Effect of Elevated Carbon Dioxide on Two Scleractinian Corals: Porites cylindrica (Dana, 1846) and Galaxea fascicularis (Linnaeus, 1767)

This study reveals the effect of elevated pCO2 on Porites cylindrica and Galaxea fascicularis. The corals responded differently under elevated pCO2. Zooxanthellae cell density, cell mitotic index, and photosynthesis rate of P. cylindrica decreased drastically under the elevated pCO2. At the end of the experiment, P. cylindrica suffered from a declining calcium carbonate precipitation rate. G. fascicularis increased its respiration rate and expelled 71% of its symbiotic zooxanthellae algae under elevated pCO2. Photosynthetic pigments in the remaining zooxanthellae algae increased from 1.85 to 11.5 times to sustain its photosynthetic outputs. At the end of the experiment, G. fascicularis managed to increase the rate of its calcium carbonate precipitation. Increase pCO2 in the atmosphere may affect species diversity of coral reefs.


Introduction
Coral reefs are among the most productive marine ecosystems serving as a feeding and breeding ground for a wide spectrum of marine organisms.Corals precipitate calcium carbonate from the seawater onto its skeletal organic matrix (SOM) and form the coral skeleton [1,2].The calcium carbonate saturation state in seawater (Ω) regulates the supply of ions for the calcification process.Any changes on the seawater carbonate system would impact the precipitation process and hence threaten the corals' health.
Intensified anthropogenic CO 2 emission from land use change, fossil fuels, cement, coal and oil combustion has caused the average partial pressure of carbon dioxide (pCO 2 ) increased from 180 to 300 ppmv (part per million by volume) in the 1800s [3] to the current 380 ppmv [4] and is expected to reach 700 ppmv or more towards the end of the 21st century [5].When Caldeira and Wickett [6] reported the ocean acidification in 2003, the impacts of the elevated pCO 2 on marine organisms were widely investigated [7][8][9][10][11][12].Nevertheless, there is no unanimous agreement on the degree of impacts among the reports.In one hand, Langdon et al. [13], Leclercq et al. [8], and Marubini et al. [14] reported declining calcification rate of reef organisms under elevated CO 2 .In the other hand, McNeil et al. [11] predicted an increase in the annual calcification rate of the coral reef in the future global warming.Lough and Barnes [12] also reported a continuous increment in the calcification of the Porites corals in the Great Barrier Reef despites of the increasing pCO 2 over the last century.Reynaud et al. [9] reported that calcification of Stylophora pistillata did not change in response to an increased pCO 2 under normal temperature.Basically, most of the reports suggested more studies are needed to reveal the effect of elevated pCO 2 on corals.Hence, this study aims to extend the understanding on the impact of elevated pCO 2 on the scrleractinian corals, Porites cylindrica (Dana 1846) and Galaxea fascicularis (Linnaeus, 1767).at an average water depth of 10 m.The coral colonies were broken into smaller coral nubbins (∼50 polyps per nubbin) and acclimatized in a 500 l aquarium supplied with running seawater for one month.The coral nubbins were supplied with 12-h light: 12-h dark cycle illumination by a metal-halide lamp at 392 ± 5 μmol m −2 s −1 .During the acclimatization, the corals nubbins were fed with newly hatched Artemia salina (Royal Artemia, 99% hatching) once for every three days, according to the recommendation made by Hii et al. [15].Twelve coral nubbins for each species were randomly selected for the experiments (six for each elevated pCO 2 and control experiment).

Experimental Setup.
Two 160 l aquaria (25 cm × 50 cm × 83 cm) were used for the experiment.One of these aquaria was used for the elevated pCO 2 and the other one as control.Six similar sizes P. cylindrica nubbin (∼50 polyps; 10 × 10 cm) were randomly selected and placed in each aquarium.Water temperature was maintained at 26 ± 1 • C by using a water chillers.The aquaria were supplied with 392 ± 5 μmol m −2 s −1 illumination by using metal-halide light on a 12-h light: 12-h dark cycle.The experiment was conducted under laboratory condition at 27 • C, salinity of 35 psu, pH T 8 and dissolved oxygen greater than 5 mg l −1 .Wave simulators were used to generate water movement in the aquarium.The elevated CO 2 aquarium was simulated by a constant pressure flow of carbon dioxide into the fed water tank.Check valves were used to prevent seawater backflow and bubble counter was used to estimate CO 2 diffusion.The control experiment was conducted under similar condition but without CO 2 stimulation.Instead of CO 2 , constant compress airflow was used in the control experiment.The CO 2 and compress air for the elevated CO 2 and control experiment was adjusted until constant seawater pH at 7.90 ± 0.05 and 8.19 ± 0.02 was obtained, respectively.Table 1 shows carbon dioxide, pH, dissolved oxygen, temperature, and salinity in the experimental chambers.The experiment was conducted for 15 days.The aquaria were cleaned once a week to remove microalgae grew on the glass.Seawater alkalinity was determined daily, photosynthesis rate of the corals was determined once every three days, while the zooxanthellae density, mitotic index, chlorophyll pigments, and surface area of the specimens were analyzed at the end of the experiment.Similar experimental setup was repeated for G. fascicularis.

