- Microbiome Announcement
- Open Access
Metagenomic analysis of the microbiota in the highly compartmented hindguts of six wood- or soil-feeding higher termites
© Rossmassler et al. 2015
- Received: 16 July 2015
- Accepted: 28 September 2015
- Published: 26 November 2015
Termites are important contributors to carbon and nitrogen cycling in tropical ecosystems. Higher termites digest lignocellulose in various stages of humification with the help of an entirely prokaryotic microbiota housed in their compartmented intestinal tract. Previous studies revealed fundamental differences in community structure between compartments, but the functional roles of individual lineages in symbiotic digestion are mostly unknown.
Here, we conducted a highly resolved analysis of the gut microbiota in six species of higher termites that feed on plant material at different levels of humification. Combining amplicon sequencing and metagenomics, we assessed similarities in community structure and functional potential between the major hindgut compartments (P1, P3, and P4). Cluster analysis of the relative abundances of orthologous gene clusters (COGs) revealed high similarities among wood- and litter-feeding termites and strong differences to humivorous species. However, abundance estimates of bacterial phyla based on 16S rRNA genes greatly differed from those based on protein-coding genes.
Community structure and functional potential of the microbiota in individual gut compartments are clearly driven by the digestive strategy of the host. The metagenomics libraries obtained in this study provide the basis for future studies that elucidate the fundamental differences in the symbiont-mediated breakdown of lignocellulose and humus by termites of different feeding groups. The high proportion of uncultured bacterial lineages in all samples calls for a reference-independent approach for the correct taxonomic assignment of protein-coding genes.
Termites are important contributors to carbon and nitrogen cycling in tropical ecosystems. Their ability to degrade lignocellulose is based on a partnership with a diverse community of microbial symbionts harbored in their intestinal tracts [1, 2].
While the evolutionarily lower termites have relatively simple guts and digest wood with the help of cellulolytic protists, the hindguts of higher termites are more strongly compartmented and contain exclusively prokaryotic microbial communities [1–3]. The individual gut compartments feature steep axial and radial gradients in physical parameters, such as pH, redox potential, and oxygen and hydrogen partial pressure [4–6], and microbial community structures along the intestinal tract strikingly differ between compartments [6–8].
Several metagenomic studies have assessed the functional potential of the gut microbiota in a few higher termites (including wood-feeding, dung-feeding, and fungus-cultivating species) but were usually restricted to the luminal contents [9–11]. These analyses revealed intriguing differences in the functional role of the microbiota in symbiotic digestion of lignocellulose, but the functional potential of the microbiota in individual compartments and differences to higher termites feeding on humus or soil are still entirely in the dark.
The wood feeder Microcerotermes parvus, the litter feeder Cornitermes sp., the humus feeders Termes hospes and Neocapritermes taracua, and the soil feeder Cubitermes ugandensis were collected in the field; the wood feeder Nasutitermes corniger was from a laboratory colony. Species were initially identified according to morphology, and the identity was corroborated by mitochondrial genome analysis . Guts of 30–50 worker termites were dissected into individual compartments, and DNA was extracted from pooled sections using a bead-beating protocol. Detailed information on the origin of the termites and sample processing can be found in the Additional file 1: Supplementary methods.
Amplicon sequencing and analysis
Summary of sample information and metagenomic library characteristics
Termite species strain mitogenomea (Diet)
Sample size (Gbp)
Assem. fraction (%)
Assy. size (Mbp)b
Contigs >50 kbp
Contigs >100 kbp
Longest contig (kbp)
SRA acc. no.d
Nasutitermes corniger Nc150 KP091691 (wood)
Microcerotermes parvus Mp193 KP091690 (wood)
Cornitermes sp. Co191 KP091688 (litter)
Termes hospes Th196 KP091693 (humus)
Neocapritermes taracua Nt197 KP091692 (humus)
Cubitermes ugandensis Cu122 KP091689 (soil)
Metagenomic sequencing and analysis
Metagenomic libraries were prepared, sequenced, quality controlled, and assembled at the Joint Genome Institute (Walnut Creek, CA, USA). DNA was sequenced on an Illumina HiSeq 2000 (Illumina Inc., San Diego, CA). Quality-controlled reads were assembled and uploaded to the Integrated Microbial Genomes (IMG/M ER) database (https://img.jgi.doe.gov/cgi-bin/mer/main.cgi) for gene identification and annotation by applying the standard operation procedure of IMG . The metagenomes are publicly available on the IMG/M ER website (see Table 1 for accession numbers). Gene functions of protein-coding genes were identified, and genes were taxonomically assigned using BLASTp (top hit) and RPS-BLAST against the COG database.
In addition to using standard precautions, we verified the reproducibility of the iTag data sets by comparing them to previously published data sets for the same termite species (or genus). We also conducted independent analyses of community structure in the same samples using libraries obtained with a different primer set (unpublished results). The absence of noteworthy differences also assured that our data sets were not contaminated.
