Point mutations in the Drosophila sodium channel gene para associated with resistance to DDT and pyrethroid insecticides

The gene para in Drosophila melanogaster encodes an α subunit of voltage-activated sodium channels, the presumed site of action of DDT and pyrethroid insecticides. We used an existing collection of Drosophila para mutants to examine the molecular basis of target-site resistance to pyrethroids and DDT. Six out of thirteen mutants tested were associated with a largely dominant, 10- to 30-fold increase in DDT resistance. The amino acid lesions associated with these alleles defined four sites in the sodium channel polypeptide where a mutational change can cause resistance: within the intracellular loop between S4 and S5 in homology domains I and III, within the pore region of homology domain III, and within S6 in homology domain III. Some of these sites are analogous with those defined by knockdown resistance (kdr) and super-kdr resistance-associated mutations in houseflies and other insects, but are located in different homologous units of the channel polypeptide. We find a striking synergism in resistance levels with particular heterozygous combinations of para alleles that appears to mimic the super-kdr double mutant housefly phenotype. Our results indicate that the alleles analyzed from natural populations represent only a subset of mutations that can confer resistance. The implications for the binding site of pyrethroids and mechanisms of target-site insensitivity are discussed. Received: 9 May 1997 / Accepted: 21 July 1997     

Knockdown resistance (kdr) to DDT and pyrethroid insecticides maps to a sodium channel gene locus in the housefly (Musca domestica)

The voltage-sensitive sodium channel is generally regarded as the primary target site of dichlorodiphenyl-trichloro-ethane (DDT) and pyrethroid insecticides, and has been implicated in the widely reported mechanism of nerve insensitivity to these compounds. This phenomenon is expressed as knockdown resistance (kdr) and has been best characterised in the housefly where several putative alleles, including the more potent super-kdr factor, have been identified. We report the isolation of cDNA clones containing part of a housefly sodium channel gene, designated Msc, which show close homology to the para sodium channel of Drosophila (99% amino acid identity within the region of overlap). Using Southern blots of insect DNA, restriction fragment length polymorphisms (RFLPs) at the Msc locus were identified in susceptible, kdr and super-kdr housefly strains. These RFLPs showed tight linkage to resistance in controlled crosses involving these strains, thus providing clear genetic evidence that kdr, and hence pyrethroid mode of action, is closely associated with the voltage-sensitive sodium channel.     

The dominant cold-sensitive Out-cold mutants of Drosophila melanogaster have novel missense mutations in the voltage-gated sodium channel gene paralytic

Here we report the molecular characterization of Out-cold (Ocd) mutants of Drosophila melanogaster, which produce a dominant, X-linked, cold-sensitive paralytic phenotype. From its initial 1.5-Mb cytological location within 13F1-16A2, P-element and SNP mapping reduced the Ocd critical region to <100 kb and to six candidate genes: hangover, CG9947, CG4420, eIF2a, Rbp2, and paralytic (para). Complementation testing with para null mutations strongly suggests Ocd and para are allelic, as does gene rescue of Ocd semilethality with a wild-type para transgene. Pesticide resistance and electrophysiological phenotypes of Ocd mutants support this conclusion. The para gene encodes a voltage-gated sodium channel. Sequencing the Ocd lines revealed mutations within highly conserved regions of the para coding sequence, in the transmembrane segment S6 of domain III (I1545M and T1551I), and in the linker between domains III and IV (G1571R), the location of the channel inactivation gate. The G1571R mutation is of particular interest as mutations of the orthologous residue (G1306) in the human skeletal muscle sodium channel gene SCN4A are associated with cases of periodic paralysis and myotonia, including the human cold-sensitive disorder paramyotonia congenita. The mechanisms by which sodium channel mutations cause cold sensitivity are not well understood. Therefore, in the absence of suitable vertebrate models, Ocd provides a system in which genetic, molecular, physiological, and behavioral tools can be exploited to determine mechanisms underlying sodium channel periodic paralyses.     

