Voltage-gated Sodium Channel Gene Research Articles

Voltage-gated sodium channels gene expression in Burning Mouth Syndrome: a case-control study

Burning mouth syndrome (BMS) is a condition characterized by painful symptoms of the oral mucosa, despite the absence of any clinical signs. Its etiology is unknown, and there is still no effective treatment to date. Current evidence has shown neuropathic impairment in BMS patients. Neuropathic pain can be related to the dysfunction of voltage-gated sodium channels, considering that these receptors regulate the induction of action potentials in nociceptive neurons. This study evaluated the gene expression of voltage-gated sodium channels Na v 1.7, Na v 1.8 and Na v 1.9 in these patients. The gene expressions of these channels were assessed by real time RT-PCR analysis of fresh-frozen tongue biopsies in a case-control study composed of 12 patients with BMS, and 5 healthy control patients, proportionally matched by sex and age, and analyzed using the 2^(-Delta Delta CT) method. There was no statistically significant difference between the analyzed groups, despite the increase in Na v 1.7 (fold-change = 3.13, p = 0.52) and decrease in Na v 1.9 (fold-change = 0.45, p = 0.36) gene expression in the BMS group. The Na v 1.8 gene was not expressed in any of the samples analyzed. Although the gene expression in the voltage-gated sodium channels in BMS under study seems to be comparable with that of the normal oral mucosa, the functionality of these channels in BMS has not yet been identified, thus suggesting that further research is needed to better understand these voltage-gated sodium channels.

Nonspiking Interneurons in the Drosophila Antennal Lobe Exhibit Spatially Restricted Activity.

Inhibitory interneurons are important for neuronal circuit function. They regulate sensory inputs and enhance output discriminability (Olsen and Wilson, 2008; Root et al., 2008; Olsen et al., 2010). Often, the identities of interneurons can be determined by location and morphology, which can have implications for their functions (Wachowiak and Shipley, 2006). While most interneurons fire traditional action potentials, many are nonspiking. These can be seen in insect olfaction (Laurent and Davidowitz, 1994; Husch et al., 2009; Tabuchi et al., 2015) and the vertebrate retina (Gleason et al., 1993). Here, we present the novel observation of nonspiking inhibitory interneurons in the antennal lobe (AL) of the adult fruit fly, Drosophila melanogaster These neurons have a morphology where they innervate a patchwork of glomeruli. We used electrophysiology to determine whether their nonspiking characteristic is because of a lack of sodium current. We then used immunohistochemsitry and in situ hybridization to show this is likely achieved through translational regulation of the voltage-gated sodium channel gene, para Using in vivo calcium imaging, we explored how these cells respond to odors, finding regional isolation in their responses' spatial patterns. Further, their response patterns were dependent on both odor identity and concentration. Thus, we surmise these neurons are electrotonically compartmentalized such that activation of the neurites in one region does not propagate across the whole antennal lobe. We propose these neurons may be the source of intraglomerular inhibition in the AL and may contribute to regulation of spontaneous activity within glomeruli.

First report of the kdr pyrethroid resistance mutation in a French population of the English grain aphid, Sitobionavenae

The lack of recent insecticide innovations and the withdrawal of efficient, but ecotoxic molecules (e.g. neonicotinoids) may lead to a shift to older insecticidal active substances, particularly pyrethroids. The evolution of resistance to this family of insecticides has long been recognized, including in many aphid species such as the grain aphid Sitobion avenae. The target-site resistance mechanism involved, named kdr (knock-down resistance), was first identified in the United Kingdom in 2014. In this study, we screened for the presence of this mutation in 25 French populations of S. avenae sampled between 2017 and 2021 on various cultivated cereals. Aphids were genotyped individually for the kdr mutation using PCR-RFLP or partial sequencing of the voltage-gated sodium channel gene. Of the 400 S. avenae individuals tested, one carried a heterozygous kdr mutation. This S. avenae kdr mutant was sampled in Northern France, the French region closest to the United Kingdom. The low frequency of the detected mutation and the geographical proximity to the UK suggest that it arose from a migration event and not an independent convergent mutation event, although this hypothesis remains to be confirmed. Our study also reveals that partial sequencing of the voltage-gated sodium-channel gene can be used for another cereal aphid species, Rhopalosiphum padi.

