臺灣博碩士論文加值系統

埃及斑蚊 (Aedes aegypti)為傳播重要蚊媒病毒感染症 (Mosquito-borne viral infection)登革熱 (dengue fever)與茲卡病毒感染症 (zika virus infection)的主要媒介，目前仍缺乏有效的登革熱及茲卡病毒感染症疫苗與處方藥劑，因此病媒蚊數量控制仍為防治疫情擴散的主要方式。目前埃及斑蚊的防治仍舊以化學藥劑防治為主，而持續的施用化學藥劑防治極易促使埃及斑蚊產生抗藥性，甚至於交互抗性，使原本數量極其有限的防治藥劑更顯侷促。因此本研究的首要目的是以抗百滅寧 (permethrin)的Per-R及抗安丹 (propoxur)的Prop-R品系埃及斑蚊為例子，探究此二品系埃及斑蚊是否對防治埃及斑蚊常用的藥劑產生交互抗性；其次利用次世代定序 (next generation sequencing)與比較轉錄體分析 (comparative transcriptome analysis)，更全面的了解埃及斑蚊的抗藥性機制，以利抗藥性抑制劑的研究。
從抗百滅寧Per-R與抗安丹Prop-R品系埃及斑蚊的幼蟲及成蟲藥劑試驗得知：(1) Per-R品系幼蟲對合成除蟲菊酯類藥劑的抗藥性皆高於800倍，但是對於安丹、陶斯松與撲滅松等乙醯膽鹼酯酶抑制劑的抗藥性則低於10倍。(2) Per-R品系雌成蟲對合成除蟲菊酯類藥劑的抗擊昏倍率較幼蟲抗藥性低，介於2.9-32倍，對安丹卻只有3.2倍的抗擊昏倍率。(3) 源自於Per-R的Prop-R品系，經過安丹約30代的篩選，雖然幼蟲只對安丹產生6.8倍的抗藥性，雌成蟲也只對安丹產生6.6倍的抗擊昏能力，但是對合成除蟲菊酯類的藥劑仍保留一定程度的幼蟲抗藥性 (21-242倍)與雌成蟲抗擊昏能力(2-27倍)。簡言之，以百滅寧篩選的Per-R品系幼蟲對其他合成除蟲菊有極高的交互抗性 (cross resistance)，對於安丹等乙醯膽鹼酯酶抑制劑的交互抗性卻很有限，但是雌成蟲對其他合成除蟲菊與安丹卻有不同程度的交互抗性，其中對賽扶寧的交互抗擊昏作用最低。另外，Per-R幼蟲抗藥程度遠高於雌成蟲的抗擊昏能力意味著，除了作用部位不敏感之外，尚有其他抗藥性機制參與其中。從NS感性品系與Per-R、Prop-R抗性品系的比較轉錄體分析 (comparative transcriptome analysis) 發現多個可能與表皮穿透能力降低、生物轉化 (biotransformation)作用增強、排泄能力增強相關的基因參與其中，例如：表皮蛋白 (cuticular protein)、細胞色素P450 (cytochrome P450, CYP)、穀胱甘肽轉移酶 (glutathione S-transferase)、酯酶 (esterase)、葡萄糖醛酸轉移酶 (UDP-glucuronosyltransferase)、ABC運輸蛋白 (ATP-binding cassette transporter)與溶質載體 (solute carrier)等基因。在這些基因產物中，表皮蛋白在Per-R與Prop-R品系埃及斑蚊幼蟲的表現量高低呈現相反趨勢，在Per-R及Prop-R品系幼蟲與雌成蟲發現過量表現的CYP多屬CYP6及CYP9。雖然大量溶質載體可能參與Per-R與Prop-R埃及斑蚊對百滅寧或安丹的抗藥性，但相關角色功能於昆蟲尚不清楚。

