Voltage-sensitive Sodium Channel Gene Research Articles

Measuring local genetic variation in permethrin-resistant head lice (Phthiraptera: Pediculidae) from Buenos Aires, Argentina.

The cosmopolitan ectoparasite human head louse, Pediculus humanus capitis (De Geer)(Phthiraptera:Pediculidae), affects mostly school-aged children, with infestations reported every year mainly due to louse resistance to pyrethroids. One of the main resistance mechanisms of pyrethroids is the target site insensitivity (kdr), which is caused by single-nucleotide point mutations (SNPs) located in the voltage-sensitive sodium channel gene. In this study, we analyzed individual head lice toxicologically via the description of their susceptibility profile to permethrin and genetically through the genotypification of their kdr alleles as well as nuclear microsatellite loci. Lice were collected from 4 schools in the city of Buenos Aires, Argentina. The resistance ratios varied from 33.3% to 71.4%, with a frequency of the T917I kdr mutation of 87.31% and with 83.6% of the head lice being homozygous resistant to pyrethroids. Microsatellite data indicated that all the louse school populations had genotype proportions that deviated from Hardy-Weinberg expectations, with FIS > 0 reflecting a deficit of heterozygotes. Bottleneck analysis suggested that all louse school populations underwent a recent reduction in population sizes, while 3 of the 4 schools had gene flow values around 1, indicating ongoing gene flow among those schools. Our study suggests that school louse populations in the city of Buenos Aires may form a metapopulation, where each school represents a small population that undergoes extinction and recolonization processes under strong permethrin selection. This is the first multilevel analysis integrating toxicological, kdr-genotyping, and microsatellite data in human louse populations.

Diversity and distribution of sodium channel mutations in Aedes albopictus (Diptera: Culicidae).

There is growing interest in insecticide resistance in the mosquito, Aedes albopictus (Skuse), as its potential for spreading diseases is increasing as urbanization and control efforts intensify. Here we review the presence and diversity of mutations in the voltage-sensitive sodium channel (Vssc) gene associated with pyrethroid resistance and report on additional surveys of these mutations in new populations with an analysis of their spread. The known diversity of these mutations has increased in recent years including the identification of 26 non-synonymous mutations, although phenotypic data associating mutations with resistance remain limited. We provide data on mutations in several new locations including those in Timor Leste, Indonesia, and Vanuatu. We use population genomic data from ddRAD analyses of target populations with the 1534C mutation to identify single nucleotide polymorphisms (SNPs) associated with the mutant to test for clustering of SNPs based on the presence of the 1534C mutation rather than population origin. Our findings suggest spread of resistance alleles via genetic invasion, which is further supported by patterns from a genome-wide principal components analysis. These data point to movement of resistance alleles across wide areas with likely impacts on local control options.

