DETERMINING THE ROLE OF VIRUSES ON HONEY BEE HEALTH.

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: TERMINATED
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1012699
• Project No.: MONB00252
• Project Start Date: Jul 1, 2017
• Project End Date: Jun 30, 2020

Project Director
Flenniken, MI, LY.

Recipient Organization
MONTANA STATE UNIVERSITY
(N/A)
BOZEMAN,MT 59717

Performing Department
Plant Sciences and Plant Pathology

Non Technical Summary
Honey bees are important pollinators of agricultural crops and plant species that enhance ecosystem biodiversity. High annual losses of US honey bee colonies (averaging 33% since 2006) have been associated with RNA viruses, but the mechanisms of honey bee host - virus interactions remain largely uncharacterized. The long-term goal of this research is to reduce honey bee colony losses caused by virus infections by advancing the epidemiologic and mechanistic understanding of the effects of viruses on bee health. To better understand the role of viruses in colony losses, we will longitudinally monitor honey bee colonies and examine the relationship between colony health and pathogen prevalence and abundance. At the individual bee level, virus infections can remain asymptomatic, cause paralysis, and/or result in death.These differential outcomes are largely dependent on host immune responses, yet the mechanisms of honey bee antiviral responses remain largely uncharacterized. To elucidate the mechanisms of honey bee host antiviral defense, we will perform laboratory-based virus infection trials in individual bees and identify candidate honey bee antiviral defense genes using high throughput sequencing. These experiments will significantly advance the understanding of the effects of viruses on honey bees at both the colony and individual levels, and may lead to the development of strategies to mitigate bee losses.

Animal Health Component

100%

Research Effort Categories

Basic

45%

Applied

45%

Developmental

10%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
211	3010	1101	100%

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

Subject Of Investigation
3010 - Honey bees;

Field Of Science
1101 - Virology;

Keywords

honey bee

apis mellifera

virus

innate immunity

host-virus interactions

rna interference

dsrna

Goals / Objectives
Objectives and HypothesesTo elucidate the impact of pathogens, including viruses, on honey bee health, this project includes colony level studies that will identify the biotic factors (i.e., viruses, microsporidia, mites, bacteria, trypanosomatids) that have the greatest impact on colony health, and studies using individual bees and cultured cells that will further our understanding of honey bee antiviral defense mechanisms.Objective 1. Identify the pathogen(s) (i.e., viruses, microsporidia, mites, bacteria) that most influence honey bee colony health by performing longitudinal monitoring of colony health, coupled with quantitative assessment of pathogen incidence/abundance.Hypothesis 1. We hypothesize pathogens, including viruses, play a major role in colony health and that the prevalence and/or abundance of particular pathogens will correlate with colony health. Objective 2. Determine the mechanism(s) of RNA-mediated antiviral responses in honey bees and examine the effects of exogenous dsRNA on honey bee gene expression. Hypothesis 2. We hypothesize that viral dsRNA triggered pathways, including RNAi and as yet uncharacterized ns-dsRNA mediated immune response (ns-dsRNA-IR), are the primary mechanisms of honey bee antiviral defense and that many of the genes involved in these antiviral defense pathways are transcriptionally regulated. Objective 3. Develop a honey bee virus - cell culture system for future investigation of honey bee host-pathogen interactions and viral pathogenesis.We expect that carrying out the project objectives, which are listed above and described in detail below, will result in a better understanding of the impacts of viruses on honey bee health and may lead to the development of strategies that minimize honey bee colony losses and/or improve colony health.

Project Methods
Methods - Objective 1Longitudinal monitoring/sampling of commercially managed honey bee coloniesAt the onset of the study, each beekeeper will identify weak (< 5 frames), average (6-8 frames), and strong colonies (> 9 frames) to participate in the study. Live honey bee samples (~ 100 per sample) will be obtained from the top of the frames in the middle of the colony. Samples will be composed of female bees of mixed age, including nurse, worker, and forager bees. The samples will be stored frozen (-80ºC) prior to analysis.Honey bee samplesTen female bees from each sample will be used for RNA extraction, cDNA synthesis, pathogen-specific PCR, and qPCR 40. The objective for pathogen screening in our study was to identify the most prevalent pathogens associated with honey bees sampled from individual colonies at each sampling event. Based on empirical data, literature values, and practical sample handling considerations, we assayed five bees per colony per sampling event. The following equation from Pirk et al. 2013, N = ln(1-D) / ln(1-P) (N=sample size, ln=natural logarithm, D=probability of detection, P=proportion of infected bees) predicts that with a sample size of ten bees, pathogenic infections affecting 25% or more of the individuals within a colony would be detected with 95% probability 83; this sample size has proven sufficient for the pathogen-specific PCR detection of highly prevalent pathogens 40,84.RNA isolationBee samples will be homogenized in 800 mL sterile H2O with sterile beads (3 mm) using a TissueLyzer (Qiagen) at 30 Hz for 2 min. Bee samples were centrifuged for 12 min at 12,000xg at 4°C to pellet debris, and supernatants were transferred to new 1.5 mL tubes containing an equal volume of Trizol reagent (Life Technologies). RNA will be extracted according to the manufacturer's instructions and suspended in sterile water.Reverse transcription / cDNA synthesiscDNA synthesis reactions will be performed by incubating 1-2 mg total RNA, M-MLV reverse-transcriptase (Promega) and 500 ng random hexamer primers (IDT) for 1 hour at 37ºC, according to the manufacturer's instructions.Polymerase Chain Reaction (PCR)PCR will be performed according to standard methods 48,85-87. In brief, 2 ml cDNA template will be combined with 10 pmol of each forward and reverse primer, and amplified with ChoiceTaq polymerase (Denville) according to the manufacturer's instructions using the following cycling conditions: 95°C for 5 min; 35 cycles of 95°C for 30 s, 57°C for 30 s, and 72°C for 30s, followed by final elongation at 72°C for 4 min. The PCR products will be visualized by gel electrophoresis/fluorescence imaging. To minimize the number of negative PCR-tests, we will pool samples (<10) for initial PCR analysis and then assess individual samples for each pathogen detected in pooled analysis. Positive and negative controls will be included for each set of reactions.Quantitative PCR (qPCR)Quantitative PCR will be utilized to analyze the relative abundance of the most prevalent pathogens in select samples to investigate the relationship between pathogen abundance and honey bee colony health. Two micrograms of RNA from each of these samples will be reverse transcribed with M-MLV as described above. All qPCR reactions will be performed in triplicate with a CFX Connect Real Time instrument (BioRad) and the following reaction conditions: 2 μL of cDNA template in 20 μL reactions containing 1X ChoiceTaq Mastermix (Denville), 0.4 µM each forward and reverse primer, 1X SYBR Green (Life Technologies), and 3 mM MgCl2. The qPCR thermo-profile consists of a single pre-incubation 95°C (1 min), 40 cycles of 95°C (10 s), 58°C (20 s), and 72°C (15 s). Plasmid standards, containing 109 to 103 copies per reaction, will be used as qPCR templates to assess primer efficiency and quantify the relative abundance of each pathogen.Methods - Objective 2 (in addition to those described above).Honey bees. Honey bees (Apis mellifera) for caged-bee studies will be obtained from honey bee colonies maintained in Montana State University's Honey Bee Research Site and Pollinator Garden. Female worker bees (all half-sisters) that emerge from a frame of brood in the laboratory will be collected daily (~ 24 hours (hrs) post-emergence). Bees will be housed in modified deli-containers at 30ºC and fed water and sucrose during the experiments55,107.Honey bee virus-infections. SINV-GFP (3,750 PFUs) will be diluted in buffer (2 µl of 10 mM Tris, pH 7.5) and injected into the thorax using a Harbo syringe equipped with disposable borosilicate needles made from capillary tubes using a micropipette puller (Narishige Model PC-10). Honey bees will be immobilized via incubation at 4ºC for 20 minutes and stabilized with insect pins and forceps during injection; after injection, bees recover at room temperature within five minutes.dsRNA preparation. dsRNA will be produced by in vitro transcription with T7 RNA polymerase (as described in 55). In brief, T7 promoter-containing dsDNA PCR-products will be used as templates for T7 polymerase transcription reactions for 10 hrs at 37ºC. DNA templates will be removed by DNAse treatment, and ssRNA products will be annealed; dsRNA quality and quantity will be assessed by agarose gel electrophoresis and spectrophotometry (NanoDrop 2000c).Sample collection and storage. Individual adult worker bees (n=10 per replicate, per time-point post-infection) from all treatment groups will be collected at 6, 48, and 72 hpi and immediately stored at -80°C for subsequent sample homogenization to obtain cellular lysates for Western blot analyses and RNA extraction.Cell culture.Commercially available insect cell lines Drosophila melanogaster S2 cell line (Invitrogen) and the Aedes albopictus C6/36 mosquito cell line (ATCC) will be propagated in Schneider.s Drosophila Medium and Leibovitz's L-15, respectively at 30°C. Honey bee cells will be cultured at 30°C with 5% CO2 and approximately 50% humidity as described in {Goblirsch:2013ba, Bergem:2006dy, Barbara:2008kg, Kreissl:1992wj, CarrilloTripp:2016ey}.Transfection of cultured cells.Effectene transfection reagent (Qiagen) is used to transfect cells with virus. Briefly, cells are seeded in 96 well plates at a density of 1.5 X 104 cells in 100 µl medium the day prior to the experiment. The LSV2 isolate (standardized inoculum amount that is equivalent to 5 ng of virus genome/transcript, which has an estimated 1.46 X 108 viral genomes per µl) or an equal volume of PBS control is transfected into each well according to the manufacturer's directions for DNA. At 0, 4, 24, 48, 72, and 96 hrs post-transfection, cells will be harvested and RNA extracted using Trizol® (Invitrogen).Virus infection of cultured cells.Cells will be seeded in 96 well plates at a density of 1.5 X 104 cells in 100 µl medium the day before introducing virus preparations (5 ng RNA equivalent), or an equal volume of PBS, into cell culture medium, and then harvesting as described above.Reverse-transcription / cDNA synthesis / qPCR. cDNA synthesis reactions will be performed by incubating 100 - 600 ng total RNA, SuperScript III (Life Technologies), and 500 ng random hexamer primers (IDT) for 10 min at 25°C, then 2 hr at 50°C, according to the manufacturer's instructions. Virus abundance and relative host gene expression will be analyzed by qPCR, using the ΔΔC(t) method and Am rpl8 as the internal control, as described above.

