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Comparative Biochemistry and Physio

Differential susceptibility to oxidative stress of two scleractinian corals:

antioxidant functioning of mycosporine-glycine

I. Yakovlevaa,b,*, R. Bhagoolia, A. Takemurac, M. Hidakaa

aDepartment of Chemistry, Biology and Marine Science, University of the Ryukyus, Nishihara, Okinawa 903-0213, JapanbInstitute of Marine Biology, Far East Branch of Russian Academy of Sciences, Palchevskogo, 17 Vladivostok 690041, Russia

cSesoko Station, Tropical Biosphere Research Center, University of the Ryukyus, Sesoko 3422, Motobu-cho, Okinawa 905-0227, Japan

Received 11 May 2004; received in revised form 21 August 2004; accepted 24 August 2004

Abstract

This study examined the importance of mycosporine-glycine (Myc-Gly) as a functional antioxidant in the thermal-stress susceptibility

of two scleractinian corals, Platygyra ryukyuensis and Stylophora pistillata. Photochemical efficiency of PSII (Fv/Fm), activity of

antioxidant enzymes, superoxide dismutase (SOD) and catalase (CAT), and composition and abundance of mycosporine-like amino acids

(MAAs) in the coral tissue and in symbiotic zooxanthellae were analyzed during 12-h exposure to high temperature (33 8C). After 6- and12-h exposures at 33 8C, S. pistillata showed a significantly more pronounced decline in Fv/Fm compared to P. ryukyuensis. A 6-h

exposure at 33 8C induced a significant increase in the activities of SOD and CAT in both host and zooxanthellae components of S.

pistillata while in P. ryukyuensis a significant increase was observed only in the CAT activity of zooxanthellae. After 12-h exposure, the

SOD activity of P. ryukyuensis was unaffected in the coral tissue but slightly increased in zooxanthellae, whereas the CAT activity in the

coral tissue showed a 2.5-fold increase. The total activity of antioxidant enzymes was significantly higher in S. pistillata than in P.

ryukyuensis, suggesting that P. ryukyuensis is less sensitive to oxidative stress than S. pistillata. This differential susceptibility of the

corals is consistent with a 20-fold higher initial concentration of Myc-Gly in P. ryukyuensis compared to S. pistillata. In the coral tissue

and zooxanthellae of both species investigated, the first 6 h of exposure to thermal stress induced a pronounced reduction in the

abundance of Myc-Gly but not in other MAAs. When exposure was prolonged to 12 h, the Myc-Gly pool continued to decrease in P.

ryukyuensis and was completely depleted in S. pistillata. The delay in the onset of oxidative stress in P. ryukyuensis and the dramatic

increase in the activities of the antioxidant enzymes in S. pistillata, which contains low concentrations of Myc-Gly suggest that Myc-Gly

provides rapid protection against oxidative stress before the antioxidant enzymes are induced. These findings strongly suggest that Myc-

Gly is functioning as a biological antioxidant in the coral tissue and zooxanthellae and demonstrate its importance in the survival of reef-

building corals under thermal stress.

D 2004 Elsevier Inc. All rights reserved.

Keywords: Coral; CAT; Mycosporine-like amino acids; Mycosporine-glycine; SOD; Thermal stress; Zooxanthellae

1. Introduction

Reef-building corals that harbor endosymbiotic dino-

flagellates (=zooxanthellae) are frequently subjected to a

variety of abiotic stresses: unusually high seawater temper-

1532-0456/$ - see front matter D 2004 Elsevier Inc. All rights reserved.

doi:10.1016/j.cbpc.2004.08.016

* Corresponding author. Institute of Marine Biology, Far East Branch

of Russian Academy of Sciences, Vladivostok 690041, Russia. Tel.: +7

4232 310931; fax: +7 4232 310900.

E-mail address: yakovleva72@mail.ru (I. Yakovleva).

atures, high doses of photosynthetically active radiation

(PAR) and ultraviolet light, changes in salinity, or a

combination of these factors, as they inhabit shallow-water

or intertidal marine environments (Brown, 1997). These

stresses can induce increased production of reactive oxygen

species (ROS) leading to significant oxidative damage to

the coral–algal complex (Lesser et al., 1990; Shick et al.,

1996; Downs et al., 2000, 2002). The susceptibility of

marine invertebrates to oxidative stress are related to

differences in the activity of specific molecular mechanisms

logy, Part B 139 (2004) 721–730

I. Yakovleva et al. / Comparative Biochemistry and Physiology, Part B 139 (2004) 721–730722

that prevent the production of ROS or protect against

their damaging effects (Regoli et al., 2000; Downs et al.,

2002).

