Molecular phylogenetic and isotopic evidence of two lineages of chemoautotrophic endosymbionts distinct at the subdivision level harbored in one host-animal type: The genus Alviniconcha (Gastropoda: Provannidae)
Extremobiosphere Research Center, Japan Agency for Marine-Earth Science and Technology, 2-15 Natsushima-cho, Yokosuka 237–0061, Japan
The hydrothermal-vent gastropod Alviniconcha hessleri from the Alice Springs deep-sea hydrothermal field in the Mariana Back-Arc Basin in the Western Pacific houses an intracellular bacterial endosymbiont in its gill. Although enzymatic analysis has revealed that the endosymbiont is a sulfur-oxidizing chemoautotroph using the Calvin–Benson cycle for the fixation of carbon dioxide, the phylogenetic affiliation of, and the trophic relationship of A. hessleri with, the chemoautotrophic endosymbiont remains undetermined. A single 16S rRNA gene sequence was obtained from the DNA extract of the gill, and phylogenetic analysis placed the source organism within the lineage of the gamma subdivision of the Proteobacteria that consists of many chemoautotrophic endosymbionts of marine invertebrates. Fluorescence in situ hybridization analysis showed the bacterium densely colonizing the gill filaments. The fatty acid profile of the symbiont-free mantle contains the high level of the 16:1 fatty acid originating from the endosymbiont, which indicates that the endosymbiont cells are digested by, and incorporated into, the host. Compound-specific carbon isotopic analysis revealed that fatty acids from the gastropod tissues are all 13C-depleted relative to the gastropod biomass. This fractionation pattern is consistent with chemoautotrophy based on the Calvin–Benson cycle and subsequent fatty-acid biosynthesis from 13C-depleted acetyl coenzyme A. The results from the present study are clearly different from those from our previous study for A. aff. hessleri from the Indian Ocean that harbors a chemoautotrophic endosymbiont belonging to the epsilon subdivision of the Proteobacteria, which mediates the reductive tricarboxylic acid cycle for carbon fixation. Thus, it is concluded here that two lineages of chemoautotrophic bacteria, phylogenetically distinct at the subdivision level, occur as the primary endosymbiont in one host-animal type, which is unknown for the other metazoans.
Nutritionally mutualistic endosymbioses between metazoans and chemoautotrophic bacteria are widespread in marine habitats [1,2]. Although few members of the delta and epsilon subdivisions of the Proteobacteria (the δ- and ε-Proteobacteria) occur inside host organisms as secondary endosymbionts [3,4], a primary chemoautotrophic endosymbiont not affiliated to the γ-Proteobacteria had been long unknown in metazoans. Recently, we have described a novel chemoautotrophic endosymbiont phylogenetically placed within the ε-Proteobacteria occurring in the deep-sea hydrothermal vent gastropod Alviniconcha aff. hessleri from the Indian Ocean .
Alviniconcha gastropods belonging to the family Provannidae inhabit deep-sea hydrothermal fields in the margins of the Western and Southwestern Pacific Ocean and on the Central Indian Ridge . It has been previously shown that A. hessleri from the Mariana Back-Arc Basin harbors a sulfur-oxidizing chemoautotrophic endosymbiont in its gill , and that the endosymbiont utilizes the Calvin–Benson cycle and ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) for carbon-dioxide fixation . Unlike the endosymbiont of A. hessleri from the Mariana Back-Arc Basin, the ε-proteobacterial endosymbiont of A. aff. hessleri from the Central Indian Ridge mediates the reductive tricarboxylic acid (rTCA) cycle for the conversion of CO2 into organic matter . This difference in carbon-fixation pathway may indicate that the chemoautotrophic endosymbionts in the two Alviniconcha hosts can be phylogenetically distinct.
In the present study, we conducted anatomical, molecular phylogenetic, fatty-acid profile, bulk and compound-specific carbon isotopic analyses in order to reveal the phylogenetic affiliation of the chemoautotrophic endosymbiont of A. hesseleri from the Mariana Back-Arc Basin in the Western Pacific, as well as the nature of the gastropod/γ-proteobacterial endosymbiosis.
