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.     

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.

     

Die stoffwechselphysiologischen Beziehungen zwischen Paramecium bursaria Ehrbg. und Chlorella spec. in der Paramecium bursaria-Symbiose

Zusammenfassung Infektionsexperimente algenfreier Paramecium bursaria mit aus diesen isolierten und unter Stickstoffmangel-Bedingungen vorkultivierten Algen deuten darauf hin, daß die Versorgung der endosymbiontischen Algen mit stickstoffhaltigen Verbindungen durch ihren Wirt in einem zu gutem Wachstum und Vermehrung der Alge ausreichendem Maße möglich ist. Die Bedeutung dieser stoffwechselphysiologischen Beziehung für die Symbiosepartner wird diskutiert.Die Vergiftung der Photosynthese der endosymbiontischen Chlorella durch 3-(3,4-Dichlorphenyl)-1,1-dimethylharnstoff (DCMU) führt in grünen Paramecium bursaria durch Beeinflussung des Kohlenstoff-Stoffwechsels zu einer Entkoppelung des symbiontischen steady state-Systems und damit zur Auflösung der Symbiose. Eine ausreichende heterotrophe Ernährung der Alge durch das Paramecium ist in der Symbiose offenbar nicht möglich.Die Anwendung von 3-(3,4-Dichlorphenyl)-1,1-dimethylharnstoff (DCMU) kann als neue Methode zur Züchtung algenfreier Paramecium bursaria dienen.

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The metabolic interactions between Paramecium bursaria Ehrbg. and Chlorella spec. in the Paramecium bursaria-symbiosisI. The nitrogen and the carbon metabolism

Symbiotic Chlorellae have been isolated from Paramecium bursaria Ehrbg. and cultivated under conditions of nitrogen deficiency. Reinfection of Chlorella-free Paramecium bursaria with these nitrogen-deficient algae resulted in a complete regeneration and multiplication of the algae within the host cells. The endosymbiotic algal cells of the Paramecium bursaria-symbiosis can be supplied by their host with nitrogen.The inhibition of photosynthesis by 3-(3,4-Dichlorophenyl)-1,1-dimethylurea (DCMU) leads in green Paramecium bursaria to a breakdown of the symbiotic steady state-system resulting in a loss of algal cells. Obviously the endosymbiotic algae cannot be fed heterotrophically by their host to such an extent that a stable symbiosis is maintained.The application of 3-(3,4-Dichlorophenyl)-1,1-dimethylurea (DCMU) can be used as a new method for culturing Chlorella-free Paramecium bursaria.

     

Identification of Chlorella viruses in Paramecium bursaria clones by pulse-field electrophoresis

The ciliates Paramecium bursaria contain endosymbiotic green algae Chlorella spp. in their cytoplasm. The algae isolated from P. bursaria are sensitive to large DNA-containing viruses of the family Phycodnaviridae. The type virus of this family is PBCV-1 (Paramecium bursaria Chlorella virus). Investigation of the total DNA of P. bursaria clones by pulse-field electrophoresis (PEGE) revealed a pronounced band on PEGE profiles of some P. bursaria clones; the band was formed by DNA molecules of approx. 300 kb. This band probably contained the DNA of Chlorella virus. Two approaches were used in the present work to confirm this hypothesis. Microbiological tests were used to scan a collection of P. bursaria clones for specific types of viruses; the 300-kb band was revealed only in the PEGE profiles of virus-containing clones. Blot hybridization of P. bursaria total DNA separated by pulse-field electrophoresis with the virus-specific probe revealed that the band under study was formed by the DNA of a Chlorella virus. Paramecium clones were shown to contain approx. 10⁵ copies of nonintegrated viral DNA.     

Cloning and characterization of endosymbiotic algae isolated fromParamecium bursaria

Summary The green parameciumParamecium bursaria has many endosymbiotic algae in its cytoplasm. Here, we cloned and characterized endosymbiotic algae fromP. bursaria and examined in detail the interaction between the cloned algae and algae-free paramecia. Homogenates ofP. bursaria were cultured on agar plates containing various kinds of media to establish clones of the endosymbiotic algae. Many algal colonies were obtained from poorly nutritious medium (CA medium) after one month in culture. Algae were picked up from these colonies and inoculations were repeated 9 times on agar plates containing CA medium. On enriched media including bacto-peptone, glucose, proteose-peptone and/or yeast extract, however, bacteria and mold grew rapidly and no algal colonies were formed. When the cloned algae were cultured in liquid CA medium, they grew faster than on agar plates and the numbers stayed constant at 1 × 10⁷ algae/ml after 7 days in culture. They revealed high infectivity to algae-free paramecia, and an incubation period of 24 h and at least 1 × 10³ algae/paramecium were required to achieve successful infection (80–90%). The growth and infection rate did not change through 74 repeated inoculations of algae in liquid CA medium. Optical microscopic observations revealed marked morphological similarity between endosymbiotic algae and free-livingChlorella, but the latter showed no infectivity to algae-free paramecia. The cloned endosymbiotic algae presented here will provide an excellent opportunity to examine the mechanism of symbiont-host interaction.     