Kdr mutations and deltamethrin resistance in house flies in Abu Dhabi, UAE

BackgroundThe house fly, Musca domestica, is a significant carrier of diseases that can impact public health. Repeated use of pyrethroid insecticides may act as a selection pressure for mutations and amino acid substitutions in the house fly voltage-sensitive sodium channel (VSSC), which ultimately confers resistance. The objectives of this study were to determine the presence of knockdown resistance (kdr) mutations using molecular tools and to set up a CDC bottle bioassay specific for house flies in the United Arab Emirates (UAE) to screen for deltamethrin resistance.MethodsAdult flies were collected from 19 locations in Abu Dhabi, UAE, and DNA was extracted, followed by PCR amplification of specific alleles (PASA) and conventional PCR using several primers to amplify regions of the VSSC gene. Sanger sequencing was performed on PCR products. We also designed primers that detect four kdr mutations using complementary DNA (cDNA) in reverse transcriptase (RT)-PCR followed by Sanger sequencing. Additionally, a CDC bottle bioassay was set up for detecting deltamethrin resistance in adult house flies.ResultsIn PASA, the primers successfully amplified the target bands (480, 280 and 200 bp). The kdr allele was found in flies collected from 18 of the 19 locations, at the highest and lowest prevalence of 46.9% and 9.4%, respectively. Resistant homozygous (RR) insects constituted 5.0% of the tested populations, and heterozygous (RS) insects accounted for 36.5%. The RR genotype was prevalent in house flies collected at 10 of 19 sampling locations. House fly populations were mostly in Hardy–Weinberg equilibrium, except in three locations. In addition to verifying the presence of the previously identified kdr mutation L1014F, in this study we detected two kdr mutations, L1014H and T929I, that have not previously been reported in the UAE. Also, for the first time in the UAE, a CDC bottle bioassay for deltamethrin resistance was used, which found that 60 min and 4.5 µg/ml were the diagnostic time and dose, respectively. Using this assay, we detected deltamethrin resistance in house flies from two of 16 locations, with a resistance level of 12.5%.ConclusionsUsing DNA sequencing, we confirmed the presence of a known kdr mutation and uncovered two new kdr mutations in house flies from Abu Dhabi. Additionally, we detected deltamethrin resistance in these flies using a CDC bottle bioassay. Further research is recommended to comprehensively identify more kdr mutations in UAE house fly populations and assess their impacts on control strategies.Graphical

The first recent case of Cimex hemipterus (Hemiptera: Cimicidae) with super-kdr mutations in the Republic of Korea.

With the increase in international travel and trade, in conjunction with the development of insecticide resistance, infestations of Cimex lectularius (L.) and Cimex hemipterus (F.) (Hemiptera: Cimicidae) have resurged globally in the last 2 decades. Recently, it was reported that C. hemipterus was also found in temperate regions, indicating the possibility of its expansion outside tropical regions. Cimex hemipterus has not been officially recorded in Korea since its initial description in 1934. Here, we report the first recent case of C. hemipterus in Korea based on morphological and molecular identification. Partial sequencing of the voltage-sensitive sodium channel gene revealed super-kdr mutations (M918I and L1014F) that are associated with pyrethroid resistance. This case report serves as a warning to intensify the bed bug surveillance system in Korea regarding the presence of C. hemipterus and to prepare effective alternative insecticides for pyrethroids.

Geographic distribution of the V1016G knockdown resistance mutation in Aedes albopictus: a warning bell for Europe

BackgroundColonization of large part of Europe by the Asian tiger mosquito Aedes albopictus is causing autochthonous transmission of chikungunya and dengue exotic arboviruses. While pyrethroids are recommended only to reduce/limit transmission, they are widely implemented to reduce biting nuisance and to control agricultural pests, increasing the risk of insurgence of resistance mechanisms. Worryingly, pyrethroid resistance (with mortality < 70%) was recently reported in Ae. albopictus populations from Italy and Spain and associated with the V1016G point mutation in the voltage-sensitive sodium channel gene conferring knockdown resistance (kdr). Genotyping pyrethroid resistance-associated kdr mutations in field mosquito samples represents a powerful approach to detect early signs of resistance without the need for carrying out phenotypic bioassays which require availability of live mosquitoes, dedicated facilities and appropriate expertise.MethodsHere we report results on the PCR-genotyping of the V1016G mutation in 2530 Ae. albopictus specimens from 69 sampling sites in 19 European countries.ResultsThe mutation was identified in 12 sites from nine countries (with allele frequencies ranging from 1 to 8%), mostly distributed in two geographical clusters. The western cluster includes Mediterranean coastal sites from Italy, France and Malta as well as single sites from both Spain and Switzerland. The eastern cluster includes sites on both sides of the Black Sea in Bulgaria, Turkey and Georgia as well as one site from Romania. These results are consistent with genomic data showing high connectivity and close genetic relationship among West European populations and a major barrier to gene flow between West European and Balkan populations.ConclusionsThe results of this first effort to map kdr mutations in Ae. albopictus on a continental scale show a widespread presence of the V1016G allele in Europe, although at lower frequencies than those previously reported from Italy. This represents a wake-up call for mosquito surveillance programs in Europe to include PCR-genotyping of pyrethroid resistance alleles, as well as phenotypic resistance assessments, in their routine activities.Graphical

Presence of the knockdown resistance (kdr) mutations in the head lice (Pediculus humanus capitis) collected from primary school children of Thailand.

