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Early life stages of the coral Seriatopora cali-endrum were used to test the hypothesis that the depression of dark respiration in coral recruits by high pCO2 is caused by perturbed protein metabolism. First, the contribution of protein anabolism to respiratory costs under high pCO2 was evaluated by measuring the aerobic respiration of S. caliendrum recruits with and without the protein synthesis inhib-itor emetine following 1 to 4 days at 45 Pa versus 77 Pa pCO2 . Second, protein catabolism under high pCO2 was evaluated by measuring the flux of ammonium (NH4+) from juvenile colonies of S. caliendrum incubated in darkness at 47 Pa and 90 Pa pCO2 . Two days after settlement, respira-tion of recruits was affected by an interaction between emetine and pCO2 , with emetine reducing respiration 63% at 45 Pa pCO2 and 27% at 77 Pa pCO2 . The interaction disappeared 5 days after settlement, when respiration was reduced 27% by emetine under both pCO2 conditions. These findings suggest that protein anabolism accounted for a large proportion of metabolic costs in coral recruits and was affected by high pCO2 , with consequences detected in aerobic respiration. Juvenile S. caliendrum showed net up-take of NH 4 ϩ at 45 Pa pCO2 but net release of NH4 + at 90 Pa pCO2 , indicating that protein catabolism, NH4+ recycling, or both were affected by high pCO2 . Together, these results are consistent with the hypothesis that high pCO2 affects protein metabolism in corals.
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Evidence That High pCO
2
Affects Protein Metabolism
in Tropical Reef Corals
PETER J. EDMUNDS
1*
AND CHRISTOPHER B. WALL
1,2
1
Department of Biology,California State University, 18111 Nordhoff Street,Northridge,
California 91330-8303; and
2
University of HawaiiatManoa,Hawaii Institute of Marine
Biology,PO Box 1346, Kaneohe,Hawaii 96744
Abstract. Early life stages of the coral Seriatopora cali-
endrum were used to test the hypothesis that the depression
of dark respiration in coral recruits by high pCO
2
is caused
by perturbed protein metabolism. First, the contribution of
protein anabolism to respiratory costs under high pCO
2
was
evaluated by measuring the aerobic respiration of S. calien-
drum recruits with and without the protein synthesis inhib-
itor emetine following 1 to 4 days at 45 Pa versus 77 Pa
pCO
2
. Second, protein catabolism under high pCO
2
was
evaluated by measuring the flux of ammonium (NH
4
) from
juvenile colonies of S. caliendrum incubated in darkness at
47 Pa and 90 Pa pCO
2
. Two days after settlement, respira-
tion of recruits was affected by an interaction between
emetine and pCO
2
, with emetine reducing respiration 63%
at 45 Pa pCO
2
and 27% at 77 Pa pCO
2
. The interaction
disappeared 5 days after settlement, when respiration was
reduced 27% by emetine under both pCO
2
conditions.
These findings suggest that protein anabolism accounted for
a large proportion of metabolic costs in coral recruits and
was affected by high pCO
2
, with consequences detected in
aerobic respiration. Juvenile S. caliendrum showed net up-
take of NH
4
at 45 Pa pCO
2
but net release of NH
4
at 90
Pa pCO
2
, indicating that protein catabolism, NH
4
recy-
cling, or both were affected by high pCO
2
. Together, these
results are consistent with the hypothesis that high pCO
2
affects protein metabolism in corals.
Introduction
Ocean acidification (OA) caused by the dissolution of
atmospheric pCO
2
in seawater is a serious threat to marine
life (Hoegh-Guldberg et al., 2007). Of the many taxa likely
to be affected by this phenomenon, those that deposit
CaCO
3
have received the most attention, because their
ability to calcify is challenged by the lower CaCO
3
satura-
tion states () associated with reduced pH (Hofmann et al.,
2010). Tropical coral reefs rely on the calcification of scler-
actinians to produce massive, wave-resistant platforms,
and form topographically complex substrata that provides
habitat to many taxa (Wild et al., 2011). Challenges to
the capacity of reef corals to serve as ecosystem engi-
neers therefore have serious implications (Silverman et al.,
2009).
Most studies of the effects of high pCO
2
on corals have
measured the outcome as perturbed rates of calcification
attributed to low without paying close attention to the
underlying mechanism (Zeebe, 2012). Promising new lines
of investigation addressing the mechanism by which coral
calcification is depressed by high pCO
2
focus on at least
four possibilities: the regulation of protons (H
) at the site
of calcification (Jokiel, 2011), most likely with active trans-
porters (Tambutte´ et al., 2011); perturbations in the activity
of enzymes that modulate host-Symbiodinium interactions
(Crawley et al., 2010); the role of nutrients (e.g., nitrogen,
sulfur, and iron) in mediating changes in the quantity of
biomass (i.e., heterotrophy [Edmunds, 2011]) and the rate of
calcification (Holcomb et al., 2010); and the capacity of
aerobic respiration to supply metabolic energy at high pCO
2
(Erez et al., 2011; Edmunds, 2012; Wall and Edmunds,
2013). The role of aerobic respiration in the response of
corals to high pCO
2
is germane to a discussion of the effects
of OA, because respiration plays a central role in metabo-
lism (Prosser, 1991), and can be affected directly by high
pCO
2
(Crawley et al., 2010; Edmunds, 2012). Further, since
a large fraction of the respiratory costs of metazoans reflects
Received 1 November 2013; accepted 11 April 2014.
* To whom correspondence should be addressed. E-mail: peter.
edmunds@csun.edu
Reference: Biol. Bull. 227: 68 –77. (August 2014)
© 2014 Marine Biological Laboratory
68
protein metabolism (Fraser and Rogers, 2007), measure-
ments of respiration in an OA context provide a means to
explore linkages among high pCO
2
, low pH, and protein-
modulated responses.
Research with model systems supports the hypothesis
that low pH affects the metabolism of amino acids and
proteins (Langenbuch and Po¨rtner, 2002, 2003; Po¨rtner,
2008; Thomsen and Melzner, 2010). Po¨rtner and colleagues
have investigated the effects of hypercapnia and reduced
extracellular pH on the marine invertebrate Sipunculus nu-
dus and on Antarctic fishes (Langenbuch and Po¨rtner, 2003;
Langenbuch et al., 2006), although their studies typically
employ lower external pH and higher pCO
2
levels (e.g.,
1.02 kPa [Langenbuch and Po¨rtner, 2002]) than those ex-
pected due to OA (100 Pa pCO
2
[van Vurren et al.,
2011]). Their research has shown for S. nudus that a low
external pH (pH
e
) of 7.20 causes a reduction of about 18%
in aerobic respiration (Reipschlager and Po¨rtner, 1996) with
a 40%– 45% reduction at pH
e
6.7 (Langenbuch and Po¨rtner,
2002); in addition, in two Antarctic fishes, respiration de-
clines 34%–37% at pH
e
6.50 (Langenbuch and Po¨rtner,
2003). Reipschlager and Po¨rtner (1996) speculated that the
effect in S. nudus is caused by low pH
e
reducing the ATP
costs of internal pH regulation between intra- and extracel-
lular compartments. Subsequently, these researchers used
the ratio of O/N released at high pCO
2
to conclude that
cellular acidosis reduces NH
4
excretion and affects the
selection of amino acids used for catabolism (Langenbuch
and Po¨rtner, 2002). By investigating the effects of high
pCO
2
on hepatocytes from Antarctic fishes incubated with
the protein synthesis inhibitor cyclohexamide, Langenbuch
and Po¨rtner (2003) demonstrated that protein biosynthesis
was impaired by cellular acidosis.
Using the aforementioned studies as motivation, and in-
terpreting modified gene expression in Acropora millepora
exposed to high pCO
2
(Kaniewska et al., 2012) as evidence
of altered protein metabolism, we tested the hypothesis that
metabolic depression in corals exposed to high pCO
2
is
associated with perturbed gross protein metabolism. We
took a two-fold approach in which the effect of high pCO
2
was first tested on the dark respiration of coral recruits
exposed to emetine to inhibit protein synthesis (Fenteany
and Morse, 1993; Pace and Manahan, 2006). Second, the
effects of high pCO
2
on the deamination of proteins and
amino acids were tested by measuring NH
4
flux between
juvenile corals and seawater in the dark. Experiments were
conducted in 2012 with the pocilloporid coral Seriatopora
caliendrum (Ehrenberg, 1834), which is common in south-
ern Taiwan where the research was conducted. In 2013, an
experiment was conducted to test for concentration-depen-
dent effects of emetine on coral respiration, with the results
used to evaluate the efficacy of the protein synthesis inhi-
bition experiment conducted in 2012.
Materials and Methods
Overview
This study was motivated by a larger effort to explore the
effects of ocean acidification on tropical reef corals, and as
part of this effort we have been incubating corals of various
life stages under different pCO
2
regimes to test for effects
on key metabolic processes. Despite advances that have
been made in this area by our group (e.g., Edmunds, 2012;
Edmunds et al., 2013) as well as others (e.g., Erez et al.,
2011), it has remained difficult to identify mechanisms
through which ocean acidification affects organismic re-
sponses in corals. The present research capitalizes on an
opportunity to advance understanding of such mechanisms
in a coral system by assembling results from different
experiments that were not designed to be presented in a
single cohesive analysis. We bring together experiments
conducted on two pocilloporid corals, using different life
stages and slightly varying physical conditions, to explore
the effects of high pCO
2
on aspects of protein metabolism.
