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REPORT
Ocean acidification has no effect on thermal bleaching in the coral
Seriatopora caliendrum
C. B. Wall •T.-Y. Fan •P. J. Edmunds
Received: 3 May 2013 / Accepted: 21 September 2013
ÓSpringer-Verlag Berlin Heidelberg 2013
Abstract The objective of this study was to test whether
elevated pCO
2
predicted for the year 2100 (85.1 Pa) affects
bleaching in the coral Seriatopora caliendrum (Ehrenberg
1834) either independently or interactively with high
temperature (30.5 °C). Response variables detected the
sequence of events associated with the onset of bleaching:
reduction in the photosynthetic performance of symbi-
onts as measured by maximum photochemical efficiency
(F
v
/F
m
) and effective photochemical efficiency (DF/F
m
0)
of PSII, declines in net photosynthesis (P
net
) and photo-
synthetic efficiency (alpha, a), and finally, reduced chlo-
rophyll aand symbiont concentrations. S. caliendrum was
collected from Nanwan Bay, Taiwan, and subjected to
combinations of temperature (27.7 vs. 30.5 °C) and pCO
2
(45.1 vs. 85.1 Pa) for 14 days. High temperature reduced
values of all dependent variables (i.e., bleaching occurred),
but high pCO
2
did not affect Symbiodinium photophysiol-
ogy or productivity, and did not cause bleaching. These
results suggest that short-term exposure to 81.5 Pa pCO
2
,
alone and in combination with elevated temperature, does
not cause or affect coral bleaching.
Keywords Ocean acidification Temperature
Bleaching Scleractinia Taiwan
Introduction
Increased atmospheric CO
2
from anthropogenic activities
is contributing to global climate change (GCC) and
threatens coral reefs through seawater warming and ocean
acidification (OA) (Hoegh-Guldberg et al. 2007). OA
caused by the dissolution of CO
2
in seawater results in a
decrease in ocean pH and a shift in the carbonate chemistry
of seawater that is unfavorable for the deposition of cal-
cium carbonate (Chan and Connolly 2013). During the
twentieth century, rising atmospheric CO
2
has resulted in a
0.7 °C increase in mean sea surface temperatures (SST)
and a decrease in sea surface pH
T
of 0.1 (Caldiera et al.
2003; Raven 2005). Atmospheric CO
2
is projected to
increase from current levels (*39.0 Pa) to between 49.6
and 86.1 Pa by the end of this century (van Vuuren et al.
2011) and likely will increase SST 2–3 °C and reduce sea
surface pH
T
by 0.3 (Meehl et al. 2007). As a result, GCC
will increase the incidence of warm-water coral bleaching,
and reef accretion will slow due to elevated SST and OA
(Pandolfi et al. 2011).
Rising SST has contributed to the decline in abundance
of corals (Hoegh-Guldberg 1999; Pandolfi et al. 2011),
largely through bleaching (Loya et al. 2001). Paling of
coral tissue (i.e., ‘‘bleaching’’), occurs in response to
multiple environmental stressors (Glynn 1996; Fitt et al.
2001), of which high light intensities and elevated tem-
perature are most prominent in causing large-scale
Communicated by Biology Editor Dr. Anastazia Banaszak
C. B. Wall (&)P. J. Edmunds
Department of Biology, California State University,
18111 Nordhoff Street, Northridge, CA 91330-8303, USA
e-mail: cbw0047@gmail.com
C. B. Wall
College of Life-Sciences, Santa Monica College,
1900 Pico Boulevard, Santa Monica, CA 90405-1628, USA
T.-Y. Fan
National Museum of Marine Biology and Aquarium, Taiwan,
Republic of China
T.-Y. Fan
Institute of Marine Biodiversity and Evolution, National Dong
Hwa University, Taiwan, Republic of China
123
Coral Reefs
DOI 10.1007/s00338-013-1085-2
bleaching (Fitt et al. 2001). Coral bleaching progresses
through a series of cellular events culminating in the
reduction in the concentrations of photopigments and
densities of Symbiodinium spp. algae within the coral, and
in some instances, results in death (Glynn 1983; Fitt et al.
2001). Photoinhibition from oxidative damage to the pho-
tosynthetic apparatus of Symbiodinium, particularly the
D1-protein, and impaired carbon fixation initiates the
events leading to symbiont expulsion (Lesser 1997; Jones
et al. 1998; Warner et al. 1999; Smith et al. 2005). These
effects can first be detected as reductions in the quantum
yield of photosystem II (PSII) (hereafter, photochemical
efficiency) in response to PSII photoinactivation (Warner
et al. 2010), and a reduced capacity to use light energy
together with reducing agents to fix CO
2
in the Calvin
cycle (hereafter, photosynthetic capacity) (Iglesias-Prieto
et al. 1992; Jones et al. 1998; Warner et al. 1999). As these
effects persist, impairment of the light and dark reactions
of photosynthesis and damage to the photosynthetic appa-
ratus by reactive oxygen species (Lesser 1997; Jones et al.
1998; Smith et al. 2005) leads to the expulsion of Symbi-
odinium, host cell detachment (Gates et al. 1992), or
apoptosis (Dunn et al. 2007).
The effects of OA on biomineralization have received
considerable attention (Chan and Connolly 2013), but the
effects on photosynthesis of Symbiodinium within corals
remain unclear. Hermatypic corals depend on Symbiodi-
nium to provide photosynthetically fixed carbon to fuel
metabolism (Muscatine et al. 1984). Some studies with
corals suggest OA reduces net photosynthesis (P
net
)
(Reynaud et al. 2003; Anthony et al. 2008), and the max-
imum rate of photosynthesis (i.e., Pnet
max) (Crawley et al.
2010), although some corals show no response of photo-
synthesis to pCO
2
enrichment (Leclercq et al. 2002;
Godinot et al. 2011). OA has also been reported to induce
bleaching in at least two corals, both alone and in concert
with elevated temperature (Anthony et al. 2008). Anthony
et al. (2008) hypothesized that the mechanism of OA-
induced bleaching involved the disruption of carbon-con-
centration mechanisms used by Symbiodinium (Weis et al.
