1. Overwintering and bee colony health: Combining temperature monitoring, hive inspection, pesticide detection and gene expression data to investigate hive health during overwintering William G. Meikle, Will Fitz, Milagra Weiss, Patrick Maes, Lucy Snyder and Kirk A. Anderson Carl Hayden Bee Research Center, USDA-ARS 2000 E. Allen Road, Tucson AZ 85719

2. Objectives : 1.Divide commercially-managed hives into four groups and subject the groups on different migratory trajectories. 2.Measure colony level parameters, including continuous internal temperature and changes in adult bee mass and brood area, to quantify differences in colony status and health. 3.Sample hives to detect differences in risk factors: pest and pathogen levels, agrochemical exposure, or gene expression, to explain observed group differences.

3. Materials and methods Four groups of 40 hives each 12 hives per group sampled intensively Colony level observations Hive weight Adult bee mass Capped brood area Continuous hive temperature Almond grade Pests and pathogens Varroa and Nosema loads Pesticide residue analysis Wax Bee bread Gene expression Immunity and virus genes Vitellogenin (nutrition) expression

4. Foraging Environment Mar.- Apr. 2014 160 splits made New Carniolan Queen Cell May 2014 160 hives moved to North Dakota Feb.-Mar. 2014 Almond pollination

5. 80 Colonies Non-Ag Dominated Forage area in North Dakota Foraging Environment 80 Colonies Non-Ag Dominated Forage area in North Dakota

6. 40 80 Colonies Canola Pollination 40 Foraging Environment 80 Colonies Non-Ag Dominated Forage area in North Dakota 80 Colonies Non-Ag Dominated Forage area in North Dakota

7. 40 Foraging Environment 80 Colonies Non-Ag Dominated Forage area in North Dakota 80 Colonies Non-Ag Dominated Forage area in North Dakota

8. 40 40 Colonies Sunflower Honey Production 40 Colonies Sunflower Honey Production Foraging Environment 80 Colonies Non-Ag Dominated Forage area in North Dakota 80 Colonies Non-Ag Dominated Forage area in North Dakota

9. 40 Foraging Environment 80 Colonies Non-Ag Dominated Forage area in North Dakota 80 Colonies Non-Ag Dominated Forage area in North Dakota

10. Foraging Environment 80 Colonies Non-Ag Dominated Forage area in North Dakota 80 Colonies Non-Ag Dominated Forage area in North Dakota

11. Hive weight, adult bee mass and brood area before and after overwintering

12. Average temperature during overwintering

13. Difference between upper and lower temperature sensors for a strong (red) hive and a weak (black) hive

14. Temperatures of live hives compared to dying hives

15. Average temperature variability during overwintering

17. Treatment group (no. hives) Honey yield Almond grade: Percent of colonies categorized by frame counts of adult bees kg per colony Primary source ≥ 65 ≥ 32 ≥ 1Dead Black (36)32 Canola & sunflowers 6.7%22.2%47.2%19.4% Blue (40)32 Canola & sunflowers 32.5%22.5%25.0%20.0% Red (40)45Clover 95.0%5.0%0.0% Yellow (40)55Clover & alfalfa 60.0%17.5%12.5%10.0% Honey yield in Sept. 2014 and colony scores in Jan. 2015 Red and Yellow groups produced more honey than Black and Blue groups. Red and Yellow groups had much higher rates of 6 frame grades than did Black and Blue groups.

18. We observed: 1.Higher adult bee and hive weight losses among Black and Blue hives than among the Red and Yellow hives; 2.Brood present in most Red and Yellow hives after overwintering, but almost no brood among the Black and Blue hives; and 3.Lower average temperatures and higher temperature variability among Black and Blue hives compared to the others, indicating that the lower amounts of adult bees and brood were consistent over time. 4.Many more hives in the Red and Yellow groups achieved high almond grades than hives in the Blue and Black groups. We considered three main reasons to explain differences observed among treatments: Different levels of Varroa, Nosema or viruses; Different levels of agrochemical exposure; Different levels of nutritional stress.

