Cloning of a horn fly cDNA, HialphaE7, 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 that expresses qualitative and quantitative differences in esterases compared with a susceptible population. The open reading frame from one of the esterase cDNAs, HialphaE7, exhibits substantial amino-acid identity to an esterase associated with diazinon resistance in Lucilia cuprina. RNA Northern blots showed that HialphaE7 mRNA was more abundant in the diazinon-resistant population than the susceptible population. DNA copy number analysis did not reveal major differences in HialphaE7 gene copy number between the two populations. The full-length cDNA to HialphaE7 was cloned and sequenced, and found to contain all of the highly conserved sequence elements associated with carboxyl/cholinesterases. The HialphaE7 homologs 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 diazinon-susceptible by topical diazinon application bioassays and total RNA isolated and hybridized to HialphaE7 probe in ribonuclease protection assays. HialphaE7 transcript was expressed at a five-fold higher level in resistant female individual flies than in susceptible female individuals.
Guerrero
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USDA-ARS Knipling-Bushland US Livestock Insects Research Lab, 2700 Fredericksburg Road, 78028, Kerrville, TX, USA. felixg@ktc.com
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001483:Base Sequence; D001681:Biological Assay; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics*; D004950:Esterases / Q000032:analysis / Q000235:genetics* / Q000378:metabolism; D005260:Female; D007305:Insecticide Resistance / Q000235:genetics; D007306:Insecticides / Q000494:pharmacology*; D008969:Molecular Sequence Data; D009117:Muscidae / Q000201:enzymology / Q000235:genetics*; D009946:Organothiophosphorus Compounds; D016133:Polymerase Chain Reaction; D014158:Transcription, Genetic
D018076:DNA, Complementary; D007306:Insecticides; D009946:Organothiophosphorus Compounds; D004950:Esterases