Fluorescent protein content and stress tolerance of two color morphs of the coral Galaxea fascicularis

Galaxea, Journal of Coral Reef Studies 17: 1-11（2015）

Abstract Fluorescent proteins in corals have been reported to have photoprotective function for algal symbionts, though it remains controversial whether the coral fluorescent proteins are actually photoprotective. Green (Gs) and brown (B) morphs of the coral Galaxea fascicularis have different contents of green fluorescent protein (GFP). To understand the function of GFP in the stress responses of the coral, we exposed polyps isolated from B and Gs morph colonies to medium or strong light (200 and 1000 μmol quanta m－2s－1) at 26 or 32℃ for 6 h. Polyps were then allowed to recover for 6 h under dim light. Although the GFP content was markedly different between Gs and B morphs, in hospite zooxanthellae in polyps of both morphs showed similar decreases in photochemical efficiency (Fv /Fm) after strong light treatment at normal or high temperature. Isolated zooxanthellae of both morphs showed similar decrease in the photochemical efficiency under light stress, indicating that they had similar tolerance to light stress. The present results suggest that fluorescent protein does not increase the tolerance of polyps to strong light and high temperature stress in G. fascicularis and that further studies are necessary to elucidate the function of GFP in this coral.

Keywords Coral, Fluorescent protein, Galaxea, Photopro tection, Symbiosis, Zooxanthellae

Introduction

Many scleractinian corals exhibit intraspecific color variation due to different contents and distribution patterns of host pigments. Fluorescent proteins and non-fluorescent chromoproteins have been identified from corals (Labas et al. 2002, Alieva et al. 2008). Corals generally have mul-tiple homologues of green fluorescent proteins (GFP) (Kao et al. 2007). Ten GFP-like protein genes, which code for GFP, CFP (cyan), RFP (red), and non-fluorescent chro-moproteins, were found in the Acropora digitifera genome (Shinzato et al. 2012).

The biological functions of GFP-like proteins remain ambiguous and controversial. Suggested roles of GFP-like proteins include (1) photoprotection (Salih et al. 2000; Dove et al. 2001; Dove, 2004; Smith et al. 2013), (2) facilitation of photosynthesis (Salih et al. 2000; Dove et al. 2008), (3) antioxidant function (Bou Abdallah et al. 2006; Palmer et al. 2009), (4) attraction of zooxanthellae (Hollingsworth et al. 2004, 2005), and (5) avoidance of herbivores (Matz et al. 2006). Additionally, possible as-sociations of fluorescent proteins with sensory functions during settlement (Kenkel et al. 2011) or metabolic path-ways related to coral growth (D’Angelo et al. 2008; Roth and Deheyn 2013) have been suggested.

Among these potential roles, photoprotective function

Fluorescent protein content and stress tolerance of two color morphs of the coral Galaxea fascicularis

Sho NAKAEMA* and Michio HIDAKA

Department of Chemistry, Biology and Marine Science, University of the Ryukyus, Nishihara, Okinawa 903-0213, Japan

* Corresponding author: E-mail: [email protected]

Communicated by M. Hatta (Editor-in-Chief)

Original paper

Nakaema and Hidaka: GFP function of Galaxea fascicularis2

has been the main focus of studies. In some shallow water corals, GFPcontaining granules are located in the epidermis, covering zooxanthellae within the gastrodermal cells. This, together with the ability of GFP to convert short wavelength light to long wavelength light, indicates that GFP functions as sunscreen for algal symbionts (Salih et al. 2000). Corals with GFP or chromoproteins showed less damage to algal photosystem II (PSII) than did corals without them (Salih et al. 2000; Dove 2004; Smith et al. 2013). On the contrary, Mazel et al. (2003) stated that the capability of fluorescent pigments to protect zooxanthellae from excess light appears to be negligibly low based on their measurements of reflectance spectra of green and brown morphs of Montastrea faveolata and of excitation spectra for chlorophyll fluorescence of strongly green-fluorescent individuals of Scolymia sp. and weakly fluorescent colonies of M. carvernosa. Leutenegger et al. (2007) also reported that green fluorescent host pigmen-tation had no influence on the degree of bleaching of two sea anemone species during a natural bleaching event.

