Axenic Cultivation of the Anaerobic Free-Living Ciliate Trimyema compressum

The strain N of Trimyema compressum , an anaerobic free-living ciliate, was cultivated axenically in a medium containing a buffered salt solution, yeast extract, trypticase, and glutathione. Dead bacteria were indispensable as food; a culture of the ciliate together with heat-killed Klebsiella pneumoniae has been established for more than one year. In the medium described, the ciliates grow to a higher cell density than in cultures with living bacteria as food. During the process of axenization, a nonmethanogenic bacterial endosymbiont was lost. In the microbodies of T. compressum , hydrogenase could be localized by the technique of indirect immunofluorescence.     

Degradation of food compounds and growth response on different food quality by the anaerobic ciliate Trimyema compressum

The biochemical composition of two food bacteria was examined on which monoxenic cultures of Trimyema compressum grew with different yields. The food bacteria were the saccharolytic fermenting bacterium Bacteroides WoCb15 and the purple nonsulfur bacterium Rubrivivax gelatinosus. Differences in composition of bacterial biomass concerned mainly the carbohydrate content. By different culture conditions for R. gelationsus and pasteurization of carbohydrate-rich cells, we were able to feed the ciliate with food mixtures of different carbohydrate content. Dry mass yields of the ciliate reached a maximum with mixtures of 80% carbohydrate-rich pasteurized cells plus 20% carbohydratepoor living cells. In the absence of degradable carbohydrate, energy metabolism depended on protein as substrate. Utilization of protein was incomplete, large amounts were converted into soluble compounds that accumulated in the culture medium. The ciliate consumed storage carbohydrate of living or pasteurized food bacteria equally well, while growth with short generation times was still dependent on a certain percentage of living bacteria as source of native protein. Lipids, nucleic acids and denatured proteins were not degradable by the ciliate. Consequences for the fermentative metabolism of Trimyema compressum are discussed.     

Effects of enrichment on protist abundances and bacterial composition in simple microbial communities

We experimentally investigated effects of nutrient enrichment and trophic structure in a microbial food web consisting of mixed bacteria, two bacterivorous ciliates ( Tetrahymena sp. and Colpidium sp.) and an omnivorous ciliate ( Blepharisma sp.) feeding on both trophic levels. We assembled all possible food webs including one or more of the ciliate species and cross-classified them with four levels of enrichment of the bacterial medium. The qualitative outcome of food web interactions was independent of enrichment and always the same: Tetrahymena strongly depressed or excluded Colpidium , and Blepharisma strongly depressed or excluded both bacterivores. Consequently, in all sub-webs only the dominant ciliate species responded positively to enrichment. The total density of bacteria increased with enrichment irrespective of food web composition. In contrast, the response of single-celled bacteria to enrichment depended on food web composition and was only weakly positive in most food webs with the omnivore. Enrichment had a positive effect on the relative success of (presumably more defended) bacterial aggregates. The outcome of interspecific interactions among ciliates could not be predicted from monoculture experiments and deviated from earlier experiments in which each bacterivore coexisted separately with the omnivore. As a potential explanation we suggest that changes in experimental protocol reduced spatial heterogeneity and increased attack rates. A simple, dynamical model shows that increased attack rates can indeed greatly decrease the upper limit and range of enrichment over which intermediate consumers can coexist with omnivores.     

