx/SOD ratio, because prior work found that unbalanced activities of the two enzymes may reflect an impaired physiological state [START_REF] Park | High SOD and low GPX activities in RBC predict susceptibility of lung cancer patients to radiation pneumonitis[END_REF][START_REF] Jayawardena | Heavy metal-induced toxicity in the Indian green frog: biochemical and histopathological alterations[END_REF]. As fixed factors, we included landscape type (more vs. less heterogeneous landscape), individual sex, and their interaction. When individuals showed high values of Cook's distance (possible outliers; [START_REF] Cook | Detection of influential observation in linear regression[END_REF] compared to the distribution of all Cook's distance values, they were excluded and the models were rerun to test whether their values were influential. Because time at which blood samples were taken varied among sites, we also ran unpaired t-tests to compare bats from the two caves (one in the more heterogeneous landscape and one in the less heterogeneous landscape) sampled at the same time of the day. We performed all analyses using SPSS Statistics 23.

Finally, we used the compute.es package [START_REF] Del Re | Compute.es: compute effect sizes. R package version 0.2[END_REF] in R (R Core Team, 2013) to calculate the standardized effect size Hedges' g from test statistics. We used the forestplots function of the metafor package in R [START_REF] Viechtbauer | Conducting meta-analyses in R with the metafor package[END_REF] to visualise values of effect size and 95% confidence interval. We considered the effect size estimates as small (Hedges g = 0.2, explaining 1% of the variance), intermediate (g = 0.5, explaining 9% of the variance) or large (g = 0.8, explaining 25% of the variance) according to [START_REF] Cohen | Power analysis for the behavioural sciences[END_REF].

Results

Bats from the more heterogeneous landscapes had higher δ 13 C and δ 15 N in erythrocytes than conspecifics from the less heterogeneous landscapes (P < 0.001, Supplementary Table S1, Fig. 2). Effect size estimates were large, and the 95% confidence intervals did not include zero for stable isotopes of both elements (Fig. 3). The significant interaction between environment and sex (P = 0.001 for δ 13 C, P = 0.002 for δ 15 N, Supplementary Table S1) indicated that sex differences in stable isotope ratios differed between environments. In the less heterogeneous landscapes, males and females had similar δ 13 C and δ 15 N values (posthoc test: P = 0.518 for δ 13 C, P = 0.899 for δ 15 N), whereas in the more heterogeneous landscapes males had higher δ 13 C (P < 0.001) and δ 15 N values (P < 0.001) than females.

Mercury concentrations in erythrocytes ranged from 16 to 602 ng/g dry weight and were similar in both sexes (P = 0.13, Supplementary Table S1), but were higher in bats from more heterogeneous landscapes compared to conspecifics from less heterogeneous landscapes (P < 0.001, Supplementary Table S1, Fig. 4). Results for mercury did not vary when a female (602 ng/g) from Lan Sak Dristrict cave was removed because of its high Cook's distance. Effect size was large, and the 95% confidence interval did not include zero (Fig. 3).

Both male and female bats living in the less heterogeneous landscapes had lower body mass (P = 0.023, Supplementary Table S1), higher SOD (P = 0.042, Supplementary Table S1), lower GPx (P = 0.048, Supplementary Table S1), and lower GPx/SOD ratios (P = 0.011, Supplementary Table S1) than bats from more heterogeneous landscapes. All other markers were similar between bats living in the two environments (all P > 0.250, Supplementary Table S1). Outcomes for lysozyme concentrations were unchanged when a male (6.29 μg/ml) from Wat Khao Wongkot cave and a female (9.46 μg/ml) from Panurangsi with high Cook's distances were removed from the analyses. Males had higher OXY (P < 0.001, Supplementary Table S1) and plasma haptoglobin (P = 0.020, Supplementary Table S1) than females irrespective of landscape. The difference in haptoglobin between sexes disappeared when a male (1.42 mg/ml) from Tha Luang District cave, and two males (2.05 and 3.19 mg/ml) and a female (1.22 mg/ml) from Wat Khao Wongkot cave with high Cook's distances were excluded from the model (P = 0.128, Supplementary Table S1).

The comparison of bats from the two caves (Lan Sak Dristrict and Wat Khao Wongkot) that were sampled at a similar time of the day provided similar results to those from the whole dataset (Table 2). Bats living in the less heterogeneous landscape (Wat Khao Wongkot) had lower body mass (P = 0.0003, Table 2; Fig. 4), higher SOD (P = 0.008, Table 2, Fig. 5), lower GPx (P = 0.014, Table 2, Fig. 5), and lower GPx/SOD ratios (P = 0.001, Table 2, Fig. 5) than bats from the more heterogeneous landscape, while all other markers were similar. The effect sizes for the significant differences were large, and the 95% confidence intervals did not overlap zero (Fig. 3). Although the effect sizes for lysozyme, haptoglobin, and BKA overlapped zero, they were intermediate and similar in sign, suggesting a potentially consistent effect of land-use on immune markers. This is more evident for the lysozyme, whose 95% confidence interval for Hedges g slightly included zero (Fig. 3).

There were small but significant negative correlations between mercury concentrations and either OXY or SOD and a significant positive correlation between mercury concentration and IgG (Supplementary Table S2). The correlation between mercury and SOD became non-significant when the individual with the highest mercury concentration (602 ng/g) was excluded (from P = 0.042 to P = 0.236, Supplementary Table S2). The positive correlation between mercury and lysozyme became significant after the exclusion of potential outliers (from P = 0.488 to P = 0.043, Supplementary Table S2). Larger bats had higher mercury concentrations in erythrocytes (P < 0.001, Supplementary Table S2).

Relationships between mercury and markers were consistent across both landscapes (the interaction between environment and mercury was not significant).

Discussion

Our work shows that the mercury contamination, foraging ecology, and immunephysiological status of a tropical bat species may vary with habitat and sex. As expected, δ 13 C values of erythrocytes reflected the origin of the primary food sources in C3-and C4dominated landscapes. Bats living in the less heterogeneous landscapes dominated by farmlands had lower δ 13 C values, indicating that the majority of consumed food sources originated from aquatic food webs such as rice paddies (Ruadreo et al., 2019). Food webs dominated by C4 plants, such as corn and cane sugar (higher δ 13 C values; De Niro and Epstein, 1978) did not largely contribute to bat diets in less heterogeneous landscapes. The less heterogeneous landscapes were dominated by rice crops (approximately 70% of rice crops in Wat Khao Wongkot; [START_REF] Srilopan | The wrinkle-lipped free-tailed bat (Chaerephon plicatus Buchannan, 1800) feeds mainly on brown planthoppers in rice fields of Central Thailand[END_REF]Ruadreo et al., 2019), which are planted during the non-reproductive season of Chaerephon plicatus (Ruadreo et al., 2019). In the less heterogeneous landscapes, bats also had lower δ 15 N, indicating that they occupy a lower trophic position (De Niro and Epstein, 1981). Prior work showed that wrinkle-lipped freetailed bats feed mainly on brown planthoppers and dipteran insects above rice fields of Central Thailand across the whole year [START_REF] Srilopan | The wrinkle-lipped free-tailed bat (Chaerephon plicatus Buchannan, 1800) feeds mainly on brown planthoppers in rice fields of Central Thailand[END_REF]Ruadreo et al., 2019). In the less heterogeneous landscapes, males and females had similar isotopic composition in erythrocytes, which is in agreement with prior work on isotopic composition measured in both fur and wing tissue of bats living in the Wat Khao Wongkot bat-cave (Ruadreo et al., 2019). In the more heterogeneous environments, the isotopic composition in erythrocytes suggested a difference between males and females in trophic position and foraging areas.

This might indicate that landscape homogenization could increase trophic overlap between males and females, possibly increasing food competition. Tracking the flight activity of bats would help to elucidate the foraging areas used by males and females.

Converse to our predictions, mercury was lower in bats living in the less heterogeneous landscapes. In Asia, soils of rice paddies may be sinks for mercury deposition and accumulation and provide an ideal environment for bacteria that methylate inorganic mercury [START_REF] Stubner | Sulfur oxidation in rice field soil: activity, enumeration, isolation and characterization of thiosulfate-oxidizing bacteria[END_REF][START_REF] Horvat | Total mercury, methylmercury and selenium in mercury polluted areas in the province Guizhou, China[END_REF][START_REF] Meng | Distribution patterns of inorganic mercury and methylmercury in tissues of rice (Oryza sativa L.) plants and possible bioaccumulation pathways[END_REF][START_REF] Meng | The process of methylmercury accumulation in rice (Oryza sativa L.)[END_REF][START_REF] Feng | Investigation of Hg uptake and transport between paddy soil and rice seeds combining Hg isotopic composition and speciation[END_REF][START_REF] Tang | Mercury in rice (Oryza sativa L.) and rice-paddy soils under long-term fertilizer and organic amendment[END_REF]. One reason for the lower concentration of mercury in bats from the less heterogeneous landscapes might relate to their lower trophic position as compared to bats from the more heterogeneous landscapes, which would be consistent with the hypothesis of a lower bioamplification effect due to shorter and less complex food webs. Mercury bioaccumulates with increasing trophic level due to the biomagnification process [START_REF] Eagles-Smith | Modulators of mercury risk to wildlife and humans in the context of rapid global change[END_REF]. Several studies show that insectivorous bats can accumulate significant amounts of mercury because of this biomagnification effect, particularly in species connected to aquatic ecosystems [START_REF] Syaripuddin | Mercury accumulation in bats near hydroelectric reservoirs in Peninsular Malaysia[END_REF]Becker et al., 2018b). However, trophic position does not explain why males and females from the more heterogeneous environments had similar mercury concentrations despite their different trophic position. Further work will be needed to elucidate the causes of individual bat variation in mercury exposure.

