Kdr Mutation Research Articles

Population dynamics and insecticide resistance in Tuta absoluta (Lepidoptera: Gelechiidae), an invasive pest on tomato in Kenya

Tuta absoluta feeds on solanaceous plants with preference on tomato. Management of the pest is mostly with chemical insecticides. This study identified insecticide resistant populations and predicted resistance to insecticides. Insecticide resistance development was modelled using system thinking, and system dynamics approaches. The model showed the pest resistance development is alarming with an exponential increase of the resistance strength mostly in recent years. Furthermore, we used seven insecticide-resistance gene markers to resolve the population structure and genetic differentiation of insecticide-resistant populations in Kenya. The genes for resistance (knockdown resistance (kdr) mutations, acetylcholinesterase (AChE) and voltage gated sodium channel (para)) were detected in all populations. Population structure analyses separated T. absoluta populations into three genetic clusters with resistant genes that are interconnected. A better insight on the population dynamics and the genetic structure T. absoluta resistant genes in Kenya will help estimate resistance strength and determine the most effective pest control strategies.

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.

Çukurova Bölgesi pamuk alanlarında Aphis gossypii Glover, 1877 (Hemiptera: Aphididae)’nin insektisit direncinin belirlenmesi

This study aimed to reveal resistance levels of Aphis gossypii Glover, 1877 (Hemiptera: Aphididae) populations against dimethoate, λ-cyhalothrin and chlorpyrifos-ethyl used in cotton fields in Çukurova region in Adana province in Türkiye. Populations were collected from 16 locations between 2020 and 2021. Bioassay, biochemical and molecular methods were used to determine resistance. Six populations were resistant according to the susceptible (Toktamış) population with leaf dip discriminating dose bioassays. Compared to the susceptible population, four populations were found at decreased susceptibility (DS) resistance levels to dimethoate and one population to chlorpyrifos-ethyl. . Only two populations resistance ratio were detected in MR (Moderate resistance) category to chlorpyrifos-ethyl. Resistance levels of other populations were observed as S (susceptible) category. Resistant populations had higher acetylcholinesterase, glutathione-S transferase and cytochrome P450 monooxygenase enzyme activities in biochemical analysis. The carboxylesterase gene transcription levels were higher in resistant populations. S431F and Kdr (knockdown) mutation were determined by the PCR-RLFP method, which is effective in organophosphate and pyrethroid insecticides resistance and 17% and 100% recessive alleles were detected in populations. The biochemical and mutation-induced resistance to dimethoate and chlorpyrifos-ethyl was detected. These results will contribute to developing strategies for resistance management of A. gossypii.

Prevalence of permethrin-resistant kdr mutation in head lice (Pediculus humanus capitis) from elementary school students in Jeddah, Saudi Arabia.

Head lice (Pediculus humanus capitis) are a major global concern, and there is growing evidence of an increase in head lice prevalence among Saudi schoolchildren. The purpose of this study is to investigate the prevalence of an insecticidal resistance mutation in head lice collected from schoolchildren. A polymerase chain reaction (PCR) was used to amplify a segment of the voltage-gated sodium channel gene subunit to assess the prevalence and distribution of the kdr T917I mutation in head lice. Subsequently, the restriction fragment length polymorphism (RFLP) patterns revealed two genotypic forms: homozygous-susceptible (SS) and homozygous-resistant (RR). The results showed that 17 (37.80%) of the 45 samples were SS, whereas 28 (62.2%) were RR and T917I and L920F point mutations were found in the nucleotide and amino acid sequences of RR. Compared to other nations, the frequency of permethrin resistance mutation in the head louse population in Saudi Arabia was low. This study provides the first evidence of permethrin resistance mutation in human head lice in Saudi Arabia. The findings of this study will highlight the rising incidence of the kdr mutation in head lice in Saudi Arabia.

