Armelle Paule

Lyautey

Fre´de´ric Garabetian

Jean-Luc Rols
email: rols@cict.fr

Autogenic versus environmental control during development of river biofilm

Keywords: Colonization experiment, community structure, grazing, microbial ecology, succession

In the natural environment, microbial community structure of river biofilm is controlled by biotic and abiotic factors. This study explored the capacity to manipulate the structure of microbial communities by modifying environmental conditions during the course of biofilm development. River epilithic biofilm was cultivated in situ on artificial substrates placed parallel to river water flow. Substrates were incubated for 3 and 5.5 weeks in river to allow natural biofilm development, at two sites with contrasting physico-chemical characteristics. The first site (Aurade´, Gers, France) was located in an agricultural watershed basin and the second site (Larroque, Haute-Garonne, France) was located in a forested watershed basin. After 3 weeks of biofilm development, a subset of substrates was collected from one site and transplanted to the second site where they remained for 2.5 further weeks. Epilithic bacterial community structure (at 3 weeks from each site and at 5.5 weeks from biofilms with and without transplantation) was assessed using PCR-DGGE of 16S rDNA fragment. Biofilm biomass was estimated using ash free dry mass (AFDM). After 3 weeks of development, biofilms from the two sites exhibited comparable AFDM values (average of 1.4 ¡ 0.2 g.m x2 ). A difference between the two sites was observed after 5.5 weeks of development: AFDM decreased for biofilms from the agricultural watershed basin (from 1.4 to 0.18 g.m x2 ) as a consequence of grazing pressure (Bithynia), and increased for biofilms from the forested agricultural watershed (from 1.4 to 2.6 g.m x2 ). Microbial community analyses revealed a differentiated community structure between biofilms from the different sites and exhibited a change of microbial community structure after 5.5 weeks of biofilm development. These observations confirm a process of ecological succession in microbial communities. Changing the incubation site during biofilm development modified the trajectory of these ecological successions, suggesting that site characteristics mainly conditioned the structure of these microbial communities.

Introduction

Epilithic biofilms are microbial aggregates constituted by heterotrophic micro-and meio-organisms (e.g., bacteria, protozoan) and phototrophic micro-organisms (e.g., diatoms, cyanobacteria), embedded in a exopolymeric substances matrix secreted by the microorganisms [START_REF] Lock | River epilithon: toward a structuralfunctional model[END_REF]. Epilithic biofilm development occurs at the interface between river bed substrates (pebbles) and the water column where hydrodynamics, light and geomorphological characteristics favor the development of a sessile biomass (Wetzel, 1983). Typically, epilithic biomass is dominated by the algal component of the community [START_REF] Peterson | Response of benthic algal communities to natural physical disturbance[END_REF]. In lotic systems, epilithic biofilms represent a major compartment involved in primary production [START_REF] Dodds | Eutrophication and trophic state in rivers and streams[END_REF], mineralization and element recycling processes (e.g., dissolved organic carbon dynamics, [START_REF] Romani | Biofilm structure and function and possible implications for riverine DOC dynamics[END_REF] and participate in autoepuration [START_REF] Sabater | The effect of biological factors on the efficiency of river biofilms in improving water quality[END_REF][START_REF] Teissier | Detailing biogeochimical N budgets in riverine epilithic biofilms[END_REF] and biodegradation of pollutants in aquatic environments [START_REF] Lawrence | Sorption and metabolism of selected herbicides in river biofilm communities[END_REF][START_REF] Sabater | Monitoring the effect of chemicals on biological communities. The biofilm as an interface[END_REF].

Epilithic biofilm functioning is conditioned by the community structure and diversity. Overall, epilithic biofilm structure can be influenced by (i) abiotic parameters such as water temperature [START_REF] Denicola | Periphyton responses to temperature at different ecological levels[END_REF], nutrient availability [START_REF] Bothwell | Algal-nutrient dynamics[END_REF][START_REF] Bella | Benthic diatom communities and their relationship to water chemistry in wetlands of central Italy[END_REF], substrate types [START_REF] Murdock | Linking benthic algal biomass to stream substratum topography[END_REF], hydrodynamics [START_REF] Biggs | Patterns in Benthic Algae of Stream[END_REF][START_REF] Battin | Contributions of microbial biofilms to ecosystem processes in stream mesocosms[END_REF], light (Wetzel, 1983;[START_REF] Boston | Photosynthesis-light relations of stream periphyton communities[END_REF][START_REF] Hill | Effects of light[END_REF] or pollutants [START_REF] Lawrence | Effects of selected pharmaceuticals on riverine biofilm communities[END_REF][START_REF] Tornes | Indicator taxa of benthic diatom communities: a case study in Mediterranean streams[END_REF][START_REF] Morin | Long-term survey of heavy-metal pollution, biofilm contamination and diatom community structure in the Riou Mort watershed, South-west France[END_REF][START_REF] Tlili | Responses of chronically contaminated biofilms to short pulses of diuron -An experimental study simulating flooding events in a small river[END_REF]; and by (ii) biotic interactions such as predation and competition [START_REF] Bourassa | Control of periphyton biomass in Laurentian streams (Quebec)[END_REF][START_REF] Jackson | Successional changes in bacterial assemblage structure during epilithic biofilm development[END_REF].

Epilithic biofilm development was demonstrated to be associated with population succession processes, both for the algal and the bacterial compartments. Colonization of bare substrates is completed by early-colonizing diatoms which will modify the microenvironment and favour the settlement and colonization of other communities [START_REF] Stevenson | Effects of current and conditions simulating autogenically changing microhabitats on benthic diatom immigration[END_REF]. However, some environmental conditions can favour initial colonization of green algae (high light intensities) or of heterotrophic bacteria (low light intensities) [START_REF] Roeselers | Heterotrophic pioneers facilitate phototrophic biofilm development[END_REF]. After initial colonization, succession of diatom populations associated with the biofilm development was recorded [START_REF] Eulin | Epilithic diatom communities during the colonization of artificial substrates in the River Garonne (France). Comparaison with the natural communities[END_REF]. Succession processes were also demonstrated for epilithic bacterial communities and were associated to biofilm maturation [START_REF] Manz | Phylogenetic composition, spatial structure, and dynamics of lotic bacterial biofilms investigated by fluorescent in situ hybridization and confocal laser scanning microscopy[END_REF][START_REF] Jackson | Successional changes in bacterial assemblage structure during epilithic biofilm development[END_REF][START_REF] Araya | Bacterial activity and community composition in stream water and biofilm from an urban river determined by fluorescent in situ hybridization and DGGE analysis[END_REF]. Biofilm maturation can ultimately promote the occurrence of micro-niches within the aggregate that will favour the development of populations involved processes such as denitrification (Lyautey et al., 2005a). According to a succession model, late development stages are associated with a decrease of the exchanges between the water column and the biofilm, and with an increase of species interaction within the aggregate [START_REF] Jackson | Changes in community properties during microbial succession[END_REF]. The main interaction is competition, which results in a decrease of the number of new species added to the community in late development stages [START_REF] Santegoeds | Structural and functional dynamics of sulfate-reducing populations in bacterial biofilms[END_REF][START_REF] Jackson | Changes in community properties during microbial succession[END_REF]Lyautey et al., 2005a).

Because of its ubiquitous character, short generation time, sessile nature, and rapid response to changes in environmental conditions, epilithic biofilms are widely used as bioindicators of water quality in lotic systems [START_REF] Burns | Potential for biofilms as biological indicators in Australian riverine systems[END_REF]. The algal compartment, and more especially diatoms, is used to assess water quality using the Diatoms Biological Index [START_REF] Prygiel | The assessment of water quality in the Artois-Picardie water basin (France) by the use of diatom indices[END_REF]. Bacteria are also sensitive to environmental or humaninduced perturbations. A spatial heterogeneity of bacterial communities was recently reported along an upland river gradient, and was related to temperature and pH variations [START_REF] Anderson-Glenna | Spatial and temporal variability in epilithic biofilm bacterial communities along an upland river gradient[END_REF]. Seasonal variations of environmental parameters proved to cause recurrent changes in community structure [START_REF] Hullar | Recurring seasonal dynamics of microbial communities in stream habitats[END_REF]. Anthropic perturbations also proved to influence bacterial community structures [START_REF] Lyautey | Bacterial diversity of epilithic biofilm assemblages of an anthropised river section using DGGE analysis of a 16S rDNA fragment[END_REF][START_REF] Bru¨mmer | Diversity and seasonal changes of uncultured Planctomycetales in river biofilms[END_REF].

In the present work, we conducted a 5.5 weeks colonization experiment: epilithic biofilm development was carried out on artificial substrates at two sites presenting contrasting environmental conditions. After 3 weeks of development, epilithic biofilms were either left for 2.5 more weeks at the same site, or were transplanted to the other study site where they were left for 2.5 more weeks of development. Bacterial community composition was assessed using 16S rDNA based PCR-DGGE on 3 and 5.5 weeks old biofilms from transplanted and unmoved communities. The objectives of this study were to (i) assess bacterial community structure changes during biofilm development; (ii) compare bacterial community structures at two contrasting sites; and (iii) evaluate the influence of a transplantation during biofilm development on bacterial communities. The transplantation process was intended to assess the relative influence of autogenic (succession) and allogenic (environmental conditions) parameters on epilithic bacterial communities.

Materials and methods

Study sites

Two sites (sites L and A) exhibiting different physicochemical parameters were chosen to carry out in situ epilithic biofilm cultures (Fig. 1). Site L on the Save River is located in a forested watershed basin. The study site is located upstream of Save Gorges and at 2 km of the city of Larroque (France) (43x11 0 43.77 00 N/00x36 0 28.59 00 E). The basin is dominated by forested and pasturage landscapes. The river bed is very rocky and wide 6.7 m. During biofilm development, the average water depth was of 50 cm and the flow velocity (flow velocity meter, Flo-mate model 2000) was about 1 m.s x1 during low water periods and 3 m.s x1 during high water periods. Site A on the Montousse´stream is located in an experimental agricultural watershed basin outlet (328 ha) of Aurade´city (France) (43x33 0 55.06 00 N/1x3 0 30.92 00 E). The area is dominated by an agricultural landscape and reported as highly contaminated by pesticides and nitrate. The main cultivated crops are wheat and sunflower. The river bed is sedimentary, confined (1.5 m wide), and woody. Water depths (50 cm) and flow velocity (0.045 m.s x1 ) were constant during the biofilm development period.

