Molecular diagnostics for detecting pyrethroid and organophosphate resistance mutations in the Q biotype of the whitefly Bemisia tabaci (Hemiptera: Aleyrodidae)

Pyrethroid and organophosphate resistance-associated mutations have been recently reported in the whitefly Bemisia tabaci (Gennadius), a major pest of protected and outdoor crops worldwide. Here, we developed simple PCR–agarose gel visualization based assays for reliably monitoring the L925I and T929V pyrethroid resistance mutations in the B. tabaci para-type voltage gated sodium channel and the iAChE F331W organophosphate resistance mutation in the acetylcholinesterase enzyme ace1.PCR-RFLP assays were developed for detecting the L925I and the F331W resistance mutations. A highly specific PASA was developed for detecting the T929V mutation. The molecular diagnostic tools were used to monitor the frequency of the resistance mutations in a large number of field caught Q biotype B. tabaci from Crete (Greece), where both organophosphates and pyrethroids are extensively used. The F331W mutation was fixed in all field individuals examined. The pyrethroid resistance mutations were detected in high frequencies: 0.38 and 0.54 for L925I and T929V, respectively. The simple diagnostics are accurate and robust, to be used alongside classical bioassays to prevent ineffective insecticide applications, and for early identification of the spreading of resistant Q biotype populations into new regions around the globe.     

Frequencies of the M918I mutation in the sodium channel of the diamondback moth in China, Thailand and Japan and its association with pyrethroid resistance

In addition to the allele frequencies of the L1014F and T929I mutations which are involved in nerve-insensitive resistance to a pyrethroid, those of the M918I mutation were examined using field strains obtained in China, Thailand, and Japan during 2009-2011. Results show that the resistance allele frequencies at the L1014F site were 89-100%, 97-100% and 65-85%, respectively, for strains in China, Thailand, and Japan. The respective allele frequencies at the T929I site were 86-100%, 70-97% and 58-84% for Chinese, Thai, and Japanese strains. With low frequencies up to 27%, M918I was found in Japan and China, but not in Thailand. The strain homozygous for the M918I and L1014F mutations was established and its resistance level to a pyrethroid was examined. The strain lacks a portion of the sodium channel gene corresponding to the 3′ portion of exon 18a, intron 18, and the 5′ portion of exon 18b. Nevertheless, the strain showed a similar level of resistance to that which was homozygous for the T929I and L1014F mutations.     

Molecular analysis of pyrethroid resistance conferred by target insensitivity and increased metabolic detoxification in Plutella xylostella

BACKGROUND: The pyrethroid resistance of the diamondback moth Plutella xylostella (L.) is conferred by increased gene expression of cytochrome P450 to detoxify the insecticide and/or through gene mutation of the sodium channel, which makes the individual insensitive to pyrethroids. However, no information is available about the correlation between the increased metabolic detoxification and the target insensitivity in pyrethroid resistance. RESULTS: Frequencies of pyrethroid‐resistant alleles (L1014F, T929I and M918I) and two resistance‐related mutations (A1101T and P1879S) at the sodium channel and expression levels of the cytochrome P450 gene CYP6BG1 were examined individually using laboratory and field strains of P. xylostella. Real‐time quantitative PCR analysis using the laboratory strains revealed that levels of larval expression of the resistant strain, homozygous for the pyrethroid‐resistant alleles other than the M918I, are significantly higher than those of the susceptible strain. In the field strains, the expression levels in insects having the same resistant alleles as those of the resistant strains varied greatly among individuals. The expression levels were not significantly higher than those in the heterozygotes. CONCLUSION: Significant correlation between the target insensitivity and the increased metabolic detoxification in pyrethroid resistance of P. xylostella was observed in the laboratory but not in the field. Copyright © 2010 Society of Chemical Industry     

Mutations in the sodium channel associated with pyrethroid resistance in the greenhouse whitefly, Trialeurodes vaporariorum

