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研究生:李瑩潔
研究生(外文):Ying-Chieh Lee
論文名稱:於高雄以中劍水蚤防治登革熱病媒蚊幼蟲之田間試驗
論文名稱(外文):Field trials using Mesocyclops spp. to control dengue mosquito larvae in Kaohsiung City
指導教授:蔡坤憲蔡坤憲引用關係
指導教授(外文):Kun-Hsien Tsai
口試委員:溫在弘
口試委員(外文):Tzai-Hung Wen
口試日期:2013-07-05
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:流行病學與預防醫學研究所
學門:醫藥衛生學門
學類:公共衛生學類
論文種類:學術論文
論文出版年:2013
畢業學年度:101
語文別:中文
論文頁數:253
中文關鍵詞:登革熱劍水蚤殺蟲劑中劍水蚤斑蚊幼蟲高雄積水地下室
外文關鍵詞:DenguecopepodMesocyclops spp.insecticideAedes larvaeflooded basementsKaohsiung
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登革熱全球發生率逐年升高,是一個重要的公共衛生議題。目前台灣病媒蚊控制策略為孳生源清除及疫情時緊急噴灑化學藥劑,然而長期密集使用化學藥劑,已使得蚊蟲產生嚴重抗藥性,因此尋求一個環境友善的登革控制策略是相當迫切需要的。從民國99年度以來,國立台灣大學公共衛生學院蟲媒傳染病實驗室已確認臺灣本土具有多種高捕食性橈足類中劍水蚤,本研究規劃將南臺灣採集到的劍水蚤實際運用於高雄的登革熱流行區,並評估其生物防治功效。首先,在埃及斑蚊分布的嘉義、台南、高雄、屏東四縣市進行劍水蚤之採集,採集樣點數分別為144、109、 179、262個,接著利用分子及型態鑑定進行物種分析,鑑定結果發現南台灣具中劍水蚤屬 (Mesocyclops spp.)、溫劍水蚤屬 (Thermocyclops spp.)、后劍水蚤屬 (Metacyclops spp.)、真劍水蚤屬 (Eucyclops spp.)、劍水蚤屬 (Cyclops spp.) 之劍水蚤,而中劍水蚤屬以北碚中劍水蚤 (M. pehpeiensis)、鄔氏中劍水蚤(M. woutersi)、奧貢中劍水蚤 (M. ogunnus) 及糙角中劍水蚤 (M. aspericornis) 最多。為了解現行殺蟲劑對於劍水蚤的影響,進行蘇力菌 (Bacillus thuringiensis israelensis,B.t.i.)、陶斯松 (Chlopyrifos)、美賜平 (Methoprene)、百滅寧 (Permethrin)、賽酚寧 (Cyphenothrin) 等五種市售殺蟲劑的感受性分析,發現斑蚊幼蟲對於殺蟲劑具高敏感性,但劍水蚤則較不受影響,比較五種殺蟲劑發現陶斯松毒殺性最高 (劍水蚤和埃及斑蚊幼蟲的半數致死濃度分別為LC50=6.2*10-4ppm和LC50=5.2*10-9ppm),對美賜平最不敏感 (劍水蚤和埃及斑蚊幼蟲的半數致死濃度分別為LC50=1.5*101 ppm和LC50=1.4*100ppm)。進一步評估劍水蚤做為生物防治可行性,在高雄積水地下室進行以劍水蚤防治登革熱病媒蚊幼蟲之田間試驗,透過研究計畫說明和取得高雄衛生局同意後,在高雄陽光家園完成三種高捕食性的中劍水蚤的大量培養,挑選單純施放中劍水蚤之列管積水地下室 (N=165),隨機取樣25%做為長期追蹤試驗點 (N=42),於試驗點以傾倒法施放中劍水蚤,定期採樣監測評估,追蹤一年多後,發現中劍水蚤可長期存活於大面積水質清澈之積水地下室,中劍水蚤成功繁殖率約45.3-60.0%,斑蚊幼蟲陽性率從65% (101年3月) 降至0 (102年7月)。綜合本研究成果發現,南台灣具有高捕食性劍水蚤物種,且劍水蚤對於殺蟲劑較不敏感,並具有顯著降低斑蚊幼蟲族群之登革熱防治功效。