Progress 07/01/17 to 06/30/20

Outputs
Target Audience:The target audiences for Dr. Flenniken's research focused on understanding honey bee host-pathogen interactions, such as determining the role of pathogens (viruses, mites, fungi, and bacteria) in honey bee colony loss/death, include: (1) commercial beekeepers, (2) scientists, (3) small-scale beekeepers, (4) growers of honey bee pollinated crops, (5) college students,(6) students (2nd-12th grade), and (7) members of the public. During this project and reporting period, Dr. Flenniken has given scientific presentations, presentations to beekeeping organizations, and engaged with the public in a range of formats including presentations and field events. Flenniken Invited Seminars / Presentations (October 1, 2019- June 30, 2020) 1. Institute on Ecosystems Seminar Series, Seminar Speaker, "Honey Bee Virus Ecology, Colony Health, and Antiviral Defense", March 4, 2020, Bozeman, MT. 2. Science, Technology, Ethics, and Society Seminar Series, Seminar Speaker, "What's Killing the Bees? The Impacts of Pathogens and other Factors on Honey Bees", March 5, 2020, Bozeman, MT. 3. Viruses 2020 - Novel Concepts in Virology, Invited Speaker, "Honey Bee Viruses, Colony Health, and Antiviral Defense", February 5, 2020, Barcelona, Spain. Select Flenniken Lab Student Presentations 1. American Bee Research Conference, Co-author, Speaker Alex McMenamin, Flenniken Lab PhD Student, "The heat shock response is antiviral in honey bees (Apis mellifera)", Jan. 9, 2020, Chicago, IL. 2. American Bee Research Conference, Co-author, Speaker, Fenali Parekh, Flenniken Lab PhD Student, "Investigating strategies to limit virus infection in honey bees", Jan. 9, 2020, Chicago, IL. Flenniken Stakeholder Events and Public Outreach (October 1, 2019- June 30, 2020 2020 Western Region Honey Bee Research Webinar, Invited Speaker, "Honey Bee Host - Virus Interactions at the Colony, Individual Bee, and Cellular Levels", Bozeman, MT (June 29, 2020). 2020 Bee Informed Bee Informed Partnership Colloquium Series, Invited Speaker, "Honey Bee Host - Virus Interactions at the Colony, Individual Bee, and Cellular Levels", Bozeman, MT (Feb. 21, 2020). This project resulted in the training of undergraduate and graduate students. Dr. Flenniken has trained/mentored 13 undergraduate students in her laboratory, including MSU Undergraduate Scholars, Howard Hughes Undergraduate Research Fellows, McNair Fellow, one Presidential Research Fellow, and Federal Work-Study Program students. She currently mentors three PhD students (two in Microbiology and two in Plant Sciences), and three students have obtained graduate degrees under the direction of Dr. Flenniken (i.e., one PhD in Microbiology, one Masters in Ecology, and one Masters in Plant Sciences. Dr. Flenniken's teaching appointment includes General Genetics and Virology (both undergraduate and graduate sections). In addition, she engaged with students in different activities including: MSU's Expanding Your Horizons (Girls in STEM Program and Native American Student Faculty Mentor Program) and the Gallatin Valley Farm Fair, where over 900 4th grade Montana students are exposed to agricultural science in 15-minute activity/interactive booths. Flenniken Publications (from 2019 - 2020 don't have exact dates; 2 out of 38 total) 1. Faurot-Daniels#, C., I., Glenny#, W., Daughenbaugh, K.F., McMenamin, A.J., Burkle, L., and Flenniken, M.L., Longitudinal monitoring of honey bee colonies reveals dynamic nature of virus abundance and indicates a negative impact of Lake Sinai virus 2 on colony health, (2020), #equal co-authorship, PLoS ONE, in press. 2. McMenamin, A.J., Daughenbaugh, and Flenniken, M.L. The Heat Shock Response in the Western Honey Bee (Apis mellifera) is Antiviral, (2020), Viruses, 12, 245; doi:10.3390/v12020245. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Dr. Flenniken has trained/mentored 18 undergraduate students in her laboratory, including MSU Undergraduate Scholars, Howard Hughes Undergraduate Research Fellows, McNair Fellow, one Presidential Research Fellow, and Federal Work-Study Program students. She currently mentors three PhD students (two in Microbiology and one in Plant Sciences), and three students have obtained graduate degrees under Dr. Flenniken (i.e., one PhD in Microbiology, one Masters in Ecology, and one Masters in Plant Sciences. All three PhD students in the Flenniken lab presented their research at national/international conferences including the American Bee Research Association Annual Meeting and the American Society for Virology conference. In addition to practicing their scientific presentation skills, these students received input from experts and further developed their scientific networks. How have the results been disseminated to communities of interest?During this project and reporting period, Dr. Flenniken has given scientific presentations, presentations to beekeeping organizations, and engaged with the public in a range of formats including presentations and field events. Flenniken Stakeholder Events and Public Outreach (October 1, 2019- June 30, 2020) 2020 Western Region Honey Bee Research Webinar, Invited Speaker, "Honey Bee Host - Virus Interactions at the Colony, Individual Bee, and Cellular Levels", Bozeman, MT (June 29, 2020). 2020 Bee Informed Bee Informed Partnership Colloquium Series, Invited Speaker, "Honey Bee Host - Virus Interactions at the Colony, Individual Bee, and Cellular Levels", Bozeman, MT (Feb. 21, 2020). Flenniken Invited Seminars / Presentations (October 1, 2019- June 30, 2020) 1. Institute on Ecosystems Seminar Series, Seminar Speaker, "Honey Bee Virus Ecology, Colony Health, and Antiviral Defense", March 4, 2020, Bozeman, MT. 2. Science, Technology, Ethics, and Society Seminar Series, Seminar Speaker, "What's Killing the Bees? The Impacts of Pathogens and other Factors on Honey Bees", March 5, 2020, Bozeman, MT. 3. Viruses 2020 - Novel Concepts in Virology, Invited Speaker, "Honey Bee Viruses, Colony Health, and Antiviral Defense", February 5, 2020, Barcelona, Spain. Select Flenniken Lab Student Presentations 1. American Bee Research Conference, Co-author, Speaker Alex McMenamin, Flenniken Lab PhD Student, "The heat shock response is antiviral in honey bees (Apis mellifera)", Jan. 9, 2020, Chicago, IL. 2. American Bee Research Conference, Co-author, Speaker, Fenali Parekh, Flenniken Lab PhD Student, "Investigating strategies to limit virus infection in honey bees", Jan. 9, 2020, Chicago, IL. What do you plan to do during the next reporting period to accomplish the goals?Throughout the duration of this project Dr. Flenniken advanced understanding of the impact of viruses on honey bee colony health at the colony, individual bee, and cellular levels as part of the MAES project objectives described below. Objectives and Hypotheses Objective 1. Determine the most influential biotic factors (i.e., viruses, microsporidia, mites, bacteria) affecting honey bee colony health by performing longitudinal monitoring of colony health, coupled with quantitative assessment of pathogen incidence/abundance. Hypothesis 1. We hypothesize pathogens, including viruses, play a major role in colony health and that the prevalence and/or abundance of particular pathogens will correlate with colony health. Objective 2. Determine the mechanism(s) of RNA-mediated antiviral responses in honey bees and examine the effects of exogenous dsRNA on honey bee gene expression. Hypothesis 2. We hypothesize that viral dsRNA triggered pathways, including RNAi and as yet uncharacterized ns-dsRNA mediated immune response (ns-dsRNA-IR), are the primary mechanisms of honey bee antiviral defense and that many of the genes involved in these antiviral defense pathways are transcriptionally regulated. Objective 3. Develop a honey bee virus - cell culture system to investigate host-pathogen interactions and viral pathogenesis. Carrying out experiments that addressed the project objectives, which are listed above advanced understanding of the impacts of viruses on honey bee health.

