Fig. 3: Mass density estimations from the segmented volumes of Junonia orithya and Bicyclus anynana scales.

Sub-volumes from the PXCT reconstructions are segmented and clustered based on mass distribution (details in the step-by-step procedural description of the mass density estimation in Methods). (A–D) The mean electron density distribution along the z-axis (from abwing (upper) to adwing (lower)) of different upper lamina sub-structures with respect to the lower lamina of J. orithya male and female scales (A, B) and B. anynana yellow mutant and wildtype scales (C, D). Insets show a schematic view of the scales with colored lines marking the transected regions measured by the corresponding-colored curves. Cyan—lower lamina, red—cross-ribs, green—trabeculae, blue—ridges, and yellow—ectopic film. We can see from the insets that the lower lamina is further away (in the z-axis) from other upper lamina structures, which is why the cyan curves are well separated and do not overlap with the other curves. (Aʹ–Dʹ) Box plots (Center line = median; box = 25th–75th percentiles; whiskers = values within 1.5 × IQR; points = outliers) of the estimated mass densities clustered as lower lamina, cross-ribs, ridges, and trabeculae reveal density variations across the scale sub-structures within each pair of differentially pigmented scales. Mass densities of α-chitin and chitin in crawfish are plotted to show the range of chitin densities reported in various organisms. The red dotted rectangle over the ectopic film density box plot in Cʹ indicates that the density estimate is unreliable, as the ectopic layer thickness is significantly smaller than the voxel-resolution limit of the PXCT. Statistics are derived from one complete PXCT tomogram per scale type (one biological replicate), and 10,000 independent voxel-column measurements within the PXCT volume (technical replicates) are used to compute the density box plots. (Aʹʹ– Dʹʹ) 2D mass density maps (upper lamina only) of the sub-volumes identified with colored contours. Source data for this figure are available in Zenodo (10.5281/zenodo.12703866).