Identifying the core microbial community in the gut of fungus‐growing termites

Gut microbes play a crucial role in decomposing lignocellulose to fuel termite societies, with protists in the lower termites and prokaryotes in the higher termites providing these services. However, a single basal subfamily of the higher termites, the Macrotermitinae, also domesticated a plant biomass‐degrading fungus (Termitomyces), and how this symbiont acquisition has affected the fungus‐growing termite gut microbiota has remained unclear. The objective of our study was to compare the intestinal bacterial communities of five genera (nine species) of fungus‐growing termites to establish whether or not an ancestral core microbiota has been maintained and characterizes extant lineages. Using 454‐pyrosequencing of the 16S rRNA gene, we show that gut communities have representatives of 26 bacterial phyla and are dominated by Firmicutes, Bacteroidetes, Spirochaetes, Proteobacteria and Synergistetes. A set of 42 genus‐level taxa was present in all termite species and accounted for 56–68% of the species‐specific reads. Gut communities of termites from the same genus were more similar than distantly related species, suggesting that phylogenetic ancestry matters, possibly in connection with specific termite genus‐level ecological niches. Finally, we show that gut communities of fungus‐growing termites are similar to cockroaches, both at the bacterial phylum level and in a comparison of the core Macrotermitinae taxa abundances with representative cockroach, lower termite and higher nonfungus‐growing termites. These results suggest that the obligate association with Termitomyces has forced the bacterial gut communities of the fungus‐growing termites towards a relatively uniform composition with higher similarity to their omnivorous relatives than to more closely related termites.     

The Cockroach Origin of the Termite Gut Microbiota: Patterns in Bacterial Community Structure Reflect Major Evolutionary Events

Termites digest wood and other lignocellulosic substrates with the help of their intestinal microbiota. While the functions of the symbionts in the digestive process are slowly emerging, the origin of the bacteria colonizing the hindgut bioreactor is entirely unknown. Recently, our group discovered numerous representatives of bacterial lineages specific to termite guts in a closely related omnivorous cockroach, but it remains unclear whether they derive from the microbiota of a common ancestor or were independently selected by the gut environment. Here, we studied the bacterial gut microbiota in 34 species of termites and cockroaches using pyrotag analysis of the 16S rRNA genes. Although the community structures differed greatly between the major host groups, with dramatic changes in the relative abundances of particular bacterial taxa, we found that the majority of sequence reads belonged to bacterial lineages that were shared among most host species. When mapped onto the host tree, the changes in community structure coincided with major events in termite evolution, such as acquisition and loss of cellulolytic protists and the ensuing dietary diversification. UniFrac analysis of the core microbiota of termites and cockroaches and construction of phylogenetic tree of individual genus level lineages revealed a general host signal, whereas the branching order often did not match the detailed phylogeny of the host. It remains unclear whether the lineages in question have been associated with the ancestral cockroach since the early Cretaceous (cospeciation) or are diet-specific lineages that were independently acquired from the environment (host selection).     

Deterministic Assembly of Complex Bacterial Communities in Guts of Germ-Free Cockroaches

The gut microbiota of termites plays important roles in the symbiotic digestion of lignocellulose. However, the factors shaping the microbial community structure remain poorly understood. Because termites cannot be raised under axenic conditions, we established the closely related cockroach Shelfordella lateralis as a germ-free model to study microbial community assembly and host-microbe interactions. In this study, we determined the composition of the bacterial assemblages in cockroaches inoculated with the gut microbiota of termites and mice using pyrosequencing analysis of their 16S rRNA genes. Although the composition of the xenobiotic communities was influenced by the lineages present in the foreign inocula, their structure resembled that of conventional cockroaches. Bacterial taxa abundant in conventional cockroaches but rare in the foreign inocula, such as Dysgonomonas and Parabacteroides spp., were selectively enriched in the xenobiotic communities. Donor-specific taxa, such as endomicrobia or spirochete lineages restricted to the gut microbiota of termites, however, either were unable to colonize germ-free cockroaches or formed only small populations. The exposure of xenobiotic cockroaches to conventional adults restored their normal microbiota, which indicated that autochthonous lineages outcompete foreign ones. Our results provide experimental proof that the assembly of a complex gut microbiota in insects is deterministic.     

