## 3 Volatile Pheromones
### Behavioural Assays to Identify Active Compounds
#### 3.4.2 Reverse Chemical Ecology Approaches
CcapOBP22 was co-crystallised with (_E,E_)-\(\alpha\)-farnesene as ligand, further supporting the potential role of this protein in semiochemical perception in this species. _In Vitro_ and _In Silico_ OBP: Ligand Binding Assays  
Several approaches have been adopted to measure the affinity of OBPs to odorants (see [350] for a review). The most common method, which is fast and requires a limited amount of protein, is based on the use of fluorescent reporters, such as 8-anilinonapthhalene-1-sulfonic acid (ANS) and \(N\)-phenylnaphthalene-1-amine (1-NPN) in competitive binding experiments [351; 352; 353]. 1-NPN is a lipophilic crystalline solid that strongly binds insect OBPs [354]; when increasing amounts of a tested ligand are added to the OBP/1-NPN system, decreasing 1-NPN fluorescence emission is inferred as 1-NPN displacement since the ligand is assumed to compete for the binding pocket initially occupied by the fluorescent reporter. This approach has been used in the medfly to evaluate the binding affinity of CcapOBP22 and CcapOBP24 to electrophysiologically-active components of the male pheromone, as well as to the two synthetic attractants trimedure and ME [223; 349]. The finding that also ME, which is a strong attractant for some _Bactrocera_ species [170; 355; 356] but not for medfly, displays binding activity (although moderate) to the above medfly proteins is intriguing. Methyl eugenol is known to induce an electrophysiological response in medfly [302]. In _Z. scutellatus_, ME elicits significant electrophysiological responses too, but it is not behaviourally active [357]. In medfly, ME has been shown to induce poor behavioural responses in binary choice bioassays, while \(o\)-eugenol was instead strongly attractive [302]. Thus, it appears that the presence of substituents on the aromatic ring can be essential to confer attraction to chemical compounds. It will be interesting to further explore the chemistry of candidate molecules able to bind tephritid OBPs to shed new light on structures that can be exploited as novel attractants. Both OBPs showed the highest binding affinity to (_E,E_)-\(\alpha\)-farnesene, which is one of the major components of medfly male pheromone bouquet, and is known to attract females [321], suggesting its role as a natural ligand for these OBPs. The verification of the behavioural responses to the presence of ligands _in vivo_ is essential to identify volatile semiochemicals with active roles in fruit fly behaviour. Ligand-binding assays have also been performed in _B. dorsalis_ using 13 chemicals, including pheromone components and attractant molecules, and six proteins with high expression in the antennae (five OBPs and one CSP) [318]. Authors showed that OBPs displayed the highest affinity to the attractants, and, in the case of BdorOBP83a-2, RNAi led to a decrease in neuronal responses to tested molecules, as shown by EAG recordings and behavioural responses. Computational reverse chemical ecology (CRCE) is another method applied to the discovery of behaviourally active chemicals [358; 359]. OBP sequences can be exploited for 3D model prediction, producing 3D structure for docking studies using specific tools. Molecular docking is commonly employed in pharmaceutical research for structure-based drug design [360; 361]. It implies the use of programs based on different algorithms applied to model the interaction between a small molecule and a protein at the atomic level. This allows the exploration of the behaviour of small molecules in the binding pocket of target proteins [362]. CRCE has been implemented in _B. dorsalis_ to screen 25 chemicals for their binding potential to a general OBP (GOBP) showing that this approach may be extremely useful to quickly predict behaviourally-active semiochemicals, for example selecting chemicals belonging to specific classes [358]. The described approach is particularly beneficial especially in tephritids given the wide absence of direct crystallographic data for OBP binding modes.