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.     

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.     

Voltage‐sensitive sodium channel mutations S989P + V1016G in Aedes aegypti confer variable resistance to pyrethroids,DDT and oxadiazines

BACKGROUND

Aedes aegypti is a vector of several important human pathogens. Control efforts rely primarily on pyrethroid insecticides for adult mosquito control, especially during disease outbreaks. A. aegypti has developed resistance nearly everywhere it occurs and insecticides are used. An important mechanism of resistance is due to mutations in the voltage‐sensitive sodium channel (Vssc) gene. Two mutations, in particular, S989P + V1016G, commonly occur together in parts of Asia.

RESULTS

We have created a strain (KDR:ROCK) that contains the Vssc mutations S989P + V1016G as the only mechanism of pyrethroid resistance within the genetic background of Rockefeller (ROCK), a susceptible lab strain. We created KDR:ROCK by crossing the pyrethroid‐resistant strain Singapore with ROCK followed by four backcrosses with ROCK and Vssc S989P + V1016G genotype selections. We determined the levels of resistance conferred to 17 structurally diverse pyrethroids, the organochloride DDT, and oxadiazines (VSSC blockers) indoxacarb (proinsecticide) and DCJW (the active metabolite of indoxacarb). Levels of resistance to the pyrethroids were variable, ranging from 21‐ to 107‐fold, but no clear pattern between resistance and chemical structure was observed. Resistance is inherited as an incompletely recessive trait. KDR:ROCK had a > 2000‐fold resistance to DDT, 37.5‐fold cross‐resistance to indoxacarb and 13.4‐fold cross‐resistance to DCJW.

CONCLUSION

Etofenprox (and DDT) should be avoided in areas where Vssc mutations S989P + V1016G exist at high frequencies. We found that pyrethroid structure cannot be used to predict the level of resistance conferred by kdr. These results provide useful information for resistance management and for better understanding pyrethroid interactions with VSSC. © 2017 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.     

Molecular studies of knockdown resistance to pyrethroids: cloning of domain II sodium channel gene sequences from insects

Knockdown resistance (kdr) is a target-site resistance mechanism that confers nerve insensitivity to DDT and pyrethroid insecticides. In the housefly, Musca domestica, molecular cloning of the para-type sodium channel gene has revealed two amino acid mutations that are associated with kdr and super-kdr resistance phenotypes. Both mutations are located in the domain II region of the channel; Leu1014 to Phe in the hydrophobic segment IIS6 and Met918 to Thr in the IIS4-IIS5 linker. To investigate whether these mutations also occur in other insects, we have designed degenerate primers based on conserved sequences in the domain II region of the sodium channel and used these to PCR amplify this region from insecticide-susceptible strains of eight diverse insect species representing four different insect Orders: Helicoverpa armigera, Plutella xylostella, Spodoptera littoralis (Lepidoptera), Blattella germanica (Dictyoptera), Tribolium castaneum (Coleoptera), Myzus persicae, Aphis gossypii and Phorodon humuli (Hemiptera). The primers amplified closely related para-type sodium channel sequences from each insect with a minimum of 85% amino acid identity between species. All of the sequences contained ‘susceptible’ Leu and Met residues at the positions associated with kdr and super-kdr resistance in the housefly. Recent results detailing the presence of a kdr-type Leu to Phe mutation in pyrethroid-resistant strains of two important agricultural pests, P. xylostella and M. persicae, are discussed. ©1997 SCI     

Resistance and cross-resistance to insecticides in human head lice from Florida and California

