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Experimental 1: Quantitative DWV transmission study: Thirty young Buckfast queens were produced in a colony with minimal Varroa- and DWV levels following the standard procedure [37] by a professional queen producer in the Reerslev, Denmark (55° 33' 21.1788'' N 11° 23' 25.4256'' E). Before grafting, the donor and rearing colonies had been confirmed to be treated against Varroa mites and free of most common bee viruses as described previously [38]. The queens were introduced to mating hives containing 250–300 Varroa-free worker bees. These mating hives were placed in a mating station (Flakkebjerg, Denmark, 55° 19' 31.278'' N 11° 23' 28.6188'' E) surrounded by drone provider colonies that had not been treated against Varroa mites for the past three years and consequently furnished drones with relatively high DWV infections for the experimental queens to mate with [31]. After mating, the queens developed DWV infections that were highly correlated among different body parts of each queen (head, thorax, abdomen, ovary, spermatheca, and sperm) and differed strongly between individual queens (DWV titers ranging from 0 to >1010) [31]. Three weeks after the onset of oviposition, one batch of 50 freshly laid eggs was collected from just constructed waxcomb into a micro-centrifuge tube from each of 25 reproductive queens. These samples were immediately stored at -80°C until RNA extraction. Micro-pestles (Eppendorf) were used to homogenize the egg samples, and total RNA was extracted from each sample using NucleoMag® 96 RNA Kit (Macherey-Nagel) on a Kingfisher Magnetic Extractor following the manufacturer’s guidelines. RNA concentration and purity were measured using a Nanodrop ND-1000 spectrophotometer (Thermo Scientific) and total RNA concentration was adjusted to 20 ng/μL with molecular grade water (Fisher Scientific). The RNA was stored at -80°C for further use.
Experiment 2: Survey of commercial population for virus transmission in eggs: A commercial population of 85 colonies headed by Italian-queens in five apiaries were surveyed for the DWV content of eggs at the beginning of beekeeping season with the kind permission of beekeeper. The apiaries belonged to a single migratory beekeeper near Mebane (LEE’s BEES Inc, North Carolina, USA). These apiaries were in 10 km distance of the main honey bee station (approximate location 36° 7' 6.4416'' N 79° 15' 13.2768'' W). The colonies were sampled on the 19th - 28th of April 2016, approximately three weeks after colony establishment from 3-lb packages. This early sampling time was chosen to provide a baseline estimate of DWV transmission through queens prior to the seasonal build-up of Varroa. Fifty freshly laid eggs were carefully collected from worker size cells of newly-produced comb and transferred into one micro-centrifuge tube per colony. The samples were transported on ice back to the laboratory where they were stored at -80°C until RNA extraction. Eggs in each micro-centrifuge tube were homogenized using micro-pestles (Fisher Scientific) and total RNA was extracted with a standard Trizol™ protocol [39]. The RNA concentration and purity were measured, adjusted and stored as above until further processing.
Experiment 3: Characterizing the mechanism of vertical transfer of DWV: Based on the results of the second experiment, five queens that laid DWV-infected eggs were transferred from the commercial beekeeping operation in Mebane, NC, to the UNCG apiary in Greensboro, NC (36° 5' 55.7448'' N 79° 53' 21.4116'' W), for further study. Queens were introduced to mini-hives (Styrofoam™ mating nucs, Mann Lake USA) with empty newly-produced worker and drone cells to induce each queen to lay simultaneously fertilized and unfertilized eggs. Three samples of 50 eggs were collected from each queen in micro-centrifuge tubes: 50 eggs from worker size cells and two batches of 50 eggs from drone size cells. One batch of drone eggs remained unmanipulated, while the other one was surface-sterilized by immersion in 5% bleach solution for five minutes followed by three rinses in sterile water [2, 33]. All samples were stored at -80°C until RNA extraction. The total RNA for each sample was extracted, its concentration and purity were measured, adjusted and stored as above until further processing.
cDNA synthesis, qPCR assays and data analysis: Using the stored RNA from all three experiments, a two-step quantitative qPCR assay was carried out to quantify the DWV viral load in the samples. For each sample, cDNA was synthesized using the High Capacity cDNA Reverse-Transcription Kit (Applied Biosystems). RNA template (10 μL) with a final concentration of 20 ng/μL was added to10 μL of the provided cDNA master mix, followed by an incubation period as recommended by the manufacturer: 10 min at 25°C, 120 min at 37°C and 5 min at 85°C. The cDNA solution was then diluted 10-fold in molecular grade water to serve as template in subsequent qPCRs to quantify DWV and other targets using unlabeled primers and SYBR Green DNA binding dye (Applied Biosystems). Quantification was performed in duplicate and in a reaction volume of 12μL for the samples in Experiment 1 and 20μL for the samples in Experiments 2 and 3. Final primer concentrations of 0.4μM were used. DWV primers used in this study quantify DWV type A [40]. The reference genes β-Actin and RPS5 were used as an internal control and for relative quantification of DWV using the ΔCt method [2]. A positive control was run in each case, and RNase-free water was added as template for a No Target Control (NTC), and a No Reverse Transcriptase (NRT) control served as an additional negative control [41]. The thermal cycling conditions using a viiA™7 apparatus (Applied Biosystems) for Experiment 1 and StepOnePlus™ (Applied Biosystems) for Experiment 2 and 3 were 10 min at 95°C, followed by 40 cycles consisting of a denaturing stage at 95°C for 15 s and as annealing/extension stage at 60°C for 1 min. Fluorescence measurements were taken at the end of each cycle. This procedure was followed by a final melt-curve dissociation analysis to confirm the specificity of the products. The primers used in this study (Table 1) have previously been validated to detect the intended targets and are commonly used in honey bees [21, 42–46]. Samples were deemed positive for a target if their melting temperature was similar to the melting temperature of the positive controls and a Ct value of 35 or lower was recorded. Our virus survey in the second experiment also screened for Sacbrood virus (SBV) and the Acute Bee Paralysis Virus complex (AKI: Acute Bee Paralysis Virus, Kashmir Bee Virus, and Israeli Acute Bee Paralysis Virus) to assess the co-occurrence of these viruses with DWV.
Table data removed from full text. Table identifier and caption: 10.1371/journal.pone.0195283.t001 Primers used to establish standard curves and analyze samples. Virus loads in each sample were quantified using absolute quantification methods based on standard curves obtained through serial dilutions of known amounts of amplicons as described before [21]. The successful amplification of reference genes (β-Actin and RPS5) was used to confirm the integrity of samples throughout the entire procedure, from RNA extraction to qPCR. The reference genes were also used for relative quantification of virus titers. Results of relative and absolute quantification did not significantly differ, thus only absolute values (copy number / μL) are presented. To improve data compliance with parametric assumptions, raw data were log10 transformed where parametric analysis was necessary [39]. Data analysis and visualization were performed using Excel and “R”, version 3.1.3.
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