西藏疟疾流行区多斑按蚊复合体传疟作用与分子生物学研究

发布时间：2018-02-09 06:08

本文关键词： 多斑按蚊复合体种型鉴定生态习性传疟媒介系统进化分析间日疟子孢子群体遗传学西藏墨脱　出处：《中国疾病预防控制中心》2009年博士论文　论文类型：学位论文

【摘要】： 目的(1)了解西藏林芝疟疾流行区墨脱县按蚊组成与多斑按蚊复合体的生态习性;鉴定多斑按蚊复合体的成员种构成;(2)检测按蚊唾腺子孢子以判定当地的传疟媒介,并结合生态习性调查探讨相关传疟按蚊的传疟能力;(3)采用分子建树方法研究多斑按蚊复合体的分子进化关系,并探讨西藏、云南和缅甸不同地理区域间伪威氏按蚊的群体遗传结构差异;为该地区的疟疾防治提供按蚊媒介的基线资料,并为制定综合媒介管理措施提供科学依据。方法(1)在西藏疟疾流行区选择墨脱县三个有代表性的自然村为调查点,调查多斑按蚊复合体的生态习性,伪威氏按蚊群体遗传学研究现场为西藏林芝地区的墨脱县和察隅县、云南勐腊县和景洪、缅甸拉咱市;(2)按蚊媒介现场调查采用人诱、牛诱和灯诱方法捕获按蚊成蚊,按蚊经形态学鉴定后放入硅胶干燥冻存备用:采用显微镜观察成蚊卵巢气管支方法判定经产蚊,并计算经产蚊比率;(3)采用多重PCR扩增ITS2序列方法对形态学鉴定为多斑按蚊复合体的成蚊进行种型鉴定;并采用混合样本和巢氏PCR方法扩增成蚊唾腺间日疟子孢子SSu rDNA以判定该地区的传疟媒介;(4)结合生态学指标,通过计算疟疾流行区伪威氏按蚊的媒介能量和昆虫学接种率以判断伪威氏按蚊的传疟作用;(5)以mtDNA-COI基因序列对多斑按蚊复合体5成员种(多斑按蚊、伪威氏按蚊、威氏按蚊、塞沃按蚊、达罗毗按蚊)进行系统进化分析;(6)以mtDNA-COI和mtDNA-Cytb基因对伪威氏按蚊进行群体遗传学分析;(7)采用Cluxtal、Chromas软件对基因序列进行核实和比对;以Bioedit、Mega和Phylip软件进行碱基组成分析和构建聚类进化树;以TCS软件计算单倍型和构建单倍型家系网络图;采用Arleiquin软件进行AMOVA分析;并将基因序列在NCBI进行BLAST比对。结果(1)西藏疟疾流行区墨脱县共捕获按蚊5 345只,其中形态学鉴定为多斑按蚊复合体97.10%(5 190/5 345),带足按蚊为2.90%(155/5 345);多重PCR方法鉴定多斑按蚊复合体种型构成,其中伪威氏按蚊为98.1%,威氏按蚊为1.9%,提示伪威氏按蚊为该地区的优势蚊种;调查期间伪威氏按蚊种群密度大,通宵均有吸血活动,通宵室内叮人率为15.80只/人·夜,并有偏吸牛血和室外吸血的习性,按蚊孳生地仅发现于稻田;(2)在360个混合样本中发现2份间日疟原虫子孢子SSu rDNA的阳性扩增,克隆测序并经NCBI BLAST同源性比对证实与间日疟原虫(AF145335)SSu rDNA基因片段100%同源,分子种型鉴定证实2份阳性混合样本均由伪威氏按蚊组成;生态学调查伪威氏按蚊的媒介能量为2.795,昆虫学接种率为0.004389,子孢子自然感染率为0.56‰;(3)UPGMA、NJ、ME、MP和ML聚类得到的亲缘关系总体趋于一致,单倍型数据显示mtDNA-COI和mtDNA-Cytb基因多态性丰富,不同群体间伪威氏按蚊mtDNA-COI和mtDNA-Cytb基因AMOVA分析Fst和Nm值分别为0.00794,31.236和0.01696,4.168。结论西藏疟疾流行区多斑按蚊复合体由伪威氏按蚊和威氏按蚊组成,其中伪威氏按蚊种群数量大,密度高,叮人率高,通宵均有吸血活动,偏吸牛血,兼吸人血,是该地区的优势蚊种,也是该地的主要传疟媒介;达罗毗按蚊、多斑按蚊与塞沃按蚊亲缘关系最近,威氏按蚊,伪威氏按蚊的关系最远;mtDNA-COI和mtDNA-Cytb均为适合于群体遗传学分析的基因序列,伪威氏按蚊西藏、云南和缅甸各群体间基因交流频繁,尚未发生群体遗传分化。
[Abstract]:Objective (1) to understand the Linzhi malaria endemic area of Tibet Medog County composition and ecological habits of Anopheles maculatus Complex; member identification of Anopheles maculatus Complex composition; (2) detection of Anopheles salivary gland sporozoites to determine local malaria vectors, and combined with the investigation to explore related ecological habits of Anopheles vectors of malaria transmission ability; (3) molecular evolution using molecular research method to build the Anopheles maculatus Complex, and explore Tibet, population genetic structure between Yunnan and Burma in different geographic regions between the pseudo lubriplate Anopheles; to provide baseline data for the prevention and treatment of malaria vector Anopheles in the region, and provide scientific basis for the comprehensive media management measures.
Methods (1) in Tibet malaria endemic region of Medog County three representative natural villages, ecological habits survey of Anopheles maculatus Complex, pseudo Anopheles population genetic study lubriplate site for the Linzhi area of Tibet and Motuo County, Chayu County, Yunnan County, Mengla and Jinghong, Burma (in Laiza city; 2) field investigation using human baited Anopheles vectors, cattle trap and light trap method to capture the Anopheles mosquito, Anopheles sinensis were identified in silica gel drying and cryopreserved by microscopic observation of mosquito ovary tracheobronchial determination method of multiparous mosquitoes, and the parous rate; (3) using multiplex PCR amplification ITS2 sequence method of morphology for the identification of mosquito Anopheles maculatus Complex for identification; and the mixed samples and nested PCR amplification of the mosquito