The global incidence of dengue has significantly increased in recent years and has become an important issue in public health. The current vector control strategy in Taiwan is source reduction. Chemical pesticides are applied only when dengue case appears. Even so, the frequent use of pesticides over a long period of time has led to development of resistance in vector mosquitoes. Therefore, a vector control method with eco-friendly approach is in demand. In 2010, NTU-CPH laboratory has identified numerous species of Mesocyclops spp. with highly predating efficiency on mosquito larvae. In this study, we further applied these local species of copepods in dengue risk areas to evaluate their performance as agents for bio-control. To start with, we collected freshwater copepods in Southern Taiwan where indigenous dengue cases frequently occur, including Chiayi (sample number, N=144), Tainan (N=109), Kaohsiung (N=179), and Pingtung (N=262). Second, we identified the species of copepods by morphology and molecular analysis. The collected freshwater copepods were recognized as Mesocyclops spp., Thermocyclops spp., Metacyclops spp., Eucyclops spp. and Cyclops spp.. Third, we investigated the chemical pesticide tolerance of copepods, and the result showed that copepods were with lower sensitivity than mosquitoes when treated with the currently used insecticides. Next, to evaluate the potential and feasibility of using Taiwanese freshwater Mesocyclops spp. as a bio-control agent of dengue virus, we cooperated with the Department of Health, Kaohsiung City Government. Inform consents were signed by the local public health centers and residents before setting sentinel sites in flooded basements in Kaohsiung (N=165). 25% of the trial sites were chosen randomly (N=42) at the following step. Sites were excluded if they were dry or too difficult to access. Eventually, 24 trial sites were targeted for long-term surveillance. After more than one year of observation (from March 2012 to July 2013), our results showed that the positive rates of larvae have decreased from 65.0% to 0, and the survival rates of copepods were 45.3% to 60.0% in clean flooded basements. In conclusion, this study has proved that copepods were not only able to be maintained and survived in the flooded basements but have functioned on decreasing the number of Aedes larvae. Our findings suggested that Taiwanese freshwater Mesocyclops could be provided as a biological agent for dengue larvae control in Kaohsiung City.

目錄
第一章 緒論 1
第一節 前言 1
第二節 登革熱流行病學及病媒蚊 4
第三節 劍水蚤之基礎生物學 7
第二章 材料與方法 9
第一節 南臺灣劍水蚤之野外採集與飼養 9
第二節 蚊蟲飼養 11
第三節 南台灣本土劍水蚤物種鑑定 12
第四節 中劍水蚤對殺蟲藥劑之感受性分析 15
第五節 中劍水蚤田野試驗 16
第三章 結果 20
第一節 南臺灣劍水蚤野外採集 20
第二節 南台灣本土劍水蚤物種鑑定 22
第三節 中劍水蚤對殺蟲藥劑之感受性分析 24
第四節 中劍水蚤田野試驗 26
第四章 討論 29
第一節 南臺灣劍水蚤之野外採集與飼養 29
第二節 南台灣本土劍水蚤物種鑑定 31
第三節 中劍水蚤對殺蟲藥劑之感受性分析 32
第四節 中劍水蚤田野試驗 33
參考文獻 37
圖目錄
圖1. 中劍水蚤雌蟲抱卵圖 44
圖2. 中劍水蚤無節幼蟲期 45
圖3. 南臺灣劍水蚤採集地點定位Google Earth圖 46
圖4. 南臺灣四縣市 (嘉義、台南、高雄、屏東) 劍水蚤採集空間分布圖 47
圖5. 南臺灣劍水蚤生態棲地 48
圖6. 嘉義縣市劍水蚤採集空間分布圖 49
圖7. 台南市劍水蚤採集空間分布圖 50
圖8. 高雄市劍水蚤採集空間分布圖 51
圖9. 屏東縣市劍水蚤採集空間分布圖 52
圖10. 北碚中劍水蚤 (Mesocyclops pehpeiensis) 顯微特徵 53
圖11. 鄔氏中劍水蚤 (Mesocyclops woutersi) 顯微特徵 54
圖12. 奧貢中劍水蚤 (Mesocyclops ogunnus) 顯微特徵 55
圖13. 南臺灣採集之劍水蚤ITS-2基因之聚合酶鏈鎖反應 (PCR) 分析電泳膠片圖 56
圖14. 南臺灣四縣市野外採集之橈足類劍水蚤ITS-2基因親緣關係樹狀圖 57
圖15. 糙角中劍水蚤及四齡埃及斑蚊幼蟲對蘇力菌以色列變種 (B.t.i.)之感受性分析 58
圖16. 糙角中劍水蚤及四齡埃及斑蚊幼蟲對陶斯松 (Chlopyrifos) 殺蟲劑之感受性分析 59
圖17. 糙角中劍水蚤及四齡埃及斑蚊幼蟲對百滅寧 (Permethrin) 殺蟲劑之感受性分析 60
圖18. 糙角中劍水蚤及四齡埃及斑蚊幼蟲對賽酚寧 (Cyphenothrin) 殺蟲劑之感受性分析 61
圖19. 糙角中劍水蚤及四齡埃及斑蚊幼蟲對美賜平 (Methoprene) 殺蟲劑之感受性分析 62
圖20. 戶外大量培養中劍水蚤之培育桶水體pH值變化圖 63
圖21. 戶外大量培養中劍水蚤之培育桶水體溫度變化圖 64
圖22. 於高雄積水地下室進行中劍水蚤的田野試驗之實驗設計 65
圖23. 中劍水蚤田野試驗之四種高雄積水地下室類型 66
圖24. 第一型積水地下室: 混濁有機質豐富之窄池 67
圖25. 第二型積水地下室:水質清徹乾淨的小面積窄池 68
圖26. 第三型積水地下室:水質清徹乾淨的大面積輕度積水 69
圖27. 第四型積水地下室:水質清澈乾淨的大面積中度積水 70
圖28. 中劍水蚤田間試驗之斑蚊幼蟲陽性率和中劍水蚤成功繁殖率變化圖 71
圖29. 以噴灑法評估不同直徑大小噴孔對糙角中劍水蚤存活之影響 72


