Ciliates as a food source for marine planktonic copepods

Copepods of the genusEurytemora, isolated from the Patuxent River, a tributary of Chesapeake Bay, were fed suspensions of the ciliateUronema isolated from the Rhode River, a subestuary of Chesapeake Bay. Grazing by copepods was determined by the decrease in numbers of ciliates, which were monitored by both direct counting and particle size analysis. Results from both methods of analysis showed significant reduction in the numbers ofUronema in the suspension whenEurytemora was present. Survival of copepods with ciliates added as food source was significantly longer than without ciliates. Analysis of field samples collected in the fall showed that ciliates comprised approximately 20% of the total plankton biomass at selected sampling sites. The results of the laboratory and field studies indicate that copepods can feed on ciliates and suggest that, in nature, ciliates may comprise an important source of food for copepods.     

Interception of small particles by flocculent structures, sessile ciliates, and the basic layer of a wastewater biofilm

We investigated attachment processes of hydrophobic and hydrophilic particles (diameter = 1 microm) to mature biofilms grown on clay marbles in a sequencing batch biofilm reactor. During a treatment cycle with filtered wastewater containing different fluorescent beads, the progression of particle density in various biofilm compartments (carrier biofilm, basic biofilm layer, biofilm flocs, and sessile ciliates) was determined by flow cytometry, confocal laser scanning microscopy and automated image analysis. Particles were almost completely removed from wastewater by typical processes of particle retention: up to 58% of particles attached to clay marbles, up to 15% were associated with suspended flocs, and up to 10% were ingested by sessile ciliates. Ingestion of particles by ciliates was exceptionally high immediately after wastewater addition (1,200 particles grazer(-1) x h(-1)) and continued until approximately 14% of the water had been cleared by ciliate filter feeding. Most probably, ciliate bioturbation increases particle sorption to the basic biofilm. Backwashing of the reactor detached pieces of biofilm and thus released approximately 50% of the particles into rinsing water. Clay marbles in the upper part of the reactor were more efficiently abraded than in the lower part. No indications for selective attachment of the applied hydrophobic and hydrophilic beads were found. As a consequence of interception patterns, organisms at elevated biofilm structures are probably major profiteers of wastewater particles; among them, ciliates may be of major importance because of their highly active digestive food vacuoles.     

Interception of Small Particles by Flocculent Structures, Sessile Ciliates, and the Basic Layer of a Wastewater Biofilm

We investigated attachment processes of hydrophobic and hydrophilic particles (diameter = 1 μm) to mature biofilms grown on clay marbles in a sequencing batch biofilm reactor. During a treatment cycle with filtered wastewater containing different fluorescent beads, the progression of particle density in various biofilm compartments (carrier biofilm, basic biofilm layer, biofilm flocs, and sessile ciliates) was determined by flow cytometry, confocal laser scanning microscopy and automated image analysis. Particles were almost completely removed from wastewater by typical processes of particle retention: up to 58% of particles attached to clay marbles, up to 15% were associated with suspended flocs, and up to 10% were ingested by sessile ciliates. Ingestion of particles by ciliates was exceptionally high immediately after wastewater addition (1,200 particles grazer⁻¹ h⁻¹) and continued until approximately 14% of the water had been cleared by ciliate filter feeding. Most probably, ciliate bioturbation increases particle sorption to the basic biofilm. Backwashing of the reactor detached pieces of biofilm and thus released approximately 50% of the particles into rinsing water. Clay marbles in the upper part of the reactor were more efficiently abraded than in the lower part. No indications for selective attachment of the applied hydrophobic and hydrophilic beads were found. As a consequence of interception patterns, organisms at elevated biofilm structures are probably major profiteers of wastewater particles; among them, ciliates may be of major importance because of their highly active digestive food vacuoles.     

Population Dynamics of Active and Total Ciliate Populations in Arable Soil Amended with Wheat

Soil protozoa are characterized by their ability to produce cysts, which allows them to survive unfavorable conditions (e.g., desiccation) for extended periods. Under favorable conditions, they may rapidly excyst and begin feeding, but even under optimal conditions, a large proportion of the population may be encysted. The factors governing the dynamics of active and encysted cells in the soil are not well understood. Our objective was to determine the dynamics of active and encysted populations of ciliates during the decomposition of freshly added organic material. We monitored, in soil microcosms, the active and total populations of ciliates, their potential prey (bacteria and small protozoa), their potential competitors (amoebae, flagellates, and nematodes), and their potential predators (nematodes). We sampled with short time intervals (2 to 6 days) and generated a data set, suitable for mathematical modeling. Following the addition of fresh organic material, bacterial numbers increased more than 1,400-fold. There was a temporary increase in the number of active ciliates, followed by a rapid decline, although the size of the bacterial prey populations remained high. During this initial burst of ciliate growth, the population of cystic ciliates increased 100-fold. We suggest that internal population regulation is the major factor governing ciliate encystment and that the rate of encystment depends on ciliate density. This model provides a quantitative explanation of ciliatostasis and can explain why protozoan growth in soil is less than that in aquatic systems. Internally governed encystment may be an essential adaptation to an unpredictable environment in which individual protozoa cannot predict when the soil will dry out and will survive desiccation only if they have encysted in time.     

