臺灣博碩士論文加值系統

以亞培松（Temephos）、陶斯松（Chlorpyrifos）、撲滅松（Fenitrothion）、亞特松（Pirimifosmethyl）等四種有機磷殺蟲劑，及必列寧（Pyrethrins）、百滅寧（Permethrin）、芬化利（Fenvalerate）、賽滅寧（Cypermethrin）四種合成除蟲菊類殺蟲劑進行埃及斑蚊幼蟲之生物檢測。測試各供試藥劑對對敏感品系、台南地區及高雄地區埃及斑蚊的半數致死濃度(LC50)，並計算抗性比值。結果顯示，台南地區及高雄地區埃及斑蚊幼蟲對有機磷殺蟲劑尚未產生抗藥性的情形，有機磷殺蟲劑對野外的埃及斑蚊幼蟲仍具有撲殺的效果。除關廟品系外，台南地區及高雄地區埃及斑蚊幼蟲對四種除蟲菊類殺蟲劑之抗性比值皆大於10，顯示台南地區及高雄地區的埃及斑蚊已對除蟲菊類殺蟲劑包括必列寧、百滅寧、賽滅寧及芬化利產生抗藥性。
在多濃度安丹抑制實驗中，97台南東區品系與高雄苓雅品系的乙醯膽鹼酯酶抑制曲線的變化，都與敏感品系相同，因此推論台灣地區埃及斑蚊的乙醯膽鹼酯酶並未產生”質”的突變，此結果與生物檢測結果相符。
在解毒酵素分析結果，高雄地區埃及斑蚊四齡幼蟲在單氧酶、酯酶及麩胺基硫轉移酶等三類解毒酵素的表現量都較敏感品系高，但是台南地區埃及斑蚊四齡幼蟲則與高雄地區品系有不同的結果，可能兩個地區對殺蟲劑的抗藥機制並不相同。
以百滅寧的亞致死劑量進行埃及斑蚊幼蟲解毒酵素誘導試驗，在台南市東區埃及斑蚊幼蟲的解毒酵素活性增加情形與接觸殺蟲劑的時間長短有關，並且與敏感品系間具有顯著性的差異。但是其他品系的實驗結果，缺乏一致性，短時間暴露殺蟲劑對不同地區品系埃及斑蚊幼蟲的影響並不一致，是否與各地區用藥習慣的不同或各地區抗藥機制差異有關，尚待進一步釐清及探討。

Field collected Aedes aegypti from each part of Tainan and Kaohsiung districts were subjected to bioassay for their susceptibilities to Temephos, Chlorpyrifos, Fenitrothion, Pirimifosmethyl, Pyrethrins, Permethrin, Fenvalerate, Cypermethrin and compared with two susceptible laboratory strains (NS strain and Bora Bora strain) by using dipping method. The resistance ratio between the 50% lethal concentration value (RR) of the field strains and the NS strain shows that the field strain were widespread, significant resistance to pyrethroid insecticides but not resistance to organophosphorus insecticides.
In TPP test (insecticide-insensitive acetylcholinesterase test), the AChE residual activities of field strain compared with susceptible strain were the same in the presence of increasing concentration of propoxur. The TPP test indicate that the point mutation of acetylcholinesterase of Aedes aegypti was not found in the southern of Taiwan.
Microplate assays were performed to measure levels of α-esterase, β-esterase, glutathione-S-transferase, monooxygenase enzymes. The patterns of elevate levels of detoxification enzymes in Kaohsiung districts and the Tainan districts strains were different. Comparison between bioassays and biochemical assays, the higher levels glutathione-S-transferase activity was significant correlation with the 50% lethal concentration value of pyrethroid insecticides.
The effect of induction detoxification enzymes activity by exposure of Aedes aegypti larvae to sub-lethal dose of permethrin was investigated. The induction detoxification enzymes activity by permethrin in the 97 Tainan East district strain were significant higher than NS strain. The induction effect was no consistency between the experimental field strains in the 1 hr, sub-lethal dose exposure treatment. The mechanisms of insecticide resistance need to be clarified and explored further.

