Nabil Majdi
email: majdi@cict.fr

Walter Traunspurger

Ste ´phanie Boyer

Benoı ˆt Mialet

Miche ` Le Tackx

Robert Fernandez

Stefanie Gehner

Loı ¨c Ten-Hage

Evelyne Buffan-Dubau

Stefano Amalfitano

Á S Boyer

Á B Mialet

Á M Tackx

Á R Fernandez

Á L Ten-Hage

Á E Buffan-Dubau

Response of biofilm-dwelling nematodes to habitat changes in the Garonne River, France: influence of hydrodynamics and microalgal availability

Keywords: Nematodes, Periphyton, Diversity, Feeding types, Algae, Environmental factors

Lotic epilithic biofilms are submitted to seasonal disturbances (e.g. flood events, self-detachment), which influence the biomass, diversity and viability of their algal and bacterial communities. The objective of this study is to examine whether (1) biofilm-dwelling nematodes respond to such seasonal changes in terms of diversity and community structure, (2) nematode species and feeding-types distribution respond to the varied trophic situations within the biofilm, since variations in biofilm microalgal composition may represent a variation in available food. The biofilm-dwelling nematode community was monitored in a temperate river over an 18 month period with a high sampling frequency. These data were linked to environmental abiotic and biofilm biotic factors. Nematode density was positively correlated to biofilm and microalgal biomass, but was dampened by floods. A clear seasonal pattern of the community was detected (summer shift), so that two nematode groups stand out: (1) the epistratefeeders Chromadorina bioculata (Schultze in Carus, 1857) and Chromadorina viridis (Linstow, 1876) were primarily related to diatom availability, and dominated the nematode assemblage most of the time, (2) seven species from various feeding types (deposit-feeders, suction-feeders and chewers) grew mainly under summer conditions concomitantly to a change of biofilm trophic status and microalgal composition. Overall, the results suggested that, in addition to abiotic disturbances, the availability of potential preys in the biofilm might represent an important driver of nematode community patterns.

Introduction

In rivers, any hard submerged substrate can be coated by a complex assemblage of organisms (e.g. bacteria, fungi, algae, heterotrophic protozoans, meiofauna and macrofauna) embedded in a mucous matrix of exopolymeric substances [START_REF] Costerton | Biofilms in the new millennium: musings from a peak in Xanadu[END_REF][START_REF] Leflaive | Temporal patterns in epilithic biofilm-relation with a putative allelopathic activity[END_REF]. This organic layer which is named either epilithic biofilm, epilithon, 'Aufwuchs' or periphyton can comprise more than 30% of microalgae in terms of biomass [START_REF] Peterson | Response of benthic algal communities to natural physical disturbance[END_REF]. Consequently, epilithic biofilms can constitute the main site of primary production in shallow water rivers harbouring hard substrates such as the Garonne in its middle part [START_REF] Ameziane | Origin and transport of phytoplankton in a large river: the Garonne, France[END_REF]. These biofilms contribute substantially to benthic food web functioning [START_REF] Liess | Invited review: direct and indirect effects in herbivore-periphyton interactions[END_REF] and to biogeochemical processes such as decomposition and nutrient retention (e.g. [START_REF] Ford | Epilithic metabolism of dissolved organic carbon in boreal forest rivers[END_REF][START_REF] Battin | Contributions of microbial biofilms to ecosystem processes in stream mesocosms[END_REF][START_REF] Teissier | Detailing biogeochemical N budgets in riverine epilithic biofilms[END_REF]. However, epilithic biofilms are unstable habitats, well-exposed to environmental perturbations. Hence they are strongly influenced by seasonal disturbances such as floods [START_REF] Biggs | Periphyton biomass dynamics in gravel bed rivers: the relative effects of flows and nutrients[END_REF] and self-detachment, a temperature-dependent bacterial degradation of the mat [START_REF] Biggs | Patterns in benthic algae of streams[END_REF][START_REF] Boule ˆtreau | Assessing the importance of a self-generated detachment process in river biofilm models[END_REF]. These disturbances are recognized to shape the biomass, diversity and viability of the algal and bacterial communities inhabiting the mat (e.g. [START_REF] Peterson | Resistance and resilience of lotic algal communities: importance of disturbance timing and current[END_REF][START_REF] Lyautey | Viability of differentiated epilithic bacterial communities in the River Garonne (SW France)[END_REF], implying important consequences on the functioning of biofilm processes [START_REF] Cardinale | Biodiversity improves water quality through niche partitioning[END_REF].

Free-living nematodes are important protagonists within biofilm communities: on the one hand, epilithic biofilms represent both a habitat and a probable important food resource for them (e.g. Peters & Traunspurger, 2005;[START_REF] Gaudes | The nematode community in cyanobacterial biofilms in the river Llobregat, Spain[END_REF][START_REF] Traunspurger | Composition and distribution of free-living aquatic nematodes: global and local perspectives[END_REF][START_REF] Caramujo | Influence of drought on algal biofilms and meiofaunal assemblages of temperate reservoirs and rivers[END_REF]. On the other hand, it has been suggested that nematode activity (e.g. through bioturbation and grazing) could affect key biofilm processes: for instance, [START_REF] Mathieu | Free-living nematodes affect oxygen turnover of artificial diatom biofilms[END_REF] indicate that nematodes influence the oxygen turnover of artificial diatom biofilms, and [START_REF] Sabater | Ecological implications of mass growth of benthic cyanobacteria in rivers[END_REF] and [START_REF] Gaudes | The nematode community in cyanobacterial biofilms in the river Llobregat, Spain[END_REF] highlight that meiofauna (mainly nematodes) can influence the release of unpleasant odorous metabolites (e.g. geosmin) by cyanobacterial biofilms, implying high economic relevance for fishing industry and drinking water production.