Head lice from Florida (SF-HL) and California (SC-HL) were resistant to permethrin compared with colonized susceptible lice from Panama (PA-HL) (5.5- and 3.4-fold, respectively) and Ecuador (EC-HL) (8.5- and 5.3-fold, respectively). Permethrin-resistant lice were cross-resistant to pyrethrum and DDT. DNA sequencing validated presence of kdr-type mutations (T929I and L932F). Permethrin resistance was synergized by piperonyl butoxide (PBO) in SC-HL. Resistance to malathion in SF-HL (1.4–2.2-fold) and SC-HL (2.1–3.6-fold) was detected. Malathion resistance in SF-HL was synergized by S,S,S-tributylphosphorotrithioate (DEF) and by PBO in SC-HL. Malathion/permethrin-resistant lice from the UK (BR-HL) were synergized by DEF but not synerziged by PBO. PBO protected BR-HL from malathion, indicating suppressed desulfuration. Abamectin resistance in SF-HL (1.7–2.5-fold) and SC-HL (1.8–2.3-fold) was detected. No resistance to lindane was found. Thus, multiple resistance mechanisms against commonly available and widely used pediculicides and insecticides are apparently occurring.     

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     

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

     

Incidence and spread of knockdown resistance (kdr) in German Colorado potato beetle (Leptinotarsa decemlineata Say) populations

The Colorado beetle, Leptinotarsa decemlineata (Say), is one of the most important pests in many potato‐producing regions. Colorado beetle infestations are normally kept under economic damage thresholds by applying insecticides such as pyrethroids. Pyrethroids are known to act on voltage‐gated sodium channels and have been used for several decades to control L. decemlineata. Their continuous and widespread use has resulted in the development of resistance, which is often linked to a L1014F target‐site mutation in the voltage‐gated sodium channel, known as knockdown resistance (kdr). Since pyrethroids are used in many potato‐growing regions in Germany, more than 140 L. decemlineata samples were collected and analysed for the presence of the kdr allele by pyrosequencing diagnostics. Results showed that kdr is present in many German L. decemlineata populations, but even its homozygous presence does not substantially compromise the efficacy of recommended label rates of pyrethroids, as demonstrated by bioassays and crossing experiments. The implications of these findings for resistance management are briefly discussed.     

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.     

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.     

Voltage-dependent Na+ channels in pyrethroid-resistant Culex pipiens L mosquitoes

In some insect species, knockdown resistance (kdr) to pyrethroids and DDT is linked to point mutations in the sequence of the para-type voltage-dependent sodium channel gene. The effects of pyrethroids were assayed on six Culex pipiens strains: two were susceptible to pyrethroids and the four others displayed various levels of resistance, but, in each case, a kdr-type mechanism was strongly suggested. Degenerate primers were designed on the basis of the corresponding sequences of the para orthologous gene reported from several orders of insects. These primers were used to amplify the region of the sodium channel gene which includes the positions where the kdr and super-kdr mutations have been found in Musca domestica. As expected, the amplified fragment was highly homologous to the para sequences. The super-kdr-like mutation (methionine to threonine at position 918 of the M domestica para sequence) was never detected in any strain. In contrast, the same kdr mutation (leucine to phenylalanine at position 1014) was present in some Culex pyrethroid-resistant samples. An alternative substitution of the same leucine to a serine was detected in one strain slightly resistant to pyrethroids but highly resistant to DDT. These data have allowed us to design a PCR-based diagnostic test on genomic DNA to determine the presence or the absence of the kdr allele in single C pipiens collected in several countries. The validity of this test was checked by comparing the frequency of the resistance allele and the toxicological data. © 1999 Society of Chemical Industry     

High-throughput approach to detection of knockdown resistance (kdr) mutation in mosquitoes, Culex quinquefasciatus, based on real-time PCR using single-labelled hybridisation probe/melting curve analysis

