Symbiotic Chlorella vulgaris of the ciliate Paramecium bursaria plays an important role in maintaining perialgal vacuole membrane functions

Treatment of symbiotic alga-bearing Paramecium bursaria cells with a protein synthesis inhibitor, cycloheximide, induces synchronous swelling of all perialgal vacuoles at about 24h after treatment under a constant light condition. Subsequently, the vacuoles detach from the host cell cortex. The algae in the vacuoles are digested by the host's lysosomal fusion to the vacuoles. To elucidate the timing of algal degeneration, P. bursaria cells were treated with cycloheximide under a constant light condition. Then the cells were observed using transmission electron microscopy. Results show that algal chloroplasts and nuclei degenerated within 9h after treatment, but before the synchronous swelling of the perialgal vacuole and appearance of acid phosphatase activity in the perialgal vacuole by lysosomal fusion. Treatment with cycloheximide under a constant dark condition and treatment with chloramphenicol under a constant light condition induced neither synchronous swelling of the vacuoles nor digestion of the algae inside the vacuoles. These results demonstrate that algal proteins synthesized during photosynthesis are necessary to maintain chloroplastic and nuclear structures, and that inhibition of protein synthesis induces rapid lysis of these organelles, after which synchronous swelling of the perialgal vacuole and fusion occur with the host lysosomes.     

Timing of perialgal vacuole membrane differentiation from digestive vacuole membrane in infection of symbiotic algae Chlorella vulgaris of the ciliate Paramecium bursaria

Each symbiotic Chlorella of the ciliate Paramecium bursaria is enclosed in a perialgal vacuole derived from the host digestive vacuole to protect from lysosomal fusion. To understand the timing of differentiation of the perialgal vacuole from the host digestive vacuole, algae-free P. bursaria cells were fed symbiotic C. vulgaris cells for 1.5min, washed, chased and fixed at various times after mixing. Acid phosphatase activity in the vacuoles enclosing the algae was detected by Gomori's staining. This activity appeared in 3-min-old vacuoles, and all algae-containing vacuoles demonstrated activity at 30min. Algal escape from these digestive vacuoles began at 30min by budding of the digestive vacuole membrane into the cytoplasm. In the budded membrane, each alga was surrounded by a Gomori's thin positive staining layer. The vacuoles containing a single algal cell moved quickly to and attached just beneath the host cell surface. Such vacuoles were Gomori's staining negative, indicating that the perialgal vacuole membrane differentiates soon after the algal escape from the host digestive vacuole. This is the first report demonstrating the timing of differentiation of the perialgal vacuole membrane during infection of P. bursaria with symbiotic Chlorella.     

Endosymbiosis of Chlorella species to the ciliate Paramecium bursaria alters the distribution of the host’s trichocysts beneath the host cell cortex

Each symbiotic Chlorella of the ciliate Paramecium bursaria is enclosed in a perialgal vacuole membrane derived from the host digestive vacuole membrane. Alga-free paramecia and symbiotic algae can grow independently. Mixing them experimentally can cause reinfection. Earlier, we reported that the symbiotic algae appear to push the host trichocysts aside to become fixed beneath the host cell cortex during the algal reinfection process. Indirect immunofluorescence microscopy with a monoclonal antibody against the trichocysts demonstrates that the trichocysts change their locality to form algal attachment sites and decrease their density beneath the host cell cortex through algal reinfection. Transmission electron microscopy to detect acid phosphatase activity showed that some trichocysts near the host cell cortex are digested by the host lysosomal fusion during algal reinfection. Removal of algae from the host cell using cycloheximide recovers the trichocyst's arrangement and number near the host cell cortex. These results indicate that symbiotic algae compete for their attachment sites with preexisting trichocysts and that the algae have the ability to ensure algal attachment sites beneath the host cell cortex.     

