Colpoda Research Articles

Identification of Differentially Expressed Water‐insoluble Proteins in the Encystment Process of Colpoda cucullus by Two‐dimensional Electrophoresis and LC‐MS/MS Analysis

In the encystment process of the ciliate protist Colpoda cucullus, we observed that the cell total protein abundance was reduced at 12 h-1 d after the onset of encystment induction subsequent to the reduction in mRNA abundance. We analyzed the alteration of the expression levels of water-insoluble proteins by two-dimensional polyacrylamide gel electrophoresis using polyoxyethylene (20) sorbitan monooleate (Tween-80), and we identified proteins whose expression levels were altered in the encystment process by a liquid chromatography tandem mass spectrometry analysis. The expression level of a 60-kDa protein (p60; heat shock protein 60) was temporarily enhanced and that of a 55-kDa protein (p55; actin) and a 49-kDa protein (p49; actin) was enhanced in the Colpoda encystment process. In mature cysts, the expression level of p55 and p49 tended to be reduced, whereas the expression level of a 50-kDa protein (p50d; α-tubulin), a 25-kDa protein (p25; α-tubulin) and a 52-kDa protein (p52c; β-tubulin) was enhanced.

Evaluation of intracellular Ca2plus concentration by Fura 2 ratiometry in encystment-induced Colpoda cucullus

In Colpoda cucullus, the signaling pathways for encystment induction involving protein phosphorylation have been believed to be triggered by an increase in the intracellular Ca 2+ concentration promoted by cell-to-cell mechanical contact due to overpopulation. By means of fura 2 ratiometry, the present study showed that the intracellular Ca 2+

Emergence of the Terrestrial Ciliate <i>Colpoda cucullus</i> from a Resting Cyst: Rupture of the Cyst Wall by Active Expansion of an Excystment Vacuole

The first sign of excysting Colpoda cucullus cells is the initiation of the pulsation of a contractile vacuole, which is then replaced by a non-pulsating vacuole (excystment vacuole) that continues to expand and finally ruptures the outermost cyst wall (ectocyst) due to inner pressure. A ciliate surrounded by flexible membranes (endocyst) thus emerges. The osmolarity of the excysting cells is estimated to be 140 mOsm L−1 from the relationship between the frequency of contractile vacuole pulsation and the external sucrose concentration. Both the expansion of the excystment vacuole and the emergence of ciliates occurred even when the cysts were immersed in hypertonic medium. In hypotonic medium containing sodium azide (NaN3, a cytochrome c oxidase inhibitor), the contractile vacuole of vegetative cells stopped pulsating and gradually expanded, causing cells to burst. When C. cucullus was induced to encyst in a hypotonic medium containing NaN3, the expansion of the excystment vacuoles was inhibited. These results suggest that the active uptake of water may be responsible for the expansion of the excystment vacuole required for the ectocyst to rupture.

EF‐1α and Mitochondrial ATP Synthase β Chain: Alteration of their Expression in Encystment‐Induced Colpoda cucullus

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the total proteins contained in encystment-induced Colpoda cucullus showed that a 50-kDa protein (p50) disappeared, whereas the expression of a 49-kDa protein (p49) was enhanced in early phase of morphogenetic transformation into the resting cyst (i.e. 2-5 h after the onset of encystment induction). Puromycin or actinomycin D inhibited the alteration in the expression of p50 and p49 by the induction of encystment. These results suggest that the encystment-specific alteration in expression of these proteins is performed by a transcriptional regulation. Liquid chromatography tandem mass spectrometry analysis revealed that p50 is mitochondrial ATP synthase β chains, and that p49 is elongation factor 1α.

Culture Age, Intracellular Ca2+ Concentration, and Protein Phosphorylation in Encystment-Induced Colpoda cucullus

In Colpoda cucullus, intracellular Ca(2+) mediates the encystment induction and protein phosphorylation that occur just prior to morphogenetic transformation into the resting form. When rapidly growing cells were stimulated to encyst, encystment was not readily induced, and the protein phosphorylation level was lower. On the other hand, in post-growing cells stimulated to encyst, the encystment rate and protein phosphorylation level were elevated. These results suggest that protein phosphorylation is closely linked to encystment induction. Why, then, are the protein phosphorylation level and encystment rate difficult to elevate in the rapidly growing cells? Fura 2 ratiometry showed that the intracellular Ca(2+) concentration (F340/F380 ratio) was raised in rapidly growing cells as well as in post-growing cells when the cells were stimulated to encyst. It is presumed that the Ca(2+)-mediated signal transduction pathways for protein phosphorylation and encystment may be triggered in rapidly growing cells, but downstream certain steps may be suppressed by certain intracellular components.