Antioxidant defenses include the enzymes superoxide

dismutase (SOD), catalase (CAT), and ascorbate and

glutathione peroxidases, the first of which detoxifies O2�

and the others H2O2 (Halliwell and Gutteridge, 1999).

Light-dependent production of ROS and its control on

SOD activity in zooxanthellate sea anemone has been well

documented (Dykens and Shick, 1982; Dykens et al.,

1992). Various lipid-soluble (carotenoids, tocopherols) and

water-soluble (ascorbic acid, uric acid, gadusol) antiox-

idant molecules scavenge oxygen radicals or quench 1O2

(Dunlap et al., 1999, 2000). In addition, in vitro experi-

ments have shown a concentration-dependent ability of

some mycosporine-like amino acids (MAAs), mycospor-

ine-glycine (Myc-Gly) and its functional analogue, myco-

sporine-taurine, to inhibit lipid peroxidation in aquatic

extracts of marine organisms (Dunlap and Yamamoto,

1995) and to scavenge 1O2 generated from certain

endogenous photosensitizers (Suh et al., 2003), although

MAAs are commonly known as UVR-screening filters

(reviewed by Shick et al., 2000). Our previous studies on

shallow-water corals showed that Myc-Gly is a major

component of MAAs in two bleaching-tolerant corals and

its concentrations decreases during daytime, suggesting

that Myc-Gly functions as an antioxidant in the corals

(Yakovleva and Hidaka, 2004). If Myc-Gly functions as

antioxidant in vivo, one would expect that high concen-

trations of MAAs, especially Myc-Gly, to potentially

provide some relief against photodynamically generated

ROS and affect the susceptibility of coral species to

environmental stresses. However, the antioxidant function

of some MAAs in vivo remains to be tested. In this study,

we compared the susceptibility of two shallow-water

scleractinian corals, Platygyra ryukyuensis and Stylophora

pistillata, to oxidative stress mediated by elevated temper-

ature and focused particularly on the changes in the

concentration of MAAs in the coral tissue and zooxan-

thellae of these species during stress. Chlorophyll a

fluorescence and activity of two enzymes—superoxide

dismutase (SOD) and catalase (CAT)—were measured in

order to assess the physiological response of corals to

elevated temperature stress.

2. Materials and methods

2.1. Collection and maintenance of corals

Colonies of S. pistillata (Esper 1797) and P.

ryukyuensis (Yabe and Sugiyama 1935) were collected

from a depth of 0.5–1 m at the reef of Aka Island and

Sesoko Island, Okinawa, Japan, respectively. Colonies

were kept in an outdoor tank supplied with running

seawater for 2 months until use to stabilize their

physiological parameters. During this period, seawater

temperature in the tank was 24–25 8C and incident

photosynthetically active radiation (PAR) did not exceed

1500 Amol photons m�2 s�1. The tank was shaded by

plastic black mesh to reduce the light intensity to 20%

of incident PAR.

2.2. Experimental design

The experiments were conducted at the Sesoko Station

of Tropical Biosphere Center of the University of the

Ryukyus in February 2003. Branches about 3 cm in length

were taken from colonies of S. pistillata, and fragments

about 4 cm2 of tissue surface area were removed from P.

ryukyuensis. Four replicate colonies were used for each

species. Coral fragments were prepared from each coral

colony and mounted on glass slides using rubber bands.

The coral pieces mounted on glass slides were left in the

same outdoor tank for 3–5 days to recover from damage

during fragmentation. Then, the initial measurements of

chlorophyll a fluorescence (Fv/Fm), activity of antioxidant

enzymes superoxide dismutase (SOD) and catalase (CAT),

and MAA content were performed with four fragments

each from a different colony for both species. Remaining

fragments were placed in transparent plastic chambers

(30�40 cm) supplied with running seawater and used as

the experimental units. The chambers with experimental

fragments were put into one of the water baths whose

temperature was regulated at 26 or 33 8C, by temperature

control units (EYELA, Thermister Tempet T-80). The

experimental fragments were exposed to 300 Amol

photons m�2 s�1 of PAR for 12 h. PAR treatment was

carried out using one 500-W incandescent lamp (NIKKO,

Tokyo, Japan). Visible irradiance was measured using a

lightmeter (LICOR, LI-250). Fv/Fm, activities of SOD and

CAT, and MAA content were again measured after 6- and

12-h stress treatment using fragments from different

colonies for both species. The number of replicates was

four.

