Determination of acaricide resistance allele frequencies in field populations of Tetranychus urticae using quantitative sequencing

Resistances to monocrotophos, fenpropathrin and abamectin in Tetranychus urticae are primarily conferred by reduced sensitivities of respective target sites [i.e., acetylcholinesterase (TuAChE), voltage-sensitive sodium channel (TuVSSC) and glutamate-gated chloride channel (TuGluCl)], which are due to point mutations (G228S and F439W in TuAChE; L1022V in TuVSSC; G323D in TuGluCl). As a population-based genotyping technique, a quantitative sequencing (QS) protocol was developed for the determination of the resistance-associated mutation frequencies in T. urticae. Standard prediction equations revealed high correlation coefficients (r² = 0.993–0.999), demonstrating that the resistant nucleotide signal ratio is highly proportional to the resistance allele frequencies. The lower and higher detection limits for the four resistance mutations were 3.7–13.1% (7.8 ± 3.3%) and 89.4–97.3% (93.3 ± 3.2%), respectively, suggesting that QS can be employed as a preliminary monitoring tool for the detection of resistance allele frequencies, which ranged approximately 7.8–93.3% at the 95% confidence level. The QS was successfully employed for the determination of resistance allele frequencies in 26 T. urticae populations. The two TuAChE mutations responsible for monocrotophos resistance were almost saturated in most field populations. The TuVSSC L1022V mutation tentatively associated with fenpropathrin resistance was also found in 9 field populations. However, the TuGluCl G323D mutation conferring abamectin resistance was found only in one field population, suggesting that abamectin resistance is not yet widespread. The QS protocol, as an alternative to traditional bioassays, will greatly facilitate resistance monitoring of T. urticae.     

A single nucleotide polymorphism in the acetylcholinesterase gene of the predatory mite Kampimodromus aberrans (Acari: Phytoseiidae) is associated with chlorpyrifos resistance

The predatory mite Kampimodromus aberrans (Oudemans) (Acari: Phytoseiidae) is one of the most important biocontrol agents of herbivorous mites in European perennial crops. The use of pesticides, such as organophosphate insecticides (OPs), is a major threat to the success of biocontrol strategies based on predatory mites in these cropping systems. However, resistance to OPs in K. aberrans has recently been reported. The present study investigated the target site resistance mechanisms that are potentially involved in OP insensitivity. In the herbivorous mite Tetranychus urticae Koch (Acari: Tetranychidae), resistance to OPs is due to a modified and insensitive acetylcholinesterase (AChE; EC: 3.1.1.7) that bears amino acid substitution F331W (AChE Torpedo numbering). To determine whether the predators and prey have evolved analogous molecular mechanisms to withstand the same selective pressure, the AChE cDNA from a putative orthologous gene was cloned and sequenced from susceptible and resistant strains of K. aberrans. No synonymous mutation coding for a G119S substitution was determined to be strongly associated with the resistant phenotype instead of the alternative F331W. Because the same mutation in T. urticae AChE was not associated with comparable levels of chlorpyrifos resistance, the role of the G119S substitution in defining insensitive AChE in K. aberrans remains unclear. G119S AChE genotyping can be useful in ecological studies that trace the fate of resistant strains after field release or in marker-assisted selection of improved populations of K. aberrans to achieve multiple resistance phenotypes through gene pyramiding. The latent complexity of the target site resistance in K. aberrans vs. that of T. urticae is also discussed in the context of data from the genome project of the predatory mite Metaseiulus occidentalis (Nesbitt) (Acari: Phytoseiidae).     

Mutation in ace1 associated with an insecticide resistant population of Plutella xylostella

Insensitive acetylcholinesterase (AChE) was determined to be involved in an EPN-resistant (ER) strain and a contaminated susceptible (CS) strain of diamondback moth (DBM, Plutella xylostella L.), as estimated by AChE inhibition assay using DDVP as a inhibitor in a nondenaturing electrophoresis gel. The ER strain exhibited very high AChE insensitivity, high resistance ratio, and two point mutations (G324A, A298S) in ace1-type AChE gene (Pxace1). The CS strain showed low AChE insensitivity, low resistance ratio, and it has only one point mutation (G324A). These findings suggest that the A298S mutation, along with reported G324A mutation (Baek et al, 2005), can be important in the development of organophosphate resistance. These results also suggest that the A298S mutation could be a good candidate for a molecular diagnosis marker for resistance monitoring. Three molecular diagnosis methods (Quantitative Sequencing; QS, PCR amplification of specific alleles; PASA and restriction fragment length polymorphism; RFLP) were developed which successfully detected specific resistance associated point mutations. Seven local population DBMs were surveyed and showed high insecticide resistance levels and a A298S mutation in Pxace1. These methods can be used to monitor the resistance allele in field population of DBMs and resistance management strategy.     

