## 2 Results and Discussion
This observation is in agreement with earlier studies of _O. insecticide_ and _O. sphegodes_, favouring headspace sorption extraction over solvent extraction [14,38]. \begin{table}
\begin{tabular}{c c c c c} \hline \hline \multirow{2}{*}{**Compound**} & \multicolumn{2}{c}{**Abundance in**} & \multicolumn{2}{c}{**Abundance in**} \\  & & _O. insecticide_ & _A. fargeii_ (**female**) & **EAD-Activity** \\ \hline
**1** & \(\bigstar\bigstar\) & & \(\bigstar\bigstar\) & \(\bigstar\bigstar\) \\
**2** & \(\bigstar\) & & \(\bigstar\bigstar\) & \(\bigstar\) \\
**3** & – & & \(\bigstar\bigstar\) & \(\bigstar\bigstar\) \\ _Arggogorytes nystacus_ visiting & **4** & – & \(\bigstar\bigstar\) & \(\bigstar\bigstar\) \\ _Othrus insecticide_ & **5** & – & \(\bigstar\bigstar\) & \(\bigstar\bigstar\) \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Occurrence of semiochemicals in _Ophrys insecifera_ (SPME extracts) and _Arggogorytes fargeii_ females (solvent extracts), with electroantennographic responses in _A. mystacus_ males. Figure 1: (**a**) Semiochemicals from _Ophrys insecifera_ (**1–2;** **1** = (Z)-8-heptadecene, **2** = _n_-pentadecene) and female _Argogorytes mystacus_ (**1–5;** **3** = (Z)-6-pentadecene, **4** = (Z)-7-pentadecene, **5** = (Z,Z)-6,9-heptadecadiene). (**b**) GC-MS total ion chromatograms of female _A. fargeii_ (upper trace) and _O. insecifera_ (lower trace). (**c**) GC-EAD of SPME extracts of _O. insecifera_ to antenna of _A. mystacus_ males. Two replicated analyses are shown. (**d**) GC-EAD of synthetic standards **1–5** to antenna of _A. mystacus_. Two replicated analyses are shown. In addition to comparing observations between various _Ophrys_ systems, it is of further interest to extend this comparison to other sexually deceptive orchids with known semiochemistry. Such cases are predominantly Australian, where the pollinator attractants in hammer orchids and spider orchids, unlike in _Ophrys_, have been found to be stored in relatively large amounts within the floral tissue [21; 23; 25]. The discovery of (_Z_)-8-heptadecene (**1**) in _O. insectifera_, detected by males of _A. mystaceus_, provides important insights about the chemistry of _Ophrys_ orchids. In earlier studies of the biosynthetic pathways for the longer chained C\({}_{25}\) and C\({}_{27}\) alkenes from _O. exalatta_ and _O. sphegodes_, C\({}_{16}\)- and C\({}_{18}\) activated carboxylic acids have been proposed as intermediates [32] (Figure 2). In fact, it has been proposed that in the plastid of the lip epidermis cell of the labellum of _O. exalatta_ and _O. sphegodes_, 16:0-ACP and 18:0-ACP are transformed to 16:1 \(\Delta^{4}\)-ACP and 18: 1 \(\Delta^{9}\)-ACP by SAD2, before being elongated in the cuticle [29]. If instead, 16:0-ACP and 18:1 \(\Delta^{9}\)-ACP are decarbonylated, the exact compounds found to be EAD-active in _O. insectifera_, \(n\)-pentadecane (**2**) and (_Z_)-8-heptadecane (**1**), would be formed (Figure 2). In a similar manner, our results can be compared to the pollinator attractants previously identified in _O. speculum_. Out of the blend of eight electrophysiologically active compounds that showed the highest pollinator attraction in field bioassays, three compounds: hexadecanal, (_Z_)-9-octadecenal, and ethyl oleate, show strong structural similarity with the hydrocarbons that we identified in _O. insectifera_. In fact, decarbonylation of these semiochemicals, in a similar way as proposed in the case of _O. sphegodes_ (Figure 2), would yield pentadecane (**2**) from hexadecanal and (_Z_)-8-heptadecene (**1**) from (_Z_)-9-octadecenal and ethyl oleate. Compared to the recent studies of Australian _Drakaea_ and _Caladenia_ orchids, where multiple, structurally diverse pollinator attractants have been identified in multiple species [21; 22; 23; 25], the structural similarities between the semiochemicals of _O. insectifera_, _O. sphegodes_, and _O. speculum_ are evident, all being clearly biosynthetically closely related carboxylic acid derived compounds.