中文摘要 I
英文摘要 III
本文目錄 V
圖 目 錄 VIII
表 目 錄 IX
縮寫表 XI
第一章 緒論 1
1.1埃及斑蚊與登革熱 1
1.2埃及斑蚊 4
1.3 防治 6
1.4昆蟲抗藥性與酵素 8
1.5 誘導 9
1.6 研究動機 11
第二章 材料方法 12
2.1 蚊子品系與飼養 12
2.2 材料 12
2.2.1殺蟲劑 12
2.2.2試藥 13
2.2.3儀器 13
2.3 殺蟲劑對埃及斑蚊幼蟲藥效之生物檢定 13
2.4 埃及斑蚊幼蟲酵素分析 14
2.4.1 酵素研磨液製備 15
2.4.2乙醯膽鹼酯酶活性分析 15
2.4.3解毒酵素活性分析 16
2.4.4 總蛋白分析 17
2.5 以百滅寧的亞致死劑量進行埃及斑蚊幼蟲解毒酵素誘導試驗 18
2.6 分子鑑定 18
2.6.1 埃及斑蚊幼蟲總RNA萃取 18
2.6.2 RNA電泳 19
2.6.3 RNA定量 19
2.6.4 RT-PCR 20
2.7 統計分析 21
第三章 結果 22
3.1. 埃及斑蚊幼蟲對殺蟲劑之感受性 22
3.2.埃及斑蚊幼蟲之酵素檢測 23
3.2.1乙醯膽鹼酯酶 23
3.2.2解毒酵素 24
3.3. 以百滅寧的亞致死劑量進行埃及斑蚊幼蟲解毒酵素誘導試驗 27
3.4. 分子鑑定 29
3.4.1抽取埃及斑蚊幼蟲RNA 29
3.4.2 RT-PCR 29
第四章 討論 31
4.1. 台灣南部地區埃及斑蚊幼蟲之藥效檢測結果 31
4.2. 台灣地區埃及斑蚊解毒酵素與抗藥性 33
4.3. 百滅寧誘導對埃及斑蚊的影響 37
第五章 結論 40
參考文獻 41
圖表 47
附錄 76

1.WHO. Dengue and dengue haemorrhagic fever. Fact sheet NO 117. 2009.
2.行政院衛生署疾病管制局。 Guidelines for dengue control. 防疫學苑。 2008。
3.CDC. 病例定義。 2009; http://www.cdc.gov.tw/file/疾病介紹/法定傳染病/第二類傳染病/登革熱/滅登練功房.files/2病例定義.pdf.
4.Gubler DJ. Dengue and Dengue Hemorrhagic Fever. Clin Microbiol Rev. 1998:480–496.
5.行政院衛生署疾病管制局。 行政院衛生署疾病管制局九十五年度科技研究發展計畫。 2006。
6.行政院衛生署疾病管制局。 台灣地區登革熱病媒蚊抗藥性現況。 登革熱防治工作檢討會。 2008。
7.Halstead SB. Pathogenesis of dengue: challenges to molecular biology. Science. 1988;239(4839):476-481.
8.Thompson M, Shotkoski F, ffrench-Constant R. Cloning and sequencing of the cyclodiene insecticide resistance gene from the yellow fever mosquito Aedes aegypti. Conservation of the gene and resistance associated mutation with Drosophila. FEBS Lett. 1993;325(3):187-190.
9.Jones JC, Madhukar BV. Effect of weight on mosquito (Aedes aegypti L.) feeding. Experientia. 1974;30(11):1255.
10.Craig GB Jr, Hickey WA. Current status of the formal genetics of Aedes aegypti. Bull World Health Organ. 1967;36(4):559-562.
11.Strode C, Wondji CS, David JP, et al. Genomic analysis of detoxification genes in the mosquito Aedes aegypti. Insect Biochem Mol Biol. 2008;38(1):113-123.
12.Ooi EE, Goh KT, Gubler DJ. Dengue prevention and 35 years of vector control in Singapore. Emerg Infect Dis. 2006;12(6):887-893.
13.謝維銓, 陳明豐, 林桂堂, 陵悀M, 馬肇義, 吳世勳。 1981 年在屏東縣琉球鄉流行的登革熱之研究。 台灣醫誌。 1982;81:1388-1395。
14.WHO. Environmental Health Criteria 9: DDT and its derivatives. 1979.
15.Lear L. Rachel Carson: Witness for Nature.: New York: Henry Hoyten.; 1997.
16.The Bald Eagle - An American Emblem. 1999; http://www.baldeagleinfo.com/eagle/eagle9.html.
17.徐爾烈。 登革熱病媒蚊對合成除蟲菊酯抗藥性之研究（Ⅱ）。 行政院衛生署科技研究發展計畫。 1996。
18.Rodriguez MM, Bisset JA, Mastrapa L, Diaz C. The association of resistance to organophosphate, carbamate and pyrethroid insecticides with the mechanisms of resistance observed in Culex quinquefasciatus strains from Ciudad de La Habana province. Rev Cubana Med Trop. 1995;47(3):154-160.
19.Fox I. Malathion resistance in Aedes aegypti of Puerto Rico induced by selection pressure on larvae. Am J Trop Med Hyg. 1980;29(6):1456-1459.
20.陳幼雪。 埃及斑蚊對藥劑之感受性試驗及登革熱病媒指數與實際斑蚊密度之關係。 2003。
21.Lines JD, Myamba J, Curtis CF. Experimental hut trials of permethrin-impregnated mosquito nets and eave curtains against malaria vectors in Tanzania. Med Vet Entomol. 1987;1(1):37-51.
