Rachael E. Bonoan

Rachael E. Bonoan is her second year as a graduate student in the Ecology, Evolution and Behavior program at Tufts University. As an undergraduate, she studied the evolution of avian cognition at the University of Massachusetts Dartmouth and nutritional resource allocation strategies in sulfur butterflies as part of the NSF REU Program at Tufts University. As a graduate student, she worked with a Tufts undergraduate to study heat dissipation within honey bee hives following localized heat stress. She has also conducted pilot studies investigating micronutrient preferences in foraging honey bees, mentoring three more undergraduates during the project. After finishing her Ph.D. at Tufts University, she plans on continuing her research and stay in academia. She is passionate about extending my research outside of academia and making it accessible to young scientists as well as the general public.

Read Rachael's Paper

HONEY BEE HEALTH AND NUTRITION: WHY DO HONEY BEES LIKE DIRTY WATER?

By Rachael E. Bonoan

INTRODUCTION

With the importance of honey bees to food security, and the threat of colony collapse disorder (CCD), it is vital to explore as many avenues of honey bee health as possible. While many studies have investigated pathogens linked to CCD and how to combat them1, there is a gap in our knowledge concerning how we can help honey bees to naturally maintain strong colonies. Much is known about the macronutrients (i.e. carbohydrates, proteins, and lipids) honey bees need to maintain a healthy hive but very little is known about the micronutrients (i.e. vitamins and minerals) that are needed.2,3,4 This gap in knowledge became apparent when, upon observing honey bees drinking water from moist soil rather than a nearby clear puddle, an undergraduate assistant asked: why do honey bees like dirty water? A literature search brought up many beekeeping forums discussing that question but only one scientific study.5 The lone study concluded that honey bees prefer dirty water sources based solely on the strong odor cue. However, given what is known about mud-puddling behavior in butterflies6,7 and sodium-specific foraging in ants8,9 and solitary bees10, I postulated that micronutrient need might be driving this behavior.

Micronutrients are essential for many physiological functions including, but not limited to, muscle movement and immune function.3 Since both nectar and pollen (carbohydrate and protein sources, respectively) only contain trace amounts of these essential vitamins and minerals,11,12,13,14 it is logical to hypothesize that to obtain a well-rounded diet, honey bees selectively forage in soil and water for minerals that the colony lacks. As such, honey bees must show a preference for those particular minerals and these preferences can be explored via experimental assays conducted in the field. As the hive is a dynamic environment and honey bees live in temperate regions, I predict that mineral preferences will vary with a hive’s needs and with forage changes across seasons. My goal is to not only illuminate why bees prefer dirty water but, importantly, to achieve a more complete understanding of a hive’s nutritional needs throughout the year.

PILOT EXPERIMENTS

Results
Pilot trials revealed that, relative to deionized water, honey bees prefer certain minerals and avoid others. During both Fall 2013 and Summer 2014, bees had the strongest preference for sodium and avoided both phosphorous and nitrogen (Figure 1). Comparing seasons revealed that while certain minerals were preferred during the fall, they were not similarly preferred during the summer (Figure 1). For example, honey bees consumed almost four times as much calcium in Fall 2013 compared with Summer 2014. This is particularly interesting because calcium can cause paralysis in too-large amounts;14 avoidance of calcium happened only during the summer when honey bees are more likely to get trace minerals from the diverse, available nutrient sources. A difference was also seen for magnesium. Overall, during the summer when resources are abundant, honey bees consumed less of the experimental solutions. My results suggest that as resource availability and micronutrient palettes change, forager preferences similarly change. However, the driving force behind this switch is likely a combination of external and internal factors: the change in season affects both the availability of nutritional resources and internal hive dynamics.

During early summer, brood rearing, and the resultant change in population size, is at its highest -- it slows down late summer/early fall.as cited in 15 In Summer 2014, I took advantage of this change to look at individual hive preferences for Bonoan, Research Proposal, 2 of 3 certain minerals by using hive-specific markers and counting bee visits to the solutions. These data will be compared to the brood development and population size of individual hives to determine if preferences correlate with the dynamic internal factors. Although these data are still being collected and organized for analysis, it is clear that a significant difference in micronutrient preferences between individual hives exists.