Human head lice are blood-sucking insects causing an infestation in humans called pediculosis capitis. The infestation is more prevalent in the school-aged population. Scalp itching, a common presenting symptom, results in scratching and sleep disturbance. The condition can lead to social stigmatization which can lead to loss of self-esteem. Currently, the mainstay of treatment for pediculosis is chemical insecticides such as permethrin. The extended use of permethrin worldwide leads to growing pediculicide resistance. The aim of this study is to demonstrate the presence of the knockdown resistance (kdr) mutation in head lice populations from six different localities of Thailand. A total of 260 head lice samples in this study were collected from 15 provinces in the 6 regions of Thailand. Polymerase chain reaction (PCR) was used to amplify the α subunit of voltage-sensitive sodium channel (VSSC) gene, kdr mutation (C→T substitution). Restriction fragment length polymorphism (RFLP) patterns and sequencing were used to identify the kdr T917I mutation and demonstrated three genotypic forms including homozygous susceptible (SS), heterozygous genotype (RS), and homozygous resistant (RR). Of 260 samples from this study, 156 (60.00%) were SS, 58 (22.31%) were RS, and 46 (17.69%) were RR. The overall frequency of the kdr T917I mutation was 0.31. Genotypes frequencies determination using the exact test of Hardy-Weinberg equilibrium found that northern, central, northeastern, southern, and western region of Thailand differed from expectation. The five aforementioned localities had positive inbreeding coefficient value (Fis > 0) which indicated an excess of homozygotes. The nucleotide and amino acid sequences of RS and RR showed T917I and L920F point mutations. In conclusion, this is the first study detecting permethrin resistance among human head lice from Thailand. PCR-RFLP is an easy technique to demonstrate the kdr mutation in head louse. The data obtained from this study would increase awareness of increasing of the kdr mutation in head louse in Thailand.

Frequency of kdr mutations in the voltage-sensitive sodium channel (VSSC) gene in Aedes aegypti from Yogyakarta and implications for Wolbachia-infected mosquito trials

BackgroundIn the inner city of Yogyakarta, Indonesia, insecticide resistance is expected in the main dengue vector, Aedes aegypti, because of the intensive local application of pyrethroid insecticides. However, detailed information about the nature of resistance in this species is required to assist the release of Wolbachia mosquitoes in a dengue control program, so that we can ensure that insecticide resistance in the strain of Ae. aegypti being released matches that of the background population.MethodsHigh-resolution melt genotyping was used to screen for kdr mutations associated with pyrethroid resistance in the voltage-sensitive sodium channel (VSSC) gene in Ae. aegypti of some areas in the inner city of Yogyakarta.ResultsThe results show that the V1016G mutation predominated, with individuals homozygous for the 1016G allele at a frequency of 82.1% and the mutant allele G at a frequency of 92%. Two patterns of co-occurrence of mutations were detected in this study, homozygous individuals V1016G/S989P; and heterozygous individuals V1016G/F1534C/S989P. We found the simultaneous occurrence of kdr mutations V1016G and F1534C at all collection sites, but not within individual mosquitoes. Homozygous mutants at locus 1016 were homozygous wild-type at locus 1534 and vice versa, and heterozygous V1016G were also heterozygous for F1534C. The most common tri-locus genotype co-occurrences were homozygous mutant 1016GG and homozygous wild-type FF1534, combined with homozygous mutant 989PP (GG/FF/PP) at a frequency of 38.28%.ConclusionsGiven the relatively small differences in frequency of resistance alleles across the city area, locality variations in resistance should have minor implications for the success of Wolbachia mosquito trials being undertaken in the Yogyakarta area.

