The sources and mechanisms of inorganic carbon transport for scleractinian coral calcification and photosynthesis were studied using a double labelling technique with H14 CO3 and 45 Ca. Clones of Stylophora pistillata that had developed into microcolonies were examined. Compartmental and pharmacological analyses of the distribution of 45 Ca and H14 CO3 in the coelenteron, tissues and skeleton were performed in dark or light conditions or in the presence of various seawater HCO3 concentrations.

For calcification, irrespective of the lighting conditions, the major source of dissolved inorganic carbon (DIC) is metabolic CO2 (70–75 % of total CaCO3 deposition), while only 25–30 % originates from the external medium (seawater carbon pool). These results are in agreement with the observation that metabolic CO2 production in the light is at least six times greater than is required for calcification. This source is dependent on carbonic anhydrase activity because it is sensitive to ethoxyzolamide. Seawater DIC is transferred from the external medium to the coral skeleton by two different pathways: from sea water to the coelenteron, the passive paracellular pathway is largely sufficient, while a DIDS-sensitive transcellular pathway appears to mediate the flux across calicoblastic cells. Irrespective of the source, an anion exchanger performs the secretion of DIC at the site of calcification. Furthermore, a fourfold light-enhanced calcification of Stylophora pistillata microcolonies was measured. This stimulation was only effective after a lag of 10 min. These results are discussed in the context of light-enhanced calcification.

Characterisation of the DIC supply for symbiotic dinoflagellate photosynthesis demonstrated the presence of a DIC pool within the tissues. The size of this pool was dependent on the lighting conditions, since it increased 39-fold after 3 h of illumination. Passive DIC equilibration through oral tissues between sea water and the coelenteric cavity is insufficient to supply this DIC pool, suggesting that there is an active transepithelial absorption of inorganic carbon sensitive to DIDS, ethoxyzolamide and iodide. These results confirm the presence of CO2-concentrating mechanisms in coral cells. The tissue pool is not, however, used as a source for calcification since no significant lag phase in the incorporation of external seawater DIC was measured.

Biomineralization is one of the most important biological processes in the living world. Nevertheless, calcification processes and, more specifically, mechanisms of ion transport largely remain a biological enigma. One of the major calcifying groups of organisms is the scleractinian corals. The rate of calcification of a coral reef is assumed to be approximately 10 kg CaCO3 m−2 year−1 (Chave et al., 1975), representing almost half the world’s CaCO3 precipitation (Smith, 1978), which has encouraged the use of corals as suitable models for biomineralization studies. Furthermore, the importance of coral reefs in the global cycling of carbonates (Smith, 1978) and their use as environmental archives (Barnes and Lough, 1996; Druffel, 1997) have stimulated research on the biology and physiology of scleractinian corals (for recent reviews, see Gates and Edmunds, 1999; Gattuso et al., 1999). Coral skeleton formation results from the delivery of Ca2+ and inorganic carbon to the site of calcification. Tambutté et al. (1996) and Marshall (1996) have demonstrated that Ca2+ is delivered to the site of calcification by a transcellular transport process through the calicoblastic epithelium, pointing out the key role of biological control of biomineralization. Ca2+ uptake by this cell layer is mediated by L-type voltage-dependent Ca2+ channels (Zoccola et al., 1999) and Ca2+ -ATPases (Ip et al., 1991). This process is very rapid, Ca2+ transport from sea water to the skeleton occurring within 1–2 min (Tambutté et al., 1996). However, there are few recent data describing the role of inorganic carbon in the biomineralization of corals.

After the pioneering work of Goreau (1961), who suggested that not all skeletal carbonate originates directly from dissolved inorganic carbon (DIC) in sea water, Pearse (1970) established that there are two different sources: (i) soluble carbonates from sea water and (ii) CO2 produced by the metabolism of coral and zooxanthella tissue. Later, Erez (1978), using 45 Ca incorporation to measure in situ calcification rates in the coral Stylophora pistillata, found them to be 5–10 times higher than those determined using H14 CO3, suggesting that metabolic CO2 may represent up to 80 % of the carbon source in the presence of light. Moreover, Barnes and Crossland (1978) suggested that carbon transport results from transcellular transport and involves at least one intermediate compound. The scarcity of results reflects the methodological problems associated with the use of radioisotopes with corals (Barnes and Crossland, 1978). Most of these problems were overcome by using coral microcolonies in which the skeleton is entirely covered by tissues, thus avoiding direct radioisotope exchange between the sea water and the skeleton (Al-Moghrabi et al., 1993).

An understanding of DIC metabolism in relation to photosynthesis and calcification is of prime importance since DIC is a common substrate for both mechanisms. Furthermore, paleoenvironmental interpretations based on the stable isotope composition of coral skeleton have to take into account the ‘vital effect’ (Goreau, 1977), since both the chemical species used (HCO3 or CO2) and the mode of transport (active versus passive) affect the isotopic fractionation. The purpose of this study was to determine the source(s) of carbon for photosynthesis and calcification, the mechanisms involved in its uptake, their kinetics and the characterization of the various carbon compartments. We have used a double labelling method (45 Ca and H14 CO3) and a compartmental and a pharmacological approach derived from a protocol that we described previously (Tambutté et al., 1996).

Biological material

Microcolonies were propagated in the laboratory from small fragments of Stylophora pistillata (Esper, 1797) collected at a depth of 5 m from the sea at the Marine Science Station, Gulf of Aqaba, Jordan. Corals were stored in an aquarium (300 l) supplied with sea water from the Mediterranean sea (exchange rate 2 % h−1 ) heated to 25±0.1 °C and illuminated with a constant irradiance of 125–150 μmol photons m−2 s−1 using metal halide lamps (Philips HQI-TS, 400 W) on a 12 h:12 h light:dark photoperiod. Microcolony propagation has been described by Al-Moghrabi et al. (1993). Briefly, terminal portions of branches (6–10 mm long) were cut from the parent colonies and placed on a nylon net (1 mm×1 mm mesh) in the same conditions of light and temperature as the parent colonies. After approximately 1 month, coral fragments became entirely covered with new tissue.

Measurements of 45 Ca2+ and H14 CO3 uptake and deposition Labelling procedure

Isotope uptake was measured using a protocol described previously for 45 Ca alone (Tambutté et al., 1996) into three compartments: coelenteron, tissue and skeleton. Experiments were either performed in the dark (after at least 12 h in the dark) or in the light with an irradiance of 250 μmol photons m−2 s−1. The composition of the solutions used for these studies is described below. Microcolonies were placed in plastic holders and incubated for 2–180 min in beakers with 10 ml of a labelled incubation medium (IM) containing 45 Ca and H14 CO3. Magnetic stirring bars maintained water movement. A 100 μl sample of IM was removed before and after each incubation for the determination of total specific radioactivity (45 Ca plus inorganic 14 C), while 1 ml samples were removed for discrimination between the specific radioactivities of 45 Ca and inorganic 14 C (see below).

Measurement of the coelenteric compartment

At the end of the labelling period, each holder and its microcolony were immersed for 20 s in a beaker containing 600 ml of unlabelled efflux medium (EM) to prevent further isotope uptake and to reduce, by isotopic dilution, isotope adsorption. Labelled microcolonies were then placed in a beaker containing 30 ml of EM for 30 min. Upon completion of the efflux, a 100 μl sample of EM was collected for the determination of total radioactivity (45 Ca, organic 14 C and inorganic 14 C) and 2 ml was removed for discrimination between 45 Ca, organic 14 C and inorganic 14 C (see below). The radioactivity collected corresponds to the coelenteric compartment (for further details, see Tambutté et al., 1996).

Measurement of tissue and skeletal compartments

To separate tissues from skeleton, the microcolonies were heated at 90 °C for 20 min in 2 ml of 1 mol l−1 NaOH. Each skeleton was then rinsed with 1 ml of distilled water, which was collected and added to the dissolved tissues. A 100 μl sample of this solution, termed the ‘tissue pool’, was counted (total radioactivity: 45 Ca, organic 14 C and inorganic 14 C), a 500 μl sample was collected for protein measurements and 1 ml was used for discrimination between 45 Ca and inorganic 14 C.

