Influence of chemosynthetic substrates availability on symbiont densities, carbon assimilation and transfer in the dual symbiotic vent mussel Bathymodiolus azoricus

High densities of mussels of the genus Bathymodiolus are present at hydrothermal vents of the Mid-Atlantic Ridge. It was already proposed that the chemistry at vent sites would a ﬀ ect their sulphide-and methane-oxidizing endosymbionts’ abundance. In this study, we conﬁrmed the latter assumption using ﬂuorescence in situ hybridization 5 on Bathymodiolus azoricus specimens maintained in a controlled laboratory environment at atmospheric pressure with one, both or none of the chemical substrates. A high level of symbiosis plasticity was observed, methane-oxidizers occupying between 4 and 39% of total bacterial area and both symbionts developing accordingly to the presence or absence of their substrates. Using H 13 CO − 3 in the presence of sulphide, 10 13 CH 4 or 13 CH 3 OH, we monitored carbon assimilation by the endosymbionts and its translocation to symbiont-free mussel tissues. Although no signiﬁcant carbon assimilation could be evidenced with methanol, carbon was incorporated from methane and sulphide-oxidized inorganic carbon at rates 3 to 10 times slower in the host muscle tissue than in the symbiont-containing gill tissue. Both symbionts thus contribute ac-15 tively to B. azoricus nutrition and adapt to the availability of their substrates. Further experiments with varying substrate concentrations using the same set-up should provide useful tools to study and even model the e ﬀ ects of changes in hydrothermal ﬂuids on B. azoricus ’ chemosynthetic nutrition.


Conclusions References
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Introduction
Mytilids of the genus Bathymodiolus are among the dominant fauna inhabiting sulphide-hydrocarbon cold seeps and hydrothermal vents worldwide (Sibuet and Olu, 1998;Van Dover, 2000).Their nutrition seems to be mainly supported by sulphideand/or methane-oxidizing bacterial (SOB, MOB) endosymbionts, located in specialized gill epithelial cells (Fisher, 1990).In fact, the coexistence of two distinct bacterial symbionts within a single cell of a multi-cellular eukaryote was demonstrated for the first time ever in the gills of Bathymodiolus spp.(Cavanaugh et al., 1987(Cavanaugh et al., , 1992;;Fisher et al., 1993).B. puteoserpentis and B. azoricus, the two mussel species present at Mid-Atlantic Ridge (MAR) vent sites, display the same general characteristics, with two distinct morphotypes of Gram-negative endosymbionts in gill bacteriocytes, and the presence of enzymes specific for sulphide and methane oxidizing metabolisms (type I ribulose 1,5-bisphosphate carboxylase/oxygenase, ATP sulfurylase, adenylyl sulfate reductase, and methanol dehydrogenase) (Robinson et al., 1998;Fiala-M édioni et al., 2002).Phylogenetic analyses show that the two Mytilid species share the same 16S rRNA phylotype of sulphide-oxidizing Gamma-proteobacterium, but harbour two distinct strains (Won et al., 2003).Duperron et al. (2006) found that the MOB 16S rRNA phylotype was also shared between the two mussel species, and that vent chemistry could affect the relative abundance of SOB and MOB.The volume occupied by each type of symbiont present within a bacteriocyte, quantified using a new 3-D fluorescence in situ hybridization (FISH) technique, varies from vent site to vent site between B. azoricus specimens (Halary et al., 2008).However, although physiological activity of the symbionts was observed in gill preparations of B. thermophilus (Nelson et al., 1995), B. puteoserpentis (Robinson et al., 1998) and in live specimens of the seep Bathymodiolid from the Gulf of Mexico (Childress et al., 1986;Fisher and Childress, 1992;Kochevar et al., 1992;Lee and Childress, 1995), it has to date not been demonstrated in live specimens of B. azoricus.
Stable isotope studies on Bathymodiolids have indicated the presence of two isotopically (and hence, nutritionally) distinct groups of mussels at the MAR (Trask and Van Dover, 1999;Fiala-M édioni et al., 2002;Colac ¸o et al., 2002a).A first group (δ 13 C∼−30‰) would depend mostly on SOB, while the heavier isotopic values of a −20‰ group could be attributed to a larger reliance on MOB.B. azoricus is a complex organism which can derive its carbon and nitrogen from two different symbionts, particulate organic matter and possibly dissolved organic matter.In such a case, the analysis of stable isotopes at natural abundance in bulk tissue, giving information on the "average" diet over a period of time, is insufficient to accurately delineate the con- tributions from the different food sources.Incubations with 13 C-enriched substrates are commonly used to study the activity of pure bacterial cultures as well as different complex samples taken from natural and artificial ecosystems and soils.Autotrophic SOB were already described performing such tracer experiments with 13 C-labelled bicarbonate (Knief et al., 2003a), as were communities of physiologically active MOB with 13 CH 4 (Knief et al., 2003b).In the present study, we investigated symbiont populations and mussel nutrition in specimens of B. azoricus collected from the MAR and kept in aquaria under various conditions in a controlled laboratory environment (LabHorta facility in the Azores, Colac ¸o and Santos, 2002b).Stable isotope tracer experiments were performed with 13 C-enriched bicarbonate, methane or methanol, followed by analysis of 13 C incorporation in the gill and muscle tissues.Densities of symbionts were estimated from FISH images using symbiont-specific probes.Results provide evidence that the symbionts develop according to the presence or absence of their substrates, and that inorganic carbon and C 1 substrates are assimilated and further translocated into symbiont-free mussel tissues.

