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Anopheles sinensis mosquito insecticide resistance: comparison of three mosquito sample collection and preparation methods and mosquito age in resistance measurements.

ABSTRACT: Insecticide resistance monitoring in malaria mosquitoes is essential for guiding the rational use of insecticides in vector control programs. Resistance bioassay is the first step for insecticide monitoring and it lays an important foundation for molecular examination of resistance mechanisms. In the literature, various mosquito sample collection and preparation methods have been used, but how mosquito sample collection and preparation methods affect insecticide susceptibility bioassay results is largely unknown. The objectives of this study were to determine whether mosquito sample collection and preparation methods affected bioassay results, which may cause incorrect classification of mosquito resistance status.The study was conducted in Anopheles sinensis mosquitoes in two study sites in central China. Three mosquito sample collection and preparation methods were compared for insecticide susceptibility, kdr frequencies and metabolic enzyme activities: 1) adult mosquitoes collected from the field; 2) F1 adults from field collected, blood-fed mosquitoes; and 3) adult mosquitoes reared from field collected larvae.Mosquito sample collection and preparation methods significantly affected mortality rates in the standard WHO tube resistance bioassay. Mortality rate of field-collected female adults was 10-15% higher than in mosquitoes reared from field-collected larvae and F1 adults from field collected blood-fed females. This pattern was consistent in mosquitoes from the two study sites. High kdr mutation frequency (85-95%) with L1014F allele as the predominant mutation was found in our study populations. Field-collected female adults consistently exhibited the highest monooxygenase and GST activities. The higher mortality rate observed in the field-collected female mosquitoes may have been caused by a mixture of mosquitoes of different ages, as older mosquitoes were more susceptible to deltamethrin than younger mosquitoes.Female adults reared from field-collected larvae in resistance bioassays are recommended to minimize the effect of confounding factors such as mosquito age and blood feeding status so that more reliable and reproducible mortality may be obtained.

SUBMITTER: Xu T

PROVIDER: S-EPMC3917893 | biostudies-literature | 2014 Jan

REPOSITORIES: biostudies-literature

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Anopheles sinensis mosquito insecticide resistance: comparison of three mosquito sample collection and preparation methods and mosquito age in resistance measurements.

Xu Tielong T Zhong Daibin D Tang Linhua L Chang Xuelian X Fu Fengyang F Yan Guiyun G Zheng Bin B

Parasites & vectors 20140128

<h4>Background</h4>Insecticide resistance monitoring in malaria mosquitoes is essential for guiding the rational use of insecticides in vector control programs. Resistance bioassay is the first step for insecticide monitoring and it lays an important foundation for molecular examination of resistance mechanisms. In the literature, various mosquito sample collection and preparation methods have been used, but how mosquito sample collection and preparation methods affect insecticide susceptibility ...[more]

PMID: 24472598

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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.

Project description:Malaria mortality rates in sub-Saharan Africa have declined significantly in recent years as a result of increased insecticide-treated bed net (ITN) usage. A major challenge to further progress is the emergence and spread of insecticide resistance alleles in the Anopheles mosquito vectors, like An. coluzzii. A non-synonymous mutation in the para voltage-gated sodium channel gene reduces pyrethroid-binding affinity, resulting in knockdown resistance (kdr). Metabolic mechanisms of insecticide resistance involving detoxification genes like cytochrome P450 genes, carboxylesterases, and glutathione S-transferases are also important. As some gene activity is tissue-specific and/or environmentally induced, gene regulatory variation may be overlooked when comparing expression from whole mosquito bodies under standard rearing conditions.We detected complex insecticide resistance in a 2014 An. coluzzii colony from southern Mali using bottle bioassays. Additional bioassays involving recombinant genotypes from a cross with a relatively susceptible 1995 An. coluzzii colony from Mali confirmed the importance of kdr and associated increased permethrin resistance to the CYP9K1 locus on the X chromosome. Significant differential expression of CYP9K1 was not observed among these colonies in Malpighian tubules. However, the P450 gene CYP6Z1 was overexpressed in resistant individuals following sublethal permethrin exposure and the carboxylesterase gene COEAE5G was constitutively overexpressed.The significant P450-related insecticide resistance observed in the 2014 An. coluzzii colony indicates that ITNs treated with the P450 inhibitor piperonyl butoxide (PBO) would be more effective in this region. The known insecticide resistance gene CYP6Z1 was differentially expressed exclusively in the context of sublethal permethrin exposure, highlighting the importance of tissue-specificity and environmental conditions in gene expression studies. The increased activity of the carboxylesterase COEAE5G in the resistant An. coluzzii colony suggests resistance to other insecticides like organophosphates. Additional gene expression studies involving other tissues (e.g. fat body) would provide a more comprehensive view of genes underlying metabolic insecticide resistance in An. coluzzii from Mali. Identifying genetic markers linked to these regulatory alleles is an important next step that would substantially improve insecticide resistance surveillance and population genetic studies in this important vector species.

