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	Figure 1. Schematic gene structures of the Paracentrotus lividus and Strongylocentrotus purpuratus metallothioneins (MTs; drawn to scale). The bent arrows indicate the putative transcription start sites (TSS). Numbers under schemes indicate base pair numbers of exons and introns; boxes represent exons: white boxes indicate untranslated regions, and coding regions are coloured. The grey diamond indicates one N stretch in the SpMTB1 intron.
	Figure 2. Multiple sequence alignment (MSA) of P. lividus and S. purpuratus MT sequences. Identities and conservative substitutions are in red font. Red shading represents identity among all sequences. Dots denote gaps. Non-conservative substitutions are in black font.
	Figure 3. Ribbon diagrams and surface representations of PlMTs. (A) General overview of the 5 PlMTs generated by homology modelling with cysteine residues labelled in orange. As far as possible, N-terminal is on the left and C-terminal is on the right; (B) Relative solvent accessibility (RSA) calculated as percentage of corresponding cysteine residues in each protein.
	Figure 4. Metal–thiolate cluster analysis and structural similarities between PlMT7, PlMT8 and SpMTA. (A) Cluster connectivities between the cysteine thiolate groups and the metal ions in the α- and β-domains of the sea urchin MTs. Cys residues are boxed in yellow, while the metal ions are specified by roman numerals; (B) Superposition of the 3D structures of PlMT7 and PlMT8 with SpMTA. Proteins are in ribbon representation. The Cys residues are in stick representation; a Gln residue is also shown. The P. lividus proteins are in ivory, the α-domain of SpMTA is shown in cyan and the β-domain is in violet. Superposition was created and rendered using Chimera package.
	Figure 5. MSA of MT protein sequences. Conservation score higher than 0.5 is highlighted in yellow. Unaligned cysteines are in orange. Cysteines aligned in all sequences are highlighted in red. Dots denote gaps. Species acronyms, taxonomy and sequence IDs are indicated in Table 1.
	Figure 6. Amino acid sequence-based neighbour-joining tree. Species acronyms and sequence IDs are indicated in Table 1. The values at nodes indicate bootstrap support greater than 50%. Tetrahymena pyriformis was used as outgroup to root the tree. Branch lengths are drawn to a scale of amino acid substitutions per site.
	Figure 7. MT gene structures of echinoderms and chordates (drawn to scale). The bent arrows indicate the putative TSSs. Numbers under schemes indicate base pair numbers of exons and introns; boxes represent exons: white boxes indicate untranslated regions, and coding regions are coloured. The grey diamond indicates one N stretch in the AjMT intron.
	Figure 8. MT expression during normal development. RT-qPCR results are shown in arbitrary units (AU) with respect to 18S RNA. The last panel (MTs) show the superposition of the other profiles, in order to display MT relative expression.
	Figure 9. Expression of constitutive MTs after metal exposure. RT-qPCR results performed on RNA extracted from 24 h embryos treated with zinc, copper, nickel or lead. C: controls grown in millipore-filtered seawater (MFSW). Metal treatments were performed with concentrations ranging from 10−8 to 10−4 M. Copper treatments ranged from 10−8 to 10−5 M.
	Figure 10. Expression of induced MTs after metal exposure. RT-qPCR results performed on RNA extracted from 24 h embryos treated with zinc, copper, nickel or lead. C: controls (MFSW). Metal treatments were performed with concentrations ranging from 10−8 (−8) to 10−4 M (−4). Copper treatments ranged from 10−8 (−8) to 10−5 M (−5). These graphics are in log2 scale.
	Figure 11. Localization of constitutive MTs during normal development. Whole-mount in situ hybridization experiments (WMISH) results, 1 h of staining. (A) MT7; (B) MT8: (a,b) gastrula stage (24 h); (c,d) prisma stage (31 h); (e–h) pluteus stage (48 h); (e,f) in the (A) panel, visualization after 15 min of staining; (e,f) in the (B) panel, different focuses of the same embryo are shown. 40× magnification. lv: lateral view; vv: ventral view; ov: oral view.
	Figure 12. Localization of induced MTs at 48 h of normal development and after metal exposure. WMISH results. (A) MT5; (B) MT6; (C) MT6 details: (a,b) untreated embryos, pluteus stage (48 h) after 3 h of staining; (c,d) 10−7 M CdCl2 prisma stage (31 h); (e) and (f) 10−6 M CuSO4 prisma stage (31 h); (g,h) 10−7 M ZnCl2 prisma stage (31 h); (C) enlargements of the (B) panel: (i,j) enlargement of (b), untreated embryos, pluteus stage (48 h); (k) 10−7 M CdCl2 prisma stage (31 h) enlargement of (e) (different focus plan); (l) 10−7 M ZnCl2 prisma stage (31 h) enlargement of (g) (different focus plan). 40× magnification. lv: lateral view; vv: ventral view; ov: oral view.
	Figure 13. Localization of constitutive MTs after metal exposure. WMISH results at 31 h (prisma stage in the controls). (A) MT7; (B) MT8; (a,b) untreated embryos (controls); (c,d) 10−7 M CdCl2; (e,f) 10−6 M CuSO4; (g,h) 10−7 M ZnCl2. 15 min of staining. 40× magnification. lv: lateral view; vv: ventral view.
	Figure 14. Multiple nucleotide sequence alignment of the probes used in WMISH experiments showing identities (red) and differences (black) among them. Red shading represents identity among all sequences. Dots denote gaps. Start and stop codons are in green.

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