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Linkage of kdr-Type resistance and the para-Homologous sodium channel gene in German cockroaches (Blattella germanica)
Insect Biochem. Molec. Biol. Vol. 24, No. 7, pp. 647 654, 1994
~
Pergamon
0965-1748(94)E0004-Z
Copyright © 1994 Elsevier ScienceLtd Printed in Great Britain. All rights reserved 0965-1748/94 $7.00 + 0.00
kdr-Type Resistance and the para-Homologous Sodium Channel Gene in German Cockroaches (Blattella germanica) Linkage of
KE DONG,t JEFFREY G. SCOTT~§ Received 27 September 1993; accepted 6 January 1994 Pyrethroids are an important class of insecticides for controlling insect pests, including the German cockroach. Unfortunately, many insects have developed resistance to pyrethroids. One of the most important mechanisms of resistance is kdr (knockdown resistance) which is characterized by neural insensitivity to pyrethroids and DDT. To investigate whether the voltage-dependent sodium channel is involved in kdr-type resistance in the German cockroach, we isolated a 120 bp DNA fragment of the para-homologous sodium channel gene from German cockroaches. Using this fragment as a probe, we identified a restriction fragment length polymorphism (RFLP) of the para-homologous sodium channel gene between susceptible and kdr-type resistant German cockroaches. RFLP analysis of F2 and backcross cockroach populations (total of 331 individuals) showed that all homozygous resistant individuals had a 3.7 kb EcoRI fragment, all homozygous susceptible individuals had a 3.0 kb EcoRI fragment, and all heterozygous individuals had both 3.7 and 3.0 kb fragments. No recombination was detected between the kdr-type resistance locus and the para-homologous sodium channel gene. This suggests that the kdr-type resistance locus and para-homologous sodium channel gene are identical or tightly linked ( < 0 . 2 c M ) in German cockroaches. Our results provide strong evidence that modification of para-homologous sodium channels is associated with kdr-type resistance. Pyrethroid resistance kdr para-homologous sodium channel gene DDT resistance
INTRODUCTION Pyrethroids are a widely used class of insecticides worldwide. Knockdown resistance (kdr) is an important mechanism by which insects develop resistance to pyrethroids and DDT (Shono, 1985). kdr was first discovered in the house fly (Musca domestica) and is characterized by neural insensitivity to pyrethroids and DDT. kdr-type resistance is widespread among many insect pests including German cockroach, Blattella germanica (Scott and Matsumura, 1981, 1983; Umeda et al., 1988), which carries human pathogens and is also the second leading cause of household allergies in the United States (Weber, 1984). The widespread existence of kdr-type resistance in insects presents a serious problem in pest management. Although kdr-type resistance in insects has been studied extensively, the molecular mechanism of this resistance remains largely unclear. Neurophysiological studies tPresent address: Department of Entomology, Agricultural Science Center, University of Kentucky, Lexington, KY 40546, U.S.A. :~Department of Entomology, Comstock Hall, Cornell University, Ithaca, NY 14853-0999, U.S.A. §Author for correspondence.
have shown that the nervous system of kdr or kdr-type insects are less sensitive to pyrethroids and DDT (Tsukamoto et al., 1965; Miller et al., 1979; Osborne and Hart, 1979; DeVries and Georghiou, 1981; Scott and Matsumura, 1981; Ahn et al., 1987; Umeda et al., 1988). Reduced sodium channel binding sites for saxitoxin in kdr house flies was reported by Rossignol (1988). However, others have failed to show any difference in sodium channel density between susceptible and kdr house flies (Grubs et al., 1988; Sattelle et al., 1988; Pauron et al., 1989) or kdr-type German cockroaches (Dong and Scott, 1991; Dong et al., 1993). Based on the enhancement of [3H]batrachotoxinin A-20-a-benzoate ([3H]BTX-B) binding by deltamethrin (a pyrethroid) in membranes from susceptible (but not resistant) strains (Pauron et al., 1989), it was suggested that a modification of the pyrethroid binding site in kdr house flies might be responsible for the resistance. However, we and others observed no enhancement of [3H]BTX-B binding by deltamethrin (Soderlund et al., 1989; Dong et al., 1993). Direct approaches using pyrethroid binding have not been successful because of the extremely high nonspecific binding associated with these compounds (Rossignol, 1988; Pauron et al., 1989; Dong, 1993).
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KE DONG and JEFFREY G. SCOTT
Changes in membrane phospholipids have also been suggested to explain the mechanism of kdr resistance by Chialiang and Devonshire (1982). Evidence for the involvement of sodium channels in kdr-type resistance in the Ectiban-R strain of German cockroaches comes from cross-resistance studies. This strain of German cockroach is cross-resistant to sodium channel site 2 neurotoxins [i.e. aconitine (16-fold) and batrachotoxin (8.7-fold)], but not to other neurotoxins (Dong and Scott, 1991), suggesting that the sodium channels of kdr-type resistant German cockroaches are qualitatively different from those of the susceptible strain (Dong and Scott, 1991). Similar cross-resistance data were obtained in kdr house flies (Osborne and Smallcombe, 1983; Salgado et al., 1983; Bloomquist and Miller, 1986). Two insect sodium channel genes, para and DSC 1, have been cloned in Drosophila melanogaster by Loughney et al. (Loughney et al., 1989) and Salkoff et al. (1987a, b), respectively. Behavioral, electrophysiological, and genetic studies demonstrated that mutations of para alter the function of sodium channels (Ganetzky and Wu, 1986; Suzuki et al., 1971; Wu and Ganetzky, 1980; Suzuki and Wu, 1984; O'Dowd et al., 1987). The deduced para polypeptide shares a striking similarity with the ~-subunit of rat brain sodium channel I in overall structure and amino acid sequence (Loughney et al., 1989). These studies suggested the para gene product was the voltage-dependent sodium channel of the D. melanogaster nervous system. Salkoff et al. (1987a) isolated DSC 1 from D. melanogaster by screening a genomic library with a cDNA probe encoding the eel sodium channel. The deduced amino acid sequence of the gene revealed an organization virtually identical to the eel sodium channel protein (Salkoff et al., 1987b). However, the physiological role of the DSCI in insects has not been demonstrated. By using degenerate oligonucleotides (based on the amino acid sequence of an extracellular loop between domains IS5 and IS6, which is identical between para and a rat brain sodium channel gene I (Noda et al., 1986), but different from that of DSC 1 ) in polymerase chain reaction (PCR), a DNA segment of the para-homologue was isolated from house fly (Knipple et al., 1991) and from seven other insect species (Doyle and Knipple, 1991). To investigate if kdr-type resistance in the German cockroach is associated with a mutation(s) in the para-homologous sodium channel gene, we cloned and sequenced a German cockroach D N A fragment corresponding to the extracellular loop between domains IS5 and IS6 of the D. melanogaster para gene, using the PCR-based method of Knipple et al. (1991). We then used this PCR product as a probe in Southern analysis and detected a restriction fragment length polymorphism (RFLP) between the susceptible and kdr-type strains. Using RFLP analysis of different resistance genotypes (F2 and backcross individuals), we demonstrated that the kdr-type locus is identical (or tightly linked) to the para-homologous gene in the German
cockroach. This provides strong evidence that a mutation(s) in the sodium channel gene is associated with kdr-type resistance in German cockroach.