Genetic structure and kdr mutations in Aedes aegypti populations along a road crossing the Amazon Forest in Amapá State, Brazil

Insecticide resistance in Aedes aegypti poses a significant threat to disease control. One form of resistance, caused by kdr mutations in the NaV gene, hinders vector control efforts in Brazil. Despite genetic differences typically accumulating among isolated populations, this mosquito can actively and passively disperse through human transportation. Our study investigated the genetic structure and spread of kdr mutations in Ae. aegypti populations across six localities in Amapá State, Brazil, within the Amazonian Forest. Using 12 microsatellite loci and qPCR methods, we assessed genetic structure and identified three common kdr mutations (V410L, V1016I, and F1534C). High prevalence of kdr alleles was observed in all localities, indicating widespread distribution in Amapá State. Microsatellite analysis revealed differentiation among mosquito populations, dividing them into two distinct clusters supported by Bayesian and DAPC analyses. Oiapoque, located along the northern border with French Guiana, exhibited the highest kdr frequencies and genetic differentiation compared to other localities. Our findings suggest genetic structure in Ae. aegypti populations in Amapá State, with some passive gene flow between clusters. The study underscores the need for continuous surveillance of Ae. aegypti populations to monitor the spread of insecticide resistance and inform effective vector control strategies.

Response to insecticides and underlying mechanisms of resistance in the field populations of Aedes aegypti Linnaeus (Diptera: Culicidae) in Puducherry, India.

Mosquito-borne diseases are major threats to human health worldwide. Successful control of vector mosquitoes requires periodic updates on their response to the insecticides that are in use. Different classes of neurotoxic insecticides have been used in vector control programs. Ae. aegypti and Ae. albopictus are the primary vectors of dengue and have developed resistance to organophosphates and synthetic pyrethroids that are used in vector control programs. Monitoring insecticide pressure and studying the underlying mechanisms of resistance in the field populations of Aedes aegypti are important to formulate resistant management strategies for their control programs. Aedes aegypti were collected from study sites Lawspet and Abishegapakkam and F1 progeny was subject to biochemical assays to determine the enzyme activity. Insecticide susceptibility tests were conducted to determine vector susceptibility/resistance to malathion and deltamethrin. Adult dried mosquitoes were subjected to multiplex PCR to detect point mutation in the VGSC gene. Insecticide susceptibility test results revealed that Aedes aegypti is resistant to malathion and incipient resistance to deltamethrin has emerged. It was observed that β-esterase and monoxygense activity were significantly higher in Lawspet sample than the laboratory strain, whereas it was comparatively lower in Abishegapakkam sample than laboratory strain. Multiplex PCR assays showed no kdr mutation in all Ae. aegypti strains. Monitoring insecticide resistance in Ae. aegypti would help the local health authorities to implement a rationalized approach for insecticide use in vector control.

Randomized detection of kdr allele frequencies in wild populations of Aedes aegypti (Diptera, Culicidae) in Colombo District, Sri Lanka

Sri Lanka is one of the most affected countries in South Asia by dengue fever, with the number of dengue cases increasing over the last five years. The main strategy for managing disease outbreaks is to reduce infected vector populations with pyrethroid insecticides. However, extensive pyrethroid exposure has resulted in an increase in the selection of knockdown resistance mutations in Aedes aegypti (Linnaeus) (Diptera, Culicidae) voltage-gated sodium channel (vgsc) gene that confer pyrethroid resistance. Colombo district records the highest dengue incidence across the country each year, thus a failed vector control program will be a major threat to public health. Multiplexed Allele-specific PCR was used to genotype kdr alleles in wild Ae. aegypti mosquitoes obtained via random sampling from Wellawatte, Borella, and Battaramulla areas in the Colombo district. This study presents the co-occurrence of F1534C and V1016G kdr mutations from a randomized population in the Colombo district. 1534C mutant allele was predominant (with a 56.7% frequency) and 1016G was prevalent in 32.5 per cent of the population. The heterozygous mutant 1016VG genotype showed the highest distribution (with a 65% frequency) and the incidence of 1534FC was 56.7 per cent. Interestingly, 1016GG was completely absent and the FC/VG mutation combination had the highest incidence with 46.7 per cent. Furthermore, 82.36 per cent of individuals with the 1534FC genotype also had the 1016VG genotype, indicating a high prevalence of pyrethroid resistance in the studied population.