Experimental protocol and sample collection

Biofilm in situ colonization was carried out between March 22 and April 30, 2007 to cover the pesticide application period in the experimental agricultural watershed basin. Artificial substrates were immersed in river water to allow in situ epilithic biofilm colonization and development. The use of artificial substrates permitted (i) to reduce the subjectivity of sample collection; (ii) to ease biofilm scraping; and (iii) to compare two stations with different physico-chemical characteristics [START_REF] Cattaneo | How artificial are artificial substrata for periphyton?[END_REF]. Moreover, no natural substrate was available at site A. Cleaned and smooth glass slides (244r 78 mm) were chosen as uniform artificial substrates. Glass slides were supported vertically within plastic trays (400r300r 100 mm) and recovered by a 5-mm mesh wire fence, as described by [START_REF] Morin | Long-term survey of heavy-metal pollution, biofilm contamination and diatom community structure in the Riou Mort watershed, South-west France[END_REF]. Every tray was made up of four glass slides which represented four replicates. Plastic trays were submerged about 20 cm below the water surface, and placed parallel to water flow, in order to avoid sedimentary deposit on glass slides and to promote micro-organisms fixation. Biofilm colonization was performed as follows: three glass slide tray tracks were placed at each of the two sites. For each site, four glass slides were sampled randomly after 3 weeks of biofilm development, and four glass slides were sampled after 5.5 weeks of biofilm development. The last set of four glass slides for which biofilm has initially developed for 3 weeks at one site were removed from their site of origin and transplanted in the other site where biofilm was left for 2.5 further weeks of development. In summary, experimental conditions included two study sites with different environmental conditions, two incubation period lengths (3 and 5.5 weeks, A and AA for site A, and L and LL for site L, respectively) and a transplantation of the biofilms during development (LA: from site L to site A, and AL : from site A to site L). After collection, glass slides were transported to the laboratory in cool bottles within moisturized garbage bags. Biofilm was removed from glass slides by scraping with a toothbrush, previously treated with NaOH 1 N in order to avoid all trace of DNA and by scraping with a microscope blade. Biofilm was suspended in 90 mL of river water previously filtered through a 0.2 mm pore size filter (cellulose acetate membrane, Whatman) and homogenised (13 500 rpm, Ultra Turrax, T25). Biofilm suspension was aliquoted for further analyses.

Physico-chemical characteristics analysis

Water physico-chemical characteristics of each study site were estimated during the biofilm development period. Temperature, conductivity, dissolved oxygen concentration and pH were measured in situ using specific electrodes. Conductivity and pH values were measured with a conductimeter Hanna HI 99 1300 and a pH meter 320 WTV (electrodes Sentix41), respectively. Dissolved oxygen concentration values were determined with an Oxi323 oxymeter (electrodes oxical-S) and temperature values were represented by the mean values that were determined by pH meter and oxymeter. In parallel, water samples were collected to assess the water quality using the following parameters: nitrate (NO 3

x ), total phosphorus and ammonium (N-NH 4 + ) concentrations. These parameters were measured by classic colorimetric methods (Secoman, Uvi Light, XT5) according to standard methods [START_REF] Apha | Standard methods for the examination of water and wastewater[END_REF]. Ammonium concentrations were determined within 10 h following sampling. Dissolved organic carbon (DOC) concentrations were determined in water filtered through a 0.45 mm pore size filter (cellulose acetate membrane, 25 mm diameter, Whatman) and analyzed using a platinum catalyzer at 680 xC (Shimadzu, Model TOC 5000). All measures and sampling were performed in the morning to allow homogeneity between samples and once or twice a week. Water samples were refrigerated during transport to the laboratory. Twenty three pesticides, commonly used in the southwest of France, among which 17 herbicides (Aclonifen, Atrazine, Atrazine desethyl, Chlorotoluron, Cyanazine, Hexazinone, Imazathabenz-methyl, Isoproturon, Linuron, Metazachlor, Metolachlor, Metoxuron, Monolinuron, Sebuthylazine, Simazine, Terbuthylazine, Trifluralin) and six fungicides (Cyproconazol, Epoxiconazol, Fenpropimorph, Flusilazol, Pendimethalin, Tebuconazol) were quantified at three times during biofilm development: T0 corresponds to the time of artificial substrates positioning, T3 and T5 correspond to 3 and 5.5 weeks of colonization, respectively. Three liters of water were collected in closed glasses bottles previously cleaned and sterilized by autoclaving. For every sample, 10 mL of dichloromethane were added to 1.5 L of water. Then, samples were kept in a cold chamber at 4 xC. Pesticides were extracted from water using dichloromethane, and pesticides levels were quantified by Gas Chromatography Mass Spectrometer (GC-MS, Thermo Fisher, Model Trace DSQ), according to [START_REF] Devault | Multi-residues analysis of pre-emergence herbicides in fluvial sediments: application to the mid-Garonne River[END_REF].

Biomass determination

Dry mass (DM) was measured by weighing the dried pellet (24 h at 80 xC) from an aliquot of 10 mL of biofilm suspension (centrifuged at 3500r g for 25 min, Heraeus Function Line). The pellet was subsequently combusted (8 h at 550 xC) to provide the ash free dry mass (AFDM). Another 10-mL aliquot of the biofilm suspension was centrifuged (12 000r g, 20 min, 4 xC). After removing the supernatant, Chlorophyll a content of the pellet was determined following an extraction in 90% acetone by spectrophotometry according to SCOR-Unesco (1966). Autotrophic index (AI) was defined as the ratio between AFDM and Chlorophyll a (APHA, 1992; [START_REF] Steinman | Biomass and pigments of benthic algae[END_REF], and indicates the relative importance of autotrophic organisms versus heterotrophic organisms and detritus. For each sample, the ratio between AFDM and DM was determined to indicate the relative importance of organic fraction in the biofilm.

Microbial community structure

After centrifugation (12 000r g at 4 xC for 20 min, Heraeus Multifuge) of an aliquot of 10 mL of the initial biofilm suspension, the pellet was stored at x80xC until further analysis. Genomic DNA extraction was performed on the pellet using Ultra Clean TM Soil DNA Isolation kit according to the manufacturer's protocol (Mobio Laboratories). The extracted DNA concentration was quantified by fluorimetry (Fluoroscan Ascent, Labsystem) using SYBR green (Sigma Aldrich). The 16S rDNA variable regions V3 to V5 were amplified using primers (Proligos) described as universal within the Bacteria domain: 341F (5 0 -CCTACGGGAGGCAGCAG-3 0 ) with a 40 bp GC sequence clamped at its 5 0 end (5 0 -CGCCCGCCGCGC-CCCGCGCCCGTCCCGCCGCCCCCGCCCG-3 0 ) and 907F (5 0 -CCGTCAATTCMTTTGAGTTT-3 0 ) [START_REF] Muyzer | Application of denaturing gel electrophoresis (DGGE) temperature gradient gel electrophoresis (TGGE) in microbial ecology[END_REF]. Amplification was carried out using an Eppendorf Mastercycleur following a protocol described elsewhere (Lyautey et al., 2005b) using 20 ng of extracted DNA as template for the PCR. Three replicate amplifications were performed for each sample. Amplified product concentrations were quantified on a 1.65% agarose gel (Eurogentec) using precision Molecular Mass Ruler (BioRad) as described previously (Lyautey et al., 2005b). DGGE was carried out using D-Code Universal Mutation Detection System (BioRad). An amount of 700 ng (approximately 234 ng from each of the three sample replicates) of PCR products were loaded onto an acrilamide gel containing a gradient of denaturant ranging from 35 to 70% (100% denaturant is 7 M urea and 40% deionized formamide). For practical purposes (only 20 samples can be processed on each DGGE gel), the four replicates of the six samples were processed on two different DGGE gels (two replicates of each sample on each DGGE gel). The replicate 2 from 5.5 week biofilm sample of site L (LL2) exhibited in the DGGE analysis a number of bands drastically lower (< 10 OTUs) than the three other replicates suggesting that a methodological problem happened with this sample, either during DNA extraction or DNA amplification. Data from this sample were thus not included in the analysis. Electrophoresis was performed at 100 V for 18 h at 60 xC. The gels were stained with SYBR Green (Sigma Aldrich) for 30 min. The gel image was captured using a CCD camera and Biocapt Software (Vilbert Lourmat) and analyzed using Bio-Numerics 5.1 software (Applied Maths, Sint-Martens-Latem, Belgium).

Data analysis

In order to avoid methodological biases due to DGGE intergel variability, each gel were analyzed separately. DGGE bands (defined as operational taxonomic units (OTUs)) were scored as present or absent from DGGE gel analysis. A matrix was constructed from the Jaccard similarity index (J= c/(a+ b+ c) where a, the number of bands found only in sample A, b, the number of bands found only in sample B and c, the number of bands shared between samples A and B). To assess changes in the microbial community structure during biofilm development, DGGE patterns were analyzed by non-metric multidimensional scaling (NMDS) analysis as described elsewhere [START_REF] Van Hannen | Changes in bacterial and eukaryotic community structure after mass lysis of filamentous cyanobacteria associated with viruses[END_REF]. NMDS analysis is a mathematical technique which provides a graphical representation of every band pattern (sample) as one plot where relative changes in community structure can be interpreted as distances between the plots. The closer the plots are to each other, the more similar are the DGGE banding patterns. NMDS was carried out using SPSS 13.0 software for Windows (SPSS Inc., Chicago, IL, USA) using a stress value < 0.1.

The difference in physical chemical characteristics between stations and the difference in DM, AFDM, Chlorophyll a, number of bands on DGGE banding patterns between biofilm samples were assessed with the Kruskal Wallis test using SPSS 13.0 software for Windows. Differences were considered statistically different at P < 0.05.

Results

Water physico-chemical parameters

For each site, water physico-chemical parameters values presented below represent average value (¡ standard deviation) from the eight water samples collected during biofilm development (Fig. 2). Water temperatures were comparable between site A (10.9 ¡ 3 xC) and site L (8.8 ¡ 2.8 xC) (P = 0.128). Dissolved oxygen concentrations measured at site A (12.3 ¡ 1 mg.L x1 ) were significantly lower than at site L (13.7 ¡ 0.8 mg.L x1 )(P< 0.01). For both sites, temperature increased and dissolved oxygen concentrations decreased during the experimental period, in accordance to seasonal variations. pH and conductivity values remained constant for both sites over the sampling period, but were significantly different between sites. pH values were above 7 for site A (7.6 ¡ 0.1) and were lower for site L (6.2¡ 0.4) (P<0.001). Conductivity was higher at site A (725 ¡ 8 mS.cm x1 ) than at site L (128 ¡ 4 mS.cm x1 )(P< 0.001). Site A is located in a calcareous watershed basin which explained conductivity and pH values. Nitrate concentrations remained constant over time for both sites with higher NO 3

x values recorded for site A (41 ¡ 1.2 mg.L x1 ) than for site L (7 ¡ 0.6 mg. L x1 )(P<0.001). No difference was observed for N-NH 4 + concentration values between site A (16 ¡ 4 mg.L x1 ) and site L (25¡ 12 mg.L x1 )( P = 0.382). Total phosphorus concentrations were significantly lower for site A (17 ¡ 5 mg.L x1 ) than for site L (63 ¡ 34 mg.L x1 )( P< 0.001). Two flood events at site L were recorded during the colonization period. DOC concentrations available were obtained for site A in 2008 (from March to April, n= 10) and for site L in 2004 (from February to May, n = 6). DOC concentrations were higher at site A (2.25 ¡ 0.07 mg.L x1 ) than at site L (1.32¡ 0.08 mg.L x1 )(P<0.05).

