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CO2 enrichment and reduced seawater pH had no
effect on the embryonic development of Acropora
palmata (Anthozoa, Scleractinia).
Pedro Medina-Rosas
a b
a
, Alina M. Szmant & Robert F. Whitehead
a
a
Center for Marine Science, University of North Carolina Wilmington, 5600 Marvin K Moss
Lane, Wilmington, NC 28409, USA
b
Centro Universitario de la Costa, Universidad de Guadalajara, Av U de G 203, Puerto
Vallarta, Jalisco 48280, Mexico
Version of record first published: 03 Jul 2012.
To cite this article: Pedro Medina-Rosas , Alina M. Szmant & Robert F. Whitehead (2013): CO2 enrichment and reduced
seawater pH had no effect on the embryonic development of Acropora palmata (Anthozoa, Scleractinia)., Invertebrate
Reproduction & Development, 57:2, 132-141
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Invertebrate Reproduction & Development, 2013
Vol. 57, No. 2, 132–141, http://dx.doi.org/10.1080/07924259.2012.704407
CO2 enrichment and reduced seawater pH had no effect on the embryonic development
of Acropora palmata (Anthozoa, Scleractinia).
Pedro Medina-Rosasab*, Alina M. Szmanta and Robert F. Whiteheada
a
Center for Marine Science, University of North Carolina Wilmington, 5600 Marvin K Moss Lane,
Wilmington, NC 28409, USA; bCentro Universitario de la Costa, Universidad de Guadalajara,
Av U de G 203, Puerto Vallarta, Jalisco 48280, Mexico
Downloaded by [Pedro Medina-Rosas] at 17:23 03 December 2012
(Received 7 October 2011; final version received 15 June 2012)
The effects of decreased pH, caused by carbon dioxide (CO2) dissolution in seawater (known as ocean
acidification (OA)), on the development of newly fertilized eggs of the Caribbean reef-building coral, Acropora
palmata, was tested in three experiments conducted during the summers of 2008 and 2009 (two repeats). Three
levels of CO2 enrichment were used: present day conditions (400 matm, pH 8.1) and two CO2-enriched conditions
(700 matm, pH 7.9, and 1000 matm, pH 7.7). No effects on the progression or timing of development, or embryo
and larval size, were detected in any of the three experimental runs. The results show that the embryos and larvae
of A. palmata are able to develop normally under seawater pH of at least 0.4 pH units lower than the present
levels. Acropora palmata larvae do not usually begin to calcify after settlement, so this study only examined the
non-calcifying part of the life cycle of this species. Most of the concern about the effects of OA on marine
organisms centers on its effect on calcification. Negative effects of OA on the embryonic development of this
species were not found and they may not manifest until the newly settled polyps begin to calcify.
Keywords: Acropora palmata; Caribbean reef coral; embryonic development; ocean acidification
Introduction
Seawater chemistry has been changing over the past
century in response to the absorption of anthropogenic
carbon dioxide (CO2) (IPCC 2007). The main
responses have been decrease in seawater pH, carbonate ion concentration, and aragonite saturation state.
Together, these seawater chemistry changes are
referred to as ocean acidification (OA). The OA has
been demonstrated to affect calcification rates of a
number of marine invertebrates with calcium carbonate skeletons, and also of some fishes (Hofmann et al.
2010). Less attention has been paid to the potential
effects of these seawater changes on the pre-calcifying
life stages of these taxa.
Most calcifying benthic invertebrates have embryonic and larval periods at the beginning of their life
cycle during which they do not secrete calcium
carbonate. This period usually lasts days to weeks.
Several of these taxa, such as mollusks and echinoderms, begin deposition of calcium carbonate during
the late planktonic stage, while in others, such as corals
and barnacles, calcification does not begin until the
larvae attach to their permanent substrate (Young
2002). Recent studies have demonstrated that growth
rates and survivorship of calcifying larvae of mollusks
*Corresponding author. Email: [email protected]
ß 2013 Taylor & Francis
and echinoderms are negatively affected by CO2
enrichment and reduced pH associated with OA
(Dupont et al. 2008; Kurihara 2008; Clark et al.
2009; Ellis et al. 2009; Byrne et al. 2011; Kimura et al.
2011). In fact, there is the potential that calcifying
marine larvae may be more susceptible to CO2
enrichment than are the sessile adults (Raven et al.
2005; Pörtner and Farrell 2008; Kurihara 2008). To
date, few studies have investigated whether the development and larval growth of marine invertebrate taxa,
that do not calcify until after they settle, are also
affected by such altered seawater chemistry.
