The brown marmorated stink bug (BMSB) is native to Asia and recently invaded the USA. RNA interference (RNAi) is a gene silencing mechanism in which the introduction of double-stranded RNA (dsRNA) inhibits gene function by degrading target mRNA. In dsRNA stability assays, the dsRNases present in the hemolymph and salivary gland secretions of BMSB showed lower activity than those in the hemolymph of Heliothis virescens. We evaluated six housekeeping genes (18S rRNA, EF1-α, Actin, Ubiquitin, 60S RP and β-Tubulin) across dsRNA treatments (injection and feeding) in nymphs and adults of BMSB and identified 18S rRNA and 60S RP as the best genes to use as a reference in reverse-transcriptase quantitative PCR (RT-qPCR). Homologs of 13 genes that were shown to function as effective RNAi targets in other insects were identified and evaluated by injecting dsRNA targeting these homologs into BMSB adults. Five out of 13 dsRNAs tested caused more than 70% mortality by seven days after injection of dsRNA. Feeding dsRNA targeting five of these genes (IAP, ATPase, SNF7, GPCR, and PPI) to nymphs caused more than 70% mortality by three of the five dsRNAs tested. These data suggest that feeding dsRNA causes target gene knockdown and mortality in BMSB.

Mosquito-borne diseases are a major threat to human health and are responsible for millions of deaths globally each year. Vector control is one of the most important approaches used in reducing the incidence of these diseases. However, increasing mosquito resistance to chemical insecticides presents challenges to this approach. Therefore, new strategies are necessary to develop the next generation vector control methods. Because of the target specificity of dsRNA, RNAi-based control measures are an attractive alternative to current insecticides used to control disease vectors. In this study, Chitosan (CS) was cross-linked to sodium tripolyphosphate (TPP) to produce nano-sized polyelectrolyte complexes with dsRNA. CS-TPP-dsRNA nanoparticles were prepared by ionic gelation method. The encapsulation efficiency, protection of dsRNA from nucleases, cellular uptake, in vivo biodistribution, larval mortality and gene knockdown efficiency of CS-TPP-dsRNA nanoparticles were determined. The results showed that at a 5:1 weight ratio of CS-TPP to dsRNA, nanoparticles of less than 200 nm mean diameter and a positive surface charge were formed. Confocal microscopy revealed the distribution of the fed CS-TPP-dsRNA nanoparticles in midgut, fat body and epidermis of yellow fever mosquito, Aedes aegypti larvae. Bioassays showed significant mortality of larvae fed on CS-TPP-dsRNA nanoparticles. These assays also showed knockdown of a target gene in CS-TPP-dsRNA nanoparticle fed larvae. These data suggest that CS-TPP nanoparticles may be used for delivery of dsRNA to mosquito larvae.

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The Colorado potato beetle (CPB; Leptinotarsa decemlineata) is one of the most notorious and difficult to control pests of potato and other solanaceous crops in North America. This insect has evolved a remarkable ability to detoxify both plant and synthetic toxins, allowing it to feed on solanaceous plants containing toxic alkaloids and to develop resistance to synthetic chemicals used for its control. RNA interference (RNAi) is a natural mechanism that evolved as an immune response to double‐stranded RNA (dsRNA) viruses where dsRNA triggers silencing of target gene expression. RNAi is being developed as a method to control CPB. Here, we evaluated four CPB‐specific genes to identify targets for RNAi‐mediated control of this insect. Out of the four dsRNAs evaluated in CPB larvae and adults, dsIAP (dsRNA targeting inhibitor of apoptosis, iap gene) performed better than dsActin, dsHSP70, and dsDynamin in inducing larval mortality. However, in adults, the mortality induced by dsActin is significantly higher than the mortality induced by dsIAP, dsHSP70, and dsDynamin. Interestingly, a combination of dsIAP and dsActin performed better than either dsIAP or dsActin alone by inducing feeding inhibition in 24 hr and mortality in 48 hr in larvae. When the dsIAP and dsActin were expressed in the Escherichia coli HT115 strain and applied as a heat‐killed bacterial spray on potato plants, it protected the plants from CPB damage. These studies show that the combination of dsIAP and dsActin shows promise as an insecticide to control CPB.

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