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.     

Determination of acaricide resistance allele frequencies in field populations of Tetranychus urticae using quantitative sequencing

Resistances to monocrotophos, fenpropathrin and abamectin in Tetranychus urticae are primarily conferred by reduced sensitivities of respective target sites [i.e., acetylcholinesterase (TuAChE), voltage-sensitive sodium channel (TuVSSC) and glutamate-gated chloride channel (TuGluCl)], which are due to point mutations (G228S and F439W in TuAChE; L1022V in TuVSSC; G323D in TuGluCl). As a population-based genotyping technique, a quantitative sequencing (QS) protocol was developed for the determination of the resistance-associated mutation frequencies in T. urticae. Standard prediction equations revealed high correlation coefficients (r² = 0.993–0.999), demonstrating that the resistant nucleotide signal ratio is highly proportional to the resistance allele frequencies. The lower and higher detection limits for the four resistance mutations were 3.7–13.1% (7.8 ± 3.3%) and 89.4–97.3% (93.3 ± 3.2%), respectively, suggesting that QS can be employed as a preliminary monitoring tool for the detection of resistance allele frequencies, which ranged approximately 7.8–93.3% at the 95% confidence level. The QS was successfully employed for the determination of resistance allele frequencies in 26 T. urticae populations. The two TuAChE mutations responsible for monocrotophos resistance were almost saturated in most field populations. The TuVSSC L1022V mutation tentatively associated with fenpropathrin resistance was also found in 9 field populations. However, the TuGluCl G323D mutation conferring abamectin resistance was found only in one field population, suggesting that abamectin resistance is not yet widespread. The QS protocol, as an alternative to traditional bioassays, will greatly facilitate resistance monitoring of T. urticae.     

Establishment of an acaricide-susceptible Tetranychus urticae strain and its species confirmation based on morphological and molecular characters

Acquisition of a reference Tetranychus strain that is completely susceptible to acaricides and retains an identical genetic background to acaricide-resistant strains is an essential step in elucidating mechanisms of resistance. To establish a strain completely susceptible to various acaricides, we collected Tetranychus mite populations from several regions in South Korea, including both remote and heavily cultivated regions. We tested their suitability as a susceptible reference strain by determining baseline susceptibility to six acaricides and by determining species identity as Tetranychus urticae. The UD strain, originally collected from a remote island region, was found to be most susceptible to all five major acaricides tested and was confirmed to as T. urticae on the basis of both morphological and molecular evidence. Moreover, molecular phylogenetic analysis revealed that the UD strain is an ancestral strain of other prevalently collected green-type strains. Taken together, we propose that the UD strain can be used as a susceptible reference strain for T. urticae resistance studies as it provides baseline susceptibility to acaricides and possesses a common genetic background with most other acaricide-resistant strains.     

Milbemectin resistance in <Emphasis Type="Italic">Tetranychus urticae</Emphasis> (Acari: Tetranychidae): selection,stability and cross-resistance to abamectin

Studies on artificial laboratory selections with milbemectin, stability of milbemectin resistance and possible cross-resistance with abamectin were carried out with Tetranychus urticae Koch to provide basic information for a milbemectin resistance management program. Selections for resistance and susceptibility to milbemectin were performed in a population of T. urticae, collected from a commercial chrysanthemum field in the State of São Paulo, Brazil. After six selections for resistance and five selections for susceptibility, susceptible (S) and resistant (R) strains of T. urticae to milbemectin were obtained. The resistance ratio (R/S) at the LC₅₀ reached 409-fold value. The stability of milbemectin resistance was also studied under laboratory conditions, using a population with initial frequency of 75% of resistant mites. The frequencies of milbemectin resistance were evaluated monthly for a period of 7 months. In order to observe possible correlation between milbemectin and abamectin resistance, the frequencies of abamectin resistance were also evaluated for that population, during the same period. The frequency of milbemectin resistance decreased from 75 to 14.5%, while the percentage of abamectin resistant mites decreased from 57 to 9.1%, in 7 months. The frequencies of milbemectin and abamectin resistance were also evaluated in 25 field populations of T. urticae, collected from several crops in the State of São Paulo. The frequencies of milbemectin resistance varied from 4.1 to 89.5%, and of abamectin, from 7.0 to 90.5%. A positive and significant correlation was observed between the frequencies of milbemectin and abamectin resistance, indicating positive cross-resistance between these acaricides. The results indicate that abamectin should be avoided for managing milbemectin resistance in T. urticae. This is the first report on milbemectin resistance in T. urticae in Brazil.     

