- Email: [email protected]
Identification of a Point Mutation in the para-Type Sodium Channel Gene from a Pyrethroid-Resistant Cattle Tick
Biochemical and Biophysical Research Communications 261, 558 –561 (1999) Article ID bbrc.1999.1076, available online at http://www.idealibrary.com on
Identification of a Point Mutation in the para-Type Sodium Channel Gene from a Pyrethroid-Resistant Cattle Tick Haiqi He,* Andrew C. Chen,* ,1 Ronald B. Davey,† G. Wayne Ivie,* and John E. George‡ *Food Animal Protection Research Laboratory, USDA-ARS, 2881 F&B Road, College Station, Texas 77845; †Cattle Fever Tick Research Laboratory, USDA-ARS, P.O. Box 969, Mission, Texas 78573; and ‡Knipling-Bushland US Livestock Insects Research Laboratory, USDA-ARS, 2700 Fredericksburg Road, Kerrville, Texas 78028
Received May 25, 1999
To investigate the molecular mechanism of resistance to pyrethroids in the southern cattle tick, Boophilus microplus, we have obtained and sequenced a partial para-homologous sodium channel cDNA from susceptible and pyrethroid-resistant tick strains. A point mutation that results in an amino acid change from Phe to Ile was identified in the highly conserved domain IIIS6 of the homologous sodium channel from ticks that are highly resistant to pyrethroid acaricides. This mutation is at a location different from those reported in the same gene in pyrethroidresistant insects. © 1999 Academic Press Key Words: Boophilus microplus; insecticide; acaricide; resistance.
Voltage-sensitive sodium channel is the primary target of pyrethroid pesticides (1). A number of studies have linked the pyrethroid resistance (knockdown resistance, kdr) to the para (Drosophila melanogaster) (2) homologous sodium channel genes in several insects, such as Musca domestica (3), Heliothis virescens (4) and Blattella germanica (5). Sequencing of the para homologous sodium channel genes from M. domestica (6, 7), B. germanica (6), and Haematobia irritans (8) showed that the kdr-type resistance was a result of point mutations (L1014F and M918T) in domain II of the sodium channel. A mutation (L1029H) in the location homologous to the kdr-mutation was also found in pyrethroid resistant H. virescens (9). A recent study revealed that new mutations (D1561V and E1565G) in the linker of the domains III and IV of the sodium channel are also associated with the expression of nerve insensitivity resistance to pyrethroid insecti1
To whom correspondence should be addressed: USDA, ARS, FAPRL, 2881 F&B Road, College Station, Texas 77845. Fax: (409) 260 –9261. E-mail: [email protected]. Sequence data from this article has been deposited with the GenBank nucleotide sequence database under Accession No. AF134216. 0006-291X/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
cides in both H. virescens and Helicoverpa armigera (10). The southern cattle tick, Boophilus microplus, transmits the protozoan parasite Babesia bovis that causes cattle fever and is considered one of the most important pests of cattle in tropical and subtropical countries. Controlling tick populations has become increasingly difficult because of the rapid development of resistance to acaricides including pyrethroids. A recent study (11) using combinations of synergists and pyrethroids and DDT showed that the kdr-type resistance was involved in some tick strains with very high levels of resistance to pyrethroids (1,000 fold). However, we have previously obtained and sequenced the cDNA encoding the kdr homologous location in domain II of the tick sodium channel and found no mutation (12). Here, we report the first genetic evidence that a point mutation in segment 6, domain III (IIIS6) of the tick sodium channel is associated with strains that are highly resistant to pyrethroid acaricides. MATERIALS AND METHODS Animals. Four strains of B. microplus, Gonzalez (G), Mexican pyrethroid resistant strain (MP), Corrales (C), and San Felipe (S), were used in this study (Table 1). These ticks were originally collected from various locations in the U.S. and Mexico and had been selected with acaricides except for the susceptible strain. Tick strains were reared as described by Davey et al. (13) and their resistance to acaricides was assayed using the FAO larval packet test (14). Unfed larvae were used for cDNA preparations