Physiological response and miRNA–mRNA interaction analysis in the head kidney of rainbow trout exposed to acute heat stress

Treatment and sampling All experimental fish (425-day-old, average weight 118 ± 5 g) were obtained from the same family. They were selected from Gansu Fisheries Research Institute (Linxia, China). Before the experiment, all the fish were adapted for 2 weeks to the indoor water-cycling system, with the water temperature maintained at 16℃, using the three sets of electric heating rods (3000 w) and automatic temperature controllers. The pH of the water was 7.4 ± 0.2 with dissolved oxygen > 7 mg L-1. This experiment consisted of two parts. One was to determine the 48h-LT50 of the rainbow trout under high temperature stress. The 48h-LT50 of the rainbow trout was determined by setting five gradient temperatures (20, 21, 22, 23, and 24℃). The water temperature was rapidly raised to each of the experimental temperatures within 2 h. Each temperature treatment consisted of 3 sets of repeats (10 fish per tank). The cumulative mortality of each group within 48 h was calculated and the 48h-LT50 of fish was obtained by linear interpolation. The 48h-LT50 was used as the next experimental water temperature. The second experiment was performed to investigate the behavior and physiological changes in the rainbow trout at 48h-LT50. The fish were randomly placed in six tanks, including three 48h-LT50 tanks (LT group) and three 16℃ tanks (CO group). The cumulative mortality in each group was calculated at 0, 2, 4, 8, 12, 24, 36, and 48 h after heat stress. Three fish were randomly selected from each group at 8 time points after heat stress. The number of WBCs and RBCs were measured (Qiang et al., 2017a) using an automatic blood cell analyzer (bc-5120, Shenzhen Mindray Bio Medical Co., Ltd., Shenzhen, China). SOD, lysozyme activity, and malondialdehyde content were measured from the head kidney tissues according to the manufacturer’s instructions (Beauchamp et al., 1971; Dominguez et al., 2015; Zhang et al., 2008). All test kits were purchased from the Nanjing Jiancheng Biological Engineering Institute (Nanjing, China). The small RNA (sRNA) was extracted from the head kidney using a mirVana™ miRNA Isolation Kit (Ambion, Austin, TX, USA) following the manufacturer’s instructions. The quality and quantity of the extracted sRNA were assessed using an Agilent 2100 (Agilent, CA, USA) Bioanalyzer. sRNA with 28S/18S ≥ 1.5 and RNA integrity number > 8.0 was selected for further analysis. Six sRNA libraries were built including CO-1, CO-2, CO-3, LT-1, LT-2, and LT-3. Six libraries were sequenced and the reads were assembled according to standard procedures (Qiang et al., 2017a)