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
2.1. Horn fly rearing and pesticide resistance assays OP-susceptible adult horn flies were obtained from a
laboratory colony maintained since 1961 at the USDA– ARS Laboratory in Kerrville, TX. Flies were reared as
described by Schmidt et al. 1976 except that the cit- rated 15 mM bovine blood used to feed adults was
treated with 250,000 units of oral-grade mycostatin and 0.5 g kanamycin sulfate per liter and the larval rearing
medium consists of two parts by weight bovine feces, one part water and one part peanut hull pellets. A field
population of diazinon-resistant horn flies at Camp Cooley in Robertson County, TX was sampled and
returned live to the laboratory, where a portion were used for topical diazinon application experiments and the
remainder frozen and stored at
280 °
C. In performing the topically applied diazinon experiments, 0.5
µ l of
diazinon dissolved in acetone was applied to the thorax of individual horn flies by using a hand microapplicator
Burkard, Rickmansworth, UK. Fifty individual adult horn flies of mixed ages were treated with 50 or 5 ng of
diazinon in acetone. These two diazinon concentrations had previously been found to be suitable for selection
of diazinon-resistant and diazinon-susceptible individ- uals from this population Guerrero et al., 1999. One
set of flies was treated with acetone alone as a negative control. As mortality occurred, time of death and sex
were recorded and the fly was frozen for subsequent analyses.
2.2. RNA preparations Total and polyA
+
RNA were purified with the RNA- gents Total RNA Isolation System and the PolyATtract
System Promega, Madison, WI. RNA concentrations were determined by absorbance spectrophotometry and
RNA analyzed by formaldehyde gel electrophoresis and Northern blotting as described in Ausubel et al. 1998.
2.3. Reverse transcriptase-PCR RT-PCR For RT-PCR, reverse transcription of polyA
+
RNA from both the laboratory OP-susceptible and the Camp
Cooley OP-resistant strains was performed on 1 µ
g of heat-denatured polyA
+
RNA at 37 °
C for 1 h in a reac- tion including 50 mM Tris–HCl pH
= 8.3, 75 mM KCl,
10 mM dithiothreitol, 3 mM MgCl
2
, 0.2 mM of each dNTP, 2
µ M of oligo dT
18
and 200 units of M-MLV reverse transcriptase Life Technologies, Gaithersburg,
MD. The RNA template was then hydrolyzed by the addition of 1.5 volumes of a 0.7 M NaOH40 mM ethy-
lenediamine-N,N,N 9,N9-tetraacetic acid EDTA sol-
ution, incubated for 10 min at 65 °
C, the solution extracted with a 25:24:1 mixture of phenol:chloro-
form:isoamyl alcohol and the cDNA recovered by etha- nol precipitation in the presence of Pellet Paint Co-Pre-
cipitant Novagen, Madison, WI. PCR was performed using a 50
µ l reaction containing 5 of the reverse tran-
scription reaction products, 1 nmol of either degenerate primer FG-144 or FG-145, 50 pmol of primer FG-146
Table 1, 10 mM Tris–HCl pH =
8.3, 50 mM KCl, 0.2 mM each dNTP, 2 mM MgCl
2
and 0.5 µ
l of a 1:1 vv mix of AmpliTaq DNA Polymerase PE Applied
Biosystems, Foster City, CA and TaqStart Antibody Clontech, Palo Alto, CA. The amplification program
Table 1 PCR primer sequences
Primer 5
9→39 DNA sequence FG-144
GGNATDCCNTAYGCNCARCCNCC FG-145
GGNGARYTNMGNTTYAARG FG-146
TGRTCYTTNARNCCNGCRTTNCC FG-158
AGATTTAAGGCTCCACAAC FG-203
GCTTCGCCACAAATGAAAC FG-206
GCTTCGCCACAAATGAAAA FG-207
GCTTCGCCACAAATGAAAG FG-208
GCTTCGCCACAAATGAAAT FG-204
CTTCGCCACAAATGAAACC FG-209
CTTCGCCACAAATGAAACG FG-210
CTTCGCCACAAATGAAACA FG-211
CTTCGCCACAAATGAAACT
1109 F.D. Guerrero Insect Biochemistry and Molecular Biology 30 2000 1107–1115
was as described in Hecker and Roux 1996 and used 96
° C for 3 min followed by 30 cycles, each consisting
of denaturation at 94 °
C for 1 min, annealing at 65 °
C for 2 min with a temperature ramp of
20.5 °
Ccycle and extension at 72
° C for 3 min, followed by 10 cycles of
denaturation at 94 °
C for 1 min, annealing at 50 °
C for 2 min and extension at 72
° C for 3 min, and a final exten-
sion at 72 °
C for 7 min. 2.4. Allele-specific PCR ASPCR
