Endomicrobia in termite guts: symbionts within a symbiont (Phylogeny, cospeciation with host flagellates, and preliminary genome analysis)

"Endomicrobia" are intracellular symbionts of termite gut flagellates that represent a distinct lineage in the novel bacterial phylum Termite Group I (TG-1). The evolutionary history of "Endomicrobia" with respect to their symbiosis with host flagellates was investigated using phylogenetic analyses and in situ identification based on small-subunit ribosomal RNA (SSU rRNA) sequences. By analyzing SSU rRNA sequences extracted from manually separated flagellate cells, "Endomicrobia" were shown to be widely distributed among termite gut flagellates. Symbionts originating from the same genus of flagellates invariably formed a host-specific monophyletic cluster in the phylogenetic tree. Their intracellular location in the cytoplasm was confirmed by fluorescent in situ hybridization (FISH) using oligonucleotide probes designed specifically for each symbiont and for the host. The phylogeny of "Endomicrobia" and host flagellates belonging to the parabasalid genus Trichonympha was investigated further in detail. SSU rRNA trees of the symbiont and the host exhibited overall congruence, which suggested cospeciation. Pairwise distance analysis and FISH corroborated the phylogenetic evidence, and these results allowed the construction of evolutionary scenarios for the divergence of "Endomicrobia" and their acquisition by flagellate hosts. "Endomicrobia" share their intracellular habitat with other bacterial symbionts. Bacterial SSU rRNA sequences originating from Trichonympha flagellates of Incisitermes marginipennis and Zootermopsis nevadensis revealed the occurrence of several distinct phylogenetic groups, representing Treponema spp., "Endomicrobia", δ-Proteobacteria, Bacteroidetes, and Mycoplasmatales. The Proteobacteria symbionts were shown to densely colonize the surface and the cytoplasm of the flagellates in high abundance. Since no pure cultures of "Endomicrobia" or their host flagellates are available, a method for the physical enrichment of "Endomicrobia" was established to gain more insights into the nature of these symbionts. "Candidatus Endomicrobium trichonymphae" (CET), the symbiont of Trichonympha flagellates, was selected as representative and enriched from gut contents of Z. nevadensis. High-molecular-weight DNA extracted from the enrichment is currently used for genome sequencing at the DOE Joint Genome Institute. A recently assembled 80-kb contig of CET revealed first insights into its metabolism, including hexuronate metabolism and the possible formation of H2. "Endomicrobia" are also present in the gut of the wood-feeding cockroach Cryptocercus punctulatus, which is considered to share a common ancestor with termites. Analysis of SSU rRNA sequences obtained from whole-gut DNA of this cockroach revealed the phylogenetic positions of six lineages (morphotypes) of parabasalid flagellates. Sequences obtained from manually isolated flagellates, which have long been assigned to the genus Trichonympha, turned out to be a previously undescribed lineage of Parabasalia. Since this new lineage may represent one of the earliest branches of parabasalid flagellates, the recovery of “Endomicrobia" sequences also from this flagellate underscores the presence of these endosymbionts already in the flagellates of the hypothetical dictyopteran ancestor of termites and cockroaches. The results of this study collectively document that "Endomicrobia" are prevalent and persistent endosymbionts of termite gut flagellates. This study also provides a better understanding of the phylogenetic properties of their biotic environment, i.e., the host flagellates and the cohabiting bacteria, which may help to explain the functional roles of "Endomicrobia" and their symbiotic interactions