Dataset Information

Establishment of an innovative and sustainable PCR technique for 1534 locus mutation of the knockdown resistance (kdr) gene in the dengue vector Aedes albopictus.

ABSTRACT: BACKGROUND:Mutation of the voltage-gated sodium channel (VGSC) gene, or knockdown resistance (kdr) gene, is an important resistance mechanism against DDT and pyrethroids for dengue vector Aedes albopictus. A phenylalanine to serine (F1534S), leucine (F1534L) and cysteine (F1534C) substitution were detected in many Ae. albopictus populations around the world, and the mutant allele frequencies have been increasing in recent years. Therefore, it is essential to establish a simple, time-saving and cost-effective procedure to monitor the alleles in large-scale studies. METHODS:Based on the mutation genotypes of the 1534 locus in the kdr gene, F/F, F/S, F/C, F/L, S/S, C/C, L/L and S/C, we designed specific forward and reverse primers and optimized the reaction conditions for establishing of the allele-specific PCR(AS-PCR) detection technique. DNA sequencing in this study was taken as the gold standard, and used to determine the accuracy of AS-PCR. RESULTS:The designed AS-PCR technique showed high specificity for distinguishing the mutations at the 1534 locus, as the accuracy for F/F, F/S, F/C, F/L, S/S, C/C and S/C were 100%, 95.35%, 100%, 100%, 100%, 100% and 100%, respectively. CONCLUSIONS:The designed AS-PCR technique effectively distinguished individual genotypes for the mutations at the 1534 locus in the kdr gene, which could facilitate the knockdown resistance surveillance in Ae. albopictus in large-scale studies.

SUBMITTER: Zhu CY

PROVIDER: S-EPMC6933705 | biostudies-literature | 2019 Dec

REPOSITORIES: biostudies-literature

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Establishment of an innovative and sustainable PCR technique for 1534 locus mutation of the knockdown resistance (kdr) gene in the dengue vector Aedes albopictus.

Zhu Cai-Ying CY Zhao Chun-Chun CC Wang Yi-Guan YG Ma De-Ling DL Song Xiu-Ping XP Wang Jun J Meng Feng-Xia FX

Parasites & vectors 20191226 1

<h4>Background</h4>Mutation of the voltage-gated sodium channel (VGSC) gene, or knockdown resistance (kdr) gene, is an important resistance mechanism against DDT and pyrethroids for dengue vector Aedes albopictus. A phenylalanine to serine (F1534S), leucine (F1534L) and cysteine (F1534C) substitution were detected in many Ae. albopictus populations around the world, and the mutant allele frequencies have been increasing in recent years. Therefore, it is essential to establish a simple, time-savi ...[more]

PMID: 31878970

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Project description:BackgroundAedes aegypti and Aedes albopictus are important vectors of human arboviruses, transmitting arboviral diseases such as yellow fever, dengue, chikungunya and Zika. These two mosquitoes coexist on Hainan Island and the Leizhou Peninsula in China. Over the past 40 years, the distribution of Ae. albopictus has gradually expanded in these areas, while the distribution of Ae. aegypti has declined dramatically mainly due to the ecological changes and some other factors such as heavy use of insecticide indoor based on endophagic bloodfeeding of the species.MethodsThis study focused on the knockdown resistance (kdr) genes of both mosquitoes, investigated their mutations, and analyzed their haplotype and evolutionary diversity combined with population genetic features based on the ND4/ND5 genes to further elucidate the molecular mechanisms underlying the development of insecticide resistance in both mosquitoes.ResultsThree mutations, S989P, V1016G and F1534C, were found to be present in Ae. aegypti populations, and the three mutations occurred synergistically. Multiple mutation types (F1534C/S/L/W) of the F1534 locus are found in Ae. albopictus populations, with the three common mutations F1534C, F1534S and F1534L all having multiple independent origins. The F1534W (TTC/TGG) mutation is thought to have evolved from the F1534L (TTC/TTG) mutation. The F1534S (TTC/TCG) mutation has evolved from the F1534S (TTC/TCC) mutation. The most common form of mutation at the F1534 locus found in this study was S1534C, accounting for 20.97%, which may have evolved from the F1534C mutation. In addition, a new non-synonymous mutation M1524I and 28 synonymous mutations were identified in Ae. albopictus populations. Correlation analysis showed that the genetic diversity of Ae. aegypti and Ae. albopictus populations did not correlate with their kdr haplotype diversity (P>0.05), but strong gene flow between populations may have contributed to the evolution of the kdr gene.ConclusionThe study of kdr gene evolution in the two mosquito species may help to identify the evolutionary trend of insecticide resistance at an early stage and provide a theoretical basis for improving the efficiency of biological vector control and subsequent research into new insecticides.

