Introduction

Symbiotic associations are mostly found in marine environments. A well-known example of which is the mutualistic symbiosis of cnidarians (coral and sea anemone) with the dinoflagellate, Symbiodinium sp1. The mutualistic association involves the translocation of 90% of the photosynthetically fixed carbon in Symbiodinium into the host cytoplasm and in return, the host provides nutrients for the dinoflagellates to live and grow2. It has been proposed that this intracellular symbiosis (i.e. endosymbiosis) plays key role in the maintenance of coral metabolism and health.

Symbodinium has been found as a symbiont in cnidaria and other invertebrates3,4,5. Endosymbiotic Symbiodinium is widely distributed throughout the host cnidarian’s gastrodermis cell layer, at a density of 106 per cm2 of colony surface area6,7. Recently, Symbiodinium was divided into nine major lineages (clade A-I) based on the analysis of the 18 S-rDNA and internal transcribed spacer regions (ITS)8,9. Symbiodinium genetic variability may reflect the different features of Symbiodinium functional biology and responses to environmental stress10. For instance, clade D is able to resilient to coral bleaching event due to the tolerance to thermal stress11. Using a proteomic approach, it has been shown that the different protein expression of the cultured and the symbiotic Symbiodinium cells were observed in sea anemone12. After examining the culture of symbiotic Symbiodinium from host, Krueger and colleague have successfully cultivated endosymbiont C15 by mimicking the host environment ex hospite13. However, the cellular and molecular mechanisms governing this mutualism are not yet fully understood.

Environmental stress, such as temperature, salinity, light intensity and disease, are causing disruption of the coral-dinoflagellate symbiotic association14,15,16. Temperature is one of the major causes affecting the stability of endosymbiotic associations17,18. Nevertheless, some previous works have shown that both low and high temperatures could induce the physiological impact on coral such as bleaching and mortality19. It has been reported that the exposure to temperature at 30–31 °C in the seawater, southern Taiwan, caused the coral bleaching20,21. Moreover, when the coral was exposed to 14 °C with full sunlight, the photosynthetic ability of the Symbiodinium was reduced and coral become bleached22. Nonetheless, the morphological profile, adaptation behavior and survival rate of the free-living Symbiodinium against thermal stress remains to be clarified.

In order to elucidate the mechanism allowing endosymbiotic Symbiodinium in clade B to adapt to ex hospite conditions and extreme temperatures, endosymbiotic Symbiodinium was isolated from host gastrodermal cells and purified with different percentages of Percoll gradient solution. Then, the Symbiodinium was inoculated onto the medium to obtain the pure Symbiodinium culture and the clade identification was carried out. Symbiodinium cell proliferation progressed more slowly when they were subjected to extreme temperatures (30 °C; 15 °C) than when they were cultured at normal temperatures (25 °C). However, morphological observations revealed that when symbiont Symbiodinium were cultivated ex hospite, their size changed. The twelve distinct protein profiles derived from the endosymbiotic Symbiodinium cells putatively included transcription translation factors, photosystem proteins and proteins associated with energy and lipid metabolism as well as defense response.

Results and Discussion

Morphological alterations of Exaiptasia pulchella in response to stress-induced bleaching

