Susceptible Mites Research Articles

Expression reduction and a variant of a P450 gene mediate chlorpyrifos resistance in Tetranychus urticae Koch

IntroductionUnderstanding how insects and mites develop resistance to chlorpyrifos is crucial for effective field management. Although extensive research has demonstrated that T. urticae exhibits high resistance to chlorpyrifos, the specific resistance mechanism remains elusive. Investigating this mechanism could provide valuable insights for pest control strategies. ObjectivesThis study aimed to reveal the mechanism of chlorpyrifos resistance in T. urticae. MethodsThe expression level of the CYP392D8 gene in T. urticae strains were analyzed using real- time quantitative PCR and western blot techniques. Functional validation of CYP392D8 was conducted through RNAi and heterogeneous expression. The production of chlorpyrifos-oxon in both resistant and susceptible strains were quantified using LC-MS/MS. Furthermore, the metabolic capabilities of CYP392D8 variants were verified using HPLC-MS and molecular docking. ResultsThe results showed the expression of CYP392D8 was reduced in some Chinese resistant populations and mites with knocked down CYP392D8 showed decreased susceptibility to chlorpyrifos. Chlorpyrifos-oxon, the active metabolite of chlorpyrifos, was generated when chlorpyrifos was incubated with recombinant CYP392D8 protein in vitro. And a higher efficiency of chlorpyrifos-oxon formation was observed with the CYP392D8-S variant from susceptible mites compared to the CYP392D8-R variant from resistant mites. After treatment with low doses of chlorpyrifos, susceptible mite extracts produced substantial amounts of chlorpyrifos-oxon, while resistant mites only showed trace amounts. In addition, molecular docking studies showed that CYP392D8-S had a higher binding capacity to chlorpyrifos than the CYP392D8-R variant. ConclusionThis study reveals a mechanism of insecticide resistance due to the bioactivation reduction in combination with the sequence variation in a pest mite. These findings provide an important theoretical bias for management strategies of mites in the field and comprehensive control.

Synergistic action and mechanism of scoparone, a key bioactive component of Artemisia capillaris, and spirodiclofen against spider mites.

Plants have numerous defensive secondary metabolites to withstand insect attacks. Scoparone, which is extracted from the medicinal plant Artemisia capillaris, has potent acaricidal effects on Tetranychus cinnabarinus. Spirodiclofen, derived from a tetronic acid derivative, is a potent commercial acaricide that is extensively used globally. However, whether scoparone has synergistic effects when used in conjunction with spirodiclofen and the underlying synergistic mechanism remains unclear. Scoparone exhibited a potent synergistic effect when it was combined with spirodiclofen at a 1:9 ratio. Subsequently, cytochrome P450 monooxygenase (P450) activity, RNA-Seq and qPCR assays indicated that the enzyme activity of P450 and the expression of one P450 gene from T. cinnabarinus, TcCYP388A1, were significantly inhibited by scoparone and spirodiclofen + scoparone; conversely, P450 was activated in spirodiclofen-exposed mites. Importantly, RNAi-mediated silencing of the TcCYP388A1 gene markedly increased the susceptibility of spider mites to spirodiclofen, scoparone and spirodiclofen + scoparone, and in vitro, the recombinant TcCYP388A1 protein could metabolize spirodiclofen. Molecular docking and functional analyses further indicated that R117, which is highly conserved in Arachnoidea species, may be a vital specific binding site for scoparone in the mite TcCYP388A1 protein. This binding site was subsequently confirmed using mutagenesis data, which revealed that this binding site was the sole site selected by scoparone in spider mites over mammalian or fly CYP388A1. These results indicate that the synergistic effects of scoparone and spirodiclofen on mites occurs through the inhibition of P450 activity, thus reducing spirodiclofen metabolism. The synergistic effect of this potent natural product on the detoxification enzyme-targeted activity of commercial acaricides may offer a sustainable strategy for pest mite resistance management. © 2024 Society of Chemical Industry.

Functional analysis of a down-regulated transcription factor-SoxNeuroA gene involved in the acaricidal mechanism of scopoletin against spider mites.

