Journal of Experimental Marine Biology and Ecology 368 (2009) 81–87

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Journal of Experimental Marine Biology and Ecology

j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j e m b e

High zooxanthella density shortens the survival time of coral cell

aggregates under thermal stress
Badrun Nesa, Michio Hidaka ⁎
Department of Chemistry, Biology, and Marine Science, University of the Ryukyus, Nishihara, Okinawa 903-0213, Japan

a r t i c l e i n f o a b s t r a c t

Article history: To investigate bleaching mechanisms in coral–zooxanthella symbiotic systems, it is important to study the
Received 9 April 2008 cellular- or tissue-level responses of corals to stress. We established an experimental system to study the
Received in revised form 14 October 2008 stress responses of coral cells using coral cell aggregates. Dissociated coral cells aggregate to form spherical
Accepted 15 October 2008
bodies, which rotate by ciliary movement. These spherical bodies (tissue balls) stop rotating and disintegrate
when exposed to a thermal stress. Tissue balls prepared from dissociated cells of Fungia sp. and Pavona
Keywords:
divaricata were exposed to either elevated temperature (31 °C, with 25 °C as the control) or elevated
Coral bleaching
Symbiodinium
temperature in the presence of exogenous antioxidants (ascorbic acid and catalase, or mannitol). The survival
Symbiosis curves of tissue balls were markedly different between 31 and 25 °C. At 31 °C, most tissue balls disintegrated
Thermal stress within 24 h, whereas at 25 °C, most tissue balls survived for more than 24 h. There was a negative correlation
Zooxanthellae between survival time and the zooxanthella density of tissue balls at 31 °C, but no significant relationship
was found at 25 °C. Antioxidants extended the survival time of tissue balls at high temperature, suggesting
that zooxanthellae produce reactive oxygen species under stress. These results indicate that zooxanthellae
produce harmful substances and damage coral cells under high-temperature stress. Tissue balls provide a
good experimental system with which to study the effects of stress and various chemical reagents on corals
cells.
© 2008 Elsevier B.V. All rights reserved.

1. Introduction (Tchernov et al., 2004) leads to the generation of excess electrons,

Coral bleaching has affected coral reefs over the last decade. In
some cases, bleaching has devastated whole reef regions or led to local
species extinction (Hoegh-Guldberg, 1999; Coles and Brown, 2003).
Coral bleaching, which is the loss of color of hermatypic corals due
to the loss of symbiotic dinoflagellates (zooxanthellae) and/or their
photosynthetic pigments, is caused by a variety of environmental
stresses (e.g., Glynn, 1993; Brown, 1997; Hoegh-Guldberg, 1999).
However, large-scale coral bleaching is usually ascribed to elevated sea
surface temperatures and/or increased solar radiation (Brown, 1997;
Hoegh-Guldberg, 1999, 2000; Fitt et al., 2001). Exposure to elevated
temperatures reduces the photosynthetic rate of zooxanthellae and
predisposes their photosynthetic apparatus to further damage by light
(Fitt and Warner, 1995; Lesser, 1996; Warner et al., 1996; Jones et al.,
1998; Bhagooli and Hidaka, 2004).
The key to understanding the underlying photo-physiologi-
cal mechanisms of coral bleaching resides in determining how
elevated temperatures exacerbate the effect of incident irradiance.
One hypothesis is that the thermal inhibition of enzymes involved
in the carbon fixation cycle (Jones et al., 1998) or a decrease in
ATP production due to a dysfunction of the thylakoid membrane
⁎ Corresponding author. Tel.: +81 98 895 8547; fax: +81 98 895 8576.
E-mail address: hidaka@sci.u-ryukyu.ac.jp (M. Hidaka).
which react with ambient oxygen to produce reactive oxygen species
(ROS). Coral hosts expel their symbionts as a protective mechanism
against ROS (Downs et al., 2002) or host cells die, releasing zoo-
xanthellae (Dunn et al., 2002). However, the cellular and molecular
mechanisms that underlie the breakdown of the symbiotic partner-
ship are still poorly understood.