salivary gland of vivax malaria sporozoite SSu rDNA to determine the area of malaria vectors; (4) combined with the ecological indicators, through the Medium energy and computing the entomological inoculation in malaria endemic area of Anopheles pseudo lubriplate rate to determine the role of malaria transmission of Anopheles pseudo lubriplate; (5) to the mtDNA-COI gene sequence of Anopheles maculatus Complex 5 member (an.maculatus, pseudo lubriplate Anopheles, Weiss Anopheles, Anopheles Saiwo, Chibi Anopheles) system evolution analysis; (6) the pseudo Williams of Anopheles population genetic analysis using mtDNA-COI and mtDNA-Cytb gene; (7) using Cluxtal and Chromas software verification and comparison of gene sequence; Bioedit, Mega and Phylip software base composition analysis and construction of phylogenetic tree; calculated by TCS software and haplotype network constructing haplotype family system; analysis was conducted using AMOVA Arleiquin software; and the BLAST gene sequence alignment in NCBI.
Results (1) Tibet malaria in Motuo County were captured only 5345 Anopheles, which were identified as Anopheles maculatus Complex 97.10% (5 190/5 345), an.peditaeniatus 2.90% (155/5 345); identification of multiple PCR maculatus Complex type structure, in which the pseudo lubriplate by mosquito was 98.1%, Williams in 1.9%, that was the dominant species of Anopheles pseudo lubriplate mosquitoes in the area; during the investigation of pseudo lubriplate Anopheles population density, were all night feeding activities, all indoor biting rate was 15.80 per person per night, and a partial suction blood and outdoor blood sucking habits, Anopheles breeding ground found only in paddy field; (2) in the 360 mixed samples found in 2 samples of Plasmodium vivax sporozoite SSu rDNA positive amplification, cloning and sequencing of NCBI and the BLAST homology with that of Plasmodium vivax (AF145335) SSu rDNA gene fragment of 100% homologous molecular types identified 2 positive samples were mixed by the pseudo Lubriplate composed of Anopheles; ecological investigation of Anopheles pseudo lubriplate medium energy is 2.795, the entomological inoculation rate was 0.004389, the natural sporozoite infection rate was 0.56 per thousand; (3) UPGMA, NJ, ME, MP and ML clustering phylogenetic relationships are generally in agreement, haplotype data show that mtDNA-COI and mtDNA-Cytb gene polymorphism rich. Different groups of pseudo lubriplate Anopheles mtDNA-COI and mtDNA-Cytb AMOVA gene analysis of Fst and Nm were 0.00794,31.236 and 0.01696,4.168.
Conclusion malaria endemic areas of Tibet Anopheles maculatus Complex by the pseudo lubriplate Anopheles and wite Anopheles composition, including the number of pseudo lubriplate Anopheles population, high density, high bite rate, both overnight feeding activities, partial suction and suction blood, blood, is the dominant species in the region, is also the main malaria vector the Anopheles; Chibi, recently an.maculatus Saiwo and Anopheles genetic relationship, lubriplate Anopheles, relationship of pseudo Wilcoxon Anopheles far; mtDNA-COI and mtDNA-Cytb are suitable for gene sequences in population genetics analysis, pseudo lubriplate Tibet Yunnan and Burma each Anopheles, gene flow between populations has not yet occurred frequently, genetic differentiation.

【学位授予单位】：中国疾病预防控制中心
【学位级别】：博士
【学位授予年份】：2009
【分类号】：R181.3

【参考文献】