Flow cytometry as a strategy to study the endosymbiosis of algae in Paramecium bursaria

BACKGROUND: The stable symbiotic association between Paramecium bursaria and algae is of interest to study such mechanisms in biology as recognition, specificity, infection, and regulation. The combination of algae-free strains of P. bursaria, which have been recently established by treating their stocks of green paramecia with herbicide paraquat (Hosoya et al.: Zool Sci 12: 807-810, 1995), with the cloned symbiotic algae isolated from P. bursaria (Nishihara et al.: Protoplasma 203: 91-99, 1998), provides an excellent clue to gain fundamental understanding of these phenomena. METHODS: Flow cytometry and light microscopy have been employed to characterize the algal cells after they have been released from the paramecia by ultrasonic treatment. Algal optical properties such as light scattering and endogenous chlorophyll fluorescence intensity have been monitored for symbiotic and free-living strains, and strains at stages of interaction with a host. RESULTS: Neither algal morphology nor chlorophyll content has been found to be altered by sonication of green paramecia. This fact allows to interpret in adequate degree changes in the optical properties of symbiont that just has been released from the association with a host (decreased forward light scatter and chlorophyll fluorescence signals). Optical characterization of both symbiotic and free-living algal strains with respect to their ability to establish symbioses with P. bursaria showed that chlorophyll content per cell volume seems to be a valuable factor for predicting a favorable symbiotic relationship between P. bursaria and algae. CONCLUSIONS: Flow cytometry combined with algae-free paramecia and cloned symbiotic algae identifies algal populations that may be recognized by host cells for the establishment of symbioses.     

Green paramecia as an evolutionary winner of oxidative symbiosis: a hypothesis and supportive data

A single cell of the green paramecia (Paramecium bursaria) harbors several hundreds of endo-symbiotic Chlorella-like algae in its cytoplasm. Removal of algae from the host organism and re-association of ex-symbiotic host paramecia with ex-symbiotic algae can be experimentally demonstrated in the laboratory. However, the mechanism precisely governing the alga-protozoan association is not fully understood, and the origin of symbiosis in the evolutionary view has not been given. Here, we propose the possible biochemical models (models 1 and 2) explaining the co-evolution between Paramecium species and algal symbionts by pointing out that algal photosynthesis in the host paramecia plays a dual role providing the energy source and the risk of oxidative damage to the host. Model 1 lays stress on the correlation between the (re)greening ability of the paramecia and the tolerance to oxidative stress whereas model 2 emphasizes the cause of evolutionary selection leading to the emergence of Paramecium species tolerant against reactive oxygen species.     

Genetics of the relationship between the ciliate Paramecium bursaria and its symbiotic algae

Abstract. Paramecium bursaria , a freshwater protozoan, typically harbors hundreds of symbiotic algae ( Chlorella sp.) in its cytoplasm. The relationship between host paramecia and symbiotic algae is stable and mutually beneficial in natural environments. We recently collected an aposymbiotic strain of P. bursaria . Infection experiments revealed that the natural aposymbiotic strain (Ysa2) showed unstable symbiosis with Chlorella sp. The algae aggregated at the posterior region of the host, resulting in aposymbiotic cell production after cell division. Cross-breeding analyses were performed to determine the heritability of the aposymbiotic condition. In crosses of Ysa2 with symbiotic strains of P. bursaria , F1 progeny were able to form stable symbioses with Chlorella sp. However, unstable symbiosis, resembling Ysa2 infection, occurred in some F2 progeny of sibling crosses between symbiotic F1 clones. Infection experiments using aposymbiotic F2 cells showed that these F2 subclones have limited ability to reestablish the symbiosis. These results indicate that the maintenance of stable symbiosis is genetically controlled and heritable, and that Ysa2 is a mutant lacking the mechanisms to establish stable symbiosis with Chlorella sp.     

Arrest of cytoplasmic streaming induces algal proliferation in green paramecia

A green ciliate Paramecium bursaria, bearing several hundreds of endosymbiotic algae, demonstrates rotational microtubule-based cytoplasmic streaming, in which cytoplasmic granules and endosymbiotic algae flow in a constant direction. However, its physiological significance is still unknown. We investigated physiological roles of cytoplasmic streaming in P. bursaria through host cell cycle using video-microscopy. Here, we found that cytoplasmic streaming was arrested in dividing green paramecia and the endosymbiotic algae proliferated only during the arrest of cytoplasmic streaming. Interestingly, arrest of cytoplasmic streaming with pressure or a microtubule drug also induced proliferation of endosymbiotic algae independently of host cell cycle. Thus, cytoplasmic streaming may control the algal proliferation in P. bursaria. Furthermore, confocal microscopic observation revealed that a division septum was formed in the constricted area of a dividing paramecium, producing arrest of cytoplasmic streaming. This is a first report to suggest that cytoplasmic streaming controls proliferation of eukaryotic cells.     

