BACKGROUND: Field resistance to malathion was reported for Mediterranean fruit fly (medfly), Ceratitis capitata, populations collected in Spain in 2004 and 2005, when medfly control mainly relied on malathion bait sprays. The mutation G328A in the acetylcholinesterase (AChE) gene (Ccace2) was then identified as the main resistance mechanism in a field-derived resistant strain. However, outdoor plant protection products containing malathion were withdrawn from the European Union in 2009 and other insecticides gained importance, such as spinosad and pyrethroids, though other organophosphates were occasionally used for medfly control for a few years. RESULTS: We have: (i) provided evidences of a novel malathion resistant mechanism in Ceratitis capitata, mediated by a heterogeneous duplication of the Ccace2 gene (RS haplotype, one of the copies bearing the mutation G328A and the other copy non-mutated)
(ii) found that individuals bearing the G328A mutation (R allele) and/or the RS haplotype were widely distributed in Spanish medfly populations during the years that malathion was used
and (iii) showed that malathion resistance reverted in field populations when analysed 8-13 years after malathion was withdrawn, but the frequencies of the genotypes containing the RS haplotype remained stable (RS/RS) or declined less (S/RS) than those containing the R allele (R/R, R/S, R/RS). CONCLUSION: This represents a scenario where the R allele and the RS haplotype are present in the field at low frequencies, but resistance may rapidly evolve if malathion or other organophosphates were used in the absence of appropriate management resistance strategies. © 2025 Society of Chemical Industry.