Endosymbiotic Methanobrevibacter species Living in Symbiotic Protists of the Termite Reticulitermes speratus Detected by Fluorescent In Situ Hybridization

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Abstract
Two species of cellulolytic protist, Dinenympha parva and Microjoenia, living in the guts of the lower termite Reticulitermes speratus are known to harbor endosymbiotic methanogens detectable with an epifluorescent microscope. DNA isolated from the guts of worker termites in a colony of R. speratus was amplified using archaea-specific primers and cloned, and partial 16S rRNA gene sequences were obtained. Archaeal PCR clones obtained from the guts of xylophagous insects in this and previous works formed four subgroups within the Methanobrevibacter branch of a phylogenetic tree; the sequences of the clones obtained in this report belonged to subgroups, designated XSAT1A and XSAT1D. Using a probe specific to each of the subgroups, 50 and 10 endosymbiotic Methanobrevibacter cells per protist respectively were detected with a probe specific to the subgroup XSAT1A by fluorescent in situ hybridization analysis in D. parva and Microjoenia. There was no observed hybridization to the endosymbiont with other subtype-specific probes including XSAT1D. Based on these results, endosymbionts in D. parva and Microjoenia sp. are proposed to belong to the Methanobrevibacter subgroup XSAT1A.