Phylogeny of 16S rRNA, ribulose 1,5-bisphosphate carboxylase/oxygenase, and adenosine 5'-phosphosulfate reductase genes from gamma- and alphaproteobacterial symbionts in gutless marine worms (oligochaeta) from Bermuda and the Bahamas

Abstract

Gutless oligochaetes are small marine worms that live in obligate associations with bacterial endosymbionts. While symbionts from several host species belonging to the genus Olavius have been described, little is known of the symbionts from the host genus Inanidrilus. In this study, the diversity of bacterial endosymbionts in Inanidrilus leukodermatus from Bermuda and Inanidrilus makropetalos from the Bahamas was investigated using comparative sequence analysis of the 16S rRNA gene and fluorescence in situ hybridization. As in all other gutless oligochaetes examined to date, I. leukodermatus and I. makropetalos harbor large, oval bacteria identified as Gamma 1 symbionts. The presence of genes coding for ribulose-1,5-bisphosphate carboxylase/oxygenase form I (cbbL) and adenosine 5'-phosphosulfate reductase (aprA) supports earlier studies indicating that these symbionts are chemoautotrophic sulfur oxidizers. Alphaproteobacteria, previously identified only in the gutless oligochaete Olavius loisae from the southwest Pacific Ocean, coexist with the Gamma 1 symbionts in both I. leukodermatus and I. makropetalos, with the former harboring four and the latter two alphaproteobacterial phylotypes. The presence of these symbionts in hosts from such geographically distant oceans as the Atlantic and Pacific suggests that symbioses with alphaproteobacterial symbionts may be widespread in gutless oligochaetes. The high phylogenetic diversity of bacterial endosymbionts in two species of the genus Inanidrilus, previously known only from members of the genus Olavius, shows that the stable coexistence of multiple symbionts is a common feature in gutless oligochaetes.

FIG. 1.

Map of sampling sites in the northwest Atlantic Ocean. I. leukodermatus worms were collected in Bermuda, and I. makropetalos specimens were collected at Lee Stocking Island (the Bahamas).

FIG. 2.

Phylogenetic placement of bacterial symbionts in I. leukodermatus and I. makropetalos based on 16S rRNA sequences. Maximum-likelihood trees of members of the (a) Gammaproteobacteria and (b) Alphaproteobacteria are shown. Symbionts of gutless oligochaetes are listed in bold type, with the I. leukodermatus symbionts boxed in light gray and I. makropetalos symbionts in dark gray. The bars represent 10% estimated sequence divergence.

FIG. 3.

Phylogenetic placement of symbiotic RubisCO large-subunit (a) and APS reductase alpha-subunit (b) sequences from I. leukodermatus and I. makropetalos based on maximum-likelihood analyses. The bars represent 10% estimated sequence divergence, except for the estimated divergence between RubisCO form I and II sequences, which corresponds to 124%. Abbreviations: SRB, sulfate-reducing bacteria; SOX, sulfur-oxidizing bacteria; SRA, sulfate-reducing archaeon. Symbionts of chemosynthetic hosts are listed in bold type.

FIG. 4.

In situ identification of bacterial symbionts in I. leukodermatus (a and b) and I. makropetalos (c and d). Epifluorescence images show a cross section through the entire worm and the symbiont-containing region of the worm's body wall (all scale bars, 10 μm). (a) Dual hybridization with InaGAM and IleuAlpha1a-IleuAlpha1b-IleuAlpha3 probes, showing gammaproteobacterial symbionts in red and alphaproteobacterial symbionts in green. (Inset) Symbionts at a higher magnification. (b) Triple hybridization with Alpha 1a, Alpha 1b, and Alpha3 probes, showing the Alpha 1a symbionts in green, Alpha 1b symbionts in red, and Alpha 3 symbionts in blue. (c) Dual hybridization with InaGAM and ImakAlpha 1b-ImakAlpha2 probes, showing gammaproteobacterial symbionts in red and alphaproteobacterial symbionts in green. (inset) Symbionts at a higher magnification. (d) Dual hybridization with ImakAlpha1b and ImakAlpha2 probes, showing the Alpha 1b symbionts in green and Alpha 2 symbionts in red.