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Reproductive traits of the cold-seep symbiotic mussel Idas modiolaeformis: gametogenesis and larval biology.

Introduction

Cold seeps, hydrothermal vents, and wood- and whale falls are reducing habitats that cover small areas and are typically dynamic and distant and isolated from one another. For these reasons they are referred to as oases in the deep sea (Lutz et al., 1984; Carney, 1994). A desire to understand how nascent habitats are quickly and densely colonized by endemic chemosynthetic fauna that in most cases are completely absent from the background deep-sea environment has raised the interest of scientists. As a result, the biogeography and population genetics of chemosynthetic metazoans are being investigated to understand their distribution patterns and speciation modes and to describe the deterministic evolutionary processes at work (Vrijenhoek, 2010a). Because the disparate nature of the sites requires long-distance dispersal of the organisms (Olu et al., 2010), the reproduction and development of chemosynthetic metazoans also became a topic of interest (Tyler et al., 2007a, 2009; Arellano and Young, 2009). However, since sampling and studying early stages of deep-sea fauna is fraught with logistical difficulties, we are still missing key information on the life-history traits and the dispersal capabilities of symbiotic metazoans. This is a key limitation in understanding the connectivity (Becker et al., 2007; Vrijenhoek, 2010a) of chemosynthetic ecosystems, and thus in evaluating the resilience disturbance, be it natural (Mullineaux et al., 2010) or anthropogenic.

Within the Mytilidae family (~250 species), a single clade (Bathymodiolinae) includes all species living in deep-sea cold seeps, hydrothermal vents, and organic falls (Lorion et al, 2010). Gill epithelial cells of chemosynthetie bivalves harbor abundant bacteria (Fujiwara et al., 2010; Duperron, 2010), in most cases chemoautotrophic sulfur-oxidizers. Within the bivalves, the Bathymodiolinae are unusual in that some species harbor methanotrophic bacteria that use methane as both carbon and energy sources. Symbionts are thought to be acquired after spawning, with juveniles being mixotrophic (Le Pennec and Beninger, 2000; Martins et al., 2008; Vrijenhoek, 2010b). To date, the best-studied life cycle is that of "Bathymodiolus" childressi, a methane-seep species from the Gulf of Mexico, in which gametogenesis, spawning, and larval development have been documented (Eckelbarger and Young, 1999; Tyler et al., 2007a; Arellano and Young, 2009). The species is gonochoric, with seasonal reproduction and potentially teleplanic larvae (Arellano and Young. 2009). A smaller mytilid, the whale-bone-associated Idas washingtonia, is a protandric hermaphrodite displaying high fecundity in females and having planktotrophic larvae (Tyler et al., 2009). Different strategies for adaptation to ephemeral habitats thus seem to exist in Bathymodiolinae.

In this study we investigated Idas modiolaeformis, a small mytilid species that was found associated with various substrates at cold seeps in the eastern Mediterranean Sea, including experimental wood colonization devices and autogenic carbonate crusts (Olu-Le Roy et ai9 2004; Duperron et al, 2008; Gaudron et al., 2010; Ritt et al., 2011; Lorion et al., 2012). Marker genes were sequenced (COI, 18S, and 28S) and indicated a very close phylogenetic relationship with I. macdonaldi from the Gulf of Mexico, suggesting a recent divergence of these species despite the distance now separating them (Lorion et al., 2012). I. modiolaeformis hosts six distinct bacteria, symbionts including sulfur- and methane-oxidizing bacteria, whereas its relative hosts only sulfur-oxidizers (Duperron et al., 2008). Despite these intriguing features, no data are currently available on the reproduction and larval development of I. modiolaeformis. We documented some features of its reproductive strategy and the type and potential dispersal abilities of larvae involved, and we tested whether symbionts occurred in the gonad tissue. For this we employed (1) histological techniques to document the reproductive status of adult specimens; (2) fluorescent in situ hybridization (FISH) techniques to test the occurrence of symbionts within gonad tissue and gametes; and (3) scanning electron microscopy (SEM) to document larval shell morphology and potential larval dispersal.

