A microtitre plate assay for characterizing insensitive acetylcholinesterase genotypes of insecticide-resistant insects

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Abstract
A rapid technique is described for characterizing and monitoring, in single insects, the insensitivity of acetylcholinesterase (AChE) to organophosphorus and carbamate insecticides. Ninety-six insects are homogenized simultaneously in a microtitre plate and portions (e.g. 0·05 for Musca domestica L.) assayed colorimetrically with acetylthiocholine in the presence and absence of diagnostic concentrations of insecticide. Reactions are monitored by a kinetic microplate reader linked to a microcomputer that determines mean AChE activities automatically by linear regressions of absorbance-time data. Mean inhibited activity is then expressed as a percentage of uninhibited activity. Several inhibitors can be tested against the same insect to yield an ‘insensitivity profile‘ of individuals and strains. In tests on M. domestica adults of known AChE genotype, the assay clearly distinguished not only between a sensitive and two slightly (3-15-fold) insensitive AChE variants but between all six genotypic combinations of these three alleles.

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