A single-cell immunoassay for phosphate stress in the dinoflagellate Prorocentrum minimum (Dinophyceae)

Current techniques for studying phytoplankton physiology in the field, such as measurements of biochemical activities, nutrient addition bioassays, an d determination of photosynthetic efficiency, are useful for assessing the physiology of the bulk community but suffer from a lack of specificity. Thi s would be improved by the development of single-cell methods for monitorin g in situ physiology, Here we develop and test an antibody-based assay for identifying phosphate stress in the model dinoflagellate Prorocentrum minim um (Pavillard) Schiller, Antiserum was raised against a cell-surface alkali ne phosphatase purified from P, minimum. Western screening indicated that t he antiserum reacted with phosphate-stressed cells but not nitrate-stressed or phosphate-replete cells in culture. Immunodepletion confirmed the ident ification of this protein as an alkaline phosphatase, Based on Western blot s, the antiserum appeared to be specific for phosphate-regulated proteins i n P, minimum because there is no discernible cross-reaction with closely re lated P, micans. A whole-cell immunofluorescence assay was used to identify phosphate stress in field populations of P, minimum from Narragansett Bay, Rhode Island. The percentage of labeled P, minimum cells in this environme nt during the summer of 1998 decreased through time as the inorganic phosph ate concentration increased. The percentage of antibody-labeled cells signi ficantly correlated with the percentage of ELF-97-labeled cells determined as another single-cell assay of phosphate stress. This is the first antibod y-based method developed for monitoring cell-specific physiology in a dinof lagellate, and the method described here may serve as a model for developin g similar tools in other species of phytoplankton.