Exploration of winter diapause stages of the emerald ash borer based on morphological and biochemical parameters

Diapause enables the critical survival of certain insects under adverse conditions, and the concealment ability of insects makes characterizing diapause traits challenging. Here, we focused on the invasive emerald ash borer (EAB, <i>Agrilus planipennis</i>), a species with obligatory winter diapause, and combined morphological, physiological, and biochemical analyses to correlate overwintering diapause stages with two pupal chamber forms: J-shaped (JL) and I-shaped (IL) larvae. Fourth-instar larvae (L4, nondiapausing, still feeding) outside the chamber and pupae (postdiapausing) inside the chamber served as controls. The results revealed that 1) the JL and IL periods overlapped, lasting up to 7 and 5 months respectively. Significant variations in physiological–biochemical parameters during JL prompted its subdivision into three substages: early (JL-E), middle (JL-M), and late (JL-L). 2) Compared with L4-stage insects, insects in the JL-E and/or JL-M stages presented significantly lower juvenile hormone (JH), trehalose, glucose, and inositol amounts, whereas the 20-hydroxyecdysone (20E) and glycerol amounts increased significantly. In the JL-L and IL stages, the amounts of JH, trehalose, glucose, and inositol no longer differed from those in L4. The IL-stage insects ultimately developed into pupae, with JH, glycerol, and inositol amounts comparable to those in the pupal stage. These findings demonstrate that the JL and IL stages in <i>A. planipennis</i> exhibit strong temporal correspondence with distinct diapause phases, as evidenced by their characteristic physiological signatures. This field-based multilevel study improves the understanding of the diapause biology of this invasive insect pest.