Symbiotic Chlorella sp. of the ciliate Paramecium bursaria do not prevent acidification and lysosomal fusion of host digestive vacuoles during infection

Summary. Each symbiotic Chlorella sp. of the ciliate Paramecium bursaria is enclosed in a perialgal vacuole derived from the host digestive vacuole, and thereby the alga is protected from digestion by lysosomal fusion. Algae-free cells can be reinfected with algae isolated from algae-bearing cells by ingestion into digestive vacuoles. To examine the timing of acidification and lysosomal fusion of the digestive vacuoles and of algal escape from the digestive vacuole, algae-free cells were mixed with isolated algae or yeast cells stained with pH indicator dyes at 25 ± 1 °C for 1.5 min, washed, chased, and fixed at various time points. Acidification of the vacuoles and digestion of Chlorella sp. began at 0.5 and 2 min after mixing, respectively. All single green Chlorella sp. that had been present in the host cytoplasm before 0.5 h after mixing were digested by 0.5 h. At 1 h after mixing, however, single green algae reappeared in the host cytoplasm, arising from those digestive vacuoles containing both nondigested and partially digested algae, and the percentage of such cells increased to about 40% at 3 h. At 48 h, the single green algae began to multiply by cell division, indicating that these algae had succeeded in establishing endosymbiosis. In contrast to previously published studies, our data show that an alga can successfully escape from the host’s digestive vacuole after acidosomal and lysosomal fusion with the vacuole has occurred, in order to produce endosymbiosis. Correspondence and reprints: Biological Institute, Faculty of Science, Yamaguchi University, Yoshida 1677-1, Yamaguchi 753-8512, Japan.     

纤毛虫与藻类共生关系的研究现状与展望

纤毛虫与藻类的共生关系在水体环境中广泛存在并有着重要的生态功能。文章回顾了国内外纤毛虫与藻类共生研究的发展历程,主要介绍了纤毛虫与藻类共生的生态功能,以及显微观察与分子生物学技术在纤毛虫与藻类共生研究中的应用;阐述了包括草履虫与小球藻共生关系建立的4个过程及其互作机制、红色中缢虫与隐藻的共生关系、宿主与共生体之间的互作等内容;提出了纤毛虫与藻类共生研究中亟待解决的科学问题,包括草履虫食物泡膜(digestive vacuole, DV)与围藻膜(perialgal vacuole, PV)发挥作用的分子机制、红色中缢虫与隐藻共生关系的建立过程、红色中缢虫在共生过程中的功能作用等,并展望未来的研究方向。     

Amino acids as a nitrogen source for Chlorella symbiotic with green hydra

Supply of amino acids may be important in controlling cell division of Chlorella symbiotic with green hydra. Freshly isolated symbionts display characteristics of N-limited algae, and low pH in perialgal vacuoles and high levels of host glutamine synthetase (GS) limit uptake of ammonium. Movement of tritiated amino acids from host to algal pools suggests that symbiotic algae utilize amino acids derived from host digestion of prey. Amounts are significant in relation to host and algal amino acids pools. During host starvation, glutamine produced by host GS may be important as a nitrogen supply to the algae, which take up this amino acid at high rates at low pH.     

Symbiotic <Emphasis Type="Italic">Chlorella variabilis</Emphasis> strain, 1 N,can influence the digestive process in the host <Emphasis Type="Italic">Paramecium bursaria</Emphasis> during early infection

The association between the ciliate Paramecium bursaria and symbiotic Chlorella spp. is mutually beneficial. However, this relationship is facultative mutualism because both the host and the symbiotic algae can grow by themselves. This association is easily re-established by mixing the two species together. Following algal mixing, some algae become enclosed in the digestive vacuole membrane of the paramecia to which both acidosomes and lysosomes fuse. To establish endosymbiosis, some algae acquire temporal resistance to the host lysosomal enzymes in the digestive vacuoles. We examined whether the algae influence the differentiation of the host digestive process using LysoSensor staining to evaluate the acidification of the digestive vacuoles. Furthermore, to assess lysosomal fusion with the digestive vacuole, Gomori’s staining was conducted. Acidification and lysosomal fusion occurred later in digestive vacuoles containing living algae than in those containing boiled algae or latex spheres. This phenomenon was observed when the living algae were maintained under a constant light condition. These results suggest that the algae release some unknown factor in response to light exposure, and the factor may be associated with the alteration of the host digestive process, indicating that the living algae can influence the host digestive processes during early algal infection.     