Encystment-inducing factors in the ciliate Euplotes elegans

Although starvation is considered one of the most important induces of ciliate encystment, its nature has been unclear. Euplotes is a well-known ciliate genus, but the relationship in Euplotes between encystment and food has not been reported. The encystment of Euplotes elegans is facilitated when it is transferred to Chalkley's solution without bacteria as food. A higher ciliate density also facilitates encystment. Thus, starvation and ciliate density needed to be examined. Ciliates were inoculated into 3 treatments: Chalkley's solution with formaldehyde-fixed bacteria as nutritive particles (FFB group), with polystyrene latex particles as non-nutritive particles (PLP group), and without particles (control group). Cysts appeared fastest and ciliate numbers increased in the FFB group. Although the encystment kinetics of the PLP group was similar to that of the control group, the encystment rate of the PLP group was lower than that of the control group in the earliest phase. This suggests that the ciliates were temporarily deceived into feeding on PLP, because they had food vacuoles containing PLP during the earliest phase of incubation. A cell-free old culture solution from a stationary phase, which probably contained excreted substances from high-density ciliates, also facilitated encystment.     

Accumulation of selenium in a model freshwater microbial food web.

The transfer of selenium between bacteria and the ciliated protozoan, Paramecium putrinum, was examined in laboratory cultures. The population growth of the ciliate was not inhibited in the presence of the highest concentrations of dissolved selenite or selenate tested (10(3) micrograms liter-1). Experiments with radioactive 75selenite or 75selenate indicated that accumulation of selenium by ciliates through time was low when feeding and metabolism were reduced by incubating at 0 degrees C. However, selenium accumulated in ciliate biomass during incubation with dissolved 75Se and bacteria at 24 degrees C and also when bacteria prelabeled with 75Se were offered as food in the absence of dissolved selenium. When 75Se-labeled bacterial food was diluted by the addition of nonradioactive bacteria, the amount of selenite and selenate in ciliates decreased over time, indicating depuration by the ciliates. In longer-term (> 5-day) fed-batch incubations with 75selenite-labeled bacteria, the selenium concentration in ciliates equilibrated at approximately 1.4 micrograms of Se g (dry weight)-1. The selenium content of ciliates was similar to that of their bacterial food on a dry-weight basis. These data indicate that selenium uptake by this ciliate occurred primarily during feeding and that biomagnification of selenium did not occur in this simple food chain.     

Detection of ingested bacteria in benthic ciliates using fluorescence in situ hybridization

Fluorescence in situ hybridization (FISH) was applied to detect ingested natural bacteria within the food vacuoles of ciliates harvested from the natural sediment. In addition to this important qualitative aspect, FISH was also successfully used to measure the bacterivory of a culture of the ciliate Tetrahymena pyriformis on natural field sediment bacteria. In this feeding experiment, we compared the FISH technique with the only available alternative technique using fluorescently stained sediment (FS-sediment). The ingestion rate of unstained sediment bacteria determined by FISH was 4.6 bacteria per ciliate and hour. In contrast, Tetrahymena pyriformis cells that fed on bacteria from FS-sediment ingested 12.7 bacteria per ciliate and hour. Bacterial abundances in the sediment were equal in both sediment types (4 x 10(8) cells g sediment dry weight(-1)) when determined by DAPI counts. However, when analyzed using DTAF-counts, the number of bacteria in the FS-sediment increased to 9.7 x 10(8) cells g sediment dry weight(-1). From our findings we conclude that bacterivory by ciliates is overestimated when FS-sediment is used because DTAF stains bacteria as well as protein-containing detritus particles, which are also ingested by many ciliates. In contrast, FISH is a direct, a posteriori method that specifically stains phylogenetic lineages, e.g. eubacteria, after ingestion and thereby avoids a false determination of the number of ingested bacteria. Thus this method can also be used for the study of natural ciliate bacterivory in benthic systems.     

Ciliate grazing on <Emphasis Type="Italic">Nitrosomonas europaea</Emphasis> and <Emphasis Type="Italic">Nitrospira</Emphasis><Emphasis Type="Italic">moscoviensis</Emphasis>: Is selectivity a factor for the nitrogen cycle in natural aquatic systems?

