Conditions facilitating infection of mosquito cell lines with Wolbachia, an obligate intracellular bacterium

  • Ann M. FallonEmail author


Factors that influence establishment of Wolbachia, an obligate intracellular bacterium, in novel insect hosts or uninfected insect cell lines are poorly understood. Infectivity of Wolbachia strain wStr was correlated with flow cytometric profiles to define optimal conditions for harvesting an infectious inoculum. Wolbachia recovered from the cell culture supernatant after gentle pipetting of infected cells represented about 1% of the total bacterial population and were more infectious than Wolbachia that remained associated with intact cells and/or membranes after low-speed centrifugation. Optimal establishment of a robust infection in naïve cells required 6 d, at a ratio of 80 to 160 bacteria per cell. Among Aedes albopictus mosquito cell lines, an aneuploid line with a 4n + 1 karyotype was more susceptible to infection than diploid lines. These findings contribute to the in vitro manipulation of Wolbachia, illustrate some of the many factors that influence infectivity, and identify areas for future investigation.


Wolbachia Mosquito cell lines Aedes albopictus Laodelphax striatellus Fluorescence microscopy Flow cytometry Transinfection 



I thank Dr. T.J. Kurtti for the helpful discussions and comments on the manuscript.

Funding information

This work was supported by NIH grant AI 081322 and by the University of Minnesota Agricultural Experiment Station, St. Paul, MN.


