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Fig. 1. Research methods and design of experiments. a example of CL-activated UV–VIS spectrum of a fragment of luminescent spine (marked with a black circle in (b) of Echinometra sp. labeled with Mn2+ at 3 mg/L showing Mn2+ emission maximum at 632 nm (Mn2+ activation in Ca2+ and Mg2+ positions in calcite), b lateral view of spine showing no growth differences during and between labeling events (orange-red skeletal regions indicate enhanced Mn2+ concentrations due to twice Mn2+ labeling; dark ragions indicate growth in normal (without Mn2+) seawater), c lateral view of spine of Paracentrotus lividus labeled with Mn2+ at 61.4 mg/L without any signs of regeneration, d, lateral view of spine of control specimen of Paracentrotus lividus without any signs of luminescence, cutting fracture is delineated with dotted line, experimental design of trial (e) and main (f) experiments; each box represents a 24 h period
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Fig. 2. CL images of polished sea urchin spines labeled during trial marking experiments. a lateral view with enlarged microregion of a spine showing regenerated tip of Paracentrotus lividus labeled with Mn2+ at 1Â mg/L, b lateral view with enlarged microregion of a spine showing regenerated tip of Paracentrotus lividus labeled with Mn2+ at 3Â mg/L, c lateral view of spine showing a regenerated tip of Echinometra sp. labeled with Mn2+ at 3Â mg/L, d cross-section of a regenerated tip of spine of Echinometra sp. labeled with Mn2+ at 3Â mg/L. Abbreviations: Th - thickening increments, Pi - pore infilling deposit Fr - cutting fracture (delineated with dotted line). Orange-red skeletal regions indicate enhanced Mn2+ concentrations due to twice Mn2+ labeling; dark regions indicate growth in normal (without Mn2+) seawater
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Fig. 3. CL and SEM images of Paracentrotus lividus labeled with Mn2+ at 1 mg/L during the second labeling experiment. a lateral view of a polished spine with enlarged microregion showing septa formation, b cross-section of a polished spine, c-e micromorphology of septa revealed after formic acid etching, f, g cross-sections showing lateral view of the polished tooth, h, i transverse cross-sections through the polished tooth (at the level of proximal shaft (∼6 mm from the aboral end and ∼8 mm from the adoral tip) and near the incisal end (∼11 mm from the aboral end and ∼3 mm from the adoral tip), respectively), j, k cross-sections near the polished ambital plates showing a contact between two test plates (adapical and interadial sutures, respectively). Abbreviations: Th - thickening increments, Pi - pore infilling deposit, Ms - microspines, Sb - stereom bridges, C - outer crust, Pb - pillar bridges, P - pisms, Cp - carinar process plates, Sp - secondary plates, Plb - plate boundary, L - lamellae, N - needles. Orange-red skeletal regions indicate enchanced Mn2+ concentrations due to Mn2+ labeling; dark regions indicate growth in normal (without Mn2+) seawater
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Fig. 4. CL images of polished ossicles of Echinometra sp. labeled with Mn2+ at 1 mg/L during the second labeling experiments. a-d transverse cross-sections through a tooth (from the level near shaft/plumula boundary (∼8 mm from the adoral tip) towards the incisal end (∼6, 4 and 2 mm from the adoral tip, respectively)), e lateral view of tooth near the proximal part of shaft, f cross-section of an ambital interambulacral plate in lateral view, g cross-section near an ambitus showing a contact (adapical suture) between two test plates, h lateral view of a growing spine. Abbreviations: 1st, 2nd, 3rd - successive labeling events (indicated by solid arrows); R - interlabeling recovery, C - outer crust, Pb - pillar bridges, P - pisms, Cp - carinar process plates, Sp - secondary plates, St - stone part, Pp - primary plates, Plb - plate boundary, L - lamellae, N - needles. Orange-red skeletal regions indicate enhanced Mn2+ concentrations due to Mn2+ labeling; dark regions indicate growth in normal (without Mn2+) seawater
