Analysis of Sodium Channel Gene Sequences in Pyrethroid-Resistant

Chapter 6

Analysis of Sodium Channel Gene Sequences in Pyrethroid-Resistant Houseflies

Downloaded by UNIV MASSACHUSETTS AMHERST on October 8, 2012 | http://pubs.acs.org Publication Date: September 27, 1996 | doi: 10.1021/bk-1996-0645.ch006

Progress Toward a Molecular Diagnostic for Knockdown Resistance (kdr) Martin S. Williamson, David Martinez-Torres, Caroline A. Hick, Nathalie Castells, and Alan L. Devonshire Biological and Ecological Chemistry Department, Institute of Arable Crops Research-Rothamsted, Harpenden, Hertfordshire, AL5 2JQ, United Kingdom Knockdown resistance (kdr) is a mechanism that confers nerve insensitivity to DDT and pyrethroid insecticides. This type of resistance is best characterized in the house fly where several kdr alleles, including the more potent super-kdr factor, have been identified. Resistance is thought to result from a modification of the voltage-sensitive sodium channel, the primary target site for these insecticides, and this is supported by genetic evidence that both kdr and super-kdr map closely to the house fly para-type sodium channel gene. To investigate the molecular basis of resistance, we have sequenced the full 6.3kb coding region of this gene from susceptible, kdr and super-kdr strains. Our results suggest that kdr is caused by a single amino acid substitution, leucine to phenylalanine, in the domainIIS6segment of the channel; while an additional methionine to threonine change in the nearby IIS4-IIS5 linker is responsible for the enhanced resistance of super-kdr. Using this information, we have developed a PCR-based diagnostic technique for detecting the kdr mutation in individual house flies. The intensive use of pyrethroid insecticides over the last 20 years has led to resistance in several important agricultural pests and this represents a significant threat to their continued effective use (7). The pyrethroids are potent neurotoxins and an important type of resistance is characterized by a marked reduction in the intrinsic sensitivity of the insect nervous system to these compounds. It confers resistance not only to all pyrethroids, but also to D D T 1,1'-(2,2,2trichloroethylidene)bis[4-chlorobenzene] which shares a similar mode of action. This mechanism was first reported (2) over forty years ago in a strain of the house fly, Musca domestica, that withstood the normally rapid knockdown effects of DDT and was subsequently termed knockdown resistance (kdr). The kdr factor was isolated genetically and found to map to a single, recessive locus on chromosome HI (3). Other kdr alleles were subsequently isolated from house fly populations in North America (JWr-Orlando) and Scandinavia (JWr-NPR) with similar resistance properties (4, 5). The kdr factor is characterized by a fairly uniform level of resistance (10 to 30-fold) to DDT and most pyrethroids (Table I), with no cross resistance to other insecticide classes. In 1978, Sawicki (6) reported 0097-6156/96/0645-0052$15.00/0 © 1996 American Chemical Society

In Molecular Genetics and Evolution of Pesticide Resistance; Brown, T.; ACS Symposium Series; American Chemical Society: Washington, DC, 1996.

6. WILLIAMSON ET AL.

Na Channel Gene Sequences in Resistant House/lies 53

a similar factor conferring broad cross resistance to D D T and pyrethroids, but with a greatly enhanced resistance to die more active type II pyrethroids that are characterized by the presence of an a-cyano-3-phenoxybenzyl alcohol. This factor, termed super-kdr, mapped to the same region of chromosome m and so appeared to be allelic to kdr, but can confer up to 500-fold resistance to pyrethroids such as deltamethrin (Table I).


Table I.

