Folia Microbiologica

, Volume 64, Issue 6, pp 789–796 | Cite as

Molecular detection and identification of Wolbachia endosymbiont in fleas (Insecta: Siphonaptera)

  • Zuhal OnderEmail author
  • Arif Ciloglu
  • Onder Duzlu
  • Alparslan Yildirim
  • Mubeccel Okur
  • Gamze Yetismis
  • Abdullah Inci
Original Article


The aim of this study was to determine the presence and prevalence of Wolbachia bacteria in natural population of fleas (Insecta: Siphonaptera) in Turkey, and to exhibit the molecular characterization and the phylogenetic reconstruction at the positive isolates with other species in GenBank, based on 16S rDNA sequences. One hundred twenty-four flea samples belonging to the species Ctenocephalides canis, C. felis, and Pulex irritans were collected from animal shelters in Kayseri between January and August 2017. All flea species were individually screened for the presence of Wolbachia spp. by polymerase chain reaction (PCR) targeting the 16S ribosomal RNA gene. According to PCR analyses, Wolbachia spp. were found prevalent in C. canis and P. irritans fleas, while it was not detected in the C. felis species. Totally, 20 isolates were purified from agarose gel and sequenced with the same primers for molecular characterization and phylogenetic analyses. The sequence analyses revealed 17 polymorphic sites and 2 genetically different Wolbachia isolates, representing two different haplotypes in two flea species. The distribution patterns, molecular characterization, and phylogenetic status of Wolbachia spp. of fleas in Turkey are presented for the first time with this study. Understanding of the role of Wolbachia in vector biology may provide information for developing Wolbachia-based biological control tools.


Compliance with ethical standards

Conflict of interest

On behalf of all authors, the corresponding author states that there is no conflict of interest.