to avoid possible contamination by the host blood. cDNA cloning. Amplification and sequencing of the tick sodium channel homologous cDNA sequence was first carried out using the susceptible (Gonzalez) strain. Total RNA was purified from tick larvae using the TRIzol Reagent (GIBCO-BRL, Gaithersburg, MD). Oligotex resin (Qiagen, Valencia, CA) was used to purify poly A 1 mRNA from the total RNA. The cDNA was synthesized using the Marathon cDNA Amplification Kit (Clontech, Palo Alto, CA). Degenerate primers (Fig. 1) derived from conserved amino acid residues of sodium channels from D. melanogaster (2) and M. domestica (7) were used for amplification of the homologous sequence from the tick cDNA. PCR was performed on the PTC-200 DNA Engine (MJ Research, Watertown, MA). PCR reactions containing cDNA (5% of
558
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS dium channel cDNA sequence was obtained, a 39RACE was performed using the Marathon cDNA as the template with gene specific primers (Fig. 1A: e and f) and cDNA adapter primers (Clontech) according to the manufacturer’s instruction to obtain the 39-end of the tick sodium channel cDNA sequence. Mutation detection by directly sequencing PCR amplified DNA. To investigate possible mutations in the sodium channel gene of pyrethroid resistant ticks, poly A 1 mRNAs from different strains were purified and cDNAs were synthesized using oligo dT and SuperScript II reverse transcriptase (SuperScript Pre-amplification System for First Strand cDNA Synthesis, GIBCO BRL). The partial homologous sodium channel cDNA sequences encoding domain III and IV were amplified by PCR with gene specific primers (Fig. 1B: g and h) from different tick strains and sequenced directly with primers i– o (Fig. 1B).
FIG. 1. Strategies for PCR amplification and sequencing of the partial cDNA sequence encoding domain III and IV of the tick sodium channel homologue. Closed boxes S1–S6 denote transmembrane segments within four homologous domains I–IV of the sodium channel. (A) Degenerate primers (a, b, c, and d) were used in the RT-PCR to amplify the sodium channel homologous sequence from tick cDNA. Primers e and f were used in 39RACE to obtain the 39-end of the tick sodium channel cDNA. (B) Primers g and h were used to amplify the tick sodium channel cDNA sequence from four different tick strains. Primers i, j, k, l, m, n, and o were used for sequencing PCR amplified DNA. The primer sequences are: (a) 59-GGI GA(AG) TGG ATI GA(AG) (AT)(CG)I ATG TGG GA-39; (b) 59-T(GT) IA(AC) ICC CAT IA(CT) I(CG)(ACT) (AG)AA (AGT)AT IA(AG) CCA-39; (c) 59-AA(CT) (CT)TI TT(CT) AT(ACT) GGI GTI AT(ACT) AT(ACT)GA(CT) AA-39; (d) 59-TT(CT) TCI A(AG)(AGT) ATI A(CT)I GC(AGT) AT(AG)TAC AT(AG) TT-39; (e) 59-GCT GTT CGA GTC CAT CCT AGA ACG-39; (f) 59-CGT CTC GTA CCT CAT CAT CAG CTT C-39; (g) 59-GAA GAT GTG GAC ACA GAC AAG CTG G-39; (h) 59-CCA AGA TGT CGA CGC AGT AGA CCA TG-39; (i) 59-CTG CTG CCC CGA CTG GTG TTA C-39; (j) 59-CGT CTC GTA CCT CAT CAT CAG CTT C-39; (k) 59-AGC ATC TGG ACG CCC ATG ATG-39; (l) 59-CCC ATT TTC TTC ATG GCG TTA TAG-39; (m) 59-GTC CTT TAG CAC CGT ACC TAG-39; (n) 59-GAA GCT GAT GAT GAG GTA CGA G-39; (o) 59-CCA AGA TGT CGA CGC AGT AGA CCA TG-39.
synthesized first strand cDNA), 200 mM each of dNTPs, 10 mM each of degenerate oligo primer pair (Fig. 1A: a and b; c and d), 0.5 ml Advantage KlenTaq Polymerase Mix, and 1x reaction buffer (Clontech) were carried out in a final volume of 50 ml. PCR started with initial denaturation at 94°C for 1 min followed by 10 cycles of 94°C for 20 s, 60°C for 30 s with decrement of 1°C per cycle, 68°C for 1 min and 30 cycles of 94°C for 20 s, 50°C for 30 s, 68°C for 1 min and a final extension at 68°C for 10 min. PCR products were analyzed on agarose gels, purified, and cloned into the pCR-Blunt vector (Invitrogen, Carlsbad, CA). Plasmids containing DNA inserts were purified using a plasmid miniprep kit (BIO-RAD Laboratories, Hercules, CA) and sequenced on an ABI-PRISM 377 automated DNA sequencer (Perkin-Elmer, Foster City, CA). After the partial homologous so-
Sequence analysis. Sequence data were analyzed using the programs provided by BCM Search Launcher (Human Genome Center, Baylor College of Medicine, Houston, TX) on the Internet (http:// kiwi.imgen.bcm.tmc.edu:8088/search-launcher/launcher.html).