Dove (2004) reported that host pigments are photo-protective at normal temperature, but that the heavily pigmented blue morph is most sensitive to bleaching at high temperature. She postulated that the protection of-fered by GFP-like proteins is reduced by thermal stress and that the shadeacclimated algal symbionts of highly pigmented corals thus suffer more severe damage when exposed to high light levels.

The objective of this study was to investigate the potential photoprotective function of coral GFP. To test whether GFP actually protects the algal symbionts from strong light, we studied susceptibility of the green fluo-rescent (Gs) morph and non-fluorescent brown (B) morph of Galaxea fascicularis Linnaeus 1767 to light stress at normal or high temperatures. We also tested the possibility that algal symbionts in pigmented colonies are shadeadapted and more sensitive to light than those in nonfluorescent colonies (Dove 2004), by comparing the degree of photoinhibition in zooxanthellae isolated from the Gs and B morphs.

Materials and Methods

Collection of coralsColonies of G. fascicularis were collected from depths

of 0.5-1 m during low tide at Zampa reef, Okinawa Island, Japan, in May 2012 and June 2013. Several color morphs of G. fascicularis have been reported from Okinawa (Hidaka and Yamazato 1985). We used the Gs and B morphs, both of which have brown polyps, but the oral disc and tentacles covering the septum are greenish in the Gs morph polyp. Five colonies of each morph were collected in each year. The colonies were kept in an outdoor tank provided with running unfiltered seawater at Sesoko Station, University of the Ryukyus, Okinawa for 10 days until use. The tank was covered with a sunshade mesh and the light intensity in the daytime was in the range of 200-800 μmol quanta m－2s－1. Polyps were iso-lated from the colonies one day before the stress exposure experiment.

Light stress treatment of in hospite and isolated zooxanthellae

Polyps with their skeletons were isolated from colonies collected in May 2012, and the basal skeletal region was removed. Isolated polyps about 2 cm long were held verti-cally using polyp stands, which were made of silicon tubing and plastic mesh. Ten polyps each from different colonies (5 Gs and 5 B morph colonies) were placed in a 5 L container filled with 3 L of filtered seawater (FSW, 0.45 μm). Three containers each with ten polyps were prepared and placed in a water bath whose temperature was regulated at 26℃ by a temperature control unit (Thermo Minder SM-05N, TAITEC, Japan). Polyps were exposed to three light conditions (0, 200, or 1000 μmol quanta m－2s－1) for 3 h at 26℃. A metal halide lamp (EYE Clean Arc MRF400D, 400W, Iwasaki Electric Co., Ltd., Japan) was used as the light source. The light intensity was measured using a light meter (Model LI-250, LI-COR Bioscience, USA) with the sensor placed at the same position as the oral disc of the polyps.

The photochemical efficiency (dark-adapted Fv /Fm) of in situ zooxanthellae was measured using a pulseamplitude-modulation (PAM) chlorophyll fluorometer

Nakaema and Hidaka: GFP function of Galaxea fascicularis 3

(MINI-PAM, Walz Inc., Germany) before and after the stress treatment and after 6 h of recovery under low light condition (5-10 μmol quanta m－2s－1). After 20 min of dark adaptation, the maximum quantum yield of the PSII (Fv /Fm) was measured at room temperature (26℃) using a custom-made black box.

To prepare isolated zooxanthellae for light stress treatment, isolated polyps were rinsed gently with filtered seawater (0.45 μm) and then coral tissue was removed from the coral skeleton using a WaterPik (Johannes and Wiebe 1970). To remove the mucus produced during the isolation procedure, the blastate was first filtered through a 180 μm nylon mesh, and next through 40 μm nylon mesh. The suspension was then homogenized with a potter homogenizer before being centrifuged at 700 g for 10 min at 4℃. The algal pellet was washed two more times by centrifugation at 700 g for 10 min at 4℃. Freshly isolated zooxanthellae were adsorbed onto Millipore filters (0.45 μm) using a syringe and a filter holder (Bhagooli and Hidaka 2003). Filters with adsorbed zooxanthellae were held on glass slides with the help of plastic tape.

