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

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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.