To investigate the bleaching mechanisms of the coral–zooxanthella
symbiotic system, it is important to study the cellular- or tissue-level
responses of corals to stressful conditions. We established an ex-
perimental system using aggregates of dissociated corals cells (tissue
balls) to study the responses of coral cells to stress and chemical
reagents such as antioxidants. Tissue balls are similar to multicellular
endothelial isolates (MEIs) from chemically dissociated coral cells
(Kopecky and Ostrander, 1999) and tissue isolates chemically deta-
ched from the skeleton of corals (Domart-Coulon et al., 2004), though
tissue balls used in this study were prepared from mechanically
dissociated cells. Tissue balls prepared from the same individual
are genetically identical and provide a useful experimental system
with which to investigate the effects of environmental stresses
and chemical reagents on the coral–zooxanthella symbiotic system.
We evaluated the use of coral cell aggregates (tissue balls) as a model
for bleaching studies. We also evaluated the hypothesis that
zooxanthellae produce harmful substances such as ROS when exposed
to thermal stress.

0022-0981/$ – see front matter © 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.jembe.2008.10.018
82 B. Nesa, M. Hidaka / Journal of Experimental Marine Biology and Ecology 368 (2009) 81–87
2. Materials and Methods
2.1. Collection and maintenance of coral specimens
Individuals of a solitary coral, Fungia sp., and fragments of the
colonial coral Pavona divaricata were collected from the reef at Sesoko
Island or Bise, northern Okinawa, Japan, and were maintained in a
neutral-density shaded (~ 30% of sea surface irradiance) outdoor
seawater table supplied with unfiltered running seawater at Sesoko
Station, Tropical Biosphere Research Center, University of the Ryukyus,
Japan. Corals were then brought to the Nishihara campus of the
University of the Ryukyus and kept in an aquarium with a subgravel
filter at 26 °C under 150 μmol photons m- 2 s- 1 on a 12:12 h light/dark
cycle for up to 2 weeks prior to use.
2.2. Preparation of tissue balls
Individuals of Fungia sp. and fragments of P. divaricata were rinsed
gently with 0.22-μm filtered seawater (FSW). Coral tissue was removed
from the skeleton using a WaterPik (Teledyne, WP-70J). To remove the
mucus produced during the isolation procedure, the coral blastate was
filtered through 180-μm nylon mesh. The blastate was then homo-
genized with a homogenizer and filtered through 40-μm nylon mesh.
The resulting filtrate was centrifuged twice at 1000 rpm for 5 min. The
supernatant was discarded, and the pellet was suspended in FSW
and dispersed by vortexing. The suspension containing dissociated coral
cells and zooxanthellae was incubated in a Petri dish overnight at room
temperature. After a few hours or overnight incubation, coral cells
Fig. 1. Degradation of a tissue ball exposed to high temperature stress. (A) A healthy and swimming Pavona divaricata tissue ball at the start of the experiment. (B) The tissue ball
stopped moving after 6 h. (C) The tissue ball stopped ciliary movement. (D) A dead tissue ball after 8 h. (E) A degraded tissue ball and dispersed zooxanthellae. (F) Completely
dispersed coral and algal cells from a tissue ball.
became aggregated and achieved a spherical shape. These spherical
bodies (tissue balls) contained various numbers of zooxanthellae and
started to rotate by ciliary movement. After overnight incubation, tissue
balls were transferred to a new Petri dish with fresh FSW. Healthy tissue
balls of similar size were chosen under a stereomicroscope (Nikon-SMZ-
10) and placed in separate wells of a 96-multiwell plate, with each well
containing 300 µl of FSW. Mean (±SD) volume of the tissue balls of
Fungia sp. and Pavona divaricata was 9.1 ± 6.2 × 105 µm3 and 2.8 ±
2.0 × 106 µm3, respectively. These preparations were allowed to recover
at room temperature for 3–6 h before the stress experiment.
