Resistance mechanisms to mitochondrial electron transport inhibitors in a field-collected strain of Tetranychus urticae Koch (Acari: Tetranychidae)

A Belgian field strain (MR-VP) of Tetranychus urticae (Koch) (Acari: Tetranychidae) exhibits different levels of resistance to four frequently used METI (mitochondrial electron transport inhibitor)-acaricides, i.e. tebufenpyrad, fenpyroximate, pyridaben and fenazaquin. Resistance factors for these compounds were 184, 1547, 5971 and 35, respectively. A 23.5-fold increase in 7-ethoxy-4-trifluoromethylcoumarin O-deethylation activity suggested that metabolic resistance through elevated levels of cytochrome P450 dependent monooxygenase-activity is a possible resistance mechanism.However, synergism studies with different metabolic inhibitors revealed some contrasting resistance mechanisms between the METI-acaricides. Tebufenpyrad resistance could only be synergized after pre-treatment with the monooxygenase inhibitor piperonyl butoxide (PBO), whereas pyridaben resistance was strongly synergized both by PBO and the esterase inhibitor S,S,S-tributylphosphorotrithioate (DEF). Resistance levels to fenpyroximate could neither be suppressed by PBO nor by DEF. Although METI-acaricides are structurally related, these findings probably reflect a different role of esterases and mono-oxygenases in metabolic detoxification between these compounds. The overall lack of synergism by diethylmaleate (DEM) suggests that glutathione-S-transferases are not an important factor in resistance to METIs.Reciprocal crosses between susceptible females and resistant males showed no maternal effect, and resistance to METI-acaricides was inherited generally as a dominant trait. Backcrosses with F1 females revealed striking differences in the mode of inheritance. Although resistance to fenpyroximate and pyridaben was under monogenic control, resistance to tebufenpyrad was under control of more than one gene.     

Multiple resistance and biochemical mechanisms of pyridaben resistance in Tetranychus urticae (Acari: Tetranychidae)

A field colony of Tetranychus urticae (Koch) (Acari: Tetranychidae) resistant to pyridaben was selected with pyridaben successively for 20 generations to produce the PR-20 strain. Resistance and multiple resistance levels of the PR-20 strain to 15 acaricides were determined using a spray bioassay. The PR-20 strain was extremely resistant to pyridaben (resistance ratio [RR] = 240]. The strain exhibited extremely strong resistance to fenpyroximate (RR=373) and acrinathrin (RR=329) and strong resistance to benzoximate (RR=84). An RR = 10-40 was observed with abamectin, fenazaquin, fenbutatin oxide, fenpropathrin, and tebufenpyrad. The PR-20 strain showed low levels of resistance (RR <10) to azocyclotin, bromopropylate, chlorfenapyr, dicofol, milbemectin, and propargite. Synergist experiments with different metabolic inhibitors revealed that piperonyl butoxide (PBO), a mixed function oxidase (MFO) inhibitor, had the greatest effect on pyridaben resistance. PBO significantly caused pyridaben resistance in the PR-20 strain to drop to the full susceptibility level of the susceptible (S) strain. However, there was no significant difference in MFO activities measured using a model substrate between the S and PR-20 strains. These results suggest that use of certain acaricides with little multiple resistance or PBO will be useful for the management of pyridaben resistance in the field.     

Mode of inheritance of resistance to three new acaricides in the Kanzawa spider mite Tetranychus kanzawai Kishida (Acari:Tetranychidae)

The genetics of resistance to three new types of acaricide tebufenpyrad, fenpyroximate and pyridaben was studied by crossing a resistant (R) with a susceptible (S) strain of Tetranychus kanzawai Kishida. The resistance ratios calculated from the LC50s of the R and S strains were 97, 1265 and 134 for tebufenpyrad, fenpyroximate and pyridaben, respectively. The responses to the three acaricides in F1 females from reciprocal crosses between the R and S strains showed that the modes of inheritance of resistance to tebufenpyrad, fenpyroximate and pyridaben were intermediate, incompletely dominant and completely recessive, respectively. Furthermore, the responses of F2 females from the reciprocal crosses indicated that the resistance to the three acaricides was under monogenic control.Exp Appl Acarol 22: 699708 © 1998 Kluwer Academic Publishers     

