Substrate Report for: Cyflumetofen

BACKGROUND: Increased expression or point mutations of carboxyl/cholinesterases (CCEs) have been involved in many cases of insecticide and acaricide resistance. However, it has been only rarely documented that downregulation of CCE genes is associated with resistance, although many insecticides and acaricides need hydrolytic activation in vivo. Previously, expression analysis of a laboratory-selected cyflumetofen-resistant strain of Tetranychus cinnabarinus indicated that resistance was associated with increased expression of a CCE gene of TcCCE04, but also the downregulation of two CCE genes, TcCCE12 and TcCCE23. RESULTS: Synergism experiments revealed the importance of ester hydrolysis in cyflumetofen toxicity, because treatment with S,S,S-tributylphosphorotrithioate (DEF) caused strong inhibition of cyflumetofen hydrolysis, in both the susceptible and resistant strains. Moreover, silencing expression of TcCCE12 and TcCCE23 via RNAi further decreased the susceptibility of mites to cyflumetofen significantly, suggesting that downregulated CCE genes could be involved in cyflumetofen resistance. In addition, it was shown that recombinant TcCCE12 protein could hydrolyze cyflumetofen effectively. CONCLUSION: Decreased esterase activity via downregulation of specific CCE genes most likely contributes to cyflumetofen resistance by decreased activation of cyflumetofen to its active metabolite. Mixtures of cyflumetofen and esterase-inhibition acaricides (e.g. organophosphates or carbamates) should be avoided in field applications.

The carmine spider mite, Tetranychus cinnabarinus is an important pest of crops and vegetables worldwide, and it has the ability to develop resistance against acaricides rapidly. Our previous study identified an esterase gene (designated TCE2) over-expressed in resistant mites. To investigate this gene's function in resistance, the expression levels of TCE2 in susceptible, abamectin-, fenpropathrin-, and cyflumetofen-resistant strains were knocked down (65.02%, 63.14%, 57.82%, and 63.99%, respectively) via RNA interference. The bioassay data showed that the resistant levels to three acaricides were significantly decreased after the down-regulation of TCE2, indicating a correlation between the expression of TCE2 and the acaricide-resistance in T. cinnabarinus. TCE2 gene was then re-engineered for heterologous expression in Escherichia coli. The recombinant TCE2 exhibited alpha-naphthyl acetate activity (483.3 +/- 71.8 nmol/mg pro. min(-1)), and the activity of this enzyme could be inhibited by abamectin, fenpropathrin, and cyflumetofen, respectively. HPLC and GC results showed that 10 mug of the recombinant TCE2 could effectively decompose 21.23% fenpropathrin and 49.70% cyflumetofen within 2 hours. This is the first report of a successful heterologous expression of an esterase gene from mites. This study provides direct evidence that TCE2 is a functional gene involved in acaricide resistance in T. cinnabarinus.

The carmine spider mite, Tetranychus cinnabarinus is an important crop and vegetable plants pest mite. As a novel acaricide, cyflumetofen is effective against Tetranychus and Panonychus mites, but its risk and biochemical mechanism of resistance in mites is not clear. In this study, the resistance against cyflumetofen was selected and its biochemical mechanisms were studied in T. cinnabarinus. After selection the susceptibility and resistance against cyflumetofen in T. cinnabarinus, the final resistance ratio reached 21.33 at LC50 (CyR-43/CyS). All the collected field populations showed low resistance against cyflumetofen, although it had never been used in China. The activity of detoxifying enzymes CarE, MFO and GSTs were significantly increased in the final selected resistance strain (CyR-43), especially that for GSTs increased more than 7-folds after selection. The resistance against cyflumetofen developed slowly when selected from the susceptible strain in laboratory, but the resistant genes already existed in field populations, and the GSTs was the most important detoxifying enzyme conferring resistance against cyflumetofen in T. cinnabarinus. These results would provide the valuable information for designing appropriate strategies for the practical application of cyflumetofen in the field and delaying resistance development.

