Ocean acidification reduces feeding rates in the scleractinian coralStylophora pistillata

Fanny Houlbreque,1* St�ephanie Reynaud,2 Claire Godinot,2 Francois Oberh€ansli,3

Riccardo Rodolfo-Metalpa,1 Christine Ferrier-Pages 2

1Laboratoire d’Excellence ‘Corail’, Institut de Recherche pour le D�eveloppement, UR 227, CoReUs, Noum�ea, New Caledonia2Centre Scientifique de Monaco, Monaco, Principality of Monaco3International Atomic Energy Agency, Environment Laboratories, Monaco, Principality of Monaco

Abstract

A general decline in coral calcification has been observed in response to ocean acidification. It has recently

been shown that heterotrophic feeding (the acquisition of organic nutrients) enables one massive coral spe-

cies to maintain its calcification rates while exposed to ocean acidification but the consequences of higher

seawater partial pressure of CO2 (PCO2) on coral feeding capacities themselves have never been investigated.

Exposing long-term acclimated starved and fed colonies of the major reef builder Stylophora pistillata to either

ambient pHT (8.06 6 0.05; 417 Pa PCO2) or low pHT (7.51 6 0.06; 1697 Pa PCO2

) showed that higher feeding

rates can counterbalance the negative effects of seawater acidification on coral calcification. Indeed, relative

to unfed corals under ambient pHT conditions, calcification rates in unfed corals decreased by 53.6% but

remained constant in fed corals under high PCO2conditions. Results also clearly show that acidification

affects the feeding capacity and feeding effort of the branching coral S. pistillata. Organic nutrient acquisition

was severely reduced under high PCO2, with a simultaneous decrease in the dissolved free amino acid uptake

rates, the alkaline phosphatase activities, and the feeding rates on Artemia salina nauplii. Considering that all

these processes are needed to fulfill the nutrient needs of the symbiotic association, on a long-term basis,

lower feeding rates will likely aggravate the already fragile physiological state of many corals under warmer,

more acidic conditions.

Reef-building corals, also called scleractinian corals, are scat-

tered throughout the oligotrophic tropical and subtropical

oceans. Their capacity to live in oligotrophic waters is probably

due to their polytrophic character, as they use both heterotro-

phy (incorporation of particulate and dissolved organic matter)

and autotrophy (photosynthates translocated from their symbi-

otic algae) as energy sources (reviewed by Houlbreque and

Ferrier-Pages 2009). Heterotrophy can account for more than

60% of the corals’ metabolic needs, depending on the coral spe-

cies and on the environmental conditions (reviewed by Houl-

breque and Ferrier-Pages 2009), although autotrophy is often

the primary nutrient source (Muscatine 1990).

Over the last decade, corals are facing several threats,

mainly due to human influences (Hughes et al. 2003). Rapid

increases in the atmospheric carbon dioxide concentration,

by driving global warming and ocean acidification, may be

the final danger to these ecosystems (Hoegh-Guldberg et al.

2007). These stressors may impair coral photosynthesis and

autotrophic carbon acquisition (Hoegh-Guldberg et al. 2007;

Anthony et al. 2008), and heterotrophy may act as a major

nutrient source for some coral species (Grottoli et al. 2006).

In this case, feeding was shown to maintain rates of calcifica-

tion, photosynthesis, tissue growth, and symbiont densities

(Grottoli et al. 2006; Ferrier-Pages et al. 2010). The positive

role of heterotrophy on coral metabolism depends on the

food concentration available to corals (Palardy et al. 2006),

as well as the feeding capacity and feeding effort, which are

species specific (Grottoli et al. 2006). Thus, some species are

able to increase their feeding rates on zooplankton when

needed, such as during bleaching events or in deep waters

while some others do not (Anthony and Fabricius 2000;

Grottoli et al. 2006). Feeding rates can also be affected by

physical factors, such as water flow and light level (Ferrier-

Pages et al. 1998).

Thus, the feeding capacity and feeding effort of individual

coral species, in normal and stress conditions, are key factors

for the determination of their resistance and resilience to

stress. The species Stylophora pistillata, is known to be “a typi-

cal heterotrophic species,” as it can use a large range of prey

size. Moreover, higher feeding rates under laboratory*Correspondence: fanny.houlbreque@ird.fr

1

LIMNOLOGYand

OCEANOGRAPHY Limnol. Oceanogr. 00, 2015, 00–00VC 2015 Association for the Sciences of Limnology and Oceanography

doi: 10.1002/lno.10003

conditions significantly stimulated its physiology (Houlbre-

que et al. 2004; Houlbreque and Ferrier-Pages 2009), both

under normal and thermal stress conditions (Ferrier-Pages

et al. 2010). However, under thermal stress, S. pistillata also

decreased its heterotrophic feeding (Ferrier-Pages et al. 2010;

Hoogenboom et al. 2012) and its acquisition of inorganic

nutrients (Godinot et al. 2011), meaning that unless food

supply is ad libitum, it will not sustain its metabolism. All

together, these first results suggest that this species, present

in most of the world’s reefs might be severely affected in the

near future if seawater temperature continues to rise.

This study aims at complementing the knowledge on the

feeding capacities and feeding effort of this species under

another main stress factor for corals, represented by seawater

acidification. It is already known that ocean acidification,

through a decrease in pH and carbonate ion concentration,

has negative effects on coral calcification, under not only

long-term but also short-term application (Anthony et al.