表目錄
表1. 台灣本土劍水蚤種源資料庫,以高雄杉林一採樣點為例 73
表2. 南臺灣四縣市之其鄉鎮區劍水蚤採集樣點數 74
表3. 南臺灣四縣市劍水蚤和蚊蟲採樣情形 75
表4. 利用多變項邏輯斯迴歸分析縣市別及有無劍水蚤(自變項) 對於蚊幼蟲孳生 (依變項) 的關係 76
表5. 南臺灣野外採集之劍水蚤型態鑑定結果 77
表6. 比較中劍水蚤和四齡埃及斑蚊幼蟲對五種殺蟲劑之感受性 79
表7. 於高雄陽光家園戶外大量培養中劍水蚤之培育桶水體溫度及水體溫度 80
表8. 進行生物防治中劍水蚤施放前,高雄市積水地下室劍水蚤普查結果 81
表9. 以直徑3mm噴孔進行噴灑法實驗,計算噴出之糙角中劍水蚤隻數及存活率 82
表10. 中劍水蚤成功繁殖試驗點數 83
表11. 各月份中劍水蚤於各類型積水地下室之成功繁殖率 84
表12. 於高雄積水地下室比較中劍水蚤施放前 (101年03月) 與施放後 (102年07月) 之斑蚊幼蟲孳生情形 85

附錄 目錄
附錄1. 西太平洋地區登革熱病媒蚊控制策略 86
附錄2. 用於防治登革熱病媒蚊的劍水蚤物種 88
附錄3. 實驗室劍水蚤培養過程及使用容器一覽 89
附錄4. 五種試驗用市售殺蟲劑之商品基礎資料 90
附錄5. 五種試驗用市售殺蟲劑之參考半數致死濃度 91
附錄6. 劍水蚤分子鑑定用之ITS2基因和分子選殖載體之引子對
序列 92
附錄7. 建立親緣關係樹狀圖之參考採用ITS2基因序列 93
附錄8. 高雄衛生局同意進行劍水蚤田間試驗之公文 94
附錄9. 大量培養中劍水蚤前準備之工作照片 95
附錄10. 各劍水蚤基因片段 96
附錄11. 其他物種 (真菌或細菌) 之基因片段 138
附錄12. 台灣本土劍水蚤種源資料庫 145


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