二斑叶螨对七种杀螨剂的抗药性测定及其机理研究

室内测定了相对敏感种群(S)和抗性种群(R)对常用7种杀螨剂的敏感性,并测定了羧酸酯酶、谷胱甘肽S-转移酶和乙酰胆碱酯酶3种酶的比活力。结果表明:二斑叶螨Tetranychus urticae Koch R种群已对甲氰菊酯和哒螨灵产生了抗性,抗性倍数分别为5.45和105.47。其中,甲氰菊酯对雌成螨的毒力最低(>3000mg/L),已远远超过田间推荐剂量,不宜继续使用。酶活测定结果表明:谷胱甘肽S-转移酶解毒活性的提高是二斑叶螨对甲氰菊酯产生抗性的原因之一;二斑叶螨对哒螨灵抗性的增强可能与羧酸酯酶有关。     

Tebufenpyrad (Pyranica®) resistance detected in two-spotted spider mite Tetranychus urticae Koch (Acari: Tetranychidae) from apples in Western Australia

A strain (WA) of Tetranychus urticae Koch was collected from apples in Western Australia following a reported field-control failure of tebufenpyrad. The WA strain had been exposed to five tebufenpyrad applications over four seasons. The tebufenpyrad resistance in the WA strain was confirmed at 63.29× which is the first reported instance of tebufenpyrad resistance in T. urticae. The tebufenpyrad resistance in T. urticae conferred a >210× cross-resistance to pryidaben, a 24.60× cross-resistance to fenpyroximate and a 2.20× cross-resistance to chlorfenapyr.Exp Appl Acarol 22: 633–641 © 1998 Kluwer Academic Publishers     

Potentiation of a propargite and fenpyroximate mixture against two-spotted spider mite, Tetranychus urticae (Acari: Tetranychidae)

Using susceptible and resistant reference strains and standard bioassay methods a new miticide product was screened for efficacy and cross-resistance against two-spotted spider mite, Tetranychus urticae Koch. Experimental product NN1 850.0 EW (fenpyroximate and propargite 3:10 ratio) was significantly (P<0.05) more toxic than either compound tested alone (co-toxicity index 1029). Non-overlapping 95% CIs at the LC₅₀ and LC₉₉ level suggest that there is no cross-resistance to NN1 850.0 EW from existing organophosphate and pyrethroid resistance. NN1 850.0 EW is worthy of further development for T. urticae control.     

Resistance to organophosphates, and the genetic background, in fruit tree red spider mite, Panonychus ulmi, from English apple orchards

Assays on adult females and eggs of Panonychus ulmi , on leaf discs of Myrobalan plum, were used to characterise and establish homogenous organophosphateresistant (OR) and susceptible strains. Reciprocal crosses of these strains, and assays on adult mites and eggs of the parent, F₁ and F₂ generations confirmed that resistance was controlled by a single major gene, the expression of which was dominant in the response to parathion, and incompletely dominant to vamidothion, dimethoate and demeton-S-methyl. Reproductive incompatibility, between strains, as in Tetranychus urticae , was not found.
Bioassays on larvae hatched from winter eggs, collected from several orchards in Kent, Essex and Somerset, were used to evaluate the spectrum of cross-resistance to a range of 20 organophosphates (OPs). Together with assays on adult females, these tests showed 10- to 100-fold resistance to most of the OPs used for several years including those noted above, but less than 10-fold to azinphos compounds and to several recently-introduced OPs, e. g. dialifos, triazophos, dioxathion. The resistance spectrum in strain OR and five field strains was similar, but some had higher levels to parathion and demeton-S-methyl, and others to dioxathion after 3–4 years' exposure. Resistance levels to individual OPs may be enhanced by specific selection. Strains resistant to OPs showed only 2- to 5-fold resistance to two carbamates, and were fully susceptible to dicofol.     