MATERIALS AND METHODS
Insects Two strains of the German cockroach (B. germanica) were used: CSMA, a pyrethroid susceptible strain (Scott and Matsumura, 1981), and Ectiban-R, a DDT and pyrethroid resistant strain selected from the VPIDLS strain (DDT-resistant) using permethrin (Scott et al., 1990). CSMA and Ectiban-R share similar genetic background (except for the kdr-type locus), as the result of repeated backcrossing and selection (Telford and Matsumura, 1970; Scott et al., 1990). Studies have shown that neural insensitivity to DDT and pyrethroids is the only resistance mechanism in Ectiban-R (Scott and Matsumura, 1981; Dong and Scott, 1991; Dong et al., 1993). This kdr-type resistance is monofactorial (Dong and Scott, 1991). The cockroach colonies were fed Purina dog chow and water ad lib. and maintained in cloth-covered plastic containers in which the rim was coated with Tree Tanglefoot (Tanglefoot Co., Grand Rapids, Mich.). The colonies were maintained at 28°C, 60% r.h. and a light:dark cycle of 14:10. Genetic analysis and bioassay Male CSMA and female Ectiban-R adults were mass crossed. Last instar female nymphs were isolated to generate virgin females for all crosses. F, adults were crossed and F2 male adults were separated into different kdr-type resistance genotypes using a residual bioassay as described by Dong and Scott (1991). 20~0 male adults were placed in a DDT-coated glass jar (2.4/~g DDT/cm 2, 250 cm 2 inner surface area). Individuals that were knocked down were collected and flash-frozen with liquid nitrogen and stored at -80°C. Knockdown was defined as being ataxic if prodded with forceps. Genomic DNA isolation For large scale isolation of the German cockroach genomic DNA, the method of Sambrook et al. (1989) was used. Genomic DNA was isolated from individual male adults by the following procedure. Individual cockroachs were homogenized in 100/~1 of homogenization buffer (0.1 mM Tris-HC1, pH 9.0, 0.1 mM EDTA and 1% SDS) in 1.5 ml microcentrifuge tubes with a pestle (Kontes, Vineland, N.J.) followed by addition of 400 #1 of the same buffer. After 20min incubation at 70°C, 70/~1 of 8 M potassium acetate was added and the mixture was left on ice for 30 min. The mixture was extracted with 400 #1 of phenol:chloroform:isoamyl alcohol (25:24:1) by gentle mixing for c. 5 min, followed by centrifugation for 3 min at 13,000 g at room temperature. The aqueous phase was collected and mixed with an equal volume of isopropanol and centrifuged for 2 min at 13,000g. The pellet was washed with 400/~1 of
kdr-TYPE RESISTANCE IN C O C K R O A C H E S
70% ethanol and dissolved in 50/~1 of TE buffer (10 mM Tris-HC1, pH 8.0 and 1 mM EDTA) with RNAase (5 #g/ml). PCR reactions and cloning of PCR products
Degenerate oligonucleotide primers were synthesized by the Analytical and Synthesis Facility of the Cornell Biotechnology Institute. The primers and PCR conditions are the same as described by Knipple et al. (1991). PCR products were extracted with phenol, precipitated with ethanol and dissolved in TE buffer. They were then digested with HindlII and XbaI. The digested PCR products were ligated with HindlII/XbaIdigested pBluescript SK ( - ) using T4 DNA ligase. The ligation mixture was used to transform Escherichia coli DH5~. Plasmids (pKDI-8) were isolated from white transformants on Luria-Bertani agar (Sambrook et al., 1989) plates supplemented with 40/~ g/ml of X-gal. The inserts were then sequenced. PCR and cloning of PCR products were repeated twice with identical results. DNA sequence analysis
Both strands of the inserts were sequenced twice using the Sequenase 2.0 kit (U.S. Biochemical, Cleveland, Ohio). Sequences were analyzed by the Blast program (Altschul et al., 1990). Southern analysis
Genomic DNA (7-8 #g) from individual cockroaches was digested with EcoR I prior to electrophoresis in 1% agarose gel and transferred to Immobilon N membrane (Millipore, Bedford, Mass.) using the method of Sambrook et al. (1989). Blots (11 × 14cm) were prehybridized and hybridized at 65°C for at least 2 and 16 h, respectively, in 25ml of prehybridization solution (6 × SSC, 1% SDS, 5 × Denhardt's solution, 100 ng/ml salmon sperm DNA, 50 mM Tris-HCl (pH 8.0) and
bp 500 - 298 - -
134 - -
F I G U R E 1. Analysis of PCR amplification products in a 4% agarose gel. The genomic D N A was used as the template for PCR. Products were analyzed in 3% NuSieve/l% SeaPlaque G T G agarose and stained with ethydium bromide. Size markers are shown in base pairs.
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2.5 mM EDTA). The blots were then washed in 0.5% SDS and 1 × SSC once for 15 min, and in 0.5% SDS and 0.4 x SSC for three times (each for 15 min at 65°C). Kodak OMAT XR5 films with intensifying screens were used for autoradiography. Probe was prepared by labeling the 525bp PvulI fragment of pKD4 with ~-32p-dCTP (6000 Ci/mmol, Amersham) using the Prime-IT II kit (Stratagene, La Jolla, Calif.). The probe was purified using a Sephadex G-50 (Sigma, St Louis, Mo) column before use.
DSCI (3-1AL) and para (ZS10.3) cDNAs from D. melanogaster were kindly provided by Dr L. Salkoff (Washington University School of Medicine) and Dr B. Ganetzky (University of Wisconsin), respectively.
RESULTS Cloning and sequencing a fragment of the parahomologous sodium channel gene from the German cockroach
To investigate the linkage of kdr-type resistance and sodium channel genes in the German cockroach, we first used the D. melanogaster DSC1 (3-1AL) and para (ZS10.3) cDNAs in Southern blotting of cockroach genomic DNAs. Neither cDNA probe showed significant hybridization, even at low stringency (45-55°C without formamide). We then used the PCR-based method developed by Knipple et al. (1991) and successfully cloned a fragment of the para-homologous sodium channel gene from the German cockroach. As shown in Fig. 1, a 120 bp DNA fragment was amplified from genomic DNAs of both susceptible and kdr-type strains. The amplification products were cloned into Bluescript SK ( - ) . Inserts (98 bp) from eight independent clones (pKD1-8, four from CSMA and four from Ectiban-R) were sequenced. The nucleotide sequences between the primers in all clones were identical (Fig. 2). However, the primer regions were heterogeneous, which is consistent with a previous report (Doyle and Knipple, 1991). As suggested by Doyle and Knipple (1991), this heterogeneity is not due to the corresponding sequence(s) present in the template DNA, but results from priming by imperfectly matched sequences in the primer mixtures during the later rounds of amplification. The deduced amino acid sequence of the amplified region (between the primers) in eight independent clones is identical to that of the American cockroach parahomologous gene, and is very similar to the para gene except for one amino acid residue (serine vs. threonine) difference (Fig. 2). Identification of a restriction fragment length polymorphism When the 525 bp PvulI fragment of pKD4 was used as the probe in hybridization with the EcoRI-digested genomic DNAs from CSMA and Ectiban-R, only a single band (3.7 kb for Ectiban-R and 3.0 kb for CSMA)
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KE D O N G and J E F F R E Y G. SCOTT
5' -AGT TTC GAT ACG TTT GGT TGG GCA TTC CTC TCG GCC TTC A G A CTG-3'
B. germanlca P. americana
D. melanogaster
NPNYGYTSFDITFGWAFLSAFRL MTQDFWE NPNYGYTSFDITIFGWAFLSAFRL MTQDFWE NPNYGYTSFDISIFGWAFLSAFRLMTQDFWE
F I G U R E 2. Comparison of the deduced amino acid sequences of the corresponding regions from German cockroach (B.
