Carbonic Anhydrase in the Scleractinian Coral Stylophora pistillata

Abstract

Carbonic anhydrases (CA) play an important role in biomineralization from invertebrates to vertebrates. Previous experiments have investigated the role of CA in coral calcification, mainly by pharmacological approaches. This study reports the molecular cloning, sequencing, and immunolocalization of a CA isolated from the scleractinian coral Stylophora pistillata, named STPCA. Results show that STPCA is a secreted form of α-CA, which possesses a CA catalytic function, similar to the secreted human CAVI. We localized this enzyme at the calicoblastic ectoderm level, which is responsible for the precipitation of the skeleton. This localization supports the role of STPCA in the calcification process. In symbiotic scleractinian corals, calcification is stimulated by light, a phenomenon called “light-enhanced calcification” (LEC). The mechanism by which symbiont photosynthesis stimulates calcification is still enigmatic. We tested the hypothesis that coral genes are differentially expressed under light and dark conditions. By real-time PCR, we investigated the differential expression of STPCA to determine its role in the LEC phenomenon. Results show that the STPCA gene is expressed 2-fold more during the dark than the light. We suggest that in the dark, up-regulation of the STPCA gene represents a mechanism to cope with night acidosis. Carbonic anhydrases (CA) play an important role in biomineralization from invertebrates to vertebrates. Previous experiments have investigated the role of CA in coral calcification, mainly by pharmacological approaches. This study reports the molecular cloning, sequencing, and immunolocalization of a CA isolated from the scleractinian coral Stylophora pistillata, named STPCA. Results show that STPCA is a secreted form of α-CA, which possesses a CA catalytic function, similar to the secreted human CAVI. We localized this enzyme at the calicoblastic ectoderm level, which is responsible for the precipitation of the skeleton. This localization supports the role of STPCA in the calcification process. In symbiotic scleractinian corals, calcification is stimulated by light, a phenomenon called “light-enhanced calcification” (LEC). The mechanism by which symbiont photosynthesis stimulates calcification is still enigmatic. We tested the hypothesis that coral genes are differentially expressed under light and dark conditions. By real-time PCR, we investigated the differential expression of STPCA to determine its role in the LEC phenomenon. Results show that the STPCA gene is expressed 2-fold more during the dark than the light. We suggest that in the dark, up-regulation of the STPCA gene represents a mechanism to cope with night acidosis. Carbonic anhydrases (CA) 4The abbreviations used are: CAcarbonic anhydraseSTPCAcarbonic anhydrase from S. pistillataanti-STPCAspecific antibody against the STPCADICdissolved inorganic carbonRACErapid amplification of cDNA endsMLmaximum likelihoodDMEMDulbecco's modified Eagle's mediumGPIglycosylphosphatidylinositolNTAnitrilotriacetic acidMCMCMCMetropolis-coupled Markov Chain Monte CarloLEClight-enhanced calcificationAAamino acids. are ubiquitous metalloenzymes that catalyze the reversible hydration of carbon dioxide into bicarbonate: CO2 + H2O ⇆ HCO3- + H+. There are at least five classes of CA with polyphyletic origin (1Hewett-Emmett D. Tashian R.E. Mol. Phylogenet. Evol. 1996; 5: 50-77Crossref PubMed Scopus (520) Google Scholar, 2Tripp B.C. Smith K. Ferry J.G. J. Biol. Chem. 2001; 276: 48615-48618Abstract Full Text Full Text PDF PubMed Scopus (450) Google Scholar, 3So A.K. Espie G.S. Williams E.B. Shively J.M. Heinhorst S. Cannon G.C. J. Bacteriol. 2004; 186: 623-630Crossref PubMed Scopus (221) Google Scholar, 4Lane T.W. Saito M.A. George G.N. Pickering I.J. Prince R.C. Morel F.M. Nature. 2005; 435: 42Crossref PubMed Scopus (467) Google Scholar, 5Supuran C.T. Scozzafava A. Bioorg. Med. Chem. 2007; 15: 4336-4350Crossref PubMed Scopus (504) Google Scholar, 6Xu Y. Feng L. Jeffrey P.D. Shi Y. Morel F.M. Nature. 