Knockdown resistance (kdr) to DDT and pyrethroid insecticides maps to a sodium channel gene locus in the housefly (Musca domestica)

The voltage-sensitive sodium channel is generally regarded as the primary target site of dichlorodiphenyl-trichloro-ethane (DDT) and pyrethroid insecticides, and has been implicated in the widely reported mechanism of nerve insensitivity to these compounds. This phenomenon is expressed as knockdown resistance (kdr) and has been best characterised in the housefly where several putative alleles, including the more potent super-kdr factor, have been identified. We report the isolation of cDNA clones containing part of a housefly sodium channel gene, designated Msc, which show close homology to the para sodium channel of Drosophila (99% amino acid identity within the region of overlap). Using Southern blots of insect DNA, restriction fragment length polymorphisms (RFLPs) at the Msc locus were identified in susceptible, kdr and super-kdr housefly strains. These RFLPs showed tight linkage to resistance in controlled crosses involving these strains, thus providing clear genetic evidence that kdr, and hence pyrethroid mode of action, is closely associated with the voltage-sensitive sodium channel.     

The Helicoverpa zea (Boddie) (Lepidoptera: Noctuidae) voltage-gated sodium channel and mutations associated with pyrethroid resistance in field-collected adult males

Helicoverpa zea is one of the most costly insect pests of food and fiber crops throughout the Americas. Pyrethroid insecticides are widely applied for its control as they are effective and relatively inexpensive; however, resistance to pyrethroids threatens agricultural systems sustainability because alternative insecticides are often more expensive or less effective. Although pyrethroid resistance has been identified in this pest since 1990, the mechanisms of resistance have not yet been elucidated at the molecular level. Pyrethroids exert their toxicity by prolonging the open state of the voltage-gated sodium channel. Here we report the cDNA sequence of the H. zea sodium channel α-subunit homologous to the para gene from Drosophila melanogaster. In field-collected males which were resistant to cypermethrin as determined by the adult vial test, we identify known resistance-conferring mutations L1029H and V421M, along with two novel mutations at the V421 residue, V421A and V421G. An additional mutation, I951V, may be the first example of a pyrethroid resistance mutation caused by RNA editing. Identification of the sodium channel cDNA sequence will allow for testing hypotheses on target-site resistance for insecticides acting on this channel through modeling and expression studies. Understanding the mechanisms responsible for resistance will greatly improve our ability to identify and predict resistance, as well as preserve susceptibility to pyrethroid insecticides.     

Differential resistance of insect sodium channels with kdr mutations to deltamethrin, permethrin and DDT

Knockdown resistance (kdr) in insects, caused by inherited nucleotide polymorphisms in the voltage-gated sodium channel (VGSC) gene, is a major threat to the efficacy of pyrethroid insecticides. Classic kdr, resulting from an L1014F substitution in the VGSC is now present in numerous pest species. Two other substitutions at the L1014 locus have also been reported, L1014S and L1014H. Here we have used expression of L1014 modified Drosophila para VGSCs in Xenopus oocytes with two-electrode voltage clamp to characterise all three mutations. The mutations L1014F and L1014H caused significant depolarizing shifts in the half activation voltage (V_50,act) from −17.3 mV (wild-type) to −13.1 and −13.5 mV respectively, whereas L1014S caused no shift in V_50,act but its currents decayed significantly faster than wild-type channels. Treatment of the wild-type channel with deltamethrin (≥1 nM), permethrin (≥30 nM) or DDT (≥1 ??M) resulted in hyperpolarizing shifts in V_50,act. Deltamethrin, permethrin and DDT also produced “tail currents” with EC₅₀s of 0.043, 0.40 and 65 ??M and maximum modifications of 837, 325 and 7% respectively. L1014F provided a high level of resistance against all insecticides for both measured parameters. L1014H most effectively combated deltamethrin induced tail currents while L1014S strongly resisted the large DDT induced shifts in V_50,act. We conclude that L1014H and L1014S may have arisen through heavy exposure to specific pyrethroids and DDT respectively.     