germanica), American cockroach (Periplaneta americana), and D. melanogaster. One amino acid residue difference is boxed, while the remaining amino acid residues are identical among the three species. The nucleotide sequences between the primers in all clones from German cockroach were identical, as shown above the amino acid sequences. Sequences for P. americana, and D. melanogaster are from Doyle and Knipple (1991) and Loughney et al. (1989), respectively. 002 and 903 were the two primers originally developed by Doyle and Knipple (1991).
was detected under high stringency hybridization down, but at different times (LTs0 = 16 h for SS and 34 h conditions (Fig. 3). When the same probe was used for RS). Because the time-mortality lines overlapped in a Southern blot of the EcoRI-digested F~ between SS and RS genotypes for c. 10 h (Fig. 4), the SS (CSMA x Ectiban-R) genomic DNA, two bands were individuals used in the linkage analysis (see below) were detected: one corresponded to the CSMA (3.0 kb) band, collected between 10 and 16h and the RS individuals the other to Ectiban-R (3.7 kb) band (Fig. 3). These were collected between 40 and 48 h after exposure to experiments demonstrated there was a R F L P of the DDT. The RR individuals were collected at the end of para-homologous gene between CSMA and Ectiban-R the bioassay (4 days). that could be used for the linkage analysis between the kdr-type locus and the para-homologous sodium Linkage analysis between the kdr-type gene and the channel gene. para-homologous sodium channel gene
Residual bioassays discriminating the susceptible, kdrtype and heterozygous cockroaches kdr-type resistance to D D T is an incompletely recessive trait (Fig. 4). As shown in Fig. 4, the three genotypes can be distinguished by a D D T residual bioassay: RR individuals were not knocked down during the period of the bioassay (4 days), whereas RS and SS were knocked
J kb 9--
An F2 population was established by crossing F~ (CSMA x Ectiban R) individuals. The F2 population was separated into three genotypes (RR, SS, and RS) using the D D T residual bioassay previously described. A total of 231 individuals (135 RR, 64 SS, 32 RS) were analyzed by genomic Southern blotting. As shown in Fig. 5 and Table 1, there was no segregation between the kdr-type locus and the para-homologous gene: all RR individuals had the 3.7 kb band, all SS individuals had the 3.0 kb band and all RS individuals had both bands. Backcross progeny (100 individuals) of F~ × Ectiban-R were also used for the linkage analysis. All 50 RS individuals had both the 3.0 and 3.7 kb bands. All 50 RR individuals had only the 3.7 kb band. Our data demonstrate that the German cockroach kdr-type gene and the para-homologous gene have extremely tight linkage ( < 0.2 cM) and are very likely the same gene (recombination occurs in both sexes of B. germanica; Ross and Cochran, 1975).
4--
DISCUSSION 3--
F I G U R E 3. Autoradiograph of a Southern blot of EcoRI-digested genomic DNAs from susceptible (CSMA), F~, and the kdr-type (Ectiban-R) German cockroaches. Conditions for gel electrophoresis, blotting, hybridization, and autoradiograph were described in Experimental Procedures. Size markers are shown in kilobases on the left.
In this paper we have shown that the kdr-type locus and the para-homologous locus have extremely tight linkage ( < 0 . 2 c M ) in Ectiban-R, a kdr-type German cockroach strain. In fact, we have not detected any recombination between the two loci in the F2 or backcross populations of 331 individuals. Therefore, it is very likely that the kdr-type mutation in Ectiban-R is within the para-homologous sodium channel gene. This pro-
kdr-TYPE RESISTANCE IN C O C K R O A C H E S
651
98 []
[]
--~ 85
.~ M
o
O
CSMA (SS)
[]
F l (RS)
,~. Ectiban-R (RR)
•
16
I l0
16
25
40
64
I ~ A 100
Time (h) F I G U R E 4. Time-mortality lines of susceptible (CSMA), F I (CSMA x Ectiban-R) and kdr-type (Ectiban-R) German cockroaches in a residual bioassay. Male adult cockroaches were placed in identical jars coated with 2.4/~g/cm 2 DDT. Numbers of knocked-down cockroaches were recorded at the times indicated. No mortality of the kdr-type German cockroaches (Ectiban-R) was observed during the period of the experiments (4 days). The bioassay was replicated three times, with a total of 150-200 cockroaches per time point.
vides strong evidence for the involvement of the parahomologous sodium channel in kdr-type resistance in German cockroaches and supports our previous finding that Ectiban-R is cross-resistant to several sodium channel neurotoxins, such as batrachotoxin (BTX) and aconitine (Dong and Scott, 1991). Although it is premature to predict the nature of the mutation(s) in the para-homologous gene that results in the kdr-type resistance, our previous results suggest that this mutation does not affect site 2 neurotoxin binding to the sodium channel (Dong et al., 1993).
RR
The amino acid sequence of the amplified region between the primers from para-homologous genes in German cockroaches is almost identical to the corresponding region from para, except for one amino acid residue difference (Fig. 2). This finding confirmed our expectation that the primers used in our PCR reactions amplify only the para-homologous sequences, but not the DSCl-homologous sequences. Interestingly, para is more similar to vertebrate sodium channel genes than to DSC1, suggesting that the para sodium channel gene may be more evolutionarily conserved among animals.
RS
SS
kl 9
F I G U R E 5. Autoradiograph of a Southern blot of EcoRl-digested genomic DNAs from individual F 2 cockroaches. F 2 cockroaches were first separated into susceptible (SS), kdr-type resitant (RR), and heterozygous (RS) according to the DDT residual bioassay (Fig. 4). Agarose gel electrophoresis, blotting, hybridization, and autoradiograph were the same as in Fig. 3. Size markers are shown in kilobases on the left. No band was detected using the Pvu II fragment from vector lacking insert.
652
KE DONG and JEFFREY G. SCOTT TABLE 1. RFLP analysis of DDT-susceptible (SS), resistant (RR) and heterozygous (RS) German cockroaches Genotype
3.0 kb band
3.7 kb band
3.0 and 3.7 kb bands
F~ SS RS RR
64 0 0
0 0 135
0 32 0
BC RS RR
0 0
0 50
50 0
Individuals were Fz of CSMA (SS) × Ectiban-R (RR) or backcross (BC, (CSMA × EctibanR) x Ectiban-R) separated by the DDT residual bioassay as described in Materials and Methods.