Culture of the Rumen Holotrich Ciliate Dasytricha ruminantium Schuberg

The successful cultivation of the anaerobic ciliate Dasytricha ruminantium is described. The cultures were established in a salts medium containing 30% clarified rumen fluid. Sucrose and extract of rumen holotrich protozoa were fed once daily for 2 to 4 hr, and Dasytricha was then transferred to medium free from these nutrients. Rumen fluid was essential. Omission of protozoal extract resulted in gradual death of the ciliates. Bovine serum satisfactorily substituted for the protozoal extract, but various rumen bacteria, extract of rumen bacteria, and extracts of plant materials could not. There was a positive correlation between formation of methane in the cultures and growth of the ciliates. It is possible that methane bacteria were ingested, but it is not excluded that survival of both dasytrichs and the methanogenic bacteria depended on a low redox potential of the medium.     

Population dynamics of active and total ciliate populations in arable soil amended with wheat

Soil protozoa are characterized by their ability to produce cysts, which allows them to survive unfavorable conditions (e.g., desiccation) for extended periods. Under favorable conditions, they may rapidly excyst and begin feeding, but even under optimal conditions, a large proportion of the population may be encysted. The factors governing the dynamics of active and encysted cells in the soil are not well understood. Our objective was to determine the dynamics of active and encysted populations of ciliates during the decomposition of freshly added organic material. We monitored, in soil microcosms, the active and total populations of ciliates, their potential prey (bacteria and small protozoa), their potential competitors (amoebae, flagellates, and nematodes), and their potential predators (nematodes). We sampled with short time intervals (2 to 6 days) and generated a data set, suitable for mathematical modeling. Following the addition of fresh organic material, bacterial numbers increased more than 1,400-fold. There was a temporary increase in the number of active ciliates, followed by a rapid decline, although the size of the bacterial prey populations remained high. During this initial burst of ciliate growth, the population of cystic ciliates increased 100-fold. We suggest that internal population regulation is the major factor governing ciliate encystment and that the rate of encystment depends on ciliate density. This model provides a quantitative explanation of ciliatostasis and can explain why protozoan growth in soil is less than that in aquatic systems. Internally governed encystment may be an essential adaptation to an unpredictable environment in which individual protozoa cannot predict when the soil will dry out and will survive desiccation only if they have encysted in time.     

Evaluation of double formalin--Lugol's fixation in assessing number and biomass of ciliates: an example of estimations at mesoscale in NE Atlantic

Ciliated protozoa are potential grazers of primary and bacterial production and act as intermediaries between picoplankton and copepods and other large suspension feeders. Accurate determination of ciliate abundance and feeding mode is crucial in oceanic carbon budget estimations. However, the impact of different fixatives on the abundance and cell volume of ciliates has been investigated in only a few studies using either laboratory cultures or natural populations. Lugol's solution and formalin are the most commonly used fixatives for the preservation of ciliates samples. In the present study, the aim was to compare 0.4% Lugol's solution and 2% borated-formalin fixation and evaluate the need of counting duplicate samples each using a different fixative. For this, a large number of samples (n = 110) from the NE Atlantic was analyzed in the frame of POMME program (Multidisciplinary Mesoscale Ocean Program). We established a statistically significant relationship (p < 0.0001) between Lugol's and formalin fixed samples for both abundance (r2 = 0.50) and biomass (r2 = 0.76) of aloricate ciliates which showed that counts were higher in Lugol's solution by a factor of 2 and a non-taxon specific cell-loss in formalin. However, loricate ciliate abundance in our samples which were represented primarily by Tintinnus spp. did not show any difference between the two treatments. Abundance and biomass of mixotrophic ciliates (chloroplast-bearing cells) were for various reasons underestimated in both treatments. Our results show that unique fixation by formalin may severely underestimate ciliates abundance and biomass although their population may not alter. For this reason, Lugol's solution is best for the estimation of their abundance and biomass. However, for counts of mixotrophs and the evaluation of the ecological role of ciliates in carbon flux, double fixation is essential. Compromises regarding the fixatives have lead to severe underestimations of mixotrophs in studies conducted by now.     