Endosymbiotic stability is dependent on the regulation of Symbiodinium cells living in the host gastrodermal cell layer15,23. Exaiptasia pulchella24 is a sea anemone that is widely distributed in the tropical and subtropical Pacific Ocean. It can be easily cultured in the laboratory and commonly used as a model organism for studying the cnidarian-Symbiodinium mutualism25,26,27. Symbiodinium cells appear brown in color and thus they bring about the apparent color of E. pulchella (Fig. 1A). In contrast, we have shown that the environmental stress caused the bleaching of sea anemone tentacles due to the loss of Symbiodinium cells, leading to the bleaching of the coral (Fig. 1C). It has been reported that unfavorable environmental conditions such as elevated temperature, extreme pH, nutrient deprivation and changes in salinity could create conditions inducing the collapse of the symbiotic relationship, resulting in the loss of Symbiodinium14,28,29,30,31,32. Bleached anemones lost 86% of their Symbiodinium symbionts32, a phenomenon that was visualized by fluorescence microscopy to detect the auto-fluorescence of Symbiodinium chlorophyll, in order to show that Symbiodinium cell density decreased in response to environmental stress (Fig. 1B,D). During bleaching, the amount of green fluorescence in the host body and tentacles increased significantly (Fig. 1D). The difference between healthy and unhealthy E. pulchella can be visualized clearly under electron microscope (Fig. 2A,D). Ultrastructural observations of healthy sea anemone tentacles revealed the presence of abundant Symbiodinium cells in the gastrodermal layer, as the Symbiodinium intracellular structures, such as chloroplasts, nuclei, pyrenoids and lipid droplets, were noted (Fig. 2). Symbiotic cells are usually in coccoid forms surrounded by a cellulose-based cell wall. They inhabit a specialized host vacuole called a symbiosome (Fig. 2B). The signs of structural degradation of the Symbiodinium cells in the bleached E. pulchella were shown. This study also revealed the occurrence of the reduction in gastrodermal Symbiodinium densities (Fig. 2A,D). Presumably, the Symbiodinium cells were degraded during the bleaching period (Fig. 2D). Interestingly, the major characteristics of these symbiotic cells included the following: the presence of only a few organelles inside of a large vacuole containing numerous lipid droplets, the presence of cytoplasmic debris in the lumen and loosened thylakoids (Fig. 2E,F). These morphological observations were similar to the previously reported observations of the morphological changes in both the host and Symbiodinium during bleaching33,34.

Figure 1
figure 1

Microscopic examination of healthy and bleached in Exaiptasia pulchella.

(A) Healthy Exaiptasia pulchella were observed under light microscopy to be brown in color throughout the column and tentacles. (B) Fluorescence microscopy under a DAPI filter shows the red auto-fluorescence representing the cellular chlorophyll of Symbiodinium sp. cells (white filled arrowheads). (C) Bleached Exaiptasia pulchella were observed using light microscopy. (D) Bleached Exaiptasia pulchella tissue observed via fluorescence microscopy revealed only a small amount of red auto-fluorescence, at the tentacle edges (white filled arrowheads).

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Figure 2
figure 2

Morphological profiles of an Exaiptasia pulchella tentacle and Symbiodinium cells, observed via Transmission Electron Microscopy.

(A,D) Ultrastructure of E. pulchella tentacle, healthy vs bleached Symbiodinium cells residing inside the host, observed via transmission electron microscopy (B,C). Symbiodinium ultrastructure in a bleached tentacle (E,F). Abbreviations: LD, lipid droplet; Ch, chloroplast; Pyr, pyrenoids; N, nucleolus; acc: accumulation body.

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Isolation of Symbiodinium cells from Exaiptasia pulchella and their culture in medium

Many symbiotic dinoflagellates can be isolated and maintained in culture, given the presence of suitable conditions. Most investigators have concluded that the morphology and life cycle of the free-living and symbiotic forms are fundamentally different to each other, but few details are known. The establishment of free-living Symbiodinium populations could help to preserve both Symbiodinium and coral during the bleaching periods. Freshly isolated Symbiodinium cells were collected from E. pulchella via a Percoll gradient35 (Fig. 3C,D). All remaining host tissue debris was removed from the Symbiodinium cells, after which the cells were incubated in f/2 medium in filtered seawater. The Symbiodinium isolates were successfully maintained by culturing them in f/2 medium (Fig. 4). After being cultured for a few months, some of the Symbiodinium cells died (appeared as blue fluorescence); however, most Symbiodinium cells in the culture medium were still alive (appeared as red fluorescence) (Fig. 3E,F). Studies have shown that the Symbiodinium clades or types associated with corals are different, depending on environmental conditions36. The Symbiodinium clade identification was conducted via RFLP, using restriction enzymes, Taq1 and Sau 3A1. The results showed that clade B was dominant in both freshly isolated and free-living cultured Symbiodinium cells. In the present study, the physiology and molecular response of symbiotic Symbiodinium to environmental stress was investigated by isolating fresh symbiotic cells from the host sea anemone. Here, the comparison of the freshly isolated with the free-living cultured cells revealed the differences in the color and size (Figs 2 and 3). Freshly isolated Symbiodinium cells had a diameter of 6.49 ± 1.06 μm, which became smaller after culturing in the medium, with a diameter of 5.6 ± 0.02 μm (Fig. 3G, Table 1). The chloroplast content in the Symbiodinium cells decreased, resulting in a more light brown color than the freshly isolated cells (Figs 2C,G and 3C). Similarly, free-living cultured Symbiodinium cells isolated from sea anemones were shown to be compact in structure and morphologically different from the Symbiodinium cells residing in the host (Fig. 2). The morphological differences of free-living cultured Symbiodinium have been reported previously37.