  1. Azarm A, Dalimi A, Mohebali M, Zarei Z (2016) Morphological and molecular characterization of Ctenocephalides spp. isolated from dogs in north of Iran. J Entomol Zool Stud 4:713–717Google Scholar
  2. Beard CB, Durvasula RV, Richards FF (1998) Bacterial symbiosis in arthropods and the control of disease transmission. Emerg Infect Dis 4:581–591PubMedPubMedCentralGoogle Scholar
  3. Blagburn BL, Dryden MW (2009) Biology, treatment, and control of flea and tick infestations. Vet Clin North Am-Small 39:1173–1200Google Scholar
  4. Casiraghi M, Bordenstein SR, Baldo L, Lo N, Beninati T, Wernegreen JJ, Werren JH, Bandi C (2005) Phylogeny of Wolbachia pipientis based on gltA, groEL and ftsZ gene sequences: clustering of arthropod and nematode symbionts in the F supergroup, and evidence for further diversity in the Wolbachia tree. Microbiol 151:4015–4022Google Scholar
  5. Da Silva TKS, Blanco CM, Ogrzewalska M, de Souza MB, Barreira JD, Moreira NS, Mares-Guia MAMM, De Lemos ES (2017) Investigation of Ehrlichia spp., Anaplasma spp. and Rickettsia spp. in ectoparasites collected from domestic animals, Rio de Janeiro State, Brazil. Virus Rev Res 22:30–36Google Scholar
  6. Dittmar K, Whiting MF (2004) New Wolbachia endosymbionts from Nearctic and Neotropical fleas (Siphonaptera). J Parasitol 90:953–957PubMedGoogle Scholar
  7. Doudoumis V, Tsiamis G, Wamwiri F, Brelsfoard C, Alam U, Aksoy E, Dalaperas S, Abd-Alla A, Ouma J, Takac P, Aksoy S, Bourtzis K (2012) Detection and characterization of Wolbachia infections in laboratory and natural populations of different species of tsetse flies (genus Glossina). BMC Microbiol 12(Suppl 1):S3PubMedPubMedCentralGoogle Scholar
  8. Dryden MW (1993) Biology of fleas of dogs and cats. Compend Contin Educ Pract Vet 15:569–579Google Scholar
  9. Eisen RJ, Gage KL (2012) Transmission of flea-borne zoonotic agents. Annu Rev Entomol 57:61–82PubMedGoogle Scholar
  10. Fischer P, Schmetz C, Bandi C, Bonow I, Mand S, Fischer K, DW B¨u (2002) Tunga penetrans: molecular identification of Wolbachia endobacteria and their recognition by antibodies against proteins of endobacteria from filarial parasites. Exp Parasitol 102:201–211PubMedGoogle Scholar
  11. Folmer O, Black M, Hoeh W, Lutz R, Vrijenhoek R (1994) DNA primers for amplification of mitochondrial cytochrome C oxidase subunit I from diverse metazoan invertebrates. Mol Mar Biol Biotechnol 3:294–299PubMedGoogle Scholar
  12. Furman DP, Catts EP (1982) Manual of medical entomology. 4. Cambridge University Press, CambridgeGoogle Scholar
  13. Gorham CH, Fang QQ, Durden LA (2003) Wolbachia endosymbionts in fleas (Siphonaptera). J Parasitol 89:283–289PubMedGoogle Scholar
  14. Gracia MJ, Calvete C, Estrada R, Castillo JA, Prebanez MA, Lucientes J (2008) Fleas parasitizing domestic dogs in Spain. Vet Parasitol 151:312–319PubMedGoogle Scholar
  15. Hilgenboecker K, Hammerstein P, Schlattmann P, Telschow A, Werren JH (2008) How many species are infected with Wolbachia? A statistical analysis of current data. FEMS Microbiol Lett 281:215–220PubMedPubMedCentralGoogle Scholar
  16. Huelsenbeck JP, Ronquist F (2001) MRBAYES: Bayesian inference of phylogenetic trees. Bioinformatics 17:754–755PubMedPubMedCentralGoogle Scholar
  17. Jeyaprakash A, Hoy MA (2000) Long PCR improves Wolbachia DNA amplification: wsp sequences found in 76% of sixty-three arthropod species. Insect Mol Biol 9:393–405PubMedPubMedCentralGoogle Scholar
  18. Kambris Z, Cook PE, Phuc HK, Sinkins SP (2009) Immune activation by life-shortening Wolbachia and reduced filarial competence in mosquitoes. Science 326:134–136PubMedPubMedCentralGoogle Scholar
  19. Kamtchum-Tatuene J, Makepeace BL, Benjamin L, Baylis M, Solomon T (2017) The potential role of Wolbachia in controlling the transmission of emerging human arboviral infections. Curr Opin Infect Dis 30(1):108–116PubMedPubMedCentralGoogle Scholar
  20. Karimian F, Vatandoost H, Rassi Y, Maleki-Ravasan N, Choubdar N, Koosha M, Arzamani K, Moradi-Asl E, Veysi A, Alipour H, Shirani M, Oshaghi MA (2018) wsp-based analysis of Wolbachia strains associated with Phlebotomus papatasi and P. sergenti (Diptera: Psychodidae) main cutaneous leishmaniasis vectors, introduction of a new subgroup wSerg. Pathog Glob Health 112:152–160PubMedPubMedCentralGoogle Scholar
  21. Kearse M, Moir R, Wilson A, Stones-Havas S, Cheung M, Sturrock S, Buxton S, Cooper A, Markowitz S, Duran C, Thierer T, Ashton B, Mentjies P, Drummond A (2012) Geneious Basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data. Bioinformatics 28:1647–1649PubMedPubMedCentralGoogle Scholar
  22. Kimura M (1980) A simple method of estimating evolutionary rate of base substitutions through comparative studies of nucleotide sequences. J Mol Evol 16:111–120PubMedGoogle Scholar
  23. Kramer F, Mencke N (2001) Flea biology and control. Springer, BerlinGoogle Scholar
  24. Librado P, Rozas J (2009) DnaSP v5: a software for comprehensive analysis of DNA polymorphism dataGoogle Scholar