Skeletons were processed according to the method described by Barnes and Crossland (1977). Briefly, clean skeletons were dissolved in 2 ml of 12 mol l−1 HCl. The resulting 14 CO2 was trapped on 200 μl of β-phenylethylamine absorbed onto two discs of Whatman GF/C filter paper in a scintillation vial connected to the test tube.

Sample processing

Measurement of the total radioactivity in the IM, EM and tissue pool samples was performed using a liquid scintillation counter (Tricarb, 1600 CA Packard) after the addition of 4 ml of the scintillation medium (Luma-gel; Packard). Since it is impossible to discriminate β emission from 45 Ca and from 14 C, the counting of a sample of double-labelled solution containing both isotopes allows the total isotope content (45 Ca, inorganic 14 C and organic 14 C) to be measured.

To discriminate labelled inorganic carbon within IM, EM and the tissue pool from 45 Ca and from the organic 14 C fraction, we performed an acid titration of the samples collected. For this purpose, 200 μl of either 1 mol l−1 HCl (for IM and EM) or 12 mol l−1 HCl (for the tissue pool) was added to the reserved samples, resulting in the formation of 14 CO2. Active bubbling with air for 15 min induces the loss of all inorganic 14 C from the medium. The radioactivity of 100 μl of the solution was then determined using a liquid scintillation counter (Tricarb, 1600 CA Packard) after the addition of the scintillation medium (4 ml of Luma-gel, Packard) and neutralisation. This fraction represents 45 Ca and 14 C-labelled organic carbon. The 14 C-labelled inorganic carbon content was then calculated by subtracting the 45 Ca and 14 C-labelled organic carbon content from the total isotope content.

For skeletons, the HCl fraction (containing 45 Ca) was counted after the addition of 4 ml of Luma-gel (Packard) and neutralisation, while the filters (containing inorganic 14 C) were counted after the addition of 4 ml of scintillation medium (Ultima-Gold XR, Packard).

Consequently, the results presented below concerning 14 C uptake relate only to inorganic carbon, whereas the results concerning 45 Ca include both 45 Ca and 14 C-labelled organic compounds. This is particularly important in the tissue compartment in the light, where 45 Ca incorporation measures both the absorption of calcium and the fixation of carbon by photosynthesis. Since we cannot discriminate in the tissue between radioactive calcium and organic carbon, we have discarded results concerning 45 Ca measurement in the tissue pool in the light. However, preliminary experiments have shown that the incorporation of 14 C-labelled organic compounds into the skeleton in the light was insignificant compared with the incorporation of inorganic carbon and can, therefore, be discounted. Similarly, secretion of 14 C-labelled organic compounds into the coelenteron is low compared with the amount of 14 C-labelled DIC.

Measurement of photosynthetic and respiration rates

The rates of photosynthesis and respiration were measured as the net rate of O2 production or consumption according to Bénazet-Tambutté et al. (1996b) using two micro-respirometers (Strathkelvin Instruments 928) each consisting of a double-walled cylindrical glass chamber (4 ml volume) and a Clark oxygen electrode (accurate to ±0.45 nmol O2 ml−1 ), with data output to a computer. Temperature was maintained at 25.0±0.5 °C using a recirculating water bath (Lauda RM20), and O2 stratification was avoided using a magnetic stirring bar.

Media and chemicals

IM and EM constituted either normal filtered sea water (FSW) or sea water (SW) depleted of or enriched in HCO3. In some experiments, inhibitors or competitors were added to the IM and EM. In each experiment, the IM and EM have the same composition, except that IM is a labelled medium containing 334–417 kBq of 45 Ca (supplied as CaCl2, 1.38 MBq ml−1 ; New England Nuclear) and 334–417 kBq of H14 CO3 (supplied as NaHCO3, 37 MBq ml−1 ; New England Nuclear) while EM is an unlabelled medium. FSW was obtained by filtering Mediterranean sea water through a 0.45 μm Millipore membrane. Bicarbonate-free FSW (0HCO3-FSW) was prepared according to Yellowlees et al. (1993). The pH of FSW was adjusted to 4.5 using 1 mol l−1 HCl to convert all DIC to CO2. The CO2 was removed by bubbling nitrogen through the FSW for approximately 1 h. The FSW was stirred throughout this procedure. The pH was then adjusted to 8.2 using 1 mmol l−1 Tris and CO2-free NaOH prepared from a saturated NaOH solution to precipitate any sodium carbonate from solution. The buffer concentration did not interfere with the measurements (results not shown). FSW containing different concentrations of dissolved inorganic carbon was obtained by adding various concentrations of NaHCO3 to 0HCO3-FSW before adjusting the pH and bubbling with air (Goiran et al., 1996). In all cases, the bicarbonate concentration was verified by calculation, according to the measured pH and total alkalinity (determined by the end-point titration acidometric technique using an alkalinity test; Merck).

4,4′-Diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS), an anion carrier inhibitor (Cabantchik and Greger, 1992), was dissolved in dimethyl sulfoxide (DMSO) to obtain a stock solution of 100 mmol l−1. The final concentration in FSW was 400 μmol l−1. Ethoxyzolamide (EZ), a carbonic anhydrase inhibitor (Palmqvist et al., 1988), was dissolved in DMSO to a concentration of 60 mmol l−1 and buffered with 1 mol l−1 Tris to pH 8.2. The final concentration in FSW was 600 μmol l−1. Although preliminary experiments demonstrated that concentrations of DMSO or ethanol up to 1 % (v/v) had no significant effect on dissolved inorganic carbon flux (results not shown), concentrations over 0.5 % were not used in the present study. Sodium iodide was used at a final concentration of 2 mmol l−1 as a competitor for HCO3 at the anion exchanger (Smith, 1988; Drechsler and Beer, 1991; Ilundain, 1992). All chemicals were obtained from Sigma or Merck.

Presentation of results

Results are expressed as nmol mg−1 protein in the NaOH-soluble pool and represent the mean ± S.E.M. for at least three replicates. Protein concentrations were measured in an autoanalyzer (Alliance Instruments) using the method of Lowry et al. (1951) and bovine serum albumin standards. One-way analysis of variance (ANOVA) with Bonferroni–Dunn post-hoc tests or Student’s t-tests were used to evaluate differences between means (95 % confidence limits).

The half-time (t1/2) of compartment loading and the affinity constant (Km) were calculated graphically according to the curve-fitting program Mac-curve fit with exponential [y=a(1—ex/b )+c] or Michaelis–Menten [y=ax(x+b)+c] equations. The flux into a given compartment was calculated according to Borle (1990) using the equations: unidirectional flux = k(compartment pool size) and k=loge2/t1/2.

Uptake and deposition of 45 Ca and 14 C in the dark

To determine the source of inorganic carbon for coral calcification and the kinetics of Ca2+ and DIC incorporation, we measured the rate of uptake of 45 Ca and 14 C into three compartments (coelenteron, tissues and skeleton). We first performed experiments in the dark to avoid any effects of photosynthesis. The rates of 45 Ca and 14 C uptake over 3 h in the dark by the coelenteric, tissue and skeletal compartments are depicted in Fig. 1. Ca2+ and DIC uptakes by both coelenteric and tissue compartments displayed saturable kinetics (Fig. 1A,B). In the coelenteron, equilibrium was reached for both isotopes after 15 min, but the t1/2 for DIC was six times the t1/2 for Ca2+. In the tissue compartment, at least 3 h was necessary before equilibrium was attained for the two isotopes. The equilibrium values in the coelenteron and in the tissues for Ca2+ and DIC and the t1/2 are summarised in Table 1. The equilibrium values represent the size of the exchangeable compartment for the two isotopes.

Table 1.

