Brooded coral larvae differ in their response to high temperature and elevated pCO2 depending on the day of release

Abstract

To evaluate the effects of temperature and pCO2 on coral larvae, brooded larvae of Pocillopora damicornis from Nanwan Bay, Taiwan (21°56.179′N, 120°44.85′E), were exposed to ambient (419–470 μatm) and high (604–742 μatm) pCO2 at ~25 and ~29 °C in two experiments conducted in March 2010 and March 2012. Larvae were sampled from four consecutive lunar days (LD) synchronized with spawning following the new moon, incubated in treatments for 24 h, and measured for respiration, maximum photochemical efficiency of PSII (F
v/F
m), and mortality. The most striking outcome was a strong effect of time (i.e., LD) on larvae performance: respiration was affected by an LD × temperature interaction in 2010 and 2012, as well as an LD × pCO2 × temperature interaction in 2012; F
v/F
m was affected by LD in 2010 (but not 2012); and mortality was affected by an LD × pCO2 interaction in 2010, and an LD × temperature interaction in 2012. There were no main effects of pCO2 in 2010, but in 2012, high pCO2 depressed metabolic rate and reduced mortality. Therefore, differences in larval performance depended on day of release and resulted in varying susceptibility to future predicted environmental conditions. These results underscore the importance of considering larval brood variation across days when designing experiments. Subtle differences in experimental outcomes between years suggest that transgenerational plasticity in combination with unique histories of exposure to physical conditions can modulate the response of brooded coral larvae to climate change and ocean acidification.

Full text

ORIGINAL PAPER
Brooded coral larvae differ in their response to high temperature
and elevated pCO
2
depending on the day of release
Vivian R. Cumbo •Peter J. Edmunds •
Christopher B. Wall •Tung-Yung Fan
Received: 30 November 2012 / Accepted: 7 June 2013
ÓSpringer-Verlag Berlin Heidelberg 2013
Abstract To evaluate the effects of temperature and
pCO
2
on coral larvae, brooded larvae of Pocillopora
damicornis from Nanwan Bay, Taiwan (21°56.1790N,
120°44.850E), were exposed to ambient (419–470 latm)
and high (604–742 latm) pCO
2
at *25 and *29 °Cin
two experiments conducted in March 2010 and March
2012. Larvae were sampled from four consecutive lunar
days (LD) synchronized with spawning following the new
moon, incubated in treatments for 24 h, and measured for
respiration, maximum photochemical efficiency of PSII
(F
v
/F
m
), and mortality. The most striking outcome was a
strong effect of time (i.e., LD) on larvae performance:
respiration was affected by an LD 9temperature interac-
tion in 2010 and 2012, as well as an LD 9pCO
2
9tem-
perature interaction in 2012; F
v
/F
m
was affected by LD in
2010 (but not 2012); and mortality was affected by an
LD 9pCO
2
interaction in 2010, and an LD 9temperature
interaction in 2012. There were no main effects of pCO
2
in
2010, but in 2012, high pCO
2
depressed metabolic rate and
reduced mortality. Therefore, differences in larval perfor-
mance depended on day of release and resulted in varying
susceptibility to future predicted environmental conditions.
These results underscore the importance of considering
larval brood variation across days when designing experi-
ments. Subtle differences in experimental outcomes
between years suggest that transgenerational plasticity in
combination with unique histories of exposure to physical
conditions can modulate the response of brooded coral
larvae to climate change and ocean acidification.
Introduction
Tropical coral reefs are threatened by global climate
change (GCC) and ocean acidification (OA), with large
numbers of corals killed by bleaching since the 1980s
(Hoegh-Guldberg 1999), and the remainder at risk from
further rises in temperature and less basic seawater (Hoe-
gh-Guldberg et al. 2007). GCC is caused by rising atmo-
spheric CO
2
concentrations, which have increased from
*280 ppm in the pre-industrial era to present day levels of
*396 ppm (http://scrippsco2.ucsd.edu), and are expected
to reach between *450 and 950 ppm by 2100 under the
representative concentration pathways (RCPs) 4.5 and 8.5,
respectively, depending on which pathway most closely
resembles reality (Detlef et al. 2011).
Approximately, 26–30 % of atmospheric CO
2
has been
absorbed by seawater over the last 200 years (Sabine et al.
2004; Orr 2011), causing surface-ocean total pH to decline
*0.1 since pre-industrial times (IPCC 2007). This process
is known as ocean acidification (OA), and it occurs when
atmospheric CO
2
mixes with seawater to form carbonic
acid, which through a cascade of chemical reactions,
decreases seawater pH and (CO
32-
), and increases
Communicated by U. Sommer.
Electronic supplementary material The online version of this
article (doi:10.1007/s00227-013-2280-y) contains supplementary
material, which is available to authorized users.
V. R. Cumbo (&)P. J. Edmunds C. B. Wall
Department of Biology, California State University,
18111 Nordhoff Street, Northridge, CA 91330-8303, USA
e-mail: vivian.cumbo@gmail.com
T.-Y. Fan
National Museum of Marine Biology and Aquarium, Pingtung,
Taiwan, ROC
T.-Y. Fan
Institute of Marine Biodiversity and Evolution, National Dong
Hwa University, Hualien, Taiwan, ROC
123
Mar Biol
DOI 10.1007/s00227-013-2280-y

(HCO
3-
) (Kleypas et al. 1999). These changes depress the
CaCO
3
saturation state (X) that defines the thermodynamic
ease with which CaCO
3
can be deposited; when Xdeclines
to \1.0, CaCO
3
dissolves (Kleypas et al. 1999). The
reduction in Xhas negative consequences for marine cal-
cifiers (Kroeker et al. 2010), most of which rely on CO
32-
for the production of calcareous shells and skeletons
(Marubini et al. 2008; Cohen et al. 2009). Declines in X,
therefore, are projected to have strong effects on marine
systems in which calcifying taxa play ecologically impor-
tant roles (Hofmann et al. 2010). Indeed, the most pro-
nounced effects of OA and declines in Xare expected on
ecologically and economically significant coral reef eco-
systems (Hoegh-Guldberg et al. 2007; Silverman et al.
2009).
Following disturbances causing widespread coral mor-
tality, the primary means of recovery for coral populations
is sexual reproduction involving pelagic larvae that recruit
to benthic surfaces (Harrison 2011). For this reason, it is of
great interest to know whether environmental challenges,
including those expected from GCC and OA, will affect
early life stages of corals (i.e., larvae and recruits) to the
same extent as adult colonies (e.g., Albright 2011; Byrne
2011). Differential susceptibility to stress in early life
stages has important consequences, either potentially act-
ing as a bottleneck to population growth (i.e., early life
stages are more sensitive than adults), or emphasizing the
success of adult colonies as the determinants of demo-
graphic rates (i.e., early life stages are less sensitive than
adults). For invertebrates, high larval mortality ([90 %)
suggests that larvae are more sensitive than adults (Goss-
elin and Qian 1997), but parental investment in cellular
defenses for offspring might confer enhanced resistance in
the larvae compared to the adults that produce them
(Hamdoun and Epel 2007).
Coral recruitment is dependent on a well-defined
sequence of events following fertilization, often occurring
in quick succession and including larval development,
dispersal, settlement, metamorphosis, and finally, growth
of the youngest benthic corals (i.e., recruits) to a size that
enhances their survival (Harrison and Wallace 1990; Har-
rison 2011). Physical conditions affect these events, for
instance, facilitating the selection of settlement surfaces
through water-borne cues (Gleason et al. 2009) and light
quality (i.e., color) (Mundy and Babcock 1998; Mason
et al. 2011), and promoting self-seeding (sensu Black 1993)
of reefs through larval retention (Cowan and Sponaugle
2009). Other physical factors have inhibitory effects on
coral recruitment. For example, coral larvae are adversely
affected by ultraviolet radiation (Gleason et al. 2006),
heavy metals (Negri et al. 2002), extreme temperatures
(Bassim and Sammarco 2003), and pesticides (Markey
et al. 2007) (reviewed in Gleason and Hofmann 2011).
More recently, studies focused on the biological conse-
quences of GCC have detected negative effects on coral
recruitment, with high temperature depressing sperm
motility (Morita et al. 2009), reducing fertilization success
(Negri et al. 2007), the development of embryos (Negri
et al. 2007; Bassim et al. 2002) and larvae (Edmunds et al.
2005; Nozawa and Harrison 2007; Putnam et al. 2008;
Randall and Szmant 2009; Heyward and Negri 2010), and
larval physiology (Edmunds et al. 2001,2011). OA alone
can negatively affect coral larvae, with steady high pCO
2
depressing larval viability, settlement (Albright et al. 2008;
Albright and Langdon 2011) and survival (Cumbo et al.
2012a), as well as post-settlement growth (Cohen et al.
2009; Suwa et al. 2010). Interestingly, oscillatory pCO
2
that is ecologically relevant to shallow back reef environ-
ments can promote growth and survival in new recruits of
Seriatopora caliendrum (Dufault et al. 2012), underscoring
the importance of temporal aspects of OA in determining
the biological responses of early life stages of corals to this
stressor.
Building from previous work (cited above), in this
study, we investigated the synergistic effect of temperature
and pCO
2
on the physiology of coral larval. The study was
designed to exploit, as an experimental strength, the rapid
development of brooded larvae (Stoddart and Black 1985;
Permata et al. 2000), as well as their phenotypic variation
within larval broods (Isomura and Nishihara 2001) and
among release days (Putnam et al. 2010; Cumbo et al.
2012b), by explicitly comparing the performance of larvae
from consecutive days over the peak release period.
Brooded larvae of Pocillopora damicornis from southern
Taiwan were studied, and were exposed to contrasts of
ambient and elevated temperature crossed with ambient
and elevated pCO
2
. Larval performance under each treat-
ment was assessed through oxygen consumption, and
photophysiology (F
v
/F
m
), with fitness evaluated through
survival measurements. We hypothesized that elevated
temperature and pCO
2
would result in metabolic depres-
sion, and a reduction in ATP supply (Edmunds et al. 2011),
which would increase larval mortality. The greatest number
of larvae is spawned during the peak release period, and for
pocilloporids in Taiwan this extends over *7 days (Fan
et al. 2002). We reasoned, therefore, that the analysis of
larvae from this peak period had the greatest ecological
relevance in understanding the effects of GCC and OA on
P. damicornis larvae in this location.
Materials and methods
Initially, we tested the effects of temperature and pCO
2
on
larval phenotypes in March 2010, but repeated the exper-
iment in March 2012 to explore the consistency of our
Mar Biol
123