Study site and animal collection
Acoustically retrievable cages were moored in August 2006 during the MOMARETO cruise (R/V "Pourquoi Pas?" IFREMER).They were positioned on diffuse venting areas at the Mid-Atlantic Ridge (MAR) hydrothermal vent field Menez Gwen (MG: 37  Kochevar et al., 1992).Seawater was replaced every second day (pH 7.5-9) and oxygenated using compressed air to 12.5-67.5%oxygen saturation.A sulphide solution (20 mmol L −1 Na 2 S in filtered seawater, pH 8.6-9.2) was dispensed discontinuously for 15 min every hour, using a peristaltic pump injecting 2 mL min −1 .Concentrations of 1 to 30 µmol L −1 were measured using the colorimetric diamine test, in the range of the 0 to 62 µmol L −1 measured at Northern MAR vent sites on mussel beds (Sarradin et al., 1998).Methane was bubbled continuously in seawater and dissolved methane monitored by sampling 60 mL seawater in a 300 mL sampling bottle rolled for 20 min before measuring the headspace concentration with a Gasurveyor 524.High methane concentrations above 300 µmol L −1 were found inhibitory for symbionts of B. childressi exhibiting high methane consumption rates (Kochevar et al., 1992), we thus tried to maintain its concentration below this level (8-91 µmol L −1 ).Mussels were sampled before the January experiments (n=3), and after 22 (n=3), 32 (n=3), 38 (n=1), 42 (n=3) and 52 (n=3) days maintenance of May mussels.The effect of maintenance was assessed statistically through Kruskal-Wallis non-parametric analyses.Temperature, pH and oxygen levels were monitored.One specimen of each experiment was sampled after 1 day enrichment, 3 specimens after 4 days (5 days in the case of the methane experiment) and 10 after 20 days (15 days with methane).At each sampling, mussels were opened and rinsed in distilled water to discard unincorporated tracers.The most anterior part of the left gill was removed from the specimens collected after 4 or 5 days experiment and fixed for FISH analysis in 2% formaldehyde.
All specimens (wild and from the maintenance and tracer experiments) were dissected into gill, mantle, muscle and remaining tissues which were frozen and lyophilized.Each tissue's dry weight was determined and a gill index (GI) was calculated from the gill tissue dry weight and shell length according to the following formula: GI = [gill tissue dry weight (g)/shell length (mm)] × 100 (1) BGD

Fatty acids extraction
Lyophilized tissues were ground to a fine powder using a mortar and pestle.Lipids were extracted using a modified version of the Bligh and Dyer method (White et al., 1979) using chloroform/methanol/phosphate buffer pH 7.4 in 1:1:0.9volume proportions.Total lipids retrieved in the chloroform phase were partitioned on silicic acid columns by sequential elution with chloroform, acetone, and methanol into apolar, neutral and polar lipids.Portions of the lipid extracts were transferred into 10 mL screw-top test tubes, previously decontaminated for 4 h at 450 • C, to which a fresh solution of methanol/hydrochloric acid/chloroform (10:1:1 volume, 3 mL) was added for a transmethylation reaction run at 90 • C for 60 min to form fatty acid methyl esters (FAMEs).

