Evaluation of topical application as a delivery method of double-stranded RNA in Rhodnius prolixus (Hemiptera, Triatominae) for salivary gene silencing

RNA interference (RNAi) techniques have been widely applied in insect pest control and genomics research, effectively targeting genes in tissues like salivary glands and the midgut in Triatominae. This study evaluated the efficacy of topical application as a delivery method for dsRNA targeting Rhodnius prolixus salivary genes, Nitrophorin 2 (NP2), and Nitric Oxide Synthase (NOS). Applying dsRNA diluted in acetone (1:1) to the dorsal side of nymphs successfully triggered RNAi, achieving transstadial mRNA reduction while preserving dsRNA integrity. Real-time quantitative polymerase chain reaction (qPCR) analysis showed significant reductions on mRNA levels of NP2 (72 % to 95.6 %) across N2 to N5 stages, with no off-target effects in other nitrophorins (NP1–NP4), confirming silencing specificity. Similarly, NOS knockdown reduced mRNA levels by 47.9 % to 91.0 % across N1 to N5 stages. These reductions were accompanied by visible color changes in salivary glands, a phenotype validated through image analysis. Functional assays further demonstrated that NP2 silencing shortened plasma coagulation time, while NOS knockdown reduced the proportion of NO-bound nitrophorins. Compared to microinjection, the topical application of 2.5 µg of dsRNA (NP2 or NOS) in N3 was less effective in reducing mRNA levels (qPCR) as well as in altering insect phenotypes (plasma recalcification time or spectrophotometry analysis). Although less efficient than microinjection, topical application as a delivery method offers advantages: it avoids physical damage to insects, simplifies application for smaller nymphs, and provides a practical alternative for studying immune-related genes. These findings highlight the potential of topical RNAi as a non-invasive, effective tool for functional genomic studies in Triatominae, particularly for salivary gland targets.