Materials and Methods

Sampling

Specimens of Idas modiolaeformis (Sturany, 1896) (Bivalve, Mytilidae, Bathymodiolinae) were sampled during two cruises at cold seeps in the eastern Mediterranean Sea (Table 1), namely the cruise MEDECO-2 aboard RV Pourquoi Pas? with the ROV Victor 6000 in 2007 and the cruise MSM 13_3/4 aboard RV Maria S. Merian with the ROV Quest 4000 in 2009. Mussels were collected from the cold-seep site Central Zone 2A (Pockmark area), from the mud volcanoes (MVs) Amon and Cheops at the Nile Deep Sea Fan area and at the MV Amsterdam in the Anaximander Mounts (Fig. 1). I. modiolaeformis specimens were collected on carbonate crusts and on artificial organic falls (large wood logs or CHEMECOLIs) (Gaudron et al., 2010; Boggemann et al., 2011) (Table 1). CHEMECOLIs are colonization devices filled either with alfalfa grass or with pine wood cubes that allow recruitment of young stages of I. modiolaeformis and were deployed for one year at the cold-seep site Central Zone 2A in Pockmark area. Data are labeled according to the PANGAEA database (http://www.pangaea.de/).

Table 1
Sampling data for specimens of Idas modiolaeformis collected
in the eastern Mediterranean

                                                  Depth
Cruise    Date   Dive   Specimens      Site      (m)

M BRIAN   30      239  Idas 1 to 5  Central      1699
          Nov                       pockmark
          2009

MSM13-3
          5 Nov   244  Idas 6 to    Amon Mud     1158
          2009         10
                                    Volcano

M BRIAN   22      252  Idas 11&12   Amsterdam    2028
          Nov                       Mud
          2009

MSM 13-4                            Volcano
          25      254  Idas 13 to   Amsterdam    2031
          Nov          15           Mud
          2009
                                    Volcano

MEDECO-2  8 Nov   336  Idas 16 to   Central      1686
          2007         20;          pockmark
                       24

          10      337  Idas 21 to   Central      1694
          Nov          23           pockmark
          2007

          20      343  Idas 26 to   Cheops Mud   3000
          Nov          29           Volcano
          2007

          17      342  Idas 30      Cheops Mud   3015
          Nov                       Volcano
          2007

          10      338  Post-larval  Central      1693
          Nov          Idas         pockmark
          2007

Cruise          Location            Substrate (label)

M BRIAN   32[degrees]53.435'N;  Sunken wood
MSM13-3   30[degrees]35.203'E   (MSM 13-3_918-1_WOOD1)
          32[degrees]36.755'N;  Carbonate crust
          31[degrees]70.450'E   (MSM13-3_944-l_GEOBOXl+2)

M BRIAN   35[degrees]20.033'N;  Carbonate crust

MSM 13-4  30[degrees]16.170'E   (MSM13-4_984_CARB)
          35[degrees]20.079'N;  Carbonate crust
          30[degrees]16.131'E   (MSM13-4_994_CARB)

MEDECO-2  32[degrees]30.030'N;  Carbonate crust
          30[degrees]15.604'E   (MEDECO2_D336_PANIER)
          32[degrees]32.060'N;  Carbonate crust
          30[degrees]21.36'E    (MEDECO2_D337_PANIER)
          32[degrees]08.484'N;  Carbonate crust
          28[degrees]09.696'E   (MEDECO2_D343_PANIER)
          32[degrees]08.489'N;  Carbonate crust
          28[degrees]09.697'E   (MEDECO2_D342_PANIER)
          32[degrees]31.977'N;  CHEMECOLI alfalfa (M70/
          30[degrees]21.178'E   2b_833_TRAC-11)CHEMECOLI
                                wood (M70/2b_833_TRAC-13)