Despite their important presence within these habitats, biofilm-dwelling nematodes still remain poorly considered as most nematological studies focus rather on sediment-dwelling nematodes [START_REF] Traunspurger | Composition and distribution of free-living aquatic nematodes: global and local perspectives[END_REF]. As a matter of fact, most information on biofilm-dwelling nematodes has issued from lentic environments: e.g. spatial distributional patterns and colonization pathways [START_REF] Traunspurger | A study of the free-living freshwater nematodes of hard substrates in the littoral of the oligotrophic Koenigssee (National Park Berchtesgaden[END_REF]Peters & Traunspurger, 2005;Peters et al., 2005). So far, only two previous studies have examined temporal distribution of biofilm-dwelling nematodes in running waters during relatively short periods [START_REF] Gaudes | The nematode community in cyanobacterial biofilms in the river Llobregat, Spain[END_REF][START_REF] Caramujo | Influence of drought on algal biofilms and meiofaunal assemblages of temperate reservoirs and rivers[END_REF]. But, long-term studies of biofilm-dwelling nematodes are still lacking, which hampers the assessment of how epilithic nematode communities react and adapt to recurrent (seasonal) abiotic disturbances and/or to fluctuations of food resources over time.

In this context, the questions put forward in this study are: (1) In temperate areas, epilithic biofilms are subject to seasonal temperature changes and hydrological events, which, as mentioned above, change their biomass and the composition of the algal and bacterial communities. Is the biofilm-dwelling nematode community influenced by such seasonal changes of their habitat? (2) As variations in composition of the microalgal community may represent a variation in available food within the mat (in terms of amount, availability and quality), do the nematode species and feeding-types distribution match with the biofilm trophic situation at a given time? With these objectives, density, biomass, diversity, age, sex and feeding types of the biofilm-dwelling nematode community was monitored over an 18 month field survey in a large temperate river: the Garonne (SW France). These data were analysed to detect potential seasonal changes, then the nematode species distribution was examined through the influence extent of both environmental abiotic drivers and biofilm biotic conditions.

Methods

Study site and sample collection

The Garonne is the largest river of south-western France with a drainage basin of 57,000 km 2 and a length of 647 km. The Garonne River displays a pluvio-nival flow regime with relatively short flashfloods caused by heavy rainfall (occurring mainly between November and January) and a long annual flood period due to snow-melt (April to June). In the Garonne, alternate cobble bars are frequently found even in channel up to the seventh-order. Between floods (i.e. low-water periods), a high epilithic biomass can grow on cobbles, being favoured by low-water velocities on the river bed and low turbidity [START_REF] Boule ˆtreau | Assessing the importance of a self-generated detachment process in river biofilm models[END_REF]. The study site was situated on one of these cobble bars located at 36 km upstream the city of Toulouse (01°17 0 53 00 E, 43°23 0 45 00 N; elevation 175 m a.s.l.), where the Garonne is of sixth-order (Fig. 1).

Samplings (N = 51) were regularly performed from September 2008 to March 2010 when hydrological conditions permitted it (sampling was only possible when discharge was lower than 175 m 3 s -1 ). On each sampling occasion, 12 immerged cobbles (mean diameter: 10 cm) were collected underwater using plastic bags to prevent any biofilm detachment during removal. To consider water level changes and depth where the biofilm typically develops [START_REF] Ameziane | Epilithic biomass in a large gravel bed river (the Garonne, France): a manifestation of eutrophication?[END_REF], cobbles were collected on a crosssection from a reference point in the riverside so that water height above cobbles remained between 30 and 50 cm. Collected cobbles were transported to the laboratory within 2 h in cool boxes with minimal disturbance. The biofilm was gathered by scraping the upper surface of each cobble with a scalpel and a toothbrush. Biofilm samples were finally suspended in MilliQ water to obtain 12 biofilm suspensions (25 ml each), in which algal aggregates were carefully crumbled with scissors. These 12 biofilm suspensions were used for the three following treatments: (1) nematode species identification and density and biomass measurements, (2) HPLC analyses of microalgal pigments and (3) epilithic ash-free dry mass (AFDM) measurements. Four replicate suspensions were used for each treatment. Scraped cobbles were photographed, and the surface of biofilm which had been removed was clearly visible and measured using ImageJ software version 1.38 [START_REF] Abramoff | Image processing with ImageJ[END_REF]. Removed Fig. 1 Location of the sampling site and cross-section view of the Garonne River at the sampling site biofilm surfaces were then reported to corresponding biofilm suspension volumes, so as densities, biomass and pigment concentrations were quantitatively expressed per area unit.

