ABSTRACT: Development, physiology, and reproduction require the precise control of hormones. Hormone bioavailability is regulated by their synthesis and transport, as well as sequestration, natural turnover, and active degradation. For most hormones, we know far less about active hormone degradation than their synthesis. Here, we use Drosophila melanogaster to investigate the enzymes that degrade the retinoic acid-like Juvenile hormones (JH). While JH titers must decrease to ensure metamorphosis, the developmental requirements of both classes of JH degradation enzymes, JH esterases (JHEs), and JH epoxide hydrolases (JHEHs), have not been determined due to genetic redundancies. To overcome this knowledge gap, we generated double knockout animals lacking both JHEs as well as triple knockout animals lacking all three JHEHs. We found that while neither class of JH degradation enzymes are required for viability, they have unexpectedly distinct requirements in developmental pacing and growth. Animals lacking JHEs pupariate at the expected time but are larger, indicating that JHEs restrain developmental growth but not pacing. In contrast, animals lacking JHEHs are a normal size but are severely delayed in their development, indicating that JHEHs regulate developmental pacing but not growth. To investigate this unexpected separation of requirements among JH degradation enzymes, we analyzed JH-producing glands, performed rescue experiments, and characterized transcriptional changes by bulk RNA-sequencing. Together, our data revealed striking differences in the developmental requirements for JHE and JHEH, yet shared requirements for regulating ecdysone-related genes. By generating these genetic tools, such hypotheses can be explored to shed light on active hormone degradation in development and beyond.
