Cécile Militon
email: cecile.militon@univ-amu.fr

Ronan Jezequel

Franck Gilbert

Yannick Corsellis

Léa Sylvi

Cristiana Cravo-Laureau

Robert Duran

Philippe Cuny

Ronan Jézéquel

Philippe Garrigues

Dynamics of bacterial assemblages and removal of polycyclic aromatic hydrocarbons in oil-contaminated coastal marine sediments subjected to contrasted oxygen regimes

Keywords: Oxic/anoxic oscillation, Aerobic and anaerobic hydrocarbon degradation, PAHs, Marine sediment, Bacterial communities, DGGE, Dual 16S rRNA/rRNA gene

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

Introduction

Polycyclic aromatic hydrocarbons (PAHs) are widespread in marine sediments as a result of both natural processes (usually occurring in the range 0.01-1 ng/g dry weight) and of human activities (e.g., industrial production, accidental leakages during polluting transport/storage, and waste incineration). These organic compounds are frequently measured at increasing levels in marine sediments, particularly in coastal areas subject to anthropogenic pressures [START_REF] Nikolaou | Levels and toxicity of polycyclic aromatic hydrocarbons in marine sediments[END_REF]. PAHs are persistent toxic molecules, several of which highly impact human and environmental health [START_REF] Cerniglia | Microbial degradation of polycyclic aromatic hydrocarbons in the aquatic environment[END_REF][START_REF] Hylland | Polycyclic aromatic hydrocarbon (PAH) ecotoxicology in marine ecosystems[END_REF]. Their fate in marine sediment has thus attracted the attention of the scientific community. Among the abiotic and biotic factors controlling hydrocarbon (HC) degradation and preservation (e.g., substrate quality, sediment character, and redox effects on microbial degradation), oxygen availability appears to be a key control [START_REF] Cuny | Biodegradation of hydrocarbons in bioturbated marine sediments[END_REF]. Indeed, this molecule, in addition to being the most favorable terminal electron acceptor during HC oxidation, is one of the key reactants for HC activation and, therefore, for HC biodegradation [START_REF] Rojo | Enzymes for aerobic degradation of alkanes[END_REF]. Removal of PAHs from contaminated aerobic ecosystems has been intensively studied for decades, and major routes for microbial aerobic biodegradation of PAHs are now well described [START_REF] Pérez-Pantoja | Aerobic degradation of aromatic hydrocarbons[END_REF]. Nevertheless, as HCs can be passively or actively buried in sediments (by sedimentation or through the action of macrobenthic organisms, respectively), they are quickly trapped in suboxic or anoxic layers. Indeed, as oxygen typically penetrates only a few millimeters into coastal sediments, these habitats are largely devoid of molecular oxygen. Moreover, due to the slow oxygen turnover in watersaturated systems, superficial sediments react rapidly to organic carbon inputs and, as a result, become mostly anoxic. By contrast, the mechanisms involved in PAH removal under anaerobic conditions have been the subject of much less investigation, despite the importance of this process in marine sediments. During the last two decades, most studies on HC degradation processes have been performed under strictly oxic or anoxic conditions (for reviews see [START_REF] Meckenstock | Anaerobic degradation of nonsubstituted aromatic hydrocarbons[END_REF][START_REF] Fuch | Microbial degradation of aromatic compounds-from one strategy to four[END_REF], and it is striking to note how little attention has been paid to oxygen-oscillating conditions, although they are widespread in these environments. In coastal marine sediments, these oxygen pulses often occur at timescales of minutes to months in response to tidal excursions, storm events, or macrofaunal burrowing and ventilation activities [START_REF] Wakeham | Degradation and preservation of organic matter in marine sediments[END_REF][START_REF] Pischedda | Imaging oxygen distribution in marine sediments. The importance of bioturbation and sediment heterogeneity[END_REF]. The impact of oxygen oscillations on HC degradation has been indirectly observed in experiments studying the influence of bioturbation on the preservation/degradation of HCs in marine sediments [START_REF] Gilbert | In situ bioturbation and fate in an experimental contaminated Mediterranean coastal ecosystem[END_REF][START_REF] Grossi | Burial, exportation and degradation of acyclic petroleum hydrocarbons following a simulated oil spill in bioturbated Mediterranean coastal sediments[END_REF][START_REF] Miralles | Alkane biodegradation and dynamics of phylogenetic subgroups of sulfate-reducing bacteria in an anoxic coastal marine sediment artificially contaminated with oil[END_REF] with no precise measurement of how the oxic/anoxic switches influence these processes. The influence of oxic/anoxic oscillations on organic matter (OM) preservation and mineralization in marine sediments has been almost exclusively studied on terrestrial-derived and marine-borne OM. These studies have highlighted that the decomposition rate of OM that has experienced switches from anoxic to oxic states may be promoted relative to steady anoxic conditions [START_REF] Sun | Effects of oxygen and redox oscillation on degradation of cell-associated lipids in surficial marine sediments[END_REF][START_REF] Bastviken | Degradation of dissolved organic matter in oxic and anoxic lake water[END_REF]) and that even if oscillating conditions do not increase OM mineralization, anoxic to oxic switches enhance OM degradation during the subsequent oxic phases [START_REF] Bastviken | Degradation of dissolved organic matter in oxic and anoxic lake water[END_REF][START_REF] Abril | In vitro simulation of oxic/suboxic diagenesis in an estuarine fluid mud subjected to redox oscillations[END_REF]. A small number of previous studies have focused on the fate of HCs under oxic/anoxic switches as an alternative cost-effective bioremediation process using simplified bacterial consortia [START_REF] Löser | Microbial degradation of hydrocarbons in soil during aerobic/anaerobic changes and under purely aerobic conditions[END_REF][START_REF] Vieira | Biodegradation of diesel oil and gasoline contaminated effluent employing intermittent aeration[END_REF][START_REF] Vitte | Effect of oxic/anoxic switches on bacterial communities and PAH biodegradation in an oil-contaminated sludge[END_REF] or native microbial communities from oil-contaminated sludge [START_REF] Vitte | Dynamics of metabolically active bacterial communities involved in PAH and toxicity elimination from oil-contaminated sludge during anoxic/oxic oscillations[END_REF]. These studies, which have focused on PAH degradation under oscillating oxygen, steady oxic, and/or steady anoxic conditions in bioreactors, have so far reached contrasting conclusions. [START_REF] Löser | Microbial degradation of hydrocarbons in soil during aerobic/anaerobic changes and under purely aerobic conditions[END_REF] and [START_REF] Vitte | Effect of oxic/anoxic switches on bacterial communities and PAH biodegradation in an oil-contaminated sludge[END_REF][START_REF] Vitte | Dynamics of metabolically active bacterial communities involved in PAH and toxicity elimination from oil-contaminated sludge during anoxic/oxic oscillations[END_REF] found no difference in PAH removal between oxic and oscillating conditions when they used, respectively, a pilot-scale plant with diesel-fuel-contaminated sand and a bioreactor filled with sludge from a contaminated environment. In contrast, [START_REF] Vieira | Biodegradation of diesel oil and gasoline contaminated effluent employing intermittent aeration[END_REF] observed that the oscillating oxygen conditions enhanced HC removal in experiments using effluent from a contaminated lagoon. Although these studies carefully addressed the role of oxygen oscillations in the fate of HCs and in their degradation, they used experimental approaches (a slurry bioreactor or a pilotscale plant) dedicated to ex situ bioremediation processes increasing mass transfer rates (i.e., pollutant desorption), increasing microorganism/pollutant/nutrient contact, and, consequently, increasing rates of pollutant biodegradation when compared to in situ bioremediation.