High-Resolution Genetic Mapping Combined with Transcriptome Profiling Reveals That Both Target-Site Resistance and Increased Detoxification Confer Resistance to the Pyrethroid Bifenthrin in the Spider Mite Tetranychus urticae.

Pyrethroids are widely applied insecticides in agriculture, but their frequent use has provoked many cases of resistance, in which mutations in the voltage-gated sodium channel (VGSC), the pyrethroid target-site, were shown to play a major role. However, for the spider mite Tetranychus urticae, it has also been shown that increased detoxification contributes to resistance against the pyrethroid bifenthrin. Here, we performed QTL-mapping to identify the genomic loci underlying bifenthrin resistance in T. urticae. Two loci on chromosome 1 were identified, with the VGSC gene being located near the second QTL and harboring the well-known L1024V mutation. In addition, the presence of an L925M mutation in the VGSC of a highly bifenthrin-resistant strain and its loss in its derived, susceptible, inbred line indicated the importance of target-site mutations in bifenthrin resistance. Further, RNAseq experiments revealed that genes encoding detoxification enzymes, including carboxyl/choline esterases (CCEs), cytochrome P450 monooxygenases and UDP-glycosyl transferases (UGTs), were overexpressed in resistant strains. Toxicity bioassays with bifenthrin (ester pyrethroid) and etofenprox (non-ester pyrethroid) also indicated a possible role for CCEs in bifenthrin resistance. A selection of CCEs and UGTs were therefore functionally expressed, and CCEinc18 was shown to metabolize bifenthrin, while teturUGT10 could glycosylate bifenthrin-alcohol. To conclude, our findings suggest that both target-site and metabolic mechanisms underlie bifenthrin resistance in T. urticae, and these might synergize high levels of resistance.

Molecular analysis of knockdown resistance (kdr) mutations in the voltage-gated sodium channel gene of Aedes aegypti populations from Saudi Arabia

BackgroundThe Aedes aegypti mosquito is the primary vector for dengue, chikungunya, yellow fever and Zika viruses worldwide. The first record of Ae. aegypti in southwestern Saudi Arabia was in 1956. However, the first outbreak and cases of dengue fever were reported in 1994, and cases have increased in recent years. Vector control for Ae. aegypti mainly uses pyrethroid insecticides in outdoor and indoor space spraying. The constant use of pyrethroids has exerted intense selection pressure for developing target-site mutations in the voltage-gated sodium channel (vgsc) gene in Ae. Aegypti against pyrethroids—mutations that have led to knockdown resistance (kdr).MethodsAedes aegypti field populations from five regions (Jazan, Sahil, Makkah, Jeddah and Madinah) of southwestern Saudi Arabia were genotyped for known kdr mutations in domains IIS6 and IIIS6 of the vgsc gene using polymerase chain reaction (PCR) amplification and sequencing. We estimated the frequency of kdr mutations and genotypes from Saudi Arabia as well as from other countries, Thailand, Myanmar (Southeast Asia) and Uganda (East Africa). We constructed haplotype networks to infer the evolutionary relationships of these gene regions.ResultsThe three known kdr mutations, S989P, V1016G (IIS6) and F1534C (IIIS6), were detected in all five regions of Saudi Arabia. Interestingly, the triple homozygous wild genotype was reported for the first time in two individuals from the highlands of the Jazan region and one from the Al-Quoz, Sahil region. Overall, nine genotypes comprising four haplotypes were observed in southwestern Saudi Arabia. The median-joining haplotype networks of eight populations from Saudi Arabia, Southeast Asia and East Africa for both the IIS6 and IIIS6 domains revealed that haplotype diversity was highest in Uganda and in the Jazan and Sahil regions of Saudi Arabia, whereas haplotype diversity was low in the Jeddah, Makkah and Madinah regions. Median-joining haplotype networks of both domains indicated selection acting on the kdr-mutation containing haplotypes in Saudi Arabia.ConclusionsThe presence of wild type haplotypes without any of the three kdr mutations, i.e. that are fully susceptible, in Saudi Arabia indicates that further consideration should be given to insecticide resistance management strategies that could restore pyrethroid sensitivity to the populations of Ae. aegypti in Saudi Arabia as part of an integrative vector control strategy.Graphical