Mechanisms of monocrotophos resistance in cotton bollworm,Helicoverpa armigera (Hübner)

Insensitive acetylcholinesterase was identified as a resistance mechanism by comparing biochemical analysis with a laboratory selected monocrotophos resistant cotton bollworm (RR: 200) and the susceptible strain. The cDNA encoding AChE was cloned by the method of RACE (rapid amplification of cDNA ends). The complete AChE gene deduced from the cDNA consisted of a putative signal peptide of 32 amino acid residues, a mature protein of 615 residues, 5' untranslated regions (UTR) of 315 bp and 3' UTR of 324 bp. The coding sequence had a high degree of homology to the AChE from other insect species reported in the GenBank. After comparing analysis of the entire AChE gene sequence from 5 resistant and 6 susceptible cotton bollworm individuals, nine mutations were identified. One of them, the Ala/Thr mutation, is likely to be responsible for the AChE insensitivity to monocrotophos.     

Management of <Emphasis Type="Italic">Tetranychus urticae</Emphasis> (Acari: Tetranychidae) in strawberry fields with <Emphasis Type="Italic">Neoseiulus californicus</Emphasis> (Acari: Phytoseiidae) and acaricides

The purpose of this study was to evaluate the performance of Neoseiulus californicus (McGregor) for the control of Tetranychus urticae Koch in commercial strawberry fields, under greenhouse conditions, in association or not with the use of acaricides. The N. californicus strain used in this study was tolerant or resistant to several pesticides. Three experiments were carried out in the State of São Paulo, Brazil. For the first experiment, the initial infestation of T. urticae was 87.1 active stages per leaflet. Two applications of propargite were made on the first and the 14th day of the experiment. Approximately 2 h after each propargite application, N. californicus was released at a rate of 3.0 and 1.9 adult mites per plant, respectively, for each application. The population of T. urticae decreased from 87.1 to 2.8 mites per leaflet in the first three weeks. After this period, the population of T. urticae was maintained at low levels (≤1.5 mites/leaflet) until the end of the experiment (10th week). Propargite and dimethoate sprayed on the strawberry field did not affect significantly the population of this predaceous mite. For the second experiment, the infestation of T. urticae was 29.1 mites per leaflet, when the acaricide chorfenapyr was applied on the strawberry field. The release of N. californicus (2 mites per plant) was made 2 weeks after spraying the acaricide. The population of T. urticae was maintained at low levels (≤2.8 mites/leaflet) for 8 weeks (evaluation period). The T. urticae infestations in plots with N. californicus were significantly lower than in non-release plots, for the experiments 1 and 2. In the third experiment, the initial infestation of T. urticae was 40.5 mites per leaflet (55.5 active stages/leaflet on release plants; 25.5 active stages/leaflet on non-release plants). Three releases of N. californicus (average rate of 3.0 adult mites/plant), without any acaricide application, were not sufficient to reduce significantly the T. urticae population in release plots (release plants + non-release plants) in 6 weeks from the first release, however, the spider mite population decreased from 55.5 to 7.8 active stages per leaflet on release plants, during this period. Interplant dispersal of N. californicus was low in this strawberry field with high infestation of T. urticae. The studies indicate the viability of the use of this strain of N. californicus for the control of T. urticae in strawberry fields under greenhouse conditions, especially in association with selective acaricides.     

Phenotypic- and Genotypic-Resistance Detection for Adaptive Resistance Management in Tetranychus urticae Koch