The positive correlation between body mass and mercury also suggests that larger bats were probably accumulating more mercury through their diet, as previously shown for little brown bats Myotis lucifugus [START_REF] Karouna-Renier | Assessment of mitochondrial DNA damage in little brown bats (Myotis lucifugus) collected near a mercury-contaminated river[END_REF]. However, [START_REF] Kumar | Presence of artisanal gold mining predicts mercury bioaccumulation in five genera of bats (Chiroptera)[END_REF] found that smaller insectivorous bat species accumulated more mercury in fur than larger insectivorous species. In our current study, larger bats were from the more heterogeneous sites. Thus, the positive correlation between body mass and mercury may be caused by the dietary difference between sites. Further work will be needed to assess the diet of Chaerephon plicatus across different land-use regimes.

Although we do not know the extent to which mercury in erythrocytes reflected that accumulated in other tissues, prior work on bats found a strong and positive correlation between mercury in whole blood and fur [START_REF] Wada | Tissue mercury concentrations and adrenocortical responses of female big brown bats (Eptesicus fuscus) near a contaminated river[END_REF][START_REF] Karouna-Renier | Assessment of mitochondrial DNA damage in little brown bats (Myotis lucifugus) collected near a mercury-contaminated river[END_REF][START_REF] Karouna-Renier | Assessment of mitochondrial DNA damage in little brown bats (Myotis lucifugus) collected near a mercury-contaminated river[END_REF], indicating that blood concentration may provide a valuable proxy for the chronic accumulation of mercury. Assuming a water content of about 80% in whole blood [START_REF] Eagles-Smith | Mercury correlations among six tissues for four waterbird species breeding in San Francisco Bay, California, USA[END_REF], values of mercury in bats living in the more heterogeneous landscapes would be equivalent to an average of 42.6 ng/g wet weight and a range from 3.2 to 120 ng/g wet weight. Although the average value is similar to that found in other bat species living far from potential sources for contamination [START_REF] Karouna-Renier | Assessment of mitochondrial DNA damage in little brown bats (Myotis lucifugus) collected near a mercury-contaminated river[END_REF], values are within the range recorded in bats living near contamination sources [START_REF] Wada | Tissue mercury concentrations and adrenocortical responses of female big brown bats (Eptesicus fuscus) near a contaminated river[END_REF][START_REF] Karouna-Renier | Assessment of mitochondrial DNA damage in little brown bats (Myotis lucifugus) collected near a mercury-contaminated river[END_REF][START_REF] Karouna-Renier | Assessment of mitochondrial DNA damage in little brown bats (Myotis lucifugus) collected near a mercury-contaminated river[END_REF]. For example, big brown bats Eptesicus fuscus from a contaminated site in the USA showed a range of mercury in blood from 50 to 200 ng/g wet weight [START_REF] Wada | Tissue mercury concentrations and adrenocortical responses of female big brown bats (Eptesicus fuscus) near a contaminated river[END_REF]. In little brown bats from three contaminated sites in the USA, mercury in blood ranged from 14 to 3,800 ng/g wet weight [START_REF] Karouna-Renier | Assessment of mitochondrial DNA damage in little brown bats (Myotis lucifugus) collected near a mercury-contaminated river[END_REF]. We are not aware of any specific sources of mercury emission near caves located in the more heterogeneous landscapes. One tentative explanation might lie with the filtering properties of forest canopy that would favor accumulation of mercury in wetlands through flooding, soil erosion, and decomposition of organic matter [START_REF] Barbosa | Mercury biomagnification in a tropical black water, Rio Negro, Brazil[END_REF][START_REF] Driscoll | Mercury contamination in forest and freshwater ecosystems in the Northeastern United States[END_REF]. In Uthai Thani, where one of the caves was located, deforestation and conversion of residual forest lands into agricultural lands is a major problem [START_REF] Waiyasusri | Monitoring and predicting land use changes in the Huai Thap Salao Watershed area, Uthaithani Province, Thailand, using the CLUE-s model[END_REF].

Moreover, both the cave in Uthai Thani and that in the Tha Luang District (i.e., those located in the more heterogeneous landscapes) were close (within a radius of 25 km from the cave) to two large watersheds (Huai Thap Salao Dam and Pa Sak Dam, respectively) that might work as sinks for mercury washed away from soil. Analyses of mercury concentration in the environment and prey will be important to ascertain the routes through which wrinkle-lipped free-tailed bats are being exposed to mercury.

Mercury is neurotoxic and can also cause many other potentially negative effects on traits like behaviour, endocrine system, immune function and cellular oxidative status and thus likely reproduction and survival [START_REF] Tan | The endocrine effects of mercury in humans and wildlife[END_REF]Whitney and Cristol, 2018). Bats with a higher concentration of mercury in erythrocytes had lower OXY in plasma and SOD in erythrocytes. This lower antioxidant protection might indicate that bats were exposed to higher cellular oxidative stress. However, we did not find any correlation between mercury and reactive oxygen metabolites. Whether mercury may increase cellular oxidative damages in wrinkle-lipped free-tailed bats remains an open question.

Our analyses also showed that bats with higher blood mercury had higher IgG and lysozyme concentrations when outliers were removed. The reasons for these correlations are currently unclear. Mercury may affect different aspects of immune function (Becker et al., 2017;Whitney and Cristol, 2018). Higher concentrations of IgG and lysozyme in bats with more blood mercury may also reflect higher parasite loads that stimulate the constitutive innate and adaptive immune responses. We observed many ectoparasites on bats in these sites (unpublished data), which might have stimulated constitutive innate and adaptive immune responses.

None of the immune markers differed largely between bats from the less or more heterogeneous landscapes. The effect size estimates for lysozyme, haptoglobin, and BKA were similar, intermediate in size, and indicated higher values in the less heterogeneous landscapes. Although the 95% confidence intervals for the effect size estimates of the three immune markers included zero, these estimates were similar in sign and size. This result stimulates further work with a larger sample size that also considers careful estimates of parasite burdens to clarify whether land-use affects immune function independently from mercury exposure. Presently, our data here suggest that some aspects of anthropogenic land use might have a stronger connection with oxidative status markers. Bats in the less heterogeneous landscapes had higher SOD and lower GPX, resulting in significant lower GPx/SOD ratios. However, conversely to our prediction, plasma reactive oxygen metabolites (marker of early derivatives of oxidative damage) did not differ between bats in the two environments.

We currently do not know the reasons for such differences in GPx and SOD and if they have any functional consequences. We might exclude a strong role of mercury contamination because the correlation with SOD was small and it was not associated with GPx nor with GPx/SOD ratio. Thus, other factors such as foraging effort, diet quality, and pesticide exposure might be involved.

Conclusions

In conclusion, our study showed that land-use was associated with foraging ecology, mercury contamination, and oxidative status in a tropical insectivorous bat species. Further, our study also showed that mercury concentrations were correlated with individual body mass and immune function. We conclude that a combination of several endogenous markers can generate a more comprehensive picture about how land-use changes may affect wildlife. Further work will be needed to ascertain the ecological relevance of the mercury concentrations we detected in bats.

Finally, our study suggests a possibly useful application to landscape planning that might inform stakeholders and policy makers on how to develop plans for land-use in a more compatible way with bats' ecological needs. As expected, our results suggested that environments with intensive agricultural practices might be less suitable for insectivorous bats than more heterogeneous environments. However, our data also showed that bottom-up effects of landscape composition may come through unexpected routes, such as mercury exposure. For example, deforestation near watersheds or other wetlands might increase mercury exposure as suggested by results from bats living in the province of Uthai Thani. This would call for a strict protection of forest patches in order to avoid soil erosion and washing out of mercury into watersheds.

for kindly correcting our English, the editor and two reviewers for providing valuable and constructive comments on our manuscript. Table 1. The fieldwork included four caves located in more or less heterogeneous landscapes. The more heterogeneous sites also included forest patches. The less heterogeneous sites did not include any forest patches and had a dominance of agricultural lands (mainly rice crops) according to data availability (Geo-Informatics and Space Technology Development Agency; Google Earth Pro; * [START_REF] Srilopan | The wrinkle-lipped free-tailed bat (Chaerephon plicatus Buchannan, 1800) feeds mainly on brown planthoppers in rice fields of Central Thailand[END_REF]; see also [START_REF] Wanger | Bat pest control contributes to food security in Thailand[END_REF]. The distance among caves ranged from ca. 80 to 230 km. All the provinces where caves were located had experienced significant changes in land cover.