To gain insight into the influence of oxygen regimes on the degradation of PAHs present in crude oil released into coastal marine environments, surficial sediment was experimentally contaminated with crude oil and incubated in field-simulating microcosms using the plug technique. This thin-layer incubation technique ensures that the incubated sediment is fully oxic, anoxic, or influenced by oxic-anoxic switches without sediment stirring [START_REF] Sun | Laboratory studies of oxic and anoxic degradation of chlorophyll-a in Long Island Sound sediments[END_REF][START_REF] Aller | Bioturbation and remineralization of sedimentary organic matter: effects of redox oscillation[END_REF][START_REF] Kristensen | Aerobic and anaerobic decomposition of organic matter in marine sediment: which is fastest?[END_REF]. The fate of PAHs was monitored during 60 days under three schemes of oxygenation (steady oxic, steady anoxic conditions, and oxic/anoxic oscillations). In this paper, we also report the changes taking place in microbenthic communities under selective pressures exerted by both crude oil and by oxygen regimes. The dynamic of the microbial community was analyzed in different oxygen regimes using a dual 16S ribosomal DNA (rRNA)/16S rRNA gene denaturing gradient gel electrophoresis (DGGE) approach, allowing the characterization of total bacteria and of metabolically active bacteria.

Materials and methods

Sediments and experimental setup Microcosms were established with mudflat sediment recovered from the Aber Benoît basin (Treglonou, France). The amount of carbon bound in organic compounds in this sediment was 6.8 %. The sediment (pH 7.7), composed of 3 % clay, 31 % silt, and 66 % sand, was contaminated with 20,000 ppm of oil (BAL 110, Arabian Light crude oil topped at 110 °C). The oiled sediment was spread in a 2-mm layer in 12 crystallizers (Ø=28 cm, h=15 cm) and covered by 9 L of synthetic sterilized seawater containing the following (g L -1 in distilled water): Tris, 2; NaCl, 23; KCl, 0.75; NH 4 Cl, 1; MgSO 4 •7H 2 O, 6.16; MgCl 2 •6H 2 O, 5.08; and CaCl 2 •2H 2 O, 1.5 (pH 7.7). Three conditions were applied over four microcosms, as illustrated in Fig. 1: (i) permanent oxygenation (Ox) and (ii) permanent anoxia (An), maintained, respectively, by continual purging of the water microcosms with sterile air and with an N 2 /CO 2 mixture and (iii) oscillation between oxic and anoxic conditions (Osc), performed by switching purging gas in water reservoirs according to the following regime: 3 days of oxia/3 days of anoxia. The incubations were carried out for 2 months at room temperature (mean 19.4 °C, std. 0.8 °C) in darkness to avoid HC photodegradation. The HC content and the microbial assemblages were monitored for the sampling times T0 (initial sampling), T1(15 days), T2 (30 days), T3 (45 days), and T4 (60 days) in order to determine the degradation kinetics and the dynamics of the microbial communities for each oxygenation regime. Thus, for each sampling time (with the exception of T0), three crystallizers were collected (one per oxygenation regime). Sediments were sampled, homogenized, and conditioned for microbiological and HC analyses.

HC analyses Two-gram sediment samples were spiked with one aliphatic perdeuterated and five aromatic perdeuterated hydrocarbon surrogate standards (LGC standard, Molsheim, France) and then extracted with 30 mL methylene chloride at 100 °C and 2000 psi for 14 min in a Dionex ASE 200 accelerated solvent extractor. Sediment extractions were performed twice for each sample. Organic extracts were dried over Na 2 SO 4 (activated at 400 °C during 4 h) and concentrated to approximately 2 mL using a Syncore (Büchi, Germany). Extracts were fractionated using a solid-phase extraction (SPE) cartridge (silica/cyanopropyl (SiO 2 /C3-CN) (1.0/0.5 g, 6 mL) (Interchim, Montluçon, France)) [START_REF] Alzaga | Fast solidphase extraction-gas chromatography-mass spectrometry procedure for oil fingerprinting. Application to the Prestige oil spill[END_REF]): fractions of saturates and aromatics were eluted simultaneously with 8 mL of methylene chloride/pentane (80:20, v/v) and concentrated to approximately 2 mL. Alkanes and aromatics were quantified using gas chromatography coupled with mass spectrometry (GC/MS). The GC was an HP 6890N (Hewlett-Packard, Palo Alto, CA, USA) equipped with a split/splitless injector (splitless time 1 min, flow 50 mL/min). The injector temperature was maintained at 300 °C. The interface temperature was 300 °C. The GC temperature gradient was from 50 °C (1 min) to 300 °C (20 min) at 5 °C/min. The carrier gas was helium at a constant flow of 1 mL/min. The capillary column used was a HP-5 MS (HP, Palo Alto, USA): 30 m× 0.25-mm ID×0.25-μm film thickness. The GC was coupled to an HP 5973 Mass Selective Detector (MSD) (electronic impact 70 eV, voltage 1200 V). n-alkanes and PAH semiquantifications were done using single ion monitoring (SIM) mode with the most representative fragment (saturates) or the molecular ion (PAH) of each compound at a minimum of 1.4 cycles/s. Nucleic acid recovery Total RNA and DNA were coextracted and purified from 2 g of sediment in triplicate using the RNA PowerSoil Total RNA Isolation kit and the RNA were prepared and incubated under permanent oxygenation (oxic) maintained by continual purging of the water microcosms with sterile air (white arrows), permanent anoxia (anoxic) maintained by continual purging of the water microcosms using an N 2 /CO 2 mixture (black arrows), and by the oscillation between oxic and anoxic conditions (Oscillation) performed by switching purging gas in water reservoirs according the following regime: 3 days of oxia/3 days of anoxia (b). At each sampling time (T0, T1, T2, T3, and T4) and for each treatment, sediments coming from one of the microcosms were sampled PowerSoil DNA Elution Accessory kit (MoBio, Carlsbad, CA, USA) according to the manufacturer's recommendations. Residual DNA in the RNA samples was removed with 4 U RNase-free DNase I (Invitrogen ™ ). The complete removal of DNA was checked by a control PCR using the bacterial primers on recovered RNA (PCR conditions described below). Total RNA was analyzed on the 2100 Bioanalyzer ® (RNA 6000 Nano Kit, Agilent Technologies) as recommended by the manufacturer. DNA purity and concentration were checked using a BioSpec-nano spectrophotometer (Shimadzu). The RNA and DNA solutions were stored at -80 and -20 °C, respectively.

