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A multiplex PCR for detection of knockdown resistance mutations, V1016G and F1534C, in pyrethroid-resistant Aedes aegypti.

ABSTRACT: Mutation of the voltage-gated sodium channel (VGSC) gene, or knockdown resistance (kdr) gene, is an important resistance mechanism of the dengue vector Aedes aegypti mosquitoes against pyrethroids. In many countries in Asia, a valine to glycine substitution (V1016G) and a phenylalanine to cysteine substitution (F1534C) are common in Ae. aegypti populations. The G1016 and C1534 allele frequencies have been increasing in recent years, and hence there is a need to have a simple and inexpensive tool to monitor the alleles in large scale.A multiplex PCR to detect V1016G and F1534C mutations has been developed in the current study. This study utilized primers from previous studies for detecting the mutation at position 1016 and newly designed primers to detect variants at position 1534. The PCR conditions were validated and compared with DNA sequencing using known kdr mutant laboratory strains and field collected mosquitoes. The efficacy of this method was also compared with allele-specific PCR (AS-PCR).The results of our multiplex PCR were in complete agreement with sequencing data and better than the AS-PCR. In addition, the efficiency of two non-toxic DNA staining dyes, Ultrapower™ and RedSafe™, were evaluated by comparing with ethidium bromide (EtBr) and the results were satisfactory.Our multiplex PCR method is highly reliable and useful for implementing vector surveillance in locations where the two alleles co-occur.

SUBMITTER: Saingamsook J

PROVIDER: S-EPMC5634894 | biostudies-literature | 2017 Oct

REPOSITORIES: biostudies-literature

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A multiplex PCR for detection of knockdown resistance mutations, V1016G and F1534C, in pyrethroid-resistant Aedes aegypti.

Saingamsook Jassada J Saeung Atiporn A Yanola Jintana J Lumjuan Nongkran N Walton Catherine C Somboon Pradya P

Parasites & vectors 20171010 1

<h4>Background</h4>Mutation of the voltage-gated sodium channel (VGSC) gene, or knockdown resistance (kdr) gene, is an important resistance mechanism of the dengue vector Aedes aegypti mosquitoes against pyrethroids. In many countries in Asia, a valine to glycine substitution (V1016G) and a phenylalanine to cysteine substitution (F1534C) are common in Ae. aegypti populations. The G1016 and C1534 allele frequencies have been increasing in recent years, and hence there is a need to have a simple a ...[more]

PMID: 29017613

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Project description:BackgroundControl of Aedes aegypti, the mosquito vector of dengue, chikungunya and yellow fever, is a challenging task. Pyrethroid insecticides have emerged as a preferred choice for vector control but are threatened by the emergence of resistance. The present study reports a focus of pyrethroid resistance and presence of two kdr mutations--F1534C and a novel mutation T1520I, in Ae. aegypti from Delhi, India.Methodology/principal findingsInsecticide susceptibility status of adult-female Ae. aegypti against DDT (4%), deltamethrin (0.05%) and permethrin (0.75%) was determined using WHO's standard insecticide susceptibility kit, which revealed resistance to DDT, deltamethrin and permethrin with corrected mortalities of 35%, 72% and 76% respectively. Mosquitoes were screened for the presence of kdr mutations including those reported earlier (I1011V/M, V1016G/I, F1534C, D1794Y and S989P), which revealed the presence of F1534C and a novel mutation T1520I. Highly specific PCR-RFLP assays were developed for genotyping of these two mutations. Genotyping using allele specific PCR and new PCR-RFLP assays revealed a high frequency of F1534C (0.41-0.79) and low frequency of novel mutation T1520I (0.13). The latter was observed to be tightly linked with F1534C and possibly serve as a compensatory mutation. A positive association of F1534C mutation with DDT and deltamethrin resistance in Ae. aegypti was established. However, F1534C-kdr did not show significant protection against permethrin.Conclusions/significanceThe Aedes aegypti population of Delhi is resistant to DDT, deltamethrin and permethrin. Two kdr mutations, F1534C and a novel mutation T1520I, were identified in this population. This is the first report of kdr mutations being present in the Indian Ae. aegypti population. Highly specific PCR-RFLP assays were developed for discrimination of alleles at both kdr loci. A positive association of F1534C mutation with DDT and deltamethrin resistance was confirmed.