BACKGROUND: Trialeurodes vaporariorum Westwood is an important pest of protected crops in temperate regions of the world. Resistance to pyrethroid insecticides is long established in this species, but the molecular basis of the mechanism(s) responsible has not previously been disclosed. RESULTS: Mortality rates of three European strains of T. vaporariorum to the pyrethroid bifenthrin were calculated, and each possessed significant resistance (up to 662‐fold) when compared with a susceptible reference strain. Direct sequencing revealed three amino acid substitutions in the para‐type voltage‐gated sodium channel (the pyrethroid and DDT target site) of bifenthrin‐resistant T. vaporariorum at positions previously implicated with pyrethroid or DDT resistance (M918L, L925I and T929I) in other related species. CONCLUSION: This study indicates that resistance to bifenthrin in T. vaporariorum is associated with target‐site insensitivity, and that the specific mutations in the sodium channel causing resistance may differ between localities. Copyright © 2012 Society of Chemical Industry     

The sodium channel gene in Tetranychus cinnabarinus (Boisduval): identification and expression analysis of a mutation associated with pyrethroid resistance

BACKGROUND: The carmine spider mite (CSM), Tetranychus cinnabarinus, is the most harmful mite pest of various crops and vegetable plants. Pyrethroid insecticide fenpropathrin has been used to control insects and mites worldwide, but CSM has developed resistance to this compound. RESULTS: Three synergists together eliminated about 50% resistance against fenpropathrin in the CSM. A point mutation was identified from the sodium channel gene of fenpropathrin‐resistant CSM (FeR) by comparing cDNA sequences between FeR and susceptible (S) sodium channel genes, which caused a phenylalanine (F) to isoleucine (I) change at amino acid 1538 position in IIIS6 of the sodium channel and has been proven to confer strong resistance to pyrethroid in other species. The mRNA expression of the sodium channel gene in the FeR and abamectin‐resistant strain (AbR), which was included as a control, were both relatively lower than in the S. CONCLUSION: These results demonstrate that a mutation (F1538I) is present in the sodium channel gene in FeR of CSM, likely playing an important role in fenpropathrin resistance in T. cinnabarinus, but that decrease in the abundance of sodium channel did not confer this resistance. The F1538I mutation could be used as a molecular marker for detecting kdr resistance in Arachnida populations. Copyright © 2011 Society of Chemical Industry     

Molecular mechanisms and monitoring of permethrin resistance in human head lice

Head lice resistance to permethrin is mainly conferred by the knockdown resistance (kdr) trait, a voltage-sensitive sodium channel (VSSC) insensitivity factor. Three VSSC mutations (M815I, T917I and L920F) have been identified. Functional analysis of the mutations using the house fly VSSC expressed in Xenopus oocytes revealed that the permethrin sensitivity is reduced by the M827I (M815I) and L932F (L920F) mutations when expressed alone but virtually abolished by the T929I (T917I) mutation, either alone or in combination. Thus, the T917I mutation is primarily responsible for permethrin resistance in head lice. Comparison of the expression rates of channel variants indicates that the M815I mutation may play a role in rescuing the decreased expression of channels containing T917I. A step-wise resistance monitoring system has been established based on molecular resistance detection techniques. Quantitative sequencing (QS) has been developed to predict the VSSC mutation frequency in head lice at a population basis. The speed, simplicity and accuracy of QS made it an ideal candidate for a routine primary resistance monitoring tool to screen a large number of wild louse populations as an alternative to conventional bioassay. As a secondary monitoring method, real-time PASA (rtPASA) has been devised for more precise determination of low resistance allele frequencies. To obtain more detailed information on resistance allele zygosity, as well as allele frequency, serial invasive signal amplification reaction (SISAR) has been developed as an individual genotyping method. Our approach of using three tiers of molecular resistance detection should facilitate large-scale routine resistance monitoring of permethrin resistance in head lice using field-collected samples.     

