L.S. Fang et al.

Proc. Natl. Sci. Counc. ROC(B)

Vol. 22, No. 4, 1998. pp. 150-158

The Subcellular Mechanism of the Release of

Zooxanthellae during Coral Bleaching
LEE-SHING FANG*,**, JIH-TERNG WANG* AND KU-LIN LIN*
*Institute of Marine Resources
National Sun Yat-Sen University
Kaohsiung, Taiwan, R.O.C.
**National Museum of Marine Biology and Aquarium, Preparatory Office
Kaohsiung, Taiwan, R.O.C.
(Received June 24, 1998; Accepted September 8, 1998)
ABSTRACT
The subcellular mechanism of how zooxanthellae leave the host cell of Acropora grandis under elevated temperature was investigated by using colchicine or cytochalasin D to deteriorate the action of microtubule and microfilament, respectively, N-ethylmaleimide to inhibit the activity of cytoplasmic myosin and
dynein, and N-(6-aminohexyl)-5-chloro-1-naphthalene sulfoamide to antagonize calmodulin. The sensitivity
of coral cell and zooxanthellae to rising temperature was also examined indirectly by studying the occurrence
of the heat-shock protein 35 kDa in them. The results showed that coral cells synthesized the heat shock
protein at a lower temperature than zooxanthellae did, suggesting it could be more sensitive to heat and could
trigger the algae releasing process. Immunofluorescence staining of microtubules revealed that when the
cytoskeleton network was disrupted by colchicine, the release of algae was also inhibited. The combination
analysis of these drug interference indicated that the zooxanthellae had to be transported within cells via the
cytoskeleton network by motor proteins, followed by host cell breakage to complete the release process. All
this information about the subcellular mechanism of the release of zooxanthellae revealed that exocytosis of
the host cell is an important mechanism of coral bleaching under mild environmental stress.
Key Words: coral bleaching, heat-shock protein, exocytosis, immunofluorescence staining, microtubules,
cytoskeleton network

I. Introduction
The frequency of coral bleaching events has increased dramatically during the past two decades.
Bleaching is associated with abnormal sea surface
temperature or salinity changes in tropical oceans
worldwide (Brown and Suharsono, 1990; Coles and
Fadlallah, 1990; Glynn and DCroz, 1990; Williams and
Bunkley-Williams, 1990; Fang et al., 1991). Bleaching
in corals could result from a reduction in the content of
chlorophyll a (Porter et al., 1989; Szmant and Gassman,
1990) and associated pigments per zooxanthella
(Kleppel et al., 1989; Fang et al., 1995), a decline in
the population density of zooxanthellae (Fisk and
Done, 1985; Hoegh-Guldberg and Smith, 1989), or
both (Glynn and D'Croz, 1990; Lesser et al., 1990).
Loss of zooxanthellae from cnidarians at the
organismal level has been extensively described
(Gates, 1990; Glynn and D'Croz, 1990; Hayes and
Bush, 1990; McCloskey et al., 1996). However, the cel-

lular and subcellular processes during bleaching

events have been much less studied (O'Brien and
Wyttenbach, 1980; Lesser et al., 1990; Fang et al.,
1997). Gates et al. (1992) summarized five major
mechanisms of the release of zooxanthellae from
coral and emphasized the role of host cell detachment.
On the other hand, the fact that bleached coral polyps
were found to still exhibit active tentacle activity and
recover fairly quickly during our field observations
(Fang et al., 1991) suggests a less damaging mechanism
for disruption of coral-algal symbiosis. Steen and
Muscatine (1987) showed that rapid exocytosis of
symbiotic algae by host cells was evoked at low temperature in the sea anemone Aiptasia pulchella; however, they also stated that a similar response was not
found in several scleractinian corals. They briefly discussed the possible subcellular mechanisms, such as
disassembly of host cell microtubules and increased
cytosolic calcium concentration, that might evoke
exocytosis of zooxanthellae during low temperature

To whom all correspondence should be addressed.

