Phagotrophic protists are key players in aquatic food webs. Although sequencing-based studies have revealed their enormous diversity, ecological information on in situ abundance, feeding modes, grazing preferences, and growth rates of specific lineages can be reliably obtained only using microscopy-based molecular methods, such as Catalyzed Reporter Deposition-Fluorescence in situ Hybridization (CARD-FISH). CARD-FISH is commonly applied to study prokaryotes, but less so to microbial eukaryotes. Application of this technique revealed that Paraphysomonas or Spumella-like chrysophytes, considered to be among the most prominent members of protistan communities in pelagic environments, are omnipresent but actually less abundant than expected, in contrast to little known groups such as heterotrophic cryptophyte lineages (e.g., CRY1), cercozoans, katablepharids, or the MAST lineages. Combination of CARD-FISH with tracer techniques and application of double CARD-FISH allow visualization of food vacuole contents of specific flagellate groups, thus considerably challenging our current, simplistic view that they are predominantly bacterivores. Experimental manipulations with natural communities revealed that larger flagellates are actually omnivores ingesting both prokaryotes and other protists. These new findings justify our proposition of an updated model of microbial food webs in pelagic environments, reflecting more authentically the complex trophic interactions and specific roles of flagellated protists, with inclusion of at least two additional trophic levels in the nanoplankton size fraction. Moreover, we provide a detailed CARD-FISH protocol for protists, exemplified on mixo- and heterotrophic nanoplanktonic flagellates, together with tips on probe design, a troubleshooting guide addressing most frequent obstacles, and an exhaustive list of published probes targeting protists.

• Klíčová slova
• CARD-FISH, aquatic microbial food webs, bacterivorous, grazing by protists, heterotrophic and mixotrophic flagellates, omnivorous and predatory flagellates, protists, unicellular eukaryotes,
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

A modified fluorescence in situ hybridization (FISH) method was used to analyze bacterial prey composition in protistan food vacuoles in both laboratory and natural populations. Under laboratory conditions, we exposed two bacterial strains (affiliated with beta- and gamma-Proteobacteria -- Aeromonas hydrophila and Pseudomonas fluorescens, respectively) to grazing by three protists: the flagellates Bodo saltans and Goniomonas sp., and the ciliate Cyclidium glaucoma. Both flagellate species preferably ingested A. hydrophila over P. fluorescens, while C. glaucoma showed no clear preferences. Differences were found in the digestion of bacterial prey with B. saltans digesting significantly faster P. fluorescens compared to two other protists. The field study was conducted in a reservoir as part of a larger experiment. We monitored changes in the bacterial prey composition available compared to the bacteria ingested in flagellate food vacuoles. Bacteria detected by probe HGC69a (Actinobacteria) and R-BT065 were negatively selected by flagellates. Bacteria detected by probe CF319a were initially positively selected but along with a temporal shift in bacterial cell size, this trend changed to negative selection during the experiment. Overall, our analysis of protistan food vacuole content indicated marked effects of flagellate prey selectivity on bacterioplankton community composition.

• MeSH
• Aeromonas hydrophila klasifikace   genetika   izolace a purifikace   MeSH
• Ciliophora mikrobiologie   fyziologie   ultrastruktura   MeSH
• DNA bakterií analýza   MeSH
• Eukaryota mikrobiologie   fyziologie   ultrastruktura   MeSH
• hybridizace in situ fluorescenční metody   MeSH
• predátorské chování fyziologie   MeSH
• Pseudomonas fluorescens klasifikace   genetika   izolace a purifikace   MeSH
• řeky mikrobiologie   parazitologie   MeSH
• ribozomální DNA analýza   MeSH
• RNA ribozomální 16S genetika   MeSH
• stravovací zvyklosti fyziologie   MeSH
• vakuoly mikrobiologie   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA bakterií MeSH
• ribozomální DNA MeSH
• RNA ribozomální 16S MeSH

An experiment designed to examine food preferences of heterotrophic nanoflagellates (HNF) grazing on bacterioplankton was performed in the freshwater Rímov reservoir (Czech Republic). Water samples were size-fractionated to obtain < 5 microm filtrate containing bacteria and HNF. To manipulate resource availability, < 5 microm treatments were incubated in dialysis bags submerged in the barrels filled with the unfiltered reservoir water amended with either orthophosphate or glucose or combination of both. We employed rRNA-targeted probes to assess HNF prey preferences by analysing bacterial prey in HNF food vacuoles compared with available bacteria. Actinobacteria (the HGC69a probe) were avoided by HNF in all treatments. Cytophaga-Flavobacterium-Bacteroidetes bacteria (the CF319a probe) were positively selected mainly in treatments in which bacteria were heavily grazed, the < 5 microm treatments, but this trend was less pronounced towards the end of the study. The members of a small subcluster of Betaproteobacteria (the R-BT065 probe) were mostly positively selected. The nutrient amendments differentially affected bacterioplankton dynamics in almost all treatments, and together with the size fractionation, altered HNF overall bacterivory as well as prey selection. Analyses of bacterivores in unfiltered treatments allowed to detect the effect of different protists on shifts in HNF selectivity observed in < 5 microm compared with unfiltered treatments.

