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First published online June 29, 2007
Journal of Experimental Biology 210, 2453-2463 (2007)
Published by The Company of Biologists 2007
doi: 10.1242/jeb.003343

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Electron and ion microprobe analysis of calcium distribution and transport in coral tissues

Alan T. Marshall^1,*, Peta L. Clode², Robert Russell³, Kathryn Prince³ and Richard Stern^2,

¹ Analytical Electron Microscopy Laboratory, Faculty of Science, Technology and Engineering, La Trobe University, Melbourne, VI 3086, Australia
² Centre for Microscopy, Characterisation and Microanalysis (M010), The University of Western Australia, 35 Stirling Hwy, Crawley, WA 6009, Australia
³ SIMS Laboratory, ANSTO, New Illawara Road, Lucas Heights, NSW 2234, Australia

View larger version (109K): [in this window] [in a new window]   Fig. 1. (A) Transverse slice through a polyp of Galaxea fascicularis showing the oral epithelia (OEp) in relation to the skeleton (SK) and extrathecal coelenteron (ETC). (B) Fluorescence image of the extrathecal epithelia in a transverse slice of freeze-substituted G. fascicularis polyp. OE, oral ectoderm; M, mesogloea; OG, oral gastrodermis; ETC, extrathecal coelenteron; AG, aboral gastrodermis; CE, calicoblastic ectoderm; SK, skeleton.

View larger version (92K): [in this window] [in a new window]   Fig. 2. Confocal images of a transverse slice (1 mm) of freeze-substituted Galaxea fascicularis showing (A) oral ectoderm (OE) and oral gastrodermis (OG) separated by mesogloea (M). In the OE, non-specialised ectoderm cells (E), mucocytes (Mu) and cnidocytes (Cn) are easily identified. The OG comprises non-specialised gastrodermis cells (G), mucocytes and zooxanthellae (Z). (B) The aboral epithelia consist of the aboral gastrodermis (AG), which is separated from the calicoblastic ectoderm (CE). Few zooxanthellae are present in the aboral gastrodermis and the epithelium consists primarily of mucocytes and non-specialised gastrodermis cells. The calicoblastic ectoderm consists of thin elongated cells containing numerous vesicles. Mucocytes are occasionally present. SK, skeleton.

View larger version (113K): [in this window] [in a new window]   Fig. 3. STEM images of 1 µm-thick, freeze-substituted sections of Galaxea fascicularis showing typical regions of the oral epithelia. (A) The oral ectoderm (OE), containing dense cnidocytes (cn), is separated from the oral gastrodermis (OG) by the mesogloea (m). Two zooxanthellae (z) are present in the OG, and a part of the extrathecal coelenteron (c) is also shown. (B) The OE and OG are shown, containing numerous dense mucocytes (mu). Part of the extrathecal coelenteron is also shown. Elemental images of Na, Cl, P, S, K and Ca are shown, with the concentration of each element represented by a thermal colour scale (in mmol kg–1). The Cl and Na images indicate the presence of NaCl in the extrathecal coelenteron and mesogloea.

View larger version (74K): [in this window] [in a new window]   Fig. 4. STEM image of a 1 µm-thick, freeze-substituted section of Galaxea fascicularis showing the oral ectoderm (OE), mesogloea (m) and oral gastrodermis (OG). Elemental images of S and Ca are shown with concentrations (in mmol kg–1) represented by a grey scale. Numerous S- and Ca-containing mucocytes (mu) are present in the oral ectoderm and gastrodermis.

View larger version (80K): [in this window] [in a new window]   Fig. 5. STEM image of a 1 µm-thick, freeze-substituted section of Galaxea fascicularis showing part of the extrathecal coelenteron (c), aboral gastrodermis (AG) and calicoblastic ectoderm (CE). Elemental images of Na, Cl, P, K and Ca are shown with the concentration (in mmol kg–1) of each element represented by a grey scale. The Ca image shows the presence of loci of nucleating calcium carbonate deposits on the organic matrix.

View larger version (31K): [in this window] [in a new window]   Fig. 6. (A) Concentrations of Ca (means ± s.e.m.) in the cells of the oral ectoderm (OE; n=60, N=6), mesogloea (M; n=37, N=4), cells of the oral gastrodermis (OG; n=51, N=5), extrathecal coelenteron (ETC; n=37, N=5), cells of the aboral gastrodermis (AG; n=34, N=3) and calicoblastic ectoderm (CE; n=33, N=3). N = number of polyps, n = number of measurements. Cellular compartments are represented by black bars and non-cellular compartments by grey bars. Values labelled with the same letter are significantly different (P<0.05). (B) Schematic diagram of coral epithelia showing Ca concentrations, as measured by x-ray microanalysis of freeze-substituted sections, in the external seawater layer (ESW), oral ectoderm (OE), mesogloea (M), oral gastrodermis (OG), extrathecal coelenteron (ETC), aboral gastrodermis (AG) and calicoblastic ectoderm (CE) adjacent to the skeleton (SK). Bulk seawater concentration (SW) is from Marshall and Clode (Marshall and Clode, 2003). (C) Cutaway diagram of a Galaxea fascicularis polyp, sectioned proximal to the mouth and tentacles, showing the organization of the extrathecal coelenteron compartments. The skeleton is shown in black. The data shown in A and summarised in B are from regions such as that shown in the circle in C.

