logical and biochemical mechanisms involved in calcium transport and compartmentalization during coral calcifica- tion. Cloned S. pistillata microcolonies were ...
Marine Biology (1995) 122: 453-459
© Springer-Verlag 1995
E. Tambutté • D. Allemand • I. Bourge • J.-P Gattuso J. Jaubert
An improved 45Ca protocol for investigating physiological mechanisms in coral calcification
Received: 22 November 1994 / Accepted: 10 January 1995
Abstract A sensitive experimental protocol using cloned corals (hereafter "microcolonies") of the branching scleractinian coral Stylophora pistillata and 45Ca has been developed to enable reproducible measurements of physiological and biochemical mechanisms involved in calcium transport and compartmentalization during coral calcification. Cloned S. pistillata microcolonies were propagated in the laboratory from small fragments of parent colonies collected in 1990 in the Gulf of Aqaba, Jordan. Cloned microcolonies have several intrinsic properties that help to reduce unwanted biological variability: (1) same genotype; (2) similar sizes and shapes; and (3) absence of macroscopic boring organisms. Errors specifically associated with long-standing problems to do with isotopic exchange were further reduced by producing microcolonies with no skeletal surfaces exposed to the radioisotope-labelled incubation medium. The value of the technique resides principally in its superior ability to elucidate transportation pathways and processes and not in its ability to quantitatively estimate calcium deposition by corals in nature. We describe here a rapidly exchangeable calcium pool in which up to 90% of the radioactive label taken up during
Communicated by M. Sarà E. Tambutté • D. Allemand ( ) • J.-P. Gattuso • J. Jaubert Observatoire Océanologique Européen, Centre Scientifique de Monaco, Avenue Saint Martin, MC-98000 Monaco, Principality of Monaco E. Tambutté Laboratoire d'Écologie Expérimentale, Université de Nice-Sophia Antipolis, F-06108 Nice Cedex 2, France E. Tambutté Commissariat à l'Énergie Atomique, Direction des Applications Militaires, Laboratoire de Géophysique, B.P. 12, D-91680 Bruyères le Châtel, France I. Bourge Laboratoire d'Océanologie, Institut de Chimie, B6, Université de Liège, B-4000 Sart Tilman par Liège 1, Belgium
incubations is located. This pool (72.9 ± 1.4 nmol Ca mg-1 protein) is presumably located within the coelenteric cavity as suggested by the following: (1) it has 4-min halftime saturation kinetics; (2) the accumulation of calcium is linearly correlated with the calcium concentration of seawater; and (3) its insensitivity to metabolic and ion transport inhibitors indicate that membranes do not isolate this compartment. Washout of this large extracellular pool greatly improved estimates of calcium deposition as evidenced by 10 to 40% reduction in coefficients of variation when compared with previous 45Ca2+ methods described in the literature. Comparisons of calcification measurements simultaneously carried out using the alkalinity anomaly technique and the 45Ca protocol described here show that the correlation coefficient of both techniques is close to 1. Unlike previous reports, our 45Ca2+-derived measurements are slightly lower than those computed from the alkalinity depletion technique.
