Jérémy Lemaire
email: jeremy.ca.lemaire@gmail.com

François Brischoux

Oliver Marquis

Rosanna Mangione

Stéphane Caut

Maud Brault-Favrou

Carine Churlaud

Paco Bustamante

Lemaire et al. : Relationships between stable isotopes and trace element concentrations in the crocodilian community of French Guiana

Keywords: Caiman, Trophic ecology, Tropical ecosystem, Blood, Contaminant

Trace elements in the blood of crocodilians and the factors that influence their concentrations are overall poorly documented. However, determination of influencing factors is crucial to assess the relevance of caimans as bioindicators of environmental contamination, and potential toxicological impact of trace elements on these reptiles. In the present study, we determined the concentrations of 14 trace elements (Ag, As, Cd, Cr, Co, Cu, Fe, Hg, Pb, Mn, Ni, Se, V, and Zn) in the blood of four French Guiana caiman species (Spectacled Caiman Caiman crocodilus [n = 34], the Black Caiman Melanosuchus niger [n = 25], the Dwarf Caiman Paleosuchus palpebrosus [n = 5] and the Smooth-fronted Caiman Paleosuchus trigonatus [n = 20]) from 8 different sites, and further investigated the influence of individual body size and stable isotopes as proxies of foraging habitat and trophic position on trace element concentrations. Trophic position was identified to be an important factor influencing trace element concentrations in the four caiman species and explained interspecific variations.

These findings highlight the need to consider trophic ecology when crocodilians are used as bioindicators of trace element contamination in environmental studies.

INTRODUCTION

All over the globe, anthropogenic activities release a variety of contaminants into ecosystems [START_REF] Bard | Global transport of anthropogenic contaminants and the consequences for the Arctic marine ecosystem[END_REF][START_REF] Pacyna | An assessment of global and regional emissions of trace metals to the atmosphere from anthropogenic sources worldwide[END_REF][START_REF] Lewis | Fate and effects of anthropogenic chemicals in mangrove ecosystems: A review[END_REF][START_REF] Tkaczyk | Synthetic organic dyes as contaminants of the aquatic environment and their implications for ecosystems: A review[END_REF]. Industrial processes, mining activities and fossil fuel combustion mainly contribute to environmental contamination by releasing trace elements [START_REF] Pacyna | Current and future emissions of selected heavy metals to the atmosphere from anthropogenic sources in Europe[END_REF][START_REF] Pirrone | Global mercury emissions to the atmosphere from anthropogenic and natural sources[END_REF][START_REF] Vereda | Assessment of heavy metal pollution from anthropogenic activities and remediation strategies: A review[END_REF]. While trace elements are naturally present in the environment, their levels drastically increase due to human activities. In South America, trace elements are naturally present in high concentrations in the soils; mining activities and industrial processes have strongly increased, which results in massive discharges of trace elements such as cadmium (Cd), mercury (Hg), and lead (Pb) into the environment [START_REF] Smolders | Effects of mining activities on heavy metal concentrations in water, sediment, and macroinvertebrates in different reaches of the Pilcomayo River, South America[END_REF][START_REF] Guédron | Mercury speciation in a tropical soil association; Consequence of gold mining on Hg distribution in French Guiana[END_REF][START_REF] Burger | Mercury, lead, cadmium, cobalt, arsenic and aelenium in the blood of semipalmated sandpipers (Calidris pusilla) from Suriname, South America: Age-related differences in wintering site and comparisons with a stopover site in New Jersey, USA[END_REF]. The major concern linked to elevated concentrations of trace elements is related to their persistence and toxicity in different ecosystem compartments: trace elements, and particularly non-essential elements such as Hg and Pb, bioaccumulate in organisms, and Hg further biomagnifies through the trophic webs, exposing top predators to relatively high concentrations of this contaminant.

High trophic-level predators such as fish, birds, reptiles or mammals have successfully been used as bioindicator organisms to monitor trace element contamination in different habitats by using a variety of tissues that integrate contaminants over different periods of time, depending on their physiological role (Silva et al., 2018;Kalisińska, 2019;[START_REF] Albuquerque | Fish tissues for biomonitoring toxic and essential trace elements in the Lower Amazon[END_REF]Lemaire et al., 2021a). More recently, the use of non-lethal sampling methods (e.g., sampling of blood, hair, scales, claws and feathers) has been emphasised as a welcome effort to decrease potential impacts of sampling on wildlife [START_REF] Carravieri | Moulting patterns drive within-individual variations of stable isotopes and mercury in seabird body feathers: implications for monitoring of the marine environment[END_REF]Guillot et al., the crocodilian community of French Guiana 2018;[START_REF] Treu | Correlation between hair and tissue mercury concentrations in Icelandic artic foxes (Vulpes lagopus)[END_REF][START_REF] Lettoof | Snake scales record environmental metal(loid) contamination[END_REF]. Additionally, the use of wildlife species to monitor environmental contamination requires information on the influence factors. In most vertebrates, foraging habitats and trophic position are important factors influencing trace element bioaccumulation [START_REF] Croizier | Trophic ecology influence on metal Lemaire et al. : Relationships between stable isotopes and trace element concentrations in the crocodilian community of French Guiana bioaccumulation in marine fish: Inference from stable isotope and fatty acid analyses[END_REF][START_REF] Sebastiano | Trophic ecology drives contaminant concentrations within a tropical seabird community[END_REF]. Stable isotope ratio of nitrogen (𝛿 15 N) has become a standardised tool to determine the trophic position of an organism in the food web [START_REF] Post | Using stable isotopes to estimate trophic position: models, methods, and assumptions[END_REF][START_REF] Boecklen | On the use of stable isotopes in trophic ecology[END_REF]. Tissues of consumers are 15 Nenriched as a function of their diet, meaning that consumers at the top of the food web show higher 𝛿 15 N values [START_REF] Minagawa | Stepwise enrichment of 15 N along food chains: further evidence and the relation between 15 N and animal age[END_REF][START_REF] Peterson | Stable isotopes in ecosystem studies[END_REF]. In a complementary manner, the stable isotope ratio of carbon (𝛿 13 C) is intensively used to determine foraging habits of organisms, allowing to discriminate the preferential feeding habitats [START_REF] Post | Using stable isotopes to estimate trophic position: models, methods, and assumptions[END_REF].

Thus, the combined use of 𝛿 15 N and 𝛿 13 C offers a powerful tool to access an organisms' trophic ecology.

Crocodilians can be used to monitor the environmental contamination through trace elements [START_REF] Nilsen | Examining toxic trace element exposure in American alligators[END_REF]. They have a lifespan of several decades leading to long-term accumulation of contaminants. Furthermore, they have a comparatively low metabolic and tissue turn-over rates which increases the bioaccumulation of contaminants [START_REF] Campbell | Ecotoxicology of crocodilians[END_REF].

Consequently, trace elements such as Hg and Pb among others, accumulate in crocodilian tissues [START_REF] Jeffree | Element concentrations in the Flesh and Osteoderms of Estuarine crocodiles (Crocodylus porosus) from the Alligator River Region, Nothern Australia: Biotic and Geographic effects[END_REF][START_REF] Almli | Hepatic and renal concentration of 10 trace elements in crocodiles (Crocodylus niloticus) in the Kafue and Luangwa rivers in Zambia[END_REF][START_REF] Warner | Blood lead concentrations in freeranging Nile crocodiles (Crocodylus niloticus) from South Africa[END_REF][START_REF] Quintela | Arsenic, lead and cadmium concentrations in caudal crest of the yacare caiman (Caiman yacare) from Brazilian Pantanal[END_REF].

Among the factors which influence Hg contamination in crocodilians, body size appears important in some species [START_REF] Jagoe | Mercury in alligators (Alligator mississippiensis) in the southeastern United States[END_REF][START_REF] Schneider | Mercury concentration in the Spectacled caiman and Black caiman (Alligatoridae) of the Amazon: Implications for human health[END_REF][START_REF] Buenfil-Rojas | Metals and Metallothioneins in Morelet's crocodile (Crocodylus moreletii) from a transboundary river between Mexico and Belize[END_REF]. The trophic position in the food web appears also as a key factor (Lemaire et al., 2021b).

However, studies combining trace elements and trophic information are lacking though it is crucial to understand species-specific contamination. the crocodilian community of French Guiana The present study considers the four caiman species living in French Guiana, the Spectacled Caiman Caiman crocodilus, the Black Caiman Melanosuchus niger, the Dwarf Caiman Paleosuchus palpebrosus and the Smooth-fronted Caiman Paleosuchus trigonatus to investigate trace element contamination at a large spatial scale in the region. We determined the concentrations of 14 trace elements in caiman blood as blood concentrations of many trace elements are good predictors of other tissues such as muscles or liver, and as blood is a dynamic matrix which reflect circulating trace elements between other tissues [START_REF] Eggins | Mercury concentrations in different tissues of turtle and caiman species from the Rio Purus, Amazonas, Brazil[END_REF][START_REF] Nilsen | Trace element biodistribution in the American alligator (Alligator mississippiensis)[END_REF]. We further investigated the influence of the body size and of the trophic ecology on trace element concentrations using stable isotopes of carbon and nitrogen as proxies.

MATERIAL AND METHODS

Sample collection

84 individuals of four caiman species were captured at 8 different sites in French Guiana (Fig. 1). Spectacled Caimans (Caiman crocodilus, n = 34) were captured at the "Pripris de Yiyi" (n = 18) and "Kaw river" (n = 16) sites, Black Caimans (Melanosuchus niger, n = 25) were captured at the "Mare Agami" site, Dwarf Caimans (Paleosuchus palpebrosus, n = 5) were captured at "Pripis de yiyi" (n = 2), "Matoury" (n = 1) and "Mana" (n = 2) sites, and Smooth-fronted Caimans (Paleosuchus trigonatus, n = 20) were captured at the sites "Nouragues station" (n = 15), "Mont Grand Matoury" (n = 4) and "French Guiana space centre" (n = 1) (Fig. 1).

Total length (TL) of all individuals was measured ventrally, and body mass was recorded. We drew blood samples (0.2 -3 mL) either through occipital venous sinus puncture, using a syringe with a 30 gauge -50 mm heparinized needle (heparin sodium), or through the lateral the crocodilian community of French Guiana tail vein with a 27 gauge -25 mm or 21 gauge -50 mm heparinized needle (heparin sodium), depending on the size of the animal. Blood samples were immediately stored at 4°C and further kept at -21°C. Whole blood was freeze-dried for 48 hours to eliminate water and then ground into a homogeneous powder before further analysis.

All individuals were released at the place of capture immediately after sampling. Capture and sample collection were performed under permits from French authorities (Direction Régionale des Territoires et de la Mer) after evaluation by the CSRPN, the regional scientific committee (Permit: N°155/DEAL/2013, N°2014114-006, N°2014114-007, N°2015034-008, R03-2019-01-09-001, R03-2019-10-24-007).