1989; Leggat et al. 1999), or impaired photoprotective
mechanisms, such as photorespiration (Crawley et al. 2010)
and nonphotochemical quenching (Hill et al. 2005). Sup-
port for these hypotheses is limited; however, as effects of
OA on the photophysiology of Symbiodinium in hospite
have not been studied in detail, and where these
effects have been investigated, the outcomes are equivocal
(Godinot et al. 2011; Iguchi et al. 2011; Edmunds 2012)
and may be specific to Symbiodinium phylotypes (Brading
et al. 2011).
The objective of the study was to test whether elevated
pCO
2
affects bleaching in corals. An experiment was
conducted in which corals were exposed to two tempera-
tures and two pCO
2
regimes, with the effects assessed
using dependent variables that detect the early stages of
bleaching (after Fitt et al. 2001): (1) the onset of reduced
photochemical efficiency (2) intermediate effects mani-
festing in reduced photosynthetic capacity and efficiency
prior to visible paling of tissue, and (3) terminal effects
culminating in reduced Symbiodinium and photopigment
densities. The experiment was conducted with the
branching pocilloporid coral Seriatopora caliendrum
(Ehrenberg 1834) that is common on the shallow reefs of
southern Taiwan (Dai and Horng 2009) where this study
was conducted and has previously been reported suscepti-
ble to thermal bleaching (Loya et al. 2001).
Materials and methods
Experimental design
Four treatments contrasted ambient temperature–ambient
pCO
2
(AT–ACO
2
), ambient temperature–high pCO
2
(AT–HCO
2
), high temperature–ambient pCO
2
(HT–ACO
2
), and
high temperature–high pCO
2
(HT–HCO
2
). Ambient tem-
perature referred to the seawater on shallow reefs in Nan-
wan Bay when the experiment was conducted in July and
August 2011 (28.0 ±0.2 °C at 3 m depth [mean ±SE,
n=21 days]) and therefore was set at 27.5 °C. Ambient
pCO
2
reflected ambient CO
2
conditions, although this
routinely was above the global mean atmospheric value of
*39.0 Pa. High temperature was relative to the maximum
seawater temperature recorded at 3 m depth on the study
reef in summer (31.0 °C) (T-Y Fan pers comm) and was
30.5 °C. High pCO
2
represented conditions projected to
occur by the year 2100 (*86 Pa) under the high CO
2
emission scenario RCP 6.0 (van Vuuren et al. 2011).
We hypothesized that OA would cause bleaching as
defined by decreased photochemical efficiency, reduced
photosynthetic capacity and efficiency, depressed chloro-
phyll acontent, and lowered Symbiodinium densities and
that these effects would be exacerbated with high temper-
ature. To test this hypothesis, corals were exposed to
treatments in 8 tanks (n=2 tanks treatment
-1
) filled with
130 L of filtered (1.0 lm) seawater. Treatments were
maintained at a salinity of 33.4 (measured with a YSI 3100
Conductivity Meter, YSI Inc., USA) with 20 % water
changes each evening. Temperatures were maintained
independently by microsensor-based regulators (Aqua-
Controller, Neptune Systems, USA) connected to a 300-W
heater (Taikong Corp.), and chiller (Aquatek, Aquasys-
tems, Taiwan), and the seawater was mixed with a pump
(1,451 L h
-1
). Light was provided to each tank by two
Coral Reefs
123
18-W fluorescent bulbs (TL-D Blue, Phillips, USA) and
two 150-W metal-halide bulbs on a 12 h light: 12 h dark
cycle that created mean irradiances ranging from 251 to
279 lmol photons m
-2
s
-1
as measured beneath the sea-
water using a spherical light sensor (LI-193, Li-Cor, USA).
Treatments of pCO
2
were maintained by bubbling
ambient air (A–CO
2
)orCO
2
-enriched air into the tanks
(H–CO
2
). To prepare high pCO
2
treatments, CO
2
was
mixed with ambient air by solenoid-controlled gas mixing
technology (Model A352, Qubit Systems, Canada). CO
2
and ambient air were mixed in a chamber, and the pCO
2
measured using an infrared (IR) gas analyzer (S151, Qubit
Systems) calibrated against certified reference gas
(1,793 ppm CO
2
, San Ying Gas Co., Taiwan). The pCO
2
treatments were maintained dynamically by the IR gas
analyzer, which regulated a solenoid valve controlling the
flow of CO
2
gas. The final pCO
2
was logged in ppm on a
PC using LabPro software (Vernier Software and Tech-
nology, USA), and a pump delivered the gas mixture to the
high-pCO
2
tanks at *15 L min
-1
. Ambient pCO
2
tanks
received ambient, non-CO
2
-enriched air at a similar flow
rate.
Treatments were monitored daily at 0900, 1200, and
1700 hrs for temperature and salinity; irradiance was
measured at 1200 hrs; pH and carbonate chemistry of the
treatments were determined daily on seawater samples
(*250 mL) taken from all tanks at 0900 hrs. Temperature
was measured using a certified digital thermometer (Fisher
Scientific 15-077-8, ±0.05 °C), and seawater was asses-
sed for total alkalinity (TA, lmol kg
-1
) and pCO
2
by
potentiometric titrations following standard operating pro-
cedures (SOP) 3 (Dickson et al. 2007); pH
T
was deter-
mined spectrophotometrically using m-cresol purple (SOP
6B, Dickson et al. 2007). Seawater samples were titrated
using an open-cell autotitrator (Model DL50, Mettler-
Toledo, USA) filled with certified acid titrant (from
A. Dickson, Scripps Institution of Oceanography) and
equipped with a DG115-SC pH probe (Mettler-Toledo).
TA was evaluated for precision and accuracy using certi-
fied reference materials (CRM) of known TA (from
A. Dickson, Scripps Institution of Oceanography) with our
analyses differing \0.9 % from certified values. pH
T
,
salinity, temperature, and TA were used in CO2SYS soft-
ware in Microsoft Excel (Fangue et al. 2010) to calculate
the components of the dissolved inorganic carbon (DIC)
system in seawater.
Coral collection
Sixty juvenile S. caliendrum (\4 cm diam.) were collected
on July 22, 2011, from Hobihu Reef (21°56.7990N,
120°44.9680E), Nanwan Bay. Colonies were collected from
3 to 4 m depth and transported to a flow-through aquarium
at the National Museum of Marine Biology and Aquarium
(NMMBA) where they were allowed to recover from col-
lection for 24 h. The recovery tank was filled with flowing,
filtered seawater (50 lm) and mixed with a pump
(1,451 L h
-1
). Temperature was maintained at ambient
conditions (28.07 ±0.10 °C, ±SE, n=24) and light was
supplied at 164 ±4lmol photons m
-2
s
-1
on a 12 h
light: 12 h dark cycle.
