Skip to main content

High-Throughput Sequencing of the 16S rRNA Gene as a Survey to Analyze the Microbiomes of Free-Living Ciliates Paramecium


Ciliates are the largest group of ubiquitous aquatic bacterivorous protists, and many species are easily cultivated. However, only few studies reported prokaryotic communities naturally associated with ciliate cells. Herein, we analyzed the microbiome composition of several strains of Paramecium (Ciliophora) originating from different locations and belonging to two morpho-species by high-throughput sequencing (HTS) of the 16S rRNA gene. Possible reasons of HTS results bias were addressed comparing DNA libraries obtained using different primers and different number of ciliate cells. Microbiomes associated with ciliates and their environments were always significantly different by prokaryotic taxonomic composition and bacterial richness. There were also pronounced differences between Paramecium strains. Interestingly, potentially pathogenic bacteria were revealed in Paramecium microbiomes.

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5


  1. Azam F, Fenchel T, Field J, Gray JS, Meyer-Reil LA, Thingstad F (1983) The ecological role of water-column microbes in the sea. Mar. Ecol. Prog. Ser. 10:257–263.

    Article  Google Scholar 

  2. Pernthaler J (2005) Predation on prokaryotes in the water column and its ecological implications. Nat. Rev. Microbiol. 3:537–546.

    Article  PubMed  Google Scholar 

  3. Hahn MW, Höfle MG (2001) Grazing of protozoa and its effect on populations of aquatic bacteria. FEMS Microbiol. Ecol. 35:113–121.

    Article  CAS  PubMed  Google Scholar 

  4. Jürgens K, Matz C (2002) Predation as a shaping force for the phenotypic and genotypic composition of planktonic bacteria. Antonie Van Leeuwenhoek 81:413:434–413:434.

  5. Jacquet S, Domaizon I, Chardon C, Personnic S (2013) Are small grazers and/or viruses a structuring factor of the free-living bacterial community in Lake Geneva? Adv Microbiol. 3:233–248.

    Article  Google Scholar 

  6. Batani G, Pérez G, Martínez G, Piccini C, Fazi S (2016) Competition and protist predation are important regulators of riverine bacterial community composition and size distribution. J. Freshw. Ecol. 31:609–623.

    Article  CAS  Google Scholar 

  7. Pierce RW, Turner JT (1992) Ecology of planktonic ciliates in marine food webs. Rev. Aquat. Sci. 6:139–181

    Google Scholar 

  8. Beaver JR, Crisman TL (1989) The role of ciliated protozoa in pelagic freshwater ecosystems. Microb. Ecol. 17:111–136.

    Article  CAS  PubMed  Google Scholar 

  9. Serra V, Fokin SI, Castelli M, Basuri C, Nitla V, Verni F, Sandeep B, Kalavati C, Petroni G (2016) “Candidatus Gortzia shahrazadis”, a novel endosymbiont of Paramecium multimicronucleatum and a revision of the biogeographical distribution of Holospora-like bacteria. Front. Microbiol.

  10. Schrallhammer M, Castelli M, Petroni G (2018) Phylogenetic relationships among endosymbiotic R-body producer: Bacteria providing their host the killer trait. Syst. Appl. Microbiol. 41:213–220.

    Article  PubMed  Google Scholar 

  11. Gong J, Qing Y, Guo X, Warren A (2014) “Candidatus Sonnebornia yantaiensis”, a member of candidate division OD1, as intracellular bacteria of the ciliated protist Paramecium bursaria (Ciliophora, Oligohymenophorea). Syst. Appl. Microbiol. 37:35–41.

    Article  CAS  PubMed  Google Scholar 

  12. Schrallhammer M, Ferrantini F, Vannini C, Galati S, Schweikert M, Görtz HD, Verni F, Petroni G (2013) “Candidatus Megaira polyxenophila” gen. nov., sp. nov.: considerations on evolutionary history, host range and shift of early divergent Rickettsiae. PLoS One 8:e72581.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Watanabe K, Nakao R, Fujishima M, Tachibana M, Shimizu T, Watarai M (2016) Ciliate Paramecium is a natural reservoir of Legionella pneumophila. Sci Rep 6, 24322.

