## 3 Results and Discussion  
### 2-Heptanone Production in Bacterial Cultures  
This study aimed at the identification and quantification, in bacterial culture filtrates, of 2-heptanone, whose protective role against honey bee pathogens has been previously reported [13]. The GC-MS analysis of CFS from the selected bacterial strainsrevealed a complex mixture of volatile compounds; among them, 2-heptanone was found at the retention time of 19.10 min (Figure 1). The gas chromatographic profile comprised many other compounds potentially interesting for their bioactivity; among which were 2,5-dimethylpyrazine at a retention time of 20.46 min, phenyl methanol (retention time of 26.90 min) and phenyl ethanol (retention time of 31.13 min). Interestingly, ant-associated bacteria have been reported to produce volatile pyrazines, including 2,5-dimethylpyrazine, previously identified as ant trail and alarm pheromones [33]. These compounds will be the object of further investigations on bioactive volatiles produced by hive-associated bacteria. Most of the other identified peaks are referred to as volatile fatty acids produced by microbial fermentation. The mass spectra of 2-heptanone from the extract of bacterial culture filtrates corresponded to that from the synthetic standard and to the spectrum in the Wiley 7n library, thus confirming the specificity of the results (Figure 2). 2-heptanone was found to be produced by both tested _A. bacterium_ strains, by one _B. thuringiensis_ and by one _A. kunkei_ in concentrations ranging from 1.5 to 2.6 ng/mL (Table 1). Traces were found in the culture filtrate of _B. asteroides_ and of the other two _A. kunkei_ strains. The production of this compound therefore seemed to be strain-specific in the case of _B. thuringiensis_ and _A. kunkei_, since it was found only in one out of three isolates in both cases. Aliphatic ketones are historically known as insect alarm pheromones. Previous quantitative data, measured on insects, gave higher concentrations of these ketones than the present ones. In extracts of heads of _Atta texama_ and _Atta cephalotes_ ants, 2-heptanone was determined at around 160 ppb and in trace amounts, respectively [34]. In hives, the role of 2-heptanone appears to depend on its concentration; it is thought to be attractive in low concentrations and repulsive in higher concentrations, but contrasting results have been reported so far. It was originally hypothesized that this compound, secreted from the glands in honey bee mandibles, acted as an alarm pheromone stimulating defensive reactions against a potential threat [35; 36]. Nevertheless, [PERSON] and colleagues (2012) [13] demonstrated that it triggered no defensive responses when applied at colony entrances in doses of 0.001, 10 and 1000 \(\mu\)L, whilst it acted as a local anesthetic of Varroa mites when 0.061 \(\mu\)L of pure compound were applied topically on mites. In hive applications of up to 500 \(\mu\)L of pure 2-heptanone, it resulted that at no time did all bees fully or permanently exit the observation hive [14]. The low concentrations of 2-heptanone produced by the bacterial strains investigated in this study (<3 ng/mL), together with the high volatility of this compound, imply no side effects on the bees. Furthermore, in hives, its evaporation due to high temperature and humidity would be so quick that an inves  
Figure 1: GC-MS chromatogram of the _Acetobacteraceae bacterium_ strain IM_G(L)3 culture filtrate. tigation was specifically performed to maintain the compound longer to use it as a mite repellent [15]. The results of this study rather suggest that 2-heptanone may have a role in microbial chemical communication, and further studies are needed for the exploitation of 2-heptanone-producing bacteria for the control of honey bee pests and pathogens. In this study, 2-heptanone production by _A. bacterium_ was reported for the first time.