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Item Data from: The Drosophila melanogaster Y-linked gene, WDY, is required for sperm to swim in the female reproductive tractHafezi, Yassi; Omurzakov, Arsen; Carlisle, Jolie A.; Caldas, Ian V.; Wolfner, Mariana F.; Clark, Andrew G. (2023-06-30)These files contain data supporting the results reported in Hafezi et al, 2023, 'The Drosophila melanogaster Y-linked gene, WDY, is required for sperm to swim in the female reproductive tract.' In Hafezi et al, 2023 we created CRISPR mutants in Drosophila of the Y-linked gene, WDY, which is required for male fertility. WDY mutants produced mature sperm with beating tails that could be transferred to females but failed to enter the female sperm storage organs. We demonstrated that WDY mutant sperm’s tails beat approximately half as fast as wild-type sperm and that sperm do not propel themselves within the male ejaculatory duct or female reproductive tract (RT). These specific motility defects likely caused the sperm storage defect and sterility of the mutants. Regional and genotype-dependent differences in sperm motility suggested that sperm tail beating and propulsion do not always correlate. Furthermore, we found significant differences in the hydrophobicity of key residues of a putative calcium-binding domain between orthologs of WDY that are Y-linked and those that are autosomal. WDY was previously shown to be under positive selection. Together these findings suggest that WDY is undergoing functional evolution that coincides with its transition from autosomal to Y-linked in Drosophila melanogaster and its most closely related species. Finally, we showed that mutants for another Y-linked gene, PRY, also show a sperm storage defect that may explain their subfertility. In contrast to WDY, PRY mutants did swim in the female RT, suggesting they are defective in yet another mode of motility, navigation, or a necessary interaction with the female RT. Overall, we provide direct evidence for the long-held presumption that protein-coding genes on the Drosophila Y regulate sperm motility.