Biol. Bull. 207: 80-86. (October 2004)
© 2004 Marine Biological Laboratory
Octocoral Chemical Signaling Selects and Controls Dinoflagellate Symbionts
Kazuhiko Koike1,
Mitsuru Jimbo1,
Ryuichi Sakai1,
Masami Kaeriyama1,
Koji Muramoto2,
Takehiko Ogata1,
Tadashi Maruyama3,
and
Hisao Kamiya1,*
1 School of Fisheries Sciences, Kitasato University, Sanriku, Ofunato, Iwate, 022-0101, Japan
2 Graduate School of Life Science, Tohoku University, Sendai, Miyagi, 981-8555, Japan
3 Marine Biotechnology Institute, Heita, Kamaishi, Iwate, 026-0001, Japan
* To whom correspondence should be addressed. E-mail: h.kamiya{at}kitasato-u.ac.jp
Symbioses between zooxanthellae (Symbiodinium spp.) and marine invertebrates, including corals, are common in shallow marine environments. The zooxanthellae contribute to host nutrition by translocating photosynthetic products and enabling them to effloresce in oligotrophic conditions. Coral mainly acquire Symbiodinium spp. by capturing free-swimming cells from the environment (1). Cultured Symbiodinium cells show a diel growth cycle with alternation between motile and non-motile cell stages once a day (2,3), and the cell divides only during the latter stage (2). When associated with a host, however, cells are arrested in a non-motile stage while healthy cell division is maintained (4). We deduced that host-directed and chemical-based mechanisms are responsible for this phenomenon since SLL-2, a lectin that binds to carbohydrate chains with a D-galactosyl moiety, produced by the octocoral Sinularia lochmodes, is localized on the cell surface of the Symbiodinium harbored in the host (5). Here we describe SLL-2 as the key chemical factor for arresting Symbiodinium in the cell-dividing, non-motile stage, while some nonsymbiotic microalgae were even destroyed by SLL-2.
Symbiotic associations between photosynthetic dinoflagellates (Symbiodinium spp.) and invertebrate hosts such as corals are crucial for the survival of the host animals since Symbiodinium supplies organic compounds to them and enables them to prosper in the oligotrophic environment that is especially common in tropical and subtropical regions. As in a case of "coral bleaching," however, evacuation of the symbiotic algae leads to the death of the hosts. Thus the host animals must acquire and properly maintain the algae for their survival.
The sexual progeny of host animals can acquire Symbiodinium in two ways: vertical transmission (acquisition by maternal inheritance) and horizontal transmission (acquisition from the environment by either larval or adult stages) (1). The latter mode, which dominates (
85%) in cnidarians (6), may allow the animals to acquire Symbiodinium cells suited to the environment where they have settled, but how these animals select and acquire Symbiodinium is not clear. For the initial infection of Symbiodinium, the two main mechanisms that have been suggested are (a) chemotactic attraction of motile Symbiodinium to the host’s mouth and gastric cavity, then subsequent transmission to the gastrodermal cells (7,8) and (b) acquisition of Symbiodinium cells that have been ingested by the hosts while feeding (8,9). Regardless of the mechanisms, Symbiodinium must at some point be selected, from other nonsymbiotic microalgae or other materials that corals have ingested, by host-directed mechanisms. In the case of Cassiopeia xamachana, Symbiodinium was phagocytosed by the endodermal cells, and only the cells capturing live Symbiodinium were able to escape digestion by avoiding lysosomal fusion (10). Moreover, some "host factors" such as a cell adhesion protein (sym32) (11,12) or the macerated tissue (6) of a sea anemone (Anthopleura elegantissima) were suspected to be involved in symbiotic events. The physiological actions of these host factors on the algae were not understood, however.
