Biochemical and molecular mechanisms of acaricide resistance in Tetranychus urticae

The two-spotted spider mite Tetranychus urticae is an economically important pest in many agricultural crops worldwide. Its high reproductive potential and extremely short life cycle, combined with the frequent acaricide applications usually required to maintain the population below economic threshold, facilitates rapid resistance build-up. This has led to the development of resistance against almost all commercially used compounds. In this study we tried to unravel the mechanisms behind the resistance against a number of acaricides with different mode of action at the toxicological, biochemical, genetic and molecular level. In a first series of experiments, we investigated pyrethroid resistance in Greek T. urticae strains. Combined data from toxicological bioassays and biochemical and synergistic studies indicated that although enhanced P450 mono-oxygenase activities were associated with resistance, target site insensitivity proved to be the major resistance component. In order to get a better insight in the latter, a 3.3 kb cDNA fragment of the T. urticae para sodium channel gene encompassing segment 4 of domain II to segment 6 of domain IV was obtained by a degenerate PCR strategy. The T. urticae sequence showed highest identity (56%) to the scabies mite, Sarcoptes scabiei, and was phylogenetically classified within the divergent group of Arachnida. Comparison of resistant and susceptible strains identified the point mutation F1538I in segment 6 of domain III, which is known to confer strong resistance to pyrethroids, along with a second mutation (A1215D) in the intracellular linker connecting domains II and III, which still has an unknown role. Also, three alternative splicing variants were identified, of which two correspond to the mutually exclusive k/l exon pair already identified in insects. The mode of inheritance of resistance was confirmed to be almost completely recessive, which is consistent with studies on the target site mechanism for pyrethroids in other organisms. In T. urticae, organophosphate and carbamate resistance is often caused by acetylcholinesterase insensitivity. By combining toxicological, biochemical and molecular data from three reference laboratory and three organophosphate resistant (OP) strains, the AChE1 mutations associated with resistance in T. urticae were characterised. The resistance ratios of the OP strains varied from 9 to 43 for pirimiphos-methyl, from 78 to 586 for chlorpyrifos, from 8 to 333 for methomyl and from 137 to 4164 for dimethoate. Compared to the reference strains, the insecticide concentration needed to inhibit 50% of the AChE1 activity in the OP strains was at least 2.7, 55, 58 and 31 times higher for the OP pirimiphos-methyl, chlorpyrifos oxon, paraoxon and omethoate respectively, and 87 times higher for the carbamate carbaryl. By comparing the AChE1 sequence between laboratory and OP strains, four amino acid substitutions were detected: (1) F331W which was present in all three OP strains; (2) T280A found in the three OP strains but not in all clones; (3) G328A, found in two OP strains; (4) A201S found in only one OP strain. F331W, G328A and A201S are possibly involved in resistance to organophosphate and carbamate insecticides. These findings were also confirmed by means of a structural model. F331W is probably the most important and the most common in T. urticae. It can be easily detected by a diagnostic PCR-RFLP assay developed in this study. We also evaluated the possible antagonism of organophosphate and carbamate insecticides on the toxicity of bifenazate in T. urticae when applied in mixtures. Bifenazate, a new and frequently used carbazate, is a pro-acaricide which needs to be activated by carboxylesterases. Two OP strains were used and several organophosphate (chlorpyrifos, azinphos methyl and phosmet) and carbamate (carbaryl and methomyl) insecticides were evaluated. Mixing chlorpyrifos with bifenazate strongly decreased bifenazate toxicity in both tested strains. However, in the strain with a higher esterase activity, antagonism decreased after 2 days. Of all other tested chemicals, only methomyl displayed an antagonistic effect 1 day after treatment. These findings indicate that mixing organophosphate and carbamate insecticides with bifenazate may inhibit bifenazate efficacy under field conditions, especially when resistant strains are present. Spirodiclofen, a recently developed and commercialized acaricide, is a selective, non-systemic tetronic acid derivative. In order to develop strategies to minimise resistance in the field to spirodiclofen, we selected a T. urticae population for spirodiclofen resistance in the laboratory. This