Microbial Ecology

, Volume 76, Issue 2, pp 467–481 | Cite as

The Microbial Community of Tardigrades: Environmental Influence and Species Specificity of Microbiome Structure and Composition

  • Matteo Vecchi
  • Irene L.G. Newton
  • Michele Cesari
  • Lorena Rebecchi
  • Roberto Guidetti
Invertebrate Microbiology


Symbiotic associations of metazoans with bacteria strongly influence animal biology since bacteria are ubiquitous and virtually no animal is completely free from them. Tardigrades are micrometazoans famous for their ability to undergo ametabolic states (cryptobiosis) but very little information is available on potential microbial associations. We characterized the microbiomes of six limnoterrestrial tardigrade species belonging to several phylogenetic lines in tandem with the microbiomes of their respective substrates. The experimental design enabled us to determine the effects of both the environment and the host genetic background on the tardigrade microbiome; we were able to define the microbial community of the same species sampled from different environments, and the communities of different species from the same environment. Our 16S rRNA gene amplicon approach indicated that the tardigrade microbiome is species-specific and well differentiated from the environment. Tardigrade species showed a much lower microbial diversity compared to their substrates, with only one significant exception. Forty-nine common OTUs (operational taxonomic units) were classified into six bacterial phyla, while four common OTUs were unclassified and probably represent novel bacterial taxa. Specifically, the tardigrade microbiome appears dominated by Proteobacteria and Bacteroidetes. Some OTUs were shared between different species from geographically distant samples, suggesting the associated bacteria may be widespread. Putative endosymbionts of tardigrades from the order Rickettsiales were identified. Our results indicated that like all other animals, tardigrades have their own microbiota that is different among species, and its assembly is determined by host genotype and environmental influences.


Endosymbiont Microbiome Rickettsiales Symbiosis Tardigrada 



We thank Kristian Hassel (Norwegian University of Science and Technology, Trondheim, Norway) and Renzo Rabacchi (Museo Civico di Ecologia e Storia Naturale di Marano sul Panaro, Modena, Italy) for the taxonomic determination of mosses in sample S7 and lichens in samples S4 and S5, respectively. We thank K. Ingemar Jönsson (Kristianstad University, Kristianstad, Sweden) for kindly providing sample S6 and Mauro Mandrioli (University of Modena and Reggio Emilia, Modena, Italy) for providing some laboratory reagents. We additionally wish to thank Kathy B. Sheehan and MaryAnn Martin (Indiana University, USA) for their support during laboratory work. Finally, we thank the anonymous reviewers and the reviewer Diane Nelson for the constructive suggestions in order to improve the manuscript.

Authors’ Contributions

This work is a part of the Ph.D. thesis of M.V. M.V. and R.G. designed and conceived the experiments. M.V. and M.C. performed the laboratory work. M.V. and I.G.N. analyzed the data. I.G.N., L.R., and R.G. provided reagents, instruments, and funds. M.V. wrote the first draft of the manuscript. M.V., I.G.N., M.C., L.R., and R.G. participated in revising the manuscript.

Funding Information

This research was supported by the Italian “Programma Nazionale Ricerche in Antartide (PNRA)–Ministero dell’Istruzione dell’Università e della Ricerca (MIUR)” as part of the project “Evolutive and phylogeographic history of Antarctic organisms and responses by ecosystems to climatic and environmental changings” (PdR 2013 B1/01) and by the “Bando per il finanziamento di azioni di mobilità nell’ambito del Programma di collaborazione scientifica e culturale dell'Università degli Studi di Modena e Reggio Emilia con Università straniere convenzionate-2016” (University of Modena and Reggio Emilia, Modena, Italy).

Compliance with Ethical Standards

Conflict of Interest

The authors declare that they have no conflict of interest.

