Final report for ONC16-019
Beekeepers are vital to our agriculture and economy as they provide honey bee colonies for pollination services and honey production. In turn, beekeeper profitability and sustainability is affected by the health of their colonies. The North Central Region (NCR) is the top honey producing area in the U.S., and many commercial colonies are transported from the NCR to other states for crop pollination. Honey bee colonies are dying from a number of causes. Commercial beekeepers report ‘queen failure’ as the top reason for colony mortality through annual surveys conducted by the Bee Informed Partnership. Queen failure will eventually lead to colony death if the bees or beekeeper do not successfully replace her. We proposed to explore potential causes of queen failure in colonies managed by commercial beekeepers in North Dakota and Minnesota. We collected and analyzed 20 pairs of healthy and failing queens to look for factors that contribute to queen failure. We found that sperm count was lower in failing queens and Deformed Wing Virus-B type was higher in failing queens. Results varied among beekeeping operations, indicating that different beekeepers may benefit from different management strategies to lower queen failure.
We collected failing and healthy pairs of queens from commercial beekeeper colonies and had them processed for mating quality, pathogens, and other health measures. We also collected samples to quantify the health of the colony: beeswax to show pesticide exposure, and adult bees to show parasite and pathogen levels in the worker bees. We provided the participating beekeepers with a full report on queens from their own colonies and the overall results. After engaging in a dialogue with these beekeepers, we will work on reaching a broader audience through targeted dialogue, writing articles for the Bee Informed blog and a beekeeper journal (American Bee Journal or Bee Culture), and developing a presentation of the project to be used at beekeeper meetings. This communication will include suggestions for management changes based on the results of this study.
Queen bees in commercial beekeeping colonies were sampled by the University of Minnesota Tech-Transfer Team, experts that routinely assess colony health for 18 commercial beekeepers who operate over 100,000 colonies throughout Minnesota and North Dakota. We worked with five commercial beekeepers over the summer of 2016, each of which wrote a letter in support of this project. Young (less than 6 months old) but failing queens, and same-aged, non-failing queens were collected. Failing queens were identified by the presence of replacement (i.e., supersedure) queens being reared in colonies, poor egg laying patterns (indicative of poor queen health), or both. For each failing queen removed for analysis, a non-failing healthy queen (no replacement queen cells, good egg laying pattern) was also removed for a paired comparison. Each failing and healthy queen pair was either located in the same apiary or nearby apiaries with similar colony and management histories. We collected 20 pairs of queens, or 40 queens total. The beekeepers worked with us to identify both the failing and healthy queens. Live queens were shipped to NCSU Queen & Disease Clinic. We provided the beekeeper with newly mated replacement queens for each sampled queen to ensure that their colonies continued to thrive, and we financially compensated the beekeepers $50 per colony to account for the potential honey production loss from those colonies.
The NCSU Queen & Disease Clinic performed tests on all queens for the following: queen morphometrics (weight, head size, thorax size, spermatheca diameter), number and viability of sperm in each queen’s spermatheca, Acute Bee Paralysis Virus, Black Queen Cell Virus, Chronic Bee Paralysis Virus, Deformed Wing Virus A and B types (DWV-A type, DWV-B type), Israeli Acute Paralysis Virus, Lake Sinai Virus, Nosema spp., trypanosomes, and the insect storage protein vitellogenin. Testing for vitellogenin was not in our original proposal, but it was suggested by one of the beekeepers since vitellogenin (as the main egg-yolk precursor protein) can be an indicator of overall queen health. Queens were immobilized by chilling and their external morphometrics were measured to quantify body size. The spermatheca in the abdomen of each queen was dissected out and stained to assess viability and number of sperm with a Nexcelom Vision® System. Levels of pathogens and vitellogenin were quantified from the remaining queen tissue by extracting RNA for qPCR analysis.
For each colony where a queen was removed for analysis, we sampled adult worker bees to quantify the adult bee infestation levels of Varroa destructor and Nosema spp. These samples were processed at the University of Minnesota. We also took a 3g sample of wax comb (empty of cell contents) from each colony for pesticide analysis. Wax samples were shipped to the USDA-AMS lab in Gastonia, North Carolina for analysis of 170 pesticide compounds. We submitted beeswax from 17 of the paired colonies (34 samples). We intended on processing only 10 pairs of beeswax (20 samples), however we were able to use some of the funds intended for the queen analysis on additional pesticide testing.
Once each type of sample was processed, the results were discussed and shared with the participating beekeepers in a readable report. To quantify the pesticide risk in each colony, hazard quotients were calculated from the beeswax results. Hazard quotients take into account the level of exposure and the LD50 to the bees (methods found in Stoner and Eitzer 2013, PLoS ONE; and Traynor et al. 2016, Scientific Reports ). We compared the metrics measured for each of the queen pairs by using paired t-tests.