In so doing, we recognize the limitations of this experimen-
tal design in establishing cause-and-effect relationships, but
we suggest that the likely conserved nature of cellular
physiology in scleractinians provides sufficient rationale for
considering our results together, and using the outcome to
identify promising hypotheses for further investigation.
To evaluate the effect of pCO
2
on protein metabolism, the
aerobic respiration of coral recruits exposed to ambient and
high pCO
2
was measured with and without emetine. This
experiment was conducted with recruits of Seriatopora cali-
endrum in March 2012. Emetine is an irreversible inhibitor
of post-translational protein synthesis in ribosomes (Groll-
man, 1968), and has been used with marine invertebrates to
investigate protein metabolism (Fenteany and Morse, 1993;
Pace and Manahan, 2007). To test for concentration-depen-
dent effects of emetine, trials were conducted to evaluate the
response of corals to different concentrations of emetine.
The objective was to determine the extent to which the
concentration of emetine was associated with metabolic
depression caused by impaired protein metabolism, versus
metabolic stimulation caused by a stress response to high
concentrations of emetine. This experiment was conducted
in March 2013 with larvae of Pocillopora damicornis (Lin-
naeus, 1758), which were the early life stage of corals
available (rather than recruits) when the experiment was
conducted. We assumed these results characterized the con-
centration-dependent response of recruits of S. caliendrum
recruits to emetine.
The effect of high pCO
2
on the flux of NH
4
to and from
juvenile Seratopora caliendrum colonies was measured as
an indirect assessment of protein catabolism in June 2012.
NH
4
is a product of protein catabolism in marine inverte-
brates (Wright, 1995); in tropical corals containing Symbio-
dinium spp., excretory NH
4
is taken up by the algae in a
69
P. J. EDMUNDS AND C. B. WALL
light-dependent manner and recycled to the host as amino
acids (Muscatine and D’Elia, 1978). Thus, while corals are
ammonotelic, NH
4
uptake by Symbiodinium typically re-
sults in no net NH
4
excretion from the holobiont (Musca-
tine and D’Elia, 1978) unless recycling is inhibited by
darkness or a metabolic inhibitor (Rahav et al., 1989). We
reasoned, therefore, that if high pCO
2
affected protein ca-
tabolism, then its effects might be evident through altered
flux of NH
4
from the holobiont (Szmant et al., 1990).
While detectable differences in NH
4
flux under high pCO
2
can reflect protein catabolism, two limitations of this ap-
proach are (1) a null result cannot be interpreted as a lack of
an effect if the change in NH
4
flux attributed to high pCO
2
is within the capacity of the Symbiodinium to absorb and
recycle NH
4
, and (2) it is not possible to resolve the causes
of net efflux of NH
4
at high pCO
2
, because the extents to
which it is caused by increased protein catabolism versus
impaired NH
4
recycling by the Symbiodinium cannot be
separated.
Incubation conditions
In March 2012 and June 2012 when the effects of pCO
2
were investigated, treatments were created by bubbling
mixtures of pure CO
2
and air into seawater, and the seawa-
ter was assayed for pH and total alkalinity (TA), with the
values used to calculate dissolved inorganic chemistry pa-
rameters. The techniques to manipulate pCO
2
and measure
dissolved inorganic chemistry parameters have been applied
in other experiments we have conducted, and the method-
ology is described elsewhere (Edmunds et al., 2013; Wall
and Edmunds, 2013). Both sets of experiments were de-
signed to contrast ambient (control) pCO
2
of about 40 Pa
with the elevated pCO
2
predicted for the end of the century
(81 Pa; van Vurren et al., 2011), but actual pCO
2
values
departed slightly from these targets (see Table 1). Seawater
in the incubation tanks was sampled daily and measured for
salinity (with a YSI 3100 conductivity meter, YSI Inc.),
temperature (0.05 °C using a certified digital thermom-
eter, Fisher Scientific 15-077-8), pH (using the dye m-
cresol, SOP 6b of Dickson et al., 2007), and TA (using
SOP3b, Dickson et al., 2007). The accuracy and precision
of the seawater analyses were evaluated by analyzing cer-
tified reference materials (CRM) of known TA (batch 110
supplied by A. G. Dickson, Scripps Institution of Oceanog-
raphy); our calculated TA values for the CRMs differed
0.6% (ranging 1.1–12.3
mol kg
1
) from certified values.
Incubation tanks were made from 130-l plastic tubs that
were individually heated and chilled and supplied with light
from fixtures containing 150-W metal halide lamps and two
18-W fluorescent bulbs. The lamps were operated on a
12:12 h light/dark cycle, and provided a light intensity of
about 300
mol and about 275
mol photons m
2
s
1
(March and June 2012, respectively) as measured with a 4-
quantum light sensor (model LI-193, Li-Cor) sensitive to
photosynthetically active radiation. Seawater temperature
was maintained at the ambient seawater temperature on the
reef from which the corals were collected when experiments
were conducted (25.3 °C in March, 27.5 °C in June),
and the tanks were filled with filtered seawater (1
m),
which was changed partially (20%) every evening. In March
and June 2012, one tank was allocated to each of the
ambient and elevated pCO
2
treatments, with ambient air or
CO
2
-enriched air delivered constantly. All experiments
were conducted at the National Museum of Marine Biology
and Aquarium (NMMBA) (Pingtung, Taiwan).
Emetine titrations (March 2013)
To evaluate the effect of emetine concentration on coral
respiration, freshly released larvae from Pocillopora dami-
cornis were incubated at three emetine concentrations from
28 to 228
mol l
1
and compared to control larvae retained
in seawater. Larvae were obtained from three colonies of P.
damicornis collected from Houbihu Reef (5-m depth) on
8 March 2013, 3 days before the new moon. Colonies were
kept in aquaria at NMMBA, where they were exposed to
sunlight and received fresh seawater that overflowed gently
into cups fitted with plankton mesh (110
m) bottoms to
retain larvae. Larvae were harvested at about 0700 h on four
Table 1
Summary of conditions in the tanks used for the incubations of corals under different pCO
2
conditions
Experiment Treatment Temperature °C
Light
mol
photons
m
2
s
1
Salinity pH pCO
2
Pa
Total alkalinity
mol kg
1
Arag
(A) Recruits Ambient pCO
2
25.3 † (7) 296 9 (4) 33.78 0.04 (6) 8.0 † (7) 45.4 0.5 (7) 2261 4 (7) 3.06 0.02 (7)
High pCO
2
25.2 † (7) 310 9 (3) 33.89 0.04 (6) 7.8 † (7) 76.9 2.5 (7) 2287 12 (7) 2.16 0.05 (7)
(B) Juveniles Ambient pCO
2
27.6 † (13) 266 5 (17) 32.69 0.1 (13) 8.0 † (13) 47.2 0.5 (13) 2205 14 (13) 3.04 0.03 (13)
High pCO
2
27.5 † (13) 264 8 (17) 32.01 0.1 (13) 7.8 † (13) 89.7 1.5 (13) 2213 15 (13) 1.94 0.02 (13)
(A) Experiments with recruits of Seriatopora caliendrum between 2 and 8 March 2012; (B) experiments with juvenile colonies of S. caliendrum between
9 and 24 June 2012. Mean SE (n) for temperature, light, salinity, and dissolved inorganic chemistry parameters; † SE 0.1.
70 PCO
2
AND PROTEIN IN CORALS
consecutive days starting 15 March, pooled among parent
colonies, gently stirred to mix parental genotypes, and al-
located to treatments.
On each day of the experiments, larvae were allocated
randomly in batches of eight to glass Wheaton vials (3.2 ml)
that were used for incubations. Vials containing larvae were
allocated to one of four treatment groups: (a) filtered sea-
water (FSW, 1
m), the control, (b) 28
mol l
1
emetine,
(c) 98
mol l
1
emetine, or (d) 228
mol l
1
emetine.
Additional vials without larvae were used for (e) FSW
control, and (f) FSW emetine control. The seawater used
to fill the vials was held at a constant temperature in a water
bath (25.3 °C, ambient seawater temperature when the ex-
periment was conducted) and equilibrated with air. Mea-
surements with a fiber-optic electrode system (described
below) revealed an O
2
concentration in this seawater that
was close to saturation (212
mol l
1
). As relatively few
larvae were released on each sampling day, it was not
possible to run all treatments on every day, and therefore a
subset of emetine treatments was applied daily until all
larvae released that day were used. Emetine (Sigma-Al-
drich, catalog D2425) was made up in three stock solutions
using 70% w/v ethanol as a carrier for the inhibitor (emetine
is soluble in ethanol, but not water). Concentrations of stock
solutions were selected to achieve final target concentra-
tions by adding 10
lor20
l of emetine stock to each vial.
The use of 10 –20
l of emetine stock ensured that the
addition of the ethanol carrier to the incubations remained
within the range of controls tested (0.2%– 0.4% v/v ethanol
in seawater) for effects on larval respiration alone.
After preparation, vials were capped with Parafilm and
placed into a single tank at about 0900 h to incubate for 3 h.
Following incubations, vials were removed from the tank,
and the seawater was siphoned out and replaced with fresh,
air-saturated seawater of known O
2
concentration together
with emetine stock solutions to maintain emetine treatment
levels. Vials again were capped with Parafilm, placed into a
darkened laboratory water bath at 25.3 °C, and left with
periodic gentle rocking to allow larval respiration to deplete
the O
2
. Previous work suggested that incubations of 1–2 h
would be necessary to deplete O
2
by about 25% (Cumbo et
al., 2012). After incubations, vials were removed from the
water bath in a random sequence relative to their treatments,
inverted to mix the seawater within, and the O
2
concentra-
tion was measured with a fiber-optic electrode.
