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Symbiosis of the hydrothermal vent gastropod Ifremeria nautilei (Provannidae) with endobacteria - structural analyses and ecological considerations.


Hydrothermal vents, which have been known since the late 1970s, have become the subject of intensive study. Biological investigations have concentrated on vents in the East Pacific and on the Mid Atlantic Ridge, where vestimentiferans, bivalve molluscs, and shrimps dominate the ecosystems (Van Dover, 1990; Tunnicliffe, 1991). It has been shown that symbioses between most of these animals and chemoautotrophic bacteria are the main source of nutrition (Fisher, 1990; Childress and Fisher, 1992). The animals supply the bacteria with reduced compounds from the vents and with oxygen from the ambient seawater. The bacteria gain energy by oxidizing sulfur species - and sometimes methane as well (Childress et al., 1986; Fisher et al. 1987, 1993; Cavanaugh et al., 1987; Distel et al., 1995) - for the fixation of organic carbon which, in turn, is utilized by their hosts.

Only in hydrothermal vent ecosystems of the Western Pacific are gastropod molluscs abundant, even attaining a dominant ecological role (Van Dover, 1990; Tunnicliffe, 1991). To date, vent areas have been investigated in the Lau Basin (Desbruyeres et al., 1994), the Manus Basin (Both et al., 1986; Tufar, 1990; Galkin, 1992), the Mariana Trough (Hessler et al., 1988; Hessler and Lonsdale, 1991), and the North Fiji Basin (Jollivet et al., 1989; Desbruyeres et al., 1994). In almost all these areas, one or both of the two prosobranch gastropods Ifremeria nautilei and Alviniconcha hessleri (Family Provannidae) occur in abundance together with several mytilid bivalve species of the genus Bathymodiolus (Waren and Bouchet, 1993; Desbruyeres et al., 1994; Cosel and Metivier, 1994). The gastropods settle in the vicinity of hydrothermal emissions where they are exposed to temperatures of about 3 [degrees] - 20 [degrees] C.

Alviniconcha hessleri was the first hydrothermal vent gastropod reported to contain endosymbiotic bacteria within its specialized gills (Ohta et al., 1988, Stein et al., 1988; Endow and Ohta, 1989a, b). The high content of elemental sulfur, the autotrophic fixation of C[O.sub.2], and the high activity of enzymes that catalize sulfide oxidation indicated that the bacteria were chemoautotrophic sulfur-oxidizing endosymbionts (Stein et al., 1988). But Ohta et al. (1988) and Endow and Ohta (1989a, b) found in one of the three specimens of Alviniconcha hessleri inspected a second morphological type of bacterial symbionts closely resembling methanotrophic bacteria.

In micrographs of A. hessleri (Stein et al., 1988), bacteria appear to be intracellular and enclosed singly in host vacuoles, although some vacuoles with several bacteria can be seen. But Endow and Ohta (1989a, b) noted that the apical vacuoles of several bacteriocytes contained narrow ducts that seemed to directly connect the bacteria to the exterior. On the other hand, they demonstrated by infiltration of ruthenium red that in the basal parts of the bacteriocytes the symbionts were truly enclosed in their vacuoles (Endow and Ohta, 1989b).

Ifremeria nautilei, the other hydrothermal gastropod species from the Lau and North Fiji Basins (Bouchet and Waren, 1991; Beck, 1991; Waren and Bouchet, 1993), deviates in several anatomical details from the related provannid A. hessleri. The hypertrophic gills, the well-developed circulatory system, and the reduced stomach lead to the assumption that symbiotic bacteria might play a more significant role in these animals. Galchenko et al. (1992), examining specimens from the Manus Basin, provided the first physiological evidence for a bacterial symbiosis. C[O.sub.2]-fixation was stimulated by reduced sulfur compounds, and radiolabeled C[H.sub.4] was metabolized and detected in the tissues. These results suggested the presence of thioautotrophic and methanotrophic bacteria in the gills, providing the gastropod with reduced carbon. The ultrastructural basis given by the authors for these results was very preliminary, and the number of specimens or ctenidial filaments inspected was not indicated. The micrographs supported the physiological data, reinforcing the conclusion that two different physiological pathways were being used by two morphologically distinct types of bacteria. Specimens of I. nautilei from the Lau and North Fiji Basin have only been studied physiologically: Desbruyeres et al. (1994) confirmed the presence of thiotrophic symbionts by the detection of ribulose-1, 5-bisphosphate carboxylase activity.

