Para Sodium Channel Gene Research Articles

A novel amino acid substitution in the para-sodium channel gene in Rhipicephalus microplus (Acari: Ixodidae) associated with knockdown resistance

Resistance acquired by the tick Rhipicephalus microplus (Canestrini) to different types of ixodicides in Mexico has had a negative impact on national and local livestock, mainly due to the transmission of diseases such as babesiosis and anaplasmosis, among others. The technique used for the diagnosis of resistance was that in the bioassays noted in the Norma Oficial Mexicana (NOM-006-ZOO-1994). The purpose of this investigation was the determination of resistance to pyrethroids through isoleucine-phenylalanine mutation in the gene KDR, in a population of ticks from Montemorelos, NL, Mexico. Preliminary bioassays demonstrated resistance to cypermethrin and deltamethrin (27.4%) and flumethrin (36.7-34.7%). To identify the mutation, DNA was extracted from 100 mg of larvae (pools), 10 pools were assessed by PCR, in which a pair of primers designed with the program Oligo 2.0 and Amplify 1.2 amplified a 136 bp fragment containing the mutation. The PCR product was subsequently sequenced to confirm the presence of the mutation. A strain susceptible to pyrethroid insecticides (Mora strain) was used as control, but it did not show the mutation. However, the mutation was detected in 4 out of 10 samples of the strain Montemorelos.

IDENTIFICATION OF MUTATION IN THE BEMISIA TABACI (GENN.) PARA SODIUM CHANNEL GENE ASSOCIATED WITH RESISTANCE TO PYRETHROIDS

Mechanism of resistance to lambda-cyhalothrin in highly resistant Bemisia tabaci strain can be explained by the voltage-gated sodium channel which is the primary target site of pyrethroid insecticides. The super knockdown resistance (super-kdr) to pyrethroids is caused by changes at specific sites on the para-type sodium channel protein domain II (IIS4-6). The B. tabaci para-type sodium channel gene was RT-PCR amplified from lab-strain (reference), Parent (first generation after treatment with the insecticide) and generation thirteen after treatment was considered. The mechanisms of resistance to Lambda-cyhalothrin (Karat 20% EC) in a Q biotype, highly resistant Bemisia tabaci strain. Analysis of the sequence of the IIS4-IIS6 region of the para sodium channel gene of lab-strain, Parent and resistant strain (G13) revealed two amino acid replacements compared to that of the SUD-S susceptible strain. One is the leucine to isoleucine substitution at position 925 (L925I) and allele r1-Q1 (GenBank accession no. DQ205206) has identical intron sequences with samples of parent and lab-strain, other is a novel kdr resistant mutation for B. tabaci, a threonine to valine substitution at position (T929V) and alleles r2-Q1 (GenBank accession no DQ205207) has identical intron sequence with G13..

Identification of pyrethroid resistance associated mutations in the para sodium channel of the two‐spotted spider mite Tetranychus urticae (Acari: Tetranychidae)

We investigated pyrethroid resistance mechanisms in Tetranychus urticae strains from Greece. Combined bioassay, biochemical and synergistic data indicated that although P450 mono-oxygenase activities were associated with the trait, target site insensitivity was the major resistance component. A 3.3 kb cDNA fragment of the T. urticae para sodium channel gene encompassing segment 4 of domain II to segment 6 of domain IV was obtained by a degenerate PCR strategy. The T. urticae sequence showed highest identity (56%) to the scabies mite, Sarcoptes scabiei, and was phylogenetically classified within the divergent group of Arachnida. Comparison of resistant and susceptible strains identified the point mutation F1538I in segment 6 of domain III, which is known to confer strong resistance to pyrethroids, along with a second mutation (A1215D) in the intracellular linker connecting domains II and III with an unknown role. Three transcripts were identified corresponding to the k and l alternative exons. The mode of inheritance of resistance was confirmed as incompletely recessive, which is consistent with a target site mechanism for pyrethroids.

Identification of a mutation in the para-sodium channel gene of the cattle tick Rhipicephalus (Boophilus) microplus associated with resistance to synthetic pyrethroid acaricides

Resistance against synthetic pyrethroid (SP) products for the control of cattle ticks in Australia was detected in the field in 1984, within a very short time of commercial introduction. We have identified a mutation in the domain II S4–5 linker of the para-sodium channel that is associated with resistance to SPs in the cattle tick Rhipicephalus (Boophilus) microplus from Australia. The cytosine to adenine mutation at position 190 in the R. microplus sequence AF134216, results in an amino acid substitution from leucine in the susceptible strain to isoleucine in the resistant strain. A similar mutation has been shown to confer SP resistance in the whitefly, Bemisia tabaci, but has not been described previously in ticks. A diagnostic quantitative PCR assay has been developed using allele-specific Taqman ® minor groove-binding (MGB) probes. Using the assay to screen field and laboratory populations of ticks showed that homozygote allelic frequencies correlated highly with the survival percentage at the discriminating concentration of cypermethrin.

