Fig. 1: Physiological response to Ca²⁺ deficiency during larval development in D. melanogaster.

a, Schematic of the methods of rearing larvae on SHM (+) or CFHM (−) from various larval stages. b–f, Larvae reared on CFHM showed reduced haemolymph Ca²⁺ levels and locomotor activity, along with elongated pupal phenotypes that correlated inversely with haemolymph Ca²⁺ concentration. b–d, The haemolymph Ca²⁺ concentration (b) and locomotor activity (c) of wandering L3 larvae and the pupal axis ratio (length/width; d) of pupae reared on SHM (+) or CFHM (−) from various larval stages. e, Representative images of wandering L3 larvae and pupae reared on SHM or CFHM from 0 h after L3 ecdysis (0 h AL3E). Scale bar, 1 mm. f, Representative images of skeletal muscles in white prepupae reared on SHM or CFHM from 0 h AL3E. Mhc-GFP was used to visualize skeletal muscles. Enlarged images correspond to the boxed areas. Scale bar, 1 mm. g–i, Larvae reared on HM containing low Ca²⁺ (0.2 mM) maintain normal circulating Ca²⁺ levels and form puparia with normal pupal axis ratios. g, Schematic of rearing larvae on HM containing varying amounts of Ca²⁺ from 0 h AL3E. h,i, The haemolymph Ca²⁺ concentration of wandering L3 larvae (h) and the pupal axis ratio of pupae (i) reared on HM containing varying amounts of Ca²⁺ from 0 h AL3E. w¹¹¹⁸ flies were used in all of the experiments except for in f. Data are mean ± s.d. Sample sizes (n) and P values (P < 0.05) are shown in the graphs. Statistical analysis was performed using two-tailed Mann-Whitney U-tests in comparison to the SHM (+) group (b–d) and one-way analysis of variance (ANOVA) with Tukey’s multiple-comparisons test (h and i); *P < 0.0001; NS, not significant (P > 0.05).

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