initial findings and strengthen our conclusions. To the
greatest extent possible, the second experiment was
designed to be similar (but not identical) to the initial
experiment, based on the treatments, collection regime of
the larvae, duration of the experiments, and dependent
variables. In the design phase of the study, we realized it
would be impossible to conduct identical experiments, in
part because the corals used in each experiment were
affected by the unique physical and biological attributes of
the year in which the analyses were conducted, and
because aspects of the initial study prompted slight changes
in the experimental design. This was most striking in the
replication within treatments, which was doubled in the
second experiment based on the variance in the initial
experiment. Further, the complexities of creating stable
treatment levels for temperature and pCO
2
allowed us to
attain precise control that was slightly inaccurate with
regard to the treatment targets. Therefore, ambient and high
temperatures were crossed with ambient and high pCO
2
in
both experiments, but the actual values of each treatment
differed slightly between years.
March 2010 experiment
Coral and larval collection
Eight P. damicornis colonies were collected at 5–7 m
depth from Hobihu Reef, Nanwan Bay, 1 week before the
new moon of March 15, 2010. P. damicornis from this
region release larvae in synchrony with lunar phases, with
release beginning near the new moon and peaking near the
first quarter moon (Fan et al. 2002). Colonies were trans-
ported to the National Museum of Marine Biology and
Aquarium (NMMBA) and placed in separate flow-through
tanks (n=8) supplied with filtered seawater (*50 lm)
and exposed to sunlight shaded to a maximum intensity of
*200 lmol quanta m
-2
s
-1
. The flow-through system
transported larvae from the corals into beakers fitted with
plankton mesh. At *0700 hours, beakers were examined
for larvae, which are released around dawn (Fan et al.
2006).
The experiments were scheduled just after the new
moon, but each spawning is unique and the day of peak
spawning can differ slightly from the peak spawning day
predicted from previous spawning events (Fan et al. 2002).
To ensure that larvae from near the peak spawning were
used, larvae were counted daily to detect the approach of
the day(s) when the greatest number of larvae was released.
The experiment was repeated using fresh larvae from four
consecutive days between 23rd and 26th of March [lunar
days (LD) 8–11] to examine the effect of day of release on
larval biology. On each day, 80 larvae colony
-1
were
pooled and stirred gently before transferring batches of 50
into each of eight 700-mL plastic tubs fitted with mesh
windows (110 lm) that were incubated individually in
each tank (n=8 tanks, 2 tanks treatment
-1
). Larvae were
collected on the day of release and incubated in treatments
for 24 h. Experiment setup times varied (\8 h) on each
day, however, there was no variation in the exposure time
to treatment conditions.
Temperature and pCO
2
treatments
The tubs containing larvae were allocated randomly to four
treatments obtained by contrasts of ambient (*25 °C for
seawater when the experiment was conducted) and high
(*29 °C) temperature crossed with ambient (*400 latm)
and high (*750 latm) pCO
2
. Treatments were selected to
gain insight into the response of larvae to seasonal extreme
temperatures in Nanwan Bay, as well as to pCO
2
condi-
tions that are feasible within *90 years (Detlef et al.
2011). Each of the four treatments was created in duplicate
tanks, and incubations lasted 24 h after which the larvae
were evaluated for respiration, dark-adapted maximum
quantum yield of PSII (F
v
/F
m
), and mortality.
Treatment tanks contained 30 L of sand-filtered (50 lm)
seawater, and were independently chilled (Aquatech Ac11
or Shyeh Duwai Enterprise), heated (Taikong Corporation),
and mixed (Rio 1100 pumps). With 50 larvae in each tank,
larval biomass was trivial compared to the volume of
seawater, with the combined larval volume [at 0.39 mm
3
each (Edmunds et al. 2011)] amounting to \10
-4
% of the
seawater volume. The seawater was partially replaced
(20 %) daily, and the tanks illuminated on a 1212-h
light:dark cycle (with lights on at 0700 hours) with metal
halide lamps. CO
2
treatments were created by bubbling air
into the tanks to create ambient pCO
2
conditions, and a
blended gas mixture (air ?CO
2
) to create high pCO
2
conditions. The gas mixture was obtained with automated
mass-flow controllers with feedback control through in-line
IR-gas analysis (Qubit Systems). This system mixed air,
delivered by a compressor and regulated at
15–20 L min
-1
, with 99 % CO
2
(120 Pa) via a solenoid
controller (Model A352, Qubit). The mixed gas (at
200 mL min
-1
) passed through two tubes, one to a pump
(P651, Qubit) and a CO
2
IR-gas analyzer (S151, Qubit) that
controlled the solenoid determining the flow of CO
2
into a
mixing chamber. The CO
2
output (ppm) of the gas analyzer
was interfaced with a PC (using LabPro, Vernier Software
and Technology) and logged using Logger Pro V3.7 soft-
ware (Vernier Software and Technology). The second tube
supplied the mixed gas to treatment tanks at
10–15 L min
-1
using a pump (Model DOA-P704-AA,
Gast Manufacturing Inc.). We used the logged pCO
2
to
verify that the gas mixture supplied to the tanks was stable
over a 24-h period, although the data are not presented.
Mar Biol
123

To determine the dissolved inorganic carbon (DIC)
chemistry of seawater in the treatments, total alkalinity
(TA), pH on the total scale, temperature, and salinity were
measured using standard procedures, and used to calculate
pCO
2
(latm), HCO
3-
(lmolkg
-1
), CO
32-
(lmolkg
-1
), and
the aragonite saturation state (X
Arag
) of seawater using
CO2SYS (Pierrot et al. 2006). TA (lmol kg
-1
) was mea-
sured through potentiometric titration (SOP 3b, Dickson
et al. 2007) using an automatic titrator (DL50, Mettler
Toledo) filled with certified acid titrant (0.1 M HCl, 0.6
NaCl, Dickson Laboratory, Scripps Institution of Ocean-
ography). The pH probe (DG101-SC, Mettler Toledo)
attached to the titrator was 3-point calibrated with pH 4.00,
7.00, and 10.00 buffers (Fisher, NBS). Certified reference
material with a known TA (Batch 98, http://andrew.ucsd.
edu/index.html) was titrated daily to determine the accu-
racy and precision of the analyses.
Seawater samples (50 mL) from the treatment tanks
were brought to 25 °C, weighed and titrated in a water-
jacketed beaker within 2–3 h of collection. The pH values
and the titrant volumes (cm
3
) obtained from the titrations
were sub-sampled for the range between pH 3.0 and 3.5
and inserted into a Microsoft Excel spreadsheet (Fangue
et al. 2010), which calculated Gran’s function as a product
of the mass of titrant added (Dickson et al. 2007). Treat-
ment tank pH was determined spectrophotometrically
using m-cresol purple dye (Sigma-Aldrich) following SOP
6b of Dickson et al. (2007) with modification (Fangue et al.
2010). Preliminary sampling of the seawater in the tanks
throughout the day confirmed that the pCO
2
treatments
were stable over a 24-h period.
Larval response to treatments
A Ruthenium-based optode (FOXY-R, 1.58 diameter,
Ocean Optics) connected to a spectrophotometer
(USB2000, Ocean Optics) and interfaced with a computer
running Ocean Optics software (OOISensor, version
1.00.08) was used to measure the respiration of the larvae.
The optode was 2-point calibrated using a zero solution
(0.01 M Na
2
B
4
O
7
10H
2
O saturated with Na
2
SO
3
) and
100 % air saturation using water-saturated air at the treat-
ment temperature. To measure respiration, 6 larvae were
removed from the treatment containers and placed into
2-mL glass Wheaton vials filled with filtered seawater from
the same treatment tank and sealed with Parafilm
TM
.A
study conducted concurrently with the present analysis
demonstrated that respiration of P. damicornis larvae in
identical vials could be measured accurately with 5 larvae
in each vial (Edmunds et al. 2011). Respiration measure-
ments were completed after the 24-h incubation period.
Larvae were dark-adapted prior to measurements so that
respiration would not be stimulated by light (Edmunds and
Davies 1988). Initial O
2
concentration in the seawater
filling the vials was determined before the vials were
sealed, and vials without larvae were used as controls.
Larvae in the sealed vials were incubated at their temper-
ature treatments for 1.5–2 h in the dark using water baths
(±0.1 °C, Hipoint, models LC-06 and LC-10). Incubation
times were selected to ensure that O
2
concentrations
remained [75 %. On completion of the incubations, vials
were removed from the chillers, gently inverted to mix the
seawater, and analyzed for O
2
saturation. O
2
saturation was
converted to concentration using gas tables [N. Ramsing
and J. Gundersen at http://www.unisense.com (based on
Garcia and Gordon 1992)] and the temperature and salinity
of the seawater, and the change in O
2
concentration con-
verted to nmol O
2
min
-1
larvae
-1
, after adjusting for
control O
2
fluxes.
Larval photophysiology was assessed using pulse
amplitude modulated (PAM) fluorometry to measure the
maximum photochemical efficiency of open reaction cen-
ters of photosystem II (RCIIs) following a period of dark
adaptation (i.e., F
v
/F
m
) of their Symbiodinium. Changes in
F
v
/F
m
can detect damage to the photosynthetic apparatus,
with declines under elevated temperature indicating dam-
age to PSII (Jones et al. 1998; Bhagooli and Hidaka 2003).
These measurements were conducted after the 24-h incu-
bation period, with larvae being dark-adapted during the
final 2 h of the incubation. After this period of darkness,
F
v
/F
m
was measured using a diving PAM (Walz, GmbH)
fitted with an 8-mm diameter probe and standardized for
measuring intensity (setting: 10) and gain (setting: 10).
Fluorescence was measured by loading 8 larvae into a drop
of seawater on the tip of the probe, with these manipula-
tions completed under weak red illumination. Two groups
of 8 larvae were measured for F
v
/F
m
from each tank, and
the average value in each tank was used for statistical
analysis.
To assess the number of larvae dying in the treatments,
at the conclusion of the incubations, tubs were removed
from the treatments and the number of swimming larvae
and settled recruits (with tissue) recorded. Due to the rapid
breakdown of dead larvae (Yakovleva et al. 2009), larvae
that could not be accounted for were assumed dead. Mor-
tality was expressed as a percentage of the number of
larvae added at the start of the experiment.
March 2012 experiment
The second experiment was designed to be virtually iden-
tical to the first (n=8 tanks, 2 tanks treatment
-1
),
although the volume of the incubation tanks was increased
to 120 L, and the sample size (number of replicate tubs
containing larvae in each tank) was doubled with the
objective of increasing statistical power and testing for tank
Mar Biol
123

