970 R. Hernandez et al. Insect Biochemistry and Molecular Biology 30 2000 969–977
activity when compared with a susceptible strain. Simi- larly, pyrethroid-hydrolyzing esterases have also been
detected in B. microplus De Jersey et al., 1985. In this study we report the complete cDNA sequences
of two esterases from B. microplus, a point mutation in one sequence, and the differences among susceptible and
resistant strains in Southern hybridization.
2. Materials and methods
2.1. Ticks Unfed larvae of six B. microplus strains originally
obtained from various locations in Texas and Mexico, and maintained at the Cattle Fever Tick Research Lab-
oratory, ARS, USDA, at Mission, TX, USA were used in this investigation Table 1. The Gonzalez and Ramiren˜o
strains were obtained from two separate outbreaks of ticks in Texas. Both strains are susceptible to OP and
pyrethroid compounds, and have been maintained with- out any insecticidal pressure. The Tuxpan and Coatza-
coalcos strains resistant to OP and pyrethroid compounds respectively, were originally obtained from Veracruz in
Mexico. Coatzacoalcos strain also exhibits a moderate level of OP resistance of 5 fold to coumaphos. The San
Felipe and Corrales strains were obtained from Tamaul- ipas and Colima, Mexico respectively, both showing
high resistance to permethrin. Assessment of suscepti- bility to acaricides was performed using the larval packet
test Stone and Haydock, 1962, with the Gonzalez strain as the susceptible reference. The resistant strains have
been maintained under constant chemical pressure since their arrival at the laboratory in Mission, Texas. Each
generation of Tuxpan strain has been exposed to increas- ing concentrations of coumaphos, at a rate of 0.05 of
active ingredient. Coatzacoalcos, San Felipe, and Cor- rales strains have been similarly exposed to permethrin.
Table 1 B. microplus strains and resistance levels to coumaphos OP and per-
methrin Pyrethroid Strain
Year and collection site Resistance index
Coumaphos Permethrin Gonzalez
1994 Zapata County, Texas Reference strain
Ramiren˜o 1995 Zapata County, Texas
1 2
Tuxpan 1982 Tuxpan, Veracruz,
8 2
Mexico Coatzacoalcos 1994 Coatzacoalcos,
5 43
Veracruz, Mexico San Felipe
1995 Tamaulipas, Mexico 2.3
3535 Corrales
1995 Colima, Mexico 1.3
2601
2.2. DNA isolation Total genomic DNA was obtained from tick larvae
by phenol extraction using a modification of the method described by Sambrook et al., 1989. Briefly, 1 g of
larvae was pulverized in liquid nitrogen with a mortar and pestle. After the liquid nitrogen had dissipated the
sample was mixed with 10 ml of extraction buffer Tris, 10 mM; EDTA, 100 mM; SDS, 0.5; pH 8.0, the DNA
was extracted with phenolchloroform and then precipi- tated with ethanol in the presence of 300 mM NaCl. The
DNA pellet was dissolved in the extraction buffer and centrifuged at 110 000g for 20 min at 4
° C to remove
glycogen. The DNA solution was then treated success- ively with RNase A, proteinase K, phenol extracted, and
ethanol precipitated. The final DNA preparation had an A
260
A
280
.1.8. 2.3. RNA isolation and cDNA synthesis
Total RNA was isolated from tick larvae using the TRIzol reagent Life Technologies, Gaithersburg, MD.
1 g of larvae was pulverized in liquid nitrogen as described above, then it was homogenized on ice in 10
ml of TRIzol reagent using a glass grinder with a Teflon pestle. Samples were incubated at room temperature for
5 min and centrifuged at 272g to remove cuticle and tissue particles. The remaining purification steps fol-
lowed the manufacturer’s protocol. Poly A
+
mRNA was purified from the total RNA using the Oligotex mRNA
purification kit Qiagen, Santa Clarita, CA. cDNA syn- thesis and rapid amplification of cDNA ends RACE
were performed using the Marathon cDNA Amplifi- cation kit Clontech, Palo Alto, CA following the manu-
facturer’s protocol.
