Extracellular β-glucan induces trained immunity in macrophages and is associated with anti-microbial defense in teleosts.

β-Glucans are widely employed as immunomodulators in the aquaculture industry and have recently been recognized for their capacity to induce trained immunity. However, most β-glucans currently used in aquaculture are derived from fungal or yeast cell walls through complex extraction processes, yielding heterogeneous products that limits both mechanistic consistency and practical applicability. Here, we evaluated the potential of an extracellular β-glucan (eBG) derived from Aureobasidium melanogenum to induce trained immunity in both macrophages and zebrafish models, and further found that macrophages are evolutionarily conserved cellular components associated with eBG-induced anti-bacterial protection. In addition, we proved that eBG-based immunostimulant could be applied to enhance disease resistance in turbot (Scophthalmus maximus). Most importantly, we compared different administration strategies and found that an intermittent "training-resting-retraining" strategy significantly outperformed continuous feeding in improving survival, alleviating clinical symptoms, and maintaining immune organ homeostasis following bacterial challenge. Our findings provide both mechanistic insights and practical guidance for developing eBG as a trained immunity-based immunostimulant in aquaculture.