DOI: 10.15482/usda.adc/29998288.v1
title: Data from: Honey bee genetic resistance outperforms a cold-storage induced halt in brood production to control mites and viruses

description: Units:Adult weight (total mass of adult bees): kilogram (kg)Brood (surface area of sealed brood cells): square centimeter (cm)Deformed wing virus genotype A (DWV-A), deformed wing virus genotype B (DWV-B) and vitellogenin (Vg) relative expression: (no units) Expression relative to actin and RP49 (see text for details).Varroa mites per bee: Varroa mites per bee in sample (number (no.) Varroa/ no. bees in sample).Varroa mite fall: no. Varroa falling per day on treated cardboard.Daily hive weight change: kgHive temperature: temperature Celsius (°C) every 5 minutes (min) at top of center frame in lower hive box (see text)Hive carbon dioxide (CO<sub>2</sub>) concentration: parts per million (ppm) every 5 min on top of middle frame in upper hive box (see text)<b>Materials and methods</b>. Two experiments were conducted, the first from August 2023 – February 2024, and the second a year later with new colonies. In August of each year ten honey bee colonies of each of three bee stocks: Pol-line, Russian and unselected Italian, for a total of thirty colonies, were selected. The colonies had been established from single box colonies received in mid-April. Bee colonies were housed in painted, two-box, 10-frame, wooden Langstroth hives (43.7 Liter (L) capacity per box). The hives were located at the University of Arizona Red Rock Agricultural Station where they were placed on stainless steel electronic scales (Tekfa model B-2418 and Avery Weigh-Tronix model BSAO1824-200, max. capacity: 100 kg, precision: ±20 grams (g) ; operating temperature: -30ºC to 70ºC) and linked to dataloggers (Hobo UX120-006M External Channel datalogger, Onset Computer Corporation, Bourne, MA) with weight recorded every 5 min. Temperature sensors (HOBO MX2201, resolution ±0.04°C, accuracy ±0.5°C) were attached to the center of the top bar on the middle frame in the bottom box and set to record every 5 min. Carbon dioxide probes (model GMP251, Vaisala Inc., Helsinki, Finland), calibrated for 0-20% concentrations, were placed on top of the center frames in the top box of each hive and linked to dataloggers (HOBO UX120-006M) set to record every 5 min.The total weight of all hive components and frames was subtracted from the total hive weight recorded during the night pre-assessment, to calculate the adult bee mass. Hives were periodically assessed to determine adult bee mass and total capped brood area. Briefly, each hive frame was lifted out sequentially, gently shaken to dislodge adult bees, photographed using a 16.3 megapixel digital camera (Canon Rebel SL1, Canon USA, Inc., Melville, NY), weighed on a portable scale (model EC15, OHaus Corp., Parsippany, NJ), and replaced in the hive. During the first assessment all hive components (i.e. lid, inner cover, box, bottom board, frames, entrance reducer) were also shaken free of bees and weighed to yield an initial mass of all hive components. The area of sealed brood per frame was measured from photographs using image analysis software, and frame values were summed to estimate colony values. Assessments took place in mid-August (pre-cold storage treatment period), late September (after the cold storage and mite treatments), mid-November (pre-winter period), and a final assessment in mid-February (post-winter period). In November and at the end of January all colonies were fed 200 g pollen patty.Half of the hives were moved into a cold storage unit (30 meters cubed (m<sup>3</sup>) internal volume, with CO<sub>2</sub> and temperature monitors, PolarKing, Fort Wayne, IN) set to 5°C with a dehumidifier and a roof-mounted exhaust fan operating 5 min per hour for ventilation, in late-August for 18 days to induce a break in brood production. Immediately after hives were moved from the cold storage unit (CSU) back to the Red Rock apiary, all colonies were treated with thymol miticide (Apiguard, Vita Bee Health, Basingstoke, UK) at 25g/hive/week for 3 weeks. Thereafter colonies were managed in an identical manner throughout the fall and winter, concluding the following February.Samples of 100 adult bees were collected from the brood nest area of the hive in August (pre-treatment), in November and in late January for gene expression analysis, and an additional approximately 150 adult bees were collected from the brood nest area in August pre-treatment and in late January to determine phoretic mite density using the alcohol wash method. Just prior to placing hives in cold storage in August, and again in October, slick paperboard coated with petroleum jelly and covered with mesh were placed onto the hive floor to monitor Varroa mite fall within the hive. The paperboards were removed after 3 days and the number of mites counted on each board.<b>Gene expression analysis. </b>For each colony and time point, pooled samples of 50 bees were homogenized in 8 milliliters (mL) of ice-cold phosphate buffered saline for three 12 second cycles at a speed of 4.5 meters per second (m/s) with a 15 second (s) dwell between each cycle in an Omni Universal Bead Mill. RNA was extracted from 50 microliters (µL) of homogenate using a Maxwell RSC SimplyRNA Tissue Kit (Promega, USA) following the manufacturer’s protocol. Complementary DNA (cDNA) synthesis was carried out using 1 microgram (µg) of RNA and QuantiTect Reverse Transcription Kits (Qiagen, USA) according to the manufacturer’s protocols. Quantitative polymerase chain reaction (qPCR) was performed using a 1:10 dilution of cDNA to quantify transcript levels of vitellogenin (Vg), DWV-A and DWV-B. All reactions were carried out as follows: initial denaturation at 95°C for 5 min; 40 cycles with denaturation at 95°C for 10 s; an annealing temperature specific to each target (Vg=58°C, DWV-A=60°C and DWV-B=62°C) and extension temperature at 60°C for 30s. The reactions were carried out using Luna<sup>® </sup>Universal qPCR Master Mix (New England Biolabs, USA) in triplicate on a CFX96 Real-Time PCR Detection System (Bio-Rad, USA). Relative expression levels were calculated based on standardized change in cycle threshold values (ΔCt) using the geometric mean of honeybee <i>β-actin</i> and RP49 for normalization.Note: The dataset includes data collected every 2 months, monthly, daily, and every 5 min, depending on the parameter.<br><br>