Vincent P Jassey
email: vincent.jassey@epfl.ch

Caroline Meyer

Christine Dupuy

Nadine Bernard

Edward Ad Mitchell

Marie-Laure Toussaint

Marc Metian

P Auriel

Daniel Chatelain

Gilbert

Vincent Ej Jassey

Auriel P Chatelain

Daniel Gilbert

To What Extent Do Food Preferences Explain the Trophic Position of Heterotrophic and Mixotrophic Microbial Consumers in a Sphagnum Peatland?

Keywords: 13 Carbon and 15 nitrogen isotopes, Feeding habit, Foraging traits, Natural stable isotopes, Microbial food web, Testate amoebae

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

Introduction

The way in which community structure and species attributes determine fluxes of energy in ecosystems is an important topic in ecology. The study of food webs provides a quantitative framework in our understanding of links between community structure and ecosystem functioning [START_REF] Thompson | Food webs: reconciling the structure and function of biodiversity[END_REF]. Food webs are networks determining the number and direction of trophic interactions in ecosystems and provide a natural framework for understanding the ecological role of species. Identifying how and to what extent these webs are structured is necessary if we hope to move beyond a "black-box" approach and toward a more mechanistic understanding of the ecosystem processes and functions that they drive [START_REF] Bascompte | Structure and dynamics of ecological networks[END_REF][START_REF] De Deyn | The response of nematodes from different trophic levels in the soil food web to the identity and diversity of plant species[END_REF][START_REF] Ings | Ecological networksbeyond food webs[END_REF][START_REF] Rooney | Integrating food web diversity, structure and stability[END_REF][START_REF] Stouffer | Evolutionary Conservation of Species' Roles in Food Webs[END_REF].

However, the presence of incompletely described feeding relationships hampers our understanding of complex and dynamic food webs in nature [START_REF] Kuwae | Variable and complex food web structures revealed by exploring missing trophic links between birds and biofilm[END_REF]. A clear example is the scarcity of existing data on microbial food webs, which, for practical reasons are often limited to experimental studies using a very limited number of taxa and often in combinations that do not reflect any natural system [START_REF] Pascal | Bacterivory of a mudflat nematode community under different environmental conditions[END_REF][START_REF] Petchey | Environmental warming alters food-web structure and ecosystem function[END_REF][START_REF] Tittel | Mixotrophs combine resource use to outcompete specialists: Implications for aquatic food webs[END_REF][START_REF] Wilken | Mixotrophic organisms become more heterotrophic with rising temperature[END_REF].

A major impediment in determining microbial food web structure probably stems from the difficulty in identifying interspecific links and collecting the required quantitative dietary data. Microbial communities are composed of numerous (and often poorly described or altogether unknown) species that can form complex food webs with numerous feeding links.

Discovering the structure of these food webs is important because they may affect processes occurring at the population, community, and ecosystem level [START_REF] Kuwae | Variable and complex food web structures revealed by exploring missing trophic links between birds and biofilm[END_REF]. The true trophic position and diversity in trophic roles of different microbial species and/or species group of different species was until recently all but impossible to determine. The increasing sensitivity of analytical tools now makes it possible to study ever-smaller organisms using stable isotope probing [START_REF] Pascal | Bacterivory of a mudflat nematode community under different environmental conditions[END_REF][START_REF] Jassey | Characterizing the Feeding Habits of the Testate Amoebae Hyalosphenia papilio and Nebela tincta along a Narrow "Fen-Bog" Gradient Using Digestive Vacuole Content and 13C and 15N Isotopic Analyses[END_REF]. Stable isotope ratios of carbon and nitrogen are increasingly used to provide time-integrated information about feeding relationships and food web structure, as showed for aquatic food webs [START_REF] Anderson | Anthropogenic alterations of lotic food web structure, evidence from the use of nitrogen isotopes[END_REF][START_REF] Carscallen | Estimating trophic position in marine and estuarine food webs[END_REF][START_REF] Mao | Food web structure of a shallow eutrophic lake (Lake Taihu, China) assessed by stable isotope analysis[END_REF]. By providing a temporally and spatially averaged estimate of carbon and nitrogen that is actually assimilated by organisms [START_REF] Schmidt | Quantitative approaches to the analysis of stable isotope food web data[END_REF], stable isotopes can improve our estimates of the major pathways of energy flows through microbial food webs [START_REF] Vander Zanden | Primary consumer delta C-13 and delta N-15 and the trophic position of aquatic consumers[END_REF].

We focused here on microbial communities living in Sphagnum-peatlands. In this diverse community a group of protozoa, the testate amoebae, are especially abundant. Testate amoebae represent an ideal model group for microbial food web studies in terrestrial ecosystems owing to their abundance, diversity, and ubiquity [START_REF] Mitchell | Testate amoebae analysis in ecological and paleoecological studies of wetlands, past, present and future[END_REF]. Testate amoebae are usually considered as feeding on a wide range of food sources, such as bacteria, fungi, microalgae, ciliates, rotifers and nematodes [START_REF] Jassey | Characterizing the Feeding Habits of the Testate Amoebae Hyalosphenia papilio and Nebela tincta along a Narrow "Fen-Bog" Gradient Using Digestive Vacuole Content and 13C and 15N Isotopic Analyses[END_REF][START_REF] Gilbert | Le régime alimentaire des thécamoebiens (Protista, Sarcodina)[END_REF][START_REF] Gilbert | Population dynamics and food preferences of the testate amoeba Nebela tincta major-bohemica-collaris complex (Protozoa) in a Sphagnum peatland[END_REF][START_REF] Laybourn | The effect of diet and temperature on reproductive rate in Arcella vulgaris ehrenberg (Sarcodina, Testacida)[END_REF][START_REF] Yeates | Testate amoebae as predators of nematodes[END_REF]. As a result, they are considered top-predators in microbial food webs [START_REF] Meyer | Effects of urban particulate deposition on microbial communities living in bryophytes, An experimental study[END_REF][START_REF] Meyer | Using Bryophyte-Testate Amoebae Microsystems as Indicators of Atmospheric Pollution at Three Different Sites (Rural, Urban and Industrial)[END_REF][START_REF] Meyer | Using testate amoebae as potential biointegrators of atmospheric deposition of phenanthrene (polycyclic aromatic hydrocarbon) on "moss/soil interface-testate amoebae community" microecosystems[END_REF]. As such, they play an important role in carbon and nutrient cycling in soils [START_REF] Aoki | Silica and testate amoebae in a soil under pineoak forest[END_REF][START_REF] Jassey | Above-and belowground linkages in Sphagnum-peatland: warming affects plant-microbial interactions[END_REF][START_REF] Schröter | C and N mineralization in the decomposer food webs of a European forest transect[END_REF][START_REF] Wilkinson | Testate amoebae and nutrient cycling with particular reference to soils[END_REF]. However, in order to understand the role of testate amoebae in carbon and nutrient cycling, key aspects of their ecology need to be understood, including how they obtain energy and nutrients. Precise information on the feeding habits of testate amoebae is limited to a few species [START_REF] Wilkinson | Testate amoebae and nutrient cycling with particular reference to soils[END_REF]. The broad range of sizes (ca. 5-500 µm in length, corresponding to volumes spanning six orders of magnitude) and high diversity of morphologies and functional traits found among testate amoebae [START_REF] Fournier | Toward the use of testate amoeba functional traits as indicator of floodplain restoration success[END_REF][START_REF] Mitchell | Testate amoebae analysis in ecological and paleoecological studies of wetlands, past, present and future[END_REF] suggest that they play different functional roles in microbial food webs. Species commonly described as omnivores probably occupy a different trophic level in the microbial network than specific feeders such as bacterivores or predators. Furthermore, many pigmented (photosynthetic) microorganisms are known or suspected to be mixotrophic (i.e. organisms that combine autotrophy and heterotrophy using endosymbiotic zoochlorellae); these include several testate amoeba species such as Hyalosphenia papilio and Archerella flavum [START_REF] Chacharonis | Observations on the ecology of protozoa associated with Sphagnum[END_REF][START_REF] Schönborn | Untersuchungen über die Zoochlorellen-Symbiose der Hochmorr-Testacean[END_REF], species that are commonly found in northern Sphagnum peatlands [e.g. [START_REF] Mitchell | Testate amoebae analysis in ecological and paleoecological studies of wetlands, past, present and future[END_REF][START_REF] Booth | Ecology of testate amoebae in a North Carolina pocosin and their potential use as environmental and paleoenvironmental indicators[END_REF][START_REF] Heal | Abundance and microdistribution of testate amoebae (Protozoa: Rhizopoda) in Sphagnum[END_REF][START_REF] Heal | Observations on the seasonal and spatial-distribution of testacea (protozoa, rhizopoda) in Sphagnum[END_REF][START_REF] Heal | The Distribution of Testate Amoebae (Rhizopoda: Testaecea) in Some Fens and Bogs in Nothern England[END_REF].

The mixotrophic life trait has significant functional and physiological implications [START_REF] Tittel | Mixotrophs combine resource use to outcompete specialists: Implications for aquatic food webs[END_REF][START_REF] Flynn | Misuse of the phytoplankton-zooplankton dichotomy: the need to assign organisms as mixotrophs within plankton functional types[END_REF][START_REF] Hartmann | Mixotrophic basis of Atlantic oligotrophic ecosystems[END_REF] and should theoretically translate into distinct isotopic ratios. Hence, the use of isotopic analyses may provide a mechanistic understanding on how testate amoebae are trophically structured, highlighting the strategy and functional role of dominant species in food web.

Here, we aim to track energy flow through microbial food chains from lower to higher trophic levels to disentangle the trophic structure of testate amoeba populations in microbial food web; and to elucidate the role of the mixotrophic strategy in microbial food web functioning. We hypothesize that the trophic level -based on isotopic enrichment-of testate amoebae within the microbial network relate to their feeding habit (bacterivory, algivory, and predation) and metabolism (heterotrophy vs. mixotrophy). Considering the key role of foraging traits such as density of prey in the feeding habit of testate amoebae [START_REF] Jassey | Characterizing the Feeding Habits of the Testate Amoebae Hyalosphenia papilio and Nebela tincta along a Narrow "Fen-Bog" Gradient Using Digestive Vacuole Content and 13C and 15N Isotopic Analyses[END_REF], we also hypothesize that prey density affects the trophic position of testate amoebae. For this, we explored the microbial food web associated with Sphagnum moss by combining predation of testate amoebae (digestive vacuole contents and predatory-prey associations), analyses of community density and biomass, and isotopic signatures (δ 13 C and δ 15 N) of testate amoebae and their potential prey, including microalgae, cyanobacteria, rotifers and nematodes.

