**1. Introduction**

The European honey bee, *Apis mellifera* (Linnaeus, 1758) is the most effective and globally distributed pollinator not only of a large number of important crops but also of wild flowering plants, some of which play an essential role in maintaining ecosystem services [1–3]. Over the past several decades, there has been a significant reduction in bee colonies, especially in some geographical regions (e.g., North America), which has raised great public and societal concern [4,5]. The decline of the honey bee population has the most tangible effect on food sources for human and livestock, disrupting wild plant pollination and diversity, altering ecological interactions and function, decreasing crop yields, reducing the yield of bee products, a large number of which have important medical value, etc. [6–9]. A honey bee colony may harbour a wide variety of disease agents and pests, bacteria, fungi, honey bee-associated viruses, parasitic mites and even other insects that try to take advantage of the rich resources contained within bee colonies [10,11]. Among them, *N. ceranae* (Fries et al. 1996) has been implicated in inflicting annually heavy

**Citation:** Shumkova, R.; Balkanska, R.; Hristov, P. The Herbal Supplements NOZEMAT HERB® and NOZEMAT HERB PLUS®: An Alternative Therapy for *N. ceranae* Infection and Its Effects on Honey Bee Strength and Production Traits. *Pathogens* **2021**, *10*, 234. https://doi.org/10.3390/ pathogens10020234

Academic Editors: Giovanni Benelli and Giovanni Cilia

Received: 31 December 2020 Accepted: 18 February 2021 Published: 19 February 2021

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losses on beekeeping [12,13]. Until 2017, two microsporidia were known—*N. apis* (Zander, 1909) (causing nosemosis Type A) and *N. ceranae* (causing nosemosis Type C) [14]. *N. ceranae* is specific for the Asiatic honey bee (*Apis cerana*, Fabricius, 1793); however, presumably after 2003, *N. ceranae* has switched its hosts and has begun to infect the European honey bee (*Apis mellifera*) [15,16]. Nowadays, *N. ceranae* has become widespread microsporidia in many regions in the world [17,18]. Based on ultra-structural and molecular investigations, a new *Nosema* species in *Apis mellifera*, namely *N. neumanni* (Chemurot et al. 2017), was described in Uganda in 2017 [19]. The importance of *N. ceranae* spillover is not limited to the honey bee but also to other insect species like the bee-eater *Merops apiaster* [20], the South American native bumblebee, *Bombus brasiliensis* (Lepeletier, 1836) [21,22], stingless bees and social wasps [23], solitary bees [24], the small hive beetle, Aethina tumida (Murray, 1867) [25], etc. Numerous studies have indicated that *N. ceranae* has become a worldwide distributed microsporidian pathogen, including in Central Italy [26,27], Croatia [28], Lithuania [29], etc. In an investigation on the prevalence of *Nosema* spp. in temperate and subtropical regions, pure *N. ceranae* infection and *N. ceranae*/*N. apis* co-infection were detected in apiaries from both regions, while pure *N. apis* infection was exclusively observed in the subtropical region [30].

It was found that *Apis cerana* showed a higher immune response and lower *N. apis* and *N. ceranae* spore loads than *A. mellifera*, suggesting that Asiatic honey bees may be better able to defend themselves against microsporidia infection [31].

The most prominent negative influences on honey bee colonies include: suppression of the honey bee immune system [32], shortening of worker bee lifespan [33], the decline in colony strength and productivity [34], queen supersedure [35], increased winter losses and colony collapse [36]. All these adverse effects of *Nosema* spp. on honey bee colonies require the search and development of effective strategies against these widespread parasites. For more than several decades, bicyclohexylammonium fumagillin (isolated from the fungus *Aspergillus fumigatus*) has been widely used as an anti-*N. ceranae* antibiotic [37]. Recent studies have shown that this antimicrobial agent is becoming less and less effective against *N. ceranae* infection [37,38]. Some researchers have further found that fumagilin is rather toxic and may provoke tumorigenic formations in humans. Moreover, it has negative effects on bee health and even leads to hyperproliferation of *N. ceranae* spores [39,40]. The observed toxic effects of fumagillin require strict measures regarding its use in many countries [41].