Nematode processing

Nematodes were extracted from four replicate biofilm suspensions using a modified gravity gradient centrifugation technique involving Ludox HS-40 after [START_REF] Pfannkuche | Sample processing[END_REF]. Nematodes so extracted were cleaned from Ludox by sieving through a 40 lm sieve, then preserved in formaldehyde (5% final concentration) and stained with 1% Rose Bengal. Nematodes were counted in a Dolfuss cell (Elvetec services, Clermont-Ferrand, France) under a Leica MZ 9.5 stereomicroscope (99-909) and their density was expressed per cm 2 . According to nematode density, between 12 and 25 individuals were randomly picked up from each replicate while counting, transferred to glycerol solution [START_REF] Seinhorst | A rapid method for the transfer of nematodes from fixative to anhydrous glycerin[END_REF], mounted on slides and identified to the best species level using a Leitz Dialux microscope at 12509 magnification.

Nematodes were classified according to their age (juveniles, fourth stage juveniles and adults), their sexual category (females, gravid females and males), and their feeding type (epistrate-feeders, depositfeeders, suction-feeders and chewers) after [START_REF] Traunspurger | Bathymetric, seasonal and vertical distribution of the feeding-types of nematodes in an oligotrophic lake[END_REF]. The Maturity Index (MI) was calculated on each sampling occasion as the weighted mean frequency of individual colonizer-persister values (cp) after [START_REF] Bongers | The maturity index: an ecological measure of environmental disturbance based on nematode species composition[END_REF]. MI ranged from 1 to 5. Nematode species with a cp = 1 were considered r-strategists (colonizers) with short-generation times, high fecundity and extreme population changes whereas those with a cp = 5 were defined as K-strategists (persisters) with lower breeding efficiency. The MI is expected to decrease during disturbed periods, when opportunistic nematodes are favoured [START_REF] Bongers | Functional diversity of nematodes[END_REF]. Over a 1-year period from September 2008 to September 2009 (N = 37), at least 100 individual nematode body dimensions (length and maximum width) were measured on each sampling occasion from microscopic pictures taken while counting. Mean individual wet weight (WW) was then determined after Andra ´ssy (1956).

Abiotic environmental factors

Mean Daily Discharge (MDD) was supplied by a gauging station of the French water management authority (DIREN Midi-Pyre ´ne ´es, Marquefave station) located at 10 km upstream the study site-with no tributary and no dam between the gauging station and the study site. The Mean Weekly Discharge (MWD) before each sampling occasion was considered in statistical analysis. To better reflect the effect of flood disturbance, days after flood (DAF), which were effective days after the last flood (MDD [ 300 m 3 s -1 ), were calculated for each sampling occasion and considered in statistical analysis. Water temperature, conductivity, pH and dissolved oxygen concentration were measured every 30 min during the whole study period with an automated multi-parameter probe (YSI 6000, YSI inc., Yellow springs, OH, USA) which was permanently settled at 5 cm above the streambed at the study site.

Biofilm microalgal composition and biomass

Microalgal pigments extraction and HPLC-analysis

On each sampling occasion, four replicate biofilm suspensions were centrifuged (3,220 g, 20 min). Pellets were freeze-dried and thoroughly homogenized. Then, 250 mg aliquots were removed from each pellet. Algal pigments from each pellet aliquot were then extracted three times (15 min at -20°C) with a total of 25 ml (10, 10 and 5 ml) 98% coldbuffered methanol (with 2% of 1 M ammonium acetate) following Buffan-Dubau & Carman (2000b). Algal pigment release was favoured at each extraction step by an ultrasonication probe (Sonifier 250A, Branson Ultrasonics corp., Danbury, CT, USA).

One millilitre of the pigment solution so obtained was then filtered on 0.2 lm PTFE syringe filter and analyzed using a high-performance liquid chromatograph (HPLC) consisting of a 100 ll loop autosampler and a quaternary solvent delivery system coupled to a diode array spectrophotometer (LC1200 series, Agilent Technologies inc., Santa Clara, CA, USA). The mobile phase was prepared and programmed according to the analytical gradient protocol described in [START_REF] Barlow | Improved resolution of mono-and divinyl chlorophylls a and b and zeaxanthin and lutein in phytoplankton extracts using reverse phase C-8 HPLC[END_REF]. Pigment separation was performed through a C8, 5 lm column (MOS-2 HYPERSIL, Thermo Fisher Scientific inc., Waltham, MA, USA). The diode array detector was set at 440 nm to detect carotenoids, and at 665 nm to detect chlorophylls and pheopigments [START_REF] Wright | Improved HPLC method for the analysis of chlorophylls and carotenoids from marine phytoplankton[END_REF]. Data analysis was performed using ChemStation software (version A.10.02, Agilent Technologies inc.). Pigments were identified by comparing their retention time and absorption spectra with those of pure standards pigments (DHI LAB products, Hørsholm, Denmark). Each pigment concentration was calculated by relating its chromatogram's peak area with the corresponding area of calibrated standard.