The present study on Ifremeria nautilei from the North Fiji Basin concentrates on an (ultra)structural survey of the bacterial symbiosis to scrutinize important details previously not sufficiently documented (Galchenko et al., 1992). These structural results are supplemented by measurements of elemental sulfur, total sulfur, and stable carbon isotope composition. They give further insight in the trophic interactions between the endosymbiotic prokaryotes and their gastropod host.

Materials and Methods

During cruise 99 of the RV Sonne in January 1995 (Auzende et al., 1995; Halbach et al., 1995), many specimens of Ifremeria nautilei (Boucher and Waren, 1991) were collected in the North Fiji Back Arc Basin at the LHOS site (Ishibashi et al., 1994) at 16 [degrees] 59.65 S-173 [degrees] 54.73 E. The sample was retrieved from a depth of 2000 m (station GTV 115) by a TV-controlled grab. The grab also contained the symbiont-harboring bivalve Bathymodiolus brevior and other hydrothermal animals. The specimens of I. nautilei examined had a shell height of about 5 cm. Five gill filaments from each of two specimens were dissected and fixed in a mixture of 4% paraformaldehyde, 3% glutaraldehyde, and 15% sucrose in 0.1 M cacodylate buffer (pH 7.4). After being rinsed in the same buffer, the tissue was postfixed in Os[O.sub.4] (1%). Samples were dehydrated in acetone, embedded in Spurr's resin, and sectioned for light and electron microscopy. For light microscopy, semithin sections were stained with toluidine blue; for electron microscopy, ultrathin sections were stained with uranyl acetate and lead citrate. Sections of filament from the two specimens were examined using a Zeiss 902A electron microscope. One filament was sequentially cut and, in intervals of 100 [[micro]meter], ultrathin sections were examined. For scanning electron microscopy, several gill filaments of two formaldehydefixed (4%) specimens were dehydrated in acetone, critical-point-dried, and gold-sputtered. A Cambridge Camscan DV 4 scanning electron microscope was used for the observations. For paraffin sections, a complete specimen was fixed in 4% formaldehyde. After dissection and standard embedding, sections were cut and stained with Azan (Romeis, 1989).

Measurements of total sulfur ([]) concentration were based on samples from three individuals (10 to 40 mg dry weight). The lyophilized gill and muscle tissues of the foot (excluding the warts, Waren and Bouchet, 1993) were digested separately for 3 h under pressure at 130 [degrees] C in 4 ml HN[O.sub.3] (65%, suprapur, Wurfels and Jackerth, 1985). The resulting solution was concentrated by evaporation to 0.5 ml, then brought to 14 ml with distilled water. In this solution the sulfur concentration was measured with an inductivity-coupled-plasma atomic emission spectrometer (ICP-AES, Perkin Elmer Plasma II). Oyster tissue SRM 1566, commonly used in mollusc studies, and a diluted standard solution were used for calibration.

For measurements of elemental sulfur ([S.sup.o]), frozen gill and foot muscle tissues of three individuals were lyophilized and analyzed by HPLC (pump: Jasco 880 PU, manual injector: Rheodyne 7105, fitted with a 20 [[micro]liter] loop; UV/VIS detector: Jasco 975-UV, set at 254 nm; column: Hamilton PRP-1 reversed phase (15 cm x 4.1 mm I.D.). The absorbance was continuously collected by a computer using the Labtech notebook software. Chromatograms were analyzed using the Labtech chrom software. Treatment of the column prior to analysis, extraction of samples in chloroform, and HPLC-protocol were according to Lauren and Watkinson (1985).

