title: Distinct contributions of A314S and novel R667Q substitutions of acetylcholinesterase 1 to carbofuran resistance of Chilo suppressalis Walker.
abstract: BACKGROUND
  In the striped stem borer, Chilo suppressalis, A314S, R667Q and H669P substitutions in acetylcholinesterase 1 (CsAChE1) have been associated with >1000-fold resistance against carbofuran. In this study, eight variants of CsAChE1 carrying different combinations of these substitutions were cloned and expressed using the Bac-to-Bac expression system to verify their contributions.
  
  
  RESULTS
  The expressed AChE1s had molecular weights of ca 160 kDa per dimer and 80 kDa per monomer. AChE kinetics and inhibition analysis showed that the A314S mutation was the key substitution responsible for a 15.1-fold decrease in hydrolytic activity to acetylthiocholine iodide and a 10.6-fold increase in carbofuran insensitivity of CsAChE. Compared with wild-type CsAChE1, this substituted CsAChE1 also showed 23.0-, 3.3- and 2.6-fold insensitivity to methomyl, triazophos and chlorpyrifos-oxon respectively. It should be noted that the R667Q substitution conferred a capability to increase the activity of wild-type and A314S-substituted CsAChE, while the A314S substitution reduced Km and compensated for overall catalytic efficiency.
  
  
  CONCLUSIONS
  With the enhancing activity of the R667Q substitution, A314S is the major CsAChE1 substitution responsible for fitness-cost compensation and increased insensitivity to AChE inhibitors. The lower insensitivity of A314S-substituted CsAChE1 to chlorpyrifos-oxon suggests that chlorpyrifos could be an alternative insecticide for managing carbofuran-resistant field C. suppressalis in Taiwan. © 2015 Society of Chemical Industry.
authors.affiliation: Department of Entomology, National Chung-Hsing University, Taichung, Taiwan, R.O.C.
authors.forename: Shu-Mei
authors.initials: SM
authors.lastname: Dai
authors.affiliation: Biotechnology Centre, National Chung-Hsing University, Taichung, Taiwan, R.O.C.
authors.forename: Cheng
authors.initials: C
authors.lastname: Chang
authors.affiliation: Department of Entomology, National Chung-Hsing University, Taichung, Taiwan, R.O.C.
authors.forename: Xin-Yi
authors.initials: XY
authors.lastname: Huang
mesh_terms: D000110:Acetylcholinesterase / Q000235:genetics*; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D002235:Carbofuran; D007305:Insecticide Resistance / Q000235:genetics; D007306:Insecticides; D009036:Moths / Q000187:drug effects* / Q000201:enzymology / Q000235:genetics
chemical_list: D007306:Insecticides; D000110:Acetylcholinesterase; D002235:Carbofuran
keywords: Baculovirus expression system; Chilo suppressalis; acetylcholinesterase 1; carbofuran resistance