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Subcellular Mechanism of Coral Bleaching

shocks. Brown et al. (1995) found in a histological
study that zooxanthellae were released from the
endoderm into the coelenteron in partially bleached
corals. Most recently, Fang et al. (1997) found that
there was a steady increase of the intracellular calcium
concentration in coral cells under heat exposure and
suggested that this may be a signal for the exocytosis of
zooxanthellae. They further determined that a continuous supply of calcium is related to the extend of
bleaching (Huang et al., 1998) which indicates that
subsequent cellular movement of host cells is required
after the initial triggering signal. However, the exact
mechanism of zooxanthellae release from coral cells
is not yet clear.
In the present study, the subcellular activity of
coral cells during experimentally-induced bleaching
was examined. Several chemicals which inhibit
cytoskeleton activity or poison cytosolic proteins were
applied to investigate their influence on the bleaching
process. Soluble protein concentration in the incubation medium was monitored as an indicator of cell
leakage. Cytoskeletal elements were immunofluorescently labeled to visualize their response to drug
treatment. Moreover, the sensitivity of coral cell and
its symbiotic algae to heat exposure was further examined using the expression of a common heat stress protein to obtain a sense of which symbiotic partner is more
sensitive to environmental stress such that the disengagement process could be activated.

II. Materials and Methods

1. Sample Collection and Maintenance
Coral (Acropora grandis) samples were collected from 3 meters deep in the Kenting area in southern
Taiwan (2156'N, 12044'E) and carried back to the
laboratory in aerated sea water. The coral colonies
were maintained in a circu-lating aquarium at 25 C under an ambient light-dark cycle (12 h L : 12 h D) and
used within a week of collection.

2. Effect of Physiological Inhibitors on Coral

Bleaching

ance (80 mol photons m2 sl ) during the experiment.

The incubation medium was removed and replaced
with pre-warmed SW-buffer by means of gentle suction using a syringe every 6 to 8 h. The removed medium was centrifuged at 500 g for 30 min at 4 C to
collect zooxanthellae. At the end of the incubation
period, zooxanthellae remaining in the coral were collected by removing the tissue with a water pike and
centrifugation described as above. The number of
zooxanthellae was counted using a hemocytometer,
and total number of the algal cells was pooled with the
number of zooxanthellae obtained from the sample of
each time interval to provide the total algal population
of the coral tip. The percentage of algae released in
each time interval was calculated based on the ratio of
the cell number in the medium to the total algal population in the coral sample. The protein content of the
supernatant obtained during each time interval was
measured using the dye-binding method (Bio-rad Co.,
U.S.A.).
In the following studies, 209 M of colchicine
(Salmon et al., 1984; Nakai et al., 1992) or 6.6 M of
cytochalasin D (Schliwa, 1982; Cooper, 1987) was
added to the SW-buffer to inhibit the cytoskeleton activity of the coral cells; 5.6 mM of N-ethylmaleimide
was used to inhibit the activity of cytoplasmic myosin
and dynein (Lye et al., 1987; Vale, 1987; McIntosh and
Porter, 1989); 44.2 M of N-(6-aminohexyl)-5-chloro1-naphthalene sulfoamide was used as a calmodulin
antagonist (Hidaka et al., 1981). The stock solutions of
physiological inhibitors were prepared by dissolving
chemicals in dimethyl sulfoxide (DMSO) to a concentration equivalent to 300 fold that of the working
solution. Triplicate coral tips (1 cm) were pre-incubated in 3 ml of SW-buffer with 10 l of DMSO (the
control) or 10 l of stock solution (inhibitory treatment) at 25 C for 1 h, and then transferred to a 32 C
water bath for 30 h under the same conditions as described above. The medium was renewed using
prewarmed SW-buffer with DMSO or chemicals every
6 h. The protein content and number of zooxanthellae
released into the medium were measured as described
above.

3. Immunofluorescence Staining of Microtubules

The first experiment was designed to monitor the
release of zooxanthellae from coral under heat stress
to establish an assay system for the following studies.
Triplicate A. grandis tips (1 cm) were incubated in 3
ml of artificial sea water buffer (SW-buffer: 420 mM
NaCl, 26 mM MgSO4, 23 mM MgCl2, 9 mM KCl, 9 mM
CaCl2, 2 mM NaHCO3, 10 mM Tris-HCl, pH 7.6) for
27 h at 25 C (the control) or 32 C (heat stress), respectively. Coral tips were exposed to constant irradi-

In order to reveal the influence of cytoskeleton

inhibitors on the host cells, coral tips were incubated
with SW-buffer supplemented with 209 M of
colchicine at 25 C for 6 h. After incubation, the coral
cells in each treatment were harvested using a modified method of Gates et al. (1992). The polyps were cut
off of the treated coral and macerated with Ca-free sea
water (omitting CaCl2 from SW-buffer and substitut-

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L.S. Fang et al.