It was not known which type of sodium channel, if either, was involved in the kdr-type resistance in German cockroach. Our approach of using a cloned fragment of the p a r a - h o m o l o g o u s gene (instead of D S C l - h o m o l o gous gene) in the linkage analysis has proven to be successful, suggesting it is the p a r a - h o m o l o g o u s sodium channel gene that is associated with kdr-type resistance in the German cockroach. Our findings are consistent with recent results from kdr house flies (Williamson et al., 1993) and pyrethroid resistant tobacco budworm (Taylor et al., 1993). Using a similar R F L P analysis, Williamson et al. (1993) reported that kdr or super-kdr and the p a r a - h o m o l o g o u s sodium channel gene are linked in the house fly. Using another R F L P analysis scheme, Taylor et al. (1993) showed that in the tobacco budworm strain RR, which has multiple pyrethroid resistance mechanisms, one of the mechanisms was linked to the p a r a - h o m o l o g o u s sodium channel gene. Amichot et al. have recently characterized a kdr-like resistance mechanism in D. melanogaster (Amichot et al., 1992). They showed that the resistant factor was associated with the second chromosome which bears the D S C 1 gene. Sequence analysis of the D S C 1 genes from both susceptible and kdr-like strains revealed one nonsilent mutation (D to N, in the region between segments $5 and $6 of the third domain). The authors suggested that this single substitution may be responsible for the kdrlike mechanism. However, they did not provide any experimental evidence to demonstrate that D S C 1 is identical or tightly linked to their kdr-like gene. Moreover, the eel and rat brain sodium channel genes have N (instead of D) in the same position and, at least in the case of rat, the species is not insensitive to pyrethroids. In addition, this kdr-like strain was only 2-fold resistant to deltamethrin. In comparison, kdr-type German cockroaches and kdr house flies have 10-fold or more resistance to deltamethrin (Farnham et al., 1987; Dong and Scott, 1993). nap ,s a D. melanogaster mutant that has a reduced sodium channel density in the nervous system (Jackson
et al., 1984), confers resistance to pyrethroids (Kasebekar and Hall, 1988; Bloomquist et al., 1989). The 3-fold deltamethrin resistance in nap '~ was correlated with reduced sensitivity of the peripheral nervous system and a 2-fold reduction in sodium channel density (Bloomquist et al., 1989). However, the cross-resistance in the nap t~ strain could be overcome with the cytochrome P-450 monooxygenase inhibitor piperonyl butoxide (Kasebekar and Hall, 1988). A m o n g 6 para mutant strains that have been tested for cross-resistance to pyrethroids, the para tsl and parat~4 mutants were resistant to fenvalerate, while para ,s2 was more sensitive to fenvalerate (Hall and Kasbekar, 1990), and para ts3, para ~ls and para sv42 showed no resistance to esfenvalerate (Dong, 1993). Loughney et al. (1989) suggested the para mutations are within introns and they result in the underproduction of sodium channel polypeptides, rather than the production of structurally aberrant channels. Therefore, reduced sodium channel density may also be responsible for pyrethroid resistance in some of the para tS mutations. It is not likely that the kdr-type resistance is related to these nap ts or para mutations, because no reduced sodium channel density was observed in the kdr-type German cockroach (Dong and Scott, 1991). Our p a r a - h o m o l o g o u s probe detected a single fragment in EcoRI-digested genomic D N A s from both CSMA and Ectiban-R in high stringency hybridization. Now it will be possible to clone the entire parahomologous genes from both strains and compare their sequences. The mutation(s) in the Ectiban-R p a r a - h o m o l o g o u s gene could then be identified. More precise molecular analysis based on sequence differences or functional assays could then be used to address which mutation(s) is responsible for the kdr-type resistance. Identification of the mutation(s) responsible for kdrtype resistance will aid the development of a new diagnostic technique to monitor kdr-type resistant individuals in the field. Because this new tool will be able to discriminate different genotypes for the resistance, it would be more accurate and sensitive than conventional methods such as diagnostic doses.
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Miller T. A., Kennedy J. M. and Collins C. (1979) CNS insensitivity to pyrethroids in the resistant kdr strain of house flies. Pestic. Biochem. Physiol. 12, 224-230. Noda M., Ikeda T., Kayano T., Suzuki H., Takeshima H., Kurasaki M., Takahashi, H. and Numa, S. (1986) Existence of distinct sodium channel messenger RNAs in rat brain. Nature (London) 320, 188-192. O'Dowd D. K., Germeraad S. and Aldrich R. W. (1987) Expression of sodium currents in embryonic Drosophila neurons: differential reduction by alleles of the para locus. Abstr. soc. Neurosci. 13, 577. Osborne M. P. and Hart R. J. (1979) Neurophysiological studies of the effects of permethrin upon pyrethroid resistant (kdr) and susceptible strains of dipteran larvae. Pestic. Sci. 10, 4074 13. Osborne M. P. and Smallcombe A. (1983) Site of action of pyrethroid insecticides in neuronal membranes as revealed by the kdr resistance factor. In IUPAC Pesticide Chemistry-Human Welfare andEnvironment pp. 103-107, Pergamon Press, Oxford.
