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

The olfactory system is pivotal for insect behavior, with odorant-binding proteins (OBPs) serving as primary detectors for semiochemicals. Previous studies demonstrated that <i>DhelOBP4</i> in <i>Dastarcus helophoroides</i> 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 <i>DhelOBP4</i> expression by binding to its promoter. RNAi knockdown revealed sex-specific regulation: <i>BarH1</i> silencing significantly reduced <i>DhelOBP4</i> expression only in females, whereas <i>BarH2</i> knockdown affected both sexes. Y-tube olfactometer assays showed that <i>BarH1</i> depletion abolished female attraction to α-pinene and 2-undecyloxy-1-ethanol, as well as male attraction to α-pinene. <i>BarH2</i> knockdown eliminated attraction in both sexes and reversed the female response to α-pinene from attraction to repulsion. These findings establish <i>DhelOBP4</i> as a key detector of these volatiles, under sex-specific regulation by <i>BarH1/2</i> to modulate behavior, revealing novel mechanisms in tritrophic interactions.