Pyrethroid resistance in the tomato red spider mite, Tetranychus evansi, is associated with mutation of the para-type sodium channel

BACKGROUND: The tomato red spider mite, Tetranychus evansi (Baker and Pritchard), is a serious pest of solanaceous crops in many African countries. In this study an investigation has been conducted to establish whether mutation of the para‐type sodium channel underlies pyrethroid resistance in T. evansi strains collected in Southern Malawi. RESULTS: Two T. evansi strains from Malawi showed tolerance to the organophosphate chlorpyrifos and resistance (20–40‐fold) to the pyrethroid bifenthrin, but were susceptible to two contemporary acaricides (abamectin and fenpyroximate) in insecticide bioassays. Cloning of a 3.1 kb fragment (domains IIS5 to IVS5) of the T. evansi para gene from pyrethroid‐resistant and pyrethroid‐susceptible strains revealed a single non‐synonymous mutation in the resistant strains that results in an amino acid substitution (M918T) within the domain II region of the channel. Although novel to mites, this mutation confers high levels of resistance to pyrethroids in several insect species where it has always been associated with another mutation (L1014F). This is the first report of the M918T mutation in the absence of L1014F in any arthropod species. Diagnostic tools were developed that allow sensitive detection of this mutation in individual mites. CONCLUSION: This is the first study of pyrethroid resistance in T. evansi and provides contemporary information for resistance management of this pest in Southern Malawi. Copyright © 2011 Society of Chemical Industry     

The pyrethroid resistance status and mechanisms in Aedes aegypti from the Guerrero state,Mexico

Dengue is one of the most important vector-borne diseases worldwide and is a public health problem in Mexico. Most programs in dengue endemic countries rely on insecticides for Aedes control. In Mexico, pyrethroid insecticides (mainly permethrin and deltamethrin) have been extensively used over a decade as adulticides and represented a strong selection for insecticide resistance for dengue vectors in several parts of the country. We studied the type, frequency and distribution of insecticide resistance mechanisms in Aedes aegypti from six municipalities in the state of Guerrero selected on the basis of historically intense chemical control and a high risk for dengue transmission. Ae. aegypti eggs were collected from October 2009 to January 2010 using ovitraps. F₁ adults, emerged from these collections, were exposed to permethrin, deltamethrin and DDT in WHO diagnostic tests and showed high resistance levels to both pyrethroids and DDT. This was consistent with the presence of increased metabolic enzyme activities and target site insensitivity due to kdr mutations. Biochemical assays showed elevated esterase and glutathione S-transferase activities in the six municipalities. The V1016I kdr mutation on the IIS6 domain of the sodium channel gene was present in an overall frequency of 0.80. A second mutation, F1534C on the IIIS6 domain of the same gene was also detected, being the first report of this mutation in Guerrero. The multiple resistance mechanisms present in Ae. aegypti from Guerrero state represent a warning for the efficacy of the pyrethroid usage and consequently for the success of the dengue control program.     

Biochemical and molecular diagnosis of insecticide resistance conferred by esterase, MACE, kdr and super-kdr based mechanisms in Italian strains of the peach potato aphid, Myzus persicae (Sulzer)

In this paper we analysed the basis of insecticide resistance in 59 Italian strains of the peach potato aphid Myzus persicae using both molecular and biochemical assays. Our data as a whole clearly indicate that most M. persicae strains (76.3%) have high or extremely high production of an esterase enzyme which sequester and detoxify insecticides with esteric group. Kdr genotypes conferring resistance towards pyrethoids are present in 57.7% of the analysed populations. Moreover, 26.5% of the kdr positive strains possess also the M918T mutation conferring super-kdr phenotype. Strains with modified AChE (MACE) are not so numerous (27.1%), although they can be found almost everywhere in Italy. Considering all the strains analysed, both MACE and kdr phenotypes are associated with high levels of esterase activity. In Central–Southern regions, kdr and MACE resistance mechanisms resulted in linkage disequilibrium. Bioassays performed in order to evaluate the efficacy of a pyrethroid insecticide against a strain possessing a F979S mutation within its para-type sodium channel gene suggests that this amino acid substitution could affect the sodium channel responsivity to pyrethroids.     

