Resistance mechanisms to mitochondrial electron transport inhibitors in a field-collected strain of Tetranychus urticae Koch (Acari: Tetranychidae)

A Belgian field strain (MR-VP) of Tetranychus urticae (Koch) (Acari: Tetranychidae) exhibits different levels of resistance to four frequently used METI (mitochondrial electron transport inhibitor)-acaricides, i.e. tebufenpyrad, fenpyroximate, pyridaben and fenazaquin. Resistance factors for these compounds were 184, 1547, 5971 and 35, respectively. A 23.5-fold increase in 7-ethoxy-4-trifluoromethylcoumarin O-deethylation activity suggested that metabolic resistance through elevated levels of cytochrome P450 dependent monooxygenase-activity is a possible resistance mechanism.However, synergism studies with different metabolic inhibitors revealed some contrasting resistance mechanisms between the METI-acaricides. Tebufenpyrad resistance could only be synergized after pre-treatment with the monooxygenase inhibitor piperonyl butoxide (PBO), whereas pyridaben resistance was strongly synergized both by PBO and the esterase inhibitor S,S,S-tributylphosphorotrithioate (DEF). Resistance levels to fenpyroximate could neither be suppressed by PBO nor by DEF. Although METI-acaricides are structurally related, these findings probably reflect a different role of esterases and mono-oxygenases in metabolic detoxification between these compounds. The overall lack of synergism by diethylmaleate (DEM) suggests that glutathione-S-transferases are not an important factor in resistance to METIs.Reciprocal crosses between susceptible females and resistant males showed no maternal effect, and resistance to METI-acaricides was inherited generally as a dominant trait. Backcrosses with F1 females revealed striking differences in the mode of inheritance. Although resistance to fenpyroximate and pyridaben was under monogenic control, resistance to tebufenpyrad was under control of more than one gene.     

Cross-resistance, inheritance, and biochemistry of mitochondrial electron transport inhibitor-acaricide resistance in Tetranychus urticae (Acari: Tetranychidae).

Resistance of the twospotted spider mite, Tetranychus urticae Koch, to acaricides acting as mitochondrial electron transport inhibitors (METIs) is an increasing problem. Because of their high levels of cross-resistance to all commercially available METI-acaricides, a Japanese strain (AKITA) and an English strain (UK-99) of T. urticae were investigated in detail. Larvae of both strains, AKITA and UK-99, showed 1,100- and 480-fold resistance against pyridaben, 870- and 45-fold resistance against fenpyroximate, and 33- and 44-fold resistance against tebufenpyrad, respectively, in a foliar spray application bioassay compared with the susceptible strain GSS. These resistance factors remained stable even when maintained in the laboratory without further selection. Furthermore, strain AKITA showed cross-resistance to dicofol. The METI resistant strains AKITA and UK-99 showed 2.4- and 1.7-fold enhanced O-ethoxycoumarin O-deethylation (cytochrome P450) activity. Increased oxidative metabolism of the METI-acaricides in the resistant strains could be partially suppressed in vivo by the monooxygenase-inhibitor piperonyl butoxide. Reciprocal crosses of homozygous, diploid females and hemizygous, haploid males of strains GSS (susceptible) and AKITA (resistant) revealed that resistance to pyridaben and fenpyroximate was inherited incompletely dominant with slight differences between maternal and paternal inheritance. This is the first attempt to mechanistically describe METI-acaricide resistance in T. urticae. The implications for resistance management strategies are discussed.     

Multiple resistance and biochemical mechanisms of pyridaben resistance in Tetranychus urticae (Acari: Tetranychidae)

A field colony of Tetranychus urticae (Koch) (Acari: Tetranychidae) resistant to pyridaben was selected with pyridaben successively for 20 generations to produce the PR-20 strain. Resistance and multiple resistance levels of the PR-20 strain to 15 acaricides were determined using a spray bioassay. The PR-20 strain was extremely resistant to pyridaben (resistance ratio [RR] = 240]. The strain exhibited extremely strong resistance to fenpyroximate (RR=373) and acrinathrin (RR=329) and strong resistance to benzoximate (RR=84). An RR = 10-40 was observed with abamectin, fenazaquin, fenbutatin oxide, fenpropathrin, and tebufenpyrad. The PR-20 strain showed low levels of resistance (RR <10) to azocyclotin, bromopropylate, chlorfenapyr, dicofol, milbemectin, and propargite. Synergist experiments with different metabolic inhibitors revealed that piperonyl butoxide (PBO), a mixed function oxidase (MFO) inhibitor, had the greatest effect on pyridaben resistance. PBO significantly caused pyridaben resistance in the PR-20 strain to drop to the full susceptibility level of the susceptible (S) strain. However, there was no significant difference in MFO activities measured using a model substrate between the S and PR-20 strains. These results suggest that use of certain acaricides with little multiple resistance or PBO will be useful for the management of pyridaben resistance in the field.     