Trace element analyses

Total mercury (THg) was quantified using an atomic absorption spectrometer AMA-254 (Advanced Mercury Analyser-254; Altec®). Two replicates of 0.5 -3.0 mg dry weight (dw) were analysed for each sample. Reproducibility for duplicate samples was approved when the relative standard deviation (RSD) was below 10%. The method was validated by the analysis of certified reference material (CRM) TORT-3 (Lobster hepatopancreas from the National Research Council of Canada (NRCC); certified Hg concentration: 0.292 ± 0.022 µg.g -1 dw) at the beginning and the end of the analytical cycle and after every 5 samples. Measured values for TORT-3 were 0.292 ± 0.006 µg.g -1 dw (n = 20), with a recovery of 99.85 ± 2.13 %. Blanks were included at the beginning of each analytical run and the limit of quantification of the AMA was 0.05 ng Hg. Silver (Ag), arsenic (As), cadmium (Cd), chromium (Cr), cobalt (Co), copper (Cu), iron (Fe), manganese (Mn), nickel (Ni), lead (Pb), selenium (Se), vanadium (V) and zinc (Zn) were determined using Inductively Coupled Plasma (ICP) Optical Emission Spectrometry (Varian the crocodilian community of French Guiana Vista-Pro ICP-OES) and Mass Spectrometry (Series II Thermo Fisher Scientific ICP-MS) on mineralized aliquots (mass: 5 -150 mg dw) as described in [START_REF] Bustamante | Metal and metalloid concentrations in the giant squid Architeuthis dux from Iberian waters[END_REF]. Aliquots were microwave-digested in a mixture of 6 mL 65% HNO3 (VWR Quality SUPRAPUR) and 2 mL 30% HCl (VWR Quality SUPRAPUR), except for samples with a weight below 100 mg where volumes of HNO3 and HCl were divided by half. Samples were then diluted to 50 mL (25 mL for samples with a weight below 100 mg) with ultrapure water. To avoid trace element contamination, all utensils used were soaked in a bath of diluted nitric acid for 48h, rinsed with ultrapure water, and dried. Two CRM (DOLT-3, Dogfish liver, NRCC, and TORT-2, Lobster hepatopancreas, NRCC) were treated and analysed in the same way as the samples. Results were in agreement with the certified values and displayed recoveries ranging from 88% to 116% (n = 10), proving repeatability of the method. All trace element concentrations are presented in µg.g -1 dw.

Isotope analysis

Except for M. niger stable isotopes, which were analysed as described in [START_REF] Caut | The dark side of the black caiman: Shedding light on species dietary ecology and movement in Agami Pond, French Guiana[END_REF], all nitrogen and carbon stable isotopes were determined in freeze-dried whole blood (aliquots mass: ~0.3mg) with a continuous flow mass spectrometer (Thermo Scientific Delta V Advantage) coupled to an elemental analyser (Thermo Scientific Flash EA1112). Results are presented in the usual 𝛿 notation relative to the deviation from standards (Pee Dee Belemnite for 𝛿 13 C and atmospheric nitrogen for 𝛿 15 N), in parts per thousand (‰) following the formula

𝛿 15 N or 𝛿 13 C = [(Rsample/Rstandard)-1]x1000, where R is 15 N/ 14 N or 13 C/ 12 C for 𝛿 15 N or 𝛿 13 C.
Replicate assays of internal laboratory standards (n = 56) indicated maximum measurement errors of ± 0.14 ‰ for nitrogen, and ± 0.18 ‰ for carbon isotope measurements. the crocodilian community of French Guiana

Statistical analysis

All statistical analyses were performed using the Software R v.3.6.1 (R core Team, 2019).

Statistical analyses were only performed on trace elements with concentrations above the limit of quantification (LOQ) in a minimum of 70% of individuals. All data were checked for normality and homogeneity of variances and log-transformed if necessary. Differences in body size between species were assessed by ANOVA. Differences in isotope composition and trace element concentrations between species were performed by ANOVAs or ANCOVAs with body size as a cofactor. Then post-hoc Tukey's honestly significant difference (HSD) was applied to evaluate the variation of contaminant and isotope values between species. Relationships between trace elements, isotopes and body size were performed by general regression models (simple or polynomial). Principal component analysis (PCA) was performed on logtransformed trace elements to detect covariance and the contaminants that reflect most of the total variance. Generalised linear models (GLM) were used to test relation of feeding ecology (using stable isotopes), sites and species on trace element concentrations. Forward selection using Akaike's Information Criterion (AICc) was applied, and the effect of variables affecting contaminants was inferred through Akaike's weights and R 2 adjusted.

RESULTS

Trace element concentrations

Among the 14 targeted trace elements, only the essential elements Cu, Fe, Mn, Se and Zn, and non-essential elements Hg and Pb were detected in the blood for more than 70% of individuals (Table S1). Ag and V always remained below the LOQ and As, Cd, Co, Cr, and Ni were only detected in few individuals (Table S1). Arsenic was exclusively detected in C. crocodilus (0.34 ± 0.15 µg.g -1 dw) which were captured in the "Kaw river" estuary, and in a single subadult M. the crocodilian community of French Guiana niger (0.24 µg.g -1 dw) from "Mare Agami" (Table S1). Additionally, Cr was found in P. trigonatus (0.34 ± 0.01 µg.g -1 dw) and C. crocodilus (0.36 ± 0.14 µg.g -1 dw). Apart from Fe concentrations, all trace element concentrations varied significantly between species (ANOVAs and ANCOVAs, all p < 0.05, Table 1). P. trigonatus showed the highest concentrations for Cu (8.32 ± 5.41 µg.g -1 dw), Mn (0.34 ± 0.26 µg.g -1 dw), Se (4.05 ± 1.43 µg.g -1 dw) and Zn (68.9 ± 53.6 µg.g -1 dw). The highest Hg concentrations were found in M. niger (1.56 ± 0.65 µg.g -1 dw) while P. palpebrosus showed the highest concentrations of Pb (1.35 ± 1.43 µg.g -1 dw, Table 1).

The Se:Hg molar ratio differed between species with the highest values for P.trigonatus (31.42 ± 14.28, Table 1).

Relationships with isotope values and sites

The PCA analysis which included Cu, Fe, Hg, Mn, Pb, Se, and Zn, explaining 60,8% of the total variance, revealed strong species segregation in the ordination space (Fig. 4). Fe and Hg were not strongly associated with other trace elements. Most parsimonious GLM models selected by AICc showed that the species and the 𝛿 15 N values explained most of the total variation in Cu, Fe, Hg, and Zn concentrations in the four caiman species while the sites and the species explained Pb concentrations and finally the sites and the 𝛿 13 C values explained Se concentrations (Table 2).

𝛿 15 N and 𝛿 13 C values varied significantly between species (ANCOVAs, all p < 0.05, Table 1); P. trigonatus showed the highest values for 𝛿 15 N (8.28 ± 0.94 ‰) and 𝛿 13 C (-26.38 ± 1.21 ‰).

The relationship between the 𝛿 15 N value and body size was positive for P. palpebrosus and C. crocodilus (R 2 = 0.781, p = 0.047 and R 2 = 0.364, p < 0.001, respectively). For 𝛿 13 C, a positive relationship with body size was only found for C. crocodilus (R 2 = 0.128, p = 0.038, Table S2). the crocodilian community of French Guiana In this species, positive relationships were found between 𝛿 13 C values and the concentrations of Mn (R 2 = 0.217, p = 0.005) and Pb (R 2 = 0.535, p < 0.001), and a negative relationship for Zn (R 2 = 0.142, p = 0.028). C. crocodilus also had negative relationships between 𝛿 15 N values and the concentrations of Cu (R 2 = 0.129, p = 0.037), Se (R 2 = 0.304, p = 0.011), and Zn (R 2 = 0.225, p = 0.005), but 𝛿 15 N values correlated positively with Mn (R 2 = 0.263, p = 0.002).

For P. trigonatus, positive relationships were found between 𝛿 13 C and 𝛿 15 N with Hg and Se concentrations (respectively R 2 = 0.229, p = 0.033 and R 2 = 0.611, p < 0.001 (Fig. 3), and R 2 = 0.290, p = 0.038 and R 2 = 0.289, p = 0.039).

For P. palpebrosus, a negative relationship was found between the 𝛿 13 C value and Cu (R 2 = 0.880, p = 0.018), a positive relationship for Hg concentration (R 2 = 0.978, p = 0.002), and a positive relationship between the 𝛿 15 N and Hg (R 2 = 0.877, p = 0.019; Fig. 3), and negative for Cu (R 2 = 0.920, p = 0.010) and Mn (R 2 = 0.935, p = 0.007).

For M. niger, a negative relationship was found between the 𝛿 15 N values and Cu concentrations (R 2 = 0.171, p = 0.040), and positive relationship for Hg (R 2 = 0.391, p < 0.001; Fig. 3).

Relationship with body size

The relationship between Hg concentration and body size was positive for P. trigonatus (R 2 = 0.353, p = 0.006) and M. niger (R 2 = 0.628, p < 0.001) (Fig. 2), and marginally positive for P. palpebrosus (R 2 = 0.768, p = 0.051) and C. crocodilus (R 2 = 0.112, p = 0.053) (Table S2).

Selenium concentrations showed a negative relationship with the body size of P. trigonatus (R 2 = 0.639, p < 0.001). In M. niger, Fe and Mn concentrations showed negative relationships with body size (respectively, R 2 = 0.229, p = 0.016 and R 2 = 0.180, p = 0.034). The relationship the crocodilian community of French Guiana between the Se:Hg molar ratio and the body size was negative for P. trigonatus (R 2 = 0.306, p = 0.042), C. crocodilus (R 2 = 0.162, p = 0.033) and M. niger (R 2 = 0.648, p < 0.001).

DISCUSSION

The present study is the first to investigate trace element concentrations and their relationship to body size and stable isotopes as proxies of foraging habitat and trophic position, in the crocodilian community of French Guiana. Our results showed that trophic ecology influence trace element concentrations, an aspect which needs to be evaluated when caimans are used as bioindicators.

Trace element concentrations

In the present study, Hg was detected in all samples, which indicates that all caiman species in French Guiana are under Hg contamination. This finding is not surprising considering that Hg is a widespread environmental contaminant that affects ecosystems worldwide (Chen et al., 2018). Additionally, forest soils of the area are known to present high natural Hg concentrations, with an average of 0.3 µg.g -1 dw [START_REF] Richard | Mercury levels of soils, sediments and fish in French Guiana, South America[END_REF]. In the present study, C. crocodilus presents a higher average blood Hg concentration than individuals of similar body size from Brazil and Colombia [START_REF] Eggins | Mercury concentrations in different tissues of turtle and caiman species from the Rio Purus, Amazonas, Brazil[END_REF]Marrugo-Negrete et al., 2019; Table 3).

Blood Hg concentrations in M. niger from French Guiana are also higher than reported values from Brazil [START_REF] Eggins | Mercury concentrations in different tissues of turtle and caiman species from the Rio Purus, Amazonas, Brazil[END_REF]Table 3). We cannot perform comparison regarding blood Hg concentration in P. trigonatus and P. palpebrosus as there is no published data for the Paleosuchus genus besides French Guiana. Mercury is well known for its ability to biomagnify through the food web and to bioaccumulate in top predators with age due to a low excretion rate [START_REF] Lavoie | Biomagnification of mercury in aquatic food webs: A worldwide meta-analysis[END_REF]. Elevated concentrations found in caimans from French Guiana, in the crocodilian community of French Guiana comparison to other studies, might be caused by high environmental Hg concentrations in the associated food chain, and/or are the result of a long trophic chain that can increase the biomagnification process [START_REF] De Almeida Rodrigues | Mercury in aquatic fauna contamination: a systematic review on its dynamics and potential health risks[END_REF].

