Identification of the Basal Bodies and Kinetodesmal Fibers in Living Cells of Paramecium tetraurelia Sonneborn, 1975 and Paramecium sonneborni Aufderheide,Daggett & Nerad, 19831

ABSTRACT. Critical use of Nomarski DIC optics and a rotocompressor permits basal bodies and kinetodesmal fibers to be visualized in the cortices of living Paramecium tetraurelia and Paramecium sonneborni. The identification of these structures is confirmed by the correspondence of the images obtained by DIC optics of living cells and by brightfield optics of fixed cells stained by the Fernández-Galiano silver technique. Examination of cells carrying cortical inversions (portions of the cortex rotated 180°) shows that inverted regions may be identified and distinguished from normal regions by the orientation of the kinetodesmal fibers of the kinetids (cortical units) within the kineties (ciliary rows). This demonstrates that both the asymmetry and the polarity of each cortical unit may be assessed in the living cell. This technique has useful applications in the study of morphogenesis and patterning in living cells and for the screening of mutations and variants conferring altered cortical phenotypes.     

Protein phosphorylation and dynamics of cytoskeletal structures associated with basal bodies in Paramecium

The presence of phosphorylated proteins associated with microtubule organizing centers in tissue culture cells during mitosis has been demonstrated by the use of monoclonal antibodies raised against mitotic HeLa cells [Vandre et al., Proc. Natl. Acad. Sci. U.S.A. 81:4439-4443, 1984]. We report here that in Paramecium two of the mitosis specific antibodies, MPM-1 and MPM-2, decorate throughout the cell cycle all the microtubule organizing centers (MTOCs) located in the cortex and in the oral apparatus (gullet). Immuno-electron microscopy showed that these antibodies labeled the electron-dense material surrounding basal bodies from which several microtubule networks as well as kinetodesmal fibers originate. During mitosis, these antibodies also stained other cortical cytoskeletal structures, the kinetodesmal fibers (MPM-1 and MPM-2) and the epiplasm (MPM-1). Among the different polypeptides recognized by the antibodies on immunoblots, three major ones of 60, 63, and 116 kDa were found to be common to the cortex (where several thousand ciliary basal bodies are anchored) and the oral apparatus (which comprises several hundred basal bodies around which various arrays of cytoplasmic microtubules are organized). Alkaline phosphatase treatment abolished the immunoreactivity of the polypeptides and the labeling observed by immunofluorescence. These results demonstrate that phosphorylated proteins are associated with all the known active microtubule organizing centers present in the cortex throughout the cell cycle of Paramecium. Furthermore they indicate that in Paramecium phosphorylation of proteins could also be involved in the cell cycle dependent dynamics of cortical cytoskeletal structures other than microtubules.     

The Ultrastructure of Specimens of Paramecium multimicronucleatum Impregnated with Silver by the Fernández-Galiano Method1

Transmission and scanning electron microscopy of specimens of Paramecium multimicronucleatum treated with the Rio-Hortega silver-impregnation method as modified by Fernández-Galiano demonstrate that considerable deposition of silver occurs around the kinetosomes, especially at the level of the basal plate and also at the proximal end of the kinetosome. In addition, silver is heavily deposited within the kinetodesmal fibers, in the fibrous matrix that surrounds the postciliary and transverse microtubules, in the connective structures observed between the two kinetosomes of a pair and between the kinetodesmal fiber and the anterior kinetosome, and in the trichocysts. Differences and similarities in sites of deposit when other methods of silver impregnation are employed are discussed and the particular value of the present technique in studies of ciliate systematics and phytogeny is stressed.     

