Gaudron Sm

SM Gaudron

Le Bris

Dr Sylvie Marylène Gaudron
email: sylvie.gaudron@snv.jussieu.fr

COLONIZATION OF ORGANIC SUBSTRATES DEPLOYED IN DEEP-SEA REDUCING HABITATS BY SYMBIOTIC SPECIES AND ASSOCIATED FAUNA

Keywords: symbiosis, settlement, colonization, larvae of invertebrates, Xylophaga spp., Idas sp., Thyasira sp., Sclerolinum contortum

In this study, our goal was to test whether typical vent/seep organisms harbouring symbionts or not, would be able to settle on organic substrates deployed in the vicinity of chemosynthetic ecosystems. Since 2006, a series of novel standardized colonization devices (CHEMECOLI: CHEMosynthetic Ecosystem COlonization by Larval Invertebrates) filled with three types of substrates (wood, alfalfa and carbonate) have been deployed in different types of reducing habitats including cold seeps in the eastern Mediterranean, a mud volcano in the Norwegian Sea, and hydrothermal vents on the Mid-Atlantic Ridge for durations of 2 weeks to 1 year. For all deployments, highest species diversities were recovered from CHEMECOLIs filled with organic substrates. Larvae from species associated with thiotrophic symbionts such as thyasirid, vesicomyid and mytilid bivalves, were recovered in the eastern Mediterranean and at the Mid-Atlantic Ridge. At the Haakon Mosby Mud Volcano, larvae of symbiotic siboglinids settled on both organic and carbonate substrates. Overall, novel colonization devices (CHEMECOLI) filled with organic substrates attracted both fauna relying on chemosynthesis-derived carbon as well as fauna relying on heterotrophy the latter being opportunistic and tolerant to sulphide.

INTRODUCTION

Deep-sea reducing ecosystems such as methane seeps and hydrothermal vents are sustained by chemosynthetic prokaryotes which use reduced compounds such as sulphide and methane, and produce organic matter from inorganic sources (Van Dover, 2000). These patchy and ephemeral marine habitats are colonized by symbiotic metazoans species (i.e. metazoans living in association with chemosynthetic bacteria) and associated fauna, depending primarily on metazoan dispersal capabilities during their early life stages (Tyler and Young, 2003), and secondly on food availability, settlement cues and predation/competition [START_REF] Micheli | Predation structures communities at deep-sea hydrothermal vents[END_REF]. Some taxa are shared among the different deep-sea reducing habitats (hydrothermal vents, cold seeps and organic falls) both among symbiotic groups (siboglinid tubeworms, vesicomyid clams, bathymodiolid mussel) as well as among non-symbiotic groups such as dorvilleid, polynoid and spionid polychaetes.

In situ colonization experiments have been carried out over two decades at hydrothermal vents, using polycarbonate plates, basalt rocks, sponges or titanium rings (Van Dover et al., 1988;[START_REF] Shank | Temporal and spatial patterns of biological community development at nascent deep-sea hydrothermal vents (9°50'N, East Pacific Rise)[END_REF][START_REF] Taylor | Rapid microbial production of filamentous sulfur mats at hydrothermal vents[END_REF][START_REF] Mullineaux | Recruitment variation during a pilot colonization study of hydrothermal vents (9°50'N, East Pacific Rise)[END_REF][START_REF] Mullineaux | Successional mechanism varies along a gradient in hydrothermal fluid flux at deep-sea vents[END_REF][START_REF] Pradillon | Colonisation habitat of newlyopened by a pioneer species, Alvinella pompejana (Polychaeta: Alvinellidae), at East Pacific Rise vent sites[END_REF][START_REF] Pradillon | Development of assemblages associated with alvinellid colonies on the walls of high-temperature vents at the East Pacific Rise[END_REF][START_REF] Kelly | Spatial and temporal patterns of colonization by deep-sea hydrothermal vent invertebrates on the Juan de Fuca Ridge, NE Pacific[END_REF][START_REF] Kelly | Diversity of invertebrate colonists on simple and complex substrates at hydrothermal vents on the Juan de Fuca Ridge[END_REF], at methane seeps using sediment trays with or without agar layers releasing sulphide [START_REF] Levin | Recruitment response of methane-seep macrofauna to sulfide-rich sediments: An in situ experiment[END_REF], at the deep-sea floor using natural or exotic sunken wood [START_REF] Pailleret | Sunken wood from the Vanuatu Islands: identification of wood substrates and preliminary description of associated fauna[END_REF]Tyler et al., 2007;Voigt et al., 2007) and finally, using natural or artificially sunken whale carcases [START_REF] Smith | Ecology of whale falls at the deep-sea floor[END_REF][START_REF] Fujiwara | Three-year investigations into sperm whalefall ecosystems in Japan[END_REF][START_REF] Lorion | Several deep-sea mussels and their associated symbionts are able to live both on wood and whale falls[END_REF]. But none of these colonization experiments involved deployment of organic substrates deployed directly on methane seeps or hydrothermal vents.

Because communities of metazoan are mostly sessile and dependant on the local chemosynthetic food web, colonization of these fragmented habitats has been assumed to be driven by larval dispersal [START_REF] Mullineaux | Dispersal mechanisms of Deep-Sea hydrothermal vent fauna Geophysical Monograph 91[END_REF]. Fertilization and development experiments at a certain pressure and temperature (see review Tyler and Young, 2003) have

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4 recently allowed an estimate of the distance some marine larvae can disperse in oceanic currents (up to 1000 km), but only a few deep-sea species were investigated. [START_REF] Mullineaux | Hydrothermal vent plumes: larval highways in the Deep-sea?[END_REF][START_REF] Mullineaux | Vertical, lateral and temporal structure in larval distributions at hydrothermal vents[END_REF] and [START_REF] Metaxas | Spatial and temporal patterns in larval supply at hydrothermal vents in the northeast Pacific Ocean[END_REF] studied larval dispersal by sampling the seawater column using tows near deep-sea reducing habitats. However, very few of the recovered larvae could be identified to the species level. Recent techniques such as in situ hybridization (ISH) using group-or speciesspecific oligonucleotide probes (targeting 18S ribosomal RNA) have recently been developed which can circumvent this difficulty [START_REF] Pradillon | Species identification of marine invertebrate early stages by whole-larvae in situ hybridisation of 18S ribosomal RNA[END_REF][START_REF] Jones | A robotic molecular method for in situ detection of marine invertebrate larvae[END_REF]. At the time of settlement for these larvae from deep-sea species that may have dispersed for weeks to months, the geochemical factors triggering settlement into these reducing habitats are not yet known.