Ca2+ and dissolved inorganic carbon pool sizes in the coelenteron and tissue compartments of Stylophora pistillata microcolonies incubated in the dark

Ca2+ and dissolved inorganic carbon pool sizes in the coelenteron and tissue compartments of Stylophora pistillata microcolonies incubated in the dark
Ca2+ and dissolved inorganic carbon pool sizes in the coelenteron and tissue compartments of Stylophora pistillata microcolonies incubated in the dark
Fig. 1.

Time course of 45 Ca (e) and 14 C (0) incorporation into the coelenteron (A), tissues (B) and skeleton (C) of Stylophora pistillata microcolonies measured in the dark. Values are expressed as means ± S.E.M. Four microcolonies were analysed for each point.

Fig. 1.

Time course of 45 Ca (e) and 14 C (0) incorporation into the coelenteron (A), tissues (B) and skeleton (C) of Stylophora pistillata microcolonies measured in the dark. Values are expressed as means ± S.E.M. Four microcolonies were analysed for each point.

The time course of Ca2+ and HCO3 deposition in the skeletal compartment was linear for both isotopes for at least 3 h (Fig. 1C). No lag phase could be detected in the dark under our experimental conditions. The rate of Ca2+ incorporation in the dark (dark calcification rate) was 12.31±1.45 nmol h−1 mg−1 protein (N=23), whereas the rate of 14 C incorporation was 3.3 times lower, i.e. 3.77±0.77 nmol h−1 mg−1 protein (N=23).

Pharmacology of 45 Ca and 14 C-labelled DIC transport in the dark

To characterise the mechanisms of inorganic carbon absorption for coral calcification, we used inhibitors of anion transport (DIDS and iodide) and of carbonic anhydrase activity (EZ). These inhibitors did not affect the general metabolic rate, as indicated by the lack of any inhibitory action on the rate of respiration of Stylophora pistillata microcolonies in the dark (Table 2).

Table 2.

Rate of respiration in the dark of Stylophora pistillata in presence of DIDS, ethoxyzolamide and iodide

Rate of respiration in the dark of Stylophora pistillata in presence of DIDS, ethoxyzolamide and iodide
Rate of respiration in the dark of Stylophora pistillata in presence of DIDS, ethoxyzolamide and iodide

Fig. 2 shows the effect of DIDS, EZ and iodide on 45 Ca and 14 C incorporation into the tissue and skeletal compartments. The inhibitors did not affect the incorporation of isotopes in the coelenteric compartment (results not shown). 45 Ca and 14 C deposition in the skeleton was almost totally inhibited by 400 μmol l−1 DIDS, a blocker of anion transport (inhibition of 86±2 % and 89±2 % respectively; ANOVA, Bonferroni–Dunn post-hoc test, P<0.0001). Moreover, 2 mmol l−1 iodide, a competitor of HCO3 at anion exchangers, inhibited 45 Ca and 14 C skeletal incorporation equally (inhibition of 67±8 % and 65±7 % respectively; ANOVA, Bonferroni–Dunn post-hoc test, P<0.0083). Finally, EZ (600 μmol l−1 ), a carbonic anhydrase inhibitor, potently blocked 45 Ca incorporation (by 56.04±6.98 %; ANOVA, Bonferroni–Dunn post-hoc test, P<0.0083), but had no effect on 14 C incorporation. In the inset of Fig. 2A, the ratio between 14 C and 45 Ca incorporation is presented as percentage of 14 C-labelled DIC versus45 Ca incorporation. In control experiments, 14 C incorporation represented 25.56±4.11 % of calcification. No significant effect on this ratio was found in response to the addition of DIDS or iodide, while EZ induced an increase in the ratio (to 62.21±15.29 %; ANOVA, Bonferroni–Dunn post-hoc test P<0.0083).

Fig. 2.

Pharmacology of 45 Ca (filled columns) and 14 C (open columns) incorporation into the skeleton (A) and tissues (B) of Stylophora pistillata microcolonies in the dark. Effects of DIDS (400 μmol l−1 ), an anion exchanger inhibitor, iodide (Io; 2 mmol l−1 ), a competitor of HCO3−1 in anion transport, and ethoxyzolamide (EZ; 300 μmol l−1 ), a carbonic anhydrase inhibitor. The inset shows the effects of these inhibitors on the ratio between 14 C incorporation and 45 Ca incorporation, reported as the percentage of 14 C-labelled dissolved inorganic carbon versus45 Ca incorporation. Values are expressed as means + s.e.m. The number of microcolonies analysed was 23 for control experiments and 9–10 for experiments in the presence of an inhibitor. Results obtained in the presence of inhibitors were compared using one-way ANOVA and a Bonferroni–Dunn post-hoc test. Asterisks indicate values statistically different from the control: *P<0.0083, **P<0.0001. C, control.

Fig. 2.

Pharmacology of 45 Ca (filled columns) and 14 C (open columns) incorporation into the skeleton (A) and tissues (B) of Stylophora pistillata microcolonies in the dark. Effects of DIDS (400 μmol l−1 ), an anion exchanger inhibitor, iodide (Io; 2 mmol l−1 ), a competitor of HCO3−1 in anion transport, and ethoxyzolamide (EZ; 300 μmol l−1 ), a carbonic anhydrase inhibitor. The inset shows the effects of these inhibitors on the ratio between 14 C incorporation and 45 Ca incorporation, reported as the percentage of 14 C-labelled dissolved inorganic carbon versus45 Ca incorporation. Values are expressed as means + s.e.m. The number of microcolonies analysed was 23 for control experiments and 9–10 for experiments in the presence of an inhibitor. Results obtained in the presence of inhibitors were compared using one-way ANOVA and a Bonferroni–Dunn post-hoc test. Asterisks indicate values statistically different from the control: *P<0.0083, **P<0.0001. C, control.

Fig. 2B shows the effect of the inhibitors on isotope incorporation into the tissues. No significant effect of DIDS, iodide or EZ on the incorporation of 45 Ca and 14 C was observed.

Uptake and deposition of 45 Ca and 14 C in light-adapted microcolonies

A second set of experiments was performed in the light (250 μmol photons m−2 s−1 ) to investigate the effects of photosynthesis on the source of inorganic carbon for calcification and on the mechanisms of carbon incorporation. Fig. 3 shows the kinetics of 45 Ca and 14 C incorporation over 1 h in the light in the coelenteric and skeletal compartments. Ca2+ and HCO3 uptake by the coelenteron displayed saturable kinetics (Fig. 3A). 45 Ca uptake equilibrated within approximately 2 min, while H14 CO3 equilibration was achieved more slowly (8.6 min). In the tissue compartment, the uptake of 14 C-labelled DIC followed a sigmoidal time course, equilibrium being reached only after 3 h (Fig. 3B and inset). The equilibrium values in the coelenteron and in the tissues for Ca2+ and HCO3 and the kinetic constants are summarised in Table 3.

Table 3.

Ca2+ and dissolved inorganic carbon pool sizes in the coelenteron and tissue compartments of Stylophora pistillata microcolonies incubated in the light

Ca2+ and dissolved inorganic carbon pool sizes in the coelenteron and tissue compartments of Stylophora pistillata microcolonies incubated in the light
Ca2+ and dissolved inorganic carbon pool sizes in the coelenteron and tissue compartments of Stylophora pistillata microcolonies incubated in the light
Fig. 3.

Time course of 45 Ca (e) and 14 C (0) incorporation into the coelenteron (A), tissues (B) and skeleton (C) of Stylophora pistillata microcolonies measured in the presence of light (250 μmol photons m−2 s−1 ). In the inset, the kinetics of incorporation of 14 Cover 3 h is presented. Values are expressed as means ± s.e.m. Four microcolonies were analysed for each point.

Fig. 3.

Time course of 45 Ca (e) and 14 C (0) incorporation into the coelenteron (A), tissues (B) and skeleton (C) of Stylophora pistillata microcolonies measured in the presence of light (250 μmol photons m−2 s−1 ). In the inset, the kinetics of incorporation of 14 Cover 3 h is presented. Values are expressed as means ± s.e.m. Four microcolonies were analysed for each point.