RESULTS AND DISCUSSION A partial cDNA sequence of the tick sodium channel was obtained by degenerate primer-mediated PCR using primer pairs of a/b and c/d (Fig. 1). The cDNA sequence was extended to the 39 end of the sodium channel by 39RACE using primers e and f. The sequences of the cDNA were determined by automated sequencing after cloning. It contains the coding region of the C-terminus, including the domain III and IV of the tick sodium channel. To investigate possible mutations in this region, cDNA sequences encoding domain III and IV of the sodium channel from 4 different tick strains (Table 1) were then amplified by PCR using gene specific primers g and h. PCR-amplified DNA (Fig. 2) was purified and sequenced directly. To further ensure the accuracy, sequencing primers were designed to space close enough to make sure that the same region was sequenced at least twice. Comparison of cDNA sequences obtained from different tick strains identified a single nucleotide change from T to A in the IIIS6 coding region of the sodium channel cDNA, which results in an amino acid change from Phe to Ile (Fig. 3). The mutation is present only in San Felipe and Corrales strains, which are highly resistant to pyrethroids and DDT. No mutations were found in the Mexican pyrethroid resistant strain, although this strain shows significant resistance
TABLE 1
B. microplus Strains Used in This Study and Resistance Index to Pyrethroid Acaricides Tick strains Gonzalez (G) Mexican pyrethroid (MP) Corrales (C) San Felipe (S)
Outbreak (year and location) 1984, 1994, 1995, 1995,
Zapata County, Texas Coatzacoalcos Vera Cruz, Mexico Los Corrales, Colima, Mexico San Felipe, Tamaulipas, Mexico 559
Resistance index Susceptible 43-fold .1000-fold .1000-fold
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FIG. 2. Agarose gel electrophoresis of PCR amplified tick sodium channel homologous cDNA products from 4 tick strains using primers g and h. M: wide-range DNA marker; G, MP, C, and S: tick strains G, MP, C, and S, respectively.
(but much less than San Felipe and Corrales strains) to pyrethroids. Preliminary results indicated that cytochrome P450 (11) and esterases (unpublished data from our lab) may be resistance mechanisms for the Mexican pyrethroid resistant tick strain. These results indicate the involvement of multiple mechanisms in resistance to pyrethroids in ticks. As indicated in Table 1, these ticks were originally obtained from Texas and different locations in Mexico. Different resistance mechanisms may have selectively evolved in particular tick populations due to geographical isolation. Pyrethroids exert the neurotoxic effects on the sodium channel primarily by slowing down the channel inactivation (15, 16). Amino acid residues in segment IIS6 as well as the intracellular segment linking do-
mains III and IV are important for the channel inactivation. These residues are believed to constitute part of the intracellular mouth of the sodium channel pore and form the inactivation gating particle (17, 18). Mutations in the IIS6 region of the para-type sodium channel from kdr strains of several insects (6 – 8) and mutations located near the start of the linkage between domains III and IV (10) have been associated with the nerve insensitivity resistance to pyrethroid insecticides. These facts suggest that these regions of the sodium channel may be target sites for pyrethroids. Structural alterations as a result of mutations may diminish the interaction of pyrethroids with sodium channels, reducing the sensitivity to pyrethroid insecticides. Amino acids involved in the interaction with neurotoxins in segment S6 may not be limited to just the domain II of the sodium channel. In this study, a mutation in the IIIS6 of the sodium channel has been identified in tick strains that are highly resistant to pyrethroids. In the rat, a mutation in IS6 of the muscle sodium channel diminished the efficacy of batrachotoxin (BTX) in eliminating the sodium channel inactivation, but did not alter the normal gating properties (19). BTX is a steroidal alkaloid that causes sodium channels to open persistently, a mode of action similar to that of pyrethroid insecticides. These results imply that in addition to the direct gating function, S6 segments of the sodium channel contain residues that are part of sites, away from the
