Massé Cécile

Garabetian Frédéric

Deflandre Bruno

Maire Olivier

Costes Laurence

Mesmer-Dudons Nathalie

Duchêne Jean-Claude

Bernard Guillaume

Ciutat Aurélie

Feeding ethology and surface sediment reworking by the ampharetid polychaete Melinna palmata Grube, 1870: effects on sediment characteristics and aerobic bacterial community composition

Keywords: Ampharetids, Melinna palmata, Feeding ethology, Surface sediment reworking, Sediment characteristics, Aerobic bacterial community composition

The present study was aiming at describing the feeding ethology and assessing surface sediment reworking together with associated effects on sediment characteristics and aerobic bacterial community composition by the ampharetid polychaete Melinna palmata, which exhibits very dense populations in the Arcachon Bay (French Atlantic coast). There was a plasticity in the main aspects (i.e., positioning of the tube at the sediment-water interface, stretching of the worms outside their tubes while feeding, location and mechanism of faeces production) constitutive of the current knowledge regarding feeding ethology in ampharetids. On average, worms dedicated 84.6% of their time to the prospection of surface sediments and produced faeces exclusively at the water sediment interface, which resulted in surface sediment reworking and induced the zonation of surface sediment in three distinct areas, namely: undisturbed sediment, prospected and faecal mound areas. Average individual surface prospected area and surface sediment reworking rate were 28 cm 2 and 6.8 mm 3 .h -1 , respectively. Surface sediments were coarser and their bulk organic contents were lower in prospected areas, intermediate in faecal mound areas and higher in undisturbed sediments. Oxygen penetrated deeper in the sediment column in faecal mound areas. Aerobic bacterial community composition associated with surface oxygenated sediments within these three areas also significantly differed, which suggests that sediment reworking per se (i.e., irrespective of changes in redox conditions) do have an effect on those compositions. These results are discussed in terms of potential food limitation in the very dense populations of M. palmata present in the Arcachon Bay. It is suggested that the high

INTRODUCTION

Once sedimented, particulate organic matter undergoes early diagenesis (i.e., a sequence of mineralization reactions taking place in the top sediment column). This process is mainly achieved by bacterial communities featuring different metabolic capacities in relation with the availability of a series of final electron acceptors. In the absence of benthic macrofauna, these receptors show a typical vertical zonation [START_REF] Froelich | Early oxydation of organic matter in pelagic sediments of the eastern equatorial Atlantic: suboxic diagenesis[END_REF]. By modifying this redox sequence and by creating microenvironments submitted to oscillatory conditions, bioturbation by benthic macrofauna disturbs oxidation processes taking place in the top sediment column [START_REF] Burdige | The biogeochemistry of manganese and iron reduction in marine sediments[END_REF]. Bioturbation activities include the mixing and spatial redistribution of both sediment particles (i.e., sediment reworking), and pore-water solutes (i.e., bioirrigation ;[START_REF] Rhoads | Organism-sediment relations on the muddy sea floor[END_REF][START_REF] Kristensen | What is bioturbation? The need for a precise definition for fauna in aquatic sciences[END_REF]. They directly affect the three components cuing the efficiency of mineralization processes taking place in the top sediment column, namely the spatial distributions of: (1) particulate organic matter; (2) final electron acceptors; and (3) bacterial community biomass (Aller and Yingst, 1985;[START_REF] Reichardt | Impact of bioturbation by Arenicola marina on microbiological parameters in intertidal sediments[END_REF], composition [START_REF] Bertics | Biodiversity of benthic microbial communities in bioturbated coastal sediments is controlled by geochemical microniches[END_REF][START_REF] Laverock | Bioturbating shrimp alter the structure and diversity of bacterial communities in coastal marine sediments[END_REF] and biogeochemical functions (Bertics et al., 2010;[START_REF] Gilbertson | Differential effects of microorganism-invertebrate interactions on benthic nitrogen cycling[END_REF]Yazdani Foshtoni et al., 2015). By doing so, bioturbation strongly influences the biogeochemical processes taking place at the sediment-water interface [START_REF] Aller | Bioturbation and remineralization of sedimentary organic matter: effects of redox oscillation[END_REF][START_REF] Aller | The effect of biogenic irrigation intensity and solute exchange on diagenetic reaction rates in marine sediments[END_REF][START_REF] Lohrer | Rapid reworking of subtidal sediments by burrowing spatangoid urchins[END_REF][START_REF] Furukawa | Interactions between macro-and microorganisms in marine sediment[END_REF][START_REF] Aller | Sedimenary diagenesis, depositional environments, and benthic fluxes[END_REF][START_REF] Braeckman | Variable importance of macrofaunal functional biodiversity for biogeochemical cycling in temperate coastal sediments[END_REF].

Sediment reworking is typically a 3D process [START_REF] Rosenberg | 3D visualization and quantification of marine benthic biogenic structures and particle transport utilizing computer-aided tomography[END_REF]. Its quantification is however usually achieved based on a reduced number of dimensions due to the difficulty in penetrating the sediment matrix. Because of the interaction with the vertical zonation of biogeochemical processes (see above) and due to the importance of the problematic of carbon burial, most attention has been devoted to the vertical dimension (see for example [START_REF] Maire | Quantification of sediment reworking rates in bioturbation research: a review[END_REF].

Classically, the assessment of sediment reworking is based on the coupling between: (1) the assessment of a vertical profile of tracer concentrations, and (2) the modelling of this profile. This whole process usually results in the computation of a vertical biodiffusion coefficient (Db) and/or nonlocal exchange functions, which account for the intensity of vertical sediment reworking through biodiffusion and non-local transport. Conversely, the quantification of horizontal sediment reworking has received much less attention although some early modelling studies have suggested that this component may be quantitatively as important as the vertical one [START_REF] Wheatcroft | A mechanistic view of the particulate biodiffusion coefficient: Step lengths, rest periods and transport directions[END_REF][START_REF] Wheatcroft | Conservative tracer study of horizontal sediment mixing rates in a bathyal basin, California borderland[END_REF] and that surface mixing is often considered as an ecological trait in studies assessing the relationship between species diversity and ecosystem functioning (e.g. [START_REF] Hewitt | Habitat variation, species diversity and ecological functioning in a marine system[END_REF]. The use of thin aquaria and luminophores (i.e., sediment particles coated with a fluorescent paint) coupled with sophisticated image acquisition and analysis techniques has only been recently introduced allowing for the 2D (i.e., vertical and horizontal) assessments of sediment reworking (e.g. [START_REF] Gilbert | 2-D optical quantification of particle reworking activities in marine surface sediments[END_REF]Maire et al., 2007bMaire et al., , 2007c[START_REF] Maire | Indirect effects of non-lethal predation on bivalve activity and sediment reworking[END_REF][START_REF] Bernard | Experimental assessment of particle mixing fingerprints in the deposit-feeding bivalve Abra alba (Wood)[END_REF].

The analysis of the effects of bioturbation on bacterial community composition and function has also favored the assessment of the vertical component due to the tight interactions between these communities and available final electron acceptors. Gallery biodiffusors (sensu [START_REF] Kristensen | What is bioturbation? The need for a precise definition for fauna in aquatic sciences[END_REF] have been mostly used to tackle this question. Through the ventilation of their burrow, these organisms enhance oxygen penetration deep in the sediment column (e.g. [START_REF] Papaspyrou | Microbial reaction rates and bacterial communities in sediment surrounding burrows of two nereidid polychaetes (Nereis diversicolor and N. virens)[END_REF][START_REF] Laverock | Bioturbating shrimp alter the structure and diversity of bacterial communities in coastal marine sediments[END_REF][START_REF] Pischedda | Characterization of specificity of bacterial community structure within the burrow environment of the marine polychaete Hediste (Nereis) diversicolor[END_REF] and thereby induce a patchy distribution of bacterial assemblages (e.g. [START_REF] Laverock | Bioturbating shrimp alter the structure and diversity of bacterial communities in coastal marine sediments[END_REF]. Moreover, and because their activity is often discontinuous, they induce short-term temporal changes in bacterial assemblage functionalities, which results in a non-equilibrium dynamics of mineralization processes (e.g. [START_REF] Wenzhöfer | Small-scale spatial and temporal variability in coastal benthic O2 dynamics: Effects of fauna activity[END_REF][START_REF] Pischedda | Imaging oxygen distribution in marine sediments. The importance of bioturbation and sediment heterogeneity[END_REF]. One difficulty when studying the effect of sediment reworking on bacterial assemblages and functions is the unraveling between the effect of sediment reworking per se and those related with changes in redox conditions (e.g. [START_REF] Laverock | Bioturbating shrimp alter the structure and diversity of bacterial communities in coastal marine sediments[END_REF]. A possibility to tackle this difficulty is to focus on benthic macrofauna performing sediment reworking without altering the oxygenation of the sediment. This is potentially the case of "Surface deposit feeding Sessile Tentaculate" worms (SST, [START_REF] Fauchald | The diet of worms: a study of polychaete feeding guilds[END_REF], which collect a very thin layer of particles at the sediment-water interface. Sediment reworking by these organisms typically creates distinct areas at the sediment surface with a clear distinction between undisturbed sediments, and prospected and faecal mound areas (e.g. [START_REF] Nowell | The foraging strategy of a subtidal and deep-sea deposit feeder[END_REF]. This allows for the establishment of a stratified sampling strategy of surface oxygenated sediment to assess the effect of sediment reworking on sediment characteristics and associated aerobic bacterial community composition (see [START_REF] Warwick | Effects of the feeding activity of the polychaete Streblosoma bairdi (Malmgren) on meiofaunal abundance and community structure[END_REF] and [START_REF] Olafsson | The impact of Melinna palmata Grube, a tube-building polychaete, on meiofaunal community structure in a soft-bottom subtidal habitat[END_REF] for a similar approach regarding meiofauna composition).

