Endosymbiosis-related changes in ultrastructure and chemical composition of Chlorella variabilis (Archaeplastida, Chlorophyta) cell wall in Paramecium bursaria (Ciliophora, Oligohymenophorea)

Chlorella variabilis , a symbiotic alga, is usually present in the cytoplasm of Paramecium bursaria , although it can be cultured in host-free conditions. Morphological and chemical properties of its cell wall were compared between its free-living and symbiotic states. Transmission electron microscopy (quick-freezing and freeze-substitution methods) revealed that the cell wall thickness of symbiotic C. variabilis was reduced to about half that of the free-living one. Chemical properties of the cell wall were examined by treatment with three ﬂuorescent reagents (calcoﬂuor white M2R, FITC-WGA, and FITC-LFA) having speciﬁc binding afﬁnities to different polysaccharides. When the algae were re-introduced into Paramecium host cells, calcoﬂuor ﬂuorescence intensity reduced by about 50%. Calcoﬂuor stains (cid:2) - d -glucopyranose polysaccharides such as cellulose, N-acetylglucosamine, sialic acid, and glycosaminoglycans. Because treatment with cellulase showed no effect on calcoﬂuor ﬂuorescence intensity, we consider that cellulose is not majorly responsible for the stainability of calcoﬂuor. Staining intensities of FITC-WGA and FITC-LFA were similar in the free-living and symbiotic conditions, suggesting that N-acetylglucosamine and sialic acid are also not responsible for the reduction in the stainability of calcoﬂuor associated with intracellular symbiosis. The amount of glycosaminoglycans on the cell wall may decrease in C. variabilis present in the cytoplasm of P. bursaria . is an the (http://


Introduction
Hundreds of Chlorella cells may be present in the cytoplasm of a single Paramecium bursaria. The host P. bursaria supplies nitrogen sources (Albers and Wiessner 1985) and CO 2 (Reisser 1980) to C. variabilis, and while living inside the host cells, C. variabilis seems to be able to avoid infection by Chlorella virus (Kawakami and Kawakami 1978). The symbiont, on the other hand, supplies maltose, a photosynthetic product, to the host (Ziesenisz et al. 1981). In addition, the host P. bursaria shows an enhanced tolerance to high temperature and UV due to the presence of the symbiotic Chlorella (Iwatsuki et al. 1998;Summerer et al. 2009). Thus, P. bursaria and C. variabilis are considered to be in a mutually beneficial symbiotic relationship. However, this symbiotic relationship is not an obligatory one; the symbiont can be artificially removed from the host (Reisser 1976 aposymbiotic white paramecia can grow in the same manner as ordinary green ones. Isolated C. variabilis can be cultured alone, and when white P. bursaria and isolated C. variabilis are mixed together, C. variabilis is ingested by white P. bursaria, and the symbiotic relationship resumes within a few days (Ziesenisz et al. 1981). From these observations, it has been considered that P. bursaria is in the very early stage of secondary symbiosis and is suitable as a model system to elucidate the mechanism of intracellular symbiosis. Despite various studies to date, however, the molecular basis involved in establishing and maintaining intracellular symbiosis in P. bursaria remains unclear. Many of the previous studies have focused on the fine structure and gene expression of host cells (Kodama et al. 2014;Song et al. 2017), but there have been only few approaches focusing on the symbiont. The only report related to this aspect showed that a component extracted from P. bursaria cells enhanced Chlorella's carbon fixation ability and activated photosynthesis (Kamako and Imamura 2006). Working from this perspective, this research aimed to analyze changes in the structure and composition of the cell wall of C. variabilis accompanying intracellular symbiosis in P. bursaria.

