An easy and rapid resistance detection protocol for the Western flower thrips Frankliniella occidentalis was established based on the residual contact vial bioassay (RCV), in which insecticide resistance levels can be estimated at 8 h-post treatment of diagnostic doses. The RDA strain was used as a reference susceptible strain, which has been reared under laboratory conditions over 10 years without exposure to any insecticides. Seven insecticides were tested for the determination of diagnostic dose. Among them, five insecticides (chlorfenapyr, acrinathrin, spinosad, emmamectin benzoate and thiamethoxam, ranged as 0.03 ~ 0.42 μg-1cm2) were applicable to the RCV. However, two insecticides (omethoate and imidacloprid) were not able to be used for the RCV because the treated inner surface of glass vials by these insecticides were too viscous, causing non-specific mortality. The RCV detection kit was employed for the estimation of resistance levels for the five insecticides in five local populations. Almost field-collected populations revealed high levels of resistance to the four insecticides (acrinathrin, spinosad, emmamectin benzoate and thiamethoxam) by showing less than 50% mortality. The baseline resistance detection by RCV method will facilitate the selection of proper insecticides for farmers to manage insecticide resistant-populations of F. occidentalis.

Establishment of rapid resistance level detection system is essential step to adopt the adaptive management for the control of various kinds of resistant pest population. Here, we established acaricides resistance detection methods based on residual contact vial bioassay (RCV) and quantitative sequencing methods (QS), and applied to determine the resistance levels from several populations in two-spotted spider mite, Tetranychus urticae, which has been considered as major notorious pest in rose cultivation area in worldwide. 12 acaricides were applicable to the RCV among 19 representative acaricides by showing the dose-dependent mortality within 8 hr, suggesting the acaricide suitability for the RCV might be varied by toxicity mechanism in each acaricides. The QS regression was established for 10 point mutations associated with five number of acaricides resistance such as organophosphate, pyrethroid, abamectin, bifenazate and etoxazol. The 95% prediction level was ranged from 10.8±5.4∼92.2±3.2%. The resistance levels were determined by above two detection methods from a total 12 strains. The laboratory-reared populations were revealed high susceptibility with low resistance allele frequencies to some acaricides, suggesting the several acaricides would be chosen for the control of those populations. However, the field-collected populations were exhibited a severe cross resistance with low susceptibility and high resistance allele frequency to almost tested acaricides, suggesting the current acaricides resistance levels are serious in rose cultivation area in Korea. The RCV and QS methods would be useful for the rapid and accurate collection of valuable information associated with acaricide resistance.

Emergence of resistant two-spotted spider mite (TSSM) can induce the over usage of standard amount of acaricides and result in various side effects. Rapid resistance monitoring is essential step for the efficient management of resistant populations by enabling the selection of appropriate acaricides. Here, we evaluated the 19 acaricides to determine its suitability for residual contact vial bioassay (RCV) by using PyriF strain as a reference. Twelve acaricides (Amitraz Abamectin, Bifenthrin, Bifenazate, Chlorfenapyr, Cyenopyrafen, Cyflumetofen, Endosulfan, Fenothiocarb, Monocrotophos, Omethoate and Tebufenpyrad) revealed the dose-dependent mortality within 8 h, whereas other remaining acaricides (Dicofol, Etoxazole, Fenbutatin oxide, Fenpyroxymate, Flufenoxuron, Spiromesifen and Pyridaben) did not. This finding suggests that the application of RCV method is limited depending on the mode of action and physicochemical properties of each acaricide. Resistance levels to 12 acaricides were determined for four field populations of TSSM by using RCV diagnostic kit. All TSSM populations showed the highest sensitivity to cyflumetofen, indicating that it would be most effective in controling field populations. RCV diagnostic kit would enable to provide crucial information for choosing the most appropriate acaricides in the field.

Emergence of resistant two-spotted spider mite (TSSM) can induce the over usage of standard amount of acaricides and result in various side effects. Rapid resistance monitoring is essential step for the efficient management of resistant populations by enabling the selection of appropriate acaricides. Here, we evaluated the 10 acaricides to determine its suitability on residual contact vial bioassay (RCV) by using PyriF strain as standard. Five acaricides (Abamectin, monocrotophos, tebufenpyrad, bifenazate and cyflumetofen) revealed the dose-dependent mortality within 8 h, whereas other remaining acaricides (Fenbutatin oxide, fenpyroxymate, flufenoxuron, spiromesifen and etoxazole) did not. This finding suggests that the application of RCV method is limited depending on the mode of action and physicochemical properties of each acaricide. Resistance levels to five acaricides (Abamectin, monocrotophos, tebufenpyrad, bifenazate and cyflumetofen) were determined for four field populations of TSSM by using RCV diagnostic kit. All TSSM populations showed the highest sensitivity to cyflumetofen, indicating that it would be most effective in controling field populations. RCV diagnostic kit would enable to provide crucial information for choosing the most appropriate acaricides in the field.

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.