The honey bee, an important, globally widespread insect pollinator, plays an important role in natural ecosystems, and as such, represents a key component in biodiversity preservation. The European honey bee (
Previous studies have confirmed the presence of viruses in pollen collected by honey bees, including clinically important viruses such as deformed wing virus (DWV), Israeli acute paralysis virus, chronic bee paralysis virus, black queen cell virus (BQCV), acute bee paralysis virus (ABPV), Kashmir bee virus (KBV) and sacbrood virus (SBV) (3, 4, 16, 19, 27, 28, 30). Furthermore, KBV and SBV have been detected in different bee food sources, such as honey and royal jelly (25), while DWV has been detected in larval food (30). High loads of such viruses in pollen and honey have been associated with increased bee mortality (23).
As a eusocial species, honey bees are highly prone to pathogen transmission, the large number of individuals and strongly organised social structure of the colony and the resultant close physical contact leading to rapid pathogen transmission. The bees may be infected directly through contaminated food or indirectly by contact with excreted faeces (3), the faecal–oral transmission route being considered the most common route of virus transmission (11). Studies have also shown that a virus can enter the hive following contact between a forager bee and a contaminated plant, potentially leading to inter-species transmission of viruses (11, 16, 27). Furthermore, the presence of a virus in bee food would bring about rapid infection of the honey bee colony with subsequent contamination of hive structures through the bee’s work activity. Viral transmission
Samples were collected during May and June 2021 from 24 different beehives at four different locations in southern Moravia, Czech Republic (Fig. 1). Five sample types were collected from each beehive,
Prior to nucleic acid extraction, the swabs from the bottom board were homogenised in 1 mL of TRI Reagent (Sigma-Aldrich, St. Louis, MO, USA) in a homogeniser with 2.8 mm ceramic beads (OMNI International, Kennesaw, GA, USA) set at 3,500 rpm for 2 min. Likewise, pooled samples of the bees from one hive (each n = 50) were homogenised in 30 mL of TRI Reagent. A 100 μL aliquot of the homogenate, which approximately corresponded to the amount obtained from one bee weighing 100 mg, was used in the extraction process. For the debris, pollen and honey samples, the homogenisation process was omitted and 1 mL of TRI reagent was added directly to 100 mg of each material. Subsequently, the homogenates, debris, pollen and honey samples were centrifuged for 15 min at 22,000 rpm and the supernatant used for extraction of viral nucleic acids.
Extraction of nucleic acids from debris was carried out according to the certified methodology of Prodělalová
Once extracted, the nucleic acids were subjected to quantitative PCR (qPCR) using the Luna Universal One-Step RT-qPCR Kit (New England BioLabs, Ipswich, MA, USA) for molecular detection of the three viruses DWV, BQCV and SBV, each sample being screened twice to rule out false positives. The reaction was carried out in accordance with the standard protocol provided by the manufacturer, with 40 amplification cycles used for each run. The amount of each component was slightly modified to achieve a total volume of 15 μL per reaction (3.75 μL of RNA). The qPCR results were then processed using the Lightcycler 480 software (Roche Diagnostics, Basel, Switzerland). The absolute quantification/2nd derivative maximum algorithm was employed to calculate the concentration of viral load based on standard viral curves. Two pairs of primers targeting two different genomic regions (helicase-coding and RNA-dependent RNA polymerase–coding) were used for real-time detection of DWV to ensure increased screening effectivity (Table 1). Owing to a lack of standards for the helicase region of DWV, viral loads were only calculated for the DWV-positive samples obtained in the reaction with RNA-dependent RNA polymerase–targeting primers.