Impacts
What was accomplished under these goals? High annual losses of honey bees, as well as range reductions and local extinctions of wild and native pollinator species, are concerning because bees are important plant pollinators. Approximately one third of the typical Western diet requires bee pollination, and honey bees (Apis mellifera) are the primary pollinators of numerous food crops including fruits, nuts, vegetables, and oil-seeds. Annually, insect-pollinated crops are valued at approximately $175 billion worldwide and $17-18 billion in North America. Research led by Dr. Michelle Flenniken is aimed at improving honey bee colony health and mitigate colony losses by understanding the effects of viruses on bee health. Honey bees are a specialty crop with dual roles in agriculture: honey production and crop pollination. Montana is an important beekeeping state, which ranked 2nd in the US for honey production in 2013 ($30 million value) and provided over 150,000 colonies for pollination services. Objective 1 - We obtained samples from honey bee colonies and performed pathogen-specific PCR and qPCR to determine the presence and abundance of pathogens, respectively. This data is couple to colony health status (using colony population size as a proxy for colony health) to evaluate the impact of pathogens on colony health. Over the duration of this MAES project funding period, research in the Flenniken lab was featured in two peer-reviewed, open-access publications that document the dynamic nature of virus infections in honey bee colonies and indicate that Lake Sinai virus 2 (LSV2) negatively impacts honey bee colony health. Objective 2 - Experiments were carried out in primary honey bee cells that were maintained in culture in the laboratory that validated the importance several genes, including a MF116383, which we determined is important to honey bee antiviral defense in individual bees. Experiments were carried out in laboratory-based studies in which honey bees were infected with a virus, and our results indicate the importance of the heat shock response (a stress response pathway) in honey bee antiviral defense. Objective 3 - We advanced our ability to use primary honey bee cells for virus-infection studies. Over the duration of this MAES project funding period, Flenniken lab members made significant advances in maintaining primary honey bee cells. Specifically, we successfully cultured primary honey bee larval hemocytes and mixed cell populations from honey bee pupae. Initial experiments indicate that several viruses, including honey bee viruses (i.e., deformed wing virus (DWV) and Lake Sinai virus 2 (LSV2) and model viruses (i.e., Flock house virus (FHV) and Sindbis-GFP) replicate in cultured honey bee cells. Specifically, Lake Sinai virus 2 exhibited modest replication in cultured hemocytes within 2-3 days post-infection, and deformed wing virus and Flock house viruses replicate in cultured pupal cells. Future studies will be aimed at investigating "core antiviral immune responses", as well as unique aspects of host - virus interactions at the cellular level.

Publications

• Type: Journal Articles Status: Accepted Year Published: 2020 Citation: Faurot-Daniels#, C., I., Glenny#, W., Daughenbaugh, K.F., McMenamin, A.J., Burkle, L., and Flenniken, M.L., Longitudinal monitoring of honey bee colonies reveals dynamic nature of virus abundance and indicates a negative impact of Lake Sinai virus 2 on colony health, (2020), #equal co-authorship, PLoS ONE, in press.
• Type: Journal Articles Status: Published Year Published: 2020 Citation: McMenamin, A.J., Daughenbaugh, and Flenniken, M.L. The Heat Shock Response in the Western Honey Bee (Apis mellifera) is Antiviral, (2020), Viruses, 12, 245; doi:10.3390/v12020245.

