Millipedes are important detritivores harbouring a diverse microbiome. Previous research focused on bacterial and archaeal diversity, while the virome remained neglected. We elucidated the DNA and RNA viral diversity in the hindguts of two model millipede species with distinct microbiomes: the tropical Epibolus pulchripes (methanogenic, dominated by Bacillota) and the temperate Glomeris connexa (non-methanogenic, dominated by Pseudomonadota). Based on metagenomic and metatranscriptomic assembled viral genomes, the viral communities differed markedly and preferentially infected the most abundant prokaryotic taxa. The majority of DNA viruses were Caudoviricetes (dsDNA), Cirlivirales (ssDNA) and Microviridae (ssDNA), while RNA viruses consisted of Leviviricetes (ssRNA), Potyviridae (ssRNA) and Eukaryotic viruses. A high abundance of subtypes I-C, I-B and II-C CRISPR-Cas systems was found, primarily from Pseudomonadota, Bacteroidota and Bacillota. In addition, auxiliary metabolic genes that modulate chitin degradation, vitamins and amino acid biosynthesis and sulphur metabolism were also detected. Lastly, we found low virus-to-microbe-ratios and a prevalence of lysogenic viruses, supporting a Piggyback-the-Winner dynamic in both hosts.

Department of Ecosystem Biology Faculty of Science University of South Bohemia in České Budějovice České Budějovice Czechia

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

Zobrazit více v PubMed

Abascal, F., Zardoya, R. & Posada, D. (2005) ProtTest: selection of best-fit models of protein evolution. Bioinformatics, 21, 2104-2105.

Ackermann, H.-W. (1998) Tailed bacteriophages: the order Caudovirales. Advances in Virus Research, 51, 135-201.

Bhunchoth, A., Blanc-Mathieu, R., Mihara, T., Nishimura, Y., Askora, A., Phironrit, N. et al. (2016) Two asian jumbo phages, ϕRSL2 and ϕRSF1, infect Ralstonia solanacearum and show common features of ϕKZ-related phages. Virology, 494, 56-66.

Byzov, B.A. (2006) Intestinal microbiota of millipedes. In: König, H. & Varma, A. (Eds.) Intestinal microorganisms of termites and other invertebrates. Berlin/Heidelberg: Springer-Verlag, pp. 89-114.

Callanan, J., Stockdale, S.R., Adriaenssens, E.M., Kuhn, J.H., Rumnieks, J., Pallen, M.J. et al. (2021) Leviviricetes: expanding and restructuring the taxonomy of bacteria-infecting single-stranded RNA viruses. Microbial Genomics, 7, 000686.

Camargo, A.P., Roux, S., Schulz, F., Babinski, M., Xu, Y., Hu, B. et al. (2023) Identification of mobile genetic elements with geNomad. Nature Biotechnology, 1-10.

Capella-Gutiérrez, S., Silla-Martínez, J.M. & Gabaldón, T. (2009) trimAl: a tool for automated alignment trimming in large-scale phylogenetic analyses. Bioinformatics, 25, 1972-1973.

Chaumeil, P.-A., Mussig, A.J., Hugenholtz, P. & Parks, D.H. (2020) GTDB-Tk: a toolkit to classify genomes with the genome taxonomy database. Bioinformatics, 36, 1925-1927.

Chen, L.-X., Méheust, R., Crits-Christoph, A., McMahon, K.D., Nelson, T.C., Slater, G.F. et al. (2020) Large freshwater phages with the potential to augment aerobic methane oxidation. Nature Microbiology, 5, 1504-1515.

Coutinho, F.H., Gregoracci, G.B., Walter, J.M., Thompson, C.C. & Thompson, F.L. (2018) Metagenomics sheds light on the ecology of marine microbes and their viruses. Trends in Microbiology, 26, 955-965.

Crawford, C.S. (1992) Millipedes as model detritivores. Berichte des Naturwissenschaftlich-Medizinischen Vereins in Innsbruck, 10, 277-288.

Danovaro, R. & Middelboe, M. (2010) Separation of free virus particles from sediments in aquatic systems. Waco, TX: The American Society of Limnololgy and Oceanography, pp. 74-81. Manual of aquatic viral ecology (MAVE).

