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Calcification and synthesis of skeletal organic material in the coral, Pocillopora damicornis (L.) (Astrocoeniidae, scleractinia)
Camp. Biochem. Physiol., 1973, Vol. 44A, pp. 669 to 672. Pergamon Press. Printed in Great Britain
SHORT COMMUNICATION CALCIFICATION AND SYNTHESIS OF SKELETAL ORGANIC MATERIAL IN THE CORAL, POCILLOPORA DAMICORNIS (L.) (ASTROCOENIIDAE, SCLERACTINIA)” STEPHEN
D. YOUNGt
Department of Zoology, University of California, Los Angeles, California 90024 (Received 1 May 1972)
Abstract-l.
Corals were incubated with Na, i4C0, in the presence of puromycin to study organic material production and calcification. 2. A significant coefficient of correlation (+0.63) was found between organic material synthesis and the deposition of calcium carbonate. 3. The effect of puromycin was not significant. 4. These data suggest an interaction, before or during deposition, of skeletal organic material and mineral carbonate.
INTRODUCTION
ARAGONITIC calcium carbonate crystals form the greater mass of the coral skeleton.
However, a significant amount of organic material of unknown function is also present (Silliman, 1846; Wainwright, 1963; Wilfert & Peters, 1969; Young, 1971a, b). Chitin was the first organic component clearly identified (Wainwright, 1963), but protein and lipid seem to be the only organic components present in all corals (Wilfert & Peters, 1969; Young, 1971a, b; Young et al., 1971). There is a controversy about the significance of these organic components. It has been suggested that one or more of these components directly initiated crystallization (Bryan & Hill, 1941; Goreau, 1956) or that they are non-functional residues trapped by rapid mineralization (Barnes, 1970). The theory that organic material directly participates in mineralization has received support from two recent studies. A soluble mucoprotein that selectively binds calcium has been isolated from the * This work formed part of a dissertation submitted to the University of California, Los Angeles in partial fulfillment of the requirements for a Ph.D. Facilities for part of this work provided by Hawaii Institute of Marine Biology and the U.S.A.E.C. Eniwetok Marine Biological Laboratory. Research support by U.S.P.H.S. grants Nos. l-Fl-DE-38 251-01 and -02 and N.S.F. Grant No. GB6438 (to Dr. L. Muscatine). Manuscript preparation support by U.S.P.H.S. Grant No. DE-02668. Contribution No. 394, Hawaii Institute of Marine Biology, University of Hawaii. t Present address: Department of Biology, Indiana University Northwest, Gary, Indiana 46408. 669
670
STEPHEND. YOUNG
shell of the clam, Mercenaria mercenaria (Crenshaw, 1972). Also, studies of rapidly growing coral, Acropora cervicornis, have shown that growth of the tip of a branch is controlled and augmented by synthetic events in subterminal areas of the branch (Pearse & Muscatine, 1971) suggesting production and transport of some required substance. However, Towe (1972) has recently outlined some of the basic problems of simple application of the matrix concept to invertebrate calcification. The present study examines the relationship between new mineral deposition and the synthesis of new skeletal organic material. An attempt was made to inhibit protein synthesis with puromycin. The species used, Pocillopora damicornis (L.), contains protein, lipid and chitin in the skeletal organic material (Young et al., 1971). MATERIALS
AND
METHODS
Tips of P. damicomis branches (approximately the first cm) were incubated in the light for 24 hr in sea water containing 0.25 &i/ml Na,WO, and either 0.0, 0.2, 0.6, 2, 6 or 20 n-moles/ml of puromycin dihydrochloride (Telser et al., 1965). Under these conditions, without puromycin, carbonate is incorporated directly into the skeletal mineral, and indirectly, through the symbiotic algae of the coral, into animal tissue and the skeletal organic material (Muscatine & Cemichiari, 1969; Young et al., 1971). Thus, both mineral and organic material become labelled with 14C. In acid, the mineral 14C is released as CO, permitting separation of radioactivity in organic material from mineral radioactivity. Contamination of skeletal samples with coral tissue was prevented by cleaning skeletons with boiling 5 N NaOH. The NaOH washes were saved for protein determination by the Lowry technique (Lowry et al., 1951). Protein determinations were used as a measure of tissue present and all results are expressed as per mg of tissue protein and are, therefore, comparable. Skeletons were prepared for fractionation by shaving 1 mm of mineral off all surfaces exposed to exchange during the incubation. After pulverization skeletons were cleaned again with 5 N NaOH to remove all traces of extraskeletal organic material. The cleaned powders were decalcified with 6 N HCI in a closed system with a CO, trap of 2aminoethanol and the trap was prepared for scintillation counting (Jeffay & Alvarez, 1961; Young et al., 1971). Radioactive skeletal organic material was precipitated and was collected and counted as described previously (Young et al., 1971).