2008; 452: 56-61Crossref PubMed Scopus (440) Google Scholar): α (vertebrates, invertebrates, bacteria, and some chlorophytes), β (eubacteria and chlorophytes), γ (archea and some eubacteria), and δ and ζ (marine diatoms). Among numerous roles in physiological processes, carbonic anhydrases play an important role in biomineralization from invertebrates to vertebrates, even if this role is not well understood (7Goreau T.F. Biol. Bull. 1959; 116: 59-75Crossref Google Scholar, 8Mitsunaga K. Akasaka K. Shimada H. Fujino Y. Yasumasu I. Numandi H. Cell Differ. 1986; 18: 257-262Crossref PubMed Scopus (40) Google Scholar, 9Kakei M. Nakahara H. Biochim. Biophys. Acta. 1996; 1289: 226-230Crossref PubMed Scopus (24) Google Scholar, 10Miyamoto H. Miyoshi F. Kohno J. Zool. Science. 2005; 22: 311-315Crossref PubMed Scopus (162) Google Scholar, 11Tohse H. Murayama E. Ohira T. Takagi Y. Nagasawa H. Comp. Biochem. Physiol. 2006; 145: 257-264Crossref Scopus (29) Google Scholar). carbonic anhydrase carbonic anhydrase from S. pistillata specific antibody against the STPCA dissolved inorganic carbon rapid amplification of cDNA ends maximum likelihood Dulbecco's modified Eagle's medium glycosylphosphatidylinositol nitrilotriacetic acid Metropolis-coupled Markov Chain Monte Carlo light-enhanced calcification amino acids. Scleractinian corals precipitate an aragonitic calcium carbonate (CaCO3) skeleton resulting from the precipitation of calcium and inorganic carbon at the site of calcification. Several studies have demonstrated the involvement of CA in coral calcification (7Goreau T.F. Biol. Bull. 1959; 116: 59-75Crossref Google Scholar, 12Isa Y. Yamazato K. Galaxea. 1984; 3: 25-36Google Scholar, 13Kingsley R.J. Watabe N. J. Exp. Zool. 1987; 241: 171-180Crossref Scopus (30) Google Scholar, 14Tambutté E. Allemand D. Mueller E. Jaubert J. J. Exp. Biol. 1996; 199: 1029-1041Crossref PubMed Google Scholar, 15Furla P. Galgani I. Durand I. Allemand D. J. Exp. Biol. 2000; 203: 3445-3457Crossref PubMed Google Scholar, 16Al-Horani F.A. Al-Moghrabi S.M. de Beer D. Mar. Biol. 2003; 142: 419-426Crossref Scopus (407) Google Scholar, 17Marshall A.T. Clode P.L. Comp. Biochem. Physiol. 2003; 136: 417-426Crossref Scopus (34) Google Scholar, 18Tambutté S. Tambutté E. Zoccola D. Caminiti N. Lotto S. Moya A. Allemand D. Adkins J. Mar. Biol. 2007; 161: 71-83Crossref Scopus (83) Google Scholar): CA could help in supplying dissolved inorganic carbon (DIC) or in removing carbonic acid from the site of skeletogenesis. One specific feature of calcification in symbiotic corals is that calcium carbonate deposition in skeleton is higher in light than in dark conditions. This phenomenon, called “light-enhanced calcification” (LEC), is due to the symbiosis that corals establish with their photosynthetic Dinoflagellates. Despite numerous studies performed on this symbiotic relationship, the mechanisms linking photosynthesis of the symbionts to coral calcification remain largely unknown (see review in Refs. 19Gattuso J.-P. Allemand D. Frankignoulle M. Amer. Zool. 1999; 39: 160-183Crossref Scopus (586) Google Scholar, 20Allemand D. Ferrier-Pagès C. Furla P. Houlbrèque F. Puverel S. Reynaud S. Tambutté É. Tambutté S. Zoccola D. C.R. Palévol. 2004; 3: 453-467Crossref Scopus (264) Google Scholar). In this study, we have cloned, sequenced, and localized an α-CA from the coral Stylophora pistillata and then determined the differential gene expression between light and dark to generate more information on the function of CA in coral calcification and in the light-enhanced calcification phenomenon. Our results allow us to propose a model to explain the role of CA in the coral calcification process. Biological Materials—Experiments were conducted in the laboratory on the branching symbiotic scleractinian coral S. pistillata (Esper, 1797). Colonies were stored in a 300-liter aquarium, supplied with seawater from the Mediterranean Sea (exchange rate 2% h-1) under controlled conditions: semi-open circuit, temperature of 26.0 ± 0.2 °C, salinity of 38.2 psu, light of 175 μmol photons m-2 s-1 (using the fluorescent tube Custom Sea Life®) on a 12:12 photoperiod. Lighting-on