The role, magnitude, and molecular nature of trans-driven expression variation underlying the upregulation of detoxification genes in pesticide resistant arthropod populations has remained enigmatic. In this study, we performed expression quantitative trait locus (eQTL) mapping (n = 458) between a pesticide resistant and a susceptible strain of the generalist herbivore and crop pest Tetranychus urticae. We found that a single trans eQTL hotspot controlled large differences in the expression of a subset of genes in different detoxification gene families, as well as other genes associated with host plant use. As established by additional genetic approaches including RNAi gene knockdown, a duplicated gene with a nuclear hormone receptor HR96-related ligand-binding domain was identified as causal for the expression differences between strains. The presence of a large family of HR96-related genes in T. urticae may enable modular control of detoxification and host plant use genes, facilitating this species’ known and rapid evolution to diverse pesticides and host plants.

The extreme adaptation potential of the generalist herbivore Tetranychus urticae (the two-spotted spider mite) to pesticides as well as diverse host plants has been associated with clade-specific gene expansions in known detoxifying enzyme families, and with extensive and rapid transcriptional responses. However, how this broad transcriptional potential is regulated remains largely unknown. Using a parental/F1 design in which four inbred strains were crossed to a common inbred strain, we assessed the genetic basis and inheritance of gene expression variation in T. urticae. Mirroring known phenotypic variation in the progenitor strains of the inbreds, we confirmed that the inbred strains we created were genetically distinct, varied markedly in pesticide resistance, and also captured variation in host plant fitness as is commonly observed in this species. By examining differences in gene expression between parents and allele-specific expression in F1s, we found that variation in RNA abundance was more often explained in trans as compared to cis, with the former associated with dominance in inheritance. Strikingly, in a gene ontology analysis, detoxification genes of the cytochrome P450 monooxygenase (CYP) family, as well as dioxygenases (DOGs) acquired from horizontal gene transfer from fungi, were specifically enriched at the extremes of trans-driven up- and downregulation. In particular, multiple CYPs and DOGs with broad substrate-specificities for pesticides or plant specialized compounds were exceptionally highly upregulated as a result of trans-regulatory variation, or in some cases synergism of cis and trans, in the most multi-pesticide resistant strains. Collectively, our findings highlight the potential importance of trans-driven expression variation in genes associated with xenobiotic metabolism and host plant use for rapid adaptation in T. urticae, and also suggests modular control of these genes, a regulatory architecture that might ameliorate negative pleiotropic effects.

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.

Dicofol has been widely used to control phytophagous mites. Although dicofol is chemically related to DDT, its mode of action has remained elusive. Here, we mapped dicofol resistance in the spider mite Tetranychus urticae to two genomic regions. Each region harbored a glutamate-gated chloride channel (GluCl) gene that contained a mutation—G314D or G326E—known to confer resistance against the unrelated acaricide abamectin. Using electrophysiology assays we showed that dicofol and other diphenylcarbinol acaricides—bromopropylate and chlorobenzilate—induce persistent currents in Xenopus oocytes expressing wild-type T. urticae GluCl3 receptors and potentiate glutamate responses. In contrast, the G326E substitution abolished the agonistic activity of all three compounds. Assays with the wild-type Drosophila GluClα revealed that this receptor was unresponsive to dicofol. Homology modeling combined with ligand-docking confirmed the specificity of electrophysiology assays. Altogether, this work elucidates the mode of action of diphenylcarbinols as mite-specific agonists of GluCl.

The extreme adaptation potential of the generalist herbivore Tetranychus urticae (the two-spotted spider mite) to pesticides as well as diverse host plants has been associated with clade-specific gene expansions in known detoxifying enzyme families, and with extensive and rapid transcriptional responses. However, how this broad transcriptional potential is regulated remains largely unknown. Using a parental/F1 design in which four inbred strains were crossed to a common inbred strain, we assessed the genetic basis and inheritance of gene expression variation in T. urticae . Mirroring known phenotypic variation in the progenitor strains of the inbreds, we confirmed that the inbred strains we created were genetically distinct, varied markedly in pesticide resistance, and also captured variation in host plant fitness commonly observed in this species. By examining differences in gene expression between parents and allele-specific expression in F1s, we found that variation in RNA abundance was more often explained in trans as compared to cis , with the former associated with dominance in inheritance. Strikingly, in a gene ontology analysis, detoxification genes of the cytochrome P450 monooxygenase (CYP) family, as well as dioxygenases (DOGs) acquired from horizontal gene transfer from fungi, were specifically enriched at the extremes of trans -driven up- and downregulation. In particular, a clade of CYPs with documented broad substrate-specificity including multiple pesticides, as well as DOGs that have recently been shown to have broad substrate specificity against plant specialized compounds, were exceptionally highly upregulated as a result of trans effects (or in some cases synergism of cis and trans ) in the most multi-pesticide resistant strains. Collectively, our findings highlight the potential importance of trans -driven expression variation in genes associated with xenobiotic metabolism and host plant use for rapid adaptation in T. urticae , and also suggest modular control of these genes, a regulatory architecture that might ameliorate negative pleiotropic effects. Author summary: The two-spotted spider mite, Tetranychus urticae, is a generalist herbivore and pest of diverse crops globally. In response to the plethora of chemicals used for its control, the species rapidly evolves pesticide resistance. Further, experimental evolution studies with T. urticae populations have demonstrated adaptation to challenging host plants in as few as five generations. The adaptation of T. urticae to pesticides and host plants has been associated with large transcriptome changes, including for genes associated with detoxification of pesticides and toxic plant compounds. Nevertheless, the basis of the observed transcriptome variation has remained largely unknown. Here, we examined the genetic control and inheritance of expression differences among five inbred T. urticae strains, including several with histories of intense pesticide selection. With a parental/F1 experimental design, we found that trans effects were common in explaining variation in detoxification gene expression, with the trans -driven upregulation of a subset of cytochrome P450 monooxygenases of broad substrate specificity especially striking in the most pesticide resistant strains. Our findings suggest that genetic variation acting with dominant or additive inheritance to impact the regulation of modules of detoxification genes may be an important target of selection during rapid pesticide and host plant evolution in herbivores.