Chewing through challenges: Exploring the evolutionary pathways to wood-feeding in insects.

Decaying wood, while an abundant and stable resource, presents considerable nutritional challenges due to its structural rigidity, chemical recalcitrance, and low nitrogen content. Despite these challenges, certain insect lineages have successfully evolved saproxylophagy (consuming and deriving sustenance from decaying wood), impacting nutrient recycling in ecosystems and carbon sequestration dynamics. This study explores the uneven phylogenetic distribution of saproxylophagy across insects and delves into the evolutionary origins of this trait in disparate insect orders. Employing a comprehensive analysis of gut microbiome data, from both saproxylophagous insects and their non-saproxylophagous relatives, including new data from unexplored wood-feeding insects, this Hypothesis paper discusses the broader phylogenetic context and potential adaptations necessary for this dietary specialization. The study proposes the "Detritivore-First Hypothesis," suggesting an evolutionary pathway to saproxylophagy through detritivory, and highlights the critical role of symbiotic gut microbiomes in the digestion of decaying wood.

Fiber-associated spirochetes are major agents of hemicellulose degradation in the hindgut of wood-feeding higher termites.

Symbiotic digestion of lignocellulose in wood-feeding higher termites (family Termitidae) is a two-step process that involves endogenous host cellulases secreted in the midgut and a dense bacterial community in the hindgut compartment. The genomes of the bacterial gut microbiota encode diverse cellulolytic and hemicellulolytic enzymes, but the contributions of host and bacterial symbionts to lignocellulose degradation remain ambiguous. Our previous studies of Nasutitermes spp. documented that the wood fibers in the hindgut paunch are consistently colonized not only by uncultured members of Fibrobacteres, which have been implicated in cellulose degradation, but also by unique lineages of Spirochaetes. Here, we demonstrate that the degradation of xylan, the major component of hemicellulose, is restricted to the hindgut compartment, where it is preferentially hydrolyzed over cellulose. Metatranscriptomic analysis documented that the majority of glycoside hydrolase (GH) transcripts expressed by the fiber-associated bacterial community belong to family GH11, which consists exclusively of xylanases. The substrate specificity was further confirmed by heterologous expression of the gene encoding the predominant homolog. Although the most abundant transcripts of GH11 in Nasutitermes takasagoensis were phylogenetically placed among their homologs of Firmicutes, immunofluorescence microscopy, compositional binning of metagenomics contigs, and the genomic context of the homologs indicated that they are encoded by Spirochaetes and were most likely obtained by horizontal gene transfer among the intestinal microbiota. The major role of spirochetes in xylan degradation is unprecedented and assigns the fiber-associated Treponema clades in the hindgut of wood-feeding higher termites a prominent part in the breakdown of hemicelluloses.

Diet is not the primary driver of bacterial community structure in the gut of litter-feeding cockroaches.

BACKGROUND: Diet is a major determinant of bacterial community structure in termite guts, but evidence of its importance in the closely related cockroaches is conflicting. Here, we investigated the ecological drivers of the bacterial gut microbiota in cockroaches that feed on lignocellulosic leaf litter. RESULTS: The physicochemical conditions determined with microsensors in the guts of Ergaula capucina, Pycnoscelus surinamensis, and Byrsotria rothi were similar to those reported for both wood-feeding and omnivorous cockroaches. All gut compartments were anoxic at the center and showed a slightly acidic to neutral pH and variable but slightly reducing conditions. Hydrogen accumulated only in the crop of B. rothi. High-throughput amplicon sequencing of bacterial 16S rRNA genes documented that community structure in individual gut compartments correlated strongly with the respective microenvironmental conditions. A comparison of the hindgut microbiota of cockroaches and termites from different feeding groups revealed that the vast majority of the core taxa in cockroaches with a lignocellulosic diet were present also in omnivorous cockroaches but absent in wood-feeding higher termites. CONCLUSION: Our results indicate that diet is not the primary driver of bacterial community structure in the gut of wood- and litter-feeding cockroaches. The high similarity to the gut microbiota of omnivorous cockroaches suggests that the dietary components that are actually digested do not differ fundamentally between feeding groups.

'Candidatus Adiutrix intracellularis', an endosymbiont of termite gut flagellates, is the first representative of a deep-branching clade of Deltaproteobacteria and a putative homoacetogen.

