Tissue and Cell 36 (2004) 379–390 Calcium localisation by X-ray microanalysis and fluorescence microscopy in larvae of zooxanthellate and azooxanthellate corals Peta L. Clode a , Alan T. Marshall b,∗ a Centre for Microscopy and Microanalysis, The University of Western Australia, Crawley, WA 6009, Australia b Department of Zoology, Analytical Electron Microscopy Laboratory, La Trobe University, Bundoora, Melbourne, Vict. 3083, Australia Received 21 October 2002; received in revised form 27 April 2004; accepted 30 June 2004 Abstract X-ray microanalysis and fluorescence microscopy (Calcium Orange TM ) was used to determine the distribution of intracellular calcium (I Ca ), in the form of total and ionic calcium respectively, in planulae and settled larvae of a zooxanthellate coral. The distribution of total calcium only was determined in larvae of an azooxanthellate coral. In azooxanthellate planulae and settled larvae, total I Ca concentration in the oral ectoderm was high and similar to that in seawater (SW). Calcium concentration did not vary (P > 0.05) between planulae and settled larvae. However, settled larvae accumulated large amounts of calcium in gastrodermal lipid-containing cells. In contrast, zooxanthellate planulae possessed significantly (P < 0.01) lower concentrations of total I Ca within ectodermal cells in comparison to settled larvae. In addition, in settled zoox- anthellate larvae total calcium concentration in the mesogloea and coelenteron was significantly (P < 0.05) higher than in the oral ectodermal and gastrodermal cells, respectively. Total I Ca concentrations in the oral ectoderm of settled larvae were also significantly (P < 0.01) lower than that of the calicoblastic ectoderm. In zooxanthellate settled larvae, ionic I Ca levels in the aboral epithelium surrounding rapidly growing septa were high. These levels increased significantly (P < 0.05) within the tissue surrounding growing septa after incubation in high-calcium SW. © 2004 Elsevier Ltd. All rights reserved. Keywords: Calcium Orange; Freeze-substitution; Intracellular calcium; Planulae; Scleractinian corals; Settled larvae 1. Introduction An understanding of the transport, accumulation and stor- age of the mineralising ions is fundamental in elucidating the mechanisms of biomineralisation in any biological sys- tem. In scleractinian corals (Phylum Cnidaria, Class Antho- zoa) however, the processes involved in formation of the calcium carbonate (CaCO 3 ) exoskeleton are not well under- stood. A fundamental aspect of biomineralisation in corals is Ca 2+ transport. Apparent differences in the mechanisms of Ca 2+ transport between zooxanthellate corals (those that pos- sess symbiotic algae—“zooxanthellae”) and azooxanthellate corals (those that do not possess symbiotic algae) (Marshall, 1996) and contradictory results reported for Ca 2+ movement ∗ Corresponding author. Tel.: +61 3 9479 2250; fax: +61 3 9479 1551. E-mail address: A.Marshall@latrobe.edu.au (A.T. Marshall). across the oral epithelium of Anthozoans (B´ enazet-Tambutt´ e et al., 1996; Clode and Marshall, 2002a), are indicative of the variation and complexity of calcification processes in scler- actinian corals. Furthermore Ca 2+ entry into coral tissues is light dependant in zooxanthellate corals (De Beer et al., 2000; Marshall and Clode, 2003) but not in azooxanthellate corals (Marshall and Clode, unpublished data). A basic requirement for unravelling the mechanisms in- volved in Ca 2+ transport is knowledge of intracellular cal- cium concentrations. Up to the present time this information has not been available. An aim of the present investigation was to provide some estimate of these concentrations. In corals, the presence of the massive CaCO 3 skeleton, extensive autofluorescence (Read et al., 1968; Logan et al., 1990; Mazel, 1997), contractile abilities of the thin epithe- lial tissue, and an ability to secrete prodigious amounts of mucus, pose considerable barriers to obtaining physiolog- 0040-8166/$ – see front matter © 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.tice.2004.06.005