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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Cold Spring Harb Protoc. Author manuscript; available in PMC 2010 October 1.
Published in final edited form as:
PMCID: PMC2916736

Curing Wolbachia infections in Nasonia (Parasitoid Wasp)

This protocol describes curing a Nasonia strain of its Wolbachia symbionts (Breeuwer and Werren 1990,Breeuwer and Werren 1993). Wolbachia cause cytoplasmic incompatibility (CI) in Nasonia. Crosses between infected and uninfected wasps, or between infected wasps of different species, will fail because of Wolbachia. The sperm modification which induces CI appears to be established before the adult stage, so it is generally not possible to cure an infection and break the CI barrier in the same generation. A Wolbachia infection can be removed from a strain over a number of generations, however, by feeding antibiotics and selecting on females who show partial CI. The current genome sequenced strains of Nasonia (AsymCX, IV7(u), RV2X(u)) are all Wolbachia-free.



  • Sucrose
  • Tetracycline
  • Culture vials
  • Filter paper
  • Wasp Cultures


  1. Create 1% tetracycline in 10% sugar water solution.
    • 1ml ddH2O
    • 0.1g sucrose
    • 0.01g tetracycline
  2. Vortex solution for 20–30 sec or until all sugar crystals have dissolved.
  3. Cut filter paper into small squares (1cm × 1cm)
  4. Dip filter paper into antibiotic solution and place 1–2 squares into a small glass/plastic vial with 5 mated females of chosen strain. Repeat for about 5–10 replicates per strain.
  5. Allow females to feed on filter paper overnight.
  6. Remove filter paper and host females individually in 12mm culture vials with 2 hosts each. Re-host females 3–4 times in new vials in two day increments.
  7. Once offspring emerge, select mated female offspring from a family which shows a progressively reduced sex ratio (increasing proportion males over time). This is evidence of CI due to reduced Wolbachia levels in eggs produced after antibiotic treatment. Females from the later hostings with high proportion males are therefore more likely to have reduced Wolbachia loads. Select females from one of these later hostings.
  8. Repeat curing procedure for at least 3 generations in order to completely eliminate the infection. Low levels of Wolbachia may not show CI but can recover over subsequent generations.
  9. Test treated strain for Wolbachia. There are two options:
    1. Phenotypically test by setting up crosses to test for cytoplasmic incompatibility. Set up replicate matings between uninfected virgin females and males from the supposedly cured strain.
    2. Test for Wolbachia with PCR (e.g. Werren and Windsor 2000)


JHW and DL acknowledge support from the NIH 1 R24 GM084917-01 and assistance from Rachel Edwards, Jon Giebel, Michael Clark and Rhitoban Raychoudhury.


  • Breeuwer JAJ, Werren JH. Microorganisms Associated With Chromosome Destruction and Reproductive Isolation Between Two Insect Species. Nature. 1990;346:558–560. [PubMed]
  • Breeuwer AJ, Werren JH. Cytoplasmic incompatibility and bacterial density in Nasonia vitripennis. Genetics. 1993;135:565–574. [PubMed]
  • Werren JH, Windsor DM. Wolbachia infection frequencies in insects: evidence of a global equilibrium? Proc R Soc Lond B Biol Sci. 2000;267:1277–1285. [PMC free article] [PubMed]