王重雄臺灣大學：昆蟲學研究所李克昌Lee, Ker-ChangKer-ChangLee2007-11-262018-06-292007-11-262018-06-292007http://ntur.lib.ntu.edu.tw//handle/246246/55062本實驗室於2005年首次證實，西方蜂會受到東方蜂微粒子蟲感染，並引發世界各地對西方蜂微粒子蟲病之病原物種的重新鑑定。本研究分析採集自南美法屬馬丁尼克島 (Martinique Island)、法國及西班牙之西方蜂樣本與台灣地區之西方蜂及東方蜂樣本之微粒子蟲種類，結果發現各地區樣本中均有感染東方蜂微粒子蟲的病蜂，尤其南美 Martiniqe Island 與台灣地區之病蜂中，僅發現東方蜂微粒子蟲，且無交叉感染現象。各地區之東方蜂微粒子蟲核醣體基因之 ITS 序列呈現高保守性，可設計為微粒子蟲病之病原診斷鑑定用的分子標誌；而 IGS 序列與 SSUrRNA 則呈現多型性。以 IGS 區段進行演化分析，顯示東方蜂微粒子蟲在不同寄主間並無演化差異；於地理演化上，不同地區間亦無隔離分化的情況。由於歐洲地區、南美及台灣地區之西方蜂族群中均受到東方蜂微粒子蟲的感染，顯示東方蜂微粒子蟲已廣泛發生於世界各地。另於法國病蜂中發現有一未定種之存在，顯示西方蜂族群中的微孢子蟲，可能有兩種以上。因此對蜜蜂的微孢子蟲病病原體診斷需更進一步鑑定確認，以避免因病原種類的混淆，造成研究結果的誤差。Since 2005, Nosema ceranae was first found in the moribund European honeybee (Apis mellifera) with nosema disease in our laboratory, this finding has evoked the worldwide reexamination of the exact caused agent of European honeybee with nosema disease. In this study, we examined the diseased European honeybees from Martiniqe Island (South America), France, Spain, and Taiwan, and also from oriental honeybee (A. cerana) from Taiwan. The results showed that only N. ceranae was detected in the collected honeybees, especially the diseased honeybees from Taiwan and Martiniqe Island (South America). In addition, no cross infection with other microsporidia was found. The ITS sequences of the N. ceranae rDNAs from the examined honey bees were highly conserved, it is suggested that this spacer sequence can be used an available marker for the detection of honey bee nosema disease. However the sequences of both IGS and SSUrRNA were polymorphic. Based on IGS sequences, there was no evolutional difference of N. ceranae from different hosts in evolution analysis, and also no allopatric differentiation occurred in the isolates from different geographic areas. N. ceranae was found in the honeybee population collected from South America, European, and Taiwan, it implied that N. ceranae has been worldwide dispersed. Furthermore, two diseased honeybees collected from France suffered from the microsporidian infection with an undefined species, it reveals that there may be more than two species of microsporidian pathogens existed in the honeybee population, so the precise identification of honeybee microsporidian pathogens is necessary. It can avoid the conflict in data caused by the misidentification of the pathogens.目 錄 誌謝…………………………………………………………………………………… i 中文摘要……………………………………………………………………………… ii 英文摘要……………………………………………………………………………… iii 壹、緒言……………………………………………………………………………… 1 貳、往昔研究………………………………………………………………………… 2 一、微孢子蟲背景簡介………………………………………………………… 2 二、蜜蜂之微粒子蟲病病原及診斷…………………………………………… 3 三、蜜蜂之微粒子蟲發育週期及發病因素…………………………………… 5 四、蜜蜂之微粒子蟲病的防治………………………………………………… 6 五、分子生物資料在微孢子蟲分類上的應用………………………………… 7 參、材料與方法……………………………………………………………………… 10 一、供試材料來源……………………………………………………………… 10 二、蜜蜂中腸DNA萃取………………………………………………………… 10 三、勝任細胞 (competent cell) 製備…………………………………………… 11 四、微粒子蟲SSUrRNA專一性引子檢測……………………………………… 11 五、ITS 序列…………………………………………………………………… 12 六、IGS 序列…………………………………………………………………… 12 七、親緣關係之分析…………………………………………………………… 13 肆、結果……………………………………………………………………………… 14 伍、討論……………………………………………………………………………… 27 陸、參考文獻………………………………………………………………………… 321802099 bytesapplication/pdfen-US東方蜂微粒子蟲西方蜂東方蜂ITSIGSNosema ceranaeApis melliferaA. ceranathesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/55062/1/ntu-96-P94632003-1.pdf