Five filters each with zooxanthellae isolated from each of five colonies were prepared for Gs and B morphs. A total of 10 filters held on glass slides were placed in a 5 L container filled with 3 L of filtered seawater. Three con-tainers each with 10 filters were prepared and were exposed to various light intensities (0, 50, and 200 μmol quanta m－2s－1) at 26℃ for 3 h. Then, the filters with isolated zooxanthellae were placed in filtered seawater and allowed to recover at 26℃ under low light conditions (5-10 μmol quanta m－2s－1) for 6 h. The maximum quan-tum yield, Fv /Fm, of isolated zooxanthellae was measured before and after the stress treatment and after recovery as described above. The glass slides mounted with filters with isolated zooxanthellae were placed in the custommade black box for Fv /Fm measurements after 20 min dark adaptation.

Light and temperature stress treatment of in hospite zooxanthellae

To compare the tolerance of Gs and B morph polyps under severer stress conditions, isolated polyps were exposed to medium or high light (200 or 1000 μmol

quanta m－2s－1) at normal or high temperatures (26℃ or 32℃) for 6 h. Then the polyps were recovered under low light (5-10 μmol quanta m－2s－1) at 26℃. The maximum quantum yield, Fv /Fm, of in hospite zooxanthellae was measured before stress treatment, 3 h and 6 h after the commencement of the stress treatment, and after 6 h recovery period. The maximum quantum yield of the PSII (Fv /Fm) was measured at room temperature (26℃) using a custom-made black box after 20 min of dark adaptation.

Polyps with their skeletons were isolated from colonies collected in June 2013 were used in this experiment. Isolated polyps about 2 cm long were held vertically using polyp stands, which were made of silicon tubing and plastic mesh. A total of 20 polyps, two polyps from each of ten colonies (5 Gs and 5 B morph colonies) were placed in a 40 L container filled with 30 L of filtered seawater (FSW, 0.45 μm). Ten polyps each from different colonies were exposed to high light (1000 μmol quanta m－2s－1) and remaining ten polyps were exposed to medium light (200 μmol quanta m－2s－1). A metal halide lamp (Funnel Lucky, 70 W, Kamihata, Japan) was used as a light source and the light intensity was set adjusting the distance between the lamp and the polyps (12 cm and 25 cm for high and medium light, respectively). Two containers each with twenty polyps were prepared. The seawater temperature of one container was kept at ambient tem-perature (26℃) and the seawater temperature of the other container was regulated at 32℃ using an aquarium heater and thermostat (Five Plan EX-003, GEX Co. Ltd., Japan).

Measurement of GFP contentTo compare the relative GFP contents of polyps of Gs

and B morphs, the fluorescence intensity per unit protein content of coral tissue extracts was measured. Coral polyps were frozen in liquid nitrogen and ground using a mortar and pestle. The powder was suspended in 5 ml of PBS (pH＝7.4). The suspension was vortexed for 20 sec and left on ice for 5 min. The extract was centrifuged at 2000 g at room temperature for 5 min and the supernatant was moved to a 2 ml tube, and centrifuged again at 12000 g for 10 min at 4℃. The supernatant was put into wells of a 96-well plate with an optically transparent bottom and black side walls (No. 165305, Nalgene Nunc International, Rochester, NY, USA). The fluorescent intensity of the


extract was measured for triplicate wells using a fluo-rescent microplate reader (GENios, Tecan, Switzerland) with excitation at 485±10 nm and emission at 535± 10 nm. The soluble protein content of polyps was deter-mined using a BIO-RAD Protein Assay kit (BIO-RAD, California, USA). Absorption at 595 nm was measured using a microplate reader (Model 550, BIO-RAD), and γ-globulin (BIO-RAD Protein Assay Standard I, 1.39 mg/ml) was used as a standard.

Zooxanthellae density and photosynthetic pigment con centration

To measure the zooxanthellae density, zooxanthellae were isolated from each polyp using a WaterPik and filtered seawater. The algal suspension was homogenized with a potter homogenizer and centrifuged at 2000 g for 5 min at the room temperature. The algal pellet was diluted in 5 ml filtered seawater and algal cells were counted using a hemocytometer. The surface area of the polyp tissue was calculated assuming that the polyp is an elliptical cylinder.

Then the number of zooxanthellae per unit surface area of the polyps was calculated. To extract photosynthetic pigments, polyps were immersed in 5 ml of 90% acetone for 48 h at 4℃. The absorbance of the extract was mea-sured at 630, 664, and 750 nm using a spectrophotometer (UV-1800, Shimadzu Scientific Instruments, Japan). The chlorophyll a and c2 concentrations were calculated following the equations of Jeffrey and Humphrey (1975). The photosynthetic pigment concentrations were norma-lized by the surface area of the polyp tissue.