Stable isotope analyses
FAMEs chloroform extracts were transferred and dried under mild conditions (37 • C, to avoid the evaporation of the most volatile FAMEs) into tin capsules (for liquids) previously decontaminated with acetone.Aliquots of lyophilized tissue powder were weighed into silver cups, previously decontaminated for 4 h at 450 • C, and acidified with a few drops of dilute HCl (5%) before analysis, to remove any possible trace of carbonates.Tissues were re-dried overnight at 60 • C. δ 13 C analyses were performed on a Flash1112 elemental analyzer, coupled to a Delta V via a Conflo III interface (Thermo Finnigan).All carbon stable isotope ratios are expressed relative to the conventional standards (VPDB limestone) as δ values, defined as: Or as Atom percent values, defined as: The reference material used as standard for carbon isotopic ratio measurement was sucrose (IAEA-CH-6).The standard followed the same analytical processes as the unknown samples.Net carbon incorporation in the mussel tissues (C inc , in µmol C g tissue −1 ) was calculated using the following formula: where A exp is the 13 C atom percent measured in the mussel after the tracer experiment, A control is the 13 C atom percent measured in the control mussel, C tissue is the carbon content of the tissue analyzed (µmol C g tissue −1 ) and A substrate the 13 C atom percent of the substrate used in the experiment (i.e.25% for CH 4 , as given by the supplier, or 16.3% for NaHCO 3 and 22.2% for CH 3 OH, as calculated from the preparation).

Fluorescence In Situ Hybridization (FISH)
FISH was performed according to the protocol used by Halary et al. (2008) with a few modifications.Upon recovery of mussel specimens, the most anterior part of their left gill was fixed in 2% formaldehyde (2-4 h, 4 • C), rinsed twice with filtered seawater, and stored at −20 • C in 1:1 ethanol/filtered seawater.In the lab, gill tissue was embedded in polyester wax and 10 µm-thick sections were cut using a microtome (JUNG, Heidelberg, Germany), collected on Superfrost ® Plus slides (Roth, Germany), and hybridized as previously described (Halary et al., 2008)  Total area occupied by bacteria and respective proportions of each phylotype were computed for the whole image.For an inter-comparison between images, bacterial areas were normalized by dividing areas by the total length of filaments visible and measured on each picture.

Maintenance conditions effects
Before the tracer experiments, mussels were maintained in aquarium at atmospheric pressure in the presence of 30±16 µmol L −1 CH 4 (8-92 µmol L −1 ) and 12±9 µmol L −1 H 2 S to support the presence of endosymbionts.Mussels collected in January were allowed to recover from the decompression stress during 5 days at atmospheric pressure (transport followed by LabHorta).A quick drop of their gill index (GI) could be noticed over this short period (Fig. 1).This drop was also observed in May mussels maintained with H 2 S and CH 4 up to 38 days before the tracer experiments (aquarium mean GI=0.46, n=11; wild animals mean GI=0.76, n=10, Kruskal-Wallis p=0.001,Fig. 1).Following this rapid loss in gill weight, no significant difference could further be evidenced between individuals collected at different time points of the maintenance period (Kruskal-Wallis p=0.475).GIs showed a significant decrease after 20 days tracer experiment (symbolized by an arrow on Fig. 1) with H 2 S (Kruskal-Wallis p=0.016, n=10), a trend also observed for mussels kept in filtered seawater with dissolved enriched CH 4 .inorganic carbon was also observed in the absence of H 2 S, although to a much lesser extent.This coupling between HCO − 3 assimilation and the presence of H 2 S was also observed in analyses of fatty acid methyl esters (FAMEs, including a small contribution from unlabeled C from the trans-methylation) from the polar lipid extracts of the different control experiments (Fig. 3).δ 13 C analysis of FAMEs from lipid fractions of mussel A (the mussel specimen displaying the highest δ 13 C values after January experiments) showed highest enrichment in polar fatty acids containing the phospholipid fatty acids (PLFA), followed by the neutral fatty acids found in part in glycolipids or as free fatty acids.A significantly lower increase in δ 13 C was found in fatty acids associated with the apolar lipid fraction among which can be found di-and tri-acylglycerols and cholesterol derivatives.

