Source: PENNSYLVANIA STATE UNIVERSITY submitted to NRP
BEHAVIORAL, PHYSIOLOGICAL AND MOLECULAR EFFECTS OF MULTIPLE FACTORS IMPACTING HONEY BEE HEALTH
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
Reporting Frequency
Annual
Accession No.
0221120
Grant No.
2010-65105-20579
Cumulative Award Amt.
$397,348.00
Proposal No.
2009-05207
Multistate No.
(N/A)
Project Start Date
Mar 1, 2010
Project End Date
Jun 30, 2014
Grant Year
2010
Program Code
[91112]- Arthropod and Nematode Biology and Management: Suborganismal Biology
Recipient Organization
PENNSYLVANIA STATE UNIVERSITY
208 MUELLER LABORATORY
UNIVERSITY PARK,PA 16802
Performing Department
Entomology
Non Technical Summary
Stress can have profound effects on behavior and physiology. The importance of stressors in agricultural systems has been highlighted in recent reports of "Colony Collapse Disorder", in which managed honey bee colonies undergo rapid and massive population declines. CCD is likely caused by a combination of stressors: pests (ie Varroa mites), pathogens, environmental toxins (including the acaricides used to control Varroa), and poor nutrition, possibly due to the use of artificial diets and monoculture cropping systems. However, in order to fully understand how different stressors undermine the health of social insects, it is necessary to examine effects on individuals and across the complete social structure. We will examine the effects of four stressors (mating number, nutrition, virus infection, and environmental toxins) on queen and worker physiology, behavior, and social interactions. The research outlined in this proposal is highly multidisciplinary and integrative, seamlessly combining apiculture, behavioral ecology, chemical ecology and genomic analyses. It will dramatically improve our understanding of the molecular, physiological and behavioral effects of stress responses in insects, in particular social insects, have significant impacts on the management of honey bee populations for agriculture, and serve as an outstanding vehicle for education and outreach. Our studies will allow us to determine if a broad array of stressors has similar or specific impacts on behavior, physiology, social communication, gene expression, and gene networks in honey bees, and thus will provide an integrative understanding of the effects of stress. Honey bees are an excellent model system for these studies because this species has large genomic resources, complex social behavior, and a number of naturally occurring stressors which are critical for both managed and unmanaged bee populations. Futhermore, honey bees are the most important pollinator of almost 100 crops and orchards grown in the U.S., and they are a producer of honey and other hive products. Our studies will highlight the importance of modifying current management practices in order to improve honey bee heath, but may also provide new diagnostic tools or methodology to ameliorate the effects of the stressor. For example, if there is a chemical "stress signature" that can be observed in the blend of volatile organic compounds of the queen, queens can be nondestructively surveyed to monitor the health of the colony. If there are specific molecular stress signatures revealed by the microarray analysis, workers can be sampled to monitor colony health or perform epidemiological analyses. Finally, if poor queen pheromone production negatively impacts worker behavior and physiology, supplements can be developed to maintain colony organization and structure until the queen can be replaced.
Animal Health Component
25%
Research Effort Categories
Basic
75%
Applied
25%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
3053010106060%
3063010106020%
3133010106020%
Goals / Objectives
Stress can have profound effects on behavior and physiology. The importance of stressors in agricultural systems has been highlighted in recent reports of "Colony Collapse Disorder", in which managed honey bee colonies undergo rapid and massive population declines. CCD is likely caused by a combination of stressors: pests (ie Varroa mites), pathogens, environmental toxins (including the acaricides used to control Varroa), and poor nutrition, possibly due to the use of artificial diets and monoculture cropping systems. However, in order to fully understand how different stressors undermine the health of social insects, it is necessary to examine effects on individuals and across the complete social structure. We will examine the effects of four key stressors experienced by honey bees: reduced queen mating number, reduced nutrition, environmental toxins (focusing on coumaphos, used to control two major bee parasites), and pathogens (focusing on two viruses). Objective 1 will characterize the effects on queen physiology, queen pheromone production and pheromone-mediated worker behavior. Objective 2 will characterize physiological parameters, including global gene expression patterns, in workers reared in these colonies. Objective 3 will determine if differences in social cues are sufficient to cause physiological differences in workers. These studies will provide critical information about the molecular, physiological and behavioral responses to an array of important stressors in an agriculturally important model system. Our results can be used to develop better management strategies for bees and other species. Furthermore, we will lay the groundwork for future studies examining the role of stressors in chemical communication.
Project Methods
Objective 1: Determine if these four factors negatively impact queen pheromone production and physiology, and pheromone-mediated worker behavior. We will fully characterize the complete blend of volatile organic compounds (VOCs) released by the queen and total chemical composition of the mandibular glands, the main source of queen pheromone in these different treatment groups. We will monitor pheromone-mediated behavioral responses of the workers to the queen, including attraction and supercedure attempts. Finally, we will monitor brood production and the levels of stored sperm. Objective 2: Determine how these four factors impact worker physiology. Marked, one-day old workers from a single genetic background will be placed in the colonies exposed to the different treatments in Objective 1. Seven days later, workers will be collected and assessed for specific physiological parameters (levels of juvenile hormone and lipids) and global brain gene expression using whole genome microarrays. The gene expression patterns will be compared across the four factors and with previous studies to determine if factors have similar effects on workers, and if expression of specific functional categories or behaviorally-relevant groups of genes are modulated. Objective 3: Determine if differences in queen mandibular gland chemical composition are sufficient to alter worker physiology in cages. Worker bees from a single genetic background will be reared in cages with mandibular gland extracts from the queens in Objective 1. After one week, workers will be collected and assessed for physiological parameters and brain expression patterns, as in Objective 2. These studies will determine if queen pheromone production alone can have significant impacts on worker physiology. The research in this proposal will substantially strengthen the ongoing education and outreach programs in the Grozinger and Tumlinson labs. First, it will provide training opportunities for two outstanding graduate students, as well as undergraduate researchers and high school students. Secondly, the data and samples produced by these studies will be incorporated into a new course that Grozinger is developing with Dr. Harland Patch, "Concepts and Techniques in Molecular Ecology". Finally, the results and methodology from this proposal will be broadly disseminated to the public and honey bee community, through connection with local K-12 educations and local, regional and national beekeeper groups. Finally, Penn State has recently formed a Center for Pollinator Research, with a mission to bring together researchers, educators, extension and outreach specialists from across the Penn State campus, the US, and the world, to begin to address the complex issues facing pollinator populations. Grozinger is the Director of this Center, and Tumlinson serves on the Steering Committee. This Center will provide an excellent platform for disseminating the research results from this study and forming additional collaborations.

