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Rainforest aquatic ecosystems include complex habitats with scarce information on their unicellular eukaryote diversity and community structure. We have investigated the diversity of ciliates in freshwater and brackish environments along the Brazilian Atlantic Forest, based on the hypervariable V4 region of the 18S-rDNA obtained by high-throughput DNA sequencing. Our analyses detected 409 ciliate taxonomic units (OTUs), mostly attributed to the classes Oligohymenophorea and Spirotrichea. A total of 11 classes, 12 subclasses, 112 genera, and 144 species were reported. We found the following: (a) the ciliate communities are more diverse in freshwater- than in Atlantic Forest-associated brackish environments; (b) the ciliate communities are composed by a small amount of highly abundant OTUs, but a high number of low-abundant or rare OTUs; (c) nearly one-third of the ciliate OTUs share less than 97% sequence identity to reference sequences and (d) phylogenetic inference supports the hypothesis that the V4 region of the Ciliophora 18S-rDNA is a suitable marker for accurate evolutionary inferences at class level. Our results showed that a considerable fraction of the HTS-detected diversity of ciliates from Brazilian Atlantic Forest is not represented in the currently available molecular databases.

Ciliates are the largest group of ubiquitous aquatic bacterivorous protists, and many species are easily cultivated. However, only few studies reported prokaryotic communities naturally associated with ciliate cells. Herein, we analyzed the microbiome composition of several strains of Paramecium (Ciliophora) originating from different locations and belonging to two morpho-species by high-throughput sequencing (HTS) of the 16S rRNA gene. Possible reasons of HTS results bias were addressed comparing DNA libraries obtained using different primers and different number of ciliate cells. Microbiomes associated with ciliates and their environments were always significantly different by prokaryotic taxonomic composition and bacterial richness. There were also pronounced differences between Paramecium strains. Interestingly, potentially pathogenic bacteria were revealed in Paramecium microbiomes.

9 pages, 3 figures, 1 table, Temporal programs synchronised with the daily cycle are of adaptive importance for organisms exposed to periodic fluctuations. This study deepens into several aspects of the exogenous and endogenous nature of microbial grazers. We investigated the diel rhythms of cell division and feeding activity of four marine protists under different light regimes. In particular, we tested if the feeding cycle of protistan grazers could be mediated by a light-aided enhancement of prey digestion, and also explored the consequences of cell division on diel feeding rhythms. Cell division occurred at night for the heterotrophic dinoflagellates Gyrodinium dominans and Oxyrrhis marina. In contrast, the mixotrophic dinoflagellate Karlodinium armiger and the ciliate Strombidium sp. mostly divided during the day. Additionally, a significant diurnal feeding rhythm was observed in all species. When exposed to continuous darkness, nearly all species maintained the cell division rhythm, but lost the feeding cycle within several hours/days (with the exception of O. marina that kept the rhythm for 9.5 days). Additional feeding experiments under continuous light also showed the same pattern. We conclude that the feeding rhythms of protistan grazers are generally regulated not by cell division nor by the enhancement of digestion by light. Our study, moreover, indicates that the cell division cycle is under endogenous control, whereas an external trigger is required to maintain the feeding rhythm, at least for most of the species studied here, This work is supported by the FERMI project (CGL2014-59227-R; MINECO/AEI/FEDER, UE) and is a contribution of the Marine Zooplankton Ecology Group (2017 SGR 87). AA was funded with a FPI fellowship (BES-2015-074092) from the MINECO of Spain, With the funding support of the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2019-000928-S), of the Spanish Research Agency (AEI)

Many chloroviruses replicate in endosymbiotic zoochlorellae that are protected from infection by their symbiotic host. To reach the high virus concentrations that often occur in natural systems, a mechanism is needed to release zoochlorellae from their hosts. We demonstrate that the ciliate predator Didinium nasutum foraging on zoochlorellae-bearing Paramecium bursaria can release live zoochlorellae from the ruptured prey cell that can then be infected by chloroviruses. The catalysis process is very effective, yielding roughly 95% of the theoretical infectious virus yield as determined by sonication of P. bursaria. Chlorovirus activation is more effective with smaller Didinia, as larger Didinia typically consume entire P. bursaria cells without rupturing them, precluding the release of zoochlorellae. We also show that the timing of Chlorovirus growth is tightly linked to the predator-prey cycle between Didinium and Paramecium, with the most rapid increase in chloroviruses temporally linked to the peak foraging rate of Didinium, supporting the idea that predator-prey cycles can drive cycles of Chlorovirus abundance.

We characterized a novel Holospora-like bacterium (HLB) (Alphaproteobacteria, Holosporales) living in the macronucleus of the brackish water ciliate Frontonia salmastra. This bacterium was morphologically and ultrastructurally investigated, and its life cycle and infection capabilities were described. We also obtained its 16S rRNA gene sequence and performed in situ hybridization experiments with a specifically-designed probe. A new taxon, “Candidatus Hafkinia simulans”, was established for this HLB. The phylogeny of the family Holosporaceae based on 16S rRNA gene sequences was inferred, adding to the already available data both the sequence of the novel bacterium and those of other Holospora and HLB species recently characterized. Our phylogenetic analysis provided molecular support for the monophyly of HLBs and placed the new endosymbiont as the sister genus of Holospora. Additionally, the host ciliate F. salmastra, recorded in Europe for the first time, was concurrently described through a multidisciplinary study. Frontonia salmastra’s phylogenetic position in the subclass Peniculia and the genus Frontonia was assessed according to 18S rRNA gene sequencing. Comments on the biodiversity of this genus were added according to past and recent literature.

