The Mitochondrial Genomes Of Culex Tritaeniorhynchus And Culex Pipiens Pallens(Diptera: Culicidae) And Comparison Analysis With Two Other Culex Species

Culex, the largest genus in Culicidae, is worldwide distributed, and its many species are important vectors of mosquito-borne diseases, including epidemic encephalitis, lymphatic filariasis and so on. The control of infectious diseases mainly depend on the control of mosquitoes, due to the stable structure, coding content conservation, maternal inheritance, rapid evolution rate, no recombination and a high copy number, the mitochondrial genome has been widely useful in the pattern analysis of molecular evolution, and in the studies on phylogeographics, phylogenetics, and population genetics. At the same time, the phylogenetic relationship study between the species has guiding significance for mosquito control. The Cx. tritaeniorhynchus and Cx.p. pallens are the major vectors of encephalitis virus and bancrofti filariasis, respectively.The study of the two mitochondrial genome can enrich the mitochondrial genome data of Culex genus, and laid the foundation for the two mosquito control research. The two mitochondrial genomes can provide a basis for the understanding of basic biology through comparatively analyzed with two others reported earlier in the genus.In this study, we extract the mitochondrial genome DNA of mosquitoes, using conventional PCR technology and gene cloning, sequencing, splicing and annotations the mitochondrial genomes of Cx. tritaeniorhynchus and Cx. p. pallens, and compared the mitogenomes with other known Culex species. Constructed the phylogenetic relationships using 13 protein-coding genes of all known Culex mitochondrial genome sequences. The main researches and results are below:1. The mitochondrial genome and analysis of Cx. tritaeniorhynchusThe mitochondrial genome of Cx. tritaeniorhynchus is a double-stranded circular molecule with 14 844 bp in length. It contains 37 genes and the non-coding region(un-obtained). The AT content of whole genome, PCGs, tRNAs and rRNAs was 77.6%、76.5% 、 78.8% and 81.8%, respectively. The nucleotide composition of the mitochondrial genome of Cx. tritaeniorhynchus showed obvious bias towards A and T.All of the 13 protein-coding genes use ATN as an initiation codon except for COI genewhich start with TCG. All protein-coding genes have complete termination codons, only COI and COII have the incomplete termination codon T. All the tRNAs, size ranged of65-72 bp, have the typical clover-leaf structure, except trnS(AGN), which does not contain the DHU arm. The secondary structure of 22 tRNAs contained 30 mismatched base pairs. The 12 S rRNAs and 16 S rRNAs secondary structures was drawn for the first time for mosquito mitochondrial genome, they also contained G-U base pairs.2. The mitochondrial genome and analysis of Cx. p. pallensThe mitochondrial genome of Cx. p. pallens is 14 844 bp in length. It contains 37 genes and a non-coding region, the structure and gene order consistent with the Cx.tritaeniorhynchus mitochondrial genome. The AT content of whole genome, PCGs,tRNAs, rRNAs and control region was 77.6%、76.6%、78.9%、82.5% and 88.8%,respectively. The nucleotide composition of the mitochondrial genome of Cx. p. pallens showed obvious bias towards A and T. The initiation codon and termination codons consistent with the Cx. tritaeniorhynchus mitochondrial genome, except the initiation codon of COII gene changed with ATA. All the tRNAs, size ranged of 64-72 bp, have the typical clover-leaf structure, except tRNASer(AGN), which does not contain the DHU arm. The secondary structure of 22 tRNAs and rRNAs all contained un-standard base pairs. The control region of Cx. p. pallens mitochondrial genome was 747 bp long, a highest AT content 88.8%, and identified four tandem repeat structures.3. Comparative analyses of known Culex mitochondrial genomesThere have been 6 mitochondrial genomes of 2 species reported in the Culex genus,are Culex pipiens pipiens and Culex quinquefasciatus species. The Cx. quinquefasciatus mitochondrial genomes is 15 587 bp length(contained one 704 bp control region), other mitochondrial genomes are 14 856 bp(un-contain control region). Comparative analysis the mitochondrial genomes of Culex species showed that their are same at the gene composition and orders. The nucleotide composition of the mitochondrial genomes all showed obvious bias towards A and T. There have three control region: Cx. p. pallens is747 bp long,AT content 88.8%; Cx. p. pipiens is 742 bp,AT content 88.8% and Cx.quinquefasciatus is 704 bp,AT content 88.5%, and all included four highly conserved tandem repeat structures.4. Phylogenetics based on Culex mitochondrial genomesThe phylogenetic relationships of Cx. tritaeniorhynchus, Cx. p. pallens and 4known Culex mitochondrial genome sequences were constructed with Anopheles gambiae as outgroup. The concatenated nucleotide sequences of 13 PCGs were used forthe phylogenetic construction using Maximum Likelihood. The results show that Cx.tritaeniorhynchus and remaining 5 mitochondrial genomes were separately. Cx. p.pallens and Cx. pipiens from Turkey were grouped. Interestingly, 2 mitochondrial genomes of Cx. quinquefasciatus were linked between Cx. p. pallens and Cx. p. pipiens.In this study, we sequenced and analyzed the mitochondrial genomes of Cx.tritaeniorhynchus and Cx. p. pallens. Their rRNA secondary structures was drawn for the first time for mosquito mitochondrial genomes. We enrich the Culex mitochondrial genome data and provide reference and basis for further research of Culex mitochondrial genomes.