Study species

The wrinkle-lipped free-tailed bat is a cave roosting species that is widespread throughout much of South-East Asia [START_REF] Csorba | Chaerephon plicatus. The IUCN Red List of Threatened Species, e.T4316A67361960[END_REF]. It occurs in large colonies that may count up to 3 million individuals (Leelapaibul et al., 2005). Populations generally forage close to roost sites (within a radius of 25 km), and have been recorded hunting above both forested areas and rice fields (Leelaipul et al., 2005;Ruadreo et al., 2019). This species is currently listed as Least Concern by IUCN [START_REF] Csorba | Chaerephon plicatus. The IUCN Red List of Threatened Species, e.T4316A67361960[END_REF]. The population is, however, declining because the habitat is being deforested for timber, firewood and conversion to agricultural use, disturbance in caves (guano mining, hunting), and eradication of colonies [START_REF] Csorba | Chaerephon plicatus. The IUCN Red List of Threatened Species, e.T4316A67361960[END_REF]. It is unknown whether contaminants or diseases play a role in driving these population

declines, yet it is assumed that contaminants, such as pesticide residues or mercury, might be involved [START_REF] Voigt | Conservation strategies for bats flying at high altitudes[END_REF]. Wrinkle-lipped free-tailed bats are expected to consume 20,000

MT of insects per year of which many are pest insects highly detrimental for rice production (Leelapaibul et al., 2005). Declines of wrinkle-lipped free-tailed bat populations are suggested to lead to drastic decreases in crop production and consequently to large economic losses [START_REF] Wanger | Bat pest control contributes to food security in Thailand[END_REF].

Immunological markers

Lysozyme, also known as muramidase or N-acetylmuramide glycanhydrolase, is an antibacterial enzyme which hydrolyses the β-(1,4)-glycosidic bond between N-al., 2017, 2018a;Ruoss et al., 2019). Protein G, a streptococcal protein, binds IgG from many wildlife taxa including bats (Schneeberger et al., 2014a). Briefly, high-binding 96-well plates were coated with 100 µl diluted plasma samples (1:5000 in 50 mM NaHCO3, pH 9.5) in duplicates and incubated for 1 hour at 37°C. After incubation, plates were washed in Trisbuffered saline/Tween-20 (TBS-T), blocked with 1% gelatine (Merck) solution and incubated for 30 min at 37°C. After washing, 100 µl of Protein G-horseradish peroxidase conjugate solution (Invitrogen, 1:12,000 in TBS-T, pH 7.4) was added to each well. The wells were washed after 30 min incubation at room temperature and submerged with 100 µl TMB [10% 3,3', 5,5'-tetramethylbenzidine (SouthernBiotech) in DMSO (Sigma-Aldrich) diluted 1:100 in phosphate-citric buffer pH 5.0 and mixed with 30% H2O2 (Hedinger)]. The reaction was stopped after 3 minutes with 1 M sulphuric acid and the absorbance was read immediately at 450 nm (Biotek; µQuant Microplate Spectrophotometer). Antibody concentration is directly proportional to the absorption, thus statistical analysis was conducted on the mean optical density (OD) of the duplicates (Schneeberger et al., 2014a;Ruoss et al., 2019).

Haptoglobin is an acute phase protein that usually occurs at low concentrations, but its production and secretion is increased in response to acute infections and trauma. To measure the concentration of haptoglobin, the standard procedure of the commercial kit "PHASE" TM Haptoglobin Assay (Tridelta, Ireland) was followed. After diluting the plasma samples (1:2) with PBS, haemoglobin was added. Haptoglobin binds to haemoglobin and maintains its peroxidase activity at a low pH. The measured peroxidase activity of haemoglobin is directly proportional to the amount of haptoglobin in the sample.

chromogen N,N-diethyl-p-phenylenediamine (ratio 100:1). The reactive oxygen metabolites that occur in the plasma react with the chromogen generating a pink colour, whose intensity is proportional to the concentration. After an incubation at 37°C for 75 minutes, we measured the optical density at 505 nm. We expressed the values as mM of H2O2 equivalents. Good parallelism between successive dilutions of plasma samples and the standard curve of the assay was obtained (R 2 =0.99).

We measured the plasma non-enzymatic antioxidant capacity using the OXYadsorbent test (Diacron International, Grosseto, Italy). This assay quantifies the in vitro reaction between the non-enzymatic antioxidants (e.g., protein thiols, vitamins C and E) and the hypochlorous acid (HOCl; pro-oxidant generated endogenously by vertebrates). All plasma samples, reference standard (300 mM) and quality controls were initially diluted 1:100 with distilled water. Then, we pipetted 5 µl of each one in duplicate in 96-well plates and we added straightaway 200 µl of HOCl using a multichannel pipette. After an incubation at 37°C for 10 minutes, we added 2 µl of N,N-diethyl-p-phenylenediamine to each well using a multichannel pipette and mixed. The N,N-diethyl-p-phenylenediamine reacts with the HOCl that did not react with the plasma antioxidants, generating a pink colour. We measured the optical density at 505 nm and we expressed the OXY values as mM of HOCl neutralized.

We measured the activity of the antioxidant enzyme glutathione peroxidase (GPx) in erythrocytes using the Ransel test (Randox Laboratories, Crumlin, UK). First, we made haemolysates by diluting an amount of erythrocytes into distilled water. We added 2 µl of haemolysate to 200 µl of a reagent (4 mM glutathione; 0.34 mM NADPH; glutathione reductase) diluted in buffer (phosphate buffer 0.05 M and pH 7.2; 4.3 mM of EDTA). Then,

  . 1) during the non-reproductive season (end of July) to avoid disturbing breeding animals. We collected blood samples from 104 individuals: 15 males and 33 females from two caves located in more heterogeneous landscapes; 22 males and 34 females from two caves located in less heterogeneous landscapes dominated by rice crops. The fieldwork complied with the current laws of Thailand and were performed as part of permit #0002/4508 granted by the National Research Council of Thailand (NRCT) and permit #108/59 granted by the Department of National Park, Wildlife and Plant Conservation (DNP).

Figure 1 .

 1 Figure 1. Map and aerial photographs illustrating the locations of caves in Thailand. Circles show a radius of 25 km from the cave, which indicates the distance within which bats can forage[START_REF] Utthammachai | Foraging habitat use by acoustic monitoring of Tadarida plicata (Buchannan, 1800) in an agricultural landscape, Ratchaburi province[END_REF][START_REF] Wanger | Bat pest control contributes to food security in Thailand[END_REF]. Sources of images: Google Maps and Google Earth Pro, ©2018 Google.

Figure 2 .

 2 Figure2. Bats living in landscapes dominated by farmlands (mostly rice crops) had lower carbon and nitrogen stable isotopes in erythrocytes than bats living in more heterogeneous environments. In agricultural landscapes, males and females had similar δ 13 C and δ 15 N, while in the more heterogeneous landscapes, males had higher δ 13 C than females. Values are shown as means and standard error.

Figure 3 .

 3 Figure3. Estimates of effect size (Hedges' g) and 95% confidence intervals from the two caves where samples were collected at a similar time of the day. Estimates are positive or negative when values of a given metric were higher or lower in bats from more heterogeneous landscapes, respectively.

Figure 4 .

 4 Figure 4. Means and standard errors for mercury concentration in erythrocytes and for body mass. * indicates a statistically significant difference.

Figure 5 .

 5 Figure 5. Means and standard errors for all markers of oxidative status analysed in the present study. ROMs = reactive oxygen metabolites; OXY = plasma non-enzymatic antioxidant capacity; GPx = glutathione peroxidase; SOD = superoxide dismutase. * indicates a statistically significant difference.

Figure 6 .

 6 Figure 6. Means and standard errors for all immune markers analysed in the present study. IgG = immunoglobulin G.

Table 2 .

 2 Outcomes of unpaired t-tests performed to compare bats from Lan Sak Dristrict cave (reference cave, more heterogeneous landscape) with those from Wat Khao Wongkot cave (less heterogeneous landscape, approximately 70% of rice crops) because they were sampled at a similar time of the day. Significant effects are shown in bold type.