Symbioses between bacteria and eukaryotes are widespread and may have significant impact on the evolutionary history of symbiotic partners. The order Rickettsiales is a lineage of intracellular Alphaproteobacteria characterized by an obligate association with a wide range of eukaryotic hosts, including several unicellular organisms, such as ciliates and amoebas. In this work, we characterized the Rickettsiales symbionts associated with two different genotypes of the freshwater ciliate Paramecium caudatum originated from freshwater environments in distant geographical areas. Phylogenetic analyses based on 16S rRNA gene showed that the two symbionts are closely related to each other (99.4% identity), belong to the family Rickettsiaceae, but are far-related with respect to previously characterized Rickettsiales. Consequently, they were assigned to a new species of a novel genus, namely “Candidatus Spectririckettsia obscura.” Screening on a database of short reads from 16S rRNA gene amplicon-based profiling studies confirmed that bacterial sequences related to the new symbiont are preferentially retrieved from freshwater environments, apparently with extremely scarce occurrence (

The psychrophilic ciliate Euplotes focardii inhabits the shallow marine coastal sediments of Antarctica, where, over millions of years of evolution, it has reached a strict molecular adaptation to such a constant-temperature environment (about -2 °C). This long evolution at sub-zero temperatures has made E. focardii unable to respond to heat stress with the activation of its heat shock protein (hsp) 70 genes. These genes can, however, be expressed in response to other stresses, like the oxidative one, thus indicating that the molecular adaptation has exclusively altered the heat stress signaling pathways, while it has preserved hsp70 gene activation in response to other environmental stressors. Since radiative stress has proved to be affine to oxidative stress in several organisms, we investigated the capability of UV radiation to induce hsp70 transcription. E. focardii cell cultures were exposed to several different irradiation regimes, ranging from visible only to a mixture of visible, UV-A and UV-B. The irradiation values of each spectral band have been set to be comparable with those recorded in a typical Antarctic spring. Using Northern blot analysis, we measured the expression level of hsp70 immediately after irradiation (0-h-labeled samples), 1 h, and 2 h from the end of the irradiation. Surprisingly, our results showed that besides UV radiation, the visible light was also able to induce hsp70 expression in E. focardii. Moreover, spectrophotometric measurements have revealed no detectable endogenous pigments in E. focardii, making it difficult to propose a possible explanation for the visible light induction of its hsp70 genes. Further research is needed to conclusively clarify this point.

Molecular surveys of eukaryotic microbial communities employing high-throughput sequencing (HTS) techniques are rapidly supplanting traditional morphological approaches due to their larger data output and reduced bench work time. Here, we directly compare morphological and Illumina data obtained from the same samples, in an effort to characterize ciliate faunas from sediments in freshwater environments. We show how in silico processing affects the final outcome of our HTS analysis, providing evidence that quality filtering protocols strongly impact the number of predicted taxa, but not downstream conclusions such as biogeography patterns. We determine the abundance distribution of ciliates, showing that a small fraction of abundant taxa dominates read counts. At the same time, we advance reasons to believe that biases affecting HTS abundances may be significant enough to blur part of the underlying biological picture. We confirmed that the HTS approach detects many more taxa than morphological inspections, and highlight how the difference varies among taxonomic groups. Finally, we hypothesize that the two datasets actually correspond to different conceptions of “diversity,” and consequently that neither is entirely superior to the other when investigating environmental protists.

Currently, the investigation of Legionella ecology falls into two distinct areas of research activity: (1) that Legionella multiply within water sources by parasitizing amoebic or ciliate hosts or (2) that Legionella grows extracellularly within biofilms. Less focus has been given to the overlaps that may occur between these two areas or the likelihood that Legionella employs multiple survival strategies to persist in water sources. It is likely that Legionella interacts with protozoa, bacteria, algae, fungi, etc., and biofilm components in a more complex fashion than multiplication or death due to the presence or absence of single components of these complex microbial systems. This paper addresses gaps that exist in the understanding of Legionella ecology and serves to pinpoint areas of future research. To assume that only one other class of organism is important to Legionella ecology may limit our understanding of how this bacterium proliferates in heated water sources and also limit our strategies for its control in the built environment.

Despite its long history, biogeography has received relatively little attention within the field of microbial ecology. Consequently, a fierce debate rages whether protists inhabit restricted geographic areas (endemism hypothesis) or are globally dispersed (ubiquitous dispersal hypothesis). The data presented in this article support the endemism hypothesis. We succeeded in isolating an oligohymenophorean ciliate from a microbial mat in a meromictic anoxic alpine lake (Alatsee) in Germany. The ciliary pattern and the morphometry of this isolate are remarkably similar to Urocentrum turbo (Mueller, 1786) Nitzsch, 1827. However, the organism does not possess trichocysts, a conspicuous and characteristic feature of U. turbo. Instead, the U. turbo-like isolate from lake Alatsee displays merely trichocyst anlagen ("ghosts") in the cytoplasm that are only visible after protargol impregnation and which become never attached to the cell's cortex. Despite the distinctness of this difference, such a morphospecies has not been described from any other environment. Thus, we suggest that the U. turbo-like isolate from lake Alatsee is a local endemic ecotype, although the sequences of the 18S rRNA, ITS1, 5.8S rRNA, and ITS2 genes are nearly identical to those of U. turbo (Mueller, 1786) Nitzsch, 1827. This indicates that neither 18S rDNA nor ITS1, ITS2, and 5.8S rDNA sequences are reliable means to conclusively resolve different morphospecies or ecotypes of ciliates. As a consequence, we argue that protist species richness can only be reliably accounted for by considering both molecular and morphological data.

