Mutations and intron polymorphisms in voltage-gated sodium channel genes of different geographic populations of Culex pipiens pallens/Culex pipiens quinquefasciatus in China.

BACKGROUND: Culex pipiens pallens and Culex pipiens quinquefasciatus are the dominant species of Culex mosquitoes in China and important disease vectors. Long-term use of insecticides can cause mutations in the voltage-gated sodium channel (vgsc) gene of mosquitoes, but little is known about the current status and evolutionary origins of vgsc gene in different geographic populations. Therefore, this study aimed to determine the current status of vgsc genes in Cx. p. pallens and Cx. p. quinquefasciatus in China and to investigate the evolutionary inheritance of neighboring downstream introns of the vgsc gene to determine the impact of insecticides on long-term evolution. METHODS: Sampling was conducted from July to September 2021 in representative habitats of 22 provincial-level administrative divisions in China. Genomic DNA was extracted from 1308 mosquitoes, the IIS6 fragment of the vgsc gene on the nerve cell membrane was amplified using polymerase chain reaction, and the sequence was used to evaluate allele frequency and knockdown resistance (kdr) frequency. MEGA 11 was used to construct neighbor-joining (NJ) tree. PopART was used to build a TCS network. RESULTS: There were 6 alleles and 6 genotypes at the L1014 locus, which included the wild-type alleles TTA/L and CTA/L and the mutant alleles TTT/F, TTC/F, TCT/S and TCA/S. The geographic populations with a kdr frequency less than 20.00% were mainly concentrated in the regions north of 38° N, and the geographic populations with a kdr frequency greater than 80.00% were concentrated in the regions south of 30° N. kdr frequency increased with decreasing latitude. And within the same latitude, the frequency of kdr in large cities is relatively high. Mutations were correlated with the number of introns. The mutant allele TCA/S has only one intron, the mutant allele TTT/F has three introns, and the wild-type allele TTA/L has 17 introns. CONCLUSIONS: Cx. p. pallens and Cx. p. quinquefasciatus have developed resistance to insecticides in most regions of China. The neighboring downstream introns of the vgsc gene gradually decreased to one intron with the mutation of the vgsc gene. Mutations may originate from multiple mutation events rather than from a single origin, and populations lacking mutations may be genetically isolated.

Pyrethroid resistance status and co-occurrence of V1016G, F1534C and S989P mutations in the Aedes aegypti population from two dengue outbreak counties along the China-Myanmar border.

BACKGROUND: Over the past two decades, dengue fever (DF) has emerged as a significant arboviral disease in Yunnan province, China, particularly in the China-Myanmar border area. Aedes aegypti, an invasive mosquito species, plays a crucial role in transmitting the dengue virus to the local population. Insecticide-based vector control has been the primary tool employed to combat DF, but the current susceptibility status of Ae. aegypti to commonly used insecticides is unknown. Assessment of Ae. aegypti resistance to pyrethroid insecticides and an understanding of the underlying mechanisms of this resistance in the China-Myanmar border region is of significant strategic importance for effectively controlling the DF epidemic in the area. METHODS: Aedes aegypti larvae collected from Ruili and Gengma counties in Yunnan Province were reared to adults in the laboratory and tested for susceptibility to three pyrethroid insecticides (3.20% permethrin, 0.08% lambda-cyhalothrin and 0.20% deltamethrin) by the standard WHO susceptibility bioassay. Genotyping of mutations in the knockdown gene (kdr), namely S989P, V1016G and F1534C, that are responsible for resistance to pyrethroid insecticides was performed using allele-specific PCR methods. A possible association between the observed resistant phenotype and mutations in the voltage-gated sodium channel gene (VGSC) was also studied. RESULTS: Aedes aegypti mosquitoes collected from the two counties and reared in the laboratory were resistant to all of the pyrethroids tested, with the exception of Ae. aegypti from Gengma County, which showed sensitivity to 0.20% deltamethrin. The mortality rate of Ae. aegypti from Ruili county exposed to 3.20% permethrin did not differ significantly from that of Ae. aegypti from Gengma County (χ2 = 0.311, P = 0.577). By contrast, the mortality rate of Ae. aegypti from Ruili County exposed to 0.08% lambda-cyhalothrin and 0.20% deltamethrin, respectively, was significantly different from that of Ae. aegypti from Gengma. There was no significant difference in the observed KDT50 of Ae. aegypti from the two counties to various insecticides. Four mutation types and 12 genotypes were detected at three kdr mutation sites. Based on results from all tested Ae. aegypti, the V1016G mutation was the most prevalent kdr mutation (100% prevalence), followed by the S989P mutation (81.6%) and the F1534C mutation (78.9%). The constituent ratio of VGSC gene mutation types was significantly different in Ae. aegypti mosquitoes from Ruili and those Gengma. The triple mutant S989P + V1016G + F1534C was observed in 274 Ae. aegypti mosquitoes (60.8%), with the most common genotype being SP + GG + FC (31.4%). The prevalence of the F1534C mutation was significantly higher in resistant Ae. aegypti from Ruili (odds ratio [OR] 7.43; 95% confidence interval [CI] 1.71-32.29; P = 0.01) and Gengma (OR 9.29; 95% CI 3.38-25.50; P = 0.00) counties than in susceptible Ae. aegypti when exposed to 3.20% permethrin and 0.08% lambda-cyhalothrin, respectively. No significant association was observed in the triple mutation genotypes with the Ae. aegypti population exposed to 3.20% permethrin and 0.20% deltamethrin resistance (P > 0.05), except for Ae. aegypti from Gengma County when exposed to 0.08% lambda-cyhalothrin (OR 2.86; 95% CI 1.20-6.81; P = 0.02). CONCLUSIONS: Aedes aegypti from Ruili and Gengma counties have developed resistance to various pyrethroid insecticides. The occurrence of multiple mutant sites in VGSC strongly correlated with the high levels of resistance to pyrethroids in the Ae. aegypti populations, highlighting the need for alternative strategies to manage the spread of resistance. A region-specific control strategy for dengue vectors needs to be implemented in the future based on the status of insecticide resistance and kdr mutations.