A mutation in the PSST homologue of complex I (NADH:ubiquinone oxidoreductase) from Tetranychus urticae is associated with resistance to METI acaricides

The acaricidal compounds pyridaben, tebufenpyrad and fenpyroximate are frequently used in the control of phytophagous mites such as Tetranychus urticae, and are referred to as Mitochondrial Electron Transport Inhibitors, acting at the quinone binding pocket of complex I (METI-I acaricides). Because of their very frequent use, resistance evolved fast more than 20 years ago, and is currently wide-spread. Increased activity of P450 monooxygenases has been often associated with resistance, but target-site based resistance mechanisms were never reported.Here, we report the discovery of a mutation (H92R) in the PSST homologue of complex I in METI-I resistant T. urticae strains. The position of the mutation was studied using the high-resolution crystal structure of Thermus thermophilus, and was located in a stretch of amino acids previously photo-affinity labeled by fenpyroximate. Selection experiments with a strain segregating for the mutant allele, together with marker-assisted back-crossing of the mutation in a susceptible background, confirmed the involvement of the mutation in METI-I resistance. Additionally, an independent genetic mapping approach; QTL analysis identified the genomic region of pyridaben resistance, which included the PSST gene. Last, we used CRISPR-Cas9 genome editing tools to introduce the mutation in the Drosophila PSST homologue.     

Multiple Resistance and Biochemical Mechanisms of Dicofol Resistance in Tetranychus urticae (Acari: Tetranychidae)

A field colony of Tetranychus urticae (Koch) resistant to dicofol was selected with dicofol successively for 20 generations to produce the DR-20 strain. Resistance and multiple resistance levels of the DR-20 strain to 15 acaricides were determined using a spray bioassay. The DR-20 strain was extremely resistant to dicofol [resistance ratio (RR), 465]. The strain showed extremely strong resistance to acrinathrin (RR, 373) and benzoximate (RR, 197) and strong resistance to bromopropylate (RR, 136), fenbutatin oxide (RR, 65), fenpropathrin (RR, 70), fenpyroximate (RR, 68), and pyridaben (RR, 63). A RR of 11–29 was observed with abamectin, fenazaquin, milbemectin, propagite, and tebufenpyrad. The DR-20 strain exhibited low levels of resistance (RR<3) to azocyclotin and chlorfenapyr. In comparative assays with detoxifying enzymes, the DR-20 strain showed 4.7-fold higher activity in p-nitroanisole-O-demethylation and 1.6-fold higher activities in both α- and β-naphthyl acetate hydrolysis. Synergist experiments with different metabolic inhibitors revealed that piperonyl butoxide, iprobenfos, triphenyl phosphate, and 4, 4-dichloro-α-methyl benzhydrol had little or no synergistic activity in the susceptible and DR-20 strains. These results suggest that employment of certain acaricides with little multiple resistance will be useful for the management of dicofol resistance in the field.     

Inheritance of pyriproxyfen resistance in the whitefly, Bemisia tabaci (Q biotype)

The inheritance of resistance to pyriproxyfen, an insect growth regulator (a juvenoid, with ovicidal and larvicidal activities), was studied in the whitefly Bemisia tabaci (Gennadius). Two parental strains, both belonging to Q biotype, were assayed with pyriproxyfen; a susceptible strain (ALM-1) originating from Spain and a pyriproxyfen-resistant one (Pyri-R) from Israel. The resistance ratio between the two parental strains was approximately 7,000-fold. Concentration-mortality lines for F(1) heterozygous females from reciprocal crosses (SS female symbol X R male symbol and RR female symbol X S male symbol ) were derived by statistical modelling and proved intermediate to those of the parents. The pooled degree of dominance from both reciprocal crosses was +0.26, indicating that resistance was incompletely or partially dominant. Mortality curves for F(2) males produced by virgin F(1) heterozygous females displayed a broad plateau at 50% mortality, indicating that resistance to pyriproxyfen in B. tabaci is conferred primarily by a mutant allele at a single locus. The role of arrhenotoky in influencing the mode of inheritance of resistance, and its selection in field populations, is discussed.     

