Title 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.
Authers Masami HAMAGUCHI, Miho SASAKI, Toyomitsu HORII, Setsuo KIYOMOTO, Satoshi OHASHI, Akihiko FUJII, Naoyuki TAKIGUCHI, Kanako HASHIMOTO and Taisuke TAKENOUCHI
Keywords H. discus discus, H. madaka, H. gigantea, species discrimination, PCRRFLP, monoclonal antibodies
Citation Bull. Fish. Res. Agen. supplement No. 5, 1-12, 2006
Abstract
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 discriminate 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 molecular 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(restriction fragment length polymorphisms)method was developed using calmodulinintrons. Fertilized eggs and planktonic larvae were analyzed by using 2 step PCR method.
Monoclonal antibodies techniques
Initially, we prepared monoclonal antibodies using samples of H. discus discus, H. madaka and H. gigantea veliger larvae as antigens, and obtained H. gigantea specific monoclonal antibodies. However, we were not able to obtain monoclonal antibodies specific to H. discus discus and H. madaka. Hence, we performed a proteome analysis between Haliotis discus discus and H. madaka and discovered several species-specific antigens. Secondly, we prepared monoclonal antibodies by using these new antigens. Consequently, we obtained monoclonal antibodies specific to each abalone species. We then surveyed the immunological specificity of these antibodies by using field samples.
Future goals
Most Haliotis species have a planktonic larval stage. To date, little is known about the ecology of Haliotis discus discus, H. madaka and H. gigantea during their early life-history stages. Consequently, the influence of the planktonic larval stage on recruitment variability is not well understood. Fine scale spatial and temporal resolution in sampling coupled with an understanding of hydrodynamics would greatly improve our knowledge of the ecology of these larvae. However, their identification, which is traditionally based on the observation of the size of larval shells and the pattern of shell surface sculptures under light and scanning electron microscopes, as well as the processing of large numbers of samples, remains a major hindrance to the study of marine invertebrate larval ecology. Antibody technique offers a powerful tool to study planktonic larval populations for two reasons: samples can be analyzed quickly and the method makes species identification easy. However, the method has a serious problem such as cross-reactivity of other mollusk species. On the other hand, PCR identification technique, although the most accurate, is time consuming and is very expensive. Therefore, we suggest a hybrid discrimination system, by combining PCR and monoclonal antibodies techniques to identify Haliotis species in their early life stages.
URI http://www.fra.affrc.go.jp/bulletin/bull/bull-b5/18.pdf