Pyrotag Sequencing of the Gut Microbiota of the Cockroach Shelfordella lateralis Reveals a Highly Dynamic Core but Only Limited Effects of Diet on Community Structure

Although blattid cockroaches and termites share a common ancestor, their diets are distinctly different. While termites consume a highly specialized diet of lignocellulose, cockroaches are omnivorous and opportunistic feeders. The role of the termite gut microbiota has been studied intensively, but little is known about the cockroach gut microbiota and its function in digestion and nutrition, particularly the adaptation to different diets. Our analyses of the bacterial gut microbiota of the blattid cockroach Shelfordella lateralis combining terminal restriction fragment length polymorphism of their 16S rRNA genes with physiological parameters (microbial metabolites, hydrogen and methane emission) indicated substantial variation between individuals but failed to identify any diet-related response. Subsequent deep-sequencing of the 16S rRNA genes of the colonic gut microbiota of S. lateralis fed either a high- or a low-fiber diet confirmed the absence of bacterial taxa that responded to diet. Instead, we found a small number of abundant phylotypes that were consistently present in all samples and made up half of the community in both diet groups. They varied strongly in abundance between individual samples at the genus but not at the family level. The remaining phylotypes were inconsistently present among replicate batches. Our findings suggest that S. lateralis harbors a highly dynamic core gut microbiota that is maintained even after fundamental dietary shifts, and that any dietary effects on the gut community are likely to be masked by strong individual variations.     

Anaerobic carbon monoxide dehydrogenase diversity in the homoacetogenic hindgut microbial communities of lower termites and the wood roach

Anaerobic carbon monoxide dehydrogenase (CODH) is a key enzyme in the Wood-Ljungdahl (acetyl-CoA) pathway for acetogenesis performed by homoacetogenic bacteria. Acetate generated by gut bacteria via the acetyl-CoA pathway provides considerable nutrition to wood-feeding dictyopteran insects making CODH important to the obligate mutualism occurring between termites and their hindgut microbiota. To investigate CODH diversity in insect gut communities, we developed the first degenerate primers designed to amplify cooS genes, which encode the catalytic (β) subunit of anaerobic CODH enzyme complexes. These primers target over 68 million combinations of potential forward and reverse cooS primer-binding sequences. We used the primers to identify cooS genes in bacterial isolates from the hindgut of a phylogenetically lower termite and to sample cooS diversity present in a variety of insect hindgut microbial communities including those of three phylogenetically-lower termites, Zootermopsis nevadensis, Reticulitermes hesperus, and Incisitermes minor, a wood-feeding cockroach, Cryptocercus punctulatus, and an omnivorous cockroach, Periplaneta americana. In total, we sequenced and analyzed 151 different cooS genes. These genes encode proteins that group within one of three highly divergent CODH phylogenetic clades. Each insect gut community contained CODH variants from all three of these clades. The patterns of CODH diversity in these communities likely reflect differences in enzyme or physiological function, and suggest that a diversity of microbial species participate in homoacetogenesis in these communities.     

Intra- and interspecific comparisons of bacterial diversity and community structure support coevolution of gut microbiota and termite host

We investigated the bacterial gut microbiota from 32 colonies of wood-feeding termites, comprising four Microcerotermes species (Termitidae) and four Reticulitermes species (Rhinotermitidae), using terminal restriction fragment length polymorphism analysis and clonal analysis of 16S rRNA. The obtained molecular community profiles were compared statistically between individuals, colonies, locations, and species of termites. Both analyses revealed that the bacterial community structure was remarkably similar within each termite genus, with small but significant differences between sampling sites and/or termite species. In contrast, considerable differences were found between the two termite genera. Only one bacterial phylotype (defined with 97% sequence identity) was shared between the two termite genera, while 18% and 50% of the phylotypes were shared between two congeneric species in the genera Microcerotermes and Reticulitermes, respectively. Nevertheless, a phylogenetic analysis of 228 phylotypes from Microcerotermes spp. and 367 phylotypes from Reticulitermes spp. with other termite gut clones available in public databases demonstrated the monophyly of many phylotypes from distantly related termites. The monophyletic "termite clusters" comprised of phylotypes from more than one termite species were distributed among 15 bacterial phyla, including the novel candidate phyla TG2 and TG3. These termite clusters accounted for 95% of the 960 clones analyzed in this study. Moreover, the clusters in 12 phyla comprised phylotypes from more than one termite (sub)family, accounting for 75% of the analyzed clones. Our results suggest that the majority of gut bacteria are not allochthonous but are specific symbionts that have coevolved with termites and that their community structure is basically consistent within a genus of termites.     