Díaz-Muñoz, S.L. & Koskella, B. (2014) Bacteria-phage interactions in natural environments. Advances in Applied Microbiology, 89, 135-183.

Dion, M.B., Oechslin, F. & Moineau, S. (2020) Phage diversity, genomics and phylogeny. Nature Reviews Microbiology, 18, 125-138.

Emerson, J.B., Roux, S., Brum, J.R., Bolduc, B., Woodcroft, B.J., Jang, H.B. et al. (2018) Host-linked soil viral ecology along a permafrost thaw gradient. Nature Microbiology, 3, 870-880.

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

Eren, A.M., Kiefl, E., Shaiber, A., Veseli, I., Miller, S.E., Schechter, M.S. et al. (2021) Community-led, integrated, reproducible multi-omics with anvi'o. Nature Microbiology, 6, 3-6.

Gan, R., Zhou, F., Si, Y., Yang, H., Chen, C., Ren, C. et al. (2022) DBSCAN-SWA: an integrated tool for rapid prophage detection and annotation. Frontiers in Genetics, 13, 885048.

Ghabrial, S.A., Castón, J.R., Jiang, D., Nibert, M.L. & Suzuki, N. (2015) 50-plus years of fungal viruses. Virology, 479-480, 356-368.

Goldberg, G.W., McMillan, E.A., Varble, A., Modell, J.W., Samai, P., Jiang, W. et al. (2018) Incomplete prophage tolerance by type III-A CRISPR-Cas systems reduces the fitness of lysogenic hosts. Nature Communications, 9, 61.

Grabherr, M.G., Haas, B.J., Yassour, M., Levin, J.Z., Thompson, D.A., Amit, I. et al. (2011) Trinity: reconstructing a full-length transcriptome without a genome from RNA-Seq data. Nature Biotechnology, 29, 644-652.

Guo, J., Bolduc, B., Zayed, A.A., Varsani, A., Dominguez-Huerta, G., Delmont TO et al. (2021) VirSorter2: a multi-classifier, expert-guided approach to detect diverse DNA and RNA viruses. Microbiome., 9, 1-13.

Hille, F. & Charpentier, E. (2016) CRISPR-Cas: biology, mechanisms and relevance. Philosophical Transactions of the Royal Society, B: Biological Sciences, 371, 20150496.

Holmes, E.C. (2011) What does virus evolution tell us about virus origins? Journal of Virology, 85, 5247-5251.

Holmfeldt, K., Odić, D., Sullivan, M.B., Middelboe, M. & Riemann, L. (2012) Cultivated single-stranded DNA phages that infect marine Bacteroidetes prove difficult to detect with DNA-binding stains. Applied and Environmental Microbiology, 78, 892-894.

Howard-Varona, C., Hargreaves, K.R., Abedon, S.T. & Sullivan, M.B. (2017) Lysogeny in nature: mechanisms, impact and ecology of temperate phages. The ISME Journal., 11, 1511-1520.

Huerta-Cepas, J., Serra, F. & Bork, P. (2016) ETE 3: reconstruction, analysis, and visualization of phylogenomic data. Molecular Biology and Evolution, 33, 1635-1638.

Hunter, M. & Fusco, D. (2022) Superinfection exclusion: a viral strategy with short-term benefits and long-term drawbacks. PLoS Computational Biology, 18, e1010125.

Joly, F.-X., Coq, S., Coulis, M., David, J.-F., Hättenschwiler, S., Mueller, C.W. et al. (2020) Detritivore conversion of litter into faeces accelerates organic matter turnover. Communications Biology, 3, 660.

Kaletta, J., Pickl, C., Griebler, C., Klingl, A., Kurmayer, R. & Deng, L. (2020) A rigorous assessment and comparison of enumeration methods for environmental viruses. Scientific Reports, 10, 18625.

Katoh, K. & Toh, H. (2008) Recent developments in the MAFFT multiple sequence alignment program. Briefings in Bioinformatics, 9, 286-298.

Kauffman, K.M., Hussain, F.A., Yang, J., Arevalo, P., Brown, J.M., Chang, W.K. et al. (2018) A major lineage of non-tailed dsDNA viruses as unrecognized killers of marine bacteria. Nature, 554, 118-122.