We collected and processed all 20 queen pairs and the accompanying samples, except 5 pairs (10 queens) which were lost before qPCR analysis. All queens tested negative for trypanosomes, Nosema spp., Acute Bee Paralysis Virus, Chronic Bee Paralysis Virus, and Israeli Acute Paralysis Virus (Table 1). There were two detections of Lake Sinai Virus in healthy queens, and one detection of Black Queen Cell virus in a healthy queen. Out of the 30 queens analyzed with qPCR, DWV-A type and DWV-B type were the most prevalent viruses: DWV-A type was found in six healthy queens and nine failing queens, and DWV-B type was found in eight healthy queens and 11 failing queens. A summary of the queen analyses by beekeeper can be found in Table 1, and the colony-level results in Table 2.
Paired t-tests showed that two factors were significantly different between the failing and healthy queen pairs. One factor was the live sperm count. Live sperm count was significantly lower in the failing queens (t = 2.69, df = 19, p-value = 0.014). Failing queens averaged 4.3 million live sperm, compared to the 5.3 million sperm average for the healthy queens. While on average the sperm count is lower in failing queens, this difference many not be biologically significant. Queens that are adequately mated are considered to have approximately >3 million sperm (Tarpy et al. 2012, J of Econ Ento), and 4.3 million sperm average is not below the threshold for “poorly mated.” However, six of the failing queens had fewer than 3 million sperm compared to only one healthy queen fewer than 3 million sperm. A low sperm count may have influenced why some of the queens met our definition of failure, but it was not true for all the failing queens sampled. As such, queens may fail for other reasons.
The second factor that was significantly different between healthy and failing queens was a high load of the virus DWV type B. DWV-B levels were significantly higher in the failing queens (3,984,788 viron copies on average) compared to the healthy queens (527,176 average) (t = -2.29, df = 14, p-value = 0.038). However, this difference was driven by DWV levels in only one beekeeper. If that beekeeper was removed from the analysis, the trend no longer held true. DWV-B may have been a cause of queen issues for that beekeeper in particular, but it is unclear if it was an underlying cause in the other operations.
It appears the beekeepers’ operation can account for some variation in the results, meaning different beekeepers appear to have different results. For example, the one beekeeper had high DWV-B type in his failing queens, another had failing queens with low sperm viability and count compared to his healthy queens, and another had much higher pesticide quotients than the other beekeepers in both healthy and failing queen colonies. A larger sample size is needed to confirm the variation in “queen failure” among beekeepers. These results also suggest that broad-scale testing for multiple factors could be useful for individual beekeepers to identify issues important to that specific operation. Different beekeepers may benefit from different management strategies to lower queen failure.
Educational & Outreach Activities
We collected queens from five beekeeping operations and discussed queen failure and the results with those beekeepers. We also discussed the project one-on-one with an additional 18 beekeepers through Tech-Transfer Teams. We presented the project at the Minnesota Honey Producers Association Meeting to an audience of approximately 50 beekeepers on July 16, 2016.
We will further use Tech-Transfer Teams as a bridge between research and extension to translate research results directly to more commercial beekeepers through on-the-farm discussions across the nation. We will continue to present at commercial beekeeper meetings, including the upcoming Minnesota and North Dakota beekeeper association meetings and other regional and national association meetings. Concise and clear articles will be written and submitted for publication in beekeeper trade journals and shared online via the Bee Informed Partnership website.
Preventing the loss of even a small percentage of the failing queens could save a beekeeper thousands of dollars, which could help make their business more sustainable. We identified low sperm count and DWV-B type as potential causes of queen failure, and we communicated verbally and in written reports with the participating beekeepers about the results in their own operations and those in the other operations anonymously. We are continuing this project to investigate if these results hold true in a larger sample size. Once we are confident in the specific factors identified, we will develop possible methods of mitigating the effects of the factor(s) based on ideas that are vetted for feasibility by commercial beekeepers. We have therefore have a high likelihood to impact the management strategies of beekeepers by sharing the results through Tech-Transfer Teams, an industry journal, presentations at beekeeper conferences, and online. Results from this project can be used to direct the hypotheses and methods of further projects investigating queen failure. The success and impact of our project will be evaluated through reporting by commercial beekeepers in ongoing annual surveys conducted by the Bee Informed Partnership. All survey results are made publically available in anonymous format on the Bee Informed website (beeinformed.org).
The participating beekeepers all expressed great interest in their results. For one beekeeper, the testing showed that his failing queens had low sperm count and viability. Because of these results, he stated that he is no longer going add inputs to colonies with poor queens, and will instead replace those queens. This will reduce his overall cost since he will no longer be spending money trying to build-up colonies with queens that will continue to fail. Another beekeeper was struck by the difference in the levels of DWV-B type in his failing queens compared to the healthy queens. He took this as an indication that the virus was a larger issue in his operation than he had previously considered.