	Variable	t-value	df	p
	ROMs	0.824	54	0.413
	OXY	0.107	54	0.915
	GPx	2.583	54	0.014
	SOD	-2.741	54	0.008
	GPx/SOD	3.357	54	0.001
	Lysozyme	-1.764	52	0.084
	IgG	0.226	53	0.822
	Haptoglobin	-1.470	35	0.150
	BKA	-1.075	22	0.294
	Body mass	3.864	54	0.0003

* Corresponding author. Email address: david.costantini@mnhn.fr

Acknowledgements

This work was supported by the Alexander-von-Humboldt-Foundation and funds from Leibniz Institute for Zoo and Wildlife Research. We are grateful to the platform "Analyses Elémentaires" of LIENSs for the access to their analytical facilities. We are also grateful to the CPER (Contrat de Projet État-Région) and the FEDER (European Regional Development Fund) for funding the AMA. The IUF (Institut Universitaire de France) is also acknowledged for its support to PB as a Senior Member. The authors wish to thank Katja Pohle and Yvonne Klaar for technical assistance in the lab, students for assistance in fieldwork, Daniel J. Becker

Data Availability

The datasets analyzed during the current study are available from the corresponding author on reasonable request.

Supplementary Material

Impacts of land use on an insectivorous tropical bat: the importance of mercury, physio-immunology and trophic position David Costantini a,b,c *, Gábor Á. Czirják d , Paco Bustamante e , Sara Bumrungsri f , Christian C. Voigt c,g a UMR 7221 CNRS/MNHN, Muséum National d'Histoire Naturelle, Sorbonne Universités, 7 rue Cuvier, Paris 75005, France acetylmuramic acid and N-acetylglucosamine residues of bacterial peptidoglycan, causing rapid bacterial lysis. The enzyme is primarily active against Gram-positive bacterial species, but cell lysis has been described both against Gram-negative microorganisms and yeasts. To measure lysozyme concentration, we used the lysoplate assay, which was adapted to low sample volumes [START_REF] Rowe | Lysozyme-associated bactericidal activity in the ejaculate of a wild passerine[END_REF]. Briefly, we prepared 1% noble agar (Sigma Aldrich) with PBS at pH = 6.3 and we added the required amount of lysozyme-sensitive bacteria Micrococcus lysodeikticus ATCC #4698 (Sigma Aldrich) to reach a bacterial concentration of 50 mg/100 ml in the agar. We poured the agar in Petri dishes on a pre-heated surface (50°C) and horizontally leveled with a water spirit to avoid quick and uneven cooling of the medium. After solidification, we inoculated 1.5 µl of plasma in test holes (1.7 mm in diameter). We used standard dilutions of hen egg white lysozyme (0.5, 0.8, 1, 2, 4, 8, 10, 20, and 40 µg/ml; Sigma-Aldrich) to prepare a standard curve in each plate. We incubated the plates at 37°C for 20 hours. During the incubation, a zone of clearing developed in the area of the gel surrounding the sample inoculation site as a result of bacterial lysis. The diameters of the cleared zones are proportional to the log of the lysozyme concentration. We photographed each plate in a photobox (Imaging system; peqlab) with a ruler next to it as a reference scale. We measured the diameter of the cleared zone digitally three times using the software ImageJ (version 1.48, http://imagej.nih.gov/ij/) and we converted the mean value on a semilogarithmic plot into hen egg lysozyme equivalents (HEL equivalents, expressed in μg/ml) according to the standard curve [START_REF] Giraudeau | Effect of restricted preen-gland access on maternal self maintenance and reproductive investment in mallards[END_REF].

We measured the concentration of immunoglobulin G (IgG), the most common antibody subtype, in plasma with a Protein G ELISA (Schneeberger et al., 2014a;Becker et Haptoglobin concentrations were calculated according to the standard curve on each plate and were expressed as mg/ml.

The BKA of plasma is an integrative functional measurement of the humoral part of the constitutive innate immunity. We performed the assay with Escherichia coli (ATCC 8739) following the method described by French and Neumann-Lee ( 2012) and adapted to bats (Becker et al., 2017(Becker et al., , 2018a;;Ruoss et al., 2019). The killing activity against this bacterial strain is mediated mainly through complement proteins [START_REF] Pap | Sexual dimorphism in immune function changes during the annual cycle in house sparrows[END_REF]Becker et al., 2019).

We diluted the plasma samples 1:8 in sterile phosphate buffered saline (PBS) and challenged them with a 10 5 /ml bacterial solution. We incubated the plasma-bacterial mixture for 30 minutes at 37°C, after which we added tryptic soy broth and we measured the background optical density at 340 nm (Biotek; µQuant Microplate Spectrophotometer). All samples were run in duplicate and all plates contained both positive (without plasma) and negative controls (without bacteria). After an incubation of 12 hours at 37°C, we measured again the optical density and we calculated the BKA value as one minus mean ODsample, divided by OD positive control (OD = optical density; Becker et al., 2018a).

Oxidative status markers

We measured the reactive oxygen metabolites (primary products of oxidative damage) in plasma using the d-ROMs test (Diacron International, Grosseto, Italy). We pipetted plasma samples, reference standards (0.225 to 1.8 mM H2O2 equivalents), blanks and quality controls (4 µl) \ in duplicate in 96-well plates. Using a multichannel pipette, we added to each well 200 µl of a solution containing acetic acid/sodium acetate buffer (0.01 M, pH 4.8) and the we added 8 µl of cumene hydroperoxide (substrate of the enzyme) to each well and we measured the optical density in kinetics after 60, 120 and 180 seconds at 340 nm. The change in optical density per minute was taken below 0.1 in order to be in the linear range of the correlation between activity of GPx and optical density. We normalized the activity of GPx by the concentration of proteins in the haemolysate and we expressed it as units per mg of proteins. We used the Bradford test with albumin as reference standard (Sigma-Aldrich) to measure the protein concentration.

We measured the activity of the antioxidant enzyme superoxide dismutase (SOD) in erythrocytes using the Ransod test (Randox Laboratories, Crumlin, UK). We added 6 µl of the haemolysate used for the GPx to 200 µl of a buffer (CAPS 40 mM and pH 10.2; 0.94 mM EDTA) containing the substrate of the enzyme (0.05 mM xanthine; 0.025 mM I.N.T.).

Then, using a multichannel pipette, we added 30 µl of xanthine oxidase to each well and we measured the optical density in kinetics after 30 seconds and 210 seconds at 505 nm. The reaction between xanthin and xanthin oxidase generates the superoxide which reacts with I.N.T. generating a red colouration. The SOD present in the sample inhibits the reaction between the superoxide and I.N.T. We normalized the activity of SOD by the concentration of proteins in the haemolysate as for the GPx and we expressed it as units per mg of proteins.

We calculated the activity of SOD using a reference standard with a known activity of the SOD enzyme.

Table S1. Outcomes of general linear models. Significant effects are shown in bold type. * indicates that the model was re-run after exclusion of statistical outliers.


Tiphaine Mille
email: tiphaine.mille@ifremer.fr

Pierre Cresson

Tiphaine Chouvelon

Paco Bustamante

Christophe Brach-Papa

Sandrine Bruzac

Emmanuelle Rozuel

Marc Bouchoucha

Mille Tiphaine

Chouvelon Tiphaine

Bustamante Paco

Brach-Papa Christophe

Bruzac Sandrine

Rozuel Emmanuelle

Bouchoucha Marc

Trace metal concentrations in the muscle of seven marine species: Comparison between the Gulf of Lions (North-West Mediterranean Sea) and the Bay of Biscay (North-East Atlantic Ocean)

Keywords: Inorganic elements, Contamination, Bioaccumulation, Fish, Shark, Norway lobster

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

Trace metals are naturally present in the Earth crust and as such they are natural components of the marine environment [START_REF] Bryan | The Effects of Heavy Metals (other than Mercury) on Marine and Estuarine Organisms[END_REF][START_REF] Mason | Trace Metals in Aquatic Systems[END_REF]. They differ fundamentally in their metabolic role and their regulation by aquatic organisms [START_REF] Amiard | Application de la spectrometrie d'absorption atomique zeeman aux dosages de huit elements traces (Ag, Cd, Cr, Cu, Mn, Ni, Pb et Se) dans des matrices biologiques solides[END_REF]. Some trace metals such as copper (Cu) or zinc (Zn) are involved in essential metabolic purposes including functional and structural role. These essential trace metals are generally regulated by aquatic organisms. On the contrary, trace metals such as cadmium (Cd) or lead (Pb) are not essential for marine life (i.e. no known biological role) and need to be detoxified or excreted when absorbed [START_REF] Rainbow | Trace metal concentrations in aquatic invertebrates: why and so what?[END_REF].

Whether essential or non-essential, they become toxic beyond a certain threshold [START_REF] Amiard | Application de la spectrometrie d'absorption atomique zeeman aux dosages de huit elements traces (Ag, Cd, Cr, Cu, Mn, Ni, Pb et Se) dans des matrices biologiques solides[END_REF]. Since marine organisms are able to accumulate trace metals, some of them like fish are sometimes considered relatively good semi-quantitative bioindicators, reflecting the occurrence and bioavailability of contaminants in areas where they live [START_REF] Raknuzzaman | Trace metal contamination in commercial fish and crustaceans collected from coastal area of Bangladesh and health risk assessment[END_REF].