Geographical distribution of pyrethroid resistance mutations in Varroa destructor across Türkiye and a European overview.

Varroa destructor Anderson & Trueman (Acari: Varroidae) is of paramount significance in modern beekeeping, with infestations presenting a primary challenge that directly influences colony health, productivity, and overall apicultural sustainability. In order to control this mite, many beekeepers rely on a limited number of approved synthetic acaricides, including the pyrethroids tau-fluvalinate, flumethrin and organophosphate coumaphos. However, the excessive use of these substances has led to the widespread development of resistance in various beekeeping areas globally. In the present study, the occurrence of resistance mutations in the voltage-gated sodium channel (VGSC) and acetylcholinesterase (AChE), the target-site of pyrethroids and coumaphos, respectively, was examined in Varroa populations collected throughout the southeastern and eastern Anatolia regions of Türkiye. All Varroa samples belonged to the Korean haplotype, and a very low genetic distance was observed based on cytochrome c oxidase subunit I (COI) gene sequences. No amino acid substitutions were determined at the key residues of AChE. On the other hand, three amino acid substitutions, (L925V/I/M), previously associated with pyrethroid resistance, were identified in nearly 80% of the Turkish populations. Importantly, L925M, the dominant mutation in the USA, was detected in Turkish Varroa populations for the first time. To gain a more comprehensive perspective, we conducted a systematic analysis of the distribution of pyrethroid resistance mutations across Europe, based on the previously reported data. Varroa populations from Mediterranean countries such as Türkiye, Spain, and Greece exhibited the highest frequency of resistance mutation. Revealing the occurrence and geographical distribution of pyrethroid resistance mutations in V. destructor populations across the country will enhance the development of more efficient strategies for mite management.

Dissection of the structure-function relationship of Nav channels.

Voltage-gated sodium channels (Nav) undergo conformational shifts in response to membrane potential changes, a mechanism known as the electromechanical coupling. To delineate the structure-function relationship of human Nav channels, we have performed systematic structural analysis using human Nav1.7 as a prototype. Guided by the structural differences between wild-type (WT) Nav1.7 and an eleven mutation-containing variant, designated Nav1.7-M11, we generated three additional intermediate mutants and solved their structures at overall resolutions of 2.9-3.4 Å. The mutant with nine-point mutations in the pore domain (PD), named Nav1.7-M9, has a reduced cavity volume and a sealed gate, with all voltage-sensing domains (VSDs) remaining up. Structural comparison of WT and Nav1.7-M9 pinpoints two residues that may be critical to the tightening of the PD. However, the variant containing these two mutations, Nav1.7-M2, or even in combination with two additional mutations in the VSDs, named Nav1.7-M4, failed to tighten the PD. Our structural analysis reveals a tendency of PD contraction correlated with the right shift of the static inactivation I-V curves. We predict that the channel in the resting state should have a "tight" PD with down VSDs.

Sodium Channel ß Subunits-An Additional Element in Animal Tetrodotoxin Resistance?

