Development of a discrimination methods by using molecular and monoclonal antibodies techniques for Haliotis discus discus, H. madaka and H. gigantea during their early life stages.

Three species of abalones, Haliotis discus discus, H. madaka and H. gigantea, are distributed along the southwest coast in Japan and are common and commercially important shellfish in the country. Survey records show a rapid decline in fisheries production of abalones in the last decade, however, few attempts have so far been made to study the early life stages of these species. One of the bottleneck problems in the study of early life stages of these species is the difficulty to discrimi- nate between species since their morphological features are very similar. In addition, a hybrid variety of H. discus discus, and H. madaka had been reported and this fact has made the species identification problem more complex. The purpose of this paper was to develop a discrimination method by using molecu- lar and monoclonal antibodies techniques for Haliotis discus discus, H. madaka and H. gigantea during their early life stages. Molecular(PCR)techniques To discriminate between Haliotis discus discus, H. madaka and H. gigantea, we analyzed some mitochondrial, 16S rDNA, COI(cytchrome c oxidase subunit I) , Cyt b (cytchrome b) and nuclear genes, 18S and 28S rDNA, ITS(internally transcribed spacer) , ferritin, and calmodulin-introns and discovered that PCR products related tocalmodulin-intron were very suitable for species discrimination. A PCR-RFLP(re- striction fragment length polymorphisms) method was developed using calmodulin- introns. Fertilized eggs and planktonic larvae were analyzed by using 2 step PCR method.