Effects of Different Application Methods of Copper-Loaded Montmorillonite on Growth Performance, Antioxidant Capacity, Gene Expression, and Intestinal Microbiota of Penaeus monodon

Penaeus monodon is a major marine aquaculture species; however, production intensification has increased water-quality deterioration and disease pressure. Copper-loaded montmorillonite (Cu-MMT) is a functional clay additive with adsorption and antimicrobial properties, yet the optimal application mode remains unclear. We compared a control (KZ), water application (PZ), and dietary inclusion (BZ) of Cu-MMT in P. monodon. BZ was associated with higher survival and a numerically higher specific growth rate, whereas final body weight did not differ among treatments. Antioxidant status improved in BZ, with higher catalase (CAT) and total superoxide dismutase (T-SOD) activities (both p < 0.05). Hepatopancreas RNA-seq identified 949 differentially expressed genes (DEGs) for KZ vs. PZ and 814 DEGs for KZ vs. BZ. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses showed that PZ was enriched for redox processes, transporter activity, and amino-acid biosynthesis—indicative of a stress-defense state—whereas BZ was enriched for proteolysis, endoplasmic-reticulum protein processing, and proteasome pathways, consistent with an anabolic, protein-quality-control–oriented mode. Intestinal 16S rRNA profiling indicated higher diversity and reduced putative pathogens in BZ. Overall, dietary Cu-MMT is the preferred application, shifting shrimp from an energy-consuming stress response to efficient anabolism and thereby improving performance and survival.