Termite gut flagellates are typically colonized by specific bacterial symbionts. Here we describe the phylogeny, ultrastructure and subcellular location of 'Candidatus Adiutrix intracellularis', an intracellular symbiont of Trichonympha collaris in the termite Zootermopsis nevadensis. It represents a novel, deep-branching clade of uncultured Deltaproteobacteria widely distributed in intestinal tracts of termites and cockroaches. Fluorescence in situ hybridization and transmission electron microscopy localized the endosymbiont near hydrogenosomes in the posterior part and near the ectosymbiont 'Candidatus Desulfovibrio trichonymphae' in the anterior part of the host cell. The draft genome of 'Ca. Adiutrix intracellularis' obtained from a metagenomic library revealed the presence of a complete gene set encoding the Wood-Ljungdahl pathway, including two homologs of fdhF encoding hydrogenase-linked formate dehydrogenases (FDHH ) and all other components of the recently described hydrogen-dependent carbon dioxide reductase (HDCR) complex, which substantiates previous claims that the symbiont is capable of reductive acetogenesis from CO2 and H2 . The close phylogenetic relationship between the HDCR components and their homologs in homoacetogenic Firmicutes and Spirochaetes suggests that the deltaproteobacterium acquired the capacity for homoacetogenesis via lateral gene transfer. The presence of genes for nitrogen fixation and the biosynthesis of amino acids and cofactors indicate the nutritional nature of the symbiosis.

Age polyethism drives community structure of the bacterial gut microbiota in the fungus-cultivating termite Odontotermes formosanus.

Fungus-cultivating termites (Macrotermitinae) possess an elaborate strategy of lignocellulose digestion. It involves a lignocellulose-degrading fungal symbiont (genus Termitomyces), a diverse gut microbiota and a characteristic labour division in food processing. In this study, using pyrotag sequencing and electron microscopy, we analysed the bacterial microbiota in the hindgut of Odontotermes formosanus and its fungus comb to investigate the spatial organization, establishment and temporal succession of the bacterial communities colonizing specific microhabitats. Our results document strong differences between the communities at the hindgut epithelium and the luminal fluid of newly moulted, young and old worker termites. The differences in community structure were consistent with the density, morphology and spatial distribution of bacterial cells and the pools of microbial metabolites in the hindgut compartment, underlining that both gut development and the age-specific changes in diet affect the composition and functional role of their gut microbiota. These findings provide strong support for the concept that changes in diet and gut environment are important determinants of community structure because they create new niches for microbial symbionts.

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.

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.

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.

Lethal disruption of the bacterial gut community in Eastern subterranean termite caused by boric acid.

The Eastern subterranean termite, Reticulitermes flavipes (Kollar) (Blattodea: Rhinotermitidae), is a significant pest, causing extensive damage to structures that amount to substantial economic losses. Boric acid is widely used for wood preservation due to its stability and broad-spectrum insecticidal properties, yet its impact on termite gut microbiomes and the implications of such effects remain understudied. Our study evaluates the dose-dependent mortality of R. flavipes upon being provided boric acid treated filter papers and investigates the resulting dysbiosis within the termite gut microbiome. Consistent with reports from other insects, mortality increased in a dose-dependent manner, with the highest boric acid concentration (203.7 µg/cm2 of filter paper) significantly reducing termite survival. 16S rRNA gene sequencing of the gut bacterial microbiome revealed notable shifts in composition, indicating boric acid-induced dysbiosis. Aside from an overall decrease in bacterial diversity, the relative abundance of some symbionts essential for termite nutrition decreased in response to higher boric acid concentrations, while several opportunistic pathogens increased. Our findings extend the understanding of boric acid's mode of action in termites, emphasizing its ability to significantly modulate the bacterial symbiont community, which can have dire effects on termite biology. Considering its ability to protect wood from further termite consumption, our study supports the continued use of boric acid and related compounds for termite-resistant treatments for wood.

Fight, retreat, repeat: The male-male agonistic behavior in the wood-feeding cockroach, Panesthia angustipennis spadica (Dictyoptera: Blattodea: Blaberidae).