Levels of cross-resistance to pyrethroids conferred by the Vssc knockdown resistance allele 410L+1016I+1534C in Aedes aegypti

Aedes aegypti is a primary vector of viral pathogens and is responsible for millions of human infections annually that represent critical public health and economic costs. Pyrethroids are one of the most commonly used classes of insecticides to control adult A. aegypti. The insecticidal activity of pyrethroids depends on their ability to bind and disrupt the voltage-sensitive sodium channel (VSSC). In mosquitoes, a common mechanism of resistance to pyrethroids is due to mutations in Vssc (hereafter referred as knockdown resistance, kdr). In this study, we found that a kdr (410L+V1016I+1534C) allele was the main mechanism of resistance in a pyrethroid-resistant strain of A. aegypti collected in Colombia. To characterize the level of resistance these mutations confer, we isolated a pyrethroid resistant strain (LMRKDR:RK, LKR) that was congenic to the susceptible Rockefeller (ROCK) strain. The full-length cDNA of Vssc was cloned from LKR and no additional resistance mutations were present. The levels of resistance to different pyrethroids varied from 3.9- to 56-fold. We compared the levels of resistance to pyrethroids, DCJW and DDT between LKR and what was previously reported in two other congenic strains that share the same pyrethroid-susceptible background (the ROCK strain), but carry different kdr alleles (F1534C or S989P + V1016G). The resistance conferred by kdr alleles can vary depending on the stereochemistry of the pyrethroid. The 410L+1016I+1534C kdr allele does not confer higher levels of resistance to six of ten pyrethroids, relative to the 1534C allele. The importance of these results to understand the evolution of insecticide resistance and mosquito control are discussed.     

Evidence for Dual Binding Sites for 1,1,1-Trichloro-2,2-bis(p-chlorophenyl)ethane (DDT) in Insect Sodium Channels

1,1,1-Trichloro-2,2-bis(p-chlorophenyl)ethane (DDT), the first organochlorine insecticide, and pyrethroid insecticides are sodium channel agonists. Although the use of DDT is banned in most of the world due to its detrimental impact on the ecosystem, indoor residual spraying of DDT is still recommended for malaria control in Africa. Development of resistance to DDT and pyrethroids is a serious global obstacle for managing disease vectors. Mapping DDT binding sites is necessary for understanding mechanisms of resistance and modulation of sodium channels by structurally different ligands. The pioneering model of the housefly sodium channel visualized the first receptor for pyrethroids, PyR1, in the II/III domain interface and suggested that DDT binds within PyR1. Previously, we proposed the second pyrethroid receptor, PyR2, at the I/II domain interface. However, whether DDT binds to both pyrethroid receptor sites remains unknown. Here, using computational docking of DDT into the K_v1.2-based mosquito sodium channel model, we predict that two DDT molecules can bind simultaneously within PyR1 and PyR2. The bulky trichloromethyl group of each DDT molecule fits snugly between four helices in the bent domain interface, whereas two p-chlorophenyl rings extend into two wings of the interface. Model-driven mutagenesis and electrophysiological analysis confirmed these propositions and revealed 10 previously unknown DDT-sensing residues within PyR1 and PyR2. Our study proposes a dual DDT-receptor model and provides a structural background for rational development of new insecticides.     

Point mutations in domain III of a Drosophila neuronal Na channel confer resistance to allethrin

Voltage-gated sodium channels are the presumed site of action of pyrethroid insecticides and DDT. We screened several mutant sodium channel Drosophila lines for resistance to type I pyrethroids. In insecticidal bioassays the para(74) and para(DN7) fly lines showed greater than 4-fold resistance to allethrin relative to the allethrin sensitive Canton-S control line. The amino acid substitutions of both mutants are in domain III. The point mutation associated with para(74) lies within the S6 transmembrane region and the amino acid substitution associated with para(DN7) lies within the S4-S5 linker region. These sites are analogous to the mutations in domain II underlying knockdown resistance (kdr) and super-kdr, naturally occurring forms of pyrethroid resistance found in houseflies and other insects. Electrophysiological studies were performed on isolated Drosophila neurons from wild type and para(74) embryos placed in primary culture for three days to two weeks. The mutant para(74) sodium currents were kinetically similar to wild type currents, in activation, inactivation and time to peak. The only observed difference between para(74) and wild-type neurons was in the affinity of the type I pyrethroid, allethrin. Application of 500 nM allethrin caused removal of inactivation and prolonged tail currents in wild type sodium channels but had little or no effect on para(74) mutant sodium channels.     