The [[Delta].sup.13]C was measured with a Finnigan Delta E mass spectrometer from gill tissue of three specimens. After decalcification with 0.1 N HCl and drying in an oven at 60 [degrees] C, samples were combusted, at 1050 [degrees] C in a stream of oxygen, to C[O.sub.2] by using a commercial Heraeus CHN analyzer attached to the mass spectrometer. Stable isotope determinations were expressed as the permille difference from the international PeeDee Belemnite standard, where [[Delta].sup.13]C = [[Ratio.sub.sample] x [[Ratio.sub.standard]].sup.-1] x [10.sup.3] ([per thousand]).


Anatomy of the gill

Ifremeria nautilei has a single ctenidium at the left side of its body, where it occupies about one whorl of the snail's shell [ILLUSTRATION FOR FIGURE 1 OMITTED]. The ctenidium consists of 1000 to 2000 serially arranged slender filaments with a length of 9 to 17 mm and a maximal width of about 0.1 to 0.2 mm. Towards their proximal and distal ends, these filaments decrease in length and width. The basal third of each filament is attached to the roof of the mantle cavity and the remaining two thirds extend free into the mantle cavity [ILLUSTRATION FOR FIGURE 2 OMITTED]. The leaflike main part of the filament is thin, its free lower edge, orientated towards the mantle cavity, is reinforced by a rigid axis [ILLUSTRATION FOR FIGURE 3A OMITTED]. In some preparations the axis was found bent towards the anterior end of the animal, resulting in an L-shaped filament [ILLUSTRATION FOR FIGURE 4 OMITTED]; this curvature may be dependent on muscle contraction or fixation. The following description is based on cross sections through the nonattached part of a ctenidial filament.

Each filament consists of two epithelial cell layers separated by a bilayered middle lamella of noncellular material [ILLUSTRATION FOR FIGURES 3, 4 OMITTED]. The internal space between these lamellar layers (width varying between 2 and 15 [[micro]meter]) harbors muscles, nerves, connective tissue, hemocoel with hemocytes, and additional extracellular material. At the distal, ventral edge of the filament the two layers of extracellular material become very thick (50 [[micro]meter]) and join, enclosing a wide hemocoelic vessel that extends all along the lower edge of the ctenidial length. In fixed material the hemocoelic space appears more or less empty, containing only some granular material, possibly remnants of the blood fluid [ILLUSTRATION FOR FIGURE 5A OMITTED]. At the boundary walls, very flat cells are abundant [ILLUSTRATION FOR FIGURE 5C OMITTED].

Each cross section through a filament (dorsal to ventral, see [ILLUSTRATION FOR FIGURE 2 OMITTED]) can be subdivided into six zones, five of which represent the symbiont-free parts, the remaining large one the symbiont-containing median part of the ctenidial filament (for numbering see [ILLUSTRATION FOR FIGURE 4 OMITTED]). Zones 1-4 form the lower (ventral) axis of the filament, zone 6 the upper (dorsal) edge.

Symbiont-free parts. The five zones that lack symbionts are described below.

Zone 1 - the ventral edge of the filament [ILLUSTRATION FOR FIGURE 5A OMITTED]. It consists of a ciliated epithelium with cells having a height of 10 to 20 [[micro]meter] and containing large nuclei (5 [[micro]meter] long). Externally, they are covered by 1.25 [[micro]meter] long microvilli and by several 5 [[micro]meter] long cilia. Glandular cells are abundant and contain electron-lucent vacuoles that harbor several homogeneous granules. Underneath the epithelium several small nerves traverse the filament (arrows in [ILLUSTRATION FOR FIGURE 5A OMITTED]).

Zone 2 - the zone enclosing the vessel [ILLUSTRATION FOR FIGURE 5B OMITTED]. Here, flat (about 4 [[micro]meter] high) nonciliated epithelial cells form a very thin cellular cover on the extracellular material. The cells contain only little electron-dark cytoplasm; at their external side they are studded with microvilli 0.5 to 0.7 [[micro]meter] in length. The mitochondria are electron dense, their cristae rarely visible.