One day following collection, the colonies of S.
caliendrum were suspended in the recovery tank using
nylon line and left to recover for 5 days. On July 28, 2011,
they were placed randomly into the treatment tanks
(n=7 tank
-1
) for incubations lasting 14 days. On August
11, 2011, corals were processed over 3 days for the
dependent variables described below and retained in
experimental conditions during this time. To maintain
comparable exposure periods among like-temperature
treatments, corals from the HT treatments were processed
first, followed by corals in the AT treatments, with corals
randomly selected for processing within each temperature
treatments.
Photochemical efficiency
The effects of temperature and pCO
2
on photochemical
efficiency were tested by measuring the maximum photo-
chemical efficiency of open RCIIs in the dark (F
v
/F
m
) and
the effective photochemical efficiency of RCII in the light
(DF/F
m
0) using pulse amplitude modulation (PAM) fluo-
rometry. PAM fluorometry is an effective tool to assess
noninvasively the photophysiology of Symbiodinium in
hospite (Warner et al. 1996,2010) and provides an indi-
cation of PSII photochemical activity and the transport of
electrons through PSII (Cosgrove and Borowitzka 2010).
F
v
/F
m
provides a measure of photochemical quenching
(qP) reflecting the rate of charge separation across PSII in
the open (i.e., dark-adapted) state, while DF/F
m
0accounts
for photochemical and nonphotochemical quenching
(NPQ), including mechanisms for the dissipation of excess
absorbed light energy as heat through the PSII antennae
complex (Hill et al. 2005). NPQ is of particular biological
importance as a mechanism of photoprotection and
avoidance of photoinhibition under peak daily irradiance
and under conditions causing bleaching (Warner et al.
1996; Jones et al. 1998; Hoegh-Guldberg and Jones 1999).
Photochemical efficiency was assessed using a Diving-
PAM (Waltz, GmbH, Effeltrich, Germany) operated at a
gain of 6, intensity of 9, and a slit width of 0.8. Prior to the
start of the experiment, PAM settings were adjusted to
obtain a range of minimum fluorescence yield (F
o
) between
200 and 400 (arbitrary units) and stabile maximum fluo-
rescence yield (F
m
). DF/F
m
0was measured to quantify
changes in quantum yield relative to the dark-adapted state
Coral Reefs
123
due to excess thermal energy dissipation and NPQ, and
F
v
/F
m
was measured to quantify the maximum efficiency
of open RCIIs in the dark-adapted state. Photochemical
efficiency was measured using a 5-mm-diameter fiberoptic
probe held *5 mm above the tissue and *1 cm behind
branch tips. DF/F
m
0was measured under actinic irradiance
(*265 lmol photons m
-2
s
-1
) and F
v
/F
m
under
weak indirect red lighting (B2.0 lmol photons m
-2
s
-1
).
DF/F
m
0was measured every second day of the incubation
at 1230 hrs, and F
v
/F
m
was measured every second day at
1730 hrs. A pilot study was used to determine the duration
of dark adaptation necessary to stabilize values of F
v
/F
m
,to
identify effects of prolonged darkness on F
v
/F
m
(i.e., dark-
induced reduction in the PQ pool; Hill and Ralph 2008),
and to test whether weak indirect red light (as produced
from small lamps used during nocturnal PAM measure-
ments) affected F
v
/F
m
. Results indicated F
o
stabilized after
\0.5 h of darkness, and F
v
/F
m
was statistically indistin-
guishable when measured following dark adaptation lasting
0.5, 1.0, or 2.0 h (F
2,27
=0.137, P=0.872), or measured
with and without weak red light (F
1,18
=0.352,
P=0.561).
Photosynthesis–irradiance (P/I) curves
To test for the effects of pCO
2
and temperature on the
ability for Symbiodinium to utilize light and perform pho-
tosynthesis, net photosynthesis (P
net
), determined from
changes in O
2
concentrations in seawater, was measured
under different irradiances using three corals selected
randomly from each treatment tank (n=6 treatment
-1
).
Two respirometers were used to measure P
net
, and each
housed a single coral in trials lasting *1.5–2.0 h. Mea-
surements of P
net
began on the 14th day of incubations, and
3 days were required to process all corals in the experi-
ment. Temperatures were maintained by placing the res-
pirometers in a water bath. Water motion in each chamber
was provided by a stir-bar, and the flow rate quantified by
photographing hydrated Artemia spp. eggs (Sebens and
Johnson 1991), revealing the mean flow rate near the center
of the respirometer to be 5.43 ±0.32 cm s
-1
(±SE,
n=20). Prior to each trial, corals were maintained in
darkness for 1 h to allow the stimulatory effect of light on
respiration to abate (Edmunds and Davies 1988). O
2
flux
then was measured at ten irradiances supplied in an
ascending sequence between 0 and 747 lmol pho-
tons m
-2
s
-1
. Light intensities were created by adjusting
the height of a 400-W metal-halide lamp (Osram Sylvania,
USA) above the respirometer, and measuring the irradiance
using a cosine-corrected light sensor recording photosyn-
thetically active radiation (PAR). The light sensor was 1.0
mm diameter and attached to a Diving-PAM (Waltz,
GmbH, Effeltrich, Germany) and was calibrated using a
Li-Cor LI-192 quantum sensor. O
2
fluxes were adjusted for
changes in O
2
concentrations in control chambers filled
with seawater alone, and controls were run at each com-
bination of temperature and pCO
2
for each irradiance, and
during darkness (n=3 treatment
-1
).