CRISPR/Cas9-mediated F1534S substitution in the voltage-gated sodium channel reveals its necessity and sufficiency for deltamethrin resistance in Aedes albopictus

Insecticide resistance in Aedes mosquitoes presents a major challenge to the control of arboviral diseases. However, resistance mechanisms for many of the insecticides remain unknown. A commonly used insecticide, deltamethrin, was used to select a resistance strain of the vector mosquito, Aedes albopictus, and we identified an F1534S substitution in the voltage-gated sodium channel (VGSC) gene product as the first event in generating resistance. Engineering an F1534S substitution using Cas9/gRNA technologies conferred deltamethrin resistance on a previously susceptible strain. Crosses that removed this mutation restored the susceptible phenotype. Predicted protein structural changes and differences in transcript accumulation levels were correlated with the resistance phenotype. Furthermore, F1534S mutations were detected in all resistant Ae. albopictus populations collected in the field. We conclude that the VGSC F1534S mutation is essential for resistance to deltamethrin in Ae. albopictus, and is a suitable molecular index for pyrethroid resistance detection and monitoring in this species.

First report of F1534C kdr mutation in deltamethrin resistant Aedes albopictus from northern part of West Bengal, India

Dengue is the most rapidly spreading vector-borne disease with an estimated 100–400 million cases each year. Control of Dengue vectors largely depends upon synthetic pyrethroids. Development of insecticide resistance in Aedes mosquitoes however, poses severe threat to insecticide-based vector management programme. Mutations in the Voltage Gated Sodium Channel gene (vgsc) serve as the primary machinery behind this resistance development. In Aedes albopictus, at least four such kdr (knock down resistance) mutations had already been documented. Here, we describe the occurrence of F1534C kdr mutation in wild population of Ae. albopictus from northern part of West Bengal, India including a novel T1520I mutation. Four populations of Ae. albopictus from the studied region were found resistant against DDT and synthetic pyrethroids, among them only one population possessed F1534C kdr mutation. A total of 200 successful amplification followed by partial sequencing of vgsc gene further revealed the presence of F1534C kdr mutation in both phenotypically susceptible and resistant mosquito specimen. Studied populations were found 81% homozygote susceptible (1534F/F), 12.5% heterozygote (1534F/C) and 6% homozygote resistant (1534C/C) for F1534C kdr mutation. The findings of the current study will help to uncover the mechanisms underlying insecticide resistance and hence to reduce errors in vector control measurements.

Identification of Knockdown Resistance Mutations in the Cimex hemipterus (Hemiptera: Cimicidae) in Iran.

The worldwide resurgence of tropical bed bug Cimex hemipterus beginning in the late 1990s has led to growing concern. Molecular data on pyrethroid resistance, which is essential for the control strategies, is unknown for C. hemipterus in Iran. The current study evaluated the deltamethrin resistance status of C. hemipterus by bioassay and molecular tests. Live bed bugs were collected from sleeping quarters (dormitories) in the city of Tehran and used for insecticide bioassay tests. For bioassay evaluation, mixed-sex pools of adult bugs were exposed to deltamethrin (0.025%)-treated paper. Polymerase chain reaction assay evaluated resistance-related mutations in the voltage-gated sodium channel gene (VGSC) gene of studied populations. On the basis of the bioassay test within the 48-h exposure to deltamethrin, C. hemipterus were determined to be resistant. Knockdown time ratios (KR50) in the studied populations of C. hemipterus was 5.5-fold compared with those of the C. lectularius Teh strain. DNA sequencing of the VGSC gene revealed the presence of mutations at M918I and L1014 in C. hemipterus. According to the bioassay and molecular results of current study, C. hemipterus showed a high degree of pyrethroid resistance. The application of multiple approaches including physical, biological, and chemical tests should be regarded in future bed bug control strategies.

