Differential enumeration and in situ localization of microorganisms in thehindgut of the lower termite Mastotermes darwiniensis by hybridization with rRNA-targeted probes

We examined the abundance and spatial distribution of major phylogenetic gr oups of the domain Bacteria in hindguts of the Australian lower termite Mas totermes darwiniensis by using in situ hybridization with group-specific, f luorescently labeled, rRNA-targeted oligonucleotide probes. Between 32.0 +/ - 7.2% and 52.3 +/- 8.2% of the DAPI-stained cells in different hindgut fra ctions were detected with probe EUB338, specific for members of the domain Bacteria. About 85% of the prokaryotic cells were associated with the flage llates of the thin-walled anterior region (P3a) and the thick wall of the p osterior region (P3b/P4) of the hindgut, as shown by DAPI staining. At most , half of the EUB338-detected cells hybridized with one of the other probes that targeted a smaller assemblage within the bacterial domain. In most fr actions, cells were found in varying numbers with probe ALF1b, which target ed members of the a-Proteobacteria, whereas substantial amounts of sulfate- reducing bacteria, gram-positive bacteria with a high DNA G+C content and m embers of the Cytophaga-Flavobacterium cluster of the Cyrophaga-Flavobacter ium-Bacteroides (CFB) phylum could be detected only in the wall fraction of P3b/P4. This clearly indicates that the hindgut microhabitats differ in th e composition of their microbial community. In situ hybridization of cryose ctions through the hindgut showed only low numbers of bacteria attached to the P3a wall. In contrast, the wall of P3b was densely colonized by rod-and coccus-shaped bacteria, which could be assigned to the Cytophaga-Flavobact erium cluster of the CFB phylum and to the group of gram-positive bacteria with a high DNA G+C content, respectively. Oxygen concentration profiles de termined with microelectrodes revealed steep oxygen gradients both in P3a a nd P3b. Oxygen was consumed within 100 mu m below the gut surface, and anox ic conditions prevailed in the central portions of both gut regions, indica ting that oxygen consumption in the hindgut does not depend on the presence of a biofilm on the hindgut wall.