Methods
Pilot studies on honey bee preferences were done at the Starks Lab at Tufts University in Medford, MA (which is equipped with eight observation hives) during Fall 2013 (September-October 2013) and Summer 2014 (July-August 2014). Given insect micronutrient requirements, I chose six specific minerals for my experiment: sodium, calcium, potassium, magnesium, phosphorous, and nitrogen.2,3 Sodium is essential for water regulation and coupled with potassium, aids in the regulation of pH in cells and body fluid. Potassium, a component in the structure of lipids and some proteins, is an important factor in the integrity of cells. Other micronutrients chosen for the study are co-factors (magnesium and calcium), and micronutrients that are important to all life forms (phosphate and nitrogen).3
Once the bees were trained to forage in a specific, open, grassy location (200 meters away from the observation hives), experimental trials were run two to five times a week (weather-permitting). For each trial, a tasting table, divided into two grids, was used. In addition to the Chart - Mean Volume Consumed in Relation to H2Osix mineral solutions, a sucrose solution (10% during fall trials, 20% during summer trials) served as the positive control and deionized water served as the negative control. There were two tubes of each solution on each grid; bees were allowed to forage at one grid while the other grid was covered with mesh to control for evaporation. At the beginning of each trial, 50 ml falcon tubes were filled with 25 ml of the appropriate solution and randomly allocated to squares on each grid. Once the tasting table was set up, bees were allowed to forage for five to seven hours (depending on foraging activity and weather) and behavioral observations were made. At the end of each trial, measurements of solution remaining in the control and experimental treatment were taken. The change in volume of the control subtracted from the experimental then yielded the total volume consumed by the foraging bees. A greater consumed volume indicates a greater preference for a particular solution.

To further assess what preferences mean for hive health and development, I gave bees a hive-specific light-weight, colored powder mark. As each hive had a unique color, I could measure number of visits of each hive to each solution. To see if hive-specific preferences correlate with internal hive dynamics, I measured each hive’s brood development and population size three times a week. This project is ongoing: I will continue both marking and brood tracking until mid-October, this will allow for a complete comparison of micronutrient preferences from mid-summer to mid-fall.

FUTURE EXPERIMENTS

Expected Results
Given the preliminary results for Fall 2013 and Summer 2014, I expect that preferences will change in the spring when flowers are just starting to bloom and hives are just starting to emerge from their over-winter cluster. I expect preferences to change depending on the geographic location of a hive and the surrounding resources. As such, I predict that the Langstroth hives my lab keeps in Grafton, MA will exhibit different preferences than the observation hives I keep in Medford, MA. While both areas are part of the Northeast region and have the same flora in general, the Grafton hives have access to large unmanaged meadows whereas the Medford hives generally exploit managed plots and small gardens. Lastly, I expect that mineral preferences will change with an immune challenge, since various minerals are important for the communication and up-regulation of immune cells.3

Methods
If awarded the 2015 Foundation Scholarship, I will use the funds to run preference assays from Spring 2015 through Fall 2015. This will allow a robust understanding of how mineral preferences change during the foraging seasons. I will also use the funds to run preference assays on a larger scale in Grafton, MA where I will manage twenty Langstroth hives. On this larger scale, I will mark bees and measure how individual hive preferences correlate with hive fitness by replicating, with modification, the marking devices I use with my observation hives. To gain a better understanding of what flora the bees bring back to the hive in Medford and Grafton, I will collect pollen from the hives and from returning foragers and run genetic analyses (RNA sequencing) to identify the plants themselves. While it is generally known what sort of plants are available in different areas, it is not clear what micronutrients Bonoan, Research Proposal, 3 of 3 these sources provide for the hive itself; this will be determined via flame photometry (equipment available at the Tufts University School of Medicine in Boston, MA) and Atomic absorption spectrometry (equipment available at the Tufts University School of Arts and Sciences in Medford, MA) of pollen samples from inside the hive.16 Understanding what pollen sources honey bees are utilizing—and what micronutrients those pollen sources provide—will reveal which micronutrients honey bees are limited by naturally and if micronutrient preferences directly correlate with this limitation.