Oxygen concentrations were measured with a fibrer-optic
system consisting of an optrode (Foxy-R, Ocean Optics)
attached to a spectrophotometer and light source (USB2000
and USB-LS-LS450, Ocean Optics), and operated by a PC
running the manufacturer’s software (OOISensor, Version
1.00.08, Ocean Optics). The optrode was calibrated in a
zero solution of 0.01 mol l
1
sodium tetraborate saturated
with sodium sulfite, and a 100% standard obtained from
water-saturated air at the measurement temperature of
25.3 °C. Oxygen saturation was converted to concentration
using tabulated values (Unisense A/S, Denmark [based on
Garcia and Gordon, 1992]) and salinity. O
2
concentrations
within the vials were measured with the vials immersed in
a water bath at 25.3 °C. Larval respiration was calculated
from the difference in O
2
between the seawater used to fill
the vials and the concentration at the end of the incubations,
corrected for controls and the ethanol carrier for the emetine
(where appropriate); rates were expressed as nmol O
2
larva
1
min
1
.
Effect of emetine on recruit respiration
To test the effects of emetine on the respiration of coral
recruits exposed to differing pCO
2
, recruits of Seriatopora
caliendrum were obtained by settling larvae on the glass of
Wheaton vials. Eight colonies of S. caliendrum were col-
lected from Houbihu reef (5-m depth) on 29 February
2012, 8 d after the new moon, and returned to NMMBA
where they were placed in separate aquaria exposed to
screened sunlight. The aquaria were supplied with running
seawater that overflowed into cups fitted with plankton
mesh bottoms in which larvae were trapped. The congener
S. hystrix shows peak larval release about 7 d after the new
moon, and therefore we suspected that the newly collected
S. caliendrum individuals were ready to release larvae.
Larvae were, indeed, released for several days starting on 1
March. Larvae were released in unequal numbers from
parent colonies, and on each of four release days (1, 2, 3,
and 5 March), larvae were collected at about 0700 h, pooled
among parent colonies, gently mixed, and allocated to in-
cubation vials.
Incubations were completed in Wheaton vials (3.2 ml),
and on the day of release, 15–20 larvae were placed into
each vial that was filled with aerated FSW (0.1
m). The
larvae were allowed to settle in vials for about 24 h in
subdued light and at room temperature (26 °C) (Edmunds
et al., 2013). After this period, the majority had settled and
metamorphosed, and most had attached sufficiently well to
avoid being dislodged by water flow. The number of vials
prepared daily varied depending on the number of larvae
released, but all larvae released each day were used, and
between 10 and 25 vials were prepared daily with larvae and
allocated at random to the two treatment tanks. Additional
vials (5 treatment
-1
) were prepared as controls (FSW alone)
and processed concurrently with the vials containing re-
cruits. Vials were placed into two custom-made incubation
systems (Edmunds et al., 2013) that supplied vials individ-
ually with a high rate of water flow (1.4 ml s
1
), and were
fitted to one of two tanks— one maintained at ambient
pCO
2
, the other at high pCO
2
(target 81 Pa) at 25.3 °C—
and illuminated at about 300
mol photons m
2
s
1
.
Larvae from the four release days were used in three
separate analyses. Larvae from 1 and 2 March were settled
71
P. J. EDMUNDS AND C. B. WALL
in vials, incubated for 24 h in the two pCO
2
treatments, and
exposed to emetine (28, 98, or 228
mol l
1
) or control
conditions in the same illuminated tanks. After 3 h their
respiration rates were measured. This experiment evaluated
the effects of emetine on the response of 2-d-old recruits to
high pCO
2
treatments. Larvae from 3 and 5 March were
processed in the same way as larvae from 1 and 2 March,
except that they were used to evaluate the response of coral
recruits to the ethanol carrier used to deliver the emetine.
This analysis contrasted the effects of 9
lversus 18
lof
70% ethanol (at a final concentration of 0.2%– 0.4% v/v)
with FSW control on the respiration of 2-d-old recruits.
Additional larvae from 3 March were settled into vials as
before, but these were exposed to pCO
2
treatments for 4 d
before being tested for the effects of emetine on respiration.
These recruits therefore were 5-d-old when tested.
The effects of emetine on respiration were tested as
described above, except that a single concentration of em-
etine was employed (100
mol l
1
). Respiration rates were
calculated from the change in O
2
concentration between the
water used to fill the vials and the final concentration after
incubations in sealed vials, corrected for FSW controls and
the ethanol carrier (where appropriate), and expressed as
nmol O
2
recruit
1
min
1
.
Effect of high pCO
2
on NH
4
flux
To test for effects of pCO
2
on NH
4
flux, eight juvenile
corals (4-cm diameter) were collected on 4 June 2012
from Houbihu Reef at a depth of 5–7 m. Corals were
transported to NMMBA and placed in a 1050-l flow-
through aquarium receiving filtered seawater (50
m) and
maintained at about 28 °C for5dtoacclimate to laboratory
conditions. Light was provided by four 150-W metal halide
lamps programmed on a 12:12 h light/dark cycle providing
a mean light intensity of 259 10
mol photons m
2
s
1
(SE, n12) measured using a 4-
spherical quantum
sensor. Previous work demonstrated this light treatment to
be sufficient to saturate photosynthesis in juvenile S. cali-
endrum (Wall et al., 2014). Corals were maintained in the
flow-through aquarium for 5 d until placed into the CO
2
-
treatment tank on 9 June to 23 June 2012.
Following acclimation, the corals were allocated ran-
domly to one of two tanks that were maintained at either
ambient or elevated pCO
2
(described above). After 14 d, the
NH
4
flux for each of the corals was measured indepen-
dently over 4 days, generally with one coral from each
treatment processed on each day to provide an overall
sample size of 5 (high CO
2
) and 3 (ambient CO
2
) corals per
treatment. Each day between 1200 and 1500 h, and after 5
to8hoflight exposure within treatments, corals were
placed individually in 0.25-l glass beakers (acid washed)
filled with 0.45
m FSW from the respective CO
2
treat-
ments. Beakers were placed in a water bath at 27.5 °C and
bubbled with either ambient air or the same CO
2
-enriched
air supplied to the treatment tank. The bubbling created a
turbulent flow estimated at about 5 cm s
1
adjacent to the
corals (as determined by photographing hydrated Artemia
eggs added to the seawater [Sebens and Johnson, 1991]).
Two beakers filled with seawater alone and bubbled with
ambient or CO
2
-enriched air served as controls.
Incubation beakers were covered with Parafilm and incu-
bated in darkness for 3 h. Thereafter, NH
4
l
1
flux was
determined as the change in NH
4
concentration in seawa-
ter of the beakers containing corals relative to controls
without corals. NH
4
concentrations in seawater were cal-
ibrated to a concentration range of 0.5–20
mol NH
4
by
using standard solutions of NH
4
Cl in distilled water. Con-
centrations were then measured with a spectrophotometer
(SP8001, Metertech, Taiwan) at 640 nm in a 10-cm-path-
length cuvette following the methods of Solorzano (1969)
and Parsons et al. (1984). NH
4
concentrations in acid-
washed beakers containing the corals at the end of the
incubations were expressed as a change (i.e., flux) relative
to controls, and normalized to time and the area of the
corals. The area of the corals was determined by wax
dipping (Stimson and Kinzie, 1991), and the final units were
nmol NH
4
cm
2
h
1
.
Statistics
The effect of emetine on the respiration of Pocillopora
damicornis larvae was tested with a one-way ANOVA
followed by post hoc analysis with the Fisher LSD method.
The effect of emetine on the respiration of Seriatopora
caliendrum recruits was evaluated with model I, two-way
ANOVA with main effects of emetine (with vs. without)
and pCO
2
(ambient vs. high); post hoc multiple contrasts
were completed using Bonferroni-adjusted t-tests The test
was repeated separately for recruits differing in age and
exposure time to pCO
2
(2dvs. 5 d). In the second experi-
ment, the effect of pCO
2
on NH
4
flux was tested with a
one-way ANOVA. The assumptions of normality and ho-
moscedasticity required for ANOVA were tested through
graphical analyses of the residuals. All statistical tests were
completed using Systat 11 running on a Windows platform.
Results
CO
2
treatment conditions
In the March 2012 and June 2012 experiments, pCO
2
treatments were regulated precisely, as reflected in very low
standard errors around the mean values for each metric. In
both experiments, CO
2
treatments differed in pH
T
and dis-
solved inorganic chemistry parameters (F241.114, df
1,12, P0.001), but total alkalinity was not different
between treatments (F0.176, df 1, 12, P0.067).
Overall, the treatments contrasted 45.4 Pa versus 76.9 Pa
72 PCO
2
AND PROTEIN IN CORALS
pCO
2
at 25.2–25.3 °C (March 2012) and 47.1 Pa versus
89.7 Pa pCO
2
at 27.5–27.6 °C (June 2012) (Table 1).
Seawater temperature did not differ between CO
2
-treat-
ments in June (F0.535, df 1, 95, P0.466) but
differed in March (F6.834, df 1, 12, P0.023),
although this difference was small (0.1 °C) (Table 1).
