High-Resolution Genetic Mapping Combined with Transcriptome Profiling Reveals That Both Target-Site Resistance and Increased Detoxification Confer Resistance to the Pyrethroid Bifenthrin in the Spider Mite Tetranychus urticae

SIMPLE SUMMARY: The two-spotted spider mite Tetranychus urticae is an important pest on agricultural crops worldwide. The widespread application of pyrethroid acaricides—such as bifenthrin—to control this pest has resulted in the development of resistance. Previous research has associated mutations in the voltage-gated sodium channel (VGSC), as well as increased detoxification, with bifenthrin resistance. Here, we performed a bulked segregant analysis to unbiasedly map resistance loci conferring resistance and found two genomic loci (QTL1 and QTL2) underlying bifenthrin resistance. The VGSC is located at QTL2, which harbors the resistance-conferring L1024V mutation. The presence of a second QTL suggested that a second resistance mechanism must be involved, and this was further investigated with a differential gene expression analysis. Multiple genes encoding detoxification enzymes, including carboxyl/choline esterases (CCEs), cytochrome P450 monooxygenases and UDP-glycosyl transferases (UGTs), were more highly expressed in bifenthrin-resistant strains. A selection of these enzymes (CCE58, CCEinc18, teturUGT10 and teturUGT29) were functionally expressed. CCEinc18 was shown to metabolize bifenthrin, while teturUGT10 could glycosylate bifenthrin-alcohol. In conclusion, our findings suggest that bifenthrin resistance in the spider mite T. urticae is mediated by both target-site and metabolic mechanisms that may act in synergy. ABSTRACT: Pyrethroids are widely applied insecticides in agriculture, but their frequent use has provoked many cases of resistance, in which mutations in the voltage-gated sodium channel (VGSC), the pyrethroid target-site, were shown to play a major role. However, for the spider mite Tetranychus urticae, it has also been shown that increased detoxification contributes to resistance against the pyrethroid bifenthrin. Here, we performed QTL-mapping to identify the genomic loci underlying bifenthrin resistance in T. urticae. Two loci on chromosome 1 were identified, with the VGSC gene being located near the second QTL and harboring the well-known L1024V mutation. In addition, the presence of an L925M mutation in the VGSC of a highly bifenthrin-resistant strain and its loss in its derived, susceptible, inbred line indicated the importance of target-site mutations in bifenthrin resistance. Further, RNAseq experiments revealed that genes encoding detoxification enzymes, including carboxyl/choline esterases (CCEs), cytochrome P450 monooxygenases and UDP-glycosyl transferases (UGTs), were overexpressed in resistant strains. Toxicity bioassays with bifenthrin (ester pyrethroid) and etofenprox (non-ester pyrethroid) also indicated a possible role for CCEs in bifenthrin resistance. A selection of CCEs and UGTs were therefore functionally expressed, and CCEinc18 was shown to metabolize bifenthrin, while teturUGT10 could glycosylate bifenthrin-alcohol. To conclude, our findings suggest that both target-site and metabolic mechanisms underlie bifenthrin resistance in T. urticae, and these might synergize high levels of resistance.