Effect of symbiotic algae on the photoaccumulation capacity of cells of the ciliate Paramecium bursaria

The character of the effect produced by symbiotic algae on photodependent behavior of their host ciliates, Paramecium bursaria, was determined. Partially alga-freed paramecia showed a reliable increase in the rates of photoaccumulation. The photoaccumulation rate gradually decreased with a further decrease in the number of zoochlorellae. Once the chlorophyll content fell down to 20-25 mu/l, the ciliates lost their capacity for photoaccululation. A mathematical model of photoaccumulation has been constructed.     

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.     

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.     

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.     

Flow cytometric studies of the host-regulated cell cycle in algae symbiotic with green paramecium

Summary. Paramecium bursaria (green paramecium) possesses endosymbiotically growing chlorella-like green algae. An aposymbiotic cell line of P. bursaria (MBw-1) was prepared from the green MB-1 strain with the herbicide paraquat. The SA-2 clone of symbiotic algae was employed to reinfect MBw-1 cells and thus a regreened cell line (MBr-1) was obtained. The regreened paramecia were used to study the impact of the hosts growth status on the life cycle of the symbiotic algae. Firstly, the relationship between the timing of algal propagation and the host cell division was investigated by counting the algal cells in single host cells during and after the host cell division and also in the stationary phase. Secondly, the changes in the endogenous chlorophyll level, DNA content, and cell size in the symbiotic algae were monitored by flow cytometry and fluorescence microscopy. The number of algae was shown to be doubled prior to or during the host cell division and the algal population in the two daughter cells is maintained at constant level until the host cell cycle reenters the cytokinesis, suggesting that algal propagation and cell cycle are dependent on the hosts cell cycle. During the hosts stationary growth, unicellular algal vegetatives with low chlorophyll content were dominant. In contrast, complexes of algal cells called sporangia (containing 1–4 autospores) were present in the logarithmically growing hosts, indicating that algal cell division leading to the formation of sporangia with multiple autospores is active in the dividing paramecia.Correspondence and reprints: Graduate School of Environmental Engineering, University of Kitakyushu, 1-1 Hibikino, Wakamatsu-ku, 808-0135 Kitakyushu, Japan.     

Brandtia ciliaticola gen. et sp. nov. (Chlorellaceae,Trebouxiophyceae) a common symbiotic green coccoid of various ciliate species

Many freshwater protists harbor unicellular green algae within their cells and these host‐symbiont relationships slowly are becoming better understood. Recently, we reported that several ciliate species shared a single species of symbiotic algae. Nonetheless, the algae from different host ciliates were each distinguishable by their different genotypes, and these host‐algal genotype combinations remained unchanged throughout a 15‐month period of sampling from natural populations. The same algal species had been reported as the shared symbiont of several ciliates from a remote lake. Consequently, this alga appears to play a key role in ciliate‐algae symbioses. In the present study, we successfully isolated the algae from ciliate cells and established unialgal cultures. This species is herein named Brandtia ciliaticola gen. et sp. nov. and has typical ‘Chlorella‐like’ morphology, being a spherical autosporic coccoid with a single chloroplast containing a pyrenoid. The alga belongs to the Chlorella‐clade in Chlorellaceae (Trebouxiophyceae), but it is not strongly connected to any of the other genera in this group. In addition to this phylogenetic distinctiveness, a unique compensatory base change in the SSU rRNA gene is decisive in distinguishing this genus. Sequences of SSU‐ITS (internal transcribed spacer) rDNA for each isolate were compared to those obtained previously from the same host ciliate. Consistent algal genotypes were recovered from each host, which strongly suggests that B. ciliaticola has established a persistent symbiosis in each ciliate species.     

Infection of algae-free Climacostomum virens with symbiotic Chlorella sp. isolated from algae-containing C. virens

The ciliate Climacostomum virens normally contains algae as symbionts in its cytoplasm and retains them over many generations. An aposymbiotic strain of C. virens which cannot re-establish a new symbiotic association by ingestion of algae derived from green Climacostomum was recently isolated in our laboratory. Results of infection experiments showed that all newly ingested, potentially symbiotic algae were digested in food vacuoles. To clarify whether these ciliates have completely lost their ability to sustain symbiosis with algae or whether this ability can eventually be re-established, infection experiments were performed using a microinjection technique. We have achieved successful infection of algae-free Climacostomum using this method. The endosymbiont population was established in ciliates from as few as 3-5 injected algae, which have retained an intact perialgal vacuole membrane around them. To our knowledge, this is the first evidence of successful infection of aposymbiotic ciliates with algae by microinjection.     