Microalgal cultures and chemotaxonomy

Algal pigment analysis by HPLC coupled with chemotaxonomic analysis using CHEMTAX program [START_REF] Mackey | CHEMTAX -a program for estimating class abundances from chemical markers: application to HPLC measurements of phytoplankton[END_REF] has proven to be a fast and precise method to determine the biomass of phytoplanktonic and microphytobenthic groups in marine and freshwater environments (e.g. [START_REF] Schlu ¨ter | Identification and quantification of phytoplankton groups in lakes using new pigment ratios -a comparison between pigment analysis by HPLC and microscopy[END_REF][START_REF] Caramujo | Influence of drought on algal biofilms and meiofaunal assemblages of temperate reservoirs and rivers[END_REF][START_REF] Lionard | Evaluation of the performance of HPLC -CHEMTAX analysis for determining phytoplankton biomass and composition in a turbid estuary (Schelde, Belgium)[END_REF]. As reported by [START_REF] Leflaive | Temporal patterns in epilithic biofilm-relation with a putative allelopathic activity[END_REF], microalgal groups inhabiting epilithic biofilms of the Garonne River are diatoms, green algae and cyanobacteria. The biomarker pigment composition found in the biofilm can be used to estimate the biomass of each of these microalgal groups by chemotaxonomy. Prior to the chemotaxonomic analysis, biomarker pigment ratio to chlorophyll a (Chl a) for each microalgal group has to be obtained. Thus, a green algae species, Pediastrum boryanum (Turpin) Meneghini (strain Pedbo01) and a diatom species, Nitzschia palea (Ku ¨tzing) W. Smith (strain Nitpa01) were isolated from the biofilm of the Garonne River and maintained on Combo medium [START_REF] Kilham | COMBO: a defined freshwater culture medium for algae and zooplankton[END_REF] at 18°C (light:dark 16:8, 45 lmol m -2 s -1 ). An aliquot of each algal culture (10 mL) was filtered on 0.7 lm glass fibre filter (GF/F, Whatman, Clifton, NJ, USA) and algal pigments were extracted and analysed from the filters following the same procedure than biofilm samples. Concerning cyanobacteria, pigment ratios calculated by [START_REF] Schlu ¨ter | Identification and quantification of phytoplankton groups in lakes using new pigment ratios -a comparison between pigment analysis by HPLC and microscopy[END_REF] for Synechococcus leopoliensis (Raciborski) Komrek (University of Toronto Culture Collection strain 102) were considered.

The biomarker pigment ratio to Chl a so obtained were used to supply the initial matrix needed for CHEMTAX analysis (Table 1). Then, CHEMTAX version 1.95 software [START_REF] Mackey | CHEMTAX -a program for estimating class abundances from chemical markers: application to HPLC measurements of phytoplankton[END_REF] was run to estimate the biomass of diatoms, green algae and cyanobacteria which were expressed as Chl a equivalents and considered as environmental biotic factors in further statistical analysis.

Total epilithic biomass and autotrophic index

On each sampling occasion, four biofilm suspensions were dried at 105°C for 18 h, weighted and then combusted at 450°C for 8 h to weight the ash-free dry mass (AFDM) of the biofilm. The Autotrophic Index (AI) was determined as the ratio AFDM/Chl a. This index is commonly used to describe the trophic status of biofilm communities, e.g. higher AI values are found in biofilms with higher proportions of heterotrophs and/or organic detritus [START_REF] Biggs | Periphyton biomass dynamics in gravel bed rivers: the relative effects of flows and nutrients[END_REF].

Statistical analysis

To investigate seasonal changes of the nematode community structure, the differences in biomass, diversity, age, sex, feeding types and MI were The influence of biotic and abiotic environmental factors on the nematode species distribution was analyzed through canonical ordination analysis with CANOCO software (version 4.5, Biometris, Wageningen, The Netherlands). Rare species (with relative occurrence \0.1%) were not considered in this analysis. Species densities were square-root transformed prior to the analysis. The distribution of nematodes was first analyzed by a detrended correspondence analysis (DCA). As the total inertia observed was less than 2.6, a predominance of linear species response curves could be expected [START_REF] Ter Braak | Ordination[END_REF][START_REF] Ter Braak | Canonical community ordination. Part I: Basic theory and linear methods[END_REF]. Therefore, a redundancy analysis (RDA) in which the ordination axes were constrained to be linear combinations of provided environmental factors was used to investigate the relationships between these factors and the distribution of main nematode species. Environmental factors were also listed (conditional effects) according to the variance they explained singly (i.e. without eventual co-variability with other factors). The statistical significance was tested with Monte Carlo permutation test (499 unrestricted permutations) with applying Bonferroni's correction (significance level set at P \ 0.005).

Results

Dynamics of the epilithic biofilm

The range and annual mean values of each measured abiotic and biotic factor are listed in Table 2. AFDM and Chl a content of the epilithic biofilm were significantly positively correlated (Spearman rank: R = 0.75; P \ 0.001) and showed considerable variations throughout the sampling period, being particularly dampened after floods (Fig. 2a). The AI was significantly higher during summer than during the other seasons (ANOVA: F = 60.2; P \ 0.001), implying globally a lower availability of microalgae within summer biofilm communities. Diatoms dominated the epilithic microalgal assemblage over the whole sampling period (Fig. 2b, Table 2). The diatom biomass was significantly higher during winter than during the other seasons (ANOVA: F = 16.1; P \ 0.001). Conversely, cyanobacterial biomass was significantly higher during summer (ANOVA: F = 4.6; P \ 0.01), and green algal biomass was significantly higher during summer and autumn (ANOVA: F = 2.8; P \ 0.05) than during the remainder of the year.

Dynamics of biofilm-dwelling nematodes

Over the whole study period, the nematode density averaged 25.4 ± 4.3 ind cm -2 and varied greatly throughout the year: the lowest density (0.36 ± 0.14 ind cm -2 ) occurred in early summer 2009 whereas the highest density (161.36 ± 52.5 ind cm -2 ) was attained during late winter 2010. As AFDM and Chl a, the nematode density was clearly dampened after flood events (Fig. 3a). Nematode density was positively correlated with DAF (Spearman rank: R = 0.36; P \ 0.01), AFDM (Spearman rank: R = 0.41; P \ 0.01) and Chl a (Spearman rank: R = 0.47; P \ 0.001). From September 2008 to September 2009, the nematode individual wet weight averaged 0.3 lg.