Primers and probes used in the qPCR and PCR reactions
Target virus | Method | Primer/probe | Target region | Reference |
---|---|---|---|---|
DWV | qPCR | qDWV-1 F,R | Helicase | 2 |
qDWV-1 P | 21 | |||
qDWV-2 F,R,P | RdRP | 12 | ||
BQCV | qPCR | qBQCV F,R | 7834–8119 bp | 15 |
qBQCV P | 20 | |||
PCR | BQCV1 F,R | 379–700 bp | 14 | |
SBV | qPCR | qSBV F,R,P | 5’NTR | 20 |
ABPV | PCR | ABPV-1 F,R | RdRP | 26 |
ABPV -2 F,R | 34–240 bp | 14 |
bp – base pairs; DWV – deformed wing virus; BQCV – black queen cell virus; SBV – sacbrood virus; ABPV – acute bee paralysis virus; F – forward; R – reverse; P – probe
The samples were additionally screened for the presence of ABPV using a standard PCR as the qPCR method for the detection of ABPV was not available at the time of testing. First, cDNA was synthesised using the Protoscript II First Strand cDNA Synthesis Kit (New England BioLabs), following which it was subjected to a PCR reaction using OneTaq Hot Start Quick-Load 2X Master Mix with standard buffer reagent (New England BioLabs) following the standard protocol provided by the manufacturer (for the primers used in the reaction) (Table 1). The PCR products then underwent electrophoresis on 1.5% agarose gel, the products being visualised by transillumination. A standard PCR reaction screened for BQCV, using a different pair of primers (Table 1).
The frequency of PCR-positive and -negative virus samples for each location matrix were compared using Pearson’s chi-squared test. In cases where the frequency did not meet the conditions of good approximation, we used Fisher’s exact test or the chi-squared test with Yates correction. The results of qPCR viral load quantification were first tested for normality using the Shapiro–Wilk test. As the data proved to be non-normally distributed, the non-parametric Kruskal–Wallis analysis of variance test with subsequent multiple comparisons to compare the copies/μL of analyte in individual matrices for each virus tested was used.
The Mann–Whitney U test was then used to compare the number of copies in pollen and bee DWV-positive samples. All statistical analyses were performed using UNISTAT v. 6.0.07 (Unistat, London, UK) or Statistica for Windows (TIBCO, Palo Alto, CA, USA), with the level of significance in each case set at P < 0.05.
All of the selected viruses except ABPV were detected in all pooled bee samples (Table 2). Deformed wing virus was present at relatively low concentrations (1.13 × 103–1.31 × 103 viral copies/μL of analyte) in all samples except one, where the concentration reached 1.86 × 108 copies/μL. In contrast, the concentrations of BQCV and SBV were substantially higher, with BQCV concentrations relatively consistent at between 106 and 108 copies/μL and concentrations of SBV more variable at between 104 and 109 copies/μL (Tables 4 and 5). The presence of ABPV was confirmed in 54.2% of bee samples, representing all samples collected from Apiary 4, levels in bees from Apiaries 1–3 being below the level of detection (Table 3). As with the bee samples, BQCV and SBV were detected in all honey samples, while DWV was recorded in 15 (62.5%) samples and ABPV in just 3 (12.5%) samples (Tables 2 and 3). Viral loads in honey analytes ranged from 104 to 106 for both BQCV and SBV, one analyte having a concentration of 107 BQCV particles (Table 5). Unfortunately, the viral loads of DWV in honey could not be calculated as viral standards for the primers targeting the helicase region were unavailable. Sacbrood virus was detected in 22 (91.7%) of the pollen samples, BQCV in 21 (87.5%) and DWV in 18 (75%).
