Figure 1.
Relief contrast images of coral cell cultures.
(A) Relief contrast of individual cells in Stylophora pistillata primary cell culture at T0. Assembly of proto-polyps after (B) 40 h (C) 50 h and (D) 73 h in culture (magnification 20×).
Figure 2.
Relief contrast, confocal and SEM images of proto-polyp.
(A) 72 h old proto-polyp (magnification 40×). (B) Z-stack of a proto-polyp at 12 d. The Symbiodinium sp. cells are seen by chlorophyll florescence and animal cells are revealed by GFP fluorescence. (C) SEM of 12 d old proto-polyp cell organization, and (D) extracellular precipitation of aragonite crystals associated with proto-polyp.
Figure 3.
SEM images and elemental composition.
SEM images of typical columnar aggregates of aragonite crystals, elongated along the c axis, formed in the dorsal surface of the cell culture (A–B) and in the S. pistillata skeleton (C). In both aggregates one can see the orthorhombic structure, characteristic of aragonite crystals. (D) ECM of a proto-polyp after cells removed by 1 M NaOH. Red circle on the SEM image indicates the sample point of the EDS. Energy-dispersive X-ray spectrum showing calcium carbonate composition of the aragonite crystal in (a) proto-polyp, in (c) coral skeleton and (d) in the ECM scaffold while (b) the composition in the cells is C, O, N and P. The Au and Pd peaks are from the gold coating.
Figure 4.
X-ray powder diffraction pattern of (A) tissue culture sample and (B) and of a powdered mother colony skeleton showing the characteristic diffraction peaks of aragonite. Composite minerals were determined by peak matching of XRD data in Jade software (MDI Products, Inc.). Peaks at 26.3 and 45.9 degree are characteristic aragonite peaks. Peaks at 21.6, 30.4, 37.4, 43.5, 49, and 54 degrees are due to LaB6, added to ensure correct peak identification of very small samples (C).
Table 1.
Relative amino acid composition of skeletal and cell culture ECM as determined by HPLC fluorometric analysis.
Figure 5.
Drawing of a proto-polyp showing the three cell layers organization.
The lower layer, the ectoderm that is attached to the plate, is covered by the endoderm containing Symbiodinium sp. cells, which in turn is covered by the upper layer of calicoblastic cells as seen in Fig. 2B. The calicoblastic layer secretes ECM as seen in Fig. 2C. Aragonite crystals form on top of the ECM (Fig. 3A and C).