However, trace metal concentrations in marine organisms result of a balance between their uptake and excretion processes [START_REF] Gobert | Trace element concentrations in the apex predator swordfish (Xiphias gladius) from a Mediterranean fishery and risk assessment for consumers[END_REF][START_REF] Guven | Acute Lethal Toxicity and Accumulation of Copper in Gammarus pulex (L.) (Amphipoda)[END_REF]. Bioaccumulation, namely the capacity of an organism to concentrate a trace metal in its tissues, depends on the uptake from water by direct contact (e.g. through epidermis, gills, digestive tract) but also from diet (e.g. [START_REF] Mathews | Dominance of dietary intake of metals in marine elasmobranch and teleost fish[END_REF][START_REF] Xu | Exposure and potential food chain transfer factor of Cd, Se and Zn in marine fish Lutjanus argentimaculatus[END_REF]. Therefore, an accurate knowledge of the organic matter sources fueling food webs and their associated contamination level is crucial to understand organisms' contamination [START_REF] Hall | Food as the Dominant Pathway of Methylmercury Uptake by Fish[END_REF]. Moreover, several biological factors such as length or age can affect diet [START_REF] Chouvelon | Species-and size-related patterns in stable isotopes and mercury concentrations in fish help refine marine ecosystem indicators and provide evidence for distinct management units for hake in the Northeast Atlantic[END_REF], and consequently affect potential dietary inputs of trace metals for consumers, in addition to the fact that the age of organisms directly influences their chronic exposure to contaminants [START_REF] Cresson | Are red mullet efficient as bio-indicators of mercury contamination? A case study from the French Mediterranean[END_REF]. Fish bioaccumulation pattern is thus a complex process that can vary according to food resources, feeding habitats, and metabolic activity of species, as well as according to the physical/chemical conditions of water column that can directly affect the bioavailability of trace metals.

In addition, some trace metals such as mercury (Hg) are well-known for their biomagnification property, namely the trend for increasing concentrations along food webs [START_REF] Bryan | Bioaccumulation of Marine Pollutants[END_REF]. This process in particular is responsible for the high trace metal concentrations generally observed in high trophic level species such as marine predators or long-lived species [START_REF] Endo | Hg, Zn and Cu levels in the muscle and liver of tiger sharks (Galeocerdo cuvier) from the coast of Ishigaki Island, Japan: Relationship between metal concentrations and body length[END_REF], which implies a high toxicological hazard for humans when consuming top predator organisms like piscivorous fishes [START_REF] Damiano | Accumulation of heavy metals to assess the health status of swordfish in a comparative analysis of Mediterranean and Atlantic areas[END_REF]. This high hazard linked with Hg in marine systems drove a major research effort on this metal, in order to understand the influence of abiotic (e.g. habitats of species; [START_REF] Chouvelon | Enhanced bioaccumulation of mercury in deep-sea fauna from the Bay of Biscay (north-east Atlantic) in relation to trophic positions identified by analysis of carbon and nitrogen stable isotopes[END_REF]) and biotic factors (e.g. fish growth, trophic functioning of ecosystems; [START_REF] Chouvelon | Oligotrophy as a major driver of mercury bioaccumulation in medium-to high-trophic level consumers: A marine ecosystem-comparative study[END_REF][START_REF] Cossa | Influences of Bioavailability, Trophic Position, and Growth on Methylmercury in Hakes (Merluccius merluccius) from Northwestern Mediterranean and Northeastern Atlantic[END_REF][START_REF] Cresson | Mercury in organisms from the Northwestern Mediterranean slope: Importance of food sources[END_REF] on Hg bioaccumulation in marine organisms, especially in commercial species. As such, several authors evidenced and partly explained the "Mediterranean Hg anomaly", i.e. the fact that species in the Mediterranean have higher Hg concentration than their counterparts from other environments [START_REF] Cossa | Influences of Bioavailability, Trophic Position, and Growth on Methylmercury in Hakes (Merluccius merluccius) from Northwestern Mediterranean and Northeastern Atlantic[END_REF][START_REF] Cossa | The Mediterranean Mercury Anomaly, a Geochemical or a BiologocalIssue[END_REF]. Contrary to Hg, such spatial differences for other trace metals were only studied for few species such as the deep-water shark Galeus melastomus [START_REF] Hornung | Trace metal content in deep-water sharks from the eastern Mediterranean Sea[END_REF] or the pelagic swordfish Xiphias gladus [START_REF] Damiano | Accumulation of heavy metals to assess the health status of swordfish in a comparative analysis of Mediterranean and Atlantic areas[END_REF].

In this context, this study aimed to document and compare the bioaccumulation of trace metals (other than Hg) in the muscle of several common species with commercial interest between the Bay of Biscay (BoB) in the north-eastern Atlantic Ocean and the Gulf of Lions (GoL) in the northwestern Mediterranean Sea. An original approach is reported in this study, relying on an interspecific comparison between several taxa including crustacean species, chondrichthyan and teleost fish, but also on the comparison of bioaccumulation patterns between essential and nonessential trace metals.

Samples were collected during two bottom trawling surveys operated by the French institute for the exploitation of the sea (Ifremer), MEDITS in the GoL in 2012 (Jadaud and Metral, 2012) and EVHOE (Leaute et al., 2008) in the BoB in 2008 (Fig. 1). These two ecosystems have been chosen for their differences in terms of trophic status (i.e. mesotrophy vs. oligotrophy; [START_REF] Liénart | Dynamics of particulate organic matter composition in coastal systems: A spatio-temporal study at multi-systems scale[END_REF] but also in terms of probable contamination, since the BoB is open onto the Atlantic Ocean while the GoL is part of the Mediterranean semi-enclosed sea. In addition, both ecosystems are major areas for fisheries, for which trace metal concentrations in commercial species may be of concern. Finally, they are two out the four French marine subregions considered and monitored by the European Marine Strategy Framework Directive. The studied species included one crustacean speciesthe Norway lobster (Nephrops norvegicus, Linnaeus, 1758)-, two sharksthe black-mouthed dogfish (Galeus melastomus, Rafinesque, 1810) and the lesser spotted dogfish (Scyliorhinus canicula, Linnaeus, 1758)-, and four teleost fishthe blackbelly rosefish (Helicolenus dactylopterus, Delaroche, 1809), the four-spot megrim (Lepidorhombus boscii, Risso, 1810), the blue whiting (Micromesistius poutassou, Risso, 1827) and the greater forkbeard (Phycis blennoides, Brünnich, 1768). These species are largely present on the shelf-edge in both ecosystems, allowing direct comparisons. They were chosen based on their importance in the communities of both systems, their relatively high trophic position [START_REF] Papiol | Food web structure and seasonality of slope megafauna in the NW Mediterranean elucidated by stable isotopes: Relationship with available food sources[END_REF] and their commercial interest. Individuals have been collected at depth ranging between 284 and 816m in the GoL and between 47 and 511m in the BoB (Table 1). Individuals were stored frozen on board and kept frozen until dissection. At the laboratory, individuals were thawed and measured to the nearest mm (cephalothorax length for N. norvegicus and total length for other species). Two muscle samples without skin and bones were taken on all individuals for isotopic (i.e. carbon and nitrogen stable isotope ratios) and trace metal analyses. For sharks and teleosts, the white dorsal muscle was used for analyses whereas for N. norvegicus, the abdominal muscle was taken out.

Due to the amount of matter needed for the analysis of trace metal concentrations (~200 mg of dry muscle tissue), several small individuals with similar parameters (sampling site, sex and size) were pooled. Mean length and isotopic ratios were used for these pools of individuals (indicated in Table 1). All muscle samples were stored frozen, before freeze-drying and grinding.