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.     

Monitoring of acaricide resistance of Tetranychus urticae (Acari: Tetranychidae) from Korean apple orchards

Tetranychus urticae populations were collected from ten commercial apple orchards and their susceptibilities to 12 acaricides were tested using a leaf disc bioassay. The resistance of each T. urticae population was reported as the LC₅₀ value, the resistance ratio (RR) and the slope of the probit–concentration regression. Cross resistances of T. urticae populations were estimated using the Spearman's rank correlation coefficient. Most local populations showed low resistance levels (RR ≤ 10). Development of resistance to METI and pyrethroid acaricides differed among local populations. The highest RR value (154.6) was found in the Uiseong population to tebufenpyrad. The Geochang population was highly resistant, especially to METI and pyrethroid acaricides. T. urticae populations collected from Suwon, Chungju, Yeongju and Geochang showed moderate resistance (10 < RR ≤ 40) to more than two acaricides. Resistance ratios to abamectin, chlorfenapyr, fenbutatin-oxide and milbemectin were low (RR ≤ 10) in all populations. The LC₅₀ values of abamectin, chlorfenapyr, fenbutatin-oxide and milbemectin ranged from 0.06 to 0.2 mg/l, from 0.67 to 3.38 mg/l, from 10.12 to 40.85 mg/l and from 0.47 to 3.01 mg/l, respectively. We discuss possible cross-resistance to acaricides using Spearman's rank correlation coefficient.     

Fitness costs associated with low-level dimethoate resistance in Phytoseiulus macropilis

Phytoseiulus macropilis Banks (Acari: Phytoseiidae) is an effective predator of tetranychid mites, but there are no data on its response to pesticides. We investigated the resistance of the predatory mite P. macropilis to the acaricides abamectin and dimethoate, and we examined the fitness costs associated with resistance. Two populations were tested: one from conventional cultivation and another from an area not commercially exploited. After the application of acaricides to the predator, we determined the lethal effects of the acaricides, the instantaneous rate of population increase (r _i ), the predation on Tetranychus urticae Koch (Acari: Tetranychidae) and its ability to locate prey in an olfactometer. P. macropilis exhibited resistance to dimethoate only. The low level of resistance (9.4x) of the predator did not affect their ability to locate prey. However, the dimethoate resistant population was not as effective in contatining prey population when in lower density and exhibited a more pronounced decrease of r _i in the presence of this acaricide, due to the reduced oviposition of the predator, a likely consequence of the different genetic background of this population.     

Influence of dispersal from almonds on the population dynamics and acaricide resistance frequencies of spider mites infesting neighboring cotton

Neighboring almond and cotton fields were sampled for spider mites in four locations in the San Joaquin Valley of California. The dominant species in the almonds wasTetranychus pacificus McGregor. In three cotton sites.T. pacificus was present in significantly higher densities near the almonds on at least one sampling date. In contrast.T. urticae Koch andT. turkestani Ugarov & Nikolski were equally abundant across the cotton fields. Almonds appeared to act as a continuous early-season source ofT. pacificus for cotton, with peaks in aerial dispersal from almonds occurring due to overcrowding, plant water stress, and applications of repellent acaricides. Cotton, which experienced little water stress, supported very high densities of spider mites and so acted primarily as a sink for spider-mite dispersal from almonds and other field crops throughout the growth-season. The frequencies of resistance expressed byT. pacificus andT. urticae were similar between neighboring crops, even if the acaricide had been registered for use only in almonds (cyhexatin) or cotton (dicofol). Thus, longterm acaricide selection and movement of spider mites between the two crops resulted in similar proportions of resistant individuals. In these study sites, large-scale dispersal ofT. pacificus from almonds rarely directly affected acaricide efficacy in cotton, because resistance frequencies were similar for spider mites from the two crops and because acaricide applications were usually made in cotton after dispersal from almonds was completed. In two cotton sites, field selection with dicofol was reversed by subsequent immigration of spider mites from neighboring field crops.     

