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Enzymatic and molecular characterization of insecticide resistance mechanisms in field populations of Aedes aegypti from Selangor, Malaysia.

ABSTRACT: BACKGROUND:Dengue is a serious public health problem worldwide, including in Selangor, Malaysia. Being an important vector of dengue virus, Aedes aegypti are subjected to control measures which rely heavily on the usage of insecticides. Evidently, insecticide resistance in Ae. aegypti, which arise from several different point mutations within the voltage-gated sodium channel genes, has been documented in many countries. Thus, this robust study was conducted in all nine districts of Selangor to understand the mechanisms of resistance to various insecticides in Ae. aegypti. Mosquitoes were collected from dengue epidemic and non-dengue outbreak areas in Selangor. METHODS:Using the Center for Disease Control and Prevention (CDC) bottle assays, the insecticide resistance status of nine different Ae. aegypti strains from Selangor was accessed. Synergism tests and biochemical assays were conducted to further understand the metabolic mechanisms of insecticide resistance. Polymerase chain reaction (PCR) amplification and sequencing of the IIP-IIS6 as well as IIIS4-IIIS6 regions of the sodium channel gene were performed to enable comparisons between susceptible and resistant mosquito strains. Additionally, genomic DNA was used for allele-specific PCR (AS-PCR) genotyping of the gene to detect the presence of F1534C, V1016G and S989P mutations. RESULTS:Adult female Ae. aegypti from various locations were susceptible to malathion and propoxur. However, they exhibited different levels of resistance against dichlorodiphenyltrichloroethane (DDT) and pyrethroids. The results of synergism tests and biochemical assays indicated that the mixed functions of oxidases and glutathione S-transferases contributed to the DDT and pyrethroid resistance observed in the present study. Besides detecting three single kdr mutations, namely F1534C, V1016G and S989P, co-occurrence of homozygous V1016G/S989P (double allele) and F1534C/V1016G/S989P (triple allele) mutations were also found in Ae. aegypti. As per the results, the three kdr mutations had positive correlations with the expressions of resistance to DDT and pyrethroids. CONCLUSIONS:In view of the above outcomes, it is important to seek new tools for vector management instead of merely relying on insecticides. If the latter must be used, regular monitoring of insecticide resistance should also be carried out at all dengue epidemic areas. Since the eggs of Ae. aegypti can be easily transferred from one location to another, it is probable that insecticide-resistant Ae. aegypti can be found at non-dengue outbreak sites as well.

SUBMITTER: Leong CS

PROVIDER: S-EPMC6521414 | biostudies-literature | 2019 May

REPOSITORIES: biostudies-literature

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Enzymatic and molecular characterization of insecticide resistance mechanisms in field populations of Aedes aegypti from Selangor, Malaysia.

Leong Cherng-Shii CS Vythilingam Indra I Liew Jonathan Wee-Kent JW Wong Meng-Li ML Wan-Yusoff Wan Sulaiman WS Lau Yee-Ling YL

Parasites & vectors 20190516 1

<h4>Background</h4>Dengue is a serious public health problem worldwide, including in Selangor, Malaysia. Being an important vector of dengue virus, Aedes aegypti are subjected to control measures which rely heavily on the usage of insecticides. Evidently, insecticide resistance in Ae. aegypti, which arise from several different point mutations within the voltage-gated sodium channel genes, has been documented in many countries. Thus, this robust study was conducted in all nine districts of Selan ...[more]

PMID: 31097010

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Project description:Aedes aegypti is the main epidemic vector of arboviruses in Africa. In Senegal, control activities are mainly limited to mitigation of epidemics, with limited information available for Ae. aegypti populations. A better understanding of the current Ae. aegypti susceptibility status to various insecticides and relevant resistance mechanisms involved is needed for the implementation of effective vector control strategies. The present study focuses on the detection of insecticide resistance and reveals the related mechanisms in Ae. aegypti populations from Senegal. Bioassays were performed on Ae. aegypti adults from nine Senegalese localities (Matam, Louga, Barkedji, Ziguinchor, Mbour, Fatick, Dakar, Kédougou and Touba). Mosquitoes were exposed to four classes of insecticides using the standard WHO protocols. Resistance mechanisms were investigated by genotyping for pyrethroid target site resistance mutations (V1016G, V1016I, F1534C and S989P) and measuring gene expression levels of key detoxification genes (CYP6BB2, CYP9J26, CYP9J28, CYP9J32, CYP9M6, CCEae3a and GSTD4). All collected populations were resistant to DDT and carbamates except for the ones in Matam (Northern region). Resistance to permethrin was uniformly detected in mosquitoes from all areas. Except for Barkédji and Touba, all populations were characterized by a susceptibility to 0.75% Permethrin. Susceptibility to type II pyrethroids was detected only in the Southern regions (Kédougou and Ziguinchor). All mosquito populations were susceptible to 5% Malathion, but only Kédougou and Matam mosquitoes were susceptible to 0.8% Malathion. All populations were resistant to 0.05% Pirimiphos-methyl, whereas those from Louga, Mbour and Barkédji, also exhibited resistance to 1% Fenitrothion. None of the known target site pyrethroid resistance mutations was present in the mosquito samples included in the genotyping analysis (performed in > 1500 samples). In contrast, a remarkably high (20-70-fold) overexpression of major detoxification genes was observed, suggesting that insecticide resistance is mostly mediated through metabolic mechanisms. These data provide important evidence to support dengue vector control in Senegal.

