Frequency of kdr Gene in House Fly Field Populations ... - BioOne

INSECTICIDE RESISTANCE AND RESISTANCE MANAGEMENT

Frequency of kdr Gene in House Fly Field Populations: Correlation of Pyrethroid Resistance and kdr Frequency JING HUANG,1, 2 MICHAEL KRISTENSEN,1 CHUANG-LING QIAO,2

AND

JØRGEN B. JESPERSEN1

J. Econ. Entomol. 97(3): 1036Ð1041 (2004)

ABSTRACT The frequency of the L1014 F kdr mutation was determined in 14 Þeld populations of house ßies, Musca domestica L., with resistance factors at LD50 for pyrethrin/piperonyl butoxide and bioresmethrin/piperonyl butoxide from 4 to 29 and 2 to 98, respectively. A polymerase chain reaction test for identifying kdr homo- or heterozygote house ßies was used to determine the frequency of kdr. The L1014 F allele was found in all populations tested. The frequency of kdr in the Þeld populations was high and varied from 0.46 to 0.99. Eleven of the populations were in HardyÐWeinberg equilibrium, whereas two strains had higher number of heterozygotes than expected, indicating a possible heterozygote advantage. The frequency of kdr was strongly correlated with the reduced mortality observed in the bioassays with pyrethrum and bioresmethrin synergized by piperonyl butoxide. This indicates that kdr is a major mechanism for pyrethroid resistance in these Þeld populations. Five Þeld populations had resistance factors ⬎25 and ⬎10 for bioresmethrin/piperonyl butoxide and pyrethrin/ piperonyl butoxide, respectively. The frequencies of kdr in these Þve populations varied from 0.89 to 0.99. The frequencies of kdr in the Þeld populations showing no or a low level of resistance had frequencies of kdr from 0.46 to 0.75, which indicates that the L1014 F kdr allele is a fully recessive genetic trait in house ßies. We have shown that the molecular diagnostic PASA method to determine the resistance phenotypes and the frequency of kdr is a powerful tool, which could be used to get information to make recommendations about pest and resistance management. KEY WORDS pyrethroid resistance, allele-speciÞc polymerase chain reaction, voltage-gated sodium channel, resistance management

THE HOUSE FLY, Musca domestica L., is a serious pest to livestock and a public health pest that acts as a transmitter of human and animal pathogens. Aerosols or space sprays with either pyrethrum (PYR) or bioresmethrin (BRM) both synergized with the cytochrome P450 inhibitor piperonyl butoxide (pbo) are commonly used for house ßy control. They are still effective on most farms in Denmark, but pyrethroid resistance is increasing (Kristensen et al. 2001). Worldwide intensive use of pyrethroids has resulted in development of resistance in many pest species (Sawicki 1985, Georghiou 1990, Hemingway and Ranson 2000), which are listed in the database of arthropod resistance to insecticides at www.cips.msu.edu/resistance/rmdb. Pyrethroid insecticides act on the nervous system by modifying the gating kinetics of voltage-sensitive sodium channels. Two major mechanisms of pyrethroid resistance are found in resistant insects: increases in the rate of metabolic detoxiÞcation of the pyrethroids, and structural mutations in the pyrethroid target site. Sometimes, both mechanisms interact to increase the level of resistance. Metabolic re1 Danish Pest Infestation Laboratory, Skovbrynet 14, 2800 Kgs Lyngby, Denmark. 2 State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of Zoology, Chinese Academy of Sciences, Beijing 100080, China.

