Coexistence of bacterial sulfide oxidizers, sulfate reducers, and spirochetes in a gutless worm (Oligochaeta) from the Peru margin

Abstract

Olavius crassitunicatus is a small symbiont-bearing worm that occurs at high abundance in oxygen-deficient sediments in the East Pacific Ocean. Using comparative 16S rRNA sequence analysis and fluorescence in situ hybridization, we examined the diversity and phylogeny of bacterial symbionts in two geographically distant O. crassitunicatus populations (separated by 385 km) on the Peru margin (water depth, approximately 300 m). Five distinct bacterial phylotypes co-occurred in all specimens from both sites: two members of the gamma-Proteobacteria (Gamma 1 and 2 symbionts), two members of the delta-Proteobacteria (Delta 1 and 2 symbionts), and one spirochete. A sixth phylotype belonging to the delta-Proteobacteria (Delta 3 symbiont) was found in only one of the two host populations. Three of the O. crassitunicatus bacterial phylotypes are closely related to symbionts of other gutless oligochaete species; the Gamma 1 phylotype is closely related to sulfide-oxidizing symbionts of Olavius algarvensis, Olavius loisae, and Inanidrilus leukodermatus, the Delta 1 phylotype is closely related to sulfate-reducing symbionts of O. algarvensis, and the spirochete is closely related to spirochetal symbionts of O. loisae. In contrast, the Gamma 2 phylotype and the Delta 2 and 3 phylotypes belong to novel lineages that are not related to other bacterial symbionts. Such a phylogenetically diverse yet highly specific and stable association in which multiple bacterial phylotypes coexist within a single host has not been described previously for marine invertebrates.

FIG. 1.

Locations of O. crassitunicatus sampling sites on the Peru margin. The worms used in this study were collected at station A at a depth of 359 m and at station B at a depth of 270 m. The two stations are separated from each other by 385 km. Station L is a site sampled by Lisa Levin in 1998 at a depth of 305 m (12°22.7′S, 77°29.1′W); this station was called station A by Levin et al. in reference and station 1 by Levin et al. in reference . Specimens from the Levin cruise were used by Giere and Krieger (23) for their ultrastructural studies of O. crassitunicatus.

FIG.2.

Phylogenetic placement of bacterial symbionts in O. crassitunicatus based on 16S rRNA sequences: maximum-likelihood trees of members of the γ-Proteobacteria (a), δ-Proteobacteria (b), and Spirochaeta (c). Symbionts of gutless oligochaetes are indicated by boldface type, and the O. crassitunicatus symbionts are enclosed in boxes. Bar = 10% estimated sequence divergence.

FIG. 3.

In situ identification of bacterial symbionts in O. crassitunicatus. The epifluorescence images show cross sections through the entire worm (a) (scale bar, 20 μm) and the symbiont-containing region of the worm's body wall (b to f) (scale bars, 10 μm). All worms shown were from station A; the only exception is the worm shown in panel c, which was from station B. (a) Dual hybridization with the GAM42a and DSS658/DSR651 probes, showing γ-proteobacterial symbionts (red) and δ-proteobacterial symbionts (green). (b) Dual hybridization with the OcraGAM1 and OcraGAM2 probes, showing the Gamma 1 symbionts (red) and Gamma 2 symbionts (green). The inset shows an enlargement of the area enclosed by a box (scale bar, 5 μm). (c) Dual hybridization with the OcraDEL1 and OcraDEL2 probes, showing the Delta 1 symbionts (green) and Delta 2 symbionts (red). (d) Dual hybridization with the OcraDEL1/OcraDEL2 and OcraDEL3 probes, showing the Delta 1 and Delta 2 symbionts (red)and the Delta 3 symbionts (green). The inset shows an enlargement of the area enclosed by a box area (scale bar, 5 μm). (e) Monohybridization with the OcraSPI probe, showing spirochete symbionts (green). (f) Triple hybridization with the GAM42a, DSS658/DSR651, and OcraSPI probes, showing the two γ-proteobacterial symbionts (red), the three δ-proteobacterial symbionts (blue), and the spirochete symbionts (yellow). The muscle tissue of the worm at the bottom of the panel appears to be bluish green because of autofluorescence at mixed wavelengths.