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

Abstract

We examined the abundance and spatial distribution of major phylogenetic groups of the domain Bacteria in hindguts of the Australian lower termite Mastotermes darwiniensis by using in situ hybridization with group-specific, fluorescently labeled, rRNA-targeted oligonucleotide probes. Between 32.0 +/- 7.2% and 52.3 +/- 8.2% of the DAPI-stained cells in different hindgut fractions were detected with probe EUB338, specific for members of the domain Bacteria. About 85% of the prokaryotic cells were associated with the flagellates of the thin-walled anterior region (P3a) and the thick wall of the posterior 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 fractions, cells were found in varying numbers with probe ALF1b, which targeted members of the alpha-Proteobacteria, whereas substantial amounts of sulfate-reducing bacteria, gram-positive bacteria with a high DNA G+C content and members of the Cytophaga-Flavobacterium cluster of the Cytophaga-Flavobacterium-Bacteroides (CFB) phylum could be detected only in the wall fraction of P3b/P4. This clearly indicates that the hindgut microhabitats differ in the composition of their microbial community. In situ hybridization of cryosections 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-Flavobacterium cluster of the CFB phylum and to the group of gram-positive bacteria with a high DNA G+C content, respectively. Oxygen concentration profiles determined with microelectrodes revealed steep oxygen gradients both in P3a and P3b. Oxygen was consumed within 100 &mgr;m below the gut surface, and anoxic conditions prevailed in the central portions of both gut regions, indicating that oxygen consumption in the hindgut does not depend on the presence of a biofilm on the hindgut wall.

Localizing transcripts to single cells suggests an important role of uncultured deltaproteobacteria in the termite gut hydrogen economy

“…Diverse microbes attached to the surface of, or resident in the cytoplasm of, protozoa may compose a large fraction of the total gut bacterial load, considering that protozoa dominate up to 90% of the hindgut volume of lower termites (21). Previous studies have proposed roles for these bacteria (26,(30)(31)(32), but the challenges faced when studying their physiology in vitro or in situ makes many proposed interactions only speculative.…”

“…The ZnD2sec FDH gene accounted for 64% of the fdhF Sec clones in the resulting libraries from total gut DNA, implying that it is perhaps the most abundant fdhF Sec -encoding member of the community. However, termite gut bacteria are known to strongly attach to the gut wall (21,22) and to associate with the surfaces or insides of gut protozoa (23), in addition to being free-swimming. Through the microfluidic device loading method, larger wood particles, protozoal particles, gut epithelial tissues, and other aggregates were routinely excluded from our initial microfluidic protocols, to prevent channel clogging in the device.…”

Molecular Phylogenetic Diversity of the Bacterial Community in the Gut of the TermiteCoptotermes formosanus

“…Studies using rRNA-based techniques such as rRNA gene clone sequencing, 5) terminal restriction fragment length polymorphisms (T-RFLP) analysis, 6) and fluorescence in situ hybridization (FISH) 7) have shown that the termite gut is a rich reservoir of novel and diverse microorganisms, and highly compartmentalized beyond our expectations, and have also indicated that our knowledge of the relevant ecosystem is still insufficient for comprehensive understanding of this symbiosis.…”

Phylogenetic and Morphological Diversity ofBacteroidalesMembers Associated with the Gut Wall of Termites

“…[10][11][12][13][14] A dense colonization of bacteria as well as methanogenic archaea is observed on the gut epithelium (simply, the gut wall). [15][16][17][18] In our recent report, 19) we investigated the spatial distribution of gut bacteria in the termite Reticulitermes speratus (Rhinotermitidae), particularly focusing on the bacteria resident on the gut wall, to demonstrate that the bacteria on the gut wall are more diverse than those in the gut lumen. As clearly shown in methanogens 18) and bacteria, 19,20) species inhabiting the gut wall are phylogenetically distinct from the other microhabitats of the gut.…”