Emetine titrations
Of the eight larvae added to each vial at the start of
incubations, all were alive and motile 3 h later, although
those exposed to 228
mol l
1
emetine were lethargic. In
the analysis of the effects of the ethanol carrier on Seriato-
pora caliendrum recruits, respiration was affected by the
addition of 9 –18
l of 70% ethanol relative to FSW (t
2.347, df 16, P0.032), and the mean respiration with
ethanol was higher by 0.017 nmol coral min
1
compared to
the mean respiration in filtered seawater (FSW). Given that
ethanol alone (at 0.2%– 0.4% v/v) increased the respiration
of early life stages of coral, estimates of the effects of
emetine delivered with an ethanol carrier on the respiration
Pocillopora damicornis larvae and S. caliendrum recruits
were downwardly adjusted by 0.017 nmol coral min
1
.
Mean respiration rates of P. damicornis larvae varied
from 0.089 0.014 nmol coral min
1
(n6) to 0.045
0.012 nmol coral min
1
depending on the concentration of
emetine (SE, n6). Respiration was reduced 41%– 49%
by emetine at 28 –98
mol l
1
, but increased again to 110%
of the control value at 228
mol l
1
. Respiration differed
among emetine concentrations (F3.695, df 3,19, P
0.030) (Fig. 1).
Effect of emetine on recruit respiration
Overall, between 9 and 21 recruits settled in the Wheaton
vials, with a mean of 16.0 0.6 recruits vial
1
in the
emetine treatments, and 17.2 0.5 recruits vial
1
in the
controls (Fig. 2) (SE, n28 and 29, respectively). In the
experiment conducted with 2-d post-settlement recruits, res-
piration ranged from 0.014 nmol O
2
recruit
1
min
1
(em-
etine treatment at ambient pCO
2
) to 0.089 nmol O
2
recruit
1
min
1
(controls at high pCO
2
), and was affected by the
interaction between emetine and pCO
2
(F13.539, df
1,32, P0.001). Bonferroni post hoc contrasts revealed
that respiration was unaffected by emetine at high pCO
2
(P0.232) but was affected by emetine at ambient pCO
2
(P0.001), where emetine reduced mean respiration 63%.
In the experiment conducted with 5-d post-settlement re-
cruits, respiration ranged from 0.049 nmol O
2
recruit
1
min
1
(emetine treatment at high pCO
2
) to 0.100 nmol O
2
recruit
1
min
1
(controls at ambient pCO
2
) and was af-
fected by emetine (F11.406, df 1,13, P0.005),
which caused a 27% reduction in mean respiration (pooled
between pCO
2
treatments). Respiration was unaffected
by pCO
2
(F0.195, df 1,13, P0.666) or the
interaction between pCO
2
and emetine (F0.620, df
1,13, P0.446).
Effect of high pCO
2
on NH
4
excretion
Corals in ambient and high pCO
2
treatments appeared
healthy and showed no signs of stress during the incuba-
tions. However, one outlier in the high CO
2
treatment was
excluded from the dataset, although this did not affect the
statistical interpretation of the data. Therefore, excretion
was measured for three corals in the ambient CO
2
treatment
and four in the high CO
2
treatment. Mean ambient [NH
4
]
in FSW supplied by the seawater system of NMMBA was
2.19 0.56
mol NH
4
l
1
(SE, n7), and bubbling
seawater during the excretion assay reduced mean ambient
[NH
4
] in control beakers by 0.24
mol NH
4
l
1
(SE,
n8), equivalent to a 20% reduction in ambient [NH
4
]
during the assay. CO
2
treatments affected NH
4
flux from
corals after 3-h incubations in darkness (F18.592, df
1,5, P0.008), with corals at ambient pCO
2
absorbing
NH
4
at 10.72 0.75 nmol NH
4
cm
2
h
1
, whereas
corals at high pCO
2
released NH
4
at 24.38 6.86 nmol
NH
4
cm
2
h
1
(both mean SE, n3– 4) (Fig. 3).
Discussion
The goal of this study was to evaluate the possibility that
high pCO
2
(ca. 85 Pa) perturbs protein metabolism in reef
corals. It was not our objective to test for roles of protein
metabolism in the reduction in calcification that occurs in
0
0.04
0.08
0.12
0 50 100 150 200 250
P = 0.03
ac
bab
c
nmol O2 larva-1 min-1
Emetine µmol L-1
Figure 1. Respiration of larvae from Pocillopora damicornis as a
function of emetine concentration. Emetine affected respiration (P
0.030). Mean SE shown (n6 for all treatments except 226
mol l
1
[n5]); treatment means marked with different letters differ significantly
(Fisher’s LSD post hoc analysis, P0.05).
73P. J. EDMUNDS AND C. B. WALL
most corals exposed to high pCO
2
(Chan and Connolly,
2013), but this possibility influenced the interpretation of
our findings. To achieve our goal, we assessed protein
metabolism in two experiments, one focused on protein
anabolism in coral recruits, and one focused on protein
catabolism in juvenile corals. Our results are consistent with
the hypothesis that exposure to about 85 Pa pCO
2
affects
protein metabolism in early life stages of corals, but they do
not demonstrate a cause-and-effect relationship. Elucidating
the causal relationship between high pCO
2
and protein
metabolism will require research that is more sophisticated
than described in the present analysis, potentially involving
radioisotope analysis of protein metabolism (e.g., Pace
et al., 2006) or studies of gene expression (sensu Kaniewska
et al., 2012), particularly in corals exposed to ocean acidi-
fication conditions and exhibiting depressed rates of calci-
fication.
The advantages of using early life stages of corals in
experimental studies lie in access to large numbers of indi-
viduals, the ability to conduct incubations in small volumes,
and the capacity to study biological events during profound
transitions in early development. A priori, therefore, the
biology of early life stages of corals can be expected to be
dynamic, as in the case of respiration for Seriatopora cali-
endrum recruits during the first5dofbenthic life (Edmunds
et al., 2013). In the present study, we measured the respi-
ration of newly recruited individuals of S. caliendrum, and
recorded values in seawater (i.e., the controls) at 2-d and 5-d
post-settlement that are similar to the trend we recorded
previously (Edmunds et al., 2013). Given the role of eme-
tine in inhibiting post-translation protein synthesis (Groll-
man, 1968; Pace and Manahan, 2006), our results imply that
0
0.02
0.04
0.06
0.08
0.10
Control Emetine
Ambient pCO2
Control Emetine
A) 2 day post-settlement B) 5 day post-settlement
Elevated pCO2
Treatment Time post-settlement (d)
nmolO2 recruit-1 min-1
Experiment 1
Experiment 2
0123456
Larval respiration
(Cumbo et al. 2012)
C) Temporal context
Ambient CO2
Elevated CO2
a
a
a
b
Figure 2. Respiration of recruits of Seriatopora caliendrum after incubations at ambient or elevated pCO
2
.
(A) Respiration after1dofsettlement,1dofexposure to ambient and high pCO
2
(Table 1), and 3-h exposure
to treatments with or without emetine (100
mol l
1
), (B) Respiration after1dofsettlement,4dofexposure
to ambient and high pCO
2
(Table 1), and 3-h exposure to treatments with or without emetine (100
mol l
1
),
and (C) Respiration of recruits in a previous experiment (Edmunds et al., 2013) conducted in March 2011 in
which corals were incubated through the early post-settlement phase (7 h–5.3 d post-settlement) at ambient (51.6
Pa) and elevated (86.4 Pa) pCO
2
during two experiments, both without emetine. Dashed line shows respiration
of free-swimming S. caliendrum larvae at 28.9 °C (Cumbo et al., 2012). Mean SE shown for all plots, with
sample sizes (A) n8 –10, (B) n4 –5), and (C) n4 6 in experiment 1 and n6 in experiment 2. Bars
in (A) marked with different letters differ significantly (Bonferroni post hoc analysis, P0.01).
Figure 3. Ammonium excretion rates of juvenile colonies of Seriato-
pora caliendrum held at ambient (A-CO
2
) and high (H-CO
2
)pCO
2
(Table
1). Negative values signify ammonia uptake from seawater; values dis-
played are mean SE (n3– 4).
74 PCO
2
AND PROTEIN IN CORALS
at ambient pCO
2
(46 Pa) protein synthesis accounts for
63% of aerobic respiration at 2-d post-settlement, but 31%
at 5-d post-settlement. High pCO
2
appears to affect the
contribution of protein metabolism to respiration at 2-d
post-settlement, as revealed by the inhibitory effect of em-
etine on respiration that was significant at ambient pCO
2
(where a 63% reduction occurred) but not at high pCO
2
(where a nonsignificant 27% decline occurred). pCO
2
had
no effect on respiration at 5-d post-settlement, but emetine
reduced respiration 27%. Thus, during the period of rapid
changes occurring within5dofsettling, protein metabolism
appears to account for the majority of metabolic costs in S.
caliendrum. High pCO
2
perturbs the contribution of protein
metabolism to respiratory costs within the first2dof
settlement, possibly by reducing the proportional contribu-
tion of protein synthesis to these costs, and increasing the
proportional contribution of “non-protein-related” processes.
