Project Title : Development of Pre-bloom Fluorimetric Assays for the Monitoring of Harmful Algal Blooms

Applicant : The Hong Kong University of Science & Technology (Professor Joseph TY WONG)

Total Approved Grant : $949,489.94

Duration : 01/07/2005 to 30/06/2007

Project Status/Remarks : Completed

Scope :
The present proposal aims to develop an array of combinatory micro-fluorimetric assays, that are based on the detection of agents that modulate membrane potential, intracellular calcium and sodium ions. As the vast majority of algal toxins involved in harmful algal blooms (HABs) belong to such modulation agents, and their assays require only a small amount of algal materials and a short sample preparation time, a combinatory micro-fluorimetric assay will generate timely information before the actual bloom is formed. The present proposal will standardize a range of sodium-binding, calcium-binding and membrane potential fluorescence dyes for reporting the effects of algal toxins and toxic algal extracts on neuronal and non-neuronal cell lines. The standard response to the different agents can then be used as references for the routine monitoring of HABs.

Summary of Findings/Outcomes :
Most known algal toxins act on ion channels either directly or indirectly, resulting in a change in intracellular ion concentrations when administered to targeted cells. The present project developed the working conditions for the use of fluorescent dyes in monitoring changes in membrane potential, intracellular calcium, and intracellular sodium levels in mammalian cell lines. Using these conditions, we were able to demonstrate specific changes in fluorescent signals in response to several purified toxins. Using a very simple extraction procedure, we were able to generate algal extracts from several local and non-local species. These extracts, when administered to the developed fluorimetric assays, were able to elicit different pattern of changes in membrane potential, intracellular calcium, and intracellular sodium levels. We also assayed algal extracts from natural seawater samples. During non-bloom conditions, only very minor responses were observed in the fluorimetric assays. However, a seasonal pattern was detected. In summary, we established the basic working conditions for using fluorimetric assays to monitor mixed species in seawater samples. The differential pattern of responses induced by the different algal toxins in the three fluorimetric assays serve as a proof of concept for the use of fluorimetric assays in the monitoring of algal blooms.