During the present study, we used the ampharetid polychaete Melinna palmata as a biological model. This species is abundant in the Zostera noltei meadows and the bare intertidal mudflats of the Arcachon Bay (France) where it has been suggested that its dense populations inhibit vertical sediment reworking [START_REF] Bernard | A comparative study of sediment particles mixing in a Zostera noltei meadow and a bare sediment mudflat[END_REF]. Although, M. palmata clearly belongs to SST [START_REF] Fauchald | The diet of worms: a study of polychaete feeding guilds[END_REF][START_REF] Jumars | Diet of worms emended: an update of Polychaete feeding guilds[END_REF], its feeding ethology has not been deeply investigated yet. The present study therefore aimed at: (1) describing the anatomy of feeding organs;

(2) establishing a typology of the different behaviors and assessing the time allocation pattern between these behaviors; (3) quantifying surface sediment reworking, and (4) assessing the effects of sediment reworking on surface sediment main characteristics, oxygen penetration and bacterial community composition. These objectives were tackled using an ex situ experimental approach. Due to the practical/technical difficulties in achieving replication (e.g. Maire et al., 2007a), this resulted in three series of experiments dedicated to one or combinations of the above mentioned specific aspects.

MATERIAL AND METHODS

Worm collection and maintenance

Melinna palmata is an ampharetid worm with a large boreo-mediterranean distribution [START_REF] Grehan | Demography and reproductive biology of Melinna palmata (Ampharetidae: Polychaeta) in inner Galway Bay on the west coast of Ireland[END_REF]. It is abundant along the Atlantic coasts from Norway to Morocco (Guillou and Hily, 1983;[START_REF] Grehan | Demography and reproductive biology of Melinna palmata (Ampharetidae: Polychaeta) in inner Galway Bay on the west coast of Ireland[END_REF][START_REF] Cacabelos | Ecological features of Terebellida fauna (Annelida, Polychaeta) from Ensenada de San Simon (NW Spain)[END_REF], in the Black Sea, the Sea of Azov, the Persian Gulf and the Mediterranean Sea [START_REF] Holthe | Polychaeta Terebellomorpha[END_REF][START_REF] Zaabi | Significations écologiques de la variabilité des polychaetes dans le golfe de Tunis[END_REF]. It has been recorded all along the metropolitan French coast (http://resomar.cnrs.fr/bases/index.php; [START_REF] Dauvin | An up to date list of polychaetous annelids from the English Channel[END_REF]. Individual worms are typically between 15 and 50 mm in length and between 2 and 3 mm in width. They bear 16 thoracic and about 60 abdominal segments. The base of their two sets of four gills is implanted on the dorsal part of their first segment [START_REF] Fauvel | Polychètes sédentaires, Faune de France[END_REF][START_REF] Rouse | Polychaetes[END_REF]. The life span is between 2 and 2.5 years in inner Galway Bay [START_REF] Grehan | Demography and reproductive biology of Melinna palmata (Ampharetidae: Polychaeta) in inner Galway Bay on the west coast of Ireland[END_REF] and secondary production in Southampton waters is 0.42 g C -2 yr -1 [START_REF] Oyenekan | Population dynamics and secondary production in Melinna palmata (Polychaeta: Ampharetidae)[END_REF].

For the present study, adult worms were collected during April 2012 (anatomy of feeding organs, 1 st and 2 nd series of experiments) and December 2013 (3 rd series of experiments and tentacles number) in the Arcachon Bay (French Atlantic Coast) at the "Germanan" site (44°42'726''N, 1°07'940''W) where extremely dense populations of M. palmata have been reported in Zostera noltei intertidal seagrass meadows [START_REF] Blanchet | Heterogeneity of macrozoobenthic assemblages within a Zostera noltii seagrass bed: diversity, abundance, biomass and structuring factors[END_REF][START_REF] Bernard | A comparative study of sediment particles mixing in a Zostera noltei meadow and a bare sediment mudflat[END_REF]. Back at the laboratory, tubes were isolated from the sediment by gently sieving on a 1 mm square mesh, and collected by hand.

Worms were then acclimatized (>10 days) in aquaria containing sieved (1 mm mesh to discard other macrofauna; e.g. [START_REF] Queiros | Can benthic community structure be used to predict process of bioturbation in real ecosystems?[END_REF] sediment from the collection site and fuelled with a continuous flow of filtered seawater from the Arcachon Bay before any subsequent manipulations/experimentations.

Anatomy of feeding organs

The tentacles of 10 worms collected in December 2013 were counted under a Nikon® SMZ25 stereomicroscope. The anatomy of the feeding organs was investigated using Scanning Electron Microscopy (SEM). Fifteen worms collected in April 2012 were left for one hour in filtered (0.22 µm) and sterilized seawater to ensure that their tentacles were deployed. Heads and tentacles were then dissected and fixed in 2.5 % glutaraldehyde with 0.4 M cacodylate buffer and 7 % NaCl for 24 h. Samples were rinsed 3 × 30 min in 0.4 M cacodylate and 4 % NaCl before being post-fixed with 2 % osmium tetroxide in 0.4 M cacodylate and 10 % NaCl. They were then dehydrated through a series of incubations in increasing alcohol concentrations (10 min in 50 %, 10 min in 70 %, 10 min in 90 %, 10 min in 95 %, 2 × 10 min in absolute and 15 min in propylene oxide). After critical-point-drying, samples were coated with gold and observed using a Quanta 200 SEM (FEI Company) at the Bordeaux Imaging Centre (University of Bordeaux, France).

Experiments

Three series of experiments were carried out during April 2012, November 2012 and February 2014 (Fig. 1). Before each experiment, worms were carefully removed from their tube and examined under a binocular microscope to check for physical integrity. All experiments were achieved on single individual worms, which were introduced at the center of a parallelepiped (11.5×17.5×6.5 cm, corresponding to a density of 50 ind.m -2 ) aquarium filled with a 6 cm layer of sediment from the collection site (sieved on 1 mm-mesh to remove other macrofauna and previously stabilized for 10 days) and placed under a continuous flow of filtered seawater from the Arcachon Bay for 10 days. This time period proved sufficient for worms to build a new tube and to efficiently burrow in the sediment.

First series of experiments

The aims of the first series of experiments (Fig. 1A) were to: (1) describe feeding ethology, (2) characterize worm behaviors, (3) assess time allocation pattern, (4) quantify the surface of sediment reworked areas, and ( 5) assess the effect of surface sediment reworking on main sediment characteristics and aerobic bacterial community composition. Thirteen worms were studied during this experiment series (temperature: 15.9 ± 2.0 °C; salinity: 30.5 ± 1.7).

Feeding ethology, typology of behaviors and time allocation pattern

A computer piloted IDS µeye UI-1580SE-C-HQ video sensor (Stemmer Imaging) was positioned 30 cm straight above the aquarium sediment surface, which was illuminated with infrared light. This system allowed for the collection of images of the sediment surface during 24 hours at a frequency of 0.1 Hz. Collected images were assembled in an AVI film, which was then visually analyzed to assess: (1) feeding ethology, (2) different types of behavior, and (3) time allocation patterns between these behaviors.

Surface sediment reworked areas

Additional images of the sediment surface were collected under ambient light on days 10, 15, 20, 25 and 30 (starting from the beginning of the acclimation period) using a Nikon ® D7000 Reflex 16.2 Mpixels camera fitted with a 18-105 mm lens. A special care was taken to insure that the image plan was strictly parallel to the sediment surface. For all worms, a zonation of the sediment surface became rapidly visible. This included: (1) a faecal mound area near the tube opening, (2) a prospected area devoid of surface particles, and (3) undisturbed sediment (Fig. 2A andB). The faecal mound and the total affected areas were manually drawn on each collected image and their surface was assessed using the Image J® software (USA National Institutes of Health) after appropriate calibration. The surfaces of prospected areas were computed as the difference between the surfaces of total affected and faecal mound areas.

Surface sediment sampling

At the end of the 20 d experiment period, the surface sediments of: (1) faecal mound areas, (2)

prospected areas, and (3) undisturbed sediments were sampled using a truncated (to allow the passage of the largest particles) pipette tip fitted to the needle port of a 10 mL syringe. All the surface sediment of each three areas was sucked by gently pulling the syringe plunger while the truncated tip was moved to sample the very top (i.e., about 2 mm) surface sediment layer. Each sample was homogenized and divided in two for the assessments of: (1) main sediment characteristics, and (2) aerobic bacterial community composition.

