*2.5. Production of Plant Extracts*

*Padina pavonica* seaweeds were collected from the Mediterranean sea during summer 2019, by hand picking method from the submerged marine rocks. The seaweeds were cleaned from impurities, dried, and powdered with a mixer. The algal flour thus obtained was extracted sequentially with hot sodium oxalate solution, hot water, 1 M and 4 M KOH solution at 20 °C according to [47] and resuspended in glycerol. The *Opuntia ficus-indica* hydroglycolic extract was obtained from fresh cladodes collected in the Malta Island during summer 2019, which were washed with distilled water and cut into small pieces before extraction. A 20:80 (*w*/*w*) water/propylene glycol mixture was used with a 1:3 (*w*/*w*) plant-solvent ratio. The cladodes were macerated for 12 h and the resultant mixture was percolated to obtain the extract [48].

#### *2.6. Treatment Administration*

Ingredients were administered in sucrose syrup (1 kg sucrose in 1 L of water) using gravity feeders starting from a day after the artificial infection with *N. ceranae* spores. The active ingredients and concentrations for each dietary treatment administered in a matrix of sucrose syrup are reported in Table 1. The tested ingredients (treatments) were supplied to honeybees a day after the artificial infection with *N. ceranae*. The treatments were available *ad libitum* during 9 days. Honey bees in every experimental cage also received tap water *ad libitum* in gravity feeders.

#### *2.7. DNA Extraction and qPCR of N. ceranae*

Extracted gut were manually macerated with plastic micro pestles in 200 μL of buffer. DNA extraction of single honey bee guts was performed with PureLink*TM* Genomic DNA Mini Kit (Invitrogen, Milan, Italy) following the manufacturer protocol with some modifications: guts were further smashed with glass beads (0.01–0.1 μm) at 50 Hz in Rotovortex. Moreover, samples were incubated at 55 °C in a water bath with 180 μL of lysis buffer and 20 μL of proteinase K per sample [49]. Fluorometric quantification of every sample was performed with Qubit Flex Fluorometer (Thermo Fisher Scientific). Extracted DNA was stored at −20 °C until further analysis. The 16S-like rRNA gene (SSU rRNA) was selected to perform *N. ceranae* specific qPCR relaying on specific primer Nc841f 5- - GAGAGAACGGTTTTTTGTTTGAGA-3 and Nc980r 5- -ATCCTTTCCTTCCTACACTGA TTG-3- [50]. The reactions were carried out on Step One thermal cycler (Applied Biosystems) with standard two-step PCR method using Fast SYBR Green PCR Master Mix (Life Technologies, Milan, Italy), according to [51,51]. The standard PCR fragment was diluted 1:10 to obtain the reference standards. The melting curve was performed in each real-time reaction to assess amplicons melting temperature (73.77 ± 0.23 °C St. Dev) according to the genetic variability of *N. ceranae*. According to Cilia et al. [52,53], the copy number of 16S-like rRNA gene in *N. ceranae* ranges from 5.7 to 11.5 per genome, therefore the obtained quantification was normalized according with the total amount of extracted DNA and divided by the average copy number of 16S-like rRNA (i.e., 8.6).