2Materials and methods
2.1Gastropod specimens and anatomy
Gastropod specimens were collected in December 1996 from the Alice Springs field in the Mariana Back-Arc Basin, the Western Pacific (18°12.8′N, 144°42.4′E), at a depth of ?3600 m, utilizing the manned submersible Shinkai 6500. Based on the mitochondrial cytochrome c oxidase subunit I gene, three individuals, examined in the present study, are identical to A. hessleri as previously described .
A. hessleri was observed and dissected into various organ systems under a binocular microscope. The gill filaments were removed from the mantle and observed with a scanning electron microscope (SEM; Hitachi S-2400) upon coating with platinum vanadium. The samples were dehydrated with a graded series of ethanol, transferred into t-butyl alcohol, and dried with a freeze dryer (Hitachi ES-2030).
Genomic DNA was extracted from the dissected gill tissues using the DNEasy Kit (QIAGEN, Valencia, Calif.) and magnetically purified using a MagExtractor Kit (TOYOBO, Osaka, Japan), in accordance with the manufacturer's instructions. The 16S rRNA gene sequences were amplified through the polymerase chain reaction (PCR) using LA Taq polymerase (TaKaRa, Tokyo, Japan) with the oligonucleotide primers Bac27F and Uni1492R . Thermal cycling was performed using a GeneAmp 9700 Thermal Cycler, with 27 cycles of denaturation at 96°C for 20 s, annealing at 53°C for 45 s, and extension at 72°C for 120 s. The amplified 16S rRNA gene-sequence products were either cloned or sequenced directly with an ABI 3100 Capillary Sequencer and a dRhodamine Sequencing Kit as per the manufacturer's recommendations (Perkin Elmer/Applied Biosystems, Foster City, CA). Bacterial clone libraries were constructed using the Original TA Cloning Kit (Invitrogen, Carlsbad, CA). The sequence similarity among all of the partial sequences, which were 500 nucleotides long, was analyzed using the FASTA program equipped with the DNASIS software (Hitachi Software, Tokyo, Japan). A single phylogenetic clone type (phylotype) was obtained from the clone type analysis, and the partial sequence was extended and manually aligned according to the secondary structures using ARB (a software environment for sequence data ). Evolutionary analysis was performed by the distance, parsimony, and maximum likelihood methods using PAUP  based on 1313 nucleotide positions (54–1471, Escherichia coli numbering), which have more than 70% homology across the γ-proteobacterial sequences.
The accession number for the bacterial 16S rRNA gene sequence from the gill endosymbiont is available at DDBJ under the Accession No. AB214932.
2.3Fluorescence in situ hybridization analysis
Using ARB , an rRNA-targeted oligonucleotide probe for a single dominant phylotype from the gill filaments was designed. The probe, hereafter referred to as Alvm577, is 16-bases long, which corresponds to E. coli position 577–592 (5′-GACCAGGCCGCCTACG-3′). The specificity of the probe was checked in part by using the Gapped-BLAST search algorithm  and by check probe analysis from the RDP-II project .
For whole-cell hybridization, dissected gill filaments from three individuals were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS, pH 7.4) for 2 h and dehydrated in an ethanol series (50%, 75%, and 100%, v/v), followed by three washes in xylene and infiltration with paraffin wax. The wax-embedded specimens were then sectioned (thickness ?3 μm) and mounted on 3-aminopropyltriethyloxysilane (APTS)-coated slides. For the deparaffinized specimens, hybridization was conducted at 46°C in a solution containing 20 mM Tris–HCl (pH 7.4), 0.9 M NaCl, 0.1% sodium dodecyl sulfate, 30% (v/v) formamide and 50 ng/μl of the Alvm577 probe and the “universal” bacterial probe EUB338 , which were labeled at the 5′ end with Cy-3 and fluorescein, respectively. After hybridization, the slide was washed at 48°C in a solution lacking the probe and formamide at the same stringency, adjusted by NaCl concentration , and subsequently stained with 4′,6-diamidino-2-phenylindole (DAPI) at 0.4 μg/ml. The slides were examined using either an Olympus BX51 microscope or an Olympus FV5000 confocal laser-scanning microscope. A negative control probe for Alvm577, in which two-base mismatches were introduced in the middle (5′-GACCATTCCGCCTACG-3′), was used for testing unspecific labeling.
