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      Co-occurrence of Point Mutations in the Voltage-Gated Sodium Channel of Pyrethroid-Resistant Aedes aegypti Populations in Myanmar

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          Abstract

          Background

          Single amino acid substitutions in the voltage-gated sodium channel associated with pyrethroid resistance constitute one of the main causative factors of knockdown resistance in insects. The kdr gene has been observed in several mosquito species; however, point mutations in the para gene of Aedes aegypti populations in Myanmar have not been fully characterized. The aim of the present study was to determine the types and frequencies of mutations in the para gene of Aedes aegypti collected from used tires in Yangon City, Myanmar.

          Methodology/Principal Findings

          We determined high pyrethroid resistance in Aedes aegypti larvae at all collection sites in Yangon City, by using a simplified knockdown bioassay. We showed that V1016G and S989P mutations were widely distributed, with high frequencies (84.4% and 78.8%, respectively). By contrast, we were unable to detect I1011M (or I1011V) or L1014F mutations. F1534C mutations were also widely distributed, but with a lower frequency than the V1016G mutation (21.2%). High percentage of co-occurrence of the homozygous V1016G/S989P mutations was detected (65.7%). Additionally, co-occurrence of homozygous V1016G/F1534C mutations (2.9%) and homozygous V1016G/F1534C/S989P mutations (0.98%) were detected in the present study.

          Conclusions/Significance

          Pyrethroid insecticides were first used for malaria control in 1992, and have since been constantly used in Myanmar. This intensive use may explain the strong selection pressure toward Aedes aegypti, because this mosquito is generally a domestic and endophagic species with a preference for indoor breeding. Extensive use of DDT for malaria control before the use of this chemical was banned may also explain the development of pyrethroid resistance in Aedes aegypti.

          Author Summary

          The use of pyrethroids with high killing activity has accelerated the development of pyrethroid resistance in vector mosquitoes. The knockdown resistance ( kdr) allele contains a single amino acid substitution in the voltage-gated sodium channel and is one of the main causative factors of pyrethroid resistance in insects. We investigated the distribution of the kdr gene in Aedes aegypti larvae collected from used tires in Yangon City, Myanmar. We detected three patterns of co-occurrence of point mutations, namely, V1016G/S989P with wide distribution, and small number of V1016G/F1534C and V1016G/F1534C/S989P, both of which were first found in the field collected Ae. aegypti population. The use of pyrethroids and/or DDT for malaria control is thought to be one of the main causes of pyrethroid resistance in Aedes aegypti. Insecticide treatment for malaria vector control seems to have been intensively conducted in the interior and along the periphery of human habitation areas, where the breeding and resting sites of Aedes aegypti are located.

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          Most cited references 26

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          Molecular characterization of pyrethroid knockdown resistance (kdr) in the major malaria vector Anopheles gambiae s.s.

          Pyrethroid-impregnated bednets are playing an increasing role for combating malaria, especially in stable malaria areas. More than 90% of the current annual malaria incidence (c. 500 million clinical cases with up to 2 million deaths) is in Africa where the major vector is Anopheles gambiae s.s. As pyrethroid resistance has been reported in this mosquito, reliable and simple techniques are urgently needed to characterize and monitor this resistance in the field. In insects, an important mechanism of pyrethroid resistance is due to a modification of the voltage-gated sodium channel protein recently shown to be associated with mutations of the para-type sodium channel gene. We demonstrate here that one of these mutations is present in certain strains of pyrethroid resistant A. gambiae s.s. and describe a PCR-based diagnostic test allowing its detection in the genome of single mosquitoes. Using this test, we found this mutation in six out of seven field samples from West Africa, its frequency being closely correlated with survival to pyrethroid exposure. This diagnostic test should bring major improvement for field monitoring of pyrethroid resistance, within the framework of malaria control programmes.
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            A mutation in the voltage-gated sodium channel gene associated with pyrethroid resistance in Latin American Aedes aegypti.

