A Sustainable Approach to Control Varroa Mites - Improving the Quality of Queens using Local Resources

For outcome 1, we ask whether feral queens have better flight ability than the commercial package bees. Our lab has developed a protocol and a scientific publication for the method (Ma et al., 2019). Virgin queens will be evaluated using flight mills (Figure 1). As the glued bees on the mill, a computer connected to the flight mill sensor recorded maximum and mean velocity, flight duration, and flight distance. At least six virgin queens from each treatment (feral or commercial) will be used and each queen will be tested for six separate trial days. Feral stocks are from our university apiary. The non-selected commercial colonies are from GA and FL as controls. All flight mills will be located in the same room, with constant light (546.2±1.0 lx) and air temperature (25±1 ∘C). Six virgin queens per treatment group (feral queen vs commercial queen) will be used in this experiment.

For outcome 2, we ask whether the level of oxidative stress in feral queens is higher than the commercial bees. Queen pupae will be produced by our lab by grafting from two treatments (feral vs commercial) and collected for tests. Reactive Oxygen Species (ROS)- mediated oxidative damage will be quantified by measuring the Malondialdehyde (MDA) level in individual queen pupae (Li-Byarlay et al., 2016, Simone-Finstrom et al., 2016, Li-Byarlay & Cleare, 2020). The assay will be conducted using the OXItek™ Thiobarbituric Acid Reactive Substances (TBARS) Assay Kit (ZeptoMetrix Corp). The Pierce™ BCA™ Protein Assay kits (Thermo Scientific) will be used to measure the total soluble protein. Oxidative damage by normalized MDA levels will be examined in three biological replicates (colony). Each colony type (feral or package) includes at least six individual queen pupae (red-eye stage). Data will be analyzed using ANOVA.

For outcome 3, we ask whether we can identify new genetic markers for queen quality using cutting-edge technology. For whole genome amplification (WGA), we will use the Primary Template-directed Amplification (PTA) (Gonzalez et al., 2020). We plan to use twelve samples, six fertilized eggs (high mite biting stock versus non-selected commercial bees), and six samples of semen for instrumental insemination from two populations, prepare single-cell sequencing libraries by the 10x Genomics Chromium controller (Traniello et al., 2020). Downstream Illumina sequencing will be carried out at Ohio State University Sequencing center. 

For outcome 4, we ask whether we can change more bee farmers on their view of local bee resources, sustainable queen rearing in Ohio. We aim to have a three-day workshop with about 33 participants each year on how to do grafting and queen rearing, how to make swarm traps and catch local feral stocks, and how to detect mite biting behavior traits using microscopes. Participating/trained bee farmers will receive a certification from Central State University. The picture on the left was our successful workshop from the previous SARE partnership grant. Figure 2 shows our 48-hr queen cell workshop organized in 2019.

For outcome 5, we plan to hold 4-5 day long field days with Purdue University and HHBBC each year on instrumental insemination events and work with queen breeders in the North Central Region to promote regional efforts on breeding for mite biters and mite resistant stocks. The field day event will be normally in May each year depending on drone and queen availabilities. Breeders will exchange genetics of queens from different feral colonies with high biting or chewing rate. Queen breeders will collect semen, conduct instrumental inseminations, and meet to discuss how to improve the breeding efforts as a group for our region. 

For outcome 6, we ask whether we can train more undergraduate students on honeybee genetics, genomics, physiology, and pollinator health. At least six students of the under-represented minority will be trained on the lab protocols to perform experiments of flight mill experiments, TBARS assays, and sequencing experiments. They will be the future workforce for sustainable apicultural and agricultural research. Figures 3 and 4 show examples of our student training for research. We have trained 13 African American college students (7 females) in the past three years.

All the data and research results will be shared by research & extension publications, newsletters to the Ohio State Beekeepers Association, CSU extension and outreach activities (American Beekeeping Federation, State of Ohio State Fair, Farm Science Review, Ohio Ecological Food and Farm Association Conference, Black Farmer Conference, Minority and Women Small Farmers Conference, and local agricultural or farmer conferences).

Reference

Gonzalez, V., Natarajan, S., Xia, Y., Klein, D., Carter, R., Pang, Y., Shaner, B., Annu, K., Putnam, D., Chen, W., Connelly, J., Pruett-Miller, S., Chen, X., Easton, J. and Gawad, C. (2020) Accurate Genomic Variant Detection in Single Cells with Primary Template-Directed Amplification. bioRxiv, 2020.11.20.391961.

Li-Byarlay, H., Huang, M.H., Simone-Finstrom, M., Strand, M.K., Tarpy, D.R. and Rueppell, O. (2016) Honey bee (Apis mellifera) drones survive oxidative stress due to increased tolerance instead of avoidance or repair of oxidative damage. Experimental Gerontology.

Li-Byarlay, H. and Cleare, X.L. (2020) Current trends in the oxidative stress and ageing of social hymenopterans. Advances in Insect Physiology, 59, 43.

Ma, C., Zhang, Y., Sun, J., Imran, M., Yang, H., Wu, J., Zou, Y., Li-Byarlay, H. and Luo, S. (2019) Impact of acute oral exposure to thiamethoxam on the homing, flight, learning acquisition and short-term retention of Apis cerana. Pest Management Science, 75, 2975-2980.

Simone-Finstrom, M., Li-Byarlay, H., Huang, M.H., Strand, M.K., Rueppell, O. and Tarpy, D.R. (2016) Migratory management and environmental conditions affect lifespan and oxidative stress in honey bees. Scientific Reports, 6.

Traniello, I.M., Bukhari, S.A., Kevill, J., Ahmed, A.C., Hamilton, A.R., Naeger, N.L., Schroeder, D.C. and Robinson, G.E. (2020) Meta-analysis of honey bee neurogenomic response links Deformed wing virus type A to precocious behavioral maturation. Scientific reports, 10, 1-12.