In the presence of light, the deposition of 45 Ca and of 14 C into the skeletal compartment was linear (Fig. 3C) and this for for at least 3 h (results not shown). No lag phase could be detected under our experimental conditions. The rate of Ca2+ incorporation in the light (light calcification rate) was 49.25±3.71 nmol h−1 mg−1 protein (N=36). The rate of 14 C incorporation was 2.9 times slower, i.e. 17.14± 1.82 nmol h−1 mg−1 protein (N=36).

Effect of HCO3 concentration in the light

Fig. 4 shows 45 Ca and 14 C uptakes at external concentrations of HCO3 ranging from 0 to 3 mmol l−1. While HCO3 uptake by the coelenteric compartment was linearly correlated (r2 =0.948, P=0.0002) with the external HCO3 concentration, coelenteric Ca2+ incorporation was not dependent (r2 =0.042, P=0.6978) on external [HCO3 ] (Fig. 4A). In tissues, 14 C-labelled DIC uptake showed typical Michaelis–Menten saturable kinetics (Km=0.2 mmol l−1 ; Fig. 4B). The plateau (22.03±2.85 nmol h−1 mg−1 protein) was reached at an external HCO3 concentration of approximately 0.5 mmol l−1. Fig. 4C shows that the rate of 45 Ca incorporation into coral skeleton became saturated at an external HCO3 concentration of approximately 1 mmol l−1, while the rate of 14 C deposition into the skeleton was linear up to 3 mmol l−1 HCO3.

Fig. 4.

External [HCO3 ]-dependence of 45 Ca (e) and 14 C (0) incorporation into the coelenteron (A), tissues (B) and skeleton (C) of Stylophora pistillata microcolonies measured in the presence of light (250 μmol photons m−2 s−1 ). Values are expressed as means ± s.e.m. Four microcolonies were analysed.

Fig. 4.

External [HCO3 ]-dependence of 45 Ca (e) and 14 C (0) incorporation into the coelenteron (A), tissues (B) and skeleton (C) of Stylophora pistillata microcolonies measured in the presence of light (250 μmol photons m−2 s−1 ). Values are expressed as means ± s.e.m. Four microcolonies were analysed.

Pharmacology of 45 Ca and 14 C-labelled DIC transport in the presence of light

Previous studies have shown that DIDS and EZ inhibit coral photosynthesis in the light (Al-Moghrabi et al., 1996), but no data are available on the effects of iodide. We therefore measured the rate of photosynthesis of microcolonies of Stylophora pistillata in the presence of 2 mmol l−1 sodium iodide. No difference in the rate of oxygen production in the absence or presence of this competitor was detected (179.71±23.08 and 172.47±8.51 nmol h−1 mg−1 protein, respectively, Student’s t-test, P>0.05; result not shown).

Fig. 5 shows the effects of DIDS, EZ and iodide on 45 Ca and 14 C incorporation (in the light) into the skeletal (Fig. 5A) and tissue (Fig. 5B) compartments. As for experiments performed in the dark, no effect of these inhibitors on the uptake of isotopes was observed in the coelenteric compartment (results not shown), consistent with previous observations (Tambutté et al., 1996).

Fig. 5.

Pharmacology of 45 Ca (filled columns) and 14 C (open columns) incorporation into the skeleton (A) and tissues (B) of Stylophora pistillata microcolonies in the presence of light (250 μmol photons m−2 s−1 ). The effects of DIDS (400 μmol l−1 ), an anion exchanger inhibitor, iodide (Io; 2 mmol l−1 ), a competitor of HCO3 in anion exchangers, and ethoxyzolamide (EZ; 300 μmol l−1 ), a carbonic anhydrase inhibitor, are shown. The inset shows the effects of these inhibitors on the ratio between 14 C incorporation and 45 Ca incorporation, reported as the percentage of 14 C-labelled dissolved inorganic carbon versus45 Ca incorporation. Values are expressed as means + s.e.m. The number of microcolonies analysed was 36 for control experiments and 5–12 for experiments in the presence of inhibitors. Results obtained in the presence of inhibitors were compared using one-way ANOVA and a Bonferroni–Dunn post-hoc test. Asterisks indicate values statistically different from the control: *P<0.0083, **P<0.0001. C, control.

Fig. 5.

Pharmacology of 45 Ca (filled columns) and 14 C (open columns) incorporation into the skeleton (A) and tissues (B) of Stylophora pistillata microcolonies in the presence of light (250 μmol photons m−2 s−1 ). The effects of DIDS (400 μmol l−1 ), an anion exchanger inhibitor, iodide (Io; 2 mmol l−1 ), a competitor of HCO3 in anion exchangers, and ethoxyzolamide (EZ; 300 μmol l−1 ), a carbonic anhydrase inhibitor, are shown. The inset shows the effects of these inhibitors on the ratio between 14 C incorporation and 45 Ca incorporation, reported as the percentage of 14 C-labelled dissolved inorganic carbon versus45 Ca incorporation. Values are expressed as means + s.e.m. The number of microcolonies analysed was 36 for control experiments and 5–12 for experiments in the presence of inhibitors. Results obtained in the presence of inhibitors were compared using one-way ANOVA and a Bonferroni–Dunn post-hoc test. Asterisks indicate values statistically different from the control: *P<0.0083, **P<0.0001. C, control.

Fig. 5A shows that the deposition of 45 Ca and of 14 C into the skeleton was almost totally inhibited by DIDS (inhibition of 85±4 % and 97±1 %, respectively; ANOVA, Bonferroni–Dunn post-hoc test, P<0.0001). EZ also had an important inhibitory effect on the deposition of both isotopes (inhibition of 67±4 % and 62±6 %, respectively; ANOVA, Bonferroni–Dunn post-hoc test, P<0.0001). In contrast, only a small non-significant inhibition was observed in the presence of iodide. In the inset of Fig. 5A, the ratio between 14 C and 45 Ca incorporation is presented as the percentage of 14 C-labelled DIC versus45 Ca incorporation. In control experiments, the incorporation of 14 C represented 36.6±3.2 % of the calcification. While no significant effect on the ratio was observed after the addition of iodide or EZ, exposure to DIDS induced a decrease in this and 0.11±0.01 nmol 14 C min−1 mg−1 protein). ratio (to 9.2±2.3 %; ANOVA, Bonferroni–Dunn post-hoc test, P<0.0083). The results presented in Fig. 5B show that uptake of 14 C-labelled DIC by the tissue was almost totally inhibited by DIDS, iodide and EZ (by 100±1 %, 90±3 % and 80±2 %, respectively; ANOVA, Bonferroni–Dunn post-hoc test, P<0.0001).

Uptake and deposition of 45 Ca and 14 C in the presence of light after a dark period

Fig. 6 shows the kinetics of the incorporation of 45 Ca and 14 C into the three compartments over 1 h when microcolonies were placed in the light after having spent 12 h in the dark. Both Ca2+ and DIC uptake by the coelenteron displayed saturable kinetics (Fig. 6A,B), with equilibrium being reached within 4–10 min for both isotopes (t1/2 =1.8 min and 5.4 min, respectively, for Ca2+ and DIC). Upon illumination of the microcolonies, 14 C-labelled DIC uptake by the tissues was linear, whereas the incorporation of 45 Ca displayed a slight lag phase of approximately 4 min (Fig. 6C,D). Finally, Fig. 6E,F shows that the deposition of 45 Ca and 14 C into the skeleton of Stylophora pistillata follows biphasic kinetics: the rates were low during the first 10 min (0.55±0.06 nmol 45 Ca min−1 mg−1 protein and 0.058±0.015 nmol 14 C min−1 mg−1 protein) but subsequently increased (0.91±0.13 nmol 45 Ca min−1 mg−1 protein

Fig. 6.

Time course of 45 Ca (e) and 14 C (0) incorporation into the coelenteron (A,B), tissues (C,D) and skeleton (E,F) of Stylophora pistillata microcolonies measured in the presence of light (250 μmol photons m−2 s−1 ) after a dark period of 12 h. Broken lines represent the extrapolated rate of Ca2+ and HCO3 incorporation into the skeletal compartment over the first 10 min of incubation. Values are expressed as means ± s.e.m. Four microcolonies were analysed for each point.

