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.

Faculty of Science University of South Bohemia České Budějovice Czechia

Institute of Soil Biology and Biogeochemistry Biology Centre CAS České Budějovice Czechia

RG Insect Gut Microbiology and Symbiosis Max Planck Institute for Terrestrial Microbiology Marburg Germany

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Alagesan P. Millipedes: diversity, distribution and ecology. In: Chakravarthy AK, Sridhara S, editors. Arthropod diversity and conservation in the tropics and sub-tropics. Singapore: Springer Singapore; 2016. pp. 119–37.

Golovatch SI, Kime RD. Millipede ( Diplopoda ) distributions: a review. Soil Org. 2009;81:565–97.

Watanabe H, Tokuda G. Cellulolytic systems in insects. Annu Rev Entomol. 2010;55:609–632. PubMed

Wybouw N, Pauchet Y, Heckel DG, Van Leeuwen T. Horizontal gene transfer contributes to the evolution of arthropod herbivory. Genome Biol Evol. 2016;8:1785–1801. PubMed PMC

Kögel-Knabner I. The macromolecular organic composition of plant and microbial residues as inputs to soil organic matter. Soil Biol Biochem. 2002;34:139–162.

Flint HJ, Bayer EA, Rincon MT, Lamed R, White BA. Polysaccharide utilization by gut bacteria: potential for new insights from genomic analysis. Nat Rev Microbiol. 2008;6:121–131. PubMed

Douglas AE. The microbial dimension in insect nutritional ecology. Funct Ecol. 2009;23:38–47.

Nicholson JK, Holmes E, Kinross J, Burcelin R, Gibson G, Jia W, et al. Host-gut microbiota metabolic interactions. Science. 2012;336:1262–1267. PubMed

Bouchon D, Zimmer M, Dittmer J. The terrestrial isopod microbiome: an all-in-one toolbox for animal–microbe interactions of ecological relevance. Front Microbiol. 2016;7:1472. PubMed PMC

Graf J. Lessons from digestive-tract symbioses between bacteria and invertebrates. Annu Rev Microbiol. 2016;70:375–393. PubMed

Huang K, Wang J, Huang J, Zhang S, Vogler AP, Liu Q, et al. Host phylogeny and diet shape gut microbial communities within bamboo-feeding insects. Front Microbiol. 2021;12:633075. PubMed PMC

Geli-Cruz O, Cafaro MJ, Santos-Flores CJ, Ropelewski AJ, Van Dam AR. Taxonomic survey of Anadenobolus monilicornis gut microbiota via shotgun nanopore sequencing. Genomics. 2019. Available from: http://biorxiv.org/lookup/doi/10.1101/560755. Accessed 30 Nov 2023. DOI

Nardi JB, Bee CM, Taylor SJ. Compartmentalization of microbial communities that inhabit the hindguts of millipedes. Arthropod Struct Dev. 2016;45:462–474. PubMed

Brune A, Dietrich C. The gut microbiota of termites: digesting the diversity in the light of ecology and evolution. Annu Rev Microbiol. 2015;69:145–166. PubMed

Sun M, Chao H, Zheng X, Deng S, Ye M, Hu F. Ecological role of earthworm intestinal bacteria in terrestrial environments: a review. Sci Total Environ. 2020;740:140008. PubMed

Byzov BA. Intestinal microbiota of millipedes. In: König H, Varma A, editors. Intestinal microorganisms of termites and other invertebrates. Berlin/Heidelberg: Springer-Verlag; 2006. pp. 89–114.

Sardar P, Šustr V, Chroňáková A, Lorenc F, Faktorová L. De novo metatranscriptomic exploration of gene function in the millipede holobiont. Sci Rep. 2022;12:16173. PubMed PMC

Heinze T, Petzold-Welcke K, van Dam JEG. Polysaccharides: molecular and supramolecular structures. Terminology. The European Polysaccharide network of Excellence (EPNOE): Research Initiatives and results. 2012. pp. 23–64.