Main sediment characteristics

Sediment granulometry was assessed using a Malvern® Master Sizer laser microgranulometer and expressed as median diameter (D50) and mud content (i.e., the volume % of particles less than 63 µm in size). Particulate organic carbon (POC) and nitrogen (PON) were measured on 10 mgDW (dry weight) freeze dried decarbonated (with 0.2N HCl according to [START_REF] Kennedy | The effects of acidification on the determination of organic carbon, total nitrogen and their stable isotopic composition in algae and marine sediment[END_REF] sediment samples using a ThermoFinnigan® Flash Elemental Analyser Series 1112. Chlorophyll a and phaeophytin a were assessed on a 6 mL 90 % acetone extracts of 400 mg WW (wet weight) sediment samples using a Perkin Elmer® spectrofluorometer [START_REF] Neveux | Spectrofluorimetric assay of chlorophylls and phaeopigments using the least squares approximation technique[END_REF]. Depending on the amount of available sediment, these measurements were achieved on 1 to 3 replicates.

Aerobic bacterial community composition

Aerobic bacterial community composition was characterized by Automated Ribosomal Intergenic Spacer Analysis (ARISA), a PCR-based whole-community fingerprinting method [START_REF] Fisher | Automated approach for ribosomal intergenic spacer analysis of microbial diversity and its application to freshwater bacterial communities[END_REF]. Within two hours after collection, 0.465 ± 0.003 g WW (Wet Weight) of sediment from each zone was placed in preservative buffer (100 mM Tris-HCl [pH 8.0], 10 0 mM EDTA [pH 8.0], 1.5 NaCl and 1 % [wt/vol] cetyltrimethylammonium bromide) [START_REF] Zhou | DNA recovery from soils of diverse composition[END_REF]. Samples were stored at -80°C until analysis. DNA extraction was achieved using 700 µL of homogenized thawed sediment sample. DNA was extracted and purified coupling a bead beating method (Lysing matrix E tubes) and Fast Prep (MP Biomedicals): two runs at 5.5 m s -1 during 30 s with the use of an extraction kit UltraClean® Soil DNA Isolation Kits (MO BIO Laboratories Inc.). The amount of extracted and purified DNA was quantified by spectrophotometry with 2 µL of DNA solution, using an Epoch microplate spectrophotometer (Biotek instruments). PCR amplification of the 16S-23S rDNA intergenic spacer was carried out using 5'FAM labelled S-D-Bact-1522-B-S-20 (5'-TGC GGC TGG ATC CCC TCC TT-3') and L-D-Bact-132-a-A-18 primers (5'-CCG GGT TTC CCC ATT CGG-3') [START_REF] Normand | ITS analysis of prokaryotes[END_REF]. The final reaction mix (25 µL) consisted of 1X PCR buffer (Promega), 1.5 mM MgCl 2 , 0.3 mg mL -1 bovine serum albumin (BSA), 5 % Dimethyl sulfoxide (DMSO), 200 µM of each deoxynucleoside triphosphate (Invitrogen), 0.5 µM of each primer (Invitrogen), 0.25 U of Taq polymerase (Promega) and 10 ng of template DNA at about 1 ng µL -1 . Amplification was performed with a Thermocycler (Eppendorf AG). After an initial denaturation at 94ºC for 5 min, 35 cycles of denaturation (94ºC, 1 min), annealing (55ºC, 1 min) and extension (72ºC, 1 min) were performed, followed by a final extension (72ºC, 10 min). For each extracted DNA sample, triplicate PCR assays were performed using 3 x 10 ng of template DNA. Amplification products of the three assays were pooled and purified using QIAquick PCR Purification Kit (QIAgen). Purified amplification products were then quantified using the spectrophotometric method previously described. Finally, 1.5 µL of amplification product adjusted by dilution to about 10 ng µL -1 were mixed with 0.1 µL GeneScan 1200 LIZ internal size standard (Applied Biosystems) and 10 µL Hi-Di formamide (Applied Biosystems). The mixture was denatured at 94°C for 4 min and fragments were discriminated using an ABI 3730XL automated sequencer (Applied Biosystems®) operated by the Plateforme Genome-Transcriptome Pierroton (a joined facility of INRA and University of Bordeaux).

Resulting electrophoregrams were analysed using the Applied Biosystems® Peak Scanner software.

Peak sizes inferior to 200 bp and superior to 1200 bp were considered as background noise and eliminated. Then, an "optimal divisor" (Od) was determined to remove fluorescence background within remaining peaks [START_REF] Osborne | New threshold and confidence estimates for terminal restriction length polymorphism analysis of complex bacterial communities[END_REF]. Peaks contributing less than 0.1% (i.e. Od value) of the total amplified DNA (as determined by relative fluorescence intensity) were indistinguishable from baseline noise and eliminated. Binning was carried out under the R software (available on http://cran.rproject.org) using the algorithm "Interactive binner" (available on http://www.ecology-research.com - [START_REF] Ramette | Quantitative community fingerprinting methods for estimating the abundance of operational taxonomic units in natural microbial communities[END_REF]. This allowed for the assessment of the relative abundance of each Operational Taxonomic Unit (OTU) in each sediment replicate. The ARISA fingerprinting method is based on 16S-23S ITS size. Since bacterial species have various numbers and types of ribosomic operons, there is no simple relationship between the occurrences of a bacterial species and the number and types of retrieved OTU [START_REF] Hill | Using ecological diversity measures with bacterial communities[END_REF]. Moreover, PCR biases may distort OTU relative abundances [START_REF] Wintzingerode Von | Determination of microbial diversity in environmental samples: pitfalls of PCR-based rRNA analysis[END_REF]. It is nevertheless assumed that OTU richness and composition realistically reflect bacterial taxonomic diversity [START_REF] Forney | Molecular microbial ecology: land of the one-eyed king[END_REF]) and support diversity pattern analyses [START_REF] Ramette | Multivariate analyses in microbial ecology[END_REF]. It is important to underline that ARISA only account for changes in OTU relative abundances.

Second series of experiments

The aim of the second series of experiments (Fig. 1B) was to assess the effect of surface sediment reworking on oxygen penetration within the sediment column. Oxygen microprofiles were achieved on five aquaria containing individual worms after an incubation period of 20 days (temperature: 16.0 ±1.6 °C; salinity: 32.5 ±0.8).

Four Unisense® A/S Clark-type microelectrodes [START_REF] Revsbech | An oxygen microsensor with a guard cathode[END_REF]; 100 µm sensor-tip diameter, 90 % response time < 7 s, stirring sensitivity < 1) were positioned on a common holder (forming a 2.5 cm side square) fixed on a MC-232, Unisense ® motorized micromanipulator. These microelectrodes were connected to a high-sensitivity Unisense® picoammeter. The whole system was connected to a portable computer and controlled by the SensorTrace® PRO v3.0 software. Oxygen profiles were performed with a vertical resolution of 100 µm and an equilibration time of 7 s down to a 1 cm depth in the sediment column. Two to four sets of 4 profiles were achieved in each aquarium.

Overall, 13 micro-profiles were located in faecal mound areas, 18 in prospected areas and 21 in undisturbed areas. Oxygen micro profiles were processed using the PRO2FLUX software [START_REF] Deflandre | PRO2FLUX -A software program for profile quantification and diffusive O2 flux calculations[END_REF] to assess oxygen penetration depths.

Third series of experiments

The aims of the third series of experiments (Fig. 1C) were to further assess worm behaviors, feeding ethology and time allocation patterns, and to quantify surface sediment reworking. Thirteen worms were studied during this series (temperature: 13.5 ± 0.9 °C; salinity: 28.9 ± 1.8).

Methodologies for the assessments of feeding ethology, worm behaviors and time allocation patterns were strictly similar to those used during the first series of experiments.

Surface sediment reworking rates were assessed for 5 individual worms (15.2 ± 5.0 mgWW) through laser telemetry [START_REF] Maire | Quantification of sediment reworking rates in bioturbation research: a review[END_REF]. This technique allowed for successive microtopography mapping of the sediment surface, which were later compared to assess the volume of reworked under a set of motorized cross tables (401XR Parker® Hannifin precision linear positioners with 5 mm ball screw) connected to Vix500 Microstepper Indexer Drives with XL-PSU power supplies. These tables were computer controlled, which allowed for precise (i.e., ±1.5 µm) positioning [START_REF] Duchêne | Hydroid and serpulid recruitment patterns using a new laser microtopography technique[END_REF]. A laser telemeter (Sick OD80) was attached to the lower Y table. The raw data (in volts) generated by the telemeter were converted in linear distances using an appropriate calibration (Maire et al., 2007b). The whole system was used for assessing the microtopography of 5 cm side square surfaces centered on tube openings and including faecal mound areas. Each scan was achieved within a 20 min time period and with a 15 µm vertical resolution. Surface sediment reworking rates were assessed by summing the differences in microtopography between two consecutive scans of the faecal mound area divided by the time duration between the beginnings of these two scans. Positive differences corresponded to an elevation, whereas negative ones corresponded to a digging of the sediment-water interface. Overall, nine couples (1-3 for each worm) of consecutive (time interval between 15 and 74.5 h) scans were achieved during this experiment.

Data processing 2.4.1. Time allocation patterns.

Time periods allocated to the different types of behaviors were assessed during the first and the third series of experiments. A Kolmogorov-Smirnov test was performed to check for possible differences in time allocation patterns between these two series.

Main sediment characteristics.

