Relative abundance of the main phyla identified in the mature mud microbiota at different temperatures and sampling sites. Phyla with abundance lower than 0.05% were grouped and are displayed as Other.

Relative abundance of the main Cyanobacteria ASVs identified in the mature mud microbiota at different temperatures and sampling sites. Only Cyanobacteria with abundance higher than 0.01% are shown.

The FT-IR spectra of M-PSs extracted from mature muds, pressed into KBr pellets. With different colours are indicated M-PSs of different spas with the relative temperature of maturation of their muds.

Monosaccharide composition and sulphate content of the six M-PSs extracted from mature muds, reported as molar percentages (mol%) and w/w percentages, respectively. Abbreviations: fuc, fucose; rha, rhamnose; galN, galactosamine; ara, arabinose; glcN, glucosamine; gal, galactose; glc, glucose; man, mannose; xyl, xylose; galA, galacturonic acid; glcA, glucuronic acid.

Recovering of normal developmental parameters of swim bladder area on larvae treated with M-PS after CuSO₄·5H₂O induced inflammation at 3 dpf. (A1,A2) Recovery from inflammation after 18 h of treatment with M-PSs extract from mud maturated at 36.6, 41.8, 49.4 °C (A1) and 46.5, 49.9, 53.5 °C (A2). (B1,B2) Recovery from inflammation after 24 h of treatment with M-PSs extract from mud maturated at 36.6, 41.8, 49.4 °C (B1) and 46.5, 49.9, 53.5 °C (B2). (C1,C2) Recovery from inflammation after 48 h of treatment with M-PSs extract from mud maturated at 36.6, 41.8, 49.4 °C (C1) and 46.5, 49.9, 53.5 °C (C2). Data are compared to control values. The swim bladder area is normalized over body length and indicated as percentages over control. Black bars represent the mean ± SD of three independent experiments conducted with 15–20 larvae per treatment. Statistical analysis was performed using GraphPad Prism 10 (Brown–Forsythe and Welch ANOVA test followed by Dunnett’s T3 multiple comparisons test with individual variances computed for each comparison). Statistical significance was set at p < 0.05 and the results of the multiple comparisons are shown with letters (different letters show differences among data). The exact adjusted p values are listed in Tables S4 and S5.

Recovering of normal developmental parameters of operculum bone area on larvae treated with M-PS after CuSO₄·5H₂O induced inflammation at 3 dpf. (A1,A2) Recovery from inflammation after 24 h of treatment with M-PSs extract from mud maturated at 36.6, 41.8, 49.4 °C (A1) and 46.5, 49.9, 53.5 °C (A2). (B1,B2) Recovery from inflammation after 48 h of treatment with M-PSs extract from mud maturated at 36.6, 41.8, 49.4 °C (B1) and 46.5, 49.9, 53.5 °C (B2). Data are compared to control values. The operculum bone area is normalized over total head area and indicated as percentages over control. Black bars represent the mean ± SD of three independent experiments conducted with 15–20 larvae per treatment. Statistical analysis was performed using GraphPad Prism 10 (Brown–Forsythe and Welch ANOVA test followed by Dunnett’s T3 multiple comparisons test with individual variances computed for each comparison). Statistical significance was set at p < 0.05 and the results of the multiple comparisons are shown with letters (different letters show differences among data). The exact adjusted p values are listed in Tables S6 and S7.

Analysis of locomotor activity of larvae treated with M-PS after CuSO₄·5H₂O inflammation at 3 dpf. (A1,A2) Recovery from inflammation after 24 h of treatment with M-PSs extract from mud maturated at 36.6, 41.8, 49.4 °C (A1) and 46.5, 49.9, 53.5 °C (A2). (B1,B2) Total distance moved by larvae depending on treatment. Lightning protocol: 10 min of light (white background) and 10 min of dark (grey background). Data were obtained considering 2 min intervals during the 60 min sessions. Black bars represent the mean ± SEM of three independent experiments conducted with 15–20 larvae per treatment in (A1,A2) and mean ± SD in (B1,B2). Statistical analysis was performed using GraphPad Prism 10 (Brown–Forsythe and Welch ANOVA test followed by Dunnett’s T3 multiple comparison test with individual variances computed for each comparison). Statistical significance was set at p < 0.05 and the results of the multiple comparisons are shown with letters (different letters show differences among data). The exact adjusted p values are listed in Tables S8 and S9.

Analysis of locomotor activity of larvae treated with M-PS after CuSO₄·5H₂O inflammation at 3 dpf. (A1,A2) Recovery from inflammation after 48 h of treatment with M-PSs extract from mud maturated at 36.6, 41.8, 49.4 °C (A1) and 46.5, 49.9, 53.5 °C (A2). (B1,B2) Total distance moved by larvae depending on treatment. Lightning protocol: 10 min of light (white background) and 10 min of dark (grey background). Data were obtained considering 2 min intervals during the 60 min sessions. Black bars represent the mean ± SEM of three independent experiments conducted with 15–20 larvae per treatment in (A1,A2) and mean ± SD in (B1,B2). Statistical analysis was performed using GraphPad Prism 10 (Brown–Forsythe and Welch ANOVA test followed by Dunnett’s T3 multiple comparisons test with individual variances computed for each comparison). Statistical significance was set at p < 0.05 and the results of the multiple comparisons are shown with letters (different letters show differences among data). The exact adjusted p values are listed in Tables S10 and S11.