2.3. Experimental protocol
2.3.1. Treatment of tissue balls
Tissue balls were exposed to normal (25 °C) or high (31 °C) tem-
perature to assess the effects of temperature on the viability of tissue
balls. In each experiment, 13-20 tissue balls were put in separate wells
of a 96-well plate for each condition. The 96-well plates with tissue
balls were placed in each of two compartments regulated at 25 or 31 °C
of an incubator (Yamato, Program Incubator IQ 820). The plates were
illuminated using a 19 W fluorescent light bulb, and the light intensity
measured using a light meter (LICOR, LI-250) was 35 µmol photons m- 2
s- 1. FSW in the wells was changed once daily, although tissue balls
incubated at 31 °C died before the first FSW change.
The experiment was repeated four times with Fungia tissue balls
using different individuals and two times with Pavona tissue balls
using different colonies. In some experiments, third 96-well plate was
used for incubation at 31 °C in the presence of antioxidant. In each
 B. Nesa, M. Hidaka / Journal of Experimental Marine Biology and Ecology 368 (2009) 81–87
Fig. 2. Survival curves of Fungia sp. (A) and Pavona divaricata (B) tissue balls at 25 °C (○) and 30 °C (●).
experiment, the number of tissue balls was the same among different
treatment conditions.
2.3.2. Survivorship of tissue balls
Tissue balls exposed to normal or high temperature were observed
under an inverted microscope (Nikon, Diaphot-300) every 2 h for the
first day, every 4 h during the second day, and then every 6 h. Tissue
balls were scored as healthy (rotating with intact morphology), stop-
ped (no rotation, but with intact morphology), or degraded at each
observation. At the initial stage of degradation, cells or aggregates of
cells dropped from the tissue ball. Finally, whole tissue balls became
degraded and coral cells and zooxanthellae were dispersed on the
bottom of the well. A tissue ball was considered dead when it became
partially degraded and ciliary movement stopped. The survival time of
tissue balls was defined as time between the start of temperature
exposure and the observation time at which the tissue ball was
considered dead. Photomicrographs of tissue balls were taken
occasionally using a 10× or 20× objective lens.
2.3.3. Zooxanthella density in tissue balls
To investigate the relationship between zooxanthella density
and the survival time of tissue balls, tissue balls containing various
numbers of zooxanthellae were exposed to normal (25 °C) or high
(31 °C) temperature and their survival time was recorded. The number
of tissue balls used in each experiment was from 13 to 20, and
experiments were repeated four and two times for Fungia sp. and Pa-
vona divaricata, respectively.
To estimate the volume of tissue balls, video images of rotating
tissue balls were taken under an inverted microscope and printed
using a video camera (Victor, TK-910) and a video printer (SONY, UP-
Fig. 3. Relationship between tissue ball size and survival time at 31 °C (A) and 25 °C (B) for Fungia sp. The dotted line indicates a non-significant correlation.
83
850) immediately before the stress treatment. The volume of tissue
balls was estimated from the video prints assuming that they are
ellipsoid. Volume was estimated using the following equation
V ¼π=6 LS2
where L = the longer diameter of the tissue ball and S = the shorter
diameter of the tissue ball.
After tissue balls became completely disintegrated, the number of
zooxanthellae within the well was counted under an inverted micro-
scope. The zooxanthella density of each tissue ball was calculated by
dividing the number of zooxanthellae by the volume of the tissue ball.
The number of dividing cells was small (2.8%) even in high temperature-
treated tissue balls, suggesting that the density of zooxanthellae was
almost constant during the experiment.
2.3.4. Effects of antioxidants
To determine whether exogenous antioxidants extend the survival
time of tissue balls at high temperature, in some experiments, 15-20
tissue balls were put into separate wells of another 96-well plate and
were incubated in FSW that contained a mixture of ascorbic acid and
catalase or FSW that contained mannitol at 31 °C. Ascorbic acid,
catalase, and mannitol were used at final concentrations of 125 μM,
250 units ml- 1, and 10 mM, respectively, following Lesser (1997). FSW
that contained the ascorbic acid and catalase mixture was changed
twice daily because ascorbic acid rapidly oxidizes in seawater. The
mannitol solution was not changed during the experimental period.