FIG. 3. Alignment of sodium channel sequences from diverse sources in the region of the point mutation Phe to Ile in pyrethroid resistant ticks. Highly conserved residues across species are shaded. Numbers are shown in relation to the Drosophila-para sodium channel sequence. The mutant residue is in reverse. Tick-C, Tick-S, Tick-MP, and Tick-G are tick strains C, S, MP, and G, respectively, as shown in Table 1. Sodium channel sequences, Musca (house fly: Musca domestica), Blattella (German cockroach: Blattella germanica), Drosophila-para (fruit fly: D. melanogaster), Cyanea (scyphozoan jellyfish: Cyanea capillata), Polyorchis (hydrozoan jellyfish: Polyorchis penicillatus), squid (Loligo opalescens), eel (Electrophorus electricus), Rat1–3 (rat brain isoforms I–III), Rat-cm and Rat-sm (rat cardiac and skeletal muscle isoforms), and Human-cm and Human-sm (human cardiac and skeletal muscle isoforms) have following accession numbers in the GenBank: U38814, U71083, P35500, L15445, AF047380, L19979, P02719, P04774, P04775, P08104, P15389, P15390, A38195, and P35499, respectively. 560
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channel mouth, that interact with neurotoxins such as BTX and pyrethroids. The interaction of these sites with the toxins indirectly affects the normal channel function in a manner much like the allosteric effect in enzymes. Under the pressure of pesticides, mutations in sodium channels may have been selected such that pesticides are no longer able to interact with these sites resulting in a decrease in the sensitivity of the animal to toxic effects of pesticides. Direct sequencing of PCR-amplified DNA provides a powerful tool for molecular genetic analyses. This approach generates more accurate DNA sequences compared to sequencing individual clones of the PCR amplified DNA because the DNA sequences generated are a consensus sequence of the majority DNA molecules and sequence errors incorporated by Taq DNA polymerase during the PCR will not be accounted for in the final sequence (20). Automated sequencing based on fluorescence of PCR products has been a useful tool for analysis of DNA variations (21). Sequence electrophoretograms can reveal polymorphism, mutations, and heterozygotes (22). We were able to identify a single point mutation using this method. In conclusion, we have identified a point mutation in the highly conserved segment IIIS6 of the sodium channel in tick strains that are highly resistant to pyrethroids and DDT. The mutation results in an amino acid change from Phe to Ile. Results indicate that S6 segments of the sodium channel are target sites of pyrethroid insecticides. Cloning and sequencing of the full-length sodium channel cDNA sequence from the tick, B. microplus, are currently underway. ACKNOWLEDGMENTS We thank Drs. A. Fallon and N. Beckage for a critical review of the manuscript. Mention of a commercial or proprietary product in this paper does not constitute an endorsement of this product by the USDA, nor does it imply the recommendation of the product by the USDA to the exclusion of similar products.
REFERENCES 1. Miller, T. A. (1988) Parasit. Today 4, S8 –S12. 2. Loughney, K., Kreber, R., and Ganetzky, B. (1989) Cell 58, 1143– 1154. 3. Williamson, M. S., Denholm, I., Bell, C. A., and Devonshire, A. L. (1993) Mol. Gen. Genet. 240, 17–22. 4. Taylor, M. F. J., Heckel, D. G., Brown, T. M., Kreitman, M. E., and Black, B. (1993) Insect Biochem. Mol. Biol. 23, 763–775. 5. Dong, K. E., and Scott, J. G. (1994) Insect Biochem. Mol. Biol. 24, 647– 654. 6. Miyazaki, M., Ohyama, K., Dunlap, D. Y., and Matsumura, F. (1996) Mol. Gen. Genet. 252, 61– 68. 7. Williamson, M. S., Martinez-Torres, D., Hick, C. A., and Devonshire, A. L. (1996) Mol. Gen. Genet. 252, 51– 60. 8. Guerrero, F. D., Jamroz, R. C., Kammlah, D., and Kunz, S. E. (1997) Insect Biochem. Mol. Biol. 27, 745–755. 9. Park, Y., and Taylor, M. F. J. (1997) Insect Biochem. Mol. Biol. 27, 9 –13. 10. Head, D. J., McCaffery, A. R., and Callaghan, A. (1998) Insect Mol. Biol. 7, 191–196. 