Lead was found in more than 90% of samples with the highest values in P. palpebrosus (1.35 ± 1.43 µg.g -1 dw) followed by P. trigonatus (0.58 ± 0.77 µg.g -1 dw). The lack of data on Pb concentrations in the blood of crocodilians does not allow for a thorough comparison.

However, the relatively high Pb concentrations we have found in P. trigonatus are concerning because the study sites seems undisturbed by any known anthropogenic activities.

Nevertheless, Pb concentrations were found in other caiman species, and our results have identified that both the sites and the species were related to Pb variation (Table 2). In French Guiana, the background levels of Pb in soil have recently been identified as elevated with very high values in some areas without any apparent relation to anthropogenic activities (S. Guédron, personal communication). The geological background appears to be the main explanation for Pb found in the blood of P. trigonatus, which may thus reflect a natural contamination. Additionally, for P. palpebrosus, such natural contamination may have been affected by anthropogenic activities at some of our study sites (i.e., French Guiana international airport and rice cultivation at "Mana", Sites #5 and #1 in Fig. 1) because of the use of specific products containing Pb (e.g., pesticides, herbicides, [START_REF] Defarge | Toxicity of formulants and heavy metals in glyphosate-based herbicides and other pesticides[END_REF].

Natural Pb contamination was already reported in the Nile Crocodile Crocodilus niloticus from a pristine environment without any known anthropogenic Pb sources, with concentrations of Pb and size of individuals being both higher than in the present study (Warner et al., 2016, Table 3). Pb concentrations, which appear to originate from natural sources, may be of concern as the metal is an extremely toxic element for human and wildlife that affects the crocodilian community of French Guiana reproductive systems, renal and hepatic functions, and endocrine processes [START_REF] Wani | Lead toxicity: a review[END_REF][START_REF] Pain | Effects of lead from ammunition on birds and other wildlife: A review and update[END_REF], Lemaire et al., 2021c). As crocodiles efficiently assimilate Pb [START_REF] Hammerton | Experimental study of blood lead kinetics in Estuarine Crocodiles (Crocodylus porosus) exposed to ingested lead shot[END_REF], they may suffer Pb poisoning. Future studies are required to determine the source of this contamination in French Guiana by using Pb stable isotopes and to further investigate the consequences of this toxic element in crocodilians.

In the present study, Se concentrations were quite different between species, with P. trigonatus and P. palpebrosus having higher Se concentrations than C. crocodilus and M. niger (Table 1). While Se is an essential trace element involved in the metabolism of living organisms [START_REF] Kieliszek | Current knowledge on the importance of selenium in food for living organisms: A review[END_REF], it negatively affects body condition in the American alligator, Alligator mississippiensis [START_REF] Finger | Dietary selenomethionine administration in the American Alligator (Alligator mississippiensis): hepatic and renal Se accumulation and its effects on the growth and body condition[END_REF]. However, Se by its antagonist interaction with Hg can play a major role in the reduction of Hg toxicity in vertebrates (Rahman et al., 2019[START_REF] Manceau | In vivo formation of HgSe nanoparticles and Hg-tetraselenolate complex from methylmercury in seabirds-Implications for Hg-Se antagonism[END_REF]. In this order, the Se:Hg molar ratio is important to inform on potential capacities to protect an organism from Hg toxicity. P. trigonatus showed the highest Se:Hg molar ratio compared to the other caiman species in French Guiana (Table 1). Sources of Se are closely related to the diet [START_REF] Rayman | Food-chain selenium and human health: emphasis on intake[END_REF], suggesting that trophic ecology can influence variation of Se concentration. The diet of P. trigonatus, which is composed of terrestrial prey, can explain differences with the other species, which mainly forage in aquatic habitats (see below; [START_REF] Magnusson | Diets of Amazonian Crocodilians[END_REF][START_REF] Villamarín | Opportunistic top predators partition food resources in a tropical freshwater ecosystem[END_REF].

Among the essential trace elements, only Fe concentrations were in the same range for all species while Cu, Mn and Zn concentrations were different between the species (Table 1). The lack of variation for Fe relates to its key role in haemoglobin, suggesting that Fe regulation is the crocodilian community of French Guiana comparable between species. As the according data is limited in crocodilians, we cannot make a robust comparison.

Influence of body size

Hg was the only non-essential trace element that showed a positive relationship with body size for all species. Crocodilians as most ectothermic vertebrates have an indeterminate growth, and consequently, age and size are generally correlated (i.e., [START_REF] Campos | Growth rates of Paleosuchus palpebrosus at the southern limit of its range[END_REF][START_REF] Eaton | Estimating age from capture data: integrating incremental growth measures with ancillary data to infer age-at-length[END_REF]. This highlights that larger -presumably older -caimans present higher Hg blood concentrations than smaller -hence younger -ones. Indeed, the positive relationship between Hg concentration and body size of M. niger is in accordance with the study from [START_REF] Eggins | Mercury concentrations in different tissues of turtle and caiman species from the Rio Purus, Amazonas, Brazil[END_REF] in Brazil, though contrasts with previous studies for C. crocodilus in Brazil and Colombia, where Hg concentrations and body size were not correlated [START_REF] Eggins | Mercury concentrations in different tissues of turtle and caiman species from the Rio Purus, Amazonas, Brazil[END_REF]Marrugo-Negrete et al., 2019). In most vertebrates, Hg blood concentration is considered to represent a relatively recent contamination [START_REF] Monteiro | Kinetics, dose-response, excretion, and toxicity of methylmercury in free-living cory's shearwater chicks[END_REF][START_REF] Fournier | The oral bioavailablility and toxicokinetics of methylmercury in common loon (Gavia immer) chicks[END_REF][START_REF] Schneider | An evaluation of the use of reptile dermal scutes as a non-invasive method to monitor mercury concentrations in the environment[END_REF] that reflects variations in the environmental Hg contamination or modifications in the origin of prey. Such variations may explain the non-consistence in the relationship between body size and Hg contamination in some cases. However, the consistence of the relationships in our results can be related to a constant environmental Hg contamination and/or a stability in the origin of prey in the examined caiman populations.

Additionally, our results show a negative relationship between the Se:Hg molar ratio and body size of C. crocodilus, M. niger and P. trigonatus. As previously discussed, body size is generally related to age in crocodilians which leads to an accumulation of Hg over time in the tissues. A Lemaire et al. : Relationships between stable isotopes and trace element concentrations in the crocodilian community of French Guiana decrease in the Se:Hg molar ratio can indicate that dietary change between juveniles and adults leads to lower Se concentrations in the prey of adults. Additionally, this decrease can be explained by the increase of Hg contamination, which needs to be detoxified to cope with its toxic effect. The main detoxification process in vertebrates involves Se for demethylation of MeHg, and its co-precipitation with Se to form tiemannite nanoparticles in the liver, but also in other tissues and organs such as muscles, kidneys and brain [START_REF] Korbas | The chemical nature of mercury in human brain following poisoning or environmental exposure[END_REF][START_REF] Manceau | In vivo formation of HgSe nanoparticles and Hg-tetraselenolate complex from methylmercury in seabirds-Implications for Hg-Se antagonism[END_REF][START_REF] Renedo | Mercury isotopes of key tissues document mercury metabolic processes in seabirds[END_REF]. Demethylation requires a substantial amount of Se and leads to a depletion of the available Se for the organism [START_REF] Manceau | In vivo formation of HgSe nanoparticles and Hg-tetraselenolate complex from methylmercury in seabirds-Implications for Hg-Se antagonism[END_REF]. The levels of the different physiochemical forms of Hg and their relation to the Se proteins deserve further studies to understand the consequences of Se:Hg molar ratio variation in blood, and potential implications on physiological processes and health status of caimans.

Concerning other trace elements, Fe and Mn concentrations negatively related to the body size of M. niger only. Mn bioconcentrates significantly in the aquatic ecosystem at low trophic levels [START_REF] Briand | Tracking trace elements into complex coral reed trophic networks[END_REF], and the change in diet between juveniles and adults may lead to such a decrease of Mn concentrations in adults compared to juveniles.

Influence of trophic ecology

Our results show that variations of Hg concentrations were explained by the species and the 𝛿 15 N (Table 2), which is a proxy of consumers' trophic position [START_REF] Post | Using stable isotopes to estimate trophic position: models, methods, and assumptions[END_REF]. Additionally, the increase of blood Hg concentrations with 𝛿 15 N for P. trigonatus, P. palpebrosus and M. niger reflects the biomagnification process which is already described in several aquatic and terrestrial ecosystems [START_REF] Cristol | The movement of aquatic mercury through terrestrial food webs[END_REF][START_REF] Rimmer | Mercury bioaccumulation and trophic transfer in the terrestrial food web of a montane forest[END_REF][START_REF] Lavoie | Biomagnification of mercury in aquatic food webs: A worldwide meta-analysis[END_REF].

Differences in 𝛿 15 N values between species can result from the interspecific competition which leads to a different trophic ecology of the four caiman species [START_REF] Magnusson | Diets of Amazonian Crocodilians[END_REF]Lemaire et al. : Relationships between stable isotopes and trace element concentrations in the crocodilian community of French Guiana [START_REF] Moldowan | Diet, gastric parasitism, and injuries of caimans (Caiman, Melanosuchus, and Paleosuchus) in the Peruvian Amazon[END_REF][START_REF] Villamarín | Opportunistic top predators partition food resources in a tropical freshwater ecosystem[END_REF] (Fig. 5). Our results highlight that trophic levels assessed via the 𝛿 15 N values are key factors driving Hg contamination in caimans. Elevated Hg concentrations are particularly concerning regarding the deleterious effects of this metal as already reported in several taxa, such as reproduction impairment and alteration of physiological functions [START_REF] Day | Relationship of blood mercury levels to health parameters in the Loggerhead Sea Turtle (Caretta caretta)[END_REF][START_REF] Evers | The Effects of Methylmercury on Wildlife: A Comprehensive Review and Approach for Interpretation[END_REF][START_REF] Morcillo | Mercury and its toxic effects on fish[END_REF]. Thus, the trophic level is an important factor that should be considered when mercury contamination and its toxicity are assessed in caimans.

