*2.1. Experimental Set up*

Cage experiments were carried out in the microbiology laboratory of the Department of Agricultural and Food Sciences, University of Bologna, in the period between October 2019 and June 2021. Newly emerged honey bees (*Apis mellifera ligustica* were obtained from multiple brood frames of emerging honey bees within the University of Bologna experimental apiary (San Lazzaro, Bologna, Italy, 210 m a.s.l.) in a continental climate. At least two independent laboratory cage tests were performed for each feed ingredient in order to validate results. The first set of assays, referred to as "First screening", was organized in order to study the effects of the feed ingredients on honey bee survival and parasite development. After this first screening, a second one, referred to as "Derived tests", was performed in order to confirm the obtained results or to test new hypotheses derived from the first one. Moreover, if the results showed a promising trend, even if not significant, the dosage of the active ingredient was increased, determining two different dosages for some of the ingredients [AA, NisA and GRA] marked as "low" (\_L) and "high" (\_H). Only para-coumaric acid and acetic acid were tested a third time using summer honey bees due to the contrasting results obtained in the two tests. All the tests were performed with the same protocol, having only differences in the season at which the newly emerged honey bees were obtaine. The ingredients assayed were chosen considering their known antimicrobial properties in both food and feed safety (organic acids and antibiotics), their immune stimulation properties (mainly plant extracts and microorganisms), but also basing the choice on traditional homemade remedies used by beekeepers (e.g., wine derivatives) that do not have a proper scientific basis. The selected compounds reported in Table 1 and are divided in four main groups: i. organic acids (acetic acid [AA], abscisic acid [ABA], p-coumaric acid [pCA]); ii. wine derivatives (ethanol [EtOH], sulphites [SUL], wine vinegar [WA]); iii. *Saccharomyces*and antibiotics (*Saccharomyces* sp. strain KIA1 [SC], a mixture of gramicidin A, B, C, and D [GRA], Nisine A [NisA]); iv. plant extracts (extract of *Opuntia ficusindica* [OPT], extract of brown alga *Padina pavonica* [PP], a mixture of manuka and tea tree oil [MT]). In all the tests, fumagillin [DCH] was used as positive control. Each assay had a dedicated control test, i.e., a group of infected bees not supplemented with any compounds in their diet [CTR]. Experimental cages had a dimension of 11 × 7 × 4 cm and were made using plastic, including a ventilation mesh. Three replicate cages, containing 50 newly emerged honey bees, were prepared for every dietary treatment and controls. Mortality in every replicate cage was registered on a daily basis, extracting the dead individuals after counting. Ten worker bees for each experimental condition were sacrificed at day 9 (experiment end) and the guts (midgut and rectum) were collected individually and stored at −20 °C until tissue analysis.

**Table 1.** Description of the different active ingredients tested in this work and its relative dose or concentration. <sup>a</sup> Dose recommended by the manufacturer. Final concentration of the ingredients are expressed as the amount of ingredient per mL of sugar syrup (1:1 *w*:*v*).

