Method for the simultaneous establishment of many axenic cultures of Paramecium

A method is described for the simultaneous treatment of 42 (or more) stocks of Paramecium, and their adaptation to growth in axenic culture. Samples of dense cultures of these ciliates growing with Enterobacter aerogenes are rendered bacteria-free by migration through 2 sets of tubes containing Adaptation Medium (Peters' salts solution, stigmaterol, vitamins, and autoclaved E. aerogenes). The 2nd set of tubes contains Adaptation Medium plus antibiotics. Bacteria-free samples containing approximately 100 animals are then transferred to test tubes containing Adaptation Medium without antibiotics. This medium also serves as a growth medium. It supports indefinite growth of all Paramecium stocks tested. After adaptation to this medium, the ciliattes can be grown in the axenic medium developed by Soldo, Godoy & van Wagtendonk. On a single trial at least half of the stocks can be expected to produce axenic cultures within 5 to 10 days by these procedures. The method has been applied successfully to several of the species of the Paramecium aurelia complex, to all syngens of Paramecium multimicronucleatum, to several stocks of Paramecium jenningsi, and to 1 stock of Paramecium caudatum and Paramecium calkinsi. A modification of the method also works for Didinium nasutum.     

Effect of Acetate on Esterase C Activity During the Growth Cycle of Paramecium*

SYNOPSIS Enhanced esterase C activity could be demonstrated by starch gel electrophoresis in various stocks of Paramecium spp. (P. primaurelia stocks 90 and 540, P. biaurelia stock 93, P. tetraurelia stock 29. P. pentaurelia stock 87, P. octaurelia stocks 31 and 300, and P. multimicronucleatum species 3, stock 8 MO) grown in Adaptation Medium. This esterase, however, was barely detectable when they were cultivated in Axenic Medium. Addition of trypticase to Adaptation Medium resulted in reduction of esterase C in the ciliates. This effect is ascribable to Na acetate present in trypticase. Since esterase C increased with the decrease in acetate concentration (as estimated by gas-liquid chromatography) during growth of Paramecium, acetate appears to be utilized by the cells. Sensitivity of esterase C to acetate occurs in all 6 species of Paramecium examined. Different stocks within a species may have different levels of sensitivity; in one case this is genetically determined. The results emphasize the importance of controlling and manipulating growth conditions for the assessment of inter- and intraspecies variations in the isozymes of Paramecium.     

Experimental Microbial Populations Thirty-five Years Later: The Influence of Food on the Symbiosis of Paramecium aurelia Syngen 4, 51.7

Changes in the nutrition of Paramecium aurelia affect its ability to serve as host for the bacteroid parasite, kappa, and the presence or absence of kappa affects its ability to grow in axenic culture. Loss of kappa, tested by the presence or absence of killer reaction, occurred in cultures of P. aurelia growing at a reduced division rate on autoclaved Enterobacter aerogenes in suspensions of lettuce and yeast autolysate 14–17 days after they had been rendered bacteria-free by washing. Killer Paramecium sterilized of bacteria by treatment with an antibiotic mixture of penicillin-G and streptomycin in combination with a nonbacterial nonliving culture medium, lost the ability to kill after from 6 to 48 hours in the sterilizing medium. The ciliates from which kappa had been lost during exposure to antibiotics could be transferred immediately and maintained in axenic culture, but those washed free of bacteria could not be maintained axenically until kappa had been lost during cultivation in a medium containing killed bacteria. It is suggested that a knowledge of the nutritional requirements of symbiotic microorganisms is essential for understanding the ecological aspects of eutrophication of aquatic environments.     

Growth of Paramecium tetraurelia in Bacterized,Monoxenic Cultures1

Wild type and mutant Paramecium tetraurelia were grown in monoxenic cultures by first growing Enterobacter aerogenes on a defined medium and then adding the Paramecium to the stationary phase bacterial culture. The bacterial growth was proportional to the concentration of the carbon source (citrate), and the Paramecium growth was dependent upon both the bacterial density and the starting density of Paramecium. The behavior, electrophysiological properties, ciliary lipid composition, and growth characteristics were similar to the commonly used bacterized medium (Cerophyl) except that 5–10 times greater Paramecium yields were reliably obtained.     