The O
2
saturation of seawater was measured using an
optrode (FOXY-R, 1.58 mm diameter, Ocean Optics,
USA) connected to a spectrophotometer (USB2000, Ocean
Optics), which logged O
2
concentrations on a PC running
Ocean Optics software (OOISensors, version 1.00.08,
Ocean Optics). The optrode was calibrated using water-
saturated air at the measurement temperature and a zero
solution of sodium sulfite (Na
2
SO
3
) and 0.01 mol L
-1
sodium tetraborate (Na
2
B
4
O
7
). O
2
saturation during the
trials was maintained between 80 and 100 % by replen-
ishing chambers with filtered (1.0 lm) seawater from
respective temperature and pCO
2
treatments. Changes in
O
2
saturation were converted to O
2
concentrations
(lmol L
-1
) using tabulated gas solubility at a known
temperature and salinity [N. Ramsing and J. Gundersen at
www.unisense.com, based on Garcia and Gordon 1992].
Rates of change in O
2
concentrations were determined by
regressing O
2
concentration against time, and standardizing
to the surface area of the coral tissue (cm
2
) as determined
by wax dipping (Stimson and Kinzie 1991). The relation-
ship between P
net
and irradiance was described with a
hyperbolic tangent function (Jassby and Platt 1976) that
included an exponent for photoinhibition (e.g., b) at high
irradiances (Platt et al. 1980) to account for photoinhibitory
effects of high-light or high-temperature exposure known
to occur in phytoplankton (Platt et al. 1980) and Symbi-
odinium (Smith et al. 2005):
Pb¼Pb
sð1eaÞebð1Þ
where a=aI/P
s
b
,b=bI/P
s
b
,P
b
is the rate of net primary
productivity (P
net
), P
s
b
is the maximum rate of net photo-
synthesis accounting for photoinhibition, ais the initial
slope of the light-limited portion of the curve, and Iis
irradiance in lmol photons m
-2
s
-1
. Hyperbolic tangent
functions were fit to the productivity data by nonlinear
regression using Systat 11 software (Systat, Inc., USA) and
used to characterize the photosynthetic efficiency (alpha, a)
and P
net
under high-light conditions. The aforementioned
curves describe the full biological relationship between P
net
and Ifor a large range of irradiances supplied in the lab-
oratory, with these intensities exceeding maximum inten-
sities found on the study reef. Mean PAR at 3 m depth on
Hobihu reef (measured between March 6 and 10, 2011,
using a 4pspherical quantum sensor (MkV-L, JFE
Advantech Co., Kobe, Japan) between 0900–1500 hrs was
660 ±30 lmol photons m
-2
s
-1
(±SE, n=148). To
compare the photosynthetic performance of corals under
ecologically relevant conditions and following exposure to
Coral Reefs
123
treatments, best-fit curves were used to calculate P
net
at
660 lmol photons m
-2
s
-1
(hereafter Pnet
660), and this met-
ric, along with a, and dark respiration, was compared
among treatments. While inclusion of an exponent for
photoinhibition (b) improved the fit of the response curves
to the empirical data, in situ irradiances were not suffi-
ciently high to elicit photoinhibition on the reef, and
therefore, bwas not employed as a dependent variable in
the analysis.
Chlorophyll a concentration and Symbiodinium density
Chlorophyll aconcentration and Symbiodinium density
were quantified by removing coral tissue from the skeleton
using an airbrush filled with filtered seawater (1.0 lm).
Colonies were airbrushed into a plastic bag, producing
8–40 mL of slurry that was homogenized (Polytron
PT2100, Kinematica, USA) prior to separating the Symbi-
odinium by centrifugation (1,5009g). The Symbiodinium
pellet was resuspended by vortexing in filtered seawater
and used to measure chlorophyll aconcentration and
symbiont density.
Symbiodinium used for chlorophyll determinations were
frozen (-4°C for 24 h) and subsequently thawed (4 °C for
24 h) and filtered onto a cellulose acetate membrane filter
(3.0 lm pore size, Critical Process Filtration, USA) to
which 3 mL of 90 % acetone was added. Samples were
refrigerated (4 °C) in darkness for 36 h, centrifuged
(1,5009gfor 3 min), and absorbances at 630 and 636 nm
measured and used to calculate chlorophyll aconcentration
using the trichromatic equations of Jeffrey and Humphrey
(1975) for dinoflagellates. Chlorophyll aconcentration was
standardized by algal cell (pg cell
-1
) and surface area
(lgcm
-2
) of the coral. Symbiodinium density (cells cm
-2
)
was determined by counting Symbiodinium in the homog-
enized slurry stripped from the coral colonies, with the
counts completed using a hemocytometer (n=4 counts).
Preliminary data showed that the mean and standard
deviation of replicate determinations of Symbiodinium
density stabilized after four counts.
Statistical analysis
Response variables were compared among treatments using
three-way nested ANOVA in which pCO
2
and temperature
were fixed effects, and tank was a random factor nested
within treatment. The physical and chemical conditions in
the treatments also were analyzed with this statistical
model. Tank was removed from the model when not sig-
nificant at PC0.25 (Quinn and Keough 2002). Significant
interactive effects were analyzed using a post hoc Tukey
test. To test the statistical assumption of ANOVA, graph-
ical analyses of residuals were employed. Analyses were
performed using Systat 11 in a Windows operating system.