  1. Aliota MT, Peinado SA, Velez ID, Osorio JE (2016) The wMel strain of Wolbachia reduces transmission of Zika virus by Aedes aegypti. Sci Rep 6:28792. CrossRefGoogle Scholar
  2. Baldridge G, Higgins LA, Witthuhn B, Markowski T, Baldridge A, Armien A, Fallon A (2017) Proteomic analysis of a mosquito host response to persistent Wolbachia infection. Res Microbiol 168:609–625. CrossRefGoogle Scholar
  3. Beckmann JF, Ronau JA, Hochstrasser M (2017) A Wolbachia deubiquitylating enzyme induces cytoplasmic incompatibility. Nature Microbiol 2:17007. CrossRefGoogle Scholar
  4. Bonneau M, Atyame C, Beji M, Justy F, Cohen-Gonsaud M, Sicard M, Weill M (2018) Culex pipiens crossing type diversity is governed by an amplified and polymorphic operon of Wolbachia. Nat Commun 9:319. CrossRefGoogle Scholar
  5. Cahn FH, Fox MS (1968) Fractionation of transformable bacteria from competent cultures of Bacillus subtilis on renografin gradients. J Bacteriol 95:867–875Google Scholar
  6. Dutra HLC, Rocha MN, Dias FBS, Mansur SB, Caragata EP, Moreira LA (2016) Wolbachia blocks currently circulating Zika virus isolates in Brazilian Aedes aegypti mosquitoes. Cell Host Microbe 19:771–774. CrossRefGoogle Scholar
  7. Fallon AM (1984) Methotrexate resistance in cultured mosquito cells. Insect Biochem 14:697–704CrossRefGoogle Scholar
  8. Fallon AM (2008) Cytological properties of an Aedes albopictus mosquito cell line infected with Wolbachia strain wAlbB. In Vitro Cell Dev Biol - Anim 44:154–161CrossRefGoogle Scholar
  9. Fallon AM (2014) Flow cytometric evaluation of the intracellular bacterium, Wolbachia pipientis, in mosquito cells. J Microbiol Methods 107:119–125CrossRefGoogle Scholar
  10. Fallon AM, Kurtti TJ (2005) Cultured cells as a tool for analysis of gene expression. In: Marquardt WC (ed) The Biology of Disease Vectors, vol 2. Elsevier, New York. Chapter 35, pp 539–549Google Scholar
  11. Fallon AM, Witthuhn BA (2009) Proteasome activity in a naïve mosquito cell line infected with Wolbachia pipientis wAlbB. In Vitro Cell Dev Biol Anim 45:460–466CrossRefGoogle Scholar
  12. Fallon AM, Baldridge GD, Higgins LA, Witthuhn BA (2013) Wolbachia from the planthopper Laodelphax striatellus establishes a robust, persistent, streptomycin-resistant infection in clonal mosquito cells. In Vitro Cell Dev Biol - Anim 49:66–73CrossRefGoogle Scholar
  13. Ferree PM, Frydman HM, Li JM, Cao J, Wieschaus E, Sullivan W (2005) Wolbachia utilizes host microtubules and dynein for anterior localization in the Drosophila oocyte. PLoS Pathog 1:e14. CrossRefGoogle Scholar
  14. Gerenday A, Fallon AM (2011) Increased levels of the cell cycle inhibitor protein, dacapo, accompany 20-hydroxyecdysone-induced G1 arrest in a mosquito cell line. Arch Insect Biochem Phyisol 78:61–73. CrossRefGoogle Scholar
  15. Hertig M (1936) The Rickettsia, Wolbachia pipientis (gen. et sp.n.) and associated inclusions of the mosquito, Culex pipiens. Parasitol 28:453–486CrossRefGoogle Scholar
  16. Hertig M, Wolbach (1924) Studies on Rickettsia-like micro-organisms in insects. J Med Res 44:329–374Google Scholar
  17. Hoffmann AA, Montgomery BL, Popovici H, Iturbe-Ormaetxe I, Johnson PH, Muzzi F, Greenfield M, Durkan M, Leong YS, Dong Y, Cook H, Axford J, Callahan AG, Kenny N, Omodei C, McGraw EA, Ritchie SA, Turelli M, O'Neill SL (2011) Successful establishment of Wolbachia in Aedes populations to suppress dengue transmission. Nature 476:454–457CrossRefGoogle Scholar
  18. Hughes G, Rivero A, Rasgon JL (2014) Wolbachia can enhance Plasmodium infection in mosquitoes: implications for malaria control? PLoS Pathog 10(9):e1004182. CrossRefGoogle Scholar
  19. Hughes GL, Koga R, Xue P, Fukatsu T, Rasgon JL (2011) Wolbachia infections are virulent and inhibit the human malaria parasite Plasmodium falciparum in Anopheles gambiae. PLoS Pathog 7(5):e1002043. CrossRefGoogle Scholar
  20. Jallepalli PV, Pellman D (2007) Aneuploidy in the balance. Science 317:904–905. CrossRefGoogle Scholar
  21. King JG, Souto-Maior C, Sartori LM, Maciel-de-Freitas R, Gomes MGM (2018) Variation in Wolbachia effects on Aedes mosquitoes as a determinant of invasiveness and vectorial capacity. Nat Commun 9:1483. CrossRefGoogle Scholar
  22. Mazzacano CA, Fallon AM (1992) Thymidine kinase deficient mutants of Aedes albopictus mosquito cells. In Vitro Cell Dev Biol Anim 28A:455–458CrossRefGoogle Scholar
  23. McMeniman CJ, Lane AM, Fong AWC, Voronin DA, Iturbe-Ormatxe I, Yamada R, McGraw EA, O'Neill SL (2008) Host adaptation of a Wolbachia strain after long-term serial passage in mosquito cell lines. Appl Environ Microbiol 74:6963–6969. CrossRefGoogle Scholar
  24. Mento SJ, Stollar V (1978) Isolation and partial characterization of drug resistant Aedes albopictus cells. Somatic Cell Genet 4:179–191CrossRefGoogle Scholar
  25. Moreira LA, Iturbe-Ormaetxe I, Jeffery JA, Lu G, Pyke AT, Hedges LM, Rocha BC, Hall-Mendelin S, Day A, Riegler M, Higo 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 139:1268–1278. CrossRefGoogle Scholar
  26. Ndiaye M, Mattei X, Thiaw OT (1995) Extracellular and intracellular rickettsia-like microorganisms in gonads of mosquitoes. J Submicrosc Cytol Pathol 27:557–563Google Scholar
  27. Noda H, Miyoshi T, Koizumi Y (2002) In vitro cultivation of Wolbachia in insect and mammalian cell lines. In Vitro Cell Dev Biol Anim 38:423–427CrossRefGoogle Scholar
  28. O’Neill SL, Pettigrew MM, Sinkins SP, Braig HR, Andreadis TG, Tesh RB (1997) In vitro cultivation of Wolbachia pipientis in an Aedes albopictus cell line. Insect Mol Biol 6:33–39CrossRefGoogle Scholar
  29. Popov VL, Korenberg EI, Nefedova VV, Han VC, Wen JW, Kovalevskii YV, Gorelova NB, Walker DH (2007) Ultrastructural evidence of the Ehrlichial developmental cycle in naturally infected Ixodes persulcatus ticks in the course of coinfection with rickettsia, Borrelia, and a flavivirus. Vector Borne Zoonotic Dis 7:699–716. CrossRefGoogle Scholar
  30. Raquin V, Valiente Moro C, Saucereau Y, Tran F-H, Potier P, Mavingui P (2015) Native Wolbachia from Aedes albopictus blocks chikungunya virus infection in cellulo. PLoS One 10(4):e0125066. CrossRefGoogle Scholar
  31. Sarver N, Stollar V (1977) Sindbis virus-induced cytopathic effect in clones of Aedes albopictus (Singh) cells. Virology 80:390–400CrossRefGoogle Scholar
  32. Shih KM, Gerenday A, Fallon AM (1998) Culture of mosquito cells in Eagle’s medium. In Vitro Cell Dev Biol Anim 34:629–630CrossRefGoogle Scholar
  33. Shotkoski FA, Fallon AM (1990) Genetic changes in methotrexate-resistant mosquito cells. Arch Insect Biochem Physiol 15:79–92CrossRefGoogle Scholar
  34. Shotkoski FA, Fallon AM (1991) An amplified insect dihydrofolate reductase gene contains a single intron. Eur J Biochem 201:157–160CrossRefGoogle Scholar
  35. Torres EM, Sokolsky T, Tucker CM, Chan LY, Boselli M, Dunham MJ, Amon A (2007) Effects of aneuploidy on cellular physiology and cell division in haploid yeast. Science 317:916–924. CrossRefGoogle Scholar
  36. Voth DE, Heinzen RA (2007) Lounging in a lysosome: the intracellular lifestyle of Coxiella burnetii. Cell Microbiol 9:829–840. CrossRefGoogle Scholar
  37. Wang ZH, Fallon AM (1997) Structural mapping of the dihydrofolate reductase amplicon in mosquito cells resistant to methotrexate. Insect Biochem Mol Biol 27:79–92CrossRefGoogle Scholar
  38. Williams JC, Weiss E (1978) Energy metabolism of Rickettsia typhi: pools of adenine nucleotides and energy charge in the presence and absence of glutamate. J Bacteriol 134:884–892Google Scholar
  39. Xi Z, Khoo CCH, Dobson SL (2005) Wolbachia establishment and invasion in an Aedes aegypti laboratory population. Science 310:326–328. CrossRefGoogle Scholar

Copyright information

© The Society for In Vitro Biology 2019

Authors and Affiliations

  1. 1.Department of EntomologyUniversity of MinnesotaSt. PaulUSA

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