First evidence of the mutations associated with pyrethroid resistance in head lice (Phthiraptera: Pediculidae) from Honduras

BackgroundThe human head louse, Pediculus humanus capitis, is a cosmopolitan blood-sucking ectoparasite affecting mostly schoolchildren in both developed and developing countries. In Honduras, chemical pediculicides are the first line of treatment, with permethrin as their main active ingredient. Despite the extended use of these products, there is currently no research investigating insecticide resistance in Honduran head lice. In head lice, the most common mechanism is knockdown resistance (kdr), which is the result of two point mutations and the associated amino acid substitutions, T917I and L920F, within the voltage-sensitive sodium channel (VSSC).MethodsGenomic DNA was extracted from 83 head lice collected in the localities of San Buenaventura and La Hicaca, Honduras. Polymerase chain reaction (PCR) was used to amplify a 332-bp fragment of the VSSC gene that contains a site affected by C/T mutation which results in a T917I amino acid substitution on each human head louse genomic DNA fragments.ResultsThe C/T non-synonymous mutation which results in the T917I kdr amino acid substitution was detected in both head lice populations at frequencies ranging between 0.45–0.5. Globally, the frequency of this substitution was 0.47. Of these, 5 (6.1%) were homozygous susceptible and 78 (93.9%) were heterozygotes. The kdr-resistant homozygote (RR) was not detected in the studied populations. Thus, 93.9% of the head lice collected in Honduras harbored only one T917I allele. Exact test for the Hardy-Weinberg equilibrium for both localities showed that genotype frequencies differed significantly from expectation. In addition, San Buenaventura and La Hicaca populations had an inbreeding coefficient (Fis) < 0, suggesting an excess of heterozygotes.ConclusionsTo our knowledge, this is the first study showing the presence of the C/T mutation responsible of the T917I kdr allele associated with pyrethroid resistance in P. h. capitis from Honduras. The PCR-restriction fragment length polymorphism (RFLP) employed here has demonstrated to be a reliable, economic, and reproducible assay that can be used to accurately genotype individual head lice for the mutation encoding the resistance-conferring T917I amino acid substitution. This highlights the necessity of proactive resistance management programmes designed to detect pyrethroid mutations before they become established within populations of head lice.

Evidence for both sequential mutations and recombination in the evolution of kdr alleles in Aedes aegypti.

BackgroundAedes aegypti is a globally distributed vector of human diseases including dengue, yellow fever, chikungunya, and Zika. Pyrethroid insecticides are the primary means of controlling adult A. aegypti populations to suppress arbovirus outbreaks, but resistance to pyrethroid insecticides has become a global problem. Mutations in the voltage-sensitive sodium channel (Vssc) gene are a major mechanism of pyrethroid resistance in A. aegypti. Vssc resistance alleles in A. aegypti commonly have more than one mutation. However, our understanding of the evolutionary dynamics of how alleles with multiple mutations arose is poorly understood.Methodology/Principal findingsWe examined the geographic distribution and association between the common Vssc mutations (V410L, S989P, V1016G/I and F1534C) in A. aegypti by analyzing the relevant Vssc fragments in 25 collections, mainly from Asia and the Americas. Our results showed all 11 Asian populations had two types of resistance alleles: 1534C and 989P+1016G. The 1534C allele was more common with frequencies ranging from 0.31 to 0.88, while the 989P+1016G frequency ranged from 0.13 to 0.50. Four distinct alleles (410L, 1534C, 410L+1534C and 410L+1016I+1534C) were detected in populations from the Americas. The most common was 410L+1016I+1534C with frequencies ranging from 0.50 to 1.00, followed by 1534C with frequencies ranging from 0.13 to 0.50. Our phylogenetic analysis of Vssc supported multiple independent origins of the F1534C mutation. Our results indicated the 410L+1534C allele may have arisen by addition of the V410L mutation to the 1534C allele, or by a crossover event. The 410L+1016I+1534C allele was the result of one or two mutational steps from a 1534C background.Conclusions/SignificanceOur data corroborated previous geographic distributions of resistance mutations and provided evidence for both recombination and sequential accumulation of mutations contributing to the molecular evolution of resistance alleles in A. aegypti.