Results
Tank parameters
Physical and chemical conditions in the treatment tanks were
maintained precisely (Table 1). Mean pCO
2
treatments were
45.1 ±0.2 and 85.1 ±0.5 Pa (±SE, n=55–56) with mean
pCO
2
across treatment tanks ranging from 43.6 to 47.0 Pa
(A–CO
2
) and 83.2 to 87.2 Pa (H–CO
2
). pCO
2
differed
between replicate tanks (F
4,103
=3.673, P=0.008) and
treatments (F
1,4
=1798.18, P=\0.001), and pH
T
dif-
fered between replicate tanks (F
4,103
=5.956, P\0.001)
and between pCO
2
treatments (F
1,4
=1120.272,
P=\0.001). The tank effects for pCO
2
and pH reflected
differences of \0.04 pH
T
and B4.0 Pa pCO
2
. For seawater
temperatures, tank effects were not detected (F
4,176
=1.261,
Table 1 Summary of physical and chemical conditions in the 8 treatment tanks between July 28 and August 11, 2011
Treatment Tank Temperature (°C) pH
total
TA (lmol kg
-1
) pCO
2
(Pa) HCO
3-
(lmol kg
-1
)CO
32-
(lmol kg
-1
)
AT–ACO
2
2 27.6 ±0.02 (23) 7.99 (14) 2196 ±8 (14) 45.4 ±0.3 (14) 1724 ±5.1 (14) 192 ±1.7 (14)
4 27.7 ±0.13 (23) 7.99 (14) 2184 ±7 (14) 44.6 ±0.3 (14) 1712 ±4.5 (14) 192 ±1.4 (14)
HT–ACO
2
3 30.4 ±0.14 (23) 8.01 (14) 2217 ±8 (14) 43.6 ±0.5 (14) 1687 ±6.3 (14) 216 ±1.8 (14)
8 30.6 ±0.02 (23) 7.98 (14) 2219 ±7 (14) 46.7 ±0.4 (14) 1711 ±5.1 (14) 207 ±1.7 (14)
AT–HCO
2
1 27.7 ±0.03 (23) 7.76 (14) 2208 ±7 (14) 86.1 ±0.6 (14) 1904 ±4.6 (14) 124 ±1.2 (14)
7 27.7 ±0.10 (23) 7.75 (13) 2200 ±8 (13) 87.1 ±0.9 (13) 1903 ±6.3 (13) 121 ±1.4 (13)
HT–HCO
2
5 30.6 ±0.13 (23) 7.77 (14) 2227 ±6 (14) 83.2 ±1.2 (14) 1884 ±3.4 (14) 141 ±2.1 (14)
6 30.5 ±0.05 (23) 7.77 (14) 2222 ±7 (14) 84.2 ±0.8 (14) 1883 ±4.7 (14) 139 ±1.5 (14)
Seawater chemistry was assessed daily and temperature three times daily (0900, 1200, 1700 h) in all tanks. Values displayed are mean ±SE (n)
TA total alkalinity, AT–ACO
2
ambient temperature–ambient pCO
2
,HT–ACO
2
high temperature–ambient pCO
2
,AT–HCO
2
ambient temperature–
high pCO
2
,HT–HCO
2
high temperature–high pCO
2
SE \0.1
Coral Reefs
123
P=0.287), and mean temperatures (±SE, n=92) were
27.65 ±0.04 °C (AT) and 30.53 ±0.05 °C (HT).
Photochemical efficiency
Corals were acclimated to laboratory conditions prior to
being placed into treatments. The progress of laboratory
acclimation was monitored through daily measures of F
v
/F
m
using 10 corals selected randomly each day. We inferred that
acclimation was complete when F
v
/F
m
did not vary among
days. After 5 days (when F
v
/F
m
stabilized), corals were
placed in treatments and DF/F
m
0and F
v
/F
m
measured every
second day (data not shown) with this regime revealing
declines at HT (but not other treatments) after 7 days. After
13 days, corals at HT experienced a 15 % (HT–HCO
2
) and
16 % (HT–ACO
2
) reduction in DF/F
m
0(P\0.05) com-
pared to corals in the AT–ACO
2
treatment (Fig. 1). Mean
F
v
/F
m
was depressed 13 and 9 % in HT–ACO
2
and HT–
HCO
2
treatments, respectively, versus AT–ACO
2
(P\0.05)
(Fig. 1). No significant effect (P[0.25) of tank was
detected for F
v
/F
m
, and tank was dropped from this analysis.
While tank also was not significant for DF/F
m
0(P=0.193),
it was retained in the analysis. DF/F
m
0was affected by
temperature (F
1,4
=96.726, P=0.001) and was reduced at
HT relative to AT. However, DF/F
m
0was not affected by
pCO
2
(F
1,4
=0.775, P=0.428), and there was no tem-
perature 9pCO
2
interaction (F
1,4
=0.0001, P=0.993).
Similarly, corals at HT exhibited reduced F
v
/F
m
and were
affected by temperature (F
1,48
=63.711, P\0.001) but not
pCO
2
(F
1,48
=2.595, P=0.114), and there was no tem-
perature 9pCO
2
interaction (F
1,48
=0.307, P=0.582).
P/I curves
Net photosynthesis standardized to area (cm
2
) increased with
irradiance and developed asymptotes at [400 lmol pho-
tons m
-2
s
-1
in 7 cases, with slight declines in P
net
at high
irradiances for 2 corals in the HT-ACO
2
treatment. Overall,
however, mean (±SE, n=5–6) values for branged from
0.030 ±0.020 (HT–ACO
2
) to 0.006 ±0.002 lmol
O
2
cm
-2
h
-1
(lmol photons m
-2
s
-1
)
-1
(HT–HCO
2
), and
these declines did not begin until ecologically relevant
irradiances from the collection depth had been exceeded.
The hyperbolic tangent functions with photoinhibition fit the
photosynthesis data well (mean r
2
=0.95) and were used to
calculate values for aand Pnet
660 for corals in each treatment
(n=5–6 treatment
-1
). Dark respiration was calculated
directly from raw data (Table 2). Tank effects were not
significant for respiration or Pnet
660 (P[0.25), and therefore,
tank was dropped from these analyses. Tank was retained in
the analysis of a(Tank P=0.216).
Area-normalized respiration ranged from 0.96 ±0.06 to
0.73 ±0.07 lmol O
2
cm
-2
h
-1
(mean ±SE, n =5–6,
Table 2) and was not affected by temperature, pCO
2
, or the
interaction between the two (Table 3). Temperature sig-
nificantly affected Pnet
660 standardized to area (P\0.001);
no effect of pCO
2
or the temperature 9pCO
2
interaction
was detected (Table 3). Area-normalized Pnet
660 decreased
95 % at HT–ACO
2
compared to AT–ACO
2
and was
affected similarly at HCO
2
, being reduced 89 % at HT
compared to AT (Fig. 2). Alpha was affected by temper-
ature (P\0.001) but not pCO
2
, or the interaction between
the two.
