Molecular characterization of ectosymbiotic bacteria from nematodes in a shallow water system

Stilbonematid nematodes can be highly abundant members of the meiofauna in coastal sediments. These nematodes are covered by sulfur-oxidizing ectosymbiotic bacteria. Cooccurring stilbonematid nematodes of various genera have been described from sandy sediments of the Mediterranean island of Elba (Italy), but nothing is known about their ectosymbiotic bacteria. The aim of this study was to characterize the nematodes and their ectosymbiotic bacteria with molecular methods. Both morphological and 18S rRNA gene phylogenetic analysis of the hosts revealed that at least four different genera co-occur in the same habitat. Each of the nematode hosts is covered by a particularly arranged bacterial coat consisting of a specific morphotype. Nematodes of the genus Catanema and Laxus for example, are covered by a monolayer of rods, Leptonemella and Stilbonema by a multilayer of coccoid bacteria, whereby Eubostrichus sp. has long filamentous bacteria on the cuticle. Phylogenetic analyses based on 3 genes (16S rRNA, ITS and partial-23S rRNA) showed that nematodes of different genera are associated with distinct ectosymbiotic bacteria that belong to the gammaproteobacterial clade of sulfur-oxidizing symbionts, typically associated with marine nematode and oligochaete worms (MONTS clade). Each of the stilbonematid nematode genera was associated with its own particular 16S-ITS-partial-23S MONTS phylotype. The individuals of two different species of Leptonemella hosted two closely related but distinct ectosymbionts, indicating that the symbiotic associations are host species-specific. While monospecific associations were shown for the genera Leptonemella and Catanema, the genera Laxus sp., Eubostrichus sp. and Stilbonema sp. had a higher diversity in their bacterial community as detected with our PCR assay. However, the symbiotic nature of these additional bacterial phylotypes remains unclear. Furthermore, we investigated the possible mode of transmission by comparing host and symbiont phylogenies. The incongruence of host and symbiont tree topologies both on the genus and species level suggests that hosts and symbionts did not cospeciate and that the hosts acquire their symbionts from their environment. Due to the highly specific association of host and symbionts coevolved recognition mechanisms to ensure a stable symbiosis over time are postulated