Table 1 Different cell types between the freshly isolated Symbiotic cells and free-living cultured Symbiodinium cells.
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Figure 3
figure 3

Freshly isolated Symbiodinium cells and free-living cultured cells were observed using light microscopy.

(A) Brown Symbiodinium residing in the Exaiptasia pulchella tentacle. (B) Fluorescence microscopy of Exaiptasia pulchella. (C,E,G) light microscopy of freshly isolated Symbiodinium, free-living cultured Symbiodinium sp., mixed cells of freshly isolated (rectangular white box) and free-living Symbiodinium (circular white box), respectively. (D,F,H) Freshly isolated Symbiodinium, free-living cultured Symbiodinium, mixed cells of freshly isolated (rectangular white box) and free-living Symbiodinium (circular white box), respectively, were observed using fluorescence microscopy. Little red auto-fluorescence was detected, representing the cellular Symbiodinium chlorophyll.

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Figure 4
figure 4

Growth curves of Symbiodinium cells cultivated at different temperatures.

Temperature treatments (15 °C, 25 °C and 30 °C) were applied to free-living Symbiodinium sp. The data represents means ± SD (n = 3).

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Extreme temperature stress induced changes in the cell morphology of cultured Symbiodinium cells

In the free-living cultures, Symbiodinium could freely approach the nutrients in the medium. Although it was likely that the nutrient uptake mechanism changed under such culturing condition, it was determined that it would not be responsible for the physiological changes observed in Symbiodinium cells37. To examine the effect of extreme temperature on Symbiodinium cellular growth, free-living Symbiodinium cells were cultivated at low or high temperatures for 7 days. The cells cultivated at low temperatures (15 °C) proliferated more slowly than those cultivated at normal temperatures (Fig. 4). Culturing at 25 °C and 30 °C did not cause any influence on the growth of Symbiodinium cells (Fig. 4), but numerous lipid droplets started to be accumulated and then they were disrupted after the 5th day of high-temperature treatment. It was similarly reported that the response to changes in temperature was an adaptive physiological adjustment of Symbiodinium28,38. Furthermore, at a lower temperature (15 °C), the Symbiodinium cells stopped proliferating (Fig. 4) and lipid droplets accumulated and occupied most of the cytoplasm in the Symbiodinium cells from day 3 (Fig. 5E–H). The Symbiodinium cells started proliferating at day 3 and were structurally degraded after 5 days at 30 °C. At this stage, the cells contained highly disrupted lipid droplets (Fig. 5K,L, arrows). Fewer lipid droplets were present in the Symbiodinium cells cultured at normal temperatures (25 °C) (Fig. 2A–D). The Symbiodinium cells were structurally degraded after 7 days at 30 °C and contained highly disrupted lipid droplets (Fig. 2I, arrows). Fewer lipid droplets were present in cells cultured at normal temperatures (Fig. 2G).

Figure 5
figure 5

TEM micrographs showing ultrastructural changes during the 7-day treatment at different temperatures in Symbiodinium cells.

Symbiodinium cells were cultured at a normal temperature (25 °C) for the indicated times (A–D). Symbiodinium cells were cultured at a low temperature (15 °C) for the indicated times (E–H). Symbiodinium cells were cultured at a high temperature (30 °C) for the indicated times (I–L). Abbreviations: LD, lipid droplet; Ch, chloroplast; Pyr, pyrenoids; N, nucleolus. The scale bars indicate 2 μm.