2008), and S. pistillata was shown to be affected by such stress

(Krief et al. 2010; Houlbreque et al. 2012). However, one study

has also recently shown that the massive species Porites spp.,

fed to repletion, was able to maintain its calcification rates

under a short-term incubation under low seawater pH

(Edmunds 2011), suggesting that feeding may counterbalance

both the negative effects of increased seawater acidification

and temperature on corals. This study has, thus, assessed both

the effect of feeding and ocean acidification on S. pistillata

metabolism, and also how ocean acidification will change the

feeding capacities of this species. This latter point has never

been investigated. Results will allow us to have a global view

on the effects of climate change on this very well studied spe-

cies and can be used in future models forecasting the effect of

climate change on branched coral species.

Methods

Aquaria setup

Five parent colonies of the scleractinian coral S. pistillata

(Esper 1797) were divided into 104 terminal portions of

branches (5-cm long fragments). Nubbins obtained were

attached to nylon wires. After three weeks, coral fragments

were entirely covered with new tissues. They were assigned

to eight 20-liter tanks (each colony being represented twice

in each tank), continuously supplied with clean carbon-

filtered seawater, pumped in front of the International

Atomic Energy Agency laboratories (Monaco). Temperature

(26.0 6 0.2�C) was kept constant in each tank using heaters

connected to electronic controllers (6 0.2�C precision), and

seawater was mixed using a submersible pump (Aquarium

systemVR , mini-jet 606). Corals received a constant irradiance

of 200 6 10 lmol photons m22 s21 (photoperiod was 12 : 12

light : dark) using metal halide lamps (PhilipsVR , HPI-T, 400

W). Ammonium, nitrate, nitrite, and phosphate were meas-

ured weekly in each tank with an AllianceVR II autoanalyser

to verify that concentrations remained low in all tanks

(<0.04 lmol L21 ammonium, 0 lmol L21 to 0.09 lmol L21

phosphate, and <0.5 lmol L21 for nitrate and nitrite).

Corals were first divided into two groups (four tanks per

condition) corresponding to two feeding levels: (1) starved

corals (SC) were not fed during the whole experiment; (2) fed

corals (FC) were fed four days per week during 1 h with Arte-

mia salina nauplii (1200 6 50 nauplii L21). The ingestion of

prey has been monitored in a previous experiment under a

dissecting microscope during feeding (Ferrier-Pages et al.

2010). After a first step of 12 weeks under these two nutri-

tional conditions, corals were either maintained for five addi-

tional weeks at ambient pHT (total scale; 8.06 6 0.05, 417 Pa

PCO2) or at low pHT (7.51 6 0.06; 1697 Pa PCO2

), with two

tanks per pHT and feeding conditions. pHT was controlled

using a pH-stat system (IKS Aquastar, precision 6 0.05 pH

unit) by independently bubbling pure CO2 in each tank. Each

tank was also continuously aerated with CO2-free air.

Seawater carbonate chemistry

The pH values of the pH-stat system were adjusted every

day from the measurements of pHT using a pH meter (Met-

rohmVR , 826pH mobile) with a glass electrode (MetrohmVR ,

electrode plus) calibrated on the total scale using Tris-HCl

buffer solution with a salinity of 38 (Dickson et al. 2007).

Mean pHT were calculated from hydrogen ion concentra-

tions of each measurement and then reconverted back to pH

(Dickson et al. 2007). Water samples were collected in glass

bottles and then passed through 0.45 lm pore size filters

(GF/F Whatman) and poisoned with 0.05 mL of 50% HgCl2(Merck, AnalarVR ) to avoid biological alteration and stored in

the dark at 4�C. Three replicated 20 mL subsamples were

analyzed at 25�C using a titration system composed of a pH

meter with a pH electrode and a 1 mL automatic burette

(MetrohmVR ). The pH was measured at 0.02 mL increments

of 0.1 mol L21 HCl. AT was calculated from the Gran func-

tion applied to pH variations from 4.2 to 3.0 as mEq L21

from the slope of the curve HCl volume vs. pH. Titrations of

total alkalinity standards provided by A.G. Dickson (batch

99 and 102) were within 0.7 lmol kg21 of the nominal

value. Mean AT of seawater was 2.482 6 0.025 mmol kg21 (n

5 20). PCO2was calculated from pHT, mean AT, temperature,

and salinity using the free access CO2 Systat package.

Physiological measurements

Calcification rates

Calcification rates were assessed at the end of the experi-

ment of incubation under the different feeding regimes and

PCO2conditions using 45Ca, according to a method adapted

from Tambutt�e et al. (1995). Five corals from each tank (n 5

10 per treatment) were randomly placed during 7 h into four

independent 7-liter tanks, where each received a spike of the

radiotracer 45Ca (45CaCl2, PerkinElmer Life and Analytical

Science Products) to reach an activity of 50 KBq L21. The

medium was continuously mixed using a submersible pump

Houlbreque et al. Acidification and Stylophora nutrition

2

(Aquarium system, mini-jet 606). Light and temperature con-

ditions were similar to those described above. One-milliliter

aliquots of seawater were removed at the beginning of the

incubation for the determination of the specific radioactivity.

At the end of the labeling period, colonies were rinsed in 30

mL unlabeled seawater for 1 h to achieve isotopic dilution of

the 45Ca contained in the coelenteron. Tissue was then dis-

solved completely in 1 mol L21 NaOH at 90�C for 20 min,

and the skeleton was rinsed twice in 1 mL NaOH and once in

distilled water. Skeletons were then dried, weighed, and dis-

solved in 1.5 mL of 12 mol L21 HCl. Liquid scintillation

medium (10 mL; UltimagoldVR Miniature Vials, Packard) was

added to the radioactive samples (seawater and dissolved skel-

etons), and b-emissions were measured using a liquid scintil-

lation counter (2100 Time-Resolved, Packard; TricarbVR ). We

considered a seawater calcium concentration of 10 mmol L21

(Tambutt�e et al. 1995). Results are expressed in nmol Ca21

g21 dry skeletal mass h21 (Houlbreque et al. 2004).