We previously found that lectin (SLL-2) isolated from the octocoral Sinularia lochmodes was localized densely on the surface of the Symbiodinium cells released from the host coral (5). Lectins can recognize specific carbohydrate structures of glycoproteins on the cell surface, and some of them are proposed to mediate interactions between hosts and symbionts. For instance, in the case of rhizobia-legume symbiosis, a number of results support the hypothesis that lectins mediate development of a symbiotic relationship between the bacteria and the plant (13). There are some examples of putative interactions between invertebrate hosts and symbiotic bacteria or algae mediated by lectin from the invertebrate (14,15). We therefore hypothesize that SLL-2 also plays a role in selecting Symbiodinium and possibly in stabilizing the symbiotic relationship. This hypothesis was tested herein by assessing the effect of purified SLL-2 on the diel cycle or cell morphology and cell division of symbiotic and nonsymbiotic microalgal cultures.
For symbiotic microalgae models, the Symbiodinium strains P083-2, JCUCS-1, and CS-156 were used. They were all in well-synchronized regular diel alternation between motile and non-motile (coccoid) forms, and more than 70% of the cells were in the motile form during the observation period (9–10 AM). The additions of 10 or 100 µg ml–1 of SLL-2 to the cultures of actively swimming Symbiodinium affected both the motility and the growth of the algae. Within 1 h after the addition of the lectin (100 µg ml–1), the cells aggregated and formed clumps. For all strains, the number of motile cells dramatically decreased at day 1 (24 h after the addition; Fig. 1a, b, c), and they had begun to transform into stationary coccoid forms (Fig. 2b). Typically, in the presence of 100 µg ml–1 protein, nearly 100% of CS-156 and P083-2 cells and 80% to 90% of JCUCS-1 cells were arrested at the coccoid form after day 3; this form persisted throughout the experimental period (one week; Fig. 1a, b, c), and dividing cells were frequently observed (arrows in Fig. 2b). The effect of SLL-2 on cell motility seemed to be concentration-dependent, as the lower concentration (10 µg ml–1) did not affect the motility of JCUCS-1 (Fig. 1b). Following the addition of 10 µg ml–1 of SLL-2 to CS-156, 65% of the cells were transformed to the coccoid form at day 1, but the effect was reversible since 50%–70% of the cells were in the motile form on day 2 and thereafter (Fig. 1a). Strain P083-2 was the most sensitive of the Symbiodinium strains tested; nearly 100% of the cells were transformed to the stationary coccoid form within 1 day after the treatment, even at 10 µg ml–1 of SLL-2 (Fig. 1c).
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Figure 1. The percentage of motile-form cells (a–c) and growth (d–f) of three Symbiodinium strains (CS-156, JCUCS-1, and P083-2) in the presence of two concentrations of the carbohydrate-binding lectin SLL-2. Circles and triangles represent 100 and 10 µg ml–1 of SLL-2, respectively; squares are the control. Each data point represents the percentage of motile-form cells out of the total cells counted (>400) for each observation period (a–c). Cell growth was monitored by in vivo chlorophyll a fluorescence measurement (d–f). All data are given as the means ± standard deviation of triplicate wells. Symbiodinium strains P083-2 and JCUCS-1 were from culture stocks maintained at the Marine Biotechnology Institute (MBI), and CS-156 was obtained from the Commonwealth Scientific & Industrial Research Organization (CSIRO). The culture was spread on agar plates (IMK liquid medium, Wako pure chemical + 1.2 % agar) with a glass rod, then the Symbiodinium colonies, formed apart from bacterial colonies, were picked up with sterilized needle and transferred to sterilized liquid IMK medium. They were initially diluted (20,000 cells ml–1) with the medium and transferred to 24-well flat-bottom plates (ultra-low protein binding; 3473, Costar). Following acclimation for 24 h (25 °C, 30 µmol photon m–2 s–1, 12:12 light:dark cycle), Symbiodinium cells were confirmed to be in a diel motile phase (more than 70%) prior to SLL-2 addition. A solution (500 µl) of SLL-2 (4) (in 8 mM Tris-HCl prepared with 60% seawater, sterilized by 0.2 µm filtration, pH 7.6) was added to the culture to provide a final concentration of SLL-2 of 100 or 10 µg ml–1. Control reactions were prepared by mixing equal volumes of the same seawater and buffer as above but in the absence of SLL-2. The swimming behavior and cell forms were observed under an inverted microscope (IX70, Olympus) daily between 9 and 10 AM (the period when the Symbiodinium strains used have the highest proportion of cells in the diel motile phase) for 6–7 days, starting right after the addition of SLL-2. Live images under magnifications of x75 and x300 were stored on a digital videotape recorder. The number of motile cells and non-motile cells (a total of more than 400 cells) in more than six observation fields (x75) of the images were counted. Cell growth in the wells was estimated by daily monitoring of in vivo chlorophyll a fluorescence (EX 485 ± 40 nm, EM 645 ± 40 nm) using a microplate fluorescence reader (FL600, Bio-tek). The first observation (day 0) was made within 1 h after the start of treatment. Note that upon the addition of both lectin (SLL-2) and control solutions, the shock of the treatment produced an initial decrease in the percentage of motile cells.
