Flubendiamide resistance and Bi-PASA detection of ryanodine receptor G4946E mutation in the diamondback moth (Plutella xylostella L.).

The extensive application of flubendiamide has led to increasingly prominent development of resistance in diamondback moth, Plutella xylostella. Here we report that the moderate and high level resistance to flubendiamide was identified in a laboratory-selected and two field-collected strains of P. xylostella. The resistance ratios were tested in the lab-selected resistant strains (R), and two field strains (BY and ZC). Compared with the S strain, the R strain showed extended larval development time, decreased pupation rate, emergencing rate, and male adult longevity. The realized heritability (h(2)=0.135) implies the high risk of flubendiamide resistance development in P. xylostella. A Bi-PASA (bi-directional PCR amplification of specific allele)-based method was successfully developed to detect the point mutation (G4946E) potentially causing flubendiamide resistance in diamondback moth, in which different fragments 866 bp + 340 bp, 866 bp+568 bp, and 866 bp+568 bp+340 bp were presented in SS, RR and RS stains, respectively. The predominant genotype was 83.33% SS homozygote in the S strain, 80.77% RR homozygote in ZC population, and 73.08% RS heterozygote in BY population, respectively. Current results showed the significant correlation between the frequencies of the allele carrying G4946E mutation (51.92%, 55.77% and 90.38% for R, BY and ZC, respectively) and the resistance ratios (40.72, 24.24 and 1779.24-folds for R, BY and ZC, respectively) in the three strains/populations. In addition, the relative PxRyR mRNA transcript level in the R strain was 2.938 ± 0.53 folds as compared with the S strain (1.0-fold).

Molecular Characterization and Bioinformatics Analysis of a Prophenoloxidase-1 (PPO1) in Plutella xylostella.

Phenoloxidase (PO) is an important enzyme in insect life, which is involved in important physical functions, such as defensive encapsulation and melanization of foreign organisms and wound healing. In this study, we obtained a cDNA sequence of 2838 bp with 2049 open reading frames encoding 682 amino acids. The protein sequence deduced from the cDNA has high homology with the known PPO1 sequences of other lepidopterous insects. There were three conserved regions, including the two copper-binding sites characteristic of arthropod PPOs. The whole PxPPO1 DNA was also obtained with 7202 bp when the five fragments were stitched together and the overlapping sequences were deleted. The PxPPO1 DNA consists of 11 introns and 12 exons, and the homology is 99.9% when the exons are compared with the above cDNA. Moreover, the gene expression levels were also determined by semiquantitative polymerase chain reaction (PCR), Western blotting, and real-time quantitative PCR; the results indicated that PxPPO1 transcripts in the eggs and the fourth instar larvae were more abundant, followed by the second and the third instar larvae, prepupae, and pupa.