Progress 05/01/24 to 04/30/25

Outputs
Target Audience: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.Goblirsch, M . The biology of theVarroamite and overwinter brood production of honey bees in the U.S. Southeast - Pine Belt Beekeepers' Association, Purvis, MS. (March 2025), Goblirsch, M.Varroa destructorand the Brood Monitoring Project - Poplarville Hobby Beekeepers' Association, Poplarville, MS. (February 2025), Goblirsch, M. Studying stressor effects on queen health and opportunities to improve honey bee genetics using a laboratory approach. Queen Health Workshop, Mississippi State University Apiculture Program and Delta Research and Extension Center, Stoneville, MS. (January 2025), Goblirsch, M. Building a research program in honeybee cell culture. Joint Minnesota and Wisconsin Honey Producers' Annual Meeting and Convention, St. Paul, MN. (July 2024), Simone-Finstrom, M. Improving bee health through integrated pest management. Louisiana Farm Bureau, New Orleans, LA. June 2024., Simone-Finstrom, M. Understanding and combatting impacts of honey bee viruses through breeding and antiviral treatments. Montgomery Beekeepers Association (PA). September, 2024, virtual presentation., Simone-Finstrom, M. Battling bees: mite resistance and other traits to promote healthier honey bees. Back Yard Beekeepers Association. Hartford, CT. October 2024., Simone-Finstrom, M. Mite resistance and other traits to promote healthier honey bees. Minnesota Honey Producers and Wisconsin State Beekeepers Association joint annual meeting. St. Paul, MN. July 2024., Simone-Finstrom, M. Mitigating impacts of viruses through bee breeding. "Strategies to Produce Better Honey Bee Queens" Meeting. Stoneville, MS. January 2024., Amiri, E. Dynamics of viruses among mite populations in hygienic and non-hygienic honey bee colonies. Iowa Honey Producers Association Annual Conference and Tradeshow, Altoona, IA, October 2024., Amiri, E. Pinpointing the critical Developmental Stages of queens to be infected by viruses. Iowa Honey Producers Association Annual Conference and Tradeshow, Altoona, IA, October 2024., Amiri, E. The role of viruses and Varroa mite in colony mortality and upcoming research plan for viral resistance breeding. 2024 the United States Russian Honey bee Breeders Association Annual convention. November, 2025, virtual presentation. Amiri, E. Pest and pathogens in honey bees; Diagnosis and control strategies. One day Mississippi and Alabama honey bee inspectors, Stoneville, MS, May 2024. Changes/Problems:In the proposal, we proposed to collected DWV symptomatic worker bees from different locations across the country (FL, MD, AZ, CA, MN, and MS) andpurify DWV-A and DWV-B to use the virus strains andtest their virulence of honey bee specific lines while controlling for environmental factors. Due to viral variation and uneven representations of DWV-A and DWV-B in samples collected from different locations the group decided to develop known cloned DWV-A and -B from the very recent DWV genomes reported from US beekeeping operations to better test the effect of each variant on honey bees. What opportunities for training and professional development has the project provided?This project has helped to hire a PhD student and a Postdoctoral fellow, who both of them are investigating different aspects of Virus-host interactions at different levels, including cellular, individual and colony levels. This activities expose them to various professional developments from basic beekeeping to virus purification, morphometric analysis,cell cullture, genomic and transcriptomic analysis. How have the results been disseminated to communities of interest?The PDand Co-PDs have been actively participating at local, state and national meeting and conferences, both in person and online to present the goals of the project and research activities that are accomplished for this project so far. The PD, has written several extension articles that have been published in the Mississippi Beekeeper Association's newsletter and planning to present some of the research results at the Mississippi Beekeeper Association anual convention, 2025 American Honey Producer Association annual convention, and 2026 American Meekeeper Federation. What do you plan to do during the next reporting period to accomplish the goals?The collaborative team will finalize the experiment I.A., I.B., and I.C. to understand the interplay between honey bee, virus and environment. We expect to accomplish experiment II.A., and II.B. to evaluate responses of of individual honey bees at different life stages, and cellular honey bees to different virus strains and species. The group will work to optimizeselect method to be able selecting resistance and susceptable queens over three generations and initiate genomic and transcriptome analysis to find selectable markers.