RESULTS
AND DISCUSSION
Figure 1 shows the results ; each point represents one sample. The ordinate and abcissa show activity in skeletal carbonate and skeletal organic material, respectively. These points are comparable since each is expressed in counts/min per mg of tissue protein. Each sample is also labelled with the amount of puromycin to which it was exposed. The coefficient of correlation between organic material counts and carbonate counts was + O-63, P-C 0.01. Neither carbonate, nor organic material radioactivity was significantly correlated with puromycin concentration (- O-56, 0.05 > P> 0.01 and - O-40, P> O-05, respectively). Thus, a sample that showed a large amount of radioactive organic material also tended to show a large amount of radioactive carbonate in the skeleton. Since no effect of the puromycin was observed, this relationship must be attributed, at least in part, to natural variation in samples.
CALCIFICATION
AND
SYNTHESIS
OF SKELETAL
ORGANIC
27-
MATERIAL
IN
CORAL
671
*(0)
24-
??
(0’6) ??
(2)
(0)
??
‘(0)
*co.21
.I21
6-
.lO.2) *co) ?? (6)
?? (s)
ACTIVITY PER
IN ORGANIC MATERIAL mg TISSUE PROTEIN (X
10-31
FIG. 1. Radioactivity in skeletal mineral and matrix for each sample treated with puromycin. Values in parentheses are puromycin dihydrochloride concentrations in n-moles/ml.
Although the cause of the correlation was not determined, these data establish that an interaction between skeletal organic material and skeletal mineral exists. This interaction may occur at the site of mineralization and represent part of the mechanism by which the animal controls calcification. Though Barnes (1970) points out that crystal growth is a physical rather than an organic phenomenon in his model of coral mineralization, he fails to consider his own statement that organic molecules and inorganic ions are necessary to favor the precipitation of aragonite instead of calcite, nor does his hypothesis fully explain the species specific structure of the coral skeleton. These results cannot be due to trapped tissue because of the methods used in preparing these samples. It does not seem likely that the results are due to carbonate incorporated by boring algae present in the skeleton for three reasons. First, it has been shown that these siphonaceous algae have very low photosynthetic rates (Kanwisher & Wainwright, 1967; Halldal, 1968) and, therefore, would not incorporate significant amounts of carbonate. Second, these algae are concentrated in older parts of skeletons (Wainwright, 1964) and are lowest in concentration in the Third, the fractions of skeletal organic growing tips used in this experiment. material labelled-chitin, protein and lipid-and the specific activities of protein
672
STEPHEN D. YOUNG
amino acids are most consistent with carbonate incorporation by the symbiotic zooxanthellae (Young et ai., 1971). It has been observed that damage to a coral colony, such as breaking off small tips, as done in this experiment, increases the metabolic activity of the colony (Kanwisher & Wainwright, 1967). It was assumed that all metabolic processes important to this experiment would have been equally affected by this increase and that the qualitative aspects of these results were not affected. REFERENCES BARNES D. J. (1971) Coral skeletons: an explanation of their growth and structure.