Deltamethrin and transfluthrin select for distinct transcriptomic responses in the malaria vector Anopheles gambiae

BackgroundThe widespread use of pyrethroid insecticides in Africa has led to the development of strong resistance in Anopheles mosquitoes. Introducing new active ingredients can contribute to overcome this phenomenon and ensure the effectiveness of vector control strategies. Transfluthrin is a polyfluorinated pyrethroid whose structural conformation was thought to prevent its metabolism by cytochrome P450 monooxygenases in malaria vectors, thus representing a potential alternative for managing P450-mediated resistance occurring in the field. In this study, a controlled selection was used to compare the dynamics of resistance between transfluthrin and the widely used pyrethroid deltamethrin in the mosquito Anopheles gambiae. Then, the associated molecular mechanisms were investigated using target-site mutation genotyping and RNA-seq.MethodsA field-derived line of An. gambiae carrying resistance alleles at low frequencies was used as starting material for a controlled selection experiment. Adult females were selected across 33 generations with deltamethrin or transfluthrin, resulting in three distinct lines: the Delta-R line (selected with deltamethrin), the Transflu-R line (selected with transfluthrin) and the Tiassale-S line (maintained without selection). Deltamethrin and transfluthrin resistance levels were monitored in each selected line throughout the selection process, as well as the frequency of the L1014F kdr mutation. At generation 17, cross-resistance to other public health insecticides was investigated and transcriptomes were sequenced to compare gene transcription variations and polymorphisms associated with adaptation to each insecticide.ResultsA rapid increase in resistance to deltamethrin and transfluthrin was observed throughout the selection process in each selected line in association with an increased frequency of the L1014F kdr mutation. Transcriptomic data support a broader response to transfluthrin selection as compared to deltamethrin selection. For instance, multiple detoxification enzymes and cuticle proteins were specifically over-transcribed in the Transflu-R line including the known pyrethroid metabolizers CYP6M2, CYP9K1 and CYP6AA1 together with other genes previously associated with resistance in An. gambiae.ConclusionThis study confirms that recurrent exposure of adult mosquitoes to pyrethroids in a public health context can rapidly select for various resistance mechanisms. In particular, it indicates that in addition to target site mutations, the polyfluorinated pyrethroid transfluthrin can select for a broad metabolic response, which includes some P450s previously associated to resistance to classical pyrethroids. This unexpected finding highlights the need for an in-depth study on the adaptive response of mosquitoes to newly introduced active ingredients in order to effectively guide and support decision-making programmes in malaria control.

Investigating the breeding habitats and kdr allele frequency of malaria vectors in Anambra Central Senatorial Zone, Southeast Nigeria

Malaria, transmitted by infected female Anopheles mosquitoes, is a parasitic disease of major public health importance in Nigeria. Various classes of insecticides have played significant role in the control and elimination of malaria by attacking the vectors. Unfortunately, the success of the use of insecticides in the control of malaria vectors is threatened by the emergence of insecticidal resistance. This study was therefore aimed at determining the resistance status of Anopheles gambiae s. l. in communities in Anambra State, southeast Nigeria. Larval samples were collected from diverse habitats and reared to adult for susceptibility test. Target site resistance assays were carried out and genotypic differentiation test was performed to assess variability of the allelic frequencies of the kdr (L1014F) mutation. The results showed that Anopheles gambiae was the predominant vector (54.2%), followed by Anopheles coluzzii (45.8%). The distribution of the frequency of resistant allele of the kdr gene F (L1014F) showed that the number of homozygous resistant genotype (RR) in Awka North LGA for An. gambiae and An. coluzzii species was highest (31), followed by Awka South (20), and Njikoka (5) LGAs. Also, the number of Heterozygous resistant genotype (Rr) was highest in Awka South (7), followed by Awka North (2), and none was recorded in Njikoka LGA. Homozygous resistant genotype (RR) was frequently observed in the three study areas of Anambra State and is a threat to the use of pyrethroids for insecticidal control of malaria vectors. There is an urgent need for implementation of insecticide resistance management and monitoring in Anambra State to assess the spread of resistance which may expand malaria vector resistance to the remaining classes of insecticides (organophosphates and carbamates) on the long run.