In contrast to Hg, Se concentrations were mostly explained by the sites and the 𝛿 13 C. As 𝛿 13 C does not vary significantly across the trophic chain and depends on the primary sources, it informs on the foraging habitat. The diet of P. trigonatus consists of more than 50% of terrestrial animals such as snakes, rodents, monkeys and other herbivorous animals; their consumption of plants, fruits and prey that are rich in Se can explain high Se concentrations in the blood of this species [START_REF] Magnusson | Diets of Amazonian Crocodilians[END_REF][START_REF] Ortiz | Paleosuchus trigonatus (Schneider's Smooth-fronted Caiman) Prey[END_REF][START_REF] Moldowan | Diet, gastric parasitism, and injuries of caimans (Caiman, Melanosuchus, and Paleosuchus) in the Peruvian Amazon[END_REF][START_REF] Villamarín | Opportunistic top predators partition food resources in a tropical freshwater ecosystem[END_REF][START_REF] Mangione | Paleosuchus trigonatus (Smooth-fronted Caiman) Predation[END_REF]. In this respect, the caimans which consume terrestrial prey may have an advantage in the Hg detoxification as discussed before, due to an elevated intake of Se.

Most of the observed trace element variations were explained by the species, and the 𝛿 13 C for Mn, and the 𝛿 15 N for Cu and Zn. Caimans with a diet composed of 𝛿 13 C-enriched prey were consequently more likely to have high Mn concentrations. The trophic level of individuals influences Cu and Zn concentrations. These results highlight that the feeding preferences of caimans drive most trace elements, as it was already demonstrated in other wild vertebrates the crocodilian community of French Guiana such as fish, seabirds or mammals [START_REF] Lahaye | Biological and ecological factors related to trace element levels in harbour porpoise (Phocoena phocoena) from European waters[END_REF][START_REF] Bodin | Trace elements in oceanic pelagic communities in the western Indian Ocean[END_REF][START_REF] Carravieri | Trace elements and persistent organic pollutants in chicks of 13 seabird species from Antarctica to the subtropics[END_REF].

CONCLUSION

In the present study, trophic position was identified to influence trace elements in the four caiman species and explains the interspecific concentrations, which highlights the necessity to evaluate trophic ecology when crocodilians are used as bioindicators of trace element contaminations in environmental studies. Additionally, Hg and Pb concentrations are relatively high in comparison to concentrations already reported in other studies and are concerning regarding their toxic effects on wildlife. In the genius Paleosuchus sp., Pb needs particular attention in French Guiana because of the high concentrations found in the blood of caimans at some sites. Lastly, the interspecific variability of the Se:Hg molar ratio deserves future study to understand the potential impact regarding protective effect against Hg and the role play by the diet. a Original data reported in wet weight, transformed in dry weight using a factor of 3.6 according to Lemaire et al., (2021a). Yiyi"; 3: "French Guiana space centre"; 4: "Mont-Grand Matoury"; 5: "Matoury"; 6: "Mare Agami"; 7: "Kaw river"; 8: "Nouragues station".

Species

Figure 1 .

 1 Figure 1. Geographic location of the 8 study sites in French Guiana and distribution of captured caiman species. Sites are 1: "Mana"; 2: "Pripris de

Figure 2 .Figure 4 .

 24 Figure 2. Relationships between body size (total length in cm) and Hg concentration (µg.g -1 dw) in the whole blood of the Black Caiman,

Table 1 .

 1 Biometric data, trace element (µg.g -1 dw), 𝛿 15 N and 𝛿 13 C (‰) values in the whole blood of the Spectacled Caiman (Caiman crocodilus), the Black Caiman (Melanosuchus niger), the Dwarf Caiman (Paleosuchus palpebrosus) the Smooth-fronted Caiman (Paleosuchus trigonatus) in French Guiana. N = number of individualsLemaire et al. : Relationships between stable isotopes and trace element concentrations in the crocodilian community of French Guiana

			Caiman crocodilus		Melanosuchus niger		Paleosuchus palpebrosus		Paleosuchus trigonatus
		N	Mean ± SD	Min / Max	N	Mean ± SD	Min / Max	N	Mean ± SD	Min / Max	N	Mean ± SD	Min / Max
	Total length (cm)	34	72.3 ± 24.7	40.6 / 176.0	25	176.4 ± 72.2	71.0 / 326.0	5	75.3 ± 44.6	35.5 / 150.0	20	82.8 ± 32.7	27.0 / 143.0
	Weight (g)	34	2154 ± 5556	173 / 33200	17	10612 ± 19559	970 /85000	4	1095 ± 1046	130 / 2500	18	3875 ± 3779	305 / 13300
	𝛿 15 N	34	6.02 ± 0.96 a	4.12 / 8.23	25	6.98 ± 0.62 b	4.93 / 7.88	5	7.30 ± 0.86 b,c	6.36 / 8.44	20	8.28 ± 0.94 c	6.10 / 9.20
	𝛿 13 C	34	̶ 27.14 ± 2.46 a,b	̶ ̶ 30.72 / ̶ ̶ 21.84	25	̶ 27.58 ± 0.74 a	̶ 29.45 / ̶ ̶ 26.36	5	̶ 27.75 ± 2.10 a	̶ 30.28 / ̶ ̶ 25.11	20	̶ 26.38 ± 1.21 b	̶ 26.20 / -̶ 23.9
	Hg	34	0.61 ± 0.39 a	0.09 / 1.53	25	1.56 ± 0.65 b	0.54 / 2.89	5	1.50 ± 1.18 b	0.54 / 3.42	20	0.35 ± 0.15 c	0.10 / 0.70
	Se	28	1.36 ± 0.28 a	0.76 / 1.92	24	1.14 ± 0.15 b	0.85 / 1.47	3	3.53 ± 1.60 c	2.45 / 5.37	15	4.05 ± 1.43 c	2.30 / 7.90
	Se:Hg (molar)	28	7.59 ± 5.27 a	1.45 / 21.54	24	2.38 ± 1.41 a,b	1.04 / 6.00	3	6.26 ± 6.01 b	1.82 / 13.09	15	31.42 ± 14.28 c	9.80 / 62.30
	Cu	34	5.95 ± 4.86 a	2.99 / 31.07	25	3.61 ± 0.68 b	2.60 / 5.21	5	6.24 ± 2.44 b,c	3.24 / 7.97	20	8.32 ± 5.41 c	4.20 / 23.10
	Fe	34	1520 ± 167 a	1201/ 1972	25	1519 ± 156 a	1281 / 1896	5	1353 ± 131 a	1172 / 1495	20	1476 ± 237 a	867 / 1856
	Mn	34	0.23 ± 0.09 a,b	0.13 / 0.52	25	0.10 ± 0.06 c	0.05 / 0.34	5	0.18 ± 0.08 b	0.09 / 0.30	19	0.34 ± 0.26 c	0.10 / 1.30
	Pb	32	0.12 ± 0.08 a	0.03 / 0.38	23	0.06 ± 0.09 b	0.02 / 0.44	5	1.35 ± 1.43 c	0.07 / 3.67	19	0.58 ± 0.77 c	0.10 / 2.70
	Zn	34	30.6 ± 8.5 a	18.5 / 51.1	25	24.7 ± 3.9 a	19.2 / 31.7	5	56.2 ± 20.9 b	38.9 / 90.8	20	68.9 ± 53.6 b	26.2 / 184.0
	Letters indicate significant differences (isotopes and trace elements: Tukey's HSD with total length in cofactor, all p < 0.05)

Table 2 .

 2 AICc model ranking of selected trace element concentrations in blood of the four French Guiana caiman species. k = number of parameters, AICc = Akaike's Information Criteria, wi = AICc weights, gdf = goodness of fit.Lemaire et al. : Relationships between stable isotopes and trace element concentrations in the crocodilian community of French Guiana

	Models	k	AICc	∆AICc	wi	R² adj	gdf
	Hg, GLM, Log-transformed
	Species + 𝛿 15 N + Species:𝛿 15 N 9	152.14	0.00	0.71	0.56	1
		Species + 𝛿 15 N 6	154.40	2.27	0.23	0.53	1
	Species + 𝛿 13 C + Species:𝛿 13 C 9	158.12	5.99	0.04	0.52	1
	Se, GLM, Log-transformed
	Sites + 𝛿 13 C + Sites:𝛿 13 C 13	-23.25	0.00	0.39	0.88	1
		Sites + 𝛿 13 C 9	-22.78	0.46	0.31	0.87	1
		Sites + Species 9	-20.41	2.84	0.09	0.87	1
	Pb, GLM, Log-transformed
		Sites + Species 10	197.04	0.00	0.48	0.58	1
	Sites + Species + Sites:Species 10	197.04	0.00	0.48	0.58	1
	Species + 𝛿 13 C + Species:𝛿 13 C 9	202.51	5.47	0.03	0.54	0.98
	Zn, GLM, Log-transformed
		Species + 𝛿 15 N 6	106.82	0.00	0.31	0.43	1
		Species 5	107.15	0.34	0.26	0.42	1
		Species + 𝛿 13 C 6	107.40	0.58	0.23	0.42	1
	Cu, GLM, Log-transformed
		Species + 𝛿 15 N 6	97.62	0.00	0.27	0.28	1
	Species + 𝛿 15 N + Species:𝛿 15 N 9	98.14	0.52	0.21	0.30	1
		Sites + 𝛿 15 N 9	99.25	1.63	0.12	0.29	1
	Mn, GLM, Log-transformed
	Species + 𝛿 13 C + Species:𝛿 13 C 9	98.15	0.00	0.57	0.57	1
	Species + 𝛿 15 N + Species:𝛿 15 N 9	99.90	1.75	0.24	0.56	1
		Species + 𝛿 13 C 6	102.99	4.84	0.05	0.53	1
	Fe, GLM, Log-transformed
		𝛿 15 N 3	-108.15	0.00	0.35	0.03	1
		𝛿 13 C 3	-107.19	0.95	0.22	0.02	1
		Species + 𝛿 13 C 6	-105.82	2.32	0.11	0.04	1

Table 3 .

 3 Review of Hg and Pb concentrations (µg.g -1 dw) reported in crocodilians. TL stands for Total Length and SVL for Snout-Vent-Length (cm).

Acknowledgements

We would like to thank the teams of the nature reserves "Kaw-Roura", "Mont-Grand-Matoury", "Nouragues" and protected area of "Pripris de Yiyi", and the "conservatoire du littoral" for logistics and assistance in the field. We would also like to thank F. Starace, F. Beau, L. Beau, M. Bacques, V. François, D. Guiral, G. Lepoint, N. Sturaro, M. Sarrazin and S. Charles for their help in the field. We are grateful to the Plateforme analyses élémentaires and to G.

Guillou from the Plateforme Analyses Isotopiques of the LIENSs laboratory for running stable isotope analysis. This work was supported by the Office de l'Eau de Guyane (OEG), the Office Français pour la Biodiversité (OFB), the Direction Générale des Territoires et de la Mer de Lemaire et al. : Relationships between stable isotopes and trace element concentrations in the crocodilian community of French Guiana Guyane (DGTM), Parc zoologique de Paris, the CNRS and the Fondation d'entreprise Hermès.

The CPER (Contrat de Projet Etat Région) and the FEDER (European regional Development Fund) are acknowledged for funding the AMA, the ICP and the IRMS of LIENSs laboratory. The Institut Universitaire de France (IUF) is acknowledged for its support to P. Bustamante as a Senior Member. We thank the Nouragues Research Field Station (managed by CNRS) which benefits from "Investissement d'Avenir" grant managed by Agence Nationale de la Recherche (AnaEE France ANR-11-INBS-0001; Labex CEBA ANR-10-LABX-25-01) and its team for assistance in the field.