Results of virus detection in bees, pollen, honey, hive debris and hive grid smears, using an end-point PCR (black queen cell virus (BQCV)) and a qPCR (deformed wing virus (DWV), black queen cell virus (BQCV) and sacbrood virus (SBV))
DWV | BQCV | SBV | |||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Sample | Bees | Pollen | Honey | Debris | Smears | Bees | Pollen | Honey | Debris | Smears | Bees | Pollen | Honey | Debris | Smears |
A1 1 | + | + | + | + | - | + | + | + | + | + | + | + | + | + | − |
A1 2 | + | + | + | + | + | + | + | + | + | − | + | + | + | + | − |
A1 3 | + | + | + | + | − | + | + | + | + | − | + | + | + | + | − |
A1 4 | + | − | + | + | + | + | + | + | + | + | + | + | + | + | − |
A2 1 | + | + | − | − | − | + | − | + | − | + | + | − | + | + | + |
A2 2 | + | − | − | − | − | + | + | + | − | − | + | + | + | + | − |
A2 3 | + | + | − | + | − | + | + | + | + | − | + | + | + | + | + |
A2 4 | + | − | − | − | − | + | + | + | − | + | + | + | + | + | + |
A2 5 | + | + | − | + | − | + | + | + | + | + | + | + | + | + | + |
A2 6 | + | − | + | + | − | + | − | + | + | + | + | + | + | + | + |
A3 1 | + | + | + | + | − | + | + | + | + | + | + | + | + | + | + |
A4 1 | + | + | + | − | − | + | + | + | − | − | + | + | + | − | − |
A4 2 | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + |
A4 3 | + | + | − | − | − | + | + | + | − | − | + | + | + | + | − |
A4 4 | + | + | − | + | − | + | + | + | − | − | + | + | + | + | − |
A4 5 | + | + | + | + | + | + | + | + | + | − | + | + | + | + | + |
A4 6 | + | − | + | − | − | + | − | + | − | + | + | − | + | + | − |
A4 7 | + | + | + | − | − | + | + | + | − | − | + | + | + | − | − |
A4 8 | + | + | − | − | − | + | + | + | − | + | + | + | + | − | + |
A4 9 | + | + | − | − | − | + | + | + | − | + | + | + | + | − | − |
A4 10 | + | − | + | − | − | + | + | + | − | − | + | + | + | − | − |
A4 11 | + | + | + | − | − | + | + | + | − | − | + | + | + | − | + |
A4 12 | + | + | + | − | − | + | + | + | − | + | + | + | + | − | + |
A4 13 | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + |
Positivity rate (%) | 100.0 | 75.0 | 62.5 | 50.0 | 20.8 | 100.0 | 87.5 | 100.0 | 45.8 | 54.2 | 100.0 | 91.7 | 100.0 | 70.8 | 50.0 |
Results of acute bee paralysis virus (ABPV) detection in bees, pollen, honey, hive debris and smears from the hive bottom board, using an end-point PCR
ABPV | |||||
---|---|---|---|---|---|
Sample | Bees | Pollen | Honey | Debris | Smears |
A1 1 | − | − | − | − | − |
A1 2 | − | + | − | − | + |
A1 3 | − | + | − | − | − |
A1 4 | − | + | − | − | − |
A2 1 | − | − | − | − | − |
A2 2 | − | − | − | − | − |
A2 3 | − | − | − | − | − |
A2 4 | − | − | − | − | + |
A2 5 | − | + | − | − | − |
A2 6 | − | + | − | − | − |
A3 1 | − | + | − | − | − |
A4 1 | + | + | − | − | − |
A4 2 | + | + | − | − | − |
A4 3 | + | − | − | − | − |
A4 4 | + | + | + | − | − |
A4 5 | + | − | − | − | + |
A4 6 | + | − | + | − | − |
A4 7 | + | − | − | − | − |
A4 8 | + | + | − | − | − |
A4 9 | + | − | + | − | + |
A4 10 | + | + | − | − | − |
A4 11 | + | − | − | − | − |
A4 12 | + | + | − | − | − |
A4 13 | + | − | − | − | − |
Positivity rate (%) | 54.2 | 50.0 | 12.5 | 0.0 | 12.5 |
Viral loads of deformed wing virus (DWV) in samples of bees, pollen, hive debris and hive grid smears
DWV | ||||
---|---|---|---|---|
Sample | Bees | Pollen | Debris | Smears |