Warren RA. Microbial hydrolysis of polysaccharides. Annu Rev Microbiol. 1996;50:183–212. PubMed

Taylor EC. Role of aerobic microbial populations in cellulose digestion by desert millipedes. Appl Environ Microbiol. 1982;44:281–91. PubMed PMC

Szabo IM, Nasser E-GA, Striganova B, Rakhmo YR, Jager K, Heydrich M, et al. Interactions among millipedes (Diplopoda) and their intestinal bacteria. 1990. p. 8.

Ramanathan B, Alagesan P. Isolation, characterization and role of gut bacteria of three different millipede species. 2012. p. 7.

Beck L, Friebe B. Verwertung von Kohlenhydraten bei Oniscus asellus (Isopoda) und Polydesmus angustus (Diplopoda). Pedobiologia. 1981.

Sardar P, Šustr V, Chroňáková A, Lorenc F. Metatranscriptomic holobiont analysis of carbohydrate-active enzymes in the millipede Telodeinopus aoutii (Diplopoda, Spirostreptida) Front Ecol Evol. 2022;10:931986.

Brune A. Symbiotic digestion of lignocellulose in termite guts. Nat Rev Microbiol. 2014;12:168–180. PubMed

Maraun M, Scheu S. Changes in microbial biomass, respiration and nutrient status of beech (Fagus sylvatica) leaf litter processed by millipedes (Glomeris marginata) Oecologia. 1996;107:131–140. PubMed

Bignell D. Relative assimilations of 14C-labelled microbial tissues and 14C-plant fibre ingested with leaf litter by the millipede Glomeris marginata under experimental conditions. Soil Biol Biochem. 1989;21:819–827.

Garcia-Rubio R, de Oliveira HC, Rivera J, Trevijano-Contador N. The fungal cell wall: Candida, Cryptococcus, and Aspergillus species. Front Microbiol. 2020;10:2993. PubMed PMC

Horváthová T, Šustr V, Chroňáková A, Semanová S, Lang K, Dietrich C, et al. Methanogenesis in the digestive tracts of the tropical millipedes Archispirostreptus gigas (Diplopoda, Spirostreptidae) and Epibolus pulchripes (Diplopoda, Pachybolidae) Appl Environ Microbiol. 2021;87:e00614–e621. PubMed PMC

Šustr V, Chroňáková A, Semanová S, Tajovský K, Šimek M. Methane production and methanogenic archaea in the digestive tracts of millipedes (Diplopoda) PLoS One. 2014;9:e102659. PubMed PMC

Ceja-Navarro JA, Nguyen NH, Karaoz U, Gross SR, Herman DJ, Andersen GL, et al. Compartmentalized microbial composition, oxygen gradients and nitrogen fixation in the gut of Odontotaenius disjunctus. ISME J. 2014;8(1):6–18. PubMed PMC

David J-F. The role of litter-feeding macroarthropods in decomposition processes: a reappraisal of common views. Soil Biol Biochem. 2014;76:109–118.

Enghoff H. East African giant millipedes of the tribe Pachybolini (Diplopoda, Spirobolida, Pachybolidae) Zootaxa. 2011;2753:1–41.

Hoess R, Scholl A. Allozyme and literature study of Glomeris guttata Risso, 1826, and G. connexa Koch, 1847, a case of taxonomic confusion (Diplopoda: Glomeridae) Zoologischer Anzeiger. 2001;240:15–33.

Gerstaecker A. Die Gliedertier - Fauna des Sansibar-Gebietes. [The arthropod fauna of the Zanzibar region]. Hansebooks; 2016.

Kocourek P, Tajovský K, Dolejš P. Mnohonožky České republiky—Příručka pro určování našich druhů [Millipedes of the Czech Republic—Guide for identification of our species].—Základní organizace Českého svazu ochránců přírody. Vlašim: English abstract; 2017. p. 256.

Unkovich M, Cadisch G, Australian Centre for International Agricultural Research . Measuring plant-associated nitrogen fixation in agricultural systems. Canberra: ACIAR; 2008.