sistance to pyrethroids in insects can be associated with increases in cytochrome P450 activity (Scott 1999), increases in general esterases and elevated glutathione S-transferases (Feyereisen 1995, Hemingway 2000). Mutations in the voltage-gated sodium channel gene are responsible for contributing to knockdown resistance (kdr) to pyrethroids and DTT (Soderlund and Knipple 2003). A replacement of leucine by phenylalanine in domain II of transmembrane segment II-S6 is the most common amino acid substitution in the kdr allele. This homologous mutation has been documented in many insects (Dong 1997, Park et al. 1997, Jamroz et al. 1998, Martinez-Torres et al. 1998) and was initially particularly well-studied in the house ßy (Williamson et al. 1993, 1996a,b). In vitro expression of the L1014 F-substituted house ßy sodium channel gene in Xenopus oocytes resulted in 10-fold less insensitivity to cismethrin, further supporting its role in kdr resistance (Smith et al. 1997). An additional replacement M918T in the house ßy sodium channel gene, termed super-kdr, can occur in presence of the kdr mutation and enhance the pyrethroid resistance 3to 20-fold the kdr level (Williamson et al. 1996a,b). A polymerase chain reaction (PCR)-based diagnostic assay has been developed to detect these resistance-causing nucleotide substitutions in several species, including M. domestica (Williamson et al. 1996a)

0022-0493/04/1036Ð1041$04.00/0 䉷 2004 Entomological Society of America

June 2004

HUANG ET AL.: FREQUENCY OF kdr GENE IN HOUSE FLY

Table 1. LD50 resistance factors for house fly field populations (Kristensen et al. 2001) Strain

PYR:pbo (1:5)

BRM:pbo (1:5)

381b3 594i2 662g 790b 791a 793a 797a 798a 799a 800a 801a 802a 806a 807a

4 4 14 25 25 4 9 6 4 29 5 12 6 6

2 4 36 87 44 9 15 5 5 98 3 40 8 12

and Hematobia irritans (L.) (Guerrero et al. 1998). The primary objective of the present research was to Þnd the frequency of kdr in Danish house ßy populations and to determine whether pyrethroid resistance of Þeld populations correlates with the frequency of kdr. Elucidating the relationship of kdr frequency with pyrethroid resistance should help in designing novel strategies to prevent or minimize the spread and evolution of resistance. Materials and Methods House flies. Field populations were collected on a variety of Danish livestock farms in 1997. A sample consisted of 200 Ð 400 house ßies. In the laboratory, the females of the collected house ßies were allowed to lay eggs. The toxicity of the synergized pyrethroids BRM:pbo and PYR:pbo and the anticholinesterases dimethoate, azamethiphos, and methomyl were tested on the Þrst generation of ßies breed in the laboratory. The level of resistance to PYR:pbo and BRM:pbo is presented in Table 1. Full details of the toxicological evaluations of the Þeld populations to neurotoxic insecticides are presented in Kristensen et al. (2001). House ßies not used for toxicological experiments were collected and stored at ⫺80⬚C. World Health Organization (WHO) is a susceptible standard reference strain developed and maintained at the University of Pavia, Pavia, Italy. The house ßies used in the current study were offspring of batch WHOij2 received at the Danish Pest Infestation Laboratory in 1988. Bioassay. The bioassay procedures at Danish Pest Infestation Laboratory has been standardized for ⬎30 yr and has been used in the development of the instructions for the WHO Standard Test for determining resistance in M. domestica. Females are preferable in the topical application bioassay, and the feeding bioassay is performed with male house ßies The contact poison effect of PYR:pbo (1:5) and BRM:pbo (1:5) was estimated by means of a topical application test in which female house ßies were treated with a range of insecticide concentrations. From the time of emergence until the analysis was completed, the test ßies were fed continuously with water and sugar. In addi-