Resistance Factors of kdr and super-kdr House Flies To DDT,Type I and Type II Pyrethroids. Data from (7, 8) House fly strain

Compound DDT Bioresmethrin Cismethrin Permethrin Fenvalerate Cypermethrin Deltamethrin

CD (I) (H) (H) 0D (H)

kdr

super-kdr

17 16 13 13 20 28 31

53 53 59 68 170 250 560

The initial characterization and availability of well defined strains for both kdr and super-kdr in the house fly has meant that most studies aimed at understanding the physiological basis of resistance have focused on this insect However, it should be noted that analogous kdr-type mechanisms showing similar cross resistance to DDT and pyrethroids have been reported in several other insect pest species (reviewed in 9). Kdr-typs resistance is ultimately defined as a mechanism that confers neuronal insensitivity to these compounds. This has been clearly established from comparative studies on nerve preparations from susceptible and kdr (or super-kdr) house flies that show the latter to be markedly (102 to 106 fold) less sensitive to the effects of pyrethroids (10). This suggests that the molecular basis of resistance resides in a modification of the nervous system that affects the normal mode of action of these insecticides. Although pyrethroids affect several neural processes, it is now generally accepted that their primary site of action is the voltage-sensitive sodium channel (see reviews 11, 12). Electrophysiological studies using voltage- and patch-clamping techniques have shown that they alter the gating kinetics of the channel, particularly to slow inactivation, and thereby to prolong the Na+ currents associated with membrane depolarizations. This results in uncontrolled bursts of action potentials leading to nerve exhaustion and death. Evidence that this type of resistance results from an alteration in the sodium channel that render it less sensitive to pyrethroids comes from cross resistance studies to certain sodium channel neurotoxins (75), and binding studies that indicate a reduced affinity for pyrethroids on the super-kdr sodium channel (74). This chapter reviews our recent progress in the cloning of sodium channel gene fragments from the house fly; the identification of single amino acid alterations in the kdr (or super-kdr) strains that correlate with resistance, and the development of a polymerase chain reaction (PCR)-based diagnostic technique for rapidly detecting the resistance mutations in individual flies.


54

MOLECULAR GENETICS AND EVOLUTION OF PESTICIDE RESISTANCE


Molecular Biology of the Voltage-Sensitive Sodium Channel The voltage-sensitive sodium channel plays a key role in nerve signalling by generating the rising phase of action potentials in die neurons of vertebrates and invertebrates (75). The channel is sensitive to localized depolarization of the nerve membrane that cause it to open, allowing sodium ions to flow down a concentration gradient across the membrane and into the cell. The open (activated) state of the channel exists for less than a millisecond as it spontaneously closes (inactivates) before returning to the resting (also closed) state. This transient opening of the channel results in further depolarization of the membrane causing more channels to open and so generates a wave of depolarization down the axon that constitutes the nerve signal. Sodium channels purified from mammalian brain comprise a large, glycosylated a subunit polypeptide (Mr 260 kD), together with two smaller associated p subunit polypeptides, p i (36 kD) and p2 (33 kD) (76). The a subunit is the main structural component of the channel and is structurally conserved across a diverse range of vertebrate and invertebrate organisms; the fl subunits have so far only been identified in mammalian tissues. The first sodium channel a subunit to be cloned was that of electric eel, encoding a single polypeptide chain of over 1800 amino acids. Similar sequences were subsequently cloned from mammalian brain (3 different genes), mammalian skeletal and cardiac muscle, and from several invertebrates including Drosophila melanogaster (reviewed in 76, 77). The cloned genes predict structurally-similar polypeptides with considerable sequence conservation, ranging from 45% amino acid homology between the vertebrate and invertebrate channel sequences to over 90% between the mammalian brain isoforms. The a subunit contains four homologous repeating domains (I-IV), with each domain containing six hydrophobic segments (S1-S6) that are thought to form membrane spanning a-helices (Figure 1). The predicted folding of these regions within the membrane has led to several related structural models of the channel with the four domains arranged as a square array about a central pore whose functional properties are formed by sequence elements within the transmembrane segments (76, 77). The role of the a subunit in forming the functional channel protein has been confirmed by the expression of cloned a subunit sequences in heterologous expression systems such as the Xenopus oocyte (78). Both eel electroplax and mammalian brain a subunits form sodium-selective channels with normal activation properties in this system, although the brain channel inactivates slowly compared to the native form; a situation that is rectified by co-expression with the 61 subunit (79). The availability of this system has enabled various aspects of the structural models to be tested directly by mutagenizing selected sequences and testing the activity of the modified channels. This has resulted in the characterization of sequence elements involved in voltage-dependent activation, ion selectivity and conductance, channel inactivation and receptor sites for the binding of certain neurotoxins (76). Since pyrethroids mainly interfere with channel inactivation, it is of interest that residues within the short intracellular peptide linking domains III and IV have been shown to be critically involved in this process. This peptide is thought to form a "ball" (the inactivation peptide) that binds to a hydrophobic site within the intracellular mouth of the activated (open) channel, so blocking the ion flow and effecting inactivation. This highlights the inactivation peptide and sequences that form the intracellular mouth of the pore as likely candidates for interaction with DDT or pyrethroids.


