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 (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.

识别鲑科鱼类对低氧（即溶解氧含量偏低）的早期基因表达应答，以此作为评估该胁迫暴露程度的手段，对其而言至关重要。这是因为受人为活动驱动的栖息地变化影响——例如全球变暖、营养盐过度输入以及持续性藻华——鲑科鱼类正日益频繁地遭遇低氧胁迫。本研究旨在发掘并验证多胁迫条件下，鲑科鱼类鳃组织中特异性响应低氧胁迫的基因表达生物标志物。研究采集了24尾淡水幼年期奇努克鲑（Oncorhynchus tshawytscha）的鳃组织，实验设置了常氧[溶解氧（DO）> 8 mg·L⁻¹]与低氧（DO = 4~5 mg·L⁻¹）两种条件，水温分别为10℃与18℃，暴露时长最长达6天。随后通过RNA测序（RNA-seq）筛选出240个在低氧与常氧条件下存在差异表达、且不受温度影响的基因。显著性最高的差异表达基因，其功能主要涉及细胞周期调控以及低氧胁迫下细胞增殖的抑制过程。选取其中显著性排名最高的30个基因，用于开发实时荧光定量PCR（qPCR）检测体系。该检测体系的验证结果显示，RNA-seq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个低氧胁迫候选生物标志物的qPCR检测，被应用于另一批322尾暴露于常氧与低氧条件下的奇努克鲑，以筛选出可精准界定低氧胁迫的最优生物标志物。多变量分析结果显示，幼鲑入海过渡期（smolt stage）、水体盐度以及发病状态，是在评估这些基因表达与低氧胁迫关联时需要考虑的相关因素。上述低氧胁迫候选基因将被应用于奇努克鲑的胁迫筛查，以明确影响该鱼类的胁迫因子种类。