The trophic potential of pollens with different botanical origin: a comprehensive proteomic and microbiological approach

Agricultural practices, pollutants and climate change can influence plant metabolic functions as well as the spatial distribution and abundance of trophic resources for pollinators. Nectar and pollen availability or changes in their dietary profile are may also occur and lead to nutritional deficit in pollinators, and trigger additional threats to ecosystem stability. The absence of a balanced diet, concerning the pollen source diversity and quantity and quality of protein content, has been proven to be detrimental for honeybee health, gut microbiome fitness and haemolymph protein content. Our research aims at investigating the potential of different trophic sources, i.e. of different botanical origin, on the stimulation of honeybee’s immune and proteomic response and modulation of the gut microbiome development. For these objectives, a holistic approach was proposed using MALDI-MS (MALDI BeeTyping) for antimicrobial peptides (AMPs) and SDS-PAGE for vitellogenin detection in haemolymph. Moreover, q-PCR was used for gut microbiome analysis. Newly-emerged caged honeybees were daily fed with a fresh suspension of pollen and sugar syrup (1:1 w/v) for a total of 40 monofloral and 13 polyfloral pollens belonging to 8 botanical families (Cistaceae, Asteraceae, Cornaceae, Boraginaceae, Fagaceae, Leguminosae, Rosaceae and Salicaceae) collected in 5 Italian regions. After 5 days, haemolymph and guts were extracted for the MALDI-BeeTyping, SDS-PAGE, and qPCR analysis after DNA extraction. Our results showed that different pollen sources can affect both AMPs and vitellogenin content in haemolymph but without significant variations. However, using a genetic algorithm on MALDI BeeTyping spectra, we developed a model able to discriminate various small size proteins correlating with vitellogenin. Finally, qPCR results highlighted that all botanical families could influence significantly the gut core microorganisms within the genera Bifidobacterium, Bombilactobacillus, Lactobacillus, Frischella, Bartonella, Gilliamella and Snodgrassella. These results represent a first step in understanding the complex interaction between pollen sources, microbiome and honeybees’ health.