To tackle this issue, we conceived a novel colonization device allowing the settlement of early-life stages of organisms that colonize deep-sea reducing habitats excluding large-size predators. We designed a standardized colonization module named CHEMECOLI (CHEMosynthetic Ecosystem COlonization by Larval Invertebrates) and filled it with organic and inorganic substrates (wood, plant material and carbonates) in order to simulate reducing habitats. Indeed, organic substrates (wood and plant) may produce sulphide through microbial degradation, while carbonate substrates (naturally present in seeps habitats) can be used as a chemically inert hard substrate for settlement. Devices were deployed in several reducing deepsea habitats, though always away from direct influence of reducing fluids. The localized distribution of hydrothermal vents enables the direct influence of the fluid to be avoided, which is not possible at cold seeps where fluid emissions are widespread. However, sulphide enrichment in these latter habitats is considered to be limited to the sediment where it is produced by sulphate-reducing bacteria (SRB) associated with the anaerobic degradation of methane [START_REF] Niemann | Novel microbial communities of Haakon Mosby Mud Volcano and their role as a methane sink[END_REF]. In all cases, methane enrichment in the water above the seafloor has been described [START_REF] Charlou | Geochemistry of high H2S and CH4 vent fluids issuing from ultramafic rocks at the Rainbow hydrothermal field (36 degrees 14'N, MAR)[END_REF](Charlou et al., , 2003) ) and its influence on the colonization device cannot be ruled out. The questions to be addressed in this paper are: 1) Are larvae from symbiotic metazoan

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5 species able to settle on natural organic substrates deployed in methane seep or hot vent areas? 2) Are vent/seep fauna larvae able to settle on natural organic substrates and hence potentially use them as stepping-stones for dispersal?

MATERIAL AND METHODS

Colonization devices

Standardized colonization devices (CHEMECOLIs, Fig. 1a) were made of a hollow PVC cylinder (14 cm diameter x10 cm high, with a total volume of 1.539 dm 3 ). The cylinder was drilled with lateral holes to permit circulation of fluids. Devices were filled either with dried alfalfa grass, natural Douglas fir wood cubes (2*2*2 cm), or carbonate cubes (2*2*2 cm) (Fig. 1b, c &d). One device could harbour roughly 100 cubes. Substrates were retained within the PVC cylinder by a plastic net of 2 mm mesh. Devices were weighted with stainless steel chain and tagged using syntactic foam. Forty eight hours prior to deployment, wood-loaded devices were soaked in cold filtered seawater in order to overcome the natural buoyancy of dry wood. At each experiment site, a set of 3 CHEMECOLIs, each filled with a different substrate, was deployed. The CHEMECOLIs were deployed and recovered in situ by ROV (Remotely Operated Vehicle) or manned submersible. For deployment and recovery, a hermetic box was used with separate compartments for each device, to avoid washing and mixing. Video and photos were recorded during these in situ manipulations (Fig. 2a &b).

Study sites

CHEMECOLIs were deployed during year 2006 in European waters exploring three distinct reducing habitats of various depth and locations (Fig. 3). Two sets of CHEMECOLIs were deployed at the cold seep site 'Central Zone 2A' in Pockmark area in the Nile Deep-Sea Fan in eastern Mediterranean (Table 1; Fig. 3) [START_REF] Dupré | Seafloor geological studies above active gas chimneys off Egypt (Central Nile deep sea fan)[END_REF][START_REF] Foucher | Structure and drivers of cold seep ecosystems[END_REF]. The first

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set was left on the bottom for two weeks during November 2006, and was then replaced with a second set that was deployed at the exact same location for one year by ROV Quest 4000 (MARUM, Bremen, Germany) (November 2006 to November 2007; see Table 1). Recovery after one year was done using the ROV Victor 6000 (Ifremer, France). Devices were deployed on outcropping authigenic carbonate crusts which are generally considered to limit the inflow of methane and sulphide from underlying sediments. However, small siboglinid tubeworms Lamellibrachia sp. nov., were observed within a crack close to the devices, meaning that direct influence of seepage, at least for the fraction of methane that is not oxidized in the sediment, could not be completely ruled out. Surrounding sediment epifauna included lucinid clams Lucinoma aff. kazani (Salas & Woodside, 2002) and Myrtea sp., mussels Idas sp., thyasirid and vesicomyid bivalves, and echinoids. Site 2A is dominated by emission of methane and heavier hydrocarbons, and there is co-occurrence of soft sedimentary and hard carbonate substrates [START_REF] Dupré | Seafloor geological studies above active gas chimneys off Egypt (Central Nile deep sea fan)[END_REF][START_REF] Foucher | Structure and drivers of cold seep ecosystems[END_REF].

A set of CHEMECOLIs was deployed for 11 months by ROV Victor 6000 (Ifremer, France) at the hydrothermal vent field Rainbow close to the Azores Triple Junction on the Mid-Atlantic Ridge (MAR), (Fig. 3; Table 1), about 10 m away from an edifice composed of several venting chimneys. No active diffuse flow was detected in the immediate surrounding of the devices, and temperature around devices equalled the seawater baseline temperature of 3.7°C.

High temperature fluids at Rainbow are highly enriched in hydrogen, methane, ferrous iron and relatively depleted in sulphide in comparison to other MAR vent sites such as Lucky Strike [START_REF] Charlou | Geochemistry of high H2S and CH4 vent fluids issuing from ultramafic rocks at the Rainbow hydrothermal field (36 degrees 14'N, MAR)[END_REF]. The high iron content in fluids leads to further depletion in bioavailable sulphide [START_REF] Schmidt | Geochemical energy sources for microbial primary production in the environment of hydrothermal vent shrimps[END_REF] with a negligible fraction of it in the form of free sulphide (Le Bris and Duperron, in press). Macrofaunal communities at Rainbow are typically dominated by the two shrimps Rimicaris exoculata (Williams & Rona, 1986) and Mirocaris fortunata (Martin & Christiansen, 1995) that form swarms around walls of large edifices, and by the mussel Finally, one set of CHEMECOLIs was deployed at the Haakon Mosby Mud volcano site (HMMV) in the Norwegian Sea (Fig. 3; Table 1). There, methane is abundant at the surface of the sediment but no sulphide was usually detected in seawater above the area of deployment, characterized by the presence of siboglinid tubeworms [START_REF] Niemann | Novel microbial communities of Haakon Mosby Mud Volcano and their role as a methane sink[END_REF][START_REF] Lösekann | Endosymbiosis between bacteria and deep-sea siboglinid tubeworms from an Arctic Cold Seep (Haakon Mosby Mud Volcano, Barents Sea)[END_REF]. Bacteria Beggiatoa spp. and siboglinid Annelid (Sclerolinum contortum (Smirnov, 2000) and Oligobrachia haakonmosbiensis (Smirnov, 2000)) constitute the dominant microorganisms and macro-fauna respectively. Both use hydrogen sulphide for chemoautotrophy, but siboglinids achieve chemosynthesis through the help of symbionts located within the trophosome [START_REF] Lösekann | Endosymbiosis between bacteria and deep-sea siboglinid tubeworms from an Arctic Cold Seep (Haakon Mosby Mud Volcano, Barents Sea)[END_REF]. Colonization devices were deployed by ROV Victor 6000 (Ifremer, France) and recovered by the ROV Quest 4000 (MARUM, Germany) after 12 months (Table 1) in the south Hummocky periphery of the mud volcano where there was generally a high density of siboglinids (>50%) and lower density of Beggiatoa spp. (<20%) [START_REF] Jerosh | Spatial distribution of mud flows, chemoautotrophic communities and biogeochemical habitats at Haakon Mosby Mud Volcano[END_REF].