  14. Peterson TS, Ferguson JA, Watral VG, Mutoji KN, Ennis DG, Kent ML (2013) Paramecium caudatum enhances transmission and infectivity of Mycobacterium marinum and M. chelonae in zebrafish Danio rerio. Dis. Aquat. Org. 106:229–239.

    Article  Google Scholar 

  15. Pushkareva VI, Ermolaeva SA (2010) Listeria monocytogenes virulence factor Listeriolysin O favors bacterial growth in co-culture with the ciliate Tetrahymena pyriformis, causes protozoan encystment and promotes bacterial survival inside cysts. BMC Microbiol 10:26.

  16. King CH, Shotts EB, Wooley RE, Porter KG (1988) Survival of coliforms and bacterial pathogens within protozoa during chlorination. Appl. Environ. Microbiol. 54:3023–3033

    CAS  PubMed  PubMed Central  Google Scholar 

  17. Erken M, Lutz C, McDougald D (2013) The rise of pathogens: predation as a factor driving the evolution of human pathogens in the environment. Microb. Ecol. 65:860–868.

    Article  PubMed  PubMed Central  Google Scholar 

  18. Sun S, Noorian P, McDougald D (2018) Dual role of mechanisms involved in resistance to predation by protozoa and virulence to humans. Front. Microbiol. 9:1017.

    Article  PubMed  PubMed Central  Google Scholar 

  19. Konstantinidis KT, Rosselló-Móra R, Amann R (2017) Uncultivated microbes in need of their own taxonomy. ISME J. 11:2399–2406.

  20. Parte AC (2014) LPSN – list of prokaryotic names with standing in nomenclature. Nucleic Acids Res. 42:613–616.

    Article  CAS  Google Scholar 

  21. Kircher M, Kelso J (2010) High-throughput DNA sequencing—concepts and limitations. Bioessays 32:524–536.

    Article  CAS  PubMed  Google Scholar 

  22. Escobar-Zepeda A, De León AVP, Sanchez-Flores A (2015) The road to metagenomics: from microbiology to DNA sequencing technologies and bioinformatics. Front Genet 6:348.

  23. Irbis C, Ushida K (2004) Detection of methanogens and proteobacteria from a single cell of rumen ciliate protozoa. J. Gen. Appl. Microbiol. 50:203–212.

    Article  CAS  PubMed  Google Scholar 

  24. Pucciarelli S, Devaraj RR, Mancini A, Ballarini P, Castelli M, Schrallhammer M, Petroni G, Miceli C (2015) Microbial consortium associated with the Antarctic marine ciliate Euplotes focardii: an investigation from genomic sequences. Microb. Ecol. 70:484–497.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Gong J, Qing Y, Zou S, Fu R, Su L, Zhang X, Zhang Q (2016) Protist-bacteria associations: Gammaproteobacteria and Alphaproteobacteria are prevalent as digestion-resistant bacteria in ciliated protozoa. Front. Microbiol. 7:498.

    Article  PubMed  PubMed Central  Google Scholar 

  26. Omar A, Zhang Q, Zou S, Gong J (2017) Morphology and phylogeny of the soil ciliate Metopus yantaiensis n. sp. (Ciliophora, Metopida), with identification of the intracellular bacteria. J. Eukaryot. Microbiol. 64:792–805.

    Article  CAS  PubMed  Google Scholar 

  27. Park T, Yu Z (2018) Do ruminal ciliates select their preys and prokaryotic symbionts? Front. Microbiol. 9:1710.

    Article  PubMed  PubMed Central  Google Scholar 

  28. Rossi A, Bellone A, Fokin SI, Boscaro V, Vannini C (2018) Detecting associations between ciliated Protists and prokaryotes with culture-independent single-cell microbiomics: a proof-of-concept study. Microb. Ecol.

