Clinically healthy Japanese flounders (average weight 16.5 ± 2.5 g) were purchased from a commercial fish farm in Shandong Province, China, and maintained at 22–24 °C in aerated seawater. Before the experiments, the fish were acclimatized in the laboratory for two weeks and verified to be free of pathogens in the liver, head kidney, and spleen, as reported previously [33]. For tissue collection, the fish were euthanized with tricaine methanesulfonate (Sigma-Aldrich Corporation, St. Louis, MO, USA), as reported previously [34].

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