Fixation rates
After 4 and 5 days of exposure to 13 C-labelled HCO − 3 (in the presence of H 2 S) or CH 4 , B. azoricus gill tissue already showed 13 C abundances significantly higher than background levels (Kruskal-Wallis p=0.049 for both treatments).At each sampling time, δ 13 C values tended to be higher in gill than in muscle tissue.Labeling of the gill tissue appeared uniform between the different mussel specimens after 4 days in H 2 S.However, strong differences in δ 13 C between individuals were observed after 15 days with enriched CH 4 and 20 days with H 2 S and enriched NaHCO 3 .In the gill tissue, carbon incorporation rates varied from 151 to 323 nmol C g dry tissue + labeled NaHCO 3 experiment, and from 56 to 228 nmol C g dry tissue −1 h −1 in the experiment with labeled CH 4 (Fig. 4, left panel).Although mussels seemed to incorporate efficiently enriched carbon from CH 3 OH during the first days of the experiment, no further enrichment could be observed after the last 16 days.No incorporation rate was thus calculated for the experiment with methanol.
Under the hypothesis that carbon is assimilated by endosymbionts located in the gill tissue and then transferred to other tissues (see discussion) rates of carbon transfer Introduction

Conclusions References
Tables Figures Printer-friendly Version Interactive Discussion can be calculated.Transfer between gill and muscle tissues ranged from 52 to 104 nmol C g dry tissue −1 h −1 in the H 2 S + labeled NaHCO 3 experiment, and from 11 to 21 nmol C g dry tissue −1 h −1 in the CH 4 experiment (Fig. 4, right panel).

Endosymbiont populations' response to experimental treatments
Gill filaments from May mussels were observed using FISH to assess the effects of the maintenance period in LabHorta and of the tracer experiments on symbiont populations (Fig. 5).Symbiont quantification results for a wild specimen, a specimen collected at the start of the experiments (after the acclimatization period) and others after 4 to 5 days in the absence or presence of either or both CH 4 and H 2 S are displayed in Table 1.Gill filaments of aquarium specimens appeared narrower than the ones from a wild mussel dissected immediately after the cage recovery, and displayed much less bacteria (Fig. 5a-b).Quantifications confirmed a decrease in area covered by bacteria by a factor of 4 (from 16.7 to 3.7 µm 2 /µm filament), and indicated a shift in symbiont relative areas, with methane-oxidizing bacteria (MOB) representing 14% of the total area occupied by bacteria after acclimatization, versus 24% before (Table 1).
At the beginning of the tracer experiments, bacterial densities remained low.A mussel kept in CH 4 +H 2 S, but at a different ratio with decreasing H 2 S and 1/3rd lower CH 4 concentrations than during acclimatization, presented an overall lower bacterial area (1.7 µm 2 /µm filaments versus 3.7 before the tracer experiments, Fig. 5c).In these conditions, sulphide-oxidizing bacteria (SOB) area appeared to decrease slower than the MOB area, when compared with the mussel collected at the end of the acclimatization period.A mussel kept four days in filtered seawater devoid of CH 4 and H 2 S also displayed a decrease in total area occupied by bacteria down to 2. bacterial area (Fig. 5f).

Discussion
Because of its capacity to recover and survive in aquaria for several months after the post-retrieval decompression stress, increasing data is being obtained from live B. azoricus maintained in aquaria at atmospheric pressure in the presence or absence of chemosynthetic substrates for the endosymbionts.Immune responses (Bettencourt et al., 2007), biochemical responses to recovery stress (Dixon et al., 2004) and to metal exposure (Company et al., 2008) have been investigated using such settings.Dixon et al. (2004) saw higher levels of DNA damage in mussels kept in the presence of methane and sulphide than in some kept without any "food supplement".In a recent study Bettencourt et al. (2008) investigated the physiological state of mussels during acclimatization to atmospheric pressure in the absence of dissolved gases over the course of several months post-capture and concluded that our laboratory set up was a suitable system to study physiological reactions.Response of symbionts to the experimental conditions was barely examined.A preliminary study done on sulphideoxidizing bacterial symbionts (SOB) showed, on transmission electron micrographs, the loss and potential re-acquisition of SOB (Kadar et al., 2005).
In this study we tried to gain insight on the symbiont densities, physiology and impact on the nutrition of B. azoricus nutrition.Results from FISH observations and GIs indicate that despite a strong post-collection initial stress, with a marked decrease in total symbiont abundances and gill dry weight, both bacterial populations can be maintained in the gill tissue of mussels kept in aquaria.Furthermore, incubation experiments indicate that the presence of either sulphide or methane alone favors the increase in absolute and relative abundance of SOB and methane-oxidizing bacterial symbionts (MOB), respectively.This supports the hypothesis that symbiont populations quickly react to changes in environmental parameters, in particular to the balance between sulphide and methane, and thus optimize the use of available compounds as suggested