Progress 10/01/18 to 09/30/19

Outputs
Target Audience:The target audiences for Dr. Flenniken's research focused on understanding honey bee host-pathogen interactions, such as determining the role of pathogens (viruses, mites, fungi, and bacteria) in honey bee colony loss/death, include: (1) commercial beekeepers, (2) scientists, (3) small-scale beekeepers, (4) growers of honey bee pollinated crops, (5) college students, (6) students (2nd-12th grade), and (7) members of the public. During this reporting period, Dr. Flenniken has given scientific presentations, presentations to beekeeping organizations, and engaged with the public in a range of formats including presentations and field events. Flenniken Invited Seminars / Presentations (October 1, 2018- September 30, 2019) 1. The Volcani Center- Agricultural Research Organization, Seminar Speaker, "Honey Bee Viruses, Colony Health, and Antiviral Defense", Sept. 18 2019, Tel Aviv, Israel. 2. Apimondia, Speaker, "The impact of viruses on honey bees at the individual, and cellular levels", Sept. 10, 2019, Montreal, Canada. 3. COLOSS Honey Bee Research Organization Virus Task Force Meeting at Apimondia, Speaker, "The impact of viruses on honey bees at the colony, individual, and cellular levels", Sept. 7 2019, Montreal, Canada. 4. Entomological Society of America, Pacific Branch Meeting, Speaker, "The impact of viruses on honey bees at the colony, individual, and cellular levels", April 4 2019, San Diego, CA. 5. American Bee Research Conference, Keynote Speaker, "The impact of viruses on honey bees at the colony, individual, and cellular levels", January 11, 2019, Tempe, AZ. Select Flenniken Lab Student Presentations 1. American Society for Virology, Annual Meeting, Co-author, Speaker Alex McMenamin, Flenniken Lab PhD Student, "AmMF116383 is important for dsRNA mediated reduction of virus infection in honey bees and honey bee immune cells", July 22, 2019, Minneapolis, MN. 2. American Society for Virology, Annual Meeting, Co-author, Speaker Fenali Parekh, Flenniken Lab PhD Student, "Investigating the efficacy of putative antiviral agents to limit virus infection in honey bees", July 22, 2019, Minneapolis, MN. 3. American Bee Research Conference, Co-author, Speaker Alex McMenamin, Flenniken Lab PhD Student, "Honey bee host-pathogen interactions at the colony, individual and cellular levels", Jan. 11, 2019, Tempe, AZ. 4. American Bee Research Conference, Co-author, Poster Presenter, Fenali Parekh, Flenniken Lab PhD Student, "Investigating the impact of honey bee genotype and Varroa destructor mites on virus prevalence and abundance", Jan. 11, 2019, Tempe, AZ. Flenniken Stakeholder Events and Public Outreach (October 1, 2018- September 30, 2019) 2019 Gallatin Valley Farm Fair (over 1,000 4th-5th graders attended this event), Belgrade, MT. Honey Bee / Pollinator Health Exhibit Coordinator; Flenniken and four Flenniken lab graduate students gave presentations, (May 7-9, 2019). 2019 Gallatin Valley Youth Environment Summit (~75 7th graders from the Gallatin Valley), Gallatin Gateway, MT. Honey Bee / Pollinator Health Exhibit April 25, 2019. 2019 Great Plains Diagnostic Network (GPDN) Webinar Series, Invited Speaker, "What's Killing the Bees? The Impacts of Pathogens and other Factors on Honey Bees", Bozeman, MT (January 23, 2019); https://msuextensionconnect.org/ppsctgjynxmb/ 2018 This Week in Virology (TWIV), Invited Guest Speaker on prominent podcast, December 2018. http://www.microbe.tv/twiv/twiv-525/ 2018 Wyoming State Beekeepers' Association Annual Meeting, Invited Speaker, "The impact of viruses on honey bees at the colony, individual, and cellular levels", Casper, Wyoming (December 6, 2018); ~ 30 attendees. 2018 Washington State Beekeepers' Association Annual Meeting, Invited Speaker, "The impact of viruses on honey bees at the colony, individual, and cellular levels", Spokane Washington (November 16, 2018); 150 attendees. 2018 Oregon State / Regional Beekeepers' Association Annual Meeting, Invited Speaker, "Honey Bee Colony Health", Salem, Oregon, (October 27, 2018); ~ 250 attendees. 2018 Montana State Beekeepers' Association Annual Meeting, Invited Speaker, "Honey Bee Colony Health", Bozeman, MT (October 18, 2018); ~ 70 attendees. 2018 MUS Science Roadshow Hi-Line Tour, seven presentations, Oct. 3-6, 2018. 2018 Produced a three-part video series about (1) honey bee research at MSU in the Flenniken Lab, (2) honey bee pathogens, and (3) honey bee pathogen detection (videos 1, 2, and 3, respectively). See: http://www.montana.edu/pollinators/resources.html Changes/Problems:n/a What opportunities for training and professional development has the project provided?Dr. Flenniken has trained/mentored 18 undergraduate students in her laboratory, including MSU Undergraduate Scholars, Howard Hughes Undergraduate Research Fellows, McNair Fellow, one Presidential Research Fellow, and Federal Work-Study Program students. She currently mentors three PhD students (two in Microbiology and one in Plant Sciences), and three students have obtained graduate degrees under Dr. Flenniken (i.e., one PhD in Microbiology, one Masters in Ecology, and one Masters in Plant Sciences. All three PhD students in the Flenniken lab presented their research at national/international conferences including the American Bee Research Association Annual Meeting and the American Society for Virology conference. In addition to practicing their scientific presentation skills, these students received input from experts and further developed their scientific networks. How have the results been disseminated to communities of interest?(see information above on stakeholder presentations) Flenniken Invited Seminars / Presentations (October 1, 2018- September 30, 2019) 1. The Volcani Center- Agricultural Research Organization, Seminar Speaker, "Honey Bee Viruses, Colony Health, and Antiviral Defense", Sept. 18 2019, Tel Aviv, Israel. 2. Apimondia, Speaker, "The impact of viruses on honey bees at the individual, and cellular levels", Sept. 10, 2019, Montreal, Canada. 3. COLOSS Honey Bee Research Organization Virus Task Force Meeting at Apimondia, Speaker, "The impact of viruses on honey bees at the colony, individual, and cellular levels", Sept. 7 2019, Montreal, Canada. 4. Entomological Society of America, Pacific Branch Meeting, Speaker, "The impact of viruses on honey bees at the colony, individual, and cellular levels", April 4 2019, San Diego, CA. 5. American Bee Research Conference, Keynote Speaker, "The impact of viruses on honey bees at the colony, individual, and cellular levels", January 11, 2019, Tempe, AZ. Select Flenniken Lab Student Presentations 1. American Society for Virology, Annual Meeting, Co-author, Speaker Alex McMenamin, Flenniken Lab PhD Student, "AmMF116383 is important for dsRNA mediated reduction of virus infection in honey bees and honey bee immune cells", July 22, 2019, Minneapolis, MN. 2. American Society for Virology, Annual Meeting, Co-author, Speaker Fenali Parekh, Flenniken Lab PhD Student, "Investigating the efficacy of putative antiviral agents to limit virus infection in honey bees", July 22, 2019, Minneapolis, MN. 3. American Bee Research Conference, Co-author, Speaker Alex McMenamin, Flenniken Lab PhD Student, "Honey bee host-pathogen interactions at the colony, individual and cellular levels", Jan. 11, 2019, Tempe, AZ. 4. American Bee Research Conference, Co-author, Poster Presenter, Fenali Parekh, Flenniken Lab PhD Student, "Investigating the impact of honey bee genotype and Varroa destructor mites on virus prevalence and abundance", Jan. 11, 2019, Tempe, AZ. Flenniken Stakeholder Events and Public Outreach (October 1, 2018- September 30, 2019) 2019 Gallatin Valley Farm Fair (over 1,000 4th-5th graders attended this event), Belgrade, MT. Honey Bee / Pollinator Health Exhibit Coordinator; Flenniken and four Flenniken lab graduate students gave presentations, (May 7-9, 2019). 2019 Gallatin Valley Youth Environment Summit (~75 7th graders from the Gallatin Valley), Gallatin Gateway, MT. Honey Bee / Pollinator Health Exhibit April 25, 2019. 2019 Great Plains Diagnostic Network (GPDN) Webinar Series, Invited Speaker, "What's Killing the Bees? The Impacts of Pathogens and other Factors on Honey Bees", Bozeman, MT (January 23, 2019); https://msuextensionconnect.org/ppsctgjynxmb/ 2018 This Week in Virology (TWIV), Invited Guest Speaker on prominent podcast, December 2018. http://www.microbe.tv/twiv/twiv-525/ 2018 Wyoming State Beekeepers' Association Annual Meeting, Invited Speaker, "The impact of viruses on honey bees at the colony, individual, and cellular levels", Casper, Wyoming (December 6, 2018); ~ 30 attendees. 2018 Washington State Beekeepers' Association Annual Meeting, Invited Speaker, "The impact of viruses on honey bees at the colony, individual, and cellular levels", Spokane Washington (November 16, 2018); 150 attendees. 2018 Oregon State / Regional Beekeepers' Association Annual Meeting, Invited Speaker, "Honey Bee Colony Health", Salem, Oregon, (October 27, 2018); ~ 250 attendees. 2018 Montana State Beekeepers' Association Annual Meeting, Invited Speaker, "Honey Bee Colony Health", Bozeman, MT (October 18, 2018); ~ 70 attendees. 2018 MUS Science Roadshow Hi-Line Tour, seven presentations, Oct. 3-6, 2018. 2018 Produced a three-part video series about (1) honey bee research at MSU in the Flenniken Lab, (2) honey bee pathogens, and (3) honey bee pathogen detection (videos 1, 2, and 3, respectively). See: http://www.montana.edu/pollinators/resources.html What do you plan to do during the next reporting period to accomplish the goals?Dr. Flenniken will continue her research as part of an individual MAES project with the objectives described below. Objectives and Hypotheses To elucidate the impact of pathogens, including viruses, on honey bee health, this project includes colony level studies that will identify the biotic factors (i.e., viruses, microsporidia, mites, bacteria, trypanosomatids) that have the greatest impact on colony health, and studies using individual bees and cultured cells that will further our understanding of honey bee antiviral defense mechanisms. Objective 1. Determine the most influential biotic factors (i.e., viruses, microsporidia, mites, bacteria) affecting honey bee colony health by performing longitudinal monitoring of colony health, coupled with quantitative assessment of pathogen incidence/abundance. Hypothesis 1. We hypothesize pathogens, including viruses, play a major role in colony health and that the prevalence and/or abundance of particular pathogens will correlate with colony health. Objective 2. Determine the mechanism(s) of RNA-mediated antiviral responses in honey bees and examine the effects of exogenous dsRNA on honey bee gene expression. Hypothesis 2. We hypothesize that viral dsRNA triggered pathways, including RNAi and as yet uncharacterized ns-dsRNA mediated immune response (ns-dsRNA-IR), are the primary mechanisms of honey bee antiviral defense and that many of the genes involved in these antiviral defense pathways are transcriptionally regulated. Objective 3. Develop a honey bee virus - cell culture system to investigate host-pathogen interactions and viral pathogenesis. We expect that carrying out the project objectives, which are listed above and described in detail below, will result in a better understanding of the impacts of viruses on honey bee health and may lead to the development of strategies that minimize honey bee colony losses and/or improve colony health.