Novaluron (Rimon), a novel IGR: potency and cross-resistance

The potency of novaluron on laboratory susceptible and field strains of S. littoralis resembles that of chlorfluazuron and both compounds are about 20-fold more potent than teflubenzuron. No appreciable resistance to novaluron or chlorfluazuron was observed in a field strain of Spodoptera littoralis collected from cucumber field in the central part of Israel. On the other hand, the field strain showed a mild resistance of about 4-fold to teflubenzuron as compared to the laboratory susceptible strain. A very resistant colony of Bemisia tabaci to pyriproxyfen (1,200- to 2,000-fold) showed no appreciable cross-resistance to novaluron. Two field colonies of B. tabaci pressurized with acetamiprid or thiamethoxam for 22 generations, resulting in a 30- to 50-fold resistance to acetamiprid and thiamethoxam, has no cross-resistance to novaluron. The above results are of special interest, indicating a possible alternation between novaluron, pyriproxyfen, and neonicotinoids in insecticide-resistance management programs aiming at preventing resistance development to these novel groups of insecticides against important pests such as whitefly and lepidopteran species.     

A mutation in the PSST homologue of complex I (NADH:ubiquinone oxidoreductase) from Tetranychus urticae is associated with resistance to METI acaricides

The acaricidal compounds pyridaben, tebufenpyrad and fenpyroximate are frequently used in the control of phytophagous mites such as Tetranychus urticae, and are referred to as Mitochondrial Electron Transport Inhibitors, acting at the quinone binding pocket of complex I (METI-I acaricides). Because of their very frequent use, resistance evolved fast more than 20 years ago, and is currently wide-spread. Increased activity of P450 monooxygenases has been often associated with resistance, but target-site based resistance mechanisms were never reported.Here, we report the discovery of a mutation (H92R) in the PSST homologue of complex I in METI-I resistant T. urticae strains. The position of the mutation was studied using the high-resolution crystal structure of Thermus thermophilus, and was located in a stretch of amino acids previously photo-affinity labeled by fenpyroximate. Selection experiments with a strain segregating for the mutant allele, together with marker-assisted back-crossing of the mutation in a susceptible background, confirmed the involvement of the mutation in METI-I resistance. Additionally, an independent genetic mapping approach; QTL analysis identified the genomic region of pyridaben resistance, which included the PSST gene. Last, we used CRISPR-Cas9 genome editing tools to introduce the mutation in the Drosophila PSST homologue.     

Susceptibility and detoxifying enzyme activity in two spider mite species (Acari: Tetranychidae) after selection with three insecticides

Changes in the susceptibility and detoxifying enzyme activity were measured in laboratory strains of Banks grass mite, Oligonychus pratensis (Banks), and twospotted spider mite, Tetranychus urticae Koch, that were repeatedly exposed to three insecticides. Three strains of each mite species were exposed to one of two pyrethroids, bifenthrin, and lambda-cyhalothrin, or an organophosphate, dimethoate, for 10 selection cycles at the LC60 for each insecticide. A reference or nonselected strain of each mite species was not exposed to insecticides. After 10 cycles of exposure, susceptibility to the corresponding insecticides, bifenthrin, lambda-cyhalothrin, and dimethoate, decreased 4.5-, 5.9-, and 289.2-fold, respectively, relative to the reference strain in the respective O. pratensis strains, and 14.8-, 5.7-, and 104.7-fold, respectively, relative to the reference strain in the respective T. urticae strains. In the bifenthrin-exposed O. pratensis strain, there was a 88.9-fold cross-resistance to dimethoate. In the dimethoate-exposed T. urticae strain, there was a 15.9-fold cross-resistance to bifenthrin. These results suggest that there may be cross-resistance between dimethoate and bifenthrin. The reduced susceptibility to dimethoate remained stable for three months in the absence of selection pressure in both mites. The decrease in susceptibility in the O. pratensis strains exposed to bifenthrin, lambda-cyhalothrin, and dimethoate was associated with a 4.7-, 3.0-, and 3.6-fold increase in general esterase activity, respectively. The decrease in susceptibility in the T. urticae strains exposed to bifenthrin and lambda-cyhalothrin was associated with a 1.3- and 1.1-fold increase in general esterase activity, respectively. The mean general esterase activity was significantly higher in the pyrethroid-exposed O. pratensis and T. urticae strains than in the nonselected strain. There was no significant increase in esterase activity in the dimethoate-exposed T. urticae strain. The decrease in susceptibility to insecticides was also associated with reduced glutathione S-transferase 1-chloro-2, 4-dinitrobenzene conjugation activity, but this did not appear to be related to changes in insecticide susceptibility. These results suggest that in these mites, the general esterases may play a role in conferring resistance to pyrethroids. However, some other untested mechanism, such as target site insensitivity, must be involved in conferring dimethoate resistance.     