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Pauron D., Barhanin J., Amichot M., Pralavorio M., Berge J.-B. and Lazdunski M. (1989) Pyrethroid receptor in the insect Na + channel: alteration of its properties in pyrethroid-resistant flies. Biochemistry 28, 1673-1677. Ross M. H. and Cochran D. G. (1975) The German cockroach, Blattella germanica. In Handbook of Genetics , Vol. 3, pp. 35~2, Plenum Press, New York. Rossignol D. P. (1988) Reduction in number of nerve membrane sodium channels in pyrethroid resistant house flies. Pestic. Biochem. Physiol. 32, 146-152. Salgado V. L., Irving S. N. and Miller T. A. (1983) Depolarization of motor nerve terminals by pyrethroids in susceptible and kdr-resistant house flies. Pestic. Biochem. Physiol. 20, 100 114. Salkoff L., Butler A., Scavarda N. and Wei A. (1987a) Nucleotide sequence of the putative sodium channel gene from Drosophila: the four homologous domains. Nucl. acids Res. 15, 85694572. Salkoff L., Butler A., Wei A., Scavarda N., Giffen K., Ifune C., Goodman R. and Mandel G. (1987b) Genomic organization and deduced amino acid sequence of a putative sodium channel gene in Drosophila. Science 237, 744-749. Sambrook J., Fritsch E. F. and Maniatis T. (1989) Molecular Cloning: a Laboratory Manual (Edited by Nolan C.), 2nd Edn. Cold Spring Harbor Laboratory Press, New York. Sattelle D. B., Leech C. A., Lummis S. C. R., Harrison B. J., Robinson H. P. C., Moores G. D. and Devonshire A. L. (1988) Ion channel properties of insects susceptible and resistant to insecticides. In Neurotox '88: Molecular Basis of Drug and Pesticide Action (Edited by Lunt G. G.) pp. 563 582. Elsevier, New York. Scott J. G. and Matsumura F. (1983) Evidence for two types of toxic actions of pyrethroids on susceptible and DDt-resistant German cockroaches. Pestic. Biochem. Physiol. 19, 141-150. Scott J. G. and Matsumura F. (1981) Characteristics of a DDT-induced case of cross-resistance to permethrin in Blattella germanica. Pestic. Biochem. Physiol. 16, 21-27. Scott J. G., Cochran D. G. and Siegfried B. D. (1990) Insecticide toxicity, synergism and resistance in German cockroach (Dictyoptera: Blattellidae), J. econ. Ent. 83, 1698 1703. Shono T. (1985) Pyrethroid resistance: importance of the kdr-type mechanism. J. pestic. Sci. 10, 141-146. Soderlund D. M., Grubs R. E. and Adams P. M. (1989) Binding of [3H]batrahotoxinin A-20-ct-benzoate to a high affinity site associated with house fly head membranes. Comp. Biochem. Physiol. 94C, 255 260. Suzuki N. and Wu C.-F. (1984) Altered sensitivity to sodium channel-specific neurotoxins in cultured neurons from temperaturesensitive paralytic mutants of Drosophila. J. Neurogenet. l, 225~38. Suzuki O. T., Grigliatti T. and Williamson R. (1971) Temperature-sensitive mutants in Drosophila melanogaster, VII. A mutation (para t~) causing reversible adult paralysis. Proc. natn. Aead. Sci. U.S.A. 68, 890-893. Taylor M. F. J., Heckel D. G., Brown T. M. Kreitman M. E. and Black B. (1993) Linkage of pyrethroid insecticide resistance to a sodium channel locus in the tobacco budworm. Insect Biochem. Molec. Biol. 23, 763-775. Telford J. N. and Matsumura F. (1970) Dieldrin binding in subcellular nerve components of cockroaches. An electron microscopic and autoradiographic study. J. econ. Ent. 63, 795-800. Tsukamoto M., Narahashi T. and Yamasaki T. (1965) Genetic control of low nerve sensitivity to DDT in insecticide-resistant houseflies. Botyu-Kagaku 31, 1-14. Umeda K., Yano T. and Hirano M. (1988) Pyrethroid-resistance mechanism in German cockroach, Blattella germanica (Orthoptera: Blattellidae). Appl. ent. Zool. 23, 373-380. Weber W. J. (1984) Fleas, Ticks and Cockroaches Disease Transmitters, 42 pp. Thomson, Fresno. Calif. Williamson M. S., Denholm I., Bell C. A. and Devonshire A. L. (1993) Knockdown resistance (kdr) to DDT and pyrethrid insecticides
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maps to a sodium channel gene locus in the housefly (Musca dornestica). Molec. Gen. Genetic. 240, 17-22. Wu C.-F. and Ganetzky B. (1980) Genetic alteration of nerve membrane excitability in temperature-sensitive paralytic mutants of Drosophila rnelanogaster. Nature (Lond.) 286, 814-816.
Acknowledgements--We thank following people: D. Nero, S.-Y. He, D. C. Knipple and T. Tomita for valuable suggestions and discussion, Mike Martin and Li Zhang for technical assistance, and B. Ganetzky and L. Salkoff for cDNA clones. This research was supported by a grant from the Johnson Wax Foundation and Hatch Project 139414.
~
Pergamon
0965-1748(94)E0004-Z
Copyright © 1994 Elsevier ScienceLtd Printed in Great Britain. All rights reserved 0965-1748/94 $7.00 + 0.00
kdr-Type Resistance and the para-Homologous Sodium Channel Gene in German Cockroaches (Blattella germanica) Linkage of
KE DONG,t JEFFREY G. SCOTT~§ Received 27 September 1993; accepted 6 January 1994 Pyrethroids are an important class of insecticides for controlling insect pests, including the German cockroach. Unfortunately, many insects have developed resistance to pyrethroids. One of the most important mechanisms of resistance is kdr (knockdown resistance) which is characterized by neural insensitivity to pyrethroids and DDT. To investigate whether the voltage-dependent sodium channel is involved in kdr-type resistance in the German cockroach, we isolated a 120 bp DNA fragment of the para-homologous sodium channel gene from German cockroaches. Using this fragment as a probe, we identified a restriction fragment length polymorphism (RFLP) of the para-homologous sodium channel gene between susceptible and kdr-type resistant German cockroaches. RFLP analysis of F2 and backcross cockroach populations (total of 331 individuals) showed that all homozygous resistant individuals had a 3.7 kb EcoRI fragment, all homozygous susceptible individuals had a 3.0 kb EcoRI fragment, and all heterozygous individuals had both 3.7 and 3.0 kb fragments. No recombination was detected between the kdr-type resistance locus and the para-homologous sodium channel gene. This suggests that the kdr-type resistance locus and para-homologous sodium channel gene are identical or tightly linked ( < 0 . 2 c M ) in German cockroaches. Our results provide strong evidence that modification of para-homologous sodium channels is associated with kdr-type resistance. Pyrethroid resistance kdr para-homologous sodium channel gene DDT resistance
INTRODUCTION Pyrethroids are a widely used class of insecticides worldwide. Knockdown resistance (kdr) is an important mechanism by which insects develop resistance to pyrethroids and DDT (Shono, 1985). kdr was first discovered in the house fly (Musca domestica) and is characterized by neural insensitivity to pyrethroids and DDT. kdr-type resistance is widespread among many insect pests including German cockroach, Blattella germanica (Scott and Matsumura, 1981, 1983; Umeda et al., 1988), which carries human pathogens and is also the second leading cause of household allergies in the United States (Weber, 1984). The widespread existence of kdr-type resistance in insects presents a serious problem in pest management. Although kdr-type resistance in insects has been studied extensively, the molecular mechanism of this resistance remains largely unclear. Neurophysiological studies tPresent address: Department of Entomology, Agricultural Science Center, University of Kentucky, Lexington, KY 40546, U.S.A. :~Department of Entomology, Comstock Hall, Cornell University, Ithaca, NY 14853-0999, U.S.A. §Author for correspondence.
have shown that the nervous system of kdr or kdr-type insects are less sensitive to pyrethroids and DDT (Tsukamoto et al., 1965; Miller et al., 1979; Osborne and Hart, 1979; DeVries and Georghiou, 1981; Scott and Matsumura, 1981; Ahn et al., 1987; Umeda et al., 1988). Reduced sodium channel binding sites for saxitoxin in kdr house flies was reported by Rossignol (1988). However, others have failed to show any difference in sodium channel density between susceptible and kdr house flies (Grubs et al., 1988; Sattelle et al., 1988; Pauron et al., 1989) or kdr-type German cockroaches (Dong and Scott, 1991; Dong et al., 1993). Based on the enhancement of [3H]batrachotoxinin A-20-a-benzoate ([3H]BTX-B) binding by deltamethrin (a pyrethroid) in membranes from susceptible (but not resistant) strains (Pauron et al., 1989), it was suggested that a modification of the pyrethroid binding site in kdr house flies might be responsible for the resistance. However, we and others observed no enhancement of [3H]BTX-B binding by deltamethrin (Soderlund et al., 1989; Dong et al., 1993). Direct approaches using pyrethroid binding have not been successful because of the extremely high nonspecific binding associated with these compounds (Rossignol, 1988; Pauron et al., 1989; Dong, 1993).
