It is widely accepted that alkaline phosphatase activity (APA) is an efficient indicator of phosphate limitation in freshwater phytoplankton communities. In this study, we investigated whether the response in APA to phosphate limitation differs among the taxa in a mixed phytoplankton assemblage. We used the new enzyme-labeled fluorescence (ELF) technique, which allows microscopic detection of phosphate limitation in individual cells of multiple species. The most prominent findings of this study were that alkaline phosphatase (AP) was induced in many, but not all taxa and that different taxa, as well as different cells within a single taxon, experienced different degrees of phosphate stress under the same environmental conditions. Our approach was to manipulate the limiting nutrient in a natural freshwater phytoplankton community by incubating lake water in the laboratory. We induced nitrogen (N) or phosphate limitation through additions of inorganic nutrients. Both the ELF assay and bulk APA indicated that the lake phytoplankton were not phosphate limited at the start of the experiment. During the experiment, several chlorophyte taxa (e.g., Eudorina and an unidentified solitary spiny coccoid) were driven to phosphate limitation when inorganic N was added, as evidenced by a higher percentage of ELF-labeled cells relative to controls, whereas other chlorophyte taxa such as Actinastrum and Dictyosphaerium were not phosphate stressed under these conditions. In the phosphate-limited treatments, little or no ELF labeling was observed in any cyanobacterial taxa. Furthermore, all taxa observed after the ELF labeling procedure (Ͼ10-m fraction) were labeled with ELF at least on one occasion, demonstrating the wide applicability of the ELF method. By using ELF labeling in tandem with bulk APA, the resolution and analysis of phosphate limitation was increased, allowing the identification of specific phosphate-stressed taxa.A common dilemma when studying nutrient limitation in phytoplankton is that a choice must be made between work-1 To whom correspondence should be sent. Present address:

No abstract

Abstract:We investigated how clearing of the tropical rainforest for cattle pasture along small, terra firme lowland streams in the Brazilian Amazon influenced stream hydraulic characteristics, solute concentrations and uptake of dissolved inorganic nitrogen and phosphorus. Measurements of stream channel structure, hydraulic properties and nutrient uptake were obtained from short-term conservative and non-conservative solute injections and an in-stream transport model in two watersheds with pairs of similar-sized forest and pasture streams in Rondônia. The pasture stream channels were deeper and had a lower cover of sandy bottom habitat and a higher cover of aquatic grass habitat than the forest streams. The pasture streams had larger transient storage zones, higher ratios of transient to channel storage and a shorter hydraulic uptake length than the forest streams. The pasture streams had lower concentrations of dissolved oxygen and NO 3 and higher concentrations of dissolved Fe 2C . Forest streams exhibited no NO 3 uptake, but one pasture stream took up NO 3 while the other did not. Uptake of NH 4 C was low and variable among streams. Uptake velocities and rates of PO 4 3 were greater in pasture streams compared to those in forest streams. In all streams, uptake lengths for NO 3 , NH 4C and PO 4 3 were long and indicated generally lower rates of uptake than in most comparably sized temperate forest streams. Uptake lengths or velocities were not correlated with stream transient storage, suggesting that other factors, such as hypoxia in pasture streams, controlled nutrient uptake. The structural differences are typical for the region, suggesting that deforestation may be altering stream hydrology and biogeochemistry over many thousands of kilometres of primary and secondary stream channels in the Amazon Basin. A better understanding of the extent and distribution of altered hydraulic and biogeochemical function in small streams is required to assess the importance of these changes for larger river networks in the Amazon basin.

A new method was utilized to study species‐specific responses of phytoplankton to phosphorus limitation in a nutrient enrichment experiment. A substrate, ELF, produces a fluorescent precipitate at the sites of alkaline phosphatase (AP), which makes it possible to visually detect phosphorus (P) limitation in individual cells of multiple species. Lake water was incubated in the laboratory to induce nitrogen (N) or P limitation. Initially, little or no ELF labeling was observed for any of the phytoplankton species, indicating a general lack of P limitation. This observation was supported by low bulk AP activity in the initial field samples. During the experiment, several chlorophyte taxa (Coelastrum, Eudorina, a solitary spiny coccoid) were driven to P limitation, as evidenced by a high percentage of cells displaying ELF labeling when inorganic N was added. Taxa such as Actinastrum and Dictyosphaerium, on the contrary, were never P limited. Little or no ELF was observed in cyanobacterial species, suggesting that P limitation was not achieved in these organisms. Using traditional bulk AP activity, significantly higher levels of AP activity were observed in treatments with inorganic N additions, compared to those with phosphate additions. ELF labeling generally followed the trend of bulk AP, except in species that did not dominate the biomass. Finally, we noted that all species observed were ELF labeled at least on one occasion, except for fragile flagellates which did not withstand the labeling procedure.

A bi-weekly seasonal study was conducted in Ashumet Pond (Cape Cod, Massachusetts). The Redfield Ratio (106C: 16N: 1P) and alkaline phosphatase activity (APA) were utilized in tandem as nutrient deficiency indicators (NDIs) for phytoplankton. The study objective was to evaluate the limiting nutrient status of the pond throughout the growing season.The development of a high throughput method for fluorometrically measuring APA allowed for a large quantity of pond-water samples to be analyzed. The new method utilized a cytofluor, a fluorescence multi-well plate reader, which increased sample throughput by 75% compared to a standard filter fluorometer method. The detection limit, capability to measure APA at different time intervals, and performance at sea were tested. APA measurements made using the cytofluor were comparable to those made using a standard filter fluorometer, thus indicating that the cytofluor is a suitable and preferred replacement to the fluorometer for APA measurements.The presence of alkaline phosphatase, an inducible phospho-hydrolytic enzyme, is commonly used as an NDI diagnostic for phosphate limitation. A nutrient enrichment incubation re-affirmed the use of APA as a robust indicator of phosphate limitation in phytoplankton. APA data indicate that the system experienced episodic periods of phosphate-deficiency, implying that the limiting nutrient regime was not static, but was changeable throughout the growing season. Seasonal trends in dissolved N:P and particulate C:P ratios often contradict the APA results, however, suggesting that the Redfield Ratio is an unreliable indicator of the overall nutrient limitation regime of the pond. The observed discrepancies between C:N:P and APA can be reconciled by taking into account seasonal changes in species composition, which played an important role in driving seasonal APA trends.