Discrimination of very closely-related insect species by molecular biological techniques

为鉴别亚洲玉米螟及其7个亲缘种（Ostrinia furnacalis、O. orientalis、O. scapulalis、O. zealis、O. zagulievi、O. latipennis、O. palustralis和O. nubilalis），寻求一种简便的分子生物学方法。(1)在日本各地采集玉米螟，对其线粒体DNA的CO II区进行了测序比较。随着样本数的增加，具有相同mtDNA单倍型的物种增多，使得玉米螟属物种的系统发育树更加复杂。未获得物种特异性序列。(2)因此采用RAPD-PCR法代替。使用80个10mer引物（Operon Inc., OPA01 - 20, OPB01 - 20, OPK02 - 20, OPL01 - 20, OPN03 - 09）进行RAPD-PCR。在检测的80个引物中，73个扩增出清晰的DNA条带。通过使用20个个体，确定了16条引物为种特异性引物。1条引物可区分东方稻蛾，2条引物可区分古稻蛾，10条引物可区分拉丁稻蛾，1条引物可区分东方稻蛾和1条新西兰稻蛾。关于肩胛骨棘球蚴和扎古利亚棘球蚴，目前还没有发现特异的引物。通过对7种Ostrinia的52个样本进行扩增，确定引物OPB-11对O、palustralis和O. latipennis具有种特异性条带。利用该引物，palustralis的条带长度为570bp，而O. latipennis的条带长度为1250bp。

A handy molecular biological method to discriminate the Asian corn borer and its 7 allied species (Ostrinia furnacalis, O. orientalis, O. scapulalis, O. zealis, O. zaguliaevi, O. latipennis, O. palustralis and O. nubilalis) was sought, (1) Ostrinia spp. were collected from various localities in Japan, and CO II region of the mitochondrial DNA was sequenced for comparison. When the sample number was increased, more species shared the same mtDNA haplotypes, causing the phylogenetic tree of Ostrinia species more complex. No species-specific sequence was obtained. (2) Therefore, RAPD-PCR method was adopted instead. Eighty 10mer primers (Operon Inc., OPA01 - 20, OPB01 - 20, OPK02 - 20, OPL01 - 20, OPN03 - 09) were used to perform RAPD-PCR. Of the 80 primers examined, 73 amplified clear DNA bands. By using 20 individuals, sixteen primers were nominated as species-specific primers. A primer discriminated O. furnacalis, two primers discriminated O. palustralis, 10 primers discriminated O. latipennis, and one each primer discriminated O. orientalis and O. zealis, respectively. Regarding O. scapulalis and O. zaguliaevi, no species-specific primer has been found until now. By increasing the sample size to 52 individuals of 7 Ostrinia species, a primer, OPB-11, was confirmed to produce a species-specific band for O, palustralis and O. latipennis. With this primer, O. palustralis showed a 570bp band, while O. latipennis produced a 1250bp band.