| Anopheles sinensis Wiedemann 1828, belonging to An. hyrcanus group of the Subgenus Anopheles, Genus Anopheles, is widely distributed in China. Anopheles sinensis is an important malaria and filariasis vector.Analysis of the genome sequence provides an important resource for analyzing the molecular evolution, genome structure and gene regulation. Due to special structure and evolution features, comparing mitochondrial genome sequences is becoming increasingly common for phylogenetic reconstruction and as a model for genome evolution. We performed illumina Hiseq sequencing and primary bioinformatics analysis of Anopheles sinensis meta-genome database. On the genome level, we made mitochondrial genome annotation and constructed phylogenetic relationship. Vector control is one of the most effective methods to prevent malaria transmission. The long term extensive utilization of pyrethroids for insecticide-treated nets and indoor residual spraying for mosquito control in the last three decades has resulted in the occurrence of resistant in many An. sinensis populations. This study identified SNPs in the coding regions of metabolic detoxification enzymes relating to metabolic resistance, and analyzed temporal and spatial variation tendency of knockdown resistance(kdr) mutation by detecting kdr genotypes of 733 individuals collecting from 17 provinces in 1996-2014. The results were as follows.1. The genome sequencing data of field An. sinensis collected from Beihu and Caoxian, Shandong was assembled and annotated. The predicted genome size was 243 Mb and 276 Mb, respectively. The number of predicted protein coding genes was 22164 or 19587, respectively.2. The mitochondrial genome of An. sinensis from Beihu and Caoxian were assembled and annotated. The complete length of mitochondrial sequence is 15076 bp, containing 37 coding genes, with 13 protein coding genes, 20 t RNA genes and 2 r RNA genes, and the control region. A phylogenetic relationship among 18 mosquito species was reconstructed by the concatenated protein coding sequences. The clusters were reasonable and stable, and the bootstrap value was high. However, the single protein coding sequence was not enough information for contributing phylogeny. The evolution analysis showed that the most recent common ancestor of all Anopheline mosquitoes appeared 98.61 myas ago. The about divergence time of Subgenus Nyssorhynchus, Cellia and Anopheles was 51.14 mya, 74.19 mya and 59.98 mya ago, respectively.3. The SNPs in the coding regions of 8 metabolic detoxification enzymes relating to metabolic resistance were identified and analyzed on the An. sinensis genome level. There was a certain degree of polymorphism in metabolic detoxification enzymes coding sequences. The range of SNP number per 1000 bp was from 8.73 to 29.94, 21.2% of them resulted amino acid substitutions.4. The kdr genotypes of 733 individuals collecting from 17 provinces in 1996-2014 were detected. At codon 1014, four alleles were identified in the An. sinensis populations. In addition to the wildtype TTG encoding leucine, three mutant alleles were detected, with TTT and TTC both encoding phenylalanine, and TGT for cysteine. A total of 10 genotypes were detected, including wildtype homozygote TTG/TTG(55.25%), mutant homozygotes TTT/TTT(17.60%), TTC/TTC(0.27%), TGT/TGT(1.91%), wildtype-mutant heterozygotes TTG/TTT(8.19%), TTG/TTC(0.82%), TTG/TGT(4.50%), TTT/TTC(0.68%), TTT/TGT(10.23%), and TTC/TGT(0.55%). The kdr mutations in An. sinensis largely occurred and accumulated progressively over time in central China. In the other regions, the kdr frequency was lower, and moreover, no kdr allele was detected in the samples from some provinces. The differential kdr distribution patterns suggest that kdr mutations play different roles in resistance to pyrethroids in different populations. |
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