Physiological and transcriptomic analyses reveal the immune and metabolic responses in gills of rainbow trout (Oncorhynchus mykiss) under moderate and severe hypoxia stress

Rainbow trout (Oncorhynchus mykiss), an important economically fish with high food value in China, is very sensitive to hypoxia. The gills are the first tissue to be attacked after hypoxia stress. However, the regulatory mechanisms in gills of rainbow trout to moderate and severe hypoxia stress are unclear. In this study, we determined the changes of biochemical parameters and gene expression in gills of rainbow trout during different periods of hypoxia (4, 8, 12, 24 h, and 1 month) and reoxygenation for 24 h, and analyzed the tissue and transcriptomic changes under moderate hypoxia for 12 h (Tm12G) and severe hypoxia for 12 h (Ts12G) compared with control (CG). The results showed that gill lamellae were bent and ILCM was reduced in the Tm12G group, while this phenomenon was more pronounced in the Ts12G group. At one month of hypoxia, we found that the gill lamellae epithelium was detached. Based on biochemical parameters and transcriptomic analysis, we found that the antioxidant and immune responses were activated, and glucose metabolism, lipid metabolism, and amino acid metabolism were enhanced, however, vitamin metabolism was suppressed under moderate hypoxia stress. Antioxidant and immune responses were activated, and glucose metabolism, lipid metabolism, and amino acid metabolism were enhanced, and nucleotide metabolism also was activated under severe hypoxia stress. From the analysis of gene expression pattern, we found that the expression of hsp90a, tnf, caspase3, mgst1, fih1, hif1a, ddit4, and egln3 showed a tendency to increase and then decrease and recovered after reoxygenation under moderate and severe hypoxia stress. Except for mgst1, the expression of other genes was significantly higher than the control at 12 h of stress. Under hypoxia stress, antioxidant and metabolic responses were first elevated and then decreased with time, and metabolic and immune pathways were activated, which coordinated with each other in response to hypoxia stress. These results provide basic information for an in-depth investigation of the physiological and molecular mechanisms in gills of rainbow trout under hypoxia stress. Overall design: Fish weighing (130 ± 5.0) g were selected and temporarily reared for two weeks in a flow-through aquaculture system with DO of 8.5 ± 0.1 mg/L, water temperature of 12 ± 0.1?. Fish were fed a commercial pellet feed twice a day. All procedures were conducted in accordance with the approval of the Animal Ethics Committee of Gansu Agricultural University and followed the protocols approved by the institutional guidelines.Healthy rainbow trout were randomly divided into three groups, including normoxia, moderate hypoxia, and severe hypoxia groups, the oxygen levels were maintained at 8.5 ± 0.1, 4.5 ± 0.1, and 3.0 ± 0.1 mg/L, respectively, and the low oxygen environment was created by mixing oxygen and nitrogen. The reoxygenation experiment was conducted immediately after 24 h of hypoxia stress and the DO level was maintained at 8.5 ± 0.1 mg/L during the experiment. Controls were divided into C (0 h control) and CM (1 month control), and the gills of the normoxia group were collected immediately before the start of the hypoxia experiment and 1 month after the start of the experiment. After hypoxia stress, gill tissues were collected at 4 h, 8 h, 12 h, 24 h, TMM (moderate hypoxia for 1 month), and TMS (severe hypoxia for 1 month), as well as R12 (hypoxia for 24 h + reoxygenation for 12 h) and R24 h (hypoxia for 24 h + reoxygenation for 24 h) in each group (moderate hypoxia group and severe hypoxia group), respectively. Gill samples were collected after fish MS-222 treatment. After collection, some samples were used for histological observation, other samples were frozen immediately in liquid nitrogen, and then transferred to -80? refrigerator for storage for biochemical parameters, gene expression and RNA-seq assays.