Trachealess, a bHLH-PAS transcription factor, is an upstream regulator of 20-hydroxyecdysone signaling essential for molting in Henosepilachna vigintioctopunctata.

The steroid hormone 20-hydroxyecdysone (20E) is the central regulator of insect molting, yet the upstream transcription factors controlling its pathway remain poorly characterized. Here, we investigate the role of the bHLH-PAS transcription factor Trachealess (HvTrh) in the 28-spotted ladybeetle, Henosepilachna vigintioctopunctata. HvTrh was ubiquitously expressed during larval development, with peak transcript levels in the pupal stage. RNAi-mediated silencing of HvTrh induced severe molting defects and high mortality across all larval instars. This was associated with a significant reduction in 20E titers and the suppression of key 20E pathway genes. The resulting molting defects were not rescued by exogenous 20E, indicating a disruption of tissue competence. Histological analysis confirmed that HvTrh knockdown impaired chitin deposition and new cuticle remodeling. Among the downregulated genes, HvE75 was the most strongly suppressed. Mechanistically, we demonstrated that HvTrh directly activates the HvE75 promoter by binding to a specific central nervous system midline element (CME), as validated by dual-luciferase reporter assays and electrophoretic mobility shift assays. Our findings establish HvTrh as a critical upstream regulator of insect molting, proposing a model where it governs the 20E signaling cascade, at least in part, through the direct transcriptional control of HvE75.