CORTICAL ULTRASTRUCTURE OF PARAMECIUM AURELIA : Studies on Isolated Pellicles

Two methods have been devised for the isolation of large quantities of purified pellicles (cortical layers) of Paramecium aurelia. Pellicles isolated by both procedures, when examined by electron microscopy, were found to contain ciliary basal bodies, two types of cortical membranes, ribbons of microtubules, kinetodesmal fibers, and elements of the infraciliary lattice system. By electron microscopy, the extent of preservation of the various cortical structures when pellicles are isolated by each method has been characterized. Pellicles isolated in both ways have been utilized to investigate cortical morphology of Paramecium. Both phase-contrast and electron microscopic observations have been made. Many new ultrastructural features were observed and are reported herein. An interesting result of this study is the discovery in stock CD that the structure of cortical territories (the territory is the functional unit of cortical morphogenesis and physiology) may vary within a single organism. Features which show variation include number of parasomal sacs, microtubular ribbons, and basal bodies (and therefore cilia) per territory, number of microtubules per ribbon, and length of kinetodesmal fibers. The possible significance of these variations, with respect to territory replication, is discussed. In addition, preliminary observations on the solubility of various cortical organelles in the presence of a number of protein-denaturing agents are reported.     

PARTIAL PURIFICATION AND PHOSPHOTUNGSTATE SOLUBILIZATION OF BASAL BODIES AND KINETODESMAL FIBERS FROM TETRAHYMENA PYRIFORMIS

Previously devised methods for the isolation of basal bodies from ciliate protozoans were found to be inadequate for chemical analysis. We have modified and expanded these procedures and developed a method which gives preparations containing mainly basal bodies and kinetodesmal fibers. This procedure involved fixation of cells in 30% ETOH followed by digitonin or Triton X-100 solubilization and homogenization with a Brinkmann Polytron. This is followed by sucrose gradient centrifugation. Negative staining and thin sectioning revealed these preparations to be substantially more pure than those of previous workers. It was also found that neutralized phosphotungstate (PTA) solubilized many of the components present in fixed Tetrahymena. Neutralized 1.0% PTA solubilized axonemes, cortical, axonemal, and basal body microtubules as well as kinetodesmal fibers. These results have been confirmed by both electron microscope observations and gel electrophoresis of 100,000 g supernatants of the PTA extracts. A solution of 0.1% PTA did not affect the fibers but did solubilize basal bodies. Running 1.0% PTA extracts from our basal body fractions on sodium dodecyl sulfate (SDS) polyacrylamide gels allowed us to tentatively identify the peptides of basal bodies and kinetodesmal fibers. The latter structures appear to consist of a single 21,000 mol wt peptide. These results also suggest that great caution should be taken in interpreting PTA images, especially of microtubules and axonemes.     

The role of cortical orientation in the control of the direction of ciliary beat in Paramecium

The swimming behavior of many ciliate protozoans depends on graded changes in the direction of the ciliary effective stroke in response to depolarizing stimuli (i.e., the avoiding reaction of Paramecium). We investigated the problem of whether the directional response of cilia with a variable plane of beat is related to the polarity of the cell as a whole or to the orientation of the cortical structures themselves. To do this, we used a stock of Paramecium aurelia with part of the cortex reversed 180 degrees. We determined the relation of the orientation of the kineties (ciliary rows) to the direction of beat in these mosaic paramecia by cinemicrography of particle movements near living cells and by scanning electron microscopy of instantaneously fixed material. We found that the cilia of the inverted rows always beat in the direction opposite to that of normally oriented cilia during both forward and backward swimming. In addition, metachronal waves of ciliary coordination were present on the inverted patch, travelling in the direction opposite to those on the normal cortex. The reference point for the directional response of Paramecium cilia to stimuli thus resides within the cilia or their immediate cortical surroundings.     

