Oral RNAi in Euschistus heros: Evaluation of Gene Silencing Using Cationic Polymer-Stabilized dsRNA

Abstract Euschistus heros , a key pest of soybean in the Neotropical region, represents a growing challenge to sustainable agricultural production. With the urgent need for more targeted and environmentally safe control strategies, RNA interference (RNAi) emerges as an innovative tool. This study investigated the gene-silencing potential of RNAi targeting V-ATPase and act , two genes involved in essential cellular processes. The sensitivity of E. heros to RNAi in adult and immature stages was evaluated by microinjection of dsRNA, as well as the ex-vivo degradation of double-stranded RNA (dsRNA) by nucleases present in saliva and hemolymph. Additionally, an oral delivery strategy based on conjugation of dsRNA to a cationic polymer was studied. Microinjection of dsRNA resulted in high sensitivity, particularly in immature individuals, and induced strong gene-silencing effects associated with increased mortality. Intense nucleolytic activity was detected in saliva, underscoring the need for protective carriers for dsRNA. Oral exposure to conjugated dsRNA effectively protected the molecules from enzymatic degradation and, in addition, exhibited effects on insect survival and gene expression. These findings highlight the strong sensitivity of E. heros to dsRNA delivered by microinjection and underscore the potential of RNAi approaches for the development of tailor-made bio-insecticides, while emphasizing the need to refine delivery strategies to fully realize their practical applications in agricultural pest management.