Angel R, Claus P, Conrad R. Methanogenic archaea are globally ubiquitous in aerated soils and become active under wet anoxic conditions. ISME J. 2012;6:847–862. PubMed PMC

Angel R, Petrova E, Lara-Rodriguez A. Total nucleic acids extraction from soil V.6. protocols.io. 2021;6. [cited 2022 May 3]. Available from: https://www.protocols.io/view/total-nucleic-acids-extraction-from-soil-bi46kgze.

Naqib A, Poggi S, Green SJ. Deconstructing the polymerase chain reaction II: an improved workflow and effects on artifact formation and primer degeneracy. PeerJ. 2019;7:e7121. PubMed PMC

Walters W, Hyde ER, Berg-Lyons D, Ackermann G, Humphrey G, Parada A, et al. Improved bacterial 16S rRNA gene (V4 and V4–5) and fungal internal transcribed spacer marker gene primers for microbial community surveys. mSystems. 2016;1:e00009–15. PubMed PMC

Angel R, Petrova E, Lara A. qPCR: Bacterial SSU rRNA 338F-516P-805R v4. 2020. Available from: https://www.protocols.io/view/qpcr-bacterial-ssu-rrna-338f-516p-805r-bqx5mxq6.

Bolger AM, Lohse M, Usadel B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics. 2014;30:2114–2120. PubMed PMC

Eren AM, Kiefl E, Shaiber A, Veseli I, Miller SE, Schechter MS, et al. Community-led, integrated, reproducible multi-omics with anvi’o. Nat Microbiol. 2021;6:3–6. PubMed PMC

Li D, Liu C-M, Luo R, Sadakane K, Lam T-W. MEGAHIT: an ultra-fast single-node solution for large and complex metagenomics assembly via succinct de Bruijn graph. Bioinformatics. 2015;31:1674–1676. PubMed

Langmead B, Salzberg SL. Fast gapped-read alignment with Bowtie 2. Nat Methods. 2012;9:357–359. PubMed PMC

Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N, et al. The sequence alignment/map format and SAMtools. Bioinformatics. 2009;25:2078–2079. PubMed PMC

Hyatt D, Chen G-L, LoCascio PF, Land ML, Larimer FW, Hauser LJ. Prodigal: prokaryotic gene recognition and translation initiation site identification. BMC Bioinformatics. 2010;11:119. PubMed PMC

Finn RD, Clements J, Eddy SR. HMMER web server: interactive sequence similarity searching. Nucleic Acids Res. 2011;39:W29–37. PubMed PMC

Kim D, Song L, Breitwieser FP, Salzberg SL. Centrifuge: rapid and sensitive classification of metagenomic sequences. Genome Res. 2016;26:1721–1729. PubMed PMC

Galperin MY, Wolf YI, Makarova KS, Vera Alvarez R, Landsman D, Koonin EV. COG database update: focus on microbial diversity, model organisms, and widespread pathogens. Nucleic Acids Res. 2020;49:D274–D281. PubMed PMC

Kanehisa M, Goto S. KEGG: Kyoto encyclopedia of genes and genomes. Nucleic Acids Res. 2000;28:27–30. PubMed PMC

Kang DD, Li F, Kirton E, Thomas A, Egan R, An H, et al. MetaBAT 2: an adaptive binning algorithm for robust and efficient genome reconstruction from metagenome assemblies. PeerJ. 2019;7:e7359. PubMed PMC

Alneberg J, Bjarnason BS, de Bruijn I, Schirmer M, Quick J, Ijaz UZ, et al. Binning metagenomic contigs by coverage and composition. Nat Methods. 2014;11:1144–1146. PubMed

Parks DH, Imelfort M, Skennerton CT, Hugenholtz P, Tyson GW. CheckM: assessing the quality of microbial genomes recovered from isolates, single cells, and metagenomes. Genome Res. 2015;25:1043–1055. PubMed PMC

RCore T . R: a language and environment for statistical computing. Vienna: R Foundation for Statistical Computing; 2016.

Martin M. Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet. J. 2011;17:10–2.

