DNA identification of two laboratory colonies of the weevils, Sitophilus oryzae (L.) and S. zeamais Motschulsky (Coleoptera: Curculionidae) in Taiwan

Abstract

DNA amplification fingerprinting (DAF) and nuclear ribosomal DNA (nrDNA) amplification were used to discriminate between two laboratory colonies of two closely related species of weevils: the rice weevil, Sitophilus oryzae (L.), and the maize weevil, S. zeamais Motschulsky. For DAF, three sets of primers (aldolase, prolactin receptor, and interleukin-1 β ) were used for identification by polymerase chain reaction (PCR) and agarose gel electrophoresis, and the highly similar patterns of the resultant amplicons reconfirmed that the two weevils are closely related. The fragments of nrDNA amplification showed that for S. oryzae and S. zeamais , the homologies of the nucleotide sequences of the internal transcribed spacer regions, ITS-1 and ITS-2, were unusually high, at 96% and 97%, respectively. Based on the ITS-1 and ITS-2 sequences, two species-specific primer sets were designed: with the primer set ITS3/So , the predicted 450 bp DNA fragment was yielded with S. oryzae genomic DNA after PCR amplification ( n =10), but no PCR product was obtained with S. zeamais ( n =10); with the primer set ITS3/Sz , the same 10 S. zeamais specimens yielded a 550 bp DNA fragment, but S. oryzae yielded no amplicons. In view of the difficulty of distinguishing between these two closely related species, the specificity and availability of these two primer sets might prove to be a useful tool for distinguishing between them. However, the nrDNA sequences of the ITS-1 and ITS-2 regions of geographically isolated populations of both weevils still need to be elucidated, and the applicability of this technique to different geographical populations will need to be confirmed by further study.