Identification of mutations in the para sodium channel of Bemisia tabaci from Crete, associated with resistance to pyrethroids

We investigated the mechanisms of resistance to α-cypermethrin in a Q biotype, highly resistant Bemisia tabaci strain (GRMAL-RP) isolated from Crete. Cytochrome P450-dependent monoxygenase activity with the substrate ethoxycoumarin, and carboxylesterase activity with the substrates α-naphthyl-acetate, β-naphthyl-acetate, and para-nitrophenol acetate were substantially elevated in the GRMAL-RP, compared to the susceptible SUD-S strain, while glutathione-S-transferase activity with the substrate 1-chloro-2,4-dinitrobenzene was not different. The metabolic inhibitors piperonyl butoxide and S,S,S-tributyl phosphorotrithioate synergised cypermethrin toxicity in the GRMAL-RP strain, however, mortality was still lower than that of the susceptible strain, indicating the presence of an additional resistance mechanism. Analysis of the sequence of the IIS4-IIS6 region of the para sodium channel gene of the GRMAL-RP strain revealed two amino acid replacements compared to that of the SUD-S susceptible strain. One is the leucine to isoleucine substitution at position 925 (L925I) previously implicated in B. tabaci pyrethroid resistance and the other is a novel kdr resistant mutation for B. tabaci, a threonine to valine substitution at position 929 (T929V). Genotype analysis showed that the L925I and T929V were present in all GRMAL-RP males tested, at an approximately 1:1 frequency, but never in combination in the same haplotype.     

Frequency and intensity of pyrethroid resistance through the CDC bottle bioassay and their association with the frequency of kdr mutations in Aedes aegypti (Diptera: Culicidae) from Mexico

BACKGROUND

The control of Aedes aegypti (L.), the main urban vector that causes arboviral diseases such as dengue, Chikungunya and Zika, has proved to be a challenge because of a rapid increase in insecticide resistance. Therefore, adequate monitoring of insecticide resistance is an essential element in the control of Ae. aegypti and the diseases it transmits. We estimated the frequency and intensity (Resistance Frequency Rapid Diagnostic Test [F‐RDT] and Resistance Intensity Rapid Diagnostic Test [I‐RDT]) of pyrethroid resistance in populations of Ae. aegypti from Mexico using the bottle bioassay and results were related to the frequencies of knockdown resistance (kdr) mutations V1016I and F1534C.

RESULTS

All populations under study were resistant to the pyrethroids: bifenthrin (99%), d‐(cis–trans)‐phenothrin (6.3% cis, 91.7% trans) and permethrin (99.5%) according to F‐RDT, and showed moderate to high‐intensity resistance at 10× the diagnostic dose (DD) in I‐RDT. Frequencies of the kdr mutation V1016I in Ae. aegypti populations were correlated with moderate permethrin resistance at 10× DD, whereas F1534C mutation frequencies were correlated with high bifenthrin resistance at 5× DD. Both I1016 and C1535 were highly correlated with high‐intensity phenothrin resistance at 1× to 10× DD.

CONCLUSIONS

This study showed that high frequencies of kdr mutations V1016I and F1534C are reflected in the results of F‐RDT and I‐RDT tests. Bioassays in conjunction with the characterization of genetic resistance mechanisms are indispensable in the strategic and rational management of resistance in mosquitoes. © 2018 Society of Chemical Industry

     

Serial invasive signal amplification reaction for genotyping permethrin-resistant (kdr-like) human head lice, Pediculus capitis

Permethrin resistance in the human head louse, Pediculus capitis De Geer (Anopulura: Pediculidae), has been reported worldwide, is associated with the knockdown phenotype, and elicits cross-resistance to DDT and the pyrethrins. Two point mutations, T929I and L932F, in the voltage-sensitive sodium channel α-subunit gene are responsible for permethrin resistance as a resistant haplotype (kdr-like). We have optimized a serial invasive signal amplification reaction (SISAR) protocol for the detection of these mutations using PCR amplified DNA fragments. SISAR distinguished all genotypes with high accuracy in a head louse population from Texas that was heterogeneous in terms of permethrin sensitivity. Using SISAR, resistance-conferring mutations are detected in a high throughput format, facilitating the efficient monitoring of permethrin resistance allele frequency in field populations.     