Specimen preservation

Aboard ship, the 29 specimens allocated to this study were fixed either in buffered 2.5% glutaraldehyde or in Trump's fixative (McDowell and Trump, 1976), except for the ones retrieved from the CHEMECOLIs, because whole wood cubes and alfalfa grass were fixed directly in 4% formaldehyde. In all cases, samples were stored at 4 [degrees]C. In the laboratory, cubes or alfalfa grass were sorted under a dissecting microscope to look for post-larvae and juveniles (Gaudron et al., 2010). The specimens were then dissected, with shells saved for SEM, and the soft tissue of 17 specimens was used for histological analyses. Each adult shell (dissoconch) was photographed and measured under an Olympus SZX12 binocular microscope equipped with Image Pro Express 6.0.

Histology

Specimens were dehydrated using an increasing ethanol series (70% to absolute), rinsed in 100% buthanol-1, then subsequently in Histoclear, and embedded in paraffin (Peel a Way, melting point 52-54 [degrees]C). Serial sections, 8-[micro]m-thick, were cut using a microtome (Jung, Heidelberg) and deposited on SuperFrost Plus slides (Euromedex Ltd.). One in every five slides was stained using hematoxylin-eosin, and a coverslip was mounted with Permount. Histological sections were examined under an Olympus BX61 microscope equipped with ImagePro. Measurements were made of the acinus diameter in male and female gonads, and of spermatozoid acrosome length. For each female (see below), the diameter of at least 100 oocytes was measured and size-distribution was investigated.

Fluorescent in situ hybridization

Non-stained slides from 8 specimens of Idas modiolaeformis (3 females and 5 males) were used in FISH experiments to look for the presence of bacteria in reproductive organs. Paraffin was removed using a serial gradient of ethanol and Histoclear. Hybridizations were performed using probes targeting the bacterial 16S rRNA of all Eubacteria (probe Eub-338: 5'-GCTGCCTCCCGTAG-GAGT-3'), and of mussel-associated methanotrophic symbionts (ImedM-138: 5'-ACCATGTTGTCCCCCACTAA-3') (Amann et al., 1990; Duperron et ai7 2005; Duperron et &L, 2008). Probe Alf968b (5'-GGTAAGGTTCTKCGCGTT-3') was used as negative control, as Alphaproteobacteria do not occur in I. modiolaeformis. Probes were labeled with Cy3 or Cy5 fluorochromes and hybridized in a buffer containing 30% formamidc as described previously (Duperron et ai, 2008). Hybridized sections were mounted in SlowFade medium (Invitrogen, CA, USA). Images were obtained for each fluorochrome using the Olympus BX61 epifluorescence microscope (Olympus, Tokyo, Japan).

Scanning electron microscopy and larval shell measurements

Shells were cleaned in 9.6% sodium hypochlorite solution, rinsed with distilled water, dried at 60 [degrees]C overnight, and mounted on adhesive carbon discs. Observations were made using a ZEISS Supra 55 scanning electron microscope. Six larval shells (prodissoconch 1: PI) and 16 post-larval shells (prodissoconch II: PII) were measured. Lengths were measured as follows: greatest dimension parallel to the provinculum (PI), and greatest anteroposterior dimension (PII and adult shells); height for PI is the greatest dimension perpendicular to the hinge line and for PII and adult shells is the greatest dorsoventral dimension (Arellano and Young, 2009). PIIs and small dissoconchs of juveniles and post-larval shells of I. modiolaeformis recovered from CHEMECOLIs were also measured under an Olympus SZX12 binocular microscope equipped with ImagePro Express 6.0. The size of PII can be considered as the settlement size within the colonization device.

