Progress 03/01/23 to 02/28/24
Outputs
Target Audience:Researchers interested in basic biology, health and behaviors of honey bees and pollinators, in general. Beekeepers and honey bee queen breeders. Commercial growers of fruits and vegetables that need honey bee pollination. General public interested in increasing health of honey bees and other pollinators. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?M. Spivak advised two graduate students that worked on this project: Hollie Wall-Dalenberg (Masters, November 2020), Maggie Shanahan (PhD, May 2023). Both received additional academic-year funding from other sources; thus this grant covered only summer salary. M. Simone-Finstrom partially supported Ally Martin Ewert, a PhD student at Louisiana State University to expand research on effects of propolis and other plant derived compounds on core honey bee microbiota. Professionals Nelson Williams and Ben Ziegler assisted with bee breeding program, and provided beekeeping training to graduate students and beekeepers. Professionals Phil Tokarz assisted with laboratory analyses. Graduate students and post-docs attended and presented findings at professional meetings, such as American Bee Research Conference and the Entomological Society of America annual meeting. How have the results been disseminated to communities of interest?For this reporting period (March 2023-March 2024, M Spivak presented research findings on propolis and social immunity as invited, or keynote, speaker at scientific and/ or beekeeping conferences in: Feb 29, 2020. Indiana Bee School (virtual): "Honey bee breeding for disease and mite resistance" and "What we know, and don't know, about the benefits of propolis to honey bees" April 15, 2020. University of Florida Two Bees in a Podcast. "Interview about propolis for bees". May 18, 2020. TheBeekeeper.org Australia, On-line educational platform. "The benefits of propolis to honey bee health" August 9, 2020. Bee Squad Bees for Veterans Program (virtual): "The benefits of propolis to bee health". Aug 29, 2020. Ist International Forum on Bees in the Time of a Pandemic, Brazil (virtual): "The health of honey bees in the U.S." November 19, 2020. Sommerset UK Beekeeping Association (virtual): "Honey bee social immunity: hygienic behavior and propolis collection" Dec 14, 2020. Palm Beach FL beekeepers (virtual): "Honey bee social immunity: hygienic behavior and propolis collection" For this reporting period, M Simone-Finstrom presented current research efforts and findings related to this project at one scientific conference (invited speaker) and as an invited speaker to three beekeeping conferences: M. Simone-Finstrom. Propolis supports social homeostasis in honey bees and supports mite resistant behaviors. International Propolis Research Group 3rd International Propolis Conference. Istanbul, Turkey, November 2023. Keynote speaker M. Simone-Finstrom. Advanced management tools. Baton Rouge Bee Lab Field Day. October 2023. M. Simone-Finstrom. Propolis and other traits that reduce mites and disease in honey bee colonies. Tri-country Pennsylvia Beekeepers meeting. September 2023. M. Simone-Finstrom. Promoting bee health with Propolis. Eastern Apicultural Society annual conference. August 2023. Invited speaker, talk as part of series as their Hambleton Research Award honoree M. Simone-Finstrom. From molecules to behavior: Taking an integrative approach to improve honey bee health and colony productivity. BeeConnected Conference, Czech Republic. April 2023. Keynote speaker Allyson Martin Ewert (PhD student) Martin, A. Natural products for honey bee health. 10-minute oral presentation. Louisiana Beekeepers Association and USDA-ARS Honey Bee Breeding, Genetics, and Physiology Laboratory Annual Fall Field Day. Oct. 2023. What do you plan to do during the next reporting period to accomplish the goals?This is last year of project.
Impacts
What was accomplished under these goals?
Impact. To help restore honey bee health, we are focusing on ways to bolster their natural behavioral defenses, or social immunity, against pathogens and parasites. Honey bees collect antimicrobial plant resins and deposit them in the nest cavity as propolis. In the lab, graduate students and post-doctoral researchers are investigating the biological mechanisms of how propolis supports bees' immune function and disease defense. In the field, we collaborated with beekeepers to quantify benefits of propolis to colony health and survivorship. We continue to select stocks of bees for multiple mechanisms of social immunity including propolis collection and hygienic behavior. In the long term these efforts will result in colonies more able to defend themselves against various parasites and pathogens, and in beekeepers having to use fewer chemical treatments to maintain colonies. Our ultimate aims are to increase the sustainability and economic viability of beekeeping to enhance food security and ecosystem vitality. Objective 1c. The continuous honey bee cell line, AmE-711, was used to test the direct effects of propolis on honey bee immune gene expression in a simplified host environment. AmE-711 cells were seeded into 24-well plates and allowed to grow until confluent. Growth medium was then replaced with 200 µL of either Schneider's Insect Medium (SIM; Medium Only), 0.7% ethanol in SIM (Solvent Control), extract containing 10 µg/mL of propolis collected from hives near Baton Rouge, LA, USA, or extract containing 10 µg/mL of propolis collected from hives in Brazil; 12 wells per treatment medium group. After 24-hours of exposure to the treatment medium, 20 ug of lipopolysaccharide (LPS) was added to 6 wells of each treatment group, and cells were then incubated for an additional 24 hours. At the end of the exposure period, RNA was extracted from cells and qPCR performed for comparative expression of the antimicrobial peptide gene, hymenoptaecin; expressed as fold change normalized to the Medium Only control group not exposed to LPS. Results show that cells exposed to LPS had significant upregulation of hymenoptaecin compared to cells not exposed to the endotoxin (F1,39 = 738.167, p < 0.0001). Cells exposed to propolis extract sourced from Brazil (but not Baton Rouge) had significantly lower hymenoptaecin expression in both the LPS+ and LPS- groups compared to the Medium Only and Solvent Control groups based on Tukey's HSD. These findings suggest that the pathogen-associated molecular pattern, LPS, is a potent inducer of an immune gene in honey bee cells, prompting additional studies to develop AmE-711 as a cell culture model for insect immunity. Furthermore, reduction in hymenoptaecin expression in the presence of propolis extract suggests this honey bee collected material has immune modulating effects at the cell level and supporting the hypothesis that it helps reduce energetic costs of immune system maintenance. Objective 2. Results and finalized analysis of the longitudinal study were finished and the manuscript was published. Findings provide clear evidence to the beekeeping community that colonies with a propolis envelope are at least as productive and have fewer disease symptoms compared to colonies without a propolis envelope. These findings establishe rough hives as a novel technique for integrated pest management for the beekeeping community, as demonstrated by a bee supply company requesting our input and feedback on a new rough hive product they developed based on this and previous research on the value of increased propolis for bee health. In addition, two additional studies were initiated following up on the reduced disease and parasites loads of colonies in the rough, high propolis boxes opening new avenues of research and new collaborations. Objective 3. We hypothesize that colonies bred for propolis collection, hygienic behavior and reduced mite population growth will demonstrate reduced pathogen prevalence and increased survivorship. We are currently evaluating colonies from diverse honey bee stocks for wintering survivorship and three behavioral traits: 1) relatively low Varroa mite population growth, both on adult bees and on pupae, between May and September; 2) hygienic behavior (assayed using a freeze-killed brood assay), and 2) propolis collection (amount of propolis deposited in commercial propolis traps). We are following survivorship and performance of 80 full-sized colonies and 75 smaller (nucleus) colonies that have not been treated for Varroa mites for two years. If a majority of these untreated colonies survive into summer of 2024, we will begin transferring the genetics (queen bees) to some commercial beekeeping operations for their continued upkeep and selection. In the long-term, this selection program will result in colonies more able to defend themselves against various parasites and pathogens and should result in beekeepers having to use fewer chemical treatments to keep colonies alive.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2024
Citation:
Shanahan, M., Simone-Finstrom, M., Tokarz, P., Rinkevich, F., Read, Q. D., & Spivak, M. (2024). Thinking inside the box: Restoring the propolis envelope facilitates honey bee social immunity. Plos one, 19(1), e0291744.
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
Martin Ewert, A., Simone-Finstrom, M., Read, Q., Husseneder, C., & Ricigliano, V. (2023). Effects of ingested essential oils and propolis extracts on honey bee (Hymenoptera: Apidae) health and gut microbiota. Journal of Insect Science, 23(6), 15.