Materials and methods

Study area and experimental design

We sampled one peat monolith (43.5 cm x 33.5 cm, 22 cm in depth) in a macroscopically homogeneous Sphagnum fallax lawn in a peatland in the Jura Mountains (Sur-les-Seignes, Frambouhans-Les Ecorces, France, 47°18'N, 6°79'E) in late-November 2010. The core was then placed under controlled conditions to quickly reactivate microbial life, average temperature of 22°C (day) and 18°C (night), a light cycle of 16h/8h (light/dark) and a light intensity of 0.029 kW.m -2 . The depth to water table (DWT) was maintained at constant level (6.5 cm below the top of the moss carpet) by adding standardized solution (Volvic water = 0.8 L every 2 days). We measured the physicochemical conditions of the peat core when the container was placed under controlled conditions. Temperature of the water table and at the surface was of 5°C and 12°C, respectively; pH = 4.4; and conductivity = 20.1 µS cm -1 . After 15 days these values increased (average temperature of the water table and at the surface of 17°C and 20°C, respectively, pH = 4.9, conductivity = 44.1 µS cm -1 ) and stabilised.

After 15 days, the Sphagnum carpet from the peat monolith was subdivided into three subsamples. For each subsample, the totality of the living apical green segments of Sphagnum shoots (top 3 cm) was collected. We randomly sampled 5 g of fresh material (ca. fifteen shoots) in each sub-plot and fixed it in 20 ml of glutaraldehyde (2% final concentration) for microbial community analysis (density, biomass, and diversity). The remaining material was used for isotopic analyses. Fully opened healthy capitula of Sphagnum (top 3 cm) were also sampled and lyophilized for isotopic analyses. No moss was taken from the marginal 5 cm border zone of the peat monolith to avoid possible border effects.

Microscopic and isotopic analyses

All microorganisms were extracted from S. fallax following the method used by Jassey et al. [START_REF] Jassey | Characterizing the Feeding Habits of the Testate Amoebae Hyalosphenia papilio and Nebela tincta along a Narrow "Fen-Bog" Gradient Using Digestive Vacuole Content and 13C and 15N Isotopic Analyses[END_REF]. Microalgae, cyanobacteria, protozoa, rotifers, nematodes and fungi were identified and quantified at 400x magnification by means of inverted microscopy (Olympus IX71) and following Uthermöhl's method [START_REF] Uthermölh | Zur vervollkommnung der quantative phytoplankton-methodik[END_REF]. Bacterial density was estimated by flow cytometry (FAC-SCalibur flow cytometer, Becton Dickinson). Fluorescent microbeads (molecular probes) 1 µm in diameter were added to each sample as an internal standard. Bacteria were stained with SYBR Green I (1/10,000 final conc.) for 15 min in the dark and samples run at medium speed (40 µL min -1 ). Bacterial size was estimated by epifluorescence microscopy using image analysis. The feeding habit of the dominant testate amoeba species, Hyalosphenia papilio, was studied in detail. To this aim, a total of 670, 715, and 500 specimens of H. papilio were observed by inverted microscopy in the three subsamples, respectively. Among active specimens, we distinguished between individuals with a prey within the shell and those that were actively feeding (determined as attached to prey or organic matter particles), to determine the feeding habit of this species, as described in Gilbert et al. [START_REF] Gilbert | Population dynamics and food preferences of the testate amoeba Nebela tincta major-bohemica-collaris complex (Protozoa) in a Sphagnum peatland[END_REF]. Such an analysis was not possible for others testate amoeba species due to their low abundance.

The biovolume of each morpho-species or group was estimated using geometrical shapes and then converted to carbon using the following conversion factors, heterotrophic bacteria, 1µm 3 = 5.6 x 10 -7 µgC [START_REF] Bratbak | Bacterial biovolume and biomass estimations[END_REF]; cyanobacteria and microalgae, 1µm 3 = 1.2 x 10 -7 µgC; ciliates and testate amoebae, 1µm 3 = 1.1 x 10 -7 µgC [START_REF] Weisse | Response of the microbial loop to the phytoplankton spring bloom in a large prealpine lake[END_REF]; fungi 1µm 3 = 2.5 x 10 -7 µgC; nematodes and rotifers, 1µm 3 = 1.25 x 10 -7 µgC [START_REF] Gilbert | The microbial loop at the surface of a peatland, Structure, function, and impact of nutrient input[END_REF]. These data were expressed as µgC per gram of S. fallax dry mass (µgC g -1 DM).

Collection and preparation of microbial samples for isotopic analyses follow Jassey et al. [START_REF] Jassey | Characterizing the Feeding Habits of the Testate Amoebae Hyalosphenia papilio and Nebela tincta along a Narrow "Fen-Bog" Gradient Using Digestive Vacuole Content and 13C and 15N Isotopic Analyses[END_REF]. We measured the isotopic signatures of cyanobacteria + microalgae (CA), fine organic matter including bacteria (FOMB), two abundant groups of micro-metazoa, small nematodes (< 200 µm) and rotifers, and the dominant testate amoeba species Arcella vulgaris, Archerella flavum, Bullinularia indica, Euglypha compressa, Hyalosphenia papilio and Nebela tincta. Microorganisms were extracted from fresh moss by six successive rinsing of S. fallax using distilled water and successively filtered at 150, 120, 70, 50, 30, 20 and 11 µm (SEFAR NITEX ®). The fraction under 11µm was used for FOMB and microalgae. For FOMB, the fraction was centrifuged at 15,000 r.min -1 for 20 min. Then, the supernatant was centrifuged again at 10,000 r.min -1 for 10 min and the pellet was used for isotopic measurements. All other categories were hand picked randomly and individually using micropipettes. Between 50 and 1,500 living testate amoeba specimens of the selected species were collected in each sub-sample, depending on their size (Table 1). In order to obtain reliable measurements, 0.1 mg of matter is required for isotopic measurement. To reach this amount, some samples had to be pooled from the three sub-plots, Arcella vulgaris, Archerella flavum, Bullinularia indica, Euglypha compressa, Nebela tincta, rotifers and nematodes, and for these no replicated measurements could be obtained. After 3 days, we also collected the CA biofilm using a micropipette. We estimated trophic positions (TP) following the method of Post [START_REF] Post | The long and short of food-chain length[END_REF]. We used S. fallax capitula as the baseline of the network for estimating trophic positions using δ 15 N, because we focused on microbial communities living in Sphagnum moss, and Sphagnum integrate temporal and spatial variation in isotopic signatures of basal resources [START_REF] Post | The long and short of food-chain length[END_REF].

Data analysis

We used linear mixed effects models to test for relationships between 1) δ 13 C and δ 15 N signatures of microbial species/groups, 2) δ 15 N and testate amoeba morphological traits (i.e. body size -length or diametershell aperture size and biovolume). These models were built while accounting for the pseudo-replication measurements in the peat monolith. We fitted all models including sub-plot as a random effect on the intercept, i.e. we corrected for the inflation of the residual degrees of freedom that would occur if we were using repeated measurements as true replicates. Regression diagnostics (leverage and outliers effects) and global validations of linear model assumptions (normality of the data distribution and homogeneity of variance) were examined and tested as described in Fox and Weisberg [START_REF] Fox | An R Companion to Applied Regression, Second Edition[END_REF].

Differences in TP of identified groups or species were assessed using non-parametric Mann Whitney tests. A hierarchical cluster analysis using the Ward distance method and Bray Curtis dissimilarity index were computed on δ 13 C and δ 15 N, isotopic nitrogen over carbon ratio, and trophic level values in order to identify feeding types of species and/or microbial groups. The robustness and the significance of the cluster were tested using bootstrap resampling (i.e. 1000 resampling from the data).

All statistical analyses were performed with R 2.15.1 using the ade4, car and pvclust packages [START_REF] Fox | An R Companion to Applied Regression, Second Edition[END_REF]45,[START_REF]R, A Language and Environment for Statistical Computing[END_REF].

Results

Microbial community structure

In term of biomass, the microbial community was composed of 53% of decomposers (bacteria and fungi), 17.3% microbial primary producers (microalgae and cyanobacteria), and 29.8% of consumers (all other groups; Fig. 1a). Bacteria accounted for almost half of the total biomass (726 ± 232 µgC g -1 DM), while fungi represented only 6.6% (103 ± 53 µgC g -1 DM; Fig. 1).

Testate amoebae represented about one-quarter of the total biomass (413 ± 176 µgC g -1 DM) and were the dominant group of consumers while rotifers made up only 3.1% of the total biomass (49 ± 37 µgC g -1 DM; Fig. 1a). Ciliates and nematodes together represented less than 1% of the total biomass (3 ± 1 and 2 ± 0 µgC g -1 DM, respectively). Primary producers were dominated by microalgae (14.7% of the total biomass), while cyanobacteria accounted for a much lower fraction of the total biomass (ca. 2.6%). The green alga Cylindrocystis brebissonii was the more abundant species of microalgae with more than 90% of the microalgal biomass, whereas Aphanothece nidulans and Anabaena cylindrica were the most abundant species of cyanobacteria (54% and 34% of cyanobacteria biomass, respectively). Hyalosphenia papilio strongly dominated the testate amoeba community and accounted for 89.8% of its biomass (Fig. 1b).

Feeding habit of Hyalosphenia papilio: digestive vacuole contents approach On average 25% of the observed Hyalosphenia papilio specimens were associated with prey.

Among the identified food sources, 80% were microbial primary producers (CA = microalgae and cyanobacteria, e.g. Aphanothece nidulans), 12% were fungi (including hyphae of ascomycetes and spores of Helicoon pluriseptatum) and 6% were ciliates (e.g. Platyophrya sphagni) (Table 2). Predation on other testate amoebae (e.g. Archerella flavum), rotifers (e.g.

Habrotrochoa sp. and Colurella sp.) and nematodes was observed only rarely (<1% of the total identified predator-prey associations). These proportions matched very closely the density of these organisms in the environment (Table 2). These frequencies represented in term of grazed biomass 9.8% of the biomass of CA, 20.1% of that of fungi and 49.3% of that of ciliates.

Isotopic composition of the microbial food web

The isotopic values of cyanobacteria and microalgae (CA) (δ 13 C = -26.8‰, δ 15 N = -6.7‰)

were almost identical to that of Sphagnum (δ 13 C = -28.0‰; δ 15 N = -5.4‰), which is therefore used as a baseline for further interpretation. By contrast, all other microbial groups yielded enriched δ 13 C and δ 15 N values relative to the baseline value determined from S. fallax shoots (Fig. 2, Table 1).