Insecticide Resistance and Metabolic Mechanisms in Aedes aegypti from Two Agrosystems (Vegetable and Cotton Crops) in Côte d'Ivoire.

Background: The emergence of insecticide resistance in Aedes vectors mosquitoes poses a real challenge for arboviral-borne disease control. In Côte d'Ivoire, data are available on phenotypic resistance and the presence of kdr mutations in Aedes aegypti. Therefore, information on metabolic resistance in Aedes populations is very scarce. Here, we assessed the insecticide resistance status of Ae. aegypti in periurban and rural areas of Côte d'Ivoire, and we investigated the role of detoxification enzymes as possible resistance mechanisms. Materials and Methods: Aedes mosquito eggs were collected between June 2019 to April 2021 in two agricultural sites. Adults of Ae. aegypti were tested using World Health Organization tube assays, with seven insecticides belonging to pyrethroids, organochlorines, carbamates, and organophosphates classes. We determined the knockdown times (KdT50, KdT95) and resistance ratios of pyrethroids in natural populations. The synergist piperonyl butoxide (PBO) was used to investigate the role of enzymes in resistance. Biochemical assays were performed to detect potential increased activities in mixed-function oxidase levels, nonspecific esterases (NSEs), and glutathione S-transferases. Results: The results showed that Ae. aegypti populations were resistant to five insecticides with mortality of 46% and 89% for 0.75% permethrin, 68% and 92% for 0.05% deltamethrin, 57% and 89% for lambda-cyhalothrin, 41% and 47% for dichlorodiphenyltrichloroethane (DDT), 82% and 91% for chlorpyrifos-methyl in Songon-Agban and Kaforo, respectively. Susceptibility to carbamates was observed in our study sites. After exposure to PBO, the susceptibility of Ae. aegypti to pyrethroids and DDT was partially restored in Songon-Agban. Whereas in Kaforo, none increase of the mortality rates of Ae. aegypti for these four insecticides was observed after exposure to PBO. Increased activity of NSE (α-esterases) was found in Songon-Agban compared with the reference susceptible strain. Conclusion: These findings provide valuable information to support decisions for vector control strategies in Cote d'Ivoire. Also, we highlight the need for the monitoring of insecticide resistance management in Aedes vectors.

Knockdown resistance mutations are common and widely distributed in Xenopsylla cheopis fleas that transmit plague in Madagascar.

Plague, caused by the bacterium Yersinia pestis, remains an important disease in Madagascar, where the oriental rat flea, Xenopsylla cheopis, is a primary vector. To control fleas, synthetic pyrethroids (SPs) have been used for >20 years, resulting in resistance in many X. cheopis populations. The most common mechanisms of SP resistance are target site mutations in the voltage-gated sodium channel (VGSC) gene. We obtained 25 collections of X. cheopis from 22 locations across Madagascar and performed phenotypic tests to determine resistance to deltamethrin, permethrin, and/or dichlorodiphenyltrichloroethane (DDT). Most populations were resistant to all these insecticides. We sequenced a 535 bp segment of the VGSC gene and identified two different mutations encoding distinct substitutions at amino acid position 1014, which is associated with knockdown resistance (kdr) to SPs in insects. Kdr mutation L1014F occurred in all 25 collections; a rarer mutation, L1014H, was found in 12 collections. There was a significant positive relationship between the frequency of kdr alleles and the proportion of individuals surviving exposure to deltamethrin. Phylogenetic comparisons of 12 VGSC alleles in Madagascar suggested resistant alleles arose from susceptible lineages at least three times. Because genotype can reasonably predict resistance phenotype, we developed a TaqMan PCR assay for the rapid detection of kdr resistance alleles. Our study provides new insights into VGSC mutations in Malagasy populations of X. cheopis and is the first to report a positive correlation between VGSC genotypes and SP resistance phenotypes in fleas. Widespread occurrence of these two SP resistance mutations in X. cheopis populations in Madagascar reduces the viability of these insecticides for flea control. However, the TaqMan assay described here facilitates rapid detection of kdr mutations to inform when use of these insecticides is still warranted to reduce transmission of plague.

