Functional diversity of long-wavelength opsins in Lepidoptera

Insect photoreceptor cells typically express G-protein-coupled opsin receptors sensitive to ultraviolet (short), blue (medium) and green (long-wavelength) radiation. Here we report on the functional diversity of lepidopteran long-wavelength (LW) opsin family members, an opsin type that has been challenging to study in part due to low expression in vitro. Using a combination of approaches, we explored the extent to which butterflies and moths have evolved red sensitive receptors that can contribute to enhancing color discrimination and visual acuity in dim-light environments. We optimized a HEK cell expression system to screen and functionally characterize LW opsins, revealing a wide range of maximal sensitivities from green (λmax 515-530 nm) to red-shifted (λmax 565-610 nm) light. Absorbance spectra from reconstituted visual pigments corroborate electrophysiological and optophysiological analyses predicting wavelength sensitivities and provide an additional tool to understand opsin functions in species lacking the eyeshine reflectance necessary for in vivo studies. We identify independent functional shifts in LW opsin gene duplicates in different species of skippers (Hesperidae) and metalmarks (Riodinidae) as an ecologically-relevant mechanism to expand the range of orange and red colors that can be concurrently discriminated. We find that long-wavelength opsins have also evolved sensitivities toward the red spectrum in certain groups of nocturnal moths. Taken together, this work identifies a substantial number of homologs to address the molecular basis for spectral tuning and diversification of LW opsin functions across Lepidoptera. Along with structural filtering by intracellular granular pigments, the dynamic evolution of visual long-wavelength opsins has contributed to expand sensing properties of the visual system to the red end of the light spectrum.