Material and Methods
Ciliate and algal cultures P. bursaria was initially collected from a pond in Kobe, Japan, and was cloned before use in this study. Aposymbiotic (white) P. bursaria was produced by mixing the cloned green P. bursaria with 0.1% (w/v) paraquat (methyl viologen: Sigma, M-2254) according to the method described by Tanaka et al. (2002). Both P. bursaria and aposymbiotic P. bursaria were cultured monoxenically with Chlorogonium capillatum (NIES-3374) as the sole food source (Omura et al. 2004). Symbiotic Chlorella cells were isolated by gently rupturing the cell membrane of P. bursaria with an ultrasonic disruptor (TOMY, UR-20P) for 1 min at room temperature. The Chlorella cells obtained were cultured on an agar plate containing C medium supplemented with 0.03% (w/v) Lserine (Kato and Imamura 2008) and were cloned using a single colony. The resulting strain (named Kb1, Fig. 1B) was cultured for more than 1 month in liquid C medium with L-serine and re-introduced into an aposymbiotic P. bursaria to yield a Kb1-bearing green P. bursaria, which was named as the Pb-Kb1 strain (Fig. 1A). The re-constructed Pb-Kb1 strain was cultured for more than one year before being used for experiments.

Transmission electron microscopy
Quick-freezing and freeze-substitution methods were employed for transmission electron microscopy because these methods proved to be superior to conventional chemical fixation for the preservation of the fine structures of the Chlorella cell wall (Song and Suzaki 2013;Song et al. 2017). Chlorella cells were harvested by low-speed centrifugation and re-suspended at a high cell density before cryofixation by impact-freezing onto a liquid nitrogen-cooled copper block using a freezing device (VFZ-1, Japan Vacuum Device, Ltd., Japan). The specimens were freeze-substituted in acetone containing 1% OsO 4 for 3 days. They were then brought back to room temperature and embedded in Spurr's resin (Polysciences, Inc., Warrington, PA). Ultrathin sections (100 nm) were cut with a diamond knife, stained with EM Stainer (Nisshin EM, Ltd., Tokyo, Japan) and Reynolds' lead citrate stain (Reynolds 1963), and observed using a transmission electron microscope (Hitachi H-7100, Hitachi Ltd., Tokyo, Japan).

Measurement of fluorescence intensity by confocal laser scanning microscopy
To investigate whether the chemical composition of Chlorella cell walls changes due to intracellular symbiosis to Paramecium, free and symbiotic Chlorella cells were stained with sugar-binding fluorescent reagents, and their staining intensities were compared.
Three sugar-binding reagents were used for fluorescent staining of the cell surface of Chlorella cells: calcofluor White M2R (Fluorescent Brightener, Sigma, St Louis, MO), FITClabeled WGA (EY Laboratories, San Mateo, CA, USA), and FITC-labeled LFA (EY Laboratories). The sugar compositions recognized by these reagents are different and are summarized in Table 1. First, Chlorella cells were centrifuged at 5000 rpm for 5 min at 20 • C, and the supernatant was discarded. Then, for calcofluor staining, an aqueous solution of the reagent (0.05%, w/v) was added to the specimen and stained for 5 min at 20 • C. For staining with WGA and LFA, Chlorella cells were mixed with 0.01% (w/v) of the reagents for 10 min at 20 • C. Thereafter, the samples were washed three times with distilled water, mixed with the same amount of anti-fading agent Aqua-Poly/Mount (Polysciences), and then placed on a glass slide and observed using a confocal laser scanning microscope (CLSM) (Olympus, FV-1000, Tokyo, Japan).
To examine whether cellulose is involved in calcofluor staining, an experiment was conducted using cellulase (cat. no. 07550-74, Nacalai Tesque Inc., Kyoto, Japan). Free-living and symbiotic Chlorella cells were treated with cellulase at 1%, 2%, or 5% (w/v) for 24 h at 20 • C. The specimens were then washed three times with distilled water and stained with calcofluor as described above. The excitation/emission wavelength combinations of the reagents used in CLSM are also listed in Table 1. The autofluorescence (ex: 405 nm, em: 630-730 nm) of Chlorella was used as a criterion for judging that the cells were alive and healthy.
Quantitative analysis of fluorescence intensity was performed using the image analysis software Image J (NIH) for  Calcofluor is a fluorescent dye that reacts with ␤-d-glucopyranose polysaccharide (Chen et al. 2007), including cellulose, chitin, sialic acid, and glycosaminoglycans (Bezares et al. 2008;Albani et al. 2000;Kapaun and Reisser 1995). WGA is a lectin that binds to N-acetylglucosamine, chitin, and sialic acid (Rodrigues et al. 2008). LFA is also a lectin that specifically reacts with sialic acid (Fischer and Brossmer 1995). fluorescence images taken by CLSM. First, the viability of Chlorella cells was evaluated based on the autofluorescence of chlorophyll. Living cells were selected, and fluorescence images of calcofluor-, WGA-, or LFA-stained cells were obtained. An area that covered a stained cell was designated, and the average brightness of the area was determined using Image J.