The individual biomass was significantly lower during summer (ANOVA: F = 14.1; P \ 0.001) than during the other seasons (Fig. 3a).

From the 2,875 nematodes identified, 28 species belonging to 11 families were found (see species list in Table 3). Two species: Chromadorina bioculata and Chromadorina viridis (family Chromadoridae) strongly dominated the assemblage accounting for 86% of all identified nematodes. Although the family Monhysteridae-particularly with species Eumonhystera dispar, Eumonhystera vulgaris and Monhystrella paramacrura-represented only 10% of all identified nematodes over the whole period, they clearly dominated the assemblage from mid-July to mid-August (Fig. 3b). Sixteen species were rare, accounting for \0.1% of all identified nematodes (Table 3).

The species richness (S) varied from 2 to 12 species averaging S = 4.23 over the whole study period. S was significantly higher during summer (ANOVA: F = 6.5; P \ 0.001) than during the other seasons.

Conversely, the Maturity Index (MI) was significantly lower (MI = 2.67) during summer (Kruskal-Wallis ANOVA: H = 31.5; P \ 0.001) than during the other seasons. This summer shift in S and MI is illustrated in Fig. 3c. Epistrate-feeders-mainly represented by C. bioculata and C. viridis-dominated representing 86% of nematodes identified over the whole sampling period. Deposit-feeders were the second most observed group representing 12% while suction-feeders and chewers were less common representing, respectively, 1.5 and 0.5%. Insect parasites (i.e. Mermithidae) represented \0.1%. During summer, the epistrate-feeders were significantly less represented (ANOVA: F = 28.5; P \ 0.001) while deposit-feeders were significantly more represented (Kruskal-Wallis ANOVA: H = 38.7; P \ 0.001) than during the other seasons (Fig. 4a).

The seasonal proportion of juveniles, fourth stage juveniles, females, gravid females and males is presented in Fig. 4b. Concerning the age structure of the community, adult nematodes averaged 70% of all identified nematodes, while fourth stage juveniles and early instar juveniles contributed, respectively, to 14 and 16%. Early instar juveniles were significantly more represented during spring (ANOVA: F = 2.8; P \ 0.05) than during the other seasons. Concerning the sex structure of the community, females represented 28% (non-gravid females) and 14% (gravid females) against 28% for males. Males contributed significantly less during summer (ANOVA: F = 3.2; P \ 0.05) than during winter.

Influence of environmental factors on nematode species distribution

The results of the redundancy analysis (RDA) testing the influence of biotic and abiotic factors on nematode species and feeding-types distribution are presented in Fig. 5 andTable 4. The temporal distribution of nematode species was significantly influenced by The proportion (%) of each species to the total number of identified nematodes (N = 2875) is provided. Each species is assigned to its corresponding colonizer-persister value (cp) after [START_REF] Bongers | Functional diversity of nematodes[END_REF] and to its corresponding feeding type (FT) after [START_REF] Traunspurger | Bathymetric, seasonal and vertical distribution of the feeding-types of nematodes in an oligotrophic lake[END_REF]: epistrate-feeders (E), deposit-feeders (D), suction-feeders (S) chewers (C) and insect parasites (P) temperature, AFDM, DAF and biomass of cyanobacteria, green algae and diatoms. The sum of all significant factor eigenvalues explained 64.1% of the variance. This analysis allowed to clearly distinguish two groups of nematode species: The first group comprised the two dominant epistrate-feeder species C. bioculata and C. viridis. These two species are situated along axis 1, scoring towards the middle right side of the biplot. Since axis 1 involved mainly factors AFDM, DAF and diatom biomass, this indicated that both species were more abundant during prolonged undisturbed periods with a high biofilm and diatom biomass. The second group comprised deposit-feeders (i.e. Eumonhystera dispar, E. vulgaris, E. barbata, Plectus aquatilis and Monhystrella paramacrura), suction-feeders (i.e. Mesodorylaimus cf. subtiliformis and Aphelenchoides sp.) and chewers (i.e.

Brevitobrilus stefanskii). These species are distributed along axis 2, scoring towards the upper part of the biplot (except for P. aquatilis). Since axis 2 involved mainly factors temperature and biomass of cyanobacteria and green microalgae, and since these both microalgal groups were significantly more represented during summer, this indicated that these nematode species were more abundant under summer conditions. No clear trend was observed for the distribution of Plectus opisthocirculus and Eumonhystera simplex.

