THE PHYSIOLOGY OF SKELETON FORMATION IN CORALS. I. A METHOD FOR MEASURING THE RATE OF CALCIUM DEPOSITION BY CORALS UNDER DIFFERENT CONDITIONS Thomas F Goreau Biol Bull 116:59-75 (1959) http://biostor.org/reference/8130 Keywords: Acropora; Acropora prolifera; Cladocora arbuscula; Hada; Manicina areolata; Oculina; Oculina diffusa; Porites divaricata; Porolithon; Zooxanthellae 8®® Page images from the Biodiversity Heritage Library, http://www.biodiversitylibrary.org/, made available under a Creative Commons Attribution-Noncommercial License http://creativecommons.org/licenses/by-nc/2.5/ THE PHYSIOLOGY OF SKELETON FORMATION IN CORALS. I. A METHOD FOR MEASURING THE RATE OF CALCIUM DEP- OSITION BY CORALS UNDER DIFFERENT CONDITIONS THOMAS F. GOREAU Department of Physiolofiy, University College of the West Indies, and The' New York Zoological Society The purpose of this study is to examine the rate of growth of reef-building corals by measuring the calcium deposition in the skeleton with the aid of a new method using radioactive calcium-45 as tracer. With this procedure it was possible to determine calcification rates in the different parts of coral colonies, and to estimate quantitatively the effect of light and darkness, zooxanthellae and carbonic anhydrase inhibitors on skeletogenesis. Numerous attempts have been made in the past to estimate the growth rates of reef-building corals, mostly by letting weighed and measured coral colonies grow in their natural habitat for periods of months to years (Agassiz, 1890; Abe, 1940; Boschma, 1936 ; Edmondson, 1929; Kawaguti, 1941; Ma, 1937; Mayor, 1924 ; Motoda, 1940 ; Stephenson and Stephenson, 1933 ; Tamura and Hada, 1932 ; Vaug- han, 1919). Recently, Kawaguti and Sakumoto (1948) tried, by a chemical method, to determine the rate of calcium uptake of corals in light and darkness. Using calcium-45 as tracer, we have developed a rapid and precise method for measuring the rate of incorporation of calcium into the coral skeleton under con- trolled laboratory conditions (Goreau, 1957). The preliminary experiments, de- scribed here, were carried out on the following coral species : Manicina areolata (Linne), Cladocora arbuscula (Lesueur), Porites divaricata (Lesueur), Acropora prolifera (Lamarck), Madracis decactis (Lyman) and Oculina diffusa (Lamarck) from Jamaica, B. W. I.; Acropora conferta (Quelch) from Eniwetok Atoll; and Montipora verrucosa (Lamarck), Porites compressa (Quelch), Pocillopora danii- cornis (Linne) and Porolithon sp., a coralline alga, from Hawaii. All the madreporarian corals used in these experiments are shallow-water forms which contain zooxanthellae. Among these, Oculina diffusa is the only species which has not been collected from reefs, but it is common in Kingston Harbour where it grows on rocks on a muddy bottom (Goreau, 1958). The Hawaiian Porolithon listed above is a calcareous alga of the family Corallinaceae, representa- tives of which are important reef builders in the Central Pacific (Emery, Tracey and Ladd, 1954). PROCEDURE Freshly collected coral colonies in good condition were put into glass vessels containing filtered sea water and fitted with tight covers. Aeration, circulation and pH were maintained by bubbling a slow stream of air through the water. The Mailing address : Department of Physiology, University College of the West Indies, Mona St. Andrew, Jamaica, B.W.I. 59 60 THOMAS F. GOREAU temperature was kept to within 1° C. during the experiments (about 25° C. in Jamaica and Hawaii, 28.5° C. in Eniwetok) by keeping the vessels partly immersed in a water bath. After allowing the coral to acclimatize for twenty-four hours, neutralized Ca45C12 was added to give about 20,000 c.p.m./ml. of sea water. The amount of calcium thus added was less than five per cent of the total dissolved Ca++ already present. The initial activity was determined by counting 60-p1 aliquots taken from each vessel after one hour, to allow for complete mixing of the isotope. In addition to the living corals, pieces of clean dead corallum from the same species were included in each vessel to act as controls for measuring the inorganic isotopic exchange rate of the coral skeleton during the experiments. Samples of coral and water were repeatedly taken, starting with three hours from the beginning of the experiment, by the following method : a coral colony, together with its control, was removed from the vessel and small pieces were cut off with scissors or cutting pliers. From five to fifteen replicate samples of about one hundred milligrams each were taken at a time. Samples were collected only from homologous parts of the colonies. This was particularly important in branch- ing corals such as Acropora and Porites where there were shown to be strong dif- ferences in the rate of calcium uptake between the apical and lateral branch polyps. The coral pieces were placed on