Sample processing methods

Within a few hours of recovery, CHEMECOLIs were sorted on board in a cold room. For each CHEMECOLI, external aspect was recorded and then the top mesh was opened to collect the colonized substrate into a bucket. One fifth of content (or 20 cubes) randomly selected from the bottom, middle and top of the device, was fixed in 4% buffered formaldehyde in twicefiltered seawater (TFSW), 2/5 (or 40 cubes) were fixed in 95% ethanol, 1/5 (or 20 cubes) was fixed for Fluorescence in situ hybridization (FISH) analyses (4% buffered formaldehyde in TFSW for few hours at 4°C, rinsed three times in TFSW then transferred into 50/50 Ethanol/TFSW and stored in 95% ethanol), the rest was shared between frozen in 10% glycerol

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or/and fixed in buffered 2.5% Glutaraldehyde. Once sorting was completed, the medium in which cubes were sorted was filtered through a 64-µm mesh to recover small fauna that was not attached to substrates, and fixed either in formaldehyde, ethanol, FISH, frozen or Glutaraldehyde.

If organisms had settled outside the device, they were fixed but not included in further analyses.

All species fixed in buffered formaldehyde were morphologically identified using a dissecting microscope. Wood cubes were dissected in thin slices using wood dissecting scissors in order to collect endofauna. For alfalfa, each piece of grass was examined including the inside of the grass itself. For carbonate cubes, only external observation was carried out. Specimens were counted, identified to the lowest taxonomic level possible, and pictures were taken. Some specimens from the dominant fauna such as polychaetes and molluscs were sent to taxonomists for species identification. Some species recovered from substrates fixed in formaldehyde from CHEMECOLIs deployed at the Nile Deep-Sea Fan and HMMV, were also recovered from substrates fixed in ethanol and used for molecular-based identification using marker genes such as COI and 18S rRNA (see below).

COI and 18S rRNA genes sequencing

DNA was extracted from soft-tissues of some specimens recovered from CHEMECOLIs deployed at the Nile Deep-Sea Fan (Table 2) and of polychaetes recovered from CHEMECOLIs deployed at HMMV, using the QIAamp DNA Micro Kit (Qiagen) following the manufacturer's protocol. Fragments of the mitochondrial cytochrome oxidase I-encoding gene (COImt DNA) were amplified using primers LCO 1490 and HCO 2198 [START_REF] Folmer | DNA primers for amplification of mitochondrial cytochrome C oxidase subunit I from metazoan invertebrates[END_REF]. Polymerase Chain Reaction (PCR) was performed as follows: an initial denaturing step of 5 min at 94°C, followed by 35 cycles at 94°C for 30s, 48°C for 40s for hybridization, then 1 min at 72°C, and a final extension for 10 min at 72°C. PCR products were sequenced by Genoscope (France).

Alternatively, a ~1800 bp fragment of the18S rRNA gene was amplified with primers 1f and [START_REF] Pradillon | Species identification of marine invertebrate early stages by whole-larvae in situ hybridisation of 18S ribosomal RNA[END_REF] using Ex Taq PCR kit (TaKaRa, Kyoto, Japan) and the following PCR protocol: initial denaturation at 96ºC for 5 min, 30 cycles of (96ºC for 1 min, 51ºC for 1 min, and 72ºC for 3 min) and a final extension at 72ºC for 10 min. Amplification products were purified using gel extraction following the manufacturer's protocol (Wizard kit Promega).

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Sequencing reactions using the PCR products as template were performed with BigDye Terminator Cycling Sequencing Ready Reaction kit (PE Applied Biosystems, Foster City, CA, USA), in both directions, using additional internal primers (see [START_REF] Pradillon | Species identification of marine invertebrate early stages by whole-larvae in situ hybridisation of 18S ribosomal RNA[END_REF]. DNA sequencing was conducted with an ABI-PRISM 3130x Genetic Analyser (Applied Biosystems Japan Ltd., Tokyo, Japan) following the manufacturer's protocols. Sequences were inspected by eye and assembled with AutoAssembler 2.1 (Applied Biosystems). All sequences were deposited in the EMBL database under given accession numbers.

Chemical analyses

For the Nile Deep-Sea Fan and Rainbow experiments, the presence of free sulphide was examined using potentiometric electrodes in situ before the recovery of CHEMECOLIs. The potentiometric probe used is equipped with a conventional Ag/Ag 2 S electrode (Le [START_REF] Bris | Autonomous potentiometric sensor for in situ sulfide monitoring in marine sulfidic media[END_REF][START_REF] Laurent | Sunken wood habitat for thirotrophic symbiosis in mangrove swamps[END_REF], which was prepared from a silver wire and calibrated in the laboratory. Above a threshold of c.a. 20 µM the electrode has a logarithmic response that allows quantitative determination of sulphide providing that the pH is known. In agreement with the theoretical response of the Ag/Ag 2 S electrode, the slope of the electrode is about 30 mV/ per decade of HS -, after correction of pH variation. Below this threshold, however, the slope tends to increase and can reach much higher values. This increase in sensitivity is combined with a lower reproducibility which does not allow fully quantitative determination of [HS -] in the lower part of this range. For this reason, only a maximum concentration could be defined when sulphide was detectable but still lower than 20-30 µM. Using the manipulator arm of the submersible, the tip

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10 of the electrodes (<5 mm total) was placed on different locations, on the top, side and base of the CHEMECOLI devices. In addition to this in situ approach, additional measurements were done on board, while the CHEMECOLIs were still in the collection box filled with bottom seawater, the electrodes were inserted among cubes or into alfalfa grass.

Data analysis

Density of species colonizing wood cubes within a given CHEMECOLI was calculated taking into account that twenty cubes represent a volume of 160 cm 3 . To compare densities among the different substrates this volume was also used for the carbonate substrate even if the substrate could not be explored in 3 dimensions. Alfalfa was colonized by organisms inside and [START_REF] Paxton | Three species of Dorvilleidae (Annelida: Polychaeta) associated with Atlantic deep-sea reducing habitats, with the description of Ophryotrocha fabriae[END_REF]. For each CHEMECOLI, we counted both the number of new taxon (N) compared to the surrounding habitat and the number of taxon (X) that was already recorded in the surrounding habitat. Biodiversity was estimated based on three different univariate indices using PRIMER v.6 (PRIMER-E): species richness (S), Shannon-Wiener index of diversity (H', log (е) such as [START_REF] Kelly | Diversity of invertebrate colonists on simple and complex substrates at hydrothermal vents on the Juan de Fuca Ridge[END_REF]), and Pielou's evenness index (J').

