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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.

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.

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.

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.

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 × 103HNF mL−1and 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 (Cyclidiumspp.) contributed only moderately (mean 26%) to total ciliate bacterivory. Unexpectedly, but highly abundant omnivorous Halteria/Pelagohalteria(Stichotrichia) and, to a lesser extent, also omnivorous Rimostrombidiumspp. (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.

Telonemiaare one of the oldest identified marine protists that for most part of their history have been recognized as a distinct incertae sedislineage. 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 Telonemiain 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 situHybridization (CARD-FISH). We recovered Telonemia18S 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 Telonemiaare 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.

The growth rate is a fundamental characteristic of bacterial species, determining its contributions to the microbial community and carbon flow. High-throughput sequencing can reveal bacterial diversity, but its quantitative inaccuracy precludes estimation of abundances and growth rates from the read numbers. Here, we overcame this limitation by normalizing Illumina-derived amplicon reads using an internal standard: a constant amount of Escherichia colicells added to samples just before biomass collection. This approach made it possible to reconstruct growth curves for 319 individual OTUs during the grazer-removal experiment conducted in a freshwater reservoir Římov. The high resolution data signalize significant functional heterogeneity inside the commonly investigated bacterial groups. For instance, many Actinobacterial phylotypes, a group considered to harbor slow-growing defense specialists, grew rapidly upon grazers’ removal, demonstrating their considerable importance in carbon flow through food webs, while most Verrucomicrobial phylotypes were particle associated. Such differences indicate distinct life strategies and roles in food webs of specific bacterial phylotypes and groups. The impact of grazers on the specific growth rate distributions supports the hypothesis that bacterivory reduces competition and allows existence of diverse bacterial communities. It suggests that the community changes were driven mainly by abundant, fast, or moderately growing, and not by rare fast growing, phylotypes. We believe amplicon read normalization using internal standard (ARNIS) can shed new light on in situ growth dynamics of both abundant and rare bacteria.

The growth rate is a fundamental characteristic of bacterial species, determining its contributions to the microbial community and carbon flow. High-throughput sequencing can reveal bacterial diversity, but its quantitative inaccuracy precludes estimation of abundances and growth rates from the read numbers. Here, we overcame this limitation by normalizing Illumina-derived amplicon reads using an internal standard: a constant amount of Escherichia colicells added to samples just before biomass collection. This approach made it possible to reconstruct growth curves for 319 individual OTUs during the grazer-removal experiment conducted in a freshwater reservoir Rímov. The high resolution data signalize significant functional heterogeneity inside the commonly investigated bacterial groups. For instance, many Actinobacterial phylotypes, a group considered to harbor slow-growing defense specialists, grew rapidly upon grazers’ removal, demonstrating their considerable importance in carbon flow through food webs, while most Verrucomicrobial phylotypes were particle associated. Such differences indicate distinct life strategies and roles in food webs of specific bacterial phylotypes and groups. The impact of grazers on the specific growth rate distributions supports the hypothesis that bacterivory reduces competition and allows existence of diverse bacterial communities. It suggests that the community changes were driven mainly by abundant, fast, or moderately growing, and not by rare fast growing, phylotypes. We believe amplicon read normalization using internal standard (ARNIS) can shed new light on in situ growth dynamics of both abundant and rare bacteria.

Different bacterioplankton species represent different food quality resources for heterotrophic nanoflagellate (HNF) communities, potentially affecting HNF growth, community dynamics and carbon flow to higher trophic levels. However, our knowledge of such dynamics is still very limited. Here, we describe the results of 11 experiments with natural HNF communities from distinct seasonal phases in two freshwater habitats. The HNF communities were released from predation pressure of zooplankton and incubated with 16 distinct ecologically relevant prey bacterial strains from important Betaproteobacteriagenera (Limnohabitans, Polynucleobacter, and Methylopumilus) and one Actinobacteriastrain from the Luna 2 cluster. We observed remarkable prey‐ and season‐specific variability in community HNF growth parameters, i.e., doubling time, volumetric gross growth efficiency (GGE), and length of lag phase. All strains, except for the actinobacterium, supported rapid HNF population growth with an average doubling time of 10 h and GGE of 29%. Our analysis revealed that 59% of the variability in flagellate GGE data was explained by the length of lag phase after prey amendments. This indicates a considerable “adaptation time,” during which the predator communities undergo compositional shifts toward flagellate bacterivores best adapted to grow on the offered prey. Importantly, the rapid HNF growth detected on various bacteria tightly corresponds to doubling times reported for fast growing bacterioplankton groups. We propose a conceptual model explaining the tight linkages between rapid bacterial community shifts and succeeding HNF community shifts, which optimize prey utilization rates and carbon flow from various bacteria to the microbial food chain.

