Health and nutritional biomarkers in honeybees: opportunities and challenges under field conditions

The decline of honeybee (Apis mellifera) populations has negative consequences not only for agriculture and beekeeping, but also for ecosystems. In human and veterinary medicine, proteomics and metabolomics provide valuable biomarkers to assess the health and nutritional status of organisms. In honeybees, the application of these techniques is still in its infancy and remains underexplored from a clinical perspective. This study aims to investigate the most abundant proteins of honeybee hemolymph as potential biomarkers of health and nutritional status at the colony level. In addition, an untargeted metabolomics-based approach was applied to honeybee extracts. Samples of hemolymph were collected from honeybees in different apiaries in the province of Bologna in different periods of the year, from April to November. Hemolymph proteins were separated and quantified by 1D SDS-PAGE. Honeybee cytosolic extracts were fractionated using size exclusion chromatography (SEC) and metabolites were analyzed in fractions using mass spectrometry (Orbitrap Exploris 480, Thermo Fisher). The five most abundant hemolymph proteins, namely vitellogenin, apolipophorin I and II, transferrin and hexamerin 70a, represent a panel of biomarkers related to key metabolic processes. These proteins are subject to interesting variations depending on many physiological and environmental factors, including the honeybees’ age (nurse bees had the highest vitellogenin concentration compared to the other two sub-castes), the season (in November, a peak of vitellogenin and transferrin concentration was observed in winter bees), and the presence of parasites (in bees parasitized by Varroa, a decrease of vitellogenin, apolipophorin II, transferrin, and hexamerin 70a was detected). One hundred and ninety-eight different pathways and more than 2000 metabolites were identified. The most abundant metabolites belonged to the flavone pathway, followed by the lipoxygenase pathway. Many metabolites were of plant origin and may be related to the environmental availability of nectar and pollen, which in turn are essential for honeybee nutrition, suggesting a possible role as biomarkers of nutritional status.