J. Phycol.

48, 117–126 (2012)

 2011 Phycological Society of America
DOI: 10.1111/j.1529-8817.2011.01099.x

VARIABILITY IN THE PRIMARY SITE OF PHOTOSYNTHETIC DAMAGE IN

SYMBIODINIUM SP. (DINOPHYCEAE) EXPOSED TO THERMAL STRESS 1

Lucy Buxton
Plant Functional Biology Climate Change Cluster, School of the Environment, University of Technology Sydney, Sydney,
New South Wales 2007, Australia

Shunichi Takahashi
ARC Centre of Excellence in Plant Energy Biology, Molecular Plant Physiology Group, Research School of Biological Sciences,
The Australian National University, Canberra, Australian Capital Territory, Australia

Ross Hill2 and Peter J. Ralph

Plant Functional Biology Climate Change Cluster, School of the Environment, University of Technology Sydney, Sydney,
New South Wales 2007, Australia

Exposure to elevated temperature is known to quenching; PAM, pulse amplitude modulated; Q A,

cause photosynthetic inhibition in the coral symbi-
ont Symbiodinium sp. Through the use of the artifi-
cial electron acceptor, methyl viologen, this study
identified how reduced photosynthetic capacity
occurs as a result of inhibition up- and ⁄ or down-
stream of ferredoxin in Symbiodinium sp. in hospite
and in culture. Heterogeneity between coral species
and symbiont clades was identified in the thermal
sensitivity of photosynthesis in the symbionts of the
scleractinian corals Stylophora pistillata and Pocillo-
pora damicornis, as well as among Symbiodinium cul-
tures of clades A, B, and C. The in hospite
symbionts of S. pistillata and the cultured clade C
Symbiodinium both exhibited similar patterns in that
their primary site of thermal inhibition occurred
downstream of ferredoxin at 32C. In contrast, the
primary site of thermal inhibition occurred
upstream of ferredoxin in clades A and B at 32C,
while at 34C, all samples showed combined up- and
downstream inhibition. Although clade C is common
to both P. damicornis and S. pistillata, the manner of
thermal inhibition was not consistent when observed
in hospite. Results showed that there is heterogeneity
in the primal site of thermal damage in Symbiodinium
among coral species and symbiont clades.
Key index words: Calvin cycle; coral bleaching;
pulse amplitude modulated fluorometry; Symbi-
odinium; thermal inhibition
Abbreviations: ATP, adenosine triphosphate; F m,
dark-acclimated maximum fluorescence; F m ’,
light-adapted maximum fluorescence; F o, dark-
acclimated minimum fluorescence; Fv ⁄ Fm, maximum
quantum yield of PSII; NPQ, nonphotochemical
1
Received 9 June 2010. Accepted 22 August 2011.
2
Author for correspondence: e-mail ross.hill@uts.edu.au.
PSII primary electron acceptor; RuBP, ribulose-
1,5- biphosphate; U PSII, effective quantum yield
of PSII
Corals harbor endosymbiotic dinoflagellate algae
known as zooxanthellae, from the genus Symbiodini-
um. Symbiodinium produces carbon-rich organic com-
pounds through photosynthesis, predominantly in
the form of glycerol, glycerides, and lipids (Muscatine
1990), of which a large proportion are transferred
to the host. Under normal conditions, corals show
brownish coloration due to photosynthetic pig-
ments, such as peridinin and chls, in Symbiodinium.
However, when corals are exposed to increased sea-
water temperature, they become pale due to a pro-
cess called coral bleaching, through the loss of algal
symbionts and degradation of photosynthetic pig-
ments. Since coral bleaching often follows a decline
of photosynthetic activity in Symbiodinium within cor-
als, impairment of photosynthesis is assumed to
cause coral bleaching (Baird et al. 2009).
In Symbiodinium, it has been proposed that
increased temperature directly damages PSII (Iglesias-
Prieto et al. 1992), the Calvin cycle enzymes (Jones
et al. 1998, Leggat et al. 2004), and ⁄ or thylakoid
membranes (Tchernov et al. 2004). Thermal stress
in conjunction with high irradiance is known to
reduce the photosynthetic efficiency of PSII (Hill
and Ralph 2005, Ulstrup et al. 2006) due to rates of
photodamage exceeding rates of counteracting
repair (Takahashi et al. 2008, 2009, Hill et al. 2011).
This net loss of functional PSII centers results in
photoinhibition, which is easily measured by a
decrease in PSII photochemical efficiency (Fv ⁄ Fm)
using pulse amplitude modulated (PAM) fluorome-
try (Hill et al. 2011). Recent studies have demonstrated