Genetic basis of resistances to chlorfenapyr and etoxazole in the two-spotted spider mite (Acari: Tetranychidae)

We studied the genetic basis of resistance to two new acaricides, chlorfenapyr and etoxazole, which have different chemical structures and modes of action in the two-spotted spider mite, Tetranychus urticae Koch. The resistance ratios calculated from the LC50s of resistant and susceptible strains were 483 for chlorfenapyr and >100,000 for etoxazole. Mortality caused by the two acaricides in F1 progeny from reciprocal crosses between the resistant and susceptible strains indicated that the modes of inheritance of resistance to chlorfenapyr and etoxazole were completely dominant and completely recessive, respectively. Mortality in F2 progeny indicated that for both acaricides, the resistance was under monogenic control. Repeated backcross experiments indicated a linkage relationship among the two acaricide resistances and malate dehydrogenase, although phosphoglucoisomerase was not linked with them. The recombination ratio between the resistances was 14.8%. From this result, we suggest that heavy spraying of the two acaricides will lead to apparent cross-resistance as a consequence of crossing over; the two resistance genes are so close to each other that it would be difficult to segregate them once they came together on the same chromosome.     

Field resistance of codling moth against Cydia pomonella granulovirus (CpGV) is autosomal and incompletely dominant inherited

The current appearance of local codling moth populations with resistance to Cydia pomonella granulovirus (CpGV) is an impediment to continuous CpGV application. Therefore, crossing experiments have been performed in order to gain information about the inheritance of resistance. Evidence is presented that the observed field resistance is stably inherited even under non-selective conditions in the laboratory. Offspring of reciprocal F(1) crosses between a susceptible ('S') and a resistant ('R') strain and backcrosses between F(1) and S were bioassayed at different CpGV concentrations. The resistant strain showed 100 times lower susceptibility in 7-day bioassays. The responses of the reciprocal crosses (male S x female R and female S x male R) did not differ significantly, indicating that resistance is autosomally inherited. The median lethal concentration for the F(1) progeny was intermediate between those of its parental strains. Mortality data obtained from the backcrosses suggested that inheritance of resistance is due to a non-additive, polygenic trait.     

二斑叶螨对七种杀螨剂的抗药性测定及其机理研究

室内测定了相对敏感种群(S)和抗性种群(R)对常用7种杀螨剂的敏感性,并测定了羧酸酯酶、谷胱甘肽S-转移酶和乙酰胆碱酯酶3种酶的比活力。结果表明:二斑叶螨Tetranychus urticae Koch R种群已对甲氰菊酯和哒螨灵产生了抗性,抗性倍数分别为5.45和105.47。其中,甲氰菊酯对雌成螨的毒力最低(>3000mg/L),已远远超过田间推荐剂量,不宜继续使用。酶活测定结果表明:谷胱甘肽S-转移酶解毒活性的提高是二斑叶螨对甲氰菊酯产生抗性的原因之一;二斑叶螨对哒螨灵抗性的增强可能与羧酸酯酶有关。     

Evaluation of Resistance Pattern to Fenpyroximate and Pyridaben in Tetranychus Urticae collected from Ggreenhouses and Apple Orchards using Lethal Concentration-slope Relationship

This study aimed to monitor the present and future developments of the resistance of Tetranychus urticae Koch to fenpyroximate and pyridaben, using the relationship of the LC₅₀ and slope of the concentration-mortality line in a probit model, for the provision of reliable resistance management tactics. Tetranychus urticae populations were collected from 16 commercial greenhouses, where various crops were cultivated, as well as from 10 apple orchards throughout Korea. The resistance to fenpyroximate and pyridaben of each population was estimated by calculating the median lethal concentration (LC₅₀), resistance ratio (RR) and slope of the concentration-mortality regression. Most of the greenhouse populations exhibited moderate levels of resistance, whereas the apple orchard populations showed only low levels, indicating that T. urticae populations in greenhouses were more strongly selected than those in apple orchards. Four population groups were established based on either the habitats (greenhouse and apple orchard) or acaricides (fenpyroximate and pyridaben). To test the hypothesis, “the slope is greatest at low and high levels of resistance,” the slopes were regressed as a function of the LC₅₀, and fitted to a polynomial regression. The polynomial regression model explained this relationship well for the four population groups (p < 0.05), indicating that the development of resistance toward fenpyroximate or pyridaben was consistent with the gradient. A laboratory selection study agreed with the results from both acaricide field populations. These results suggest that the gradient was a good indicator of the susceptibility of T. urticae to genetic variations, which was related to the LC₅₀. The application of these findings is also discussed in relation to the resistance management of T. urticae.     