effects for the dependent biological variables. As described
below, experimental difficulties affecting a single tank
made it problematic to include tank in the statistical anal-
yses, and therefore, replicates were pooled between tanks
in each treatment combination. Experimental procedures
were identical except as noted below.
Coral and larval collection
In 2012, eight colonies of P. damicornis were again col-
lected from 5 to 7 m depth on Hobihu Reef, *1 week
before the new moon on 21 February. The experiment was
conducted using larvae freshly released on four consecu-
tive days between 2nd and 5th of March (LD 9–12);
logistical constraints prevented collection of larvae on
identical LDs to 2010, but the discrepancy was only 1 day
(later in 2012 compared to 2010). Of the larvae released
from the colonies, 160 larvae colony
-1
were pooled among
colonies and stirred gently to produce a homogeneous
mixture. Two tubs containing 50 larvae selected randomly
from the mixture were placed one of 8 tanks exhibiting the
conditions described below.
Temperature and pCO
2
treatments
The mesocosm assembly employed in 2012 was similar to
that used in 2010, and again the objective was to contrast
ambient (*25 °C for ambient seawater when the experi-
ment was conducted) and high (*29 °C) temperature
crossed with ambient (*400 latm) and high (*750 latm)
pCO
2
. The experimental system allowed temperature and
pCO
2
to be regulated with precision, but treatment levels
were slightly inaccurate with regard to duplicating the
treatments created in 2010.
Larval response to treatments
Larvae were sampled from each replicate tub, and their
response to the treatment conditions was assessed using
dark respiration, photophysiology (F
v
/F
m
), and mortality,
and each dependent variable was measured as described
above.
General timing of larval release during lunar March
To place the present results in a broader context, the timing
of larval release from P. damicornis in Taiwan was
examined over multiple years to determine when the peak
larval release occurred in March, relative to the preceding
new moon. The analysis was used to weight the outcome of
the present analysis for ecological significance based on the
LD when the majority of larvae are released. The analysis
was accomplished by compiling data on lunar March
timing of larval release for P. damicornis for 2003, 2005,
2007, and 2008 (Fan et al. 2006; TY Fan unpublished data).
In each of 4 years, coral colonies from the same location as
in this study were transported to NMMBA and placed in
flow-through aquaria as described above. In 2003 and
2005, 8 colonies month
-1
were used, but in 2007 and
2008, 4 colonies month
-1
were used. During these analy-
ses, the beakers collecting larvae released from each col-
ony were inspected daily (at 0900 hours) and the number
of larvae counted. The number of larvae released daily
from each colony in each year was summed to give the
total number released on each LD over 4 years.
Statistical analysis
In 2010, to determine whether duplicate tanks within each
treatment represented the same conditions, pCO
2
(latm)
and temperature (°C) were analyzed using a 3-way ANOVA
with tank nested within the fixed effect of temperature and
pCO
2
. This design was changed in 2012 because experi-
mental difficulties resulted in extreme outlying values for
dependent variables in one tank in the ambient temperature/
ambient pCO
2
treatment. These values were exceptionally
high—bigger than we had recorded in previous experiments
and unlikely to be biologically feasible—and the between-
tank variation was fourfold greater than in any other treat-
ment. Using the aforementioned extreme values as a
rationale, the tank generating the outlying values was
removed from the primary analyses, although a secondary
analysis was also conducted to consider the bias this might
introduce (describe below). To evaluate physical and
chemical conditions in tanks during 2012, where present,
duplicate tanks in each treatment were compared by ttests
with respect to pCO
2
(latm) and temperature (°C). For both
years, larval respiration, F
v
/F
m
, and mortality were com-
pared among the fixed effects of temperatures, pCO
2
, and
LD using a 3-way Model I ANOVA in which temperature,
pCO2, and LD were fixed effects.
In 2010, the statistical analysis was completed with one
replicate tank
-1
and two replicates treatment
-1
. In 2012,
the statistical analysis of response variables was completed
with 4 replicate treatments
-1
(i.e., with results pooled
between tanks), except for ambient temperature/ambient
pCO
2
for which one tank was dropped from the analyses
(leaving 2 replicates in one tank). To evaluate the extent to
which the outcome of the 2012 experiment was biased by
exclusion of one tank from the experimental design and the
inability to use tank as a nested effect in the statistical
model, the analysis was repeated with imputed values
(following Zar 2010) obtained from the means values in the
tank remaining in this treatment (Supplementary Material).
For all analyses, the residuals were graphed to test the
statistical assumptions of ANOVA, and percent mortality
Mar Biol
123

was arcsine transformed to meet these assumptions. Inter-
action terms that were not significant at P[0.25 were
removed sequentially from the analysis, with the ANOVA
repeated with each omission (Quinn and Keough 2002).
Where significant main effects of LD were detected, Tukey
post hoc tests were used to determine which pairs of LD
differed significantly. Statistical analyses were carried out
using Systat 11 running in a Microsoft Windows
environment.
Results
March 2010 experiment
Temperature and pCO
2
treatments
The mean temperatures for each treatment were
25.4 ±0.1 °C [ambient temperature, ambient pCO
2
(ATAC)], 25.5 ±0.1 °C [ambient temperature, high pCO
2
(ATHC)], 29.3 ±0.2 °C [high temperature, ambient pCO
2
(HTAC)], and 29.6 ±0.2 °C (high temp, high pCO
2
(HTHC) (all ±SE, n=4). Temperature differed between
duplicate tanks within each treatment (F=30.994,
df =4.95, P\0.001), but this effect reflected the accuracy
of the temperature probe (±0.01 °C; digital thermometer
15-077-8, probe 15-077-7, Control Company) rather than a
difference that was likely to be biologically meaningful
(i.e., paired tanks differed \0.1 °C in mean temperature).
TA determinations of the CRM were on average 5 % higher
than the certified values, and therefore, TA values of the
treatment seawater were downwardly adjusted by the same
amount (Table 1). Mean pCO
2
in the treatment tanks were
459 ±28 latm (ATAC), 419 ±10 latm (HTAC),
604 ±19 latm (ATHC), and 712 ±21 latm (HTHC) (all
±SE, n=4, Table 1). pCO
2
did not differ between dupli-
cate tanks within each CO
2
treatment (F=1.133,
df =4.15, P=0.378), it differed between each CO
2
treatment at each temperature (F=8.559, df =1.4,
P=0.042). Hereafter, temperature and pCO
2
averaged
across tanks within each treatment are reported. Light was
supplied at 370 ±6lmol quanta m
-2
s
-1
(±SE, n=16).
Coral larvae physiology
Pocillopora damicornis larvae were actively swimming in
all treatments after 24 h, and \2 % settled. All statistical
results for respiration, F
v
/F
m
, and mortality are displayed
in Table 2; there were no main effects of pCO
2
for any
dependent variable (P[0.314), and two-way interactive
effects of pCO
2
were detected for mortality (described
below).
Larval respiration ranged from 0.068 to 0.262 nmo-
lO
2
larva
-1
min
-1
and varied between temperatures in a
pattern that differed between LDs (P\0.001); respiration
was also affected significantly by the main effects of
temperature (P=0.007) and LD (P\0.001) (Table 2).
Separation of the results by LD revealed that the temper-
ature 9LD interaction was driven largely by the high
respiration of larvae exposed to high temperature on LD 9;
these rates were 1.75-fold (HTAC) and 1.60-fold (HTHC)
higher than the respiration averaged across all treatments
[0.146 ±0.008 nmol O
2
larva
-1
min
-1
(±SE, n=32)]
(Fig. 1). Post hoc analysis of the LD effect showed that
respiration on LD8 was lower than on LD9 or LD10
(P\0.001), and that respiration on LD9 was higher than
on LD10 or LD11 (PB0.001); no other contrasts were
significant (P[0.123).
Table 1 Carbonate chemistry in the experiments conducted in 2010 and 2012 for four treatments: *25 °C, ambient pCO
2
(ATAC), *29 °C,
ambient pCO
2
(HTAC), *25 °C, high pCO
2
(ATHC), and *29 °C, high pCO
2
(HTHC)
Experiment Treatment pH T(°C) Salinity TA
(lmol
-1
kg
-1
)
pCO
2
(latm)
HCO
3-
(lmol
-1
kg
-1
)
CO
32-
(lmol
-1
kg
-1
)
X
Arag
March 2010 ATAC 7.98
a
25.4 ±0.1 34.0 ±0.1 2,164 ±8 459 ±28 1,728 ±22 176 ±7 2.82 ±0.12
HTAC 8.02
a
29.3 ±0.2 35.4 ±0.2 2,318 ±9 419 ±10 1,748 ±7 235 ±5 3.79 ±0.07
ATHC 7.88
a
25.5 ±0.1 34.1 ±0.2 2,177 ±10 604 ±19 1,813 ±12 147 ±3 2.36 ±0.05
HTHC 7.82
a
29.6 ±0.2 35.2 ±0.4 2,243 ±15 714 ±21 1,854 ±12 158 ±4 2.56 ±0.06
March 2012 ATAC 8.00
a
25.3
b
33.8
c
2,269 ±3 454 ±6 1,802 ±4 190 ±2 3.05 ±0.03
HTAC 7.99
a
29.5
b
34.2 ±0.1 2,305 ±7 470 ±4 1,780 ±7 215 ±1 3.50 ±0.02
ATHC 7.82
a
25.3
b
33.9 ±0.1 2,278 ±9 742 ±13 1,945 ±9 136 ±2 2.18 ±0.02
HTHC 7.82
a
29.4
b
34.3 ±0.1 2,310 ±19 741 ±15 1,921 ±16 159 ±3 2.60 ±0.05
Mean ±SE shown [n=4 and 8 for all variables in 2010 and 2012 (respectively) except 2012, ATAC 25 °C where n=4]. Tanks were
illuminated at a mean intensity of 370 ±6lmol quanta m
-2
s
-1
(±SE, n=16) in 2010, and 307 ±2lmol quanta m
-2
s
-1
(±SE, n=24) in
2012. pH (
a
SE B0.02), temperature (T°C,
b
SE B0.03 °C), total alkalinity (TA), and salinity (
c
SE \0.1) were measured, and the remaining
carbon parameters estimated using CO2SYS (Pierrot et al. 2006). The saturation state of aragonite (X
Arag
) assumes a calcium concentration of
10 mg kg SW
-1
Mar Biol
123