Reverse transcription polymerase chain reaction of 16S rRNA Denatured total RNA (125 ng) was reverse transcribed using the MasterScript kit (5 Prime) according to the manufacturer's recommendations and the 16S reverse primer Uni_1492R (5′-TACGYTACCTTGTTACGACTT-3′) [START_REF] Lane | Rapid determination of 16S ribosomal RNA sequences for phylogenetic analyses[END_REF]. PCR amplifications were performed using the primer set Uni_1492R and 27F (5′-AGAGTTTGATCC TGGCTCAG-3′) [START_REF] Lane | Rapid determination of 16S ribosomal RNA sequences for phylogenetic analyses[END_REF]. The 50-μL reaction mixture contained 1 μM of each primer, 0.2 μM deoxynucleotide triphosphate (dNTP), 20 ng of RT products, 0.75 U of Taq polymerase (5 Prime), and 1× of the recommended buffer (5 Prime). The reactions were performed in an iCycler thermocycler (Bio-Rad) using a program with an initial denaturation step for 3 min at 94 °C, followed by 30 cycles of three steps (94 °C for 1 min, 50 °C for 1 min, 72 °C for 2 min) and a final extension step for 5 min at 72 °C. Nested PCR was then performed as described previously on fulllength 16S rRNA genes (5 μL) using the bacterial primer set 907RA (5′-CCGTCAATTCMTTTRAGTTT-3′) [START_REF] Lane | Rapid determination of 16S ribosomal RNA sequences for phylogenetic analyses[END_REF] and GML5F containing a GC clamp (underlined) (5′-CGCCCGCCGCGCCCCGCGCCCGTC CCGCCGCCCCCGCCCGCCGCCTACGGGAGGCAGG AG-3′) [START_REF] Muyzer | Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16S rRNA[END_REF]. The reactions were performed using a touchdown program with an initial denaturation step for 5 min at 94 °C, followed by 20 cycles of three steps (94 °C for 1 min, 60 °C for 1 min that reduced onehalf degree per cycle, 72 °C for 2 min). Finally, 14 cycles of three steps were performed (94 °C for 1 min, 50 °C for 1 min, 72 °C for 2 min) followed by a final extension step for 5 min at 72 °C. PCR products were visualized and quantified by agarose gel electrophoresis.

PCR amplification of the 16S rRNA genes PCR amplifications were performed using the bacterial primer set and the programs used for the nested PCR amplification (described above). The 50-μL reaction mixture contained 1× tuning buffer, 1 μM of each primer, 0.2 μM dNTP, 1.5 U PCR extender polymerase (5 Prime), 2 μg T4 gene 32 protein (Roche), and 10 ng DNA. PCR products were visualized and quantified by agarose gel electrophoresis.

DNA fingerprinting by DGGE Eight hundred nanograms of PCR products was loaded on a 6 % acrylamide gel with a 30-50 % denaturing gradient for the bacterial fingerprints (where 100 % denaturant contained 7 M urea and 40 % deionized formamide). Electrophoresis was conducted in 7 L of TAE 1× (40 mM Tris acetate, pH 8.0, 1 mM Na 2 EDTA) in a Bio-Rad Dcode system ® (Bio-Rad, USA) at 180 V at 60 °C for 280 min. After electrophoresis, the gel was stained with 20 μg of ethidium bromide diluted in 400 mL of TAE 1× and visualized on a UV transilluminator. Bacterial profiles were numerized using the Gel Doc 2000 ® system (Bio-Rad).

DGGE band isolation and sequencing DNA bands were cut out of the DGGE gel and eluted overnight in 20 μL of molecular-grade water at 4 °C. Two microliters of elution was re-amplified as described previously (nested PCR amplification protocol) using primer sets without the GC clamp under the following conditions: (i) an initial denaturation step for 3 min at 94 °C followed by (ii) 30 cycles of three steps (94 °C for 30 s, 55 °C for 30 s, 72 °C for 40 s) followed by a final extension step for 2 min at 72 °C. PCR products were visualized using agarose gel electrophoresis and purified with the Wizard ® SV Gel and PCR Clean-Up System (Promega) as suggested by the manufacturer. Purified DNA was sequenced by GATC Biotech (Konstanz, Germany).

Sequence treatment and statistical analyses Molecular data treatment was performed using the MEGA5 program [START_REF] Tamura | MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods[END_REF]). The sequences were compared with the GenBank nucleotide database using the BLASTN algorithm [START_REF] Altschul | Basic local alignment search tool[END_REF]). Sequences were checked for chimeric PCR artifacts using DECIPHER [START_REF] Wright | DECIPHER, a searchbased approach to chimera identification for 16S rRNA sequences[END_REF]. Multiple sequence alignment was performed using the CLUSTAL W program [START_REF] Thompson | CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice[END_REF]. Phylogenies were reconstructed using the neighbor-joining method [START_REF] Saitou | The neighbour-joining method: a new method for reconstructing phylogenetic trees[END_REF]) and Kimura's two-parameter model [START_REF] Kimura | A simple method for estimating evolutionary rate of base substitutions through comparative studies of nucleotide sequences[END_REF]. Bootstrap analysis was performed with 1000 resampled datasets to determine the statistical significance of the branching order. DGGE band profiles were analyzed with GelComparII (Applied Maths, Belgium). Fingerprints of the bacterial communities were subjected to principal component analysis (PCA) with the FactoMineR package [START_REF] Lê | FactoMineR: an R package for multivariate analysis[END_REF]) with software R.2.11.1 to summarize and simplify the data by reducing the dimensionality of the community structures. Similarity coefficient calculations based upon band positions and their relative intensity (Pearson coefficient) allowed the creation of dendrograms using the arithmetic mean algorithm (UPGMA).

Nucleotide sequence accession numbers

The sequences determined in this study have been submitted to the GenBank database under Accession Nos. KM405449 to KM405514.

Results and discussion

Effect of oxygenation regimes on removal of PAH Due to the small quantity of sediment used in each microcosm (plug technique), after sampling the sediment for the nucleic acids extractions, all the remaining sediment had to be used for the chemical analysis and results are given from only one replicate. However, [START_REF] Stauffert | Impact of oil on bacterial community structure in bioturbated sediments[END_REF], using sediments from the same tidal flat and the same analytical procedure, obtained highly reproducible results for the HC analyses between replicate samples. We therefore assume that the presented chemical results are representative of the dynamics of HCs under the different oxygenation regimes. The concentrations of n-alkanes (n-C10 to n-C38) and total PAHs in the sediment at the initial sampling (T0) were 2,620 and 466 μgg -1 of dry weight, respectively. Medium chain-length n-alkanes (n-C10 to n-C17) represented 40 % of the alkanes, whereas two-, three-, four-and five-aromatic-ring HCs represented 20.5, 62.0, 16.9, and 0.1 % of total PAHs, respectively (Fig. 2).

