###### Abstract  
In the study of the chemical communication of adults of the Melolonithidae family, bacteria have been observed in the epithelium of the genital chamber; possibly, bacteria are involved in the production of sex attractants in their hosts. Therefore, it is important to identify the volatile organic compounds from bacteria (VOCsB) released by these microorganisms and study the biological activity stimulated by VOBCs in adults of Melolonithidae. In this study, bacteria were isolated from the genital chamber of _Cyclocephala lunulata_ and _Cyclocephala barrerai_, from which VOCsB were extracted using static headspace solid-phase microextraction (SHS-SPME) and dynamic headspace Super Q solid-phase extraction (DHS-SPE) and analyzed using gas chromatography-mass spectrometry. The effect of VOCsB on the hosts and conspecifics was evaluated utilizing an olfactometer and electroantennography (EAG). Two species of Enterobacteria were isolated from the genital chamber of each female species, and VOCsB derived from sulfur-containing compounds, alcohols, esters, and fatty acids were identified. An attraction response was observed in olfactometry studies, and antennal responses to VOCsB were confirmed in EAG bioassays. With these results, new perspectives on the relationship between these beetles and their bacteria emerge, in addition to establishing a basis for management programs in the future. Cytocephala; microbial natural products discovery; volatile organic compounds from bacteria; attraction; bioassays; EAG +
Footnote †: journal: molecules  
Article  
pastures [2; 5; 6]. For the monitoring and management of these beetle species, studies have been carried out to take advantage of the substances involved in intraspecific interactions [7; 8; 9; 10], focusing on volatile organic compounds involved in attracting a partner for copulation, such as sex pheromones [11; 12]. Bacteria have been observed in the reproductive system of females of Melolonthidae, specifically in the accessory glands and the genital chamber-structures associated with sex pheromones production [10; 11; 12; 13; 14]. Based on this evidence, it has been suggested that bacteria participate in the production of these chemicals, such as in the case of _Morganella morganii_ found in the accessory glands of female individuals of melolonthid _Costelytra zealandica_ beetles. These microorganisms produce volatile organic compounds from bacteria (VOCsB) such as phenol, which has been reported as a component of the sex pheromone of _C. zealandica_[15; 16]. The presence of the enterobacteria _Klebsiella oxytocin_ and _Klebsiella michiganensis_ have also been recorded in the genital chamber of _Phylophaga obselata_, where a similar relationship may occur [17]. These relationships have not been studied in _Cyclocephala_ (Coleoptera: Scarabaeoidea: Melolonthidae) [3]. Bacterial growth conditions, such as culture media, influence VOCsB diversity and abundance, so it is not appropriate to use general culture media to carry out studies of the production of VOCsB. Bacteria must be cultivated in specific culture media [18; 19; 20] containing 10% glucose and sulfur compounds, as the triple-sugar medium iron (TSI) allows the synthesis of a greater diversity of VOCsB [21]. In the extraction of VOCsB, static headspace solid-phase microextraction (SHS-SPME) has been used to capture these compounds; due to the sensitivity and how they adapt to the bacterial culture, the fibers that have been used in various studies are those of polydimethylsiloxane (PDMS), polyacrylate (PA), and/or divinylbenzene (DVB) for VOCsB of Enterobacteriaceae [22; 23; 24]. However, SHS-SPME is a destructive test, which limits its use in bioassays, so dynamic headspace Super Q solid-phase extraction (DHS-SPE) [25; 26] is an alternative to obtain samples that can be tested in attraction bioassays; the two forms of extraction are followed by VOCsB identification by gas chromatographic mass-spectrometry (GC-MS) [19; 24].