Insight into the mode of action of plant-derived acaricides will help in the development of sustainable control strategies for mite pests. Scopoletin, a promising plant-derived bioactive compound, displays prominent acaricidal activity against Tetranychus cinnabarinus. The transcription factor SoxNeuroA plays a vital role in maintaining calcium ion (Ca2+ ) homeostasis. Down-regulation of SoxNeuroA gene expression occurs in scopoletin-exposed mites, but the functional role of this gene remains unknown. A SoxNeuroA gene from T. cinnabarinus (TcSoxNeuroA) was first cloned and identified. Reverse transcription polymerase chain reaction (RT-PCR), quantitative real-time polymerase chain reaction (qPCR), and Western blotting assays all confirmed that the gene expression and protein levels of TcSoxNeuroA were significantly reduced under scopoletin exposure. Furthermore, RNA interference silencing of the weakly expressed SoxNeuroA gene significantly enhanced the susceptibility of mites to scopoletin, suggesting that the acaricidal mechanism of scopoletin was mediated by the weakly expressed SoxNeuroA gene. Additionally, yeast one-hybrid (Y1H) and dual-luciferase reporter assays revealed that TcSoxNeuroA was a repressor of Orai1 Ca2+ channel gene transcription, and the key binding sequence was ATCAAAG (positions -361 to -368 of the Orai1 promoter). Importantly, site-directed mutagenesis and microscale thermophoresis assays further indicated that ASP185, ARG189, and LYS217, which were key predicted hydrogen-bonding sites in the molecular docking model, may be the vital binding sites for scopoletin in TcSoxNeuroA. These results demonstrate that the acaricidal mechanism of scopoletin involves inhibition of the transcription factor SoxNeuroA, thus inducing the activation of the Orai1 Ca2+ channel, eventually leading to Ca2+ overload and lethality. Elucidation of the transcription factor-targeted mechanism for this potent plant-derived acaricide has vital implications for the design of next-generation green acaricides with novel targets. © 2023 Society of Chemical Industry.

Failure of scabies treatment: a systematic review andmeta-analysis.

Treatment failure is considered to be an important factor in relation to the increase in scabies incidence over the last decade. However, the regional and temporal differences, in addition to the predictors of therapy failure, are unclear. We aimed to conduct a systematic review of the prevalence of treatment failure in patients with scabies and investigation of associated factors. We searched MEDLINE, EMBASE, CINAHL, Web of Science, Scopus, Global Health and the Cochrane Central Register of Controlled Trials from inception to August 2021 for randomized and quasi-randomized trials, in addition to observational studies that enrolled children or adults diagnosed with confirmed or clinical scabies treated with permethrin, ivermectin, crotamiton, benzyl benzoate, malathion, sulfur or lindane, and measured treatment failure or factors associated with treatment failure. We performed a random effects meta-analysis for all outcomes reported by at least two studies. A total of 147 studies were eligible for inclusion in the systematic review. The overall prevalence of treatment failure was 15.2% [95% confidence interval (CI) 12.9-17.6; I2 = 95.3%, moderate-certainty evidence] with regional differences between World Health Organization regions (P = 0.003) being highest in the Western Pacific region (26.9%, 95% CI 14.5-41.2). Oral ivermectin (11.8%, 95% CI 8.4-15.4), topical ivermectin (9.3%, 95% CI 5.1-14.3) and permethrin (10.8%, 95% CI 7.5-14.5) had relatively lower failure prevalence compared with the overall prevalence. Failure prevalence was lower in patients treated with two doses of oral ivermectin (7.1%, 95% CI 3.1-12.3) compared with those treated with one dose (15.2%, 95% CI 10.8-20.2; P = 0.021). Overall and permethrin treatment failure prevalence in the included studies (1983-2021) increased by 0.27% and 0.58% per year, respectively. Only three studies conducted a multivariable risk factor analysis; no studies assessed resistance. A second dose of ivermectin showed lower failure prevalence than single-dose ivermectin, which should be considered in all guidelines. The increase in treatment failure over time hints at decreasing mite susceptibility for several drugs, but reasons for failure are rarely assessed. Ideally, scabicide susceptibility testing should be implemented in future studies.

