2.5.2. Bee Treatment Feeding Trials

The experiment on infected bees aimed to measure the effect on bee palatability, tolerability, and N. ceranae infection of DSM patties from B. nigra (N) and E. sativa (R), prepared as described above (Section 2.4). Treatments started one-day post-infection (dpi) and included four treated groups fed with DSM patties enriched with two doses of Brassicaceae DSM, 2 and 4%, both for B. nigra and for E. sativa (N2, N4, R2, R4), and two controls each receiving unadded candy (CTRL−) or sugar water added with 25 µg/mL fumagillin (CTRL+). The six cages containing the infected bees (N = 25) were randomly assigned to the groups and incubated at 33 °C throughout the trial. Three replicates were made for each treatment. Solid and liquid feeds were supplied ad libitum and integrated with fresh food on the need. Unlimited water availability was secured by a syringe until the end of the trial.

DSM patty palatability was performed at a 1–3-day interval after inoculation by weighting the feeders and calculating the food consumption by the difference of weight. The calculated amount was divided by the number of days to obtain the daily consumption, and by the number of bees that were present in the cage at the beginning of the interval, to calculate the average individual daily intake. This amount, calculated a daily basis for each cage, was summed up for the first seven days of the treatments (8 dpi) to calculate the weekly cumulative individual ingestion. Multiplying the food ingestion data by the respective GSL concentration allowed calculating the average individual GSL intake that occurred in each cage. Ingestion data of the CTRL+ were not reported as this group received syrup instead of solid food.

The cages were inspected at 1–3-day intervals to determine even bee survival, by counting the dead bees. After counting, the dead bees were removed from the cages.

Living bees were sampled from each cage the 6, 8, 10, 13, and 15 dpi, and frozen at −20 °C until the molecular analysis was performed (Section 2.6). Whenever possible, five bees were collected per cage, even if the bee mortality prevented a complete sampling plan in all groups.

During the inspections, three and five bees escaped, respectively, from the cages with infected and uninfected groups. Those individuals were removed from the dataset and further ignored.