Fig. 6.

Time course of 45 Ca (e) and 14 C (0) incorporation into the coelenteron (A,B), tissues (C,D) and skeleton (E,F) of Stylophora pistillata microcolonies measured in the presence of light (250 μmol photons m−2 s−1 ) after a dark period of 12 h. Broken lines represent the extrapolated rate of Ca2+ and HCO3 incorporation into the skeletal compartment over the first 10 min of incubation. Values are expressed as means ± s.e.m. Four microcolonies were analysed for each point.

Sources of inorganic carbon for calcification

In the present study, sources of inorganic carbon for calcification of Stylophora pistillata have been characterised and compared for microcolonies incubated in the dark or in the presence of light. Under both conditions, the rate of 45 Ca deposition into the coral skeleton was greater than the rate of 14 C incorporation (Figs 1C, 3C). The ratio 14 C/45 Ca expresses the incorporation of DIC originating from the external medium: a ratio of 1 (100 %) means that the same amounts of seawater DIC and Ca2+ are incorporated into the skeleton, while a lower value means that an unlabelled source of DIC is also used. In the present experiments, the ratio in the dark did not differ significantly from that in the light (25.56±4.11 % in the dark and 36.60±3.22 % in the light; Student’s t-test, P>0.05). These results demonstrate that the inorganic carbon originating from the surrounding medium does not constitute the major source of DIC for coral calcification in either light or dark conditions. The labelled seawater DIC is likely to be diluted by unlabelled DIC originating from coral tissue. This unlabelled DIC could be either intracellular bicarbonate or metabolic CO2, as suggested by Goreau (1961), Pearse (1970) and Erez (1978). The calcification rate remains constant for periods of at least 180 min (Figs 1, 3), ruling out the involvement of the tissue pool, the t1/2 of which is less than 100 min (Tables 1, 3). Consequently, the unlabelled source of DIC must be the metabolic CO2 produced by the symbiotic association.

A role for this metabolic CO2 as source of carbon for calcification has already been demonstrated in non-symbiotic gorgonians (Allemand and Grillo, 1992; Lucas and Knapp, 1997) and in symbiotic corals (Crossland, 1980). Supporting this hypothesis, the calicoblastic cells, which are responsible for skeletogenesis, contain numerous mitochondria (Johnston, 1980; E. Tambutté, personal communication). Furthermore, measured respiration rates in Stylophora pistillata microcolonies (Table 2) show that metabolic CO2 production (193.92±13.04 nmol h−1 mg−1 protein) is sufficient to support the metabolic inorganic carbon requirements for calcification in both dark and light conditions (8.54±1.64 nmol h−1 mg−1 protein and 32.11±4.13 nmol h−1 mg−1 protein, respectively, i.e. from 4.4 to at least 16.5 % of metabolic CO2 production). Moreover, the respiration rate probably increases in the light (Shick, 1990). While Goreau (1961) and Erez (1978) reported a 14 C/45 Ca ratio in the light similar to the ratio that we measured here (21 % and 22 %, respectively), their results differed in the dark. Erez (1978) determined that the major source of DIC in the dark was the external pool (mean dark 14 C/45 Ca ratio 93 %). However, his experiments were performed in situ, and there was marked variability in the results obtained, with the ratio in the dark varying between 28 and 100 %.

Our results demonstrate that the major source of DIC for calcification is the metabolic DIC pool irrespective of lighting conditions and, therefore, of photosynthesis. This result should be taken into account for paleoenvironmental studies using stable carbon isotopes since a predominant incorporation of metabolic CO2 leads to the formation of a skeleton with a light isotopic composition, which does not reflect the situation in sea water.

Compartmental analysis of Ca2+ and dissolved inorganic carbon

Coelenteric Ca2+ and DIC analysis

Both in the dark and in the light, the equilibrium constant (t1/2) for DIC in the coelenteron was higher than that for Ca2+ (Tables 1, 3). This is probably a consequence of the higher transepithelial permeability to Ca2+ compared with HCO3 (Bénazet-Tambutté et al., 1996a; Furla et al., 1998a). In the light, both equilibrium constants increased slightly compared with dark conditions (Tables 1, 3), suggesting that the permeability of the tentacles is probably reduced by light. These changes are probably due to a modification of the tissue thickness induced by light (Furla et al., 1998a). Absorption of both Ca2+ and DIC into the coelenteric cavity was insensitive to inhibitors of ion transport, suggesting that it occurs via passive diffusion. This was confirmed by the linear correlation between the external [HCO3 ] and the coelenteric DIC pool (Fig. 4A). These results support the finding of Tambutté et al. (1996), who demonstrated that calcium uptake in the coelenteron is mediated by a paracellular pathway across the oral tissue.

From the size of the coelenteric DIC pool and the kinetic constant, it is possible to calculate the transepithelial DIC flux (see Materials and methods). In the dark, the passive trans-epithelial flux through oral tissues is 2.2 nmol min−1 mg−1 protein (i.e. 132 nmol h−1 mg−1 protein), while it is theoretically 1.03 nmol min−1 mg−1 protein (i.e. 62 nmol min−1 mg−1 protein) in the light. This passive transepithelial flux of DIC through oral tissues appears to be enough to support calcification even in the light (49 nmol min−1 mg−1 protein). However, this flux is far too small to support symbiont photosynthesis (180 nmol min−1 mg−1 protein), suggesting the need for active uptake of DIC by the oral tissue, in accordance with previous findings (Furla et al., 2000).

In the presence of light, the coelenteric DIC pool was decreased by approximately 44 % compared with dark conditions (Tables 1, 3). When equilibrium is reached between the Ca2+ concentration within the coelenteron and the sea water, it is possible to extrapolate the water volume of the coelenteron (Tambutté et al., 1996). From the data presented in Tables 1 and 3 concerning the coelenteric Ca2+ pool, and the specific radioactivity, water volumes of 10.76±0.78 μl mg−1 protein in the dark and 8.61±0.45 μl mg−1 protein in light were calculated. From these values, and knowing the size of the coelenteric HCO3 pool, it is possible to calculate the coelenteric concentration of HCO3 : 2.29±0.15 mmol l−1 in the dark and 1.29±0.05 mmol l−1 in the light. A similar light-dependent decrease in DIC concentration within body fluids has been described in symbiotic clams (Leggat et al., 1999) and may reflect the continuous uptake of DIC for both calcification and photosynthesis. Activation of photosynthesis in the presence of light probably induced, during the first few minutes of illumination, an instantaneous uptake of DIC from the coelenteric medium by endodermal cells, as previously demonstrated in the Mediterranean sea anemone Anemonia viridis. In this species, a comparable decrease in HCO3 concentration in the light (dark coelenteric DIC concentration 2.43±0.02 mmol l−1, P. Furla and D. Allemand, unpublished data; light coelenteric DIC concentration 1.31±0.18 mmol l−1 ; P<0.05, Furla et al., 1998b) and a simultaneous increase in the coelenteric pH to 9.0 (Furla et al., 1998b) have been measured. Since the DIC used for calcification necessarily originates from the coelenteron, the dependence of calcification on DIC (Fig. 4) can be redrawn using the calculated DIC concentration within the coelenteron (Fig. 7). Now, calcification appears to be saturated at low coelenteric HCO3 concentrations (approximately 200 μmol l−1 ; Km 60 μmol l−1 ).

Fig. 7.

Coelenteric [HCO3 ]-dependence of 45 Ca (e) and 14 C (0) incorporation into the skeleton of Stylophora pistillata microcolonies measured in the presence of light (data from Fig. 4C). Values are expressed as means ± s.e.m. Four microcolonies were analysed.

Fig. 7.

Coelenteric [HCO3 ]-dependence of 45 Ca (e) and 14 C (0) incorporation into the skeleton of Stylophora pistillata microcolonies measured in the presence of light (data from Fig. 4C). Values are expressed as means ± s.e.m. Four microcolonies were analysed.