Tolo Harbor, a subtropical semi-enclosed coastal water body, is surrounded by an expanding urban community, which contributes to large concentrations of nutrient runoff, leading to algal blooms and localized hypoxic episodes. Present knowledge of protist distributions in subtropical waters during hypoxic conditions is very limited. In this study, therefore, we combined parallel 454 pyrosequencing technology and denaturing gradient gel electrophoresis (DGGE) fingerprint analyses to reveal the protist community shifts before, during, and after a 2-week hypoxic episode during the summer of 2011. Hierarchical clustering for DGGE demonstrated similar grouping of hypoxic samples separately from oxic samples. Dissolved oxygen (DO) concentration and dissolved inorganic nitrogen:phosphate (DIN:PO4) concentrations significantly affected OTU distribution in 454 sequenced samples, and a shift toward a ciliate and marine alveolate clade II (MALV II) species composition occurred as waters shifted from oxic to hypoxic. These results suggest that protist community shifts toward heterotrophic and parasitic tendencies as well as decreased diversity and richness in response to hypoxic outbreaks.

Protistan grazing on biofilms is potentially an important conduit enabling energy flow between microbial trophic levels. Contrary to the widely held assumption that protistan feeding primarily involves ingestion of biofilm cells, with negative consequences for the biofilm, this study demonstrated preferential grazing on the noncellular biofilm matrix by a ciliate, with selective ingestion of yeast and bacterial cells of planktonic origin over attached and biofilm-derived planktonic cells. Introducing a ciliate to two biofilm-forming Cryptococcus species, as well as two bacterial species in a model biofilm system, fluorescent probes were applied to determine ingestion of cellular and noncellular biofilm fractions. Fluoromicroscopy, as well as photometric quantification, confirmed that protistan grazing enhanced yeast biofilm metabolism, and an increase in biofilm biomass and viability. We propose that the extracellular polymeric matrix of biofilms may act as an interface regulating interaction between predator and prey, while serving as source of nutrients and energy for protists.

We still have a rudimentary understanding about the mechanism by which plant roots may stimulate soil microbial interactions. A biochemical model involving plant-derived biochemical fractions, such as exudates, has been used to explain this "rhizosphere effect" on bacteria. However, the variable response of other soil microbial groups, such as protozoa, to the rhizosphere suggests that other factors could be involved in shaping their communities. Thus, two experiments were designed to: (1) determine whether stimulatory and/or inhibiting factors associated with particular plant species regulate ciliate diversity and abundance and (2) obtain a better understanding about the mechanism by which these plant factors operate in the rhizosphere. Bacterial and chemical slurries were reciprocally exchanged between two plant species known to differ in terms of ciliate species richness and abundance (i.e., Canella winterana and plantation Tectona grandis). Analysis of variance showed that the bacteria plus nutrients and the nutrients only treatment had no significant effect on overall ciliate species richness and abundance when compared to the control treatment. However, the use of only colpodean species increased the taxonomic resolution of treatment effects revealing that bacterial slurries had a significant effect on colpodean ciliate species richness. Thus, for particular rhizosphere ciliates, biological properties, such as bacterial diversity or abundance, may have a strong influence on their diversity and possibly abundance. These results are consistent with a model of soil bacteria-mediated mutualisms between plants and protozoa.

The quantitative importance and composition of protozoan communities was investigated in sandy and silty intertidal sediments of a polyhaline and a freshwater site in the Schelde estuary. Total biomass of the protozoans studied, integrated over the upper 4 cm of the sediment, ranged from 41 to 597 mg C m(-2) and was in the same order of magnitude at the polyhaline and the freshwater intertidal site. Nanoheterotrophs were the dominant protozoans, in terms of both abundance and biomass. Ciliate abundances appeared to be largely determined by physical constraints, namely, the amount of interstitial space and hydrodynamic disturbances. It remains unclear which factors control nanoheterotrophic abundances and biomasses, which showed comparatively little seasonal and between-site fluctuations. Salinity differences were clearly reflected in the protozoan community composition. The dominant role of sessile ciliates is a unique feature of sediments in the freshwater tidal reaches, which can be attributed to the dynamic nature of sedimentation and resuspension processes associated with the maximum turbidity zone. Based on biomass ratios and estimated weight-specific metabolic rates, protozoa possibly accounted for approximately 29 to 96% of the estimated combined metabolic rate of protozoan and metazoan consumers at our sampling stations in late spring/early autumn. The contribution of protozoa to this combined metabolic rate was higher at the sandy than at the silty stations and was mainly accounted for by the nanoheterotrophs. These data emphasize the potential importance of small protozoa in sediments and suggest that protozoa are important components of benthic food webs.

Three aspects of size selective feeding by the scuticociliate Cyclidium glaucoma were studied in continuous cultivation systems. Firstly, grazing-induced changes in abundance, biomass, and size structure of a bacterial community were investigated. Secondly, we studied possible grazing-protection mechanisms of bacteria as a response to permanent presence of the predator. And finally, we were looking for potential feedback mechanisms within this predator-prey relationship, i.e., how the ciliate population reacted to a changed, more grazing-protected bacterial community. The first stage of the cultivation system consisted of the alga Cryptomonas sp. and the accompanying mixed bacterial community. These organisms were transferred to two second stage vessels, a control stage without ciliates and a second one inoculated with C. glaucoma. After the first week, the abundance of bacteria in the latter decreased by 60% and remained stable until the end of the experiment (65 d), whereas bacterial biomass was less affected (393 mg C L-1 during days 0-7, 281 mg C L-1 afterwards). The mean bacterial cell volume doubled from 0.089 mm3 to 0.167 mm3, which was mainly due to increasing cell widths. During the whole investigation period formation of colonies or filaments was not observed, but we found a clear feedback of ciliates on bacterial size. An increase in bacterial cell volume was always followed by a decline of the predator population, resulting in a yet undescribed type of microbial predator-prey relation. Literature and our own data on the optimal food size range grazed by C. glaucoma showed that bacterial cell width rather than length was responsible for that observed phenomenon. Finally, we suggest that uptake rates of spherical latex beads give only limited information on truly ingestible prey volumes and that prey geometry should be considered in future studies on size selective feeding of protists.

