ABSTRACT
The primary structure of the second transmembrane (M2) segment of resistant to dieldrin (RDL), an ionotropic γ-aminobutyric acid receptor (GABAR) subunit, and the structure-function relationships in RDL are well conserved among insect species. An amino acid substitution at the 2' position in the M2 segment (Ala to Ser or Gly) confers resistance to non-competitive antagonists (NCAs) of GABARs. Here, a cDNA encoding RDL was cloned from the two-spotted spider mite Tetranychus urticae Koch. Unlike insect homologs, native TuRDL has His at the 2' position (H305) and Ile at 6' (I309) in the M2 segment and is insensitive to NCAs. Single and multiple mutations were introduced in the M2 segment of TuRDL, and the mutant proteins were expressed in Xenopus oocytes and examined for the restoration of sensitivity to NCAs. The sensitivity of a double mutant (H305A and I309T in the M2 segment) was greatly increased but was still considerably lower than that of insect RDLs. We therefore constructed chimeric RDLs consisting of TuRDL and Drosophila melanogaster RDL and examined their sensitivities to NCAs. The results show that the N-terminal region containing the Cys-loop as well as the M2 segment confers functional specificity; thus, our current understanding of the mechanism underlying NCA binding to GABARs requires reappraisal.
Subject(s)
Chloride Channels/genetics , Drosophila Proteins/chemistry , Receptors, GABA-A/chemistry , Tetranychidae/genetics , gamma-Aminobutyric Acid/pharmacology , Amino Acid Sequence , Animals , Aphids , Brassica , Chloride Channels/metabolism , Dose-Response Relationship, Drug , Drosophila Proteins/genetics , Drosophila melanogaster , Drug Resistance/drug effects , Drug Resistance/genetics , Female , Male , Phaseolus , Protein Structure, Secondary , Receptors, GABA-A/genetics , Tetranychidae/drug effects , Xenopus laevis , gamma-Aminobutyric Acid/metabolismABSTRACT
BACKGROUND: Cyflumetofen is a novel acaricide developed by Otsuka AgriTechno Co., Ltd. It affects only spider mites and has no effect on insects, crustaceans or vertebrates under conditions of practical use. The mode of action of cyflumetofen, its selectivity for mites and its safety for insects and vertebrates were investigated. RESULTS: The research showed that cyflumetofen inhibited mitochondria complex II in mites. In addition, the de-esterified form (AB-1) of cyflumetofen inhibited mitochondria complex II at extremely low concentrations. AB-1 was also detected as the main metabolite in mites. CONCLUSION: The mode of action of cyflumetofen is to inhibit mitochondria complex II by affecting its action site after being metabolised to AB-1. However, inhibition by cyflumetofen and AB-1 in other organisms was very weak. Selectivity for other organisms has contributed to differences in action site activities.
Subject(s)
Acaricides/toxicity , Propionates/toxicity , Tetranychidae/drug effects , Animals , Arthropod Proteins/genetics , Arthropod Proteins/metabolism , Insecta/drug effects , Species Specificity , Tetranychidae/genetics , Tetranychidae/metabolismABSTRACT
Octopamine receptors are attractive insecticide targets. To screen compounds acting at octopamine receptors simply and rapidly, we constructed a chemiluminescent reporter gene assay system that detects secreted placental alkaline phosphatase transcriptionally regulated by the cAMP response element for a silkworm octopamine receptor. This system proved useful in high-throughput screening to develop octopamine receptor-specific insecticides.