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Identification of specifically expressed proteins from freshly isolated symbiotic Symbiodinium cells

Stochaj and Grossman12 reported that the protein profiles of free-living and endosymbiotic Symbiodinium cells were different to each other because the latter was obtained from sea anemone Exaiptasia pallida12. A proteomic approach was employed in order to obtain the protein profiles of freshly isolated Symbiodinium cells (Table 2). The protein expression of Symbiodinium cells was analyzed and various differentially regulated proteins were detected. The SDS-PAGE of free-living cultured Symbiodinium cell proteins was compared to that of the freshly isolated cells (Fig. 6; Fig. S1). Twelve proteins with a variety of cellular functions were identified, including transcription and translation factors (“EF-1 alpha-like protein” from protein band I1). EF-1α (also known as EF1A) plays an essential role in translation, binding aminoacyl-tRNAs (aa-tRNAs) and bringing them to the A site of the ribosome. EF-1α is also known to be involved in several other cellular processes, including the ubiquitin-dependent proteolytic system39. A number of proteins that are stress-induced at the translation level have been identified in endosymbionts. “WRKY transcription factor” from protein band I2 was found to play an important role in regulating nutrient deficiency tolerance40. Previous studies have shown that these endosymbionts could survive in nutrient-limited environments, such as those with limited nitrogen levels37. In endosymbionts, nutrient limitation may induce the expression of the WRKY transcription factor for the tolerance control. “Dehydration responsive element binding protein (DREB)” from protein band I4. DREB transcription factors induce a set of abiotic stress-response genes responsible for maintaining the organism’s water balance and imparting tolerance to abiotic stress41. The “pre-mRNA splicing factor ATP-dependent RNA helicase-like protein” was determined as protein band I5. Furthermore, we detected that photosystem II D2 protein (PsbD2) and glutamate 1-semialdehyde (GSA) aminotransferase were overexpressed in endosymbiotic cells (“Photosystem II D2 protein” from protein band I1 and “glutamate-1-semialdehyde aminotransferase” from protein band I2). GSA aminotransferase catalyzes the last step of the conversion of glutamate to δ-aminolevulinate and eight molecules of which are needed in order to synthesize a single chlorophyll molecule42. Other proteins associated with energy metabolism (“NADH dehydrogenase subunit F” from protein band I2, “Oxidoreductase” from protein band I2 and “ARF-related small GTPase” from protein band I5) and lipid metabolism (“acyl-coA thioesterase 13” from protein band I3 and “7-dehydrocholesterol reductase-like” from protein band I4), as well as a defense response protein (“LRR-kinase protein” from protein band I5), were also detected (Table 2). Furthermore, the result showed that the discovery of the presence of ARF-related small GTPase in endosymbiotic Symbiodinium is similar to the study of the lipid droplets in the nitrogen-deprived free-living Symbiodinium cells37, demonstrating that the changes in the energy metabolism of the symbiotic association with Cnidaria such as free-living Symbiodinium cultured in nitrogen-deficient environments.

Table 2 Functional classification of proteins identify from freshly isolated Symbiotic Symbiodinium cells.
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Figure 6
figure 6

Different proteins expressed in free-living cultured and freshly isolated symbiotic Symbiodinium cells.

Total protein (40 μg) was extracted from both freshly isolated and free-living cultured Symbiodinium sp. resolved in SDS-PAGE. Five protein bands were excised for mass spectrometric analysis.

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Conclusion

This study revealed the morphological variability and distinct protein profiles of the free-living cultured and endosymbiotic Symbiodinium cells isolated from Exaiptasia pulchella. Temperature-induced morphological changes and the variable cell proliferation were shown in free living cultured Symbiodinium cells. Proteomic analysis of major proteins in freshly isolated cells were found to be involved in regulation of transcription and translation, photosystem and metabolism of energy and lipid.

