**1. Introduction**

Requirements for food production and agricultural intensification are resulting in a growing demand for insect pollination services [1]. Bumblebees (*Bombus* spp.) are vital and important insect pollinators to both the agricultural crops and wild plants in natural ecosystems, worldwide [2]. Their commercial rearing has boosted the economic importance of this insect in crop pollination [3]. Free-living bumblebee colonies are integral pollinators within native plant communities throughout temperate ecosystems. Adult bumblebees' robust size, long tongues, and buzz-pollination behavior result in their great pollination effectiveness. Therefore, they are indispensable, particularly for some plant species.

The decline in the number of insects pollinators is determined by various factors, such as climatic niche changes combined with reductions in natural food availability, the lack of the nesting materials, landscape alteration, agricultural intensification, and the spread of pathogens [4,5]. Reports of pollinator numbers declining due to the increased mortality in recent years are alarming on a global level [5–7].

Currently, pollinators are facing increased vulnerability to infectious diseases and other negative environmental stressors, such as harmful pesticides [8]. A reported decline in the abundance and diversity of beneficial insect pollinators can be caused by virus infections. The wild bumblebee population is also under the threat of viral infections [9]. Viruses are spilling over from managed and imported honeybees to free-living insect pollinators, including bumblebees [10,11]. Consequently, there is the possibility of disease occurrence after direct transmission through shared contaminated floral resources or

Toplak, I. The First Detection and Genetic Characterization of Four Different Honeybee Viruses in Wild Bumblebees from Croatia. *Pathogens* **2021**, *10*, 808. https://doi.org/ 10.3390/pathogens10070808

**Citation:** Tlak Gajger, I.; Šimenc, L.;

Academic Editor: Giovanni Cilia

Received: 29 May 2021 Accepted: 24 June 2021 Published: 25 June 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

facilitated by changes in host immune status and susceptibility [1]. Moreover, recently, genetically identical strains of deformed wing virus (DWV) were detected in honeybee colonies (*Apis mellifera*) and in *Varroa destructor*, their obligate parasitic mite, making it a vector of this virus disease [10,12].

The importation and deployment of managed honeybee and bumblebee colonies may be a source of pathogen introductions in new geographical areas or alterations in the dynamics of native parasites and causative agents of secondary diseases, e.g., viruses, that ultimately increase disease prevalence in wild bees [1]. Insect pollinator decline has become a worldwide issue [4,5], causing increased concerns over effects on global food production [5], the stability of pollination services [13], and the disruption of the plant–pollinator link [14].

It is known that RNA honeybee viruses, due to their short generation of life and high mutation rates, have already crossed species barriers and have successfully infected a wide range of new insect hosts, such as free-living wild bees—solitary bees and bumblebees, wasps, hoverflies, and ants [9,15].

In recent years, there have been several reports of bumblebees infected with viral honeybee pathogens: DWV, black queen cell virus (BQCV), Israeli acute paralysis virus (IAPV), chronic bee paralysis virus (CBPV), Kashmir bee virus (KBV), and Sacbrood bee virus (SBV) [10,15,16]. In Croatia, the prevalence and regional distribution patterns of seven different honeybee viruses was studied, and simultaneous infection of adult honeybee samples with two to four different viruses was identified [17,18].

The aim of this research was to determine the presence and quantification of different honeybee viruses (ABPV, CBPV, BQCV, and DWV) in wild bumblebee samples originating from 27 geographically different locations. This is the first record of molecular viral examinations, as well as important new phylogenetic comparison information for endemic honeybee virus strains circulating in bumblebees in the territory of Croatia.