Surveillance of a kdr Resistance Mutation in Culex pipiens (Diptera: Culicidae) and Culex quinquefasciatus in California.

Culex pipiens Linnaeus and Culex quinquefasciatus Say are the primary vectors of West Nile and St. Louis encephalitis viruses in California. Pyrethrins and pyrethroids (synthetic pyrethrins) are the most widely used insecticides to control adult stage mosquitoes to prevent disease transmission. The most abundant and widespread mutation associated with pyrethroid resistance is the L1014F mutation of the voltage-sensitive sodium channel gene. Statewide, based on the testing of almost 2,000 mosquitoes from 14 counties, the resistant allele frequency was 71%. Although the L1014F mutation was found in all counties assessed, the resistance allele profiles differed between regions of California. The highest resistant allele frequency occurred in the Central region and lowest frequencies were from the Northern and Southern regions. Resistance allele frequencies observed in 2014–2016 are nearly 1.5 times higher than those from pre-2012, indicating that resistance profiles can change over time. Regular monitoring of the L1014F kdr mutation will help aid in operational decisions.

Insecticide resistance status of Aedes aegypti and Aedes albopictus mosquitoes in Papua New Guinea

BackgroundAedes aegypti and Ae. albopictus are important vectors of infectious diseases, especially those caused by arboviruses such as dengue, chikungunya and Zika. Aedes aegypti is very well adapted to urban environments, whereas Ae. albopictus inhabits more rural settings. Pyrethroid resistance is widespread in these vectors, but limited data exist from the Southwest Pacific Region, especially from Melanesia. While Aedes vector ecology is well documented in Australia, where incursion of Ae. albopictus and pyrethroid resistance have so far been prevented, almost nothing is known about Aedes populations in neighbouring Papua New Guinea (PNG). With pyrethroid resistance documented in parts of Indonesia but not in Australia, it is important to determine the distribution of susceptible and resistant Aedes populations in this region.MethodsThe present study was aimed at assessing Aedes populations for insecticide resistance in Madang and Port Moresby, located on the north and south coasts of PNG, respectively. Mosquitoes were collected using ovitraps and reared in an insectary. Standard WHO bioassays using insecticide-treated filter papers were conducted on a total of 253 Ae. aegypti and 768 Ae. albopictus adult mosquitoes. Subsets of samples from both species (55 Ae. aegypti and 48 Ae. albopictus) were screened for knockdown resistance mutations in the voltage-sensitive sodium channel (Vssc) gene, the target site of pyrethroid insecticides.ResultsHigh levels of resistance against pyrethroids were identified in Ae. aegypti from Madang and Port Moresby. Aedes albopictus exhibited susceptibility to pyrethroids, but moderate levels of resistance to DDT. Mutations associated with pyrethroid resistance were detected in all Ae. aegypti samples screened. Some genotypes found in the present study had been observed previously in Indonesia. No Vssc mutations associated with pyrethroid resistance were found in the Ae. albopictus samples.ConclusionsTo our knowledge, this is the first report of pyrethroid resistance in Ae. aegypti mosquitoes in PNG. Interestingly, usage of insecticides in PNG is low, apart from long-lasting insecticidal nets distributed for malaria control. Further investigations on how these resistant Ae. aegypti mosquito populations arose in PNG and how they are being sustained are warranted.

Frequency of kdr and kdr-his Alleles in Stable Fly (Diptera: Muscidae) Populations From the United States, Costa Rica, France, and Thailand.