Identification of zooxanthellae cladeThe clade type of the zooxanthellae was determined by

PCR-RFLP analysis using the restriction enzyme TaqI. Polyps isolated from five Gs morph colonies and five B morph colonies that were used for combined light and stress treatment described above were immersed in CHAOS solution (4 M guanidine thiocyanate, 0.5% salkosyl, 25 mM Tris-HCl (pH 8.0), 0.1 M β-mercapto-ethanol) to preserve the DNA and stored at －20℃ until use. DNA was extracted from the CHAOS solution (100 μl) using a DNeasy Blood and Tissue kit (Qiagen, Hilden, Germany) according to manufacturer's protocol.

Amplifications were performed in a volume of 25 μl containing 0.01 mM primers (Lsu-UFP1: 5′-CCCGCTA-ATTTAAGCATATAAGTA-3′; Lsu-URP1: 5′-TTAGAC-TCCTTGGTCGTGTTTCA-3′) Zardoya et al. 1995) and 10 ng template DNA, 10×PC buffer, 0.1 mM dNTPs, and 0.5 U EX Taq polymerase (TaKaRa Bio, Otsu, Japan). Thermalcycling was performed under the following condition: 1 cycles of 94℃ for 5 min, 35 cycles of 94℃ for 45 s, 52℃ for 45 s, and 90 s at 72℃, and a final 7 min extension at 72℃. For RFLP analysis, the PCR products were digested with TaqI at 65℃ for 3 h. Restriction frag-ments were separated by electrophoresis on 4% agarose.

Fluorescence microscopy of polyps and cryosections of tentacles

Fluorescent photomicrographs of the oral surface of polyps of the Gs and B morphs were taken under a fluo-rescent stereoscope AZ100 (Nikon) equipped with a GFP-B filter using a digital camera (Digital Sight DS-Fi1, Nikon). To compare fluorescence intensities, the fluores-cence photomicrographs were taken under the same set-tings.

To examine the distribution of GFP within a tentacle, frozen sections of tentacles were observed under a fluo-rescence microscope. Tentacles with green fluorescent pigment were easily dissected from polyps of the Gt morph (Hidaka and Yamazato 1985). Frozen sections (about 20 μm thick) of tentacles were made using a CM1100 cryostat (Leica Biosystems Nussloch GmbH, Germany) and were observed under a fluorescence micro-scope (OPTIPHOTO-2 equipped with GFP-B filter, Nikon). Fluorescent photomicrographs were obtained using a digital camera (Digital Sight DS-5M, Nikon).

Statistical analysesStatistical analyses were conducted using R software

version 3.0.1. The equality of variances was tested by F-test or Levene’s test, and the normality was tested by Shapiro-Wilk normality test. The extents of damage and recovery of the PSII of in hospite and isolated zoo xanthellae were compared between the Gs and B morphs using a two-tailed unpaired t-test. Since the data set of Fv /Fm values among different treatment of the same time points did not meet the parametric requirements, a Steel-


Dwass post hoc test was used for multiple group comparisons with absolute values of Fv /Fm. The photosynthe-tic pigment concentrations was compared between the two morphs using a twotailed unpaired ttest, while GFP content and zooxanthellae density were compared between the two morphs using a Mann-Whitney U-test.

Results

GFP contents of the Gs and B morphs of Galaxea fascicularis

Green fluorescence was observed in the tentacles

covering the septa and in part of the oral disc in the Gs morph of G. fascicularis, but no such fluorescence was observed in the B morph (Fig. 1). The GFP content of the Gs morph was significantly higher than that of the B morph (Fig. 2A), while chlorophyll a content of the B morph was significantly higher than that of the Gs morph (Fig. 2B). No significant differences in zooxanthellae cell density were observed between the two morphs (Fig. 2C).