The bacterial community of the P1 compartment of most termite species was dominated by Firmicutes, which is in agreement with previous reports on the microbiota of this sometimes highly alkaline hindgut compartment [6–8]; the high proportions of Spirochaetes and Actinobacteria in certain termite species are exceptional but not unprecedented [6, 8]. The bacterial communities in the P4 were generally more diverse than in the other compartments and displayed an increasing abundance of Bacteroidetes, which matches previous observations with Nasutitermes and Cubitermes species [6, 7]. The detailed classification results for all taxonomic ranks down to the genus level are shown in Additional file 2: Table S1.
Metagenomic sequencing of the major hindgut compartments (P1, P3, and P4) of the six termite species yielded an average library size of 42 Gbp (range, 30–70 Gbp), with 90 % of the bases (range, 68–99 %) in the assembled fraction (Table 1). The large number of bacterial contigs longer than 100 kbp and the strong size reduction of the assemblies to 1.4 Gbp (range, 0.6–2.1 Gbp) after dereplication indicate a relatively low diversity of the respective communities. In a pilot experiment with N. corniger and Cubitermes ugandensis, we also obtained smaller libraries (3–5 Gbp) for the crop (foregut), midgut, and P5 compartments, with only 50 % of the bases in the assembled fraction. Because assembly sizes after dereplication were about tenfold smaller (0.1–0.4 Gbp) (Table 1), these datasets were not included in the following analyses.
A comparison of the bacterial community structure determined by iTag analysis with the phylogenetic classification of protein-coding genes in the metagenomes revealed large discrepancies already at the phylum level (Additional file 2: Table S1). While Fibrobacteres and the TG3 phylum were highly abundant in bacterial communities of wood- and litter-feeding termites, they were strongly underrepresented (Fibrobacteres) or undetected (TG3 phylum) in the taxonomic assignments of the protein-coding genes (exemplified in Additional file 3: Figure S1). This discrepancy is explained by the lack of appropriate reference genomes in public databases. The only sequenced genome from Fibrobacteres, the rumen isolate Fibrobacteres succinogenes, is only distantly related to Fibrobacteres detected in this study , and the draft genome of Chitinivibrio alkaliphilus, the first isolate of the TG3 phylum , was not included in public databases at the time of analysis. The high abundance of genes assigned to Proteobacteria, which contrasts strongly with their low proportion in the iTag datasets, is also likely caused by the bias introduced by incorrect assignment due to the lack of reference genomes.
The results of this preliminary analysis show that microbial structure and function are correlated with both the digestive strategy of the host and corresponding microhabitats. The large metagenomic datasets will allow an in-depth analysis of the microbial functions in the homologous gut compartments and a comparison between hosts with diverging digestive strategies. Of particular interest will be the gene functions related to the digestion of lignocellulose and the putative peptidic substrates in the diet of the humivorous host . To overcome the bias in the taxonomic assignment of the genes, we are currently using a reference-independent approach to reconstruct population genomes for the major lineages of uncultivated symbionts.
Availability of supporting data
This study was supported by the Max Planck Society, a grant of the Deutsche Forschungsgemeinschaft (DFG) in the Collaborative Research Center SFB 987, and the LOEWE program of the state of Hessen (Synmikro). The work conducted by the U.S. Department of Energy Joint Genome Institute, a DOE Office of Science User Facility, is supported by the Office of Science of the U.S. Department of Energy under Contract No. DE-AC02-05CH11231. The authors thank all JGI staff and particular their project manager Tijana Glavina del Rio for their excellent service.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
- Brune A. Symbiotic digestion of lignocellulose in termite guts. Nat Rev Microbiol. 2014;12:168–80.View ArticlePubMedGoogle Scholar
- Brune A, Dietrich C. The gut microbiota of termites: digesting the diversity in the light of ecology and evolution. Annu Rev Microbiol. 2015;69. in press.Google Scholar
- Dietrich C, Köhler T, Brune A. The cockroach origin of the termite gut microbiota: patterns in bacterial community structure reflect major evolutionary events. Appl Environ Microbiol. 2014;80:2261–9.PubMed CentralView ArticlePubMedGoogle Scholar
- Brune A, Emerson D, Breznak JA. The termite gut microflora as an oxygen sink: microelectrode determination of oxygen and pH gradients in guts of lower and higher termites. Appl Environ Microbiol. 1995;61:2681–7.PubMed CentralPubMedGoogle Scholar