Tetrodotoxin (TTX) is a neurotoxic molecule used by many animals for defense and/or predation, as well as an important biomedical tool. Its ubiquity as a defensive agent has led to repeated independent evolution of tetrodotoxin resistance in animals. TTX binds to voltage-gated sodium channels (VGSC) consisting of α and ß subunits. Virtually all studies investigating the mechanisms behind TTX resistance have focused on the α subunit of voltage-gated sodium channels, where tetrodotoxin binds. However, the possibility of ß subunits also contributing to tetrodotoxin resistance was never explored, though these subunits act in concert. In this study, we present preliminary evidence suggesting a potential role of ß subunits in the evolution of TTX resistance. We gathered mRNA sequences for all ß subunit types found in vertebrates across 12 species (three TTX-resistant and nine TTX-sensitive) and tested for signatures of positive selection with a maximum likelihood approach. Our results revealed several sites experiencing positive selection in TTX-resistant taxa, though none were exclusive to those species in subunit ß1, which forms a complex with the main physiological target of TTX (VGSC Nav1.4). While experimental data validating these findings would be necessary, this work suggests that deeper investigation into ß subunits as potential players in tetrodotoxin resistance may be worthwhile.

Lidocaine Inhibits Rat Prostate Cancer Cell Invasiveness and Voltage-Gated Sodium Channel Expression in Plasma Membrane.

There is increasing evidence, mostly from breast cancer, that use of local anaesthetics during surgery can inhibit disease recurrence by suppressing the motility of the cancer cells dependent on inherent voltage-gated sodium channels (VGSCs). Here, the possibility that lidocaine could affect cellular behaviours associated with metastasis was tested using the Dunning cell model of rat prostate cancer. Mostly, the strongly metastatic (VGSC-expressing) Mat-LyLu cells were used under both normoxic and hypoxic conditions. The weakly metastatic AT-2 cells served for comparison in some experiments. Lidocaine (1-500 µM) had no effect on cell viability or growth but suppressed Matrigel invasion dose dependently in both normoxia and hypoxia. Used as a control, tetrodotoxin produced similar effects. Exposure to hypoxia increased Nav1.7 mRNA expression but VGSCα protein level in plasma membrane was reduced. Lidocaine under both normoxia and hypoxia had no effect on Nav1.7 mRNA expression. VGSCα protein expression was suppressed by lidocaine under normoxia but no effect was seen in hypoxia. It is concluded that lidocaine can suppress prostate cancer invasiveness without effecting cellular growth or viability. Extended to the clinic, the results would suggest that use of lidocaine, and possibly other local anaesthetics, during surgery can suppress any tendency for post-operative progression of prostate cancer.

Voltage-gated sodium channel gene mutation and P450 gene expression are associated with the resistance of Aphis spiraecola Patch (Hemiptera: Aphididae) to lambda-cyhalothrin.

Aphis spiraecola Patch is one of the most economically important tree fruit pests worldwide. The pyrethroid insecticide lambda-cyhalothrin is commonly used to control A. spiraecola. In this 2-year study, we quantified the resistance level of A. spiraecola to lambda-cyhalothrin in different regions of the Shaanxi province, China. The results showed that A. spiraecola had reached extremely high resistance levels with a 174-fold resistance ratio (RR) found in the Xunyi region. In addition, we compared the enzymatic activity and expression level of P450 genes among eight A. spiraecola populations. The P450 activity of A. spiraecola was significantly increased in five regions (Xunyi, Liquan, Fengxiang, Luochuan, and Xinping) compared to susceptible strain (SS). The expression levels of CYP6CY7, CYP6CY14, CYP6CY22, P4504C1-like, P4506a13, CYP4CZ1, CYP380C47, and CYP4CJ2 genes were significantly increased under lambda-cyhalothrin treatment and in the resistant field populations. A L1014F mutation in the sodium channel gene was found and the mutation rate was positively correlated with the LC50 of lambda-cyhalothrin. In conclusion, the levels of lambda-cyhalothrin resistance of A. spiraecola field populations were associated with P450s and L1014F mutations. Our combined findings provide evidence on the resistance mechanism of A. spiraecola to lambda-cyhalothrin and give a theoretical basis for rational and effective control of this pest species.

Correlation between pyrethroid knockdown resistance and mutation frequency of voltage-gated sodium channel and its application in Aedes aegypti management.

