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. 2014 Aug:51:52-61.
doi: 10.1016/j.ibmb.2014.05.004. Epub 2014 May 22.

High resolution genetic mapping uncovers chitin synthase-1 as the target-site of the structurally diverse mite growth inhibitors clofentezine, hexythiazox and etoxazole in Tetranychus urticae

Peter Demaeght  1 , Edward J Osborne  2 , Jothini Odman-Naresh  3 , Miodrag Grbić  4 , Ralf Nauen  5 , Hans Merzendorfer  3 , Richard M Clark  6 , Thomas Van Leeuwen  7
Affiliations

High resolution genetic mapping uncovers chitin synthase-1 as the target-site of the structurally diverse mite growth inhibitors clofentezine, hexythiazox and etoxazole in Tetranychus urticae

Peter Demaeght et al. Insect Biochem Mol Biol. 2014 Aug.

Abstract

The acaricides clofentezine, hexythiazox and etoxazole are commonly referred to as 'mite growth inhibitors', and clofentezine and hexythiazox have been used successfully for the integrated control of plant mite pests for decades. Although they are still important today, their mode of action has remained elusive. Recently, a mutation in chitin synthase 1 (CHS1) was linked to etoxazole resistance. In this study, we identified and investigated a Tetranychus urticae strain (HexR) harboring recessive, monogenic resistance to each of hexythiazox, clofentezine, and etoxazole. To elucidate if there is a common genetic basis for the observed cross-resistance, we adapted a previously developed bulk segregant analysis method to map with high resolution a single, shared resistance locus for all three compounds. This finding indicates that the underlying molecular basis for resistance to all three compounds is identical. This locus is centered on the CHS1 gene, and as supported by additional genetic and biochemical studies, a non-synonymous variant (I1017F) in CHS1 associates with resistance to each of the tested acaricides in HexR. Our findings thus demonstrate a shared molecular mode of action for the chemically diverse mite growth inhibitors clofentezine, hexythiazox and etoxazole as inhibitors of an essential, non-catalytic activity of CHS1. Given the previously documented cross-resistance between clofentezine, hexythiazox and the benzyolphenylurea (BPU) compounds flufenoxuron and cycloxuron, CHS1 should be also considered as a potential target-site of insecticidal BPUs.

Keywords: Chitin synthesis inhibitors; Fukui function; Genetic mapping; Insecticide; Mite growth inhibitors.

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Figures

Fig. 1
Fig. 1. Chemical structures of the mite growth inhibitors used in this study
Clofentezine (a), hexythiazox (b), diflovidazin (c) and etoxazole (d).
Fig. 2
Fig. 2. Genetics of clofentezine, hexythiazox and etoxazole resistance
Concentration response relationship of clofentezine (A), hexythiazox (B) and etoxazole toxicity (C) on London, HexR and cross progeny. Resistance to all three compounds is (incompletely) recessive as evidenced by high mortality in the F1 generation (triangles). The mortality plateau at 50% within the range of discriminating pesticide doses for F2 haploid males (squares) demonstrates that resistance to all three compounds segregates as a single locus.
Fig. 3
Fig. 3. Bulk segregant genetic mapping of a shared resistance locus for etoxazole, hexythiozox, and clofentezine
(A) Sliding window analysis examining the deviation in allele frequency between each of the various selected, and the unselected, F6 intercross progeny for all SNP positions in 150kb windows across the T. urticae genome (with 10kb offset). Genomic data derived from pooled mites treated with either etoxazole, hexythiazox or clofentezine are marked in red, black or blue respectively. Scaffolds are presented in order of length and are marked by alternating grey rectangles. The position of chitin synthase (CHS1) is highlighted with an arrow. (B) Fixation of HexR-specific SNPs across the pesticide treated progeny. The percent of parent-of-origin informative SNPs segregating in the unselected population that became fixed for resistant parent alleles in the selected lines is plotted in 150kb windows across the genomic scaffolds (plotting parameters are same as in A). (C) Peak of differentiation centers on CHS1. A large proportion HexR-specific alleles become fixed in the selected progeny on T. urticae scaffold 3 and centers nearby CHS1. (D) Distance of the sliding window peak to previously identified CHS1 I1017F variant associated with etoxazole resistance. The distance from the midpoint of the most differentiated genomic window to CHS1, and specifically to the I1017F SNP (also see Table S2), is shown for each of the selected lines. Individual genes are demarcated with boxes (exons) connected by lines segments (introns) with vertical position indicating gene strand.
Fig. 4
Fig. 4. Cuticular chitin in cryosections from different T. urticae strains
Cryosections were prepared from sensitive control strain treated with water (C), hexythiazox- and clofentezine resistant strain HexR (R), and hexythiazox- and clofentezine-treated sensitive strain London (S). The specimens were stained with 0.01% (w/v) CFW to visualize chitin deposition in the cuticle. Fluorescence was recorded using identical settings for exposure time and grayscale profiles. Above, the representative images show the effects of hexythiazox (left) and clofentezine (right) on chitin deposition in the cuticle. Scale bar, 100 μm. below, the densitometric analysis of CFW fluorescence in mite cuticles is shown. Significant differences in fluorescence intensities compared with sensitive control strains; p < 0.001 (Student’s t-test).
Fig. 5
Fig. 5. Location of the I-to-F mutation associated with etoxazole, hexythiazox and clofentezine resistance
(A) Schematic of CHS1 domains from T. urticae. The I1017F mutation is located in the last transmembrane helix. LB, lipid bilayer; 5TMS, cluster of five transmembrane segments; and CC, coiled-coil motif. Rectangular boxes represent trans-membrane domains (Van Leeuwen et al., 2012a). (B) The single I-to-F mutation is universally shared at CHS1 in geographically diverse strains conferring resistance to etoxazole, hexythiazox and clofentezine (see also Supplemental Table 3; Van Leeuwen et al. (2012a)).
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References

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