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Evidence for gene duplication in the voltage-gated sodium channel gene of Aedes aegypti.

ABSTRACT: BACKGROUND AND OBJECTIVES:Mutations in the voltage-gated sodium channel gene (NaV), known as kdr mutations, are associated with pyrethroid and DDT insecticide resistance in a number of species. In the mosquito dengue vector Aedes aegypti, besides kdr, other polymorphisms allowed grouping AaNaV sequences as type 'A' or 'B'. Here, we point a series of evidences that these polymorphisms are actually involved in a gene duplication event. METHODOLOGY:Four series of methods were employed: (i) genotypying, with allele-specific PCR (AS-PCR), of two AaNaV sites that can harbor kdr mutations (Ile1011Met and Val1016Ile), (ii) cloning and sequencing of part of the AaNaV gene, (iii) crosses with specific lineages and analysis of the offspring genotypes and (iv) copy number variation assays, with TaqMan quantitative real-time PCR. RESULTS:kdr mutations in 1011 and 1016 sites were present only in type 'A' sequences, but never in the same haplotype. In addition, although the 1011Met-mutant allele is widely disseminated, no homozygous (1011Met/Met) was detected. Sequencing revealed three distinct haplotypes in some individuals, raising the hypothesis of gene duplication, which was supported by the genotype frequencies in the offspring of specific crosses. Furthermore, it was estimated that a laboratory strain selected for insecticide resistance had 5-fold more copies of the sodium channel gene compared with a susceptible reference strain. CONCLUSIONS AND IMPLICATIONS:The AaNaV duplication here found might be a recent adaptive response to the intense use of insecticides, maintaining together wild-type and mutant alleles in the same organism, conferring resistance and reducing some of its deleterious effects.

SUBMITTER: Martins AJ

PROVIDER: S-EPMC3868448 | biostudies-literature | 2013 Jan

REPOSITORIES: biostudies-literature

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Evidence for gene duplication in the voltage-gated sodium channel gene of Aedes aegypti.

Martins Ademir Jesus AJ Brito Luiz Paulo LP Linss Jutta Gerlinde Birggitt JG Rivas Gustavo Bueno da Silva GB Machado Ricardo R Bruno Rafaela Vieira RV Lima José Bento Pereira JB Valle Denise D Peixoto Alexandre Afranio AA

Evolution, medicine, and public health 20130101 1

<h4>Background and objectives</h4>Mutations in the voltage-gated sodium channel gene (NaV), known as kdr mutations, are associated with pyrethroid and DDT insecticide resistance in a number of species. In the mosquito dengue vector Aedes aegypti, besides kdr, other polymorphisms allowed grouping AaNaV sequences as type 'A' or 'B'. Here, we point a series of evidences that these polymorphisms are actually involved in a gene duplication event.<h4>Methodology</h4>Four series of methods were employe ...[more]

PMID: 24481195

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Project description:BACKGROUND:Yellow fever is endemic in some countries in Africa, and Aedes aegpyti is one of the most important vectors implicated in the outbreak. The mapping of the nation-wide distribution and the detection of insecticide resistance of vector mosquitoes will provide the beneficial information for forecasting of dengue and yellow fever outbreaks and effective control measures. METHODOLOGY/PRINCIPAL FINDINGS:High resistance to DDT was observed in all mosquito colonies collected in Ghana. The resistance and the possible existence of resistance or tolerance to permethrin were suspected in some colonies. High frequencies of point mutations at the voltage-gated sodium channel (F1534C) and one heterozygote of the other mutation (V1016I) were detected, and this is the first detection on the African continent. The frequency of F1534C allele and the ratio of F1534C homozygotes in Ae. aegypti aegypti (Aaa) were significantly higher than those in Ae. aegypti formosus (Aaf). We could detect the two types of introns between exon 20 and 21, and the F1534C mutations were strongly linked with one type of intron, which was commonly found in South East Asian and South and Central American countries, suggesting the possibility that this mutation was introduced from other continents or convergently selected after the introgression of Aaa genes from the above area. CONCLUSIONS/SIGNIFICANCE:The worldwide eradication programs in 1940s and 1950s might have caused high selection pressure on the mosquito populations and expanded the distribution of insecticide-resistant Ae. aegypti populations. Selection of the F1534C point mutation could be hypothesized to have taken place during this period. The selection of the resistant population of Ae. aegypti with the point mutation of F1534C, and the worldwide transportation of vector mosquitoes in accordance with human activity such as trading of used tires, might result in the widespread distribution of F1534C point mutation in tropical countries.