Aedes aegypti, the primary vector responsible for transmitting dengue fever in southern Taiwan, has developed a relatively high resistance to synthetic pyrethroids. It has evolved four amino acid substitutions in the voltage-gated sodium channel (VGSC), namely S996P, V1023G, F1565C, and D1794Y. To unveil the distribution and correlation of VGSC mutations and pyrethroid resistance among different field populations, Ae. aegypti collected from various districts in Kaohsiung and Tainan Cities underwent tests for resistance development against different pyrethroids and frequency of S996P, V1023G, F1565C, and D1794Y substitutions. The adult knockdown assay revealed a relatively high knockdown resistance in the Ae. aegypti populations from Kaohsiung and Tainan against permethrin, cypermethrin, and fenvalerate (averaging >50-fold). Conversely, less resistance was observed against α-cypermethrin, deltamethrin, λ-cyhalothrin, cyfluthrin, and etofenprox (averaging <35-fold). Using Polymerase Chain Reaction/restriction fragment length polymorphism analysis, four mutant haplotypes were identified in these field populations. Notably, the SIAVFD and SIBVFD wild haplotypes were absent. Analysis utilizing IBM SPSS Statistics 20.0 and Spearman's rank correlation coefficient indicated that Haplotype C (PIAGFD), especially P allele, frequency displayed a significant positive correlation with five Type II pyrethroid resistance, while 1023G and 1023G/G exhibited a significant association with permethrin and fevalerate resistance. Conversely, Haplotype E (SIBVCD) negatively correlated with pyrethroid resistance, particularly fenvalerate resistance (-0.776). Haplotype C and E were the most prevalent and widely distributed among the investigated field populations. This prevalence of haplotype C is likely tied to the extensive and excessive use of Type II pyrethroids for dengue control over the past three decades. Given the significant positive correlation, the best-fit lines and R2 values were established to facilitate the swift prediction of knockdown resistance levels to various pyrethroids based on VGSC mutation frequency. This predictive approach aims to guide insecticide usage and the management of pyrethroid resistance in the field populations of Ae. aegypti in Taiwan.

Kdr genotyping and the first report of V410L and V1016I kdr mutations in voltage-gated sodium channel gene in Aedes aegypti (Diptera: Culicidae) from Iran.

BACKGROUND: Aedes aegypti is the main vector of arboviral diseases worldwide. The species invaded and became established in southern Iran in 2020. Insecticide-based interventions are primarily used for its control. With insecticide resistance widespread, knowledge of resistance mechanisms is vital for informed deployment of insecticidal interventions, but information from Iranian Ae. aegypti is lacking. METHODS: Fifty-six Ae. aegypti specimens were collected from the port city of Bandar Lengeh in Hormozgan Province in the South of Iran in 2020 and screened for kdr mutations. The most common kdr mutations in Latin America and Asia (V410L, S989P, V1016G/I and F1534C), especially when present in combinations, are highly predictive of DDT and pyrethroid resistance were detected. Phylogenetic analyses based on the diversity of S989P and V1016G/I mutations were undertaken to assess the phylogeography of these kdr mutations. RESULTS: Genotyping all four kdr positions of V410L, S989P, V1016G/I and F1534C revealed that only 16 out of the 56 (28.57%) specimens were homozygous wild type for all kdr mutation sites. Six haplotypes including VSVF (0.537), VSVC (0.107), LSVF (0.016), LSIF (0.071), VPGC (0.257) and LPGC (0.011) were detected in this study. For the first time, 11 specimens harbouring the V410L mutation, and 8 samples with V1016I mutation were found. V410L and V1016I were coincided in 8 specimens. Also, six specimens contained 1016G/I double mutation which was not reported before. CONCLUSIONS: The relatively high frequency of these kdr mutations in Iranian Ae. aegypti indicates a population exhibiting substantial resistance to pyrethroid insecticides, which are used widely in control operations and household formulations. The detection of the 410L/1016I kdr mutant haplotype in Iranian Ae. aegypti suggests possible convergence of invasive populations from West Africa or Latin America. However, as Iran has very limited maritime/air connections with those African countries, a Latin American origin for the invasive Ae. aegypti in Iran is more plausible.

Functional diversity of voltage-gated sodium channel in Drosophila suzukii (Matsumura).

BACKGROUND: The larvae of Drosophila suzukii Matsumura feed directly inside the fruit, causing catastrophic damage to orchards. The misuse of pyrethroid insecticides during the control period has led to increasing resistance of D. suzukii to pyrethroids acting on the voltage-gated sodium channel (VGSC). RESULTS: In this study, the sodium channel of D. suzukii was cloned (DsNav 5 GenBank number: OQ871532). The results of multiple-sequence alignment showed that the homology of sodium channel between D. suzukii and Drosophila melanogaster was as high as 95.3%. Analysis of transcripts from 62 variants of D. suzukii VGSC revealed a total of six alternative splicing sites (exons u, j, a, b, e, and h) and 33 RNA editing. Exons j, a, b, e, and h are conserved in D. melanogaster and other insects, whereas exon u has never been reported before. The number of A-to-I was distinctly more than that of U-to-C for RNA editing. All D. suzukii VGSC variants were expressed in Xenopus oocytes, but only one (type 5) was able to produce robust currents and nine produce weak currents. DsNav 5 with TipE of D. melanogaster co-expresses current better than its own TipE. Subsequently, tetrodotoxin was verified to be a blocker of VGSC, and the gating properties of DsNav 5 were investigated. CONCLUSION: These findings proved that the VGSC of D. suzukii has not only the basic gating properties, but also the diversity of gating properties. This study also laid a foundation for the study of pyrethroid resistance mechanism of VGSC in D. suzukii. © 2023 Society of Chemical Industry.

