Tetrastichus brontispae exploits host-derived On-miR-281-3p to suppress the phenoloxidase pathway and facilitate its development.

Parasitoid wasps rely on precise immune manipulation to establish infections in their hosts. Tetrastichus brontispae must overcome the robust melanization response of its palm pest host Octodonta nipae, yet the molecular mechanisms regulating to this process remain poorly characterized, particularly regarding microRNA (miRNA)-mediated control of the phenoloxidase (PO) cascade. Bioinformatic analysis of the O. nipae genome identified 11 melanization-associated miRNAs targeting key immune genes. Dual luciferase detection system assay and RNA binding protein immunoprecipitation results revealed that On-miR-182-5p and On-miR-281-3p regulate the melanization pathway through distinct mechanisms: On-miR-182-5p binds the 5' untranslated region (UTR) of OnSPN1, while On-miR-281-3p targets the 3'CDS of OnPPO1. Antagomir-mediated knockdown of miR-281-3p increased OnPPO1 messenger RNA (mRNA) levels by 52% and elevated PO activity by 68%, whereas miR-182-5p agomir treatment unexpectedly up-regulated OnSPN1 expression 2.4-fold. Disrupting this regulatory network significantly impaired T. brontispae fitness, reducing emergence rates by 27.3%, skewing offspring sex ratios to 3.8 females per male, and prolonging development time by 48 h. Our study reveals a sophisticated immune manipulation strategy where T. brontispae hijacks host miR-182-5p (activator) and miR-281-3p (suppressor) to bidirectionally regulate the PO cascade, simultaneously inhibiting melanization while maintaining microbial defense capacity. These findings provide novel targets for improving biological control strategies against invasive palm pests through miRNA-based interventions.