蚊蟲共生物Wolbachia之親緣關係及其與蟲媒病毒在蚊蟲之交互作用

Phylogeny of the Symbiont Wolbachia in Mosquitoes and Its Potential Interactions with Arbovirus in the Mosquito

蔡坤憲(Kun-Hsien Tsai)

指導教授 ： 陳維鈞

共同指導教授 ： 吳文哲(Wen-Jer Wu)

國立臺灣大學/生物資源暨農學院/昆蟲學研究所/博士(2004年)

https://doi.org/10.6342/NTU.2004.02490

摘要

中文摘要
Wolbachia是一類共生性的立克次體，在大多數節肢動物和絲蟲的生殖組織中均可發現。Wolbachia的存在導致寄主生殖現象的改變，包括種間、種內不同品系間的細胞質不親和性 (cytoplasmic incompatibility; CI)、孤雌生殖的誘導 (parthenogenesis induction; PI) 以及生殖偏向雌性 (feminization)。本文就台灣地區田間蚊蟲感染Wolbachia的情形進行檢測，結果顯示Wolbachia在台灣蚊蟲種類的盛行率為51.72 % (15/29)，在蟲體各組織趨性亦呈現系統性感染。由Wolbachia的wsp (Wolbachia surface protein) 基因的分子親緣系統分析顯示，蚊蟲Wolbachia可分成二個主要類群 (A群和B群) 和九個亞群 (AlbA, Ann, Con, Gen, Mur, Neo, Omi, Pap, Pip)，其中A群計有6種，分屬3個亞群 (AlbA, Gen, Omi)；B群計有9種，分屬5個亞群 (AlbA, Con, Mur, Neo, Pip)，由分子親緣系統所建立的系統樹顯示仍有新亞群存在的可能性。過程中，為了細胞生物學研究上的便利性，本實驗利用分子基因轉殖的技術，成功表現一段Wolbachia wsp基因的表面蛋白，以該蛋白免疫兔子所生產的抗血清，適用在南方氏墨點法和免疫螢光偵測法。此外，為瞭解Wolbachia與蟲媒病毒 (arbovirus) 間的交互作用，本實驗以病毒液中胸注射的方法進行感染，利用螢光顯微鏡觀察日本腦炎病毒和Wolbachia在組織內及細胞內的分布，結果顯示，Wolbachia族群主要聚集分布於卵巢的卵細胞內，以及零散分布於唾腺細胞中；日本腦炎病毒則主要感染蚊蟲唾腺的側葉近端和卵巢的輸卵管總管段。由電子顯微鏡觀察結果發現，唾腺中Wolbachia的分布包括唾腺側葉的近端和遠端，以及中葉的遠端。在側葉的近端，Wolbachia和日本腦炎病毒二種微生物同時分布於相同的唾腺細胞中。此外，白腹叢蚊感染Wolbachia與否對其族群以及F1子代的病毒感染率在統計上未達顯著性差異。在日本腦炎病毒感染效價的比較上，唾腺組織病毒效價遠大於卵巢輸卵管，統計上達顯著性差異；然而，在Wolbachia感染與否二組比較試驗中，相同組織部位則呈現統計上差異不顯著。

關鍵字

Wolbachia ； 共生物 ； 蚊蟲 ； 交互作用 ； 蟲媒病毒 ； 親緣關係

並列摘要

Abstract
Wolbachia are maternally inherited bacteria that infect a wide range of arthropods as well as filarial worms. The infection usually results in reproductive distortions of the host, primarily cytoplasmic incompatibility, parthenogenesis, and feminization. This study showed that Wolbachia infection (15/29; 51.72%) was prevalent among field-caught mosquitoes in Taiwan. Each Wolbachia isolate derived from specific mosquitoes can be classified as a specific group and even a subgroup. Because there were still some isolates which did not belong to any known subgroup, more subgroups were expected to be identified. Investigation on tissue tropism of Wolbachia in either Aedes albopictus or Armigeres subalbatus revealed that Wolbachia were extensively distributed within the host. Of which, the ovary was the organ most susceptible to Wolbachia infection. Among 15 mosquito species infected by Wolbachia, 6 and 9 species were assigned to group A and group B, respectively. The 6 species belonging to group A Wolbachia were further classified into 3 sub-groups (AlbA, Gen, and Omi) while 9 species of group B Wolbachia were classified into 5 sub-groups (Ann, Con, Mur, Neo and Pip). The phylogenetic analysis also revealed the occurrence of several novel sub-groups of Wolbachia in mosquitoes used in this study. In addition, wsp gene has been identified in mosquitoes collected from different geographic locations in Taiwan. In this study, anti-Wolbachia antiserum was produced by using Wolbachia wsp protein, that had been expressed from a conserved region of wsp gene of Ar. subalbatus. Existence of Wolbachia either in salivary glands or ovaries was recognized by indirect immunofluorescent antibody test (IFA), demonstrating that the induced antiserum is suitable for identifying localization of Wolbachia in mosquito tissues. Intracellular bacteria have been described in several species of mosquitoes, but their relationships with the hosts and effects on virus transmission have not yet been elucidated; which have been examined by using intrathoracic inoculation of JE virus into Ar. subalbatus in this study. Based on examinations by both immunofluorescence antibody test (IFA) and electron microscopy (EM), Wolbachia was mainly distributed in ovaries where as JE virus was in salivary glands. On salivary glands, Wolbachia were observed in both distal and proximal parts of lateral lobes, and distal part of the median lobe. Meanwhile, both Wolbachia and JE virus can also be seen within the same cell of salivary glands; which has provided a model to elucidate the relationships between Wolbachia and JE virus. In fact, infection rates between mosquitoes with or without Wolbachia infection showed no difference in either salivary glands or ovaries. The infection rates were usually low in JE virus-infected F1 progenies of Ar. subalbatus with or without infection of Wolbachia. The results of plague assay showed that daily titers of JE virus were not significantly different in either Wolbachia-infected or free Ar. subalbatus.

並列關鍵字

mosquitoes ； phylogeny ； interaction ； Wolbachia ； arbovirus

參考文獻

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