Final report for LNC21-459

LNC21-459 (project overview)

Project Type: Research and Education

Funds awarded in 2021: $249,998.00

Projected End Date: 10/29/2025

Grant Recipient: Central State University

Region: North Central

State: Ohio

Project Coordinator:

Dr. Hongmei Li-Byarlay

Central State University

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Project Information

Summary:

Wild feral honeybee colonies are one of the best local resources for improving the genetic diversity of honeybees. It is important to educate bee farmers about queen rearing and utilize local resources for sustainable apiculture instead of importing thousands of packages of bees with pathogens and parasites in the United States. The quality of the queen of a honeybee colony is essential for survival. Based on our data, feral queens tend to live longer than commercial queens. We hypothesized that feral queens may tolerate oxidative stress by living longer.

By comparing feral stocks with commercial package colonies, the aims listed below will be achieved:

1) to reveal the quality of flight ability and mating ability, we will compare the flight abilities of virgin queens using flight mills in the lab;

2) to analyze levels of oxidative stress of queen pupae using physiological assays;

3) to reveal the difference in the population, we aim to investigate the genetic variation among different queens, worker, and drones via single-cell sequencing technology with the best accuracy;

4) to educate bee farmers and beekeepers on queen rearing and grafting, the Central State University Research and Extension programs provide hands-on workshops in 2023 and 2024 and customized classes (webinars and field day) on queen grafting, genetic diversity, and queen disease;

5) to work with Purdue University (1862 Land-Grant Institution) and regional queen breeders to select mite biters, and

6) to train students on pollinator health and agricultural research. We work with beekeepers and queen breeders from across the north central region (OH, IN, IL, WV, KY, and PA) on queen breeding efforts.

CSU agricultural and natural resources extension supports farmers in using feral bees with high grooming & mite biting behavior. This research and education program provided by an HBCU and 1890 Land-Grant Institution will improve the genetic diversity of honeybees significantly for the North Central Region. By using local resources of feral colonies, we aim to improve environmental quality by reducing the risks of new pathogens and parasites from importing honey bee colonies to Ohio.

Project Objectives:

Our objectives/outcomes are:

1) test the flight abilities of virgin queens to achieve the outcome of the knowledge of the flight ability;

2) analyze levels of oxidative stress of pupae;

3) identify new transcriptomic variation among different castes;

4) to provide field days and work with regional queen breeders on instrumental inseminations and breeding for mite-resistant/mite-biter stocks;

5) to organize hands-on workshops and field days for local beekeepers to learn about grafting and queen rearing; and,

6) to train underrepresented minority students on apiculture, pollinator health, honeybee genetics, and breeding.

Introduction:

Honeybees are the most important managed pollinators, contributing $15 billion to the US economy. However, managed bee colonies are in a 60% annual decline in Ohio. The ectoparasitic mite Varroa destructor has been the most severe factor for disease and weakness of the colonies since 1987. Promoting mite-resistant bees is one of the most effective ways to mitigate the bee decline. We have a collection of bee stocks from feral colonies that are mite-resistant. However, the limiting factor for local honeybee resources is fewer queens and nuclei available to fulfill the demand of beekeepers in the region. Working with experienced queen producers, we will investigate how to improve the queen quality of honeybee stocks. Our research aims to study the flight abilities of virgin queens, levels of oxidative stress of queen pupae, differentially expressed genes between queen and workers using single-cell sequencing technology, and providing hands-on field workshops and training for beekeepers and bee farmers on queen rearing, grafting, and, work with regional queen breeders on instrumental inseminations and breeding for mite-resistant/mite-biter stocks. Students at Central State University, as first-generation college students or from low-income families, are trained on apiculture, pollinator health, honeybee genetics, and breeding.

