本文關(guān)鍵詞： 多斑按蚊復(fù)合體 種型鑒定 生態(tài)習(xí)性 傳瘧媒介 系統(tǒng)進(jìn)化分析 間日瘧 子孢子 群體遺傳學(xué) 西藏 墨脫　出處：《中國(guó)疾病預(yù)防控制中心》2009年博士論文　論文類型：學(xué)位論文

【摘要】： 目的(1)了解西藏林芝瘧疾流行區(qū)墨脫縣按蚊組成與多斑按蚊復(fù)合體的生態(tài)習(xí)性;鑒定多斑按蚊復(fù)合體的成員種構(gòu)成;(2)檢測(cè)按蚊唾腺子孢子以判定當(dāng)?shù)氐膫鳢懨浇?并結(jié)合生態(tài)習(xí)性調(diào)查探討相關(guān)傳瘧按蚊的傳瘧能力;(3)采用分子建樹方法研究多斑按蚊復(fù)合體的分子進(jìn)化關(guān)系,并探討西藏、云南和緬甸不同地理區(qū)域間偽威氏按蚊的群體遺傳結(jié)構(gòu)差異;為該地區(qū)的瘧疾防治提供按蚊媒介的基線資料,并為制定綜合媒介管理措施提供科學(xué)依據(jù)。 方法(1)在西藏瘧疾流行區(qū)選擇墨脫縣三個(gè)有代表性的自然村為調(diào)查點(diǎn),調(diào)查多斑按蚊復(fù)合體的生態(tài)習(xí)性,偽威氏按蚊群體遺傳學(xué)研究現(xiàn)場(chǎng)為西藏林芝地區(qū)的墨脫縣和察隅縣、云南勐臘縣和景洪、緬甸拉咱市;(2)按蚊媒介現(xiàn)場(chǎng)調(diào)查采用人誘、牛誘和燈誘方法捕獲按蚊成蚊,按蚊經(jīng)形態(tài)學(xué)鑒定后放入硅膠干燥凍存?zhèn)溆?采用顯微鏡觀察成蚊卵巢氣管支方法判定經(jīng)產(chǎn)蚊,并計(jì)算經(jīng)產(chǎn)蚊比率;(3)采用多重PCR擴(kuò)增ITS2序列方法對(duì)形態(tài)學(xué)鑒定為多斑按蚊復(fù)合體的成蚊進(jìn)行種型鑒定;并采用混合樣本和巢氏PCR方法擴(kuò)增成蚊唾腺間日瘧子孢子SSu rDNA以判定該地區(qū)的傳瘧媒介;(4)結(jié)合生態(tài)學(xué)指標(biāo),通過計(jì)算瘧疾流行區(qū)偽威氏按蚊的媒介能量和昆蟲學(xué)接種率以判斷偽威氏按蚊的傳瘧作用;(5)以mtDNA-COI基因序列對(duì)多斑按蚊復(fù)合體5成員種(多斑按蚊、偽威氏按蚊、威氏按蚊、塞沃按蚊、達(dá)羅毗按蚊)進(jìn)行系統(tǒng)進(jìn)化分析;(6)以mtDNA-COI和mtDNA-Cytb基因?qū)瓮习次眠M(jìn)行群體遺傳學(xué)分析;(7)采用Cluxtal、Chromas軟件對(duì)基因序列進(jìn)行核實(shí)和比對(duì);以Bioedit、Mega和Phylip軟件進(jìn)行堿基組成分析和構(gòu)建聚類進(jìn)化樹;以TCS軟件計(jì)算單倍型和構(gòu)建單倍型家系網(wǎng)絡(luò)圖;采用Arleiquin軟件進(jìn)行AMOVA分析;并將基因序列在NCBI進(jìn)行BLAST比對(duì)。 結(jié)果(1)西藏瘧疾流行區(qū)墨脫縣共捕獲按蚊5 345只,其中形態(tài)學(xué)鑒定為多斑按蚊復(fù)合體97.10%(5 190/5 345),帶足按蚊為2.90%(155/5 345);多重PCR方法鑒定多斑按蚊復(fù)合體種型構(gòu)成,其中偽威氏按蚊為98.1%,威氏按蚊為1.9%,提示偽威氏按蚊為該地區(qū)的優(yōu)勢(shì)蚊種;調(diào)查期間偽威氏按蚊種群密度大,通宵均有吸血活動(dòng),通宵室內(nèi)叮人率為15.80只/人·夜,并有偏吸牛血和室外吸血的習(xí)性,按蚊孳生地僅發(fā)現(xiàn)于稻田;(2)在360個(gè)混合樣本中發(fā)現(xiàn)2份間日瘧原蟲子孢子SSu rDNA的陽(yáng)性擴(kuò)增,克隆測(cè)序并經(jīng)NCBI BLAST同源性比對(duì)證實(shí)與間日瘧原蟲(AF145335)SSu rDNA基因片段100%同源,分子種型鑒定證實(shí)2份陽(yáng)性混合樣本均由偽威氏按蚊組成;生態(tài)學(xué)調(diào)查偽威氏按蚊的媒介能量為2.795,昆蟲學(xué)接種率為0.004389,子孢子自然感染率為0.56‰;(3)UPGMA、NJ、ME、MP和ML聚類得到的親緣關(guān)系總體趨于一致,單倍型數(shù)據(jù)顯示mtDNA-COI和mtDNA-Cytb基因多態(tài)性豐富,不同群體間偽威氏按蚊mtDNA-COI和mtDNA-Cytb基因AMOVA分析Fst和Nm值分別為0.00794,31.236和0.01696,4.168。 結(jié)論西藏瘧疾流行區(qū)多斑按蚊復(fù)合體由偽威氏按蚊和威氏按蚊組成,其中偽威氏按蚊種群數(shù)量大,密度高,叮人率高,通宵均有吸血活動(dòng),偏吸牛血,兼吸人血,是該地區(qū)的優(yōu)勢(shì)蚊種,也是該地的主要傳瘧媒介;達(dá)羅毗按蚊、多斑按蚊與塞沃按蚊親緣關(guān)系最近,威氏按蚊,偽威氏按蚊的關(guān)系最遠(yuǎn);mtDNA-COI和mtDNA-Cytb均為適合于群體遺傳學(xué)分析的基因序列,偽威氏按蚊西藏、云南和緬甸各群體間基因交流頻繁,尚未發(fā)生群體遺傳分化。
[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.

【學(xué)位授予單位】：中國(guó)疾病預(yù)防控制中心
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2009
【分類號(hào)】：R181.3

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