Most cited article - PubMed ID 16329920
Food selection by bacterivorous protists: insight from the analysis of the food vacuole content by means of fluorescence in situ hybridization
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
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.
Heterotrophic nanoflagellates (HNF) and ciliates are major protistan planktonic bacterivores. The term HNF, however, describes a functional guild only and, in contrast to the morphologically distinguishable ciliates, does not reflect the phylogenetic diversity of flagellates in aquatic ecosystems. Associating a function with taxonomic affiliation of key flagellate taxa is currently a major task in microbial ecology. We investigated seasonal changes in the HNF and ciliate community composition as well as taxa-specific bacterivory in four hypertrophic freshwater lakes. Taxa-specific catalyzed reporter deposition-fluorescence in situ hybridization probes assigned taxonomic affiliations to 51%-96% (average ±SD, 75 ± 14%) of total HNF. Ingestion rates of fluorescently labelled bacteria unveiled that HNF contributed to total protist-induced bacterial mortality rates more (56%) than ciliates (44%). Surprisingly, major HNF bacterivores were aplastidic cryptophytes and their Cry1 lineage, comprising on average 53% and 24% of total HNF abundance and 67% and 21% of total HNF bacterivory respectively. Kinetoplastea were important consumers of bacteria during summer phytoplankton blooms, reaching 38% of total HNF. Katablepharidacea (7.5% of total HNF) comprised mainly omnivores, with changing contributions of bacterivorous and algivorous phylotypes. Our results show that aplastidic cryptophytes, accompanied by small omnivorous ciliate genera Halteria/Pelagohalteria, are the major protistan bacterivores in hypertrophic freshwaters.
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.
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.
- Keywords
- Cercozoa, Cryptophyceae, Katablepharidacea, bacterivorous and predatory flagellates, ciliates, freshwater microbial food webs,
- MeSH
- Cercozoa * MeSH
- Cryptophyta MeSH
- In Situ Hybridization, Fluorescence MeSH
- Food Chain * MeSH
- Fresh Water MeSH
- Animals MeSH
- Check Tag
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
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.
- MeSH
- Bacteria classification genetics isolation & purification metabolism MeSH
- Cryptophyta microbiology MeSH
- Phylogeny MeSH
- Heterotrophic Processes MeSH
- In Situ Hybridization, Fluorescence MeSH
- Plankton microbiology MeSH
- Food Chain MeSH
- Seasons MeSH
- Fresh Water microbiology parasitology MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
We studied the diversity of Limnohabitans using reverse line blot hybridization with Limnohabitans lineage-specific probes in the freshwater canyon-shaped Římov reservoir (Czech Republic). To examine the succession of distinct lineages, we performed (i) a study of an intensive spring sampling program at the lacustrine part of the Římov reservoir (from ice melt through a phytoplankton peak to the clear-water phase), and (ii) a seasonal study (April to November) when the occurrence of distinct Limnohabitans lineages was related to the inherent longitudinal heterogeneity of the reservoir. Significant spatiotemporal changes in the compositions of distinct Limnohabitans lineages allowed for the identification of "generalists" that were always present throughout the whole season as well as "specialists" that appeared in the reservoir only for limited periods of time or irregularly. Our results indicate that some phytoplankton groups, such as cryptophytes or cyanobacteria, and zooplankton composition were the major factors modulating the distribution and dynamics of distinct Limnohabitans lineages. The highest Limnohabitans diversity was observed during the spring algal bloom, whereas the lowest was during the summer cyanobacterial bloom. The microdiversity also markedly increased upstream in the reservoir, being highest at the inflow, and thus likely reflecting strong influences of the watershed.IMPORTANCE The genus Limnohabitans is a typical freshwater bacterioplankton and is believed to play a significant role in inland freshwater habitats. This work is unique in detecting and tracing different closely related lineages of this bacterial genus in its natural conditions using the semiquantitative reverse line blot hybridization method and in discovering the factors influencing the microdiversity, subtype alternations, and seasonality.