RESULTS

Species recovered within CHEMECOLIs

For each experiment we tried to assign organisms to described species [START_REF] Sibuet | Biogeography, biodiversity and fluid dependance of deep sea cold seep communities at active and passive margins[END_REF][START_REF] Desbruyères | Variations in deep-sea hydrothermal vent communities on the Mid-Atlantic Ridge near the Azores plateau[END_REF][START_REF] Gebruk | Methane seep community of the Haakon Mosby Mud Volcano (the Norwegian Sea): composition and trophic aspects[END_REF][START_REF] Olu-Leroy | Cold seep communities in the deep eastern Mediterranean Sea: composition, symbiosis, and spatial distribution on mud volcanoes[END_REF]Desbruyères et 2). COImt DNA sequences were compared with the NCBI nucleotide database. The sequence from Idas sp. matched 100% against Genbank accession no EF210072-1b obtained from adult Idas sp. specimens from the Central Zone in the eastern Mediterranean during the 2003 Nautinil cruise [START_REF] Duperron | Unexpected co occurence of 6 bacterial symbionts in the gill of the cold seep mussel Idas sp. (Bivalvia: Mytilidae)[END_REF]. Other sequences did not match 100% with any published sequence. The 18S rRNA sequence obtained from one polychaete species, Paramphinome sp. (J. Blake) recovered within both organic substrates from CHEMECOLIs deployed in HMMV, was compared with the Genbank database and was identified as identical with a sequence from Paramphinome jeffreysi (McIntosh, 1868).

Overall, a total of 33 taxa were recovered from all CHEMECOLIs deployed in the three study areas hosting reducing habitats. Main groups recovered were Mollusca (bivalves and gastropods) and Annelida (polychaetes), followed by Arthropod Crustacean, Nemertea, Sipuncula, Foraminifera, Actinopodia and Echinoderma (Table 3). Species recovered varied among study sites, but also among the different CHEMECOLI substrates deployed within a given study site (Table 3). For both the Rainbow and the Nile Deep-sea Fan 1-year experiments, more than 50% of the species encountered within both organic and carbonate substrates were not previously reported in the surrounding chemosynthetic communities. In the HMMV experiment, only 30% of the fauna recovered from CHEMECOLIs was not documented in the surrounding habitat. Overall densities of specimens (Table 3) were the highest for organic substrates and especially for the wood substrate, reaching 14,987.5 specimens per dm 3 in the CHEMECOLI deployed at HMMV due to the high number of wood-borers that colonized a cube (~117 specimens).

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12 Mollusc dominated as colonists within CHEMECOLIs deployed for 2-weeks and 1-year experiments at the Nile Deep-Sea Fan (Fig. 4a). High densities of bivalves were counted within the wood substrates due to the abundance of wood-boring bivalves (Xylophaga dorsalis (Turton, 1819)) (~287.5 specimens per dm 3 for the short-term 2-week experiment and 525 specimens per dm 3 for the long-term 1-year experiment), while gastropods were more abundant on the alfalfa substrate (~300 specimens per dm 3 ), along with polychaetes (~ 200 specimens per dm 3 ). In the long-term experiment, polychaetes as well densely colonized the wood substrate (~200 specimens per dm 3 ). Lower densities of crustaceans (max ~60 specimens per dm 3 ) and Sipuncula were also present on organic substrates in the long-term experiment. Carbonate substrates deployed for one year were colonized only by low densities of gastropods Clelandella myriamae, hesionid polychaetes and sipuncle (Table 3). Young settler-stages (juveniles) of symbiotic bivalve species harbouring sulphur-oxidizing bacteria were successfully recovered. These included Idas sp., Vesicomyid sp. and Thyasira sp. (Fig. 5a). These three species were all recovered within the wood substrate after the two-weeks and after the one-year experiments. Idas sp. was also recovered in the alfalfa substrate after the one-year experiment (Fig. 5a).

CHEMECOLIs recovered after one year at the Rainbow site, were poorly colonized compared to CHEMECOLIs deployed into cold seep habitats for one year. Wood-boring bivalves (Xylophaga atlantica (H.G. Richards, 1942)) dominated in the wood-containing device, and juveniles of symbiotic bivalves were also recovered (~200 specimens per dm 3 , Table 3, Fig. 5b) similar morphologically to vesicomyid juveniles, however a molecular identification is necessary to ascertain this observation such as ISH (see introduction). Some polychaetes (Prionospio sp. nov. 3, ~40 specimens per dm 3 ) were recovered within both wood and alfalfa substrates (Fig. 4b).

CHEMECOLIs deployed for one year at the HMMV were densely colonized, especially the wood and alfalfa substrates (Fig. 4c). Wood-boring bivalves (Xyloredo ingolfia (Turner, 1972)) were dominant in the wood substrate reaching the highest density documented in our

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13 study with 14,412.5 specimens per dm 3 . The gastropod Alvania aff. griegi occurred on the alfalfa substrate reaching ~220 specimens per dm 3 . Polychaetes occurred on all substrates, more abundant on the wood (400 specimen per dm 3 ) but were as well present in high densities in alfalfa and carbonate substrates (>100 specimens per dm 3 ). Juveniles of the chemosymbiotic species Sclerolinum contortum (Fig. 5c) were recovered inside all three CHEMECOLIs, displaying tube lengths ranging from few mms to few cms (Table 3). Adult siboglinid tubeworms were attached to the outside mesh of each colonization device that was in contact with the soft sediment (but not counted as not within the mesh). Among crustaceans (Fig. 4c), amphipods and copepods occurred on wood and alfalfa substrates, while Metacaprella sp. occurred on all three substrates (Table 3).

Species richness (S), Shannon-Wiener diversity (H') and Pielou's evenness (J') indices differed among substrates at all deployment sites (Table 4). No statistical support can be summoned due to the lack of replicates, however for each study site, S and H' were the highest for wood and alfalfa substrates compared to carbonate substrates, except for the wood substrate in HMMV where the species richness is the highest but Shannon-Wiener diversity index is very low due to the dominance of the species Xyloredo ingolfia that gives an equitability of 0.1 (J').

Chemical data

In situ punctual sulphide measurements on colonization devices after one year deployment at the Nile Deep-Sea Fan did not reveal any sulphide enrichment in their immediate surrounding before recovery (data not shown). Yet very low sulphide levels, micromolar to submicromolar in alfalfa and wood respectively, were detected inside the devices after recovery suggesting that sulphide had been produced (Table 5). Measurement in the immediate vicinity (<1 m from colonization device) conversely revealed that local free sulphide enrichment could be

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14 detected in the cracks of the underlying carbonate pavement (data not shown). Sulphide was however not detected in the water above these cracks.

At the Rainbow hydrothermal vent, no free sulphide was detected in situ at the top, side or basis of the devices. On board, neither the alfalfa pieces, nor the wood cube devices displayed significant sulphide enrichment when the electrodes were inserted deeper inside. Compared to Nile deep-sea fan, the situation is however quite different in Rainbow since free sulphide is also extremely low even in the habitat of Bathymodiolus mussels or shrimps in the immediate vicinity of the venting chimneys.

DISCUSSION

The goal of this study was to test whether organic substrates within CHEMECOLIs supported the colonization of marine invertebrates' endemic from geologically-driven reducing habitats such as hydrothermal vents and cold seeps, and to test whether larvae of symbiontbearing metazoans were able to settle on these substrates hence potentially use them as steppingstones for dispersal. For both questions we can respond positively.

