Mucus transcriptional profiling as a minimally invasive approach to identify thermal stress in a stenothermal salmonid

Global climate change has increased the frequency and severity of stressful temperatures that freshwater fishes experience, necessitating rapid and sensitive methods to monitor wild populations. Tissues used to measure transcriptional responses traditionally involved invasive or lethal sampling, which may be undesirable for imperilled species. Epidermal mucus offers a non-lethal and minimally invasive alternative, but whether thermal thresholds can be detected in mucus to identify fish experiencing thermal stress is unclear. Bull trout (Salvelinus confluentus) are a legally protected salmonid and cold-water specialist, generally occupying waters 12 {degrees}C and below, with higher temperatures resulting in cellular stress. Therefore, we measured a suite of 56 genes using high-throughput qPCR to compare machine learning classifiers developed with transcriptional profiles of epidermal mucus, gill, liver, and muscle to classify laboratory reared juvenile bull trout as below (9 {degrees}C, 12 {degrees}C) or above (15 {degrees}C, 18 {degrees}C) cellular thermal thresholds. Mucus profiles most resembled gills but represented an intermediate transcriptional response to all tissues. A reduced biomarker panel of 10 genes in mucus assigned fish to stress categories with 94.1% (95% CI = 71.3-99.9%) accuracy, which was comparable to gill (100.0%, CI = 82.4- 100%), liver (95.0%, CI = 75.1-99.9%), and muscle (100.0%, CI = 80.5-100.0%). Sex-specific temperature effects were evident in all tissues, but less pronounced in mucus and gill than in liver and muscle. Our findings demonstrate that transcriptional profiling of mucus can reliably identify individuals experiencing thermal stress, highlighting the promise of this non-lethal approach for monitoring at-risk species.