Changes in main sediment characteristics within undisturbed sediment, faecal mound and prospected areas were described using a non-metric Multi Dimensional Scaling (nMDS; [START_REF] Clarke | Change in marine communities: An approach to statistical analysis and interpretation[END_REF] based on untransformed data (i.e., median grain size, POC, PON, chlorophyll a and phaeophytin a) and using Euclidean distance. Mud content was not used for this analysis because of its strong redundancy with D50. The sediment characteristics mainly responsible for the difference between areas were identified using the SIMiliarity PERcentage analysis (SIMPER) procedure [START_REF] Clarke | Change in marine communities: An approach to statistical analysis and interpretation[END_REF]. Differences in the values of each characteristic between areas were looked for using univariate Friedman ANOVAs for paired samples.

Aerobic bacterial community composition.

Aerobic bacterial community composition was described using nMDS [START_REF] Clarke | Change in marine communities: An approach to statistical analysis and interpretation[END_REF] based on untransformed data and Bray-Curtis dissimilarities. Significant differences between community composition were looked for using a multivariate One-Way PERMANOVA [START_REF] Anderson | A new method for non-parametric multivariate analysis of variance[END_REF][START_REF] Mcardle | Fitting multivariate models to community data: a comment on distance-based redundancy analysis[END_REF]) with the three above-mentioned "sediment areas" as fixed factor.

Bacterial diversity was assessed using the complementary of the Simpson index (1-D) and associated equitability (E 1/D ) with:

D = ∑ 2 E 1/D = /
where p i is the relative abundance of OTU i and S is OTU richness Differences in the values of D and E 1/D between areas were looked for using univariate Friedman ANOVAs for paired samples.

Oxygen

Significant differences between oxygen penetration depths between the three considered areas were looked for using a Friedman ANOVA for paired samples.

All statistics, except for Friedman ANOVAs (Excel®), were performed using the PRIMER® 6 package [START_REF] Clarke | Change in marine communities: An approach to statistical analysis and interpretation[END_REF] and its PERMANOVA add on [START_REF] Anderson | A new method for non-parametric multivariate analysis of variance[END_REF].

RESULTS

Anatomy of feeding organs and feeding ethology

Examined worms bore from 11 to 15 tentacles (12.6 ± 1.3 mean ± sd). Tentacles were implanted side by side around the upper lip of the mouth (Fig. 3A andB). On the inner side of each tentacle, a groove was densely covered with cilia (Fig. 3C andD). Tentacle outer side consisted in an accordion shaped structure with only a few dispersed clumps of cilia (Fig. 3E). A particular structure without cilia was present at the tip of each tentacle (Fig. 3F).

The position and the elevation of the anterior part of the tube varied between worms. On some occasions, the anterior part of the tube was vertical and only slightly emerging from the sediment surface. On some others, the anterior part of the tube was lying horizontal at the sediment surface.

None of the observed worms left their tube during our experiments. Moreover, worms did not always stretch their anterior part out of the tube while feeding (Fig. 4A andB) but they occasionally did (Fig 4C). While feeding tentacle tips prospected the sediment surface. For worms 2.0 ± 0.2 cm in length, everted tentacles stretched up to 3.9 ± 0.2 cm over the sediment surface and active tentacles could be almost four times longer than resting ones. We did not notice any evidence of particle-size selection on tentacles. Conversely, we observed that worms were able to detach mineral fragments of several millimeters in size from the sediment and to transport them along the ciliary grooves of their tentacles to their mouth. We only occasionally observed active tentacle retractions toward tube openings. We also observed the presence of an area where large mineral particles were deposited near tube openings (see the tip of the red arrow on Fig. 2B). In spite of numerous attempts using thin aquaria and/or transparent substrates, we never observed the production of faeces within the sediment column (CM personal observation). Faeces were violently expulsed from the tube and catapulted on the sediment surface ca 1 cm away from tube openings (Fig. 4D-F). This defecation mode resulted in the creation of ca 1 cm high faecal mounds where we never observed prospection by tentacles while worms were feeding.

Typology of behaviors and time allocation pattern

Seven types of behaviors were identified: (1) Prospection when tentacles explored the sediment surface to collect particles and gills are visible outside the tube; (2) Gill movements when gill waving could be seen outside the tube opening and when no tentacle is stretched; (3) Head movements when head could be seen quickly entering or exiting the tube; (4) Maintenance when worms agglomerated particles at the opening of the tube using their tentacles; ( 5) Immobility when gills were visible outside the tube but remained immobile; (6) Hidden in the tube when worms were not visible at all at the sediment surface; and (7) Faeces egestion which occurred concomitantly with the six abovementioned behaviors.

Time allocation patterns between behaviors did not significantly differ between the first and the third series of experiments (Kolmogorov-Smirnov test, p > 0.05) allowing for the pooling of the results of these two series. Overall, monitored individuals were active (i.e., all behavior types but Immobility and Hidden in the tube) 89.5 ± 2.4 % of the time (Fig. 5). Otherwise, they were either immobile (7.0 ± 8.9 % of the time) or hidden in their tube (3.5 ± 3.6 % of the time). During activity phases, Prospection was by far the most dominant behavior (84.6 ± 10.1 % of the time); followed by

Gill movements (3.3 ± 3.8 % of the time); Maintenance (1.2 ± 1.2 % of the time); and Head movements (0.4 ± 0.8 % of the time). Faeces egestion occurred on average every 45.4 ± 3.9 min.

Surface sediment reworking

During the first series of experiments, average total surface sediment reworked area increased from 37 to 49 cm² between the beginning and the end of the "experiment periods" (Friedman ANOVA for paired samples, p<0.01). During the same time, average faecal mound surface increased from 9 to 22 cm² (Friedman ANOVA for paired samples, p<0.01). Conversely, the average surface of the prospected area remained stable at 28 cm² (Friedman ANOVA for paired samples p > 0.10). Changes recorded for individual worms globally showed the same pattern with one exception characterized by a transitory decline in total and prospected areas (Fig. 6).

An example of a microtopography scan taken during the third series of experiments is shown in Fig. 7. One can distinguish the faecal mound, which reaches 11 mm in height and a prospected area, which extends down to 4 mm in depth relative to the undisturbed area. The average surface sediment reworking rate (+4.9 10 -6 ± 1.3 10 -5 mm 3 h -1 ) and the mean change in the height of the undisturbed area (3.3 10 -7 mm) were negligible. Average surface sediment reworking rates were -4.6 ± 7.1 mm 3 h -1 and 6.8 ± 5.1 mm 3 h -1 in the prospected and the faecal mound areas, respectively. In the prospected area, surface sediment reworking induced a sediment excavation of 1.9 ± 0.2 mm in average. Conversely, it resulted in a mean elevation of the faecal mound area of 10.5 ± 1.9 mm. Considering a simple surface sediment transport between the prospected and the faecal mound areas, and the fact that the whole prospected areas may not have been covered in totality by our scans, the average surface sediment reworking rate of M. palmata was estimated to be 6.8 mm 3 .h -1 .

Sediment characteristics and oxygen penetration depths

Overall, the granulometrical and main biochemical characteristics of surface sediments significantly differed between the undisturbed sediment and the prospected and faecal mound areas (multivariate One-Way PERMANOVA, p < 0.05; Fig. 8). The SIMPER analysis showed that POC accounted for 59.2 % of the dissimilarity between faecal mound and prospected areas, to 62.8 % of the dissimilarity between faecal mound areas and undisturbed sediments, and to 64.3 % of the dissimilarity between prospected areas and undisturbed sediments. However, all characteristics significantly differed between areas (Friedman ANOVA for paired samples, p < 0.01; Table 1).

Undisturbed sediments showed the finest mean D50 (13.8 ± 2.0 µm), the second highest mud content (83.8 ± 4.8 %), the highest mean POC and PON contents (33.5 ± 4.0 mg g -1 DW and 3.2 ± 0.8 mg g - 1 DW, respectively) and the highest mean pigments contents (1.4 ± 0.5 µg g -1 DW in chlorophyll a and 6.8 ± 2.3 mg g -1 DW in phaeophytin a). Conversely, prospected areas were characterized by the coarsest sediment particles (D50 = 17.1 ± 2.2 µm, mud content = 76.6 ± 3.9 %) the lowest mean POC and PON contents (27.0 ± 5.4 mg g -1 DW and 2.2 ± 0.7 mg g -1 DW, respectively) and the lowest mean pigment contents (1.2 ± 0.8 µg g -1 DW of chlorophyll a (not significantly different from the faecal mound area) and 4.0 ± 2.3 µg g -1 DW of phaeophytin a, respectively). Faecal mound areas showed intermediate D50 (14.5 ± 2.3 µm), highest mud content (84.3 ± 4.6 %), mean POC and PON contents (30.0 ± 3.6 mg g -1 DW and 2.7 ± 0.6 mg g -1 DW, respectively), and mean pigment contents (1.0 ± 0.4 µg g -1 DW of chlorophyll a and 6.7 ± 1.4 µg g -1 DW of phaeophytin a). Overall, there was a clear opposition between D50 on one-side and POC and PON contents on the other side. This opposition segregated most the three considered spatial areas. Changes in pigment contents were largely independent and did not clearly differentiate these areas (Fig. 8).

Oxygen penetration depths significantly differed between areas (Friedman ANOVA for paired samples, p<0.01). Mean oxygen penetration depths were shallower in prospected areas (4.0 ± 0.1 mm) than in undisturbed sediments (4.9 ± 0.2 mm) and in faecal mound areas (7.2 ± 0.4 mm).