While inhibitors of protein synthesis can support an ef-
fective coarse-grained analysis of protein anabolism, it is
more challenging to investigate protein catabolism in sym-
biotic corals because the product of deamination (i.e.,
NH
4
) is recycled through the Symbiodinium cells (Musca-
tine and D’Elia, 1978). In ammonotelic organisms that
cannot recycle NH
4
, it is more straightforward to measure
protein synthesis and NH
4
excretion in order to prepare
nitrogen budgets (Hawkins, 1985) and evaluate protein me-
tabolism (Langenbuch and Po¨rtner, 2003). In symbiotic
corals, however, nutrient recycling by Symbiodinium can
mask shifts in protein catabolism (Szmant et al., 1990),
although this effect can be prevented with metabolic inhib-
itors. For instance, azaserine can directly inhibit NH
4
uptake by inhibiting glutamate synthase in Symbiodinium
(Rahav et al., 1989), and herbicides like DCMU (3-(3,4-
dichlorophenyl)-1,1-dimethylurea) can inhibit photosyn-
thetic electron transport (Calvayrac et al., 1979), or cessa-
tion of NH
4
uptake can be induced in darkness (Muscatine
and D’Elia, 1978); under such conditions symbiotic corals
release NH
4
(Rahav et al., 1989). We explored the use of
DCMU and azaserine to inhibit NH
4
uptake by Seriato-
pora caliendrum, but the results were unsatisfactory relative
to the results of keeping the corals in darkness to achieve the
same outcome. With the concentrations of DCMU em-
ployed, we found the inhibition of NH
4
uptake to be
incomplete; moreover, azaserine (over a range of concen-
trations) resulted in tissue necrosis.
Ammonium concentrations [NH
4
] in tropical seawater
are typically low (0.05– 0.30
mol NH
4
l
1
) (Rahav et al.,
1989; Grover et al., 2002). While ambient [NH
4
]insea
-
water reported here is elevated (2
mol NH
4
l
1
), it is
within values reported for tropical reefs during summer
months (Hatcher and Frith, 1985). Juvenile Seriatopora
caliendrum maintained in ambient seawater (47.1 Pa pCO
2
)
took up NH
4
at 10.72 0.75 nmol NH
4
cm
2
h
1
during short-term exposure to darkness. Ammonium uptake
by symbiotic reef corals is expected in the light (Muscatine
and D’Elia, 1978), with the processes mediated by both the
coral host (Yellowlees et al., 1994) and Symbiodinium
(Muscatine and D’Elia, 1978) and decaying in extended
periods of darkness (Muscatine and D’Elia, 1978). Eventu-
ally, prolonged darkness (24 h) reverses the direction of
flux, and NH
4
is excreted (Muscatine and D’Elia, 1978;
Rahav et al., 1989). In the present case, it is unlikely that 3 h
of darkness was sufficient for the uptake of NH
4
by
Symbiodinium in S. caliendrum to decay appreciably (Mus-
catine and D’Elia, 1978), and therefore it is reasonable to
infer that juvenile S. caliendrum exposed to 47.1 Pa pCO
2
in
the light were taking up NH
4
at about 11 nmol NH
4
cm
2
h
1
. This rate is approximately half the NH
4
uptake
rate of Stylophora pistillata (Rahav et al., 1989), but is
within the range reported for S. pistillata by Godinot et al.
(2011) and similar to values reported for Pocillopora capi-
tata (Muscatine and D’Elia, 1978). However, when juvenile
S. caliendrum were maintained at 89.7 Pa pCO
2
for 14 d and
the flux of NH
4
measured in darkness, high rates of
excretion (24 nmol NH
4
cm
2
h
1
) were detected. As
the NH
4
flux was again recorded over 3 h of darkness, the
rates probably were indicative of those occurring in the
light, and therefore it is reasonable to infer that incubation
at high pCO
2
was associated with impaired NH
4
recycling.
The net NH
4
excretion rates for Seriatopora caliendrum
in darkness are 2-fold greater than those of Stylophora
pistillata under conditions inhibiting NH
4
uptake (9.0 –
2.0 nmol NH
4
cm
2
h
1
, Rahav et al., 1989), but are
similar to those reported for Pocillopora damicornis follow-
ing 19 h of darkness [1.0
mol NH
4
coral
1
h
1
(Mus-
catine and D’Elia, 1978), this study 0.7
mol NH
4
coral
1
h
1
]. Based on a protein content of 6 mg coral
1
(C. B.
Wall, unpubl. data) for the small colonies of S. caliendrum
used in the present study, and a 17% N fraction in proteins
of aquatic animals (Gnaiger and Bitterlich, 1984), the ex-
cretion rates reported here at 89.7 Pa pCO
2
represent hourly
protein catabolism of about 1% of the total protein content
(57
g protein coral
1
h
1
) in the tissue of S. caliendrum.
Proportionately similar rates of protein catabolism have
been reported in other corals (Szmant et al., 1990), and
therefore the estimated rates of protein loss in S. caliendrum
exposed to high pCO
2
are ecologically relevant. However, it
is important to note that they could not be sustained without
incurring a loss of protein biomass. Evaluating the implica-
tions of the NH
4
excretion reported in the present study, as
well as the inferred increase in deamination of protein and
amino acids, will require tests of hypotheses that can ex-
plain the results observed. While it was beyond the scope of
the present study to begin such work, there are two nonex-
clusive hypotheses that deserve attention: (1) catabolism of
proteins and amino acids is accelerated at high pCO
2
so that
75
P. J. EDMUNDS AND C. B. WALL
the deamination product (i.e., NH
3
) overwhelms the capac-
ity of the Symbiodinium to recycle nitrogen, or (2) the
capacity of Symbiodinium to take up NH
4
is impeded.
In summary, the present study reports two new findings to
the field considering the effects of high pCO
2
on corals: (1)
respiration of Seriatopora caliendrum recruits is depressed
by emetine, and 2 days after settlement, this effect is mod-
ulated by high pCO
2
; and (2) the flux of ammonium from
juvenile colonies of S. caliendrum changes from uptake to
efflux at high pCO
2
. These results support the hypothesis
that the effects of high pCO
2
on corals can include per-
turbed protein metabolism, although this conclusion re-
quires caution in interpretation because metabolic inhibitors
(like emetine) can have undesired (and unknown) effects on
processes other than the ones they are intended to inhibit
(here, post-translational protein synthesis). Nevertheless,
while our evidence is circumstantial, when interpreted
against what is currently known of the physiological basis
of the biological effects of high pCO
2
in living organisms
(Somero, 2012), we suggest it makes a stimulating argu-
ment for further investigations of this topic on corals and
other marine organisms.
Acknowledgments
This project was funded by the U.S. National Science
Foundation (OCE 08-44785 to PJE). We thank T.-Y. Fan
for hosting us at the National Museum of Marine Biology
and Aquarium (NMMBA); G. Baghdasarian, L. Bramanti,
S. Zamudio, and J. Smolenski for lab and field assistance;
and our friends and colleagues at NMMBA who made this
work enjoyable and productive. A portion of this work was
submitted in partial fulfillment of the MS degree at Califor-
nia State University, Northridge (CSUN), to CBW. This is
contribution number 213 of the CSUN Marine Biology
Program.
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77P. J. EDMUNDS AND C. B. WALL
... As ocean acidification increases the energetic cost of calcification (Cohen and Holcomb, 2009), a higher energy production potential (ETSA) could allow for a stronger maintenance of the aragonitic saturation state at the site of calcification, enabling the maintenance of inorganic growth even under elevated pCO 2 (McCulloch et al., 2012). This increased energetic potential relative to biomass would additionally support other metabolic processes important for the resistance against ocean acidification, such as high fecundity (Albright, 2011), protein synthesis (Edmunds and Wall, 2014), or the maintenance of other anabolic pathways (Kaniewska et al., 2015). ...
... In the present study, coral species that are sensitive to ocean acidification showed a decrease in their biomass regardless of whether they were acclimatized for 4-5 months or exposed for their entire lifetime to elevated pCO 2 . Similar decreases in biomass have been observed across multiple studies (Edmunds and Wall, 2014;Strahl et al., 2015;Wall et al., 2017) with the response attributed to an impairment of protein anabolism due to elevated pCO 2 . Two of the losing coral species (A. ...
Article
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Coral communities around the world are projected to be negatively affected by ocean acidification. Not all coral species will respond in the same manner to rising CO 2 levels. Evidence from naturally acidified areas such as CO 2 seeps have shown that although a few species are resistant to elevated CO 2 , most lack sufficient resistance resulting in their decline. This has led to the simple grouping of coral species into “winners” and “losers,” but the physiological traits supporting this ecological assessment are yet to be fully understood. Here using CO 2 seeps, in two biogeographically distinct regions, we investigated whether physiological traits related to energy production [mitochondrial electron transport systems (ETSAs) activities] and biomass (protein contents) differed between winning and losing species in order to identify possible physiological traits of resistance to ocean acidification and whether they can be acquired during short-term transplantations. We show that winning species had a lower biomass (protein contents per coral surface area) resulting in a higher potential for energy production (biomass specific ETSA: ETSA per protein contents) compared to losing species. We hypothesize that winning species inherently allocate more energy toward inorganic growth (calcification) compared to somatic (tissue) growth. In contrast, we found that losing species that show a higher biomass under reference p CO 2 experienced a loss in biomass and variable response in area-specific ETSA that did not translate in an increase in biomass-specific ETSA following either short-term (4–5 months) or even life-long acclimation to elevated p CO 2 conditions. Our results suggest that resistance to ocean acidification in corals may not be acquired within a single generation or through the selection of physiologically resistant individuals. This reinforces current evidence suggesting that ocean acidification will reshape coral communities around the world, selecting species that have an inherent resistance to elevated p CO 2 .
... Also, increased rates of ammonia excretion can indicate enhanced rates of protein catabolism. Studies on mussel and coral report elevated ammonia excretion rates under experimental ocean acidification (Michaelidis et al., 2005;Thomsen and Melzner, 2010;Edmunds and Wall, 2014). Another indirect assay of altered protein metabolism is to measure relative changes in oxygen consumption in the presence and absence of a protein synthesis inhibitor. ...
