Identification and Functional Characterization of an Omega-Class Glutathione S-Transferase Gene PcGSTO1 Associated with Cyetpyrafen Resistance in Panonychus citri (McGregor).

Cyetpyrafen is a recently developed acaricide. The citrus red mite, Panonychus citri (McGregor), has developed significant resistance to cyetpyrafen. However, the molecular mechanism underlying the cyetpyrafen resistance in P. citri remains unclear. Glutathione S-transferases (GSTs) play a critical role in arthropod pesticide resistance. This study showed that GSTs were potentially related to the resistance of P. citri to cyetpyrafen through synergistic experiments and enzyme activity analysis. An omega-family GST gene, PcGSTO1, was significantly up-regulated in the egg, nymph, and adult stages of the cyetpyrafen-resistant strain. Additionally, silencing of PcGSTO1 significantly increased the mortality of P. citri to cyetpyrafen and recombinant PcGSTO1 demonstrated the ability to metabolize cyetpyrafen. Our results indicated that the overexpression of PcGSTO1 is associated with cyetpyrafen resistance in P. citri, and they also provided valuable information for managing resistance in P. citri.

Contrasting roles of cytochrome P450s in amitraz and chlorfenapyr resistance in the crop pest Tetranychus urticae.

The molecular mechanisms of amitraz and chlorfenapyr resistance remain only poorly understood for major agricultural pests and vectors of human diseases. This study focusses on a multi-resistant field strain of the crop pest Tetranychus urticae, which could be readily selected in the laboratory to high levels of amitraz and chlorfenapyr resistance. Toxicity experiments using tralopyril, the active toxophore of chlorfenapyr, suggested decreased activation as a likely mechanism underlying resistance. Starting from the same parental strain, transcriptome profiling revealed that a cluster of detoxifying genes was upregulated after amitraz selection, but unexpectedly downregulated after chlorfenapyr selection. Further functional validation associated the upregulation of CYP392A16 with amitraz metabolism and the downregulation of CYP392D8 with reduced activation of chlorfenapyr to tralopyril. Genetic mapping (QTL analysis by BSA) was conducted in an attempt to unravel the genetic mechanisms of expression variation and resistance. This revealed that chlorfenapyr resistance was associated with a single QTL, while 3 QTLs were uncovered for amitraz resistance. Together with the observed contrasting gene expression patterns, we argue that transcriptional regulators most likely underly the distinct expression profiles associated with resistance, but these await further functional validation.

CYP4CL2 Confers Metabolic Resistance to Pyridaben in the Citrus Pest Mite Panonychus citri.

The citrus red mite Panonychus citri has developed strong resistance to acaricides. Cytochrome P450 monooxygenases (P450s) can detoxify pesticides and are involved in pesticide resistance in many insects. Here, a pyridaben-resistant P. citri strain showed cross-resistance to cyenopyrafen, bifenazate, fenpyroximate, and tolfenpyrad. Piperonyl butoxide, a P450 inhibitor, significantly increased the toxicity of pyridaben to resistant (Pyr_Rs) and susceptible (Pyr_Control) P. citri strains. P450 activity was significantly higher in Pyr_Rs than in Pyr_Control. Analyses of RNA-Seq data identified a P450 gene (CYP4CL2) that is potentially involved in pyridaben resistance. Consistently, it was up-regulated in two field-derived resistant populations (CQ_WZ and CQ_TN). RNA interference-mediated knockdown of CYP4CL2 significantly decreased the pyridaben resistance in P. citri. Transgenic Drosophila melanogaster expressing CYP4CL2 showed increased pyridaben resistance. Molecular docking analysis showed that pyridaben could bind to several amino acids at substrate recognition sites in CYP4CL2. These findings shed light on P450-mediated pyridaben resistance in pest mites.

Effects of acaricides on the activities of monooxygenases in bovine liver microsomes.