Discussion

To the best of our knowledge, the present study is the first long-term monitoring of nematode assemblages inhabiting lotic epilithic biofilms. Although the biofilm-dwelling nematode community was not diversified, two groups of species showing different dynamics were clearly distinguished and seemed to adapt to biofilm composition and seasonality: the first group, consisting of the strongly dominating Chromadorina bioculata and C. viridis, was mainly related to biofilm composition (i.e. age, thickness and diatom content) whereas the second group of species mainly grew under summer conditions. The nematode density averaged 25.4 ind cm -2 and ranged from 0.4 to 161.4 ind cm -2 in the epilithic biofilm over the whole study period. This result lies within the range of values reported for lake epilithic biofilms, i.e. 2.8-161.5 ind cm -2 (Peters & Traunspurger, 2005) and for river epilithic biofilms, i.e. 10-100 ind cm -2 [START_REF] Gaudes | The nematode community in cyanobacterial biofilms in the river Llobregat, Spain[END_REF]. In our study, the nematode community constituted a permanent component of river epilithic biofilms. [START_REF] Mathieu | Free-living nematodes affect oxygen turnover of artificial diatom biofilms[END_REF] suggested that nematode activity could affect the oxygen turnover of diatom biofilms at density values C50 ind cm -2 . This threshold value of density was reached on several occasions during the study period suggesting that this influence was substantial in the epilithic biofilms of the Garonne River.

Nematode density positively correlated with AFDM and Chl a. This strengthens the hypothesis that the amount of microalgae and organic matter favour meiobenthic organisms-such as nematodes-in epilithic biofilms [START_REF] Hillebrand | Control of microbenthic communities by grazing and nutrient supply[END_REF]Peters & Traunspurger, 2005). However, nematode density and biofilm biomass were both clearly dampened after floods (Figs. 2a,3a). Moreover, the positive relation found between nematode density and DAF pointed out the negative impact of floods on nematode populations. It is well-known that epilithic biofilms are detached by shear stress, substratum instability and abrasive effects of suspended solids during flood events [START_REF] Biggs | Periphyton biomass dynamics in gravel bed rivers: the relative effects of flows and nutrients[END_REF][START_REF] Boule ˆtreau | Assessing the importance of a self-generated detachment process in river biofilm models[END_REF]. It is thus obvious that nematodes were swept away with the biofilm when flood occurred. This corroborates the studies of [START_REF] Robertson | Hydraulic habitat and the assemblage structure of stream benthic microcrustacea[END_REF] and [START_REF] Palmer | Disturbance and patch-specific responses: the interactive effects of woody debris and floods on lotic invertebrates[END_REF] showing that floods are important factors shaping meiobenthic communities in rivers.

The species richness observed in the present study (i.e. 28 species over the whole study period) agreed with those observed for several lake epilithic biofilms, i.e. 29 and 8-34 species (in, respectively, Traunspurger, 1992;Peters & Traunspurger, 2005). However, higher species richness values were often reported for sediment-dwelling nematodes (see review [START_REF] Traunspurger | Nematoda[END_REF]. As previously shown in lakes (Peters & Traunspurger, 2005), our results suggest that, also in rivers, nematode diversity is lower in biofilms than in sediments. Reasons for this diversity difference remain complex and unclear [START_REF] Hodda | Nematode diversity in terrestrial, freshwater aquatic and marine systems[END_REF]. A possible explanation might be that, in the Garonne river, nematodes had to totally re-colonize the biofilm after critical floods several times a year (e.g. in January, April-May and November 2009, Fig. 3a). Conversely, in sediments, meiobenthic organisms can migrate deeper towards less disturbed sediment layers to shelter against increasing discharge conditions (Dole- [START_REF] Dole-Olivier | Response of invertebrates to lotic disturbance: is the hyporheic zone a patchy refugium?[END_REF]. Thus, biofilm-dwelling nematodes could be more exposed than sediment-dwelling nematodes to flood disturbances, which are known to decrease benthic invertebrate diversity [START_REF] Death | Diversity patterns in stream benthic invertebrate communities: the influence of habitat stability[END_REF].

While diatoms dominated biofilm algal assemblages in terms of biomass, two epistrate-feeder species Chromadorina bioculata and Chromadorina viridis dominated strongly the nematode assemblage. This observation supports the trend previously hypothesized that, in freshwater benthic environments, nematode communities are generally dominated by few species (e.g. [START_REF] Zullini | Bdelloid rotifers and nematodes in a small Italian stream[END_REF]Michiels & Traunspurger, 2005a;Peters & Traunspurger, 2005). Furthermore, this corroborates a previous study indicating that the epistrate-feeder Chromadorita leuckarti (de Man, 1876) dominates the nematode assemblages in diatom-dominated biofilms of the Llobregat River, Spain [START_REF] Gaudes | The nematode community in cyanobacterial biofilms in the river Llobregat, Spain[END_REF]. C. bioculata and C. viridis were clearly segregated from the other nematode species (Fig. 5) and primarily positively related to diatom biomass. Due to their high content of polyunsaturated fatty acids [START_REF] Phillips | Role of different microbes and substrates as potential suppliers of specific, essential nutrients to marine detritivores[END_REF], diatoms are known to represent a high-quality food resource often selected by benthic primary consumers (e.g. [START_REF] Goedkoop | Pelagic-benthic coupling: profundal benthic community response to spring diatom deposition in mesotrophic Lake Erken[END_REF]Buffan-Dubau & Carman, 2000a). Furthermore, it has been evidenced that a marine nematode belonging to the Chromadorina genus: Chromadorina germanica (Bu ¨tschli, 1874) feeds on benthic diatoms (e.g. [START_REF] Tietjen | Feeding behaviour of marine nematodes[END_REF][START_REF] Deutsch | Gut ultrastructure and digestive physiology of two marine nematodes, Chromadorina germanica (Bu ¨tschli, 1874) and Diplolaimella sp[END_REF]. Therefore, it is likely that the presence of large amounts of a potential food resource may favour C. bioculata and C. viridis. This finding strengthens that nematode feeding strategies match with the availability of their preys within the biofilm.

