Combination of restriction endonuclease digestion with the ΔΔCt method in real-time PCR to monitor etoxazole resistance allele frequency in the two-spotted spider mite.

Monitoring resistance allele frequency at the early stage of resistance development is important for the successful acaricide resistance management. Etoxazole is a mite growth inhibitor to which resistance is conferred by an amino acid substitution in the chitin synthase 1 (CHS1; I1017F) in T. urticae. If the susceptible allele can be specifically digested by restriction endonuclease, the ΔΔCt method using real-time PCR for genomic DNA (RED-ΔΔCt method) may be available for monitoring the resistance allele frequency. We tested whether the etoxazole resistance allele frequency in a pooled sample was accurately measured by the RED-ΔΔCt method and validated whether the resistance variant frequency was correlated with etoxazole resistance phenotype in a bioassay. Finally, we performed a pilot test using field populations. Strong linearity of the measures by the RED-ΔΔCt method with practical resistance allele frequencies; resistance allele frequency in the range between 0.5% to at least 0.75% was strictly represented. The strong linear relationship between hatchability of haploid male eggs after the etoxazole treatments (phenotype) and resistance allele frequencies in their mothers provided direct evidence that I1017F is a primary resistance factor to etoxazole in the strains used for experiments. The pilot test revealed a significant correlation between egg hatchability (including both diploid female eggs and haploid male eggs) and estimators in field populations. Consequently, we concluded that the RED-ΔΔCt method is a powerful tool for monitoring a resistance allele in a pooled sample.