Determination of Net Photosynthesis
Rate.Coral nubbins were carefully transferred into six (3 transparent and 3 opaque) 1.5 L airtight chambers (one nubbin in each chamber).The chambers were filled with seawater at the desired pCO 2 , tighten without headspace and air bubble, and incubated in the experimental aquarium for two hours.Dissolved oxygen (DO) in the glass chamber was measured before and after the incubation by using Winkler's method [16].Gross primary production rate was assessed by summation of oxygen produced by net photosynthesis and respiration rate of the corals [17].Six empty chambers (3 transparent and 3 opaque) were also incubated in the experimental aquaria to serve as controls.Data were standardized using surface area of the specimens.Surface area of the specimens was measured using paraffin wax technique [18].Briefly, paraffin wax was used to coat the specimen.Surface area of the specimens could be obtained by referring the weight of the paraffin wax coated on the specimen to the standard curve of paraffin wax versus surface area.The standard curve was generated by regressing weight of the paraffin wax to known surface area density blocks.

Zooxanthellae Density and Mitotic Index.
Coral tissue was extracted by using modified Water Pik method [19] by brushing off the coral surfaces and flushed with filtered seawater (0.2 μm milipore).The process was repeated until the coral specimen turned white.The volume of the extracts was recorded and later determined for the zooxanthellae density.The zooxanthellae cell was counted using an improved Neubauer haemacytometer.Zooxanthellae density was determined from the total zooxanthellae cell extracted from the coral over the total surface area of the specimen.Mitotic Index (MI %) was determined from the number of cell division over the population of the cell density [20].Dividing cell is defined as any cell between the points of initial appearance of cell walls in the mother cells to the formation of their own cell envelope in the daughter cells [21].

Chlorophyll Analysis.
Chlorophyll pigment was analysed by using spectrophotometric method [16].Three aliquots of coral tissue extracts were used to determine the chlorophylls content.The extract was first filtered through a GFC filter paper (Milipore) at half the atmospheric pressure (0.5 atm); a few drops of magnesium carbonate (Merck) were added during the filtration to prevent the formation of phaeopigments.The GFC filter paper was then transferred into a 15 mL centrifuge tube; 10 mL of 90% acetone (Merck) was added into the tube and shaken thoroughly.The sample was stored at −20 • C in the dark for 24 hours before they were centrifuged at 875 g for 10 minutes.The supernatant was decanted into a 1 cm glass cuvette and measured at 664 nm, 647 nm, and 630 nm by using a double beam UV-Visible spectrophotometer (Shimadzu 1601).The chlorophyll a, b, and c content were determined by using the equations reported by Parsons et al. [16].

Coral Calcification, Surface Area, and CO 2 Speciation.
Coral calcification rate was measured by using method developed by Smith and Kinsey [22] based on alkalinity anomaly.This technique assumed that each mole of CaCO 3 precipitated the total alkalinity was lowered by two equivalents.Alkalinity was determined by using titration method as described by APHA [23].Concentration of bicarbonate ion, carbonate ion, and dissolved carbon dioxide was calculated based on the total alkalinity and pH to examine the dynamics of the entire carbonate system for the experiment.Seawater pH was measured in total scale.Measurement and preparation of the calibration buffer for total scale pH was based on the procedures described by Wedborg et al. [24].Coral calcification against time was tested using regression analysis.SAS and Microsoft Excel with analysis tool-pack module were used for the statistical analysis.

Effect of Elevated CO 2 on Net Photosynthesis and Respiration.
The effect of elevated carbon dioxide in seawater on photosynthesis and respiration rate of the corals was dependent on species (Figure 1).Photosynthesis (P = .02)and respiration rate (P = .04) of P. cylindrica decreased significantly under the elevated pCO 2 .On average, the net photosynthesis rate of P. cylindrica under elevated pCO 2 and control experiment was 0.156 ± 0.07 μmol O 2 cm −2 h −1 and 0.307 ± 0.07 μmol O 2 cm −2 h −1 , respectively.Oxygen consumption rate of P. cylindrica was also significantly lower (P = .04).On average, the oxygen consumption rate of P. cylindrica under elevated pCO 2 and control experiment was 0.77 ± 0.06 μmol O 2 cm −2 h −1 and 0.97 ± 0.06 μmol O 2 cm −2 h −1 , respectively.Elevated pCO 2 in the seawater did not lead to a significant change in the net photosynthesis rate of G. fascicularis (P = .48).Average net photosynthesis rate of the coral under normal and elevated CO 2 was 1.18 ± 0.13 and 1.17 ± 0.11 μmol O 2 cm −2 h −1 , respectively.Unlike P. cylindrica, respiration rate of G. fascicularis in the elevated CO 2 environment was significantly higher than the control (P = .004).The respiration rate of G. fascicularis in normal and the elevated pCO 2 was 0.66 ± 0.11 and 1.10 ± 0.19 μmol O 2 cm −2 h −1 , respectively.Gross photosynthesis rate of G. fascicularis was also significantly higher than that of the control (P = .001).On average, gross photosynthesis rate of G. fascicularis under normal and the elevated pCO 2 was 2.27 ± 0.25 and 1.83 ± 0.14 μmol O 2 cm −2 h −1 , respectively.