Cytidine Analogues and Stomatogenic Recovery in Amicronucleate Paramecium tetraurelia and Paramecium jenningsi

Removal of the micronuclei of Paramecium tetraurelia and Paramecium jenningsi by micropipetting generates amicronucleate cell lines. These cell lines go through a period of growth depression for several dozen fissions, but they gradually recover. Amicronucleate cells in the depression period characteristically exhibit abnormal oral development, particularly reduction in the length of the buccal cavity and an abnormal pattern of the oral membranelles. To test the notion that the macronucleus is involved in the recovery of amicronucleate cell lines, DNA demethylation drugs were administered to amicronucleates in the depression period. After at least 4 fissions, the treated amicronucleates were assessed for their progress in recovery by scoring the proportion of cells with normal oral membranelles. Cvtidine analogues which demethylate cytosine specifically at the 5 position, namely 5-azacytidine, 5-aza-2'- deoxycytidine and 5-fluoro-2'-deoxycytidine. promoted recovery of the amicronucleates. Cytidine, 6-azacytidine, 2'-fluoro-2'-deoxy-cytidine and cytosine-β-D-arabinofuranoside did not. These results suggest that (i) 5-methylcytosine is present in the macronucleus of these Paramecium species, probably in small amounts and (ii) recovery of amicronucleates involves demethylation of macronuclear DNA. This implies that in normal cells the micronuclei are involved in maintaining the macronuclear DNA in a methylated state and hence the inactivation of the macronuclear sequences that are to be employed for stomatogenic recovery. A general mechanism for the control of gene expression may therefore be employed for the regulation of specific sequences.     

Internalization of cycloheptaamylose-dansyl chloride complex during labelling of surface membrane in living Paramecium aurelia cells

Internalization of cycloheptaamylose-dansyl chloride complex during surface labelling of living long-term starved Paramecium aurelia cells has been observed. This process may be inhibited by pretreatment of the ciliates with dichloroisoproterenol. Uptake of cycloheptaamylose-dansyl chloride may be visualized only after UV preirradiation: the appearance of orange-fluorescing vacuoles of diameter 2.3-4.5 micron may then be observed. Microspectrographic analysis performed on the cells and dansyl derivatives indicates that this fluorescence is produced by a photochemical reaction of dansyl chloride - released from CDC complex inside the digestive vacuoles-under the influence of UV irradiation.     

温度休克法诱导草履虫的同步分裂

运用细胞周期原理,采用温度休克法,对尾草履虫进行分裂周期同步化的研究,实验中草履虫经过3－5h的处理后,就能观察到大量不同阶段的无性生殖横分裂状态,并获得了大量处于分裂阶段的草履虫。运用这种技术取材容易,获取率稳定,可达61％,可为细胞生理学等领域的研究提供大量的同步分裂个体。     

Chromogranin A-like proteins in the secretory granules of a protozoan, Paramecium tetraurelia

The ciliate protozoan Paramecium tetraurelia produces secretory granules (trichocysts) which release needle-like structures composed of small, acidic proteins. Using antibodies against isolated chromogranin A (CGA) and against trichocyst proteins, we found cross-reactive proteins in chromaffin granules and trichocysts. Four independently derived sera against isolated CGA stained bands of the Mr 15,000-25,000 family of trichocyst proteins on immunoblots. A positive response was also obtained with antiserum against chemically synthesized peptides (PL26 and GE25) corresponding to defined regions of the CGA amino acid sequence. In extracts of whole Paramecium, larger proteins (Mr 53,000 and 49,000) also reacted with antibodies against CGA and the related synthetic peptides. These larger proteins may represent unprocessed precursors to the smaller proteins of mature trichocysts. Antiserum to trichocysts recognized CGA in chromaffin granule lysates. Further evidence of a Paramecium protein related to CGA was provided by hybridization of Paramecium mRNA with cloned cDNA for bovine CGA. Our results suggest striking conservation in evolution of CGA-like proteins that may play some role, as yet unknown, in secretion.     