Thirteen of the 23 pesticides tested were detected with a concentration above 0.1 mg.L x1 at least once during the study period. Cumulated pesticide concentrations are presented in Table 1. Contamination levels by pesticides on both sites were from 1 to 5 mg.L x1 during biofilm development. Concentrations were in the same order of magnitude for both sites at each period.

Biomass descriptors

AFDM values ranged from 0.18 to 2.6 g.m x2 (Fig. 3). After 3 weeks of development, biofilms from sites L and A exhibited comparable AFDM values around 1.4 g.m x2 (P = 0.773). After 5.5 weeks of development, biofilms from site L (AL and LL) (up to 2.6 g.m x2 ) exhibited higher AFDM levels than 5.5-weeks biofilms from site A (LA and AA) (down to 0.18 g.m x2 )( P<0.001). At site L, important biomass were consistent with the growth process and biofilm accretion, whereas the low biomass recorded at site A were consistent with the occurrence of grazing as revealed by the presence of several Bithynia molluscs (Prosobranchia, Bithyniidae) on glass slides. AFDM/DM ratio values ranged from 6.0 to 17.8%. Low values reveal a biofilm rich in detritus and sedimentary particles. These sedimentary particles are likely to have been imported by the two flood events observed in site L and by the presence of sediments on river bed in site A. Important standard deviation values were caused by the presence of filamentous algae which decreased the homogeneity of biofilm samples. The autotrophic index (AI) values ranged between 42 and 258. Three weeks old biofilms exhibited higher AI levels than 5.5 weeks old biofilms (P< 0.001), indicating that late stages were essentially constituted by autotrophic organisms.

Microbial community structure

The number of bands (OTUs) obtained for each experimental conditions are presented in Table 2. Analysis of DGGE banding patterns revealed a total of 66 and 94 OTUs for gel 1 (replicates numbers 3 and 4 of each samples) and gel 2 (replicates 1 and 2), respectively. The average number of OTUs per sample varied from 34 and 56 for communities from 3-week biofilm to 40 and 55 for 5.5-week biofilm communities, for gel 1 and gel 2, respectively, indicating a constant richness along with biofilm maturation (P= 0.371) and the transplanted experiment (P = 0.752). Along with biofilm development new species appeared in the communities. An average of 15 OTUs appeared at site A between 3 week old and 5.5 week old communities and an average of 12.5 OTUs appeared at site L. At the same time, the disappearance of OTUs along with the aggregate development was observed, with an average number of 9 OTUs disappearing for both sites. The transplanted experiment also induced the appearance of news OTUs: 12.5 at site A and 10 at site L. Mature biofilms both from the transplanted communities and from the untransplanted biofilms only shared six common OTUs. NMDS analysis was carried out on the presenceabsence matrix separately for each DGGE gel following by a superposition of both dimension representation plots (Fig. 4). NMDS takes the community-level similarity of the samples. The stress values of final configuration were 0.123 and 0.105 and the proportions of variance explained were 0.93 and 0.94. The proximity between replicates on the plot revealed an important similarity of the DGGE banding patterns. Three and 5.5 week old biofilm communities from sites A and L exhibited differentiated structures according mainly to the different incubation sites and then to the state of maturation of the aggregate. Bacterial communities from the transplanted samples differed according to their site of origin (three first weeks of development) and of the site they were transplanted to. NMDS analysis showed that differentiated bacterial community structures were mainly mediated by the environmental conditions. conditions on epilithic biofilm bacterial community dynamics.

Discussion

Theprimaryobjectivesofthisworkweretoevaluatethe relative importance of development and of environmental

The choice of the two study sites proved to be relevant for the purpose of this work since different environmental conditions were recorded during the study period. Hydrodynamics, which discriminated the two sites, generally causes substrate instability, and can generate a loss of biomass during flood events [START_REF] Biggs | Patterns in Benthic Algae of Stream[END_REF]. To minimise the importance of this parameter and to homogenize growth between our two study sites, biofilms were cultivated on artificial substrates. Artificial substrates prevent the occurrence of biases due to intra-and inter-site variations during sampling. Although it has been suggested that substrate properties such as surface roughness or hydrophobicity can influence bacterial community structure [START_REF] Anderson-Glenna | Spatial and temporal variability in epilithic biofilm bacterial communities along an upland river gradient[END_REF], it was shown that artificial substrate support the development of algal community composition representative of natural communities [START_REF] Eulin | Epilithic diatom communities during the colonization of artificial substrates in the River Garonne (France). Comparaison with the natural communities[END_REF][START_REF] Barbiero | A multi-lake comparaison of epilithic diatom communities on natural and artificial substrates[END_REF][START_REF] Lane | A comparison of diatom community structure on natural and artificial substrata[END_REF]. However, artificial substrate ability to reproduce natural communities remains controversial [START_REF] Cattaneo | How artificial are artificial substrata for periphyton?[END_REF]. The water physicochemical parameters of the two study sites were very contrasted. Site A, located in an agricultural watershed basin, was characterized by high levels of pH, conductivity, nitrate and DOC concentrations. Site L, located in a forested watershed basin, was mainly characterized by its higher total phosphorus concentrations. Concerning pesticides, the two sites presented similar levels of contamination. On the basis of environmental factors, sites A and L are clearly discriminated by their trophic states.

The choice of the incubation period length was based on the dynamic of epilithic biofilm growth occurring in large rivers (Lyautey et al., 2005a;[START_REF] Bouleˆtreau | Assessing the importance of a self-generated detachment process in river biofilm models[END_REF]. Epilithic biofilm growth is defined by an accretion phase related to colonization and growth processes and characterized by an increase of AFDM resulting in a biomass peak. Observed AFDM variations were consistent with previous experiments: at site L, epilithic biofilms presented a biomass increase over time (colonization and growth) whereas at site A, a biomass decrease was observed after three weeks. The presence of aquatic Bithynia molluscs (Prosobranchia, Bithyniidae) on glass slides suggests that the disturbance was due to a grazing event. The biomass removal was more important for biofilms transplanted from site L to site A suggesting that they might have been more attractive for the invertebrates. The maximum biomasses reached in the present work were slightly below those previously recorded in the literature (up to 12 g AFDM.m x2 in [START_REF] Lyautey | Bacterial diversity of epilithic biofilm assemblages of an anthropised river section using DGGE analysis of a 16S rDNA fragment[END_REF] and between 15 and 25.6 g AFDM.m x2 in [START_REF] Biggs | Patterns in Benthic Algae of Stream[END_REF]). These lower biomasses could be explained by (i) a shorter growth period in the present work (5.5 weeks) as opposed to up to 11 weeks in [START_REF] Biggs | Patterns in Benthic Algae of Stream[END_REF]; (ii) a limited adhesion of cells due to nature of the substrates used; or (iii) a limited nutrient availability [START_REF] Dodds | Suggested classification of stream trophic state: distributions of temperate stream types by chlorophyll, total nitrogen, and phosphorus[END_REF]. The low AFDM/DM values recorded in the present work suggest that biofilms were rich in detritus and sedimentary particles despite artificial supports being parallel to flow. Sedimentary particles were likely imported by the two flood events observed in site L and by the presence of sediments on river bed in site A. The autotrophic index indicates the relative importance of autotrophic versus heterotrophic organisms and detritus. With values between 42 and 258, epilithic biofilms described here were stable and constituted by equivalent proportions of photoautotrophic and heterotrophic microorganisms [START_REF] Bourassa | Control of periphyton biomass in Laurentian streams (Quebec)[END_REF].

Bacterial community composition was assessed using 16S rDNA based PCR-DGGE. Methodological biases (chimera and heteroduplex formation, template annealing, detection of the dominant populations) are well known and have been widely discussed in the literature [START_REF] Muyzer | Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of PCR-amplified genes coding for 16S rRNA[END_REF]. Applied to epilithic biofilms, the use of Eubacteria-specific primers was proved to over-estimate bacterial richness by allowing the amplification of cyanobacteria and plastid DNA (Lyautey et al., 2005b). With averages of 40 (replicates 3 and 4) to 55 (replicates 1 and 2) OTUs per sample of 5.5-week biofilm, bacterial richness was higher than the richness observed for epilithic biofilm grown on artificial substrates [START_REF] Cody | Characterization of benthic bacterial assemblages in a polluted stream using denaturing gradient gel electrophoresis[END_REF][START_REF] Araya | Bacterial activity and community composition in stream water and biofilm from an urban river determined by fluorescent in situ hybridization and DGGE analysis[END_REF][START_REF] Dorigo | Lotic biofilm community structure and pesticide tolerance along a contamination gradient in a vineyard area[END_REF], natural substrates (Lyautey et al., 2005a), and bacterial communities from phytoplankton in tropical water bodies [START_REF] Dumestre | Changes in bacterial and archaeal assemblages in an equatorial river induced by the water eutrophisation of Petit Saut dam reservoir (French Guiana)[END_REF] or in marine environment [START_REF] Schauer | Spatial differences in bacterioplankton composition along the Catalan coast (NW Mediterranean) assessed by molecular fingerprinting[END_REF]. This could reflect limited niches and resource availability or much harsher environment caused by continual grazing and sloughing processes [START_REF] Anderson-Glenna | Spatial and temporal variability in epilithic biofilm bacterial communities along an upland river gradient[END_REF]. The use of artificial substrates did not seem to affect bacterial community richness.

Dynamics of biofilm bacterial community were assessed at two sampling dates (at 3 and 5.5-week development) which reveals constant richness values during biofilm maturation but the appearance or the disappearance of species suggest changing bacterial communities and then a species succession consistent with theoretical models of biofilms succession [START_REF] Jackson | Changes in community properties during microbial succession[END_REF]. Observed succession is thus mostly driven by autogenic processes. Allogenic factors also influence bacterial community composition, and can explain the differences observed between the two sampling sites. In freshwater environments, it was suggested that pH, temperature, and nutrient availability were correlated with variations of structure [START_REF] Lyautey | Bacterial diversity of epilithic biofilm assemblages of an anthropised river section using DGGE analysis of a 16S rDNA fragment[END_REF][START_REF] Hullar | Recurring seasonal dynamics of microbial communities in stream habitats[END_REF]. Turbulent flow was also demonstrated to influence bacterial community composition during the biofilm initial growth phase, by selecting the pioneer algal species that will create the biofilm microenvironment [START_REF] Besemer | Biophysical controls on community succession in stream biofilms[END_REF].

The transplant experiment was intended to assess the relative influence of autogenic (succession) and allogenic (environmental conditions) parameters on epilithic bacterial communities. Epilithic biofilm community development initially occurred in two separate stream environments characterized by their proper trophic states, and then some of the biofilms were transplanted to the second environment for further development. It appeared that transplantation contributed to modifications of the bacterial communities, and modified the trajectory of the epilithic biofilm succession. However, the fact that some OTUs were commons between 5.5 week biofilms with and without transplantation indicates that succession was also involved in the temporal differentiation process by allowing common populations, either already present at time 3 weeks but below detection level or inoculated from surface waters from both sites, to develop in the aggregate. This implicates an influence of allogenic factors over autogenic ones. Our results suggest that the three initial weeks of biofilm development were too long to already determine the bacterial community composition and the 2.5 weeks of development following transplantation allowed to modify the community. Hypothetically, grazing might favour the addition of new species by decreasing the number of competitive species or by increasing vacant microhabitats. However, such a phenomenon was not observed, probably because the period of time between grazing and sample collection was too short to allow the initiation of a secondary ecological succession.