As expected, the alkylated derivatives of PAHs were more abundant than the parent compounds in the studied families.

After 2 months, the determination of residual HCs showed removal efficiencies of n-alkanes of 32, 21, and 13 % under permanent oxic, oxic/anoxic oscillating, and permanent anoxic conditions, respectively (Fig. 3). As shown in Fig. 2, PAHs were partially depleted at the end of the experiment in the oxic condition (69 %) although they were weakly or not depleted in the oscillating (18 %) and the anoxic (12 %) conditions. Different trends have been reported in previous studies focusing on the effect of oxygenation regimes in determining the fate of HCs [START_REF] Löser | Microbial degradation of hydrocarbons in soil during aerobic/anaerobic changes and under purely aerobic conditions[END_REF][START_REF] Vieira | Biodegradation of diesel oil and gasoline contaminated effluent employing intermittent aeration[END_REF][START_REF] Vitte | Effect of oxic/anoxic switches on bacterial communities and PAH biodegradation in an oil-contaminated sludge[END_REF][START_REF] Vitte | Dynamics of metabolically active bacterial communities involved in PAH and toxicity elimination from oil-contaminated sludge during anoxic/oxic oscillations[END_REF]. [START_REF] Löser | Microbial degradation of hydrocarbons in soil during aerobic/anaerobic changes and under purely aerobic conditions[END_REF] observed that less than 50 % of diesel fuel was removed in 4 weeks, irrespective of the condition (oxic and oscillating conditions) when using a pilot-scale plant with diesel-fuel-contaminated sand (3 g/kg). The results obtained by [START_REF] Löser | Microbial degradation of hydrocarbons in soil during aerobic/anaerobic changes and under purely aerobic conditions[END_REF] are in accordance with those reported by [START_REF] Vitte | Effect of oxic/anoxic switches on bacterial communities and PAH biodegradation in an oil-contaminated sludge[END_REF][START_REF] Vitte | Dynamics of metabolically active bacterial communities involved in PAH and toxicity elimination from oil-contaminated sludge during anoxic/oxic oscillations[END_REF] who examined sludge from a contaminated environment using a bioreactor. They concluded that removal efficiency was the same under permanent oxygenation and anoxic/oxic alternations, obtaining removal of 98 % of PAHs in 3 weeks and up to 80 % under permanent anoxic conditions. In contrast, [START_REF] Vieira | Biodegradation of diesel oil and gasoline contaminated effluent employing intermittent aeration[END_REF] used effluent from a contaminated lagoon to reveal improved efficiency of diesel fuel and gasoline removal under conditions of intermittent aeration (90 % removal of total HCs) rather than under constant aeration or without aeration (84 and 70 % removal, respectively) after 3 weeks. Among the biodegradation results for identifiable HC classes, these authors also observed n-alkane removals of 99.6, 98.5, and 76.8 % under conditions of intermittent, constant, and no aeration, respectively. In comparison to results obtained in our study, all these works show, on one hand, greater HC removal irrespective of aeration condition and, on the other hand, nearidentical removal efficiencies under permanent oxygenation and oxic/anoxic alternations. The different results obtained by our study and the previous ones are mainly due to the lack of contaminated sediment resuspension in the thin-layer experimental device (Fig. 1). Indeed, sediment resuspension has been shown to increase the remobilization of sediment-sorbed pollutants [START_REF] Latimer | Mobilization of PAHs and PCBs from in-place contaminated marine resuspension events[END_REF]) and the bacterial mineralization of HCs [START_REF] Leblanc | The influence of sediment resuspension on the degradation of phenanthrene in flow-through microcosms[END_REF]) due to the increase of the desorbed fraction of PAH compounds available for bacterial uptake [START_REF] Cerniglia | Microbial degradation of polycyclic aromatic hydrocarbons in the aquatic environment[END_REF]. Under permanent oxygenation, the fate of the different PAH families followed the same downward trend during the experiment, with an acceleration of PAH removal from 45 to 60 days (T3 to T4) (Fig. 2). Benzo(b)thiophene (Fig. 2a) and naphthalene (Fig. 2b) (two-aromatic fused ring PAHs) showed the greatest depletion with 75 and The order of susceptibility of PAH families to degradation was in accordance with those generally reported [START_REF] Leahy | Microbial degradation of hydrocarbons in the environment[END_REF][START_REF] Head | Marine microorganisms make a meal of oil[END_REF]). Low-molecularweight (LMW) PAHs (<3 rings) are degraded and volatilized more rapidly than high-molecular-weight (HMW) PAHs (>3 rings) because as molecular weight increases, vapor pressure and solubility decrease while hydrophobicity increases. HMW PAHs have more recalcitrant structures and tend to be strongly adsorbed to sediment particles, thus increasing their persistence in the environment. Under permanent anoxia, PAHs were weakly depleted after 60 days (T4) (Fig. 2). PAH biodegradation is normally initiated by the introduction of oxygen atoms into the aromatic nucleus by oxygenases, but novel strategies that cope with the absence of dioxygen under anoxic conditions have recently been discovered [START_REF] Fuch | Microbial degradation of aromatic compounds-from one strategy to four[END_REF]).

According to field (e.g., [START_REF] Venosa | Bioremediation of an experimental oil spill on the shoreline of Delaware Bay[END_REF] and fieldsimulating studies (e.g., [START_REF] Coates | Anaerobic hydrocarbon degradation in petroleum contaminated harbor sediments under sulfate-reducing and artificially imposed ironreducing conditions[END_REF][START_REF] Bregnard | Degradation of weathered diesel fuel by microorganisms from a contaminated aquifer in aerobic and anaerobic microcosms[END_REF], anaerobic biodegradation exists for LMW PAHs but is rarely observed for HMW PAHs in natural anoxic environments [START_REF] Coates | Anaerobic degradation of polycyclic aromatic hydrocarbons and alkanes in petroleum-contaminated marine harbor sediments[END_REF]. Moreover, [START_REF] Hayes | Role of prior exposure on anaerobic degradation of naphthalene and phenanthrene in marine harbor sediments[END_REF] suggested that microorganisms inhabiting sediments not contaminated with petroleum did not significantly oxidize PAHs under anaerobic conditions. Although the Aber Benoît basin of the northwestern coast of Brittany was polluted by an oil tanker spill in 1978 (Amoco-Cadiz), chemical analysis performed in the upper layer (2 cm) of the studied sediment detected only 4 mg of biogenic HCs per gram of dry sediment [START_REF] Stauffert | Impact of oil on bacterial community structure in bioturbated sediments[END_REF]. Even though the presence of HC induces selective pressures on microbial communities and may stimulate the community's tolerance to toxicants by the selection of favorable genotypes [START_REF] Blanck | Pollution-induced community tolerance (PICT) in marine periphyton in a gradient of tri-n-butyltin (TBT) contamination[END_REF], the absence of petrogenic HC in the sediment appears to explain the very low self-purification capacity of the LMW PAHs observed under anoxic conditions.

