Introduction
Gill structural integrity and functionality are essential for fish survival, performance, and overall fitness in wild and aquaculture environments. Inside open sea cages, farmed Atlantic salmon are vulnerable to a variety of gill-damaging stressors, such as jellyfish, harmful algae blooms, farm management practices, and infection. Gill lesions and diseases have led to significant economic losses in Atlantic salmon aquaculture globally.
Fortunately, recent findings show that Atlantic salmon gill can regenerate following damage, restoring respiratory and ion regulatory functions (Ghanizadeh-Kazerouni et al., 2024) . Building on this discovery, the present study investigates transcriptomic changes in regenerating gill filaments over 20 weeks following resection.
Materials and Methods
Post-smolt Atlantic salmon maintained in freshwater were lightly anaesthetised with MS-222 and subjected to gill filament resections of either 30% or 50% of filament length. Control fish underwent anaesthesia without resection. Fish from the Control and Resected groups were terminally sampled at 1, 2, 4, 8, 12, 16, and 20 weeks post-resection to collect intact and resected filaments, respectively. T he 50% resected filament group was chosen for RNA sequencing as that group was expected to show more robust transcriptomic responses.
Four to seven filaments per condition and time point were processed for total RNA isolation and submitted to Canada’s Michael Smith Genome Sciences Centre (Vancouver, BC) for library preparation and sequencing on an Illumina NovaSeq 6000 (PE150, targeting ≥50 million reads per library). Raw data were quality-checked and analysed using an established bioinformatics pipeline (Eslamloo et al., 2022), and differential expression analyses were performed with DESeq2 comparing Control and Resected groups at each time point, using an adjusted p-value < 0.01 and requiring expression in at least half the replicates of at least half the groups.
Results and Discussion
During the trial, gill filament regeneration became evident by week 8 and reached an average of 34.2% regrowth by week 20, accompanied by restored Na⁺/K⁺-ATPase activity (Ghanizadeh-Kazerouni et al., 2024) . In parallel, t ranscriptomic differences between Resected and Control filaments diminished as healing and regeneration progressed, with 2,574 differentially expressed genes (DEGs) at week 1 post-resection, 970 DEGs at week 2, 782 DEGs at week 4, 610 DEGs at week 8, 526 DEGs at week 12, 216 DEGs at week 16, and 273 DEGs at week 20.
A h ierarchical clustering analysis helped identify distinctive regulation patterns over time among the DEGs, linked to specific biological processes via Gene Ontology enrichment analyses . Inflammation-related transcripts were markedly elevated at week 1 but declined rapidly thereafter. Genes involved in cell proliferation, migration, and differentiation—including pluripotent, epithelial, and neuroepithelial cells—were also highly expressed in early stages (weeks 1 & 2), consistent with immunohistochemical observations (Ghanizadeh-Kazerouni et al., 2024). A similar temporal pattern was observed for genes related to angiogenesis, mesenchymal cell transition, cytoskeletal organisation, and cell junction assembly.
Notably, up-regulated DEGs associated with cellular energy metabolism and organ development displayed a progressive rise in transcript levels between weeks 1 and 8 —coinciding with the occurrence of blastema-like structures (Ghanizadeh-Kazerouni et al., 2024)— followed by a decline and stabilisation by week 16. These patterns suggest a phased regeneration process, beginning with rapid immune resolution and activation of progenitor cells, followed by tissue patterning and structural remodelling.
DEGs with reduced transcript levels in the Resected filaments exhibited a progressive—though often incomplete—return towards Control-like expression over time . T he most robustly over-represented biological processes among these genes included ion transport (e.g. chloride, potassium, and sodium), synaptic signalling and potentiation, and lipid metabolism—processes critical in establishing functionality.
Interestingly, a distinct cluster of downregulated genes showed transcript levels rising rapidly after week 2 , surpassing those in Control filaments by week 4. This cluster was enriched for fibroblast growth factor (FGF) signalling and associated processes , including glycosaminoglycan metabolism, cartilage development, extracellular matrix (ECM) components, and ECM remodelling. This pattern might reflect parallel recruitment and activity of fibroblasts and chondrocytes during structural remodelling, supporting the re-establishment of filament architecture.
In summary, our RNA sequencing results describe the transcriptomic changes underlying gill filament regeneration in Atlantic salmon as a phased process, including immune resolution, progenitor cell activation, cell proliferation, and tissue remodelling and development. Further analyses will focus on genes correlated with regeneration rates to identify molecular drivers and potential markers of successful tissue restoration.
References
Eslamloo, K., Kumar, S., Xue, X., Parrish, K.S., Purcell, S.L., Fast, M.D. and Rise, M.L. 2022. Global gene expression responses of Atlantic salmon skin to Moritella viscosa. Scientific Reports 12.1 (2022): 4622.
Ghanizadeh-Kazerouni, E., Wilson, J.M., Jones, S.R.M. and Brauner, C.J. 2024. Characteristics of a gill resection – Regeneration model in freshwater laboratory-reared Atlantic salmon ( Salmo salar ). Aquaculture 579, 740210.