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

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.  

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

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 did face challenges of low number of worker bees available for the testing. In the summer of 2025, student will optimize the protocol more, and aim to collect more queen bees from both high and low grooming colonies to compare their flight ability and their oxidative stress for objective 2. 

for Objective 3,  we modified the goal 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-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 same hive were pooled to increase the number of cells. To minimize the cell mortality, the brains were dissected at 4 °C walk-in chamber and immediately proceeded to cell disassociation.

Library construction and sequencing:

To maximize the number of cell being analyzed, we used all the disassociated brain cells from 2 brains to construct 10X single cell library. 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 sequenced at Singulomics using 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 profile within each cell. Then cells were clustered into categories based on the similarity of gene expression profiles.

Progresses and Results:

Library construction:

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

Table 1. Brain single-cell libraries

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

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 were taken place for those genes (Table 3).

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

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

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 cluster claimed by Cell Ranger, based on expression profile similarity

For Objective 4, we have successfully organized three queen workshops for beekeepers from Ohio and nearby regions in June 22nd, 24th, August 11th-12th 2022, May 25th -26th, 2023, and May 16th -17th, 2024. We changed bee farmers on their view 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 high, they had received certifications from Central State University. The picture on the left was our successful workshop from 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

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 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 genetics of queens from different feral colonies with 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 5 undergraduate students (K Clark, C Woods, K Major, L Ambrister, K. Briscoe) on honeybee genetics, genomics, physiology, and pollinator health. They were trained on the lab protocols to perform experiments of flight mill experiments, TBARS assays, preparing DNA and RNA molecular samples for sequencing experiments. They career goals are in sustainable apicultural and agricultural research.