A New Method For Axenic Mass Cultivation of Paramecium Tetraurelia*

SYNOPSIS A method is described for the axenic mass cultivation of Paramecium tetraurelia strains 51s and 299s. the ciliate is grown in an enriched axenic medium developed by Soldo, Godoy & van Wagtendonk. Under continuous shaking on a rotary shaker, cultures were grown in one-liter Erlenmeyer flasks with 330 ml medium yield cell densities of 32,000 cell/ml and 20,000 cells/ml for strains 299s and 51s respectively. Doubling time is considerably shorter under these conditions than in the conventional static cultures. A 20-liter airlift bioreactor is described in detail which can be used successfully to otain up to 100 g wet weight of Paramecium in a single run; in this reactor the cell density reaches 38,000 cells/ml for strain 299s. and 23,000 cells/ml for 51s. This technic should facilitate the study of minor protein components of the ciliate.     

Morphological and molecular investigations of Paramecium schewiakoffi sp. nov. (Ciliophora, Oligohymenophorea) and current status of distribution and taxonomy of Paramecium spp.

Paramecium schewiakoffi sp. nov. is described from a pond in Shanghai, China. It is a freshwater species belonging to the “aurelia” subgroup of the genus. It is of similar size and shape to P. jenningsi, but has a single large micronucleus of the “chromosomal” morphological type, while P. jenningsi has two smaller micronuclei. The general morphology, morphometric characteristics and nuclear reorganization pattern, a random amplified polymorphic DNA (RAPD) fingerprint pattern, and the small subunit rRNA gene sequence are presented for the species. Comparison of P. schewiakoffi with the other species of Paramecium indicates that it is a valid new species of the genus. Geographical locations reported for many Paramecium species do not support the theory that all ciliates have a cosmopolitan distribution. It is proposed that, in an extension of Jankowski's earlier suggestion, the genus Paramecium should be subdivided into four subgenera: Chloroparamecium, Helianter, Cypriostomum and Paramecium, on the basis of morphometric, biological and molecular differences.     

Establishment of axenic endosymbiotic strains of Japanese Paramecium bursaria and the utilization of carbohydrate and nitrogen compounds by the isolated algae

Cells of the green paramecium, Paramecium bursaria, contain several hundred endosymbiont cells. The properties of the symbionts are considered to vary depending on the collection site of the host. Difficulties in achieving axenic cells and maintenance of axenic strains for long periods have been reported for symbiotic algae from P. bursaria isolated in Japan. To establish axenic algal strains from such Japanese P. bursaria, symbionts were isolated carefully, and isolated axenic strains were grown on an agar medium containing organic nitrogen compounds. Symbiotic algal strains were obtained from three Japanese P. bursaria strains and their axenicity was confirmed by DAPI staining, cultural tests of bacterial contamination, and DGGE-PCR. These axenic strains have been maintained for over 2 years. Utilization of carbohydrates and nitrogen compounds by symbionts was examined. Monosaccharides (glucose and fructose) increased the growth of the symbiont but disaccharides (maltose and sucrose) did not. Japanese axenic symbionts were able to use ammonia and amino acids, but not nitrate or nitrite. While potent nitrite reductase activity was stimulated by nitrate induction, nitrate reductase activity was not. Nitrate utilization of Japanese symbionts differed from that reported for European and American symbionts.     

