It has been suggested that food storage inside the nest may offer termites with a nutritional provision during low resource availability. Additionally, feces employed as construction material provide an excellent environment for colonization by microorganisms and, together with the storage of plant material inside the nest, could thus provide some advantage to the termites in terms of lignocellulose decomposition. Here, we conducted for the first time a comprehensive study of the microbial communities associated to a termite exhibiting food storage behavior using Illumina sequencing of the 16S and (ITS2) regions of rRNA genes, together with enzymatic assays and data collected in the field. Cornitermes cumulans (Syntermitinae) stored grass litter in nodules made from feces and saliva located in the nest core. The amount of nodules increased with nest size and isolation, and interestingly, the soluble fraction of extracts from nodules showed a higher activity against hemicellulosic substrates compared to termite guts. Actinobacteria and Sordariales dominated microbial communities of food nodules and nest walls, whereas Spirochetes and Pleosporales dominated gut samples of C. cumulans. Within Syntermitinae, however, gut bacterial assemblages were dissimilar. On the other hand, there is a remarkable convergence of the bacterial community structure of Termitidae nests. Our results suggest that the role of nodules could be related to food storage; however, the higher xylanolytic activity in the nodules and their associated microbiota could also provide C. cumulans with an external source of predigested polysaccharides, which might be advantageous in comparison with litter-feeding termites that do not display food storage behavior.

The evolution of the symbiotic association with microbes allowed termites to decompose ingested lignocellulose from plant-derived substrates, including herbivore dung and soil humus. Representatives of the Syntermitinae (Termitidae) range in their feeding habits from wood and litter-feeding to humus-feeding species. However, only limited information is available about their feeding ecology and associated microbial communities. Here we conducted a study of the microbial communities associated to the termite Procornitermes araujoi using Illumina sequencing of the 16S and ITS rRNA genes. This species has been previously included in different feeding guilds. However, most aspects of its feeding ecology are unknown, especially those associated to its symbiotic microbiota. Our results showed that the microbial communities of termite guts and nest substrates of P. araujoi differed significantly for bacteria and fungi. Firmicutes dominated the bacterial gut community of both workers and soldiers, whereas Actinobacteria was found in higher prevalence in the nest walls. Sordariomycetes was the most abundant fungal class in both gut and nest samples and distinguish P. araujoi from the grass/litter feeding Cornitermes cumulans. Our results also showed that diversity of gut bacteria were higher in P. araujoi and Silvestritermes euamignathus than in the grass/litter feeders (C. cumulans and Syntermes dirus), that could indicate an adaptation of the microbial community of polyphagous termites to the higher complexity of their diets.

Lignocellulose digestion in termites is achieved through the functional synergy between gut symbionts and host enzymes. However, some species have evolved additional associations with nest microorganisms that collaborate in the decomposition of plant biomass. In a previous study, we determined that plant material packed with feces inside the nests of Cornitermes cumulans (Syntermitinae) harbors a distinct microbial assemblage. These food nodules also showed a high hemicellulolytic activity, possibly acting as an external place for complementary lignocellulose digestion. In this study, we used a combination of ITS sequence analysis, metagenomics, and metatranscriptomics to investigate the presence and differential expression of genes coding for carbohydrate-active enzymes (CAZy) in the food nodules and the gut of workers and soldiers. Our results confirm that food nodules express a distinct set of CAZy genes suggesting that stored plant material is initially decomposed by enzymes that target the lignin and complex polysaccharides from fungi and bacteria before the passage through the gut, where it is further targeted by a complementary set of cellulases, xylanases, and esterases produced by the gut microbiota and the termite host. We also showed that the expression of CAZy transcripts associated to endoglucanases and xylanases was higher in the gut of termites than in the food nodules. An additional finding in this study was the presence of fungi in the termite gut that expressed CAZy genes. This study highlights the importance of externalization of digestion by nest microbes and provides new evidence of complementary digestion in the context of higher termite evolution.