*4.2. Experimental Design*

The study was conducted during the period of 11 months (5 August 2019–8 June 2020) at the Experimental Apiary of the Research Center of Stockbreeding and Agriculture— Smolyan, Bulgaria (41◦35- 7.01-- N, 24◦41- 30.98-- E). The apiary is located in Smolyan municipality—the Perelik-Prespa part of the Western Rhodope Mountains. The apiary consists of 60 colonies of *Apis mellifera rodopica* (local ecotype of *A. m. macedonica*) housed in Langstroth Rut hives. All hives have exposure to the same environment and the same forage conditions.

For the purposes of our investigation, 45 of those colonies were selected based on the presence of *N. ceranae* infection, which was detected in the 25 forager bees sampled from each colony both microscopically and with PCR analysis. Forager bees can be distinguished from house bees based on appearance (with less, darker hairs in the chest area), presence of visible pollen load on their legs, and location in the hive (mainly in the part of the combs occupied by food supplies). Considering the presence of *N. ceranae* infection, the 45 honey bee colonies were divided into three groups—two experimental groups NH (*n* = 15) and NHP (*n* = 15), and a control group C (*n* = 15). The bee colonies were equalized in terms of bee colony strength, sealed worker brood area, amount of honey and stored pollen area.

The treatment of the bee colonies with herbal supplements was carried out once in the autumn (5 August 2019) and once in the spring (9 April 2020). The bee colonies in the experimental groups were treated 4 times at 7-day intervals with NOZEMAT HERB® and NOZEMAT HERB PLUS® (Extract Pharma, Sofia, Bulgaria) at a dose of 10 mL of the product dissolved in 100 mL of sugar syrup (1:1, w/w), according to the manufacturer's instructions (Extract Pharma Ltd., Sofia, Bulgaria). The solution was sprayed with a syringe onto the bee combs in each experimental hive. The bee colonies of the control C group were sprayed only with sugar syrup (1:1, w/w), at the same dose as the experimental groups.

Before each pre-treatment (August 2019 and April 2020) and about two months posttreatment (October 2019 and June 2020), 20 forager honey bees were sampled from each colony from the three investigated groups for microscopic (stored at 4 ◦C) and PCR analysis (stored at −20 ◦C) for *N. ceranae* examination.
