## 4 Discussion  
Here, we present original results of the MVOCs emitted by Enterobacteriaceaeome commonly associated with QFF and provide evidence of olfactory and behavioral modulation in virgin females. While previous MVOC-related studies generally used SPME fibers and were carried out with bacteria associated with _A. ludens_ and/or _Rhugoletis pomonella_[18; 26; 30; 31; 32; 33], we used and compared the efficiency of two different headspace collection methods, SPME and Tenax(r). Given that both methods are composed of different adsorbent materials, their efficiency at collecting MVOCs was complementary and used for different bioassays (i.e., solvent Tenax(r) extracts for behavioral assays and SPME extracts for electrophysiology). In line with previous studies, Tenax(r) filters adsorbed a larger range of compounds and exhibited a higher sensitivity, while SPME was more efficient at trapping short-chain hydrocarbons [34; 35]. Given that certain compounds (e.g., short-chain and volatile molecules) may have had a breakthrough volume based on our Tenax(r) collection parameters, and because some MVOCs (e.g., 2.5-dimethyl-pyrazine and 3 ethyl-2,5-dimethyl-pyrazine) were identified by both methods, while others were detected by only one method, our study highlights the importance of using and comparing various headspace collection methods when investigating MVOCs. Overall, the chemical composition of the bacterial extracts revealed the presence, in high abundance, of various aldehydes, alcohols, esters, terpenes, and pyrazines, as previously shown [36; 37]. Terpenes, esters, aldehydes, and alcohols are the most common chemical classes identified among the wide array of plant volatiles [38] and food lures used against Tephritid flies and detected by the fly antennae [39]. Pyrazines, which are attractive to _A. ludens_ and produced by the bacteria _K. pneumoniae_ and CF [18], were also found, among amides, in the male rectal gland of _B. cucurbiae_, as well as in orchids attractive to males [40]. Figure 7: Absolute number of virgin females _Bactrocen tryoni_ choosing between individual compounds (black bars) versus the control solvent (grey bars) in a Y-tube olfactometer. (n = 20 for each compound). Statistical significance: \(p\leq 0.05\) (*), \(p\leq 0.01\) (**), \(p\leq 0.001\) (***). Although amides were suggested to be involved in the response to sex-specific pheromones in QFF [41], the role of pyrazines remains unclear. In this study, while bacteria were not necessarily responsible for the production of all the detected pyrazines, a wider diversity of these compounds was found in bacteria, when compared to the control media, supporting the findings that bacteria can synthesize certain pyrazines [42]. While most compounds were found across the four studied bacteria, which advocates for the existence of common biosynthetic pathways across Enterobacteriaceaeae, our chemical analyses also revealed species-specific MVOC production. Although it is still unknown whether Tephritidae flies seek particular bacteria species or strains based on specific MVOC profiles, females were more attracted to indole and 2-phenylethanol, which were detected in all bacteria but CF, than phenol, which was identified in CF extracts only. We showed that the production of MVOCs also depended on the pH of the culture medium, as previously reported [43; 44]. For instance, phenol was more abundant in acidic growth media, while KO produced more 2-phenylethanol and indole when cultured in neutral and alkaline pH. Similarly, we reported that chemical profiles were specific to the nature of the growing medium. For instance, esters prevailed in apples, while hydrocarbons and terpenes were mostly present in kiwis and oranges, respectively.