• MeSH
• Actinobacteria MeSH
• druhová specificita MeSH
• Eukaryota fyziologie   MeSH
• fosfor metabolismus   MeSH
• glukosa metabolismus   MeSH
• hybridizace in situ fluorescenční MeSH
• predátorské chování fyziologie   MeSH
• Proteobacteria MeSH
• RNA ribozomální 16S MeSH
• sladká voda mikrobiologie   MeSH
• stravovací zvyklosti fyziologie   MeSH
• zooplankton fyziologie   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• fosfor MeSH
• glukosa MeSH
• RNA ribozomální 16S MeSH

Bacterioplankton from a meso-eutrophic dam reservoir was size fractionated to reduce (<0.8-microm treatment) or enhance (<5-microm treatment) protistan grazing and then incubated in situ for 96 h in dialysis bags. Time course samples were taken from the bags and the reservoir to estimate bacterial abundance, mean cell volume, production, protistan grazing, viral abundance, and frequency of visibly infected cells. Shifts in bacterial community composition (BCC) were examined by denaturing gradient gel electrophoresis (DGGE), cloning and sequencing of 16S rDNA genes from the different treatments, and fluorescence in situ hybridization (FISH) with previously employed and newly designed oligonucleotide probes. Changes in bacterioplankton characteristics were clearly linked to changes in mortality rates. In the reservoir, where bacterial production about equaled protist grazing and viral mortality, community characteristics were nearly invariant. In the "grazer-free" (0.8-microm-filtered) treatment, subject only to a relatively low mortality rate (approximately 17% day(-1)) from viral lysis, bacteria increased markedly in concentration. While the mean bacterial cell volume was invariant, DGGE indicated a shift in BCC and FISH revealed an increase in the proportion of one lineage within the beta proteobacteria. In the grazing-enhanced treatment (5-microm filtrate), grazing mortality was approximately 200% and viral lysis resulted in mortality of 30% of daily production. Cell concentrations declined, and grazing-resistant flocs and filaments eventually dominated the biomass, together accounting for >80% of the total bacteria by the end of the experiment. Once again, BCC changed strongly and a significant fraction of the large filaments was detected using a FISH probe targeted to members of the Flectobacillus lineage. Shifts of BCC were also reflected in DGGE patterns and in the increases in the relative importance of both beta proteobacteria and members of the Cytophaga-Flavobacterium cluster, which consistently formed different parts of the bacterial flocs. Viral concentrations and frequencies of infected cells were highly significantly correlated with grazing rates, suggesting that protistan grazing may stimulate viral activity.

• MeSH
• Bacteria klasifikace   virologie   MeSH
• ekosystém * MeSH
• Eukaryota růst a vývoj   MeSH
• fylogeneze MeSH
• mikrobiologie vody * MeSH
• molekulární sekvence - údaje MeSH
• plankton MeSH
• predátorské chování MeSH
• ribozomální DNA genetika   MeSH
• RNA ribozomální 16S genetika   MeSH
• voda parazitologie   MeSH
• zásobování vodou * MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• ribozomální DNA MeSH
• RNA ribozomální 16S MeSH
• voda MeSH

We document a remarkable morphological transformation, attributable to grazing by nanoflagellate Ochromonas sp. DS, of a phycocyanin-rich freshwater Cyanobium sp. (10-NR 98.2% similar 16S rRNA gene sequence to the type species Cyanobium gracile). The single cells aggregated into microcolonies (average size 40 microm) in the presence of the protist. Colonies were characterized by hundreds of tubes (spinae), 100 nm to 1 microm long and 63 +/- 6 nm wide, on the surfaces of the Cyanobium cells co-cultured with Ochromonas. Spinae production, previously unknown for the freshwater Cyanobium species, suggests that picocyanobacterial life strategies are more flexible than previously thought.