Aedes aegypti is the main vector that transmits the viruses for dengue fever and zika virus infection. Since there is no effective vaccine and prescription for dengue virus and zika virus, the management of vector population is still the primary way to prevent the epidemic of dengue fever and zika fever, and chemical control is the most efficient and frequent used method for vector control. However, long-term and frequent use of chemical control has speeded up the evolution of insecticide resistance and cross-resistance, which will impact the efficacy of vector management. The purpose of this study is using permethrin-resistant (Per-R) and propoxur-resistant (Prop-R) of A. aegypti as examples to study the cross-resistance of Per-R and Prop-R A. aegypti to insecticides used frequently for vector control and comparative transcriptome analysis using next generation sequence to understand more about the permethrin-resistant and propoxur-resistant mechanisms in A. aegypti.
According to the bioassay of Per-R and Prop-R larval and adult A. aegypti, three main results were observed. (1) The resistant ratios of Per-R larvae to pyrethroids were higher than 800-fold, but lower than 10-fold to propoxur, chlorpyrifos and fenitrothion. (2) The knockdown-resistant ratios of Per-R adult female to pyrethroids were much lower, 2.9-32 fold, than resistance ratios of larvae, but there was 3.2-fold knockdown resistance to propoxur. (3) Prop-R strain, which was derived from Per-R strain and has been selected by propoxur for about 30 generations, developed, only 6.8-fold resistance in larvae while 6.6-fold knockdown resistance in adult female to propoxur. Furthermore, Prop-R strain still retained certain pyrethroid resistance in larvae (21-242 fold) and knockdown resistance in adult female (2-27 fold). In briefly, Per-R larvae have highly cross-resistance to other pyrethroids and limited cross-resistance to acetylcholinesterase inhibitors, e.g. propoxur. However, adult female have different levels of cross-resistance to other pyrethroids and propoxur, with the lowest cross-resistance to cyfluthrin. The fact that resistance ratios of Per-R larvae to various pyrethroids were much higher than knockdown-resistant ratios of adult female indicates there are other mechanisms involved in permethrin resistance in addition to target site insensitivity. Based on the comparative transcriptome analysis of NS, Per-R and Prop-R A. aegypti, a number of genes were found to be associated with decrease of cuticle penetration, increase of biotransformation and excretion, such as cuticular protein, cytochrome P450 (CYP), glutathione S-transferase, UDP-glucuronosyltransferase, esterase, ATP-binding cassette transporter and solute carrier. The expression of cuticular protein gene in larvae of Per-R and Prop-R A. aegypti showed the opposite trend. In addition, the over expressed CYPs found in the Per-R and Prop-R larvae and adult female are mostly CYP6 and CYP9. Although solute carrier genes found in Per-R and Prop-R may be involved in the resistance of permethrin and propoxur, their roles in insect are not clear.

目次

中文摘要 i
英文摘要 ii
目次 iv
表目次 vii
圖目次 viii
附錄目次 ix
壹、 前言 1
貳、 前人研究 3
一、 埃及斑蚊 3
二、 埃及斑蚊傳播的主要疾病介紹 4
(一) 登革熱 4
1. 登革病毒 4
2. 登革病毒的複製與感染方式 4
3. 登革熱臨床症狀與指標轉變 4
4. 登革熱的疫情變化 6
5. 登革病毒的預防與治療發展現況 6
(二) 茲卡病毒感染症 8
1. 茲卡病毒 8
2. 茲卡感染症的臨床症狀 8
3. 茲卡病毒感染症疫情變化 8
4. 茲卡病毒的預防與治療發展現況 9
三、 埃及斑蚊防治手段 10
(一) 物理防治 10
(二) 生物性防治 10
(三) 生物轉殖技術防治 11
(四) 化學防治 12
四、抗藥性 13
五、抗藥性機制 14
(一) 表皮的阻隔 14
(二) 生物轉化作用與排泄作用 15
(三) 作用部位不敏感 16
六、轉錄體分析 16
參、 材料與方法 18
一、 供試埃及斑蚊品系簡介 18
二、 埃及斑蚊飼養 18
三、 使用藥劑 18
四、 藥膜製備 19
五、 藥劑試驗 19
(一) 埃及斑蚊幼蟲基礎感受性試驗 19
(二) 埃及斑蚊成蟲擊暈試驗 19
六、 抗性品系篩選 20
七、 總量RNA萃取 20
八、 RNA濃度測定 21
九、 DNase處理及RNA保存 21
十、轉錄組De novo分析 21
(一) RNA定序 21
(二) 定序數據篩選 22
(三) De novo組裝 22
(四) 功能註釋 23
(五) Unigene的編碼序列預测 23
(六) Unigene表現量計算 23
(七) 差異表達基因檢測 24
肆、 結果 25
一、 埃及斑蚊抗藥性品系篩選 25
二、 Per-R與Prop-R品系幼蟲對常用防治藥劑的交互抗性測試 25
三、 Per-R與Prop-R品系成蟲對常用防治藥劑的交互抗擊昏測試 25
四、 次世代定序資料分析 26
(一) 差異表達基因檢測 26
(二) Nt與KEGG代謝途徑(pathway)的比對註釋分析 27
伍、 討論 28
一、 Per-R與Prop-R對常用防治藥劑的感受性 28
二、 抗藥性基因的DEG數目與表現量分析 29
三、 次世代定序技術對昆蟲抗藥性帶來的突破 32
陸、 結論 33
柒、 引用文獻 34
圖表 45
附錄 69

