Development of molecular diagnostics of the two point mutations in acetylcholinesterase gene associated with insecticide resistance in the cotton aphid, Aphis gossypii Glover, (Homoptera: Aphididae) and a survey of genotypic frequency in field populations

In a previous study, we found two amino acid substitutions, Ser431 Phe and Ala302Ser, in the Drosophila Ace paralogous acetyleholinesterase (AP-AChE) genes from pirimicarb-resistant Aphis gossypii. Ser43 I Phe is responsible for the development of pirimicarb resistance and Ala302Ser may play a role in insensitivity to organophosphorus insecticides. In this study, we developed the two types of molecular diagnostics for detecting these mutations. A polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP)-based method was developed to detect the Ser431Phe and Ala302Ser mutations. A PCR amplification of specific alleles (PASA) method, designated to detect Ser43 I Phe mutations allowed the three genotypes to be discriminated more rapidly and simply than by PCR-RFLP. Genotyping of samples collected from Japanese pear and Satsuma mandarin orchards in Japan was carried out using PCR-RFLP. We revealed that the genetic composition of the A. gossypii orchard population changes with time without exposure to insecticide and that molecular diagnostics are useful for monitoring the development of insecticide resistance. Although both mutations were frequently detected in field populations, pirimicarb-resistant aphids did not seem to always constitute a large proportion in field populations in Japan. We found several individuals which had Ser302 without Phe431. This finding denies the concept that this genotype is inviable from the viewpoint of enzymatic kinetics.