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.

BACKGROUND: High-levels of etoxazole resistance have not yet been frequently reported in Panonychus citri. Although a highly resistant strain was discovered in 2014, etoxazole resistance has not become a significant problem in areas of citrus production in Japan. A target site mutation in chitin synthase 1 (CHS1), I1017F, is a major etoxazole-resistance factor in Tetranychus urticae.To investigate the mechanisms of etoxazole resistance and the dispersal of resistance genes, we analyzed target-site mutations in a highly resistant strain and their geographical distribution in Japan.RESULTS: High-level etoxazole resistance was completely recessive. The I1017F mutation was detected in CHS1 of the highly resistant strain, and its frequency was correlated with the hatchability of eggs treated with etoxazole. Sequencing and variant frequency analyses of local populations by quantitative polymerase chain reaction revealed that I1017F is restricted to the Ariake Sea area of Kyushu Island. Although a new nonsynonymous substitution, S1016L, accompanied by I1017F was found in CHS1 of the highly resistant strain, CRISPR/Cas9 engineering of flies showed that S1016L had no effect on the etoxazole resistance conferred by I1017F.CONCLUSION: I1017F is a major target site mutation that confers high-level etoxazole resistance on P. citri. Dispersion of I1017F possibly was suppressed as a result of the completely recessive inheritance of resistance together with low gene flow between local populations.