Investigating male sex determination and embryonic genetic functions of Medfly to develop novel sexing strains: Drosophila as resource and an in vivo test tube.

As a proof of principle of a functional screen for Medfly tsl putative genes, we planned to use a Drosophila tsl mutant strain, elav, encoding a neuronal RNA-binding protein, and perform embryos injections of a BAC genomic tsl+ fragment (100 kb long) to confirm a transient functional rescue. We first confirmed that the elavts1 stock was inviable at 25oC, by placing vials of flies at this non-permissive temperature. Adults did not die immediately and some even lived long enough to lay a few eggs. Some of these survived long enough to hatch as larvae and a small number survived to adulthood. However, no F1 adults lived long enough to propagate a viable stock at 25oC. Adults were also allowed to lay eggs at the permissive temperature of 18oC. After 24 hours of egg-laying, vials were cultured in parallel at 18oC and 25oC. Larvae and a small number of adults were observed following continuous culture at 25oC. A transient heat-shock of 1 hr at 37 oC were also applied to 24 hour embryo collections. Again, larvae and a small number of adults were observed in the vials. Therefore, under these conditions elavts does not offer the proper temperature-sensitivity required to test the ability of a BAC to rescue ts-induced embryonic lethality. We started to explore alternative Drosophila embryonic tsl mutant strains to be used within the next months. Medfly XX-only embryos injections of recombinant Medfly synthetic MOY protein induced phenotypic and molecular intersexuality, confirming the maintenance of function of the synthetic molecule. A first series of Medfly XX-only injections of recombinant BdMOY and BoMOY proteins led to mild transient shift of Cctra splicing toward male-specific one, and in XX/XY embryos not phenotypic effects at adult stages. A first series of embryos injections of a MoY transgenic vector (PiggyBac based) led to a dozen adult flies which were crossed and the subsequent progeny was screened for transgenics. No fluorescent individuals in F1 were detected. We attempted to integrate by Cas9 a dsRED transgene into MoY locus to obtain detectable XY females to be maintained by crossing them with XX reverted males (by Cctra RNAi). We obtained few XY reverted females but lacking integration of the tramsgene.