Mutation Report for: I214V/G488S_bacol-ACHE

In the present study, the frequencies of three organophosphate (OP) resistance-associated mutations in acetylcholinesterase gene of Bactrocera oleae (BoAce) populations collected from 8 different important olivegrowing areas in the west part of Turkey were determined. Populations were sampled from the areas that have been treated with only the pyrethroid alpha-cypermethrin; pyrethroids plus OPs; deltamethrin with pheromone eco-traps, and no insecticide treatment applied areas for many years. For Ile214Val and Gly488Ser point mutations PCR-RFLP and for Delta3Q deletion mutation PCR diagnostic tests were carried out. Seventy-two percent of the total individuals analyzed in the study were exhibited heterozygous genotype (RS) for both Ile214Val and Gly488Ser point and homozygous susceptible genotype (SS) for Delta3Q deletion mutations. This RS/RS/SS combination together with RS/RR/SS with the frequency of 13% were the most common two combinations observed in all of the populations under different insecticide regimes, even in the populations under no insecticide pressure for many years. Independent evaluation of the three mutations resulted in 0.450, 0.534 and 0.037 frequency values for the resistant alleles of 214Val, 488Ser and Delta3Q mutations, respectively. Among the studied populations, the frequencies of resistant alleles for the positions of 214 and 488 were not differed from each other. However, in 3 of the populations the frequency of the R allele of Delta3Q was zero and it changed between 0.025 and 0.100 in the remaining five populations. Results of this study contributed to the distribution pattern of the two point mutations in Europe and a pattern for Delta3Q mutation was determined for the first time in the field collected olive fly samples.

We have recently identified two resistance-associated point mutations of organophosphate (OP)-insensitive acetylcholinesterase in the olive fruit fly Bactrocera oleae, the most important olive orchard pest world-wide. We have developed simple PCR-restriction fragment length polymorphism assays for each mutation utilising an AccI restriction site created by Ile214Val, and a BssHII restriction site destroyed by a neutral change always accompanying the second mutation Gly488Ser. Samples from Greece homozygous for both mutations proved the most insensitive to dimethoate. The frequencies of these mutations in field-collected samples from several countries were investigated. Ninety-three percent of samples from Greece and Albania, where OPs have been extensively used in B. oleae control, were homozygous for both mutations. Resistance-associated alleles were detected at lower frequencies, but still with both mutations in conjunction in the majority of cases, in western Mediterranean countries with limited use of OPs. Samples from South Africa, however, did not have either of the resistance-associated mutations. The double mutation haplotype clearly confers a strong selective advantage in field populations of B. oleae exposed to OPs.

A 2.2-kb full length cDNA containing an ORF encoding a putative acetylcholinesterase (AChE) precursor of 673 amino acid residues was obtained by a combined degenerate PCR and RACE strategy from an organophosphate-susceptible Bactrocera oleae strain. A comparison of cDNA sequences of individual insects from susceptible and resistant strains, coupled with an enzyme inhibition assay with omethoate, indicated a novel glycine-serine substitution (G488S), at an amino acid residue which is highly conserved across species (G396 of Torpedocalifornica AChE), as a likely cause of AChE insensitivity. This mutation was also associated with a 35-40% reduction in AChE catalytic efficiency. The I199V substitution, which confers low levels of resistance in Drosophila, was also present in B. oleae (I214V) and in combination with G488S produced up to a 16-fold decrease in insecticide sensitivity. This is the first agricultural pest where resistance has been associated with an alteration in AChE, which arises from point mutations located within the active site gorge of the enzyme.