臺灣博碩士論文加值系統

地下家蚊 (Culex pipiens molestus) 為尖音家蚊種群 (Culex pipiens complex) 的物種之一，主要分佈在溫帶地區的國家，有著不吸血即可產卵的自生型生物特性，並由連日清等人於1996年首度在北台灣被紀錄，與本土的熱帶家蚊共存於都市環境之中。這個被認為是外來入侵的蚊種，不僅滋擾民眾生活，同時也被指出為傳播疾病的病媒蚊之一，因此在環境與公共衛生上值得深入探究。然而，二十年後的今日，對於台灣本島出現的地下家蚊了解甚少，本研究嘗試藉由採集台北及高雄這南北兩大都會區內的地下家蚊，分析其族群內的粒線體、內共生菌Wolbachia及其噬菌體，以了解兩地族群的基因概況。本研究自2015年12月至2019年5月期間，於建築物內部採集蚊蟲樣本。樣本初步先以形態學外觀辨識，後續再利用acetylcholinesterase (Ace) 基因上的變異進行分子生物學物種鑑定。同時，藉由分析粒線體DNA、蚊蟲內共生菌Wolbachia及其感染的噬菌體的基因片段，以了解族群之中蚊蟲的基因多樣性。經調查分析來自於台北81棟及高雄30棟建物內的家蚊樣本，結果顯示：建物內部有地下家蚊出沒的比例，台北與高雄分別為84% (68/81)及63% (19/30)。定序兩地共87條COI基因，可發現2種基因多型性(AA&GA)，其中台北的族群兩型皆存在，高雄則皆為AA。分析其內共生菌Wolbachia上的wsp基因發現，家蚊皆感染Wolbachia pipientis (wPip)，另用ank基因分析, 其感染之Wolbachia皆分類至wPip-IV。在噬菌體orf7基因感染情況，台北的地下家蚊族群顯示有不同程度的感染狀況 (二重感染1.6%；4/253，三重感染83.4%；211/253及無感染15.0%；38/253)，而在高雄的族群皆為三重感染(70.8%；51/72)或無感染(29.2%；21/72)。此外，不同基因多型性的地下家蚊彼此交配皆能有效產下自生型的卵筏。本研究藉由至建築物內部進行採樣，證實了在住家內部出現的家蚊族群以地下家蚊佔多數，此外，基因多樣性的結果顯示台灣地下家蚊族群來源推測有多次引入的可能。

Culex pipiens molestus is an autogenous (produce their first eggs without a blood meal), hypogenous (develop underground), stenogamous (mate in a confined space) and non-diapause (not affect by photoperiod) mosquito which recognized as an invasive species initially documented in 1996 by Lien et al. and coexisted with common species Cx. quinquefasciatus in our urban city. This species is not only nuisance people’s daily life, but plays a potential role in disease transmission; thus, making it an environmental and public health concern. However, little is known about its distribution and genetic diversity since it has been found two decades later in Taiwan. In this study, Culex samples were collected from the internal of the buildings in Taipei and Kaohsiung from December 2015 to May 2019. The collected samples were initially discriminated based on their morphological features, and a subset of samples was further analyzed molecularly targeting the acetylcholinesterase (Ace) gene. Further, several genes were characterized to evaluate population genetic diversity including Cytochrome oxidase c subunit I (COI), Wolbachia surface protein (wsp), ankyrin domain protein (ank2), and phage WO (orf7, pk1). In total, a pool of Culex mosquitoes was collected in 81 and 30 buildings of Taipei and Kaohsiung respectively. The presence of Cx. p. molestus was 84% (68/81) in Taipei and in 63% (19/30) Kaohsiung. Analyzing the barcode region of mtDNA revealed two haplotype differences (AA, GA) in one substitute site, and all the samples in Kaohsiung were cline to haplotype AA. The Wolbachia-related gene showed identically in all collected population, and was clustered into wPip-IV; however, the phage orf7 sequences analysis showed double (1.6%; 4/253) or triple (83.4%; 211/253) or non-infection (15.0%; 38/253) in Taipei population, contrasted to all triple (70.8%; 51/72) or non-infection (29.2%; 21/72) in Kaohsiung. Crossing experiments showed Cx. p. molestus in Taipei are fully compatible, neither COI nor orf7 gene differences have affected the results. After the survey of Cx. p. molestus population in Taipei and Kaohsiung, Cx. p. molestus was the majority in indoor building mosquitoes. Furthermore, the northern population showed more genetic diversity than in the southern, and the diverse genotypes of the populations were probably driven from multi-introduction.

致謝 i
Abstract ii
摘要 iv
Study Framework v
Content vi
List of Figures viii
List of Tables ix
Chapter 1 Introduction 1
1.1 Overview of Culex pipiens complex taxonomy 1
1.2 Biological characteristics 2
1.3 Morphological and molecular identification 3
1.4 Medical importance on Culex pipiens complex 4
1.5 Endosymbiont Wolbachia and its phage (WO) 4
1.6 Study aims 6
Chapter 2 Materials & Methods 8
2.1 Sample collection 8
2.2 Molecular analysis 8
2.2.1 DNA extraction 8
2.2.2 Culex species taxa identification 9
2.2.3 Barcode COI gene analysis 10
2.2.4 Determination of Wolbachia strain and typing 11
2.2.5 Determination of bacteriophage WO 12
2.3 Sequence analysis 13
2.4 Mosquito rearing 14
2.5 Reciprocal crossing type 14
Chapter 3 Results 18
3.1 Distribution of Culex mosquitoes in urban city 18
3.2 Polymorphism of the DNA sequence of the mitochondrial gene COI 19
3.3 Wolbachia strain and typing 21
3.4 Bacteriophage WO infection in Culex mosquitoes 21
3.5 Reciprocal cross results 22
Chapter 4 Discussion 24
References 30
Appendix 59

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