Physiology of diapause and cold hardiness in overwintering pupae of the apple leaf miner Phyllonorycter ringoniella in Japan

The apple leaf miner Phyllonorycter ringoniella (Matsumura) (Lepidoptera: Gracillariidae) overwinters as a diapausing pupa. The diapause rate reaches 100% in early October. Diapause intensity decreases gradually from early October and diapause terminates in early February. The fresh body weight of diapausing pupae is 1.6 times that of non-diapausing pupae. The main cryoprotectant in P. ringoniella pupae is trehalose. Three stages are distinguishable as indicated by the correlations between diapause intensity, levels of cold hardiness and the trehalose content: diapause induction occurred in October, diapause development from November to December, and post-diapause quiescence from January to April. During diapause induction, the pupae accumulate low levels of trehalose and do not survive exposure to -15 degreesC. During diapause development, the pupae gradually accumulate more trehalose and show some ability to survive exposure to -15 degreesC, but not to -20 degreesC. During post-diapause quiescence, the pupae accumulate relatively more trehalose and cold hardiness fully develops, but decreases quickly in April. The trehalose content in pupae sampled in December is unaffected by acclimation temperatures in the range 0-30 degreesC, but decreases in pupae sampled in March after acclimation at temperatures from 5 to 15 degreesC. These results suggest that overwintering pupae of P. ringoniella have the ability to accumulate trehalose and develop a high level of cold hardiness during diapause development.