Assessing the analytical reliability of traditional and novel sampling methods for the study of flower rewards quality

Pollinator insects are declining worldwide also due to the alteration of their diet which plays a pivotal role in influencing their health status. Despite interspecific and intraspecific diversity in the diet, pollinators completely or partly rely on pollen and nectar as food sources. A precise characterization of the chemical composition of these flower resources represents a key step in the definition of pollinators' nutritional ecology. However, pollen and nectar represent challenging sources to collect and analyze, especially due to their small amounts per flower, and the application of suitable sampling and analysis tools is a pivotal step to perform dedicated studies and comparisons. Here, we compared a recently proposed tool based on a portable vacuum cleaner for floral pollen collection (E-PoSa, i.e., Electronic Pollen Sampler) with traditional pollen sampling methods (i.e., anther collection and anther sieving) together with the evaluation of different nectar sampling techniques (i.e., centrifugation, microcapillaries, washing, and microrinsing) by looking at the differences in their quantitative recovery as well as their chemical profiling. Pollen and nectar were collected from three model flower species each. Our results demonstrated that different collection methods introduce biases in the nutritional profiling of floral rewards and specifically: (1) underestimation of the pollen protein and lipid content in the anther collection method; (2) reduction in the volume of recovered nectar by centrifugation; (3) overestimation of the glucose content in the nectar collected by flower washing and underestimation of the glucose content by microrinsing; and (4) relevant biases in the phytochemical profiles of pollen and nectar by analyzing the whole anthers and the nectar collected by washing the entire flower. Differences in methods were not directly related to the different productivity of pollen and nectar across species. The final goal of the study is to propose standardized, comparable, and easily accessible strategies for the study of flower resources that ultimately impact on pollinators' nutritional ecology. Acknowledging the potential influences of the sampling techniques and moving toward shared field protocols will advance the comprehension of species interactions, foraging patterns, and pollinators' nutritional needs.