Photosynthesis, respiration, and photosystem II

photochemistry

Photosynthesis and respiration rates were measured at the

end of the experiment on five corals taken from each tank (n

5 10 per treatment) according to the same protocol as in

Houlbreque et al. (2012), using StrathkelvinVR 928R electrodes

and incubating nubbins at the right pHT and at the culture

irradiance. Data were normalized to skeletal surface area.

Chlorophyll a and c2 were extracted twice in 99% acetone (24

h at 4�C). The extracts were centrifuged at 11,000 3 g for 10

min at 4�C and the absorbances measured at 630 nm, 663

nm, and 750 nm. Chlorophyll contents were computed

according to the equations of Jeffrey and Humphrey (1975).

Surface area was determined using the wax-dipping method

described in Stimson and Kinzie (1991). In addition, their

photosynthetic efficiency (Fv : Fm), electron transport rate

(ETR) of the photosystem II (PSII) of zooxanthellae in hospite

as well as Quantum yield of nonphotochemical fluorescence

quenching other than caused by downregulation of the light-

harvesting function (Y(NO)) and quantum yield of nonphoto-

chemical fluorescence quenching due to downregulation of

the light-harvesting function (Y(NPQ)) were measured using a

Diving Pulse Amplitude Modulated fluorometerVR (Walz),

according to Ralph and Gademann (2005). In vivo, chloro-

phyll takes the form of pigment-protein complexes that are

embedded in thylakoid membranes and funnel excitation

energy to photosynthesis reaction centers (RCs). In darkness,

when all RCs are open, chlorophyll fluorescence from PSII is

minimal (denoted F0). Conversely, when all RCs are closed

after illumination with a saturating pulse, chlorophyll fluores-

cence is maximal (denoted Fm) and the ratio of Fm to F0 indi-

cates the maximum quantum yield of PSII. Under

illumination with photosynthetically active radiation (PAR),

when a subsequent saturating pulse is applied, the maximal

and minimal fluorescence yields (F0m and F

0

0) changes due to

the induction of nonphotosynthetic quenching mechanisms.

The saturation pulse method allows the partitioning of

absorbed excitation energy between light used for photo-

chemistry, Y(II) and excitation that is dissipated. The latter

component is split into regulated and nonregulated processes.

Regulated dissipation, Y(NPQ), is thought to function to pro-

tect the photosynthetic units from damage due to excess light

absorption. Nonregulated dissipation, Y(NO), is a conse-

quence of PSII RC closures due to inhibition of electron trans-

port. A high value of Y(NPQ) indicates that irradiance is

excessive but shows that the sample has retained the regula-

tory mechanisms to protect its photosynthetic apparatus. A

high value of Y(NO) indicates that both the photochemical

energy conversion and the protective regulatory mechanisms

are insufficient to process excitation energy (Hoogenboom

et al. 2012).

Total organic carbon fluxes

The beaker incubation technique (Wild et al. 2005) was

used to quantify net total organic carbon (TOC) flux rates in

the different conditions. For this purpose, at the end of the

experiment six corals in each condition (n 5 3 for each

tank) were transferred without aerial exposure into acid-

washed and seawater-rinsed 250-mL glass beakers filled with

200 mL 0.4 lm filtered seawater at the right pH. Control

beakers contained only seawater (n 5 3 for each pH condi-

tion). Stirring was applied by Teflon-coated magnetic stirring

bars. After 6 h, corals were transferred back to the experi-

mental tanks. Before and after incubations, seawater subsam-

ples (17 mL, n 5 3 per beaker and sampling) were drawn

with sterile syringes from the homogenized incubation

media into precombusted (450�C, 5 h) glass vials. Vials were

acidified with phosphoric acid (20%, 250 lL) to pH < 2 and

kept frozen (220�C) until analysis by high-temperature cata-

lytic oxidation using a TOC analyzer (ShimadzuVR TOC-VCPH;

CV maximum �1.5%, i.e., 6 1 lmol C L21; referenced by

Consensus Reference Materials of Hansell Research Labora-

tory, University of Miami). For calculation of TOC net flux

rates, concentration differences measured from the control

beakers were subtracted from those measured in coral-

containing beakers, and the results were normalized to incu-

bation time and corals-specific skeletal surface area.

Feeding measurements

To assess feeding capacity of S. pistillata under normal

and low pH conditions, several measurements were per-

formed on the same samples used for TOC flux rates. Alka-

line phosphatase activity (APA) and uptake of dissolved free

amino acids (DFAA) were measured to estimate the utiliza-

tion of dissolved organic phosphorus and nitrogen, respec-

tively. Grazing rates on A. salina nauplii provided a measure

of the capacity to feed on particulate organic matter.

