Phosphate stress in cultures and field populations of the dinoflagellate Prorucentrum minimum detected by a single-cell alkaline phosphatase assay

Alkaline phosphatase activity is a common marker of phosphate stress in man y phytoplankton, but it has been difficult to attribute alkaline phosphatas e activity to specific organisms or groups of phytoplankton in the field wi th traditional biochemical procedures, A ne cv alkaline phosphatase substra te, ELF-97 (enzyme-labeled fluorescence), shows promise in this regard. Whe n a phosphate group is cleaved from the ELF-97 reagent, the remaining molec ule precipitates near the site of enzyme activity, thus fluorescently taggi ng cells with alkaline phosphatase activity. We characterized ELF-97 labeli ng in axenic cultures of a common dinoflagellate, Prorocentrum minimum, in order to understand ELF-97 labeling dynamics when phosphate nutrition varie s. Enzyme activity, as detected by ELF-97 labeling, appears to be induced i n late-log- or early-stationary-phase cultures if cells are grown in low-ph osphate media and is lost when phosphate-stressed cells are refed with phos phate, ELF-97 appears to label an inducible intracellular alkaline phosphat ase in P, minimum based on confocal microscopy studies. This may limit the use of this reagent to organisms that lack high levels of constitutive intr acellular phosphatases, After laboratory cultures were characterized, ELF-9 7 was used to assay field populations of P, minimum in Narragansett Bay dur ing two 1-week periods, and 12 to 100% of the P. minimum cells were labeled . The level of cell labeling was reduced by 3 days of incubation with added inorganic phosphate. Our results indicate that ELF-97 is an excellent new tool for monitoring phytoplankton phosphate stress in the environment when the data are supported by appropriate laboratory studies.