Anecdotal evidence of pyrethroid insecticide product failure for the control of stable fly [Stomoxys calcitrans (L.)] populations in the United States and worldwide prompted us to evaluate the frequency of knockdown resistance (kdr)-type polymorphisms within the voltage-sensitive sodium channel (Vssc) gene of field collected specimens from the United States, France, Costa Rica, and Thailand. The kdr-his allele (L1014H), associated with permethrin resistance, was detected in stable flies from the 10 states sampled in the United States, as well as from Costa Rica and France (Toulouse). Field collections of stable flies from California (Modesto) and New York (Cliffton Springs) exhibited reduced susceptibility upon exposure to a diagnostic permethrin concentration of 10× LC99, but survival did not appear to strictly associate with frequency of the kdr-his allele. This suggests that there are additional resistance mechanisms contributing to the phenotype in these states. The kdr allele (L1014F) was detected for the first time in stable flies originating in France and Thailand, and an improved, DNA-based diagnostic assay was developed and validated for use in future screens for kdr and kdr-his allele frequencies from field collections. The absence of kdr in United States and Costa Rica populations suggests that the allele is currently restricted to Europe and Asia.

Two novel house fly Vssc mutations, D600N and T929I, give rise to new insecticide resistance alleles

The house fly, Musca domestica, is a serious pest because it transmits a large diversity of human and veterinary diseases. Insecticides, particularly pyrethroids, are commonly used to control house flies. However, the evolution of pyrethroid resistance has reduced the effectiveness of these insecticides. A major mechanism of resistance to pyrethroids is target site insensitivity caused by the mutations in the voltage-sensitive sodium channel (Vssc) gene (e.g. kdr [L1014F] and super-kdr [M918T+L1014F]). Recently, two novel Vssc alleles, super-kdr+D600N and kdr+T929I were detected in a field collected resistant house fly population in Kansas, USA in 2013. To determine the levels of resistance that these new alleles confer to pyrethroids, we isolated strains having the unique Vssc alleles, but being otherwise congenic to the susceptible strain, aabys. We compared levels of resistance conferred to 14 pyrethroids and determined the inheritance of resistance to 8 pyrethroids. Our results revealed that super-kdr+D600N conferred higher levels of resistance to seven pyrethroids relative to super-kdr, and kdr+T929I showed super-kdr-like levels of resistance in house flies. Our results are compared with previous studies and reveal that addition of T929I to the kdr mutation (L1014F) increased resistance to all pyrethroids (except etofenprox), and enhanced resistance by ~1000-fold to acrinathrin and flumethrin. The implications of these results on the evolution of resistance are discussed.

Overcoming super-knock down resistance (super-kdr) mediated resistance: multi-halogenated benzyl pyrethroids are more toxic to super-kdr than kdr house flies.

Target site insensitivity because of mutations in the voltage-sensitive sodium channel gene (Vssc) is a major mechanism of resistance to pyrethroid insecticides in the house fly, Musca domestica. There are three known Vssc alleles that confer resistance to pyrethroids in the house fly: knock down resistance (kdr; L1014F), super-kdr (M918T + L1014F) and kdr-his (L1014H), but there has been no side-by-side comparison of the resistance levels that they confer. We compared the levels of resistance conferred by the three Vssc alleles in congenic strains to 19 structurally diverse pyrethroids, and compared the full-length Vssc cDNA sequences from each strain. Generally, the levels of resistance conferred were kdr-his < kdr < super-kdr. However, there was significant variation in this pattern, especially for super-kdr, for which both high and low resistance ratios were observed for several pyrethroids. We also examined the levels of resistance in heterozygotes. Resistance in each of the hybrids was generally inherited as an incompletely recessive trait, except for the kdr-his/kdr hybrids, which showed incompletely to completely dominant resistance (ie had resistance levels comparable to kdr homozygotes). The importance of these results to understanding the frequencies of these resistance alleles in natural populations, the evolution of insecticide resistance and resistance management strategies are discussed.

