Abstract
BACKGROUND: Many arthropods rely on their gut microbiome to digest plant material, which is often low in nitrogen but high in complex polysaccharides. Detritivores, such as millipedes, live on a particularly poor diet, but the identity and nutritional contribution of their microbiome are largely unknown. In this study, the hindgut microbiota of the tropical millipede Epibolus pulchripes (large, methane emitting) and the temperate millipede Glomeris connexa (small, non-methane emitting), fed on an identical diet, were studied using comparative metagenomics and metatranscriptomics. RESULTS: The results showed that the microbial load in E. pulchripes is much higher and more diverse than in G. connexa. The microbial communities of the two species differed significantly, with Bacteroidota dominating the hindguts of E. pulchripes and Proteobacteria (Pseudomonadota) in G. connexa. Despite equal sequencing effort, de novo assembly and binning recovered 282 metagenome-assembled genomes (MAGs) from E. pulchripes and 33 from G. connexa, including 90 novel bacterial taxa (81 in E. pulchripes and 9 in G. connexa). However, despite this taxonomic divergence, most of the functions, including carbohydrate hydrolysis, sulfate reduction, and nitrogen cycling, were common to the two species. Members of the Bacteroidota (Bacteroidetes) were the primary agents of complex carbon degradation in E. pulchripes, while members of Proteobacteria dominated in G. connexa. Members of Desulfobacterota were the potential sulfate-reducing bacteria in E. pulchripes. The capacity for dissimilatory nitrate reduction was found in Actinobacteriota (E. pulchripes) and Proteobacteria (both species), but only Proteobacteria possessed the capacity for denitrification (both species). In contrast, some functions were only found in E. pulchripes. These include reductive acetogenesis, found in members of Desulfobacterota and Firmicutes (Bacillota) in E. pulchripes. Also, diazotrophs were only found in E. pulchripes, with a few members of the Firmicutes and Proteobacteria expressing the nifH gene. Interestingly, fungal-cell-wall-degrading glycoside hydrolases (GHs) were among the most abundant carbohydrate-active enzymes (CAZymes) expressed in both millipede species, suggesting that fungal biomass plays an important role in the millipede diet. CONCLUSIONS: Overall, these results provide detailed insights into the genomic capabilities of the microbial community in the hindgut of millipedes and shed light on the ecophysiology of these essential detritivores. Video Abstract.