Tandem duplication has enabled functional differentiation in an olfactory gene cluster of Anastatus japonicus

Clustered odorant-binding protein (OBP) genes are observed across insect species, yet the functional principles governing their organization remain elusive. Here, we identify a lineage-specific expansion to 62 OBPs in the generalist egg parasitoid wasp Anastatus japonicus, predominantly organized as tandem arrays on chromosome 4. Focusing on a representative cluster of four tandem duplicates (AjapOBP16-19), integrative analyses reveal this quartet as a partially differentiated chemosensory unit. AjapOBP16/18 show specialized binding toward specific sesquiterpenoids, AjapOBP19 exhibits broad-spectrum binding with preference for aromatics, and AjapOBP17 displays only moderate to low affinity for all tested ligands. This functional divergence is rooted in distinct binding-pocket architectures. Key ligands identified including β-caryophyllene trigger dose-dependent antennal responses and elicit behavioral attraction. RNAi-mediated knockdown confirms that AjapOBP16/18 are collectively required for β-caryophyllene recognition. Our findings reveal that tandem OBP clusters can evolve as cooperative yet partially differentiated modules, providing insights into how gene duplication shapes insect olfaction.