  29. Shapiro E, Biezuner T, Linnarsson S (2013) Single-cell sequencing-based technologies will revolutionize whole-organism science. Nat. Rev. Genet. 14:618–630.

    Article  CAS  PubMed  Google Scholar 

  30. Yoon HS, Price DC, Stepanauskas R, Rajah VD, Sieracki ME, Wilson WH, Yang EC, Duffy S, Bhattacharya D (2011) Single-cell genomics reveals organismal interactions in uncultivated marine protists. Science 332:714–717.

    Article  CAS  PubMed  Google Scholar 

  31. Martinez-Garcia M, Brazel D, Poulton NJ, Swan BK, Gomez ML, Masland D, Sieracki ME, Stepanauskas R (2012) Unveiling in situ interactions between marine protists and bacteria through single cell sequencing. ISME J 6:703–707.

    Article  CAS  PubMed  Google Scholar 

  32. Hugerth LW, Andersson AF (2017) Analysing microbial community composition through amplicon sequencing: from sampling to hypothesis testing. Front. Microbiol. 8:1561.

    Article  PubMed  PubMed Central  Google Scholar 

  33. Kodama Y, Fujishima M (2016) Paramecium as a model organism for studies on primary and secondary endosymbioses. Biocommunication of Ciliates: 277–304.

  34. Klindworth A, Pruesse E, Schweer T, Jörg Peplies, Quast C, Horn M, Glöckner F (2013) Evaluation of general 16S ribosomal RNA gene PCR primers for classical and next-generation sequencing-based diversity studies. Nucleic Acids Res 41:e1.

  35. Zhou J, Wu L, Deng Y, Zhi X, Jiang YH, Tu Q, Xie J, Van Nostrand JD, He Z, Yang Y (2011) Reproducibility and quantitation of amplicon sequencing-based detection. ISME J 5:1303–1313.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Wen C, Wu L, Qin Y, Van Nostrand JD, Ning D, Sun B, Xue K, Liu F, Deng Y, Liang Y, Zhou J (2017) Evaluation of the reproducibility of amplicon sequencing with Illumina MiSeq platform. PLoS One 12:e0176716.

  37. Yang B, Wang Y, Qian P-Y (2016) Sensitivity and correlation of hypervariable regions in 16S rRNA genes in phylogenetic analysis. BMC Bioinformatics 17:135.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Almeida A, Mitchell AL, Tarkowska A, Finn RD (2018) Benchmarking taxonomic assignments based on 16S rRNA gene profiling of the microbiota from commonly sampled environments. Gigascience 7:giy054.

  39. Zhang J, Kobert K, Flouri T, Stamatakis A (2014) PEAR: a fast and accurate Illumina paired-end reAd mergeR. Bioinformatics 30:614–620.

    Article  CAS  PubMed  Google Scholar 

  40. Edgar RC (2013) UPARSE: highly accurate OTU sequences from microbial amplicon reads. Nat. Methods 10:996–998.

    Article  CAS  PubMed  Google Scholar 

  41. Konstantinidis KT, Tiedje JM (2005) Genomic insights that advance the species definition for prokaryotes. Proc. Natl. Acad. Sci. 102:2567–2572.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Kim M, Oh H-S, Park S-C, Chun J (2014) Towards a taxonomic coherence between average nucleotide identity and 16S rRNA gene sequence similarity for species demarcation of prokaryotes. Int. J. Syst. Evol. Microbiol. 64:346–351.

    Article  PubMed  Google Scholar 

  43. Edgar RC, Haas BJ, Clemente JC, Quince C, Knight R (2011) UCHIME improves sensitivity and speed of chimera detection. Bioinformatics 27:2194–2200.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Cole JR, Wang Q, Fish JA, Chai B, McGarrell DM, Sun Y, Brown CT, Porras-Alfaro A, Kuske CR, Tiedje JM (2014) Ribosomal Database Project: data and tools for high throughput rRNA analysis. Nucleic Acids Res 42:D633–D642.