Keen, E.C. & Dantas, G. (2018) Close encounters of three kinds: bacteriophages, commensal bacteria, and host immunity. Trends in Microbiology, 26, 943-954.

Kieft, K., Zhou, Z. & Anantharaman, K. (2020) VIBRANT: automated recovery, annotation and curation of microbial viruses, and evaluation of viral community function from genomic sequences. Microbiome., 8, 1-23.

Kirsch, J.M., Brzozowski, R.S., Faith, D., Round, J.L., Secor, P.R., & Duerkop, B.A. (2021). Bacteriophage-bacteria interactions in the gut: from invertebrates to mammals. Annual Review of Virology, 8(1), 95-113.

Knowles, B., Silveira, C., Bailey, B., Barott, K., Cantu, V., Cobián-Güemes, A. et al. (2016) Lytic to temperate switching of viral communities. Nature, 531, 466-470.

Koonin, E.V. & Makarova, K.S. (2009) CRISPR-Cas: an adaptive immunity system in prokaryotes. F1000 Biology Reports, 1, 95.

Koubová, A., Lorenc, F., Terézia Horváthová, A. & Chroňáková, V.Š. (2023) Millipede gut-derived microbes as a potential source of cellulolytic enzymes. World Journal of Microbiology and Biotechnology, 39, 169.

Krupovic, M., Varsani, A., Kazlauskas, D., Breitbart, M., Delwart, E., Rosario, K. et al. (2020) Cressdnaviricota: a virus phylum unifying seven families of rep-encoding viruses with single-stranded, circular DNA genomes. Journal of Virology, 94, e00582-20. Available from: https://doi.org/10.1128/jvi.00582-20

Larralde, M. (2022) Pyrodigal: python bindings and interface to prodigal, an efficient method for gene prediction in prokaryotes. Journal of Open Source Software., 7, 4296.

Lefkowitz, E.J., Dempsey, D.M., Hendrickson, R.C., Orton, R.J., Siddell, S.G. & Smith, D.B. (2018) Virus taxonomy: the database of the international committee on taxonomy of viruses (ICTV). Nucleic Acids Research, 46, D708-D717.

Li, C.-X., Shi, M., Tian, J.-H., Lin, X.-D., Kang, Y.-J., Chen, L.-J. et al. (2015) Unprecedented genomic diversity of RNA viruses in arthropods reveals the ancestry of negative-sense RNA viruses. Goff SP, editor. eLife, 4, e05378.

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

Li, H. & Durbin, R. (2010) Fast and accurate long-read alignment with Burrows-Wheeler transform. Bioinformatics, 26, 589-595.

López-García, P., Gutiérrez-Preciado, A., Krupovic, M., Ciobanu, M., Deschamps, P., Jardillier, L. et al. (2023) Metagenome-derived virus-microbe ratios across ecosystems. The ISME Journal., 1-12, 1552-1563.

Makarova, K.S., Wolf, Y.I. & Koonin, E.V. (2018) Classification and nomenclature of CRISPR-Cas systems: where from here? The CRISPR Journal., 1, 325-336.

Marynowska, M., Goux, X., Sillam-Dussès, D., Rouland-Lefèvre, C., Halder, R., Wilmes, P. et al. (2020) Compositional and functional characterisation of biomass-degrading microbial communities in guts of plant fibre- and soil-feeding higher termites. Microbiome., 8, 96.

Mihara, T., Nishimura, Y., Shimizu, Y., Nishiyama, H., Yoshikawa, G., Uehara, H. et al. (2016) Linking virus genomes with host taxonomy. Viruses, 8, 66.

Mirzaei, M.K. & Maurice, C.F. (2017) Ménage à trois in the human gut: interactions between host, bacteria and phages. Nature Reviews Microbiology, 15, 397-408.

Münch, P.C., Franzosa, E.A., Stecher, B., McHardy, A.C. & Huttenhower, C. (2021) Identification of natural CRISPR systems and targets in the human microbiome. Cell Host & Microbe, 29, 94-106.e4.

Nayfach, S., Camargo, A.P., Schulz, F., Eloe-Fadrosh, E., Roux, S. & Kyrpides, N.C. (2021) CheckV assesses the quality and completeness of metagenome-assembled viral genomes. Nature Biotechnology, 39, 578-585.