11. Miller, R., Davey, R. B., and George, J. E. (1999) J. Med. Entomol., in press. 12. He, H., Chen, A. C., Davey, R. B., Ivie, G. W., and George, J. E. (1999) J. Med. Entomol., in press. 13. Davey, R. B., Garza, J., Jr., Thompson, G. D., and Drummond, R. O. (1980) J. Med. Entomol. 17, 287–289. 14. Stone, B. F., and Haydock, K. P. (1962) Bull. Entomol. Res. 53, 563–578. 15. de Weille, J. R., Brown, L. D., and Narahashi, T. (1990) Brain Res. 512, 26 –32. 16. Narahashi, T. (1992) Trends Pharmacol. Sci. 13, 236 –241. 17. Moorman, J. R., Kirsch, G. E., Brown, A. M., and Joho, R. H. (1990) Science 250, 688 – 691. 18. Catterall, W. A. (1993) Trends Neurosci. 16, 500 –506. 19. Wang, S. Y., and Wang, G. K. (1998) Proc. Natl. Acad. Sci. USA 95, 2653–2658. 20. Bao, V. B. (1995) in PCR Primer: A Laboratory Manual (Dieffenbach, C. W., and Dveksler, G. S., Eds.), pp. 509 –526, CSHL Press, New York. 21. Kwok, P. Y., Carlson, C., Yager, T. D., Ankener, W., and Nickerson, D. A. (1994) Genomics 23, 138 –144. 22. Parker, L. T., Zakeri, H., Deng, Q., Spurgeon, S., Kwok, P. Y., and Nickerson, D. A. (1996) BioTechniques 21, 694 – 699.
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Identification of a Point Mutation in the para-Type Sodium Channel Gene from a Pyrethroid-Resistant Cattle Tick Haiqi He,* Andrew C. Chen,* ,1 Ronald B. Davey,† G. Wayne Ivie,* and John E. George‡ *Food Animal Protection Research Laboratory, USDA-ARS, 2881 F&B Road, College Station, Texas 77845; †Cattle Fever Tick Research Laboratory, USDA-ARS, P.O. Box 969, Mission, Texas 78573; and ‡Knipling-Bushland US Livestock Insects Research Laboratory, USDA-ARS, 2700 Fredericksburg Road, Kerrville, Texas 78028
Received May 25, 1999
To investigate the molecular mechanism of resistance to pyrethroids in the southern cattle tick, Boophilus microplus, we have obtained and sequenced a partial para-homologous sodium channel cDNA from susceptible and pyrethroid-resistant tick strains. A point mutation that results in an amino acid change from Phe to Ile was identified in the highly conserved domain IIIS6 of the homologous sodium channel from ticks that are highly resistant to pyrethroid acaricides. This mutation is at a location different from those reported in the same gene in pyrethroidresistant insects. © 1999 Academic Press Key Words: Boophilus microplus; insecticide; acaricide; resistance.
Voltage-sensitive sodium channel is the primary target of pyrethroid pesticides (1). A number of studies have linked the pyrethroid resistance (knockdown resistance, kdr) to the para (Drosophila melanogaster) (2) homologous sodium channel genes in several insects, such as Musca domestica (3), Heliothis virescens (4) and Blattella germanica (5). Sequencing of the para homologous sodium channel genes from M. domestica (6, 7), B. germanica (6), and Haematobia irritans (8) showed that the kdr-type resistance was a result of point mutations (L1014F and M918T) in domain II of the sodium channel. A mutation (L1029H) in the location homologous to the kdr-mutation was also found in pyrethroid resistant H. virescens (9). A recent study revealed that new mutations (D1561V and E1565G) in the linker of the domains III and IV of the sodium channel are also associated with the expression of nerve insensitivity resistance to pyrethroid insecti1
To whom correspondence should be addressed: USDA, ARS, FAPRL, 2881 F&B Road, College Station, Texas 77845. Fax: (409) 260 –9261. E-mail: [email protected]. Sequence data from this article has been deposited with the GenBank nucleotide sequence database under Accession No. AF134216. 0006-291X/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
