Symbiotic relationships between invertebrates and bacteria are a prominent feature in marine systems. Investigations of these marine symbiotic relationships have been spurred on by the discovery of their dominance in invertebrates from extreme environments, such as hydrothermal vents. One of the first symbioses described in hydrothermal vent environments was that of the endosymbiotic chemoautotrophic sulfur-oxidizing bacteria associated with the deep-sea hydrothermal vent giant tubeworm,
Riftia pachyptila (
8). While these bacteria have not been cultured, their role in the symbiosis has been determined in detail by using a variety of physiological and molecular methods (
16,
22). Other hydrothermal vent symbiotic associations that have been described include chemoautotrophic endosymbionts associated with other vestimentiferans and bivalves, as well as the episymbionts associated with the shrimp
Rimicaris exoculataand the polychaetes
Alvinella pompejana and
Alvinella caudata (
5,
7,
13,
15,
19,
28).
A. pompejana is considered to be the most thermotolerant and eurythermal metazoan yet described (
6,
9). It forms tubes and colonizes the walls of high-temperature chimneys (40 to 105°C) at vent sites along the East Pacific Rise (EPR) from 32°S to 21°N latitude (11; C. A. Di Meo, G. Luther, and S. C. Cary, unpublished data).
A. pompejana is characterized by a dense, specific epibiotic microflora associated with the dorsal integument of the worm (
5,
12). Although electron microscopy studies of the
A. pompejana epibionts suggest that they are highly diverse, two bacterial morphotypes, a filamentous sheathed form and a rod-shaped form, appear to predominate (
17,
18). The filamentous form is integrated into specialized expansions of the intersegmentary parts, while the rod-shaped form appears to be less abundant but evenly distributed on the dorsal surface (
17,
18). The role of these epibionts in the symbiotic association with
A. pompejana is unclear. Ribulose-1,5-biphosphate carboxylase/oxygenase (RuBisCO),
14C labeling, and bicarbonate uptake assays have not implicated the dominant filamentous morphotype in autotrophic CO
2 fixation (
1,
2). However, it has been suggested that the symbionts may provide a food source for the worm (
5), as hypothesized for the ectosymbionts of
R. exoculata (
28,
31), detoxify the immediate environment surrounding the worm, or possibly provide thermal insulation (
11,
29).
In previous investigations, a combination of restriction fragment length polymorphism (RFLP) in conjunction with sequence analysis of a 16S ribosomal DNA (rDNA) clone library and in situ hybridizations performed with clone-specific oligonucleotides demonstrated that the
A. pompejana community is dominated by 4 families (of 32 families) that form a tight clade within the epsilon subdivision of the
Proteobacteria, at least 2 of which are made up of filamentous organisms (
5,
19). In the present study, a novel spirochete phylotype was discovered after further screening of the same 16S rDNA clone library. Spirochete A appeared to be much more dominant in the community when two separate DNA fingerprinting methods were used. However, spirochete A was not found in other
A. pompejana specimens when spirochete-specific primers were used. We also demonstrated that a second, unique spirochete (spirochete B), while accounting for only a small percentage of the community, was a consistent component of the
A. pompejana epibiont population throughout the geographic range of the worm.
DISCUSSION
A. pompejana is characterized by a dense coating of epibionts whose role in the symbiotic association remains unknown. A previous study of the complexity of this association revealed the prevalence of two filamentous epsilon-proteobacterial phylotypes as determined by both 16S rDNA library screening and in situ hybridization, confirming previous morphological evidence of dominant filamentous symbionts (
5,
19). Although these two closely related bacterial phylotypes dominate the community, there is morphological and molecular evidence of a more diverse assemblage of bacteria on the dorsal surface of
A. pompejana (
5,
10,
17-19,
29). In this investigation, besides confirming the diversity of members of the epsilon subdivision of the
Proteobacteria by DGGE analysis, we determined the presence of spirochetes in the
A. pomepjana community and found that one of these spirochetes is a minor, but consistent member.
Spirochete B was detected in all A. pompejana epibiont communities surveyed (at latitudes from 13°N to 32°S along the EPR), although it was a minor member of the community as determined by DGGE analysis, accounting for (∼1%) of the population. More importantly, its close phylogenetic relationship throughout almost the entire geographic range of A. pompejana (more than 97% similarity) and the fact that identical spirochetes were found at the same vent sites in different years suggest that this spirochete may be involved in an integrated symbiosis with A. pompejana. The fidelity of this relationship has yet to be determined. None of the other spirochetes detected were consistently present in the A. pompejana epibiont community, and most were found in the hydrothermal vent environment separate from the worm.