De novo transcriptome profiling and characterization of voltage-sensitive sodium channel gene of Tropilaelaps mercedesae parasitizing honey bees

Tropilaelaps mercedesae (Acari: Laelapidae) is an ectoparasite of the Western honey bee Apis mellifera and has become a major threat to the apiculture in Asia, including Korea. To establish gene resources of T. mercedesae, the whole transcriptome of mixed developmental stages of mites was analyzed by RNA sequencing. An mRNA-focused library was generated from total RNA extracted from the mixed stages (adults, nymphs and larvae) of T. mercedesae using the TruSeq RNA Library Preparation kit and sequenced using the HiSeq 2000 platform. A total of 59,041,198 reads were obtained with 85% Q30 value. Trimmed sequence data was de novo assembled into 64,868 contigs with an average sequence length of 878bp. Transcriptome annotation to the NCBI non-redundant database resulted in a total of 14,336 Blast hits (22.1%). Functional analysis demonstrated that 11,919 contigs were grouped into 45 Gene Ontology (Conesa et al.) terms, and 1226 contigs were assigned to Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Based on the transcriptome dataset, a large number of contigs associated with binding, cellular process and catalytic activity were identified. In order to provide basic information for efficient management of potential resistance to fluvalinate, a pyrethroid acaricide most widely used for the control of T. mercedesae, the gene encoding the voltage-sensitive sodium channel α-subunit (vssc), which is the molecular target of fluvalinate, was annotated and characterized.

A point mutation (L1015F) of the voltage-sensitive sodium channel gene associated with lambda-cyhalothrin resistance in Apolygus lucorum (Meyer–Dür) population from the transgenic Bt cotton field of China

In China, the green mirid bug, Apolygus lucorum (Meyer–Dür), has caused severe economic damage to many kinds of crops, especially the cotton and jujubes. Pyrethroid insecticides have been widely used for controlling this pest in the transgenic Bt cotton field. Five populations of A. lucorum collected from cotton crops at different locations in China were evaluated for lambda-cyhalothrin resistance. The results showed that only the population collected from Shandong Province exhibited 30-fold of resistance to lambda-cyhalothrin. Neither PBO nor DEF had obvious synergism when compared the synergistic ratio between SS and RR strain which was originated from the Shandong population. Besides, there were no statistically significant differences (p>0.05) in the carboxylesterase, glutathione S-transferase, or 7-ethoxycoumarin O-deethylase activities between the Shandong population and the laboratory susceptible strain (SS). The full-length sodium channel gene named AlVSSC encoding 2028 amino acids was obtained by RT-PCR and rapid amplification of cDNA ends (RACE). One single point mutation L1015F in the AlVSSC was detected only in the Shandong population. Our results revealed that the L1015F mutation associated with pyrethroid resistance was identified in A. lucorum populations in China. These results will be useful for the rational chemical control of A. lucorum in the transgenic Bt cotton field.

Association between Three Mutations, F1565C, V1023G and S996P, in the Voltage-Sensitive Sodium Channel Gene and Knockdown Resistance in Aedes aegypti from Yogyakarta, Indonesia.

Mutations in the voltage-sensitive sodium channel gene (Vssc) have been identified in Aedes aegypti and some have been associated with pyrethroid insecticide resistance. Whether these mutations cause resistance, alone or in combination with other alleles, remains unclear, but must be understood if mutations are to become markers for resistance monitoring. We describe High Resolution Melt (HRM) genotyping assays for assessing mutations found in Ae. aegypti in Indonesia (F1565C, V1023G, S996P) and use them to test for associations with pyrethroid resistance in mosquitoes from Yogyakarta, a city where insecticide use is widespread. Such knowledge is important because Yogyakarta is a target area for releases of Wolbachia-infected mosquitoes with virus-blocking traits for dengue suppression. We identify three alleles across Yogyakarta putatively linked to resistance in previous research. By comparing resistant and susceptible mosquitoes from bioassays, we show that the 1023G allele is associated with resistance to type I and type II pyrethroids. In contrast, F1565C homozygotes were rare and there was only a weak association between individuals heterozygous for the mutation and resistance to a type I pyrethroid. As the heterozygote is expected to be incompletely recessive, it is likely that this association was due to a different resistance mechanism being present. A resistance advantage conferred to V1023G homozygotes through addition of the S996P allele in the homozygous form was suggested for the Type II pyrethroid, deltamethrin. Screening of V1023G and S996P should assist resistance monitoring in Ae. aegypti from Yogyakarta, and these mutations should be maintained in Wolbachia strains destined for release in this city to ensure that these virus-blocking strains of mosquitoes are not disadvantaged, relative to resident populations.