The present work confirmed that bacterial community structure was initially controlled by allogenic factors and then followed a succession pattern dominated by autogenic factors. Transplantation modified the structure and dynamics of epilithic bacterial communities that revealed the importance of allogenic factors on bacterial community successions. It could have been of interest to link the functional diversity with bacterial community structure during epilithic biofilm development and following transplantation, by identifying the populations that appeared in the community. A minimal number of species is responsible for stability and function in ecosystem processes, and all changes in this biodiversity could cause processes changes [START_REF] Chapin | Consequences of changing biodiversity[END_REF][START_REF] Loreau | Biodiversity and ecosystem functioning: current knowledge and future challenges[END_REF]. According to the experimental design used, it could be hypothesized that bacterial community structure changes would reflect variations of functional diversity. Epilithic biofilm could thus represent a powerful model to study relationships between structure and function in aquatic ecosystem.

Fig. 1 .

 1 Fig. 1. Location of the two sites (black dots) chosen for in situ epilithic biofilm cultures. Site L is located in a forested watershed and site A is located in an agricultural watershed.

Fig. 2 .

 2 Fig. 2. Weekly evolution of dissolved oxygen concentrations and temperatures for site A (black dots) and site L (white dots) during the period of in situ biofilm colonization. " Week = 0 " indicates the date where artificial substrates were placed in situ (March 22, 2007).

Table 1 .

 1 Cumulated pesticide concentrations measured in river water from study sites A and L for three dates (T0, T3, and T5). T0 corresponds to the time of artificial substrates positioning, T3 and T5 correspond to 3 and 5.5 weeks of colonization, respectively. All concentrations are expressed in mg.L x1 .

Fig. 3 .

 3 Fig. 3. Average ¡ SD value plots of ash free dry mass (AFDM), autotrophic index (AI, white bars) and AFDM/DM ratio (black bars) for biofilm samples according to the different experimental conditions: A, L, AA, LA, AL and LL (refer to materials and methods for the letters meaning).

Fig. 4 .

 4 Fig. 4. Two dimension representation plot of non-metric multidimensional scaling (NMDS) analysis of DGGE banding patterns from the two gels analyzed in the present work for bacterial communities from 3 and 5.5 week biofilms from site L (white dots) and from site A (black dots) and from transplanted biofilms (dark squares). Plots issued from the first DGGE gel were rotated (45x counterclockwise -axis represented by dashed lines) to allow superposition of plots from gels 1 and 2. The numbers 1, 2, 3 and 4 indicate replicates from one same experimental condition.

Table 2 .

 2 Number of bands (OTUs) obtained from DGGE pattern analysis for different experimental conditions (A, L, AA, LL, AL and LA, refer to materials and methods for the letters meaning). Four replicates (numbers 1 to 4) were analyzed for each condition.

		A	L	AA	LL	AL	LA
	15 85 2	5 7	6 1	5 4	5 4
	2	56	60	51	-	54	51
	33 63 3	3 9	4 0	4 8	3 7
	43 43 1	4 2	3 9	4 8	3 8

Acknowledgements. We thank D. Dalger and G. Merlina for water chemistry analysis, J.L. Probst for DOC values and Y. Nicaise for PCR-DGGE analysis. We also thank the " Association des Agriculteurs d'Aurade´" for the access to site A.


Alice Huertas

Ly Tu

Natalia Gambaryan

Barbara Girerd

Frédéric Perros

David Montani

Dominique Fabre

Elie Fadel

Saadia Eddahibi

Sylvia Cohen-Kaminsky

Christophe Guignabert

Marc Humbert
email: marc.humbert@abc.aphp.fr

Prof Humbert Marc

Leptin and regulatory T-lymphocytes in idiopathic pulmonary arterial hypertension

Keywords: dysimmunity, endothelial dysfunction, leptin, pulmonary arterial hypertension, regulatory T lymphocytes

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

Introduction

Idiopathic pulmonary arterial hypertension (IPAH) corresponds to precapillary pulmonary hypertension in which there is neither a family history of the disease, nor an identified risk factor and is defined as a resting mean pulmonary arterial pressure mPAP 5 mm(g with a normal pulmonary artery wedge pressure 5 mm(g 1.

Although the pathophysiology of IPAH has been extensively studied in the past decades and several new pathways have been identified, the etiology of this disease is still not clearly understood. It is now well established that inflammation plays an important role in IPAH 2-6 and increasing data are also supporting the hypothesis that immunologic disorders could be present in IPAH patients: circulating autoantibodies have been detected 7-8 and recent evidence are indicating that regulatory T cells (Treg) could play a role in PAH 9. Despite these findings, little is known about the exact role of inflammation and dysimmunity in the development of IPAH and it remains unclear how immune mechanisms contribute to the pathogenesis of IPAH.

Recent evidence has demonstrated a detrimental involvement of leptin, a cytokine-like hormone mainly secreted by adipocytes, in promoting the pathogenesis of various autoimmune diseases 10-11. Although leptin, product of the obese (ob) gene, has been discovered as the appetite regulator, it regulates a wide range of physiological functions 10-13. Interestingly, it has also been shown that leptin can promote chronic autoimmune disorders by regulating Treg onset and/or function 14. Intriguingly, little is known about the pulmonary effects of leptin, and in particular its inflammatory and/or immunologic role in IPAH.

Treg are T lymphocytes known to dampen autoreactive responses and are able to delay the onset and progression of autoimmune disorders: reduced frequency of Treg and/or defective suppressor function have been observed in these diseases 14-15. So far, little is known about the role of leptin on the lung and in particular about its effects on the pulmonary vessels.

IPAH is characterized by small-sized pulmonary arteries/arterioles in which there is intimal hyperplasia with medial and adventitial hypertrophy, hyperplasia and fibrosis. Cells from the vessel wall are known to play an important role in the pathogenesis of IPAH. In particular, pulmonary endothelial cells (P-EC) represent a critical cell type. Endothelial dysfunction is characterized by an altered vasoconstriction/vasodilatation balance and disorganized pro-proliferative and apoptotic-resistant phenotype that can lead to the formation of plexiform lesions mostly described in IPAH patients 16.

We hypothesized that leptin, synthesized by dysfunctional P-EC, might play a role in the immunopathology of IPAH by regulating circulating Treg function. We addressed this issue using freshly recovered circulating Treg from IPAH patients. Our findings indicate that Treg function is reduced in IPAH patients compared to controls in a leptindependent manner. Furthermore, using cultured cells recovered after lung transplantation in IPAH patients, we demonstrated that there is a major increase in leptin synthesis from IPAH P-EC compared to controls. Taken together, our data suggest that leptin could derive from endothelial cells and may play a role in the immunopathogenesis and/or progression of IPAH.

Methods

Subjects

Blood samples were collected in patients with IPAH and in sclerodermaassociated PAH (SSc-PAH) during usual follow-up and in control subjects (

Leptin, leptin receptor and pro-inflammatory measurements

Serum samples were collected and stored at -80°C. For the measurement of leptin, C-reactive protein (CRP), tumor necrosis factor (TNF)-, monocyte chemoattractant protein (MCP)-1, interleukin-6 and 1, human AlphaLISA or ELISAs kits were used accordingly to manufacturer's instructions PerkinElmer and R&D Systems.).

Circulating soluble leptin receptor (sObR) levels were measured using ELISAs (as recommended by the manufacturer; R&D Systems).

Flow cytometry analysis

After blood samples withdrawal, peripheral blood mononuclear cells (PBMCs) It is now well known that CD127 expression inversely correlates with FoxP3 activation 17. To test whether Treg analysis and cell count were similar using these different markers, we randomly selected 5 subjects from each group and compared the two Treg phenotypes , ie CD4 + CD25 + CD127 low FoxP3 + , and CD4 + CD25 + CD127 low (the subgroup characteristics are presented as online supplemental data). The cell count was similar by using these 2 different surface markers (Figure 4C and Table 2). Based on these preliminary tests and to preserve the intracellular space, we avoided PBMCs and defined Treg as CD4 + CD25 + CD127 low (Figure 3A). Treg cell count was expressed as the percentage of total CD4 + cells.

In order to evaluate the ObR expression, PBMCs were also stained under nonpermeabilized conditions with a monoclonal antibody against ObR fluorescently conjugated with PE (R&D Systems). Treg expressing ObR were defined as CD4 + CD25 + CD127 low ObR + and were quantified as the percentage of CD4 + CD25 + CD127 low .

To determine the functional status of the Treg expressing ObR, we stained them under permeabilized conditions with a monoclonal antibody against phosphorylated STAT3 (pSTAT3) conjugated with Alexa 488 (Cell Signaling Technology).

To assess the effect of leptin on Treg functional status, we stimulated freshly isolated PBMCs from healthy controls with recombinant human leptin at a concentration of 250ng/ml for 5 minutes (R&D Systems, 18). Then, we stained and analyzed the Treg and pSTAT3 as described above.

Flow cytometry data were acquired with a flow cytometer (MACSQuant) and analyzed by FlowJo software program (Tree Star, Inc).

Human pulmonary tissues and immunohistostaining

Lung specimens were obtained at the time of lung transplantation from patients with IPAH (n=10), at the Marie Lannelongue Hospital, Le Plessis-Robinson, France.

Control-lung specimens were obtained from patients without any evidence of pulmonary vascular disease who underwent lobectomy or pneumonectomy for localized lung cancer (n=15). Lung specimens were fixed in 4% paraformaldehyde and embedded in Paraffin. 5-μm sections were dewaxed and rehydrated progressively. Citrate buffer pH6 was used for antigen retrieval, and endogenous peroxidase was quenched with hydrogen peroxide. Sections were blocked with 5% of bovine serum albumin and incubated overnight with the following antibodies: polyclonal antibody against leptin (dilution: 1/100; Santa Cruz Biotechnologies, Santa Cruz, CA, USA) and monoclonal antibody against leptin receptor (dilution: 1/50; R&D systems Europe, Lille, France).

Biotin/Streptavidin-peroxidase systems, DAB substrate was used for relevation (Universal LSAB HRP Kit, Dako, Trappes, France). Nuclei were counterstained with hematoxylin and mounted. Controls used for these antibodies included omission of the primary antibody and substitution of the primary antibody by isotype control.

Endothelial cell isolation and leptin measurement

Human microvascular P-EC isolated from lung tissue fragments using immunomagnetic purification were cultured as previously described 19. To quantify leptin in conditioned media, the isolated cells were seeded on 6-well plates at a density of 1×10 5 cells/well. After 24 hours, cells were washed twice with PBS then incubated for 24h in serum-free MCDB131 (without the addition of growth factors). Leptin was measured in conditioned media using ELISAs (as recommended by the manufacturer; R&D Systems).