Project description:BackgroundAedes albopictus (Skuse) is an important vector of chikungunya, dengue, yellow fever and Zika viruses. In the absence of anti-viral medication and with limited availability of a commercial vaccine for public health use, vector control remains an effective means for reducing Aedes-borne disease morbidity. Knowledge about genetic mutations associated with insecticide resistance (IR) is a prerequisite for developing rapid resistance diagnosis, and the distribution and frequency of IR conferring mutations is important information for making smart vector control decisions.MethodsPartial DNA sequences of domain II and domain III of Ae. albopictus voltage gated sodium channel (VGSC) gene were amplified from a total of 426 individuals, collected from 17 sites in the Beijing municipality. These DNA fragments were sequenced to discover the possible genetic mutations mediating knockdown resistance (kdr) to pyrethroids. The frequency and distribution of kdr mutations were assessed in the 17 Ae. albopictus populations. The origin of kdr mutations was investigated by haplotype clarification and phylogenetic analysis.ResultsSequence alignments revealed the existence of multiple mutations (V1016G, I1532T, F1534S and F1534L) in VGSC. The highest frequency of the mutant 1016G allele (0.647) was found in Haidian, while 1016G was not detected in Huai Rou, Yan Qing, Ping Gu and Shun Yi. The frequency of 1532T was highest (0.537) in the population from the Olympic Forest Park (OFP, Chao Yang District), but not detectable in Huai Rou and Mi Yun. Two mutations were observed at codon 1534 with different distribution patterns: 1534L was only found in Tong Zhou (TZ) with a frequency of 0.017, while 1534S was distributed in TZ, OFP, Fang Shan, Da Xing and Shi Jing Shan with frequencies ranging from 0.019 (OFP) to 0.276 (TZ). One 1016G, one 1532T, one 1534L and two 1534S haplotypes were identified.ConclusionsMultiple mutations (V1016G, I1532T, F1534L/S) in VGSC were found in Ae. albopictus in Beijing. This represents the first report of V1016G in Ae. albopictus. Sequence alignment and phylogenetic analysis revealed multiple origins of 1534S. The spatial heterogeneity in distribution and frequency of kdr mutations calls for a site-specific strategy for the monitoring of insecticide resistance. The relatively high frequencies of V1016G warn of a risk of pyrethroid resistance in mosquitoes in the urban zones.

Project description:Aedes aegypti and Ae. albopictus are the vectors of dengue, the most important arboviral disease of humans. To date, Aedes ecology studies have assumed that the vectors are truly absent from sites where they are not detected; since no perfect detection method exists, this assumption is questionable. Imperfect detection may bias estimates of key vector surveillance/control parameters, including site-occupancy (infestation) rates and control intervention effects. We used a modeling approach that explicitly accounts for imperfect detection and a 38-month, 55-site detection/non-detection dataset to quantify the effects of municipality/state control interventions on Aedes site-occupancy dynamics, considering meteorological and dwelling-level covariates. Ae. aegypti site-occupancy estimates (mean 0.91; range 0.79-0.97) were much higher than reported by routine surveillance based on 'rapid larval surveys' (0.03; 0.02-0.11) and moderately higher than directly ascertained with oviposition traps (0.68; 0.50-0.91). Regular control campaigns based on breeding-site elimination had no measurable effects on the probabilities of dwelling infestation by dengue vectors. Site-occupancy fluctuated seasonally, mainly due to the negative effects of high maximum (Ae. aegypti) and minimum (Ae. albopictus) summer temperatures (June-September). Rainfall and dwelling-level covariates were poor predictors of occupancy. The marked contrast between our estimates of adult vector presence and the results from 'rapid larval surveys' suggests, together with the lack of effect of local control campaigns on infestation, that many Aedes breeding sites were overlooked by vector control agents in our study setting. Better sampling strategies are urgently needed, particularly for the reliable assessment of infestation rates in the context of control program management. The approach we present here, combining oviposition traps and site-occupancy models, could greatly contribute to that crucial aim.