Prevalence ofkdr-like Mutations Associated with Pyrethroid Resistance in Human Head Louse Populations in Japan

Pyrethroid insecticides play very important roles in the control of pediculosis, which is caused by human head louse (Pediculus humanus capitis De Geer) worldwide. The development of head louse resistance to pyrethroid pediculicides has become a global issue during the last two decades. Pyrethroid resistance is associated with amino acid substitutions in the alpha-subunit of the para-sodium channel gene; these substitutions are termed kdr-like mutations. The frequencies of four mutations (D11E, M815I, T929I, and L932F) in the sodium channel gene were studied in human head louse colonies collected from Japan by using SNaPshot method. A total of 630 head lice collected from 282 infested people were analyzed, and it was found that 55 lice of 19 colonies homozygously or heterozygously possessed kdr-like genes, in which the four mutations existed concomitantly. This suggested that the quadruple mutant haplotype is a common feature of the kdr genes of pandemic head lice. The frequencies of the occurrence of resistant genes in the total individuals tested and resistant gene-carrying colonies were 8.7 (55/630) and 6.7% (19/263), respectively. Because the resistant gene was detected in the colonies collected from 11 of the 22 prefectures surveyed, it is speculated that resistant head lice are already spread extensively in Japan. This was the first large-scale survey of pyrethroid resistant head lice in Japan.

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 F(3) 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 F(3) 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.

Role for para sodium channel gene 3′ UTR in the modification of Drosophila seizure susceptibility

Voltage-gated sodium channel genes are highly regulated at the level of transcription or translation. In this study, we have utilized the combination of genetic, electrophysiological, and molecular analyses to identify a 7-kb 3'-untranslated region (UTR) of the Drosophila para sodium channel gene. Disruption of this segment by P-element insertion causes reduction of para primary transcript, but not Rbp2 transcripts. The identification of this novel 3'-UTR is based on a P-insertion mutation called para(JS1), which was isolated from a P-element mutagenesis screen for seizure suppressors in a Drosophila model of epilepsy. The para(JS1) mutation was identified 6845 bp downstream of the para gene, which resides in an intergenic region that lies between para and Rbp2 (RNA-binding protein 2) genes. Interestingly, reverse-transcription PCR showed that the region containing para(JS1) is substantially transcribed and this transcribed region is associated with para coding region. We discussed possible mechanisms of how reduced transcription of the para gene or alterations in sodium channel subunit composition might be indicated by the para(JS1) mutation and implications for para 3' UTR function.

Pyrethroid-resistant diamondback moth expresses alternatively spliced sodium channel transcripts with and without T929I mutation

This study revealed two distinct alternatively spliced exons, A1 and A2, encoding part of the domain IIS4–IIS5 of the para-sodium channel gene in the diamondback moth (DBM). Exons A1 and A2, respectively, revealed 79% and 91% identity at the nucleotide and amino acid levels. Both alternative exons included the T929I site, which has been associated with pyrethroid resistance in DBM. In the pyrethroid-resistant strain, susceptible (Thr) and resistant (Ile) amino acids were encoded at the T929I site in exons A1 and A2, respectively, but in the pyrethroid-susceptible strain, only Thr was encoded at the site in both exons. The transcripts containing exon A1 were expressed constitutively in all developmental stages. The transcripts containing exon A2 were also detected in all developmental stages, but the levels were significantly lower in the 3rd and 4th instar larvae. Tissue-specific data from the 4th instar larvae and adults showed that the expression of transcripts containing exon A2 was higher in heads than in bodies. These findings suggest that alternative splicing of the para-sodium channel gene might produce distinct channels with different sensitivities to pyrethroids, possibly in a tissue-specific manner.