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Fig. 5. CL and optical images of polished ossicles of Prionocidaris baculosa labeled with Mn2+ at 1 mg/L during the second marking experiments. a cross-section of an ambitus interambulacral plate in lateral view, b cross-section of a young interambulacral plate near the apical disc in meridional view, c cross-section of an ambitus interambulacral plates with enlarged central tubercle in lateral view, d lateral view of tooth near the proximal part of the shaft, e lateral view of tooth near the incisal end, f-i transverse cross-sections through a tooth (from the level near shaft/plumula boundary (∼7 mm from the adoral tip) towards the incisal end (∼5, 3 and 1 mm from the adoral tip, respectively)), j lateral view of a distal end of growing spine, k lateral view of a growing spines showing an initial phase of cortex formation at the middle height of the spine, l lateral view (at the middle height of the spine) of a growing spines showing later phase of cortex formation with enlargement of selected microregion, m-o cross-sections of a growing spine showing later phase of cortex formation (at the middle height of spine) under CL, optical and polarizing microscope views, respectively, p-s lateral views (at the middle height of spine) of a growing spine showing late phase of cortex formation. Abbreviations: Th - thickening increments, Res? - possible deposition of calcite on previously resorbed stereom, Ms - microspines, C - outer crust, Pb - pillar bridges, P - prisms, Pp - primary plates, L - lamellae, N - needles, Spi - spinule, Pro - protuberances, Sc - stromatic channels, Ic - inner cortex, Oc - outer cortex. Orange-red skeletal regions indicate enchanced Mn2+ concentrations due to Mn2+ labeling; dark regions indicate growth in normal (without Mn2+) seawater
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Fig. 6. Barcharts showing the average extension rates (±SD) during and between the labeling events for four selected microstructural areas in three examined species. Results of the Wilcoxon tests indicate no statistically significant differences. a longitudinal extension rates measured during the first labeling event and subsequent recovery period in Paracentrotus lividus (spine illustrated on Fig. 2a), b extension rates of test plate in meridional direction during the first labeling event and subsequent recovery period in Paracentrotus lividus (plate illustrated on Fig. 3j), c longitudinal extension rates of regenerating spine measured during the first labeling event and subsequent recovery period in Echinometra sp. (spine illustrated on Fig. 2c), d lateral extension rates of inner cortex measured during the first labeling event and subsequent recovery period in Prionocidaris baculosa (spine illustrated on Fig. 5m)
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Fig. 7. Model of cortex development. a SEM image showing basic morphological features of cortex, b enlargement of fine irregular polycrystalline structure from the outermost cortex, c enlargement of the layered microspine from the inner cortex. d-i successive phases of cortex formation: microspine development (d), thickening of microspines (e), deposition of more massive stereom with conchoidal fracture on layered microspines between stromatic channels (f), development of spinules (g) formation of protuberances from spinules (h), eventual fusion of neighboring protuberances (i). Abbreviations: Spin - spinule, Pro - proturberances, Epi - epibionts, S - stereom, Ic - inner cortex, Oc - outer cortex, Ms - microspines, Firc - fine irregular crystal structure, Sc - stromatic channels, M - medulla, Rs - rectilinear stereom
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Fig. 8. Model of tooth development (compiled and modified after [45–49]). a lateral view of a tooth of Paracentrotus lividus, b enlargement of plumula region, c cone-like tooth elements, sticking alternately one within the other, d-f growth of individual tooth element from initial growth phase (d) to late stage (f). Abbreviations: E - epidermis, Pp - primary plate, L - lamellae, N - needles, P - prism, Sp - secondary plate, H - pillar bridges, M - median plane, Hf - main fold of pp., U - umbo, zA - central part of pp., mA - middle part of pp., sA - side part of pp.
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Fig. 9. Model of interambulacral ambital plate development (modified after [58]). a tangential view, b meridional view. Arrows indicate directions of the growth fronts. Mean growth rates per each species in a given direction are highlighted in color (black = Paracentrotus lividus, red = Echinometra sp., green = Prionocidaris baculosa)
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