  25. Marshall AG (1981) The sex ratio in ectoparasitic insects. Ecol Entomol 6:155–174Google Scholar
  26. Moreira LA, Iturbe-Ormaetxe I, Jeffery JA, Lu G, Pyke AT, Hedges LM, Rocha BC, Hall-Mendelin S, Day A, Riegler M, Hugo LE, Johnson KN, Kay BH, McGraw EA, van den Hurk AF, Ryan PA, O’Neill SL (2009) A Wolbachia symbiont in Aedes aegypti limits infection with dengue, Chikungunya, and Plasmodium. Cell 139(7):1268–1278PubMedGoogle Scholar
  27. O’Neill SL, Giordano R, Colbert AM, Karr TL, Robertson HM (1992) 16S rRNA phylogenetic analysis of the bacterial endosymbionts associated with cytoplasmic incompatibility in insects. Proc Natl Acad Sci U S A 89:2699–2702PubMedPubMedCentralGoogle Scholar
  28. Oteo JA, Portillo A, Portero F, Zavala-Castro J, Venzal JM, Labruna MB (2014) Candidatus Rickettsia asemboensis and Wolbachia spp. in Ctenocephalides felis and Pulex irritans fleas removed from dogs in Ecuador. Parasit Vectors 7:455PubMedPubMedCentralGoogle Scholar
  29. Otranto D, Wall R (2008) New strategies for the control of arthropod vectors of disease in dogs and cats. Med Vet Entomol 22:291–302PubMedGoogle Scholar
  30. Pan X, Pike A, Joshi D, Bian G, McFadden MJ, Lu P, Liang X, Zhang F, Raikhel AS, Xi Z (2018) The bacterium Wolbachia exploits host innate immunity to establish a symbiotic relationship with the dengue vector mosquito Aedes aegypti. ISME J 12(1):277–288PubMedGoogle Scholar
  31. Posada D (2008) jModelTest: phylogenetic model averaging. Mol Biol Evol 25:1253–1256PubMedGoogle Scholar
  32. Pourali P, Roayaei Ardakani M, Jolodar A, Razi Jalali MH (2009) PCR screening of the Wolbachia in some arthropods and nematodes in Khuzestan Province. Iran J Vet Res 10:216–222Google Scholar
  33. Rolain JM, Franc M, Davoust B, Raoult D (2003) Molecular detection of Bartonella quintana, B. koehlerae, B. henselae, B. clarridgeiae, Rickettsia felis, and Wolbachia pipientis in cat fleas, France. Emerg Infect Dis 9:338–342PubMedGoogle Scholar
  34. Rust MK (2005) Advances in the control of Ctenocephalides felis (cat flea) on cats and dogs. Trends Parasitol 21:232–236PubMedGoogle Scholar
  35. Sazama EJ, Bosch MJ, Shouldis CS, Ouellette SP, Wesner JS (2017) Incidence of Wolbachia in aquatic insects. Ecol Evol 7(4):1165–1169PubMedPubMedCentralGoogle Scholar
  36. Tamura K, Stecher G, Peterson D, Filipski A, Kumar S (2013) MEGA7: molecular evolutionary genetics analysis version 6.0. Mol Biol Evol 30:2725–2729PubMedPubMedCentralGoogle Scholar
  37. Tavassoli M, Ahmadi A, Imani A, Ahmadiara E, Javadi S, Hadian M (2010) Survey of flea infestation in dogs in different geographical regions of Iran. Korean J Parasitol 48:145–149PubMedPubMedCentralGoogle Scholar
  38. Tay ST (2013) Wolbachia endosymbionts, Rickettsia felis and Bartonella species, in Ctenocephalides felis fleas in a tropical region. J Vect Ecol 38:200–202Google Scholar
  39. Vivero RJ, Cadavid-Restrepo G, Herrera CXM, Soto SIU (2017) Molecular detection and identification of Wolbachia in three species of the genus Lutzomyia on the Colombian Caribbean coast. Parasit Vectors 10:220Google Scholar
  40. Wall R, Shearer D (2001) Veterinary ectoparasites, biology, pathology and control, 2nd edn. Blackwell Science, LondonGoogle Scholar
  41. Weinert LA, Araujo-Jnr EV, Ahmed MZ, Welch JJ (2015) The incidence of bacterial endosymbionts in terrestrial arthropods. Proc R Soc Lond B Biol Sci 282:20150249Google Scholar
  42. Werren JH, Baldo L, Clark ME (2008) Wolbachia: master manipulators of invertebrate biology. Nat Rev Microbiol 6:741–751PubMedGoogle Scholar
  43. Werren JH, Windsor DM (2000) Wolbachia infection frequencies in insects: evidence of a global equilibrium? Proc Biol Sci 267(1450):1277–1285PubMedPubMedCentralGoogle Scholar
  44. Whiting MF, Whiting AS, Hastriter MW, Dittmar K (2008) A molecular phylogeny of fleas (Insecta: Siphonaptera): origins and host associations. Cladistics 24:677–707Google Scholar
  45. Xi Z, Gavotte L, Xie Y, Dobson SL (2008) Genome-wide analysis of the interaction between the endosymbiotic bacterium Wolbachia and its Drosophila host. BMC Genomics 9(1):1PubMedPubMedCentralGoogle Scholar
  46. Zhou W, Rousset F, O’Neill SL (1998) Phylogeny and PCR-based classification of Wolbachia strains using wsp gene sequences. Proc R Soc Lond Ser B 265:509–515Google Scholar
  47. Zug R, Hammerstein P (2012) Still a host of hosts for Wolbachia: analysis of recent data suggests that 40% of terrestrial arthropod species are infected. PLoS One 7(6):e38544PubMedPubMedCentralGoogle Scholar

Copyright information

© Institute of Microbiology, Academy of Sciences of the Czech Republic, v.v.i. 2019

Authors and Affiliations

  • Zuhal Onder
    • 1
    Email author
  • Arif Ciloglu
    • 1
  • Onder Duzlu
    • 1
  • Alparslan Yildirim
    • 1
  • Mubeccel Okur
    • 1
  • Gamze Yetismis
    • 1
  • Abdullah Inci
    • 1
  1. 1.Faculty of Veterinary Medicine, Parasitology DepartmentErciyes UniversityKayseriTurkey

Personalised recommendations