T929I Mutation Research Articles

Pyrethroid-resistant diamondback moth expresses alternatively spliced sodium channel transcripts with and without T929I mutation

This study revealed two distinct alternatively spliced exons, A1 and A2, encoding part of the domain IIS4–IIS5 of the para-sodium channel gene in the diamondback moth (DBM). Exons A1 and A2, respectively, revealed 79% and 91% identity at the nucleotide and amino acid levels. Both alternative exons included the T929I site, which has been associated with pyrethroid resistance in DBM. In the pyrethroid-resistant strain, susceptible (Thr) and resistant (Ile) amino acids were encoded at the T929I site in exons A1 and A2, respectively, but in the pyrethroid-susceptible strain, only Thr was encoded at the site in both exons. The transcripts containing exon A1 were expressed constitutively in all developmental stages. The transcripts containing exon A2 were also detected in all developmental stages, but the levels were significantly lower in the 3rd and 4th instar larvae. Tissue-specific data from the 4th instar larvae and adults showed that the expression of transcripts containing exon A2 was higher in heads than in bodies. These findings suggest that alternative splicing of the para-sodium channel gene might produce distinct channels with different sensitivities to pyrethroids, possibly in a tissue-specific manner.

Identification of mutations associated with pyrethroid resistance in the para-type sodium channel of the cat flea, Ctenocephalides felis

Knockdown resistance (kdr) to pyrethroid insecticides is caused by point mutations in the pyrethroid target site, the para-type sodium channel of nerve membranes. This most commonly involves alterations within the domain II (S4–S6) region of the channel protein where five different mutation sites have been identified across a range of insect species. To investigate the incidence of this mechanism in cat fleas, we have cloned and sequenced the IIS4–IIS6 region of the para sodium channel gene from seven laboratory flea strains. Analysis of these sequences revealed two amino acid replacements at residues previously implicated in pyrethroid resistance. One is the ‘common’ kdr mutation, a leucine to phenylalanine substitution (equivalent to L1014F of housefly) reported previously in several other insects. The other is a threonine to valine substitution (equivalent to T929V) and is a novel variant of the T929I mutation first identified in diamondback moth. The L1014F mutation was found at varying frequency in all of the laboratory flea strains, whereas the T929V mutation was found only in the highly resistant Cottontail strain. We have developed rapid PCR-based diagnostic assays for the detection of these mutations in individual cat fleas and used them to show that both L1014F and T929V are common in UK and US flea populations. This survey revealed a significant number of fleas that carry only the V929 allele indicating that co-expression with the F1014 allele is not necessary for flea viability.

Knockdown resistance allele frequency in field populations of Plutella xylostella in Korea

Two separate PCR amplification of specific allele (PASA) protocols have been developed and optimized for the rapid detection of the T929I and L1014F mutations, known to confer pyrethroid knockdown resistance by a nerve insensitivity mechanism in the diamondback moth, Plutella xylostella. To investigate the current status of pyrethroid resistance mediated by the nerve insensitivity mechanism in Korean populations of P. xylostella, PASA genotyping was conducted with 10 representative regional field populations in conjunction with the bioassay using fenvalerate and cypermethrin. Moderate to high levels of pyrethroid resistance were determined in all the regional populations investigated as judged by LC 50 values. Individual genotyping by PASA revealed that the T929I and L1014F mutations exist en bloc as a haplotype, and the frequencies of the L1014F and T929I mutations were 100% and 70–100% in all examined field populations, respectively, correlating well with pyrethroid resistance levels. To investigate when the two mutations first arose and how quickly they spread, we examined the frequency of the mutations in dry P. xylostella adult specimens collected from 1974 to 1995. Unexpectedly, the L1014F was observed even in the specimens collected in 1974 long before the selection of P. xylostella population with pyrethroids, whereas the T929I mutations began to show in 1995 after extensive selection with pyrethroids. These findings indicate that the L1014F mutation occurred first, perhaps due to initial selection by DDT, whereas the T929I mutation was selected subsequently by pyrethroids as a second mutation. In summary, high frequency of the knockdown resistance alleles indicates that pyrethroid resistance by the nerve insensitivity mechanism is at an almost saturated level in the representative Korean populations of P. xylostella, demanding more intensive regulation of pyrethroid use for its control. Use of efficient genotyping techniques, such as PASA, should greatly facilitate the monitoring of P. xylostella resistance in the field.

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 ( r 2=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 ( r 2=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.

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.