2.3. Chlorophyll a fluorescence measurements

Chlorophyll fluorescence was measured using a pulse-

amplitude-modulation fluorometer (MINI-PAM, Waltz,

Germany). Coral fragments were taken from the exper-

imental chamber and placed in a custom-made black box

with filtered (0.45 Am) seawater and dark adapted for 20

min. The initial fluorescence (F0) was measured by

exposing the coral to weak red light (b1 Amol photons

m�2 s�1). Maximum fluorescence (Fm) was determined by

applying a 0.8-s saturation pulse of intense white light

(8000 Amol photons m�2 s�1). The ratio of variable (Fv) to

maximum fluorescence (Fm), Fv/Fm, was used as an

indicator of the maximum potential quantum yield. The

dark-adapted quantum yield (Fv/Fm, Schreiber et al., 1994)

provides a good approximation of the maximum photo-

I. Yakovleva et al. / Comparative Biochemistry and Physiology, Part B 139 (2004) 721–730 723

chemical efficiency of photosystem II (PSII) (Oquist et al.,

1992). The Fv/Fm values of coral fragments before stress

treatment ranged from 0.58 to 0.65, typical of those in

corals experiencing no damage to PSII (e.g., Fitt et al.,

2001).

2.4. Isolation of zooxanthellae

Coral fragments were divided in two pieces. One of them

was used for MAA analysis in the intact coral and the other

for the isolation of symbiotic algae. For isolation procedure,

coral fragments were rinsed gently with 0.45-Am filtered

seawater (FSW) and then coral tissue was removed from the

coral skeleton by Water-Pik with FSW (Johannes and

Wiebe, 1970). To remove the mucus produced during the

isolation procedure, coral blastate was first filtered through a

350-Am nylon mesh. The blastate was then homogenized

with a potter homogenizer. Following centrifugation at

1500�g for 5 min, filtration through 40-Am mesh was

performed. The algal pellet was washed five times by

centrifugation [1500�g for 5 min, 2000�g for 10 min

(twice) and 3000�g for 15 min (twice)]. This method of

isolating and cleaning zooxanthellae yields intact algal cells.

This was checked microscopically under 400� magnifica-

tion using a Nikon OPTIPHOT-2 microscope. Freshly

isolated zooxanthellae were adsorbed on Millipore filters

(0.45 Am) by use of a syringe and processed for MAA

analysis.

2.5. MAA analysis

Coral fragments were extracted with 5 ml of 70% of

HPLC-grade absolute methanol for 16 h at 4 8C. Similar

extraction procedures were used on isolated, cleaned

zooxanthellae. Methanol extracted skeleton and filters with

algal cells were stored at �20 8C for later analysis of total

protein content, following the procedure of Bradford (1976),

with bovine gamma globulin as a standard. The UV spectra

of methanol extracts were recorded with a Hitachi model

2000 scanning spectrophotometer for the presence of

absorption peaks in the wavelength range 250–400 nm.

Extracts were then centrifuged for 5 min at 3000�g to

remove particles and passed through two C-18 Sep-Pak Plus

cartridges (Water) in series to remove chromatographically

intractable material. The supernatant was then evaporated to

dryness at 30 8C using a centrifugal vacuum evaporator

(CVE-100, EYELA) and temporarily stored at �20 8Cbefore analysis.

MAA analysis was performed by slightly modifying the

procedures described in Dunlap and Chalker (1986) and

Shick et al. (1992). Using a Shimadzu HPLC system,

MAAs were separated by reverse-phase isocratic HPLC on

a Nucleosil 100 C8 column (25 cm, 4.6 Am) protected

with a Nucleosil C8 guard (4.6 mm i.d.�10 mm length)

filled with the same material, in an aqueous mobile phase

of 0.1% acetic acid and 25% methanol. The flow rate was

0.7 ml min�1, and absorbance of the eluate was measured

online at 313 and 330 nm. Individual peaks were

identified by absorption, retention time and co-chromatog-

raphy with prepared standards from the zoanthid Palythoa

tuberculosa (mycosporine-glycine, palythine and palythi-

nol), the sea anemone Anthopleura uchidai (mycosporine-

2 glycine), and the red algae Porphyra sp. (shinorine and

phorphyra-334), Chondrus crispus (palythine, asterina-

330), and Mastocarpus stellatus (shinorine). Peaks were

baseline corrected and integrated before concentrations

were calculated using available extinction coefficients

(Shick et al., 1992; Karsten et al., 1998). The MAA

concentration in each sample was normalized to the

amount of protein in the coral fragments or in the isolated

algal cells.

2.6. Analysis of antioxidant enzymes

For the analyses of superoxide dismutase (SOD) and

catalase (CAT), fragments of corals were crashed in liquid

nitrogen and homogenized in ice-cold 50 mM potassium

phosphate buffer (pH 7.8) containing protease inhibitor

cocktail (Sigma cat. no. P8340). The pellet containing the

isolated zooxanthellae was prepared similarly as zooxan-

thellae for MAA analysis, except that phosphate buffer

was used instead of filtered seawater and cleaned

zooxanthellae were resuspended in phosphate buffer.