Warmer temperatures reduce chemical tolerance in the redlegged earth mite (Halotydeus destructor), an invasive winter-active pest.

BACKGROUNDQuantifying how chemical tolerance of pest arthropods varies with temperature is important for understanding the outcomes of chemical control, for measuring and monitoring resistance, and for predicting how pesticide resistance will evolve under future climate change. We studied the redlegged earth mite, Halotydeus destructor (Tucker), a winter‐active invasive agricultural pest in Australia. Using a replicated block experiment, we tested the effect of different thermal conditions on the expression of chemical tolerance to a pyrethroid and two organophosphates. Our chemical bioassays were conducted on two redlegged earth mite populations: one possessed organophosphate resistance, whilst the other was susceptible to pesticides. Mites were first acclimated at cool (4 °C) and warm (14 °C) conditions and then exposed to pesticides in both cool (11 °C) and warm (18 °C) test conditions.RESULTSWarm test conditions generally reduced chemical tolerance to all pesticides relative to cool test conditions. Median lethal dose (LD50) values of mites tested under cool conditions were 1.12–3.57‐fold greater than of mites tested under warm conditions. Acclimation had a variable and small impact on chemical responses. Thermal factors (ratio between test temperatures) were similar between populations for each active ingredient. Despite reduced chemical tolerances under warm test conditions for individual mite populations, resistance factors (ratio between resistant and susceptible mite populations) were relatively consistent.CONCLUSIONOur data provides context for prior theoretical work demonstrating climatically constrained pesticide resistances in Australian redlegged earth mites. Estimates of temperature dependent toxicity measured in this study may be useful in parameterizing models of redlegged earth mite control under an increasingly warm and more variable climate. © 2022 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Comparing the efficiency of RNAi after feeding and injection of dsRNA in spider mites

Pesticide resistance in spider mites drives the development of acaricides with novel mode of action, which could benefit from RNAi as a screening tool in search of new molecular targets. RNAi via oral delivery of dsRNA has been frequently reported in spider mites, but injection of dsRNA is rarely reported. We compare here the efficiency of oral delivery versus injection of dsRNA in female adult mites. When comparing silencing efficiency, oral delivery of dsRNAs silenced 40.6 ± 8.9% of CPR, 63.8 ± 6.9% of CHMP2A, and 37.7 ± 5.7% of CHMP3 genes. Similar silencing efficiencies were found for injection (48.6 ± 3.7% of CPR, 70.2 ± 4.1% of CHMP2A, 59.8 ± 2.2% of CHMP3), but with much lower quantities of dsRNAs. Oral delivery of dsRNA failed to silence the expression of the CHMP4B gene, but this could be accomplished by injection of dsRNA (23.1 ± 1.0%). When scoring the phenotypic effects of silencing, both oral delivery and injection of CHMP2A- and CHMP3-dsRNA influenced the locomotion speed of mites significantly. For CPR, silencing could only be accomplished by dsRNA injection, not by feeding. CPR silencing significantly impacted the toxicity of a typical acaricide, pyridaben, as the susceptibility of mites raised 2.75-fold. Last, injection of Eya-dsRNA in adults produced transgenerational phenotypic effects on 3.59% of offspring, as quantified by an observed deviation in eye development, while oral delivery of Eya-dsRNA did not. In conclusion, injection of dsRNA is superior to oral delivery in silencing the expression of the selected genes in this study and could be considered the method of choice to study gene function in reverse genetic approaches.

Fitness Costs Associated with Pyrethroid Resistance in Halotydeus destructor (Tucker) (Acari: Penthaleidae) Elucidated Through Semi-field Trials.