To test whether protist grazing selectively affects the composition of aquatic bacterial communities, we combined high-throughput sequencing to determine bacterial community composition with analyses of grazing rates, protist and bacterial abundances and bacterial cell sizes and physiological states in a mesocosm experiment in which nutrients were added to stimulate a phytoplankton bloom. A large variability was observed in the abundances of bacteria (from 0.7 to 2.4 × 106cells per ml), heterotrophic nanoflagellates (from 0.063 to 2.7 × 104cells per ml) and ciliates (from 100 to 3000 cells per l) during the experiment (~3-, 45- and 30-fold, respectively), as well as in bulk grazing rates (from 1 to 13 × 106bacteria per ml per day) and bacterial production (from 3 to 379?µg per C?l per day) (1 and 2 orders of magnitude, respectively). However, these strong changes in predation pressure did not induce comparable responses in bacterial community composition, indicating that bacterial community structure was resilient to changes in protist predation pressure. Overall, our results indicate that peaks in protist predation (at least those associated with phytoplankton blooms) do not necessarily trigger substantial changes in the composition of coastal marine bacterioplankton communities.

To test whether protist grazing selectively affects the composition of aquatic bacterial communities, we combined high-throughput sequencing to determine bacterial community composition with analyses of grazing rates, protist and bacterial abundances and bacterial cell sizes and physiological states in a mesocosm experiment in which nutrients were added to stimulate a phytoplankton bloom. A large variability was observed in the abundances of bacteria (from 0.7 to 2.4 × 106cells per ml), heterotrophic nanoflagellates (from 0.063 to 2.7 × 104cells per ml) and ciliates (from 100 to 3000 cells per l) during the experiment (∼3-, 45- and 30-fold, respectively), as well as in bulk grazing rates (from 1 to 13 × 106bacteria per ml per day) and bacterial production (from 3 to 379 μg per C l per day) (1 and 2 orders of magnitude, respectively). However, these strong changes in predation pressure did not induce comparable responses in bacterial community composition, indicating that bacterial community structure was resilient to changes in protist predation pressure. Overall, our results indicate that peaks in protist predation (at least those associated with phytoplankton blooms) do not necessarily trigger substantial changes in the composition of coastal marine bacterioplankton communities.

In aquatic microbial ecology, it has been considered that most extracellular enzymes except phosphatases are of bacterial origin. We tested this paradigm by evaluating the relationship between bacterial cell number and the activity of three glycolytic enzymes from 17 fresh waters and also from a laboratory experiment. Our large sets of pooled data do not seem to support such a simple explanation, because we found only a weak correlation of bacterial number with activity of α-glucosidase (rs = 0.63), β-glucosidase (rs = 0.45), and β-N-acetylhexosaminidase (rs = 0.44). We also tested relations of the enzymatic activities to potential sources of natural substrates: dissolved organic carbon (DOC) and phytoplankton (as chlorophyll a). Their correlations with the enzymatic activities tested were very weak or insignificant. On the other hand, we found evidence for distinct producers of extracellular enzymes by analysing enzyme kinetics. The kinetics usually did not follow the simple Michaelis-Menten model but a more complex one, indicating a mixture of two enzymes with distinct affinity to a substrate. In combination with size fractionation, we could sometimes even distinguish three or more different enzymes. During diatom blooms, the diatom biomass tightly correlated with β-N-acetylhexosaminidase activity (>4 μm fraction). We also documented very tight relationships between activity of both glucosidases and dry weight of Daphnia longispina (rs = 1.0 and 0.60 for α- and β-glucosidases, respectively) in an alpine clear-water lake. Our data and evidence from other studies indicate that extracellular glycosidic activities in aquatic ecosystems cannot generally be assigned only to bacteria. Also invertebrate animals and other eukaryotes (fungi, diatoms, protozoa etc.) should be considered as potentially very important enzyme producers. (© 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)

Bacterivorous protists are known to induce changes in bacterial community composition (BCC). We hypothesized that changes in BCC could be related quantitatively to a measure of grazing: the ratio of bacterial mortality to growth rate. To test this hypothesis, we analyzed time-course changes in BCC, protistan grazing rate, and bacterial production from 3 in situstudies conducted in a freshwater reservoir and three laboratory studies. In the field experiments, samples were manipulated to yield different levels of protistan bacterivory and incubated in dialysis bags. Laboratory investigations were continuous cultivation studies in which different bacterivorous protists were added to bacterial communities. BCC was assessed using 4–6 different rRNA-targeted oligonucleotide probes for community analysis. Change in BCC (Δ BCC) was estimated as the sum of changes in the proportions of the two phylogenetic groups that showed the largest shifts. Analysis of a set of 22 estimates of shifts in the ratio of grazing to production rate over periods of 48–72 h and Δ BCC showed that Δ BCC was positively and tightly correlated (r2= 0.784) with shifts in the ratio of grazing mortality to cell production. While the nature of a shift in BCC is unpredictable, the magnitude of the change can be related to changes in the balance between bacterial production and protistan grazing.

