# Chemical Composition and Antimicrobial Properties of Honey Bee Venom
## 2 Results and Discussion  
### Chemical Composition of Volatile Compounds  
The complex social behavior of honey bees includes the collective protection of the nest, which is carried out thanks to an effective system of chemical communication with the participation of VOCs. VOCs produced by [PERSON]'s gland are secreted by worker bees during alarm behavior [6; 37]. The study of these VOCs has a long history, and it was initially found that the main component that induces aggressive behaviour in bees is 3-methylbutyl acetate (isoamyl acetate) [38]. However, this volatile compound alone elicits a weaker reaction than bee stinging apparatus extracts [39]. Further studies of the chemical composition of these extracts led to the discovery of new compounds in them [3; 40; 41]. In particular, [PERSON] et al. [41], who subjected hexane extracts to chromatography, identified 22 organic C\({}_{6}\)-C\({}_{36}\) compounds. However, this analysis technique does not allow one to determine the most volatile components with fewer than six carbon atoms per molecule. On the other hand, extracts contain a number of low-volatility compounds with more than 20 carbon atoms. In this study, a more modern analysis technique was used: SPME of VOCs from the vapor phase, which is in equilibrium with the object under study, and subsequent determination of the extracted compounds by GC-MS. Figure 1 shows typical VOC chromatogram profiles of dry BV and freshly extracted BV, recorded under the same conditions. As can be seen, the volatile secretions of fresh bee venom are characterized by an increased relative content of components with a retention time of more than 30 min. In the chromatograms of dry bee venom, 89 peaks of C\({}_{1}\)-C\({}_{19}\) organic compounds of various classes were registered, the share of which in the TIC of the chromatogram was at least 0.01% of TIC, while the chromatograms of fresh venom were richer and contained 139 peaks. In the first case, 83 compounds (93%) were positively identified, and in the second case, the level of identification was lower and amounted to 77% (107 compounds). All registered components were divided into ten groups depending on their chemical structure. Table 1 presents the group composition of the volatile components of two samples of dry (Dv-1 and Dv-2) and two samples of freshly extracted bee venom (Fv-1 and Fv-2). Along with individual groups of compounds, their main representatives are given. A complete list of compounds registered on chromatograms is given in Table S1 in the Supplementary Materials. Differences in the composition of samples of dry BV obtained in two consecutive years, one after the other, are predominantly quantitative. As can be seen from Table 1, especially strong quantitative discrepancies were found in the case of aliphatic alcohols and acids. The reasons for the observed differences are not clear. Quantitative differences in the composition of fresh BV collected from overwintered and summer bees also occur, but they are not so significant. When comparing the composition of dry and fresh BV, attention is drawn to a much larger number of individual compounds and the share in the TIC of terpenids in fresh venom. The same applies to lactones, the content of which in the secretions of dry bee venom was insignificant, and in the case of fresh it was at the level of 5-6% TIC. In summary, this study significantly expands the range of VOCs contained in BV, since in an earlier study using the HS-SPME/GC-MS technique, the number of components identified was 45 [36]. Some of the compounds reported in the last cited work were not found by us. These compounds were butyl nitrile, anethole, (_E_)-cinnamaldehyde, 1-methyl-3-cyclohexene-1-carbaldehyde, cyclooctanol, geranyl acetone, geranic acid, and tetradecanoic acid. Figure 1: Chromatographic profile of volatiles in dry bee venom (**top**) and fresh extracted venom (**bottom**).