Density of species and species richness recovered within CHEMECOLIs

The most abundant taxa recovered from CHEMECOLIs were molluscs (bivalves and gastropods) and annelid polychaetes. This observation fits with the results of previous colonization experiments in deep-sea reducing habitats (Van Dover et al., 1988;[START_REF] Shank | Temporal and spatial patterns of biological community development at nascent deep-sea hydrothermal vents (9°50'N, East Pacific Rise)[END_REF][START_REF] Mullineaux | Recruitment variation during a pilot colonization study of hydrothermal vents (9°50'N, East Pacific Rise)[END_REF][START_REF] Smith | Ecology of whale falls at the deep-sea floor[END_REF][START_REF] Levin | Recruitment response of methane-seep macrofauna to sulfide-rich sediments: An in situ experiment[END_REF][START_REF] Fujiwara | Three-year investigations into sperm whalefall ecosystems in Japan[END_REF][START_REF] Pailleret | Sunken wood from the Vanuatu Islands: identification of wood substrates and preliminary description of associated fauna[END_REF][START_REF] Kelly | Diversity of invertebrate colonists on simple and complex substrates at hydrothermal vents on the Juan de Fuca Ridge[END_REF][START_REF] Pradillon | Development of assemblages associated with alvinellid colonies on the walls of high-temperature vents at the East Pacific Rise[END_REF]. In the Nile Deep-Sea Fan (short-term and long-term), HMMV and MAR experiments, organic substrates (wood and alfalfa) displayed higher metazoan abundances compared to carbonate substrates. The high bivalve abundances reported for wood-filled CHEMECOLIs are explained by the high densities

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15 of wood-boring bivalves represented by a single species per site. Overall, thirty to sixty percent of species which have colonized wood substrates deployed in the eastern Mediterranean, in the Mid-Atlantic Ridge and in the Norwegian Sea, have not been described previously from the surrounding seep or vent communities (Table 3).

Several physical and chemical factors may influence the settlement of larvae of species endemic to deep-sea reducing habitats. [START_REF] Kelly | Spatial and temporal patterns of colonization by deep-sea hydrothermal vent invertebrates on the Juan de Fuca Ridge, NE Pacific[END_REF] suggested that settlement of colonists on basaltic plates deployed at hydrothermal vents was correlated with temperature and hydrogen sulphide. Similarly, temperature was presented as a cue for colonists to locate preferred habitat conditions in a gradient of hydrothermal vent flow (Bates et al., 2005). Both these parameters are tracers of the impact of vent fluids on the environment. It is therefore impossible to discriminate a direct influence of sulphide from other factors correlated to the vent fluid ratio. CHEMECOLIs at the vent site Rainbow were deployed ten meters away from chimneys and species abundances were very low compared to the two other experiments carried out in cold seep habitats. A low supply of larvae in the particular period 2006-2007 at Rainbow may explain this result as shown in Pacific hydrothermal vent colonization experiments for particular years [START_REF] Kelly | Spatial and temporal patterns of colonization by deep-sea hydrothermal vent invertebrates on the Juan de Fuca Ridge, NE Pacific[END_REF].

The distance from active venting results in no significant signature of fluid emission in the surrounding water, as evidenced by the low background temperature measured (3.5°C). The lack of any attractive signature of fluid venting could be an alternative explanation for the low rates of colonization observed at Rainbow. [START_REF] Kelly | Spatial and temporal patterns of colonization by deep-sea hydrothermal vent invertebrates on the Juan de Fuca Ridge, NE Pacific[END_REF] nevertheless demonstrated that vent species were recovered at a similar distance from vents, even though settlement and postsettlement survival rates were lower compared to basaltic plates deployed closer to the vent edifice. The difference in fluid geochemistry (low free H 2 S and high rate of precipitation close to vent sources at Rainbow) might also contribute to the difference between those experiments.

M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT

16 Species richness calculated from our three in situ experiments (Table 4) were in the order of magnitude reported by [START_REF] Kelly | Diversity of invertebrate colonists on simple and complex substrates at hydrothermal vents on the Juan de Fuca Ridge[END_REF] from colonization experiments at hydrothermal vents, where S ranged from 11 on sponge substrates to 6 on basaltic plates. Species richness from our experimental study were however, lower than those obtained from colonization experiments carried at methane seeps by [START_REF] Levin | Recruitment response of methane-seep macrofauna to sulfide-rich sediments: An in situ experiment[END_REF] where S values were between 16-19 species and where sulphide added to tray experiments increased the species richness. [START_REF] Kelly | Diversity of invertebrate colonists on simple and complex substrates at hydrothermal vents on the Juan de Fuca Ridge[END_REF] hypothesised that the physical structure may have influenced the species richness in their experiments, with complex physical structure of the sponge favouring a more diverse faunal assemblage by creating interstitial spaces that could provide protection against predation.

In our experiments, degradation of wood by wood-boring bivalves increased the area available for colonization by other species such as spionid polychaetes at the Nile Deep-Sea Fan or at Rainbow (pers. observ.). Similarly, available surfaces in alfalfa-filled colonizers were large. In both organic substrates, micro-niches offered a variety of chemical micro-environments similar to sponges in [START_REF] Kelly | Diversity of invertebrate colonists on simple and complex substrates at hydrothermal vents on the Juan de Fuca Ridge[END_REF] allowing more species to colonize and therefore increasing the species richness. These probably differentiate compared to carbonate substrates, where species richness was more similar to that recovered on basaltic plates in [START_REF] Kelly | Diversity of invertebrate colonists on simple and complex substrates at hydrothermal vents on the Juan de Fuca Ridge[END_REF].

Occurrence of species harbouring sulphur-oxidizing bacteria in CHEMECOLIs

Results from both the two-weeks and one-year experiments at the Nile Deep-Sea Fan suggested a sulfophilic stage, which is a particular stage in the faunal succession, documented from whale carcasses, during which chemosynthetic fauna harbouring thiotrophic symbionts establishes [START_REF] Smith | Ecology of whale falls at the deep-sea floor[END_REF], as indicated by the presence of Vesicomyid sp., Thyasira sp. and Idas sp. These taxa, also reported from whale falls and methane seeps, are typical from reducing ecosystems (Kiel and Godaert, 2006). Indeed, all known members of families

M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT

Vesicomyidae and Thyasiridae harbour sulphur-oxidizing bacterial symbionts [START_REF] A N U S C R I P T A C C E P T E D Accepted Manuscript 30 Peek | Cospeciation of chemoautotrophic bacteria and deep sea clams[END_REF][START_REF] Taylor | Evolutionary relationships of the bivalve family Thyasiridae (Mollusca: Bivalvia), monophyly and superfamily status[END_REF][START_REF] Dubilier | Symbiotic diversity in marine animals: the art of harnessing chemosynthesis[END_REF]. Regarding Idas sp., barcoding demonstrated that it was the same species previously documented to harbour 6 distinct bacterial symbionts, including sulphur-and methane-oxidizing bacteria and found at cold seeps in the eastern Mediterranean [START_REF] Duperron | Unexpected co occurence of 6 bacterial symbionts in the gill of the cold seep mussel Idas sp. (Bivalvia: Mytilidae)[END_REF]. Phylogenetically, Idas sp. clusters within the large group which includes all large mussels (Bathymodiolus) associated with deep-sea seeps and vents as well as many smaller species associated with organic falls worldwide [START_REF] Duperron | Unexpected co occurence of 6 bacterial symbionts in the gill of the cold seep mussel Idas sp. (Bivalvia: Mytilidae)[END_REF][START_REF] Lorion | Several deep-sea mussels and their associated symbionts are able to live both on wood and whale falls[END_REF]. In our in situ experiment, Idas sp. was found associated with the two organic substrates, wood and alfalfa whereas Thyasira sp. and Vesicomyid sp. juveniles settled on wood cubes but were not recovered in the alfalfa.

