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 used inhibitors to disrupt the gut microbiota of juvenile Epibolus pulchripes (tropical, CH4-emitting) and Glomeris connexa (temperate, non-CH4-emitting) and isotopic labelling. Feeding the millipedes sterile or antibiotics-treated litter 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 in E. pulchripes, but it resumed after returning to normal feeding. Employing 13C-labeled leaf litter and RNA-SIP revealed a slow and gradual prokaryote labelling, indicating a significant density shift only by day 21. 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.

• MeSH
• Bacteria metabolism   genetics   MeSH
• Arthropods * microbiology   metabolism   MeSH
• Feces microbiology   MeSH
• Fermentation * MeSH
• Plant Leaves metabolism   microbiology   MeSH
• Gastrointestinal Microbiome * MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH

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.

• Keywords
• Acetogens, Ecosystem engineers, Glycoside hydrolases, Hindgut microbiota, Millipede holobiont, Nutrient cycling, Polysaccharide degradation, Symbiosis,
• MeSH
• Bacteria MeSH
• Bacteroidetes genetics   MeSH
• Arthropods * genetics   MeSH
• Nitrogen metabolism   MeSH
• Phylogeny MeSH
• Metagenome MeSH
• Metagenomics MeSH
• Proteobacteria genetics   MeSH
• Carbohydrates MeSH
• Sulfates metabolism   MeSH
• Gastrointestinal Microbiome * genetics   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Video-Audio Media MeSH
• Journal Article MeSH
• Names of Substances
• Nitrogen MeSH
• Carbohydrates MeSH
• Sulfates MeSH

Methane emission by terrestrial invertebrates is restricted to millipedes, termites, cockroaches, and scarab beetles. The arthropod-associated archaea known to date belong to the orders Methanobacteriales, Methanomassiliicoccales, Methanomicrobiales, and Methanosarcinales, and in a few cases also to non-methanogenic Nitrososphaerales and Bathyarchaeales. However, all major host groups are severely undersampled, and the taxonomy of existing lineages is not well developed. Full-length 16S rRNA gene sequences and genomes of arthropod-associated archaea are scarce, reference databases lack resolution, and the names of many taxa are either not validly published or under-classified and require revision. Here, we investigated the diversity of archaea in a wide range of methane-emitting arthropods, combining phylogenomic analysis of isolates and metagenome-assembled genomes (MAGs) with amplicon sequencing of full-length 16S rRNA genes. Our results allowed us to describe numerous new species in hitherto undescribed taxa among the orders Methanobacteriales (Methanacia, Methanarmilla, Methanobaculum, Methanobinarius, Methanocatella, Methanoflexus, Methanorudis, and Methanovirga, all gen. nova), Methanomicrobiales (Methanofilum and Methanorbis, both gen. nova), Methanosarcinales (Methanofrustulum and Methanolapillus, both gen. nova), Methanomassiliicoccales (Methanomethylophilaceae fam. nov., Methanarcanum, Methanogranum, Methanomethylophilus, Methanomicula, Methanoplasma, Methanoprimaticola, all gen. nova), and the new family Bathycorpusculaceae (Bathycorpusculum gen. nov.). Reclassification of amplicon libraries from this and previous studies using this new taxonomic framework revealed that arthropods harbor only CO2 and methyl-reducing hydrogenotrophic methanogens. Numerous genus-level lineages appear to be present exclusively in arthropods, suggesting long evolutionary trajectories with their termite, cockroach, and millipede hosts, and a radiation into various microhabitats and ecological niches provided by their digestive tracts (e.g., hindgut compartments, gut wall, or anaerobic protists). The distribution patterns among the different host groups are often complex, indicating a mixed mode of transmission and a parallel evolution of invertebrate and vertebrate-associated lineages.

• Keywords
• Bathyarchaeia, Nitrososphaerales, archaea, cockroaches, gut microbiota, methanogens, millipedes, termites,
• Publication type
• Journal Article MeSH

In this paper, the growth requirements, fermentation pattern, and hydrolytic enzymatic activities of anaerobic ciliates collected from the hindgut of the African tropical millipede Archispirostreptus gigas are described. Single-cell molecular analysis showed that ciliates from the millipede hindgut could be assigned to the Nyctotherus velox and a new species named N. archispirostreptae n. sp. The ciliate N. velox can grow in vitro with unspecified prokaryotic populations and various plant polysaccharides (rice starch-RS, xylan, crystalline cellulose20-CC, carboxymethylcellulose-CMC, and inulin) or without polysaccharides (NoPOS) in complex reduced medium with soluble supplements (peptone, glucose, and vitamins). Specific catalytic activity (nkat/g of protein) of α amylase of 300, xylanase of 290, carboxymethylcellulase of 190, and inulinase of 170 was present in the crude protein extract of N. velox. The highest in vitro dry matter digestibility was observed in RS and inulin after 96 h of fermentation. The highest methane concentration was observed in xylan and inulin substrates. The highest short-chain fatty acid concentration was observed in RS, inulin, and xylan. In contrast, the highest ammonia concentration was observed in NoPOS, CMC, and CC. The results indicate that starch is the preferred substrate of the N. velox. Hydrolytic enzyme activities of N. velox showed that the ciliates contribute to the fermentation of plant polysaccharides in the gut of millipedes.