Absence of known knockdown resistance mutations but fixation of CYP337B3 was detected in field populations of Helicoverpa armigera across China

The cotton bollworm (Helicoverpa armigera) is a worldwide agricultural pest that infests many important crops. Pyrethroids targeting the voltage-gated sodium channel (VGSC) have been long used in the control of the cotton bollworm. Two amino acid substitutions (D1561V and E1565G) in H. armigera VGSC (HaVGSC) and the presence of a chimeric P450 gene (CYP337B3) have been documented to be associated with pyrethroid resistance. To understand the current occurrence of kdr mutations and the CYP337B3 gene in Chinese H. armigera populations, high-throughput amplicon sequencing was adopted to detect potential nucleotide variations in three fragments of the VGSC gene that cover 10 reported knockdown resistance (kdr) sites in insects, and gene-specific PCR was performed to examine the presence of CYP337B3 gene in H. armigera samples collected across China. The nucleotide variation analysis revealed a wealth of nucleotide variations in not only exons but also introns in the VGSC gene in Chinese H. armigera populations. However, neither previously reported kdr-conferring amino acid replacements nor other non-synonymous mutations were observed in a total of 1439 examined individuals. Population genetic analysis suggested that the H. armigera population in Nanchang, Jiangxi Province (JNC) had a moderate genetic differentiation from other populations, while no significant divergence was observed in other populations in northern and northwestern China. The CYP337B3 was present in all the examined individuals, indicating that CYP337B3 is extensively fixed in H. armigera populations across China. These results support that point mutations in VGSC are not a major factor involved in the current pyrethroid resistance in H. armigera. Instead, CYP337B3 plays a prevalent role in the development of resistance to pyrethroids in H. armigera.

Myzus persicae (Sulzer, 1776) (Hemiptera: Aphididae) popülasyonlarında neonikotinoid ve sentetik piretroid insektisitlerin tekli ve karışımlarına karşı direnç durumu

The peach potato aphid, Myzus persicae (Sulzer, 1776) (Hemiptera: Aphididae), a vector of many plant virus diseases, causes damage to its wide range of hosts by direct feeding. Chemical control has been the primary method to control this species, and the intensive use of insecticides has led to the development of resistance. In this study, conducted between the years 2017-2019, firstly resistance ratio of five M. persicae populations from Antalya, Türkiye were determined by leaf-dip bioassay method. The field populations showed significant resistance to thiamethoxam (between 201-332 fold) and lambda-cyhalothrin (between 50-103 fold) when compared to susceptible population. To identify whether resistance mediated by mutations in sodium channel and nicotinic acetylcholine receptor, DNA regions that encompass “mutation hot-spot” were sequenced. This revealed no population contained R81T mutation that has been previously linked with neonicotinoid resistance. As to synthetic pyrethroid resistance, the L1014F kdr mutation was fixed in all field populations. This study is the first description of kdr mutation in M. persicae populations from Türkiye. Bioassay results also indicated that the toxicity of thiamethoxam and lambda-cyhalothrin mixture was higher than that of lambda-cyhalothrin alone. Our findings can make significant contributions to M. persicae resistance management.

Inference of selection pressures that drive insecticide resistance in Anopheles and Culex mosquitoes in Korea

Pyrethroids are primarily used for mosquito control in Korea. However, high frequencies of mutations conferring resistance to not only pyrethroids but also to other insecticides have been found in mosquito populations. This study aimed to examine the hypothesis that insecticides used outside of public health may play a role in selection. Briefly, the resistance mutation frequencies to three insecticide groups (pyrethroids, organophosphates, and cyclodienes) were estimated in two representative groups of mosquito species (Anopheles Hyrcanus Group and Culex pipiens complex). The relationship between these frequencies and the land-use status of the collection sites was investigated through multiple regression analysis. In the Anopheles Hyrcanus Group, the frequencies of both ace1 (organophosphate resistance) and rdl (cyclodiene resistance) mutations were positively correlated with ‘proximity to golf course’, possibly be due to the insecticides used for turf maintenance. They also showed positive correlations with field area and rice paddy area, respectively, suggesting the role of agricultural insecticides in the selection of these resistance traits. For the Cx. pipiens complex, the kdr (pyrethroid resistance), ace1, and rdl mutations were positively correlated with the residential area, field, and rice paddy, respectively. Therefore, pyrethroids used for public health could serve as a direct source of resistance selection pressure against kdr, whereas non-public health insecticides may pose primary selection pressure against the ace1 and rdl traits. The current findings suggest that the insecticides used in agriculture and the golf industry play a significant role in mosquito selection, despite variations in the extent of indirect selection pressure according to the mosquito groups and insecticide classes.

