## 3 Volatile Pheromones
### Analytical Approaches to Unravel Pheromone Composition
#### 3.4.1 Conventional Chemical Ecology Approaches
oleae_, olean(1,7-dioxaspiro[5.5]undecane) was isolated and identified as the major component of the female-produced sex pheromone in 1980 [79]. (4\(R^{\ast}\),6\(R^{\ast}\))-4-hydroxyoleane and (3\(R^{\ast}\),6\(R^{\ast}\))-3-hydroxyoleane (the asterisks indicate the chiral centers) were then isolated as minor components [79]. Bioassays involving the synthetic enantiomers of oleane showed that (\(R\))-oleane is active on males, while (\(S\))-oleane on females, and GC analyses showed that females can produce (\(\pm\))-oleane [207]. The synthesis of enantiomers of 4-hydroxyoleanes was conducted by Mori and co-workers [286] and a recent study identified and synthesised 11 new \(B\). _oleae_ female-specific components [287]. Knowledge of pheromone composition in _Rhugoletis_ is still limited to \(R\). _cerasi_ and \(R\). _batava_. In this last species, Buda and colleagues recently applied GC-MS analyses of fly headspace and found that males emit (-)-\(\delta\)-heptalactone. The authors then synthesised the two enantiomers of (-)-\(\delta\)-heptalactone using enantioselective synthesis and found that only (-)-\(\delta\)-heptalactone elicited an electrophysiological response in both sexes, proposing that this chemical may act as aggregation pheromone [61]. Despite these research efforts, similar studies focusing on the optimisation of chemical synthesis of pheromone components are completely missing for the genus _Anastrepha_, as well as for many other species belonging to the _Bactrocera_, _Ceratitis_, _Rhugoletis_ and _Zeugodacus_ genera. Identification of Electrophysiological-Active Volatiles  
GC-EAD uses the electrophysiological response from a dissected insect antenna to assign activity to a gas chromatographic peak. The GC eluent is normally split between a flame ionization detector (FID) and the insect antenna, which is connected to an amplifier and recorded by silver or platinum electrodes. The response from each detector is recorded simultaneously so that an EAD response can be correlated with a specific peak in the FID chromatogram. This technique is particularly useful for the assignment of activity to chemicals which may be present as minor components in complex mixtures, although it provides no information about the function of the active compounds. GC-EAD analyses of male pheromone or rectal glands extracts have been performed on several tephrited species, including _C. fascicentris_, _C. anonne_ and _C. rosa_[214], _C. capitata_[221; 241], _B. oleae_[288], _A. serpentina_[193], _A. fracterulus_[177; 289; 290; 291], _A. obliqua_[243] and _A. striata_[292]. Additional studies performed GC-EAD analyses on the headspace volatiles of both sexes, in the species \(R\). _batav_[61], _Bactrocera fruenfeldi_ (Schiner) [293], _B. musae_[76], and _C. capitata_[223]. In addition to GC-EAD, gas chromatography-electropalpogram detection (GC-EPD) has begun to be used to test the responses of tephritid maxillary palps to the pheromone emissions [293]. Furthermore, electroantennography-based experiments can be performed independently from gas chromatography. In this case, pure compounds are employed to assess the response to a certain stimulus and the required dose to elicit a detectable response. In this case, chemicals are delivered to insect antennae/palps in controlled conditions by pumping the odour from a reservoir [256]. Since early works in the '80s [244; 294; 295], numerous EAG-based studies have been performed in tephriids to test the electrophysiological response to volatiles of fruits and flowers [296; 297; 298; 299], artificial attractants [300; 301; 302; 303; 304], as well as pheromone components [305; 306]. The use of GC-EAD brought the EAG-based approaches to a higher level of sophistication by using the antenna/palp of the target insect as a detector for the gas chromatograph.