Organophosphates (OPs), pyrethrins and fipronil, are acaricides commonly used in cattle, mainly as pour on formulations. Scant information is available on their potential interactions with hepatic xenobiotic metabolizing enzymes. This work aimed to evaluate in vitro the potential inhibitory effects of widely employed acaricides on catalytic activities mediated by hepatic cytochrome P450 (CYP) and flavin-monooxygenase (FMO) enzymes in cattle. Bovine (n = 4) liver microsomes were incubated in the absence (control assays) and in presence of different OPs (fenthion, chlorpyrifos, ethion, diazinon and dichlorvos), fipronil and cypermethrin at 0.1-100 µm. Five oxidative enzyme activities were assayed by spectrofluorimetric or HPLC methods: 7-ethoxyresorufin O-deethylase (for CYP1A1), methoxyresorufin O-demethylase (for CYP1A2), benzyloxyresorufin O-debenzylase (for CYP2B), testosterone 6-beta hydroxylase (for CYP3A) and benzydamine N-oxidase (for FMO). All acaricides, particularly phosphorothionate-containing OPs, inhibited to some extent more than one enzyme activity. The most frequent inhibitor was fenthion, which inhibited (p < .05) all enzyme activities tested (from 22% at 1 µm to 72% at 100 µm). However, low inhibitory potencies (IC50s higher than 7 µm) of all acaricides studied were observed against the catalytic activities assayed. Therefore, the risk of in vivo metabolic interactions due to inhibition of monooxygenases would be low under common husbandry conditions.

Determination of histopathological effects and myoglobin, periostin gene-protein expression levels in Danio rerio muscle tissue after acaricide yoksorrun-5EC (hexythiazox) application.

Although pesticides are essential agrochemicals to annihilate harmful organisms in agriculture, their uncontrolled use has become an important threat to environmental health. Exposure to pesticides can affect many biological systems including immune system, endocrine system, and nervous system. However, the potential side effects of pesticides to skeletal muscle system remain unclear. Present study has focused on the evaluation of this issue by using an acaricide, yoksorrun-5EC (hexythiazox), in an aquatic model organism, Danio rerio. The histological analyses revealed that increased concentrations of the acaricide cause degradation of skeletal muscle along with increased necrosis and atrophy in myocytes, intercellular edema, and increased infiltrations between perimysium sheaths of muscle fibers. The effects of acaricide on myoglobin and periostin, which are associated with oxygen transport and muscle regeneration, respectively, were investigated at the gene and protein levels. RT-PCR results suggested that high concentration yoksorrun-5EC (hexythiazox) can induce myoglobin and periostin genes. Similar results were also obtained in the protein levels of these genes by western blotting analysis. These results suggested that yoksorrun-5EC (hexythiazox)-dependent disruption of skeletal muscle architecture is closely associated with the expression levels of myoglobin and periostin genes in Danio rerio model.

Interaction between anti-tick vaccine and a macrocyclic lactone improves acaricidal efficacy against Rhipicephalus (Boophilus) microplus (Canestrini) (Acari: Ixodidae) in experimentally infested cattle.

The southern cattle fever tick (SCFT) Rhipicephalus (Boophilus) microplus, is considered the most important ectoparasite of livestock in the world because of high financial losses associated with direct feeding and transmission of the hemoparasites Babesia bovis, B. bigemina, and Anaplasma marginale. Unfortunately, SCFT in many parts of the world have evolved resistance to all market-available pesticides thus driving development of new control technologies. Vaccination against ticks using the tick gut protein Bm86 has been shown to be effective against acaricide-resistant ticks. This technique has been successfully implemented in Puerto Rico for the control of acaricide-resistant R. microplus on dairy and beef cattle. Observations from Puerto Rico indicate a potentially positive interaction between anti-tick vaccination when used in conjunction with systemic acaricide treatment. In this project, controlled animal studies were completed directly comparing efficacy of anti-tick vaccination with and without systemic acaricide. Results show that the Bm86 anti-tick vaccine in combination with the macrocyclic lactone, Moxidectin, expressed a synergistic interaction, providing greater and longer efficacy than either treatment alone.

