Two female-lethal driver constructs for New World screwworm, Cochliomyia hominivorax: Similar expression, different performance

The New World screwworm, Cochliomyia hominivorax, eradication program has traditionally used mixed-sex sterile releases for population control. However, modeling suggests releasing only males could be up to five times more effective in controlling wild populations and significantly reduce diet and labor costs if females are removed early in development. While genetic sexing systems have been successfully developed in other species by selectively breeding for sex-linked traits such as embryo heat tolerance or pupa color, these methods are not feasible for C. hominivorax. Therefore, Tet-off transgenic sexing systems were developed for C. hominivorax including single insertion pupa-lethal and two-component embryo-lethal constructs. Here, strains created with new drivers selected for expected embryonic female lethality, DR6 and DR7, utilizing Nullo or CmCg14427 promoters respectively, were created and evaluated for biological performance and feasibility of use for mass-production. However, these strains were unreliable and had low yields when tetracycline was used for transgene suppression. Switching to doxycycline improved strain health and allowed for comprehensive characterization. Interestingly, both drivers had similar gene expression patterns, but DR6 caused pupal lethality while DR7 killed female embryos. Although strain performance was generally acceptable, it was lower than the non-transgenic production strain (J-06). Notably, within the two DR6 strains, higher activity resulted in lower biological yields. These findings highlight that selected promoters may not always perform as intended, and using excessively active promoters can lead to inconsistent or diminished strain performance compared to those with moderate activity.