Zone 3 - the zone of strong ciliation [ILLUSTRATION FOR FIGURE 5C OMITTED]. It consists of two layers of large, strongly ciliated cells (15 [[micro]meter] high), based on thin (1 [[micro]meter]) noncellular lamellae that enclose a separating space about 2 [[micro]meter] in width. This lacunar space is nearly completely filled with cells that probably represent hemocytes (not shown in micrograph). In each ciliated cell three areas can be recognized: The apical area, 3.5 [[micro]meter] high, is characterized by abundant cilia up to 20 [[micro]meter] long. Here, in addition to mitochondria, the cytoplasm contains rough endoplasmatic reticulum and spherical, electron-dense vacuoles 0.5 [[micro]meter] in width. The middle area of the cells is dominated by globular, large nuclei of about 3.5 [[micro]meter] diameter. The basal cell area is completely filled with tightly packed oval mitochondria. These are about 0.5 [[micro]meter] in length and contain many parallel, well-developed cristae.

Zone 4 - the hinge zone [ILLUSTRATION FOR FIGURE 6A OMITTED]. Located between the ciliated cells (zone 3) and the bacteriocytes (zone 5), this region is characterized by large (15 [[micro]meter] high), nonciliated cells. In comparison to the surrounding tissue, here both the cell membranes and the thin (0.5 [[micro]meter]) extracellular lamellae are strongly folded. This might indicate the function of this zone as a hinge between the axis and the rest of the filament. The two central lamellae are separated by an interlamellar space that is about 15 to 20 [[micro]meter] in width and traversed by two groups of muscles, two large nerves, and a hemocoelic lacuna. Minor nerves are located at the base of the epithelial cells.

Zone 6 - the upper filament edge [ILLUSTRATION FOR FIGURE 6B OMITTED]. In the nonattached part of the ctenidial filament this zone forms the dorsal edge, characterized by an epithelium consisting of glandular and ciliated cells. Three morphotypes of glandular cells with apparently different secretions can be distinguished. Interspersed between these glandular cells are intercalary, multiciliated cells with cilia 5 [[micro]meter] long.

Bacteriocytes and bacterial symbionts in ctenidial filament. The symbiont-containing median part of the ctenidial filament is Zone 5.

Zone 5 - the symbiont zone [ILLUSTRATION FOR FIGURES 3, 4, 7A, 9 OMITTED]. Representing nearly the entire flattened part of the filament, this dominant region is characterized by numerous symbiont-containing epithelium cells (bacteriocytes). About 250 of these cylindrical to pear-shaped bacteriocytes (about 30 to 50 [[micro]meter] long, maximum diameter about 8 [[micro]meter]) are visible on each cross section through the filament.

The cytological structure of the bacteriocytes is fairly uniform. Their surface is studded with 0.7 [[micro]meter]-long microvilli. The outer parts of these cells are densely filled with inclusions that resemble bacteria [ILLUSTRATION FOR FIGURES 3C, 7, 8 OMITTED]. Most are about 2.5 [[micro]meter] (length) by 0.5 [[micro]meter] (width), but some may reach a size of 4 [[micro]meter] by 1 [[micro]meter]. Their cell envelope is of the gram-negative type with clearly visible inner and outer membranes [ILLUSTRATION FOR FIGURE 7B OMITTED]. A peripherally located dense cytoplasm and a central network of condensed DNA betray the prokaryotic nature of these cells [ILLUSTRATION FOR FIGURES 7C, D, E OMITTED]. In one of the two specimens inspected the bacteria contained membrane-bound electron-lucent vesicles [ILLUSTRATION FOR FIGURE 7D OMITTED]. Typical bacterial division stages were not found. Since transitional stages between all bacterial sizes were found, there are no reasons to assume the existence of different morphological types of bacteria in the examined sections. Membrane stacks, characteristic for the methanotrophic bacteria, could never be discerned.

