Cellulose fermentation by the gut microbiota is likely not essential for the nutrition of millipedes

Millipedes are thought to depend on their gut microbiome for processing plant-litter-cellulose through fermentation, similar to many other arthropods. However, this hypothesis lacks sufficient evidence. To investigate this, we disrupted the gut microbiota of juvenile Epibolus pulchripes (tropical, CH4-emitting) and Glomeris connexa (temperate, non-CH4-emitting) using chemical inhibitors and isotopic labelling. Feeding the millipedes sterile or antibiotics-treated litter notably reduced faecal production and microbial load without major impacts on survival or weight. Bacterial diversity remained similar, with Bacteroidota dominant in E. pulchripes and Pseudomonadota in G. connexa. Sodium-2-bromoethanesulfonate treatment halted CH4 emissions and reduced the faecal mcrA copies in E. pulchripes after 14 days, but emissions resumed after returning to normal feeding. Methanogens in the order Methanobacteriales and Methanomasscilliicoccales associated with protists were detected using Catalysed Reporter Deposition Fluorescence In situ Hybridization (CARD-FISH) on day 21, despite suppressed CH4-emission. Employing 13C-labeled leaf litter and RNA-SIP revealed a slow and gradual prokaryote labelling, indicating a significant density shift only by day 21. In addition to labelling of taxa from orders well-recognized for their role in (ligno)cellulose fermentation (e.g., Bacteroidales, Burkholderiales, and Enterobacterales), others, such as members of Desulfovibrionales were also labelled. Surprisingly, labelling of the fungal biomass was somewhat quicker. Our findings suggest that fermentation by the gut microbiota is likely not essential for the millipedes nutrition. ImportanceMillipedes (Diplopoda) constitute the third most significant group of detritivores after termites and earthworms, yet they have been comparatively understudied. Traditionally, it was believed that millipedes gain energy from fermenting cellulose using their gut microbiota, similar to wood-feeding termites, but this belief lacks evidence. This study used two model millipede species, Epibolus pulchripes (large, tropical, and methane emitter) and Glomeris connexa (small, temperate, and non-methane emitter) to test this belief. We used chemical manipulation experiments, stable isotope labelling, and DNA sequencing to comprehend the microbiotas role in the millipedes nutrition. The findings suggest that cellulose fermentation by the gut microbiota may not be essential for millipede nutrition; instead, bacteriovory and fungivory might be the dominant feeding strategies of millipedes.