2.4Bulk carbon isotopic analysis
Three gastropod individuals were dissected into gill and mantle tissues, and the dissected tissues were lyophilized. A small portion of each lyophilized tissue was powdered and then acid-fumed for 6 h (53). The rest of the untreated lyophilized tissue was stored at −80°C for fatty-acid extraction. The carbon isotopic compositions of the cultures and the gastropod tissues were analyzed by a Thermo Electron DELTAplus Advantage mass spectrometer connected to an elemental analyzer (EA1112) through a ConFlo III interface.
2.5Analysis of the fatty-acid methyl-ester profiles
For the extraction of cellular fatty acids (FA), a method described in  was used. Approximately 20 mg of the gastropod tissues were incubated in 1 ml of anhydrous methanolic hydrochloric acid at 100°C for 3 h. After the addition of 1 ml of deionized, distilled water (DDW) to the cooled aliquots, the fatty-acid methyl-esters (FAMEs) were extracted three times with 3 ml of n-hexane. The n-hexane fractions are washed with an equal volume of DDW and dehydrated with anhydrous Na2SO4. The concentrated FAMEs were stored at −20°C for subsequent carbon isotopic analyses.
The identities of the FAMEs were determined by comparison of the retention times and spectra to those of known FAME standards by gas chromatography–mass spectrometry (GC–MS), using a Shimadzu GCQ GC–MS system. The oven temperature was set to 140°C for 3 min and then increased to 250°C at a rate of 4°C/min with He at a constant flow of 1.1 ml/min through a DB-5MS column (30 m × 0.25 μ× 0.25 mm; J&W Scientific). The standard nomenclature for fatty acids was used. FA are designated X:Y, where X is the number of carbon atoms and Y is the number of double bonds.
2.6Compound-specific carbon isotopic analysis
The δ13C values of the FAMEs were determined by the GC-carbon-isotope ratio MS using a Thermo Electron DELTAplus Advantage mass spectrometer connected to a GC (Agilent 6890) through a GC/C/C/III interface. The oven temperature was set to 120°C for 3 min and then increased to 300°C at a rate of 4°C/min with He at a constant flow of 1.1 ml/min through a HP-5 column (30 m × 0.25 μ× 0.25 mm; Agilent). The isotopic compositions of the FAMEs were measured with an internal isotopic standard (19:0, δ13C =−29.80‰), and the additional carbon atom from the methanol-derivatizing reagent (δ13C =−39.04‰) was corrected. The internal isotopic standard produced measurement errors within 1‰ for all isotopic analyses.
The animal of A. hessleri is globular, spirally coiled and consists chiefly of the head-foot, the pallial cavity and the visceral mass. Most of the area of the pallial cavity is occupied by numerous ctenidial (gill) lamellae which are vertically hung from the mantle (m: Fig. 1A). Each lamella is divisible into several zones including the vertical ridges (vr), the transverse groove (tg), the lateral cilia (lc), a stiffened zone containing skeletal rods (sr), and vessels and blood sinus (Fig. 1A). The vertical ridges are formed of a layer of granular bacteriocytes, which densely contain vermicular bacteria inside (Fig. 1B–D).
The phylogenetic affiliation of the microbial cells in the gill filaments was determined based on the 16S rRNA gene sequences. The examination of 16 clones generated from a 16S rRNA gene-sequence library from the gill filaments of a single gastropod showed only one 16S rRNA gene sequence type. The occurrence of a single endosymbiont in the gill filaments was also supported by the results of the direct sequencing of the PCR amplified 16S rRNA gene of the phylotype from two other gastropods. As shown in Fig. 2, phylogenetic analysis placed the phylotype within the γ-Proteobacteria, which is closely related with the endosymbionts of vestimentiferan tubeworms and bivalve mollusks. The closest relative of the phylotype was the endosymbiont of the hydrothermal-vent gastropod Ifremeria nautilei belonging to the same family Provannidae.