Cooperators

Click linked name(s) to expand/collapse or show everyone's info

Dr. Zixiao Zhao
Postdoctoral Associate
Central State University (1890 Land Grant)
Danielle Kroh
dkroh@centralstate.edu
Field Technician
Central State University (1890 Land Grant)
1400 brush row road
wilberforce, OH 45384
Dr. Zhaorigetu Hubhachen
zhubhachen@centralstate.edu
Postdoctoral Associate
Central State University (1890 Land Grant)
1400 brush row road
wilberforce, OH 45384
Dr. Rameshwor Pudasaini
rpudasaini@centralstate.edu
Postdoctoral Research Associate
Central State University (1890 Land Grant)
1400 brush row road
Wilberforce, OH 45384

Research

Hypothesis:

Queen bees from high-grooming stocks have a set of genetic markers for selection, and different levels of oxidative stress and flight abilities compared to low-grooming or commercial stocks.

Materials and methods:

Figure 1. A demonstration of one flight mill in our lab.

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. Six virgin queens from each treatment (feral or commercial) were used, and each queen was tested for six separate trial days. Feral stocks were from our university apiary. The non-selected commercial colonies were package bees from GA. All flight mills were located in the same room of CSU Bee Research Field Lab, with constant light (546.2±1.0 lx) and air temperature (23±2 ∘C). Six virgin queens per treatment group (feral queen vs commercial queen) was used in this experiment.

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

For outcome 3, we ask whether we can identify new genetic markers for queen quality using cutting-edge technology. We have prepared queen and worker bee samples from high-biting stock and non-selected commercial bees, prepared single-cell sequencing libraries using the 10x Genomics Chromium controller (Traniello et al., 2020). The sequencing was carried out at the Singulomics company.

For outcome 4, we ask whether we can change more bee farmers ' views on local bee resources, sustainable queen rearing in Ohio. We organized two-days workshop and field demo with 18-25 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 received the certiﬁcations from Central State University.

For outcome 5, we held a week-long field work with Heartland Honey Bee Breeders Cooperative (HHBBC) and Kentucky Queen Bee Breeders Association (KQBBA) each year on instrumental insemination events and work with queen breeders in the North Central Region to promote regional efforts on breeding for high mite biting and mite-resistant stocks. The field day events were on May 26-30, 2025, May 27-31, 2024, June 1-6, 2023 with drone and queen availabilities. Breeders exchanged genetics of queens from different feral colonies (KY and OH) with high biting or chewing rates. Queen breeders collected semen, conducted instrumental inseminations, and discussed 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. Fifteen CSU (1890 Land Grant Institution) undergraduate students were trained on how to perform flight mill experiments, assays for oxidative stress, and prepare RNA samples for the sequencing experiments. The training prepared students for a future career in sustainable apicultural and agricultural research. Figures 3 and 4 show examples of our student training for research.

Figure 3. Undergraduate student Heaven Strachan was presenting her research at the Entomological Society of America 2025 in Portland, OR.

Figure 4. Undergraduate student Camiyah Woods was doing a lab experiment (CSU, 2025).

We have submitted our research results as one revised manuscript for peer-reviewed publication, 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 beekeepers associations).

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.

Research results and discussion:

Regarding the objective 1 on the flight ability, for the year of 2022, we have started the grant as the first year. As to the objectives/outcomes, we have worked on establishing a protocol to test the flight abilities of virgin queens, hired a postdoctoral researcher to work on the genomic variations of queens for breeding; provided a field day with regional queen breeders on instrumental inseminations and breeding for mite-resistant/mite-biter stocks; organized hands-on workshop and field day for local beekeepers to learn about grafting and queen rearing; and trained 2 CSU students on apiculture, pollinator health, honeybee genetics, and breeding.

For the year of 2023, we have established a lab protocol for the flight ability experiments, and equipped with 6 flight mills in the molecular lab and 4 mills in our field lab. As to Objective 1, CSU Student Kristan Major has established a protocol for testing fly abilities of virgin queens. The comparison is between virgin queens handled before instrumental insemination (II) and after II.