Chlorophyll a and Symbiodinium density
After 2 weeks in the treatments, corals in AT treatments
appeared a normal color while corals in the HT treatment
showed signs of bleaching, although no corals died. When
normalized to area, mean concentration of chlorophyll
aranged from 13.35 ±0.43 lgcm
-2
in AT–ACO
2
to
3.73 ±0.43 lgcm
-2
in HT–ACO
2
(±SE, n=12–14)
(Fig. 3a). The interaction of temperature and pCO
2
was
significant (F
1,48
=5.074, P=0.029) due to a 13 %
reduction in chlorophyll acm
-2
between AT–ACO
2
and
AT–HCO
2
, and a 25 % increase in HT–HCO
2
compared to
HT–ACO
2
(Fig. 3a). Chlorophyll aconcentration was
affected by temperature (F
1,48
=193.646, P\0.001), but
not pCO
2
(F
1,48
=0.457, P=0.502), and post hoc analyses
revealed A–CO
2
and H–CO
2
conditions within temperature
treatments (i.e., 27.7 and 30.5 °C) were not significantly
different from each other (P[0.05) (Fig. 3a). When nor-
malized to Symbiodinium cells, chlorophyll aconcentration
was unaffected by temperature (F
1,28
=0.143, P=0.709),
Fig. 1 Effective photochemical efficiency of RCIIs in actinic light
(DF/F
m
0)(*266 lmol photons m
-2
s
-1
) and maximum photochem-
ical efficiency of open RCIIs (F
v
/F
m
) for juvenile Seriatopora
caliendrum exposed for 14 days to combinations of temperature and
pCO
2
(Table 1). Values displayed are mean ±SE
(n=13–14 treatment
-1
)
Coral Reefs
123
pCO
2
(F
1,28
=0.181, P=0.673), or the interaction
between the two (F
1,28
=0.001, P=0.975) (Fig. 3a-inset).
Symbiodinium densities standardized to area were
affected by temperature (F
1,28
=104.676, P\0.001) but
not pCO
2
(F
1,28
=0.315, P=0.579), or the interaction
between the two (F
1,28
=0.343, P=0.563). Mean Sym-
biodinium densities decreased 69 % in HT–ACO
2
com-
pared to AT–ACO
2
, and 65 % in HT–HCO
2
compared to
AT–HCO
2
(Fig. 3b).
Discussion
OA and coral bleaching
Large-scale thermal bleaching is a major cause of declining
coral cover (Wilkinson 2008), and therefore, reports that
elevated pCO
2
can induce bleaching at a magnitude
equivalent to thermally induced bleaching (Anthony et al.
2008) have received much attention. Despite this attention,
there has not been a rigorous test of the hypothesis that OA
affects the same physiological processes that underpin
thermal bleaching (Warner et al. 1996; Fitt et al. 2001; but
see Brading et al. 2011). In the present study, bleaching of
S. caliendrum at elevated temperature and high pCO
2
was
evaluated at three functional levels corresponding to the
sequence of events taking place during the onset of
bleaching (Fitt et al. 2001). High pCO
2
(85.1 Pa) did not
cause bleaching, either individually, or interactively with
high temperature.
Our results draw attention to the inconsistencies in the
reported effects of high pCO
2
in directly (or indirectly)
causing coral bleaching. For instance, our findings are
contrary to reports that pCO
2
as high as 152.0 Pa causes
Table 2 Dark respiration and parameters describing the relationship between net photosynthesis (lmol O
2
cm
-2
h
-1
) and irradiance (lmol
photons m
-2
s
-1
)(P/I) standardized by area for juvenile Seriatopora caliendrum
Parameter Treatments
AT–ACO
2
AT–HCO
2
HT–ACO
2
HT–HCO
2
R(lmol O
2
cm
-2
h
-1
) 0.96 ±0.06 (6) 0.75 ±0.04 (5) 0.73 ±0.07 (5) 0.75 ±0.08 (6)
Pnet
660 (lmol O
2
cm
-2
h
-1
) 1.80 ±0.44 (6) 1.96 ±0.29 (6) 0.08 ±0.09 (5) 0.22 ±0.09 (6)
a(lmol O
2
cm
-2
h
-1
)
(lmol photons m
-2
s
-1
)
-1
0.013 ±0.0007 (6) 0.011 ±0.0004 (6) 0.004 ±0.0005 (5) 0.004 ±0.0003 (6)
Corals were incubated for 14 days in combinations of temperature (°C) and pCO
2
(Pa) (Table 1). Parameters were obtained from the best-fit
hyperbolic tangent with an exponent for photoinhibition (Platt et al. 1980). Values displayed are mean ±SE (n=number of corals)
Rrespiration; Chl a chlorophyll a,Pnet
660 rate of net photosynthesis as measured at 660 lmol photons m
-2
s
-1
,athe initial slope of the light-
limited portion of the curve
Table 3 Statistical analysis of photosynthesis versus irradiance (P/I) curve parameters standardized by surface area for juvenile Seriatopora
caliendrum exposed to different treatments
Dependent variable Effect df MS F P
Respiration (lmol O
2
cm
-2
h
-1
) pCO
2
1 0.044 1.823 0.194
Temp 1 0.070 2.930 0.104
pCO
2
9Temp 1 0.070 2.912 0.105
Error 18 0.024
Pnet
660 (lmol O
2
cm
-2
h
-1
) pCO
2
1 0.132 0.291 0.596
Temp 1 17.078 32.728 <0.001
pCO
2
9Temp 1 0.001 0.001 0.974
Error 19 0.453
apCO
2
1 4.00 910
-6
2.162 0.215
(lmol O
2
cm
-2
h
-1
) Temp 1 3.43 910
-4
171.417 <0.001
(lmol photons m
-2
s
-1
)
-1
pCO
2
9Temp 1 2.00 910
-6
1.103 0.353
Tank (pCO
2
9Temp) 4 2.00 910
-6
1.639 0.216
Error 15 1.00 910
-6
Analyses were performed using a partly nested ANOVA with two fixed factors (pCO
2
and Temperature) and one nested factor (Tank). Tank was
dropped from the analysis when P[0.25; significant values (P\0.05) are in bold
Chl a chlorophyll a, df degrees of freedom, MS mean sum of squares, parameter definitions in Table 2
Coral Reefs
123
bleaching in Acropora intermedia and Porites lobata
(Anthony et al. 2008) and leads to declines in net pro-
ductivity of Stylophora pistillata (Reynaud et al. 2003), A.
