Genome editing of the vermilion locus generates a visible eye color marker for Oncopeltus fasciatus.

Insects display a vast array of eye and body colors. Genes encoding products involved in biosynthesis and deposition of pigments are ideal genetic markers, contributing, for example, to the power of Drosophila genetics. Oncopeltus fasciatus is an emerging model for hemimetabolous insects, a member of the piercing-sucking feeding order Hemiptera, that includes pests and disease vectors. To identify candidate visible markers for O. fasciatus, we used parental and nymphal RNAi to identify genes that altered eye or body color while having no deleterious effects on viability. We selected Of-vermilion for CRISPR/Cas9 genome editing, generating three independent loss-of-function mutant lines. These studies mapped Of-vermilion to the X-chromosome, the first assignment of a gene to a chromosome in this species. Of-vermilion homozygotes have bright red, rather than black, eyes and are fully viable and fertile. We used these mutants to verify a role for Of-xdh1, ortholog of Drosophila rosy, in contributing to red pigmentation using RNAi. Rather than wild-type-like red bodies, bugs lacking both vermilion and xdh1 have bright yellow bodies, suggesting that ommochromes and pteridines contribute to O. fasciatus body color. Our studies generated the first gene-based visible marker for O. fasciatus and expanded the genetic toolkit for this model system.