Introduction
Calcium fluxes on the order of 2.5 µmol h-1 per 3 to 4 cm branch segments (Barnes and Chalker 1990) demonstrate that scleractinian corals are among the most rapidly calcifying organisms. Although many studies of coral calcification have been published in the past 20 yr (see reviews by Buddemeier and Kinzie 1976 and Barnes and Chalker 1990), calcium transport by coral tissues is still poorly understood and calcium pools of coral tissue have never been studied. This lack of data is largely the result of experimental difficulties associated with the combined use of radio-isotopes and freshly cut coral samples (Buddemeier and Kinzie 1976). Coefficients of variation of calcification rates measured with 45Ca typically ranged from 20 to 50% (reviewed by Barnes and Crossland 1982) because of : ( 1 ) population variability (Barnes and Crossland 1982), (2) stressrelated metabolic anomalies (Hidaka and Yamazato 1982) and (3) isotopic exchange between the radioactive calcium
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of the incubation medium and the non-radioactive calcium of the coral skeleton (Clausen and Roth 1975; Barnes and Crossland 1977). Similar problems that led to exaggerated estimates of calcification in algae led Borowitzka and Larkum (1976) to suggest that 45Ca was not an appropriate technique for investigation of CaCO3 deposition. Variation in protocols used to process the labelled samples also increased the magnitude of experimental errors (Crossland and Barnes 1977; Bôhm 1978). The duration of rinsing steps for eliminating extracellular and non-precipitated radioisotopic fractions was generally short and typically variable (Clausen 1971; Clausen and Roth 1975; Krishnaveni et al. 1989; Ip and Krishnaveni 1991), or not specified at all (Chalker and Taylor 1975; Chalker 1976; Crossland and Barnes 1977; Barnes and Crossland 1977, 1982; Rinkevich and Loya 1984). Media used to rinse samples also varied; for example, Clausen and Roth (1975) and Barnes and Crossland (1982) used natural seawater while Chalker (1976) and Krishnaveni et al. (1989) used calcium-free artificial seawater. Consequently, 45Ca techniques are seldom used at present and other techniques such as alkalinity anomaly and buoyant weight methods (see Buddemeier and Kinzie 1976) are typically preferred. Unfortunately, neither method possesses sufficient sensitivity for measuring short-term variations in calcification rate or investigating the kinetics of calcium transport. Kinetic analysis of calcium uptake by calcareous algae led Borowitzka and Larkum (1976) and Bôhm (1978) independently to describe a fast component corresponding to calcium exchange between intercellular spaces and the external medium. Barnes and Crossland (1977) and Allemand and Grillo (1992) recognized similar processes in corals and suggested that complete wash-out of a large extracellular compartment is necessary for accurate estimation of 45Ca skeletal incorporation. The 45Ca protocol described here makes two significant changes in previously used methods. The first consists of using laboratory-cultured microcorals of Stylophora pistillata (Esper 1797), called "microcolonies", rather than freshly cut coral samples. The coral microcolony is a new biological model, the specific properties of which reduce the biological variability of experimental results; it has fixed size, shape and genotype and an absence of macro-boring organisms. Furthermore, its skeleton is entirely covered by animal tissues, thus avoiding isotopic exchange (Al-Moghrabi et al. 1993). The second is an elaborated experimental protocol adapted from the procedure recently developed by Allemand and Grillo (1992) to investigate the calcification of the mediterranean red coral that includes a thorough rinsing of the labelled samples. This protocol led us to describe on the basis of kinetics and pharmacological evidence an extracellular fast Ca2+-exchanging compartment. This improved 45Ca technique has been compared with that of the alkalinity anomaly which was recently validated by Chisholm and Gattuso (1991).
Table 1 Stylophora pistillata. Biological parameters of S. pistillata microcolonies (n = 8)