Genome shrinkage and loss of nutrient-providing potential in the obligate symbiont of the primitive termite Mastotermes darwiniensis

Beneficial microbial associations with insects are common and are classified as either one or a few intracellular species that are vertically transmitted and reside intracellularly within specialized organs or as microbial assemblages in the gut. Cockroaches and termites maintain at least one if not both beneficial associations. Blattabacterium is a flavobacterial endosymbiont of nearly all cockroaches and the termite Mastotermes darwiniensis and can use nitrogenous wastes in essential amino acid and vitamin biosynthesis. Key changes during the evolutionary divergence of termites from cockroaches are loss of Blattabacterium, diet shift to wood, acquisition of a specialized hindgut microbiota, and establishment of advanced social behavior. Termite gut microbes collaborate to fix nitrogen, degrade lignocellulose, and produce nutrients, and the absence of Blattabacterium in nearly all termites suggests that its nutrient-provisioning role has been replaced by gut microbes. M. darwiniensis is a basal, extant termite that solely retains Blattabacterium, which would show evidence of relaxed selection if it is being supplanted by the gut microbiome. This termite-associated Blattabacterium genome is ～8% smaller than cockroach-associated Blattabacterium genomes and lacks genes underlying vitamin and essential amino acid biosynthesis. Furthermore, the M. darwiniensis gut microbiome membership is more consistent between individuals and includes specialized termite gut-associated bacteria, unlike the more variable membership of cockroach gut microbiomes. The M. darwiniensis Blattabacterium genome may reflect relaxed selection for some of its encoded functions, and the loss of this endosymbiont in all remaining termite genera may result from its replacement by a functionally complementary gut microbiota.     

Intracolony variation of bacterial gut microbiota among castes and ages in the fungus-growing termite Macrotermes gilvus

The fungus-growing termites Macrotermes cultivate the obligate ectosymbiontic fungi, Termitomyces. While their relationship has been extesively studied, little is known about the gut bacterial symbionts, which also presumably play a crucial role for the nutrition of the termite host. In this study, we investigated the bacterial gut microbiota in two colonies of Macrotermes gilvus, and compared the diversity and community structure of bacteria among nine termite morphotypes, differing in caste and/or age, using terminal restriction fragment length polymorphism (T-RFLP) and clonal analysis of 16S rRNA. The obtained molecular community profiles clustered by termite morphotype rather than by colony, and the clustering pattern was clearly more related to a difference in age than to caste. Thus, we suggest that the bacterial gut microbiota change in relation to the food of the termite, which comprises fallen leaves and the fungus nodules of Termitomyces in young workers, and leaves degraded by the fungi, in old workers. Despite these intracolony variations in bacterial gut microbiota, their T-RFLP profiles formed a distinct cluster against those of the fungus garden, adjacent soil and guts of sympatric wood-feeding termites, implying a consistency and uniqueness of gut microbiota in M. gilvus. Since many bacterial phylotypes from M. gilvus formed monophyletic clusters with those from distantly related termite species, we suggest that gut bacteria have co-evolved with the termite host and form a microbiota specific to a termite taxonomic and/or feeding group, and furthermore, to caste and age within a termite species.     