Association of Paramecium bursaria Chlorella viruses with Paramecium bursaria cells: ultrastructural studies

Paramecium bursaria Chlorella viruses were observed by applying transmission electron microscopy in the native symbiotic system Paramecium bursaria (Ciliophora, Oligohymenophorea) and the green algae Chlorella (Chlorellaceae, Trebouxiophyceae). Virus particles were abundant and localized in the ciliary pits of the cortex and in the buccal cavity of P. bursaria. This was shown for two types of the symbiotic systems associated with two types of Chlorella viruses - Pbi or NC64A. A novel quantitative stereological approach was applied to test whether virus particles were distributed randomly on the Paramecium surface or preferentially occupied certain zones. The ability of the virus to form an association with the ciliate was investigated experimentally; virus particles were mixed with P. bursaria or with symbiont-free species P. caudatum. Our results confirmed that in the freshwater ecosystems two types of P. bursaria -Chlorella symbiotic systems exist, those without Chlorella viruses and those associated with a large amount of the viruses. The fate of Chlorella virus particles at the Paramecium surface was determined based on obtained statistical data and taking into account ciliate feeding currents and cortical reorganization during cell division. A life cycle of the viruses in the complete symbiotic system is proposed.     

Multigene family encoding 3',5'-cyclic-GMP-dependent protein kinases in Paramecium tetraurelia cells

In the ciliate Paramecium tetraurelia, 3',5'-cyclic GMP (cGMP) is one of the second messengers involved in several signal transduction pathways. The enzymes for its production and degradation are well established for these cells, whereas less is known about the potential effector proteins. On the basis of a current Paramecium genome project, we have identified a multigene family with at least 35 members, all of which encode cGMP-dependent protein kinases (PKGs). They can be classified into 16 subfamilies with several members each. Two of the genes, PKG1-1 and PKG2-1, were analyzed in more detail after molecular cloning. They encode monomeric enzymes of 770 and 819 amino acids, respectively, whose overall domain organization resembles that in higher eukaryotes. The enzymes contain a regulatory domain of two tandem cyclic nucleotide-binding sites flanked by an amino-terminal region for intracellular localization and a catalytic domain with highly conserved regions for ATP binding and catalysis. However, some Paramecium PKGs show a different structure. In Western blots, PKGs are detected both as cytosolic and as structure-bound forms. Immunofluorescence labeling shows enrichment in the cell cortex, notably around the dense-core secretory vesicles (trichocysts), as well as in cilia. Immunogold electron microscopy analysis reveals consistent labeling of ciliary membranes, of the membrane complex composed of cell membrane and cortical Ca2+ stores, and of regions adjacent to ciliary basal bodies, trichocysts, and trafficking vesicles. Since PKGs (re)phosphorylate the exocytosis-sensitive phosphoprotein pp63/pf upon stimulation, the role of PKGs during stimulated exocytosis is discussed, in addition to a role in ciliary beat regulation.     

Cortical alveoli of Paramecium: a vast submembranous calcium storage compartment

The plasma membrane of Paramecium is underlain by a continuous layer of membrane vesicles known as cortical alveoli, whose function was unknown but whose organization had suggested some resemblance with muscle sarcoplasmic reticulum. The occurrence of antimonate precipitates within the alveoli first indicated to us that they may indeed correspond to a vast calcium storage site. To analyze the possible involvement of this compartment in calcium sequestration more directly, we have developed a new fractionation method, involving a Percoll gradient, that allows rapid purification of the surface layer (cortex) of Paramecium in good yield and purity and in which the alveoli retain their in vivo topological orientation. This fraction pumped calcium very actively in a closed membrane compartment, with strict dependence on ATP and Mg2+. The pumping activity was affected by anti-calmodulin drugs but no Triton-soluble calmodulin binding protein could be identified, using gel overlay procedures. The high affinity of the pump for calcium (Km = 0.5 microM) suggests that it plays an important role in the normal physiological environment of the cytosol. This may be related to at least three calcium-regulated processes that take place in the immediate vicinity of alveoli: trichocyst exocytosis, ciliary beating and cytoskeletal elements dynamics during division.     