Effects of light stress on the photochemical efficiency of in hospite and isolated zooxanthellae

The photochemical efficiency (dark-adapted Fv /Fm) of in hospite zooxanthellae decreased markedly after a 3 h

Fig. 1　Bright field and epifluorescence photographs of Gs and B morphs of G. fascicularis. (A) Colony of Gs and B morphs. Scale bar, 5 mm. (B) and (C) Photomicrographs of polyps under low voltage halogen lamp or excitation light (460-500 nm). Scale bar, 2 mm.


exposure to high light levels (1000 μmol quanta m－2s－1) in both the Gs and B morphs (Fig. 3A). No significant dif-ference in the absolute Fv /Fm was observed between the Gs and B morphs after 3 h of exposure to either high light, low light or dark conditions (Fig. 3B). After 6 h of re-covery, the Fv /Fm of in hospite zooxanthellae in both the Gs and B morphs which had been exposed to high light had recovered almost completely (Fig. 3B). High light (200 μmol m－2s－1) treatments caused significant declines

in the Fv /Fm of isolated zooxanthellae compared with dark condition in both morphs (Fig. 4A). There was no sig-nificant difference in the Fv /Fm between zooxanthellae isolated from the Gs and B morphs after 3 h exposure to high light conditions or after 6 h recovery (Fig. 4B). How-ever, in two conditions, there was significant difference in the Fv /Fm between zooxanthellae isolated from the Gs and B morphs; after 3 h exposure to dark conditions and after 6 h recovery in low light condition (Fig. 4B).

Effects of light and heat stress on the photochemical efficiency of in hospite zooxanthellae

The Fv /Fm of in hospite zooxanthellae exposed to NTHL (normal temperature and high light) and HTHL (high temperature and high light) treatments was significantly lower than those kept at the control condition NTNL (normal temperature and normal light) in both morphs (Fig. 5A). There was no significant difference in the absolute Fv /Fm of in hospite zooxanthellae between Gs and B morphs after 6 h exposure to all conditions (Fig. 5B). After 6 h recovery, significant difference was ob-served between both morphs in the condition HTNL (Fig. 5B). After 6 h stress treatment, some polyps exposed to high light treatment (NTHL and HTHL condition) opened their mouth and spew out their mesenterial filament from their mouth and tips of septal tentacles.

Zooxanthellae cladePCR-RFLP analysis showed that zooxanthellae assoc-

iated with nine colonies (4 Gs morph colonies and 5 B morph colonies) were clade D, while one Gs morph colony was associated with clade C.

Fluorescent microscopy of Gt morph tentaclesGFPcontaining cells were located in the epidermis of

the green tentacles of the Gt morph (Fig. 6A, B). The GFP were distributed just above the zooxanthellae, which were located in the gastrodermis. This was also the case for the green tentacles covering the septa of the Gs morph (photo not shown). In addition, a higher intensity of green fluo-rescence, and hence higher content of GFP, was observed on the sunlit side of the tentacles (Fig. 6C).

Fig. 2　GFP and chlorophyll contents and zooxanthellae density of the polyps of the Gs and B morphs of G. fascicularis. (A) GFP content. The GFP content was ex pressed as relative fluorescent intensity per unit soluble pro tein. (B) Chlorophyll a and c2 contents. (C) Zooxanthellae density. Means±SD (n＝5 colonies for both Gs and B morphs). The asterisk in A and B indicates significant difference (Mann-Whitney U-test, p＜0.01 and t-test, p＜0.05 respectively).


Discussion

This study showed that zooxanthellae of the highly pigmented Gs morph and non-fluorescent B morph of G. fascicularis suffered similar damage to PSII when exposed to high light, high temperature, and multiple stresses of both high light and high temperature. This suggests that the GFP of G. fascicularis is not photo protective. An alternative explanation is that the GFP has a photoprotective function, but the algal symbionts in the highly pigmented Gs morph are acclimated to shade or low light stress and

are thus more sensitive to light stress than are algal symbionts in the non-fluorescent B morph. Under this sce-nario, as a result of the opposite effects of increased host protection and increased algal symbionts sensitivity to light stress, the Gs morph may have showed similar sensitivity to light stress as the B morph. However, the present results that freshly isolated zooxanthellae from the Gs and B morphs showed similar sensitivities to light stress appear to refute the possibility that algal symbionts in the highly pigmented Gs morph are shade adapted.

