Source: PENNSYLVANIA STATE UNIVERSITY submitted to
MOLECULAR MECHANISMS OF HONEY BEE MATING
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
NRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
0218405
Grant No.
2006-35607-06064
Project No.
PEN04333
Proposal No.
2009-02063
Multistate No.
(N/A)
Program Code
51.3
Project Start Date
Jan 15, 2009
Project End Date
Nov 30, 2010
Grant Year
2009
Project Director
Grozinger, C. M.
Recipient Organization
PENNSYLVANIA STATE UNIVERSITY
208 MUELLER LABORATORY
UNIVERSITY PARK,PA 16802
Performing Department
Entomology
Non Technical Summary
Honey bees have a number of reproductive states, including virgin queens, laying virgin queens, instrumentally inseminated queens, and naturally mated queens (Winston 1987) . For these different reproductive states, we can monitor behavior (ie, egg-laying behavior or taking mating flights) pheromone production, ovary activation, and gene expression in both the brain and ovaries using microarray analysis. Queen pheromone production, in particular, is critical to colony organization and health, since it regulates many aspects of worker behavior (including worker reproduction and foraging) and inhibits rearing of new queens and swarming, which can reduce colony strength (Slessor et al. 2005) . Ultimately, our goal is to understand the physiological processes that cause post-mating changes in queens. At the molecular level, we hope to match gene expression patterns with specific physiological or behavioral changes. These studies will identify candidate genes and pathways that can serve as the basis for further functional analyses or as markers for breeding programs. This research has been conducted by two graduate students (Sarah Kocher and Elina Lastro Nino) and a post-doctoral associate (Freddie-Jeanne Richard, now an assistant professor at University of Poitiers, France), in collaboration with Professor David Tarpy (NCSU). We have found that brain gene expression and queen pheromone profiles are significantly modified by insemination quantity, in studies using instrumentally inseminated and natural mated queens (Kocher et al. 2008; Richard et al. 2007) . Furthermore, workers are attuned to these differences and are preferentially attracted to the pheromone produced by multiply mated queens. Our studies have demonstrated that both stretch receptors in the oviducts and seminal proteins appear to trigger post-mating changes (Richard et al. in prep, Kocher et al submitted) . Given that instrumental insemination is critical for honey bee breeding and selection (particularly to prevent accidental mating with Africanized bees), these results will allow us to develop modifications to the instrumental insemination process to further improve queen quality and colony health. These studies suggest that the production of queen pheromone is exquisitely sensitive to factors associated with reproduction and mating.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
3053110113050%
3053110108050%
Knowledge Area
305 - Animal Physiological Processes;

Subject Of Investigation
3110 - Insects;

