Species identification of the mud snails Batillaria multiformis and B. attramentaria by PCR-RFLP method—the advantages of two restriction enzymes

The habitats of Batillaria multiformis (Near Threatened species) and Batillaria attramentaria are often overlapping on a mudflat, and young individuals of two Batillaria snails were difficult to identify by shell morphology. This is one of the causes that the study, such as population dynamics of two Batillaria species, did not advance. Recently, species identification by DNA analysis for B. multiformis and B. attramentaria was developed using a PCR-RFLP method. However, using only one restriction enzyme and universal primer may lead to misidentification. Therefore, this study examined a more precise method using two restriction enzymes for the PCR-RFLP technique. The mitochondrial DNA gene encoding cytochrome c oxidase subunit I (COI) was amplified, and two restriction enzymes, PstI and HinfI, were selected for this method. The number and position of bands after the agarose gel electrophoresis enable us to identify the two Batillaria species. After the effectiveness of this method was confirmed by adult B. multiformis and B. attramentaria that had each typical shell shape, young Batillaria snail samples that were difficult for morphological identification were identified by this PCR-RFLP method. We proved that the proposed PCR-RFLP identification method is simple and reliable in identifying the morphologically similar B. multiformis and B. attramentaria.