Pyrethroid resistance in the redlegged earth mite, Halotydeus destructor (Tucker), is primarily attributed to a kdr (knockdown resistance) mutation in the parasodium channel gene. To assess fitness costs associated with this resistance, adult resistant and susceptible populations were mixed in different proportions in microcosm tubs and placed in a shade-house simulating field conditions. Three separate experiments were undertaken whereby parental mites were collected from the field and offspring were followed for two to three generations. The association between fitness costs and kdr-mediated resistance was investigated by examining differences in mite numbers and changes in resistant allele frequencies across generations. In two (of the three) experiments, the population fitness measure of mites was significantly lower in microcosms containing a higher proportion of resistant individuals compared with treatments containing susceptible mites. No differences in mite fitness were observed between treatments in the third experiment; in this instance, the starting proportion of individuals homozygous for the resistant mutation was much lower (~40%) than in the other experiments (>90%). In all three experiments, a decrease in the resistant allele frequency across mite generations was observed. These findings indicate a potential deleterious pleiotropic effect of the kdr mutation on the fitness of H. destructor and have implications for resistance management strategies aimed at this important agricultural pest. Further experiments investigating fitness costs directly in the field are warranted.

Defense Responses in the Interactions between Medicinal Plants from Lamiaceae Family and the Two-Spotted Spider Mite Tetranychus urticae Koch (Acari: Tetranychidae)

This study aimed to determine the effects of plant species on the biological parameters of Tetranychus urticae Koch and the time of mite infestation on plant physiology in Ocimum basilicum L., Melissa officinalis L. and Salvia officinalis L. Mite infestation induced various levels of oxidative stress depending on plant species and the duration of infestation. Host plants affected T. urticae life table parameters. The low level of susceptibility was characteristic of S. officinalis, which appeared to be the least infected plant species and reduced mites demographic parameters. Infested leaves of S. officinalis contained elevated levels of hydrogen peroxide (H2O2) and malondialdehyde (MDA) compared to control. In addition, higher membrane lipid peroxidation and higher activity of guaiacol peroxidase (GPX) and lower activity of catalase (CAT) were recorded with a longer mite infestation. In contrast, O. basilicum appeared to be a suitable host on which T. urticae could develop and increase in number. In basil leaves, increasing levels of hydrogen peroxide and MDA with elevated GPX activity and strongly decreased catalase activity were recorded. Knowledge of the differences in mite susceptibility of the tested medicinal plants described in this study has the potential to be applied in breeding strategies and integrated T. urticae pest management in medicinal plant cultivations.

소석회와 식물 추출물의 닭진드기에 대한 구충 효과

This study evaluated calcium hydroxide (slaked lime) and a combination of plant extracts (‘natural product’; clove, cinnamon, and saponin; 1:1:1 ratio) as acaricidal control mechanisms for poultry red mites. Red mite susceptibility was evaluated after treatments with 10% slaked lime, 20% slaked lime, and 1% natural product. The duration of the acaricidal effect was also tested at 0, 10, 30, and 60 min after treatment using 20% slaked lime, 1% natural product, or a mixture of both. In the <i>in vitro</i> experiment, the slaked lime treatments were 73.2% (10% slaked lime) and 85.1% (20% slaked lime) effective on red mites. In acaricidal effect of control materials over times, with 20% slaked lime, the acaricidal effect decreased to 50.7% after 30 min, and 12.7% after 60 min (<i>P</i>&lt;0.05). With 1% natural product, there was no acaricidal effect after 30 min (<i>P</i>&lt;0.05). With 20% slaked lime +1% natural product, all of poultry red mites died until 30 min, and 92.9% after 60 min (<i>P</i>&lt;0.05). On the farm, poultry red mites were observed that the number of poultry red mites increased 7,923 from 36 to 45 weeks, but then decreased to 483 after 20% slaked lime plus 1% natural product treatment. These results indicate that combining slaked lime and plant extracts effectively control poultry red mites.

Physiological responses of plants and mites to salicylic acid improve the efficacy of spirodiclofen for controlling Tetranychus urticae (Acari: Tetranychidae) on greenhouse tomatoes.