Callahan BJ, Sankaran K, Fukuyama JA, McMurdie PJ, Holmes SP. Bioconductor workflow for microbiome data analysis: from raw reads to community analyses. F1000Res. 2016;5:1492. PubMed PMC

Parks DH, Chuvochina M, Waite DW, Rinke C, Skarshewski A, Chaumeil P-A, et al. A standardized bacterial taxonomy based on genome phylogeny substantially revises the tree of life. Nat Biotechnol. 2018;36:996–1004. PubMed

McMurdie PJ, Holmes S. phyloseq: an R package for reproducible interactive analysis and graphics of microbiome census data. PLoS One. 2013;8:e61217. PubMed PMC

Davis NM, Proctor DM, Holmes SP, Relman DA, Callahan BJ. Simple statistical identification and removal of contaminant sequences in marker-gene and metagenomics data. Microbiome. 2018;6:226. PubMed PMC

Lozupone C, Knight R. UniFrac: a new phylogenetic method for comparing microbial communities. Appl Environ Microbiol. 2005;71:8228–8235. PubMed PMC

Kopylova E, Noé L, Touzet H. SortMeRNA: fast and accurate filtering of ribosomal RNAs in metatranscriptomic data. Bioinformatics. 2012;28:3211–3217. PubMed

Grabherr MG, Haas BJ, Yassour M, Levin JZ, Thompson DA, Amit I, et al. Trinity: reconstructing a full-length transcriptome without a genome from RNA-Seq data. Nat Biotechnol. 2011;29:644–652. PubMed PMC

Pronk LJU, Medema MH. Whokaryote: distinguishing eukaryotic and prokaryotic contigs in metagenomes based on gene structure. Microb Genom. 2022;8:mgen000823. PubMed PMC

von Meijenfeldt FAB, Arkhipova K, Cambuy DD, Coutinho FH, Dutilh BE. Robust taxonomic classification of uncharted microbial sequences and bins with CAT and BAT. Genome Biol. 2019;20:217. PubMed PMC

Chaumeil P-A, Mussig AJ, Hugenholtz P, Parks DH. GTDB-Tk: a toolkit to classify genomes with the genome taxonomy database. Bioinformatics. 2020;36:1925–1927. PubMed PMC

Luo C, Rodriguez-R LM, Konstantinidis KT. MyTaxa: an advanced taxonomic classifier for genomic and metagenomic sequences. Nucleic Acids Res. 2014;42:e73. PubMed PMC

Glendinning L, Stewart RD, Pallen MJ, Watson KA, Watson M. Assembly of hundreds of novel bacterial genomes from the chicken caecum. Genome Biol. 2020;21:34. PubMed PMC

Bengtsson-Palme J, Hartmann M, Eriksson KM, Pal C, Thorell K, Larsson DGJ, et al. METAXA2: improved identification and taxonomic classification of small and large subunit rRNA in metagenomic data. Mol Ecol Resour. 2015;15:1403–1414. PubMed

Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. J Mol Biol. 1990;215:403–410. PubMed

Zhou Z, Tran PQ, Breister AM, Liu Y, Kieft K, Cowley ES, et al. METABOLIC: high-throughput profiling of microbial genomes for functional traits, metabolism, biogeochemistry, and community-scale functional networks. Microbiome. 2022;10:33. PubMed PMC

Drula E, Garron M-L, Dogan S, Lombard V, Henrissat B, Terrapon N. The carbohydrate-active enzyme database: functions and literature. Nucleic Acids Res. 2021;50:D571–D577. PubMed PMC

Sayers EW, Bolton EE, Brister JR, Canese K, Chan J, Comeau DC, et al. Database resources of the National Center for Biotechnology Information. Nucleic Acids Res. 2021;50:D20–D26. PubMed PMC

Wickham H. ggplot2: elegant graphics for data analysis. Springer-Verlag New York; 2016. Available from: https://ggplot2.tidyverse.org.

