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
3.1. Esterase cDNA cloning The degenerate primers FG-144, FG-145 and FG-146
were designed from conserved regions of esterase sequences noted by Newcomb et al. 1997b. RT-PCR
using FG-144 and FG-146 and polyA
+
RNA from diaz- inon-resistant and diazinon-susceptible flies resulted in
the amplification of a 396 bp fragment [Fig. 1A] that was found to have substantial DNA sequence homology
and amino-acid identity to LcaE7 and DmaE7 from L. cuprina and Drosophila melanogaster, respectively. In
L. cuprina, this region of the LcaE7 gene contains an amino-acid substitution, Gly
137
→Asp, which confers diazinon resistance Newcomb et al., 1997a. Therefore,
1110 F.D. Guerrero Insect Biochemistry and Molecular Biology 30 2000 1107–1115
Fig. 1. Alignment of the deduced amino-acid sequence from the horn fly HiaE7 and HiaE8 cDNAs with previously characterized sequences
from L. cuprina and D. melanogaster. The deduced amino-acid sequence of Hi α
E7 was aligned with Lc α
E7 and Dm α
E7 from L. cuprina and D. melanogaster, respectively A, and Hi
α E8 was aligned with Lc
α E8 and Dm
α E8 from L. cuprina and D. melanogaster, respectively B. Alignment
was performed with the pam250S scoring matrix of the MacVector Software Ver. 5.0. GenBank accession numbers for LcaE7, LcaE8, DmaE7 and DmaE8 are U56636, U49423, U51052 and U51050, respectively. Asterisks indicate an amino-acid identity compared with the Hi
α E7 or
Hi α
E8 sequences while a dot . indicates a conservative amino-acid substitution. An underscored residue F
indicates that an amino acid was eliminated from the sequence string to maximize alignment to Hi
α E7 or Hi
α E8. The positions of the two degenerate primers successfully used in
the RT-PCR experiment to clone a fragment of HiaE7, FG-144 and FG-146, are noted by lines over the positions of the corresponding amino acids.
1111 F.D. Guerrero Insect Biochemistry and Molecular Biology 30 2000 1107–1115
to check for the presence of an amino-acid substitution at the same position in the horn fly esterase, 11 clones
resulting from the RT-PCR experiments on the diazinon- resistant
Camp Cooley
population sample
were sequenced. No differences in nucleotide sequence of this
396 bp fragment were found between the susceptible and resistant populations. This fragment was used to probe
a cDNA library from the diazinon-resistant Camp Cooley adult fly population, and a 2175 bp clone was
isolated and designated HiaE7 GenBank accession no. AF139082. HiaE7 contains a 281 bp 5
9-untranslated region, a 1713 bp open reading frame, a 181 bp 3
9- untranslated region and a polyadenylated 3
9 end. The open reading frame is preceded by several in-frame stop
codons and encodes a 570 amino-acid protein with 74 and 58 amino-acid identity to the LcaE7 and DmaE7
esterase-encoding genes from L. cuprina and D. mel- anogaster,
respectively. A
polyadenylation signal
sequence AAUAAA is located 140 bp downstream from the UAG stop codon. ASPCR assays of 50 individ-
ual flies from the diazinon-resistant population showed no sequence differences at codon 137 of HiaE7 between
the susceptible and resistant flies data not shown.
The conceptual translation product of HiaE7 was examined by alignment to the Torpedo californica ace-
tylcholinesterase sequence GenBank accession no. X03439 and found to contain 23 of the 24 invariant
residues found in 29 lipases and esterases examined by Cygler et al. 1993, including three residues that could
serve as the active-site motif, Ser
218
, G1u
351
and His
371
data not shown. The contextual sequence surrounding these invariant residues was also conserved in Hi
α E7.
