Mutations in cardinal are responsible for the red-1 and peach eye color mutants of the red flour beetle Tribolium castaneum

Ommochromes are the major pigments found in the eyes, eggs, wings and epidermis of insects. Here, we report the identification and characterization of the gene responsible for red-1 locus of Tribolium, whose mutants have white eyes due to lack of ommochrome pigments in the eyes. Using a candidate gene approach, we demonstrated that red-1 and peach mutants have molecular defects in the cardinal gene, which encodes a haem peroxidase that is considered to convert 3-hydroxykynurenine into ommochromes in pigment granules. Our experiments showed that the expression pattern of cardinal correlates well with the progression of eye pigmentation during pupal stages. We performed gene editing experiments using the Receptor-Mediated Ovary Transduction of Cargo (ReMOT) Control technique to disrupt the cardinal gene by adult injection, and were able to establish a novel cardinal mutant line. Our complementation test provided definitive genetic evidence that cardinal is located at the red-1 locus. The present study will lead to a greater understanding of the function and diversity of ommochrome pathway genes in insects. Our successful use of ReMOT Control in beetles will facilitate the development of more efficient and versatile systems for insect genome editing by simple adult injection.