AN IMPROVED CA-45 PROTOCOL FOR INVESTIGATING PHYSIOLOGICAL-MECHANISMSIN CORAL CALCIFICATION

A sensitive experimental protocol using cloned corals (hereafter ''mic rocolonies'') of the branching scleractinian coral Stylophora pistilla ta and Ca-45 has been developed to enable reproducible measurements of physiological and biochemical mechanisms involved in calcium transpor t and compartmentalization during coral calcification. Cloned S. pisti llata microcolonies were propagated in the laboratory from small fragm ents 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. Er rors specifically associated with long-standing problems to do with is otopic exchange were further reduced by producing microcolonies with n o skeletal surfaces exposed to the radioisotope-labelled incubation me dium. The value of the technique resides principally in its superior a bility to elucidate transportation pathways and processes and not in i ts 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 incubations is loca ted. This pool (72.9 +/- 1.4 nmol Ca mg-l protein) is presumably locat ed within the coelenteric cavity as suggested by the following: (1) it has 4-min halftime saturation kinetics; (2) the accumulation of calci um is linearly correlated with the calcium concentration of seawater; and (3) its insensitivity to metabolic and ion transport inhibitors in dicate that membranes do not isolate this compartment. Washout of this large extracellular pool greatly improved estimates of calcium deposi tion as evidenced by 10 to 40% reduction in coefficients of variation when compared with previous Ca-45(2+) methods described in the literat ure. Comparisons of calcification measurements simultaneously carried out using the alkalinity anomaly technique and the Ca-45 protocol desc ribed here show that the correlation coefficient of both techniques is close to 1. Unlike previous reports, our Ca-45(2+)-derived measuremen ts are slightly lower than those computed from the alkalinity depletio n technique.