Nanoscale cuticle mass density variations influenced by pigmentation in butterfly wing scales

How pigment distribution influences the cuticle density within a microscopic butterfly wing scale, and how both impact each scale's final reflected color, remains unknown. We use ptychographic X-ray computed tomography to quantitatively determine, at nanoscale resolutions, the three-dimensional mass density of scales with pigmentation differences. By comparing cuticle densities between two pairs of scales with pigmentation differences, we determine that the density of the lower lamina is inversely correlated with pigmentation. In the upper lamina structure of Junonia orithya and Bicyclus anynana, low pigment levels also correlate with sheet-like chitin structures as opposed to rod-like structures. Within each scale, we determine that the lower lamina in all scales has the highest density, and distinct layers within the lower lamina help explain reflected color. We hypothesize that pigments, in addition to absorbing specific wavelengths, can affect cuticle polymerization, density, and refractive index, thereby impacting reflected wavelengths that produce colors.