Group	Distance	Total Time	Halt Time	Flying Time	Mean Velocity	Velocity Max
Non-Inseminated Queen	90.1637m	340.814s	13.128s (4)	327.686s	0.13793m/s	0.521
Inseminated Queen	63.7743m	481.898s	38.467s (3)	443.431s	0.14314m/s	0.44248

She also collected data and tested 6 queen bees between high and low biting colonies of CSU stocks.

Group	Distance	Total Time	Halt Time	Flying Time	Mean Velocity	Velocity Max
Low Biting Queen	240.332m	580.295s	33.385s	546.91s	0.40193m/s	0.45073
High Biting Queen	12.8805m	339.263s	559.036s	-219.773s	0.09349m/s	0.45663

Group	Distance	Total Time	Halt Time	Flying Time	Mean Velocity	Velocity Max
Low Biting Workers (Trial 1)	119.381m	5359.04s	37597s	-32238s	0.22658m/s	0.44624
High Biting Workers (Trial 1)	140.743m	51.5s	77964.8s	-77913.3s	0.35666m/s	0.77757
Low Biting Workers (Trial 2)	878.215m	85215.7s	483.591s	84732.1s	0.30881m/s	0.67273
High Biting Worker (Trial 2)	530.615m	11676.5s	3814.92	7861.54s	0.27799m/s	0.81178

For the year of 2024 summer, CSU student Laverne Ambrister was able to collect worker bees from both high and low grooming colonies, and worked on the comparison of flight abilities between the two stocks. Student has presented her summer research at our lab's Research Symposium in July 2024. The slides with data are attached here.

Laverne Ambrister Research Presentation 2024 Summer

We faced challenges of a low number of worker bees available for the testing. In the summer of 2025, students tried to optimize the protocol more, and aimed to collect more queen bees from both high and low grooming colonies to compare their flight ability and their oxidative stress for objective 2. However, with the temperature changes during the field season, we were not able to collect more queens to test.

For the summer of 2025, CSU students Laverne Ambrister and D'Anastacia Hutchinson were able to collect and mark worker bees from both high and low grooming colonies. The data is showing below:

	Flight Distance	Flight Time	Mean Velocity	Max Velocity
High biting (x̄)	1,521.32	4,356.46	0.34	0.45
Low biting (x̄)	1,524.93	5,773.62	0.34	0.38
F (1, 7) statistic	0.00	0.11	0.01	2.00
p-value	1.00	0.76	0.91	0.20

With the limited data we collected, we did not find a significant difference in flight distance, flight time, mean velocity, or maximum velocity between high and low biting colonies. Student D'Anastacia Hutchinson presented her research at the 2025 Entomological Society of America National conference at Portland, Oregon. The presentation is attached here.D'Anastacia Hutchinson's ESA Presentation

for Objective 3, our goal was to study the gene expression and genomic difference among different reproductive castes, including the queen (reproductive), worker (non-reproductive), and drone (reproductive) castes by using the spatial transcriptomics and single-cell RNA-seq technologies.

Bees: We used three distinct A. mellifera colonies as biological control: H119, H148 and H156. Each one contained two deep hive boxes. Each box contained 8 frame of bees. These colonies were managed at the Central State University campus by State Road 42 in Xenia, Ohio. Two workers and two drones, and two sister queens were collected. Workers and drones were directly collected from the hive. For queens, neonates within 3 days old were grafted into queen cups and placed in a queenless cell builder, which consists of workers from the same colony. Before adult queens were hatched, the queen cells were transferred to individual queen cages and were continued to be raised in the queenless hive for the experiment.

Brain dissections and cell disassociation: The brain dissection and cell disassociation were followed by the revised protocol (supplementary 1). Brains of two individuals from the same caste of the same hive were pooled to increase the number of cells. To minimize the cell mortality, the brains were dissected at a 4 °C walk-in chamber and immediately proceeded to cell disassociation.

Library construction and sequencing: To maximize the number of cells being analyzed, we used all the dissociated brain cells from 2 brains to construct 10X single-cell libraries. 3’ Single-cell RNA-seq libraries were prepared using Chromium Next GEM Automated workflow (10X Genomics). The prepared libraries went through quality control (QC) procedures and were sequenced at Singulomics using the Illumina NovaSeq X Plus platform.