Paternal inheritance of egg traits in mice: a case of genomic imprinting

Eggs from reciprocal hybrids between the C57BL/6By and BALB/cBy strains were tested for their susceptibility to attack by hyaluronidase and pronase. There were significant reciprocal differences between the F1 females in the responses of their unfertilized eggs to both enzymes. The F1 hybrids from BALB mothers showed the increased susceptibility characteristic of C57BL whilst the F1 hybrids with C57BL mothers were more resistant to both enzymes, like BALB mice. Eggs from the four kinds of reciprocal F2 hybrid females also showed patroclinous patterns of susceptibility. A patroclinous difference was found between reciprocal crosses of the CXBD and CXBE recombinant inbred strains but not in crosses between recombinant inbred strains with similar phenotypes. Cross fostering did not alter the phenotypes of the C57BL and BALB females or those of their reciprocal F1 hybrids. The findings are interpreted in terms of differential genomic imprinting of paternally inherited information. The possible general usefulness of patroclinous differences between reciprocal F1 females in revealing differences in imprinting is noted.     

Life Table and Sensitivity Analysis as Fitness Evaluation Method of Fenpyroximate and Pyridaben Resistant Twospotted Spider Mite (Tetranychus urticae Koch)

Fitness associated with pesticide resistance was assessed in twospotted spider mites, Tetranychus urticae Koch, using life table analysis. Two strains resistant to fenpyroximate (FR) and pyridaben (PR), and one susceptible strain (S) were included in this study. Two reciprocal crosses (FR♂xS♀ and PR♂xS♀) were established by conventional crossing procedure. Intrinsic rate of natural increase (r_m) was used as a comparative parameter of reproductive fitness among 3 parent strains (FR, PR, and S) and two reciprocal crosses. Sensitivity analysis was performed to evaluate significances of biological factors contributing to r_m. The r_m values were not significantly different from each other, with an exception of FR♂xS♀ cross and ranged from 0.250 to 0.274 per day. Sensitivity analysis revealed that birth rate (b) and mortality (μ) effects could account for the effect on r_m in the two resistant strains. Contributions of b and μ to r_m was markedly different in two resistant strains; high contribution in b and low in μ. Age at the first reproduction (t) and age at the last reproduction (t_ω) made roughly equal contributions to r_m between the strains and the crosses. This study clearly showed that how r_m was affected by various biological factors and provided a basic analytical tool for pesticide resistant management.     

截形叶螨对哒螨灵、阿维菌素和阿维·哒螨灵的抗性选育和抗性稳定性研究

【目的】明确截形叶螨Tetranychus truncatus Ehara对哒螨灵、阿维菌素和阿维·哒螨灵3种田间常用药剂产生抗性的速率和稳定性,为叶螨的抗性综合治理提供一定的理论依据。【方法】采用室内生测法,对截形叶螨进行药剂的抗性筛选、衰退和再恢复规律研究。【结果】经过连续30代的药剂汰选,截形叶螨对哒螨灵、阿维菌素和阿维·哒螨灵3种药剂产生了不同程度的抗药性,抗性指数分别达到197.50、19.56和12.57;停止喷药后,其抗性都有所下降,其中截形叶螨对哒螨灵的抗性最不稳定,培育至30代后,抗性衰退率达到63.54%,对阿维菌素的抗性较为稳定,抗性衰退率为23.30%;再次恢复用药后,截形叶螨对哒螨灵、阿维菌素和阿维哒螨灵抗性再度回升,以抗哒螨灵品系的抗性恢复最快,药剂汰选30代后,增长率达到了58.47%,阿维·哒螨灵次之(增长率为38.67%),抗阿维菌素的品系抗性恢复最慢,增长率仅为22.86%。【结论】截形叶螨对哒螨灵抗性不稳定,停止用药后,敏感性易恢复,对阿维菌素和阿维·哒螨灵的抗性较稳定,一旦抗性产生不易衰退,故田间应用时应交替轮换用药。     