Field Of Science
1080 - Genetics; 1130 - Entomology and acarology;
Goals / Objectives
Honey bees have a number of reproductive states, including virgin queens, laying virgin queens, instrumentally inseminated queens, and naturally mated queens (Winston 1987). For these different reproductive states, we can monitor behavior (ie, egg-laying behavior or taking mating flights) pheromone production, ovary activation, and gene expression in both the brain and ovaries using microarray analysis. Queen pheromone production, in particular, is critical to colony organization and health, since it regulates many aspects of worker behavior (including worker reproduction and foraging) and inhibits rearing of new queens and swarming, which can reduce colony strength (Slessor et al. 2005). Ultimately, our goal is to understand the physiological processes that cause post-mating changes in queens. At the molecular level, we hope to match gene expression patterns with specific physiological or behavioral changes. These studies will identify candidate genes and pathways that can serve as the basis for further functional analyses or as markers for breeding programs. Furthermore, these studies will allow us to better understand the processes and factors involved in stimulating post-mating changes in queens, which will undoubtedly lead to improvements in instrumental insemination process and breeding operations that can produce healthier queens. This research has been conducted by two graduate students in my group (Sarah Kocher and Elina Lastro Nino) and a post-doctoral associate (Freddie-Jeanne Richard), and in collaboration with Professor David Tarpy (NCSU).
Project Methods
We have found that brain gene expression and queen pheromone profiles are significantly modified by reproductive state. Comparisons of virgin queens, incompletely mated and fully mated, laying queens revealed that incompletely mated queens were intermediate in terms of ovary activation, ovary gene expression patterns, and pheromone profiles (Kocher et al 2008). However, brain expression patterns in these queens more closely resembled virgin queens, suggesting that brain expression patterns track behavioral changes, while ovary expression patterns track ovary activation and changes in pheromone profiles. A second study comparing virgin, saline-, semen-inseminated and naturally mated queens revealed that again brain expression patterns tracked behavior (Kocher et al in prep). However, ovary development and pheromone profiles were closely linked (Kocher et al submitted). Interestingly, workers were more attracted to the chemical extracts of queens with higher ovary activation scores. This suggests that queen pheromone may be an honest signal of queen fecundity. Another series of studies revealed that insemination quantity is important for regulating post-mating changes in queens (Richard et al 2007). Queens inseminated with semen from a single drone (single drone inseminated, SDI) vs 10 drones (multi-drone inseminated) have significant differences in brain gene expression and pheromone profiles. Workers are more attracted to the chemical extracts of MDI queens. Furthermore, both volume and insemination substance (semen vs saline) are important for producing these changes (Richard et al in prep). Since these studies were conducted shortly after insemination, we have repeated these studies using queens maintained in small colonies and collected after egg-laying has initiated (Nino, Tarpy and Grozinger, unpublished data). Finally, since the instrumental insemination protocol involved anesthetizing queens with carbon dioxide, we compared virgin queens, CO2-treated queens, and queens that were treated with CO2 and also physically manipulated but not inseminated (Nino, Tarpy and Grozinger, unpublished data) with chemical, behavioral and microarray analyses. We will also complete a study of the supercedure rates of queens inseminated with low vs high volumes of semen in colonies. Studies conducted in 2008 (and partially supported by a grant from the Florida Department of Agriculture) demonstrated that colonies headed by low volume-inseminated queens built more queen cells and cups, and these queens had a significantly lower life span than their high-volume inseminate sisters. We will complete a second replicate. Finally, preliminary studies have revealed that SDI vs MDI queens have differential expression of candidate immune genes in their fat bodies, suggesting that their responses to infection may also differ. Given the importance of honey bee viruses to bee health, we will investigate this further, by comparing the effects of deformed wing virus infection on SDI and MDI queens. We will monitor the duration of infection, the molecular immune response, and the effects on chemical communication between the queen and workers.

