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

<i>Trichogramma</i> 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 <i>T. dendrolimi</i>, we developed the first cross-species RNAi system for both <i>T. dendrolimi</i> and the previously intractable <i>T. ostriniae</i>. Temporal expression profiling identified <i>white</i> and <i>laccase 2</i> 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 <i>T. dendrolimi</i> prepupae/pupae, while microinjection was essential for <i>T. ostriniae</i> to bypass prepupal mortality during soaking. Concentration-dependent RNAi targeting the <i>white</i> gene achieved 85.61% transcript reduction in <i>T. dendrolimi</i> via soaking and 89.36% in <i>T. ostriniae</i> 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 <i>laccase 2</i> gene, soaking at 2000 ng/μL induced 88.35% transcript reduction in <i>T. dendrolimi</i> and 73.31% in <i>T. ostriniae</i>, leading to incomplete cuticle tanning and sclerotization. This study resolves the long-standing challenge of genetic manipulation in <i>Trichogramma</i> wasps, providing a universally applicable framework to decipher parasitoid-host interactions at the molecular scale, which is useful for sustainable pest management strategies.