First report of classical knockdown resistance (kdr) mutation, L1014F, in human head louse Pediculus humanus capitis (Phthiraptera: Anoplura).

There are at least three known knockdown resistance (kdr) mutations reported globally in the human head louse Pediculus humanus capitis De Geer (Phthiraptera: Anoplura) that are associated with reduced sensitivity to pyrethroids. However, the prevalence of kdr mutation in head lice is not known in the Indian subcontinent. To identify kdr mutations in the Indian head lice population, the genomic region of the voltage-gated sodium channel gene encompassing IIS1-2 linker to IIS6 segments was PCR-amplified and sequenced from P. humanus capitis samples collected from different geographic localities of India. DNA sequencing revealed the presence of four kdr mutations: M827I, T929I, L932F and L1014F. The presence of a classical kdr mutation L1014F, the most widely reported mutation across insect-taxa associated with the kdr-trait, is being reported for the first time in P. humanus capitis.

Novel real-time PCR assay detects widespread distribution of knock down resistance (kdr) mutations associated with pyrethroid resistance in the mosquito, Culex quinquefasciatus, in Thailand

Susceptibility to pyrethroids in the mosquito Culex quinquefasciatus, the major vector of lymphatic filariasis, is being seriously threatened worldwide. Knockdown resistance (kdr), caused by mutations in the voltage gated sodium channel (VGSC) gene, particularly the L1014F mutation, is an important resistance mechanism. Our aim was to develop a real-time PCR with melt curve analysis to evaluate the distribution of the L1014F mutation in Cx. quinquefasciatus throughout Thailand and to determine the polymorphism pattern of a VGSC gene fragment spanning the L1014F mutation. A total of 3760 females from 18 localities across five regions of Thailand were bio-assayed by exposure to 0.05% deltamethrin WHO papers, showing mortality rates ranging from 2.4% to 83.0%. Genotyping of 753 dead and surviving mosquitoes using our novel real-time PCR assay with melt curve analysis and tetra-primer allele-specific PCR revealed the mutant F1014 allele is closely associated with the deltamethrin resistance phenotype. The L1014F mutation was found at high frequency throughout Thailand, particularly in the North. However, some survivors were homozygous for wild type L1014 allele, which were further sequenced for the IIP-IIS6 region of VGSC gene. The haplotype network of phenotypically characterized individuals indicated the presence of other possible kdr alleles/resistance mechanisms at play including two novel mutations, V978E and D992E. The finding of new putative kdr alleles and widespread distribution of the F1014 allele emphasizes the significant role of kdr mutations in pyrethroid resistance in Thai Cx. quinquefasciatus populations. Monitoring kdr variations and phenotypic resistance is critical for managing resistance in Cx. quinquefasciatus.

Voltage-gated sodium channel scn8a is required for innervation and regeneration of amputated adult zebrafish fins

Teleost fishes and urodele amphibians can regenerate amputated appendages, whereas this ability is restricted to digit tips in adult mammals. One key component of appendage regeneration is reinnervation of the wound area. However, how innervation is regulated in injured appendages of adult vertebrates has seen limited research attention. From a forward genetics screen for temperature-sensitive defects in zebrafish fin regeneration, we identified a mutation that disrupted regeneration while also inducing paralysis at the restrictive temperature. Genetic mapping and complementation tests identify a mutation in the major neuronal voltage-gated sodium channel (VGSC) gene scn8ab. Conditional disruption of scn8ab impairs early regenerative events, including blastema formation, but does not affect morphogenesis of established regenerates. Whereas scn8ab mutations reduced neural activity as expected, they also disrupted axon regrowth and patterning in fin regenerates, resulting in hypoinnervation. Our findings indicate that the activity of VGSCs plays a proregenerative role by promoting innervation of appendage stumps.

