Optix Regulates Abdominal Melanin Pigmentation in the Tobacco Hawkmoth Manduca sexta

Research on butterflies has uncovered a conserved "toolkit" of genes for color pattern development and evolution. One of these genes is optix, a homeobox transcription factor that regulates ommochrome and melanin pigmentation, as well as structural coloration, in nymphalid butterflies. It remains unclear, however, whether optix plays any roles in color patterning outside of the Nymphalidae. We used CRISPR-Cas9 to disrupt optix in the tobacco hornwormmoth Manduca sexta and observed a dramatic abdominal pigmentation phenotype, where orange pigmentation was replaced by black eumelanin. Chemical assays suggest that the orange pigment is not an ommochrome, indicating that optix modulates an alternative, uncharacterized pigment pathway in M. sexta. RNA-seq and chemical analyses of orange and black abdominal scales lead us to speculate that the orange pigment may be a type of melanin, perhaps N-β-alanyldopamine (NBAD) sclerotin. Our results suggest that optix plays a deeply ancestral role in pigment regulation in Lepidoptera, and demonstrates evolutionary flexibility in how it interfaces with pigment chemistry across moths and butterflies.