NUTRITIONAL DEFICITS: THE IMPACTS OF FUNGICIDES AND LARVAL QUEEN NUTRITION ON HONEY BEE COLONY HEALTH

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: AFRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 1008626
• Grant No.: 2016-67012-24684
• Cumulative Award Amt.: $150,000.00
• Proposal No.: 2015-03505
• Project Start Date: Dec 15, 2015
• Project End Date: Dec 14, 2018
• Grant Year: 2016
• Program Code: [A7201]- AFRI Post Doctoral Fellowships

Recipient Organization
UNIV OF MARYLAND
(N/A)
COLLEGE PARK,MD 20742

Performing Department
Research Administration

Non Technical Summary
Honey bees are the most prolific pollinators of agricultural crops, pollinating 100+ food crops. Beekeepers currently experience unsustainable colony losses, losing 30% of their colonies over the winter and 45% annually. These high losses are due in part to poor nutrition, increased pesticide contamination, and queen losses. Neonicotinoids have received the most attention in the press, but they rarely appear in colony food stores. In stark contrast fungicide contamination is abundant, but the risks to colony health are poorly understood. Fungicides may negatively impact colony wide nutrition by killing the beneficial microorganisms in stored pollen that help make this protein source more nutritious. Honey bee colonies also replace poor quality queens and this turnover is linked with increased colony mortality. Improving larval nutrition of queens could significantly reduce queen turnover and thus colony losses. My proposal outlines three experiments on colony nutrition:1) Fungicide Impacts on Colony Health: determine the impact of fungicides on honey bee health.2) Determine if larval pheromones indicate future queen potential: examine if a natural chemical signal given off by young larvae stimulates queen rearing.3) Improve Larval Queen Nutrition: determine if honey bee larvae supplemented with a natural chemical signal receive better larval nutrition and so improves the quality and longevity of the queen.By addressing these significant issues facing beekeepers, we improve colony health and reduce losses. This ensures a continuous supply of honey bee colonies for pollination in the United States, protecting America's agricultural independence and food security.

Animal Health Component

70%

Research Effort Categories

Basic

25%

Applied

70%

Developmental

5%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
211	3010	1060	50%
211	3010	1010	50%

Knowledge Area
211 - Insects, Mites, and Other Arthropods Affecting Plants;

Subject Of Investigation
3010 - Honey bees;

Field Of Science
1060 - Biology (whole systems); 1010 - Nutrition and metabolism;

Keywords

fungicides

honey bee

nutrition

pollen

pollination

queen quality

royal jelly

Goals / Objectives
Objective 1: determine the impacts of fungicides on colony development. Beekeepers report experiencing increased colony losses shortly after pollinating high-fungicide crops, such as blueberries and watermelon. Currently fungicides are largely deemed safe for bees, but they may impact colony development if they disrupt the beneficial microbial communities in hives. Microorganisms make proteins, lipids and amino acids more available in stored pollen, potentially improving the nutrition of nurse bees, who canalize the protein into honey bee larvae. Disruptions to microbial colony symbionts may result in inadequate nurse bee nutrition and thus increased brood mortality and colony morbidity. Should fungicides prove harmful to colony health, beekeepers could trap incoming pollen in high-risk crops to protect colony health.Objective 2: improve larval queen nutrition. Colonies regularly replace poor quality queens, costing beekeepers money and time. These queen replacement events are significantly tied to colony losses, reducing the number of hives available for pollination. My PhD research suggests that e-beta-ocimene (eβ), the pheromone emitted by both young larvae and well-mated queens may be a "feed me" signal that results in increased feeding. We need a better understanding of how inadequate queen nutrition impacts queen longevity and replacement, and if naturally occurring pheromones can be used to augment queen quality by increasing larval nutrition

