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ADF 20130111 Progress Report 28 April 2015

Graham Parsons, Dr. Medhat Nasr, Dr. Rob Currie and Dr. Suresh Desai

1. Project Title ADF File Number and Reporting Period

Honey Bee Health: Management of Varroa mites and VirusesADF File # 20130111Reporting for 1 April 2014 to 31 March 2015

2. Specify project activities undertaken during this reporting period.

A) Methodology - include approaches, experimental design, tests, materials, sites, etc. Please note any significant changes from the original work plan will require approval from the Ministry.

Objective 1. “Developing alternative new acaricides to enhance the rotation system for sustainable control of Varroa mites and vectored viruses in honey bee colonies.”

a) Evaluation of HopGuard II as a potential new acaricide for Varroa control. The varroa mite is considered the most serious parasite of honey bees. Recent research reported by Vandervalk et. al. 2014 reported that HopGuard, a biopesticide made from hops (Humulus lupulus L.) extracts might have a potential to be used for varroa mites’ control. HopGuard showed efficacy within 40% under Alberta Conditions. These results were encouraging. It encouraged our team to consider this acaricide for further development and testing a new formulation for applying HopGuard under this objective to enhance the rotation system for Varroa management.

A large field experiments was conducted in Edmonton, Alberta, Canada. The objective of this experiment is to: 1) study the dose response of applying once 1/2, 1, 1.5 and 2 HopGuard strips per 5 frames of bees on Varroa mite mortality and the side effects on honey bees, 2) study the efficacy of applying a second application using the same rates used in study 1 to determine the efficacy of two consecutive applications on Varroa mite mortality and side effects on bees.

The bee population and brood areas and Varroa mite infestation levels were initially measured in 48 bee colonies. A complete randomized block experimental design was used when assigning treatments to account for variations in colony strength, brood area and initial mite infestation. A block of 6 bee colonies (replicates) was assigned randomly to each tested treatment. The tested treatments are as follows:

1. ½ a strip of Hopguard II for every 5 frames of bees applied once.2. ½ a strip of Hopguard II for every 5 frames of bees applied twice, 10 days apart.3. 1 strip of Hopguard II for every 5 frames of bees applied once.4. 1 strip of Hopguard II for every 5 frames of bees applied twice, 10 days apart.5. 1.5 strips of Hopguard II for every 5 frames of bees applied once.6. 1.5 strips of Hopguard II for every 5 frames of bees applied twice, 10 days apart.7. 2 strips of Hopguard II for every 5 frames of bees applied once.8. No treatment.

For finishing treatment the standard acaricide, Apivar was applied to all colonies as a finishing treatment. Colonies receiving one application or no treatment had Apivar placed in them on June 26, 2014. All colonies receiving a second application of Hopguard II had Apivar placed in them on July 6, 2014. Apivar was left in bee colonies for 42 days as recommended on the approved label.

The bee population and the capped brood areas were determined in all experimental colonies on June 24-25, July 3-4, when the final Apivar strip was removed from colonies on August 18-19, 2014. This scoring process involved removal and examination of each frame in the colony, where the square inches of capped brood was determined using a frame fitted with square inches, and the percentage of the frame covered in bees was determined to the 1/4th of the frames.

Samples of approximately 300 bees were collected from brood combs and varroa mites were determined in each sample of bees. Varroa mite mortality was also evaluated by collecting dead mites falling through a screen bottom board fitted with a sticky trap every 1, 2, 3, and 5 days after first application of Hopguard and second application of Hopguard. Then, sticky traps were replaced every 5 days throughout the Apivar treatment period. All sticky boards were examined visually and all collected mites were counted throughout the experimental period to determine the daily mite mortality during the testing and finishing treatment period.

Statistical Analysis: The efficacy of tested treatments was calculated according to the following formula:

Efficacy = ((#mites killed by treatment) / ((# of mites killed by treatment) + (# of mites killed by finishing treatment)))*100

A comparison of frames covered with bees and calculated brood areas was conducted between pre-treatment and after treatment to determine any side effects of applied treatments on honey bees. All data for dose response are currently being subjected for further statistical analyses.

