Unexpected enrichment of antibiotic resistance genes in recirculating aquaculture effluents of rainbow trout (Oncorhynchus mykiss): microbial community assembly and mobile genetic elements as key drivers.

INTRODUCTION: Recirculating aquaculture systems (RAS) are increasingly used in intensive aquaculture because of their capacity to reduce nitrogen, phosphorus, and organic matter discharges. However, whether improved conventional effluent quality is accompanied by a reduced antibiotic resistance gene (ARG) burden remains unclear. METHODS: During the mid-cultivation stage, effluents were collected from four RAS farms and four paved flow-through raceway systems (PFRS). Water quality parameters, including total nitrogen (TN), total phosphorus (TP), chemical oxygen demand (COD), chlorophyll-a (Chl-a), and dissolved oxygen (DO), were measured. Microbial community composition was characterized using 16S rRNA gene sequencing, while ARGs and mobile genetic elements (MGEs) were quantified using GeoChip 5.0. Multivariate statistics and co-occurrence network analyses were used to evaluate associations among environmental conditions, microbial communities, ARG profiles, and MGE profiles. RESULTS: RAS effluents showed substantially lower nutrient and organic loads than PFRS, confirming improved conventional water quality. However, total ARG abundance was significantly higher in RAS than in PFRS, increasing from 35.39 ± 11.43 × 106 to 63.99 ± 15.33 × 106, corresponding to a 1.81-fold increase. In contrast, total MGE abundance was numerically higher in RAS but did not differ significantly between systems. Microbial alpha diversity was lower in RAS, and beta diversity analysis showed distinct community structuring between cultivation modes, with both compositional differences and dispersion effects contributing to group separation. ARG class- and subtype-level profiles showed heterogeneous abundance patterns, with several major ARG categories remaining highly abundant in RAS, although most feature-level differences were not significant after FDR correction. Co-occurrence network analysis further revealed structured associations among microbial species, ARGs, and MGEs, with modular organization and highly connected resistance-associated nodes. CONCLUSION: This study demonstrates a "low-pollution but non-reduced ARG burden" pattern in RAS effluents. Improved removal of conventional pollutants did not correspond to a proportional reduction in ARG or MGE profiles. These findings indicate that conventional water-quality indicators alone are insufficient to evaluate resistance-associated genetic pollution in engineered aquaculture systems, and that ARGs, MGEs, microbial community structure, and species-ARG/MGE association patterns should be integrated into future RAS monitoring and management.