The genus
Spirochaeta is comprised of obligately and facultatively chemoheterotrophic anaerobes that are generally free-living freshwater and marine species (
26). However, Dubilier et al. demonstrated the presence of three symbionts associated with cuticular invaginations of
O. loisae, an annelid inhabiting marine sediments, one of which is a member of the
Spirochaeta family (
14). Until our study, only one spirochete had been discovered in a hydrothermal vent environment (
21). Although this spirochete was not phylogenetically characterized, its growth characteristics were consistent with those of other anaerobic chemoheterotrophic
Spirochaeta strains, the majority of which ferment available carbohydrates to produce acetate, CO
2, and H
2 (
21). Therefore, the metabolism of the
A. pompejana spirochete epibiont most likely is fermentative, and the spirochete probably uses available carbohydrates in the mucus surrounding the worm and potentially supplies carbon sources and electron donors to the other epibionts (members of the epsilon subdivision of the
Proteobacteria).
The
A. pompejana epibiont community described in this study and many other symbiotic and nonsymbiotic communities have been characterized by using PCR. The drawbacks to PCR-based analyses of microbial communities have been reviewed at length (
33). Two of the major concerns lie with PCR bias and different resolution capabilities of the methods. A determination of whether a member is considered dominant or minor in a community by PCR-based techniques may be questioned if only a single parameter is used for analysis. We felt that this study was a unique opportunity to characterize the spirochetes in a microbial community by using three separate, PCR-based methods. For each analysis (16S rDNA library screening, DGGE, and T-RFLP analysis) we used different primers, cycling conditions, and resolution techniques. DGGE and T-RFLP analyses, in which 16S rDNA amplification products that were approximately 171 and 918 bp long, respectively, were used, resulted in similar relative frequencies of spirochete A. The frequency of the corresponding clone was significantly different in the 16S rDNA library, where the entire 16S rDNA gene had been amplified (
19). Alignment of the sequence of the 3′ primer used for the 16S rDNA library with the 10 sequences most similar to spirochete A (= APG10) demonstrated that 7 of 10 members of the spirochete family had four or more mismatches in the 3′ end of the primer sequence. Thus, many of the spirochetes could not be amplified representatively with this primer and were underrepresented in the full-length 16S rDNA community analysis. Like the results of other investigators, our results indicate that primer bias may be extremely important in a comparison of the members of a community (
27,
30,
34).
There have been only a few previous molecular biology-based studies that described the microbial diversity observed at hydrothermal vents, and in none of them were spirochetes found (
20,
24,
32). This may have been due to absence of the organisms or, as suggested by this study, to the choice of primers used for PCR amplification. During this investigation, we discovered several novel spirochetes not only in the
A. pompejana epibiont community but also in the surrounding tubes and chimney samples from other hydrothermal vent environments. Because of the difficulty of isolating and cultivating spirochetes, detailed molecular analysis of similar environments with spirochete-specific primers would most likely reveal other, previously unknown members of the spirochete division. We are currently screening other hydrothermal vent habitats with such primers to identify additional novel spirochetes that are most likely important components of the microbial communities associated with deep-sea hydrothermal vent environments.
Unlike many other symbiotic associations in marine systems, the bacterial community associated with
A. pompejana is morphologically and phylogenetically diverse. A group of closely related members of the epsilon subdivision of the
Proteobacteria dominates the community (
19; this study), while a smaller percentage of the
A. pompejanaepibiont community members from a wide geographic range studied in this work consists of more diverse members of the epsilon subdivision of the
Proteobacteria and of a closely related
Spirochaeta phylogenetic cluster. The role of the spirochetes remains unclear, but our results support the hypothesis that some of the epibionts of
A. pompejana are chemoheterotrophic. Further research is currently being performed in our laboratory to characterize the mixture of
A. pompejanaepibionts collected from geographically, thermally, and chemically diverse environments and to decipher their metabolic capabilities in an effort to discover their role in this integrated episymbiosis.