The δ 13 C and δ 15 N signatures of consumers covered a broad range of values, from -27.3‰ to -21.6‰ and -1.9‰ to +1‰, respectively. Isotopic values were significantly higher in consumers (testate amoebae, rotifers and nematodes) as compared to primary producers (S. fallax and CA; P = 0.04, r = 0.5). For instance, the isotopic diagram showed that Archerella flavum was more enriched in δ 13 C (+ 1.6‰) and δ 15 N (+ 2.9‰) than FOMB (Fig. 2). Isotopic values of Arcella vulgaris (δ 13 C = + 0.3‰; δ 15 N = + 2.1‰), B. indica (δ 13 C = + 0.7‰; δ 15 N = + 3‰), and N. tincta (δ 13 C = + 0.3‰; δ 15 N = + 1.7‰) were also consistently enriched relative to those of their supposed diet (CA) (Fig. 2, Table 1). Our results also showed that the mixotrophic species H. papilio was highly enriched in δ 13 C (+ 5.2‰), as compared to its diet (CA) (Fig. 2, Table 2). This was not the case for the other mixotrophic species studied (A. flavum).

Translating these results into trophic position, primary producers (CA) had the same trophic level as S. fallax in the microbial food web (TP = 1), while FOMB (TP = 2) and consumers (TP ranged from 2.3 to 2.9) had significant higher trophic level than primary producers (P < 0.05) (Table 1, Fig. 3b). In the cluster analysis, microbial groups were split into primary producers and consumers. The microbial consumers were further split into five sub-groups corresponding to different feeding strategies (Fig. 3a,b, Table 1). The first subgroup corresponded to mixotrophic algivores (C1) composed by H. papilio (Fig. 3). The second sub-group included decomposers (FOMB; C2), while the third and fourth sub-groups corresponded to bacterivores (C3) including A. flavum and nematodes, and heterotrophic algivores (C4) including A. vulgaris, B. indica, N. tincta (Fig. 3, Table 1). The fifth sub-group (C5) included E. compressa and rotifers and corresponded to a mix between algivores and bacterivores (Fig. 3, Table 1). Furthermore, we found that the trophic level of H. papilio was linked to the contribution of ciliates and CA to its total diet (Fig. 4). We found that the trophic level of H. papilio increased with the rise of ciliates and the decrease of CA in its total diet (Fig. 4a,b).

The δ 15 N values of testate amoebae were negatively correlated to simple morphological traits such as size of their shell aperture (r = -0.86, P = 0.02) and their biovolume (r = -0.88, P = 0.01), while no significant correlation was found with their body size (length or diameter) (r = -0.72, P = 0.07, ANOVA).

Discussion

We investigated the trophic structure of the microbial food web associated to Sphagnum moss by combining analyses of microbial density and biomass, stable isotopes (δ 13 C and δ 15 N) of microbial groups and digestive vacuole content of key microbial consumers. We found that the trophic level of microbial organisms inferred from stable isotopes was in line with expectations; bacteria (FOMB) have a higher trophic level than autotrophs (S. fallax, CA), and microbial consumers a higher trophic level than both decomposers and autotrophs. We further showed that isotopic signatures of moss and CA did not differ and that both autotrophic microorganisms and Sphagnum mosses have similar directional shifting in food web space. This agrees with our assumption that autotrophs can be used as the baseline of the microbial food web associated to Sphagnum moss.

Trophic structure of the microbial food web

In most cases, we found isotopic enrichments in line with those of aquatic and soil food web in which consumer δ 15 N signatures were higher than 3‰ [START_REF] Post | The long and short of food-chain length[END_REF][START_REF] Kohzu | Food webs in Mongolian grasslands : The analysis of 13C and 15N natural abundances[END_REF]. Based on these isotopic enrichments and identified feeding habit, we showed two trophic pathways in our microbial network: a herbivorous pathway and a detritivorous pathway. Firstly, we found a significant enrichment in δ 15 N between microbial autotrophs (CA) and microbial consumers such as Nebela tincta, Euglypha compressa, Hyalosphenia papilio, Arcella vulgaris and rotifers, characterizing the herbivorous pathway. These isotopic enrichments indicated that these consumers mainly fed on CA in the current study. This agrees with our feeding observations on H. papilio, as well as with previous observations of feeding habits for Arcella vulgaris, Euglypha compressa and rotifers [START_REF] Gilbert | Le régime alimentaire des thécamoebiens (Protista, Sarcodina)[END_REF][START_REF] Laybourn | The effect of diet and temperature on reproductive rate in Arcella vulgaris ehrenberg (Sarcodina, Testacida)[END_REF][START_REF] Segers | Global diversity of rotifers (Rotifera) in freshwater[END_REF][START_REF] Sharma | Rotifer communities of floodplain lakes of the Brahmaputra basin of lower Assam (NE India), biodiversity, distribution and ecology[END_REF]. Secondly, the significant δ 15 N enrichment between FOMB and consumers such as Archerella flavum and nematodes suggests the existence of a detritivorous pathway. A. flavum has been described as exclusively autotrophic [START_REF] Schönborn | Untersuchungen über die Zoochlorellen-Symbiose der Hochmorr-Testacean[END_REF], but its isotopic signature rather indicated that this species mainly fed on bacteria or fine organic particles (FOMB). Although food particles are rarely observed in A. flavum vacuoles, this does not exclude the possibility that bacteria occur in their vacuole. Indeed, bacteria are difficult to identify in testate amoeba vacuoles when using direct observation with epifluorescence microcopy (pers. observation). Obviously, the recorded isotopic signatures do not exclude that microbial consumers feed on additional food sources and/or intermediate trophic level [START_REF] Ponsard | Assessing top-down and bottom-up control in a litterbased soil macroinvertebrate food chain[END_REF][START_REF] Scheu | The soil food web beech forests (Fagus sylvatica) of contrasting hulus type: stable isotope analysis of a macro-and a mesofauna-dominated community[END_REF], as clearly showed for H. papilio.

More surprising is the difference between the isotopic signatures of H. papilio and N. tincta between this study and the one of Jassey et al. [START_REF] Jassey | Characterizing the Feeding Habits of the Testate Amoebae Hyalosphenia papilio and Nebela tincta along a Narrow "Fen-Bog" Gradient Using Digestive Vacuole Content and 13C and 15N Isotopic Analyses[END_REF]. These results reflect inter-site differences, showing that the isotopic signatures of a given species can be quite variable in space and/or time depending on environmental conditions [START_REF] Ponsard | Assessing top-down and bottom-up control in a litterbased soil macroinvertebrate food chain[END_REF]. However, the similar signature of the baseline (i.e. Sphagnum fallax) between the two studies also underlines differences of inter-site food web functioning. The linkages between the trophic position of H. papilio and the contribution of main categories of prey to its total diet agree the hypothesis that differences in prey-availability influence trophic position of testate amoebae. Indeed, isotopic signatures and trophic levels of top-predators, H. papilio and N. tincta (TPH. papilio, 2.3 and TPN. tincta, 2.4), were significantly lower than those recently obtained by Jassey et al.

(2012; TPH. papilio, 3.5 and TPN. tincta, 4.2). Such results are not surprising when we consider the low density of the potential energetic prey for H. papilio and N. tincta recorded in this study (e.g. ciliates, rotifers and nematodes). Because of the very low relative abundances of its preferential food sources (i.e. ciliates; [START_REF] Jassey | Characterizing the Feeding Habits of the Testate Amoebae Hyalosphenia papilio and Nebela tincta along a Narrow "Fen-Bog" Gradient Using Digestive Vacuole Content and 13C and 15N Isotopic Analyses[END_REF]), our results suggest that H. papilio adapts its feeding behaviour by targeting the most abundant and available food sources (e.g. CA). This agrees with the predictions of optimal foraging theory, which states that organisms forage in such a way as to maximize their net energy intake. Optimally-foraging organisms can minimize the energy cost of food uptake by targeting the most abundant and easily captured prey [START_REF] Akin | Body size and trophic position in a temperature estuarine food web[END_REF][START_REF] Stephens | Foraging Theory[END_REF]. We estimated that predation of H. papilio on ciliates represented up to 50% of the total standing biomass of ciliates. This suggests that the density of ciliates was probably lower than a threshold allowing higher active predation of H. papilio on this otherwise energetically optimal prey. Our results reveal that testate amoeba species occupy different trophic positions in the microbial food web and that these differences could be related to their feeding type.

Conversely, this study illustrates that it is difficult to reliably infer trophic position of testate amoebae using relatively simple morphological traits without taking into account prey stocks.

Food webs usually exhibit a surprisingly consistent topological architecture across different ecosystem types [START_REF] Ings | Ecological networksbeyond food webs[END_REF][START_REF] Schneider | Body mass constraints on feeding rates determine the consequences of predator loss[END_REF] mediated by fundamental constraints where species feed on nearly contiguous ranges of mostly lower ranked species [START_REF] Stouffer | Quantitative analysis of the local structure of food webs[END_REF]. In general, species with large body size occupy high trophic levels and consume many food sources, whereas smaller species share low trophic levels with more specific food strategies (e.g. bacterivory; [START_REF] Brose | Climate change in size-structured ecosystems Introduction[END_REF]). Body size is thus a fundamental indicator of trophic level in food webs because it places constraints on many biological variables, including crucial foraging traits (energy content of resources, handling times, attack rates, and prey density) [START_REF] Barnes | Global patterns in predator-prey size relationships reveal size dependency of trophic transfer efficiency[END_REF][START_REF] Heckmann | Interactive effects of body-size structure and adaptive foraging on food-web stability[END_REF][START_REF] Layman | Body size and trophic position in a diverse tropical food web[END_REF][START_REF] Thierry | The consequences of size dependent foraging for food web topology[END_REF]. However, strong relationship between body size (here testate amoebae shell length or diameter) and trophic position cannot be found for consumers that exploit a wide range of prey body sizes [START_REF] Kuwae | Variable and complex food web structures revealed by exploring missing trophic links between birds and biofilm[END_REF][START_REF] Layman | Body size and trophic position in a diverse tropical food web[END_REF], as shown here with H. papilio and N. tincta. Nevertheless, we found that the size of shell aperture and biovolume were inversely proportional to the trophic position of testate amoebae in the microbial network, while usually these morphological traits are considered as indicators of high trophic level [START_REF] Jassey | Characterizing the Feeding Habits of the Testate Amoebae Hyalosphenia papilio and Nebela tincta along a Narrow "Fen-Bog" Gradient Using Digestive Vacuole Content and 13C and 15N Isotopic Analyses[END_REF][START_REF] Meyer | Using testate amoebae as potential biointegrators of atmospheric deposition of phenanthrene (polycyclic aromatic hydrocarbon) on "moss/soil interface-testate amoebae community" microecosystems[END_REF][START_REF] Jassey | Above-and belowground linkages in Sphagnum-peatland: warming affects plant-microbial interactions[END_REF]. Again, such results illustrate the importance of foraging traits such as the density of preferential food sources on trophic position of consumers such as H. papilio and N. tincta, and therefore on the food web structure. Indeed, if we compare these results to those of Jassey et al. [START_REF] Jassey | Characterizing the Feeding Habits of the Testate Amoebae Hyalosphenia papilio and Nebela tincta along a Narrow "Fen-Bog" Gradient Using Digestive Vacuole Content and 13C and 15N Isotopic Analyses[END_REF], these data indicate that two morphologically similar populations of the same species (e.g. H. papilio) may occupy very different trophic positions, thus altering any overall relationship between morphology and trophic position in Sphagnumassociated food web. Furthermore, the inverse relationship between morphological trait-sizes and trophic levels showed that smaller testate amoeba species such as A. flavum -found as a consumer of FOMB-occupied the top of the food web. This suggested that bacterivores received more translocated nitrogen from the detritivorous pathway than herbivores from the herbivorous pathway, giving a greater functional role to bacterivores. This isotopic signature of bacteria -and thus their trophic level-depends of the signature of the detrital source, which may sometimes be higher than that of autotrophs [START_REF] Ponsard | Assessing top-down and bottom-up control in a litterbased soil macroinvertebrate food chain[END_REF]. Therefore, a switch from an herbivorous to a detritivorous dominant pathway could break down the morphological relationships between trait-size and trophic level.

