## 3 Volatile Pheromones
### Behavioural Assays to Identify Active Compounds  
Bioassays play key roles in the study of rephritid semiochemicals [271]. Bioassays allow to assess the behavioural effect(s) (e.g., lek formation, male signalling, courtship, copulation, host finding for oviposition, HMP deposition) of synthesised active compounds (pure or in mixture) with respect to host or mate interactions, differently from EAG assays that provide the electrophysiological responses of isolated organs, structures or olfactory receptors (ORs). The biological response measured by a bioassay is essential to attribute to a molecule or a mixture of chemicals the pheromone identity. Behavioural effects in rephritid species can be evaluated in laboratory, semi-field and field conditions where both the stimuli and the background in which the stimuli are presented are tightly controlled [314]. Techniques for insect bioassays have been widely reviewed (see for example [256; 315; 316; 317]) and widely applied to rephritid species, for which most experiments have been so far performed in laboratory conditions. Initial studies were done in close-range cage bioassays estimating medfly female attraction to male pheromone components based on landings on filter paper soaked with olfactory stimuli [218]. Currently used setups adopted for the study of rephritid olfaction include (i) two-choice systems, (ii) flight tunnels, and (iii) multi-arm olfactometers. Two-choice systems, using arenas (i.e., observation chambers) [174; 318; 319], as well as Y-tubes/T-maze [85; 231; 293; 320], have been used to evaluate attractive or repulsive responses following exposure to pheromone (isolated from headspace or rectal glands); in this framework, dynamic systems like the Y-tubes, allowing an air flow carrying the chemicals to be evaluated for the chemo-ecological role should be preferred, since they avoid the risk of saturating receptors of the tested flies, which is common in still-air arenas. Flight tunnels, also known as wind tunnels, are extremely useful to monitor medium-distance flight responses to mate-derived chemicals [201; 270; 321]. Their use, for example, allowed to assess the attraction of females to male emissions in _C. capitata_[270] and _A. serpentina_[193], or of both sexes to ME-fed _B. dorsalis_ males [70]. Wind tunnels also permitted to prove that pheromone components can exert behavioural effects in _A. ludens_ females [181]. These systems can integrate different components for the simultaneous identification of released volatile chemicals and the assessment of their attractiveness, as well as recording environmental parameters and fly activity [201], including the observation of flight patterns in females responding to male-derived volatiles [321]. Lastly, multi-arm olfactometers have been mainly used so far to test fruit odours and with flies released into an area composed of multiple chambers from which airflow-containing odour flows [322]. Moving laboratory results to the field for real-world applications is a timely challenge in rephritid research. Therefore, field assays to evaluate rephritid attraction should be considered after successful laboratory evaluation of a given compound. Field studies can be performed either using live insects/glands extracts as a source of pheromone or traps with different types of dispensers releasing pheromone pure chemicals or mixtures, as described especially for the medfly and the olive fruit fly. In the case of _C. capitata_, a synthetic blend releasing three male pheromone compounds (i.e., ethyl (_E_)-oct-3-enoate, geranyl acetate and (_E_,_E_)-\(\alpha\)-farresene), in a ratio similar to that observed in natural conditions, was effective in attracting females, as shown by trap catches [225]. Another study found that trimmedure was more effective than pheromone individual components or mixtures in trapping flies [323]. For _B.