Picophytoplankton growth rates and nutrient limitation estimated by flow cytometry

Béatrice Bec, Yves Collos and André Vaquer

Laboratoire "Ecosystèmes lagunaires" UMR 5119 - CNRS, Université Montpellier II, case 093, Place Eugène Bataillon, 34095 MONTPELLIER cedex 05
FRANCE

Corresponding author : bbec@univ-montp2.fr

Flow cytometry

Different populations of picophytoplankton can be distinguished by flow cytometry on the basis of their light scatter signals, which is related to cell size and structure; and their autofluorescence resulting from excitation with 488 nm laser and related to photosynthetic pigment composition. In fact, two types of natural fluorescence can be differentiated by flow cytometry: the red fluorescence emission due to chlorophyll ( > 650 nm) and the orange fluorescence due to phycoerythrin (PE, = 542-582 nm). Picophytoplankton abundances were estimated with a FACSCalibur flow cytometer (Becton Dickinson). All samples were analyzed with a mixture of fluorescent beads of 0.96, 1.8 and 2.97µm diameter ("Fluoresbrite" YG, Polysciences, Inc.) in order to normalize all parameters.

Data examples

In two marine Mediterranean coastal lagoons, the combination of dot plots of light scatter vs red fluorescence, and dot plots of light scatter vs orange fluorescence allowed to discriminate picoeukaryotes and PE-rich cyanobacteria, which were examined by transmission electron microscopy (Figure 1) as Synechococcus spp. (Waterbury et al, 1979). In both lagoons, the red fluorescence and light scatter of eukaryotic picoplankton were slightly higher than those of cyanobacteria populations.

Figure 1. Electron micrographs of Synechococcus spp. showing typical structures of the organism; T, thylakoids; C, carboxysomes; SL, surface layer.

In Thau lagoon (Figure 2), picophytoplankton was represented by a minuscule photosynthetic picoplankton, Ostreococcus tauri , which is the smallest autotrophic picoeukaryote identified to date (<1µm, Courties et al, 1994) and dominant in the picoplankton community of the lagoon (Vaquer et al 1996). However, during summer, picocyanobacteria were abundant and outnumbered the picoeukaryote population.

Figure 2. Flow cytograms corresponding to dot plots of side light scatter (abscissa axis) versus red or orange fluorescence (ordinate axis) of Thau lagoon water sample. Red and orange fluorescence histograms are presented below cytograms. The eukaryotic picoplankton Ostreococcus tauri and PE-rich cyanobacteria are identified by the green and orange cluster respectively. Grey dots correspond to non-photosynthetic particles such as detritus.

In Salses-Leucate lagoon (Figure 3), two sub-populations of cyanobacteria could be distinguished from differences in orange fluorescence (Cyan 1 & Cyan 2 with weak and strong fluorescence respectively) and a eukaryotic population was also identified but it was less abundant.

Figure 3. Flow cytograms corresponding to dot plots of red/orange fluorescence (ordinate axis) versus side light scatter (abscissa axis) of Salses-Leucate lagoon water sample. Red and orange fluorescence histograms are represented below cytograms. Two sub-populations of cyanobacteria (orange and yellow clusters) are discriminated by their orange fluorescence. The picoeukaryote population (green cluster) is less abundant than the Ostreococcus tauri population in Thau lagoon.

In both lagoons, growth rates of various picoplanktonic groups were estimated from in situ incubations with differential enrichments (Andersen et al, 1991), and were calculated from temporal changes (24h) of cell densities according to the dilution method (Landry and Hassett, 1982). Although flow cytometry characteristics of picophytoplankton seemed to be very similar in both Mediterranean lagoons (Figure 4), growth rates responses were different.

Figure 4. Comparison of growth rates (d^-1) responses of picophytoplankton to different nutrient enrichments: Controls (open bar), enrichment without nitrogen (large hatched bar) or without phosphorus (fine hatched bar), complete enrichment (filled bar). Horizontal bars above histograms indicate treatments not significantly different from each other based on PLSD Fisher comparison test (p<0.05).

In fact, the picoplankton community (picoeukaryotes and cyanobacteria) appeared nitrogen limited in the Thau lagoon (figure 4A). Growth rates ranged from 0.10 d^-1 in enrichment without nitrogen to 1.30 d^-1 in complete enrichment for Ostreococcus tauri, and from 0.39 d^-1 in control to 1.24 d^-1 in complete enrichment for cyanobacteria. In contrast, in Salses-Leucate lagoon (figure 4B), the picoplankton community was not nutrient limited. Picoeukaryotes growth rates ranged from 0.96 d^-1 in control to 2.17 d^-1 in enrichment without nitrogen whereas both cyanobacteria sub-populations (Cyan 1&2) seemed more competitive in enrichment without phosphorus (1.1 and 1.29 d^-1 respectively).
Thus flow cytometry allows the discrimination of various groups within the picophytoplankton, and permits to compare growth capacities and to detect possible nutrient limitation of this photosynthetic community based on cell density, their light scatter and autofluorescence.

Cited references

Andersen, T., Schartau, A.K.L., Paasche, E. 1991. Quantifying external and internal nitrogen and phosphorus pools, as well as nitrogen and phosphorus supplied through remineralization, in coastal marine plankton by means of a dilution technique. Mar Ecol Prog Ser 69: 67-80.

Courties C., Vaquer A., Troussellier M., Lautier J., Chrétiennot-Dinet M. J., Neveux J., Machado C., H. Claustre. 1994. Smallest eukaryotic organism. Nature 370 : 255.

Landry, M. R., Hassett, R. P. 1982. Estimating the grazing impact of micro-zooplankton. Mar. Biol.67: 283-288.

Vaquer A., Troussellier M., Courties C., Bibent B. 1996. Standing stock and dynamics of picophytoplankton in the Thau lagoon (northwest Mediterranean coast). Limnol. Oceanogr. 41:1821-1828.

Waterbury J. B., Watson S. W., Guillard R. R. L., Brand L. E. 1979. Widespread occurrence of a unicellular, marine, planktonic cyanobacterium. Nature 277: 293-294.