Materials and methods Biological material Microcolonies were propagated in the laboratory from small fragments of Stylophora pistillata collected in September 1990 at a depth of 5 m in front of the Marine Science Station, Gulf of Aqaba (Jordan). Colonies were packed in humidified plastic bags and transported to the Musée océanographique de Monaco (14 h transport time). Corals were stored in a 300-liter aquarium supplied with seawater from the Mediterranean (exchange rate: 2% H-1) heated to 26 ± 0.1 °C and illuminated with constant irradiance of 175 µmol m-2.s-1] using metal halide lamps (Philips HQI-TS, 400 W) on a 12:12 photo-period. Microcolony propagation Terminal portions of branches (6 to 10 mm long) were cut with pliers from parent colonies, placed on a nylon net (1x1 mm mesh) tensioned across a PVC frame and maintained under the previously stated conditions of light and temperature. They were rotated three times a week to prevent adhesion and allow tissue to grow over the exposed skeleton. After about 1 mo coral fragments became entirely covered with new tissues. Microcolonies characteristics are summarized in Table 1. Measurement of 45Ca uptake and deposition Measurements were made at equivalent times of day in order to avoid possible variation caused by endogenous circadian rhythms (Buddemeier and Kinzie 1976). Microcolonies were placed in plastic holders and incubated for 5 to 300 min in 6-ml beakers containing 240 kBq of 45Ca (as CaCl2, 1.38 MBq ml-1, New England Nuclear) dissolved in seawater filtered using 0.45 µm Millipore membranes (filtered seawater, FSW). Water motion was provided during each incubation by small stirring bars in order to reduce as much as possible diffusion limitation by boundary layers. Exposure to air was limited to less than 5 s during transfer to the incubation beakers. Incubations of varying duration were carried out on at least three samples under light and temperature conditions similar to those described during culture. 100-µl aliquots of seawater were removed during each incubation for determination of specific radioactivity. Preliminary experiments using an O2 electrode (Clark-type electrode, Rank Brothers Ltd., UK) showed that rates of photosynthesis remained constant during incubations. At the end of the labelling period each holder and its microcolony were immersed for 20 s in a beaker containing 600 ml FSW, then rinsed five times with 5 ml of ice-cold glycine-high calcium medium (50 mM CaCl2, 950 mM glycine, pH adjusted to 8.2) to prevent further calcium uptake and reduce, by isotopic dilution, the 45Ca adsorbed on to the external surface of both the microcolony and the holder. The total duration of the rinsing procedure was less than 1 min. Labelled microcolonies were then incubated in a beaker containing 20 ml FSW for 30 min to monitor 45Ca efflux into the rinse medium. Water motion was provided in the efflux medium by small stirring bars. Upon completion of the efflux, microcolony tissues were dissolved completely over a period of 20 min in 1 ml of 2 N
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NaOH at 90 °C. The supernatant (hereafter "NaOH-soluble fraction") was collected and the skeleton was rinsed first in 1 ml of distilled water and, subsequently, five times in 5 ml FSW. The first rinse solution was added to the NaOH fraction; the remaining five were discarded since they did not contain proteins. Finally, the skeletons were dried and dissolved in 1.5 ml 12 N HC1 overnight ("HCl-soluble fraction"). Radioactive samples were added to 4 ml Luma-gel (Packard) and -emissions were measured using a liquid scintillation counter (Tricarb, 1600 CA Packard).
Unless otherwise specified, all chemicals were obtained from Sigma and were of analytical grade. DIDS (4,4'-diisothiocyanatostilbene-2,2/-disulfonic acid), verapamil, methoxyverapamil (D600), diltiazem, ethoxyzolamide were dissolved as stock solution in dimethyl sulfoxide (DMSO). Flunarizine was dissolved in methanol. Other inhibitors (NaCN, lanthanum, cobalt, cadmium, nickel) were dissolved in distilled water. The final DMSO or methanol concentration never exceeded 1% (v:v). Preliminary experiments showed that this concentration had no effect on calcium uptake (results not shown). A 15-min pre-incubation step was completed whenever inhibitors were used. The inhibitor was present at the same concentration during the pre-incubation, incubation and efflux periods.
Measurement of microcolony extracellular space 14
C-dextran(MW70000;8 kBqml-1,15.7 GBq g-1, Amersham) was used as an extracellular marker. The experimental procedure was identical to that described above for measurement of radioactive calcium uptake. Pulse-chase experiment Pulse-chase experiments to characterize calcium pool kinetics were performed following 1-h long incubations in labelled seawater. For this purpose, the amount of radioactivity remaining in the NaOHand HCl-soluble fractions was measured over efflux periods ranging from 10 min to 2 h into 20 ml of unlabelled FSW.
Statistical analysis and curve fitting Exponential [y = a + b exp (-c x)] and linear [y = a + b x] functions were fitted to the experimental data using the Igor data analysis package (Wave Metrics, Inc.). Results are presented as mean ± SD of at least three measurements. The relationship between TA and 45Ca incorporation estimates of calcium deposition was investigated using functional linear regression analysis (Jacques and Pilson 1980). Statistical parameters are not significantly different from 0 unless otherwise stated: *** p < 0.001. The coefficient of variation (%) corresponds to SD/mean x 100.