Project description:BACKGROUND:Aedes aegypti is a primary vector of dengue, chikungunya and Zika infections in India. In the absence of specific drugs or safe and effective vaccines for these infections, their control relies mainly on vector control measures. The emergence of insecticide resistance in vectors, especially against pyrethroids, is a serious threat to the insecticide-based vector control programme. This study reports the presence of multiple knockdown resistance (kdr) mutations present in an Ae. aegypti population from Bengaluru (India), including a new mutation F1534L. METHODS:Aedes aegypti collected from Bengaluru were subjected to insecticide susceptibility tests with DDT, deltamethrin and permethrin. The DNA sequencing of partial domain II, III and IV of the voltage-gated sodium channel (VGSC) was performed to screen kdr mutations present in the population and PCR-based assays were developed for their detection. Genotyping of kdr mutations was done using PCR-based assays, allelic frequencies were determined, and tests of genetic association of kdr mutations with the insecticide resistance phenotype were performed. RESULTS:The Ae. aegypti population was resistant to DDT, deltamethrin and permethrin. The DNA sequencing of the VGSC revealed the presence of four kdr mutations, i.e. S989P and V1016G in domain II and two alternative kdr mutations F1534C and F1534L in domain III. Allele-specific PCR assays (ASPCR) were developed for the detection of kdr mutations S989P and V1016G and an existing PCR-RFLP based strategy was modified for the genotyping of all three known kdr mutations in domain III (F1534L, F1534C and T1520I). Genotyping of Ae. aegypti samples revealed a moderate frequency of S989P/V1016G (18.27%) and F1534L (17.48%), a relatively high frequency of F1534C (50.61%) and absence of T1520I in the population. Mutations S989P and V1016G were in complete linkage disequilibrium in this population while they were in linkage equilibrium with kdr mutations F1534C and F1534L. The alleles F1534C and F1534L are genetically associated with permethrin resistance. CONCLUSIONS:A new kdr mutation, F1534L, was found in an Ae. aegypti population from Bengaluru (India), co-occurring with the other three mutations S989P, V1016G and F1534C. The findings of a new mutation have implications for insecticide resistance management.

Project description:BACKGROUND:Aedes borne viral diseases, notably dengue, are increasingly reported in Cameroon with Aedes aegypti being a major vector. Data on insecticide resistance of this vector and underlying mechanisms needed for outbreak preparedness remain scarce in Cameroon. Here, we present the nationwide distribution of insecticide resistance in Ae. aegypti and investigate the potential resistance mechanisms involved. METHODS:Immature stages of Ae. aegypti were collected between March and July 2017 in 13 locations across Cameroon and reared until G1/G2/G3 generation. Larval, adult bioassays, and piperonyl butoxide (PBO) synergist assays were carried out according to World Health Organization guidelines. F1534C mutation was genotyped using allele specific polymerase chain reaction in field collected adults (Go) and the polymorphism of the sodium channel gene was assessed. The ?2 test was used to compare the mortality rate between bioassays with insecticides only and bioassays after preexposure to PBO synergist. RESULTS:Larval bioassay revealed that all the three populations tested with temephos were susceptible. Adult bioassays showed a good level of susceptibility toward both pyrethroids tested, 0.25% permethrin and 0.05% deltamethrin, with six out of 10 populations susceptible. However, two populations (Douala and Edéa) were resistant (deltamethrin [73.2-92.5% mortality], permethrin [2.6-76.3% mortality]). The resistance to 4% dichlorodiphenyltrichloroethane was observed in four out of 10 populations tested (16.8-87.1% mortality). Resistance was also reported to carbamates including 0.1% propoxur (60.8-87.1% mortality) and to 0.1% bendiocarb (82.9% mortality). All populations tested were fully susceptible to 1% fenitrothion. A partial recovery of susceptibility was observed in the pyrethroid resistant population of Douala after pre-exposed to PBO suggesting the implication of cytochrome P450 monoxygenases permethrin resistance. Genotyping and sequencing detected the F1534C kdr mutation in the two pyrethroid resistant locations of Edéa and Douala, with allelic frequency of 3.3% and 33.3% respectively. However, the high genetic diversity of the sodium channel gene supports the recent introduction of this mutation in Cameroon. CONCLUSIONS:This study revealed the contrasting resistance profiles to insecticides of Ae. aegypti populations in Cameroon suggesting that, instead of a unique nationwide control approach, a regionally adapted strategy will be needed to control this vector. The localised distribution of the F1534C kdr mutation supports this region-specific control strategy.

Dataset Information

A multiplex PCR for detection of knockdown resistance mutations, V1016G and F1534C, in pyrethroid-resistant Aedes aegypti.

Publications

A multiplex PCR for detection of knockdown resistance mutations, V1016G and F1534C, in pyrethroid-resistant Aedes aegypti.

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