Molecular Characterization and Development of Molecular Probes for Harmful Algal Bloom causing Raphidophyceae from Hong Kong  

Project Title: Molecular Characterization and Development of Molecular Probes for Harmful Algal Bloom causing Raphidophyceae from Hong Kong

Applicant: Hong Kong University of Science and Technology

Project Proponent: Dr. Joseph Wong Tin-yum

Total Approved Grant: $668,244.13 (jointly funded by ECF and WWGF on a 50/50 basis)

Duration: 1 January 2003 - 31 December 2004

Project Status/Remarks: Completed

Scope:
The project aims to characterize and develop molecular probes for the detection of Raphidophyceae, which reportedly causes harmful algal bloom (HAB). The project involves compilation of a database of ribosomal DNA sequence for certain genera of Raphidophyceae, isolation and characterization of LsurDNA sequence from certain species isolated in Hong Kong, and development of DNA probes and protocols for the molecular detection of those species. The proposed work is expected to enhance rapid identification of causative species of HABs.

Summary of the Findings/Outcomes :
Harmful algal blooms caused by members of the raphidophytes always cause massive fish kills. The present project characterized three Hong Kong strains of raphidophytes at the molecular level by isolation and sequencing of two genetic markers (D1/D2 and ITS). The sequences of the two Chattonella species were highly similar with each other, but differ significantly from H.akashiwo. The data generated in the present study also allow the comparison of the Hong Kong isolates with other strains in the world. We were able to design DNA primers that are Chattonella-specific and do not generate products with H.akashiwo by Polymerase Chain Reaction (PCR). The two species of Chattonella are distinguishable from each other via Random Amplification of Polymorphic DNA (RAPD) analysis. Direct PCR with whole cell did not generate observable results, probably because of the resistance of the raphidophyte cell wall. We developed, for the first time, detergent-based single-cell PCR protocols for the raphidophytes, after testing a whole series of PCR conditions. Based on this success, we further developed single-cell nested-PCR and single-cell RAPD protocols for the molecular identification of the three Hong Kong raphidophytes. These technologies newly developed in this study will allow rapid identification of this important group of algae.