3.3Fluorescence in situ hybridization
In order to ensure that the phylotype revealed by direct sequencing and 16S rRNA gene-sequence clone-library analysis originates from the endosymbiont in the gill filaments, we conducted fluorescence in situ hybridization (FISH) analysis using the probe Alvm577. The sections of the gill filaments from three gastropods were hybridized with the EUB338 and Alvm577 probes, followed by DNA staining with DAPI. Representative epifluorescence micrographs are shown in Fig. 3. The presence of dense aggregates of bacterial cells as well as host nuclei (labeled “N” in Fig. 3A) in the gill filaments was confirmed by FISH with the EUB338 probe together with DAPI staining (Fig. 3A,C,D and F). Hybridization with the Alvm577 probe specific to the endosymbiont gave a signal pattern almost identical to that of the EUB338 probe (Fig. 3B and C), indicating that most of the bacterial cells are affiliated to the phylotype. A negative control probe, in which two-base mismatches were introduced in the middle of the Alvm577, was not hybridized with the bacterial cells under the same conditions as described above, which precluded the possibility of unspecific labeling (Fig. 3E and F).
3.4Bulk carbon isotopic analysis
The δ13C values of the gastropod gill and mantle tissues were measured. In good agreement with the carbon isotopic data from a previous study in which A. hessleri from the same field was investigated , the gastropod tissues had a δ13C range from −28.4‰ to −29.7‰ as shown in Table 1. Despite the abundance of endosymbiont cells in the gill, the carbon isotopic compositions of the symbiont-free tissues are nearly identical to that of the gill, indicating that the endosymbiont biomass was as 13C-depleted as the symbiont-free gastropod tissues.
Table 1. Carbon isotopic compositions of the total biomass and FAME of the gastropod tissues
Carbon isotopic compositions are reported as δ13C values (‰).
aMean ± SD. At least duplicate measurements were conducted for each of the tissue parts. Three gastropod individuals were analyzed.
bThe isotopic compositions of the total FAMEs were calculated on the basis of the FAME compositions.
Alviniconcha hessleri from the Mariana Back-Arc Basin
−29.7 ± 0.4a
−38.6 ± 0.9
−39.5 ± 0.6
−37.7 ± 0.5
36.5 ± 0.7
−36.9 ± 0.4
−28.4 ± 0.5
−36.1 ± 0.4
−36.8 ± 0.1
−36.4 ± 0.5
−35.7 ± 0.4
−36.5 ± 0.9
Alviniconcha aff. hessleri from the Central Indian Ridge in 
−11.0 ± 0.1
−5.1 ± 0.9
−6.5 ± 0.9
−7.7 ± 0.8
−8.5 ± 1.4
−7.6 ± 0.8
−10.7 ± 0.1
−8.3 ± 0.9
−9.1 ± 1.4
−9.2 ± 0.9
−8.6 ± 2.0
It is well established that the FAME profiles of marine mollusks are similar to those of the organisms they feed on [18–20]. As shown in Fig. 4, the FAME profiles from the symbiont-free mantle and the symbiont-bearing gill of the gastropod are similar, and the symbiont-free mantle tissue contains the high level of the 16:1 FA that is considered to originate from the endosymbiotic cell [21,22].
3.6Compound-specific carbon isotopic analysis
The carbon isotopic compositions of some FAMEs from the gastropod tissues were measured; the 13C values of the FAMEs after correction for the methanol-derivatizing reagent and the total FAMEs calculated on the basis of the FAME compositions are shown in Table 1. The FAMEs analyzed in this study were all 13C-depleted relative to the biomass. The saturated and monounsaturated C16 FAs in the gill tissue were particularly 13C-depleted, by 8.9‰ and 9.8‰, relative to the biomass, respectively (Table 1).