Consequently, the chemistry of inorganic carbon within the coelenteron is profoundly altered upon illumination. If we assume that the coelenteric pH varies at least from 7.5 (in the dark) to 8.5 (in the light), as measured by Kühl et al. (1995) and by Furla et al. (1998b), and taking into account changes in total DIC measured in the present study, it is possible to calculate the carbon species involved. In Table 4, we have summarised the main metabolic characteristics of Stylophora pistillata. These data show that CO2 concentrations vary from 66 μmol l−1 in the dark to 3 μmol l−1 in the light, and that the saturation states of calcite and aragonite are increased by a factor 4.7 in the light.

Table 4.

Summary of the main characteristics of Ca2+ s and dissolved inorganic carbon metabolism in Stylophora pistillata microcolonies in the dark or in light

Summary of the main characteristics of Ca2+ s and dissolved inorganic carbon metabolism in Stylophora pistillata microcolonies in the dark or in light
Summary of the main characteristics of Ca2+ s and dissolved inorganic carbon metabolism in Stylophora pistillata microcolonies in the dark or in light

Tissue Ca2+ and DIC analysis

The DIC equilibrium value in the tissues was reached in the light, as in the dark, after approximately 3 h (Fig. 1B, inset of Fig. 3B). Nevertheless, the DIC pool in the light was approximately 39 times larger than in the dark (Table 3). If we consider the cell water space measured by Bénazet-Tambutté et al. (1996a), i.e. 1.56 μl mg−1 protein, we can calculate the cellular concentration of DIC in the light and in the dark from the DIC tissue pool: 147 and 3.7 mmol l−1, respectively.

Consequently, the ratio [DIC]intracellular/[DIC]extracellular increases from 1.6±0.2 in the dark to 61.3±0.2 in the light. This increase is another argument supporting the proposition that a CO2-concentrating mechanism is stimulated upon illumination, as previously suggested by Allemand et al. (1998). Furthermore, the ratio measured in the light (61.3) is within the range found in phototrophs possessing a CO2-concentrating mechanism, which varies between 10 in some macrophytes to 16 000 in some cyanobacteria (Aizawa and Miyachi, 1986; Bowes and Salvucci, 1989).

Mechanisms of DIC transport for coral photosynthesis and calcification

In the presence of light, DIC accumulation is sensitive to DIDS, iodide and EZ, suggesting the involvement of an anion exchanger and carbonic anhydrase in the absorption of DIC for photosynthesis (Fig. 5B). While Furla et al. (2000) recommended that care should be taken regarding the specificity of DIDS in corals, the inhibitory effect of iodide confirms the involvement of anion exchangers. However, because the inhibitory effect of DIDS is greater than that of iodide, we cannot exclude the participation of an H+ -ATPase, as has been described previously in other symbiotic, but non-calcifying, cnidarians (Furla et al., 2000).

Pharmacological experiments performed in the dark allowed the determination of mechanisms by which DIC is transported for coral calcification independently of symbiont photosynthesis. The results presented in Fig. 2A show that the target of DIDS also plays an important role in the mechanisms of coral calcification, since the skeletal incorporation of Ca2+ and DIC was greatly inhibited by this blocker, as previously shown by Tambutté et al. (1996). However, previous studies have demonstrated that this inhibitor affects not only anion exchangers but also anion conductances and P-type H+ -ATPases (Furla et al., 2000). Consequently, it is possible that DIDS could also inhibit the Ca2+ -ATPase, another P-type ATPase (Forgac, 1989), responsible for the extrusion of Ca2+ from calicoblastic cells (Ip et al., 1991; Tambutté et al., 1996).

Nevertheless, the role of anion transport in CaCO3 precipitation was confirmed by the inhibitory action of iodide. This competitor affected both Ca2+ incorporation into the skeleton and DIC incorporation, demonstrating that its target is involved in both external and metabolic DIC incorporation. Finally, EZ alters the ratio of seawater/metabolic DIC supply in the dark (inset of Fig. 2A) but is without effect on 14 C incorporation, suggesting that only the metabolic source of DIC is dependent on carbonic anhydrase activity. The inhibition of the metabolic source was partially compensated by the predominant absorption of external DIC (62 % of total CaCO3 deposition instead of 25 %). In the light, similar results were obtained, suggesting that the mechanisms of calcification in the light are similar to those in the dark. However, while EZ had only a slight and insignificant stimulatory effect on the 14 C/45 Ca ratio, DIDS inhibited the supply of DIC from external sea water, and this was offset by an increase in the supply of metabolic CO2 (inset of Fig. 5).

The present data suggest that, irrespective of its sources (external or metabolic), DIC uptake for symbiont photosynthesis and secretion by calicoblastic cells into the skeleton is dependent on an anion transport mechanism, indicating that HCO3 is the ionic species transported. Our results also demonstrate that the metabolic source of DIC is dependent on carbonic anhydrase, which catalyses the hydration of CO2 to HCO3 in the calicoblastic cells, to prevent leakage of gaseous CO2. After paracellular diffusion of DIC across the oral tissue, the uptake of external DIC into calicoblastic cells occurs by a mechanism that remains to be characterised.

Light-enhanced calcification

Numerous studies of coral calcification have demonstrated stimulation of CaCO3 deposition in the light compared with the dark (for a review, see Gattuso et al., 1999). Similarly, in the present study, we have shown that the rate of calcification of Stylophora pistillata microcolonies in the light is 4.00±0.08 times greater than the rate of calcification in the dark (Figs 1C, 3C). Moreover, the application of blockers of photosynthesis led to an inhibition of this light-enhanced calcification. Incorporation of both isotopes was similarly stimulated. Interestingly, the enhancement of calcification was apparent only 10 min after the onset of illumination (Fig. 6). The kinetics of light-enhanced calcification has not been extensively studied. Barnes and Crossland (1978) measured a lag period of 35–45 min before inorganic carbon deposition was stimulated in the skeleton of Acropora acuminata. These authors suggested that this lag phase was an artefact arising from the dilution of 14 C by an unlabelled pool of DIC in the tissue. Our results are not consistent with such a hypothesis since we measured a lag phase for both 45 Ca and 14 C-labelled DIC deposition. Moreover, in the tissues, this lag phase was shorter (2–4 min) for both isotopes (Fig. 6C,D). Finally, the inorganic carbon present in the tissues was saturated after 60 min in the dark and after 150 min in the light, whereas the rate of calcification remained constant for at least 3 h. These results suggest the activation of mechanisms for DIC and Ca2+ absorption and/or deposition following illumination (Mueller, 1984).

Numerous hypotheses have been proposed to explain the stimulation of calcification by light (for a review, see Barnes and Chalker, 1990). The most relevant are light-enhanced calcification (Goreau, 1959; Allemand et al., 1998), dark-repressed calcification (Marshall, 1996) and trans-calcification (McConnaughey, 1995; McConnaughey and Whelan, 1997). Goreau (1959) suggested that H+ secretion produced by calcification led to the production of CO2 within the coelenteron, which was removed by photosynthesis according to the following equations:
formula
formula
The hypothesis of McConnaughey and Whelan (1997) is similar to that of Goreau (1959) (coelenteric H+ released by CaCO3 precipitation is used for coelenteric HCO3 dehydration to produce CO2), but suggests that, in this way, calcification may stimulate photosynthesis by supplying CO2. However, this last model was recently contradicted by studies performed by Gattuso et al. (2000), who demonstrated that it is possible to inhibit light-induced calcification without influencing photosynthesis. Allemand et al. (1998) have noted that all these hypotheses assume that the DIC source for photosynthesis is supplied from the coelenteron, which is probably not the case (Furla et al., 1998b; present study). They suggested an alternative hypothesis for light-enhanced calcification based on the titration of H+ produced by calcification with the OH produced by photosynthesis (Furla et al., 1998b).