Ultrastructure of the proximal region of somatic cilia in Paramecium tetraurelia

The morphology of the transition zone between the terminal plate of the basal body and the 9 + 2 region of the somatic (non-oral) cilium has been examined in Paramecium tetraurelia. Freeze-fracture and thin- section techniques disclosed both membrane specializations and various internal structural linkages. Freeze-fracture material revealed sets of particles interrupting the unit membrane. The more distal of these form plaquelike arrays while the proximal set of particles forms the ciliary "necklace." The plaque regions correspond to anionic sites on the outer membrane surface as revealed by binding of polycationic ferritin. Both the plaque particles and the necklace particles appear to be in contact with outer doublet microtubules via a complex of connecting structures. In the interior of the transition zone an axosomal plate supports an axosome surrounded by a ring of lightly packed material. Only one of the two central tubules of the axoneme reaches and penetrates the axosome. Below the axosomal plate four rings, each approx. 20 nm wide, connect adjacent outer doublets. An intermediate plate lies proximal to these rings, and a terminal plate marks the proximal boundary of this zone. Nine transitional fibers extend from the region of the terminal plate to the plasmalemma. The observations described above have been used to construct a three-dimensional model of the transition region of "wild-type" Paramecium somatic cilia. It is anticipated that this model will be useful in future studies concerning possible function of transition-zone specializations, since Paramecium may be examined in both normal and reversed ciliary beating modes, and since mutants incapable of reverse beating are available.     

Expression of the green fluorescent protein in Paramecium tetraurelia

In this paper we describe the expression of green fluorescent protein (GFP) as a reporter in vivo to monitor transformation in Paramecium cells. This is not trivial because of the limited number of strong promoters available for heterologous expression and the very high AT content of the genomic DNA, the consequence of which is a very aberrant codon usage. Taking into account differences in codon usage we selected and modified the original GFP open reading frame (ORF) from Aequorea victoria and placed the altered ORF into the Paramecium expression vector pPXV. Injection of the linearized plasmid into the macronucleus resulted in a cytoplasmic fluorescence signal in the clonal descendants, which was proportional to the number of copies injected. Southern hybridization indicated the establishment and replication of the plasmid during vegetative growth. Expression was also monitored by Northern and Western analysis. The results indicate that the modified GFP can be used in Paramecium as a reporter for transformation as an alternative to selection with antibiotics and that it may also be used to construct and localize fusion proteins.     

Responses to hypergravity in proliferation of Paramecium tetraurelia

It has been reported that Paramecium proliferates faster when cultured under microgravity in orbit, and slower when cultured under hypergravity. This shows that the proliferation rate of Paramecium affected by gravity. The effect of gravity on Paramecium proliferation has been argued to be direct in a paper with an axenic culture under hypergravity. To clear up uncertainties with regard to the effect of gravity, Paramecium tetraurelia was cultured axenically under hypergravity (20 x g) and the time course of the proliferation was investigated quantitatively by a new non-invasive method, laser-beam optical slice, for measuring the cell density. This method includes optical slicing a part of the culture and computer-aided counting of cells in the sliced volume. The effects of hypergravity were assessed by comparing the kinetic parameters of proliferation that were obtained through a numerical analysis based on the logistic growth equation. Cells grown under 20 x g conditions had a significantly lower proliferation rate, and had a lower population density at the stationary phase. The lowered proliferation rate continued as long as cells were exposed to hypergravity (> one month). Hypergravity reduced the cell size of Paramecium. The long and short axes of the cell became shorter at 20 x g than those of control cells, which indicates a decrease in volume of the cell grown under hypergravity and is consistent with the reported increase in cell volume under microgravity. The reduced proliferation rate implies changes in biological time defined by fission age. In fact the length of autogamy immaturity decreased by measure of clock time, whereas it remained unchanged by measure of fission age.     