Progress 03/01/10 to 06/30/14

Outputs
Target Audience: The ongoing research projects are of significant interest to members of the scientific community interested in plant-pollinator interactions, bee behavior and health, as well as to beekeepers and other stakeholder groups interested in developing approaches to conserving and supporting honey bee and bumble bee populations. Changes/Problems: Since our genomic studies revealed a strong impact of metabolic and nutrition-sensing pathways in response to parasite infection and exposure to pesticides, we decided to explore the role of these pathways in more detail in response to harsh overwintering conditions, when bees are unable to actively foraging for food. Since this is the time of year when beekeepers experience the greatest losses, providing insights into the mechanisms regulating overwintering failure or success can improve management practices. Furthermore, since diet greatly decreased the toxicity of pesticides on honey bees, we are exploring the role of diet in impacting viral and parasite loads in bees. What opportunities for training and professional development has the project provided? During this reporting period, the project provided partial or complete support for the research conducted by four graduate students (David Galbraith, Holly Holt, Anthony Vaudo and Mehmet Doke) and two postdoctoral researchers (Elina Lastro Nino, Etya Amsalem). Three undergraduate students were also engaged as research assistants for the project. The students and postdoctoral fellows thus had the opportunity to engage in multidisciplinary research spanning genomics, physiology, behavior and ecology, and presented their research finding at multiple scientific conferences and stakeholder group meetings. Note that both postdoctoral fellows and two students has their salaries fully supported by fellowships from other sources. How have the results been disseminated to communities of interest? Results from these studies were reported by the PI, students and postdoctoral fellows at four invited talks at scientific conferences, five invited seminars in the US and Europe, contributed talks and posters at three scientific conferences, presentations (including hands-on workshops) at three regional and national beekeeper conferences, and at the 2013 and 2014 Honey Bee Queen Rearing Workshop, a two day workshop hosted by the PI. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? We completed the analysis of the impacts of chronic parasitization (with Nosema) on gene expression patterns in multiple tissues over a time course in worker honey bees. The results of these studies revealed that Nosema infection has immediate impacts on metabolic and nutrition-sensing pathways and only later impacts expression of genes involved in behavioral maturation and immunity. These data provided a model for understanding the pleiotropic effects of chronic parasitization and resulted in one manuscript, published in 2013. Ongoing studies are examining the impact of Nosema of gene expression, physiology and behavior of drone bees, to determine if similar pathways are operating in males. Interestingly, our data thus far suggest drones are more resistant to the impact of Nosema. We completed the analysis of the impacts of pesticide exposure (coumaphos and fluvalinate) on global gene expression patterns, queen pheromone production, and worker responses to queen pheromone. The genomic data revealed that pesticide-induced changes in gene expression overlapped significantly with expression differences in bees fed poor (sucrose only) or rich (pollen/honey) diets. Additional studies revealed that diet strongly impacted the effect of pesticides and reduced mortality. The results of the genomic study were incorporated into a manuscript which is now in review. Interestingly, there was little impact of pesticide exposure on pheromone production or responses to pheromones; these data will be incorporated into a manuscript for submission in late-2014. We have completed the analysis of the impacts of viral infection on global gene expression patterns, DNA methylation levels, queen pheromone production, and worker responses to queen pheromone. We demonstrated that viral infection results in transcriptional and methylation changes in distinct groups of genes, and these genes do not overlap with those regulated by Nosema or pesticide, suggesting there is a distinct viral immune response pathway. The manuscript describing these results will be incorporated into a manuscript which will be submitted in August 2014. There was little impact of pesticide exposure on pheromone production or responses to pheromones; these data will be incorporated into a manuscript (along with the pesticide data above) for submission in late-2014. Note that these studies were partially supported by funding from NSF on epigenetic mechanisms regulation bee behavior and physiology. We have also assessed the effects of nutrition on queen pheromone production and found little effect; these data will be incorporated into a manuscript with the other queen pheromone production/response data sets described above. However, given the strong association of diet and metabolic/nutrient sensing pathways in response to stressors, we are further exploring the role of nutrition in shaping bee behavior and response to stressful conditions. We are examining the function of diet and these pathways in overwintering behavior in honey bees and bumble bees (one of the most stressful periods, where beekeepers lose ~30% of their colonies) and bumble bee pathogen loads. Note that these studies were partially supported by funding from NAPPC.