F
v
/F
m
of Symbiodinium within the larvae ranged from
0.570 to 0.708 (averaged between replicates in each tank)
across treatments over the 4-day experiment, and differed
significantly among LDs (P\0.001) (Table 2). Post hoc
analysis of the LD effect revealed that F
v
/F
m
on LD8 was
lower than on LD9 or LD11 (PB0.011), with no other
contrasts significant (P[0.086). Additionally, there was a
trend for F
v
/F
m
to be affected by the LD 9tempera-
ture 9pCO
2
interaction (P=0.058) and the main effect of
temperature (P=0.053) (Table 2). These trends reflected
an effect of high pCO
2
that depressed F
v
/F
m
at ambient
temperatures on LD8 but increased it on LD10, and an
effect at high temperature that depressed F
v
/F
m
on LD10
but had no effect on LD11; the temperature effect indicated
a tendency for F
v
/F
m
to increase with temperature.
Overall, B48 % of the larvae died during each incuba-
tion, with mean mortality varying from 0 to 32 %
depending on conditions (Fig. 2). A strong LD 9pCO
2
interaction (P=0.006; Table 2) revealed differential
mortality effects of pCO
2
on LD8 and LD9 (when high
pCO
2
decreased mortality 56–91 %) versus LD10 [when
high pCO
2
increased mortality from 0 to 7 % (*25 °C)
and from 3 to 29 % (*29 °C)] and LD 11 (when mortality
was unaffected by pCO
2
).
Table 2 Results of three-way Model I ANOVAs comparing the
effects of LD (lunar day) of release, temperature (*25 and *29 °C)
and pCO
2
(ambient and high) on larval respiration
(nmol min
-1
larvae
-1
), dark-adapted maximum quantum yield of
PSII (F
v
/F
m
), and mortality in experiments conducted in March 2010
and March 2012
Response variable Source 2010 2012
df MS FPdfMS FP
Respiration LD 3 0.0132 29.196 <0.001 3 0.0054 2.514 0.072
Temp 1 0.0066 14.594 0.007 1 0.0043 19.770 <0.001
pCO
2
1 0.0005 1.061 0.314 1 0.0014 6.622 0.013
LD 9temp 3 0.0036 7.995 <0.001 3 0.0007 3.053 0.039
Temp 9pCO
2
1 0.0015 3.399 0.079 1 0.0004 1.878 0.178
LD 9pCO
2
3 0.0006 2.526 0.071
LD 9pCO
2
9temp

3 0.0010 4.539 0.008
Error 22 0.0005 40 0.0010
F
v
/F
m
LD 3 0.0058 8.680 0.001 3 0.0003 0.524 0.668
Temp 1 0.0029 4.353 0.053 1 0.0003 0.599 0.443
pCO
2
1\0.0001 0.006 0.938 1 \0.0001 0.043 0.838
LD 9temp 3 \0.0001 0.352 0.788 3

LD 9pCO
2
3 0.0002 0.262 0.852 3

Temp 9pCO
2
1 0.0015 2.372 0.143 1 0.0007 1.407 0.241
LD 9temp 9pCO
2
3 0.0021 3.061 0.058 3

Error 16 0.0007 49 0.0005
Mortality LD 3 0.0750 2.606 0.076 3 0.0211 2.126 0.113
Temp 1 0.0981 3.407 0.078 1 0.0201 2.012 0.164
pCO
2
1 0.0145 0.503 0.487 1 0.3178 31.900 <0.001
LD 9pCO
2
3 0.1568 5.446 0.006 3 0.0025 0.252 0.418
LD 9temp

3 0.0648 6.504 <0.001
Temp 9pCO
2
1 0.0067 0.669 0.418
LD 9temp 9pCO
2
3 0.0186 1.869 0.151
Error 23 0.0288 39 0.0100
Interactions that were not significant (at P[0.25) were sequentially removed (

) from the analysis with the ANOVA re-run with each omission
(Quinn and Keough 2002). In 2010, the experiment was conducted with duplicate tanks in each treatment, with one replicate tank
-1
; thus, it was
not possible to test for tank effects. In 2012, the experiment was doubled in size with duplicate determinations for each tank, with the objective of
testing for tank effects. Due to logistical constraints, one tank in the ATAC treatment was dropped from the analysis, and again it was not
possible to test for tank effects. The 2012 results were pooled between tanks in treatments where 2 tanks remained, and analyzed as an
unbalanced design using Type III sum of squares; significant effects are in bold. Repeating the analysis with imputed values to restore tank as a
nested factor in this design did not appreciably alter the statistical outcomes (Table S1)
Mar Biol
123

March 2012 experiment
Temperature and pCO
2
treatments
The mean temperatures for each treatment were
25.3 ±0.03 °C (ATAC), 25.3 ±0.02 °C (ATHC),
29.5 ±0.03 °C (HTAC), and 29.4 ±0.03 °C (HTHC) (all
±SE, n=8 except ATAC where n=4). Temperature
differed between duplicate tanks within each treatment
(t=4.371, df =6, P[0.005), but again this effect
reflected the accuracy of the temperature probe (±0.01 °C)
rather than a difference that was likely to be biologically
meaningful (i.e., paired tanks differed \0.1 °C in mean
temperature). TA determinations of the CRM were on
average 0.6 % higher than the certified values. Mean pCO
2
in the treatments were 454 ±6latm (ATAC),
470 ±4latm (HTAC), 742 ±13 latm (ATHC), and
741 ±15 latm (HTHC) (all ±SE, n=8 except ATAC
where n=4, Table 1); pCO
2
did not differ between
duplicate tanks within each pCO
2
treatment (t=1.526,
df =6, P[0.178), but it differed between each pCO
2
treatment at each temperature (t=1.526, df =6,
P\0.003). Hereafter, temperature and pCO
2
averaged
across tanks within each treatment are reported. Light was
supplied at 307 ±2lmol quanta m
-2
s
-1
(±SE, n=24).
Coral larvae physiology
In 2012, P. damicornis larvae were actively swimming in
all treatments after 24 h. Refer to Table 2for statistical
results for respiration, F
v
/F
m
, and mortality. pCO
2
had
main effects for both respiration and mortality
Lunar Day 8 9 10 11 12
2010
2012
2010
2012
nmol O
2
min
-1
larvae
-1
Year
Fv / Fm
High CO2
Amb CO2
0.05
0.10
0.15
0.20
0.25
0.05
0.10
0.15
0.20
0.25
0.56
0.60
0.64
0.68
0.72
0.56
0.60
0.64
0.68
0.72
~25 ~29
~25 ~29
~25 ~29 ~25 ~29 ~25 ~29 ~25 ~29
~25 ~29
Temperature (oC)
Fig. 1 Interaction plots illustrating the statistical relationships for
respiration and maximum photochemical efficiency [of open RCIIs
(quantum yield, F
v
/F
m
)] in P. damicornis larvae incubated for 24 h
under combinations of two temperatures crossed with two pCO
2
regimes (Table 1). Experiments were conducted with larvae from 4
consecutive days close to the peak release in March 2010 and March
2012, and larvae from each day were used in independent experiments
with results corresponding with the columns of graphics (LD 8–12).
Upper two rows display respiration and lower two rows display F
v
/
F
m
; mean ±SE shown with n=2 in 2010 and n=4 in 2012, except
for ATAC on LD 9 (n=2). Note: dependent variables for pCO
2
treatments are offset laterally for clarity
Mar Biol
123