2.4. Oligonucleotide primers Degenerate oligonucleotide primers Ace1, Ace2, and
Ace3 were chosen based on sequences of amino acids conserved in acetylcholinesterase AChE from other
organisms, following the previously described methods Arpagaus et al., 1994. Also specific primers were
derived from clones 8 and 13 and used in the synthesis of probes labeled with digoxigenin, and in detection of
a point mutation Table 2.
2.5. Polymerase Chain Reaction PCR PCR was carried out in a GeneAmp 9600 thermal
cycler Perkin Elmer Cetus, Norwalk, CT. The reaction mixture to obtain the first fragment with the degenerate
primers contained the 4 deoxyribonucleoside 5 9-triphos-
phates 200 µ
M each, degenerate primers Ace1 and Ace2 or Ace3 2
µ M each, adaptor-ligated cDNA as
template, Taq DNA polymerase 1.25 units, Tris-HCl
971 R. Hernandez et al. Insect Biochemistry and Molecular Biology 30 2000 969–977
Table 2 Primers used in the PCR reactions
a
Primer ID Sequence
Nucleotide position
b
Degenerate oligonucleotide primers: Ace1
5 9-GAAG GACT TGCT CTI TACT CTI AA-39
Derived from known Ace2
5 9-CC IGC IGA CTTC ICC AGAA-39
AChE sequences Ace3
5 9-CC IGC AGCT CTTC ICC AGAA -39
Oligonucleotide primers used in the synthesis of Dig-probes: GS81
5 9-CTAGCGATGCGTTTCCGTTGAACGC-39 antisense
589-613 clone 8 GS82
5 9-CAATAGCACCAAGCCCGTAATGGTG-39 sense 351-375
clone 8 GS131
5 9-TCGCCCCCGAAGTGTTCGATGTTAC-39 antisense
590-614 clone 13 GS132
5 9-CTGGGTTCCCGAGAAAGCCATGAAC-39 sense
336-360 clone 13 Oligonucleotide primers used to detect the point mutation in PCR:
MU1G 5
9-GAAGCAATCCTGAAAAACTTAAGG-39 sense wild type 1097-1120 clone 13
MU1A 5
9-GAAGCAATCCTGAAAAACTTAAGA-39 sense mutant 1097-1120 clone 13
GS138CR 5
9-TCGGACAAGTAGTCCAGGTAC-39 antisense 1230-1250 clone 13
a
I =
deoxyinosine. Ace1 =
sense primer derived from the amino acid sequence EDCLYLN conserved in the AChE family. Ace2 and Ace3 =
antisense primers derived from the amino acid sequence FGESAG conserved in the AChE family, differing in the codon for serine TCI or AGTC underlined. MU1G and MU1A different in the last nucleotide, underlined.
b
Based on nucleotide sequences from clones 8 and 13, considering ATG start codon as nucleotide one.
10 mM, pH 8.3, KCl 50 mM, MgCl
2
1.5 mM, and gelatin 0.001 in a final volume of 25
µ l. The reaction
mixture was initially heated for 3 min at 94 °
C, then incu- bated for 40 cycles 94
° C for 1 min, 46
° C for 1 min,
and 72 °
C for 1.5 min, and a final extension for 5 min at 72
° C. All reagents for PCR were obtained from Perkin
Elmer, except primers, which were obtained from Inte- grated DNA Technologies Inc. Coralville, IA. PCR
products were separated by electrophoresis on 1 aga- rose gels SeaKem ME, FMC, Rockland, ME in TBE
buffer Tris 45 mM, borate 45 mM, EDTA 1 mM, pH 8.0. The resulting bands were visualized by ethidium
bromide staining.
