UNLABELLED: In aquatic microbial food webs, ciliates represent an important trophic link in the energy transfer from prokaryotes, algae, and heterotrophic nanoflagellates (HNFs) to higher trophic levels. However, the trophic role of abundant small ciliates (<20 µm) is not clearly understood. To unveil their trophic linkages, we conducted two experiments manipulating both top-down and bottom-up controlling factors, thus modulating the trophic cascading and bacterial prey availability for protists during contrasting spring and summer seasons with samples collected from a freshwater meso-eutrophic reservoir. Water samples were size fractionated to modify food web complexity, i.e., 10 µm, 20 µm, and unfiltered control and amended with bacterial prey additions. The samples were analyzed by morphological and sequencing techniques. The bacterial amendments triggered strong ciliate growth following the peaks of HNFs in the 10 and 20 µm treatments, reflecting a trophic cascading from HNFs to raptorial prostome ciliates (Balanion planctonicum and Urotricha spp.) in spring. In summer, HNFs and ciliates peaked simultaneously, suggesting the important trophic cascade also from bacteria to bacterivorous scuticociliates (Cyclidium glaucoma and Cinetochilum margaritaceum) and HNFs. In spring, unfiltered treatments showed stronger ciliate top-down control by zooplankton than in summer. The sequence analysis revealed season-specific manipulation-induced shifts in ciliate communities and their large cryptic diversity. However, morphological and molecular analyses also revealed considerable discrepancies in the abundance of major ciliate taxa. The ciliate communities responded to our experimental manipulations in season-specific fashions, thereby highlighting the different roles of ciliates as an intermediate trophic link between prokaryotes and higher trophic levels. IMPORTANCE: Ciliates represent an important trophic link in aquatic microbial food webs. In this study, we used the food web manipulation techniques to reveal their complex trophic interactions during seasonally different plankton scenarios occurring in spring and summer. Manipulating top-down controlling factors (grazing pressure of micro- and metazooplankton grazers) and bottom-up factors (an availability of bacterial prey) shaped distinctly the complexity and dynamics of natural plankton communities and thus yielded significant changes in ciliate community dynamics. The experimentally simplified plankton and ciliate communities responded to our manipulations in season-specific fashions, reflected in different roles of ciliates as an intermediate trophic link between prokaryotes and higher trophic levels. This study also demonstrates that the combination of molecular and morphological analyses is essential to gain more realistic insights into the structure of ciliate community and for providing robust, ecologically meaningful results.

• Keywords
• aquatic food web, ciliates, experimental manipulations, freshwater reservoir, long-read amplicon sequencing, microbial loop, quantitative protargol staining,
• MeSH
• Bacteria MeSH
• Ciliophora * genetics   classification   physiology   MeSH
• Food Chain MeSH
• Seasons MeSH
• Fresh Water * microbiology   parasitology   MeSH
• Publication type
• Journal Article MeSH

Deep, cold, and dark hypolimnia represent the largest volume of water in freshwater lakes with limited occurrences of phototrophs. However, the presence of prokaryotes supports populations of bacterivorous ciliates and heterotrophic nanoflagellates (HNF). Nevertheless, protistan bacterivory rates and the major hypolimnetic ciliate bacterivores are poorly documented. We conducted a high frequency sampling (three-times a week) in the oxic hypolimnion of a stratified mesoeutrophic reservoir during summer, characterized by stable physicochemical conditions and low water temperature. Using fluorescently labeled bacteria we estimated that ciliates and HNF contributed, on average, 30% and 70% to aggregated protistan bacterivory, respectively, and collectively removed about two thirds of daily hypolimnetic prokaryotic production. The ciliate community was analyzed by the quantitative protargol staining method. One scuticociliate morphotype dominated the hypolimnetic ciliate community, accounting for 82% of total ciliates and over 98% of total ciliate bacterivory, with average cell-specific uptake rate of 202 prokaryotes per hour. Moreover, long-amplicon sequencing revealed that the scuticociliate belongs to an unidentified clade closely related to the Ctedoctematidae and Eurystomatellidae families. The high-resolution sampling, microscopic, and sequencing methods allowed uncovering indigenous microbial food webs in the hypolimnetic environment and revealed a functional simplification of ciliate communities, dominated by a new bacterivorous scuticociliate lineage.

• Keywords
• bacterivorous protists, cold hypolimnetic layer, freshwater reservoir, new lineage of scuticociliates, protistan bacterivory rates,
• MeSH
• Bacteria * classification   isolation & purification   genetics   MeSH
• Ciliophora * classification   genetics   MeSH
• Phylogeny MeSH
• Lakes microbiology   MeSH
• Oligohymenophorea * classification   genetics   isolation & purification   MeSH
• Sequence Analysis, DNA MeSH
• Fresh Water * microbiology   parasitology   MeSH
• Publication type
• Journal Article MeSH