Algal-endosymbiont contribution to host Hyalosphenia papilio diet

The highly enriched δ 13 C content of H. papilio (i.e. -22‰ on average) suggested that this species used another non-identified 13 C baseline than other testate amoeba species (i.e. for which the average was -26‰), explaining why this species showed such distinct isotopic patterns. An alternative hypothesis is that H. papilio used another source of carbon than CA and ciliates such as their endosymbiotic algae. Few studies have attempted to quantify the energetic benefits of endosymbiotic algae of mixotrophic species but Schönborn [START_REF] Schönborn | Untersuchungen über die Zoochlorellen-Symbiose der Hochmorr-Testacean[END_REF] suggested that H. papilio could probably live without food sources using these endosymbiotic algae as alternate energy source. The low availability of energetic resources probably led H. papilio to use its endosymbionts as complementary energy source, and thus to receive more translocated carbon from the stimulated activity of its endonsymbionts than from CA, explaining why δ 13 C signature of H. papilio was so high. By analogy to our results, Bergschneider and Muller-Parker [START_REF] Bergschneider | Nutritional role of two algal symbionts in the temperate sea anemone Anthopleura elegantissima Brandt[END_REF] showed that the 13 C signature of zoochlorellae increased from -25‰ to -20‰ when they were used as complementary energy source in Anemona elegantissima. These authors also showed that zoochlorellate anemone exhibited distinctly lower patterns of 15 N values than non-zoochlorellate anemone indicating that symbiotic anemones received nutrition from both external (heterotrophic) and internal (translocated algal products) sources. Although our results do not allow drawing strong conclusion about the use of endonsymbionts as a complementary source of energy in H. papilio specimens, they suggest that resource availability may play a fundamental role in the use of endosymbionts as energy source.

The difference between the isotopic signatures of the two-mixotrophic species studied (H. papilio and A. flavum) provides evidence in support the hypothesis that resource availability may play a fundamental role in the use of endosymbionts as energy source.

Although obvious morphological differences in overall size and size of shell aperture occur between these mixotrophic species, their range of prey available is likely to explain their different isotopic signatures. These results suggest that mixotrophic species may become more heterotrophic with abundant and available food sources, which alters their functional role in food webs and the carbon cycle. Indeed, a shift towards more heterotrophic nutrition may change the isotopic signature of mixotrophic specimens, and therefore their functional role from primary production towards consumers [START_REF] Wilken | Mixotrophic organisms become more heterotrophic with rising temperature[END_REF]. Such results highlight that dynamics in prey-predator abundance could modify the functional role of mixotrophic species in the microbial food web. Furthermore, these results also suggest that mixotrophic species can easily outcompete other competitors when food source are rare in networks by shifting on their endonsymbionts [START_REF] Tittel | Mixotrophs combine resource use to outcompete specialists: Implications for aquatic food webs[END_REF]. Hence, our results provide reason to expect that mixotrophic life history trait play key role for both prey abundance and microbial food web structure associated to Sphagnum.

In conclusion, our study demonstrates that nitrogen and carbon isotopic signatures of microorganisms are useful for estimating food web structure of microbial network associated to Sphagnum. These results show that testate amoeba consumers occupy different trophic levels within the microbial food web, depending to their feeding behavior, the density of their food resources and their metabolism (i.e. mixotrophy vs. heterotrophy). Our findings provide experimental support for the novel hypothesis that mixotrophy has key implications in food web structure and functioning [START_REF] Lopez-Urrutia | Scaling the metabolic balance of the oceans[END_REF][START_REF] Yvon-Durocher | Warming alters the metabolic balance of ecosystems[END_REF]. Given the key position of mixotrophs in microbial community in peatlands, changes in their functional role towards primary producers to consumers (or vice versa) may cascade through food webs, altering species interactions as well as the magnitude and direction of carbon fluxes.

  Food sources: B = bacteria; Fu = Fungi; CA = cyanobacteria and microalgae; Fl = flagellates; Ci = ciliates; T = testate amoebae; M = micrometazoan (rotifers, nematodes) Feeding type: 0A = photosynthetic organism (protist); 1 = OM feeders; 2A = bacterivores; 2B = primary producer feeders; 2C = bacteria and primary producer feeders

Figure 1 :

 1 Figure 1: (a) Relative biomass of different microbial groups (% of the total microbial

Figure 2 :

 2 Figure 2: Stable isotope (δ 13 C and δ 15 N) food web diagram of the major microbial groups and

Figure 3 :

 3 Figure 3: (a) Hierarchical clustering (Bray and Curtis index method) of microbial groups and testate amoeba species from Sphagnum fallax based on their δ 13 C and δ 15 N, isotopic nitrogen

Figure 4 :

 4 Figure 4: (a) Contribution of food sources to the diet of Hyalosphenia papilio. Error bars,

Table 1 :

 1 Number of specimen sampled (n), weight of species sampled (mg, mean ± standard error), isotopic ratios (‰, mean ± standard error) of carbon and nitrogen in Sphagnum fallax, microbial groups and testate amoeba species. Trophic level, feeding type and strategy of microbial groups and testate amoeba species.

	553

Table 2 :

 2 Relative proportion of the identified prey ingested by Hyalosphenia papilio.Relative proportion of the abundance of microbial groups in Sphagnum fallax, excluding bacteria. CA = microalgae and cyanobacteria. Relative proportion of the biomass of the identified prey ingested by H. papilio.

		Frequency of the preys ingested by H. papilio (%)	Frequency of identified prey (%)	Frequency of ingested organisms in term of biomass (%)
	CA	80%	82.4%	9.8%
	Fungi	12%	11.8%	20.1%
	Ciliates	6%	0.4%	49.3%
	Testate amoebae	0%	4.7%	< 0.1%
	Rotifers	0.6%	0.5%	0.8%
	Nematods	0%	0.2%	< 0.1%

Acknowledgments

This research is a contribution of the ANR PEATWARM project (Effect of moderate warming on the functioning of Sphagnum peatlands and their function as carbon sink) and the µPOL-AIR project (Use of Sphagnum-peatlands to quantify the deposition of long-range air pollutants and to evaluate their impact on environment). PEATWARM is supported by the French National Agency for Research under the ''Vulnerability, Environment-Climate'' Program (ANR-07-VUL-010) and µPOL-AIR by PRIMEQUAL program (support of French Ministry of Environment and ADEME) (2010-Q.3-Chorus2100082984). Further funding was provided to VEJ.J by the Franche-Comté Region, to CM by µPOL-AIR project and to EAD.M and AP.C. by the University of Neuchâtel. The authors would like to thank T. Sime-Ngando and J. Colombet (University of Clermont-Ferrand, FR) for their help with the flow cytometry and two anonymous reviewers for their valuable comments on the manuscript.


S Como

C Lefrancois

E Maggi

F Antognarelli

C Dupuy

Behavioral responses of juvenile golden grey mullet Liza aurata to changes in coastal temperatures and consequences for benthic food resources

Keywords: Grey mullets, Liza aurata, feeding behavior, temperature, grazing, microphytobenthos, spatial variance

Temperature is an important factor for fish. Yet, little is known about its effects on the feeding behavior of fish and the subsequent consequences of these behavioral changes on the spatial distribution of resources. We analyzed the differences in the feeding behavior of two sizes of juvenile Liza aurata at two water temperatures (i.e. 10°C and 20°C), using laboratory mesocosms. We also examined whether temperature-induced changes in feeding behavior of the smaller size of L. aurata would affect the spatial distribution of the microphytobenthos (MPB) biomass, an important resource in coastal systems.

Both the number of feeding events and the swimming velocity during feeding in juvenile L. aurata were higher at 20°C than at 10°C, independently of the fish size. The time spent feeding did not vary between 10°C and 20°C, while the distance covered during feeding was significantly smaller at 20°C than at 10°C. Grazing did not affect the mean MPB biomass, but did increase its spatial variance at the smaller scale (i.e. a few centimeters) at 20°C.

A high number of feeding events, a high swimming velocity during feeding and a small distance covered during feeding in 20°C-acclimated L. aurata most likely represented an adaptation to an increase in metabolism, as well as to the need to reduce the energy costs of feeding at 20°C. Results also indicated that changes in feeding behavior of the 20°C-acclimated L. aurata were responsible for the increase in small-scale spatial variability in the MPB biomass. We suggested that the enhanced spatial patchiness due to grazing by fish at 20°C might yield a local increase in the mean MPB biomass, probably increasing photosynthetic efficiency of cells and algal growth that counterbalance the negative effect of algal removal by fish, and resulting in the lack of an overall significant effect on the mean.