117
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that thermal acceleration of photoinhibition in Sym-
biodinium is primarily associated with inhibition of
the repair of photodamaged PSII (Takahashi et al.
2004, 2009), or overwhelming rates of photodamage
exceeding any increase in rates of repair (Hill et al.
2011). Since damages to PSII (Allakhverdieva and
Murata 2004), Calvin cycle enzymes (Jones et al.
1998, Leggat et al. 2004, Lilley et al. 2010), and thy-
lakoid membrane (Mizusawa et al. 2009) all have a
potential to inhibit the repair of photodamaged
PSII, it is still uncertain which of them is the pri-
mary site of thermal damage and which is most
strongly associated with acceleration of photoinhibi-
tion in Symbiodinium during bleaching events. Sensi-
tivity of photoinhibition to heat stress differs among
Symbiodinium clades and coral species (Bhagooli and
Hidaka 2003). Furthermore, it is likely that the sen-
sitivity differs between Symbiodinium in hospite and
in culture (Iglesias-Prieto et al. 1992, Bhagooli and
Hidaka 2003). Although thermal sensitivity was
assumed to be linked to symbiont genotype,
research has shown that sensitivity to photoinhibi-
tion differs among subclades of Symbiodinium
(Tchernov et al. 2004, Sampayo et al. 2007).
The components of photosynthetic electron trans-
port can be isolated and manipulated by addition of
artificial electron acceptors such as methyl viologen
(N,N’-dimethyl-4,4’-bipyridinium dichloride; MV).
MV accepts electrons from PSI and oxidizes ferre-
doxin (Fig. 1). Thus, the application of MV allows
the separation of the light-dependant reactions
(upstream of ferredoxin) and those reactions occurring
downstream such as the Calvin cycle (downstream
FIG. 1. Diagram depicting the site of action of the competitive electron acceptor methyl viologen at PSI (adapted from Taiz and Zeiger
2002).
LUCY BUXTON ET AL.
of ferredoxin). Comparative measurements of pho-
tosynthetic electron flow using PAM fluorometry in
the presence of MV and in its absence allow us to
examine the effect of thermal stress on these two
components of photosynthesis. While MV is a useful
chemical for preventing overreduction of PSII elec-
tron transport, it is also a known herbicide and can
lead to oxidative damage (Falkowski and Raven
1997). Therefore, a comprehensive bioassay was
conducted to identify optimal working concentra-
tions, incubation times, interactive effects of temper-
ature, and possible inhibitory effects of MV on
Symbiodinium sp. In addition, the effectiveness of MV
as an artificial electron acceptor was characterized
through the inhibition of the Calvin cycle by glycol-
aldehyde (GA) and subsequent addition of MV.
These preliminary experiments were essential first
steps, required for the subsequent characterization
of impacts upstream or downstream of ferredoxin in
Symbiodinium under thermal stress.
To examine the primal target of heat stress in
Symbiodinium, we examined the effect of increased
temperature on the photosynthetic efficiency of PSII
(FPSII) and the thermal energy dissipation (nonpho-
tochemical quenching [NPQ]). The experiments
were carried out in the presence and absence of MV
using two different coral species (Stylophora pistillata
and Pocillopora damicornis) and four different cul-
tured Symbiodinium clades.
MATERIALS AND METHODS
Coral and Symbiodinium sp. growth conditions. Colonies of
the branching corals Pocillopora damicornis and Stylophora
 P H O T O S Y N T H E S I S I N S Y MB I O D I N I UM
pistillata were collected from Heron Island lagoon (<2 m deep)
(15206¢ E, 2029¢ S) (Great Barrier Reef Marine Park Author-
ity collection permit number G05 ⁄ 14083.1) and transported to
the University of Technology, Sydney. Eight colonies of each
coral species were cultured in the laboratory for 1 month in a
500 L recirculating aquarium using artificial seawater (Red Sea
Coral Pro in reverse osmosis water) with carbonate of 140 ppm,
salinity 33 ppt, 26 ± 1C and irradiance of 250 lmol pho-
tons Æ m)2 Æ s)1. Coral nubbins 2 cm3 were established from
each colony and acclimated for a further month prior to use in
experiments. For each experiment, five nubbins were used per
treatment (n = 5). Genetic analysis of zooxanthellae isolated
from P. damicornis and S. pistillata collected from Heron Island
by our laboratory have identified the population in both
species as clade C (Hill et al. 2009). This is in agreement with
other studies from Heron Island (LaJeunesse et al. 2003,
Magalon et al. 2007).
Four different cultured Symbiodinium strains were used in
these experiments. Cells were grown in f ⁄ 2 media (Guillard
and Ryther 1962) made up in artificial seawater (as above) and