Scleractinian corals are a prime example of an
ecologically important group of calcifying animals that
are sensitive to seawater acidification as adults
(Kleypas and Langdon 2006; Cohen and Holcomb
2009), which do not calcify before settlement. Most
corals are broadcast spawners (Harrison 2010).
Fertilization and development occur in the water
column where the non-calcifying embryos and larvae
live until reaching competency (2–8 days range), and
then settle into the benthos to begin their sessile
(calcifying) life. Aside from calcification, developmental processes that could be affected by OA include
fertilization, cell division rates, gastrulation, larval
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Invertebrate Reproduction & Development
metamorphosis, and settlement. Processes that affect
coral developmental success are important in determining the larval supply, and thus dispersal and
connectivity potential.
This study reports an investigation of the effects of
CO2 enrichment and reduced pH seawater on the
embryonic development of the Caribbean reef coral,
Acropora palmata (Lamarck 1816). This species, along
with its congener, A. cervicornis, was listed in 2006
as threatened under the U.S. Endangered Species
Act, and critically endangered by the International
Union for Conservation of Nature (IUCN). A number
of recent studies have reported the effects of OAassociated conditions on coral embryonic development
and early life stages. Albright et al. (2010) reported that
increased pCO2 levels decreased successful fertilization
and larval settlement of A. palmata, but embryonic
development was not assessed in that study. Studies on
other Acropora species found no effect on the embryonic development of A. tenuis at reduced pH conditions (pH 7.6; Kurihara 2008), or on survival and
oxygen consumption of A. digitifera larvae at pH
7.6–7.3 (Suwa et al. 2010; Nakamura et al. 2011).
However, the polyp size and zooxanthella infection
rates in A. digitifera decreased at reduced pH levels
(Suwa et al. 2010). In another study, low saturation
state conditions (ca pH 7.8) had no effect on the larval
development, metamorphosis, or settlement of Porites
astreoides, a Caribbean coral that broods its larvae
(Albright et al. 2008). In experiments using adult
colonies of Montipora capitata, there was no effect on
neither the number of gametes and gamete bundles
produced by colonies maintained at pH 7.2 compared
to colonies at pH 8.2, nor on the settlement of larvae
released by Pocillopora damicornis under the same
conditions (Jokiel et al. 2008).
In addition to OA, elevated atmospheric CO2 levels
are responsible for global warming and elevated
seawater temperatures that stress corals to the point
of causing a phenomenon known as ‘bleaching’ and
coral mass mortality during anomalously warm
summer months (Glynn 1996; IPCC 2007). In fact,
the devastating effects that elevated seawater temperatures have had on corals and coral reef health were
observed long before the potential effects of OA on
coral calcification became a concern. An earlier study
on the effects of elevated seawater temperatures on the
development of A. palmata, using temperature levels
similar to those that cause bleaching and mass mortality, accelerated the embryonic development rate,
caused increased percentages of abnormal embryos,
decreased survivorship, and reduced larval settlement
(Randall and Szmant 2009). That study served as a
partial model for examining the effects of elevated CO2
on the development of A. palmata. While it has been
observed that warming is deleterious to the development of this species (Randall and Szmant 2009), effects
133
of OA are not known. Newly fertilized eggs of
A. palmata were cultured to competency under three
CO2 levels, representing present day conditions
(400 matm, control, pH 8.1) and two CO2-enriched
conditions predicted to occur during this century
(700 matm, mid pH 7.9, and 1000 matm, low pH 7.7)
by the Intergovernmental Panel on Climate Change
(IPCC 2007), to determine whether the experimental
conditions altered the development. The experiment
was repeated thrice over 2 years, but no effect was
observed on the development.
Materials and methods
Background on Acropora palmata development
A. palmata broadcast spawns over a few nights after
the full moon of August and/or September (Szmant
1986). Embryonic and larval development of A.
palmata takes about 6 days (Randall and Szmant
2009), and follows a pattern similar to those previously
described for several Pacific acroporid species
(Hayashibara et al. 1997; Gilmour 1999; Ball et al.
2002; Okubo and Motokawa 2007). Briefly, eggs and
sperms are compressed into a bundle, with a single
bundle released by any given polyp during a spawning
event. Once bundles rise to the sea surface, they break
down and fertilization takes place within 1 h. Cleavage
is holoblastic, with irregular divisions until they reach
the morula stage. After 8 h, embryos flatten into an
irregular shape, known as the prawn chip, which is the
blastula in this species (Okubo and Motokawa 2007;
Pace and Szmant, unpublished). Gastrulation occurs
by the thickening and folding of the prawn chip into a
bowl-like shape. As gastrula formation proceeds, the
embryos become rounded and smooth with a small
blastopore. This takes ca 2 days after fertilization. The
gastrulae gradually take a pear shape and become
motile by ca 80–100 h after fertilization. By days 5 or 6,
the planulae become competent, and begin to settle on
the substrate, metamorphose into polyp, and secrete
their first CaCO3 skeleton. This information was used
to guide the sampling described below.
