Fusion dsRNA targeting ecdysteroid signaling provides efficient and selective RNAi ‐based control of spider mites

BACKGROUND Tetranychus urticae is a major agricultural pest mite whose rapid evolution of pesticide resistance necessitates innovative control strategies. RNA interference (RNAi) offers a promising alternative to chemical pesticides for its high specificity, efficiency, and environmental safety. Ecdysteroid signaling genes, which regulate molting and development in arthropods, represent potential RNAi targets for inducing lethal molting defects. This study investigated the roles of ecdysteroid signaling pathway genes in T. urticae molting and evaluated the potential of fusion double‐stranded RNA (dsRNA) constructs targeting multiple genes to enhance RNAi efficacy. RESULTS The expression profiling revealed that TuE74 , TuECR , TuHR4 , TuE93 , and TuHR3 were highly expressed during the molting stage of T. urticae . RNAi‐mediated silencing of these genes, particularly TuECR , TuHR3 , TuHR4 , and TuE74 , caused distinct molting defects and high mortality. Fusion dsRNAs targeting combinations of these genes ( ECR‐HR3‐HR4 or ECR‐HR3‐E74 ) achieved stronger and more coordinated gene silencing, lower median lethal concentration (LC 50 ) and LC 90 values than single‐gene dsRNA (ds TuHR3 ), and exhibited cross‐species efficacy against Tetranychus cinnabarinus and Tetranychus evansi . Importantly, the fusion dsRNAs showed no adverse effects on the predatory mite Neoseiulus californicus , supporting their biosafety. Spray‐based delivery of fusion dsRNA significantly delayed molting, confirming its biological activity, although optimization of delivery efficiency is still required to achieve complete developmental arrest. CONCLUSION The present study demonstrates that multi‐target fusion dsRNA can substantially enhance RNAi efficacy, broaden control spectra across pest mite species, and maintain environmental safety. These results provide a molecular basis and applied framework for the development of next‐generation RNAi‐based acaricides for sustainable spider mite management.