Cloning of a horn fly cDNA, Hi alpha E7, encoding an esterase whose transcript concentration is elevated in diazinon-resistant flies

Reverse transcriptase-polymerase chain reaction (PCR) was used to clone two esterase cDNAs from a diazinon-resistant field population of horn flies th at expresses qualitative and quantitative differences in esterases compared with a susceptible population. The open reading frame from one of the este rase cDNAs, Hi alpha E7, exhibits substantial amino-acid identity to an est erase associated with diazinon resistance in Lucilia cuprina. RNA Northern blots showed that Hi alpha E7 mRNA was more abundant in the diazinon-resist ant population than the susceptible population. DNA copy number analysis di d not reveal major differences in Hi alpha E7 gene copy number between the two populations. The full-length cDNA to Hi alpha E7 was cloned and sequenc ed, and found to contain all of the highly conserved sequence elements asso ciated with carboxyl/cholinesterases. The Hi alpha E7 homologs in diazinon- resistant strains of L. cuprina and Musca domestica have been shown to poss ess an amino-acid substitution conferring diazinon hydrolytic activity to t he esterase enzyme. This amino-acid substitution was not found in diazinon- resistant horn flies examined by allele-specific PCR. Individual flies from the resistant field population were phenotyped as diazinon-resistant or di azinon-susceptible by topical diazinon application bioassays and total RNA isolated and hybridized to Hi alpha E7 probe in ribonuclease protection ass ays. Hi alpha E7 transcript was expressed at a five-fold higher level in re sistant female individual flies than in susceptible female individuals. Pub lished by Elsevier Science Ltd.