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Progress 03/01/18 to 02/28/24
Outputs
Target Audience:Researchers interested in basic biology, health and behaviors of honey bees and pollinators, in general. Beekeepers and honey bee queen breeders. Commercial growers of fruits and vegetables that need honey bee pollination. General public interested in increasing health of honey bees and other pollinators. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Graduate Students M. Spivak advised two graduate students that worked on this project: Hollie Wall-Dalenberg worked on Obj 1 (Masters, November 2020), and Maggie Shanahan worked on Obj 2 (PhD, May 2023). M. Simone-Finstrom co- advised Master's student, Sarah Lang, who expanded research on effects of propolis on core honey bee microbiota from March 2020 to August 2020 (Obj 1). This work was then advanced by Allyson Martin, PhD student, who also conducted studies related to understanding the effects of propolis exposure on larval resistance to pathogens (Obj 2). M. Simone-Finstrom partially supported Ally Martin Ewert, a PhD student at Louisiana State University to expand research on effects of propolis and other plant derived compounds on core honey bee microbiota (Obj 1). Graduate students attended and presented findings at professional meetings, such as American Bee Research Conference, the Entomological Society of America annual meeting and the International Propolis Research Group. Post-doctoral Researchers M. Spivak supervised Dr. Mike Goblirsch (was post-doctoral researcher, but now is Research Scientist at USDA-ARS lab in Mississippi), who mentored and assisted graduate students and technicians in the lab to conduct molecular and microbiological experiments related to propolis and cell cultures (Obj 1) . Beginning in 2021, M. Spivak hired a new post-doctoral researcher, Sofia Nekulin-Levin, to analyze seasonal viral loads in colonies in the breeding program (Obj 3). M. Simone-Finstrom supervised post-doctoral researcher, Perot Saelao, a bioinformatician who learned and worked on pipelines for analyses related to the honey bee microbiota (Obj 1). Post-doctoral researchers attended and presented findings at professional meetings, such as American Bee Research Conference, the Entomological Society of America annual meeting and the International Propolis Research Group. Undergraduates Over the course of the grant, M. Simone-Finstrom advised 6 undergraduate students, each worked on small aspects of the studies to develop skills in molecular biology and field collections and performed analyses related to scoring propolis deposition. Professionals M. Spivak supervised honey bee field technicians Gary Reuter and Yuuki Metread in MN from 2019-2021 to assist with bee breeding program, and provide beekeeping training to graduate students and beekeepers. Beginning in 2021, M. Spivak also supervised field technicians Héctor Morales, Nelson Williams and Ben Sallmann and Ben Ziegler (all partially supported by project) to assist with beekeeping and breeding for Obj 3. M. Simone-Finstrom supervised honey bee field technicians Phil Tokarz, Bob Cox, David Dodge, Amanda Frake, Allyson Martin, Sarah Lang and Victor Rainey in LA to assist with field trials and laboratory analyses. Visiting ScientistsM. Simone- Finstrom supported a visiting Australian researcher, J. Gerdts, for one month to learn additional breeding and management techniques and methods for assessing propolis deposition. J. Gerdts assisted with field experiments and molecular analyses. How have the results been disseminated to communities of interest?# Invited Regional Stakeholder meetings - 53 # National Stakeholder meetings - 6 # Conferences/ Seminars - 13 # International Stakeholder -16 # International Conferences - 23 What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Impact. Honey bee (Apis mellifera) populations are experiencing high and unsustainable annual losses due to a number of stressors that weaken bees' innate defenses resulting in a downward spiral of health. To help restore honey bee health, we are focusing on ways to bolster their natural behavioral defenses, or social immunity, against pathogens and parasites. Honey bees collect antimicrobial plant resins and deposit them in the nest cavity as propolis. In the lab, graduate students and postdoctoral researchers are investigating the biological mechanisms of how propolis supports bees' immune function and disease defense. In the field, we are collaborating with beekeepers to quantify benefits of propolis to colony health and survivorship. We have begun selecting from lines of bees for multiple mechanisms of social immunity including propolis collection and hygienic behavior. In the long term these efforts will result in colonies more able to defend themselves against various parasites and pathogens, and in beekeepers having to use fewer chemical treatments to maintain colonies. Our ultimate aims are to increase the sustainability and economic viability of beekeeping to enhance food security and ecosystem vitality. Objective 1 Determine the role of propolis volatiles, and bees' contact with propolis in the nest, on modulating honey bee immune response and pathogen defense As bees have evolved with the antimicrobial properties of propolis within the nest, we hypothesized that propolis exposure would reduce the abundance and diversity of opportunistic and pathogenic microbes in and on bees, allowing for reduced investment in individual bee immune gene expression and increased abundance of bees' core microbiome community. Two experiments, now published, confirmed this hypothesis. First, honey bee microbiota was more consistent between bees collected from P+ colonies, while those from P- colonies exhibited greater microbial diversity. Secondly, core microbiome species significantly increased in absolute abundance in the bees from P+ compared to P- colonies, whereas opportunistic and pathogenic bacteria were at significantly lower relative abundance in P+ colonies. Additionally, we explored if bee specific microbes are susceptible or resilient to compounds in propolis. Overall. the bee gut microbiota appears robust to perturbation by even high concentrations of PNPs, including propolis. Using the AmE-711 honey bee cells (a continuous honey bee cell line) we found a reduction in immune gene, hymenoptaecin, expression in the presence of propolis extract, demonstrating immune modulating effects at the cell level and supporting the hypothesis that it helps reduce energetic costs of immune system maintenance. Objective 2. Quantify the potential benefit of propolis to health and productivity of colonies in a commercial beekeeping operation When wild honey bee colonies (Apis mellifera) nest in hollow tree cavities, they coat the rough cavity walls with a continuous layer of propolis, a substance comprised primarily of plant resins. We assessed different surface texture treatments (rough wood boxes, boxes outfitted with propolis traps, and standard, smooth wood boxes) in terms of their ability to stimulate propolis collection, and we examined the effect of propolis on colony health, pathogen loads, immune gene expression, bacterial gene expression, survivorship, and honey production in both stationary and migratory beekeeping contexts. We found that rough wood boxes are the most effective box type for stimulating propolis deposition. Results from the commercial operation over two years of study show that colonies with custom-made boxes deposited more propolis and have larger colony populations in February during almond pollination (p=0.03, N=134). The use of rough wood boxes did correspond with decreased honey production in year one migratory colonies but had no effect during year two. Although the use of rough wood boxes did not improve colony survivorship overall, Melissococcus plutonius detections via gene expression were significantly lower in rough wood boxes, and viral loads for multiple viruses tended to decrease as propolis deposition increased. Finally, in both stationary and migratory operations, propolis deposition was correlated with a seasonal decrease and/or stabilization in the expression of multiple immune and bacterial genes, suggesting that propolis-rich environments contribute to hive homeostasis. Findings provide clear evidence to the beekeeping community that colonies with a propolis envelope are at least as productive and have fewer disease symptoms compared to colonies without a propolis envelope. These findings established rough hives as a novel technique for integrated pest management for the beekeeping community, as demonstrated by a bee supply company requesting our input and feedback on a new rough hive product they developed based on this and previous research on the value of increased propolis for bee health. In addition, two additional studies were initiated following up on the reduced disease and parasites loads of colonies in the rough, high propolis boxes opening new avenues of research and new collaborations. Objective 3. Initiate a comprehensive breeding program for colony health, with emphasis on selecting for behavioral mechanisms of social immunity against parasites and pathogens. We hypothesize that colonies bred for low mite population growth, propolis collection, and hygienic behavior will demonstrate reduced pathogen prevalence and increased productivity. We are currently evaluating colonies from diverse honey bee stocks for wintering survivorship and three behavioral traits: 1) relatively low Varroa mite population growth, both on adult bees and on pupae, between May and September over two summers; 2) hygienic behavior (assayed using a freeze-killed brood assay), and 2) propolis collection (amount of propolis deposited in commercial propolis traps). In 2022, we also included analysis of virus loads. After four years of selection, we wintered 139 colonies in 2022-2023 from six different queen stocks that had maintained very low mite levels and had low or nondetectable virus loads. 75% of these colonies were alive in 2023, and from them, we continue to follow the survivorship and performance of 80 full-sized colonies and 75 smaller (nucleus) colonies that have not been treated for Varroa mites for two years. We anticipate that a majority of these untreated colonies will survive into summer of 2024, and we will begin transferring the genetics (queen bees) to some commercial beekeeping operations for their continued selection and propagation. In the long-term, this selection program will result in colonies more able to defend themselves against various parasites and pathogens and should result in beekeepers having to use fewer chemical treatments to keep colonies alive.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Bankova, V., Bertelli, D., Borba, R., Conti, B. J., da Silva Cunha, I. B., Danert, C., & Zampini, C. (2019). Standard methods for�Apis mellifera�propolis research.�Journal of Apicultural Research,�58(2), 149. https://doi.org/10.1080/00218839.2016.1222661
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2020
Citation:
Simone-Finstrom M, Ni�o EL, Flenniken ML, Arrowsmith H, Wu-Smart J. Proceedings of the 2019 American Bee Research Conference. Insects. 2020; 11(2):88. https://doi.org/10.3390/insects11020088
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Progress 03/01/22 to 02/28/23
Outputs
Target Audience:Researchers interested in basic biology, health and behaviors of honey bees and pollinators, in general. Beekeepers and honey bee queen breeders. Commercial growers of fruits and vegetables that need honey bee pollination. General public interested in increasing health of honey bees and other pollinators. Changes/Problems:Continuing covid-related maximized telework posture and delays prevented being able to fully onboard a graduate student and tsetup funding agreement for the student's stipend and project through the subaward. Since those delays continued through spring 2022 the student was not able to complete projects as outlined and needs another field season and 12 months to conduct the studies and subsequent analyses for Objective 1. What opportunities for training and professional development has the project provided?In 2022, M. Spivak advised post-doctoral researcher Dr. Sofia Nikulin who developed new techniques to analyze virus loads in the breeder colonies (Objective 3). M. Simone-Finstrom supported PhD student Allyson Martin to expand research on effects of propolis on core honey bee microbiota and conduct studies related to understanding the effects of propolis exposure on larval resistance to pathogens. Professionals Nelson Williams and Ben Sallmann assisted with bee breeding at Univ MN beginning in 2021; they also provided beekeeping training to graduate students and beekeepers. Professionals Phil Tokarz, Amanda Frake and Victor Rainey assisted with field trials and laboratory analyses. Graduate students and post-docs attended and presented findings at professional meetings, such as American Bee Research Conference and the Entomological Society of America annual meeting. How have the results been disseminated to communities of interest? For this reporting period (March 2022-March 2023, M Spivak presented research findings on propolis and social immunity as invited, or keynote, speaker at scientific and/ or beekeeping conferences in: International Propolis Research Group "How to Encourage Honey Bees to Construct a Propolis Envelope" May 5 Association of Southeastern Biologists, R.H. Martin Plenary Address. "Honey Bee Health: From Socialized Medicine to Nutritional Landscapes" March 30 Denver Highland Beekeeping Club "Why Care About Propolis" Oct 20 Virginia Beekeepers Association "The benefits of hygienic behavior and propolis to honey bee health" Oct 13 Association of Education and Research Greenhouse Curators annual 2022 conference, UMN. "Pollinators and Climate Change" July 27 Bees Beyond Borders, International Bee Development "Why Care About Propolis" June 1 Chatham County North Carolina Beekeepers Association "Benefits of Propolis to Honey Bee Health" April 10 Missouri State Beekeeping Association "Honey Bee Social Immunity" March 11 Bee Improvement and Bee Breeders Association, UK "Social Immunity" March 8 Scottish Beekeeping Association "The Wonders of Propolis and an Introduction to Honey Bee Hygienic Behavior" March 3 For this reporting period, M Simone-Finstrom presented current research efforts and findings related to this project at 2 scientific conferences (1 as invited speaker) and findings were included in the Baton Rouge Bee Lab research updates presented at three beekeeping meetings: M. Simone-Finstrom, M. Shanahan, M. Spivak. Social homeostasis in honey bees is supported by resin use. International Union for the Study of Social Insects conference. San Diego, CA. July 2022. Invited presentation (virtual, and presented by M. Shanahan) M. Simone-Finstrom, M. Shanahan, M. Spivak. Increased resin deposition in hives alters immune response of managed colonies in stationary and migratory beekeeping operations. International Propolis Research Group Conference, May 2022. (virtual) Louisiana State Beekeepers Association Annual Convention. Alexandria, LA. August 2022. Louisiana State Beekeepers and USDA-ARS Baton Rouge Field Day. Baton Rouge, LA. October 2022. American Honey Producers Association. Tucson, AZ. Dec 2021. Goblirsch, M,Avalos A, Simone-Finstrom M, Spivak M. 2022. RNA-seq analysis of honey bee AmE-711 cells exposed to propolis extract. International Propolis Research Group Virtual Meeting. Maggie Shanahan (PhD student) Shanahan M,(2022)El propóleo y la salud de las abejas Apis mellifera.Beekeeping meeting in Emiliano Zapata, Chiapas, Mexico. Shanahan M,(2022)La importancia del propóleo para las abejas Apis mellifera e implicaciones para practicesapícolas.VIIJornadasde Apicultura Sierra de Cádiz, Andalucía, España (virtual format). Shanahan M, (2022).Propolis collection and social immunity in the stingless bee Scaptotrigona mexicana. International Union for the Study of Social Insects conference (virtual format). Shanahan M,(2022).Supporting propolis collection to bolster honey bee health.Snohomish Beekeeping Club (virtual format). Allyson Martin (PhD student) A. Martin, V. Ricigliano, & M. Simone-Finstrom. Effects of plant natural products on the health and gut microbiota of the honey bee Apis mellifera. American Bee Research Conference, Dec 2021 (virtual) A. Martin. Effects of propolis and other plant natural products on honey bee health, detoxification and gut microbiota. International Propolis Research Group conference, May 2022 (virtual). A. Martin, V. Ricigliano, & M. Simone-Finstrom. Effects of propolis and other plant natural products on honey bee health, detoxification and gut microbiota. Entomological Society of America Annual Convention. November 2022. What do you plan to do during the next reporting period to accomplish the goals?Objective 1. Due to restrictions related to COVID and maximized telework posture of USDA employees through May 2022, progress continued to have some delays. In spring 2023, we will finalize experiments related to the effects of propolis exposure on the antimicrobial activity of brood food (Objective 1b) and on larval immune response to bacterial and fungal pathogens (1b) and viral infection (1c), as described in the proposal. Larval rearing trials will be led by Baton Rouge, LA by M. Simone-Finstrom and performed by PhD student Allyson Martin in LA and M. Goblirsch in MS. An additional field-experiment will confirm laboratory-based results and further examine and evaluate the antimicrobial activity and components of brood food in propolis-rich and propolis-poor colonies relative to pathogen exposure. Objective 2: Two manuscripts will be submitted detailing the final analyses and progress based on the stationary and migratory studies of the effect of propolis deposition in colonies in real-world settings.
