The need for sustainable and efficient aquaculture production to meet global food demand has led to the development of new technologies, such as recirculating aquaculture systems (RAS). In recent years, advancement in Norwegian land-based aquaculture has been highlighted by the implementation of RAS to produce Atlantic salmon (Salmo salar ) in a more controlled environment, thereby addressing recurrent issues such as diseases and escapes. Despite its promise of a superior rearing environment compared to that of the traditional flow-through systems, several challenges have presented themselves, one of which is hydrogen sulphide (H2S)-related mass mortalities . Currently, we have a significant knowledge gap on the physiological mechanisms of how Atlantic salmon respond to prolonged exposure to H2S .
M ucosal organs such as gills, skin and olfactory organs represent the primary barrier against noxious compounds present in the aquatic environment. They can mount strong immune responses against immunotoxicants, including H2S . Previously we have shown that mucosal organs of Atlantic salmon exhibit differential sensitivity to transient exposure to H2S. Also, d istinct molecular and structural alterations have been observed in these organs after transient exposure to H2S. However, whether these organs exhibit the same sensitivity to prolonged sub-lethal H2S exposure remains to be elucidated . Therefore, this study investigated how prolonged exposure to H2 S could impact the barriers at mucosal sites of Atlantic salmon.
Materials and Methods
A group of Atlantic salmon post-smolts (35 ppt) were reared under three H2 S level conditions – 0 µM/L (Control), 0.05±0.02 µM/L (Low) and 0.12 ± 0.02 µM/L (High) – for 12 days. Each treatment group had 3 replicate tanks (Figure 1). After the exposure period, s amples from the gills, skin and olfactory organ were collected for gene expression analysis, histology and RNAscope/in situ hybridisation. Moreover, mucus samples from the gills and skin were also collected for proteomics analysis.
Results and Discussion
The prolonged exposure to H2 S led to changes in gene expression, mainly affecting genes crucial for stress response, xenobiotic detoxification and immunity (Figure 2). Multi-tissue a nalysis revealed that the gills and olfactory organs were highly sensitive to H2 S where more than half of the genes analys ed were altered by both H2 S doses . The expression of these marker genes showed minimal variations in the skin. These transcriptional profiles were not in agreement with previous transient exposure studies suggesting that the differential sensitivity of mucosal organs to H2 S was influenced by the duration of exposure. Mucosal structural integrity remained favourable following H2 S exposure since histological and morphological analyses did not show any relevant changes in H2S-exposed fish compared with the control group. RNAScope identified and localised the transcripts important for sulphide detoxification in mucosal organs, including suox , sqor1 , and sqor2. High throughput proteomics of gill and skin mucus revealed significant changes in mucosal proteome following H2S exposure. 129 and 160 proteins were differentially altered in gill and skin mucus, respectively. Reactome pathway analysis revealed significant alterations in ribosomal processes and reactive oxygen species detoxification in the skin mucus, while drug metabolism was affected in gill mucus. Gene Ontology analysis further elucidated that some of the substantially affected proteins were crucial for toll-like 9 signalling and hydrogen peroxide catabolism, while affected proteins in gill mucus were important for lipoprotein metabolic process and regulation of endocytosis.
Overall, the results demonstrate that prolonged exposure to sub-lethal doses of H2 S altered the mucosal defences of Atlantic salmon. The mucosal changes were reflected in the gene and protein expression profiles of molecules important for stress, immunity and xenobiotic metabolism. Nonetheless, H2 S exposure revealed no substantial structural changes in mucosal organs. These results indicate that Atlantic salmon are capable of coping with low levels of H2S , but it remained to be investigated whether this inherent mechanism at mucosal sites can persist for longer period . The study significantly advances our understanding of how Atlantic salmon interacts with H2S at mucosal surfaces.
This work was supported by the Norwegian Research Council No. 300825 and Erasmus Mundus Joint Master Degree Scholarship.