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

Abstract

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 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.