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This protocol covers most procedures used by the Nasonia geneticist, including virgin collection. Nasonia are easily sexed as pupae, so collecting virgins is easily done with a window of several days. The female-specific ovipositor is visible at even the earliest pupal stages as a longitudinal ridge on the ventral posterior abdomen. To prolong generations, yellow pupae can be stored at 4-8C for up to two months and adults for two to three weeks. Haplo-diploid crossing methods are introduced in the example of making a new Nasonia vitripennis mutant homozygous, using an aunt-nephew mating approach. The protocol can be extended to inbreeding strains. Inbreeding depression is generally not a problem in Nasonia unless the mutation itself is female sterile. Mother-son mating and sibmating are also reasonable approaches to inbreeding.
12mm culture vials and cotton plugs
Honey water (40% honey, 60% sterile H2O)
Dissecting needle bent at 45 degrees 2cm from tip (“hockey stick”)
Index card (fold long edges to 90 degrees, 1cm from edge, to form a tray)
No female pupae seen.
Female parent may not have mated, or asymmetric Wolbachia infections caused cytoplasmic incompatibility. Unmated (virgin) Nasonia females will produce all-male broods. Recently emerged females (< 3 hr) from stocks may not have mated. Collect virgin females from 4C backup and resume cross. Check Wolbachia infection status of stocks. In general, infected males can only successfully fertilize eggs that have the same Wolbachia types. Virgins from an infected lab strain such as LabII can be used if male infection status is unknown.
homozygous females do not lay eggs or do not mate.
The mutation may be female-sterile. Maintain in heterozygous fashion (Steps 12-16 above) or discard.
JHW and DL acknowledge support from the NIH 1 R24 GM084917-01 and assistance from Rachel Edwards, Jon Giebel, Michael Clark and Rhitoban Raychoudhury.
Citation: Werren JH, Loehlin DW. Virgin collection and haplodiploid crossing methods in Nasonia (parasitoid wasp). CSH Protoc. 2009