intermedia and P. lobata (Anthony et al. 2008; Crawley
et al. 2010). Additionally, while elevated pCO
2
reduced
photochemical efficiency in Porites australiensis (F
v
/F
m
;
Iguchi et al. 2011) and massive Porites spp. (F
v
/F
m
and
DF/F
m
0; Edmunds 2012), and increased concentration of
chlorophyll aper algal cell in S. pistillata (e.g., photo-
acclimation; Crawley et al. 2010), we found no effect of
pCO
2
on photochemical efficiency and the content of
chlorophyll acm
-2
or chlorophyll acell
-1
. However, our
results are consistent with several other studies showing
that pCO
2
as high as 227 Pa has no effect on photosyn-
thesis in Acropora eurystoma (Schneider and Erez 2006)or
S. pistillata (Godinot et al. 2011). Further, high pCO
2
has
no effect on F
v
/F
m
in S. pistillata (Godinot et al. 2011), or
chlorophyll content (a?c
2
) and Symbiodinium density in
P. australiensis (Iguchi et al. 2011). While 14-day expo-
sure to elevated pCO
2
in the present study did not result in
bleaching, elevated temperature reduced both photosyn-
thetic performance and symbiont and photopigment den-
sity, as has previously been reported (Hoegh-Guldberg and
Smith 1989; Warner et al. 1999; Anthony et al. 2008). This
result reaffirms the importance of rising SST in negatively
affecting corals (Hoegh-Guldberg et al. 2007) and under-
scores the possibility that corals could succumb to the
effects of high temperature before they are impacted seri-
ously by high pCO
2
(Hoegh-Guldberg et al. 2007).
Onset of bleaching
Previous evidence that OA directly affects one of the
proximal processes (i.e., photochemical efficiency) driving
coral bleaching is equivocal, but the present results clearly
support a null effect for high pCO
2
. It is unclear what
factors are responsible for conflicting pCO
2
effects on
Symbiodinium photochemical efficiency among studies;
however, OA effects on coral calcification are modulated
by light intensity (Dufault et al. 2013; Suggett et al. 2013)
and light intensity may also provide insight into the dis-
parate effects of OA on photochemical efficiency.
Numerous OA studies have been performed under subsat-
urating low light intensities (\10–150 lmol photons
-1
0
1
2
3
0 200 400 600 800
Irradiance (µmol photons m-2 s-1)
Net photosynthesis (µmol O
2
cm
-2
h
-1
)
Fig. 2 Net photosynthesis (P
net
) versus irradiance (P/I) curves for
juvenile Seriatopora caliendrum exposed for 14 days to combinations
of temperature and pCO
2
as described in Table 1. Symbols
correspond to treatments AT–ACO
2
(dark circles), HT–ACO
2
(open
circles), AT–HCO
2
(dark triangles), and HT–HCO
2
(open triangles).
At each irradiance, values are mean P
net
±SE (n=5–6); best-fit
lines are fit to mean P
net
values
2
6
10
14
chlorophyll a ( g) cm
µ-2
(a)
0
2
4
6
HCO2 ACO2 HCO2
27.7°C 30.5°C
-1
pg Chl a cell
ACO2
0
1
2
3
Symbiodinium x 10 cm
AT-ACO AT-HCO HT-ACO HT-HCO
22 22
Treatments
6
-2
(b)
CO
2
Treatments
a
a
b
b
Fig. 3 Chlorophyll anormalized to area and Symbiodinium cells, and
the density of Symbiodinium spp. in juvenile Seriatopora caliendrum
exposed for 14 days to combinations of temperature and pCO
2
(Table 1). aChlorophyll a(lgcm
-2
)(n=12–14 treatment
-1
) with
inset showing chlorophyll acontent per Symbiodinium cell at 27.7 °C
(white columns), 30.5 °C(gray columns), and 45.1 and 85.1 Pa CO
2
;
(b)Symbiodinium spp. density per surface area of coral tissue (cm
-2
)
(n=8 treatment
-1
). Values displayed are mean ±SE; letters
indicate post hoc multiple comparisons with dissimilar letters
marking treatments that differed (P\0.05)
Coral Reefs
123
m
-2
s
-1
) (e.g., Crawley et al. 2010; Iguchi et al. 2011) that
may not be ecologically relevant to corals in situ. We
acknowledge light intensities employed here (265 lmol
photons m
-2
s
-1
) were below field irradiances; however, light
intensity exceeded the saturation irradiance (I
k
=P
max
/a)
for photosynthesis in these corals (I
k
=144 ±3lmol
photons m
-2
s
-1
[±SE, n=3], C.B. Wall pers comm).
Under subsaturating irradiances, energy dissipation path-
ways (e.g., NPQ) are less active than under saturating
irradiances. However, OA appears to prematurely activate
energy dissipation pathways (Crawley et al. 2010), which
may result in reduced photochemical efficiency and pro-
ductivity. At low light intensities, the effects of OA on
photochemical efficiency may be masked due to various
factors, including the coral’s previous light history, low
levels of NPQ (i.e., reducing DF/F
m
0) and PSII photoin-
activation (i.e., reducing F
v
/F
m
), and efficient turnover of
the D1 protein of PSII. Therefore, CO
2
effects under low
light intensities may only manifest under prolonged incu-
bations, or under short incubation at high irradiances
(Edmunds 2012). It is clear that further research on the
interaction of light and OA in affecting photochemical
efficiency will be required to resolve these issues.
Intermediate events associated with bleaching
The pCO
2
-enrichment alone, or in combination with high
temperature (HT), did not affect the dark respiration of S.
caliendrum, or their photosynthetic efficiency (a) and
photosynthetic capacity (Pnet
660). However, in HT treatments,
concurrent with decreased F
v
/F
m
and DF/F
m
0, large
reduction in Pnet
660 and awas observed, while dark respira-
tion remained unchanged. Under prolonged exposure to
high light intensities or elevated temperatures, photopro-
tective mechanisms that normally shift energy away from
PSII begin to breakdown, resulting in photo-oxidative
damage to PSII, reduced photosynthetic productivity, and
ultimately, bleaching (Lesser 1997; Warner et al. 1999;
Smith et al. 2005). In the present study, a reduction in Pnet
660
associated with elevated temperature may be attributed to
several processes including reduced densities of Symbi-
odinium or the number of photosynthetic units (PSU), and a
slower turnover rate of PSII and the D1 reaction center
protein (Falkowski and Raven 1997; Warner et al. 1999).