(PB0.013), and a three-way interactive effect for respi-
ration (described below). These outcomes largely were
unchanged when imputed values were used to restore tank
as a nested effect in the statistical design (Table S1).
Larval respiration ranged from 0.081 to 0.179
nmol O
2
larva
-1
min
-1
and varied among LDs in a pattern
that differed between pCO
2
and temperature treatments
(P=0.008), and between temperatures (P=0.039)
(Table 2). Separation of the results by LD revealed that
interactions were driven by depressed respiration of larvae
exposed to high pCO
2
on LD9, no effect of pCO
2
on LD 10
and 11, and an effect of high pCO
2
that caused respiration
to be depressed at *25 °C but accelerated at *29 °C
(Fig. 1). Respiration was also affected by the main effects
of temperature (P\0.001) and pCO
2
(P=0.013), tending
to increase with temperature and decline at high pCO
2
. The
grand mean respiration (i.e., pooled among treatments) was
0.126 ±0.003 nmol O
2
larva
-1
min
-1
(n=56) and this
differed significantly (t=2.737, df =86, P=0.008)
from the 2010 grand mean of 0.146 ±0.008 nmol O
2
larva
-1
min
-1
(±SE, n=32); overall, respiration was
14 % lower in 2012.
F
v
/F
m
of the Symbiodinium within the larvae ranged
from 0.615 to 0.759 across treatments over the 4-day
experiment, but it was unaffected by any of the main
effects or their interactions (P[0.241). The grand mean
F
v
/F
m
(i.e., pooled among treatments) was 0.653 ±0.003
(n=56) and this differed significantly (t=2.083,
df =86, P=0.040) from the 2010 grand mean of
0.640 ±0.006 (±SE, n=32), although the effect size was
small (2 %).
Overall, B30 % of the larvae died during each incuba-
tion, and mean mortality varied from 0 to 13 % depending
on conditions (Fig. 1). A strong LD 9temperature inter-
action (P\0.001; Table 2) revealed differential thermally
mediated mortality for larvae released on LD9 and LD10
compared to LD11 and LD12. Temperature had equivocal
effects on mortality on LD9, and no effect on LD10. On
LD 11, *29 °C increased mortality 6.5-fold compared to
*25 °C (pooled between pCO
2
levels), but on LD12,
mortality was reduced 46 % at *29 °C versus *25 °C.
Additionally, mortality was strongly affected by pCO
2
(F=31.900, df =1.39, P\0.001) and was depressed at
high pCO
2
(2 %) compared to ambient pCO
2
(8 %) (both
pooled over LDs and temperatures).
Lunar March larval release
Larval release from P. damicornis during lunar March in
2003, 2005, 2007, and 2008 showed a consistent pattern
with release beginning on LD4, low numbers released for
several days, and then a large increase to peak release on
LD 9 (when 31 % of larvae were released) (Fig. 2).
Thereafter, larval release declined, with 14 % released on
LD10, 11 % on LD11, and 7 % on LD12; few larvae were
found after LD13. In the present study, the four days
Lunar Day 8 9 10 11 12
2010
2012
Year
High CO2
Amb CO2
0.0
0.2
0.4
0.6
0.0
0.2
0.4
0.6
Mortality
~25 ~29
~25 ~29 ~25 ~29 ~25 ~29 ~25 ~29
Temperature (oC)
Fig. 2 Interaction plots illustrating the statistical relationships for
proportional mortality of P. damicornis larvae incubated for 24 h
under combinations of two temperatures crossed with two pCO
2
regimes (Table 1), using larvae from 4 consecutive days close to the
peak release in March 2010 and March 2012. Larvae from each LD
were used in independent experiments with results corresponding
with the columns of graphics (LD 8–12). Upper row displays
mortality in March 2010, and lower row displays mortality in March
2012. Mean ±SE shown for back transformed data with n=2in
2010 and n=4 in 2012, except for ATAC on LD 9 (n=2). Note:
dependent variables for pCO
2
treatments are offset laterally for clarity
Mar Biol
123

sampled began on LD8 (2010) or LD9 (2012) and, there-
fore, relative to the historic records of larval release, the
results from LD9 of both experiments are most relevant to
31 % of larvae released in the environment.
Discussion
Our results from two experiments in different years reveal
that P. damicornis larvae are affected by high pCO
2
and
elevated temperature. However, they also show that the
responses to pCO
2
and temperature often depend on the
lunar day the larvae were released, with some effects dis-
appearing or reversing for larvae released on consecutive
days, and others arising from complex interactions among
pCO
2
, temperature, and LD. Evidence that the day of
release affects the response of brooded coral larvae to
environmental challenges demonstrates that time should be
integrated into studies of this coral life stage, both to
reduce residual variance attributed to unrecognized effects
of release day, and to explicitly address the biological
implications of variation among larvae released on differ-
ent days.
The dynamic responses of P. damicornis larvae to the
aforementioned conditions provide new insights into the
biology of brooding corals. The implications of these
responses are enhanced by the strength of the temporal
signal for three traits in two separate experiments. For
larval respiration, in 2010, rates were elevated on LD9
compared to the other 3 days, and on LD9 it was strongly
stimulated by high temperature; in 2012, rates were
depressed by high pCO2 on LD9, but not on the other
3 days, and were stimulated by high temperature except at
ambient pCO
2
on LD12. For F
v
/F
m
, in 2010, values were
low on LD8 compared to LD9 and 11, but in 2012, values
did not vary among LDs. Finally, for mortality, in 2010,
rates were affected by high pCO
2
, with reductions on LD8
and LD9, increases on LD10, and no effect on LD11; in
2012, rates were not clearly affected by high temperature
on LD9 and LD10, but were stimulated by high tempera-
ture on LD11, and depressed by high temperature on LD12.
These results demonstrate that functional variations exist in
brooded coral larvae (Cumbo et al. 2012b; Edmunds et al.
2001; Isomura and Nishihara 2001; Putnam et al. 2010),
with differences depending on day of release.
Coral larvae are well known to differ within a brood,
both for brooding (Edmunds et al. 2001; Isomura and
Nishihara 2001; Gaither and Rowan 2010; Putnam et al.
2010; Yeoh and Dai 2010; Cumbo et al. 2012b; Rivest and
Hofmann 2012) and broadcast spawning (Nozawa and
Okubo 2011) corals. However, relatively little is known of
the functional implications of this variation (Isomura and
Nishihara 2001; Nozawa and Okubo 2011), despite a
history of examining similar effects in other taxa (Levitan
1996; Marshall and Keough 2003). For corals, this over-
sight is related in part to the challenges of working with
larvae, which encourages researchers [including ourselves
(Edmunds et al. 2001; Putnam et al. 2008)] to pool results
from larvae released over multiple days to obtain sufficient
replicates. An unfortunate consequence of this practice is
the inability to detect nuanced effects of treatments
attributed to larvae differing among days for a variety of
traits including size, symbiont density, respiration, com-
petency, and photophysiology (Edmunds et al. 2001; Put-
nam et al. 2010; Cumbo et al. 2012b). While some of these
traits can independently affect larval success, for instance,
with bigger larvae having a longer pelagic larval duration
(PLD) and an enhanced likelihood of finding more distant
habitats (Isomura and Nishihara 2001; but see Nozawa and
Okubo 2011), the effects of variation in these traits on the
response to physical and chemical conditions have not been
explored.
The causes of within-brood variation in coral larvae are
unknown, but they are likely to reflect the chronology of
development within the maternal polyp. In this regard,
corals differ little from other marine invertebrate in which
larval development coincides with rapid (over hours-days)
changes in physiology, structure, and behavior (Strathmann
1985; Hadfield 2000). In the case of reef corals, there is
reason to suspect that these changes might be large, as they
include those affecting both the animal host and its Sym-
biodinium, and they culminate in the unique functional
attributes of this mutualistic symbiosis (Stambler 2011).
Morphological aspects of these trends have been described
for a few coral species, for example, within 96 h of fer-
tilization in Acropora millepora,Pocillopora meandrina
and Fungia scutaria (Ball et al. 2002; Marlow and Mar-
tindale 2007), and behavioral aspects have been described
in a few other species, for instance, over a few days in
Agaricia humilis and Diploria strigosa (Raimondi and
Morse 2000; Bassim and Sammarco 2003). Larval devel-
opment in P. damicornis appears also to be rapid, because
the time between fertilization and larval release is only
*14 days (Stoddart and Black 1985; Permata et al. 2000),
and their larvae grow at *62 lm days
-1
(Stoddart and
Black 1985).
In brooding corals, however, development as a means to
account for variation in larvae is augmented by potential
effects arising from the initial development within the
maternal polyp (i.e., transgenerational effects, sensu
Agrawal et al. 1999). In this context, brooding is biologi-
cally significant because the conditions within the maternal
polyp differ from the external environment in terms of
oxygen (Harland and Davies 1995), light intensity (Salih
et al. 2000; Enriquez et al. 2005), and microbial flora
(Herndl and Velimirov 1985; Agostini et al. 2012), thereby
Mar Biol
123

