Flow Cytometry as a Strategy to Study the Endosymbiosis of Algae in Paramecium bursaria

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1 2000 Wiley-Liss, Inc. Cytometry 41: (2000) Flow Cytometry as a Strategy to Study the Endosymbiosis of Algae in Paramecium bursaria Bogdan I. Gerashchenko, 1 Naohisa Nishihara, 1 Toshiko Ohara, 2 Hiroaki Tosuji, 2 Toshikazu Kosaka, 1 and Hiroshi Hosoya 1,3 * 1 Department of Biological Science, Faculty of Science, Hiroshima University, Higashi-Hiroshima, Japan 2 Department of Chemistry and Bioscience, Faculty of Science, Kagoshima University, Kagoshima, Japan 3 PRESTO, Japan Science and Technology Corporation (JST), Higashi-Hiroshima, Japan Received 13 April 2000; Revision Received 13 July 2000; Accepted 21 July 2000 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: , 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. Cytometry 41: , Wiley-Liss, Inc. Key terms: cloned symbiotic algae; Paramecium bursaria; symbiosis; light scattering; chlorophyll; fluorescence; flow cytometry; microscopy Symbiotic associations are excellent models for studying cell-to-cell interaction, mechanisms of immunity, and evolution of eukaryotic cells. Endosymbioses of freshwater hosts and algae, i.e., green ciliates, achieve stability through such complex phenomena as recognition, specificity, and regulation (1). Actually, green protozoa are widespread in different types of freshwater and seawater habitats. They, particularly green ciliates, were among the first organisms ever observed by light microscopy. The ciliate, Paramecium bursaria, exists as a green paramecium because each animal cell carries in its cytoplasm several hundred unicellular green algal cells that are morphologically similar to the genus, Chlorella. One of the remarkable and not well-understood peculiarities of this system is the steady state in number of algae per protozoan cell. Although P. bursaria and algae coexist symbiotically (2 5), these two partners can be separated, cultured independently, and recombined to reestablish a symbiotic relationship (6 11; Fig. 1). Recently, endosymbiotic algae isolated from P. bursaria were cloned and their infectivity to algae-free paramecia examined (10 12). In the present study, using flow cytometry, algal cell size and chlorophyll content were used to evaluate the symbiont interaction with a host. This is because both cell growth and chlorophyll metabolism are presumed to be readily affected by changes in environmental conditions. In addition, these two criteria are informative enough to discriminate algal populations on their ability to establish symbioses with host cells. For this purpose, we employed clones of symbiotic as well as free-living (non-symbiotic) algae. Grant sponsor: Japan Science and Technology Corporation (JST). *Correspondence to: Dr. Hiroshi Hosoya, Department of Biological Science, Graduate School of Science, Hiroshima University, Kagamiyama, Higashi-Hiroshima , Japan. hhosoya@sci.hiroshima-u.ac.jp

2 210 GERASHCHENKO ET AL. FIG. 1. Schematic diagram showing the reestablishment of symbiotic unit (1) after separation of the host (P. bursaria) and the symbiont (algae; 2 and 3) for independent culturing. Symbiotic algae are ingested by algae-free P. bursaria (4). After ingestion, only a few are still alive. They are enclosed in special perialgal vacuoles (5) and they expand (6) until the system reaches a steady state in symbiont number per host cell (7, 8). MATERIALS AND METHODS Experimental Organisms One strain of P. bursaria syngen 1 (KSK-103, mating type IV) was isolated from the Kawasa-kyo river in Fuchu City (Hiroshima, Japan) in It was maintained in lettuce infusion supplemented with Klebsiella pneumoniae as a food with a 12- h light and 12-h dark (LD) regimen at 23 1 C. Five strains of cloned symbiotic algae (SA-1, SA-3 and SA-3a, SA-4, and SA-9) were obtained from the following strains of P. bursaria singen 1: OK-312, I (Okuda-Oike pond, Higashi-Hiroshima City, Hiroshima, Japan, 1991); KSK-103, IV (Kawasa-kyo river, 1995); BS-4, II (a river in Bessiyama Village, Ehime, Japan, 1994); and OZ-3, III (Ozegawa river, Otake City, Hiroshima, Japan, 1994; respectively (12). Five strains of free-living algae (Chlorella vulgaris [c-27], Chlorella sorokiniana [c-43 and c-212], and Chlorella kessleri [c-208 and c-531]) were obtained in 1998 from the Institute of Applied Microbiology (IAM) culture collection at the University of Tokyo. Before the experiment, both symbiotic and free-living algae were transferred from agar plates containing CA medium (12) to the liquid CA medium and cultured under constant light (approximately 2,000 lux) at 24 C as described previously (12). At the stationary phase of growth (10-day culture), the algal cells in suspension were centrifuged at 720g for 5 min, washed three times in phosphate-buffered saline (PBS; 137 mm NaCl, 2.7 mm KCl, 1.5 mm KH 2 PO 4, 8.0 mm Na 2 HPO 4, ph 7.3), brought to a concentration of cells/ml, and further analyzed by flow cytometry. Reinfection In order to estimate the effects of symbiosis with respect to the cloned algal cells, one strain of symbiotic algae (SA-3) was employed to reinfect (procedure: 12) the algae-free P. bursaria (KSKw-103) obtained from the green paramecia (KSK-103) using the herbicide, paraquat (10). Both the algae and the algae-free paramecia were washed three times with lettuce infusion and mixed at a ratio of about algae:1 paramecium. One hundred paramecia were used for reinfection. The mixture was incubated for 24 h under the same conditions as the stock culture. After incubation, the paramecia were individually isolated from the reaction mixture, washed three times in depression slides, and transferred for culturing to the hanging drops of bacterized lettuce infusion (one paramecium per drop). The ratio of reinfection obtained in paramecia after 5-day culturing in the hanging drops reached 95%. Finally, reinfected ( re-green ) paramecia (KSKr-103) were expanded in lettuce infusion after they had been individually separated from noninfected cells. Sonication Conditions Both green (KSK-103) and re-green (KSKr-103) paramecia at the late logarithmic phase of growth (10-day culture) were washed three times with lettuce infusion followed by ultrasonic treatment (Branson Sonifier model 450, Branson Ultrasonics, Danbury, CT). Approximately 3,000 cells in 1 ml of lettuce infusion were sonicated in 1.5-ml plastic tubes surrounded by ice at the output control setting of 3 for 90 s. Under these conditions, paramecia were completely destroyed, but symbiotic algae were not affected in appearance. Prior to flow cytometry, the homogenates of paramecia were centrifuged at 720g for 5 min. The released algae were washed three times in PBS and brought to a concentration of cells/ml. The control experiment with algae (SA-3) that were not used for reinfection of the host cells, using various treatment conditions such as output control settings of 2 and 3 and a sonication time of 0, 45, 90, or 135 s, gave similar results in the algal numbers as well as in algal morphology and chlorophyll content according to the microscopic and flow cytometric observations (data not shown).

