Visualizing the 3D morphological structure and dynamic defensive characteristics of teleost mucus barrier through an ex vivo approach in flounder (Paralichthys olivaceus).

The fish mucus barrier serves as the first line of defense against pathogen invasion, and its structural and functional changes are key indicators for assessing fish health. However, due to limitations in in situ observation techniques, the three-dimensional (3D) architecture and dynamic barrier functions of the fish mucus layer have long remained poorly understood. In this study, using teleost flounder (Paralichthys olivaceus) as a model, we established an integrated approach combining ex vivo tissue culture with lectin fluorescence labeling and confocal laser scanning microscopy, which maintained high cell viability within 6 h, achieving high-fidelity 3D reconstruction of the mucus layer on the gills, hindgut, and skin, and revealed tissue-specific differences in mucus layer thickness, surface area, ultrastructure, and 3D morphology. Using fluorescent microbeads (FMBs) of varying diameters, we demonstrated that the mucus layer functions as a size-dependent sieving barrier. By simulating immersion exposure to inactivated and live Edwardsiella tarda, and 1 μm FMBs, we further elucidated the differential handling of distinct particles by the mucus layer during 1 h: inactivated bacteria were partially retained within the mucus network, partially penetrated to the epithelial surface, and partially cleared via shedding of mucus clumps; live bacteria, although partially captured, progressively traversed to the epithelial surface while triggering mucus layer elevation and shedding of bacteria-containing mucus clumps; while 1 μm microbeads rapidly penetrated but subsequently became anchored within the mesh, showing minimal further displacement and rarely undergoing clearance. These results demonstrated that the mucus layer acted as a dynamic barrier system, exerting its defensive function through the synergistic action of physical entrapment and active clearance and at the same time promoting the adhesion of the biotic/abiotic particles. This study provides new insights into the structural and dynamic defensive functions of the teleost mucus barrier.