Spawn collection
Spawn from A. palmata colonies was collected at Tres
Palmas Reef, in Rincón, Puerto Rico, where this
species is particularly abundant and appears healthy.
Spawn was collected on three dates over two reproductive seasons: 19 August 2008, 10 August 2009, and
8 September 2009. Bundles were collected from at least
four to six different colonies using inverted conical nets
attached over the coral colonies; nets were equipped
with collecting bottles and floats at the cod-end.
Additional spawn was collected using plankton nets
towed by divers. While we do not know the exact
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134
P. Medina-Rosas et al.
number of genets represented in each spawn collection,
estimates are in the range of 5 to 412. The various
batches of spawn were mixed together and gently
agitated to break up bundles, and allowed to fertilize.
Excess sperm was washed away with fresh seawater
after 1 h. Sperm concentrations were not determined.
The embryos were then transported by car to the Isla
Magueyes, La Parguera, field laboratory of the
Department of Marine Science, University of Puerto
Rico, Mayaguez, a drive of ca 1.5 h. Within 3 h after
spawning, 6 mL of the spawn mix, containing an
estimated 12,000 developing embryos, were placed into
each of six 20-L JellyquariumÕ [Midwater Systems,
California]. Three sets of duplicate aquaria were
supplied with filtered flow-through seawater as follows: one set with ambient seawater (control treatment
ca pH 8.1), one set with seawater bubbled with CO2 to
produce a pH of 7.9 (moderate treatment), and one set
seawater bubbled with CO2 to produce a pH of 7.7
(low treatment). The JellyquariumÕ aquaria were
selected for culturing the embryos because they are
designed to provide a gentle circular water movement,
by the continuous introduction of fresh seawater
through a flow-bar that keeps the embryos in suspension. The flow-through design reduces the amount of
culture maintenance because the metabolic and embryonic waste is continuously flushed from the cultures
reducing the frequency of more extensive cleaning and
handling of embryos to every other day. Flow rates
were adjusted to 20 L/h, for a turnover rate of once per
hour. A high turnover rate was important to maintain
the desired experimental CO2/pH conditions within
the tanks.
Experimental seawater physicochemical conditions
The seawater supply on Isla Magueyes is pumped from
the shallow reef fringe on the west side of the island.
The intake is located in a shallow well, and the water
flows into a small concrete holding tank from where it is
pumped throughout the island. Residence time in this
tank is estimated to be a few hours at most. Water was
filtered through a sand filter followed by 20 and 0.5 mm
membrane filters. The filtered seawater was then split
into three lines: one ambient control and two CO2treated controls. Pure CO2 gas was injected into two of
the water streams using clear PVC standpipes to control
water flow and variable area gas flow meters to control
CO2 flow. CO2 was injected at the bottom of the vertical
standpipes and the cross-flow configuration of downflowing water and up-flowing gas ensured complete
dissolution of CO2 before the water exited through an
in-line static mixer into the aquaria. Gas flow rates were
adjusted to produce seawater of ca pH 7.9 (calculated
CO2 of 600 matm) and ca pH 7.7 (calculated CO2 of
1000 matm), levels of CO2 enrichment, and pH expected
to occur in the near future (Gattuso et al. 2011). The
control treatment used the same type of cross-flow set
up, but was bubbled only with ambient air and had a pH
of ca 8.1 (calculated CO2 of 400 matm). The pH and total
alkalinity (TA) values of the incoming water were not
altered by these treatments, and varied in response to
ambient conditions on the reef. For comparison, pCO2
concentrations in the surface waters measured on Cayo
Enrique reef, in front of La Parguera, ranged from 350
to 500 matm during 2009 (Gledhill et al. 2011).
Water samples to determine pHTotal were collected
thrice a day, and analyzed within 10 min of collection
using the m-cresol purple indicator method and an
Ocean Optics spectrophotometer according to Clayton
and Byrne (1993). The precision of the method was
ca 0.0004 pH units. Throughout the experiments, pH
was also measured every few hours with an Orion pH
meter equipped with a Ross Ultra electrode and
calibrated daily with NBS buffers. pHNBS measurements were 0.12 0.01 higher than pHTotal. Meter
measurements were only used as a rapid way to
monitor the consistency of the treatment systems; the
spectrophotometric pH values were always used for the
chemical calculations described below.