Conclusions References
Tables Figures Printer-friendly Version Interactive Discussion by previous authors (Trask and Van Dover, 1999;Colac ¸o et al., 2002;Salerno et al., 2005;Duperron et al., 2006).The range of variation observed in symbiont relative abundances, with MOB occupying between 4 and 39% of total bacterial area, confirms the high level of plasticity of the dual symbiosis.In the absence of both sulphide and methane, MOB seem less affected than SOB, which could be due to their ability to use alternate carbon sources, for example by expressing ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) as demonstrated in a vent Bathymodiolid from the Mid-Okinawa Trough (Elsaied et al., 2006).The rate of digestion of each symbiont by the mussel, combined with their respective size (1.25 µm mean diameter for MOB, 0.35 µm for SOB; Fiala-M édioni et al., 2002) could also have a considerable impact on MOB/SOB surface ratios evolution with time.Although data must be taken with caution as only one individual per experimental condition was analyzed, the fact that observations qualitatively agree with expected results is a strong indication that our set-up is appropriate for the enrichment of each endosymbiont.Recently, a 3-D-FISH technique was employed to quantify the effect of a sulphide pulse in a similar experimental set-up, on the volume and relative abundance of symbionts in mussel gills (Halary et al., 2008).We herein provide the first indication that MOB also respond to a methane pulse.
Stable isotope tracer experiments evidenced that H 2 S is needed for the assimilation of inorganic carbon and that, within the gill lipid fractions, this carbon is first incorporated into phospholipids which are major components of Gram-negative bacterial membranes (Kates, 1964).This confirms the chemoautotrophic sulphide-oxidizing activity of one of the endosymbionts in our experimental setting.We could also see a slight assimilation of H 13 CO − 3 in the absence of H 2 S.This could be explained by the hypotheses that i) Sulphur or thiosulfate reserves are present in the gill tissue, although granules of elemental sulphur were never observed in B. azoricus; ii) MOB would harbor a RubisCO activity as observed in type X-methane oxidizers (Baxter et al., 2002) and in Bathymodiolus sp.(Elsaied et al., 2006); or iii) SOB is capable of using ATP from the host mussel to assimilate inorganic carbon.However, this carbon incorporation could be considered negligible when the δ carbon incorporated, and more data is needed to confirm that it is significant.Distribution of labeled carbon in tissues suggests that in the case of carbon uptake by SOB, inorganic carbon is first incorporated by bacteria within the gill tissue, then transferred to the rest of the tissues including the digestive system and vital organs, muscle and mantle tissues being the last ones to get this carbon.Although enrichment of the different tissues is only displayed for a single mussel here, we could observe the same pattern in 2 additional mussels from other enrichment attempts with H 2 S and NaH 13 CO 3 (data not shown).This hypothesis of carbon transfer from the gills to symbiont-free tissue was suggested for a seep Bathymodiolus sp. by Fisher and Childress (1992) who could observe an increase of radiolabel coming from 14 CH 4 in symbiont-free tissues during a chase period.The latter authors suggested that the transfer might occur by intracellular digestion of the symbionts, from the observation of partially degraded bacteria in bacteriocyte bodies with lysosomal activity (also observed in B. azoricus by Fiala-M édioni et al., 2002).This could explain the fact that tissues in direct connection with the gill would benefit first from the carbon assimilated by SOB.Alternatively, the symbionts, grown into the mussel cell, could have been released in the food grove of the gills and passed through the digestive system.Slow transfer to muscle tissue, also observed with carbon incorporated by MOB, may also reflect differences in turnover: the gill tissue harbors bacteria that are continuously doubling and being digested, and hence show a 13 C abundance more like that of the symbionts; muscle tissue, in contrast, contains exclusively C accumulated through the entire life of the mussel, and its 13 C abundance, therefore, is less responsive to recently ingested food.Rough calculations gave carbon incorporation rates 3 times lower in muscle tissue than in symbiont-containing gill tissue for SOB