Results Measurement of calcification using the alkalinity anomaly technique
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A further series of experiments involved dual measurement of alkalinity depletion (Smith and Kinsey 1978) and 45Ca incorporation to provide comparative estimates of calcium deposition. Microcolonies of Stylophora pistillata were incubated at 25.5 ± 0.1 °C in 7 ml 45Ca (40 kBq mol~') labelled FSW. Incubations were performed at least in duplicate under illumination (175|µmol m-2 s-1) for 0.25, 0.5, 1, 1.5, 2 and 3 h in glass beakers covered by a transparent plastic film to reduce evaporation. A total of 23 incubations were carried out. Upon completion of experiments the microcolonies were processed to determine 45Ca incorporation as described before. For determination of total alkalinity (TA), water samples were taken at the beginning and end of each incubation, the latter being filtered prior to measurement (0.45-µm membranes). Samples were stored in darkness at 4 °C and processed within 24 h of collection. TA was measured by potentiometric titration of 5-ml aliquots (Mettler titrator DL 70) using 0.01 N HC1 (Merck Titrisol, 9974), fortified with NaCl (Merck, 6404) to yield an ionic strength equivalent to that of seawater (salinity = 38). The pH electrode (Orion, 81 02) was calibrated daily against National Bureau of Standards (NBS) buffers (4.008 and 7.000 at 25 °C). Titrant additions were not made until pH had stabilised to ±0.003 unit for more than 5 s. Samples, HC1 and the titration vessel were maintained at 25 °C. TA was computed using the Gran equation corrected for sulphate and fluorides (Hansson and Jagner 1973) with pH values ranging from 3.0 to 4.2. Calcification was computed using the stoichiometric relationship between the deposition of calcium carbonate and the decrease in TA: TA decreases by two equivalents for each mole of CaCO3 precipitated (see Smith and Kinsey 1978).
Efflux of 45Ca from microcolonies loaded during 1 h is shown in Fig. 1. 45Ca released from the microcolony display saturation kinetics and reached a plateau within 10 min. Increasing the volume of the rinse medium did not alter the plateau. Semi-logarithmic treatment of the results (inset of Fig. 1) allowed us to determine a Tl/2 (half-life) for calcium washout of 4 min corresponding to a rate constant of 0.17 min-1. In order to characterize this pool, its size (i.e., the total amount of 45Ca released in the efflux medium during the
Ca Efflux from coral microcolonies
Media and chemicals For experiments with varying calcium concentrations, artificial seawater (ASW) was prepared from distilled water as described by Allemand et al. (1984). CaCl2 was replaced by NaCl in order to provide equivalent osmotic potential. Proteins were measured in an autoanalyzer (Alliance Instruments) using the method of Lowry et al. (1951) and standards of bovine serum albumin.
Fig. 1 Stylophora pistillata. Kinetics of 45Ca efflux from a microcolony of S. pistillata. The microcolony was loaded in 45Ca-labelled seawater for 1 h. Radioactive efflux was performed after a brief rinse (1 min) in 20 ml fresh seawater. Each data point represents mean ± SD of eight to ten determinations. Inset: semi-logarithmic plot for determination of T1/2 (4 min). (Q amount of tracer in the efflux medium at time t). Equilibrium value (Qeq)was taken as 16 535 dpm mg-1 protein
456 Table 2 Stylophora pistillata. Effect of various inhibitors on the size of the calcium efflux pool (expressed as nmol Ca mg-1 protein). Microcolonies were incubated for 1 h in 45Ca-filtered seawater. Results presented as mean ± SD with the number of determination in
parentheses
Fig. 2 Kinetics characterization of the "efflux compartment". Each data point represents mean ± SD of eight to ten determinations. A Size of this pool (i.e., total amount of calcium contained in this pool as determined by the value of the plateau shown in Fig. 1) as a function of the external calcium concentration during the incubation period. B Time course of the pool loading in filtered seawater
30-min rinse period) was plotted against the concentration of calcium ranging from 0 to 20 mM during the incubation step. Fig. 2 A shows that these two parameters are linearly correlated, suggesting a passive equilibration of this compartment. Fig. 2B shows that the calcium content of this compartment plotted against incubation time follows a saturable function; upon equilibration, the calcium content of this compartment did not vary significantly as a function of the incubation time during at least 5 h. Determination of the microcolony seawater space After 1 h of incubation with 14C-dextran, microcolonies were rinsed and the radioactive efflux monitored as for 45 Ca experiments. The amount of radioactivity released, taking into account the external radioactivity concentration during the labelling period, corresponds to an extracellular seawater space of 10.1 ±0.5 µl mg-1 protein. Pharmacological characteristics of the calcium efflux pool To further characterize this calcium compartment, the effects of various inhibitors on its loading were tested. A