Rapid resistance detection is necessary for the adaptive management of acaricide-resistant populations of Tetranychus urticae. Detection of phenotypic and genotypic resistance was conducted by employing residual contact vial bioassay (RCV) and quantitative sequencing (QS) methods, respectively. RCV was useful for detecting the acaricide resistance levels of T. urticae, particularly for on-site resistance detection; however, it was only applicable for rapid-acting acaricides (12 out of 19 tested acaricides). QS was effective for determining the frequencies of resistance alleles on a population basis, which corresponded to 12 nonsynonymous point mutations associated with target-site resistance to five types of acaricides [organophosphates (monocrotophos, pirimiphos-methyl, dimethoate and chlorpyrifos), pyrethroids (fenpropathrin and bifenthrin), abamectin, bifenazate and etoxazole]. Most field-collected mites exhibited high levels of multiple resistance, as determined by RCV and QS data, suggesting the seriousness of their current acaricide resistance status in rose cultivation areas in Korea. The correlation analyses revealed moderate to high levels of positive relationships between the resistance allele frequencies and the actual resistance levels in only five of the acaricides evaluated, which limits the general application of allele frequency as a direct indicator for estimating actual resistance levels. Nevertheless, the resistance allele frequency data alone allowed for the evaluation of the genetic resistance potential and background of test mite populations. The combined use of RCV and QS provides basic information on resistance levels, which is essential for choosing appropriate acaricides for the management of resistant T. urticae.     

Interactions in the biological control of western flower thrips Frankliniella occidentalis (Pergande) and two-spotted spider mite Tetranychus urticae Koch by the predatory bug Orius insidiosus Say on beans

An omnivore shows preference to its preys and thus its control efficiency could be altered in different mix infestation system. The efficiency of Orius insidiosus for biocontrol of either Frankliniella occidentalis or Tetranychus urticae alone or for the two pests in combinations was studied on beans. When only mites or thrips were offered as prey, 1 or 2 O. insidiosus could considerably suppress pest populations at an initial density of 20, 40, and 80 adult female mites, and 100 and 160 thrips larvae, respectively. A single O. insidiosus was able to reduce mite populations by 52.9, 38.7, and 25.8% at initial densities of 20, 40, and 80 mites, respectively, two bugs achieved control levels of 60.6, 63.1, and 38.4%. Releases of 1 and 2 O. insidiosus resulted in corrected mortalities of 62.5 and 87.9%, and 46.3 and 71.9% in F. occidentalis at initial larval densities of 100 and 160, respectively. When two pests were simultaneously offered, the efficiency of O. insidiosus in controlling T. urticae markedly decreased. Furthermore, mite control decreased with increasing T. urticae densities and was also affected by the density of O. insidiosus. The presence of mites at initial densities of 20–80 females did not significantly influence thrips control by O. insidiosus. The presence of F. occidentalis resulted in higher oviposition by O. insidiosus females than the presence of mites, indicating that thrips are a more suitable resource than T. urticae for O. insidiosus. The implications for biocontrol of F. occidentalis and T. urticae are discussed.     

A novel point mutation of acetylcholinesterase in a trichlorfon-resistant strain of the oriental fruit fly Bactrocera dorsalis (Diptera: Tephritidae)

Acetylcholinesterase (AChE) is the target enzyme of organophosphorus and carbamate insecticides. We applied trichlorfon to select resistant strains of Bactrocera dorsalis Hendel in the laboratory. Two trichlorfon-resistant strains, the Tri-R₁ strain with 18.23-fold resistance and the Tri-R₂ strain with 69.5-fold resistance, were obtained. Three known mutations, I159V, G433S and Q588R were identified in AChE of two resistant strains, and a novel mutation, G365A, was identified in the more resistant Tri-R₂ strain. The modeled 3-D-structure of AChE showed that G365A and G433S are closely adjacent in the gorge above the catalytic site S235. Mutations of G365A and G433S resulted in a steric hindrance by stronger Van der Waals force between two sites. Such a minor structural change might block insecticides from squeezing through the gorge to reach the active site, but not the natural substrate. Compared with the susceptible strain, the AChE activity of the Tri-R₁ strain and the Tri-R₂ strain was 0.87- and 0.67-fold, the K _m value of the Tri-R₁ strain and the Tri-R₂ strain was 0.11- and 0.10-fold, the V _max value of two resistant strains was 0.26- and 0.15-fold, whereas, the I ₅₀ to trichlorfon significantly increased by 9.07- and 13.19-fold. These results suggested that the novel point mutation G365A of AChE might be involved in increasing resistance to trichlorfon in the resistant strain of oriental fruit fly.     

