ABSTRACT
The planktonic food web in the oligotrophic Mediterranean Sea is dominated by small-sized (<20 µm) microbes, with nanoflagellates being the major bacterial grazers and the main participants in nutrient cycling. Phosphate is a key nutrient in the P-limited Cretan Sea (NE Mediterranean) and P-availability can affect its trophic dynamics. Here, we examined the grazing potential of heterotrophic (HF) and pigmented (PF) nanoflagellates as a response mechanism to phosphate amendment. Flagellate grazing effect on bacteria was quantified in P-amended nutrient-depleted water from the Cretan Sea over the course of 4 days using microcosm experiments. P-addition positively affected HF abundance, while PF abundance remained unchanged. At the community level, P-addition had a negative effect on PF bacterial removal rates. In the control, PF-grazing rate was significantly higher than that of HF throughout the experiment. Pigment analysis showed no changes in phytoplankton community composition as a result of P-addition, indicating that PF grazing rate declined as a physiological response of the cells. The present study emphasizes the dominant grazing role of PF under P-depleted conditions and reveals that during the late stratified season PF respond to P-addition by lowering their grazing rates, enhancing the relative importance of bacterial removal by HF.
Subject(s)
Phosphates , Seawater , Bacteria , Humans , Mediterranean Sea , PhytoplanktonABSTRACT
Chlorophyll a (Chl a) is the predominant pigment in every single photosynthesizing organism including phytoplankton and one of the most commonly measured water quality parameters. Various methods are available for Chl a analysis, but the majority of them are of limited throughput and require considerable effort and time from the operator. The present study describes a high-throughput, microplate-based fluorometric assay for rapid quantification of Chl a in phytoplankton extracts. Microplate sealing combined with ice cooling was proved an effective means for diminishing solvent evaporation during sample loading and minimized the analytical errors involved in Chl a measurements with a fluorescence microplate reader. A set of operating parameters (settling time, detector gain, sample volume) were also optimized to further improve the intensity and reproducibility of Chl a fluorescence signal. A quadratic regression model provided the best fit (r 2 = 0.9998) across the entire calibration range (0.05-240 pg µL-1). The method offered excellent intra- and interday precision (% RSD 2.2 to 11.2%) and accuracy (% relative error -3.8 to 13.8%), while it presented particularly low limits of detection (0.044 pg µL-1) and quantification (0.132 pg µL-1). The present assay was successfully applied on marine phytoplankton extracts, and the overall results were consistent (average % relative error -14.8%) with Chl a concentrations (including divinyl Chl a) measured by high-performance liquid chromatography (HPLC). More importantly, the microplate-based method allowed the analysis of 96 samples/standards within a few minutes, instead of hours or days, when using a traditional cuvette-based fluorometer or an HPLC system. Graphical abstract TChl a concentrations (i.e. sum of Chl a and divinyl Chl a in ng L-1) measured in seawater samples by HPLC and fluorescence microplate reader.
