The Honey bee (
Hygienic behavior is a potential struggle mechanisms against
Although several diagnostic techniques have been used to detect honeybee viruses, it is very difficult or impossible to differentiate and quantify virus infections through field symptoms (Desai, Kumar, & Currie, 2016). Reverse transcriptase-quantitative PCR (RT-qPCR), which is 1000 times more sensitive than ELISA and 100 times more sensitive than conventional non-nested RT-PCR (de Miranda, Cordoni, & Budge, 2010), can be rapidly implemented in independent laboratories after the preparation of primary protocol and primer sequences (Simeunović et al., 2014). RT-qPCR has been preferred more recently with TaqMan (Cirkovic et al., 2018) due to highly reliable, fast and accurate results (Chantawannakul et al., 2006) even if a little bit expensive. In the present study, we investigated the success of the selection in terms of hygienic behavior in the struggle against DWV and
Sampling was carried out during April 2017 in Muğla-Fethiye-İncirköy which is located at 601 meters above sea level in the south-western region of Turkey. This area was isolated for breeding Muğla honey bees to study hygienic behavior. The hygienic bee breeding programe has been carried out using the pin test (Newton & Ostasiewski, 1986) and artificial insemination according to Laidlaw & Page (1997) since 2013 by the Muğla Beekeping Association. Samples were provided from fifty hygienic colonies (Oskay et al., 2019), and fifty non-hygienic colonies, which were used as the control, and is Muğla honey bees ecotype (
For the first three years starting in 2012, the hygenic bee breeding program was conducted as a project supported by by Republic of Turkey Ministry of Agriculture and Forestry General Directorate of Agricultural Research and Policies (Oskay et al., 2019). After the project was completed, the breeding program was carried out by Muğla Beekeeping Association (MAYBIR). Colonies were evaluated twice for hygienic behavior in April each year. Because cold nitrogen was not easily found in land conditions far from the city center, the pin-killed brood (PKB) assay was preferred to determine the hygienic feature levels of all the colonies in this program. The breeding program started with two-hundred colonies in an area closed to other bekeepers far away from the city center in order (Oskay et al., 2019). During the tests, 11- to 15-day-old individuals in the pupae period in 100 eyes in the hatching frames were killed by Pin and kept in hives for twenty-four hours. At the end of this period, colonies where worker bees cleaned 95% or more of the dead pupae from the eyes were determined. Hygienic behavior increased in the population from 43% in 2012; 63% in 2013; 91.7% in 2014 to 96.8% in 2015 (Oskay et al., 2019). The samples were collected in 2017, and MAYBIR announced the hygenic behavior ratio as 97% from. Anti-varroa treatment by Rulamit VA (seven doses of smoke were pressed from the flight hole of each hive) was applied in early or mid February according to seasons in the Hygenic breeding programme and repeated three times with a three-day interval in November and February.
To determine the varroa loads of sampled colonies, the bottom board method was used (Dietemann et al., 2013). After applying acaraside (rulamit-containing incense), mites dropped to white sheets of paper that laid on the bottom board in the hygienic and non-hygienic hives. The sheets were collected, sealed in plastic bags and brought to the laboratory. The mites were counted using a magnifying glass and saved in the laboratory.
Total RNAs of each bee and pupae were extracted using high pure viral nucleic acid kit version 19 (Roche, Switzerland) according to the manufacturer's protocol. Ten worker bees and ten pupae samples from each colony were extracted separately as a bulk. Each bulk sample was crushed by a hand drill and homogenized in a 5 ml microcentrifuge tube. Then the RNA was extracted, and the samples were measured using the BioDropDuo (BioDrop Ltd, UK) and checked using gel electrophoresis (Reliant Gel systems). The RNA samples found to be sufficient in terms of quality and quantity ranging from 239.20 ng/ul to 343.30 ng/ul from hygienic (worker=46 and pupa=42) and non-hygienic (worker=50 and pupa=46) colonies were stored at −80°C until further use.
For reverse transcription, one-step RT-qPCR (reverse transcriptase quantitative polymerase chain reaction) assay was performed using the Real Time ready RNA virus master kit (Roche). TaqMan technology utilizing a fluorescent probe (FAM-TAMRA) was used to identify the amount of DWV, and the LightCycler® 96 (Roche) instrument was used for fluorescence detection. The RT-qPCR reactions were performed in 20 μl volumes containing 0.8 μl PP mix (0.1 μl 100 pmol each of the gene-specific forward and the reverse primer, 0.15 μl 20 pmol each of the gene-specific probe, and 1.65 μl PCR-grade H2O), 0.3μl enzyme blend (50×), 2.5 μl ready buffer (5×), 2 μl RNA and 4.4 μl H2O. The thermocycling profile for this assay was as follows: 5 min at 50°C and 5 min 58°C for cDNA synthesis, 1 min at 95°C for inactivation of the reverse transcriptase following 40 cycles of 10 sec at 95°C for denaturation and 30 sec at 60°C for annealing and data collection. For assays, a six-fold dilution series (104–109 copies) of synthetic positive control of a known concentration was also run on each reaction plate to constitute a standard curve (Sup. Fig. 1). Calculating the R value as 1 indicates that the 6-fold solution we use is sufficient. We used a null reaction mix as a negative control, and a for second time we checked out the DWV positive samples using RT-qPCR in order to confirm the quantitation. Also, DWV load per colony was determined by taking the average of the calculated values as a result of both RT-qPCR.
