Extracellular phosphatase activity (PA) has been used as an overall indicator of P depletion in lake phytoplankton. However, detailed insights into the mechanisms of PA regulation are still limited, especially in the case of acid phosphatases. The novel substrate ELF97 phosphate allows for tagging PA on single cells in an epifluorescence microscope. This fluorescence-labeled enzyme activity (FLEA) assay enables for autecological studies in natural phytoplankton and algal cultures. We combined the FLEA assay with image analysis to measure cell-specific acid PA in two closely related species of the genus Coccomyxa (Trebouxiophyceae, Chlorophyta) isolated from two acidic lakes with distinct P availability. The strains were cultured in a mineral medium supplied with organic (beta-glycerol phosphate) or inorganic (orthophosphate) P at three concentrations. Both strains responded to experimental conditions in a similar way, suggesting that acid extracellular phosphatases were regulated irrespectively of the origin and history of the strains. We found an increase in cell-specific PA at low P concentration and the cultures grown with organic P produced significantly higher (ca. 10-fold) PA than those cultured with the same concentrations of inorganic P. The cell-specific PA measured in the cultures grown with the lowest organic P concentration roughly corresponded to those of the original Coccomyxa population from an acidic lake with impaired P availability. The ability of Coccomyxa strains to produce extracellular phosphatases, together with tolerance for both low pH and metals can be one of the factors enabling the dominance of the genus in extreme conditions of acidic lakes. The analysis of frequency distribution of the single-cell PA documented that simple visual counting of 'active' (labeled) and 'non-active' (non-labeled) cells can lead to biased conclusions regarding algal P status because the actual PA of the 'active' cells can vary from negligible to very high values. The FLEA assay using image cytometry offers a strong tool in plankton ecology for exploring P metabolism.

Department of Ecology Faculty of Science Charles University Prague Czechia

Department of Ecosystem Biology Faculty of Science University of South Bohemia České Budějovice Czechia

Department of Zoology Fisheries Hydrobiology and Apiculture Faculty of AgriSciences Mendel University Brno Czechia

Institute of Hydrobiology Biology Centre CAS České Budějovice Czechia

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Barcytė D., Nedbalová L. (2017). Coccomyxa: a dominant planktic alga in two acid lakes of different origin. Extremophiles 21 245–257. 10.1007/s00792-016-0899-6 PubMed DOI

Berman T., Wynne D., Kaplan B. (1990). Phosphatases revisited: analysis of particle-associated activities in aquatic systems. Hydrobiologia 207 287–294. 10.1007/BF00041467 DOI

Bischoff H. W., Bold H. C. (1963). Phycological Studies. IV. Some Soil Algae from Enchanted Rock and Related Algal Species. Austin, TX: University of Texas Publications.

Boavida M. J., Heath R. T. (1984). Are the phosphatases released by Daphnia magna components of its food? Limnol. Oceanogr. 29 641–645. 10.4319/lo.1984.29.3.0641 DOI

Cao X., Song C., Zhou Y., Štrojsová A., Znachor P., Zapomělová E., et al. (2009). Extracellular phosphatases produced by phytoplankton and other sources in shallow eutrophic lakes (Wuhan, China): taxon-specific versus bulk activity. Limnology 10 95–104. 10.1007/s10201-009-0265-9 DOI

Cao X. Y., Štrojsová A., Znachor P., Zapomělová E., Liu G. X., Vrba J., et al. (2005). Detection of extracellular phosphatases in natural spring phytoplankton of a shallow eutrophic lake (Donghu, China). Eur. J. Phycol. 40 251–258. 10.1080/09670260500192760 DOI

Carr O. J., Goulder R. (1990). Fish-farm effluents in rivers. I. Effects on bacterial populations an alkaline phosphatase activity. Water Res. 24 631–638. 10.1016/0043-1354(90)90196-D DOI

Cembella A. D., Antia N. J., Harrison P. J. (1984). The utilization of inorganic and organic phosphorus compounds as nutrients by eukaryotic microalgae: a multidisciplinary perspective: Part I. Crit. Rev. Microbiol. 10 317–391. 10.3109/10408418209113567 PubMed DOI

Chróst R. J. (1991). “Environmental control of the synthesis and activity of aquatic microbial ectoenzymes,” in Microbial Enzymes in Aquatic Environments ed. Chróst R. J. (New York, NY: Springer-Verlag; ) 29–59.

