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

## TL;DR

Researchers developed a new RNAi system to study gene function in tiny Trichogramma wasps, enabling better pest control strategies.

## Contribution

The first cross-species RNAi system for T. dendrolimi and T. ostriniae was developed with stage-specific protocols.

## Key findings

- RNAi targeting white and laccase 2 genes achieved significant transcript reduction in both species.
- Soaking and microinjection methods were effective but species and stage-specific.
- The system enables functional gene studies in miniature parasitoid wasps for precision biocontrol.

## Abstract

Trichogramma wasps, as environmentally friendly biocontrol agents, are widely employed to control lepidopteran pests. In recent years, people have attempted to study their gene function through molecular means in order to improve the control efficiency against agricultural and forestry pests of Trichogramma wasps. However, the progress has been severely hindered by their minute body size (<1 mm) and complex host egg-dependent parasitic behavior. In this study, we established a universal RNA interference (RNAi) technology system applicable to T. dendrolimi and T. ostriniae, targeting the white gene (regulating eye pigment deposition) and laccase 2 gene (involved in cuticle sclerotization). Furthermore, we analyzed the effects of different dsRNA treatment concentrations and treatment periods on interference efficiency, overcoming the technical barriers associated with cross-species applications in conventional methods. Phenotypic analyses revealed that both non-invasive soaking and microinjection approaches achieved efficient gene silencing in T. dendrolimi and pupal-stage T. ostriniae. Notably, to avoid high mortality rates, pre-pupal T. ostriniae is required to use the microinjection method. This work establishes standardized protocols for functional gene studies in miniature parasitoid wasps and provides methodological foundations for developing precision biocontrol technologies through genetic engineering.

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