4.1The trophic interactions between A. hessleri and the γ-proteobacterial endosymbiont
Within the gill filaments, the gastropod harbors the endosymbiont that phylogenetically falls into the γ-proteobacterial lineage, many members of which are the chemoautotrophic endosymbionts of marine invertebrates inhabiting sulfide-rich environments. In order to better understand the nutritional interactions between the host gastropod and the γ-proteobacterial endosymbiont, we attempted to compare our results obtained for the gastropod/γ-proteobacterial endosymbiosis to those of previous studies on relatively well-established chemoautotrophic endosymbioses between marine invertebrates and some γ-proteobacterial endosymbionts . Marine bivalves, which nutritionally depend on intracellular chemoautotrophic γ-Proteobacteria, have FA profiles similar to those of their endosymbionts, indicating that the endosymbiont cells are digested by, and incorporated into, the host bivalves [20,22,23]. Similarly, A. hessleri has monounsaturated FAs originating from the γ-proteobacterial endosymbiont in the symbiont-free tissue as well as the symbiont-bearing gill. In addition, the carbon isotopic compositions of the biomass and FAs of the symbiont-bearing and symbiont-free tissues are nearly identical, which strongly suggests that the endosymbiont cells are digested by, and incorporated into, the host gastropod.
4.2Chemoautotrophy in the γ-proteobacterial endosymbiont
If an organism assimilates carbon from a small molecule, either CO2, CH4 or acetate, significant isotope fractionation is likely to accompany the carbon assimilation . The fractionation of carbon isotopes is also associated with the biosynthesis of acetyl-coenzyme A (acetyl-CoA), a precursor of FA synthesis [24,25]. The range of the fractionations varies with the pathways involved in carbon assimilation and acetyl-CoA synthesis. Therefore, the fractionation patterns of the carbon source relative to the biomass, and of the biomass relative to the fatty acids of a given organism provide diagnostic information regarding the carbon metabolism of the organism .
The δ13C value of the gastropod biomass is distinct from that of marine organisms nutritionally depending on photosynthetically derived organic matter which typically ranges from −8‰ to −25‰. The isotope fractionation pattern of the gastropod biomass relative to the CO2 from the hydrothermal vents at the Alice Springs field with a δ13C value of −4.3‰ is similar to that exhibited by other hydrothermal-vent invertebrates which derive their nutrition from the chemoautotrophy in the γ-proteobacterial endosymbionts using the Calvin–Benson cycle and rubisco [24,27]. The FAs of A. hessleri are all depleted in 13C relative to the biomass, which is consistent with the chemoautotrophy based on the Calvin–Benson cycle and subsequent biosynthesis of FAs from 13C-depleted acetyl-CoA. These isotope fractionation patterns support the inference that the γ-proteobacterial endosymbiont of A. hessleri from the Alice Springs field in the Mariana Back-Arc Basin is a chemoautotroph mediating the Calvin–Benson cycle as previously revealed by enzymatic analysis .
4.3γ- and ε-proteobacterial endosymbionts harbored in one host-animal type
The δ13C value of the biomass of A. hessleri is apparently different from that of A. aff. hessleri from the Indian Ocean with a δ13C value of ?11‰ (Table 1), which nutritionally depends on the chemoautotrophy in the ε-proteobacterial endosymbiont using the rTCA cycle. The difference in carbon-fixation pathway between the γ- and ε-proteobacterial endosymbionts of the Alviniconcha gastropods is also supported by the difference in carbon isotope fractionation pattern of fatty acids relative to the gastropod biomass. A. aff. hessleri from the Indian Ocean has the FAs all enriched in 13C relative to the gastropod biomass (Table 1), which is also characteristic of the chemoautotrophy mediated by the rTCA cycle and subsequent biosynthesis of FAs from 13C-enrhiched acetyl-CoA. Additionally, the endosymbiont FA is almost absent in the symbiont-free tissues of A. aff. hessleri, which indicates that the endosymbiont might translocate organic material across the cell membrane, rather than the endosymbiont cells being digested by the host . The results from the present study provide the molecular phylogenetic and isotopic evidence that two lineages of chemoautotrophic bacteria phylogenetically distinct at the subdivision level occur as the primary endosymbiont in one host-animal type, the genus Alviniconcha. It should be also addressed that the Alviniconcha gastropods are currently the only known organisms capable of forming endosymbiotic relationships with two phylogenetically and physiologically distinct chemoautotrophic bacteria.
We thank the captains and crews of the R/V Yokosuka and the Shinkai 6500 for their technical expertise.