Results

Gonad morphology

In all 17 specimens investigated (Table 1), male and female gametes were localized within distinct acini occurring in the dorsal part of the visceral mass and in the region of the foot or the pedal retractor muscles surrounding the digestive tract (Fig. 2A, B). Shell heights ranged between 3.2 mm and 10.8 mm. Three specimens (17.6%) were females, displaying about 15 acini, each filled with either fully grown oocytes (in the most dorsal acini) or with oogonia at different stages of oogenesis (in the ventral acini). These females were rather large specimens (Table 1) with shell heights of 10.8 mm (Idas 1), 9.2 mm (Idas 8), and 7.3 mm (Idas 24). The 14 other specimens (82.4%) contained 6 to 8 acini filled with male gametes (stages from spermatogonia to spermatozoid) localized in the dorsal region, whereas the ventral region displayed about 15 female acini ([+ or -]S.D. = 131 [+ or -]56 [micro]m) showing signs of atresia. Indeed, the lumen was either empty or displaying evidence of lyzed gametes including disintegrated oocytes in which nucleus and nucleoli were absent (Fig. 3E). For this reason, these specimens were considered as physiologically male. Among these transition males, a single specimen (Idas 6, Table 1) contained some acini in the dorsal region where oogonia were observed at the periphery along with spermatids, where both gametes were present within the same single acinus (Fig. 3B).

Gametogenesis

The overall morphology of male and female acini was more or less similar. For male acini, germinal cells sat within the inner wall of the acinus, with spermatocysts, spermatids, and spermatozoids developing toward the lumen. Only two stages of maturity were observed, and no immature or spent stages were seen. The first stage was the ripe stage, in which the acinus was compressed and elongated with an irregular shape ([+ or -]S.D. = 186 [+ or -] 60 [micro]m; n = 6), was full of spermatocysts and spermatids, and displayed mature spermatozoids (Fig. 3A). The second stage was the spawning stage, where acini were deflated and displayed numerous spermatozoids and few spermatocysts (Fig. 3C, D). Specimens at this stage were either ready to spawn or in the process of spawning. The only "true" hermaphrodite (Idas 6, Table 2) had male acini at this stage, dominated by spermatozoids ([+ or -]S.D. = 3.2 [+ or -] 0.4 [micro]m; n = 19) and some spermatids ([+ or -]S.D. = 4.4[+ or -] 0.6 [micro]m; n = 13), co-occurring with vitellogenic oocytes ([+ or -]S.D. = 7[+ or -] 0.7 [micro]m; n = 5) located at the periphery of the acinus (Fig. 3b).

Table 2
Post-larval and adult shell dimensions of Idas modiolaetbrmis
specimens

                                PI length           PII([micro]m)
Species          Habitat        ([micro]m)             Length

Idas             Eastern        71.7-95.0           300-426

modiolaeformis   Mediterranean  78.6 [+ or -] 3.4   379.4 [+ or -] 11.7
                 seeps          (n = 6)             (n = 14)

"Bathymodiolus"  Northern Gulf  113.3 [+ or -] 2.0  432.7-453.6
childressi       of Mexico
                 seeps

Mytilus edulis   Shallow water  95                  260-320
                                   Dissoconch ([micro]m)

Species          Height            Length

Idas             276-395           5996 [+ or -] 4085

modiolaeformis   344 [+ or -] 5.0  406-14005
                 (n = 27)          (n = 44)

"Bathymodiolus"  --                --
childressi

Mytilus edulis   --                --

                                     Estimated
                                     minimal
                                     growth rate in
Species          Height              [micro]m 30 d.sup.-1    Teeth

Idas             5880 [+ or -] 3776  117.2 [+ or -] 34.6  18

modiolaeformis   363-12722           12.7-523.2
                 (n = 41)            (n = 14)

"Bathymodiolus"  --                  1440 [+ or -] 0.3    29-31
childressi

Mytilus edulis   --                  --                   --

Species          References

Idas             This study
modiolaeformis

"Bathymodiolus"  Arellano and
childressi       Young,
                 2009

Mytilus edulis   Lutz et al.,
                 1980

PI and PII are the Prodissoconch I and the Prodissoconch II.
Where applicable, mean, standard deviation, and minimum and maximum
values measured are cited; n is the total number of specimens measured.