Cross-resistance between strains of Bacillus sphaericus but not B. thuringiensis israelensis in colonies of the mosquito Culex quinquefasciatus

Two colonies of Culex quinquefasciatus Say (Diptera: Culicidae) were selected with Bacillus sphaericus strains C3-41 and IAB59 in the laboratory for 13 and 18 generations; they attained 145,000- and 48.3-fold resistance, respectively, in comparison with a susceptible laboratory colony (SLCq) and showed very high levels of cross-resistance (8500- to 145,000-fold) to B. sphaericus strains C3-41, 1593, 2297 and 2362. They were relatively susceptible to B. sphaericus strains LP1-G and 47-6B (only 0.8- to 2.8-fold tolerance), with 24.8- to 48.3-fold cross-resistance to strain IAB59. B. sphaericus-resistant mosquito colonies remained highly susceptible to B. thuringiensis israelensis, suggesting that B.t.i. would be of value in the management of B. sphaericus-resistant Cx. quinquefasciatus colonies. The demonstration of low or no cross-resistance of two selected resistant Cx. quinquefasciatus colonies to IAB59, LP1-G and 47-6B strains of B. sphaericus and the finding of a major 49 kDa protein in these strains suggest that there is likely to be another mosquitocidal factor in the three strains.     

Multiple Resistance and Biochemical Mechanisms of Dicofol Resistance in Tetranychus urticae (Acari: Tetranychidae)

A field colony of Tetranychus urticae (Koch) resistant to dicofol was selected with dicofol successively for 20 generations to produce the DR-20 strain. Resistance and multiple resistance levels of the DR-20 strain to 15 acaricides were determined using a spray bioassay. The DR-20 strain was extremely resistant to dicofol [resistance ratio (RR), 465]. The strain showed extremely strong resistance to acrinathrin (RR, 373) and benzoximate (RR, 197) and strong resistance to bromopropylate (RR, 136), fenbutatin oxide (RR, 65), fenpropathrin (RR, 70), fenpyroximate (RR, 68), and pyridaben (RR, 63). A RR of 11–29 was observed with abamectin, fenazaquin, milbemectin, propagite, and tebufenpyrad. The DR-20 strain exhibited low levels of resistance (RR<3) to azocyclotin and chlorfenapyr. In comparative assays with detoxifying enzymes, the DR-20 strain showed 4.7-fold higher activity in p-nitroanisole-O-demethylation and 1.6-fold higher activities in both α- and β-naphthyl acetate hydrolysis. Synergist experiments with different metabolic inhibitors revealed that piperonyl butoxide, iprobenfos, triphenyl phosphate, and 4, 4-dichloro-α-methyl benzhydrol had little or no synergistic activity in the susceptible and DR-20 strains. These results suggest that employment of certain acaricides with little multiple resistance will be useful for the management of dicofol resistance in the field.     