647
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KE DONG and JEFFREY G. SCOTT
Changes in membrane phospholipids have also been suggested to explain the mechanism of kdr resistance by Chialiang and Devonshire (1982). Evidence for the involvement of sodium channels in kdr-type resistance in the Ectiban-R strain of German cockroaches comes from cross-resistance studies. This strain of German cockroach is cross-resistant to sodium channel site 2 neurotoxins [i.e. aconitine (16-fold) and batrachotoxin (8.7-fold)], but not to other neurotoxins (Dong and Scott, 1991), suggesting that the sodium channels of kdr-type resistant German cockroaches are qualitatively different from those of the susceptible strain (Dong and Scott, 1991). Similar cross-resistance data were obtained in kdr house flies (Osborne and Smallcombe, 1983; Salgado et al., 1983; Bloomquist and Miller, 1986). Two insect sodium channel genes, para and DSC 1, have been cloned in Drosophila melanogaster by Loughney et al. (Loughney et al., 1989) and Salkoff et al. (1987a, b), respectively. Behavioral, electrophysiological, and genetic studies demonstrated that mutations of para alter the function of sodium channels (Ganetzky and Wu, 1986; Suzuki et al., 1971; Wu and Ganetzky, 1980; Suzuki and Wu, 1984; O'Dowd et al., 1987). The deduced para polypeptide shares a striking similarity with the ~-subunit of rat brain sodium channel I in overall structure and amino acid sequence (Loughney et al., 1989). These studies suggested the para gene product was the voltage-dependent sodium channel of the D. melanogaster nervous system. Salkoff et al. (1987a) isolated DSC 1 from D. melanogaster by screening a genomic library with a cDNA probe encoding the eel sodium channel. The deduced amino acid sequence of the gene revealed an organization virtually identical to the eel sodium channel protein (Salkoff et al., 1987b). However, the physiological role of the DSCI in insects has not been demonstrated. By using degenerate oligonucleotides (based on the amino acid sequence of an extracellular loop between domains IS5 and IS6, which is identical between para and a rat brain sodium channel gene I (Noda et al., 1986), but different from that of DSC 1 ) in polymerase chain reaction (PCR), a DNA segment of the para-homologue was isolated from house fly (Knipple et al., 1991) and from seven other insect species (Doyle and Knipple, 1991). To investigate if kdr-type resistance in the German cockroach is associated with a mutation(s) in the para-homologous sodium channel gene, we cloned and sequenced a German cockroach D N A fragment corresponding to the extracellular loop between domains IS5 and IS6 of the D. melanogaster para gene, using the PCR-based method of Knipple et al. (1991). We then used this PCR product as a probe in Southern analysis and detected a restriction fragment length polymorphism (RFLP) between the susceptible and kdr-type strains. Using RFLP analysis of different resistance genotypes (F2 and backcross individuals), we demonstrated that the kdr-type locus is identical (or tightly linked) to the para-homologous gene in the German
cockroach. This provides strong evidence that a mutation(s) in the sodium channel gene is associated with kdr-type resistance in German cockroach.
MATERIALS AND METHODS
Insects Two strains of the German cockroach (B. germanica) were used: CSMA, a pyrethroid susceptible strain (Scott and Matsumura, 1981), and Ectiban-R, a DDT and pyrethroid resistant strain selected from the VPIDLS strain (DDT-resistant) using permethrin (Scott et al., 1990). CSMA and Ectiban-R share similar genetic background (except for the kdr-type locus), as the result of repeated backcrossing and selection (Telford and Matsumura, 1970; Scott et al., 1990). Studies have shown that neural insensitivity to DDT and pyrethroids is the only resistance mechanism in Ectiban-R (Scott and Matsumura, 1981; Dong and Scott, 1991; Dong et al., 1993). This kdr-type resistance is monofactorial (Dong and Scott, 1991). The cockroach colonies were fed Purina dog chow and water ad lib. and maintained in cloth-covered plastic containers in which the rim was coated with Tree Tanglefoot (Tanglefoot Co., Grand Rapids, Mich.). The colonies were maintained at 28°C, 60% r.h. and a light:dark cycle of 14:10. Genetic analysis and bioassay Male CSMA and female Ectiban-R adults were mass crossed. Last instar female nymphs were isolated to generate virgin females for all crosses. F, adults were crossed and F2 male adults were separated into different kdr-type resistance genotypes using a residual bioassay as described by Dong and Scott (1991). 20~0 male adults were placed in a DDT-coated glass jar (2.4/~g DDT/cm 2, 250 cm 2 inner surface area). Individuals that were knocked down were collected and flash-frozen with liquid nitrogen and stored at -80°C. Knockdown was defined as being ataxic if prodded with forceps. Genomic DNA isolation For large scale isolation of the German cockroach genomic DNA, the method of Sambrook et al. (1989) was used. Genomic DNA was isolated from individual male adults by the following procedure. Individual cockroachs were homogenized in 100/~1 of homogenization buffer (0.1 mM Tris-HC1, pH 9.0, 0.1 mM EDTA and 1% SDS) in 1.5 ml microcentrifuge tubes with a pestle (Kontes, Vineland, N.J.) followed by addition of 400 #1 of the same buffer. After 20min incubation at 70°C, 70/~1 of 8 M potassium acetate was added and the mixture was left on ice for 30 min. The mixture was extracted with 400 #1 of phenol:chloroform:isoamyl alcohol (25:24:1) by gentle mixing for c. 5 min, followed by centrifugation for 3 min at 13,000 g at room temperature. The aqueous phase was collected and mixed with an equal volume of isopropanol and centrifuged for 2 min at 13,000g. The pellet was washed with 400/~1 of
kdr-TYPE RESISTANCE IN C O C K R O A C H E S
70% ethanol and dissolved in 50/~1 of TE buffer (10 mM Tris-HC1, pH 8.0 and 1 mM EDTA) with RNAase (5 #g/ml). PCR reactions and cloning of PCR products
Degenerate oligonucleotide primers were synthesized by the Analytical and Synthesis Facility of the Cornell Biotechnology Institute. The primers and PCR conditions are the same as described by Knipple et al. (1991). PCR products were extracted with phenol, precipitated with ethanol and dissolved in TE buffer. They were then digested with HindlII and XbaI. The digested PCR products were ligated with HindlII/XbaIdigested pBluescript SK ( - ) using T4 DNA ligase. The ligation mixture was used to transform Escherichia coli DH5~. Plasmids (pKDI-8) were isolated from white transformants on Luria-Bertani agar (Sambrook et al., 1989) plates supplemented with 40/~ g/ml of X-gal. The inserts were then sequenced. PCR and cloning of PCR products were repeated twice with identical results. DNA sequence analysis
Both strands of the inserts were sequenced twice using the Sequenase 2.0 kit (U.S. Biochemical, Cleveland, Ohio). Sequences were analyzed by the Blast program (Altschul et al., 1990). Southern analysis
Genomic DNA (7-8 #g) from individual cockroaches was digested with EcoR I prior to electrophoresis in 1% agarose gel and transferred to Immobilon N membrane (Millipore, Bedford, Mass.) using the method of Sambrook et al. (1989). Blots (11 × 14cm) were prehybridized and hybridized at 65°C for at least 2 and 16 h, respectively, in 25ml of prehybridization solution (6 × SSC, 1% SDS, 5 × Denhardt's solution, 100 ng/ml salmon sperm DNA, 50 mM Tris-HCl (pH 8.0) and
bp 500 - 298 - -
134 - -
F I G U R E 1. Analysis of PCR amplification products in a 4% agarose gel. The genomic D N A was used as the template for PCR. Products were analyzed in 3% NuSieve/l% SeaPlaque G T G agarose and stained with ethydium bromide. Size markers are shown in base pairs.