Introduction

Temperature is one of the most important environmental factors for the physiology and metabolism of fish [START_REF] Mckenzie | The effects of environmental factors on the physiology of aerobic exercise[END_REF]. Consequently, it can affect all the behavioral traits of fish, including feeding behavior, as well as influence the outcomes of the consumer-resource interactions. If few studies have explicitly investigated the effect of temperature on the feeding behavior of fish (e.g., [START_REF] Nowicki | Interactive effects of elevated temperature and CO2 on foraging behavior of juvenile coral reef fish[END_REF][START_REF] Theodorou | Reaching the limit: Constrained behavioural flexibility of juvenile Atlantic cod (Gadus morhua) at current coastal temperatures[END_REF], even less has been done to understand how these behavioral changes influence the spatial or temporal distribution of resources in coastal areas [START_REF] Polunin | Algal food supply and grazer demand in a very productive coral-reef zone[END_REF][START_REF] Smith | Temperature effects on herbivory for an Indo-Pacific parrotfish in Panamá: implications for coral-algal competition[END_REF]. Furthermore, most of these studies have been focused on tropical herbivorous fish in coral reef systems. Very little is known about the effects of temperature on the feeding behavior of other fish inhabiting coastal systems. An example is the grey mullet (Mugilidae), one one of the most abundant fish in coastal areas worldwide [START_REF] Laffaille | Can thin lipped mullet directly exploit the primary and detritic production of European macrotidal salt marshes?[END_REF][START_REF] Cardona | Habitat selection by grey mullets (Osteichthyes: Mugilidae) in Mediterranean estuaries: the role of salinity[END_REF][START_REF] Lebreton | Trophic ecology of mullets during their spring migration in a European saltmarsh: a stable isotope study[END_REF][START_REF] Whitfield | A global review of the cosmopolitan flathead mullet Mugil cephalus Linnaeus 1758 (Teleostei: Mugilidae), with emphasis on the biology, genetics, ecology and fisheries aspects of this apparent species complex[END_REF].

Rises in temperature (within naturally occurring ranges) are usually accompanied by an increase in metabolism and energy demands [START_REF] Ferreira | Seasonal grazing rates and food processing by tropical herbivorous fishes[END_REF][START_REF] Biro | Mechanisms for climate-induced mortality of fish populations in whole-lake experiments[END_REF][START_REF] Biro | Small within-day increases in temperature affects boldness and alters personality in coral reef fish[END_REF] and, consequently, in food consumption and feeding activity [START_REF] Polunin | Algal food supply and grazer demand in a very productive coral-reef zone[END_REF][START_REF] Smith | Temperature effects on herbivory for an Indo-Pacific parrotfish in Panamá: implications for coral-algal competition[END_REF]. In particular, an increase in the number of feeding events [START_REF] Polunin | Ecological correlates of foraging periodicity in herbivorous reef fishes of the Coral Sea[END_REF][START_REF] Ferreira | Seasonal grazing rates and food processing by tropical herbivorous fishes[END_REF][START_REF] Floeter | Geographical gradients of marine herbivorous fishes: patterns and processes[END_REF][START_REF] Smith | Temperature effects on herbivory for an Indo-Pacific parrotfish in Panamá: implications for coral-algal competition[END_REF][START_REF] Nowicki | Interactive effects of elevated temperature and CO2 on foraging behavior of juvenile coral reef fish[END_REF], the amount of time spent feeding [START_REF] Biro | Mechanisms for climate-induced mortality of fish populations in whole-lake experiments[END_REF]) and the distances covered during feeding [START_REF] Biro | Small within-day increases in temperature affects boldness and alters personality in coral reef fish[END_REF][START_REF] Theodorou | Reaching the limit: Constrained behavioural flexibility of juvenile Atlantic cod (Gadus morhua) at current coastal temperatures[END_REF] have been observed with temperature increases.

In general, it has been shown through experiments that the addition of grazers or predators results in the reduction of the mean biomass (or density) of their resources [START_REF] Berlow | Strong effects of weak interactions in ecological communities[END_REF][START_REF] Armitage | Decoupling of nutrient and grazer impacts on a benthic estuarine diatom assemblage[END_REF]. If a consumer reduces the mean biomass of a resource, this should also cause a reduction in spatial variance of the biomass itself, due to the relationship between the mean and the variance [START_REF] Taylor | Aggregation, variance and the mean[END_REF]. In coral reef systems, [START_REF] Polunin | Algal food supply and grazer demand in a very productive coral-reef zone[END_REF] showed that the increase in the number of feeding events at increasing temperatures can lead to increases in algal consumption. It is therefore possible that the amount of algae removed and the amount of variance lost in an area both increase as water temperatures increase, even if the density of grazers and the availability of their resources do not vary in that area [START_REF] Polunin | Ecological correlates of foraging periodicity in herbivorous reef fishes of the Coral Sea[END_REF][START_REF] Beukers-Stewart | Behavioural and developmental responses of predatory coral reef fish to variation in the abundance of prey[END_REF]. However, whether or not grazing has negative effects on the mean and the spatial variance of the algal biomass depends on the time-scale of measurement and the interplay between the rates of algal removal and algal growth. Grazing is effective in reducing the mean and the spatial variance of algal biomass only if the rates of algal removal exceed the rates of algal growth. Furthermore, whether or not grazing decreases the overall spatial variance of a resource biomass depends not only on the mean effect of the trophic interaction, the overall biomass of the resource and the mathematical relationship between the mean and the variance, but also on the variance of the trophic interaction as well as the residual variability of the resource (i.e. the component of variation that is not due to grazing) [START_REF] Benedetti-Cecchi | Variance in ecological consumer-resource interactions[END_REF],2003;[START_REF] Benedetti-Cecchi | Interactive effects of spatial variance and mean intensity of grazing on algal cover in rock pools[END_REF]. The overall spatial variance of the resource is expected to decrease if the loss of variance caused by the reduction in the mean abundance of the resource is larger than the residual variability in the resource abundance, and if the negative effect caused by the reduction in the mean abundance of the resource is larger than the positive effect due to increased spatial variance of grazing.

In this study, we tested the effects of changes in water temperature on the feeding behavior of juvenile golden grey mullet Liza aurata, one of the most abundant European grey mullets inhabiting the Northwestern Atlantic coasts and the Mediterranean Sea [START_REF] Cardona | Habitat selection by grey mullets (Osteichthyes: Mugilidae) in Mediterranean estuaries: the role of salinity[END_REF][START_REF] Lebreton | Trophic ecology of mullets during their spring migration in a European saltmarsh: a stable isotope study[END_REF]. The feeding behavior was analyzed at 10°C and 20°C, two temperatures representing low and high seasonal values (which occur naturally during winter and summer, respectively) in the geographic distribution of L. aurata [START_REF] Cardona | Habitat selection by grey mullets (Osteichthyes: Mugilidae) in Mediterranean estuaries: the role of salinity[END_REF][START_REF] Lebreton | Trophic ecology of mullets during their spring migration in a European saltmarsh: a stable isotope study[END_REF]. We were also interested in understanding whether temperature-induced changes in the feeding behavior of L. aurata would affect the mean and the spatial variance of the microphytobenthos (MPB) biomass, an important benthic food resource for L. aurata [START_REF] Laffaille | Can thin lipped mullet directly exploit the primary and detritic production of European macrotidal salt marshes?[END_REF][START_REF] Pasquaud | Determination of fish trophic levels in an estuarine system[END_REF][START_REF] Lebreton | Trophic ecology of mullets during their spring migration in a European saltmarsh: a stable isotope study[END_REF]. We used a series of laboratory experiments in mesocosm with natural sediment, where we measured the feeding behavior of juvenile L. aurata and the mean and the spatial variance of MPB at each temperature (i.e. 10°C and 20°C). As juveniles must meet higher metabolic rates and energy demands at increasing temperatures, we expected (i) the number of feeding events, (ii) the total time spent feeding and/or (iii) the distance covered during feeding to be higher at increasing temperatures; as temperature can affect swimming performance in fish [START_REF] Claireaux | Effect of temperature on maximum swimming speed and cost of transport in juvenile European sea bass (Dicentrarchus labrax)[END_REF][START_REF] Deslauriers | The effects of temperature on swimming performance of juvenile shortnose sturgeon (Acipenser brevirostrum)[END_REF], we also expected (iv) the velocity of swimming during feeding to be higher at higher temperatures. Whether or not the grazing of grey mullets operates with large spatial or temporal variance able to enhance the patchiness of MPB is not known. Grey mullets, however, feed in schools [START_REF] Whitfield | A global review of the cosmopolitan flathead mullet Mugil cephalus Linnaeus 1758 (Teleostei: Mugilidae), with emphasis on the biology, genetics, ecology and fisheries aspects of this apparent species complex[END_REF] and their gregarious behavior should increase the likelihood of homogeneous grazing in an area [START_REF] Adler | The effect of grazing on the spatial heterogeneity of vegetation[END_REF]. At each experimental temperature, it is possible that the amount of variance in the MPB biomass caused by a heterogeneous grazing would be lower than the amount of MPB variance lost due to the reduction in the mean MPB biomass. Therefore, we expected grazing to have a negative effect (v) on the mean of the MPB biomass and (vi) on the amount of spatial variability of MPB but we also expected that (vii) the amount of microalgae removed and the amount of variance lost due to grazing would be higher at 20°C than at 10°C. Because feeding activity in juveniles often varies with fish size [START_REF] Choat | Daily feeding rates in herbivorous labroid fishes[END_REF][START_REF] Buckle | Ontogeny of space use and diet of two temperate damselfish species, Parma microlepis and Parma unifasciata[END_REF], tests on the feeding behavior were repeated with two sizes of juveniles (i.e. J0 and J1). However, only J0 was taken into account to test for the grazing effects on MPB, as this size dominates the natural populations in shallow coastal areas [START_REF] Hotos | Age and growth of the golden grey mullet Liza aurata (Actinopterygii: Mugiliformes: Mugilidae) in the Messolonghi-Etoliko lagoon and the adjacent Gulf of Patraikon, Western Greece[END_REF][START_REF] Lebreton | Trophic ecology of mullets during their spring migration in a European saltmarsh: a stable isotope study[END_REF].

Material and Methods

Experimental fish

Thirty-six J0 juveniles and 36 J1 juveniles (mean total length (TL) ± SE: 9.71± 0.23 cm and 16.10 ± 0.12 cm, respectively) were used in this experiment. The fish were collected along the Sardinian coast of Italy and then transported to the Marine Station of the University of La Rochelle (France), where the experiment was carried out. Fish were maintained for one month in 1000 liter holding tanks (1 × 1 × 1 m) filled with re-circulated filtered sea water. Temperatures and salinity ranged between 14 and 19°C (mean ± S.E: 17 °C ± 0.2, n=30) and 29 and 35 psu (mean ± S.E: 32 psu ± 0.3, n=30), respectively. After the mullets were transferred into experimental tanks, water temperatures were adjusted by 0.5 °C per day, until experimental temperatures were reached (10°C and 20°C). The fish were then allowed to acclimate to the experimental temperature for at least three months. During this period fish were under natural photoperiod of the location (46°10 N) and fed with natural periphyton daily and with dry pellets (BioMar) twice a week as an integration of proteins and vitamins. The use of natural periphyton allowed us to maintain the fish in laboratory conditions without compromising their natural foraging behavior [START_REF] Richard | Influence of periphyton substrates and rearing density on Liza aurata growth and production in marine nursery ponds[END_REF]. Natural periphyton was developed on artificial substrates in an adjacent pond, following the methods described in [START_REF] Richard | Influence of periphyton substrates and rearing density on Liza aurata growth and production in marine nursery ponds[END_REF].