Dynamics of bacterial assemblages under different oxygen regimes following a simulated oil spill

The microbial assemblages of the sediments were studied at the beginning of the experiment (T0) and 15, 30, 45, and 60 days (T1, T2, T3, and T4, respectively) after oil addition under the three different oxygen regimes. Changes in bacterial community structure were investigated using PCR-DGGE based on the 16S rRNA gene. DGGE fingerprinting analysis is a useful method for following the dynamics of microbial communities in different ecosystems (e.g., [START_REF] Gomariz | From community approaches to single-cell genomics: the discovery of ubiquitous hyperhalophilic Bacteroidetes generalists[END_REF][START_REF] Zhen | Effects of manure compost application on soil microbial community diversity and soil microenvironments in a temperate cropland in china[END_REF]) because it allows the direct determination of occurrence and relative frequency of the microbial ribotypes present at each sampling date [START_REF] Fromin | Statistical analysis of denaturing gel electrophoresis (DGE) fingerprinting patterns[END_REF]. Furthermore, associated ecological factors explaining the fingerprints can be determined based on the interpretation of these biological data (e.g., [START_REF] Bagi | Naphthalene biodegradation in temperate and arctic marine microcosms[END_REF].

The PCA ordination based on the DGGE profiles of the total bacteria is shown in Fig. 4a. The first two principal components (PC1 and PC2) of the PCA explained 69 % of the total variance. Separation of the bacterial communities along PC1 was significantly affected by the input of oxygen during the incubations. The PCA further revealed strong changes in the bacterial communities during the first 15 days of incubation, according to the oxygen regime to which they are subject, thus revealing an adaptation of the community to oxia, anoxia, and oscillating oxygen conditions. All samples subjected to anoxic conditions at 15,30,45,and 60 days (T1An,T2An,T3An,T4An,respectively) were clustered together (average similarity 61 %). DGGE patterns from anoxic samples were markedly different to those representing the first stages of bacterial assemblage dynamics under permanent oxic conditions. Permanently oxygenated samples were distributed in four different clusters, indicating significant changes in the composition of the bacterial communities during the first 45 days with a resilience of a portion of the bacterial populations at 60 days (Fig. 4a). These observations are in accordance with previous studies that showed rapid and significant changes of bacterial communities after HC addition in oxygenated environments [START_REF] Röling | Robust hydrocarbon degradation and dynamics of bacterial communities during nutrient-enhanced oil spill bioremediation[END_REF][START_REF] Alonso-Gutiérrez | Bacterial communities from shoreline environments (Costa da Morte, northwestern Spain) affected by the prestige oil spill[END_REF][START_REF] Vitte | Effect of oxic/anoxic switches on bacterial communities and PAH biodegradation in an oil-contaminated sludge[END_REF][START_REF] Vitte | Dynamics of metabolically active bacterial communities involved in PAH and toxicity elimination from oil-contaminated sludge during anoxic/oxic oscillations[END_REF]. Resilience of bacterial populations after an oil spill event concomitant with a decrease of HC content in the contaminated environment has also been reported [START_REF] Delille | Effectiveness of bioremediation of crude oil contaminated subantarctic intertidal sediment: the microbial response[END_REF][START_REF] Bordenave | Effects of heavy fuel oil on the bacterial community structure of a pristine microbial mat[END_REF]. Under oscillating oxygen conditions, an important shift of the bacterial community was observed only after 30 days. It is interesting to note that after 45 and 60 days under these conditions, the bacterial communities not only did show the highest similarity with the bacterial community observed after 15 days under permanent oxic conditions but also exhibited similar PAH removal. At the beginning of the experiment (T0), DGGE patterns revealed the species evenness of the bacterial population as evidenced by the lack of dominant phylotypes (Fig. 5). Sequence analyses (Fig. 6) showed the presence of an unidentified phylotype (B9) and of two phylotypes affiliated with Bacteroidetes: B5 with Lutibacter and B4 distantly related to Owenweeksia hongkongensis (KF528159: Flavobacteriia; Flavobacteriales; Cryomorphaceae; 92 % identity). Monitoring of the bacterial communities under permanent oxic conditions showed a shift due to the setting up of the microcosms (including HC contamination) with four main phylotypes, which became dominant after 15 days (T1Ox). These early responders belong to the genera Labrenzia (B12), Lewinella (B1), and Marinobacterium (B3) and to the phylum Deltaproteobacteria (B11). With the exception of B3 and B11, these phylotypes were transiently dominant in the bacterial assemblage with a return to the basal level after 30 days. Blooms of several phylotypes belonging to Proteobacteria (e.g., Marinobacterium) and Bacteroidetes may have temporarily but strongly contributed to the microbial community in contaminated sites, as has been previously observed [START_REF] Röling | Bacterial community dynamics and hydrocarbon degradation during a field-scale evaluation of bioremediation on a mudflat beach contaminated with buried oil[END_REF][START_REF] Yakimov | Natural microbial diversity in superficial sediments of Milazzo Harbor (Sicily) and community successions during microcosm enrichment with various hydrocarbons[END_REF]. From 30 days (T2), the relative abundance of the unidentified phylotype (B9) increased to become dominant after 45 days (T3). At the end of the experiment (T4), the DGGE pattern revealed a strong decrease of the dominant phylotypes in the bacterial assemblage. In microcosms subjected to oscillating oxic/ anoxic conditions, the early responders (with the exception of Marinobacterium) identified under continuously oxic conditions (T1Ox) were observed only after 30 days and were associated with a phylotype belonging to the Bacteroidetes (B21) and with two phylotypes observed during the initial sampling (unidentified Bacteroidetes: B4 and unidentified bacterium: B9). Throughout the experiment, the bacterial community was mainly composed of the unidentified Bacteroidetes (B21), Lewinella (B1), and the unidentified bacterium (B9). Under permanent anoxic conditions, specific phylotypes flourished during the experiment and were associated with phylotypes (B4 and B21) belonging to the Bacteroidetes observed under the other conditions (Fig. 4b). F r o m 1 5 d a y s ( T 1 ) , p h y l o t y p e s a f f i l i a t e d w i t h Marinobacterium (B6), as well as an unidentified Bacteroidetes (B22), distantly related to O. hongkongensis (KF528159; 89 %), were observed (Fig. 4b). By the end of the experiment, a noticeable increase was recorded in the relative abundance of the deltaproteobacterial phylotype (B27), which is close to the sulfate-reducing hydrocarbonoclastic Desulfobacula toluolica (NR_119006; 96 % identity). Thus, the presence of dioxygen in oily sediments drives the selection of specific phylotypes. During our incubations, the dynamics of the bacterial communities seems to be due to a combination of selective pressures exerted by the oxygen regime together with the utilization of HC substrates by adapted bacteria.