Dove (2004) observed that the heavily pigmented blue morph of Acropora aspera was most sensitive to bleaching

Fig. 3　Effects of light stress on the photochemical efficiency (Fv /Fm) of in hospite zooxanthellae photosystem II of Gs and B morphs of G. fascicularis. All values are shown by the absolute Fv /Fm values. (A) Changes in the Fv /Fm of in hospite zooxanthellae of Gs morph (left) and B morph (right). Light intensities during stress treatment are 0 (◇), 200 (□), 1000 (△) μmol quanta m－2s－1. Black bars above the x-axis represent 3 h stress treatment and white bars present 6 h recovery period. (B) Impact of light stress on the Fv /Fm of in hospite zooxanthellae after 3 h stress treatment (left) and after 6 h recovery (right) are shown. Light conditions were dark (D), low light (LL) and high light (HL). Means±SD (n＝5 colonies for Gs morphs and n＝5 colonies for B morphs). There was no significant difference between the two morphs (t-test, p＞0.05).


at high temperature, whereas the host pigments were photoprotective at normal temperatures. She proposed that, in highly pigmented corals, algal symbionts are accli mated to shade, and if the photoprotection offered by host pigments is lost due to thermal stress, the shadeacclimated algal symbionts become highly sensitive to bleaching stress. Since fluorescent protein expression is downregulated by thermal stress in corals (SmithKeune and Dove 2007; Desalvo et al. 2008; Rodriguez-Lanetty et al. 2009), the above explanation is plausible in the case of A. aspera. However, the present results show that GFP had no influence on the degree of photoinhibition to PSII

of the algal symbionts, even at normal temperature (26℃), and that freshly isolated zooxanthellae from the Gs and B morphs showed similar sensitivities to light stress. The present findings are not consistent with the above expla-nation.

Four Gs and five B morph colonies of G. fascicularis used for the combined light and temperature stress experiment were associated with clade D zooxanthellae, and only one Gs morph colony was associated with clade C zooxanthellae. Even if the data of the clade C containing Gs morph colony was excluded from the analysis, results were similar and no significant difference was found in

Fig. 4　Effects of light stress on the photochemical efficiency (Fv /Fm) of isolated zooxanthellae photosystem II. All values are shown by the absolute Fv /Fm. (A) Changes in the Fv /Fm of isolated zooxanthellae of Gs morph (left) and B morph (right). Light intensities during stress treatment are 0 (◇), 50 (□), 200 (△) μmol quanta m－2s－1. Black bar above the x-axis represent 3 h stress treatment and white bar present 6 h recovery period. (B) Impacts of light stress on the Fv /Fm of isolated zooxanthellae after 3 h stress treatment (left) and after 6 h recovery (right) are shown. Light conditions were dark (D), low light (LL) and high light (HL). Means±SD (n＝5 colonies for Gs morphs and n＝5 colonies for B morphs). The asterisk indicates significant difference between the two morphs (ttest, p＜0.05).


the Fv /Fm values after high light or combined high light and high temperature stress treatment between the two morphs. This also supports that zooxanthellae associated with the two color morphs have similar stress sensitivity and that the GFP is not photoprotective in the coral.

Two species of sea anemones, Anemonia sulcata and A. rustica, had markedly different contents of GFP, but both species suffered similar extents of bleaching during a natural bleaching event (Leutenegger et al. 2007). It was concluded that GFP had no influence on the degree of bleaching between the two anemone species. Various color morphs of the same coral species have been observed under the same light conditions side by side (Takabayashi

and Hoegh-Guldberg 1995; Salih et al. 2000). Further-more, Mazel et al. (2003) found no significant correlation be tween GFP concentration and depth, while the concentration of mycosporine-like amino acids (MAAs), UV-absorbing pigments, was significantly correlated with depth (Dunlap et al. 1986). These observations do not support a photoprotective function of GFP and are consistent with the present results.

However, there are several studies that support a photoprotective function of GFP. GFP is reported to protect algal symbiont PSII from photoinhibition caused by excess sunlight (Salih et al. 2000). Since chlorophyll a has its absorption peak at a wavelength of around 430 nm

Fig. 5　Effects of light and heat stress on the photochemical efficiency (Fv /Fm) of in hospite zooxanthellae photosystem II. All values are shown by the absolute Fv /Fm. (A) Changes in the Fv /Fm of in hospite zooxanthellae of Gs morph (left) and B morph (right). Stress conditions are NTNL (○), HTNL (◇), NTHL (□), HTHL (△). Black bar above the x-axis represent 6 h stress treatment and white bar present 6 h recovery period. (B) Impacts of light stress on the Fv /Fm of in hospite zooxanthellae after 6 h stress treatment (left) and after 6 h recovery (right) are shown. Means±SD (n＝5 colonies for Gs morphs and n＝5 colonies for B morphs). The asterisk indicates significant difference between the two morphs (t-test, p＜0.05).


