The role of symbiotic bacteria in chemical signaling in reptilian host species

Every animal hosts diverse microbial communities, including bacteria, fungi, and protozoa, which together constitute its microbiome. Symbiotic bacteria play a pivotal role in many vital host processes. Despite their relevance, studies exploring bacterial involvement in mate choice are scarce and fragmentary. Many lizards possess femoral glands producing a rich mixture of chemicals, including volatile compounds crucial for mate choice. This project will use the sand lizard (Lacerta agilis) to understand how symbiotic bacteria mediate intraspecific chemical communication. We will study microbial communities from both sexes to (task 1) characterize the bacteria associated with femoral glands, and (task 2) explore bacterial dynamics during the reproductive cycle to determine whether a stable functional core microbiome exists and which bacterial groups are overrepresented during mating season. We will also isolate and culture femoral gland bacteria to (task 3) characterize the volatile organic compounds (VOCs) they release and test whether more diverse microbiomes yield richer chemical cocktails. Functional groups of bacteria involved in volatile production will be identified. The final goal (task 4) will test whether mate choice is mediated by scents produced by glandular microbes. For this, male lizard microbiomes will be altered with antibiotics, and behavioral bioassays will compare female responses to treated vs untreated males. We will use modern NGS (metabarcoding and shotgun metagenomics) to decipher the femoral gland microbiome, expected to be distinct and diverse, enriched in anaerobes and lipophilic bacteria adapted to hydrophobic conditions. A stable core microbiome is predicted to ensure pheromone production. Additionally, bacterial cultures combined with SPME-GC/MS will reveal volatile profiles and bacteria-compound relationships. Microbial diversity should correlate with the number of volatiles produced, as many bacteria (i.e. ca 50-80%) can synthesize them. Certain volatile classes, e.g., fatty acids, should link to specific taxa like fermentative anaerobes. Finally, behavioral assays will test whether females prefer males with unaltered microbiomes capable of producing normal scent cues. The project will advance understanding of microbiome function in animals and determine whether pheromone-based mate choice is modulated by host-associated microbes. For the first time, the inherent microbiome of reptile epidermal glands will be characterized, identifying bacterial volatiles and experimentally testing their role in sexual selection.