APA assays

APA was performed according to Godinot et al. (2013),

using the nonbiological substrate p-nitrophenyl phosphate

Houlbreque et al. Acidification and Stylophora nutrition

3

(pNPP), which yields a colored product, p-nitrophenolate, on

hydrolysis. For this purpose, corals were individually

incubated in 250-mL beakers filled with 200 mL of filtered

(0.2 lm) seawater at the pH corresponding to their mainte-

nance tanks (either pHT 8.06 or 7.51) and continuously agi-

tated with a magnetic stirrer. Beakers without coral served as

a control for APA activity of the seawater and for any nonen-

zymatic hydrolysis of the pNPP substrate. At the end of the

experiments, corals were frozen for subsequent normaliza-

tion of the data to the protein and skeletal surface area. Pro-

teins were extracted in 1 mol L21 NaOH for 30 min at 90�C,

measured using the commercially available protein Assay Kit

(BiCinchoninic acid Assay Interchim kit) (Hoogenboom

et al. 2012).

DFAA depletion experiments

An algal mix containing 19 different amino acids (Algal

Amino Acid Mixture, Reference ULM-2314-1, Lot PR-19236,

Larodan Fine Chemicals ABVR ) was used for these measure-

ments, according to Tremblay et al. (2012). Corals were indi-

vidually incubated in beakers filled with 200 mL filtered

seawater, enriched with a solution of 3.0 6 0.1 lmol L21

DFAA, and continuously mixed using a magnetic stirrer. The

final concentration was precisely determined taking into

account the DFAA concentration already present in seawater.

Three additional beakers per pH were also kept as controls

(without corals). Corals were incubated for 3 h at a constant

temperature of 26�C in a water bath and at the same irradi-

ance and pH as for the culture. Five milliliters of subsamples

were taken every hour, filtered through a 0.2-lm syringe fil-

ter (Minisart, 16532, Sartorius Stedim BiotechVR ) to remove

any particles that could interfere, and DFAA concentrations

were quantified using a spectrofluorometer XeniusVR (Safas)

according to Grover et al. (2008). Standard solutions of

DFAA from 0.5 lmol L21 to 4.0 lmol L21 were prepared for

internal calibration and to set up the photomultiplier volt-

age. Results were normalized to the skeletal surface area or

protein content and expressed as lmol DFAA or lg C or lg

N. For this purpose, the C and N contents as well as the per-

centage of each amino acid present in the algal mix were

taken into account (ca., 58.2 lg C per lmol DFAA, ca., 17 lg

N per lmol DFAA).

Zooplankton feeding rates

Feeding rates were assessed using PlexiglasVR flow tanks

following Ferrier-Pages et al. (2010), with a mean flow rate of

3.6 cm s21 and 1200 Artemia nauplii L21 in total. Corals

were incubated during 1 h at a constant temperature of 26�C

in individual tanks and at the same irradiance and pH as for

the culture. One tank was kept as a control (without coral).

In preliminary experiments, we checked that high PCO2levels

did not affect the short-term mobility and growth of Artemia

nauplii. Feeding rates were calculated as the decrease in prey

concentration during this hour and were normalized to the

skeletal surface area. They were also expressed as lg of car-

bon or lg nitrogen ingested. For this purpose, about 1200

nauplii were filtered on precombusted GF/F glass microfiber

filters (25 mm, WhatmanVR , 1825-025), and carbon and

nitrogen contents were determined using a Perkin Elmer

2400 Series II CHNS-O Elemental Analyzer (Perkin ElmerVR ).

The calibration was conducted with standards (K-factor) of

glycine (32.00% of carbon and 18.66% of nitrogen) and the

carbon and nitrogen value of the filters (blank) was also

determined. The nauplii contained 1.25 6 0.30 lg C

nauplii21 and 0.25 6 0.01 lg N nauplii21.

Daily carbon acquisition in our culture conditions

Given our quantification of photosynthetic and grazing

rates, we compared total daily carbon acquisition via auto-

trophy and heterotrophy for colonies maintained under the

different pHs. Total daily acquisition of carbon through

autotrophy (PC) was calculated using the rates of gross pho-

tosynthesis measured at 200 lmol photons m22 s21, consid-

ering a 12-h daylight period. Oxygen fluxes were converted

to carbon equivalents based on molar weights, as: PC 5 lmol

O2 produced 3 12/PQ and RC 5 lmol O2 consumed 3 12 3

RQ (Anthony and Fabricius 2000), where PQ and RQ are

photosynthetic and respiratory quotients equal to 1.1 and

0.8, respectively (Muscatine 1981). The percentage contribu-

tion of zooxanthellae acquired C to daily animal respiration

(CZAR; adapted from Muscatine 1981; Grottoli et al. 2006)

was subsequently calculated by Eq. 1, for corals maintained

under both pHs and feeding conditions:

CZAR 5 PC=RCð Þ3 100% (1)

Potential daily carbon acquisition from heterotrophy (HC)

was calculated by Eq. 2:

HC 5 GR 3 t (2)

where GR is the grazing rate on A. salina nauplii for each

incubation condition, expressed in lg C cm22 and t is the

duration of feeding every day for S. pistillata. In our experi-

mental conditions, we considered 1 h. DFAA uptake was not

taken into account as concentrations are usually very low in

the tanks.

We calculated the percentage contribution of hetero-

trophically acquired carbon to daily animal respiration

(CHAR, Grottoli et al. 2006) by Eq. 3:

CHAR 5 HC=RCð Þ3 100%: (3)

Statistical analysis

All data were tested for assumptions of normality using

Shapiro Wilk’s test and homogeneity of variances was

checked using Cochran’s test and were transformed when

required. After verification of the absence of significant dif-

ferences between tanks in each pH treatment (three-way

ANOVA, p > 0.05), data were pooled. Two-way ANOVAs

Houlbreque et al. Acidification and Stylophora nutrition

4

were used to test the effect of pHT (7.51 and 8.06) and feed-

ing (fed, starved) on the different physiological parameters

and feeding measurements. STATISTICAVR software (StatSoft)

was used. When ANOVA determined a significant difference,

Tukey honest significant difference (HSD) test was used. All

data are expressed as the mean 6 standard deviation (SD).