(blue light), strong blue light causes photoinhibition (decrease in the Fv /Fm) to a greater extent than do green or red light (Oguchi et al. 2011). Since GFP absorbs blue light and emits green light, GFP is considered to have photoprotective function, especially against blue light. In the greenpigmented tentacles of G. fascicularis, GFP is located in the epidermis above the zooxanthellae and the content of GFP was higher on the sunlit side of the tentacles than on the shaded side. This is consistent with the report by Roth et al. (2010) that strong light up-regulates the expression of fluorescent proteins in Acropora yongei. It is likely that GFP expression in G. fascicularis is influenced by light and that GFP may have a function related to the response of the coral to its light environment. Zooxanthellae of the two color morphs of G. fascicularis with different GFP contents suffered similar photoinhibition by high light stress. This suggests that GFP does not have photoprotective function in the coral G. fascicularis. Further studies are needed to clarify the function of fluorescent proteins in G. fascicularis.

Acknowledgements

The authors would like to thank the staff of Sesoko Station, Tropical Biosphere Research Center, University of the Ryukyus, for allowing us to use the facilities. This work was partly supported by International Research Hub Project for Climate Change and Coral Reef/Island Dy-

namics of University of the Ryukyus.

References

Alieva NO, Konzen KA, Field SF, Meleshkevitch EA, Hunt ME, Beltran-Ramirez V, Miller DJ, Wiedenmann J, Salih A, Matz MV (2008) Diversity and evolution of coral fluo-rescent proteins. PLoS One 3: e2680

Bhagooli R, Hidaka M (2003) Comparison of stress susceptibil-ity of in hospite and isolated zooxanthellae among five coral species. J Exp Mar Biol Ecol 291: 181-197

Bou-Abdallah F, Chasteen ND, Lesser MP (2006) Quenching of superoxide radicals by green fluorescent protein. Biochim Biophys Acta 1760: 1690-1695

DeSalvo MK, Voolstra CR, Sunagawa S, Schwarz JA, Stillman JH, Coffroth MA, Szmant AM, Medina M (2008) Differ-ential gene expression during thermal stress and bleaching in the Caribbean coral Montastraea faveolata. Mol Ecol 17: 3952-3971

Dove S, Hoegh-Guldberg O, Ranganathan S (2001) Major colour patterns of reefbuilding corals are due to a family of GFP-like proteins. Coral Reefs 19: 197-204

Dove S, (2004) Scleractinian corals with photoprotective host pigments are hypersensitive to thermal bleaching. Mar Ecol Prog Ser 272: 99-116

Dove SG, Lovell C, Fine M, Deckenback J, Hoegh-Guldberg O, Iglesias-Prieto R, Anthony KRN (2008) Host pigments: potential facilitators of photosynthesis in coral symbioses. Plant Cell Environ 31: 1523-1533

Fig. 6　Gt morph colony and tentacles of G. fascicularis. (A) A Gt morph colony. Scale bar, 5 mm. (B) Epifluorescence micrograph of a frozen section of a Gt morph tentacle. Scale bar, 50 μm. EC, ectoderm (epider-mis); En, endoderm. (C) Bright field (upper) and epifluorescence (lower) photomicrographs of Gt morph tentacles. Arrows indicate the sunlit side. Scale bar, 0.5 mm. Epifluorescence microphotographs were taken under excitation light (460-500 nm).


Dunlap W, Chalker B, Oliver J (1986) Bathymetric adaptations of reefbuilding corals at Davies Reef Great Barrier Reef Australia III UV-B absorbing compounds. J Exp Mar Biol Ecol 104: 239-248

D’Angelo C, Denzel A, Vogt A, Matz M, Oswald F, Salih A, Nienhaus G, Wiedenmann J (2008) Blue light regulation of host pigment in reef-building corals. Mar Ecol Prog Ser 364: 97-106

Hidaka M, Yamazato K (1985) Color morph of Galaxea fascicularis found in the reef around the Sesoko Marine Science Center. Galaxea 4: 33-35

