Functional Role of Odorant-Binding Proteins in Response to Sex Pheromone Component Z8-14:Ac in Grapholita molesta (Busck)

The plum fruit moth (PFM), <i>Grapholita funebrana</i>, and the oriental fruit moth (OFM), <i>G. molesta</i>, are closely related fruit moth species that severely damage fruit trees in Rosaceae. Both species share common primary sex pheromone components <i>Z</i>8-12:Ac and <i>E</i>8-12:Ac. The secondary sex pheromone components of PFMs consist of <i>Z</i>8-12:OH, <i>Z</i>8-14:Ac, and <i>Z</i>10-14:Ac, while those of OFMs include <i>Z</i>8-12:OH and 12:OH. Previous researchers have proved that the inclusion of <i>Z</i>8-14:Ac and <i>Z</i>10-14:Ac did not augment PFM catches but inhibited OFM catches in orchards in Europe, thereby maintaining the species-specificity of the PFM sex attractant. However, which of these components, <i>Z</i>8-14:Ac or <i>Z</i>10-14:Ac, plays the major role in inhibiting OFM attraction remains unclear. In the current study, electroantennogram (EAG) assays indicated that both OFM and PFM males exhibited a moderate EAG response to <i>Z</i>8-14:Ac and <i>Z</i>10-14:Ac. Rubber septa loaded with varying ratios of <i>Z</i>8-14:Ac (1% to 30%) or <i>Z</i>10-14:Ac (5% to 110%) combined with a constant dose of <i>Z</i>8-12:Ac and <i>E</i>8-12:Ac produced diverse trapping effects. Sex attractants containing <i>Z</i>8-14:Ac did not significantly affect the trapping of PFM males but drastically reduced the capture of OFM males, with the reduction reaching up to 96.54%. Attractants containing more than 10% of <i>Z</i>10-14:Ac simultaneously reduced the number of OFM and PFM males captured. <i>Z</i>8-14:Ac was indispensable for maintaining the specificity of sex pheromones. Fluorescence competitive binding assays of recombinant GmolPBP2 showed the lowest <i>K_i</i> value (0.66 ± 0.02 μM) among the PBPs/GOBPs from OFMs, suggesting that it is the most likely target for <i>Z</i>8-14:Ac. Molecular dynamic simulation and site-directed mutagenesis assays confirmed that the Phe12 residue, which forms a π-alkyl interaction with <i>Z</i>8-14:Ac, was crucial for GmolPBP2 binding to <i>Z</i>8-14:Ac. In conclusion, <i>Z</i>8-14:Ac is vital to the specificity of PFM sex pheromones inhibiting OFM attractants when added to <i>Z</i>8-12:Ac and <i>E</i>8-12:Ac. This could be potentially used to develop species-specific sex attractants for the PFM.