Evidence of de novo synthesis of maltose excreted by the endosymbiotic Chlorella from Paramecium bursaria

The endosymbiotic Chlorella sp. from Paramecium bursaria excretes maltose both in the light and in the dark. Experiments on photosynthetic ¹⁴CO₂ fixation and ¹⁴CO₂ pulse-chase experiments show that maltose is synthesized in the light directly from compounds of the Calvin cycle, whereas in the dark it results from starch degradation.     

Zur Taxonomie einer auxotrophen Chlorella aus Paramecium bursaria Ehrbg

Zusammenfassung Auf Grund der physiologischen Merkmale einer in Paramecium bursaria Ehrbg. auftretenden Chlorella ergibt sich eine systematische Zuordnung in den Formenkreis um Chlorella vulgaris f. tertia Fott et Nováková und Chlorella vulgaris var. vulgaris Beijerinck. Hiervon abweichende Befunde anderer Autoren werden diskutiert.

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On the taxonomy of an auxotrophic Chlorella isolated from Paramecium bursaria ehrbg

An auxotrophic Chlorella has been isolated from Paramecium bursaria Ehrbg. and cultivated in mass culture in an inorganic medium supplied with vitamins B₁ and B₁₂. With regard to its physiological properties it is not identical with either one of the so far known Chlorella species. It belongs, however, to the group of Chlorella vulgaris f. tertia Fott et Novaková and Chlorella vulgaris var. vulgaris Beijerinck.

     

Comparative studies on photosynthetic enzymes of the symbiotic Chlorella from Paramecium bursaria and other symbiotic and non-symbiotic Chlorella strains

The activities of ribulose 1,5-bisphosphate carboxylase and of carbonic anhydrase were studied in cell-free extracts of two symbiotic Chlorella strains isolated from Paramecium bursaria and from Spongilla sp., and of two nonsymbiotic strains of Chlorella (Chlorella fusca and Chlorella vulgaris) cultivated at varied CO₂-concentrations. The symbiotic Chlorella of Paramecium bursaria differs distinctly from the other Chlorella strains by a higher activity of ribulose 1,5-bisphosphate carboxylase, which is independent of the actual CO₂-concentration, and by a lack of carbonic anhydrase activity. These properties are discussed with respect to their ecological significance.Abbreviations CA carbonic anhydrase - Pbi Paramecium bursaria isolate - RuBP ribulose 1,5-bisphosphate Dedicated to Prof. Dr. André Pirson on the occasion of his 70th birthday     

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.     

Correlation between circadian periods and cellular activities in Paramecium bursaria

Paramecium bursaria shows a circadian rhythm of photoaccumulation: photoaccumulation is stronger during the day than at night. We obtained five strains of P. bursaria having different circadian periods under continuous light conditions, ranging from 20.9 to 27.9 h. Various physiological activities were compared in the cells of these strains. The periods of contractile vacuole contraction were in the range 10–15 s, which was almost proportional to the periods of the circadian rhythm in each strain. Swimming velocities were inversely proportional to the circadian period; i.e. swimming velocities were high in strains whose circadian periods were short. Resting membrane potential was more depolarized in strains with longer circadian periods. Finally, the membrane resistance of the resting state was reduced in proportion to the increase of the circadian period. Such correlation between the cellular properties and the circadian period suggests that the circadian clock mechanism is associated with various physiological activities of the cell.     

Photoadaptation Alters the Ingestion Rate of Paramecium bursaria, a Mixotrophic Ciliate

Bacteriovorous protozoa harboring symbiotic algae are abundant in aquatic ecosystems, yet despite a recent interest in protozoan bacterivory, the influence of light on their ingestion rates has not been investigated. In this study, Paramecium bursaria containing endosymbiotic Chlorella was tested for the effect of light on its ingestion rate. P. bursaria was grown for 4 to 6 days under five different light fluxes ranging from 1 to 90 microeinsteins s^-1 m^-2. Ingestion rates were determined by using 0.77-μm-diameter fluorescent microspheres. 4′,6-Diamidino-2-phenylindole dihydrochloride-labeled Enterobacter cloacae was used in one experiment to confirm differences in uptake rates of bacteria by P. bursaria. Unlike phagotrophic phytoflagellates, the ciliates demonstrated different ingestion rates in response to different light intensities. Although symbionts contribute carbon to their host via photosynthesis, the paramecia of the present study fed faster after exposure to higher light intensities, whereas their aposymbiotic counterparts (lacking endosymbionts) were unaffected. Light-induced changes in ingestion rates were not immediate, but corresponded to the period of time required for endosymbiont populations to change significantly. This strongly suggests that the symbionts, stimulated by higher light levels, may dictate the feeding rates of their hosts. Thus, light, apart from temperature, may influence the impact of certain protists on natural bacteria and may affect laboratory-based determinations of protistan feeding rates.     

