Flow cytometric measurements of cellular responses in a toxic dinoflagellate, Alexandrium minutum, upon exposure to thermal, chemical and mechanical stresses

Abstract

The purpose of the present study was to provide a flow-cytometric (FCM) approach evaluating Alexandrium minutum cellular responses to mechanical, thermal and chemical stresses. Coupling SYBR-Green I and SYTOX-Green staining, FCM analysis and sorting, and microscopic observations permitted identification and characterization of five cellular states/forms of A. minutum; (1) vegetative cells, (2) pellicle cysts, (3) degraded cells, (4) empty theca, and (5) dead cells. Storage on ice resulted in the transformation of a portion of vegetative cells into dead cells, pellicle cysts, and “degraded” cells; however, centrifugation resulted mostly in pellicle cysts and a few degraded cells. After these thermal and mechanical treatments, control and stressed cultures were monitored for 14 days. Stressed A. minutum cultures appeared to grow at the same rates as control cultures during the first seven days. During the last week of monitoring, however, cell densities of stressed cultures reached their stationary phase earlier than control cultures, suggesting incomplete recovery. Additional experiment assessing kinetics of excystment indicated that it can occur less than 9 h following mechanical stress (centrifugation) and that 75% of the culture can excyst within 24 h. Upon 30 min of exposure to chemical stressors (saponine and H 2O 2), only vegetative cells, pellicle cysts, and dead cells were detected. For both chemicals, encystment was dose-dependent. Counts of pellicle cysts increased with increasing saponine or H 2O 2 concentration. Pellicle cysts were the most-predominant cell type at chemical treatments above 0.05 g L −1 saponine and above 0.015% H 2O 2. Occurrence of dead cells appeared to follow an all-or-none response as dead-cell percentage increased from 3% at 0.015% H 2O 2 to 81% at 0.03% H 2O 2 without pellicle cyst formation. Overall, encystment–excystment of A. minutum upon changes of environmental conditions can occur very rapidly but can be monitored using FCM and SYBR-Green I staining.