Water samples to determine TA were collected twice
daily, and analyzed within 24 h of sampling. TA was
analyzed using the method of Yao and Byrne (1998),
with 0.1 N HCl standardized against calibrated seawater (Standard batch 81; A. Dickson, Scripps Institute of
Oceanography, La Jolla, CA). Replicate seawater
samples of ca 30 g were weighed to 1 mg, and then
sufficient acid to reduce the pH to 4.0 was added and
the sample was re-weighed. The acidified sample was
aerated vigorously for 5 min to drive-off excess CO2
before adding bromocresol green indicator. The absorbance of the sample was read using an Ocean Optics
spectrophotometer. This method yields a precision of ca
2 mmol kg1 in replicate seawater samples.
Seawater chemical parameters, including carbonate, bicarbonate and carbon dioxide concentrations,
and aragonite saturation state, were calculated with the
program CO2SYS (Lewis and Wallace 1998), using the
measured values of TA, pHTotal, salinity, and temperature. Salinity was determined with a hand refractometer readable to 0.5 units, and temperature was
measured using a HOBO Pro V2 data logger programmed to record temperature every 10 min on each
tank. Pressure and nutrient concentration effects were
assumed to be negligible. The seawater physicochemical conditions of each of the three experiments are
summarized in Table 1.
Quantification of embryonic development patterns
The starting time for the three replicate experiments
was set at midnight of the night of spawn collection.
Control
Mid pH
Low pH
Control
Mid pH
Low pH
Control
Mid pH
Low pH
20–24 August 2008
35 1
35 1
35 1
35 1
35 1
35 1
35 1
35 1
35 1
S
28.6 0.1
29.0 0.1
29.3 0.1
29.3 0.6
29.5 0.6
29.5 0.6
29.7 0.3
30.1 0.3
30.1 0.3
T
( C)
2285 8
2284 9
2289 10
2285 12
2276 12
2305 24
2260 14
2268 13
2271 7
TA
(mmol/kg)
1940 20
2069 22
2159 26
1981 12
2069 12
2177 13
1952 18
2061 11
2168 12
TCO2
(mmol/kg)
8.07 0.04
7.83 0.04
7.64 0.06
7.99 0.03
7.81 0.04
7.63 0.03
8.00 0.03
7.80 0.02
7.56 0.03
pHT
368 36
711 80
1199 193
456 35
752 80
1223 82
443 32
757 37
1445 107
pCO2
(matm)
1678 34
1890 31
2018 33
1752 21
1894 22
2036 11
1721 24
1866 12
2037 14
HCO3
(mmol/kg)
243 15
161 12
111 13
217 12
156 13
110 9
219 9
156 5
96 5
CO3
(mmol/kg)
10 1
18 2
31 5
12 1
19 2
31 2
11 1
19 1
36 3
CO2
(mmol/kg)
3.93 0.3
2.60 0.2
1.80 0.2
3.51 0.2
2.53 0.2
1.79 0.1
3.56 0.1
2.54 0.1
1.56 0.1
arag
Notes: Salinity (S), temperature (T), pH and TA were measured as described in the methods. The remaining values were calculated using CO2SYS (Lewis and Wallace 1998).
Measurements of TA and pHTotal are means of replicates collected thrice a day during five days of experiments. Temperatures are the means of daily means of measurements taken
every 10 min by logging thermographs.
9–13 September 2009
11–15 August 2009
Treatment
Experiment
Table 1. Mean (SD) seawater physicochemical values during the three experiments in which embryos of A. palmata were cultured from 2 h post-fertilization to larval settlement
under high CO2/reduced pH conditions.
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Invertebrate Reproduction & Development
135
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136
P. Medina-Rosas et al.
Samples of ca 100 embryos were taken at the time the
newly fertilized and developing embryos were introduced into the Jellyquaria (ca 3 h after spawning), and
at eight additional times over the following 5 days of
development. For specific times in each experiment, see
Table 3 (one sample per Jellyquarium, two samples per
treatment). Samples were fixed in 2% glutaraldehyde
in 0.05 M sodium cacodylate buffer at pH 7.5, and
stored in the refrigerator until examined under the
microscope.
To identify stages and quantify the developmental
progression of embryos, each sample was examined
under a dissecting microscope at magnification up to
90. The number of embryos at each developmental
stage, as well as the percentage of abnormally shaped
embryos (based on descriptions and photographs in
Randall and Szmant (2009)) were counted and the
respective percentages were calculated.