Ciliated protists are important predators of bacteria in many aquatic habitats, including sediments. Since, many biochemical transformations within the nitrogen cycle are performed by bacteria, ciliates could have an indirect impact on the nitrogen cycle through selective grazing on nitrogen-transforming bacteria. As a case study, we examined ciliate grazing on nitrifying bacteria of the genera Nitrosomonas and Nitrospira. All experiments were designed as in vitro-experiments with cultures of different bacteria and ciliate species. The nitrifying bacteria used in our experiments were Nitrosomonas europaea [Winogradsky 1892] and Nitrospira moscoviensis [Ehrich 2001]. The ciliates comprised of four species that are known as efficient bacterivores and common members of the protist community in aquatic systems: Paramecium aurelia [Müller 1773], Euplotes octocarinatus [Carter 1972], Tetrahymena pyriformis [Ehrenberg 1830] and Cyclidium glaucoma [Müller 1786]. Our experimental approach, using a combination of DAPI and FISH staining, was successful in allowing the observation of ingestion of specific bacteria and their detection within ciliate food vacuoles. However, the ciliates in this study showed no significant selective grazing. No food preferences for a any bacterial taxon or any size class or morphotype were detected. Correlation with time between ciliate abundance and bacterial abundance or biovolume, using log transformed growth rates of ciliates and bacteria, showed no significant results. On the bacterial side, neither an active defence mechanism of the nitrifying bacteria against ciliate grazing, such as changes in morphology, nor competition for resources were observed. These results suggest that in our in vitro-experiments grazing by ciliates has no influence on abundance and growth of nitrifying bacteria and nitrification.     

Functional integration of Methanobacterium formicicum into the anaerobic ciliate Trimyema compressum

Monoxenic cultures of the anaerobic, endosymbiont-free ciliate Trimyema compressum were incubated with low numbers of Bacteroides sp. strain WoCb15 as food bacteria and two strains (DSM 3636 and 3637) of Methanobacterium formicicum, which originally had been isolated from the anaerobic protozoa Metopus striatus and Pelomyxapalustris. The ciliate which had lost its original endosymbiotic methanogens ingested both strains of M. formicicum. The methanogenic bacteria were found intact in large vacuoles in contrast to the food bacteria which were digested. Single methanogens were separated from the vacuoles and appeared surrounded by a membrane in the cytoplasm of the ciliate. After 2 months of incubation, the methanogenic bacteria still exhibited the typical bluish fluorescence and the new symbiotic association of M. formicicum and T. compressum excreted methane. Increasing the growth rate of the ciliates by large numbers of food bacteria resulted in a loss of the methanogenic bacteria, due to statistical outgrowth.     

Size-selective feeding by Cyclidium sp. on bacterioplankton and various sizes of cultured bacteria

Abstract Size-selective grazing by Cyclidium sp., isolated as a dominant ciliate bacterivore from the Římov Reservoir (South Bohemia), was examined using fluorescent labelled bacteria (FLB) produced from natural bacterioplankton or pure bacterial cultures. Sizes of ingested bacteria in food vacuoles were measured directly. Three experimental arrangements were used: (1) Ciliates were grown on the pure culture of Alcaligenes xylosoxidans and fed with various proportions of ‘large’ and ‘small’ FLB (mean biovolume, 0.377 and 0.202 μm³, respectively) prepared from the same bacterial species. Results clearly showed significant selection of larger bacteria. (2) Ciliates were grown on natural bacterioplankton from the reservoir and subsequently fed on FLB prepared from the reservoir bacterioplankton (mean biovolume, 0.065 μm³). Independent of either prey or predator abundance, larger FLB (> 0.100 μ m³, and especially those > 0.200 μ m³) were ingested with much higher frequency than their occurrence i the natural assemblage. (3) Ciliates were grown on the reservoir baterioplankton and fed by FLB prepared from the culture of Pseudomonas sp. In contrast with previous results, no size selection of the ciliate was found when FLB were different from the bacterial food used to grow the ciliate. Ecological impacts of size-selective bacterivory are suggested.     