Target-site resistance to pyrethroids in European populations of pollen beetle, Meligethes aeneus F.

Pollen beetle, Meligethes aeneus F. (Coleoptera: Nitidulidae) is a major univoltine pest of oilseed rape in many European countries. Winter oilseed rape is cultivated on several million hectares in Europe and the continuous use of pyrethroid insecticides to control pollen beetle populations has resulted in high selection pressure and subsequent development of resistance. Resistance to pyrethroid insecticides in this pest is now widespread and the levels of resistance are often sufficient to result in field control failures at recommended application rates. Recently, metabolic resistance mediated by cytochrome P450 monooxygenases was implicated in the resistance of several pollen beetle populations from different European regions. Here, we have also investigated the possible occurrence of a target-site mechanism caused by modification of the pollen beetle para-type voltage-gated sodium channel gene. We detected a single nucleotide change that results in an amino acid substitution (L1014F) within the domain IIS6 region of the channel protein. The L1014F mutation, often termed kdr, has been found in several other insect pests and is known to confer moderate levels of resistance to pyrethroids. We developed a pyrosequencing-based diagnostic assay that can detect the L1014F mutation in individual beetles and tested more than 350 populations collected between 2006 and 2010 in 13 European countries. In the majority of populations tested the mutation was absent, and only samples from two countries, Denmark and Sweden, contained pollen beetles heterozygous or homozygous for the L1014F mutation. The mutation was first detected in a sample from Denmark collected in 2007 after reports of field failure using tau-fluvalinate, and has since been detected in 7 out of 11 samples from Denmark and 25 of 33 samples from Sweden. No super-kdr mutations (e.g. M918T) known to cause resistance to pyrethroids were detected. The implications of these results for resistance management strategies of pollen beetle populations in oilseed rape crops are discussed.     

Determination of knockdown resistance allele frequencies in global human head louse populations using the serial invasive signal amplification reaction

BACKGROUND: Pediculosis is the most prevalent parasitic infestation of humans. Resistance to pyrethrin‐ and pyrethroid‐based pediculicides is due to knockdown (kdr)‐type point mutations in the voltage‐sensitive sodium channel α‐subunit gene. Early detection of resistance is crucial for the selection of effective management strategies. RESULTS: Kdr allele frequencies of lice from 14 countries were determined using the serial invasive signal amplification reaction. Lice collected from Uruguay, the United Kingdom and Australia had kdr allele frequencies of 100%, while lice from Ecuador, Papua New Guinea, South Korea and Thailand had kdr allele frequencies of 0%. The remaining seven countries investigated, including seven US populations, two Argentinian populations and populations from Brazil, Denmark, Czech Republic, Egypt and Israel, displayed variable kdr allele frequencies, ranging from 11 to 97%. CONCLUSION: The newly developed and validated SISAR method is suitable for accurate monitoring of kdr allele frequencies in head lice. Proactive management is needed where kdr‐type resistance is not yet saturated. Based on sodium channel insensitivity and its occurrence in louse populations resistant to pyrethrin‐ and pyrethroid‐based pediculicides, the T917I mutation appears to be a key marker for resistance. Results from the Egyptian population, however, indicate that phenotypic resistance of lice with single or double mutations (M815I and/or L920F) should also be determined. Copyright © 2010 Society of Chemical Industry     

Increased frequency of the T929I and L932F mutations associated with knockdown resistance in permethrin-resistant populations of the human head louse, Pediculus capitis, from California, Florida, and Texas