Gut Bacterial Community of the Xylophagous Cockroaches Cryptocercus punctulatus and Parasphaeria boleiriana

Cryptocercus punctulatus and Parasphaeria boleiriana are two distantly related xylophagous and subsocial cockroaches. Cryptocercus is related to termites. Xylophagous cockroaches and termites are excellent model organisms for studying the symbiotic relationship between the insect and their microbiota. In this study, high-throughput 454 pyrosequencing of 16S rRNA was used to investigate the diversity of metagenomic gut communities of C. punctulatus and P. boleiriana, and thereby to identify possible shifts in symbiont allegiances during cockroaches evolution. Our results revealed that the hindgut prokaryotic communities of both xylophagous cockroaches are dominated by members of four Bacteria phyla: Bacteroidetes, Firmicutes, Proteobacteria, and Actinobacteria. Other identified phyla were Spirochaetes, Planctomycetes, candidatus Saccharibacteria (formerly TM7), and Acidobacteria, each of which represented 1–2% of the total population detected. Community similarity based on phylogenetic relatedness by unweighted UniFrac analyses indicated that the composition of the bacterial community in the two species was significantly different (P < 0.05). Phylogenetic analysis based on the characterized clusters of Bacteroidetes, Spirochaetes, and Deltaproteobacteria showed that many OTUs present in both cockroach species clustered with sequences previously described in termites and other cockroaches, but not with those from other animals or environments. These results suggest that, during their evolution, those cockroaches conserved several bacterial communities from the microbiota of a common ancestor. The ecological stability of those microbial communities may imply the important functional role for the survival of the host of providing nutrients in appropriate quantities and balance.     

Diet is the primary determinant of bacterial community structure in the guts of higher termites

The gut microbiota of termites plays critical roles in the symbiotic digestion of lignocellulose. While phylogenetically ‘lower termites’ are characterized by a unique association with cellulolytic flagellates, higher termites (family Termitidae) harbour exclusively prokaryotic communities in their dilated hindguts. Unlike the more primitive termite families, which primarily feed on wood, they have adapted to a variety of lignocellulosic food sources in different stages of humification, ranging from sound wood to soil organic matter. In this study, we comparatively analysed representatives of different taxonomic lineages and feeding groups of higher termites to identify the major drivers of bacterial community structure in the termite gut, using amplicon libraries of 16S rRNA genes from 18 species of higher termites. In all analyses, the wood‐feeding species were clearly separated from humus and soil feeders, irrespective of their taxonomic affiliation, offering compelling evidence that diet is the primary determinant of bacterial community structure. Within each diet group, however, gut communities of termites from the same subfamily were more similar than those of distantly related species. A highly resolved classification using a curated reference database revealed only few genus‐level taxa whose distribution patterns indicated specificity for certain host lineages, limiting any possible cospeciation between the gut microbiota and host to short evolutionary timescales. Rather, the observed patterns in the host‐specific distribution of the bacterial lineages in termite guts are best explained by diet‐related differences in the availability of microhabitats and functional niches.     

Intra- and Interspecific Comparisons of Bacterial Diversity and Community Structure Support Coevolution of Gut Microbiota and Termite Host

We investigated the bacterial gut microbiota from 32 colonies of wood-feeding termites, comprising four Microcerotermes species (Termitidae) and four Reticulitermes species (Rhinotermitidae), using terminal restriction fragment length polymorphism analysis and clonal analysis of 16S rRNA. The obtained molecular community profiles were compared statistically between individuals, colonies, locations, and species of termites. Both analyses revealed that the bacterial community structure was remarkably similar within each termite genus, with small but significant differences between sampling sites and/or termite species. In contrast, considerable differences were found between the two termite genera. Only one bacterial phylotype (defined with 97% sequence identity) was shared between the two termite genera, while 18% and 50% of the phylotypes were shared between two congeneric species in the genera Microcerotermes and Reticulitermes, respectively. Nevertheless, a phylogenetic analysis of 228 phylotypes from Microcerotermes spp. and 367 phylotypes from Reticulitermes spp. with other termite gut clones available in public databases demonstrated the monophyly of many phylotypes from distantly related termites. The monophyletic “termite clusters” comprised of phylotypes from more than one termite species were distributed among 15 bacterial phyla, including the novel candidate phyla TG2 and TG3. These termite clusters accounted for 95% of the 960 clones analyzed in this study. Moreover, the clusters in 12 phyla comprised phylotypes from more than one termite (sub)family, accounting for 75% of the analyzed clones. Our results suggest that the majority of gut bacteria are not allochthonous but are specific symbionts that have coevolved with termites and that their community structure is basically consistent within a genus of termites.     