Cycloheximide induces synchronous swelling of perialgal vacuoles enclosing symbiotic Chlorella vulgaris and digestion of the algae in the ciliate Paramecium bursaria

Cycloheximide is known to inhibit preferentially protein synthesis of symbiotic Chlorella of the ciliate Paramecium bursaria, but to hardly host protein synthesis. Treatment of algae-bearing Paramecium cells with cycloheximide induces synchronous swelling of all perialgal vacuoles that are localized immediately beneath the host's cell membrane. In this study, the space between the symbiotic algal cell wall and the perialgal vacuole membrane widened to about 25 times its normal width 24 h after treatment with cycloheximide. Then, the vacuoles detached from beneath the host's cell membrane, were condensed and stained with Gomori's solution, and the algae in the vacuoles were digested. Although this phenomenon is induced only under a fluorescent light condition, and not under a constant dark condition, this phenomenon was not induced in paramecia treated with cycloheximide in the light in the presence of the photosynthesis inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea. These results indicate that algal proteins synthesized in the presence of algal photosynthesis serve some important function to prevent expansion of the perialgal vacuole and to maintain the ability of the perialgal vacuole membrane to protect itself from host lysosomal fusion.     

An experimental test of the symbiosis specificity between the ciliate Paramecium bursaria and strains of the unicellular green alga Chlorella

The ciliate Paramecium bursaria living in mutualistic relationship with the unicellular green alga Chlorella is known to be easily infected by various potential symbionts/parasites such as bacteria, yeasts and other algae. Permanent symbiosis, however, seems to be restricted to Chlorella taxa. To test the specificity of this association, we designed infection experiments with two aposymbiotic P. bursaria strains and Chlorella symbionts isolated from four Paramecium strains, seven other ciliate hosts and two Hydra strains, as well as three free-living Chlorella species. Paramecium bursaria established stable symbioses with all tested Chlorella symbionts of ciliates, but never with symbiotic Chlorella of Hydra viridissima or with free-living Chlorella. Furthermore, we tested the infection specificity of P. bursaria with a 1:1:1 mixture of three compatible Chlorella strains, including the native symbiont, and then identified the strain of the newly established symbiosis by sequencing the internal transcribed spacer region 1 of the 18S rRNA gene. The results indicated that P. bursaria established symbiosis with its native symbiont. We conclude that despite clear preferences for their native Chlorella, the host-symbiont relationship in P. bursaria is flexible.     

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.     

Endosymbionts in paramecium

Paramecium species are extremely valuable organisms to enable experiments for the reestablishment of endosymbiosis. This is investigated in two different systems, the first with Paramecium caudatum and the endonuclear symbiotic bacterium Holospora species. Although most endosymbiotic bacteria cannot grow outside the host cell as a result of their reduced genome size, Holospora species can maintain their infectivity for a limited time. We found that an 89-kDa periplasmic protein has an important function for Holospora's invasion into the target nucleus, and that Holospora alters the host gene expression; the host thereby acquires resistance against various stresses. The second system is the symbiosis between P. bursaria and symbiotic Chlorella. Alga-free P. bursaria and the algae retain the ability to grow without a partner. Consequently, endosymbiosis between the aposymbiotic host cells and the symbiotic algae can be reestablished easily by mixing them. We now found four checkpoints for the reestablishment of the endosymbiosis between P. bursaria and the algae. The findings in the two systems provide excellent opportunities for us to elucidate not only infection processes but also to assess the associations leading to eukaryotic cell evolution. This paper summarizes recent progresses on reestablishment of the primary and the secondary endosymbiosis in Paramecium.     

Comparison of two aposymbiotic ciliate clones of Climacostomum virens by their ability for reinfection

The ability of two aposymbiotic (algae-free) subclones of the same green clone of C. virens to establish a stable symbiotic association with Chlorella sp. has been studied by light and electron microscopy. Alga-free subclone No. 1 was obtained from the original green clone by a long-term cultivation in darkness, while subclone No. 2 originated from one cell that spontaneously lost the algae and was found among normal green cells during daily inspection. For infection, algae isolated from ciliates with chlorellae of parental clone of C. virens were used. 5-10 minutes after feeding with Chlorella, specimens of both subclones show numerous algae mostly inside food vacuoles, but some rare algae (3-4 per cell) may occur in individual perialgal vacuoles. Later on, the number of symbiotic chlorellae in ciliates of subclone No. 1 increased, and a stable symbiotic association was reestablished. Unlike, in specimens of subclone No. 2 all newly ingested algae were seen digested within food vacuoles. Within 24-28 h all the ciliates investigated appeared free of algae. However, obviously stable symbiotic ciliate-algae systems in this subclone were obtained after improving the microinjection technique. Injection of algae into alga-free ciliates resulted in maintenance of intact chlorellae in these ciliates. The algae were seen to be located individually within perialgal vacuoles, being presumably protected against host lytic enzyme attack. The endosymbiont population in ciliates was established from as many as 3-5 originally injected algae. The number of symbiotic chlorellae increased steadily reaching the value equal to that in the parental clone 28-30 days after the start of experiment.     

