Candidatus Anadelfobacter veles and Candidatus Cyrtobacter comes, Two New Rickettsiales Species Hosted by the Protist Ciliate Euplotes harpa (Ciliophora, Spirotrichea)

The order Rickettsiales (Alphaproteobacteria) is a well-known group containing obligate endocellular prokaryotes. The order encompasses three families (Rickettsiaceae, Anaplasmataceae, and Holosporaceae) and a fourth, family-level cluster, which includes only one candidate species, Candidatus Midichloria mitochondrii, as well as several unnamed bacterial symbionts. The broad host range exhibited by the members of the Candidatus Midichloria clade suggests their eventual relevance for a better understanding of the evolution of symbiosis and host specificity of Rickettsiales. In this paper, two new bacteria belonging to the Candidatus Midichloria clade, hosted by two different strains of the ciliate protist Euplotes harpa, are described on the basis of ultrastructural observations, comparative 16S rRNA gene sequence analysis, and an estimation of the percentage of infection. Ultrastructure of these bacteria shows some unusual features: one has an electron-dense cytoplasm, and the other one lacks a symbiosomal membrane. The latter was up to now considered an exclusive feature of bacteria belonging to the family Rickettsiaceae. 16S rRNA gene phylogenetic analysis unambiguously places the new bacteria in the Candidatus Midichloria clade, although their phylogenetic relationships with other members of the clade are not clearly resolved. This is the first report of a ciliate-borne bacterium belonging to the Candidatus Midichloria clade. On the basis of the data obtained, the two bacteria are proposed as two new candidate genera and species, Candidatus Anadelfobacter veles and Candidatus Cyrtobacter comes.