creating the potential for larval acclimatization to brood
conditions within the maternal polyp prior to release. As
the influence of age at release on larval phenotype is woven
closely with the possible effects of within-polyp acclima-
tization, further studies of this topic might benefit from
characterizing the environment within polyps prior to lar-
val release, and testing for effects of these conditions on
larvae through manipulative experiments. Agostini et al.
(2012) provide a good example of the potential value of
such studies, for they recorded biological and chemical
conditions within the gastrovascular cavity of Galaxea
fascicularis and found them to be very different from
ambient seawater.
Multiple years are dissimilar in terms of conditions (e.g.,
seawater temperature, light regimes, seawater flow)
affecting coral reef environments, particularly in locations
like Nanwan Bay where there are rapid changes of a large
magnitude in select physical conditions [here, temperature
(Lee et al. 1999)]. Therefore, acclimatization of P. dami-
cornis larvae to brood conditions prior to release in dif-
ferent years (2010 and 2012) is a plausible driver of
diversity in response to unique chronologies of physical
conditions, and probably explains why the grand means of
all three measured traits differed between years. Such
effects provide a parsimonious explanation for the differ-
ences in results obtained from our two experiments, and
highlight the potential importance of transgenerational
plasticity (TGP) in modulating the response of marine
organisms to the effects of climate change (Marshall 2008;
Salinas and Munch 2012). In the present study, TGP dif-
fering among years is an intriguing possibility to account
for the appearance of significant effects of pCO
2
in the
2012 experiment (for mortality and respiration) that were
absent in 2010. It is premature to explore this possibility in
the present study, in part because the history of the
P. damicornis colonies used for larval release in 2010 and
2012 is unknown, but more importantly, TGP remains
unexplored in reef corals. The form of the pCO
2
effects that
emerged in 2012 provide a strong incentive to study the
causal processes, as high pCO
2
in this year promoted short-
term larval survival, perhaps by depressing metabolic rate.
Coral larvae are sensitive to elevated temperature, with
the effects dependent on the magnitude and duration of the
thermal challenge. They respond to temperature within
24 h of exposure (Edmunds et al. 2005; Rodriguez-Lanetty
et al. 2009; Heyward and Negri 2010), with moderate
increases in temperature accelerating development and
respiration (Edmunds et al. 2005; Rodriguez-Lanetty et al.
2009; Heyward and Negri 2010), and favoring shorter PLD
and more restricted dispersal (Heyward and Negri 2010;
O’Connor et al. 2007). As the upward thermal challenge
becomes acute, mortality rises (Edmunds et al. 2001; Baird
et al. 2006) and the capacity to feed autotrophically
declines (Edmunds et al. 2001). The response of coral
larvae to pCO
2
is known from only a few studies, but the
emerging results are equivocal with regard to the effects on
swimming stages [e.g., Albright and Langdon (2011),
Nakamura et al. (2011), Chua et al. (in press)]; the effects
of high pCO
2
become more consistently negative once
settlement and metamorphosis have occurred (Suwa et al.
2010; de Putron et al. 2011; Moya et al. 2012).
A summary of the known effects of high pCO
2
on early
life stages of corals, from gametes to recruits, underscores
the diverse of responses that have been reported. For
instance, prior to fertilization, low pH (7.8) impedes sperm
mobility in Acropora digitifera (Morita et al. 2009), and
high pCO
2
(673 and 998 latm) reduces fertilization suc-
cess in A. palmata, particularly at low sperm concentration
(Albright et al. 2010). Following development to a swim-
ming larval stage, high pCO
2
(560 and 800 latm) reduces
respiration 27–63 % in larvae of Porites astreoides
(Albright and Langdon 2011), yet the effects of high pCO
2
(331–397, 1,172–1,683, and 2,011–3,100 latm) on the
respiration of A. digitifera larvae were not statistically
discernable (Nakamura et al. 2011). Nakamura et al. (2011)
and Chua et al. (in press) were also unable to detect effects
of high pCO
2
(up to 3,100 latm, Nakamura et al. 2011)on
the survival of Acropora sp. larvae, and more recently
Nakamura et al. (2012) found that heat shock protein
expression in A. digitifera larvae were unaffected by
926 latm pCO
2
. Anlauf et al. (2011) reported the per-
centage of P. panamensis larvae swimming or dead was
unaffected by 861 latm pCO
2
.
Equivocal effects of CO
2
on coral larvae have also been
reported at settlement, with high pCO
2
(560 and 800 latm)
reducing settlement 42–60 % in P. astreoides (Albright
and Langdon 2011), and 45–69 % in Acropora palmata
(Albright et al. 2010), and low pH (7.3) reducing larval
metamorphosis by 15 % after 2 h exposure, and by 89 %
after 7 days exposure in A. digitifera (Nakamura et al.
2011). In contrast, 560 and 800 latm pCO
2
had no effect
on settlement of P. astreoides in an earlier experiment by
Albright et al. (2008), and high pCO
2
(861 latm) had no
statistically discernable effects on the settlement of
P. panamensis (Anlauf et al. 2011). Further, high pCO
2
(917 latm) had no effect on metamorphosis of A. mille-
pora and A. hyacinthus larvae (Chua et al. in press).
Albright and Langdon (2011) and Albright et al. (2010)
suggest the effects of pCO
2
on coral larval settlement can
be mediated indirectly through the impacts of pCO
2
on the
flora associated with settlement surfaces and, therefore,
trials at high pCO
2
will generate dissimilar results
depending on whether the settlement surfaces have been
conditioned at ambient or elevated pCO
2
(see also Webster
et al. 2012). Albright and Langdon (2011) reported that
limestone tiles conditioned at high pCO
2
(560 or 800 latm
Mar Biol
123

pCO
2
) were coated in flora having distinctive profiles of
photosynthetic accessory pigments compared to tiles con-
ditioned at 380 latm and, therefore, it is reasonable to infer
that they present settlement cues to coral larvae that indi-
rectly affect their settlement at high pCO
2
(Albright and
Langdon 2011).
Once coral larvae have settled and metamorphosed into
recruits, however, the effects of high pCO
2
appear to be
more consistently negative. For instance, growth (increase
in planar area) declined 16–35 % in P. astreoides and
39–50 % in A. palmata when exposed to 560 and 800 latm
pCO
2
(Albright et al. 2010; Albright and Langdon 2011),
and growth of newly settled Favia fragum, and P. astreo-
ides was depressed at low X
Arag
(i.e., \2.5) (Cohen et al.
2009; de Putron et al. 2011). Most recently, Moya et al.
(2012) demonstrated that high pCO
2
(1,000 and 750 vs.
380 ppm) affected gene expression in newly recruited
A. millepora, specifically suppressing metabolism but
enhancing the synthesis of organic matrix. In the only study
that has simultaneously explored the impacts of tempera-
ture and pCO
2
on coral recruits, Anlauf et al. (2011) found
that calcification and biomass in P. panamensis were both
reduced 28 % at 29.5 °C and a pH of 7.83.
Using the aforementioned studies as a context, the
present results are consistent with the notion that the effects
of pCO
2
on coral larvae can be equivocal (Chua et al. in
press), and that brooded coral larvae differ strikingly among
days of release. Our results convey dissimilar outcomes
depending on the lunar day from which the larvae were
harvested. This large variation in larval performance will
likely have implications for the response of P. damicornis
populations to future environmental conditions. While it
was beyond the scope of this study to explicitly evaluate the
implications of strong temporal variation in larval perfor-
mance, particularly with regard to how it affects recruitment
success and the contribution to population growth, it is
interesting to note that this variation in response crosses
gradient of unequal larval production among different LDs
(Fig. 3). In southern Taiwan, 31 % of larvae released from
P. damicornis over 4 years were released on LD9, which
may correspond to larvae with specific phenotypes. Eluci-
dating the phenotype of these larvae from our data is
problematic, as larval release does not necessarily corre-
spond perfectly with the same lunar days in different years
(Fan et al. 2002). Nevertheless, it is intriguing to note the
correspondence of the historical peak larval release with
LD9, which in the present study yielded larvae with rela-
tively unusual phenotypes; the respiration of LD9 larvae
was remarkably sensitive to temperatures in 2010, and
subject to strong depression at high pCO
2
, and LD9 larvae
displayed low mortality at high pCO
2
in 2010 and 2012.
Such phenotypic variation has strong potential benefits for
larval success—for example, by controlling catabolism of
energy reserves and modulating pelagic larval duration—
and it may be productive in the future research to test for
TPG-mediated fitness consequences to the release of vary-
ing numbers of phenotypically diverse larvae by reef corals.
Acknowledgments This research was funded through the US
National Science Foundation (BIO-OCE 08-44785) and contributes to
the collaboration supported under the Memorandum of Understanding
between California State University, Northridge, USA and National
Dong Hwa University, Hualien, Taiwan. We thank A. Dufault, H.M.
Putnam, S. Zamudio, A. Mayfield, P.J. Liu, Y.H Chen and O. Chan
for field and laboratory assistance. Comments from four anonymous
reviewers and H. Po
¨rtner improved an earlier draft of this paper. This
is contribution number 194 of the CSUN Marine Biology Program.
References
Agostini S, Suzuki Y, Higuchi T, Casereto BE, Yoshinaga K, Nakano
Y, Fujimura H (2012) Biological and chemical characteristics of
the coral gastric cavity. Coral Reefs 31:147–156
Agrawal AA, LaForsch C, Tollrian R (1999) Transgenerational
induction of defenses in animals and plants. Nature 401:60–63
Albright R (2011) Reviewing the effects of ocean acidification on
sexual reproduction and early life history stages of reef-building
corals. J Mar Biol. doi:10.1155/2011/473615
Albright R, Langdon C (2011) Ocean acidification impacts multiple
early life history processes of the Caribbean coral Porites
astreoides. Glob Change Biol 17:2478–2487
Albright R, Mason B, Langdon C (2008) Effect of aragonite
saturation state on settlement and post-settlement growth of
Porites astreoides larvae. Coral Reefs 27:485–900
0
1000
2000
3000
4000
5000
6000
7000
8000
1 8 15 22 29
No. larvae released
Lunar March
Lunar day
2012 sampling
2010 sampling
Fig. 3 Compilation of data describing larval release from P. dami-
cornis during lunar March of 2003, 2005, 2007, and 2008 in southern
Taiwan. Data are from 8 colonies in 2003 and 2005, and 4 in 2007 and
2008 and describe the summed larval release over 4 years; time is
expressed in lunar days beginning with the preceding new moon (LD
1). Days from which larvae were harvested for this study in 2010 and
2012 indicated (gray bars), with sampling days marked with a
horizontal line
Mar Biol
123