Concentrations of three essential trace metals: copper (Cu), nickel (Ni) and zinc (Zn), and three non-essentials: silver (Ag), cadmium (Cd), lead (Pb), were measured by Inductively Coupled Plasma-Mass Spectrometry (ICP-MS; Thermo X-Series I), after several steps of sample preparation. Briefly, subsamples (~200 mg) of homogenized dry muscle tissue were digested with a mixture of 3.5 mL HNO3 and 5 mL HCl Suprapur quality, and then heated in a microwave oven. After the mineralization process, each sample was completed to 50 mL with milli-Q quality water, and a known quantity of internal standard (indium) was added before analysis. Samples were re-diluted when necessary before analyses. Trace metal analyses were run according to a thorough quality control program including the analysis of Certified Reference Materials (CRMs): DORM-3 and DORM-4 (fish protein, National Research Council Canada), and NIST-2976 (mussel tissue, National Institute of Standards and Technology USA). CRMs were analyzed in the same conditions as the samples and results were in good agreement with the certified values. Ag, Cu, Cd, Ni, Pb, andZn were 0.05, 2.5, 0.05, 0.25, 0 was set to 2.3 for sharks, 3.2 for teleosts and 3.4 for N. norvegicus in both ecosystems. These values were consistent with literature and results of dedicated experiments, as reviewed by [START_REF] Chouvelon | Enhanced bioaccumulation of mercury in deep-sea fauna from the Bay of Biscay (north-east Atlantic) in relation to trophic positions identified by analysis of carbon and nitrogen stable isotopes[END_REF]. Regarding the trophic baseline, most recent approaches recommend using primary consumers rather than primary producers, as isotopic ratios of primary producers may be highly variable, whereas primary consumers (i.e. consumers at theoretical trophic level

Limits of quantification (LQ) for

2) provide a time-integrated information on the organic matter sources actually incorporated in food webs [START_REF] Jennings | Trophic levels of marine consumers from nitrogen stable isotope analysis: estimation and uncertainty[END_REF][START_REF] Trueman | Stable isotope-based location in a shelf sea setting: accuracy and precision are comparable to light-based location methods[END_REF]. In the Gulf of Lions, the mean value measured for 200-300 μm zooplankton by [START_REF] Espinasse | Patterns of variations in C and N stable isotope ratios in size-fractionated zooplankton in the Gulf of Lion, NW Mediterranean Sea[END_REF] was used as the trophic baseline (i.e. δ 15 Nbaseline=3.69‰). Indeed, previous studies demonstrated that shelf-edge Mediterranean food webs are based on falling phytoplanktonic production [START_REF] Cresson | Mercury in organisms from the Northwestern Mediterranean slope: Importance of food sources[END_REF], justifying the use of a pelagic trophic baseline for the Mediterranean organisms considered here.

In the BoB, the scallop Pecten maximus was used as the trophic baseline, consistently with the formula developed by [START_REF] Chouvelon | Enhanced bioaccumulation of mercury in deep-sea fauna from the Bay of Biscay (north-east Atlantic) in relation to trophic positions identified by analysis of carbon and nitrogen stable isotopes[END_REF] for this ecosystem, to correct for the onshore offshore effect on the consumers' isotopic ratios (δ 15 Nbaseline = 33.47 + 1.556 × δ 13 Cconsumer).

The relationships of metal concentrations with length and trophic level were investigated by a linear model including these continuous variables as two potential biological factors driving bioaccumulation and biomagnification, respectively. This analysis allowed to explain both potentially large inter-individual variability but also to avoid bias in the measure of the ecosystem effect. The later was added in the model as factor as well as the interaction with length and trophic level. The model was reduced by a bidirectional elimination procedure based on the Akaike information criteria (AIC) [START_REF] Borcard | Numerical Ecology With R[END_REF]. In order to determine active factors, significance of effects in the reduced model was tested by F-tests between nested models respecting marginality of the effects [START_REF] Fox | An {R} Companion to Applied Regression[END_REF]. In the case where the size was correlated with the trophic level (as tested by the Pearson correlation coefficient test), only the total length was included in the model. Only trace elements for which>70% of the concentrations were above the LQ have been modeled.

In addition, in order to complete the approach element by element, an index of global contamination was calculated from all element concentrations including concentration above the LQ. The Trace Element Pollution Index (TEPI) proposed by [START_REF] Richir | A reassessment of the use of Posidonia oceanica and Mytilus galloprovincialis to biomonitor the coastal pollution of trace elements: New tools and tips[END_REF] has been chosen because this index allows a reliable comparison of global trace element contamination between environments, whatever the studied trace element. The TEPI values were calculated for each species and each environment as follow: TEPI=(Cf1×Cf2…Cfn)1/n where Cfn is the mean normalized concentration (individual concentration divided by the mean concentration) of the trace element n. All statistical analyses were performed to the error threshold of 5%, using the "car" [START_REF] Fox | An {R} Companion to Applied Regression[END_REF]) "MASS" [START_REF] Venables | Modern Applied Statistics with S[END_REF] and "PMCMR" [START_REF] Pohlert | The Pairwise Multiple Comparison of Mean Ranks Package (PMCMR)[END_REF] packages in the statistical environment [START_REF] Core | R: A Language and Environment for Statistical Computing[END_REF].

Overall, this study provides baseline information on the concentrations of some trace metals, both essential (Cu, Ni, Zn) and nonessential (Ag, Cd, Pb), in several marine species from the GoL and the BoB (Table 2). The results are consistent with the pattern of low metal concentrations (metals other than Hg) usually found in the muscle tissue, in comparison with other tissues/organs such as liver or kidneys [START_REF] Canli | The relationships between heavy metal (Cd, Cr, Cu, Fe, Pb, Zn) levels and the size of six Mediterranean fish species[END_REF][START_REF] Dural | Bioaccumulation of some heavy metals in different tissues of Dicentrarchus labrax L, 1758, Sparus aurata L, 1758 and Mugil cephalus L, 1758 from the Camlik lagoon of the eastern coast of Mediterranean (Turkey)[END_REF][START_REF] El-Moselhy | Bioaccumulation of heavy metals in some tissues of fish in the Red Sea, Egypt. Egypt[END_REF][START_REF] Endo | Hg, Zn and Cu levels in the muscle and liver of tiger sharks (Galeocerdo cuvier) from the coast of Ishigaki Island, Japan: Relationship between metal concentrations and body length[END_REF][START_REF] Long | Metallothionein induction and bioaccumulation kinetics of Cd and Ag in the marine fish Terapon jarbua challenged with dietary or waterborne Ag and Cu[END_REF][START_REF] Mormede | Heavy metal concentrations in commercial deep-sea fish from the Rockall Trough[END_REF]. Consistently, in the present study, all muscle samples from teleosts and sharks (i.e. fish) displayed Ag and Cd concentrations below the quantification limits (Table 2). In general, gills, intestinal viscera, liver and kidneys contain relatively high metal burden, indicating that they likely constitute major organs for metal uptake (gills and intestinal viscera) and final deposition or excretion (liver, kidneys) [START_REF] Xu | Exposure and potential food chain transfer factor of Cd, Se and Zn in marine fish Lutjanus argentimaculatus[END_REF]. Trace element accumulation indeed differs according to organs and as such, Cd tends to be preferentially accumulated in liver and kidney, owing to higher levels of metallothioneins (chelating proteins) that act as sites for Cd storage in order to protect fish from cellular metal toxicity [START_REF] Dural | Bioaccumulation of some heavy metals in different tissues of Dicentrarchus labrax L, 1758, Sparus aurata L, 1758 and Mugil cephalus L, 1758 from the Camlik lagoon of the eastern coast of Mediterranean (Turkey)[END_REF][START_REF] Endo | Hg, Zn and Cu levels in the muscle and liver of tiger sharks (Galeocerdo cuvier) from the coast of Ishigaki Island, Japan: Relationship between metal concentrations and body length[END_REF]. Consequently, higher Cd concentrations are generally observed in the liver than in the muscle in fish [START_REF] Eisler | Chapter 3 -Fishes[END_REF]. Similarly, Ag concentrations in intestinal viscera were shown to be higher than in muscle tissues in the marine fish Terapon jarbua, for instance [START_REF] Long | Metallothionein induction and bioaccumulation kinetics of Cd and Ag in the marine fish Terapon jarbua challenged with dietary or waterborne Ag and Cu[END_REF]. Here, the muscle was chosen as the edible part mostly consumed in marine organisms, and thus hazardous if metal contamination is high. In the present study, any sample has metal concentrations above the food safety thresholds determined by the European commission regulation, i.e. 0.05 μg g -1 wet mass and 0.5 μg g -1 wet mass for Cd in fish and crustaceans respectively, 0.3 μg g -1 wet mass and 0.5 μg g -1 wet mass for Pb in fish and crustaceans respectively (EC, 2006). This corresponds to threshold values of 0.25 μg g -1 dry mass for Cd in fish and 2.5 μg g -1 dry mass for Cd in crustaceans, 1.5 μg g -1 dry mass for Pb in fish and 2.5 μg g -1 dry mass in crustaceans, if an empirical wet to dry conversion factor of 5 is used.

Within a given ecosystem, significant differences between species were observed (Fig. 2). In addition, the species-dependent pattern was similar between both ecosystems. The highest concentrations were observed for the crustacean N. norvegicus, especially for Ag, Cd, and Cu.

Among fish, the flatfish L. boscii had the highest mean Cu and Pb concentrations (despite high standard deviations), and the shark S. canicula had the highest Zn concentrations. These results are in agreement with [START_REF] Younis | Bioaccumulation of Heavy Metals in Fish, Squids and Crustaceans from the Red Sea, Jeddah Coast, Saudi Arabia[END_REF] who showed higher Cd, Cu, Pb and Zn concentrations in the muscle of 3 crustaceans such as the spiny lobster Panulirus penicillatus (same suborder that N. norvegicus) than in the muscle of 10 demersal fish species in the Red Sea. The high values of Cu and Zn for the studied crustacean appear driven by the specific metabolism of this taxon.