  45. Salter SJ, Cox MJ, Turek EM, Calus ST, Cookson WO, Moffatt MF, Turner P, Parkhill J, Loman NJ, Walker AW (2014) Reagent and laboratory contamination can critically impact sequence-based microbiome analyses. BMC Biol 12:87.

  46. Delafont V, Bouchon D, Héchard Y, Moulin L (2016) Environmental factors shaping cultured free-living amoebae and their associated bacterial community within drinking water network. Water Res. 100:382–392.

    Article  CAS  PubMed  Google Scholar 

  47. Rajendhran J, Gunasekaran P (2011) Microbial phylogeny and diversity: small subunit ribosomal RNA sequence analysis and beyond. Microbiol. Res. 166:99–110.

    Article  CAS  PubMed  Google Scholar 

  48. Newton RJ, Jones SE, Eiler A, McMahon KD, Bertilsson S (2011) A guide to the natural history of freshwater lake bacteria. Microbiol. Mol. Biol. Rev. 75:14–49.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Castelli M, Sassera D, Petroni G (2016) Biodiversity of “non-model” Rickettsiales and their association with aquatic organisms. Rickettsiales: Biology, Molecular biology, Epidemiology, and Vaccine development: 59–91, Springer.

  50. Selivanova EA, Poshvina DV, Khlopko YA, Gogoleva NE, Plotnikov AO (2018) Diversity of prokaryotes in planktonic communities of saline Sol-Iletsk lakes (Orenburg oblast, Russia). Microbiology 87:569–582.

    Article  CAS  Google Scholar 

  51. García-Bayona L, Comstock LE (2018) Bacterial antagonism in host-associated microbial communities. Science 361:eaat2456.

    Article  CAS  PubMed  Google Scholar 

  52. Thomas V, McDonnell G (2007) Relationship between mycobacteria and amoebae: ecological and epidemiological concerns. Lett. Appl. Microbiol. 45:349–357.

    Article  CAS  PubMed  Google Scholar 

  53. Delafont V, Mougari F, Cambau E, Joyeux M, Bouchon D, Héchard Y, Moulin L (2014) First evidence of amoebae-mycobacteria association in drinking water network. Environ Sci Technol 48:11872–11882.

    Article  CAS  PubMed  Google Scholar 

  54. Denoncourt AM, Paquet VE, Charette SJ (2017) Packaging of Mycobacterium smegmatis bacteria into fecal pellets by the ciliate Tetrahymena pyriformis. FEMS Microbiol Lett 364:fnx237.

  55. Barker J, Brown MRW (1994) Trojan horses of the microbial world: Protozoa and the survival of bacterial pathogens in the environment. Microbiology 140:1253–1259.

    Article  CAS  PubMed  Google Scholar 

Download references


This study was funded by the Russian Science Foundation (grant number 16-14-10157), except the pilot analysis of the P. aurelia samples, which was supported by the Russian Foundation for Basic Research (grant number 14-04-01796).

Author information

Authors and Affiliations


Corresponding author

Correspondence to Andrey O. Plotnikov.

Ethics declarations

Conflict of Interest

The authors declare that they have no conflict of interest.

Electronic Supplementary Material


(XLSX 98 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Plotnikov, A.O., Balkin, A.S., Gogoleva, N.E. et al. High-Throughput Sequencing of the 16S rRNA Gene as a Survey to Analyze the Microbiomes of Free-Living Ciliates Paramecium. Microb Ecol 78, 286–298 (2019).

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI:


  • Ciliates
  • Microbiomes
  • Bacterial communities
  • Single cell sequencing
  • Metabarcoding
  • Human pathogens and commensals