|Budget Amount *help
¥2,000,000 (Direct Cost : ¥2,000,000)
Fiscal Year 1992 : ¥700,000 (Direct Cost : ¥700,000)
Fiscal Year 1991 : ¥1,300,000 (Direct Cost : ¥1,300,000)
Toxigenic dinoflagellates belonging to Alexandrium catenella and A. tamarense produce potent neurotoxins responsible for paralytic shellfish poisoning (PSP) in coastal waters and cause serious economic and public health problems. However, the morphological taxonomy of these organisms remains controversial because of the very similar and changeable features and also toxin content is different among strains. Accordingly, a simple, easy and objective method for identification is a pressing need. In this study I attempted to identify both species by means of monoclonal antibodies, and to develop DNA probe specific for each species. The results are as follows;
(1) Large scale cultures of A. catenella and A. tamarense with clonal and axenic conditions were established by the isolation of germinated cells from the cysts isolated from Tanabe Bay and Ofunato Bay.
(2) PSP troxins of all strains were analyzed by HPLC-fluorometric analysis and then the typical strains showing different toxin content
and composition were selected for following studies.
(3) Monoclonal antibodies specific to toxic Alexandrium, which were prepared by us, could immunologically discriminate and identify inter- and intraspecies of these microalgae.
(4) In toxic Alexandrium, the purification method of whole cell DNA with high molecular weight (>50kbp) was established for the first time. Amplification with two primers positioned 3'end of 17SrDNA and 5'end of 24SrDNA yielded a PCR product of 610pb in all strains of both species. The DNA fragments corresponded to the regions of ITS(internal spacer)1, 5.8S and ITS2. Digestion analysis with AluI and RsaI showed the different restriction fragment length polymorphism in both species. The identification results by the DNA fragments correspond to that of immunological analysis. From these results and also low degree of sequence similarity in the ITS regions of both species, DNA probe specific for this region is definitely an effective tool for the identification of toxic dinoflagellate Alexandrium species.