Impacts
What was accomplished under these goals? Objective 1 - We obtained samples from honey bee colonies and performed pathogen-specific PCR and qPCR to determine the presence and abundance of pathogens, respectively. This data is couple to colony health status (using colony population size as a proxy for colony health) to evaluate the impact of pathogens on colony health. Objective 2 - Experiments were carried out in primary honey bee cells that were maintained in culture in the laboratory that validated the importance a gene (i.e., MF116383) that we determined is important to honey bee antiviral defense in individual bees. Experiments were carried out in laboratory-based studies in which honey bees were infected with a virus, and our results indicate the importance of the heat shock response (a stress response pathway) in honey bee antiviral defense. Objective 3 - We advanced our ability to use primary honey bee cells for virus-infection studies. Specifically, we successfully cultured primary honey bee larval hemocytes and mixed cell populations from honey bee pupae. In cultured hemocytes Lake Sinai virus 2, a common honey bee infecting virus, exhibited modest replication within 2-3 days post-infection.

Publications

• Type: Journal Articles Status: Published Year Published: 2019 Citation: Grozinger, C.M. and Flenniken, M.L. (2019) Bee Viruses: Ecology, Pathogenicity, and Impacts. Annual Review of Entomology Vol. 64:205-226, doi.org/10.1146/annurev-ento-011118-111942
• Type: Journal Articles Status: Published Year Published: 2018 Citation: McMenamin, A., Daughenbaugh, K.D., Parekh, F., Pizzorno, M.C., and Flenniken, M.L. Honey Bee and Bumble Bee Antiviral Defense, (2018), Viruses, 10(8), 395; doi:10.3390/v10080395. Flenniken, M.L., Antiviral Defense in Invertebrates (2018), Viruses, 10, 403; doi:10.3390/v10080403.
• Type: Journal Articles Status: Published Year Published: 2018 Citation: McMenamin, A. and Flenniken, M.L. (2018). Recently identified bee viruses and their impact on bee pollinators, Current Opinion in Insect Science, 26:120129 https://doi.org/10.1016/j.cois.2018.02.009.
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Piccolomini, A. M., Whiten, S.R., Flenniken, M.L., O'Neill, K., Peterson, R. Acute Toxicity of Permethrin, Deltamethrin, and Etofenprox to the Alfalfa Leafcutting Bee, (2018) 1-5. Journal of Economic Entomology, doi: 10.1093/jee/toy014.

