Submitted to: Insect Biochemistry and Molecular Biology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: April 20, 2000
Publication Date: N/A
Interpretive Summary: Horn flies are blood-feeding flies which parasitize cattle. The fly has begun to develop resistance to insecticides in many regions of the United States. As resistance to organophosphate (OP) insecticides is still uncommon in horn flies, this class of insecticides is becoming increasingly relied upon for control. Horn flies selected from field populations displaying OP resistance have been shown to overproduce esterases, metabolic proteins which can detoxify OPs. A gene from a fly which parasitizes sheep, Lucilia cuprina, encodes an esterase, LcaE7, which has been shown to confer OP resistance. The gene region encoding a specific horn fly esterase, designated HiaE7, has been cloned and sequenced and displays 85% similarity to LcaE7. By using diazinon applied topically to individual flies and selecting those individuals showing diazinon susceptibility and resistance, the HiaE7 gene has been shown to be more active in resistant horn flies.
Technical Abstract: RT-PCR was used to clone two esterase cDNAs from a diazinon resistant field population of horn flies which express qualitative and quantitative differences in esterases compared to a susceptible population. The open reading frame from one of the esterase cDNAs, HiaE7, exhibits substantial amino acid identity to an esterase associated with diazinon resistance in Lucilia cuprina. RNA Northern blots showed that HiaE7 mRNA was more abundant in the diazinon resistant population than the susceptible population. DNA copy number analysis did not reveal major differences in HiaE7 gene copy number between the two populations. The full-length cDNA to HiaE7 was cloned and sequenced and found to contain all the highly conserved sequence elements associated with carboxyl/cholinesterases. The HiaE7 homologues in diazinon resistant strains of L. cuprina and Musca domestica have been shown to possess an amino acid substitution conferring diazinon hydrolytic activity to the 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 susceptible by topical diazinon application bioassays and total RNA isolated and hybridized to HiaE7 probe in ribonuclease protection assays. HiaE7 transcript was expressed at a 5-fold higher level in resistant female individual flies than susceptible female individuals.