In the ventral region of the visceral mass of female specimens, female gonads consisted of rounded acini containing pre-vitellogenic and vitellogenic (pedunculated) primary oocytes attached to the inner wall of the acinus (Fig. 4A). Fully grown vitellogenic oocytes were in prophase I of meiosis with developed nuclei and visible, eccentrically located nucleoli occurring within misshaped acini (the delimitation was difficult to identify), localized in the dorsal part of the mantle (Fig. 4b). Idas 1 (Table 1) harbored two cohorts of oocytes, one grouping fully grown vitellogenic oocytes from 14 to 43 /xm in diameter ([+ or -]S.D. = 32.5 [+ or -] 7.50 [micro]m; n = 40) and the second grouping oocytes undergoing pre-vitellogenesis and vitellogenesis ([+ or -]S.D. = 3.2 [+ or -] 0.9 [micro]m; n = 170) (Fig. 5). Idas 8 (Table 1) was at a more advanced maturing stage than Idas 1. The mean diameter of fully grown vitellogenic oocytes was 40.7 [+ or -] 7.8 [micro]m ([+ or -]S.D.; n = 35), and the mean oocyte diameter across oocytes undergoing pre-vitellogenesis and vitellogenesis was 3.8 [+ or -]1.6 /xm ([+ or -]S.D.; n = 151) (Fig. 5). Finally, Idas 24 was riper than Idas 8, with a mean fully grown vitellogenic oocyte diameter of 41.5 [+ or -] 7.6 [micro]m (n = 34) and a mean oocyte diameter during pre- and vitellogenesis of 5.0 [micro]m [+ or -]1.5 [micro]m ([+ or -]S.D.; n = 144) (Fig. 5).

Presence of symbionts

Specimens fixed in Trump's fixative or glutaraldehyde and embedded in paraffin were used for FISH. For future studies it must be noted that Trump-fixed tissue produced stronger signals with reduced autofluorescence. Positive, unambiguous signals were obtained on each section (n -- 8) when using the Eubacteria-targeting probe Eub338, whereas no signal was seen using probe Alf968b (n = 8). Strong Eub338 fluorescence was observed not only within the gills of I. modiolaefarmis, but also on the epithelium in the ventral part of the visceral mass, on the foot (Fig. 6A). In addition, positive signals were observed within the digestive tract, associated with boluses (not shown). No signal was observed within female (Fig. 6C) or male acini. Only in Idas 5 was a reduced signal identified in the dorsal part where male acini occur that appeared to be spirochaetes (Fig. 6E). The probe targeting methanotrophic symbionts (ImedM-138) hybridized with bacteria located in the gills of I. modiolaefonnis (n = 8) (Fig. 6D). This fluorescence was not observed within the male or female acini nor within any other location in I. modiolaeformis within the same histological section.

Larval biology

New recruits of I. modiolaeformis collected within CHEMECOLIs showed a clear reddish prodissoconch II compared to a whitish dissoconch (Fig. 7C, D). Length of prodissoconch I (Fig, 7A) ranged from 71.7 to 95 [micro]m

([+ or -]S.D. = 78.6 [+ or -] 3.4 [micro]m; n = 6). Eighteen teeth adorning one provinculum from a single adult specimen were counted under the scanning electron microscope (Table 2). Length of prodissoconch II ranged from 300 to 426 [micro]m ([+ or -]S.D. = 379.4 [+ or -] 11.7 [micro]m; n = 14).

Discussion

This paper is the first attempt to document reproductive features of a chemosynthetic metazoan from cold seeps in the Mediterranean Sea. To date, most of the research effort has focused on geology (Dupre et al., 2007; Foucher et al., 2009), biodiversity (Olu-Leroy et al., 2004; Gaudron et al., 2010; Ritt et al., 2011), trophic dynamics (Carlier et al., 2010), symbioses (Duperron et al., 2008; Brissac et al., 2011), and phylogeny (Lorion et al., 2012).