Characterization of metabolically active bacteria

Only a minority of the phylotypes present in the 16S rRNA gene fingerprints were identical to those present in the fingerprints obtained from 16S rRNA. In general, metabolically active and growing bacteria contain more rRNA molecules from ribosomes than do resting or starved bacterial cells [START_REF] Nomura | Regulation of synthesis of ribosomes and ribosomal components[END_REF]. Thus, the use of a dual 16S rRNA/ 16S rRNA gene approach allowed the identification of microorganisms that were presumably metabolically active, particularly those involved in the response to oil within the whole bacterial assemblages. Clustering analysis comparing the DGGE patterns from active bacteria revealed a separation of the samples into five clusters when an average similarity of 60 % was considered (Fig. 7). The first cluster (cluster II) was composed solely of the time zero sample (T0) and showed an average similarity of 50 % with the Bearly-stage cluster^(cluster I), which was made up of highly similar band profiles (average similarity: 94 %) from the first sampling times (T1 and T2, irrespective of the oxygenation regime). This observation indicates a shift of the active bacterial communities during the first 2 weeks, certainly due to crude oil contamination and an adaptation of the community to the experimental conditions. After 2 weeks (T1), the most active phylotypes were affiliated with Pseudomonas (B33, B34), Labrenzia (B12), and with an unidentified Deltaproteobacteria (B11) (Fig. 6). Thirty days after contamination (T2), these phylotypes were associated with an unidentified bacterium (B38) under both oscillating oxic/anoxic and permanent oxic conditions. B38 is distantly related to Alkaliphilus metalliredigens (NR_074633: Firmicutes; Clostridia; Clostridiales; Clostridiaceae; 87 %) and Desulfomicrobium baculatum (CP001629: Proteobacteria; Deltaproteobacteria; Desulfovibrionales; Desulfomicrobiaceae; 83 %) suggesting that this phylotype is a novel lineage. Under these oxygenation regimes, a sharp modification of the relative abundance of the dominant active phylotypes was observed at 45 and 60 days. This observation was in accordance with the clustering analysis showing that the samples T3Ox, T3Osc, T4Ox, and T4Osc were grouped in the cluster III when B38 was highly active (Fig. 7). Coincidentally, the greatest change in the abundance of phylotype B38 occurred during the final 15 days, the period during which LMW PAH biodegradation rates were the most significant for the permanent oxic and the oscillating oxygen conditions. Nonetheless, without uncontaminated control, it cannot be discarded that these changes in the community are not also linked to an adaptation to the experimental conditions. It should be noted, however, that other studies provide evidence of a drastic increase of 16S rRNA fingerprints associated with Pseudomonas spp. in the initial phase of oil degradation [START_REF] Röling | Bacterial community dynamics and hydrocarbon degradation during a field-scale evaluation of bioremediation on a mudflat beach contaminated with buried oil[END_REF][START_REF] Vitte | Effect of oxic/anoxic switches on bacterial communities and PAH biodegradation in an oil-contaminated sludge[END_REF]. Several strains of Pseudomonas have shown ability to degrade a number of aliphatic and (poly)aromatic HCs in pure culture [START_REF] Mulet | Pseudomonas diversity in crude-oil-contaminated intertidal sand samples obtained after the Prestige oil spill[END_REF], in simplified communities [START_REF] Singleton | Stable-isotope probing of bacteria capable of degrading salicylate, naphthalene, or phenanthrene in a bioreactor treating contaminated soil[END_REF] or in complex in situ communities [START_REF] Kostka | Hydrocarbon-degrading bacteria and the bacterial community response in Gulf of Mexico beach sands impacted by the Deepwater Horizon oil spill[END_REF]). More recently, members of the genus Labrenzia were isolated under aerobic conditions from beach sands contaminated during the Deepwater Horizon oil spill [START_REF] Overholt | Draft genome sequences for oil-degrading bacterial strains from beach sands impacted by the Deepwater Horizon oil spill[END_REF]. Members of this genus are all considered to be chemoheterotrophic and non-fermentative facultative anaerobes. Under permanent anoxic conditions at T2, active bacteria were associated with Pseudomonas (B33, B 3 4 ) , L a b re n z i a ( B 1 2 ) , a n d w i t h m e m b e r s o f Deltaproteobacteria (B11). Other phylotypes belonged to t h e o r d e r P s e u d o m o n a d a l e s a n d t h e g e n u s Marinobacterium: B35 (Pseudomonas stutzeri CP007509; 91 %), B44, (Pseudomonas sp. KJ765934; 99 %), and B43 (Marinobacterium rhizophilum GQ245896; 96 %). Finally, cluster IV and cluster V were related to last incubation times (T3An and T4An, respectively), underlining the fact that a succession of active bacteria occurred in anoxia during the last 15 days, when the LMW PAH removal began. The most active bacteria belonged to Marinobacterium (B43) and to the Pseudomonadales (B44 and B35) after 45 days. After 60 days, sequences related to Pseudomonadales (B35) and to an unidentified Bacteroidetes (B51), showing closest sequence similarity to O. hongkongensis (KF528159: Flavobacteriia; Flavobacteriales; Cryomorphaceae; 86 %), were the most active. Further study is needed to confirm that these phylotypes are involved in HC removal and, in any case, to determine the succession from aliphatic HC-degrading bacteria to those utilizing aromatic HCs.

Conclusions

Under field-realistic conditions and after 60 days of incubation, permanent oxygenation resulted in the greatest PAH removal from oiled sediments, and oscillating regimes produced results similar to continuous anoxic conditions. Under oscillating oxygen conditions, 60 days were required to remove as much PAH as after 15 days under permanent oxic conditions. At the same time, very low PAH removal was observed after 60 days under permanent anoxic conditions. Moreover, without sediment resuspension, the effectiveness of intermittent aeration on HC removal seemed to be drastically reduced when compared with the oil sludge process [START_REF] Vieira | Biodegradation of diesel oil and gasoline contaminated effluent employing intermittent aeration[END_REF][START_REF] Vitte | Effect of oxic/anoxic switches on bacterial communities and PAH biodegradation in an oil-contaminated sludge[END_REF][START_REF] Vitte | Dynamics of metabolically active bacterial communities involved in PAH and toxicity elimination from oil-contaminated sludge during anoxic/oxic oscillations[END_REF]. Pseudomonas spp., Labrenzia sp., and an unidentified Deltaproteobacteria were involved in the early response to perturbation due to oil contamination and the setting up of the microcosm irrespective of the oxygen regime, followed by the selection of late responders which were specifically adapted to the oxygen regime. The phylotype B38, distantly related to A. metalliredigens and D. baculatum, was highly active during the last stage of the experiment when the LMW PAH biodegradation rates were significant for permanent oxic and oscillating oxygen conditions, suggesting that this novel phylotype could play a role in biodegradation. This work has provided complementary knowledge on HC removal by potential HC-degrading candidates in coastal marine sediment as subjected to permanent oxic conditions, to relatively slow oxic-anoxic switches (3 days oxic/3 days anoxic), and to permanent anoxic conditions without sediment stirring. Because coastal marine sediments are subject to contrasted oxic conditions depending on tidal excursions, hydrodynamics, and the presence of bioturbating organisms, it is critical to determine HC fate in these ecosystems under different oxygenation schemes. Through bioturbation, dwelling macrofauna induces faster patterns of oxygen introduction in the anoxic sediments (e.g., 10 min oxic/20 min without oxygen input, Alitta virens irrigation rhythm; [START_REF] Kristensen | Oxygen and carbon dioxide exchange in the polychaete Nereis virens: influence of ventilation activity and starvation[END_REF]. For this purpose, further studies are required to decipher microbial interactions and their role in HC degradation in coastal marine sediments subject to faster oxygen fluctuations.