Albright R, Mason B, Miller M, Langdon C (2010) Ocean acidifi-
cation compromises recruitment success of the threatened
Caribbean coral Acropora palmata. Proc Natl Acad Sci USA
107:20400–20404
Anlauf H, D’Croz L, O’Dea A (2011) A corrosive concoction: the
combined effects of ocean warming and acidification on the
early growth of a stony coral are multiplicative. J Exp Mar Biol
Ecol 397:13–20
Baird AH, Gilmour JP, Kamiki TM, Nonaka M, Pratchett MS,
Yamamoto H, Yamasaki H (2006) Temperature tolerance of
symbiotic and non-symbiotic coral larvae. Proceedings of the
10th international coral reef symposium, pp 38–42
Ball EE, Hayward DC, Reece-Hoyes JS, Hislop NR, Samuel G, Saint
R, Harrison PL, Miller DJ (2002) Coral development: from
classical embryology to molecular control. Int J Dev Biol
46:671–678
Bassim KM, Sammarco PW (2003) Effects of temperature and
ammonium on larval development and survivorship in a
scleractinian coral (Diploria strigosa). Mar Biol 142:241–252
Bassim KM, Sammarco PW, Snell TL (2002) Effects of temperature
on success of (self and non-self) fertilization and embryogenesis
in Diploria strigosa (Cnidaria, Scleractinia). Mar Biol
140:479–488
Bhagooli R, Hidaka M (2003) Comparison of stress susceptibility of
in hospite and isolated zooxanthellae among five coral species.
J Exp Mar Biol Ecol 291:181–197
Black KP (1993) The relative importance of local retention and inter-
reef dispersal of neutrally buoyant material on coral reefs. Coral
Reefs 12:43–53
Byrne M (2011) Impact of ocean warming and ocean acidification on
marine invertebrate life history stages: vulnerabilities and
potential for persistence in a changing ocean. Oceanogr Mar
Biol Annu Rev 49:1–42
Chua MC, Leggat B, Moya A, Baird AH (in press) Near-future
reductions in pH will have no consistent ecological effects on the
early life history stages of reef corals. Mar Ecol Prog Ser. doi:
10.3354/meps10318
Cohen AL, McCorkle DC, de Putron S, Gaetani GA, Rose KA (2009)
Morphological and compositional changes in the skeletons of
new coral recruits reared in acidified seawater: insights into the
biomineralization response to ocean acidification. Geochem
Geophys Geosyst 10:Q07005. doi:10.1029/2009GC002411
Cowan RK, Sponaugle S (2009) Larval dispersal and marine
population connectivity. Ann Rev Mar Sci 1:443–466
Cumbo VR, Fan TY, Edmunds PJ (2012a) Effects of exposure
duration on the response of Pocillopora damicornis larvae to
elevated temperature and high pCO
2
. J Exp Mar Biol Ecol 439:
100–107
Cumbo VR, Fan TY, Edmunds PJ (2012b) Physiological development
of brooded larvae from two pocilloporid corals in Taiwan. Mar
Biol. doi:10.1007/s00227-012-2046-y
de Putron SJ, McCorkle DC, Cohen AL, Dillon AB (2011) The
impact of seawater saturation state and bicarbonate ion concen-
tration on calcification by new recruits of two Atlantic corals.
Coral Reefs 30:321–328
Detlef PV, Edmonds J, Kainuma M, Riahi K, Thomason A, Hibbard
K, Hurtt GC, Kram T, Krey V, Lamarque J-F, Masui T,
Meinshausen M, Nakicenovic N, Smith SJ, Rose SK (2011) The
representative concentration pathways: an overview. Clim
Change 109:5–31
Dickson AG, Sabine CL, Christian JR (eds) (2007) Guide to best
practices for ocean CO
2
measurements. PICES Special Publica-
tion 3, p 191
Dufault AM, Cumbo VR, Fan TY, Edmunds PJ (2012) Effects of
diurnally oscillating pCO2 on calcification and survival of coral
recruits. Proc R Soc Lond B. doi:10.1098/rspb.2011.2545
Edmunds PJ, Davies PS (1988) Post-illumination stimulation of the
respiration rate in the coral Porites porites. Coral Reefs 7:7–9
Edmunds PJ, Gates RD, Gleason DF (2001) The biology of larvae
from the reef coral Porites astreoides, and their response to
temperature disturbances. Mar Biol 139:981–989
Edmunds PJ, Gates RD, Leggat W, Hoegh-Guldberg O, Allen-Requa
L (2005) The effect of temperature on the size and population
density of dinoflagellates in larvae on the reef coral Porites
astreoides. Invert Biol 124:185–193
Edmunds PJ, Cumbo VR, Fan TY (2011) Effects of temperature on
the respiration of brooded larvae from tropical reef corals. J Exp
Biol 214:2783–2790
Enriquez S, Mendez ER, Iglesias-Prieto R (2005) Multiple scattering
on coral skeletons enhances light absorption by symbiotic algae.
Limnol Oceanogr 50:1025–1032
Fan TY, Li JJ, Ie SX, Fang LS (2002) Lunar periodicity of larval
release by pocilloporid corals in southern Taiwan. Zool Stud
41:288–294
Fan TY, Lin KH, Kuo FW, Soong K, Liu LL, Fang LS (2006) Diel
patterns of larval release by five brooding scleractinian corals.
Mar Ecol Prog Ser 321:133–142
Fangue NA, O’Donnell MJ, Sewell MA, Matson PG, MacPherson
AC, Hoffman GE (2010) A laboratory-based, experimental
system for the study of ocean acidification effects on marine
invertebrate larvae. Limnol Oceanogr Methods 8:441–452
Gaither MR, Rowan R (2010) Zooxanthellar symbiosis in planula
larvae of the coral Pocillopora damicornis. J Exp Mar Biol Ecol
386:45–53
Garcia HE, Gordon LI (1992) Solubility in seawater: better fitting
solutions. Limnol Oceanogr 37:1307–1312
Gleason DF, Hofmann DK (2011) Coral larvae: from gametes to
recruits. J Exp Mar Biol Ecol 408:42–57
Gleason DF, Edmunds PJ, Gates RD (2006) Ultraviolet radiation
effects on the behavior and recruitment of larvae from the reef
coral Porites astreoides. Mar Biol 148:503–512
Gleason DF, Danilowicz BS, Nolan CJ (2009) Reef waters stimulate
substratum exploration in planulae from brooding Caribbean
corals. Coral Reefs 28:549–554
Gosselin LA, Qian PY (1997) Juvenile mortality in benthic marine
invertebrates. Mar Ecol Prog Ser 46:265–282
Hadfield MG (2000) Why and how marine-invertebrate larvae
metamorphose so fast. Cell Dev Biol 11:437–443
Hamdoun A, Epel D (2007) Embryo stability and vulnerability in an
always changing world. Proc Natl Acad Sci USA
104:1745–1750
Harland AD, Davies PS (1995) Symbiont photosynthesis increases
both respiration and photosynthesis in the symbiotic sea
anemone Anemonia viridis. Mar Biol 123:715–722
Harrison PL (2011) Sexual reproduction of scleractinian corals. In:
Dubinsky Z, Stambler N (eds) Coral reefs: an ecosystem in
transition. Springer, Netherlands, pp 59–85
Harrison PL, Wallace CC (1990) Reproduction, dispersal and
recruitment of scleractinian corals. In: Dubinsky Z (ed) Ecosys-
tems of the world, vol 25., Coral reefsElsevier, Amsterdam,
pp 133–207
Herndl GJ, Velimirov B (1985) Bacteria in the coelenterons of
Anthozoa: control of coelenteric bacterial density by the
coelenteric fluid. J Exp Mar Biol Ecol 93:115–130
Heyward AJ, Negri AP (2010) Plasticity of larval pre-competency in
response to temperature: observations on multiple broadcast
spawning coral species. Coral Reefs 29:631–636
Hoegh-Guldberg O (1999) Climate change, coral bleaching and the
future of the world’s coral reefs. Mar Fresh Res 50:839–866
Hoegh-Guldberg O, Mumby PJ, Hooten AJ, Steneck RS, Greenfield
P, Gomez E, Harvell CD, Sale PF, Edwards AJ, Caldeira K,
Knowlton N, Eakin CM, Iglesias-Prieto R, Muthuga N, Bradbury
Mar Biol
123