Population genetic structure and evolutionary genetics of Anopheles sinensis based on knockdown resistance (kdr) mutations and mtDNA-COII gene in China–Laos, Thailand–Laos, and Cambodia–Laos borders

BackgroundVector control is still a pivotal method for preventing malaria, and its potency is weakened by the increasing resistance of vectors to chemical insecticides. As the most abundant and vital malaria vector in Southeast Asia, the chemical insecticide resistance status in Anopheles sinensis remains elusive in Laos, which makes it imperative to evaluate the true nature of chemical insecticide resistance-associated genetic mutations in An. sinensis in Laos.MethodsAdult An. sinensis were collected from three border regions in Laos. DNA was extracted from individual mosquitoes. PCR amplification and DNA sequencing of a fragment containing codon 1014 of the voltage-gated sodium channel (vgsc) gene were completed to study the kdr allele frequency distribution, kdr intron polymorphism, population genetic diversity, and the evolutionary status of the kdr codon. The mitochondrial cytochrome c oxidase subunit II gene (COII) was amplified and sequenced to examine population variations, genetic differentiation, spatial population structure, population expansion, and gene flow patterns.ResultsNine wild kdr haplotypes of the vgsc gene were detected in this study, and eight of them, namely 1014L1, 1014L2, 1014L4, 1014L7, 1014L9, 1014L10, 1014L11, and 1014L21, were discovered in the China–Laos border (northern Laos), while 1014L3 was only detected in the Thailand–Laos border (northwestern Laos) and Cambodia–Laos border (southern Laos). The newly identified haplotype, 1014L21, was uniquely distributed in the China–Laos border and was not identified in other countries. Based on sequence analysis of the mitochondrial COII genes, significant genetic differentiation and limited gene flow were detected between the China–Laos and Cambodia–Laos An. sinensis populations, which suggested that those two regions were genetically isolated. The distinct distribution of the kdr haplotype frequencies is probably the result of geographical isolation in mosquito populations.ConclusionsLack of kdr mutations in the vgsc gene was probably due to genetic isolation and the absence of intense selection pressure in the three border regions of Laos. This study reveals that pyrethroid-based chemical insecticides are still appropriate for battling An. sinensis in parts of Laos, and routine monitoring of chemical insecticide resistance should be continuously implemented and focused on more restricted areas as part of chemical insecticide resistance management.Graphical

Divergent cis-regulatory evolution underlies the convergent loss of sodium channel expression in electric fish.

South American and African weakly electric fish independently evolved electric organs from muscle. In both groups, a voltage-gated sodium channel gene independently lost expression from muscle and gained it in the electric organ, allowing the channel to become specialized for generating electric signals. It is unknown how this voltage-gated sodium channel gene is targeted to muscle in any vertebrate. We describe an enhancer that selectively targets sodium channel expression to muscle. Next, we demonstrate how the loss of this enhancer, but not trans-activating factors, drove the loss of sodium channel gene expression from muscle in South American electric fish. While this enhancer is also altered in African electric fish, key transcription factor binding sites and enhancer activity are retained, suggesting that the convergent loss of sodium channel expression from muscle in these two electric fish lineages occurred via different processes.

Prevalence of Knock-Down Resistance F1534S Mutations in Aedes albopictus (Skuse) (Diptera: Culicidae) in North Carolina.

Knock-down resistance (kdr) mutations in the voltage-gated sodium channel gene of Aedes species mosquitoes are biomarkers for resistance to pyrethroid insecticides. In the United States, few studies have reported kdr mutations among Aedes albopictus (Skuse) (Diptera: Culicidae) populations. In this study, we sought to compare the presence of kdr alleles among Ae. albopictus mosquitoes collected from Fort Bragg and Wake County, North Carolina. We collected 538 Ae. albopictus mosquitoes, including 156 from 4 sites at Fort Bragg, North Carolina and 382 from 15 sites in Wake County, North Carolina to compare the prevalence of kdr mutations. Of those successfully sequenced, we identified 12 (3.0%) mosquitoes with kdr mutations, all of which were attributed to variants at position 1534 within domain 3. All mutations were found in mosquitoes collected at Wake County sites; no mutations were identified in collections from Fort Bragg. There was a focus of mutations observed at the Wake County sites with approximately 92% (11 of 12) of the mosquitoes with the mutation coming from one site, where kdr mutations represented 24.4% (11 of 45) of all mosquitoes collected. We observed highly focal resistance in a suburban area of Raleigh, which may be attributable to peri-domestic mosquito control activities that involve area dispersal of pyrethroid insecticides. More robust surveillance is needed to monitor the emergence and spread of resistance.

