Development of a Stage- and Species-Specific RNAi System for Molecular Insights in Wasps.

wasps, egg parasitoids widely used to control lepidopteran pests, have long eluded in-depth molecular mechanistic studies due to their minute size and genetic tool scarcity. While previous RNAi efforts were restricted to , we developed the first cross-species RNAi system for both and the previously intractable . Temporal expression profiling identified and as stage-specific RNAi targets, peaking during prepupal/pupal stages, which were tested across species and developmental stages using microinjection and soaking dsRNA delivery methods. Survival analysis prioritized soaking for prepupae/pupae, while microinjection was essential for to bypass prepupal mortality during soaking. Concentration-dependent RNAi targeting the gene achieved 85.61% transcript reduction in via soaking and 89.36% in via microinjection at 2000 ng/μL, correlating with 64.06% and 32.09% white-eyed pupae, causing a significant reduction in eye pigments. For the gene, soaking at 2000 ng/μL induced 88.35% transcript reduction in and 73.31% in , leading to incomplete cuticle tanning and sclerotization. This study resolves the long-standing challenge of genetic manipulation in wasps, providing a universally applicable framework to decipher parasitoid-host interactions at the molecular scale, which is useful for sustainable pest management strategies.