Identification of mutations in the para sodium channel of Bemisia tabaci from Crete, associated with resistance to pyrethroids

We investigated the mechanisms of resistance to α-cypermethrin in a Q biotype, highly resistant Bemisia tabaci strain (GRMAL-RP) isolated from Crete. Cytochrome P450-dependent monoxygenase activity with the substrate ethoxycoumarin, and carboxylesterase activity with the substrates α-naphthyl-acetate, β-naphthyl-acetate, and para-nitrophenol acetate were substantially elevated in the GRMAL-RP, compared to the susceptible SUD-S strain, while glutathione- S-transferase activity with the substrate 1-chloro-2,4-dinitrobenzene was not different. The metabolic inhibitors piperonyl butoxide and S, S, S-tributyl phosphorotrithioate synergised cypermethrin toxicity in the GRMAL-RP strain, however, mortality was still lower than that of the susceptible strain, indicating the presence of an additional resistance mechanism. Analysis of the sequence of the IIS4–IIS6 region of the para sodium channel gene of the GRMAL-RP strain revealed two amino acid replacements compared to that of the SUD-S susceptible strain. One is the leucine to isoleucine substitution at position 925 (L925I) previously implicated in B. tabaci pyrethroid resistance and the other is a novel kdr resistant mutation for B. tabaci, a threonine to valine substitution at position 929 (T929V). Genotype analysis showed that the L925I and T929V were present in all GRMAL-RP males tested, at an approximately 1:1 frequency, but never in combination in the same haplotype.

Involvement of a sodium channel mutation in pyrethroid resistance in Cydia pomonella L, and development of a diagnostic test

Populations of the codling moth, Cydia pomonella L (Lepidoptera, Tortricidae) have developed resistance to several classes of insecticide such as benzoylureas, juvenile hormone analogues, ecdysone agonists and pyrethroids, but the corresponding resistance mechanisms have not been extensively studied. Knockdown resistance (kdr) to pyrethroid insecticides has been associated with point mutations in the para sodium channel gene in a great variety of insect pest species. We have studied two susceptible strains (S and Sv) and two resistant strains (Rt and Rv) of C pomonella that exhibited 4- and 80-fold resistance ratios to deltamethrin, respectively. The region of the voltage-dependent sodium channel gene which includes the position where kdr and super-kdr mutations have been found in Musca domestica L was amplified. The kdr mutation, a leucine-to-phenylalanine replacement at position 1014, was found only in the Rv strain. In contrast, the super-kdr mutation, a methionine-to-threonine replacement at position 918, was not detected in any C pomonella strain. These data allowed us to develop a PCR-based diagnostic test (PASA) to monitor the frequency of the kdr mutation in natural populations of C pomonella in order to define appropriate insecticide treatments in orchards.

Identification of mutations associated with pyrethroid resistance in the para-type sodium channel of the cat flea, Ctenocephalides felis

Knockdown resistance (kdr) to pyrethroid insecticides is caused by point mutations in the pyrethroid target site, the para-type sodium channel of nerve membranes. This most commonly involves alterations within the domain II (S4–S6) region of the channel protein where five different mutation sites have been identified across a range of insect species. To investigate the incidence of this mechanism in cat fleas, we have cloned and sequenced the IIS4–IIS6 region of the para sodium channel gene from seven laboratory flea strains. Analysis of these sequences revealed two amino acid replacements at residues previously implicated in pyrethroid resistance. One is the ‘common’ kdr mutation, a leucine to phenylalanine substitution (equivalent to L1014F of housefly) reported previously in several other insects. The other is a threonine to valine substitution (equivalent to T929V) and is a novel variant of the T929I mutation first identified in diamondback moth. The L1014F mutation was found at varying frequency in all of the laboratory flea strains, whereas the T929V mutation was found only in the highly resistant Cottontail strain. We have developed rapid PCR-based diagnostic assays for the detection of these mutations in individual cat fleas and used them to show that both L1014F and T929V are common in UK and US flea populations. This survey revealed a significant number of fleas that carry only the V929 allele indicating that co-expression with the F1014 allele is not necessary for flea viability.

Alternative splicing of an insect sodium channel gene generates pharmacologically distinct sodium channels.

Alternative splicing is a major mechanism by which potassium and calcium channels increase functional diversity in animals. Extensive alternative splicing of the para sodium channel gene and developmental regulation of alternative splicing have been reported in Drosophila species. Alternative splicing has also been observed for several mammalian voltage-gated sodium channel genes. However, the functional significance of alternative splicing of sodium channels has not been demonstrated. In this study, we identified three mutually exclusive alternative exons encoding part of segments 3 and 4 of domain III in the German cockroach sodium channel gene, para(CSMA). The splice site is conserved in the mouse, fish, and human Na(v)1.6 sodium channel genes, suggesting an ancient origin. One of the alternative exons possesses a stop codon, which would generate a truncated protein with only the first two domains. The splicing variant containing the stop codon is detected only in the PNS, whereas the other two full-size variants were detected in both the PNS and CNS. When expressed in Xenopus oocytes, the two splicing variants produced robust sodium currents, but with different gating properties, whereas the splicing variant with the stop codon did not produce any detectable sodium current. Furthermore, these two functional splicing variants exhibited a striking difference in sensitivity to a pyrethroid insecticide, deltamethrin. Exon swapping partially reversed the channel sensitivity to deltamethrin. Our results therefore provide the first evidence that alternative splicing of a sodium channel gene produces pharmacologically distinct channels.