Extracts were sonicated for 30 min on ice and centrifuged

at 14000�g for 5 min at 3 8C. The resulting supernatants

were frozen in liquid nitrogen and stored at �80 8C for

later analysis of total protein content, following the

procedure of Bradford (1976), and for assay of SOD and

CAT activities.

Catalase was measured by the depletion of H2O2 at 240

nm (Beers and Sizer, 1952). Measurements were carried

out with a Hitachi model 2000 scanning spectrophotometer

at a constant temperature of 25 8C. The assay was

performed with 30% H2O2 in 50 mM potassium phosphate

buffer pH 7.8.

The activity of total SOD was estimated according to the

method of Elstner and Heupel (1976) and Oyanagui (1984).

Standards were prepared using bovine erythrocyte SOD

(Wako cat. no. 190-08771). It was determined by its ability

to inhibit the reduction of cytochrome c by superoxide

anions generated from the xanthine oxidase/hypoxanthine

system. The reduction of cytochrome c was monitored in 50

mM potassium phosphate buffer pH 7.8, 0.5 mM EDTA, 1

mM hypoxanthine, 10�5 M cytochrome c, and 2.5 mU/ml

xanthine oxidase, and different volume of samples were

used to determine the 50% inhibition of reaction rate. The

rate of reduction of cytochrome c was followed spectro-

photometrically at 550 nm, 25 8C. One unit of SOD was

defined as the amount of enzyme inhibiting by 50% the

reduction of cytochrome c. Results for SOD and CAT are

expressed as units (U) of enzyme activity per milligram

protein.

Fig. 1. Changes in the relative Fv/Fm in the in hospite zooxanthellae of P.

ryukyuensis and S. pistillata exposed to 33 8C for 6- or 12- h under

moderate light (300 Amol photons m�2 s�1). Relative Fv/Fm values,

normalized to the initial values before stress treatment, are shown.

MeansFS.D. (n=4).

I. Yakovleva et al. / Comparative Biochemistry and Physiology, Part B 139 (2004) 721–730724

2.7. Statistical analyses

Differences between experimental and initial mean

values were analyzed using a t-test. For assessing the

Fig. 2. Effects of temperature on the activity of superoxide dismutase (SOD) in the co

(C, D) under moderate light (300 Amol photons m�2 s�1). SOD activities before an

effects of temperature on Fv/Fm, SOD and CAT activities,

and MAA concentrations ANOVA followed by Tukey

multiple comparisons were used. In some cases, data were

log-transformed.

3. Results

3.1. Changes in dark-adapted Fv/Fm

Coral fragments of P. ryukyuensis and S. pistillata

exposed to 26 8C under moderate light (300 Amol photons

m�2 s�1) showed no significant change (t-test, pN0.05) in

photochemical efficiency of PSII, Fv/Fm, during the

experimental period (data not shown). However, after

exposure at 33 8C, Fv/Fm decreased significantly ( pb0.05)

in both corals (Fig. 1). The reduction in Fv/Fm was

significantly (ANOVA, pb0.05) more pronounced in S.

pistillata than in P. ryukyuensis. After 6-h exposure, the Fv/

Fm was 85% of the initial value in P. ryukyuensis and 55%

in S. pistillata (Fig. 1). After 12-h exposure, the Fv/Fm was

about 70% and 20% of the initial value in P. ryukyuensis

and S. pistillata, respectively (Fig. 1).

3.2. Changes in antioxidant enzymes

To determine if acute thermal stress causes oxidative

stress in P. ryukyuensis and S. pistillata, we measured the

ral tissue (A) and zooxanthellae (B) ofP. ryukyuensis and those of S. pistillata

d after 6- or 12-h treatment at 26 or 33 8C are shown. MeansFS.D. (n=4).

Fig. 3. Effects of temperature on the activity of catalase (CAT) in the coral tissue (A) and zooxanthellae (B) of P. ryukyuensis and those of S. pistillata (C, D)

under moderate light (300 Amol photons m�2 s�1). CAT activities before and after 6- or 12-h treatment at 26 or 33 8C are shown. MeansFS.D. (n=4).

I. Yakovleva et al. / Comparative Biochemistry and Physiology, Part B 139 (2004) 721–730 725

activity of SOD (Fig. 2) and CAT (Fig. 3) in the coral tissue

and isolated zooxanthellae after exposure at ambient (26 8C)and elevated temperature (33 8C). For both coral species,

there were no significant differences in SOD and CAT

activities of the coral tissue (t-test, pN0.05) and zooxanthel-

lae (t-test, pN0.05) before and after exposure at 26 8C (Figs.