Gu Z, Gu L, Eils R, Schlesner M, Brors B. “Circlize” implements and enhances circular visualization in R. 2014. PubMed

Letunic I, Bork P. Interactive Tree Of Life (iTOL) v5: an online tool for phylogenetic tree display and annotation. Nucleic Acids Res. 2021;49:W293–W296. PubMed PMC

Li H. Minimap2: pairwise alignment for nucleotide sequences. Bioinformatics. 2018;34:3094–3100. PubMed PMC

Li H. Aligning sequence reads, clone sequences and assembly contigs with BWA-MEM. arXiv; 2013. [cited 2023 Nov 30]. Available from: http://arxiv.org/abs/1303.3997.

Paetzel M. Bacterial signal peptidases. In: Kuhn A, editor. Bacterial cell walls and membranes. Cham: Springer International Publishing; 2019. pp. 187–219.

Rabouille C. Pathways of unconventional protein secretion. Trends Cell Biol. 2017;27:230–240. PubMed

Lin H, Castro NM, Bennett GN, San K-Y. Acetyl-CoA synthetase overexpression in Escherichia coli demonstrates more efficient acetate assimilation and lower acetate accumulation: a potential tool in metabolic engineering. Appl Microbiol Biotechnol. 2006;71:870–874. PubMed

Wolfe AJ. The acetate switch. Microbiol Mol Biol Rev. 2005;69:12–50. PubMed PMC

Ikeda-Ohtsubo W, Strassert JFH, Köhler T, Mikaelyan A, Gregor I, McHardy AC, et al. ‘Candidatus Adiutrix intracellularis’, an endosymbiont of termite gut flagellates, is the first representative of a deep-branching clade of Deltaproteobacteria and a putative homoacetogen. Environ Microbiol. 2016;18:2548–2564. PubMed

Dhivya A, Alagesan P. Isolation and identification of microbial load in the gut and faeces of millipede Spinotarsus colosseus. World J Zool. 2018;13:04–9.

Ineson P, Anderson JM. Aerobically isolated bacteria associated with the gut and faeces of the litter feeding macroarthropods Oniscus asellus and Glomeris marginata. Soil Biol Biochem. 1985;17:843–849.

Engel P, Moran NA. The gut microbiota of insects – diversity in structure and function. FEMS Microbiol Rev. 2013;37:699–735. PubMed

Knapp BA, Seeber J, Rief A, Meyer E, Insam H. Bacterial community composition of the gut microbiota of Cylindroiulus fulviceps (diplopoda) as revealed by molecular fingerprinting and cloning. Folia Microbiol. 2010;55:489–496. PubMed

Bredon M, Herran B, Bertaux J, Grève P, Moumen B, Bouchon D. Isopod holobionts as promising models for lignocellulose degradation. Biotechnol Biofuels. 2020;13:49. PubMed PMC

Delhoumi M, Catania V, Zaabar W, Tolone M, Quatrini P, Achouri MS. The gut microbiota structure of the terrestrial isopod Porcellionides pruinosus (Isopoda: Oniscidea) Eur Zool J. 2020;87:357–368.

Xu L, Sun L, Zhang S, Wang S, Lu M. High-resolution profiling of gut bacterial communities in an invasive beetle using PacBio SMRT sequencing system. Insects. 2019;10:248. PubMed PMC

Suárez-Moo P, Cruz-Rosales M, Ibarra-Laclette E, Desgarennes D, Huerta C, Lamelas A. Diversity and composition of the gut microbiota in the developmental stages of the dung beetle Copris incertus Say (Coleoptera, Scarabaeidae) Front Microbiol. 2020;11:1698. PubMed PMC

Berlanga M, Llorens C, Comas J, Guerrero R. Gut bacterial community of the xylophagous cockroaches Cryptocercus punctulatus and Parasphaeria boleiriana. PLoS One. 2016;11:e0152400. PubMed PMC

Dietrich C, Köhler T, Brune A. The Cockroach origin of the termite gut microbiota: patterns in bacterial community structure reflect major evolutionary events. Appl Environ Microbiol. 2014;80:2261–2269. PubMed PMC

Lampert N, Mikaelyan A, Brune A. Diet is not the primary driver of bacterial community structure in the gut of litter-feeding cockroaches. BMC Microbiol. 2019;19:238. PubMed PMC

