## 3 Volatile Pheromones
### Behavioural Assays to Identify Active Compounds
#### 3.4.2 Reverse Chemical Ecology Approaches
Identification and Functional Analysis of OR Genes  
Although OBPs are excellent study targets to either understanding the molecular and biochemical mechanisms of odour perception in insects, and to explore the development of pest control agents, they have broad binding specificity, are also distributed in non-olfactory tissues and have different functions [363]. Conversely, ORs are transmembrane proteins showing high specificity and sensitivity. Thus, genes encoding for chemosensory receptors are also becoming to be identified and characterised in tephritids, such as _B. dorsalis_[338; 364; 365; 366], _B. minax_[341], _B. oleae_[367], _B. latiftrons_ and _Z. cucurbate_[368], and _C. capitata_[344]. Olfactory receptors have been described as heteromeric ligand-gated ion channels consisting of a specific OR and the highly-conserved co-receptor Orco [369]. Olfactory receptors are transmembrane proteins for which no 3D structure is available yet, and they are a more difficult target than OBPs to be expressed and purified in heterologous systems. So far, the only three-dimensional structure currently available, obtained using a cryo-electron microscopy-based approach, is for a tetramer of Orco, described in the parasitic fig wasp _Apocarppta bakeri_ (Joseph) [370]. Thus, only limited data on the functional activity of tephritid ORs are available. In a recent study, ten _B. dorsalis_ ORs were co-expressed with their essential co-receptor BdorORCO in _Xenopus laevis_ Daudin (Anura: Pipidae) oocytes. Two-electrode voltage clamp was then used to record currents from injected oocytes when ligands (i.e., 1-octen-3-ol, geranyl acetate, farnesenes, and linalyl acetate) were diluted in the assay buffer [365]. Some of the identified ORs have been shown to respond to plant volatiles [365] or ME [371]. Further research efforts oriented to clarify the structure of tephritid ORs are essential to understand the molecular recognition mechanisms they are involved in, as well as their interactions with OBPs, and thus their functional roles. Insect ORs display a different topology from those of other animal G protein-coupled receptors (GPCRs) [372], with a C-terminal faced to the extracellular section and the N-terminal to the intracellular section. This feature makes insect ORs ideal targets to be explored for the development of insect-specific pest control strategies. These may include the inhibition of either Orco or the ORx/Orc complex by antagonists able to, for example, disrupt mating behaviour through the manipulation of pheromone receptivity. Interestingly, in _B. oleae_, transient knockdown via RNAi gene silencing in adult individuals showed that knockdown of Orco expression reduces the mating ability in both sexes and completely inhibits oviposition [367]. Tephritid CSPs have been identified [338; 336; 337], but their variable pattern of tissue distribution, the different potential functions, the still unproven binding ability [318] and unavailability of structural information [363] is locating them in a less attractive field of investigation.