Sub‐micrometer insights into the cytoskeletal dynamics and ultrastructural diversity of butterfly wing scales

ABSTRACT Background The color patterns that adorn lepidopteran wings are ideal for studying cell type diversity using a phenomics approach. Color patterns are made of chitinous scales that are each the product of a single precursor cell, offering a 2D system where phenotypic diversity can be studied cell by cell, both within and between species. Those scales reveal complex ultrastructures in the sub‐micrometer range that are often connected to a photonic function, including iridescent blues and greens, highly reflective whites, or light‐trapping blacks. Results We found that during scale development, Fascin immunostainings reveal punctate distributions consistent with a role in the control of actin patterning. We quantified the cytoskeleton regularity as well as its relationship to chitin deposition sites, and confirmed a role in the patterning of the ultrastructures of the adults scales. Then, in an attempt to characterize the range and variation in lepidopteran scale ultrastructures, we devised a high‐throughput method to quickly derive multiple morphological measurements from fluorescence images and scanning electron micrographs. We imaged a multicolor eyespot element from the butterfly Vanessa cardui (V. cardui), taking approximately 200 000 individual measurements from 1161 scales. Principal component analyses revealed that scale structural features cluster by color type, and detected the divergence of non‐reflective scales characterized by tighter cross‐rib distances and increased orderedness. Conclusion We developed descriptive methods that advance the potential of butterfly wing scales as a model system for studying how a single cell type can differentiate into a multifaceted spectrum of complex morphologies. Our data suggest that specific color scales undergo a tight regulation of their ultrastructures, and that this involves cytoskeletal dynamics during scale growth. Key Findings Butterfly wing scales each derive from the cytoskeletal extension of individual cells, with sub‐micrometer structures deriving from localized chitin synthesis around transient F‐actin bundles. Chitin ridge distances are stable throughout pupal development and are accurately predicted by F‐actin patterning. Fascin‐like punctae are consistent with a role in sub‐cellular actin patterning. A new high‐throughput method enables the automated quantification of adult scale cell ultrastructures on large scanning electron micrographs. Phenomics approach reveals continuous variatio