Progress 06/01/23 to 05/31/24

Outputs
Target Audience:The Target audiences reached during the Progress Interval, include beekeepers, queen breeders, entomologists, apiary inspectors and honey bee biologists that were in attendance at the follwing presentations. Note that the American Bee Research Conference occurred in conjunction with the American Beekeeping Federation Meeting in New Orleans, LA., where there were substantial numbers of beekeepers and queen breeders attending the American Bee Research Conference presentations and discussions. Simone-Finstrom, M. Harnessing Genetic Tools to Combat Honey Bee Stressors. Hambleton Research Award presentation at the annual meeting of the Eastern Apicultural Society. Amherst, MA. August 2023. Simone-Finstrom, M. & Ricigliano, V. Advancing Technologies to Combat Honey Bee Viruses Through Breeding and Antiviral Treatments. Eastern Apicultural Society conference. Amherst, MA. August 2023. Simone-Finstrom, M. Disease, parasite and viral resistance through management and breeding. Presentation at the annual meeting of the Apiary Inspectors of America. January 2024. Simone-Finstrom, M. Mitigating impacts of viruses through bee breeding. Strategies to Produce Better Honeybee Queens Workshop, Stoneville, MS. January 2024. Simone-Finstrom, M. Improving Bee Health through Integrated Pest Management. Louisiana Farm Bureau annual convention. New Orleans, LA. June 2024. Simone-Finstrom, M. Harnessing Genetic Tools to Combat Honey Bee Stressors. Battling bees: mite resistance and other traits to promote healthier honey bees. Minnesota Honey Producers Association and Wisconsin State Beekeepers Association joint annual convention. St. Paul, MN. July 2024. Keynote speaker. Conference presentations: Simone-Finstrom, M., Walsh, E., Slater, G. Evans, J. & Weaver, D. "Drone variation in susceptibility to Deformed wing virus as a target for selection." Presentation at the annual Entomological Society of America conference. Washington, D.C. November 2023. Simone-Finstrom, M., Walsh, E., Slater, G. Evans, J. & Weaver, D. Variation in susceptibility to Deformed wing virus in honey bee drones in relation to quality metrics. Presentation at the annual American Bee Research Conference. New Orleans, LA. January 2024. In addition, B. Weaver Apiaries, Inc., conducts beekeeping and honey bee queen production seminars and workshops throughout the year, and the Virus Resistance research being funded by this grant was presented and discussed during those events too. Changes/Problems:As described in the previous section, we plan to change from single-drone inseminations, to insemination with semen from multiple related drones (multiple drones produced by the same queen). This will provide inseminated queens with much greater longevity, and facilitate our production of multiple generations with increasing levels of DWV resistance. Moreover, we will begin our drone collection, screening and semen collection much earlier in the spring than we have previously. Again, this will allow us to produce a second generation of DWV resistant queens earlier in the year, and confirm the tenative technical progress we have achieved. Finally, we will only move drones and virgin queens from Navasota to Baton Rouge via hand courier, to avoid the multiple extreme mortality events caused by repeated failure of UPS and USPS to provide reliable, on-time delivery and protection from environmental extremes of heat and cold. What opportunities for training and professional development has the project provided?Several students and technicians were also trained as part of these efforts, including one partially funded by this project through an ORISE internship. One other student, an undergraduate from the University of Texas San Antonio, a Hispanic Serving Institution, worked on this project as part of a new collaboration with Dr. Ferhat Ozturk at UTSA funded through National Institute of Food and Agriculture's "From Learning to Leading: Cultivating the Next Generation of Diverse Food and Agriculture Professionals Program. Two undergraduate students at Texas A&M University's Departments of Entomology and Agriculture Communications spent significant time providing technical assistance on the project, and one of them was a successful applicant toPhD program in Honey Bee Biology at the University of Florida, with her efforts on the project providing proof of her field research experience. How have the results been disseminated to communities of interest?A variety of presentations and talks, previously detailed above, disseminated the results of the project during the reporting interval. To reitierate, they included the following: Simone-Finstrom, M. Harnessing Genetic Tools to Combat Honey Bee Stressors. Hambleton Research Award presentation at the annual meeting of the Eastern Apicultural Society. Amherst, MA. August 2023. Simone-Finstrom, M. & Ricigliano, V. Advancing Technologies to Combat Honey Bee Viruses Through Breeding and Antiviral Treatments. Eastern Apicultural Society conference. Amherst, MA. August 2023. Simone-Finstrom, M. Disease, parasite and viral resistance through management and breeding. Presentation at the annual meeting of the Apiary Inspectors of America. January 2024. Simone-Finstrom, M. Mitigating impacts of viruses through bee breeding. Strategies to Produce Better Honeybee Queens Workshop, Stoneville, MS. January 2024. Simone-Finstrom, M. Improving Bee Health through Integrated Pest Management. Louisiana Farm Bureau annual convention. New Orleans, LA. June 2024. Simone-Finstrom, M. Harnessing Genetic Tools to Combat Honey Bee Stressors. Battling bees: mite resistance and other traits to promote healthier honey bees. Minnesota Honey Producers Association and Wisconsin State Beekeepers Association joint annual convention. St. Paul, MN. July 2024. Keynote speaker. Conference presentations: Simone-Finstrom, M., Walsh, E., Slater, G. Evans, J. & Weaver, D. "Drone variation in susceptibility to Deformed wing virus as a target for selection." Presentation at the annual Entomological Society of America conference. Washington, D.C. November 2023. Simone-Finstrom, M., Walsh, E., Slater, G. Evans, J. & Weaver, D. Variation in susceptibility to Deformed wing virus in honey bee drones in relation to quality metrics. Presentation at the annual American Bee Research Conference. New Orleans, LA. January 2024. What do you plan to do during the next reporting period to accomplish the goals?During the next reporting period we will begin drone injections with nanoluciferase tagged DWV as soon as drones are produced in Texas and Lousiana in the sping of 2025. After injection, drones will be screened with UV radiation, and semen from drones with low post-injection DWV replication will be collected and used to inseminate virgin queens graftedfrom DWV resistant and DWV susceptible queens. Worker progeny of these inseminated queens will be screened to confirm queen progeny phenotype as more or less DWV resistant than worker progeny of the inseminated queens' mothers. Next drones produced inthose colonies headed by inseminated queens exhibiting enhanced DWV resistance (as compared to their mothers) will be collected and used to inseminate daughter queens grafted from other queens heading other colonies exhibiting enhanced DWV resistance. Importantly, we will be collecting semen from multiple brotherdrones once DWV resistance or susceptibility is confirmed, and virgin queens will be inseminated with semen from multiple resistant drones. This will provide inseminated queens that will produce worker and drone offspring for much longer periods of time. In the pastweinseminated with semen from only single drones, but the severely reduced longevityof single-drone inseminated queens has hampered our ongoingefforts to produce multiple iterations and generations of queens exhibiting increasedDWV resistanceover earlier generations. We are confident that this approach of using semen from multiple related drones to inseminate queens will dramatially accelerate our progress in 2025.

