The wings of butterflies and moths (Lepidoptera) are typically covered with thousands of flat, overlapping scales that endow the wings with colorful patterns. Yet, numerous species of Lepidoptera have evolved highly transparent wings, which often possess scales of altered morphology and reduced size, and the presence of membrane surface nanostructures that dramatically reduce reflection. Optical properties and anti-reflective nanostructures have been characterized for several ‘clearwing’ Lepidoptera, but the developmental processes underlying wing transparency are unknown. Here, we apply confocal and electron microscopy to create a developmental time-series in the glasswing butterfly, Greta oto, comparing transparent and non-transparent wing regions. We find that during early wing development, scale precursor cell density is reduced in transparent regions, and cytoskeletal organization during scale growth differs between thin, bristle-like scale morphologies within transparent regions and flat, round scale morphologies within opaque regions. Next, we show that nanostructures on the wing membrane surface are composed of two layers: a lower layer of regularly arranged nipple-like nanostructures, and an upper layer of irregularly arranged wax-based nanopillars composed predominantly of long-chain n-alkanes. By chemically removing wax-based nanopillars, along with optical spectroscopy and analytical simulations, we demonstrate their role in generating anti-reflective properties. These findings provide insight into morphogenesis and composition of naturally organized micro- and nanostructures and may provide bioinspiration for new anti-reflective materials.

The wing reflection measurements were performed on a Cary 5000 UV-Vis-NIR spectrophotometer, equipped with a light source of tungsten halogen and an integrating sphere diffuse reflectance accessory (Internal DRA 1800). Wing measurements from the dorsal wing surface were recorded using three different individuals for untreated and three different individuals for hexane treatments with unpolarized light with a spot size of 100 µm for an incident angle of 8o to avoid the loss of direct specular reflectance component through the aperture. All measurements were taken in the dark to avoid possible stray illumination from the surrounding environment and we performed two technical replicates for each individual wing. A reference measurement was done with a calibrated commercial white spectralon standard to calculate the relative diffuse reflectance. The reflectance measurements and mean data are presented in S2 Table.