List of primers used in this study.

<div><p>Phosducin-like proteins (PhLP) are thioredoxin domain-containing proteins that are highly conserved across unicellular and multicellular organisms. PhLP family proteins are hypothesized to function as co-chaperones in the folding of cytoskeletal proteins. Here, we present the initial molecular, biochemical, and functional characterization of <i>CG4511</i> as <i>Drosophila melanogaster PhLP3</i>. We cloned the gene into a bacterial expression vector and produced enzymatically active recombinant PhLP3, which showed similar kinetics to previously characterized orthologues. A fly strain homozygous for a P-element insertion in the 5’ UTR of the <i>PhLP3</i> gene exhibited significant downregulation of <i>PhLP3</i> expression. We found these male flies to be sterile. Microscopic analysis revealed altered testes morphology and impairment of spermiogenesis, leading to a lack of mature sperm. Among the most significant observations was the lack of actin cones during sperm maturation. Excision of the P-element insertion in <i>PhLP3</i> restored male fertility, spermiogenesis, and seminal vesicle size. Given the high level of conservation of PhLP3, our data suggests PhLP3 may be an important regulator of sperm development across species.</p></div>