The candidate phylum 'Termite Group 1' of bacteria: phylogenetic diversity, distribution, and endosymbiont members of various gut flagellated protists

The candidate phylum 'Termite Group 1' (TG1) of bacteria, which is abundant in termite guts but has no culturable representative, was investigated with respect to the in situ localization, distribution, and diversity. Based on the 16S rRNA gene sequence analyses and FISH in termite guts, a number of lineages of TG1 members were identified as endosymbionts of a variety of gut flagellated protists from the orders Trichonymphida, Cristamonadida, and Oxymonadida that are mostly unique to termites. However, the survey in various environments using specific PCR primers revealed that TG1 members were also present in termites, a cockroach, and the bovine rumen that typically lack these protist orders. Most of the TG1 members from gut flagellates, termites, cockroaches, and the rumen formed a monophyletic subcluster that showed a shallow branching pattern in the phylogenetic tree, suggesting their recent diversification. Although endosymbionts of the same protist genera tended to be closely related, the endosymbiont lineages were often independent of the higher level classifications of their host protist and were dispersed in the phylogenetic tree. It appears that their cospeciation is not the sole rule for the diversification of TG1 members of endosymbionts.     

Formyltetrahydrofolate synthetase gene diversity in the guts of higher termites with different diets and lifestyles

In this study, we examine gene diversity for formyl-tetrahydrofolate synthetase (FTHFS), a key enzyme in homoacetogenesis, recovered from the gut microbiota of six species of higher termites. The "higher" termites (family Termitidae), which represent the majority of extant termite species and genera, engage in a broader diversity of feeding and nesting styles than the "lower" termites. Previous studies of termite gut homoacetogenesis have focused on wood-feeding lower termites, from which the preponderance of FTHFS sequences recovered were related to those from acetogenic treponemes. While sequences belonging to this group were present in the guts of all six higher termites examined, treponeme-like FTHFS sequences represented the majority of recovered sequences in only two species (a wood-feeding Nasutitermes sp. and a palm-feeding Microcerotermes sp.). The remaining four termite species analyzed (a Gnathamitermes sp. and two Amitermes spp. that were recovered from subterranean nests with indeterminate feeding strategies and a litter-feeding Rhynchotermes sp.) yielded novel FTHFS clades not observed in lower termites. These termites yielded two distinct clusters of probable purinolytic Firmicutes and a large group of potential homoacetogens related to sequences previously recovered from the guts of omnivorous cockroaches. These findings suggest that the gut environments of different higher termite species may select for different groups of homoacetogens, with some species hosting treponeme-dominated homoacetogen populations similar to those of wood-feeding, lower termites while others host Firmicutes-dominated communities more similar to those of omnivorous cockroaches.     

Origin of termite eusociality: trophallaxis integrates the social,nutritional, and microbial environments

1. Numerous cladistic analyses have converged: termites are a monophyletic clade embedded within the paraphyletic cockroaches, and sister group to the biparental, wood‐feeding cockroach Cryptocercus. The latter is, therefore, an appropriate model for testing assumptions regarding early termite evolution. 2. The ground plan of the termite ancestor is reviewed based on shared characters of ecology, life history, and behaviour in Cryptocercus and incipient termite colonies, and includes two levels of dependence: a reliance of all individuals on gut microbiota, and dependence of early instars on parental care. Both these conditions co‐evolved with parent‐to‐offspring proctodeal trophallaxis. 3. The termite ancestor lived in a single log serving as food and nest. This ‘one‐piece’ nesting ecology prioritises nitrogen conservation and strongly influences interacting social, nutritional, and microbial environments. Each of these environments individually and in combination profoundly affect cockroach development. 4. Proctodeal trophallaxis integrates the social, nutritional, and microbial environments. A change in trophallactic behaviour, from parental to alloparental, can, therefore, shift developmental trajectories, ultimately adding a third level of dependence. The death of gut protists during the host molting period and consequent interdependence of family members shifted the hierarchical level at which selection acted; fixation of eusociality quickly followed. 5. The basic nesting ecology did not change when termites evolved eusociality, the change occurred in the allocation and use of existing resources within the social group, driven by nitrogen scarcity, mediated by trophallaxis, and made possible by a strongly lineage‐specific set of life history characteristics.     

