Fluorescently labelled bacteria in combination with FACS successfully discriminate actively-feeding mixotrophs in a lake water sample

Mixotrophic protists are capable to act both as primary producers and primary consumers at the base of the aquatic food web, thus constituting key organisms in ecosystems where they are abundant. However, their identity, abundance, ecological dynamics and biogeochemical impact in aquatic ecosystems remains understudied in comparison to those organisms matching the classical dichotomy between heterotrophy and autotrophy. In this study, we present a method based on fluorescently labelled prey and fluorescence-activated cell sorting to taxonomically identify actively-feeding individual mixotrophic flagellates in natural lakewater samples. A proof-of-concept experiment was carried out where water from an oligotrophic lake was exposed to several types of fluorescently labelled prey and a complementary treatment based on food-vacuole staining. A total of 996 individual isolates were successfully recovered, and all treatments exhibited high sensitivity for putative mixotrophic taxa: overall, 87% of the occurrences could be assigned to dictyochophytes, 9% to chrysophytes and 3% to dinoflagellates. In addition, taxonomic profiling of the sample showed no presence of other putative mixotrophic species, indicating high specificity of the method. We argue that this methodological approach can be a valuable tool to uncover relevant and unexpected active mixotrophic species in a wider range of aquatic environments, and could easily be coupled to other techniques to describe the finer details of the trophic status of aquatic microbial communities.