• Keywords
• Nyctotherus, ciliates, enzymatic activities, hindgut protozoa, in vitro growth, polysaccharide fermentation, tropical millipedes,
• Publication type
• Journal Article MeSH

Invertebrate-microbial associations are widespread in the biosphere and are often related to the function of novel genes, fitness advantages, and even speciation events. Despite ~ 13,000 species of millipedes identified across the world, millipedes and their gut microbiota are markedly understudied compared to other arthropods. Exploring the contribution of individual host-associated microbes is often challenging as many are uncultivable. In this study, we conducted metatranscriptomic profiling of different body segments of a millipede at the holobiont level. This is the first reported transcriptome assembly of a tropical millipede Telodeinopus aoutii (Demange, 1971), as well as the first study on any Myriapoda holobiont. High-throughput RNA sequencing revealed that Telodeinopus aoutii contained > 90% of the core Arthropoda genes. Proteobacteria, Bacteroidetes, Firmicutes, and Euryarchaeota represented dominant and functionally active phyla in the millipede gut, among which 97% of Bacteroidetes and 98% of Firmicutes were present exclusively in the hindgut. A total of 37,831 predicted protein-coding genes of millipede holobiont belonged to six enzyme classes. Around 35% of these proteins were produced by microbiota in the hindgut and 21% by the host in the midgut. Our results indicated that although major metabolic pathways operate at the holobiont level, the involvement of some host and microbial genes are mutually exclusive and microbes predominantly contribute to essential amino acid biosynthesis, short-chain fatty acid metabolism, and fermentation.

• MeSH
• Bacteroidetes MeSH
• Arthropods * genetics   MeSH
• Amino Acids, Essential MeSH
• Fatty Acids, Volatile MeSH
• Gastrointestinal Microbiome * genetics   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Amino Acids, Essential MeSH
• Fatty Acids, Volatile MeSH

Methanogens represent the final decomposition step in anaerobic degradation of organic matter, occurring in the digestive tracts of various invertebrates. However, factors determining their community structure and activity in distinct gut sections are still debated. In this study, we focused on the tropical millipede species Archispirostreptus gigas (Diplopoda, Spirostreptidae) and Epibolus pulchripes (Diplopoda, Pachybolidae), which release considerable amounts of methane. We aimed to characterize relationships between physicochemical parameters, methane production rates, and methanogen community structure in the two major gut sections, midgut and hindgut. Microsensor measurements revealed that both sections were strictly anoxic, with reducing conditions prevailing in both millipedes. Hydrogen concentration peaked in the anterior hindgut of E. pulchripes. In both species, the intestinal pH was significantly higher in the hindgut than in the midgut. An accumulation of acetate and formate in the gut indicated bacterial fermentation activities in the digestive tracts of both species. Phylogenetic analysis of 16S rRNA genes showed a prevalence of Methanobrevibacter spp. (Methanobacteriales), accompanied by a small fraction of so-far-unclassified "Methanomethylophilaceae" (Methanomassiliicoccales), in both species, which suggests that methanogenesis is mostly hydrogenotrophic. We conclude that anoxic conditions, negative redox potential, and bacterial production of hydrogen and formate promote gut colonization by methanogens. The higher activities of methanogens in the hindgut are explained by the higher pH of this compartment and their association with ciliates, which are restricted to this compartment and present an additional source of methanogenic substrates. IMPORTANCE Methane (CH4) is the second most important atmospheric greenhouse gas after CO2 and is believed to account for 17% of global warming. Methanogens are a diverse group of archaea and can be found in various anoxic habitats, including digestive tracts of plant-feeding animals. Termites, cockroaches, the larvae of scarab beetles, and millipedes are the only arthropods known to host methanogens and emit large amounts of methane. Millipedes are ranked as the third most important detritivores after termites and earthworms, and they are considered keystone species in many terrestrial ecosystems. Both methane-producing and non-methane-emitting species of millipedes have been observed, but what limits their methanogenic potential is not known. In the present study, we show that physicochemical gut conditions and the distribution of symbiotic ciliates are important factors determining CH4 emission in millipedes. We also found close similarities to other methane-emitting arthropods, which might be associated with their similar plant-feeding habits.

• Keywords
• Methanobrevibacter, Methanomassiliicoccales, digestive tract, methane, methanogenesis, methanogenic community, physicochemical parameters, tropical millipedes,
• MeSH
• Bacteria genetics   metabolism   MeSH
• Arthropods microbiology   MeSH
• Formates metabolism   MeSH
• Phylogeny MeSH
• Gastrointestinal Tract metabolism   MeSH
• Hydrogen-Ion Concentration MeSH
• Oxygen analysis   MeSH
• Methane metabolism   MeSH
• Oxidation-Reduction MeSH
• RNA, Ribosomal, 16S genetics   MeSH
• Gastrointestinal Microbiome * genetics   MeSH
• Hydrogen metabolism   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Formates MeSH
• formic acid MeSH Browser
• Oxygen MeSH
• Methane MeSH
• RNA, Ribosomal, 16S MeSH
• Hydrogen MeSH