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. Overall design: 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).