微粒子特有基因 sr22 應用於蜜蜂微粒子病之診斷

Abstract

蜂群衰竭失調症 (CCD) 之主要症狀為西方蜂 (Apis mellifera) 族群中，造成外勤蜂之大量損失，這種失調現象不只影響了蜜蜂產業，也同時影響授粉行為。到目前為止仍尚未找出確切的發生原因。東方蜂微粒子 (Nosema ceranae) 被認為是造成該失調症的可能元凶之一。東方蜂微粒子早期被認為只感染東方蜂 (A. ceranae)，但目前已被發現普遍感染於世界各地西方蜂族群。蜜蜂之中腸為主要之感染部位。傳統上，診斷蜜蜂之微粒子病是利用光學顯微鏡檢查中腸是否有孢子。本研究之感染試驗中，以光學顯微鏡觀察只可在感染後第九天發現孢子的存在。為了發展感染初期的診斷以及偵測東方蜂微粒子於中腸組織的數量，利用抑制性扣減雜合法(suppression subtractive hybridization, SSH) 找出東方蜂微粒子的特有基因 sr22 , sr28, sr71和sr85 (大小分別: 798, 414, 1128 and 1023 nts)，並利用這些基因設計引子對進行 DNA 的偵測以及基因表現之時序變化。根據實驗結果， sr22 被選為做為後續研究基因，sr22 DNA 可在感染後第 3 天被偵測出，而 RNA 在感染後 84 小時，即可偵測出。這項結果可推測 sr22 基因為一個晚期基因 (latter gene)，且根據 NCBI 資料庫的比對，該基因可能與孢壁蛋白的組成有關。利用 qPCR 計算出 sr22 基因於東方蜂微粒子基因體中的拷貝數，其結果為 1.09。這項結果可用以了解感染組織中確切的微粒子數量以及微粒子感染程度之關聯。除此之外， sr22 同時可在西方蜂微粒子與家蠶微粒子基因體中被偵測出，顯示出該基因在這 Nosema 屬中具有高保守性。

Colony Collapse Disorder (CCD) is a syndrome described as a mass loss of foraging workers in the colonies of western honey bee (Apis mellifera). This syndrome impacts not only on the honeybee products and also the pollination. However causing factor is still undetermined. The microsporidiun Nosema ceranae,is being suggested that it plays an important role on CCD. This parasite is originally found from Asian honey bee, A. ceranae, but now it is a prevalent parasite and worldwide distributed in A. mellifera colonies. The midgut of honey bee is the main target organ. Traditionally, the diagnosis of honey beemicrosporiosis is based on spore examination in the midgut by light microscopy. In our experimental infection of N. ceranae to 3-day-old workers, the spores could be detected initially at 9-day post infection by light microscopy. In order to develop an early diagnosis method and to detect the number of parasite individuals in the examined midguts, four microsporidian specificgenes, sr22 , sr28, sr71 and sr85 were cloned and identified by SSH (suppression subtractive hybridization), the size of these genes were 798, 414, 1128 and 1023 nts, respectively. The sr22 was selected for this study, this gene is a hypothetical protein of N. ceranae. The sr22 DNA could be detected at 3-day post infection by PCR, while the sr22 expression was detected initially at 84-hour post infection by RT-PCR. The latter showed that this gene is a very latter gene which is suggested that this gene may involve the construction of spore wall. The former showed that this gene could be a molecular marker for early diagnosis of microsporidian infection. Due to the complication of microsporidian life cycle, the copy number of each N. ceranae individual, about 1.09, was also estimated by qPCR. This result will be used to reveal the number of parasite individuals in the infection sample and also implied the infection degree of this parasite. Furthermore, sr22 could also be detected in the genomes of N. apis and N. bombycis, indicating that this gene is highly conserved at least in the genus of Nosema.