Nile crocodile

Crocodylus niloticus

KwaZulu


Élise Lacoste
email: eliz.lacoste@gmail.com

Fehmi Boufahja

Corinne Pelaprat

Patrik Le Gall

Tom Berteaux

Gregory Messiaen

Serge Mortreux

Jocelyne Oheix

Vincent Ouisse

Emmanuelle Roque D'orbcastel

Nabila Gaertner-Mazouni

Marion Richard

First simultaneous assessment of macro-and meiobenthic community response to juvenile shellfish culture in a Mediterranean coastal lagoon (Thau, France)

Keywords: Environmental Impact Assessment, Aquaculture, Benthic indicators, Trait based Approach, Macrofauna, Meiofauna

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

Introduction

Coastal ecosystems are facing drastic environmental changes due to many human pressures that threat the integrity of communities and may have major impacts on ecosystem functioning and its ability to sustain ecosystem services [START_REF] Halpern | A global map of human impact on marine ecosystems[END_REF][START_REF] Cardinale | Biodiversity loss and its impact on humanity[END_REF]. In shallow costal ecosystems, benthic communities play a central ecological role in benthic-pelagic coupling through their feeding and movements that promote nutrient and oxygen exchange throughout the sediment layer. Thus, any alteration of these communities may have implications for ecosystem structure and performance [START_REF] Thrush | Functional role of large organisms in intertidal communities: Community effects and ecosystem function[END_REF][START_REF] Kristensen | Influence of benthic macrofauna community shifts on ecosystem functioning in shallow estuaries[END_REF]) and understanding how they respond to disturbance is crucial to predict future changes and help maintain and improve the quality of marine ecosystems. To date, most studies have focused on the response of benthic macrofaunal communities to disturbances [START_REF] Pratt | Detecting subtle shifts in ecosystem functioning in a dynamic estuarine environment[END_REF][START_REF] Dimitriou | Response of benthic macrofauna to eutrophication in a mesocosm experiment: Ecosystem resilience prevents hypoxic conditions[END_REF][START_REF] Ellis | Multiple stressor effects on marine infauna: Responses of estuarine taxa and functional traits to sedimentation, nutrient and metal loading[END_REF]. Recently, interest has been growing in the study of meiofauna, since it has been shown that these organisms may modulate biological interactions in the sediment layer [START_REF] Bonaglia | Meiofauna increases bacterial denitrification in marine sediments[END_REF], Lacoste et al. 2018a) and play a significant role in benthic ecosystem processes and services [START_REF] Schratzberger | Meiofauna matters: The roles of meiofauna in benthic ecosystems[END_REF]. Due to their widespread distribution, short life and reproductive cycles, high abundance and diversity, meiofauna are thus beginning to be used as biological indicators for the assessment and monitoring of aquatic ecosystems [START_REF] Semprucci | A review of Italian research on free-living marine nematodes and the future perspectives on their use as Ecological Indicators (EcoInds)[END_REF][START_REF] Zeppilli | Is the meiofauna a good indicator for climate change and anthropogenic impacts?[END_REF], Bianchelli et al. 2018).

Bivalve aquaculture has various environmental impacts [START_REF] Mckindsey | Influence of suspended and off-bottom mussel culture on the sea bottom and benthic habitats: a review[END_REF][START_REF] Brenner | Bivalve aquaculture transfers in Atlantic Europe. Part B: environmental impacts of transfer activities[END_REF] including the accumulation of biodeposits on the bottom under culture sites that may results in local organic enrichment. Such disturbance typically affect benthic communities according to the [START_REF] Pearson | Macrobenthic succession in relation to organic enrichment and pollution of the marine environment[END_REF] model of organic enrichment with a progressive increase in the abundance of enrichment-tolerant species in the vicinity of farms, whereas communities directly under the farming structures are dominated by a few opportunistic species [START_REF] Callier | Multi-scale spatial variations in benthic sediment geochemistry and macrofaunal communities under a suspended mussel culture[END_REF], 2009[START_REF] Mckindsey | Influence of suspended and off-bottom mussel culture on the sea bottom and benthic habitats: a review[END_REF]. Several studies have reported lower richness and/or abundance of organisms, as well as differential community composition of macrofauna [START_REF] Christensen | Impacts of longline mussel farming on oxygen and nitrogen dynamics and biological communities of coastal sediments[END_REF], Harstein and Rowden 2004, Lacoste et al. 2018b), or meiofauna [START_REF] Mirto | Microbial and meiofaunal response to intensive mussel-farm biodeposition in coastal sediments of the Western Mediterranean[END_REF][START_REF] Mahmoudi | Mussel-farming effects on Mediterranean benthic nematode communities[END_REF][START_REF] Netto | The response of nematode assemblages to intensive mussel farming in coastal sediments (Southern Brazil)[END_REF] under farming structures compared with reference sites. One notable feature of these studies is the focus on structural attributes of the benthos (e.g. taxonomic composition, number of species) whereas functional changes are disregarded. Only a few studies have used a functional approach to investigate the impact of interactions between the environment and fish (Papageorgiou et al. 2009, Dimitriadis and[START_REF] Dimitriadis | Functional diversity and species turnover of benthic invertebrates along a local environmental gradient induced by an aquaculture unit: The contribution of species dispersal ability and rarity[END_REF], or mussel [START_REF] Lacoste | Changes in infaunal assemblage structure influence nutrient fluxes in sediment enriched by mussel biodeposition[END_REF] aquaculture.

Growing awareness that changes in biodiversity may modify ecosystem functioning (e.g. [START_REF] Loreau | Partitioning selection and complementarity in biodiversity experiments[END_REF], Naeem et al. 2009) has led to the recognition that impacts on the functional diversity of benthic assemblages, as opposed to their structural features, should be better quantified (e.g. [START_REF] Strong | Marine biodiversity and ecosystem function relationships: The potential for practical monitoring applications[END_REF][START_REF] Cernansky | The biodiversity revolution[END_REF]. Due to functional redundancies in communities, observed changes in species composition may be buffered [START_REF] Walker | Biodiversity and Ecological Redundancy[END_REF][START_REF] Snelgrove | The biodiversity of macrofaunal organisms in marine sediments[END_REF]) such that structural impacts on benthic assemblages are not always matched by their functional counterparts [START_REF] Bolam | Impacts of dredged material disposal on macrobenthic invertebrate communities: A comparison of structural and functional (secondary production) changes at disposal sites around England and Wales[END_REF]. Otherwise, trophic pathways (indicator of ecosystem functioning) may be impacted without apparent changes of the overall benthic diversity [START_REF] Dubois | Effects of oyster farming on macrofaunal assemblages associated with Lanice conchilega tubeworm populations: A trophic analysis using natural stable isotopes[END_REF]). Thus, functional-based approaches, including biological trait analysis and the computation of functional diversity (FD) indices, provide useful proxies to investigate the effect of human disturbances at the ecosystem-functioning level [START_REF] Mouillot | Alternatives to taxonomic-based approaches to assess changes in transitional water communities[END_REF][START_REF] Mouillot | A functional approach reveals community responses to disturbances[END_REF]) and to anticipate the potential loss of ecosystem services associated with biodiversity erosion [START_REF] Cardinale | Biodiversity loss and its impact on humanity[END_REF]. Moreover, this approach can be used to compare communities that have no species in common but do share traits.

Thau Lagoon is a Mediterranean marine ecosystem of notable economic importance due to the many ecosystem services it supports, in particular, the significant oyster farming activity (about 7 000 tons per year, near 10% of the national production). A few authors have described changes in macrofaunal communities in relation to oyster culture [START_REF] Mazouni | Nutrient and oxygen exchanges at the water-sediment interface in a shellfish farming lagoon (Thau, France)[END_REF][START_REF] Duport | Benthic macrofauna and sediment reworking quantification in contrasted environments in the Thau Lagoon[END_REF][START_REF] Thouzeau | Spatial and temporal variability of benthic biogeochemical fluxes associated with macrophytic and macrofaunal distributions in the Thau lagoon (France)[END_REF], and one study showed spatial variation of meiofauna abundance [START_REF] Guelorget | Structure et organisation de l'étang de Thau d'après la faune benthique (macrofaune, meiofaune). Relations avec le confinement[END_REF]). But to date, no list has been published of meiofauna assemblages in the lagoon, even though these communities may respond more rapidly to biodeposition than macrofauna do and could be good indicators of lagoon ecological status. Potential associated functional changes in macro-and meiofauna remain unknown. Thus, in a global context of increasing intensity and frequency of climatic and anthropogenic disturbances, there is a need to describe and predict changes in functional community structure to anticipate potential losses of ecosystem services associated with biodiversity modifications in coastal areas such as Thau Lagoon.

The aims of this study were therefore 1/ to document macrobenthic and meiobenthic (nematode) communities in relation to oyster culture in Thau Lagoon and 2/ to compare the response of some selected taxonomic and functional attributes to disturbances caused by shellfish culture for macro-and meiofauna assemblages. To our knowledge, this is the first time the impact of bivalve aquaculture on macro-and meiobenthic communities has been studied simultaneously using a traditional taxonomic based approach coupled with a functional approach.

Materials and Methods

Study area and sampling strategy

Thau Lagoon is a French Mediterranean semi-enclosed shallow lagoon (mean depth 4 m) covering an area of 68 km 2 (Fig. 1). It is the most important French Mediterranean shellfish farming zone with an annual production of about 7 330 tons of oysters in 2016 (DDTM34 2017) reared on spatially organized structures, called "tables". Each table is 50 × 12 m long and supports between 800 and 1200 suspended nets or ropes holding juvenile or adult oysters.

Sixteen tables form a 'block' and the blocks are separated from each other by a distance of 150m. Annual variations in water temperature and salinity in the lagoon are large, varying from 3 °C to 30 °C and from 33 to 40 PSU respectively [START_REF] Messiaen | Marine environmental station database of Thau lagoon[END_REF]).

The study was conducted in a dedicated shellfish-farming zone in the western part of the lagoon (Fig. 1). As a part of a research program (see [START_REF] Richard | Influence of OSHV-1 oyster mortality episode on dissolved inorganic fluxes: An ex situ experiment at the individual scale[END_REF][START_REF] Richard | Changes in planktonic microbial components in interaction with juvenile oysters during a mortality episode in the Thau lagoon (France)[END_REF], the study focused on juvenile production, whose impact on ecosystem has never previously been evaluated in Thau Lagoon. Samples were collected at two sites in the farmed zone directly below juvenile tables (TO and TE sites) and at two additional sites located at a distance of 6 200 m outside the farming zone (CO and CE sites) (Fig. 1). Some characteristics of the four sites are given in Table 1, the main difference being algal cover (Halopytis sp.) which was greater at TO and CO sites. Sampling was done during three surveys in 2015 (September 29-October 2; October 28-November 4 and December 1-December 4, hereafter referred to as October, November and December) covering the whole juvenile production period. Each date, three replicate sediment cores (9 cm diameter, 10 cm depth) were randomly collected by scuba divers at each site. The samples were preserved in 10% buffered formalin and later transferred into 70% alcohol.

