The candidate phylum 'Termite Group 1' (TG1) of bacteria, which is abundant in termite guts but has no culturable representative, was investigated with respect to the in situ localization, distribution, and diversity. Based on the 16S rRNA gene sequence analyses and FISH in termite guts, a number of lineages of TG1 members were identified as endosymbionts of a variety of gut flagellated protists from the orders Trichonymphida, Cristamonadida, and Oxymonadida that are mostly unique to termites. However, the survey in various environments using specific PCR primers revealed that TG1 members were also present in termites, a cockroach, and the bovine rumen that typically lack these protist orders. Most of the TG1 members from gut flagellates, termites, cockroaches, and the rumen formed a monophyletic subcluster that showed a shallow branching pattern in the phylogenetic tree, suggesting their recent diversification. Although endosymbionts of the same protist genera tended to be closely related, the endosymbiont lineages were often independent of the higher level classifications of their host protist and were dispersed in the phylogenetic tree. It appears that their cospeciation is not the sole rule for the diversification of TG1 members of endosymbionts.

Rs-N31, a 16S rRNA phylotype affiliated with the genus Desulfovibrio, has frequently been detected from the gut of the wood-feeding termite Reticulitermes speratus. In this study, we designed a probe specifically targeting phylotype Rs-N31 and performed fluorescence in situ hybridization to identify the corresponding cells. The signals were detected exclusively inside the cells of the flagellate Trichonympha agilis, which simultaneously harbours another intracellular bacterium belonging to the candidate phylum Termite Group 1 (TG1). The detected cells were coccoid or short rods and specifically localized in the cortical layer of mainly, the anterior part of the flagellate cell. Approximately 1800 cells were contained in a single host cell, accounting for, in total, 2% of the whole prokaryotic gut microbiota. The genes dsrAB and apsA for sulfate reduction and a gene-encoding H(2)-uptake hydrogenase, both possessing a high sequence identity with those of known desulfovibrios, were obtained by polymerase chain reaction (PCR) from the host cells isolated using a micromanipulator, and their expression was verified by reverse-transcription PCR. Thus, we suggest that this endosymbiont acts as a sink for the hydrogen generated by both the flagellates and possibly TG1 symbionts. For this uncultured bacterium, we propose a novel species, 'Candidatus Desulfovibrio trichonymphae'.

The flagellate Caduceia versatilis in the gut of the termite Cryptotermes cavifrons reportedly propels itself not by its own flagella but solely by the flagella of ectosymbiotic bacteria. Previous microscopic observations have revealed that the motility symbionts are flagellated rods partially embedded in the host cell surface and that, together with a fusiform type of ectosymbiotic bacteria without flagella, they cover almost the entire surface. To identify these ectosymbionts, we conducted 16S rRNA clone analyses of bacteria physically associated with the Caduceia cells. Two phylotypes were found to predominate in the clone library and were phylogenetically affiliated with the "Synergistes" phylum and the order Bacteroidales in the Bacteroidetes phylum. Probes specifically targeting 16S rRNAs of the respective phylotypes were designed, and fluorescence in situ hybridization (FISH) was performed. As a result, the "Synergistes" phylotype was identified as the motility symbiont; the Bacteroidales phylotype was the fusiform ectobiont. The "Synergistes" phylotype was a member of a cluster comprising exclusively uncultured clones from the guts of various termite species. Interestingly, four other phylotypes in this cluster, including the one sharing 95% sequence identity with the motility symbiont, were identified as nonectosymbiotic, or free-living, gut bacteria by FISH. We thus suggest that the motility ectosymbiont has evolved from a free-living gut bacterium within this termite-specific cluster. Based on these molecular and previous morphological data, we here propose a novel genus and species, "Candidatus Tammella caduceiae," for this unique motility ectosymbiont of Caducaia versatilis.Termites harbor complex gut microbiota, which comprises unicellular eukaryotes, bacteria, and archaea. The majority of the symbiotic flagellates in termite guts are characterized by both their cellulolytic activity and an abundance of prokaryotic symbionts within and/or attached to the outsides of their cell bodies. Whereas the ecological roles that these physically associated symbionts fulfill for their host protists are mostly unknown, that of ectosymbionts of the flagellate Caduceia versatilis (family Devescovinidae, order Cristamonadida, phylum Parabasalia) in the gut of the termite Cryptotermes cavifrons was clearly demonstrated by Tamm in 1982; the ectosymbiotic bacteria confer motility on the host Caduceia protists with their flagella (36). Although the host protists possess their own flagella, their rapid gliding movement is provided solely by the flagellated ectosymbiotic bacteria and never by their own flagella. This arrangement is comparable to the only other known example of motility symbiosis between prokaryotes and eukaryotic hosts, that is, between the devescovinid flagellate Mixotricha paradoxa in the gut of the termite Mastotermes darwiniensis and its ectosymbiotic spirochetes (6,19,41).Detailed microscopic observations have revealed that the motility ectosymbionts of C. versatilis are uniform rod-shaped bacteria...