BACKGROUND: Knockdown resistance (kdr) mutation (L1014F) is a well‐defined mechanism of resistance to pyrethroids and DDT in many insect species. Sensitive detection of the mutations associated with resistance is a prerequisite for resistance management strategies. The authors have developed a new real‐time molecular diagnostic assay based on SimpleProbe^®/melting curve analysis for large‐scale kdr genotyping in the wild population of Culex quinquefasciatus Say, the principal vector of bancroftian filariasis. Melting curve analysis is based on the thermal stability difference between matched and mismatched DNA duplexes. The application of SimpleProbe^® chemistry in insects described here is novel in entomology research. RESULTS: The mosquitoes homozygous for knockdown‐resistant and knockdown‐susceptible allele showed melting peaks at 60.45 °C ( ± 0.25) and 64.09 °C ( ± 0.24) respectively. The heterozygous mosquitoes yielded both peaks at approximately 60.5 °C ( ± 0.2) and 64.20 °C ( ± 0.23). Among the 92 samples genotyped, 16 were found to be homozygous resistant, 44 homozygous susceptible and 32 heterozygous. Comparative assessments were made of all the reported methods for kdr genotyping. CONCLUSION: The present method is cheaper, faster, more reliable and versatile than other alternatives proposed in detecting correct kdr genotypes in mosquitoes. This is the first report using a single‐labelled hybridisation probe to detect point mutations in insect populations. Copyright © 2010 Society of Chemical Industry     

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.     

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     

First molecular genotyping of voltage gated sodium channel alleles in Culex quinquefasciatus populations in Malaysia

A nationwide investigation was performed to detect the presence of 1014 mutation(s) in voltage gated sodium channel (kdr) gene of Culex quinquefasciatus from 14 residential areas across 13 states and a federal territory in Malaysia. Molecular genotyping of kdr mutation was performed via a modified three tubes allele-specific-polymerase chain reaction (AS-PCR) and direct sequencing of kdr gene. Based on the results of AS-PCR, homozygous susceptible (SS) genotype was found in nine out of 14 populations with 38 individuals from a total sample size of 140. Heterozygous (RS) genotype was most predominant (99 individuals) and distributed across all study sites. Homozygous resistance (RR) genotype was detected in Perak (one individual) and Selangor (two individuals). The resistance kdr allele frequencies ranged from 0.1 to 0.55, with the highest being detected in Cx. quinquefasciatus population from Selangor. This study has documented the first field-evolved instance of 1014F mutation in Malaysian mosquitoes and the findings of this study could be utilized in the implementation of strategic measures in vector control programs in Malaysia.     

The Knockdown Resistance (kdr) Mutation in Pyrethroid-Resistant German Cockroaches

A point mutation in thepara-homologous sodium channel gene has been shown to be associated with knockdown resistance (kdr) in several insect species including the German cockroach. In this study, we analyzed the genomic organization of the region where thekdrmutation resides and then performed polymerase chain reaction (PCR) and sequencing using genomic DNA as the template to detectkdrmutation in 24 pyrethroid-resistant German cockroach strains, most of which have been collected recently from the field. Thekdrmutation, G to C at nt 2979 resulting in a leucine to phenylalanine amino acid substitution, was detected in 20 strains including 2 strains from overseas (China and Germany). Our results clearly indicate that thekdrmutation is widespread in German cockroach populations. However, the super-kdrmutation detected in super-kdrhouse flies was not found in any of the 4 strains that showed higher levels of knockdown resistance. Little correlation was observed between the presence of thekdrmutation and the level of knockdown resistance, suggesting the existence of multiple resistance mechanisms in many of these strains.     

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     

Sodium channel mutations (T929I and F1534S) found in pyrethroid-resistant strains of the cigarette beetle,Lasioderma serricorne (Coleoptera: Anobiidae)

RNA-seq data analysis of cigarette beetle (Lasioderma serricorne) strains having different sensitivities to pyrethroids identified sodium channel mutations in strains showing pyrethroid resistance: the T929I and F1534S mutations. These results suggest that reduced sensitivity of the sodium channel confers the pyrethroid resistance of L. serricorne. Results also showed that the F1534S mutation mostly occurred concurrently with the T929I mutation. The functional relation between both mutations for pyrethroid resistance is discussed.     

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.     

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.