Introduction
Rising summer sea temperatures, driven by climate change, pose a significant challenge to the salmon aquaculture industry. Prolonged exposure to temperatures above the optimum range adversely impacts appetite, growth, metabolism, and disease resistance. Although the physiological effects of thermal stress on fishes are well-documented, there is limited knowledge on the molecular mechanisms underlying the thermal stress response in the fish brain. The brain, as the central organ regulating the stress response through the hypothalamus-pituitary-interrenal (HPI) axis and the brain-sympathetic-chromaffin cell (BSC) axis, is highly sensitive to environmental stressors. Moreover, it is a complex organ comprising distinct regions with specific functions, including growth, reproduction, cognition, movement, and metabolism. Investigating various brain regions of salmon exposed to thermal stress conditions would provide insights into region-specific and common stress adaptation mechanisms. Therefore, in this study, we investigated the thermal stress impacts on cerebellum (CBE), hypothalamus (HYP), medulla oblongata (MED), optic tectum (OPT), pituitary gland (PIT), and telencephalon (TEL) of salmon using quantitative proteomics . Additionally, we examined the proteomic profiles of two immune organs, the head kidney (HK) and spleen (SP), to elucidate their roles under thermal stress conditions.
Materials and methods
A total of 150 Atlantic salmon post-smolts (mean weight: ~300 g) were randomly allocated to six 500 L seawater flow-through tanks. Three tanks were assigned to the control group ( maintained at 15°C) and three to the thermal stress (TS) treatment (temperature increased by 1°C over four weeks and then maintained at 19°C for four weeks). The dissolved oxygen satu ration was maintained at 100 % ± 5 % throughout the experiment in both control and TS treatment tanks. At the end of the experiment, six distinct brain regions -CBE, HYP, MED, OPT, PIT , and TEL were collected from six fish per treatment. Additionally, HK and spleen SP were collected from five fish per treatment. Proteomic analysis of samples was conducted using Liquid Chromatography-Mass Spectrometry (LC-MS/MS). Follow ing LC-MS/MS analysis, statistical analysis (p- value <0.05, f old change > ± 1.5) were performed to identify differentially expressed proteins (DEPs) between the control (15°C) and TS group (19°C). Finally, bioinformatics tools (DAVID & ClueGO) were used for functional enrichment analysis of the DEPs to identify gene ontology terms and KEGG pathways.
Results
An average of over 9000 proteins were identified across the six brain regions. A comparative analysis of DEPs indicated that only two proteins were common across all regions, with the majority of DEPs being unique to specific regions. SERPIN H1 (heat shock protein 47), a well-validated thermal stress biomarker was found to be upregulated in the TEL, HYP, and PIT regions of the TS group. Proteins related to several key pathways and processes, such as amino acid metabolism and protein folding in PIT; endopeptidase activity in TEL, HYP, and PIT; and cardiac muscle contraction, RNA binding, and glucose metabolism in MED, were upregulated. Furthermore, in most regions except MED and OPT, proteins involved in mRNA processing , cytoplasmic translation, spliceosome, RNA binding, and ribosome were downregulated.
In HK and SPL, the molecular chaperones SERPIN H1 and HSP90a were upregulated under TS conditions. Bioinformatics analysis revealed that upregulated proteins in HK were enriched in unfolded protein binding and protein processing in the endoplasmic reticulum. In the spleen, notable changes were observed in proteins related to lipid metabolism and protein degradation. Consistent with the brain regions, proteins downregulated in both the spleen and HK were linked to pathways and processes associated with translation and transcription.
Conclusion
This study represents the first investigation of the thermal stress response of distinct brain regions (CBE, HYP, MED, OPT, PIT, and TEL) in Atlantic salmon at the proteome level. The differential expression analysis revealed that protein expression varies among regions in response to sub-optimal water temperatures, suggesting that each region may possess a unique adaptation mechanism to cope with thermal stress. Notably, the upregulation of SERPIN H1 across multiple brain regions, as well as in HK and SPL, underscores its critical role in protecting cells from thermal stress-induced damage. Additionally, the downregulation of the transcription and translation machinery in all tissues indicates a possible reduction in the efficiency of the splicing process.
Overall, this proteomic study provides an enhanced understanding of the tissue-specific molecular mechanisms underlying thermal stress adaptation in salmon . These insights have the potential to support the industry in enhancing the resilience of salmon populations to increasing water temperatures induced by climate change . Furthermore, our region-specific brain proteome analysis may serve as a foundational basis for future research investigating the role of distinct brain regions on various physiological processes including behaviour, cognitive function, appetite regulation, energy metabolism, and neuroendocrine responses under varying environmental conditions.