Phylogenetic Characterization of Episymbiotic Bacteria Hosted by a Hydrothermal Vent Limpet (Lepetodrilidae,Vetigastropoda)

Marine invertebrates hosting chemosynthetic bacterial symbionts are known from multiple phyla and represent remarkable diversity in form and function. The deep-sea hydrothermal vent limpet Lepetodrilus fucensis from the Juan de Fuca Ridge complex hosts a gill symbiosis of particular interest because it displays a morphology unique among molluscs: filamentous bacteria are found partially embedded in the host's gill epithelium and extend into the fluids circulating across the lamellae. Our objective was to investigate the phylogenetic affiliation of the limpet's primary gill symbionts for comparison with previously characterized bacteria. Comparative 16S rRNA sequence analysis identified one y- and three e-Proteobacteria as candidate symbionts. We used fluorescence in situ hybridization (FISH) to test which of these four candidates occur with the limpet's symbiotic gill bacteria. The y-proteobacterial probes consistently hybridized to the entire area where symbiotic bacteria were found, but fluorescence signal from the y-proteobacterial probes was generally absent. Amplification of the y-proteobacterial 16S rRNA gene using a specific forward primer yielded a sequence similar to that of limpets collected from different ridge sections. In total, direct amplification or FISH identified a single y-proteobacterial lineage from the gills of 23 specimens from vents separated by a distance up to about 200 km and collected over the course of 2 years, suggesting a highly specific and widespread symbiosis. Thus, we report the first filamentous y-proteobacterial gill symbiont hosted by a mollusc.