臺灣博碩士論文加值系統

研究背景: 沃爾巴氏共生菌(endosymbiont Wolbachia)之潛在應用於蚊媒介傳染病防治，已成為一種對抗蚊蟲之絕佳新興生物防治策略，雖然家蚊(Culex spp.)在印尼已被認定為傳播淋巴性絲蟲及日本腦炎病毒之主要病媒蚊種，然有關此蚊體內微生物相之組成及結構(尤其是沃爾巴氏菌)，則仍不清楚。
研究目的: 本研究目的將確認採集自印尼蘇門達臘家蚊之基因種，並鑑定其共生沃爾巴氏菌之存在性、盛行率及親緣演化關係分析。
研究方法: 本研究運用蚊蟲野外採集技術進行印尼蘇門達臘之家蚊採集，以便篩檢家蚊感染沃爾巴氏菌之狀況，並以聚合酶鏈反應(PCR)技術針對蚊蟲粒腺體之COI及ND5基因，以確認蘇門達臘家蚊之基因種，並針對沃爾巴氏菌之體表蛋白基因 (wsp; wolbachia surface protein)，以偵測蘇門達臘家蚊感染沃爾巴氏菌之狀況，而它們的親緣關係則應用鄰接法(neighbor-joining method)進行親源遺傳之演化分析。
研究結果: 結果顯示沃爾巴氏菌之DNA可以在33.8% (106/314)的野外採集家蚊體中檢測到，其雌蚊感染率在城市及鄉村地區分別為52.4%及36.4%，然而雄蚊感染率在城市及鄉村地區則分別為9.4%及20%。針對蚊蟲粒腺體基因(Cytochrome c oxidase subunit 1, COI; NADH Dehydrogenase 5, ND5)之親源分析，則確認家蚊基因種為熱帶家蚊(Cx quinquefasciatus)，而針對wsp基因之親源分析結果亦顯示這些印尼熱帶家蚊存在之共生沃爾巴氏菌株屬於與Cx. pipiens complex相同之Wolbachia group B及新發現之group A。
結論： 本研究乃首次提供有關沃爾巴氏菌存在於印尼熱帶家蚊之分子診斷證據，並依據沃爾巴氏菌wsp基因之親源分析結果亦確認其基因種株group B與新發現基因種株group A。

Background: The potential application of the endosymbiotic bacteria, Wolbachia, to the control of mosquito-borne infections has emerged as a novel biological control strategy against mosquitoes. Although Culex mosquitoes play the role as the primary vector for lymphatic filariasis and Japanese encephalitis in Indonesia, little is known about the composition and structure of their microbiota, specifically Wolbachia.
Objectives: The objectives of this study are to determine the genetic identity of Culex mosquitoes collected from Sumatera Utara, Indonesia, and to identify the presence, frequency and phylogenetic analysis of its symbiotic Wolbachia.
Methods: Field surveys were conducted to screen the infection status of Wolbachia in field-collected Culex mosquitoes from Sumatera Utara, Indonesia. We performed the PCR assays to identify the mosquito species based on the mitochondrial COI and ND5 genes, and to detect Wolbachia wsp (wolbachia surface protein) gene in Culex mosquitoes. The phylogenetic relationships were analyzed by neighbor-joining method.
Results: Wolbachia DNA were detected in 33.8% (106/314) of wild-caught mosquitoes. The rate of infection in adult females were detected with 52.4% in urban area and 36.4% in rural area. While in males, the infections were detected with 9.4% in urban area and 20% in rural area. The Blast search and phylogenetic analysis of the COI and ND5 genes identify the genetic identity of Culex quinquefasciatus from Indonesia. The wsp gene sequence analysis revealed that the Wolbachia strain from Indonesian Cx. quinquefasciatus was identical to the Wolbachia strains of supergroup B previously reported in members of the Cx. pipiens complex and newly identified Wolbachia strains of supergroup A.
Conclusions: This study provides the first molecular evidence of Wolbachia infection in Cx. quinquefasciatus populations of Indonesia. Based on wsp sequences, this study found Cx. quinquefasciatus were singly or superinfected with A and B Wolbachia strain. The phylogenetic analysis revealed a new discovery of Wolbachia group A in Cx. quinquefasciatus.

Acknowledgment……………………………………………...…………....... iii
Table of Contents…………………………………………………………….. iv
List of Tables………………………………………………………………… v
List of Figures…………………………………………...…………………… vi
Abstract…………………………………………………………...…...…...… vii

Chapter 1 Introduction……………………………………………………….. 1
1.1. Background…………………………………………………………. 1
1.2. The Objectives……………………………………………………… 3
Chapter 2 Literature Review…………………………………………………. 4
2.1. Culex Mosquito as a Vector of Diseases…………………………… 4
2.2. Distribution of Culex Mosquito in Indonesia and Its Impact………. 5
2.3. Wolbachia as a Novel Biological Mosquito Control……………….. 7
2.4. Genetic Analysis of Wolbachia…………………………………….. 8
Chapter 3 Materials and Methods……………………………………………. 11
3.1. Study Sites………………………………………………………….. 11
3.2. Mosquito Collection………………………………………………... 11
3.3. DNA Extraction and PCR Analysis…………..………………...….. 11
3.4. Sequencing and Phylogenetic Analysis……………………..….….. 14
3.5. Statistics Analyzing………………………………………………… 15
Chapter 4 Results…………………………………………………………….. 16
4.1. Wolbachia Detection in Culex Mosquitoes…………………………. 16
4.2. Wolbachia Infection Rate…………………………………………… 16
4.3. Phylogenetic Analysis of Wolbachia…………………….....…...….. 17
4.4. Phylogenetic Analysis of Culex Mosquitoes…………………..…… 17
Chapter 5 Discussions…………………………………………………….….. 19
References……………………………………………………………………. 23
Appendix……………………………………………………………………... 45

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