Monitoring of carbamate and organophosphate resistance levels in Nilaparvata lugens based on bioassay and quantitative sequencing

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 of N. lugens 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' ranges are ca. 0.0–51.7%, 0.0–88.9%, 5.1–56.0% and 6.7–57.3%, respectively. The F331H and I332L were tightly linked to each other, suggesting that these mutations may occur simultaneously. In 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 could therefore be employed as a supportive tool for the rapid monitoring of CB insecticide resistance levels in N. lugens.     

Management of <Emphasis Type="Italic">Tetranychus urticae</Emphasis> (Acari: Tetranychidae) in strawberry fields with <Emphasis Type="Italic">Neoseiulus californicus</Emphasis> (Acari: Phytoseiidae) and acaricides

The purpose of this study was to evaluate the performance of Neoseiulus californicus (McGregor) for the control of Tetranychus urticae Koch in commercial strawberry fields, under greenhouse conditions, in association or not with the use of acaricides. The N. californicus strain used in this study was tolerant or resistant to several pesticides. Three experiments were carried out in the State of São Paulo, Brazil. For the first experiment, the initial infestation of T. urticae was 87.1 active stages per leaflet. Two applications of propargite were made on the first and the 14th day of the experiment. Approximately 2 h after each propargite application, N. californicus was released at a rate of 3.0 and 1.9 adult mites per plant, respectively, for each application. The population of T. urticae decreased from 87.1 to 2.8 mites per leaflet in the first three weeks. After this period, the population of T. urticae was maintained at low levels (≤1.5 mites/leaflet) until the end of the experiment (10th week). Propargite and dimethoate sprayed on the strawberry field did not affect significantly the population of this predaceous mite. For the second experiment, the infestation of T. urticae was 29.1 mites per leaflet, when the acaricide chorfenapyr was applied on the strawberry field. The release of N. californicus (2 mites per plant) was made 2 weeks after spraying the acaricide. The population of T. urticae was maintained at low levels (≤2.8 mites/leaflet) for 8 weeks (evaluation period). The T. urticae infestations in plots with N. californicus were significantly lower than in non-release plots, for the experiments 1 and 2. In the third experiment, the initial infestation of T. urticae was 40.5 mites per leaflet (55.5 active stages/leaflet on release plants; 25.5 active stages/leaflet on non-release plants). Three releases of N. californicus (average rate of 3.0 adult mites/plant), without any acaricide application, were not sufficient to reduce significantly the T. urticae population in release plots (release plants + non-release plants) in 6 weeks from the first release, however, the spider mite population decreased from 55.5 to 7.8 active stages per leaflet on release plants, during this period. Interplant dispersal of N. californicus was low in this strawberry field with high infestation of T. urticae. The studies indicate the viability of the use of this strain of N. californicus for the control of T. urticae in strawberry fields under greenhouse conditions, especially in association with selective acaricides.     

Determination of organophosphate and carbamate resistance allele frequency in diamondback moth populations by quantitative sequencing and inhibition tests

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 (AChE) locus, putatively involved in organophosphate (OP) and carbamate (CB) insecticide resistance. The nucleotide resistant signal ratio at each mutation site was generated from sequencing chromatograms and plotted against the corresponding resistance allele frequency. Frequency prediction equations were generated from the plots by linear regression, and the signal ratios were 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, implying the prevalent state of these resistance-associated mutations. In the AChE inhibition assay, all populations showed reduced sensitivity to paraoxon, DDVP, carbaryl, and carbofuran, supporting the notion that DBM resistance to OPs and CBs is widespread in Korea.     

Evaluation of the poultry red mite, Dermanyssus gallinae (Acari: Dermanyssidae) susceptibility to some acaricides in field populations from Italy

Red mite field populations from seven naturally infested Italian caged laying poultry farms were investigated for their susceptibility to acaricide formulations available on the market, containing amitraz, carbaryl and permethrin. A minimum of 3,000 mites of all stages were collected from each farm and were tested with five acaricide concentrations (5, 10, 20, 50, 100%) plus an untreated control (0%). Field red mite populations were found to be tolerant even with the highest concentrations with carbaryl and permethrin for six (86%) and three (42%) of the investigated farms, respectively (P < 0.05). Furthermore, six (86%) of the investigated farms showed a red mite population susceptible to amitraz at any concentration. Out of the seven field populations tested with amitraz, one population is becoming less tolerant whereas another was the most tolerant to carbaryl and permethrin at any concentration. Data show that the lack of effectiveness of some acaricides is spreading in Europe and call for the adoption of alternative management strategies to avoid development of resistance.     