B. Screening new active ingredients for Varroa mite control. A review of literature was conducted to determine potential acaricide to be included in the first laboratory screening test. Two graduate students will be starting this year at University of Alberta to work on this project. Thus, we can continue meeting our objective in the project in addition to the reported first study.

Objective 2. “Assess and determine the level of virulence of different strains of viruses found in bee colonies that are infested with Varroa, mite free, and winterkilled within the province of Saskatchewan.”

This project objective was put on hold due to recent reports of incursion of Varroa mites to this region. The Provincial Apiarist, Geoff Wilson met with beekeepers on May 16th 2014 at which time they reported Varroa mites in the Meadow Lake area. He subsequently verified this report with a visit to the area in June. As a result there is not sufficient Varroa-free bee source for the project’s needs. The co-investigators are currently working on modifying this objective and use the newly developed status of varroa mites to study: 1) the dynamics of the epidemiology of varroa and viruses development after new incursion of mites to honey bee colonies occurred in Meadow lake area, and 2) Varroa – viruses interrelationship to colony survivorship based on Varroa and viruses levels in surviving and dead colonies after winter. This objective will provide more insights on how virus and Varroa populations will spread throughout the population overtime. Meanwhile how the complex relationship can determine the survivorship of honey bee colony.

Objective 3. “Evaluation of virus presence and concentration in bee colonies throughout the year to establish their relation to Varroa populations, treatment time impacts on bee population growth and colony mortality.”

During the month of May and June donor colonies for the project were collected from beekeepers throughout Saskatchewan. The collected colonies were placed in one of five bee yard locations in and around the Prince Albert/MacDowall area of Saskatchewan. During the spring and summer these colonies were evaluated for strength and thoroughly assessed for disease. Numerous inspections were performed for the brood disease American foul brood (Paenibacillus larvae larvae) as such an infection would severely hamper the project.

At the end of July, once the colonies were confirmed to be without any symptoms of American foulbrood, 50 were chosen for inclusion in the experiment. Fifty double brood chamber Langstroth colonies were split with queen excluders after having the bees driven out of the top box with Bee Repel®. Twenty four hours later, after workers had returned to the top box, it was removed to the location of the experiment. This left a queen right donor colony that was not used for the experiment and a queenless, brood and bee filled top box. The 50 top boxes were shook free of bees into two large screened bulk boxes. While the brood chambers were nearly free of bees the frames were sorted between colonies to roughly equalize the brood area of the boxes. Next the two large screened bulk boxes of known weight were then reweighed to determine the amount of bees they contained. Then each of the two bulk boxes was sampled in triplicate for the percent Varroa infection using an alcohol wash. The two bulk boxes were found to have a different % Varroa infection, so one scoop from each bulk box was placed in each of the experimental colonies. Each scoop of bees was weighed with a kitchen scale to 450 grams for a total of 900 grams of bees per brood chamber. The resulting colonies were then screened in for 24 hours. After 24 hours they received a 10 day old queen cell and were unscreened. The colonies were then left for three weeks until August 22nd to give the queens time to hatch, mate and begin laying in the colonies.

After the August 22nd assessment, some colonies were removed from the experiment because of queen issues or other problems with the colonies. This left 43 colonies to be randomly divided into one of 4 treatment groups: Fall treatment, spring treatment, spring and fall treatment and no treatment. The treatment group without Varroa had to be dropped due to the lack of bees for this group (See objective 2 for details). On September 9th and 10th the colonies were first assessed and treated. Assessment included measurement of brood area, counting of bee spaces filled by the bee cluster, collecting 300 bees in alcohol for Varroa sampling, and collection of 30 bees into test tubes for later virus sampling by the University of Manitoba. Treatment groups that required treating (Fall treatment and spring and fall treatment groups) were treated to label directions with Apivar® (amitraz). Sticky board traps were placed under the colonies continuously from September 6th to October 20th and changed every 7 to 10 days. Colonies were assessed again on September 24th, October 6th, and October 20 and 21st. Colonies were weighed on October 23rd and scored for adult population by counting filled bee spaces top and bottom on October 24th. Colonies were them moved into indoor wintering facilities where they were maintained at approximately 5°C and 60% relative humidity. During the experiment all colonies were managed as commercial colonies would be. This included placing and removing honey supers as needed as well as feeding with sugar syrup in preparation for the winter.