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Figure 2. Light micrographs from the video-captured images of Symbiodinium sp. (strain CS-156) after SLL-2 treatment and in control wells. (a) Actively swimming CS-156 cells are in rapid motion and are blurred on this image (control). (b) CS-156 with SLL-2 treatment (day 3, 100 µg ml–1) shows only coccoid-form cells and numerous dividing cells (arrows).
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At these concentrations (10 or 100 µg ml–1), the growth of CS-156 and JCUCS-1 (Fig. 1d, e)—as indicated by the increase of the in vivo chlorophyll a fluorescence—was not affected, whereas that of P083-2 gradually became inhibited, even at the lower concentration (Fig. 1f). At both 10 and 100 µg ml–1 SLL-2, growth slowed on day 4 relative to the control, and significantly (P < 0.05; Student’s t test) smaller biomasses (ca. 50% and 60% of control, respectively) were observed at day 6. The effects of SLL-2 on the physiology of the Symbiodinium cells were completely inhibited in the presence of an inhibitor; i.e., CS-156 cells were not arrested in the coccoid form when 0.2 M of melibiose, the most effective inhibitor of the hemagglutination of SLL-2 (5), was added to the culture. It should be noted that the other inhibitors of SLL-2 showed some toxicity to the culture at the effective concentrations. This result suggested that the property of the lectin to bind to the D-galactosyl moiety in the carbohydrate chain is required for its activity. Interestingly, however, the addition of a D-galactose-binding lectin from Arachis hypogaea (peanut) to three Symbiodinium strains did not show any coccoid-arresting activity even at higher concentrations (300 µg ml–1). Thus, the carbohydrate-binding property alone did not explain the unique actions of SLL-2 on the algal cells.
Similarly, we tested the effects of the lectin on three nonsymbiotic dinoflagellates (Alexandrium minutum, Gymnodinium catenatum, and Prorocentrum micans) and on a non-dinoflagellate control (the chlorophyte Tetraselmis sp.) (Fig. 3). The responses of these nonsymbiotic microalgae to SLL-2 differed from those observed for Symbiodinium. The addition of SLL-2 had no significant effect on either the motility or growth of A. minutum (Fig. 3a, e). The addition of SLL-2 (100 µg ml–1) to cultures of G. catenatum and P. micans resulted in cell death after the cells inflated and burst within 24 h (Fig. 3b, c; Fig. 4b, d). Even at 10 µg ml–1, a similar significant effect was observed on G. catenatum, but not on P. micans. Cells of Tetraselmis sp., the chlorophyte control, aggregated and formed cell clumps (Fig. 4f) with complete loss of cell motility and significant inhibition of growth after day 4, even at 10 µg ml–1 (ca. 60% inhibition on day 4; Fig. 3d, h).