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tion, milk was provided for 3Ð 4 d immediately before treatment. Flies were kept in a controlled atmosphere room at 25Ð26⬚C, 60 Ð 65% RH with continuous light before and after the topical application. Tests were carried out for 6 Ð7 d after emergence. Immediately before treatment, house ßies were anesthetized with CO2. One microliter of the insecticide in acetone solution was then applied to the dorsal thorax of each house ßy. Mortality, i.e., the number of house ßies dead or paralyzed, was recorded after 24 h. Two or three replicates of 20 house ßies were tested at six to nine concentrations for each test. In addition, two replicates of 20 house ßies were treated with acetone as control. Insecticides. The insecticides used were all of technical grade and diluted with analytical grade acetone. Pyrethrum (15.01% pyrethrin I, 9.65% pyrethrin II) was from Mortalin (Haslev, Denmark). Bioresmethrin (98%) was a gift from Rothamsted Research (Harpenden, UK). Piperonyl butoxide (98%) was from Cheminova (Lemvig, Denmark). DNA Purification. Genomic DNA was prepared from individual male adult house ßies stored at ⫺80⬚C. Male house ßies were used to avoid mated or gravid females. A house ßy was placed in microcentrifuge tube and kept on ice. Four hundred microliters of TEN extraction buffer (10 mM Tris-HCl, 2 mM EDTA, 0.4 M NaCl, pH 8.0) was added, and the ßy was homogenized by using sterile plastic pestle. Ten microliters of 10% SDS and 8 ␮l of 20 mg ml⫺1 Proteinase K were added to each tube. The solution was mixed well and incubated at 55⬚C for 1 h. Then, 300 ␮l of 6 M NaCl was added to each tube. After centrifugation (20 min; 14,000 rpm), 500 ␮l of supernatant was transferred to a tube, and DNA was precipitated by adding 500 ␮l of 2-propanol (⫺20⬚C for 20 min). After centrifugation (20 min; 14,000 rpm), the pellet was washed by icecold 70% ethanol and then dried. The pellet was suspended in 50 ␮l of H2O. One microliter was used for the PCR assay. PCR Amplification of Specific Allele (PASA). An assay based on Williamson et al. (1996b) to genotype each house ßy for the presence of the Leu to Phe replacement in the sodium channelÕs S6 transmembrane segment of domain II was established. Two outer (kdr1 and kdr4) and two inner allele-speciÞc (kdr2 and kdr3) primers were designed for PASA to detect the house ßy kdr mutation (L1014 F; CTT to TTT), so that both homozygotes and heterozygotes can be discriminated in a single PCR reaction. The sequence of sense outer primer, kdr1, was 5⬘-AAGGATCGCTTCAAGG-3⬘, whereas the antisense outer primer, kdr4, was 5⬘-TTCACCCAGTTCTTAAAACGAG-3⬘. The sense inner allele-speciÞc primer kdr2, ending in a T to match the mutant TTT codon, was used to detect the kdr allele. The sequence of kdr2 was 5⬘-GTCGTGATCGGCAATT-3⬘. The antisense inner primer, kdr3, ended in a G to match the CTT codon of the susceptible allele, sequence was 5⬘-CGTCAACTTACCACAAG-3⬘. AmpliÞcation of susceptible allele resulted in a 480-bp fragment and a 200-bp fragment. Instead of the 200-bp fragment, a 280-bp fragment

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Piscataway, NJ). AmpliÞcation was initiated by 95⬚C for 2 min followed by 40 cycles, 94⬚C for 45 s, 54⬚C for 30 s, 72⬚C for 90 s, and a Þnal extension step at 72⬚C for 10 min. AmpliÞed fragments were analyzed by electrophoresis on a 3% high-resolution agarose gel (Cambrex Bio Science Walkersville, Inc., Walkersville, MD) and were visualized by ethidium bromide staining under UV light. Results and Discussion

Fig. 1. Genotype kdr mutation by PASA. (A) kdr1 and kdr3 were used to amplify 200-bp susceptible allele fragment. The 280-bp kdr allele fragment was ampliÞed by kdr2 and kdr4. A control fragment was the result of ampliÞcation by using kdr1 and kdr4. (B) PCR products from individual house ßies after separation on a 3% agrose gel.

could be ampliÞed with 480-bp fragment from kdrtype allele (Fig. 1). PASA was performed in thin-walled microcentrifuge tubes by using 25-␮l reactions. The following are the optimized reaction conditions: 1 ␮l of genomic DNA prepared as described above from single male house ßy, 10 pmol of each outer primer, 40 pmol of each inner primer, 2.5 ␮l of 10⫻ PCR buffer, 1 pmol of dNTP mix, 1 mg ml⫺1 bovine serum albumin, 1 U of TaqDNA polymerase (Amersham Biosciences, Inc., Table 2.