Endosymbiotic bacteria were observed to inhabit the cytoplasm of the freshwater ciliate Paramecium novaurelia. Transmission electron microscopy and toxicity tests with sensitive paramecia showed that the endosymbionts belong to the genus Caedibacter. The bacteria conferred a killer trait to their host paramecia. The production of a proteinaceous inclusion body ("R-body") in the bacterial cell makes them toxic to other paramecia after they become enclosed in food vacuoles. R-bodies of Caedibacter sp were associated with phages, which are known in most other Caedibacter species to code for the R-body proteins. The killer-effect of P. novaurelia on sensitive P. caudatum strains was of the "paralysis" type, which is a characteristic of the symbiont species Caedibacter caryophila. Until now C. caryophila was known to inhabit the macronucleus of Paramecium caudatum only. Sequencing of the 16S rRNA-gene proved that Caedibacter sp from the cytoplasm of P. novaurelia belongs to the species C. caryophila as well. The rDNA-sequence of 1695 bp length differed in a total of only 1 bp from the corresponding gene in C. caryophila from the macronucleus of P. caudatum. The results indicate that the infection of specific host cell compartments may depend on host genes, but not on different traits of the infecting symbiont species. The occurrence of killer and sensitive paramecia strains together in one pond is discussed with respect to the competitive advantage of the killer trait.

Grazing behavior of both individual cells and populations of the marine hypotrich Euplotes mutabilis, a largely benthic ciliate that feeds on suspended particles, was studied using fluorescent latex microspheres. Microspheres of sizes 0.57-, 1.90-, 3.06-, 5.66-, and 10.0-μm diam were offered at concentrations from 102 to 106 ml−1. Their uptake in a ten-min period was determined. Food particles within such ranges of size and concentration are found under natural conditions. The ciliates ingested particles of all sizes offered. Uptake rates at all concentrations were dependent upon particle size, with 1.90- and 3.06-μm diam microspheres having the highest uptake rate in all cases. For all sizes, there was an increase in the percentage of feeding cells and in the uptake rate (the number of particles ingested cell−1 h−1), with increasing particle concentration. When uptake was expressed as the volume ingested, maximum values were obtained for 5.85-μm diam microspheres at a concentration of 106 ml−1. By taking a small number of large particles, present at a low concentration in the medium, a ciliate can ingest a greater biovolume than by taking a high number of small particles which are abundant in the medium. These results demonstrate that some ciliates can graze particles of a wide range of sizes. Also, its nutrition can be affected by changing ambient concentrations of different prey, both through effects on the proportion of feeding cells and through the relative food content of the particles. The data can also add to the understanding of food niche partitioning between species. At low particle concentrations, particularly, it is important to consider the behavior of individual ciliates as well as of the whole population.

The addition of ciliated protozoa to aquatic microcosms and bench-scale sewage treatment plants increases decomposition rates. This is surprising, inasmuch as protozoa consume bacteria, which are the primary decomposers. One possible mechanism of the increase in decomposition rate is the direct consumption of dissolved organic matter by protozoa that are feeding primarily on bacteria. This possibility was explored experimentally in two-stage continuous cultures, with glucose limitingEscherichia coli in the first stage andE. coli limitingTetrahymena pyriformis in the second. Glycine and histidine were the test nutrients. The results of adding them to the second stages suggested that direct uptake by ciliates does not affect the dynamics of dissolved amino acids in pelagic environments or activated sludge plants. Ciliates might, however, affect the dynamics of amino acid pools in environments high in nutrients and ciliates, perhaps including some microenvironments near decomposing material or in benthic sediments. Direct uptake of dissolved amino acids by ciliates probably does not affect ciliate or bacterial populations substantially.

The relative importance of autotrophic flagellates, desmids, cyanobacteria, and ciliates as food forDaphnia magna was examined using cohort life tables. Each cohort was fed a single food type at a given concentration, and comparisons among each type were made. Algal feeding treatments included three levels of young (7 to 14 days old)Chlamydomonas reinhardi (Chlorophyta, Chlamydomonadacae), two levels of senescent (14 days old)C. reinhardi, two levels ofCryptomonas sp. (Chlorophyta, Cryptomonadacae), two levels ofStaurastrum sp. (Chlorophyta, Desmidacae), four levels of young (7 to 15 days old) or senescent (15 days old)Microcystis aeruginosa (Cyanophyta, Chlorococcacae), and a no-food treatment. The ciliatesCyclidium sp. andParamecium caudatum were also presented at concentrations of 1 or 10(2) cells/ml, as well as mixtures ofC. reinhardi (10(3)/ml) andCyclidium (1/ml) orP. caudatum (1/ml).Daphnia growth, reproduction, and survivorship were highest whenC. reinhardi orCryptomonas were the food source, while those starved or fedM. aeruginosa had shorter survivorship and lower growth and reproduction.Daphnia grew and had high survivorship when fedP. caudatum, but even though eggs were produced, most were aborted after 2 or 3 days.Staurastrum andCyclidium produced intermediate growth and survivorship, but reproduction was seen only in the 10(3) Staurastrum/ml treatment. Carbon and nitrogen content were general indicators of nutritional value. However, growth, reproduction, and survivorship were higher in some cohorts fed treatments containing relatively low levels of carbon and nitrogen. Other cohorts were short-lived and did not reproduce, despite being fed much higher levels of carbon and nitrogen. The results also suggest that green algae are nutritionally valuable forDaphnia, whereas cyanobacteria are not. As measured by life-table parameters, the nutritional value of ciliates was variable, with some being poor food sources. Thus, the potential of ciliates as a trophic link between microbial production and higher trophic levels may vary with the ciliate community structure. Our results suggest that ciliates alone were insufficient as a food source to supportDaphnia population growth.

