Objective This paper aims to determine the physiological adaptation mechanism of the 4-age wintering larva of M. alternatus to low temperature stress.

Method The 4-age wintering larva of M. alternatus were collected from Dexing, Jiangxi Province of eastern China in mid-March of the 2021, and seven temperature gradients of 25 ℃ (control), 4, 0, −5, −10, −15 and −20 ℃ and three time gradients of 30 min, 1 h and 3 h were set, the cold tolerance, content of cold-resistant substances, activity of antioxidant enzymes and the damage of midgut under low temperature stress were determined.

Result (1) Near the supercooling point (−9.27 ± 0.90) ℃, the 50% lethal time was about 1.9 h (114.97 min), and the 99% lethal time was about 2.8 d (4 061.86 min). Under low temperature stress below the supercooling point (−15 ℃), the lethal time of 50% and 99% was about 1/4 of that under low temperature treatment of −10 ℃. (2) After low temperature stress treatment, the water content of the 4-age wintering larvae did not change significantly, the fat content and glycogen mass concentration were significantly decreased after 3 h at 0 ℃ (P < 0.05), and the sorbitol mass concentration was significantly increased after 1 h at −5 ℃ (P < 0.05), and significantly decreased after 1 h at −20 ℃ (P < 0.05). (3) After low temperature stress treatment, the activities of antioxidant enzymes (SOD, POD, CAT) were significantly increased (P < 0.05). (4) The midgut of the 4-age wintering larvae of M. alternatus was decomposed after −15 ℃ treatment for 3 h, and vacuoles appeared between cells. After −20 ℃ treatment for 3 h, the midgut cells were ruptured, and the cell vacuoles escaped and filled the whole intestinal cavity, and the vacuoles disappeared.

Conclusion This study clarified the physiological response of oxidative stress of the fourth instar overwintering larvae to low temperature stress, which could enrich and improve the study on cold tolerance mechanism of M. alternatus.