**4. Materials and Methods**

In August 2017 and August 2018, a total of 147 clinically healthy bumblebee workers of different species: *Bombus terrestis/ lucorum*, *B. lapidarious*, *B. sylvarum*, *B. pascuorum*, *B. hortorum* and *B. humilis* (Table 4) were individually collected on flowers in nature. Sampling was carried out at four different locations in Slovenia (Figure 5): 24 and 30 bumblebees were collected in Sevno in 2017 and 2018, 10 and 15 in Lukovica, 20 and 17 in Naklo and 11 and 20 in Ljubljana, respectively. At the same locations (Sevno, Lukovica, Naklo and Ljubljana) on the day of bumblebee sampling, also 10 clinically healthy honeybee workers (*Apis mellifera carnica*) were collected on flowers, for a total of 80 clinically healthy honeybee workers. In April 2018, also 15 clinically healthy bumblebee queens were collected on the same way as bumblebee workers on flowers, 10 samples at the Sevno site (5 samples of *B. terrestris/lucorum* and 5 samples of *B. lapidarious*) and 5 samples of *B. pascuorum* at the Ljubljana site. All samples were frozen and stored at minus 60 ◦C until use.


**Table 4.** Number of tested bumblebee workers of six different species collected in Slovenia in 2017 and 2018.

In the laboratory, each bumblebee was placed in an Ultra-Turrax DT-20 tube (IKA, Germany) and 3 mL of RPMI 1640 medium was added. Clinically healthy honeybees collected on the same day at each location were pooled (10 bees from the same location and at the same time of sampling in one pool) and in laboratory 5 mL of RPMI 1640 medium (Gibco, UK) was added to each sample. The samples were homogenised, and 1 mL of the suspension was taken for isolation of DNA before centrifugation. The remainder was centrifuged at 2500× *g* for 5 min. Total RNA was isolated from each sample using the QIAamp viral RNA mini kit (Qiagen, Germany) according to the manufacturer's instructions.

DNA was isolated using a commercial isolation kit (Institute of Metagenomics and Microbial Technologies-IMMT, Slovenia). Briefly, 1 mL of the mixture was added to a 2-mL tube containing ≤106-μm-diameter glass beads (Sigma-Aldrich, St. Louis, MI, USA) and centrifuged at 10,000× *g* for 5 min. The pellet was resuspended in 392 μL of lysis buffer and 8 μL of proteinase K (Sigma-Aldrich, St. Louis, MI, USA). This was followed by bead beating on a MagNALyser device (Roche, Basel, Switzerland), at 6400 rpm for 60 s and incubation at 56 ◦C for 15 min. Bead beating and incubation were repeated three times and twice, respectively. The rest of the isolation was performed according to the manufacturer's protocol.

The RNA of six honeybee viruses in bumblebee workers: ABPV, BQCV, CBPV, DWV, SBV and LSV was detected by specific reverse transcription and polymerase chain reaction method (RT-PCR) as previously described [27,48]. Results were considered positive based on the size of the RT-PCR products in the agarose gel when the expected product size was present (ABPV 452 nt, BQCV 770 nt, CBPV 570 nt, DWV 504 nt, SBV 814 nt and LSV 603 nt). Isolated RNA from bumblebee queens was tested for ABPV, BQCV, CBPV and DWV as described above.

Isolated DNA from each bumblebee worker and queen was used to detect *N. bombi*, *N. ceranae*, *N. apis*, *A.s bombi*, *C. bombi* and *L. passim*. Polymerase chain reactions (PCR) and real-time polymerase chain reaction (qPCR) were performed according to previously published protocols [49–51].

#### **5. Conclusions**

In 147 bumblebees tested, the prevalence of ABPV, BQCV, DWV, SBV, LSV and *N. ceranae*, *N. bombi*, *C. bombi* and *A. bombi* was detected for the first time in Slovenia, while honeybees sampled at the same time and locations were positive for ABPV, BQCV, CBPV, DWV, SBV, LSV, *N. ceranae*, *C. bombi*, *A. bombi* and *L. passim*. In bumblebee queens, only BQCV, *C. bombi* and *A. bombi* were diagnosed.

The study raised some new questions regarding the transmission of pathogens between honeybees and bumblebees. However, it must be kept in mind that many factors can have an impact on surviving pollinators, including the transmission of pathogens from managed bees to wild pollinators. The evident spillover is why we need to put more attention also in good care of managed bees in order to preserve wild bees.

**Author Contributions:** Conceptualisation: M.P.O. and D.B.; methodology: M.P.O., I.T., U.Z. and D.B.; investigation: M.P.O., I.T., U.Z. and D.B., in-field activity: D.B.; laboratory activity: I.T. and U.Z.; formal analysis: M.P.O., I.T. and U.Z.; data curation: M.P.O., I.T. and U.Z.; writing—original draft preparation: M.P.O.; writing—review and editing: I.T., U.Z. and D.B.; funding acquisition: M.P.O. and D.B. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Slovenian Research Agency (research core funding P4- 0092 and P1-0255) and the CRP project V4-1622 (The importance of wild pollinators in agricultural crop cultivation and sustainable agricultural management in order to ensure reliable pollination), financed by the Slovenian Research Agency and Ministry of Agriculture, Forestry and Food.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

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

**Acknowledgments:** The authors thank all students for sampling of bumblebees and honeybees and technicians for laboratory work, also thank Slovenian Research Agency and Ministry of Agriculture, Forestry and Food for financial support.

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

#### **References**

