Across species, animals have diverse sex determination pathways, each consisting of a hierarchical cascade of genes and its associated regulatory mechanism. Houseflies have a distinctive polymorphic sex determination system in which a dominant male determiner, the M-factor, can reside on any of the chromosomes. We identified a gene, (), as the M-factor. originated from a duplication of the spliceosomal factor gene (). Targeted disruption results in complete sex reversal to fertile females because of a shift from male to female expression of the downstream genes and The presence of on different chromosomes indicates that translocated to different genomic sites. Thus, an instructive signal in sex determination can arise by duplication and neofunctionalization of an essential splicing regulator.

14 15 Sex determination, the developmental process by which organismal sex is established, evolves 16 fast, often due to changes in the master regulators at the top of the pathway. Additionally, in 17 species with polygenic sex determination, multiple different master regulators segregate as 18 polymorphisms. Understanding the forces that maintain polygenic sex determination can be 19informative of the factors that drive the evolution of sex determination. The house fly, Musca 20 domestica, is a well-suited model to those ends because natural populations harbor male-21 determining loci on each of the six chromosomes and a bi-allelic female-determiner. To 22 investigate how natural selection maintains polygenic sex determination in house fly, we assayed 23 the phenotypic effects of proto-Y chromosomes by performing RNA-seq experiments to measure 24 gene expression in house fly males carrying different proto-Y chromosomes. We find that the 25 proto-Y chromosomes have similar effects as a non-sex-

Sex determination, the developmental process by which organismal sex is established, evolves fast, often due to changes in the master regulators at the top of the pathway. Additionally, in species with polygenic sex determination, multiple different master regulators segregate as polymorphisms. Understanding the forces that maintain polygenic sex determination can be informative of the factors that drive the evolution of sex determination. The house fly, Musca domestica, is a well-suited model to those ends because natural populations harbor male-determining loci on each of the six chromosomes and a biallelic female determiner. To investigate how natural selection maintains polygenic sex determination in the house fly, we assayed the phenotypic effects of proto-Y chromosomes by performing mRNA-sequencing experiments to measure gene expression in house fly males carrying different proto-Y chromosomes. We find that the proto-Y chromosomes have similar effects as a nonsex-determining autosome. In addition, we created sex-reversed males without any proto-Y chromosomes and they had nearly identical gene expression profiles as genotypic males. Therefore, the proto-Y chromosomes have a minor effect on male gene expression, consistent with previously described minimal X-Y sequence differences. Despite these minimal differences, we find evidence for a disproportionate effect of one proto-Y chromosome on male-biased expression, which could be partially responsible for fitness differences between males with different proto-Y chromosome genotypes. Therefore our results suggest that, if natural selection maintains polygenic sex determination in house fly via gene expression differences, the phenotypes under selection likely depend on a small number of genetic targets.

The classic brown body (bwb) mutation in the housefly Musca domestica impairs normal melanization of the adult cuticle. In Drosophila melanogaster, a reminiscent pigmentation defect results from mutations in the yellow gene encoding dopachrome conversion enzyme (DCE). Here, we demonstrate that the bwb locus structurally and functionally represents the yellow ortholog of Musca domestica, MdY. In bwb Musca strains, we identified two mutant MdY alleles that contain lesions predicted to result in premature truncation of the MdY open reading frame. We targeted wildtype MdY by CRISPR-Cas9 RNPs and generated new mutant alleles that fail to complement existing MdY alleles, genetically confirming that MdY is the bwb locus. We further found evidence for Cas9-mediated interchromosomal recombination between wildtype and mutant bwb alleles. Our work resolves the molecular identity of the classic bwb mutation in Musca domestica and establishes the feasibility of Cas9-mediated genome editing in the Musca model.

The classic brown body (bwb) mutation in the housefly