To prepare genomic DNA, individual frozen adult flies were crushed in 1.5 ml microcentrifuge tubes using
a disposable pellet pestle PGC Scientifics, Gaithersburg, MD. Next, 25
µ l of buffer [500 mM KCl, 100 mM
Tris–HCl pH =
8.3] was added and the tube contents incubated for 2 min in a boiling water bath. Following
a 5 min centrifugation at 15,000g, an aliquot of the supernatant was diluted 1:10 in water. The ASPCR assay
thermocycling parameters consisted of an initial denatur- ation at 96
° C for 2 min followed by 35 cycles, each con-
sisting of denaturation at 94 °
C for 1 min, annealing at 66
° C for 1 min and extension at 72
° C for 1 min, and a
final extension at 72 °
C for 7 min. Reactions contained 1
µ l of the diluted fly DNA, 10 mM Tris–HCl pH
= 8.3,
50 mM KCl, 0.05 mM each dNTP, 2 mM MgCl
2
, and 0.1
µ l of a 1:1 vv mix of AmpliTaq DNA Polymerase
and TaqStart Antibody, and 20 pmol of both primer FG- 158 and one of the allele-specific primers, FG-203, FG-
204, FG-206, FG-207, FG-208, FG-209, FG-210 or FG- 211 Table 1. Amplification products were detected by
agarose gel electrophoresis Ausubel et al., 1998.
2.5. Cloning and sequencing For subcloning and sequencing experiments, DNA
products from the RT-PCR experiments were purified using agarose gel electrophoresis, extracted with the
Qiaex Gel Extraction Kit Qiagen, Inc., Chatsworth, CA and subcloned into pT7Blue-2 using the Perfectly Blunt
Cloning Kit Novagen, Inc., Madison, WI. DNA was prepared from selected clones using the QIAprep spin
miniprep kit and inserts purified by digestion with restriction enzyme, agarose gel electrophoresis and gel
extraction. Probes were labeled with
32
P-dATP New England Nuclear, Boston, MA using the Strip-EZ DNA
Labeling Kit Ambion Inc., Austin, TX. Autoradio- grams were analyzed by densitometric scanning and
quantified by
Collage Image
Analysis Software
Fotodyne, Inc., Hartland, WI. The full-length HiaE7 cDNA clone was obtained by screening a cDNA library
synthesized in the Uni-ZAP XR vector Stratagene, La Jolla, CA from polyA
+
RNA from the Camp Cooley adults. The library was screened with the partial HiaE7
fragment from the RT-PCR experiment using standard procedures Ausubel et al., 1998 and in vivo excision
performed according to the cDNA library kit manufac- turer’s protocols. Manual sequencing of both DNA
strands was done with the T7 Sequenase Version 2.0 DNA Sequencing Kit Amersham Life Sciences, Inc.,
Cleveland, OH. The deduced amino-acid sequence alignments were performed with the pam250S scoring
matrix of the MacVector Software Ver. 5.0 Oxford Molecular Group, Oxford, UK.
2.6. Ribonuclease protection assays Gene expression assays were performed with gene-
specific probes synthesized from restriction-enzyme- digested HiaE7 and HiaE8 cDNA clones. T3 RNA
polymerase, the MAXIscript In Vitro Transcription Kit and the HybSpeed RPA Kit Ambion, Inc., Austin, TX
were used to generate
32
P-labeled antisense strand RNA probes and perform ribonuclease protection assays
RPA. Total RNA was isolated from individual adult horn flies by a miniprep procedure Verwoerd et al.,
1989. Ultraviolet UV absorbance values were used to ensure that equal amounts of total RNA from each RNA
miniprep were present in the RPA probe hybridization reactions. Human glyceraldehyde 3-phosphate dehydro-
genase GAPDH was used as a control probe to verify that RNA concentration was approximately constant
between samples. GAPDH had previously been shown to have high levels of expression in all life stages of the
horn fly data not shown. The RPA hybridizations used 0.5
µ g of total RNA and 30,000 cpm each of radiolab-
eled GAPDH, HiaE7 and HiaE8 RNA probe in a single assay. The template sources for the probes were the
396 bp HiaE7 and 326 bp HiaE8 cDNAs isolated during the degenerate primer RT-PCR experiments and sub-
cloned into pT7Blue-2. Molecular biology grade chemi- cals were obtained from GIBCO BRL Gaithersburg,
MD, Sigma Chemical Co. St. Louis, MO or Fisher Scientific Pittsburgh, PA when available.
3. Results
