Abstract

Melanised focal changes (MFCs) in the skeletal muscle of Atlantic salmon (Salmo salar) represent an increasing quality concern in aquaculture, as they are associated with chronic inflammation, pigment accumulation, and significant economic losses. Although the histopathological features of MFCs have been extensively described, their underlying molecular mechanisms remain poorly understood. In this study, we employed an integrative approach combining transcriptomic profiling, oxidative stress evaluation, apoptosis assessment, and in vitro functional assays to elucidate the pathways involved in MFCs development in salmon flesh. RNA-seq analysis revealed 2084 upregulated transcripts in the MFC group, primarily enriched in pathways related to cytokine-cytokine receptor interaction, NOD-like receptor signaling, and C-type lectin receptor signaling. Conversely, 337 transcripts were downregulated, predominantly associated with autophagy, insulin signaling, and mTOR signaling pathways. RT-qPCR validation of selected differentially expressed transcripts (DETs), along with oxidative damage measurements and western blot analysis of caspase-cleaved proteins, confirmed elevated oxidative stress, inflammation, and apoptosis in MFC-affected muscle. To further evaluate the role of TNF-alpha in mediating oxidative stress in skeletal muscle, we conducted an in vitro assay using S. salar myotubes. Exposure of myotubes to recombinant Atlantic salmon TNF-alpha induced a dose-dependent increase in reactive oxygen species (ROS) production via NF-kappa B activation, leading to elevated transcript expression of Tumor necrosis factor receptor (tnfr1). Our results support a model in which MFCs arise from persistent oxidative stress and localized inflammation, culminating in chronic immune activation, apoptosis, and pigment accumulation in skeletal muscle. This study advances our understanding of the signaling pathways involved in muscle melanisation and identifies potential molecular targets for mitigation strategies in salmonid aquaculture.