Other metrics that were used to determine whether
the CO2/pH treatments had an effect on the development of A. palmata were the diameters and volumes of
gastrula and planula stages, respectively. Gastrulae
were treated as spheres and planulae as oblongs for
these analyses. The measurements were made by taking
photographs of the embryos and using Image J to
measure the length and width of an average of 80
individuals per sample (range of 54–98). The means of
two diameter measurements per embryo (in mm) were
used to estimate the diameters of the gastrulae;
planulae volumes, in mm3, were calculated by measuring the length and width of the larvae and using the
formula of an oblate spheroid, 4/3ab2, where a equals
half of the length and b is half of the width.
Data analysis
Statistical comparisons of embryonic development
among CO2/pH treatments were conducted by chisquared statistic (Zar 1999) on the percentages of
developmental stages at individual time points.
Percentage data were arcsine transformed to produce
data sets with acceptable normality and equality of
variance. The distribution of embryos among stages at
pH 8.1 (control treatment) was designated as the
expected distribution, and the distribution at the two
CO2/pH treatments were compared against it.
Differences were considered significant at p 5 0.05.
The embryo and planula size data were analyzed by
Kruskal–Wallis one-way analysis of variance
(ANOVA) to compare gastrula diameter and planulae
volume among pH treatments at a selected time point
for each stage within each of the three experiments
(Zar 1999). Comparisons of the three experimental
runs (August 2008, August 2009, and September 2009)
for each of the pH treatments individually were made
using the same one-way ANOVA.
Results
Time course of embryonic development
Microscopic analysis of the nine sets of samples per
experimental run showed that the development of the
embryo of A. palmata in the present experiments
(Table 2) followed the same basic time-course as
previously reported for control samples by Randall
and Szmant (2009). Specifically, embryos in the 3 h
samples were in the early cleavage stages (before
morula); at 8 h, embryos were in the prawn chip/
blastula stage; samples collected at 20, 32, 44 and 60 h
after fertilization were in various stages of gastrulation;
by 80 h, embryos had reached the early planula stage;
and samples collected at 90 and 100 þ h after fertilization consisted of fully formed planulae. The number of
embryos counted in the samples for each run varied
slightly and averaged: August 2008, 97 individuals
(25.8
Standard
Deviation
(SD);
min ¼ 50;
max ¼ 152); August 2009, 139 individuals (34.6 SD;
min ¼ 90; max ¼ 253); and September 2009, 112 individuals (31.03 SD; min ¼ 53; max ¼ 181). Chisquared statistical analysis of the percentages of the
various embryonic stages present at any given time
during the development showed that there was no
difference in the progression of development under the
three CO2/pH treatments (Table 3).
Randall and Szmant (2009) found that, especially
during gastrulation, many embryos failed to progress
through development normally, taking on irregular
shapes, and that the percentage of embryos failing to
develop normally increased with increasing temperature. Thus, the percentage of abnormal embryos in a
culture can be used as an indicator of a variable
negatively affecting the development. The percentage
of abnormal embryos or larvae were low, generally
510% (lower than in Randall and Szmant (2009))
(Table 4), and again there was no significant difference
in the percentage of abnormal embryos among treatments and experiments.
Gastrula and planula dimensions
The diameter of newly fertilized A. palmata eggs is in
the range of 600–630 mm (Szmant, unpublished).
Gastrulae in the present experiments ranged from
610–630 mm (Figure 1a). Within each treatment, there
was no significant difference in the gastrula diameter
among the embryos used in the three experimental runs
(Chi-squared analysis; control, H2 ¼ 4.571, p ¼ 0.067;
mid pH, H2 ¼ 0.857, p ¼ 0.800; low pH, H2 ¼ 2.000,
p ¼ 0.533). Further, within runs, no significant differences among CO2/pH treatments were found for the
diameters of A. palmata gastrulae in the three pH
experiments (August 2008, H2 ¼ 1.143, p ¼ 0.667;
August 2009, H2 ¼ 3.714, p ¼ 0.200; September 2009,
H2 ¼ 2.000, p ¼ 0.533).
Invertebrate Reproduction & Development
137
Table 2. Percentages of A. palmata embryos in each of the seven development stages in samples of larval cultures grown at the
three nominal seawater CO2/pH treatments: pH 8.1 (control), mid pH 7.9, and low pH 7.7.