Evaluation of hepatotoxicity and nephrotoxicity induced by fenpyroximate in subchronic-orally exposed Wistar rats.

BACKGROUNDS: Fenpyroximate (FEN) is an acaricide that inhibits the complex I of the mitochondrial respiratory chain. The aim of this work was to explore the hepatotoxic and nephrotoxic effects of FEN on Wistar rats. METHODS: The study involved five groups: a control group and four groups treated with FEN at 1, 2, 4, and 8 mg/Kg bw for 28 consecutive days. Histological examination and biochemical analysis of hepatic and renal biomarkers were performed. The malondialdehyde (MDA), protein carbonyl levels, and antioxidant enzymes activities were measured. Comet assay was conducted to explore FEN genotoxicity. RESULTS: FEN induced a disturbance of the hepatic and renal functions as evidenced by an increase in AST, ALT, ALP, creatinine, and uric acid levels and histopathological modifications in the two examined tissues. FEN increased hepatic and renal lipid peroxidation and protein oxidation. The activities of liver and kidney SOD, CAT, GPX, and GST are increased significantly in FEN-treated rats at doses of 2 and 4 mg/kg bw. However, with the dose of 8 mg/kg bw of FEN, these activities are decreased. Moreover, FEN increased DNA damage in a dose-dependent manner. CONCLUSION: FEN was hepatotoxic and nephrotoxic very likely through induction of oxidative stress.

A H258Y mutation in subunit B of the succinate dehydrogenase complex of the spider mite Tetranychus urticae confers resistance to cyenopyrafen and pyflubumide, but likely reinforces cyflumetofen binding and toxicity.

Succinate dehydrogenase (SDH) inhibitors such as cyflumetofen, cyenopyrafen and pyflubumide, are selective acaricides that control plant-feeding spider mite pests. Resistance development to SDH inhibitors has been investigated in a limited number of populations of the spider mite Tetranychus urticae and is associated with cytochrome P450 based detoxification and target-site mutations such as I260 T/V in subunit B and S56L in subunit C of SDH. Here, we report the discovery of a H258Y substitution in subunit B of SDH in a highly pyflubumide resistant population of T. urticae. As this highly conserved residue corresponds to one of the ubiquinone binding residues in fungi and bacteria, we hypothesized that H258Y could have a strong impact on SDH inhibitors toxicity. Marker assisted introgression and toxicity bioassays revealed that H258Y caused high cross resistance between cyenopyrafen and pyflubumide, but increased cyflumetofen toxicity. Resistance associated with H258Y was determined as dominant for cyenopyrafen, but recessive for pyflubumide. In vitro SDH assays with extracted H258 mitochondria showed that cyenopyrafen and the active metabolites of pyflubumide and cyflumetofen, interacted strongly with complex II. However, a clear shift in IC50s was observed for cyenopyrafen and the metabolite of pyflubumide when Y258 mitochondria were investigated. In contrast, the mutation slightly increased affinity of the cyflumetofen metabolite, likely explaining its increased toxicity for the mite lines carrying the substitution. Homology modeling and ligand docking further revealed that, although the three acaricides share a common binding motif in the Q-site of SDH, H258Y eliminated an important hydrogen bond required for cyenopyrafen and pyflubumide binding. In addition, the hydrogen bond between cyenopyrafen and Y117 in subunit D was also lost upon mutation. In contrast, cyflumetofen affinity was enhanced due to an additional hydrogen bond to W215 and hydrophobic interactions with the introduced Y258 in subunit B. Altogether, our findings not only highlight the importance of the highly conserved histidine residue in the binding of SDH inhibitors, but also reveal that a resistance mutation can provide both positive and negative cross-resistance within the same acaricide mode of action group.

High-resolution genetic mapping reveals cis-regulatory and copy number variation in loci associated with cytochrome P450-mediated detoxification in a generalist arthropod pest.