Impacts
What was accomplished under these goals?
Impact. To help restore honey bee health, we are focusing on ways to bolster their natural behavioral defenses, or social immunity, against pathogens and parasites. Honey bees collect antimicrobial plant resins and deposit them in the nest cavity as propolis. In the lab, graduate students and post-doctoral researchers are investigating the biological mechanisms of how propolis supports bees' immune function and disease defense. In the field, we are collaborating with beekeepers to quantify benefits of propolis to colony health and survivorship. We are selecting a new line of bees for multiple mechanisms of social immunity including propolis collection and hygienic behavior. In the long term these efforts will result in colonies more able to defend themselves against various parasites and pathogens, and in beekeepers having to use fewer chemical treatments to maintain colonies. Our ultimate aims are to increase the sustainability and economic viability of beekeeping to enhance food security and ecosystem vitality. Objective 1. Experiments are in progress,to be completed prior to the end of the grant,where AmE-711 honey bee cells were pretreated with extracts prepared from propolis from Brazil and Baton Rouge. The cells were then exposed to bacteria-derived lipopolysaccharides (LPS) to stimulate an immune response.Based on preliminary experiments,we expect propolis pretreatment will lead to a more robust immune response compared to cells treated with LPS only. This result will supportour published evidencethat propolis has a modulating effect on immune gene expression inadulthoney bees. It will also demonstrate the utility of a continuous honey bee cell line as a tool for enhancing understanding of honey bee immune signaling at the cellular level. Work was done toward testing direct and indirect effects of propolis exposure on royal jelly (larval food) antimicrobial activity and larval resistance to pathogens. Initial studies determined the doses of propolis extract that can be used in an in vitro rearing setting for pathogen challenge studies where larval mortality is unaffected by the incorporation of propolis in diet or rearing environment. Additional experiments were conducted following up on previously published work that found that propolis exposure influences microbiota of honey bees. For this work, the focus was understanding if bee specific microbes are susceptible or resilient to compounds in propolis. Bees, maintained in laboratory cages, were fed propolis extracts or other plant natural products (PNPs) to examine effects on lifespan and gut microbiota. The sucrose control, Brazilian propolis, and lemongrass-fed cages had the longest median lifespans while the thyme and spearmint-fed cages had the shortest. Analyses of whole bee abdomens revealed some differences in the abundances of prominent bacterial gut taxa in bees fed antibiotic, spearmint, and thyme relative to the controls, but overall the bee gut microbiota appears robust to perturbation by even high concentrations of PNPs, including propolis. Objective 2. We compared propolis deposition in colonies hived in standard, smooth boxes or custom-made, textured boxes for propolis deposition in MN and LA, and in a migratory, commercial beekeeping operation. Results from the commercial operation over two years of study provide demonstrable evidence to the beekeeping community that colonies with a propolis envelope are at least as productive and have fewer disease symptoms compared to colonies without a propolis envelope. This work has been presented at scientific and industry meetings and two manuscripts are in the final stages of preparation for submission to peer-reviewed journals. Objective 3. We hypothesize that colonies bred for propolis collection, hygienic behavior and reduced mite population growth will demonstrate reduced pathogen prevalence and increased survivorship. We are currently evaluating colonies from diverse honey bee stocks for wintering survivorship and three behavioral traits: 1) relatively low Varroa mite population growth, both on adult bees and on pupae, between May and September the previous summer; 2) hygienic behavior (assayed using a freeze-killed brood assay), and 2) propolis collection (amount of propolis deposited in commercial propolis traps). In 2022, we also included analysis of virus loads (Deformed wing virus; analyzed by post-doc Sofia Nikulin). After four years of selection, we wintered 148 colonies from six different queen stocks that did not require treatment for Varroa mites, and had low or non-detectable virus loads by late summer. These colonies are located in an area isolated from other beekeepers, and queens are mating naturally in this area with drones from our selected stocks. We will be testing daughter colonies from the stocks this summer in areas more densely populated with honey bee colonies to determine how they perform under higher mite pressure from surrounding colonies. We will continue to propagate these stocks after the termination of this grant. If they survive two years with no treatments, we will begin working with some commercial queen producers to ensure they are propagated by the beekeeping industry.
Publications
- Type:
Book Chapters
Status:
Awaiting Publication
Year Published:
2023
Citation:
Shanahan M, Simone-Finstrom M, Spivak M. Resin Use and Social Immunity In: Pot-Propolis in Stingless Bee Ecology. (ed) D. Roubik, Springer Nature
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Progress 03/01/21 to 02/28/22
Outputs
Target Audience:Researchers interested in basic biology, health and behaviors of honey bees and pollinators, in general. Beekeepers and honey bee queen breeders. Commercial growers of fruits and vegetables that need honey bee pollination. General public interested in increasing health of honey bees and other pollinators. Changes/Problems:We will be requesting a no-cost extension to complete experiments that were held up due to COVID restrictions in 2020-2021. What opportunities for training and professional development has the project provided?M. Spivak is advising Maggie Shanahan (current PhD candidate) who is working on Objective 1Ab and Objective 2 as part of her thesis (summer salary support from the project), a new post-doctoral researcher, Sofia Nekulin-Levin, who is analyzing seasonal viral loads in colonies in the breeding program (Objective 3), and a part-time undergraduate, Isabell Dyrbye Wright, who was trained to analyze Varroa loads and extract RNA for viral analysis. M. Spivak also supervises field technicians Héctor Morales, Nelson Williams and Ben Sallmann (all partially supported by project) to assist with beekeeping and breeding for Objective 3. M. Simone-Finstrom supported Allyson Martin, hired as a research assistant and then as an incoming PhD student through Louisiana State University to expand research on effects of propolis on core honey bee microbiota and to conduct the in vitro reared larval trials (Obj 1B) from August 2020 to present. Due to COVID restrictions undergraduates have been unable to be recruited or conduct work in M. Simone-Finstrom's research laboratory. Professional Phil Tokarz assisted with laboratory analyses. Graduate students and post-docs attended and presented findings at professional meetings, such as American Bee Research Conference and the Entomological Society of America annual meeting. How have the results been disseminated to communities of interest?For this reporting period (March 2021-Jan 2022), M Spivak presented research findings on propolis and social immunity as invited, or keynote, speaker at scientific and/ or beekeeping conferences in: March 6, 2021. Danish Beekeepers Association (virtual): "Bee Breeding" March 13, 2021. Chester Co, PA Beekeepers Association. "Honey Bee Social Immunity: Hygienic Behavior and Propolis Collection" March 30, 2021. Biology & Genomics of Social Insects, Cold Spring Harbor Virtual Meeting. "Benefits of Propolis to Social Immunity of Apis mellifera" May 4, 2021. Toronto Beekeepers Association. "Honey Bee Social Immunity: Hygienic Behavior and Propolis Collection" May 6, 2021. IPM4Bees. "Honey Bee Breeding for Disease and Mite Resistance" June 12, 2021. Maryland Beekeeping Association "Honey Bee Social Immunity: Hygienic Behavior and Propolis Collection" May 27, 2021. International Propolis Research Group. "Propolis and the Honey Bee Microbiome" June 24, 2021. Norwegian Beekeepers Association. "Benefits of Propolis to Honey Bees" September 8, 2021. Honey Bee Veterinarian Consortium, North Carolina State University. "Honey Bee Social Immunity and Breeding" October 29, 2021. British Columbia Beekeepers 100th Anniversary Meeting. "Social Immunity: Bee Health Care in Changing Times(keynote) Oct 29 November 17, 2021. UK Central Association for Beekeepers "The Benefits of Propolis to Honey Bees" December 1, 2021. Harvey Mudd College Biology Colloquium. "Social Immunity" Dec 1 December 2, 2021. V Congreso Nacional de Apicultores de Peru - Cusco. "Breeding Honey Bees for Disease and Mite Resistance" For this reporting period, M Simone-Finstrom presented current research efforts and findings related to this project at one scientific conference (invited speaker) and as an invited speaker to three beekeeper based organizations: · October 28, 2021. Montgomery County Beekeepers Association, Pennsylvania. "Propolis: its benefits to the colony and how we can stimulate the process" · August 17, 2021. Inside the Hive. "Propolis in beekeeping: Multifaceted effects of resin use by honey bees" https://www.youtube.com/watch?v=LZc3ouD-pak · May 27, 2021. International Propolis Research Group. "Impact of propolis on colony heath and pesticide exposure in agroecosystems" M Goblirsch (propolis and social immunity talks) Social Immunity for Honey Bee Health. Alameda County Beekeepers' Association, San Francisco, CA. October, 2021. Mixed effects of propolis extract on honey bee viruses and immunity in vitro. International Propolis Research Group Virtual Meeting, UK. May, 2021. Social Immunity for Colony Health. At Home Beekeeping Series, Distance Learning for Beekeepers. Auburn University Extension Service. April, 2021. Maggie Shanahan (PhD student) January 21, 2022. American Bee Research Conference. "Thinking inside the box: building beehives that stimulate propolis collection and support honey bee health" October 7, 2021. UMN Social Insects course: Saint Paul, Minnesota."Stingless bees: Natural history, social behavior, and who gets to be queen" May 27, 2021. International Propolis Research Group. "Resin use by stingless bees" What do you plan to do during the next reporting period to accomplish the goals?Objective 1: Concurrent exposure of AmE-711 to a honey bee virus and propolis extract had no effect on levels of introduced ABPV (or persistent DWV infection). However, our finding that propolis extract led to increased expression of immune signaling molecules in the absence of challenge starting at 12 hours of exposure warrants further investigation. We will pretreat cells with propolis extract for 24 hours and then inoculate cultures with a low dose of ABPV to determine whether propolis has an antiviral priming effect. We will also expose the virus inoculate directly to extract prior to infecting cells to determine if propolis has a virucidal effect. Due to restrictions related to COVID and maximized telework posture of USDA employees, progress in 2021 continued to be delayed. In spring 2022, we also will complete experiments related to the effects of propolis exposure on the antimicrobial activity of brood food (Objective 1b) and on larval immune response to bacterial and fungal pathogens (1b), as described in the proposal. Larval rearing trials will be done in Baton Rouge, LA by M. Simone-Finstrom and M. Goblirsch in MS. Experiments to determine how propolis exposure influences the antimicrobial activity of worker glandular secretions will be done by injecting newly emerged bees with LPS to stimulate the immune system, they will then be maintained in cages in propolis-rich or propolis-poor colonies for 7 days at which point their hypopharyngeal glands, which produce larval food, will be dissected. The glands will then be analyzed for antimicrobial activity via qRT-PCR for GOX and Defensin1 expression and glucose oxidase enzyme activity, as these are known to be major factors involved in royal jelly antimicrobial activity. This work will be done in coordination with both MN and Baton Rouge research groups. For objective 1Ab, melanization and survivorship experiments will be repeated, with minor modifications in experimental design to rule out the possible negative effects of exposure to finely ground propolis vs. whole propolis pieces, and to determine whether the reduced melanization response observed in caged bees post-propolis exposure has any impact on survivorship. For Objective 1c, we will continue to investigate the effects of propolis on immune gene expression and virus infection in honey bee cell cultures using different methods of exposure. We will also conduct experiments to determine whether pre-conditioning the medium with propolis extract will lead to a reduction in subsequent viral load after AmE-711 are inoculated with ABPV. Objective 2: This objective is complete and graduate student, Maggie Shanahan, will be submitting results for publication soon. Objective 3. We will continue selection for colonies that demonstrate behavioral mechanisms of social immunity against parasites and pathogens, and quantification of progress.