Statistical analysis

Results are expressed as mean ± sem. A p<0.05 level of statistical significance was used for all analyses. The Shapiro-Wilk test was used to ensure that data had a normal distribution. All between-groups comparisons were assessed using One Way ANOVA;

post-hoc analysis of significant variables was performed using Tukey Test with all pairwise multiple comparisons. Differences between two selected groups (controls and IPAH) were compared using unpaired t-test. Pearson correlations were used to establish associations between the dependent variables (i.e., leptin, ObR expression on Treg) and relevant independent variables. All statistical procedures were carried out using GraphPad Prism version 5.0 (GraphPad Software Inc.)

Results

Leptin and pro-inflammatory cytokines serum concentration

To investigate the role of leptin in PAH pathogenesis, we first quantified serum leptin concentration and found that both IPAH and SSc-PAH patients had a significantly higher leptinemia than controls. No difference was found between IPAH and SSc-PAH (Figure 1A and Table 2).

In order to compare the relative free leptin index and the leptin bioavailability in these 3 groups of subjects, we measured sObR levels. We found a similar level in the 3 groups without any statistically significant difference (Figure 1B and Table 2). These 2). MCP-1, IL-6 and IL-1 were significantly increased in IPAH and SSc-PAH patients compared to controls (Table 1). Furthermore, there was no correlation between the pro-inflammatory cytokines and leptin levels (Figure 2B-C).

These data indicate that in PAH patients, in IPAH and in SSc-PAH, serum leptin levels are increased independently of pro-inflammatory cytokines.

ObR expression on Treg cell membrane

Since our findings indicated a possible role of leptin in PAH pathogenesis, we analyzed the expression of its receptor ObR on Treg. After withdrawing peripheral venous blood samples from controls and PAH patients, we first selected Treg among the fluorescently tagged PBMCs by flow cytometry analysis.

We selected Treg and analyzed the expression of ObR on their membrane (Figure 3A). Flow cytometry analysis revealed that ObR expression was markedly increased in PAH patients, as compared to controls (Figure 3B-C and Table 2), either when expressed as percentage of Treg cells (Figure 3C and Table 2), or as absolute numbers (Figure 3D and Table 2). ObR expression was similar in IPAH and SSc-PAH. Interestingly, the expression of ObR on Treg cell membrane did not correlate with BMI or serum leptin levels (Figure 3E-H).

Taken altogether, these findings demonstrate that ObR expression is markedly increased in IPAH patients compared to controls and to a similar extent as in SSc-PAH.

Interestingly, the level of ObR expression is independent of the patients' BM) or serum leptin levels.

Circulating Treg levels

To further explore the role of leptin on Treg in PAH, we first examined the Treg cell count. Flow cytometry analysis revealed a normal Treg cell count in all groups, controls and PAH patients. We expressed the Treg as percentage of CD4 + cells as well as in absolute numbers and also found a normal Treg cell count (Figure 4A-B and Table 2).

These data indicate that leptin does not seem to influence Treg numbers in PAH patients.

Functional status of Treg

Our next objective was to determine the extent to which leptin could play a role on Treg function. As readout of ObR activity, we measured pSTAT3 amounts, because STAT3 is known to participate in the intracellular signaling pathways of ObR 11. After

Treg permeabilization, we quantified pSTAT3 by flow cytometry (Figure 5A).

Interestingly, the number of Treg-pSTAT3 + was markedly decreased in PAH patients compared to controls, and similarly in IPAH and SSc-PAH (Figure 5B-C and Table 2). In order to further investigate the relationship between leptin and Treg function, we stimulated freshly isolated PBMCs from healthy controls with recombinant leptin, we stained them as described above and measured pSTAT3 amounts by flow cytometry.

Treg-pSTAT3+ were significantly decreased when stimulated by leptin, clearly showing a leptin-dependent effect on Treg functional status (Figure 5D). Taking these results together, we hypothesize that leptin regulates Treg in PAH by inhibiting them through the ObR binding.

Leptin secretion by endothelial cells

Leptin has recently been detected in lung tissue 20. Since our findings suggested a mechanistic role for leptin in Treg functional regulation and P-EC represent a critical cell type in IPAH patients 16, our next aim was to investigate whether P-EC could represent one of the sources of leptin in PAH. To test this hypothesis, after exclusion of inflammatory mechanisms as a source of leptin (Figure 2B-C), we performed immunohistochemistry on lung specimens from controls and IPAH patients and we stained them for leptin and ObR. A more intense immunoreactivity was noted for leptin in the endothelium of distal pulmonary arterial walls in IPAH patients versus controls. In contrast, no significant changes in ObR were found between IPAH and control patients. Therefore, we assessed that P-EC are an important source of leptin in the lung. To further assess whether the synthesis and the release of leptin by P-EC were increased in PAH, we isolated microvascular P-EC from lung specimens from IPAH patients and from controls. We measured leptin protein amount in the cell supernatant and found a significant increase in leptin in IPAH patients compared to controls (Figure 6). These findings indicate that P-EC contribute to the increased secretion of leptin measured in IPAH patients.

Discussion

Leptin, Treg function and ObR expression upregulation

Despite strong evidence for the role of leptin in autoimmunity, the precise mechanism of its activity is still controversial. Much of the difficulty relies on the highly pleiotropic activities of leptin on both the neuroendocrine and immune systems. In the

Leptin and inflammation

Leptin production is not only regulated by food intake, but also by various hormones, as well as by several inflammatory mediators, both in humans and in experimental models 10-11. The pro-inflammatory properties of leptin are similar to those of other acute-phase reactants. Generally, leptin increases in the course of acute infection, sepsis and inflammation 10-11. It is now clear that inflammation plays an important role in PAH pathogenesis. On the other hand, some studies suggested an association between leptin levels and inflammatory markers such as soluble TNF receptors or CRP 23 that was not confirmed by others 24. We approached this controversial question by measuring serum pro-inflammatory cytokine levels in PAH and controls. Conversely to recent data published by Quark and colleagues 25, we found normal levels of CRP without any difference within the groups. We confirmed our previous data showing increase in IL-6 and 1and normal levels of TNF- in PAH patients 2. We showed no correlation between leptin levels and inflammatory markers.

Of note, patients did not receive therapies susceptible to decrease inflammatory markers, such as statins, and there was no active systemic disease in the patients with SSc-PAH we studied, as evidenced by the lack of steroid and/or immunosuppressant therapy at the time of SSc-PAH management, and the normal CRP levels measured in these subjects. This suggests that in the PAH patients we studied, increased levels of leptin were not linked to markers of systemic inflammation.

Pulmonary endothelial cells as a source of leptin?

Leptin expression and secretion is constitutively produced by adipocytes and has been first restricted to this tissue only, but recent reports have demonstrated that it is also produced at low levels by other tissues 26. Values are expressed as mean ± sem. NS: non significant.

  from IPAH, SSc-PAH patients and controls were obtained by standard Ficoll gradient centrifugation. The cells were carefully washed with PBS and resuspended in a staining buffer containing 10% of human serum. The cells were then fluorescently labeled with the following monoclonal antibodies: anti-CD4 conjugated with either Alexa 488 (Becton Dickinson) or Pacific Blue (BioLegend); anti-CD25 conjugated with Alexa 647 (BioLegend); anti-CD127 conjugated with PerCp-Cy5.5 (Becton Dickinson); and anti-FoxP3 conjugated with Pacific Blue (BioLegend), under non-permeabilized or permeabilized conditions (IntraPrep, following manufacturer's instructions, Beckman Coulter), to enable surface or intracellular staining, respectively. Flow cytometry gating conditions and the mean fluorescence intensity (MFI) were set and normalized, respectively, against isotype-and fluorophore-matched non-immune IgGs.

  data indicate that circulating active leptin levels are increased in both IPAH and SSc-PAH. Correlations between serum leptin levels and body mass index (BMI) in controls and PAH patients are shown in Figure 1C-D. Furthermore, no correlation was found between circulating leptin levels and disease severity expressed as haemodymic and/or functional parameters. Since leptin has been shown to act as a key player in inflammatory conditions, we tested whether serum leptin concentration in PAH correlated with serum pro-inflammatory cytokines. CRP and TNF- concentrations were normal in all groups, without any statistically significant difference among the groups (Figure 2A and Table

  One of the central unanswered questions in PAH pathogenesis relates to the role of inflammation and dysimmunity in the development and/or progression of IPAH. It remains unclear how immune mechanisms contribute to the pathogenesis of IPAH. We approached this question by determining the role of leptin and endothelial dysfunction in circulating Treg regulation. Our data suggest, for the first time to our knowledge, that leptin may play a role in the immunopathogenesis of IPAH by inhibiting Treg function, through its receptor upregulation on circulating Treg surface. Furthermore, we are also showing, using P-EC isolated from PAH patients and lung tissues, that P-EC could represent one of the sources of leptin.

  immune system, leptin can directly affect the activity of numerous immune cell types of both the innate and the adaptive systems. A critical protective role for Treg in several autoimmune diseases is now well established 14-15, 17 but it is still controversial whether leptin induces Treg anergy and/or hyporesponsiveness in autoimmune diseases. In order to identify Treg by flow cytometry, we have chosen to use surface markers because recent studies have indicated that, in humans, expression of endogenous FoxP3 is not sufficient to induce Treg activity or to identify Treg cells 21, 22 In this study, we establish a unique link between leptin and Treg cells in PAH, by showing that leptin can modulate the hyporesponsiveness of Treg in vivo. Freshly isolated Treg from PAH patients display higher expression of ObR on their surface compared to healthy controls. At the molecular level, circulating PAH Treg cells express low level of pSTAT3, which represents the major signalling pathway in Treg. In contrast, the number of circulating Treg is in the normal range and it is similar in PAH patients as well as in controls. Interestingly, these results were similar in the two major PAH subgroups tested, IPAH and SSc-PAH, in which there is an established associated immunological disorder. Furthermore, when stimulated by recombinant leptin, Treg cells express lower levels of pSTAT3, clearly indicating a direct effect of leptin on Treg functional status. It appears that, in PAH, leptin controls Treg function rather than Treg numbers that could participate to the immunopathogenesis of PAH.

Figure 1 :

 1 Figure 1: Leptin measurements

Figure 2 :

 2 Figure 2: C-reactive protein measurements

Figure 3 :

 3 Figure 3: Leptin receptor (ObR) expression

Figure 4 :

 4 Figure 4: Regulatory T lymphocytes (Treg)

Figure 5 :

 5 Figure 5: Functional status of regulatory T lymphocytes (Treg) expressing leptin

Figure 6 :

 6 Figure 6: Leptin production by human pulmonary endothelial cells

Figure 1 BMI

 1 Figure 1

Table

  ). All patients were treated with PAH specific treatments. Inclusion criteria were age above 18 years and PAH diagnosis confirmed by right-heart catheterization with a stable clinical and heamodynamic status for the last 3 months. Exclusion criteria were an heritable form of PAH, anaemia, thyroid dysfunction, diabetes, metabolic syndrome, and immunosuppressive or corticosteroid therapies in the last 6 months for SSc-PAH..

Characteristics at diagnosis and follow-up were stored in the Registry of the French Network of Pulmonary Hypertension set up in agreement with French bioethics laws (French Commission Nationale de l'Informatique et des Libertés) and all patients gave their written informed consent.

