Peng Wei1,2, Peter Demaeght2, Kristof De Schutter2, Linda Grigoraki3,4, Vassiliki Labropoulou5, Maria Riga3, John Vontas3,4, Ralf Nauen6, Wannes Dermauw2, Thomas Van Leeuwen2. 1. Key Laboratory of Entomology and Pest Control Engineering, College of Plant Protection, Southwest University, Chongqing, China. 2. Laboratory of Agrozoology, Department of Plants and Crops, Faculty of Bioscience Engineering, Ghent University, Ghent, Belgium. 3. Molecular Entomology Lab, Institute of Molecular Biology and Biotechnology (IMBB), Foundation for Research and Technology (FORTH), Heraklion, Greece. 4. Pesticide Science Laboratory, Department of Crop Science, Agricultural University of Athens, Athens, Greece. 5. Insect Molecular Genetics and Biotechnology, Institute of Biosciences and Applications, National Centre for Scientific Research, Athens, Greece. 6. Bayer AG, CropScience Division, R&D, Pest Control, Monheim, Germany.
Abstract
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, CCE04SR-VP and CCE04London , with CCE04SR-VP 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 CCE04SR-VP overexpression was more likely due to selection for the CCE04SR-VP 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 (CCE04SR-VP ) in two genetically independent strains, which is highly overexpressed. Based on kinetic enzyme data, however, quantitative rather than qualitative differences between CCE04SR-VP and CCE04London 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 CCE04SR-VP being a likely hypothesis.
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, CCE04SR-VP and CCE04London , with CCE04SR-VP 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 CCE04SR-VP overexpression was more likely due to selection for the CCE04SR-VP 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 (CCE04SR-VP ) in two genetically independent strains, which is highly overexpressed. Based on kinetic enzyme data, however, quantitative rather than qualitative differences between CCE04SR-VP and CCE04London 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 CCE04SR-VP being a likely hypothesis.
Authors: Robert Greenhalgh; Wannes Dermauw; Joris J Glas; Stephane Rombauts; Nicky Wybouw; Jainy Thomas; Juan M Alba; Ellen J Pritham; Saioa Legarrea; René Feyereisen; Yves Van de Peer; Thomas Van Leeuwen; Richard M Clark; Merijn R Kant Journal: Elife Date: 2020-10-23 Impact factor: 8.140
Authors: Claudia Tschesche; Michaël Bekaert; Joseph L Humble; James E Bron; Armin Sturm Journal: Comp Biochem Physiol C Toxicol Pharmacol Date: 2021-06-10 Impact factor: 3.228