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研究生:林鶯熹
研究生(外文):Ying-Hsi Lin
論文名稱:台灣埃及斑蚊對合成除蟲菊酯殺蟲劑的抗藥性
論文名稱(外文):Insecticide Resistance to Pyrethroid in Aedes aegypti
指導教授:徐爾烈徐爾烈引用關係
指導教授(外文):Err-Lien Hsu
學位類別:博士
校院名稱:國立臺灣大學
系所名稱:昆蟲學研究所
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:中文
論文頁數:101
中文關鍵詞:殺蟲劑抗藥性埃及斑蚊酯酶氧化酶P450基因
外文關鍵詞:Aedes aegyptimicrosomal monooxygenaseinsecticide resistanceesteraseP450 gene
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高雄市前鎮區、苓雅區(2002)和持續以百滅寧篩選的苓雅篩藥(1990R)品系埃及斑蚊與感性品系的抗性比,成蟲皆大於312.5,於幼蟲則分為20.8、37.5和102.1,故野外品系的蚊蟲抗藥性仍有提升之空間。以WHO的合成除蟲菊酯殺蟲劑藥膜測試2002年台南和高雄埃及斑蚊(Aedes aegypti)成蟲的半數擊昏時間(KT50)和24小時死亡率。高雄巿前鎮區和苓雅區(2002)品系對賽飛寧具抗藥性。高雄巿前鎮區和苓雅區(2002)品系對第滅寧具抗藥性。高雄巿前鎮區、苓雅篩藥(1990R)、苓雅區(2002)和新興區,高雄縣林園鄉和鳳山巿品系對依芬寧具抗藥性。高雄巿前鎮區、苓雅區(2002)、新興區、三民區和左營區,高雄縣林園鄉、岡山鎮和鳳山巿,台南縣歸仁鄉、新巿鄉,和台南巿南區、西區和中區品系對百滅寧產生抗藥性。而高雄巿三民區、左營區和高雄縣岡山鎮品系對賽洛寧具抗藥性。本研究推測埃及斑蚊對百滅寧的抗藥性為2002年南台灣登革熱疫情難以控制的原因之一。2003年高雄市前鎮區、苓雅區和高雄縣鳳山市品系埃及斑蚊對依芬寧和百滅寧仍具抗藥性,但前鎮區和苓雅區對於賽飛寧和第滅寧的抗藥性降低。台南市南區埃及斑蚊對百滅寧和賽洛寧感藥性皆低,為抗藥性較嚴重地區。台南縣歸仁鄉埃及斑蚊對百滅寧和賽洛寧具抗藥性。協力劑測試則顯示埃及斑蚊幼蟲對百滅寧的抗性與氧化�@和酯�@相關性較高。
分別以α-naphthyl acetate和β-naphthyl acetate為受質時,成蟲的酯�@活性最高者皆為前鎮區品系,分為0.249�b0.011和0.248�b0.009 m mole/min/mg protein。幼蟲的酯�@活性不論以α-naphthyl acetate或β-naphthyl acetate為受質時,皆未顯示與抗藥性相關。以α-naphthyl acetate或β-naphthyl acetate為受質染色埃及斑蚊的酯�@電泳結果相近;成蟲的酯�@band數目比幼蟲少,野外品系和感性品系間成蟲的酯�@band並無明顯差異。苓雅篩藥(1990R, F31)品系幼蟲的細胞色素P450含量最高,為276.9�b23.0 p mole/mg protein。苓雅篩藥(1990R)品系成蟲的細胞色素b5含量最高,為22.043�b13.854 pmole/mg protein。百滅寧代謝�@作用中成蟲和幼蟲的微粒體皆可使百滅寧的量減少,顯然埃及斑蚊成蟲和幼蟲的微粒體中含可代謝百滅寧的成分,皆以苓雅區(2002)品系的百滅寧減少率最大,分為89.047�b85.794%和58.125�b6.500 %。將感性品系和抗性品系分別配單對,連續3代後獲得一感性品系(N2-8-4)和三個抗性品系(L12-10-4、L12-10-25和L12-10-18)。抗性品系(L12-10-4、L12-10-25和L12-10-18) 與感性品系(N2-8-4)的抗性比分別為27.0、46.7和64.7。感性品系的酯�@帶只有band 2,而抗性品系則只有band 3,或由band 2和band 3分別組成。不論以α-naphthyl acetate或β-naphthyl acetate為受質,感性品系的酯�@活性皆低於三個抗性品系。以paraoxon抑制感性品系和抗性品系埃及斑蚊成蟲的酯�@後仍有部分的酯�@活性存在。感性品系之處女雌蚊皆會吸血,約45%處女雌蚊吸血未產卵,平均產卵量為104.6�b9.1個卵,其單隻處女雌蚊最高產卵量為169個卵。抗性品系之處女雌蚊亦皆會吸血,約43%處女雌蚊吸血未產卵,平均產卵量為88.5�b5.8個卵,其單隻處女雌蚊最高產卵量為120個卵。未交尾雌蚊所產的卵不孵化,且在乾燥後皆呈現乾扁狀態,置入水中呈縱裂。感性品系�e感性品系,只有3.5%雌蚊吸血後未產卵,平均產卵量為125.4�b9.9個卵,其單隻雌蚊最高產卵量為175個卵,所產的卵之孵化率為64.3�b15.7%。抗性品系�e抗性品系,40.7%雌蚊吸血後未產卵,平均產卵量為86.1�b39.7個卵。單隻雌蚊最高產卵量為143個卵,所產的卵之孵化率為41.4�b37.5%。感性品系雌蚊�e抗性品系雄蚊,17.9%雌蚊吸血後未產卵,平均產卵量為117.4�b20.1個卵,其單隻雌蚊最高產卵量為182個卵,所產的卵之孵化率為85.2�b14.2%。感性品系雄蚊�e抗性品系雌蚊,約23.1%雌蚊吸血後未產卵,平均產卵量為86.4�b10.5個卵,其單隻雌蚊最高產卵量為149個卵,所產的卵之孵化率為70.1�b26.7%。