• MeSH
• buněčné výběžky fyziologie   MeSH
• fenotyp * MeSH
• Ochromonas fyziologie   MeSH
• sinice ultrastruktura   MeSH
• stravovací zvyklosti fyziologie   MeSH
• transmisní elektronová mikroskopie MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH

Heterotrophic nanoflagellates (HNF) are considered as major planktonic bacterivores, however, larger HNF taxa can also be important predators of eukaryotes. To examine this trophic cascading, natural protistan communities from a freshwater reservoir were released from grazing pressure by zooplankton via filtration through 10- and 5-µm filters, yielding microbial food webs of different complexity. Protistan growth was stimulated by amendments of five Limnohabitans strains, thus yielding five prey-specific treatments distinctly modulating protistan communities in 10- versus 5-µm fractions. HNF dynamics was tracked by applying five eukaryotic fluorescence in situ hybridization probes covering 55-90% of total flagellates. During the first experimental part, mainly small bacterivorous Cryptophyceae prevailed, with significantly higher abundances in 5-µm treatments. Larger predatory flagellates affiliating with Katablepharidacea and one Cercozoan lineage (increasing to up to 28% of total HNF) proliferated towards the experimental endpoint, having obviously small phagocytized HNF in their food vacuoles. These predatory flagellates reached higher abundances in 10-µm treatments, where small ciliate predators and flagellate hunters also (Urotricha spp., Balanion planctonicum) dominated the ciliate assemblage. Overall, our study reports pronounced cascading effects from bacteria to bacterivorous HNF, predatory HNF and ciliates in highly treatment-specific fashions, defined by both prey-food characteristics and feeding modes of predominating protists.

• Klíčová slova
• Cercozoa, Cryptophyceae, Katablepharidacea, bacterivorous and predatory flagellates, ciliates, freshwater microbial food webs,
• MeSH
• Cercozoa * MeSH
• Cryptophyta MeSH
• hybridizace in situ fluorescenční MeSH
• potravní řetězec * MeSH
• sladká voda MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Aerobic Anoxygenic Phototrophic (AAP) bacteria are bacteriochlorophyll (BChl) a -containing organisms which use light energy to supplement their predominantly heterotrophic metabolism. Here, we investigated mortality and growth rates of AAP bacteria in three different freshwater lakes in Central Europe: the mountain lake Plešné, the oligo-mesotrophic Lake Stechlin and the forest pond Huntov. The mortality of AAP bacteria was estimated from diel changes of BChl a fluorescence. Net and gross growth rates were calculated from the increases in AAP cell numbers. The gross growth rates of AAP bacteria ranged from 0.38 to 5.6 d-1 , with the highest values observed during summer months. Simultaneously, the rapidly growing AAP cells have to cope with an intense grazing pressure by both zooplankton and protists. The presented results document that during the day, gross growth usually surpased mortality. Our results indicate that AAP bacteria utilize light energy under natural conditions to maintain rapid growth rates, which are balanced by a generally intense grazing pressure.

• MeSH
• aerobióza MeSH
• aerobní bakterie klasifikace   genetika   růst a vývoj   izolace a purifikace   MeSH
• biodiverzita MeSH
• fototrofní procesy MeSH
• jezera mikrobiologie   MeSH
• roční období MeSH
• Publikační typ
• časopisecké články MeSH
• Geografické názvy
• Evropa MeSH

Diplonemids are one of the most abundant groups of heterotrophic planktonic microeukaryotes in the world ocean and, thus, are likely to play an essential role in marine ecosystems. So far, only few species have been introduced into a culture, allowing basic studies of diplonemid genetics, morphology, ultrastructure, metabolism, as well as endosymbionts. However, it remains unclear whether these heterotrophic flagellates are parasitic or free-living and what are their predominant dietary patterns and preferred food items. Here we show that cultured diplonemids, maintained in an organic-rich medium as osmotrophs, can gradually switch to bacterivory as a sole food resource, supporting positive growth of their population, even when fed with a low biovolume of bacteria. We further observed remarkable differences in species-specific feeding patterns, size-selective grazing preferences, and distinct feeding strategies. Diplonemids can discriminate between low-quality food items and inedible particles, such as latex beads, even after their ingestion, by discharging them in the form of large waste vacuoles. We also detected digestion-related endogenous autofluorescence emitted by lysosomes and the activity of a melanin-like material. We present the first evidence that these omnipresent protists possess an opportunistic lifestyle that provides a considerable advantage in the generally food resource-limited marine environments.

• MeSH
• Bacteria genetika   MeSH
• ekosystém * MeSH
• Eukaryota * MeSH
• plankton MeSH
• stravovací zvyklosti MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

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.

• Klíčová slova
• aquatic food web, ciliates, experimental manipulations, freshwater reservoir, long-read amplicon sequencing, microbial loop, quantitative protargol staining,
• MeSH
• Bacteria MeSH
• Ciliophora * genetika   klasifikace   fyziologie   MeSH
• potravní řetězec MeSH
• roční období MeSH
• sladká voda * mikrobiologie   parazitologie   MeSH
• Publikační typ
• časopisecké články MeSH