二点叶螨对甲氰菊酯、氧乐果和四螨嗪抗药性的选育、衰退和恢复

以兰州吐鲁沟公园的二点叶螨Tetranychusurticae为敏感品系 ,分别用氧乐果、甲氰菊酯、四螨嗪喷雾处理 15次 ,其抗性水平分别为 38 5、 479 8和 6 7 3倍。将抗性品系分别与敏感品系进行杂交和回交的结果表明 :抗氧乐果品系的显性系数DRS (R♀×S♂ )为 0 470 0、DSR (S♀×R♂ )为 0 4749;抗甲氰菊酯品系的DRS (R♀×S♂ )为 0 5 15 5 ,DSR 为0 5 2 37;抗四螨嗪品系的DRS为 0 3134 ,DSR 为 0 2 46 6。表明二点叶螨对这 3种药剂的抗性均是由单个不完全显性基因所控制。在连续 10个月不接触药剂的情况下 ,3个抗性品系的抗药性都有下降 ,抗氧乐果品系的抗性下降最快 ,只有敏感品系的抗性倍数的 3 6倍 ;抗甲氰菊酯种群的抗性下降较慢 ,为敏感品系的 95 9倍。再经甲氰菊酯、氧乐果、四螨嗪分别连续15次喷雾处理后 ,3个抗性种群的抗性水平又再度回升 ,抗甲氰菊酯品系回升较快 ,抗性为敏感品系的 5 2 3 5倍 ,抗四螨嗪品系次之 ,抗氧乐果的品系抗性恢复最慢。     

Genetic analysis and cross-resistance spectrum of a laboratory-selected chlorfenapyr resistant strain of two-spotted spider mite (Acari: Tetranychidae)

A laboratory susceptible strain of Tetranychus urticae was selected with chlorfenapyr resulting in a resistant strain. After 12 cycles of exposure, the resistance ratio (RR) calculated from the LC50s of susceptible and selected strain was 580. The resistant strain was screened with 16 currently used acaricides for cross-resistance. Cross-resistance was detected with amitraz (RR = 19.1), bifenthrin (RR = 1.3), bromopropylate (RR = 7.5), clofentezine (RR = 29.6) and dimethoate (RR = 17.6). No cross-resistance was detected with the new molecules acequinocyl, bifenazate and spirodiclofen. Mortality caused by chlorfenapyr in the F1 progeny from reciprocal crosses between both strains indicated that the mode of inheritance was incomplete recessive. Mortality in F2 progeny indicated that the resistance was under the control of more than one gene. Synergist experiments with S,S,S-tributylphosphorotrithioate (DEF), piperonylbutoxide (PBO) and diethylmaleate (DEM), which are inhibitors of esterases, monooxygenases and glutathion-S-transferases respectively, suggested a major role of esterases in the resistance to chlorfenapyr.     

Enhanced activation is the mechanism of negative cross-resistance to Chlorpyrifos in the Dicofol-IR strain of Tetranychus urticae (Acari: Tetranychidae).

The mechanism responsible for negative cross-resistance to chlorpyrifos was examined in isogenic dicofol susceptible (Orchard-12) and resistant (Dicofol-IR) two-spotted spider mites, Tetranychus urticae Koch. The acetylcholinesterase of both strains was equally sensitive to inhibition by chlorpyrifos oxon. However, the Dicofol-IR strain showed increased oxidative activation of chlorpyrifos to chlorpyrifos oxon relative to the Orchard-12 strain, suggesting this mechanism is responsible for the observed negative cross-resistance.     