Competition is one of the most critical factors affecting animal behaviors. Aggressive interactions are central to acquiring resources or mating partners. Agonistic behavior is more common among males than females. Although laboratory observations of these behaviors give detailed descriptions under controlled conditions, field observations without human intervention are required because those supply information that provides insights into their function. In this paper, we report on the field observation and auxiliary laboratory experiments of male-male agonistic behavior of a wood-feeding cockroach, Panesthia angustipennis, and discuss its strategy. In the field, a male pushed the opponent with the horn on the pronotum out of a gap between two logs, under which a female was. After pushing, the male repeatedly returned to a place close to the female, even if it did not subdue the opponent entirely. It suggests that the male-male agonistic behavior in P. angustipennis has both attack and avoidance. The bout was repeated as the ejected male reapproached the male. In contrast, the inferior male often escaped in the laboratory recording after field observation. Keeping the fighting experience for several days may contribute to the males avoiding a "losing battle." This study significantly enhances our understanding of the mating strategy of P. angustipennis through male-male agonistic behavior and provides possibilities for its cognitive aspects from the fighting experience.

The Genome of Arsenophonus sp. and Its Potential Contribution in the Corn Planthopper, Peregrinus maidis.

The co-evolution between symbionts and their insect hosts has led to intricate functional interdependencies. Advances in DNA-sequencing technologies have not only reduced the cost of sequencing but, with the advent of highly accurate long-read methods, have also enabled facile genome assembly even using mixed genomic input, thereby allowing us to more easily assess the contribution of symbionts to their insect hosts. In this study, genomic data recently generated from Peregrinus maidis was used to assemble the genome of a bacterial symbiont, Pm Arsenophonus sp. This ~4.9-Mb assembly is one of the largest Arsenophonus genomes reported to date. The Benchmarking Universal Single-Copy Orthologs (BUSCO) result indicates that this Pm Arsenophonus assembly has a high degree of completeness, with 96% of the single-copy Enterobacterales orthologs found. The identity of the Pm Arsenophonus sp. was further confirmed by phylogenetic analysis. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis indicates a major contribution by Pm Arsenophonus sp. to the biosynthesis of B vitamins and essential amino acids in P. maidis, where threonine and lysine production is carried out solely by Pm Arsenophonus sp. This study not only provides deeper insights into the evolutionary relationships between symbionts and their insect hosts, but also adds to our understanding of insect biology, potentially guiding the development of novel pest control methods.

Wood fibers are a crucial microhabitat for cellulose- and xylan- degrading bacteria in the hindgut of the wood-feeding beetle Odontotaenius disjunctus.

Introduction: Wood digestion in insects relies on the maintenance of a mosaic of numerous microhabitats, each colonized by distinct microbiomes. Understanding the division of digestive labor between these microhabitats- is central to understanding the physiology and evolution of symbiotic wood digestion. A microhabitat that has emerged to be of direct relevance to the process of lignocellulose digestion is the surface of ingested plant material. Wood particles in the guts of some termites are colonized by a specialized bacterial fiber-digesting microbiome, but whether this represents a widespread strategy among insect lineages that have independently evolved wood-feeding remains an open question. Methods: In this study, we investigated the bacterial communities specifically associated with wood fibers in the gut of the passalid beetle Odontotaenius disjunctus. We developed a Percoll-based centrifugation method to isolate and enrich the wood particles from the anterior hindgut, allowing us to access the wood fibers and their associated microbiome. We then performed assays of enzyme activity and used short-read and long-read amplicon sequencing of the 16S rRNA gene to identify the composition of the fiber-associated microbiome. Results: Our assays demonstrated that the anterior hindgut, which houses a majority of the bacterial load, is an important site for lignocellulose digestion. Wood particles enriched from the anterior hindgut contribute to a large proportion of the total enzyme activity. The sequencing revealed that O. disjunctus, like termites, harbors a distinct fiber-associated microbiome, but notably, its community is enriched in insect-specific groups of Lactococcus and Turicibacter. Discussion: Our study underscores the importance of microhabitats in fostering the complex symbiotic relationships between wood-feeding insects and their microbiomes. The discovery of distinct fiber-digesting symbionts in O. disjunctus, compared to termites, highlights the diverse evolutionary paths insects have taken to adapt to a challenging diet.

Reevaluating Symbiotic Digestion in Cockroaches: Unveiling the Hindgut's Contribution to Digestion in Wood-Feeding Panesthiinae (Blaberidae).