Die stoffwechselphysiologischen Benziehungen Zwischen Paramecium bursaria Ehrbg. und Chlorella spec. in der Paramecium bursaria-Symbiose

Zusammenfassung Der endosymbiontische Verband von Paramecium bursaria Ehrbg. mit Chlorella spec. (grünes Paramecium) wurde physiologisch und cytologisch untersucht. Ein Vergleich der Eigenschaften der Symbiosecinheit mit denen der getrennt kultivierten Symbiosepartner ergab die folgenden Merkmale und Unterschiede: 1. Der symbiontische Verband hat bis zu einer Beleuchtungsstärke von 6000 lux eine stärkere Photosyntheseleistung als die aus ihm isolierte und in Massenkultur in einem definierten Medium kultivierte Alge. Algenfreie P. bursaria zeigen nur eine minimale Fähigkeit zur CO₂-Fixierung. 2. Der Kompensationspunkt der Photosynthese liegt beim algenhaltigen Paramecium bei ca. 4000–5000 lux, derjenige der getrennt kultivierten Alge bei ca. 200–400 lux. 3. Die Symbioseeinheit hat im Dunkeln im Vergleich mit algenfreien P. bursaria einen niedrigeren, im Vergleich mit der frei kultivierten Alge jedoch einen höheren Sauerstoffbedarf. 4. Das grüne Paramecium nimmt weniger Kohlenhydrate aus dem Medium auf als algenfreie Paramecien, hat aber eine höhere Aufnahmeleistung als die isoliert gezogenen Algen. 5. Im Symbioseverband besitzt die symbiontische Alge im Licht eine kompakte Lagerung der photosynthetischen Membranen und eine massive Stärkeablagerung. Die Vergiftung der Photosynthese durch 3-(3,4-Dichlorphenyl)-1,1-dimethylharnstoff (DCMU) oder die Kultur im Dunkeln führt in algenhaltigen Paramecien zu einer aufgelockerten Lagerung der Thylakoide und einer Verringerung der Stärkeablagerung. Die Algen-population unterliegt im symbiontischen Verband einem komplexen Regulationsmechanismus, bei dem u. a. der intracelluläre Kohlenhydratspiegel eine Rolle spielt. Die geschilderten Ergebnisse werden im Zusammenhang mit der Ökologie des grünen P. bursaria diskutiert.

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The metabolic interactions between Paramecium bursaria Ehrbg. and Chlorella spec. in the Paramecium bursaria-symbiosisII. Symbiosis-specific properties of the physiology and the cytology of the symbiotic unit and their regulation

The endosymbiotic association of Paramecium bursaria Ehrbg. with Chlorella spec. (green Paramechim) was studied both physiologically and cytologically. Comparison of the properties of the symbiotic unit with those of the symbiotic partiners which bad been isolated from it revealed the following features and differences: 1. Up to 6000 lux the photosynthetic capacity of the symbiotic unit is higher than that of the isolated symbiotic algae grown independently in mass culture under defined conditions. Alga-free. Paramecium bursaria (colourless Paramecium) show a very low rate of CO₂-fixation. 2. The green Paramecium has a higher compensationpoint of photosynthesis (4000–5000 lux) than the isolated alga (200–400 lux). 3. Green paramecia consume less oxygen in darkness than colourless organisms but more than the isolated algae. 4. The uptake of carbohydrates from the culture medium by green paramecia is lower than the uptake by alga-free P. bursaria but higher than the one of the isolated algae. 5. Symbiotic algae within the intact symbiotic unit show tightly packed photosynthetic membranes and an intense deposition of starch. In the presence of 3-(3,4-Dichlorophenyl)-1,1-dimethylurea (DCMU) or in darkness the arrangement of thylakoids is less compact and the deposition of starch is reduced. The growth and the number of the symbiotic algae in situ is regulated by a complex mechanism to which the intracellular level of carbohydrates belongs. The results are discussed in connection with ecological aspects of the Paramecium bursaria-endosymbiosis.

     

Economic Mass Cultivation of Paramecium tetraurelia on a 200-Liter Scale1

ABSTRACT. A new and inexpensive medium is described for axenic mass cultivation of Paramecium tetraurelia stock 51s and the double mutant pawn A/pawn B. Skim milk powder is the major carbon and nitrogen source in this medium. Growth characteristics (proliferation rate, final cell density, and cell size) are similar to those observed with other axenic culture media. Cultures were run in one-liter erlenmeyer flasks, a 20-liter, and a 250-liter airlift bioreactor. The yield of a large bioreactor is 750 g (wet wt.) Paramecium or 5 × 10⁹ cells. This easy, economical culture technique will greatly facilitate the use of Paramecium as a model organism for extensive biochemical studies.     