Impacts
What was accomplished under these goals? Project activity from Aug 1, 2023-Aug 30-2024 Research progress: Progress has been made on several fronts related to project objectives. Specific methodologies and approaches were adjusted from the proposed work due to limitations in transporting live specimens from the beekeeping operation in Texas to the laboratory in Louisiana. Additional questions regarding method optimization to increase the success of the project were also addressed based on atypical issues that arose in the artificial insemination process with honey bee queens. In August-September 2023, 200 drone honey bees collected in the Weaver beekeeping operation were brought to Baton Rouge and injected with deformed wing virus (DWV) solution. Another 200 drones collected from a mite-resistant population bred in Baton Rouge were also tested. Semen was collected from the drones and they were analyzed for viral loads to indicate whether they were susceptible to DWV (viral load increased) or resistant (viral load remained low) after 48h post-injection. Overall the Weaver population exhibited a bi-modal distribution with drones showing a mix of resistance and susceptibility in this group. However, the Baton Rouge population displayed a skewed distribution with most of the drones being susceptible to DWV infection. From both populations, semen from drones that were determined to be susceptible and drones assessed as resistant was used to artificially inseminate queens as a starting point for bi-directional selection for virus resistance. However, there were unforeseen issues in the insemination process that unfortunately resulted in most of the queens dying and none of the queens overwintered successfully to be able to be tested in spring 2024. This was in part due to the atypical process that we had to rely on for the drone phenotyping where the semen was individually collected from each drone and then had to be maintained until after the viral assessments could be completed. We revised this plan to improve the likelihood of success in 2024. From the 2023 samples, we were able to make some other discoveries. We determined that drone size, which is typically thought of as a measure of drone quality, was unrelated to viral resistance. We did however, find that some recently identified genetic markers for drone quality (e.g. sperm quality, flight activity, maturation). One of these genes (GB55703, indicative of sperm quality) showed higher expression of this gene in resistant drones from the Weaver population suggesting that viral resistance may have a positive association with drone sperm quality. Other results suggested that in the Baton Rouge population, but not the Weaver population, other viral infections influenced the drones ability to fight off the DWV challenge. This finding, coupled with the fact that honey bees often are co-infected naturally with several viruses, indicated that we have to pre-screen test populations for other viruses and/or include a full viral screen panel in our assessments of resistance and susceptibility. In 2024, the focus has been to determine best practices for methodology to improve the success of the screening process and make it more accessible to conduct with and in beekeeping operations. The screening method itself is straightforward and quick to conduct, however, honey bee drones are fragile and difficult to maintain in the lab post-injection because they require special care and treatment. Additionally the need to inject, then collect semen from all individuals, and then determine viral load creates a significant bottleneck in the process. As part of best practice determination, we aimed to determine if we can simply determine a colony level phenotype and then use drones later collected from those colonies (but not tested themselves) to conduct standard artificial insemination. For this we had to examine within colony variation for drones, determine what age of drone to test and then ensure that responses of the determined age correctly predicts the resistant/susceptible phenotype of sexually mature drones. We first screened immature drones, adult drones that are unable to fly and so are 5-10 days old, for response to DWV injection from 16 different colonies. From this we were able to determine that drones from the same colonies have extremely consistent responses. To better determine the age to proceed with testing, we paint-marked newly emerged drones (at 1d old) from six different colonies, with 3 being identified as resistant and 3 susceptible based on the initial screening. These drones were maintained in a shared host colony to ensure that environmental factors (e.g. nutritional access, etc.) did not influence viral susceptibility.. Drones were then collected and injected with DWV at 1d, 5d, 7d, 10d and 14d old from inside the host colony and then also returning from a flight, which is indicative of sexual maturity. 1d old drones survived the injection process at ~90%, which is significantly better than the 50%-60% survival noted in mature drones. However these young drones were all extremely susceptible to viral infection regardless of colony origin, indicating that this age would not work for the test perhaps due to an immature immune system. 5d-10d old drones survived infection well (>80% survival) and exhibited consistent responses within colonies. Current analyses are still pending the comparison of immature to mature drones, but will inform whether we can use assessments of immature drones to then determine the resistance phenotype for drones used in insemination. This work is ongoing and will continue over the next few months to produce successful breeding lines for further study. Several students and technicians were also trained as part of these efforts, including one partially funded by this project through an ORISE internship. The other student, an undergraduate from the University of Texas San Antonio, a Hispanic Serving Institution, worked on this project as part of a new collaboration with Dr. Ferhat Ozturk at UTSA funded through National Institute of Food and Agriculture's "From Learning to Leading: Cultivating the Next Generation of Diverse Food and Agriculture Professionals Program."

