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We present an economical imaging system with integrated hardware and software to capture multispectral images of Lepidoptera with high efficiency. This method facilitates the comparison of colors and shapes among species at fine and broad taxonomic scales and may be adapted for other insect orders with greater three-dimensionality. Our system can image both the dorsal and ventral sides of pinned specimens. Together with our processing pipeline, the descriptive data can be used to systematically investigate multispectral colors and shapes based on full-wing reconstruction and a universally applicable ground plan that objectively quantifies wing patterns for species with different wing shapes (including tails) and venation systems. Basic morphological measurements, such as body length, thorax width, and antenna size are automatically generated. This system can increase exponentially the amount and quality of trait data extracted from museum specimens.

The wings of Lepidoptera contain a matrix of living cells whose function requires appropriate temperatures. However, given their small thermal capacity, wings can overheat rapidly in the sun. Here we analyze butterfly wings across a wide range of simulated environmental conditions, and find that regions containing living cells are maintained at cooler temperatures. Diverse scale nanostructures and non-uniform cuticle thicknesses create a heterogeneous distribution of radiative cooling that selectively reduces the temperature of structures such as wing veins and androconial organs. These tissues are supplied by circulatory, neural and tracheal systems throughout the adult lifetime, indicating that the insect wing is a dynamic, living structure. Behavioral assays show that butterflies use wings to sense visible and infrared radiation, responding with specialized behaviors to prevent overheating of their wings. Our work highlights the physiological importance of wing temperature and how it is exquisitely regulated by structural and behavioral adaptations.

Color vision has evolved multiple times in both vertebrates and invertebrates and is largely determined by the number and variation in spectral sensitivities of distinct opsin subclasses. However, because of the difficulty of expressing long-wavelength (LW) invertebrate opsins in vitro, our understanding of the molecular basis of functional shifts in opsin spectral sensitivities has been biased toward research primarily in vertebrates. This has restricted our ability to address whether invertebrate G_qprotein-coupled opsins function in a novel or convergent way compared to vertebrate G_topsins. Here we develop a robust heterologous expression system to purify invertebrate rhodopsins, identify specific amino acid changes responsible for adaptive spectral tuning, and pinpoint how molecular variation in invertebrate opsins underlie wavelength sensitivity shifts that enhance visual perception. By combining functional and optophysiological approaches, we disentangle the relative contributions of lateral filtering pigments from red-shifted LW and blue short-wavelength opsins expressed in distinct photoreceptor cells of individual ommatidia. We use in situ hybridization to visualize six ommatidial classes in the compound eye of a lycaenid butterfly with a four-opsin visual system. We show experimentally that certain key tuning residues underlying green spectral shifts in blue opsin paralogs have evolved repeatedly among short-wavelength opsin lineages. Taken together, our results demonstrate the interplay between regulatory and adaptive evolution at multiple G_qopsin loci, as well as how coordinated spectral shifts in LW and blue opsins can act together to enhance insect spectral sensitivity at blue and red wavelengths for visual performance adaptation.