Table 1 : Subjects anthropometric and hemodynamic characteristics

 1 The question of the existence of another source of leptin involved in the immune regulation in humans remains unanswered. Endothelial dysfunction represents one the major cellular PAH characteristics that could contribute to increased leptin secretion 3.By using P-EC isolated from PAH and lung specimens, we are showing here for the first time, that PAH pulmonary endothelial cells produce more leptin than controls. This can explain, at least in part, the increased serum leptin levels in PAH patients compared to controls, despite the normal number of Treg cells. Further studies are needed to confirm that endothelial cells represent the source of leptin and the role of leptin on Treg in PAH.In conclusion, our findings address a central unanswered question in PAH pathogenesis, namely that relating to the role of dysimmunity in the development and/or progression of IPAH. Our evidence that leptin and endothelial cells play a determining role in the regulation of Treg in IPAH reveals a previously unknown mechanism in the IPAH immunopathogenesis, which can contribute to the onset and/or progression of the disease. Our findings indicate for the first time that leptin must be considered as an actor of immunological disorders in IPAH.

	Tables
		Controls	IPAH	SSc-PAH
	number (n)	20	25	11
	sex female/male (ratio)	12/8 (0.66%)	20/5 (0.25%)	9/2 (0.22%)
	age (years)	38 ± 3*	52 ± 3	68 ± 3 #
	BMI (kg/m 2 )	25 ± 1	26 ± 1	27 ± 1
	NYHA class	NA	II-III	II-IV
	mPAP (mmHg)	NA	45 ± 3	41 ± 4
	PcwP (mmHg)	NA	8 ± 1	9 ± 1
	PVR (Wood unit)	NA	7 ± 1	7 ± 1
	cardiac output (ml/min)	NA	5.8 ± 0.3	5.5 ± 0.6
	cardiac index (l/min/m 2 )	NA	3.0 ± 0.2	3.0 ± 0.3
	6-MWT distance (m)	NA	459 ± 21	314 ± 44 #
	BNP (ng/ml)	NA	103 ± 28	250 ± 92
	CRP (mg/l)	0.05 ± 0.003	0.05 ± 0.002	0.04 ± 0.005
	TNF- (pg/ml)	0.5 ± 0.4	4.2 ± 2.0	2.2 ± 2
	MCP-1 (pg/ml)	273 ± 19*	682 ± 158	460 ± 42
	IL-6 (pg/ml)	1 ± 0.4*	25 ± 9	8 ± 2
	IL-1(pg/ml)	0.01 ± 0.01*	32 ± 8	17 ± 12
	PAH specific treatment:
	ERA		11	3
	PDE-5i:		0	1
	ERA+PDE-5i ERA+PGI2	NA	8 2	4 0
	PDE-5i+PGI2		2	0
	ERA+PDE-5i+PGI2		2	3
	no treatment		0	0

Interestingly, it has been shown that another important source of leptin are the Treg cells themselves, which both secrete leptin and express leptin receptor 10. Thus, leptin can mediate a negative autocrine loop in Treg, which can promote the onset and/or the progression of autoimmune diseases 22. Theoretically, the blockade of leptin by antagonists, antibodies or soluble receptors should inhibit leptin bioavailability and reduces its effects. So far, this has been shown in animal models with experimental conditions 10. In humans, leptin administration does not efficiently improve immune function in the normal or obese individual but only in individuals with congenital leptin deficiency and lipodystrophy 27.

Table 2 : Regulatory T lymphocytes, leptin receptor and serum leptin data

 2

		Controls	IPAH	SSc-PAH
	Treg (% CD4 + )	4 ± 0.5	4 ± 0.4	3 ± 0.4
	Treg (/ml)	67 ± 8	61 ± 8	51 ± 9
	Treg FoxP3+ (% CD4 + )	5 ± 0.8	3 ± 0.4	4 ± 0.2
	ObR (% Treg)	16 ± 5*	39 ± 4	36 ± 9
	ObR (/ml)	6 ± 2*	19 ± 2	22 ± 8
	ObR (MFI)	378 ± 46	313 ± 58	466 ± 21
	pSTAT3 (% Treg ObR + )	8 ± 1.6*	4 ± 0.5	4 ± 0.6
	pSTAT3 (MFI)	1090 ± 72	1152 ± 58	1134 ± 46
	leptin (ng/ml)	2 ± 0.3*	6 ± 0.9	6 ± 1.4
	circulating soluble ObR (ng/ml)	27 ± 1	27 ± 2	22 ± 2

FoxP3: forkhead box P3; IPAH: idiopathic pulmonary arterial hypertension; MFI: mean fluorescence intensity; ObR: leptin receptor; pSTAT3: phosphorylated Signal Transducer

Acknowledgements

Dr. Huertas is supported by the Josso Award 2010 from the French Medical Research Foundation.


Jean-François Carriasa
email: j-francois.carrias@univ-bpclermont.fr

Olivier Brouarda

Céline Leroyc

Régis Céréghinod

Laurent Pélozuelod

Alain Dejeanc

Bruno Corbaraa

An ant-plant mutualism induces shifts in the protist community structure of a tank-bromeliad

Keywords: Obgleich Ameisen gemeinschaftsübergreifende Effekte auslOsen konnen, indem sie in terrestrischen Lebensriiumen Ânderungen des physikalischen Habitats bewirken, ist ihr Einfiuss auf aquatische Gemeinschaften von Phytotelmata weitgehend femoratus bzw. Pachycondyla goeldii angelegt werden, vor. Ihre um deren relative Bedeutung für Unterschiede zwischen den mit Bromelien, die von verschiedenen Ameisenarten betreut wurden, legt nahe, dass die mutualistischen Ameisen indirekt Ânderungen im mikrobiellen Nahrungsnetz vermitteln Aechmea mertensii, Ant-garden, Protists, Biodiversity, Camponotus femoratus, Pachycondyla goeldii, Phytotelmata, French Guiana

Although ants may induce community-wide effects via changes in physical habitats in terrestrial environments, their influence on aquatic communities living in plant-held waters remains largely underexplored. The neotropical tank-bromeliad Aechmea mertensii (Bromeliaceae) occurs along forest edges in ant-gardens initiated by Camponotus femoratus or by Pachycondyla goeldii. Its leaves form wells that hold rainwater and provide suitable habitats for many aquatic organisms. We postulated that these ant-plant mutualisms indirectly affect the microbial community structure via changes in the environmental conditions experienced by the plants. To test this hypothesis, we analyzed the protist communities from 63 tank-bromeliads associated with either C. femoratus or P. goeldii (hereafter Cf-Aechmea and Pg-Aechmea) along a forest edge in French Guiana. For each plant, a large number of environmental variables (including habitat structure, food resources, incident radiation and the presence of aquatic invertebrates) were quantified to determine their relative importance in driving any observed differences across ant-associated plants. Pg-Aechmea are located in sun-exposed areas and hold low volumes of water and low amounts of detritus, whereas Cf-Aechmea are located in partially shaded areas and accumulate higher amounts of water and detritus. Protists (i.e., protozoa and algae) inhabiting Cf-Aechmea exhibit greater richness and abondances than those in Pg-Aechmea. Variations in detritus content, number of leaves, incident radiation, and the epiphyte richness of the ant-garden were the main factors explaining the variation in protist richness. A shift in the functional group composition of protists between bromeliads tended by different ant species suggested that mutualistic ants indirect! y mediate changes in the microbial food web.

Introduction

Ants are involved in an interlocking set of mutualisms [START_REF] Mckey | The trophic structure of tropical ant-plant-herbivore interactions: Community consequences and coevolutionary dynarnics[END_REF]) and ant-gardens are recognized as belonging to the most sophisticated of ali symbioses between ants and flowering plants in lowland Amazonia [START_REF] Orivel | The diversity and ecology of ant gardens (Hymenoptera: Formicidae; Spermatophyta: Angiospermae)[END_REF]. Ant-gardens are initiated by a few species of ants whose founding queens and/or workers build arboreal carton nests. Ants collect and incorporate into the walls of their nests the seeds of selected epiphyte species which then germinate and grow (Orivel & Dejean 1999), so that the plant roots stabilize and anchor the entire structure in the supporting tree. The seeds of these epiphytes attract ants through nutritional rewards called elaiosomes, but even in their absence these seeds remain attractive to ant-gardenbuilding ants thanks to compounds on the seed cuticle (Orivel & Dejean 1999;Youngsteadt, Nojima, Haberlein, Schulz, & Scha12008). In tum, the plants bene fit from seed dispersal and protection from defoliating insects [START_REF] Orivel | The diversity and ecology of ant gardens (Hymenoptera: Formicidae; Spermatophyta: Angiospermae)[END_REF] ). Among the Neotropical epiphytes, the tank-bromeliad Aechmea mertensii Schult.f. (Bromeliaceae) only occurs (over its full range) in association with ant-gardens [START_REF] Benzing | Bromeliaceae: Profile of an adaptive radiation[END_REF]. In French Guiana, A. mertensii is found in pioneer growths in ant-gardens initiated either by Camponotus femoratus Fabr. or by Pachycondyla goeldii Forel (Corbara & Dejean 1996). The leaves of A. mertensii are tightly interlocking, forming severa! wells that collect water and organic detritus. These wells are characteristic of tank-forming bromeliads acting as phytotelmata. The aquatic communities inhabiting tank-bromeliads consist of micro-and macroinvertebrates, occasionally vertebrates (i.e., anurans) [START_REF] Kitching | Food webs and container habitats: The natural history and ecolo gy ofphytotelmata[END_REF][START_REF] Frank | lnsects and allies associated with bromeliads: A review[END_REF], and diverse microorganisms [START_REF] Brouard | Understory environments influence functional diversity in tank-bromeliad ecosystems[END_REF]). Since A. mertensii is both a phytotelm-and an ant-garden bromeliad, it was deemed a relevant model for studying the role of interspecific mutualistic interactions in shaping aquatic communities in nature [START_REF] Céréghino | Ants mediate the structure of phytotelm communities in an ant-garden bromeliad[END_REF][START_REF] Céréghino | Ant-plant mutualisms promote functional diversity in phytotelm communities[END_REF].

We investigated the effect of these ant-plant mutualisms on aquatic microorganisms by analyzing protist (protozoa and algae) communities from 63 bromeliads associated with either C. femoratus or P. goeldii. In aquatic environments, protozoa control bacterial prey and release large amounts of dissolved inorganic nutrients [START_REF] Sherr | Significance of predation byprotists in aquatic microbial food webs[END_REF], while algae are frequently dominant primary producers and have recently been identified as a ubiquitous and relevant component of the aquatic communities of tank-bromeliads [START_REF] Brouard | Are algae relevant to the detritus-based food web in tank-bromeliads?[END_REF][START_REF] Brouard | Understory environments influence functional diversity in tank-bromeliad ecosystems[END_REF]. A previous study bas shown that, as dispersal agents for A. mertensii, P. goeldii and C. femoratus indirect! y influence the physical characteristics of the plant by determining the location of the seedling in areas ranging from sun-exposed to partially shaded [START_REF] Leroy | Ants mediate foliar structure and nitrogen acquisition in a tankbromeliad[END_REF]. We thus hypothesized that the density of algae would increase with the plant's exposure (i.e., from shaded to exposed areas), and thus would be more relevant to the microbial food web in Pg-Aechmea than in Cf-Aechmea. The latter constitute more complex and larger habitats and receive larger amounts of leaf litter from overhanging trees. Therefore, we expected larger densities and a greater richness of protists in Cf-Aechmea compared to Pg-Aechmea. Finally, we projected that ants would indirectly influence the microbial food web due to changes in the numerical importance of the functional groups of protists.