Chlorella variabilis and Micractinium reisseri sp. nov. (Chlorellaceae,Trebouxiophyceae): Redescription of the endosymbiotic green algae of Paramecium bursaria (Peniculia,Oligohymenophorea) in the 120th year

Symbiotic algae of the ciliate Paramecium bursaria (Ehrenberg) Focker are key species in the fields of virology and molecular evolutionary biology as well as in the biology of symbiotic relationships. These symbiotic algae were once identified as Zoochlorella conductrix Brandt by the Dutch microbiologist, Beijerinck 120 years ago. However, after many twists and turns, the algae are today treated as nameless organisms. Recent molecular analyses have revealed several different algal partners depending on P. bursaria strains, but nearly all P. bursaria contains a symbiont belonging to either the so‐called ‘American’ or ‘European’ group. The absence of proper names for these algae is beginning to provoke ill effects in the above‐mentioned study areas. In the present study, we confirmed the genetic autonomy of the ‘American’ and ‘European’ groups and described the symbionts as Chlorella variabilis Shihira et Krauss and Micractinium reisseri Hoshina, Iwataki et Imamura sp. nov., respectively (Chlorellaceae, Trebouxiophyceae).     

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.     

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.     

Symbiotic Chlorella enhances the thermal tolerance in Paramecium bursaria

Symbiotic Chlorella enhanced the tolerance to high temperature in Paramecium bursaria. We found that 50% of Chlorella-free P. bursaria died within 85 s of exposure to 41°C in a standard saline solution, while the presence of Chlorella almost doubled the survival time of P. bursaria (160 s, P<0.001). The degree of tolerance in 3-(3,4-dichlorophenyl)-1,1-dimethylurea (an inhibitor for photosynthesis) treated Chlorella-containing P. bursaria and Chlorella-containing organisms kept in the dark for 24 h was as low as in Chlorella-free organisms. The degree of tolerance to high temperature in Chlorella-free P. bursaria in solutions containing maltose, glucose, fructose or O₂, was as high as that of normal Chlorella-containing organisms. The degree of thermal tolerance in Chlorella-containing P. bursaria was not affected in the presence of these carbohydrates or oxygen.     

The Spatial and Temporal Distribution of Paramecium bursaria in the Littoral Zone1

The spatial and seasonal distribution of Paramecium bursaria in two small Indiana ponds was studied using a sampling grid. Very small (5.0 ml) samples were taken so that the individual microhabitats could be studied. The results were evaluated in comparison to the data collected for the P. aurelia complex collected in the same manner and at the same sites. It was found that P. bursaria exist in a clumped distribution, but that the distribution was not very different from random. Paramecium bursaria also exist at the surface and at the mud-water interface. Temperature does not seem to play a statistically significant role in determining population size. The breeding system of P. bursaria is optimized for an outbreeding population of low density. In comparison, the species of the P. aurelia complex exist in a very clumped distribution, are found only at the mud-water interface, and are inbreeders. The evolutionary strategies of the two types of paramecia are discussed.     

Mechanism of Photoaccumulation in Paramecium bursaria

Responses of Paramecium bursaria to light intensity changes were investigated. The resting paramecia show a direction changing response (photophobic response) to a sudden decrease of light intensity, whereas no response was shown to an increase in intensity. The critical intensity decrease dI_c necessary to show the response was measured at various values of initial light intensity, and the ratio dI_c/I was found to be equal to ～0.15. The swimming paramecia show different behavior depending on their swimming direction in the spatial gradient of light intensity. Paramecia show direction change more frequently when they are swimming down the gradient than in the opposite direction. This difference in the rate of direction changing is 13–17%. These results may offer an explanation for the mechanism of photoaccumulation.     

Comments on the taxonomic treatment of Micractinium reisseri (Chlorellaceae,Trebouxiophyceae), a common endosymbiont in Paramecium

Micractinium reisseri Hoshina, Iwataki et Imamura (Chlorellaceae, Trebouxiophyceae), an alga symbiotic with the ciliate Paramecium bursaria (Ehrenberg) Focker, was recently renamed M. conductrix (K. Brandt) Pröschold et Darienko. The name ‘conductrix’ was originally given to an algal endosymbiont of Hydra, a genus of Hydrozoa. Here, I outline key differences between symbionts of hydra and Paramecium, and therefore, argue against the validity of the taxonomic treatment of M. reisseri by Pröschold and colleagues.