cides in both H. virescens and Helicoverpa armigera (10). The southern cattle tick, Boophilus microplus, transmits the protozoan parasite Babesia bovis that causes cattle fever and is considered one of the most important pests of cattle in tropical and subtropical countries. Controlling tick populations has become increasingly difficult because of the rapid development of resistance to acaricides including pyrethroids. A recent study (11) using combinations of synergists and pyrethroids and DDT showed that the kdr-type resistance was involved in some tick strains with very high levels of resistance to pyrethroids (1,000 fold). However, we have previously obtained and sequenced the cDNA encoding the kdr homologous location in domain II of the tick sodium channel and found no mutation (12). Here, we report the first genetic evidence that a point mutation in segment 6, domain III (IIIS6) of the tick sodium channel is associated with strains that are highly resistant to pyrethroid acaricides. MATERIALS AND METHODS Animals. Four strains of B. microplus, Gonzalez (G), Mexican pyrethroid resistant strain (MP), Corrales (C), and San Felipe (S), were used in this study (Table 1). These ticks were originally collected from various locations in the U.S. and Mexico and had been selected with acaricides except for the susceptible strain. Tick strains were reared as described by Davey et al. (13) and their resistance to acaricides was assayed using the FAO larval packet test (14). Unfed larvae were used for cDNA preparations to avoid possible contamination by the host blood. cDNA cloning. Amplification and sequencing of the tick sodium channel homologous cDNA sequence was first carried out using the susceptible (Gonzalez) strain. Total RNA was purified from tick larvae using the TRIzol Reagent (GIBCO-BRL, Gaithersburg, MD). Oligotex resin (Qiagen, Valencia, CA) was used to purify poly A 1 mRNA from the total RNA. The cDNA was synthesized using the Marathon cDNA Amplification Kit (Clontech, Palo Alto, CA). Degenerate primers (Fig. 1) derived from conserved amino acid residues of sodium channels from D. melanogaster (2) and M. domestica (7) were used for amplification of the homologous sequence from the tick cDNA. PCR was performed on the PTC-200 DNA Engine (MJ Research, Watertown, MA). PCR reactions containing cDNA (5% of
558
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS dium channel cDNA sequence was obtained, a 39RACE was performed using the Marathon cDNA as the template with gene specific primers (Fig. 1A: e and f) and cDNA adapter primers (Clontech) according to the manufacturer’s instruction to obtain the 39-end of the tick sodium channel cDNA sequence. Mutation detection by directly sequencing PCR amplified DNA. To investigate possible mutations in the sodium channel gene of pyrethroid resistant ticks, poly A 1 mRNAs from different strains were purified and cDNAs were synthesized using oligo dT and SuperScript II reverse transcriptase (SuperScript Pre-amplification System for First Strand cDNA Synthesis, GIBCO BRL). The partial homologous sodium channel cDNA sequences encoding domain III and IV were amplified by PCR with gene specific primers (Fig. 1B: g and h) from different tick strains and sequenced directly with primers i– o (Fig. 1B).
FIG. 1. Strategies for PCR amplification and sequencing of the partial cDNA sequence encoding domain III and IV of the tick sodium channel homologue. Closed boxes S1–S6 denote transmembrane segments within four homologous domains I–IV of the sodium channel. (A) Degenerate primers (a, b, c, and d) were used in the RT-PCR to amplify the sodium channel homologous sequence from tick cDNA. Primers e and f were used in 39RACE to obtain the 39-end of the tick sodium channel cDNA. (B) Primers g and h were used to amplify the tick sodium channel cDNA sequence from four different tick strains. Primers i, j, k, l, m, n, and o were used for sequencing PCR amplified DNA. The primer sequences are: (a) 59-GGI GA(AG) TGG ATI GA(AG) (AT)(CG)I ATG TGG GA-39; (b) 59-T(GT) IA(AC) ICC CAT IA(CT) I(CG)(ACT) (AG)AA (AGT)AT IA(AG) CCA-39; (c) 59-AA(CT) (CT)TI TT(CT) AT(ACT) GGI GTI AT(ACT) AT(ACT)GA(CT) AA-39; (d) 59-TT(CT) TCI A(AG)(AGT) ATI A(CT)I GC(AGT) AT(AG)TAC AT(AG) TT-39; (e) 59-GCT GTT CGA GTC CAT CCT AGA ACG-39; (f) 59-CGT CTC GTA CCT CAT CAT CAG CTT C-39; (g) 59-GAA GAT GTG GAC ACA GAC AAG CTG G-39; (h) 59-CCA AGA TGT CGA CGC AGT AGA CCA TG-39; (i) 59-CTG CTG CCC CGA CTG GTG TTA C-39; (j) 59-CGT CTC GTA CCT CAT CAT CAG CTT C-39; (k) 59-AGC ATC TGG ACG CCC ATG ATG-39; (l) 59-CCC ATT TTC TTC ATG GCG TTA TAG-39; (m) 59-GTC CTT TAG CAC CGT ACC TAG-39; (n) 59-GAA GCT GAT GAT GAG GTA CGA G-39; (o) 59-CCA AGA TGT CGA CGC AGT AGA CCA TG-39.
