Identification of Hypoxia-Specific Biomarkers in Salmonids Using RNA-Sequencing and Validation Using High-Throughput qPCR

Abstract

Identifying early gene expression responses to hypoxia (i.e., low dissolved oxygen) as a tool to assess the degree of exposure to this stressor is crucial for salmonids, because they are increasingly exposed to hypoxic stress due to anthropogenic habitat change, e.g., global warming, excessive nutrient loading, and persistent algal blooms. Our goal was to discover and validate gill gene expression biomarkers specific to the hypoxia response in salmonids across multi-stressor conditions. Gill tissue was collected from 24 freshwater juvenile Chinook salmon (Oncorhynchus tshawytscha), held in normoxia [dissolved oxygen (DO) > 8 mg L-1] and hypoxia (DO = 4‒5 mg L-1) in 10 and 18°C temperatures for up to six days. RNA-sequencing (RNA-seq) was then used to discover 240 differentially expressed genes between hypoxic and normoxic conditions, but not affected by temperature. The most significantly differentially expressed genes had functional roles in the cell cycle and suppression of cell proliferation associated with hypoxic conditions. The most significant genes (n = 30) were selected for real-time qPCR assay development. These assays demonstrated a strong correlation (r = 0.88; p < 0.001) between the expression values from RNA-seq and the fold changes from qPCR. Further, qPCR of the 30 candidate hypoxia biomarkers was applied to an additional 322 Chinook salmon exposed to hypoxic and normoxic conditions to reveal the top biomarkers to define hypoxic stress. Multivariate analyses revealed that smolt stage, water salinity, and morbidity status were relevant factors to consider with the expression of these genes in relation to hypoxic stress. These hypoxia candidate genes will be put into application screening Chinook salmon to determine the identity of stressors impacting the fish.

The Optimization and Biological Significance of a 29-Host-Immune-mRNA Panel for the Diagnosis of Acute Infections and Sepsis

“…The GWAS catalog includes C-reactive protein measurement and monocyte count. Differential expression of this gene may be related to hypoxia-stress [ 43 ].…”

Endangered Cultus Lake sockeye salmon exhibit genomic evidence of hypoxic and thermal stresses while rearing in degrading freshwater lacustrine critical habitat

“…Although sockeye salmon juveniles may not have been directly exposed to either hypoxic or thermal stressors at the time of sampling, the selected biomarkers are more highly activated under chronic than acute stresses (e.g. Jeffries et al , 2014 ; Anttila et al , 2015 ; Houde et al , 2019 ; Akbarzadeh et al , 2018 , 2020 ), and thus should reflect the stress signatures of their recent environmental experiences. For instance, the induction of hypoxia and thermal responsive genes in fish exposed to either water temperature and hypoxia stresses remains upregulated hours after returning to CTs and normal oxygen (i.e.…”

“…Our previous validation of hypoxia biomarkers performed using a stress-challenge study in juvenile Chinook salmon ( Oncorhynchus tshawytscha ) showed that the combinations of biomarkers most strongly differentiating normoxic and hypoxic states varied between salinity environments. Hence, in this study, to identify signals of hypoxia in fish using the freshwater lake habitat, we combined seven hypoxia genes (biomarkers) that previously showed the strongest differential activity for fish exposed to hypoxic conditions in freshwater ( Akbarzadeh et al , 2020 ) with four general hypoxia biomarkers from the literature ( Houde et al , 2019 ) and applied these to the juvenile sockeye salmon samples obtained from Cultus and Chilliwack lakes ( Table 2 ). To examine thermal stress, 10 biomarkers previously validated in salmonids ( Akbarzadeh et al , 2018 ; Houde et al , 2019 ) were applied ( Table 2 ).…”

“…A genomic tool called the ‘Salmon Fit-Chip’ has been recently developed by the DFO Molecular Genetics Laboratory to identify specific stressors (e.g. thermal, hypoxic, osmotic and general stress) and diseases (infectious agents, viral disease, inflammation, immune stimulation) in salmon and trout that is based on targeted host response profiling of gill tissue ( Akbarzadeh et al , 2018 , 2020 ; Miller et al , 2016 , 2017 ; Houde et al , 2019 ). Gill is an ideal tissue to monitor environmental responses because of its direct contact with water and can be sampled non-lethally.…”

Seasonal near-surface hypoxia in a temperate fjord in Clayoquot Sound, British Columbia

“…A growing body of research demonstrates that salmon are vulnerable to and influenced by low oxygen conditions, particularly in conjunction with additional stressors such as water temperature (Houde et al, 2019;Akbarzadeh et al, 2020). Shoaled hypoxic waters and elevated sea-surface temperatures cause vertical habitat compression (Bakun et al, 2015), which may increase interaction with predators, parasites, and pathogens, and possibly decrease feeding opportunities (Breitburg et al, 2018).…”