Suspension feeding in ciliated protozoa: Feeding rates and their ecological significance

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 (> 1–5m) compare favorably with metazoan suspension feeders with respect to the ability to concentrate dilute suspensions of particles. Species specialized on smaller food particles (0.2–1m), 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.     

Endosymbiotic Methanogenic Bacteria In Anaerobic Ciliates: Significance For the Growth Efficiency of the Host

ABSTRACT Endosymbiotic methanogenic bacteria of three species of anaerobic ciliates (Plagiopyla frontata, Metopus conforms , and M. palaeformis) were inactivated with the specific methanogen inhibitor 2-bromoethanesulfonic acid. the absence of endosymbiont methanogens reduced growth rate and growth yield by about 30% in P. frontata and M. contortus , while no significant change in fitness was observed in M. palaeformis. In Plagiopyla the growth rate constant is not affected by an artificially increased pH₂ neither in normal nor in methanogen-free ciliates. the energetic advantage conferred by endosymbiont methanogens in Plagiopyla and in Metopus contortus probably is due to excretion of organic material from the bacteria at the expense of bacterial reproduction. It is unlikely that the maintenance of a low pH₂ within the cells due to H₂-consumption by the bacteria is important to the ciliates.     

Excessive expression of the plant kinesin TBK5 converts cortical and perinuclear microtubules into a radial array emanating from a single focus

TBK5 is a plant-specific kinesin constantly expressed in tobacco BY-2 cells. An analysis of the distribution of green fluorescent protein-tagged TBK5 (GFP-TBK5) transiently expressed in BY-2 protoplasts revealed that TBK5 could associate with microtubules in vivo. GFP-TBK5 often assembled to form a single particle when accumulated in cells. The particle was located in close proximity to the nucleus, and its formation was accompanied by the development of a radial array of microtubules emanating from it and the loss of cortical microtubules. Microtubule depolymerization by treatment with propyzamide inhibited particle formation and stimulated the formation of dispersed aggregates of GFP-TBK5. Through expression of different TBK5 mutants as GFP fusions, the motor domain, two separated coiled-coil domains and the C-terminal domain of TBK5 were identified as the domains playing essential roles in particle formation. Mutants with putatively non-motile motor domains or lacking the C-terminal domain were localized to cortical and perinuclear microtubules, whereas those lacking either of the coiled-coil domains were preferentially distributed around the nucleus and along perinuclar microtubules. Further, the deletion of one of the coiled-coil domains or the C-terminal domain was sufficient to inhibit the propyzamide-induced formation of dispersed aggregates, whereas the mutation in the motor domain was not. These results led us to propose a model in which the particle is formed through the microtubule-based movement of GFP-TBK5 toward the nucleus and subsequent microtubule-independent aggregation based on coiled-coil interactions. The dramatic microtubule rearrangement would be explained if GFP-TBK5 relocated and gathered newly formed microtubules and/or microtubule-nucleating units.     

Microbial composition and structure of aerobic granular sewage biofilms

Aerobic activated sludge granules are dense, spherical biofilms which can strongly improve purification efficiency and sludge settling in wastewater treatment processes. In this study, the structure and development of different granule types were analyzed. Biofilm samples originated from lab-scale sequencing batch reactors which were operated with malthouse, brewery, and artificial wastewater. Scanning electron microscopy, light microscopy, and confocal laser scanning microscopy together with fluorescence in situ hybridization (FISH) allowed insights into the structure of these biofilms. Microscopic observation revealed that granules consist of bacteria, extracellular polymeric substances (EPS), protozoa and, in some cases, fungi. The biofilm development, starting from an activated sludge floc up to a mature granule, follows three phases. During phase 1, stalked ciliated protozoa of the subclass Peritrichia, e.g., Epistylis spp., settle on activated sludge flocs and build tree-like colonies. The stalks are subsequently colonized by bacteria. During phase 2, the ciliates become completely overgrown by bacteria and die. Thereby, the cellular remnants of ciliates act like a backbone for granule formation. During phase 3, smooth, compact granules are formed which serve as a new substratum for unstalked ciliate swarmers settling on granule surfaces. These mature granules comprise a dense core zone containing bacterial cells and EPS and a loosely structured fringe zone consisting of either ciliates and bacteria or fungi and bacteria. Since granules can grow to a size of up to several millimeters in diameter, we developed and applied a modified FISH protocol for the study of cryosectioned biofilms. This protocol allows the simultaneous detection of bacteria, ciliates, and fungi in and on granules.     