occurred at 9:00 AM. Corals were fed three times weekly with a mix of Artemia salina nauplii, A. salina frozen adults, and frozen krill. RNA Extraction—Extractions of RNA were performed at 12:00 AM for the light condition and at 12:00 PM for the dark condition, because we have previously shown that calcification rates were stable in the light and dark with a 2.6-fold enhancement of calcification rates in light (21Moya A. Tambutté S. Tambutté E. Zoccola D. Caminiti N. Allemand D. J. Exp. Biol. 2006; 209: 3413-3419Crossref PubMed Scopus (100) Google Scholar). Total RNAs were extracted with Trizol® reagent (Invitrogen) according to the Chomczynski method (22Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (63190) Google Scholar), which was slightly modified (23Moya A. Tambutté S. Béranger G. Gaume B. Scimeca J.C. Allemand D. Zoccola D. Marine Biotechnol. 2008; 10.1007/s10126-008-9101-1Google Scholar). Briefly, five grams of coral apexes were ground into powder in a cryogrinder (Freezer/Mill 6770, Spex Sample Prep®) and dissolved in 20 ml of Trizol® reagent. Three centrifugations (10 min, 15,000 × g, 4 °C) were performed. 4 ml of chloroform were added to the last supernatant and incubated 2–3 min at room temperature. A centrifugation (15 min, 12,000 × g, 4 °C) allowed separation of an and a The was to a chloroform ml of were added to the and incubated for min at room temperature. A centrifugation (15 min, 12,000 × g, 4 °C) the of the This was in ml of min, × g, 4 min at room and then in of The RNA was × g, min, 4 °C) to the last of was performed to real-time from was the according to the of the we performed with and results to the of not cDNA and was performed (Invitrogen) according to the of the on of RNA with an for the of carbonic anhydrase and on of for real-time amplification was (Invitrogen) and for the and for the The amplification was into a were and on with and were and for the of the of three was on with and by the and of the rapid amplification of cDNA ends experiments were performed the and for and and for ends were in and and were conducted on the in the The was determined the S. G. H. 2007; PubMed Scopus Google and were performed on the for Biological N. S. S. J. Mol. Biol. 1999; PubMed Scopus Google Scholar). site was performed the B. P. F. J. Mol. Biol. 1999; PubMed Scopus Google and and the of the was performed the K. Biol. Chem. and The of carbonic anhydrase amino acid were performed with in A was with the and The model of was F. D. 2005; PubMed Scopus Google a of of the amino acid and with a with rate a of and amino acid the with a for of the with a S. Biol. 2003; PubMed Scopus Google or a 2001; PubMed Scopus Google were used to under a model the amino acid model and the previously by The was and were performed with used 4 Metropolis-coupled Markov Chain Monte Carlo were from the of the the determined by the of likelihood with were under the of a of and of were performed by The to the of the with the Briefly, the with and to was into to an to a from was to were with the of Three the the the specific antibody against the carbonic anhydrase from S. pistillata, were into and stored at for with of were for immunolocalization previously by Puverel S. E. Zoccola D. I. A. Lotto S. Allemand D. S. 2005; Scopus Google Scholar). Briefly, apexes of S. pistillata were in in at 4 and then in at 4 were then in an and in were and on was performed according to the previously by Puverel S. E. Zoccola D. I. A. Lotto S. Allemand D. S. 2005; Scopus Google Scholar). Briefly, of were incubated for in medium 20 in at room temperature. The were then incubated with the or the at room temperature and at 4 in in were incubated with at room with were for 20 min with and were in medium and with a of STPCA in were from coral and into expression The was with the and The a the of the STPCA to a at the sequencing, the was into with the reagent A by was used to STPCA. of the were the was with medium was the supernatant of medium was a to which the of the The was with and and the was with the and The of was by of then the with a The was the of the the was and was a and STPCA was incubated