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

Abstract Background: In 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.Methods: High-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.Results: The 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%.Conclusions: Given 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.

Download Full-text

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

10.21203/rs.3.rs-17263/v5 ◽

2020 ◽

Author(s):

Juli R. Wuliandari ◽

Ary A. Hoffmann ◽

Warsito Tantowijoyo ◽

Nancy M. Endersby-Harshman

Keyword(s):

Inner City ◽

Insecticide Resistance ◽

Sodium Channel ◽

Pyrethroid Resistance ◽

Infected Mosquito ◽

Simultaneous Occurrence ◽

Wild Type ◽

Pyrethroid Insecticides ◽

Background Population ◽

Homozygous Mutant

Download Full-text

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

10.21203/rs.3.rs-17263/v3 ◽

2020 ◽

Author(s):

Juli R. Wuliandari ◽

Ary A. Hoffmann ◽

Warsito Tantowijoyo ◽

Nancy M. Endersby-Harshman

Keyword(s):

Aedes Aegypti ◽

Inner City ◽

Insecticide Resistance ◽

Sodium Channel ◽

Pyrethroid Resistance ◽

Infected Mosquito ◽

Simultaneous Occurrence ◽

Pyrethroid Insecticides ◽

Background Population ◽

Homozygous Mutant

Abstract Background : In 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. Methods: High-resolution melt genotyping was used to screen for kdr mutations associated with pyrethroid resistance in the voltage-sensitive sodium channel (V SSC ) gene in Ae. aegypti of some areas in the inner city of Yogyakarta. Results: The 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 wildtype 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%. Conclusions: Given 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.

Download Full-text

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

10.21203/rs.3.rs-17263/v2 ◽

2020 ◽

Author(s):

Juli R. Wuliandari ◽

Ary A. Hoffmann ◽

Warsito Tantowijoyo ◽

Nancy M. Endersby-Harshman

Keyword(s):

Inner City ◽

Insecticide Resistance ◽

Sodium Channel ◽

Pyrethroid Resistance ◽

Infected Mosquito ◽

Simultaneous Occurrence ◽

High Resolution Melt ◽

Pyrethroid Insecticides ◽

Background Population ◽

Homozygous Mutant

Abstract Background: In 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 A. aegypti being released matches that of the background population.Methods: High-resolution melt genotyping was used to screen for kdr mutations associated with pyrethroid resistance in the voltage-sensitive sodium channel (VSSC) gene in A. aegypti of some areas in the inner city of Yogyakarta.Results: The 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 wildtype 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%.Conclusions: Given 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.

Download Full-text

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

10.21203/rs.3.rs-17263/v1 ◽

2020 ◽

Author(s):

Juli R. Wuliandari ◽

Ary A. Hoffmann ◽

Warsito Tantowijoyo ◽

Nancy M. Endersby-Harshman

Keyword(s):

Aedes Aegypti ◽

Inner City ◽

Insecticide Resistance ◽

Sodium Channel ◽

Pyrethroid Resistance ◽

Infected Mosquito ◽

Simultaneous Occurrence ◽

High Resolution Melt ◽

Pyrethroid Insecticides ◽

Homozygous Mutant

Abstract Background: In 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.Methods: High-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.Results: The 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 wildtype 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%.Conclusions: Given 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.

Download Full-text

Characterization of the M918T sodium channel gene mutation associated with strong resistance to pyrethroid insecticides in the peach-potato aphid, Myzus persicae (Sulzer)

Bulletin of Entomological Research ◽

10.1017/s0007485307005524 ◽

2007 ◽

Vol 98 (2) ◽

pp. 183-191 ◽

Cited By ~ 30

Author(s):

I. Eleftherianos ◽

S.P. Foster ◽

M.S. Williamson ◽

I. Denholm

Keyword(s):