Our results indicate that a clear shift of the nematode community occurred during summer (Fig. 3b). Such seasonal variations of species composition were previously reported for sediment-dwelling nematode communities in lakes [START_REF] Traunspurger | Fischbiologie des Ko ¨nigssees. Nahrungsangebot und Nahrungswahl. Band I: Das Meiobenthos des Ko ¨nigssees[END_REF]Michiels & Traunspurger, 2005c) and in rivers [START_REF] Beier | Seasonal distribution of free-living nematodes in the Ko ¨rsch, a coarse-grained submountain carbonate stream in southwest Germany[END_REF]. In our study, the summer nematode community is more diversified with a higher proportion of deposit-feeders: e.g. Monhysteridae (Figs. 3c,4a). Concomitantly, the proportion of microalgae in the biofilm (AI) was reduced, but the microalgal community became more diversified. Several hypotheses can be advanced to account for this summer shift:

Firstly, the RDA analysis (Fig. 5) evidenced that a diversified group of nematode species (mainly deposit-feeding species) grew under summer conditions. It is known that summer temperatures enhance the proportion of diversified bacterial assemblages inside epilithic biofilms of the Garonne River [START_REF] Boule ˆtreau | Assessing the importance of a self-generated detachment process in river biofilm models[END_REF][START_REF] Lyautey | Viability of differentiated epilithic bacterial communities in the River Garonne (SW France)[END_REF]. Depositfeeding nematodes can show species-specific feeding response to bacterial and cyanobacterial diversity and availability [START_REF] Moens | Selective attraction of marine bacterivorous nematodes to their bacterial food[END_REF][START_REF] Ho ¨ckelmann | Odor compounds from cyanobacterial biofilms acting as attractants and repellents for free-living nematodes[END_REF][START_REF] Schroeder | Fluctuating food availability may permit coexistence in bacterivorous nematodes[END_REF]. Therefore, it can be suggested that the higher nematode diversity observed during summer could result from a decrease of interspecific competition while the microbial food resources are more diversified (e.g. cyanobacteria, green microalgae and potentially bacteria), confirming that resource availability can structure nematode species composition and diversity (Michiels & Traunspurger, 2005b;[START_REF] Ristau | Relation between nematode communities and trophic state in southern Swedish lakes[END_REF].

Secondly, [START_REF] Michiels | Maintenance of biodiversity through predation in freshwater nematodes?[END_REF], 2004) observed that the density of predators can increase the number of co-existing nematode species by preventing competitive exclusion due to dominant species. In the present study, the density of the predatory nematode Brevitobrilus stefanskii was positively linked to summer conditions (Fig. 5). However, preventing competitive exclusion could also have resulted from macrobenthic predators and grazers (e.g. insect larval stages of Plecoptera, Trichoptera and Ephemeroptera), which are particularly abundant during summer (peaking in early July) in the Garonne River (Leflaive et al., 2008, Majdi et al., unpubl. data).

Thirdly, temperature is known to strongly influence benthic communities in running waters [START_REF] Hawkins | Channel morphology, water temperature, and assemblage structure of stream insects[END_REF][START_REF] Stead | All creatures great and small: patterns in the stream benthos across a wide range of metazoan body size[END_REF]. When temperature is high, the biomass of the epilithic biofilm remains severely controlled by self-generated detachment processes and grazers [START_REF] Boule ˆtreau | Assessing the importance of a self-generated detachment process in river biofilm models[END_REF][START_REF] Hillebrand | Meta-analysis of grazer control of periphyton biomass across aquatic ecosystems[END_REF]. Moreover, [START_REF] Lawrence | Microscale evaluation of the effects of grazing by invertebrates with contrasting feeding modes on river biofilm architecture and composition[END_REF] experimentally showed that grazing of phototrophic biofilm by macrobenthic invertebrates resulted in a significant reduction of autotrophic biomass with an increase of bacterial biomass within grazed regions, corroborating the first hypothesis described above. Thus, these disturbances can lead to a thin summer biofilm layer with a high proportion of heterotrophic organisms where intensive competition for space and resources may create harsh life conditions for epibenthic invertebrates. This suggestion is supported by the decrease of the algal proportion in the biofilm observed during this period. Therefore, it makes sense that typical opportunistic and bacterial-feeding nematodes with a small body size and a low MI (e.g. Monhysteridae) could benefit from these harsh conditions. Moreover, Monhysteridae species-especially genus Eumonhystera-are known to reproduce parthenogenetically [START_REF] Traunspurger | Fischbiologie des Ko ¨nigssees. Nahrungsangebot und Nahrungswahl. Band I: Das Meiobenthos des Ko ¨nigssees[END_REF]. This reproductive strategy probably accounted for the significant reduction of the male proportion observed during summer (Fig. 4b). Overall, summer nematode species lifestyle fits well with corresponding biofilm biotic conditions, suggesting that a close coupling occurs between nematode assemblage functional structure and biofilm characteristics.