Science, N. Y. 170,1305-1308. BRYAN W. H. & HILL D. (1941) Spherulitic crystallization as a mechanism of skeletal growth in the hexacorals. Proc. R. Sot. Queensland 52, 78-91. CRENSHAW M. A. (1972) The soluble matrix from Mercenaria mercenaria shell. Biomineralisation 5. (In press.) GOREAUT. F. (1956) A study of the biology and histochemistry of corals. Ph.D. dissertation, Yale University. HALLDALP. (1968) Photosynthetic capacities and photo synthetic action spectra of endozoic algae of the massive coral Favia. Biol. Bull. mar. biol. Lab., Woods Hole 134, 411-424. JEFFAY H. & ALVAREZJ. (1961) Liquid scintillation counting of carbon-14: use of ethanolamine-ethylene glycol monomethyl ether-toluene. Analyt. Chem. 33, 612-615. KANWISHERJ. W. & WAINWRIGHTS. A. (1967) Oxygen balance in some reef corals. Biol. Bull, mar. biol. Lab., Woods Hole 133, 378-390. LOWRY 0. H., ROSEBROUGH N. J., FARR A. L. & RANDALLR. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193,265-275. MUSCATINEL. & CERNICHIARI E. (1969) Assimilation of photosynthetic products of zooxanthellae by a reef coral. Biol. Bull. mar, biol. Lab., Woods Hole 137, -506-523. PEARCEV. B. & MUSCATINEL. (1971) Role of symbiotic algae (zooxanthellae) in coral calcification. Biol. Bull. mar. biol. Lab;, Woods Hole 141, 350-363. SILLIMANB., JR. (1846) On the composition of the calcareous corals. Am. J. Sci. Arts 51, 189-199. TELSER A., ROBINSONH..C..&. DORFI& A. (1965) The biosynthesis of chondroitin-sulfate protein complex. Proc. natn. Acad. Sci. U.S.A. 54, 912-919. Towz K. M. (1972) Invertebrate‘shell structure and the organic matrix concept. Biomineralisation 4, l-14. WAINWRIGHTS. A: (1963) Skeletal organization in the coral, %cillopora damicornis. Q. Jl microsc. Sci. 104,169-183. WAINWRIGHTS. A. (1964). Studies of the-mineral phase of coral skeleton. Expl cell Res. 34,213-230. WILFERT M. & PETERSW. (1969) Vorkommen von Chitin bei Coelenteraten. 2. Morph. Tiere 64,77-84. YOUNG S. D. (1971a) Organic material from scleractinian coral skeletons-I. Variation in composition between several species. Comp. Biochem. Physiol. 4OB,.l13-120. YOUNGS. D. (1971b) Organic matrices associated with CaCO, skeletons of several species of hermatypic corals. In Experimental Coeleneterate Bioloky (Edited by LENHOFF H., MUSCATINEL. & DAVISL.), pp. 260-264. University of Hawaii Press, Honolulu. YOUNG S. D., O’CONNORJ. D. & MUSCATINEL. (1971) Organic material from scleractinian coral skeletons-II. Incorporation of 14C into protein, chitin and lipid. Comp. Biochem. Physiol. 40B, 945-958. -Key Word Index-Calcification; organic matrix ; aragonite.
puromycin;
Pocillopora
damicornis;
zooxanthellae;
SHORT COMMUNICATION CALCIFICATION AND SYNTHESIS OF SKELETAL ORGANIC MATERIAL IN THE CORAL, POCILLOPORA DAMICORNIS (L.) (ASTROCOENIIDAE, SCLERACTINIA)” STEPHEN
D. YOUNGt
Department of Zoology, University of California, Los Angeles, California 90024 (Received 1 May 1972)
Abstract-l.
Corals were incubated with Na, i4C0, in the presence of puromycin to study organic material production and calcification. 2. A significant coefficient of correlation (+0.63) was found between organic material synthesis and the deposition of calcium carbonate. 3. The effect of puromycin was not significant. 4. These data suggest an interaction, before or during deposition, of skeletal organic material and mineral carbonate.
INTRODUCTION
ARAGONITIC calcium carbonate crystals form the greater mass of the coral skeleton.