BACKGROUND: Increased expression or point mutations of carboxyl/cholinesterases (CCEs) have been involved in many cases of insecticide and acaricide resistance. However, it has been only rarely documented that downregulation of CCE genes is associated with resistance, although many insecticides and acaricides need hydrolytic activation in vivo. Previously, expression analysis of a laboratory-selected cyflumetofen-resistant strain of Tetranychus cinnabarinus indicated that resistance was associated with increased expression of a CCE gene of TcCCE04, but also the downregulation of two CCE genes, TcCCE12 and TcCCE23. RESULTS: Synergism experiments revealed the importance of ester hydrolysis in cyflumetofen toxicity, because treatment with S,S,S-tributylphosphorotrithioate (DEF) caused strong inhibition of cyflumetofen hydrolysis, in both the susceptible and resistant strains. Moreover, silencing expression of TcCCE12 and TcCCE23 via RNAi further decreased the susceptibility of mites to cyflumetofen significantly, suggesting that downregulated CCE genes could be involved in cyflumetofen resistance. In addition, it was shown that recombinant TcCCE12 protein could hydrolyze cyflumetofen effectively. CONCLUSION: Decreased esterase activity via downregulation of specific CCE genes most likely contributes to cyflumetofen resistance by decreased activation of cyflumetofen to its active metabolite. Mixtures of cyflumetofen and esterase-inhibition acaricides (e.g. organophosphates or carbamates) should be avoided in field applications.

The carmine spider mite, Tetranychus cinnabarinus is an important pest of crops and vegetables worldwide, and it has the ability to develop resistance against acaricides rapidly. Our previous study identified an esterase gene (designated TCE2) over-expressed in resistant mites. To investigate this gene's function in resistance, the expression levels of TCE2 in susceptible, abamectin-, fenpropathrin-, and cyflumetofen-resistant strains were knocked down (65.02%, 63.14%, 57.82%, and 63.99%, respectively) via RNA interference. The bioassay data showed that the resistant levels to three acaricides were significantly decreased after the down-regulation of TCE2, indicating a correlation between the expression of TCE2 and the acaricide-resistance in T. cinnabarinus. TCE2 gene was then re-engineered for heterologous expression in Escherichia coli. The recombinant TCE2 exhibited alpha-naphthyl acetate activity (483.3 +/- 71.8 nmol/mg pro. min(-1)), and the activity of this enzyme could be inhibited by abamectin, fenpropathrin, and cyflumetofen, respectively. HPLC and GC results showed that 10 mug of the recombinant TCE2 could effectively decompose 21.23% fenpropathrin and 49.70% cyflumetofen within 2 hours. This is the first report of a successful heterologous expression of an esterase gene from mites. This study provides direct evidence that TCE2 is a functional gene involved in acaricide resistance in T. cinnabarinus.

Carboxylesterases (CarEs) play important roles in metabolism and detoxification of dietary and environmental xenobiotics in insects and mites. On the basis of the Tetranychuscinnabarinus transcriptome dataset, 23 CarE genes (6 genes are full sequence and 17 genes are partial sequence) were identified. Synergist bioassay showed that CarEs were involved in acaricide detoxification and resistance in fenpropathrin- (FeR) and cyflumetofen-resistant (CyR) strains. In order to further reveal the relationship between CarE gene's expression and acaricide-resistance in T. cinnabarinus, we profiled their expression in susceptible (SS) and resistant strains (FeR, and CyR). There were 8 and 4 over-expressed carboxylesterase genes in FeR and CyR, respectively, from which the over-expressions were detected at mRNA level, but not DNA level. Pesticide induction experiment elucidated that 4 of 8 and 2 of 4 up-regulated genes were inducible with significance in FeR and CyR strains, respectively, but they could not be induced in SS strain, which indicated that these genes became more enhanced and effective to withstand the pesticides' stress in resistant T. cinnabarinus. Most expression-changed and all inducible genes possess the Abhydrolase_3 motif, which is a catalytic domain for hydrolyzing. As a whole, these findings in current study provide clues for further elucidating the function and regulation mechanism of these carboxylesterase genes in T. cinnabarinus' resistance formation.

The carmine spider mite, Tetranychus cinnabarinus is an important crop and vegetable plants pest mite. As a novel acaricide, cyflumetofen is effective against Tetranychus and Panonychus mites, but its risk and biochemical mechanism of resistance in mites is not clear. In this study, the resistance against cyflumetofen was selected and its biochemical mechanisms were studied in T. cinnabarinus. After selection the susceptibility and resistance against cyflumetofen in T. cinnabarinus, the final resistance ratio reached 21.33 at LC50 (CyR-43/CyS). All the collected field populations showed low resistance against cyflumetofen, although it had never been used in China. The activity of detoxifying enzymes CarE, MFO and GSTs were significantly increased in the final selected resistance strain (CyR-43), especially that for GSTs increased more than 7-folds after selection. The resistance against cyflumetofen developed slowly when selected from the susceptible strain in laboratory, but the resistant genes already existed in field populations, and the GSTs was the most important detoxifying enzyme conferring resistance against cyflumetofen in T. cinnabarinus. These results would provide the valuable information for designing appropriate strategies for the practical application of cyflumetofen in the field and delaying resistance development.