Study On The Capacity Of Malaria Transmission And Molecularbiology Of Anopheles Maculatus S.L. In Malaria Endemic Area Of Xizang Autonomous Region

Objective(1) To explore the Anopheles composition and ecological behaviour of An.maculatus s.l.in malaria endemic area of Xizang.(2) To identify the species of An. maculatus s.l.and find the malaria transmission vector by amplifing the sporozoites DNA in Anopheles salivary glands and study the transmission capacity of malaria vector.(3) To construct the phyletic evolution tree of 5 members of An.maculatus s.l. with mtDNA-COI and analyze the variance of population genetics constitution in the different geography area with mtDNA-COI and mtDNA-Cytb,Thus to provide the vector baseline data for malaria control and the scientific evidence for integrated malaria vector control programme.Methods(1) Three representative villages were selected for vector investigation and Xizang,Yunnan and Lazan city of Myanmar were picked up to collect An. pseudowillmori for population genetics analysis.(2) Human baited net traps(HBNT), cow baited traps(CBT) and CDC light traps were used for catching of mosquito adults,and oviposited moquitoes were decided by ootheca tracheal branches with microscopy.(3) Amplifing the ITS2 gene according to the variance of 5 members of An.maculatus s.l.by multiple PCR was adopted to identify the species.Nested PCR with mixed sample was applied to decide the malaria vector by amplifing sporozoites SSu rDNA of Plasmodium vivax.(4) Integrating the ecological behaviour,malaria vector capacity and entomology inoculation rate(EIR) were calculated to evalue the transmission ability of An.pseudowillmori.(5) Phyletic evolution analysis of 5 members(An.pseudowilomori,An.willmori,An.maculatus s.s.,An.sawadwongporni, An.dravidicus) of An.maculatus s.l.was performed with mtDNA-COI.(6) Population genetics analysis was executed with mtDNA-COI and mtDNA-Cytb gene. (7) Sequence comparision and blast were carry out by Cluxtal and Chromas software, Bioedit,Mega and Phylip software were used for the calculation of the basepair feature and contruct the phylogenetic tree.Haplotype family lattice map and AMOVA analysis were implemented by TCS and Arlequin software,respectively.Internet sequence comparision was blasted through NCBI web.Results(1) 5 345 anopheline moquito adults were collected and of which 97.10%(5 190/5 345) were An.maculatus s.l.identified by morphology,and An.peditaeniatus accounted for the remainder.An.pseudowillmori(98.1%) and An.willmori(1.9%) were identified by the method of molecularbiology among An.macualtus group.An. pseudowillmori was dominant with high density and whole night biting rate.The human biting rate was 15.80 per man per night and only ricefied found the anopheline larvae.(2) Two mixed samples were amplified out the positive SSu rDNA fragment.After clone sequencing and web blast,it was found 100% homologization compared with P.vivax(AF145335),and further investigation confirmed the two mixed samples each included 10 mosquitoes were composed of An. pseudowillmori.The vector capacity,entomology inoculation rate(EIR) and sporozoites natural infection rate were 2.795,0.004389 and 0.56‰,respectively.(3) Coincident results were obtained by UPGMA,NJ,MP,ME and ML clustering methods.Gene polymorphism was found in mtDNA-COI and mtDNA-Cytb with haplotype data,AMOVA analysis found Fst were 0.00794 and 0.01696 with mtDNA-COI and mtDNA-Cytb between different An.pseudowillmori populations.Conclusion An.maculatus s.l.was composed of An.pseudowillmori and An.willmori, and An.pseudowillmori was the dominant anopheline mosquito with high density and biting rate and was the primary malaria vector.An.sawadwongporni,An.dravidicus and An.maculatus s.s.with close relationship,and An.pseudowillmori with the remote distance to the others.The two molecular marks of mtDNA-COI and mtDNA-Cytb beseem to population genetics analysis.An.pseudowillmori gene flow was frequently and no obvious genetics variance was found among population between China(Xizang,Yunnan) and Myanmar(Lazan).