Fig. 1

 1 Fig.1Experimental setup. For each oxygen regime, four thinlayer incubation microcosms (a) were prepared and incubated under permanent oxygenation (oxic) maintained by continual purging of the water microcosms with sterile air (white arrows), permanent anoxia (anoxic) maintained by continual purging of the water microcosms using an N 2 /CO 2 mixture (black arrows), and by the oscillation between oxic and anoxic conditions (Oscillation) performed by switching purging gas in water reservoirs according the following regime: 3 days of oxia/3 days of anoxia (b). At each sampling time (T0, T1, T2, T3, and T4) and for each treatment, sediments coming from one of the microcosms were sampled

Fig. 2

 2 Fig.2PAHs remaining in the sediments exposed to permanent oxygenation (white circle), to oscillation between oxic and anoxic conditions (white up-pointing triangle) and to permanent anoxia (black circle). Studied families (parent and alkylated forms) are

Fig. 3 N

 3 Fig.3N-alkanes (n-C10 to n-C38) remaining in the sediments exposed to permanent oxygenation (white circle), to oscillation between oxic and anoxic conditions (white uppointing triangle), and to permanent anoxia (black circle)

Fig. 4

 4 Fig. 4 Ordination analysis of banding pattern data in permanent oxic (Ox), permanent anoxic (An), and in oscillating oxicanoxic (Osc) samples for sampling times T0 (initial sampling), T1 (15 days), T2 (30 days), T3 (45 days), and T4 (60 days). a Principal component analysis (PCA) based on bacterial class relative abundance. b Arrows point in the direction of maximal variation in species abundance, and arrow length is proportional to maximal rate of species change. Straight line indicates 60 % similarity; broken line indicates 50 % similarity

Fig. 5

 5 Fig. 5 DGGE patterns of bacterial communities in permanent oxic (Oxic), permanent anoxic (Anoxic), and in oscillating oxic-anoxic (Osc) samples for sampling times T0 (initial sampling), T1 (15 days), T2 (30 days), T3 (45 days), and T4 (60 days). Numbers indicate excised and sequenced bands

Fig. 7

 7 Fig. 7 Cluster analysis of DGGE profiles of active bacterial communities in permanent oxic (Ox), permanent anoxic (anoxic), and in oscillating oxic-anoxic (Osc) samples for sampling times T0 (initial sampling), T1

Acknowledgments This research was funded by the French National Agency (ANR) under the DHYVA (ANR 2006 SEST 09) and DECA PAGE (ANR 2011 CESA 006 01) projects. We thank Patricia Bonin for many fruitful and interesting exchanges.

Conflict of interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Florence Helft
email: florence.helft@gmail.com

Claudie Doums

Thibaud Monnin

No evidence of pre-copulatory mate choice by gynes in the facultatively parthenogenetic ant Cataglyphis cursor

No evidence of pre-copulatory mate choice by gynes in the facultatively parthenogenetic ant Cataglyphis cursor

KEYWORDS

Eusocial insects, Sexual selection, Inclusive mate choice, Multiple mating.

Sexual selection favours phenotypic traits affecting reproductive success, for instance through better access to a sexual partner resulting from increased competitiveness among individuals of the same sex (intra-sexual selection) or from increased attractiveness towards individuals of the opposite sex (inter-sexual selection). While sexual selection has received considerable attention in solitary organisms it has been the subject of less attention in social species, especially in social insects [START_REF] Davidson | Sexual selection in harvester ants (Hymenoptera: Formicidae: Pogonomyrmex)[END_REF][START_REF] Boomsma | The evolution of male traits in social insects[END_REF][START_REF] Boomsma | kin selection versus sexual selection: why the ends do not meet[END_REF][START_REF] Den Boer | Seminal fluid mediates ejaculate competition in social insects[END_REF][START_REF] Boomsma | Beyond promiscuity: mate-choice commitments in social breeding[END_REF]). This might be linked to the fact that in the latter sexual partners are committed for life, which minimizes the potential for sexual selection [START_REF] Boomsma | Beyond promiscuity: mate-choice commitments in social breeding[END_REF]. Furthermore, the widespread occurrence of nuptial flights associated with a high mortality of sexuals favours the evolution of traits that optimize and access to partners (e.g. flight abilities, partner detection) but not those linked with pre-copulatory sexual selection (e.g. weaponry, ornaments, courtship behaviours) [START_REF] Boomsma | The evolution of male traits in social insects[END_REF][START_REF] Baer | The copulation biology of ants (Hymenoptera: Formicidae)[END_REF]).

However, some eusocial insects mate multiply on the ground near the nest entrance. This lowers the mortality of gynes (young virgin queens) since they are in the presence of protective workers and can retreat into the nest. Moreover, workers may directly participate in gyne mate choice, i.e. exercise a form of inclusive mate choice [START_REF] Cronin | Opportunities for mate choice in the fissionperforming ant Cataglyphis cursor[END_REF]. Indeed, gynes and workers have the same interests in choosing the "best" males. This is because workers typically do not reproduce and obtain indirect fitness by helping related queens. Workers would therefore benefit from helping related gynes to choose good mates, and could achieve this by physically preventing males of poor quality from accessing gynes.

Gynes would benefit from deferring mate choice to workers if doing so minimises their exposure to desiccation and predation. In addition, workers may collectively be in a better position to make an optimal choice than hard-pressed gynes.