Sensitivity to diethylether anesthesia of fruit flies primarily depends on the genotypes of the sodium channel gene rather than the states of the membranes and the mechanisms might be different from heat-induced paralysis

The para locus of Drosophila melanogaster encodes the α subunit of a voltage-sensitive sodium channel. Many of the mutants develop paralysis at the high temperature (37°C) and are hypersensitive to diethylether anesthesia. We examined whether the two aspects of the phenotype are mediated by a same mechanism that involves the sodium channel molecule by investigating properties of the three para alleles ( para hd838 , para ts1 and para ts3 ). Larvae of the all para strains showed almost normal sensitivities to diethylether anesthesia while adult flies of them showed hypersensitivities to that in the following manner: para hd838 ≦ para ts1 < para ts3 < Canton-S. Larvae of the two para strains showed hypersensitivities to heat-induced paralysis in the following manner: para ts1 = para ts3 < para hd838 = Canton-S, while adult flies of the all para strains showed hypersensitivities to that in the following manner: para ts1 = para ts3 < para hd838 < Canton-S. The distinct phenotype of para hd838 from para ts1 or para ts3 observed in the larval and adult heat-induced paralysis, would be the reflection of the difference of the mutation sites between in para hd838 and para ts1 or para ts3 . In addition, because the rank of sensitivity in the adult anesthesia and heat-induced paralysis were reversed between para hd838 and para ts1 or para ts3 , the mechanisms of diethylether anesthesia and heat-induced paralysis are not the same. There would be the anesthesia-specific mechanisms because the sensitivity to anesthesia became more remarkable in para hd838 than in para ts1 or para ts3 . para hd838 has an insertion of P-element, one of a transposable element, in the second intron of the para sodium channel gene. Excision of the P-element from the para locus of the para hd838 conferred the flies the wild-typic phenotype and reduction of the para gene in para hd838 dramatically enhanced the hypersensitivity to anesthesia, suggesting that the phenotype of the strain was caused exclusively by the mutation of the para locus. The susceptibility to anesthesia also depends on the temperature at which the flies were assayed, not at which they were cultured, but the dependence on the assay temperature was smaller than that on the genotype. By these findings we could assure that the sensitivity to diethylether anesthesia of para hd838 primarily depends on the genotypes of the sodium channel gene rather than other reasons such as the fluidity of the membranes.

Novel point mutations in the German cockroach para sodium channel gene are associated with knockdown resistance ( kdr) to pyrethroid insecticides

Knockdown resistance ( kdr) to pyrethroid insecticides has been attributed to point mutations in the para sodium channel gene in more than a half dozen insect pest species. In this study, we identified two novel para mutations in five highly resistant kdr-type German cockroach strains. The two mutations, from glutamic acid (E434) to lysine (K434) and from cysteine (C764) to arginine (R764), respectively, are located in the first intracellular linker connecting domains I and II. E434K is located near the beginning of the linker (closest to domain I), whereas C764R is found toward the end of the linker (closest to domain II). Two additional mutations from aspartic acid (D58) to glycine (G58), and from proline (P1880) to leucine (L1888), respectively, were found in one of the resistant strains. The four mutations coexist with the previously identified leucine to phenylalanine (L993F) kdr mutation in IIS6, and are present only in the highly resistant individuals of a given strain. These findings suggest that these mutations might be responsible for high levels of knockdown resistance toward pyrethroid insecticides in the German cockroach.

Molecular mapping of insecticide resistance genes in the yellow fever mosquito (Aedes aegypti).

