Intestinal adaptation of crayfish to pond and rice paddy farming: a microbiome-enzyme perspective

The red swamp crayfish ( Procambarus clarkii ) is a globally significant aquaculture species, yet the biological trade-offs between intensive pond monoculture and integrated rice-crayfish co-culture systems remain poorly understood at the intestinal level. This study investigated the intestinal physiological and microbial adaptations of P. clarkii reared in pond (high-input formulated feed) and rice paddy (low-input natural diet) systems for 60 days. We integrated analyses of intestinal digestive, antioxidant, immune barrier, and energy metabolism enzymes with 16S rRNA sequencing of the gut content-associated microbiota. Rice paddy-cultured crayfish exhibited significantly higher amylase activity, enhanced antioxidant enzyme activities (SOD, CAT, GSH-Px), and lower oxidative damage (MDA), alongside higher immune barrier enzyme activities (AKP, ACP) and reduced Na + /K + -ATPase activity compared to pond-cultured crayfish. Conversely, pond-cultured crayfish showed elevated lipase and total protease activities, lower immune barrier enzyme activities, and greater energy transport workload. Microbial analysis of intestinal lumen contents revealed higher lumen-associated microbial diversity in the rice paddy group, with enrichment of Bacteroidota and taxa commonly associated with polysaccharide degradation, whereas the pond group showed enrichment of Pseudomonadota (formerly Proteobacteria) and taxa that include potential opportunistic pathogens such as Citrobacter and Acinetobacter . BugBase phenotype prediction indicated higher abundance of “Potentially pathogenic” phenotypes in the pond group. These findings suggest that the two aquaculture systems are associated with distinct, coordinated intestinal adaptation patterns: a “stress-resistance-oriented” phenotype in rice paddies characterized by enhanced antioxidant defense, efficient carbohydrate utilization, lower physiological burden, and a more diverse lumen-associated microbiota with greater abundance of potentially beneficial taxa, versus a “growth-oriented” phenotype in ponds characterized by elevated metabolic load, oxidative stress, altered immune status, and opportunistic pathogen enrichment. This study provides a basis for further hypothesis-driven research and may help inform sustainable aquaculture practices, balancing productivity with animal health and welfare.