at in with or were to and a were with the or the the and were supplied by CA and was used for the CO2 hydration J. Biol. Chem. Full Text PDF PubMed Google Scholar). a of 0.2 was used an at the of with or and 20 or 20 the CO2 hydration for a of The CO2 from to for the of the and at least of the of the were used for the The rates were determined in the and from the of were in with is not at and to were with and enzyme were for min at room temperature to the to allow for the of the The were by from and the from at least three was performed on the genes of S. STPCA in the study, A. Tambutté S. Béranger G. Gaume B. Scimeca J.C. Allemand D. Zoccola D. Marine Biotechnol. 2008; 10.1007/s10126-008-9101-1Google Scholar, and D. Tambutté E. E. Puverel S. Scimeca J.C. Allemand D. Tambutté S. Biochim. Biophys. Acta. 2004; PubMed Scopus Google Scholar, of and and and and were the was according to the that is in was performed the and in in a in of cDNA 0.2 of (10 of and of was performed with the for min, for min, then for for min for a RNA were determined by the method of of between and and genes (23Moya A. Tambutté S. Béranger G. Gaume B. Scimeca J.C. Allemand D. Zoccola D. Marine Biotechnol. 2008; 10.1007/s10126-008-9101-1Google and by the with the light were into form by 2001; PubMed Scopus Google Scholar). of experiments are was performed a Results are of a Carbonic from the S. a S. pistillata A that the from S. pistillata to a carbonic experiments were used to The of with a from the This for a of amino and a molecular of The of the STPCA is on the with in numerous with and α-CA A on the of the carbonic anhydrase catalytic the three for the by an in and the important amino in the by an in A is in the STPCA with between and that the is secreted or and a or could we suggest that the STPCA is a secreted form of are in the STPCA in and and (10 and of α-CA and method similar for and are shown the in are in this with the α-CA from the The STPCA with the of and of the STPCA enzyme in were with the antibody S. pistillata is a coral by the of numerous by a to the The in with seawater is the the to the skeleton is the The of named ectoderm and The ectoderm is a that is in with the skeleton. This calcium is to the the calicoblastic ectoderm Refs. S. E. Zoccola D. I. A. Lotto S. Allemand D. S. 2005; Scopus Google Scholar, E. Allemand D. Zoccola D. A. Lotto S. Caminiti N. Tambutté S. 2007; Scopus Google Scholar). a of S. pistillata in the with the the seawater and the the skeleton. is a of the and the of the calicoblastic was in of the Carbonic form of the carbonic anhydrase was in The form with a at the was in the medium and on STPCA was to and on A with or was performed. The possesses an molecular of the is by the In the the STPCA was to a with or Results show that are on the CA and were performed on the STPCA. The for the CO2 hydration by the well for the human are shown in The results show that the STPCA a catalytic than of the of that of the In this is similar to the which is a secreted shown in and STPCA have a similar and a to × from the of that STPCA is by similar to and for the CO2 hydration are for the CO2 hydration J. Biol. Chem. Full Text PDF PubMed Google by the human the and the secreted and the and the STPCA at 20 and in and their with C.T. 2008; PubMed Scopus Google Scholar), a used in a determine the role of STPCA in the “light-enhanced calcification” phenomenon, expression of the STPCA gene were by real-time under light and dark conditions. Results are expression with the genes and (23Moya A. Tambutté S. Béranger G. Gaume B. Scimeca J.C. Allemand D. Zoccola D. Marine Biotechnol. 