大黑點白蠶蛾微孢子之分類地位及親緣關係之分析

Abstract

從台灣榕樹害蟲-大黑點白蠶蛾 (Ocinara lida Moore) 分離出新微孢子：大黑點白蠶蛾變形微孢子 (Vairimorpha ocinarae n. sp.)。此微孢子會造成幼蟲之系統性感染，主要感染位置為馬氏管及中腸肌肉，感染的幼蟲其脂肪體遭受到嚴重地萎縮。此微孢子之孢子有二型：雙核孢子具有 11 至 13 圈極管；單核孢子常呈八孢子聚集，具有 12 圈極絲。電子緻密之上孢子內涵體充斥上孢子腔，此特徵與來自家蠶與火蟻之微孢子 (分別為 Vairimorpha sp. NIS-M11 及 V. invictae) 類似，但有異於其它變形微孢子種類。根據 SSUrDNA 序列，此微孢子與分離自家蠶之 Vairimorpha sp. NIS-M11 及 Vairimorpha sp. NIK-3h 關係密切。形態和遺傳特性上，此微孢子應屬變形微孢子屬之新種。變形微孢子種群 (Vairimorpha complex) 內之種類及分離株，利用核糖體 RNA 基因 (Small subunit ribosomal DNA, SSUrDNA 及 Large subunit ribosomal DNA)、內轉錄區間各區段 (HG4F-HG4R 區段、ITS+580R 區段及 580R 區段)、特定蛋白質之基因 (alpha-tubulin、beta-tubulin 及 hsp70) 與多基因統合序列進行親緣關係之分析。變形微孢子種群之種和分離株可分成四群，並據以評估各群間及群內之相關性。基於基因統合序列之分析及 SSUrDNA 之 PCR-RFLP，發現第三群與第四群關係較第二群密切，而疏於第一群。本研究顯示所有分析的基因和區段之親緣分析及核苷酸序列相似度的分析，內轉錄區間 (Internal transcribed spacer，ITS) 是最適合用於群間及群內的鑑識區段，其次為蛋白質基因。在內質網微孢子種群中，基於 SSUrDNA 序列分析可分成兩群，群間及群內相似度皆為 98~99%。在分群分析上，仍需要有較多的其他基因序列才能證實群間與群內的相關性。

A new microsporidium, Vairimorpha ocinarae n. sp., was isolated from the moribund larvae of Ocinara lida Moore (Lepidoptera: Bombycidae), a pest of Ficus spp. in Taiwan. This isolate causes a systemic infection of O. lida larvae. The midgut epithelium, midgut muscle tissues, and Malpighian tubules were the main target tissues for this isolate, and the atrophied fatty body was found in the seriously infected larvae. Two types of spores were found: binuclear spores possessed 11 to 13 polar-tube coils; mononuclear spores possessed 12 coils polar tube and aggregated usually to form octospores. Electron-dense episporontal inclusions filled in the episporontal space, and this structure was similar to that of Vairimorpha sp. NIS-M11 isolated from Bombyx mori and V. invictae isolated from Solenopsis invicta, but different from other Vairimorpha species. Based on the phylogenetic analysis of SSUrDNA sequences, this isolate is closely related to Vairimorpha sp. NIS-M11 and Vairimorpha sp. NIK-3h. Morphological and genetic characters showed that this isolate is a new species placed in the genus Vairimorpha. The species and isolates in the Vairimorpha complex were analyzed phylogenetically based on the sequences of rDNA (SSUrDNA and LSUrDNA), ITS (HG4F-HG4R region, ITS+580R region, and 580R region), protein coding genes (alpha-tubulin, beta-tubulin and hsp70 genes), and total sequenced genes. The four groups were identified and the relationships of inter-groups and intra-groups were evaluated. Based on phylogenetic analyses of total sequence genes and RFLP-PCR of SSUrDNA, the Group 3 and 4 are closer to Group 2 than Group 1. The results of the phylogenetic analyses based on the sequences of each examined gene or region were evaluated for the group identification. The internal transcribed spacer (ITS) is the best candidate for identification of inter-groups and intra-groups. The second candidate is protein coding genes. In the Endoreticulatus complex, there were two groups formed based on the phylogenetic analyses of SSUrDNA. The identities of inter-groups and intra-groups were both 98~99%. It still needs more gene sequence evidences to confirm the relationships between intra-groups and inter-groups.