Results

There was a significant difference in chlorophyll and pro-

tein concentrations between feeding treatments (ANOVA, p <

0.05; Table 1; Fig. 1a) with higher values found in FC (Tukey

HSD, p < 0.05). Protein contents (data not shown) averaged

across pH treatments is 1.00 6 0.31 mg cm22 and 2.12 6 0.35mg cm22 for starved and FC, respectively. Symbiont density(Fig. 1b) was significantly affected by feeding with lower val-ues on FC. However, feeding had an opposite effect amongpH treatments as showed by interactions with pH (ANOVA,pH 3 Feeding: F1,36 5 10.20, p 5 0.03; Table 1).

Rates of net photosynthesis (Pn) but not respiration,

expressed in lmol O2 h21 cm22, were significantly affected

by feeding (ANOVA, F1,36 5 15.98, p 5 0.00; Table 1). In

terms of carbon, gross photosynthesis (Pg) provided from

13.1 lmol C cm22 d21 to 15.1 lmol C cm22 d21 to corals (or

157 lg C cm22 d21 to 181 lg C cm22 d21), respectively, for

FC at pHT 7.51 and SC at pHT 8.06. The contribution of the

Table 1. Summary of two-way ANOVAs followed by Tukey honest significant difference post hoc testing the effect of pH (7.5 and8.1 pHT units) and feeding regime (fed [F] and starved [S]) on S. pistillata main physiological parameters. Bold face numbers indicatesp < 0.05.

Source of variation df MS F p Post hoc df MS F p Post hoc

Chlorophyll NPQmax

pH 1 18.45 1.65 0.206 1 0.061 27.86 0.000

Feeding (F) 1 96.14 8.63 0.006 F>S 1 0.212 96.00 0.000

pH3F 1 2.42 0.22 0.644 1 0.086 39.12 0.000

Error 36 401.06 36 0.079

Protein Gross calcification

pH 1 0.012 0.10 0.757 1 13E105 34.8 0.000 7.5<8.1

Feeding (F) 1 7.498 61.42 0.000 F>S 1 88E104 22.8 0.000 F>S

pH3F 1 0.004 0.03 0.865 1 52E103 1.34 0.255

Error 20 2.440 36 14E105

Symbiont density TOC

pH 1 13E111 2.94 0.095 1 1.754 24.20 0.000

Feeding (F) 1 14E112 32.37 0.000 1 0.401 5.51 0.029

pH3F 1 45E111 10.20 0.003 1 8.190 113.22 0.000

Error 36 1.6E113 20 1.440

Net photosynthesis APA

pH 1 0.044 2.10 0.156 1 34E104 293.7 0.000

Feeding (F) 1 0.338 15.98 0.000 F<S 1 3550 3.0 0.097

PCO23T 1 0.009 0.42 0.520 1 7528 6.4 0.020

Error 36 0.761 19 22E103

Respiration DFFA

pH 1 0.002 0.79 0.781 1 0.027 98.02 0.000 7.5<8.1

Feeding (F) 1 0.064 3.02 0.091 1 0.003 10.85 0.004 F<S

pH3F 1 0.027 1.30 0.262 1 0.000 1.09 0.308

Error 36 0.760 20 0.005

ETRmax Grazing rates

pH 1 449E102 183.3 0.000 7.5<8.1 1 65E103 27.42 0.000 7.5<8.1

Feeding (F) 1 393E102 160.3 0.000 F>S 1 13E103 5.59 0.028 F<S

pH3F 1 677,6 2.8 0.105 1 19E102 0.80 0.382

Error 36 8818.3 20 47E103

Y(NO)max

pH 1 0.133 88.07 0.000

Feeding (F) 1 0.133 88.07 0.000

pH3F 1 0.089 58.59 0.000

Error 36 0.054

Houlbreque et al. Acidification and Stylophora nutrition

5

zooxanthellae to the respiration of the holobiont (CZAR)

was, therefore, respectively, equal to about 228% 6 33% for

SC and 191% 6 31% for FC, maintained at both pHT. Con-

cerning the photosynthetic efficiency, there was a significant

effect of feeding and pH (ANOVA, p < 0.05; Table 1). ETRmax

values were higher in FC at low pH compared to starved

ones at normal pH, respectively (Tukey HSD, p < 0.001; Fig.

2a). Feeding, pH, and their interaction significantly affected

the nonphotochemical quenching, both Y(NO)max and

Y(NPQ)max (ANOVA, p < 0.001; Table 1; Fig. 2b,c). The two

nonphotochemical quenching had an opposite response to

treatments, as showed by post hoc comparisons of the inter-

action between feeding and pH. Although the regulated

nonphotochemical quenching Y(NPQ) decreased on FC kept

at normal pH, Y(NO)max increased for the same treatments

(Fig. 2c). For both nonphotochemical quenching parameters,

the interaction of pH and feeding were significant (Table 1).

Significant differences in the calcification rates were

found between feeding and pH (ANOVA, p < 0.001; Table 1)

but not in their interaction. Under lower pH, calcification

was higher of 80% in FC compared to unfed ones

(from 253.9 6 87.9 nmol Ca21 g21 dry Skeleton h21 to 478.7

6 87.9 nmol Ca21 g21 dry Skeleton h21, respectively, for

starved and FC) and of 30% under normal pH (from 547.5 6

156.5 to 917.5 6 211.1, respectively, for starved and FC; Fig.