The present study confirms that, despite a decrease in coelenteric DIC concentration, there is a light-induced stimulation of calcification in Stylophora pistillata. Figs 4C and 7 reveal that the rate of calcification in the light is limited neither by external DIC concentration (Km 220 μmol l−1 ) nor by coelenteric DIC concentration (Km 60 μmol l−1 ), suggesting that there is no competition between photosynthesis and calcification for the external DIC source. These results contrast with the recent findings of Marubini and Thake (1999), who described stimulation of the calcification of the coral Porites porites by approximately 62 % after the addition of 2 mmol l−1 HCO3 in sea water.

Our results demonstrate that light-enhanced calcification is not dependent on a change in the supply of DIC, which remains mainly metabolic CO2. Moreover, it is not an instantaneous phenomenon, but the stimulation of calcification requires activation of some unknown physiological mechanisms. This activation is responsible for the lag period observed. We hypothesise that changes in the pH/carbon state within the coelenteric cavity are part of this mechanism.

Concluding remarks

This paper clarifies such aspects of coral calcification as DIC sources, the mechanisms of DIC deposition into the skeleton, the interactions between photosynthesis and calcification and the activation of light-enhanced calcification. The conclusions, summarised in Fig. 8, are that the major source of DIC for coral calcification is metabolic CO2 (independent of lighting conditions), and that DIC availability correlates with the presence of carbonic anhydrase activity probably localised within the calicoblastic cells, as previously suggested by Isa and Yamazato (1984). The secretion of DIC at the site of calcification is performed by an anion exchanger. After paracellular diffusion across the oral epithelial layers, 25–30 % of the DIC that originates from the external medium enters the calicoblastic cells by a DIDS-sensitive mechanism, which remains uncharacterised. There is no competition for DIC between photosynthesis and calcification of Stylophora pistillata microcolonies, calcification being saturated at low external and coelenteric DIC concentrations. Our results demonstrate the absence of an internal DIC pool for coral calcification. However, there is a 61-fold accumulation of DIC within the tissue in the light compared with the DIC concentration of the external medium. This accumulation occurs through the CO2-concentrating mechanism used for symbiont photosynthesis. Finally, we report a fourfold stimulation of calcification of Stylophora pistillata microcolonies in the presence of light, which appears after 10 min of illumination. We suggest that the coelenteric secretion of OH consecutive to external HCO3 absorption for symbiont photosynthesis favours stimulation of light-induced calcification.

Fig. 8.

Model of dissolved inorganic carbon (DIC) absorption for coral calcification and photosynthesis. Details are given in the text. Cal. Cell, calicoblastic cell; Zoox, zooxanthella; Pi, inorganic phosphate; CA, carbonic anhydrase.

Fig. 8.

Model of dissolved inorganic carbon (DIC) absorption for coral calcification and photosynthesis. Details are given in the text. Cal. Cell, calicoblastic cell; Zoox, zooxanthella; Pi, inorganic phosphate; CA, carbonic anhydrase.

We are grateful to G. Albini for performing the preliminary experiments. We also thank the Marine Station of Aqaba for facilitating the initial collection of corals, and the staff of the public aquarium of the Oceanographic Museum. We are also very grateful to Dr M. Frankignoulle for the free use of his ‘CO2’ program and to Drs F. Marubini and R. J. Wilkins for helpful comments on the manuscript. This study was conducted as part of the Centre Scientifique de Monaco 1996–2000 research program. It was supported by the government of the Principality of Monaco and the Council of Europe (Open Partial Agreement on Major Natural and Technological Disasters).

