Chlorella-mediated methionine-copper bioencapsulation in Artemia nauplii modulates the Keap1/Nrf2 antioxidant axis, innate immunity, and appetite regulation in hybrid grouper larvae

Copper (Cu) serves as an essential trace element and obligate cofactor of Cu/Zn superoxide dismutase and ceruloplasmin; however, its nutritional delivery to marine fish larvae via live prey has not been fully investigated. This study assessed the dose-response effects of trophic Cu transfer on hybrid grouper ( Epinephelus fuscoguttatus ♀ × E. lanceolatus ♂) larvae over a 15-day period, employing a three-tier bioencapsulation system where Chlorella was primarily enriched with methionine-copper (Met-Cu; 0, 5, 10, 15, and 20 mg/L) and delivered via Artemia nauplii. The Cu content in enriched Artemia ranged from 173.92 to 606.68 mg/kg dry weight (DW), resulting in dose-dependent increases in larval whole-body Cu (140.9–178.71 mg/kg DW). Larvae fed with 15 mg/L Met-Cu enrichment (Cu15) exhibited peak survival (71.67 ± 2.08%), final body weight (250.37 ± 43.58 mg), and specific growth rate (14.17 ± 2.97%/d). This optimal dose significantly enhanced digestive capacity (pepsin, trypsin, lipase, and amylase) and antioxidant enzyme activities (GSH-Px, CAT, and SOD), while concurrently minimizing malondialdehyde (MDA) accumulation. Gene expression analysis suggested activation of the Keap1/Nrf2 pathway in the Cu15 treatment, leading to the upregulation of downstream antioxidant genes ( CAT , GPX , GR , LZM ). A balanced innate immune response was achieved, exemplified by upregulation of IL-1β , IL-8 , and IL-10 , alongside suppression of TNF-α and TGF-β . Additionally, optimal Cu enrichment influenced appetite regulation by downregulating anorexigenic signals ( CGRP , NUCB2 ) and upregulating the orexigenic receptor NPY8R . Conversely, 20 mg/L Met-Cu induced signs of Cu toxicity, as indicated by reduced Nrf2 expression, decreased SOD activity, and increased TNF-α levels. In conclusion, 15 mg/L Met-Cu represents the optimal Artemia enrichment concentration for hybrid grouper larviculture. This study offers new mechanistic insights into the effects of live-prey Cu biofortification on antioxidant signaling, innate immune homeostasis, and neuroendocrine appetite control.