Chlorophyll Pigments, Zooxanthellae Cell Density, and
Mitotic Index.Chlorophyll pigments, zooxanthellae cell density, and mitotic index of P. cylindrica and G. fascicularis responded differently under elevated pCO 2 (Table 2).Photosynthetic pigments of the zooxanthellae algae in P. cylindrica seem to be increased under elevated pCO 2 ; however the increment was not significant (chlorophyll-a, P = .088;chlorophyll-b, P = .386;chlorophyll-c, P = .109).Nevertheless, elevated pCO 2 reduced the zooxanthellae cell density (P = .043)and the mitotic index (P = .010)significantly in P. cylindrica.
Photosynthetic pigments of the zooxanthellae algae in G. fascicularis increased significantly under elevated pCO 2 (chlorophyll-a, P = .016;chlorophyll-b, P = .004;chlorophyll-c, P = .006).Under the elevated pCO 2 , zooxanthellae cell density in G. fascicularis declined significantly (P = .002)as compared to the control.However, The mitotic cell division index was remained as that in the control experiment. 2 showed the effect of elevated pCO 2 on the calcium carbonate precipitation rate of P. cylindrica and G. fascicularis.P. cylindrica suffered from a significant decline in the calcium carbonate precipitation rate.On average, the calcium carbonate precipitation rate of P. cylindrica declined from 0.203 ± 0.137 μmol CaCO 3 cm −2 d −1 (control) down to −0.150 ± 0.196 μmol CaCO 3 cm −2 d −1 .At the end of the experiment, P. cylindrica suffered from a net dissolution of calcium carbonate from its skeleton.G. fascicularis was able to tolerate the pCO 2 stress.

Calcification. Figure
G. fascicularis sustained a higher calcium carbonate precipitation rate under the elevated pCO 2 .On average, the CaCO 3 precipitation rate of G. fascicularis in the control and elevated CO 2 environment was 0.112 ± 0.099 and 0.256 ± 0.209 μmol CaCO 3 cm −2 d −1 , respectively.The calcium carbonate precipitation rate of G. fascicularis under elevated CO 2 environment overtook the control after four days.