Localization of attachment area of the symbiotic Chlorella variabilis of the ciliate Paramecium bursaria during the algal removal and reinfection

Chlorella spp. and ciliate Paramecium bursaria share a mutual symbiosis. However, both alga-removed P. bursaria and isolated symbiotic algae can grow independently. Additionally, mixing them experimentally can cause algal reinfection through host phagocytosis. Although the symbiotic algal localization beneath the host cell cortex is a prerequisite phenomenon for maintenance of the relationship of their endosymbiosis, how and where the algae locate beneath the host cell cortex remains unknown. To elucidate this phenomenon, algal distribution patterns during algal removal and reinfection were observed. During algal removal, algae at the host anterior cortex were easier to remove than at the posterior and ventral or dorsal cortex areas. During algal reinfection, the algae after separation from the host digestive vacuoles tended to localize beneath the host ventral or dorsal cortex more readily than that at other cortices. Algae that reinfected trichocyst-removed paramecia didn’t show this localization. Trichocyst-discharge experiments clarified that trichocysts of the anterior cortex are difficult to remove. In 14 strains of P. bursaria, some of the paramecia lacked their symbiotic algae at the anterior cortex. These observations demonstrate that symbiotic algae of P. bursaria are difficult to localize at the anterior cortex and that they are easy to remove from the area.     

Infectivity of Chlorella species for the ciliate Paramecium bursaria is not based on sugar residues of their cell wall components, but on their ability to localize beneath the host cell membrane after escaping from the host digestive vacuole in the early infection process

Summary. Paramecium bursaria cells harbor several hundred symbiotic algae in their cytoplasm. Algae-free cells can be reinfected with algae isolated from algae-bearing cells or cultivated Chlorella species through the digestive vacuoles. To determine the relationship between the infectivity of various Chlorella species and the nature of their cell wall components, algae-free P. bursaria cells were mixed with 15 strains of cultivated Chlorella species and observed for the establishment of endosymbiosis at 1 h and 3 weeks after mixing. Only 2 free-living algal strains, C. sorokiniana C-212 and C. kessleri C-531, were maintained in the host cells, whereas free-living C. sorokiniana C-43, C. kessleri C-208, C. vulgaris C-27, C. ellipsoidea C-87 and C-542, C. saccharophila C-183 and C-169, C. fusca var. vacuolata C-104 and C-28, C. zofingiensis C-111, and C. protothecoides C-150 and C-206 and the cultivated symbiotic Chlorella sp. strain C-201 derived from Spongilla fluviatilis could not be maintained. These infection-incapable strains could escape from the host digestive vacuole but failed to localize beneath the host cell membrane and were eventually digested. Labeling of their cell walls with Alexa Fluor 488-conjugated wheat germ agglutinin, GS-II, or concanavalin A, with or without pretreatment with 0.4 N NaOH, showed no relationship between their infectivity and the stainability with these lectins. Our results indicate that the infectivity of Chlorella species for P. bursaria is not based on the sugar residues on their cell wall and on the alkali-insoluble part of the cell wall components, but on their ability to localize just beneath the host cell membrane after escaping from the host digestive vacuole. Correspondence and reprints: Environmental Science and Engineering, Graduate School of Science and Engineering, Yamaguchi University, Yoshida 1677-1, Yamaguchi 753-8512, Japan.     

Photosynthesis dependent acidification of perialgal vacuoles in theParamedum bursaria/Chlorella symbiosis: Visualization by monensin

Summary After treatment with the carboxylic ionophore monensin theChlorella containing perialgal vacuoles of the greenParamecium bursaria swell. TheParamecium cells remain motile at this concentration for at least one day. The swelling is only observed in illuminated cells and can be inhibited by DCMU. We assume that during photosynthesis the perialgal vacuoles are acidified and that monensin exchanges H⁺ ions against monovalent cations (here K⁺). In consequence the osmotic value of the vacuoles increases. The proton gradient is believed to drive the transport of maltose from the symbiont into the host. Another but light independent effect of the monensin treatment is the swelling of peripheral alveoles of the ciliates, likewise indicating that the alveolar membrane contains an active proton pump.Abbreviations HEPES N-(2-hydroxyethyl)piperazine-N-2-ethane sulfonic acid - DCMU 3-(3, 4-dichlorophenyl)-1,1-dimethylurea     

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.     