A1 1 | 1.86 × 108 | 1.03 × 107 | 4.98 × 104 | − |
A1 2 | 5.26 × 102 | 7.60 × 105 | 2.90 × 104 | 3.50 × 105 |
A1 3 | 2.05 × 102 | 2.49 × 106 | 1.67 × 103 | − |
A1 4 | 1.13 × 10 | − | 2.54 × 103 | 1.01 × 105 |
A2 1 | 1.16 × 102 | 3.57 × 105 | − | − |
A2 2 | 9.64 × 10 | − | − | − |
A2 3 | 4.95 × 10 | − | 9.54 × 102 | − |
A2 4 | 7.58 × 10 | − | 7.15 × 102 | − |
A2 5 | 1.14 ×102 | 2.67 × 105 | 2.02 × 103 | − |
A2 6 | 8.49 × 10 | − | − | − |
A3 1 | 7.08 × 10 | 8.58 × 105 | 5.52 × 103 | − |
A4 1 | − | 1.42 × 106 | − | − |
A4 2 | 2.61 × 10 | − | 3.57 × 103 | − |
A4 3 | 1.94 × 10 | − | − | − |
A4 4 | − | − | 6.59 × 102 | − |
A4 5 | 3.83 × 10 | − | 1.80 × 102 | − |
A4 6 | − | − | − | − |
A4 7 | − | 1.11 × 106 | − | − |
A4 8 | 4.55 × 10 | 6.59 × 106 | − | − |
A4 9 | 7.41 × 10 | − | − | − |
A4 10 | 2.68 × 102 | − | − | − |
A4 11 | 2.72 × 102 | − | − | − |
A4 12 | 1.31 × 103 | − | − | − |
A4 13 | 6.96 × 10 | − | − | − |
Mean | 7.75 × 106 a | 1.01 × 106 ab | 4.03E × 103 ab | 1.89 × 104 bc |
SD | 3.80 × 107 | 2.43 × 106 | 1.14 × 104 | 7.41 ×104 |
SD – standard deviation. Data represent numbers of virus copies/μL of analyte. Viral loads of some DWV-positive samples could not be calculated because of a lack of viral standards for the helicase-coding region targeted by one of the primer pairs used. – denotes a number of viral copies under the detection limit. Mean viral loads of materials with different lowercase letters are statistically significantly different (P < 0.05)
Viral loads of BQCV and SBV in samples of bees, pollen, honey, hive debris and hive grid smears
BQCV | SBV | ||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|
Sample | Bees | Pollen | Honey | Debris | Smears | Bees | Pollen | Honey | Debris | Smears | |
A1 1 | 4.23 × 107 | 1.58 × 107 | 2.83 × 106 | 7.21 × 105 | − | 1.76 × 104 | 5.86 × 106 | 1.01 × 106 | 8.39 × 106 | − | |
A1 2 | 5.05 × 108 | 2.40 × 106 | 3.08 × 106 | 2.10 × 106 | − | 1.53 × 105 | 2.00 × 105 | 2.30 × 106 | 6.74 × 106 | − | |
A1 3 | 5.24 × 107 | 3.28 × 106 | 3.22 × 106 | 1.43 × 105 | − | 1.64 × 105 | 2.06 × 106 | 3.81 × 106 | 3.98 × 106 | − | |
A1 4 | 5.57 × 107 | 2.85 × 105 | 2.15 × 106 | 7.58 × 106 | 1.36 × 105 | 3.10 × 104 | 4.53 × 104 | 1.70 × 106 | 1.04 × 107 | − | |
A2 1 | 5.66 × 106 | − | 1.03 × 106 | − | 7.87 × 105 | 3.95 × 104 | − | 4.96 × 105 | 4.26 × 108 | 2.27 × 104 | |
A2 2 | 3.14 × 106 | 5.00 × 106 | 2.03 × 105 | − | − | 1.41 × 109 | 1.22 × 106 | 7.19 × 104 | 3.31 × 107 | − | |
A2 3 | 7.56 × 106 | 5.42 × 106 | 3.78 × 105 | 5.94 × 106 | − | 9.00 × 108 | 4.41 × 106 | 2.09 × 105 | 1.08 × 109 | 1.19 × 108 | |
A2 4 | 5.66 × 107 | 7.62 × 105 | 2.30 × 106 | − | 4.18 × 105 | 2.30 × 108 | 3.00 × 105 | 1.99 × 106 | 1.20 × 109 | 2.46 × 105 | |
A2 5 | 2.10 × 107 | 8.70 × 106 | 2.23 × 106 | 1.27 × 106 | 7.98 × 105 | 8.48 × 109 | 1.57 × 107 | 1.79 × 106 | 2.94 × 109 | 4.41 × 106 | |
A2 6 | 9.89 × 107 | − | 3.59 × 106 | 7.24 × 105 | 7.99 × 105 | 3.06 × 106 | 7.91 × 104 | 1.48 × 106 | 8.68 × 106 | 3.53 × 105 | |
A3 1 | 1.40 × 107 | 2.81 × 106 | 4.17 × 106 | 7.70 × 106 | − | 4.73 × 105 | 1.08 × 106 | 2.33 × 106 | 5.06 × 107 | 2.14 × 104 | |
A4 1 | 3.94 × 107 | 3.71 × 107 | 4.94 × 106 | − | − | 2.96 × 105 | 8.30 × 106 | 8.07 × 105 | − | − | |
A4 2 | 1.90 × 108 | 2.37 × 107 | 1.75 × 106 | 2.63 × 106 | 2.28 × 105 | 4.20 × 108 | 4.20 × 106 | 1.71 × 105 | 8.60 × 107 | − | |
