**3. Discussion**

About 15–20 years after the detection of *N. ceranae* in the Eastern honey bee (*Apis cerana*), these microsporidia became a globally distributed pathogen [81–84]. Currently, *N. ceranae* is considered one of the main causes of severe annual losses in beekeeping worldwide [85]. For this reason, various strategies have been developed for the control of nosemosis type C caused by *N. ceranae*. It is of crucial importance that the applied approaches are not toxic to honey bees and also to humans (through residues remaining in honey bee products), and are not polluting the environment.

One of the allopathic therapies related to the fight against nosemosis is the use of ecologic phytotherapy [86,87]. It is a suitable remedy against nosemosis due to the low toxicity to bees and the safety of the environment and human health.

A number of studies have focused on the impact of some herbal extracts on the regulation of the expression of certain genes in honey bees in order to reduce the damage caused by *N. ceranae*. For example, it has been shown that plant extracts or decoction from *Andrographis paniculate* promote Wnt and JNK pathways by upregulating the expression of certain genes (including armadillo, basket, frizzled and groucho) in intestinal cells [57]. These results have demonstrated that this Chinese herb can provide protection from *N. ceranae* infection under laboratory conditions, suggesting that it can be used in apiculture to control *N. ceranae*. Another study has revealed that *Eleutherococcus senticosus* extract contains eleutherosides (eleutheroside B + E), which have an impact on the honey bee immune system [88]. These eleutherosides increase phenoloxidase (PO, a major defense enzyme in many invertebrates) in hemolymph and inhibit the development of fungal spores. The piperine (an alkaloid in the roots of the Piperaceae family) and curcumin (a natural phenol produced by *Curcuma longa*) are known as natural supplements which increase the activity of the antioxidant system in honey bees [89,90]. They promote the activities of antioxidant enzymes such as superoxide dismutase, peroxidase, catalase and glutathione S-transferase, which reduces oxidative stress [91]. Moreover, it is observed that these herbal supplements exhibit hydroxyl radical scavenger action in honey bees, suppressing the destructive effects of the free radicals and reactive oxygen species [92]. Because *N. ceranae* is able to induce oxidative stress in bees [93], piperine and curcumin are potential candidates regarding antinosemosis therapy.

The present study is the first report of the in vivo application of two plant extracts, NOZEMAT HERB® (NH) and NOZEMAT HERB PLUS® (NHP), against *N. ceranae* and their

impact on honey bee colonies. These products are patented herbal supplements for honey bees. NH contains herbal extracts, vegetable glycerin, water, citric acid, and preservative potassium sorbate. NHP contains additional herbal extract as well as vitamin C (ascorbic acid). The exact quantitative composition of these two supplements is patent-protected and thereby not disclosed in this paper.

After a sequence analysis of the part of the 16S rDNA gene, we obtained a fragment with 219 bp. Then, we performed a BLAST analysis in the GenBank genetic sequence database to identify regions of local similarity between the available sequences for the *N. ceranae* 16S rDNA gene. We found the highest homology of our sequences (Acc. no. MG657260) with *N. ceranae* isolates from Argentina (Acc. no. KX024757) and Lithuania (Acc. no. JQ639314). Besides the conventional PCR analysis for detection of *N. ceranae* based on sequence analysis of 16S rDNA gene, a recently developed new method not only provides rapid and reliable detection of the presence of these microsporidia but also allows the quantification of *N. ceranae* (qPCR assay) based on the highly-conserved protein coding gene Hsp70 [94].

In both years of our study (2019–2020), a significant reduction of *N. ceranae* spore loads was observed after NH and NHP administration, compared to the pre-treatment level. Among the benefits from these two supplements is their positive effect on the intestinal microflora of honey bees. Numerous studies have indicated that microbial communities have an essential role in the resistance to pests and pathogens, environmental toxins and pesticide exposure [77,95–97]. As for the *N. caranae* infection, there is evidence that these microsporidia disrupt the microbiota, causing dysbiosis, which may have consequences on bee development and immune suppression [97–99]. As an intestinal infection, *N. ceranae* causes significant changes in the composition of honey bee gut microfauna, and the plant extracts that we use contain biologically active substances that have a beneficial effect on gut bacterial communities. In fact, both products (NH and NHP) contain basic polyphenols—flavonoids and phenolic acids—as biologically active compounds. According to some authors, the antimicrobial activity of plant extracts is not due to a single biologically active substance (flavonoids vs phenolic acid), but rather to the totality of all, with potentially synergistic effects [59,100]. Phenolic compounds extracted from *Artemisia dubia* and *Aster scaber* have shown a clear anti-nosemosis effect, which is a promising strategy for controlling nosemosis [101,102]. As far as flavonoids are concerned, the studies carried out so far have not identified a single representative that can be effective on its own against nosemosis [59,65]. Unfortunately, the mechanism of action of both flavonoids and phenolic compounds against *N. ceranae* has not been elucidated yet. The latter would certainly help to speed up the process against these microsporidia.