The lone absent invariant residue was a cysteine puta- tively involved in disulfide bridge formation with Cys
111
. However, there are two other cysteines at positions 88
and 89 in the primary sequence which could serve in bridge formation with Cys
111
. There are three regions of Hi
α E7 that exhibit higher deviation from the amino-acid
sequences of Lc α
E7 and Dm α
E7. These regions are Cys
89
→Ser
106
, Met
283
→His
295
and Val
358
→Val
437
, which have 56 and 44, 62 and 38, and 75 and 49
amino-acid identity plus conservative substitutions with Lc
α E7 and Dm
α E7, respectively. Outside these three
regions Hi α
E7 exhibits 90 and 87 amino-acid ident- ity plus conservative substitutions with Lc
α E7 and
Dm α
E7, respectively. Using the analysis of Cygler et al. 1993 and amino-acid alignment with the T. californica
acetylcholinesterase, these three regions of Hi α
E7 corre- spond to regions noted as possessing high variability
within the esterase family of enzymes. Cys
89
→Ser
106
corresponds to loop L
1,2
, a region near the conserved Trp of the active site involved in substrate binding Harel et
al., 1993. Met
283
→His
295
appears to correspond to the a
3 6,7
helix, while the long region between Val
358
→Val
437
encompasses the a
1–4 7,8
helices of which the a
1 7,8
and a
2 7,8
helices display high variability among the esterases. RT-PCR using FG-145 and FG-146 with polyA
+
RNA from diazinon-resistant and diazinon-susceptible flies resulted in the amplification of a 326 bp cDNA
GenBank accession no. AF139081. This cDNA, desig- nated HiaE8, contained an open reading frame spanning
the entire 326 bp that encoded a protein with 84 and 75 amino-acid identity to the LcaE8 and DmaE8
esterase-encoding genes from L. cuprina and D. mel- anogaster, respectively [Fig. 1B]. Eight independent
clones from the RT-PCR experiment with both the sus- ceptible and the resistant fly mRNA were sequenced and
no nucleotide differences were found.
A 221 bp cDNA GenBank accession no. AF139080 was amplified during the RT-PCR with FG-144 and FG-
146 and RNA from diazinon-resistant flies. This cDNA, designated HiaE1, was found to have substantial DNA
sequence homology to a number of esterases from D. melanogaster. The open reading frame from HiaE1
data not shown had from 55 to 60 amino-acid ident- ity
to esterase-encoding
genes DmaE1,
DmaE2, DmaE5, DmaE8, DmaE9 and DmaE10 from D. mel-
anogaster, and LcaE8 and LcaE9 from L. cuprina. Although the highest amino-acid identity was to
DmaE1, no putative esterase designation could be reliably ascertained from this short fragment. During the
RNA Northern blot experiments, the HiaE1 cDNA hybridized to a broad region of the Northern blot instead
of a discrete band data not shown. This result was later duplicated using a blot that had previously been shown
to contain intact mRNA. Additionally, it was found that the HiaE1 probe could not be stripped from the blot
and interfered with subsequent reprobings with HiaE7. Because of these difficulties, further investigations of the
HiaE1 cDNA were not done.
3.2. Analysis of HiaE7 and HiaE8 expression To investigate possible resistance-associated quanti-
tative differences in esterase gene expression, RNA from the susceptible and resistant populations was examined
by Northern analysis. RNA was isolated from a sample of unfed, newly emerged, susceptible control flies, a
second sample from the same population that had been fed for 3–5 days, and the diazinon-resistant Camp
Cooley population. HiaE7 expression was elevated in the resistant population compared with both susceptible
samples Fig. 2, while HiaE8 expression levels were comparable in the unfed susceptible and the resistant
Camp Cooley flies. HiaE8 expression was depressed in the flies that had been fed for 3-5 days. HiaE7
hybridized to a single RNA species of approximately 2.8 kb in both the susceptible and the resistant samples,
while the HiaE8 blot showed hybridization to two tran- scripts of approximately 4.4 and 3.6 kb in the susceptible
sample and a single band of approximately 4.2 kb in the resistant sample.
1112 F.D. Guerrero Insect Biochemistry and Molecular Biology 30 2000 1107–1115
Fig. 2. Northern analysis of RNA from diazinon-resistant and -sus-
ceptible horn flies. PolyA
+
RNA was purified from susceptible SUS unfed, newly emerged adult horn flies U, susceptible 3–5 day old
flies fed citrated bovine blood F, and diazinon-resistant CC adults of mixed age that had been feeding on cattle at the Camp Cooley
ranch F. The RNA was fractionated by denaturing formaldehyde gel electrophoresis and Northern blots probed with
32
P-labeled 396 bp HiaE7 E7 and 326 bp HiaE8 E8 RT-PCR cDNA fragments. 3
µ l
and 1 µ
g per lane of polyA
+
were used for the HiaE7 and HiaE8 blots, respectively. Autoradiography of both blots was under identical
conditions using BioMax MS film and a Transcreen-HE intensifying screen Kodak, Rochester, NY.
The enhanced expression of the HiaE7 transcript in the resistant population could be due to an increase in
gene copy number. To evaluate this possibility, restric- tion-enzyme-digested genomic DNA from both the sus-
ceptible and resistant populations was fractionated by agarose gel electrophoresis and analyzed by Southern
blotting. Probing the blot with the HiaE7 cDNA did not reveal major differences between the OP-susceptible and
OP-resistant samples, indicating a similar HiaE7 gene copy number in both populations Fig. 3.