Materials and methods

Study site and ant-gardens

This study was conducted in French Guiana in March 2009 in a secondary forest located along an 11-km-long dirt road near the Petit-Saut Dam (latitude: 5°03'43"N; lon- gitude: 53°02'46"W; elevation a.s.l.: 80 rn). The climate is tropical moist, with 3000 mm of yearly precipitation distributed over 280 d. There is a major drop in rainfall between September and November (dry season) and another shorter, more irregular dry period in March. The maximum monthly temperature averages 33.5 °C, and the monthly minimum is 20.3°C.

All samples were taken from A. mertensii bromeliads rooted on well developed ant-gardens inhabited either by C. femoratus and Crematogaster levior (n = 31) or by P.

goeldii (n = 32). A. mertensii shifts from a funnel-like, crateriform shape (Cf-Aechmea) to a bulbous, amphora shape (Pg-Aechmea) along a gradient from partially shaded to sunexposed locations within the same area of forest edge. C. femoratus is a polygynous arboreal formicine species living in a parabiotic association with the myrmicine species Cr. levior; that is to say, they share the same nests and trails but shelter in different cavities of the nests [START_REF] Orivel | The diversity and ecology of ant gardens (Hymenoptera: Formicidae; Spermatophyta: Angiospermae)[END_REF]. Their large, polydomous colonies and aggressiveness identify them as territorially dominant arboreal species in Neotropical rainforest canopies. Conversely, P. goeldii is a monogynous (single queen) arboreal ponerine species with comparatively smaller populations, although the colonies may be polydomous [START_REF] Corbara | Arboreal nest building and antgarden initiation by a ponerine ant[END_REF][START_REF] Dejean | The importance of ant gardens in the pioneer vegetal formations of French Guiana (Hymenoptera: Formicidae)[END_REF].

Habitat variables and sampling

To avoid the influence of ontogenetic gradients, all sampled bromeliads were at the flowering stage of the plant life cycle. For each tank-bromeliad, we recorded fifteen variables. Plant height (cm) was measured as the distance from the bottom of the body to the top of the crown. Plant width (cm) was the maximum distance between the tips of the leaves (mean of two measurements at 90°). After recording the total number of leaves and number of distinct wells constituting the tank (see Appendix A: Figure 1), the leaf display was estimated as the proportion of horizontal and verticalleaves (% ). The length and width of the longest leaf were also recorded, as well as the height and diameter (two random measurements at 90°) of the reservoir (cm). This fust set of ten variables described the vegetative traits of the bromeliads. We then recorded the elevation above the ground (rn) and the number of epiphyte species (including A. mertensii) rooted on the ant-garden. The percentages of total incident radiation above the bromeliads were calculated using hemispherical photographs, GPS locations, and an image processing software (Gap Light Analyzer 2.0; Frazer, Canham, & Lertzman 1999), as described in [START_REF] Leroy | Ants mediate foliar structure and nitrogen acquisition in a tankbromeliad[END_REF]. This set of three variables described the local conditions of the bromeliads in the supporting ant-gardens. The water volume extracted (mL) and the amount of fine particulate organic matter (FPOM; 1000--0.45 J.LID in size) were recorded. These two variables were chosen to describe the amount of water available to freshwater organisms and the amount of food resources at the base of the food webs.

To sample the water retained in A. mertensii, we carefully emptied the wells (4--13 wells per plant) using 5-mL and 10-mL micropipettes with the end trimmed to widen the orifice. This technique allowed us to collect most of the water (Jocqué, Kernahan, Nobes, Willians, & Field 2010). We then recorded the volume extracted from each bromeliad as well as the amount of FPOM, which was expressed as preserved volume (mm 3 after decantation in graduated test-tubes; see also [START_REF] Harlan | Do habitat size and shape modify abiotic factors and communities in artificial treeholes?[END_REF]. A subsample of 20% of the collected volume was fixed with 2% (v/v) glutaraldehyde to count the protists and other microorganisms. The remaining water (and its contents) was preserved in the field in 4% formalin (final concentration) to count the aquatic invertebrates (see below).

Protist diversity

Subsamples (1-5 mL) forcounting algae andheterotrophic nanoflagellates (hereafter, HNF) were stained with primulin, collected onto 0.8 J.LID pore-size black Nuclepore filters, mounted with oil between a glass slide and a cover slip, and then stored at -20 oc. The slides were exarnined under UV light (340-380nm) at llOOx magnification using a Leica DC 300F epifluorescence microscope. Pigmented protists were distinguished by detecting the red autofluorescence of chlorophyll a under blue light ( 450-490 nm) excitation.

Subsamples of 2 mL were placed into plankton chambers to count ciliates and naked or testate amoebae. Counting was conducted using an inverted Leica microscope at 400x magnification by scanning the entire chamber area. Protists were identified to morphospecies based on morphological characteristics (i.e., size, cell shape, pigmentation, flagella, cilla, shell), and the abundance of each species in different functional groups (i.e., algae: phototrophs; HNF: raptorial feeders of small bacteria; ciliates: filter feeders of suspended bacteria; naked and testate amoebae: consumers ofbacteria, algae, and non-living organic material using pseudopodia) was estimated for each sampled plant. Protist diversity was expressed as species richness and through the Shannon index. Species richness (S) was determined as the number of protist morphospecies in each bromeliad. The Shannon index (H') was calculated as:

H' =-LPilnpi
where Pi is the relative biomass of species i and is calculated as the ratio between the individual biovolume of a species and the biovolume of the complete protist community in each sample.

Table 1. Differences in habitat characteristics and protist variables between Aechmea mertensii associated with Camponotus femoratus (Cf-Aechmea) and those associated with Pachycondyla goeldii (Pg-Aechmea). IR: incident radiation, WV: water volume, FPOM: fine particulate organic matter, NW: number of wells, and HNF: heterotrophic nanofiagellates.

Cf-Aechmea

Mean±SE Range

Habitat characteristics IR(%) 25.1 ± 9.9 12.5-48.2 WV(mL) 129.0 ± 89.9 26.0-428.0 FPOM(mL) 10.9 ± 12.6 0.6-51.6 NW 9.3 ± 2.2 6.0-13.0

Protist abondances HNF(10 4 mL-1 ) 4.5 ± 6.1 0.9-35.8 Ciliates (10 2 mL -1 ) 4.3 ± 5.6 0.3-29.3 Algae (1Q4 mL - 1 ) 10.1 ± 29.4 0.1-134.6 Testate amoebae (1 0 2 mL -1 )

1.9 ± 5.3 0.01-29.0 Naked amoebae (10 2 mL -1 ) 0.5 ± 0.8 0.0-2.5

Protist richness (S) 13.2 ± 2.1 9.0-18.0 Protist diversity (Jf)

1.8 ± 0.6 0.3-2.8

Counting other aquatic organisms Bacteria and metazoa (i.e., rotifers and macroinvertebrates) were also counted and constituted a set of five "ecological" explanatory variables in subsequent analyses. For bacterial abundance, a 1 mL subsample was stained with SYBR Green (10-4 M) and analyzed through flow cytometry (Becton Dickinson FACS Calibur). Rotifers were counted after sedimentation in plankton chambers using the same method as described for ciliates and testate amoebae. Macroinvertebrates were sorted and preserved in 70% ethanol. They were identified to genus, species or morphospecies and theo partitioned into "functional feeding groups" (FFGs) based on morphological and behavioral adaptations to food acquisition [START_REF] Merritt | An introduction to the aquatic insects of North America[END_REF]. These functional groups were: collector-filterers, collector-gatherers, and predators. Because shredders were only found in a few samples and in low abundances, this FFG was omitted. FFG density was expressed as ind/mL.

Data analyses

Differences in protist diversity, the abundances of the different functional groups of protists, the amount of FPOM, water volume, the number of wells per plant, and the percentage of total incident radiation between Cf-Aechmea and Pg-Aechmea (n = 31 and n = 32, respectively) were tested using a one-way ANOVA. These analyses were conducted on log transformed data to meet the assomptions of normality and homogeneity.

Relationships between all environmental variables (including the five "ecological" explanatory variables), the bromeliads, and protist abundance data were examined using

Results

Pg-Aechmea were located in the sunniest part of the forest edge and thus, as was found in a previous study [START_REF] Leroy | Ants mediate foliar structure and nitrogen acquisition in a tankbromeliad[END_REF]), received significantly more light than Cf-Aechmea (Table 1 ). They contained lower water volumes, lower amounts ofFPOM, and lower number of wells per plant than Cf-Aechmea. The abundances of HNF, algae and testate amoebae were significantly higher in Cf-Aechmea than in Pg-Aechmea. The mean abundances of ciliates and naked amoebae were not significantly different between the bromeliads (Table 1 ). Of the 25 protist morphospecies recorded, 22 were shared by both ant-gardens, while the three remaining were exclusive to Cf-Aechmea (See Appendix A: Table 1). The protist richness in Cf-Aechmea was higher than in Pg-Aechmea but protist diversity, expressed by the Shannon (H') indice, was lower in Cf-Aechmea than in Pg-Aechmea (Table 1).

Axes 1 and 2 of the RDA accounted for 18.8% of the total species variance and 83.2% of the species-environment relationship. Bigenvalues for axes 1 and 2 were 0.15 and 0.04, respectively. Species-environment correlations were 0.840 for axis 1 and 0.759 for axis 2. Forward selection identified four variables as explaining a significant amount of the species variance (arrows in Fig. l A): FPOM (P=0.002), number of leaves (P=0.002), incident radiation (P=O.Ol), and number of epiphyte species (P= 0.01). None of the five eoological variables was significant in determining species distribution. RDA-scores for ail of the variables tested are provided in the supplem.entary material (Appendix A: Table 2). FPOM accounted for the greatest proportion of the total canonicaleigenvalues (12.5%;F= 8.73; P=0.002). Thescatterplot of the RDA allowed us to distinguish two main subsets along axis 1 when bromeliads were more specifically grouped according to the ant partner (Fig. l A); i.e., the Pg-Aechmea area (left part of the scatterplot) and the Cf-Aechmea area (right area). Pg-Aechmea were less scattered than Cf-Aechmea in the scatterplot indicating a more homogenous protist community within individuals ofPg-Aechmea. Axis 1 displayed a gradient of phytotelm habitat conditions. There was a gradient of habitat complexity (total number ofleaves) and amount of FPOM made available to the microorganisms from low (left side of the scatterplot) to high (right), and a gradient of incident radiation ranging from low (right) to high (left). Within these gradients, Pg-Aechmea individuals were found in sun-exposed areas and contained greater abundances of small-sized ciliates (Fig. lB,see Appendix A: Table 1 ). These bromeliads exhibited low abondance for the majority of the algal taxa. In contrast to Pg-Aechmea, Cf-Aechmea showed lower abundances of large-sized ciliates but higher abondances of the majority of other protist taxa including HNF, algal and testate amoebae morphospecies (Fig. l B, see Appendix A: Table 1). There was a higbly significant difference in sample coordinates accordmg to bromeliad species (left cluster=Pg-Aechmea, right cluster=Cf-Aechmea) along axis 1 (P<O.OOOl). Finally, it appeared that ant-gardens associated with C. femoratus hold a higher diversity of epiphyte species than the ant-gardens associated with Pg-Aechmea.