Published results of studies based on samples size fractionated by sequential filtration (e.g. 0.2–3 μm) indicate that many ciliate, dinoflagellate and rhizarian phylotypes are found among marine picoeukaryotes. This is somewhat surprising as these protists are typically known as being large organisms (often >10 μm) and no picoplanktonic species have so far been identified. Here, the abundances of ciliate and dinoflagellate phylotypes in published molecular studies of picoeukaryotes are shown to correlate negatively with the pore size chosen for the end filter in the sequential filtrations (i.e. the filter used to collect the microbial biomass). This suggests that extracellular DNA adhering to small particles may be the source of ciliate and dinoflagellate phylotypes in picoplanktonic size fractions. This hypothesis was confirmed using real-time qPCR, which revealed significantly less dinoflagellate 18S rDNA in a 0.8–3-μm size fraction compared to 0.2–3 μm. On average, the abundance of putative extracellular phylotypes decreased by 84–89 % when a 0.8- μm end filter was used rather than a 0.2-μm end filter. A 0.8-μm filter is, however, not sufficient to retain all picoeukaryotic cells. Thus, selection of filter pore size involves a trade-off between avoiding artefacts generated by extracellular DNA and sampling the entire picoeukaryotic community. In contrast to ciliate and dinoflagellate phylotypes, rhizarian phylotypes in the picoplankton size range do not display a pattern consistent with an extracellular origin. This is likely due to the documented existence of picoplanktonic swarmer cells within this group.

Francisella are facultative intracellular bacteria causing severe disease in a broad range of animals. Two species are notable: Francisella tularensis, the causative organism of tularemia and a putative warfare agent, and Francisella noatunensis, an emerging fish pathogen causing significant losses in wild and farmed fish. Although various aspects of Francisella biology have been intensively studied, their natural reservoir in periods between massive outbreaks remains mysterious. Protists have been suspected to serve as a disguised vector of Francisella and co-culturing attempts demonstrate that some species are able to survive and multiply within protozoan cells. Here, we report the first finding of a natural occurrence of Francisella sp. as a protist endosymbiont. By molecular and morphological approaches, we identified intracellular bacteria localized in a strain of the marine ciliate Euplotes raikovi, isolated from the coast of Adriatic Sea. Phylogenetic analysis placed these endosymbionts within the genus Francisella, in close but distinct association with F. noatunensis. We suggest the establishment of a novel subspecies within F. noatunensis and propose the cytoplasmatic endosymbiont of E. raikovi as “Candidatus F. noatunensis subsp. endociliophora” subsp. nov.

Protozoan grazers play an important role in controlling the density of crude-oil degrading marine communities as has been evidenced in a number of microcosm experiments. However, small bioreactors contain a low initial titre of protozoa and the growth of hydrocarbon-depleting bacteria is accompanied by the fast depletion of mineral nutrients and oxygen, which makes microcosms rather unsuitable for simulating the sequence of events after the oil spill in natural seawater environment. In the present study, the population dynamics of marine protozoan community have been analysed in a 500 l mesocosm experiment involving bioaugmented oil booms that contained oil sorbents and slow-release fertilisers. A significant increase in numbers of marine flagellates and ciliates on biofilms of oil-degrading microbes was microscopically observed as early as 8 days after the start of the experiment, when protozoa exhibited a population density peak making up to 3,000 cells ml(-1). Further, the protozoan density varied throughout the experiment, but never dropped below 80 cells ml(-1). An 18S rRNA gene-based fingerprinting analysis revealed several changes within the eukaryotic community over the whole course of the experiment. Initial growth of flagellates and small ciliates was followed by a predominance of larger protozoa. According to microscopic observations and SSU rRNA molecular analyses, most predominant were the ciliates belonging to Euplotidae and Scuticociliatia. This is the first study to characterise the eukaryotic communities specifically in a large-scale oil bioremediation trial using both microscopy-based and several molecular techniques.

We found a recurrent seasonal pattern in abundance and composition of planktonic ciliates in Lake Constance, FRG, over a three-year period. Abundance peaks occurred in early spring and summer/autumn, while ciliate numbers were low in late spring (clear-water phase) and winter. Prostomatida and Oligotrichida dominated in early spring. They responded immediately to the phytoplankton spring bloom, while Haptorida, Peritrichida, and large Scuticociliatida (Histiobalantium) were delayed by 1 to 2 weeks. The spring community broke down at the onset of the clear-water phase.Pelagohalteria viridis containing symbiontic algae appeared shortly after this event. A highly diverse community was recorded in summer/autumn. Peritrichida, small Oligotrichida, and large Scuticociliatida reached their maxima during this season. Small Scuticociliatida were rare throughout the year and contributed moderately to total ciliate numbers only during the cold season. The observed seasonal sequence of pelagic ciliates in Lake Constance is discussed in relation to simultaneously collected data on potential food organisms and grazers.

