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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