Study On Immune Genes Polymorphism In The Malaria Mosquito Anopheles Sinensis (Diptera: Culicidae)

Anopheles sinensis Wiedemann,1828, are belonging to An. hyrcanus group of theSubgenus Anopheles, Genus Anopheles, is widely distributed in China, and it is importantmalaria and filariasis vector. The investigations showed there was distict ecological habitsand vector efficiency among the geographical areas. So the genetic basis of these needs tobe an explanation. In this study, the population genetic variation and divergence of An.sinensis will be detected inferred by mtDNA-COI, Toll6and SRPN14immune genes, andthe molecular evolution of the two genes in the An. hyrcanus species group.The research strategy was as follows: The454sequencing result from the An.sinensis genome will be analyzed, and the contigs homologous to the An. gambiae immunegenes hope to be found. Immune genes of primers and PCR assay were designed andestablished in An. hyrcanus group. Polymorphism of mtDNA-COI, Toll6and SRPN14genes were detected and the level of An. sinensis population genetic variation anddivergence were estimated. The molecular evolution of Toll6and SRPN14genes in theAn. hyrcanus group was studied. The results were as follows:1. Nineteen contigs of An. sinensis were obtained, which were high homology withimmune-related genes in the GenBank database, are among them17contigs was from An.gambiae immune-related genes.2. Part of2immune genes in An. sinensis were amplified and sequenced. One of themwas signal pathway gene Toll6. Another one was inhibiting protein serine protease geneSRPN14.3. Anopheles sinensis mtDNA-COI sequences from459individuals of16populationswere analyzed. The total haplotypes were251, and41of them shared between populations.The genealogical relationship among haplotypes was at a high level of homoplasy. Thelevel of sequence difference within the population was highest in Henan population (11.83246±3.59614,6.71265±3.57225), while the lowest occured in Liaoning population(4.30562±1.88608，2.60952±1.65685). The range of haplotype diversity was from0.709(Yunnan) to0.998(Anhui). The genetic divergence between Yunnan with the others was allgreat, and the highest was in Sichuan population (Fst=0.6220). The Fst values werenegative in9pairwise populations, which suggested that their genetic divergence was verylow. The AMOVA result indicated that the variance within populations (83.14%) wasgreater than among them (16.86%). Fst value for all populations was0.168, whichsuggested that level of population genetic variation of An. sinensis was medium. Manteltest results indicated that the level of genetic distance was positively correlated withgeographic distance (R2=0.006, P=0.073). The population genetic structure fits theisolation-by-distance model. The neutral test and mismatch analysis results showed that theAn. sinensis population was expanding process recently.4. The polymorphism of Toll6and SRPN14genes were detected in16An. sinensispopulations. The results showed that there were a number of sequences types, but rarelyshared between populations. The level of sequence variation between populations was verylow, as5.05%with Toll6, and5.37%with SRPN14. The major difference existed amongindividuals within populations. The Fst value was0.055(Toll6) and0.054(SRPN14) all ofthe populations, which suggested that the level of population genetic divergence was verylow. The evolution of Toll6and SRPN14genes was under the purification selection;resulted from them both employed a huge role in immune reactions in insects.5. The polymorphism of Toll6and SRPN14genes from9species in Hyrcanus groupwere detected. The inter-species variation was larger than intra-species. The phylogenetictree showed that the topologic structure by Toll6gene was similar with by ITS2andmorphology, but by SRPN14gene not. The paiwise species evolution pattern of Toll6andSRPN14were all under the purification selection by their restricted function.Anopheles immune response against the parasite may determine its survival and development in mosquitoes, so the Anopheles immune-related genes should be the mostpromising new malaria control methods target genes, Anopheles immune-related genepolymorphism will provide a new interpretation of pathogens-vector coevolutionaryrelationship, and a theoretical basis for the formulation of effective vector control strategy.