Genetic analysis and cross-resistance spectrum of a laboratory-selected chlorfenapyr resistant strain of two-spotted spider mite (Acari: Tetranychidae)

A laboratory susceptible strain of Tetranychus urticae was selected with chlorfenapyr resulting in a resistant strain. After 12 cycles of exposure, the resistance ratio (RR) calculated from the LC50s of susceptible and selected strain was 580. The resistant strain was screened with 16 currently used acaricides for cross-resistance. Cross-resistance was detected with amitraz (RR = 19.1), bifenthrin (RR = 1.3), bromopropylate (RR = 7.5), clofentezine (RR = 29.6) and dimethoate (RR = 17.6). No cross-resistance was detected with the new molecules acequinocyl, bifenazate and spirodiclofen. Mortality caused by chlorfenapyr in the F1 progeny from reciprocal crosses between both strains indicated that the mode of inheritance was incomplete recessive. Mortality in F2 progeny indicated that the resistance was under the control of more than one gene. Synergist experiments with S,S,S-tributylphosphorotrithioate (DEF), piperonylbutoxide (PBO) and diethylmaleate (DEM), which are inhibitors of esterases, monooxygenases and glutathion-S-transferases respectively, suggested a major role of esterases in the resistance to chlorfenapyr.     

Resistance of Brevipalpus phoenicis (Geijskes) (Acari: Tenuipalpidae) to acaricides that inhibit cellular respiration in citrus: cross-resistance and fitness cost

Acaricides that inhibit cellular respiration play an important role in the control of Brevipalpus phoenicis (Geijskes) in citrus groves in Brazil. Studies were conducted to evaluate: (a) the variability in the susceptibility among B. phoenicis populations collected from citrus groves to cyhexatin, azocyclotin, propargite and sulphur; (b) cross-resistance relationships between propargite and azocyclotin, cyhexatin, dinocap, pyridaben and sulphur; and (c) the fitness cost associated with propargite resistance in B. phoenicis under laboratory conditions. A residual-type contact bioassay was used to characterize the susceptibility. The susceptibility was estimated with diagnostic concentrations based LC(95) of each acaricide. The cross-resistance was evaluated by characterizing the concentration-mortality responses of susceptible (S) and propargite-resistant (Propargite-R) strains. The fitness cost was evaluated by measuring the biological parameters of S and Propargite-R strains on citrus fruits at 25 +/- 1 degrees C and fotophase of 14h. Significant differences in the susceptibility of B. phoenicis were detected at diagnostic concentration of cyhexatin (survivorship from 16.3% to 80.5%), azocyclotin (from 3.0% to 15.0%), propargite (from 1.0% a 71.6%) and sulphur (from 9.0% to 82.6%). A low intensity of cross-resistance was detected between propargite and the acaricides azocyclotin (1.8-fold), cyhexatin (4.6-fold), dinocap (3.5-fold) and pyridaben (3.5-fold). On the other hand, the intensity of cross-resistance to sulphur (> 111-fold) was very high. There was no fitness cost associated with B. phoenicis resistance to propargite, based on biological parameters evaluated. Therefore, the use of these acaricides should also be done very carefully in resistance management of B. phoenicis to acaricides.     

Tebufenpyrad (Pyranica®) resistance detected in two-spotted spider mite Tetranychus urticae Koch (Acari: Tetranychidae) from apples in Western Australia

A strain (WA) of Tetranychus urticae Koch was collected from apples in Western Australia following a reported field-control failure of tebufenpyrad. The WA strain had been exposed to five tebufenpyrad applications over four seasons. The tebufenpyrad resistance in the WA strain was confirmed at 63.29× which is the first reported instance of tebufenpyrad resistance in T. urticae. The tebufenpyrad resistance in T. urticae conferred a >210× cross-resistance to pryidaben, a 24.60× cross-resistance to fenpyroximate and a 2.20× cross-resistance to chlorfenapyr.Exp Appl Acarol 22: 633–641 © 1998 Kluwer Academic Publishers     

The Effect of Patch Size and Persistence of Host Plants on the Development of Acaricide Resistance in the Two-spotted Spider Mite Tetranychus Urticae (Acari: Tetranychidae)

Spatial and temporal characteristics of host plants can influence the population biology of the herbivores feeding on them. In this study, I examined the effect of variation in host plant characteristics on the development of acaricide resistance in the two-spotted spider mite Tetranychus urticae, a widely distributed agricultural pest. This investigation examined the geographic variation in the degree of resistance to two new types of acaricide, pyridaben and fenpyroximate. From mortality tests at field-level concentrations of the acaricides, many populations collected from fruit trees and roses had a high frequency of resistant individuals for acaricides while almost all populations collected from herbaceous crops had low frequencies of resistant individuals. These results, combined with those from a previous allozyme study, indicate that patch size and persistence of host plants regulate the population structure of the mites including gene flow between populations and, by extension, the development of acaricide resistance.     