Distribution and frequency of ace-1 and kdr mutations of Culex pipiens subgroup in the Republic of Korea.

Mosquitoes in the Culex pipiens subgroup are the primary vectors of the West Nile virus. Two members, Culex pallens and Culex pipiens f. molestus, are present in the Republic of Korea (ROK). Because the Culex pipiens subgroup occurs in large amounts, often near human habitation, it is frequently exposed to various insecticides, which is probably responsible for the rapid evolution of insecticide resistance traits. Experiments related to insecticide resistance in the Culex pipiens subgroup conducted in the ROK have been performed without discrimination below the species level. This study categorized Culex pipiens mosquitoes subgroup from 13 regions in the ROK into Culex pallens and Culex pipiens f. molestus, and target site genotypes for acetylcholinesterase-1 (ace-1) and voltage-gated sodium channel (vgsc) genes were identified for each taxon. Screening for ace-1 did not identify a resistant allele (G119S) in Cx. pipiens f. molestus, and heterozygous resistance (AGC/GGC) was identified in one Cx. pallens collected in Mokpo. In vgsc, knockdown resistance (kdr) mutations [TTT(L1014F) and TCA(L1014S)] were present in both taxa, with Cx. pipiens f. molestus having homozygous resistance (TTT/TTT): 44%, heterozygous resistance (TTT/TTA): 28%, and homozygous susceptibility (TTA/TTA): 28%, whereas Cx. pallens showed homozygous resistance (TTT/TTT or TCA/TCA): 26%, heterozygous resistance (TTT/TTA, TTT/TCA, or TCA/TTA): 26%, and homozygous susceptibility (TTA/TTA): 48%. Furthermore, the unique vgsc allele was present in both Cx. pipiens f. molestus and Cx. pallens. This was the first experiment to analyze the Culex pipiens subgroup living in the ROK below the species level, and its results could be used in the future for more detailed mosquito control.

LRRC37B is a human modifier of voltage-gated sodium channels and axon excitability in cortical neurons.

The enhanced cognitive abilities characterizing the human species result from specialized features of neurons and circuits. Here, we report that the hominid-specific gene LRRC37B encodes a receptor expressed in human cortical pyramidal neurons (CPNs) and selectively localized to the axon initial segment (AIS), the subcellular compartment triggering action potentials. Ectopic expression of LRRC37B in mouse CPNs in vivo leads to reduced intrinsic excitability, a distinctive feature of some classes of human CPNs. Molecularly, LRRC37B binds to the secreted ligand FGF13A and to the voltage-gated sodium channel (Nav) ß-subunit SCN1B. LRRC37B concentrates inhibitory effects of FGF13A on Nav channel function, thereby reducing excitability, specifically at the AIS level. Electrophysiological recordings in adult human cortical slices reveal lower neuronal excitability in human CPNs expressing LRRC37B. LRRC37B thus acts as a species-specific modifier of human neuron excitability, linking human genome and cell evolution, with important implications for human brain function and diseases.

First report of kdr mutations in the voltage-gated sodium channel gene in the arbovirus vector, Aedes aegypti, from Nouakchott, Mauritania.