22.Mathenge EM, Gimnig JE, Kolczak M, Ombok M, Irungu LW, Hawley WA. Effect of permethrin-impregnated nets on exiting behavior, blood feeding success, and time of feeding of malaria mosquitoes (Diptera: Culicidae) in western Kenya. J Med Entomol. 2001;38(4):531-536.
23.Hemingway J, Hawkes NJ, McCarroll L, Ranson H. The molecular basis of insecticide resistance in mosquitoes. Insect Biochem Mol Biol. 2004;34(7):653-665.
24.Alout H, Berthomieu A, Hadjivassilis A, Weill M. A new amino-acid substitution in acetylcholinesterase 1 confers insecticide resistance to Culex pipiens mosquitoes from Cyprus. Insect Biochem Mol Biol. 2007;37(1):41-47.
25.Vulule JM, Beach RF, Atieli FK, et al. Elevated oxidase and esterase levels associated with permethrin tolerance in Anopheles gambiae from Kenyan villages using permethrin-impregnated nets. Med Vet Entomol. 1999;13(3):239-244.
26.Brogdon WG, McAllister JC, Corwin AM, Cordon-Rosales C. Independent selection of multiple mechanisms for pyrethroid resistance in Guatemalan Anopheles albimanus (Diptera: Culicidae). J Econ Entomol. 1999;92(2):298-302.
27.楊藹華。 漫談生物技術在作物抗蟲之應用。 台南區農業專訊。 2000;32。
28.Yu SJ. Induction of detoxification enzymes by triazine herbicides in the fall armyworm, Spodoptera frugiperda. Pestic Biochem Physiol. 2004;80:113-122.
29.Willoughby L, Chung H, Lumb C, Robin C, Batterham P, Daborn PJ. A comparison of Drosophila melanogaster detoxification gene induction responses for six insecticides, caffeine and phenobarbital. Insect Biochem Mol Biol. 2006;36(12):934-942.
30.Willoughby L, Batterham P, Daborn PJ. Piperonyl butoxide induces the expression of cytochrome P450 and glutathione S-transferase genes in Drosophila melanogaster. Pest Manag Sci. 2007;63(8):803-808.
31.Vontas J, Blass C, Koutsos AC, et al. Gene expression in insecticide resistant and susceptible Anopheles gambiae strains constitutively or after insecticide exposure. Insect Mol Biol. 2005;14(5):509-521.
32.Suwanchaichinda C, Brattsten LB. Induction of microsomal cytochrome P450s by tire-leachate compounds, habitat components of Aedes albopictus mosquito larvae. Arch Insect Biochem Physiol. 2002;49(2):71-79.
33.Poupardin R, Reynaud S, Strode C, Ranson H, Vontas J, David JP. Cross-induction of detoxification genes by environmental xenobiotics and insecticides in the mosquito Aedes aegypti: impact on larval tolerance to chemical insecticides. Insect Biochem Mol Biol. 2008;38(5):540-551.
34.WHO. Instructions for determing the susceptibility of resistance of mosquito larvae to insecticides. World Health Organ Tech Rep Ser. 1981.
35.Finney D. Probit Analysis. Cambridge University Press. 1971.
36.Hemingway J. Techniques to detect insecticide resistance mechanisms : field and laboratory manual. World Health Organ Tech Rep Ser. 1998.
37.Bourguet D, Pasteur N, Bisset J, Raymond M. Determination of Ace.1 Genotypes in Single Mosquitoes: Toward an Ecumenical Biochemical Test. Pestic Biochem Physiol. 1996;55(2):122-128.
38.白秀華。 您用過殺蟲劑嗎？。 2001; http://www.kmuh.org.tw/www/kmcj/data/9003/4671.htm。
39.Hougard JM, Zaim SD, Guillet P. Bifenthrin: a useful pyrethroid insecticide for treatment of mosquito nets. J Med Entomol. 2002;39(3):526-533.
40.Berge JB, Chevillon C, Raymond M, Pasteur N. Resistance of insects to insecticides. Molecular mechanisms and epidemiology. C R Seances Soc Biol Fil. 1996;190(4):445-454.
41.Bardin PG, van Eeden SF, Moolman JA, Foden AP, Joubert JR. Organophosphate and carbamate poisoning. Arch Intern Med. 1994;154(13):1433-1441.
42.Nene V, Wortman JR, Lawson D, et al. Genome sequence of Aedes aegypti, a major arbovirus vector. Science. 2007;316(5832):1718-1723.
43.Bai H, Ramaseshadri P, Palli SR. Identification and characterization of juvenile hormone esterase gene from the yellow fever mosquito, Aedes aegypti. Insect Biochem Mol Biol. 2007;37(8):829-837.