Inheritance of resistance to Bacillus thuringiensis subsp. kurstaki in Trichoplusia ni

The genetic inheritance of resistance to a commercial formulation of Bacillus thuringiensis subsp. kurstaki was examined in a Trichoplusia ni colony initiated from a resistant population present in a commercial vegetable greenhouse in British Columbia, Canada. Progeny of F(1) reciprocal crosses and backcrosses between F(1) larvae and resistant (P(R)) and susceptible (P(S)) populations were assayed at different B. thuringiensis subsp. kurstaki concentrations. The responses of progeny of reciprocal F(1) crosses were identical, indicating that the resistant trait was autosomal. The 50% lethal concentration for the F(1) larvae was slightly higher than that for P(S), suggesting that resistance is partially recessive. The responses of both backcross progeny (F(1) x P(R), F(1) x P(S)) did not correspond to predictions from a single-locus model. The inclusion of a nonhomozygous resistant parental line in the monogenic model significantly increased the correspondence between the expected and observed results for the F(1) x P(R) backcross but decreased the correspondence with the F(1) x P(S) backcross results. This finding suggests that resistance to B. thuringiensis subsp. kurstaki in this T. ni population is due to more than one gene.     

朱砂叶螨对三种杀螨剂的抗性选育与抗性风险评估

为评价朱砂叶螨Tetranychus cinnabarinus对3种杀螨剂的抗性风险,在实验室抗性品系选育基础上,应用数量遗传学中的域性状分析法,研究了朱砂叶螨北碚种群对甲氰菊酯、阿维菌素和哒螨灵3种杀螨剂的抗性现实遗传力,并对3种药剂在不同杀死率下抗性发展的速率进行了预测。结果表明：分别单一连续汰选16代后,朱砂叶螨对甲氰菊酯、阿维菌素的抗性倍数分别达26.54和4.51倍,对哒螨灵表现为敏感性降低（抗性倍数为1.16倍）;朱砂叶螨对甲氰菊酯、阿维菌素和哒螨灵的抗性现实遗传力分别为0.2472,0.1519和0.0160。在室内选择条件下,杀死率为50%～90%时,要获得10倍抗性,甲氰菊酯仅需要13～6代,阿维菌素需要约21～10代;哒螨灵需要约197～89代;在田间选择,三种药剂都将需要更长的时间。抗性筛选16代结果表明,抗性风险较高的是菊酯类的甲氰菊酯,其次是生物源农药阿维菌素,杂环类的哒螨灵抗性风险较小。试验结果可为朱砂叶螨抗性治理提供参考。     

Residual contact vial bioassay for the on-site detection of acaricide resistance in the two-spotted spider mite

To develop a less technique-dependent bioassay technique that can be conveniently used by practitioners or farmers in the field for the monitoring of acaricide resistance of the two-spotted spider mite, Tetranychus urticae Koch, a residual contact vial (RCV) method was established using a 5-ml glass vial coated with acaricides. The RCV bioassay procedures were optimized by using abamectin and tebufenpyrad, two widely used acaricides. The diagnostic concentrations causing 100% mortality within 8 h post-treatment in a susceptible strain of T. urticae was set at 30 and 60 ppm for abamectin and tebufenpyrad, respectively. The vial-coated pesticides were stable at least one year when stored at ? 20 °C as determined by HPLC. There was no significant difference in the bioassay results in repeated RCV bioassay by three different experimenters, indicating its high reproducibility and reliability. RCV-based resistance monitoring of 15 field populations of mites revealed that abamectin resistance begins to spread but tebufenpyrad resistance is already prevalent in Korea. The RCV diagnostic kit, when used by practitioners or farmers on site, should provide crucial information for the selection of most suitable acaricides for different field populations of T. urticae.