3 SYMBIOTIC ALGAE AND FLOW CYTOMETRY 211 FIG. 2. Two-parameter light scatter plots (FSC versus SSC) and histograms of chlorophyll fluorescence (FL3) of control algal cells (SA-3), which were not used for reinfection of paramecia (A,B) and algal cells (SA-3) after they were collected from reinfected paramecia (KSKr-103; C,D). E,F: Wild-type algae of green paramecia (KSK-103). Events with low light scatter (E) and fluorescence signal (F) may correspond to algae of the clone SA-3a shown in Figure 3A. Optical Measurements The algal samples were analyzed on a FACSCalibur flow cytometer (Becton-Dickinson Immunocytometry Systems, San Jose, CA) equipped with a 15-mW argon-ion laser (488 nm). The fluorescence of endogenous chlorophyll was measured in the red fluorescence channel (FL 3) through a 650-nm longpass filter with logarithmic amplification. The forward (FSC) and 90 side scatter (SSC) signals were collected in linear mode. Approximately events were measured. Analysis of the data was performed with CELLQuest software (Becton Dickinson). Cells were gated on the chlorophyll fluorescence signals to eliminate debris from the analysis. In addition, algae were characterized morphologically by bright field light microscopy (BH-2 microscope, Olympus, Japan). RESULTS Comparison of control SA-3 cells with algal cells collected from reinfected P. bursaria (KSKr-103) did not show a change in SSC, but showed a decrease in FSC (Fig.

4 FIG. 3. Strains of cloned symbiotic (A) and free-living (B) algae (10-day culture in liquid CA medium) characterized by flow cytometry and bright field light microscopy. Light micrographs of the cloned algae of each strain are shown with their dual-parameter histograms of FSC against SSC and histograms of chlorophyll fluorescence intensity (FL3). Scale bar 10 m.

