Insect Biochemistry and Molecular Biology 30 2000 1107–1115 www.elsevier.comlocateibmb
Cloning of a horn fly cDNA, HiaE7, encoding an esterase whose transcript concentration is elevated in diazinon-resistant flies
Felix D. Guerrero
USDA–ARS Knipling–Bushland US Livestock Insects Research Lab, 2700 Fredericksburg Road, Kerrville, TX 78028, USA Received 17 November 1999; received in revised form 18 April 2000; accepted 19 April 2000
Abstract
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, 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
of the highly conserved sequence elements associated with carboxylcholinesterases. The HiaE7 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 HiaE7 probe in ribonuclease protection assays. HiaE7 transcript was expressed at a five-fold higher level in resistant female individual flies than in susceptible female individuals.
Published by Elsevier Science Ltd.
Keywords: Diazinon resistance; Ribonuclease protection assays; Sequence; Esterases
1. Introduction
The horn fly, Haematobia irritans L. Diptera: Muscidae, is a hematophageous parasite of cattle. There
is a long history of reliance upon the use of insecticides for control of this pest and, as a consequence, there have
been outbreaks of resistance in various fly populations Sparks et al., 1985. Resistance to organophosphates
OPs had been reported as early as 1963 Burns and Wilson, 1963. Byford et al. 1999 found that horn flies
developed moderate levels of resistance to diazinon in both laboratory and field experiments employing con-
tinuous insecticide pressure. Carboxylesterases are a major mechanism of OP resistance in mosquitoes, gener-
ally causing resistance by sequestration of the OP before it can reach the target site within the insect Hemingway
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and Karunaratne, 1998. Likewise, the OP resistance of other insects exhibiting a metabolic resistance mech-
anism to OPs has been shown to be due to the overpro- duction of esterase as in the saw-toothed grain beetle
Conyers et al., 1998 and the German cockroach Scharf et al., 1997, among others. However, in Lucilia cuprina,
a specific mutation in an esterase gene has been found to confer OP resistance Newcomb et al., 1997a. The
mutated gene, LcaE7, contains a Gly
137
→Asp substi- tution within the active site of the esterase conferring
OP hydrolase activity. In fact, a second mutated LcaE7 allele has been identified in malathion-resistant popu-
lations of L. cuprina. Resistant individuals from these populations possess a Trp
251
→Leu substitution which confers malathion carboxylesterase activity leading to
malathion resistance Campbell et al., 1998. Recently, an OP-resistant strain of Musca domestica was found to
possess the Gly
137
→Asp amino-acid substitution in the M. domestica ortholog to LcaE7 Claudianos et al.,
1999.
1108 F.D. Guerrero Insect Biochemistry and Molecular Biology 30 2000 1107–1115
Several horn fly populations were recently discovered in Texas that were resistant to the OP diazinon. Bio-
chemical investigations implicated the overexpression of esterases as a mechanism of resistance Guerrero et al.,
1999. Using topical applications of diazinon to pheno- type individual flies as resistant or susceptible, native
polyacrylamide gel electrophoresis PAGE analysis found that resistant flies were qualitatively and quantitat-
ively different from susceptible flies in their esterase activity gel profiles. Some resistant flies had a higher
amount of total esterase activity while others appeared to have a total esterase activity similar to the levels
found in susceptible flies but possessed unique gel bands of esterase activity. Since OPs are becoming increasingly
relied upon for horn fly control, it seemed prudent to study specific mechanisms of OP resistance in order to
develop strategies to maximize the effectiveness of OPs and the period of time before resistance becomes wide-
spread. This report describes the cloning and sequencing of two horn fly esterase cDNAs, designated HiaE7 and
HiaE8. HiaE7 encodes the horn fly homolog to the L. cuprina OP-resistance-conferring esterase, LcaE7. The
expression of HiaE7 and HiaE8 mRNA in individual OP-resistant and OP-susceptible horn flies was assayed
to determine if either of the esterase mRNAs was differ- entially expressed in association with OP resistance.
2. Materials and methods