2008; 10.1007/s10126-008-9101-1Google Scholar), and the in the light condition a according to the and for the amplification of and The of was by Results show that in the expression of STPCA is higher during the dark ± than during the light ± A Carbonic from the S. on the α-CA we and a carbonic anhydrase in the scleractinian coral S. This α-CA, named a of amino The STPCA a CA catalytic site of three that the and amino in the for (1Hewett-Emmett D. Tashian R.E. Mol. Phylogenet. Evol. 1996; 5: 50-77Crossref PubMed Scopus (520) Google Scholar). coral CA have cloned, in the coral F. M. Biol. Bull. 2006; PubMed Scopus Google Scholar). named a and and amino M. Biol. Bull. 2006; PubMed Scopus Google Scholar). are than the STPCA The a and a catalytic site the three and amino from the M. Biol. Bull. 2006; PubMed Scopus Google Scholar). to a and the of a that the STPCA is not secreted or The STPCA not a site or that this is secreted than of the STPCA in that the STPCA to the α of carbonic we a with α-CA This that the STPCA with of the F. and N. of F. have isolated from and N. not establish symbiosis with that to We that the STPCA to the and not to the symbiotic of S. The is in with the results of L. J. G. Science. 2007; PubMed Scopus Google Scholar). By have shown that the α-CA from a gene gene in and L. J. G. Science. 2007; PubMed Scopus Google Scholar). In to we that a with the if we not have this that than the One specific feature of is that are to a The S. pistillata to a or secreted F. that STPCA is not a form of of N. and F. not a similar to that this a that was in that invertebrates the of and and is a between and the from the invertebrates and are in the of CA Physiol. Biochem. Zool. 1999; PubMed Scopus Google Scholar, Evol. 2007; PubMed Scopus Google Scholar, S. S. J. 2007; PubMed Scopus Google Scholar). This a coral CA that was from the of S. P. M. T.W. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). shown in the of L. J. G. Science. 2007; PubMed Scopus Google Scholar), we that the of carbonic anhydrases is a or secreted which then into and Tashian (1Hewett-Emmett D. Tashian R.E. Mol. Phylogenet. Evol. 1996; 5: 50-77Crossref PubMed Scopus (520) Google that and form a that than the CA The of STPCA to the secreted form of CA is by CA and the STPCA an similar to that of secreted STPCA is a CO2 hydration is by similar to of CA in the symbiotic S. pistillata, CA shown to play a role in CO2 to symbiont photosynthesis Smith L. Mar. Biol. Scopus Google Scholar, S. C. Allemand D. N. Jaubert J. J. Exp. Mar. Biol. 1996; 199: Scopus Google Scholar, P. Allemand D. Amer. J. Physiol. 2000; PubMed Google and calcification (7Goreau T.F. Biol. Bull. 1959; 116: 59-75Crossref Google Scholar, 14Tambutté E. Allemand D. Mueller E. Jaubert J. J. Exp. Biol. 1996; 199: 1029-1041Crossref PubMed Google Scholar, 15Furla P. Galgani I. Durand I. Allemand D. J. Exp. Biol. 2000; 203: 3445-3457Crossref PubMed Google Scholar, 16Al-Horani F.A. Al-Moghrabi S.M. de Beer D. Mar. Biol. 2003; 142: 419-426Crossref Scopus (407) Google Scholar, 17Marshall A.T. Clode P.L. Comp. Biochem. Physiol. 2003; 136: 417-426Crossref Scopus (34) Google Scholar). CA in the study to to the is the calicoblastic from and similar were in symbiotic and The of the role of CA in coral calcification from the study of (7Goreau T.F. Biol. Bull. 1959; 116: 59-75Crossref Google Scholar). the CA calcification this that the enzyme in removing carbonic acid by calcification from the numerous have that CA calcification rates and have that a CA is in the inorganic carbon for calcification the of at the calcification site E. Allemand D. Mueller E. Jaubert J. J. Exp. Biol. 1996; 199: 1029-1041Crossref PubMed Google P. Galgani I. Durand I. Allemand D. J. Exp. Biol. 2000; 203: 3445-3457Crossref PubMed Google Scholar, 16Al-Horani F.A. Al-Moghrabi S.M. de Beer D. Mar. Biol. 2003; 142: 419-426Crossref Scopus (407) Google Scholar, 17Marshall A.T. Clode P.L. Comp. Biochem. Physiol. 2003; 136: 417-426Crossref Scopus (34) Google Scholar). A role for STPCA in calcification is by the specific localization of this enzyme in the calicoblastic which is the the skeleton. is well that this a role in calcification S. E. Zoccola D. I. A. Lotto S. Allemand D. S. 2005; Scopus Google Scholar, E. Allemand D. Zoccola D. A. Lotto S. Caminiti N. Tambutté S. 2007; Scopus Google Scholar, Scopus Google Scholar). localization is in with the results of and Yamazato Y. Yamazato K. Galaxea. 