Protein phosphorylation in encystment-induced Colpoda cucullus: localization and identification of phosphoproteins

In Colpoda cucullus, the morphogenetic transformation was preceded by an enhancement of the in vivo protein phosphorylation level. Immunofluorescence microscopy using antiphosphoserine antibody showed that these phosphorylated proteins were localized in the macronucleus and other cytoplasmic regions. Biotinylated Phos-tag/ECL assays of isolated macronuclei showed that a 33-kDa protein (p33) was localized within them. The p33 obtained from isolated macronuclei was tentatively identified as ribosomal P0 protein by LC-MS/MS analysis. In addition, among the encystment-specific phosphoproteins obtained by phosphate-affinity chromatography, the 29-, 31-, and 33-kDa proteins (p29, p31, and p33) were tentatively identified as ribosomal P0 protein, whereas the 24-kDa phosphoprotein (p24) was tentatively identified as ribosomal S5 protein.

Resting cyst formation of unicellular organism Colpoda and phosphorylation analyses by Phos-tag

In the unicellular eukaryote Colpoda cucullus, resting cyst formation (encystment), which is a cellular morphogenetic process, is mediated by intracellular signaling pathways, which are triggered by an inflow of Ca2+ due to cell-to-cell mechanical contact. The enhanced chemiluminescence detection (ECL) for phosphorylated proteins using biotinylated Phos-tag showed that the phosphorylation level in several proteins was enhanced in Ca2+-dependent manner prior to the beginning of the cyst formation (within 1 h after the onset of encystment induction). The cAMP enzyme immunoassay (EIA) showed that the intracellular cAMP concentration was elevated in encystment-induced cells. The encystment induction and the phosphorylation of some proteins are slightly promoted by the addition of membrane-permeable derivatives of cAMP or non-selective phosphodiesterase inhibitor, while they tend to be suppressed by the addition of an inhibitor of cAMP-dependent kinase (PKA). These results suggest that a Ca2+-activated signaling pathway involving cAMP/PKA-dependent protein phosphorylation may be responsible for the encystment induction of C. cucullus. Encystment-specific phosphorylated proteins were isolated by phosphate-affinity chromatography using Phos-tag agarose beads, and some of them were tentatively identified by mass spectrometry analysis.

Allelopathy of diterpenoids on three species of soil ciliates

Allelopathy of diterpenoids extracted from plants of the genus Robdosia on three common species of soil ciliates, Colpoda inflata, Colpoda cucullus and Euplotes muscicola, was studied by acute toxicity test, sub-lethal effect test and morphological observation. Acute toxicity test showed that there was remarkable toxicity of the diterpenoids on the individuals of the three soil ciliate species, and there was close correlation between toxicity and concentration of the diterpenoids. 12 h-LC 50 values of the diterpenoids on the individuals of C. inflata, C. cucullus and E. muscicola were 161.40 mg L −1, 94.80 mg L −1 and 83.70 mg L −1 respectively, and 24 h-LC 50 values were 114.90 mg L −1, 92.30 mg L −1and 65.80 mg L −1 separately. Sub-lethal effect test of soil ciliates suggested that there existed significant inhibition of the diterpenoids on population growth of the three ciliates with dose-dependant relationships, population density and growth rate of the test group was obviously lower than that of the control group. Morphological observation indicated that diterpenoids affected the body shapes of the three ciliates and made them shorter and thicker, and the higher the concentration of diterpenoids, the greater the affection. The results are of great significance for understanding the functions of ciliates and their relationships to other organisms, and for the application of allelopathy in biological pest control in the soil ecosystems.