Cotner J. B., Wetzel R. G. (1991). 5′-Nucleotidase activity in a eutrophic lake and an oligotrophic lake. Appl. Environ. Microbiol. 57 1306–1312. PubMed PMC

Cotner J. B., Wetzel R. G. (1992). Uptake of dissolved inorganic and organic phosphorus compounds by phytoplankton and bacterioplankton. Limnol. Oceanogr. 37 232–243. 10.4319/lo.1992.37.2.0232 DOI

Currie D. J., Kalff J. (1984). A comparison of the abilities of freshwater algae and bacteria to acquire and retain phosphorus. Limnol. Oceanogr. 29 298–310. 10.4319/lo.1984.29.2.0298 DOI

Dell Inc. (2016). Dell Statistica (Data Analysis Software System), Version 13.2.

Dignum M., Hoogveld H. L., Matthijs H. C. P., Laanbroek H. J., Pel R. (2004). Detecting the phosphate status of phytoplankton by enzyme-labelled fluorescence and flow cytometry. FEMS Microbiol. Ecol. 48 29–38. 10.1016/j.femsec.2003.12.007 PubMed DOI

Dyhrman S. T., Palenik B. (1999). Phosphate stress in cultures and field populations of the dinoflagellate Prorocentrum minimum detected by a single-cell alkaline phosphatase assay. Appl. Environ. Microbiol. 65 3205–3212. PubMed PMC

Dyhrman S. T., Ruttenberg K. C. (2006). Presence and regulation of alkaline phosphatase activity in eukaryotic phytoplankton from the coastal ocean: implications for dissolved organic phosphorus remineralization. Limnol. Oceanogr. 51 1381–1390. 10.4319/lo.2006.51.3.1381 DOI

González-Gil S., Keafer B. A., Jovine R. V. M., Aguilera A., Lu S., Anderson D. M. (1998). Detection and quantification of alkaline phosphatase in single cells of phosphorus-starved marine phytoplankton. Mar. Ecol. Prog. Ser. 164 21–35. 10.3354/meps164021 DOI

Healey F. P., Hendzel L. L. (1979). Fluorometric measurement of alkaline-phosphatase activity in algae. Freshwat. Biol. 9 429–439. 10.1111/j.1365-2427.1979.tb01527.x DOI

Healey F. P., Hendzel L. L. (1980). Physiological indicators of nutrient deficiency in lake phytoplankton. Can. J. Fish. Aquat. Sci. 37 442–453. 10.1139/f80-058 DOI

Hoppe H. G. (1983). Significance of exoenzymatic activities in the ecology of brackish water: measurements by means of methylumbelliferyl substrates. Mar. Ecol. Prog. Ser. 11 299–308. 10.3354/meps011299 DOI

Hoppe H. G. (2003). Phosphatase activity in the sea. Hydrobiologia 493 187–200. 10.1023/A:1025453918247 DOI

Hrdinka T., Šobr M., Fott J., Nedbalová L. (2013). The unique environment of the most acidified permanently meromictic lake in the Czech Republic. Limnologica 43 417–426. 10.1016/j.limno.2013.01.005 DOI

Huang B. Q., Huang S. Y., Wen Y., Hong H. S. (2000). Effects of dissolved phosphorus on alkaline phosphatase activity in marine microalgae. Acta Oceanol. Sin. 19 29–35.