Impacts
What was accomplished under these goals?
Impact. To help restore honey bee health, we are focusing on ways to bolster their natural behavioral defenses, or social immunity, against pathogens and parasites. Honey bees collect antimicrobial plant resins and deposit them in the nest cavity as propolis. In the lab, graduate students and post-doctoral researchers are investigating the biological mechanisms of how propolis supports bees' immune function and disease defense. In the field, we are collaborating with beekeepers to quantify benefits of propolis to colony health and survivorship. We are selecting a new line of bees for multiple mechanisms of social immunity including propolis collection and hygienic behavior. In the long term these efforts will result in colonies more able to defend themselves against various parasites and pathogens, and in beekeepers having to use fewer chemical treatments to maintain colonies. Our ultimate aims are to increase the sustainability and economic viability of beekeeping to enhance food security and ecosystem vitality. Objective 1A: In order to determine how propolis exposure affects immune-challenged bees, we placed newly emerged bees in an incubator in trough cages containing varying amounts of finely ground propolis (high = 2.5g, medium = 0.5g, low = 0.1g, and control = 0g). Five cages were set up per treatment, and each cage contained bees from three different colonies (10 bees/colony). Cages were outfitted with hardware cloth to prevent bees from coming into direct contact with propolis. Survivorship was monitored for seven days to determine whether exposure to high doses of propolis impacts honey bee survival. After seven days, bees were implanted with a nylon thread for four hours (i.e., immune challenge), to measure the strength of their melanization (wound-healing) response. Preliminary results indicate that, at high doses, propolis can negatively impact honey bee survivorship, and that bees exposed to propolis melanize less. Follow-up studies will be conducted in summer of 2022 to determine whether observed impacts on honey bee survivorship and immune response resulted from propolis presentation (i.e., propolis pieces collected from colonies, vs. finely ground propolis), and whether decreased melanization confers fitness benefits to bees with propolis exposure. Objective 1B. To address the question on the direct or indirect of a propolis-enriched environment on larval survival when exposed to pathogens, we aimed to rear honey bee larvae in vitro using artificial diets under laboratory conditions. As our first step, dose response curves were completed for larval exposure to two different propolis types (Brazilian green propolis or Louisiana propolis) and for three different routes of exposure (volatile, mixed into diet, propolis lining cell wall) that mimicked potential natural exposures. Overall the Brazilian propolis exhibited higher toxicity than the Louisiana propolis and propolis mixed directly into larval diet resulted in significant mortality. It is highly unlikely that propolis is mixed directly into larval diet, and these results suggest that including that treatment during pathogen challenge trials may not be needed. With now having established appropriate doses, we plan to complete this objective in the coming months. Objective 1C.We previously found that exposure of AmE-711 honey bee cells to either Brazilian or Louisiana propolis extracts led to increased expression of key molecules in the Toll and Imd signaling pathways beginning at 12 hours after exposure in the absence of pathogen challenge.This suggests that immune signaling cascades may be stimulated by propolis exposure and could lead to a more robust immune response upon pathogen challenge. However, propolis-induced immune stimulation may be restricted to specific pathogens, as levels of persistent DWV-A or a second, introduced virus, ABPV, were unaffected by the presence of extract in the culture medium. RNA-seq analysis is currently being conducted on cell culture samples exposed to either Brazil or Louisiana extracts without pathogen challenge to provide a more complete picture of effects on immune and other cellular processes. Objective 2. We hypothesize that stimulating colonies to build a natural propolis envelope will affect honey production and colony survival over the course of a year in beekeeping operations. We compared propolis deposition in colonies hived in boxes custom-made to stimulate propolis deposition and in the standard, smooth wood boxes that most beekeepers currently use in both stationary (MN) and migratory, commercial beekeeping contexts. Results from the commercial operation over two years of study show that colonies with custom-made boxes deposited more propolis and exhibit a near-significant reduction in European foul brood. Honey production was lower for propolis colonies as compared to control colonies on average in year 1 but only significant for apiaries that had lower resource availability suggesting a trade-off for honey production vs. propolis collection in certain circumstances. However, in year 2 when propolis was already present in the boxes there was no difference between control or propolis colonies, indicating that any potential cost to honey production is alleviated after an initial setup period. Analysis of immune gene expression from the first year of study showed less variable and reduced gene expression in the propolis-rich environment, consistent with our previous studies. Analysis of core microbiota via qRT-PCR found similar results to the 16S sequencing projects. Results provide demonstrable evidence to the beekeeping community that colonies with a propolis envelope are at least as productive and have fewer disease symptoms compared to colonies without a propolis envelope. If the propolis treatment increases honey bee lifespan and colony-level health, productivity and survival, this will provide additional support to our breeding program and provide a novel technique for integrated pest management for the beekeeping community. Objective 3. We hypothesize that colonies bred for propolis collection, hygienic behavior and reduced mite population growth will demonstrate reduced pathogen prevalence and increased survivorship. We are evaluating colonies from diverse honey bee stocks for wintering survivorship and the following metrics: 1) relatively low Varroa mite population growth, both on adult bees and on pupae, between May and September; 2) low viral loads throughout the season (DWV, ABPV, IAPV and others); 3) hygienic behavior (assayed using a freeze-killed brood assay), and 4) propolis collection (amount of propolis deposited in commercial propolis traps). Last winter, 2020-21, 28 of 67 colonies survived without treatment for Varroa. The highest scoring colonies, including those selected for high propolis collection, were used as breeder stock for the next generation in 2021. 50 new colonies are wintering (2021-22) without treatment. Samples (preserved in -80 freezer) from another 50, the parent colonies of the untreated stock, will provide future genetic information on haplotypes that persist in surviving stock. We expect higher survival as we continue the multi-pronged selection program into the future.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Shanahan M, Spivak M. 2021. Resin use by stingless bees: A review. Insects. 12, 719. https://doi.org/10.3390/insects12080719
- Type:
Other
Status:
Published
Year Published:
2021
Citation:
Metz BN, Wu-Smart J, Simone-Finstrom M. Proceedings of the 2020 American Bee Research Conference. Insects. 2020; 11(6):362. https://doi.org/10.3390/insects11060362
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Wagoner K, Miller JG, Keller J, Bello J, Waiker P, Schal, Spivak M. Rueppell O. 2021. Hygiene-eliciting brood semiochemicals as a tool for assaying honey bee (Hymenoptera: Apidae) colony resistance to Varroa (Mesostigmata: Varroidae). J. Insect Sci. 21( 6) 4, doi.org/10.1093/jisesa/ieab064
|
Progress 03/01/20 to 02/28/21
Outputs
Target Audience:Researchers interested in basic biology, health and behaviors of honey bees and pollinators, in general. Beekeepers and honey bee queen breeders. Commercial growers of fruits and vegetables that need honey bee pollination. General public interested in increasing health of honey bees and other pollinators. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?M. Spivak advised two graduate students that worked on this project: Hollie Wall-Dalenberg (graduated with a Masters degree in November 2020), Maggie Shanahan (current PhD candidate). Both received additional academic-year funding from other sources; thus this grant covered only summer salary. M. Simone-Finstrom supported Sarah Lang, a recent Masters graduate was supported by this project to expand research on effects of propolis on core honey bee microbiota from March 2020 to August 2020. This work was then advanced by a technician (Allyson Martin) who was hired by M. Simone-Finstrom in August 2020. Dr. Mike Goblirsch (former post-doctoral researcher in Spivak lab) is now Research Scientist at USDA-ARS lab in Mississippi. Due to COVID restrictions, M. Goblirsch was unable to hold in-person workshops on the establishment and use of honey bee cell cultures in research. Due to COVID restrictions undergraduates have been unable to be recruited or conduct work in M. Simone-Finstrom's research laboratory. Professional Gary Reuter and technician Yuuki Metread in MN assisted with bee breeding program, and provided beekeeping training to graduate students and beekeepers. Professionals Phil Tokarz, Bob Cox, and David Dodge and technicians Sarah Lang and Allyson Martin in LA assisted with field trials and laboratory analyses. Graduate students and post-docs attended and presented findings at professional meetings, such as American Bee Research Conference and the Entomological Society of America annual meeting. How have the results been disseminated to communities of interest?For this reporting period (March 2020-March 2021, M Spivak presented research findings on propolis and social immunity as invited, or keynote, speaker at scientific and/ or beekeeping conferences in: Feb 29, 2020. Indiana Bee School (virtual): "Honey bee breeding for disease and mite resistance" and "What we know, and don't know, about the benefits of propolis to honey bees" April 15, 2020. University of Florida Two Bees in a Podcast. "Interview about propolis for bees". May 18, 2020. TheBeekeeper.org Australia, On-line educational platform. "The benefits of propolis to honey bee health" August 9, 2020. Bee Squad Bees for Veterans Program (virtual): "The benefits of propolis to bee health". Aug 29, 2020. Ist International Forum on Bees in the Time of a Pandemic, Brazil (virtual): "The health of honey bees in the U.S." November 19, 2020. Sommerset UK Beekeeping Association (virtual): "Honey bee social immunity: hygienic behavior and propolis collection" Dec 14, 2020. Palm Beach FL beekeepers (virtual): "Honey bee social immunity: hygienic behavior and propolis collection" Feb 1, 2021. Georgia Beekeepers Association (virtual): "Honey bee social immunity: hygienic behavior and propolis collection" Feb 13, 2021. Swedish Beekeepers Association (virtual): "Benefits of propolis to honey bee health" March 6, 2021. Danish Beekeepers Association (virtual): "Bee Breeding" For this reporting period, M Simone-Finstrom presented current research efforts and findings related to this project at one scientific conference (invited speaker) and as an invited speaker to three beekeeping conferences: · M. Simone-Finstrom, Propolis in beekeeping: Multifaceted effects of resin use by honey bees. Randolph County (NC) Beekeepers Association. October 2020. (virtual) · M. Simone-Finstrom, F. Rinkevich, M. Spivak. Pesticides and Propolis: The influence of honey bee-collected plant resins on pesticide exposure and sensitivity in agroecosystems. Entomological Society of America Annual Convention. November 2020. (virtual) · M. Simone-Finstrom. Factors influencing colony survival in migratory beekeeping: A tale of two longitudinal studies. American Honey Producers Association. Annual convention, December 2020. (virtual). · M. Simone-Finstrom, K. Ihle. Nutrition, Social Immunity and New Trait Selection: Current research on breeding and management in Baton Rouge. Great Plains Master Beekeepers. June 2020 (virtual) M Goblirsch (propolis and social immunity talks) · Social Immunity for Colony Health. At Home Beekeeping Webinar. Distance Learning