maintained under an irradiance of 40–60 lmol pho-
tons Æ m)2 Æ s)1 at 26C. To remove the influence of culture
age on physiological responses, all samples were taken from
cultures in the exponential growth phase (21 d of growth). To
identify the genetic clade of each culture, 4 mL of each was
centrifuged at 3,400 rpm (1,300g; Labogene, Lynge, Denmark,
1580 MGR) for 10 min and the supernatant discarded. The
algal pellet was resuspended in 1 mL of salt-saturated DMSO
and stored at room temperature for subsequent genetic
identification. Symbiodinium DNA was extracted using the
Bioline Genomic DNA Isolate Mini Kit (London, UK). Symbi-
odinium genotypes were distinguished using the 28S rRNA gene
using a primer set designed by Zardoya et al. (1995), forward
5¢-CCC GCT GAA TTT AAG CAT ATA AGT AAG CGG-3¢ and
reverse 5¢-GTT AGA CTC CTT GGT CCG TGT TTC AAG A-3¢.
Reactions were performed in 100 lL containing PCR buffer,
40 lg BSA, 0.2 mM deoxynucleotide triphosphate (Bioline),
0.25 lM concentrations of each primer, 1 lL of Taq DNA
polymerase (New England), and 1 lL of template DNA.
Amplification conditions for PCR were 94C for 5 min followed
by 30 cycles of 94C for 1 min, 65C for 1 min, and 72C for
1 min, with a final extension step at 72C for 7 min. All
amplifications were carried out in a PerkinElmer 2400 thermal
cycler. PCR products were purified using a GFX PCR DNA and
gel band purification kit (Amersham Biosciences, Bucking-
hamshire, UK) and were sequenced in both directions at the
Macrogen sequencing facility (Seoul, Korea). Results were
compared to existing sequences stored in GenBank (http://
www.ncbi.nlm.nih.gov). Culture CS-73 (from the Common-
wealth Scientific and Industrial Research Organisation,
CSIRO) was identified as clade A (GenBank accession number
U63480) and the additional three monocultures were identi-
fied as clade A (accession number AF427455), B (accession
number GQ984268), and C (accession number AF427462).
Throughout the remainder of this article, these four cultures
will be referred to as CS-73, clade A, clade B, and clade C,
respectively.
Fluorescence measurements. A Diving-PAM Fluorometer (Walz
GmbH, Effeltrich, Germany) was used to measure photosyn-
thetic responses of symbionts in coral samples, while a Water-
PAM Fluorometer (Walz GmbH) was used to measure algae
suspensions. To confirm that actinic light levels stimulated
sufficient photosynthetic activity in both whole corals and
cultured zooxanthellae, steady-state light curves were con-
ducted prior to commencement of experimental assays (Sero-
dio et al. 2006, Ulstrup et al. 2006). Prior to the start of each
steady-state light curve, samples were dark-incubated for
30 min, after which a saturating pulse was applied and
fluorescence levels Fo and Fm were measured. Samples were
119
then exposed to 12 increasing PAR irradiance levels, from 12 to
920 lmol photons Æ m)2 Æ s)1. Under each irradiance level, Fs
and Fm¢ were determined each 90 s, until a steady-state in
DF ⁄ Fm¢ was reached (minimum 5 min). The time required to
reach a new steady-state varied with the difference in irradiance
levels of consecutive light steps but was typically reached within
10 min. The final effective quantum yield of PSII (FPSII = [Fm¢–
Ft] ⁄ Fm¢) was recorded once it stabilized at a given irradiance
(5 min). Experimental light intensity was set at the highest
irradiance at which no decline in FPSII was observed during the
steady-state light curve (50 lmol photons Æ m)2 Æ s)1 cultured
Symbiodinium sp., 80 lmol photons Æ m)2 Æ s)1 whole corals).
Induction curves were performed on all samples, with dark-
acclimation applied for 10 min and minimum fluorescence
(Fo) measured via the application of weak pulsed measuring
light. Preliminary experiments identified that 10 min dark-
acclimation allowed maximum Fm and minimum Fo to be
achieved (data not shown). A saturating pulse of light
(>4,000 lmol photons Æ m)2 Æ s)1) enabled the measurement
of Fm. Maximum quantum yield of PSII can be calculated as
(Fm–Fo) ⁄ Fm = Fv ⁄ Fm. Immediately following the saturating
pulse, the sample was exposed to actinic light (80 and 50 lmol
photons Æ m)2 Æ s)1 for whole corals and cultures, respectively)
and a series of saturating flashes to measure Fm¢. These
saturating flashes allowed for the determination of effective
quantum yield of FPSII and NPQ (= [Fm–Fm¢] ⁄ Fm¢) can be
calculated (Schreiber et al. 1986).
Temperature treatments. Experiments were conducted at the
control temperature of 26C and then repeated at the elevated
temperatures of 32C and 34C. Symbiodinium sp. and the whole
corals used in this study were cultured at 26C. Coral bleaching
is known to occur between 30C and 34C (Iglesias-Prieto et al.
