Identification and functional characterization of key ecdysteroid biosynthesis and signalling genes regulating moulting and metamorphosis in Dastarcus helophoroides

Dastarcus helophoroides is an important ectoparasitoid used in the biological control of longhorn beetles. Its first-instar larvae actively seek hosts and progress through four larval instars before pupation. Throughout these developmental stages, 20-hydroxyecdysone (20E) plays a pivotal role in initiating and regulating moulting and metamorphosis; however, the specific genes and underlying molecular mechanisms in D. helophoroides remain uncharacterized. In this study, comparative transcriptome analysis across larval development identified differentially expressed genes (DEGs) associated with cuticle remodelling, hormone metabolism and stage-specific metabolic and cellular reprogramming. RT-qPCR validation revealed stage-specific expression patterns of 20E biosynthesis and signalling pathway genes, with EcR, Br-C, E93 and HR39 peaking during the larval-prepupal transition, whereas CYP18A1, E74A, E75B, HR3 and FTZ-F1 were highly expressed in early instars. RNAi targeting the eight key genes (CYP315A1, CYP314A1, EcR, USP, Br-C, E93, HR39 and FTZ-F1) severely disrupted metamorphosis and reduced adult eclosion by 47.83-100%. The developmental arrests were accompanied by stage-specific morphological abnormalities, including abnormal pigmentation, wing deformities and malformed prepupae and pupae. This study identifies key ecdysteroid biosynthesis and signalling genes as critical regulators of D. helophoroides development, providing insights into growth regulation and artificial rearing optimization in this parasitoid biocontrol agent.