For my PhD, I am interested in how the preferred micronutrients affect immune function in immune challenged colonies. Raising bees on both monofloral and polyfloral diets, Alaux et al.17showed that diet diversity is vital to the strength of baseline immune function in honey bees. Immune challenged colonies, however, have yet to be studied. Within the next two years, I plan to run an experiment similar to Alaux et al.17 but on colonies challenged with pathogens that have been linked to CCD (e.g. Nosema apis18). As another addition to the Alaux et al.17 design, I will measure more immune parameters in order to achieve a complete understanding of how diet diversity affects immune function. Furthermore, there is not a complete understanding of which nutrients in this “diverse diet” are important. I am interested in which micronutrients the honey bees raised on a polyfloral diet are taking advantage of and which micronutrients those raised on a monofloral diet are lacking. It is possible that bees raised on a polyfloral diet lack micronutrients as well; just because a diet is diverse, does not mean that the bees have everything they need.

BROADER IMPLICATIONS
In the short-term, my current results can help backyard beekeepers to better manage their hives via supplemental diets. At this point, it can be preliminarily concluded that honey bees in the Medford area are looking for supplemental sodium, which is very easy to mix into a sugar solution for supplemental feeding. Currently, these results can be used to convince backyard beekeepers and gardeners that diversity in supplemental diets and in the garden is best. I will be sharing my results with the general public at this fall’s Tufts Community Day where I will have an exhibit of an observation hive and pollen slides made from pollen I collected this summer. I also plan to pass this information along this coming spring as I have been invited to speak at the monthly Cape Cod Beekeeper’s Association meeting as well as a Garden Club meeting in Portsmouth, RI. I am closely involved with the Boston Area Beekeeping Association and have shared my research with various backyard beekeepers in the area via the Tufts Beehive Tour and other local bee-themed events.

In the long term, these results will allow for the development of artificial diets tailored for specific hive characteristics (e.g. amount of brood, honey, etc.), season-specific diet supplements, and better overall nutrition throughout the year. On the commercial side, it will allow for inexpensive supplementation of hives that are subjected to large monocultures—the antithesis of a diverse diet. If we know that honey bees require a certain micronutrient (e.g. based on the pilot data, sodium) or micronutrient palette for proper hive development and that the specific monoculture (e.g. almonds) they are currently pollinating lacks that micronutrient(s), then that micronutrient(s) can easily be added as a supplement to that hive’s diet via a salt solution. This will allow for healthier hives with minimal disruption to current pollination practices. Aside from commercial pollination, naturally healthy, chemical-free hives are especially important to the apicultural industry where honey production is concerned. For the backyard beekeeper, this study will provide insight for the production of more nutritious, well-rounded supplemental diets and therefore naturally healthier and stronger colonies throughout the year.

1. vanEngelsdorp, D., J. D. Evans, C. Saegerman,et al. 2009. PLoS one. 2.Haydak, M. H. 1970. Annu Rev Entomol. 3.Cohen, A. C. 2004. Insect Diets: Science and Technology, CRC Press LLC. 4.Huang, Z. 2010. Am Bee J. 5.Butler, G. C. 1940. J Evolution Biol. 6.Arms, K., P. Feeny and R. C. Lederhouse 1974. Science. 7.Beck, J., E. Muhlenberg and K. Fielder. 1999. Oecologia. 8.Kaspari, M., S. P. Yanoviak, R. Dudley, et al. 2009. PNAS. 9.Chavarria Pizarro, L., H. F. McCreery, S. P. Lawson, et al. 2012. Ecol Entomol. 10.Barrows, E. M. 1974. Fla Entomol. 11.Herbert, E. W. J. and N. J. Miller-Ihli. 1987. Am Bee J. 12. Brodschneider, R. and K. Crailsheim. 2010. Apidologie. 13.Keller, I., P. Fluri and A. Imdorf. 2005. Bee World. 14.Somerville, D. 2005. Fat bees, skinny bees - a manual on honey bee nutrition for beekeepers, Rural Industries Research and Development Corporation. 15.Winston, M.L. 1987. The Biology of the Honey Bee, Harvard University Press. 16.Manzoor, M., G.H.N. Shah, V. Mathivanan, et al. 2013. IJANS. 17.Alaux, C., F. Ducloz, D. Crauser et al. 2010. Biol Letters. 18.Cornman, R. S., D. R. Tarpy, Y. Chen, et al. 2012. PLoS one.

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Foundation for the Preservation of Honey Bees, Inc.
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