Temporal Associations Between Serotonin‐Related Signals and Feeding Rhythm in Red Swamp Crayfish ( Procambarus clarkii ) Reared in Recirculating Aquaculture Systems: Insights From Untargeted Metabolomics

Recirculating aquaculture systems (RAS) permit precise environmental control, but high‐density culture may intensify competition for feed in red swamp crayfish ( Procambarus clarkii ). Serotonin‐related signals are potentially involved in feeding regulation, but how they vary with feeding over a 24 h postfeeding cycle in this species under RAS conditions remains unclear. This study therefore characterized feeding behavior and explored feeding‐associated metabolic signals. A total of 120 crayfish were assigned to three 350 L tanks (40 crayfish per tank). Feeding events were recorded hourly for seven consecutive days. Based on the observed peaks and troughs, six crayfish per tank were sampled at 1, 4, 9, 13, 16, and 24 h after feeding (18 crayfish per time point; three tank‐level biological replicates). Serum was analyzed by untargeted metabolomics, and serotonin concentration and serotonin receptor mRNA expression were measured in neuroendocrine tissue. Metabolites were screened using an unadjusted p   < 0.05 and a variable importance in projection (VIP) value > 1.0; no false‐discovery‐rate correction was applied, so these findings are exploratory. Feeding frequency showed peaks at T1, T9, and T16 and troughs at T4, T13, and T24. Among 427 putative differential metabolites, serotonin had a VIP value of 2.3459 and was positively, but not significantly, correlated with feeding frequency ( r  = 0.78, p = 0.067). Serotonin‐related pathways, including tryptophan metabolism and serotonergic synapse, were enriched, and temporal variation in neuroendocrine serotonin and 5htr2a expression was observed. These exploratory and associative findings nominate serotonin‐related signaling, including 5‐HT2A, as a candidate pathway associated with the postfeeding temporal pattern. Functional perturbation studies are required to establish causality and receptor mediation.