Niche heterogeneity determines bacterial community structure in the termite gut (Reticulitermes santonensis)

Differences in microenvironment and interactions of microorganisms within and across habitat boundaries should influence structure and diversity of the microbial communities within an ecosystem. We tested this hypothesis using the well characterized gut tract of the European subterranean termite Reticulitermes santonensis as a model. By cloning and sequencing analysis and molecular fingerprinting (terminal restriction fragment length polymorphism), we characterized the bacterial microbiota in the major intestinal habitats - the midgut, the wall of the hindgut paunch, the hindgut fluid and the intestinal protozoa. The bacterial community was very diverse (> 200 ribotypes) and comprised representatives of several phyla, including Firmicutes (mainly clostridia, streptococci and Mycoplasmatales-related clones), Bacteroidetes, Spirochaetes and a number of Proteobacteria, all of which were unevenly distributed among the four habitats. The largest group of clones fell into the so-called Termite group 1 (TG-1) phylum, which has no cultivated representatives. The majority of the TG-1 clones were associated with the protozoa and formed two phylogenetically distinct clusters, which consisted exclusively of clones previously retrieved from the gut of this and other Reticulitermes species. Also the other clones represented lineages of microorganisms that were exclusively recovered from the intestinal tract of termites. The termite specificity of these lineages was underscored by the finding that the closest relatives of the bacterial clones obtained from R. santonensis were usually derived also from the most closely related termites. Overall, differences in diversity between the different gut habitats and the uneven distribution of individual phylotypes support conclusively that niche heterogeneity is a strong determinant of the structure and spatial organization of the microbial community in the termite gut.     

Oxygen Affects Gut Bacterial Colonization and Metabolic Activities in a Gnotobiotic Cockroach Model

The gut microbiota of termites and cockroaches represents complex metabolic networks of many diverse microbial populations. The distinct microenvironmental conditions within the gut and possible interactions among the microorganisms make it essential to investigate how far the metabolic properties of pure cultures reflect their activities in their natural environment. We established the cockroach Shelfordella lateralis as a gnotobiotic model and inoculated germfree nymphs with two bacterial strains isolated from the guts of conventional cockroaches. Fluorescence microscopy revealed that both strains specifically colonized the germfree hindgut. In diassociated cockroaches, the facultatively anaerobic strain EbSL (a new species of Enterobacteriaceae) always outnumbered the obligately anaerobic strain FuSL (a close relative of Fusobacterium varium), irrespective of the sequence of inoculation, which showed that precolonization by facultatively anaerobic bacteria does not necessarily favor colonization by obligate anaerobes. Comparison of the fermentation products of the cultures formed in vitro with those accumulated in situ indicated that the gut environment strongly affected the metabolic activities of both strains. The pure cultures formed the typical products of mixed-acid or butyrate fermentation, whereas the guts of gnotobiotic cockroaches accumulated mostly lactate and acetate. Similar shifts toward more-oxidized products were observed when the pure cultures were exposed to oxygen, which corroborated the strong effects of oxygen on the metabolic fluxes previously observed in termite guts. Oxygen microsensor profiles of the guts of germfree, gnotobiotic, and conventional cockroaches indicated that both gut tissue and microbiota contribute to oxygen consumption and suggest that the oxygen status influences the colonization success.     

Nest specificity of the bacterial community in termite guts (Hodotermes mossambicus)

While recent results have provided strong evidence for the presence of a stable gut microbiota among several termite species, little is known about variations at the colony or individual level. Using a cultivation-independent approach, we investigated the structure of the bacterial community in the gut of termites from four different colonies of Hodotermes mossambicus. 16S rRNA-based terminal restriction fragment length polymorphism (T-RFLP) analysis of the bacterial gut microbiota revealed (1) a high consistency of the gut microbiota among nestmates and (2) subtle but distinct differences in community structure between individuals from different colonies. Since products of bacterial metabolism may contribute to a colony odor that can be used as discriminatory signal, the presence of a colony-specific bacterial community adds support to the hypothesis that the gut microbiota of termites is involved in nestmate recognition. Received 12 July 2005; revised 10 February and 15 March 2006; accepted 7 April 2006.     

