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 populationlevel 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.

Fitness costs of key point mutations that underlie acaricide target‐site resistance in the two‐spotted spider miteTetranychus urticae

“…Resistance to mite growth inhibitors and chitin synthase inhibitors has been attributed to the I1017F mutation in chitin synthase (Demaeght et al., 2014; Douris et al., 2016; Grigoraki et al., 2017; Riga et al., 2017; Van Leeuwen et al., 2012). As the I1017F mutation is located in a highly conserved region of the CHS1 gene (Douris et al., 2016; Grigoraki et al., 2017; Suzuki, Shiotsuki, Jouraku, Miura, & Minakuchi, 2017; Yamamoto, Yoneda, Hatano, & Asada, 1995) , this substitution could impair the enzymatic function.…”

“…The analysis of the temporal dynamics of etoxazole resistance in mixed population experiment (Figure 3) showed that in all three populations (R30, R50, and R70), the frequency of resistant homozygotes dropped below 10% in a period of 8 months (Figure 3). Interestingly, the expression of T. urticae CHS1 is the highest in the obligate chrysalis stage wherein the mite molts to complete its development (Van Leeuwen et al., 2012). Mite development time could thus be affected by the I1017F mutation by inefficient molting and a longer chrysalis stage.…”

“…A commercial formulation of etoxazole (Borneo, 120 g/L SC) was used in this study. The discriminating etoxazole concentration is based on previously obtained mortality data (Van Leeuwen et al., 2012). Mortality was assessed after 48 hr.…”

Benzoylurea resistance in western flower thrips <i>Frankliniella occidentalis</i> (Thysanoptera: Thripidae): the presence of a point mutation in <i>chitin synthase 1</i>

“…In the presence of piperonyl butoxide (PBO), a cytochrome P450 inhibitor, the insecticidal activity against novaluron tended to be enhanced. In field strains, the analysis of the nucleotide sequence of CHS1 cDNA revealed a nonsynonymous substitution from isoleucine to methionine at the same position reported in T. urticae 18) and in P. xylostella, 19) suggesting that this might account for benzoylurea resistance in the field strains.…”

“…11,17) A recent study revealed that etoxazole resistance in the two-spotted spider mite Tetranychus urticae was attributed to a point mutation, I1017F, in CHS1. 18) Furthermore, a mutation, I1042M/F, in the CHS1 of the benzoylurea-resistant diamondback moth Plutella xylostella was discovered at the same position as the I1017F of T. urticae, and in vivo functional analysis using a genome-editing approach revealed that this mutation conferred resistance to benzoylureas in D. melanogaster. 19) These recent studies have provided evidence that CHS1 could be the target of benzoylureas and etoxazole.…”