Quantitative trait loci mapping of genome regions controlling permethrin resistance in the mosquito Aedes aegypti

The mosquito Aedes aegypti is the principal vector of dengue and yellow fever flaviviruses. Permethrin is an insecticide used to suppress Ae. aegypti adult populations but metabolic and target site resistance to pyrethroids has evolved in many locations worldwide. Quantitative trait loci (QTL) controlling permethrin survival in Ae. aegypti were mapped in an F₃ advanced intercross line. Parents came from a collection of mosquitoes from Isla Mujeres, México, that had been selected for permethrin resistance for two generations and a reference permethrin-susceptible strain originally from New Orleans. Following a 1-hr permethrin exposure, 439 F₃ adult mosquitoes were phenotyped as knockdown resistant, knocked down/recovered, or dead. For QTL mapping, single nucleotide polymorphisms (SNPs) were identified at 22 loci with potential antixenobiotic activity including genes encoding cytochrome P450s (CYP), esterases (EST), or glutathione transferases (GST) and at 12 previously mapped loci. Seven antixenobiotic genes mapped to chromosome I, six to chromosome II, and nine to chromosome III. Two QTL of major effect were detected on chromosome III. One corresponds with a SNP previously associated with permethrin resistance in the para sodium channel gene and the second with the CCEunk7o esterase marker. Additional QTL but of relatively minor effect were also found. These included two sex-linked QTL on chromosome I affecting knockdown and recovery and a QTL affecting survival and recovery. On chromosome II, one QTL affecting survival and a second affecting recovery were detected. The patterns confirm that mutations in the para gene cause target-site insensitivity and are the major source of permethrin resistance but that other genes dispersed throughout the genome contribute to recovery and survival of mosquitoes following permethrin exposure.     

Multiple Origins of kdr-type Resistance in the House Fly,Musca domestica

Insecticide resistance is a model phenotype that can be used to investigate evolutionary processes underlying the spread of alleles across a global landscape, while offering valuable insights into solving the problems that resistant pests present to human health and agriculture. Pyrethroids are one of the most widely used classes of insecticides world-wide and they exert their toxic effects through interactions with the voltage-sensitive sodium channel (Vssc). Specific mutations in Vssc (kdr, kdr-his and super-kdr) are known to cause resistance to pyrethroid insecticides in house flies. In order to determine the number of evolutionary origins of kdr, kdr-his and super-kdr, we sequenced a region of Vssc from house flies collected in the USA, Turkey and China. Our phylogenetic analysis of Vssc unequivocally supports the hypothesis of multiple independent origins of kdr, super-kdr and kdr-his on an unprecedented geographic scale. The implications of these evolutionary processes on pest management are discussed.     

Investigating the Molecular Mechanisms of Organophosphate and Pyrethroid Resistance in the Fall Armyworm Spodoptera frugiperda

The fall armyworm Spodoptera frugiperda is an economically important pest of small grain crops that occurs in all maize growing regions of the Americas. The intensive use of chemical pesticides for its control has led to the selection of resistant populations, however, to date, the molecular mechanisms underlying resistance have not been characterised. In this study the mechanisms involved in the resistance of two S. frugiperda strains collected in Brazil to chlorpyrifos (OP strain) or lambda-cyhalothrin (PYR strain) were investigated using molecular and genomic approaches. To examine the possible role of target-site insensitivity the genes encoding the organophosphate (acetylcholinesterase, AChE) and pyrethroid (voltage-gated sodium channel, VGSC) target-site proteins were PCR amplified. Sequencing of the S. frugiperda ace-1 gene identified several nucleotide changes in the OP strain when compared to a susceptible reference strain (SUS). These result in three amino acid substitutions, A201S, G227A and F290V, that have all been shown previously to confer organophosphate resistance in several other insect species. Sequencing of the gene encoding the VGSC in the PYR strain, identified mutations that result in three amino acid substitutions, T929I, L932F and L1014F, all of which have been shown previously to confer knockdown/super knockdown-type resistance in several arthropod species. To investigate the possible role of metabolic detoxification in the resistant phenotype of the OP and PYR stains all EST sequences available for S. frugiperda were used to design a gene-expression microarray. This was then used to compare gene expression in the resistant strains with the susceptible reference strain. Members of several gene families, previously implicated in metabolic resistance in other insects were found to be overexpressed in the resistant strains including glutathione S-transferases, cytochrome P450s and carboxylesterases. Taken together these results provide evidence that both target-site and metabolic mechanisms underlie the resistance of S. frugiperda to pyrethroids and organophosphates.     