Progress 10/01/17 to 09/30/18

Outputs
Target Audience:Target audience(s) reached: The target audiences for Dr. Flenniken's research focused on understanding honey bee host-pathogen interactions, such as determining the role of pathogens (viruses, mites, fungi, and bacteria) in honey bee colony loss/death, include: (1) commercial beekeepers, (2) scientists, (3) small-scale beekeepers, (4) growers of honey bee pollinated crops, (5) college students, (6) students (2nd-12th grade), and (7) members of the public. During this reporting period, Dr. Flenniken has given scientific presentations, presentations to beekeeping organizations, and engaged with the public in a range of formats including presentations and field events. Flenniken Invited Seminars / Presentations (June 30, 2018- September 30, 2018) 1. Universita? degli Studi di Udine, Invited Speaker, "Host vs. Virus: The Case of Honey Bees", April 13, 2018, Udine, Italy. 2. Entomological Society of America, Invited Speaker, "Honey Bee Viruses, Colony Health, and Antiviral Defense", November 7 2017, Denver, CO. 3. Society for Invertebrate Pathology of America, Invited Speaker, "Bee Viruses and Honey Bee Health", August 2017, San Diego, CA Select Flenniken Lab Student Presentations 1. American Society for Virology, Annual Meeting, Co-author, Speaker Alex McMenamin, Flenniken Lab PhD Student, "Honey bee host-pathogen interactions at the colony, individual and cellular levels", July 26, 2018, College Park, MD. 2. American Bee Research Conference, Co-author, Speaker Alex McMenamin, Flenniken Lab PhD Student, "Honey bee host-pathogen interactions at the colony, individual and cellular levels", Jan. 12, 2018, Reno, NV. Flenniken Stakeholder Events and Public Outreach (June 30, 2018- September 30, 2018) 2018 Pollinator Symposium, Flenniken Lab Graduate Students (i.e., Fenali Parekh, Alex McMenamin, and Sandra Barroso Arevelo) gave short presentations to the public on their research efforts aimed at understanding the role of pathogens on honey bee health at both the colony and individual bee levels, Bozeman, MT (Aug. 9, 2018); ~ 50 community members. 2018 Big Sky Gallatin Country Women's Club (WGWC), Invited Speaker, "Honey Bee Research at MSU", Big Sky, MT (June 13, 2018); ~ 60 community members. 2018 Expanding Your Horizons, Flenniken lab students (i.e., Vanessa Orcutt and Marie Pizzorno) interacted with 6th-8th grade girls interested in STEM, Bozeman, MT (April 21, 2018); ~ 60 students. 2018 Science Inquiry Lecture Series sponsored by Gallatin Valley Friends of the Sciences, Speaker, "Honey Bee Health and Your Health", Bozeman, MT (March 21, 2018), approximately 180 community members. 2018 Honey Bee Pathogens sponsored by Gallatin Valley Beekeeping Club, Speaker, "Honey Bee Pathogens", Bozeman, MT (March 10, 2018), 75 attendees. 2018 Beekeeping Master Course University of British Columbia, Invited Speaker, "Bee Pathogens 501", and, "Honey Bee Pathogen and Pathway Discovery", Vancouver, BC, Canada, (February 2018). 2018 Montana State University Ag Connects, Speaker, "Honey Bee Health and Your Health", Bozeman, MT (January 15, 2018). 2017 Montana State Beekeepers' Association Annual Meeting, Invited Speaker, "Honey Bee Colony Health", Bozeman, MT (October 20, 2017). 2017 National Resource Conservation Service (NRCS) Pollinator Plantings and Monitoring meeting, discussion and tour of MSU's Honey Bee Research Site and Pollinator Garden (July 10, 2017); ~ 15 people. Dr. Flenniken is an Assistant Professor in the Department of Plant Sciences and Plant Pathology (PSPP) at Montana State University with a 60% Research, 30% Teaching, and 10% Service appointment. Dr. Flenniken investigates honey bee host-pathogen interactions; research projects include (1) determining the mechanisms and contributions of RNA-triggered pathways in honey bee antiviral defense, (2) honey bee pathogen monitoring, detection and discovery, and (3) investigating the pathogenesis of the recently discovered Lake Sinai viruses. In addition, and in collaboration with Dr. Nina Zidak, Dr. Flenniken is examining differential susceptibly and immune responses of several potato cultivar and seed sources to Potato virus Y (PVY). Target audience(s) reached: The target audiences for Dr. Flenniken's research focused on understanding honey bee host-pathogen interactions, such as determining the role of pathogens (viruses, mites, fungi, and bacteria) in honey bee colony loss/death, include: (1) commercial beekeepers, (2) scientists, (3) small-scale beekeepers, (4) growers of honey bee pollinated crops, (5) college students, (6) students (2nd-12th grade), and (7) members of the public. During this reporting period, Dr. Flenniken has given scientific presentations, presentations to beekeeping organizations, and engaged with the public in a range of formats including presentations and field events. Flenniken Invited Seminars / Presentations (June 30, 2018- September 30, 2018) 1. Universita? degli Studi di Udine, Invited Speaker, "Host vs. Virus: The Case of Honey Bees", April 13, 2018, Udine, Italy. 2. Entomological Society of America, Invited Speaker, "Honey Bee Viruses, Colony Health, and Antiviral Defense", November 7 2017, Denver, CO. 3. Society for Invertebrate Pathology of America, Invited Speaker, "Bee Viruses and Honey Bee Health", August 2017, San Diego, CA Select Flenniken Lab Student Presentations 1. American Society for Virology, Annual Meeting, Co-author, Speaker Alex McMenamin, Flenniken Lab PhD Student, "Honey bee host-pathogen interactions at the colony, individual and cellular levels", July 26, 2018, College Park, MD. 2. American Bee Research Conference, Co-author, Speaker Alex McMenamin, Flenniken Lab PhD Student, "Honey bee host-pathogen interactions at the colony, individual and cellular levels", Jan. 12, 2018, Reno, NV. Flenniken Stakeholder Events and Public Outreach (June 30, 2018- September 30, 2018) 2018 Pollinator Symposium, Flenniken Lab Graduate Students (i.e., Fenali Parekh, Alex McMenamin, and Sandra Barroso Arevelo) gave short presentations to the public on their research efforts aimed at understanding the role of pathogens on honey bee health at both the colony and individual bee levels, Bozeman, MT (Aug. 9, 2018); ~ 50 community members. 2018 Big Sky Gallatin Country Women's Club (WGWC), Invited Speaker, "Honey Bee Research at MSU", Big Sky, MT (June 13, 2018); ~ 60 community members. 2018 Expanding Your Horizons, Flenniken lab students (i.e., Vanessa Orcutt and Marie Pizzorno) interacted with 6th-8th grade girls interested in STEM, Bozeman, MT (April 21, 2018); ~ 60 students. 2018 Science Inquiry Lecture Series sponsored by Gallatin Valley Friends of the Sciences, Speaker, "Honey Bee Health and Your Health", Bozeman, MT (March 21, 2018), approximately 180 community members. 2018 Honey Bee Pathogens sponsored by Gallatin Valley Beekeeping Club, Speaker, "Honey Bee Pathogens", Bozeman, MT (March 10, 2018), 75 attendees. 2018 Beekeeping Master Course University of British Columbia, Invited Speaker, "Bee Pathogens 501", and, "Honey Bee Pathogen and Pathway Discovery", Vancouver, BC, Canada, (February 2018). 2018 Montana State University Ag Connects, Speaker, "Honey Bee Health and Your Health", Bozeman, MT (January 15, 2018). 2017 Montana State Beekeepers' Association Annual Meeting, Invited Speaker, "Honey Bee Colony Health", Bozeman, MT (October 20, 2017). 2017 National Resource Conservation Service (NRCS) Pollinator Plantings and Monitoring meeting, discussion and tour of MSU's Honey Bee Research Site and Pollinator Garden (July 10, 2017); ~ 15 people. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project resulted in the training of undergraduate and graduate students. Dr. Flenniken has trained/mentored 13 undergraduate students in her laboratory, including MSU Undergraduate Scholars, Howard Hughes Undergraduate Research Fellows, McNair Fellow, one Presidential Research Fellow, and Federal Work-Study Program students. She currently mentors three PhD students (two in Microbiology and one in Plant Sciences), and three students have obtained graduate degrees under the direction of Dr. Flenniken (i.e., one PhD in Microbiology, one Masters in Ecology, and one Masters in Plant Sciences. Dr. Flenniken's teaching appointment includes General Genetics and Virology (both undergraduate and graduate sections). In addition, she engaged with students in different activities including: MSU's Expanding Your Horizons (Girls in STEM Program and Native American Student Faculty Mentor Program) and the Gallatin Valley Farm Fair, where over 900 4th grade Montana students are exposed to agricultural science in 15-minute activity/interactive booths. How have the results been disseminated to communities of interest?This project resulted in the training of undergraduate and graduate students. Dr. Flenniken has trained/mentored 13 undergraduate students in her laboratory, including MSU Undergraduate Scholars, Howard Hughes Undergraduate Research Fellows, McNair Fellow, one Presidential Research Fellow, and Federal Work-Study Program students. She currently mentors three PhD students (two in Microbiology and one in Plant Sciences), and three students have obtained graduate degrees under the direction of Dr. Flenniken (i.e., one PhD in Microbiology, one Masters in Ecology, and one Masters in Plant Sciences. Dr. Flenniken's teaching appointment includes General Genetics and Virology (both undergraduate and graduate sections). In addition, she engaged with students in different activities including: MSU's Expanding Your Horizons (Girls in STEM Program and Native American Student Faculty Mentor Program) and the Gallatin Valley Farm Fair, where over 900 4th grade Montana students are exposed to agricultural science in 15-minute activity/interactive booths. What do you plan to do during the next reporting period to accomplish the goals?During this reporting period, Dr. Flenniken has given scientific presentations, presentations to beekeeping organizations, and engaged with the public in a range of formats including presentations and field events. Flenniken Invited Seminars / Presentations (June 30, 2018- September 30, 2018) 1. Universita? degli Studi di Udine, Invited Speaker, "Host vs. Virus: The Case of Honey Bees", April 13, 2018, Udine, Italy. 2. Entomological Society of America, Invited Speaker, "Honey Bee Viruses, Colony Health, and Antiviral Defense", November 7 2017, Denver, CO. 3. Society for Invertebrate Pathology of America, Invited Speaker, "Bee Viruses and Honey Bee Health", August 2017, San Diego, CA Select Flenniken Lab Student Presentations 1. American Society for Virology, Annual Meeting, Co-author, Speaker Alex McMenamin, Flenniken Lab PhD Student, "Honey bee host-pathogen interactions at the colony, individual and cellular levels", July 26, 2018, College Park, MD. 2. American Bee Research Conference, Co-author, Speaker Alex McMenamin, Flenniken Lab PhD Student, "Honey bee host-pathogen interactions at the colony, individual and cellular levels", Jan. 12, 2018, Reno, NV. Flenniken Stakeholder Events and Public Outreach (June 30, 2018- September 30, 2018) 2018 Pollinator Symposium, Flenniken Lab Graduate Students (i.e., Fenali Parekh, Alex McMenamin, and Sandra Barroso Arevelo) gave short presentations to the public on their research efforts aimed at understanding the role of pathogens on honey bee health at both the colony and individual bee levels, Bozeman, MT (Aug. 9, 2018); ~ 50 community members. 2018 Big Sky Gallatin Country Women's Club (WGWC), Invited Speaker, "Honey Bee Research at MSU", Big Sky, MT (June 13, 2018); ~ 60 community members. 2018 Expanding Your Horizons, Flenniken lab students (i.e., Vanessa Orcutt and Marie Pizzorno) interacted with 6th-8th grade girls interested in STEM, Bozeman, MT (April 21, 2018); ~ 60 students. 2018 Science Inquiry Lecture Series sponsored by Gallatin Valley Friends of the Sciences, Speaker, "Honey Bee Health and Your Health", Bozeman, MT (March 21, 2018), approximately 180 community members. 2018 Honey Bee Pathogens sponsored by Gallatin Valley Beekeeping Club, Speaker, "Honey Bee Pathogens", Bozeman, MT (March 10, 2018), 75 attendees. 2018 Beekeeping Master Course University of British Columbia, Invited Speaker, "Bee Pathogens 501", and, "Honey Bee Pathogen and Pathway Discovery", Vancouver, BC, Canada, (February 2018). 2018 Montana State University Ag Connects, Speaker, "Honey Bee Health and Your Health", Bozeman, MT (January 15, 2018). 2017 Montana State Beekeepers' Association Annual Meeting, Invited Speaker, "Honey Bee Colony Health", Bozeman, MT (October 20, 2017). 2017 National Resource Conservation Service (NRCS) Pollinator Plantings and Monitoring meeting, discussion and tour of MSU's Honey Bee Research Site and Pollinator Garden (July 10, 2017); ~ 15 people.