1992, Fitt and Warner 1995, Hoegh-Guldberg 2011), and it has
been clearly demonstrated that in situ corals exposed to
temperatures above 32C show reduced fluorescence yield and
other effects that point to a significant reduction in photosyn-
thetic activity (Fitt and Warner 1995, Warner et al. 1996,
Iglesias-Prieto 1997, Jones et al. 1998). Therefore, these
treatments were chosen to represent ecologically relevant
conditions.
Developing a methyl viologen bioassay. Preliminary experi-
ments were performed to test (a) the optimum conditions
under which MV should be used with Symbiodinium sp. (i.e., MV
concentration, length of exposure and temperature interac-
tion), (b) chemical inhibition of the Calvin cycle using GA
(Sicher 1984), and (c) the effectiveness of MV as an electron
acceptor in the reversal of GA inhibition (Falkowski and Raven
1997).
Optimal concentration of methyl viologen: To assess the toxic
effects of MV and identify the optimum working concentration
of MV with Symbiodinium in hospite (P. damicornis and
S. pistillata) (2 cm3, ca. 17 lg Æ mL)1 chl a) and cultures
(1 mL cells concentrated to ca. 1 lg Æ mL)1 chl a in 2 mL
0.45 lm filtered seawater [FSW]), samples were exposed to a
range of MV concentrations (0, 5, 10, 20, 30, 40 mM) for 4 h in
the dark. Induction curves were conducted every 30 min (80
and 50 lmol photons Æ m)2 Æ s)1 for whole corals and cultures,
respectively). Induction curves were performed as previously
described, and Fm, FPSII, and NPQ and were recorded. The
testing procedure showed 20 mM to be optimal concentration
at 26C and under 50 and 80 lmol photons Æ m)2 Æ s)1.
Length of methyl viologen exposure: Coral nubbins of S. pistil-
lata (2 cm3, ca. 17 lg Æ mL)1 chl a) were incubated in 10 mL of
0.45 lm FSW in 20 mL scintillation vials at 26C and FPSII was
measured as described above. MV (20 mM) was added to each
vial and left in the dark (n = 4) as determined from the
previous experiment. Fv ⁄ Fm was measured every 5 min for 2 h.
This test was repeated on P. damicornis and each test strain of
Symbiodinium sp. (1 mL cells concentrated to ca. 1 lg Æ mL)1
120 LUCY BUXTON ET AL.
chl a in 2 mL 0.45 lm FSW). Preliminary experiments showed
the optimum incubation time for MV was 40 min for in hospite
Symbiodinium sp. and 75 min for cultured Symbiodinium sp.
(data not shown).
Temperature interaction with methyl viologen: The interaction
of temperature and MV was tested at 26, 32, and 34C.
Populations of cultured Symbiodinium sp. (3 mL FSW) were
maintained in a glass 20 mL scintillation vial at 26C and FPSII
was measured under actinic light of 50 lmol pho-
tons Æ m)2 Æ s)1. A known concentration of MV was added to
each sample (10, 20, 30 mM). FPSII was measured after 30, 60,
and 120 min of incubation with MV. This test was repeated
on each test strain of Symbiodinium sp. For the whole corals,
P. damicornis and S. pistillata, 2 cm3 nubbins were placed in the
20 mL scintillation vial with 10 mL artificial seawater (ASW)
and the experiment repeated as above (with 10, 20, 30 mM
MV) at 80 lmol photons Æ m)2 Æ s)1 with measurements after
30, 60, and 120 min of incubation.
To test the interaction of temperature and MV, experiments
were repeated as above at 32C and 34C. Temperatures were
increased gradually at a rate of 1C per 10 min to the final test
temperature using a temperature-controlled water bath (Julabo,
MV, Germany).
Methyl viologen as an effective electron acceptor: Inhibition of
the Calvin cycle in Symbiodinium sp. was achieved through
additions of GA. The effectiveness of MV as an artificial
electron acceptor acting to ameliorate the inhibition by GA was
also tested. Coral nubbins of S. pistillata (2 cm3, ca.
17 lg Æ mL)1 chl a) were incubated in 10 mL 0.45 lm FSW in
20 mL scintillation vials at 26C, and FPSII was measured as
described above. Half the samples were incubated in the dark
with 20 mM GA for 1 h and Fv ⁄ Fm measurements taken.
Varying concentrations of MV (0, 5, 10, 20, 30, 40 mM) were
added to all the samples and incubated in the dark for a
further hour. At the end of incubation (2 h total), Fv ⁄ Fm was
remeasured. This test was repeated on P. damicornis and each
test strain of Symbiodinium sp. (1 mL cells concentrated to ca.
1 lg Æ mL)1 chl a in 2 mL 0.45 lm FSW).
Investigating thermal inhibition of the dark reactions in Symbi-
odinium sp. Utilizing the findings of the MV bioassay, exper-
iments investigating photosynthetic impacts upstream or
downstream of ferredoxin in Symbiodinium under thermal
stress were conducted. Populations of cultured Symbiodinium
sp. strain CS-73 were concentrated to 1 lg Æ mL)1 chl a (equal
to 6.03 · 105 cells ± 5.3 · 104). One mL of cell concentrate