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.

Activation of the ambusher tick Rhipicephalus microplus (Acari: Ixodidae) exposed to different stimuli.

The present study aimed to evaluate the behaviour of larvae of Rhipicephalus microplus exposed to different stimuli. A Y-olfactometer was positioned vertically and R. microplus larvae were exposed to environmental air, CO2 alone, N,N-diethyl-3-methylbenzamide (DEET) alone, and CO2 combined with the repellents DEET and (E)-2-octenal. Tests were also conducted with the olfactometer positioned horizontally; in this case, however, only CO2 was tested. In all tests conducted with the Y-olfactometer positioned vertically, CO2 activated R. microplus larvae even in the presence of DEET and (E)-2-octenal, although activation was lower when these repellents were used. In the absence of CO2 , larval behaviour against DEET was similar to that of the larvae in the control group. In the tests performed with the olfactometer positioned horizontally, the larvae had no significant response to the presence of CO2 . The larvae were not attracted to or repelled by any compound tested in either the vertical or horizontal position of the olfactometer. The lack of horizontal displacement, attraction or repellence may have been a result of the ambush behaviour of this tick species. However, when larvae were exposed to stimuli and the olfactometer was positioned vertically, the interference of attractant and repellent stimuli in larval behaviour was assessed.

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.

Substrate specificity and promiscuity of horizontally transferred UDP-glycosyltransferases in the generalist herbivore Tetranychus urticae.

Uridine diphosphate (UDP)-glycosyltransferases (UGTs) catalyze the addition of UDP-sugars to small hydrophobic molecules, turning them into more water-soluble metabolites. While their role in detoxification is well documented for vertebrates, arthropod UGTs have only recently been linked to the detoxification and sequestration of plant toxins and insecticides. The two-spotted spider mite Tetranychus urticae is a generalist herbivore notorious for rapidly developing resistance to insecticides and acaricides. We identified a set of eight UGT genes that were overexpressed in mites upon long-term acclimation or adaptation to a new host plant and/or in mite strains highly resistant to acaricides. Functional expression revealed that they were all catalytically active and that the majority preferred UDP-glucose as activated donor for glycosylation of model substrates. A high-throughput substrate screening of both plant secondary metabolites and pesticides revealed patterns of both substrate specificity and promiscuity. We further selected nine enzyme-substrate combinations for more comprehensive analysis and determined steady-state kinetic parameters. Among others, plant metabolites such as capsaicin and several flavonoids were shown to be glycosylated. The acaricide abamectin was also glycosylated by two UGTs and one of these was also overexpressed in an abamectin resistant strain. Our study corroborates the potential role of T. urticae UGTs in detoxification of both synthetic and natural xenobiotic compounds and paves the way for rapid substrate screening of arthropod UGTs.

Isolation and Characterization of New 16-Membered Macrolides from the aveA3 Gene Replacement Mutant Strain Streptomyces avermitilis TM24 with Acaricidal and Nematicidal Activities.

Polyketides represent an important class of biologically active and structurally diverse compounds found in nature. They are biosynthesized from acyl CoA precursors by polyketide synthases (PKSs). The use of combinatorial biosynthesis to form hybrid PKSs is considered to be an excellent approach for the development of novel polyketides. Here, 10 new 16-membered macrolide compounds were isolated from the broth of the genetically engineered strain Streptomyces avermitilis TM24, in which the PKS gene aveA3 was seamlessly replaced by the milbemycin PKS gene milA3. Their structures were elucidated on the basis of NMR and MS spectroscopic analyses. The acaricidal and nematicidal activities of them against Tetranychus cinnabarinus and Bursaphelenchus xylophilus were tested. The results indicated that compound 1 had potent acaricidal activity against adult mites with an LC50 value of 0.0022 mg L-1, while compounds 5 and 7 possessed potent nematicidal activity with LC50 values of 4.56 and 4.30 mg L-1, respectively.