Phenotypic resistance to pyrethroid associated to metabolic mechanism in Vgsc-L995F-resistant Anopheles gambiae malaria mosquitoes.

Background: The indiscriminate use of insecticides in agriculture and public health lead to a selection of resistance mechanisms in malaria vectors compromising vector control tools and strategies. This study investigated the metabolic response in the Vgsc-L995F Anopheles gambiae Tiassalé resistance strain after long-term exposure of larvae and adults to deltamethrin insecticide. Methods: Vgsc-L995F An. gambiae Tiassalé strain larvae were exposed over 20 generations to deltamethrin (LS) and adults to PermaNet 2.0 (AS) and combining exposure at larvae and adult stages (LAS) and compared to unexposed (NS) group. All four groups were subjected to the standard World Health Organization (WHO) susceptibility tube tests using deltamethrin (0.05%), bendiocarb (0.1%) and malathion (5%). Vgsc-L995F/S knockdown-resistance ( kdr) mutation frequency was screened using multiplex assays based on Taqman real-time polymerase chain reaction (PCR) method. Additionally, expression levels of detoxification enzymes associated to pyrethroid resistance, including CYP4G16, CYP6M2, CYP6P1, CYP6P3, CYP6P4, CYP6Z1 and CYP9K1, and glutathione S-transferase GSTe2 were measured. Results: Our results indicated that deltamethrin resistance was a response to insecticide selection pressure in LS, AS and LAS groups, while susceptibility was observed in NS group. The vectors showed varied mortality rates with bendiocarb and full susceptibility to malathion throughout the selection with LS, AS and LAS groups. Vgsc-L995F mutation stayed at high allelic frequency level in all groups with a frequency between 87% and 100%. Among the overexpressed genes, CYP6P4 gene was the most overexpressed in LS, AS and LAS groups. Conclusion: Long-term exposure of larvae and adults of Vgsc-L995F resistant- An. gambiae Tiassalé strain to deltamethrin and PermaNet 2.0 net induced resistance to deltamethrin under a significant effect of cytochromes P450 detoxification enzymes. These outcomes highlight the necessity of investigating metabolic resistance mechanisms in the target population and not solely kdr resistance mechanisms prior the implementation of vector control strategies for a better impact.

Frequency and spatial distribution of knock-down resistance (kdr) to pyrethroids in multiple oilseed rape pest species of the genus Ceutorhynchus.

The protection of European oilseed rape (OSR) from damaging insects relies on pyrethroid insecticides, but the development of resistance in key coleopteran pests such as the pollen beetle (Brassicogethes aeneus) and the cabbage stem flea beetle (Psylliodes chrysocephala) has resulted in reduced effectiveness of these insecticides. The sodium channel gene mutation L1014F knock-down resistance (kdr) is a contributing factor in resistance to pyrethroids in B. aeneus and P. chrysocephala, but little is known about the status of resistance in weevils of the genus Ceutorhynchus (Coleoptera: Curculonidae). Therefore, the present study investigated pyrethroid susceptibility and the presence of the kdr mutation in four Ceutorhynchus species. The kdr mutation in either its heterozygous or homozygous form was found in all investigated Ceutorhynchus species (C. picitarsis, C. pallidactylus, C. napi and C. obstrictus). Samples where pyrethroids in bioassays still provided control at 100% field rate or below contained kdr at frequencies of ≤12.5%, whilst bioassays using 100% field rate that did not control Ceutorhynchus populations contained homozygous resistant individuals at frequencies of greater than 55%. Field sampling demonstrated that kdr frequencies in populations of C. picitarsis and C. obstrictus collected from across France and Germany ranged from 0 to 100%. The present study demonstrated the potential of all four Ceutorhynchus species tested to develop pyrethroid resistance via the L1014F (kdr) mutation. Although kdr frequency varies among species and geographic locations, the risk of loss of pyrethroid insecticide effectiveness is high. Integration of other control tools for resistance management is therefore needed. © 2023 Society of Chemical Industry.