filter paper to remove excess radioactive sea water, then washed in five two-minute changes of slightly alkaline distilled water. After this, each sample was dissolved in a separate tube containing two milliliters dilute HC1, and heated to boiling. The coral suspension was homogenized to disperse the organic matter. The contents of each tube were made up to five mil- liliters with distilled water, and a 500-pl aliquot was taken for Kjeldahl nitrogen determination. The calcium in each tube was precipitated as the oxalate by the method of Vogel (1943), and filtered out on pre-weighed Whatman No. 42 filter paper planchets, using a cone to spread the precipitate in circles of uniform diameter. The dried and weighed samples were counted with an end window G-M tube, and the observed activity corrected for self-absorption. In the early stages of these investigations, the question arose of choosing a suitable parameter on the basis of which the calcium uptake could be expressed. For example, Mayor (1924) measured coral respiration in terms of tissue weight after the corallum had been dissolved with nitric acid ; Odum and Odum (1955) deter- mined biomass by loss on ignition at 600° C. ; and Kawaguti and Sakumoto (1948) measured calcification rates per gram coral. None of these methods was con- sidered satisfactory. The writer had previously used organic nitrogen as a measure of total cellular matter in corals (Goreau, 1956). The relationship of organic nitrogen to tissue weight was determined for the polyps of Massa angulosa, a coral from which fairly large skeleton-free pieces of tissue could be readily obtained. In this species nitrogen constituted 2 per cent of the wet weight and 11.2 per cent of the dry weight. All results, save those of the exchange controls which lacked tissue, were expressed in terms of calcium deposited per milligram of nitrogen, on the assumption that the nitrogen is a measure of the total coral (plus zooxanthellae) protein present. Nitrogen was determined by the micro-Kjeldahl method of Ma and Zuazaga (1942). The amount of calcium taken up by the coral was calculated from the specific activity of the sea water in the vessels. This was determined by counting 60-id SKELETON FORMATION IN CORALS 61 water aliquots spread to a constant diameter in lens paper circles mounted on micro- scope coverslips and dried under a lamp. The observed count was corrected for self-absorption and the specific activity of the water calculated from its calcium content. THE CALCIUM EXCHANGE IN THE SKELETON CONTROLS Equilibrium exchanges of calcium between the skeleton and sea water were determined on samples of dead coral devoid of tissue, and run at the same time as the living experimental colonies. Isotopic equilibrium appeared to be established 2000 2 -J 2 1000 1.L.1 900 900 700 600 500 - D 0 4 00 )— 2 > 300 1— 5 200 — Li 11.1 0 100 0 10 20 30 40 50 60 TO 80 90 100 TEMPERATURE IN °C FIGURE 1. Calcium-45 exchange of small pieces of corallum from Marticina areolata with sea water at 4° C., 28° C., 58° C. and 100° C. The coral was carefully cleaned to remove all organic matter, and the experiments ran for twenty-four hours. The ordinate is the specific activity plotted on a logarithmic scale. 62 THOMAS F. GOREAU 1000 800 A 600 2 0= a_1 0 400 LEGEND 2 < CURVE A, LIVING CORAL cr (..o CURVE 8 , SKELETON CONTROL :71_I cc 200 W a_ w 1— D Z_ cc I 0 0 L.LJ B80 U) I— z D 60 0 (..) z 40 B i--: (...) < (.) LT_ 20 (7) w a_ u) 1 1 101 1 1 1 1 0 5 10 15 20 25 30 TIME IN HOURS FIGURE 2. SKELETON FORMATION IN CORALS 63 rather slowly, but in most species tested, the process was sixty to eighty per cent complete at the end of twenty-four hours. As expected, the rate of exchange with sea water was strongly temperature-dependent. This is demonstrated in Figure 1, which shows the specific activity of small pieces of Manicina areolata which have been allowed to equilibrate at different temperatures in sea water containing calcium- 45. In most species tested, the rate of calcium-45 deposition in the living coral was much faster than in the skeleton controls. This is shown in Figure 2 for Acropora prolifera, in which the specific activity of the dead corallum is about five per cent that of the living coral at the end of twenty-nine hours. In water of a given specific activity the equilibration rate appears to be much slower in imperforate corals such as Oculina or Phyllangia than in perforate species such as Acropora or Porites. The effect of the total skeletal surface on the exchange rate is being studied. There is some evidence that the living coenosarc forms a barrier which re- stricts calcium exchange of the skeleton with the sea water. In a number of experi- ments in which the calcium rate of the experimental colonies was very low, it was noted that the specific activity of the skeleton controls was higher than that of the living coral.