for Beekeeping Clubs. Alabama Extension Service. April 2021 Maggie Shanahan (PhD student) El Colegio de la Frontera Sur (invited seminar speaker): Tapachula, Chiapas, Mexico. March 5, 2020.El uso de propóleo por la abeja Scaptotrigona mexicana y posibles implicaciones para la salud de su colmena. Mesoamerican Native Bee Conference:Cholula, Puebla, Mexico. Noviembre 28, 2019.La inmunidad social y el uso de propóleo en Apis mellifera y abejas sin aguijón. UMN Social Insects course: Saint Paul, Minnesota. Fall 2019.Stingless bees: Natural history, social behavior, and who gets to be queen. Apimondia conference:Montreal, Canada, September 2019.Sticking together: Resin use and social immunity in honey bees and stingless bees. Minnesota Hobby Beekeepers Association: Saint Paul, Minnesota, April 2019.Sticking together: Resin use in honey bees and stingless bees. What do you plan to do during the next reporting period to accomplish the goals?Objective 1: To build on our understanding on the effects of propolis on the honey bee immune system we will use a simplified host environment, the AmE-711 cell line, to examine the response of the honey bee immune with and without pathogen challenge. Preliminary evidence shows that the AmE-711 cell line expresses the antimicrobial peptide, Hymenoptaecin. AmE-711 cells will be cultured with or without the addition to propolis to the culture medium. We will then challenge AmE-711 cells with pathogen associated molecular patterns, including LPS and beta-glucan, and quantify signaling and effector molecules associated honey bee humoral immunity using qRT-PCR and sequencing. These experiments will provide much needed information on the direct effects of propolis on honey bee immunity. Due to restrictions related to COVID and maximized telework posture of USDA employees, progress in 2020 was delayed. In spring 2021, we also will begin experiments related to the effects of propolis exposure on the antimicrobial activity of brood food (Objective 1b) and on larval immune response to bacterial and fungal pathogens (1b) and viral infection (1c), as described in the proposal. Larval rearing trials will be done in Baton Rouge, LA by M. Simone-Finstrom and M. Goblirsch in MS. Experiments to determine how propolis exposure influences the antimicrobial activity of worker glandular secretions will be done by injecting newly bees with LPS to stimulate the immune system, they will then be maintained in cages in propolis-rich or propolis-poor colonies for 7days at which point their hypopharyngeal glands, which produce larval food, will be dissected. The glands will then be analyzed for antimicrobial activity via qRT-PCR for GOX and Defensin1 expression and glucose oxidase enzyme activity, as these are known to be major factors involved in royal jelly antimicrobial activity. This work will be done in coordination with both MN and Baton Rouge research groups. For Objective 1c, we will continue to investigate the effects of propolis on immune gene expression and virus infection in honey bee cell cultures using different methods of exposure. Our preliminary studies show that non-contact exposure of an inoculum containing a second virus (ABPV) to volatiles from propolis extract had no effect on virus infection when cells were exposed to the treated inoculum for 24hrs. Further experiments will be conducted to determine if direct contact of the ABPV inoculum to propolis extract has a negative effect on virus infectivity in AmE-711 cells. This experiment will help answer the question of whether direct contact of virus with propolis will inactivated virus particles and provides a simulation of what may occur in the hive environment. We will also conduct experiments to determine whether pre-conditioning the medium with propolis extract will lead to a reduction in subsequent viral load after AmE-711 are inoculated with ABPV. Objective 2: We have completed the analysis of the comparison of propolis deposition in colonies hived in boxes with rough wood interior wall and smooth surfaced interior walls to determine which treatment provides the most benefit to colony health and survivorship and will submit a manuscript to a peer-reviewed journal, based largely on PhD candidate M. Shanahan's work. We have conducted a second year of trials in collaboration with a migratory beekeeper to test whether a rough wood interior to stimulate propolis collection influences colony health and productivity as compared to the typical smooth surface interior that the beekeeping industry uses as its standard currently. Samples collected in February 2020 will undergo analysis of immune gene expression and relative abundance of core microbiota to confirm the results of the first year of the longitudinal study. Objective 3. We will continue selection for colonies that demonstrate behavioral mechanisms of social immunity against parasites and pathogens, and quantification of progress.
Impacts
What was accomplished under these goals?
Impact. To help restore honey bee health, we are focusing on ways to bolster their natural behavioral defenses, or social immunity, against pathogens and parasites. Honey bees collect antimicrobial plant resins and deposit them in the nest cavity as propolis. In the lab, graduate students and post-doctoral researchers are investigating the biological mechanisms of how propolis supports bees' immune function and disease defense. In the field, we are collaborating with beekeepers to quantify benefits of propolis to colony health and survivorship. We are selecting a new line of bees for multiple mechanisms of social immunity including propolis collection and hygienic behavior. In the long term these efforts will result in colonies more able to defend themselves against various parasites and pathogens, and in beekeepers having to use fewer chemical treatments to maintain colonies. Our ultimate aims are to increase the sustainability and economic viability of beekeeping to enhance food security and ecosystem vitality. Our previous published work has shown that in-hive propolis exposure reduces individual adult bee investment in physiological immunity, however the mechanism explaining these effects was not yet clear. A bees have evolved with the antimicrobial properties of propolis within the nest, we hypothesized that propolis exposure would reduce the abundance and diversity of opportunistic and pathogenic microbes in and on bees, allowing for reduced investment in individual bee immune gene expression and increased abundance of bees' core microbiome community. Two experiments, now published, were performed; bees for both experiments were collected from large field colonies that either had a propolis envelope (P+ colonies) or did not (P- colonies). DNA extractions and 16S amplicon sequencing was conducted to determine bacterial community structure and composition within whole bees or just on the bee mouthparts. In both experiments, honey bee microbiota was more consistent between bees collected from P+ colonies, while those from P- colonies exhibited greater microbial diversity. Several key members of the honey bee gut microbiota were significantly altered in P- colonies suggesting that propolis may play an important role in maintaining favorable levels of commensal bacteria (Saelao et al. 2020). OTU analysis of the mouthparts showed that core microbiome species significantly increased in absolute abundance in the bees from P+ compared to P- colonies, whereas opportunistic and pathogenic bacteria were at significantly lower relative abundance in P+ colonies (Wall-Dalenberg et al. 2020). These findings support our hypothesis that a propolis envelope reduces pathogenic or opportunistic microbes and promote the proliferation of beneficial core bacterial species on and in the honey bees, allowing for reduced investment in individual bee immune gene expression. Objective 1c. The continuous honey bee cell line, AmE-711, was used to test the direct effects of propolis on honey bee immune gene expression and determine whether exposure to propolis affects levels of a persistent (DWV-A) and introduced viral infection (Acute bee paralysis virus; ABPV) in a simplified host environment. AmE-711 cells were incubated in the presence of a noncytotoxic dose of propolis extract from Brazil or Louisiana and then cells were harvested at 0, 3, 6, 12, and 24 hrs after exposure for total RNA. RT-qPCR results revealed that PGRP had increased expression 12 hrs after exposure to both Brazil and Louisiana propolis (p≤0.008), while dorsal and relish showed increased expression 24 hrs after exposure to both Brazil and Louisiana propolis (p≤0.007 and p<0.0001, respectively). Defensin levels were not different between cells exposed to either source of propolis extract and untreated controls at all timepoints. We tested whether exposure of cells to propolis could reduce levels of persistent DWV-A and a second, introduced virus, ABPV. Cells were infected with a LC50 mixture of ABPV:DWV, followed by addition of propolis extract to the culture medium. Cells were harvested at 0, 3, 6, 12, and 24 hrs of exposure to propolis, and levels of both viruses were quantified using RT-qPCR. Levels of persistent DWV-A were unaffected by treatment over time (p=0.117). ABPV was significantly reduced in both propolis treatment groups, but only at timepoint 0 (i.e., 15 minutes after exposure to extract) (p≤0.028), with 5 of 7 replicates from the propolis treatment groups having ABPV levels below the limit of detection. Additional studies are underway that will look at the effects of propolis on lower doses of the ABPV:DWV inoculum before and after introduction into culture. Objective 2. We hypothesize that lining colonies with a propolis envelope will affect honey production and colony survival over the course of a year in beekeeping operations. We compared propolis deposition in colonies hived in custom-made boxes for propolis deposition and in control colonies not provided textured surface in the boxes in MN and LA, and in a migratory, commercial beekeeping operation. Results from the commercial operation over two years of study show that colonies with custom-made boxes deposited more propolis and have larger colony populations in February during almond pollination(p=0.03, N=134). Honey production was lower for propolis colonies as compared to control colonies on average in year 1 but only significant for apiaries that had lower resource availability suggesting a trade-off for honey production vs. propolis collection in certain circumstances. However, in year 2 when propolis was already present in the boxes there was no difference between control or propolis colonies, indicating that potentially any potential cost to honey production is alleviated after an initial setup period. Analysis of immune gene expression from the first year of study showed less variable and reduced gene expression in the propolis-rich environment, consistent with our previous studies. Analysis of core microbiota via qRT-PCR found similar results to the 16S sequencing projects. This finding highlights the utility of using real-time PCR to assess microbial loads as an additional way to assess effects of the nest environment on honey bee health. Results will provide demonstrable evidence to the beekeeping community that colonies with a propolis envelope are at least as productive and have fewer disease symptoms compared to colonies without a propolis envelope. If the propolis treatment increases honey bee lifespan and colony-level health, productivity and survival, this will provide additional support to our breeding program and provide a novel technique for integrated pest management for the beekeeping community. Objective 3. We hypothesize that colonies bred for propolis collection, hygienic behavior and reduced mite population growth will demonstrate reduced pathogen prevalence and increased survivorship. We are currently evaluating colonies from diverse honey bee stocks for wintering survivorship and three behavioral traits: 1) relatively low Varroa mite population growth, both on adult bees and on pupae, between May and September; 2) hygienic behavior (assayed using a freeze-killed brood assay), and 2) propolis collection (amount of propolis deposited in commercial propolis traps). We are following survivorship and performance of 50 full-sized colonies and 75 smaller (nucleus) colonies, culling colonies that do not meet our standards. In the long-term, this breeding program will result in colonies more able to defend themselves against various parasites and pathogens and should result in beekeepers having to use fewer chemical treatments to keep colonies alive.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Spivak M, Danka RG. 2020. Perspectives on hygienic behavior in Apis mellifera and other social insects. Apidologie DOI: 10.1007/s13592-020-00784-z
