Population bulk segregant mapping uncovers resistance mutations and the mode of action of a chitin synthesis inhibitor in arthropods

Abstract

Because of its importance to the arthropod exoskeleton, chitin biogenesis is an attractive target for pest control. This point is demonstrated by the economically important benzoylurea compounds that are in wide use as highly specific agents to control insect populations. Nevertheless, the target sites of compounds that inhibit chitin biogenesis have remained elusive, likely preventing the full exploitation of the underlying mode of action in pest management. Here, we show that the acaricide etoxazole inhibits chitin biogenesis in Tetranychus urticae (the two-spotted spider mite), an economically important pest. We then developed a population-level bulk segregant mapping method, based on high-throughput genome sequencing, to identify a locus for monogenic, recessive resistance to etoxazole in a field-collected population. As supported by additional genetic studies, including sequencing across multiple resistant strains and genetic complementation tests, we associated a nonsynonymous mutation in the major T. urticae chitin synthase (CHS1) with resistance. The change is in a C-terminal transmembrane domain of CHS1 in a highly conserved region that may serve a noncatalytic but essential function. Our finding of a target-site resistance mutation in CHS1 shows that at least one highly specific chitin biosynthesis inhibitor acts directly to inhibit chitin synthase. Our work also raises the possibility that other chitin biogenesis inhibitors, such as the benzoylurea compounds, may also act by inhibition of chitin synthases. More generally, our genetic mapping approach should be powerful for high-resolution mapping of simple traits (resistance or otherwise) in arthropods.

Fig. 1.

CFW staining of cryosections from T. urticae. Cryosections from control (C) or etoxazole-treated mites (T) were stained with 0.01% (wt/vol) CFW to visualize chitin deposition in the cuticle. Fluorescence was recorded using identical settings for exposure time and grayscale profiles. The representative image shows an inhibitory effect of etoxazole on chitin formation. BF, bright field. (Scale bar, 100 μm.)

Fig. 2.

Genetics and mapping of etoxazole resistance in T. urticae. (A) Concentration response relationship of etoxazole toxicity on London, EtoxR, and crosses. High resistance is inherited recessively and is not maternal [compare reciprocal F1s (triangles) with parental strains (circles)]. A pronounced mortality plateau at 50% for haploid males that are the progeny of unfertilized F1 females (squares) demonstrates control by a single major factor. (B) Fixation of EtoxR SNPs in the selected population, as assessed with a sliding window of 150 kb (windows were sequentially offset by 10 kb; the percent of fixed EtoxR SNPs in a widow following selection is plotted; see Figs. S2 and S3 for details). Scaffolds 1, 2, and 4 are representative of the genome average (no evidence of selection); only scaffolds 3, 23, and 40 show marked evidence of shifts in allele frequencies in response to etoxazole selection (Fig. S3). The position of CHS1 is indicated at top.

Fig. 3.

A nonsynonymous SNP in CHS1 is associated with etoxazole resistance. (A) Etoxazole resistance in strains of diverse geographical origin as documented by mortality at a discriminating dose. Error bars represent SE. (B) A single I-to-F variant is associated with etoxazole resistance, and becomes fixed in a segregating population (Strain005) after a single selection (Strain005R). Strains from different regions display different haplotypes. Only nonsingleton nonsynonymous variants are displayed (see Fig. S3 for additional information). (C) Concentration response relationship of etoxazole toxicity in London, EtoxR, Strain005R, and crosses (see legend). Because Strain005R × EtoxR progeny are not susceptible, recessive resistance in the two strains (Fig. 2A and Fig. S5) must share the same genetic basis. Strain origins are Belgium (MR-VL), Canada (London), Holland (Strain005/005R), Japan (EtoxR), Greece (TuSB9), and Germany (GSS).

Fig. 4.

Location of the mutation conferring etoxazole resistance, and evaluation of its effect on chitin biosynthesis in yeast. (A) Schematic representation of domain architecture of CHS1 from T. urticae. NTR, N-terminal region; CD, catalytic domain; CTR, C-terminal region; 5TMS, cluster of five transmembrane segments; and CC, coiled-coil motif. Rectangular boxes represent transmembrane domains. Arrows point to signature sequences QRRRW (catalytic domain) and WGTR (N-terminal region). (B) Aligned amino acid sequences of helix 5 in the 5TMS clusters of CHS1 of representative insect species, T. urticae, and the corresponding helix 3 of S. cerevisiae. Universally conserved residues are marked in bold. The position of the I1017F substitution in etoxazole-resistant mites is indicated in gray. S, etoxazole susceptible; R, etoxazole resistant; Tc, Tribolium castaneum; Lm, Locusta migratoria; Ms, Manduca sexta; Dm, D. melanogaster; Aa, Aedes aegypti; Nv, Nasonia vitripennis; Tu, T. urticae; Sc, S. cerevisiae. (C) Complementation of the CFW resistance phenotype in chs3Δ strains. chs3Δ cells were transformed with pRS415 plasmids containing the ORFs for CHS3 or its mutant version CHS3^L1094F. WT, chs3Δ, chs3ΔCHS3, and chs3ΔCHS3^L1094F cells were grown overnight in liquid YPD medium and spotted after dilution with water onto YPD plates with or without 50 μg/mL CFW. In contrast to chs3Δ CHS3, the CFW phenotype in chs3Δ CHS3^L1094F cells is not restored, indicating that CHS3^L1094F is nonfunctional.