Symbiotic spirochetes in the termite hindgut: phylogenetic identification of ectosymbiotic spirochetes of oxymonad protists

Some species of protists inhabiting the hindgut of lower-termites have a la rge number of ectosymbiotic spirochetes oil the cell surface. The phylogene tic positions of the ectosymbiotic spirochetes of three oxymonad protists, Dinenympha porteri in the gut of Reticulitermes speratus, and Pyrsonympha s p. and Dinenympha sp. in Hodotermopsis sjoestedti, were investigated withou t cultivation of these organisms. Protist fractions carefully collected wit h a micromanipulator were used as templates for the amplification of small subunit ribosomal RNA genes (SSU rDNA). The phylogenetic tree inferred from the nucleotide sequences of the SSU rDNA showed that they were affiliated with the Treponema cluster of spirochetes and they were divided into two cl usters. One was grouped together with the spirochetal sequences reported pr eviously from the gut of termites and the other was related to the Trepoema bryantii subgroup of treponemes (denoted as termite Treponema clusters I a nd II, respectively). Whole-cell in situ hybridization using a fluorescent- labeled oligonucleotide probe specific for the group of sequences in cluste r II identified most of the ectosymbiotic spirochetes of the oxymonad proti sts in the gut of R. speratus and H. sjoestedti. However, not all of the ec tosymbiotic spirochetes could be detected by means of this cluster II group -specific probe and the population of ectosymbiotic spirochetes of cluster II was different among the oxymonad species. In the case of D. porteri, an oligonucleotide probe specific for one member of cluster II recognized a po rtion of the ectosymbiolic spirochetes of cluster II, and their population was also different depending on the cell-type of D. porteri in terms of the attachment of ectosymbiotic spirochetes. The results indicate that the spi rochetes of cluster II and probably those of a parr of cluster I can be ass igned to ectosymbiotic species of oxymonad protists and that the population of ectosymbiotic spirochetes associated with a single protist consists of at least three species of phylogenetically distinct spirochetes. (C) 2000 F ederation of European Microbiological Societies. Published by Elsevier Scie nce B.V. All rights reserved.