Reaction mixture for PCR to obtain specific fragments with the wild type or the mutant allele contained the
4 deoxyribonucleoside 5 9-triphosphates 200
µ M each,
specific primers GS138CR and MU1G or MU1A 0.2 µ
M, genomic DNA as template, Advantage cDNA polymerase mix 0.5 units, Tricine-KOH 40 mM,
KOAc 15 mM, MgOAc
2
3.5 mM, and bovine serum albumin 3.75
µ gml in a final volume of 25
µ l. Tem-
perature conditions included 3 min at 94 °
C, then 35 cycles 94
° C for 20 sec, 63
° C for 30 sec, and 68
° C for
30 sec, and a final extension for 5 min at 68 °
C. All reagents for PCR were obtained from Clontech except
primers, which were obtained from Integrated DNA Technologies Inc. Coralville, IA. PCR products were
separated and visualized as above.
2.6. cDNA cloning PCR products were isolated and purified from gel
slices using the Advantage PCR-Pure kit Clontech. Purified products were end-polished using the PCR Pol-
ishing kit Stratagene, La Jolla, CA. The polished DNA fragments were cloned using the Zero Blunt PCR clon-
ing kit, and transformed into TOPO10 competent cells Invitrogen, Carlsbad, CA. Colonies were screened by
PCR with M13 universal primers for the presence of DNA inserts. Plasmid DNA was purified from positive
colonies using a plasmid miniprep kit BIO-RAD Labs, Hercules, CA.
2.7. cDNA sequencing Recombinant plasmids were sequenced on an auto-
mated DNA sequencer ABI-PRISM 377, Perkin Elmer, Foster City, CA using the dye-terminator at the DNA
Sequencing and Oligo Lab, Department of Veterinary Pathobiology, Texas AM University, College Station,
TX. Sequences were analyzed using the BCM Search Launcher Human Genome Center, Baylor College of
Medicine, Houston, TX on the Internet :8088search- launcherlauncher.html. From
plasmid inserts that showed homology with published sequences of AChE,
we designed specific primers that were used for 5 9- and
3 9-RACE to obtain the entire cDNA sequence.
2.8. Southern hybridization Genomic DNA digested with either restriction endon-
uclease EcoRI or double digested with EcoRI and NsiI was separated in 1 agarose gel by electrophoresis
stained with ethidium bromide and photographed. The DNA in the gel was denatured, neutralized and trans-
ferred to a Nylon membrane Nytran
, Schleicher and
Schuell, Keene, NH as described by Sambrook et al. 1989. The membrane was then rinsed with 2
× SSC and
baked at 80 °
C for 2 h in a vacuum oven. Two probes of 263 and 279 bp were made using specific primers
972 R. Hernandez et al. Insect Biochemistry and Molecular Biology 30 2000 969–977
Table 2 with the PCR DIG Probe Synthesis Kit Boehringer Mannheim, Indianapolis, IN, and using
Gonzalez cDNA as the template. PCR conditions included denaturation at 95
° C for 5 min, then 5 cycles
of 94 °
C for 30 s and 72 °
C for 4 min, 5 cycles of 94 °
C for 30 s and 70
° C for 4 min, 35 cycles of 94
° C for 20
s and 68 °
C for 4 min, and a final extension of 68 °
C for 5 min. Hybridization and signal detection were carried
out using the DIG High Prime DNA Labeling and Detec- tion Starter kit II Boehringer-Mannheim. Briefly, the
baked Nylon membrane was prehybridized with a stan- dard hybridization buffer containing formamide for 30
min at 48
° C; the probe was denatured by boiling for 5
min, added to the prehybridization solution and incu- bated in a hybridization oven for 12 h at 48
° C with
gentle agitation. High stringency conditions included 2 post-hybridization washes of 5 min each in 2
× SSC and
0.1 SDS at room temperature and 2 washes of 15 min each in 0.5
× SSC and 0.1 SDS at 68
° C under constant
agitation. Finally the labeled DNA bands were detected following the manufacturer’s protocol, after exposing an
X-ray film BioMax MR film, Eastman Kodak Com- pany, Rochester, N.Y. to the membrane for 18 h.
3. Results