metabolic inhibitor (1 mM NaCN), calcium channel inhibitors (100 µM verapamil, D600 or diltiazem; 10 µM flunarizine; heavy metals such as 200 µM lanthanum, 500 µM cobalt, 1 mM cadmium or nickel), an anion transport inhibitor (300 µM BIDS), and a carbonic anhydrase inhibitor (300 µM ethoxyzolamide) did not significantly change the amount of 45Ca released from this compartment into the efflux medium (Table 2). Effect of rinsing the efflux calcium pool on the measurement of calcium uptake by tissues and calcium deposition on skeleton The effect of rinsing this compartment on the measurement of calcium uptake and incorporation in skeleton was determined. Microcolonies were incubated in 45Ca-FSW for 1 h. After the 30-min rinse period the distribution of calcium in the microcolony was 53% in the extracellular compartment (72.9±1.4 nmol mg-1 protein), 5% in the NaOHsoluble fraction (7.1±0.7 nmol mg-1 protein) and 42% in the HCl-soluble fraction (58.5±6.7 nmol mg-1 protein). Fig. 3 shows the comparison of the data obtained after a complete rinse of the extracellular compartment (present method) or after the brief 1-min rinse as generally stated in literature. Our results show that inadequate rinsing causes overestimation of NaOH-soluble and HCl-soluble fractions by factors of 8 and 1.5, respectively, in comparison to our protocol. Fig. 4 illustrates exponential decrease of radioactivity in the NaOH-soluble fraction in the pulse-chase experiment. The semi-logarithmic treatment of the data (inset of Fig. 4) demonstrates the presence of two calcium pools, the first one with a high turnover rate (Tl/2 of 4 min) identical to that determined in the efflux experiment (see Fig. 1) and the second one with a Tl/2 of about 20 min. The calcium content of the HCl-soluble pool did not vary significantly after 30 min of chase period (result not shown).
Fig. 3 Comparison of methods currently used in the literature to the method set up in the present work. Amount of calcium taken up during a 1-h incubation by the two (three) compartments studied was plotted without (standard procedure) or with (present method) the efflux procedure (mean ± SD of 6 to 21 determinations). Inset: distribution of radioactive calcium after microcolony processing by these two procedures. (Hatched bars HCl-soluble pool; open bars NaOH-soluble pool; filled bars "efflux" pool)
Fig. 4 Time course of the radioactive calcium content of the NaOHsoluble pool as a function of the duration of the efflux period. Inset: semi-logarithmic plot shows presence of two calcium pools (Tl/2 of 4 min and 22 min). (Q and Qeq same as in Fig. 1)
Comparison with the alkalinity anomaly technique
intercellular space and the external medium is also in the range of several minutes (ca. 6 min, Borowitzka and Larkum 1976). Therefore, we suggest that the 45Ca efflux from the coral microcolony shown in Fig. 1 corresponds to the washout of equivalent extracellular space, probably the coelenteric cavity. Barnes and Grassland (1977) and Allemand and Grillo (1992) reached similar conclusions on work with zooxanthellate scleractinian corals and the octocoral Corallium rubrum, respectively. Several facts confirm this hypothesis: its saturationtime (Fig. 2B), its linear dependence on extracellular calcium concentration (Fig. 2 A) and its total insensitivity to carrier inhibitors rules out a carrier-mediated step. Efflux of 45Ca reported in gorgonians (Velimerov and King 1979; Kingsley and Watabe 1984) has been interpreted as a passive leakage or active efflux from cells by calcium pumps. The total amount of 45Ca released in the medium enables the estimation of the size of this compartment which contains 72.9±1.4 nmol Ca mg-1 protein when the microcolony is equilibrated with seawater (Fig. 1). Assuming that the 45Ca concentration of the internal medium at equilibrium is equal to that of 45Ca-labelled seawater, we can estimate the volume of the gastrovascular cavity of the microcolony as 7.3±1.2 µl mg-1 protein. This value may be compared to that obtained with 14C-dextran, an impermeant molecule with a high molecular weight (70000) classically used to label extracellular space, i.e., 10.1±0.5 µl mg-1 protein. These two values are in the same range, the higher value obtained with dextran may be due to binding phenomena of dextran with glycosaminoglycans on the cell surface (Ehrenfeld and Cousin 1982). This agreement rules out the hypothesis of calcium binding processes and supports arguments for distribution of the calcium pool in an extracellular volume.