Materials and Methods

Collection of sea anemone and isolation of Symbiodinium cells from E. pulchella tissues

The sea anemone Exaiptasia pulchella was collected from National Museum of Marine Biology Aquarium. They were maintained in filtered seawater (FSW) at room temperature under a photosynthetically active radiation (PAR) of 40 μmol m−2s−1 in a 12-h light/12-h dark (12L/12D) cycle. Symbiodinium cells isolated from its host tissue were subjected to further isolation method developed by Pasaribu et al.35. E. pulchella was then blended for releasing the Symbiodinium cells in FSW (filtered seawater) and homogenized with a glass grinder. The homogenized solution was then filtered through the 20-μm mesh and centrifuged at 4,000 × g for 5 min. The pellet collected after the centrifugation contained mostly Symbiodinium cells, which were resuspended in FSW and vortexed for 2 min. The mixture was centrifuged at 2,000 × g for 5 min. The pellet was retained and then resuspended in fresh FSW containing 1% (v/v) of triton X-100. The mixture was vortexed again for 2 min.

Symbiodinium clade identification

The genetic identity (18S rDNA) of the cultured Symbiodinium was examined by PCR-RFLP (Polymerase chain reaction-Restriction fragment length polymorphism) analysis41 and shown to be clade B. Symbiodinium DNA was extracted using a plant genomic DNA extraction miniprep system (VIOGENE, Taipei). Basically, Symbiodinium nuclear small subunit (n18S-rDNA) was amplified by PCR from 3 replicate extracts of each of the two cultures using the primers, ss5z (an equimolar mixture of the oligonucleotides 5′-GCAGTTATAATTTATTTGATGGTCACTGCTAC-3′ and 5′-GCAGTTATAGTTTATTTGATGGTTGCTGCTAC-3′) and ss3z (5′-AGCACTGCGTCAGTCCGAATAATTCAC CGG-3′) and digested by the restriction enzymes, Taq I and Sau3A I (Promega, USA). Digestion products were separated by electrophoresis on 1.5% 0.5x TAE (Amresco, USA) agarose gels, to generate the RFLP pattern. RFLP pattern analysis was compared to the literature41 to assign each culture to one of the established Symbiodinium n18S-rDNA RFLP clades.

Symbiodinium culture and treatment

The free-living Symbiodinium sp. (clade B) were cultured in the f/2 medium in filtered seawater (FSW) at room temperature under a photosynthetically active radiation (PAR) of 40 μmol m−2s−1 in a 12-h light/12-h dark (12L/12D) cycle. For treatment, three batch cultures were grown in the f/2 medium with temperatures 15 °C, 25 °C and 30 °C, respectively.

Cell density determination

The Symbiodinium cell density was examined with haemocytometer based cell counting. Cell densities were determined daily by placing an aliquot of well-mixed culture suspension on a Neubauer hemocytometer (Marienfel, Germany) under an Axioskop2 Plus microscope (Zeiss, Germany) connected to a CCD camera (Photometrics, USA).

The transmission electron microscopy and imaging analysis

To investigate the morphological variability of Symbiodinium cells within the host cells or in the free-living form, Symbiodinium cells were collected and fixed in 2.5% glutaraldehyde and 2% paraformaldehyde in 100 mM sodium phosphate containing 5% sucrose (pH 7.3) for 2.5 h at 4 °C. They were then rinsed with 100 mM sodium phosphate buffer at 4 °C. Cells were post-fixed in 1% OsO4 in 50 mM sodium phosphate (pH 7.3) for 1 h at 4 °C. The cell aliquots were then washed 3 times for 15 min each with the same buffer and dehydrated by a graded ethanol series (50, 70, 80, 90, 95 and 100%) before embedding in LR white Resin. Thin sections (70 nm) cut by a Leica Reichert Ultracut R were collected on nickel grids, post-stained with 2.5% uranyl acetate and 0.4% lead citrate, rinsed 3 times with water and the samples were viewed on a JEM-1400 transmission electron microscope (JEOL, Japan). In order to determine the lipid droplet area from the acquired images, the ratio of the actual length to pixel was first determined by distance calibration using the scale bar of the acquired TEM image. Individual lipid droplet was selected by threshold adjustment and the area (μm2) of each lipid droplet was calculated by using the region measurement function of Metamorph.