BACKGROUND: Since 2014, dengue epidemics have occurred almost annually in Nouakchott, the capital city of Mauritania, coinciding with the recent establishment of Aedes aegypti, the primary vector of dengue, in the city. Anopheles arabiensis, the primary vector of malaria, is also abundant not only in Nouakchott but also in most areas of the country. Resistance to insecticides has been studied in An. arabiensis but not in Ae. aegypti in Mauritania. The objective of the present study was to establish the baseline data on the frequencies of knockdown resistance (kdr) mutations in the voltage-gated sodium channel (vgsc) gene in Ae. aegypti collected in Nouakchott to improve vector control. METHODS: Resting Ae. aegypti mosquitoes were collected in 2017 and 2018 in Teyarett and Dar Naim districts in Nouakchott using a battery-powered aspirator. Polymerase chain reaction (PCR) and DNA sequencing were performed to detect the presence of five kdr mutations known to be associated with pyrethroid resistance: L982W, S989P, I1011M/G, V1016G/I, and F1534C. RESULTS: A total of 100 female Ae. aegypti mosquitoes were identified among collected resting culicid fauna, of which 60% (60/100) were unfed, 12% (12/100) freshly blood-fed, and 28% (28/100) gravid. Among the mutations investigated in this study, 989P, 1016G, and 1534C were found to be widespread, with the frequencies of 0.43, 0.44, and 0.55, respectively. Mutations were not found in codons 982 and 1011. No other mutations were detected within the fragments analyzed in this study. Genotype distribution did not deviate from Hardy-Weinberg equilibrium. The most frequent co-occurring point mutation patterns among Ae. aegypti mosquitoes were the heterozygous individuals 989SP/1016VG/1534FC detected in 45.1% of mosquitoes. In addition, homozygous mutant 1534CC co-occurred simultaneously with homozygous wild type 989SS and 1016VV in 30.5% of mosquito specimens. Inversely, homozygous wild-type 1534FF co-occurred simultaneously with homozygous mutant 989PP and 1016GG in 19.5% of the mosquitoes. CONCLUSIONS: To our knowledge, this is the first study reporting the presence of three point mutations in the vgsc gene of Ae. aegypti in Mauritania. The findings of the present study are alarming because they predict a high level of resistance to pyrethroid insecticides which are commonly used in vector control in the country. Therefore, further studies are urgently needed, in particular phenotypic characterization of insecticide resistance using the standardized test.

Mutations Linked to Insecticide Resistance Not Detected in the Ace-1 or VGSC Genes in Nyssorhynchus darlingi from Multiple Localities in Amazonian Brazil and Peru.

Indoor residual spray (IRS), mainly employing pyrethroid insecticides, is the most common intervention for preventing malaria transmission in many regions of Latin America; the use of long-lasting insecticidal nets (LLINs) has been more limited. Knockdown resistance (kdr) is a well-characterized target-site resistance mechanism associated with pyrethroid and DDT resistance. Most mutations detected in acetylcholinesterase-1 (Ace-1) and voltage-gated sodium channel (VGSC) genes are non-synonymous, resulting in a change in amino acid, leading to the non-binding of the insecticide. In the present study, we analyzed target-site resistance in Nyssorhynchus darlingi, the primary malaria vector in the Amazon, in multiple malaria endemic localities. We screened 988 wild-caught specimens of Ny. darlingi from three localities in Amazonian Peru and four in Amazonian Brazil. Collections were conducted between 2014 and 2021. The criteria were Amazonian localities with a recent history as malaria hotspots, primary transmission by Ny. darlingi, and the use of both IRS and LLINs as interventions. Fragments of Ace-1 (456 bp) and VGSC (228 bp) were amplified, sequenced, and aligned with Ny. darlingi sequences available in GenBank. We detected only synonymous mutations in the frequently reported Ace-1 codon 280 known to confer resistance to organophosphates and carbamates, but detected three non-synonymous mutations in other regions of the gene. Similarly, no mutations linked to insecticide resistance were detected in the frequently reported codon (995) at the S6 segment of domain II of VGSC. The lack of genotypic detection of insecticide resistance mutations by sequencing the Ace-1 and VGSC genes from multiple Ny. darlingi populations in Brazil and Peru could be associated with low-intensity resistance, or possibly the main resistance mechanism is metabolic.

The voltage-gated sodium channel, para, limits Anopheles coluzzii vector competence in a microbiota dependent manner.

The voltage-gated sodium channel, para, is a target of DDT and pyrethroid class insecticides. Single nucleotide mutations in para, called knockdown resistant or kdr, which contribute to resistance against DDT and pyrethroid insecticides, have been correlated with increased susceptibility of Anopheles to the human malaria parasite Plasmodium falciparum. However, a direct role of para activity on Plasmodium infection has not yet been established. Here, using RNA-mediated silencing, we provide in vivo direct evidence for the requirement of wild-type (wt) para function for insecticide activity of deltamethrin. Depletion of wt para, which is susceptible to insecticide, causes deltamethrin tolerance, indicating that insecticide-resistant kdr alleles are likely phenocopies of loss of para function. We then show that normal para activity in An. coluzzii limits Plasmodium infection prevalence for both P. falciparum and P. berghei. A transcriptomic analysis revealed that para activity does not modulate the expression of immune genes. However, loss of para function led to enteric dysbiosis with a significant increase in the total bacterial abundance, and we show that para function limiting Plasmodium infection is microbiota dependent. In the context of the bidirectional "enteric microbiota-brain" axis studied in mammals, these results pave the way for studying whether the activity of the nervous system could control Anopheles vector competence.