2 and 3). In contrast, elevated temperature had a significant

effect on enzymatic activity of P. ryukyuensis (ANOVA,

pb0.05) and S. pistillata (ANOVA, pb0.05) but the response

was different between these coral species.

In P. ryukyuensis, the SOD activity of the coral tissue

was unaffected (ANOVA, pN0.05) by elevated temperature

during the whole exposure time (Fig. 2A), while in S.

pistillata, it increased by 4-fold after the first 6 h of stress

exposure and remained at that level for the next 6 h (Fig.

2C). In zooxanthellae of P. ryukyuensis, the SOD activity

increased slightly (but significantly, pb0.05) only after the

12-h stress treatment (Fig. 2B). Zooxanthellae of S.

pistillata showed a more pronounced increment (6-fold,

pb0.05) in the SOD activity already after the 6-h stress

treatments, with a further 50% decline ( pb0.05) after 12 h

exposure (Fig. 2D).

The response of the CAT activity in the coral tissue and

zooxanthellae to elevated temperature also differed signifi-

cantly (ANOVA, pb0.05) between species investigated (Fig.

3). In P. ryukyuensis, the CAT activity of the coral tissue

remained unaffected (ANOVA, pN0.05) during the first 6 h

of stress treatment, but after 12 h, a 2.5-fold increase

(ANOVA, pb0.05) was observed (Fig. 3A). In the coral

tissue of S. pistillata, the CAT activity was increased by

about 1.8-fold of the initial value within 6 h and further by

2.7-fold ( pb0.05) after 12 h (Fig. 3C). The CAT activity in

the zooxanthellae of both coral species was also signifi-

cantly ( pb0.05) increased under elevated temperature. After

6-h exposure, the enhancement of CAT activity was

significantly ( pb0.05) higher in the algae of S. pistillata

than those of P. ryukyuensis (Fig. 3B,D). Exposure for 12 h

resulted in a slight (but significant, pb0.05) reduction in

zooxanthellae CAT activity for both species investigated but

it was still significantly (ANOVA, pN0.05) higher in S.

pistillata than in P. ryukyuensis.

3.3. MAA profiles and changes in MAA content

Table 1 shows the composition and initial content of

MAAs in the coral tissue and isolated zooxanthellae of P.

ryukyuensis and S. pistillata. The typical set of MAAs

identifiable in the coral tissue of both species included

shinorine, porphyra-334, mycosporine-glycine (Myc-Gly),

mycosporine-2glycine (Myc-2Gly), palythine, palythinol,

and asterina-330. Additionally, P. ryukyuensis tissue con-

tained palythene and an unidentified UV-absorbing sub-

stance, which was not studied further. The extracts of

symbiotic algae of S. pistillata contained four MAAs:

porphyra-334, Myc-Gly, Myc-2Gly, and palythine. How-

ever, only two MAAs were presented in quantifiable

Table 1

Composition and initial content of mycosporine-like amino acids in the coral tissue and isolated zooxanthellae (Zx) of two scleractinian corals P. ryukyuensis

and S. pistillata from Okinawa, Japan

Species Mycosporine-like amino acids (nmol/mg protein)

SH PR M2G PN PL AS PE MG

Platygyra ryukyuensis Tissue 0.14F0.06 2.08F0.14 0.14F0.02 34.23F6.16 1.75F0.42 1.31F0.49 0.86F0.30 12.67F4.64

Zx – – – 2.84F1.27 – – – 1.22F0.30

Stylophora pistillata Tissue 4.03F.83 7.15F3.78 12.30F0.99 5.52F0.46 2.41F0.83 10.51F3.31 – 0.52F0.01

Zx – 0.20F0.22 0.25F0.04 1.18F0.87 – – – 0.55F0.19

SH—shinorine; PR—porphyra-334; M2GUmycosporine-2-glycine; PN—palythine; PL—palythinol; AS—asterina-330; PE—palythene; MG—mycosporine-

glycine.

I. Yakovleva et al. / Comparative Biochemistry and Physiology, Part B 139 (2004) 721–730726

amounts in isolated zooxanthellae of P. ryukyuensis: Myc-

Gly and palythine.