selection yielded a strain with a resistance ratio of 274, determined on the larval stage. This strain was used to determine genetic, toxicological, biochemical and cross-resistance data. The egg stage of the resistant strain remained far more susceptible than the mobile stages. No cross-resistance was found against other acaricides, except for spiromesifen, another tetronic acid derivative. Based on synergist experiments and enzyme assays, it appeared that especially P450 mono-oxygenases, but also esterases and glutathione-S-transferases, could be involved in the metabolic detoxification of spirodiclofen. Pre-treatment of the resistant females with the synergists PBO or DEF could increase the inhibitory effect of spirodiclofen on reproduction, again demonstrating the possible involvement of mono-oxygenases and esterases. Among the esterases of T. urticae, probably only a few are more expressed in spirodiclofen resistant strains, and these apparently prefer the purpose-designed substrate 1-naphthyl 2,2-dimethylbutyrate. This was also confirmed by separating esterase isozymes by native isoelectric focusing. Because spirodiclofen interferes with lipid biosynthesis, total lipid content was measured in female adults. No significant differences between treated and non-treated female adults were found, both in the susceptible and resistant strain. However, the total lipid content in the resistant females was significantly higher than in susceptible females. The genetic analysis using crossing experiments showed that the resistance is inherited as an intermediate trait under control of more than one gene. Resistance to spirodiclofen in the laboratory selected strain exceeded by far the recommended field rate, showing the capacity for quick resistance development in the field. A good acaricide resistance management programme is necessary to prevent fast resistance build-up in the field. However, without selection pressure, resistance tends to be unstable and can decrease in the presence of susceptible individuals owing to the intermediate, polygenic inheritance mode. No evidence was found to support the existence of other resistance mechanisms, like mutations in the target site (ACCase) or overproduction of the target enzyme. In order to facilitate designing new ACCase inhibitor insecticides and understanding mechanism of resistance to currently used ACCase inhibitors, we tried to clone and express T. urticae ACCase using different expression systems. The most promising results were achieved by a baculovirus expression system and also by heat-shocking Codon Rare E. coli cells with the pET-ACC vector. More efforts should be done to overcome several problems in expressing this enzyme e.g. by optimizing conditions to get higher levels of expression, protein solubility, activity and stability. We also performed a practical screening in Dutch cut rose glasshouses due the fact that growers complained on failing spider mite control. In order to check whether control failure was caused by resistance, the susceptibility of 15 strains, sampled from infested roses, was tested to 11 acaricides: tebufenpyrad, milbemectin, abamectin, cyflumetofen, bifenthrin, spiromesifen, hexythiazox, etoxazole, bifenazate, acequinocyl and chlorpyrifos. Three doses were used to discern between resistance and susceptibility: the recommended field dose (FD), FD/5 and FD x 5. Ten of these strains were screened for known resistance mechanism such as elevated detoxifying enzyme activities and previously reported resistance mutations using biochemical and molecular diagnostics. Potential cross-resistance between acaricides was estimated by correlation analysis. The strains showed different levels of resistance to tested acaricides. Twelve out of 15 strains showed resistance against hexythiazox, and 9 out of 15 against bifenthrin and tebufenpyrad. Two strains were found which showed high levels of resistance to most tested insecticides, even against one which has not been registered in Europe. A high correlation was found between some acaricides, e.g. between abamectin and milbemectin. Without selection pressure, resistance seemed to be unstable in most cases, after one year. All field strains had an increased metabolic activity, compared to the susceptible strains. A number of amino acid substitutions known to be involved in resistance to different groups were present in different strains. The best example was the presence of the F331W substitution in the acetylcholine esterase, which was present in all field strains tested. The results obtained in this research may all together lead to a better understanding of acaricide resistance in T. urticae, which, in turn, may lead to more efficient resistance management programs