Experimental Approach to the Role of Different Ecological Types of Protozoa in the Activated-sludge System

Relationship between bacteria and ciliates was investigated in continuous cultures with and without the recycling of flocculated biomass. Monocultures of ciliates of various ecological types were used together with mixed cultures of bacteria or bacteria and flagellates. The effect of ciliates on the distribution of flocculated and dispersed, biomass was tested with respect to characterizing an optimum mixed culture for activated-sludge treatment. Monoprotozoal populations were not successful in stabilizing the morphology of a bacterial community in a system with recycling of flocculated biomass. Stabilization was observed (with particular ecological types of ciliates) in the system with a high residence time without recycling and/or in the system with the recycling inoculated by both flagellates and ciliates.     

Heat shock protein of the Hsp70 family in the euryhaline cilate Paramecium nephridiatum and its role in adaptation to salinity changes

The level of the Hsp70 heat shock protein was studied in the cells of euryhaline ciliates Paramecium nephridiatum after environmental salinity changes. Two types of treatment were used: “shock” and “adaptation.” In the former case the ciliates were placed for 1 h into medium with stress salinity, and subsequently returned for 2 h to the medium they were acclimated to. In the latter case the ciliates were placed for 3 h into the medium with the stress level of salinity. The ciliates acclimated to fresh water (0‰ salinity) were shown to have a higher constitutive level of Hsp70 than those acclimated to 10‰. Transfer of the protists from fresh water to medium with 10‰ salinity (the shock medium) did not increase Hsp70 synthesis, whereas the return transfer resulted in induction of Hsp70 in the cells. In both directions of salinity change, “adaptation” led to induction of Hsp70. The obtained results support the hypothesis that salinity of 10‰ is less harmful for the eurihaline ciliate P. nephridiatum, than fresh water is. We also presume that the ability of euryhaline ciliates to survive in a wide salinity spectrum might be determined by the higher constitutive level of their Hsp70 in comparison with that of stenohaline representatives of the same genus.     

Enumeration of small ciliates in culture by flow cytometry and nucleic acid staining

We developed a fast and simple protocol for accurate quantification of small freshwater ciliates by flow cytometry (FCM). The ciliates were stained with several nucleic acid stains such as TO-PRO-1, YO-YO-1 and PicoGreen, and analysed by a commercially available flow cytometer. The method was tested with cultures of the prostomatid species Urotricha farcta and Balanion planctonicum, including the small cryptophyte Cryptomonas sp. as food. Of the dyes tested, TO-PRO-1 gave the best results. Flow cytometric results agreed well with microscopic counts. Due to its greater speed and accuracy, FCM was superior to light microscopy. FCM was also superior to electronical particle counting and sizing (EPCS). Of particular importance, FCM in combination with TO-PRO-1 staining allowed unequivocal discrimination in cases of overlapping size distributions between the target population (i.e., the ciliate predators) and other particles (the cryptophyte prey, detritus).     

Ciliate–adenovirus interactions in experimental co-cultures of Euplotes octocarinatus and in wastewater environment

The aim of the present work was to study the dynamics of the interactions between human adenovirus and ciliates under both experimental and field conditions. Experimental co-cultures of the ciliated protozoan Euplotes octocarinatus and human adenovirus (HAdV) type 2 were established and virus internalization was investigated using nested PCR and direct immunofluorescence (IF). In addition, to study protozoa-virus interactions in the field, wild ciliates were isolated from active sludges of a wastewater treatment plant and analyzed for the presence of adenovirus using direct IF. In vitro experiments revealed HAdV type 2 inside Euplotes cells after 15 min of contact and its persistence until at least 35 days post infection. In addition, our results showed the adsorption of adenovirus on the surface of wild ciliates. We conclude that HAdV is taken up by ciliates, however more studies are necessary in order to better investigate the mechanisms, the infectivity of internalized virus and the protective effects of internalization against disinfection.     