was diluted in 2 mL of 0.45 lm FSW and maintained in a glass
20 mL scintillation vial at 26C. Dark-light induction curves
were performed (actinic light of 50 lmol photons Æ m)2 Æ s)1)
before placing the vial into a thermostatically controlled water
bath. Temperatures were ramped as before, and the samples
left in darkness. FPSII was measured after 120 min of incuba-
tion (actinic light of 50 lmol photons Æ m)2 Æ s)1) followed by
the addition of 20 mM MV to half the samples, while the
controls did not receive MV. All samples were then incubated
in darkness at temperature for a further 120 min. The control
experiment samples were held at ambient temperature (26C).
This test was repeated on each test strain of Symbiodinium sp.
(clades A, B, and C). For the whole corals, P. damicornis and S.
pistillata, a 2 cm3 (ca. 17 lg Æ mL)1 chl a) nubbin was placed
in the 20 mL scintillation vial with 10 mL FSW and the
experiment repeated as above (actinic light of 80 lmol
photons Æ m)2 Æ s)1).
Chl analysis. For whole coral pieces, tissue was removed
from the skeleton using an air brush and compressed air. The
tissue slurry was suspended in 15 mL of 0.45 lm FSW and
centrifuged at 1,000g, 26C for 10 min. The pellet was
resuspended in 15 mL FSW, homogenized and filtered through
20 lm mesh to remove insoluble host material, then centri-
fuged at 800g, 26C for 5 min. The pellet was resuspended in a
final volume of 5 mL 90% acetone for 20 h at 4C in darkness.
For cultured zooxanthellae, the cell suspension was spun at
1,000g, 26C for 10 min and resuspended in 5 mL 90% acetone
for 20 h at 4C in darkness. After pelleting the cells by
centrifugation (5 min at 1,000g), an absorption spectrum
(400–800 nm) of the acetone supernatant was measured using
a spectrophotometer (LKB Biochrom Ultraspec II, Cambridge,
England). Chl a and c2 concentrations were calculated accord-
ing to the equations of Jeffrey and Humphrey (1975).
Statistical analysis. Experiments in the MV bioassay
employed one-way analysis of variance (ANOVA) tests with
Tukey-Kramer post hoc tests (a = 0.05) to detect any
significant differences between (a) FPSII under different MV
concentrations, (b) Fv ⁄ Fm in control and MV-treated samples
under different temperatures, (c) FPSII under different GA
concentrations, and (d) FPSII under different MV concentra-
tions in the presence of GA. In the subsequent experiments
investigating thermal inhibition of the dark reactions in
Symbiodinium, repeated measures ANOVA tests with Tukey-
Kramer post hoc tests (a = 0.05) were used to detect changes
in FPSII and NPQ over the length of the experiment between
treatments at a single temperature in the presence or absence
of MV. One-way ANOVA tests were also performed to detect
any significant differences in FPSII and NPQ between the + and
)MV treatments at each temperature. The assumptions of
normality (Kolmogorov-Smirnov test) and equal variance
(Levene’s test) were met in all cases. These analyses were
performed using SPSS statistical software (version 11.0, 2001,
Chicago, IL, USA).
RESULTS
Developing a methyl viologen bioassay. At the control
temperature (26C), results show that FPSII did not
significantly change by additions of MV (5–40 mM)
(P-values > 0.05). When incubated at 32C and
34C, there were some concentrations of MV for
which a significant increase in FPSII was observed
compared to the control at that temperature
(Fig. 2, B and C, as denoted by asterisk, P < 0.005).
Overall, a concentration of 20 mM MV was the most
effective in increasing FPSII under thermal stress
(Fig. 2, A–C). MV at a concentration of 20 mM was
shown to sufficiently saturate FPSII in both whole
corals and cultured Symbiodinium sp. at 32C and
34C (Fig. 2, A–C). Preliminary experiments showed
that the optimum incubation time for MV was
40 min for in hospite Symbiodinium sp. or 75 min for
cultured Symbiodinium sp. (data not shown). Results
show that Fv ⁄ Fm remained unaffected, or signifi-
cantly increased in the presence of 20 mM MV at all
temperatures (Fig. 3, A–C, significant increase com-
pared to the control denoted by asterisk, P < 0.005).
Interestingly, at 26C, Fv ⁄ Fm significantly increased
in clades B and C upon addition of 20 mM MV
(Fig. 3A), although measures of FPSII did not detect
any significant change at this temperature and MV
concentration (Fig. 2A). Both whole corals and cul-
tured Symbiodinium sp. incubated with GA at concen-
trations greater than 10 mM (26C, 1 h) showed a
significant decrease in FPSII (Fig. 4, P < 0.005). In
the in hospite symbionts of P. damicornis, a significant
decrease in FPSII was found following inhibition of
 P H O T O S Y N T H E S I S I N S Y MB I O D I N I UM 121