We studied the planktonic food web in eutrophic Sau Reservoir (Catalonia, NE Spain). Along the longitudinal axis from the Ter River downstream to the dam, we characterized a microbial succession of food web dominance of bacteria‐HNF‐ciliates. The Ter River transports a large load of organic material into the reservoir, with a bacterial density of ∼9 · 106large cells per ml. While at the first lacustrine station of the Reservoir HNF were the dominant bacterial consumers, at the others, an oligotrich ciliate, Halteria grandinella, was the main protozoan bacterivore. Most of the bacterial production in the reservoir epilimnion was consumed by grazing. The spatial succession of the reservoir microbial food webs was followed downstream by maximum densities of their potential predators among zoo‐plankters – rotifers, and early developmental stages of copepods.

The microbial loop of a naturally acidic bog lake, Große Fuchskuhle (Northeastern Germany), that had been artificially divided into 4 basins, was investigated. In the northeast (NE) and southwest (SW) basins, which differ strongly in chemistry and primary production, we conducted intensive studies of the main carbon fluxes through microbial food webs. In the less acidic, NE basin, much higher phytoplankton as well as bacterial biomass and production were found in parallel with negligible numbers of larger zooplankters. Weakly top‐down controlled populations of protists were characterized by an exceptionally low numerical proportion of heterotrophic nanoflagellates (HNF) to ciliates (‐1.5‐3.5). The ciliate community was dominated by a scuticociliate, Cyclidiumsp. (>95% of total ciliates), with an estimated grazing rate equal to 46–80% of heterotrophic bacterial production. In contrast, in the more humic, SW basin, both phyto‐ and bacterioplankton dynamics seemed to be top‐down controlled by abundant populations of small fine‐filter feeding cladocerans, Ceriodaphnia quadrangulaand Diaphanosoma brachyurum. Consequently, ciliates disappeared from the food web structure of the SW basin, HNF dropped to negligible numbers and bacteria showed very uniform morphology, dominated by small cocci or short rods. Our investigations have shown that the division of the lake into separate compartments can lead to very different microbial food web structures with extreme species compositions.

We examined the hypothesis that different types of ingested matter may be processed at different rates by a marine microzooplankter. Ciliates were exposed to a prey item and then diluted at 1:100. Disappearance or digestion of inert (fluorescent microspheres) and digestible prey items (heat‐killed and fluorescently labeled Synechococcus, natural Synechococcus, and Isochrysis galbana) were determined from changes in cell contents using epifluorescence microscopy. Ingested prey declined exponentially, and prey analogues were processed like natural prey. There were no significant differences in rates of disappearance or decay constants for the different prey items or in feeding vs. nonfeeding ciliates or between ciliates cultured on (or naive to) natural Synechococcus. The overall average t½of cell contents at 22°C was 75 min. The exponential declines in cell contents indicated that some ingested chlorophyll a, whether in Synechococcusor Isochrysis, can be long‐lived. The relatively invariant decay constant for ingested matter opens the possibility of using a gut passage approach to estimate instantaneous rates of grazing for natural populations of oligotrich ciliates.

Size fractionated ( < 0.23, 0.23-2.5, 2.5-100 µm) apparent α-glucosidase activity (αglca), β-glucosidase activity (βglca), and β-n-acetylglucosaminidase activity (βglcnaca) were measured using muf-substrates in the eutrophic římov reservoir during one season. the total αglca (0.7-5.0 nmol · l-1 · h-1) correlated with chlorophyll -a, bacterial and cladoceran biomasses, and abundance of heterotrophic nanoflagellates. The total βGlcA (2.0-7.9 nmol · l-1 · h-1) showed a relationship to cladoceran biomass. The particle-bound βGlcA correlated with bacterial and cladoceran biomasses and chlorophyll-a. The total βGlcNAcA (1.3-7.4 nmol · l-1 · h-1) correlated with copepod biomass. Ecological significance of βGlcNAcA was supported by N-acetyl-14-Cglucosamine incorporation (Vmax: 0.42-1.98 nmol · l-1 · h-1), which indicates the ability of microorganisms to utilize the liberated N-acetylglucosamine. Relationships found for the above enzyme activities to zooplankton might be explained by subsequent bacterial hydrolysis of nondigested algal or chitinaceous remains egested by cladocerans and copepods.

Thymidine incorporation into DNA is widely used to estimate rates of bacterial growth and secondary production in aquatic systems. The procedure requires the use of several conversion factors and assumptions to convert rates of thymidine uptake to rates of carbon production. Perhaps the most controversial of the conversion factors is that which converts rates of incorporation to the rate of cell production. During a year-long study in Lake Arlington, Texas, we empirically determined conversion factors from bacterial growth in filtered (1.0 µm porosity) and diluted (1:9) lake water. Bacterial growth rate constants determined from changes in cell abundance were compared to growth rate constants estimated from empirically derived (both instantaneous and annually averaged), theoretical, and modeled conversion factors. Single value conversion factors (i.e., theoretical, or average of 19 empirically determined conversion factors) did not yield estimates of bacterial growth rate constants that compared favorably to growth estimates from changes in bacterial abundance. Conversion factors, determined from a regression model based on empirically determined conversion factors, gave annual growth estimates that were similar to those obtained from changes in cell abundance.