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This protocol describes a method to use RNA interference (RNAi) to knock down genes in Nasonia larvae. Unlike Drosophila, RNAi in Nasonia is systemic. Lynch and Desplan (2006) have injected double stranded RNA (dsRNA) in female pupae and successfully knocked down genes in offspring embryos. By injecting dsRNA against the eye color gene cinnabar in last-instar Nasonia larvae, we have successfully produced adult red-eye-color phenotypes.
Injected larvae of earlier stages must be returned to parasitized hosts to feed. Foster larvae will continue to eat the host and reduce the risk of injected larvae drowning. To avoid ambiguous results, set up foster hosts with females marked by known mutants that differ from the expected results of RNAi knockdown. We used bl13,or123 which have blue bodies and orange eyes (readily distinguished from the scarlet eyes of the cn knockdown). Desiccated or rotted foster hosts are the main causes of larval loss after injection. To prevent this, use sterile techniques when handling foster hosts and be very careful not to puncture the fly carcass.
Problem: Injected wasps do not pupate.
Solution: dsRNA treatment or injection stress may have killed the larva or induced diapause. Compare survivorship of treatment and buffer-injected controls. Diapause larvae will continue to wiggle. Diapause can be broken by refrigeration for >8 weeks.
JHW and DL acknowledge support from the NIH 1 R24 GM084917-01 and assistance from Rachel Edwards, Jon Giebel, Michael Clark and Rhitoban Raychoudhury.