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.

The stable fly, Stomoxys calcitrans L., is an important pest of livestock. Stable flies are considered as mechanical vectors of veterinary disease. Pyrethroids and organophosphates have been widely used for stable fly control. To establish resistance monitoring molecular tool, we isolated the partial cDNA and genomic fragments of voltage-sensitive sodium channel and acetylcholinesterase genes encompassing the well known conserved sites for resistance-associated mutations. To examine the current status of stable fly resistance to pyrethroids and organophosphates mediated by the nerve insensitivity mechanism in Korean population of S. calcitrans, DNA-based genotyping in conjunction with residual contact vial (RCV) bioassay were conducted with 11 representative regional field populations. No resistance-associated mutations were detected in these S. calcitrans populations, suggesting that these populations are likely still susceptible to both pyrethroids and organophosphates. Establishment of RCV bioassay protocol and availalbility of target site sequence information will greatly facilitate resistance monitoring of S. calcitrans in the field.