The male determining M-factor of the global disease vector Musca domestica is a paralog of the generic splice factor CWC22

Sex determination mechanisms are bewilderingly diverse. A cascade of genes with hierarchical regulation characterizes sex determination pathways in insects. How such pathways evolve is poorly understood, partly due to a lack of comparative data. Houseflies are well known for their polymorphic sex determination with populations carrying a dominant male determiner M on the Y chromosome or any of the five autosomes. We identified the male determining gene Mdm responsible for splicing regulation of the key switch transformer by exploiting the existence of housefly populations with different sex determination mechanisms. We demonstrate that Mdm originated from duplication of CWC22/nucampholin, a generic and essential splicing regulator across Metazoans with a crucial role in exon-junction complex assembly and non-sense mediated decay. We show that strains with the M-locus on the Y and on different autosomes carry multiple copies of Mdm indicating that the same male determiner translocated to different genomic sites in the genome. We found that embryonic RNAi-based silencing of Mdm leads to differentiation of ovaries in males, while targeted Mdm disruption with CRISPR/CAS-9 resulted in complete sex reversal to fertile females. Our study reveals how a duplicate of a gene with a general splice-regulation role during development can be recruited to serve a specific function in the determination of male sex. Mdm appears to be unique to the housefly representing a compelling example for the plasticity at the instructive level of sex determination hierarchies.