FIG. 2. The effect of methyl viologen on the effective quantum

yield of whole corals and cultured Symbiodinium sp. Figure shows
the results from a range of concentrations of methyl viologen,
and the effects of temperature. Asterisks denote a significant
increase in effective quantum yield from the control (no MV).
Error bars represent ±standard error (n = 5). MV, methyl
viologen.
FIG. 3. The effect of 20 mM methyl viologen on Fv ⁄ Fm. Aster-
isks denote significant effect of MV compared to the control
the Calvin cycle by 15 mM GA compared to the con- ()MV) at a given temperature. Error bars represent ±standard
error (n = 5). MV, methyl viologen.
trol (Fig. 5, P < 0.001). Additions of MV caused a
significant increase in FPSII at MV concentrations
15 mM and greater compared to those with GA, but
no MV (as denoted by asterisk, Fig. 5, P < 0.001).
Results also show that there was no significant dif-
ference between FPSII of the control and FPSII fol-
lowing incubation with MV at concentrations
greater than 15 mM (Fig. 5, P > 0.05). Results from
P. damicornis provide a representative response,
equivalent to the findings from in hospite symbionts
of S. pistillata and all Symbiodinium clades (data not
shown).
Evidence of thermal inhibition downstream of ferredoxin
in Symbiodinium sp. At 32C in the absence of MV,
significant decreases in FPSII were evident in the FIG. 4. The effect of glycolaldehyde on the effective quantum
symbionts of S. pistillata and Symbiodinium sp. clades yield of whole corals and cultured Symbiodinium sp. at 26C. Error
A, B, and C (Fig. 6, B–D, E) when compared to the bars represent ±standard error (n = 5).
122 LUCY BUXTON ET AL.
FIG. 5. Reversal of photosynthetic inhibition by methyl violo-
gen following incubation with glycolaldehyde for Pocillopora dami-
cornis at 26C. Asterisks denote a significant increase in effective
quantum yield in the GA + MV treatment compared to those with
only GA. Error bars represent ±standard error (n = 5). GA, glycol-
aldehyde; MV, methyl viologen.
control (26C) (as denoted by A, B, C). At 32C, P.
damicornis symbionts showed no significant change
in FPSII (P > 0.05). Incubation with MV at 32C
caused a significant increase in FPSII compared to
treatments without MV in S. pistillata symbionts and
cultured Symbiodinium clade C (Fig. 6, B and E, as
denoted by *). Symbionts of S. pistillata exhibited a
significant rise in FPSII from 0.377 to 0.553 (Fig. 6B),
as did clade C from 0.422 to 0.556 (Fig. 6E) (32C
)MV and 32C +MV, respectively). In both these
samples, FPSII was not significantly different from
the control, 26C +MV (as denoted by a, b, c). In
the absence of MV, incubation at higher tempera-
ture (34C) caused significant declines in FPSII in
all samples compared to the control (26C) (Fig. 6).
In symbionts of S. pistillata, and Symbiodinium cul-
tures of clades A, B, and C, the further reduction in
FPSII was also significantly lower than at 32C
(Fig. 6, B–D, E). Comparison between the mono-
cladal cultures revealed that clade C expressed the
largest percentage decrease in FPSII at 34C com-
pared to the control (63% decrease in FPSII,
Fig. 6E). Addition of MV at 34C caused a signifi-
cant increase in FPSII in all samples (Fig. 6). How-
ever, FPSII did not recover to a level comparable
with that of the control (26C +MV) in any of the
samples. The largest relative increase in FPSII was
exhibited by clade C upon addition of MV (49%,
Fig. 6E).
At 32C, NPQ increased in CS-73 only, from
0.082 to 0.516 (Fig. 7F, as denoted by A, B). Incuba-
tion of CS-73 with MV at 32C elicited no significant
change in NPQ (Fig. 7F). However, incubation with
MV at the same temperature caused a significant
decrease in NPQ in S. pistillata symbionts and the
clade C culture (Fig. 7, B and E, as denoted by *)
from 0.133 to 0.025 in S. pistillata, and from 0.127
to 0.011 in clade C (32C )MV and 32C +MV,
respectively). At 34C, NPQ was significantly higher
than 26C in the symbionts of P. damicornis, S. pistil-
lata, and cultures A, C, and CS-73 (Fig. 7, A–C, E,
and F). However, CS-73 showed no further increase
in NPQ compared to 32C (0.516 ± 0.042 at 32C
)MV and 0.486 ± 0.002 at 34C )MV) (Fig. 7F).
The greatest level of NPQ at 34C was exhibited by
the symbionts of P. damicornis (0.744). Addition of
MV at 34C caused a significant decrease in NPQ in
P. damicornis and S. pistillata symbionts, and clade A
and C cultures. Furthermore, NPQ returned to
the level of the control (26C +MV) in all these
samples.
Evidence of thermal inhibition upstream of ferredoxin in
Symbiodinium sp. Under moderate heat stress
(32C), clade A and B cultures showed a significant
decline in FPSII compared to the control (Fig. 6, C
and D). FPSII did not significantly change with the
addition of MV (P > 0.05).
DISCUSSION
Exposure to elevated temperature is known to
cause photosynthetic inhibition in the coral symbi-
ont Symbiodinium sp.; however, it is yet to be conclu-
sively resolved whether thermal bleaching occurs
due to a breakdown at a single site within the pho-
tosynthetic apparatus. Several models of photophysi-
ological thermal damage in corals have been
proposed and fall into two broad groups; those
where the damage takes place at the site of the light
reactions (Warner et al. 1999, Hill et al. 2004,
2011), and those that occur further down the elec-
tron transport chain in the Calvin cycle (Jones et al.
1998, Wooldridge 2009). To differentiate between
these two models, this study applied the artificial
electron acceptor, MV, to provide evidence of pri-
mary thermal inhibition occurring either up- or
downstream of PSI in free-living Symbiodinium sp.
and those in hospite under conditions of moderate
(32C) and high (34C) thermal stress. Results
clearly show that additions of MV at all tempera-
tures caused FPSII and Fv ⁄ Fm to either increase or
remain unchanged (Figs. 2 and 3). It is therefore
possible to conclude that 20 mM MV used here had
no significant inhibitory effect on cultured Symbiodinium
sp. or those in hospite.
To determine the presence of significant thermal
inhibition of photosynthesis, FPSII and NPQ values
at 32C and 34C were compared to that of the con-
trol (26C) in the absence of MV (Figs. 6 and 7, as
denoted by capital letters). To assess the incidence
of this photosynthetic inhibition occurring at a site
downstream of ferredoxin, it was necessary to com-
pare FPSII and NPQ values before and after the
addition of MV at a given temperature (Figs. 6 and
7, as denoted by an asterisk). Interestingly, these
results showed that in cases where NPQ significantly
decreased following addition of MV under elevated
temperature (Fig. 7), there was a concurrent
increase in FPSII (Fig. 6). This finding suggests that
by allowing for more efficient electron transport,
MV reduces the activation of NPQ processes. Finally,
to assess the extent to which down-stream inhibition
 P H O T O S Y N T H E S I S I N S Y MB I O D I N I UM 123