Publications

  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Holt, H.L., Aronstein, K. and C.M. Grozinger. Chronic parasitization by the microsporidian Nosema causes global expression changes in core nutritional, metabolic, and behavioral pathways in honey bee workers (Apis mellifera) BMC Genomics 14:799 (2013).
  • Type: Journal Articles Status: Under Review Year Published: 2014 Citation: Schmehl, D. R., Teal, P.E.A., Frazier, J.F. and C. M. Grozinger. " Genomic analysis of the interaction between pesticide exposure and nutrition in honey bees (Apis mellifera)". Journal of Insect Physiology. (in review).
  • Type: Journal Articles Status: Under Review Year Published: 2014 Citation: Richards, J., Carr-Markell, M., Hefetz, A., Grozinger, C.M. and H. Mattila. "Volatile production by honey bee (Apis mellifera) queens changes dynamically during swarming". Apidologie (in review)
  • Type: Journal Articles Status: Other Year Published: 2015 Citation: Galbraith, G.A.*, Yang. X.*, Nino, E.L., Yi, S., and C. M. Grozinger. "Dual genomic response pathways to systemic viral infection in honey bees (Apis mellifera)". Submission planned for August 2014.


Progress 02/29/12 to 02/27/13

Outputs
Target Audience: The results of these studies 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. Changes/Problems: We had originally planned to study the effects of viruses on honey bees in small colonies. Because of difficulty in obtaining consistent, high levels of infection in our treated colonies, we moved this to a cage study. We originally did not plan to examine the effects of parasite infection (Nosema) as part of this project, but the interests of the student and our ability to leverage funding from a subcontact of a USDA-CAP grant allowed us to incorporate these studies into the overall project. What opportunities for training and professional development has the project provided? This grant as supported the research, education and training of four graduate students and two undergraduates in 2012. Three of the students presented their research at international conferences, and one undergraduate presented her research at an undergraduate research exhibition. Elina Lastro Nino (PhD student, completed PhD in 2012), Daniel Schmehl (PhD student, completed PhD in 2012), David Galbraith (PhD Student), Holly Holt (PhD Student), Caroline Hozza (Undergraduate), Megan Snyder (Undergraduate). How have the results been disseminated to communities of interest? In 2012, this research was presented by the PI at two invited talks at national conferences, five invited seminars at universities, one talk at a beekeeper conference and one talk (which is available for streaming online) the the public, as well as three talks at an international conference by the associated graduate students. The grant also supports an annual Honey Bee Queen Rearing Workshop, which trains 14 beekeepers in honey bee queen rearing, breeding, and evaluation for productivity and disease resistance, in an effort to improve local breeding programs. This Workshop is a cornerstone of the PA Honey Bee Stock Improvement Program, which is a partnership between PA beekeepers and PSU researchers. What do you plan to do during the next reporting period to accomplish the goals? We are in the process of confirming microarray results for the effects of pesticides using qRT-PCR. Once this is completed, the data will be compiled into a manuscript and submitted for publication. We have completed the analyses of RNA-Seq and bisulfite seq data for the virus studies, and are currently synthesizing the two data sets. We are repeating the study in order to confirm expression changes in candidate genes using qRT-PCR. Once this is complete, the data will be compiled into a manuscript and submitted for publication. For the studies of the effects of Nosema in workers, the manuscript is in revision. We are performing additional studies/replicates of the effects of Nosema infection on drones, and plan to have these analyses completed by spring 2014. We will combine data from our studies of the effects of pesticides, viruses, and nutriton on queen pheromone and queen-worker interaction into a single manuscript, and submit this for publication in fall 2013.