Huang Z., Terpetschnig E., You W., Haugland R. P. (1992). 2-(2′-phosphoryloxyphenyl)-4(3H)-quinazolinone derivates as fluorogenic precipitating substrates of phosphatases. Anal. Biochem. 207 32–39. 10.1016/0003-2697(92)90495-S PubMed DOI

Jansson M., Olsson H., Pettersson K. (1988). Phosphatases; origin, characteristics and function in lakes. Hydrobiologia 170 157–175. 10.1007/BF00024903 DOI

Jones J. G. (1972). Studies on freshwater micro-organisms: phosphatase activity in lakes of differing degrees of eutrophication. J. Ecol. 60 777–791. 10.2307/2258564 DOI

Knoll L. B., Morgan A., Vanni M. J., Leach T. H., Williamson T. J., Brentrup J. A. (2016). Quantifying pelagic phosphorus regeneration using three methods in lakes of varying productivity. Inland Waters 6 509–522. 10.1080/IW-6.4.866 DOI

Litchman E., Nguyen B. L. V. (2008). Alkaline phosphatase activity as a function of internal phosphorus concentration in freshwater phytoplankton. J. Phycol. 44 1379–1383. 10.1111/j.1529-8817.2008.00598.x PubMed DOI

Mindl B., Sonntag B., Pernthaler J., Vrba J., Psenner R., Posch T. (2005). Effects of phosphorus loading on the interactions of algae and bacteria: a reinvestigation of the “phytoplankton-bacteria paradox” in a continuous cultivation system. Aquat. Microb. Ecol. 38 203–213. 10.3354/ame038203 DOI

Nagata T., Kirchman D. L. (1992). Release of macromolecular organic complexes by heterotrophic marine flagellates. Mar. Ecol. Prog. Ser. 83 233–240. 10.3354/meps083233 DOI

Nedoma J., García J., Comerma M., Šimek K., Armengol J. (2006). Extracellular phosphatases in a Mediterranean reservoir: seasonal, spatial and kinetic heterogeneity. Freshwat. Biol. 51 1264–1276. 10.1111/j.1365-2427.2006.01566.x DOI

Nedoma J., Padisák J., Koschel R. (2003a). Utilisation of 32P-labelled nucleotide- and non-nucleotide dissolved organic phosphorus by freshwater plankton. Arch. Hydrobiol. Adv. Limnol. 58 87–99.

Nedoma J., Porcalová A., Komárková J., Vyhnálek V. (1993). Phosphorus deficiency diagnostics in the eutrophic Římov reservoir. Water Sci. Technol. 28 75–84.

Nedoma J., Štrojsová A., Vrba J., Komárková J., Šimek K. (2003b). Extracellular phosphatase activity of natural plankton studied with ELF97 phosphate: fluorescence quantification and labelling kinetics. Environ. Microbiol. 5 462–472. 10.1046/j.1462-2920.2003.00431.x PubMed DOI

Nedoma J., Vrba J. (2006). Specific activity of cell-surface acid phosphatase in different bacterioplankton morphotypes in an acidified mountain lake. Environ. Microbiol. 8 1271–1279. 10.1111/j.1462-2920.2006.01023.x PubMed DOI

Novotná J., Nedbalová L., Kopáček J., Vrba J. (2010). Cell-specific extracellular phosphatase activity of dinoflagellate populations in acidified mountain lakes. J. Phycol. 46 635–644. 10.1111/j.1529-8817.2010.00858.x DOI

Ou L. J., Huang B. Q., Hong H. S., Qi Y. Z., Lu S. H. (2010). Comparative alkaline phosphatase characteristics of the algal bloom dinoflagellates Prorocentrum donghaiense and Alexandrium catenella, and the diatom Skeletonema costatum. J. Phycol. 46 260–265. 10.1111/j.1529-8817.2009.00800.x DOI

Ren L. X., Wang P. F., Wang C., Chen J., Hou J., Qian J. (2017). Algal growth and utilization of phosphorus studied by combined mono-culture and co-culture experiments. Environ. Pollut. 220 274–285. 10.1016/j.envpol.2016.09.061 PubMed DOI

Rengefors K., Pettersson K., Blenckner T., Anderson D. M. (2001). Species-specific alkaline phosphatase activity in freshwater spring phytoplankton: application of a novel method. J. Plankton Res. 23 435–443. 10.1093/plankt/23.4.435 DOI

Rengefors K., Ruttenberg K. C., Haupert C. L., Taylor C., Howes B. L. (2003). Experimental investigation of taxon-specific response of alkaline phosphatase activity in natural freshwater phytoplankton. Limnol. Oceanogr. 48 1167–1175. 10.4319/lo.2003.48.3.1167 DOI

Reynolds C. S. (1997). Vegetation Processes in the Pelagic: A Model for Ecosystem Theory. Oldenburg: Ecology Institute.