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Goblirsch M, Warner JF, Sommerfeldt BA, Spivak M. 2020. Social fever or general immune response? Revisiting an example of social immunity in honey bees. Insects 11: 528 doi:10.3390/insects11080528
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Dalenberg H, Maes P, Mott B, Anderson KE, Spivak M. 2020. Propolis envelope promotes beneficial bacteria in the honey bee (Apis mellifera) mouthpart microbiome. Insects 11, 453. doi:10.3390/insects1107/0453
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Saelao P, Borba RS, Ricigliano V, Spivak M, Simone-Finstrom M. 2020. Honeybee microbiome is stabilized in the presence of propolis. Biology Letters 16: 202003. doi.org/10.1098/rsbl.2020.0003
|
Progress 03/01/19 to 02/29/20
Outputs
Target Audience:Researchers interested in basic biology, health and behaviors of honey bees and pollinators, in general. Beekeepers and honey bee bee breeders. Commercial growers of fruits and vegetables that need honey bee pollination. General public interested in increasing health of honey bees and other pollinators Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?M. Spivak is advising two graduate students: Hollie Wall-Dalenberg (Masters student) and Maggie Shanahan (PhD student). Both received additional academic-year funding from other sources (Wall-Dalenberg received Diversity Fellowship from Univ MN; Shanahan received a Graduate Scholarship from NSF); thus this grant covers only their summer salary. M. Simone-Finstrom supported a visiting Australian researcher, J. Gerdts, for one month. M. Simone-Finstrom was a co-advisor for J. Gerdts' PhD thesis and after she graduated she came to the US and, specifically M. Simone-Finstrom's lab, to learn additional breeding and management techniques and methods for assessing propolis deposition. J. Gerdts assisted with field experiments and molecular analyses. M. Simone-Finstrom supervises one post-doctoral researcher, Perot Saelao, a bioinformatician who learned and worked on pipelines for analyses related to the honey bee microbiota. M. Simone-Finstrom also advises 5 undergraduate students, each worked on small aspects of the studies to develop skills in molecular biology and field collections and performed analyses related to scoring propolis deposition. M. Simone-Finstrom also co-advises Master's student, Sarah Lang, who after her thesis defense in Jan 2020 was supported by this project to expand research on effects of propolis on core honey bee microbiota. M. Spivak supervised Dr. Mike Goblirsch (was post-doctoral researcher, but now is Research Scientist at USDA-ARS lab in Mississippi), who mentored and assisted graduate students and technicians in the lab to conduct molecular and microbiological experiments. M. Goblirsch led a two-day international workshop in July 2019 on cell culture. Two scientists, one from USDA-ARS Bee Research Lab in Beltsville, MD, and a graduate student from the Univ of CT were provided with hands on instruction in initiating and using primary cultures and established cell lines from honey bee tissues. Professional Gary Reuter and technician Yuuki Metread in MN assist with bee breeding program, and provide beekeeping training to graduate students and beekeepers. Professionals Phil Tokarz, Bob Cox, and David Dodge in LA assist with field trials and laboratory analyses. Undergrad? Graduate students and post-docs attend and present findings at professional meetings, such as American Bee Research Conference and the Entomological Society of America annual meeting. How have the results been disseminated to communities of interest?For this reporting period, M Spivak presented research findings on propolis and social immunity as invited, or keynote, speaker at scientific and/ or beekeeping conferences in: International Union for the Study of Social Insects, European Section, (Keynote): "Honey Bee Social Immunity" Klosterneuberger Austria, March 19-22. Massachusetts Beekeeping Association, "Bee Health in the USA" Wooster, MA, March 2 Eagle Bluff Environmental Learning Center, "Getting Bees Back on Their Own Six Feet" Lansboro, MN April 16. University of Missouri, Department of Biology CV Riley Lecture Series, "Socialized Medicine in Honey Bee Colonies" April 17 MN Honey Producers Association, "Research Update" Fargo, ND, July 11-12. Apimondia International Bee Conference, "Breeding for Disease and Mite Resistance" Montreal, Canada, Sept 9-12. MN Hobby Beekeeping Assoc, "Breeding Bees for Disease and Mite Resistance" Nov 12. Idaho Beekeeping Association "Benefits of Propolis to Bee Health" and "Breeding Bees for Disease and Mite Resistance" Dec 5. For this reporting period, M Simone-Finstrom presented current research efforts and findings related to this project at one scientific conference and as an invited speaker to two beekeeping conferences: M. Simone-Finstrom, K. Ihle, A. Avalos. From Genetic Characterization to New Trait Selection: Baton Rouge Efforts in Bee Breeding. Small Interest Group: Queen Breeders and Package Producers Workshop at the annual convention of the American Beekeeping Federation. January 2020. Schaumburg, IL. M. Simone-Finstrom, F. Rinkevich, M. Shanahan, & M. Spivak. Propolis in beekeeping: Understanding multifaceted effects of resin use by honey bees. American Bee Research Conference. January 2020. Schaumburg, IL. M. Simone-Finstrom. Breeding better bees in Baton Rouge: Integrative approach to improve colony health. Annual meeting of the Russian Honey Bee Breeders Association. October 2019, Baton Rouge, LA. M Goblirsch (propolis and social immunity talks) Honey bee research at the University of Minnesota. La Crosse Area Beekeepers' Regional Meeting. La Crosse WI. March 2020. What is social fever? Pine Belt Beekeepers' Association. Purvis MS. March 2020. Beekeeping moving forward: Recognizing the challenges, exploring the opportunities. Jordan Beekeeping Association. Amman and Irbid Jordan. January 2020. Advanced beekeeping techniques, a three-day workshop. Jordanian Beekeepers Association. Irbid Jordan. January 2020. Beekeeping Workshop. USAID Partners of the Americas Farmer to Farmer Program and the Guyana Livestock Development Authority. Georgetown Guyana. November 2020. Another look at social fever in honey bees. XLVIth Apimondia International Apicultural Congress. Montréal Québec Canada. September 2019. The evidence for social fever, you be the judge? Minnesota Hobby Beekeepers' Association. St. Paul, MN. July 2020. Maggie Shanahan (PhD student) El Colegio de la Frontera Sur (invited seminar speaker): Tapachula, Chiapas, Mexico. March 5, 2020.El uso de propóleo por la abeja Scaptotrigona mexicana y posibles implicaciones para la salud de su colmena. Mesoamerican Native Bee Conference:Cholula, Puebla, Mexico. Noviembre 28, 2019.La inmunidad social y el uso de propóleo en Apis mellifera y abejas sin aguijón. UMN Social Insects course: Saint Paul, Minnesota. Fall 2019.Stingless bees: Natural history, social behavior, and who gets to be queen. Apimondia conference:Montreal, Canada, September 2019.Sticking together: Resin use and social immunity in honey bees and stingless bees. Minnesota Hobby Beekeepers Association: Saint Paul, Minnesota, April 2019.Sticking together: Resin use in honey bees and stingless bees. Hollie Wall-Dalenberg (Masters student) Western Colorado Beekeepers Association, Grand Junction, CO, November, 2019 - "The effects of propolis on the honey bee (Apis mellifera) mouthpart microbiome" Entomological Society of America, St. Louis, MO, November 13, 2019 - "The effects of propolis on the honey bee (Apis mellifera) mouthpart microbiome" What do you plan to do during the next reporting period to accomplish the goals?Objective 1: We have submitted two manuscripts for publication reporting on results from Objective 1A and plan to submit a third this year. To build on our understanding on the effects of propolis on the honey bee immune system we will use a simplified host environment, the AmE-711 cell line, to examine the response of the honey bee immune with and without pathogen challenge. Preliminary evidence shows that the AmE-711 cell line expresses the antimicrobial peptide, Hymenoptaecin. AmE-711 cells will be cultured with or without the addition to propolis to the culture medium. We will then challenge AmE-711 cells with pathogen associated molecular patterns, including LPS and beta-glucan, and quantify signaling and effector molecules associated honey bee humoral immunity using qRT-PCR and sequencing. These experiments will provide much needed information on the direct effects of propolis on honey bee immunity. We also will begin experiments related to the effects of propolis exposure on the antimicrobial activity of brood food (Objective 1b) and on larval immune response to bacterial and fungal pathogens (1b) and viral infection (1c), as described in the proposal. Larval rearing trials will be done in Baton Rouge, LA by M. Simone-Finstrom and M. Goblirsch in MS. Experiments to determine how propolis exposure influences the antimicrobial activity of worker glandular secretions will be done by injecting newly bees with LPS to stimulate the immune system, they will then be maintained in cages in propolis-rich or propolis-poor colonies for 7days at which point their hypopharyngeal glands, which produce larval food, will be dissected. The glands will then be analyzed for antimicrobial activity via qRT-PCR for GOX and Defensin1 expression and glucose oxidase enzyme activity, as these are known to be major factors involved in royal jelly antimicrobial activity. This work will be done in coordination with both MN and Baton Rouge research groups. For Objective 1c, we will continue to investigate the effects of propolis on virus infection in honey bee cell cultures using different methods of exposure. To simulate conditions that may occur in the hive environment, we will treat an inoculum containing a second honey bee virus, Acute bee paralysis virus (ABPV), via non-contact exposure to propolis volatiles for different incubation periods. We will then inoculate AmE-711 cells with the treated ABPV. ABPV causes rapid cell death when added to AmE-711 cell cultures; therefore, we hypothesize that non-contact exposure to propolis volatiles will inactivate the virus, resulting in lower ABPV replication and less cytopathic effect compared to untreated controls. Objective 2: We will complete the analysis of the comparison of propolis deposition in colonies hived in boxes with rough wood interior wall and smooth surfaced interior walls to determine which treatment provides the most benefit to colony health and survivorship and submit a manuscript to a peer-reviewed journal, based largely on PhD candidate M. Shanahan's work. We will also conduct a second year of trials in collaboration with a migratory beekeeper to test whether a rough wood interior to stimulate propolis collection influences colony health and productivity as compared to the typical smooth surface interior that the beekeeping industry uses as its standard currently. Samples collected in February 2020 will also undergo analysis of immune gene expression and relative abundance of core microbiota. The colonies from the first year's trial return from CA almond pollination in mid-March 2020, and they will be assessed for colony strength metrics upon their return. As data suggested a potential trade-off in resin collection vs. honey production using new boxes, rough boxes with propolis already deposited will be used to see if there is a difference once propolis has already been established in these hives. Starting in April 2020, a second 120 colonies will be followed in the commercial beekeepers migratory route from March 2020 through March 2021, half having rough hive interiors with propolis already deposited from the previous year and half with smooth interiors. Samples will be collected in August 2020 in South Dakota to assess honey production and February 2021 during almond pollination to determine bee health and colony survival. Objective 3. Selection for colonies that demonstrate behavioral mechanisms of social immunity against parasites and pathogens will continue. We expect to begin to see results of selection efforts this year.
