Diapause is a critical survival strategy for insects, enabling them to overcome unfavorable environments and maintain life cycle consistency (Togashi, 2021, Schebeck et al., 2024). Pests such as Ips typographus, Rhagoletis cerasi, Mindarus abietinus, and Lymantria dispar successfully endure extremely low temperatures and food shortages through diapause (Dolezal and Sehnal, 2007, Doherty et al., 2018, Moraiti et al., 2020, Schebeck et al., 2022, Barker et al., 2023). Among pests, wood-boring pests spend most of their life cycle hidden within tree trunks, minimizing exposure to external environmental conditions. This concealment, combined with diapause, enhances their ability to resist threats. Although understanding their developmental biology and environmental adaptability is crucial for developing targeted population control measures, research on the diapause characteristics of wood-boring pests remains limited because of their strong concealment ability and complex life cycles (Luo et al., 2023).

Insect diapause is a dynamic process, encompassing stages such as prediapause, diapause (induction, maintenance, and termination), and postdiapause (Schebeck et al., 2024). At each stage, insects undergo corresponding changes in external morphology and physiological–biochemical processes, with subtle internal changes occurring even within the same stage (Sgolastra et al., 2010). External morphological traits, such as body color, eye spots, and body shape, often serve as the most intuitive indicators of diapause stages (Dhillon and Hasan, 2017, Zerbino et al., 2020, Amozegarfard et al., 2023). However, when external morphology alone is insufficient for accurate determination, changes in physiological–biochemical processes related to diapause can serve as critical auxiliary indicators (Ding et al., 2011, Huang et al., 2022). Juvenile hormone (JH) and 20-hydroxyecdysone (20E) are key regulators of diapause and are associated with physiological changes (Jiang et al., 2011, Subta et al., 2017, Hutfilz, 2022). Additionally, biochemicals such as trehalose, glucose, glycerol, and inositol play vital roles in lowering the freezing point of diapausing insects, protecting cells from low-temperature damage, and providing metabolic support throughout diapause. These biochemicals are important reference parameters for distinguishing diapause stages (Nomura and Ishikawa, 2001, Crosthwaite et al., 2011, Ding et al., 2011, Treanore and Amsalem, 2020, Huang et al., 2022, Xiao et al., 2022).

The emerald ash borer (EAB), Agrilus planipennis (Coleoptera: Buprestidae), is a highly destructive wood-boring pest that feeds primarily on ash trees (Fraxinus spp.). Its larvae feed on the phloem and cambium, girdling the tree and disrupting water and nutrient transport, typically leading to tree death within approximately three to five years (Tussey et al., 2018). Native to China, Japan, South Korea, and the Russian Far East, A. planipennis has invaded the United States, Canada, European Russia, and Ukraine since 2002, severely damaging landscape ash species such as white ash (F. americana), green ash (F. pennsylvanica), velvet ash (F. velutina), and European ash (F. excelsior) (Pellecchia, 2020). The pest has killed millions of ash trees in the United States, causing significant economic and ecological losses, with local government management costs alone reaching up to $800 million (Hudgins et al., 2024). Currently, A. planipennis populations continue to expand across affected regions, with their distribution extending from 31°N (USDA, 2024) to 60°N (Selikhovkin et al., 2023), highlighting their strong environmental adaptability and extensive damage potential.

Agrilus planipennis is generally univoltine but can become semivoltine in areas with low population density or high latitude (Cappaert et al., 2005, Tluczek et al., 2011, Christianson and Venette, 2018, Nalepa et al., 2021). In univoltine populations, the larval stage is the most damaging and longest phase of the life cycle. First- to fourth-instar larvae feed on ash tree phloem (Wang et al., 2010) before entering obligatory winter diapause in xylem pupal chambers in late summer or autumn. Within the chamber, they progress through three forms: J-shaped larvae (JL), I-shaped larvae (IL), and pupae (Jennings et al., 2014, Pellecchia, 2020, Duan et al., 2021). Although research to date has focused on the basic biology and cold resistance of A. planipennis (Crosthwaite et al., 2011, Sobek-Swant et al., 2012, Vermunt et al., 2012, Jones et al., 2020), studies by Discua Duarte (2013) and Duan et al. (2021) have explored its diapause characteristics, identifying the JL form as the primary diapause induction phase and low temperatures as necessary for diapause termination. However, these studies did not clarify the relationship between the IL form and diapause stages. Furthermore, although some studies have acknowledged the existence of JL and IL forms in the pupal chamber, definitions of the JL and IL forms vary, complicating efforts to understand the diapause characteristics of the borer. Wang et al., 2010, Christianson and Venette, 2018, and Pellecchia (2020) described the IL form as prepupae, where A. planipennis straightens its body between the JL and pupal forms. In contrast, Sobek-Swant et al., 2012, Chamorro et al., 2012, Lyons, 2015, and Jones et al. (2020) collectively referred to both the JL and IL forms as prepupae, encompassing all nonfeeding stages before pupation. These discrepancies highlight the need to resolve key questions about the overwintering diapause of wild A. planipennis populations in pupal chambers, as well as the relationships between different chamber forms (JL versus IL) and the physiological–biochemical processes regulating diapause.

Similar to previous findings, our preliminary experiments revealed that A. planipennis remains in the JL and IL forms for several months before pupation within the pupal chamber. To understand its overwintering diapause accurately and avoid confusion caused by the term “prepupae”, we distinguished and described the two forms based on morphology: the J-shaped form (JL) and the straightened form (IL). Focusing on field univoltine populations, A. planipennis samples were collected regularly during autumn, winter, and spring, and their developmental stages and morphology were recorded. Physiological and biochemical parameters—such as hormone, carbohydrate, and alcohol amounts—were measured to clarify the relationships among these forms during overwintering diapause.