Identification of three Aulacaspis species (Hemiptera: Coccoidea: Diaspididae) by rapid molecular diagnostic technique for quarantine application in Taiwan

Abstract

Scale insects are small and similar in morphology, and the identification of species especially in the immature stages is difficult. In this study, a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique was developed to identify three Aulacaspis species, A. murrayae, A. yabunikkei and A. yasumatsui. Primers ITS5 and ITS4 were used to amplify the internal transcribed spacer (ITS) regions of the ribosomal DNA (rDNA). The genomic DNA isolated from A. yasumatsui yielded a single fragment after PCR amplification. The fragment size was about 0.6 kb. The other species, A. murrayae and A. yabunikkei, gave two PCR products. The sizes of the two fragments were about 0.6 kb and 0.67 kb. These three Aulacaspis species could be easily discriminated according to the species-specific patterns by digesting the PCR products with endonucleases TaqI, MspI and CfoI. 介殼蟲蟲體微小且外形相似，幼體時期特徵不明顯，在鑑定上不易確定，造成在檢疫工作上困難重重。本研究利用基因組核酸標誌 (genomic DNA markers)，以分子診斷技術鑑別三種白輪盾介殼蟲屬 (Aulacaspis spp.) 之介殼蟲，分別為蘇鐵白輪盾介殼蟲 (A. yasumatsui Takagi)、月橘白輪盾介殼蟲 (A. murrayae Takahashi) 和樟白輪盾介殼蟲 (A. yabunikkei Kuwana)。以單隻標本 DNA 進行聚合酶連鎖反應-限制酶片段長度多態型 (polymerase chain reaction-restriction fragment length polymorphism, PCR-RFLP) 的結果分析，利用 PCR 引子對 (ITS5 和 ITS4) 增幅 rDNA 之 ITS 區段，蘇鐵白輪盾介殼蟲增幅出單一片段，大小約 0.6 kb；月橘白輪盾介殼蟲和樟白輪盾介殼蟲則增幅出二片段，大小約 0.6 kb 和 0.67 kb。利用 TaqI、MspI 和 CfoI 核酸內切酶進行 ITS/rDNA PCR 產物酶切，所得圖譜可容易區別三種普遍分布且外型進似的白輪盾介殼蟲。