Molecular and Biochemical Detection of Insecticide Resistance in the Leishmania Vector, Phlebotomus papatasi (Diptera: Psychodidae) to Dichlorodiphenyltrichloroethane and Pyrethroids, in Central Iran.

The aim of the present study was to explore resistance markers and possible biochemical resistance mechanisms in the Phlebotomine sand fly Phlebotomus papatasi in Esfahan Province, central Iran. Homogenous resistant strains of sand flies were obtained by exposing P. papatasi collected from Esfahan to a single diagnostic dose of DDT. The adults from the colony were tested with papers impregnated with four pyrethroid insecticides: Permethrin 0.75%, Deltamethrin 0.05%, Cyfluthrin 0.15%, and Lambdacyhalothrin 0.05% to determine levels of cross-resistance. To discover the presence of mutations, a 440 base pair fragment of the voltage gated sodium channel (VGSC) gene was amplified and sequenced in both directions for the susceptible and resistant colonies. We also assayed the amount of four enzymes that play a key role in insecticide detoxification in the resistant colonies. A resistance ratio (RR) of 2.52 folds was achieved during the selection of resistant strains. Sequence analysis revealed no knockdown resistance (kdr) mutations in the VGSC gene. Enzyme activity ratio of the resistant candidate and susceptible colonies were calculated for α-esterases (3.78), β-esterases (3.72), mixed function oxidases (MFO) (3.21), and glutathione-S-transferases (GST) (1.59). No cross-resistance to the four pyrethroids insecticides was observed in the DDT resistant colony. The absence of kdr mutations in the VGSC gene suggests that alterations in esterase and MFO enzymes are responsible for the resistant of P. papatasi to DDT in central Iran. This information could have significant predictive utility in managing insecticide resistant in this Leishmania vector.

Detection of pyrethroid resistance mutations and intron variants in the voltage-gated sodium channel of Aedes (Stegomyia) aegypti and Aedes (Stegomyia) albopictus mosquitoes from Lao People's Democratic Republic.

In Lao People's Democratic Republic, Aedes aegypti (Linnaeus 1762) and Aedes albopictus (Skuse 1894) mosquitoes (Diptera: Culicidae) are vectors of arboviral diseases such as dengue. As the treatment for these diseases is limited, control of the vectors with the use of pyrethroid insecticides is still essential. However, mutations in the voltage-gated sodium channel (vgsc) gene giving rise to pyrethroid resistance are threatening vector control programs. Here, we analysed both Ae. aegypti and Ae. albopictus mosquitoes, which were collected in different districts of Laos (Kaysone Phomvihane, Vangvieng, Saysettha and Xaythany), for vgsc mutations commonly found throughout Asia (S989P, V1016G and F1534C). Sequences of the vgsc gene showed that the F1534C mutation was prevalent in both Aedes species. S989P and V1016G mutations were detected in Ae. aegypti from each site and were always found together. In addition, the mutation T1520I was seen in Ae. albopictus mosquitoes from Saysettha district as well as in all Ae. aegypti samples. Thus, mutations in the vgsc gene of Ae. aegypti are prevalent in the four districts studied indicating growing insecticide resistance throughout Laos. Constant monitoring programmes and alternative strategies for controlling Aedes should be utilized in order to prolong the effectiveness of pyrethroids thereby maximizing vector control.

