BarH1 and BarH2 Regulate the Expression of Odorant-Binding Protein 4 to Mediate the Recognition of Two Volatiles in Dastarcus helophoroides

The olfactory system is pivotal for insect behavior, with odorant-binding proteins (OBPs) serving as primary detectors for semiochemicals. Previous studies demonstrated that DhelOBP4 in Dastarcus helophoroides is essential for detecting herbivore-induced plant volatiles (HIPVs). Here, we demonstrated through dual-luciferase reporter assays, yeast one-hybrid (Y1H) assays, and electrophoretic mobility shift assays (EMSA) that transcription factors BarH1/2 directly activate DhelOBP4 expression by binding to its promoter. RNAi knockdown revealed sex-specific regulation: BarH1 silencing significantly reduced DhelOBP4 expression only in females, whereas BarH2 knockdown affected both sexes. Y-tube olfactometer assays showed that BarH1 depletion abolished female attraction to α-pinene and 2-undecyloxy-1-ethanol, as well as male attraction to α-pinene. BarH2 knockdown eliminated attraction in both sexes and reversed the female response to α-pinene from attraction to repulsion. These findings establish DhelOBP4 as a key detector of these volatiles, under sex-specific regulation by BarH1/2 to modulate behavior, revealing novel mechanisms in tritrophic interactions.