Impacts
What was accomplished under these goals? We examined the effects of queen insemination volume. We found significant effects of insemination volume on queen-worker behavioral interactions, worker physiological responses to queen pheromone, and queen longevity. This work was published in 2013. We studied the effects of two chemicals (coumophos and fluvalinate) commonly used by beekeepers to control Varroa mites on queen pheromone production and worker behavior and physiology. We found little effect of these pesticides on queen pheromone production and queen-worker interactions, suggesting this chemical communication system is very sensitive to mating quality but is otherwise surprisingly buffered. We found pesticide exposure causes significant changes in gene expression in workers based on a microarray analysis; detoxification, metabolism, immune, and nutrition genes are especially affected. We then tested the impacts of diet on resistance to pesticides and found bees provided a pollen diet were much more resistant that bees fed sucrose and/or soy-derived protein. We have examined the effect of Nosema infection on worker gene expression profiles (this work was also supported by a USDA-CAP grant). Using a timecourse of Nosema infection and examining effects in different tissues, we demonstrated that Nosema appears to primarily impact nutrition-sensing pathways, and the previously observed effects on immunity and behavioral maturation appear to be a consequence of the impact on nutrition. This manuscript is currently in revision. We have extended these studies to examine the impacts of Nosema on drones. We have examined gene expression, drone physiology, and drone performance during mating flights. While Nosema does impact expression of candidate genes, it appears to cause little effect on drone physiology, behavior and performance, suggesting that drones have mechanisms that allow them to be buffered from the effects of Nosema. Finally, we examined the impacts of viral infection on worker bees. We again found little effect on queen-worker interactions, but did find significant changes in gene expression. These changes were largely distinct from the molecular responses we observed in previously published studies of acute immune responses to wounding and bacteria, and distinct from the results of or Nosema-and pesticide challenges.

Publications

  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Ni�o, E.L., Malka, O., Hefetz, A., Teal, P., Hayes, J. and C.M. Grozinger. Long-term effects of honey bee queen (Hymenoptera: Apis mellifera) insemination volume on queen-worker interactions Journal of Insect Physiology 58(8):1082-1089 (2012).