Aerobic bacterial community composition

Overall, 296 OTUs were found, ranging from 200 to 1166 bp. In undisturbed sediments, we significantly differed between the three areas (multivariate One-Way PERMANOVA, p < 0.05 -Fig. 9). Within-group average Bray-Curtis similarity was 61.2 % in undisturbed sediments, 66.2 % in faecal mound and 66.0 % in prospected areas. These values were clearly higher than between-groups average similarities (i.e., 48.9 % between undisturbed sediments and faecal mound areas, 48.3 % between undisturbed sediments and prospected areas, and 52.9 % between faecal mounds and prospected areas).

Discussion

Anatomy of feeding organs and feeding ethology

The observations achieved during the present study allow refining the current knowledge regarding the anatomy of the feeding organs and the feeding ethology in ampharetid polychaetes. Our morphological observations confirm that the upper lip of Melinna palmata, which bears the tentacles is eversible as already described in Melinna pacifica [START_REF] Zhadan | Comparative morphology of the feeding apparatus in the Terebellida (Annelida: Polychaeta)[END_REF]. Conversely to what had been previously observed for the terebellid Eupolymnia nebulosa [START_REF] Grémare | Feeding, Tube-building and particle-size selection in the terebellid Eupolymnia nebulosa[END_REF], they showed:

(1) the accordion structure of the tentacle, and (2) the existence of a particular structure near the tip of each tentacle that could possibly play the role of an adhesive cup. [START_REF] Bacescu | Substratum-Animals[END_REF] pictured the positioning of M. palmata while feeding. He described a tube, with a posterior part vertically penetrating within the sediment column and an anterior part largely emerging from the sediment surface, which is fastened obliquely by the worm when feeding at the sedimentwater interface. Jumars et al. ( 2015) described a more general pattern for tube positioning in ampharetids with the posterior part of the tube curving down in the sediment and the anterior part usually lying parallel to the sediment surface allowing for a horizontal posture of the worm with its ventral side down. According to [START_REF] Bacescu | Substratum-Animals[END_REF], while feeding, M. palmata "stretches out of the tube spreading the tentaculate palate over the substratum". Our own observations clearly show that there is not a unique pattern of tube positioning. Most often, tube openings remained vertical only slightly emerging from the sediment surface with worms positioned head up. On some occasions, however, we observed anterior parts of the tube lying on the sediment surface. Interestingly, [START_REF] Buchanan | The bottom fauna communities and their sediment relationship off the coast of Northumberland[END_REF] already reported vertical positioning of tube openings in dense populations of Melinna cristata and [START_REF] Fauchald | The diet of worms: a study of polychaete feeding guilds[END_REF] related this positioning with the scarcity of available food. The lack of a unique general pattern is true as well for the extension of worm outside of their tubes while feeding since most of the time, only a few tentacles and the very extremities of the gills were visible at the sediment surface. When everted, tentacles stretched up to ca 4 cm (for 2 cm long worms), which is rather limited as is the case in ampharetids compared to terebellids [START_REF] Warwick | Effects of the feeding activity of the polychaete Streblosoma bairdi (Malmgren) on meiofaunal abundance and community structure[END_REF][START_REF] Grémare | Feeding, Tube-building and particle-size selection in the terebellid Eupolymnia nebulosa[END_REF][START_REF] Jumars | Diet of worms emended: an update of Polychaete feeding guilds[END_REF]. While extended, tentacle tips prospected the sediment surface to collect particles, which fully supports that ampharetids use their retractable and ciliated tentacles to pick up food particles on the sediment surface [START_REF] Fauchald | The diet of worms: a study of polychaete feeding guilds[END_REF]. Overall, and besides differences in their number and extension, the functioning of the tentacles appeared very similar to the one described for Eupolymnia nebulosa [START_REF] Grémare | Feeding, Tube-building and particle-size selection in the terebellid Eupolymnia nebulosa[END_REF]Maire et al., 2007a). However, an important difference is that on some occasions we observed a combination of ciliary entrainments along and muscular contractions of the tentacles to convey particles to the mouth as already reported for Hobsonia florida [START_REF] Taghon | Optimal foraging by deposit-feeding invertebrates: roles of particle size and organic coating[END_REF]. Conversely to this author, we were, however, not able to observe the retraction of mucous coated tentacles into the mouth. During our experiments, worms only produced faeces at the water sediment interface, which has already been observed for several other ampharetids (e.g. [START_REF] Nowell | The foraging strategy of a subtidal and deep-sea deposit feeder[END_REF], including M. palmata [START_REF] Olafsson | The impact of Melinna palmata Grube, a tube-building polychaete, on meiofaunal community structure in a soft-bottom subtidal habitat[END_REF]. Faeces were violently expulsed from tubes, which resulted in the deposition of fresh faeces ca one centimeter away from tube openings. [START_REF] Nowell | The foraging strategy of a subtidal and deep-sea deposit feeder[END_REF] observed a similar pattern in the deep-sea ampharetid Amphicteis scaphobranchiata. These authors described the expulsion mechanism, which includes gills, mucous and body binding, and takes much less than 10 s (i.e., the time lag between the acquisitions of two consecutive images during our own experiments). We were not able to depict this process for M. palmata based on the experiments described in the present paper. However, other video recordings show that worms bind their body prior egestion so that their pygidia are located close to tube openings and directly expelled faeces (CM personal observation). According to [START_REF] Nowell | The foraging strategy of a subtidal and deep-sea deposit feeder[END_REF], faeces expulsion constitutes an adaptation to maintain a feeding pit, which enhances new particle deposition within feeding area of individual A. scaphobranchiata living in the deep sea (see also [START_REF] Taghon | Optimal foraging by deposit-feeding invertebrates: roles of particle size and organic coating[END_REF]. This hypothesis is not necessarily appropriate for shallower ampharetids due to stronger hydrodynamism. Moreover, in M. Palmata faecal pellets were expulsed within the ca 4 cm radius of prospected areas even though worms never actively transported particles from their faecal mound area (see also below).

Evidence for and quantification of surface sediment reworking

Queiros et al. ( 2015) stated that M. palmata "is a tube-dweller exhibiting conveyor-belt transport of particulates". During our own experiments, we observed that faeces egestion only occurred at the sediment-water interface. Together with the collection of surface particles by the tentacles, and the lack of motility in ampharetids [START_REF] Jumars | Diet of worms emended: an update of Polychaete feeding guilds[END_REF]; confirmed in the case of M. palmata by our own observations), this is likely to result in mostly horizontal particle displacements at the sediment-water interface and thus in surface sediment reworking.

To our knowledge, vertical sediment reworking by M. palmata has only been experimentally quantified once through ex situ luminophore experiments and associated image analysis techniques [START_REF] Queiros | Can benthic community structure be used to predict process of bioturbation in real ecosystems?[END_REF]. These authors reported vertical biodiffusion coefficients (Dbs) less than 1cm 2 .y - 1 with maximal penetration depth typically less than 2cm. Among the 4 species studied by [START_REF] Queiros | Can benthic community structure be used to predict process of bioturbation in real ecosystems?[END_REF], M. palmata was clearly the one with the lowest Dbs. Moreover, M. palmata was also the only species not featuring any marked seasonal changes in Dbs, thereby suggesting that its vertical sediment reworking intensity is not cued by seasonally changing parameters but rather limited in itself.

Although caution should be taken when comparing Dbs derived from different studies since their assessments can be greatly affected both by technological (e.g. incubation duration, vertical resolution…) and environmental parameters (e.g. temperature, food availability, animal density…), this interpretation is further supported by the analysis of literature data regarding Dbs derived from luminophore experiments (see for example Maire et al., 2007c). The Dbs found for M. palmata [START_REF] Queiros | Can benthic community structure be used to predict process of bioturbation in real ecosystems?[END_REF] are clearly among the lowest ever reported. They are for example much lower than those reported for the deposit-feeding bivalve Abra ovata during summertime (i.e., up to ca 31 cm 2 .y -1 in the absence of food addition) and equivalent to those reported during wintertime when this bivalve is considered almost totally inactive due to low temperature (Maire et al., 2007c; Fig. 7). [START_REF] Bernard | A comparative study of sediment particles mixing in a Zostera noltei meadow and a bare sediment mudflat[END_REF] carried out a series of in situ luminophore experiments in both seagrass beds and adjacent bare sediments within the Arcachon Bay where M. palmata is present. They reported a significant negative correlation between M. palmata abundances and Dbs and attributed this effect to the stabilization of the sediment induced by dense populations of this species [START_REF] Brenchley | Mechanisms of spatial competition in marine soft-bottom communities[END_REF]. Here again, this supports weak vertical sediment reworking by M. palmata. Moreover, while running out these experiments, Bernard (personal communication) observed the penetration of luminophores 2 to 4 cm deep in the sediment immediately following their introduction at the sediment surface. This was attributed to passive transfers within the tubes of M. palmata. Similar transfers also likely occurred within siphonal galleries during the A. ovata experiments mentioned above and probably partly accounted for the decrease in Dbs with incubation duration reported by Maire et al. (2007c) during their wintertime experiments. Anyhow, this type of potential artefactual luminophore penetration may clearly contribute to an overestimation of (already weak) Dbs in M. palmata. Based on all this set of rationale and our own observations, our conclusion is that M. palmata does not belong to any existing functional group of sediment reworking. Indeed, the unique characteristics of the particle mixing of M.

palmata, conveying particles only on the sediment surface, led us to propose a new functional group of sediment reworking: the surface conveyors. Further studies would validate this hypothesis.