Identification of Kb1
The sequence of Kb1 (1263 bp) was obtained (GenBank Accession No., LC420163), which includes ITS1-5.8S-ITS2 rDNA and a group I intron at position L200. The ITS2 sequence of Kb1 was identical to several sequences of Chlorella variabilis including the authentic strain SAG 211-6 and the most famous strain ATCC 50258/CCAP 211/84 (NC64A). The sequence was separated from the other species (Fig. 2), therefore, Kb1 can be identified as C. variabilis. As a side note, the L200 introns have been found only from C. variabilis (Hoshina and Imamura 2009).

Thickness and fine structure of the cell wall of free-living and symbiotic C. variabilis
Free-living (cultured alone for >1 month) and symbiotic Chlorella living in P. bursaria were observed using a transmission electron microscope. Twenty-one individuals of free-living and symbiotic C. variabilis were randomly selected, and the thickness of the cell wall was measured at an arbitrary position of each cell. Because the outer surface of the cell wall showed a fluffy appearance ( Fig. 3B and C), the thickness of the cell wall was measured as the distance from the base of the fluffy structure to the innermost position of the cell wall. The thickness of the cell wall of the freeliving C. variabilis was 11-17 nm and that of the symbiotic C. variabilis was 6-10 nm (Fig. 3A).
The fluffy structure was observed on the surface of cell walls in both free-living and symbiotic C. variabilis cells. The fluffy filaments have a uniform length of about 15 nm.

Fig. 2.
Neighbor-joining tree based on ITS2 sequences. The tree was constructed via ClustalX2 (Larkin et al. 2007) program with the option of Exclude positions with gaps. The alignment was based on that of Heeg and Wolf (2015). Numbers at each node represent bootstrap probabilities of the NJ analysis; only values above 50% are shown.

Changes in carbohydrate composition in the cell wall
Fluorescence intensity of calcofluor-stained cells was measured on free-living and symbiotic C. variabilis cells. The cell wall was stained with calcofluor, which is known to react with sialic acid, chitin, cellulose, and glycosaminoglycans (Table 1). The cell wall in the endosymbiotic state showed weaker fluorescence as compared with that in the free-living state (Fig. 4), apparently indicating a decrease in a calcofluor-reacting carbohydrate as a result of symbiosis. Staining intensities of two types of lectins (LFA and WGA) were also examined to further characterize the molecular composition in the cell wall. These lectins also stained the cell wall well, but unlike calcofluor, no significant changes were detected between free-living and symbiotic cells (Fig. 5), indicating that chitin and sialic acid may not account for the reduction of calcofluor staining that accompanies endosymbiosis.
To examine the possible involvement of cellulose in calcofluor staining, free-living C. variabilis was treated with different concentrations of cellulase for 24 h. The cellulasetreated cells were then subjected to calcofluor staining and examined by CLSM. As shown in Fig. 5C, the fluorescence  intensity was not influenced by cellulase treatment, even at a very high concentration (5%, at which complete degradation of plant cell walls usually occurs). These results suggested that cellulose is not a major sugar component stained by calcofluor and that it may be glycosaminoglycans that are responsible for the reduction of calcofluor staining in the endosymbiotic C. variabilis cells.

