Introduction
Smoltification represents a critical developmental transition in Atlantic salmon (Salmo salar) aquaculture, with successful adaptation to seawater directly impacting production outcomes. Despite its importance, the molecular mechanisms underlying this complex physiological transformation remain incompletely understood. This study employed a comprehensive proteomic analysis to elucidate the temporal progression of protein expression changes during smoltification to inform targeted nutritional strategies.
Methods
Blood samples were collected from a single cohort of Atlantic salmon at four developmental stages: pre-smolt, smolt, 3-week post-smolt, and 3-month post-smolt. Proteomic analysis identified ~ 3,500 proteins per sample, with 2,588 proteins retained for differential expression analysis. Principal Component Analysis (PCA) was performed to distinguish developmental stages based on proteome signatures. Weighted correlation network analysis (WGCNA) identified protein modules with stage-specific expression patterns. Gene Ontology (GO) and KEGG pathway enrichment analyses characterised the biological processes and pathways altered during each developmental transition.
Results
PCA revealed distinct proteome signatures for each developmental stage, with PC1 (11.1%) primarily separating pre-smolt/smolt from post-smolt stages (Fig. 1). WGCNA identified seven distinct protein modules with stage-specific expression patterns showing moderate to weak correlations (-0.16 to 0.12) with developmental stages, indicating complex regulatory networks underlying the smoltification process. Six modules demonstrated highly significant (p <0.001) differences in expression across developmental stages.
The pre-smolt to smolt transition was characterised by extensive transcriptional reprogramming, with significant enrichment of RNA splicing (FDR < 1.0e-45) and spliceosome pathways (FDR < 1.0e-40). This stage also showed activation of necroptosis and NOD-like receptor signalling pathways, indicating immune system involvement in initial adaptation. The MEgreen module exhibited the highest expression in smolt stage (0.122 ±0.077), suggesting involvement in smoltification-specific processes.
The smolt to 3-week post-smolt period revealed marked upregulation of translation (FDR < 1.0e-64) and ribosomal processes (FDR < 1.0e-85), indicating intensive protein synthesis. This transition was characterised by significant enrichment of carbon metabolism, oxidative phosphorylation, and tight junction pathways essential for osmotic regulation. The MEbrown module peaked at the 3-week post-transfer stage (0.095 ±0.022), showing inverse correlations between pre-smolt (-0.15) and post-transition stages, suggesting involvement in seawater adaptation processes.
The final transition to 3-month post-smolt involved upregulation of protein quality control mechanisms, including protein folding and regulation of proteolysis (FDR < 1.0e-11). Phagosome pathways showed the highest enrichment (FDR < 1.0e-11), alongside necroptosis and protein processing in the endoplasmic reticulum. The MEred and MEyellow modules showed the strongest positive correlation with the 3-month post-transition samples (0.12), indicating involvement in long-term seawater adaptation mechanisms.
Discussion
This study reveals that smoltification requires substantial energetic investment and precise protein regulation, evidenced by coordinated expression of energy metabolism modules (MEyellow, MEred) and protein homeostasis modules (MEturquoise, MEgreen, MEbrown) across different developmental stages. The significant upregulation of translation and ribosomal processes during the smolt to post-smolt transition suggests this period represents a critical window where nutritional support for protein synthesis could enhance adaptation success. These findings align with previous studies reporting increased protein synthesis in Atlantic salmon during smoltification but provide a more comprehensive temporal characterisation of the process.
The concurrent activation of immune-related processes throughout smoltification, particularly complement activation and NOD-like receptor signalling pathways, suggests that immune system modulation is an integral component of the adaptation process rather than a secondary response. This finding has important implications for feed formulation, suggesting that immunostimulants or immune-supporting nutrients might be beneficial during smoltification. The enrichment of tight junction pathways during seawater transition further emphasises the importance of intestinal barrier function in osmotic adaptation, pointing to potential benefits of dietary components that support epithelial integrity. Additionally, the enrichment of cellular detoxification and stress response pathways in later stages suggests potential benefits from antioxidant supplementation during long-term seawater adaptation.
This proteomic analysis provides a detailed molecular framework for understanding smoltification in Atlantic salmon, highlighting critical periods of metabolic demand and protein synthesis that could be targeted with specific nutritional strategies. These findings may inform the development of stage-specific feed formulations to support the substantial energetic and metabolic requirements revealed during the smoltification process, potentially improving production outcomes in salmon aquaculture.