Four experiments covering different seasons were performed to test the impact of increased benthic and planktonic resource availability on the structure of biofilm-dwelling ciliate communities which were cultivated in river bypass systems. The growth of benthic bacteria was stimulated by the addition of dissolved organic carbon. The enrichment of the planktonic resource was achieved by supplementation with suspended bacteria. It was shown that both resource enrichments can differentially influence abundance and taxonomic structure of ciliate communities. Furthermore, both resources can influence different stages during biofilm colonization. Increased benthic bacterial growth mainly resulted in both an accumulation of primarily grazing-resistant bacterial filaments and in an increase in the number of vagile heterotrophic flagellates. This can stimulate nanophagous ciliates (feeding on flagellates) in addition to the direct stimulation of bacteriovorous ciliates. The effects of the planktonic bacteria enrichments were twofold: They could have been utilized either directly by suspension-feeding ciliates or indirectly through an enhanced growth of suspension-feeding attached heterotrophic flagellates, which were then in turn grazed upon by ciliates. The magnitude of responses of the total ciliate abundance to the two resource enrichments further depended on the background conditions, thereby showing temporarily variable limitations of these resources. Furthermore, the particular taxonomic groups stimulated by one resource type sometimes differed between the experiments, an observation which demonstrates that the response depends on different environmental factors and is not easily predictable based simply on resource type. Taken together, our results emphasize the need of a differentiated view on the effects of resources on complex biofilm-dwelling consumer communities with respect to both the origin of carbon source as well as the particular environmental conditions.

The anaerobic free-living ciliate, Trimyema compressum, is known to harbor both methanogenic archaeal and bacterial symbionts in the cytoplasm. To clarify their phylogenetic belongings, a full-cycle rRNA approach was applied to this symbiosis. Phylogenetic analysis showed that the methanogenic symbiont was related to Methanobrevibacter arboriphilicus, which was distantly related to symbionts found in other Trimyema species. This result suggested that Trimyema species do not require very specific methanogenic symbionts, and symbiont replacement could have occurred in the history of Trimyema species. On the other hand, the bacterial symbiont was located near the lineage of the family Syntrophomonadaceae in the phylum Firmicutes. The sequence similarity between the bacterial symbiont and the nearest species was 85%, indicating that bacterial symbionts may be specific to the Trimyema species. The elimination of bacterial symbionts from the ciliate cell by antibiotic treatment resulted in considerably decreased host growth. However, it was not restored by stigmasterol addition (2 microg ml(-1)), which was different from the previous report that showed that the symbiont-free strain required exogenous sterols for growth. In addition, the decline of host growth was not accompanied by host metabolism shift toward the formation of more reduced products, which suggested that the contribution of bacterial symbionts to the host ciliate was not a dispose of excessive reducing equivalent arising from the host's fermentative metabolism as methanogenic symbionts do. This study showed that bacterial symbionts make a significant contribution to the host ciliate by an unknown function and suggested that interactions between bacterial symbionts and T. compressum are more complicated than hitherto proposed.

The extent of induced morphological defense in Euplotes daidaleos correlates to this ciliate's predation risk from the defense-inducing predator species. Euplotes daidaleos responded by morphological transformation only to organisms that are able to feed on typically formed Euplotes cells (63 ± 5 μm cell width in E. daidaleos). Three of those potential predator species caused defensive changes to various degrees (Student's t-test, P < 0.1 to P < 0.0001): Lembadion bullinum (Ciliata) induced 82 ± 6 μm cell width in E. daidaleos; Chaetogaster diastrophus (Oligochaeta) induced 85 = 6 μm width; and Stenostomum sphagnetorum (Turbellaria) induced 89 ± 8 μm width (at a density of 10 predators per milliliter, respectively). At higher predator densities (50 or 100 organisms per milliliter), Euplotes developed a correspondingly larger width (to a maximum of 103 ± 10 μm in the presence of S. sphagnetorum). Euplotes did not respond to organisms (e.g., Blepharisma japonicum, Colpidium campylum, Didinium nasutum, Paramecium caudatum, Spirostomum ambiguum, Stentor coeruleus) that cannot feed on this ciliate species. Daphnia longispina and Bursaria truncatella predators, which can feed on large prey of ≥125, or ≥200 μm in diameter, respectively, also had no effect on the morphology of Euplotes. The extent of defense in Euplotes that was induced by 10 predators per milliliter during 24 h decreased the predation risk from those predators to 67% in the presence of S. sphagnetorum, to 50% with L. bullinum, and to 15% with C. diastrophus, compared to the typical form of Euplotes. In a natural population, the defensive form of E. daidaleos was found with average cell widths of 88 ± 8 μm. The results indicate that predator-induced defense in natural Euplotes populations is beneficial to this prey and that it is adapted to the predation abilities of Euplotes predators, whereby energetical costs related to defensive changes may be saved.

Surface-associated algivorous ciliated protozoa are common in the benthos of streams, but little is known about the feeding ecology of these organisms. We compared the functional responses of two algivorous ciliated protozoa, Oxytricha fallax (a filter feeder) and Trithigmostoma cucullulus (an encounter feeder). The ciliates were fed (14)C-labeled Navicula cryptocephala in laboratory feeding experiments to determine their potential to consume significant amounts of algal prey. Logistic regression, and plots of the proportion of N. cryptocephala ingested vs. the total number offered, indicated functional responses of a typical rectangular hyperbolic (type II) form for both ciliates. Ingestion rates were estimated from regressions of the number of (14)C-labeled N. cryptocephala cells ingested per ciliate vs. time. Maximum feeding rates and half-saturation concentrations were estimated by fitting the observed ingestion rates and experimental algal densities to a function of the Michaelis-Menten enzyme kinetics form using nonlinear regression. For O. fallax, the maximum feeding rate was estimated to be 1.07 N. cryptocephala cells per minute, and the half-saturation concentration was 3.9 × 102 N. cryptocephala per square centimeter. For T. cucullulus the maximum feeding rate was estimated to be 0.2 N. cryptocephala per minute, and the half-saturation concentration was 5.4 × 10(3) N. cryptocephala per square centimeter. The data were also fitted using only the number of cells ingested at 60 and 120 min, by converting the endpoint consumption to rates. For O. fallax, the estimated maximum feeding rates were 1.3 and 1.0 N. cryptocephala per minute for 60 and 120 min, respectively, and estimated half-saturation concentrations were 5.1 × 10(2) and 3.5 × 10(2) N. cryptocephala per square centimeter. For T. cucullulus, estimated maximum feeding rates were 0.6 and 0.4 N. cryptocephala per minute for 60 and 120 min, respectively, and estimated half-saturation concentrations were 1.5 × 10(4) and 1.1 × 104 N. cryptocephala per square centimeter. These results suggest that kinetic methods for estimating ingestion rates are more accurate than endpoint determinations. Based on field observations of periphyton densities, these ciliates potentially are consuming 4.8% of the total available standing crop of diatom biomass per day and this could represent up to 16% of total available daily primary production.