The relationship between estimates of calcification measured by TA determination and incorporation of 45Ca is shown in Fig. 5. The correlation coefficient is 0.99*** (n = 23) and the geometric relationship is: y=u + v x, with u = -243.6±36.1 and v = 0.88 ± 0.06. The regression line cannot be forced through the origin because the y-intercept (u) is significantly different from 0 (p — 0.003). When standardized by protein, the x-intercept value corresponds to 31.8 nmol Ca mg-1 protein.
Discussion Methodological aspects Non-specific exchange between labelled and unlabelled calcium has been overcome for the first time using scleractinian coral microcolonies which have the advantage of a skeleton entirely covered by animal tissue, thus avoiding any contact between the extracellular tracer and the skeleton. Böhm (1978) has stressed that the existence of different calcium pools complicates the application of radioactive uptake data. We have described here a rapidly exchangeable calcium compartment suggesting either the washout of an extracellular compartment (Borle 1969; Claret-Berthon et al. 1977) or binding to tissue surface. The latter hypothesis can be ruled out since the microcolonies were rinsed rapidly (1 min) in a high calcium medium which removed non-specific Ca2+-binding (Milne and Coukell 1991). In the green algae Halimeda cylindracea and H. tuna, Tl/2 of the calcium exchange between the
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Therefore, the first step of the washing procedure must eliminate this medium, which contains the largest proportion of exchangeable Ca, in order to avoid overestimation and high variability of the tissue and skeleton calcium pools. Indeed, the distribution of radioactive calcium in non-rinsed microcolonies appears to be largely modified in comparison to rinsed ones: the extracellular calcium pool, which represents 53% of the radioactivity taken up during 1 h of incubation, is released during the dissolution of tissues, and this induces a large overestimation of the NaOH-soluble fraction and an increase of isotopic exchange with the skeleton (Fig. 3). Consequently, the coefficient of variation using our rinsing procedure is 11.5% (as calculated from data of Fig. 3), while the most recent reported in the literature are in the range 20 to 30% (Barnes and Crossland 1982). As the turnover rate of this compartment is 4 min, 30 min was chosen as a suitable time for rinsing. Fig. 4 shows that such a period does not induce important changes in the NaOH-soluble pool. However, a limitation of such a long washing procedure is that it makes our method inadequate for accurate estimation of rapidly (less than 10 min) exchangeable tissue compartments. Comparison with the alkalinity anomaly technique The alkalinity depletion technique and the present 45Ca fixation method are strongly correlated with a correlation coefficient of nearly 1. The slope of the line, however, is less than 1 (0.88) and there is an offset between the two techniques used: the alkalinity-derived estimates are positive when the radioisotope estimates are 0 (x-intercept of 276.8 nmol). The radioisotope estimates are, therefore, lower than the alkalinity-derived estimates. Since the alkalinity anomaly technique has recently been validated (Chisholm and Gattuso 1991), these results suggest that our 45Ca technique slightly underestimates the calcification rate. There have been a few other attempts to evaluate the 45 Ca technique to measure calcification rates of corals.