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Figure 3. The percentage of motile cells (a–d) and the growth (e–h) of three nonsymbiotic dinoflagellates (Alexandrium minutum, Gymnodinium catenatum, and Prorocentrum micans) and a non-dinoflagellate chlorophyte (Tetraselmis sp.) in the presence of SLL-2. All experimental procedures were the same as those for Symbiodinium (Fig. 1), except for the initial cell concentrations: 2,000 cells ml–1 for the dinoflagellates and 20,000 cells ml–1 for Tetraselmis sp. Both A. minutum (Cu-AM1) and Tetraselmis sp. (Cu-Ch) were isolated and identified by Dr. L. Thaithaworn, Department of Marine Science, Chulalongkorn University, Thailand; G. catenatum (GC-27-1) and P. micans (P-A4) were isolated and identified by Dr. S. Sakamoto at the National Research Institute of Fisheries and Environment of Inland Sea, Japan. All the strains except Tetraselmis sp. and P. micans are axenic, and even in the nonaxenic cultures, no bacterial growth was apparent during the experiments.
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Figure 4. Light micrographs from the video-captured images of microalgae after SLL-2 treatment and in control wells. (a) Gymnodinium catenatum forming cell chains (control). (b) G. catenatum 24 h after SLL-2 (100 µg ml–1) treatment showing a swollen cell (an arrow) and debris from destroyed cells (arrowheads). (c) Actively swimming Prorocentrum micans (control). (d) P. micans 24 h after SLL-2 (100 µg ml–1) treatment showing cytoplasm ejected from the theca (arrows) and numerous empty thecae (arrowheads). (e) Tetraselmis sp. cells are in rapid motion and are blurred on this image (control). (f) Tetraselmis cells forming cell clumps without motility.
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In microalgae treated with SLL-2, immunochemical staining using the anti-SLL-2 antibody showed that the SLL-2 was localized on the surfaces of all Symbiodinium strains and other microalgae tested (Fig. 5). Although the cells of G. catenatum and P. micans burst, their debris were also clearly stained (Fig. 5c, d). These results indicate that the lectin binds to sites, probably to glycoproteins on the microalgal cell surface, in its mode of action.
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Figure 5. Fluorescent micrographs of immunostained cells at day 6 after treatment (100 µg ml–1). Binding of anti-SLL-2 antibody is shown by green fluorescence; red fluorescence indicates the presence of chlorophyll a in the plastids. (a) Symbiodinium sp. (CS-156); (b) Alexandrium minutum (note: cell is not destroyed.); (c) destroyed cell of Gymnodinium catenatum with stained debris; and (d) burst cell of Prorocentrum micans. Immunostaining was conducted as follows: cells were harvested and washed several times by repeated centrifugation (1000 x g, 10 min) and resuspended in filtered seawater to remove unbound SLL-2. Then the cells were fixed in 4% paraformaldehyde in cacodylate buffer (0.1 M cacodylate/0.1 M sucrose, pH 7.6) for 4 h at 4 °C, and washed with the same buffer three times. Cells were then blocked with 2% bovine serum albumin (BSA) for 1 h, reacted with anti-SLL-2 antibody (5) diluted 1000 times with 20 mM Tris-HCl-buffer (pH 7.4) containing 0.2% BSA for 2 h at room temperature, and washed three times. Then the cells that reacted to the primary antibody were stained with fluorescein-isothiocyanate–labeled anti-rabbit IgG goat antibody (Funakoshi) diluted 1000 times with 20 mM Tris HCl buffer for 2 h at room temperature, and then washed three times with the same buffer. Observation was conducted under a fluorescent microscope (EX 455–490 nm, BS 500 nm, BH2-RFC, Olympus). The same immunostaining procedures were used on the cells in the control wells (without adding SLL-2), but no antibody bindings were observed.