He S, Ivanova N, Kirton E, Allgaier M, Bergin C, Scheffrahn RH, et al. Comparative metagenomic and metatranscriptomic analysis of hindgut paunch microbiota in wood- and dung-feeding higher termites. PLoS One. 2013;8:e61126. PubMed PMC

Tokuda G, Mikaelyan A, Fukui C, Matsuura Y, Watanabe H, Fujishima M, et al. Fiber-associated spirochetes are major agents of hemicellulose degradation in the hindgut of wood-feeding higher termites. Proc Natl Acad Sci U S A. 2018;115:E11996–E12004. PubMed PMC

Egert M, Wagner B, Lemke T, Brune A, Friedrich MW. Microbial community structure in midgut and hindgut of the humus-feeding larva of Pachnoda ephippiata (Coleoptera: Scarabaeidae) Appl Environ Microbiol. 2003;69:6659–6668. PubMed PMC

Mohammed WS, Ziganshina EE, Shagimardanova EI, Gogoleva NE, Ziganshin AM. Comparison of intestinal bacterial and fungal communities across various xylophagous beetle larvae (Coleoptera: Cerambycidae) Sci Rep. 2018;8:10073. PubMed PMC

Voříšková J, Baldrian P. Fungal community on decomposing leaf litter undergoes rapid successional changes. ISME J. 2013;7:477–486. PubMed PMC

Carta LK, Thomas WK, Meyer-Rochow VB. Two nematodes (Nematoda: Diplogastridae, Rhabditidae) from the invasive millipede Chamberlinius hualienensis Wang, 1956 (Diplopoda, Paradoxosomatidae) on Hachijojima Island in Japan. J Nematol. 2018;50:479–486. PubMed PMC

Nagae S, Sato K, Tanabe T, Hasegawa K. Symbiosis of the millipede parasitic nematodes Rhigonematoidea and Thelastomatoidea with evolutionary different origins. BMC Ecol Evol. 2021;21:120. PubMed PMC

Purdy KJ. The distribution and diversity of euryarchaeota in termite guts. Advances in applied microbiology. Academic Press; 2007. p. 63–80. Available from: https://www.sciencedirect.com/science/article/pii/S0065216407620036. Cited 2023 Nov 9. PubMed

Hongoh Y, Ohkuma M. Termite gut flagellates and their methanogenic and eubacterial symbionts. 2010. pp. 55–79.

Lichtwardt RW. Trichomycetes and the Arthropod Gut. In: Brakhage AA, Zipfel PF, editors. Human and animal relationships. Berlin: Springer; 2008. p. 3–19.

Gibson LJ. The hierarchical structure and mechanics of plant materials. J R Soc Interface. 2012;9:2749–2766. PubMed PMC

Wei H, Xu Q, Taylor LE, Baker JO, Tucker MP, Ding S-Y. Natural paradigms of plant cell wall degradation. Curr Opin Biotechnol. 2009;20:330–338. PubMed

Zhang H, Yohe T, Huang L, Entwistle S, Wu P, Yang Z, et al. dbCAN2: a meta server for automated carbohydrate-active enzyme annotation. Nucleic Acids Res. 2018;46:W95–101. PubMed PMC

Carvalho DB, Paixão DA, Persinoti GF, Cota J, Rabelo SC, Grandis A, et al. Degradation of sugarcane bagasse by cockroach consortium bacteria. Bioenerg Res. 2022;15:1144–1156.

Ni J, Tokuda G. Lignocellulose-degrading enzymes from termites and their symbiotic microbiota. Biotechnol Adv. 2013;31:838–850. PubMed

Wormit A, Usadel B. The multifaceted role of pectin methylesterase inhibitors (PMEIs) Int J Mol Sci. 2018;19:2878. PubMed PMC

The CAZypedia Consortium Ten years of CAZypedia: a living encyclopedia of carbohydrate-active enzymes. Glycobiology. 2018;28:3–8. PubMed

Cromack K, Sollins P, Todd RL, Crossley DA, Fender WM, Fogel R, et al. Soil microorganism—arthropod interactions: fungi as major calcium and sodium Sources. In: Mattson WJ, et al., editors. The role of arthropods in forest ecosystems. Berlin, Heidelberg: Springer; 1977. pp. 78–84.

