Landscape transcriptomics identify

Rapid heating events, such as heatwaves, are becoming more frequent and intense as a result of climate change. Importantly, such extreme weather events can be more important drivers of extirpation and selection than changes in annual or seasonal averages and they pose a particularly large threat to poikilothermic organisms. In this study, we evaluate the thermal stress response of a coldwater adapted fish species, the eastern brook trout (Salvelinus fontinalis), to two successive heatwaves during July and August 2022. We sampled brook trout at eight time points from four streams (N=116 fish) that differed in thermal habitat, sequenced mRNA from gill samples using TagSeq, and quantified expression levels of 32,670 unique transcripts. Multivariate analyses found that overall expression patterns in response to water temperature change were similar among streams. These analyses futher detected groups of genes involved in immune response and oxygen carrier activity that were upregulated and downregulated respectively at higher temperatures. We also detected 43 genes that were differentially expressed at different time points and followed the same expression pattern during the two heatwaves. Of these genes, 42 covaried with water temperature and most (27, 62.8%) exhibited responses that varied by stream. Some of the differentially expressed genes, including heat shock proteins and cold-inducible RNA binding proteins, have been widely linked to temperature responses in experimental studies, whereas other genes we identified have functions that have not been well-studied in relationship to temperature (e.g., collagen organization) or have unknown functions. This study shows the utility of landscape transcriptomic approaches to identify important biological processes governing wild organismal responses to short-term stressors. The results of this study can guide future investigations to identify phenotypic and genetic diversity that contribute to adaptive response to heatwaves and improve predictions of how populations will respond to climate change. Samples were taken from gill filaments of brook trout captured in four streams near State College, PA, USA during July & August 2022. Each stream was sampled on eight occasions which coincided with different points of two successive heatwave events. Streams were sampled on three occasions 3-4 days apart starting at the onset of the heatwaves, with time points representing periods of rising temperatures, peak temperatures, and declining post-peak temperatures. A fourth sample was taken 6-7 days after the third sample during a period of non-stressful temperatures to capture post-heatwave recovery responses. Three to five fish were sampled during each sampling event. Samples were only taken from individuals >120mm total length, and all individuals were sexed based on PCR amplification of sdY primers developed by Rud and Buchatsky (2014) and Webster et al. (2021).受气候变化影响，热浪等快速升温事件的发生频率与强度正逐年攀升。尤为关键的是，相较于年际或季节平均气温变化，这类极端天气事件对物种局域灭绝与适应性选择的驱动作用更为显著，且对变温生物构成的威胁尤为严峻。本研究以适应冷水环境的东部溪红点鲑（Salvelinus fontinalis）为研究对象，探究其在2022年7-8月间遭遇两次连续热浪时的热应激响应。研究团队从4个热生境存在差异的溪流中，于8个时间点采集了溪红点鲑样本（共116尾），利用TagSeq技术对鳃组织样本中的mRNA进行测序，并定量分析了32670个独特转录本的表达水平。多变量分析表明，各溪流间鱼类响应水温变化的整体基因表达模式较为相似。进一步分析发现，高温环境下，参与免疫应答与氧载体活性的基因簇分别呈现上调与下调表达趋势。此外，本研究还鉴定出43个在不同采样时间点存在差异表达的基因，且两次热浪期间它们的表达模式均保持一致。其中42个基因的表达水平与水温呈共变关系，且多数（27个，占比62.8%）的响应模式存在溪流间差异。部分差异表达基因（如热休克蛋白（heat shock proteins）与冷诱导RNA结合蛋白（cold-inducible RNA binding proteins））的温度响应功能已在实验研究中得到广泛验证；但本研究鉴定出的其他基因，其功能与温度胁迫的关联尚未得到充分研究（例如胶原组织构建相关基因），甚至部分基因功能尚不明确。本研究证实了景观转录组学方法在解析野生生物应对短期胁迫的核心生物学过程方面的应用价值。本研究结果可为未来探究驱动物种适应热浪的表型与遗传多样性提供研究思路，并助力优化种群对气候变化响应的预测模型。 实验样本采集自美国宾夕法尼亚州斯泰特科利奇附近4条溪流中的东部溪红点鲑，采样时间为2022年7-8月。每条溪流共完成8次采样，覆盖两次连续热浪过程的不同阶段：热浪起始后，每3-4天采样1次，共完成3次采样，分别对应水温上升期、峰值期以及峰后降温期；第三次采样结束后6-7天，在非胁迫水温条件下完成第4次采样，以捕捉热浪后的恢复响应特征。每次采样共采集3-5尾溪红点鲑，且仅选取总长度超过120mm的个体。所有个体均通过Rud与Buchatsky（2014）以及Webster等人（2021）开发的sdY引物进行PCR扩增，以鉴定其性别。