Reproduction

Idas modiolaeformis specimens display both male and female acini simultaneously, although all specimens except one displayed only male or female gametes. This suggests hermaphrodism over the course of an individual's life, but with relatively abrupt changes in sex rather than extended periods of simultaneous hermaphrodism. In our sample, out of the 17 specimens studied using classical histology, 13 were functionally male, 3 were functionally female, and one seemed to be a simultaneous hermaphrodite. These data are consistent with results from I. washingtonia, a wood- and whale-fall mussel, in which sex ratio was also skewed toward males, with only 10% of test organisms being females (Tyler et al., 2009). In that study, most putative female I. washingtonia shell lengths were larger (> 8 mm) than putative male shell lengths (< 4 mm), between which all sexual stages were identified, suggesting protandric hermaphrodism with possible switching of sex a number of times in between. In the present study, shell lengths for the three female I. modiolaeformis individuals were larger than 7 mm, whereas transition males shell lengths were all between 3.6 and 11.6 mm. Therefore a protandric hermaphrodism may also be the reproductive mode in this species, where bigger females will typically have higher fecundity and the availability of numerous males will increase the chance of external fertilization.

However, almost all transition males displayed female acini containing disintegrated oocytes, with only one specimen exhibiting limited remains of oogonia within developed male acini, which would tend to support a switch from female to male (proterogyn hermaphrodism) or repeated switches of sex. Immature oocytes in the periphery of a male acinus were previously documented in the hydrothermal vent mussel Bathymodiolus elongatus (Le Pennec and Beninger. 1997) and in the whalebone-associated I. washingtonia (Tyler et al., 2009). In these two species, numerous primary oocytes occurred within a male acinus. Atretic male gametes were observed in B. elongatus, where lysed spermatozoids were observed close to lipid-like substances and phagocytes (Le Pennec and Beninger, 1997). Germinal cells of acini in these species are therefore not likely to be genetically determined, and the sex of a specimen must be controlled by some epigenetic factor. This is seen in the echiurian Bonellia viridis (Berec et al., 2005). A possible epigenetic factor for I. modiolaeformis could be the proximity of fluid emissions as suggested by Tyler el al. (2009) for I. washingtonia, where the gender may be a function of the position on the whale skeleton with its concomitant access to hydrogen sulfide and oxygen.

In marine polychaetes, opportunistic sex change may be expected when the local habitat is characterized by severe fluctuations in food or mate availability, favoring one sex over the other-and where sex-switching may prove to be a beneficial strategy in species that are capable of wide larval dispersal but exhibit low mobility in adults (Sella, 2006). When individuals of the sulfide-tolerant polychaete species Capitella sp. I, which is known to occur within chemosynthetic habitats (Gaudron el al., 2010), are reared in small groups with a low proportion of females, males can develop eggs and function as either sex. This change from male to hermaphrodite was seen as an adaptation to living in small groups with strong local competition (Petraitis, 1985). Idas modiolaeformis docs not form the dense aggregates often observed in large Bathymodiolus spp.; instead, it occurs at low densities on any one given carbonate crust sample.

Another important adaptation to patchy and ephemeral habitats is rapid development to attain reproductive maturity. This has been demonstrated in the opportunistic wood-boring bivalve Xylophaga depalmai, with males reaching maturity within 50 days of wood-panel colonization (Tyler et al., 2007b). While size (and age) at maturation will need to be further explored in Idas modiolaeformis, some evidence is already available. All specimens investigated, including the smallest (3.2 mm in shell height), were mature. Recently, two opportunistic vent-colonizing gastropods, Ctenopelta porifera and Lepeiodrilus tevnianus, yielded size at first maturity above 2 mm when investigated, corresponding to only 1.5 months after colonization (Bayer et al., 2011). This emphasizes the need to investigate small specimens in greater detail.