Project description:Aedes albopictus is the only vector that can transmit the dengue virus in Zhejiang Province, central China, and it can develop insecticide resistance due to long-term exposure to pyrethroids. The presence of knockdown resistance (kdr) mutations is one of the mechanisms responsible for pyrethroid resistance, and has been reported in some Ae. albopictus populations in southern China. However, little is known about the DNA diversity of the voltage-gated sodium channel (VGSC) gene in Ae. albopictus populations in central China. Four Ae. albopictus field populations were collected, in Yiwu (YW), Quzhou (QZ), Wenzhou (WZ), and Jiaxing (JX) from Zhejiang Province, central China. The susceptibility of Ae. albopictus adults to three pyrethroids (beta-cypermethrin, deltamethrin, and permethrin) was tested using the WHO tube assay, and Kdr mutations were identified via PCR and sequencing. The relationship between kdr mutations and pyrethroid phenotypes was also analyzed. Of the four populations, none was sensitive to any pyrethroid tested, and the YW population showed the strongest pyrethroid resistance. Non-synonymous kdr mutations were detected in codons 1532 and 1534, domain III. At codon 1534, one mutant allele, TCC(S), was detected in the four populations with a frequency of 42.08%, while at codon 1532, one mutant allele, ACC(T), was detected in the JX and QZ populations, with frequencies of 4.22 and 3.03%, respectively. The F1534S mutant allele was positively correlated with both beta-cypermethrin and deltamethrin resistance phenotypes (OR > 1, P < 0.05), whereas the I1532T mutant allele was possibly negatively correlated with beta-cypermethrin, deltamethrin, and permethrin resistance phenotypes (OR < 1, P > 0.05). In conclusion, resistance and resistance mutations regarding to three pyrethroids are already present in the Ae. Albopictus populations from Zhejiang, central China, which prompts the need to use non-insecticide-based methods of insect control.

Project description:Recent outbreaks of locally transmitted dengue and Zika viruses in Florida have placed more emphasis on integrated vector management plans for Aedes aegypti (L.) and Aedes albopictus Skuse. Adulticiding, primarily with pyrethroids, is often employed for the immediate control of potentially arbovirus-infected mosquitoes during outbreak situations. While pyrethroid resistance is common in Ae. aegypti worldwide and testing is recommended by CDC and WHO, resistance to this class of products has not been widely examined or quantified in Florida. To address this information gap, we performed the first study to quantify both pyrethroid resistance and genetic markers of pyrethroid resistance in Ae. aegypti and Ae. albopictus strains in Florida. Using direct topical application to measure intrinsic toxicity, we examined 21 Ae. aegypti strains from 9 counties and found permethrin resistance (resistance ratio (RR) = 6-61-fold) in all strains when compared to the susceptible ORL1952 control strain. Permethrin resistance in five strains of Ae. albopictus was very low (RR<1.6) even when collected from the same containers producing resistant Ae. aegypti. Characterization of two sodium channel kdr alleles associated with pyrethroid-resistance showed widespread distribution in 62 strains of Ae. aegypti. The 1534 phenylalanine to cysteine (F1534C) single nucleotide polymorphism SNP was fixed or nearly fixed in all strains regardless of RR. We observed much more variation in the 1016 valine to isoleucine (V1016I) allele and observed that an increasing frequency of the homozygous V1016I allele correlates strongly with increased RR (Pearson corr = 0.905). In agreement with previous studies, we observed a very low frequency of three kdr genotypes, IIFF, VIFF, and IIFC. In this study, we provide a statewide examination of pyrethroid resistance, and demonstrate that permethrin resistance and the genetic markers for resistance are widely present in FL Ae. aegypti. Resistance testing should be included in an effective management program.

Dataset Information

Establishment of an innovative and sustainable PCR technique for 1534 locus mutation of the knockdown resistance (kdr) gene in the dengue vector Aedes albopictus.

Publications

Establishment of an innovative and sustainable PCR technique for 1534 locus mutation of the knockdown resistance (kdr) gene in the dengue vector Aedes albopictus.

Similar Datasets

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