synthesized first strand cDNA), 200 mM each of dNTPs, 10 mM each of degenerate oligo primer pair (Fig. 1A: a and b; c and d), 0.5 ml Advantage KlenTaq Polymerase Mix, and 1x reaction buffer (Clontech) were carried out in a final volume of 50 ml. PCR started with initial denaturation at 94°C for 1 min followed by 10 cycles of 94°C for 20 s, 60°C for 30 s with decrement of 1°C per cycle, 68°C for 1 min and 30 cycles of 94°C for 20 s, 50°C for 30 s, 68°C for 1 min and a final extension at 68°C for 10 min. PCR products were analyzed on agarose gels, purified, and cloned into the pCR-Blunt vector (Invitrogen, Carlsbad, CA). Plasmids containing DNA inserts were purified using a plasmid miniprep kit (BIO-RAD Laboratories, Hercules, CA) and sequenced on an ABI-PRISM 377 automated DNA sequencer (Perkin-Elmer, Foster City, CA). After the partial homologous so-
Sequence analysis. Sequence data were analyzed using the programs provided by BCM Search Launcher (Human Genome Center, Baylor College of Medicine, Houston, TX) on the Internet (http:// kiwi.imgen.bcm.tmc.edu:8088/search-launcher/launcher.html).
RESULTS AND DISCUSSION A partial cDNA sequence of the tick sodium channel was obtained by degenerate primer-mediated PCR using primer pairs of a/b and c/d (Fig. 1). The cDNA sequence was extended to the 39 end of the sodium channel by 39RACE using primers e and f. The sequences of the cDNA were determined by automated sequencing after cloning. It contains the coding region of the C-terminus, including the domain III and IV of the tick sodium channel. To investigate possible mutations in this region, cDNA sequences encoding domain III and IV of the sodium channel from 4 different tick strains (Table 1) were then amplified by PCR using gene specific primers g and h. PCR-amplified DNA (Fig. 2) was purified and sequenced directly. To further ensure the accuracy, sequencing primers were designed to space close enough to make sure that the same region was sequenced at least twice. Comparison of cDNA sequences obtained from different tick strains identified a single nucleotide change from T to A in the IIIS6 coding region of the sodium channel cDNA, which results in an amino acid change from Phe to Ile (Fig. 3). The mutation is present only in San Felipe and Corrales strains, which are highly resistant to pyrethroids and DDT. No mutations were found in the Mexican pyrethroid resistant strain, although this strain shows significant resistance
TABLE 1
B. microplus Strains Used in This Study and Resistance Index to Pyrethroid Acaricides Tick strains Gonzalez (G) Mexican pyrethroid (MP) Corrales (C) San Felipe (S)
Outbreak (year and location) 1984, 1994, 1995, 1995,
Zapata County, Texas Coatzacoalcos Vera Cruz, Mexico Los Corrales, Colima, Mexico San Felipe, Tamaulipas, Mexico 559
Resistance index Susceptible 43-fold .1000-fold .1000-fold
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FIG. 2. Agarose gel electrophoresis of PCR amplified tick sodium channel homologous cDNA products from 4 tick strains using primers g and h. M: wide-range DNA marker; G, MP, C, and S: tick strains G, MP, C, and S, respectively.