Abundance and species richness

Back to the laboratory, the content of each core sample was passed through a 1mm mesh sieve. Macrofauna in the > 1mm fraction was sorted and identified to the lowest taxonomic resolution possible, up to species in most cases. The < 1 mm fraction was decanted over a 40µm sieve. All nematodes were sorted under a stereo-microscope, transferred to glycerol through a series of ethanol-glycerine solutions, mounted on glycerine slides [START_REF] Somerfield | Soft sediment meiofaunal community structure in relation to a long-term heavy metal gradient in the Fal Estuary system[END_REF]) and identified to the lowest taxonomic level possible using the generic keys of Platt andWarwick (1983, 1988) and [START_REF] Warwick | Free-living Marine Nematodes. Part III. British Monhysterida[END_REF] and species descriptions in the literature. Abundance (N) and species richness (S) of macrofauna and nematodes were characterized.

The Shannon-Wiener diversity index (H') and Pielou's eveness (J') were calculated for each site. Taxa that were observed only once were removed from analyses [START_REF] Clarke | Change in marine communities. An approach to statistical analysis and interpretation[END_REF]; all results are expressed per core (63 cm 2 ).

Functional analysis

The common step in all functional ecology studies is to characterize the functional strategy of each species of interest by identifying a relevant combination of functional traits [START_REF] Violle | Let the concept of trait be functional![END_REF]. There is currently no consensus on the methodology for selecting the most appropriate traits for a given study [START_REF] Marchini | Functions and ecological status of eight Italian lagoons examined using biological traits analysis (BTA)[END_REF]. As the aim of the present study was to use traits as a proxy for assemblage response to shellfish culture, we decided to focus on the biological traits and ecological groups of macrofauna that have already been shown to be good indicators of organic enrichment in this specific context [START_REF] Borja | A Marine Biotic Index to establish the ecological quality of soft-bottom benthos within European estuarine and coastal environments[END_REF][START_REF] Lacoste | Changes in infaunal assemblage structure influence nutrient fluxes in sediment enriched by mussel biodeposition[END_REF]). To our knowledge, this is the first study to simultaneously evaluate the functional response of both macrofauna and meiofauna (nematodes) to bivalve aquaculture. We thus selected nematodes biological traits that have been shown to respond to organic enrichment [START_REF] Alves | Benthic meiofauna as indicator of ecological changes in estuarine ecosystems: The use of nematodes in ecological quality assessment[END_REF][START_REF] Kandratavicius | Response of estuarine free-living nematode assemblages to organic enrichment: an experimental approach[END_REF], with the assumption that they are comparable with those of macrofauna. Thus, selected traits for both assemblages were related to trophic guilds, ecological strategy, and interaction with sediments (Table 2). Each selected trait was subdivided into several modalities (Table 2). Organisms identified up to species level were classified in functional groups based on existing literature (e.g. [START_REF] Wieser | Die Bezichung swischen Mundho¨ hlengestalt, Erna¨hrungsweise und Vorkommen bei freilebenden marinen Nematoden[END_REF][START_REF] Thistle | Nematode tail-shape groups respond to environmental differences in the deep sea[END_REF][START_REF] Queiros | A bioturbation classification of European marine infaunal invertebrates[END_REF], WoRMS Editorial Board 2018). When reliable information was not available, data from the closest phylogenetic neighbor were used or the taxon was excluded from subsequent analysis. All nematodes and 44 macrofaunal (accounting for 91% of all individuals identified) taxa were thus retained for functional analysis. The subsequent 'trait per taxon' matrix was combined with the 'taxa per sample' matrix (abundance) to create a 'trait per sample' matrix. The relative abundance of the functional groups was then calculated by summing abundance across all taxa allocated to each functional group at a given site per sampling date. Finally, the two matrices were used to calculate functional indices (functional richness: FRic, functional eveness: FEve and functional dispersion: FDis) as outlined by [START_REF] Villéger | New multidimensional functional diversity indices for a multifaceted framework in functional ecology[END_REF] and [START_REF] Laliberté | FD: measuring functional diversity from multiple traits, and other tools for functional ecology[END_REF] for both macrofauna and nematodes.

Using only categorical traits, FRic was defined as the number of unique trait combinations (equivalent to singular species).

Although its results are ambiguous [START_REF] Semprucci | A review of Italian research on free-living marine nematodes and the future perspectives on their use as Ecological Indicators (EcoInds)[END_REF], the Index of Trophic Diversity (ITD, [START_REF] Heip | The Ecology of Marine Nematodes[END_REF] is popular and widely used for nematodes. We thus calculated ITD for both nematodes and macrofauna, using their respective trophic guilds, with the aim of evaluating its consistency in the case of disturbances caused by shellfish culture. The index was calculated following [START_REF] Heip | The Ecology of Marine Nematodes[END_REF]: ITD=∑ 2 , where is the percentage contribution of each feeding type. Thus, a low ITD value corresponds to high trophic diversity (each group accounting for the same percentage).

Statistical analysis

The effect of Date × Condition (Farm vs Reference) on univariate measurements of benthic communities (taxonomic and functional indices) was tested using linear mixed effect models (lMEM; modelling normally distributed responses) with site as a random factor nested in Condition. The effect of factors was considered as significant when the null vs main effect models (using maximum likelihood estimation) differed significantly (ANOVA, p < 0.05).

Assumptions were assessed visually by examination of residual plots [START_REF] Quinn | Experimental Design and Data Analysis for Biologists[END_REF]Keough 2002, Bates et al. 2015). All mixed effect models were constructed in R using lme4 [START_REF] Bates | Nematode biodiversity and benthic trophic state are simple tools for the assessment of the environmental quality in coastal marine ecosystems[END_REF]) and Tukey's HSD tests as implemented in library emmeans [START_REF] Lenth | emmeans: Estimated Marginal Means, aka Least-Squares Means[END_REF] were used as post hoc tests to compare conditions at each date if lMEM showed a significant effect of the interaction. Variation in multivariate assemblage structure (abundance data) was visualized using model-based ordination (generalized linear latent variable model) with the R package gllvm (Niku et al. 2019) and evaluated using multivariate linear modelling with the R package mvabund [START_REF] Wang | Mvabund -an R package for model-based analysis of multivariate abundance data[END_REF], assuming a negative binomial distribution. Species that contributed significantly to differences among conditions were identified using the p.uni argument that allows univariate 'species-by-species' comparisons in mvabund. Variation in assemblage functional composition between dates and conditions ('traits by site' matrix) was also compared using the same multivariate approach.

Results

Taxonomic indices

A total of 2 679 organisms belonging to 118 taxa were identified in the macrofaunal community in this study. Annelida (36.7%) and Mollusca (35.5%) were the most widely represented group followed Arthropoda (25.8%). The mollusk Musculus subpictus alone represented 24.2% of the total sample, whereas we found a single individual of 42 taxa, which were subsequently removed from analyses.

A total of 778 nematodes belonging to 41 taxa were identified. Two taxa, Paracomesma dubium and Spirina gerlachi, represented 34% of the organisms recorded and 6 taxa were represented by a single individual. As this is the first time the nematodes have been described

for Thau Lagoon, a complete list of taxa is provided in Table S1.

Table 3 lists the values of each index averaged per Condition (for all sampling dates).

Statistical results are those of the Condition effect (pairwise comparison), except in case of a significant effect of Date × Condition interaction. Although there was a trend of more taxa and greater abundance of macrofauna at reference sites, the Condition effect was not statistically significant (Table 3). The effect of Condition on the diversity indices was neither significant (Table 3).

A similar pattern was observed for nematodes, with the greatest abundance and richness values observed at reference sites (Table 3). lMEM revealed a significant effect of Date × Condition on S, H' and J', with contrasts indicating higher values of S and H' in December at reference sites (S = 11 ± 3 and H = 2.1 ± 0.1) compared to farm sites (S = 4 ± 1 and H = 1.3 ± 0.2).

Community structure

The model-based projection of macrofauna community structure (Fig. 2A) showed a clear distinction between conditions. The multivariate test confirmed the significant difference between communities at the farm vs at the reference sites (Table 4). Univariate comparisons were significant for five taxa; three of which were more abundant at the farm sites (Chaetozone gibber, Notomastus sp. and Neanthes acuminata) and two that were observed only at reference sites (Phascolion strombus and Pusillina lineolata).

The ordination based on nematodes abundance (Fig. 2B) also showed a distinction between communities from the two conditions (Farm vs Reference), confirmed by the significance of the multivariate test (Table 4). Differences between sites within conditions were less clear relative to macrofauna as there was a major overlap of symbols representing samples from different sites (especially TO and TE) in the ordination graph (Fig. 2B). Nematode assemblages also varied among dates (Table 4). Pairwise comparisons indicated that assemblages differed significantly between October and November (p = 0.041). Among species that best explained variation among sites, Paracomesoma dubium, Anticomopsis longicaudata and Spirina gerlachi, were more abundant at the two reference sites.

Conversely, one taxon was more abundant under farming structures than at the reference sites (Daptonema fallax) and three taxa were present only at the two farm sites (Anticoma eberthi, Eurystomina sp. and Metalinhomoeus sp.).

Functional analysis

Macrofauna functional composition

Figure 3 shows the relative abundance of each biological trait. Although there was some variability within conditions and between dates, macrofauna at the reference sites was mainly characterized by filter-feeder organisms (ca 50%: Fig. 3), which are mostly sensitive to organic enrichment (ecological group I, Fig. 3). The proportion of filter-feeders was lower in the farming zone, whereas the proportion of deposit feeders and grazers increased (Fig. 3).

These communities were dominated by animals that are mostly tolerant to organic enrichment or first-order opportunistic (ecological groups III and IV: Fig. 3). Mobility and reworking types did not allow us to clearly distinguish farming and reference sites (Fig. 3), except that upward-downward conveyors (UC and UDC: Fig. 3) appeared to be more abundant at the farm sites. The high abundance of surficial reworkers (S) at site TO (Fig. 3) was mainly due to the presence of many Arthropoda, also belonging to grazers (Gr) and ecological group I, and the the bivalve Musculus subpictus. These taxa were mainly found in association with the algae Halopithys sp. The GLM indicated a significant effect of Condition on the functional composition of assemblage (df = 1, Dev. = 225.45, p < 0.001), with no significant effect of the interaction (Date × Condition, df = 2, Dev. = 48.39, p = 0.393).

Nematode functional composition

The most representative feeding group at reference sites was epigrowth feeders (2A) whereas the percentage of detritivores (1B) was increased at the farm sites (Fig. 4). Overall, the community at the two reference sites was dominated by taxa with a cp score of 3 (Fig. 4) and amphids with a spiral shape (Sp: Fig. 4). At the two farm sites, the percentage of opportunistic nematodes (cp score of 2: Fig. 4) with circular amphid (Cr) increased (Fig. 4). The adult shape did not clearly differ betweenfarming zone sites and those outside the farming zone (Fig. 4). The high percentage of long/thin adults (lt) at site TO in September was mainly due to the presence of Metalinhomoeus longiseta. The GLM indicated a significant effect of Condition on assemblage functional composition (df = 1, Dev. = 154.49, p < 0.001), with no significant effect of the interaction (Date × Condition, df = 2, Dev. = 48.22, p = 0.212). and at the reference sites (CE, CO) on the three sampling dates (D1: October, D2: November, D3: December). Feeding groups: 1A: selective or 1B: non selective deposit-feeder, 2A: epigrowth feeder, 2B: predators; Life history: opportunistic (2) to persister (5); Adult shape: lt: long/thin, sl: slender ; Amphid shape: Cr: circular, Id: indistinct, Pk: pocket-like, REL: rounded or elongate, Sp: spiral.