3). Additionally, for both fed and SC, calcification rates were

up to two fold higher at normal than at low pH.

TOC fluxes (data not shown) were all positive, showing a

release of organic carbon into seawater. TOC significantly

Fig. 1. (a) Chlorophyll content per unit surface area of skeleton(lg cm22); (b) Symbiont density per unit surface skeleton (zooxanthellae3 106 cm22); (c) Oxygen production and consumption rates per unit

surface area of skeleton (lmol O2 h21 cm22) for corals maintained fiveweeks under the two pH (pHT57.51 or 8.06) and feeding conditions.

F5fed corals, S5starved corals. Data are presented as means (n 5 5) 6

standard deviation.Fig. 2. (a) ETR vs. irradiance (PAR); (b) Y(N0); (c) Y(NPQ) for fed and

starved corals maintained under pHT 5 7.51 or pHT 5 8.06. Data arepresented as means (n 5 5) 6 standard deviation.

Houlbreque et al. Acidification and Stylophora nutrition

6

differed between pH and feeding and their interaction

(ANOVA, p < 0.001; Table 1). This release seems higher in

starved (1.2 6 0.3 lg cm22 h21 and 1.7 6 0.2 lg cm22 h21)

than in FC (0.5 6 0.4 lg cm22 h21 and 0.6 6 0.1 lg cm22

h21) exposed to pHT 5 8.06 and 7.51, respectively.

The activity of the alkaline phosphatase enzyme (i.e.,

APA; Fig. 4a), involved in the transformation of dissolved

organic phosphorus into dissolved inorganic phosphorus

(phosphate), was not significantly influenced by the feeding

regime, but significantly decreased by more than 50% under

low pH (ANOVA F1,19 5 293.7, p 5 0.000; Table 1). Uptake

of dissolved free amino acids (i.e., DFFA, Fig. 4b) was signifi-

cantly different between feeding and pH treatments

(ANOVA, p < 0.01). A decrease in pH, as well as feeding,

induced a significant decrease in the DFAA uptake rate corals

(Tukey HSD, p < 0.01; Table 1; Fig. 4b). In terms of carbon,

1 h of DFAA uptake at 3 lmol L21 supplied 7.19 6 0.05 lg C

cm22 to the corals maintained at pHT 5 8.06 and only 3.4 6

0.03 lg C cm22 to the corals maintained at pHT 5 7.51.

There was a significant effect of feeding and pH on grazing

rates (Fig. 4c; ANOVA, p < 0.05, Table 1). Grazing rates were

lower in SC at low pH (Tukey HSD, p < 0.05; Table 1). In

terms of carbon, 1 h of grazing on A. salina nauplii supplied

159 6 20 lg C cm22 to corals maintained at natural pHT of

8.06 and only 113 6 23 lg C cm22 to corals maintained at

low pH. The calculated CHAR varied between 134% and

172%, for FC maintained at low and normal pH, respectively.

Table 2 summarizes the carbon budget in the corals sub-

mitted to the different conditions. Considering that carbon

used for calcification comes mostly from respiration or from

external dissolved inorganic carbon (Furla et al. 2000), calci-

fication is not taken into account in this carbon budget.

For SC, the total carbon input was equal to 189 6 29 lg C

cm22 d21 and 186 6 27 lg C cm22 d21 for pHT 5 8.06 and

7.51, respectively. Respiration consumed 76 6 11 lg C cm22

d21 to 81 6 21 lg C cm22 d21 and organic carbon release

accounted for 17 6 2 lg C cm22 d21 to 11 6 3 lg C cm22

d21, for pH 5 8.06 and 7.51, respectively, considering only a

release during the 12 h of daylight. Thus, under these cul-

ture conditions, 96 lg C cm22 d21 and 94 lg C cm22 d21

remained for other processes.

For FC, under pH 5 8.06, autotrophic carbon contributed

for 169 6 21 lg C cm22 d21 while the heterotrophic carbon

contributed for 159 lg C cm22 d21 or a total of 328.4 lg C

cm22 d21. Respiration consumed 92 6 10 lg C cm22 d21

while release of organic carbon contributed for 6 6 8 lg C

cm22 d21, leaving about 230 lg C cm22 d21 for other proc-

esses. At pH 5 7.51, as the feeding rates were lower, the total

carbon remaining was 189 lg C cm22 d21.

Discussion

This study is consistent with the previous and unique

observation of Edmunds (2011) that feeding can

Fig. 3. Rates of calcification per skeleton dry mass (nmol Ca21 g21 skel-etal dry mass h21) for fed and starved corals maintained under two pH

conditions (pHT 5 7.51 or 8.06) for 7 h. Data are presented as means (n5 5) 6 standard deviation.

Fig. 4. (a) APA expressed in nmol mg protein21 h21; (b) DFAA uptake innmol mg protein21 h21; (c) Grazing rates of A. salina nauplii in cells mgprotein21 h21 for corals maintained five weeks under the two pH

(pHT 5 7.51 or 8.06) and the two feeding conditions. F 5 fed corals, S 5

starved corals. Data are presented as means (n 5 5) 6 standard deviation.

Houlbreque et al. Acidification and Stylophora nutrition

7

counterbalance the negative effects of seawater acidification

on coral calcification. It, however, clearly shows that acidifi-

cation influences the feeding capacity and feeding intensity

of the branching coral S. pistillata. Considering that hetero-

trophy brings essential nutrients to the symbiotic associa-

tion, on a long-term basis, lower feeding rates will likely

weaken the already fragile physiological state of many corals

under more acidic and warmer conditions.