Experiment
August 2008
August 2009
September 2009
pH treatment
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Sample
haf
Control
Mid
Low
Control
Mid
Low
Control
Mid
Low
1
2
3
8
3
20
(A) 100
(A) 44.6
(B) 55.4
(D) 100
(A) 100
(A) 39.3
(B) 60.7
(D) 100
(A) 100
(A) 32.9
(B) 67.1
(D) 100
4
5
6
32
45
60
7
80
8
90
(D) 100
(D) 100
(E) 90.1
(F) 9.9
(D) 11.1
(E) 8.5
(F) 80.4
(E) 4.4
(F) 95.6
(F) 92.1
(G) 7.9
(A) 100
(A) 10.6
(B) 89.4
(C) 82.0
(D) 18.0
(D) 100
(D) 100
(E) 89.5
(F) 10.5
(D) 7.1
(E) 0.0
(F) 92.9
(F) 100
(A) 100
(A) 16.3
(B) 83.7
(C) 86.5
(D) 13.5
(D) 100
(D) 100
(E) 70.6
(F) 29.4
(D) 11.5
(E) 22.1
(F) 66.4
(F) 100
(A) 100
(A) 24.2
(B) 75.8
(C) 87.1
(D) 12.9
(D) 100
(D) 100
(E) 77.5
(F) 22.5
(D) 5.1
(E) 11.1
(F) 83.8
(F) 100
(A) 100
(A) 25.9
(B) 74.1
(C) 81.0
(D) 19.0
(D) 100
(D) 100
(E) 86.4
(F) 13.6
(D) 5.4
(E) 14.1
(F) 80.5
(F) 100
104
(D) 100
(D) 100
(E) 91.5
(F) 8.5
(D) 10.3
(E) 0.6
(F) 89.1
(E) 4.7
(F) 95.3
(F) 93.6
(G) 6.4
(A) 100
(A) 11.9
(B) 88.1
(C) 87.7
(D) 12.3
(D) 100
(D) 100
(E) 84.4
(F) 15.6
(D) 8.3
(E) 0.0
(F) 91.7
(F) 100
9
(D) 100
(D) 100
(E) 93.6
(F) 6.4
(D) 14.8
(E) 10.8
(F) 74.5
(E) 5.3
(F) 94.7
(F) 87.1
(G) 12.9
(A) 100
(A) 12.3
(B) 87.7
(C) 91.7
(D) 8.3
(D) 100
(D) 100
(E) 76.1
(F) 23.9
(D) 6.4
(E) 0.0
(F) 93.6
(F) 100
(F) 77.9
(G) 22.1
(F) 90.6
(G) 9.4
(F) 89.1
(G) 10.9
(F) 81.0
(G) 19.0
(F) 87.6
(G) 12.4
(F) 91.7
(G) 8.3
Notes: The experiment was repeated three times: August 2008, August 2009, and September 2009. Samples were collected at nine
intervals during development from 3 hours after fertilization to larval maturity (planula stage). For specific times, see Table 3.
Percentages were calculated using embryos exhibiting normal stages of development (i.e excluding irregular embryos which are
reported separately in Table 4). Stage designations: (A) Early cell division up to morula; (B) Prawn chip (blastula); (C) Midgastrulation; (D) Gastrula, (E) Early planula, (F) Late planula, (G) Pre-settlement planula. haf ¼ hours after fertilization. Values
are means of two replicate samples per treatment/time point (one per Jellyquarium).
Planula volumes ranged from 103 to 134 mm3
(Figure 1b) and again, no significant difference
was found among the three experimental runs
(Control, H2 ¼ 3.714, p ¼ 0.200; mid pH, H2 ¼ 0.200,
p ¼ 0.533; low pH, H2 ¼ 4.571, p ¼ 0.067) or among
CO2/pH treatments within each experiment
(August 2008, H2 ¼ 3.714, p ¼ 0.200; August 2009,
H2 ¼ 0.286, p ¼ 0.933; September 2009, H2 ¼ 2.571,
p ¼ 0.400).
Since there were no significant differences among
experiments, another ANOVA was run using all data
to increase replication, and again no significant was
found in gastrula (F ¼ 0.909, p ¼ 0.424) and planula
(F ¼ 1.007, p ¼ 0.389) dimensions.
Discussion
No significant effect of CO2 enrichment and reduced
seawater pH was detected on the timing of embryonic
development, or on the dimensions of gastrulae and
planulae, of the Caribbean coral A. palmata. The
experiment was repeated thrice over a 2-year period,
and thus the results were repeatable as well as
statistically robust within each experimental run. The
pH conditions tested ranged from present-day ambient
levels on Puerto Rican coral reefs, pH 8.1, to pH levels
of 7.9 and 7.7, predicted to occur by the end of the
twenty-first century if present rates of anthropogenic
CO2 emissions continue (IPCC 2007). While not
experimentally quantified, there was no observable
difference in settlement of larvae under the three
experimental conditions as they continued to develop
within the Jellyquaria.
