Expression and standardized measurement of hemolytic activity in Heterosigma akashiwo

Heterosigma akashiwo (Hada) is a marine Raphidophyte responsible for fish deaths in temperate coastal waters. Factors leading to Heterosigma species dominance or levels of toxicity remain unresolved. This study investigated the role of several factors on the hemolytic activity of the ichthyotoxic alga Heterosigma akashiwo. Hemolytic activity was measured using the erythrocyte lysis assay, which involves incubation of an algal sample with erythrocytes and subsequent photometric measurement of hemoglobin released from lysed erythrocytes. Preliminary experiments determined the optimal assay conditions (e.g. algal sample type, cell density, incubation conditions) to detect maximal hemolytic activity in H. akashiwo. Subsequent experiments determined the roles of light; isolate variation, and growth stage. The expression of hemolytic assay was light dependent, dependent on the stage of growth and but varied little among isolates. Dramatic variations in the measurement of hemolytic activity were evident depending on the handling and preparation of the sample.