Each bacterium was tightly embedded in a vacuole, but the vacuoles often appeared interconnected. Therefore there seems to exist a complex "network" of anastomosing vacuoles harboring the bacteria. [ILLUSTRATION FOR FIGURES 7C, 8B OMITTED]. Moreover, in all the examined sections, some peripheral vacuoles were observed to have an opening towards the outside environment [ILLUSTRATION FOR FIGURES 7C, 8A OMITTED].

Whereas the distal part of the bacteriocytes contained only bacteria and a few mitochondria, the basal part was dominated by the nucleus and several large, non-membrane-bound droplets of a homogeneous, electron-lucent material [ILLUSTRATION FOR FIGURE 8C OMITTED]. This internal cell area also harbored many 1 [[micro]meter]-long electron-dark mitochondria that were rich in cristae. Inclusions resembling phagolysosomes (Fiala-Medioni et al., 1994), which would represent stages of bacteria in digestion, could often be observed in the basal part of the bacteriocytes [ILLUSTRATION FOR FIGURE 8D OMITTED].

Interspersed between the bacteriocytes, but in much lower abundance, were secretory cells and a few intercalary cells. The two lamellae between the epithelial layers of bacteriocytes enclosed a space that was filled by hemocoel, putative hemocytic cells, thick cords of collagen, and connective tissue [ILLUSTRATION FOR FIGURES 3B, 6A OMITTED].

Sulfur and 13C measurements

In the gill and foot tissue of Ifremeria nautilei, sulfur was detected (Table I) in concentrations comparable to those in other symbiont-containing molluscs, but somewhat lower than those reported for Vestimentifera (see compilation in Stein et al., 1988). Concentration of elemental sulfur ([S.sup.o]) differed by a factor of 8 among the individuals examined. The average concentrations of [S.sup.o] and [] were significantly higher in the gill than in the foot (Table I).

The stable carbon isotopic values measured from three specimens of I. nautilei equaled -31.4 [per thousand], -31.4 [per thousand], and -33.7 [per thousand]. Thus, they were much more depleted than those in marine animals that are linked to a photosynthetically based food chain (Fry and Sherr, 1984).
Table I

Percentages ([+ or -]SD) of elemental sulfur ([S.sup.o]) and total
sulfur ([]) (based on mg dry weight) in gill and foot of
three individuals (115-1, 115-2, 115-4) of Ifremeria nautilei

Sample      Organ       S[double prime]        []

115-1       gill       0.54 [+ or -] 0.02           2.23
            foot       0.25 [+ or -] 0.21           1.43
115-2       gill       3.98 [+ or -] 0.93           2.49
            foot       0.13 [+ or -] 0.13           1.14
115-4       gill       1.88 [+ or -] 0.14           2.84
            foot       0.03 [+ or -] 0.01           1.05

Values for [S.sup.o] and [] are significantly higher in
the gill than in the foot (Mann-Whitney U-test). In gill sample
115-2, the higher value of [S.sup.o] than [] may account
for the patchy localization of elemental sulfur in the tissue.
Additionally, the resulting variance of data may be altered by the
different sample sizes required for the two methods of sulfur


Comparison with other provannid gastropods

The first hydrothermal vent gastropod shown to contain thiotrophic bacteria in its gills was Alvinoconcha hessleri (Stein et al., 1988). This provannid species is often found around the same hydrothermal outlets as Ifremeria nautilei, to which it is closely related (Both et al., 1986; Jollivet et al., 1989; Tufar, 1990; Galchenko et al., 1992; Desbruyeres et al., 1994). Despite some differences in gross morphology - e.g., in shell structure, nervous system, and pallial margin, as well as in the presence of oesophageal pouches and wartlike tubercles on the foot of L. nautilei (Waren and Bouchet, 1993) - the two species apparently have a very similar type of bacterial symbiosis. This conclusion is based on ultrastructural data from the preliminary work by Galchenko et al. (1992) and from the present study.