Nepal, R., Houtak, G., Wormald, P.-J., Psaltis, A.J. & Vreugde, S. (2022) Prophage: a crucial catalyst in infectious disease modulation. The Lancet Microbe., 3, e162-e163.

Nishimura, Y., Yoshida, T., Kuronishi, M., Uehara, H., Ogata, H. & Goto, S. (2017) ViPTree: the viral proteomic tree server. Bioinformatics, 33, 2379-2380.

Nobrega, F.L., Walinga, H., Dutilh, B.E. & Brouns, S.J.J. (2020) Prophages are associated with extensive CRISPR-Cas auto-immunity. Nucleic Acids Research, 48, 12074-12084.

Nweze, J.E., Šustr, V., Brune, A. & Angel, R. (2024) Functional similarity despite taxonomical divergence in the millipede gut microbiota points to a common trophic strategy. Microbiome, 12, 16.

Parikka, K.J., Le Romancer, M., Wauters, N. & Jacquet, S. (2017) Deciphering the virus-to-prokaryote ratio (VPR): insights into virus-host relationships in a variety of ecosystems. Biological Reviews, 92, 1081-1100.

Pasin, F., Daròs, J.-A. & Tzanetakis, I.E. (2022) Proteome expansion in the Potyviridae evolutionary radiation. FEMS Microbiology Reviews, 46, fuac011.

Puxty, R.J., Evans, D.J., Millard, A.D. & Scanlan, D.J. (2018) Energy limitation of cyanophage development: implications for marine carbon cycling. The ISME Journal., 12, 1273-1286.

Quistad, S.D., Grasis, J.A., Barr, J.J. & Rohwer, F.L. (2017) Viruses and the origin of microbiome selection and immunity. The ISME Journal., 11, 835-840.

Riechmann, J.L., Laín, S. & García, J.A. (1992) Highlights and prospects of potyvirus molecular biology. Journal of General Virology, 73, 1-16.

Rohwer, F. & Edwards, R. (2002) The phage proteomic tree: a genome-based taxonomy for phage. Journal of Bacteriology, 184, 4529-4535.

Rollie, C., Chevallereau, A., Watson, B.N.J., Chyou, T., Fradet, O., McLeod, I. et al. (2020) Targeting of temperate phages drives loss of type I CRISPR-Cas systems. Nature, 578, 149-153.

Roux, S., Adriaenssens, E.M., Dutilh, B.E., Koonin, E.V., Kropinski, A.M., Krupovic, M. et al. (2019) Minimum information about an uncultivated virus genome (MIUViG). Nature Biotechnology, 37, 29-37.

Roux, S., Camargo, A.P., Coutinho, F.H., Dabdoub, S.M., Dutilh, B.E., Nayfach, S. et al. (2023) iPHoP: an integrated machine learning framework to maximize host prediction for metagenome-derived viruses of archaea and bacteria. PLoS Biology, 21, e3002083.

Roux, S., Krupovic, M., Daly, R.A., Borges, A.L., Nayfach, S., Schulz, F. et al. (2019) Cryptic inoviruses revealed as pervasive in bacteria and archaea across Earth's biomes. Nature Microbiology, 4, 1895-1906.

Russel, J., Pinilla-Redondo, R., Mayo-Muñoz, D., Shah, S.A. & Sørensen, S.J. (2020) CRISPRCasTyper: automated identification, annotation, and classification of CRISPR-Cas loci. The CRISPR Journal., 3, 462-469.

Sakaguchi, S., Urayama, S., Takaki, Y., Hirosuna, K., Wu, H., Suzuki, Y. et al. (2022) NeoRdRp: a comprehensive dataset for identifying RNA-dependent RNA polymerases of various RNA viruses from metatranscriptomic data. Microbes and Environments, 37, ME22001.

Sardar, P., Šustr, V., Chroňáková, A. & Lorenc, F. (2022) Metatranscriptomic holobiont analysis of carbohydrate-active enzymes in the millipede Telodeinopus aoutii (Diplopoda, Spirostreptida). Frontiers in Ecology and Evolution, 10, 10.

Sardar, P., Šustr, V., Chroňáková, A., Lorenc, F. & Faktorová, L. (2022) De novo metatranscriptomic exploration of gene function in the millipede holobiont. Scientific Reports, 12, 16173.