This finding contrasts with the results of experiments testing the effects of elevated seawater temperatures associated with global warming, another
independent consequence of anthropogenic CO2
enrichment, on the embryonic development of A.
palmata. Randall and Szmant (2009) found accelerated
development, increased percentages of abnormal
embryos, and greatly reduced survivorship of embryos
raised at moderately elevated seawater temperatures of
30 C and 31.5 C. In contrast to the detrimental effects
of temperature on A. palmata embryos (Randall and
Szmant 2009; Portune et al. 2010), the CO2/pH
treatments in this study did not increase the percentage
of abnormal embryos or change the developmental
timing. Thus, these two conditions resulting from the
anthropogenically induced global change have differing effects when tested independently. Comparison of
the two studies, which in 2008 were conducted with
sub-samples of the same spawn event, indicates that
warming appears to have more severe effect on the
population dynamics and recovery potential of this
ecologically important species. It would be interesting
138
P. Medina-Rosas et al.
Table 3. Statistical comparisons of percentages of embryos in the various development stages of A. palmata embryos reared at
three seawater CO2/pH levels (data in Table 2).
Downloaded by [Pedro Medina-Rosas] at 17:23 03 December 2012
Sample #
Hours
after
fertilization
Chi-squared
p value
Chi-squared
p value
3
7
23
30.5
46
60
79.5
91
104
0.0555
0.0284
0.0145
0.0050
–
0.0077
0.2435
0.0025
0.0451
0.9996
0.9859
0.9041
0.9436
–
0.9300
0.8853
0.9601
0.8318
0.0173
0.0567
0.0114
0.0510
–
0.0195
0.0173
0.0022
0.0248
0.9996
0.9720
0.9149
0.8213
–
0.8889
0.9913
0.9625
0.8748
3
8
20
32
45
60
80
94
104
0.0088
0.0025
0.0183
–
–
0.0322
0.0059
–
0.0928
1.0000
0.9999
0.8923
–
–
0.8575
0.9387
–
0.7606
0.1700
0.0172
0.0902
–
–
0.0948
0.0009
–
0.0690
0.9994
0.9994
0.7639
–
–
0.7581
0.9760
–
0.7927
1.5
8
20
32
44
60
80
92
104
0.0104
0.0308
0.0002
–
–
0.0170
0.1285
–
0.0256
0.9948
0.9847
0.9887
–
–
0.8962
0.9377
–
0.8728
0.0015
0.0432
0.0196
–
–
0.1050
0.0851
–
0.0772
0.9992
0.9786
0.8886
–
–
0.7459
0.9583
–
0.7811
August 2008
1
2
3
4
5
6
7
8
9
August 2009
1
2
3
4
5
6
7
8
9
September 2009
1
2
3
4
5
6
7
8
9
7.9 pH compared to 8.1 pH
7.7 pH compared to 8.1 pH
Notes: Each of the three experimental runs was analyzed separately. Chi-squared analyses were used for treatment comparisons.
Distributions of embryos at the control pH 8.1 samples were designated as the expected distributions, and the distributions at the
mid (7.9) and low (7.7) pH levels were compared to the expected. There were two replicate samples per treatment per time point.
Significance level is p 0.05 level.
Table 4. Percentages of abnormal or irregular A. palmata embryos in each sample of larval cultures grown at the three nominal
seawater CO2/pH treatments: pH 8.1 (control), mid pH 7.9 and low pH 7.7 (see Table 2).
Experiment
August 2008
August 2009
September 2009
pH treatment
Sample
2
3
4
5
6
7
8
9
hours
Control
Mid
Low
Control
Mid
Low
Control
Mid
Low
8
20
32
45
60
80
90
104
12.8
18.5
9.1
11.1
8.8
6.2
9.0
2.7
12.9
18.0
9.8
11.5
8.4
6.0
8.0
2.1
11.8
17.9
10.4
11.9
8.3
6.3
8.7
1.9
4.8
7.2
4.0
5.9
5.9
5.4
5.9
2.5
5.1
6.8
4.6
5.2
5.1
6.5
5.4
2.5
4.4
5.7
3.9
5.7
6.4
4.4
6.2
3.2
10.5
12.4
9.9
11.0
7.8
7.3
9.7
2.8
11.3
12.5
10.0
10.3
7.6
7.1
9.5
3.2
10.2
13.2
10.2
10.5
8.6
7.8
9.1
2.9
Notes: The experiment was repeated thrice: August 2008, August 2009, and September 2009.