Progress 01/15/09 to 11/30/10

Outputs
OUTPUTS: Honey bees have a number of reproductive states, including virgin queens, laying virgin queens, instrumentally inseminated queens, and naturally mated queens (Winston 1987). For these different reproductive states, we can monitor behavior (ie, egg-laying behavior or taking mating flights) pheromone production, ovary activation, and gene expression in both the brain and ovaries using microarray analysis. Queen pheromone production, in particular, is critical to colony organization and health, since it regulates many aspects of worker behavior and inhibits rearing of new queens and swarming, which can reduce colony strength (Slessor et al. 2005). Ultimately, our goal is to understand the physiological processes that cause post-mating changes in queens. At the molecular level, we hope to match gene expression patterns with specific physiological or behavioral changes. These studies will identify candidate genes and pathways that can serve as the basis for further functional analyses or as markers for breeding programs. Furthermore, these studies will allow us to better understand the processes and factors involved in stimulating post-mating changes in queens, which will undoubtedly lead to improvements in instrumental insemination process and breeding operations that can produce healthier queens. This research has been conducted by two graduate students in my group (Sarah Kocher and Elina Lastro Nino) and a post-doctoral associate (Freddie-Jeanne Richard), and in collaboration with Professor David Tarpy (NCSU). We examined the association between brain gene expression, pheromone profiles and queen reproductive state (Richard et al 2007, Kocher et al 2008). We dissected the effects of ovary activation on pheromone production and worker responses to the pheromone (Kocher 2009), and the effects of insemination volume and substance of pheromone production, brain gene expression, and ovary activation (Kocher 2010, Nino et al 2011, Richard et al submitted, Nino et al in prep). We performed analyses shortly after mating (2-5 days) and after egg-laying had initiated (10 days after insemination). From 2005-2010, data from these studies was disseminated by the PI at 15 invited departmental seminars and 14 invited talk at symposia at national and international scientific conferences. Several additional talks were presented at conferences and beekeeper meetings by the coPI, students and postdoc associated with this project. PARTICIPANTS: Christina Grozinger (PI; Associate Professor, Penn State) David Tarpy (Co-PI; Associate Professor, NCSU) Freddie-Jeanne Richard (Assistant Professor, University of Poitiers; postdoctoral research at NCSU from 2005-2008) Elina Lastro Nino (PhD student, Penn State) Sarah Kocher (PhD student, NCSU; Postdoctoral Associate, Penn State) Bernardo Nino (Senior Research Technician, Penn State) Joe Flowers (Research Technician, NC State) David Galbraith (Undergraduate Researcher, Penn State) Stephanie Narvaez (Undergraduate Researcher, Penn State) Dave Overman (Undergraduate Research, NCSU) Philip Durham (Undergraduate Research, NCSU) Kelly Hutcherson (Undergraduate Research, NCSU) This project provided opportunities for the students and postdoc involved to attend and present at a variety of conferences, including the annual Entomology Society of America Meetings, the Congress of the International Society for the Study of Social Insects, and the Honey Bee Biology and Genomics meeting at Cold Spring Harbor. TARGET AUDIENCES: The research outcomes are relevant to the scientific community, including social insect researchers, insect physiologists, behavioral ecologists, chemical ecologists, and evolutionary biologists. It is also of substantial importance to beekeepers and the public, especially given the great interest in conservation of honey bees and other pollinator species. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
We found that ovary activation and pheromone production were linked, and could be dissociated from behavioral changes during the transition from virgin to mated queen (Kocher 2008). Ovary activation was strongly linked to changes in pheromone blend, and queens that had more activated ovaries were more attractive to workers (Kocher 2009). Insemination volume altered pheromone production, and workers were more attracted to the extracts of multiply inseminated queens than singly inseminated queens (Richard 2007). Inseminated queens differed from naturally mated queens in pheromone profiles and brain gene expression (Kocher 2009, 2010). Insemination volume appeared to play a role in triggering short-term changes (Kocher 2010), while insemination substance may play a role in regulating long-term changes (Nino et al in prep). Treatment with CO2 and physical manipulation during instrumental insemination also caused long-term changes in gene expression and pheromone profiles (Nino 2011). The effects of instrumental insemination treatment, volume and substance can be observed even after queens have initiated egg-laying, and thus the effects of the mating process can be relatively long-lasting, even after queens reach their "final" reproductive state.

Publications

  • Nino, E. L., D. R. Tarpy, and C. M. Grozinger. 2011. Dissection of factors that trigger post-mating changes in honey bee queens (Apis mellifera L.). Insect Molecular Biology (In Press).
  • Kocher, S. D., F. J. Richard, D. R. Tarpy, and C. M. Grozinger. 2008. Genomic analysis of post-mating changes in the honey bee queen. BMC Genomics 9:232.
  • Richard, F. J., D. R. Tarpy, and C. M. Grozinger. 2007. Effects of insemination quantity on honey bee queen physiology. PLoS ONE 2(10):e980.


