Progress 05/01/24 to 04/30/25
Outputs
Target Audience:United States (US) beekeepers represent the primary target audience of our research. In 2024, over 55% of managed honey bee colonies in the US were lost, representing unsustainable losses to the industry and threatening the delivery of agricultural pollination services. Pests and pathogens are key drivers of honey bee colony losses. Beekeepers and diagnosticians screen for these biotic stressors visually, mechanically, or using molecular approaches, but these techniques are often time consuming, highly variable, and can be unreliable. Beekeepers urgently need better screening tools to mitigate the impact of established pests and pathogens and limit the introduction of new threats. Our research into developing novel eDNA and eRNA methodologies could give beekeepers the tools needed to detect emerging threats to their colonies rapidly and accurately. Entomologists and laboratory diagnosticians represent key additional target audiences for our project. These scientists can use the techniques we develop to screen for pests and pathogens of apiculture importance, and study biotic communities associated with key environments. Our methods will be relevant in other fields as well. Changes/Problems:As described in last year's report, an important change in our grant objectives involved the unexpected introduction of the yellow-legged hornet (Vespa velutina), a honey bee predator of serious economic concern, in Savannah, Georgia. Given the proximity of this introduction to the University of Florida, we were uniquely situated to shift Objective 2a to focus primarily on validating eDNA methodologies through monitoring spread of the yellow-legged hornet in Georgia, rather than the giant northern hornet (Vespa mandarinia) in Washington. This decision was further strengthened by the giant hornet's official eradication from the US since the grant was awarded. We no longer intend to swab nest materials from unoccupied giant hornet nests in Washington, as we have validated this eDNA technique with yellow-legged hornet nests in Georgia. The active threat of the yellow-legged hornet represented a timely opportunity to verify the use of eDNA as a screening tool for emergent honey bee pests during the first wave of invasion. Additionally, due to delays in receiving international research permits, we had to postpone our sampling trip to Thailand, originally planned for May 2024, to December 2024. We still were able to collect all the samples we intended. However, there was no hornet activity this time of year in the regions of Thailand where we sampled and thus, we did not observe either Vespa mandarinia or V. velutina, which were two of our focal species with high invasion potential in the US. Despite this, we sampled colonies hosting other potentially invasive species, including those we intended to target (other Apis species, Tropilaelaps spp.) and those we had not anticipated to be present in our original planning (Euvarroa spp., Aethina tumida). We believe our work with the hornet in Georgia, combined with the opportunity to test the eDNA detection of additional species of concern, will offset any setbacks associated with this modification. We do not anticipate the change in the sampling trip dates will limit our ability to complete the objective successfully. What opportunities for training and professional development has the project provided?We hired Dr. Kaitlin Deutsch to manage the day-to-day project components. In the last reporting period, Dr. Deutsch has learned eDNA/eRNA sampling techniques, bioinformatic analysis skills, and Bash coding language. Additionally, she has presented results from this project at two international conferences, allowing her to build her professional network with other scientists in the field. She has also engaged in over ten outreach and extension events in the last year, gaining valuable experience sharing science with different communities and stakeholders. Dr. Ellis and Dr. Deutsch created a formal professional development plan for the next year, including experience with project planning/execution, collaborator networking, grant writing and administration, manuscript development and submission, student mentorship, and project oversight. Drs. Ellis and Boardman will work closely with Dr. Deutsch to ensure her continued professional growth and development. How have the results been disseminated to communities of interest?The results described in this report have been disseminated to colleagues at academic conferences and stakeholders at various outreach events. Results from Objectives 1 and 2a (eRNA and yellow-legged hornet eDNA) were presented at two international and one national academic conferences (see "Products" for full details). Additionally, results have been shared with beekeepers at four beekeeping conferences (Florida State Beekeepers Association October 2024; Illinois State Beekeepers Association November 2024, American Beekeeping Federation, January 2025, Michigan State Beekeepers Association March 2025), and five outreach events: Memphis Area Beekeepers Association monthly meeting (September 2024), Chipola Beekeepers Association (October 2024), the University of Florida (UF) Master Beekeeper Program monthly meeting (April 2025), UF Honey Bee Research and Extension Laboratory Spring Bee College (March 2025), and UF Honey Bee Research and Extension Laboratory Summer Bee College (August 2024). The initial eDNA research was also discussed in a Two Bees in a Podcast, episode 166 (University of Florida Honey Bee Research and Extension Laboratory) released in May 2024 - https://podcasters.spotify.com/pod/show/ufhbrel/episodes/Episode-166-Environmental-DNA-e2k67rk. Collectively, this information reached more than 5,000 beekeepers globally. What do you plan to do during the next reporting period to accomplish the goals?In the next reporting period, we will continue to accomplish goals outlined in Objectives 1 and 2 and initiate data collection for Objective 3. Specifically, for Objective 1, we will scale up our optimized eRNA methodologies and analyses to perform next-generation sequencing on a high volume of eRNA samples collected from hive environs. This reporting period, we developed a standardized eRNA sampling methodology and a robust NGS workflow using two pilot samples, as well as evaluated which surfaces most reliably yielded virus RNA using conventional primer-based screening methods. In the next reporting period, we will scale up our analyses by collecting and sequencing eRNA samples across multiple honey bee colonies and using our bioinformatic pipeline to characterize the full viral community for a greater number of samples more representative of real-world monitoring. For Objective 2a, we will draft and submit for review the manuscript describing our eDNA methodology for monitoring the yellow-legged hornet. This reporting period, we finalized sample collection, processing, and analysis for this project. In the next reporting period, we will write the manuscript and publish this dataset. For Objective 2b, we will perform metabarcoding on eDNA samples collected from Thailand to determine the bacterial, fungal, and arthropod associates in bee colonies in this region. This reporting period, we traveled to Thailand and sampled from multiple bee colonies within the native range of potential pest and predator species. In the next reporting period, we will process samples and acquire all sequencing data to be prepared to begin drafting the manuscript. For Objective 2c, co-PI Boardman and postdoc Deutsch will travel to the Cape Town region of South Africa in October 2025 to sample from wild and managed Apis mellifera capensis colonies. As before, we will collect swab samples from the hive environment and extract DNA from these samples. After collection, we will use a targeted eDNA approach to determine if diagnostic assays to detect A. m. capensis can be conducted on DNA collected from the environment and not the honey bees themselves. Finally, for Objective 3, we will conduct the national ring test of our eDNA/eRNA sampling methodology for honey bee colonies. We will develop kits with sampling supplies and detailed instructions to mail to our collaborators around the country. The collaborators will collect eDNA samples per our instructions and then mail them back to us for analysis. Samples will be sequenced, and we will evaluate the utility of our eDNA/eRNA technique as a standardized sampling tool across multiple laboratories.
Impacts
What was accomplished under these goals?
The goal of our work is to provide beekeepers with a more effective method to monitor emergent pests and pathogens, prevent the establishment of new threats, and mitigate the impact of known pests and pathogens. For Objective 1, we developed standardized eRNA sampling methods and a next generation sequencing pipeline for characterizing the full community of honey bee viruses using environmental RNA from the hive environs. We collected samples from seven hive surfaces across multiple honey bee colonies using sterile swabs. Using targeted screening, we detected at least one honey bee-associated virus on 86% of all samples. Swabs of frame top bars and honey had the greatest prevalence and richness of viruses, indicating some places in thehive are more informative than are others for reliably detecting viruses. Further, we conducted a full shotgun RNA sequencing pilot project on two eRNA samples (swabs of hive tool and hive entrance) as an untargeted method to characterize the full virus community on eDNA samples. This included the development of a complete bioinformatic pipeline to analyze sequence data. We found that virus enrichment (ribodepletion) successfully increased the proportion of viral genetic material in our samples, and that the hive tool had greater viral richness than the hive entrance. On the hive tool, we identified over 12 honey bee viruses, including those not commonly screened for in routine diagnostic testing. Thus, the development of an eRNA monitoring tool will allow beekeepers and honey bee diagnosticians to identify a suite of viral pathogens rapidly and less invasively. Importantly, this untargeted approachwill allow the detection of uncommon, new, and/or emerging viruses. For Objective 2a, we tested eDNA approaches to monitor the recent invasion of the yellow-legged hornet (Vespa velutina), in Savannah, Georgia. A honey bee predator, the yellow-legged hornet, was first detected in apiaries in Savannah in August 2023. We collected samples every month during April-November 2024 from sentinel apiaries (apiaries in the vicinity of hornet nests but without confirmed sightings) and rapid-response apiaries (apiaries where the invasive hornets were confirmed present). At all sites, we swabbed hive surfaces and collected samples of soil beneath the colonies. We detected yellow-legged hornet DNA on one out of 175 eDNA samples at sentinel apiaries (0.6% detection rate). At rapid-response apiaries, we detected yellow-legged hornet DNA on seven out of 65 eDNA samples (11% detection rate). This infrequent detection of hornet DNA from apiary samples, even where hornets were present, matches hornet behavior, as adults catch their prey in flight