Frouz J, Kristůfek V, Li X, Santrůcková H, Sustr V, Brune A. Changes in amount of bacteria during gut passage of leaf litter and during coprophagy in three species of Bibionidae (Diptera) larvae. Folia Microbiol (Praha) 2003;48:535–542. PubMed

McKee LS, La Rosa SL, Westereng B, Eijsink VG, Pope PB, Larsbrink J. Polysaccharide degradation by the Bacteroidetes: mechanisms and nomenclature. Environ Microbiol Rep. 2021;13:559–581. PubMed

Vera-Ponce de León A, Jahnes BC, Duan J, Camuy-Vélez LA, Sabree ZL. Cultivable, host-specific Bacteroidetes symbionts exhibit diverse polysaccharolytic strategies. Appl Environ Microbiol. 2020;86:e00091–20. PubMed PMC

Bozorov TA, Rasulov BA, Zhang D. Characterization of the gut microbiota of invasive Agrilus mali Matsumara (Coleoptera: Buprestidae) using high-throughput sequencing: uncovering plant cell-wall degrading bacteria. Sci Rep. 2019;9:4923. PubMed PMC

Pester M, Brune A. Hydrogen is the central free intermediate during lignocellulose degradation by termite gut symbionts. ISME J. 2007;1:551–565. PubMed

Ragsdale SW. Pyruvate ferredoxin oxidoreductase and its radical intermediate. Chem Rev. 2003;103:2333–2346. PubMed

Gagen EJ, Padmanabha J, Denman SE, McSweeney CS. Hydrogenotrophic culture enrichment reveals rumen Lachnospiraceae and Ruminococcaceae acetogens and hydrogen-responsive Bacteroidetes from pasture-fed cattle. FEMS Microbiol Lett. 2015;362:fnv104. PubMed

Schuchmann K, Müller V. Autotrophy at the thermodynamic limit of life: a model for energy conservation in acetogenic bacteria. Nat Rev Microbiol. 2014;12:809–821. PubMed

Kane MD, Brauman A, Breznak JA. Clostridium mayombei sp. nov., an H2/CO2 acetogenic bacterium from the gut of the African soil-feeding termite Cubitermes speciosus. Arch Microbiol. 1991;156:99–104.

Kane MD, Breznak JA. Acetonema longum gen nov. sp. nov., an H2/CO2 acetogenic bacterium from the termite Pterotermes occidentis. Arch Microbiol. 1991;156:91–8. PubMed

Arora J, Kinjo Y, Šobotník J, Buček A, Clitheroe C, Stiblik P, et al. The functional evolution of termite gut microbiota. Microbiome. 2022;10:78. PubMed PMC

Søndergaard D, Pedersen CNS, Greening C. HydDB: a web tool for hydrogenase classification and analysis. Sci Rep. 2016;6:34212. PubMed PMC

Martins M, Pereira IAC. Sulfate-reducing bacteria as new microorganisms for biological hydrogen production. Int J Hydrogen Energy. 2013;38:12294–12301.

Dröge S, Limper U, Emtiazi F, Schönig I, Pavlus N, Drzyzga O, et al. In vitro and in vivo sulfate reduction in the gut contents of the termite Mastotermes darwiniensis and the rose-chafer Pachnoda marginata. J Gen Appl Microbiol. 2005;51:57–64. PubMed

Greening C, Geier R, Wang C, Woods LC, Morales SE, McDonald MJ, et al. Diverse hydrogen production and consumption pathways influence methane production in ruminants. ISME J. 2019;13:2617–2632. PubMed PMC

Brune A, Ohkuma M. Role of the termite gut microbiota in symbiotic digestion. In: Bignell DE, Roisin Y, Lo N, editors. Biology of termites: a modern synthesis. Dordrecht: Springer Netherlands; 2011. p. 439–75. 10.1007/978-90-481-3977-4_16.Cited 2022 Nov 18.