19. Varroa: Avg. no. mites per 100 bees ± SE Nosema: Avg. no. spores per bee ± SE (values x 10 6 ) Treatment group (no. colonies) BeforeAfter BeforeAfter Black (12)0.23 ± 0.160.16 ± 0.10 1.21 ± 0.435.67 ± 0.85 Blue (12)0.04 ± 0.040.44 ± 0.34 2.22 ± 0.613.68 ± 1.54 Red (12)0.12 ± 0.120.58 ± 0.50 0.93 ± 0.351.44 ± 0.41 Yellow (12)0.12 ± 0.090.26 ± 0.19 0.45 ± 0.275.00 ± 1.67 Varroa and Nosema loads before and after overwintering Varroa and Nosema loads were low and not significantly different among treatment groups in Fall 2014. Varroa loads did not change appreciably over winter, but Nosema loads were significantly higher in January.

20. Oct. 2014 Jan. 2015 Pesticide (ppb) BlackBlueRedYellow BlackBlueRedYellow Boscalid (fungicide) 25.7 Carbendazim (fungicide) 7.515.424.719.9 15.342.458.246.0 Chlorferone (acaricide) 146 TR5.78.37.4 Chlorothalonil (fungicide) 246204173340 1080 Chloropyrifos (insecticide) 16.0 215 171170 Coumaphos 14520221.014.0 14.310.9 10.2 Coumaphos ox. 23111754.731.3 1164116.913.4 Cyprodinil (fungicide) 15.732.4 60.0 78.016.419.4 Fenpyroximate (acaricide) 12424.558.973.1 15.4 Fluridone (herbicide) 1740 531750 43.666.311758.3 Pendimethalin (herbicide) 90.2 484371 Pyraclostrobin (fungicide) 19.1 Tebuconazole (fungicide) 22.3 20.456.737.1 Thymol 1950352030202250 17.5 Vinclozolin (fungicide) 6.7 1790225028704320 2,4 DMPF (from amitraz) 4272631070723 Pesticide Analysis of Wax (combined sample per treatment group) No major differences were observed between groups with respect to the presence/absence or concentration of any pesticide residues

21. Pollen DatePesticide (ppb)BlackBlueRedYellow Oct. 20142,4 DMPF (amitraz) 10-25.716.4-56.924.9-18624.9-94.3 Chloropyrifos (insecticide) 0-1020-71.20-33.80-25.0 Coumaphos 00-100-10.50-18.3 Tebuconazole (fungicide) 0-47.70-41.20-50.20-139 Thymol 00-2450-2090-294 Jan. 20152,4 DMPF (amitraz) 0-620-29.216.3-31.916.5-52.7 Chloropyrifos (insecticide) 0-19.10-12.90-85.50 Coumaphos 00100-26.3 Tebuconazole (fungicide) 0-30.00-73.10-34.80-215 Thymol 0-19700-2860-268 Pesticide Analysis of Bee Bread (range of 3 samples per treatment group) No major differences were observed between groups with respect to the presence/absence or concentration of any pesticide residues

22. Immunity and Nutrition Gene Expression Abaecin (antimicrobial peptide) Deformed Wing Virus (pathogen) Defensin 1 (antimicrobial peptide)Hymenoptacin (antimicrobial peptide)

23. Lysozyme 2 (antibacterial peptide) Expression Pre-overwintering Y values show the amount of gene expression relative to the Yellow group Immunity and Nutrition Gene Expression

24. Vitellogenin Expression Fall ‘14 Y values show the amount of gene expression relative to the Yellow group Immunity and Nutrition Gene Expression

25. Forage In North Dakota 2002 2010 (Gallant et. al. 2014)

26. We observed: 1.No significant differences among groups in terms of Varroa or Nosema loads. Nosema loads were higher after overwintering. 2.Few differences among groups with respect to the types and concentrations of agrochemicals found in bee bread and wax. There are always possibilities of synergism among pesticides. 3.Few differences among groups with respect to expression of genes for immunity. 4.A significant difference in vitellogenin expression between the Red and Yellow groups, with high levels of expression, and the Black and Blue groups, with relatively low levels of expression. 5.It is possible that nutrition issues interfere with the genetic responses to challenges from pathogens and/ or pesticides. Future plans: Repeat this study, to the extent possible, increasing the number of colonies involved and confirming the links between internal hive temperature, colony status, and risk factors.

27. Acknowledgements: Many thanks to Zac Browning of Browning Honey, Jamestown, ND, for supplying and transporting hives, and to Brendon Mott, Michael Giansiracusa and Ian Carstensen for technical assistance.