5 SYMBIOTIC ALGAE AND FLOW CYTOMETRY 213 FIG. 3.CONT.

6 214 GERASHCHENKO ET AL. 2A versus 2C). There was also an alteration in the profile of the fluorescence histogram (bimodality with the tendency to trimodality) with a slight decrease of the relative fluorescence intensity (Fig. 2B versus 2D). The same optical parameters were utilized to monitor the wild type, i.e., noncloned, algae from green paramecia (KSK-103; Figs. 2E and 2F). Together, the appearance of events with lower light scatter and fluorescence signal, and the shape of the major peak of the fluorescence histogram, may reflect the fact that a population of algae naturally inhabiting the green paramecia (KSK-103) is composed of various cell clones. Figure 3 presents the flow cytometric and microscopic characterization of the cloned algae of different strains at the stationary phase of growth. The algae of both symbiotic and free-living strains were highly diverse in their morpho-optical parameters. Because the algal cells are coccoid, the FSC signals from algae in general were correlated with their size. These algal strains were further characterized by the dependency between cell sizes and fluorescence intensities of endogenous chlorophyll. Their distribution based on the mean magnitude of FSC signals is demonstrated in the relationship with mean chlorophyll fluorescence per cell (Fig. 4A). The majority of algal strains were distributed in accordance with a logarithmic type curve, except for three clones of symbiotic algae (SA-1, SA-3, and SA-9). Thus, algal cell size may not always correlate with chlorophyll fluorescence intensity. The same three clones were also distinguishable in the graph of clonal dispersion where the FSC parameter was substituted for the SSC parameter (Fig. 4B). DISCUSSION We have shown that reinfection of P. bursaria by cloned symbiotic algae (SA-3) results in changes in the algal light scatter and in autofluorescence characteristics (reduced forward light scatter and fluorescence signals). This may be due to the fact that the algae in the host decrease in size with concordant decrease of the relative chlorophyll content per cell. A number of growth and metabolic parameters in algae, including chlorophyll content, are affected by changes in environmental conditions (e.g., nutrition, light intensity, and temperature; 13). The bimodal character of the algal fluorescence histogram (Fig. 2D) may reflect the distinct redistribution of the chlorophyll content within the population of algae inhabited in the host. To determine whether or not this phenomenon is cell division cycle dependent, further investigations are needed. The same optical properties of algae (light scattering and fluorescence of endogenous chlorophyll) have been examined to evaluate the algal potential for the establishment of symbioses with P. bursaria. For this purpose, strains of free-living and symbiotic algae were screened together as a reference, because little is known about the perfect symbiotic relationships between free-living algae and P. bursaria. In general, they are not infective for P. bursaria, or they have low infectivity with no permanent symbioses. Although there are no principal differences in FIG. 4. Optical mapping of algal strains based on the relationship between chlorophyll fluorescence intensity per cell and magnitude of FSC (A) and SSC (B) signals. Their values are expressed as mean channel numbers. ( ), ( ), ( ), and (-) represent clones with high, intermediate, low, and no infectivity, respectively. morphology or cytology between symbiotic and free-living algae, they differ by some physiological properties (14). Symbiotic algae can excrete monosaccharides and disaccharides such as glucose, fructose, xylose, maltose, and trehalose, and this function is not induced by external factors. Moreover, they produce more oxygen at low light fluence rates than do free-living algae. The ability of algae to release sugar and the algal cell surface organization are the only criteria known to date by which P. bursaria selects its symbiotic partner (1,15,16). We demonstrate that by two-parametric analysis of mean channel numbers of the optical signals (FSC versus chlorophyll fluorescence and SSC versus chlorophyll fluorescence) from algae of

7 SYMBIOTIC ALGAE AND FLOW CYTOMETRY 215 different strains with different infectivity for P. bursaria (KSKw-103) an algal symbiotic capability is likely to be reliably assessed by the additional two criteria such as cell size/volume and chlorophyll content. The infectivities of various algal strains with respect to P. bursaria (KSKw- 103) were previously examined and expressed as a percentage of reinfected paramecia (KSKr-103) among the total number of paramecia taken for reinfection after a 20-day incubation of reaction mixture (our unpublished data). Algae capable of reinfecting % of host cells were designated as highly infective; 40 69% of host cells algae with intermediate infectivity; less than 40% of host cells algae with low infectivity. Algae that did not infect paramecia at all were designated as noninfective. Among the highly infective algal strains demonstrated in optical maps (Fig. 4), the clones of symbiotic algae such as SA-1, SA-3, and SA-9 were located separately from the major group of less infective and noninfective clones: SA-3a; SA-4; C. vulgaris (c-27); C. sorokiniana (c-43 and c-212); and C. kessleri (c-208 and c-531) with a logarithmic character of dispersion. Although the algae of SA-1, SA-3, and SA-9 clones have smaller sizes than the freeliving algae of clones such as C. sorokiniana (c-43) and C. kessleri (c-208 and c-531), they autofluoresce brighter except C. kessleri (c-208). At the same time, they are larger and brighter than SA-3a, SA-4, C. vulgaris (c-27), and C. sorokiniana (c-212). These findings prompted us to conclude that algae with smaller sizes/volumes with larger amounts of chlorophyll have the higher probability to reinfect a host with a subsequent stable symbiosis. In this regard, a factor such as chlorophyll content per cell volume is valuable for predicting favorable symbiotic relationships between P. bursaria and algae. Interestingly, the algae of other symbiotic clones, such as SA-3a and SA-4, which have smaller sizes and less autofluorescence intensities, are known to be noninfective. Moreover, the SA-4 clone showed no infectivity with respect to its stock host, BS-4 (our unpublished data). We propose that algae of these two clones, and possibly algae of other unknown symbiotic clones, which became smaller and chlorophyll deficient, became low or noninfective, were generated later and survived the evolution/selection process. Thus, optical mapping of algal strains by means of flow cytometry is an easy, quick, and reliable tool for monitoring chlorophyll content/cell volume state and assessing their symbiotic potential. ACKNOWLEDGMENTS We thank Y. Ishizaka for her helpful technical assistance. 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