and rarely, if ever, contact hive surfaces. Thus, current eDNA approaches in apiaries do not seem to be effective at monitoring the spread of the yellow-legged hornet. Eliminating eDNA surveillance as a potential monitoring strategy for the hornet allows researchers and resource managers to investigate alternative methods of monitoring the yellow-legged hornet, methods such as harmonic radar tracking and camera traps. For Objective 2b, we traveled to Thailand to collect eDNA samples from honey bee colonies within the native range of potentially invasive pest species. With collaborators from Burapha University, we collected over 225 swab samples from colonies of Apis mellifera, A. cerana, A. dorsata, A. florea, and at least six species of stingless bee from central Thailand. We also collected nearly 125 samples of whole bees, pests and other nest associates for DNA barcoding. Following all USDA-APHIS permitting regulations, these samples were brought back to the US where they are being stored until processing and sequencing. The analysis of these samples will allow us to test the ability of eDNA samples to detect the arthropod species that are organisms of concern in the US, benefiting regulatory agencies that routinely monitor for invasive pests. Finally, the postdoctoral scholar leading this project gained invaluable laboratory and bioinformatics skills while collecting, processing, and analyzing these environmental samples and associated sequence data. We will use the results and expertise developed from these first two objectives in following years for Objective 3: to sample standardized hive surfaces that consistently yield a high diversity of eDNA and eRNA and continue to develop this method as a scalable tool to monitor for emergent pests and pathogens.
Publications
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Progress 05/01/23 to 04/30/24
Outputs
Target Audience:U.S. beekeepers represent the primary target audience of our research. In 2023, nearly 50% of honey bee colonies in the United States were lost, representing unsustainable losses to the industry and threatening the delivery of agricultural pollination services. Pests and pathogens are key drivers of honey bee colony losses. Beekeepers and diagnosticians screen for these biotic stressors visually, mechanically, or using molecular approaches, but these techniques are often time consuming, highly variable, and can be unreliable. Beekeepers urgently need better screening tools to mitigate the impact of established pests and pathogens and limit the introduction of new threats. Our research into developing novel eDNA and eRNA methodologies will give beekeepers the tools needed to detect emerging threats to their colonies rapidly and accurately. Entomologists and laboratory diagnosticians represent key additional target audiences for our project. Entomologists and other scientists can use the techniques we develop to study biotic communities associated with any environment. Diagnosticians can use the techniques to screen for pests and pathogens of apiculture importance, but the methods will be relevant in other fields as well. Changes/Problems:An important change in our grant objectives involved the unexpected introduction of the yellow-legged hornet (Vespa velutina), a honey bee predator of serious economic concern, in Savannah, Georgia. Given the proximity of this introduction to the University of Florida, we were uniquely situated to shift Objective 2a to focus primarily on validating eDNA methodologies through monitoring spread of the yellow-legged hornet in Georgia, rather than the giant northern hornet (Vespa mandarinia) in Washington. This decision was further strengthened by the giant hornet's likely eradication from the United States, given it has not been seen in Washington in over two years. While we still intend to swab nest materials from unoccupied giant hornet nests in Washington from the initial invasion in 2020, the active threat of the yellow-legged hornet represented a timely opportunity to verify the use of eDNA as a screening tool for emergent honey bee pests during the first wave of invasion.Additionally, we had intended to have eRNA (Obj 1) sequencing completed by Q4 of Y1. However, our postdoctoral scholarwas only able to join the project in Q3 (January 2024), pushing the anticipated completion of this objective to Year 2. Nevertheless, we do not anticipate this will limit our ability to complete the objective successfully. What opportunities for training and professional development has the project provided?We hired Dr. Kaitlin Deutsch to manage the day-to-day project components. She has only been a member of the project team since January 2024, but has already learned eDNA/eRNA sampling techniques, networked with other scientists in the field, and gained experience with a new invasive species threat to honey bee colony health. Drs. Ellis and Boardman will work closely with Dr. Deutsch to ensure her continued professional growth and development. For example, Dr. Ellis will work with Dr. Deutsch to create a formal professional development plan. This plan will include experience with project planning/execution, collaborator networking, grant writing and administration, manuscript development and submission, student mentorship, and project oversight. How have the results been disseminated to communities of interest?We have not begun formal results dissemination for this project. We are in the data collection and analysis stages of the project. Dissemination will start once results are available and vetted for release. What do you plan to do during the next reporting