Protocol

Grey mullets are a gregarious fish and exhibit schooling behavior [START_REF] Whitfield | A global review of the cosmopolitan flathead mullet Mugil cephalus Linnaeus 1758 (Teleostei: Mugilidae), with emphasis on the biology, genetics, ecology and fisheries aspects of this apparent species complex[END_REF]. Therefore, we used 3 fish for each run of the experiment. Each fish was used only once. Before being transferred into the mesocosm (4 × 1 × 1 m; Fig. 1), fish were marked on the opercula with plastic tags of different colors using Krazy glue, following the procedure described in [START_REF] Lefrancois | The effect of hypoxia on locomotor performance and behaviour during escape in Liza aurata[END_REF]. For marking, fish were lightly sedated with MS222 (0.08 g l -1 ). Each tagging procedure took <2 min and fish ventilated continuously with no noticeable decrease in ventilation rate. Fish never lost balance and resumed swimming as soon as they were transferred to the acclimatization chamber (Como pers. obs.).

Each fish group was placed in the acclimatization chamber of the mesocosm under natural photoperiod and was left undisturbed for 12 hours before the experiment in order to allow them to recover from any stress caused by this handling. Fish were not fed during this period.

An hour and a half before the beginning of the experiment, the water level of the working chamber of the mesocosm was raised by about 50 cm and the fish were encouraged to move from the acclimatization to the working chamber (where the sediment was positioned; see below). This was done by opening the sliding door and lifting up the floor of acclimatization chamber (Fig. 1). In general, the fish entered rapidly (less than 3 min for transfer). The experiment began when the fish started feeding on the sediment placed on the floor and lasted for 3 hours. The cameras were turned on as the fish entered the working chamber and recorded their foraging for the entire duration of the experiment. At the end, the fish were encouraged to move back to the acclimatization chamber and the water level was progressively reduced in the working chamber All the experimental runs were performed in the same period of the day (in the morning) in order to avoid a bias in the results done by daily variations in the fish feeding activity [START_REF] Almeida | Feeding ecology of Liza ramada (Risso, 1810) (Pisces, Mugilidae) in a southwestern estuary of Portugal[END_REF]. All precautions were taken to limit fish stress during the experiments. Each fish manipulation was done with careful handling. None of the fish showed abnormal behaviour or died during the experiment.

The sediment used in this study was collected on an intertidal mudflat in Aiguillon Bay (France; 46°10'N, 1°15'W) close to the Marine Station, by scraping off the upper 2 mm. The sediment was sieved through 5 mm screens to eliminate plant debris and then kept on plastic plates (0.60 × 0.40 × 0.05 m), laid on the bottom of a tank, which had the same shape and area of the working chamber of the mesocosm. Tanks were positioned inside an external greenhouse, so that the sediment experienced the same natural light conditions as in the field. Sediment was exposed to the natural tidal cycles of the area of collection, through a series of pumps, which controlled the flow of water in and out of the tanks. The water in the tanks was renewed daily to avoid excessive increases in water temperature and salinity due to water evaporation. Sediment was kept in the greenhouse for a period of three days before being used in the mesocosm. This was a sufficient period of time to recover from disturbance for both microalgal biomass and community structure [START_REF] Peterson | Resistance and resilience of lotic algal communities: importance of disturbance timing and current[END_REF]. For each run of the experiment, a different series of sediment plates was used. The mesocosm was set up in a room with natural sources of light in order to allow for photosynthesis to be carried out by microalgae during each run of the experiment.

Experimental designs

The experiment on the effect of changes in temperature on the feeding behavior of juvenile L. aurata consisted in the manipulation of two fixed, crossed factors: Temperature (T; T 10 and T 20 ) and Size (S; J0 and J1). For each combination, there were 6 replicates (i.e. replicate runs) with 3 juvenile fish. In order to compare the feeding behavior of fish of different sizes, the same ratio between fish biomass and foraging area (i.e. the area of the working chamber) was used for both fish sizes. A constant biomass of fish per unit area was thus used, ≈ 55 g m -2 . To attain this biomass, the surface area of the working chamber was set to 0.48 m² (i.e. 1.20 × 0.40 m) and to 1.44 m 2 (1.80 × 0.80 m) for J0 and J1, respectively. It corresponded to two and six adjacent plastic plates respectively (with the reconstructed sediment), which perfectly fitted on the bottom of the working chamber.

The influence of temperature on the effects of grazing on MPB biomass was assessed through the manipulation of two fixed, crossed factors: Temperature (T; T 10 and T 20 ) and Grazing. At each temperature, two out of the six replicate runs belonging to the J0 level of the factor Size were used in this experiment. Grazing effects were assessed, at both 10°C and 20°C, by comparing these two replicate runs of the grazed mesocosm (hereafter called grazed treatment, G) with two replicate runs of the mesocosm without L. aurata (hereafter called Un-grazed treatment, U). For U, the same experimental procedure was followed as for G except that fish were not introduced into the mesocosm (i.e. the surface area of the working chamber was set to 0.48 m² and two plastic plates with sediment were positioned on the bottom). U enabled us to estimate both the mean biomass of MPB in absence of grazers and the spatial components of variance in the distribution of MPB biomass that was not due to the grazing by L. aurata (i.e. residual variability). The fish density used in this experiment, which corresponded to 6.25 individual m -2 , was ecologically realistic, as it is within the natural range of densities of grey mullets observed in marine coastal and transitional systems [START_REF] Pais | Spatial variability of fish fauna in sheltered and exposed shallow rocky reefs from a recently established Mediterranean Marine Protected Area[END_REF][START_REF] Mwandya | Distribution patterns of striped mullet Mugil cephalus in mangrove creeks, Zanzibar, Tanzania[END_REF][START_REF] Nicolas | Functional diversity in European estuaries: Relating the composition of fish assemblages to the abiotic environment[END_REF].

Since only one mesocosm was available, only one replicate of the experimental treatments was run each day and the study lasted 28 days (between the end of March and the end of June 2009). Each day, the replicate should have been randomly assigned to one of the six experimental combinations (T 10 J0=T 10 G, T 20 J0=T 20 G, T 10 J1, T 20 J1, T 10 U, T 20 U). However, changing the grazing area, the arrangement of cameras or the water temperature in the water reserve tank of the mesocosm every day was practically impossible. Therefore, the replicates for each combination were grouped together and done over a period of 6-8 consecutive days. Such a temporal stratification could have introduced a bias in the results, if the MPB availability had varied naturally in the sediment used during the study period.

Changes in availability or nutritional values of food are known to affect the feeding behavior of fish [START_REF] Polunin | Ecological correlates of foraging periodicity in herbivorous reef fishes of the Coral Sea[END_REF][START_REF] Beukers-Stewart | Behavioural and developmental responses of predatory coral reef fish to variation in the abundance of prey[END_REF]. To check for this potential bias, 6 sediment samples were collected from the experimental replicates of each combination of T × S before each run, in order to estimate the MPB biomass (as a concentration of Chlorophyll-a; µg chl-a cm -2 ; see paragraph below for the description of the methodology). Data were analyzed through a one-way ANOVA with Time as a fixed factor (4 levels, which corresponded to the combinations of T × S). The analysis did not reveal any difference among the four times (see Appendix A).

Temporal segregation could have also introduced a bias in the results of the experiment done to test for the effects of Grazing and T on MPB biomass. To check for this potential bias, sediment samples were collected in two additional tanks located in the greenhouse where the MPB was maintained during both periods when the replicates of G and U for the two levels of T (i.e. T 10 and T 20 ) were run. Sediment samples were collected following the same sampling design used in G and U (see paragraph below for the description of the sampling collection) and the mean and the components of variance were estimated accordingly. Data were then analyzed through a one-way ANOVA with Time as a fixed factor (2 levels, which corresponded to the two times when T 10 and T 20 replicates were run, respectively). Both the mean and the components of variance of MPB biomass did not vary between times (see Appendix B). These results indicated that the availability of MPB biomass and its spatial distribution did not vary in the sediment used during the study period; we can therefore reasonably exclude any bias due to the temporal segregation of the replicate runs of both experiments.

Data collection and analysis

Feeding behavior of L. aurata

We assessed the grazing behavior throughout the total time spent feeding, the number of feeding events, swimming velocity during feeding and the distance covered during feeding. The total time spent feeding (in min) was calculated as the sum of the time lengths of all grazing events for each fish whereas the number of feeding events was estimated as the number of times a fish fed on the sediment. These variables were estimated for each individual and analyzed with the open source ODRec (Observational Data Recorder). The tags positioned on the opercula of the fish allowed us to monitor each single individual for the entire duration of the experiment. These variables were estimated during the 3 hours of the video tracking, minus the first 20 minutes. A pre-analysis of the videos showed that during this initial phase fish exhibited an abnormal feeding activity. This represents a compensatory response to the 12-hours of food deprivation they underwent before the experiment [START_REF] Ali | Compensatory growth in fishes: a response to growth depression[END_REF].

For these two variables, the mean values of the test group were then calculated. Differently, the distance covered during feeding was calculated as the distance (in cm) covered on the sediment surface at each grazing event. This was assessed during 20 grazing events of one individual from each group test (i.e. from each run of the experiment) and analyzed with WINanalyze software. Since the position of the video cameras provided a lateral view along the longest side of the working chamber, the position of the fish could only be followed on a X-axis. Therefore, in order to increase the accuracy of the assessment of the distance travelled by the fish, the 20 grazing events were randomly selected from those that occurred parallel to the longest axis of the working chamber. The swimming velocity during feeding was then calculated by summing the horizontal vectors of distance travelled by the fish (cm), divided by the total duration of the 20 grazing events considered and is expressed as centimeters per second (cm • sec -1 ).

As additional data, droppings left on the sediment surface were collected at the end of each run of the experiment (i.e. for each combination of Temperature and Size factors) for determination of the content of Chlorophyll-a (i.e. µg chl-a g -1 ) in fish droppings.