Inhibition of lysosomal fusion with symbiont-containing vacuoles in Paramecium bursaria

Paramecium bursaria harbors several hundred intracellular Chlorella symbionts which remain undigested at the same time that the host cell phagocytizes and digests other organisms. Using electron microscopy and thorotrast labelling, we have shown that secondary lysosomes fuse with food vacuoles, but do not fuse with vacuoles containing symbiotic algae. From these and other data we suggest that the symbiotic algae alter the membrane of the vacuole which surrounds them, thus inhibiting fusion with secondary lysosomes.     

The endosymbiotic unit ofClimacostomum virens andChlorella sp. I. Morphological and physiological studies on the algal partner and its localization in the host cell

Summary The ciliateClimacostomum virens forms an endosymbiotic association with coccoid chlorophycean algae which can be isolated and grown as sterile mass cultures with an inorganic medium. According to both morphological and physiological properties, the algae probably belong to the genusChlorella and have some features in common with symbiotic chlorellae isolated from the ciliatesParamecium bursaria andStentor polymorphus. These and other endosymbiotic chlorellae studied so far excrete maltose or glucose, but algae fromClimacostomum virens show a different excretion pattern by releasing glucose, fructose and xylose. Possible biosynthetic pathways are discussed. Algae inClimacostomum virens are either located individually in special perialgal vacuoles where they are probably protected against attack by host lytic enzymes or to a lesser extent in food vacuoles in different states of digestion. The endosymbiotic character of theClimacostomum virens-Chlorella sp.-association is discussed.Dedicated to Prof. H. A.von Stosch on the occasion of his 75th birthday.     

Separation of two cell signalling molecules from a symbiotic sponge that modify algal carbon metabolism

Two distinct cell signals have been isolated from the sponge host of the tropical sponge/macroalga symbiotic association Haliclona cymiformis/Ceratodictyon spongiosum. These water soluble cell signals (M(r) between 500 and 1000) modify separate steps in the carbon metabolism in both C. spongiosum and the microalga, Symbiodinium from the coral Plesiastrea versipora. The first signal, host release factor (HRF), stimulates the release of compounds derived from algal photosynthesis; the second signal, photosynthesis inhibiting factor (PIF), partially inhibits photosynthesis. Both HRF from the sponge H. cymiformis and HRF from the coral P. versipora stimulated the release of glycerol from Symbiodinium suggesting that they act at a similar step in the metabolism of this alga. This is the first time that such cell signals have been isolated from a sponge. We suggest that they belong to a family of similar cell signals from symbiotic invertebrates that modify algal carbon metabolism.     

Multiple origins of the symbioses in Paramecium bursaria

Many organisms have symbioses with photosynthetic algae as typified by corals, clams, lichens, and some protozoa. Paramecium bursaria contains green algal symbionts and this unicellular ciliate is a textbook example used for microscopic observation in junior high school science projects. We have determined molecular phylogenies for the green algal symbionts. The symbiotic algae are the main constituent of the Paramecium cytoplasm, and we have recognized a total of four species, of which two were newly discovered in the present study. One should be regarded genetically as Chlorella vulgaris, and it belongs phylogenetically to the Chlorella clade (Chlorellaceae, Trebouxiophyceae) as well as "American" and "European" groups, which we previously introduced. Their genetic dissimilarities are 0.50-0.83% in 18S rDNA comparisons, but those of the internal transcribed spacer 2 (ITS2) reach an unambiguous level (22.6-26.6%). These dissimilarities suggest that they are equivalent to discrete species derived from multiple origins as paramecian symbionts. Another newcomer was clearly separated from the Chlorellaceae, and this alga clustered with Coccomyxa spp. in ITS2 analyses. These symbiotic relations indicate multiple origins of symbionts.     

On the developmental dependence between Cyanophora paradoxa and Cyanocyta korschikoffiana in symbiosis

The prokaryote Cyanocyta korschikoffiana was isolated from the eukaryote Cyanophora paradoxa. The synthesis of several thylakoid proteins in these cyanelles is influenced by light and darkness and is sensitive to cycloheximide, the inhibitor of the eukaryotic host's translation. The possibility of a direct coordination between the translations of the host and of the cyanelles is discussed.Abbreviations CHM treatment addition of cycloheximide - CPN chlorophylline - PBN phycobiline - SDS-PAGE sodium-dodecylsulphate-polyacrylamide gelelectrophoresis     

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.