Project description:BACKGROUND:Recent outbreaks of dengue and other Aedes aegypti-borne arboviruses highlight the importance of a rapid response for effective vector control. Data on insecticide resistance and underlying mechanisms are essential for outbreak preparedness, but are sparse in much of Africa. We investigated the levels and heterogeneity of insecticide resistance and mechanisms of Ae. aegypti from contrasting settings within and around Ouagadougou, Burkina Faso. METHODOLOGY/PRINCIPAL FINDINGS:Bioassays were performed on larvae and adults to diagnose prevalence of resistance, and to assess levels where resistance was detected. Investigation of resistance mechanisms was performed using synergist bioassays, knockdown resistance (kdr) target site mutation genotyping and quantitative PCR expression analysis of candidate P450 genes. Larval dose-response assays indicated susceptibility to the organophosphates tested. Adult females were also susceptible to organophosphates, but resistance to carbamates was suspected in urban and semi-urban localities. Females from all localities showed resistance to pyrethroids but resistance prevalence and level were higher in urban and especially in semi-urban areas, compared to the rural population. Environment was also associated with susceptibility: adults reared from larvae collected in tires from the semi-urban site were significantly less resistant to pyrethroids than those collected from large outdoor drinking water containers ('drums'). Susceptibility to both pyrethroids tested was largely restored by pre-exposure to Piperonyl Butoxide (PBO), suggesting a strong metabolic basis to resistance. The 1534C kdr mutation was nearly fixed in semi-urban and urban areas but was far less common in the rural area, where the 1016I kdr mutation frequency was also significantly lower. P450 gene analysis detected limited over-expression of single candidates but significantly elevated average expression in the semi-urban site compared to both a susceptible laboratory colony, and females from the other collection sites. CONCLUSIONS/SIGNIFICANCE:Our results reveal pyrethroid resistance and paired kdr mutations in both urban and semi-urban sites at levels that are unprecedented for mainland Africa. The combination of target site and metabolic mechanisms is common in Ae. aegypti populations from other continents but is a worrying finding for African populations. However, organophosphate insecticides are still active against both larvae and adults of Ae. aegypti, providing useful insecticidal options for control and resistance management.

Project description:Owing to the increased reports in Aedes-borne diseases in the Caribbean and Latin America, the United States Agency for International Development assisted the Jamaican Ministry of Health and Wellness in conducting insecticide susceptibility tests on Aedes aegypti populations. Sentinel sites were established in seven parishes of Jamaica (St. Catherine, Kingston and St. Andrew, St. Thomas, Portland, St. Mary and St. Ann) and Aedes aegypti eggs were collected, reared to adults per collected population and their susceptibility to varying pyrethroids and organophosphates were tested using the World Health Organization paper bioassays for these insecticides. The Centers for Disease Control and Prevention bottle bioassay was used to assess susceptibility to the carbamate, bendiocarb. The voltage gated sodium channel gene mutations V1016I and I1011V, normally associated with pyrethroid resistance, were also analysed. The results showed that Aedes aegypti collected from all parishes exhibited resistance to pyrethroids at the following concentrations, permethrin 0.25-2.5%; deltamethrin 0.03-0.15%; lambda-cyhalothrin 0.03-0.3%; and etofenprox 0.5-2.5%. The insecticide deltamethrin at concentration 0.3% was the only pyrethroid tested that resulted in high mortality, 94.9 ± 0.34% knockdown within 1 hour of exposure and 98.95 ± 0.01% mortality (p <0.01) at 24 hours post exposure. The frequency of the voltage gated sodium channel gene mutation V1016I was high in the tested population, possibly accounting for the reduced sensitivity to pyrethroids. Organophosphate resistance was also observed in all populations tested. Mortality rates for 0.8% Malathion was 0.8 ± 0.70-60.68 ± 0.38% after 24 hour and 0.00-47.10 ± 3.02%, for pirimiphos-methyl 0.21%. Bendiocarb applied as 12.5 μg/ bottle resulted in mortality rates of 76.25 ± 4.30-100 ± 0.00% after 30 minutes of exposure. The results showed that Ae. aegypti from the seven parishes analysed demonstrated resistance to the insecticides tested. Deltamethrin and bendiocarb at concentrations 0.3% and 12.5μg respectively, were considered most effective, causing high mortality in the local populations. Routine monitoring and evaluations of Ae. aegypti populations from the included parishes are recommended. Additionally, the study results represent the most comprehensive testing to date with local Aedes aegypti populations distributed across different parishes of Jamaica and should be useful to guide national and sub national strategies for vector control and surveillance.

