Taxonomic Research Of The Genus Anopheles(Diptera: Culicidae) In China And Sequencing And Analysis Of Two Anopheles Mtgenomes

The genus Anopheles is one group of the most important medical insects, many of which are important vectors of malaria and filariasis. This study summarized the taxonomic status, research status and geographic distribution of the genus Anopheles in China based on the research literature. The Anopheles we collected were identified morphologically. Anopheles minimus and Anopheles culicifacies species B are important vectors of human malaria in southern China and Southeast Asia. The phylogenetics of mosquitoes has not been well resolved, and the mitochondrial genome has been proven to be an important marker in the study of evolutionary biology. In this study, the complete mtgenome of An. minimus and An. culicifacies species B were sequenced for the first time. The mtgenome of An. minimus and An. culicifacies species B were assembled and annotated compared with the Anopheles mtgenome published, and the genome structure, base composition, protein-coding genes, tRNA secondary structure and control region were analyzed in detail. The phylogenetics of An. minimus and An. culicifacies species B with 25 other Anopheles species was constructed by maximum likelihood and Bayesian inference, based on concatenated PCG sequences. The main results are as followed:1. 2590 Anopheles mosquitoes that collected from 21 distributed sites in 11 provinces of China that contains Yunnan, Anhui, Chongqing, Jilin, Shanxi, Shandong, Heilongjiang, Xinjiang, Neimenggu, Xizang and Sichuan were identified morphologically. A total of ten kinds of subgenus Anopheles and six kinds of subgenus Cellia were found. Statistics of the geographic distribution of the genus Anopheles in China showed that there are 48 and 44 mosquito species distributed in Southwest and south China area, respectively. The species in the central China area is relatively small, which distributed 28 mosquito species. The northeast region, north China region, mongo-xingjiang region and qinghai tibet region are located in the temperate zone and frigid zone with a big temperature difference, resulting in a less distributed of the mosquito species.2. The complete mtgenome of An. minimus was 15 395 bp long and contains 37 genes and a non-coding region. Gene organization was compact with no rearrangement compared with other mosquitoes, only had some short intergenic spacers and gene overlap. The mtgenome performs a clear bias in nucleotide composition with a positive AT-skew and a negative GC-skew, which illustrated that the mtgenome of An. minimus preferences using A base and C base. All 13 protein-coding genes prefer to use the codon UUA(Leu), and the 13 protein-coding genes use ATN as an initiation codon with an exception of COI and ND5 genes start with TCG and GTG, respectively. Most protein-coding genes have the complete termination codons, but COI, COII, COIII and ND4 have the incomplete termination codon T. The length of 22 tRNA genes were ranging from 64 to 72 bp, and 19 G-U unmatched base pairs were found in the An. minimus mtgenome. The length of the control region was 537 bp long and has the highest A+T content reaching up to 93.1%. In addition, the control region also include a conserved T-stretch and a(TA)n stretch.3. The complete mtgenome of An. culicifacies species B was 15 330 bp long and contains 37 genes and a non-coding region. Gene organization was compact with no rearrangement compared with other mosquitoes, only had some short intergenic spacers and gene overlap. The mtgenome performs a clear bias in nucleotide composition with a positive AT-skew and a negative GC-skew, which illustrated that the mtgenome of An. culicifacies species B preferences using A base and C base. All 13 protein-coding genes prefer to use the codon UUA(Leu), and the 13 protein-coding genes use ATN as an initiation codon with an exception of COI and ND5 genes start with TCG and GTG, respectively. Most protein-coding genes have the complete termination codons, but COI, COII, COIII and ND4 have the incomplete termination codon T. The length of 22 tRNA genes were ranging from 64 to 72 bp, and 16 G-U unmatched base pairs were found in the An. culicifacies species B mtgenome. The length of the control region was 498 bp long and has the highest A+T content reaching up to 92.6%. In addition, the control region also include a conserved T-stretch and a(AATATTTATATATT)n stretch.4. Phylogenetic analyses of An. minimus and An. culicifacies species B with all known 25 Anopheles species were performed by ML and BI based on concatenated protein-coding gene sequences, with Culex pipiens pipiens as outgroup. The ML and Bayesian analysis yielded similar phylogenetic topology structures, except for the two species in the subgenus Anopheles. As shown in the phylogenetic tree, all Anopheles were mainly divided into two branches. One branch contains subgenus Cellia and subgenus Anopheles, the other branch contains subgenus Nyssorhynchus and subgenus Kerteszia. The phylogenetic tree showed that subgenus Anopheles, subgenus Nyssorhynchus and subgenus Kerteszia were monophyletic group, respectively. The subgenus Nyssorhynchus and subgenus Kerteszia were sister relationship.Punctulatus Group, Funestus Group, Albitarsis Group and Leucosphyrus Group were monophyletic group. The An. minimus and An. culicifacies species B belong to different groups in the Myzomyia Series, they were clustered together both in ML tree and BI tree, which was consistent with the traditional classification standard. The position of two species in the subgenus Anopheles in the phylogenetic tree had a slight difference, the reason may be due to different phylogenetic methods or historical evolutionary, so more subgenus Anopheles mtgenome are needed to elucidate the phylogenetic relationship of mosquitoes.