Conclusion

Biomass of epilithic microalgae constituting potential food sources for nematodes was plainly identified as an important predictor of nematode community dynamics. Overall, our results strongly suggest that variations in microalgal composition and proportion in the biofilm might drive the observed changes in nematode diversity and functional feeding group composition. This supports the hypothesis that nematodes are involved in a strong trophic coupling with their microbial habitat and should be taken into consideration in further studies on biofilm dynamics and functioning. Notably, studies of nematode feeding behaviour could disentangle trophic interactions in epilithic biofilms and their potential feedback on biofilm's structure and composition.

Fig. 2 Fig. 3

 23 Fig. 2 Temporal dynamics of a epilithic chlorophyll a (Chl a) concentration (±SE, N = 4), ash-free dry mass (AFDM) of the biofilm (±SE, N = 4) and mean daily discharge (MDD), and b the relative proportion (%) of epilithic microalgal groups to

Fig. 4

 4 Fig. 4 Seasonal variations of the nematode community structure in the biofilm: a seasonal proportion of epistratefeeders (E), deposit-feeders (D), suction-feeders (S) and chewers (C), and b seasonal proportion of males (#), females ($), gravid females (g$), fourth stage juveniles (J4) and juveniles (J)

Fig. 5

 5 Fig.5Biplot from the redundancy analysis (RDA) explaining the distribution of nematode species densities according to environmental factors. Ordination axes were rescaled to range from -1 to 1. Slim dotted arrows are non-significant factors. Bold arrows are significant factors (Monte Carlo permutation test with Bonferroni's correction, P \ 0.005). The eigenvalues (k) are indicated for main ordination axes. Environmental factor abbreviations: biomass of diatoms (Diatoms), green algae (GreenAlg) and cyanobacteria (Cyano), epilithic ash-free

Table 1

 1 CHEMTAX pigment ratio matrix Zea), b-carotene (b-car), chlorophyll b (Chl b) and chlorophyll c (Chl c) versus chlorophyll a (Chl a) concentrations from corresponding microalgal cultures.For green algae and diatoms these ratios were obtained from pure cultures of, respectively, Pediastrum boryanum and Nitzschia palea. For cyanobacteria, pigment ratios were obtained from Synechococcus leopoliensis[START_REF] Schlu ¨ter | Identification and quantification of phytoplankton groups in lakes using new pigment ratios -a comparison between pigment analysis by HPLC and microscopy[END_REF]

	Algal group	Species	Biomarker pigment ratios to Chl a
			Fuco	Lut	Viola	Diad	Zea	b-car	Chl a	Chl b	Chl c
	Green algae	P. borianum		0.143	0.049		0.014	0.043	1	0.088
	Diatoms	N. palea	0.477			0.102		0.002	1	0.121
	Cyanobacteria	S. leopoliensis					0.411	0.011	1
	Ratios were calculated considering the relative concentrations of fucoxanthin (Fuco), lutein (Lut), violaxanthin (Viola),
	diadinoxanthin (Diad), zeaxanthin (

Table 2

 2 Measured abiotic and biofilm biotic factors

			Annual mean ± SE	Min	Max
		Temperature (°C)	14.6 ± 0.05	1.7	27.3
		O 2 (mg l -1 )	11.5 ± 0.02	7.4	22.1
		pH (-)	7.6 ± 0.004	6.7	9.1
	Annual means refer to 2009. For temperature, O 2 , pH	Conductivity (lS cm -1 ) Mean daily discharge (m 3 s -1 )	270.9 ± 0.001 124.7 ± 6.0	154 18	493 814
	and conductivity	Days after flood (day)	89.4 ± 11.1	7	233
	(N = 17507). For days after flood and the biotic factors (N = 31). Minimum and	AFDM (g m -2 ) Chlorophyll a (mg m -2 )	27.4 ± 2.7 321.5 ± 50	4.4 10.7	79.7 1012.8
	maximum values refer to	Green algae (%)	17.1 ± 2.3	0	36.3
	the whole sampling period (i.e. September 2008-March 2010)	Cyanobacteria (%) Diatoms (%)	2.2 ± 0.6 80.7 ± 2.7	0 50.6	14.6

Table 3

 3 Biofilm-dwelling nematode species in the study site between September 2008 and March 2010

	Nematode taxa

Table 4

 4 Conditional effects from the redundancy analysis (RDA)

	Factors	k	P
	Diatoms	0.149	0.002**
	T	0.138	0.002**
	DAF	0.104	0.002**
	AFDM	0.102	0.002**
	Cyano	0.084	0.004**
	GreenAlg	0.064	0.004**
	Cond	0.015	0.122
	pH	0.013	0.154
	MWD	0.006	0.502
	O 2	0.003	0.786
	Each environmental factor is listed by its eigenvalue (k)
	indicating the importance of its own contribution (i.e. without
	co-variabiliy, see ''Methods'') to explain the distribution
	variance of nematodes species. Significant factors (**) at
	P \ 0.005 (see ''Methods''). Biomass of diatoms (Diatoms),
	green algae (GreenAlg) and cyanobacteria (Cyano), epilithic
	ash-free dry mass (AFDM), water temperature (T), pH,
	dissolved O 2 (O 2 ), conductivity (Cond), mean weekly
	discharge (MWD) and days after flood (DAF)

Acknowledgments We thank J. Ferriol for helping with algal cultures. We are grateful to K. Martens and to two anonymous reviewers for their helpful comments on the manuscript. This study was funded by a national CNRS EC2CO-CYTRIX program. Nabil Majdi received a doctoral grant no. 31381-2008 by the French department of higher education and research (MESR).