Data analysis: The software Cell Ranger (version 8.0.1) was adopted for analyzing 10X single-cell data. Sequences underwent a series of quality control and filtering process and aligned to A. mellifera reference genome (Amel_Hav3.1). Cell counting and unique molecular identifier (UMI) counting were performed to determine cell and gene expression profiles within each cell. The,n cells were clustered into categories based on the similarity of gene expression profiles.

Library construction: A total of 9 libraries were constructed from pooled 2 brains of each group (Table 1).

Table 1. Brain single-cell libraries

Sample Name	Colony	Caste	i7 Index	i5 index	Load volume (ul)	cDNA mid QC (pg/ul)	Library concentration (ng/ul)
H148Q	Hive 148	Queen	ATGGCTTGTG	GAATGTTGTG	32	3220	17.2
H148D	Hive 148	Drone	TCTTACTTGC	TGACCTCTAG	32	846	12.7
H148W	Hive 148	Worker	CGTCAAGGGC	TAGGTCACTC	32	2620	11.7
H119Q	Hive 119	Queen	TTCTCGATGA	TGTCGGGCAC	32	3590	17.9
H119D	Hive 119	Drone	TCCGTTGGAT	ACGTTCTCGC	32	912	31.7
H119W	Hive 119	Worker	ACGACTACCA	ACGACCCTAA	32	4250	22
H156Q	Hive 156	Queen	AGAATGGTTT	GAGGGTGGGA	32	2170	51.2
H156D	Hive 156	Drone	ATGGGTGAAA	CTTGGGAATT	32	2460	61.4
H156W	Hive 156	Worker	TCGTCAAGAT	GCAACTCAGG	32	3070	16.3

Sequencing: NovaSeq X Plus platform provided sequencing with deep sequence depth. About 300 – 450 million reads were generated (Table 2).

Table 2. Sequencing depth of each single-cell RNA-Seq library

Sample	Number of Reads
H148Q	315,626,297
H148D	340,892,933
H148W	337,192,257
H119Q	288,232,877
H119D	430,845,989
H119W	373,787,953
H156Q	330,185,359
H156D	406,436,566
H156W	421,692,134

Reads alignment, cell and UNI counting and cell clustering: Sequencing reads from all libraries report high rate of mapping to A. mellifera reference genome. The overall mapping rates are between 84.20% and 96.30%. The majority of confidently aligned reads were mapped to exons, while a small portion were aligned to introns and intergenic regions. There are 2-10% Reads were mapped to antisense strands, suggesting RNAi regulation mechanisms took place for those genes (Table 3).

Table 3. The alignment result of single cell RNA-Seq sequencing reads

Sample	Mapped to Genome	Mapped Confidently to Genome	Mapped Confidently to Intergenic Regions	Mapped Confidently to Intronic Regions	Mapped Confidently to Exonic Regions	Mapped Confidently to Transcriptome	Reads Mapped Antisense to Gene
H148Q	90.40%	77.30%	9.50%	16.10%	51.70%	63.60%	4.10%
H148D	88.80%	75.60%	8.40%	10.30%	56.90%	63.90%	3.20%
H148W	95.10%	76.20%	9.80%	15.60%	50.80%	61.50%	4.70%
H119Q	84.20%	74.40%	10.50%	15.90%	48.00%	58.40%	5.30%
H119D	90.50%	84.90%	9.80%	8.90%	66.20%	71.40%	3.60%
H119W	91.70%	86.50%	11.70%	18.60%	56.20%	69.60%	5.10%
H156Q	95.00%	73.00%	12.50%	13.80%	46.70%	51.10%	9.40%
H156D	95.40%	92.30%	6.40%	2.50%	83.30%	83.50%	2.30%
H156W	96.30%	93.40%	7.60%	7.20%	78.60%	82.80%	3.00%

All the samples showed similar total number of gene detected, suggesting that the same set of genes were expressed regardless the differences of colony and caste. The number of living cell called from CellRanger varies from 2,000 (H148W) to 81,695 (H119D). Cells from each sample show gene number median from 228 to 1661 and RNA molecule from 4,228 to 152,992. This is probably due to the variation among samples during sample and library preparations It is predicted that sample with less viable cell tend to show lower number of cell and more genes and molecules within each cell.