Cockroaches of the subfamily Panesthiinae (family Blaberidae) are among the few major groups of insects feeding on decayed wood. Despite having independently evolved the ability to thrive on this recalcitrant and nitrogen-limited resource, they are among the least studied of all wood-feeding insect groups. In the pursuit of unraveling their unique digestive strategies, we explored cellulase and xylanase activity in the crop, midgut, and hindgut lumens of Panesthia angustipennis and Salganea taiwanensis. Employing Percoll density gradient centrifugation, we further fractionated luminal fluid to elucidate how the activities in the gut lumen are further partitioned. Our findings challenge conventional wisdom, underscoring the significant contribution of the hindgut, which accounts for approximately one-fifth of cellulase and xylanase activity. Particle-associated enzymes, potentially of bacterial origin, dominate hindgut digestion, akin to symbiotic strategies observed in select termites and passalid beetles. Our study sheds new light on the digestive prowess of panesthiine cockroaches, providing invaluable insights into the evolution of wood-feeding insects and their remarkable adaptability to challenging, nutrient-poor substrates.

The use of Beauveria bassiana for the control of the larger grain borer, Prostephanus truncatus, in stored maize: Semi-field trials in Ghana.

Laboratory research in Ghana demonstrated the effectiveness of an isolate of Beauveria bassiana (IMI 389521) from the United Kingdom against the larger grain borer Prostephanus truncatus (Horn) (Coleoptera: Bostrichidae), a major pest of stored maize. The minimum effective concentration, following artificial infestation trials on maize, was between 109 and 1010 cfu/kg maize. Before moving out to village-level control, a major requirement was to determine if the product could effect control in artificially infested maize held under real environmental conditions in several locations in Ghana. Therefore, this study investigated the efficacy of formulated conidia of B. bassiana, IMI 389521, at two concentrations (1 × 109 and 3.16 × 109 cfu/kg maize) to control P. truncatus on stored maize kernels under semi-field conditions in Ghana. Maize ('Obatanpa' cultivar) kernels were treated with the formulated B. bassiana product and stored in polypropylene woven bags in cribs in Southern Ghana. After 24 h, one hundred adults of P. truncatus were placed into each bag containing the treated maize. Mortality and the percent of weight loss of kernels were assessed every two weeks for three months. The semi-field trials revealed the possibility of successfully controlling adult P. truncatus on maize kernels treated with B. bassiana at 3.16 × 109 cfu/kg maize. However, due to the minimal protection of kernels after four weeks, re-treating maize kernels after this period is recommended to ensure maximum protection during prolonged storage.

'Candidatus Arthromitus' revised: segmented filamentous bacteria in arthropod guts are members of Lachnospiraceae.

The name Arthromitus has been applied collectively to conspicuous filamentous bacteria found in the hindguts of termites and other arthropods. First observed by Joseph Leidy in 1849, the identity of these filaments has remained contentious. While Margulis and colleagues declared them to be a life stage of Bacillus cereus, others have assumed them to belong to the same lineage as the segmented filamentous bacteria (SFB) from vertebrate guts, a group that has garnered much attention due to their unique ability to specifically modulate their host's immune response. Both SFB and Arthromitus filaments from arthropod guts were grouped under provisional name 'Candidatus Arthromitus' by Snel and colleagues as they share a striking similarity in terms of their morphology and close contact to the host gut wall. While SFB form a distinct lineage within the family Clostridiaceae, the identity of the filaments from arthropod guts remains elusive. Using whole-genome amplification of single filaments capillary picked from termite guts and fluorescence in situ hybridization of 16S rRNA with group-specific oligonucleotide probes, we show that they represent a monophyletic lineage within the family Lachnospiraceae distinct from that of SFB. Therefore, 'Candidatus Arthromitus' can no longer be used for both groups. Given the historic precedence, we propose to reserve this name for the filaments that were originally described by Leidy. For the SFB from vertebrate guts, we propose the provisional name 'Candidatus Savagella' in honour of the American gut microbiologist Dwayne C. Savage, who was the first to describe that important bacterial group.

Comparative Efficacy of a Fungal Entomopathogen with a Broad Host Range against Two Human-Associated Pests.

The ability of a fungal entomopathogen to infect an insect depends on a variety of factors, including strain, host, and environmental conditions. Similarly, an insect's ability to prevent fungal infection is dependent on its biology, environment, and evolutionary history. Synanthropic pests have adapted to thrive in the indoor environment, yet they arose from divergent evolutionary lineages and occupy different feeding guilds. The hematophagous bed bug (Cimex lectularius) and omnivorous German cockroach (Blattella germanica) are highly successful indoors, but have evolved different physiological and behavioral adaptations to cope with the human-built environment, some of which also reduce the efficacy of fungal biopesticides. In order to gain greater insight into the host barriers that prevent or constrain fungal infection in bed bugs and German cockroaches, we tested different doses of Beauveria bassiana GHA through surface contact, topical application, feeding, and injection. Bed bugs were generally more susceptible to infection by B. bassiana with the mode of delivery having a significant impact on infectivity. The German cockroach was highly resilient to infection, requiring high doses of fungal conidia (>8.8 × 104) delivered by injection into the hemocoel to cause mortality. Mortality occurred much faster in both insect species after exposure to surfaces dusted with dry conidia than surfaces treated with conidia suspended in water or oil. These findings highlight the importance of developing innovative delivery techniques to enhance fungal entomopathogens against bed bugs and cockroaches.