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Invertebrate Reproduction & Development
139
Figure 1. Dimensions of Acropora palmata gastrulae (a; diameter in mm) and planulae (b; volume in mm3) raised under three
CO2/pH treatments (control, pH 8.1, white bars; mid pH 7.9, gray bars; low pH 7.7, black bars). The experiment was repeated
thrice, during August 2008, August 2009, and September 2009. Sample size (n) values for each treatment are included on
each bar.
to attempt a multifactorial experiment to test whether
there is any synergistic effect between the elevated
temperature and elevated CO2/reduced pH. Warmer
water, below the thermal threshold, may ameliorate the
effect of more acidic waters on the calcification process
for some invertebrates, including abalone and sea
urchins (Sheppard Brennand et al. 2010; Byrne et al.
2011). In contrast, a study using Porites panamensis,
the only coral species to date on which larvae were
studied under OA and warming conditions, showed
that primary polyp growth not only slightly reduced
under acidic seawater (pH 7.8) conditions alone, but
was also significantly reduced (30%) when pH was
low and temperature was elevated (Anlauf et al. 2011).
An additional significant reduction in the biomass was
also observed under the combined warm OA conditions (Anlauf et al. 2011).
In a study on the effects of OA on the fertilization
in A. palmata, Albright et al. (2010) found that
fertilization success decreased under similar pCO2
conditions (468, 673, and 998 matm) used in this
study. For the experiments, we fertilized embryos in
the field immediately after spawning, within 1 h of
collecting bundles, under an ambient pCO2 estimated
to be in the neighborhood of 450 matm (Gledhill et al.
2010). No sperm concentration was estimated for this
study, but fertilization success was estimated before the
experiments to ensure viable embryos for the
three runs.
A number of studies have reported that growth
of some (but not all) taxa that calcify as adults,
and a number of calcifying larval stages are affected
when exposed to experimentally CO2-enriched or
reduced pH conditions (Fabry et al. 2008; Doney
et al. 2009; Ries et al. 2009; Yu et al. 2011). Recent
studies have shown that the effects are species-specific.
For example, survivorship at reduced pH of A. tenius
was higher than for A. digitifera, showing that two
species of Acropora in the same region of Japan
respond in different ways (Suwa et al. 2010). The
effects of OA on most species in the ocean are still
unknown. It is generally considered that early life
history stages are the most sensitive to environmental
stressors, including CO2-induced OA (Kurihara 2008;
Raven et al. 2005; Pörtner and Farrell 2008; Byrne
2011), however very few taxa have been examined
to date.
In summary, our results show that A. palmata
embryos and larvae are able to tolerate decrease in the
pH of seawater of at least 0.4 pH units. This study
covers for the first time (see reviews of Albright (2011)
and Byrne (2011)) the ontogenic stages, from fertilization to competent larvae, of this species, under
enriched CO2 and reduced pH conditions. However,
this is only one part of the life cycle of this organism,
before calcification begins. Most concern about the
effect of OA on calcifying marine organisms centers on
its effect on calcification, and skeleton secretion in this
species does not begin until planulae settle and
metamorphose into a polyp. Thus, negative effects of
OA on this species may not manifest until settlement,
where OA could affect growth rates and survivorship
of the juvenile corals. Further, we were not able to test
the simultaneous effects of CO2 enrichment and
elevated seawater temperatures, which in the natural
world are co-occurring (Turley and Findlay 2009;
Byrne 2011; Pandolfi et al. 2011). While on its own
CO2 enrichment/decreased pH did not have had a
demonstrable effect on the development of A. palmata,
it is possible that under conditions of elevated seawater temperatures, OA could make a bad situation
worse.
140
P. Medina-Rosas et al.
Acknowledgments
Thanks to Patrick Erwin, Carly Randall, and Andy Miller
for field and laboratory assistance. Authors also thank Dr
Ernesto Weil for the use of laboratory space, and Katie
Flynn and beach volunteers in Puerto Rico for field
assistance. This project was partially supported by funds
from the World Bank Coral Reef Targeted Research
Program to AMS, and UNCW Academic Affairs funds to
support coral reef research at UNCW. PhD program support
for PMR was provided by CONACYT (#117687) and
PROMEP SEP. We thank the three reviewers who helped
improve this article.
Downloaded by [Pedro Medina-Rosas] at 17:23 03 December 2012
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