ing Trisma with 1.67 mM NaH 2 PO 4 and 8.10 mM
Na2HPO4) for 4.5 h at 4 C with three times of fresh
buffer replacement. The polyps were then incubated
with 0.05% trypsin in Ca-free sea water overnight at
4 C with gentle shaking. Coral cells were collected
from the supernatant (cell suspension) after centrifugation at 1200 g and 4 C for 10 min. These cells
were put into 4 C high salt phosphate buffer solution
(PBS 0.1 M sodium phosphate buffer pH 7.4 containing 3% NaCl and 0.004% CaCl2) at a concentration of approximate 104 to 105 cells.
The coral cell suspension was dropped on polylysine treated slides and allowed to sit for 30 min at
room temperature. The slides were then fixed in 3.7%
paraformalin for 10 min and in 20 C methanol : acetone mixture (1:1) for 30 min. Then, the slides
were macerated with a series of solutions including:
PBS (a solution containing 136.87 mM NaCl, 2.68 mM
KCl, 1.67 mM NaH2PO4H2O, and 8.10 mM Na2HPO4
12H2O) for 3 min and 0.2% Triton X-100/ PBS for 2
min, followed by a wash with PBS. Non-specific reactions in the cells were blocked using 1% bovine serum
albumin for 30 min at room temperature. Finally, the
cells were washed with PBS, and incubated with 20 ml
of anti- -tubulin antibody (Boehringer Mannheim
Biochemica, Germany) in 1: 50 dilution for 60 min at
room temperature. The cells were further rinsed with
0.05% Tween-20/ PBS plus a PBS wash and incubated
with 20 ml of fluorescent isothiocyanate-conjugated
goat anti-rabbit immunoglobulin G (Boehringer
Mannheim Biochemica, Germany) in a 1 : 20 dilution
for 60 min at room temperature. The slides were then
rinsed, mounted using glycine-buffered glycerol, and
observed using a microscope equipped with
epifluorescence optics. The fluorescent cells were photographed using Kodak Ektachrome P1600 film
(1600 ASA).

4. The Response of 35 kDa HSP in Coral Cell and

Zooxanthellae to Elevated Temperature
The occurrence of the 35 kDa heat shock protein
in coral cells and their zooxanthellae (Fang et al.,
1993, 1997) was used as an index of temperature stress.
This protein is likely the same as the novel environmentally-regulated 33 kDa protein obtained from
cnidarian symbiotic algae, described by Bythell et al.
(1995) although amino acid sequence analysis was not
performed in this experiment.
Coral tips approximately 5 cm long were incubated in pre-warmed sea water at 25, 27, 29, 31, 33 and
35 C. After incubation, the corals were transferred
to liquid nitrogen to stop the cellular processes then
thawed at 4 C prior to analysis. Coral tissue was col-

lected from the skeleton using a water pik, and the

coral cells and zooxanthellae were separated as
described by Muscatine et al. (1989). Proteins of
these cells were first extracted with 0.05 M Tris-HCl
buffer for water soluble protein; then, the remainder
was extracted with Tris-HCl buffer containing 2%
sodium dedocyl sulfate (SDS). Both of the extraction
buffers contained a mixture of protease inhibitors,
including 10 mM of EDTA, 2 mM of benzamide HCl,
0.1 mM of pepstatin, 0.1 mM of leupeptin, 0.1 mM of
chloroquine, 5 mM of phenylmethylsulfonyl fluoride,
10 mM of iodoacetate, and 0.2 mM of chymostatin.
The extracts were separated using a SDS-PAGE system (Hames, 1990) with 10 to 15% gradient polyacryamide gel.

III. Results
1. Effect of Different Chemicals on Coral Bleaching
The number of zooxanthellae released into the
surrounding water during each typical coral bleaching
experiment followed a sigmoid curve over time (Fig.
1(a)). The greatest release coincided with the peak
amount of protein in the medium, which indicated host
cell membrane breakage (Fig. 1(b)).
Figure 2(a) shows that the cytoskeleton poisons
colchicine and cytochelasin D inhibited the release of
zooxanthellae. Although protein leakage happened
around 12 h after incubation, the release of zooxanthellae had not increased.
N-ethylmaleimide and N-(6-aminohexyl)-5chloro-1-napthalenesulfonamide were both effective
inhibitors of coral bleaching (Figs. 3 and 4). This
showed that the normal function of myosin, dynein and
calmodulin is necessary for coral bleaching. The treatment of N-ethylmaleimide produced very early
protein leakage but resulted in very little zooxanthellae release at the same time.

