RNA interference (RNAi) has been widely adopted as a primary reverse genetic tool to determine the physiological function of genes of interest. Nevertheless, the lack of optimized RNA delivery method has been a major obstacle for non-model organisms, such as Cimex lectularis. In this study, we have established a RNAi protocol for the silencing of C. lectularis salivary gland-specific cholinesterase (SChE) gene based on micro-injection of double stranded RNA (dsRNA). An aliquot (20 nl) of dsRNA solution (4.5 ng/nl) was injected to body cavity through the arthrodial membrane between metathoracic coxa and sternum of adult females. Observed mortality was less than 5% and at 6-day post injection, while the gene silencing efficiency reached 97~99% at 2-6 day post injection. This result demonstrates the efficacy of injection RNAi via the arthrodial membrane in C. lectularius.

Two point mutations (V419L and L925I) in the voltage-sensitive sodium channel (VSSC) α-subunit gene have been identified in deltamethrin-resistant bedbugs. To predict resistance allele frequencies of sodium channel mutations (V419L and L925I) in bedbugs at a population level, we developed quantitative sequencing (QS) protocol. The signal ratios between resistant and susceptible nucleotides were generated from sequencing chromatogram and plotted against the corresponding resistance allele frequencies. Linear regression coefficients of the plots were close to 1 (r2 = 0.9928 and 0.9998), suggesting that the signal ratios are reliable correlated with the resistance allele frequencies. To enable on-site monitoring of pyrethroid resistance in bed bugs, residual contact vial (RCV) bioassay method was established and used to determine median lethal concentration (LC50) values to deltamethrin for various bed bug strains. Resistance allele frequencies in these bedbug strains predicted by QS were correlated well with the RCV bioassay results, confirming the roles of two mutations in pyrethroid resistance. Taken together, employment of QS in conjunction with RCV bioassay should greatly facilitate resistance monitoring of bedbugs in the field.