At Rainbow, juveniles bivalve morphologically similar to vesicomyid specimens were recovered within the wood substrate. Interestingly, vesicomyid clams are not, to date, reported from the Northern Mid-Atlantic Ridge [START_REF] Desbruyères | Variations in deep-sea hydrothermal vent communities on the Mid-Atlantic Ridge near the Azores plateau[END_REF] and only further studies using DNA sequencing [START_REF] Comtet | Molecular and morphological identification of settlement-stage mussel larvae, Bathymodiolus azoricus (Bivalvia: Mytilidae) preserved in situ at active vent fields on the Mid-Atlantic Ridge[END_REF] may ascertain their identification.

Sclerolinum contortum (Monolifera siboglinid) was recovered from all 3 substrates at HMMV. According to [START_REF] Sahling | The physicochemical habitat of Sclerolinum sp. at Hook Ridge hydrothermal vent, Bransfield Strait, Antartica[END_REF], the genus Sclerolinum occurs at hydrothermal vents, methane seeps, decaying wood and other organic falls. S. contortum occurs buried 15 cm deep in the sediment at HMMV, where sulphide concentrations are around 0.15 mM, and harbours sulphide-oxidizing autotrophic symbionts within its trophosome [START_REF] Lösekann | Endosymbiosis between bacteria and deep-sea siboglinid tubeworms from an Arctic Cold Seep (Haakon Mosby Mud Volcano, Barents Sea)[END_REF]. We herein confirm that S. contortum can settle and grow in decaying plant debris, in wood and mineral substrates. Specimens recovered within the wood substrate were very tiny (only few mms) compared to those from alfalfa and carbonate substrates (few cms). The high densities of wood-boring bivalves per wood cube may have limited the development of S. contortum in the latter CHEMECOLI. Concentrations of sulphide are certainly different within these three substrates and may also have affected the growth, survival rate and development of this documented to be inversely correlated with the abundance of other benthic fauna [START_REF] Dando | Interactions between sediment chemistry and frenulate pogonophores (Annelida) in the north-east Atlantic[END_REF]. Besides, frenulates usually 'mine" insoluble sulphide into an anoxic zone within the sediment by their root that is deeply buried in this area [START_REF] Dando | Interactions between sediment chemistry and frenulate pogonophores (Annelida) in the north-east Atlantic[END_REF].

New species, opportunistic species and tolerance to sulphide

The biodiversity of Nile Deep-Sea Fan and HMMV cold seeps is still under investigation (Vanreusel et al., 2009), and some of the species recovered within CHEMECOLIs may not yet be documented from the background fauna. [START_REF] Gebruk | Methane seep community of the Haakon Mosby Mud Volcano (the Norwegian Sea): composition and trophic aspects[END_REF] have listed 19 species so far from HMMV and Olu-Le Roy et al. ( 2004) have numerated 27 species from 4 mud volcanoes located in the eastern Mediterranean. At least some recovered species specialized in the exploitation of organic substrates are probably not present as adults in the surrounding habitat in our experiment.

Wood-boring bivalves from the family Xylophagainae were for example recovered from all wood samples, albeit with different species (T. Haga). Xylophaga spp. are opportunistic species in the deep-sea which settle exclusively on sunken wood [START_REF] Turner | Wood, mollusks, and deep-sea food chains[END_REF]Tyler et al., 2007).

Small cocculiniform gastropods identified as Coccopigya sp. (maybe 2 new species according to A. Warén) and rissoid gastropods identified as Alvania aff. griegi (A. Warén) were recovered within the alfalfa-filled CHEMECOLIs from the Nile Deep-Sea Fan and HMMV, respectively.

Juveniles of a new Alvania species (sp.1) were recovered at the HMMV site in dense aggregations by [START_REF] Gebruk | Methane seep community of the Haakon Mosby Mud Volcano (the Norwegian Sea): composition and trophic aspects[END_REF] and could be the same species as retrieved from the alfalfafilled colonizer. The genus Alvania is commonly found around hot vents, on sulphidic rocks or at the base of black smokers [START_REF] Desbruyères | Handbook of Deep Sea Hydrothermal Vents: Denisia 18[END_REF] and the species A. aff. griegi was described Three new species of Spionidae polychaetes (within genus Prionospio) were identified within devices containing organic substrates: two from CHEMECOLIs deployed at the Nile Deep-Sea Fan, and a third from devices deployed at Rainbow (J. Blake). According to [START_REF] Sigvaldadottir | Two new species of Spionidae (Annelida: Polychaeta) from Mid-Atlantic Ridge hydrothermal vents[END_REF], only five polychaetes species occurred at Rainbow including two species of Spionidae P. unilamellata [START_REF] Sigvaldadottir | Two new species of Spionidae (Annelida: Polychaeta) from Mid-Atlantic Ridge hydrothermal vents[END_REF] and Laonice asaccata [START_REF] Sigvaldadottir | Two new species of Spionidae (Annelida: Polychaeta) from Mid-Atlantic Ridge hydrothermal vents[END_REF]. Spionid polychaetes are considered as opportunistic species in the deep-sea fauna (Van Dover et al., 1988) and very tolerant to sulphide exposure [START_REF] Desbruyères | Variations in deep-sea hydrothermal vent communities on the Mid-Atlantic Ridge near the Azores plateau[END_REF][START_REF] Levin | Recruitment response of methane-seep macrofauna to sulfide-rich sediments: An in situ experiment[END_REF]. A new species of Glyceridae, Glycera sp.

nov. (M. Böggemann) was recovered from biogenic substrates deployed in Nile Deep-Sea Fan.

Glyceridae polychaete such as G. dibranchiate (Ehlers, 1868) from mudflats has been shown to be highly tolerant to sulphide exposure [START_REF] Hance | Cytotoxicity from sulfide exposure in a sulfide-tolerant marine invertebrate[END_REF] and this may explain why a new species of Glycera was recovered from both biogenic substrates in CHEMECOLIs deployed at the Nile Deep-Sea Fan.