Sodium Channel ◽

Myzus Persicae ◽

Pyrethroid Resistance ◽

Type I ◽

Pyrethroid Insecticides ◽

Potato Aphid ◽

Channel Gene ◽

Sodium Channel Gene ◽

Wide Range ◽

Peach Potato Aphid

AbstractRecent advances in the characterisation of insect sodium channel gene sequences have identified a small number of point mutations within the channel protein that are implicated in conferring target-site resistance to pyrethroid insecticides (so-called knockdown resistance or kdr). The L1014F (leucine-to-phenylalanine) mutation located in the centre of segment 6 of the domain II region (IIS6) of the sodium channel (the so-called kdr trait) has been detected in the peach-potato aphid, Myzus persicae (Sulzer), and is considered to be the primary cause of pyrethroid resistance in this species. Here we report on the characterisation of a second mutation, M918T (methione-to-threonine), within the nearby IIS4–S5 intracellular linker (the so-called super-kdr trait) in a field clone also possessing L1014F, with both mutations present in heterozygous form. The resistance phenotype of M. persicae clones possessing various combinations of L1014F and M918T to a wide range of pyrethroids (both Type I and II) was assessed in leaf-dip bioassays and to lambda-cyhalothrin applied at up to ten times the recommended field rate as foliar sprays to aphids feeding on whole plants. Bioassay results demonstrated that presence of both mutations was associated with extreme resistance to all the pyrethroids tested relative to aphids lacking the mutations. Furthermore, this resistance well exceeded that shown by aphids that were homozygous for L1014F but lacking M918T. However, pre-treatment with piperonyl butoxide in the leaf-dip bioassays failed to suppress pyrethroid resistance in aphids carrying one or both of the mutations. The relevance of these findings for monitoring and managing pyrethroid resistance in M. persicae populations in the field is discussed.

Download Full-text

High-throughput genotyping of a full voltage-gated sodium channel gene via genomic DNA using target capture sequencing and analytical pipeline MoNaS to discover novel insecticide resistance mutations

10.1101/564609 ◽

2019 ◽

Author(s):

Kentaro Itokawa ◽

Koji Yatsu ◽

Tsuyoshi Sekizuka ◽

Yoshihide Maekawa ◽

Osamu Komagata ◽

...

Keyword(s):

Amino Acid ◽

Insecticide Resistance ◽

Sodium Channel ◽

Genomic Dna ◽

Genomic Structure ◽

Voltage Gated Sodium Channel ◽

Pyrethroid Insecticides ◽

Voltage Gated ◽

Probe Set ◽

Capture Sequencing

AbstractInsects’ voltage-gated sodium channel (VGSC) is the primary target site of pyrethroid insecticides. Various amino acid substitutions in the VGSC protein are known to confer insecticide resistance and are selected under insecticide pressure. In the genome, the VGSC gene consists of more than 30 exons sparsely distributed across a large genomic region, which often exceeds 100 kbp. Due to this complex genomic structure of gene VGSC, it is usually challenging to genotype full coding nucleotide sequences (CDSs) of VGSC from individual genomic DNA (gDNA). In this study, we designed biotinylated oligonucleotide probes via annotated CDSs of VGSC of Asian tiger mosquito, Aedes albopictus. The probe set effectively concentrated (>80,000-fold) all targeted regions of gene VGSC from pooled barcoded Illumina libraries each constructed from individual A. albopictus gDNAs. The probe set also captured all homologous VGSC CDSs except tiny exons from the gDNA of other Culicinae mosquitos, A. aegypti and Culex pipiens complex, with comparable efficiency by virtue of the high conservation of VGSC at the nucleotide level. Furthermore, we developed an automated bioinformatic analysis pipeline to genotype VGSC after capture sequencing—MoNaS (Mosquito Na+ channel mutation Search)—which conducts mapping of reads, variant calling, and variant annotation for nonsynonymous mutations. The proposed method and our bioinformatic tool should facilitate the discovery of novel amino acid variants conferring insecticide resistance on VGSC and population genetics studies on resistance alleles with respect to the origin, selection, and migration of both clinically and agriculturally important insect pests.

Download Full-text

Molecular Insight into the Knockdown Resistance (kdr) in the Voltage Gated Sodium Channel (vgsc) Gene of the Main Dengue Vector, Aedes aegypti (Diptera: Culicidae) and the Discovery of Novel Regional Specific Point Mutation A1007G in Malaysia.

10.21203/rs.3.rs-102568/v2 ◽

2021 ◽

Author(s):

Mas Azlin M. Akhir ◽

Mustafa F. F. Wajidi ◽

Sébastien Lavoué ◽

Ghows Azzam ◽

Izhan Shahrin Jaafar ◽

...