Fig. 5 Relationship between total alkalinity estimates and 45Ca incorporation estimates. Lines shown are the geometric regression line for the present study (continuous line; y=-243.6 + 0.88 x) and the functional regression line obtained by Smith and Roth (dotted line; in Smith and Kinsey 1978)
Smith and Roth (in Smith and Kinsey 1978) compared the alkalinity anomaly and the 45Ca techniques for measuring the calcification rates of corals (Fig. 5). It seems that their data were analysed using predictive regression, which should not be used when both variables are subject to measurement error (Jacques and Pilson 1980). The slope of the geometric regression line can be calculated using the functional regression parameters given by Smith and Roth (in Smith and Kinsey 1978). It is 0.86 (slope of the functional regression/correlation coefficient = 0.81/0.94), a value close to the one reported in the present study. Smith and Roth (in Smith and Kinsey 1978) found that the relationship between alkalinity-depletion estimates and 45 Ca estimates displays a positive y-intercept, which they interpreted as the uptake of radioisotope through inorganic exchange or physical adsorption at the broken base of coral tips. Such passive, non-biologically mediated processes are clearly suppressed using our protocol. We found instead that the alkalinity depletion technique detects a low CaCO3 deposition when there is no apparent fixation of 45 Ca (positive Jt-intercept). This could result from: (1) a loss of radioactivity during the 30-min wash period due to isotopic dilution; (2) a loss of radioactivity during the cleaning process in sodium hydroxide; and/or (3) a time lag due to the loading of intermediate pools (extracellular and tissue compartments). The x-intercept value of 13.8 nmol Ca mg-1 protein is lower than the calcium coelenteric pool (72.9 nmol mg-1 protein) but remains, however, in the same range. Calcium transport and coelenteron
between
external
seawater
One intriguing point is the mechanism of calcium efflux from the coelenteron. Gladfelter (1983) has shown that the gastrovascular cavity is lined by flagellated endodermal cells and assumed that the mechanism of fluid movement is by flagellar action. However, the washout of the coelenteron appears to be metabolic energy-independent, since it is not inhibited by cyanide. This result rules out the hypothesis of flagellar movement which is ATP-dependent (Gibbons 1981). Since the equilibration of the coelenteron with external seawater results from a passive diffusion (linear correlation between gastrovascular and seawater calcium, energy-independent process), it is suggested that the oral epithelium is leaky with respect to calcium ions. This would ensure a constant and large supply of calcium ions for calcification. Further experiments are in progress to resolve this point. By using a new biological model for studying scleractinian coral physiology, the microcolony of Stylophorapistillata, we have characterized a large and rapidly exchangeable calcium compartment in order to set up a washing procedure. It is demonstrated that the understanding of the compartmentation of the isotopes is necessary before developing a method for measuring radioisotope uptake and incorporation by scleractinian corals. The method described appears to be valid in comparison to that of the al-
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kalinity anomaly technique and may enable us to gain better insight into calcium transport mechanisms and the calcification process in corals (Tambutté et al. in preparation). Acknowledgements This study was conducted as part of the O. O. E. 1991 -1995 research program. It was supported by the Council of Europe (Open Partial Agreement on Major Natural and Technological Disasters), the Programme National Récifs Coralliens (PRCO) and a grant from Tournesol, a cooperative program between France and the French-speaking Belgian community. We thank Dr. J. Chisholm as well as the three anonymous referees for helpful comments on the manuscript and C. Emery for his technical assistance. Part of this work has been presented at the 7th International Symposium on Biomineralization (Tambutté et al. 1995).