Ca 2+-dependent in vivo protein phosphorylation and encystment induction in the ciliated protozoan Colpoda cucullus

Encystment induction of Colpoda cucullus is promoted by an increase in external Ca 2+ and overpopulation of Colpoda vegetative cells. Using phos-tag detection assays, the present study revealed that the in vivo phosphorylation level in several proteins [33 kDa, 37 kDa, 37.5 kDa, 43 kDa, 47 kDa, 49 kDa, etc.] was raised when the vegetative cells were stimulated by overpopulation to encyst in a medium containing 0.1 mM Ca 2+ or without the addition of Ca 2+. Both overpopulation-mediated encystment induction and protein phosphorylation were suppressed by the addition of EGTA. Ca 2+/overpopulation-stimulated encystment induction and protein phosphorylation were also suppressed by the addition of BAPTA-AM. These results suggest that the Ca 2+ inflow promoted by cell-to-cell stimulation due to overpopulation may activate signaling pathways involving protein phosphorylation and encystment induction. In the presence of cAMP-AM, the phosphorylation levels of 33 kDa, 37 kDa, 37.5 kDa, 43 kDa, 47 kDa and 49 kDa proteins were enhanced, and encystment induction was promoted. Enzyme immunoassays (EIAs) showed that intracellular cAMP concentration was raised prior to encystment when the cells were stimulated by overpopulation. These results suggest that cAMP/PKA-dependent protein phosphorylation, which is an event on Ca 2+-triggered signaling pathways, may be involved in encystment induction.

Chromatin extrusion in resting encystment of Colpoda cucullus: A possible involvement of apoptosis‐like nuclear death

The extrusion of macronuclear chromatin is a remarkable characteristic during encystment in Colpoda, but the biological significance of this phenomenon has not been fully elucidated. Here we demonstrate that chromatin extrusion occurs with high frequency when encystment was induced by increasing Ca(2+) in growing cells in various stages of the cell cycle. The Feulgen-DNA reaction revealed that vegetatively growing cells have more macronuclear DNA than cells in the stationary phase, suggesting an association of macronuclear DNA content with the execution of chromatin extrusion. Using 4',6-diamidino-2-phenylindole (DAPI), we found that the size of the macronuclear extrusion body was reduced with time and eventually disappeared approximately 24h after encystment induction. In addition, oligonucleosome-sized DNA cleavage was confirmed to occur concomitant with the size reduction, suggesting that the extrusion body is selectively degraded, while the normal macronucleus remains alive. Combined use of acridine orange and Hoechst 33342 demonstrated that the extruded body was increasingly acidified before final resorption. These features are reminiscent of the nuclear degradation process in conjugating Tetrahymena, and therefore we conclude that chromatin extrusion in Colpoda might occur to adjust the macronuclear DNA content prior to encystment. In this way, it is similar to the apoptotic-like nuclear death that occurs during the conjugation of other ciliates.

The effects of resource enrichment, dispersal, and predation on local and metacommunity structure

Community structure is the observable outcome of numerous processes. We conducted a laboratory experiment using a microbial model system to disentangle effects of nutrient enrichment, dispersal, and predation on prey species richness and predator abundance at local and metacommunity scales. Prey species included: Chilomonas sp., Colpidium striatum, Colpoda cucullus, Paramecium tetraurelia, P. caudatum, Philodina sp., Spirostomum sp., Tetrahymena thermophila, and Uronema sp., and Stentor coeruleus was the predator used. We hypothesized that: (1) increased basal resources should maintain greater species richness and higher predator abundance; (2) dispersal should maintain greater species richness; and (3) predation should reduce species richness, especially in the high resource treatments relative to no-predator treatments. Our results support all three hypotheses. Further, we show that dispersal affects richness at the local community scale but not at the metacommunity scale. However, predation seems to have major effects at both the local and metacommunity scale. Overall, our results show that effects of resource enrichment, dispersal, and predation were mostly additive rather than interactive, indicating that it may be sometimes easier to understand their effects than generally thought due to complex interactive effects.

Morphological study on the encystment of the ciliated protozoan Colpoda cucullus

Morphological study on the encystment of the ciliated protozoan <i>Colpoda cucullus</i>

Studies on the resting cyst of ciliated protozoan Colpoda cucullus: resistance to temperature and additional inducing factors for en-or excystment

Studies on the resting cyst of ciliated protozoan <i>Colpoda cucullus</i>: resistance to temperature and additional inducing factors for en-or excystment

Ciliates (Protozoa: Ciliophora) of Selected Ephemeral Flushson Tropical Inselbergs