Sodium channel genes and their differential genotypes at the L-to-F kdr locus in the mosquito Culex quinquefasciatus

The para-type sodium channel in insects is the primary target of pyrethroid and DDT insecticides. However, modifications in the target protein structure such as point mutations or substitutions, resulting from single nucleotide polymorphisms (SNP), cause insensitivity of the insect’s nervous system to pyrethroids and DDT and, in turn, result in insecticide resistance. Among these mutations, substitution of leucine to phenylalanine (L to F) in the 6th segment of domain II (IIS6) has been clearly associated with pyrethroid and DDT resistance in many insect species, including mosquitoes. Here, multiple copies of the sodium channel gene were identified in the mosquito Culex quinquefasciatus by Southern blot analysis and polymerase chain reaction (PCR) analysis. Two genomic DNA fragments of the mosquito sodium channel gene (509 and 181 bp) were detected by a single PCR primer pair. Sequence analysis indicated the lack of an intron sequence in the 181 bp sodium channel fragment. Single nucleotide polymorphism (SNP) analysis revealed a strong correlation among the frequencies of L-to-F allelic (T) expression at the RNA level, the frequencies and resistance allele (T) at the L-to-F site of the 509 bp genomic DNA fragment, which did include an intron sequence, and the levels of insecticide resistance. Taking together, this study, for the first time, not only revealed multiple copies of the sodium channel gene presented in the Culex mosquito genome but also suggested that the one with the intro sequence may be a functional copy of the sodium channel gene in the Culex mosquitoes.     

Molecular modelling studies of kdr mutations in voltage gated sodium channel revealed significant conformational variations contributing to insecticide resistance

Voltage gated sodium channels (VGSC) of mosquito vectors are the primary targets of dichlorodiphenyltrichloroethane (DDT) and other synthetic pyrethroids used in public health programmes. The knockdown resistant (kdr) mutations in VGSC are associated with the insecticide resistance especially in Anophelines. The present study is aimed to emphasize and demarcate the impact of three kdr-mutations such as L1014S, L1014F and L1014H on insecticide resistance. The membrane model of sodium transport domain of VGSC (STD-VGSC) was constructed using de novo approach based on domain and trans-membrane predictions. The comparative molecular modelling studies of wild type and mutant models of STD-VGSC revealed that L1014F mutant was observed to be near native to the wild type model in all the respects, but, L1014S and L1014H mutations showed drastic variations in the energy levels, root mean square fluctuations (RMSF) that resulted in conformational variations. The predicted binding sites also showed variable cavity volumes and RMSF in L1014S and L1014H mutants. Further, DDT also found be bound in near native manner to wild type in L1014F mutant and with variable orientation and affinities in L1014S and L1014H mutants. The variations and fluctuations observed in mutant structures explained that each mutation has its specific impact on the conformation of VGSC and its binding with DDT. The study provides new insights into the structure–function-correlations of mutant STD-VGSC structures and demonstrates the role and effects of kdr mutations on insecticide resistance in mosquito vectors.     

A sodium channel mutation identified in Aedes aegypti selectively reduces cockroach sodium channel sensitivity to type I, but not type II pyrethroids

Voltage-gated sodium channels are the primary target of pyrethroid insecticides. Numerous point mutations in sodium channel genes have been identified in pyrethroid-resistant insect species, and many have been confirmed to reduce or abolish sensitivity of channels expressed in Xenopus oocytes to pyrethroids. Recently, several novel mutations were reported in sodium channel genes of pyrethroid-resistant Aedes mosquito populations. One of the mutations is a phenylalanine (F) to cysteine (C) change in segment 6 of domain III (IIIS6) of the Aedes mosquito sodium channel. Curiously, a previous study showed that alanine substitution of this F did not alter the action of deltamethrin, a type II pyrethroid, on a cockroach sodium channel. In this study, we changed this F to C in a pyrethroid-sensitive cockroach sodium channel and examined mutant channel sensitivity to permethrin as well as five other type I or type II pyrethroids in Xenopus oocytes. Interestingly, the F to C mutation drastically reduced channel sensitivity to three type I pyrethroids, permethrin, NRDC 157 (a deltamethrin analogue lacking the ??-cyano group) and bioresemthrin, but not to three type II pyrethroids, cypermethrin, deltamethrin and cyhalothrin. These results confirm the involvement of the F to C mutation in permethrin resistance, and raise the possibility that rotation of type I and type II pyrethroids might be considered in the control of insect pest populations where this particular mutation is present.     

