**2. Results and Discussion**

To identify semiochemicals in *O. insectifera*, and sex pheromone candidates in *Argogorytes fargeii* pollinators, solvent extractions of flowers and insects, and floral headspace sampling, were conducted. Samples of *O. insectifera* labella were extracted in solvents of increasing polarity, from *n*-hexane, to dichloromethane, to methanol. Headspace volatile sampling was performed using solid phase extraction (SPME). Furthermore, whole females of *A. fargeii* were extracted in dichloromethane. Due to the very limited number of pollinators available, we were restricted to evaluating biological activity using gas chromatography coupled with electroantennography (GC-EAD). Since we were unable to locate males of *A. fargeii*, GC-EAD was used to detect which components of the various extracts were detected by *A. mystaceus*, a closely related species that is the second main pollinator of *O. insectifera* [9]. Two compounds from the floral extracts were repeatedly EAD-active (elicited responses in six out of 10, and two out of 10 EAD experiments). These two compounds were tentatively identified by mass spectrometry (GC-MS) as a C17 alkene and *n*-pentadecane. In previous studies on *O. insectifera*, *n*-pentadecane (**2**, Figure 1a) was indeed found to be active in EAG experiments, while no alkenes were isolated or identified [11]. Here, we found that *n*-pentadecane and the C17 alkene were present in the female *A. fargeii* (six extracts of individual insects) and were also present in only minor amounts in floral solvent extracts (three extracts of 10 flowers). We investigated the double bond location by dimethyldisulfide (DMDS) microderivatisation of a semi-preparative GC purified compound that was extracted from the wasp. The observation of identical retention times and mass spectra between the semiochemical isolated from the wasp and the synthesized (*Z*)-8-heptadecene (**1**), before and after treatment with DMDS, meant that the double bond position and configuration of the natural product could be confirmed. Furthermore, a floral extract was treated analogously, and was confirmed to contain identical mass fragments at the same relative intensity and retention time, confirming that the compound detected by *A. mystaceus* was shared between *O. insectifera* and female *A. fargeii.* In addition to the semiochemicals identified from flowers, another two C15-alkenes and one C17-diene were identified from females of *A. fargeii*. These compounds were also isolated by semi-preparative GC and treated with DMDS. Candidate compounds were synthesized and co-injected with natural extracts (on two GC columns) and tested with GC-EAD. The monoenes were subsequently confirmed as (*Z*)-6-pentadecene (**3**) and (*Z*)-7-pentadecene (**4**), while the diene was identified as (*Z*,*Z*)-6,9-heptadecadiene (**5**) (Figure 1).

**Figure 1.** (**a**) Semiochemicals from *Ophrys insectifera* (**1**–**2**; **1** = (*Z*)-8-heptadecene, **2** = *n*-pentadecane) and female *Argogorytes mystaceus* (**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. insectifera* (lower trace). (**c**) GC-EAD of SPME extracts of *O. insectifera* to antenna of *A. mystaceus* males. Two replicated analyses are shown. (**d**) GC-EAD of synthetic standards **1**–**5** to antenna of *A. mystaceus*. Two replicated analyses are shown.

The GC-EAD and GC-MS analyses of the floral extracts showed that *n*-pentadecane (**2**) was of low abundance and was electrophysiologically active in only two experiments, while (Z)-8-heptadecene (**1**) was active in six experiments. When tested as synthetics at higher concentrations (100 ng to 1 μg), both compounds were strongly EAD-active in replicated experiments. However, the additional alkenes **3**–**5** from *A. fargeii*, when tested as synthetic samples at the higher concentration, elicited consistently less frequent and/or weaker EAD responses compared to the orchid-produced **1** and **2** (Figure 1, Table 1).


**Table 1.** Occurrence of semiochemicals in *Ophrys insectifera* (SPME extracts) and *Argogorytes fargeii* females (solvent extracts), with electroantennographic responses in *A. mystaceus* males.

✔✔= very abundant compound (>20% of base peak area); repeated (6 extracts, >6 synthetic samples) strong EAD-responses. ✔ = abundant compound (>10% of base peak area); repeated EAD-responses (2 extracts, >6 synthetic samples). (✔) = occasional weaker EAD-response (generally less than 50% of response of orchid semiochemicals, >3 synthetic samples). Photo A.M. Weinstein.

By analysing the GC-MS traces of floral extracts, it was observed that larger amounts of compounds **1** and **2** were present in headspace samples of flowers compared with solvent extracts. Although headspace extractions and solvent extractions are not directly comparable, our findings indicate that the flowers likely continuously produce compounds (indicated by increasing quantity with an increase in SPME sampling time), rather than depend on stored compounds (indicated by very low amounts in solvent extracts) in the floral tissue. This observation is in agreement with earlier studies of *O. insectifera* and *O. sphegodes,* favouring headspace sorption extraction over solvent extraction [14,38]. 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 C25 and C27 alkenes from *O. exaltata a*nd *O. sphegodes*, C16- and C18 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. exaltata* and *O. sphegodes*, 16:0-ACP and 18:0-ACP are transformed to 16:1 Δ4-ACP and 18: 1 Δ9-ACP by SAD2, before being elongated in the cuticle [29]. If instead, 16:0-ACP and 18:1 Δ9-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).

**Figure 2.** Proposed biosynthesis of bioactive alkenes in *Ophrys sphegodes* (from [32]) and *O. insectifera.*

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 [21–23,25], 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. It is also interesting to note the difference in volatility compared to the widely studied Australian systems, where "traditional" volatiles are used as long-range attractants, while the European systems utilise less volatile cuticular hydrocarbons, such as the C27–C29 alkenes in *O. sphegodes,* which have been proven sufficiently volatile to lure pollinators from a distance as attractants [15]. Furthermore, it is relevant to note that in the case of *O. insectifera* and *A. fargeii*, the orchid and pollinator share the exact same semiochemicals, which is in agreement with other investigated *Ophrys* systems, including *O. sphegodes* [39] and *O. speculum* [17], as well as with most Australian systems [4] (but see [40]).

In conclusion, we have identified (*Z*)-8-heptadecene (**1**) and pentadecane (**2**) as shared semiochemicals from *O. insectifera* and *A. fargeii*. Access to denser populations of *A. fargeii* or *A. mystaceus* would be required to undertake bioassays testing the field activity of these compounds as pollinator attractants. Nevertheless, this study provides an important first step in the identification of key compounds that, once pollinator populations have been located, are available to be tested in field behavioural bioassays. Furthermore, the identification of these semiochemicals and comparison with related species within the genus shows strong commonalities in structures and suggests a conserved biosynthetic pathway for semiochemical production within *Ophrys*.