Progress 01/15/09 to 01/14/10

Outputs
OUTPUTS: Honey bees have a number of reproductive states, including virgin queens, laying virgin queens, instrumentally inseminated queens, and naturally mated queens (Winston 1987). For these different reproductive states, we can monitor behavior (ie, egg-laying behavior or taking mating flights) pheromone production, ovary activation, and gene expression in both the brain and ovaries using microarray analysis. Queen pheromone production, in particular, is critical to colony organization and health, since it regulates many aspects of worker behavior and inhibits rearing of new queens and swarming, which can reduce colony strength (Slessor et al. 2005). Ultimately, our goal is to understand the physiological processes that cause post-mating changes in queens. At the molecular level, we hope to match gene expression patterns with specific physiological or behavioral changes. These studies will identify candidate genes and pathways that can serve as the basis for further functional analyses or as markers for breeding programs. Furthermore, these studies will allow us to better understand the processes and factors involved in stimulating post-mating changes in queens, which will undoubtedly lead to improvements in instrumental insemination process and breeding operations that can produce healthier queens. This research has been conducted by two graduate students in my group (Sarah Kocher and Elina Lastro Nino) and a post-doctoral associate (Freddie-Jeanne Richard), and in collaboration with Professor David Tarpy (NCSU). In previous years, we found that brain gene expression and queen pheromone profiles are significantly modified by reproductive state (Richard et al 2007, Kocher et al 2008). In subsequent studies, we more closely examined the effects of ovary activation on pheromone production and worker responses to the pheromone, and dissected the effects of insemination volume and substance of pheromone production, brain gene expression, and ovary activation. We performed analyses shortly after mating (2-5 days) and after egg-laying had initiated (10 days after insemination). We have initiated a project to examine even longer term effects of insemination volume by monitoring egg-laying queens in small colonies for several weeks in the field. We monitored queen longevity, queen-worker interactions, and worker physiology. We will complete the field portion of this study in the summer of 2010. In 2009, the data from these studies was disseminated at 3 invited departmental seminars, two invited symposia presentations at the Entomological Society of America and Society of Molecular Biology and Evolution Annual Meetings, and an invited talk and workshop on queen breeding at the PA State Beekeeper's Meeting. PARTICIPANTS: Christina Grozinger (PI; Associate Professor, Penn State). David Tarpy (Co-PI; Associate Professor, NCSU). Freddie-Jeanne Richard (Assistant Professor, University of Poitiers; postdoctoral research at NCSU from 2005-2008). Elina Lastro Nino (PhD student, Penn State). Sarah Kocher (PhD student, NCSU; Postdoctoral Associate, Penn State). Bernardo Nino (Senior Research Technician, Penn State). David Galbraith (Undergraduate Researcher, Penn State). Stephanie Narvaez (Undergraduate Researcher, Penn State). This project provided support for Ms. Lastro Nino to attend the Entomological Society of American Annual Meeting in December 2009 and present her research, and for Mr. Nino to attend the American Beekeeping Federation Annual meeting in January 2010. TARGET AUDIENCES: The research outcomes are relevant to the scientific community, including social insect researchers, insect physiologists, behavioral ecologists, chemical ecologists, and evolutionary biologists. It is also of substantial importance to beekeepers and the public, especially given the great interest in conservation of honey bees and other pollinator species. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
We completed analysis of and published a study comparing virgin, saline-inseminated, semen-inseminated and naturally mated queens, two days after insemination or mating. This study revealed that brain expression patterns tracked flight behavior (Kocher et al in press, 2009). Overall brain expression patterns of the inseminated groups clustered together, while the brain expression patterns in virgin and naturally mated queens were more similar based on a hierarchical clustering analysis. However, ovary activation and pheromone profiles were intermediate for the two inseminated groups (Kocher et al 2009). Interestingly, in a choice test, workers were more attracted to the chemical extracts of naturally mated queens compared to either inseminated group, and more attracted to the chemical extracts of the two inseminated groups compared to the virgins. This suggests that pheromone production tracks ovary activation, and workers are sensitive to slight changes in chemical profiles. It also suggests that queen pheromone may be an honest signal of queen fecundity. We have been analyzing data related to three other studies. The first examined the effects of CO2 (an anesthetic used during instrumental insemination) and physical manipulation on queen behavior, physiology and brain gene expression; the second examined the effects of insemination volume and substance on brain gene expression and pheromone production, and the third examined the effects of these two factors on fat body gene expression, behavior and physiology. Overall our results suggest that CO2 treatment has significant effects on mating flight behavior and weakly effects ovary development, while physical manipulation has stronger effects on ovary activation. Both substance and volume play an important role in regulate gene expression patterns and pheromone production. These effects can be observed even after queens have initiated egg-laying, and thus the effects of the mating process can be relatively long-lasting, even after queens reach their "final" reproductive state.

Publications

  • Kocher, S.D., F.J. Richard, D.R. Tarpy, and C.M. Grozinger. 2009. The effects of mating and instrumental insemination on honey bee behavior, physiology, and brain gene expression. Insect Molecular Biology. Published online December 11, 2009. http://dx.doi.org/10.1111/j.1365-2583.2009.00965.x
  • Kocher, S.D., F.J. Richard, D.R. Tarpy, and C.M. Grozinger. 2009. Queen reproductive state modulates queen pheromone production and queen-worker interactions in honey bees. Behavioral Ecology 20:1007-1014