Table 4. Cell and UMI counting results for each sample

Sample	Estimated Number of Cells	Mean Reads per Cell	Median Genes per Cell	Valid Barcodes	Sequencing Saturation	Total Genes Detected	Median UMI Counts per Cell
H148Q	23,669	13,335	1,626	97.10%	31.40%	11,826	3,675
H148D	32,593	10,459	681	96.80%	47.90%	11,812	2,270
H148W	2,205	152,922	1,661	96.30%	83.00%	11,548	8,158
H119Q	68,165	4,228	556	96.40%	30.30%	11,839	1,189
H119D	81,695	5,274	342	98.10%	50.50%	11,732	1,602
H119W	69,287	5,395	589	97.80%	46.10%	11,860	1,380
H156Q	17,913	18,433	876	96.40%	77.00%	11,736	1,829
H156D	51,514	7,890	228	98.40%	42.70%	11,407	3,568
H156W	63,251	6,667	273	98.20%	52.80%	11,659	2,209

Based on the similarity of expression profile, the cells of each sample can be clustered into several groups. Our data demonstrates except two samples shows low number of clusters are likely due to the low number of cell count. Other presented 14-23 clusters, based on the algorithm.

Table 5. Number of clusters claimed by Cell Ranger, based on expression profile similarity

Sample	# of Cluster
H119D	21
H119Q	23
H119W	23
H148D	14
H148Q	23
H148W	5
H156D	18
H156Q	7
H156W	19

We are working on a new manuscript to publish the results of Objective 3.

For Objective 4, we have successfully organized four queen workshops for beekeepers from Ohio and nearby regions in June 22nd, 24th, August 11th-12th 2022, May 25th -26th, 2023, May 16th -17th, 2024, and May 22-23th, 2025. We changed bee farmers' views of local bee resources, sustainable queen rearing in Ohio. Two-day hands-on workshop with about 20 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 took quizzes, and with a grade of 80% or higher, they received certiﬁcations from Central State University.

All participants and beekeepers were learning how to do queen grafting during the queen rearing workshop of 2025 in CSU Bee Research Lab.

A participant /beekeeper was learning how to graft a larva to a queen cup during the queen rearing workshop of 2024

Feedback and survey from participants are attached here, showing that 93% of participants are satisfied or very satisfied with our workshop.

Post-Survey summary data 2024 Pre-Survey morecommnents 2024

2025 CSU 6th Queen Workshop Pre-Survey (Li-Byarlay Lab) jan2026

2025 CSU 6th Queen Workshop Post-Survey (Li-Byarlay Lab) jan2026

For objective 5, we held 5 day long field days with Heartland Honey Bee Breeding Coop (HHBBC) and Kentucky Queen Bee Breeding Association (KQBBA) in May 30-June 3rd 2022, June 1st -6th 2023, May 27th - 31st, 2024, and May 26-30, 2025 on instrumental insemination events and worked with queen breeders in the North Central Region to promote regional efforts on breeding for mite biters and mite resistant stocks. Breeders exchanged the genetics of queens from different feral colonies with a high biting rate. Queen breeders collected bee semen from CSU high biting stocks, conducted instrumental inseminations, and met to discuss how to improve the breeding efforts as a group.

For objective 6, we have trained 15 undergraduate students on honeybee genetics, genomics, physiology, and pollinator health. They were trained on the lab protocols to perform experiments of ﬂight mill experiments, TBARS assays, and preparing DNA and RNA molecular samples for sequencing experiments. Their career goals are in sustainable apicultural and agricultural research.