Supplementation with sterols improves food quality of a ciliate for Daphnia magna

Experimental results provide evidence that trophic interactions between ciliates and Daphnia are constrained by the comparatively low food quality of ciliates. The dietary sterol content is a crucial factor in determining food quality for Daphnia. Ciliates, however, presumably do not synthesize sterols de novo. We hypothesized that ciliates are nutritionally inadequate because of their lack of sterols and tested this hypothesis in growth experiments with Daphnia magna and the ciliate Colpidium campylum. The lipid content of the ciliate was altered by allowing them to feed on fluorescently labeled albumin beads supplemented with different sterols. Ciliates that preyed upon a sterol-free diet (bacteria) did not contain any sterols, and growth of D. magna on these ciliates was poor. Supplementation of the ciliates' food source with different sterols led to the incorporation of the supplemented sterols into the ciliates' cells and to enhanced somatic growth of D. magna. Sterol limitation was thereby identified as the major constraint of ciliate food quality for Daphnia. Furthermore, by supplementation of sterols unsuitable for supporting Daphnia growth, we provide evidence that ciliates as intermediary grazers biochemically upgrade unsuitable dietary sterols to sterols appropriate to meet the physiological demands of Daphnia.     

Feeding response of a benthic copepod to ciliate prey type,prey concentration and habitat complexity

1. Protozoans are important consumers within microbial food webs and, in turn, they represent potential prey for small metazoans. However, feeding interactions within these food webs are rarely characterised and this is especially true for freshwater sediments. 2. We aimed to quantify the feeding links between a freshwater meiofaunal copepod and ciliates in two laboratory experiments. The first experiment addressed the response of Eucyclops serrulatus towards ciliate density and type (two ciliate species of the same genus differing in terms of body size). A second experiment assessed the effect of habitat structure on feeding rates by introducing different structural complexity into the feeding arena. In contrast to the first experiment, which was run only for one time period, this experiment also tested three different total feeding times (4, 7 and 9 h). 3. Eucyclops serrulatus exhibited high ingestion rates, with 3–69 ciliates copepod^?1 h^?1 consumed depending on food concentration, food type and habitat complexity. Copepods exhibited a preference for the smaller ciliate when total ciliate concentration was low, but selected both ciliates equally when food concentrations were medium or high. However, at very high food concentration, Eucyclops preferred the larger ciliate (which was 1/3 of its own body size), suggesting that the longer handling times of the larger prey are rewarding when the large prey is present in high numbers. In terms of total numbers consumed, copepods fed on more small ciliates, but in terms of carbon units both ciliates were selected equally when total prey concentration was low or medium. However, copepods derived more carbon from the larger prey at high and very high prey concentrations (up to 0.7 μgC out of a maximum of 1.1 μgC copepod^?1 h^?1). Habitat complexity influenced the feeding of copepods when it was observed over time. 4. The copepod–ciliate link is well known from the pelagic zone of both marine and freshwater habitats. We have shown its potential importance within the benthos, where it can be influenced by food identity, food quantity and possibly by habitat complexity.     

Virus particle production in lysogenic bacteria exposed to protozoan grazing

Electron microscopy was used to investigate the apparent induction of virus particle production in bacteria undergoing digestion by ciliates. Results showed that numbers of bacteria containing virus particles increased by a factor of 25 when enclosed within ciliate food vacuoles. It was also found that 10% of these particles survived the digestion process to be released back into the aquatic habitat within faecal pellets. The possibility of virus gene transfer occurring between lysogenically infected bacteria that survive the ciliate digestive processes, is also considered.     

