## 3 Volatile Pheromones
### Behavioural Assays to Identify Active Compounds
#### 3.4.2 Reverse Chemical Ecology Approaches  
In addition to the above-described techniques to identify tephritid semiochemicals, recent studies are increasingly showing that RCE has great importance in understanding the molecular basis of insect chemical perception and identifying the active volatile semiconductors [257]. Studies began to be performed to identify genes involved in chemosensory perception in tephritids, including olfactory, ionotrophic and gustatory receptors (ORs, IRs and GRs, respectively), OBPs, odorant degrading enzymes (ODEs), and chemosensory proteins (CSPs) [331]. Insect OBPs are small soluble proteins mostly found in the chemosensitive lymph of sensory organs where they bind molecules of odorants and pheromones (see [332] for a review). Thus, OBPs are considered ideal molecular targets for binding assays to identify chemicals with a potential behaviourally-active role in tephritid biology. The expression of OBPs, followed by their purification and structural analysis, is indeed adopted to perform OBP ligand binding studies; ligands are screened from sets of volatiles emitted from host plants, pheromones, or synthetic attractants used in field applications, and once identified, behavioural responses are evaluated _in vivo_[256]. Identification and Functional Analysis of OBP Genes  
Putative OBP genes have been initially identified through expressed sequence tag (EST) approaches in C. capitata [333], _B. dorsalis_[334; 335], _R. suavis_[336], and _R. pomorella_[337] and their transcriptional profile started to be explored. Subsequently, with the advent of next generation sequencing, more OBPs have been discovered through mining of RNA-seq data and whole genome sequencing in several petritid species, including _B. dorsalis, B. minimax_[104; 338; 339; 340; 341], _A. fraterculus, A. obliquau_[106; 342; 343], and _C. capitata_[344]. Antrenal proteomics profiling has been applied to _B. dorsalis_ to identify differentially expressed genes, including OBP genes, in ME-responsive males [345]. Functional studies have been performed to assess their role in odour perception. These include tracing OBP genes expression profiles in different tissues, developmental stages and in response to maturation and mating, RNA interference (RNAi) combined to electrophysiology to assess the involvement of target OBPs in odour detection, followed by behavioural assays [335; 341; 342; 343; 344; 345; 346; 347]. Purification and 3D-Structural Analysis of Identified/Expressed OBPs  
The binding specificity of OBPs expressed in the main chemosensory organs, i.e., antennae and maxillary palps, may help in the identification of pheromone/pheromone components that are still uncharacterised. Thus, OBPs that are (i) abundant in olfactory tissues, or, ideally, specific to these tissues, and (ii) showing sequence similarities to already characterised proteins known to be involved in chemical communication in other insects can be functionally characterised by using ligand-binding assays. OBPs are expressed in bacterial or yeast systems and the recombinant proteins purified with chromatographic steps using different techniques, including anion-exchange or gel filtration chromatography, or affinity chromatography on nickel columns when histidine-tags are added to the OBP sequence (see [348] for a review). Purified proteins can then be used for ligand-binding experiments and to solve their structure through X-ray crystallography or NMR spectroscopy [348]. The first, and, so far, the only available, structural characterisation of a tephritid OBP was recently obtained in the medfly using X-ray crystallography [349]. The structure of CcapOBP22 is characterised by six \(\alpha\)-helical elements, a typical feature of insects' OBPs, interconnected by three disulphide bridges. Differently from other insect OBP structures, CcapOBP22 also carries a 7 th \(\alpha\)-helix at the C-terminus, which contributes to delimit the ligand-binding pocket.