Detection of Knockdown-Resistance Homozygous Mutant C1534C using Allele-Specific Polymerase Chain Reaction in Aedes Albopictus and Aedes Aegypti

Highlights: This research analyzed a previously understudied subject in Surabaya, Indonesia, and discovered knockdown resistance (kdr) mutations in Aedes albopictus. The findings of this study can prompt further research, including bioassay testing and the development of more potent insecticides. Abstarct : Dengue hemorrhagic fever (DHF) has been endemic in Indonesia for decades. Insecticides are necessary to manage the transmission of the dengue virus. However, prolonged use of insecticides can lead to insecticide resistance. This study aimed to examine the genotype of mosquitoes using the allele-specific polymerase chain reaction (ASPCR) method. The ASPCR method was chosen for genotype detection due to its high sensitivity, affordability, and ease of design. Five mosquitoes were collected from human habitation in four different areas of Surabaya, Indonesia, namely Kranggan, Ulul Azmi Mosque, Ploso, and Kalijudan. Among them, three samples were identified as Aedes albopictus (A1, A2, and A5) and two samples were identified as Aedes aegypti (A3 and A4). The frequency of resistant alleles was analyzed using the Hardy-Weinberg package in RStudio version 2023.03.1. This study revealed that two mosquitoes carried homozygous mutant alleles with a band of 113 bp and three mosquitoes carried homozygous wild-type alleles with a band of 93 bp. Cysteine-to-cysteine (C/C) mutations and phenylalanine-to-phenylalanine (F/F) mutations at codon 1534 were observed in Aedes aegypti and Aedes albopictus mosquitoes. The homozygous mutant alleles were found in Kranggan, Surabaya, Indonesia. Further research is required to assess insecticide resistance and knockdown resistance (kdr)-like mutation by collecting more representative samples from larger areas within the region of Surabaya. Nevertheless, this study can be used as a reference for vector control and early prevention of dengue fever.

Leucine to tryptophane substitution in the pore helix IIP1 confer sodium channel resistance to pyrethroids and DDT

Aedes aegypti is responsible for transmitting a variety of arboviral infectious diseases such as dengue and chikungunya. Insecticides, particularly pyrethroids, are used widely for mosquito control. However, intensive used of pyrethroids has led to the selection of kdr mutations on sodium channels. L982W, locating in the PyR1 (Pyrethroid receptor site 1), was first reported in Ae. aegypti populations collected from Vietnam. Recently, the high frequency of L982W was detected in pyrethroid-resistant populations of Vietnam and Cambodia, and also concomitant mutations L982W + F1534C was detected in both countries. However, the role of L982W in pyrethroid resistance remains unclear. In this study, we examined the effects of L982W on gating properties and pyrethroid sensitivity in Xenopus oocytes. We found that mutations L982W and L982W + F1534C shifted the voltage dependence of activation in the depolarizing direction, however, neither mutations altered the voltage dependence of inactivation. L982W significantly reduced channel sensitivity to Type I pyrethroids, permethrin and bifenthrin, and Type II pyrethroids, deltamethrin and cypermethrin. No enhancement was observed when synergized with F1534C. In addition, L982W and L982W + F1534C mutations reduced the channel sensitivity to DDT. Our results illustrate the molecular basis of resistance mediates by L982W mutation, which will be helpful to understand the interacions of pyrethroids or DDT with sodium channels and develop molecular markers for monitoring pest resistance to pyrethroids and DDT.