3.3. Ribonuclease protection assay of individual flies Following the Northern analysis, which suggested an
increase in HiaE7 expression in the resistant population, coupled with information from previous studies which
showed qualitative and quantitative variation in esterase protein expression among individual diazinon-resistant
flies Guerrero et al., 1999, individual fly HiaE7 gene expression was assayed. Topical diazinon application to
flies from the resistant population was used to phenotype individuals as diazinon-resistant or diazinon-susceptible.
Total RNA was purified from one susceptible and resist- ant male, and four susceptible and resistant female flies,
and probed in ribonuclease protection assays Fig. 4. The susceptible male lane 2 was one of four male indi-
viduals that died approximately 2 h after the application of 5 ng of diazinon. Twenty-one male flies remained
very active after 2 h at that dose. The resistant male lane 3 survived 77 min after the application of 50 ng diazi-
non. The other 24 flies in that treatment group died within 30 min after receiving this dose. The susceptible
Fig. 3. HiaE7 copy number analysis of horn fly genomic DNA. Gen-
omic DNA 10 µ
l from diazinon-resistant CC and diazinon-suscep- tible SUS horn flies was digested with restriction enzyme XbaI X,
HindIII H or EcoRI E, fractionated on a 0.7 agarose gel, trans- ferred to a nylon membrane by Southern blotting and probed with the
32
P-labeled 396 bp HiaE7 RT-PCR cDNA fragment.
female flies in lanes 4 and 5 were two of the three indi- vidual females that were barely alive 2 h after receiving
5 ng of diazinon. The remaining 22 females that received this dosage were very active after 2 h. The susceptible
females in lanes 6 and 7 died 17 and 24 min, respect- ively, after receiving 50 ng diazinon. Of the 55 female
flies receiving the 50 ng dose, all but five died within 40 min. Resistant female flies in lanes 8, 9, 10 and 11
survived for 52, 47, 93 and 89 min, respectively, after receiving the 50 ng diazinon dose. The transcript levels
of both HiaE7 and HiaE8 were elevated in the resistant female individuals compared with the susceptible indi-
viduals from the same population. The HiaE7 and HiaE8 gene expression levels shown on the autoradio-
gram of Fig. 4 were quantified by densitometry and the values normalized by expressing the data relative to the
expression of the GAPDH control hybridization probe Table 2. There is about a five-fold increase in HiaE7
expression in the resistant females compared with the susceptible females, while HiaE8 is expressed about two
times higher in the resistant females. Because only a sin- gle resistant male fly was obtained from this field sam-
ple, no firm conclusions can be drawn about expression levels in males.
1113 F.D. Guerrero Insect Biochemistry and Molecular Biology 30 2000 1107–1115
Fig. 4. RPA of HiaE7 and HiaE8 transcript levels in individual adult horn flies. Total RNA was purified from individual diazinon-resistant R,
lanes 3 and 8–11 and diazinon-susceptible S, lanes 2 and 4–7 Camp Cooley male lanes 2–3 and female lanes 4–11 adult flies that were phenotyped by topical diazinon application. Each lane contained 0.5
µ g total RNA probed with 30,000 cpm each of
32
P-labeled antisense HiaE7 E7, HiaE8 E8 and GAPDH probes labeled to specific activities of 6.4
× 10
8
cpm µ
g, 5.3 ×
10
8
cpm µ
g and 4.5 ×
10
8
cpm µ
g, respectively. Prelimi- nary experiments were performed to ensure the RPAs were done under conditions of probe excess. Lane 1 consists of a control reaction containing
all three probes hybridized in the absence of RNA. Table 2
Relative levels of HiaE7 and HiaE8 transcripts in individual diazinon-resistant Res and diazinon-susceptible Sus horn flies Lane ID
a
Sex Phenotype
E7
b
E8
b
2 M
Sus 0.41
0.11 3
M Res
0.43 0.009
4 F
Sus 0.008
0.04 5
F Sus
0.09 0.06
6 F
Sus 0.09
0.05 7
F Sus
0.08 0.06
Sus F mean ±
standard deviation: 0.06
± 0.04
0.05 ±
0.01 8
F Res
0.17 0.089
9 F
Res 0.38
0.13 10
F Res
0.30 0.13
11 F
Res 0.43
0.14 Res F mean
± standard deviation:
0.32 ±
0.12 0.12
± 0.02
a
From analysis of Fig. 4.
b
Esterase mRNA transcript level relative to GAPDH.
4. Discussion
