Differences in gene expression between high and low tolerance rainbow trout (Oncorhynchus mykiss) to acute thermal stress

Understanding the mechanisms that underlie the adaptive response of ectotherms to rising temperatures is key to mitigating the effects of climate change.  We assessed the molecular and physiological processes that differentiate between rainbow trout (Oncorhynchus mykiss) with high and low tolerance to acute thermal stress. To achieve our goal, we used a critical thermal maximum trial in two strains of rainbow trout to elicit loss of equilibrium responses to identify high and low-tolerance fish. We then compared the hepatic transcriptome profiles of high and low-tolerance fish relative to untreated controls common to both strains to uncover patterns of differential gene expression and to gain a broad perspective on the interacting gene pathways and functional processes involved.  We observed some of the classic responses to increased temperature (e.g., induction of heat shock proteins) but these responses were not the defining factors that differentiated high and low-tolerance fish. Instead, high-tolerance fish appeared to suppress growth-related functions, enhance certain autophagy components, better regulate neurodegenerative processes, and enhance stress-related protein synthesis, specifically spliceosomal complex activities, mRNA regulation, and protein processing through post-translational processes, relative to low-tolerance fish. In contrast, low-tolerance fish had higher transcript diversity and demonstrated elevated developmental, cytoskeletal, and morphogenic, as well as lipid and carbohydrate metabolic processes, relative to high-tolerance fish. Our results suggest that high-tolerance fish engaged in processes that supported the prevention of further damage by enhancing repair pathways, whereas low-tolerance fish were more focused on replacing damaged cells and their structures.

Methods
A description of the Procedures used is available at: https://www.protocols.io/private/48F2FF00AB4811EE819C0A58A9FEAC02

Synopsis as follows:

Fish from 27 families (derived from a half-sib step chain cross design) from the Lyndon strain, and similarly, fish from 28 families from the Alma strain of rainbow trout were reared at 8.5°C at the Ontario Aquaculture Research Centre (OARC). Fish were maintained at OARC until around 38 weeks post-fertilization before being transported to the Hagen Aqualab at the University of Guelph for temperature-controlled thermal trials. Fish were acclimated to 12°C for 12 days prior to the initiation of the trial, and food was withheld 72 hours prior to the start of the trial.

At around 40 weeks post-fertilization, an average of 880 fish from each strain were tested for their acute thermal tolerance in 8 replicate tanks within the experimental room in Hagen Aqualab. The acute thermal trials lasted for 18 hours where the fish was subjected to stages of thermal increase. The first stage of the trial involved a ramping of temperature from 12 to 24°C at a rate of ~3.4°C/hour in the Lyndon trial, and ~2.7°C/hour in the Alma trial. These differences were not intentional but resulted from accidental differences in water flow rates between the two trials. After the ramping interval, water was maintained at the target temperature for three hours. Following the three-hour stasis period, the temperature was increased by 1°C over a one-hour interval and maintained for a further three hours at the new level. This stepwise procedure was repeated until the temperature reached 28°C. When each fish lost equilibrium (see description in Procedures document), it was euthanized, and the time from the onset of the thermal trial to that at which it experienced loss of equilibrium (LOE) was recorded. Very few fish (< 1%) maintained equilibrium to the 27-28°C plateau. The sequential rank order of the fish’s time to LOE was used to assign a low tolerance versus high tolerance status to the rainbow trout.

Whole liver including connective, nervous, and vascular tissue was dissected immediately from low- and high-tolerance fish (N = 6 fish/group), as well as those of six control fish sampled from the 12°C environment and were stored in RNAlater (Invitrogen, Burlington, ON, Canada). mRNA was isolated using the Qiagen RNAeasy Mini Kit (QIAGEN, Toronto, ON, Canada.

An Illumina NovaSeq 6000 RNA-Seq analysis (100 bp paired-end reads) was conducted by Génome Québec (GQ; Montreal, QC, Canada), and we processed the subsequent *.fastq files provided by them using Qiagen CLC Genomics Workbench (RRID:SCR_011853). The *.fastq reads were aligned to the first release version of the current reference rainbow trout OmykA_1.1 genome build (GCF_013265732.2) available from the NCBI genome database resources (RRID:SCR_002474). Gene expression comparisons were made between high and low-tolerance rainbow trout to control fish, as well as between high and low-tolerance fish.

Greater details on the methods various analysis steps and findings from the study are provided in Turner et al. (2024). Reference to be provided upon publication.