period to accomplish the goals? In the next reporting period, we will continue to accomplish goals outlined in Objectives 1 and 2. Specifically, for Objective 1, we will use next-generation sequencing techniques to characterize full viral communities from eRNA samples collected from hive environs. This reporting period, we demonstrated that eRNA can be collected and used to screen for honey bee viruses non-invasively using conventional primer-based screening methods. While our goal was to have total RNA sequencing data for Objective 1 by the end of Year 1, we took additional time to 1) optimize our RNA collection & extraction methodologies from environmental samples with low yield and 2) ensure our pilot data was free of contamination. In the next reporting period, we will sequence eRNA samples and perform bioinformatic analyses to characterize the full viral community, including pathogens that are not commonly screened for in routine diagnostic testing. For Objective 2a, we will continue to work with the Florida and Georgia State Departments of Agriculture to monitor the spread of the invasive yellow-legged hornet using eDNA from swab and soil samples collected from apiaries within the invaded range of the hornet. For Objective 2b and 2c, we will travel to Thailand and South Africa to test our eDNA and eRNA methods in locations where potentially invasive species exist. Specifically, in summer 2024, we will travel to Burapha University in Chon Buri, Thailand to collect soil and swab samples from honey bee colonies within the native ranges of multiple organisms of concern (Vespa spp., Tropilaelaps spp., other Apis species, etc.) that are likely to cause significant damage to honey bee colonies in the U.S. should they be introduced or establish. Subsequently, we will perform next-generation sequencing on these samples to ensure we are able to detect all emergent threats via an eDNA monitoring approach. In spring 2025 (our current target date), we will travel to the Cape Town region of South Africa to sample from wild and managed Apis mellifera capensis colonies. As before, we will collect soil and swab samples from the hive environment and extract DNA from these samples. The samples will be collected in the second reporting period but processed in the third reporting period (Year 3). After collection, we will use a targeted eDNA approach to determine if diagnostic assays to detect A. m. capensis can be conducted on DNA collected from the environment and not the honey bees themselves.
Impacts
What was accomplished under these goals?
The goal of our work is to provide beekeepers with a more effective method to monitor emergent pests and pathogens, prevent the establishment of new threats, and mitigate the impact of known pests and pathogens. For Objective 1, we validated and optimized methodologies to detect honey bee viruses using environmental RNA from the hive environs. We collected samples from five hive substrates using two different types of swabs. We reliably detected a suite of the most important honey bee RNA viruses from swabs of (a) hive tools, (b) honey cells, and (c) in-hive sugar feeders, regardless of the type of swab used. Viruses could also be detected from swabs of the hive entrance, but this seemed dependent on the type of swab used. Surprisingly, we did not detect any virus RNA from swabs of the bottom board no matter the type of swab used. Our next steps involve next-generation sequencing of these RNA samples to characterize the full viral community within hives, including those pathogens not commonly screened for in routine diagnostic testing. These findings confirm that, despite concerns over rapid RNA degradation, eRNA can be collected and used to screen non-invasively for honey bee viruses. Thus, the development of an eRNA monitoring tool will allow beekeepers and honey bee diagnosticians to identify a suite of viral pathogens rapidly and less invasively. For Objective 2, we successfully utilized eDNA approaches to monitor the recent invasion of the yellow-legged hornet (Vespa velutina), in Savannah, Georgia. A honey bee predator, the yellow-legged hornetwas first detected in apiaries in Savannah in August 2023. At apiaries where the invasive hornets were confirmed present, we swabbed hive substrates and collected samples of soil beneath the colonies. We were able to detect yellow-legged hornet DNA on hive tools and in soil samples reliably, validating our eDNA methodology as an effective way to monitor the continued invasion of the hornet.Having an eDNA monitoring tool for the hornet will allow State Departments of Agriculture to identify areas where the hornet has established and efficiently allocate resources toward nest discovery and destruction, thereby minimizing the impact of the predatory hornet on local beekeepers' colonies. Not only can eDNA monitoring be employed to mitigate the effect of the yellow-legged hornet, but it also confirms that eDNA/eRNA can be used to detect emergent honey bee pests and predators via non-invasive sampling. Additionally, the postdoctoral scholar leading this project gained invaluable laboratory skills while collecting, processing, and analyzing these environmental samples and developed significant expertise in honey bee pest and pathogen diagnostics. We will use the results from these first two objectives in following years for Objective 3 to sample standardized hive substrates that consistently yield a high diversity of eDNA and eRNA and continue to develop this method as a scalable tool to monitor for emergent pests and pathogens.
Publications
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