Experimental investigation of taxon‐specific response of alkaline phosphatase activity in natural freshwater phytoplankton

Abstract

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:

Limitations of using extracellular alkaline phosphatase activities as a general indicator for describing P deficiency of phytoplankton in Chinese shallow lakes

“…They were always positive to ELF substrate in Lake Erken (Rengefors et al 2001). When induced phosphate limitation through additions of inorganic N in lake water in the laboratory, several chlorophyte taxa (e.g., Eudorina and an unidentified solitary spiny coccoid) began to be driven to phosphate limitation as evidenced by a higher percentage of ELF-labeled cells (Rengefors et al 2003). While in the phosphate-limited treatment, little or no ELF-labeling was observed in any cyanobacterial taxa (Rengefors et al 2003).…”

“…When induced phosphate limitation through additions of inorganic N in lake water in the laboratory, several chlorophyte taxa (e.g., Eudorina and an unidentified solitary spiny coccoid) began to be driven to phosphate limitation as evidenced by a higher percentage of ELF-labeled cells (Rengefors et al 2003). While in the phosphate-limited treatment, little or no ELF-labeling was observed in any cyanobacterial taxa (Rengefors et al 2003). Because of their small size and large cell-specific surfaces, it is possible that cyanobacteria did not experience any P stress (Reynolds 1997).…”

Flow cytometric detection of phosphatase activity combined with 13C-CO2 tracer-based growth rate assessment in phytoplankton populations from a shallow lake

“…The objections to bulk AP activity measurements are largely met by use of a novel phosphatase substrate, a member of the enzyme-labelled fluorescence substrate family (ELF-97 TM ) that yields highly fluorescent precipitates of ELF alcohol (ELFA) at the site of enzymatic activity (Huang et al 1993, Haugland 1995. ELF phosphate (ELFP) has been used for the detection of endogenous phosphatases in complex mixtures of marine phytoplankton by FCM (González-Gil et al 1998) and fluorescence microscopy (Dyhrman & Palenik 1999), and is also applicable to freshwater phytoplankton (Rengefors et al 2001, Nedoma et al 2003, Rengefors et al 2003, Dignum et al 2004. In this study we apply ELFP in FCM to assess the P i -sensing status of individual cells and trichomes in the phytoplankton community of Lake Loosdrecht (The Netherlands) and link this information to 13 C-CO 2 tracerbased growth rates derived from FCM-sorted fractions of selected populations.…”

“…group itself is known to include several closely related species with trichomes of highly variable length and width (Zwart et al 2004). Differences in the thickness of the cells' outer layers and subsequent longer diffusion time may have caused uneven staining among cells (see also Rengefors et al 2003). Another explanation, however, is that population heterogeneity arises from the cells being in different growth phases.…”

Specific-detection of alkaline phosphatase activity in individual species of marine phytoplankton

“…ELF-based assays have already been applied to field samples of phytoplankton in order to assess phosphorus-state at a single-species level (Dyhrman & Palenik 1999Rengefors et al 2001Rengefors et al , 2003Štrojsová et al 2003). Presence and absence of ELF-labeled cells may represent the phosphorusstarved and not starved state of the AP-producing species, respectively.…”