Biochemical composition of algivorous freshwater ciliates: you are not what you eat

The focus of our study was to determine whether the biochemical composition of two algivorous ciliates, both fed the same alga, resembles that of their diet. By comparing both ciliated protozoa we intended to identify species-specific differences in the metabolic features of these ciliates. Carbon- and cell-specific concentrations of fatty acids and essential amino acids were investigated for the ciliates Balanion planctonicum and Urotricha farcta grown on the cryptomonad Cryptomonas phaseolus. Stepwise discriminant analyses (SDA) indicated differences in the biochemical composition between ciliates and their diet and between the two ciliated protozoa. Carbon-specific fatty acid concentrations were usually higher in the ciliates than in their diet, especially concentrations of monounsaturated and some polyunsaturated fatty acids. Except for tryptophan, valine, and lysine, amino acid concentrations were higher in the ciliates than in C. phaseolus. Furthermore, differences in the polyunsaturated fatty acids accounted for the largest discrepancies between the two ciliated protozoa. The higher concentrations in the ciliates compared to their diet suggest that these species are capable of efficiently ingesting, assimilating or possibly synthesizing some fatty acids and amino acids. We conclude that dietary fatty acid and amino acid composition influences the composition of the two ciliated protozoa to a minor extent, and that species-specific differences in fatty acid and amino acid metabolism may be more important determinants of the biochemical composition of the studied ciliates. Moreover, the metabolism of polyunsaturated fatty acids seems to differ more profoundly between the two ciliated protozoa than the metabolism of other fatty acid classes or amino acids.     

Biomass and feeding activity of phagotrophic mixotrophs in the northwestern Black Sea during the summer 1995

Biomass and activities of planktonicmicroorganisms (bacteria, nanoplankton andmicroplankton) were measured in the northwestern BlackSea during summer 1995. The method based on theuptake of fluorescently labeled prey was chosen todetermine the ingestion rate of bacteria andnanoplankton by phagotrophic microorganisms. Thismethod revealed the presence of mixotrophic organismssuch as ’plastid-retaining ciliates‘ in the wholecoastal area. Mixotrophic ciliates were dominated bymicro-sized forms and maximum biomasses were recorded inthe water masses characterised by low nutrientconcentrations but high food particle concentrations. Mixotrophic nanoflagellates were absentand mixotrophic dinoflagellates were observed at onestation only. Mixotrophic ciliates were shown to ingestpreferably bacteria while mixotrophic dinoflagellateswere grazing almost exclusively on nanoflagellates.Although the biomass of mixotrophic organisms weresignificantly lower than those of aplastidic protozoa,their feeding activity contributed to 14 and 24% ofthe ingestion of bacteria and nanoplankton, respectively.This is due to the high specificingestion rate of mixotrophic micro-sized ciliates anddinoflagellates, which were two and three times higher,respectively, than the specific ingestion rate ofbacteria and nanoplankton by aplastidic protozoa. Thissuggests a significant contribution of phagotrophicmixotrophs to the microbial network of thenorthwestern Black Sea. This revised version was published online in July 2006 with corrections to the Cover Date.     

Isolation and identification of herbivorous ciliates from contaminated microalgal cultures

Ciliates are a common but understudied group of grazers that can invade microalgal cultures. To estimate the potential impact of ciliates on microalgal culture productivity, the identification of species that can invade these cultures is essential. Furthermore, isolation of these herbivorous ciliates allows to use them in experiments that investigate the impact of ciliate grazing on the productivity of microalgal cultures. The main aims of this study were to isolate and identify ciliates that invade cultures of the freshwater microalgae Chlorella and Chlamydomonas, and to establish a live collection of these ciliates for usage in future experiments. To this end, we optimized a method for isolating ciliates from contaminated microalgal cultures and we developed a new PCR primer set for amplifying the partial 18S rDNA of ciliates belonging to the classes Spirotrichea, Oligohymenophorea and Colpodea. As a result, we isolated 11 ciliates from microalgal enrichment cultures inoculated with non-sterile dust and various freshwater sources. Of these 11 species, 7 were found to be feeding on Chlamydomonas. Ciliate species that fed on Chlorella could not be isolated in this study. Ciliate species feeding on Chlamydomonas were identified based on a combination of morphological observations and molecular analyses of partial 18S rDNA sequences.     

Continuous Culture of Ruminal Microorganisms in Chemically Defined Medium: II. Culture Medium Studies

Ruminal ciliates have been grown in continuous culture in chemically defined media and in the absence of viable bacteria. Oligotrichic ruminal ciliates seem to require insoluble carbohydrates for growth; the holotrichic ciliates require soluble carbohydrates, but at low concentrations. Both groups of ciliates utilize amino acids as their principal nitrogen source when these are supplied in micromolar concentrations; at millimolar concentrations, amino acids are toxic, possibly from excessive ammonia formation arising from ciliate deaminase activity. Holotrichic ruminal ciliates are destroyed by overdeposition of amylopectin when glucose is present above 0.1% concentration in the medium. Ecological requirements of ruminal ciliates are also described.     

Simultaneous consumption of bacteria and dissolved organic matter byTetrahymena pyriformis

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.