Palythine was the most abundant MAA both in the coral

tissue and isolated zooxanthellae of P. ryukyuensis

(64.4F7.4% and 70.0F9.7%, respectively), followed by

Myc-Gly (23.8F4.1% and 30.1F0.1%, respectively). In

contrast, the most abundant MAA in the coral tissue of S.

pistillata was Myc-2Gly (29.0F3.2%), followed by aster-

ina-330 (24.8F2.6%), porphyra-334 (16.9F0.8%), and

palythine (13.0F1.2%). The concentration of the remaining

MAA species were low and collectively did not contribute

more than 11.8F0.3% and 16.4F0.3% to the total pool in

the coral tissue of P. ryukyuensis and S. pistillata,

respectively. In symbiotic algae of S. pistillata, palythine

Fig. 4. Effects of temperature on the content of individual mycosporine-like amino

moderate light (300 Amol photons m�2 s�1). Coral fragments were exposed to 2

exposures, respectively. Relative MAA values, normalized to the initial values be

made the highest contribution to the MAA pool

(54.1F8.7%).

For both investigated coral species, there were no

significant ( pN0.05) changes in the concentration of MAAs

in the coral tissue and zooxanthellae after 6- and 12-h

exposure at 26 8C (Figs. 4A,B and 5A,B). However, when

P. ryukyuensis and S. pistillata were treated at 33 8C, asignificant ( pb0.05) decline in the abundance of oxo-

carbonyl MAA, Myc-Gly, was observed both in the coral

tissue and zooxanthellae already after the 6-h stress treat-

ment, while the concentrations of imino-MAAs were almost

constant (Figs. 4C,D and 5C,D). In P. ryukyuensis, Myc-Gly

decreased only to 40% and 60% in the coral tissue and

zooxanthellae, respectively. In S. pistillata, the decrease of

acids (MAAs) in the coral tissue and zooxanthellae of P. ryukyuensis under

6 8C (A, B) or 33 8C (C, D). Light and black bars represent 6- and 12-h

fore stress treatment, are shown. MeansFS.D. (n=4).

Fig. 5. Effects of temperature on the content of individual mycosporine-like amino acids (MAAs) in the coral tissue and zooxanthellae of S. pistillata under

moderate light (300 Amol photons m�2 s�1). Details as in Fig. 4.

I. Yakovleva et al. / Comparative Biochemistry and Physiology, Part B 139 (2004) 721–730 727

Myc-Gly was drastic, about 80–90% in both the coral tissue

and the algae. Exposure to 33 8C for 12 h induced a

significantly ( pb0.05) more pronounced degradation of

Myc-Gly both in the coral tissue and symbiotic algae of P.

ryukyuensis compared to the 6-h stress treatment, represent-

ing a decrease by 60% and 75%, respectively.

HPLC analysis did not show any traces of Myc-Gly both

in the coral tissue and zooxanthellae of S. pistillata after 12-

h stress treatment. The concentration of imino-MAAs did

not show any significant ( pN0.05) change both in the coral

tissue and zooxanthellae of P. ryukyuensis exposed to 33 8Cfor 12 h, while it showed some tendency to drift downward

in the coral tissue and zooxanthellae of S. pistillata.

4. Discussion

Our results clearly showed that P. ryukyuensis was more

tolerant to the 12-h thermal stress compared with S.

pistillata. This is evident because, despite a significant

reduction in photochemical efficiency of PSII, Fv/Fm, in

zooxanthellae of both species under elevated temperature,

the decrease in Fv/Fm was greater in S. pistillata than in P.

ryukyuensis. Similar differential decline in Fv/Fm in the

two studied corals has also been reported previously

(Bhagooli and Hidaka, 2004). Such a decrease in Fv/Fm

during stress has been considered to represent damage to

photosynthetic machinery of zooxanthellae (Jones et al.,

2000; Warner et al., 1996, 1999) via an imbalance between

degradation and resynthesis of D1 reaction center protein,

causing accumulation of nonfunctional PSII (Vasilikiotis

and Melis, 1994). Damage to the D1 protein of PSII has

been shown to involve active oxygen (Tschiersch and

Ohmann, 1993). Furthermore, oxygen toxicity has been

demonstrated to be a mechanism for the decrease in

photosynthetic efficiency observed in zooxanthellae

exposed to thermal stress because elevated temperature

causes an increase in the flux of these reduced oxygen

products (Lesser, 1996). In fact, both tested species

responded to elevated temperature exposure by exhibiting

increased activities of the protective enzymes superoxide

dismutase (SOD) and catalase (CAT) (Figs. 2 and 3).

Increases in the activities of these enzymes are considered

as a response to high concentrations of reactive oxygen

species (ROS), indicating oxidative stress (Halliwell and

Gutteridge, 1999). This was clearly demonstrated by the

early findings that, in the temperate endosymbiotic sea

anemone Anthopleura elegantissima (Dykens and Shick,

1982; Dykens et al., 1992) and various algal–invertebrate

symbioses (Shick and Dykens, 1985), host SOD activity

changed in response to the amount of O2 generated

photosynthetically by its algal symbionts. Under exposure

to thermal stress, the SOD and CAT activities were more

than 2-fold higher in the coral tissue and zooxanthellae of

S. pistillata than those in P. ryukyuensis. Thus, our

observations suggest a higher level of oxidative stress for

S. pistillata, matching a higher sensitivity of this species to

elevated temperature than P. ryukyuensis.