Supplementary material

248_2017_1134_MOESM1_ESM.docx (2 mb)
ESM 1 (DOCX 2074 kb)
248_2017_1134_MOESM2_ESM.xlsx (88 kb)
ESM 2 (XLSX 88 kb)


  1. 1.
    Guidetti R, Altiero T, Rebecchi L (2011) On dormancy strategies in tardigrades. J Insect Physiol 57(5):567–576CrossRefPubMedGoogle Scholar
  2. 2.
    Nelson DR, Guidetti R, Rebecchi L (2015) Phylum Tardigrada. 17. Ecology and general biology: Thorp and Covich’s freshwater invertebrates4th edn. Academic Press (Elsevier Inc.), Amsterdam, pp 347–380CrossRefGoogle Scholar
  3. 3.
    Goldstein B, King N (2016) The future of cell biology: emerging model organisms. Trends Cell Biol 26(11):818–824CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Horikawa DD, Kunieda T, Abe W et al (2008) Establishment of a rearing system of the extremotolerant tardigrade Ramazzottius varieornatus: a new model animal for astrobiology. Astrobiology 8(3):549–556CrossRefPubMedGoogle Scholar
  5. 5.
    Jönsson KI, Rabbow E, Schill RO et al (2008) Tardigrades survive exposure to space in low Earth orbit. Curr Biol 18(17):R729–R731CrossRefPubMedGoogle Scholar
  6. 6.
    Rebecchi L, Altiero T, Guidetti R et al (2009) Tardigrade resistance to space effects: first results of experiments on the LIFE-TARSE mission on FOTON-M3 (September 2007). Astrobiology 9(6):581–591CrossRefPubMedGoogle Scholar
  7. 7.
    Rebecchi L, Altiero T, Cesari M et al (2011) Resistance of the anhydrobiotic eutardigrade Paramacrobiotus richtersi to space flight (LIFE–TARSE mission on FOTON-M3). J Zool Syst Evol Res 49(s1):98–103CrossRefGoogle Scholar
  8. 8.
    Persson D, Halberg KA, Jørgensen A et al (2011) Extreme stress tolerance in tardigrades: surviving space conditions in low earth orbit. J Zool Syst Evol Res 49(s1):90–97CrossRefGoogle Scholar
  9. 9.
    Guidetti R, Rizzo AM, Altiero T, Rebecchi L (2012) What can we learn from the toughest animals of the Earth? Water bears (tardigrades) as multicellular model organisms in order to perform scientific preparations for lunar exploration. Planet Space Sci 74(1):97–102CrossRefGoogle Scholar
  10. 10.
    Rebecchi L (2013) Dry up and survive: the role of antioxidant defences in anhydrobiotic organisms. J Limnol 72(1s):8Google Scholar
  11. 11.
    Wang C, Grohme MA, Mali B et al (2014) Towards decrypting cryptobiosis—analyzing anhydrobiosis in the tardigrade Milnesium tardigradum using transcriptome sequencing. PLoS One 9(3):e92663CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Kondo K, Kubo T, Kunieda T (2015) Suggested involvement of PP1/PP2A activity and de novo gene expression in anhydrobiotic survival in a tardigrade, Hypsibius dujardini, by chemical genetic approach. PLoS One 10(12):e0144803CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Boothby TC, Tapia H, Brozena AH et al (2017) Tardigrades use intrinsically disordered proteins to survive desiccation. Mol Cell 65(6):975–984CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Garey JR, Schmidt-Rhaesa A (1998) The essential role of “minor” phyla in molecular studies of animal evolution. Am Zool 38(6):907–917CrossRefGoogle Scholar
  15. 15.
    Gabriel WN, McNuff R, Patel SK et al (2007) The tardigrade Hypsibius dujardini, a new model for studying the evolution of development. Dev Biol 312(2):545–559CrossRefPubMedGoogle Scholar
  16. 16.
    Edgecombe GD, Legg DA (2014) Origins and early evolution of arthropods. Paléo 57(3):457–468Google Scholar
  17. 17.
