Endocrine and molecular dynamics during diapause termination and post-diapause reproductive development in adult Dendroctonus rufipennis.

In temperate and boreal ecosystems, insect diapause is a critical adaptive strategy that synchronizes life history transitions with seasonal environmental conditions. Temperature, in particular, plays an important role in diapause maintenance and termination, yet its mechanistic effects remain less resolved. Dendroctonus rufipennis, a major bark beetle in North American spruce (Picea) forests, exhibits an obligate adult diapause terminated by prolonged cold exposure. To characterize diapause termination, we examined reproductive development, endocrine dynamics, and gene expression in male and female adults exposed to low-temperature conditions, based on a refined definition of diapause phases. During cold exposure, reproductive systems in both sexes remained immature, with morphological development initiated only after temperature recovery and dispersal. Endocrine analyses showed that 20-hydroxyecdysone (20E) increased during cold exposure, with a peak at day 38 in males and sustained elevation between days 38 and 75 in females, whereas juvenile hormone (JH) accumulated gradually to a peak at day 75; both returned to baseline levels after temperature recovery. At the molecular level, the expression of 20E biosynthesis gene Shade was significantly increased at day 25, while JH signaling-related genes (Kr-h1 and Schlank) exhibited pronounced sex-specific expression patterns. However, these transcriptional differences did not translate into differences in reproductive development timing, suggesting a decoupling between gene expression and phenotypic outcomes. Collectively, these findings link environmental cues to developmental and molecular processes underlying diapause transitions and highlights D. rufipennis as a valuable model for studying insect diapause.