... Another indirect assay of altered protein metabolism is to measure relative changes in oxygen consumption in the presence and absence of a protein synthesis inhibitor. In coral, the effect of the inhibitor emetine on respiration rates was dependent on pCO 2 (Edmunds and Wall, 2014). Similarly, under acidification, the relative effect of the inhibitor cyclohexamide on metabolic rate of hepatocyte cells of Antarctic fish is depressed at pH 6.5 (vs. ...
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Exogenous environmental factors alter growth rates, yet information remains scant on the biochemical mechanisms and energy trade-offs that underlie variability in the growth of marine invertebrates. Here we study the biochemical bases for differential growth and energy utilization (as adenosine triphosphate [ATP] equivalents) during larval growth of the bivalve Crassostrea gigas exposed to increasing levels of experimental ocean acidification (control, middle, and high pCO2, corresponding to ∼400, ∼800, and ∼1100 µatm, respectively). Elevated pCO2 hindered larval ability to accrete both shell and whole-body protein content. This negative impact was not due to an inability to synthesize protein per se, because size-specific rates of protein synthesis were upregulated at both middle and high pCO2 treatments by as much as 45% relative to control pCO2. Rather, protein degradation rates increased with increasing pCO2. At control pCO2, 89% of cellular energy (ATP equivalents) utilization was accounted for by just 2 processes in larvae, with protein synthesis accounting for 66% and sodium-potassium transport accounting for 23%. The energetic demand necessitated by elevated protein synthesis rates could be accommodated either by reallocating available energy from within the existing ATP pool or by increasing the production of total ATP. The former strategy was observed at middle pCO2, while the latter strategy was observed at high pCO2. Increased pCO2 also altered sodium-potassium transport, but with minimal impact on rates of ATP utilization relative to the impact observed for protein synthesis. Quantifying the actual energy costs and trade-offs for maintaining physiological homeostasis in response to stress will help to reveal the mechanisms of resilience thresholds to environmental change.
... As precipitators of an external CaCO 3 skeleton, stony corals are thought to be under considerable stress from increasing atmospheric pCO 2 (Kleypas et al. 1999;Erez et al. 2011;Pandolfi et al. 2011). A variety of studies have shown that calcification rate (e.g., Anthony et al. 2008;Drenkard et al. 2013;Strahl et al. 2015) and related metabolic processes (ex; Edmunds and Wall 2014) are depressed in experimentally increased pCO 2 treatments for some coral species. Arguably, however, increased CO 2 levels do not always have adverse effects on coral calcification (Ries et al. 2009;Comeau et al. 2013;Strahl et al. 2015), and some taxa persist in naturally high CO 2 environments, such as near vents (Fabricius et al. 2011;Crook et al. 2012) or in bays that experience low aragonite saturation due to high calcification and respiration (Shamberger et al. 2014). ...
... In our study, a doubling of glucose resulted in increased gene transcription and protein production, but this did not translate to increased calcification, nor was there a boron isotope effect. This reinforces the important role of a fixed carbon source for SOM protein production, whose synthesis may be perturbed under increased pCO 2 (Edmunds and Wall 2014). Previous studies have shown, however, that addition of fixed carbon as glucose or glycerol leads to increased calcification (Holcomb et al. 2014a); this was not observed in this study. ...
Article
Understanding the cellular and molecular responses of stony corals to ocean acidification is key to predicting their ability to calcify under projected high CO2 conditions. Of specific interest are the links between biomineralization proteins and the precipitation of new calcium carbonate (CaCO3), which potentially can provide a better understanding of the biomineralization process. We have assessed the effects of increased CO2 on the calcification process in cell cultures of the stony coral, Stylophora pistillata, reared in nutrient-enriched artificial seawater at four pCO2 levels and two glucose concentrations. Dispersed S. pistillata cells grown at low (400 ppmV) and moderate (700 ppmV) pCO2 re-aggregate into proto-polyps and precipitate CaCO3. When grown at pCO2 levels of 1000 ppmV and 2000 ppmV, the cells up-regulate genes for two highly acidic proteins as well as a carbonic anhydrase, but down-regulate long term cadherin protein production and minimize proto-polyp formation, and exhibit a significant decrease in measurable CaCO3 precipitation. However, cell cultures precipitate CaCO3 in all treatments, even at slightly undersaturated conditions (Ωaragonite < 0.95). Glucose addition does not influence either biomineralization gene expression or calcification rate. Measured δ11B of the mineral phase, as a proxy of the pH at the calcifying sites, is out of equilibrium with the ambient seawater medium surrounding the cells and proto-polyps, suggesting pH is elevated in the micro-environment of the precipitating mineral. Our results suggest that coral cells possess molecular mechanisms to help compensate for the effects of ocean acidification within the bounds projected in the coming century.
... Towle et al. (2015) indicated that coral can buffer OA effects by increasing feeding rates under pH 7.8-8.0 and Edmunds and Wall (2014) showed that catabolism of proteins and amino acids is accelerated by OA (pH 7.8-8.0), supporting that OA may perturb protein metabolism. ...
Article
Ocean acidification has emerged as a major concern in the last fifteen years and studies on the impacts of seawater acidification on marine organisms have multiplied accordingly. This review aimed at synthesizing the literature on the effects of seawater acidification on tropical scleractinians under laboratory-controlled conditions. We identified 141 articles (published between 1999 and 2021) and separated endpoints into 22 biological categories to identify global trends for mitigation and gaps in knowledge and research priorities for future investigators. The relative number of affected endpoints increased with pH intensity (particularly for endpoints associated to calcification and reproduction). When exposed to pH 7.6-7.8 (compared to higher pH), 49% of endpoints were affected. The diversity in experimental designs prevented deciphering the modulating role of coral life stages, genera or duration of exposure. Finally, important bias in research efforts included most experiments on adult corals (68.5%), in 27 out of 150 (18%) coral ecoregions and exclusively from shallow-waters.
... (email: amy.briggs@uga.edu) of photosynthetic 9 and non-photosynthetic taxa 10 . These effects may not influence metabolic rates like photosynthesis, but could have significant consequences at the cellular level, affecting resource allocation among biological processes and potentially leading to shifts in organismal performance under certain environmental conditions. ...
Article
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Ocean acidification (OA) is predicted to enhance photosynthesis in many marine taxa. However, photophysiology has multiple components that OA may affect differently, especially under different light environments, with potentially contrasting consequences for photosynthetic performance. Furthermore, because photosynthesis affects energetic budgets and internal acid-base dynamics, changes in it due to OA or light could mediate the sensitivity of other biological processes to OA (e.g. respiration and calcification). To better understand these effects, we conducted experiments on Porolithon onkodes, a common crustose coralline alga in Pacific coral reefs, crossing pCO2 and light treatments. Results indicate OA inhibited some aspects of photophysiology (maximum photochemical efficiency), facilitated others (α, the responsiveness of photosynthesis to sub-saturating light), and had no effect on others (maximum gross photosynthesis), with the first two effects depending on treatment light level. Light also exacerbated the increase in dark-adapted respiration under OA, but did not alter the decline in calcification. Light-adapted respiration did not respond to OA, potentially due to indirect effects of photosynthesis. Combined, results indicate OA will interact with light to alter energetic budgets and potentially resource allocation among photosynthetic processes in P. onkodes, likely shifting its light tolerance, and constraining it to a narrower range of light environments.
... In the present study, total biomass (mg AFDW cm −2 ) was not affected by treatments, yet area-normalized protein (a common proxy for biomass; [41]) was reduced approximately 20% under 957 µatm pCO 2 and 15.7 mol photons m −2 d −1 . Together, high light and high pCO 2 may interfere with aspects of protein metabolism [36] or turnover [29] in P. acuta manifesting in reduced protein per skeletal surface [21,85]. However, in our study the total organic fraction of P. acuta biomass (i.e. ...
Article
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Ocean acidification (OA) is predicted to reduce reef coral calcification rates and threaten the long-term growth of coral reefs under climate change. Reduced coral growth at elevated pCO2 may be buffered by sufficiently high irradiances, however, the interactive effects of OA and irradiance on other fundamental aspects of coral physiology, such as the composition and energetics of coral biomass, remain largely unexplored. This study tested the effects of two light treatments (7.5 vs. 15.7 mol photons m-2 d-1) at ambient- or elevated-pCO2 (435 vs. 957 μatm) on calcification, photopigment and symbiont densities, biomass reserves (lipids, carbohydrates, proteins), and biomass energy content (kJ) of the reef coral Pocillopora acuta from Kāne‘ohe Bay, Hawai‘i. While pCO2 and light had no effect on either area- or biomass-normalized calcification, tissue lipids gdw-1 and kJ gdw-1 were reduced 15% and 14% at high pCO2, and carbohydrate content increased 15% under high light. The combination of high light and high pCO2 reduced protein biomass (per unit area) by ~ 20%. Thus, under ecologically relevant irradiances, P. acuta in Kāne‘ohe Bay does not exhibit OA-driven reductions in calcification reported for other corals; however, reductions in tissue lipids, energy content, and protein biomass suggest OA induced an energetic deficit and compensatory catabolism of tissue biomass. The null effects of OA on calcification at two irradiances support a growing body of work concluding some reef corals may be able to employ compensatory physiological mechanisms that maintain present-day levels of calcification under OA. However, negative effects of OA on P. acuta biomass composition and energy content may impact the long-term performance and scope for growth of this species in a high pCO2 world.