I. Yakovleva et al. / Comparative Biochemistry and Physiology, Part B 139 (2004) 721–730728

Compared to the ambient temperature treatment, expo-

sure to thermal stress induced a pronounced reduction in the

abundance of Myc-Gly in the coral tissue and zooxanthellae

of both species investigated within the first 6 h. Moreover,

the Myc-Gly pool decreased further when exposure was

prolonged to 12 h (Figs. 4 and 5). Although Myc-Gly is

known as a precursor of other MAAs in some coral reef

organisms (Dunlap and Shick, 1998), it is not likely that

Myc-Gly was consumed to synthesize other MAAs because

the concentrations of other MAAs, i.e., imino-carbonyl

MAAs (shinorine, porthyra-334, Myc-2Gly, palythine,

palythinol, asterina-330 and palythene), did not change

significantly during the experimental period, irrespective of

temperature. This invariance of imino-MAA concentrations

under the high pressure of ROS produced during exposure

to elevated temperature is consistent with the previous

findings of their oxidative stability (Dunlap and Yamamoto,

1995). In contrast, Myc-Gly is known to have antioxidant

activity and to be consumed during reactions with ROS

(Dunlap and Yamamoto, 1995; Dunlap and Shick, 1998;

Suh et al., 2003). A clear relationship between the levels of

oxidative stress and decomposition of Myc-Gly in the tissue

and zooxanthellae of P. ryukyuensis and S. pistillata might

be evident for its functioning as antioxidant. Moreover,

differences in the relative abundance of Myc-Gly concen-

trations between these species may, at least partly, explain

the differential susceptibility of P. ryukyuensis and S.

pistillata to the acute thermal stress.

A higher tolerance to elevated temperature in P.

ryukyuensis is consistent with the 20- and 2-fold higher

concentrations of Myc-Gly in its tissue and zooxanthellae,

respectively, compared to those of S. pistillata. Although

after 6-h stress treatment, Myc-Gly in both coral species

decreased in equal proportions (Table 1; Figs. 4 and 5), this

decrease averaged only 40–60% for the coral tissue and

zooxanthellae of P. ryukyuensis, respectively, while the pool

of Myc-Gly in both components of S. pistillata was almost

depleted (Figs. 4 and 5). In terms of Myc-Gly functioning as

antioxidant, these results may explain the delay in the onset

of oxidative stress in P. ryukyuensis during the first 6 h of

exposure to elevated temperature (Figs. 1–3) and signifi-

cantly higher level of oxidative stress in S. pistillata than

that in P. ryukyuensis when the thermal stress exposure was

prolonged to 12 h.

Interestingly, the 6-h stress treatment caused a pro-

nounced reduction in the concentrations of Myc-Gly in the

tissue and zooxanthellae of P. ryukyuensis without signifi-

cant effect on the activity of SOD in both components.

Superoxide anion (�O2), which can be scavenged by SOD

(Campa, 1991), is the primary product of the reduced

molecular oxygen mediating oxygen toxicity (Halliwell and

Gutteridge, 1999). It seems that the host and symbionts of

P. ryukyuensis were not stressed sufficiently to initiate the

antioxidant enzyme system under these conditions. This is

probably because Myc-Gly, as a small molecule antiox-

idant, provides a more rapid protection against superoxide

anions before antioxidant enzymes are induced. Thus,

oxidative stress can be relieved without the relatively high

costs of enzyme induction as far as the concentration of

Myc-Gly is sufficiently high to cope with oxidative stress.

In P. ryukyuensis exposed to high temperature, the SOD

activity increased significantly in symbionts but not in

animal tissue, which contain 10 times higher concentration

of Myc-Gly than algal component (Table 1). This finding

also supports the above idea that Myc-Gly functions as first

aid to oxidative stress. Furthermore, S. pistillata, which

does not contain detectable amounts of Myc-Gly after 12-h

exposure to high temperature, suffered sever damage to

PSII (Fig. 1) and higher levels of oxidative stress than P.

ryukyuensis. Thus, the lower levels of oxidative stress in

the tissue of P. ryukyuensis than in its zooxanthellae as well

as the greater capacity of P. ryukyuensis to tolerate

oxidative stress compared with S. pistillata are concomitant

with higher concentrations of Myc-Gly and strongly

indicate this compound confers temporary protection

against oxidative stress. These results are consistent with

the fact that antioxidant activity of Myc-Gly is concen-

tration-dependent (Dunlap and Shick, 1998), and may

explain why corals growing in shallow water under

numerous stresses, causing the generation of ROS, gen-

erally have disproportionately greater quantity of Myc-Gly

than deep water conspecifics (Dunlap et al., 2000).