Simulating effects of environmental factors on biological control of Tetranychus urticae by Typhlodromus pyri in apple orchards

Successful biological control of mites is possible under various conditions, and identifying what are the requirements for robust control poses a challenge because interacting factors are involved. Process-based modeling can help to explore these interactions and identify under which conditions biological control is likely, and when not. Here, we present a process-based model for population interactions between the phytophagous mite, Tetranychus urticae, and its predator, Typhlodromus pyri, on apple trees. Temperature and leaf nitrogen concentration influence T. urticae rates of development and reproduction, while temperature and rate of ingestion of prey and pollen influence T. pyri rates of survival and reproduction. Predator and prey population dynamics are linked through a stage structured functional response model that accounts for spatial heterogeneity in population density throughout the trees. T. urticae biomass-days (BMD’s), which account for sizes of larvae, nymphs and adults, indicate level of mite-induced leaf damage. When BMD’s exceed 290 per leaf, there are economic losses. When BMD’s exceed 350 per leaf, T. urticae population growth is curbed and eventually the population decreases. Simulations were run to determine which conditions would lead to current year economic loss and increased risk of loss in the following year, i.e. where more T. urticae than T. pyri are present at the end of September. Risk was high with one or more of the following initial conditions: a high prey: predator ratio (10:1 or more); a low to intermediate (0.04–0.2 T. urticae per leaf) initial density; T. urticae with a higher initial proportion of adult females than T. pyri; and a delayed first detection of mites, whether in late July, or sometimes in late June, but not in early June. Warm summer weather, higher leaf nitrogen and T. urticae immigration into trees were also risk factors. Causes for these patterns based on biological characteristics of T. urticae and T. pyri are discussed, as are counter measures which can be taken to reduce risk.     

Milbemectin resistance in <Emphasis Type="Italic">Tetranychus urticae</Emphasis> (Acari: Tetranychidae): selection,stability and cross-resistance to abamectin

Studies on artificial laboratory selections with milbemectin, stability of milbemectin resistance and possible cross-resistance with abamectin were carried out with Tetranychus urticae Koch to provide basic information for a milbemectin resistance management program. Selections for resistance and susceptibility to milbemectin were performed in a population of T. urticae, collected from a commercial chrysanthemum field in the State of São Paulo, Brazil. After six selections for resistance and five selections for susceptibility, susceptible (S) and resistant (R) strains of T. urticae to milbemectin were obtained. The resistance ratio (R/S) at the LC₅₀ reached 409-fold value. The stability of milbemectin resistance was also studied under laboratory conditions, using a population with initial frequency of 75% of resistant mites. The frequencies of milbemectin resistance were evaluated monthly for a period of 7 months. In order to observe possible correlation between milbemectin and abamectin resistance, the frequencies of abamectin resistance were also evaluated for that population, during the same period. The frequency of milbemectin resistance decreased from 75 to 14.5%, while the percentage of abamectin resistant mites decreased from 57 to 9.1%, in 7 months. The frequencies of milbemectin and abamectin resistance were also evaluated in 25 field populations of T. urticae, collected from several crops in the State of São Paulo. The frequencies of milbemectin resistance varied from 4.1 to 89.5%, and of abamectin, from 7.0 to 90.5%. A positive and significant correlation was observed between the frequencies of milbemectin and abamectin resistance, indicating positive cross-resistance between these acaricides. The results indicate that abamectin should be avoided for managing milbemectin resistance in T. urticae. This is the first report on milbemectin resistance in T. urticae in Brazil.     

Both the anterior and posterior eyes function as photoreceptors for photoperiodic termination of diapause in the two-spotted spider mite

Photoreceptors involved in photoperiodism in insects and mites can be either the retinal photoreceptors in the visual system or nonvisual extraretinal photoreceptors. Mites with no eyes have a clear photoperiodic response, suggesting the involvement of extraretinal photoreceptors in mite photoperiodism. In mites equipped with eyes, however, it is not known whether the retinal or extraretinal photoreceptors are involved in photoperiodism. The two-spotted spider mite Tetranychus urticae possesses two pairs of eyes. Adult females of this species terminate diapause in response to long days. To investigate whether the eyes function as photoperiodic photoreceptors in T. urticae, their eyes were ablated using a laser ablation system. Mites with their eyes intact terminated diapause under long days after low temperature exposure, whereas they remained in diapause under short days. Under constant darkness, they did not terminate diapause. When all eyes were removed, the mites remained in diapause even when they were maintained under long days. In contrast, the mites showed clear photoperiodic response when only the anterior or posterior eyes were removed. These results indicate that both the anterior and posterior eyes function as photoreceptors in photoperiodic termination of diapause in T. urticae.     