FIG. 6. The effect of tempera-

ture on FPSII at 26, 32, and 34C
in the absence (solid bars) and
presence (cross-hatched bars) of
methyl viologen. Those bars
marked with * denote a signifi-
cant difference in FPSII following
the addition of methyl viologen
at a given temperature. Signifi-
cant differences between the con-
trol (26C) and temperature
treatments (32C and 34C) are
denoted by capital letters in the
absence of MV. Lowercase letters
denote significant differences
between the 26C +MV and tem-
perature treatments (32C and
34C +MV). Error bars represent
±standard error (n = 5). MV,
methyl viologen.

contributed to photosynthetic inhibition as a whole,
it was necessary to examine the recovery of FPSII fol-
lowing addition of MV compared to the control.
This was assessed by comparing results from 32C
+MV and 34C +MV to 26C +MV (Figs. 6 and 7 as
denoted by lowercase letters).
Primary impact of moderate heat stress in Symbiodinium
within corals. The moderate thermal stress treatment
(32C) caused a significant decline in FPSII in the
symbionts of S. pistillata, but not those in P. damicornis
(Fig. 6B). This result is consistent with the previous
report showing that P. damicornis is less thermally sen-
sitive than S. pistillata (Loya et al. 2001).
The thermally induced decline in FPSII in S. pistil-
lata symbionts at 32C was completely reversed fol-
lowing addition of MV and the value returned to a
level comparable to that of the control (26C +MV).
This result identifies that (a) thermal photosyn-
thetic inhibition in S. pistillata symbionts is occur-
ring downstream of ferredoxin, and that (b) this
mode of inhibition comprises the majority of overall
FPSII inhibition. Therefore, we are able to conclude
that the primary site of thermal inhibition in
S. pistillata symbionts at 32C is a result of reduced
electron transport at a site downstream of ferre-
doxin and not as a result of inhibition occurring at
124 LUCY BUXTON ET AL.
PSII or PSI. These findings are consistent with those
of Jones et al. (1998) who speculated that the pri-
mary site of thermal inhibition of S. pistillata symbio-
nts was via Calvin cycle inhibition accompanied by a
characteristic increase in NPQ.
Although past studies on Heron Island have
found P. damicornis and S. pistillata both harbor
clade C Symbiodinium (LaJeunesse et al. 2003, Mag-
alon et al. 2007, Hill et al. 2009), the different
responses in primary sites of impact during bleach-
ing, may be due to distinctions in subcladal geno-
type. Indeed, Sampayo et al. (2007) demonstrated
FIG. 7. The effect of tempera-
ture on non-photochemical
quenching at 26, 32, and 34C in
the absence (solid bars) and pres-
ence (cross-hatched bars) of
methyl viologen. Those bars
marked with * denote a signifi-
cant difference in FPSII following
the addition of methyl viologen
at a given temperature. Signifi-
cant differences between the con-
trol (26C) and temperature
treatments (32C and 34C) are
denoted by capital letters in the
absence of MV. Lowercase letters
denote significant differences
between the 26C +MV and tem-
perature treatments (32C and
34C +MV). Error bars represent
±standard error (n = 5). MV,
methyl viologen.
specific subcladal partitioning, which were distinct
between the two coral species, and such fine-scale
differences may be responsible for the disparity
found between P. damicornis and S. pistillata in this
study (Sampayo et al. 2007, 2008, 2009). Alterna-
tively, it is possible that the discrepancy between
P. damicornis and S. pistillata may reflect a distinct
property of the host, such as tissue thickness or pig-
mentation (Baird et al. 2009).
Difference in primary sites of thermal damage among
cultured Symbiodinium spp. Intercladal distinctions
in the primary site of thermal damage are evident
 P H O T O S Y N T H E S I S I N S Y MB I O D I N I UM
Table 1. Summary of significant changes in FPSII and in
the presence of methyl viologen at 26, 32, and 34C.
32C
Upstream Downstream Upstream Downstream
Sample of PSI of PSI of PSI
Pocillopora damicornis No No Yes
Stylophora pistillata No Yes Yes
Clade A Yes No Yes
Clade B Yes No Yes
Clade C No Yes Yes
CS-73 No No Yes
among the cultured Symbiodinium clades. Although
clades A, B, and C all exhibited decreases in FPSII at
32C (Fig. 6, C, D, and E), MV caused complete
reversal of FPSII decline in cultured clade C only,
indicating that primary site of thermal inhibition is
downstream of ferredoxin in clade C and not
upstream (Fig. 6E). In clades A and B, FPSII signifi-
cantly declined from the controls at 32C, but MV
did not significantly increase photosynthetic effi-