The resistance levels of different human head louse populations from the USA to 1% permethrin were evaluated using permethrin-impregnated, filter paper disk-contact bioassay. Populations from southern California, south Florida and south central Texas showed 1.5-, 3.1-, and 1.5- to 5.1-fold resistance compared to insecticide-susceptible head louse populations from Panama or Ecuador. Permethrin-resistant or permethrin-susceptible homozygous or heterozygous genotypes were determined from sequences of PCR-amplified genomic DNA fragments of the voltage-sensitive sodium channel α-subunit gene by the presence of a T or C, or both, respectively, at nucleotide positions 36 and 44 in the sequence. The presence of a T at both these positions resulted in the amino acid substitutions, T929I and L932F, respectively. Of the 424 louse samples examined that had the T929I mutation, all also possessed the L932F mutation, indicating that the two mutations were tightly linked. The southern California population was phenotypically determined by bioassay to be comprised of 45% resistant individuals and had a resistant allele frequency of 0.53 by DNA sequence analysis. The south Florida population was phenotypically determined to consist of 87% resistant individuals and had a resistant allele frequency of 0.97. The four Texas populations varied in the level of resistance and in resistant allele frequency. The Mathis population was phenotypically determined to consist of 15% resistant individuals and had a resistant allele frequency of 0.33. However, the populations from San Antonio, Mansfield, and Corpus Christi were likewise phenotyped to have 91%, 94%, and 100%, respectively, resistant individuals and a 0.98, 1.00 and 1.00, respectively, resistant allele frequency. The log survival time versus logit mortality regression lines of susceptible-homozygotes, resistant-homozygotes, and heterozygotes determined that the resistance trait was complete recessive. Thus, the presence of homozygotes of the T929I and L932F mutations in the voltage-sensitive sodium channel correlated well with increased survival time following exposure to permethrin and indicates that a knockdown-type nerve insensitivity mechanism is functioning as the major mechanism causing permethrin resistance in USA head louse populations. Our results substantiate that permethrin resistance in human head louse population in the USA is widespread but variable. Permethrin resistance is highly correlated with the presence of the T929I and L932F point mutations, which are suitable for detection by a variety of DNA-based diagnostic techniques [Pest Manag. Sci. 57 (2001) 968]. Large-scale monitoring of permethrin resistance is possible utilizing these techniques and would provide critical information necessary for the development of an effective resistance management program for pediculosis.     

Diversity and frequencies of genetic mutations involved in insecticide resistance in field populations of the house fly (Musca domestica L.) from China

Insecticides have been extensively used for house fly control in China, with dichlorvos and deltamethrin being widely used. Knowledge about the current status of insecticide resistance and the underlying genetic changes is crucial for developing effective fly control strategies. The susceptibility to dichlorvos and deltamethrin, and the frequencies of genetic mutations involved in insecticide resistance were studied in five field populations of the house fly collected across China. Bioassay results show that flies exhibit 14- to 28-fold resistance to dichlorvos and 41- to 94-fold resistance to deltamethrin, indicating that dichlorvos and deltamethrin resistance are common in house fly populations in China. Molecular analysis reveals that flies from the five various locations carry resistance alleles at multiple loci and have diverse allelic types, different relative frequencies and combinations of each allele. Four non-synonymous single nucleotide polymorphisms (SNPs) (i.e. V260L, G342A/V, F407Y) in acetylcholinesterase (Ace) and two mutations (W251L/S) in a carboxylesterase (MdαE7) were commonly present in the field house flies. The L1014H rather than L1014F mutation in the voltage sensitive sodium channel gene (Vssc) was widely distributed in Chinese house flies. CYP6D1v1, which confers pyrethroid resistance, was found in all the five tested populations in China, although its frequency in house fly from Shandong province was very low. Our results suggest that resistance monitoring and management of house flies should be customized for a given location.     

