Padronização da técnica de RNA de interferência pela aplicação tópica de RNA de fita dupla em Rhodnius prolixus (Hemiptera, Triatominae) utilizando o gene da Nitroforina 2 salivar

Abstract

The triatomine bug Rhodnius prolixus, a significant vector of Trypanosoma cruzi, the causative agent of Chagas disease in South and Central America, serves as an ideal model for integrated physiology research in disease vectors. However, despite the extensive knowledge accumulated regarding its physiology and the availability of its genome, there remain numerous gaps in our understanding of gene function in these vectors. To address these questions, we chose to explore the topical application of dsRNA as an RNAi delivery strategy. This approach was selected for its minimally invasive nature, speed, and cost-effectiveness, even though it has been underexplored in triatomines. Our primary objective in this study was to assess the effectiveness of topical RNAi application in R. prolixus nymphs and comprehend its impact on gene expression and the phenotype of their salivary glands. The most significant results revealed that topical RNAi application on the dorsal side of the nymphs resulted in significant gene silencing, with effects extending to subsequent developmental stages. A notable feature of this technique is its non-invasiveness, preserving the insects' integrity for future investigations in behavior and physiology. In comparison with injection, where there was lighter staining in the insects' salivary glands, nymphs treated with dsRNA through topical application in the previous stage exhibited similar staining to the control group. Given the substantial reduction in NP2 mRNA expression achieved through topical dorsal application (95.6% to 71.5%) and the effective transmission of the silencing effect to the next stage, this promising methodology presents significant advantages for future research focused on salivary gene silencing through RNAi.