High DDT resistance without apparent association to kdr and Glutathione-S-transferase (GST) gene mutations in Aedes aegypti population at hotel compounds in Zanzibar

Global efforts to control Aedes mosquito-transmitted pathogens still rely heavily on insecticides. However, available information on vector resistance is mainly restricted to mosquito populations located in residential and public areas, whereas commercial settings, such as hotels are overlooked. This may obscure the real magnitude of the insecticide resistance problem and lead to ineffective vector control and resistance management. We investigated the profile of insecticide susceptibility of Aedes aegypti mosquitoes occurring at selected hotel compounds on Zanzibar Island. At least 100 adults Ae. aegypti females from larvae collected at four hotel compounds were exposed to papers impregnated with discriminant concentrations of DDT (4%), permethrin (0.75%), 0.05 deltamethrin (0.05%), propoxur (0.1%) and bendiocarb (0.1%) to determine their susceptibility profile. Allele-specific qPCR and sequencing analysis were applied to determine the possible association between observed resistance and presence of single nucleotide polymorphisms (SNPs) in the voltage-gated sodium channel gene (VGSC) linked to DDT/pyrethroid cross-resistance. Additionally, we explored the possible involvement of Glutathione-S-Transferase gene (GSTe2) mutations for the observed resistance profile. In vivo resistance bioassay indicated that Ae. aegypti at studied sites were highly resistant to DDT, mortality rate ranged from 26.3% to 55.3% and, moderately resistant to deltamethrin with a mortality rate between 79% to and 100%. However, genotyping of kdr mutations affecting the voltage-gated sodium channel only showed a low frequency of the V1016G mutation (n = 5; 0.97%). Moreover, for GSTe2, seven non-synonymous SNPs were detected (L111S, C115F, P117S, E132A, I150V, E178A and A198E) across two distinct haplotypes, but none of these were significantly associated with the observed resistance to DDT. Our findings suggest that cross-resistance to DDT/deltamethrin at hotel compounds in Zanzibar is not primarily mediated by mutations in VGSC. Moreover, the role of identified GSTe2 mutations in the resistance against DDT remains inconclusive. We encourage further studies to investigate the role of other potential insecticide resistance markers.

Spatial heterogeneity of knockdown resistance mutations in the dengue vector Aedesalbopictus in Guangzhou, China

BackgroundThe city of Guangzhou has been the epicenter of dengue fever in China since the 1990s, with Aedesalbopictus being the primary vector. The main method used to control vectors and prevent dengue fever has been the application of chemical insecticides; however, this control strategy has resulted in the development of resistance to these insecticides in mosquitoes. Here we report our investigation of the patterns of knockdown resistance (kdr) mutations in 15 field populations of Ae.albopictus collected from 11 districts in Guangzhou.ResultsFour mutant alleles (V1016G, F1534S, F1534C, F1534L) were detected in domain II and III of the voltage-gated sodium channel (VGSC) gene. Various allele frequencies of kdr mutations were observed (3.1–25.9% for V1016G, 22.6–85.5% for F1534S, 0–29.0% for F1534L, 0.6–54.2% for F1534C). Seven kdr haplotypes (VF, VS, VL, VC, GF, GC, GS) were identified; the highest frequency of haplotypes was found for the single mutant haplotype VS (50.8%), followed by the wild-type VF haplotype (21.7%) and the single mutant haplotype VC (11.9%). Of the three double mutant haplotypes, GF was the most frequent (8.8%), followed by GC (1.2%) and GS (0.8%). Aedesalbopictus showed spatial heterogeneity in deltamethrin resistance in populations collected in Guangzhou. We also observed significant differences in haplotype frequency. The frequency of the VC haplotype was significantly higher in high-risk dengue areas than in low-risk ones.ConclusionsThe kdr allele V1016G was discovered for the first time in Guangzhou. Genetic isolation in mosquito populations and long-term insecticide selection seem to be responsible for the persistent, patchy distribution of kdr mutant alleles. The small-scale spatial heterogeneity in the distribution and frequency of kdr mutations may have important implications for vector control operations and insecticide resistance management strategies.Graphical

Community structure and insecticide resistance of malaria vectors in northern-central Myanmar