Hollingsworth L, Krupp D, Lewis T, Leong J (2004) Early onset and expression of fluorescent protein in the larvae of the mashroom coral Fungia scutaria Lamarck 1801. Proc 10 Int Coral Reef Symp: 99-105

Hollingsworth LL, Kinzie RA, Lewis TD, Krupp DA, Leong J-AC (2005) Phototaxis of motile zooxanthellae to green light may facilitate symbiont capture by coral larvae. Coral Reefs 24: 523-523

Jeffrey SW, Humphrey GF (1975) New spectrophotometric equations for determining chlorophylls a, b, c1 and c2 in higher plants, algae and natural phytoplankton. Biochem Physiol Pflanz 167: 191-194

Johannes RE, Wiebe WJ (1970) A method for determination of coral tissue biomass and composition. Limnol Oceanogr 21: 540-547

Kao H, Sturgis S, DeSalle R, Tsai J, Davis D (2007) Dynamic regulation of fluorescent proteins from a single species of coral. Mar Biotechnol 9: 733-746

Kenkel CD, Traylor MR, Wiedenmann J, Salih A, Matz MV (2011) Fluorescence of coral larvae predicts their settlement response to crustose coralline algae and reflects stress. Proc R Soc B 278: 2691-2697

Labas YA, Gurskaya NG, Yanushevich YG, Fradkov AF, Lukyanov KA, Lukyanov SA, Matz MV (2002) Diversity and evolution of the green fluorescent protein family. Proc Natl Acad Sci USA 99: 4256-4261

Leutenegger A, Kredel S, Gundel S, D’Angelo C, Salih A, Wiedenmann J (2007) Analysis of fluorescent and non-fluorescent sea anemone from Mediterranean Sea during a bleaching event. J Exp Mar Biol Ecol 353: 221-234

Mazel CH, Lesser MP, Gorbunov MY, Barry TM, Farrel JH, Wyman KD, Falkowski PG (2003) Green-fluorescent pro-

teins in Caribbean corals. Limnol Oceanogr 48: 402-411 345-350

Oguchi R, Terashima I, Kou J, Chow WS (2011) Operation of dual mechanisms that both lead to photoinactivation of photosystem II in leaves by visible light Physiol Plant 142: 47-55

Palmer CV, Modi CK, Mydlarz LD (2009) Coral fluorescent proteins as antioxidants. PloS One 4: e7298

Rodriguez-Lanetty M, Harii S, Hoegh-Guldberg O (2009) Early molecular responses of coral larvae to hyperthermal stress. Mol Ecol 18: 5101-5114

Roth MS, Latz MI, Goericke R, Deheyn DD (2010) Green fluorescent protein regulation in the coral Acropora yongei during photoacclimation. J Exp Biol 213: 3644-3655

Roth MS, Deheyn DD (2013) Effect of cold stress and heat stress on coral fluorescence in reef-building corals. Sci Rep 3: 1421 doi: 101038/srep01421

Salih A, Larkum A, Cox G, Kuhl M, Hoegh-Guldberg O (2000) Fluorescent pigments in corals are photoprotective. Nature 408: 850-853

Shinzato C, Shoguchi E, Tanaka M, Satoh N (2012) Fluorescent protein candidate genes in the coral Acropora digitifera genome. Zool Sci 29: 260-264

Smith EG, D’Angelo C, Salih A, Wiedenmann J (2013) Screen-ing by coral green fluorescent protein (GFP)-like chromo-proteins supports a role in photoprotection of zooxanthellae. Coral Reefs 32: 463-474

Smith-Keune C, Dove S (2008) Gene expression of a green fluorescent protein homolog as a host-specific biomarker of heat stress within a reef-building coral. Mar Biotechnol 10: 166-180

Takabayashi M, Hoegh-Guldberg O (1995) Ecological and physiological differences between two colour morphs of the coral Pocillopora damicornis. Mar Biol 123: 705-714

Zardoya R, Costas E, Lopez-Rodas V, Garrido-Pertierra A, Bautista JM (1995) Revised Dinoflagellate phylogeny in-ferred from molecular analysis of largesubunit ribo somal RNA gene sequences. Mole Evol 41: 637-645

Received: 23 May 2014Accepted: 2 December 2014

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Fluorescent protein content and stress tolerance of two color morphs of the coral Galaxea fascicularis