Discussion
The very first effect of the elevated pCO 2 was on the thermodynamic equilibrium of the seawater carbonate.The seawater pH T declined from pH T 8.19 ± 0.02 in the control experiment down to pH T 7.90 ± 0.04 in the experimental tank.Decrease seawater pH would have an effect on the intracellular pH of the corals [25].Changes in seawater pH    would subsequently affect protein properties, cell membrane permeability and other biological functions including the calcium carbonate precipitation rate [26,27].Although decrease seawater pH would have an effect on the aragonite saturation state (Ω aragonite ), Gattuso et al. [28] suggested that calcification of Stylophora pistillata and Acropora sp. may not be affected significantly at the level corresponding to 560 μatm pCO 2 .
When incubated under high pCO 2 , P. cylindrica showed a lower respiration rate, photosynthesis rate, zooxanthellae density, and the cell mitotic index and there was no significant different in terms of the photosynthesis pigments in the zooxanthellae cells incubated under normal and elevated pCO 2 .The calcium carbonate precipitation rate of the coral's declined significantly under the elevated pCO 2 .At the end of the experiment, the corals were dying and produced substantial amount of mucous.G. fascicularis reacted differently under elevated pCO 2 .The zooxanthellae cells contained in the coral were expelled by 71% under the elevated pCO 2 .There is no significant change in the mitotic index of the zooxanthellae cell incubated under elevated pCO 2 and control experiment.The photosynthetic pigments of the remaining zooxanthellae cells increased significant under the elevated pCO 2 .Under the elevated pCO 2 , the corals increased gross photosynthesis rate to offset the increased respiration rate.The net photosynthesis remained no change in the experiments.At the end of the incubation, G. fascicularis sustained a positive calcium carbonate precipitation rate in the elevated pCO 2 experiment.The G. fascicularis remained healthy under the elevated pCO 2 condition until the end of the experiments.
In terms of corals' calcification rate, G. fascicularis was able to withstand the pCO 2 stress while P. cylindrica failed to tolerate elevated pCO 2 .The mechanisms of how corals precipitate calcium carbonate onto their skeletal organic matrix are not fully understood yet.It is commonly recognized that, calcification is a highly biologically control mechanisms and simultaneous supply of ions and the formation of skeletal organic matrices are essential controls for the coral's calcification.In order to build the skeleton, corals have to supply calcium and inorganic carbon from the seawater to the deposition site and also remove protons produced during the process.The transcellular pathway for transporting ions through the epithelia is an active process which required energy to against the concentration gradient; especially in the calicoblastic ectoderm [2].The elevated pCO 2 depressed the respiration rate of P. cylindrica.As a result, lower energy supply retarded the calcium carbonate precipitation in the coral.Under elevated pCO 2 , G. fascicularis expelled its zooxanthellae cell in order to counter the stress.Zooxanthellae cell density was significantly declined and the photosynthetic pigments (chlorophyll-a, b and c) of the remaining zooxanthellae cells increased significantly (1.85-11.5 fold) to compensate the photosynthetic ration of the expelled cells.Higher respiration rate of G. fascicularis under the elevated pCO 2 enhanced higher energy supply to the coral and drove higher calcium carbonate precipitation rate for the coral.
Although photosynthesis of the symbiotic algae is highly coupled with the coral calcification, the full mechanisms underlying the interaction are not known [1,2].Allemand et al. [2] proposed photosynthesis-induced OH − secretion that neutralized H+ produced during the calcium carbonate precipitation.By removing the hydrogen ion, the process of the calcification could be driven the precipitation process.G. fascicularis in this study was able to sustain their photosynthesis rate and the physiological adaptation of the coral made them to withstand the CO 2 stress.Other than that, thick tissue biomass of G. fascicularis may also help the coral's calcification as this process took place in the cell cytoplasm.Loya et al. [29] also hypothesized that under environmentally stressful conditions, thick tissue could protect the underlying zooxanthellae from severe stress.The thin layer tissue and dense skeleton of P. cylindrica do not have this advantages and this may be contributed to the skeletal loss of the coral.
This study found that P. cylindrica suffered from the elevated pCO 2 while G. fascicularis was able to deal with the stress.The impact of elevated pCO 2 on corals is species specific.Different species of scleractinian corals in the reefs may have their own physiological adaptation to overcome the pCO 2 stress.Coral calcification is a biological controlled and energy-demanding mechanism.It is believed that, the status of the coral in terms of its energy reserves and the feeding strategies should have a role in their ability to tolerate external stress.

Conclusion
Elevated pCO 2 in seawater stressed P. cylindrica and G. fascicularis.Degree of the CO 2 stress on the corals was species dependant.High pCO 2 declined zooxanthellae cell density, mitotic cell index, respiration, and photosynthesis rate of P. cylindrica and, as a result, reduced calcium carbonate precipitation rate of P. cylindrica.G. fascicularis expelled its zooxanthellae cell under high pCO 2 .Photosynthetic pigments contained in the remaining symbiotic zooxanthellae increased to sustain the photosynthesis.High pCO 2 also increased respiration rate of G. fascicularis.At the end of the experiment, G. fascicularis incubated under high pCO 2 increased its calcium carbonate precipitation rate.G. fascicularis showed better tolerance towards high pCO 2 as compared to P. cylindrica.The species-dependent reaction of the corals suggested that increase atmospheric pCO 2 would affect species diversity of the coral reefs.

Figure 1 :
Figure 1: Changes in net photosynthesis, respiration, and gross photosynthesis in P. cylindrica and G. fascicularis under normal and elevated pCO 2 .Open bar ( ) indicates elevated pCO 2 while dotted bar ( ) indicates control condition.

Figure 2 :
Figure 2: Calcium carbonate precipitation rate for P. cylindrica and G. fascicularis under control and elevated pCO 2 experiment.Dashed line and solid circle indicate control while solid line and open circle indicate elevated pCO 2 .

Table 1 :
Water quality parameters (mean ± standard deviation) in the control and experimental aquaria.The reading was based on day 0 and subsequent daily measurements for fifteen days (n = 16).The effect of elevated CO 2 on photosynthesis, calcification, chlorophyll, mitotic index, and the zooxanthellae cell density of the scleractinian corals was tested by using paired sample t-test.Paired sample t-test was conducted to analyze data collected in day 0 and day 14.

Table 2 :
Chlorophyll pigments, zooxanthellae cell density, and mitotic index of P. cylindrica and G. fascicularis (mean ± standard deviation) under control and elevated pCO 2 .Each reading was based on six coral nubbins analysed at the end of the experiment.Pair t-test was conducted to compare the parameters under control and elevated pCO 2 .Bolded figures indicate a significant different and vice versa.