However, a significant amount of organic material of unknown function is also present (Silliman, 1846; Wainwright, 1963; Wilfert & Peters, 1969; Young, 1971a, b). Chitin was the first organic component clearly identified (Wainwright, 1963), but protein and lipid seem to be the only organic components present in all corals (Wilfert & Peters, 1969; Young, 1971a, b; Young et al., 1971). There is a controversy about the significance of these organic components. It has been suggested that one or more of these components directly initiated crystallization (Bryan & Hill, 1941; Goreau, 1956) or that they are non-functional residues trapped by rapid mineralization (Barnes, 1970). The theory that organic material directly participates in mineralization has received support from two recent studies. A soluble mucoprotein that selectively binds calcium has been isolated from the * This work formed part of a dissertation submitted to the University of California, Los Angeles in partial fulfillment of the requirements for a Ph.D. Facilities for part of this work provided by Hawaii Institute of Marine Biology and the U.S.A.E.C. Eniwetok Marine Biological Laboratory. Research support by U.S.P.H.S. grants Nos. l-Fl-DE-38 251-01 and -02 and N.S.F. Grant No. GB6438 (to Dr. L. Muscatine). Manuscript preparation support by U.S.P.H.S. Grant No. DE-02668. Contribution No. 394, Hawaii Institute of Marine Biology, University of Hawaii. t Present address: Department of Biology, Indiana University Northwest, Gary, Indiana 46408. 669
670
STEPHEND. YOUNG
shell of the clam, Mercenaria mercenaria (Crenshaw, 1972). Also, studies of rapidly growing coral, Acropora cervicornis, have shown that growth of the tip of a branch is controlled and augmented by synthetic events in subterminal areas of the branch (Pearse & Muscatine, 1971) suggesting production and transport of some required substance. However, Towe (1972) has recently outlined some of the basic problems of simple application of the matrix concept to invertebrate calcification. The present study examines the relationship between new mineral deposition and the synthesis of new skeletal organic material. An attempt was made to inhibit protein synthesis with puromycin. The species used, Pocillopora damicornis (L.), contains protein, lipid and chitin in the skeletal organic material (Young et al., 1971). MATERIALS
AND
METHODS
Tips of P. damicomis branches (approximately the first cm) were incubated in the light for 24 hr in sea water containing 0.25 &i/ml Na,WO, and either 0.0, 0.2, 0.6, 2, 6 or 20 n-moles/ml of puromycin dihydrochloride (Telser et al., 1965). Under these conditions, without puromycin, carbonate is incorporated directly into the skeletal mineral, and indirectly, through the symbiotic algae of the coral, into animal tissue and the skeletal organic material (Muscatine & Cemichiari, 1969; Young et al., 1971). Thus, both mineral and organic material become labelled with 14C. In acid, the mineral 14C is released as CO, permitting separation of radioactivity in organic material from mineral radioactivity. Contamination of skeletal samples with coral tissue was prevented by cleaning skeletons with boiling 5 N NaOH. The NaOH washes were saved for protein determination by the Lowry technique (Lowry et al., 1951). Protein determinations were used as a measure of tissue present and all results are expressed as per mg of tissue protein and are, therefore, comparable. Skeletons were prepared for fractionation by shaving 1 mm of mineral off all surfaces exposed to exchange during the incubation. After pulverization skeletons were cleaned again with 5 N NaOH to remove all traces of extraskeletal organic material. The cleaned powders were decalcified with 6 N HCI in a closed system with a CO, trap of 2aminoethanol and the trap was prepared for scintillation counting (Jeffay & Alvarez, 1961; Young et al., 1971). Radioactive skeletal organic material was precipitated and was collected and counted as described previously (Young et al., 1971).
RESULTS
AND DISCUSSION
Figure 1 shows the results ; each point represents one sample. The ordinate and abcissa show activity in skeletal carbonate and skeletal organic material, respectively. These points are comparable since each is expressed in counts/min per mg of tissue protein. Each sample is also labelled with the amount of puromycin to which it was exposed. The coefficient of correlation between organic material counts and carbonate counts was + O-63, P-C 0.01. Neither carbonate, nor organic material radioactivity was significantly correlated with puromycin concentration (- O-56, 0.05 > P> 0.01 and - O-40, P> O-05, respectively). Thus, a sample that showed a large amount of radioactive organic material also tended to show a large amount of radioactive carbonate in the skeleton. Since no effect of the puromycin was observed, this relationship must be attributed, at least in part, to natural variation in samples.
CALCIFICATION
AND
SYNTHESIS
OF SKELETAL
ORGANIC
27-
MATERIAL
IN
CORAL
671
*(0)
24-
??
(0’6) ??
(2)
(0)
??
‘(0)
*co.21
.I21
6-
.lO.2) *co) ?? (6)
?? (s)
ACTIVITY PER
IN ORGANIC MATERIAL mg TISSUE PROTEIN (X
10-31
FIG. 1. Radioactivity in skeletal mineral and matrix for each sample treated with puromycin. Values in parentheses are puromycin dihydrochloride concentrations in n-moles/ml.
