Lysine Triggers Acute Oviposition by Activating the 20E-ETH-JH Signaling Cascade in Drosophila melanogaster

Reproductive performance is closely linked to nutrient availability, yet the specific nutrient-derived cues governing oviposition decision-making remain inadequately defined. Chemically defined holidic media used in Drosophila melanogaster studies provide precise control over dietary components; however, the mechanisms by which individual nutrients exert acute effects on inter-organ endocrine signaling to trigger oviposition behavior are not well understood. In a two-choice oviposition assay, where substrates are presented within the same chamber, we observed that HM induces a rapid increase in egg laying in D. melanogaster compared to grape/apple juice media (GJM), yielding 4.02× and 5.27× more eggs at 2 h and 8 h, respectively. Systematic nutrient omission and subsequent supplementation identify lysine as the key essential amino acid driving this rapid oviposition response. Notably, this phenomenon reflects a short-term oviposition reaction, rather than long-term nutritional modulation of ovarian development. Lysine supplementation results in elevated levels of 20-hydroxyecdysone (20E) and juvenile hormone (JH), as well as the induction of ecdysis-triggering hormone (ETH) expression. Pharmacological interventions support a model in which a 20E–ETH–JH endocrine cascade underlies the lysine-dependent phenotype: methoprene phenocopies the effect, whereas precocene suppresses the HM-induced increase in egg laying. A 20E analog similarly enhances oviposition. Consistent with the role of JH in reproductive tissues, activation of the JH pathway correlates with increased expression of extracellular matrix genes in the ovarian muscle. Collectively, these results demonstrate that lysine acts as a critical nutritional cue to activate the 20E–ETH–JH endocrine axis and acutely promote egg laying behavior in D. melanogaster over a short time period. This provides a defined framework for investigating the short-term nutrient hormone regulation of ovulation and offers a practical strategy for efficient egg collection.