    Levin M, Anavy L, Cole AG et al (2016) The mid-developmental transition and the evolution of animal body plans. Nature 531(7596):637–641CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Smith FW, Boothby TC, Giovannini I et al (2016) The compact body plan of tardigrades evolved by the loss of a large body region. Curr Biol 26(2):224–229CrossRefPubMedGoogle Scholar
  19. 19.
    Boothby TC, Tenlen JR, Smith FW et al (2015) Evidence for extensive horizontal gene transfer from the draft genome of a tardigrade. Proc Natl Acad Sci 112(52):15976–15981CrossRefPubMedGoogle Scholar
  20. 20.
    Arakawa K, Yuki Y, Masaru T (2016) Genome sequencing of a single tardigrade Hypsibius dujardini individual. Sci Data 3:160063CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Bemm F, Weiß CL, Schultz J, Förster F (2016) Genome of a tardigrade: horizontal gene transfer or bacterial contamination? Proc Natl Acad Sci 113(22):E3054–E3056CrossRefPubMedGoogle Scholar
  22. 22.
    Boothby TC, Goldstein B (2016) Reply to Bemm et al. and Arakawa: identifying foreign genes in independent Hypsibius dujardini genome assemblies. Proc Natl Acad Sci 113(22):E3058–E3061CrossRefPubMedGoogle Scholar
  23. 23.
    Delmont TO, Eren AM (2016) Identifying contamination with advanced visualization and analysis practices: metagenomic approaches for eukaryotic genome assemblies. PeerJ 4:e1839CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Koutsovoulos G, Kumar S, Laetsch DR et al (2016) No evidence for extensive horizontal gene transfer in the genome of the tardigrade Hypsibius dujardini. Proc Natl Acad Sci 113(18):5053–5058CrossRefPubMedGoogle Scholar
  25. 25.
    Nicholson JK, Holmes E, Kinross J et al (2012) Host-gut microbiota metabolic interactions. Science 336(6086):1262–1267CrossRefPubMedGoogle Scholar
  26. 26.
    Kamada N, Seo SU, Chen GY, Núñez G (2013) Role of the gut microbiota in immunity and inflammatory disease. Nat Rev Immunol 13(5):321–335CrossRefPubMedGoogle Scholar
  27. 27.
    Ezenwa VO, Gerardo NM, Inouye DW et al (2012) Animal behavior and the microbiome. Science 338(6104):198–199CrossRefPubMedGoogle Scholar
  28. 28.
    Brucker RM, Bordenstein SR (2013) The hologenomic basis of speciation: gut bacteria cause hybrid lethality in the genus Nasonia. Science 341(6146):667–669CrossRefPubMedGoogle Scholar
  29. 29.
    Jasmin C, Anas A, Nair S (2015) Bacterial diversity associated with Cinachyra cavernosa and Haliclona pigmentifera, cohabiting sponges in the coral reef ecosystem of Gulf of Mannar, Southeast coast of India. PLoS One 10(5):e0123222CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Roder C, Bayer T, Aranda M et al (2015) Microbiome structure of the fungid coral Ctenactis echinata aligns with environmental differences. Mol Ecol 24(13):3501–3511CrossRefPubMedPubMedCentralGoogle Scholar
  31. 31.
    Lanan MC, Rodrigues PAP, Agellon A et al (2016) A bacterial filter protects and structures the gut microbiome of an insect. ISME J 10:1866–1876CrossRefPubMedPubMedCentralGoogle Scholar
  32. 32.
    Gerdts G, Brandt P, Kreisel K et al (2013) The microbiome of North Sea copepods. Helgoland Mar Res 67(4):757CrossRefGoogle Scholar
  33. 33.
    Derycke S, De Meester N, Rigaux A et al (2016) Coexisting cryptic species of the Litoditis marina complex (Nematoda) show differential resource use and have distinct microbiomes with high intraspecific variability. Mol Ecol 25(9):2093–2110CrossRefPubMedGoogle Scholar
  34. 34.