Diversity and resilience of the wood‐feeding higher termite Mironasutitermes shangchengensis gut microbiota in response to temporal and diet variations

Termites are considered among the most efficient bioreactors, with high capacities for lignocellulose degradation and utilization. Recently, several studies have characterized the gut microbiota of diverse termites. However, the temporal dynamics of the gut microbiota within a given termite with dietary diversity are poorly understood. Here, we employed 16S rDNA barcoded pyrosequencing analysis to investigate temporal changes in bacterial diversity and richness of the gut microbiota of wood‐feeding higher termite Mironasutitermes shangchengensis under three lignocellulose content‐based diets that feature wood, corn stalks, and filter paper. Compositions of the predominant termite gut residents were largely constant among the gut microbiomes under different diets, but each diet caused specific changes in the bacterial composition over time. Notably, microbial communities exhibited an unexpectedly strong resilience during continuous feeding on both corn stalks and filter paper. Members of five bacterial phyla, that is, Spirochaetes, Firmicutes, Actinobacteria, Tenericutes, and Acidobacteria, were strongly associated with the resilience. These findings provide insights into the stability of the gut microbiota in higher termites and have important implications for the future design of robust bioreactors for lignocellulose degradation and utilization.     

Phylogenetic and morphological diversity of Bacteroidales members associated with the gut wall of termites

The microbial community adherent directly or indirectly to the gut wall of termites is distinct from that of the other habitats in the gut. The bacterial 16S rRNA genes were identified from the fractionated gut walls of two termite species, Hodotermopsis sjoestedti and Neotermes koshunensis, and compared with those previously identified from Reticulitermes speratus. Surprisingly, the bacterial constituents were almost entirely different among the termites at the phylotype level (the criterion of the phylotype was >97% nucleotide identity). Bacteria in the order Bacteroidales, which were commonly abundant symbionts on gut walls, were phylogenetically analyzed. They were dispersed in a number of clusters formed by phylotypes from the guts of various termites. In situ hybridization with probes specific for some phylotypes and a phylogenetic cluster detected the cells of several Bacteroidales members with a significant variety of cell morphology in the gut wall fractions, which reflects the phylogenetic diversity of this order.     

Molecular phylogenetic profiling of prokaryotic communities in guts of termites with different feeding habits

Termites are an important group of terrestrial insects that harbor an abundant gut microbiota, many of which contribute to digestion, termite nutrition and gas (CH(4), CO(2) and H(2)) emission. With 2200 described species, termites also provide a good model to study relationships between host diet and gut microbial community structure and function. We examined the relationship between diet and gut prokaryotic community profiles in 24 taxonomically and nutritionally diverse species of termites by using nucleic acid probes targeting 16S-like ribosomal RNAs. The relative abundance of domain-specific 16S-like rRNAs recovered from gut extracts varied considerably (ranges: Archaea (0-3%); Bacteria (15-118%)). Although Bacteria were always detectable and the most abundant, differences in domain-level profiles were correlated with termite diet, as evidenced by higher relative abundances of Archaea in guts of soil-feeding termites, compared to those of wood-feeding species in the same family. The oligonucleotide probes also readily distinguished gut communities of wood-feeding taxa in the family Termitidae (higher termites) from those of other wood-feeding termite families (lower termites). The relative abundances of 16S-like archaeal rRNA in guts were positively correlated with rates of methane emission by live termites, and were consistent with previous work linking high relative rates of methanogenesis with the soil (humus)-feeding habit. Probes for methanogenic Archaea detected members of only two families (Methanobacteriaceae and Methanosarcinaceae) in termite guts, and these typically accounted for 60% of the all archaeal probe signal. In four species of termites, Methanosarcinaceae were dominant, a novel observation for animal gut microbial communities, but no clear relationship was apparent between methanogen family profiles and termite diet or taxonomy.