649
2.5 mM EDTA). The blots were then washed in 0.5% SDS and 1 × SSC once for 15 min, and in 0.5% SDS and 0.4 x SSC for three times (each for 15 min at 65°C). Kodak OMAT XR5 films with intensifying screens were used for autoradiography. Probe was prepared by labeling the 525bp PvulI fragment of pKD4 with ~-32p-dCTP (6000 Ci/mmol, Amersham) using the Prime-IT II kit (Stratagene, La Jolla, Calif.). The probe was purified using a Sephadex G-50 (Sigma, St Louis, Mo) column before use.
DSCI (3-1AL) and para (ZS10.3) cDNAs from D. melanogaster were kindly provided by Dr L. Salkoff (Washington University School of Medicine) and Dr B. Ganetzky (University of Wisconsin), respectively.
RESULTS Cloning and sequencing a fragment of the parahomologous sodium channel gene from the German cockroach
To investigate the linkage of kdr-type resistance and sodium channel genes in the German cockroach, we first used the D. melanogaster DSC1 (3-1AL) and para (ZS10.3) cDNAs in Southern blotting of cockroach genomic DNAs. Neither cDNA probe showed significant hybridization, even at low stringency (45-55°C without formamide). We then used the PCR-based method developed by Knipple et al. (1991) and successfully cloned a fragment of the para-homologous sodium channel gene from the German cockroach. As shown in Fig. 1, a 120 bp DNA fragment was amplified from genomic DNAs of both susceptible and kdr-type strains. The amplification products were cloned into Bluescript SK ( - ) . Inserts (98 bp) from eight independent clones (pKD1-8, four from CSMA and four from Ectiban-R) were sequenced. The nucleotide sequences between the primers in all clones were identical (Fig. 2). However, the primer regions were heterogeneous, which is consistent with a previous report (Doyle and Knipple, 1991). As suggested by Doyle and Knipple (1991), this heterogeneity is not due to the corresponding sequence(s) present in the template DNA, but results from priming by imperfectly matched sequences in the primer mixtures during the later rounds of amplification. The deduced amino acid sequence of the amplified region (between the primers) in eight independent clones is identical to that of the American cockroach parahomologous gene, and is very similar to the para gene except for one amino acid residue (serine vs. threonine) difference (Fig. 2). Identification of a restriction fragment length polymorphism When the 525 bp PvulI fragment of pKD4 was used as the probe in hybridization with the EcoRI-digested genomic DNAs from CSMA and Ectiban-R, only a single band (3.7 kb for Ectiban-R and 3.0 kb for CSMA)
650
KE D O N G and J E F F R E Y G. SCOTT
5' -AGT TTC GAT ACG TTT GGT TGG GCA TTC CTC TCG GCC TTC A G A CTG-3'
B. germanlca P. americana
D. melanogaster
NPNYGYTSFDITFGWAFLSAFRL MTQDFWE NPNYGYTSFDITIFGWAFLSAFRL MTQDFWE NPNYGYTSFDISIFGWAFLSAFRLMTQDFWE
F I G U R E 2. Comparison of the deduced amino acid sequences of the corresponding regions from German cockroach (B.
germanica), American cockroach (Periplaneta americana), and D. melanogaster. One amino acid residue difference is boxed, while the remaining amino acid residues are identical among the three species. The nucleotide sequences between the primers in all clones from German cockroach were identical, as shown above the amino acid sequences. Sequences for P. americana, and D. melanogaster are from Doyle and Knipple (1991) and Loughney et al. (1989), respectively. 002 and 903 were the two primers originally developed by Doyle and Knipple (1991).
was detected under high stringency hybridization down, but at different times (LTs0 = 16 h for SS and 34 h conditions (Fig. 3). When the same probe was used for RS). Because the time-mortality lines overlapped in a Southern blot of the EcoRI-digested F~ between SS and RS genotypes for c. 10 h (Fig. 4), the SS (CSMA x Ectiban-R) genomic DNA, two bands were individuals used in the linkage analysis (see below) were detected: one corresponded to the CSMA (3.0 kb) band, collected between 10 and 16h and the RS individuals the other to Ectiban-R (3.7 kb) band (Fig. 3). These were collected between 40 and 48 h after exposure to experiments demonstrated there was a R F L P of the DDT. The RR individuals were collected at the end of para-homologous gene between CSMA and Ectiban-R the bioassay (4 days). that could be used for the linkage analysis between the kdr-type locus and the para-homologous sodium Linkage analysis between the kdr-type gene and the channel gene. para-homologous sodium channel gene
Residual bioassays discriminating the susceptible, kdrtype and heterozygous cockroaches kdr-type resistance to D D T is an incompletely recessive trait (Fig. 4). As shown in Fig. 4, the three genotypes can be distinguished by a D D T residual bioassay: RR individuals were not knocked down during the period of the bioassay (4 days), whereas RS and SS were knocked
J kb 9--
An F2 population was established by crossing F~ (CSMA x Ectiban R) individuals. The F2 population was separated into three genotypes (RR, SS, and RS) using the D D T residual bioassay previously described. A total of 231 individuals (135 RR, 64 SS, 32 RS) were analyzed by genomic Southern blotting. As shown in Fig. 5 and Table 1, there was no segregation between the kdr-type locus and the para-homologous gene: all RR individuals had the 3.7 kb band, all SS individuals had the 3.0 kb band and all RS individuals had both bands. Backcross progeny (100 individuals) of F~ × Ectiban-R were also used for the linkage analysis. All 50 RS individuals had both the 3.0 and 3.7 kb bands. All 50 RR individuals had only the 3.7 kb band. Our data demonstrate that the German cockroach kdr-type gene and the para-homologous gene have extremely tight linkage ( < 0.2 cM) and are very likely the same gene (recombination occurs in both sexes of B. germanica; Ross and Cochran, 1975).
4--
DISCUSSION 3--
F I G U R E 3. Autoradiograph of a Southern blot of EcoRI-digested genomic DNAs from susceptible (CSMA), F~, and the kdr-type (Ectiban-R) German cockroaches. Conditions for gel electrophoresis, blotting, hybridization, and autoradiograph were described in Experimental Procedures. Size markers are shown in kilobases on the left.