Functional diversity of sodium channel variants in common eastern bumblebee, Bombus impatiens.

For the past decade, Colony Collapse Disorder has been reported worldwide. Insecticides containing pyrethroids may be responsible for a decline in bees, which are more sensitive to pyrethroids compared with other insects. Voltage-gated sodium channels (Nav ) are the major target sites of pyrethroids, and the sodium channel diversity is generated through extensive alternative splicing and RNA editing. In this study, we cloned and analyzed the function of variants of the Nav channel, BiNav , from Bombus impatiens. BiNav covers a 46 kb genome region including 30 exons. Sequence analysis of 56 clones showed that the clones can be grouped into 22 splice types with 11 optional exons (exons j, w, p, q, r, b, e, t, l/k, and z). Here, a special alternative exon w is identified, encoding a stretch of 31 amino acid resides in domain I between S3 and S4. RNA editing generates 18 amino acid changes in different positions in individual variants. Among 56 variants examined, only six variants generated sufficient sodium currents for functional characterization in Xenopus oocytes. In the presence of B. impatiens TipE and TEH1, the sodium current amplitude of BiNav 1-1 increased by fourfold, while TipE of other insect species had no effect on the expression. Abundant alternative splicing and RNA editing of BiNav suggests the molecular and functional pharmacology diversity of the Nav channel for bumblebees. This study provides a theoretical basis for the design of insecticides that specifically target pests without affecting beneficial insects.

Prokaryotic voltage-gated sodium channels are more effective than endogenous Nav1.5 channels in rescuing cardiac action potential conduction: an in silico study.

Methods to augment Na+ current in cardiomyocytes hold potential for the treatment of various cardiac arrhythmias involving conduction slowing. Because the gene coding cardiac Na+ channel (Nav1.5) is too large to fit in a single adeno-associated virus (AAV) vector, new gene therapies are being developed to enhance endogenous Nav1.5 current (by overexpression of chaperon molecules or use of multiple AAV vectors) or to exogenously introduce prokaryotic voltage-gated Na+ channels (BacNav) whose gene size is significantly smaller than that of the Nav1.5. In this study, based on experimental measurements in heterologous expression systems, we developed an improved computational model of the BacNav channel, NavSheP D60A. We then compared in silico how NavSheP D60A expression vs. Nav1.5 augmentation affects the electrophysiology of cardiac tissue. We found that the incorporation of BacNav channels in both adult guinea pig and human cardiomyocyte models increased their excitability and reduced action potential duration. When compared with equivalent augmentation of Nav1.5 current in simulated settings of reduced tissue excitability, the addition of the BacNav current was superior in improving the safety of conduction under conditions of current source-load mismatch, reducing the vulnerability to unidirectional conduction block during premature pacing, preventing the instability and breakup of spiral waves, and normalizing the conduction and ECG in Brugada syndrome tissues with mutated Nav1.5. Overall, our studies show that compared with a potential enhancement of the endogenous Nav1.5 current, expression of the BacNav channels with their slower inactivation kinetics can provide greater anti-arrhythmic benefits in hearts with compromised action potential conduction.NEW & NOTEWORTHY Slow action potential conduction is a common cause of various cardiac arrhythmias; yet, current pharmacotherapies cannot augment cardiac conduction. This in silico study compared the efficacy of recently proposed antiarrhythmic gene therapy approaches that increase peak sodium current in cardiomyocytes. When compared with the augmentation of endogenous sodium current, expression of slower-inactivating bacterial sodium channels was superior in preventing conduction block and arrhythmia induction. These results further the promise of antiarrhythmic gene therapies targeting sodium channels.

A novel strategy for screening mutations in the voltage-gated sodium channel gene of Aedes albopictus based on multiplex PCR-mass spectrometry minisequencing technology.