Such a sediment reworking mode results in the zonation of the surface sediment in three distinct areas: (1) faecal mounds resulting from faeces accumulation, (2) prospected areas that may be conversely depressed relative to the general sediment surface [START_REF] Nowell | The foraging strategy of a subtidal and deep-sea deposit feeder[END_REF], and (3) undisturbed sediments. Such a zonation has already been observed for several surface tentaculate deposit-feeders (eg [START_REF] Nowell | The foraging strategy of a subtidal and deep-sea deposit feeder[END_REF][START_REF] Warwick | Effects of the feeding activity of the polychaete Streblosoma bairdi (Malmgren) on meiofaunal abundance and community structure[END_REF] including M. palmata [START_REF] Olafsson | The impact of Melinna palmata Grube, a tube-building polychaete, on meiofaunal community structure in a soft-bottom subtidal habitat[END_REF]. During the present study, we observed a radius of ca 4 cm (for 2 cm long worms) for prospected areas, which is much higher than the 2 cm reported by [START_REF] Gibbs | Copper accumulation by the polychaete Melinna palmata: an antipredation mechanism?[END_REF] for 25-35 mm long worms. Conversely, the distances between tube openings and the areas of faeces deposition were smaller during our experiments (i.e., typically close to 1cm) than the 4-5 cm reported by Olafsson et al. (1990, Fig. 1) based on in situ observations of a Scottish M. palmata population. To our knowledge, the only assessment of surface sediment excavation by an ampharetid is qualitative (i.e., several mm) for Amphicteis scaphobranchiata [START_REF] Taghon | Optimal foraging by deposit-feeding invertebrates: roles of particle size and organic coating[END_REF], which is fully compatible with the 1.9 mm reported during the present study for time-limited experiments.

The use of laser telemetry to infer surface sediment reworking rates has only been introduced recently (Maire et al., 2007b;[START_REF] Duchêne | Hydroid and serpulid recruitment patterns using a new laser microtopography technique[END_REF], and to our knowledge the only comparable surface sediment reworking rates to ours are therefore those measured by Maire et al. (2007b) in the deposit-feeding bivalve Abra ovata. These authors reported major short-term temporal changes in surface sediment reworking rates in relation with the intensity of inhalant siphon activity. Nevertheless, average values over 27 h time periods were equal or superior to 50 mm 3 .h -1 , which is about 8 times the average value (i.e., 6.8 mm 3 .h -1 ) found during the present study. Irrespective of differences in experimental conditions that we did not control for, this suggests that the aspiration by sediment siphons is a more efficient mechanism of surface deposit feeding than the use of tentacles. This is supported by the comparison of the average proportions of time allocated to feeding in:

(1) M. palmata (84.6%, present study), ( 2) another surface tentaculate feeder the terebellidae Eupolymnia nebulosa (74.2 %, Maire et al., 2007a), (3) Abra ovata (40.5 %, [START_REF] Grémare | Feeding behavior and functional response of Abra ovata and A. nitida compared by image analysis[END_REF], and (4) Abra nitida (54.6 %, [START_REF] Grémare | Feeding behavior and functional response of Abra ovata and A. nitida compared by image analysis[END_REF]. [START_REF] Duchêne | Marine benthic faunal activity patterns on a sediment surface assessed by video numerical tracking[END_REF] quantified the activity of Melinna cristata at the surface of a large sediment core incubated ex situ. They also concluded to the investment of a large proportion of time in (feeding) activity at the sediment-water interface by this species. All these results/observations suggest that the meeting of nutritional requirements required less time allocation in siphon than in tentaculate surface deposit-feeders although additional studies are clearly required to further tackle this point.

Effect on surface sediment granulometry biochemical characteristics and oxygen penetration depth

Sediment reworking by Melinna palmata induced an increase in the median size diameter (D50) of surface sediment in prospected areas. It is not fully clear, however, whether this resulted from a selection for finer particles or from the simple depletion of finer surface particles and then the ingestion of coarser particles initially located slightly deeper in the sediment column. Positive selection for finer particles by deposit-feeders has classically been put in relation with the optimal foraging strategy [START_REF] Taghon | Predicting particle selection by deposit feeders: a model and its implications[END_REF] and the fact that these particles are the one featuring the highest organic matter content due to their higher surface/volume ratio [START_REF] Mayer | Organic matter in small mesopores in sediments and soils[END_REF]. Although, this paradigm is currently under debate for deposit-feeders as a whole (see [START_REF] Jumars | Diet of worms emended: an update of Polychaete feeding guilds[END_REF] for a review), it is still generally accepted that surface tentaculate deposit feeders do indeed tend to preferentially feed on particles smaller than the available median grain size [START_REF] Jumars | Diet of worms emended: an update of Polychaete feeding guilds[END_REF].

Moreover, it has been suggested that in this feeding guild, particle size selection is mostly mechanical and results from three opposite processes taking place at the tips and along the tentacles [START_REF] Jumars | Mechanics of particle selection by tentaculate deposit feeders[END_REF], namely: (1) particle encounter by the tentacles, which would lead to a positive selection toward larger particles according to the De Lesse's principle;

(2) detachment of particles from the sediment, which would result in a selection toward smaller particles because of their higher surface/volume ratio, which better counteracts gravity forces; and (3) differential loss during the transfer of particles within the ciliary groove to the mouth, which here again favors the selection of finer particles due to their higher surface/volume ratio [START_REF] Jumars | Mechanics of particle selection by tentaculate deposit feeders[END_REF] and to their quicker speed of displacement along the tentacles (Maire et al., 2007a). This theoretical mechanical model of particle selection has been tested on the terebellid Eupolymnia nebulosa [START_REF] Grémare | Feeding, Tube-building and particle-size selection in the terebellid Eupolymnia nebulosa[END_REF]Maire et al., 2007a). Based on: (1) direct observations of natural particles transiting along the tentacles, and (2) comparison of particle sizeselection during feeding (with a positive selection for smaller particles) and tube building (with a positive selection for larger particles), these authors concluded that tentacles are likely not the only organs involved in particle-size selection and that another selection process probably occurs at the level of the mouth as already observed in the spionid Streblospio benedicti [START_REF] Kihslinger | Food patches and a surface deposit feeding spionid polychaete[END_REF]. During our own experiments, we did not notice any direct evidence of particle-size selection on the tentacles, which proved able to transport particles several millimeters in size. Conversely, (1) the presence of a structure that may potentially constitute an adhesive cup near the tip of each tentacle may contribute to reduce the mechanical positive selection for finer particles during their detachment from the sediment surface, (2) the limited maximal extension of the tentacles may also contribute to limit the preferential loss of larger particles during their transit along the tentacles so as occasional retraction of tentacles toward the mouth. Moreover, the occurrence of intermediate (i.e., between the unaffected sediment and the prospected area) D50 in faecal mound areas suggests that the size of the particles ingested by M. palmata may more rely on their availability at the sediment surface than on their size. Conversely, the observation of the deposition of large mineral particles at the immediate vicinity of tube openings supports the occurrence of a selection process after the transit of particles along the tentacles. Indeed such accumulation probably does not result from the preferential egestion of large inorganic particles [START_REF] Jumars | Diet of worms emended: an update of Polychaete feeding guilds[END_REF] since, during our experiments, faecal pellets were catapulted away from tube openings (see above).

During the present study, we assessed two bulk characteristics of sedimentary organics, namely POC and PON. Both parameters correlated negatively with D50, which at least partly reflects the general negative relationship linking sediment grain size and associated organic matter concentrations [START_REF] Mayer | Organic matter in small mesopores in sediments and soils[END_REF]; see above). Chlorophyll a and phaeophytin a are derived from (fresh or degraded, respectively) plant materials and are therefore not representative of bulk sedimentary organics. During the present study, changes in their concentrations were largely independent of those of D50, POC and PON, which may reflect the fact that these concentrations result largely from the presence of individual particles (e.g. diatoms and or plant-derived detritus) rather than from the coating of organic matter to the surface of sediment grains. We identified POC as the parameter contributing most to differences between undisturbed sediments, prospected and faecal mound areas. Chlorophyll a and phaeophytin a concentrations contributed much less to these differences. Our conclusion is thus that the main effect of M. palmata on the biochemical characteristics of surface sediment is quantitative and probably mainly result from changes in sediment granulometry.

During the present study, mean oxygen penetration depths were between 4.0 (prospected areas) and 7.2 mm (faecal mound areas). These values are slightly higher than those (i.e., typically between 2 and 4 mm) measured both ex and in situ at the Germanan site [START_REF] Delgard | Etude des effets cet du rôle des herbiers à Zostera noltii sur la biogéochimie des sédiments intertidaux[END_REF][START_REF] Rigaud | Transient biogeochemistry in intertidal sediments: New insights from tidal pools in Zostera noltei meadows of Arcachon Bay (France)[END_REF].