In Molecular Genetics and Evolution of Pesticide Resistance; Brown, T.; ACS Symposium Series; American Chemical Society: Washington, DC, 1996. I

IV

pSCP2

Figure 1. Diagram of the voltage-sensitive sodium channel showing its proposed membrane folding (76,77). The region covered by the house fly c D N A (pSCP2) that was used for RJFLP linkage mapping is highlighted and positions of the two resistance-associated amino acid mutations Met9ig to Thr and Leu 1014 to Phe in the kdr or super-kdr channel sequences are marked (*). The full sequence of the house fly sodium channel will appear in the EMBIVGenBank databases under the accession number X96668.

II


56



Sodium Channel Sequences in Susceptible and kdr House Flies The cloning of sodium channel sequences from vertebrates and D. melanogaster has provided the opportunity to study the corresponding sequences of susceptible, kdr and super-kdr house flies in order to look for changes that might confer resistance. In Drosophila two sodium channel genes have been cloned, DSC1 located on chromosome II (20) and para on the X chromosome (21). The a subunit sequences encoded by these two genes are surprisingly divergent, being no more similar to each other (~ 50% amino acid homology) than they are to the vertebrate sequences. The para gene is known to encode a physiologically important sodium channel within die Drosophila nervous system and was cloned by selecting the mutant phenotypes that result from P element insertion at this locus (21). In contrast, DSC1 was cloned by low stringency hybridization using a vertebrate sodium channel probe and since no neural mutants have been identified that map to this locus, the functional significance of this gene is at present unclear. For this reason, our studies of the kdr resistance mechanism have focused on the cloning and analysis of para-homologous gene sequences from the house fly. In order to clone the house fly para-type gene we used a para fragment from Drosophila to screen a house fly adult head c D N A library at low stringency. A c D N A clone was recovered (pSCP2) that contained domain TV and C-terminal sequences of a House Fly sodium channel (Figure 1) with close homology to the published domain IV region of the para sequence (22). This c D N A was used to probe Southern blots of EcoRl-digested house fly genomic D N A and detected restriction fragment length polymorphisms (RFLPs) in the D N A of susceptible, kdr and super-kdr strains. These RFLPs serve as allele-specific D N A markers for the sodium channel gene and were therefore used to analyze the offspring of controlled genetic crosses involving these strains to establish linkage between the resistance phenotypes and the sodium channel locus. From the combined analysis of over 300 F progeny we found that both kdr and super-kdr factors were closely linked to the sodium channel gene locus (22). These results consolidated physiological evidence that the sodium channel is the primary target of pyrethroid action and implicated the para-type sodium channel rather than DSC1 as the site of resistance. Similar studies in other insects have confirmed this linkage of foir-type resistance to the para sodium channel. Taylor et al. (23) cloned a region extending across domains HI and IV of the para-type sodium channel from the tobacco budworm, Heliothis virescens, and using a PCR-based R F L P technique showed evidence of linkage in a strain carrying multiple pyrethroid resistance factors. Dong and Scott (24) amplified a region from domain I of the para-type gene of German cockroach, Blattella germanica, and identified an R F L P in a kdr-type strain that also showed tight linkage to resistance. Finally, Knipple et al. (25) carried out an independent analysis using a kdr house fly strain, also using an R F L P within the domain I fragment of the para-type gene, and showed similar linkage to resistance. Taken together, these studies not only provide overwhelming support for the hypothesis that the kdr mechanism results from modification^) of the para-type sodium channel, but also infer that this mechanism is likely to be highly conserved across a range of insect species. To investigate further the molecular alterations that cause kdr and super-kdr resistance^we have cloned the full 6.3 kb coding region of the house fly para-type gene within five overlapping cDNA clones from the reference susceptible strain Cooper (26). The cDNAs predict a polypeptide of 2108 amino acids with 92% sequence identity to the para sodium channel of Drosophila and around 50% homology to vertebrate sodium channels. Using this (wild-type) sequence as a template, we designed sequence-specific oligonucleotide primers to selectively amplify the corresponding coding sequences of both kdr and super-kdr strains and 2