Salicylic acid (SA) is a signaling molecule that can induce plant resistance to certain herbivores. Although the role of jasmonic acid in mediating mite-tomato plant interactions has been well studied, the role of salicylic acid has not. This study examined how the application of exogenous SA, via its effects on tomato plant physiology, alters the activity of mite digestive enzymes, mite energy reserves, and mite susceptibility to spirodiclofen. Enzymatic activity-including superoxide dismutase, ascorbate peroxidase, guaiacol peroxidase, polyphenol oxidase, and phenylalanine ammonia-lyase-along with contents of total phenolic, hydrogen peroxide, and total chlorophyll significantly increased in plants 24h after treatment with 2mM of SA. In contrast, catalase activity significantly decreased in treated plants, and malondialdehyde content was unaffected. Mites fed on tomato plants treated with SA had significantly lower glutathione S-transferase, esterase, α-amylase, and aminopeptidase activities than those fed on control plants. Energy reserve analyses demonstrated a significant decrease in contents of lipid, protein, and glycogen in mites fed on SA-treated plants, whereas carbohydrate content significantly increased. The LC50 of spirodiclofen was decreased 1.8-fold for Tetranychus urticae fed on SA-treated tomato plants compared to controls. Treatment of adult mites with 2mM SA on leaf discs did not cause any direct mortality after 24h. Finally, a greenhouse bioassay confirmed that spider mite mortality following exposure to spirodiclofen was significantly higher on SA plants than on control plants. Mortality of mites exposed to half of the recommended rate of spirodiclofen was similar to those exposed to the recommended rate when they were held on treated plants. These results have valuable implications for T. urticae management programs in tomato production.

Functional analysis of an upregulated calmodulin gene related to the acaricidal activity of curcumin against Tetranychus cinnabarinus (Boisduval).

Curcumin is a promising botanical acaricidal compound with activity against Tetranychus cinnabarinus. Calmodulin (CaM) is a key calcium ion (Ca2+ ) sensor that plays a vital role in calcium signaling. Overexpression of the CaM gene with inducible character occurs in curcumin-treated mites, but its functional role remains to be further analyzed by RNA interference (RNAi) and protein expression. A CaM gene was cloned from T. cinnabarinus (designated TcCaM). TcCaM was upregulated and the protein was activated in mites by curcumin. The susceptibility of mites to curcumin was decreased after inhibiting CaM function with anti-CaM drug trifluoperazine (TFP) and silencing CaM transcription with RNAi, suggesting that the CaM gene is involved in the acaricidal activity of curcumin against mites. Moreover, the TFP pre-treated Sf9 cells were resistant to curcumin-mediated increase in [Ca2+ ]i levels, indicating that CaM-mediated Ca2+ homeostasis was disturbed by curcumin. TcCaM was then re-engineered for heterologous expression in Escherichia coli. Strikingly, our results showed that the recombinant CaM protein was directly activated by curcumin via inducing its conformational changes, its half-maximal effective concentration (EC50 ) value is 0.3 μmol L-1 in vitro, which is similar to curcumin against CaM-expressing Sf9 cells (0.76 μmol L-1 ) in vivo. These results confirm that the overexpressed CaM gene is involved in the acaricidal activity of curcumin, and the mode of action of curcumin may be via activating CaM function, and thereby disrupting Ca2+ homeostasis in T. cinnabarinus. This study highlights the novel target mechanism of new acaricides, promoting our understanding of the molecular mechanism of CaM-mediated acaricide targets in mites.

Global stability analysis of a general nonlinear scabies dynamics model

Abstract In this work we present a general nonlinear model of scabies infection dynamics. The dynamics is described by a five-dimensional system of ordinary differential equations that expresses the transmissions between susceptible, infectious/infective individuals and adult scabiei mites. The intrinsic growth rate for susceptible individuals, the infection rates as well as the removal and transmission rates of infected individuals and adult mites are modeled by general nonlinear functions. Based on a set of conditions on these general functions we investigate and analyze our model. Nonnegativity and boundedness of solutions of the model are conducted. A basic reproduction number, ℜ 0 M , is calculated for the model which ensures the existence and stability of all corresponding equilibria. Using candidate Lyapunov functions, it is shown that whenever the basic reproduction number is less than or equal unity, the model has an associated disease-free equilibrium, Q 0 M , that is globally asymptotically stable. In addition, when the threshold exceeds unity the model has a globally asthmatically stable endemic equilibrium, Q ¯ M . Finally, using some parameter values related to the scabies infection dynamics, numerical simulation results are demonstrated to clarify the strength of our main theoretical results. Sensitivity analysis of the endemic equilibrium has been performed.

