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Knockdown resistance of Anopheles sinensis in Henan province, China.

ABSTRACT: Vivax malaria was historically epidemic in Henan Province of China and Anopheles sinensis was the main vectors and poor farming communities bare the greatest burden of disease. Knockdown resistance in An. sinensis is one of the mechanisms of resistance against pyrethroids. In the present study, the frequency of mutations from An. sinensis was examined in Henan province, China.Anopheles was collected from Kaifeng, Tongbai, Tanghe, Pingqiao, Shihe, and Yongcheng counties of Henan province in 2013. Molecular identification of Anopheles species was conducted by polymerase chain reaction (PCR) amplifying the internal transcribed spacer 2 (ITS2). Part of the IIS6 domain of the para-type sodium channel protein gene was polymerase chain reaction-amplified and directly sequenced. Frequency and geographic difference of kdr gene mutant types were analysed.208 Anopheles were received molecular identification, of which 169 (81.25%) were An. sinensis, 25 (12.02%) were Anopheles yatsushiroensis, and 12 (5.77%) were Anopheles lesteri. A 325 bp fragment of the para-type sodium channel gene including position 1014 was successfully sequenced from 139 Anopheles, of which 125 (89.93%) were An. sinensis, 12 (8.63%) were An. yatsushiroensis, 2 (1.44%) were An. lesteri. The molecular analyses revealed that mutations existed at codon 1014 in An. sinensis but not in An. yatsushiroensis and An. lesteri. Frequency of kdr mutation was 73.60% (92/125) from population of An. sinensis in Henan province, of which L1014F (TTT?+?TTC) allele frequencies accounted for 46.40% (58/125), and was higher than that of L1014C(TGT) which accounted for 27.20% (34/125) ( ?2?=?55.423, P?

SUBMITTER: Zhang HW

PROVIDER: S-EPMC4381423 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Project description:BackgroundHistorically, malaria due to Plasmodium vivax has been epidemic in Henan Province, China, with Anopheles sinensis as the main vector. The most effective measures to prevent malaria transmission are based on vector control through the use of insecticides. However, insecticides exert a strong selective pressure on mosquito populations for insecticide resistance. The aim of this study was to investigate the susceptibility profile and population genetic characteristics of An. sinensis to provide basic data and scientific guidance for the study of resistance mechanisms and the control of An. sinensis in Henan Province.MethodsAdult Anopheles mosquitoes were collected at sites near local farmers' sheepfolds, pigsties and/or cowsheds located in Pingqiao, Xiangfu, Xiangcheng and Tanghe counties/districts of Henan Province during July-September 2021 for insecticide susceptibility testing. Molecular identification of collected mosquitoes as belonging to genus Anopheles was by PCR, and the frequencies of mutations in the knockdown resistance gene (kdr) and acetylcholinesterase-1 gene (ace-1) were detected using gene amplification. The mitochondrial DNA cytochrome oxidase subunit I (COI) gene was amplified in deltamethrin-resistant and deltamethrin-sensitive mosquitoes to analyze the genetic evolutionary relationship.ResultsA total of 1409 Anopheles mosquitoes were identified by molecular identification, of which 1334 (94.68%) were An. sinensis, 28 (1.99%) were An. yatsushiroensis, 43 (3.05%) were An. anthropophagus and four (0.28%) were An. belenrae. The 24-h mortality rates of An. sinensis in Pingqiao, Tanghe, Xiangcheng and Xiangfu counties/districts exposed to deltamethrin were 85.85%, 25.38%, 29.73% and 7.66%, respectively; to beta-cyfluthrin, 36.24%, 70.91%, 34.33% and 3.28%, respectively; to propoxur, 68.39%, 80.60%, 37.62% and 9.29%, respectively; and to malathion, 97.43%, 97.67%, 99.21% and 64.23%, respectively. One mutation, G119S, was detected in the ace-1 gene. The frequencies of the main genotypes were 84.21% of specimens collected in Xiangfu (G/S), 90.63% of speciments collected in Xiangcheng (G/G) and 2.44% of speciments collected in Tanghe (S/S). Significantly higher G119S allele frequencies were observed in both propoxur- and malathion-resistant mosquitoes than in their sensitive counterparts in the Tanghe population (P < 0.05). Three mutations, L1014F (41.38%), L1014C (9.15%) and L1014W (0.12%), were detected in the kdr gene. The genotypes with the highest frequency in the populations of An. sinensis in Xiangfu and Tanghe were the mutant TTT (F/F) and wild-type TTG (L/L), at 67.86% (57/84) and 74.29% (52/70), respectively. In Pingqiao and Xiangfu, higher frequencies of the L1014F allele and lower frequencies of the L1014C allele were observed in mosquitoes resistant for beta-cyfluthrin than in those which were sensitive for this insecticide (P < 0.05). The results of Tajima's D and of Fu and Li's D and F were not significantly negative (P > 0.10), and each haplotype was interlaced and did not form two distinct branches.ConclusionsHigh resistance to pyrethroids and propoxur was observed at four sites, but the resistance to malathion varied according to the location. Anopheles belenrae and the L1014W (TGG) mutation in An. sinensis were first discovered in Henan Province. The deltamethrin-resistant and deltamethrin-sensitive mosquito populations showed no genetic differentiation. The generation of resistance might be the result of the combination of multiple factors.

Project description:Anopheles sinensis is a major vector of malaria in China. The gamma-aminobutyric acid (GABA)-gated chloride channel, encoded by the RDL (Resistant to dieldrin) gene, is the important target for insecticides of widely varied structures. The use of various insecticides in agriculture and vector control has inevitably led to the development of insecticide resistance, which may reduce the control effectiveness. Therefore, it is important to investigate the presence and distribution frequency of the resistance related mutation(s) in An. sinensis RDL to predict resistance to both the withdrawn cyclodienes (e.g. dieldrin) and currently used insecticides, such as fipronil.Two hundred and forty adults of An. sinensis collected from nine locations across Guangxi Zhuang Autonomous Region were used. Two fragments of An. sinensis RDL (AsRDL) gene, covering the putative insecticide resistance related sites, were sequenced respectively. The haplotypes of each individual were reconstructed by the PHASE2.1 software, and confirmed by clone sequencing. The phylogenetic tree was built using maximum-likelihood and Bayesian inference methods. Genealogical relations among different haplotypes were also analysed using Network 5.0.The coding region of AsRDL gene was 1674 bp long, encoding a protein of 557 amino acids. AsRDL had 98.0% amino acid identity to that from Anopheles funestus, and shared common structural features of Cys-loop ligand-gated ion channels. Three resistance-related amino acid substitutions (A296S, V327I and T345S) were detected in all the nine populations of An. sinensis in Guangxi, with the 296S mutation being the most abundant (77-100%), followed by 345S (22-47%) and 327I (8-60%). 38 AsRDL haplotypes were identified from 240 individuals at frequencies ranging from 0.2 to 34.8%. Genealogical analysis suggested multiple origins of the 345S mutation in AsRDL.The near fixation of the 296S mutation and the occurrence of the 327I and 345S mutations in addition to 296S, in all the nine tested An. sinensis populations in Guangxi, strongly indicate a risk of multiple insecticide resistance. The haplotype diversity plus genetic heterogeneities in the geographical distribution, and multiple origins of AsRDL alleles call for a location-customized strategy for monitoring and management of insecticide resistance.

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Knockdown resistance of Anopheles sinensis in Henan province, China.

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