2. Immunofluorescence Staining of Microtubules

of Chemical Treated Coral Cell
Figure 5 shows the fluorescence image of the
microtubules of normal coral cells and that after
colchicine treatment, respectively. This chemical
showed effectiveness in inhibiting coral bleaching
(Fig. 2).
The image reveals the normal distribution of
microtubules of coral cells which happened to be in
the anaphase of the cell cycle (Fig. 5(a)), and the
occurrence of many spots in the intracellular network of microtubules after colchicine treatment
(Fig. 5(b)).

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Subcellular Mechanism of Coral Bleaching

(a)
(a)

(b)
Fig. 1. The release of zooxanthellae (a) and soluble protein (b) into
medium during 32 C thermal bleaching of coral.

(b)
Fig. 2. The effects of cytoskeleton poisons on thermal bleaching of coral
as shown by the release of zooxanthellae (a) and protein (b).

3. The Sensitivity of Coral Cells and Zooxanthellae to Temperature Elevation

Figure 6 shows a typical protein pattern in the
form of an electrophoretic diagram of the coral
samples incubated in 31 C. It can be seen that hsp 35
appeared after incubation for 3 h. However, after 22 h,
many of the protein bands faded or disappeared. This
was due to cell leakage as shown in Fig. 1. Table 1
shows the temperature at which hsp 35 occurred in the
coral cells and in the zooxanthellae after 3 h of treatment at each temperature, respectively. Hsp 35 occurred in coral at 29 C while it did not occur in algae
until 33 C was reached. This reveals that the coral
had a more sensitive biochemical response to elevated
temperature than did zooxanthellae.

IV. Discussion
Within a cell, microtubules and actin filaments
play major roles in organelle transportation (Grafstein
and Forman, 1980; Beckerle and Porter, 1983; Hayden

and Allen, 1984). Microtubules have also been found

to be required in vesicle-mediated secretion (Cho and
Garant, 1981; Boyd et al., 1982; Bussonmabillot et al.,
1982) and in delivery of endocytic vesicles to lysosomes
(Oka and Weigel, 1983; Wolkoff et al ., 1985). It is
clear from Fig. 2 that both microtubules and actin
filaments are involved in the process of coral bleaching. In Fig. 1, it is seen that during a coral bleaching
event, due to the elevated temperature, the tendency
of bleaching, or the change in the amount of zooxanthellae discharged, and protein release coincide.
This indicates that coral bleaching is related to the
integrity of cell membranes (Gates et al., 1992). However, Fig. 2 shows that the protein release of the
chemical treated corals did not coincide with the discharge of zooxanthellae. This suggests that without
the normal participation of cytoskeletons, the algae
would not move to the apex of the host cell as it was
found to do in sea anemone (Steen and Muscatine,
1987). The zooxanthellae remain in the host cell de-

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L.S. Fang et al.

(a)

(a)

(b)

(b)

Fig. 3. The effect of myosin and dynein inhibitor on thermal bleaching of

coral as shown by the release of zooxanthellae (a) and protein (b).

Fig. 4. The effect of a calmodulin antagonist on thermal bleaching of coral

as shown by the release of zooxanthellae (a) and protein (b).

spite the rupture of cell membranes. In fact, all the

chemical treated corals (Figs. 2-4) revealed the
similar extend of zooxanthellae released, which were
significantly lower than untreated ones; but these corals still had a protein release under the elevated temperature. It could be that elevated temperature was
the major factor causing the breakage of coral membranes and protein release, but release of zooxanthellae would require more intracellular activity.
The immunofluorescence image of microtubules
of coral cells after colchicine treatment shows what
happened in the cell (Fig. 5(b)). The network was
transformed into many spots. Cytoskeletons change
their conformation during thermal stress (Glass et al.,
1985; Walter et al., 1990). The aggregation of the
microtubules in coral cell was found to be similar to
the change of cells after heat treatment (Welch and
Feramisco, 1985). This suggests that the track func-

tion of this cytoskeleton during endocytic vesicle

movement is distorted.
The intracellular transportation is related to the
combined action of cytoskeletons and motor proteins
of force generating enzymes (Vale, 1987). These enzymes are ATPases, which bind on microtubules and
provide energy that enables the vesicles to move on
the microtubules. Figure 3 shows that the treatment
of N-ethylmaleimide, an inhibitor of two motor proteins, myosin and dynein, greatly suppressed the
release of zooxanthellae. The figure also shows that
the algae did not release coincide with the protein release, as in the bleaching process without inhibitors.
The above results strongly suggest that coral
bleaching under the stress conditions employed in this
experiment requires motor proteins to transport
zooxanthellae-containing vesicles via the cytoskeleton
track to the cell membrane. If this process is not com-