In this paper we have shown that the kdr-type locus and the para-homologous locus have extremely tight linkage ( < 0 . 2 c M ) in Ectiban-R, a kdr-type German cockroach strain. In fact, we have not detected any recombination between the two loci in the F2 or backcross populations of 331 individuals. Therefore, it is very likely that the kdr-type mutation in Ectiban-R is within the para-homologous sodium channel gene. This pro-
kdr-TYPE RESISTANCE IN C O C K R O A C H E S
651
98 []
[]
--~ 85
.~ M
o
O
CSMA (SS)
[]
F l (RS)
,~. Ectiban-R (RR)
•
16
I l0
16
25
40
64
I ~ A 100
Time (h) F I G U R E 4. Time-mortality lines of susceptible (CSMA), F I (CSMA x Ectiban-R) and kdr-type (Ectiban-R) German cockroaches in a residual bioassay. Male adult cockroaches were placed in identical jars coated with 2.4/~g/cm 2 DDT. Numbers of knocked-down cockroaches were recorded at the times indicated. No mortality of the kdr-type German cockroaches (Ectiban-R) was observed during the period of the experiments (4 days). The bioassay was replicated three times, with a total of 150-200 cockroaches per time point.
vides strong evidence for the involvement of the parahomologous sodium channel in kdr-type resistance in German cockroaches and supports our previous finding that Ectiban-R is cross-resistant to several sodium channel neurotoxins, such as batrachotoxin (BTX) and aconitine (Dong and Scott, 1991). Although it is premature to predict the nature of the mutation(s) in the para-homologous gene that results in the kdr-type resistance, our previous results suggest that this mutation does not affect site 2 neurotoxin binding to the sodium channel (Dong et al., 1993).
RR
The amino acid sequence of the amplified region between the primers from para-homologous genes in German cockroaches is almost identical to the corresponding region from para, except for one amino acid residue difference (Fig. 2). This finding confirmed our expectation that the primers used in our PCR reactions amplify only the para-homologous sequences, but not the DSCl-homologous sequences. Interestingly, para is more similar to vertebrate sodium channel genes than to DSC1, suggesting that the para sodium channel gene may be more evolutionarily conserved among animals.
RS
SS
kl 9
F I G U R E 5. Autoradiograph of a Southern blot of EcoRl-digested genomic DNAs from individual F 2 cockroaches. F 2 cockroaches were first separated into susceptible (SS), kdr-type resitant (RR), and heterozygous (RS) according to the DDT residual bioassay (Fig. 4). Agarose gel electrophoresis, blotting, hybridization, and autoradiograph were the same as in Fig. 3. Size markers are shown in kilobases on the left. No band was detected using the Pvu II fragment from vector lacking insert.
652
KE DONG and JEFFREY G. SCOTT TABLE 1. RFLP analysis of DDT-susceptible (SS), resistant (RR) and heterozygous (RS) German cockroaches Genotype
3.0 kb band
3.7 kb band
3.0 and 3.7 kb bands
F~ SS RS RR
64 0 0
0 0 135
0 32 0
BC RS RR
0 0
0 50
50 0
Individuals were Fz of CSMA (SS) × Ectiban-R (RR) or backcross (BC, (CSMA × EctibanR) x Ectiban-R) separated by the DDT residual bioassay as described in Materials and Methods.
It was not known which type of sodium channel, if either, was involved in the kdr-type resistance in German cockroach. Our approach of using a cloned fragment of the p a r a - h o m o l o g o u s gene (instead of D S C l - h o m o l o gous gene) in the linkage analysis has proven to be successful, suggesting it is the p a r a - h o m o l o g o u s sodium channel gene that is associated with kdr-type resistance in the German cockroach. Our findings are consistent with recent results from kdr house flies (Williamson et al., 1993) and pyrethroid resistant tobacco budworm (Taylor et al., 1993). Using a similar R F L P analysis, Williamson et al. (1993) reported that kdr or super-kdr and the p a r a - h o m o l o g o u s sodium channel gene are linked in the house fly. Using another R F L P analysis scheme, Taylor et al. (1993) showed that in the tobacco budworm strain RR, which has multiple pyrethroid resistance mechanisms, one of the mechanisms was linked to the p a r a - h o m o l o g o u s sodium channel gene. Amichot et al. have recently characterized a kdr-like resistance mechanism in D. melanogaster (Amichot et al., 1992). They showed that the resistant factor was associated with the second chromosome which bears the D S C 1 gene. Sequence analysis of the D S C 1 genes from both susceptible and kdr-like strains revealed one nonsilent mutation (D to N, in the region between segments $5 and $6 of the third domain). The authors suggested that this single substitution may be responsible for the kdrlike mechanism. However, they did not provide any experimental evidence to demonstrate that D S C 1 is identical or tightly linked to their kdr-like gene. Moreover, the eel and rat brain sodium channel genes have N (instead of D) in the same position and, at least in the case of rat, the species is not insensitive to pyrethroids. In addition, this kdr-like strain was only 2-fold resistant to deltamethrin. In comparison, kdr-type German cockroaches and kdr house flies have 10-fold or more resistance to deltamethrin (Farnham et al., 1987; Dong and Scott, 1993). nap ,s a D. melanogaster mutant that has a reduced sodium channel density in the nervous system (Jackson
et al., 1984), confers resistance to pyrethroids (Kasebekar and Hall, 1988; Bloomquist et al., 1989). The 3-fold deltamethrin resistance in nap '~ was correlated with reduced sensitivity of the peripheral nervous system and a 2-fold reduction in sodium channel density (Bloomquist et al., 1989). However, the cross-resistance in the nap t~ strain could be overcome with the cytochrome P-450 monooxygenase inhibitor piperonyl butoxide (Kasebekar and Hall, 1988). A m o n g 6 para mutant strains that have been tested for cross-resistance to pyrethroids, the para tsl and parat~4 mutants were resistant to fenvalerate, while para ,s2 was more sensitive to fenvalerate (Hall and Kasbekar, 1990), and para ts3, para ~ls and para sv42 showed no resistance to esfenvalerate (Dong, 1993). Loughney et al. (1989) suggested the para mutations are within introns and they result in the underproduction of sodium channel polypeptides, rather than the production of structurally aberrant channels. Therefore, reduced sodium channel density may also be responsible for pyrethroid resistance in some of the para tS mutations. It is not likely that the kdr-type resistance is related to these nap ts or para mutations, because no reduced sodium channel density was observed in the kdr-type German cockroach (Dong and Scott, 1991). Our p a r a - h o m o l o g o u s probe detected a single fragment in EcoRI-digested genomic D N A s from both CSMA and Ectiban-R in high stringency hybridization. Now it will be possible to clone the entire parahomologous genes from both strains and compare their sequences. The mutation(s) in the Ectiban-R p a r a - h o m o l o g o u s gene could then be identified. More precise molecular analysis based on sequence differences or functional assays could then be used to address which mutation(s) is responsible for the kdr-type resistance. Identification of the mutation(s) responsible for kdrtype resistance will aid the development of a new diagnostic technique to monitor kdr-type resistant individuals in the field. Because this new tool will be able to discriminate different genotypes for the resistance, it would be more accurate and sensitive than conventional methods such as diagnostic doses.
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Acknowledgements--We thank following people: D. Nero, S.-Y. He, D. C. Knipple and T. Tomita for valuable suggestions and discussion, Mike Martin and Li Zhang for technical assistance, and B. Ganetzky and L. Salkoff for cDNA clones. This research was supported by a grant from the Johnson Wax Foundation and Hatch Project 139414.