橘全爪螨对双甲脒的抗性选育及其P450基因的表达差异分析

为了对双甲脒进行抗性风险评估, 弄清P450基因在橘全爪螨Panonychus citri抗药性中的作用, 在室内用双甲脒对橘全爪螨进行了抗性选育和交互抗性研究, 同时分析了橘全爪螨双甲脒抗性和敏感品系P450基因表达差异。经过12代抗性选育, 获得了橘全爪螨双甲脒抗性品系, 与敏感品系比较, 橘全爪螨对双甲脒的抗性倍数达到26.32倍。抗性风险评估表明, 橘全爪螨对双甲脒抗性遗传力h²为0.148。螺螨酯、 丁醚脲、 炔螨特和三唑锡对抗性品系的LC₅₀分别为敏感品系的16.85, 4.98, 2.13和2.05倍, 表明双甲脒抗性品系对螺螨酯、 丁醚脲、 炔螨特和三唑锡具有明显的交互抗性。阿维菌素、 苯丁锡、 哒螨灵、 矿物油对抗性品系LC₅₀分别为敏感品系的1.10, 1.21, 0.67和0.99倍, 表明双甲脒抗性品系对上述4种药剂没有显著的交互抗性。基因差异性分析发现, 抗性品系中有16条P450基因发生了上调, 27条P450基因发生了下调, 其中CYP389A6上调倍数最高［log₂ratio (RS/SS)=11.526］, CYP389A2下调倍数最高［log2ratio(RS/SS) =-12.683］, 由此推断, CYP389A6上调和CYP389A2下调可能是橘全爪螨对双甲脒产生抗性的重要原因。     

Various levels of cross-resistance to Bacillus sphaericus strains in Culex pipiens (Diptera: Culicidae) colonies resistant to B. sphaericus strain 2362.

We studied the cross-resistance to three highly toxic Bacillus sphaericus strains, IAB-59 (serotype H6), IAB-881 (serotype H3), and IAB-872 (serotype H48), of four colonies of the Culex pipiens complex resistant to B. sphaericus 2362 and 1593, both of which are serotype H5a5b strains. Two field-selected highly resistant colonies originating from India (KOCHI, 17,000-fold resistance) and France (SPHAE, 23,000-fold resistance) and a highly resistant laboratory-selected colony from California (GeoR, 36,000-fold resistance) showed strong cross-resistance to strains IAB-881 and IAB-872 but significantly weaker cross-resistance to IAB-59 (3- to 43-fold resistance). In contrast, a laboratory-selected California colony with low-level resistance (JRMM-R, 5-fold resistance) displayed similar levels of resistance (5- to 10-fold) to all of the B. sphaericus strains tested. Thus, among the mosquitocidal strains of B. sphaericus we identified a strain, IAB-59, which was toxic to several Culex colonies that were highly resistant to commercial strains 2362 and 1593. Our analysis also indicated that strain IAB-59 may possess other larvicidal factors. These results could have important implications for the development of resistance management strategies for area-wide mosquito control programs based on the use of B. sphaericus preparations.     

Assessment of cross-resistance potential to neonicotinoid insecticides in Bemisia tabaci (Hemiptera: Aleyrodidae)

Laboratory bioassays were carried out with four neonicotinoid insecticides on multiple strains of Bemisia tabaci (Gennadius) to evaluate resistance and cross-resistance patterns. Three imidacloprid-resistant strains and field populations from three different locations in the southwestern USA were compared in systemic uptake bioassays with acetamiprid, dinotefuran, imidacloprid and thiamethoxam. An imidacloprid-resistant strain (IM-R) with 120-fold resistance originally collected from Imperial Valley, California, did not show cross-resistance to acetamiprid, dinotefuran or thiamethoxam. The Guatemala-resistant strain (GU-R) that was also highly resistant to imidacloprid (RR=109-fold) showed low levels of cross-resistance when bioassayed with acetamiprid and thiamethoxam. However, dinotefuran was more toxic than either imidacloprid or thiamethoxam to both IM-R and GU-R strains as indicated by low LC50s. By contrast, a Q-biotype Spanish-resistant strain (SQ-R) of B. tabaci highly resistant to imidacloprid demonstrated high cross-resistance to the two related neonicotinoids. Field populations from Imperial Valley (California), Maricopa and Yuma (Arizona), showed variable susceptibility to imidacloprid (LC50s ranging from 3.39 to 115 microg ml(-1)) but did not exhibit cross-resistance to the three neonicotinoids suggesting that all three compounds would be effective in managing whiteflies. Yuma populations were the most susceptible to imidacloprid. Dinotefuran was the most toxic of the four neonicotinoids against field populations. Although differences in binding at the target site and metabolic pathways may influence the variability in cross-resistance patterns among whitefly populations, comparison of whitefly responses from various geographic regions to the four neonicotinoids indicates the importance of ecological and operational factors on development of cross-resistance to the neonicotinoids.     

