*4.3. Microscopic Detection of N. ceranae*

The bees were sent to the National Reference Laboratory for Bee Diseases at the National Diagnostic Science and Research Veterinary Medical Institute (Sofia, Bulgaria) in a cooler bag. The abdomens of 20 forager honey bees from each colony of the experimental groups and the control group were macerated in 3 mL of distilled water, and the pellets were filtered and centrifuged for 10 min at 1000× *g*. A 100 μL aliquot was placed on a microscope slide and covered with a coverslip. *N. ceranae* spores were counted at ×400 magnification. Positive samples were recounted for an accurate spore count using a Neubauer hemocytometer on (0.1 μL volume). One *N. ceranae* spore observed in the entire hemocytometer's grid (25 × 16 = 400 small squares) was equal to an average of 1500 spores per bee. The reported information was the number of spores per bee [112].

#### *4.4. DNA Extraction, PCR Amplification and Sequencing*

After light microscopy, a total of 150 spore samples from each investigated colony were investigated by a PCR analysis in the experimental groups and the control group in the pre-treatment period. For each of the suspensions of isolated *N. ceranae* spores, an aliquot of 50 μL was transferred to a new tube and centrifuged at 15,000× *g* for 10 min. The total DNA was isolated from the obtained supernatant by using a GeneMATRIX Tissue DNA purification kit (Cat. no. E3550, EURx Ltd., Gdansk, Poland) as per the manufacturer's instruction. Briefly, the pellet was resuspended in a buffer Lyse T, and 20 μL of Proteinase K were added and incubated overnight at 56 ◦C with shaking. The quality and quantity of the isolated DNA were checked by 1% agarose gel electrophoresis and then visualized under UV trans-illuminator gel documentation systems after staining with SimpliSafe™ (cat. no. E4600; EURx Ltd., Gdansk, Poland). The isolated DNA was stored at −20 ◦C before analysis.

Considering that *N. ceranae* is becoming a globally distributed pathogen, we decided to perform molecular detection on these microsporidia.

The small-subunit (SSU) rRNA (*16S rDNA*) gene was chosen for molecular detection of *N. ceranae*, using the primers 218MITOC—FOR (5- -CGGCGACGATGTGATATGAAAATATTAA-3- ) and 218MITOC—REV (5- -CCCGGTCATTCTCAAACAAAAAACCG-3- ) designed by Martín-Hernández et al. [113]. Negative controls were included in all PCR experiments. As a positive control, cytochrome c oxidase subunit 1 (*coI*) gene fragment of *Apis mellifera* was

used in all the studied samples. The sequence of primers used for positive control was CoI2- F (5- -CCTGATATAGCATTTCCTCG-3- ) and CoI2-R (5- -TGTGAATGATCTAAAGGTGG-3- ) designed on the basis of the complete mitochondrial genome of *A. m. ligustica* (Acc. no. L06178) [114].

All PCR reactions were performed with 10 ng template DNA in a final volume of 50 μL (NZYTaq II 2 × Colourless Master Mix, cat. no. MB354; NZYTech, Lda.—Genes and Enzymes, Lisbon, Portugal). The PCR conditions were as follows: initial denaturation at 94 ◦C for 5 min; 30 cycles of denaturation at 94 ◦C for 30 s, primers hybridization at 50 ◦C for 30 s, elongation at 72 ◦C for 1 min, and final elongation at 72 ◦C for 10 min. The successfully amplified products for *N. ceranae* were purified with a GeneMATRIX PCR/DNA Clean-Up Purification Kit (cat. no. E3520; EURx Ltd., Gdansk, Poland) and sequenced in both directions using a PlateSeq kit (Eurofins Genomics Ebersberg, Germany).