Publications

Progress 06/01/22 to 05/31/23

Outputs
Target Audience:Dr. Michael Simone Finstrom made a presentation at the American Bee Research Conference and Daniel Weaver made a presentation at the Texas Beekeeper's Association Conference regarding the project. Changes/Problems:We plan to begin drone collections earlier in the spring and initiate subsequent rounds of DWV injection, screening for DWV resistance and semen collection so that more queens may be inseminated earlier in the year when the natural levels of drone production and worker production are higher, to enable us to assess the extent of the differences in DWV resistance exhibited by queens inseminated with semen from drones with low DWV replication and queens inseminated with semen from drones with high DWV replication. Further efforts will be made to confirm our early evidence that qRT-PCR results are tightly correlated with nanoluciferase luminescense evidence. What opportunities for training and professional development has the project provided?Several undergraduate University students employed by B. Weaver Apiaries, Inc. have been trained in queen rearing techniques, participated in drone collection, drone collection method optimization, drone screening, data collection and drone storage technique optimization. Additional exposure to laboratory methods of molecular and luminescent measurements, as well as semen collection and insemination are planned for the future. How have the results been disseminated to communities of interest?Two presentations have been made, one to the honey bee research community at the Bee Research Conference, and to beekeepers in Texas at the Texas Beekeepers Association meeting. What do you plan to do during the next reporting period to accomplish the goals?We will start our drone collection, injection, screening, semen collection and queen insemination efforts earlier in the year, and hopefully avoid the extensive drone mortality in transit prior to DWV clone injection, and unfortunate queen mortality post-insemination, as well as early queen failure due to limited quantities on semen used for insemination in the reported period.

Impacts
What was accomplished under these goals? Inthe period covered by this progress report, drones were collected from colonies that demonstrated phenotypic resistance to DWV, and colonies that did not exhibit significant phenotypic resistance to DWV. These drones were then injected with either: 1) DWV viral constructs containing a luciferase reporter gene; or 2) a phosphate buffer solution devoid of DWV. After 48 hours the surviving injected drones were screened for the presence of DWV and the level of DWV replication was quantified by qRT-PCR and also by illumination with UV light that would cause the luciferase to flouresce. Semen was collected from DWV-clone-injected drones that exhibited both high and low levels of DWV replication, presumably representing the extremes of the distribution of DWV resistance in the injected drones. Semen from single drones selected as either demonstrating low or high DWV replication were used to inseminate virgin queens, and inseminated queens were introduced to colony nuclei. Unfortunately, the single drone inseminated queens experienced high levels of over-winter mortality and early failure during the subsequent spring where brood rearing surged initially but then was suppressed by prolonged cool /wet weather followed by extremely hot and dry summer weather. Consequently, were unable to evaluate the progeny of the inseminated queens for differing levels of DWV resistance because too few survived and were laying long enough to enable collection of statitistically significant data.

Publications