An array of structural features of I. nautilei indicates that the bacterial symbiosis is of high functional importance for the host.

* The most obvious trait is the extremely high number and unusual length of ctenidial filaments. I. nautilei has 15 to 20 times more ctenidial filaments than the 30 to 89 found in its aposymbiotic relative Provanna spp. (Waren and Ponder, 1991).

* The ctenidial filaments in monopectinate prosobranch gastropods are usually triangular, with their base fully attached to the mantle roof (Demian, 1965). A ciliary ridge extending from the efferent edge of the filaments creates water currents for irrigation (Lutfy and Demian, 1965). In I. nautilei this efferent edge is highly elongated and a rigid axis has developed that bears the extended bacteriocytic epithelia and encloses a wide hemocoelic vessel. This axis may enhance the stability of the flimsy leaflike filaments. It also may act as a flexible hinge that, in cooperation with longitudinal and transverse muscle fibers, would allow movement of the whole filament. The "blood" vessel is presumably responsible for nutrition and gas exchange.

* The bacteria occur in extremely high numbers in the bacteriocytes and occupy a large area of the filaments. The stomach of the host is reduced to only 1/10 the size of the stomachs of related nonsymbiotic provannid gastropods (Bouchet and Waren, 1991). This points to the relevance of the symbiosis for the animals' nutrition. Furthermore, even though bacteriocytes represent the dominant structures in the gill tissues of other molluscs with a bacterial symbiosis, the bacterial zone in I. nautilei is nearly twice as voluminous as in these species (Le Pennec and Hily, 1984; Southward, 1986; Fiala-Medioni et al., 1986; Fiala-Medioni and Metivier, 1986; Fiala-Medioni, 1988; Fiala-Medioni and Felbeck, 1990). Whether this difference mirrors a larger dependence on chemoautotrophic nutrition is an open question.

The black-tipped warts on the foot of I. nautilei have been suspected, without closer analysis, to be "involved" in the symbiosis (Waren and Bouchet, 1993). Our analyses of ultrathin sections through the warts (data not shown) did not demonstrate the presence of any bacteria, thus refuting such an assumption.

Position of the bacteria in a vacuolar network

In most molluscs with chemoautotrophic bacteria, the prokaryotes are intracellular although their detailed arrangement may vary. In I. nautilei the position of the symbiotic bacteria in vacuoles of ctenidial cells is essentially the same as that described by Endow and Ohta (1989a, b) for the related gastropod A. hessleri. These studies indicate that peripheral vacuoles have some contact to the ambient seawater via small ducts. This topological arrangement seems to be specific to these two related gastropod species and might indicate a common origin of the symbiosis. In A. hessleri it could be demonstrated by staining with ruthenium red that this connection via "open" vacuoles was restricted to the externally located bacteria (Endow and Ohta, 1989b). Regrettably, the fresh material needed for this experiment was not retained in I. nautilei.

The chemoautotrophic nature of the bacteria

In specimens of I. nautilei from the Lau and Fiji Basins (Desbruyeres et al., 1994) and from the Manus Basin (Galchenko et al., 1992), ribulose-1, 5-bisphosphate carboxylase/oxygenase activity provided evidence for the chemoautotrophy of the symbiotic bacteria. The stable carbon isotope values measured for I. nautilei in this study are higher than those reported for specimens from the Manus Basin (Galchenko et al., 1992). They stay in the range of values found in symbiont-containing bivalves known to depend fully on their symbionts for nutrition (Childress and Fisher, 1992). Supporting evidence for the thiotrophic nature of the symbionts is provided by the high concentration of sulfur in the gills; this is regarded as a reliable criterion for the presence of sulfur-oxidizing bacteria (Childress and Fisher, 1992). It is, of course, still possible that this sulfur is a product of purely chemical oxidation of sulfide. The concentration of elemental sulfur in I. nautilei (0.5%-3.9% dry weight) is comparable to that in the gastropod A. hessleri (Stein et al., 1988) or in the bivalves Lucinoma annulata (0.2%-2.1% dw; Vetter, 1985) and Calyptogena magnifica (4.4% dw, Stein et al., 1988). Similarly, the concentration of total sulfur (2.2%-2.8% of dry weight) in the gills of I. nautilei is in the same range as in the gills of L. annulata (2.5%-5.6% dw; Vetter, 1985) and C. magnifica (3.8% dw; Roesijadi and Crecelius, 1984) which have been proven to harbor thiotrophic bacteria. The foot, which has never been found to harbor bacteria in any mollusc with bacterial symbionts, contained about 1% dry weight total sulfur in I. nautilei (see Table I).