BACKGROUND: The current prevention and control strategy for Aedes albopictus heavily relies on comprehensive management, such as environmental management and chemical control. However, the wide application of pyrethroids has facilitated the development of insecticide resistance, primarily via mutations in the voltage-gated sodium channel (VGSC) gene. This study aims to develop a novel strategy for detecting mutations in the VGSC gene in Ae. albopictus using multiplex PCR-mass spectrometry (MPCR-MS) minisequencing technology. METHODS: We established a new strategy for detecting mutations in the VGSC gene in Ae. albopictus using MPCR-MS minisequencing technology. MPCR amplification and mass probe extension (MPE) were first used, followed by single nucleotide polymorphism (SNP) typing mass spectrometry, which allows the simultaneous detection of multiple mutation sites of the VGSC gene in 96 samples of Ae. albopictus. A total of 70 wild-collected Ae. albopictus were used to evaluate the performance of the method by comparing it with other methods. RESULTS: Three target sites (1016, 1532, 1534) in the VGSC gene can be detected simultaneously by double PCR amplification combined with matrix-assisted laser desorption ionization-time-of-flight mass spectrometry, achieving a detection limit of 20 fg/µl. We applied this method to 70 wild-collected Ae. albopictus, and the obtained genotypes were consistent with the routine sequencing results, suggesting the accuracy of our method. CONCLUSIONS: MPCR-MS minisequencing technology provides a sensitive and high-throughput approach to Ae. albopictus VGSC gene mutation screening. Compared with conventional sequencing, this method is economical and time-saving. It is of great value for insecticide resistance surveillance in areas with a high risk of vector-borne disease.

Frequencies and distribution of kdr and Ace alleles that cause insecticide resistance in house flies in the United States.

House flies (Musca domestica L) are nuisances and vectors of pathogens between and among humans and livestock. Population suppression has been accomplished for decades with pyrethroids and acetylcholinesterase (AChE) inhibitors, but recurrent selection has led to increased frequency of alleles conferring resistance to those two classes of active ingredients (Geden et al., 2021). A common mechanism of resistance to both classes involves an altered target site (mutations in Voltage gated sodium channel (Vgsc) for pyrethroids or in Ace for AChE inhibitors). As part of ongoing efforts to understand the origin, spread and evolution of insecticide resistance alleles in house fly populations, we sampled flies in 11 different US states, sequenced, and then estimated frequencies of the Vgsc and Ace alleles. There was substantial variation in frequencies of the four common knockdown resistance alleles (kdr (L1014F), kdr-his (L1014H), super-kdr (M918T + L10414F) and 1B (T929I + L1014F) across the sampled states. The kdr allele was found in all 11 states and was the most common allele in four of them. The super-kdr allele was detected in only six collections, with the highest frequencies found in the north, northeast and central United States. The kdr-his allele was the most common allele in PA, NC, TN and TX. In addition, a novel super-kdr-like mutation in mutually exclusive exon 17a was found. The overall frequencies of the different Ace alleles, which we name based on the amino acid present at the mutation sites (V260L, A316S, G342A/V and F407Y), varied considerably between states. Five Ace alleles were identified: VAGF, VAVY, VAGY, VAAY and VSAY. Generally, the VSAY allele was the most common in the populations sampled. The susceptible allele (VAGF) was found in all populations, ranging in frequency from 3% (KS) to 41% (GA). Comparisons of these resistance allele frequencies with those previously found suggests a dynamic interaction between the different alleles, in terms of levels of resistance they confer and likely fitness costs they impose in the absence of insecticides.

MicroRNA-335-5p suppresses voltage-gated sodium channel expression and may be a target for seizure control.

There remains an urgent need for new therapies for treatment-resistant epilepsy. Sodium channel blockers are effective for seizure control in common forms of epilepsy, but loss of sodium channel function underlies some genetic forms of epilepsy. Approaches that provide bidirectional control of sodium channel expression are needed. MicroRNAs (miRNA) are small noncoding RNAs which negatively regulate gene expression. Here we show that genome-wide miRNA screening of hippocampal tissue from a rat epilepsy model, mice treated with the antiseizure medicine cannabidiol, and plasma from patients with treatment-resistant epilepsy, converge on a single target-miR-335-5p. Pathway analysis on predicted and validated miR-335-5p targets identified multiple voltage-gated sodium channels (VGSCs). Intracerebroventricular injection of antisense oligonucleotides against miR-335-5p resulted in upregulation of Scn1a, Scn2a, and Scn3a in the mouse brain and an increased action potential rising phase and greater excitability of hippocampal pyramidal neurons in brain slice recordings, consistent with VGSCs as functional targets of miR-335-5p. Blocking miR-335-5p also increased voltage-gated sodium currents and SCN1A, SCN2A, and SCN3A expression in human induced pluripotent stem cell-derived neurons. Inhibition of miR-335-5p increased susceptibility to tonic-clonic seizures in the pentylenetetrazol seizure model, whereas adeno-associated virus 9-mediated overexpression of miR-335-5p reduced seizure severity and improved survival. These studies suggest modulation of miR-335-5p may be a means to regulate VGSCs and affect neuronal excitability and seizures. Changes to miR-335-5p may reflect compensatory mechanisms to control excitability and could provide biomarker or therapeutic strategies for different types of treatment-resistant epilepsy.