Primary cardiac angiosarcoma: a clinicopathological and molecular genetic analysis of thirteen cases

Objective: To investigate the clinicopathological and molecular features of primary cardiac angiosarcoma (PCAS), and to analyze the correlation between KDR mutation and the clinicopathological features of PCAS. Methods: Thirteen cases of PCAS were collected at Beijing Anzhen Hospital, Capital Medical University from January 2007 to December 2021. The clinicopathological features, diagnosis, differential diagnosis and outcome were retrospectively analyzed. KDR mutation was detected by next-generation sequencing (NGS) and then the expression of KDR (VEGFR2) was determined by immunohistochemistry (IHC), with review of relevant literatures. Results: There were eight males and five females with a mean age of 45 years. The primary tumor was in the right atrium in 10 cases, left atrium in two cases and right ventricle in one case. The histomorphology was mainly poorly differentiated angiosarcoma (11 cases), with highly pleomorphic spindle or round cells in solid sheets, brisk mitotic activity and extensive necrosis. Vascular lumen formation was observed in two cases of high to moderate differentiation, and biphenotypic differentiation was seen in five cases. IHC staining showed CD34, CD31, Fli1, ERG and vimentin were diffusely positive, pan-cytokeratin was positive, Ki-67 index ranged from 3% to 90%, which was positively correlated with the differentiation degree and grade of the PCASs (P<0.05). At the end of follow-up period, one patient was alive, two patients were lost to follow-up, and the remaining 10 patients had an average survival time of 4.6 months. Finally, NGS sequencing was performed on seven samples after screening, and the results showed that KDR and NF1 mutations were both present in three cases. VEGFR2 expression had no significant correlation with the differentiation degree and grade of PCAS (P>0.05), and it was not related to KDR mutation. Conclusions: PCASs mainly occur in the right atrium, and are mainly poorly differentiated. Ki-67 index is helpful to assess the degree and grade of tumor differentiation. The occurrence and development of PCAS may be related to the pathway involved in KDR mutation, but KDR mutation has no clear correlation with clinicopathological characteristics of PCAS, and immunohistochemical staining can not replace gene detection to determine whether the tumor had KDR mutation.

High frequency of Voltage-gated sodium channel (VGSC) gene mutations in Aedes albopictus (Diptera: Culicidae) suggest rapid insecticide resistance evolution in Shanghai, China.

Dengue fever is an infectious disease that is imported into Shanghai, China and requires prevention and control measures. Controlling the vector Aedes albopictus through insecticide use is a key approach to dengue control. However, the rapid evolution of insecticide resistance in Ae. albopictus has raised concerns about the failure of dengue control efforts. Knockdown resistance (kdr) caused by point mutations in the voltage-gated sodium channel (VGSC) gene is a primary mechanism of pyrethroid resistance. In this study, we investigated the kdr mutations of Ae. albopictus in Shanghai and evaluated the trend in its evolution. We collected 17 populations of Ae. albopictus from 15 districts in Shanghai in 2020, extracted genomic DNA from individual mosquitoes, and amplified Domain II, III, and IV in VGSC using PCR. Following sequencing, we obtained 658 VGSC sequences. We detected the nonsynonymous mutations V1016G, I1532T, and F1534S/C/I, among which V1016G and F1534C/I were reported in Shanghai for the first time and F1534I was a novel mutant allele in Ae. albopictus. The overall mutation frequency was 84.65%, with individual mutation frequencies ranging from 46.81% to 100%, excluding the Fengxian District population, which had a frequency of 0%. The V1016G and I1532T mutation types accounted for 7.14% and 3.42%, respectively. The mutant allele at codon 1534 accounted for 63.98% of all mutations, including TCC/S (62.77%), TGC/C (1.06%), and ATC/I (0.15%). We identified and classified five intron types in Domain III by length, including A (83 bp, 12.07%), B (68 bp, 87.30%), C (80 bp, 0.16%), D (72 bp, 0.16%), and E (70 bp, 0.31%). Individuals with intron B had a significant mutation tendency at codon 1534 relative to intron A (chi-square test, p < 0.0001). We found no correlation between mutation frequency and the amount of pyrethroid used (Pearson correlation, p = 0.4755). In recent years, kdr mutations in the Ae. albopictus population in Shanghai have rapidly evolved, as evidenced by an increase in mutation types and significantly increased mutation frequency. The F1534I/ATC mutant allele was found to be a novel mutation, F1534C/TGC was reported for the first time in Shanghai, and intron B in Domain III was significantly associated with mutation frequency at codon 1534. Continuous monitoring of resistance changes and strict regulation of insecticide use are required.