Impacts
What was accomplished under these goals? Since funding of our proposal, we have been in contact with the United States Russian Honey Bee Breeders Association establishing our collaborative network and plan corresponding experiments outlined in our proposal. We initially presented our needs at the 2024 meeting of the association where Drs. Arian Avalos and Esmaeil Amiri outlined goals, expected deliverables, and benefits to US Russian Honey bee Breeders Association members, and broadly US beekeepers. In addition, PD and Co-PDs also met with leadership of the Russian Honey bee Breeders Association in Mississippi in January 2025 to outline experiments and resolve logistical needs. Since then, project members have been working toward the defined experiments as outlined below: Objective I: Using a model virus system to understand the effect size of host genetics, environmental factors, and virulence during infection in honey bees. I.A: When controlling for environment and virus strain, how do honey bees from different genetic lines respond to virus infection? Postdoc McMenamin and Co-PD Simone-Finstrom have initialized experiments assaying for signatures of virus resistance among the panel of Russian bee matrilines as compared to commercial Italian stock as the susceptible control. Overall, 80 colonies, with queens represented from 10 different lines derived from the US Russian Honey Bee Breeders Association were established across two apiaries. Eggs were collected from 5 colonies per line to examine vertical transmission of viruses through the oviposition process with samples presently being analyzed. To determine adult viral susceptibility in honey bee workers, white eyed pupae from 3 randomly selected colonies per line were injected with a luminescent molecular clone of DWV. Source colonies were classified as either resistant or susceptible based on luminescence results. Of the matrilines examined, WR, WO, and YW were identified as interesting candidates. However, the dominant effect on observed variation in susceptibility in our experiments was colony of origin (i.e. environment) as opposed to parental lineage. Follow-up experiments are focusing on select parent colonies which displayed the strongest effect on viral load to examine whether brood in these colonies develop wing deformities at lower rates as compared to susceptible candidates when injected with DWV. I.B: When controlling for host genetics and environment, how do honey bees respond to virus strains derived from geographically distinct locations? We collected DWV symptomatic worker bees from different locations across the country (FL, MD, AZ, CA, MN, and MS) to purify DWV-A and DWV-B to test their virulence in specific lines while controlling for environmental factors. Due to viral variation and uneven representations of DWV-A and DWV-B in samples collected from different locations the group decided to develop known cloned DWV-A and -B from the very recent DWV genomes reported from US beekeeping operations to better test the effect of each variant on honey bees. Postdoc McMenamin and Co-PI Simone-Finstrom have been working on it and as soon as preparation of cloned DWV-A and -B will continue to test them in the selected colonies from the I.A. I.C: When controlling for host genetics and virus strain, how do honey bees located in different environments respond to virus infection? We established agreements with our collaborators (Drs. Judy Chen, Jay Evans, and Julia fine) to provide the known cloned DWV-A and DWV-B, as well as genetically related Pol-line queens to test the effect of environment on infection. Including our apiary at Stoneville, MS and USDA-ARS, Beltsville, MD, and USDA-ARS, Davis, CA locations our experiment provides a representative transect of beekeeping in the US. This experiment is in progress and on target for our 2026 projected start. Our focus has been to communicate with collaborators to being to establish apiary sites and introduce the Pol-line queens in Spring 2026. Objective II: Characterizing variation in honey bee responses to viral infections based on host and virus genetic background to inform identification of genetic markers of virus resistance. II.A: Evaluate responses of individual honey bees to different virus species at different life stages. PhD student, Gokul Dhanapati and PI Esmaeil Amiri are using CBPV and evaluate the response of different ages of worker honey bees (1day, 1 week, 2 weeks, 3 weeks old) to virus in three different seasons (Spring, Summer and Fall). The experiment evaluates the mortality rate after virus injection. PhD student, Gokul Dhanapati is presently hosted by the USDA-ARS Baton Rouge and in collaboration with Postdoc McMenamin and Co-PI Simone-Finstrom conducting an experiment to assess the age-matched adult bees from candidate colonies for resistance to DWV infection at 0, 3, 7 and 14 days old following aging in either an incubator or colony environment. The immune response from individual injected honey bees at different time points are collected and pending molecular analysis. II.B: Evaluate responses of honey bee cell lines to different viral strains and species. Postdoc McMenamin and co-PI Goblirsch have set up the queen monitoring cage system (QMCs) for establishment of primary cell cultures from individual and pooled egg sources. Whereas pooled egg sources enable higher throughput due to being easier technically, single-egg cultures provide higher resolution of genetic markers contributing to observed suppression of viral replication in the case of multiply-mated queens. Countable single-cell suspensions from pooled egg homogenates provide replicable establishment of tissue cultures in multi-well format with standardized tissue amount per experimental replicate (10^6 cells per well). Additionally, a protocol for establishing single-egg cultures has been devised, though standardization of tissue biomass seeded into wells remains a challenge. Present efforts are focusing on the standardization to ensure an effectively uniform multiplicity of infection during assays. Ensuing experiments will determine permissibility of cultures to infection, which may arise at a later stage in culture development as tissues continue to differentiate in vitro. In the future, QMCs will be used to source larvae to be reared in vitro for the establishment of hemocyte and pupal tissue culture models as well. Objective III: Selection for reduced viral infections in commercial queen producing operations and validation of genetic markers for resistance against common honey bee viruses. III.A: Quantify viral infection in breeder colonies from the Russian Honey Bee Breeders, select and propagate breeder queens with low and high virus infections. PhD student, Gokul Dhanapati and PI Esmaeil Amiri visited three queen producing operations who are producing Russian honey bee queens in Mississippi, early spring and collected eggs and worker bees from their breeder colonies. After molecularly evaluation of breeder queens, progeny queens were produced from 7 different Russian honey bee genetic lines, the progeny queens are introduced in 40 new colonies, and their health status will be evaluation before and after winter to produce following generations to be selected for high and low viral infection.

Publications