and 5 to 10 times lower for MOB.Kochevar et al. (1992) described in more details the characteristics inherent to MOB from a seep Bathymodiolid in a study using 14 CH 4 .They showed that about 70% of the consumed methane was being incorporated as organic compounds and the highest net carbon incorporation rate was obtained at 250 µmol L −1 dissolved methane, with 5 µmoles carbon incorporated per gram whole mussel soft tissue wet weight per hour.Our experiments were performed at 5 times lower methane concentration, and as rates depend upon concentration of substrates and are probably influenced by the physiological state of the animals and bacteria, we can not extrapolate our results to mussels in their home environment.However, these rates estimates, reported as amounts of carbon incorporated per dry specific tissue weight per hour, appear realistic in view of the results reported for B. childressi (Kochevar et al., 1992).
In an attempt to describe the physiology of MOB symbionts in more detail, we tested the possibility of C incorporation from methanol.Indeed, methanol dehydrogenase, necessary for growth on either methane or methanol, was found to be active in the gills of B. azoricus (Fiala-M édioni et al., 2002), and putative methanol-oxidizing symbionts were identified in the seep mussel B. heckerae by Duperron et al. (2007).Methanol, if available, would seem a better carbon and energy source compared to methane because the first step of methanotrophy, producing methanol, does not yield energy.However, no significant carbon uptake from methanol was observed, questioning whether symbionts can even get access to this potential resource.
Although working at atmospheric pressure, the LabHorta aquarium setting can thus be employed to study the dynamics of symbiont populations.In the future, pulses of varying intensities and durations or parallel measurements of rates of symbiont digestion by the host should be foreseen.Together with the identification of biomarkers for the endosymbionts following tracer experiments, such experiments should provide useful tools to study the effects of changes in hydrothermal fluids on the chemosynthetic nutrition of the Mid-Atlantic Ridge mussel and gain an insight on the extent to which the mussel beds interact with and impact their surroundings.Mid-Atlantic Ridge: evidence for a dual symbiosis, Mar. Biol., 141, 1035-1043, 2002. Fisher, C. R.: Chemoautotropic and methanotrophic symbioses in marine invertebrates, Crit. Rev. Aquat. Sci., 2, 399-436, 1990.Fisher, C. R. and Childress, J. J.: Organic carbon transfer from methanotrophic symbionts to the host hydrocarbon-seep mussel, Symbiosis, 12, 221-235, 1992.
using the ATTO488labelled ImedM probe (5 ′ -ACCAGGTTGTCCCCCACTAA-3 ′ ,Duperron et al., 2008) specific for the methane-oxidizing symbiont and the Cy5-labelled BangT-642 probe (5 ′ -CCTATACTCTAGCTTGCCAG-3 ′ ,Duperron et al., 2005) specific for the sulphideoxidizing symbiont.Images were acquired using a BX61 microscope (Olympus Optical Co., Tokyo, Japan) under a 10× objective (NA 1.30) and analyzed using the ImageJ software(Abramoff, 2001).The green channel corresponding to the ImedM probe (methane-oxidizers) and the blue channel corresponding to the BangT probe (sulphideoxidizers) were binarized by applying a luminosity threshold to distinguish between background fluorescence and probe signal, and numbers of pixels belonging to each type of symbiont were computed.

3. 2
Tracer repartition in tissues and specific compound classes Assimilation of 13 C from HCO −

Fig. 1 .
Fig.1.Variations in gill index with time spent in aquarium and with the presence of methane and/or sulphide (mean±SD).Empty triangles: January fresh and control mussels exposed to methane and sulphide, filled circles: May fresh and control exposed to methane and sulphide, empty circles: May sulphide + bicarbonate tracer mussels, filled triangles: May methane tracer mussels.
−1 ).Two control experiments were run in parallel with either no addition of sulphide or the addition of sulphide with un-enriched bicarbonate.

Table 1 .
Results from rough endosymbiont quantifications obtained by FISH image processing.CH 4 / H 2 S ratios are indicative, and in the case of fresh mussels, the value of 1.2 reported is a mean from end-member fluids at Menez Gwen (0.67-1.75, Desbruy ères et al., 2000), which might not be representative of the situation in the immediate environment of mussels.CH 4 / H 2 S Surf Symb µm 2 / % MOB % SOB MOB/SOB Fil length µm