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° 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.

鉴定对缺氧（即低溶解氧）的早期基因表达反应作为评估暴露于此应激源的程度的工具对于鲑鱼至关重要，因为鲑鱼由于人为栖息地变化（例如，全球变暖）而日益暴露于低氧胁迫下，营养物过多，藻类持续开花。我们的目标是发现和验证在多应激条件下鲑鱼中低氧反应所特有的g基因表达生物标记。收集nor鱼组织，取自24只淡水少年奇努克鲑（Oncorhynchus tshawytscha），使其处于常氧[溶解氧（DO）> 8 mg L ^-1 ]和缺氧（DO = 4-5 mg L ^-1）在10和18°的温度下最多工作六天。然后使用RNA测序（RNA-seq）来发现缺氧和常氧条件下240个差异表达的基因，但不受温度的影响。最明显差异表达的基因在细胞周期和与缺氧条件相关的细胞增殖抑制中具有功能性作用。选择最重要的基因（n = 30）用于实时qPCR分析开发。这些分析显示出很强的相关性（r = 0.88; PRNA seq的表达值与qPCR的倍数变化之间的差异<0.001）。此外，将30种候选缺氧生物标记的qPCR应用于暴露于低氧和常氧条件下的322头奇努克鲑鱼，以揭示定义低氧应激的顶级生物标记。多变量分析表明，这些阶段的表达与低氧胁迫有关，是软体动物阶段，水盐度和发病率的相关因素。这些缺氧候选基因将用于筛选奇努克鲑鱼，以确定影响鱼类的应激源的身份。