Simultaneous determination of nine acaricides and two metabolites in comb honey by LC/MS/MS.

We developed a method for the simultaneous determination of acaricides in comb honey using LC/MS/MS. Because methods for honey analysis had not previously been applied to comb honey, we modified three techniques for sample preparation and LC/MS/MS conditions. First, we used a modified QuEChERS method that changed the extraction solution from ethyl acetate to acetonitrile. Second, we replaced the InertSep® MA-1 (30 mg, 1 ml) clean-up cartridge with an Oasis® HLB (60 mg, 3 ml). Third, we changed the ionisation mode from ESI to atmospheric pressure chemical ionisation (APCI). With these modifications, sample matrices had no effect on the identification and quantification of analytes, using an external solvent calibration curve. We verified this new method with nine acaricides and two metabolites on comb honey and honey samples from three different honey origins. The trueness ranged from 74.0 to 99.4%. The relative standard deviation of repeatability (RSDr) ranged from 0.8 to 14.8% and that of within-laboratory reproducibility (RSDWR) ranged from 1.3 to 14.8%. All criteria met Japanese validation guidelines. The LOQ was 1.0 µg kg-1 for all analytes. We applied this method to 10 comb honey and 31 honey samples commercially available in Tokyo. From the results of the analysis of 41 samples, we observed that amitraz remained as N-(2,4-dimethylphenyl)-N-methylformamidine (DMPF) in 9 comb honey and 23 honey samples and that their residual concentrations were less than 20 µg kg-1. Using this new method, we improved recovery and precision, which enabled precise quantitative determination. Furthermore, the residual amitraz value in honey determined by both this new and the previous method were in good agreement.

Acaricidal Activity of Cyclodipeptides from Bacillus amyloliquefaciens W1 against Tetranychus urticae.

Bioassay-guided fractionation of the supernatant of the biocontrol strain Bacillus amyloliquefaciens W1 led to the isolation of eight acaricidal cyclodipeptides from the active fractions by column chromatography separation and HPLC purification. The chemical structures of these compounds were identified as cyclo-(Gly-l-Phe), 2, cyclo-(l-Phe- trans-4-OH-l-Pro), 3, cyclo-(Gly-l-Tyr), 4, cyclo-(l-Ala-l-Pro), 5, cyclo-(l-Pro- trans-4-OH-l-Pro), 6, cyclo-(Gly-l-Pro), 7, cyclo-(l-Pro-l-Pro), 8, and cyclo-(l-Tyr- trans-4-OH-l-Pro), 9. Those cyclodipeptides displayed significant acaricidal activities with LC50 values of 13.85-98.24 µM. Cyclo-(l-Tyr- trans-4-OH-l-Pro) (LC50 13.85 µM) was five times more effective than the positive control abamectin (LC50 72.06 µM). The results indicated that the hydroxyl group is an important component. This is the first report on the acaricidal capabilities of cyclodipeptides against Tetranychus urticae. The results revealed that the acaricidal activity of the biocontrol strain B. amyloliquefaciens W1 was dependent on its constituent cyclodipeptides, which have the potential to be safe and environmentally friendly acaricides.

Glutathione S-transferases play a role in the detoxification of flumethrin and chlorpyrifos in Haemaphysalis longicornis.