Inventory of Varroa destructor susceptibility to amitraz and tau-fluvalinate in France.

Varroa destructor is one of the greatest threats for the European honeybee, Apis mellifera. Acaricides are required to control mite infestation. Three conventional chemical acaricide substances are used in France: tau-fluvalinate, flumethrin and amitraz. Tau-fluvalinate was used for over 10years before experiencing a loss of effectiveness. In 1995, bioassay trials showed the first mite resistance to tau-fluvalinate. In some countries, amitraz was widely used, also leading to resistance of V. destructor to amitraz. In France, some efficiency field tests showed a loss of treatment effectiveness with amitraz. We adapted the bioassay from Maggi and collaborators to determine mite susceptibility to tau-fluvalinate and amitraz in France in 2018 and 2019. The lethal concentration (LC) which kills 90% of susceptible mite strains (LC90) is 0.4 and 12µg/mL for amitraz and tau-fluvalinate, respectively. These concentrations were chosen as the determining factors to evaluate mite susceptibility. Some mites, collected from different apiaries, present resistance to amitraz and tau-fluvalinate (71% of the mite samples show resistance to amitraz and 57% to tau-fluvalinate). As there are few active substances available in France, and if mite resistance to acaricides continues to increase, the effectiveness of the treatments will decrease and therefore more treatments per year will be necessary. To prevent this situation, a new strategy needs to be put in place to include mite resistance management. We suggest that a bioassay would be a good tool with which to advise the policymakers.

Population-specific effect of Wolbachia on the cost of fungal infection in spider mites.

Many studies have revealed the ability of the endosymbiotic bacterium Wolbachia to protect its arthropod hosts against diverse pathogens. However, as Wolbachia may also increase the susceptibility of its host to infection, predicting the outcome of a particular Wolbachia‐host–pathogen interaction remains elusive. Yet, understanding such interactions and their eco‐evolutionary consequences is crucial for disease and pest control strategies. Moreover, how natural Wolbachia infections affect artificially introduced pathogens for biocontrol has never been studied. Tetranychus urticae spider mites are herbivorous crop pests, causing severe damage on numerous economically important crops. Due to the rapid evolution of pesticide resistance, biological control strategies using entomopathogenic fungi are being developed. However, although spider mites are infected with various Wolbachia strains worldwide, whether this endosymbiont protects them from fungi is as yet unknown. Here, we compared the survival of two populations, treated with antibiotics or naturally harboring different Wolbachia strains, after exposure to the fungal biocontrol agents Metarhizium brunneum and Beauveria bassiana. To control for potential effects of the bacterial community of spider mites, we also compared the susceptibility of two populations naturally uninfected by Wolbachia, treated with antibiotics or not. In one population, Wolbachia‐infected mites had a better survival than uninfected ones in absence of fungi but not in their presence, whereas in the other population Wolbachia increased the mortality induced by B. bassiana. In one naturally Wolbachia‐uninfected population, the antibiotic treatment increased the susceptibility of spider mites to M. brunneum, but it had no effect in the other treatments. These results suggest that natural Wolbachia infections may not hamper and may even improve the success of biological control using entomopathogenic fungi. However, they also draw caution on the generalization of such effects, given the complexity of within‐host–pathogens interaction and the potential eco‐evolutionary consequences of the use of biocontrol agents for Wolbachia‐host associations.

Inter- and intraspecific variation of spider mite susceptibility to fungal infections: Implications for the long-term success of biological control.