Photobehaviour of Paramecium bursaria infected with different symbiotic and aposymbiotic species of Chlorella

The endosymbiotic unit of Paramecium bursaria and Chlorella spec. shows two types of photobehaviour: 1) A step-up photophobic response which possibly depends on photosensitive agents in the ciliate cell itself — as is also shown by alga-free Paramecium bursaria - and can be drastically enhanced by photosynthetic activity of symbiotic algae; and 2) a step-down photophobic response. The step-down response leads to photoaccumulation of green paramecia. Both types of photobehaviour in Paramecium bursaria do not depend on any special kind of algal partners: The infection of alga-free Paramecium bursaria with different Chlorella species results in new ciliatealgae-associations. They are formed not only by combination of the original symbiotic algae with their host, but also by infection with other symbiotic or free-living (aposymbiotic) chlorellae, respecitively. Systems with other than the original algae are not permanently stable — algae are lost under stress conditions — but show the same types of photobehaviour. Photoaccumulation in general requires algal photosynthesis and occurs only with ciliates containing more than fifty algae/cell. It is not mediated by a chemotactic response to oxygen in the medium, since it occurs at light fluence rates not sufficient for a release of oxygen by the symbiotic system, e.g., below its photosynthetic compensation point. Photoresponses can be inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). Sensory transduction does not depend on any special symbiotic features of the algae, e.g., sugar excretion. The participation of oxygen in the Paramecium cell, of its cytoplasmic pH and of ions released or taken up by endosymbiotic algae in sensory transduction is discussed.     

Particle-Based Axenic Media for Tetrahymenids*

SYNOPSIS. Autoclavable, natural particulate media simplify axenic cultivation of tetrahymenid ciliates and presumably favor selection for phagotrophy. Viability is at least 2 months at room temperature (24–26 C) for the lipid-sensitive tetrahymenids Tetrahymena setosa, T. corlissi, T. paravorax, T. limacis, and T. patula, also for T. rostrata and (at 12 C), for strains of the T. pyriformis complex and Glaucoma chattoni. A typical medium consists of crude soy “lecithin”+ skim milk powder +Saccharomyces cerevisiae cells. Other useful particules readily available commercially are: whole liver powder, cells of Micrococcus lysodeikticus and Escherichia coli, and powdered residue of liver which had been extracted with 70% ethanol (liver #2). Preliminary experiments indicate that some of these media are suitable for the maintenance of Paramecium octaurelia stock 299S and Colpidium campylum. Such mixtures may serve as points of departure for devising media for more fastidious phagotrophs.     

Transfer of Photosynthetically Produced Carbohydrate from Endosymbiotic Chlorellae to Paramecium bursaria*

Paramecium bursaria (Ehrenberg) and an endozoic zoochlorella Chlorella conductrix (Brandt) live in a symbiotic relationship. Uptake of NaH¹⁴CO₃ was studied to determine if carbohydrate products of photosynthesis are transferred to the host paramecium. Paramecium bursaria containing the algal symbionts took up NaH¹⁴CO₃ but those without the algal symbionts did not. Radioactive maltose, glucose, fructose and malate were identified from the ethanolic extract of paramecia. Transfer of materials from Paramecium to Chlorella and the transfer of other materials from Chlorella to Paramecium, led to the conclusion that this is a mutualistic relationship, both organisms benefiting from the relationship.     

Biochemical Taxonomy of Symbiotic Chlorella Strains from Paramecium and Acanthocystis*

Biochemical and physiological characters of 5 symbiotic Chlorella strains, 4 of them from Paramecium (Ciliata) and one from Acanthocystis (Rhizopoda, Heliozoa), were studied. Four strains (3 from Paramecium and one from Acanthocystis) belong to the same species. This is characterized by the presence of hydrogenase, no formation of secondary carotenoids, no growth on mannitol, requirements for thiamine and vitamin B₁₂, and a G + C content of the DNA of 66.4–68.4 mol%; the limits of growth are at pH 5.5, at up to 1% NaCl, and at 26–30°C. The strains are somewhat heterogeneous in their utilization of inorganic nitrogen sources: only two of them are able to use nitrate, whereas all can grow with nitrite and ammonium. Thus, in two strains the nitrate-reducing system — in contrast to nitrite reductase — seems to be defective. Another strain, which has been claimed to be from Paramecium, belongs to C. protothecoides.     