Food vacuole contents in the ciliate, Balantidium jocularum (Balantididae), a symbiont in the intestine of the surgeonfish, Naso tonganus (Acanthuridae)

During the past 16 years, the ciliate Balantidium jocularum has been collected from the intestines of many specimens of its fish host, Naso tonganus, all collected from the Great Barrier Reef near Lizard Island, Australia. Ciliates for this study of food consumption were isolated in 1988, 1989, 2003, and 2005. Nineteen specimens of B. jocularum were examined in the transmission electron microscope to determine the contents of both food vacuoles and a putative discharging cytoproct vacuole. Food vacuoles contained rod-shaped bacteria, tightly coiled spirilliform bacteria, and one or more euglenid flagellates. In several balantidia of somewhat different form than the type species of B. jocularum, the large bacterium, Epulopiscium fishelsoni, was observed in light microscope protargol preparations. Some putative phagolysosomes retained spirilliform bacteria that were apparently intact, and others contained partially digested flagellates. Food in a single discharging cytoproct vacuole consisted of normal appearing spirilliform bacteria, some other bacteria, and no flagellates. The results argue for non-selective ingestion of food and selective digestion; hence, somewhat inefficient food processing.     

Bacterivory by the ciliate Euplotes in different states of hunger

Abstract: The feeding of the marine ciliate Euplotes mutabilis was studied using bacteria ( Vibrio natriegens ) doubly labelled with ³H-thymidine and ¹⁴C-leucine. In the presence of abundant bacteria (30 × 10⁶ bacteria ml⁻¹), an average Euplotes cell (initially without food vacuoles) with a protein content of 12 ng consumed 16 × 10³ bacteria in the first hour and 27 × 10³ bacteria over four hours, accumulating about 60% of the bacterial protein into ciliate macromolecules. Euplotes which had been starved or under-fed to reduce cell protein biomass to 7 or 9 ng consumed significantly fewer bacteria, but the gross growth efficiency for protein did not change. The rate of consumption of bacteria by large Euplotes of protein content 15 ng was initially less than that of 12 ng cells, and it decreased markedly before the end of a 4-hour experiment. Recently divided cells ingested bacteria rapidly, but showed a reduced gross growth efficiency of about 40%. At low bacterial concentrations (6 × 10⁶ bacteria ml⁻¹) the rates of ingestion were markedly reduced to between     and     of maximal levels; the smallest cells could not sustain feeding activity at the low prey concentration and gross growth efficiency fell from 43 to 20% during a 4-hour experiment. The strategy adopted by Euplotes in response to local fluctuations in food supply involves rapid consumption with high growth efficiency in times of plenty, but slow shrinkage without cell division to survive in times of shortage.     

End products of metabolism in the anaerobic ciliate Trimyema compressum

Abstract The products of anaerobic and micro-aerobic (0.8% O₂) metabolism of the sapropelic ciliate Trimyema compressum strain N were studied. Under anaerobic conditions ethanol was formed in large amounts representing 44% of the total carbon excreted. Acetate, lactate, formate, CO₂ and H₂ were minor products, while succinate was formed in hardly detectable amounts. Under micro-aerobic conditions O₂ was consumed, CO₂ and formate were produced as major end products and no H₂, ethanol and succinate were formed.     

Contrasting dynamics of ciliate communities in sandy and silty sediments of an estuarine intertidal flat

During a 1-year study of the ciliate faunas of a silty and a sandy site on an intertidal flat in the Westerschelde estuary, a total number of 107 taxa were recorded belonging to at least 52 genera and 15 orders. Our results suggest that physical properties of the sediment were more important in regulating ciliate abundance, diversity and community composition than food availability, predation, temperature or oxygen concentration. Ciliate abundance and diversity were positively related to sediment grain size and the ciliate community of silty sediments was found to be a subset of that of sandy sediments. At the sandy site, where the sediment composition was stable, seasonal changes in the ciliate community were related to changes in food availability and/or temperature. At both the sandy and silty sites, a clear vertical gradient in the ciliate community was observed that appeared to be linked to gradients in food availability and oxygen concentration. These vertical gradients in ciliate community composition, however, were less steep than the measured oxygen gradients, probably due to the presence of oxic microniches in the anoxic zone.     