由感性品系雌蚊�e抗性品系雄蚊的後代數量,較感性品系雄蚊�e抗性品系雌蚊的後代數多。利用EIA Reader和微量盤測定感性品系(NS)單隻雌成蚊酯�@活性平均為0.22�b0.14μmole/min/mg protein,酯�@活性的頻率分佈於0.03-0.88μmole/min/mg protein,其中以0.1-0.19μmole/min/mg protein的個體數最多,佔50.57%。苓雅篩藥(1990R) (F29) 單隻雌成蚊酯�@活性平均為0.26�b0.03μmole/min/mg protein,酯�@活性的頻率分佈集中於0.21-0.35μmole/min/mg protein,以0.2-0.29μmole/min/mg protein的個體數最多,佔87.4%。單對飼養所得到的族群中,N2-8-4族群單隻雌成蚊酯�@活性平均為0.26�b0.08μmole/min/mg protein,酯�@活性的頻率分佈於0.17-0.68μmole/min/mg protein,其中以0.2-0.29μ mole/min/mg protein的個體數最多,佔70.3%。L12-10-4族群單隻雌成蚊酯�@活性平均為0.22�b0.03μmole/min/mg protein,其酯�@活性的頻率分佈集中於0.18-0.32μmole/min/mg protein,以0.2-0.29μmole/min/mg protein的個體數最多,佔84.2%。在L12-10-25族群單隻雌成蚊酯�@活性平均為0.22�b0.02μmole/min/mg protein,其酯�@活性的頻率分佈集中於0.18-0.30μmole/min/mg protein,以0.2-0.29μmole/min/mg protein的個體數最多,佔92.6%。L12-10-18族群單隻雌成蚊酯�@活性平均為0.23�b0.05μmole/min/mg protein,其酯�@活性的頻率分佈集中於0.17-0.33μmole/min/mg protein,以0.2-0.29μmole/min/mg protein的個體數最多,佔55.6%,而23.3%為0.1-0.19μmole/min/mg protein。於埃及斑蚊細胞色素P450基因選殖中,RT-PCR於約250bp處可獲得一P450基因片段,與熱帶家蚊抗百滅寧相關基因CYP6F1部分片段具雜合作用,選殖出六個序列,一個為249bp(W103),五個(w102、W105、W108、W111和W112)為231bp。W103與另外五個序列相似性較低,為63.1-66.2%,但與已知雙翅目氨基酸序列相似性為六個序列中較高者,為45.1-59.0%,其中與矮小瘧蚊之CYP6B5的相似性最高,為59.0%。Semi-quantitative RT-PCR analysis共獲得6個片段,分為1.4kb、750bp、600bp、350bp、300bp和250bp。其中300bp片段DNA量在百滅寧抗性品系中比感性品系高,可能與抗藥性有關,但選殖該片段並非P450 gene。另250bp即為W103片段。由W103片段設計專一性primer,與oligo-dT執行3’RACE,得到700bp、600bp、500bp、450bp、380bp、300bp和100bp。其中在約450-600bp片段之間與W103合成的probe具雜合作用。選殖600bp後並無colony hybridization。而直接以不同片段的萃取液直接滴加作雜合作用,則以500bp的雜合較強,可能500bp為埃及斑蚊幼蟲體內與P450相關的W103片段之3’端。


The resistance ratios of adult mosquitoes of the Chianjen, Lingya (2002), and Lingya (1900R) strains for permethrin compared to susceptible strains were above 312.5X, while those of the 4th instar larvae were 20.8, 37.5, and 102.1, respectively. We used WHO insecticide-impregnated papers to determine the insecticide susceptibility of adult Aedes aegypti in 2002.. The Chianjen and Lingya (2002) strains showed resistance to cyfluthrin. The Chianjen, Lingya (2002), and Shinshing strains showed resistance to deltamethrin. The Chianjen, Lingya (1990R), Lingya (2002), Shinshing, Linyuan and Fengshan strains showed resistance to etofenprox. The Chianjen, Lingya (2002), Shinshing, Sanmin, Tzuoying, Linyuan, Gangshan, Fengshan, Gueiren, Shinshi, S. District, W. District, and C. District strains showed resistance to permethrin. The Sanmin, Tzuoying, and Gangshan strains showed resistance to lambdacyhalothrin. 