Invertebrates that are endemic to the highly sulfidic, reducing environments at deep-sea hydrothermal vents and cold seeps are commonly associated with chemosynthetic endosymbiotic bacteria (7
). These bacteria oxidize the reduced sulfur compounds that are abundant in hydrothermal fluid (8
). The resultant energy produced by the endosymbiont is coupled to the production of carbon sources that support the growth and maintenance of the invertebrate hosts (6
). The metabolic link between the invertebrate hosts and their endosymbionts has obvious implications for the dispersal and colonization strategies employed by the host organisms.
Vent endemic host organisms may employ one of three different mechanisms for transmission of their endosymbionts to the next generation. Previous studies have shown that the Vesicomyid bivalves form species-specific associations with endosymbiotic bacteria (12
). Host specificity is maintained in these associations by vertical transmission of the bacteria through the egg from the parent to the offspring (3
). The endosymbiont and host phylogenies are congruent, which is consistent with a vertical mode of symbiont transmission (3
). In contrast, results from several studies suggest that endosymbiont transmission in vestimentiferan tubeworms occurs horizontally (5
), possibly through ingestion of bacteria upon larval settlement (28
). It is also possible that horizontal transmission in vestimentiferan-bacterial symbioses could occur through infection of new recruits with bacteria previously associated with established congeners (20
), although potential mechanisms for this transfer have not been determined. If horizontal transmission is indeed the mechanism for bacterial acquisition in vestimentiferans, we expect that biogeographic variation would exist among symbionts acquired from the same host species collected in geographically isolated vent and seep sites. Conversely, if vestimentiferan larvae settle in the same locations where their symbionts are acquired, we expect that vestimentiferans living at the same site would harbor identical symbionts.
In a recent study, genetic variation in vestimentiferan endosymbionts was investigated by comparing sequences of the 16S rRNA genes of bacteria collected from various species of hydrothermal vent and cold seep tubeworms (20
). The results showed a marked phylogenetic distinction between vestimentiferan endosymbionts collected from hydrothermal vents and soft-bottom, cold seep environments. This study suggested that vestimentiferans acquire one of two distinct species of free-living bacteria depending on whether they settle on basaltic, hydrothermal vent sites or sedimented, cold seep sites. A third, unique bacterial species was discovered in a tubeworm that inhabited a sedimented whale fall. Significant genetic variation was not found among symbionts found within each of the habitat types. Symbionts from three different host genera collected from five different hydrothermal vent sites along the East Pacific Rise (EPR) had identical 16S rRNA gene sequences.
The observation of so little sequence variation among endosymbiont 16S rRNA genes suggests that all tubeworm endosymbionts may belong to a single species. However, the conservative nature of the 16S rRNA gene often renders it inadequate for distinguishing among conspecific strains of bacteria (50
). Therefore, it is not surprising that Feldman et al. (20
) did not find significant genetic variation within the habitat clades. It is possible that significant genetic variation exists between endosymbionts of geographically distributed vestimentiferan hosts but that these species- or strain-level differences have not been detected by approaches that have been employed to date.
The goal of our study was to resolve the question of genetic variation among widely distributed vestimentiferan endosymbionts by utilizing two molecular techniques that have historically proven useful for comparing closely related strains of bacteria. Our first approach entailed sequencing of the internal transcribed spacer (ITS) region, a noncoding region within the rRNA that often contains genetic variation sufficient for differentiating species of prokaryotes (2
). Our second approach involved using a DNA fingerprinting technique that utilizes repetitive extragenic palindromes (REPs) as priming sites to generate diagnostic banding patterns from prokaryotic genomic DNA (25
). The REP-PCR technique is particularly useful for investigations of bacterial symbiosis because REP sequences have been found only in bacterial DNA; phage and eukaryotes do not contain REP elements (25
). By virtue of these repetitive elements, symbiont DNA can be selectively amplified from a mixed population of symbiont and host DNA to allow analysis of bacterial population diversity. Our results provide information on the extent of genetic variation among the symbionts, as well as further evidence to support a horizontal mode of symbiont acquisition among the vestimentiferan tubeworms.