Carbon flow dynamics in the pelagic community of the Sau Reservoir (Catalonia,NE Spain)

Changes in the pelagic community structure and activity along the longitudinal axis of the eutrophic Sau Reservoir (Catalonia, NE Spain) were studied between 1996 and 1999. Samples were taken from several transects from river to dam, measuring dissolved organic carbon (DOC), bacterial abundance and production, chlorophyll a concentration, heterotrophic nanoflagelate (HNF) and ciliate abundances and their grazing rates, and zooplankton density. The role of microbial and classical food chains (i.e., based directly on phytoplankon) were compared in the Sau Reservoir by analysing river-to-dam gradients in biomass and carbon and their temporal changes. The detritic metabolic pathway was more important near to the inflow, due to high allochthonous organic matter loads allowing the rapid development of the microbial food web. Protozoans (HNF and ciliates) consumed most of the bacterial production (i.e., >50%) in the reservoir. As opposed to the systems of lower trophic status ciliate carbon biomass and bacterivory contributions were larger than those of the HNF. We estimated species-specific ciliate growing rates on bacteria and distinguished several periods with high importance of distinct ciliate communities.     

Some lower food web organisms in the nutrition of marine harpacticoid copepods: an experimental study

Some lower food web organisms from the marine littoral environment were studied as food for harpacticoid copepods. In laboratory experiments, it could be shown that, among the ciliates, the slow-movingUronema sp. was taken up while the fast-movingEuplotes sp. was not.Asterionella glacialis, a pennate diatom with spiny projections, was unsuitable as food. The centric diatomSkeletonema costatum was ingested by all harpacticoid species tested, includingTisbe holothuriae, Paramphiascella vararensis, Amphiascoides debilis andDactylopodia vulgaris. All are epibenthic and phytal species occurring in the shallow waters of Helgoland (North Sea). The amount of ciliate and algal carbon taken up was less than that provided by bacteria under laboratory conditions. However, some diatom food may be essential for the development ofD. vulgaris.     

Observations on Bacterial Feeding by the Rumen Ciliate Isotricha prostoma

SYNOPSIS. Starvation of Isotricha prostoma for 72–96 hours decreased the cellular amylopectin granules and facilitated the microscopic search for bacterial feeding. I. prostoma selected and ingested only certain rods from among many types of rumen bacteria. In order to isolate the bacteria important as a food source for Isotricha , the starved protozoa were allowed to feed on mixed rumen bacteria, washed, and the crushed protozoan contents quickly cultured for bacteria. Several strains of bacteria were isolated in pure culture. Three of the rod strains isolated were rapidly ingested by I. prostoma when fed to the ciliate. In a monobacterial culture I. prostoma divided once before succumbing.     

Feasibility of using GFP-expressing Escherichia coli, coupled with fluorimetry, to determine protozoan ingestion rates

The feasibility of using a live Escherichia coli population, which had been engineered to express the green fluorescent protein (GFP), coupled with fluorimetry, was tested as a means for determining protozoan ingestion rates. Its potential use was based on evidence that once cells are acidified, e.g. in a food vacuole, the fluorescence is lost. Of the 29 protozoa tested, over 85% ingested the GFP-expressing E. coli and a detailed experiment with the ciliate Tetrahymena pyriformis was carried out, principally to assess the performance of the live bacterium against two commonly used surrogate prey, i.e. fluorescently labelled bacteria (FLB) and fluorescently labelled microspheres (FLMs). A decrease in GFP-expressing E. coli fluorescence and, hence, concentration, was recorded by fluorimetry and epifluorescence microscopy, with calculated ingestion rates being equivalent. A higher ingestion rate was determined by counting the number of fluorescent E. coli within the ciliate over 120 s, but this was equivalent to that obtained for the stained E. coli using the same direct method of analysis. However, the ciliate was shown to process the stained and unstained E. coli cells differently, with only the latter resulting in an increase in ciliate abundance.