Progress 03/01/11 to 02/28/12

Outputs
OUTPUTS: Honey bees colonies are exposed to a variety of stressors, including parasites, pathogens, pesticides, poor nutrition and management practices. Social stressors can also impact colony function and health; colonies that supercede (replace) their queens lose productivity. Here, we examine the effects of different stressors (viruses, pesticides, nutrition, queen mating quality) on the worker bees and queen bees. In worker bees, we examine molecular and physiological responses, while in queen bees, we examine effects of these stressors on production of social pheromones and the resulting responses in workers. These studies will provide key information about the molecular, physiological and behavioral responses to an panel of important stressors in a critical model system, and determine if responses to these stressors are specific or general. These results will not only shed light on the basic biology of the system, but also allow for the development of improved management strategies. In 2011, this research was presented by the PI at four invited talks at national conferences, three invited seminars at universities, and talks at three beekeeper meetings. The grant also supported the development of a Queen Rearing Workshop for beekeepers in May 2011. PARTICIPANTS: Christina Grozinger (PI; Associate Professor), James Tumlinson (co-PI; Professor), Ramesh Sagili (collaborator, Oregon State), Elina Lastro Nino (PhD student), Daniel Schmehl (PhD student), Jessica Richards (MSc Student), David Galbraith (PhD Student), Bernardo Nino (Senior Research Technician, Penn State), and Janet Teeple (Undergraduate) 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 studied the effects of coumophos and fluvalinate on queen pheromone production and worker behavior and physiology. Both pesticides significantly change the composition of queen pheromone; behavioral assays of worker responses to these pheromones will be completed in May 2012. Pesticide exposure causes significant changes in gene expression in workers based on a microarray analysis; detoxification and metabolism genes are especially affected. We also examined the effects of queen insemination volume. Insemination volume significantly impacted worker attraction to the queen, worker chemical profiles in the Dufours glands (which is linked to worker reproduction), and queen longevity in small colonies. It did not alter queen egg-laying behavior or worker hormone levels (which is associated with worker behavioral maturation). The manuscript resulting from these studies is currently in review. A second study attempted to control the behavior of naturally mated queens in order to produce queens with high vs low insemination levels, but this study did not result in queens that differed in terms of their sperm quantity, attraction to workers, egg-laying rates, etc. We treated small colonies of bees with extracts from virus infected bees and monitored viral loads. We were unable to establish significantly different viral titers or populations in our treated vs control colonies. Treated colonies did not have any detectable differences in health or productivity. Studies using caged workers demonstrated that young workers infected with viruses had increased mortality. Molecular studies of the effects of these viruses on gene expression and DNA methylation are underway. In collaboration with Ramesh Sagili, Oregon State University, we examined the effect of feeding colonies of bees pollen from a single source (almond) or multiple sources. No differences in brood production, retinue response to the queen, or hypopharyngeal gland content was observed.

Publications

  • No publications reported this period


Progress 03/01/10 to 02/28/11

Outputs
OUTPUTS: Stress can have profound effects on behavior and physiology. The importance of stressors in agricultural systems has been highlighted in recent reports of "Colony Collapse Disorder", in which managed honey bee colonies undergo rapid and massive population declines. CCD is likely caused by a combination of stressors: pests (ie Varroa mites), pathogens, environmental toxins (including the acaricides used to control Varroa), and poor nutrition, possibly due to the use of artificial diets and monoculture cropping systems. However, in order to fully understand how different stressors undermine the health of social insects, it is necessary to examine effects on individuals and across the complete social structure. We will examine the effects of four key stressors experienced by honey bees: reduced queen mating number, reduced nutrition, environmental toxins (focusing on coumaphos and fluvalinate, used to control two major bee parasites), and pathogens (focusing on viruses). Objective 1 will characterize the effects on queen physiology, queen pheromone production and pheromone-mediated worker behavior. Objective 2 will characterize physiological parameters, including global gene expression patterns, in workers reared in these colonies. Objective 3 will determine if differences in social cues are sufficient to cause physiological differences in workers. These studies will provide critical information about the molecular, physiological and behavioral responses to an array of important stressors in an agriculturally important model system. Our results can be used to develop better management strategies for bees and other species. Furthermore, we will lay the groundwork for future studies examining the role of stressors in chemical communication. This research was presented in a plenary lecture and invited symposium presentation at the International Union for the Study of Social Insects Congress (August 2010, Copenhagan, DK), and a poster and talk at the Entomological Society of America Annual Meeting (December 2010, San Diego). It was also presented at invited talks at the Western PA Beekeeper Meeting (February 2011), the Eastern Branch Entomological Society meeting (March 2011), and Rutgers University (March 2011). PARTICIPANTS: Christina Grozinger (PI; Associate Professor), James Tumlinson (co-PI; Professor), Elina Lastro Nino (PhD student), Daniel Schmehl (PhD student), and Bernardo Nino (Senior Research Technician, Penn State). This project provided support for Ms. Lastro Nino and Mr. Schmehl to attend the Entomological Society of American Annual Meeting in December 2010 and present their research. 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
In the summer of 2010, we studied the effects of coumophos and fluvalinate on queen pheromone production and worker behavior and physiology. Fluvalinate significantly reduced worker attraction to the queen and altered queen pheromone production. Molecular analyses of effects on worker gene expression are ongoing. We also examined the effects of queen insemination volume. Insemination volume significantly impacted worker attraction to the queen, worker chemical profiles in the Dufour's glands, and queen longevity. It did not alter queen egg-laying behavior or worker hormone levels. In the summer of 2011, we will continue to examine the effects of fluvalinate, nutrition (in collaboration with Ramesh Sagili, Oregon State University), and queen mating number (using naturally mated queens).

Publications

  • No publications reported this period