Aizawa
,
K.
and
Miyachi
,
S.
(
1986
).
Carbonic anhydrase and CO2-concentrating mechanisms in microalgae and cyanobacteria
.
FEMS Microbiol.
39
,
215
233
.
Al-Moghrabi
,
S.
,
Allemand
,
D.
and
Jaubert
,
J.
(
1993
).
Valine uptake by the scleractinian coral Galaxea fascicularis: characterization and effect of light and nutritional status
.
J. Comp. Physiol. B
163
,
355
362
.
Al-Moghrabi
,
S.
,
Goiran
,
C.
,
Allemand
,
D.
,
Speziale
,
N.
and
Jaubert
,
J.
(
1996
).
Inorganic carbon uptake for photosynthesis by the symbiotic coral/dinoflagellate association. II. Mechanisms for bicarbonate uptake
.
J. Exp. Mar. Biol. Ecol.
199
,
227
248
.
Allemand
,
D.
,
Furla
,
P.
and
Bénazet-Tambutté
,
S.
(
1998
).
Mechanisms of carbon acquisition for endosymbiont photosynthesis in Anthozoa
.
Can. J. Bot.
76
,
925
941
.
Allemand
,
D.
and
Grillo
,
M.-C.
(
1992
).
Biocalcification mechanisms in gorgonians. 45 Ca uptake and deposition by the Mediterranean red coral Corallium rubrum
.
J. Exp. Zool
.
292
,
237
246
.
Barnes
,
D. J.
and
Chalker
,
B. E.
(
1990
).
Calcification and photosynthesis in reef-building corals and algae
. In
Coral Reefs
(ed.
Z.
Dubinsky
), pp.
109
131
. Amsterdam: Elsevier.
Barnes
,
D. J.
and
Crossland
,
C. J.
(
1977
).
Coral calcification: sources of error in radioisotope techniques
.
Mar. Biol.
42
,
119
129
.
Barnes
,
D. J.
and
Crossland
,
C. J.
(
1978
).
Diurnal productivity and apparent 14 C calcification in the staghorn coral Acropora acuminata
.
Comp. Biochem. Physiol.
59A
,
133
138
.
Barnes
,
D. J.
and
Lough
,
J. M.
(
1996
).
Coral skeletons: storage and recovery of environmental information
.
Global Change Biol.
2
,
569
582
.
Bénazet-Tambutté
,
S.
,
Allemand
,
D.
and
Jaubert
,
J.
(
1996a
).
Permeability of the oral epithelial layers in Cnidarians
.
Mar. Biol.
126
,
43
53
.
Bénazet-Tambutté
,
S.
,
Allemand
,
D.
and
Jaubert
,
J.
(
1996b
).
Inorganic carbon supply to symbiont photosynthesis of the sea anemone, Anemonia viridis: role of the oral epithelial layers
.
Symbiosis
20
,
199
217
.
Borle
,
A. B.
(
1990
).
Measurement of calcium movement across membranes: kinetic analysis and conceptualization
. In
Intracellular Calcium Regulation
(ed.
F.
Bronner
), pp.
19
75
.
New York
:
Wiley-Liss
.
Bowes
,
G.
and
Salvucci
,
M. E.
(
1989
).
Plasticity in the photosynthetic carbon metabolism of submersed aquatic macrophytes
.
Aquat. Bot.
34
,
233
266
.
Cabantchik
,
Z. I.
and
Greger
,
R.
(
1992
).
Chemical probes for anion transporters of mammalian cell membranes
.
Am. J. Physiol.
262
,
C803
C827
.
Chave
,
K. E.
,
Smith
,
S. V.
and
Roy
,
K. J.
(
1975
).
Carbonate production by coral reefs
.
Mar. Geol
.
12
,
123
140
.
Crossland
,
C. J.
(
1980
).
Release of photosynthetically-derived organic carbon from a hermatypic coral, Acropora cf. acuminata
.
Endocytobiol.
1
,
163
171
.
Drechsler
,
Z.
and
Beer
,
S.
(
1991
).
Utilization of inorganic carbon by Ulva lactuca
.
Plant Physiol.
97
,
1439
1444
.
Druffel
,
E. R. M.
(
1997
).
Geochemistry of corals: Proxies of past ocean chemistry, ocean circulation and climate
.
Proc. Natl. Acad. Sci. USA
94
,
8354
8361
.
Erez
,
J.
(
1978
).
Vital effect on stable-isotope composition seen in foraminifera and coral skeletons
.
Nature
273
,
199
202
.
Forgac
,
M.
(
1989
).
Structure and function of vacuolar class of ATP-driven proton pumps
.
Physiol. Rev.
69
,
765
796
.
Furla
,
P.
,
Bénazet-Tambutté
,
S.
,
Jaubert
,
J.
and
Allemand
,
D.
(
1998a
).
Diffusional permeability of dissolved inorganic carbon through the isolated oral epithelial layers of the sea anemone, Anemonia viridis
.
J. Exp. Mar. Biol. Ecol.
221
,
71
88
.
Furla
,
P.
,
Bénazet-Tambutté
,
S.
,
Jaubert
,
J.
and
Allemand
,
D.
(
1998b
).
Functional polarity of the tentacle of the sea anemone Anemonia viridis: role in inorganic carbon acquisition
.
Am. J. Physiol.
274
,
R303
R310
.
Furla
,
P.
,
Orsenigo
,
M. N.
and
Allemand
,
D.
(
2000
).
Involvement of H+ -ATPase and carbonic anhydrase in inorganic carbon absorption for endosymbiont photosynthesis
.
Am. J. Physiol.
278
,
R870
R881
.
Gates
,
R. D.
and
Edmunds
,
P. J.
(
1999
).
The physiological mechanisms of acclimatization in tropical reefs corals
.
Am. Zool.
39
,
30
43
.
Gattuso
,
J.-P.
,
Allemand
,
D.
and
Frankignoulle
,
M.
(
1999
).
Photosynthesis and calcification at cellular, organismal and community levels in coral reefs: A review on interactions and control by carbonate chemistry
.
Am. Zool
.
39
,
160
183
.
Gattuso
,
J.-P.
,
Reynaud
,
S.
,
Bourge
,
I.
,
Furla
,
P.
,
Romaine-Lioud
,
S.
,
Frankignoulle
,
M.
and
Jaubert
,
J.
(
2000
).
Calcification does not stimulate photosynthesis in the zooxanthellate scleractinian coral Stylophora pistillata
.
Limnol. Oceanogr
.
45
,
246
250
.
Goiran
,
C.
,
Al-Moghrabi
,
S.
,
Allemand
,
D.
and
Jaubert
,
J.
(
1996
).
Inorganic carbon uptake for photosynthesis by the symbiotic coral/dinoflagellate association. I. Photosynthetic performances of symbionts and dependence on sea water bicarbonate
.
J. Exp. Mar. Biol. Ecol.
199
,
207
225
.
Goreau
,
T. F.
(
1959
).
The physiology of skeleton formation in corals. I. A method for measuring the rate of calcium deposition by corals under different conditions
.
Biol. Bull.
116
,
59
75
.
Goreau
,
T. F.
(
1961
).
On the relation of calcification to primary productivity in reef building organisms
. In
The Biology of Hydra and of Some Other Coelenterates
(ed.
H. M.
Lenhoff
and
W. F.
Loomis
), pp.
269
285
. Coral Gables, Florida: University of Miami Press.
Goreau
,
T. J.
(
1977
).
Coral skeletal chemistry: physiological and environmental regulation of stable isotopes and trace metals in Montastrea annularis
.
Proc. R. Soc. Lond. B
196
,
291
315
.
Ilundain
,
A.
(
1992
).
Intracellular pH regulation in intestinal and renal epithelial cells
.
Comp. Biochem. Physiol.
101A
,
413
424
.
Ip
,
Y. K.
,
Lim
,
A. L. L.
and
Lim
,
R. W. L.
(
1991
).
Some properties of calcium-activated adenosine triphosphatase from the hermatypic coral Galaxea fascicularis
.
Mar. Biol.
111
,
191
197
.
Isa
,
Y.
and
Yamazato
,
K.
(
1984
).
The distribution of carbonic anhydrase in a staghorn coral Acropora hebes (Dana)
.
Galaxea
3
,
25
36
.
Johnston
,
I. S.
(
1980
).
The ultrastructure of skeletogenesis in zooxanthellate corals
.
Int. Rev. Cytol
.
67
,
171
214
.
Kühl
,
M.
,
Cohen
,
Y.
,
Dalsgaard
,
T.
,
Jorgensen
,
B. B.
and
Revsbech
,
N. P.
(
1995
).
Microenvironment and photosynthesis of zooxanthellae in scleractinian corals studied with microsensors for O2, pH and light
.
Mar. Ecol. Prog. Ser
.
117
,
159
172
.
Leggat
,
W.
,
Badger
,
M. R.
and
Yellowlees
,
D.
(
1999
).
Evidence for an inorganic carbon-concentrating mechanism in the symbiotic dinoflagellate Symbiodinium sp
.
Plant Physiol.
121
,
1247
1255
.
Lowry
,
O. H.
,
Rosebrough
,
N. J.
,
Farr
,
A. L.
and
Randall
,
R. J.
(
1951
).
Protein measurement with the folin phenol reagent
.
J. Biol. Chem.
193
,
265
275
.
Lucas
,
J. M.
and
Knapp
,
L. W.
(
1997
).
A physiological evaluation of carbon sources for calcification in the octocoral Leptogorgia virgulata (Lamarck)
.
J. Exp. Biol.
200
,
2653
2662
.
Marshall
,
A. T.
(
1996
).
Calcification in hermatypic and ahermatypic corals
.
Science
271
,
637
639
.
Marubini
,
F.
and
Thake
,
B.
(
1999
).
Bicarbonate addition promotes coral growth
.
Limnol. Oceanogr.
44
,
716
720
.
McConnaughey
,
T.
(
1995
).
Ion transport and the generation of biomineral supersaturation
.
Bull. Inst. Oceanogr
.
14
,
1
18
.
McConnaughey
,
T. A.
and
Whelan
,
J. F.
(
1997
).
Calcification generates protons for nutrient and bicarbonate uptake
.
Earth-Sci. Rev.
42
,
95
117
.
Mueller
,
E.
(
1984
).
Effects of a calcium channel blocker and an inhibitor of phosphodiesterase on calcification in Acropora formosa
.
Adv. Reef Sci. Book of abstracts, Miami, Florida. 87–88 (abstracts)
.
Palmqvist
,
K.
,
Sjöberg
,
S.
and
Samuelsson
,
G.
(
1988
).
Induction of inorganic carbon accumulation in the unicellular green algae Scenedesmus obliquus and Chlamydomonas reinhardtii
.
Plant Physiol.
87
,
437
442
.
Pearse
,
V. B.
(
1970
).
Incorporation of metabolic CO2into coral skeleton
.
Nature
228
,
383
.
Shick
,
J. M.
(
1990
).
Diffusion limitation and hyperoxic enhancement of oxygen consumption in zooxanthellate sea anemones, zoanthids and corals
.
Biol. Bull.
179
,
148
158
.
Smith
,
L.
(
1978
).
Coral reef area and the contributions of reefs to processes and resources of the world’s oceans
.
Nature
273
,
225
226
.
Smith
,
R. G.
(
1988
).
Inorganic carbon transport in biological systems
.
Comp. Biochem. Physiol.
90B
,
639
654
.
Tambutté
,
É.
,
Allemand
,
D.
,
Mueller
,
E.
and
Jaubert
,
J.
(
1996
).
A compartmental approach to the mechanism of calcification in hermatypic corals
.
J. Exp. Biol.
199
,
1029
1041
.
Yellowlees
,
D.
,
Dionisio-Sese
,
M. L.
,
Masuda
,
K.
,
Maruyama
,
T.
,
Abe
,
T.
,
Baillie
,
B.
,
Tsuzuki
,
M.
and
Miyachi
,
S.
(
1993
).
Role of carbonic anhydrase in the supply of inorganic carbon to the giant clam–zooxanthellate symbiosis
.
Mar. Biol
.
115
,
605
611
.
Zoccola
,
D.
,
Tambutté
,
É.
,
Sénégas-Balas
,
F.
,
Michiels
,
J.-F.
,
Failla
,
J.-P.
,
Jaubert
,
J.
and Allemand., D
. (
1999
).
Cloning of a calcium channel α1 subunit from the reef-building coral, Stylophora pistillata
.
Gene
227
,
157
167
.