Molecular Identification and Phylogeny of China Main Mosquito Species Based on DNA Barcoding
|School||PLA Military Academy of Medical Sciences|
|Keywords||Culicidae mtDNA rDNA Molecular identification Phylogeny|
Culicidae belongs to Diptera, Nematocera. The harm of mosquito is not only blood-sucking, but some species is important vector of many serious diseases. Mosquito is an important medical insect, so, it has been attached great importance to entomologists and physicians, and has had extensive and deep research. As so far, there are 40 genus and 3200 species (subspecies) in the world, and 18 genus and 390 species (subspecies) in China. At present, the classification system of mosquito based on the morphological characteristics established by taxonomists is relatively mature, and it explains the genetic relationship of mosquito specieses, but for phylogeny, although many taxonomists reconstruct the phylogeny of mosquito based on morphological characteristics, the choice of the morphological characteristics, whether plesiomorphy or not, influenced by researchers. And for some features, such as the length of male and female antennae, the length of breathing tube, color scales, bristles of stigma and so on, it difficult to estimate the significance and relative importance in evolutionary process. Moreover, taxonomists rarely take ecological habits into the current classification system, so we have not in-depth understanding of the mosquito.Moreover, for control mosquito-born diseases and eliminating malaria by 2020, the identification and classification is very important. At present, there are two limitations for morphological classification. Firstly, the inherent defect of morphological classification: 1.Phenotypic plasticity and Genetic variability lead to incorrect identification easily, 2.Tranditional identification can not find the cryptic species, and 3. Morphological identification restrained by sex and developmental stage of specimen, therefore many specimens can not be identified. Secondly, the lack of senior taxonomists is common. In this case, we need urgently a fast, precise, automated and global tool for classification and identification.On this basis, we reconstructed the phylogeny of China mosquito with 5 molecular markers: mtDNA-COI、mtDNA-COII、mtDNA-Cytb、rDNA-28S-D2 and rDNA-28S-D3, and for screening the DNA Barcoding gene, we analyzed the characteristics of each marker, and designed the molecular identification system of China mosquitoes.In our research, we collected totally 15 genus and 126 mosquito species (subspecies), genomic DNA was extracted from single specimen, and the mtDNA-COI、mtDNA-COII、mtDNA-Cytb、rDNA-28S-D2 and rDNA-28S-D3 were amplified and sequenced, for some abnormal samples, we cloned it and then sent to sequencing again. Using SeqVerter、Clustal X 1.8、Primer Primiser 5.0、ModelTest 3.7、MrMTgui、MEGA 4.0、PAUP*4.0b10(PC)and MrBayes 3.1 for sequence correction, alignment, signature analysis, model test, phylogeny reconstruction, screening DNA Barcoding gene, establishing database of molecular identification system and so on. Phylogenetic analyses under the Neighbor-Joining (NJ),maximum Parsimony (MP) and maximum likelihood (ML) optimal criteria were performed by PAUP*4.0bl0 software. Bayesian inference also was employed to reeonstruct the Phylogenetic tree by MrBayes V3.0 software, and developed the“Molecular identification system of China mosquito”with VB 6.0 software.The sequence 15 genus and 124 species of COI, 15 genus and 99 species of COII, 15 genus and 92 species of Cytb, 14 genus and 81species of all mitochondrial genes, 13 genus and 62 species of D2+D3, 10 genus and 46 species of all sequences were obtained in this research. We reconstructed 27 Phylogenetic trees inferred by 6 dataset, and combined the molecular data with morphologic characteristics and ecological habits, we found the BI tree based on COI and COII gene is the best reasonable tree. And analyzed the characteristics of sequence and genetic distance, we selected finally COI gene as the DNA Barcoding gene, and analyzed the all 415 COI gene sequences, confirmed the intraspecific parameter of molecular identification system as 2%, nucleotide substitution model as p-distance model.The results of the research as follows:(1) More than 98% intraspecific distance of COI gene of mosquito is less than 2%, and there is no overlapping within intraspecies.(2) We found cryptic species is common in mosquito based on the COI gene, such as Ae.dorsalis, Ae.vexans, Tr.aranoides, Tx.splendens and Cx.modestus. Besides, Molecular differentiation emerged in Cq.crassipes and An.sinensis.(3) The data of COI gene hint that Ae.craggi and Ae.annandalei, Cx.Spiculosus and Cx.minor could not rise to specific level. (4) Combining molecular data with morphologic characteristics and Ecological habits, we got a cladogram of China mosquito, and the cladogram shows: the Genus Anopheles and Genus Orthopodomyiini is the most primitive group, and the Genus Toxorhychites, Genus Uranotaenia, Genus Heizmannia, Genus Amigeres, Genus Topomyia, Genus Malaya and Genus Tripyeroides is more evolutive group. Besides, we thought maybe Genus Heizmannia is just a subgenus of Genus Aedes.(5) We reconfirmed the status of former Tribe Aedini and former Genus Mansonia, and thought the Culex Subgenus Lutzia and Culex Subgenus Barraudius belong to Culex Subgenus Culex with the all 5 molecular markers.(6) We established the database of molecular identification system based on the COI gene, and developed the“Molecular identification system of China mosquito”software. And the main function of software is able to identify mosquito at specific level, the results include not only the species name but also the geographical distribution, Ecological habits and Medical importance of the mosquito which under inspection.