... These studies included microalgae (e.g., Gómez et al., 2001), macroalgae (e.g., Jover y Lake, 2008;Jover et al., 2012;Diez et al., 2013), gorgonians (e.g., Olivera et al., 2010aOlivera et al., , 2010b, molluscs (e.g., Espinosa et al., 2012;Diez y Jover, 2013b), and other invertebrates (e.g., Gómez et al., 2009;Diez, 2013;Diez y Jover 2013a). The phylum Cnidaria is one of the best represented in tropical seas (Calder, 2013;Fautin et al., 2013;Fernandez et al., 2014), mainly corals have been studied (Scleractinea) (e.g., Anderson and Pratech, 2014;Edmunds and Wall, 2014;Palumbi et al., 2014). ...
Article
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The Caribbean cnidarians are well studied, mainly the builders of reefs (Scleractinea). Cuban marine biodiversity is representative of the Caribbean Sea. However, many localities remain understudied to this moment. In this work, we present the checklist of 22 species of soft corals from the southeastern coast of Cuba. The specimens were collected or observed in shallow waters up to 25 m depth, in 11 localities in Granma, Santiago de Cuba and Guantánamo provinces. The studied ecosystems include rocky, sandy and muddy bottoms, coral reefs, seagrass and mangroves. The number of recorded species of soft corals in the southeastern coast represent the highest richness in the Cuban platform. Bunodosoma cavernatum is a new record to Cuba. Eighteen species are represented in Marine Protected Areas. It is necessary to assess the conservation status of the populations of these animals, and their use for monitoring the status of MPAs. RESUMEN Los cnidarios del Caribe están bien estudiados, en mayoría los forma-dores de arrecifes (Scleractinea). La biodiversidad marina cubana es rep-resentativa del Mar Caribe. No obstante, muchas localidades no han sido suficientemente estudiadas hasta el momento. En este trabajo se presenta la lista de 22 especies de corales blandos de la costa suroriental de Cuba. Los especímenes fueron recolectados u observados en aguas someras has-ta 25 m de profundidad, en 11 localidades de las provincias Granma, Santiago de Cuba y Guantánamo. Los ecosistemas estudiados incluy-eron los fondos rocosos, arenosos y fangosos, arrecifes coralinos, praderas de angiospermas y manglares. El número de especies de corales blan-dos registrado en la costa suroriental representa la mayor riqueza en la plataforma cubana. Bunodosoma cavernatum es un nuevo registro para Cuba. En las Áreas Marinas Protegidas están representadas 18 especies. Es necesario evaluar el estado de conservación de las poblaciones de estos organismos y su uso para monitorear el estado de las AMP.
... There is a long history of investigating the effects of damage on corals (Cole et al., 2008;Rotjan and Lewis, 2008;Bruckner and Bruckner, 2016), and recent interest in the response of corals to ocean acidification (Chan and Connolly, 2013;Kroeker et al., 2013), but little is known of the interaction between these stressors in terms of their effects on coral performance (but see Renegar et al., 2008;Horwitz and Fine, 2014;Hall et al., 2015). A prior, it was reasonable to hypothesize that regeneration of coral tissue and skeleton following damage would be affected by ocean acidification, potentially because the physical and chemical consequences of this phenomenon can alter the use of metabolic energy by metazoans (Pan et al., 2015), and in reef corals, can modulate holobiont respiration and photochemical efficiency (Anthony et al., 2008;Crawley et al., 2010;Edmunds, 2012;Kaniewska et al., 2012; but see Schneider and Erez, 2006;Comeau et al., 2016), calcification (Erez et al., 2011;Chan and Connolly, 2013), and protein metabolism (Kaniewska et al., 2012;Edmunds and Wall, 2014). In contrast to this (21) 3.36 ± 0.07 (21) 6 ...
Article
Scleractinian corals on tropical reefs are exposed to many natural and anthropogenic disturbances, and while much is known about their responses to such conditions, it is unclear whether the responses will remain the same in a future affected by climate change and ocean acidification. To evaluate how one aspect of these effects — wound repair — might be influenced by ocean acidification, small colonies of massive Porites spp. from the back reef of Moorea, French Polynesia, were damaged to simulate the effects of single bites by corallivorous fishes, and healing was measured under contrasting PCO2 regimes. Using experiments lasting 19–20 d and employing superficial (2013) or deep (2014) lesions, the effects of damage were evaluated at ~ 400 μatm (ambient) and ~ 1000 μatm PCO2 (both at ~ 28.5 °C) using calcification and healing as dependent variables. Damage reduced calcification of massive Porites spp. in 2013, but not in 2014, and PCO2 had no effect on area-normalized calcification or healing in both years, although biomass-normalized calcification was reduced by high PCO2 in 2014. Overall these results reveal the physiological resilience of this functional group of corals to the extent of ocean acidification expected by the end of this century, and suggest that over this period small colonies will remain capable of recovery from minor damage arising from fish corallivory.
... For example, massive Porites responds to high pCO 2 (~800 µatm) with changes in biomass (Edmunds 2011), and shifts in the energy content of tissue characterize the response of at least 2 coral species to elevated pCO 2 (Schoepf et al. 2013). Moreover, there is indirect evidence that high pCO 2 (760 µatm) affects the protein metabolism of Seriatopora caliendrum (Edmunds & Wall 2014). ...
Article
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We tested the hypothesis that ocean acidification (OA) affects spatial competition among scleractinian corals. Competitive ability was evaluated indirectly by linear extension of Porites lutea and Montipora aequituberculata placed in intraspecific, interspecific, and control pairings (paired with dead coral skeleton) and exposed to ambient (similar to 400 mu atm) and elevated (similar to 1000 mu atm) pCO(2) in experiments conducted in Moorea, French Polynesia, and Okinawa, Japan. High pCO(2) had no effect on linear extension of M. aequituberculata in Moorea, but in Okinawa, it reduced linear extension 37%; high pCO(2) had no significant effect on linear extension of P. lutea in Okinawa. Much of the negative effect of high pCO(2) on linear extension for M. aequituberculata in Okinawa was due to reduced extension in control pairings, with corals engaged in intra- and interspecific competition unaffected by OA. Linear extension of M. aequituberculata and P. lutea in interspecific pairings decreased relative to control pairings at ambient pCO(2) by 39 and 71%, respectively, indicating a strong effect of competition on extension rates. These differences however disappeared at elevated pCO(2) when the linear extension of controls was depressed. Together, our results show that OA can negatively affect the linear extension of corals not engaged in competition, as shown in the control pairings, and suggest that OA does not directly affect the ability of corals to compete with one another for space.
Article
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The absorbtion of human-emitted CO2 by the oceans (elevated PCO2) is projected to alter the physiological performance of coral reef organisms by perturbing seawater chemistry (i.e. ocean acidification). Simultaneously, greenhouse gas emissions are driving ocean warming and changes in irradiance (through turbidity and cloud cover), which have the potential to influence the effects of ocean acidification on coral reefs. Here, we explored whether physiological impacts of elevated PCO2 on a coral–algal symbiosis (Pocillopora acuta–Symbiodiniaceae) are mediated by light and/or temperature levels. In a 39 day experiment, elevated PCO2 (962 versus 431 µatm PCO2) had an interactive effect with midday light availability (400 versus 800 µmol photons m−2 s−1) and temperature (25 versus 29°C) on areal gross and net photosynthesis, for which a decline at 29°C was ameliorated under simultaneous high-PCO2 and high-light conditions. Light-enhanced dark respiration increased under elevated PCO2 and/or elevated temperature. Symbiont to host cell ratio and chlorophyll a per symbiont increased at elevated temperature, whilst symbiont areal density decreased. The ability of moderately strong light in the presence of elevated PCO2 to alleviate the temperature-induced decrease in photosynthesis suggests that higher substrate availability facilitates a greater ability for photochemical quenching, partially offsetting the impacts of high temperature on the photosynthetic apparatus. Future environmental changes that result in moderate increases in light levels could therefore assist the P. acuta holobiont to cope with the ‘one–two punch’ of rising temperatures in the presence of an acidifying ocean.
Article
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Juvenile colonies of massive Porites spp. were exposed to manipulated pH and bicarbonate ([HCO3(-)]) in situ to test the hypothesis that ocean acidification (OA) does not affect respiration and calcification. Incubations lasted 28 h and exposed corals to ambient temperature and light with ecologically relevant water motion. Three treatments were applied: (1) ambient conditions of pH 8.04 and 1751 μmol HCO3(-) kg(-1) (Treatment 1), (2) pCO2-induced ocean acidification of pH 7.73 and 2011 μmol HCO3(-) kg(-1) (Treatment 2), and (3) pCO2 and HCO3(-)-enriched seawater of pH 7.69 and 2730 μmol HCO3(-) kg(-1) (Treatment 3). The third treatment providing elevated [HCO3(-)] was used to test for stimulatory effects of dissolved inorganic carbon on calcification under low pH and low saturation of aragonite (Ωarag), but it does not reflect conditions expected to occur under CO2-driven OA. Calcification of juvenile massive Porites spp. was affected by treatments, with an 81% elevation in Treatment 3 versus Treatment 1, but no difference between Treatments 1 and 2; respiration and the metabolic expenditure concurrent with calcification remained unaffected. These findings indicate that juvenile massive Porites spp. are resistant to short exposures to OA in situ, and separately, that they can increase calcification at low pH and low Ωarag if [HCO3(-)] is elevated. Juvenile Porites spp. may therefore be limited by dissolved inorganic carbon under ambient pCO2 conditions.