Schliep, K.P. (2011) phangorn: phylogenetic analysis in R. Bioinformatics, 27, 592-593.

Shang, J., Tang, X. & Sun, Y. (2023) PhaTYP: predicting the lifestyle for bacteriophages using BERT. Briefings in Bioinformatics, 24, bbac487.

Shi, M., Lin, X.D., Tian, J.H., Chen, L.J., Chen, X., Li, C.X. et al. (2016) Redefining the invertebrate RNA virosphere. Nature, 540, 539-543.

Shkoporov, A.N. & Hill, C. (2019) Bacteriophages of the human gut: the “known unknown” of the microbiome. Cell Host & Microbe, 25, 195-209.

Silveira, C. & Rohwer, F. (2016) Piggyback-the-winner in host-associated microbial communities. npj Biofilms and Microbiomes, 2, 16010. Available from: https://doi.org/10.1038/npjbiofilms.2016.10

Simmonds, P., Adriaenssens, E.M., Zerbini, F.M., Abrescia, N.G., Aiewsakun, P., Alfenas-Zerbini, P. et al. (2023) Four principles to establish a universal virus taxonomy. PLoS Biology, 21, e3001922.

Stamatakis, A. (2014) RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics, 30, 1312-1313.

Steinegger, M. & Söding, J. (2017) MMseqs2 enables sensitive protein sequence searching for the analysis of massive data sets. Nature Biotechnology, 35, 1026-1028.

Taylor, E.C. (1982) Role of aerobic microbial populations in cellulose digestion by desert millipedes. Applied and Environmental Microbiology, 11, 281-291.

Thingstad, T.F. (2000) Elements of a theory for the mechanisms controlling abundance, diversity, and biogeochemical role of lytic bacterial viruses in aquatic systems. Limnology and Oceanography, 45, 1320-1328.

Tikhe, C.V. & Husseneder, C. (2018) Metavirome sequencing of the termite gut reveals the presence of an unexplored bacteriophage community. Frontiers in Microbiology, 8, 2548.

Touchon, M., Bernheim, A. & Rocha, E.P. (2016) Genetic and life-history traits associated with the distribution of prophages in bacteria. The ISME Journal, 10, 2744-2754.

Wagner, P.L. & Waldor, M.K. (2002) Bacteriophage control of bacterial virulence. Infection and Immunity, 70, 3985-3993.

Wang, M., Fu, S., Xu, H., Wang, M. & Shi, L. (2018) College of Environment and Planning, Henan University, Kaifeng, Henan 475004. Ecological functions of millipedes in the terrestrial ecosystem. Biodiversity Science, 26, 1051-1059.

Watson, B.N., Steens, J.A., Staals, R.H., Westra, E.R. & van Houte, S. (2021) Coevolution between bacterial CRISPR-Cas systems and their bacteriophages. Cell Host & Microbe, 29, 715-725.

Weissman, J.L., Laljani, R.M.R., Fagan, W.F. & Johnson, P.L.F. (2019) Visualization and prediction of CRISPR incidence in microbial trait-space to identify drivers of antiviral immune strategy. The ISME Journal, 13, 2589-2602.

Wheeler, T.J. & Eddy, S.R. (2013) Nhmmer: DNA homology search with profile HMMs. Bioinformatics, 29, 2487-2489.

Wickham, H. (2016) ggplot2: elegant graphics for data analysis. New York: Springer-Verlag.

Wilkins, D. (2023) gggenes: draw gene arrow maps in ‘ggplot2.’ R package version 0. 5. 1.

Winter, C., Bouvier, T., Weinbauer, M.G. & Thingstad, T.F. (2010) Trade-offs between competition and defense specialists among unicellular planktonic organisms: the “killing the winner” hypothesis revisited. Microbiology and Molecular Biology Reviews, 74, 42-57.

Zhang, J., Gao, Q., Zhang, Q., Wang, T., Yue, H., Wu, L. et al. (2017) Bacteriophage-prokaryote dynamics and interaction within anaerobic digestion processes across time and space. Microbiome., 5, 5.

Zhang, Y.-Z., Shi, M. & Holmes, E.C. (2018) Using metagenomics to characterize an expanding virosphere. Cell, 172, 1168-1172.

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