2003, the Chianjen, Linya and Fengshan strains of Aedes aegypti were resistant to both etofenprox and permethrin, but the Chianjen and Lingya (2002) reduced resistance to cyfluthrin and deltamethrin. The Tainan South District strain of Aedes aegypti showed the resistant to permethrin and cyhalothrin. The Tainan Gueiren strain of Aedes aegypti showed the resistant to permethrin and cyhalothrin. Results of a synergism study on larvae resistant to permethrin showed correlations to the activities of microsomal monooxygenases and esterases.
The highest specific activities of esterase are 0.249�b0.011 and 0.248�b0.009 m mole/min/mg protein in Chianjen strain adults with ?naphthyl acetate andβ-naphthyl acetate, respectively. The specific activities of esterase in larvae are not related with resistance when using ?naphthyl acetate or β-naphthyl acetate as substrate.
We cloning of cytochrome P450 from Ae. aegypti resistant to permethrin, and amplified a partial cDNA fragment about 250bp fragment.in RT-PCR. Southern hybridization using a CYP6F1 cDNA fragment as a probe showed a signal. We got six partial P450 sequence, one was 249bp and the other five were 231bp. W103 was low similarity with the others from 63.1% to 66.2%. W103 was higher similarities with the sequences of dipterean that has known than the other five sequences, 45.1-59.0%. The highest similarity with CYP6P5 from Anopheles minimus, 59.0%. There were 6 bands using semi-quantitative RT-PCR analysis, 1.4kb, 750bp, 600bp, 350bp, 300bp, and 250bp. 300bp fragment transcript was greater in the permethrin-resistant Ae. aegypti compared to the susceptible strain.


目錄.................................. 1
表次.................................. 2
圖次.................................. 3
壹、前言.................................. 5
貳、前人研究.................................. 8
參、材料方法............................. 17
肆、結果............................... 32
伍、討論............................... 43
陸、中文摘要..................................
54
柒、英文摘要.................................. 57
捌、參考文獻.................................. 59
玖、誌謝.................................. 70
拾、表.................................. 71
拾壹、圖.................................. 87


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