RH, Dubi A, Hatziolos ME (2007) Coral reefs under rapid
climate change and ocean acidification. Science 318:1737–1742
Hofmann GE, Barry JP, Edmunds PJ, Gates RD, Hutchins DA,
Klinger T, Sewell MA (2010) The effect of ocean acidification
on calcifying organisms in marine ecosystems: an organism-to-
ecosystem perspective. Annu Rev Evol Syst 41:127–147
IPCC (2007) Climate change 2007: synthesis report. Contribution of
Working Groups I, II and III to the Fourth Assessment Report of the
Intergovernmental Panel on Climate Change [Core Writing Team,
Pachauri RK, Reisinger A (eds)]. IPCC, Geneva, Switzerland, 104 pp
Isomura N, Nishihara M (2001) Size variation of planulae and its
effect on the lifetime of planulae in three pocilloporid corals.
Coral Reefs 20:309–315
Jones RJ, Hoegh-Guldberg O, Larkum AWD, Schreiber U (1998)
Temperature-induced bleaching of corals begins with impair-
ment of the CO
2
fixation mechanism in zooxanthellae. Plant,
Cell Environ 21:1219–1230
Kleypas JA, Buddemeier RW, Archer D, Gattuso JP, Langdon C,
Opdyke BN (1999) Goechemical consequences of increased
atmospheric carbon dioxide on coral reefs. Science 284:118–120
Kroeker KJ, Kordas RL, Crim RN, Singh GG (2010) Meta-analysis
reveals negative yet variable effects of ocean acidification on
marine organisms. Ecol Lett 13:1419–1434
Lee HJ, Chao SY, Fan KL, Kuo TY (1999) Tide-induced eddies and
upwelling in a semi-enclosed basin: Nan Wan. Estuar Coast
Shelf Sci 49:775–787
Levitan DR (1996) Predicting optimal and unique egg sizes in free-
spawning marine invertebrates. Am Nat 148:174–188
Markey KL, Baird AH, Humphrey C, Negri AP (2007) Insecticides
and fungicide affect multiple coral life stages. Mar Ecol Prog Ser
330:127–137
Marlow HQ, Martindale MQ (2007) Embryonic development in two
species of scleractinian coral embryos: Symbiodinium localiza-
tion and mode of gastrulation. Evol Dev 9:355–367
Marshall DJ (2008) Transgenerational plasticity in the sea: context-
dependent maternal effects across the life history. Ecology
89:418–427
Marshall DJ, Keough MJ (2003) Variation in the dispersal potential of
non-feeding invertebrate larvae: the desperate larva hypothesis
and larval size. Mar Ecol Prog Ser 255:145–153
Marubini F, Ferrier-Page
`s C, Furla P, Allemand D (2008) Coral
calcification responds to seawater acidification: a working
hypothesis towards a physiological mechanism. Coral Reefs
27:491–499
Mason B, Beard M, Miller MW (2011) Coral larvae settle at a higher
frequency on red surfaces. Coral Reefs. doi:10.1007/s00338-
011-0739-1
Morita M, Suwa R, Iguchi A, Nakamura M, Shimada K, Sakai K,
Suzuki A (2009) Ocean acidification reduces sperm flagellar
motility in broadcast spawning reef invertebrates. Zygote
18:103–107
Moya A, Huisman L, Ball EE, Hayward DC, Grasso LC, Chua CM,
Woo HN, Gattuso JP, Foret S, Miller DJ (2012) Whole
transcriptome analysis of the coral Acropora millepora reveals
complex responses to CO
2
-driven acidification during the
initiation of calcification. Mol Ecol 21:2440–2454
Mundy CN, Babcock RC (1998) Role of light intensity and spectral
quality in coral settlement: implications for depth-dependent
settlement? Mar Ecol Prog Ser 223:235–255
Nakamura M, Ohki S, Suzuki A, Sakai K (2011) Coral larvae under
ocean acidification: survival, metabolism and metamorphosis.
PLoS ONE 6:e14521
Nakamura M, Morita M, Kurihara H, Mitarai S (2012) Expression of
hsp70, hsp90 and hsf1 in the reef coral Acropora digitifera under
prospective acidified conditions over the next several decades.
Biol Open 1:75–81
Negri AP, Smity LD, Webster NS, Heyward AJ (2002) Understanding
ship-grounding impacts on a coral reef: potential effects of anti-
foulant paint contamination on coral recruitment. Mar Pollut
Bull 44:111–117
Negri AP, Marshall PA, Heyward AJ (2007) Differing effects of
thermal stress on coral fertilization and early embryogenesis in
four Indo Pacific species. Coral Reefs 26:759–763
Nozawa Y, Harrison PL (2007) Effects of elevated temperature on
larval settlement and post-settlement survival in scleractinian
corals, Acropora solitaryensis and Favites chinensis. Mar Biol
152:1181–1185
Nozawa Y, Okubo N (2011) Survival dynamics of reef coral larvae
with special consideration of larval size and the genus Acropora.
Biol Bull 220:15–22
O’Connor MI, Bruno JF, Gaines SD, Halpern BS, Lester SE, Kinlan
BP, Weiss JM (2007) Temperature control of larval dispersal and
the implications for marine ecology, evolution, and conservation.
Proc Natl Acad Sci USA 104:1266–1271
Orr JC (2011) Recent and future changes in ocean carbonate
chemistry. In: Gattuso J-P, Hansonn L (eds) Ocean acidification.
Oxford University Press, Oxford, pp 41–66
Permata WD, Kinzie RA, Hidakal M (2000) Histological studies on
the origin of planulae of the coral Pocillopora damicornis. Mar
Ecol Prog Ser 200:191–200
Pierrot D, Lewis E, Wallace DWR (2006) MS excel program developed
for CO
2
system calculations. ORNL/CDIAC-105a. Carbon Diox-
ide Information Analysis Center, Oak Ridge National Laboratory,
US Department of Energy, Oak Ridge, Tennessee
Putnam HM, Edmunds PJ, Fan TY (2008) Effects of temperature on
the settlement choice and photophysiology of larvae from the
reef coral Stylophora pistillata. Biol Bull 215:135–142
Putnam HM, Edmunds PJ, Fan TY (2010) Effect of a fluctuating thermal
regime on adult and larval reef corals. Invert Biol 129:199–209
Quinn GP, Keough MJ (2002) Experimental design and data analysis
for biologists. Cambridge University Press, Cambridge
Raimondi PT, Morse ANC (2000) The consequence of complex larval
behavior in a coral. Ecology 81:3193–3211
Randall CJ, Szmant AM (2009) Elevated temperature reduces
survivorship and settlement of the larvae of the Caribbean
scleractinian coral, Favia fragum (Esper). Coral Reefs 28:537–
545
Rivest E, Hofmann G (2012) Metabolic plasticity in coral larvae
under ocean acidification and warming. Proceedings of the 12th
international coral reef symposium (abstracts) 158
Rodriguez-Lanetty M, Harii S, Hoegh-Guldberg O (2009) Early
molecular responses of coral larvae to hyperthermal stress. Mol
Ecol 18:1501–5114
Sabine CL, Feely RA, Gruber N, Key RM, Lee K, Bullister JL,
Wanninkhof R, Wong CS, Wallace DWR, Tilbrook B, Millero
FJ, Peng TH, Kozyr A, Ono T, Rios AF (2004) The ocean sink
for anthropogenic CO
2
. Science 305:367–371
Salih A, Larkum A, Cox G, Ku
¨hl M, Hoegh-Guldberg O (2000)
Fluorescent pigments in corals are photoprotective. Nature
408:850–853
Salinas S, Munch SB (2012) Thermal legacies: transgenerational
effects of temperature on growth in a vertebrate. Ecol Lett
15:159–163
Silverman J, Lazar B, Cao L, Caldeira K, Erez J (2009) Coral reefs
may start dissolving when atmospheric CO
2
doubles. Geophys
Res Lett 36:L05606
Stambler N (2011) Zooxanthellae: the yellow symbionts inside
animals. In: Dubinsky Z, Stambler N (eds) Coral reefs: an
ecosystem in transition. Springer, Netherlands, pp 87–106
Stoddart JA, Black R (1985) Cycles of gametogenesis and planulation
in the coral Pocillopora damicornis. Mar Ecol Prog Ser 23:
153–164
Mar Biol
123

Strathmann RR (1985) Feeding and nonfeeding larval development
and life-history evolution in marine invertebrates. Annu Rev
Ecol Syst 16:339–361
Suwa R, Nakamur M, Morita M, Shimada K, Iguchi A, Sakai K,
Suzuki A (2010) Effects of acidified seawater on early life stages
of scleractinian corals (Genus Acropora). Fish Sci 76:93–99
Webster NS, Uthicke S, Botte ES, Flores F, Negri A (2012) Ocean
acidification reduces induction of coral settlement by crustose
coralline algae. Glob Change Biol. doi:10.1111/gcb.12008
Yakovleva IM, Baird AH, Yamamoto HH, Bhagooli R, Nonaka M,
Hidaka M (2009) Algal symbionts increase oxidative damage
and death in coral larvae at high temperatures. Mar Ecol Prog
Ser 378:105–112
Yeoh S-R, Dai C-F (2010) The reproduction of sexual and asexual
larvae within single broods of the scleractinian coral, Pocillo-
pora damicornis. Mar Biol 157:351–359
Zar JH (2010) Biostatistical analysis. Prentice Hall, Englewood
Cliffs, NJ
Mar Biol
123