Phylogenetic diversity, abundance, and axial distribution of bacteria in the intestinal tract of two soil-feeding termites (Cubitermes spp.)

Abstract

The hindgut of soil-feeding termites is highly compartmentalized and characterized by pronounced axial dynamics of the intestinal pH and microbial processes such as hydrogen production, methanogenesis, and reductive acetogenesis. Nothing is known about the bacterial diversity and the abundance or axial distribution of the major phylogenetic groups in the different gut compartments. In this study, we showed that the variety of physicochemical conditions is reflected in the diversity of the microbial communities in the different gut compartments of two Cubitermes species (TERMITIDAE: Termitinae). 16S rRNA gene clones from the highly alkaline first proctodeal segment (P1) of Cubitermes orthognathus represented almost exclusively gram-positive bacteria with low G+C content (LGC bacteria). In the posterior gut segments, their proportion decreased progressively, and the clone libraries comprised a variety of phyla, including the Cytophaga-Flexibacter-Bacteroides group, various subgroups of Proteobacteria, and the spirochetes. Phylogenetic analysis revealed that many of the clones clustered with sequences from the guts of other termites, and some even formed clusters containing only clones from C. orthognathus. The abundance and axial distribution of major phylogenetic groups in the gut of Cubitermes ugandensis were determined by fluorescence in situ hybridization with group-specific oligonucleotide probes. While the results were generally in good agreement with those of the clonal analysis, direct counts with probes specific for the Planctomycetales revealed a severe underestimation of representatives of this phylum in the clone libraries. Results obtained with newly designed FISH probes directed against two clusters of LGC clones from C. orthognathus indicated that the clones were restricted to specific gut regions. A molecular fingerprinting analysis published in a companion paper (D. Schmitt-Wagner, M. W. Friedrich, B. Wagner, and A. Brune, Appl. Environ. Microbiol. 69:6018-6024, 2003) corroborated the presence of compartment-specific bacterial communities in the gut of different Cubitermes species.

FIG. 1.

Gut morphology of a Cubitermes sp. worker termite. Gut sections were separated at the indicated positions. The gut was drawn in its unraveled state to illustrate the sequence of the individual segments: C, crop; M, midgut; ms, mixed segment; and P1, P3, P4, and P5, the proctodeal segments (53). Hydrogen partial pressure in the individual gut segments was determined with hydrogen-sensitive microsensors for Cubitermes orthognathus (63). The average luminal pH of the major gut segments was determined with glass pH microelectrodes for Cubitermes speciosus (18).

FIG. 2.

Relative clone frequencies in major phylogenetic groups of the clone libraries of the four major hindgut sections of C. orthognathus. The different subphyla of Proteobacteria are differentiated by Greek letters. The number of clones in each clone library is given in parentheses. CFB, Cytophaga-Flexibacter-Bacteroides.

FIG. 3.

Phylogenetic position of clones obtained in this study (shown in bold) affiliated with gram-positive bacteria with low G+C content (LGC bacteria), inferred by maximum-likelihood analysis of 1,318 valid alignment positions. Shorter sequences (shown in italics) were added to the framework tree with the ARB parsimony tool (42). The scale bar indicates approximately 10% sequence difference. All marked nodes were reproducibly present in all phylogenetic analyses (including maximum-likelihood, maximum-parsimony, and neighbor-joining algorithms). Only nodes with bootstrap values (DNAPARS, 1,000 replicates) of >90% (•) and >50% (○) are marked. Clusters of clones containing only sequences from C. orthognathus were grouped; numerals indicate the numbers of clones in a particular cluster. Roman numerals on the right indicate the major clostridial clusters defined by Collins et al. (19). The tree was rooted with Escherichia coli (accession no. J01695), Holophaga foetida (accession no. X77215), Agrobacterium tumefaciens (accession no. M11223), and Verrucomicrobium spinosum (accession no. X90515) as outgroup.

FIG. 4.

Phylogenetic position of clones obtained in this study (shown in bold) affiliated with the Cytophaga-Flexibacter-Bacteroides phylum, inferred by maximum-likelihood analysis of 1,311 valid alignment positions. For details on tree calculation, evaluation, and notation, see the legend to Fig. 3.

FIG. 5.

Phylogenetic position of clones obtained in this study (shown in bold) affiliated with the Proteobacteria, inferred by maximum-likelihood analysis of 1,348 valid alignment positions. Greek letters represent the different subgroups. For details on tree calculation, evaluation, and notation, see the legend to Fig. 3. The tree was rooted with Thermus aquaticus (accession no. L09663), Aquifex pyrophilus (accession no. M83548), Thermotoga maritima (accession no. M21774), and Verrucomicrobium spinosum (accession no. X90515) as outgroup.

FIG. 6.

Rarefaction analysis of all bacterial 16S rRNA gene clones recovered from the hindgut of C. orthognathus. The expected number of clones was calculated from the number of clones analyzed at the species level with 97% sequence similarity (○) and at a sequence similarity level of 95% (▪) and 90% (□), arbitrarily defined as the genus or group level. The slope of the curves indicates whether the diversity was covered (zero or low slope) or whether new taxa can be expected if additional clones were to be analyzed (steep slope).