The frequency of insecticide/acaricide target‐site resistance is increasing in arthropod pest populations and is typically underpinned by single point mutations that affect the binding strength between the insecticide/acaricide and its target‐site. Theory predicts that although resistance mutations clearly have advantageous effects under the selection pressure of the insecticide/acaricide, they might convey negative pleiotropic effects on other aspects of fitness. If such fitness costs are in place, target‐site resistance is thus likely to disappear in the absence of insecticide/acaricide treatment, a process that would counteract the spread of resistance in agricultural crops. Hence, there is a great need to reliably quantify the various potential pleiotropic effects of target‐site resistance point mutations on arthropod fitness. Here, we used near‐isogenic lines of the spider mite pest Tetranychus urticae that carry well‐characterized acaricide target‐site resistance mutations to quantify potential fitness costs. Specifically, we analyzed P262T in the mitochondrial cytochrome b, the combined G314D and G326E substitutions in the glutamate‐gated chloride channels, L1024V in the voltage‐gated sodium channel, and I1017F in chitin synthase 1. Five fertility life table parameters and nine single‐generation life‐history traits were quantified and compared across a total of 15 mite lines. In addition, we monitored the temporal resistance level dynamics of populations with different starting frequency levels of the chitin synthase resistant allele to further support our findings. Three target‐site resistance mutations, I1017F and the co‐occurring G314D and G326E mutations, were shown to significantly and consistently alter certain fitness parameters in T. urticae. The other two mutations (P262T and L1024V) did not result in any consistent change in a fitness parameter analyzed in our study. Our findings are discussed in the context of the global spread of T. urticae pesticide resistance and integrated pest management.

Chemical control strategies are driving the evolution of pesticide resistance in pest populations. Understanding the genetic mechanisms of these evolutionary processes is of crucial importance to develop sustainable resistance management strategies. The acaricide pyflubumide is one of the most recently developed mitochondrial complex II inhibitors with a new mode of action that specifically targets spider mite pests. In this study, we characterize the molecular basis of pyflubumide resistance in a highly resistant population of the spider mite Tetranychus urticae. Classical genetic crosses indicated that pyflubumide resistance was incompletely recessive and controlled by more than one gene. To identify resistance loci, we crossed the resistant population to a highly susceptible T. urticae inbred strain and propagated resulting populations with and without pyflubumide exposure for multiple generations in an experimental evolution set-up. High-resolution genetic mapping by a bulked segregant analysis approach led to the identification of three quantitative trait loci (QTL) linked to pyflubumide resistance. Two QTLs were found on the first chromosome and centered on the cytochrome P450 CYP392A16 and a cluster of CYP392E6-8 genes. Comparative transcriptomics revealed a consistent overexpression of CYP392A16 and CYP392E8 in the experimental populations that were selected for pyflubumide resistance. We further corroborated the involvement of CYP392A16 in resistance by in vitro functional expression and metabolism studies. Collectively, these experiments uncovered that CYP392A16 N-demethylates the toxic carboxamide form of pyflubumide to a non-toxic compound. A third QTL coincided with cytochrome P450 reductase (CPR), a vital component of cytochrome P450 metabolism. We show here that the resistant population harbors three gene copies of CPR and that this copy number variation is associated with higher mRNA abundance. Together, we provide evidence for detoxification of pyflubumide by cytochrome P450s that is likely synergized by gene amplification of CPR.

BACKGROUND In spider mites, mutations in the mitochondrial cytochrome b Qo pocket have been reported to confer resistance to the Qo inhibitors bifenazate and acequinocyl. In this study, we surveyed populations of the two‐spotted spider mite Tetranychus urticae for mutations in cytochrome b, linked newly discovered mutations with resistance and assessed potential pleiotropic fitness costs. RESULTS We identified two novel mutations in the Qo site: G132A (equivalent to G143A in fungi resistant to strobilurins) and G126S + A133T (previously reported to cause bifenazate and acequinocyl resistance in Panonychus citri). Two T. urticae strains carrying G132A were highly resistant to bifenazate but not acequinocyl, whereas a strain with G126S + A133T displayed high levels of acequinocyl resistance, but only moderate levels of bifenazate resistance. Bifenazate and acequinocyl resistance were inherited maternally, providing strong evidence for the involvement of these mutations in the resistance phenotype. Near isogenic lines carrying G132A revealed several fitness penalties in T. urticae; a lower net reproductive rate (R0), intrinsic rate of increase (rm) and finite rate of increase (LM); a higher doubling time (DT); and a more male‐biased sex ratio. CONCLUSIONS Several lines of evidence were provided to support the causal role of newly discovered cytochrome b mutations in bifenazate and acequinocyl resistance. Because of the fitness costs associated with the G132A mutation, resistant T. urticae populations might be less competitive in a bifenazate‐free environment, offering opportunities for resistance management. © 2019 Society of Chemical Industry

