Community heterogeneity and single-cell digestive activity of estuarine heterotrophic nanoflagellates assessed using lysotracker and flow cytometry.

Heterotrophic nanoflagellates (HNFs) are an essential component of all aquatic microbial food webs, and yet the exploration of the numerical and single-cell responses of these organisms in mixed assemblages still represents a major technical challenge. LysoTracker Green staining combined with flow cytometry was recently proposed for the enumeration of aquatic HNFs. Here we show that LysoTracker Green not only allows the enumeration of HNFs in estuarine samples with a wide range of HNF abundances, but also allows the discrimination of distinct HNF populations in mixed assemblages. In addition, the resulting cytometric parameters can be used to characterize cell size and the level of activity of the cells in the different populations that are detected. LysoTracker Green accumulates preferentially in lysosomes, and we demonstrate that the green fluorescence emission from HNF cells stained with LysoTracker strongly correlates with cell-specific beta-glucosaminidase (beta-Gam) activity, a key digestive enzyme of lysosomal origin in eukaryotic cells. Our results further show that different populations that develop in estuarine regrowth cultures are characterized by different intrinsic ranges of size and of feeding activity, and that there is a wide range of single-cell responses within these HNF populations. We found a large degree of uncoupling between cell size and feeding activity, both between and within HNF populations, and there appears to be no clear allometric scaling of feeding activity. We were able to reconstruct the succession of distinct HNF populations that developed during the regrowth experiments, and explore the complex interactions that occurred between numerical (change in abundance of the cytometric populations) and single-cell HNF responses.