A flavin-dependent monooxgenase confers resistance to chlorantraniliprole in the diamondback moth, Plutella xylostella

Abstract

The diamondback moth, Plutella xylostella, is a damaging pest of cruciferous crops, and has evolved resistance to many of the insecticides used for control, including members of the diamide class. Previous work on the molecular basis of resistance to diamides has documented mutations in the target-site, the ryanodine receptor, in resistant populations of P. xylostella worldwide. In contrast the role of metabolic resistance to this insecticide class is significantly less clear. Here we show that overexpression of a flavin-dependent monooxgenase (FMO) confers resistance to the diamide chlorantraniliprole in P. xylostella. Transcriptome profiling of diamide resistant strains, with and without target-site resistance, revealed constitutive over-expression of several transcripts encoding detoxification enzymes compared to susceptible strains. Two of these, CYP6BG1, and PxFMO2 were particularly highly overexpressed (33,000 and 14,700-fold, respectively) in a resistant strain (HAW) lacking target-site resistance. After 17 generations without diamide selection the resistance of the HAW strain fell by 52-fold and the expression of PxFMO2 by > 1300-fold, however, the expression of CYP6BG1 declined by only 3-fold. Generation of transgenic Drosophila melanogaster expressing these genes demonstrated that PxFMO2, but not CYP6BG1, confers resistance in vivo. Overexpression of PxFMO2 in the HAW strain is associated with mutations, including a putative transposable element insertion, in the promoter of this gene. These enhance the expression of a reporter gene when expressed in a lepidopteran cell line suggesting they are, at least in part, responsible for the overexpression of PxFMO2 in the resistant strain. Our results provide new evidence that insect FMOs can be recruited to provide resistance to synthetic insecticides.

Keywords: Chlorantraniliprole; Diamide; Flavin monooxygenase; Insecticides; Plutella xylostella; Resistance.

Graphical abstract

Fig. 1

Transcriptome profiling of diamide resistant and susceptible P. xylostella strains. (A) Venn diagram showing numbers of differentially expressed genes in multiple microarray comparisons of P. xylostella. (B) Numbers of differentially expressed genes in each treatment comparison. (C) Relative expression (fold change) of candidate resistance genes in different strains of P. xylostella measured by qPCR. Error bars display 95% CLs. Significant differences (p < 0.05) in expression between resistant strains and the relevant susceptible reference strain is denoted using asterisks above bars as determined by One-way ANOVA with post hoc testing (Tukey HSD) (CM versus CHL and FLU) or unpaired t tests (ROTH versus HAW). ROTH: long-term lab susceptible strain, HAW: chlorantraniliprole resistant strain from Hawaii, CM: susceptible field strain from Thailand, CHL: diamide resistant field strain from Thailand, FLU: diamide resistant field strain from Thailand. XDH: xanthine dehydrogenase, ST: sugar transporter, SCD: short-chain dehydrogenase, CE: carboxylesterase.

Fig. 2

Insecticide bioassays of transgenic D. melanogaster expressing P. xylostella candidate resistance genes to select diamide insecticides. A) Sensitivity of transgenic flies to chlorantraniliprole. B) Sensitivity of transgenic flies to flubendiamide. XDH: xanthine dehydrogenase, SCD: short-chain dehydrogenase. Error bars indicate 95% CLs. Significant changes in sensitivity of control and transgenic lines are indicated using an asterisk and are based on non-overlapping 95% fiducial limits of LC₅₀ values.

Fig. 3

Chlorantraniliprole susceptibility and expression of candidate resistance genes in selected and unselected lines of the HAW P. xylostella strain over the course of 17 generations. A) Calculated LC₅₀ values for chlorantraniliprole for the two lines over the course of the experiment. Error bars indicate 95% CLs. B) Expression of candidate resistance genes in the two lines over the experiment as measured by QPCR. Error bars indicate SD.

Fig. 4

Alterations in the PxFMO2 promoter of the HAW strain enhance expression. A) Alignment of the sequences immediately upstream of PxFMO2 derived from the HAW and ROTH strains. The terminal inverted repeats (TIR) present at the boundaries of a putative transposable element insertion are indicated in red. The start of the coding sequence of PxFMO2 is shown in yellow. B) Alignment of the PxFMO2 genome against 5 representative scaffolds taken from the P. xylostella genome that share the repetitive element. In both A and B grey regions indicate similarity between sequences and black regions indicate sequence differences. Indels are represented by gaps in the sequence. The identity plot above the alignment displays the identity across all sequences for every position. Green indicates that the residue at the position is the same in all sequences. Yellow indicates less than complete identity and red refers to very low identity for the given position. C) Reporter gene activity (normalised to renilla fluorescence) of the ROTH and HAW PxFMO2 promoter variants. Letters above bars indicate significant differences, P < 0.001; one-way ANOVA with post-hoc Tukey HSD. Error bars indicate 95% CLs. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)