Molecular survey of knockdown resistance to pyrethroids in human scabies mites

There are growing concerns about the emergence of resistance of Sarcoptes scabiei to permethrin and other pyrethroid derivatives. Sarcoptes scabiei mites collected from 40 patients visiting two hospitals near Paris, France, were genotypically characterized considering two parts of their voltage-sensitive sodium channel gene. No nucleotide polymorphism resulting in a codon change at codon 733 or other positions associated with knockdown resistance in other arthropods has been identified. These data may establish a reference line for future surveys of the susceptibility of S. scabiei in the area.

Multiple evolutionary origins of knockdown resistance (kdr) in pyrethroid-resistant Colorado potato beetle, Leptinotarsa decemlineata

The Colorado potato beetle (CPB), Leptinotarsa decemlineata, is a serious agricultural pest that is difficult to control due to its rapid evolution of resistance to most insecticides. Knockdown resistance (kdr) has been identified as the chief mechanism of resistance against pyrethroid insecticides in CPB and is due to a single nucleotide polymorphism, L1014F, of the voltage-sensitive sodium channel gene, LdVssc1. In order to determine whether kdr had a single or multiple evolutionary origins in CPB, 96 larvae from 10 locations (nine from across the United States and one in Bulgaria) were collected, and partial genomic sequences for LdVssc1 were obtained for each individual. The associated intron, three base pairs downstream from the mutation site, was used in the identification of novel resistant (F1014) and susceptible (L1014) alleles. In addition, genotyping was performed to investigate the presence of the super-kdr allele (L1014F+M918T). In total, 8 resistant haplotypes (all kdr) and 21 susceptible haplotypes were identified. While no M918T was found, a T929I mutation, which is also associated with pyrethroid resistance in other species, was present in 16 larvae from Bulgaria. A novel T929N mutation was found in conjunction with L1014F. Within the United States, collections with only susceptible haplotypes exhibited higher allelic diversity than collections with primarily resistant haplotypes. Phylogenetic analysis suggests 2–3 independent origins of kdr.

Diversity and frequencies of genetic mutations involved in insecticide resistance in field populations of the house fly (Musca domestica L.) from China

Insecticides have been extensively used for house fly control in China, with dichlorvos and deltamethrin being widely used. Knowledge about the current status of insecticide resistance and the underlying genetic changes is crucial for developing effective fly control strategies. The susceptibility to dichlorvos and deltamethrin, and the frequencies of genetic mutations involved in insecticide resistance were studied in five field populations of the house fly collected across China. Bioassay results show that flies exhibit 14- to 28-fold resistance to dichlorvos and 41- to 94-fold resistance to deltamethrin, indicating that dichlorvos and deltamethrin resistance are common in house fly populations in China. Molecular analysis reveals that flies from the five various locations carry resistance alleles at multiple loci and have diverse allelic types, different relative frequencies and combinations of each allele. Four non-synonymous single nucleotide polymorphisms (SNPs) (i.e. V260L, G342A/V, F407Y) in acetylcholinesterase (Ace) and two mutations (W251L/S) in a carboxylesterase (MdαE7) were commonly present in the field house flies. The L1014H rather than L1014F mutation in the voltage sensitive sodium channel gene (Vssc) was widely distributed in Chinese house flies. CYP6D1v1, which confers pyrethroid resistance, was found in all the five tested populations in China, although its frequency in house fly from Shandong province was very low. Our results suggest that resistance monitoring and management of house flies should be customized for a given location.