Other opportunistic species were encountered within CHEMECOLIs such as the Amphinomidae polychaete Paramphinome jeffreysi identified to the species level as a result of 18S rRNA sequencing (A. Nunes Jorge). A great number of Amphinomidae have been reported associated with the siboglinid Sclerolinum contortum at HMMV [START_REF] Gebruk | Methane seep community of the Haakon Mosby Mud Volcano (the Norwegian Sea): composition and trophic aspects[END_REF] but they were not identified to the species level. This opportunistic species is generally associated with other polychaete species such as Prionospio steenstrupi (Malmgren, 1867), Capitella capitata (Fabricius, 1780) and Heteromastus filliformis (Claparède, 1864) in soft marine sediment enriched in organic matter and associated with the symbiotic thyasirid bivalve Thyasira sarsii (Philippi, 1845) [START_REF] Kutti | Effects of organic effluents from a salmon farm on a fjord system. II. Temporal and spatial patterns in infauna community composition[END_REF]. At HMMV, a nematode Halomonhystera disjuncta (Bastian,

M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT 20
1865) that is usually described in intertidal organically enriched mudflats was also found in high abundance in the microbial mats area (Vanreusel et al., 2009).

Another opportunistic polychaete species, Ophryotrocha sp., was found within wood substrate deployed at HMMV and the genus Ophryotrocha is common in organics-enriched habitats [START_REF] Rouse | Polychaetes[END_REF]. This species is morphologically similar to the one newly described by [START_REF] Paxton | Three species of Dorvilleidae (Annelida: Polychaeta) associated with Atlantic deep-sea reducing habitats, with the description of Ophryotrocha fabriae[END_REF], O. cf. spatula (Fournier & Conlan, 1994) that lives in the white microbial mats and nearby sediments at HMMV. [START_REF] Levin | Recruitment response of methane-seep macrofauna to sulfide-rich sediments: An in situ experiment[END_REF] also recovered one species of dorvilleid polychaete within tray with sulphide deployed within the seep: O. platykephale (Blake, 1985) that lives as well on bacterial mats in the methane seeps at the northern California margin. This species can tolerate up to 1mM concentration of sulphide [START_REF] Levin | Recruitment response of methane-seep macrofauna to sulfide-rich sediments: An in situ experiment[END_REF].

Overall non-symbiotic fauna associated with symbiotic fauna recovered within CHEMECOLIs filled with wood and alfalfa substrates, were opportunistic and tolerant to sulphide confirming that CHEMECOLIs actually mimic reducing habitats.

Sulphide production from organic substrates and potential role in the colonization process

Sulphide enrichment in sunken woods has been reported in a number of studies related to archaeological shipwreck and sulphide concentration exceeding hundreds of micromolar have been recently documented at the surface in naturally sunken wood in a mangrove swamp [START_REF] Laurent | Sunken wood habitat for thirotrophic symbiosis in mangrove swamps[END_REF]). Sulphide production from wood decomposition is considered to result of the activity of anaerobic sulphate-reducing bacteria (SRB). Palacio et al. (2006) Only micromolar to submicromolar levels of sulphide were recorded within both organic substrates from CHEMECOLI deployed in eastern Mediterranean recovered on board, much less than measured on natural sunken wood in a tropical shallow water mangrove [START_REF] Laurent | Sunken wood habitat for thirotrophic symbiosis in mangrove swamps[END_REF]. The fact that sulphide was undetectable in situ in contact with the CHEMECOLI indicates that the influence of substrate degradation on the micro-environment was insignificant with respect to this parameter at the time of recovery (i.e. after one year). The presence of species known to harbour sulphide-oxidizing symbionts and of numerous sulphide-tolerant species characteristic of sulphidic habitats suggests that the presence of these species is not necessarily related to high sulphide content. Two hypotheses can be proposed to explain this result: 1) the microenvironment has been enriched to a larger extent over a limited period of time preceding the recovery and colonizers still reflect these earlier sulphide-enriched stages; 2) sulphide production by SRB is just starting and thiotrophic species settlement is triggered by indirect clues correlated to sulphide production, such as compounds produced by cellulose-degrading bacteria. This stresses the need for a better characterisation of the variability of physico-chemical conditions over time in future sunken wood deep-sea experiments. The fact that taxa such as thyasirid, vesicomyid and mytilid bivalves were already recovered after 2 weeks in the Nile Deep-Sea Fan wood experiment, whereas these species were absent from the carbonate colonization device, supports the latter hypothesis. However, the idea that the presence of organic matter is the only factor triggering the settlement of these species should be taken with caution since methane, and even sulphide enrichment in the local environment can not be excluded and may have influenced the settlement of these bivalves. A 2-week record on the carbonate at the base of one colonization device indeed revealed pulsated sulphide enrichment, likely originating from a crack located beneath it (Le [START_REF] Bris | Autonomous potentiometric sensor for in situ sulfide monitoring in marine sulfidic media[END_REF]. HMMV, and Rainbow were mostly colonized by molluscs (gastropods or bivalves) and annelid (polychaetes), and juvenile stages were successfully recovered. Organic substrates were more densely colonized than the carbonate substrate. Wood-boring bivalves were found on every wood substrate at all sites, although with different species. Organic substrates yielded several species presumably harbouring chemosynthetic bacterial symbionts, as well as associated heterotrophic fauna common at hot vents, methane seeps and organic falls and tolerant to sulphide. Species that were not previously reported in these reducing habitats, but that were common in other type of reducing habitats, were also present. fellowship from HERMES (EC). We are indebted to taxonomists for their precious expertises: J. Blake, M. Böggemann, and P. Hutchings for the polychaetes, A. Warén for the gastropods, T.

Haga for the wood-boring bivalves. Genoscope, J. Lambourdière, M-C. Boisselier and S. Samadi are acknowledged for their technical expertise in COI sequencing. We are also grateful to A.

Nunes Jorge and the JAMSTEC (Japan) that did the 18S rRNA sequencing.

Bates, A. E., Tunnicliffe, V., Lee, R. W., 2005. Role of thermal conditions in habitat selection by hydrothermal vent gastropods. Marine Ecology Progress Series 305, 1-15. Charlou, J.L., Donval, J.P., Zitter, T., Roy, N., Jean-Baptiste, P., Foucher, J.P., Woodside, J.,

7

  Bathymodiolus azoricus(Cosel & Comtet in Cosel, Comtet & Krylova, 1999) which occurs in beds on chimneys and around diffuse vent flows. Recovery of CHEMECOLIs was done by the manned submersible Nautile (Ifremer, France).

  stems and therefore the 3D was explored. Species recovered from CHEMECOLIs from the Nile Deep-Sea Fan were compared with what was known from Sibuet et al. (1998) and Olu-Le Roy et al. (2004); species recovered from CHEMECOLIs from Rainbow were compared with what was known from Desbruyères et al. (2001) and Desbruyères et al. (2006); species recovered from CHEMECOLIs from HMMV were compared with what was known from[START_REF] Gebruk | Methane seep community of the Haakon Mosby Mud Volcano (the Norwegian Sea): composition and trophic aspects[END_REF]

  al., M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT 11 2006 and Paxton and Morineaux, 2009). DNA barcode sequences (COI mitochondrial gene) were obtained from specimens of the two mollusc gastropods Coccopigya sp. and Clelandella myriamae (Gofas, 2005), for Idas sp., and for the Annelid Prionospio sp., all from modules deployed in Nile Deep-Sea Fan (Table