Besides biological activity, the two main factors affecting oxygen penetration within the sediment column are: (1) sediment granulometry, which largely controls sediment porosity and sediment organic content, and (2) organic mineralization processes, which largely control oxygen consumption in the sediment column. During the present study, deeper oxygen penetration were recorded in faecal mound areas despite the fact that they presented intermediate D50 and bulk (i.e.,POC and PON) organic content. This may partly result from the fact that faecal mound areas are the ones showing the lowest concentrations of chlorophyll a, which is representative of a highly labile component of sedimented POM. Moreover, faecal mound areas are indeed basically constituted by large particle aggregates (i.e., faecal pellets at different stages of dislocation; see Fig. 2 and4), which are destroyed during microgranulometrical analyses. Sediment microgranulometry measurements therefore probably not constitute a sound proxy of sediment porosity in this particular case. Unfortunately, sediment porosity was not directly assessed during the present study. A clear possibility is thus that sediment porisity is especially high in faecal mound areas, which would also contribute to enhance oxygen penetration within the sediment column.

Potential intraspecific interactions and food limitation in field populations

In the English Channel and the Celtic sea, the density of subtidal Melinna palmata populations is typically only several hundred of individuals per meter square or less [START_REF] Gage | Community structure of the benthos in Scottish sea-lochs. I. Introduction and species diversity[END_REF][START_REF] Oyenekan | Population dynamics and secondary production in Melinna palmata (Polychaeta: Ampharetidae)[END_REF][START_REF] Olafsson | The impact of Melinna palmata Grube, a tube-building polychaete, on meiofaunal community structure in a soft-bottom subtidal habitat[END_REF][START_REF] Grehan | Demography and reproductive biology of Melinna palmata (Ampharetidae: Polychaeta) in inner Galway Bay on the west coast of Ireland[END_REF][START_REF] Dauvin | The presence of Melinna palmata (Annelida: Polychaeta) and Ensis directus (Mollusca: Bivalvia) related to sedimentary changes in the Bay of Seine (English Channel, France)[END_REF]. To our knowledge, the highest recorded density was 1613 ind.m -2 in the Bay of Morlaix [START_REF] Ibanez | Long-term changes (1977 to 1987) in a muddy fine sand Abra alba -Melinna palmata community from the Western English Channel: multivariate time series analysis[END_REF]. As for the Bay of Biscay, the highest recorded density, except for the Arcachon Bay, is apparently 1000 ind.m -2 [START_REF] Cacabelos | Ecological features of Terebellida fauna (Annelida, Polychaeta) from Ensenada de San Simon (NW Spain)[END_REF]. Several studies suggest that M. palmata populations positively respond to an enrichment in fine particles and/or organic matter. Some populations have apparently benefited from the vicinity of a local sewage output in the Bay of Brest (Guillou and Hily, 1983) and from an enrichment of the Bay of Seine in fine particles [START_REF] Dauvin | The presence of Melinna palmata (Annelida: Polychaeta) and Ensis directus (Mollusca: Bivalvia) related to sedimentary changes in the Bay of Seine (English Channel, France)[END_REF]. At a larger scale, the spread and the increase of M.

palmata during the 1970s in Romanian waters was attributed to an increase in the frequency of phytoplanktonic blooms due to eutrophication [START_REF] Gomoiu | On the populations of Melinna palmata Grube at the Romanian littoral of the Black Sea[END_REF]cited by Grehan, 1991).

During the present study, worms were collected from a very dense (i.e., up to 6745 ind.m -2 during spring; [START_REF] Bernard | A comparative study of sediment particles mixing in a Zostera noltei meadow and a bare sediment mudflat[END_REF] intertidal population associated with a Zotera noltei meadow.

These authors also reported high (i.e., up to 2000 ind.m -2 during spring) densities in adjacent bare sediments. To our knowledge, these densities are the highest ever reported for M. palmata. They respectively correspond ca 1.5 and 5.0 cm 2 surfaces available per individual worm, respectively. These values are much smaller than those of individual prospected areas measured during the present study (i.e., mean value of 28 cm 2 ), which suggests the occurrence of intraspecific interactions in the populations of the Arcachon Bay. [START_REF] Massé | Effets de la bioturbation sur la diversité des communautés bactériennes du sédiment. Approches expérimentales et in-situ. De Melinna palmata aux communautés de la vasière Ouest-Gironde[END_REF] assessed the effects of worm density on the feeding ethology and time allocation patterns of M. palmata using the same methodologies as in the present study. She reported that at high (i.e., >1184 ind.m -2 ) densities, worms did not only forage in prospected areas but also in neighboring faecal mound areas. Together with the occurrence of lower bulk sedimentary organics in prospected areas (see above), this change in feeding behavior tends to suggest that food limitation may occur in field populations. Ampharetids are classified as discretely motile by [START_REF] Jumars | Diet of worms emended: an update of Polychaete feeding guilds[END_REF]. However, the main mechanism involved in motility is tube elongation, which apparently cannot take place in dense populations when tube anterior parts are vertical [START_REF] Fauchald | The diet of worms: a study of polychaete feeding guilds[END_REF] as was most often the case during the present study and in the Germanan Z. noltei meadow (Bernard, personal observations). In such cases, taking into account sediment transport to feeding (i.e., prospected) areas is essential when elaborating foraging theories [START_REF] Nowell | The foraging strategy of a subtidal and deep-sea deposit feeder[END_REF]. In this context, it is worthwhile to notice that the two populations of the Arcachon Bay are both intertidal, and that tidal currents probably contribute to sedimentary movements between undisturbed sediments, prospected and faecal mounds areas thereby allowing for the continuous renewal of the food resources available for M. palmata. Along the same line, the difference in densities between the Z. noltei and the bare sediment populations may result from the enhancement of particle trapping by seagrass meadows [START_REF] Gacia | An approach to measurement of particle flux and sediment retention within seagrass (Posidonia oceanica) meadows[END_REF][START_REF] Gacia | Sediment retention by a Mediterranean Posidonia oceanica meadow: the balance between deposition and resuspension[END_REF][START_REF] Hendricks | Experimental assessment and modeling evaluation of the effects of the seagrass Posidonia oceanica on flow and particle trapping[END_REF].

Effect on aerobic bacterial community composition

Many studies have assessed the effect of macrobenthic bioturbators on sediment bacteria (see review in Table 2). These studies have concerned a large variety of macrobenthic species belonging to different phylla. They have been carried out either ex or in situ and for some of them have involved experimental incubations. They also differ by the parameters used to assess bacterial responses. Most studies have dealt with abundance, biomass or viability assessments. As far as ampharetids are concerned, the only available study is the one by Aller and Aller (1986) on Amphicteis sp. It has been achieved in situ by sampling surface sediments around a single worm at a 4827m depth and has shown an increase in surface sediment bacterial abundances from the prospected area to the tube insertion at the sediment-water interface.

Studies assessing bacterial community composition have been mostly carried out in situ and have involved samplings at different depths within the sediment column. Observed differences in bacterial community composition thus potentially resulted from changes in oxygen and other electron acceptors availabilities. Only several studies have tackled the effect of macrobenthic bioturbators on bacterial community composition in surface oxygenated sediments. Most of them were however based on the comparison of bacterial community composition in field stations [START_REF] Bertics | Biodiversity of benthic microbial communities in bioturbated coastal sediments is controlled by geochemical microniches[END_REF] or experimental enclosures [START_REF] Laverock | Bioturbating shrimp alter the structure and diversity of bacterial communities in coastal marine sediments[END_REF] with different bioturbator densities but not from the direct sampling of reworked and non-reworked surface sediments. [START_REF] Wilde | Spatial heterogeneity of bacterial assemblages in marine sediments: the influence of deposit feeding by Balaglonossus aurantiacus[END_REF] directly compared bacterial community composition in the faecal mounds of Balaglonossus aurantiacus and ambient surface sediments. Based on functional parameters, they reported qualitative differences that were transitory since microbial assemblage composition of degrading faeces rapidly converged with those of ambient surface sediments. These patterns were interpreted as resulting from: (1) the differential digestion of ingested bacteria, and (2) the stimulation/injection of non culturable bacteria during gut passage.

Our own results also show that aerobic bacterial community composition differ in undisturbed sediments, faecal mound and prospected areas by Melinna palmata. In this sense, they are in good agreement with previous works based on spatial assessments of biogeochemical processes [START_REF] Reichardt | Impact of bioturbation by Arenicola marina on microbiological parameters in intertidal sediments[END_REF]Bertics and Ziebis, 2010;Bertics et al., 2010[START_REF] Bertics | Denitrification and nitrogen fixation dynamics in the area surrounding an individual ghost shrimp (Neotrypaea californiensis) burrow system[END_REF]. As mentioned above, changes in sediment bacterial community composition are often related to changes in oxygenation. Sediment reworking is not the only activity of benthic macrofauna that can produce such changes. Hydraulic activities (i.e., the induction of water transports within biogenic structures and sediment interstices by benthic macrofauna) could as well be involved [START_REF] Woodin | Infaunal hydraulic ecosystem engineers: cast of characters and impacts[END_REF][START_REF] Woodin | Same pattern, different mechanism: locking onto the role of key species in seafloor ecosystem process[END_REF][START_REF] Volkenborn | Oscillatory porewater bioadvection in marine sediments induced by hydraulic activities of Arenicola marina[END_REF].