BACKGROUND: Haemaphysalis longicornis is a tick of importance to health, as it serves as a vector of several pathogens, including Theileria orientalis, Babesia ovata, Rickettsia japonica and the severe fever with thrombocytopenia syndrome virus (SFTSV). Presently, the major method of control for this tick is the use of chemical acaricides. The glutathione S-transferase (GST) system is one mechanism through which the tick metabolizes these acaricides. Two GSTs from H. longicornis (HlGST and HlGST2) have been previously identified. RESULTS: Enzyme kinetic studies were performed to determine the interaction of acaricides with recombinant H. longicornis GSTs. Recombinant HlGST activity was inhibited by flumethrin and cypermethrin, while recombinant HlGST2 activity was inhibited by chlorpyrifos and cypermethrin. Using real-time RT-PCR, the upregulation of the HlGST gene was observed upon exposure to sublethal doses of flumethrin, while the HlGST2 gene was upregulated when exposed to sublethal doses of chlorpyrifos. Sex and strain dependencies in the induction of GST gene expression by flumethrin were also observed. Knockdown of the HlGST gene resulted in the increased susceptibility of larvae and adult male ticks to sublethal doses of flumethrin and the susceptibility of larvae against sublethal doses of chlorpyrifos was increased upon knockdown of HlGST2. CONCLUSIONS: HlGST could be vital for the metabolism of flumethrin in larvae and adult male ticks, while HlGST2 is important in the detoxification of chlorpyrifos in larval ticks.

The first assessment of the stress inducible defense of Leucaena leucocephala with acaricidal potential effect against Rhipicephalus (Boophilus) microplus (Acari: Ixodidae).

Plants respond to wounding caused by mechanical stress or herbivory by synthesizing defense proteins. There are no studies reporting the action of induced plant proteins against ticks. The aim of this study was to investigate the effect of mechanically wounded Leucaena leucocephala leaves against Rhipicephalus (Boophilus) microplus. Initially, we carried out time course experiments to evaluate the impact of mechanical wounding on the protein content and the peroxidase, catalase and protease inhibitor activities in L. leucocephala. We then evaluated the acaricidal activity on R. (B.) microplus from protein extract collected from L. leucocephala after mechanical wounding. L. leucocephala leaves were artificially wounded, and after 6, 12, 24 and 48h, the leaves were collected for protein extraction. Quantitative and qualitative analyses of the proteins were performed. The protein content and peroxidase and protease activities increased 12h after wounding, and the acaricidal activity of this protein extract was evaluated using engorged R. (B.) microplus females. The protein extract obtained after wounding reduced egg production (8.5%) compared to those without wounding. Furthermore, the extract reduced egg hatching by 47.7% and showed an overall efficacy of 56.3% at 0.1 mgP/mL of the protein. We demonstrated that L. leucocephala defensive proteins could be effective against R. (B.) microplus.

The first assessment of the stress inducible defense of Leucaena leucocephala with acaricidal potential effect against Rhipicephalus (Boophilus) microplus (Acari: Ixodidae) / Primeira avaliação de extratos proteicos de Leucaena leucocephala induzidos por injúria mecânica com atividade carrapaticida sobre Rhipicephalus (Boophilus) microplus (Acari: Ixodidae)

Abstract Plants respond to wounding caused by mechanical stress or herbivory by synthesizing defense proteins. There are no studies reporting the action of induced plant proteins against ticks. The aim of this study was to investigate the effect of mechanically wounded Leucaena leucocephala leaves against Rhipicephalus (Boophilus) microplus. Initially, we carried out time course experiments to evaluate the impact of mechanical wounding on the protein content and the peroxidase, catalase and protease inhibitor activities in L. leucocephala. We then evaluated the acaricidal activity on R. (B.) microplus from protein extract collected from L. leucocephala after mechanical wounding. L. leucocephala leaves were artificially wounded, and after 6, 12, 24 and 48h, the leaves were collected for protein extraction. Quantitative and qualitative analyses of the proteins were performed. The protein content and peroxidase and protease activities increased 12h after wounding, and the acaricidal activity of this protein extract was evaluated using engorged R. (B.) microplus females. The protein extract obtained after wounding reduced egg production (8.5%) compared to those without wounding. Furthermore, the extract reduced egg hatching by 47.7% and showed an overall efficacy of 56.3% at 0.1 mgP/mL of the protein. We demonstrated that L. leucocephala defensive proteins could be effective against R. (B.) microplus.

