Study on the Diversity of the <scp>CXCL8</scp> Family in <i>Carassius auratus</i> : Homologue Characteristics and Tissue‐Specific Immune Responses Under <i>H. doneci</i> Infection

Chemokines are essential for vertebrate immune regulation, and teleosts possess diverse CXCL8 lineages. However, studies on CXCL8 in C. auratus remain limited. In the present study, four CXCL8 homologues (CaCXCL8-1 to -4) were successfully cloned and identified from C. auratus, and their expression patterns were analysed in the tissues of healthy and those naturally infected with H. doneci. Bioinformatic analyses revealed that all homologues possess the conserved structural features of CXC chemokines. However, the divergence was observed in the N-terminal ELR motif: CaCXCL8-1 and CaCXCL8-2 harboured a QLR motif, whereas CaCXCL8-3 and CaCXCL8-4 possessed a DPR motif. Phylogenetic analysis classified CaCXCL8-1 and CaCXCL8-2 into the CXCL8_L2 lineage, and CaCXCL8-3 and CaCXCL8-4 into the teleost-specific CXCL8_L1b lineage. Tertiary structure predictions indicated that CaCXCL8-1, -3, and -4 conserve key receptor-binding sites, whereas CaCXCL8-2 retained only GAG-binding sites, suggesting potential functional divergence. Under healthy conditions, the four homologues were all constitutively expressed but displayed divergent basal profiles. Following H. doneci infection, they exhibited distinct tissue-specific expression patterns: CaCXCL8-1 was significantly upregulated in the gills; CaCXCL8-2 was specifically induced in the spleen; CaCXCL8-3 was markedly upregulated in both the gills and head kidney; and CaCXCL8-4 was upregulated in the liver and head kidney but downregulated in the spleen. These divergent expression patterns suggest functional specialisation among the CaCXCL8 homologues, with different paralogs potentially coordinating localised versus systemic immune responses. Our findings provide evidence for the functional diversification of the CXCL8 chemokine in teleosts and contribute to understanding the evolution of complex immune systems in vertebrates.