*4.5. Evaluation of Honey Bee Strength, Sealed Worker Brood and Food Supplies*

During the investigation period, 12 measurements were performed in all investigated groups (experimental and control) to determine the strength of the colonies (based on mass) and 11 measurements to determine the sealed worker brood area, the amount of honey and the stored pollen area. These measurements were performed once pre-treatment (August 2019) and four times post-treatment, at 12-day intervals, until the end of September (2019). After the winter period, the same measurements were performed once before the second pre-treatment (April 2020) and four times post-treatment, at 12-day intervals, until the beginning of June (2020).

The following parameters characterizing the development of the bee colonies were determined:


#### *4.6. Statistical Analysis*

The dependent variables studied and normalized by linear transformation were: number of *N. ceranae* spores, strength (mass) of honey bee colonies, sealed worker brood area, amount of honey, and stored pollen area. These variables in the investigated groups were compared using F-statistic, a one-way ANOVA analysis of variance (IBM SPSS Statistics 23.0 for Windows) with a post hoc Tukey HSD test for multiple comparisons with Bonferroni and Holm correction.

The obtained sequences (219 bp the part of *16S rRNA* gene) were deposited in the GenBank database under accession numbers MG657260.

#### **5. Conclusions**

This is the first study to evaluate in vivo the effects of NOZEMAT HERB® and NOZE-MAT HERB PLUS® on *N. ceranae* spore loads, honey bee strength and production traits for two consecutive years. Both herbal supplements increase the strength of the bee colonies, with NOZEMAT HERB® showing a stronger impact. The sealed worker brood area does not seem to be significantly affected by the treatment. However, both supplements seriously increase the amount of capped honey, and this effect is maintained throughout

the year. The supplements also increase the stored pollen area throughout the year, with NOZEMAT HERB PLUS® having a greater effect.

Both herbal supplements can be successfully used as an alternative therapy against nosemosis. Furthermore, they are not toxic to bees and are easily ingested by the latter. As plant extracts, they are completely safe for humans, animals, and the environment. Therefore, they can be fed as natural food supplements all year round in combination with sugar syrup or as honey bread for winter feeding.

**Author Contributions:** Conceptualization, R.S. and R.B.; methodology, R.S., R.B. and P.H.; investigation, R.S. and R.B.; data curation, R.S., R.B. and P.H.; writing—original draft preparation, R.S., R.B. and P.H.; writing—review and editing, R.S., R.B. and P.H.; All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was funded by the National Scientific Fund of the Bulgarian Ministry of Education and Science, (grant numbers 06/10 17.12.2016). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

**Institutional Review Board Statement:** The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Ethics Committee of the RESEARCH CENTRE OF STOCKBREEDING AND AGRICULTURE, Smolyan, Bulgaria (protocol code 627/30.03.2015).

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author.

**Acknowledgments:** We thank Ilian Gechev for kindly providing both herbal supplements. We also thank Maya Marinova (IBPhBME-BAS, Bulgaria) for the English editing.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