The involvement of workers in mate choice has been suggested in army ants [START_REF] Franks | Sexual competition during colony reproduction in army ants[END_REF], Argentine ants (in the invasive range, [START_REF] Sunamura | Workers select mates for queens: a possible mechanism of gene flow restriction between supercolonies of the invasive Argentine ant[END_REF]) and the facultatively parthenogenetic ant Cataglyphis cursor [START_REF] Cronin | Opportunities for mate choice in the fissionperforming ant Cataglyphis cursor[END_REF][START_REF] Helft | First evidence of inclusive sexual selection in the ant Cataglyphis cursor: worker aggressions differentially affect male access to virgin queens[END_REF]. In the latter two species, workers are aggressive towards all foreign males who enter or come near the nest searching for a receptive gyne. Worker aggression may be a form of inclusive sexual selection by workers if they differentially affect some male phenotypes. This is indeed what occurs in C. cursor. Gynes are produced by the multiply mated queen [START_REF] Lenoir | Reproduction and dispersal in the ant Cataglyphis cursor (Hymenoptera, Formicidae)[END_REF][START_REF] Pearcy | Conditional use of sex and parthenogenesis for workers and queen production in ants[END_REF]) and workers are highly related to both parthenogenetically produced gynes (clones of their mother, r ~ 0.5) and sexually produced gynes (r = 0.44 on average) [START_REF] Doums | Facultative use of thelytokous parthenogenesis for queen production in the polyandrous ant Cataglyphis cursor[END_REF], and a recent study showed that worker aggression toward males affected male relative mating potential, increasing it for lighter males and decreasing it for heavier males [START_REF] Helft | First evidence of inclusive sexual selection in the ant Cataglyphis cursor: worker aggressions differentially affect male access to virgin queens[END_REF]. The present study therefore aims at determining whether C. cursor gynes exercise pre-copulatory mate choice or not. We observed mating behaviour of gynes in the presence of two males under laboratory conditions. The males differed in the level of harassment they had experienced from workers, with one of the two males previously exposed to worker aggression for 10 minutes while the other was not exposed to workers.

We used 115 sexuals (46 males and 69 females) from 22 colonies which had been produced in nature but which emerged in the laboratory, allowing us to be certain of their virginity (see Supplementary material). We carried out 23 mating tests, each using one focal gyne and two males. In addition, two other gynes were used during the exposure phase of males to worker aggression immediately prior to the mating test (below). The focal gyne was placed alone in a mating arena to which it could habituate. Simultaneously, one male was placed in another box containing a second gyne in a mesh cage, so that he could perceive her presence but could not mate (treatment without workers or W-). A second male was placed in a third box containing the third gyne, also encaged, but with the addition of 50 of the gyne's nestmate workers that could aggress the male as shown in [START_REF] Helft | First evidence of inclusive sexual selection in the ant Cataglyphis cursor: worker aggressions differentially affect male access to virgin queens[END_REF] (male W+). After 10 minutes, the two males were moved to the mating arena containing the focal gyne and matings were observed for 10 minutes. All individuals were used only once.

The three gynes used in each test were sisters and at least 19 days old, since previous observations suggest that most gynes are sexually active at this age. The two males were brothers and unrelated to the gynes. We selected males of the same age and size (see Supplementary material), hence they differed only in their previous exposure to worker harassment.

Mating attempts were unambiguous. The male mounted above the gyne with his front and middle legs embracing her thorax and only his hind legs remaining on the ground, made frenzied antennae contacts and attempted to copulate by rubbing the tip of his abdomen around that of the gyne. A successful attempt ended by the intromission of the male's genitalia into the female gaster for several seconds and was thus recorded as a mating even though we do not know whether sperm transfer occurred. Using BioLogic software (Dimitri Missoh, http://swarmy.free.fr/wordpress/2009) we recorded the latency before the first mating attempt, the number of mating attempts, and the number and duration of mating (i.e. duration of copulation). All statistical analyses were conducted using R-Gui software (version 3.0.2, 2008).

The gyne refused to mate with either male in nine out of the 23 tests. This may be because she was not receptive or stressed by the experimental setup, e.g. by the absence of nestmate workers or colony odour. Twenty matings occurred in the remaining 14 tests. They lasted 1 min 10 s ± 30 s (mean ± SD). C. cursor queens mate multiply [START_REF] Lenoir | Reproduction and dispersal in the ant Cataglyphis cursor (Hymenoptera, Formicidae)[END_REF][START_REF] Pearcy | Conditional use of sex and parthenogenesis for workers and queen production in ants[END_REF]) and multiple mating by gynes was indeed observed during our 10 minutes tests. Nine gynes mated once, four mated twice (two with the same male and two with both males) and one mated thrice (with both males). Similarly, males can mate multiply in this species [START_REF] Cronin | Opportunities for mate choice in the fissionperforming ant Cataglyphis cursor[END_REF]) and multiple mating by males was also recorded, with 14 males mating once and three mating twice (Fig. 1).

Although all W+ males were aggressed by workers during the exposure phase that immediately preceded the mating test (from 22 to 201 aggressions in 10 minutes, median = 94), this had no effect on the outcome of mating tests. W+ and W-males did not differ in latency before the first mating attempt (Wilcoxon test: p = 0.9), number of mating attempts (Wilcoxon test: p = 0.7), number of matings (Fisher exact test: p = 0.54) or duration of matings (Wilcoxon test: p = 0.7) (Fig. 1).

Both males attempted mating in 11 out of the 14 tests where mating occurred. The gyne always mated with the first male who attempted mating, usually on his second or third attempt, and she always mated before the second male attempted to mate (Fig. 1). This suggests that gynes mated indiscriminately, at least for their first mating, and supports the hypothesis that gynes largely defer pre-copulatory mate choice to workers [START_REF] Helft | First evidence of inclusive sexual selection in the ant Cataglyphis cursor: worker aggressions differentially affect male access to virgin queens[END_REF]. Note, however, that this does not preclude post-copulatory sperm selection [START_REF] Baer | The copulation biology of ants (Hymenoptera: Formicidae)[END_REF]. For instance, cryptic mate choice by gynes could occur by rejecting the sperm of less fit partners and/or by controlling the duration of mating [START_REF] Oppelt | Dynamics of sperm transfer in the ant Leptothorax gredleri[END_REF]. In several instances we observed that gynes in copula turned around and bit the mounting male and that this ended the copulation. This suggests that gynes have at least some degree of control over mating duration, and it is interesting in this context to note that mating duration was indeed very variable, lasting from 10 s to 1 min 57 s (Fig. 1).

The indiscriminate mating by gynes observed in our experiment is in apparent contradiction with previous field observations that gynes can refuse to mate and indeed actively avoid most mating attempts [START_REF] Lenoir | Reproduction and dispersal in the ant Cataglyphis cursor (Hymenoptera, Formicidae)[END_REF], Cronin et al. 2011). However, in these field observations gynes were likely already mated since most had lost their wings and wing loss is a good predictor of being mated [START_REF] Doums | Facultative use of thelytokous parthenogenesis for queen production in the polyandrous ant Cataglyphis cursor[END_REF]), hence they were likely not seeking to mate but were seeking to be transported by workers to a new colony [START_REF] Lenoir | Reproduction and dispersal in the ant Cataglyphis cursor (Hymenoptera, Formicidae)[END_REF][START_REF] Chéron | Unequal resource allocation among colonies produced by fission in the ant Cataglyphis cursor[END_REF]. Further studies should investigate whether all matings result in the transfer of sperm and/or seminal secretions, whether mating duration affects this transfer, and whether gynes can adjust mating duration according to male quality or mating status.

Figure 1 :

 1 Figure 1: Timing of mating attempts and duration of matings. The x-axis shows the duration

ACKNOWLEDGEMENTS

We thank C. Haussy for helping with colony collection, R. Péronnet for helping in rearing the ants, and A. L. Cronin for linguistic help.