Resistance to Organophosphates and Biochemical Genotyping of Acetylcholinesterases in Tetranychus urticae (Acari: Tetranychidae)

Resistance to organophosphorus acaricides in the two-spotted spider mite Tetranychus urticae is largely due to the reduced sensitivity of the target enzyme acetylcholinesterase (AChE). The insensitivity of AChE to the organophosphates (OPs) demeton-S-methyl, ethyl paraoxon, and chlorpyrifos oxon and the carbamate carbofuran was identified in a German laboratory strain of T. urticae (WI) selected with oxydemeton-methyl and a field-collected strain from Florida, T. urticae (VB). Depending on the inhibitor used, AChE in the resistant strains was 34- to 380-fold less sensitive than the AChE of a German susceptible strain, T. urticae (GSS), as revealed by conventional microtiter plate assays. A modified, highly sensitive microfluorometric assay enabled us to measure the AChE activity in a quarter of a single mite. The assay is based on the reaction of thiocholine with a fluorogenic coumarin phenylmaleimide, yielding an intensely fluorescent product. This technique was used to genotype individual spider mites and to examine the frequency of sensitive and insensitive AChE alleles in several strains. The kinetic parameters (Km and Vmax) of AChE from susceptible (GSS) and resistant (Wl and VB) strains of T. urticae were compared for different substrates. In a foliar spray application bioassay, larvae of strains Wl and VB showed only moderate resistance levels in contrast to their high degree of AChE insensitivity relative to the GSS strain. While resistance ratios to ethyl parathion were comparable in strains Wl and VB, the difference in chlorpyrifos resistance was remarkable. The field-collected strain VB and the laboratory selected strain Wl exhibited 78- and 9.5-fold resistance against chlorpyrifos, respectively. As there is no obvious difference in AChE inhibition kinetics between the two strains, this result suggests an additional resistance mechanism in the VB strain against chlorpyrifos.