White pupae genes in the Tephritids Ceratitis capitata, Bactrocera dorsalis and Zeugodacus cucurbitae: a story of parallel mutations

Authors: WARD, C - University Of Adelaide; AUMANN, R - Justus-Liebig University; WHITEHEAD, M - University Of Liverpool; NIKOLOULI, K - Food And Agriculture Organization Of The United Nations (FAO); LEVEQUE, G - McGill University - Canada; GOUVI, G - Food And Agriculture Organization Of The United Nations (FAO); FUNG, E - South Australian Research And Development Institute; REILING, S - McGill University - Canada; DJAMBAZIAN, H - McGill University - Canada; HUGHES, M - University Of Liverpool; WHITEFORD, S - University Of Liverpool; CACERES-BARRIOS, CARLOS - Food And Agriculture Organization Of The United Nations (FAO); NGUYEN, T - University Of Adelaide; CHOO, AMANDA - University Of Adelaide; CRISP, PETER - University Of Adelaide; Sim, Sheina; Geib, Scott; MAREC, F - Academy Of Science Of Czech Republic; HACKER, I - Justus-Liebig University; RAGOUSSIS, J - McGill University - Canada; DARBY, ALLISTAIR - University Of Liverpool; BOURTZIS, KOSTANTINOS - Food And Agriculture Organization Of The United Nations (FAO); BAXTER, SIMON - University Of Melbourne; SCHETELIG, MARC - Justus-Liebig University

Submitted to: Nature Biotechnology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/14/2020
Publication Date: 1/21/2021
Citation: Ward, C.M., Aumann, R.A., Whitehead, M.A., Nikolouli, K., Leveque, G., Gouvi, G., Fung, E., Reiling, S.J., Djambazian, H., Hughes, M.A., Whiteford, S., Caceres-Barrios, C., Nguyen, T.N., Choo, A., Crisp, P., Sim, S.B., Geib, S.M., Marec, F., Hacker, I., Ragoussis, J., Darby, A.C., Bourtzis, K., Baxter, S.W., Schetelig, M.F. 2021. White pupae genes in the Tephritids Ceratitis capitata, Bactrocera dorsalis and Zeugodacus cucurbitae: a story of parallel mutations. Nature Biotechnology. 12. Article 491. https://doi.org/10.1038/s41467-020-20680-5.
DOI: https://doi.org/10.1038/s41467-020-20680-5

Interpretive Summary: Mass releases of sterilized male insects within sterile insect technique programs have helped suppress insect pest populations since the 1970s. In the major horticultural pests Bactrocera dorsalis, Ceratitis capitata, and Zeugodacus cucurbitae, a key phenotype white pupae (wp) has been used for decades to selectively remove females before releases, yet the gene responsible remained unknown. In a large collaborative effort between academic and national laboratories, we used classical and modern genetic approaches to identify and functionally characterize the mutation conferring the white pupal trait and demonstrated our ability to create novel genetic sexing strains which can be applied to human health pathogens and other agricultural pests.

Technical Abstract: Flooding insect pest populations with huge numbers of sterilized males is an effective mean of biological control as they mate with, but cannot fertilize, wild females. The greatestchallenge of the sterile insect technique (SIT) is the removal of unrequired factory reared females prior to sterilization and release. Spontaneous white-pupae (wp-) color mutations have been integrated as a dimorphic selectable marker into historical SIT strains for three major tephritid fruit fly pests, Bactrocera dorsalis, Ceratitis capitata and Zeugodacus cucurbitae. Here we identify parallel genetic mutations causing the phenotype in all three species using diverse experimental approaches. The B. dorsalis wp- locus was introgressed into a species, Bactrocera tryoni, and whole-genome sequencing identified a 37 bp truncating mutation within a gene containing a Major Facilitator Superfamily domain (MFS). In C. capitata and Z. cucurbitae, cytogenetics, comparative genomics and transcriptomic analysis of strains carrying brown and white pupae phenotypes identified an 8,150 bp insertion of a putative transposon into the C. capitata MFS ortholog and a 13 bp deletion in the Z. cucurbitae ortholog. In B. tryoni CRISPR/Cas9 mediated knock-out of the putative Bt_wp developed mosaic white and brown puparium colors in G0, and G1 progeny with recessive white pupae phenotypes. In C. capitata, complementation crosses of CRISPR-induced wp- mutants to flies carrying the naturally occurring recessive wp- mutation confirmed the role of the gene. Gene editing technology carries the potential for engineering white pupae phenotypes and generating dimorphic SIT strains in other tephritids or insect pest species.