The frequency of the L1014 F kdr mutation was determined in 14 Þeld populations of house ßies representing different levels of pyrethroid resistance. The ßies were collected in 1997 to determine the status and development of insecticide resistance in Danish house ßy populations (Kristensen et al. 2001). Resistance factors (RFs) at LD50 for PYR:pbo and BRM:pbo varied from 4 to 29 and 2 to 98, respectively (Table 1). A simple, single PCR test for identifying kdr homoor heterozygote house ßies was used to determine the frequency of kdr. Two primers asymmetrically ßank the mutated region and two primers are speciÞc for susceptible and kdr genotype, respectively (see Materials and Methods for details). Genomic DNA was extracted from individual house ßies; 49 Ð54 ßies from each population were tested. The L1014 F allele was found in all the populations tested but was not present in the susceptible laboratory strain WHO. The frequency of kdr in the Þeld populations was high and varied from 0.46 to 0.99 (Table 2). The ßies tested were the Þrst generation offspring from laboratory breeding of ßies collected in the Þeld. After random mating of 200 Ð 400 ßies, we would thus expect the genotypic proportions of kdr to be at HardyÐWeinberg equilibrium. This was tested by a ␹2 test comparing the observed numbers of kdr homo- and heterozygotes with the expected numbers calculated from the kdr frequency. Eleven populations were in HardyÐWeinberg equilibrium, whereas 381b3, 594i2, and 799a deviated from equilibrium (Table 2). In 381b3 and 799a, we Þnd a higher number of heterozy-

Phenotypes of the pyrethroid knockdown resistance allele, kdr (L1014F), and the susceptible allele, sus (L1014)

Strain

n

Observed kdr/kdr

Observed kdr/sus

Observed sus/sus

Frequency of kdr

H.W. ␹2

WHO 381b3 594i2 662g 790b 791a 793a 797a 798a 799a 800a 801a 802a 806a 807a

20 54 54 49 54 54 51 54 53 54 54 53 54 53 54

0 9 12 39 51 51 16 30 10 7 53 14 51 29 20

0 42 24 9 3 3 28 21 29 36 1 22 3 21 25

20 3 18 1 0 0 7 3 14 11 0 17 0 3 9

0.00 0.56 0.50 0.89 0.97 0.97 0.59 0.75 0.46 0.46 0.99 0.47 0.97 0.75 0.60

17.90 3.93 0.30* 0.04* 0.04* 0.91* 0.07* 0.54* 6.27 0.00* 1.48* 0.04* 0.10* 0.06*

Field populations were tested for HardyÐWeinberg (H.W.) equlibrium by ␹2 test (*P ⫽ 0.05; ␹2 ⫽ 3.84).

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Fig. 2. Correlation between the kdr allele and the frequencies of surviving adult house ßies treated with either 320 ng of pyrethrum or 160 ng of bioresmethrin.

Fig. 3. Relationship of resistance factor at LD50 for pyrethrum/piperonyl butoxide and bioresmethrin/piperonyl butoxide and of the kdr allele, as determined by the PASA test.

gotes, indicating a possible heterozygote advantage in the absence of selection, whereas 594i2 had more homozygote susceptible individuals than expected. To examine the relationship between the kdr mutation and the toxicity of pyrethroid in the Þeld populations in a like-for-like comparison, the frequencies of survivors were calculated from the original bioassay (Kristensen et al. 2001). The house ßies were tested at either 160 ng of bioresmethrin or 320 ng of pyrethrum, both synergized with piperonyl butoxide, and the number of individuals tested varied between 40 and 60 house ßies. The increasing frequency of kdr was strongly correlated with the reduced mortality observed in the bioassays as shown in Fig. 2 (PYR:pbo: y ⫽ 129.3x Ð 45.0, r2 ⫽ 0.85 and BRM:pbo: y ⫽ 141.3x Ð 59.4, r2 ⫽ 0.89). The correlation between the frequencies of kdr and the number of survivors was determined by calculating the product moment correlation coefÞcient. The correlation coefÞcients for PYR:pbo and BRM:pbo were r ⫽ 0.92 and r ⫽ 0.94, respectively, indicating signiÞcant correlation at P ⬍ 0.01 (df ⫽ 12). This indicates that kdr is a major mechanism for pyrethroid resistance in these Þeld populations. Many years of Þeld experiments and reports on control failures received from growers and pest control ofÞcers has resulted in deÞnition of a threshold level, indicating which level of resistance could give control problems (Kristensen et al. 2001). The threshold levels were RF50 ⫽ 10 for PYR:pbo and RF50 ⫽ 25 for BRM:pbo, which can be translated to 500 ng of pyrethrum and 250 ng of bioresmethrin per house ßy, respectively (Kristensen et al. 2001). Five Þeld populations, 662 g, 790b, 791a, 800a, and 802a, had RF50 to PYR:pbo and BRM:pbo above 10 and 25, respectively (Table 1). The frequencies of kdr in these Þve populations varied from 0.89 to 0.99 (Table 2; Fig. 3). The frequencies of kdr should thus be ⬎0.89 before we reach a level of resistance to pyrethrum or bioresmethrin, which could lead to control failure. The frequencies of kdr in the Þeld populations showing no or a low level of resistance to PYR:pbo and BRM:pbo with RF50 ⬍10 and ⬍25, respectively, had frequencies of kdr from 0.46 to 0.75. The highest level of resistance, RF50 of 9 and 15, in these strains were observed in 797a, which had kdr frequency of 0.75. This indicates that