Impacts
What was accomplished under these goals?
Impact. To help restore honey bee health, we are focusing on ways to bolster their natural behavioral defenses, or social immunity, against pathogens and parasites. Honey bees collect antimicrobial plant resins and deposit them in the nest cavity as propolis. In the lab, graduate students and post-doctoral researchers are investigating the biological mechanisms of how propolis supports bees' immune function and disease defense. In the field, we are collaborating with beekeepers to quantify benefits of propolis to colony health and survivorship. We have begun selecting a new line of bees for multiple mechanisms of social immunity including propolis collection and hygienic behavior. In the long term these efforts will result in colonies more able to defend themselves against various parasites and pathogens, and in beekeepers having to use fewer chemical treatments to maintain colonies. Our ultimate aims are to increase the sustainability and economic viability of beekeeping to enhance food security and ecosystem vitality. Objective 1a. As bees have evolved with the antimicrobial properties of propolis within the nest, we hypothesized that propolis exposure would reduce the abundance and diversity of opportunistic and pathogenic microbes in and on bees, allowing for reduced investment in individual bee immune gene expression and increased abundance of bees' core microbiome community. Two experiments were performed; bees for both experiments were collected from large field colonies that either had a propolis envelope (P+ colonies) or did not (P- colonies). Samples previously generated from colonies in MN were used to analyze effects of a propolis rich environment (P+ colonies) on microbial communities from whole abdomen samples. Analysis was conducted in Baton Rouge, LA. DNA extractions and 16S amplicon sequencing was conducted to determine microbiota community structure and composition. Honey bee microbiota was more consistent between bees collected from P+ colonies, while those from P- colonies exhibited greater microbial diversity. Several key members of the honey bee gut microbiota were significantly altered in the absence of propolis suggesting that it may play an important role in maintaining favorable levels of commensal bacteria (In Review). In another experiment, bacterial microbiomes of DNA extracted from honey bee mouthparts were sequenced at the University of AZ through a collaboration with Dr. Kirk Anderson (USDA-ARS Bee Lab, Tucson, AZ). The mouthparts of worker bees in P+ colonies had significantly lower bacterial diversity and significantly higher bacterial abundance compared to the mouthparts of bees in P- colonies. OTU analysis showed that core microbiome species significantly increased in absolute abundance in the bees from P+ compared to P- colonies, whereas opportunistic and pathogenic bacteria were at significantly lower relative abundance in P+ colonies (submitted). The immune gene transcript abundance of hymenoptaecin was significantly lower in bees from P+ colonies These findings support our hypothesis that a propolis envelope appears to reduce pathogenic or opportunistic microbes and promote the proliferation of beneficial core bacterial species on and in the honey bees, allowing for reduced investment in individual bee immune gene expression. . Objective 1c. The AmE-711 cell line is persistently infected with DWV-A. We used RT-qPCR to compare levels of endogenous DWV-A to levels of the host reference gene, RPS5. We observed a strong positive correlation between the CT values for the RPS5 host gene and the CT values for DWV-A, supporting the observation that DWV-A exists in equilibrium with AmE-711 host cells under culture conditions that do not induce a cytopathic effect. We tested if the addition of propolis extract to the culture medium at a non-cytotoxic concentration could reduce viral load. AmE-711 cells were incubated for 0, 1, 3, 7, 21, or 30 hrs in culture medium that was untreated, or was supplemented with either propolis extract or ethanol alone. The relative amount of endogenous DWV-A was unaffected by the interaction of treatment and duration of exposure (submitted). Objective 2. We hypothesize that lining colonies with a propolis envelope will affect honey production and colony survival over the course of a year in beekeeping operations. We compared propolis deposition in colonies hived in custom-made boxes for propolis deposition and in control colonies not provided textured surface in the boxes in MN and LA, and in a migratory, commercial beekeeping operation. Results from the commercial operation to date show that colonies with custom-made boxes deposited more propolis and tended to have larger colony populations in February 2020 (7 ± 0.45 vs. 6 ± 0.45 frames of bees, N=83). Honey production was lower for propolis colonies as compared to control colonies on average but only significant for apiaries that had lower resource availability suggesting a trade-off for honey production vs. propolis collection. Analysis of immune gene expression showed less variable and reduced gene expression in the propolis-rich environment, consistent with our previous studies. Analysis of core microbiota via qRT-PCR found similar results to the 16S sequencing projects. This finding highlights the utility of using real-time PCR to assess microbial loads as an additional way to assess effects of the nest environment on honey bee health. Results will provide demonstrable evidence to the beekeeping community that colonies with a propolis envelope are more productive and have fewer disease symptoms compared to colonies without a propolis envelope. If the propolis treatment increases honey bee lifespan and colony-level health, productivity and survival, this will provide additional support to our breeding program and provide a novel technique for integrated pest management for the beekeeping community. Objective 3. We hypothesize that colonies bred for propolis collection, hygienic behavior and reduced mite population growth will demonstrate reduced pathogen prevalence and increased survivorship. We are currently evaluating colonies from diverse honey bee stocks in MN and LA for wintering survivorship and two behavioral traits: 1) hygienic behavior (currently measured using a freeze-killed brood assay), and 2) propolis collection (amount of propolis deposited in commercial propolis traps stapled to the inside of boxes). Due to our relatively short summer season, we found the third trait -- low Varroa mite population growth (measured by sampling mites on adult bees and on brood at beginning and end of the season) - was difficult to quantify in time to raise and mate queens before winter so will modify our selection plan and timing to better quantify this trait for this summer. We have identified colonies with the highest scores for hygienic behavior and propolis collection in 2018 and 2019, and, Spivak instrumentally inseminated daughter queens from these colonies with semen collected from other top scoring colonies each year. In 2020, will evaluate the colonies headed by the inseminated queens and continue to breed from them. At this date, winter survivorship (2019-2020) is 75% in the selected colonies. In the long-term, this breeding program will result in colonies more able to defend themselves against various parasites and pathogens and should result in beekeepers having to use fewer chemical treatments to keep colonies alive.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Spivak M, Goblirsch, M, Simone-Finstrom M. 2019. Social-medication in bees: The line between individual and social regulation. Curr. Opinion Insect Science 33:49-55. doi.org/10.1016/j.cois.2019.02.009
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Wagoner K, Spivak M, Hefetz A, Reams R, Rueppell O. 2019. Stock-specific chemical brood signals are induced by Varroa and Deformed Wing Virus, and elicit hygienic response in the honey bee. Sci Reports. 9:8753. doi.org/10.1038/s41598-019-45008-2
- Type:
Other
Status:
Published
Year Published:
2020
Citation:
Spivak M, Mendel B. 2020. Propolis for Bees. 2millionblossoms.com
- Type:
Other
Status:
Published
Year Published:
2019
Citation:
Starr, Christopher K. 2019. Encyclopedia of social insects. Springer Nature Living Reference. https://doi.org/10.1007/978-3-319-90306-4
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
5. L�pez-Uribe MM, Ricigliano VA, Simone-Finstrom M. 2020. Defining Pollinator Health: A Holistic Approach Based on Ecological, Genetic, and Physiological Factors. Annual Review of Animal Biosciences. 8:1, 269-294
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Progress 03/01/18 to 02/28/19
Outputs
Target Audience:Researchers interested in basic biology, health and behaviors of honey bees and pollinators, in general. Beekeepers and honey bee bee breeders. Commercial growers of fruits and vegetables that need honey bee pollination. General public interested in increasing health of honey bees and other pollinators Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?M. Spivak is advising two graduate students working on this project: Hollie Wall-Dalenberg (Masters student) and Maggie Shanahan (PhD student). M. Simone-Finstrom is advising one undergraduate student who is working on this project to develop skills in molecular biology and field collections. M. Spivak supervises post-doctoral researcher Mike Goblirsch, who in turn mentors and assists graduate students and technicians in the lab to conduct molecular and microbiological experiments. M. Goblirsch led a two-day international workshop in July 2018 on cell culture. Five early-career scientists were invited to participate and were provided with hands on instruction in initiating and using primary cultures and established cell lines from honey bee tissues. Professionals Gary Reuter and Yuuki Metread in MN assist with bee breeding program, and provide beekeeping training to graduate students and beekeepers. Professionals Phil Tokarz and Bob Cox in LA assist with field trials and laboratory analyses. Graduate students and post-docs attend and present findings at professional meetings, such as American Bee Research Conference and the Entomological Society of America annual meeting. How have the results been disseminated to communities of interest?For this reporting period, M Spivak presented research findings on propolis and social immunity as invited, or keynote, speaker at scientific conferences in: Sofia, Bulgaria: 2nd International Conference on the Benefits of Propolis to Bee and Human Health, Sept 2018 Klosterneuberger, Austria: Central European Section, International Union for the Study of Social Insects. March 2019 And at beekeeping association meetings in (some examples): Kansas Beekeepers June 2018 Heartland Apicultural Society, July 2018 MN Honey Producers Association, July 2018 Ohio State Beekeeping Association, Nov, 2018 Lima, Peru: IV Congreso Nacional Apícola and I Encuentro International en Apicultura, Universidad Nacional Agraria la Molina (UNALM), Nov 2018 Myrtle Beach, South Carolina: American Beekeeping Federation meeting, Jan 2019 Northern Ireland: Ulster Beekeeping Association, Feb 2019 Worcester, Mass: Massachusetts Beekeeping