Hypoxia_Biomarker_qPCR data.xlsx

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 (<i>Oncorhynchus tshawytscha</i>), held in normoxia [dissolved oxygen (DO) &gt; 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 &lt; 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.

将低氧（即溶解氧浓度过低）下的早期基因表达响应作为评估该胁迫暴露程度的工具，对鲑科鱼类（salmonids）而言至关重要——由于人为活动导致的栖息地改变，例如全球变暖、营养盐过度富集以及持续性藻华，这类鱼类正日益暴露于低氧胁迫之中。本研究旨在发掘并验证多胁迫条件下鲑科鱼类体内特异性响应低氧胁迫的鳃组织基因表达生物标志物（biomarker）。研究采集了24尾淡水幼年期奇努克鲑（*Oncorhynchus tshawytscha*）的鳃组织，实验设置常氧[溶解氧（dissolved oxygen, DO）>8 mg·L⁻¹]与低氧（DO=4~5 mg·L⁻¹）两种条件，分别在10℃与18℃水温下养殖长达6天。随后通过RNA测序（RNA-sequencing, RNA-seq）筛选出240个在低氧与常氧条件下存在差异表达、且不受水温影响的差异表达基因（differentially expressed genes）。表达差异最显著的基因主要参与细胞周期调控以及与低氧条件相关的细胞增殖抑制过程。选取其中表达差异最显著的30个基因（n=30）用于开发实时定量PCR（real-time quantitative PCR, qPCR）检测体系。该检测体系验证显示，RNA-seq得到的基因表达量与qPCR得到的基因表达倍数变化之间存在极强的相关性（r=0.88；p<0.001）。此外，针对这30个低氧候选生物标志物，研究团队使用qPCR对额外322尾暴露于低氧与常氧条件下的奇努克鲑进行检测，以筛选出可用于界定低氧胁迫的最优生物标志物。多元统计分析结果显示，幼鲑入海过渡期（smolt stage）、水体盐度以及发病状态是与这类基因表达及低氧胁迫关联相关的关键影响因素。这些低氧候选生物标志物将被应用于奇努克鲑的胁迫因子筛查，以明确影响该鱼类的胁迫因子种类。