ciency, suggesting that inhibition occurred at a site
up-stream of ferredoxin. It may also suggest that
there was no inhibition downsteam of ferredoxin in
clade A and B; however, any damage downstream
may not have been apparent using our protocol as
the damaged upstream site would have restricted
electron flow.
At higher temperatures, all clades (A, B, and C)
exhibited further decline in FPSII compared to
32C. Although this was alleviated by MV, FPSII was
still significantly lower than the control (26C
+MV), indicating that there is residual inhibition
unaffected by the presence of MV. It is therefore
possible to conclude that both up- and downstream
inhibition is present at 34C. Clade A and C exhib-
ited elevated NPQ at 34C compared to the control
(26C), and this trend was reversed in the presence
of MV. As all cultures were kept under the same
conditions during batch culture, this difference
could not be attributed to the past thermal history.
Therefore, we speculate that there is a difference in
clade-specific primary sites of thermal inhibition in
cultured Symbiodinium, with damage occurring to
the photosynthetic apparatus upstream of ferre-
doxin in clades A and B, whereas it takes place
downstream in clade C at 32C (Table 1).
The Symbiodinium strain CS-73 (identified as clade
A) exhibited greater thermal tolerance than the
other three cultures, with no change in FPSII at
32C. However, NPQ was strongly induced in CS-73
at 32C and reached levels greater than those from
whole corals or other cultures (NPQ = 0.516).
Although exposure to 34C caused declines in FPSII
in CS-73, values were still greater than any other of
the samples tested and correlated with very strong
NPQ. The differences in the responses from the
clade A culture and CS-73 culture (both from the
same Symbiodinium clade) may reflect a fine-scale,
125
subclade, genetic disparity between the two strains
of Symbiodinium.
How does heat stress accelerate photoinhibition in
34C Symbiodinium? The sequence of events preceding
photosynthetic inhibition in Symbiodinium is com-
of PSI plex and results presented here highlight differen-
Yes tial sites of primary thermal inhibition occurring
Yes between coral species and clades. This study is the
Yes first to demonstrate differential thermal inhibition
Yes of sites up- and downstream of ferredoxin in Symbi-
Yes
Yes odinium. Our present results are unable to ascertain
where exactly downstream this inhibition is occur-
ring. RUBISCO in the Calvin cycle was proposed as
a likely target for thermal dysfunction (Jones et al.
1998, Leggat et al. 2004, Lilley et al. 2010). The
results of this study clearly indicate that there is
more than one primary site of thermal impact to
the photosynthetic apparatus during thermal stress.
Furthermore, this impact is coral species dependant,
and variable amongst Symbiodinium cultures, high-
lighting variability in thermal sensitivity in the
photosynthetic machinery.
CONCLUSION
This study has identified cladal and coral species-
specific heterogeneity in photosynthetic sensitivity
to thermal stress in Symbiodinium. Firstly, the primary
site of inhibition in S. pistillata and cultured clade C
occurred at a site downstream of ferredoxin under
moderate thermal stress, and secondly, the primary
site of inhibition differed across Symbiodinium clades.
This investigation has demonstrated that closely
related Symbiodinium taxa can exhibit significant dif-
ferences in physiology (Hennige et al. 2009), and it
also suggests greater complexity in the coral host-
symbiont relationship in determining bleaching sus-
ceptibility of the holobiont (Abrego et al. 2008).
This study highlights the necessity for evaluating the
responses of many species and clades, to gain a
holistic picture on the impacts of thermal stress to
the photosynthetic apparatus, as investigation into
one clade or coral species does not illuminate a
general mechanism that is apparent for all coral
species. Extrapolations from experimental results
obtained from cultured Symbiodinium to zooxanthel-
lae in hospite are not consistent. Differences
observed here in the photosynthetic functionality of
C in symbiosis with S. pistillata and P. damicornis may
indicate either a distinctive property of the host
or some other pathway of the algae that changes
during growth in hospite and warrants further
investigation.
We thank Dee Carter and Tien Bui from the University of
Sydney for performing Symbiodinium clade analyses, and two
anonymous reviewers whose comments greatly improved this
manuscript.
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