Contrasting Roles of Mobile Genetic Elements and Metal Resistance Genes in Shaping the Gut Resistome of Wild Fish from the Qiantang River

The dissemination of antibiotic resistance genes (ARGs) in riverine ecosystems poses a pressing public health threat, while the mechanisms governing the assembly of the gut resistome in wild fish remain poorly elucidated. This study aimed to elucidate the distributional patterns of ARGs across multiple environmental compartments and to identify factors associated with their variation, particularly the contributions of mobile genetic elements (MGEs) and metal resistance genes (MRGs) to gut resistome variation. Metagenomic sequencing was conducted on 60 samples, comprising water, sediment, and gut contents from three wild fish species (Megalobrama terminalis, Aristichthys nobilis, and Coilia nasus) with distinct feeding habits, collected from four reaches of the Qiantang River basin. A total of 305 ARG subtypes belonging to 23 classes were identified. ARG composition differed significantly across environmental media and host species (permutational multivariate analysis of variance, PERMANOVA; p < 0.01), with host species identity as the primary structuring factor. Variance partitioning analysis (VPA) revealed that MGEs independently explained the largest fraction of ARG variation in A. nobilis (33.8%, p = 0.006), whereas MRGs dominated in C. nasus (33.3%, p = 0.005); in M. terminalis, MGEs and MRGs together accounted for 47.9% of the variation. Metagenomic assembly recovered 2622 ARG-carrying contigs, of which 28.3% (743) were predicted as plasmid sequences; physical co-localization among ARGs, MGEs, and MRGs was detected on both chromosomes and plasmids. Metagenomic binning validated the physical co-localization of ARG-MGE-MRG modules in genera such as Morganella and Burkholderia at the genome level, while plasmid-borne high-risk ARGs were identified in Aeromonas. Risk ranking further revealed significant enrichment of Rank II potentially high-risk ARGs (e.g., mcr-7.1, blaZ) in fish guts, carried by potential pathogens. These findings suggest that horizontal gene transfer involving MGEs and co-selection related to MRGs are closely associated with the fish gut resistome composition in a manner dependent on host ecology, providing a scientific basis for shifting riverine resistance management from concentration-based control toward the interruption of dissemination pathways.