The regular occurrence of degradation stages of symbiotic bacteria could indicate a digestive utilization of the prokaryotes by their host. This "harvesting" via phagocytotic degradation has also been found in Riftia pachyptila (Bosch and Grasse, 1984), in symbiont-harboring oligochaeta (Giere and Langheld, 1987), and in bivalves (Fiala-Medioni and Felbeck, 1990).

Thiotrophic vs. methanotrophic bacteria

Methanotrophic bacteria have been reported in I. nautilei from the Manus Basin (Galchenko et al., 1992). The identification was based on the activity of the methanol-oxidizing enzyme methanol dehydrogenase, the incorporation of labeled carbon from methane, the very low [[Delta].sup.13]C value of the gills (-38.3[per thousand]), and the presence of intracytoplasmic membranes in some of the bacteria. This is in contrast to the present study on specimens of the same species retrieved in the Fiji Basin, some 3000 km away. We could not discover any bacteria with the internal membranes typical of methanotropic bacteria (Childress et al., 1986; Fisher et al., 1987; Cavanaugh et al., 1987; Cavanaugh, 1992; Fisher et al., 1993; Distel et al., 1995). Although, at least in free-living methanotrophs, the occurrence of membranes is variable depending on culture conditions (Prior and Dalton, 1985; Collins et al., 1991), the higher [[Delta].sup.13]C values found in our specimens might also indicate an absence of methanotrophs. For a more conclusive interpretation, the [[Delta].sup.13]C values of the methane sampled at the North Fiji hydrothermal vents are required.

Our samples, taken from only a few specimens and ctenidial filaments, might have missed material containing methanotrophic bacteria due to an inhomogeneous distribution in the large gills. Alternatively, the small-scale spatial and temporal variability in the physical-chemical composition of the environment (Chevaldonne et al., 1991) might have caused the gastropod population to show local variations in bacterial symbiont stock. Both options have also been considered by Ohta et al. (1988), who postulated that methanotrophic symbionts were present only in low abundance (up to 10% of the symbionts). They also discussed the possibility that the methanotrophic bacteria might not be present in all members of the gastropod population, depending on the ambient conditions. Endow and Ohta (1989b) suggested that the putative methanotrophic symbionts of A. hessleri, as well as the prevailing thiotrophic type, might have been acquired "horizontally" from the outside. This would relate the symbiosis to environmental conditions, i.e., the presence of the symbionts in the environment. From an adaptive perspective, it would lend the host a favorable flexibility to cope with the high spatial and temporal variability of the vent ecosystem and might explain why A. hessleri and I. nautilei are mixotrophs that have retained a functional, though reduced, digestive tract (Bouchet and Waren, 1991; Waren and Bouchet, 1993; Desbruyeres et al., 1994). It could then be assumed that the composition of thiotrophic and methanotrophic bacteria reflects the composition of the reduced compounds released from the vents (Distel et al., 1995). This variability would also explain the inconsistency in our measurements of [S.sup.o] in I. nautilei, which varied by a factor of 8. These considerations imply that assessment of one or two types of symbionts might depend on fortuitous individual temporal or local conditions. Physiological assessment through diagnostic enzymes might be problematical because low concentrations can escape detection (Endow and Ohta, 1989b). Hence, one probably has to apply molecular techniques to reliably demonstrate the presence or absence of different symbiont types. The occurrence of more than one type of symbiotic bacteria in one host has been shown to exist in several populations of the mytilid bivalves (Childress et al., 1986; Fisher et al., 1987; Cavanaugh et al., 1987; Fisher et al., 1993; Distel et al., 1995) and in gutless oligochaetes (ultrastructural evidence: Giere et al., 1995; molecular evidence: Dubilier et al., unpubl. data).