(but much less than San Felipe and Corrales strains) to pyrethroids. Preliminary results indicated that cytochrome P450 (11) and esterases (unpublished data from our lab) may be resistance mechanisms for the Mexican pyrethroid resistant tick strain. These results indicate the involvement of multiple mechanisms in resistance to pyrethroids in ticks. As indicated in Table 1, these ticks were originally obtained from Texas and different locations in Mexico. Different resistance mechanisms may have selectively evolved in particular tick populations due to geographical isolation. Pyrethroids exert the neurotoxic effects on the sodium channel primarily by slowing down the channel inactivation (15, 16). Amino acid residues in segment IIS6 as well as the intracellular segment linking do-
mains III and IV are important for the channel inactivation. These residues are believed to constitute part of the intracellular mouth of the sodium channel pore and form the inactivation gating particle (17, 18). Mutations in the IIS6 region of the para-type sodium channel from kdr strains of several insects (6 – 8) and mutations located near the start of the linkage between domains III and IV (10) have been associated with the nerve insensitivity resistance to pyrethroid insecticides. These facts suggest that these regions of the sodium channel may be target sites for pyrethroids. Structural alterations as a result of mutations may diminish the interaction of pyrethroids with sodium channels, reducing the sensitivity to pyrethroid insecticides. Amino acids involved in the interaction with neurotoxins in segment S6 may not be limited to just the domain II of the sodium channel. In this study, a mutation in the IIIS6 of the sodium channel has been identified in tick strains that are highly resistant to pyrethroids. In the rat, a mutation in IS6 of the muscle sodium channel diminished the efficacy of batrachotoxin (BTX) in eliminating the sodium channel inactivation, but did not alter the normal gating properties (19). BTX is a steroidal alkaloid that causes sodium channels to open persistently, a mode of action similar to that of pyrethroid insecticides. These results imply that in addition to the direct gating function, S6 segments of the sodium channel contain residues that are part of sites, away from the
FIG. 3. Alignment of sodium channel sequences from diverse sources in the region of the point mutation Phe to Ile in pyrethroid resistant ticks. Highly conserved residues across species are shaded. Numbers are shown in relation to the Drosophila-para sodium channel sequence. The mutant residue is in reverse. Tick-C, Tick-S, Tick-MP, and Tick-G are tick strains C, S, MP, and G, respectively, as shown in Table 1. Sodium channel sequences, Musca (house fly: Musca domestica), Blattella (German cockroach: Blattella germanica), Drosophila-para (fruit fly: D. melanogaster), Cyanea (scyphozoan jellyfish: Cyanea capillata), Polyorchis (hydrozoan jellyfish: Polyorchis penicillatus), squid (Loligo opalescens), eel (Electrophorus electricus), Rat1–3 (rat brain isoforms I–III), Rat-cm and Rat-sm (rat cardiac and skeletal muscle isoforms), and Human-cm and Human-sm (human cardiac and skeletal muscle isoforms) have following accession numbers in the GenBank: U38814, U71083, P35500, L15445, AF047380, L19979, P02719, P04774, P04775, P08104, P15389, P15390, A38195, and P35499, respectively. 560
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
channel mouth, that interact with neurotoxins such as BTX and pyrethroids. The interaction of these sites with the toxins indirectly affects the normal channel function in a manner much like the allosteric effect in enzymes. Under the pressure of pesticides, mutations in sodium channels may have been selected such that pesticides are no longer able to interact with these sites resulting in a decrease in the sensitivity of the animal to toxic effects of pesticides. Direct sequencing of PCR-amplified DNA provides a powerful tool for molecular genetic analyses. This approach generates more accurate DNA sequences compared to sequencing individual clones of the PCR amplified DNA because the DNA sequences generated are a consensus sequence of the majority DNA molecules and sequence errors incorporated by Taq DNA polymerase during the PCR will not be accounted for in the final sequence (20). Automated sequencing based on fluorescence of PCR products has been a useful tool for analysis of DNA variations (21). Sequence electrophoretograms can reveal polymorphism, mutations, and heterozygotes (22). We were able to identify a single point mutation using this method. In conclusion, we have identified a point mutation in the highly conserved segment IIIS6 of the sodium channel in tick strains that are highly resistant to pyrethroids and DDT. The mutation results in an amino acid change from Phe to Ile. Results indicate that S6 segments of the sodium channel are target sites of pyrethroid insecticides. Cloning and sequencing of the full-length sodium channel cDNA sequence from the tick, B. microplus, are currently underway. ACKNOWLEDGMENTS We thank Drs. A. Fallon and N. Beckage for a critical review of the manuscript. Mention of a commercial or proprietary product in this paper does not constitute an endorsement of this product by the USDA, nor does it imply the recommendation of the product by the USDA to the exclusion of similar products.
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