Beating Them with Their Own Stick-Tick Uses Amidase of Bacterial Origin as Part of Its Immune Arsenal.

As vectors of microbial diseases in vertebrates, ticks are excellent at regulating bacterial proliferation in and around them. In a recent issue of Cell, Hayes et al. (2020) reveal acarid toxins of bacterial origin that help eliminate microbes that are pathogenic to black-legged ticks but commensal to their vertebrate hosts.

Genomes of Gut Bacteria from Nasonia Wasps Shed Light on Phylosymbiosis and Microbe-Assisted Hybrid Breakdown.

Phylosymbiosis is a cross-system trend whereby microbial community relationships recapitulate the host phylogeny. In Nasonia parasitoid wasps, phylosymbiosis occurs throughout development, is distinguishable between sexes, and benefits host development and survival. Moreover, the microbiome shifts in hybrids as a rare Proteus bacterium in the microbiome becomes dominant. The larval hybrids then catastrophically succumb to bacterium-assisted lethality and reproductive isolation between the species. Two important questions for understanding phylosymbiosis and bacterium-assisted lethality in hybrids are (i) do the Nasonia bacterial genomes differ from other animal isolates and (ii) are the hybrid bacterial genomes the same as those in the parental species? Here, we report the cultivation, whole-genome sequencing, and comparative analyses of the most abundant gut bacteria in Nasonia larvae, Providencia rettgeri and Proteus mirabilis Characterization of new isolates shows Proteus mirabilis forms a more robust biofilm than Providencia rettgeri and that, when grown in coculture, Proteus mirabilis significantly outcompetes Providencia rettgeri Providencia rettgeri genomes from Nasonia are similar to each other and more divergent from pathogenic, human associates. Proteus mirabilis from Nasonia vitripennis, Nasonia giraulti, and their hybrid offspring are nearly identical and relatively distinct from human isolates. These results indicate that members of the larval gut microbiome within Nasonia are most similar to each other, and the strain of the dominant Proteus mirabilis in hybrids is resident in parental species. Holobiont interactions between shared, resident members of the wasp microbiome and the host underpin phylosymbiosis and hybrid breakdown.IMPORTANCE Animal and plant hosts often establish intimate relationships with their microbiomes. In varied environments, closely related host species share more similar microbiomes, a pattern termed phylosymbiosis. When phylosymbiosis is functionally significant and beneficial, microbial transplants between host species and host hybridization can have detrimental consequences on host biology. In the Nasonia parasitoid wasp genus, which contains a phylosymbiotic gut community, both effects occur and provide evidence for selective pressures on the holobiont. Here, we show that bacterial genomes in Nasonia differ from other environments and harbor genes with unique functions that may regulate phylosymbiotic relationships. Furthermore, the bacteria in hybrids are identical to those in parental species, thus supporting a hologenomic tenet that the same members of the microbiome and the host genome impact phylosymbiosis, hybrid breakdown, and speciation.

Gut microbial compositions mirror caste-specific diets in a major lineage of social insects.

Social insects owe their ecological success to the division of labour between castes, but associations between microbial community compositions and castes with different tasks and diets have not been extensively explored. Fungus-growing termites associate with fungi to degrade plant material, complemented by diverse gut microbial communities. Here, we explore whether division of labour and accompanying dietary differences between fungus-growing termite castes are linked to gut bacterial community structure. Using amplicon sequencing, we characterize community compositions in sterile (worker and soldier) and reproductive (queen and king) termites and combine this with gut enzyme activities and microscopy to hypothesise sterile caste-specific microbiota roles. Gut bacterial communities are structured primarily according to termite caste and genus and, in contrast to the observed rich and diverse sterile caste microbiotas, royal pair guts are dominated by few bacterial taxa, potentially reflecting their specialized uniform diet and unique lifestyle.