Use of live, fluorescently-labeled algae for measuring microzooplankton grazing in natural communities

Here, our goal was to develop a technique for staining live algae with vital fluorochromes and to further test whether this method may serve as a tool for examining the trophic roles and functional diversity of microzooplankton. We tested 4 fluorochromes on a total of 10 phytoplankton species, out of which only 3 proved effective, and only on some of the species tested. The fluorochrome Vybrant did not dye any algal species, CellTracker Blue successfully dyed 2 nanoflagellate species (Isochrysis galbana and Tetraselmis sp.) and one dinoflagellate (Heterocapsa sp.), and LysoSensor and LysoTracker each dyed 2 diatom species (Thalassiosira weissflogii and Skeletonema costatum). Further experiments with the 2 most successful fluorochromes (CellTracker and LysoSensor) indicated that optimum incubation times ranged from 4 to 8 h and that the percentage of stained cells was not improved at concentrations higher than 10 μM and 2 μM for CellTracker and LysoSensor, respectively. The residence times of the fluorochromes under natural light conditions were greater than 24 h (60-80% of stained algae). Labeling algae with CellTracker had no significant effect on their growth rate or C:N molar ratio. LysoSensor, however, had minor (although significant) effects on the growth rates of stained vs. unstained algae. Bottle grazing experiments showed that Oxyrrhis marina grazed on unstained nanoflagellate species at equal rates to those stained with CellTracker; however, a positive discrimination for stained cells was detected when Gyrodinium dominans was used as the grazer. We also measured microzooplankton ingestion rates in natural algal communities by combining the dilution method with the addition of live algae into a natural plankton suspension. The addition of stained algae did not significantly affect phytoplankton growth or mortality rates due to microzooplankton grazing. The low toxicity of fluorochromes and the easy visualization of labeled algae inside predators make this method a useful tool for estimating grazing rates of microzooplankton and for the quantification of different trophic interactions among protists in the microbial food web. However, given the limited number of algae species successfully stained further research is needed to obtain more universal dyes.