Certain plant species have become specialized for life in nutrient-poor habitats by supplementing their diet with soil-dwelling animals. One such habitat is found on tropical inselbergs, namely the ephemeral flushs. Among the carnivorous plants living here is Genlisea, which is known to prey on ciliates (BARTHLOTT et al., 1998). We have now examined the edaphon of ephemeral flushs of ten Brazilian inselbergs and five inselbergs of the Ivory Coast and found more than 50 ciliate species at 23 sampling sites. The smallest number of species found at a single site was 3 and the largest 23 species. The most frequent species were Cyclidium muscicola, Colpoda inflata, Drepanomonas revoluta, Gonostomum affine and Colpoda cucullus. To quantify the food supply of the carnivores, the potential number of individuals and the wet masses of some ciliate communities were determined by the culture technique of BUITKAMP (1979). The quantity range was between 35 and 250 ciliates per g dry weight of soil and the wet masses of the ciliate community extended from 0.2 to 4.3 mg/kg dry weight of soil.

DNA methylation in ciliates: implications in differentiation processes.

Much experimental evidence on the role of DNA methylation in gene expression has been reported. Here we review reports on DNA methylation in ciliated protozoa, emphasizing its implications in cell differentiation processes. Both types of methylated bases (adenine and cytosine) can be found in macronuclear DNA. The division cycle and conjugation have been studied with regard to adenine methylation, and several different functions have been assigned to the methylation changes detected in these processes. Cytosine methylation changes were analyzed during stomatogenesis of Paramecium and encystment of Colpoda inflata. A comparative analysis with other similar microbial eukaryotic differentiation processes is carried out.

Growth and survival of tench larvae fed under different feeding strategies

Two feeding experiments were conducted to evaluate the growth and survival rates of tench Tinca tinca L. larvae when initially fed with a combination of three different brands of Artemia nauplii under two conditions: (A) in the laboratory and (B) on a commercial farm. At the same time, a protozoan culture of the freshwater ciliate Colpoda cucullus was additionally tested in one of the experiments to possibly enhance the initial hunting behaviour of the larvae. The larvae were fed every 4 h from the onset of exogenous feeding up to 14 days of age. Three types of commercial Artemia products, mainly differing in size and high unsaturated fatty acid (HUFA) content, were used. One group was fed with C. cucullus as a starter. The different combinations of Artemia nauplii were used to evaluate possible effects on larval growth. The final growth at 26.3 °C, expressed in length and weight, did not show significant differences, suggesting the use of the most economically feasible Artemia strain studied. The experiments confirmed that using smaller prey during the first 2 days of feeding increases their survival rate, although the mean final survival rate was high (89%). In the experiment carried out at the commercial fish farm facilities, experimental groups were also fed with Artemia nauplii, using the EG type either enriched or not enriched with HUFA. Again, one of the groups was offered the ciliate C. cucullus as initial feed. Final growth showed significant differences when using Colpoda culture as a starter feed, although this test resulted in the lowest survival rate (69%), indicating that further studies on the management of its culture should be undertaken to improve the applicability of the technique. The mean final survival rate was 83%.

RESTING CYST WALL GLYCOPROTEINS ISOLATED FROM TWO COLPODID CILIATES ARE GLYCINE-RICH PROTEINS

Two glycoproteins bands isolated from the cyst wall protein pattern of two colpodid ciliates,Colpoda inflata (gp46CI) and Colpoda cucullus (gp46CC) were analysed for their amino acid composition. Both glycoproteins are very rich in glycine and have a relatively high hydrophobicity, containing additionally many leucine and alanine residues. Their high degree of similarity is both quantitative and qualitative. Compared with just two previously published reports, their amino acid compositions are similar to those found in the hydrolysed cyst wall total proteins from the ciliatesC. steinii and Paraurostyla spp. The amino acid composition corroborates that they are indeed glycoproteins, because asparagine, an amino acid residue suitable for the attachment to N-acetylglucosamine by its amide group (N-glycan), is abundant in both proteins. We discuss our data in relation to other glycine-rich proteins and a comparison with amino acid composition protein databases is carried out.