Axenic Paramecium caudatum. I. Mass Culture and Structure*

SYNOPSIS. To establish and grow Paramecium caudatum in mass axenic culture the culture medium of Soldo, Godoy & van Wagtendonk was modified by substituting phosphatidylethanolamine (PE) for TEM-4T and by a 10-fold increase in folic acid. Population densities of 4000 to 6000 cells/ml and a generation time of 20–26 h are regularly obtained. Optimal growth is obtained with PE-stigmasterol ratios between 40:1 to 400:1. Cells from 1-day-old axenic cultures have many lipid bodies aggregated in clumps (which disappear in 2 to 3 days) as well as foci of rough endoplasmic reticulum bordered by dictyosomes. The latter suggests a very active metabolism. Crystalline sheets found in both food vacuoles and lysosomes presumably play a role in digestion. Axenically grown cells also have abundant Golgi bodies (dictyosomes) and by late log phase become filled with lysosomes.     

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.     

Studies on the Macronuclei of Doublet Paramecium tetraurelia: Distribution of Macronuclei and DNA at Fission*

SYNOPSIS. Doublet Paramecium tetraurelia would be expected to contain 2 macronuclei if their nuclear complement were strictly analogous to that of singlets. However, most doublets are unimacronucleate. It is shown in this study that dimacronucleate cells are present only in young clones. Unimacronucleate cells arise either through abnormalities in the determination and distribution of macronuclear anlagen during the first cell cycle after conjugation, or from dimacronucleate cells through abnormal division and segregation of macronuclei during the fission process. When a change in the number of macronuclei occurs through abnormalities in the division and segregation of daughter macronuclei, the daughter cells produced typically have DNA contents more similar than those expected from either random segregation of daughter macronuclei, or from the normal segregation pattern in ciliates in which changes in the number of macronuclei in progeny cells do not occur. This suggests that part of the regulation process of macronuclear DNA content in Paramecium may occur through control of the segregation pattern of daughter macronuclei.     

The Purification of DNA from the Genomes of Paramecium aurelia and Tetrahymena pyriformis1

SYNOPSIS. Methods are described for the recovery of highly purified DNA from Paramecium aurelia and Tetrahymena pyriformis in high yields. Our DNA is only slightly contaminated with RNA and carbohydrate, and little or no protein can be detected. We could not reduce the RNA (orcinol-positive material) by further treatment (sephadex or hydroxyapatite chromatography and preparative CsCl gradients). At the extreme our DNA is contaminated with 15–20% RNA but the real value is most likely considerably lower than this. The DNA we have prepared from Paramecium and Tetrahymena shows all the properties of double-stranded, high molecular weight DNA when characterized by temperature melting, CsCl density gradient centrifugation and hydroxyapatite and sephadex chromatography. When denatured, it absorbs to nitrocellulose filters. The 2 major results of importance from our work reported here are: (1) There is similarity in base composition of DNA from different syngens of Paramecium (28% G+C for syngens 1, 2, 4, 5, and 9 and 29–30% G+C for syngen 8) while there is variation between the syngens of Tetrahymena (24–31% G+C for syngens 1, 4, 7, 10, 11, and 12); (2) the density of any Paramecium DNA varies depending upon whether the cells are grown in the presence of bacteria or in axenic medium. Our results are compatible with observations previously reported for Tetrahymena but contradict those made for Paramecium. The earlier reports of differences in base composition between syngens of Paramecium are probably due in large part to the use of stocks grown on bacteria.     

