Development Of Techniques For Identification And Counting Of Toxic Alexandrium Species Along Chinese Coast

In this study, two highly variable regions of rDNA, the 5.8S ribosomal RNA gene and flanking internal transcribed spacers 1 and 2, and the 5' portion of the large-subunit rDNA (encompasses the so-called "D1" and "D2" hypervariable domains) of several strains of Alexandrium spp. isolated from the East China Sea and the South China Sea, were amplified, sequenced, and subjected to phylogenetic analysis. Besides, the same region of rDNA of several other strains of Alexandrium spp. originated from other countries or regions were also sequenced to serve as quality control of the experiment. On the basis of results above, we developed a method basing on fluorescence i n situ hybridization ( FISH) technique t o d etect t he t oxic A lexandrium species in China using two specific FITC-labeled probes targeted on Large Sub-Unit ribosomal RNA(LSU rRNA). And we try to do the counting work using Flow Cytometry( FCM) as a detector. The sequences were compared and analyzed with Clustal X and MEGA 2 software. It was found that the sequences of all the toxic A. tamarense and A. catenella isolated from Chinese coast were the same. Compared with the sequence information of Alexandrium spp. from GenBank, the sequences of A. tamarense and A. catenella from China are similar to genotype catenella. The sequences of two other strains of Alexandrium, which haven't been nominated by morphotype identification, are similar to genotype affine. The sequence of A. minutum isolated from Taiwan is very different from those of the species(LAC27 or 181NT strain) isolated from Western European, but similar with the sequence of the strain(Anakoha bay isolate) isolated from New Zealand. The FISH experiment showed that the probe SPEC-PROBE2 could identify the toxic species specifically very well. The cells labled by probe SPEC-PROBE2 could be easily identified using fluorescence microscope or flow-cytometry. The application of the technique would improve the accuracy and efficiency in routine monitoring on toxic Alexandrium species in China.