View larger version (51K): [in this window] [in a new window]   Fig. 7. Low spatial resolution SIMS images of a 2 µm-thick, freeze-substituted section of Galaxea fascicularis showing the distribution of (A) 40Ca and (B) 44Ca after 1 min incubation in 44Ca artificial seawater in the light. The 44Ca/40Ca ratio image (C) and line profile (E) measured over A–B in (C) indicate that relatively little 44Ca had entered the cells and extrathecal coelenteron, with the highest relative concentration being in the external seawater layer. Grey values in E represent pixel intensity values along A–B in (C) as a function of distance (µm). (D) Adjacent section, stained with Toluidine Blue, showing the external seawater layer (SW), oral ectoderm (OE), mesogloea (M), oral gastrodermis (OG), extrathecal coelenteron (ETC), aboral gastrodermis (AG) and calicoblastic ectoderm (CE).

View larger version (57K): [in this window] [in a new window]   Fig. 8. Low spatial resolution SIMS images of a 2 µm-thick, freeze-substituted section of Galaxea fascicularis showing the distribution of (A) 40Ca and (B) 44Ca after 8 min incubation in 44Ca artificial seawater in the light. The 44Ca/40Ca ratio image (C) and line profile (E) measured over A–B in (C) indicate that 44Ca has reached a higher relative concentration in the mesogloea and extrathecal coelenteron than in the external seawater layer. Grey values in E represent pixel intensity values along A–B in (C) as a function of distance (µm). (D) Adjacent section, stained with Toluidine Blue, showing external seawater layer (SW), oral ectoderm (OE), mesogloea (M), oral gastrodermis (OG), extrathecal coelenteron (ETC), epithelia (AE) and skeleton (SK).

View larger version (54K): [in this window] [in a new window]   Fig. 9. Low spatial resolution SIMS images of a 2 µm-thick, freeze-substituted section of Galaxea fascicularis showing the distribution of (A) 40Ca and (B) 44Ca after 8 min incubation in Ca44 artificial seawater in the dark. The 44Ca/40Ca ratio image (C) and line profile (E) measured over A–B in (C) indicate that 44Ca has reached a higher relative concentration in the mesogloea and extrathecal coelenteron than in the external seawater layer. The grey values, however, are lower than in Fig. 7. Grey values in E represent pixel intensity values along A–B in (C) as a function of distance (µm). (D) Adjacent section, stained with Toluidine Blue, showing the external seawater layer (SW), oral ectoderm (OE), mesogloea (M), oral gastrodermis (OG), extrathecal coelenteron (ETC), aboral gastrodermis (AG) and calicoblastic ectoderm (CE).

View larger version (25K): [in this window] [in a new window]   Fig. 10. High-resolution NanoSIMS image of a 2 µm-thick, freeze-substituted section of Galaxea fascicularis after 1 min incubation in 44Ca artificial seawater in the light. The 44Ca/40Ca ratio image (A) and line profile (B) indicate that 44Ca had reached a high relative concentration in the unspecialised oral ectodermal cells but little exchange had occurred in the mucocytes (Mu) or cnidocytes (Cn). Natural (unenriched) levels are also indicated. SW, external seawater layer. In B the ratio 44Ca/40Ca along B–A in (A) is plotted against distance (µm).

View larger version (101K): [in this window] [in a new window]   Fig. 11. High-resolution NanoSIMS image of a 2 µm-thick, freeze-substituted section of Galaxea fascicularis after 8 min incubation in 44Ca artificial seawater in the light. The 40Ca image (A) shows very high levels of 40Ca in cnidocytes and mucocytes in the oral ectoderm (OE) and oral gastrodermis (OG) compared with unspecialised ectodermal cells (E) and mesogloea (M). ETC, extrathecal coelenteron. The 44Ca/40Ca ratio image (B) indicates that 44Ca had reached a high relative concentration in the unspecialised oral ectodermal cells (E) and mesogloea but relatively little exchange had occurred in the mucocytes (Mu) or cnidocytes (Cn).