Rychtecký P., Řeháková K., Kozlíková E., Vrba J. (2015). Light availability may control extracellular phosphatase production in the turbid environment. Microb. Ecol. 69 37–44. 10.1007/s00248-014-0483-5 PubMed DOI

Schindler D. W. (2012). The dilemma of controlling cultural eutrophication of lakes. Proc. Biol. Sci. 279 4322–4333. 10.1098/rspb.2012.1032 PubMed DOI PMC

Schindler D. W., Carpenter S. R., Chapra S. C., Hecky R. E., Orihel D. M. (2016). Reducing phosphorus to curb lake eutrophication is a success. Environ. Sci. Technol. 50 8923–8929. 10.1021/acs.est.6b02204 PubMed DOI

Siuda W., Chróst R. J. (2001). Utilization of selected dissolved organic phosphorus compounds by bacteria in lake water under non-limiting orthophosphate conditions. Pol. J. Environ. Stud. 10 475–483.

Sommer U. (1981). The role of r- and K-selection in the succession of phytoplankton in Lake Constance. Acta Oecol. 2 327–342.

Sommer U. (1985). Comparison between steady state and non-steady state competition: experiments with natural phytoplankton. Limnol. Oceanogr. 30 335–346. 10.4319/lo.1985.30.2.0335 DOI

Štrojsová A., Nedoma J., Štrojsová M., Cao X., Vrba J. (2008). The role of cell-surface-bound phosphatases in species competition within natural phytoplankton assemblage: an in situ experiment. J. Limnol. 67 128–138. 10.4081/jlimnol.2008.128 DOI

Štrojsová A., Vrba J. (2006). Phytoplankton extracellular phosphatases: investigation using ELF (Enzyme Labelled Fluorescence) technique. Pol. J. Ecol. 54 715–723.

Štrojsová A., Vrba J. (2009). Short-term variation in extracellular phosphatase activity: possible limitations for diagnosis of nutrient status in particular algal populations. Aquat. Ecol. 43 19–25. 10.1007/S10452-007-9154-7 DOI

Štrojsová A., Vrba J., Nedoma J., Komárková J., Znachor P. (2003). Seasonal study on expression of extracellular phosphatases in the phytoplankton of an eutrophic reservoir. Eur. J. Phycol. 38 295–306.

Štrojsová A., Vrba J., Nedoma J., Šimek K. (2005). Extracellular phosphatase activity of freshwater phytoplankton exposed in different in situ phosphorus concentrations. Mar. Freshw. Res. 56 417–424. 10.1071/MF04283 DOI

Vrba J., Komárková J., Vyhnálek V. (1993). Enhanced activity of alkaline phosphatases – phytoplankton response to epilimnetic phosphorus depletion. Water Sci. Technol. 28 15–24.

Vrba J., Kopáček J., Bittl T., Nedoma J., Štrojsová A., Nedbalová L., et al. (2006). A key role of aluminium in phosphorus availability, food web structure, and plankton dynamics in strongly acidified lakes. Biologia 61 S441–S451. 10.2478/s11756-007-0077-5 DOI

Vrba J., Kopáček J., Fott J., Kohout L., Nedbalová L., Pračáková M.et al. (2003). Long-term studies (1871–2000) on acidification and recovery of lakes in the Bohemian Forest (central Europe). Sci. Total Environ. 310 73–85. 10.1016/S0048-9697(02)00624-1 PubMed DOI

Wetzel R. G. (1991). “Extracellular enzymatic interactions: storage, redistribution, and interspecific communication,” in Microbial Enzymes in Aquatic Environments ed. Chróst R. J. (New York, NY: Springer-Verlag; ) 6–28.

Young E. B., Tucker R. C., Pansch L. A. (2010). Alkaline phosphatase in freshwater Cladophora-epiphyte assemblages: regulation in response to phosphorus supply and localization. J. Phycol. 46 93–101. 10.1111/j.1529-8817.2009.00782.x DOI