2.3.5. Statistical analyses
Differences in survivorship among treatments were tested using
the log-rank test for each experiment with each tissue ball as
84 B. Nesa, M. Hidaka / Journal of Experimental Marine Biology and Ecology 368 (2009) 81–87

Fig. 4. Relationship between zooxanthella density and survival time of Fungia sp. tissue balls at 31 °C (A) and 25 °C (B) and of Pavona divaricata tissue balls at 31 °C (C) and 25 °C (D).
Solid and dotted lines indicate significant and non-significant correlations, respectively.

statistical unit. Correlation analysis was used to describe the relation- survival curve of P. divaricata tissue balls was similar to that of Fungia
ship between zooxanthella density or tissue ball volume and survival sp., although P. divaricata tissue balls survived longer than Fungia
time for each experiment with each tissue ball as statistical unit. A tissue balls both at 31 °C and especially at 25 °C (Fig. 2B).
p b 0.05 was considered significant.
3.3. Effect of size on the survival time of tissue balls
3. Results
In most cases, no significant correlation was observed between
3.1. Degradation of tissue balls survival time and the size of tissue balls in either species. Typical plots
of survival time of Fungia tissue balls against the size of the tissue balls at
At the start of the experiment with P. divaricata tissue balls, the tissue 25 and 31 °C in FSW are shown in Fig. 3. The plots are from one ex-
balls rotated (Fig. 1A). After several hours at 31 °C, they stopped rotating, perimental run at 31 °C (Fig. 3A) and 25 °C (Fig. 3B) and each point
but in most cases, ciliary movement could be observed on the surface, represents single tissue ball. We analyzed a total of 16 cases (survival time
and sometimes the balls started to move again. At this point, tissue balls vs size plots obtained under various conditions: 4 plots at both 25 and
were considered stopped, but still alive (Fig. 1B). Later, tissue balls 31 °C and additional 2 plots in the presence of antioxidants with Fungia
started to degrade, losing some coral and algal cells (Fig. 1C). When tissue balls and 2 plots at both 25 and 31 °C and additional 2 plots in the
degradation proceeded further, considerable amounts of coral cells and presence of antioxidant with Pavona tissue balls). In only 2 of 16 cases was
zooxanthellae were sloughing off, and tissue balls did not maintain their
original spherical shape (Fig. 1D). At this stage, ciliary movement was no Table 1
longer observed, and the tissue balls were considered dead. Later, more Correlations between zooxanthella density and survival time of tissue balls
coral cells and zooxanthellae became dispersed, leaving only remnants Species Treatment Exp. No r-value Significance Zoox. density
of tissue balls (Fig. 1E). Finally, coral and algal cells became completely (104 cells/mm3)
dispersed on the bottom of the well (Fig. 1F). Fungia sp. 25 °C I 0.678 (19) ⁎ 0.0 - 1.00
II 0.205 (13) NS 0.57 - 3.04
3.2. Survival of tissue balls at different temperatures III - 0.144 (15) NS 0.024 - 1.57
IV - 0.053 (16) NS 0.0 - 1.21
31 °C I - 0.632 (19) ⁎ 0.006 - 2.02
The survival curves of tissue balls were markedly different between II - 0.607 (13) ⁎ 0.57 - 4.08
the high (31 °C) and normal (25 °C) temperatures (Fig. 2). Tissue balls of III - 0.691 (15) ⁎ 0.019 - 2.01
both Fungia sp. and P. divaricata died significantly more rapidly at 31 °C IV - 0.517 (17) ⁎ 0.0072 - 2.5
Pavona divaricata 25 °C V 0.001 (20) NS 0.083 - 2.44
than at 25 °C (log-rank test, p b 0.001). Each survivorship was from a
VI - 0.067 (19) NS 0.065 - 1.72
single run starting from 13-20 tissue balls. The same results were 31 °C V - 0.646 (20) ⁎ 0.007 - 1.38
obtained in four and two replicated experiments using Fungia and VI - 0.429 (20) NS 0.041 - 1.286
Pavona tissue balls, respectively. At 31 °C, most Fungia sp. tissue balls The range of zooxanthella density is also shown.
died within 24 h, whereas most tissue balls survived for longer than 24 ⁎p b 0.05; NS = not significant.
h and more than half tissue balls survived for 52 h at 25 °C (Fig. 2A). The Number in the parenthesis indicates the number of tissue balls.