One preprint of our data is deposited in BIORXIV with a title of "Genetic Variation in Honey Bee Queen Reproductive Performance: Implications for Colony Growth and Sustainability", Zhaorigetu Hubhachen, et al., bioRxiv 2025.05.09.653176; doi: https://doi.org/10.1101/2025.05.09.653176.

Research conclusions:

The overarching goal of this project was to evaluate the queen bee quality of high mite-biting and grooming behavior in honey bees using physiological or behavioral measurements, while simultaneously strengthening regional capacity for sustainable queen breeding. Specifically, we sought to (1) quantify flight performance of queens and workers from high- and low-grooming stocks, (2) establish molecular tools to investigate caste- and reproduction-related gene expression, and (3) translate research findings into practical breeding and management outcomes through education, training, and collaboration.

To address these objectives, we developed standardized flight mill protocols for both queens and workers and established the necessary laboratory and field infrastructure. Flight performance was measured across multiple years in virgin queens, instrumentally inseminated queens, and workers originating from colonies selected for high or low mite-biting behavior. Although early datasets were limited by sample size and field constraints, the optimized protocols implemented in later years allowed for statistical comparisons of flight traits in bees. These analyses showed no significant differences in flight distance, flight duration, or flight velocity between high- and low-biting worker or queen stocks, indicating that selection for mite-biting behavior does not negatively affect worker flight performance under the tested conditions. This result supports the hypothesis that mite-biting traits can be incorporated into breeding programs without compromising key aspects of worker mobility and foraging potential.

We also tested the oxidative stress of queen bees between high and low biting breeding stocks, but the data collected were limited due to technical difficulties. We plan to continue to work on this research question in our future projects. In parallel, we applied single-cell RNA sequencing to characterize brain gene expression across reproductive castes (queens, workers, and drones). High-quality sequencing data were generated from nine libraries across three colonies, revealing consistent gene detection and robust cellular clustering. These results establish a foundational molecular resource for understanding caste-specific neural and reproductive biology and provide a platform for future mechanistic studies linking behavior, reproduction, and resilience to environmental stressors.

The project achieved substantial applied impact. Four annual queen-rearing workshops and multiple multi-day instrumental insemination field events trained beekeepers and queen breeders in sustainable queen production, grafting, and selection for mite-resistant traits. Over 90% of participants reported satisfaction with the training, and certified participants adopted local queen rearing and breeding practices that reduce reliance on imported queens. These practices are expected to improve colony survival and reduce Varroa treatment costs, supporting long-term farm and apiary sustainability.

Overall, the project met its primary objectives by demonstrating that mite-biting selection does not incur measurable flight performance penalties, establishing advanced genomic tools for honey bee research, and facilitating tangible improvements in regional breeding capacity and beekeeper education. These outcomes collectively advance sustainable apiculture and pollinator health in the North Central region.

Participation summary

10 Farmers/Ranchers participating in research

15 Others participating in research

Education

Educational approach:

The educational approach of this project integrated experiential learning, stakeholder engagement, and workforce development to translate research findings into practical outcomes for beekeepers and future professionals. Education was delivered through a combination of hands-on workshops, field days, and mentored research experiences. Annual queen-rearing workshops provided beekeepers with direct training in grafting, sustainable queen production, swarm trapping, and identification of mite-biting behavior, emphasizing locally adapted breeding strategies. Multi-day instrumental insemination field events facilitated peer-to-peer learning among queen breeders and promoted regional collaboration and genetic exchange.

Undergraduate students were trained through mentored, inquiry-based research, gaining hands-on experience with flight performance assays, molecular biology techniques, and data analysis. Students presented findings at institutional symposia and national scientific conferences, reinforcing communication skills and professional development. Surveys and assessments were used to evaluate learning outcomes and guide iterative improvement. This integrated educational model ensured that research, training, and outreach reinforced one another, accelerating adoption of sustainable apicultural practices while developing the next generation of pollinator scientists and practitioners.

Pictures are from annual queen-rearing workshops.

In 2023, we have 20 participants from OH and nearby states.

