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.

• Klíčová slova
• bacterivorous protists, cold hypolimnetic layer, freshwater reservoir, new lineage of scuticociliates, protistan bacterivory rates,
• MeSH
• Bacteria * klasifikace   izolace a purifikace   genetika   MeSH
• Ciliophora * klasifikace   genetika   MeSH
• fylogeneze MeSH
• jezera mikrobiologie   MeSH
• Oligohymenophorea * klasifikace   genetika   izolace a purifikace   MeSH
• sekvenční analýza DNA MeSH
• sladká voda * mikrobiologie   parazitologie   MeSH
• Publikační typ
• časopisecké články 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.

• Klíčová slova
• 18S and 16S amplicon sequencing, CARD-FISH, Epilimnion, Freshwater, Hypolimnion, Metalimnion, Microbial food webs, Protists, Spring succession,
• Publikační typ
• časopisecké články 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.

• Klíčová slova
• CARD-FISH, Telonemia, freshwater lakes, metagenomics, microbial food webs, predatory flagellate,
• MeSH
• biodiverzita MeSH
• fylogeneze * MeSH
• hybridizace in situ fluorescenční * MeSH
• jezera mikrobiologie   parazitologie   MeSH
• metagenom MeSH
• metagenomika MeSH
• RNA ribozomální 18S * genetika   MeSH
• sladká voda * mikrobiologie   parazitologie   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• RNA ribozomální 18S * MeSH

BACKGROUND: The phytoplankton spring bloom in freshwater habitats is a complex, recurring, and dynamic ecological spectacle that unfolds at multiple biological scales. Although enormous taxonomic shifts in microbial assemblages during and after the bloom have been reported, genomic information on the microbial community of the spring bloom remains scarce. RESULTS: We performed a high-resolution spatio-temporal sampling of the spring bloom in a freshwater reservoir and describe a multitude of previously unknown taxa using metagenome-assembled genomes of eukaryotes, prokaryotes, and viruses in combination with a broad array of methodologies. The recovered genomes reveal multiple distributional dynamics for several bacterial groups with progressively increasing stratification. Analyses of abundances of metagenome-assembled genomes in concert with CARD-FISH revealed remarkably similar in situ doubling time estimates for dominant genome-streamlined microbial lineages. Discordance between quantitations of cryptophytes arising from sequence data and microscopic identification suggested the presence of hidden, yet extremely abundant aplastidic cryptophytes that were confirmed by CARD-FISH analyses. Aplastidic cryptophytes are prevalent throughout the water column but have never been considered in prior models of plankton dynamics. We also recovered the first metagenomic-assembled genomes of freshwater protists (a diatom and a haptophyte) along with thousands of giant viral genomic contigs, some of which appeared similar to viruses infecting haptophytes but owing to lack of known representatives, most remained without any indication of their hosts. The contrasting distribution of giant viruses that are present in the entire water column to that of parasitic perkinsids residing largely in deeper waters allows us to propose giant viruses as the biological agents of top-down control and bloom collapse, likely in combination with bottom-up factors like a nutrient limitation. CONCLUSION: We reconstructed thousands of genomes of microbes and viruses from a freshwater spring bloom and show that such large-scale genome recovery allows tracking of planktonic succession in great detail. However, integration of metagenomic information with other methodologies (e.g., microscopy, CARD-FISH) remains critical to reveal diverse phenomena (e.g., distributional patterns, in situ doubling times) and novel participants (e.g., aplastidic cryptophytes) and to further refine existing ecological models (e.g., factors affecting bloom collapse). This work provides a genomic foundation for future approaches towards a fine-scale characterization of the organisms in relation to the rapidly changing environment during the course of the freshwater spring bloom. Video Abstract.

• MeSH
• Bacteria MeSH
• Eukaryota genetika   MeSH
• metagenom * MeSH
• plankton MeSH
• sladká voda MeSH
• viry * genetika   MeSH
• voda MeSH
• Publikační typ
• audiovizuální média MeSH
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• voda MeSH

Morphology-based microscopic approaches are insufficient for a taxonomic classification of bacterivorous heterotrophic nanoflagellates (HNF) in aquatic environments since their cells do not display reliably distinguishable morphological features. This leads to a considerable lack of ecological insights into this large and taxonomically diverse functional guild. Here, we present a combination of fluorescence in situ hybridization followed by catalyzed reporter deposition (CARD-FISH) and environmental sequence analyses which revealed that morphologically indistinguishable, so far largely cryptic and uncultured aplastidic cryptophytes are ubiquitous and prominent protistan bacterivores in diverse freshwater ecosystems. Using a general probe for Cryptophyceae and its heterotrophic CRY1 lineage, we analyzed different water layers in 24 freshwater lakes spanning a broad range of trophic states, sizes and geographical locations. We show that bacterivorous aplastidic cryptophytes and the CRY1 lineage accounted for ca. 2/3 and ¼ of total HNF, respectively, in both epilimnetic and hypolimnetic samples. These heterotrophic cryptophytes were generally smaller and more abundant than their chloroplast-bearing counterparts. They had high uptake rates of bacteria, hinting at their important roles in channeling carbon flow from prokaryotes to higher trophic levels. The worldwide ubiquity of Cryptophyceae and its CRY1 lineage was supported by 18S rRNA gene sequence analyses across a diverse set of 297 freshwater metagenomes. While cryptophytes have been considered to be mainly plastidic "algae", we show that it is the aplastidic counterparts that contribute considerably to bacterial mortality rates. Additionally, our results suggest an undiscovered diversity hidden amongst these abundant and morphologically diverse aplastidic cryptophytes.

• MeSH
• Bacteria genetika   MeSH
• Cryptophyta * genetika   mikrobiologie   MeSH
• ekosystém * MeSH
• fylogeneze MeSH
• heterotrofní procesy MeSH
• hybridizace in situ fluorescenční MeSH
• jezera MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Planktonic ciliate species form multiple trophic guilds and are central components of freshwater food webs. Progress in molecular analytical tools has opened new insight into ciliate assemblages. However, high and variable 18S rDNA copy numbers, typical for ciliates, make reliable quantification by amplicon sequencing extremely difficult. For an exact determination of abundances, the classical morphology-based quantitative protargol staining is still the method of choice. Morphotype analyses, however, are time consuming and need specific taxonomic expertise. Catalyzed reporter deposition fluorescence in situ hybridization (CARD-FISH) may represent a promising tool for the analysis of planktonic ciliates by combining molecular identification with microscopic quantification. We tested the applicability of CARD-FISH using nine cultured ciliate species. Eight species- and three genus-specific oligonucleotide probes were designed based on their 18S rRNA genes. The CARD-FISH protocol was adapted and the specificity of probes was established. We subsequently examined the precision of quantitation by CARD-FISH on single cultures and mock assemblages. Successful tests on lake water samples proved that planktonic ciliates could be identified and quantified in field samples by CARD-FISH. Double hybridizations allowed studying interspecific predator prey interactions between two ciliate species. In summary, we demonstrate that CARD-FISH with species-specific probes can facilitate studies on the population dynamics of closely related, small sized or cryptic species at high sampling frequencies.

• Klíčová slova
• CARD-FISH, ciliate quantification, fluorescence in situ hybridization, lake water samples, planktonic ciliates, quantitative protargol staining,
• Publikační typ
• časopisecké články MeSH