In crustaceans, Zn is a key component of many enzymes such as carbonic anhydrase, and Cu is a functional part of the respiratory protein hemocyanin [START_REF] Rainbow | Trace metal concentrations in aquatic invertebrates: why and so what?[END_REF]. As such, hemolymph, the circulatory liquid of crustaceans is Cu-rich and represents>50% of the Cu in crustaceans, being also fairly constant with the exception of starvation periods [START_REF] Depledge | Haemolymph protein composition and copper levels in decapod crustaceans[END_REF].

Similarly, high Zn values measured in the muscle of S. canicula are also consistent with previous studies. Indeed, authors showed experimentally that the bioaccumulation capacity of Zn from seawater was particularly pronounced in this shark species, resulting in a higher concentration factor for S. canicula than for the teleost turbot Psetta maxima, for instance [START_REF] Jeffree | Comparison of the bioaccumulation from seawater and depuration of heavy metals and radionuclides in the spotted dogfish Scyliorhinus canicula (Chondrichthys) and the turbot Psetta maxima (Actinopterygii: Teleostei)[END_REF][START_REF] Jeffree | Phylogenetic consistencies among chondrichthyan and teleost fishes in their bioaccumulation of multiple trace elements from seawater[END_REF]. However, the other chondrichthyan species G. melastomus presented lower Zn concentrations but higher Cu and Pb concentrations. Several studies have shown heterogeneity in metal bioaccumulation between chondrichthyan species (e.g. [START_REF] Glover | Concentrations of arsenic, selenium and ten heavy metals in school shark, Galeorhinus australis (Macleay), and gummy shark, Mustelus antarcticus (Günther), from South-eastern Australian Waters[END_REF][START_REF] Jeffree | Phylogenetic consistencies among chondrichthyan and teleost fishes in their bioaccumulation of multiple trace elements from seawater[END_REF].

Inferring such a pattern for G. melastomus may explain the differences observed between the two shark species, but actual pattern could only be explained by dedicated experiments. Thus, the peculiar metabolism of S. canicula regarding Zn may explain the high values measured excluding local contamination.

In this study, only 7 element concentrations have been modeled, i.e. Zn concentration for S.

canicula and H. dactylopterus and all element concentrations except Ni for N. norvegicus. The crustacean species presented a significant increase of Ag and Cd concentrations with increasing trophic level of the individuals and also a significant biomagnification of Cu that differ according to the ecosystem (significant interaction Ecosystem×Trophic level). In addition, a significant bioaccumulation was observed for Cu that differed also between ecosystems. In general for crustaceans, Cd is mainly taken up from water [START_REF] Rainbow | Phylogeny of trace metal accumulation in crustaceans[END_REF]. This bioaccumulation pattern has been described for different crustacean species such as the barnacle Elminius modestus and the shrimp Palaemon elegans. Thus Cd is bioaccumulated in a metabolically available form along life and is stored in a detoxified form without excretion process [START_REF] Rainbow | Trace metal concentrations in aquatic invertebrates: why and so what?[END_REF]. As such, the pattern observed in this study for Ag and Cd in N. norvegicus seems to follow this accumulation pattern. However, all relationships found in the present study must still be confirmed as it is not supported by a large number of samples. For all the fish species studied (both sharks and teleosts), there was no relationship between element concentrations and the total length and/or trophic level except for Zn concentrations in H. dactylopterus, which presented a significant decrease of Zn concentrations with increasing fish length. This likely suggests low bioaccumulation and trophic transfer of the studied trace metals for these high-trophic level consumers, and/or that their inputs over time are lower than the outputs through excretion, or than the dilution of metal burden with growth, for instance. The absence of relationship between fish size and trace element concentrations, whether essential or non-essential, has been indeed already described in previous studies [START_REF] Canli | The relationships between heavy metal (Cd, Cr, Cu, Fe, Pb, Zn) levels and the size of six Mediterranean fish species[END_REF]. The absence of relationships for the essential elements that are Cu and Zn in particular likely indicates that the body concentrations are regulated and maintained at a certain concentration [START_REF] Canli | The relationships between heavy metal (Cd, Cr, Cu, Fe, Pb, Zn) levels and the size of six Mediterranean fish species[END_REF][START_REF] Hornung | Trace metal content in deep-water sharks from the eastern Mediterranean Sea[END_REF]. However, a negative relationship was already observed [START_REF] Endo | Hg, Zn and Cu levels in the muscle and liver of tiger sharks (Galeocerdo cuvier) from the coast of Ishigaki Island, Japan: Relationship between metal concentrations and body length[END_REF], and may be explained by a higher metabolic activity in fast-growing young individuals, requiring higher amount of these elements than older individuals. Similarly, concentrations of non-essential elements such as Cd or Pb tend to decrease or to show non-significant trends with increasing fish size and/or trophic level, probably due to higher excretion rate and/or dilution of metal burden with growth, and lower metabolism [START_REF] Eisler | Chapter 3 -Fishes[END_REF]. In the present study, for both ecosystems, the fish size range represented through the sampling rather concerned fishes with large size (i.e. more adults than juveniles), which could explain the absence of this significant relationship due to the lack of juvenile fish in our sampling.

A similar absence of significant relationship was nevertheless recently observed for a Mediterranean scorpionfish over a larger size range, demonstrating that the pattern observed in the present study may not be so biased by the sample size (Ourgaud et al., 2018). Finally, biomagnification likely depends on the bioavailability of metals in prey or food sources, on the trace metal assimilation efficiency of predators, on food web complexity and on its species composition [START_REF] Wang | Interactions of trace metals and different marine food chains[END_REF]. Most trace metals (with the exception of cesium (Cs) and Hg) do not biomagnify along aquatic food webs [START_REF] Xu | Exposure and potential food chain transfer factor of Cd, Se and Zn in marine fish Lutjanus argentimaculatus[END_REF], which is supported here by the absence of relationship between element concentrations and trophic level. In addition, the pattern observed here in muscle tissues may be blurred by the preferential bioaccumulation of metals in liver or kidneys, as commented above.

For each species, mean concentrations of most of the trace metals studied were higher in the GoL than in the BoB (Table 3, Fig. 2). Moreover, this trend is confirmed by the TEPI values. All species combined, the TEPI values were higher in the GoL (1.13) than in the BoB (0.70), as it was the case for the majority of TEPI calculated when each species was considered separately (Table 2). This pattern is consistent with previous studies on trace metal contamination such as Hg [START_REF] Chouvelon | Oligotrophy as a major driver of mercury bioaccumulation in medium-to high-trophic level consumers: A marine ecosystem-comparative study[END_REF][START_REF] Cossa | The Mediterranean Mercury Anomaly, a Geochemical or a BiologocalIssue[END_REF][START_REF] Harmelin-Vivien | Difference of mercury bioaccumulation in red mullets from the north-western Mediterranean and Black seas[END_REF] but also Cd and Pb [START_REF] Damiano | Accumulation of heavy metals to assess the health status of swordfish in a comparative analysis of Mediterranean and Atlantic areas[END_REF]. In our study, only H. dactylopterus and N. norvegicus presented significant difference of metal concentrations between both locations (Table 3). For H.

dactylopterus, mean Zn a concentration was 18.7 ± 2.7 μg•g -1 in the GoL and was significantly higher than in the BoB, where the values were all below the quantification limit. Mean Ag concentration and Cu concentration for N. norvegicus were significantly higher in the GoL (1.060 ± 0.477 μg•g -1 and 61.9 ± 25.1 μg•g -1 , respectively) than in the BoB (0.512 ± 0.191 μg•g -1 and 12.6 ± 4.7 μg•g -1 , respectively). These results could be explained by three potential hypotheses.