A horizontally transferred cyanase gene in the spider mite Tetranychus urticae is involved in cyanate metabolism and is differentially expressed upon host plant change

The genome of the phytophagous two-spotted spider mite Tetranychus urticae was recently sequenced, representing the first complete chelicerate genome, but also the first genome of a highly polyphagous agricultural pest. Genome analysis revealed the presence of an unexpected high number of cases of putative horizontal gene transfers, including a gene that encodes a cyanase or cyanate lyase. In this study we show by recombinant expression that the T. urticae cyanase remained functionally active after horizontal gene transfer and has a high affinity for cyanate. Cyanases were also detected in other plant parasitic spider mites species such as Tetranychus evansi and Panonychus citri, suggesting that an ancient gene transfer occurred before the diversification within the Tetranychidae family. To investigate the potential role of cyanase in the evolution of plant parasitic spider mites, we studied cyanase expression patterns in T. urticae in relation to host plant range and cyanogenesis, a common plant defense mechanism. Spider mites can alter cyanase expression levels after transfer to several new host plants, including the cyanogenic Phaseolus lunatus. However, the role of cyanase is probably not restricted to cyanide response, but likely to the plant nutritional quality as a whole. We finally discuss potential interactions between cyanase activity and pyrimidine and amino acid synthesis.     

Sequence of a cDNA encoding acetylcholinesterase from susceptible and resistant two-spotted spider mite,Tetranychus urticae

Acetylcholinesterase (AChE) from two-spotted spider mites, Tetranychus urticae was compared between an organophosphate susceptible (TKD) and a resistant (NCN) strain. The AChE of TKD had lower affinity to acetylthiocholine and propionylthiocholine than that of NCN, and the inhibition of AChE by DDVP, ambenonium, eserine and n-methyl-eserine showed that NCN was more insensitive than TKD. AChE cDNA sequence was determined, and the 687 amino acids of primary structure were deduced. There were six replacements of amino acid residues in TKD and two in NCN. #F331(439)C was the only substitution unique to NCN, however, this mutation existed homozygously in only two out of nine mites. This residue is one of the gorge lining components, and #F331(439)C might act an important role in the sensitivity of AChE to the inhibitors.     

Interactions in a tritrophic acarine predator-prey metapopulation system V: Within-plant dynamics of Phytoseiulus persimilis and Tetranychus urticae (Acari: Phytoseiidae, Tetranychidae)

To investigate the relative contributions of bottom-up (plant condition) and top-down (predatory mites) factors on the dynamics of the two-spotted spider mite (Tetranychus urticae), a series of experiments were conducted in which spider mites and predatory mites were released on bean plants. Plants inoculated with 2, 4, 8, 16, and 32 adult female T. urticae were either left untreated or were inoculated with 3 or 5 adult female predators (Phytoseiulus persimilis) one week after the introduction of spider mites. Plant area, densities of T. urticae and P. persimilis, and plant injury were assessed by weekly sampling. Data were analysed by a combination of statistical methods and a tri-trophic mechanistic simulation model partly parameterised from the current experiments and partly from previous data. The results showed a clear effect of predators on the density of spider mites and on the plant injury they cause. Plant injury increased with the initial number of spider mites and decreased with the initial number of predators. Extinction of T. urticae, followed by extinction of P. persimilis, was the most likely outcome for most initial combinations of prey and predators. Eggs constituted a relatively smaller part of the prey population as plant injury increased and of the predator population as prey density decreased. We did not find statistical evidence of P. persimilis having preference for feeding on T. urticae eggs. The simulation model demonstrated that bottom-up and top-down factors interact synergistically to reduce the density of spider mites. This may have important implications for biological control of spider mites by means of predatory mites.     

Testing for reproductive interference in the population dynamics of two congeneric species of herbivorous mites