Project Methods
Experiment 1: Four contained flight cages will be set-up. Eight colonies established from packages on foundationless frames will be set-up per flight cage (32 total) and fed sugar syrup and pollen. Each flight cage will receive one of the following four pollen treatments: 1) insecticide laced, 2) fungicide laced, 3) insecticide & fungicide laced, 4) uncontaminated control.For treatment group 1, the pollen will be laced with 3 commonly found insecticides chlorpyrifos, fenpropathrin, and pyridaben, providing 10% of an individual bee's LD50 based on average pollen consumption rates of 100 mg during the nursing phase. These are field-relevant contamination rates found in 15% of stored pollen samples taken from commercial beekeepers. Treatment group 2 will be contaminated with two fungicides, chlorothalonil and propiconazole tied to colony losses in blueberries. Because fungicides are currently rated relatively harmless, the pollen will be contaminated at 2% of an individual bee's LD50. Treatment group 3 will be laced with both the 3 pesticides and the 2 fungicides. The control group will receive pesticide-free pollen collected from the desert. In short we will feed colonies pesticide levels based on real world observations.Colonies will be kept inside the flight cages for 1 month, ensuring that a full brood cycle is reared on the four different pollen treatments. Bees will be allowed to forage at a 50% sucrose feeder inside the flight cage. Pollen will be fed in patties inside the colonies and replaced 2x per week. Queen egg laying rates and brood development will be monitored 2x per week, by removing frames and photographing each side so that successful transition from egg to larvae and capped pupae can be followed. Frames of brood will be measured weekly. The queen will be confined for 1 day on a specific comb to lay eggs. The comb will be removed into an incubator prior to adult bee emergence, the bees marked and returned to their colonies. At 7 days of age, this marked cohort will be collected to measure hypopharyngeal gland development. After 1 month, the colonies will be removed from the flight cages and allowed to forage normally. Colonies will be inspected weekly for brood development and disease. Foraging strength will be monitored weekly via multiple 4-min entrance counts. Honey and pollen stores will also be measured and compared across treatment groups.Experiment 2: To determine the specific larval instar preferentially reared into queens, a laying queen will be caged onto a section of comb for 4 hours and the resultant eggs allowed to develop into larvae of known age. This will be repeated over time, so that a single comb contains all five larval instars. The comb will then be placed into a strong queenless colony, where bees will reroute worker-destined larvae into queens. The number of queens cells reared from each larval instar will be recorded, reared to queen emergence and the queen quality evaluated.Experiment 3: Queens will be caged for 4 hours to provide eggs of known age. Shortly after hatching, the larvae will be grafted into queen cups using standard queen rearing techniques. For pheromone studies, eβ is typically suspended in paraffin oil. One subset of grafted larvae will be supplemented with different doses of eβ corresponding to 1, 10, 100 and 1000 larval equivalents emitted by normal 2nd instar worker larvae. The remainder will serve as 1) paraffin oil controls and 2) un-manipulated controls. We will measure queen cell acceptance rate and the amount of royal jelly deposited in a subset of cells. The remaining queen cells will be reared to emergence. A subset of reared queens will be evaluated for queen quality (weight, thorax width, head width, and ovariole number). The remainder will be open-air mated and installed in colonies to measure supersedure rates and longevity. A subset of the mated queens will be evaluated for insemination success using standard evaluation techniques. All together 30 queens reared for each eβ treatment dose will be evaluated for queen quality; 20 will be evaluated for supersedure rates and longevity; and 10 will be evaluated for insemination success.

Progress 12/15/15 to 12/14/18

Outputs
Target Audience:Results from experimental trials have been presented to other scientists at conferences and targeted stakeholders at local, state and regional beekeeper meetings. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The grant provided opportunities to network with other scientists involved in parallel fields and develop my oral presentation skills. How have the results been disseminated to communities of interest?The results have been presented at a scientific conference, we've submitted an abstract to Apimondia in Montreal in 2019, and we are finalizing a manuscript. We've given several talks to local, state and regional beekeeping meetings to raise awareness of the results with stakholders. The research on pesticides in pollen was presented at the national meeting of American Honey Producers in Phoenix, AZ in January 2019. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Objective 1: Pesticide residues in bee bread were analyzed for six consecutive years from apiaries in over 35 states. Two insecticides (chlorpyrifos and fenpropathrin), and two fungicides (chlorothalonil and propiconazole) were frequently detected during the survey. We ran a large scale field trial in 2018 feeding colonies sublethal doses of the most commonly occuring pesticides. We applied field relevant doses, at a) 10% of LD50 of the two insecticides, or b) 2% of the LD50 for the two fungicides or c) a combination of both to treatment groups over a 30 day period. The contaminants were fed to the treatment groups through pollen patties, and incoming pollen was restricted by a pollen trap installed at the entrance of every colony. Foraging activity was recorded weekly at the hive entrance. All treatment groups sent out a higher proportion of pollen foragers compared to controls, though engaged in less overall foraging activity. Additionally, each treatment group consumed less of the pollen patty than the control group. Brood development was tracked from egg to emergence, before and after treatments. Following treatment, total loss of brood prior to emergence was 11.4% for the control group, 20.4%, 29.3%, and 20.8% for fungicide, insecticide and both contaminant groups respectfully. Brood cannibalization was significantly higher in all treatment groups. Queen loss through supersedure occurred in the treatment groups as well. We are finalizing our data analysis and preparing the manuscript for publication. We have planned future experiments to focus on the impacts of these fungicides on microsymbionts in the colony. Objective 2: We found that low level fungicide exposure reduced queen acceptance and caused queen rejection. The pheromone supplementation experiments were inconclusive and we are designing additional trials to investigate other nutritional supplementation to improve queen health.

