Photoperiod regulation of diapause development in Loxostege sticticalis : coordinated 20‐hydroxyecdysone dynamics and PTTH / EcR / USP

BACKGROUND The beet webworm, Loxostege sticticalis , a globally notorious agricultural pest enters diapause as mature larvae to overwinter in the northern temperate zone. The moth develops normally at 16L: 8D and 22 ± 1 °C, and enters diapause at 22 ± 1 °C and 12L:12D. While juvenile hormone (JH) regulates larval diapause in this insect species, the role of photoperiod‐20‐hydroxyecdysone (20E) Axis remains enigmatic. RESULTS Photoperiod‐driven diapause progression is orchestrated through synchronized changes in 20E titers and expression of PTTH, EcR, and USP . Under short‐day conditions, mature larvae exhibited 20E peaks followed by sustained lower levels (≈50 ng/mg) during diapause, PTTH expression was suppressed at diapause while EcR/USP co‐downregulation in maintained diapause. Diapause terminating individuals showed pre‐activation of PTTH/EcR/USP transcription before 20E resurgence. However, non‐diapause larvae under long‐day photoperiod, steadily rising 20E titer were mirrored by progressive PTTH/EcR/USP upregulation. Functional validation revealed that microinjection of 20E agonist‐‐tebufenozide at early fifth‐instar significantly prolonged larval development by 5.8 days but accelerated pupation by 11.9 days. Conversely, RNAi‐mediated knockdown of PTTH, EcR, or USP dramatically shortened the fifth‐instar duration but led to a significant 9.70 days extension of the mature larval phase. CONCLUSION Our results establish a causal chain wherein photoperiod disrupts 20E biosynthesis via PTTH suppression, leading to EcR/USP silencing that sustains diapause. Pharmacological and genetic interventions demonstrate that the PTTH‐20E‐EcR/USP axis governs developmental transitions during both diapause and non‐diapause trajectories.