Goevert D, Conrad R. Carbon isotope fractionation by sulfate-reducing bacteria using different pathways for the oxidation of acetate. Environ Sci Technol. 2008;42:7813–7817. PubMed

Kuhnigk T, Branke J, Krekeler D, Cypionka H, König H. A feasible role of sulfate-reducing bacteria in the termite gut. Syst Appl Microbiol. 1996;19:139–149.

Bar-Shmuel N, Behar A, Segoli M. What do we know about biological nitrogen fixation in insects? Evidence and implications for the insect and the ecosystem. Insect Sci. 2020;27:392–403. PubMed

Loiret FG, Ortega E, Kleiner D, Ortega-Rodés P, Rodés R, Dong Z. A putative new endophytic nitrogen-fixing bacterium Pantoea sp. from sugarcane. J Appl Microbiol. 2004;97:504–11. PubMed

Walterson AM, Stavrinides J. Pantoea: insights into a highly versatile and diverse genus within the Enterobacteriaceae. FEMS Microbiol Rev. 2015;39:968–984. PubMed

Hongoh Y. Toward the functional analysis of uncultivable, symbiotic microorganisms in the termite gut. Cell Mol Life Sci. 2011;68:1311–1325. PubMed PMC

Breznak JA, Switzer JM. Acetate synthesis from H2 plus CO2 by termite gut microbes. Appl Environ Microbiol. 1986;52:623–630. PubMed PMC

Ngugi DK, Ji R, Brune A. Nitrogen mineralization, denitrification, and nitrate ammonification by soil-feeding termites: a 15N-based approach. Biogeochemistry. 2011;103:355–369.

Horn MA, Mertel R, Gehre M, Kästner M, Drake HL. In vivo emission of dinitrogen by earthworms via denitrifying bacteria in the gut. Appl Environ Microbiol. 2006;72:1013–1018. PubMed PMC

Šustr V, Šimek M, Faktorová L, Macková J, Tajovský K. Release of greenhouse gases from millipedes as related to food, body size, and other factors. Soil Biol Biochem. 2020;144:107765.

Ayayee P, Bhattacharyya S, Arnold T, Werne J, Leff L. Experimental investigation of potential biological nitrogen provisioning by freshwater insect gut microbiomes using 15N isotope analysis. Preprints; 2019. Available from: https://www.preprints.org/manuscript/201908.0034/v1. Cited 2023 Mar 22.

Wang H, Gunsalus RP. The nrfA and nirB nitrite reductase operons in Escherichia coli are expressed differently in response to nitrate than to nitrite. J Bacteriol. 2000;182:5813–5822. PubMed PMC

Kuwahara H, Yuki M, Izawa K, Ohkuma M, Hongoh Y. Genome of ‘Ca. Desulfovibrio trichonymphae’, an H2-oxidizing bacterium in a tripartite symbiotic system within a protist cell in the termite gut. ISME J. 2017;11:766–76. PubMed PMC

López-Sánchez MJ, Neef A, Peretó J, Patiño-Navarrete R, Pignatelli M, Latorre A, et al. Evolutionary convergence and nitrogen metabolism in Blattabacterium strain Bge, primary endosymbiont of the cockroach Blattella germanica. PLoS Genet. 2009;5:e1000721. PubMed PMC

Sabree ZL, Kambhampati S, Moran NA. Nitrogen recycling and nutritional provisioning by Blattabacterium, the cockroach endosymbiont. Proc Natl Acad Sci. 2009;106:19521–19526. PubMed PMC

Ma Z, Liu H, Liu C, He H, Zhao J, Wang X, et al. Streptosporangium sonchi sp. nov. and Streptosporangium kronopolitis sp. nov., two novel actinobacteria isolated from a root of common sowthistle (Sonchus oleraceus L.) and a millipede (Kronopolites svenhedind Verhoeff) Antonie Van Leeuwenhoek. 2015;107:1491–9. PubMed

Disruption of millipede-gut microbiota in E. pulchripes and G. connexa highlights the limited role of litter fermentation and the importance of litter-associated microbes for nutrition