Several loci conferring insecticide resistance in the yellow fever mosquito (Aedes aegypti) have previously been mapped by simple recombinational mapping. Here we describe correlation of these resistance phenotypes with molecular gene probes for insecticide target sites by RFLP mapping. The para sodium channel gene homologue and the GABA receptor gene Resistance to dieldrin map to the same genome regions as the DDT/pyrethroid and cyclodiene resistance loci, respectively. Although the acetylcholinesterase (target site of organophosphorus and carbamate insecticides) gene Ace does not map to any known resistance locus, it maps very close to the sex-determining locus. We discuss the possibilities that, if identified, Ace-mediated resistance in A. aegypti will be sex linked or that, as suggested for anopheline mosquitoes, two independent Ace loci may exist, one of which is autosomal. These results support the importance of target site insensitivity as an insecticide resistance mechanism in mosquitoes.

Multiple Allelic Variation in a Sodium Channel Gene from Populations of Australian Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) Detected via Temperature Gradient Gel Electrophoresis

Helicoverpa armigera, the cotton bollworm; is a significant pest of Australian field and vegetable crops and has a widespread incidence of resistance to pyrethroid insecticides. Naturally occurring variation in the para sodium channel gene was assayed in H. armigera. the polymerase chain reaction (PCR) was used to amplify part of the gene and temperature gradient gel electrophoresis (TGGE) used to detect differences in the melting properties of the amplified DNA fragments. A gene fragment of approximately 890 bp was characterised in a total of 66 individuals, comprising four sample groups: untreated controls and fenvalerate‐treated individuals from both the Namoi valley, NSW and the St. George irrigation area, Qld. At least sixteen alleles were detected for the 3′‐end, 250 bp, Hpall digest fragment of the amplified part of the gene. the Namoi and St. George allele distributions were significantly heterogeneous, making this locus a suitable marker for use, with others, in migratory studies among H. armigera populations. Since this gene is a candidate for pyrethroid resistance in the related American pest Heliothis virescens (F.) we looked for a difference in allele distributions between insecticide treated and untreated groups. No association between molecular variation in this gene and fenvalerate resistance was detected.

A single amino acid change in the para sodium channel protein is associated with knockdown-resistance ( kdr) to pyrethroid insecticides in German cockroach

Previous genetic and pharmacological studies suggest that knockdown-resistance ( kdr) to pyrethroid insecticides likely results from a mutation(s) in the para sodium channel gene. In this study, para sodium channel genes from an insecticide-susceptible German cockroach strain, CSMA, and a kdr-type German cockroach strain, Ectiban-R, were cloned and sequenced. Comparison of the nucleotide sequences of para CSMA and para Ectiban-R revealed two nucleotide differences at nt 1491 and nt 2979, respectively. Only the difference at nt 2979 (G in para CSMA and C in para Ectiban-R) resulted in an amino acid change (Leu 993 in CSMA and Phe 993 in Ectiban-R). Leu 993/Phe 993 is located in the highly conserved membrane-spanning segment 6 of domain II (IIS6). Furthermore, reverse transcription-polymerase chain reaction (RT-PCR) sequence analysis using another two insecticide-susceptible and one kdr German cockroach strain, revealed that C 2979 was specifically associated with kdr resistant strains, whereas only G 2979 was present in all susceptible strains. These results suggest that a single amino acid change from Leu 993 to Phe 993 is responsible for the kdr-type resistance in German cockroach. The sequences reported in this paper have been deposited in the Genbank database (accession nos U73583 and U73584).

Developmental Analysis of the smellblind Mutants: Evidence for the Role of Sodium Channels in Drosophila Development

We have recently found evidence that two smellblind mutants, originally identified as olfactory mutants, define a new class of mutation in the para (paralytic) sodium channel gene (Lilly et al., in press). These two mutants have previously been shown to be heat-sensitive developmental lethals: animals raised at elevated temperatures die as embryos or as young larvae (Lilly and Carlson, 1990, Genetics 124, 293-302). This article shows, surprisingly, that both mutants are not only heat-sensitive lethals, but also cold-sensitive lethals: when raised at reduced temperatures, the mutants develop to the adult stage, but then die prematurely. Moreover, this effect is sexually dimorphic, in that lethality is substantially greater for females than males. The association of cold-sensitivity with mutations of a sodium channel gene has recently been found in the case of a cold-sensitive human muscle disease, paramyotonia congenita, in which sodium channel function is defective (Ptacek et al., 1992, Neuron 8, 891-897). The heat-sensitive and cold-sensitive periods for the two smellblind mutants are determined through a series of reciprocal temperature shift experiments and are found to be different. Most, if not all, of the contribution to the heat-sensitive lethality occurs before metamorphosis, whereas the cold-sensitivity is particularly pronounced after the onset of metamorphosis. These results are discussed in terms of the developmental role of the para sodium channel and the role of electrical activity in Drosophila development.