CHARACTERIZATION OP GYPSY-MOTH POPULATIONS AND RELATED SPECIES USING A NUCLEAR-DNA MARKER

The discovery of egg masses of Asian gypsy moth onboard Russian freighters in Pacific ports of North America and the capture of adult male gypsy moths in pheromone traps underscored the need for a positive identification of Asian and European gypsy moth males. We have devised a method for differentiation of these two populations based on restriction endonuclease cleavage of a polymerase chain reaction (PCR) generated product. DNA primers for the 5S and 28S rDNA were employed to amplify the ITS2 region using PCR. The ends of the amplified DNA products were sequenced and gypsy moth specific oligonucleotide primers were designed to amplify the gypsy moth ITS2 region. Eleven of 28 restriction enzymes tested cleaved the amplified product. The restriction enzymes ClaI, PvuI, and TaqI generated distinct restriction fragment patterns for the Asian and European gypsy moth PCR product. This diagnostic was applied to samples of field-collected gypsy moths in a double blind test and correctly identified them as Asian or European. Additional studies demonstrated that these primers and restriction enzymes could be used to differentiate among Lymantria dispar, L. monacha, and L. mathura.