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

## TL;DR

Adding copper-loaded montmorillonite to shrimp diets improves their growth, antioxidant activity, and gut health compared to other application methods.

## Contribution

This study identifies dietary inclusion as the optimal application method for Cu-MMT in shrimp, based on gene expression and microbiota changes.

## Key findings

- Dietary Cu-MMT (BZ) improved shrimp survival and antioxidant enzyme activities compared to control and water application.
- BZ treatment shifted gene expression toward anabolic processes and protein quality control, unlike water application (PZ).
- BZ increased gut microbial diversity and reduced potential pathogens compared to other treatments.

## Abstract

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.