Article
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The objective of this study was to test whether elevated pCO2 predicted for the year 2100 (85.1 Pa) affects bleaching in the coral Seriatopora caliendrum (Ehrenberg 1834) either independently or interactively with high temperature (30.5 °C). Response variables detected the sequence of events associated with the onset of bleaching: reduction in the photosynthetic performance of symbionts as measured by maximum photochemical efficiency (F v/F m) and effective photochemical efficiency (ΔF/F m′) of PSII, declines in net photosynthesis (P net) and photosynthetic efficiency (alpha, α), and finally, reduced chlorophyll a and symbiont concentrations. S. caliendrum was collected from Nanwan Bay, Taiwan, and subjected to combinations of temperature (27.7 vs. 30.5 °C) and pCO2 (45.1 vs. 85.1 Pa) for 14 days. High temperature reduced values of all dependent variables (i.e., bleaching occurred), but high pCO2 did not affect Symbiodinium photophysiology or productivity, and did not cause bleaching. These results suggest that short-term exposure to 81.5 Pa pCO2, alone and in combination with elevated temperature, does not cause or affect coral bleaching.
Article
The water flow régimes on a coral reef change with depth, distance from shore, and orientation to incoming waves. Low flow conditions may limit the ability of corals to capture prey, because the rate of encounter with particles suspended in the water column depends on the transport of water past the corals’ feeding structures. High flow speeds, however, cause deformation or collapse of feeding structures, and prey capture success is likely to decrease under such conditions. The specific relationship between flow speed and particle capture by passive suspension feeders like anthozoans may be determined by tentacle size and shape, tentacle and polyp stiffness and colony morphology. The importance of particle capture, as well as the types (zooplankton, detritus) and the size range of particles captured by corals, are almost unknown. The only published information for any species to date is Porter’s (1974) report on coelenteron contents of Montastrea cavernosa. The relationship between water flow and particle (hydrated Artemia cysts) capture has been studied for two octocoral (Alcyonium) species by Patterson (1984) and McFadden (1986) and for one scleractinian coral (Meandrina meandrites, Johnson & Sebens, unpubl.) in laboratory flume studies. The present study is an attempt to relate prey capture to water flow under field conditions for two species (Meandrina meandrites, Madracis decactis) in Salt River Canyon, St Croix, U.S.V.I., using the underwater habitat ‘Aquarius’ in July 1988. Concurrent measurements of water flow and wave height were made at several positions on the reef between 7 and 45 m using recording electromagnetic current meters (Interocean S4) mounted on rigid posts 0.5 m off the substrate. During the same two-week period, particle release (hydrated Artemia cysts) experiments were carried out over colonies of Meandrina and Madracis in the field. Particles were released upstream of the corals for 3 min, after which corals were caused to retract their tentacles, and flow above the coral was measured using macrovideo photography of moving cysts in a slit beam of light parallel to the flow. The corals were then collected and preserved under water for coelenteron content analysis, including both captured cysts and natural prey (Zooplankton). Particle concentrations were determined from samples collected by pumping water from directly over the coral during the experiment, using intake heads that allowed omnidirectional lateral sampling.
Article
Environmental stresses such as hypoxia or hypercapnia are known to cause acid-base disturbances and in several organisms they lead to metabolic depression. The present study was undertaken to quantify the influence of these changes in acid-base parameters on metabolic rate. We determined the rate of oxygen consumption in a non-perfused preparation of the body wall musculature of the marine worm Sipunculus nudus at various levels of extra- and intracellular pH (pHe and pHi, respectively), P(CO2) and [HCO3-]. The acid-base status of the tissue was modified and clamped by long- term exposure to media set to specific values of extracellular pH, P(CO2) and [HCO3-]. At a pile of 7.90, which is equivalent to the normoxic normocapnic in vivo extracellular pH, and an ambient P(CO2) of 0.03 kPa (control conditions), pHi was 7.26±0.02 (mean ± S.D., N=5). A reduction of extracellular pH from 7.90 to 7.20 resulted in a significant decrease of phi to 7.17±0.05 at 0.03 kPa P(CO2) (normocapnia) and to 7.20±0.02 at 1.01 kPa P(CO2) (hypercapnia). At the same time, the rate of oxygen consumption of the tissue was significantly depressed by 18.7±4.7% and 17.7±3.0%, respectively. A significant depression of oxygen consumption by 13.7±4.7 % also occurred under hypercapnia at pHe 7.55 when pHi was elevated above control values (7.32±0.01). No significant changes in oxygen consumption were observed when pile was either drastically elevated to 8.70 under normocapnia (pHi 7.36±0.05) or maintained at 7.90 during hypercapnia (pHi 7.37±0.03). ATP and phospho-L-arginine concentrations, as well as the Gibbs free energy change of ATP hydrolysis (dG/dξ(ATP)), were maintained at high levels during all treatments, indicating an equilibrium between energy supply and demand. We conclude that the depression of aerobic energy turnover in isolated body wall musculature of S. nudus is induced by low extracellular pH. A model is proposed which could explain a reduced ATP cost of phi regulation during extracellular acidosis, thus contributing to metabolic depression.
Article
I tested the hypothesis that the effects of high pCO2 and temperature on massive Porites spp. (Scleractinia) are modified by heterotrophic feeding (zooplanktivory). Small colonies of massive Porites spp. from the back reef of Moorea, French Polynesia, were incubated for 1 month under combinations of temperature (29.3°C vs. 25.6°C), pCO2 (41.6 vs. 81.5 Pa), and feeding regimes (none vs. ad libitum access to live Artemia spp.), with the response assessed using calcification and biomass. Area-normalized calcification was unaffected by pCO2, temperature, and the interaction between the two, although it increased 40% with feeding. Biomass increased 35% with feeding and tended to be higher at 25.6°C compared to 29.3°C, and as a result, biomass-normalized calcification statistically was unaffected by feeding, but was depressed 12-17% by high pCO2, with the effect accentuated at 25.6°C. These results show that massive Porites spp. has the capacity to resist the effects on calcification of 1 month exposure to 81.5 Pa pCO2 through heterotrophy and changes in biomass. Area-normalized calcification is sustained at high pCO2 by a greater biomass with a reduced biomass-normalized rate of calcification. This mechanism may play a role in determining the extent to which corals can resist the long-term effects of ocean acidification.
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Humans are continuing to add vast amounts of carbon dioxide (CO2) to the atmosphere through fossil fuel burning and other activities. A large fraction of the CO2 is taken up by the oceans in a process that lowers ocean pH and carbonate mineral saturation state. This effect has potentially serious consequences for marine life, which are, however, difficult to predict. One approach to address the issue is to study the geologic record, which may provide clues about what the future holds for ocean chemistry and marine organisms. This article reviews basic controls on ocean carbonate chemistry on different timescales and examines past ocean chemistry changes and ocean acidification events during various geologic eras. The results allow evaluation of the current anthropogenic perturbation in the context of Earth's history. It appears that the ocean acidification event that humans are expected to cause is unprecedented in the geologic past, for which sufficiently well-preserved records are available.
Article
I tested the hypothesis that high pCO2 (76.6 Pa and 87.2 Pa vs. 42.9 Pa) has no effect on the metabolism of juvenile massive Porites spp. after 11 days at 28 °C and 545 μmol quanta m−2 s−1. The response was assessed as aerobic dark respiration, skeletal weight (i.e., calcification), biomass, and chlorophyll fluorescence. Corals were collected from the shallow (3–4 m) back reef of Moorea, French Polynesia (17°28.614′S, 149°48.917′W), and experiments conducted during April and May 2011. An increase in pCO2 to 76.6 Pa had no effect on any dependent variable, but 87.2 Pa pCO2 reduced area-normalized (but not biomass-normalized) respiration 36 %, as well as maximum photochemical efficiency (F v/F m) of open RCIIs and effective photochemical efficiency of RCIIs in actinic light (∆F/ $ F_{\text{m}}^{\prime } $ ); neither biomass, calcification, nor the energy expenditure coincident with calcification (J g−1) was effected. These results do not support the hypothesis that high pCO2 reduces coral calcification through increased metabolic costs and, instead, suggest that high pCO2 causes metabolic depression and photochemical impairment similar to that associated with bleaching. Evidence of a pCO2 threshold between 76.6 and 87.2 Pa for inhibitory effects on respiration and photochemistry deserves further attention as it might signal the presence of unpredictable effects of rising pCO2.
Article
We tested the effects of pCO2 on Seriatopora caliendrum recruits over the first 5.3 d of post-settlement existence. In March 2011, 11–20 larvae were settled in glass vials (3.2 mL) and incubated at 24.0 °C and ~ 250 μmol quanta m− 2 s− 1 while supplied with seawater (at 1.4 mL s− 1) equilibrated with 51.6 Pa pCO2 (ambient) or 86.4 Pa pCO2. At 51.6 Pa pCO2, mean respiration 7 h post-settlement was 0.056 ± 0.007 nmol O2 recruit− 1 min− 1, but rose quickly to 0.095 ± 0.007 nmol O2 recruit− 1 min− 1 at 3.3 d post-settlement, and thereafter declined to 0.075 ± 0.002 nmol O2 recruit− 1 min− 1 at 5.3 d post-settlement (all ± SE). Elevated pCO2 depressed respiration of recruits by 19% after 3.3 d and 12% overall (i.e., integrated over 5.3 d), and while it had no effect on corallite area, elevated pCO2 was associated with weaker adhesion to the glass settlement surface and lower protein biomass. The unique costs of settlement and metamorphosis for S. caliendrum over 5.3 d are estimated to be 257 mJ recruit− 1 at 51.6 Pa pCO2, which is less than the energy content of the larvae and recruits.