表目次

表一、抗藥性篩選與感受性試驗的使用藥劑 45
表二、雌成蟲抗擊昏試驗所使用的藥劑種類與藥膜濃度 46
表三、抗百滅寧Per-R品系的篩選過程與抗藥性變化情形 47
表四、抗安丹Prop-R品系的篩選過程與抗藥性變化情形 48
表五、感性NS品系埃及斑蚊幼蟲對各種除蟲菊殺蟲劑與乙醯膽鹼抑制劑的感受性基礎資料 49
表六、抗性Per-R品系埃及斑蚊幼蟲對各種除蟲菊殺蟲劑與乙醯膽鹼抑制劑的感受性基礎資料 50
表七、抗性Prop-R品系埃及斑蚊幼蟲對各種除蟲菊殺蟲劑與乙醯膽鹼抑制劑的感受性基礎資料 51
表八、感性NS品系埃及斑蚊成蟲對各種除蟲菊殺蟲劑與安丹的感受性基礎資料 52
表九、抗性Per-R品系埃及斑蚊成蟲對各種除蟲菊殺蟲劑與安丹的感受性基礎資料 53
表十、抗性Prop-R品系埃及斑蚊成蟲對各種除蟲菊殺蟲劑與安丹的感受性基礎資料 54
表十一、埃及斑蚊雌成蟲轉錄體基因庫功能註釋摘要 55
表十二、埃及斑蚊雌成蟲轉錄體基因庫功能註釋摘要 56

圖目次

圖一、Per-R抗百滅寧與Prop-R抗安丹埃及斑蚊幼蟲對九種常用防治藥劑的抗藥性比 57
圖二、Per-R抗百滅寧與Prop-R抗安丹埃及斑蚊雌成蟲對七種常用防治藥劑的抗擊昏比 58
圖三、抗百滅寧Per-R、抗安丹Prop-R與感性NS埃及斑蚊幼蟲表現量有差異的基因數量 59
圖四、抗百滅寧Per-R、抗安丹Prop-R與感性NS埃及斑蚊雌成蟲表現量有差異的基因數量 60
圖五、抗百滅寧Per-R與感性NS埃及斑蚊幼蟲表現量有差異的基因依KEGG代謝途徑分類 61
圖六、抗安丹Prop-R與感性NS埃及斑蚊幼蟲表現量有差異的基因依KEGG代謝途徑分類 62
圖七、抗百滅寧Per-R與抗安丹Prop-R埃及斑蚊幼蟲表現量有差異的基因依KEGG代謝途徑分類 63
圖八、抗百滅寧Per-R與感性NS埃及斑蚊雌成蟲表現量有差異的基因依KEGG代謝途徑分類 64
圖九、抗安丹Prop-R與感性NS埃及斑蚊雌成蟲表現量有差異的基因依KEGG代謝途徑分類 65
圖十、抗百滅寧Per-R與抗安丹Prop-R埃及斑蚊雌成蟲表現量有差異的基因依KEGG代謝途徑分類 66
圖十一、與百滅寧及安丹抗藥性相關且表現量具有顯著性差異的埃及斑蚊幼蟲基因數量 67
圖十二、與百滅寧及安丹抗藥性相關且表現量具有顯著性差異的埃及斑蚊雌成蟲基因數量 68

附錄目次

附錄一、Per-R品系F35至F42篩選結果 69
附錄二、Bora、NS及Per-R品系埃及斑蚊的Aa-para突變基因頻度 70
附錄三、轉錄組分析簡要流程示意圖 71
附錄四、Unigene組裝示意圖 72

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