6.

WILLIAMSON ET AL. Na Channel Gene Sequences in Resistant Houseflies 57

carried out a full comparison of the wild-type and resistant channel sequences (26). The kdr strain showed only one amino acid change within the main body of the channel from the start of homology domain I through to the end of domain TV. This was the replacement Leuiow to Phe (CTT to T I T ) which was located within the transmembrane S6 segment of domain II (Figure 1). Super-kdr contained the same L e u i o u to Phe mutation together with an additional Met9ig to Thr replacement (ATG to A T A ) in the nearby S4-S5 intracellular loop of domain II (Figure 1). To assess the importance of these mutations in conferring the resistance phenotype, we then amplified the domain II region encompassing these changes from a second, unrelated kdr strain, six other super-kdr strains collected in different parts of the world, and five additional susceptible strains (26). The same Leu to Phe mutation was found in the second kdr strain, and both mutations (Leu to Phe and Met to Thr) in each of the six super-kdr strains. Neither mutation was present in the five pyrethroid-susceptible strains. The detection of only two amino acid changes within the main body of the sodium channel and their correlation across a range of kdr and super-kdr strains provide strong evidence that we have identified the molecular changes that underline these resistance phenotypes, and functional expression studies to confirm this are in progress. A n important role for these mutations is further supported by their localization within the S4-S5 and S6 transmembrane regions of the channel protein. Recent studies of vertebrate channels suggest that these regions are located at the intracellular mouth of the channel pore where they form a receptor site for the inactivation peptide that blocks the channel during the inactivation process. This is consistent with the known physiological role of pyrethroids and DDT in delaying channel inactivation and suggests that the kdr mutations we have found define part of the binding site for these insecticides at the intracellular mouth of the channel. A more detailed discussion of this and the possible effects of these mutations on pyrethroid binding is given elsewhere (26). The identification of these mutations has interesting implications for the evolution of the kdr and super-kdr resistance factors. The presence of the kdr (Leu to Phe) mutation in the super-kdr strains together with the additional (Met to Thr) mutation suggests that super-kdr arises sequentially from kdr rather than independently of it, since the likelihood of both mutations arising simultaneously from a wild type background is extremely low. Super-kdr would therefore only be predicted to arise from a population in which kdr is already established and would result from selection pressure with type II pyrethroids such as deltamethrin to which super-kdr offers a significant enhancement in the level of resistance. It will be interesting to determine whether the super-kdr (Met to Thr) mutation can also confer a level of resistance on its own, either by identifying field populations that contain only this mutation, or through site-directed mutagenesis and in vitro expression of the modified gene. Development of a Molecular Diagnostic for Knockdown Resistance (kdr) Our initial attempts to develop a DNA-based molecular diagnostic for resistance centered on exploiting the RFLPs associated with the sodium channel that were used as genetic markers in the linkage mapping (see above). Analysis of a wider range of kdr and super-kdr house fly strains did indeed reveal many similarities in their RFLPs patterns (27), however this technique was unreliable for diagnosing resistance in field populations since none of the bands occurred consistently in all strains. This is not surprising since the mutations associated with resistance are located in the central, domain II region of the sodium channel, whereas the probe used in these RFLP studies (pSCP2) detects changes in the C terminal and 3' noncoding regions of the gene (Figure 1). Hence, variability within this 3' region will not necessarily affect die resistance status of the gene.


58


380bp


Cooper

1

G

Analysis of Sodium Channel Gene Sequences in Pyrethroid-Resistant

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