## 3 Volatile Pheromones
### Analytical Approaches to Unravel Pheromone Composition
#### 3.4.1 Conventional Chemical Ecology Approaches  
The components of the complex blends of tephritid pheromones can be isolated, identified and tested using interdisciplinary approaches involving bioassays, sensory physiology, analytical and organic chemistry, and biochemistry. Recent breakthroughs in analytical techniques allow the rapid screening of semiochemicals with more sensitive bioassays and their isolation and identification from relatively smaller amounts of material. Several non-destructive and artefact-free methods are available for collecting natural odorants from living organisms. Examples include the adsorption of odours on different polymer matrices contained in cartridges or filters. Trapped odours can be desorbed thermally or eluted with organic solvents followed by analysis using gas chromatography. The direct coupling of a chemical (flame ionization detector and/or mass spectrometer) and biological detector (e.g., the use of an insect antenna) permits simultaneous isolation and identification of bioactive components from trapped odours. Furthermore, the discovery of more efficient chemical synthetic methods now allows the state-of-art synthesis of semiochemicals of high purity whose field activity may provide answers to ecological and evolutionary questions associated with the importance of the chemical in the behaviour of the target insect [259]. Volatile Collection and Analytical Techniques for Their Identification  
Chemical identification of tephritid volatiles requires a chromatographic separation followed by detection using spectrometric analytical methods. Although a wide range of methods is available in principle, the number which is suitable in practice depends upon the amount of insect material that can be obtained. Solvent extraction of tephritid rectal glands using standard solvents such as heptane, hexane, methanol, ethanol, dichloromethane and ethyl ether is commonly used [166; 213; 231; 232]. More recently, mixtures of solvents (acetonitrile/water and methanol/acetonitrile/water) were applied for the extraction of semiochemicals from the whole body of adult medflies [260]. Solid phase microextraction (SPME) is a solvent free, pre-concentration technique developed by Arthur and Pawliszyn [261] for application in solid, liquid, or gaseous samples (reviewed in [262]). The results obtained using SPME fibres are similar to those obtained with solvent extraction [263; 264; 265]. SPME is a known and effective alternative to liquid-liquid extraction. It provides some advantages over liquid-liquid extraction process because of reduced solvent consumption. In tephritids, different SPME coating materials have been used for volatile collections. A polydimethylsiloxane/divinylbenzene fibre (PDMS/DVB) was applied for pre-concentration of volatile compounds emitted by male and female medflies in different mating status (virgin or mated), and age (3 or 9 days old) [222]. In total, 70 compounds of diverse chemical classes such as alcohols, acids, aldehydes, terpen, branched hydrocarbons and esters were reported and identified by gas chromatography coupled with mass spectrometric detection (GC-MS) [222]. Similarly, PDMS fibres have been used to compare the composition of the pheromone of medfly males from a standard laboratory strain reared as larvae on laboratory media and fresh fruits [220]. Five and 30 day-old males have been used, with 36 and 27 chemicals (mostly belonging to terpen, amides, esters and alkanes) identified to be emitted by these two age-classes, respectively [220]. In a recent work on _C. capitata_ volatiles, divinylbenzene/carboxen/polydimethylsiloxane (DVB/CAR/PDMS) combined with GC-MS and gas chromatography-flame ionization detection (GC-FID) techniques resulted in the identification of 27, 23 and 29 compounds from larvae, pupae and adults, respectively [266]. PDMS/DVB SPME fibres have been applied to collect volatiles produced by _B. zonata_ males and females and _R. batwa_ males, respectively [61,213].