 B. Nesa, M. Hidaka / Journal of Experimental Marine Biology and Ecology 368 (2009) 81–87
Fig. 5. (A) Survival curve of Fungia sp. tissue balls at 25 °C (▲), 31 °C (●), and 31 °C in the presence of 125 μM ascorbic acid and 250 U/ml catalase (○). (B) Survival time of Pavona
divaricata tissue balls at 25 °C (▲), 31 °C (●), 31 °C in the presence of 10 mM mannitol (□). A + C indicates ascorbic acid and catalase and MN indicates mannitol.
there a significant correlation between survival time and the size of tissue
balls: a significant positive correlation in one experiment at 25 °C with
Fungia sp. and a significant negative correlation in one experiment at 31 °C
with P. divaricata, both in the absence of antioxidant.
3.4. Effect of zooxanthella density on the survival time of tissue balls
There was a significant negative correlation between zooxanthella
density and the survival time of tissue balls at 31 °C with Fungia sp.
(Fig. 4A). There was a significant negative correlation in all four
replicates at 31 °C (Table 1). Similarly, a negative correlation between
zooxanthella density and survival time was found at 31 °C with P.
divaricata, although it was significant for only one of two replications
(Table 1). In contrast, at normal temperature (25 °C), there was no
significant correlation between zooxanthella density and the survival
time of tissue balls in three of four replications with Fungia sp. and
two replications with P. divaricata (Table 1). A significant positive
correlation was found between zooxanthella density and survival time
at 25 °C in one case with Fungia sp. (Fig. 4B).
3.5. Effect of antioxidants on the survival time of tissue balls
The addition of antioxidants extended the survival time of tissue
balls in some cases at 31 °C, but the effectiveness of antioxidant
reagents differed between the two species. The addition of ascorbic
acid and catalase significantly extended the survival time of Fungia
tissue balls (log-rank test, p b 0.01; Fig. 5A). However, mannitol had no
effect on the survivorship of Fungia tissue balls at 31 °C (p = 0.878). On
the other hand, the addition of mannitol significantly extended the
survival time of P. divaricata (log-rank test, p b 0.001; Fig. 5B). The
ascorbic acid and catalase mixture, however, had no effect on the
survivorship of P. divaricata tissue balls at 31 °C (p = 0.218).
4. Discussion
4.1. Use of tissue balls to study coral bleaching
We demonstrated that the survivorship of tissue balls (aggregates
of dissociated coral cells) differs markedly under normal and high
temperatures. Tissue balls prepared from dissociated coral cells might
provide a good experimental system to assess the effects of
environmental stresses on corals. The size of tissue balls had no
significant effect on survival time in most cases. Thus, the size of tissue
balls did not affect their survival, at least in the size range used here.
The degradation of tissue balls was a continuous process beginning
with the cessation of rotation, followed by the partial loss of cells from
the tissue ball, and finally, the complete dispersion of coral and algal
85
cells. However, we considered that tissue balls were dead when they
no longer maintained their original spherical shape as a result of the
release of cells and cellular fragments. At this stage, no ciliary move-
ment was observed on the surface of tissue balls.
Kopecky and Ostrander (1999) obtained multicellular endothelial
isolates (MEI), that is, multicellular tissue fragments, from coral cells
dissociated in calcium-free seawater. The tissue balls described here are
similar to MEI, although the coral cells were dissociated mechanically
using a Waterpik. Both tissue balls and MEI consist of multiple cell types,
including zooxanthellae. Both tissue balls and MEI rotated continuously,
survived for up to a few hundred hours, and had similar degradation
processes. Kopecky and Ostrander (1999) suggested that MEI could act
as materials for in vitro studies of the effects of chemicals or disease
vectors on corals. Domart-Coulon et al. (2004) reported that soft tissue
detached from coral skeletons using calcium-free seawater can be
cultured for a short time (3 days) and is useful for physiological studies.