the L1014 F kdr allele is a fully recessive genetic trait in house ßies. The PASA analysis of the Þeld house ßy populations signiÞcantly indicates that kdr mutation is a mechanism for pyrethroid resistance. But does the super-kdr allele also inßuence the pyrethroid resistance in the Þeld populations? Jamroz et al. 1998 found that, the super-kdr mutation was less associated with resistance in Þeld populations of H. irritans than in laboratory strains with equivalent level of resistance. In ⬎550 wild horn ßies assayed, there were no super-kdr homozygotes and only 23 heterozygotes, whereas superkdr allele frequency was 0.42 in a resistant laboratory population. Maybe the different ways of pyrethroid selection in the Þeld and in the laboratory contribute to the differences in super-kdr allele frequency. Extensive analysis relating super-kdr allele frequency to pyrethroid resistance in house ßy Þeld populations should be done in the future. It has been stated that the kdr mutated sodium channel alleles confer little or no effect on overall Þtness, which would result in resistant populations returning slowly to susceptibility (Roush and McKenzie 1987). This has been indicated in studies, where pyrethroid resistance was maintained in horn ßy Þeld populations after absence or restriction of pyrethroid use (Weinzierl et al. 1990, Jamroz et al. 1998, Guglielmone et al. 2002). Target site insensitivity to pyrethroids has on the other side been reported to have a Þtness cost in horn ßies. When selection was stopped in the autumn, there was an increase of susceptible sodium channel alleles, resulting in a decrease in resistance in early season populations (Scott et al. 1997, Guerrero et al. 2002). In our study the Þeld populations, 381b3 and 799a show a higher proportion of kdr heterozygotes than the other susceptible populations after HardyÐWeinberg equilibrium analyses. The farm where 381b3 was collected have omitted insecticides since the beginning of the 1990s but had a high level of resistance to multiple insecticides in the 1970s (Keiding 1979, Farnham et al. 1987). Does the deviation from HardyÐWeinberg equilibrium reßect history of the farm? Even after many years without selection, the percentage of homozygous-susceptible house ßies is low, whereas the proportion of heterozy-