Pyflubumide is a novel selective carboxanilide acaricide that inhibits mitochondrial complex II of mainly spider species such as Tetranychus urticae. We explored the baseline toxicity and potential crossresistance risk of pyflubumide in a reference panel of T. urticae strains resistant to various acaricides with different modes of action. A cyenopyrafen resistant strain (JPR) was identified as the only strain with low-to-moderate level of cross-resistance to pyflubumide (LC50 = 49.07 mg/L). In a resistance risk assessment approach, JPR was subsequently selected which lead to two highly resistant strains JPR-R1 (LC50 = 1437.48 mg/L) and JPR-R2 (LC50 = 1893.67 mg/L). Interestingly, compared to adult females, resistance was much less pronounced in adult males and eggs of the two JPR-R strains. In order to elucidate resistance mechanisms, we first sequenced complex II subunits in susceptible and resistant strains, but target-site insensitivity could not be detected. In contrast, synergism/ antagonism experiments strongly suggested that cytochrome P450 monooxygenases are involved in pyflubumide resistance. We therefore conducted genome-wide gene expression experiments to investigate constitutive and induced expression patterns and documented the overexpression of five cytochrome P450 and four CCE genes in JPR-Rs after pyflubumide exposure. Together, we provide a thorough resistance risk assessment of a novel complex II inhibitor, and provide first evidence for metabolic resistance mediated by cytochrome P450s in T. urticae.

Pomegranate (Punica granatum L.) orchards in the Middle East are typically composed of a mix of different cultivars in which variation in fruit infestation by carob moth Ectomyelois ceratoniae (Zeller) (Lepidoptera: Pyralidae) has been observed. However, seasonal variation in infestation and adaptation of the carob moth to this cropping system have not been explored. We monitored the progress of fruit infestation in 10 pomegranate cultivars during the growing season of two consecutive years in pomegranate orchards of Iran. Overall, levels of infestation in fruits were strongly correlated with susceptibility to fruit cracking in pomegranate, so that cracked fruits and cracking-susceptible cultivars were infested the most. However, this pattern changed during the season. Infestation was first observed on crackingsusceptible cultivars. At this point almost all cracked fruits were infested. Towards the end of the season, infestation in uncracked fruits and cracking-resistant cultivars increased. Uncracked fruits seem better overwintering sites for carob moth as under simulated winter conditions, survival of insect larvae in uncracked fruits was >3 times higher than in cracked fruits. Taken together, our data reveal that cracked fruits of pomegranate are the better host during the growing season, while uncracked fruits better sustain carob moth population in winter. It seems therefore advisable not to grow cracking-susceptible and cracking-resistant cultivars together in the same area.

Chemical control strategies are driving the evolution of pesticide resistance in pest populations. Understanding the genetic mechanisms of these evolutionary processes is of crucial importance to develop sustainable resistance management strategies. The acaricide pyflubumide is one of the most recently developed mitochondrial complex II inhibitors with a new mode of action that specifically targets spider mite pests. In this study, we characterize the molecular basis of pyflubumide resistance in a highly resistant population of the spider mite Tetranychus urticae . Classical genetic crosses indicated that pyflubumide resistance was incompletely recessive and controlled by more than one gene. To identify resistance loci, we crossed the resistant population to a highly susceptible T. urticae inbred strain and propagated resulting populations with and without pyflubumide exposure for multiple generations in an experimental evolution set-up. High-resolution genetic mapping by a bulked segregant analysis approach led to the identification of three quantitative trait loci (QTL) linked to pyflubumide resistance. Two QTLs were found on the first chromosome and centered on the cytochrome P450 CYP392A16 and a cluster of CYP392E6-8 genes. Comparative transcriptomics revealed a consistent overexpression of CYP392A16 and CYP392E8 in the experimental populations that were selected for pyflubumide resistance. We further corroborated the involvement of CYP392A16 in resistance by in vitro functional expression and metabolism studies. Collectively, these experiments uncovered that CYP392A16 N-demethylates the toxic carboxamide form of pyflubumide to a non-toxic compound . A third QTL coincided with cytochrome P450 reductase ( CPR ), a vital component of cytochrome P450 metabolism. We show here that the resistant population harbors three gene copies of CPR and that this copy number variation is associated with higher mRNA abundance . Together, we provide evidence for detoxification of pyflubumide by cytochrome P450s that is likely synergized by gene amplification of CPR .