Spider mites are severe pests of several annual and perennial crops worldwide, often causing important economic damages. As rapid evolution of pesticide resistance in this group hampers the efficiency of chemical control, alternative control strategies, such as the use of entomopathogenic fungi, are being developed. However, while several studies have focused on the evaluation of the control potential of different fungal species and/or isolates as well as their compatibility with other control methods (e.g., predators or chemical pesticides), knowledge on the extent of inter‐ and intraspecific variation in spider mite susceptibility to fungal infection is as yet incipient. Here, we measured the mortality induced by two generalist fungi, Beauveria bassiana and Metarhizium brunneum, in 12 spider mite populations belonging to different Tetranychus species: T. evansi, T. ludeni, and T. urticae (green and red form), within a full factorial experiment. We found that spider mite species differed in their susceptibility to infection by both fungal species. Moreover, we also found important intraspecific variation for this trait. These results draw caution on the development of single strains as biocontrol agents. Indeed, the high level of intraspecific variation suggests that (a) the one‐size‐fits‐all strategy may fail to control spider mite populations and (b) hosts resistance to infection may evolve at a rapid pace. Finally, we propose future directions to better understand this system and improve the long‐term success of spider mite control strategies based on entomopathogenic fungi.

Kinome Analysis of Honeybee (Apis mellifera L.) Dark-Eyed Pupae Identifies Biomarkers and Mechanisms of Tolerance to Varroa Mite Infestation

The mite Varroa destructor is a serious threat to honeybee populations. Selective breeding for Varroa mite tolerance could be accelerated by biomarkers within individual bees that could be applied to evaluate a colony phenotype. Previously, we demonstrated differences in kinase-mediated signaling between bees from colonies of extreme phenotypes of mite susceptibility. We expand these findings by defining a panel of 19 phosphorylation events that differ significantly between individual pupae from multiple colonies with distinct Varroa mite tolerant phenotypes. The predictive capacity of these biomarkers was evaluated by analyzing uninfested pupae from eight colonies representing a spectrum of mite tolerance. The pool of biomarkers effectively discriminated individual pupae on the basis of colony susceptibility to mite infestation. Kinome analysis of uninfested pupae from mite tolerant colonies highlighted an increased innate immune response capacity. The implication that differences in innate immunity contribute to mite susceptibility is supported by the observation that induction of innate immune signaling responses to infestation is compromised in pupae of the susceptible colonies. Collectively, biomarkers within individual pupae that are predictive of the susceptibility of colonies to mite infestation could provide a molecular tool for selective breeding of tolerant colonies.

P8 nuclear receptor responds to acaricides exposure and regulates transcription of P450 enzyme in the two-spotted spider mite, Tetranychus urticae

Spider mites are destructive arthropod pests on many crops and they have developed resistance to nearly all acaricides. In recent years, along with the application of high throughput sequencing, the molecular mechanisms of mite resistance had made a series of progress. But, the response in molecular level of mite exposure to acaricides, as well as the original mechanism of resistance development was still unclear. To disclose the deeply mechanisms, we used RNA sequencing to analyze the responses of mite exposure to a sublethal concentration (LC30) treatment of the three different action mode acaricides (Abamectin, Fenpropathrin, and Tebufenpyrad). A high number of differentially expressed genes may well be involved in detoxification and regulatory, with extensive overlap in differentially expressed genes between the three insecticide treatments. Two cytochrome P450 genes were co-up-regulated and one glutathione S-transferase genes were co-down-regulated in all the treatments, while carboxylesterase genes only had a response to abamectin. This interesting phenomenon revealed that P450 enzymes play an important role in the early stage of mite exposure to acaricide. Moreover, a P8 nuclear receptor gene was in response to stress caused by exposure to acaricides and RNA interference (RNAi) experiment indicated P8 nuclear receptor regulates the P450 enzyme activity and susceptibility of mites to acaricide. The differential response information of gene expression based on a large-scale sequence would provide some useful clues for studying the molecular mechanisms of mite resistance formation and development.