Functional indices

The lowest FRic values of both macrofauna and nematodes were recorded under the tables (TE and TO) (Table 5). The Date × Condition interaction had a significant effect for nematodes, with contrasts indicating significantly higher FRic values at reference sites in December (9.2 ± 1.6) compared with the farm sites (4 ± 0.6). Macrofauna FEve was higher under the tables in November (0.72 ± 0.06) and December (0.72 ± 0.04) than at the reference sites (0.57 ± 0.04 and 0.56 ± 0.04 respectively in November and December) whereas no difference was found for nematodes. The Date × Condition interaction had a significant effect on nematodes FDis but with no difference between conditions each date.

The ITD for macrofauna was significantly lower at the farm sites (Table 5) indicating that these sites presented a higher trophic diversity than did reference sites. For nematodes, the Date × Condition interaction had a significant effect on ITD but no difference was found between conditions at each date.

Discussion

First description of nematodes in Thau Lagoon

With the exception of marine benthic ecosystems in Italy and Tunisia [START_REF] Semprucci | ADENOPHOREA -NEMATODI MARINI. Checklist della Fauna Marina Italiana[END_REF], Boufahja et l. 2014), there is a serious gap in knowledge concerning the Mediterranean meiofauna. This is particularly true for coastal lagoons, for which the spatial and temporal distribution of meiofauna, including free-living nematodes, is not well documented or is underestimated, despite they often greatly contribute to the biodiversity of coastal areas [START_REF] Balsamo | Meiofauna as a tool for marine ecosystem biomonitoring[END_REF]. Nematodes can provide critical information on the functioning of ecosystems and have already been proposed as potential indicators of anthropogenic disturbance [START_REF] Schratzberger | Structure and taxonomic composition of subtidal nematode and macrofauna assemblages at four stations around the UK coast[END_REF][START_REF] Danovaro | Exponential decline of deep-sea ecosystem functioning linked to benthic biodiversity loss[END_REF][START_REF] Moreno | The use of nematodes in assessing ecological quality status in the Mediterranean coastal ecosystems[END_REF][START_REF] Pusceddu | Assessment of benthic trophic status of marine coastal ecosystems: Significance of meiofaunal rare taxa[END_REF]). Our study is the first to provide a complete list of nematode taxa observed in the French Mediterranean Thau lagoon. Compared to the few studies that have documented other Mediterranean coastal areas, the number of taxa reported here from Thau Lagoon is similar but abundances were in the lower range [START_REF] Mirto | Microbial and meiofaunal response to intensive mussel-farm biodeposition in coastal sediments of the Western Mediterranean[END_REF][START_REF] Danovaro | Sustainable impact of mussel farming in the Adriatic Sea (Mediterranean Sea): Evidence from biochemical, microbial and meiofaunal indicators[END_REF][START_REF] Mahmoudi | Mussel-farming effects on Mediterranean benthic nematode communities[END_REF]. The community structure showed many similarities with checklists of Italian and Tunisian lagoons, published respectively by [START_REF] Semprucci | ADENOPHOREA -NEMATODI MARINI. Checklist della Fauna Marina Italiana[END_REF] and [START_REF] Boufahja | More than 35 years of studies on marine nematodes from Tunisia: a checklist of species and their distribution[END_REF]. Ten of the common species found in Thau Lagoon have also been found in Italy (e.g.

Anticoma eberthi, Longicyatholaimus longicaudatus) and 18 species were recorded in both Thau Lagoon and Tunisia (e.g. Spirinia gerlachi, S. parasitifera, Thalassironus britannicus, for details, see Table S1). These shared records could be linked to common environmental parameters among areas and to water exchanges between the two Mediterranean basins, which make large-scale dispersal between Tunisia, Italy and France possible. The checklist of nematodes resulting from this study could be the basis to further documenting meiobenthic diversity in Mediterranean coastal areas, with the aim to advance our understanding of its spatio-temporal variability.

Response of macrofaunal and nematode assemblages to shellfish culture

Overall, community structure (taxonomic and functional) differed between conditions, with lower abundance and richness (some significant, some not) at farm sites, as expected from previous studies [START_REF] Mirto | Microbial and meiofaunal response to intensive mussel-farm biodeposition in coastal sediments of the Western Mediterranean[END_REF][START_REF] Christensen | Impacts of longline mussel farming on oxygen and nitrogen dynamics and biological communities of coastal sediments[END_REF][START_REF] Callier | Evaluation of indicators used to detect mussel farm influence on the benthos: two case studies in the Magdalen Islands, Eastern Canada[END_REF]. The presence of opportunistic species under the farm tables (e.g. Neanthes acuminata, Prionospio fallax, Daptonema sp.) whose abundance is favored under organically enriched conditions, showed that biodeposition by juvenile oysters impacts the benthic environment below farm structures in the study area. However, the high and similar species evenness values (J) at the farm and reference sites revealed the absence of strongly dominant species that would be expected in an organically enriched environment [START_REF] Pearson | Macrobenthic succession in relation to organic enrichment and pollution of the marine environment[END_REF]. The organic enrichment due to the presence of juvenile oysters is likely low compared with that of adult oysters, but the continuous exploitation of the area for farming may have modified community characteristics compared with outside of the farming zone.

The results of the analysis of nematodes revealed differences in both univariate indices and community structure at the farm and reference sites. Our results for nematodes are in agreement with the study of [START_REF] Mirto | Microbial and meiofaunal response to intensive mussel-farm biodeposition in coastal sediments of the Western Mediterranean[END_REF] who reported decreased meiofauna richness and abundance in aquaculture sites relative to reference areas [START_REF] Mirto | Microbial and meiofaunal response to intensive mussel-farm biodeposition in coastal sediments of the Western Mediterranean[END_REF]. Inversely, a study in Bizerta Lagoon indicated increased nematode abundance at farm sites relative to control sites [START_REF] Mahmoudi | Mussel-farming effects on Mediterranean benthic nematode communities[END_REF]. The latter authors suggested that increased abundance was due to differential tolerance to organic matter, decreased competition, and a subsequent competitive release of more resistant species due to sediment organic enrichment. However, other factors (biotic and abiotic) could also explain the distribution of species since, for example, Paracomesma dubium was more abundant at farm sites in the study by [START_REF] Mahmoudi | Mussel-farming effects on Mediterranean benthic nematode communities[END_REF], whereas in our study, we observed a higher abundance at reference sites.

Temperature variations may have contributed to explain temporal changes since when significant, the effect of Condition was mostly detected in December.

In this study, macrofaunal taxonomic indices failed to detect a significant effect of shellfish culture whereas the multivariate approach did, as already reported in other studies [START_REF] Grant | Influence of 'bouchot' mussel culture on the benthic environment in a dynamic intertidal system[END_REF], Lacoste et al. 2018a). Previous studies in Thau Lagoon produced conflicting results

on the impact of aquaculture on macrofauna, ranging from low [START_REF] Mazouni | Nutrient and oxygen exchanges at the water-sediment interface in a shellfish farming lagoon (Thau, France)[END_REF][START_REF] Duport | Benthic macrofauna and sediment reworking quantification in contrasted environments in the Thau Lagoon[END_REF]) to severe with a completely defaunated site in a deep shellfish area [START_REF] Thouzeau | Spatial and temporal variability of benthic biogeochemical fluxes associated with macrophytic and macrofaunal distributions in the Thau lagoon (France)[END_REF]). These authors highlighted the concomitant role of depth, algal cover and aquaculture in community composition. Our study also suggests that algal cover played an important role, at least for the macrofauna community. Some arthropods, (e.g. Gammarella sp., Cymodoce truncata) were found attached to the algae Halopithys sp., so the TO and CO sites had specific assemblages structure due to these taxa.

The general trend thus supports the hypothesis that juvenile oysters slightly influenced both macrobenthic and nematode communities, although the variability between sites within conditions may have masked statistical differences in some indicators. Although this may require a labor-intensive sampling effort, it is recommended to sample several farm and reference sites to disentangle the influence of aquaculture on benthic communities, if such an influence exists, from the natural variability. This is particularly important in transitional waters as coastal lagoons where high natural variability may exist due to rapid environmental changes.

Insights from the functional approach

To date, the effect of aquaculture on benthic communities has usually been evaluated using traditional taxonomic approaches [START_REF] Lacoste | Biodiversity-Ecosystem Functioning (BEF) approach to further understanding aquaculture-environment interactions with application to bivalve culture and benthic ecosystems[END_REF]. Here, we demonstrate that some species traits can be used to predict the response of macro-and meiofauna to disturbances caused by shellfish culture since the distribution of traits and functional indices performed well in distinguishing between the farm and reference sites.

For both assemblages, trophic group and ecological group/life history were the traits that best responded to the presence of oyster tables. As reported in other studies, the presence of aquaculture facilities often decreases the abundance and diversity of filter-feeding organisms [START_REF] Forrest | Benthic impacts of intertidal oyster culture, with consideration of taxonomic sufficiency[END_REF][START_REF] Dubois | Effects of oyster farming on macrofaunal assemblages associated with Lanice conchilega tubeworm populations: A trophic analysis using natural stable isotopes[END_REF][START_REF] Fabi | Impact of an open-sea suspended mussel culture on macrobenthic community (Western Adriatic Sea)[END_REF]) whereas increased organic supply in farm zones favors the presence of non-selective deposit feeders. Following the classification of Grall and Glemarec (1997) and [START_REF] Borja | A Marine Biotic Index to establish the ecological quality of soft-bottom benthos within European estuarine and coastal environments[END_REF], farming zones favored the presence of tolerant and opportunistic taxa whereas reference sites were dominated by sensitive organisms. Among the meiofauna, epigrowth feeders were the most abundant organisms at reference sites and were replaced by non-selective deposit feeders in the farming zone. The reduction in the abundance of epigrowth feeders at the farm sites may be due to excluding competition with other groups but we can also hypothesize a lower abundance of diatoms under oyster tables due to a shading effect [START_REF] Mckindsey | Influence of suspended and off-bottom mussel culture on the sea bottom and benthic habitats: a review[END_REF].

Amphids are complex sensorial organs of nematodes used in the search of food and partners, and are useful for taxonomical identification. To date, there is no information linking amphids to a particular type of marine environment, except [START_REF] Semprucci | Do the morphological and functional traits of free-living marine nematodes mirror taxonomical diversity?[END_REF], who described a potential link between inconspicuous amphids and rich food sources areas. Accordingly, our results suggest that the more intense and regular food supply at farm sites may limit the advantage of large amphids (REL, Sp) and hence that nematodes with smaller amphids (Cr) are more abundant in farming zones. However, this preliminary result requires further investigation to highlight potential links between morphological features of nematodes and their marine habitat.