Total protein extraction

The protocol was operated according to the work by Hurkman and Tanaka43. Symbiodinium cell was ground into powder in the mortar with liquid N2 The powder was mixed completely with 10 ml of the extraction buffer (0.7 M sucrose, 0.5 M Tris base, 30 mM HCl, 50 mM EDTA, 0.1 M KCl and 2% β-mercaptoethanol) and 10 ml of phenol.  The upper phenol layer was separated from the lower buffer layer by centrifugation at 8,000 × g for 15 min at 4 °C and transferred the phenol layer into a new tube. To remove contaminants, the phenol layer was mixed with equal volume of fresh extraction buffer again. After complete mixing, the phenol layer was separated from the buffer again. After complete mixing, the phenol layer was separated from the buffer by centrifugation at 8,000 × g for 15 min at 4 °C and transferred into a new tube containing five-fold volume of pre-cold 0.1 M NH4OAc/methanol to precipitate the proteins at −20 °C overnight. The pellet was collected by centrifugation at 8,000 × g for 15 min at 4 °C and washed with 10 ml of 0.1 NH4OAc/methanol for three times followed by 10 ml of acetone for two times. Finally the pellet was dissolved in the lysis buffer (8M Urea, 2% CHAPS, 30 mm 1,4-dithiothretol (DTT)) and stored at −20 °C.

Protein quantification

Proteins were quantified with the Quant-iTTM protein assay kit (Invitrogen Molecular Probes, Italy), using the Qubit fluorometer according to the manufacturer’s instructions (the kit is used for quantify the protein concentrations ranging from 12.5 μg/ml to 5 mg/ml).

SDS-PAGE

Protein extracted from the symbiotic Symbiodinium cells collected from E. pulchella and free-living Symbiodinium cells were suspended in an equal volume of 2 × sample buffer according to the suggestion in the Bio-Rad instruction manual and resolved by SDS-PAGE using 15% (w/v) polyacrylamide in the separating gel and 4.75% polyacrylamide in the stacking gel44. After electrophoresis, the gel was stained with Coomassie Blue R-250 and then destained.

In-gel digestion of the major proteins in Symbiodinium cell

The several bands of Symbiodinium cell proteins resolved by SDS-PAGE were manually excised from the gel and ground into pieces. After washing with 50% acetonitrile and 50% acetonitrile/25 mM ammonium bicarbonate, the protein was reduced and alkylated at 56 °C for 45 min in 10 mM dithiothreitol and 55 mM iodoacetamide in 25 mM ammonium bicarbonate, followed by overnight in-gel digestion with 0.1 μg in 15 μl of TPCK-treated modified porcine trypsin (Promega, USA) in the same buffer at 37 °C. The supernatant containing tryptic peptides was combined with two more extracts of the gel by 50% acetonitrile/5% formic acid. The sample was analyzed by matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) and MALDI-MS/MS. All data were acquired by quadrupole-time-of-flight (Q-TOF) hybrid mass spectrometers (Micromass Q-Tof Ultima, Manchester, UK and Applied Biosystems QSTAR, USA), in which α-cyano-4-hydroxycinnamic acid was used as the matrix. The low-energy collision-induced dissociation MS/MS product ion spectra acquired from Q-TOF Ultima and QSTAR were analyzed by Micromass ProteinLynxTM Global Server 2.0 and Applied Biosystems BioAnalystTM data processing software, respectively. For protein identification, the acquired MS/MS spectra were automatically searched against the NCBInr database using the Mascot search program (www.matrixscience.com) restricted to all entries taxonomy. The mass tolerance parameter was 20 ppm, the MS/MS ion mass tolerance was 1 Da and up to one missed cleavage was allowed. Variable modifications considered were methionine oxidation and cysteine carboxyamidomethylation. Positive identification of proteins was confirmed by observation of at lest one of the following criteria: (i) the total number of matched peptides (mps) is more than 2, or (ii) the mps equals 2 with two different matched peptides, or (iii) the MOWSE score has to be higher than 67 which indicates identity or extensive homology (p < 0.05).

Additional Information

How to cite this article: Pasaribu, B. et al. Morphological Variability and Distinct Protein Profiles of Cultured and Endosymbiotic Symbiodinium cells Isolated from Exaiptasia pulchella. Sci. Rep. 5, 15353; doi: 10.1038/srep15353 (2015).