Phylogenetic placement of "zoochlorellae" (Chlorophyta), algal symbiont of the temperate sea anemone Anthopleura elegantissima

At northern latitudes the sea anemones Anthopleura elegantissima and its congener A. xanthogrammica contain unidentified green chlorophytes (zoochlorellae) in addition to dinophytes belonging to the genus Symbiodinium. This dual algal symbiosis, involving members of distinct algal phyla in one host, has been extensively studied from the perspective of the ecological and energetic consequences of hosting one symbiotic type over the other. However, the identity of the green algal symbiont has remained elusive. We determined the phylogenetic position of the marine zoochlorellae inhabiting A. elegantissima by comparing sequence data from two cellular compartments, the nuclear 18S ribosomal RNA gene region and the plastid-encoded rbcL gene. The results support the inclusion of these zoochlorellae in a clade of green algae that form symbioses with animal (Anthopleura elegantissima), fungal (the lichen genus Nephroma), and seed plant (Ginkgo) partners. This clade is distinct from the Chlorella symbionts of Hydra. The phylogenetic diversity of algal hosts observed in this clade indicates a predisposition for this group of algae to participate in symbioses. An integrative approach to the study of these algae, both within the host and in culture, should yield important clues about how algae become symbionts in other organisms.     

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.     

Behavior of a Virus in a Symbiotic System, Paramecium bursaria—Zoochlorella

SYNOPSIS In a culture system of Paramecium bursaria , virus particles were found in large number. The particle was able to infect and multiply in certain cells of the zoochlorella, an intracellular symbiotic alga of P. bursaria. The infective particle, designated as zoochlorella cell virus (ZCV), was icosahedral and 120–180 nm in edge to edge diameter. The ZCV particle was found to differ from any of the already established viruses attacking the green and the blue-green algae. Within the system where P. bursaria cells were growing, ZCV particles were detected in the depression of the pellicle, among the cilia growing in the cytopharynx, and in the food vacuole of P. bursaria. ZCV particles were infective only for the zoochlorella cells which were recently released from the cytoplasm of P. bursaria. The multiplication process of ZCV comprised the adsorption of the particle to the cell wall of the zoochlorella, the penetration of nucleic acid into the host cell interior, the replication of viral constituents, the maturation of viral particles and their final release by the burst of the zoochlorella cell. ZCV particles appeared only in the cytoplasmic region of the zoochlorella cell in which many ribosomes were distributed. A possible ecosystem among the 3 members consisting of P. bursaria , zoochlorella and ZCV is discussed.     

Growth kinetics of algal populations exsymbiotic from Paramecium bursaria by flow cytometry measurements.

BACKGROUND: The ciliate Paramecium bursaria normally exists as a green paramecium system because each animal cell carries several hundred, unicellular, green, algal cells in its cytoplasm. One of the remarkable and poorly understood pecularities of this system is the steady state in the number of algae per protozoan cell. A major point in the study of mechanisms governing the persistence of symbiont numbers is adequate understanding of the algal life cycle. METHODS: Asynchronously growing cell populations of several algal strains (SA-1, SA-3, and SA-9) exsymbiotic from P. bursaria were characterized by flow cytometry. Algal endogenous chlorophyll and DNA contents were monitored to analyze cell growth kinetics at logarithmic and stationary culture phases. Cell sorting visualized the morphology of algae corresponding to the hyperhaploid (2C and 4C) DNA peaks. RESULTS: Cell-division cycle-dependent changes in chlorophyll and DNA content distributions were most dramatic in logarithmically growing algal populations (an increase in the number of S-phase cells and cells with more chlorophyll), which are thought to be associated with accelerated DNA and chlorophyll metabolism in log-phase algal cultures. Upon reaching the stationary phase of growth, algal populations distinctly showed, in addition to one haploid (1C) DNA peak, two hyperhaploid peaks (2C and 4C) corresponding mainly to cells with two and four nuclei, respectively. CONCLUSIONS: Growth characteristics of algae exsymbiotic from P. bursaria monitored by flow cytometry provide valuable information for the analysis of the algal life cycle, which is important for understanding the regulation mechanisms of symbiont numbers.