Genetic differences between specimens from the Manus and the North Fiji Basin might also develop in response to the spatial and temporal instability of hydrothermal vents. Genetic separation in populations of neighboring vent fields has been shown to occur in Alviniconcha hessleri (Denis et al., 1993) and in the bivalves Calyptogena spp. (Vrijenhoek et al., 1994) and Bathymodiolus spp. (Craddock et al., 1995).

Ecological considerations and evolutionary aspects

It is commonly argued that there is an evolutionary trend from extra- to intracellular symbioses (Smith, 1979; Southward, 1986; Craddock et al., 1995; Giere, 1996; Ott, 1996). In gastropods, the first step might be represented by Lepetodrilus fucensis from the northeastern Pacific. In all sections of this vent limpet, bacteria originating from the dense colonies of bacteria on the epithelial surface of the gill have been observed to become endocytosed and digested in lysosome-like organelles (De Burgh and Singla, 1984). This has been interpreted as a routine process that occurs in addition to the normal grazing on the ambient bacterial population. In Alviniconcha hessleri and Ifremeria nautilei the symbiotic bacteria are enclosed in vacuoles, but still appear to have some contact with the outside. This would represent an intermediate step in a morphological series. The most advanced step, complete incorporation of symbionts into bacteriocytes, is common in many bivalve molluscs, but has not yet been described from symbiont-harboring gastropods.

The bacterial symbioses in A. hessleri and I. nautilei are structurally very similar, and the two hosts often settle close together in hydrothermal fields. Taxonomically, however, the snails have been assigned to different gastropod genera. This taxonomic difference might reflect a slight ecological or physiological niche diversification. Usually, A. hessleri has been found a little closer to the hydrothermal outlets than I. nautilei (Bouchet and Waren, 1991; Galchenko et al., 1992; Waren and Bouchet, 1993; Desbruyeres et al., 1994). It seems possible that this pattern is temperature dominated and mirrors specific differences in temperature preference. In the North Fiji Basin, Chevaldonne et al. (1991) obtained data on temperature gradients and the distribution of the gastropod colonies. But the complex temperature pattern seems to change locally very much and, for the area investigated here, it is not possible to relate the distribution pattern of the two gastropods to the temperature gradients around the hydrothermal outlets.

In molluscs, symbiosis obviously developed several times independently (Distel et al., 1994). The gills of bivalves and gastropods harbor different types of bacteria, sometimes even in an individual host. The present study indicates that separate populations of the same species may differ in their bacterial partners. An intraspecific symbiotic variability between bacterial groups (methanotrophs and thioautotrophs) may have contributed to the success of these gastropods in the unstable and ecologically diverse ecosystems of Pacific hydrothermal vents (Van Dover, 1990). The reasons for their distributional restriction to the Southwest Pacific remain unclear, but a more detailed picture of the symbiotic variability and ecological versatility may help to clarify it.


We would like to thank the crew of the RV Sonne 99 cruise for their good cooperation. We are indebted to Prof. Dr. W. Dannecker and Dr. F. Meyberg from the University of Hamburg for the ICP-AES analysis, Dr. B. Vismann from the Marine Biological Laboratory in Helsingor for HPLC analysis of [S.sup.o], and Dr. M. Segl from the University of Bremen for 13C measurements. We thank G. Wegener for her valuable assistance in the microscopical preparations. Our special thanks go to two anonymous colleagues for their efforts in reviewing the manuscript. Their work has much improved this paper. This study was financially supported by the Ministry of Education, Science, Research and Technology (BMBF) of Germany, grant no. 03G0099A.

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Author:Windoffer, R.; Giere, O.
Publication:The Biological Bulletin
Date:Dec 1, 1997
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