Unfortunately, the two cruises on which specimens for this study were collected occurred at the same period of the year (October to December), precluding any examination of seasonality. Nevertheless, mature specimens were found, indicating that breeding occurs in Idas modiolaeformis during this period, whether it is seasonal or continuous. For example, Ctenopelta porifera and Lepetodrilus tevnianus mentioned above both employ quasi-continuous spawning (Bayer et al., 2011). The three I. modiolaeformis females carried two cohorts of oocytes, suggesting that while one batch of oocytes undergoes spawning, another one is maturing in preparation for subsequent release. Similarly, several stages of spermatogonia were observed within male acini, indicating that while the mature spermatozoids are released there is the capacity for new batches of spermatogonia to be produced. It was thought in the past that chemosynthetic species would display continuous reproduction due to the continuous supply of energy at vent and seeps, but seasonal variations in reproductive investment have subsequently been documented in many species, including the vent mussels Bathxmodiohts azoricus and "Bahymodiolus" childressi (Colaco et al., 2006; Tyler et al., 2007a), and in I. washingtonia (Tyler et al., 2009). Seasonality in gamete production has been attributed lo variations in the food available to the planktotrophic larvae, which in turn would be related to the vertical downward flux of spring bloom production. In the eastern Mediterranean, the major phytoplankton bloom occurs in winter from October to March (Krom et al., 2010), and this likely increases food availability for the I. modiolaeformis larvae released during the spawning period from October to December in this study.

Reproduction and bacterial symbiosis

1. modiolaeformis hosts six distinct bacteria associated with its gill epithelial cells, dominated by oxidizers of methane and sulfur (Duperron et al., 2008; Lorion et al., 2012). In this study we confirmed the occurrence of bacteria within gills of all specimens. As anticipated, within any one section the positive control was visible in the form of FISH signals in the gills. Although male and female acini, as well as gametes, were examined, no signal was identified from the application of either the general bacterial probe or the methanotroph-specific probe. This indicates that bacterial symbionts are acquired at a later stage in their lifecycle. Environmental acquisition in Bathymodiolinae is supported by several lines of evidence. For example, ultrastructural investigation of gonads did not show any prokaryotes in male and female gametes in "Bathymodiohts" childressi (Eckelbarger and Young, 1999), and prokaryotes were also absent from the male gametes of the three vents species B. thermophilus, B. puteaserpensis, and B. elongatus (Le Pennec and Beninger, 1997). However, bacterial symbionts may be acquired at an early stage, as supported by ultrastructural evidence indicating the occurrence of methane-and sulfur-oxidizers in gills of post-larvae and juveniles (shell lengths between 0.6 and 12 mm) of B. azoricus and B. heckerae (Salerno et al., 2005). Phylogeny-based studies also support environmental acquisition of symbionts, since host and symbiont phylogenies are not similar (Won et al., 2003). Given that the symbionts of two very closely related hosts often tend to be very closely related themselves, it is hypothesized that symbionts in this circumstance are acquired early, from the parental population (Duperron, 2010).

Larval biology

The greatest diameters measured for oocytes within female I. modiolaeformis fall within the range reported for other Bathymodiolinae (50 to 80 [micro]m) and shallow-water mussels (60 to 90 [micro]m) that have planktotrophic larvae (Arellano and Young, 2009). Likewise, the relatively minute prodissoconch I (72 to 95 [micro]m) produced by the energy reserves of the eggs and the comparatively large size of the prodissoconch II (300 to 426 [micro]m) produced during feeding in the plankton are signs of planktotrophy (Ockelmann, 1965; Lutz et al., 1980). The size of the PI of I. modiolaeformis is more related to the size of the PI in shallow-water mytilid species (85-120 [micro]m) (Arellano and Young, 2009). However, the size of the PII is closer to seep and vent mytilid species such as "Bathymodiolus" childressi (385-548 [micro]m) than to the shallow-water species Mytilus edulis (120-252 [micro]m) (Arellano and Young, 2009). The diameter of PII reflects the time spent in the plankton, and based on data from other species (Table 2), we can hypothesize that the time spent in the plankton is between the 4 weeks inferred from M. edulis (Lutz et al., 1980) and the 5 months inferred from B. childressi (Arellano and Young, 2009). Recent work reports specimens resembling I. modiolaeformis in the Canyon Lacaze-Duthiers (western Mediterranean, N. Le Bris, University Pierre and Marie Curie, France; pers. comm.) and in CHEMECOLIs deployed in the Gulf of Cadiz in the Atlantic Ocean (C. Rodrigues, University Aveiro, Portugal, and University Pierre and Marie Curie, France; pers. comm.). Currently, I. modiolaeformis has been reported only in those natural habitats of cold seeps in the eastern Mediterranean (Duperron et al., 2008; Ritt et al., 2011) but not in the Atlantic Ocean nor the western Mediterranean. Coupling biological data with modeling approaches would certainly help estimating how far larvae can disperse.