Impacts
What was accomplished under these goals? To generate and disseminate objective, scientifically-based knowledge about plant sciences, including plant pathology, plant genetics, crop science, plant biology and horticulture. * What was accomplished under these goals? High annual losses of honey bees, as well as range reductions and local extinctions of wild and native pollinator species, are concerning because bees are important plant pollinators. Approximately one third of the typical Western diet requires bee pollination, and honey bees (Apis mellifera) are the primary pollinators of numerous food crops including fruits, nuts, vegetables, and oil-seeds. Annually, insect-pollinated crops are valued at approximately $175 billion worldwide and $17-18 billion in North America, Research led by Dr. Michelle Flenniken is aimed at improving honey bee colony health and mitigate colony losses by understanding the effects of viruses on bee health. Honey bees are a specialty crop with dual roles in agriculture: honey production and crop pollination. Montana is an important beekeeping state, which ranked 2nd in the US for honey production in 2013 ($30 million value) and provided over 150,000 colonies for pollination services. (for sharing of scientific based knowledge - see below)

Publications

• Type: Journal Articles Status: Published Year Published: 2018 Citation: McMenamin, A., Daughenbaugh, K.D., Parekh, F., Pizzorno, M.C., and Flenniken, M.L. Honey Bee and Bumble Bee Antiviral Defense, (2018), Viruses, 10(8), 395; doi:10.3390/v10080395. Flenniken, M.L., Antiviral Defense in Invertebrates (2018), Viruses, 10, 403; doi:10.3390/v10080403.
• Type: Journal Articles Status: Published Year Published: 2018 Citation: McMenamin, A. and Flenniken, M.L. (2018). Recently identified bee viruses and their impact on bee pollinators, Current Opinion in Insect Science, 26:120129 https://doi.org/10.1016/j.cois.2018.02.009

Progress 07/01/17 to 09/30/17

Outputs
Target Audience:The target audiences for Dr. Flenniken's research focused on understanding honey bee host-pathogen interactions, such as determining the role of pathogens (viruses, mites, fungi, and bacteria) in honey bee colony loss/death, include: (1) commercial beekeepers, (2) scientists, (3) small-scale beekeepers, (4) growers of honey bee pollinated crops, (5) college students, (6) students (2nd-12th grade), and (7) members of the public. During this reporting period, Dr. Flenniken has given seven scientific presentations, four presentations to beekeeping organizations, and engaged with the public five times in a range of formats including presentations and field events. Flenniken Invited Seminars / Presentations (2016-2017) 1. Society for Invertebrate Pathology of America, Pacific Branch Meeting, Invited Speaker, "Bee Viruses and Honey Bee Health", August 2017, San Diego, CA 2. Entomological Society of America, Pacific Branch Meeting, Speaker, "Honey Bee RNA-mediated antiviral responses", April 4 2017, Portland, Oregon. 3. Montana Tech, Speaker, "Honey Bee Pathogen and Immune Pathway Discovery", Nov. 17 2016, Butte, MT Select Flenniken Lab Student Presentations 1. American Society for Virology, Annual Meeting, Co-author, Speaker Alex McMenamin, Flenniken Lab PhD Student, "Transcriptional level characterization of honey bee antiviral defense mechanisms at the individual bee and cellular levels", June 24, 2017, Madison, WI. 2. American Bee Research Conference, Co-author, Speaker Laura Brutscher, Flenniken Lab PhD Student, "Honey Bee Transcriptional Response to Virus Infection", Jan. 8th 2016, Ponte Verde, Florida. Flenniken Stakeholder Events and Public Outreach 2017 Montana State Beekeepers' Association Annual Meeting, Invited Speaker, "Honey Bee Colony Health", Bozeman, MT (October 20, 2017); Flenniken Lab Tour October 21, 2017. 2017 Gallatin Gardeners Club, Invited Speaker, "Honey Bee Health", Bozeman, MT (May 1, 2017); ~ 100 community members. 2017 Gallatin Valley Farm Fair (over 1,000 4th-5th graders attended this event), Belgrade, MT. Honey Bee / Pollinator Health Exhibit Coordinator; Flenniken and four Flenniken lab graduate students gave presentations, (May 9-11, 2017). 2017 Bozeman High School Biomed Students Lab Visit, Michelle Flenniken interacted with 11th and 12th grade students interested in careers in STEM fields, Bozeman, MT (April 21, 2017); ~ 20students. 2017 Expanding Your Horizons, Flenniken lab students (i.e., Laura Brutscher and Shantelle Smolik) interacted with 6th-8th grade girls interested in STEM, Bozeman, MT (April 22, 2017); ~ 60 students. 2017 Pollinator Symposium, Flenniken Lab Graduate Students (i.e., Laura Brutscher, Alex McMenamin, and Will Glenny) gave short presentations to the public on their research efforts aimed at understanding the role of pathogens on honey bee health at both the colony and individual bee levels, Bozeman, MT (April 19, 2017); ~ 50 community members. 2017 INBRE, Café Scientifique Invited Speaker, "What's Killing the Bees? The Impact of Pathogens and Other Factors on Honey Bee Colony Health", Bozeman, MT (March 28, 2017); ~ 100 community members. 2017 MSU's Science Roadshow 10x10 Talks, Invited Presenter, "Honey Bee Health and Your Health", Bozeman, MT, ~ 450 community members, (March 23, 2017) 2016 Museum of the Rockies, Invited Speaker, "Honey Bee Pathogens and Colony Health", Bozeman, MT (November 15, 2016). 2016 Alberta Beekeepers' Association Annual Meeting, Invited Speaker, "Honey Bee Pathogens and Colony Health", Edmonton, Alberta, Canada (November 8, 2016). 2016 Montana State Beekeepers' Association Annual Meeting, Invited Speaker, "Honey Bee Colony Health", Whitefish, MT (October 20, 2016). Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Dr. Flenniken has trained/mentored 13 undergraduate students in her laboratory, including MSU Undergraduate Scholars, Howard Hughes Undergraduate Research Fellows, McNair Fellow, one Presidential Research Fellow, and Federal Work-Study Program students. She currently mentors two PhD students (one in Microbiology and one in Plant Sciences), and three students have obtained graduate degrees under Dr. Flenniken (i.e., one PhD in Microbiology, one Masters in Ecology, and one Masters in Plant Sciences. How have the results been disseminated to communities of interest?Target audience(s) reached: The target audiences for Dr. Flenniken's research focused on understanding honey bee host-pathogen interactions, such as determining the role of pathogens (viruses, mites, fungi, and bacteria) in honey bee colony loss/death, include: (1) commercial beekeepers, (2) scientists, (3) small-scale beekeepers, (4) growers of honey bee pollinated crops, (5) college students, (6) students (2nd-12th grade), and (7) members of the public. During this reporting period, Dr. Flenniken has given seven scientific presentations, four presentations to beekeeping organizations, and engaged with the public five times in a range of formats including presentations and field events. Flenniken Invited Seminars / Presentations (2016-2017) 1. Society for Invertebrate Pathology of America, Pacific Branch Meeting, Invited Speaker, "Bee Viruses and Honey Bee Health", August 2017, San Diego, CA 2. Entomological Society of America, Pacific Branch Meeting, Speaker, "Honey Bee RNA-mediated antiviral responses", April 4 2017, Portland, Oregon. 3. Montana Tech, Speaker, "Honey Bee Pathogen and Immune Pathway Discovery", Nov. 17 2016, Butte, MT. Select Flenniken Lab Student Presentations 1. American Society for Virology, Annual Meeting, Co-author, Speaker Alex McMenamin, Flenniken Lab PhD Student, "Transcriptional level characterization of honey bee antiviral defense mechanisms at the individual bee and cellular levels", June 24, 2017, Madison, WI. 2. American Bee Research Conference, Co-author, Speaker Laura Brutscher, Flenniken Lab PhD Student, "Honey Bee Transcriptional Response to Virus Infection", Jan. 8th 2016, Ponte Verde, Florida. Flenniken Stakeholder Events and Public Outreach 2017 Montana State Beekeepers' Association Annual Meeting, Invited Speaker, "Honey Bee Colony Health", Bozeman, MT (October 20, 2017); Flenniken Lab Tour October 21, 2017. 2017 Gallatin Gardeners Club, Invited Speaker, "Honey Bee Health", Bozeman, MT (May 1, 2017); ~ 100 community members. 2017 Gallatin Valley Farm Fair (over 1,000 4th-5th graders attended this event), Belgrade, MT. Honey Bee / Pollinator Health Exhibit Coordinator; Flenniken and four Flenniken lab graduate students gave presentations, (May 9-11, 2017). 2017 Bozeman High School Biomed Students Lab Visit, Michelle Flenniken interacted with 11th and 12th grade students interested in careers in STEM fields, Bozeman, MT (April 21, 2017); ~ 20students. 2017 Expanding Your Horizons, Flenniken lab students (i.e., Laura Brutscher and Shantelle Smolik) interacted with 6th-8th grade girls interested in STEM, Bozeman, MT (April 22, 2017); ~ 60 students. 2017 Pollinator Symposium, Flenniken Lab Graduate Students (i.e., Laura Brutscher, Alex McMenamin, and Will Glenny) gave short presentations to the public on their research efforts aimed at understanding the role of pathogens on honey bee health at both the colony and individual bee levels, Bozeman, MT (April 19, 2017); ~ 50 community members. 2017 INBRE, Café Scientifique Invited Speaker, "What's Killing the Bees? The Impact of Pathogens and Other Factors on Honey Bee Colony Health", Bozeman, MT (March 28, 2017); ~ 100 community members. 2017 MSU's Science Roadshow 10x10 Talks, Invited Presenter, "Honey Bee Health and Your Health", Bozeman, MT, ~ 450 community members, (March 23, 2017) 2016 Museum of the Rockies, Invited Speaker, "Honey Bee Pathogens and Colony Health", Bozeman, MT (November 15, 2016). 2016 Alberta Beekeepers' Association Annual Meeting, Invited Speaker, "Honey Bee Pathogens and Colony Health", Edmonton, Alberta, Canada (November 8, 2016). 2016 Montana State Beekeepers' Association Annual Meeting, Invited Speaker, "Honey Bee Colony Health", Whitefish, MT (October 20, 2016). What do you plan to do during the next reporting period to accomplish the goals?Continue performing honey bee host -pathogen interaction research at the colony, individual bee, and cellular levels. Experiments, sample collection, and data analyses are ongoing.