A lot of research has been carried out on the application of plant extracts supplementation against various pathogens in bee colonies, e.g., *V. destructor* and honey bee-associated viruses, *N. ceranae*, American foulbrood (*Paenibacillus larvae*) [66,87,103–105], or pests, e.g., the greater wax moth *Galleria mellonella* [106]. In addition to the effect of plant extracts, the potential beneficial effects of herbal extracts on honey bee colonies have been tracked as well. Certain researchers purposefully reflect on the impact of various plant extracts on honey bee performance [107–111].

Our study aimed to evaluate the impact of the herbal supplements NOZEMAT HERB® and NOZEMAT HERB PLUS®—on the one hand, as an alternative therapy against nosemosis, and on the other hand, considering the influence of the two products on honey bee colony strength and honey production and pollen collection for two consecutive years (2019 and 2020).

We found a significant reduction of *N. ceranae* spores load after administration of NH and NHP, compared to the pre-treatment period both in 2019 and in 2020. In contrast to the experimental groups, the control C group exhibited a less clear decrease of *N. ceranae* spores levels (Figure 1). These data are consistent with other studies in which a significant decrease of *N. ceranae* spores load was established after the application of other commercial food supplements, such as HiveAlive™ [64] and ApiHerb [65]. A similar effect was found in Chinese herbal extracts (in particular *Andrographis paniculata*), when applied as a decoction to treat *N. ceranae* infections in *A. mellifera* [55]. These results indicate that plant extracts represent a potent alternative therapy against nosemosis.

During the first year (2019) of our research, the administration of the two herbal supplements did not reveal significant differences between the experimental NH and NHP groups compared to the control C group with regard to colony strength (Figure 2). These results are an indicator of a decrease in the strength of bee colonies as autumn approaches. These data support the findings of Charistos et al. [64], who also did not observe differences between the groups in terms of colony strength after the administration of HiveAlive™ food supplement during the autumn. The first informative review in the spring of the following year (2020) showed a significant increase in colony strength in both experimental groups in contrast to the control group, which is an indicator of the more successful overwintering of the treated bee colonies. This trend continued after the second treatment (April 2019) until the beginning of the autumn period. These data support previous investigations about the positive influence of herbal supplements such as HiveAlive™, *Laurus nobilis* L. and *Agaricus brasiliensis* extracts on honey bee colony strength [64,104,107].

Similar to the indicator of bee colony strength, the sealed worker brood area did not show significant differences in all the groups after the first herbal supplement administration (Figure 3). The first informative review in the spring of the following year (2020) showed a larger sealed worker brood area in the NH group, but as a whole, this indicator was not affected by the applied plant extracts.

The amount of capped honey was affected most significantly after the second treatment of the bee colonies in April 2020 (Figure 4). After that, the amount of capped honey was significantly higher in the two experimental NH and NHP groups compared to the control C group. This indicator maintained significantly higher levels in the two experimental groups throughout the summer, which is a prerequisite for successful wintering of the bee colonies and reducing the costs associated with additional feeding during the winter.

It is interesting to note that the stored pollen area was significantly affected after the first treatment (August 2019) despite the presence of sparse flowering vegetation (Figure 5). After the second treatment the following year, a larger stored pollen area was observed in the experimental groups, and it seemed that the NHP group had an advantage over the NH and the control C group in terms of this indicator.

Although the findings of the present study clearly demonstrate the benefits from applying the two plant extracts for the reduction of *N. ceranae* spore counts as well as for honey bee performance in general, these encouraging results call for further research in order to clarify the impact of NOZEMAT HERB® and NOZEMAT HERB PLUS® on *N. ceranae* spore loads as well as on honey bees.

#### **4. Materials and Methods**