Project description:BACKGROUND:Anopheles sinensis is one of the most abundant vectors of malaria and other diseases in Asia. Vector control through the use of insecticides is the front line control method of vector-borne diseases. Pyrethroids are the most commonly used insecticides due to their low toxicity to vertebrates and low repellency. However, the extensive use of insecticides has imposed strong selection pressure on mosquito populations for resistance. High levels of resistance to pyrethroid insecticides and various mutations and haplotypes in the para sodium channel gene that confers knockdown resistance (kdr) have been detected in An. sinensis. Despite the importance of kdr mutations in pyrethroid resistance, the evolutionary origin of the kdr mutations is unknown. This study aims to examine the evolutionary genetics of kdr mutations in relation to spatial population genetic structure of An. sinensis. METHODS:Adults or larvae of Anopheles sinensis were collected from various geographic locations in China. DNA was extracted from individual mosquitoes. PCR amplification and DNA sequencing of the para-type sodium channel gene were conducted to analyse kdr allele frequency distribution, kdr codon upstream and downstream intron polymorphism, population genetic diversity and kdr codon evolution. The mitochondrial cytochrome c oxidase COI and COII genes were amplified and sequenced to examine population variations, genetic differentiation, spatial population structure, population expansion and gene flow patterns. RESULTS:Three non-synonymous mutations (L1014F, L1014C, and L1014S) were detected at the kdr codon L1014 of para-type sodium channel gene. A patchy distribution of kdr mutation allele frequencies from southern to central China was found. Near fixation of kdr mutation was detected in populations from central China, but no kdr mutations were found in populations from southwestern China. More than eight independent mutation events were detected in the three kdr alleles, and at least one of them evolved multiple times subsequent to their first divergence. Based on sequence analysis of the mitochondrial COI and COII genes, significant and large genetic differentiation was detected between populations from southwestern China and central China. The patchy distribution of kdr mutation frequencies is likely a consequence of geographic isolation in the mosquito populations and the long-term insecticide selection. CONCLUSION:Our results indicate multiple origins of the kdr insecticide-resistant alleles in An. sinensis from southern and central China. Local selection related to intense and prolonged use of insecticide for agricultural purposes, as well as frequent migrations among populations are likely the explanations for the patchy distribution of kdr mutations in China. On the contrary, the lack of kdr mutations in Yunnan and Sichuan is likely a consequence of genetic isolation and absence of strong selection pressure. The present study compares the genetic patterns revealed by a functional gene with a neutral marker and demonstrates the combined impact of demographic and selection factors on population structure.