However, several rationale suggest that this is probably not the case in Melinna palmata. As stated above, the gills are most of the time out of the tube, and no ventilation behavior of the tube was observed during the present experiments. Second, dedicated experiments using fluoresceine [START_REF] Pascal | Influence of the mud shrimp Upogebia pusilla (Decapoda: Gebiidea) on solute and porewater exchanges in an intertidal seagrass (Zostera noltei) meadow of Arcachon Bay: an experimental assessment[END_REF] have shown that bioirrigation rates are negligible [START_REF] Massé | Effets de la bioturbation sur la diversité des communautés bactériennes du sédiment. Approches expérimentales et in-situ. De Melinna palmata aux communautés de la vasière Ouest-Gironde[END_REF]. Third, the transfers of water and inorganic solutes across the mucous lining of the tubes of Melinna cristata are clearly inhibited [START_REF] Hannides | Diffusion of organic and inorganic solutes through macrofaunal mucus secretions and tube linings in marine sediments[END_REF]. Overall, our interpretation is that surface sediment reworking is thus indeed responsible of the differences in aerobic bacterial community composition recorded during the present study.

During our experiments, aerobic bacterial community composition seemed to differ most between undisturbed sediments and prospected areas and were intermediate in faecal mound areas.

Diversity and equitability also tended to be lower in undisturbed sediments than in prospected and faecal mound areas. This last result is in contradiction with those of [START_REF] Wilde | Spatial heterogeneity of bacterial assemblages in marine sediments: the influence of deposit feeding by Balaglonossus aurantiacus[END_REF] who reported a lower diversity in faecal mounds than in ambient sediments. Discrepancies between the two studies may clearly result from differences in the methodological approaches used to assess bacterial community composition. As far as the present study is concerned, higher bacterial diversity in faecal mounds may result from: (1) the egestion of resident (i.e., enteric) bacteria [START_REF] Harris | The presence, nature, and role of gut microflora in aquatic invertebrates: a synthesis[END_REF], (2) quick changes in faeces bacterial community composition in faeces as suggested by [START_REF] Wilde | Spatial heterogeneity of bacterial assemblages in marine sediments: the influence of deposit feeding by Balaglonossus aurantiacus[END_REF], which would result in the presence of different bacterial communities in faecal mounds, and

(3) the possible ingestion (and then the further egestion) of bacteria associated with the surface sediment of prospected areas after excavation (see also the section of the discussion regarding the effect on sediment granulometry). The occurrence of higher diversity in prospected areas than in undisturbed sediments may result from the continuous disturbance experienced by the former due to feeding by M. palmata. The intermediate disturbance hypothesis (IDH; [START_REF] Grime | Competitive exclusion in Herbaceous vegetation[END_REF][START_REF] Connell | Diversity in tropical rain forests and coral reefs[END_REF] indeed predicts higher biodiversity in areas submitted to intermediate frequency of discrete disturbing events. In prospected areas, the periodic removal of bacteria attached to organic particles from the sediment surface might constitute such events. In the theoretical framework of IDH, the species richness peak results from co-occurrence of K-and r-strategists (Mac [START_REF] Arthur | The theory of Island biogeography[END_REF]. These concepts are still in debate for micro-organisms [START_REF] Sousa | Disturbance in marine intertidal boulder fields: the nonequilibrium maintenance of species diversity[END_REF], with special respect to general trends in bacterial carbon use in ready biodegradability tests (Vásquez-Rodriguez et al., 2007) or soil community [START_REF] Fierer | Toward an ecological classification of soil bacteria[END_REF]. For bacteria, K-strategist species might be slow growing, specialized, exoenzyme producing so-called oligotrophic bacteria while r-strategist species might be fast growing, generalist, opportunist so-called copiotrophic bacteria [START_REF] Fierer | Toward an ecological classification of soil bacteria[END_REF]. Accordingly, by removing part of the bacterial biomass, M. palmata would promote an optimal degradation of the sediment organic matter in the oxic layer. Further studies could take advantage in testing this hypothesis by addressing community level substrate utilization in prospected areas.

  recorded 209 OTUs ranging from 200 to 1032 bp, versus 218 ranging from 200 to 1032 bp in faecal mound areas and, 238 ranging from 200 to 1166 bp in prospected areas. Mean values of 1-Dsignificantly differed between areas (Friedman ANOVA for paired samples, p<0.01). They were lower in undisturbed sediments (0.909 ± 0.043) than in faecal mound (0.938 ± 0.012) and prospected (0.948 ± 0.012) areas. E 1/D also significantly differed between areas (Friedman ANOVA for paired samples, p<0.05) with lower values in undisturbed sediments (0.177 ± 0.049) than in prospected (0.220 ± 0.078) and faecal mound (0.234 ± 0.041) areas. Aerobic bacterial community composition

Fig. 1 :

 1 Figure legends: Fig. 1: General flowcharts of the three series of experiments carried out during the present study.

Fig. 2 :

 2 Fig. 2: First series of experiments. Top views of the sediment surface after reworking by Melinna palmata; A & B differ by the location of the faecal mound relative to the tube opening. FM: Faecal Mound area; PA: Prospected Area; TO: Tube Opening; US: Undisturbed Sediment. The red arrow shows large the accumulation of large mineral particles near the tube opening.

Fig. 3 :Fig. 4 :

 34 Fig. 3: Picture of the buccal cavity and the tentacles of Melinna palmata obtained by stereomicroscopy (A) and scanning electron microscopy (B -F). Gi: gills; T: tentacles; L: lip; Bc: buccal cavity; As: Accordion structure; Gr: ciliary groove; Tip: tip of the tentacle; Ci: cilia.

Fig. 5 :

 5 Fig. 5: First and third series of experiments. Time allocation pattern of Melinna palmata between its different behaviors. Vertical bars are standard deviations.

Fig. 6 :

 6 Fig. 6: First series of experiments. Temporal changes in the: (A) total surface of reworked sediment, (B) surface of the faecal mound area, and (C) surface of the prospected area by individual worms(see text for details). Each symbol corresponds to an individual worm.

Fig. 7 :

 7 Fig. 7: Third series of experiments. Example of a microtopography scan. FM: Faecal Mound, PA: Prospected Area.

Fig. 8 :

 8 Fig. 8: First series of experiments. nMDS plot based the granulometrical and biogeochemical characteristics of surface sediments (see text for details).

Fig. 9 :

 9 Fig. 9: First series of experiments. nMDS plot based on aerobic bacterial community composition (see text for details).

Fig. 1

 1 Fig. 1

Fig. 3

 3 Fig. 3

Fig. 4

 4 Fig. 4

Fig. 5

 5 Fig. 5

  Fig. 6

Table legends :

 legends Table1: Mean ± Standard deviation of several parameters measured in the three areas defined by the sediment reworking of Melinna palmata: median grain size (D50), mud content, chlorophyll and phaeophytin a, particulate organic carbon (POC) and nitrogen (PON) and oxygen penetration depth

Table 2 :

 2 Review of studies on effects of macrofaunal biotubation on bacterial abundance, biomass, activity or composition with the bioturbator used and the study type. Each number refers to a reference TABLE 2 1

		Study type		Effect on bacteria				Studied layers
	Bioturbators	Ex-situ (Experimental)	In-situ	Abundance Biomass Viability	Production	Activity	Composition	Depth (gradient, burrow vs ambient)	Surface (horizontal gradient)
	Polychaeta
	Alitta virens	14	12	12, 14			12	12, 14
	Amphicteis sp.		3	3				3	3
	Arenicola marina		4, 5, 7, 11	4, 5, 7, 11		4		4, 5, 7, 11	4, 11
	Branchyoasicus americana		6	6			6	6
	Capitella capitata	2		2	2			Tubes (2)
	Diopatra cuprea		9	9			9	Tubes (9)
	Hediste diversicolor	8, 10, 13	12, 15	8,10, 12		8, 10	12, 13, 15	8, 10, 12, 13, 15
	Heteromastus filiformis	1		1				1
	Notomastus lobatus		6	6			6	6
	Perinereis aibuhitensis		33	33			33	33
	Bivalvia
	Arctica atlantica	16		16				16
	Cerastoderma edule	8, 10	7	7, 8, 10		8, 10		7, 8, 10
	Macoma balthica	1, 14		1, 14				1, 14
	Meretrix meretrix		33	33			33	33
	Mya arenaria	14	17	14, 17		17		14, 17
	Tellina texana	1		1				1
	Crustacea
	Biffarius arenosus		26	26		26	26	26
	Callianassa kraussi
	Callianassa subterranea	19					19	19	19
	Callianassa trilobata		28	28			28	28
	Corophium volutator	8, 10		8, 10		8, 10		8, 10
	Neotrypaea californiensis	23, 24, 25	22, 24, 25	22		23, 24, 25	22, 24	22	22, 23, 24, 25
	Nihonotrypaea harmandi		32	32			32	32
	Pestarella tyrrhena		18	18			18	18
	Uca crenulata		22	22			22	22	22
	Upogebia deltaura	21	20	20, 21		21	19	20, 21	19
	Upogebia major	28	28	28		28		28
	Hemichordata
	Balaglonossus aurantiacus		6, 30	6, 30		30	6	6	30
	Ptychodera bahamensis		31	31			31		31

AKNOWLEGMENTS

Cécile Massé was supported by a doctoral fellowship from French Ministère de l'Enseignement Supérieur et de la Recherche. We thank Isabelle Svahn from the Bordeaux Imaging Center (BIC) -Electronic Microscopy Pole of the University of Bordeaux. We also thank Franck Salin, head of Plateforme Genome-Transcriptome Pierroton (INRA, Bordeaux, France) for ARISA analyses.