Project description:BackgroundKnockdown resistance (kdr) to dichlorodiphenyltrichloroethane (DDT) and pyrethroids is known to link amino acid substitutions in the voltage-gated sodium channel (VGSC) in Aedes aegypti. Dengue fever primarily transmitted by Ae. aegypti is an annual public health issue in Taiwan. Accordingly, pyrethroid insecticides have been heavily used for decades to control mosquito populations in the summer and autumn. In Taiwan, an Ae. aegypti population with two VGSC mutations, V1016G and D1763Y, was described previously.Methodology/principal findingAedes aegypti (G0) were collected in Tainan and Kaohsiung in southern Taiwan. The VGSC gene polymorphisms of the kdr mutations and the intron flanked by exons 20 and 21 were verified. The first generation offspring (G1) were used to measure the resistance level to cypermethrin, a pyrethroid insecticide currently used in Taiwan. In addition to V1016G and D1763Y, we describe two new mutations, S989P and F1534C, which have not been reported in Taiwan. Moreover, we also identify two types (groups A and B) of introns between exons 20 and 21. Intriguingly, the kdr mutations S989P, V1016G and D1763Y are strictly located on the haplotype harboring the group A intron, whereas F1534C links to the group B intron. When those data were taken together, we proposed the following six haplotypes for VGSC genes in Taiwan today: (i)S989-intron A-V1016-F1534-D1763, (ii)S989-intron A-V1016G-F1534-D1763, (iii)S989P-intron A-V1016G-F1534-D1763, (iv)S989-intron A-V1016G-F1534-D1763Y, (v)S989-intron B-V1016-F1534-D1763 and (vi)S989-intron B-V1016-F1534C-D1763. Triple heterozygous mutations of either S989P/V1016G/F1534C or V1016G/F1534C/D1763Y can be found in one single Ae. aegypti mosquito. The proportions of the VGSC mutations were relevant to cypermethrin resistance. Notably, the presence of S989P and V1016G in the population could be a helpful reference to predict the resistance level to cypermethrin. This is the first study to demonstrate the coexistence of four kdr mutations in a population of Ae. aegypti.Conclusions/significanceFour kdr mutations (S989P, V1016G, F1534C and D1763Y) and two intron forms (Group A and B) were commonly found in local Ae. aegypti populations in Taiwan.

Project description:BACKGROUND:Resistance of Aedes aegypti to photostable pyrethroid insecticides is a major problem for disease-vector control programs. Pyrethroids target the voltage-gated sodium channel on the insects' neurons. Single amino acid substitutions in this channel associated with pyrethroid resistance are one of the main factors that cause knockdown resistance in insects. Although kdr has been observed in several mosquito species, point mutations in the para gene have not been fully characterized in Ae. aegypti populations in Vietnam. The aim of this study was to determine the types and frequencies of mutations in the para gene in Ae. aegypti collected from used tires in Vietnam. METHODS AND FINDINGS:Several point mutations were examined that cause insensitivity of the voltage-gated sodium channel in the insect nervous system due to the replacement of the amino acids L1014F, the most commonly found point mutation in several mosquitoes; I1011M (or V) and V1016G (or I), which have been reported to be associated to knockdown resistance in Ae. aegypti located in segment 6, domain II; and a recently found amino acid replacement in F1269 in Ae. aegypti, located in segment 6, domain III. Among 756 larvae from 70 locations, no I1011M or I1011V nor L1014F mutations were found, and only two heterozygous V1016G mosquitoes were detected. However, F1269C mutations on domain III were distributed widely and with high frequency in 269 individuals among 757 larvae (53 collection sites among 70 locations surveyed). F1269C frequencies were low in the middle to north part of Vietnam but were high in the areas neighboring big cities and in the south of Vietnam, with the exception of the southern mountainous areas located at an elevation of 500-1000 m. CONCLUSIONS:The overall percentage of homozygous F1269C seems to remain low (7.4%) in the present situation. However, extensive and uncontrolled frequent use of photostable pyrethroids might be a strong selection pressure for this mutation to cause serious problems in the control of dengue fever in Vietnam.

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.

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Evidence for gene duplication in the voltage-gated sodium channel gene of Aedes aegypti.

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Evidence for gene duplication in the voltage-gated sodium channel gene of Aedes aegypti.

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