V‐ATPase subunit M9.7‐d is essential for sperm motility in <i>Drosophila melanogaster</i>

V-ATPases are crucial for animal development and survival, but their functions in fertility are largely unknown. Here, we found that knockdown of VhaM9.7-d in germ cells induced complete sterility in male Drosophila melanogaster, but had no effects on female fertility. Depletion of VhaM9.7-d did not severely impair spermatogenesis, as the mature sperm appeared in the seminal vesicles (SVs) of the testes. However, the sperm released from the SVs of the VhaM9.7-d-knockdown males rapidly lost their motility and were unable to move over long distances. These sperm could be transferred to the female's uterus during copulation, but failed to be stored in the seminal receptacle (SR) and fertilize the egg. Tandem mass tag (TMT) proteomic analyses of SVs, including their contents, identified 434 differentially expressed proteins (DEPs) when comparing the control group to the VhaM9.7-d-knockdown group. Many downregulated proteins were enriched in phagosome and oxidative phosphorylation (OxPHOS) pathways. Subsequent experiments, encompassing the LysoSensor Probe assay, CMXRos staining, and ATP measurement, confirmed that the knockdown of VhaM9.7-d significantly disrupted phagolysosomal and mitochondrial functions, leading to diminished acidity, heightened levels of reactive oxygen species (ROS), and a decrease in both mitochondrial membrane potential and ATP contents in the SVs. These results suggest that VhaM9.7-d plays an essential role in maintaining the homeostasis of sperm energy metabolism by regulating phagolysosomal activity and mitochondrial OxPHOS. Our data provide valuable insights for the further study of the mechanisms of related diseases such as human asthenospermia.