We demonstrated that tissue balls provide a good experimental system
with which to examine the effects of environmental stress on the coral–
zooxanthella symbiotic system. Coral larvae can also be used to study
mechanisms of coral bleaching. Coral larvae have differential survivor-
ship at 28, 32, and 36 °C (Baird et al., 2006). The advantage of tissue balls
is that they can be prepared easily and are genetically identical. Fur-
thermore, tissue balls can be used regardless of the season, whereas
coral larvae can be used only during the reproductive season.
The survivorship of the control tissue balls decreased past 75-100
h, suggesting a continuous degradation of the physiological integrity
of the cells. Thus the utility of the tissue balls is limited to 3-4 days in
the present experimental condition. But tissue balls are more sensitive
to thermal stress than coral nubbins and the experimental system
using tissue balls enables us to study the effect of environmental
stresses in a shorter period (less than 2-3 days) than when coral
nubbins are used. Furthermore a large number of tissue balls can be
incubated in multi-well plates and can be easily observed under an
inverted microscope. Tissue balls are also ideal material for assays for
cellular damage such as Comet Assay (alkaline single-cell gel
electrophoresis for detection of DNA damage) as they can be easily
dissociated to single cells (Nesa and Hidaka, in preparation). In our
preliminary study, some Pavona tissue balls survived more then two
weeks, indicating that the survivorship of tissue balls under normal
condition might be extended by technical improvement.
4.2. Relationship between zooxanthella density and survival time of
tissue balls
In most cases, there was a significant negative correlation between
zooxanthella density and survival time of tissue balls at 31 °C in both
Fungia sp. and P. divaricata. In contrast, there was no significant
86 B. Nesa, M. Hidaka / Journal of Experimental Marine Biology and Ecology 368 (2009) 81–87
correlation between zooxanthella density and survival time at the
normal temperature. In only one of four replicate experiments with
Fungia tissue balls was there a significant positive correlation between
zooxanthella density and survival time. Under high temperature
stress, tissue balls that had a higher density of zooxanthellae tended to
die earlier than those that had a low density of zooxanthella. These
results suggest that zooxanthellae became a burden for the coral hosts
under high temperature stress. It is possible that at high temperatures,
zooxanthellae produced harmful substances that induced the degra-
dation of tissue balls.
4.3. Involvement of reactive oxygen species in tissue ball degradation
To examine whether zooxanthellae produce harmful substances
under thermal stress, we studied effects of antioxidants on the survival
of tissue balls at high temperature. Exogenous antioxidants extended
the survival time in some replicates, supporting the idea that harmful
substances produced by symbiotic algae under thermal stress are ROS.
Oxidative stress is thought to play a role in coral bleaching (Lesser, 1996,
1997; Downs et al., 2002). Several studies have indicated that thermal
stress can damage the photosynthetic system of symbiotic algae in the
presence of light through the production of ROS (reviewed by Stat et al.,
2006). Thermal stress may directly damage photosystem II or inhibit the
D1 protein re-synthesis that is necessary to repair photosystem II
(Warner et al., 1999; Takahashi et al., 2004). Alternatively, damage to
photosystem II may be a secondary effect of ROS produced through over-
reduction of the electron transport chain due to thermal inhibition of the
Calvin cycle in the presence of light (Jones et al., 1998; Bhagooli and
Hidaka, 2004; Yakovleva and Hidaka, 2004). The inhibition of photo-
system II repair by ROS is accelerated by the deceleration of the Calvin
cycle (Nishiyama et al., 2006). Tchernov et al. (2004) suggested that the
loss of the integrity of the thylakoid membrane caused by thermal
stress reduces ATP generation and leads to the restriction of carbon
assimilation, which results in ROS generation through the Mehler re-
action (i.e., the photochemical reduction of O2 in photosystem I). ROS
produced in algae leak out of algal cells and are transferred to the animal
host, inducing physiological stress (Downs et al., 2002; Tchernov et al.,
2004). ROS are likely involved in both damage to the photosynthetic
apparatus of symbiotic algae and in the DNA damage, apoptosis, auto-
phagy, and necrosis of host cells (Lesser and Farrell, 2004; Richier et al.,
2006; Dunn et al., 2007).