  Adults of S. contortum were recovered at the bottom of the three CHEMECOLIs deployed at HMMV. Larvae must have been released in the three devices by mature reproductive adults from below. In soft sediments, abundance of siboglinid frenulates is

  from a piece of sunken driftwood (Friiele, 1879, A. Warén personal communication).

  have measured cellulolytic activities in Fir wood after 2 months in laboratory experiments using Mediterranean seawater at 14°C (same temperature recorded at the Nile Deep-Sea Fan). The dynamics of this process is still poorly understood. Especially, nothing is known for the deep environments considered in this study (> 1500 m).

  standardized, colonization device, CHEMECOLI, was designed to recover young stages of endemic fauna from reducing habitats and to study colonization processes in the absence of large predators. Overall, colonization devices deployed at the Nile Deep-Sea Fan,

  to thank the people who have deployed and recovered some of the CHEMECOLIs: Olivier Gros, Mélina Laurent, Mélanie Bergmann, Catherine Pierre and Delphine Cottin. We are grateful to chief scientists, captains and crews of RVs Pourquoi pas?, Meteor, L'Atalante, Polarstern, and teams operating ROVs Victor 6000 (Ifremer, France), Quest 4000 (MARUM, Bremen, Germany), and submersible Nautile (Ifremer, France). Sample collection was funded by CHEMECO (European Sciences Foundation (ESF)/Eurocores/EURODEEP), HERMES (EC) and DIWOOD (EC) programs, and by IFREMER, CNRS, and MPI institutes. Analysis and interpretation of data was supported by CHEMECO (ESF/Eurocores/EURODEEP) and UPMC. Marie Pailleret and Florence Pradillon were supported respectively by a Ph D grant from ANR Deepoases and by a Post-doctoral

  Party, M.S., 2003. Evidence of methane venting and geochemistry of brines on mud volcanoes of the eastern Mediterranean Sea. Deep-Sea Research I 50:941-958.

Fig. 2 .

 2 Fig.2. CHEMECOLIs deployed in situ in 2006 at Haakon Mosby Mud Volcano during the

Fig. 3 .

 3 Fig.3. Map (after Interridges website) of study sites where CHEMECOLIs were deployed: at

Fig. 4 .

 4 Fig.4. Densities of the 4 dominant groups of invertebrates that have colonized the CHEMECOLI

Fig. 5 .

 5 Fig.5. Densities of thiotrophic species versus non thiotrophic species that have colonized the

Table 1 .

 1 Sampling details of deployment and recovery of CHEMECOLIs including dates, cruises, latitude & longitude, depth and substrates.

	Substrates	Deployment date	Cruises	Sites	Details of sites	Recovery date &	Cruises
		and dive				dive
	Alfalfa	06/06/2006	VICKING	Haakon	72°00'14''N,	29/06/2007	ARK XXII/1b
	Wood	Dive 277-7		Musby Mud	14°43'22''E,	Dive 166-3
	Carbonate			Vulcano	1257m
				(HMMV)
	Alfalfa	21/08/2006,	MOMARETO	Rainbow/	36°13'76''N,	14,15/07/2007	MOMARDREAM
	Wood	Dive 292-9		MAR	33°54'19''W,	Dives 1676&1677
	Carbonate				2300m
	Wood	4/11/2006	BIONIL	Nile Deep-	32° 31'97''N,	18/11/2006	BIONIL
	Carbonate	Dive 120		Sea	30°21'18''E,	Dive 126
				Fan/eastern	1693m
				Mediterranean
	Alfalfa	18/11/2006	BIONIL	Nile Deep-	32°31'97''N,	10/11/2007	MEDECO
	Wood	Dive 126		Sea Fan/	30°21'18''E,	Dive 338-17
	Carbonate			eastern	1693m
				Mediterranean

Table 2 .

 2 Details of the specimens from the MEDECO cruise sampled in eastern Mediterranean at the Nile Deep-Sea Fan, which mitochondrial partial COI gene for cytochrome oxidase subunit I have been sequenced and recorded into the EMBL database with GenBank accession number.

	Species	Specimens Substrates Site details	COI
					accession
					number
	Clelandella myriamae 1	Alfalfa	32°31'97''N,	FM212782
	Clelandella myriamae 2	Alfalfa	30°21'17''E,	FM212783
	Clelandella myriamae 3	Alfalfa	1693m	FM212784
	Coccopigya sp.	1	Alfalfa		FM212785
	Coccopigya sp.	2	Alfalfa		FM212786
	Idas sp.	1	Alfalfa		FM212787
	Prionospio sp.	1	Wood		FM212788
	Prionospio sp.	2	Alfalfa		FM212789
	Prionospio sp.	3	Alfalfa		FM212790
	Prionospio sp.	4	Alfalfa		FM212791
	Prionospio sp.	5	Alfalfa		FM212792

Table 4 .

 4 Univariate indices of colonist assemblage structure from the different substrates withinCHEMECOLIs deployed: at the Nile Deep-Sea Fan: short-term experiment, MW1 (wood substrate), MC1 (carbonate substrate), and long-term experiment, MW2 (wood substrate), MA2 (alfalfa substrate) and MC2 (carbonate substrate); at Rainbow: RW (wood substrate), RA (alfalfa substrate) and RC (carbonate substrate); at Haakon Mosby Mud Volcano (HMMV): HW (wood substrate), HA (alfalfa substrate) and HC (carbonate substrate). S: species richness, N: the number of individuals, H': Shannon-Wiener diversity and J': Pielou's evenness. Description of a CHEMECOLI: a: colonization device filled with cubes of Douglas fir (scale bar equalled to 3.3 cm); b: carbonate substrate; c: wood substrate; d: alfalfa dried substrate.

		S	N	J'	H'
	Nile Deep-sea Fan
	2 weeks
	MW1	8	75 0.64 1.33
	MC1	1	3	-	-
	1 year
	MW2	10 123 0.54 1.24
	MA2	12 86 0.73 1.80
	MC2	3	5 0.86 0.95
	Rainbow
	1 year
	RW	5	46 0.76 1.23
	RA	3	10 0.73 0.80
	RC	2	4 0.81 0.56
	HMMV
	1 year
	HW	10 2398 0.01 0.22
	HA	9	78 0.74 1.62
	HC	3	19 0.6 0.66

M A N U S C R I P T A C C E P T E D

Table 3. List of taxa identified within CHEMECOLIs and their estimated densities per dm 3 within the device. N: is for taxon that is a new record for the site and X is for taxon that has been recorded already in the surrounding habitat. 1) short-term experiment at the Nile Deep-Sea Fan: MW1 (wood substrate) and MC1 (carbonate substrate); 2) long-term experiment at the Nile Deep-Sea Fan: MW2 (wood substrate), MA2 (alfalfa substrate) and MC2 (carbonate substrate);

3) long-term experiment at Rainbow: RW (wood substrate), RA (alfalfa substrate) and RC (carbonate substrate; 4) long-term experiment at HMMV: HW (wood substrate), HA (alfalfa substrate) and HC (carbonate substrate).