Mean and variance of MPB biomass

At the end of the two replicate runs of both G, and U treatments, at both temperatures, the top 5 millimeters of the sediment layer were collected using a hand corer (12 mm inner diameter) for the determination of MPB biomass. Three sediment cores were collected 3 cm apart in each of three plots chosen approximately 10 cm apart, at each of four sites spaced about 20 cm apart. A total of thirty-six sediment cores were therefore collected at the end of each replicate run. Samples were immediately frozen, freeze-dried and then kept in the dark at -80°C until further processing. The chlorophyll-a (chla) concentration (i.e. µg chl-a cm -2 ) in the sediment was analysed following the method described in [START_REF] Guarini | Dynamics of spatial patterns of microphytobenthic biomass: inferences from a geostatistical analysis of two comprehensive surveys in Marennes-Oléron Bay (France)[END_REF] and used as proxy of MPB biomass.

Statistical analyses

To test for the effects of temperature and fish size on the feeding behavior of L. aurata, a twoway analysis of variance (ANOVA, [START_REF] Underwood | Experiments in ecology[END_REF] was used, with Temperature (T; T 10 and T 20 ) and Size (S; J0 and J1) as crossed, fixed factors, and 6 replicates (i.e. replicate runs). The same model of ANOVA was used to analyze mean values of chl-a content (µg chl-a g -1 ) in the droppings found within each mesocom.

The mean biomass of MPB was estimated for each replicate run (n=36 cores) and data were analyzed using a two-way ANOVA with Temperature (T; T 10 and T 20 ) and Grazing (G and U) as fixed crossed factors and two replicates (i.e. replicate runs). The components of variance in MPB biomass were estimated over a range of three spatial scales: Cores (3 cm apart), Plots (10 cm apart) and Sites (20 cm apart), according to a fully nested sampling design. They were calculated following the method described in [START_REF] Underwood | Experiments in ecology[END_REF], in order to provide independent measures of the amount of variability of MPB biomass at each spatial scale. Occasionally, negative estimates were obtained. In these cases, variances were set to zero and the other components were recalculated following the "pool-theminimum-violator" procedure, as recommended by [START_REF] Fletcher | How to cope with negative estimates of components of variance in ecological field studies[END_REF]. As the components of variance in MPB biomass at each scale (i.e. Cores, Plots and Sites) were not independent from each other, they were analyzed separately, using the same model of ANOVA as for mean biomass of MPB.

Homogeneity of variances was checked using Cochran's C-test [START_REF] Winer | Statistical principles in experimental design[END_REF]. All analyses were done using Statistica (StatSoft 6.1, 1994).

Results

Effect of Temperature and Size on feeding behavior of L. aurata

The analysis of variance showed a significant effect of Size on the total time spent feeding, independently of the level of Temperature (Table 1). The total time spent feeding by J0 individuals was higher than that spent by J1 ones during the three hours of the experiment (i.e. about 14 and 5 minutes, respectively) (a posteriori multiple comparison, Student-Newman-Keuls, SNK, p = 0.05; test, Fig. 2).

Results also highlighted significant main effects of both Temperature and Size on the number of feeding events, the swimming velocity during feeding and the cumulative distance covered during feeding (Table 1). SNK tests revealed that the number of feeding events and swimming velocity attained during feeding were consistently higher in 20°C-acclimated L. aurata than in 10°C-acclimated ones. In contrast, the cumulative distance covered during feeding events was consistently lower in 20°Cacclimated L. aurata than in 10°C-acclimated L. aurata, by an average factor of three (i.e. 287 and 102 cm, respectively) (Fig. 2). In addition, the swimming velocity and distance covered during feeding was higher in J1 than in J0 individuals, while the opposite trend was observed in the number of feeding events (Fig. 2).

Finally, a significant main effect of Temperature (F 1,12 = 40.47, p<0.00) on the mean chl-a content in the droppings was found. In particular, values were higher at 10°C than at 20°C (mean chl-a content [mg chl-a g sed -1 ] ± SE: 10°C = 1815. 05 ± 158.84; 20°C = 524.85 ± 124.16).

Effect of Grazing and Temperature on mean and spatial variance of MPB biomass

The analysis of variance did not detect any effect of Grazing, Temperature or Grazing × Temperature interaction on the mean values of MPB biomass or on the spatial variance of MPB biomass at the largest spatial scales (i.e. Sites and Plots) (Table 2; Fig. 3a,c-d). On the other hand, a significant Grazing × Temperature effect was observed on the spatial variance of MPB biomass at the smallest spatial scale (i.e. Cores) (Table 2; Fig. 3b). SNK tests showed that the spatial variance of MPB biomass on the scale of cores was three-times higher in the presence of J0 L. aurata (G) than in controls (U) at 20°C (G>U), while no differences between G and U were found at 10°C. In addition, in the presence of grazing (G), variance was higher at 20°C than at 10°C, while no differences were found in U between the two temperatures.

Discussion

Results showed that both the number of feeding events and the swimming velocity during feeding were higher in 20°C-acclimated L. aurata than in 10°C-acclimated ones, independent of their size. In addition, results showed the swimming velocity and the distance covered during feeding to be higher in J1 than J0 individuals, while the opposite was observed for the total time spent feeding and the number of feeding events.

Contrary to our expectations, grazing did not have any negative effect on the mean biomass of MPB at either acclimation temperature. However, it increased the spatial variance in the MPB biomass at the smaller spatial scale (i.e. a few centimeters) at 20°C, indicating a relationship with the observed changes in feeding behavior at this temperature.

Effect of Temperature and Size on feeding behavior of L. aurata

Rising temperatures increase metabolism, thus influencing feeding activity of fish [START_REF] Biro | Mechanisms for climate-induced mortality of fish populations in whole-lake experiments[END_REF][START_REF] Biro | Small within-day increases in temperature affects boldness and alters personality in coral reef fish[END_REF]). The higher number of feeding events in juvenile L. aurata at 20°C than at 10°C was in accordance with studies on other species (both in the laboratory and in the field), showing a positive relationship between the number of feeding events and water temperature [START_REF] Polunin | Ecological correlates of foraging periodicity in herbivorous reef fishes of the Coral Sea[END_REF][START_REF] Ferreira | Seasonal grazing rates and food processing by tropical herbivorous fishes[END_REF][START_REF] Floeter | Geographical gradients of marine herbivorous fishes: patterns and processes[END_REF][START_REF] Smith | Temperature effects on herbivory for an Indo-Pacific parrotfish in Panamá: implications for coral-algal competition[END_REF][START_REF] Theodorou | Reaching the limit: Constrained behavioural flexibility of juvenile Atlantic cod (Gadus morhua) at current coastal temperatures[END_REF]. It is worth noting that an interpretation of this result in the laboratory, however, needs to be made with caution. The number of feeding events that an individual displays in a mesocosm may, in fact, be influenced by behavioral constraints due to the experimental set up (i.e. the tank size). In particular, if in nature an individual prefers to feed by doing one single trace instead of many distinct ones, this behavior could be modified within a mesocom, if the swimming velocity increases (as observed in our study at 20°C), and if it is due to the increased distance covered during feeding. In this case, in fact, fish should be forced to interrupt their feeding within the mecosom, resulting in an increase in the number of feeding events.

However, the cumulative distance covered during feeding was found to be three times lower at 20°C than at 10°C in our study. Therefore, the increase in swimming velocity at 20°C was not due to the increased distance covered at each feeding event, but to an even larger decrease in the time spent feeding per event at that temperature. This suggests that the higher number of feeding events in 20°Cacclimated L. aurata than in 10°C-acclimated ones represents a voluntary modification of the individual behavior of L. aurata at 20°C, as a consequence of the need to maximize food consumption at this temperature. The observed reduction in the distance covered during feeding at 20°C is in contrast with other studies on different species [START_REF] Biro | Small within-day increases in temperature affects boldness and alters personality in coral reef fish[END_REF][START_REF] Theodorou | Reaching the limit: Constrained behavioural flexibility of juvenile Atlantic cod (Gadus morhua) at current coastal temperatures[END_REF]. In particular, [START_REF] Theodorou | Reaching the limit: Constrained behavioural flexibility of juvenile Atlantic cod (Gadus morhua) at current coastal temperatures[END_REF] showed a concomitant increase in the number of feeding events and in the distance covered during feeding as temperature increased in the Atlantic cod Gadus morhua. Reducing distance and time spent feeding while increasing the number of feeding events in L. aurata may represent a foraging strategy, which contributes to decreasing the energy costs involved in feeding activity as metabolism increases [START_REF] Rice | Roles of locomotion in feeding[END_REF]. The suction feeding strategy typical of grey mullets involves the integrated mechanical act of swimming, jaw movement and related muscle activity and it is energetically costly [START_REF] Rice | Roles of locomotion in feeding[END_REF]. At 20°C, feeding by making long feeding traces might not pay off in terms of energy expenditures with respect to energy gains. In fact, ingesting a large amount of sediment at one time might not provide any energetic advantages due to the rapid gastric evacuation and intestinal translocation which reduce the time of interaction between ingested food and enzymatic activities involved in the digestion and assimilation processes. Indeed, the gut transit time in fish decreases with increasing temperatures [START_REF] Halver | Fish nutrition[END_REF]Evans and Clairborne, 2006), so that long feeding traces can even represent a disadvantage. It is worth noting, however, that the enzymatic activities involved in the extraction of nutrients from food (i.e. digestion) and nutrients absorption through the intestinal walls (i.e. assimilation) are known to increase with temperature [START_REF] Halver | Fish nutrition[END_REF]Evans and Clairborne, 2006), so that this higher efficiency in digestion and assimilation can counterbalance the short gut transit time. The efficiency of such a process is highlighted by the lower amount of MPB found in droppings at 20°C than in those at 10°C, suggesting a more complete algal digestion in 20°C-acclimated L. aurata. Grey mullets are generally active swimming foragers.

Maintaining the capacity for locomotion is more important in active fishes than in sit-and-wait foragers because the former are more likely to encounter predators and need to be able to avoid capture, and they continue to hunt or graze while still digesting [START_REF] Fu | The behavioural, digestive and metabolic characteristics of fishes with different foraging strategies[END_REF]. Therefore, grey mullets should be able to modulate their feeding behavior in order to save energy for other activities such as locomotion.

In accordance with other fish species [START_REF] Choat | Daily feeding rates in herbivorous labroid fishes[END_REF][START_REF] Videler | Fish swimming[END_REF][START_REF] Buckle | Ontogeny of space use and diet of two temperate damselfish species, Parma microlepis and Parma unifasciata[END_REF], size influenced the feeding behavior of juvenile L. aurata in this study. In general, the swimming velocity was observed to increase [START_REF] Videler | Fish swimming[END_REF] and the number of feeding events to decrease with size in herbivorous fish [START_REF] Choat | Daily feeding rates in herbivorous labroid fishes[END_REF][START_REF] Buckle | Ontogeny of space use and diet of two temperate damselfish species, Parma microlepis and Parma unifasciata[END_REF]. This latter result has been related to the fact that the amount of food ingested during each feeding event increases with size so that a fish fulfils its energetic requirement after a lower number of feeding events at increasing size [START_REF] Buckle | Ontogeny of space use and diet of two temperate damselfish species, Parma microlepis and Parma unifasciata[END_REF].

