Symbioses between bacteria and eukaryotes are ubiquitous, yet our understanding of the interactions driving these associations is hampered by our inability to cultivate most host-associated microbes. Here, we used a metagenomic approach to describe four co-occurring symbionts from the marine oligochaete Olavius algarvensis, a worm lacking a mouth, gut, and nephridia. Shotgun sequencing and metabolic pathway reconstruction revealed that the symbionts are sulfur-oxidizing and sulfate-reducing bacteria, all of which are capable of carbon fixation, providing the host with multiple sources of nutrition. Molecular evidence for the uptake and recycling of worm waste products by the symbionts suggests how the worm could eliminate its excretory system, an adaptation unique among annelid worms. We propose a model which describes how the versatile metabolism within this symbiotic consortium provides the host with an optimal energy supply as it shuttles between the upper oxic and lower anoxic coastal sediments which it inhabits. 3 Symbiosis plays a major role in shaping the evolution and diversity of eukaryotic organisms 1 . Remarkably, only recently has there been an emerging recognition that most eukaryotic organisms are intimately associated with a complex community of beneficial microbes that are essential for their development, health, and interactions with the environment 2 . This renaissance in symbiosis research stems from advances in molecular approaches that have enabled the study of natural microbial consortia using cultivationindependent methods [3][4][5] . Metagenomic analyses have provided a new dimension in the study of community organization and metabolism in natural microbial communities [6][7][8][9][10] .To date, however, genomic analyses of symbiotic microbes from eukaryotes have been confined to individual strains (for the only exception see Wu et al. 11), limiting our ability to understand the intricate interactions involving communication, competition, and resource partitioning that shape symbiotic microbial communities.Here, we used random shotgun sequencing and nucleotide-signature based binning to study the symbiotic community in Olavius algarvensis. This marine worm belongs to a group of oligochaetes (phylum Annelida) that lack a mouth, gut, and anus, and are unique among annelid worms in having reduced their nephridial excretory system 12 . They live in obligate and species-specific associations with multiple extracellular bacterial endosymbionts located just below the worm cuticle 12 . Since the symbionts have yet to be grown in culture, their phylogeny has only been accessible through 16S rRNA analysis and fluorescence in situ hybridization (FISH) 13,14 . O. algarvensis lives in coastalMediterranean sediments and harbors a chemoautotrophic sulfur-oxidizing Gammaproteobacterium ( 1 symbiont) and a deltaproteobacterial sulfate reducer ( 1 symbiont), recently shown to be engaged in an endosymbiotic sulfur cycle 14. An additional gamma-and deltaproteobacterial symbiont ( 3 and 4 symbionts) of ...

Gutless oligochaete worms are found worldwide in the pore waters of marine sediments and live in symbiosis with chemoautotrophic sulfur-oxidizing bacteria. In the Mediterranean, two species of gutless oligochaete worms, Olavius algarvensis and O. ilvae, co-occur in sediments around sea grass beds. These sediments have extremely low sulfide concentrations (< 1 microM), raising the question if O. ilvae, as shown previously for O. algarvensis, also harbours sulfate-reducing symbionts that provide its sulfur-oxidizing symbionts with reduced sulfur compounds. In this study, we used fluorescence in situ hybridization (FISH) and comparative sequence analysis of genes for 16S rRNA, sulfur metabolism (aprA and dsrAB), and autotrophic carbon fixation (cbbL) to examine the microbial community of O. ilvae and re-examine the O. algarvensis symbiosis. In addition to the four previously described symbionts of O. algarvensis, in this study a fifth symbiont belonging to the Spirochaetes was found in these hosts. The symbiotic community of O. ilvae was similar to that of O. algarvensis and also included two gammaproteobacterial sulfur oxidizers and two deltaproteobacterial sulfate reducers, but not a spirochete. The phylogenetic and metabolic similarity of the symbiotic communities in these two co-occurring host species that are not closely related to each other indicates that syntrophic sulfur cycling provides a strong selective advantage to these worms in their sulfide-poor environment.

SummaryThe marine oligochaete worm Tubificoides benedii is often found in high numbers in eutrophic coastal sediments with low oxygen and high sulfide concentrations. A dense biofilm of filamentous bacteria on the worm's tail end were morphologically described over 20 years ago, but no further studies of these epibiotic associations were done. In this study, we used fluorescence in situ hybridization and comparative sequence analysis of 16S rRNA and proteincoding genes to characterize the microbial community of the worm's tail ends. The presence of genes involved in chemoautotrophy (cbbL and cbbM) and sulfur metabolism (aprA) indicated the potential of the T. benedii microbial community for chemosynthesis. Two filamentous ectosymbionts were specific to the worm's tail ends: one belonged to the Leucothrix mucor clade within the Gammaproteobacteria and the other to the Thiovulgaceae within the Epsilonproteobacteria. Both T. benedii ectosymbionts belonged to clades that consisted almost exclusively of bacteria associated with invertebrates from deepsea hydrothermal vents. Such close relationships between symbionts from shallow-water and deep-sea hosts that are not closely related to each other are unusual, and indicate that biogeography and host affiliation did not play a role in these associations. Instead, similarities between the dynamic environments of vents and organic-rich mudflats with their strong fluctuations in reductants and oxidants may have been the driving force behind the establishment and evolution of these symbioses.

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