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Alkaline phosphatase (AP) activity in marine and freshwater phytoplankton has been associated with phosphorus (P) limitation whereby the enzyme functions in the breakdown of exogenous organic P con~pounds to utilizable inorganic forms. Current enzyme assays to determine the P status of the phytoplankton measure only the AP activity of the whole community and do not yield information on ~ndiv~dual species. A new insoluble fluorogenic substrate for AP, termed ELF (EnzymeLabeled Fluorescence), yields a stable, highly fluorescent precipitate at the site of enzyme act~vity and thus has the capability to determine the P status of indimdual cells. In t h~s study, ELF was utilized for in situ detection and quantification of AP in marine phytoplankton cultures and a comparison was made between the insoluble ELF substrate and several soluble AP substrates [3-0-methylfluorescein phosphate (MFP), 3,6-fluorescein diphosphate (FDP) and Attophos]. Non-axenic batch cultures of Alexandrium fundyense, Arnphidln~um sp, and Isochrysis galbana were grown in different media types using orthophosphate as an inorganic source and sodium-glycerophosphate as an organic source, with final phosphate concentrations ranging from 38.3 to 3 p M (i.e. f/2, f/40, f/80, plus ambient P). Epifluorescence microscopy was used to determine if and where the cells were labeled with ELF, while flow cytometry was used to quantify the amount of ELF retained on individual cells. The detection of the soluble substrates utilized a multiwell fluorescence plate reader (CytofluorTh4). Only cells grown in low phosphate concentrations (f/40, f/80) exhibited the bright green fluorescence signal of the ELF precipItate. This signal was always observed for P-starved Amphidiniurn sp. and I galbana cells, but was seen in some A, fundyense cells only during the late stationary phase. Cells grown in high phosphate concentrations (i.e. at f/2 levels) showed no ELF fluorescence. Slightly positive soluble substrate assays suggest that these species may have produced small amounts of AP constitutively that were not detected with the precipitable substrate. Similar results were obtained when the cultures were analyzed by flow cytometry. Except for A. fundyense, cells grown In low phosphate concentrations showed high ELF fluorescence. However, no positive ELF fluorescence was detected with the Cytofluor for all 3 species due to lack of instrument sensitivity. Comparable analysis using the soluble substrates MFP, FDP, and Att~phos-l-~' on the Cytofluor showed little activity for A , fundyense, but high fluorescence for P-starved Amphldiniun? sp. and I. galbana. Insoluble ELF thus provides a means to detect and quantify AP in individual cells using visual observations or flow cytometry. This technique offers a new level of resolution and sensitivity at the single cell level that can provide insights into the P nutrition of phytoplankton and other microorganisms in natural waters.

During late spring and early summer of 2005, large-scale (>15 000 km 2 ), mixed dinoflagellate blooms developed along the the coast of the East China Sea. Karenia mikimotoi was the dominant harmful algal bloom species in the first stage of the bloom (late May) and was succeeded by Prorocentrum donghaiense approximately 2 wk later. Samples were collected from different stations along both north-south and west -east transects, from the Changjiang River estuary to the south Zhejiang coast, during 3 cruises of the Chinese Ecology and Oceanography of Harmful Algal Blooms Program, before and during the bloom progression. Nitrogen isotope tracer techniques were used to measure rates of NO 3 -, NH 4 + , urea, and glycine uptake during the blooms. High inorganic nitrogen (N), but low phosphorus (P) loading from the Changjiang River led to high dissolved inorganic N:dissolved inorganic P ratios in the sampling area and indicate the development of P limitation. The rates of 15 N-uptake experiments enriched with PO 4 3 -were enhanced compared to unamended samples, suggesting P limitation of the N-uptake rates. The bloom progression was related to the change in availability of both organic and inorganic N and P. Reduced N forms, especially NH 4 + , were preferentially taken up during the blooms, but different bloom species had different rates of uptake of organic N substrates. K. mikimotoi had higher rates of urea uptake, while P. donghaiense had higher rates of glycine uptake. Changes in the availability of reduced N and the ratios of N:P in inorganic and organic forms were suggested to be important in the bloom succession. Nutrient ratios and specific uptake rates of urea were similar when compared to analogous blooms on the West Florida Shelf.

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