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Subcellular Mechanism of Coral Bleaching

Table 1. The Temperature at which hsp 35 Appeared in Coral Cell
(Acropora grandis) and its Symbiotic Zooxanthellae
T C

25

27

29

31

33

35

Coral
Zooxanthellae

+
+

+
+

Note: +: with hsp35

: without hsp35

(a)

Fig. 5. (a) An immunofluorescence image of microtubules in normal coral

cells. (b) Microtubules of coral cells treated with colchicine. Many
spots are observed when compared to normal cells.

94 (KD)
67
43
hsp3 5
30
20.1
14
M

12

22

Fig. 6. An electrophoretic diagram of proteins from coral cells showing the

appearance of hsp 35. M are the markers. The numbers in each
sample column indicate the number of hours of incubation of coral
at 31 C.

pleted, cell membrane breakage alone will not result

in the release of zooxanthellae. This is very similar to
the process of cell exocytosis.
Huang et al. (1998) demonstrated that a calcium
signal essential for coral bleaching could be a triggering signal for host cell exocytosis. They further
showed that a continuous supply of calcium ions was
significantly related to the extend of bleaching. This
suggests that calcium-related cellular activities for
exocytosis will be further activated with the participation of calmodulin. Figure 4 shows the effectiveness of N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide, a calmodulin antagonist, in the inhibition
of zooxanthellae release. When the protein which binds
calcium ion and regulates its molecular activity is
blocked, the release of zooxanthellae is also stopped.
In other words, the function of calmodulin is as a link
between the calcium signal and the transportation of
zooxanthellae containing vacuoles on cytoskeleton.
Our experiment using the hsp 35 kDa as a marker
to examine the sensitivity of coral and zooxanthellae to temperature is followed the idea developed
by Hofmann and Somero (1996). Because there is a
long dispute about whether coral or zooxanthellae
plays an active role in terminating the symbiotic relationship, the finding showing the occurrence of 35 kDa
in coral cells at lower temperature indicates that the
host is more sensitive to environmental stress, and that
there may be an earlier cellular response to adjust the
symbiotic relationship. The other subcellular activities
examined in this study, which revealed that exocytosis is an important mechanism of coral bleaching,
further suggests that the host cell plays a more active
role. This bleaching mechanism provides wider flexibility for coral in the symbiotic relationship. Once
settled, coral is less mobile than its symbiotic algae.
Therefore, it is advantageous for coral to adapt to
environmental change by adjusting the symbiotic
relationship using normal cell activities. It is unlikely
that cross contamination of hsp 35 occured since a
checking procedure similar to that described by
Bythell et al. (1995) was performed; if it had happened,
the 35 kDa protein should have appeared on the gels
of zooxanthellae samples treated at 29 C and 31 C.
In summary, the mechanism of zooxanthellae

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L.S. Fang et al.

research was supported by the National Science Council of the Republic of China (NSC 85-2321-B-291-001 BH).

References

Fig. 7. Schematic representation of the discharge of zooxanthellae from

the host cell as one of the bleaching mechanisms of coral under
environmental stress (e.g., elevated temperature). (1) The coral cell
senses environmental stress (rising temperature) and produces a
cytosolic calcium signal; (2) the signal is mediated by calmodulin,
which activates the process of transporting the vacuole that contains
zooxanthellae to the host cell membrane; (3) the vacuole is anchored on cytoskeletons, and its movement is powered by motor
proteins; (4) zooxanthellae move closer to the membrane via
cytoskeleton network. Its final release is achieved through breakup
of the host membrane. Abbreviations: CS: cytoskeleton; CCS:
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The vacuole is anchored on cytoskeletons and its
movement is powered by motor proteins. When the
zooxanthellae moves closer to the membrane through
the cytoskeleton network, its final release is achieved
through breakup of the host membrane.

Acknowledgment
We thank Dr. C.L. Tsai for helping with the fluorescence photograph, Miss Y.H. Wu for performing electrophoresis analysis and
Dr. C. A. Chen and Mr. Y. C. Lin for preparing the manuscript. This

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