A two-locus molecular characterization of Paramecium calkinsi

Paramecium calkinsi (Ciliophora, Protozoa) is a euryhaline species which was first identified in freshwater habitats, but subsequently several strains were also collected from brackish water. It is characterized by clockwise spiral swimming movement and the general morphology of the "bursaria type." The present paper is the first molecular characterization of P. calkinsi strains recently collected in distant regions in Russia using ITS1-5.8S- ITS2-5'LSU rDNA (1100bp) and COI (620bp) mtDNA sequenced gene fragments. For comparison, our molecular analysis includes P. bursaria, exhibiting a similar "bursaria morphotype" as well as species representing the "aurelia type," i.e., P. caudatum, P. multimicronucleatum, P. jenningsi, and P. schewiakoffi, and some species of the P. aurelia species complex (P. primaurelia, P. tetraurelia, P. sexaurelia, and P. tredecaurelia). We also use data from GenBank concerning other species in the genus Paramecium and Tetrahymena (which used as an outgroup). The division of the genus Paramecium into four subgenera (proposed by Fokin et al. 2004) is clearly presented by the trees. There is a clear separation between P. calkinsi strains collected from different regions (races). Consequently, given the molecular distances between them, it seems that these races may represent different syngens within the species.     

Filamentous actin in Paramecium cells: mapping by phalloidin affinity labeling in vivo and in vitro

In living Paramecium cells, microinjected rhodaminyl (R)-phalloidin rapidly labels a thin cortical layer. This can be more clearly resolved with microinjected and fixed cells (allowing for better resolution) as well as with isolated pellicles (surface membrane complexes with trichocysts, microfilaments, and mitochondria attached). Labeling of a longitudinal and perpendicular pattern, reflecting the relief of the cell surface, and labeling of ciliary basal bodies then becomes clearly visible. Other structures labeled by R-phalloidin are the surfaces of food vacuoles of different sizes and, although inconsistently, the borders of the buccal cavity. Small acidic compartments (as identified by acridine orange fluorescence vital staining), probably representing acidosomes and small lysosomes, were not labeled. F-actin on food vacuole surfaces may somehow be involved in intracellular transport or fusion processes. No labeling was observed in association with the osmoregulatory system (contractile vacuoles and their ampullae and radial canals). The specificity of in vivo labeling obtained was supported by the abolition of R-phalloidin labeling when isolated pellicles were pretreated with unlabeled phalloidin or with DNAse I. It was also possible to discriminate among different layers of R-phalloidin binding in the cortex by detaching different layers of the surface complex from each other. Since localization of F-actin in ciliates has raised a considerable amount of dispute in the past, we also repeated all these experiments with RITC-labeled HMM, but we obtained essentially the same labeling pattern as with R-phalloidin. Ciliary basal bodies therefore clearly contain some F-actin. Our data shed some light on aspects of surface structuring and motility in these cells.     

Organization and closing of mitochondrial deoxyribonucleic acid from Paramecium tetraaurelia and Paramecium primaurelia.

Previously we showed that the mitochondrial deoxyribonucleic acid (DNA) from Paramecium aurelia consists of a linear genome and that replication of this genome is initiated at one terminus and proceeds unidirectionally to the other terminus. Analyses of mitochondria from four closely related species (1, 4, 5, and 7) indicated that the species 1, 5, and 7 DNAs are essentially completely homologous but that the species 4 mitochondrial DNA is only 40 to 50% homologous with that from species 1. The major regions of homology are those containing the genes for ribosomal ribonucleic acid (RNA). To understand the replication and organization of the linear mitochondrial genome better, we compared species 1 (Paramecium primaurelia) and 4 (Paramecium tetraaurelia) DNAs with regard to restriction fragment mapping and homology between initiation regions; we also identified the sites of the genes for ribosomal RNA. In general, the structures of the species 1 and 4 mitochondrial genomes were quite similar. Each ribosomal RNA gene was present in one copy per genome, with the large ribosomal RNA gene located near the terminal region of replication and the small ribosomal RNA gene located more centrally. These two genes were separated by about 10 kilobases in the species 1 genome and by about 12 kilobases in the species 4 genome. In contrast to our previous findings, by using nonstringent hybridization conditions we detected homology between the species 1 and 4 DNA fragments containing the initiation regions. We constructed recombinant DNA clones for many fragments, especially those containing the initiation region and the ribosomal RNA genes. We also constructed restriction enzyme maps for six enzymes for both P. primaurelia and P. tetraaurelia.     

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.