The jumping behavior of Strobilidium velox and Halteria grandinella was analyzed videographically. On average, undisturbed cells of these species jumped 1.7–3.6 and 8 times per minute and spent 0.8 and 1.0% of their time jumping, respectively. Both ciliate species initiated jumps after encounters with rotifer predators. S. velox jumped on contact with Asplanchna girodi, traveling a mean distance of 1.5 mm (33 body lengths) at a mean velocity of 7 mm/s (154 body lengths/s) at 17°C. H. grandinella jumped on contact or near contact with Synchaeta pectinata, traveling a mean distance of 0.37 mm (18 body lengths) at a mean velocity of 2.76 mm/s (131 body lengths/s) at 20°C. The maximum velocity recorded during these escape jumps was 16.07 mm/s for S. velox and 3.70 mm/s for H. grandinella. In S. velox, swimming velocity during jumps was not significantly correlated either with swimming velocity just before jumping (mean = 0.15 mm/s) or with distance traveled. In H. grandinella, jumping velocity and distance also were not significantly correlated. Jumping in S. velox and H. grandinella was calculated to require approximately 149% and 41 % of total metabolic rate, respectively. Jumping seemed to be an effective defense against rotifer predation. Only 3% of 93 S. velox cells contacted by A. girodi were captured, and only 12% of 92 H. grandinella cells contacted or closely approached by S. pectinata were captured; all other cells jumped away. A predation experiment showed that A. girodi was about twice as, and significantly more, likely to ingest Paramecium tetraurelia as S. velox in a mixture of equal numbers of these ciliates. The swimming velocity of S. velox during jumps is the highest one so far reported for an oligotrich, and equals the highest one reported for any ciliate (Mesodinium rubrum).

16 pages, 5 figures, 3 tables, Despite the recognized importance of protozoans (flagellates and ciliates) as predators of bacteria, there are very few estimates of their abundance in fine sediments of freshwater lakes. This is due, in part, to the lack of a standard methodology. Because of the low concentration of protists in relation to particles, epifluorescence counts can not always be used. Instead, dilution followed by live counting was used to solve the masking by sediment particles. One to twenty μ1 sample aliquots were diluted with filtered lake water in a Palmer-Maloney counting slide. Four to eight replicates were sufficient to minimize the counting error, while minimizing effort. The method is highly replicable and could potentially be calibrated for different sediment types because sediment masking depends on the mean particle size of the sediment. When this method was applied in a survey of benthic sites in Quebec lakes, flagellate abundances were found to range from 100 to 180,000 cells ml−1, while ciliate numbers ranged from 26 to 11,000 cells ml−1. Bacteria are 105 to 107 times more abundant than protists and, thus, the impact of these protists on sediment bacterial dynamics is likely to be minimal

Bacterial biovolumes of hypertrophic Humboldt Lake (total dissolved solids = 3.3 g liter(-1); 6 m deep) and oligotrophic Redberry Lake (total dissolved solids = 20.9 g liter(-1); 17 m deep), Saskatchewan, were measured concurrently with a variety of environmental variables to identify the major factors correlated with volume changes. There was no difference (P0.05) in mean bacterial volume between Redberry Lake (0.084 ± 0.034 μm(3) SD) and Humboldt Lake (0.083 ± 0.021 μm(3) SD). Statistical analyses suggested there were marked differences in the factors associated with the pronounced seasonality of bacterial cell volumes in these two lakes. Variance in bacterial volume in the epilimnion of Redberry Lake was best explained by a multivariate regression model which included ciliate abundance and chlorophyll concentration (r (2) = 0.96). The model accounting for changes in hypolimnetic bacterial volume included ciliate numbers and primary production (r (2) = 0.94), of the measured variables. Bacterial volume in Humboldt Lake was most highly correlated with primary production (r (2) = 0.59). Bacterial production (estimated as the rate of thymidine incorporation into DNA) and growth (thymidine incorporation rate normalized to cell numbers) were not correlated to cell volume, with the exception of cocci volume in Humboldt Lake.

Experimental studies of a microbial food chain involving organic carbon substrates,Enterobacter aerogenes, and ciliate protozoansParamecium primaurelia andDldinium nasutum were conducted in stirred, aerated batch cultures. Quantitative measurements were made of organic carbon levels and of cell numbers, mean cell volumes, and total biovolumes for all three microbial populations. A mathematical model based on Monod kinetics was developed to describe this four-trophic level predator-prey system. The model was formulated in terms of biovolume, which is the product of cell numbers and mean cell size, and includes terms for bio-volume decay. Batch culture data were used to derive parameter values, and model simulations were compared to experimental results. Despite the significance ofParamecium-Didinium studies in ecological literature, the entire food chain has not been previously studied or modeled.

