Methodology for Single Bee and Bee Brain ¹H-NMR Metabolomics

The feasibility of metabolomic ¹H NMR spectroscopy is demonstrated for its potential to help unravel the complex factors that are impacting honeybee health and behavior. Targeted and non-targeted ¹H NMR metabolic profiles of liquid and tissue samples of organisms could provide information on the pathology of infections and on environmentally induced stresses. This work reports on establishing extraction methods for NMR metabolic characterization of Apis mellifera, the European honeybee, describes the currently assignable aqueous metabolome, and gives examples of diverse samples (brain, head, body, whole bee) and biologically meaningful metabolic variation (drone, forager, day old, deformed wing virus). Both high-field (600 MHz) and low-field (80 MHz) methods are applicable, and ¹H NMR can observe a useful subset of the metabolome of single bees using accessible NMR instrumentation (600 MHz, inverse room temperature probe) in order to avoid pooling several bees. Metabolite levels and changes can be measured by NMR in the bee brain, where dysregulation of metabolic processes has been implicated in colony collapse. For a targeted study, the ability to recover 10-hydroxy-2-decenoic acid in mandibular glands is shown, as well as markers of interest in the bee brain such as GABA (4-aminobutyrate), proline, and arginine. The findings here support the growing use of ¹H NMR more broadly in bees, native pollinators, and insects.