However, other biochemical defenses, such as heat-shock

proteins (Fang et al., 1997; Downs et al., 2000; Brown

et al., 2002) and antioxidant enzymes, ascorbate and

glutathione peroxidases (Lesser et al., 1990; Richier

et al., 2003), might also be involved in determining the

susceptibility of coral–algal symbiosis to ROS-mediated

effects of elevated temperature. Furthermore, marine

invertebrates often contain various water-soluble antiox-

idants, 2-O-methilascorbate, uric acid, and 4-deoxygadusol

(reviewed by Dunlap et al., 2000). These molecules possess

higher antioxidant activity than oxo-carbonyl MAAs as

demonstrated by a comparison of their electrochemical

properties (Shick and Dunlap, 2002), and, thus, may

provide more efficient protection against ROS damage in

the tissues of algal-invertebrate symbiosis than Myc-Gly.

However, metabolic fate and possible cycling of these

antioxidants in symbiotic cnidarians are largely unexplored

and need further investigation.

Although the two corals used in this study were

collected from shallow-water areas and further acclimated

to the same environment for 2 months, they showed

different initial abundance of Myc-Gly (Table 1). It is

likely that the differences in the concentrations of Myc-Gly

and other MAAs are due to genetic differences between P.

ryukyuensis and S. pistillata rather than to the different

history of environmental stresses they experienced. Shick

et al. (1992) postulated intergenetic differences in the MAA

concentrations among coral reef asymbiotic sea cucumbers.

Furthermore, Banaszak et al. (2000) reported differences in

the ability to synthesize MAAs between cultured zooxan-

I. Yakovleva et al. / Comparative Biochemistry and Physiology, Part B 139 (2004) 721–730 729

thellae of different phylotypes. If we assume that MAAs in

algal–invertebrate symbiosis originate in the phototrophic

partner, the differences in MAA abundance between P.

ryukyuensis and S. pistillata may be due to the presence of

different genotypes of zooxanthellae in the corals. This is in

agreement with the different patterns of MAA biosynthesis

observed in the coral zooxanthellae with P. ryukyuensis

symbionts only having palythine and Myc-Gly while algae

of S. pistillata additionally contained significant amounts of

porphyra-334 and Myc-2Gly (Table 1). However, LaJeu-

nesse et al. (in press) observed only genotype C Symbio-

dinium spp. in P. ryukyuensis and S. pistillata collected

from Okinawa and Aka Islands coasts. This fact put our

data on MAA composition in zooxanthellae in a contra-

diction to the findings of Banaszak et al. (2000) demon-

strated a high predilection for the MAA synthesis only in

the phylotypes A Symbiodinium spp. but not in the

phylotypes B and C. Nevertheless, although experimental

coral colonies that we used were collected in the same

areas, they did not originate from particular coral colonies

that LaJeunesse and colleagues studied. Moreover, a given

symbiosis often contains a genotypically diverse assem-

blage of zooxanthellae (LaJeunesse, 2001).

5. Conclusions

A pronounced reduction in the concentration of

oxocarbonyl-MAA, Myc-Gly, in contrary to stable levels

of imino MAAs, was observed in both the coral tissue and

symbionts of P. ryukyuensis and S. pistillata exposed to

elevated temperature. This, together with a clear relation-

ship between the levels of oxidative stress and Myc-Gly

decomposition, provide a corroborate evidence that Myc-

Gly is functioning as biological antioxidant in the coral

tissue and zooxanthellae of shallow-water scleractinian

corals. Despite the similarity in the pattern of changes in

Myc-Gly concentrations, P. ryukyuensis was less sensitive

to oxidative stress compared to S. pistillata. The differ-

ential sensitivity to oxidative stress might be, at least

partly, accounted for by the different initial concentrations

of Myc-Gly in the corals. The present results suggest that

a high Myc-Gly abundance may render corals more

tolerant to oxidative toxicity and increase their surviv-

ability in habitats with lethal temperature perturbations, or

under the pressure of other stresses that mediate produc-

tion of ROS.

Acknowledgements

This research was supported by Grants in Aid for

Scientific research from the Japan Society for Promotion

of Science to IY. RB is thankful to the Ministry of

Education, Culture, Sports, Science and Technology,

Japan, for a scholarship. We are grateful to the staff of

Sesoko Station, Tropical Biosphere Research Center,

University of the Ryukyus, Okinawa, Japan for use of

facilities.

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