Photodamage and decreased turnover rates of the D1 pro-
tein are associated with the onset of thermal bleaching in
corals (Warner et al. 1999), as well as in heat-stressed
Symbiodinium in vitro (Iglesias-Prieto et al. 1992) and
more generally, in marine diatoms and natural phyto-
plankton assemblages undergoing photoinhibition in situ
(Behrenfeld et al. 1998). Decreases in aof colonies of S.
caliendrum exposed to HT treatments indicates a
diminished integrity of RCIIs, potentially arising from
photo-oxidative damage to the D1 protein of RCIIs (Lesser
1997; Warner et al. 1999), and fewer photons absorbed by
the antennae complex being transferred to the acceptor side
of PSII (Falkowski and Raven 1997).
The null effect of pCO
2
treatments on coral respiration
agrees with studies, showing no effect of pCO
2
(up to
217 Pa) on the dark respiration of S. pistillata (Reynaud
et al. 2003; Crawley et al. 2010; Godinot et al. 2011),
Acropora intermedia or Porites lobata (120.1 Pa)
(Anthony et al. 2008), A. eurystoma (56.0 Pa) (Schneider
and Erez 2006), and Porites spp. at 76.6 Pa (Edmunds
2012) and 99 Pa (Wall and Edmunds 2013), but conflict
with studies reporting OA reduces coral respiration
(Edmunds 2012). In the present study, high flow rates may
have stimulated coral respiration across treatments by
reducing the diffusion boundary layer (DBL) across the
corals tissue (Lesser et al. 1994), thereby reducing the
ability to detect a treatment effect on respiration. Alterna-
tively, the disparity in OA effects on coral respiration may
reflect differential responses to elevated external CO
2
or
reduced extracellular pH, or the magnitude of the pCO
2
treatments (Edmunds 2012).
The decrease in aand Pnet
660 of S. caliendrum kept at
30.5 °C is consistent with studies showing a decrease in the
photosynthetic performance of Symbiodinium in hospite
(Coles and Jokiel 1977) and in vitro (Iglesias-Prieto et al.
1992) for corals exposed to elevated temperatures
([28 °C). However, 85.1 Pa pCO
2
did not affect the light-
limited efficiency of photosynthesis (a) or the rate of
photosynthesis of S. caliendrum at mean in situ irradiances
(i.e., Pnet
660). These findings are in agreement with studies
with corals showing no effect of pCO
2
(up to 152 Pa) on
the capacity for photosynthesis (Langdon and Atkinson
2005; Schneider and Erez 2006) and photosynthetic effi-
ciency (Crawley et al. 2010)ofSymbiodinium. While coral
photosynthesis appears insensitive to changes in pH (Goi-
ran et al. 1996; Schneider and Erez 2006), it has been
suggested that rates of Symbiodinium photosynthesis are
carbon limited under ambient [DIC] of *2,000 lmol kg
-1
(Herfort et al. 2008), and therefore, increase dissolved CO
2
or elevated [DIC] (*200 lmol kg
-1
primarily in the form
of HCO
3-
) from OA could stimulate photosynthesis,
although the general consensus is that Symbiodinium pro-
ductivity is not stimulated by 10 % increase in [DIC].
Variability in abiotic factors, such as PAR or seawater
temperature, may influence the impacts of OA on corals
and the performance of their Symbiodinium, particularly in
short- versus long- term experiments. However, disparities
in effects of OA on photosynthesis of corals may also
reflect genetic variability in the response of Symbiodinium
to environmental disturbance (Ragni et al. 2010; Putnam
Coral Reefs
123
et al. 2012), or differential reliance of the algae on CCMs
versus CO
2(aq)
to supply carbon for photosynthesis (sensu
Brading et al. 2011). For instance, the response of four
cultured Symbiodinium phylotypes (Brading et al. 2011)to
high pCO
2
is phylotype-specific. However, the
role(s) played by Symbiodinium phylotypes in the response
of corals to OA is uncertain, first because it is unknown
what phylotypes associate with S. caliendrum in southern
Taiwan (although, S. hystrix on the Great Barrier Reef
associates with 5 phylotypes of clade C Symbiodinium,
Bongaerts et al. 2010), and further, the microenvironment
surrounding Symbiodinium in hospite differs from the
external environment (Venn et al. 2009). The coral host is
thought to exert strong control over the photosynthetic
performance of Symbiodinium (Gates et al. 1999), and
therefore, the effects of OA on Symbiodinium in hospite are
likely to be modulated by the host.
Terminal stages of bleaching
The terminal stages of coral bleaching involve a reduction
in photopigment content of Symbiodinium and the expul-
sion of symbionts (Fitt et al. 2001). In the present study,
14 days at 30.5 °C led to declines in the density of Sym-
biodinium and reductions in their chlorophyll acontent, but
neither trait was affected by high pCO
2
. Crawley et al.
(2010) reported no change in Symbiodinium densities, but
an increase in chlorophyll acontent cell
-1
in response to
high pCO
2
(38.5 vs. 70.9 and 111.5 Pa), and suggested that
OA elicits photoacclimation by Symbiodinium. While
chlorophyll concentration in S. caliendrum was not affec-
ted by pCO
2
alone, the pCO
2
9temperature interaction led
to reduction in chlorophyll aat 27.7 °C and increases at
30.5 °C relative to 45.1 Pa. Similarly, 77.0 Pa pCO
2
decreased chlorophyll a(mg protein
-1
)inS. pistillata at
25.2 °C and increased chlorophyll aat 28.3 °C, relative to
46.6 Pa (Reynaud et al. 2003). However, in the present
study, chlorophyll a(pg) cell
-1
and Symbiodinium densi-
ties did not change in response to pCO
2
; therefore, the
change in chlorophyll acontent is likely to be caused by
symbiont expulsion and not photoacclimation. Using S.
caliendrum, our results suggest OA interacts with temper-
ature to affect the chlorophyll content of Symbiodinium,but
this modulation of pigment content does not result in
increased photosynthetic performance or ameliorated
effects of thermal bleaching on the holobiont.
Acknowledgments This research was funded by the US National
Science Foundation through Grant BIO-OCE 08-44785 (to PJE) and
was submitted in partial fulfillment of the MS degree for CBW. We
thank two anonymous reviewers for comments that improved an
earlier draft of this paper, V.R. Cumbo, A.M. Dufault, E. Rivest and
S. Zamudio for field assistance, and NMMBA for logistical support.
This is contribution number 200 of the Marine Biology Program of
California State University, Northridge.
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