Development of genetic diagnosis method of toxic microalgae

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Recent paralytic shellfish poisoning caused by toxic dinoflagellates which have very similar morphological features are serious problems in aquatic farms and public health. The purpose of this study are the establishment of molecular identification of toxic dinoflagellates Alexandrium spp. and development of genetic diagnosis method of toxic dinoflagellates(1) Phylogenetic analysis based on 18S rDNA sequences and the D1/D2 region in 28S rDNA were examined among the many isolates of toxic A.tamarense, A.catenella, A.tamiyavanichii, A.ostenferdii and nontoxic A.affine, A.fraterculus, A.insuetum, A.psedogonyaulax.(2) A rapid and precise identification method was established using a fluorescent, rRNA-targeted, oligonucleotide probes for toxic A.tamiyavanichii which are recently observed in Harima-Nada and the nontoxic A.affine and A.frateruculus. Each probe was species specific when applied using fluorescence in situ hybridization (FISH).(3) Real-time PCR assay was designed, evaluated and established for rapid detection and quantification of the vegetative cells and cysts (hypnozygotes) of toxic dinoflagellates A.tamarense and A.catenella.(4) The screening methods of the specific genes expressed in differential stages, such as cyst formation and germination of A.tamarense and A.catenella, were developed by use of differential display and subtractive hybridization methods. As a result, some genes specific for the sexual fusion and germination stages were detected.