    Schmidt VT, Smith KF, Melvin DW, Amaral-Zettler LA (2015) Community assembly of a euryhaline fish microbiome during salinity acclimation. Mol Ecol 24(10):2537–2550CrossRefPubMedGoogle Scholar
  35. 35.
    Vecchi M, Vicente F, Guidetti R et al (2016) Interspecific relationships of tardigrades with bacteria, fungi and protozoans, with a focus on the phylogenetic position of Pyxidium tardigradum (Ciliophora). Zool J Linn Soc-Lond 178(4):846–855CrossRefGoogle Scholar
  36. 36.
    Krantz SL, Benoit TG, Beasley CW (1999) Phytopathogenic bacteria associated with Tardigrada. Zool Anz 238(3–4):259–260Google Scholar
  37. 37.
    Bright M, Bulgheresi S (2010) A complex journey: transmission of microbial symbionts. Nat Rev Microbiol 8(3):218–230CrossRefPubMedPubMedCentralGoogle Scholar
  38. 38.
    Weinert LA, Araujo-Jnr EV, Ahmed MZ, Welch JJ (2015) The incidence of bacterial endosymbionts in terrestrial arthropods. Proc R Soc Lond B Biol 282:20150249CrossRefGoogle Scholar
  39. 39.
    Baquiran JP, Thater B, Sedky S et al (2013) Culture-independent investigation of the microbiome associated with the nematode Acrobeloides maximus. PLoS One 8(7):e67425CrossRefPubMedPubMedCentralGoogle Scholar
  40. 40.
    Wilson CG (2011) Desiccation-tolerance in bdelloid rotifers facilitates spatio-temporal escape from multiple species of parasitic fungi. Biol J Linn Soc 104(3):564–574CrossRefGoogle Scholar
  41. 41.
    Guil N, Giribet G (2009) Fine scale population structure in the Echiniscus blumi-canadensis series (Heterotardigrada, Tardigrada) in an Iberian mountain range—when morphology fails to explain genetic structure. Mol Phylogenet Evol 51(3):606–613CrossRefPubMedGoogle Scholar
  42. 42.
    Truett GE, Heeger P, Mynatt RL et al (2000) Preparation of PCR-quality mouse genomic DNA with hot sodium hydroxide and tris (HotSHOT). BioTechniques 29(1):52–54PubMedCrossRefGoogle Scholar
  43. 43.
    Jørgensen A, Møbjerg N, Kristensen RM (2007) A molecular study of the tardigrade Echiniscus testudo (Echiniscidae) reveals low DNA sequence diversity over a large geographical area. J Limnol 66(1s):77–83CrossRefGoogle Scholar
  44. 44.
    Altiero T, Giovannini I, Guidetti R, Rebecchi L (2015) Life history traits and reproductive mode of the tardigrade Acutuncus antarcticus under laboratory conditions: strategies to colonize the Antarctic environment. Hydrobiologia 761(1):277–291CrossRefGoogle Scholar
  45. 45.
    Cesari M, McInnes SJ, Bertolani R et al (2016) Genetic diversity and biogeography of the south polar water bear Acutuncus antarcticus (Eutardigrada: Hypsibiidae)—evidence that it is a truly pan-Antarctic species. Invertebr Syst 30(6):635–649Google Scholar
  46. 46.
    Guidetti R, Rebecchi L, Bertolani R et al (2016) Morphological and molecular analyses on Richtersius (Eutardigrada) diversity reveal its new systematic position and lead to the establishment of a new genus and a new family within Macrobiotoidea. Zool J Linn Soc-Lond 178(4):834–845CrossRefGoogle Scholar
  47. 47.
    Rebecchi L, Rossi V, Altiero T et al (2003) Reproductive modes and genetic polymorphism in the tardigrade Richtersius coronifer (Eutardigrada, Macrobiotidae). Invertebr Biol 122(1):19–27CrossRefGoogle Scholar
  48. 48.
    Cesari M, Giovannini I, Bertolani R, Rebecchi L (2011) An example of problems associated with DNA barcoding in tardigrades: a novel method for obtaining voucher specimens. Zootaxa 3104(1):42–51Google Scholar
  49. 49.