Although the cause of the correlation was not determined, these data establish that an interaction between skeletal organic material and skeletal mineral exists. This interaction may occur at the site of mineralization and represent part of the mechanism by which the animal controls calcification. Though Barnes (1970) points out that crystal growth is a physical rather than an organic phenomenon in his model of coral mineralization, he fails to consider his own statement that organic molecules and inorganic ions are necessary to favor the precipitation of aragonite instead of calcite, nor does his hypothesis fully explain the species specific structure of the coral skeleton. These results cannot be due to trapped tissue because of the methods used in preparing these samples. It does not seem likely that the results are due to carbonate incorporated by boring algae present in the skeleton for three reasons. First, it has been shown that these siphonaceous algae have very low photosynthetic rates (Kanwisher & Wainwright, 1967; Halldal, 1968) and, therefore, would not incorporate significant amounts of carbonate. Second, these algae are concentrated in older parts of skeletons (Wainwright, 1964) and are lowest in concentration in the Third, the fractions of skeletal organic growing tips used in this experiment. material labelled-chitin, protein and lipid-and the specific activities of protein
672
STEPHEN D. YOUNG
amino acids are most consistent with carbonate incorporation by the symbiotic zooxanthellae (Young et ai., 1971). It has been observed that damage to a coral colony, such as breaking off small tips, as done in this experiment, increases the metabolic activity of the colony (Kanwisher & Wainwright, 1967). It was assumed that all metabolic processes important to this experiment would have been equally affected by this increase and that the qualitative aspects of these results were not affected. REFERENCES BARNES D. J. (1971) Coral skeletons: an explanation of their growth and structure.
Science, N. Y. 170,1305-1308. BRYAN W. H. & HILL D. (1941) Spherulitic crystallization as a mechanism of skeletal growth in the hexacorals. Proc. R. Sot. Queensland 52, 78-91. CRENSHAW M. A. (1972) The soluble matrix from Mercenaria mercenaria shell. Biomineralisation 5. (In press.) GOREAUT. F. (1956) A study of the biology and histochemistry of corals. Ph.D. dissertation, Yale University. HALLDALP. (1968) Photosynthetic capacities and photo synthetic action spectra of endozoic algae of the massive coral Favia. Biol. Bull. mar. biol. Lab., Woods Hole 134, 411-424. JEFFAY H. & ALVAREZJ. (1961) Liquid scintillation counting of carbon-14: use of ethanolamine-ethylene glycol monomethyl ether-toluene. Analyt. Chem. 33, 612-615. KANWISHERJ. W. & WAINWRIGHTS. A. (1967) Oxygen balance in some reef corals. Biol. Bull, mar. biol. Lab., Woods Hole 133, 378-390. LOWRY 0. H., ROSEBROUGH N. J., FARR A. L. & RANDALLR. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193,265-275. MUSCATINEL. & CERNICHIARI E. (1969) Assimilation of photosynthetic products of zooxanthellae by a reef coral. Biol. Bull. mar, biol. Lab., Woods Hole 137, -506-523. PEARCEV. B. & MUSCATINEL. (1971) Role of symbiotic algae (zooxanthellae) in coral calcification. Biol. Bull. mar. biol. Lab;, Woods Hole 141, 350-363. SILLIMANB., JR. (1846) On the composition of the calcareous corals. Am. J. Sci. Arts 51, 189-199. TELSER A., ROBINSONH..C..&. DORFI& A. (1965) The biosynthesis of chondroitin-sulfate protein complex. Proc. natn. Acad. Sci. U.S.A. 54, 912-919. Towz K. M. (1972) Invertebrate‘shell structure and the organic matrix concept. Biomineralisation 4, l-14. WAINWRIGHTS. A: (1963) Skeletal organization in the coral, %cillopora damicornis. Q. Jl microsc. Sci. 104,169-183. WAINWRIGHTS. A. (1964). Studies of the-mineral phase of coral skeleton. Expl cell Res. 34,213-230. WILFERT M. & PETERSW. (1969) Vorkommen von Chitin bei Coelenteraten. 2. Morph. Tiere 64,77-84. YOUNG S. D. (1971a) Organic material from scleractinian coral skeletons-I. Variation in composition between several species. Comp. Biochem. Physiol. 4OB,.l13-120. YOUNGS. D. (1971b) Organic matrices associated with CaCO, skeletons of several species of hermatypic corals. In Experimental Coeleneterate Bioloky (Edited by LENHOFF H., MUSCATINEL. & DAVISL.), pp. 260-264. University of Hawaii Press, Honolulu. YOUNG S. D., O’CONNORJ. D. & MUSCATINEL. (1971) Organic material from scleractinian coral skeletons-II. Incorporation of 14C into protein, chitin and lipid. Comp. Biochem. Physiol. 40B, 945-958. -Key Word Index-Calcification; organic matrix ; aragonite.
puromycin;
Pocillopora
damicornis;
zooxanthellae;