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.

The resistance levels to carbamate (CB) and organophosphate (OP) insecticides were determined by topical application in 14 field strains of Nilaparvata lugens. The resistance levels to CB and OP were 1.3~47.5-fold and 1.4~14.4-fold higher than a susceptible strain, respectively. A quantitative sequencing (QS) protocol was established to determine the allele frequencies of four acetylcholinesterase point mutations putatively associated with CB and OP resistance. The allele frequencies of the four mutations (G119A, F/Y330S, F331H and I332L) in field strains ranged from ca. 0.0~51.7%, 0.0~88.9%, 2.5~47.7%, 5.1~56.0% and 6.7~57.3%, respectively. The F331H and I332L were tightly linked each other, suggesting these mutations may occur simultaneously. In the correlation analysis, G119A was not well correlated with actual resistance levels (r2= < 0.232), whereas F331H and I332L showed a better correlation with the resistance levels of benzofuranyl methylcarbamates (r2= 0.595). This finding indicates that F331H and I332L mutation frequencies may be used as molecular markers for detecting carbamate resistance in N. lugens. A QS protocol detecting the F331H and I332L mutation frequencies would be employed as a supportive tool for rapid monitoring of CB insecticide resistance levels in N. lugens

The resistance levels against carbamates (CB) and organophosphates (OP) were determined through bioassay and quantitative sequencing (QS) methods in 16 field populations of Nilaparvata lugens. The resistance levels to CB and OP by bioassay were 1.3~47.5-fold and 1.4~14.4-fold higher than a susceptible strain, respectively. The QS protocol was established to determine the allele frequencies of eight point mutations on acetylcholinesterase putatively associated CB and OP resistance. The allele frequencies of four mutations in local populations (G119A, F/S330Y, F331H and I332L) ranged from ca. 0.0~51.7%, 1.0~44.3%, 8.5~57.3% and 7.12~56.6%, respectively. The average prediction limits were –9.6±5.1~7.7±2.9%. The F330Y, F331H and I332L were tightly linked each other, suggesting these mutations may occur simultaneously. In the correlation analysis, G119A was not well correlated with both insecticides (r2= less 0.25), whereas F/S330Y, F331H and I332L showed better correlation with the resistance levels of carbamate (r2=0.590) than organophosphate (r2=0.235). This finding indicates that F/S330Y, F331H and I332L mutation frequencies are suitable for detecting carbamate resistance in N. lugens. QS will be applicable for the rapid monitoring of resistance levels to CB insecticides in N. lugens.

A quantitative sequencing (QS) protocol was established for predicting the frequencies of the A298S and G324A mutations in the diamondback moth (Plutella xylostella) type-1 acetylcholinesterase locus, putatively involved with organophosphate and carbamate insecticideresistance. The nucleotide signal ratio at each mutation site was generated from sequencing chromatograms and plotted against the corresponding resistance allele frequency. Frequency prediction equations were generatedfrom the plots by linear regression, and the signal ratios were shown to highly correlated with resistance allele frequencies (r2>0.987). QS analysis of 15 representative regional field populations of DBM in Korea revealed that the allele frequencies of both A298S and G324A were over 70% in most field populations. As judged by inhibition assay, all populations showed resistance to paraoxon, DDVP, carbaryl, and carbofuran. In addition, different DBM strains exhibited differential sensitivities to both OPs and CBs depending on the structure of inhibitor, implying that the resistance of DBM against OPs and CBs is saturated and widespread in Korea.

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.

A quantitative sequencing (QS) protocol that detects the frequencies of sodium channel mutations (M815I, T917I and L920F) responsible for knockdown resistance in permethrin-resistant head lice was tested as a population genotyping method. Genomic DNA fragments of the sodium channel α-subunit gene that encompass the three mutation sites were PCR-amplified from individual head lice with either resistant or susceptible genotypes, and combined together in various ratios to generate standard DNA template mixtures for QS. Following sequencing, the signal ratios between resistant and susceptible nucleotides were calculated and plotted against the corresponding resistance allele frequencies. Quadratic regression coefficients of the plots were close to 1, demonstrating that QS is highly reliable for the prediction of resistance allele frequencies. Prediction of resistance allele frequencies by QS in several globally collected lice samples including 12 Korean lice populations suggested that permethrin resistance varied substantially amongst different geographical regions. Three local populations of Korean lice were determined to have 9.8-36.7% resistance allele frequencies, indicating that an urgent resistance management is needed. QS should serve as a preliminary resistance monitoring tool for proper management strategies by allowing early resistance detection.