RT-qPCR amplification results were taken into account of the standard curve of the samples designed as the synthetic positive control. Here, the number of amplified viruses in each sample was calculated according to the six-fold dilution of known values as 104–109 (Sup. Fig. 1). All quantitative data were subjected to the normal distribution test (Kolmogorov-Smirnov). Indepentent t-test was performed for Cq (quantification cycle) and
Statistical comparison of control and hygienic colonies in terms of DWV loads, Cq values, prevalence of DWV, and Varroa loads
N | Mean±St.E RT-qPCR | N | Mean±St.E Cq | % Prevelances | Varroa Counts | ||
---|---|---|---|---|---|---|---|
H | Worker | 46 | 181627.69±64074.74** | 19 | 20.10±1.15* | 41.3 | 28.92±5.41** |
Pupa | 42 | 234.38±112.34 | 29 | 26.36±0.55 | 69.0 | ||
C | Worker | 50 | 241982.35±65972.63** | 33 | 17.13±0.59* | 66.0 | 108.90±9.80** |
Pupa | 46 | 937.70±578.55 | 28 | 25.56±0.66 | 60.8 |
H: Hygienic, C: Control
Means with different superscripts are significantly different in terms of p<0.05 p<and 0.01, respectively.
To determine whether the selection in terms of hygienic behavior is effective on
DWV loads (a) and the number of quantification cycles (b) of worker bees and pupae from control and hygienic colonies regarding qPCR. By black lines and circles, respectively represent worker bees and pupae.
In recent decades in Turkey, Gülmez, Bursalı, & Tekin (2009) and Tozkar et al. (2015) have studied Deformed Wing Virus, and Muz & Muz (2009, 2017) studied how both DWV and
A comparison of colonies showed that DWV prevalence was higher in pupae (69.0%) but in contrast lower in worker bees (41.3%) from hygienic colonies (Tab. 1). This supports a previous study by Gauthier et al. (2007) who suggested that pupae were more susceptible to DWV replication and found differences between DWV loads in adults and pupae, which might result from the fact that diseased adults have much shorter lifespans. Moreover, depending on the higher hygienic behavior of hygienic colonies, bees could remove the infected pupae from colonies, and by doing so, the number of healthy adults might be higher which would contribute to a lowering of virus concentration in hygienic colonies Gauthier et al. (2007). We also found the amount of DWV of hygienic colonies to be lower than the control (Tab. 1). Yañez et al., (2015) and Li et al., (2012) drew attention to the higher prevalence of DWV in
Spivak & Reuter (1998) compared hygienically selected and unselected
The virus prevalence was unexpectedly higher at the pupae phase in the hygienic colonies in the present study. While hygienic colonies have fewer viruses in their worker bees and pupae, it is not known why the virus prevalence is higher in terms of pupae in hygienic colonies than the control (Tab. 1). While there are a few possible responses to explain this situation, the sperm pool may be used in artificial insemination to obtain hygienic colonies. Namely, when sperm is collected, the somatic cells of virus-infected drones might also be collected. Even if there is no virus in the sperm, somatic cells can carry the infection to the pool. If there is an infection even in a single drone, the sperm pool used to fertilize the queens may be contaminated. More populations may have been infected with viruses due to fertilized queens using the sperm pool in the hygienic colonies. Results that support this have been pointed out in previous studies that viruses can translate horizontally through individuals in the same generation and can be transferred from one generation to another vertically through their queens (Chen, Evans, & Feldlaufer, 2006; Yue et al., 2007; Tantillo et al., 2015). The venereal passing of DWV through artificial insemination with infected semen in DWV-free virgin queens has been already testified fully by de Miranda & Fries (2008). The key result of the present study may be an indication that hygienic colonies remove the infected pupae from the hive and thereby reduce the spread of the virus in adult bees, and if so, this is a success of hygienic colony breeding.
Like all other bee viruses, DWV is harmful for beekeeping and is considered the most serious secondary pathogen associated with varroosis (Yue & Genersch, 2005), although many studies have concluded that the