BACKGROUND: Protists are essential contributors to eukaryotic diversity and exert profound influence on carbon fluxes and energy transfer in freshwaters. Despite their significance, there is a notable gap in research on protistan dynamics, particularly in the deeper strata of temperate lakes. This study aimed to address this gap by integrating protists into the well-described spring dynamics of Římov reservoir, Czech Republic. Over a 2-month period covering transition from mixing to established stratification, we collected water samples from three reservoir depths (0.5, 10 and 30 m) with a frequency of up to three times per week. Microbial eukaryotic and prokaryotic communities were analysed using SSU rRNA gene amplicon sequencing and dominant protistan groups were enumerated by Catalysed Reporter Deposition-Fluorescence in situ Hybridization (CARD-FISH). Additionally, we collected samples for water chemistry, phyto- and zooplankton composition analyses. RESULTS: Following the rapid changes in environmental and biotic parameters during spring, protistan and bacterial communities displayed swift transitions from a homogeneous community to distinct strata-specific communities. A prevalence of auto- and mixotrophic protists dominated by cryptophytes was associated with spring algal bloom-specialized bacteria in the epilimnion. In contrast, the meta- and hypolimnion showcased a development of a protist community dominated by putative parasitic Perkinsozoa, detritus or particle-associated ciliates, cercozoans, telonemids and excavate protists (Kinetoplastida), co-occurring with bacteria associated with lake snow. CONCLUSIONS: Our high-resolution sampling matching the typical doubling time of microbes along with the combined microscopic and molecular approach and inclusion of all main components of the microbial food web allowed us to unveil depth-specific populations' successions and interactions in a deep lentic ecosystem.

• Keywords
• 18S and 16S amplicon sequencing, CARD-FISH, Epilimnion, Freshwater, Hypolimnion, Metalimnion, Microbial food webs, Protists, Spring succession,
• Publication type
• Journal Article MeSH

BACKGROUND: Aerobic anoxygenic phototrophic (AAP) bacteria are heterotrophic bacteria that supply their metabolism with light energy harvested by bacteriochlorophyll-a-containing reaction centers. Despite their substantial contribution to bacterial biomass, microbial food webs, and carbon cycle, their phenology in freshwater lakes remains unknown. Hence, we investigated seasonal variations of AAP abundance and community composition biweekly across 3 years in a temperate, meso-oligotrophic freshwater lake. RESULTS: AAP bacteria displayed a clear seasonal trend with a spring maximum following the bloom of phytoplankton and a secondary maximum in autumn. As the AAP bacteria represent a highly diverse assemblage of species, we followed their seasonal succession using the amplicon sequencing of the pufM marker gene. To enhance the accuracy of the taxonomic assignment, we developed new pufM primers that generate longer amplicons and compiled the currently largest database of pufM genes, comprising 3633 reference sequences spanning all phyla known to contain AAP species. With this novel resource, we demonstrated that the majority of the species appeared during specific phases of the seasonal cycle, with less than 2% of AAP species detected during the whole year. AAP community presented an indigenous freshwater nature characterized by high resilience and heterogenic adaptations to varying conditions of the freshwater environment. CONCLUSIONS: Our findings highlight the substantial contribution of AAP bacteria to the carbon flow and ecological dynamics of lakes and unveil a recurrent and dynamic seasonal succession of the AAP community. By integrating this information with the indicator of primary production (Chlorophyll-a) and existing ecological models, we show that AAP bacteria play a pivotal role in the recycling of dissolved organic matter released during spring phytoplankton bloom. We suggest a potential role of AAP bacteria within the context of the PEG model and their consideration in further ecological models.

• Keywords
• pufM gene, Aerobic anoxygenic phototrophs, Aquatic microbial ecology, Freshwaters, Long-term sampling, Microbial seasonal succession, PEG model, Photoheterotrophs,
• MeSH
• Bacteria, Aerobic genetics   metabolism   MeSH
• Bacteria genetics   MeSH
• Biomass MeSH
• Phototrophic Processes * MeSH
• Phytoplankton genetics   MeSH
• Lakes * microbiology   MeSH
• Publication type
• Journal Article MeSH

Telonemia are one of the oldest identified marine protists that for most part of their history have been recognized as a distinct incertae sedis lineage. Today, their evolutionary proximity to the SAR supergroup (Stramenopiles, Alveolates, and Rhizaria) is firmly established. However, their ecological distribution and importance as a natural predatory flagellate, especially in freshwater food webs, still remain unclear. To unravel the distribution and diversity of the phylum Telonemia in freshwater habitats, we examined over a thousand freshwater metagenomes from all over the world. In addition, to directly quantify absolute abundances, we analyzed 407 samples from 97 lakes and reservoirs using Catalyzed Reporter Deposition-Fluorescence in situ Hybridization (CARD-FISH). We recovered Telonemia 18S rRNA gene sequences from hundreds of metagenomic samples from a wide variety of habitats, indicating a global distribution of this phylum. However, even after this extensive sampling, our phylogenetic analysis did not reveal any new major clades, suggesting current molecular surveys are near to capturing the full diversity within this group. We observed excellent concordance between CARD-FISH analyses and estimates of abundances from metagenomes. Both approaches suggest that Telonemia are largely absent from shallow lakes and prefer to inhabit the colder hypolimnion of lakes and reservoirs in the Northern Hemisphere, where they frequently bloom, reaching 10%-20% of the total heterotrophic flagellate population, making them important predatory flagellates in the freshwater food web.

• Keywords
• CARD-FISH, Telonemia, freshwater lakes, metagenomics, microbial food webs, predatory flagellate,
• MeSH
• Biodiversity MeSH
• Phylogeny * MeSH
• In Situ Hybridization, Fluorescence * MeSH
• Lakes microbiology   parasitology   MeSH
• Metagenome MeSH
• Metagenomics MeSH
• RNA, Ribosomal, 18S * genetics   MeSH
• Fresh Water * microbiology   parasitology   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• RNA, Ribosomal, 18S * MeSH