Project description:Malaria is one of the most important public health problems in Southeast Asia, including Hainan Island, China. Vector control is the main malaria control measure, and insecticide resistance is a major concern for the effectiveness of chemical insecticide control programs. The objective of this study is to determine the resistance status of the main malaria vector species to pyrethroids and other insecticides recommended by the World Health Organization (WHO) for indoor residual sprays.The larvae and pupae of Anopheles mosquitoes were sampled from multiple sites in Hainan Island, and five sites yielded sufficient mosquitoes for insecticide susceptibility bioassays. Bioassays of female adult mosquitoes three days after emergence were conducted in the two most abundant species, Anopheles sinensis and An. vagus, using three insecticides (0.05% deltamethrin, 4% DDT, and 5% malathion) and following the WHO standard tube assay procedure. P450 monooxygenase, glutathione S-transferase and carboxylesterase activities were measured. Mutations at the knockdown resistance (kdr) gene and the ace-1 gene were detected by DNA sequencing and PCR-RFLP analysis, respectively.An. sinensis and An. vagus were the predominant Anopheles mosquito species. An. sinensis was found to be resistant to DDT and deltamethrin. An. vagus was susceptible to deltamethrin but resistant to DDT and malathion. Low kdr mutation (L1014F) frequency (<10%) was detected in An. sinensis, but no kdr mutation was detected in An. vagus populations. Modest to high (45%-75%) ace-1 mutation frequency was found in An. sinensis populations, but no ace-1 mutation was detected in An. vagus populations. Significantly higher P450 monooxygenase and carboxylesterase activities were detected in deltamethrin-resistant An. sinensis, and significantly higher P450 monooxygenase, glutathione S-transferase and carboxylesterase activities were found in malathion-resistant An. vagus mosquitoes.Multiple insecticide resistance was found in An. sinensis and An. vagus in Hainan Island, a malaria-endemic area of China. Cost-effective integrated vector control programs that go beyond synthetic insecticides are urgently needed.

Project description:BackgroundVector surveillance is essential in determining the geographical distribution of mosquito vectors and understanding the dynamics of malaria transmission. With the elimination of human malaria cases, knowlesi malaria cases in humans are increasing in Malaysia. This necessitates intensive vector studies using safer trapping methods which are both field efficient and able to attract the local vector populations. Thus, this study evaluated the potential of Mosquito Magnet as a collection tool for Anopheles mosquito vectors of simian malaria along with other known collection methods.MethodsA randomized 4 × 4 Latin square designed experiment was conducted to compare the efficiency of the Mosquito Magnet against three other common trapping methods: human landing catch (HLC), CDC light trap and human baited trap (HBT). The experiment was conducted over six replicates where sampling within each replicate was carried out for 4 consecutive nights. An additional 4 nights of sampling was used to further evaluate the Mosquito Magnet against the "gold standard" HLC. The abundance of Anopheles sampled by different methods was compared and evaluated with focus on the Anopheles from the Leucosphyrus group, the vectors of knowlesi malaria.ResultsThe Latin square designed experiment showed HLC caught the greatest number of Anopheles mosquitoes (n = 321) compared to the HBT (n = 87), Mosquito Magnet (n = 58) and CDC light trap (n = 13). The GLMM analysis showed that the HLC method caught significantly more Anopheles mosquitoes compared to Mosquito Magnet (P = 0.049). However, there was no significant difference in mean nightly catch of Anopheles mosquitoes between Mosquito Magnet and the other two trapping methods, HBT (P = 0.646) and CDC light traps (P = 0.197). The mean nightly catch for both An. introlatus (9.33 ± 4.341) and An. cracens (4.00 ± 2.273) caught using HLC was higher than that of Mosquito Magnet, though the differences were not statistically significant (P > 0.05). This is in contrast to the mean nightly catch of An. sinensis (15.75 ± 5.640) and An. maculatus (15.78 ± 3.479) where HLC showed significantly more mosquito catches compared to Mosquito Magnet (P < 0.05).ConclusionsMosquito Magnet has a promising ability to catch An. introlatus and An. cracens, the important vectors of knowlesi and other simian malarias in Peninsular Malaysia. The ability of Mosquito Magnet to catch some of the Anopheles mosquito species is comparable to HLC and makes it an ethical and safer alternative.

Dataset Information

Anopheles sinensis mosquito insecticide resistance: comparison of three mosquito sample collection and preparation methods and mosquito age in resistance measurements.

Publications

Anopheles sinensis mosquito insecticide resistance: comparison of three mosquito sample collection and preparation methods and mosquito age in resistance measurements.

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