Considering the above, it seems essential to develop new, alternative approaches against nosemosis. Until now, there have been several new basic approaches to control nosemosis in honey bees.

#### *1.1. Use of Small Molecules*

The use of biologically active small molecules represents a promising approach against nosemosis [38]. A large number of organic compounds have been tested for control of nosemosis. These include: porphyrins (Porphyrin: PP(Asp)2 and Porphyrin: TMePyP), inhibitors of the enzyme methionine aminopeptidase type 2 (MetAP2), phenolic acids (formic acid, oxalic acid, etc.), polyphenol compounds (resveratrol and thymol), etc. [42–44]. Although they represent a reliable alternative therapy in the combat against nosemosis, a disadvantage of these compounds is that after their use viable spores remain in beehives, combs, and feces [38]. Thus, there is a real danger of infection or re-infection in the treated honey bee colonies.

#### *1.2. RNA Interference as a Gene Regulating Expression Approach*

Another approach for treating *N. ceranae* infection is associated with the use of RNA interference (RNAi). RNAi represents a biological process in which small RNA molecules (microRNA (miRNA) and small interfering RNA (siRNA)) inhibit gene expression or translation, by degrading targeted messenger RNA (mRNA) molecules via post-transcriptional gene silencing [45]. RNAi is widely used in human medicine and represents a promising

new anticancer approach [46]. In beekeeping, RNAi technology has been used to protect honey bees from infection by various pathogens and parasites [47]. In vitro studies have shown that RNAi can be applied successfully against some honey bee-associated viruses and the ectoparasitic mite *Varroa destructor* (Anderson and Trueman, 2000) [48,49].

Using an RNAi strategy to reduce the expression of some honey bee genes (gene silencing) has been one of the key measures against nosemosis [50,51]. An example of this is the upregulation of the mRNA levels of the naked cuticle gene (nkd) in adult bees by means of *N. ceranae* infection provoking a suppressed host immune function [52]. It has been found that the oral application of nkd double-stranded RNA (dsRNA) in *N. ceranae*infected bees, i.e., silencing the host nkd gene, can activate the immune response, suppress the reproduction of *N. ceranae*, and improve honey bees' health status [52]. Another similar strategy, but this time with the use of RNAi-based gene silencing on parasitic DNA, is the downregulation of the gene encoding *N. ceranae* polar tube protein 3 (ptp3) through the application of dsRNA that is homologous in gene sequence [50]. The ptp3 is the part of the polar tube structure relevant to host–parasite interaction, contributing to the parasitic invasion [53]. It has been demonstrated that the oral application of a dsRNA corresponding to the sequences of *N. ceranae* ptp3 gene silences the expression of the corresponding ptp3 in *N. ceranae*-infected bees. As a result, *N. ceranae* load reduction, improvement of host physiological status, and extension of lifespan in infected bees have been observed [49]. The application of this therapy has also its drawbacks. One of them is related to the degradation of the dsRNA molecules inside insects' guts, which is associated with additional costs regarding the protection of the dsRNA molecules from insect gut nucleases [54]. Other limiting factors include: the insects' gut pH and the related activity of the restricted enzyme (affecting the stability of dsRNA), the amount of the dsRNA molecules when administered orally in target insects, the length of the dsRNA molecules, and the life stage of the insects (larvae, pupae or adults) [54]. To overcome these obstacles, dsRNAs have been incorporated in liposomes or nanoparticles, and then these particles have been delivered to insects through feeding on an artificial diet [55]. Nanoparticles/liposomes stabilize the dsRNA molecules, thus ensuring the greater efficiency of the RNAi process.

The results obtained from the use of RNAi technology have clearly demonstrated the prospects of its applications in anti-nosemosis therapy, but more research is needed in order to be widely implemented in beekeeping practice.