This study has demonstrated that Idas modiolaeformis is a hermaphroditic species with the capacity to switch from male to female, in which symbionts are not transmitted through gametes or gonad tissue, and in which larval development is most probably planktotrophic. Representatives of the genus Idas occur on wood falls, whale falls, and cold seeps worldwide, which are ephemeral and reducing habitats (Tyler et al., 2009; Lorion et al., 2010). However, because Idas is not a monophyletic genus, great variability in reproductive and physiological traits can be expected, necessitating the repeated investigation of various species (Lorion et al., 2010). Factors controlling larval settlement are not yet understood, though sulfur or methane emissions could be important, especially if symbionts are acquired during early larval stages. In addition, it is clear that deterministic factors governing dispersal and settlement may be complex for this species, given that rapid colonization has previously been recorded in I. modiolaeformis upon CHEMECOLIs that were deployed for only a period of 10 days in the central pockmark within the Nile Deep Sea Fan in 2006 (Gaudron et al., 2010), where little sulfide emission was recorded (Le Bris et al., 2008).

Acknowledgments

We thank Olivier Gros, Christian Borowski, and Clara Rodrigues, who collected and fixed Idas modiolaeformis and CHEMECOLIs samples on board. We are indebted to Omar Boudouma for his help with the SEM at University Pierre and Marie Curie. We thank the captains and crews of RVs Pourquoi Pas? and Maria S. Merian and teams operating ROVs Victor 6000 (Ifremer, France) and Quest 4000 (MARUM, Bremen, Germany). We thank Catherine Pierre, Antje Boetius, and Frank Wenzhofer for the provision of ship time and samples collected on the following research expeditions: MEDECO-2, M70/2b and MSM13/3 + 4 with RVs Pourquoi Pas?, Meteor, and Maria S. Merian, funded by IFREMER, the DFG, and the Excellence cluster MARUM. Sample collections were funded by DIWOOD (GDRE of CNRS and MPG), HERMES (EC), and CHEMECO (European Sciences Foundation (ESF)/Euro-cores/EURODEEP). Data analyses were supported by ANR Deepoases and HERMIONE EC (FP7/2007-2013-no 226354). We acknowledge Sven Laming for editing this manuscript.

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SYLVIE MARYLENE GAUDRON *, EMILE DEMOYENCOURT, AND SEBASTIEN DUPERRON

Universite Pierre et Marie Curie--Paris VI, CNRS, UMR7138, Systematique, Adaptations, Evolution, AMEX, 7 Quai St. Bernard, 75252 Paris, France

Received 15 September 2011; accepted 29 November 2011.

* To whom correspondence should be addressed. E-mail: sylvie.gaudron@snv.jussieu.fr

Abbreviations: CHEMECOLI, chemosynthetic ecosystem colonization by larval invertebrates; FISH, fluorescent in situ hybridization; PI, prodissoconch I; PII, prodissoconch II.
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Author:Gaudron, Sylvie Marylene; Demoyencourt, Emile; Duperron, Sebastien
Publication:The Biological Bulletin
Article Type:Report
Geographic Code:4EUFR
Date:Feb 1, 2012
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