Effect of feeding and seawater acidification on

S. pistillata metabolism

Although feeding was shown to enhance rates of photo-

synthesis in some previous studies (Houlbreque and Ferrier-

Pages 2009), here the photosynthetic rates of FC have been

slightly decreased. Conversely, feeding changed the photo-

chemistry and enhanced ETRmax, under both pH conditions,

suggesting a better photosynthetic efficiency of PSII, prob-

ably due to a higher content of PSI and PSII proteins (PsbA

and PsaC), as previously observed (Hoogenboom et al. 2012).

Moreover, under normal PCO2conditions, feeding changed

the functioning of PSII, as it increased the unregulated non-

photochemical quenching Y(NO), which reflects the fraction

of energy that is passively dissipated in the form of heat and

fluorescence, and thus decreased the regulated nonphoto-

chemical quenching Y(NPQ), corresponding to the fraction

of energy dissipated in the form of heat via the photoprotec-

tive mechanisms. In addition, photosynthetic rates of S. pis-

tillata did not significantly change with the seawater

carbonate chemistry and/or the decrease in pH, as already

noticed in previous short-term studies conducted on other

scleractinian corals (Reynaud et al. 2003). These conclusions

are consistent with the assertion that corals do not rely on

dissolved CO2 for their photosynthesis (Gattuso et al. 1999).

A reduction in areal symbiont concentration, and subse-

quently in the areal rates of gross photosynthesis, was how-

ever observed after a long-term exposure to very low pH

(7.2; Tremblay et al. 2013), but the amount of carbon

acquired by the host remained unchanged due to a higher

percentage of carbon translocation from the symbionts.

Despite the lack of photosynthesis stimulation by feeding

under high PCO2condition, feeding maintained calcification

at a high rate (comparable to unfed corals under normal

conditions), whereas calcification rates significantly

decreased by 53.6% in unfed corals, relative to unfed corals

under ambient CO2 conditions. Such decline in calcification

rates observed in unfed corals under seawater acidification is

a common feature (Langdon and Atkinson 2005). One of the

main explanations is a greater energetic cost of mineralizing

at low Xarag, which under normal conditions, accounts for

13–30% of the energy supplied by respiration (Allemand

et al. 2011). Seawater acidification may thus increase the

energetic demand for CaCO3 production (Holcomb et al.

2010). Even if respired heterotrophic carbon might be also

used as carbon for calcification (Allemand et al. 2011), nutri-

ent supply, in either the inorganic dissolved form, or in the

particulate form as in this study, may reduce the influence

of seawater acidification on calcification, by offsetting the

additional energy demand. This process was suggested both

on adult corals (Langdon and Atkinson 2005; Holcomb et al.

2010), as well as on juveniles (Drenkard et al. 2013). The

additional energy can be used through an increase in tissue

biomass (Edmunds 2011; Drenkard et al. 2013), although

this is not always the case, as in this study. Such feeding

enhancement of calcification in S. pistillata was already

observed under normal culture conditions (seawater temper-

ature around 25�C and actual level of PCO2; Reynaud et al.

2003; Houlbreque et al. 2004; Houlbreque and Ferrier-Pages

2009) and is also valid under thermal or acidification stress,

as demonstrated in this study. Maintaining optimal levels of

calcification seems thus to be the priority in corals when

possible. It has to be noticed, however, that although rates

of calcification of FC at pH 5 7.51 were similar to the rates

measured in unfed corals at pH 5 8.06, they were signifi-

cantly lower (by 33%) than those measured in FC at pH 5

8.1. This may be due to lower grazing rates of S. pistillata on

A. salina at high PCO2, which would be consistent with the

energetic theory of the effect of feeding and seawater acidifi-

cation on coral calcification. An important question, still

unsolved, is the amount of food needed to sustain coral cal-

cification during stress events, as well as the amount actually

available for corals in situ. Prey levels added in our experi-

mental tanks (1200 Artemia nauplii L21 or a total of 24,000

Table 2. Percentage CZAR, percentage CHAR, and carbon left for other processes than respiration (noted C left), in starved and fedcorals (noted S and F, respectively) and submitted to a pHT of 8.06 or 7.51. Autotrophic and heterotrophic carbons are the quantityof carbon supplied via autotrophy and heterotrophy. Respiration (Rc) and release of TOC are the quantity of carbon consumed bythe respiration process and lost via the release of mucus, respectively. They are all expressed in lg C cm22 d21.

Incubation

conditions CZAR% CHAR%

C

left

Autotrophic

carbon

Heterotrophic

carbon RC

Release of

TOC

7.5 S 218620 94 186627 81621 1163

8.1 S 238642 96 189629 76611 1762

7.5 F 194638 134 189 165615 113623 84621 564

8.1 F 188625 172 230 169621 159620 92610 668

Houlbreque et al. Acidification and Stylophora nutrition

8

Artemia in the 20-liter tanks) are higher than natural reef

concentrations, but they were available for only 1 h, provid-

ing in total up to 24 mg C d21, if we consider that the total-

ity was ingested, which was not the case. Considering that

Heidelberg et al. (2004) estimated that zooplankton abun-

dance during nighttime is around 5 mg C m3 or 5 lg C L21,

and that in some reefs, 12,000 m3 flow over 1-m strip of a

reef during 24 h, or 6000 m3 during the 12-h night, (Ayukai

et al. 1995), a total of 30,000 mg C m3 (or 30 mg C L21)

should be available for corals at the end of the night, a value

close to what corals received in our tanks. Although this cal-

culation has to be considered with care, and although no

real in situ measurement of prey availability is available, we

might consider that calcification in some coral species can

be sustained by feeding under natural conditions. However,

as demonstrated in this study (and discussed below) with the

branching scleractinian coral S. pistillata, the capacity of prey

capture in some coral species is severely and negatively

affected by stress conditions, which mitigates the positive

effect of heterotrophy in the sustainment of coral metabo-

lism under stress conditions. This is especially true because

global productivity levels are predicted to decrease in the

future (Behrenfeld et al. 2006; Steinacher et al. 2010).