- Schmitt-Wagner D, Brune A. Hydrogen profiles and localization of methanogenic activities in the highly compartmentalized hindgut of soil-feeding higher termites (Cubitermes spp.). Appl Environ Microbiol. 1999;65:4490–6.PubMed CentralPubMedGoogle Scholar
- Köhler T, Dietrich C, Scheffrahn RH, Brune A. High-resolution analysis of gut environment and bacterial microbiota reveals functional compartmentation of the gut in wood-feeding higher termites (Nasutitermes spp.). Appl Environ Microbiol. 2012;78:4691–701.PubMed CentralView ArticlePubMedGoogle Scholar
- Schmitt-Wagner D, Friedrich MW, Wagner B, Brune A. Axial dynamics, stability, and interspecies similarity of bacterial community structure in the highly compartmentalized gut of soil-feeding termites (Cubitermes spp.). Appl Environ Microbiol. 2003;69:6018–24.PubMed CentralView ArticlePubMedGoogle Scholar
- Thongaram T, Hongoh Y, Kosono S, Ohkuma M, Trakulnaleamsai S, Noparatnaraporn N, et al. Comparison of bacterial communities in the alkaline gut segment among various species of higher termites. Extremophiles. 2005;9:229–38.View ArticlePubMedGoogle Scholar
- Warnecke F, Luginbühl P, Ivanova N, Ghassemian M, Richardson TH, Stege JT, et al. Metagenomic and functional analysis of hindgut microbiota of a wood-feeding higher termite. Nature. 2007;450:560–5.View ArticlePubMedGoogle Scholar
- He S, Ivanova N, Kirton E, Allgaier M, Bergin C, Scheffrahn RH, et al. Comparative metagenomic and metatranscriptomic analysis of hindgut paunch microbiota in wood- and dung-feeding higher termites. PLoS One. 2013;8, e61126.PubMed CentralView ArticlePubMedGoogle Scholar
- Liu N, Zhang L, Zhou H, Zhang M, Yan X, Wang Q, et al. Metagenomic insights into metabolic capacities of the gut microbiota in a fungus-cultivating termite (Odontotermes yunnanensis). PLoS One. 2013;8, e69184.PubMed CentralView ArticlePubMedGoogle Scholar
- Dietrich C, Brune A. The complete mitogenomes of six higher termite species reconstructed from metagenomic datasets (Cornitermes sp., Cubitermes ugandensis, Microcerotermes parvus, Nasutitermes corniger, Neocapritermes taracua, and Termes hospes ). Mitochondrial DNA. 2014, early online (http://dx.doi.org/10.3109/19401736.2014.987257).
- Degnan PH, Ochman H. Illumina-based analysis of microbial community diversity. ISME J. 2012;6:183–94.PubMed CentralView ArticlePubMedGoogle Scholar
- Mikaelyan A, Köhler T, Lampert N, Rohland J, Boga H, Meuser K, et al. Classifying the bacterial gut microbiota of termites and cockroaches: a curated phylogenetic reference database (DictDb). Syst Appl Microbiol. in revision.Google Scholar
- Markowitz VM, Chen I-MA, Chu K, Szeto E, Palaniappan K, Pillay M, et al. IMG/M 4 version of the integrated metagenome comparative analysis system. Nucleic Acids Res. 2014;42:D568–73.PubMed CentralView ArticlePubMedGoogle Scholar
- Hongoh Y, Deevong P, Inoue T, Moriya S, Trakulnaleamsai S, Ohkuma M, et al. Intra- and interspecific comparisons of bacterial diversity and community structure support coevolution of gut microbiota and termite host. Appl Environ Microbiol. 2005;71:6590–9.PubMed CentralView ArticlePubMedGoogle Scholar
- Costa PS, Oliveira PL, Chartone-Souza E, Nascimento AMA. Phylogenetic diversity of prokaryotes associated with the mandibulate nasute termite Cornitermes cumulans and its mound. Biol Fertil Soils. 2013;49:567–74.View ArticleGoogle Scholar
- Hongoh Y, Deevong P, Hattori S, Inoue T, Noda S, Noparatnaraporn N, et al. Phylogenetic diversity, localization, and cell morphologies of members of the candidate phylum TG3 and a subphylum in the phylum Fibrobacteres, recently discovered bacterial groups dominant in termite guts. Appl Environ Microbiol. 2006;72:6780–8.PubMed CentralView ArticlePubMedGoogle Scholar
- Mikaelyan A, Strassert JFH, Tokuda G, Brune A. The fibre-associated cellulolytic bacterial community in the hindgut of wood-feeding higher termites (Nasutitermes spp.). Environ Microbiol. 2014;16:2711–22.View ArticleGoogle Scholar
- Brauman A, Doré J, Eggleton P, Bignell D, Breznak JA, Kane MD. Molecular phylogenetic profiling of prokaryotic communities in guts of termites with different feeding habits. FEMS Microbiol Ecol. 2001;35:27–36.View ArticlePubMedGoogle Scholar
- Noirot C. The gut of termites (Isoptera): comparative anatomy, systematics, phylogeny. II. Higher termites (Termitidae). Ann la Société Entomol Fr. 2001;37:431–71.Google Scholar
- Suen G, Weimer PJ, Stevenson DM, Aylward FO, Boyum J, Deneke J, et al. The complete genome sequence of Fibrobacter succinogenes S85 reveals a cellulolytic and metabolic specialist. PLoS One. 2011;6, e18814.PubMed CentralView ArticlePubMedGoogle Scholar
- Sorokin DY, Gumerov VM, Rakitin AL, Beletsky AV, Damsté JSS, Muyzer G, et al. Genome analysis of Chitinivibrio alkaliphilus gen. nov., sp. nov., a novel extremely haloalkaliphilic anaerobic chitinolytic bacterium from the candidate phylum Termite Group 3. Environ Microbiol. 2014;16:1549–65.View ArticlePubMedGoogle Scholar