Publications

• Type: Conference Papers and Presentations Status: Accepted Year Published: 2018 Citation: Traynor et al (2018) Pesticide Residues in Bee Bread from the Natonal Honey Bee Disease Survey. EurBee 8. Ghent, Belgium. Sept 18-20, 2018.
• Type: Journal Articles Status: Other Year Published: 2019 Citation: Lamas, Z.S., D. vanEnglesdorp, K.S. Traynor. Queen replacement and brood cannibalism in colonies exposed to sublethal pesticides in pollen.
• Type: Journal Articles Status: Other Year Published: 2019 Citation: Traynor, K. S., S. Tosi, N. Steinhauer, R. Rose, D. vanEngelsdorp. Pesticide residues in stored pollen from the National Honey Bee Disease Survey.

Progress 12/15/16 to 12/14/17

Outputs
Target Audience:The target audience were stakeholders, including beekeepers, farmers and home owners. Also other scientists working in this field of research. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?It has allowed me to present results at a national conference. It has allowed me to refine and develop protocols. It allowed me to increase my skill set in instrumental insemination. How have the results been disseminated to communities of interest?Public presentations to stakeholders and at scientific conferences; published papers in open access journals What do you plan to do during the next reporting period to accomplish the goals?Redo both experiments to see if trends are significant over additional replicates.

Impacts
What was accomplished under these goals? Objective 1: We ran this experiment, but due to inclement weather the results were inconclusive. We experienced too much swarming. We intend to repeat the experiment this spring. Objective 2: We designed a protocol for supplementing larvae to improve queen quality. The intial results looks promising, but were insignificant. We intend to repeat the experiment this spring and summer to determine if pheromone supplementation does increase queen quality.

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Traynor & vanEnglesdorp (2017) The impacts of commonly applied insecticides and fungicides on Apis mellifera nutrition and colony development. Entomological Society of America. Denver, CO.
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Traynor, K. S., Pettis, J. S., Tarpy, D. R., Mullin, C. A., Frazier, J. L., & Frazier, M. (2016). In-hive Pesticide Exposome: Assessing risks to migratory honey bees from in-hive pesticide contamination in the Eastern United States. Scientific reports, 6, 33207.
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Traynor, K. S., Wang, Y., Brent, C. S., Amdam, G. V., & Page, R. E. (2017). Young and old honeybee (Apis mellifera) larvae differentially prime the developmental maturation of their caregivers. Animal Behaviour, 124, 193-202.

Progress 12/15/15 to 12/14/16

Outputs
Target Audience:The target audience reached during this year included several beekeeping organizations, as I was invited to speak by the Long Island Beekeepers Association, the Virginia State Beekeepers Association, and several regional beekeeping clubs. Changes/Problems:We experienced a very cool, wet spring, which significantly delayed colony development and hampered queen rearing opportunities. The early summer was then frequently punctuated by rain, once again making queen rearing difficult. Despite these poor weather conditions, I was able to develop protocols for supplementing queen larvae with pheromones and will repeat the experiments in Spring 2017. What opportunities for training and professional development has the project provided?The project has allowed me to improve my statistical analysis skills. It has also increased my public speaking abilities, as I presented to numerous beekeeping clubs. It has allowed me to network with well-established professors in my field, building a solid research network. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?I will address objective 1 with a long-term feeding and survival experiment to be initiated this spring. I will redo the experiments for objective 2, now that I have developed reliable queen rearing protocols.

Impacts
What was accomplished under these goals? This season I addressed Objective 2, developing appropriate protocols for improving queen larval nutrition. This required testing multiple methods of application to determine if the larvae would be reared to completion. The weather unfortunately did no co-operate well this season, making queen rearing more difficult than usual. The experiments with the perfected protocol will thus be redone next spring.

Publications

• Type: Journal Articles Status: Accepted Year Published: 2017 Citation: Traynor, KS, Wang Y., Brent C.S., Amdam G.V., Page, R.E. (in press) Young and old honey bee (Apis mellifera) larvae differentially prime the developmental maturation of their caregivers. Animal Behavior