Discussion
In this study, we compared the ultrastructure and chemical composition of the cell wall of C. variabilis in free-living and symbiotic states. To establish a stable coexistence within the cytoplasm of P. bursaria, Chlorella cells need to avoid lysosomal attacks by host cells (Dolan 1992). The symbiotic Chlorella within the host cell is enclosed by a membrane called the peri-algal vacuole (PV) membrane (Karakashian et al. 1968;Meier et al. 1984). Using a novel cryofixation technique, we have recently found that the PV membrane is closely attached to the tips of filamentous appendages on the outer surface of the cell wall of symbiotic C. variabilis strain Kb1 cells (Song and Suzaki 2013). We also reported that mitochondria of host cells frequently form intimate connections with the symbiont cell wall via the PV membrane (Song et al. 2017). These observations suggest the importance of the interaction between the Chlorella cell wall and the PV membrane in the formation and maintenance of the symbiotic relationship.
Observation by electron microscopy revealed that the cell wall of Chlorella became thinner in the symbiotic condition. In C. vulgaris, it has been reported that the cell wall thickness remains constant even when culture conditions are changed (Martínez et al. 1991). To the authors' knowledge, there are no reports stating that the thickness of the cell wall changes depending on environmental conditions. The decrease in the cell wall associated with symbiosis found in this study may be attributed to the result of the symbiotic interaction between the host and symbiont. Because the main function of the algal cell wall is to provide structural support and protection against mechanical stress, it is not necessary for endosymbiotic algal cells to retain their rigid cell walls in the endosymbiotic condition, and reduction of cell wall thickness in endosymbiosis may be a reasonable consequence.
In the present study, we showed that the staining intensity of calcofluor in the cell wall of C. variabilis changes owing to symbiosis. Because calcofluor is a fluorescent dye that reacts with ␤-d-glucopyranose polysaccharide (Chen et al. 2007), our results indicate that the amount of ␤-d-glucopyranose polysaccharide on Chlorella cell wall might have reduced in the symbiotic state. Therefore, we examined the amount In both LFA-and WGA-stained Chlorella cells, fluorescence intensities were not significantly different between free-living and symbiotic conditions. Cellulase treatment did not result in changes in calcofluor staining (C). Error bars show standard deviations, and Welch'st test was used for statistical analysis. The numbers in the bars represent sample numbers. of chitin, sialic acid, and glycosaminoglycans, which can be stained with calcofluor (Bezares et al. 2008;Albani et al. 2000;Kapaun and Reisser 1995) and which are known to be present in the Chlorella cell wall (Kapaun and Reisser 1995;Correll 1964). Because calcofluor stains cellulose well, we also assessed the possibility of the presence of cellulose in the Chlorella cell wall. As shown in Fig. 5, the experiment involving cellulase digestion demonstrated that calcofluor staining might not be attributed to the presence of cellulose. Although it is a conjecture based on the elimination method, our experiments demonstrated that glycosaminoglycans remained as the only possible substances considered to be the cause of reduction in calcofluor stainability among substances that can be stained by calcofluor. Glycosaminoglycans are polysaccharides composed of sugars to which sulfate groups are added, including chondroitin sulfate and hyaluronic acid. Karakashian and Rudzinska (1981) suggested that some polyanionic component present in the cell wall of Chlorella aids in preventing lysosomal attack to the PV membrane. Glycosaminoglycans are negatively-charged polyanionic polysaccharide chains, and it is therefore plausible that glycosaminoglycans in the Chlorella cell wall may pass through the PV membrane, thereby acting to avoid lysosomes from the PV membrane. To fully understand such an avoidance mechanism, molecular characterization of the PV membrane would be necessary. Takeda et al. (1998) reported that the sugar composition of the cell walls differs between symbiotic and free-living species of Chlorella: symbiotic species are rich in glucosamine, one of the component sugars of glycosaminoglycans. A glycoprotein on the cell wall of a symbiotic alga has been reported to be important for establishing symbiosis with sea anemone (Lin et al. 2000). When Legionella bacteria infects animal cells, it releases vesicles made of lipopolysaccharide inside the phagosome and inhibits fusion by lysosomes (Fernandez-Moreira et al. 2006). Based on these similarities and our results, cell wall polysaccharides are suggested to be important in facilitating intracellular symbiosis of C. variabilis in the cytoplasm of P. bursaria.