Project description:BackgroundArboviral diseases, including dengue and chikungunya, are major public health concerns in Bangladesh where there have been unprecedented levels of transmission reported in recent years. The primary approach to control these diseases is to control the vector Aedes aegypti using pyrethroid insecticides. Although chemical control has long been practiced, no comprehensive analysis of Ae. aegypti susceptibility to insecticides has been conducted to date. The aim of this study was to determine the insecticide resistance status of Ae. aegypti in Bangladesh and investigate the role of detoxification enzymes and altered target site sensitivity as resistance mechanisms.MethodsEggs of Aedes mosquitoes were collected using ovitraps from five districts across Bangladesh and in eight neighborhoods of the capital city Dhaka, from August to November 2017. CDC bottle bioassays were conducted for permethrin, deltamethrin, malathion, and bendiocarb using 3- to 5-day-old F0-F2 non-blood-fed female mosquitoes. Biochemical assays were conducted to detect metabolic resistance mechanisms, and real-time PCR was performed to determine the frequencies of the knockdown resistance (kdr) mutations Gly1016, Cys1534, and Leu410.ResultsHigh levels of resistance to permethrin were detected in all Ae. aegypti populations, with mortality ranging from 0 to 14.8% at the diagnostic dose. Substantial resistance continued to be detected against higher (2×) doses of permethrin (5.1-44.4% mortality). Susceptibility to deltamethrin and malathion varied between populations while complete susceptibility to bendiocarb was observed in all populations. Significantly higher levels of esterase and oxidase activity were detected in most of the test populations as compared to the susceptible reference Rockefeller strain. A significant association was detected between permethrin resistance and the presence of Gly1016 and Cys1534 homozygotes. The frequency of kdr (knockdown resistance) alleles varied across the Dhaka Aedes populations. Leu410 was not detected in any of the tested populations.ConclusionsThe detection of widespread pyrethroid resistance and multiple resistance mechanisms highlights the urgency for implementing alternate Ae. aegypti control strategies. In addition, implementing routine monitoring of insecticide resistance in Ae. aegypti in Bangladesh will lead to a greater understanding of susceptibility trends over space and time, thereby enabling the development of improved control strategies.

Project description:Vector control largely relies on neurotoxic chemicals, and insecticide resistance (IR) directly threatens their effectiveness. In some cases, specific alleles cause IR, and knowledge of the genetic diversity and gene flow among mosquito populations is crucial to track their arrival, rise, and spread. Here we evaluated Aedes aegypti populations' susceptibility status, collected in 2016 from six different municipalities of Rio de Janeiro state (RJ), to temephos, pyriproxyfen, malathion, and deltamethrin. We collected eggs of Ae. aegypti in Campos dos Goytacazes (Cgy), Itaperuna (Ipn), Iguaba Grande (Igg), Itaboraí (Ibr), Mangaratiba (Mgr), and Vassouras (Vsr). We followed the World Health Organization (WHO) guidelines and investigated the degree of susceptibility/resistance of mosquitoes to these insecticides. We used the Rockefeller strain as a susceptible positive control. We genotyped the V1016I and F1534C knockdown resistance (kdr) alleles using qPCR TaqMan SNP genotyping assay. Besides, with the use of Ae. aegypti SNP-chip, we performed genomic population analyses by genotyping more than 15,000 biallelic SNPs in mosquitoes from each population. We added previous data from populations from other countries to evaluate the ancestry of RJ populations. All RJ Ae. aegypti populations were susceptible to pyriproxyfen and malathion and highly resistant to deltamethrin. The resistance ratios for temephos was below 3,0 in Cgy, Ibr, and Igg populations, representing the lowest rates since IR monitoring started in this Brazilian region. We found the kdr alleles in high frequencies in all populations, partially justifying the observed resistance to pyrethroid. Population genetics analysis showed that Ae. aegypti revealed potential higher migration among some RJ localities and low genetic structure for most of them. Future population genetic studies, together with IR data in Ae aegypti on a broader scale, can help us predict the gene flow within and among the Brazilian States, allowing us to track the dynamics of arrival and changes in the frequency of IR alleles, and providing critical information to improving vector control program.

Dataset Information

Enzymatic and molecular characterization of insecticide resistance mechanisms in field populations of Aedes aegypti from Selangor, Malaysia.

Publications

Enzymatic and molecular characterization of insecticide resistance mechanisms in field populations of Aedes aegypti from Selangor, Malaysia.

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