Modelling insecticide-binding sites in the voltage-gated sodium channel

A homology model of the housefly voltage-gated sodium channel was developed to predict the location of binding sites for the insecticides fenvalerate, a synthetic pyrethroid, and DDT an early generation organochlorine. The model successfully addresses the state-dependent affinity of pyrethroid insecticides, their mechanism of action and the role of mutations in the channel that are known to confer insecticide resistance. The sodium channel was modelled in an open conformation with the insecticide-binding site located in a hydrophobic cavity delimited by the domain II S4-S5 linker and the IIS5 and IIIS6 helices. The binding cavity is predicted to be accessible to the lipid bilayer and therefore to lipid-soluble insecticides. The binding of insecticides and the consequent formation of binding contacts across different channel elements could stabilize the channel when in an open state, which is consistent with the prolonged sodium tail currents induced by pyrethroids and DDT. In the closed state, the predicted alternative positioning of the domain II S4-S5 linker would result in disruption of pyrethroid-binding contacts, consistent with the observation that pyrethroids have their highest affinity for the open channel. The model also predicts a key role for the IIS5 and IIIS6 helices in insecticide binding. Some of the residues on the helices that form the putative binding contacts are not conserved between arthropod and non-arthropod species, which is consistent with their contribution to insecticide species selectivity. Additional binding contacts on the II S4-S5 linker can explain the higher potency of pyrethroid insecticides compared with DDT.     

Allelic Variation of Cytochrome P450s Drives Resistance to Bednet Insecticides in a Major Malaria Vector

Scale up of Long Lasting Insecticide Nets (LLINs) has massively contributed to reduce malaria mortality across Africa. However, resistance to pyrethroid insecticides in malaria vectors threatens its continued effectiveness. Deciphering the detailed molecular basis of such resistance and designing diagnostic tools is critical to implement suitable resistance management strategies. Here, we demonstrated that allelic variation in two cytochrome P450 genes is the most important driver of pyrethroid resistance in the major African malaria vector Anopheles funestus and detected key mutations controlling this resistance. An Africa-wide polymorphism analysis of the duplicated genes CYP6P9a and CYP6P9b revealed that both genes are directionally selected with alleles segregating according to resistance phenotypes. Modelling and docking simulations predicted that resistant alleles were better metabolizers of pyrethroids than susceptible alleles. Metabolism assays performed with recombinant enzymes of various alleles confirmed that alleles from resistant mosquitoes had significantly higher activities toward pyrethroids. Additionally, transgenic expression in Drosophila showed that flies expressing resistant alleles of both genes were significantly more resistant to pyrethroids compared with those expressing the susceptible alleles, indicating that allelic variation is the key resistance mechanism. Furthermore, site-directed mutagenesis and functional analyses demonstrated that three amino acid changes (Val¹⁰⁹Ile, Asp³³⁵Glu and Asn³⁸⁴Ser) from the resistant allele of CYP6P9b were key pyrethroid resistance mutations inducing high metabolic efficiency. The detection of these first DNA markers of metabolic resistance to pyrethroids allows the design of DNA-based diagnostic tools to detect and track resistance associated with bednets scale up, which will improve the design of evidence-based resistance management strategies.     

Genetic Studies of Membrane Excitability in Drosophila: Lethal Interaction between Two Temperature-Sensitive Paralytic Mutations

Two mutants of Drosophila melanogaster, para ^ts1 (1-53.9) and nap^ts (2-56.2) both display similar temperature-sensitive paralysis associated with blockage in the conduction of nerve action potentials, suggesting that the two gene products have a similar function. This idea is supported by the observation that the double mutant is unconditionally lethal. Genetic analysis of this synergistic interaction has revealed the following: 1) it specifically involves the para and nap loci; (2) all para alleles interact with nap^ts, but the strength of the interaction varies in an allele-dependent fashion; (3) lethality of the double mutant occurs during the first larval instar with para^ts1 but differs with other para alleles; (4) hypodosage of para ⁺ causes lethality in a nap^ts background. These results together with previous electrophysiological, behavioral and pharmacological studies of these mutants suggest that both para and nap affect sodium channels and possibly encode different subunits.