- Keywords
- ITS, Limnohabitans, bacterial diversity, canyon-shaped freshwater reservoir, reverse line blot hybridization, subtypes,
- MeSH
- Comamonadaceae classification genetics isolation & purification MeSH
- Cryptophyta growth & development MeSH
- Ecosystem MeSH
- Eutrophication MeSH
- Phytoplankton growth & development MeSH
- Seasons MeSH
- Cyanobacteria growth & development MeSH
- Fresh Water microbiology MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Geographicals
- Czech Republic MeSH
Because their large growth potential is counterbalanced with grazing by heterotrophic nanoflagellates (HNF), bacteria of the genus Limnohabitans, which are common in many freshwater habitats, represent a valuable model for examining bacterial carbon flow to the grazer food chain. We conducted experiments with natural HNF communities taken from two distinct habitats, the meso-eutrophic Římov Reservoir and the oligo-mesotrophic Lake Cep (South Bohemia). HNF communities from each habitat at distinct seasonal phases, a late April algal bloom and a late May clear water phase, were each fed 3 Limnohabitans strains of differing cell sizes. Water samples were prefiltered (5 μm) to release natural HNF communities from zooplankton control and then amended with the Limnohabitans strains L. planktonicus II-D5 (medium sized, rod shaped), Limnohabitans sp. strain T6-5 (thin, long, curved rod), and Limnohabitans sp. strain 2KL-3 (large solenoid). Using temporal sampling and prey treatment, we determined HNF growth parameters such as doubling time, growth efficiency, and length of lag phase prior starting to exponential growth. All three Limnohabitans strains supported HNF growth but in significant prey-, site-, and season-dependent fashions. For instance, addition of the moderately large T6-5 strain yielded very rapid HNF growth with a short lag phase. In contrast, the curved morphology and larger cell size of strain 2KL-3 made this prey somewhat protected against grazing by smaller HNF, resulting in slower HNF growth and longer lag phases. These trends were particularly pronounced during the late May clear-water phase, which was dominated by smaller HNF cells. This may indicate a longer "adaptation time" for the flagellate communities toward the large prey size offered.
- MeSH
- Comamonadaceae cytology growth & development MeSH
- Heterotrophic Processes MeSH
- Lakes microbiology parasitology MeSH
- Food Chain MeSH
- Seasons MeSH
- Fresh Water microbiology parasitology MeSH
- Zooplankton growth & development MeSH
- Animals MeSH
- Check Tag
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Geographicals
- Czech Republic MeSH
Different bacterial strains can have different value as food for heterotrophic nanoflagellates (HNF), thus modulating HNF growth and community composition. We examined the influence of prey food quality using four Limnohabitans strains, one Polynucleobacter strain and one freshwater actinobacterial strain on growth (growth rate, length of lag phase and growth efficiency) and community composition of a natural HNF community from a freshwater reservoir. Pyrosequencing of eukaryotic small subunit rRNA amplicons was used to assess time-course changes in HNF community composition. All four Limnohabitans strains and the Polynucleobacter strain yielded significant HNF community growth while the actinobacterial strain did not although it was detected in HNF food vacuoles. Notably, even within the Limnohabitans strains we found significant prey-related differences in HNF growth parameters, which could not be related only to size of the bacterial prey. Sequence data characterizing the HNF communities showed also that different bacterial prey items induced highly significant differences in community composition of flagellates. Generally, Stramenopiles dominated the communities and phylotypes closely related to Pedospumella (Chrysophyceae) were most abundant bacterivorous flagellates rapidly reacting to addition of the bacterial prey of high food quality.
- MeSH
- Actinobacteria physiology MeSH
- Burkholderiaceae physiology MeSH
- Time Factors MeSH
- Comamonadaceae physiology MeSH
- Eukaryota growth & development metabolism physiology MeSH
- Bacterial Physiological Phenomena MeSH
- Genes, rRNA genetics MeSH
- Heterotrophic Processes MeSH
- Food Chain MeSH
- Fresh Water microbiology MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