Discussion

Although plant-insect interactions are pervasive in the tropics and elsewhere (Mc Key et al. 2005;[START_REF] Rico-Gray | The ecolo gy and evolution of ant-plant interactions[END_REF], the influence of cross-scale interactions on microorganisms (i.e., how plant-insect interactions affect the microbial components of natural systems) bas not yet been investigated. This is due to the fact that ecologists have primarily focused on the outcomes of predation by grazerslfilter-feeders (including their cascading impacts on 2). microbial food webs) [START_REF] Sommer | Trophic cascades in marine and freshwater plankton[END_REF]) and/or resource availability (Hoekman, Wmston, & Mitchell 2009), while the indirect impacts of non-trophic interactions between plants and/or animais (e.g., competition, mutualisms, chemical eues) on the entire community have been less often addressed [START_REF] Adl | Protists in soil ecology and forest nutrient cycling[END_REF]. It is, however, weil known that organisms may exert community-wide effects via physical changes to habitats with consequences for the structural organization of the occupants and their fonctions [START_REF] Jones | Organisms as ecosystem engineers[END_REF]. As dispersal agents for the tankbromeliad A. mertensii in the Neotropics [START_REF] Corbara | Les '1ardins de fourmis", une association plantes-fourmis originale[END_REF], the ant-garden ants C. femoratus and P. goeldii determine the location of its seedlings, and, because these ant species have different nesting preferences as regards light intensity, they indirectly influence the phenotype of the plant [START_REF] Leroy | Ants mediate foliar structure and nitrogen acquisition in a tankbromeliad[END_REF]. A. mertensii reveals a significant plasticity in its vegetative traits, which influences both habitat characteristics (e.g., tank shape and size, nomber of leaves; [START_REF] Leroy | Ants mediate foliar structure and nitrogen acquisition in a tankbromeliad[END_REF], present study) and resource availability (e.g., water and litter) of the aquatic microecosystem. These changes induce an important shift in the structure of the protist community. Our results therefore demonstrate the decisive, indirect influence of the ant partner on the densities, diversity and functional structure of a complete microbial community via changes in habitat and abiotic conditions. Among the 20 variables tested, we found four abiotic variables explaining the shift in protist richness between antgarden bromeliads. The quantity of FPOM explained most of the variance in community structure. The partially shaded sites selected by C. femoratus permit the development oflarge tank-bromeliads that receive large amounts of organic matter from overhanging trees. Most of these bromeliads are a very favorable site for HNFs and testate amoebae which are known to consume both suspended and attached bacteria and organic matter. In addition, testate amoebae are frequently associated with organic surfaces and larger reservoirs which provide a more diverse range of microhabitats. In accordance with previous studies [START_REF] Richardson | The bromeliad rnicrocosm and the assessment of faunal diversity in a Neotropical forest[END_REF]Armbruster, Hutchinson, & Cotgreave 2002), the large amounts of organic matter found in these individuals sustain a higher diversity of macroinvertebrate FFGs (see Appendix A: Table 3). By fragmenting detrital organic matter particles, detritivorous invertebrates are known to enhance bacterial activity [START_REF] Cummins | Feeding ecology of stream invertebrates[END_REF] and thus the predators of these bacteria, such as HNF and testate amoebae. Although we observed congruent patterns in the abondances of protists and macroinvertebrate FFGs, none of the biotic variables was significant in our analysis, suggesting that the putative positive impact of invertebrates on protists was likely minor.

H a I セ @ ..- CX) • CI-Aechmea o Pg-Ae<:hmea IR d セ @ 1 セ M M M M セ M M M M M M j M M M M M M M セ ] M M M セ @ (B) セ @ cq 9 H9o セ M Q セ N P セ M M M M M M M M M M セ M M M M M M M M M M Q M N P セ @
Habitat complexity (number of leaves) also had a strong impact on the protist community by increasing the ahundance and richness of the community. This result is consistent with numerous previous studies dealing with fauna from both non-ant-garden [START_REF] Richardson | The bromeliad rnicrocosm and the assessment of faunal diversity in a Neotropical forest[END_REF]Armbruster et al. 2002;[START_REF] Srivastava | Why are predators more sensitive to habitat size than their prey? lnsights from bromeliad insect food webs[END_REF][START_REF] Jabiol | Structure of aquatic insect communities in tank-bromeliads in a East-Amazonian rainforest in French Guiana[END_REF]) and ant-garden bromeliads [START_REF] Céréghino | Ants mediate the structure of phytotelm communities in an ant-garden bromeliad[END_REF]). In addition to larger quantities of detritus, larger numbers of sub-reservoirs per plant result in greater habitat complexity providing more micro-habitats and resources, reducing competition, and permit more species to coexist. As a result, protist richness was significantly higher in Cf-Aechmea individuals compared to Pg-Aechmea individuals. Our results also indicate that ant-gardens that had the highest epiphyte richness (4-5 species) were all associated with C. femoratus, and this richness positively affected the protist community. lt should be noted that the abondances of large-sized protozoa (i.e., large-sized ciliate taxa and all of the testate amoebae taxa) increased with the number of epiphytes. These characteristics show that mature ant-gardens associated with C. femoratus are miniature spots of biodiversity containing a high level of richness of epiphytes and aquatic invertebrates [START_REF] Céréghino | Ant-plant mutualisms promote functional diversity in phytotelm communities[END_REF], protists (this study), and almost certainly terrestrial arthropods (persona! observations).

The percentage of total incident radiation received by the bromeliads strongly affects the morphology of the plant [START_REF] Montero | The phytotelmata and foliage macrofauna assemblages of a bromeliad species in different habitats and seasons[END_REF]. A. mertensii individuals associated with P. goeldii grew in sun-exposed areas and, as such, their rosettes had a small width and were amphora shaped [START_REF] Leroy | Ants mediate foliar structure and nitrogen acquisition in a tankbromeliad[END_REF]. The low quantities of FPOM and the lower complexity of these phytotelmata reduce the abondance and richness of most heterotrophic protists but also the abondance and richness of the algae. Thus, in contrast to our predictions, algae showed high abondances in the largest Cf-Aechmea which receive incident radiation two times lower than Pg-Aechmea. This result conflicts with the patterns of algal abondance in "non-ant-garden" bromeliads [START_REF] Brouard | Are algae relevant to the detritus-based food web in tank-bromeliads?[END_REF] ) and suggests that the nutrients provided by the decomposition of organic matter, rather than light, are a key factor controlling algal growth in A. mertensii. The abondant bacteria and protozoa in the largest Cf-Aechmea might stimulate the rate of decomposition of organic matter, thus releasing more nutrients in forms available to algae [START_REF] Ngai | Predators accelerate nutrient cycling in a bromeliad ecosystem[END_REF]. In contrast, the small amounts of organic matter and the resulting low abondances of microbial decomposers and their predators in Pg-Aechmea revealed a nutrient-limited microcosm likely to restrict algal growth as light is not a limiting factor for photosynthesis in this case. Also, it is likely that the trichome-coated Pg-Aechmea leaves outcompete algae for nutrient uptak:e in water-and nutrientstressed conditions. The predominance of ciliate taxa within the protist community of Pg -Aechmea reinforced the idea of a low level of productivity in these phytotelmata. Indeed, owing to their higher volume-specifie clearance rates in comparison to other protozoa, ciliates are undoubtedly better adapted to consuming suspended prey in a diluted environment such as the phytotelmata of Pg-Aechmea.

Because ant-plant interactions determine the lightexposure conditions, the structure of the container habitat, and the amount of detritus at the base of the aquatic food web in the bromeliad A. mertensii, functional shifts (mostly abondance patterns) occur as the protist community structure changes from one ant partner to the other. This study therefore underscores how one level of interspecifie interaction (an ant-bromeliad mutualism) can affect another level of interaction (the bromeliad food web), thereby influencing the functional groups of protists and microbial food web structure. Because A. mertensii is common and has never been found growing outside ant-gardens in the Neotropics, the role of ants in creating microcosms and maintaining aquatic microbial diversity along forest edges is cri ti cal.

  Protist abundances were log(n + 1) transformed prior to analyses. An initial Detrended Correspondence Analysis (DCA) in CANOCO v4.5 showed that a linear model was most applicable because of low species turnover along axis 1 (gradient= 0.924,Leps & Smilauer 2003); thereafter, a Redundancy Analysis (RDA) was used to examine protist relationships with the 20 environmental variables. Forward selection was employed to test which of the environmental variables explained a significant (P < 0.05) proportion of the species variance. The significance of explanatory variables was tested against 500 Monte-Carlo permutations. Mann-Whitney tests were used to test significant differences in bromeliad distribution in the RDA according to the ant partner (Cf-Aechmea vs Pg-Aechmea) by using the coordinates of samples on the most significant axis.

Fig. 1 .

 1 Fig. 1. Redundancy Analysis (RDA) biplots. (A) Bromeliads and environmental variables. Different symbols were used to indi- cate ant-garden species. Environmental variables are represented as vectors; directions show the gradients, the lengths of arrows represent the sttengths of the variables in the ordination space. Only the variables explaining a significant (P < 0.05) proportion of the species variance are represented. Marloers were used to identify the associated ant-species. Cf-Aechmea = bromeliads associated with Camponotus fernoratus, Pg-Aechmea = bromeliads associated with Pachycondyla goeldii. (B) Distribution of protist taxa in the ordination space. The abbreviations for environmental variables are: NL =total number of leaves; NE = number of epiphyte species rooted on the ant-garden; IR= percentage of total incident radiation above the bromeliads; FPOM = amount of fine particulate organic matter (mL). The abbreviations for protist taxa are: H = heterotrophic nanoflagellates; C = ciliates: A= algae; T = testate amoebae; nA= naked amoebae. Details on the morphological characteristics and putative affiliation of protist taxa are provided in the supplementary material (Appendix A, Table2).

Acknowledgments

We are grateful to the members of the 'Laboratoire Hydréco de Petit-Saut' for their logistical help and to three anonymous reviewers for helpful comments and suggestions. Special thanks to Andrea Yockey-Dejean forproofreading the manuscript. Financial support was provided by the French Centre National de la Recherche Scientifique (Project 2ID of the programme Amazonie Il), and the Fondation pour la Recherche sur la Biodiversité (Project MICBROME, AAP-IN-2009-038).

Appendix A. Supplementary data

Supplementary data associated with this article can be found, in the online version, at http:l/dx.doi.org/10.1016/ j.baae.2012.10.002.