Keyword(s):

Insecticide Resistance ◽

Sodium Channel ◽

Vector Control ◽

Resistance Mechanism ◽

Point Mutations ◽

Voltage Gated Sodium Channel ◽

Pyrethroid Insecticides ◽

Voltage Gated ◽

Control Programmes ◽

Pirimiphos Methyl

Abstract Background: Characterization of the insecticide resistance mechanism imparts the society with the information on the evolutionary process involved in the adaptation of Aedes aegypti mosquito to environmental changes. Investigating the phenotypic status of the target mosquitoes, their resistance level as well as elucidating the genotypic profile provides information about the involvement of insecticide resistance mechanism, in terms of portraying the evolution of resistance in the field, to eventually managing vector control programmes. In this current study, we investigated the quantification responses for the phenotypic and genotypic resistance of Ae. aegypti population from different states in Malaysia. Methods: We tested insecticide susceptibility status of adult Ae. aegypti from populations of States of Penang, Selangor and Kelantan (Peninsular Malaysia) against 0.25% permethrin and 0.25% pirimiphos-methyl through WHO bioassay kit. Permethrin-resistant and permethrin susceptible samples were then genotyped for domains II and III in the voltage gated sodium channel (vgsc) gene using allele specific PCR (AS-PCR) for the presence of diagnostic single nucleotide mutations. AS-PCR results were then validated in sequencing these two domains to identify any possible additional point mutations. Results: Adult WHO bioassay revealed that populations of Ae. aegypti from these three states were highly resistant towards 0.25% permethrin and 0.25% pirimiphos-methyl. Genotyping results showed that three knockdown (kdr) mutations (i.e. S989P, V1016G and F1534C) were associated with pyrethroid resistance in these populations. We also report for the first time the presence of the A1007G mutation in Malaysian populations of Ae. aegypti.Conclusions: This study brings an insight on the occurrence and association of point mutations with insecticide resistance in Malaysian populations of Ae. aegypti. The results reveal the widespread of several kdr mutations in the field with the consequence to compromise the use of pyrethroid insecticides in vector control programmes. Knowledge on the distribution of target site resistance throughout Malaysia is vital to ensure the success of the insecticide-based vector control programme.

Download Full-text

Identification of a rapidly-spreading triple mutant for high-level metabolic insecticide resistance in Anopheles gambiae provides a real-time molecular diagnostic for anti-malarial intervention deployment

10.1101/2021.02.11.429702 ◽

2021 ◽

Author(s):

Harun Njoroge ◽

Arjen van’t Hof ◽

Ambrose Oruni ◽

Dimitra Pipini ◽

Sanjay C. Nagi ◽

...

Keyword(s):

Insecticide Resistance ◽

Anopheles Gambiae ◽

Pyrethroid Resistance ◽

Border Region ◽

Malaria Vectors ◽

Molecular Diagnostic ◽

Triple Mutant ◽

Pyrethroid Insecticides ◽

High Level ◽

Mutant Haplotype

AbstractInsecticide resistance provides both an increasingly pressing threat to the control of vector-borne diseases and insights into the remarkable capacity of natural populations to show rapid evolutionary responses to contemporary selection. Malaria control remains heavily dependent on deployment of pyrethroid insecticides, primarily in long lasting insecticidal nets (LLINs), but resistance in the major malaria vectors has increased over the last 15 years in concert with dramatic expansion of LLIN distributions. Identifying genetic mechanisms underlying high-level resistance in mosquitoes, which may almost entirely overcome pyrethroid efficacy, is crucial for the development and deployment of potentially resistance-breaking tools. Using the Anopheles gambiae 1000 genomes (Ag1000g) data we identified a very recent selective sweep in mosquitoes from Uganda which localized to a cluster of cytochrome P450 genes, including some commonly implicated in resistance. Further interrogation revealed a haplotype involving a trio of mutations, a nonsynonymous point mutation in Cyp6p4 (I236M), an upstream insertion of a partial Zanzibar-like transposable element (TE) and a duplication of the Cyp6aa1 gene. The mutations appear to have originated recently in An. gambiae from the Kenya-Uganda border region around Lake Victoria, with stepwise replacement of the double-mutant (Zanzibar-like TE and Cyp6p4-236M) with the triple-mutant haplotype (including Cyp6aa1 duplication), which has spread into the Democratic Republic of Congo and Tanzania. The triple-mutant haplotype is strongly associated with increased expression of genes able to metabolise pyrethroids and is strongly predictive of resistance to pyrethroids most notably deltamethrin, a commonly-used LLIN insecticide. Importantly, there was increased mortality in mosquitoes carrying the triple-mutation when exposed to nets co-treated with the synergist piperonyl butoxide (PBO). Frequencies of the triple-mutant haplotype remain spatially variable within countries, suggesting an effective marker system to guide deployment decisions for limited supplies of PBO-pyrethroid co-treated LLINs across African countries. Duplications of the Cyp6aa1 gene are common in An. gambiae across Africa and, given the enzymes metabolic activity, are likely to be a useful diagnostic for high levels of pyrethroid resistance.