Individuals of the sea anemone Anthopleura elegantissima that
contain zooxanthellae have superoxide dismutase (SOD) activity that
is nearly two orders of magnitude greater than in individuals that lack
zooxanthellae (Dykens and Shick, 1982). Cells of the symbiotic sea
anemone Anemonia viridis have higher SOD activity than do cells of
the non-symbiotic sea anemone Anemonia schmidti (Richier et al.,
2005). Dykens et al. (1992) directly observed light-dependent ROS
production in zooxanthellate anemones and noted the necessity of
defense against photooxidative stress, a cost that is seldom considered
in mutualistic symbioses. Our finding that the survival time of tissue
balls was negatively correlated with zooxanthella density suggests
that symbiotic algae can become a source of photooxidative stress
under high temperature conditions.
We attempted to demonstrate that the harmful substances from
zooxanthellae were ROS through the use of exogenous antioxidants.
The survival time of Fungia tissue balls kept at 31 °C was extended by
the addition of a mixture of ascorbic acid and catalase (Fig. 5A),
although mannitol was not effective. In contrast, the survival time of
Pavona tissue balls was not affected by the addition of ascorbic acid
and catalase, but was extended significantly by mannitol. It is unclear
why ascorbic acid and catalase were effective only for Fungia tissue
balls and mannitol was effective only for Pavona tissue balls. Ascorbic
acid and mannitol are scavengers of hydroxyl radicals, whereas
catalase scavenges hydrogen peroxide. These compounds are effective
antioxidants and should reduce the harmful effects of ROS activity.
Variable effects of antioxidants on corals under light and thermal
stress have been reported. The incubation of the coral Agaricia te-
nuifolia with exogenous antioxidants (125 μM ascorbic acid and 250 U
ml- 1 catalase) resulted in improved photosynthetic rates and de-
creased algal expulsion under elevated temperature conditions
(Lesser, 1997). Exogenous antioxidants (500 μM ascorbic acid and
200 U ml- 1 catalase) ameliorated the detrimental effects of toxic oxy-
gen on the photoinhibition of cultured zooxanthellae (Lesser, 1996).
However, Franklin et al. (2006) found that the addition of exogenous
antioxidants (150 μM ascorbic acid and 10 mM mannitol) to water
surrounding coral had no effect on either photoinhibition or symbiont
mortality in Stylophora pistillata. The species-specific effects of an-
tioxidants might reflect physiological differences among coral–
zooxanthella symbiotic complexes. If this is the case, it is possible
that different species have different strategies for protection against
oxidative stress. Further studies are needed to understand the pro-
tection provided by various antioxidants and the cause of variation in
antioxidant activity on corals and tissue balls.
5. Conclusions
We established an experimental system with which to study the
responses of coral cells to stress and chemical reagents such as
antioxidants. Many tissue balls that are genetically identical can be
prepared and used as replicates for stress experiments. Our results
suggest that zooxanthellae produce harmful substances under high
temperature stress. In some cases, antioxidants increased the survival
time of Fungia sp. and P. divaricata tissue balls, indicating that the
harmful substances from zooxanthellae might be ROS.
Acknowledgements
This study was partly supported by a Grant-in-Aid for Scientific
Research C (17570023) and by the 21st Century COE program of the
University of the Ryukyus. We thank the staff of the Tropical Biosphere
Research Center (Sesoko Station) for allowing us to use the facilities.
Badrun Nesa is grateful to Monbukagakusho (Japanese Ministry of
Education, Culture, Sports, Science and Technology) for a scholarship.
[SS]
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