Functional diversity (FD) indices were computed for macrofauna and nematodes to evaluate the relevance of these indices in the case of aquaculture-induced disturbances. Nematode

FRic was reduced at the farm sites and FD indices revealed the influence of aquaculture on macrofauna assemblages that was not detected using taxonomic indices. This confirms the usefulness of the functional approach, and more generally of multi-index approaches to detect the influence of farming [START_REF] Borja | Assessing the suitability of a range of benthic indices in the evaluation of environmental impact of fin and shellfish aquaculture located in sites across Europe[END_REF][START_REF] Lacoste | Changes in infaunal assemblage structure influence nutrient fluxes in sediment enriched by mussel biodeposition[END_REF]. Interestingly, macrofauna FD indices indicated a higher trophic diversity and functional evenness at farm sites whereas no changes were detected at the richness level. The actual level of disturbance may therefore have shifted community composition that promoted a more even use of the trophic niche, which, in turn, may have increased the productivity of the system [START_REF] Mason | Functional richness, functional evenness and functional divergence: The primary components of functional diversity[END_REF].

Compared with the taxonomic approach, functional analysis not only offers the opportunity to study the response of communities to the environment but also the subsequent impact of the changes on ecosystem properties [START_REF] Lavorel | Predicting changes in community composition and ecosystem functioning from plant traits: Revisiting the Holy Grail[END_REF]Garnier 2002, Lacoste et al. 2020), which is essential to evaluate the sustainable exploitation of aquaculture systems.

Limitation and future outlook of the functional approach

This work provides insights into the functional response of benthic communities to aquaculture-induced disturbance. We proposed the use of four selected traits that may respond both for macrofauna and nematode assemblages, that we assumed referred to similar behavior. The choice of biological traits may strongly influence the calculation of functional indices [START_REF] Petchey | Functional diversity: back to basics and looking forward[END_REF]. It was not our objective to test the most appropriate traits to use from a wide selection of possible choices. Rather we wished to highlight the potential of functional analysis to evaluate the impact of aquaculture using various kinds of benthic organisms (macrofauna and nematodes) and traits that have already been shown to respond to organic enrichment [START_REF] Borja | A Marine Biotic Index to establish the ecological quality of soft-bottom benthos within European estuarine and coastal environments[END_REF][START_REF] Alves | Benthic meiofauna as indicator of ecological changes in estuarine ecosystems: The use of nematodes in ecological quality assessment[END_REF][START_REF] Kandratavicius | Response of estuarine free-living nematode assemblages to organic enrichment: an experimental approach[END_REF][START_REF] Lacoste | Changes in infaunal assemblage structure influence nutrient fluxes in sediment enriched by mussel biodeposition[END_REF]. Future experiments that examine multiple traits in different regions and systems will be needed to evaluate if functional analysis is a reliable tool to assess the benthic impacts of aquaculture.

The number of taxa and the completeness and accuracy of the species trait data play a significant role in the estimation of functional trait metrics [START_REF] Pakeman | Functional trait metrics are sensitive to the completeness of the species' trait data?[END_REF]Quested 2007, Pakeman 2014). In this exploratory study, we chose to use the maximum number of taxa for which traits were available. However, the inclusion of arthropods, for example, is questionable since it appears that these organisms were mostly associated with Halopithys sp.

algae and are very mobile, so they could also have been classified as epifauna instead of infauna and disregarded. When we excluded them from the functional analysis (data not shown), we obtained differences in the representation of relative abundances based on traits, but no difference with respect to FD indices.

Although further studies are needed to identify the traits that react most consistently in a given manner or the taxonomic groups to considered, this work provides interesting information on the potential of the trait based approach to detect disturbances caused by aquaculture. Along with theoretical work, we think that further studies encompassing different kinds of disturbance across ecosystems will enable the implementation of a framework intended to include the functional approach in benthic monitoring programs.

Conclusion and Perspectives

This study documented the macrobenthic and the nematode communities in relation with juvenile oyster culture in Thau Lagoon. The trait-based approach we propose in this work

shows the feasibility of detecting the impact of shellfish culture on the functional structure of both macro-and meiofauna communities in coastal lagoons, although further studies are needed to identify general patterns, within and across biogeographical regions and aquaculture systems. Combining taxonomic and functional approaches may add relevant ecological information and help obtain a clearer understanding of the functioning of benthic communities that are impacted by anthropogenic activities. For aquaculture, as well as other human-induced disturbance, the functional approach will undoubtedly enhance our ability to monitor activities and aid compliance with conservation strategies such as the Water Framework Directive Strategy (EC 2000), which requires an understanding of both how function varies spatially and how it responds to human disturbances. Several gaps that represent many research opportunities should however be addressed to 'operationalize' such approach. As examples i) new tools such as eDNA would allow to get rapid identification of organisms once taxonomic validation of database will be improved, ii) further description of benthic organisms biological and ecological traits should be completed to feed databases.

Figure 1 .

 1 Figure 1. Location of the four sampling sites in Thau lagoon. The two T sites (TE & TO) were directly under the tables and the two reference sites (CE & CO) were located at a distance of 200 m from the shellfish farming area.

Figure 2 .

 2 Figure 2. Model based biplot (unconstrained ordination) of abundance data for (A) macrofauna and (B) nematodes. Samples (cores) are shown in colors indexed by Site and Date (symbols).

Figure 3 .

 3 Figure 3. Relative abundance of the different macrofauna functional groups at the farm sites (TE, TO) and at the reference sites (CE, CO) on the three sampling dates (D1: October, D2: November, D3: December). Feeding groups: F: filter-feeder, Gr: grazer, D: deposit-feeder, SubD: subsurface depositfeeder, C: carnivorous; Ecological groups from very sensitive (I) to opportunistic (V) ; Mobility from fixed (I) to burrower (IV) ; Reworking types: E: epifauna, S: surficial modifier, B: biodiffuser, UC: upward conveyor, UDC: up-/downward conveyor.

Figure 4 .

 4 Figure 4. Relative abundance of the different nematode functional groups at the farm sites (TE, TO)

Table 1 .

 1 Summary description of the 4 sampled sites

			TE
			CE
		TO
			CO
		Reference sites		Farm sites
		CO	CE	TO	TE
	Depth	3.7 m	4.2 m	3.6 m	4.2 m
	Bottom
	observation
	Culture system	-	-	Juvenile oysters ca. 1.1 t per table *	Juvenile oysters ca. 1.4 t per table

*Calculation from initial measurements on a sub-sample

Table 2 .

 2 List of biological traits and respective categories selected for macrofauna and nematodes.

	Category	Level	Code
	Feeding group	Filter/suspension-feeder	F
		Grazer/Detritivorous	Gr
		Deposit-feeder	D
		Subsurface deposit-feeder	SubD
		Carnivorous	C
	Ecological group	Very sensitive	I
		Tolerant	II
		Indifferent	III
		2 d -order opportunistic	IV
	Macrofauna	1 st -order opportunistic	V
	Mobility	Fixed in a tube	I
		Limited movement	II
		Slow movement	III
		Free movement (burrow system)	IV
	Reworking type	Epifauna	E
		Surficial modifier	S
		Biodiffusers	B
		Upward conveyors	UC
		Upward/downward conveyors	UDC
	Feeding groups	Selective deposit-feeder	1A
		Non selective deposit-feeder	1B
		Epigrowth feeder	2A
		Predators/omnivores	2B
	Cp-score	Opportunistics	2
		to	3 4
	Nematodes	Persisters	5
	Adult shape	Long/thin	Lt
		Slender	Sl
	Amphid shape	Circular	Cr
		Indistinct	Id
		Pocket-like	Pk
		Rounded or elongate loop	REL
		Spiral	Sp

Table 3

 3

			Farm	Reference	Fc	df	L-ratio	p
	Macrofauna	N	40±8	106±16	Cd	3	5.592	0.133
		S	13±1	21±2	Cd	3	2.941	0.401
		H'	2.1±0.1	2.3±0.1	Cd	3	2.740	0.480
		J'	0.8±0.1	0.8±0.1	Cd	3	5.791	0.122
	Meiofauna	N	12±1	31±5	Cd	3	5.962	0.113
		S	6±1	10±1	D×Cd 5	12.356	0.030*
		H'	1.5±0.1	1.9±0.1	D×Cd 5	20.704	<0.001*
		J'	0.9±0.1	0.8±0.1	D×Cd 5	12.538	0.028*

. Macrofauna and nematodes characteristics (mean ± SE, n = 3) under juvenile oyster tables (Farm sites) and beyond the farming zone (Reference sites). Abundance (number of individuals, N) and richness (number of taxa, S), Shannon diversity (H') and Pielou's eveness (J'). Statistical effect of factors (Fc) Condition (Cd: Farm vs Reference) or Date × Condition interaction (D × Cd). *Significant difference at the 0.05 level.

Table 4 .

 4 Results of the GLM testing the effect of Date, Condition and their interaction on macrofauna and nematode assemblages composition (abundance). *Significant difference at the 0.05 level.

	df	Dev.	p

Table 5 .

 5 Macrofauna and nematodes functional characteristics (mean ± SE, n = 3) at sites under the juvenile oyster tables (Farm sites) and outside the farming zone (Reference sites). (FRic: functional richness, FEve:

			Farm	Reference	Fc	df	L-ratio	p
	Macrofauna	FRic	8±1	12±1	D×Cd	5	10.657	0.05
		FEve	0.7±0.1	0.5±0.1	D×Cd	5	11.805	<0.05*
		FDis	0.4±0.1	0.4±0.1	Cd	3	1.520	0.677
		ITD	0.4±0.1	0.7±0.1	Cd	3	9.237	<0.05*
	Meiofauna	FRic	6±1	9±1	D×Cd	5	12.326	<0.05*
		FEve	0.7±0.1	0.7±0.1	Cd	3	2.481	0.479
		FDis	0.3±0.1	0.4±0.1	D×Cd	5	13.69	0.017*
		ITD	0.5±0.1	0.5±0.1	D×Cd	5	13.043	<0.05*

functional evenness, FDis: functional dispersion, ITD: Index of Trophic Diversity). Statistical effect of factors (Fc) Condition (Cd: Farm vs Reference) or Date × Condition interaction (D × Cd) *Significant difference at the 0.05 level.

Acknowledgements

This work is a contribution to the MORTAFLUX program, funded by the Scientific Direction of Ifremer and by the EC2CO BIOHEFECT action (Coordinator: M. Richard). The PostDoctoral fellowship of É. Lacoste was funded by the Ifremer and the University of French Polynesia. Ifremer and IRD funded two missions for Dr Fehmi Boufahja in Sète in 2019 to facilitate nematode determination analysis and French-Tunisian exchanges.

Macrofauna analysis were funded by the MARBEC UMR. The authors thank Nicolas Cimiterra for the realization of the map (Figure 1). Many thanks to Geneviève Guillouet and Zoely Rakotomonga-Rajaonah for their administrative help, and Daphne Goodfellow for revising the English of the manuscript. We finally thank the anonymous reviewers for the helpful comments on a previous version of this manuscript.