Effects of ocean acidification on feeding capacities and

carbon budget

Contrarily to what was shown for the uptake of dissolved

inorganic nitrogen and phosphorus, which were not influ-

enced by seawater acidification in S. pistillata (Godinot et al.

2011), this study shows that the organic nutrient acquisition

of that species was negatively affected under high PCO2, with a

simultaneous decrease in the DFAA uptake rates, in the APA,

and in the feeding rates, processes essential to fulfill the nitro-

gen and phosphorus needs of corals (Grover et al. 2006; Godi-

not et al. 2013). It has to be noticed that this latter effect was

independent of the trophic status of the coral colonies.

Uptake of DFAA involves a carrier-mediated transepithelial

transport (Grover et al. 2008), often proton-gradient depend-

ent (Tsuji et al. 1994), which might thus be influenced, under

low pH, by the increase in proton (H1) concentration at the

carrier surface. The transformation of dissolved organic phos-

phorus into dissolved inorganic phosphorus, readily usable by

corals, is mediated by metalloenzymes called phosphatases,

which are also pH-dependent. The alkaline phosphatase con-

sidered in this study, which has an optimum efficiency under

a basic pH (Godinot et al. 2013), had thus its activity lowered

by 50% under low pH conditions.

Finally, low seawater pH also affected the capture rates of

A. salina nauplii, but the process involved remains to be

determined. The decrease in capture rates could be due to a

polyp retraction or a slow down of their mobility due to

acidic waters. While it has been demonstrated that some

bleached corals (e.g., Montipora capitata) submitted to a ther-

mal stress were able to increase their feeding rates and to

acquire large quantities of heterotrophic carbon (Grottoli

et al. 2006), this is not the case for S. pistillata, whose capture

rates were halved when stressed by high temperatures (Fer-

rier-Pages et al. 2010) or here by seawater acidification. This

suggests that S. pistillata will lose part of its heterotrophic

capacities in the future. Although the uptake rates of dis-

solved inorganic nitrogen and phosphorus were not affected

by seawater acidification alone (Godinot et al. 2011), S. pistil-

lata will undoubtedly lack essential nutrients in the future, as

it thrives in oligotrophic waters in which inorganic nutrients

are in nanomolar concentrations (Ducklow 1990).

Concerning the carbon budget of S. pistillata, results

acquired in this study show that the acquisition of autotro-

phic carbon, and its consumption through respiration, should

not be changed by an increase in PCO2. Conversely, heterotro-

phy will be negatively affected, since the calculated CHAR

dropped from 172% at normal pHT to 134% under low pHT.

As autotrophic carbon was demonstrated to be junk food for

corals (Falkowski et al. 1984), mostly respired (Bachar et al.

2007), and as biomass is rather build through heterotrophy

(Houlbreque et al. 2004), corals like S. pistillata will likely see

their tissue and skeletal growth highly decreased in the future.

This experimental study has demonstrated that feeding sus-

tains calcification rates in the branching coral S. pistillata, sub-

mitted to seawater acidification, as it was demonstrated in the

massive coral species Porites sp. (Edmunds 2011). It has thus

the same positive effect on coral metabolism as during a ther-

mal stress induced bleaching (Grottoli et al. 2006; Ferrier-Pages

et al. 2010). Assuming calcification is an energy-requiring pro-

cess, the mechanism by which feeding sustains calcification

may very well be via a supply of energy. However, acidification

also decreased the feeding rates on dissolved and particulate

organic matter, impairing the heterotrophic acquisition of

energy by this coral species. Although recent studies suggested

that FC could maintain high rates of skeletal growth and

CaCO3 production under ocean acidification (Edmunds 2011;

Drenkard et al. 2013), this proposed mechanisms for resilience

will be in vain if rising CO2 conditions inhibit coral feeding to

the extent that they are unable to maintain calcification rates.

The major threat is that not only will ocean acidification

reduce coral feeding effort in some coral species but there will

also be less food available to corals in a warmer future (Behren-

feld et al. 2006; Steinacher et al. 2010). Under the most

extreme scenario for the likely trajectory for reef ecosystems,

corals will transition from “ambient CO2, fed” to “high CO2,

unfed” conditions, which would induce a large drop in calcifi-

cation rate.

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Acknowledgments

Thanks are due to Andy Foggo for his precious advice in the statisticalinterpretation of the data. We also thank Beat Gasser for his help in thesetting up the experiment and to three anonymous reviewers for their

helpful comments and constructive remarks on this paper.The international Atomic Energy Agency and the Centre Scientifique

de Monaco are grateful to the Government of the Principality of Mon-aco for the support provided to their laboratories.

Submitted 19 February 2014

Revised 16 September 2014

Accepted 17 September 2014

Associate editor: John Albert Raven

Houlbreque et al. Acidification and Stylophora nutrition

11