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

Abstract

AbstractMillipedes 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 juvenileEpibolus pulchripes(tropical, CH4-emitting) andGlomeris 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, withBacteroidotadominant inE. pulchripesandPseudomonadotainG. connexa. Sodium-2-bromoethanesulfonate treatment halted CH4emissions and reduced the faecalmcrAcopies inE. pulchripesafter 14 days, but emissions resumed after returning to normal feeding. Methanogens in the orderMethanobacterialesandMethanomasscilliicoccalesassociated with protists were detected using Catalysed Reporter Deposition FluorescenceIn situHybridization (CARD-FISH) on day 21, despite suppressed CH4-emission. Employing13C-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, andEnterobacterales), others, such as members ofDesulfovibrionaleswere 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 millipede’s 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) andGlomeris 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 microbiota’s role in the millipede’s 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.