            Pyrethroids are commonly used as mosquito adulticides and evolution of resistance to these compounds is a major threat to public health. 'Knockdown resistance' to pyrethroids (kdr) is frequently caused by nonsynonymous mutations in the voltage-gated sodium channel transmembrane protein (para) that reduce pyrethroid binding. Early detection of kdr is critical to the development of resistance management strategies in mosquitoes including Aedes aegypti, the most prevalent vector of dengue and yellow fever viruses. Brengues et al. described seven novel mutations in hydrophobic segment 6 of domain II of para in Ae. aegypti. Assays on larvae from strains bearing these mutations indicated reduced nerve sensitivity to permethrin inhibition. Two of these occurred in codons Iso1011 and Val1016 in exons 20 and 21 respectively. A transition in the third position of Iso1011 encoded a Met1011 replacement and a transversion in the second position of Val1016 encoded a Gly1016 replacement. We have screened this same region in 1318 mosquitoes in 32 additional strains; 30 from throughout Latin America. While the Gly1016 allele was never detected in Latin America, we found two new mutations in these same codons. A transition in the first position of codon 1011 encodes a Val replacement while a transition in the first position of codon 1016 encodes an Iso replacement. We developed PCR assays for these four mutations that can be read either on an agarose gel or as a melting curve. Selection experiments, one with deltamethrin on a field strain from Santiago de Cuba and another with permethrin on a strain from Isla Mujeres, Mexico rapidly increased the frequency of the Iso1016 allele. Bioassays of F(3) offspring arising from permethrin susceptible Val1016 homozygous parents and permethrin resistant Iso1016 homozygous parents show that Iso1016 segregates as a recessive allele in conferring kdr. Analysis of segregation between alleles at the 1011 and 1016 codons in the F(3) showed a high rate of recombination even though the two codons are only separated by a ~250 bp intron. The tools and information presented provide a means for early detection and characterization of kdr that is critical to the development of strategies for resistance management.
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              Pyrethroid and DDT cross-resistance in Aedes aegypti is correlated with novel mutations in the voltage-gated sodium channel gene.

              Samples of the dengue vector mosquito Aedes aegypti (L.) (Diptera: Culicidae) were collected from 13 localities between 1995 and 1998. Two laboratory strains, Bora (French Polynesia) and AEAE, were both susceptible to DDT and permethrin; all other strains, except Larentuka (Indonesia) and Bouaké (Ivory Coast), contained individual fourth-instar larvae resistant to permethrin. Ten strains were subjected to a range of biochemical assays. Many strains had elevated carboxylesterase activity compared to the Bora strain; this was particularly high in the Indonesian strains Salatiga and Semarang, and in the Guyane strain (Cayenne). Monooxygenase levels were increased in the Salatiga and Paea (Polynesia) strains, and reduced in the two Thai strains (Mae Kaza, Mae Kud) and the Larentuka strain. Glutathione S-transferase activity was elevated in the Guyane strain. All other enzyme profiles were similar to the susceptible strain. The presence of both DDT and pyrethroid resistance in the Semarang, Belem (Brazil) and Long Hoa (Vietnam) strains suggested the presence of a knock-down resistant (kdr)-type resistance mechanism. Part of the S6 hydrophobic segment of domain II of the voltage-gated sodium channel gene was obtained by RT-PCR and sequenced from several insects from all 13 field strains. Four novel mutations were identified. Three strains contained identical amino acid substitutions at two positions, two strains shared a different substitution, and one strain was homozygous for a fourth alteration. The leucine to phenylalanine substitution that confers nerve insensitivity to pyrethroids in a range of other resistant insects was absent. Direct neurophysiological assays on individual larvae from three strains with these mutations demonstrated reduced nerve sensitivity to permethrin or lambda cyhalothrin inhibition compared to the susceptible strains.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS Negl Trop Dis
                PLoS Negl Trop Dis
                plos
                plosntds
                PLoS Neglected Tropical Diseases
                Public Library of Science (San Francisco, USA )
                1935-2727
                1935-2735
                July 2014
                31 July 2014
                : 8
                : 7
                Affiliations
                [1 ]Department of Vector Ecology and Environment, Institute of Tropical Medicine, Nagasaki University, Nagasaki, Japan
                [2 ]Medical Entomology Research Division, Department of Medical Research (Lower Myanmar), Yangon, Myanmar
                [3 ]The Global Center of Excellence Program, Nagasaki University, Nagasaki, Japan
                Mahidol University, Thailand
                Author notes

                The authors have declared that no competing interests exist.

                Conceived and designed the experiments: HK SZMO KZT NM. Performed the experiments: HK SZMO ST EK YNMM HMT. Analyzed the data: HK SZMO ST EK. Contributed reagents/materials/analysis tools: YNMM HMT. Wrote the paper: HK. Provided critical comment on the manuscript and final approval of the version to be published: KZT NM.

                Article
                PNTD-D-14-00371
                10.1371/journal.pntd.0003032
                4117438
                25077956

                This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                Page count
                Pages: 8
                Funding
                This study was funded by Japan Initiative for Global Research Network on Infectious Diseases. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Medicine and Health Sciences
                Infectious Diseases
                Infectious Disease Control
                Vector-Borne Diseases
                Custom metadata
                The authors confirm that all data underlying the findings are fully available without restriction. The unique DNA haplotype sequences were deposited in GenBank (DDBJ, accession  =  AB914689, AB914690, AB914687, AB914688).

                Infectious disease & Microbiology

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