Seven known species of rumen ciliates and mixedEntodinium spp. showed association with plant particles in rumen fluid in vitro. Association was greater with fresh particles than with hay, and substantially decreased when the water-soluble components of the particles were removed, suggesting that the water-soluble components may be responsible for the association. The association was rapid and maximal between 5 and 35 min (depending on the ciliate species) after exposure to the particles, and involved major transfers of ciliate populations and biomass from the liquid phase to the solid phase of the system. The most rapid and largest population transfers to the particles from the rumen fluid were shown by the holotrich ciliates, where transfers of up to 97% of the population were recorded. Association with plant particles by all species examined occurred within the pH range 5.5-7.5, and decreased with time when the particles were incubated in rumen contents in vivo. The ciliate biomass transferring from the liquid to the solid phase varied with the composition of the ciliate population.

Abundance, biovolume, and species composition of pelagic ciliates in Lake Constance were recorded over two annual cycles (1987/88). Production was estimated from mean annual biovolumes and size-specific growth rates obtained from the literature. Cell concentrations and biovolumes ranged from 0.1 to 120 cells ml(-1) and from 3 to 1,200 mm(3) m(-3), respectively. Mean annual values were, respectively, 6.8 cells ml(-1) and 94 mm(3) m(-3) in 1987, and 12.0 cells ml(-1) and 130 mm(3) m(-3) in 1988. In both years, prostome nanociliates (20μm) dominated numerically, while strobiliids in the size range 20-35μm contributed most significantly to ciliate production. Ciliate community production, according to a crude calculation, yielded approximately 10-15 g C m(-2) year(-1).

The quantitative uptake of suspended particles has been studied in 14 species of ciliated protozoa in terms of the maximum rate of water cleared at low particle concentrations and of the maximum ingestion rate at high particle concentrations. The results, supported by data from the literature, show that ciliates which feed on larger particles (> 1-5μm) compare favorably with metazoan suspension feeders with respect to the ability to concentrate dilute suspensions of particles. Species specialized on smaller food particles (0.2-1μm), the size range of most bacteria in natural environments, require a higher concentration of particles. Bacterial population densities which can sustain ciliate growth are found in sediments, waters rich in organic material, and in the early successional stages of decomposing organic material. This is not the case in open waters in general where bacterivorous ciliates cannot play a role as grazers of bacteria.

The growth rate or numerical response of five species of bactivorous ciliates to the abundance ofEnterobacter aerogenes was examined in monoxenic culture. The ciliatesColpidium campylum, C. colpoda, Glaucoma scintillons, G. frontata, andCyclidium glaucoma were isolated from a small pond. Four were grown in shaken cultures, while three were grown in cultures in which the bacteria were allowed to settle on the bottom of the culture vessel. Of the seven response curves generated, four had distinct thresholds, so that the Michaelis-Menten model usually fitted to ciliate numerical response curves was not appropriate. In shaken cultures, half-saturation prey densities ranged from 5.5 × 10(6) to 42.9 × 10(6) bacteria/ml. In unshaken cultures, half-saturation densities ranged from 0.057 × 10(6) to 14.6 × 10(6) bacteria/cm(2). Two species grown on both suspended and settled bacteria attained higher growth rates and had lower half-saturation prey densities feeding on settled bacteria.

The ciliateUronema nigricans was found to acquire tolerance to mercury after being fed mercury-laden bacteria followed by exposure of washed suspensions of these ciliates to various concentrations of mercury in solution. Significant differences in percent mortality were observed for ciliates fed mercury-laden bacteria compared with control suspensions fed mercury-free bacteria. The phenomenon of acquired mercury tolerance was demonstrated within a single generation time. Ciliates fed mercury-free bacteria and subsequently exposed to increasing levels of mercury in solution showed an elevated tolerance to concentrations which, on initial testing, resulted in mortality of 83% of the ciliate population. The effect of ingesting mercury-laden bacteria on growth rate ofUronema was examined, and results showed no significant differences in growth rates of both 3- and 14-day-old cultures of protozoa that had been fed mercury-laden and mercury-free bacteria under controlled conditions.

We report the characterization of the bacterial consortium associated to Euplotes focardii, a strictly psychrophilic marine ciliate that was maintained in laboratory cultures at 4 °C after its first isolation from Terra Nova Bay, in Antarctica. By Illumina genome analyser, we obtained 11,179 contigs of potential prokaryotic origin and classified them according to the NCBI’s prokaryotic attributes table. The majority of these sequences correspond to either Bacteroidetes (16 %) or Proteobacteria (78 %). The latter were dominated by gamma- (39 %, including sequences related to the pathogenic genus Francisella), and alpha-proteobacterial (30 %) sequences. Analysis of the Pfam domain family and Gene Ontology term variation revealed that the most frequent terms that appear unique to this consortium correspond to proteins involved in “transmembrane transporter activity” and “oxidoreductase activity”. Furthermore, we identified genes that encode for enzymes involved in the catabolism of complex substance for energy reserves. We also characterized members of the transposase and integrase superfamilies, whose role in bacterial evolution is well documented, as well as putative antifreeze proteins. Antibiotic treatments of E. focardii cultures delayed the cell division of the ciliate. To conclude, our results indicate that this consortium is largely represented by bacteria derived from the original Antarctic sample and may contribute to the survival of E. focardii in laboratory condition. Furthermore, our results suggest that these bacteria may have a more general role in E. focardii survival in its natural cold and oxidative environment. Electronic supplementary material The online version of this article (doi:10.1007/s00248-015-0568-9) contains supplementary material, which is available to authorized users.