Functional genomics implicates ebony in the black pupae phenotype of tephritid fruit flies

Authors: PAULO, DANIEL - University Of Hawaii; NGUYEN, THU - University Of Melbourne; WARD, CHRIS - University Of Adelaide; Corpuz, Renee; Kauwe, Angela; RENDON, PEDRO - International Atomic Energy Agency (IAEA); RUANO, ROCIO - International Atomic Energy Agency (IAEA); CARDOSO, AMANDA - International Atomic Energy Agency (IAEA); GOUVI, GEORGIA - International Atomic Energy Agency (IAEA); FUNG, ELISABETH - University Of Adelaide; CRISP, PETER - South Australian Research And Development Institute; OKADA, ANZU - University Of Adelaide; CHOO, AMANDA - University Of Adelaide; STAUFFER, CHRISTIAN - University Of Natural Resources And Life Sciences, Vienna; BOURTZIS, KOSTAS - International Atomic Energy Agency (IAEA); Sim, Sheina; BAXTER, SIMON - University Of Melbourne; Geib, Scott

Submitted to: Communications Biology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/8/2025
Publication Date: 1/15/2025
Citation: Paulo, D.F., Nguyen, T.N.M., Ward, C.M., Corpuz, R.L., Kauwe, A.N., Rendon, P., Ruano, R.E.Y., Cardoso, A.A.S., Gouvi, G., Fung, E., Crisp, P., Okada, A., Choo, A., Stauffer, C., Bourtzis, K., Sim, S.B., Baxter, S.W., Geib, S.M. 2025. Functional genomics implicates ebony in the black pupae phenotype of tephritid fruit flies. Communications Biology. 8. Article 60. https://doi.org/10.1038/s42003-025-07489-y.
DOI: https://doi.org/10.1038/s42003-025-07489-y

Interpretive Summary: We utilized a combination of genetic and genomic techniques to characterize the mutation causing the black pupae phenotype in the mass rearing strain of the mexican fruit fly. Using targeted mutagenesis (CRISPR), we re-created this mutation in several species of pest fruit flies across 4 genera, demonstrating the conservation of this gene as associated with a black pupae phenotype. The black pupae phenotype is the foundation of a genetic sexing strain for the mexican fruit fly, allowing for the separation of male and female pupae towards generation of male only sterile release for the Sterile Insect Technique, and the ability to rapidly re-create the black pupae mutation in new strains and species opens the door to non-transgenic genetic sexing systems to expand across pest fruit fly species.

Technical Abstract: The remarkable diversity of insect pigmentation offers a captivating avenue for exploring evolution and genetics. In tephritid fruit flies, decoding the molecular pathways underlying pigmentation traits also plays a central role in applied entomology. Mutant phenotypes like the black pupae (bp) have long been used as a component of genetic sexing strains, allowing male-only release in tephritid sterile insect technique applications. However, the genetic basis of bp remains largely unknown. Here, we present independent evidence from classical and modern genetics revealing that the bp phenotype in the GUA10 strain of the Mexican fruit fly, Anastrepha ludens, is caused by a large deletion at the ebony locus resulting in the removal of the entire protein-coding region of the gene. CRISPR/Cas9-mediated knockout of ebony orthologues produces analogous bp phenotypes in diverse species, functionally validating our findings and enabling further exploration of Ebony's role in pigmentation and development in tephritids. Our discoveries offer fundamental knowledge for developing new sexing strains based on the bp marker and for future evolutionary developmental biology studies in tephritid flies.