Diagnostic Molecular Markers for Phosphine Resistance in U.S. Populations of Tribolium castaneum and Rhyzopertha dominica

Stored product beetles that are resistant to the fumigant pesticide phosphine (hydrogen phosphide) gas have been reported for more than 40 years in many places worldwide. Traditionally, determination of phosphine resistance in stored product beetles is based on a discriminating dose bioassay that can take up to two weeks to evaluate. We developed a diagnostic cleaved amplified polymorphic sequence method, CAPS, to detect individuals with alleles for strong resistance to phosphine in populations of the red flour beetle, Tribolium castaneum, and the lesser grain borer, Rhyzopertha dominica, according to a single nucleotide mutation in the dihydrolipoamide dehydrogenase (DLD) gene. We initially isolated and sequenced the DLD genes from susceptible and strongly resistant populations of both species. The corresponding amino acid sequences were then deduced. A single amino acid mutation in DLD in populations of T. castaneum and R. dominica with strong resistance was identified as P45S in T. castaneum and P49S in R. dominica, both collected from northern Oklahoma, USA. PCR products containing these mutations were digested by the restriction enzymes MboI and BstNI, which revealed presence or absence, respectively of the resistant (R) allele and allowed inference of genotypes with that allele. Seven populations of T. castaneum from Kansas were subjected to discriminating dose bioassays for the weak and strong resistance phenotypes. Application of CAPS to these seven populations confirmed the R allele was in high frequency in the strongly resistant populations, and was absent or at a lower frequency in populations with weak resistance, which suggests that these populations with a low frequency of the R allele have the potential for selection of the strong resistance phenotype. CAPS markers for strong phosphine resistance will help to detect and confirm resistant beetles and can facilitate resistance management actions against a given pest population.     

Development of acaricide resistance in Pacific spider mite (<Emphasis Type="Italic">Tetranychus pacificus</Emphasis>) from California vineyards

In recent years, grape growers in California reported failures of acaricides against Tetranychus pacificus McGregor. We collected T. pacificus populations from four vineyards and tested them for resistance to bifenazate, propargite and pyridaben. In addition, we sequenced part of the cytochrome b gene of bifenazate-resistant and -susceptible T. pacificus to test for the presence of mutations reported to confer resistance to the congeneric T. urticae. None of the mutations conferring resistance to bifenazate in T. urticae were present in resistant T. pacificus. Resistance levels ranged from full susceptibility to statistically significant 11-fold resistance to pyridaben, sevenfold resistance to bifenazate and fourfold resistance to propargite compared to a susceptible population. Despite the relatively low levels of resistance detected, we estimated that under the conditions of our study the highest field rates of bifenazate and pyridaben application would cause less than 58 and 66% mortality of adult females in the most resistant populations, respectively. In contrast, field rates of propargite application would cause close to 100% mortality in the least susceptible population. These results highlight a potential link between resistance development and reduced field effectiveness for bifenazate and pyridaben. Finally, T. pacificus may be more tolerant to bifenazate and propargite than T. urticae, since the LC₅₀ values for the susceptible population of T. pacificus were several times higher than LC₅₀’s reported for susceptible T. urticae.     

Variable fitness and reproductive effects of Wolbachia infection in populations of the two-spotted spider mite Tetranychus urticae Koch in China

Maternally inherited Wolbachia bacteria are widely distributed among insects, and their presence usually causes modifications of the host. To understand the evolutionary history of diverse host-Wolbachia associations, we investigated the symbiosis between Wolbachia and the two-spotted spider mite Tetranychus urticae Koch in China. The cytoplasmic incompatibility (CI) level, fecundity, female ratio, host longevity and host development time were examined. Our results indicate that Wolbachia bacteria had variable effects on the reproduction and fitness of Chinese populations of T. urticae. Variability of CI expression within T. urticae ranged from no CI to a strong level of CI in spite of the low variability of the wsp gene. Relative to uninfected mites, infected females in one of the three populations showed enhanced fecundity associated with the infection of Wolbachia. This is the first report of a Wolbachia infection promoting the fecundity of infected females in T. urticae. Furthermore, we found both positive and negative effects of Wolbachia infection on longevity and the development time. The differences in ecological characters may be attributed to both Wolbachia and host genotype.     

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.     