A4 3 | 6.86 × 107 | 2.62 × 107 | 1.69 × 105 | − | − | 1.01 × 105 | 4.21 × 106 | 4.26 × 104 | 1.40 × 107 | − | |
A4 4 | 4.74 × 107 | 5.59 × 106 | 3.40 × 105 | − | − | 2.09 × 105 | 1.33 × 106 | 6.47 × 104 | 1.64 × 107 | − | |
A4 5 | 1.16 × 108 | 1.52 × 107 | 3.57 × 106 | 1.05 × 106 | − | 1.22 × 105 | 3.29 × 106 | 6.65 × 105 | 1.08 × 106 | 8.41 × 103 | |
A4 6 | 4.93 × 107 | − | 4.86 × 106 | − | 6.87 × 104 | 2.91 × 105 | − | 1.69 × 106 | 5.34 × 106 | − | |
A4 7 | 1.07 × 108 | 3.50 × 107 | 1.48 × 106 | − | − | 1.16 × 105 | 4.60 × 106 | 2.38 × 105 | − | − | |
A4 8 | 1.48 × 108 | 8.35 × 107 | 1.10 × 106 | − | 5.03 × 105 | 1.12 × 105 | 4.61 × 106 | 1.07 × 105 | − | 1.05 × 105 | |
A4 9 | 1.62 × 108 | 2.45 × 107 | 7.64 × 104 | − | 2.42 × 105 | 6.43 × 108 | 1.95 × 106 | 1.58 × 104 | − | − | |
A4 10 | 9.39 × 107 | 2.40 × 107 | 1.90 × 106 | − | − | 3.51 × 105 | 2.93 × 106 | 7.64 × 105 | − | − | |
A4 11 | 7.00 × 107 | 2.25 × 107 | 1.16 × 107 | − | − | 4.20 × 105 | 2.19 × 107 | 4.43 × 106 | − | 9.20 × 103 | |
A4 12 | 2.82 × 108 | 3.74 × 107 | 3.55 × 105 | − | 1.60 × 105 | 4.10 × 105 | 1.10 × 107 | 5.64 × 104 | − | 4.56 × 104 | |
A4 13 | 1.00 × 108 | 3.32 × 107 | 1.15 × 106 | 5.84 × 105 | 2.59 × 105 | 1.72 × 109 | 8.10 × 106 | 2.88 × 105 | 4.19 × 108 | 7.95 × 105 | |
Mean | 9.73 × 107 a | 1.72 × 107 b | 2.44 × 106 bc | 1.27 × 106 cd | 1.83 × 105 d | 5.75 × 108 a | 4.47 × 106 a | 1.11 × 106 a | 2.63 × 108 a | 5.21 × 106 b | |
SD | 1.09 × 108 | 1.93 × 107 | 2.46 × 106 | 2.37 × 106 | 2.75 × 105 | 1.75 × 109 | 5.38 × 106 | 1.22 × 106 | 6.58 × 108 | 2.43 × 107 |
SD – standard deviation. Data represent numbers of virus copies/μL of analyte. – denotes a number of viral copies in samples under the detection limit. Mean viral loads of materials with different lowercase letters are statistically significantly different (P < 0.05)
Surprisingly, qPCR-positive pollen samples harboured significantly higher (P < 0.001) DWV loads (105–107 copies/μL) than honeybee samples (101–103, except one sample). Acute bee paralysis virus was detected in 12 (50%) samples by at least one of the primer pairs used (Tables 4 and 5). However, the ABPV-positive pollen samples were not consistent with the ABPV-positive bee samples, presumably because the ABPV-positive bees all had all come from one location (Apiary 4), while ABPV-positive pollen samples had been collected at all sampling locations (Table 3). Deformed wing virus was detected in 12 (50%) of the debris samples and BQCV and SBV in 17 (70.8%) of them, ABPV not being present in detectable quantities (Tables 2 and 3). While the viral loads of DWV detected in debris were lower than those in the pollen samples, ranging from 102 to 104 copies/μL, BQCV loads were comparable with those in all other types of materials. Surprisingly, SBV loads in the debris reached 109 copies/μL in three samples. As expected, detection of viruses in smear samples proved least successful because of the nature of the material used. Deformed wing virus was detected in just five (20.8%) of the smear samples, BQCV in 13 (54.2%), SBV in 12 (20%) and ABPV in 4 (16.6%) (Tables 2 and 3). As in bees, the frequency of positive DWV, BQCV and SBV samples was significantly higher in pollen and honey than in smears and debris (Table 2).