     

Characteristics of the digestive vacuole membrane of the alga-bearing ciliate Paramecium bursaria

Cells of the ciliate Paramecium bursaria harbor symbiotic Chlorella spp. in their cytoplasm. To establish endosymbiosis with alga-free P. bursaria, symbiotic algae must leave the digestive vacuole (DV) to appear in the cytoplasm by budding of the DV membrane. This budding was induced not only by intact algae but also by boiled or fixed algae. However, this budding was not induced when food bacteria or India ink were ingested into the DVs. These results raise the possibility that P. bursaria can recognize sizes of the contents in the DVs. To elucidate this possibility, microbeads with various diameters were mixed with alga-free P. bursaria and traced their fate. Microbeads with 0.20μm diameter did not induce budding of the DVs. Microbeads with 0.80μm diameter produced DVs of 5-10μm diameter at 3min after mixing; then the DVs fragmented and became vacuoles of 2-5μm diameter until 3h after mixing. Each microbead with a diameter larger than 3.00μm induced budding similarly to symbiotic Chlorella. These observations reveal that induction of DV budding depends on the size of the contents in the DVs. Dynasore, a dynamin inhibitor, greatly inhibited DV budding, suggesting that dynamin might be involved in DV budding.     

A cell signal from the coral Plesiastrea versipora reduces starch synthesis in its symbiotic alga, Symbiodinium sp

We have previously shown that the coral cell signal, host release factor (HRF) from the scleractinian coral Plesiastrea versipora (Lamarck) stimulates the release of glycerol from its symbiotic dinoflagellate, Symbiodinium sp. Glycerol is a precursor for algal triacylglycerol (TG) and starch, and we have previously observed that HRF reduces the amount of newly synthesized TG in Symbiodinium sp. We have now examined the effect of P. versipora HRF on starch synthesis in isolated Symbiodinium. HRF had no effect on starch synthesis after 2 h photosynthesis (16.3+/-3.0 microg starch per 10(6) algae) compared with algae in seawater (13.9+/-1.2 microg starch per 10(6) algae). However, after 4 h incubation in HRF, there was a reduction (0-76%), in the amount of newly synthesized starch which was correlated with the amount of HRF (10-76 microg/ml). Reducing algal synthesis of both TG and starch in parallel with stimulating glycerol release may provide a mechanism to regulate the population density of intracellular symbiotic algae while still ensuring the transfer of photosynthetically fixed carbon to the animal host in the form of glycerol.     

Metatrancriptomic analysis from the Hepatopancreas of adult white leg shrimp (<Emphasis Type="Italic">Litopenaeus vannamei)</Emphasis>

The amoeba, Mayorella viridis contains several hundred symbiotic green algae in its cytoplasm. Transmission electron microscopy revealed strong resemblance between symbiotic algae from M. viridis the symbiotic Chlorella sp. in the perialgal vacuoles of Paramecium bursaria and other ciliates. Although it is thought that the M. viridis and symbiotic algae could be model organisms for studying endosymbiosis between protists and green algae, few cell biological observations of the endosymbiosis between M. viridis and their symbiotic algae have been published. In this study, we characterized the specificity of endosymbiotic relationships between green algae and their hosts. Initially, we established stable cultures of M. viridis in KCM medium by feeding with Chlorogonium capillatum. Microscopic analyses showed that chloroplasts of symbiotic algae in M. viridis occupy approximately half of the algal cells, whereas those in P. bursaria occupy entire algal cells. The symbiotic algae in P. bursaria contain several small spherical vacuoles. The labeling of actin filaments using Acti-stain? 488 Fluorescent Phalloidin revealed no relationship between host actin filaments and symbiotic algal localization, although the host mitochondria were localized around symbiotic algae. Symbiotic algae from M. viridis could infect algae-free P. bursaria but could not support P. bursaria growth without feeding, whereas the original symbiotic algae of P. bursaria supported its growth without feeding. These data indicated the specificity of endosymbiotic algae relationships in M. viridis and P. bursaria.