RNAi‐based functional genomics tools for the beet leafhopper using microinjection and nanoparticle‐based topical spray

The beet leafhopper (BLH), Neoaliturus tenellus, is a major agricultural pest and vector of beet curly top virus (BCTV), a pathogen responsible for severe economic losses in vegetable and field crops in the Western U.S. Despite its economic importance, the genetic basis of BLH survival, reproduction, and virus-vector interactions remains unexplored mainly due to the lack of functional genomics tools in this species. In this study, we assessed the feasibility of RNA interference (RNAi) in BLHs by targeting two genes, protein gustavus isoform X2 (Gus) and pumilio homolog 3 (Pum3), which were previously identified as differentially expressed following BCTV infection. Double-stranded RNA (dsRNA) targeting conserved domains of each gene was delivered via microinjection or star polycation nanoparticle-mediated topical spray. Our results show that microinjection of dsRNA resulted in >90% knockdown of both Gus and Pum3 by 5 days post-treatment. Silencing Pum3 significantly reduced BLH survival and fecundity, whereas silencing Gus reduced fecundity without affecting adult survival, suggesting gene-specific fitness consequences. Nanoparticle-mediated dsRNA spray resulted in 88% reduction in Gus and 62% reduction in Pum3 expression, yet both produced biological effects on survival and/or reproduction, comparable to those observed with microinjection. To our knowledge, this study provides the first demonstration of the effectiveness of microinjection and nanoparticle-mediated RNAi in the BLH. Together, these findings demonstrate the critical roles of Gus and Pum3 in vector fitness and establish microinjection and nanoparticle-based dsRNA spray as robust platforms for functional genomics and developing potential scalable, non-invasive RNAi strategies for sustainable vector and disease management.