    Kristensen RM (1987) Generic revision of the Echiniscidae (Heterotardigrada), with a discussion of the origin of the family. In Biology of tardigrades. Selected symposia and monographs UZI, Vol. 1, no. 261, p C335. Mucchi Modena, ItalyGoogle Scholar
  50. 50.
    Caporaso JG, Lauber CL, Walters WA et al (2012) Ultra-high-throughput microbial community analysis on the Illumina HiSeq and MiSeq platforms. ISME J 6(8):1621–1624CrossRefPubMedPubMedCentralGoogle Scholar
  51. 51.
    Schloss PD, Westcott SL, Ryabin T et al (2009) Introducing mothur: open-source, platform-independent, community-supported software for describing and comparing microbial communities. Appl Environ Microbiol 75(23):7537–7541CrossRefPubMedPubMedCentralGoogle Scholar
  52. 52.
    Pruesse E, Quast C, Knittel K et al (2007) SILVA: a comprehensive online resource for quality checked and aligned ribosomal RNA sequence data compatible with ARB. Nucleic Acids Res 35(21):7188–7196CrossRefPubMedPubMedCentralGoogle Scholar
  53. 53.
    Price MN, Dehal PS, Arkin AP (2009) FastTree: computing large minimum evolution trees with profiles instead of a distance matrix. Mol Biol Evol 26(7):1641–1650CrossRefPubMedPubMedCentralGoogle Scholar
  54. 54.
    Miller MA, Pfeiffer W, Schwartz T (2010) Creating the CIPRES Science Gateway for inference of large phylogenetic trees. In Gateway computing environments workshop (GCE), New Orleans, pp 1–8Google Scholar
  55. 55.
    McMurdie PJ, Holmes S (2013) phyloseq: an R package for reproducible interactive analysis and graphics of microbiome census data. PLoS One 8(4):e61217CrossRefPubMedPubMedCentralGoogle Scholar
  56. 56.
    Oksanen J, Kindt R, Legendre P et al (2009) vegan: community ecology package. R package version 1.17–9. Available at:
  57. 57.
    Revell LJ (2012) phytools: an R package for phylogenetic comparative biology (and other things). Methods Ecol Evol 3(2):217–223CrossRefGoogle Scholar
  58. 58.
    White JR, Nagarajan N, Pop M (2009) Statistical methods for detecting differentially abundant features in clinical metagenomic samples. PLoS Comput Biol 5(4):e1000352CrossRefPubMedPubMedCentralGoogle Scholar
  59. 59.
    Knights D, Kuczynski J, Charlson ES et al (2011) Bayesian community-wide culture-independent microbial source tracking. Nat Methods 8(9):761–763CrossRefPubMedPubMedCentralGoogle Scholar
  60. 60.
    Münkemüller T, Lavergne S, Bzeznik B et al (2012) How to measure and test phylogenetic signal. Methods Ecol Evol 3(4):743–756CrossRefGoogle Scholar
  61. 61.
    Blomberg SP, Garland T, Ives AR (2003) Testing for phylogenetic signal in comparative data: behavioral traits are more labile. Evolution 57(4):717–745CrossRefPubMedGoogle Scholar
  62. 62.
    Katoh K, Misawa K, Kuma KI, Miyata T (2002) MAFFT: a novel method for rapid multiple sequence alignment based on fast Fourier transform. Nucleic Acids Res. 30(14):3059–3066CrossRefPubMedPubMedCentralGoogle Scholar
  63. 63.
    Bertolani R, Guidetti R, Marchioro T et al (2014) Phylogeny of Eutardigrada: new molecular data and their morphological support lead to the identification of new evolutionary lineages. Mol Phylogenet Evol 76:110–126CrossRefPubMedGoogle Scholar
  64. 64.
    Nawrocki E (2009) Structural RNA homology search and alignment using covariance models. Washington University, St. LouisGoogle Scholar
  65. 65.
    Ronquist F, Huelsenbeck JP (2003) MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19(12):1572–1574CrossRefPubMedGoogle Scholar
  66. 66.
