Development of pheromonal tools for honeybee breeding

2014 Annual Report for GNE14-090

Project Type: Graduate Student
Funds awarded in 2014: $14,998.00
Projected End Date: 12/31/2015
Grant Recipient: Pennsylvania State University
Region: Northeast
State: Pennsylvania
Graduate Student:
Faculty Advisor:
Dr. Thomas Baker
Pennsylvania State University
Faculty Advisor:

Development of pheromonal tools for honeybee breeding


The purpose of this project is to lay the groundwork for the development of natural, large-scale stock improvement programs necessary for the conservation of a principal crop pollinator, the honey bee. Currently, the decline of populations of honey bees (Apis mellifera), the primary providers of pollination services in agriculture, is a major threat to food security in the United States, given that a majority of our fruit, vegetable, and nut crops are largely or entirely (i.e. almonds) dependent on honey bee pollination (Gallai et al. 2009; Winfree, 2009). This decline is attributed to the multiple challenges these pollinators currently face, which include pressure from parasites and pathogens, exposure to pesticides, reduced nutrition, and even climate fluctuations (Potts et al. 2010). In addition, honey bee breeding currently requires a high level of technical expertise and expensive equipment to facilitate artificial insemination. Our goal is to find/develop accessible tools and methods to facilitate controlled natural mating.


Because many honey bee behaviors are mediated by pheromones, we have begun to explore the effects of a major queen pheromone (9-ODA) on the physiology and behavior of males. This pheromone has previously shown to regulate worker behavior, development/physiology, thus potentially similar impacts on drones could provide ways on regulating drone reproductive behavior. A second objective seeks to identify whether a male-produced pheromone exists and its role in mediating male mating aggregations. Currently, we are near completion with our first objective, having found and characterized an effect of 9-ODA on both drone physiology and reproductive behavior. Progress on additional objectives is ongoing, and remains in accordance with the previously proposed timeline.

Objectives/Performance Targets

Objective 1: Characterize the effect of the queen pheromone, 9-ODA, on the rate of drone sexual maturation.



    1. Characterize Vg/Juvenile hormone levels in 9-ODA exposed/unexposed male drones. (Completed)


    1. Characterize the effects of 9-ODA exposure on the reproductive behavior of drones under natural colony conditions. (Completed)





Objective 2: Identify and characterize a putative drone-produced pheromone involved in the formation of drone aggregations.



    1. Dissect putative pheromone glands from male drones and extract the contents. (Completed)


    1. Analyze gland contents using GC-MS. Identify compounds produced in the glands. (Ongoing)


    1. Ascertain the biological activity of identified gland compounds using behavioral assays. (Expected completion: Summer 2015)





Objective 3: Disseminate the practical outcomes of these objectives to research and stakeholder communities



    1. Present ongoing findings at professional and stakeholder meetings. (ABS 2014; Ongoing)


    1. Publish findings in professional research journals. (Expected, Spring 2015; Fall 2015)



Experiments for objectives 1 and 2 were performed in the Summer of 2014 on The Pennsylvania State University campus. To address objective 1, we performed two experiments to assess the effects of 9-ODA on male physiology and behavior. A replicated cage study assay showed that 9-ODA exposure affected the expression of the egg-yolk protein Vitellogenin (Vg) (Figure 1A) and blood hemolymph levels of the critical behavioral regulator, Juvenile hormone (Figure 1B). This is exciting for multiple reasons. First, the role of drones in the colony has been largely overlooked and perhaps underestimated by researchers due to the generalization that males do not contribute to colony productivity. The finding that a queen pheromone may function to regulate critical features of male physiology may suggest a larger role of drones as members of the colony. Second, the impact of a pheromone on male physiology is indicative of a “primer” effect, the first such finding in male honey bees, and a rare example in males within all of the animal kingdom. This opens up new areas of inquiry in terms of identifying the biological function and relevance of this effect by 9-ODA.


A replicated colony level study showed that 9-ODA exposure had an impact on both the latency to begin reproductive mating flights (Figure 2A) in drones and on the number of flights males took (Figure 2B). These findings complement the results of the first experiment, suggesting that drone physiology is impacted by 9-ODA exposure in such a way as to delay and/or retard reproductive behavior. The surprise in the result of this second experiment is that the more profound impacts are not on the latency to initiate reproductive behavior, but rather in the frequency of attempted reproduction on a colony level, both of which are retarded by 9-ODA exposure.


Work on objective 2 also began this past summer, with the dissection and solvent extraction of mandibular glands from sexually mature drones. GC-MS analysis of the extracts is ongoing and will continue through the spring. We expect these analyses to yield candidate compounds for which we will characterize their biological activity in the coming year. Completion of objective 1 is nearly complete, requiring the processing of the samples for a second replicate and a complete statistical analysis of the data. Work on objective 2 has begun and is ongoing.

Impacts and Contributions/Outcomes

This project remains in its infancy, but has already yielded exciting and potentially useful results. We now have evidence that 9-ODA may be regulating specific features of reproductive physiology and behavior in males, providing a potential avenue for developing practical and accessible breeding methods for stock improvement programs. If the effect we produced could be scaled to a commercial breeding operation, it would allow a breeder to make the males in selected colonies more reproductively competitive than others in the same area, allowing for male stock selection to take place in a natural setting.

Our ongoing work on objective 2 has the potential to identify compounds which could be used to regulate the formation of mating congregations in drones. Taken together, the outcomes herein may yield methods for controlling male reproductive behavior both in and out of honey bee colonies.


Recently, our progress on objective 1 was presented at the Annual Meeting of the Animal Behavior Society (ABS) in Princeton, NJ.


Dr. Thomas Baker
Distinguished Professor
Pennsylvania State University
Chemical Ecology Laboratory
University Park, PA 16802
Dr. Christina Grozinger
Pennsylvania State University
Chemical Ecology Laboratory - Room 1
University Park, PA 16802