Alkali exposure induces autophagy through activation of the MAPKpathway by ROS and inhibition of mTOR in Eriocheir sinensis

Saline-alkali water is widely distributed around the world, and salinization of water ecosystems has occurred in some areas of the world, comprising approximately one-third of the total land area, which has drawn much attention to the conservation of water ecosystems and aquatic animals. However, due to the complex compo-sition of saline-alkaline water, many aquatic animals are unable to survive, which greatly limits the development and utilization of saline-alkaline waters. Carbonate alkalinity is an important stress factor for aquatic animals in saline-alkaline water. Exposure to highly alkaline carbonate can induce oxidative stress. For this study, we used Eriocheir sinensis as a model organism to evaluate the effects of alkaline stress on oxidative stress and autophagy. The trial was divided into five alkali level treatment groups (control, 4.375 mmol/L, 8.75 mmol/L, 17.5 mmol/L, and 35 mmol/L, respectively), and liver tissues were assessed by antioxidant enzyme kits, real-time quantitative PCR assays and ultrastructural observations at 3 time points (24 h, 48 h, 96 h). Compared with the control group, the activities of antioxidant enzymes (superoxide dismutase (SOD), glutathione reductase (GSH), glutathione peroxidase (GSH-PX) and total antioxidant capacity (T-AOC)) in the alkali stress group increased and then decreased with increasing time, while the content of malondialdehyde (MDA) increased. At the molecular level, the expression of MAPK pathway-related genes (P38, MAPK, JNK) in the alkaline stress group showed a dose -dependent increase at 48 and 96 h and was significantly higher than that in the control group. The expression of autophagy-related genes (ATG5, ATG12, ATG7 and GABARAP) increased dose-dependently at 24, 48, and 96 h and was significantly higher than that in the control group. In contrast, mTOR expression was always in a suppressed state. These results suggest that alkaline stress induces activation of the MAPK pathway via ROS and inhibits mTOR expression, thereby inducing autophagy in the liver tissue of Eriocheir sinensis. This study investigated the stress mechanism of carbonate on Eriocheir sinensis and provided a theoretical basis for the continued exploitation of saline-alkaline water aquaculture.