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The most intriguing aspect of these results is that the lectin affected the physiological condition of symbiotic algae, while either causing the deterioration of or having no effect on nonsymbiotic microalgal strains. These effects were distinct at the concentration level of 100 µg ml–1. On the basis of the hemagglutination activity of the coelomic fluid obtained from fresh coral, we estimated the concentration of SLL-2 to be about 300 µg g–1 of the wet weight of S. lochmodes. We also found that the effective concentration of SLL-2 saturated at 100 µg ml–1 and the addition of 300 µg ml–1 did not show any toxicity to the Symbiodinium cells. The results of this study provided some experimental evidence for the as-yet-unidentified ecological interaction between the microalgae and the coral. In our model, lectin stored in the nematocyst of the coral (5) is probably released when algal cells are introduced to the gastric cavity and the microalgae are then chemically selected by their varying responses to lectin; that is, the lectin is toxic to some nonsymbiotic algae—as in the cases of G. catenatum, P. micans, and Tetraselmis sp.—but others—as represented by A. minutum in this study—can sometimes escape unaffected. On the other hand, the lectin changes the physiology of Symbiodinium, creating conditions that may be favorable to the symbiosis between the algae and the coral.
Interestingly, our observation of the discrete effects of SLL-2 on different types of algae suggests that further selection may occur among the different clades of Symbiodinium. In gorgonians, infection by several clades of Symbiodinium occurs at the onset of symbiosis; however, the population eventually converges into a single clade of algae (16). The strains of Symbiodinium (P083-2, JCUCS-1, and CS-156) used in the present experiments belong to different clades (A, B, and F, respectively) (17,18). The actual Symbiodinium harbored in the coral could not be used due to the difficulty of culturing it; clade C, a ribotype of symbionts in S. lochmodes determined on the basis of its chloroplast large subunit (23S) rDNA sequence (DDBJ Accession No. AB159231) (19), was not available. For these reasons, our experimental model is somewhat indirect for assessing the chemical recognition that actually occurs between the host and the algae. However, the facts that the responses of Symbiodinium to the lectin varied significantly among the strains used and that the clade of Symbiodinium in S. lochmodes was uniform strongly suggest that some selection analogous to our experiment had taken place in the coral-algae system. Therefore, further experiments using an actual Symbiodinium isolate of the coral or a culture of clade C will be of interest.
Of note, our results indicated that the action of SLL-2 toward Symbiodinium is mediated not only by carbohydrate-binding activity, but also by more specific but still unidentified mechanisms, since the peanut lectin that recognizes a D-galactosyl moiety such as SLL-2 was inert in our models.
Our finding of lectin-mediated chemical recognition and physiological modulation between symbiotic dinoflagellates and a coral is a novel one. Identifying the protein responsible for this process can illuminate physiological and possibly also evolutionary attributes of symbiosis, one of the coral reef’s most important ecological aspects. Further investigation of the physiological and ecological aspects of these symbioses might allow us to better understand globally important ecological problems such as coral bleaching, which is triggered by the evacuation of zooxanthellae from the host.
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Acknowledgments
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The authors express their thanks to Mr. Saburo Hosaka, a chairman of the Establishment of Tropical Marine Ecological Research (ETMER), and the staff of Akajima Marine Science Laboratory (AMSL) for access to their collection of specimens. Thanks are also due to Dr. Lirdwitayaprasit Thaithaworn, Chulalongkorn University, and Dr. Setsuko Sakamoto, National Research Institute of Fisheries and Environment of Inland Sea, for supplying cultures. This study is supported in part by a Study-In-Aid for Scientific Research (B) from the Ministry of Education, Culture, and Science, Japan to H.K. (80011964 and 15380145).
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Footnotes
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Received 28 January 2004; accepted 20 July 2004.
Current address: Marine Biology and Ecology Research Program, Japan Agency for Marine-Earth Science and Technology (JAMSTEC), Natsushima, Yokosuka, Kanagawa, 237-0061, Japan. 
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