References Allemand D, De Renzis G, Ciapa B, Girard JP, Payan P (1984) Characterization of valine transport in sea urchin eggs. Biochim Biophys Acta 772:337-346 Allemand D, Grille MC (1992) Biocalcification mechanism in gorgonians: 45Ca uptake and deposition by the Mediterranean red coral Corallium rubrum. J exp Zool 262:237-246 Al-Moghrabi S, Allemand D, Jaubert J (1993) Valine uptake by the scleractinian coral Galaxeafascicularis: characterization and effect of light and nutritional status. J Comp Physiol 163:355-362 Barnes DJ, Chalker BE (1990) Calcification and photosynthesis in reef-building corals and algae. In: Dubinsky Z (ed) Coral reefs, ecosystems of the world. Elsevier, Amsterdam, pp 109-131 Barnes DJ, Crossland CJ (1977) Coral calcification: sources of error in radioisotope techniques. Mar Biol 42:119-129 Barnes DJ, Crossland CJ (1982) Variability in the calcification rate of Acropora acuminata measured with radioisotopes. Coral Reefs 1:53-57 Böhm L (1978) Application of the 45Ca tracer method for determination of calcification rates in calcareous algae: effect of calcium exchange and differential saturation of algal calcium pools. Mar Biol 47:9-14 Borle AB (1969) Kinetic analysis of calcium movements in HeLa cell cultures. I. Calcium influx. J gen Physiol 53:43-56 Borowitzka MA, Larkum AWD (1976) Calcification in the green alga Halimeda. II. The exchange of Ca2+ and the occurrence of age gradients in calcification and photosynthesis. J exp Bot 27: 864-878 Buddemeier RW, Kinzie RA (1976) Coral growth. Oceanogr mar Biol A Rev 14:183-225 Chalker BE (1976) Calcium transport during skeletogenesis in hermatypic corals. Comp Biochem Physiol 54A:455-459 Chalker BE, Taylor DL (1975) Light-enhanced calcification, and the role of oxidative phosphorylation in calcification of the coral Acropora cervicornis. Proc R Soc Lond (Ser B) 190:323331 Chisholm JRM, Gattuso J-P (1991) Validation of the alkalinity anomaly technique for investigating calcification and photosynthesis in coral reef communities. Limnol Oceanogr 36:1232-1239
Claret-Berthon B, Claret M, Mazet JL (1977) Fluxes and distribution of calcium in rat liver cells: kinetic analysis and identification of pools. J Physiol 272:529-552 Clausen CD (1971) Effects of temperature on the rate of 45Calcium uptake by Pocillopora damicornis. In: Lenhoff HM, Muscatine L, Davis LD (eds) Experimental coelenterate biology. Univ of Hawaii Press, Honolulu, pp 246-259 Clausen CD, Roth AA ( 1975) Estimation of coral growth-rates from laboratory 45Ca-incorporation rates. Mar Biol 33:85-91 Crossland CJ, Barnes DJ (1977) Calcification in the staghorn coral Acropora acuminata: variations in apparent skeletal incorporation of radioisotopes due to different methods of processing. Mar Biol 43:57-62 Ehrenfeld J, Cousin J-L (1982) Ionic regulation of the unicellular green alga Dunaliella tertiolecta. J Membrane Biol 70:47-57 Gibbons IR (1981) Cilia and flagella of eukaryotes. J Cell Biol 91(3):107-124 Gladfelter EH (1983) Circulation of fluids in the gastrovascular system of the reef coral Acropora cervicornis. Biol Bull mar biol Lab, Woods Hole 165:619-636 Hansson I, Jagner D ( 1973) Evaluation of the accuracy of Gran plots by means of computer calculations. Application to the potentiometric titration of the total alkalinity and carbonate content of seawater. Analytica chim Acta 65:363-373 Hidaka M, Yamazoto K (1982) Effect of light on budding of isolated polyps of the scleractinian coral Galaxeafascicularis. Galaxea 1:65-75 Ip YK, Krishnaveni P ( 1991 ) Incorporation of strontium (90Sr++) into the skeleton of hermatypic coral Galaxeafascicularis. J exp Zool 258:273-276 Jacques TG, Pilson MEQ (1980) Experimental ecology of the temperature scleractinian coral Astrangia danae. I. Partition of respiration, photosynthesis and calcification between host and symbionts. Mar Biol 60:167-178 Kingsley RJ, Watabe N (1984) Calcium uptake in the gorgonian Leptogorgia virgulata. The effects of ATPase inhibitors. Comp Biochem Physiol 79A:487-491 Krishnaveni P, Chou LM, Ip YK (1989) Deposition of calcium (45Ca2+) in the coral, Galaxeafascicularis. Comp Biochem Physiol 94A:509-513 Lowry OH, Rosebrough NJ, Farr AL, Randall RJ ( 1951 ) Protein measuement with the folin phenol reagent. J biol Chem 193:265-275 Milne JL, Coukell MB (1991) A Ca2+ transport system associated with the plasma membrane of Dictyostelium discoideum is activated by different chemoattractant receptors. J Cell Biol 112:103-110 Rinkevich B, Loya Y (1984) Does light enhance calcification in hermatypic corals? Mar Biol 80:1-6 Smith SV, Kinsey DW (1978) Calcification and organic carbon metabolism as indicated by carbon dioxide. In: Stoddart DR, Johannes RE (eds) Coral reefs: research methods. Unesco, Paris, pp 469-484 Tambutté E, Allemand D, Jaubert J (1995) The Stylophora pistillata coral microcolony: a model for studying transport process during coral biomineralization. Proc 7th int Biomin Symp Bull Inst Oceanogr n° spécial 14 (in press) Velimerov B, King J (1979) Calcium uptake and net calcification rates in the octocoral Eunicella papillosa. Mar Biol 50:349-358