Cloning and different expression of ATP synthase genes between propargite resistant and susceptible strains of Tetranychus cinnabarinus (Acarina: Tetranychidae)

The carmine spider mite, Tetranychus cinnabarinus (Boisduval), is a serious phytophagous mite damaging important crops and can rapidly develop resistance to acaricides. Mitochondrial ATP synthase (F1F0 ATP synthase) is an important target site of acaricides. The role of ATP synthase in acaricide resistance remains unclear at the molecular level. In this study, twelve full-length cDNAs of ATP synthase genes were cloned and characterized from T. cinnabarinus and their expression levels were determined for both progargite-resistant and susceptible strains. The effect of propargite exposure on gene expression was also evaluated. Analyses of gene expression revealed that TcATPsynU-2, TcATPsynF0-2 and TcATPsynF0-4 were significantly down-regulated in the progargite-resistant strain. TcATPsynF0-2 and TcATPsynF0-4 had a strong response to progargite exposure. The results suggest that lower levels of TcATPsynU-2, TcATPsynF0-2 and TcATPsynF0-4 expression might be related to propargite-resistance observed in the resistant T. cinnabarinus. This is the first attempt to identify specific ATPase genes involved in propargite resistance in T. cinnabarinus.     

Functional characterization of the Tetranychus urticae CYP392A11, a cytochrome P450 that hydroxylates the METI acaricides cyenopyrafen and fenpyroximate

Cyenopyrafen is a Mitochondrial Electron Transport Inhibitor (METI) acaricide with a novel mode of action at complex II, which has been recently developed for the control of the spider mite Tetranychus urticae, a pest of eminent importance globally. However, some populations of T. urticae are cross-resistant to this molecule, and cyenopyrafen resistance can be readily selected in the lab. The cytochrome P450s genes CYP392A11 and CYP392A12 have been strongly associated with the phenotype. We expressed the CYP392A11 and the CYP392A12 genes with T. urticae cytochrome P450 reductase (CPR) in Escherichia coli. CYP392A12 was expressed predominately as an inactive form, witnessed by a peak at P420, despite optimization efforts on expression conditions. However, expression of CYP392A11 produced a functional enzyme, with high activity and preference for the substrates Luciferin-ME EGE and ethoxycoumarin. CYP392A11 catalyses the conversion of cyenopyrafen to a hydroxylated analogue (k_cat = 2.37 pmol/min/pmol P450), as well as the hydroxylation of fenpyroximate (k_cat = 1.85 pmol/min/pmol P450). In addition, transgenic expression of CYP392A11 in Drosophila melanogaster, in conjunction with TuCPR, confers significant levels of fenpyroximate resistance.The overexpression of CYP392A11 in multi-resistant T. urticae strains, not previously exposed to cyenopyrafen, which had been indicated by microarray studies, was confirmed by qPCR, and it was correlated with significant levels of cyenopyrafen and fenpyroximate cross-resistance. The implications of our findings for insecticide resistance management strategies are discussed.     

Geographical distribution and origin of acetylcholinesterase mutations conferring acaricide resistance in Tetranychus urticae populations from Turkey

Two-spotted spider mite, Tetranychus urticae Koch (Acari: Tetranychidae), is a cosmopolitan pest species that can feed on more than 1000 host plant species. Historically, organophosphate (OP) and carbamate insecticides have been used to control this extremely polyphagous pest. However, its ability to develop acaricide resistance rapidly has led to failure in control. Mutations in acetylcholinesterase gene (ace), the target-site of OP and carbamate insecticides, have been reported to be one of the major mechanisms underlying this developing resistance. In this study, mutations previously associated with resistance (G119S, A201S, T280A, G328A, F331W/Y) in ace have been screened in 37 T. urticae populations collected across Turkey. All mutations were found in various populations, except G119S. Almost all populations had F331W/Y mutation (being fixed in 32 populations), whereas only two populations harboured A201S mutation, but not fixed. On the other hand, more than half of the populations contained T280A and G328A mutations. In addition, the presence of same haplotypes in populations originating from distinct geographic locations and a wide variety of ace haplotypes might indicate multiple origins of F331W and F331Y mutations; however, this needs further investigation. The results of area-wide screening showed that ace mutations are widely distributed among T. urticae populations. Therefore, the use of this group of insecticides should be limited or only rotational use might be regarded as a resistance management tool due to its different mode of action from other main acaricide groups in T. urticae control across Turkey.