Intracellular bacteria associated with the ascidian Ecteinascidia turbinata:: phylogenetic and in situ hybridisation analysis

The ascidian Ecteinascidia turbinata (Herdman) is a colonial sea squirt found in the Caribbean and Mediterranean Seas. In the present study, the bacterial complement of E. turbinata has been assessed by 16S rRNA gene analysis and the most commonly occurring strains identified by restriction fragment length polymorphism. Three strains were found to predominate using this approach, with one representing >50% of clones from both larval and adult material. The 16S rRNA gene sequence of the most commonly occurring strain did not match with any known bacterial sequences and could only be assigned to the gamma-proteobacteria subdivision. The two other frequently occurring strains were assigned to the Mollicutes. In situ hybridisation analysis with eubacterial probes to 16S rRNA revealed the presence of apparently endosymbiotic bacteria in adult and larval tissue, and electron microscopy confirmed the presence of putative bacteriocytes in the larval tissue. The presence of the same bacteria in the brooded larvae suggested that they were vertically transmitted from parent to offspring. Further hybridisation using a novel probe designed to be specific to the 16S rRNA sequence of the dominant strain, highlighted the same cell types as that revealed by the eubacterial probe. The results suggest that the bacteria represent a novel strain, denoted 'Candidatus Endoecteinascidia frumentensis', and that they may have an important role in the biology of E. turbinata.