Download Full-text

First detection of the kdr mutation (L1014F) in the plague vector Xenopsylla cheopis (Siphonaptera: Pulicidae)

Parasites & Vectors ◽

10.1186/s13071-019-3775-2 ◽

2019 ◽

Vol 12 (1) ◽

Author(s):

Nian Liu ◽

Xiangyang Feng ◽

Mei Li ◽

Xinghui Qiu

Keyword(s):

Network Analysis ◽

Insecticide Resistance ◽

Dna Sequences ◽

Pyrethroid Resistance ◽

Control Strategies ◽

Effective Control ◽

Phenotypic Effect ◽

Pyrethroid Insecticides ◽

Xenopsylla Cheopis ◽

Flea Control

Abstract Background The oriental rat flea, Xenopsylla cheopis, is the most efficient vector of the plague. Pyrethroid insecticides such as cypermethrin, cyhalothrin and deltamethrin have been often used to limit plague transmission via controlling the vector during outbreaks. However, this strategy is threatened by the development of insecticide resistance. Understanding the mechanisms underlying pyrethroid resistance is the prerequisite for successful flea control. Methods Partial DNA sequences of X. cheopis voltage gated sodium channel (VGSC) gene were amplified from a total of 111 individuals, collected from a natural plague epidemic foci in Baise City, Guangxi Zhuang Autonomous Region of China. These DNA fragments were sequenced. The frequency and distribution of kdr mutations were assessed in four X. cheopis populations. The origin of kdr mutations was investigated by phylogenetic and network analysis. Results The classical knockdown resistance (kdr) mutation (L1014F) was detected in four field populations at frequencies ranging between 0.021–0.241. The mutant homozygote was observed only in one of the four populations. Seven haplotypes were identified, with two of them carrying the resistance L1014F mutation. Phylogenetic tree and network analysis indicated that the L1014F allele was not singly originated. Based on polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) profiling, an easy-to-use and accurate molecular assay for screening individual fleas for the L1014F mutation was developed. Conclusions To our knowledge, this work represents the first report of the L1014F mutation in the plague vector X. cheopis. The incidence of the L1014F allele highlights the need of further studies on the phenotypic effect of this mutation in this plague vector. Early detection and monitoring of insecticide resistance is suggested in order to make effective control strategies in case of plague outbreaks in this region.

Download Full-text

Characterization of Sodium Channel Mutations in the Dengue Vector Mosquitoes Aedes aegypti and Aedes albopictus within the Context of Ongoing Wolbachia Releases in Kuala Lumpur, Malaysia

Insects ◽

10.3390/insects11080529 ◽

2020 ◽

Vol 11 (8) ◽

pp. 529

Author(s):

Noor Afizah Ahmad ◽

Nancy M. Endersby-Harshman ◽

Nur Ruqqayah Mohd Mazni ◽

Nur Zatil Aqmar Mohd Zabari ◽

Siti Nor Syazwani Amran ◽

...

Keyword(s):

Sodium Channel ◽

Pyrethroid Resistance ◽

Control Measure ◽

Mosquito Species ◽

Gene Mutations ◽

Laboratory Culture ◽

Target Area ◽

Type I ◽

Kuala Lumpur ◽

Pyrethroid Insecticides

Specific sodium channel gene mutations confer target site resistance to pyrethroid insecticides in mosquitoes and other insects. In Aedes mosquito species, multiple mutations that contribute to resistance vary in their importance around the world. Here, we characterize voltage sensitive sodium channel (Vssc) mutations in populations of Aedesaegypti from Kuala Lumpur, Malaysia, and look at their persistence in populations affected by ongoing Wolbachia releases (a dengue control measure). We also describe a Vssc mutation in Aedesalbopictus (F1534L) found for the first time in Malaysia. We show that there are three predominant Vssc haplotypes in Aedesaegypti in this region, which all persist with regular backcrossing, thereby maintaining the original genetic composition of the populations. We identify changes in genotype frequency in closed populations of Ae. aegypti maintained for multiple generations in laboratory culture, suggesting different fitness costs associated with the genotypes, some of which may be associated with the sex of the mosquito. Following population replacement of Ae. aegypti by Wolbachia in the target area, however, we find that the Vssc mutations have persisted at pre-release levels. Mosquitoes in two genotype classes demonstrate a type I pyrethroid resistance advantage over wildtype mosquitoes when exposed to 0.25% permethrin. This resistance advantage is even more pronounced with a type II pyrethroid, deltamethrin (0.03%). The results point to the importance of these mutations in pyrethroid resistance in mosquito populations and the need for regular backcrossing with male mosquitoes from the field to maintain similarity of genetic background and population integrity during Wolbachia releases.

Download Full-text