Ciliates as engineers of phototrophic biofilms

1. Phototrophic biofilms consist of a matrix of phototrophs, non‐photosynthetic bacteria and extracellular polymeric substances (EPS) which is spatially structured. Despite widespread exploitation of algae and bacteria within phototrophic biofilms, for example by protozoans, the ‘engineering’ effects of these ciliates on the spatial heterogeneity of phototrophic biofilms are poorly studied. 2. We studied the potential engineering effects of two ciliates, Urostyla sp. and Paramecium bursaria, on the spatial heterogeneity of synthetic multispecies biofilms. Biomass of phototrophic organisms, EPS and bacteria was analysed three dimensionally using confocal laser scanning microscopy. Spatial heterogeneity and cover of the phototrophs, bacteria and EPS were determined at several depths within the biofilm. 3. Ciliate species did not interfere with the overall development of phototrophic microorganisms, because the thickness of the biofilm was equal whether the ciliates were present or not, even though their abundance did affect spatial heterogeneity of biofilm components. When Urostyla was present, it reduced aggregation in EPS and bacteria and increased EPS biovolume. This implies a local facilitating effect of ciliates on photosynthetic activity. Biofilms to which Paramecium was added did not differ from controls in terms of phototrophs, EPS cover and biovolume. Nevertheless, ciliates affected the spatial heterogeneity of these components as phototrophs and EPS became more evenly distributed. 4. This study shows that ecosystem engineering by organisms does not only occur at large spatial scales, as in grasslands and estuaries, but also plays a role at the microscopic scale of biofilms. This effect on spatial heterogeneity was not driven by substantial exploitation of biofilm components, but via the subtle engineering effects of ciliates.     

Monoclonal Antibody to a Bacterial Endonuclear Symbiont Holospora Cross Reacts with Proteins of Contractile Vacuole Radial Canals of Paramecium Species

ABSTRACT A monoclonal antibody (mAb) IR-2-1 was raised against a 67-kDa protein purified from the macronucleus-specific bacterial symbiont Holospora obtusa of Paramecium caudatum. The mAb was found to react with two bands (31 and 67-kDa) on gels of H. obtusa. Indirect immunofluorescence microscopy showed that these antigens were distributed inside the cells. However, unexpectedly, this mAb also cross reacted with the radial arms of the contractile vacuole in P. caudatum, P. tetraurelia, P. multimicronucleatum, P. jenningsi and P. bursaria as well as with their cytoplasm. Immunoelectron microscopy showed that the antigens were located on the decorated spongiome of the radial arms. In immunoblots, mAb IR-2-1 reacted with a band of 67 kDa in all Paramecium species examined. However, no band appeared in the immunoblot of isolated macronuclei of H. obtusa-free P. caudatum and no label was seen in the nuclear matrix of the macronucleus of air-dried P. caudatum. These results suggest that the 67-kDa antigen found in H. obtusa was not imported from the host macronucleus and the same antigen in the host contractile vacuoles and cytoplasm were not derived from the symbiont. These results also showed that an epitope on the decorated spongiome of the Paramecium species is shared by its bacterial symbiont. In contrast to the decorated tubule-specific mAb, DS-1, the antigens for IR-2-1 appeared to be loosely membrane bound as they were lost in paraformaldehyde fixed and acetone permeabilized Paramecium. Supplementary key words. Contractile vacuole complexes, Holospora obtusa, monoclonal antibody, Paramecium.     

L'appareil de Golgi des Ciliés. Ultrastructure, particulièrement chez Paramecium

Electronmicroscopic study of Coleps, Colpidium, Stylonychia, and especially of Paramecium confirmed the presence of the Golgi complex in these fresh-water ciliates. The complex consisted of numerous dictyosomes scattered throughout the cytoplasm. Each dictyosome included a few flat, partly reticulated saccules lying parallel to a cistern of rough endoplasmic reticulum which was free of ribosomes on the side exposed to the dictyosome. A unique layer of vesicles, characterized by constant size and a thick wall, separated the endoplasmic reticulum from the dictyosomes. The vesicles could be regarded as transition vesicles. Coated vesicles were seen in continuity with some of the flattened saccules. The possible role of the Golgi complex in the physiology of ciliates is discussed.