Resumo As plantas respondem a injúria causada por estresse mecânico ou por ataque de herbívoros através da síntese de proteínas de defesa. Não há estudos de proteínas induzidas de plantas contra carrapatos. O objetivo deste estudo foi verificar a atividade acaricida de extratos protéicos de folhas Leucaena leucocephala após injúria mecânica, sobre Rhipicephalus (Boophilus) microplus. Inicialmente foram conduzidos experimentos em diferentes intervalos de tempo para avaliar o impacto da injúria mecânica no conteúdo de proteína, atividade de peroxidase, catalase e inibidor de protease de L. leucocephala. Em seguida foi avaliada a atividade acaricida sobre R. (B.) microplus de um extrato protéico após injúria mecânica. Folhas de L. leucocephala foram artificialmente feridas e após 6, 12, 24 e 48h, as folhas foram coletadas para extração de proteínas. Análises quantitativas e qualitativas das proteínas foram realizadas. A quantidade de proteína e atividades de peroxidase e protease aumentaram 12h após a injúria. O extrato proteico obtido após injúria (12h) reduziu a produção de ovos (8,5%) em comparação com extratos de plantas sem injúria. O extrato reduziu 47,7% a eclosão de ovos e apresentou eficácia geral de 56,3% a 0,1 miligrama de proteína por mL (mgP/mL). Apresentamos que proteínas de defesa de L. leucocephala podem ter atividade sobre R. (B.) microplus.

Intraspecific variation among Tetranychid mites for ability to detoxify and to induce plant defenses.

Two genotypes coexist among Kanzawa spider mites, one of which causes red scars and the other of which causes white scars on leaves, and they elicit different defense responses in host plants. Based on RNA-Seq analysis, we revealed here that the expression levels of genes involved in the detoxification system were higher in Red strains than White strains. The corresponding enzyme activities as well as performances for acaricide resistance and host adaptation toward Laminaceae were also higher in Red strains than White strains, indicating that Red strains were superior in trait(s) of the detox system. In subsequent generations of strains that had survived exposure to fenpyroximate, both strains showed similar resistance to this acaricide, as well as similar detoxification activities. The endogenous levels of salicylic acid and jasmonic acid were increased similarly in bean leaves damaged by original Red strains and their subsequent generations that inherited high detox activity. Jasmonic acid levels were increased in leaves damaged by original White strains, but not by their subsequent generations that inherited high detox activity. Together, these data suggest the existence of intraspecific variation - at least within White strains - with respect to their capacity to withstand acaricides and host plant defenses.

Amitraz and its metabolite differentially activate α- and ß-adrenergic-like octopamine receptors.

BACKGROUND: Amitraz is a formamidine acaricide and insecticide used to control ticks, mites and fleas. N2 -(2,4-Dimethylphenyl)-N1 -methyformamidine (DPMF), a metabolite of amitraz, is thought to be an active agent that exerts acaricidal and insecticidal effects by acting as an agonist on octopamine receptors. The emergence of cattle ticks resistant to amitraz is a serious problem that requires urgent attention. The objective of this research was to determine which type of octopamine receptor is the primary target of amitraz and thereby understand the molecular mechanisms of action and resistance to amitraz. RESULTS: Amitraz and DPMF potently activated Bombyx mori α- and ß-adrenergic-like octopamine receptors (α- and ß-AL OARs) that were stably expressed in HEK-293 cells. Notably, DPMF elevated intracellular cAMP levels, with an EC50 of 79.6 pm in ß-AL OARs, the transcripts of which were prevalently and widely localised in B. mori body parts. Furthermore, DPMF elevated the intracellular Ca2+ levels, with an EC50 of 1.17 nm in α-AL OARs. CONCLUSION: Although both amitraz and DPMF acted as OAR agonists, the metabolite DPMF was more potent than amitraz and differentially activated α- and ß-AL OARs. The present findings provide a basis for studies to examine the mechanism of amitraz resistance and to develop novel acaricides and insecticides. © 2016 Society of Chemical Industry.