Effect of grazing and temperature on mean and spatial variance of MPB biomass

Our results provide the first evidence that the temperature-induced changes in the feeding behavior of L. aurata can influence the spatial distribution of their main food resource, MPB. However, the mechanisms linking the behavioral changes of L. aurata to the spatial distribution of the MPB biomass are more complex than previously thought [START_REF] Polunin | Algal food supply and grazer demand in a very productive coral-reef zone[END_REF][START_REF] Smith | Temperature effects on herbivory for an Indo-Pacific parrotfish in Panamá: implications for coral-algal competition[END_REF]. We had predicted that the direct negative effects of grazing on the mean and spatial variance of the MPB biomass would be higher at 20°C than at 10°C because of an increase both in the amount of algae removed and in the amount of variance lost due to grazing by fish at increasing water temperatures, as was found to be the case for herbivorous tropical fish [START_REF] Polunin | Algal food supply and grazer demand in a very productive coral-reef zone[END_REF]. Our results showed that grazing at 20°C did not induce any change in the mean MPB biomass but it significantly increased the variability in the spatial distribution of the MPB biomass at the smallest spatial scale. This latter result was in accordance with studies showing that grazing can increase the spatial patchiness of resources [START_REF] Hillebrand | Grazing regulates the spatial variability of periphyton biomass[END_REF]. In our study, it is likely that the enhanced spatial patchiness of MPB due to grazing at 20°C counterbalanced the direct negative effect of algal removal by fish through an enhanced growth rate of algae. Spatial variability at a scale of a few cm is known to favor primary productivity of microalgae [START_REF] Cardinale | The influence of substrate heterogeneity on biofilm metabolism in a stream ecosystem[END_REF] by affecting the flow and uptake of nutrients [START_REF] Riber | Boundary-layer and internal diffusion effects on phosphorus fluxes in lake periphyton[END_REF], as well as the ability of small benthic grazers to recognize and select food patches [START_REF] Kawata | Perceptual scales of spatial heterogeneity of periphyton for freshwater snails[END_REF]. For instance, the small-scale spatial variability of MPB biomass generated by grazing at 20°C may have increased the photosynthetic efficiency of cells and algal growth by preventing nutrient depletion or CO 2 limitation at the base of the biofilm [START_REF] Riber | Boundary-layer and internal diffusion effects on phosphorus fluxes in lake periphyton[END_REF]. In our mesocosms, it is likely that all these effects yielded a local increase in the mean MPB biomass that counterbalanced the direct negative effects of grazing, resulting in the lack of an overall significant effect [START_REF] Berlow | Strong effects of weak interactions in ecological communities[END_REF].

Theoretical and empirical studies have shown that trophic interactions that have weak effects on a resource may enhance the patchiness of that resource, if foraging operates with large spatial or temporal variance [START_REF] Berlow | Strong effects of weak interactions in ecological communities[END_REF][START_REF] Benedetti-Cecchi | Variance in ecological consumer-resource interactions[END_REF][START_REF] Sommer | Benthic microalgal diversity enhanced by spatial heterogeneity of grazing[END_REF][START_REF] Adler | The effect of grazing on the spatial heterogeneity of vegetation[END_REF][START_REF] Flecker | Tropical fishes as biological bulldozers: density effects on resource heterogeneity and species diversity[END_REF][START_REF] Hillebrand | Grazing regulates the spatial variability of periphyton biomass[END_REF]) and if the amount of variance generated by grazing activity is larger than the residual variability in the resource abundance [START_REF] Benedetti-Cecchi | Variance in ecological consumer-resource interactions[END_REF]. Most of these studies have focused on the grazing pattern of benthic invertebrates while little is known about the ability of epibenthic fish to cause variability in the spatial distribution of the resources [START_REF] Hillebrand | Grazing regulates the spatial variability of periphyton biomass[END_REF]. [START_REF] Flecker | Tropical fishes as biological bulldozers: density effects on resource heterogeneity and species diversity[END_REF] showed that the heterogeneous grazing of the epibenthic fish Parodon apolinari significantly increased the spatial variability in biofilm abundance in stream. As with P. apolinari, our results provided indications of a heterogeneous grazing of L. aurata at 20°C, able to enhance the patchiness of MPB within the mesocosm.

Although previous studies have investigated the effects of grazing on the spatial patchiness of resources [START_REF] Hillebrand | Grazing regulates the spatial variability of periphyton biomass[END_REF], little attention has been given to the effects of behavioral plasticity of grazers. The effects of temperature-induced behavioral changes of grazing fish on the spatial patchiness of their resources, like those observed in our study, should be taken into account in future ecological studies in natural systems. Indeed, grazing fish such as grey mullets are one of the most abundant fish in coastal areas [START_REF] Laffaille | Can thin lipped mullet directly exploit the primary and detritic production of European macrotidal salt marshes?[END_REF][START_REF] Cardona | Habitat selection by grey mullets (Osteichthyes: Mugilidae) in Mediterranean estuaries: the role of salinity[END_REF][START_REF] Lebreton | Trophic ecology of mullets during their spring migration in a European saltmarsh: a stable isotope study[END_REF][START_REF] Whitfield | A global review of the cosmopolitan flathead mullet Mugil cephalus Linnaeus 1758 (Teleostei: Mugilidae), with emphasis on the biology, genetics, ecology and fisheries aspects of this apparent species complex[END_REF].

Maintaining high level of spatial patchiness of MPB by affecting the behavior of grazing fish may be a way through which high seasonal temperatures favor primary productivity in areas characterized by high biomass or during spring blooms, when nutrient depletion or CO 2 limitation inhibits the photosynthetic efficiency of cells and algal growth [START_REF] Guarini | Dynamics of spatial patterns of microphytobenthic biomass: inferences from a geostatistical analysis of two comprehensive surveys in Marennes-Oléron Bay (France)[END_REF][START_REF] Jesus | Spatial dynamics of microphytobenthos determined by PAM fluorescence[END_REF].

Conclusion

In summary, this study provides the first evidence of clear-cut changes in the feeding behavior of juvenile L. aurata at high seasonal temperatures. These changes most likely reflect not only the need to maximize food consumption due to the temperature-induced increases in metabolism, but also the ability of L. aurata to modulate individual feeding behavior, in order to save energy for other activities, like locomotion, which are energetically costly.

Our results also provide the first evidence that the temperature-induced changes in the feeding behavior of L. aurata influence the spatial distribution of MPB, their main food resource. However, the mechanisms linking the behavioral changes of L. aurata to the response of the MPB biomass to grazing are more complex than those expected from a mere increase of algal consumption at higher water temperatures. We suggest that the changes in the feeding behavior of L. aurata at high seasonal temperatures enhance the spatial patchiness of the MPB biomass at the smallest spatial scale, thus increasing photosynthetic efficiency of cells and algal growth and favoring primary productivity. Table 1 Results of ANOVAs to test for the effect of Temperature and Size on the total time spent feeding (min), the number of feeding events (n. traces), the swimming velocity during feeding (cm sec -1 ) and the distance covered during feeding (cm). Significant P values (<0.05) are indicated with an asterisk.

Tables

Fig

  Fig. 2 Como et al.

Table 2

 2 Results of ANOVAs to test for the effect of Grazing and Temperature on mean MPB biomass (estimated as chlorophyll-a concentration; µg cm -2 ) and components of variance (CV) in MPB biomass at the scales of Cores, Plots and Sites. Significant P values (<0.05) are indicated with an asterisk.

			Time (min)		Events (n. traces)
	Source	DF	MS	F	P	MS	F	P
	Temperature = T	1	49.65	4.03 0.06	514.54	21.94 0.00*
	Size = S	1 539.41 43.76 0.00*	819.22	34.93 0.00*
	T × S	1	14.08	1.14 0.30	3.18	0.14 0.72
	residual A	20	12.33			23.45
	Total	23
	Cochran's C		0.54 (P>0.05)		0.47(P>0.05)
	Transformation			None		None
			Velocity (cm sec -1 )	Distance (cm)
	Source	DF	MS	F	P	MS	F	P
	Temperature = T	1 325.98 102.85 0.00*	205873.88 103.94 0.00*
	Size = S	1	84.71 26.73 0.00*	15898.57	8.03 0.01*
	T × S	1	3.83	1.21 0.28	2039.28	1.03 0.32
	residual A	20	3.17			1980.66
	Total	23
	Cochran's C		0.49 (P>0.05)		0.58(P>0.05)
	Transformation		Sqrt(X+1)			none

A denominator for T, S and T × S

Acknowledgements

The authors are grateful Lisandro Benedetti-Cecchi and Paolo Domenici for their comments and discussion during various stages of this work, to Samuel Péan for the technical support for the video analysis, and Michel Prineau, Francoise Mornet, Andrea Satta and Marcella Cannas for their help in the laboratory. We sincerely thank S. Thrush and an anonymous referee for their helpful comments on the manuscript. The work was supported by the French ANR (National Research Agency) through the VASIREMI project "Trophic significance of microbial biofilms in tidal flats" (contract ANR-06-BLAN-0393-01).

Captions:

Figure 1 Schematic illustration of the mesocosm (viewed from the side) and the video-tracking system, which consisted of four polychrome video cameras (25 image sec -1 ), a multichannel video interface and a DVD recorder. The cameras were placed along the longest side of the working chamber in order to provide a lateral view. Not shown: a series of lamps connected to a timer which provided a supplementary light source in addition to the natural illumination, and a buffer tank connected to a thermostat which controlled temperature, circulation and the water level inside the mesocosm.

Figure 2 Mean values (n=6 ± SE) of the total time spent feeding (min), the number of feeding events (n. traces), the swimming velocity during feeding (cm sec -1 ) and the distance covered during feeding (cm) for each acclimation temperature (i.e. 10°C and 20°C) and fish size (i.e. J0 and J1). Asterisk (*) indicates where significant differences (P<0.05) between the two acclimation temperatures and the two fish sizes occur.

Appendices

Appendix A Results of the 1-way ANOVA on the effect of Time (4 levels, which corresponded to the combinations of the two levels of Temperature = T and Size = S) on mean MPB biomass (estimated as chlorophyll-a concentration; µg cm -2 ). The number of experimental replicates for each time (i.e. each combination of T × S) was six. Samples were collected before each run.