When phylogenetically close, two competing species may reproductively interfere, and thereby affect their population dynamics. We tested for reproductive interference (RI) between two congeneric haplo-diploid spider mites, Tetranychus evansi and Tetranychus urticae, by investigating their interspecific mating and their population dynamics when they competed on the same plants. They are both pests of tomato, but differ in the host plant defences that they suppress or induce. To reduce the effect of plant-mediated interaction, we used a mutant tomato plant lacking jasmonate-mediated anti-herbivore defences in the competition experiment. In addition, to manipulate the effect of RI, we introduced founder females already mated with conspecific males in mild RI treatments or founder, virgin females in strong RI treatments (in either case together with heterospecific and conspecific males). As females show first-male sperm precedence, RI should occur especially in the founder generation under strong RI treatments. We found that T. urticae outcompeted T. evansi in mild, but not in strong RI treatments. Thus, T. evansi interfered reproductively with T. urticae. This result was supported by crossing experiments showing frequent interspecific copulations, strong postmating reproductive isolation and a preference of T. evansi males to mate with T. urticae (instead of conspecific) females, whereas T. urticae males preferred conspecific females. We conclude that interspecific mating comes at a cost due to asymmetric mate preferences of males. Because RI by T. evansi can improve its competitiveness to T. urticae, we propose that RI partly explains why T. evansi became invasive in Europe where T. urticae is endemic.     

Development of Neoseiulus womersleyi (Schicha) and Euseius ovalis (Evans) feeding on four tetranychid mites (Acari: Phytoseiidae,Tetranychidae) and pollen

The development of the predatory mites, Neoseiulus womersleyi (Schicha) and Euseius ovalis (Evans), feeding on four tetranychid mites (Tetranychus urticae, Tetranychus kanzawai, Oligonychus mangiferus, Panonychus citri), maize pollen or Chinese loofah pollen was studied at 25 °C. Immature stages of N. womersleyi feeding on T. urticae and T. kanzawai had shorter developmental duration (4.71 and 5.02 days for females, 4.77 and 5.19 days for males, respectively) than those feeding on other food sources. Immature stages of E. ovalis females feeding on O. mangiferus and T. urticae developed in 4.99 and 5.13 days, respectively, the shortest developmental duration measured. Immature stages of E. ovalis males feeding on O. mangiferus and T. urticae developed in 5.12 and 5.37 days, respectively. The longevity of N. womersleyi males (13.31 to 14.51 days) and females (17.67 to 21.81 days) feeding on T. urticae, T. kanzawai or maize pollen was longer than the longevity of N. womersleyi feeding on O. mangiferus, P. citri or loofah pollen. E. ovalis males (12.91 to 16.74 days) and females (16.24 to 23.77 days) feeding on O. mangiferus, T. urticae or maize pollen lived longer than E. ovalis males and females feeding on T. kanzawai, P. citri or loofah pollen.     

Evolutionary adaptation to host plants in a laboratory population of the phytophagous mite Tetranychus urticae Koch

Summary For evolutionary expansion of host range to occur in an herbivore population, genetic variation in ability to survive on and/or accept new hosts must be present. To determine whether a population of the phytophagous mite Tetranychus urticae contained such variation, I established lines from the population on two hosts on which mites initially showed both high juvenile mortality and low acceptance, tomato and broccoli. In less than ten generations, mites from the line kept on each host showed both lower mortality and greater acceptance on it than mites from a control line kept on lima bean, a favorable host for T. urticae. Host acceptance was measured by the proportion of mites attempting to disperse from leaves of the host. The line kept on tomato but not the one kept on broccoli also increased in development rate on its host. These results and those of a similar previous experiment on cucumber indicate that T. urticae populations can adapt to a diversity of initially unfavorable hosts. T. urticae populations therefore should be able to respond to temporal and spatial variation in host availability by adapting to the most abundant hosts.     

Multiple resistance mechanisms to abamectin in the two-spotted spider mite

The two-spotted spider mite, Tetranychus urticae, is one of the most destructive pests of various orchard trees and garden plants. Biochemical mechanisms of abamectin resistance in two T. urticae strains (PTF, 239-fold resistance; AbaR, ca. 4753-fold resistance) were investigated. The involvement of both esterase (Est) and mixed function oxidase (MFO) in abamectin resistance was suggested by synergistic bioassays, in which median lethal time (LT₅₀) values were significantly reduced by pretreatment with triphenyl phosphate (TPP) and piperonyl butoxide, respectively. Detoxification enzyme assays confirmed that Est and MFO were related to abamectin resistance as metabolic factors. Moreover, some Est bands on a native isoelectric focusing gel were specifically inhibited by TPP, implying their association with resistance. Pretreatment with verapamil in synergistic bioassays did not reduce the LT₅₀ to abamectin, suggesting that the ABC transporter is not likely involved in resistance. However, enhanced MFO and Est activities in the AbaR strain were not enough to account for the extremely high level of abamectin resistance, which suggests the involvement of additional resistance mechanisms, such as target site insensitivity.