Association, March 2019 M Simone-Finstrom (propolis talks) Central Victorian Apiarist Association, Australia, August 2018 California Master Beekeeper Program, August 2018 Mississippi State Beekeepers Association, October 2018 USDA Honey Bee Breeding, Genetics and Physiology Laboratory and the Louisiana State Beekeepers Association will hold the 22nd Annual Field Day, October 2018 M Goblirsch (propolis and social immunity talks) Sofia, Bulgaria: 2nd International Conference on the Benefits of Propolis to Bee and Human Health, Sept 2018 Door County Beekeepers Association, Sturgeon Bay, WI, May 2018 Department of Natural Sciences, Metropolitan State University, St. Paul, MN, April 2018 Maggie Shanahan (PhD student) Entomological Society of America: "Optimizing the benefits of propolis to honey bee health in commercial beekeeping operations" poster presentation. November 2018 American Bee Research Conference: Optimizing the benefits of propolis to honey bee health in commercial beekeeping operations" Oral presentation. January 2019 What do you plan to do during the next reporting period to accomplish the goals?Objective 1: Experiments for Objective 1a, on the mode of action of propolis on adult bee immune function are underway. We will begin experiments on Objective 1b, related to the effects of propolis exposure to larval immune response and brood pathogen defense, as described in the proposal. Objective 1c: We will continue to investigate the effects of propolis on virus infection in honey bee cell cultures. Since AmE-711 cells can tolerate addition of a relatively high amount of propolis to culture medium (≤ 1µg/mL) without a producing a toxic effect, we plan to expose cells for a longer duration (i.e., days to weeks) and collect culture medium at regular intervals during exposure to determine if there is a reduction in DWV titer over time. Additionally, we will pre-expose AmE-711s to propolis followed by inoculation with a mixture of two viruses, Acute bee paralysis virus and DWV. We will use an archived inoculum harvested from cells showing cytopathic effects and positive for both viruses by RT-qPCR. We expect that the propolis exposure will reduce infectivity of the viruses but endogenous levels of DWV will remain unaffected. We are adding an experiment to this objective to explore the effects of propolis exposure on the cuticular microbiome of honey bee mouthparts using DNA sequencing. The hypothesis is that the antimicrobial properties of propolis may not affect bacterial and fungal abundance, but may affect community composition of bacteria and fungi on the mouthparts. This work will be collaboration with Dr. K. Anderson at USDA-ARS Bee Lab, Tucson, AZ, and funding for sequencing will come from other sources. This work will comprise the Master's thesis of Hollie Wall-Dalenberg. Objective 2: We will complete the comparison of propolis deposition in colonies hived in boxes with rough wood interior wall and smooth surfaced interior walls to determine which treatment provides the most benefit to colony health and survivorship. We will also begin trials in collaboration with a migratory beekeeper to test whether a rough wood interior to stimulate propolis collection influences colony health and productivity as compared to the typical smooth surface interior that the beekeeping industry uses as its standard currently. 120 colonies will be followed in the commercial beekeepers migratory route from March 2019 through March 2020, half having rough hive interiors and have with smooth interiors. Samples will be collected in August 2019 in South Dakota during honey production and February 2020 during almond pollination to determine bee health and colony survival. Objective 3: Selection for colonies that demonstrate behavioral mechanisms of social immunity against parasites and pathogens will continue as planned.
Impacts
What was accomplished under these goals?
Impact. Honey bee (Apis mellifera) populations are experiencing high and unsustainable annual losses due to a number of stressors that weaken bees' innate defenses resulting in a downward spiral of health. To help restore honey bee health, we are focusing on ways to bolster their natural behavioral defenses, or social immunity, against pathogens and parasites. Honey bees collect antimicrobial plant resins and deposit them in the nest cavity as propolis. In the lab, graduate students and post-doctoral researchers are investigating the biological mechanisms of how propolis supports bees' immune function and disease defense. In the field, we are collaborating with beekeepers to quantify benefits of propolis to colony health and survivorship. We have begun selecting a new line of bees for multiple mechanisms of social immunity including propolis collection and hygienic behavior. In the long term these efforts will result in colonies more able to defend themselves against various parasites and pathogens, and in beekeepers having to use fewer chemical treatments to maintain colonies. Our ultimate aims are to increase the sustainability and economic viability of beekeeping to enhance food security and ecosystem vitality. Objective 1. Objective 1a. We initiated studies to measure the immune response of adult bees held in laboratory cages with and without propolis exposure. We hypothesize that propolis exposure has an indirect modulatory role on honey bee immune function whereby the decrease in microbial loads allows for reduced investment in immune function. Bees were exposed to propolis either through direct contact with propolis within the cages, or through volatiles from propolis outside the cages (no direct contact). The levels of immune gene expression (hymenoptaecin), and 16S eubacterial transcript abundance, are being measured in 7d-old bees from the cages (each cage contained bees from three colonies, three cages per treatment). Data is currently being analyzed using real-time quantitative PCR. Objective 1c. We confirmed that the AmE-711 honey bee cell line is persistently infected with deformed wing virus (DWV) using RT-qPCR and the level of infection remains static over time. Since the AmE-711 cells have endogenous virus, yet maintain a healthy phenotype, it eliminates the challenge of purifying virus from other sources and testing inoculates for infectivity. To test the hypothesis that propolis reduces infection with DWV at the cell level, we cultured AmE-711 cells and exposed them to propolis dissolved in culture medium. We then measured DWV levels using RT-qPCR on RNA extracted from AmE-711 cells at two timepoints after exposure, 3 and 6 hours. We observed a reduction in DWV titer in cultures exposed to propolis over time; however, this reduction was comparable to control cultures exposed to solvent alone. Moreover, levels of DWV were higher in cultures exposed to propolis and solvent compared to untreated cultures, suggesting that chemical exposure may have an effect on the host-virus interaction, at least in the short-term. Objective 2. We hypothesize that lining colonies with a propolis envelope will affect honey production and colony survival over the course of a year in a commercial, migratory beekeeping operation. We began by conducting a pilot experiment to optimize methods for stimulating bees to build a propolis envelope within commercial bee boxes. We began by testing bee box surface textures that promote propolis collection, to ultimately quantify how much propolis is needed to provide significant colony health benefits. This pilot experiment indicated that the interior texture of the wooden bee boxes needs to be extremely rough for bees to deposit a propolis envelope equivalent in depth as colonies do within tree cavities in nature. Thus, we had new boxes constructed by a private company with very rough interior texture (add photo?). This summer, 2019, we will compare propolis deposition in 12 colonies hived in these custom made boxes with propolis deposition in 12 colonies fitted with commercial propolis traps, and 12 control colonies not provided textured surface in the boxes. We will collect colony health measures (colony population, Varroa loads over the season, signs of disease, honey production and winter survival), quantify propolis deposition, and collect individual adult bees from immune gene testing to compare immune gene transcript abundance across the treatments. Results will provide demonstrable evidence to the beekeeping community that colonies with a propolis envelope are more productive and have fewer disease symptoms compared to colonies without a propolis envelope. If the propolis treatment increases honey bee lifespan and colony-level health, productivity and survival, this will provide additional support to our breeding program and provide a novel technique for integrated pest management for the beekeeping community. Objective 3. We hypothesize that colonies bred for propolis collection, hygienic behavior and reduced mite population growth will demonstrate reduced pathogen prevalence and increased productivity. We are currently evaluating colonies from diverse honey bee stocks (mixtures of beekeeping stocks derived from A. mellifera ligustica and carnica) in MN and LA. We are evaluating each colony for gentleness (scored from most to least gentle during routine management), honey production (kg produced), wintering survivorship (frames of bees and brood in early spring), and three behavioral traits: 1) hygienic behavior (currently measured using a freeze-killed brood assay), 2) propolis collection (amount of propolis deposited in commercial propolis traps stapled to the inside of boxes), and 3) low Varroa mite population growth (measured by sampling mites on adult bees and on brood at beginning and end of the season). We have identified colonies with the highest scores for all measures, and in 2018, Spivak instrumentally inseminated daughter queens from these colonies with semen collected from other top scoring colonies. This summer, 2019, we will evaluate the colonies headed by the inseminated queens, and continue to evaluate new stocks of bees. In the long-term, this breeding program will result in colonies more able to defend themselves against various parasites and pathogens, and should result in beekeepers having to use fewer chemical treatments to keep colonies alive.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Wagoner K, Spivak M, Rueppell O. 2018. Brood affects hygienic behavior in the honey bee (Hymenoptera: Apidae). J. Econ. Entomol. 111(6): 2520-2530. doi.org/10.1093/jee/toy266
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2018
Citation:
Spivak, M. 2nd International Conference on the Benefits of Propolis to Bee and Human Health. Sofia, Bulgaria. What we know and dont know about the benefits of propolis for honey bee health. Sept 28-29.
- Type:
Other
Status:
Awaiting Publication
Year Published:
2019
Citation:
Spivak, M; Simone-Finstrom, M. Propolis. In: Encyclopedia of Social Insects. Springer.
- Type:
Journal Articles
Status:
Awaiting Publication
Year Published:
2019
Citation:
Spivak M, Goblirsch, M, Simone-Finstrom M. Social-medication in bees: The line between individual and social regulation. Curr. Opinion Insect Science. In Press
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