Estimation of knockdown resistance in diamondback moth using real-time PASA

We have developed a simple and accurate real-time PASA (PCR amplification of specific allele) (rtPASA) protocol for the estimation of pyrethroid resistance allele frequency in pooled DNA samples using the T929I-mutated allele of sodium channel α-subunit gene from the diamondback moth, Plutella xylostella, as a model. Conditions for the rtPASA for the detection of the T929I mutation were optimized by adjusting annealing temperature, template, and primer concentrations. Using standard DNA mixtures of susceptible and resistant alleles in various ratios, a plot of allele frequency versus cycle threshold value (C_t value) was generated for the prediction of allele frequency. The semi log plot was linear within the 1-80% range of resistance allele frequencies with a high correlation coefficient (r²=0.997) when highly stringent conditions (67 °C annealing temperature and 2 ng template DNA) were used. For the detection of lower frequencies of the resistant allele (0.004-1%), another hyperbolic semi log plot was constructed (r²=0.983) using the C_t values obtained from the rtPASA with less stringent conditions (66 °C annealing temperature and 5 ng template DNA). The rtPASA was able to detect the resistant allele at frequency as low as 0.02%. The performance of the rtPASA was evaluated by comparing with the data generated from 50 individual genotypings. We demonstrated that the actual resistance allele frequency of a field population of P. xylostella precisely matched the predicted frequency deduced by our protocol. The rtPASA format is applicable for the detection of additional mutations associated with resistance in other insect pest species and will allow rapid and efficient monitoring of resistance in field populations in a high throughput format.     

Frequency detection of pyrethroid resistance allele in Anopheles sinensis populations by real-time PCR amplification of specific allele (rtPASA)

To investigate the level of pyrethroid resistance in Anopheles sinensis Wiedemann 1828 (Diptera: Culicidae), a major malaria vector in Korea, we cloned and sequenced the IIS4-6 transmembrane segments of the sodium channel gene that encompass the most widely known kdr mutation sites. Sequence analysis revealed the presence of the major Leu-Phe mutation and a minor Leu-Cys mutation at the same position in permethrin-resistant field populations of An. sinensis. To establish a routine method for monitoring resistance, we developed a simple and accurate real-time PCR amplification of specific allele (rtPASA) protocol for the estimation of resistance allele frequencies on a population basis. The kdr allele frequency of a field population predicted by the rtPASA method (60.8%) agreed well with that determined by individual genotyping (61.7%), demonstrating the reliability and accuracy of rtPASA in predicting resistance allele frequency. Using the rtPASA method, the kdr allele frequencies in several field populations of An. sinensis were determined to range from 25.0 to 96.6%, suggestive of widespread pyrethroid resistance in Korea.     

Acetylcholinesterase point mutations putatively associated with monocrotophos resistance in the two-spotted spider mite

Molecular mechanisms of monocrotophos resistance in the two-spotted spider mite (TSSM), Tetranychus urticae Koch, were investigated. A monocrotophos-resistant strain (AD) showed ca. 3568- and 47.6-fold resistance compared to a susceptible strain (UD) and a moderately resistant strain (PyriF), respectively. No significant differences in detoxification enzyme activities, except for the cytochrome P450 monooxygenase activity, were found among the three strains. The sensitivity of acetylcholinesterase (AChE) to monocrotophos, however, was 90.6- and 41.9-fold less in AD strain compared to the UD and PyriF strains, respectively, indicating that AChE insensitivity mechanism plays a major role in monocrotophos resistance. When AChE gene (Tuace) sequences were compared, three point mutations (G228S, A391T and F439W) were identified in Tuace from the AD strain that likely contribute to the AChE insensitivity as predicted by structure analysis. Frequencies of the three mutations in field populations were predicted by quantitative sequencing (QS). Correlation analysis between the mutation frequency and actual resistance levels (LC₅₀) of nine field populations suggested that the G228S mutation plays a more crucial role in resistance (r² = 0.712) compared to the F439W mutation (r² = 0.419). When correlated together, however, the correlation coefficient was substantially enhanced (r² = 0.865), indicating that both the F439W and G228S mutations may work synergistically. The A391T mutation was homogeneously present in all field populations examined, suggesting that it may confer a basal level of resistance.