Pre-molt cholesterol regulation of ecdysone receptor (EcR) signaling in the Y-organ contributes to dietary control of molting in Scylla paramamosain.

Molting death syndrome (MDS), characterized by incomplete ecdysis and failed shell hardening, severely constrains mud crab (Scylla paramamosain) aquaculture. Cholesterol is an essential precursor for ecdysteroid synthesis, but its stage-specific roles and endocrine targets during the molt cycle remain unclear. Here we examined how dietary cholesterol and ecdysone receptor (EcR) signaling interact to regulate molting physiology and MDS risk. Juvenile crabs were fed a basal diet (0% cholesterol added) or a diet supplemented with 0.75% cholesterol (air-dry basis) and followed through a complete molt cycle, while hemolymph hormones, chitinolytic enzymes and expression of cholesterol-transport and steroidogenic genes/proteins in the Y-organ and hepatopancreas were analyzed across molt stages. Pre-molt in vivo dsEcR interference and 20-hydroxyecdysone (20E) injection were used to test EcR dependence. Isolated Y-organ cultures were used to monitor genes and proteins involved in ecdysone biosynthesis and the EcR signaling cascade, whereas Drosophila S2 cells were used to assess EcR cascade-related genes and proteins, cholesterol metabolism-related proteins, and EcR immunofluorescence and subcellular localization. Cholesterol supplementation increased molting rate, selectively enhanced pre-molt hemolymph ecdysteroids, and up-regulated Y-organ cholesterol-trafficking and ecdysteroidogenic genes, together with EcR/RXR/BR-C signaling and CHIT/NAG/PO activities. Pre-molt dsEcR knockdown largely abolished cholesterol- and 20E-induced endocrine and transcriptional responses, caused Y-organ ultrastructural damage and reduced EcR expression, and triggered MDS arrest at pre-molt substages. In vitro, cholesterol directly stimulated the expression of genes involved in ecdysone activation and degradation (nvd, dib, and cyp18a1) and enhanced the expression of EcR-mediated molting response markers, including ecr/EcR, br-c/BR-C, and ftz-f1, in isolated Y-organs. In Drosophila S2 cells, the EcR agonists 20-hydroxyecdysone (20E) and methoxyfenozide (MEO) primarily activated the EcR-dependent molting pathway (ecr/EcR, br-c/BR-C, usp, e74, e75, hr4) and EcR immunofluorescence, whereas cholesterol mainly induced lysosomal and sterol-trafficking pathways (NPC1, Lamp1). Overall, our findings suggest that moderate dietary cholesterol improves molting performance, potentially by enhancing pre-molt cholesterol delivery to the Y-organ and promoting EcR-dependent molting cascades. These results provide mechanistic insight into how dietary cholesterol availability may influence molting regulation.