Molecular Adjuvant Potential of and in Starry Flounder Challenged with .

In fish, the innate immune system is crucial for rapid defense against pathogens. In this study, we performed transcriptome sequencing using next-generation sequencing (NGS) to identify and characterize granulocyte colony-stimulating factor () and macrophage colony-stimulating factor () in starry flounder (). The gene (594 bp, 198 aa) features a conserved IL-6 domain, while the gene (621 bp, 207 aa) contains a predicted transmembrane region. Phylogenetic analysis confirmed high evolutionary conservation with other marine species. Quantitative real-time PCR revealed that is highly expressed in the skin, peripheral blood leukocytes, and muscle, with significant up-regulation in immune organs following infection; exhibited a similar tissue-specific expression pattern. Recombinant () was produced using a cell-free system and effectively enhanced leukocyte phagocytic activity at an optimal concentration of 150 μg/mL, without causing cytotoxicity in hemolytic assays. In contrast, exhibited folding issues during purification. These findings highlight the potential of as a molecular adjuvant to enhance immune responses in aquaculture. This study provides foundational knowledge for developing cytokine-based adjuvants, which could reduce antibiotic dependency and enhance vaccine efficacy in sustainable aquaculture systems.