Heat stress hardening of oriental armyworms is induced by a transient elevation of reactive oxygen species during sublethal stress

Pre‐exposure to mild heat stress enhances the thermotolerance of insects. Stress hardening is a beneficial physiological plasticity, but the mechanism underlying it remains elusive. Here we report that reactive oxygen species (ROS) concentrations were quickly and transiently elevated in the armyworms, Mythimna separata , by exposing them to 40°C, but not other tested temperatures. Larvae exposed to 40°C had subsequently elevated antioxidant activity and the highest survival of all tested heating conditions. The elevation of ROS after lethal heating at 44°C for 1 h was approximately twofold compared to heating at 40°C. Injection of an optimal amount of hydrogen peroxide (H 2 O 2 ) similarly caused sequential elevation of ROS and antioxidant activity in the test larval hemolymph, which led to significantly enhanced survival after lethal heat stress. The H 2 O 2 ‐induced thermotolerance was abolished by coinjection of potent antioxidants such as ascorbic acid or N ‐acetylcysteine. Both preheating at 40°C and H 2 O 2 injection enhanced expression of genes encoding superoxide dismutase 1, catalase, and heat shock protein 70 in the fat body of test larvae, indicating the adequate heat stress induced a transient elevation of ROS, followed by upregulation of antioxidant activity. We infer that thermal stress hardening is induced by a small timely ROS elevation that triggers a reduction–oxidation signaling mechanism.