BackgroundMyanmar is one of the six countries in the Greater Mekong Subregion (GMS) of Southeast Asia. Malaria vectors comprise many Anopheles species, which vary in abundance and importance in malaria transmission among different geographical locations in the GMS. Information about the species composition, abundance, and insecticide resistance status of vectorial systems in Myanmar is scarce, hindering our efforts to effectively control malaria vectors in this region.MethodsDuring October and November 2019, larvae and adult females of Anopheles mosquitoes were collected in three sentinel villages of Banmauk township in northern Myanmar. Adult female mosquitoes collected by cow-baited tent collection (CBTC) and adults reared from field-collected larvae (RFCL) were used to determine mortality rates and knockdown resistance (kdr) against deltamethrin using the standard WHO susceptibility test. Molecular species identification was performed by multiplex PCR and ITS2 PCR, followed by DNA sequencing. The kdr mutation at position 1014 of the voltage-gated sodium channel gene was genotyped by DNA sequencing for all Anopheles species tested.ResultsA total of 1596 Anopheles mosquitoes from seven morphologically identified species groups were bioassayed. Confirmed resistance to deltamethrin was detected in the populations of An. barbirostris (s.l.), An. hyrcanus (s.l.), and An. vagus, while possible resistance was detected in An. annularis (s.l.), An. minimus, and An. tessellatus. Anopheles kochi was found susceptible to deltamethrin. Compared to adults collected by CBTC, female adults from RFCL had significantly lower mortality rates in the four species complexes. A total of 1638 individuals from 22 Anopheles species were molecularly identified, with the four most common species being An. dissidens (20.5%) of the Barbirostris group, An. peditaeniatus (19.4%) of the Hyrcanus group, An. aconitus (13.4%) of the Funestus group, and An. nivipes (11.5%) of the Annularis group. The kdr mutation L1014F was only detected in the homozygous state in two An. subpictus (s.l.) specimens and in a heterozygous state in one An. culicifacies (s.l.) specimen.ConclusionsThis study provides updated information about malaria vector species composition and insecticide resistance status in northern Myanmar. The confirmed deltamethrin resistance in multiple species groups constitutes a significant threat to malaria vector control. The lack or low frequency of target-site resistance mutations suggests that other mechanisms are involved in resistance. Continual monitoring of the insecticide resistance of malaria vectors is required for effective vector control and insecticide resistance management.Graphical

Non-canonical endogenous expression of voltage-gated sodium channel NaV 1.7subtype by the TE671 rhabdomyosarcoma cell line.

The human TE671 cell line was originally used as a model of medulloblastoma but has since been reassigned as rhabdomyosarcoma. Despite the characterised endogenous expression of voltage‐sensitive sodium currents in these cells, the specific voltage‐gated sodium channel (VGSC) subtype underlying these currents remains unknown. To profile the VGSC subtype in undifferentiated TE671 cells, endpoint and quantitative reverse transcription–PCR (qRT‐PCR), western blot and whole‐cell patch clamp electrophysiology were performed. qRT‐PCR profiling revealed that expression of the SCN9A gene was ∼215‐fold greater than the SCN4A gene and over 400‐fold greater than any of the other VGSC genes, while western blot confirmed that the dominant SCN9A RNA was translated to a protein with a molecular mass of ∼250 kDa. Elicited sodium currents had a mean amplitude of 2.6 ± 0.7 nA with activation and fast inactivation V 50 values of −31.9 ± 1.1 and −69.6 ± 1.0 mV, respectively. The currents were completely and reversibly blocked by tetrodotoxin at concentrations greater than 100 nm (IC50 = 22.3 nm). They were also very susceptible to the NaV1.7 specific blockers Huwentoxin‐IV and Protoxin‐II with IC50 values of 14.6 nm and 0.8 nm, respectively, characteristic of those previously determined for NaV1.7. Combined, the results revealed the non‐canonical and highly dominant expression of NaV1.7 in the human TE671 rhabdomyosarcoma cell line. We show that the TE671 cell line is an easy to maintain and cost‐effective model for the study of NaV1.7, a major target for the development of analgesic drugs and more generally for the study of pain.Key points Undifferentiated TE671 cells produce a voltage‐sensitive sodium current when depolarised.The voltage‐gated sodium channel isoform expressed in undifferentiated TE671 cells was previously unknown. Through qRT‐PCR, western blot and toxin pharmacology, it is shown that undifferentiated TE671 cells dominantly (>99.5%) express the NaV1.7 isoform that is strongly associated with pain. The TE671 cell line is, therefore, a very easy to maintain and cost‐effective model to study NaV1.7‐targeting drugs.