Ohkuma, Moriya

Abstract In 1944, Harold Kirby described microorganisms living within nuclei of the protists Trichonympha in guts of termites; however, their taxonomic assignment remains to be accomplished. Here, we identified intranuclear symbionts of Trichonympha agilis in the gut of the termite Reticulitermes speratus. We isolated single nuclei of T. agilis, performed whole-genome amplification, and obtained bacterial 16S rRNA genes by PCR. Unexpectedly, however, all of the analyzed clones were from pseudogenes of 16S rRNA with large deletions and numerous sequence variations even within a single-nucleus sample. Authentic 16S rRNA gene sequences were finally recovered by digesting the nuclear DNA; these pseudogenes were present on the host Trichonympha genome. The authentic sequences represented two distinct bacterial species belonging to the phylum Verrucomicrobia, and the pseudogenes have originated from each of the two species. Fluorescence in situ hybridization confirmed that both species are specifically localized, and occasionally co-localized, within nuclei of T. agilis. Transmission electron microscopy revealed that they are distorted cocci with characteristic electron-dense and lucent regions, which resemble the intranuclear symbionts illustrated by Kirby. For these symbionts, we propose a novel genus and species, ‘Candidatus Nucleococcus trichonymphae’ and ‘Candidatus Nucleococcus kirbyi’. These formed a termite-specific cluster with database sequences, other members of which were also detected within nuclei of various gut protists, including both parabasalids and oxymonads. We suggest that this group is widely distributed as intranuclear symbionts of diverse protists in termite guts and that they might have affected the evolution of the host genome through lateral gene transfer.

Summary 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’.

Termites harbor diverse symbiotic gut microorganisms, the majority of which are as yet uncultivable and their interrelationships unclear. Here, we present the complete genome sequence of the uncultured Bacteroidales endosymbiont of the cellulolytic protist Pseudotrichonympha grassii, which accounts for 70% of the bacterial cells in the gut of the termite Coptotermes formosanus . Functional annotation of the chromosome (1,114,206 base pairs) unveiled its ability to fix dinitrogen and recycle putative host nitrogen wastes for biosynthesis of diverse amino acids and cofactors, and import glucose and xylose as energy and carbon sources. Thus, nitrogen fixation and cellulolysis are coupled within the protist's cells. This highly evolved symbiotic system probably underlies the ability of the worldwide pest termites Coptotermes to use wood as their sole food.

Summary Many reports have stated that flagellated protists in termite guts harbour ectosymbiotic spirochetes on their cell surface. In this study, we describe another bristle‐like ectosymbiont affiliated with the order Bacteroidales . The 16S rRNA phylotype Rs‐N74 predominates among Bacteroidales clones obtained from the gut of the termite Reticulitermes speratus . An Rs‐N74 phylotype‐specific probe was designed in this study and used for detection of the corresponding bacteria in the gut by fluorescence in situ hybridization (FISH) analysis. Surprisingly, the signals were detected specifically from the bristle‐like ‘appendages’ of various flagellate species belonging to the genus Dinenympha ; these ‘appendages’ had been believed to be spirochetal ectosymbionts or structures of the protists. The Rs‐N74 bacteria attached to the cell surface of the protists by a tip and coexisted with the spirochetal ectosymbionts. An electron micrograph revealed their morphology to be similar to a typical Bacteroidales bacterium. This bacterium is proposed to represent a novel genus and species, ‘ Candidatus Symbiothrix dinenymphae’, phylogenetically affiliated with a cluster consisting exclusively of uncultured strains from termite guts. A Bacteroidales ‐specific probe for FISH further revealed that this type of symbiosis exists also in various other protists, including parabasalids and oxymonads, and is widespread in termite guts.

ABSTRACTThe flagellateCaduceia versatilisin the gut of the termiteCryptotermes cavifronsreportedly 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 theCaduceiacells. Two phylotypes were found to predominate in the clone library and were phylogenetically affiliated with the “Synergistes” phylum and the orderBacteroidalesin theBacteroidetesphylum. 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; theBacteroidalesphylotype 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, “CandidatusTammella caduceiae,” for this unique motility ectosymbiont ofCaducaia versatilis.