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gotes is ⬎77%. Maybe the heterozygote has a Þtness advantage through a pleiotropic effect, as recently indicated by Foster et al. (2003) or maybe homozygote-susceptible house ßies are rare in the area of populations 381b3 and 799a and will migrate infrequently into the populations. House ßy genotypes possessing the kdr mutation were shown to exhibit behavioral differences in comparison with susceptible ßies. The resistant individuals showed no positional preference along a temperature gradient, whereas susceptible genotypes exhibited a strong preference for warmer temperatures (Foster et al. 2003). Temperature preference is an important Þtness parameter for house ßies. Warmer temperatures enhance egg production and development. Temperature is the primary determinant for infection by Entomophthora and additionally fungus-infected ßies can cure themselves by behavioral fever by selecting a hot spot, e.g., a heat lamp (Kalsbeek et al. 2001). The preference for warmer temperatures in susceptible house ßies will thus increase the breeding success of susceptible individuals and lower the frequency of the resistance gene. Standardized bioassay methods for evaluating the status and development of insecticide resistance are valuable in determining phenotype of populations to insecticides. However, compared with PCR assay, bioassays are always time-consuming and requiring a large number of samples. Another advantage of molecular assays over standardized bioassay is that it can successfully identify heterozygotes, which is important in monitoring populations showing a susceptible phenotype while bearing a large proportion of heterozygotes, which could signiÞcantly affect the control efÞciency of pyrethroids. Although molecular diagnostics are powerful tools to determine the frequency of resistant alleles and the changes in allelic frequency in response to management tactics, they are effective only in those cases in which resistance-conferring mutations are already identiÞed and their relation to control failure is established. In light of the diversity of mutations capable of conferring knockdown resistance, the results of molecular diagnostic assays designed to detect a single previously identiÞed mutation must be interpreted with caution (Soderlund and Knipple 2003). We have shown that the molecular diagnostic PASA method to determine the resistance phenotypes and the frequency of kdr is a powerful tool, which could be used to provide information to make recommendation about pest and resistance management.

Acknowledgments We thank Martin S. Williamson (Rothamsted Research, United Kingdom) for valuable information to implement the PASA test. This work was supported by the Sino-Danish ScientiÞc and Technological Cooperation, Danida Fellowship Programme to J.H.

Vol. 97, no. 3 References Cited

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Roush, R. T., and J. A. McKenzie. 1987. Ecological genetics of insecticide and acaricide resistance. Annu. Rev. Entomol. 32: 361Ð380. Sawicki, R. M. 1985. Resistance to pyrethroid insecticides in arthropods, pp. 143Ð192. In D. H. Hutson and T. R. Roberts [eds.], Progress in pesticide biochemistry and toxicology. Wiley, New York. Scott, J. G. 1999. Cytochromes P450 and insecticide resistance. Insect Biochem. Mol. Biol. 29: 757Ð777. Scott, J. A., F. W. Plapp, and D. E. Bay. 1997. Pyrethroid resistance associated with decreased biotic Þtness in horn ßies (Diptera: Muscidae). Southw. Entomol. 22: 405Ð 410. Smith, T. J., S. H. Lee, P. J. Ingles, D. C. Knipple, and D. M. Soderlund. 1997. The L1014F point mutation in the house ßy Vssc1 sodium channel confers knockdown resistance to pyrethroids. Insect Biochem. Mol. Biol. 27: 807Ð 812. Soderlund, D. M., and D. C. Knipple. 2003. The molecular biology of knockdown resistance to pyrethroid insecticides. Insect Biochem. Mol. Biol. 33: 563Ð577. Weinzierl, R. A., C. D. Schmidt, D. B. Faulkner, G. F. Cmarik, and G. D. Zinn. 1990. Chronology of per-

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methrin resistance in a southern Illinois population of the horn ßy (Diptera: Muscidae) during and after selection by pyrethroid use. J. Econ. Entomol. 83: 690 Ð 697. Williamson, M. S., I. Denholm, C. A. Bell, and A. L. Devonshire. 1993. Knockdown resistance (kdr) to DDT and pyrethroid insecticides maps to a sodium channel gene locus in the house ßy (Musca domestica). Mol. Gen. Genet. 240: 17Ð22. Williamson, M. S., D. Martinez-Torres, C. A. Hick, N. Castells, and A. L. Devonshire. 1996a. Analysis of sodium channel gene sequences in pyrethroid-resistant houseßies, pp. 52Ð 61. In T. M. Brown [ed.], Molecular genetics and evolution of pesticide resistance. American Chemical Society, Washington, DC. Williamson, M. S., D. Martinez-Torres, C. A. Hick, and A. L. Devonshire. 1996b. IdentiÞcation of mutations in the houseßy para-type sodium channel gene associated with knockdown resistance (kdr) to pyrethroid insecticides. Mol. Gen. Genet. 252: 51Ð 60. Received 19 November 2003; accepted 22 January 2004.