Pyrethroid resistance in the pest mite, Halotydeus destructor: Dominance patterns and a new method for resistance screening

An L1024F substitution in the para gene, which encodes a subunit of the voltage-gated sodium channel, has been implicated in pyrethroid resistance in a mite pest, Halotydeus destructor, which attacks rape and other grain crops. A high-resolution melt (HRM) genotyping assay was developed for testing the relative pyrethroid susceptibility of different para genotypes and for high-throughput field screening of resistant alleles. The L1024F mutation was found to be incompletely recessive in phenotypic laboratory bioassays with the pyrethroid pesticide, bifenthrin. While the resistance ratio of heterozygotes (RS) to susceptible homozygotes (SS) was <6 in 24 h bioassays, the resistant homozygotes (RR) (with a resistance ratio > 200,000) survived the recommended field rate of bifenthrin (100 mgL−1). HRM genotyping of mites from field populations across Australia indicated the presence of resistant alleles in Western Australia and South Australia, but not in Victoria and New South Wales. The assay developed will be useful for routine screening of pyrethroid resistance, and the dominance relationships established here point to useful resistance management strategies involving the maintenance of reservoirs of susceptible mites to dilute resistant homozygotes in a population.

Downregulation of carboxylesterase contributes to cyflumetofen resistance in Tetranychus cinnabarinus (Boisduval).

Increased expression or point mutations of carboxyl/cholinesterases (CCEs) have been involved in many cases of insecticide and acaricide resistance. However, it has been only rarely documented that downregulation of CCE genes is associated with resistance, although many insecticides and acaricides need hydrolytic activation in vivo. Previously, expression analysis of a laboratory-selected cyflumetofen-resistant strain of Tetranychus cinnabarinus indicated that resistance was associated with increased expression of a CCE gene of TcCCE04, but also the downregulation of two CCE genes, TcCCE12 and TcCCE23. Synergism experiments revealed the importance of ester hydrolysis in cyflumetofen toxicity, because treatment with S,S,S-tributylphosphorotrithioate (DEF) caused strong inhibition of cyflumetofen hydrolysis, in both the susceptible and resistant strains. Moreover, silencing expression of TcCCE12 and TcCCE23 via RNAi further decreased the susceptibility of mites to cyflumetofen significantly, suggesting that downregulated CCE genes could be involved in cyflumetofen resistance. In addition, it was shown that recombinant TcCCE12 protein could hydrolyze cyflumetofen effectively. Decreased esterase activity via downregulation of specific CCE genes most likely contributes to cyflumetofen resistance by decreased activation of cyflumetofen to its active metabolite. Mixtures of cyflumetofen and esterase-inhibition acaricides (e.g. organophosphates or carbamates) should be avoided in field applications. © 2019 Society of Chemical Industry.

Host Plant-Induced Susceptibility of Two-Spotted Spider Mite Tetranychus urticae (Acari: Tetranychidae) to Some Reduced-Risk Acaricides

The two-spotted spider mite Tetranychus urticae (Acari: Tetranychidae) is a cosmopolitan pest of a large number of host plants having extensive damage potential and the terrific ability of pesticide resistance. In the present study, spider mite population were reared continuously on the leaves of three susceptible host plants namely country bean (Phaseolus vulgaris L), papaya (Carica papaya L) and jute (Corchorus olitorius L) and their susceptibility to some reduced-risk acaricides were determined by leaf disc bioassay method. Abamectin was found to be the most toxic acaricide (LC50 values were 0.432, 0.342 and 0.324 mg L-1 on the country bean, papaya and jute leaf discs respectively at 24 h after treatment application) which was followed by azadirachtin, emamectin benzoate, spinosad and hexythiazox respectively. For all the tested acaricides, toxicity increased proportionately with increasing time duration after treatment application. Moreover, spider mites reared on country bean leaves were comparatively less susceptible to all acaricides followed by mites reared on papaya and jute leaves respectively. Based on the present study, it might be inferred that host plants play a considerable role in the susceptibility of spider mites to acaricides and abamectin could be used as a puissant chemical for controlling spider mites.