Amidase, a novel detoxifying enzyme, is involved in cyflumetofen resistance in Tetranychus cinnabarinus (Boisduval)

Amidase is an important hydrolytic enzyme in detoxification metabolism. Amidase hydrolyzes a wide variety of nonpeptide carbon-nitrogen bonds by attacking a cyano group or carbonyl carbon. However, little is known about the relationship between amidase and insecticides. In this study, the amidase activity was significantly higher in cyflumetofen-resistant strain (CyR) than in the susceptible strain (SS) of Tetranychus cinnabarinus, and diethyl-phosphoramidate (an amidase inhibitor) significantly decreased cyflumetofen resistance in T. cinnabarinus. More importantly, an amidase gene, TcAmidase01, was identified in T. cinnabarinus, and the TcAmidase01 overexpression was detected in both two cyflumetofen-resistant strains (CyR and YN-CyR), indicating that it is involved in cyflumetofen resistance in mites. A phylogenetic analysis showed that TcAmidase01 was clustered with deaminated glutathione amidases, which possess hydrolytic activity. The recombinant TcAmidase01 protein showed amidase activity toward succinamate, and the activity could be inhibited by cyflumetofen. High-performance liquid chromatography-mass spectrometry (HPLC-MS) analysis provided evidence that recombinant TcAmidase01 could decompose cyflumetofen by hydrolysis, and the potential metabolites (2-(4-(tert-butyl) phenyl)-2-cyanoacetate and 2-(trifluoromethyl) benzoic acid) were identified. These results show that TcAmidase01 contribute to cyflumetofen-resistance in T. cinnabarinus by hydrolyzing cyflumetofen, and this is the first study to suggest that amidase has a role in insecticides resistance in arthropods.