    Darriba D, Taboada GL, Doallo R, Posada D (2012) jModelTest 2: more models, new heuristics and parallel computing. Nat Methods 9(8):772–772CrossRefPubMedPubMedCentralGoogle Scholar
  67. 67.
    Spor A, Koren O, Ley R (2011) Unravelling the effects of the environment and host genotype on the gut microbiome. Nat Rev Microbiol 9(4):279–290CrossRefPubMedGoogle Scholar
  68. 68.
    Dirksen P, Marsh SA, Braker I et al (2016) The native microbiome of the nematode Caenorhabditis elegans: gateway to a new host-microbiome model. BMC Biol 14:38CrossRefPubMedPubMedCentralGoogle Scholar
  69. 69.
    Eichmiller JJ, Hamilton MJ, Staley C et al (2016) Environment shapes the fecal microbiome of invasive carp species. Microbiome 4(1):44CrossRefPubMedPubMedCentralGoogle Scholar
  70. 70.
    Franzenburg S, Walter J, Künzel S et al (2013) Distinct antimicrobial peptide expression determines host species-specific bacterial associations. Proc Natl Acad Sci 110(39):E3730–E3738CrossRefPubMedGoogle Scholar
  71. 71.
    Kinchin IM (1994) The biology of tardigrades. Portland Press, Chapel Hill, p 186Google Scholar
  72. 72.
    Yu XJ, Walker D (2006) The order Rickettsiales. In: Dworkin MS, Falkow S, Rosenberg E et al (eds) The Prokaryotes. Springer, New York, pp 493–528CrossRefGoogle Scholar
  73. 73.
    Guidetti R, Altiero T, Marchioro T et al (2012) Form and function of the feeding apparatus in Eutardigrada (Tardigrada). Zoomorphology 131(2):127–148CrossRefGoogle Scholar
  74. 74.
    Werren JH, Baldo L, Clark ME (2008) Wolbachia: master manipulators of invertebrate biology. Nat Rev Microbiol 6(10):741–751CrossRefPubMedGoogle Scholar
  75. 75.
    Bertolani R (2001) Evolution of the reproductive mechanisms in tardigrades—a review. Zool Anz 240(3):247–252CrossRefGoogle Scholar
  76. 76.
    Darby AC, Cho NH, Fuxelius HH et al (2007) Intracellular pathogens go extreme: genome evolution in the Rickettsiales. Trends Genet 23(10):511–520CrossRefPubMedGoogle Scholar
  77. 77.
    McCutcheon JP, Moran NA (2012) Extreme genome reduction in symbiotic bacteria. Nat Rev Microbiol 10(1):13–26CrossRefGoogle Scholar
  78. 78.
    Nakabachi A, Ishida K, Hongoh Y et al (2014) Aphid gene of bacterial origin encodes a protein transported to an obligate endosymbiont. Curr Biol 24(14):R640–R641CrossRefPubMedGoogle Scholar
  79. 79.
    Fitzpatrick DA, Creevey CJ, McInerney JO (2006) Genome phylogenies indicate a meaningful α-proteobacterial phylogeny and support a grouping of the mitochondria with the Rickettsiales. Mol Biol Evol 23(1):74–85CrossRefPubMedGoogle Scholar
  80. 80.
    Artamonova II, Lappi T, Zudina L, Mushegian AR (2015) Prokaryotic genes in eukaryotic genome sequences: when to infer horizontal gene transfer and when to suspect an actual microbe. Environ Microbiol 17(7):2203–2208CrossRefPubMedGoogle Scholar
  81. 81.
    Chaisiri K, McGarry J, Morand S, Makepeace BL (2015) Symbiosis in an overlooked microcosm: a systematic review of the of the bacterial flora of mites. Parasitology 142:1152–1162CrossRefPubMedGoogle Scholar
  82. 82.
    Hansen AK, Moran NA (2014) The impact of microbial symbionts on host plant utilization by herbivorous insects. Mol Ecol 23(6):1473–1496CrossRefPubMedGoogle Scholar

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Authors and Affiliations

  1. 1.Department of Life SciencesUniversity of Modena and Reggio EmiliaModenaItaly
  2. 2.Department of BiologyIndiana UniversityBloomingtonUSA

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