Real-time volatilomics reveals microbiota and pathogen fingerprints in the honey bee

Understanding the complex relationship between gut microbiota and their hosts often relies on invasive sampling techniques. Honey bees provide a tractable model for host-microbe studies. Here we establish single-bee volatilomics using secondary electrospray ionization (SESI-HRMS) to examine volatile organic compounds released to the air around an individual live honey bee. Specifically, we focused on primary gut microbiota metabolites present in gnotobiotic bees. Our findings reveal distinct volatilome profiles in honey bees that depend on their gut bacterial colonization state. We cross-validated our findings using an established metabolomics technique, LC-HRMS, to compare and contrast the metabolites detectable with each mass spectrometry-based method. Finally, we assessed the ability of SESI-HRMS to detect colonization with the bee pathogen S. marcescens . By comparing the volatile signature of this bacterium grown in liquid culture with that of infected honey bee headspace, we identified overlapping compounds, including butane-2,3-diol, that were absent in uninfected bees. SESI-HRMS volatilomics, paired with LC-HRMS, therefore has potential to identify non-invasive biomarkers of bee microbiome composition and infection at the level of individual insects. These biomarkers represent practical targets for the development of simple, field-ready diagnostic tools for monitoring pollinator health.