Here are the surveys to show the demographics and change of views of our participants.

2025 CSU 6th Queen Workshop Pre-Survey (Li-Byarlay Lab) jan2026

2025 CSU 6th Queen Workshop Post-Survey (Li-Byarlay Lab) jan2026

Pre-Survey morecommnents 2024

Post-Survey summary data 2024

Student D'Anastacia Hutchinson's 2025 ESA Presentation

Student Heaven Strachan's 2025 ESA Poster Presentation

Project Activities

2022 CSU Queen Rearing workshop

2023 CSU Queen Rearing workshop

2024 CSU Queen Rearing workshop

2024 bee research mini - sympsoium

2023 student presentation

Educational & Outreach Activities

1 Curricula, factsheets or educational tools

1 Journal articles

10 On-farm demonstrations

1 Online trainings

1 Published press articles, newsletters

3 Tours

4 Webinars / talks / presentations

4 Workshop field days

Participation summary:

100 Farmers/Ranchers

3 Agricultural service providers

15 Others

Education/outreach description:

11/9/2025 The connection between mite-biting ability and flight performance, 2025 ESA, Portland, OR;
10/17/2025 Oxidative Stress in Honey Bees and Small Carpenter Bees, BeeCon 2025 virtual talk;
11/20/2024 Bee breeding and IPM for better pollinator health Northeastern IPM Center webinar, invited virtual talk, Northeastern IPM center;
1/9/2024 Genes and Genomics underlying the Social Behavior of Honey Bees University of Florida Dept of Biology seminar Gainesville, FL invited talk;
9/28/2023 Genetics and breeding of mite-biting bees for resilience to Varroa mites 2023 USDA Pollinator Health PD Meeting virtual talk;
2/15/2023 New insights into the grooming and biting behavior of honey bees from the mandible transcriptomics, Kentucky Queen breeding Association workshop, invited talk;
1/18/2023 Genetics and Breeding of Mite-Resistant Honey Bees Stocks in Ohio and the Midwest, Central Ohio Beekeeper Association invited talk;
11/13/2022 Testing new physical control on Varroa mites in honeybee colonies Joint ESA/ESC/ESBC annual meeting Vancouver, Canada;
10/1/2022 How Do I Become a Bee Scientist: the ABCs and XYZs of Women Doing Bee Science, Bee Culture Conference, Medina, OH, invited talk;
4/25/2022 Mandibles from mite-biting bees: a multifaceted approach, Entomological Society of Ameria International Branch symposium virtual;
4/25/2022 Differences in Varroa Mite size: Feral vs Commercial Honeybees, Entomological Society of Ameria International Branch symposium virtual;
6/24/2022 CSU queen rearing workshop at CSU,
5/31/2022 Queen insemination field day, CSU campus, with Heartland honey bee breeding coop and Kentucky Queen Bee Breeding Association.

Learning Outcomes

56 Farmers/Ranchers gained knowledge, skills and/or awareness

20 Agricultural service providers gained knowledge, skills and/or awareness

Project Outcomes

80 Farmers/Ranchers changed or adopted a practice

Key practices changed:

Bee farmers and beekeepers in Ohio learned how to do grating and queen rearing using local resources
Bee farmers and beekeepers in Ohio learned about the basic principles on how to do queen breeding
Bee farmers and beekeepers in Ohio learned how to make queen cell starter and finisher colonies.
Bee farmers and beekeepers in Ohio are educated on honeybee genetics, behavior, queen bee genetics and biology.

2 Grants applied for that built upon this project

1 Grant received that built upon this project

2 New working collaborations

Success stories:

testimony of beekeepers bobbie oct 2023

Quote from an Urban Beekeeper Dayton Ohio:

"The information gained from the 2024 workshop was invaluable in helping us to better understand the biology of queen rearing. The classroom instruction was clear and concise also, the field demonstrations provided great hands-on experience."

Recommendations:

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Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the author(s) and should not be construed to represent any official USDA or U.S. Government determination or policy.