1984; 3: 25-36Google localized CA in the calicoblastic ectoderm of the coral in the the CA was localized in the and on the of the R.J. Watabe N. J. Exp. Zool. 1987; 241: 171-180Crossref Scopus (30) Google Scholar), an important role of CA in the calcification process. This not the of at least CA for CO2 to symbiont previously Smith L. Mar. Biol. Scopus Google Scholar, P. Allemand D. Amer. J. Physiol. 2000; PubMed Google Scholar, D. Furla P. S. J. Scholar). The localization of STPCA with the and of the STPCA gene suggest that the enzyme is secreted by the calicoblastic shown by J. Nature. Scopus Google and Furla P. Galgani I. Durand I. Allemand D. J. Exp. Biol. 2000; 203: 3445-3457Crossref PubMed Google that CO2 is the of carbon for calcification. In this the secreted CA help in this CO2 from the to the because of the of this F. Al-Moghrabi S.M. de Beer D. J. Exp. Mar. Biol. 2003; Scopus Google into The by this of then from the site of calcification by the the calicoblastic D. Tambutté E. E. Puverel S. Scimeca J.C. Allemand D. Tambutté S. Biochim. Biophys. Acta. 2004; PubMed Scopus Google Scholar), which the is well that light calcification in corals S. D. Bull. Scholar). we have shown that the calcification rate is times higher than the dark calcification rate in S. pistillata (21Moya A. Tambutté S. Tambutté E. Zoccola D. Caminiti N. Allemand D. J. Exp. Biol. 2006; 209: 3413-3419Crossref PubMed Scopus (100) Google Scholar). This enhancement of calcification by light is due to the symbiosis between corals and their photosynthetic its mechanism largely unknown (see review in Refs. 19Gattuso J.-P. Allemand D. Frankignoulle M. Amer. Zool. 1999; 39: 160-183Crossref Scopus (586) Google Scholar, 20Allemand D. Ferrier-Pagès C. Furla P. Houlbrèque F. Puverel S. Reynaud S. Tambutté É. Tambutté S. Zoccola D. C.R. Palévol. 2004; 3: 453-467Crossref Scopus (264) Google Scholar). In the study, we investigated the differential expression of the STPCA gene during light and dark conditions. Our results show that the STPCA gene is during the dark with light a in and in CA is important to that is the that a is in corals for a CA gene well for A similar was previously in in which CA a of gene expression with of expression at night with expression H. Murayama E. Ohira T. Takagi Y. Nagasawa H. Comp. Biochem. Physiol. 2006; 145: 257-264Crossref Scopus (29) Google Scholar). a with and during the night and this are not well understood (see review in Refs. P. H. G. N. C.R. 2004; 3: Scopus Google Scholar, D. N. de H. G. P. E. of Biological and Scholar). explain the differential expression of CA between light and dark, H. Murayama E. Ohira T. Takagi Y. Nagasawa H. Comp. Biochem. Physiol. 2006; 145: 257-264Crossref Scopus (29) Google that CA to the of by the inorganic and the of in the In corals, of at the and an during dark with light F. Al-Moghrabi S.M. de Beer D. J. Exp. Mar. Biol. 2003; Scopus Google Scholar, P. S. Jaubert J. Allemand D. J. Physiol. Comp. PubMed Google Scholar). during the dark, by calcification the by the is photosynthesis P. Galgani I. Durand I. Allemand D. J. Exp. Biol. 2000; 203: 3445-3457Crossref PubMed Google Scholar). In the coral S. pistillata, we suggest that the up-regulation of STPCA expression during the dark the of from CO2 an in into the in the light to in the dark F.A. Al-Moghrabi S.M. de Beer D. Mar. Biol. 2003; 142: 419-426Crossref Scopus (407) Google a is in with of (7Goreau T.F. Biol. Bull. 1959; 116: 59-75Crossref Google and Furla P. Galgani I. Durand I. Allemand D. J. Exp. Biol. 2000; 203: 3445-3457Crossref PubMed Google that the of carbonic anhydrase on calcification is higher in the dark than in the light, an of carbonic anhydrase in the This that an enhancement of CA expression during the dark, a in CA expression is not to in light-enhanced calcification, which to at least in on a more in the light P. Galgani I. Durand I. Allemand D. J. Exp. Biol. 2000; 203: 3445-3457Crossref PubMed Google Scholar). In the STPCA in this study is a secreted enzyme by the calicoblastic ectoderm a role in calcification. We propose that this CA the hydration rate of CO2 into This is the to show an up-regulation of a coral gene during a This up-regulation of STPCA then help to the CO2 hydration rate during dark conditions. We for the of F. and for help in