Honey bee viruses were detected in all four types of non-invasive material tested. None of these materials are routinely used for detection of honey bee viruses, the majority of previous research on transmission of viruses
In addition to facilitating non-invasive screening, another advantage of some of the materials studied in the present work is easier processing. While the hard cuticles of the bee samples, for example, needed to be crushed during the homogenisation process (7), there was no need for homogenisation of the honey and pollen samples due to their natural consistency. On the other hand, each material carried some disadvantages, with the smear samples being the least reliable sample type. Swabs are routinely used in veterinary medicine to obtain virus-containing samples from different areas according to the clinical symptoms (
Detection and quantification of viruses in debris was much less successful than detection in honey or pollen. Hive debris consists mostly of wax from uncapped honey cells or wax particles that fall when the newly emerging bees chew through the cell capping. However, it also commonly contains varying proportions of other materials, such as pollen loads lost by the bees, sugar crystals or small parts of dead bees that fall through the grid in the bottom board. The heterogeneity of such materials could lead to inconsistent results during virus detection. However, the main drawback of virus detection from debris is the laboriousness of the nucleic extraction process, particularly as regards the wax particles, which melt easily, making it very difficult to work with. Consequently, the extracted nucleic acids need to undergo a secondary refining step in order for qPCR to run correctly; detection being far less successful without this step (20).
Of all the non-invasive materials tested, the honey and pollen samples provided best results, with the viral loads for BQCV and SBV being particularly high and almost reaching those detected in the control (bee) samples. In comparison, the detection levels of the other viruses were lower, DWV, for example, being omnipresent in the bee samples but not detected in any honey or pollen samples. This could have been due to viral titres being below the detection threshold, as some of the bee samples contained very low concentrations of viral particles per μL. The results for ABPV detection were the least consistent, bee samples testing positive for ABPV at just one location, Apiary 4, and the few honey samples where ABPV was detected also originating from the same apiary. In the case of pollen samples, however, the results were completely different, with at least one pollen sample from Apiaries 1, 2 and 3 found to be ABPV-positive, despite bees from these localities being ABPV-negative. There are several possible explanations for this inconsistency. Both DWV and ABPV can infect not only honey bees but other insect pollinators as well (5, 9, 11). As the bees share the local pastures with other wild pollinators and honey bee colonies, they are likely to come into direct contact with pollen and flowering plant structures visited by infected pollinators, which act as a transmission route for the bee-infecting viruses (1). Unlike nectar, however, pollen does not undergo any enzymatic processes inside the honey sac and does not come into direct contact with the forager’s digestive system before storage; thus, potentially contaminated pollen can be brought inside the hive and stored without infecting the bee. This may explain the absence of a detectable ABPV load in the pooled bee samples and its presence in beehive pollen samples. Moreover, the foraging bees collected in this study only consume very low amounts of the pollen compared to the larvae and nurse bees (6). Another partial explanation may be the nutritional quality and antiviral activity of phytochemicals found in the pollen (17). While high-quality nutrition is closely linked with honey bee wellness and immunity, there is no correlation between pollen quality and the presence of pathogens (including viruses),
Acute bee paralysis virus is strongly associated with the parasitic
It is also possible that the bees in Apiary 4 were influenced by some factor that provided improved conditions for ABPV multiplication, or that the ABPV strain present at this locality was more successful in replication and/or matched our primers better. However, all the bee samples used came from apparently healthy and strong colonies, indicating that the detectable (
In conclusion, honey bee viruses were successfully detected in a range of non-invasive materials sourced from the hive (