Overexpression of an alternative allele of carboxyl/choline esterase 4 (CCE04) of Tetranychus urticae is associated with high levels of resistance to the keto-enol acaricide spirodiclofen.

Background: Spirodiclofen is an acaricide that targets lipid biosynthesis by inhibiting acetyl-coenzyme A carboxylase. Spirodiclofen resistance in spider mites has been previously documented and was associated with overexpression of CYP392E10, a cytochrome P450 mono-oxygenase that metabolizes spirodiclofen. However, additional mechanisms have been suggested in several studies and a carboxyl/choline esterase gene, CCE04, was shown to be overexpressed in two genetically different strains, SR-VP and SR-TK, both exhibiting high spirodiclofen resistance levels.

Results: We identified two different CCE04 alleles in both resistant strains, CCE04 and CCE04 , with CCE04 being highly overexpressed. Isoelectric focusing analysis confirmed the overexpression of a single esterase isozyme, while copy number and random fragment length polymorphism analysis revealed that CCE04 overexpression was more likely due to selection for the CCE04 allele rather than gene amplification. Both CCE04 alleles were functionally expressed using the Pichia expression system. Functional enzyme assays revealed only limited kinetic differences between CCE04 isoforms for model substrates. In addition, inhibition/competition experiments with spirodiclofen suggested a similar interaction with both enzymes, whereas its active metabolite, spirodiclofen enol, did not inhibit enzyme activity.

Conclusion: Our study suggests that selection with spirodiclofen results in enrichment of a specific allele of CCE04 (CCE04 ) in two genetically independent strains, which is highly overexpressed. Based on kinetic enzyme data, however, quantitative rather than qualitative differences between CCE04 and CCE04 seem more likely to be involved in resistance. Our findings are discussed in the light of a possible spirodiclofen resistance mechanism, with sequestration of spirodiclofen by CCE04 being a likely hypothesis. © 2019 Society of Chemical Industry.