First, differences of trace metal concentrations may result from higher sediment concentrations in the GoL than in the BoB. Bentho-demersal species as the ones used in the present study forage near or at the surface of the sediment, and may thus be influenced by chemical contamination in the sediment. Mean values measured in the sediment within the French network of chemical contamination monitoring ("ROCCh", ex "RNO") were found to be higher in the GoL (Cu=16.5 μg•g -1 , Ni=30μg•g -1 , Pb=31 μg•g -1 and Zn=78.5 μg•g -1 dry weight) than in the south of the BoB (the Landes and the Basque coasts: Ni=13.3 μg•g -1 , Cu=7.1 μg•g -1 , Pb=24.1 μg•g -1 and Zn=79.3 μg•g -1 dry weight), which might support this hypothesis. A similar pattern was observed in the present study for Cu and Ni in organisms (crustaceans and fish), with concentrations two times higher in the GoL than in the BoB. Secondly, this pattern of generally higher metal concentrations in individuals from the GoL than from the BoB is consistent with previous studies on other contaminants such as Hg, which highlighted the prevalent effect of oligotrophy, driving higher contamination levels, lower organisms' size and lower C and N stable isotope ratios in the Mediterranean (i.e. lower bio-dilution effect of contaminants in general through the food web) [START_REF] Chouvelon | Oligotrophy as a major driver of mercury bioaccumulation in medium-to high-trophic level consumers: A marine ecosystem-comparative study[END_REF][START_REF] Cossa | Influences of Bioavailability, Trophic Position, and Growth on Methylmercury in Hakes (Merluccius merluccius) from Northwestern Mediterranean and Northeastern Atlantic[END_REF][START_REF] Harmelin-Vivien | Difference of mercury bioaccumulation in red mullets from the north-western Mediterranean and Black seas[END_REF]. This is also observed in this study where the majority of metal concentrations were on average higher in the GoL than in the BoB, along with lower total lengths and trophic levels, likely revealing the crucial role of sources of organic matter and primary producers at the base of the food web in the bioaccumulation and trophic transfer of metals (for more details, see [START_REF] Chouvelon | Oligotrophy as a major driver of mercury bioaccumulation in medium-to high-trophic level consumers: A marine ecosystem-comparative study[END_REF].

Thirdly, an increasing number of studies have documented relatively high levels of trace metal contamination in shelf-edge or deep species (Cronin et al., 1998;[START_REF] Hornung | Trace metal content in deep-water sharks from the eastern Mediterranean Sea[END_REF], especially when compared to neritic species [START_REF] Chouvelon | Enhanced bioaccumulation of mercury in deep-sea fauna from the Bay of Biscay (north-east Atlantic) in relation to trophic positions identified by analysis of carbon and nitrogen stable isotopes[END_REF]. In this study, samples from the GoL were caught deeper than BoB samples. This may also explain the highest species contamination observed in the GoL by a potential effect of the depth on trace metal concentrations, where remineralization and/or transformation and modification of the bioavailability of scavenging elements may occur (e.g. for Hg, Heimbürger et al., 2010).

In summary, this study investigated and compared the concentrations of six trace metals in the muscle of seven species including one crustacean, two chondrichthyan and four teleost species between the Atlantic Ocean and the Mediterranean Sea. The highest concentrations were found in samples from the GoL (i.e. Mediterranean), confirmed by the use of a more global pollution index (TEPI). Although the difference is less marked than the pattern previously observed for Hg [START_REF] Chouvelon | Oligotrophy as a major driver of mercury bioaccumulation in medium-to high-trophic level consumers: A marine ecosystem-comparative study[END_REF], these results seem to confirm the peculiarity of the Mediterranean Sea concerning trace metals. Additionally, relationships between the concentrations of the six analyzed trace metals and total length or trophic level were never significant for fish, except one for one fish (Zn concentrations in H. dactylopterus). A significant increase of Ag, Cd and Cu concentrations in the muscle with increasing trophic level, and a significant decrease of Cu with increasing length were observed for the crustacean only. The absence of clear patterns may be largely due to the confounding effects of element integration, metabolic regulation and excretion mechanisms, more complex for these trace metals than for exclusive contaminants like Hg or Cs.

Further work based on the concentrations of these trace metals in the muscle, measured in other species for instance, would both support these results obtained and provide more data for a better understanding of metal bioaccumulation processes and transfers in food webs. In addition, in the present study, only species with a relatively high trophic level were considered, thus it would be also highly relevant to examine other species or compartments with lower trophic level such as plankton. 2. Mean metal concentrations taking account of values<LQ (two significant figures) with standard deviation (μg•g -1 dry mass) in the muscle of the studied species from the Gulf of Lions (GoL) and the Bay of Biscay (BoB). The Trace Element Pollution Index (TEPI) calculated from the same data set is also indicated.

  .05 and 15 μg•g -1 dry weight, respectively. Sample concentrations are given with two significant figures.Isotopic analyses were performed on a small subsample of grinded muscle with a Thermo Scientific Delta V Advantage mass spectrometer coupled to a Thermo Scientific Flash EA1112 elemental analyzer. The results are presented in the classical δ notation 13 C or 15 N and R the ratio between heavy and light isotopes. Standard is Pee Dee Belemnite for δ 13 C and atmospheric nitrogen for δ 15 N. Based on replicate measurements of internal laboratory standards, the experimental precision was<0.20‰ for both δ 13 C and δ 15 N. The trophic level of each individual i was calculated from isotope values, with the following formula, 𝑇𝐿 𝑖 = 𝑇𝐿 𝑏𝑎𝑠𝑒𝑙𝑖𝑛𝑒 + 𝛿 15 𝑁 𝑖 -𝛿 15 𝑁 𝑏𝑎𝑠𝑒𝑙𝑖𝑛𝑒 𝑇𝐸𝐹 TEF (Trophic Enrichment Factor, i.e. the δ 15 N change between two consecutive trophic levels)

<Figure 1 .

 1 Figure 1. Map of the sampling stations in the both ecosystems.

Figure 2 .

 2 Figure 2. Boxplot of metal concentrations taking account of values<LQ in the muscle (μg g -1 dry mass), according to the studied species and ecosystem (Bay of Biscay in black and Gulf of Lions in grey). The bottom and top of the box are the first and the third quartiles of the data distribution, the horizontal segment is the median, the whiskers represent the most extreme data point within 1•5 interquartile range and points are data points out of this range. Gm: Galeus melastomus; Sc: Scyliorhinus canicula; Hd: Helicolenus dactylopterus; Lp: Lepidorhombus boscii; Mp: Micromesistius poutassou; Pbl: Phycis blennoides; Nn: Nephrops norvegicus.

Table 1 .

 1 Sample description according to ecosystems and species, with the sample number (N), the sampling depth range, the total (fish) or cephalothorax (crustacean) length range , and the trophic level range. The number of pools included in the total sample number is indicated between brackets.

	Ecosystem & Species	N	Depth (m) Mean (min-max)	Length(mm) Mean (min-max)	Trophic level Mean (min-max)
	NW Mediterranean Sea (GoL)	94
	Chondrichthyan fishes
	Galeus melastomus	21 (6)	520±190 (284-816)	426±83 (265-558)	4.17±0.28 (3.77-4.82)
	Scyliorhinus canicula	10 (1)	470±163 (284-665)	473±56 (369-569)	4.25±0.11 (4.01-4.44)
	Teleost fishes
	Helicolenus dactylopterus	20 (7)	445±146 (284-679)	228±52 (141-309)	3.68±3.30 (3.3-4.02)
	Lepidorhombus boscii	6 (1)	355±72 (284-426)	258±43 (204-326)	3.40±0.08 (3.32-3.54)
	Micromesistius poutassou	12	379±67 (284-426)	256±29 (222 -311)	3.54±0.12 (3.35-3.77)
	Phycis blennoides	20 (5)	548±179 (284-816)	254±50 (196-377)	3.76±0.21 (3.06-4.01)
	Crustaceans
	Nephrops norvegicus	5 (2)	426±0 (426-426)	41±8 (30-50)	2.93±0.07 (2.84-3.03)
	NE Atlantic Ocean (BoB)	58
	Chondrichthyan fishes
	Galeus melastomus	12	289±109 (47-337)	606±72 (500-720)	4.35±0.14 (4.06-4.61)
	Scyliorhinus canicula	10	126.00±4 (122-130)	579±30 (530-630)	4.45±0.13 (4.25-4.66)
	Teleost fishes
	Helicolenus dactylopterus	5	492±0 (492-492)	370±20 (340-400)	4.09±0.06 (4.01-4.16)
	Lepidorhombus boscii	5	128±0 (128-128)	302±28 (260-340)	3.31±0.04 (3.25-3.36)
	Micromesistius poutassou	16	239±154 (109-511)	250±48 (200-330)	3.91±0.12 (3.65-4.12)
	Phycis blennoides	5	259±168 (124-461)	510±60 (440-580)	4.04±0.13 (3.9-4.2)
	Crustaceans
	Nephrops norvegicus	5	60±0 (60-60)	64±6 (57-74)	2.77±0.07 (2.65-2.85)

Table

Acknowledgments

Authors are particularly grateful to the "Agence de l'Eau Rhône Méditerranée Corse" for the financial support of RETROMED project that has allowed to finance Mediterranean analyses.

Analyses relative to the BoB area have been financed by the "Conseil Régional de Poitou-Charentes" and the "Contrat de projet Etat-Région" (CPER 2013). We would also like to thank J. Knoery from IFREMER, and P. Richard, G. Guillou and B. Lebreton from the LIENSs laboratory for trace metal and isotopic analyses, respectively. Thanks are also due to the chief scientists and the crew of the R/V "Thalassa" and "L'Europe" during EVHOE 2008 and MEDITS 2012 surveys as well as to F. Chavanon and C. Ravel for their help during sampling and laboratory work. The IUF (Institut Universitaire de France) is also acknowledged for its support to PB as a Senior Member.