5.2. Stimuli preparation
The prior knowledge regarding the chemical cues (source, nature and etc.) determines the procedure for stimuli preparation. If the glandular origin of the secretion is known, then a chemical extract of the gland/s can be used following these steps:
- Dissect the glands of interest under double distilled water.
- Clean unwanted tissues attached to the gland.
- Wash once or twice with double distilled water.
- Transfer the gland into a glass vial
- Extract with solvent.
As described by Millar and Sims (1998) the glands can often be extracted by soaking for a few minutes to hours. Furthermore, the extraction is solvent dependent with solvents such as alcohols penetrating membranes more effectively and yielding greater extraction of cellular content than less polar solvents (e.g. hexane). Although this approach provides relatively large quantities of compounds in the extracts, it might not correctly represent the composition of the emitted odour from the gland. The latter can be better assessed by in situ or ex situ head space collections (Millar and Sims, 1998) (see sections 2 and 3). The crude extracts can be tested directly or after fractionation using a hand-made column (Katzav-Gozansky et al., 2001) (see section 4.2.2, steps 1-5) or commercially available columns.
Analysis and quantification of the extracted components can be achieved by GC-MS with an internal standard added during the preparation of the extract as previously described in section 126.96.36.199. The amount of the glandular secretion is calculated based on the average amount of several analysed queens, all components included. This amount is considered as queen equivalent (Qeq). Where the chemical nature of the suspected mixture is known, a synthetic blend of it prepared in the natural ratio of the individual components can be used. For example in the case of Dufour's gland, esters can be synthesized from commercially available alcohols and acid chlorides following standard procedures as described by Francke et al., 2000 and Katzav-Gozansky et al. (2001), detailed below. The blend of the esters is prepared based on the relative proportions of the esters present in the queens’ total glandular constituents. The doses used are calculated as queen equivalents (Qeq) see Table 1.