二斑叶螨对阿维菌素、哒螨灵和甲氰菊酯的抗性选育及其解毒酶活力变化

在室内模拟田间药剂的选择压力,用阿维菌素、哒螨灵和甲氰菊酯对二斑叶螨Tetranychuc urticae逐代处理,以选育其抗性种群。选育至12代,对阿维菌素抗性增长到6.72倍,对哒螨灵抗性增长到12.1倍,对甲氰菊酯抗性增长到19.9倍。酶抑制剂和离体酶活性的测定结果表明,阿维菌素抗性种群的多功能氧化酶和谷胱甘肽S-转移酶的活性均有所提高;二斑叶螨对哒螨灵的抗性可能与多功能氧化酶、羧酸酯酶的活性增强有关;而羧酸酯酶、多功能氧化酶和谷胱甘肽S-转移酶活性的增强可能是二斑叶螨对甲氰菊酯产生抗性的主要原因。     

Complete maternal inheritance of bifenazate resistance in Tetranychus urticae Koch (Acari: Tetranychidae) and its implications in mode of action considerations

Bifenazate is a selective hydrazine carbazate acaricide launched in 1999 and reported to be neurotoxic, since preliminary studies on the mode of action suggested that bifenazate may act on GABA-gated chloride channels. However, this information has not yet been supported by mechanistic studies. Therefore bifenazate is still considered as a neuronal inhibitor, but with unknown mode of action. Here we report an alternative hypothesis on the mode of action of bifenazate, i.e. its possible interference with a non-neuronal target site. An acaricide susceptible strain of Tetranychus urticae Koch (Acari: Tetranychidae), LS-VL, was artificially selected for bifenazate resistance, and after 36 generations an extremely high resistance ratio (RR) of >164,000 was obtained. This bifenazate-resistant strain (BR-VL) lacks cross-resistance to many different chemical classes and modes of action of other acaricides. In order to check for metabolic resistance mechanisms, synergists known to inhibit well-known detoxification routes were used together with in vitro enzymatic assays. No synergism or highly increased detoxification activity was observed in the resistant strain. However, the organophosphorous esterase inhibitor S,S,S-tributylphosphorotrithioate (DEF) applied to the susceptible strain could completely antagonise the acaricidal efficacy of bifenazate, suggesting that bifenazate is a pro-acaricide, not active by itself, that needs in vivo activation by esterases. Reciprocal crosses of diploid females and haploid males of strains LS-VL (susceptible) and BR-VL (bifenazate resistant) revealed that bifenazate resistance was inherited completely maternally, i.e. resistance is fully dominant when susceptible males were crossed with resistant females, and fully recessive when resistant males were crossed with susceptible females. Such an inheritance pattern has to our knowledge never been observed before in the case of insecticide/acaricide resistance. This observation may suggest a target-site for bifenazate encoded by the mitochondrial genome. Further evidence supporting such a hypothesis was obtained when measuring the ATP-level in spider mites treated with bifenazate. The ATP content in bifenazate treated mites declined progressively between 0 and 4h after treatment, similarly to mites treated with the complex I inhibitor fenpyroximate, an acaricide known to interfere with mitochondrial function. The obtained results suggest a target-site other than GABA-gated chloride channels, most likely encoded by and located in the mitochondria.