Diversity and geographic distribution of riboprints from three cosmopolitan species of Colpoda müller (Ciliophora: Colpodea)

Summary Ribosomal RNA gene restriction site polymorphisms were determined for 114 isolates of three cosmopolitanly distributed species of Colpoda , a common soil ciliate genus. Isolates were obtained from soil samples and identifications confirmed using silver staining techniques. Based on restriction site analysis of a 1095 bp region of the large subunit (LSU) rRNA gene, thirty-one distinct riboprints were observed; 11 unique to Colpoda steinii , 8 unique to Colpoda inflata , 10 unique to Colpoda cucullus , and two riboprints shared by several isolates of C. inflata and C. cucullus . The average intraspecific genetic distance estimated from riboprint data was greatest among C. steinii isolates (0.0790, n = 37), intermediate among C. cucullus isolates (0.0519, n = 37), and the least among C. inflata isolates (0.0383, n = 40). Phylogenetic analysis of riboprint data using distance methods revealed no geographic pattern in the distribution of riboprints in any of the species examined. Furthermore, many of the riboprints were globally distributed, suggesting no equatorial barrier to species dispersal in the species examined. Several riboprints, however, appeared to be restricted on the basis of temperature, suggesting the possibility of fitness differences among riboprints with respect to environmental variables. Shared riboprints between two species of Colpoda indicates the limitations of riboprints in species discrimination within in this group, possibly due to the retention of ancestral polymorphisms or the inadequate resolution of the region of LSU rRNA gene that we examined. Additional molecular markers will be needed in order to resolve this issue.

Die Auswirkungen von Düngung und Kalkung auf die Bodenciliaten (Protozoa: Ciliophora) eines Fichtenbestandes im Schwarzwald (Süddeutschland)

Summary The influence of fertilization and liming on the ciliate communities of very acidified spruce forests was tested. Investigations were conducted in the Black Forest, Southern Germany. Experimental areas were treated with different substances: S1 = control area, S2 = magnesium-sulphate area, S3 = ammonia-sulphate area, S4 = lime area. Fertilization led to no change in H + -concentration, but liming showed a significant rise in pH-value. The qualitative-quantitative analysis of the ciliate communities were carried out in laboratory microcosms. Various culture methods yielded 47 species. The abundance measurements were conducted with a modified “Buitkamp-culture-method”: dry soil samples were mixed with destilled water and the ciliate communities were analysed after 1, 2, 3, 5, 6, 7, 8, 10, 13, 17, 20, 30, 50, 70, and 90 days. Species composition and abundances showed continued and substantial changes during the experiment. First appeared the little colpodid ciliates Pseudoplatyophrya nana, Colpoda steinii , and Colpoda inflata , all of them pioneer-species, which were frequent only for a few days. These species showed characteristics of r-selection and they are well adapted for the extreme habitat “spruce forest”. They were in S1 more frequent than in S2–S4. The larger colpodid ciliates of the Colpoda cucullus / C. henneguyi group were frequent only on the limed area S4; they were substantial longer active than their smaller relatives. Whereas Cyclidium muscicola and Leptopharynx costatus were on all areas active during the whole experimental period, Drepanomonas revoluta appeared very late. It was in S2–S4 more frequent than in S1. The hypotrich ciliate fauna is just quantitatively important in S4. Tetrahymena edaphoni was only active in S4; in cultures from this bacteria rich area T. edaphoni occurred on a massive scale. Liming led to a greater change in the ciliate communities than fertilization with magnesium-sulphate and ammonia-sulphate. The investigation of the ciliate succession in laboratory microcosms offers next to relevant aut-ecological facts, aspects of the condition of a habitat. The method can be used as an instrument for bioindication in terrestrial ecosystems.

Growth of Antarctic and temperate terrestrial Protozoa under fluctuating temperature regimes

Population growth rates of Antarctic and temperate isolates of the microflagellate Heteromita globosa Stein and of the ciliate Colpoda cucullus Müller were determined during incubation both under constant 3.5° and 8.0°C, and under temperature regimes fluctuating on 24-hour cycles, between 0° and 15°C, to simulate Antarctic microclimates. Fluctuating temperatures did not inhibit growth. No growth of Colpoda occurred at 3.5°C. It is suggested that successful growth in nature depends upon the microclimate providing sufficient degree-hours per day above encystment/excystment threshold temperature (+1.5°C for Heteromita about +4°C for Colpoda). The minimum number of degree-hours above threshold needed for growth of Colpoda is estimated to lie between 48 and 96. Monitored microclimates in the maritime Antarctic do not provide enough degree-hours per day, though subantarctic microclimates are more favourable, as may be exceptionally warm localities in the continental Antarctic where daily insolation is high in summer. These data are consistent with the recorded presence of Colpoda spp. in the subantarctic and rarely in the continental Antarctic, and their absence from the maritime Antarctic.