Small bacterivorous eukaryotes play a cardinal role in aquatic food webs and their taxonomic classification is currently a hot topic in aquatic microbial ecology. Despite increasing interest in their diversity, core questions regarding predator-prey specificity remain largely unanswered, e.g., which heterotrophic nanoflagellates (HNFs) are the main bacterivores in freshwaters and which prokaryotes support the growth of small HNFs. To answer these questions, we fed natural communities of HNFs from Římov reservoir (Czech Republic) with five different bacterial strains of the ubiquitous betaproteobacterial genera Polynucleobacter and Limnohabitans. We combined amplicon sequencing and catalyzed reporter deposition fluorescence in situ hybridization (CARD-FISH) targeting eukaryotic 18 S rRNA genes to track specific responses of the natural HNF community to prey amendments. While amplicon sequencing provided valuable qualitative data and a basis for designing specific probes, the number of reads was insufficient to accurately quantify certain eukaryotic groups. We also applied a double-hybridization technique that allows simultaneous phylogenetic identification of both predator and prey. Our results show that community composition of HNFs is strongly dependent upon prey type. Surprisingly, Cryptophyta were the most abundant bacterivores, although this phylum has been so far assumed to be mainly autotrophic. Moreover, the growth of a small lineage of Cryptophyta (CRY1 clade) was strongly stimulated by one Limnohabitans strain in our experiment. Thus, our study is the first report that colorless Cryptophyta are major bacterivores in summer plankton samples and can play a key role in the carbon transfer from prokaryotes to higher trophic levels.

Despite the importance of shallow lakes worldwide, knowledge of microbial components, the base of their food webs, remains scarce. To close this gap, we investigated planktonic microbial food webs, in particular protistan bacterivory (for both ciliates and heterotrophic nanoflagellates [HNF]), in 10 shallow hypertrophic fishponds in South Bohemia (Czech Republic). We used fluorescently labeled bacteria as bacterivory tracers to estimate how abundant protistan populations in fishponds (4–25 × 103 HNF mL−1 and 55–770 ciliates mL−1) contribute to total bacterial mortality. Fluorescence microscopy, innovative image processing tools, and quantitative protargol staining were combined to detect major bacterivorous and omnivorous ciliate taxa. We quantified bacterial production, bacterivory by individual ciliate species, total ciliates, and total protistan bacterivory in all fishponds. On average, ciliate bacterivory was comparable to that of HNF, accounting for 56% and 44% of total protistan grazing, respectively. We found that primarily bacterivorous Peritrichia (genera Vorticella, Epistylis) and Scuticociliata (Cyclidium spp.) contributed only moderately (mean 26%) to total ciliate bacterivory. Unexpectedly, but highly abundant omnivorous Halteria/Pelagohalteria (Stichotrichia) and, to a lesser extent, also omnivorous Rimostrombidium spp. (Oligotrichia) contributed significantly more (mean 71%) to total ciliate bacterivory than typical bacterivorous taxa. This suggests that unselective grazers, which feed on a broader size spectrum from bacteria to small algae, may have a considerable competitive advantage in hypertrophic environments rich in small particles. Moreover, a meta‐analysis of available literature data supports our hypothesis that the role of ciliate bacterivory increases significantly, relative to HNF bacterivory, along a trophic gradient toward hypertrophic habitats.