A study in ebony: Functional genomics evidence linking the ebony gene to the black pupae phenotype in tephritid fruit flies

Tephritidae (‘true fruit flies’) is a family of Diptera that comprises some of the most invasive and destructive agricultural pests worldwide. Area-wide integrated pest management programs (AW-IPM) incorporating sterile insect technique (SIT) have been successful in suppressing populations of fruit flies in the wild. In addition, the development of genetic sexing strains (GSS) has allowed for sterile male-only releases, which improves the efficacy and cost-effectiveness of SIT programs. In tephritids, the selectable phenotypic markers named white pupae and black pupae were isolated and successfully used to remove females before SIT releases. While the gene responsible for the white pupae phenotype was recently discovered, the genetic basis of the black pupae mutation has yet to be resolved. In this study, we demonstrated the integration of genetics, genomics, transcriptomics, and CRISPR/Cas9 genome-editing tools to identify loci responsible for phenotypic variations in insect populations. Using this integrated framework, we collected robust functional evidence supporting the link between the evolutionarily conserved ebony gene (e) and the black pupae phenotype in tephritids. This allowed us to recreate the black pupae phenotype in major agricultural fruit fly pests, including Anastrepha, Ceratitis, Bactrocera, and Zeugodacus, using our previous optimized protocol for CRISPR/Cas9-mediated gene knockout. The framework described here can assist the discovery and transference of well-established selectable genetic markers into species without robust genetic sexing systems.