Impacts
What was accomplished under these goals? High annual losses of honey bees, as well as range reductions and local extinctions of wild and native pollinator species, are concerning because bees are important plant pollinators. Approximately one third of the typical Western diet requires bee pollination, and honey bees (Apis mellifera) are the primary pollinators of numerous food crops including fruits, nuts, vegetables, and oil-seeds. Annually, insect-pollinated crops are valued at approximately $175 billion worldwide and $17-18 billion in North America, Research led by Dr. Michelle Flenniken is aimed at improving honey bee colony health and mitigate colony losses by understanding the effects of viruses on bee health. Honey bees are a specialty crop with dual roles in agriculture: honey production and crop pollination. Montana is an important beekeeping state, which ranked 2nd in the US for honey production in 2013 ($30 million value) and provided over 150,000 colonies for pollination services. Flenniken Publications (from 2016 - 2017; 6 out of 30 total) 1. Brutscher, L.M., Daughenbaugh, K.D., and Flenniken, M.L., Virus and dsRNA-triggered transcriptional responses reveal key components of honey bee antiviral defense, (2017), Scientific Reports 7 doi:10.1038/s41598-017-06623-z. 2. Glenny#, W., Cavigli#, I., Daughenbaugh, K.F., Radford, R., Kegeley, S., and Flenniken, M.L., Honey bee (Apis mellifera) colony health and pathogen composition in migratory beekeeping operations involved in California almond pollination, (2017), #equal co-authorship, PLoS ONE 12(8): e0182814 doi.org/10.1371/journal.pone.0182814 3. Doublet, V., Poeschle, Y., Gogol-Doring, Alaux, C., Annoscia, D., Aurori, C., Barribeau, S.M., Bedoya-Reina, O., Brown, M.J.F., Bull, J.C., Flenniken, M.L, Galbraith, D.A., Genersch, E., Gisder, S., Grosse, I., Holt, H.L., Hultmark, D., Lattorff, H.M.G, Le Conte, Y., Manfredini, F., McMahon, D.P., Moritz, R.F.A., Nazzi, F., Niño, E.L.,Nowick, K., van Rij, R.P., Paxton, R.J., and Grozinger, C.M., Unity in defence: honeybee workers exhibit conserved molecular responses to diverse pathogens, (2017), BMC Genomics, 18:207 doi: 10.1186/s12864-017-3597-6. 4. Brutscher, L.M., McMenamin A.J., and Flenniken, M.L., The buzz about bee viruses, (2016), PLoS Pathogens 12(8): e1005757. doi:10.1371/journal.ppat.1005757. 5. McMenamin A.J., Brutscher, L.M., Glenny, W., and Flenniken, M.L., Environmental factors affecting the replication and pathogenicity of bee viruses, (2016), Current Opinion in Insect Science (16):14-21. 6. Engel P., et al. Flenniken, M.L., Dainat, B., The bee microbiome: impact on bee health and model for evolution and ecology of host-microbe interactions, (2016), mBio 7(2): e02164-15

Publications

• Type: Journal Articles Status: Published Year Published: 2017 Citation: Glenny#, W., Cavigli#, I., Daughenbaugh, K.F., Radford, R., Kegeley, S., and Flenniken, M.L., Honey bee (Apis mellifera) colony health and pathogen composition in migratory beekeeping operations involved in California almond pollination, (2017), #equal co-authorship, PLoS ONE 12(8): e0182814 doi.org/10.1371/journal.pone.0182814
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Doublet, V., Poeschle, Y., Gogol-Doring, Alaux, C., Annoscia, D., Aurori, C., Barribeau, S.M., Bedoya-Reina, O., Brown, M.J.F., Bull, J.C., Flenniken, M.L, Galbraith, D.A., Genersch, E., Gisder, S., Grosse, I., Holt, H.L., Hultmark, D., Lattorff, H.M.G, Le Conte, Y., Manfredini, F., McMahon, D.P., Moritz, R.F.A., Nazzi, F., Ni�o, E.L., Nowick, K., van Rij, R.P., Paxton, R.J., and Grozinger, C.M., Unity in defence: honeybee workers exhibit conserved molecular responses to diverse pathogens, (2017), BMC Genomics, 18:207 doi: 10.1186/s12864-017-3597-6.
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Brutscher, L.M., Daughenbaugh, K.D., and Flenniken, M.L., Virus and dsRNA-triggered transcriptional responses reveal key components of honey bee antiviral defense, (2017), Scientific Reports 7 doi:10.1038/s41598-017-06623-z.