University of Manitoba Sample Processing

Project funding for viral analysis started in January 2015. Prior to this honey bee samples were collected by Graham Parsons from 43 colonies and shipped to Manitoba on dry ice. The samples were received on 11th, 26th of September, 8th and 22nd October of 2014 for a total of 172 samples. Samples of 30 bees per colony were stored in -80 °C for further analysis.

In the month of January, reagents and equipment needed for virus analysis were identified and required items were ordered. During the months of January –March, a new protocol was prepared to allow direct quantification of bee viruses (deformed wing virus (DWV), black queen cell virus (BQCV), sac brood virus (SBV), Israeli acute paralysis virus (IAPV), Kashmir bee virus (KBV), acute bee paralysis virus (ABPV), and chronic bee paralysis virus (CBPV),) concentrations that will provide for efficient extraction and quantification of the bee samples that will allow for cross comparison of viral loads between years and seasons. To quantify virus load, the frozen bee samples will be crushed in a mortar under liquid nitrogen according to our standard lab protocol or through automated crushing. The total RNA will be extracted from 30 mg of ground honey bee material using an RNeasy Mini Kit (Qiagen, Valencia, CA, USA) following the manufacturer’s instructions.

RNA samples will be dissolved in molecular grade water in the presence of ribonuclease inhibitor and stored at -80 °C for further analysis. The RNA quantity and purity will be determined by spectrophotometer by measuring the absorbance at 260 nm and 280 nm. An average of 2 μg of total RNA will be reverse transcribed to produce cDNA using iScript™ Reverse Transcription Supermix for RT-qPCR (Bio-rad) according to the supplier’s recommendations.

Currently, the bee samples are being crushed for the RNA extraction. All the sample crushing and RNA extraction should be completed by April 2015 for samples taken in fall of 2014. Real Time PCR reactions will be performed and compared to a standard curve so the number of virus particles can be quantified. The efficiency of each primer set and gBlocks will be validated prior to analysis. The virus quantification by real time PCR will be carried out in triplicate on a CFX96 Touch™ Real-Time PCR Detection System (Bio-rad) using SsoFast™ EvaGreen® Supermix kit (Bio-rad). Viral gene copies will be calculated using gBlocks as the standard curve in Bio-rad CFX manager software. We anticipate that the analysis for the first project year’s bee sampling will be completed by July 1, 2015.

Objective 4 “Developing best management practices for production of healthy nucleus bee colony (3-10 frames of bees) with acaricides’ rotation strategies to replace dead colonies.”

Saskatchewan Replicate

During the month of May and June donor colonies for the project were collected from beekeepers throughout Saskatchewan. The collected colonies were placed in one of five bee yard locations in and around the Prince Albert/MacDowall area of Saskatchewan. During the spring and summer these colonies were evaluated for strength and thoroughly assessed for disease. Numerous inspections were performed for the brood disease American foul brood (Paenibacillus larvae larvae) as such an infection would severely hamper the project.

On July 24th and 25th the nucleus colonies (nucs) were created from the donor colonies. For the 3 frame nucs, 1 frame of bees with brood, 1 frame of bees with honey and one empty brood frame were combined in a 3 frame nuc box. In the 5 frame nucs 2 frames of bees with brood, one frame of bees and honey were combined with one honey filled dark comb frame and one

empty drawn dark brood frame. Frames and bees were screened into the colonies for 24 hours and had 10 day old queen cells added on July 27th.

On August 14th and 15th the nucs were assessed for their queen-right status. The cell was first checked for hatch success, then the colony was checked for all brood stages including eggs, larva and capped brood. All queens, if successfully raised, were found in the colony and marked with green paint on the abdomen for easy identification.

On September 10th the successfully raised nucs were evaluated for brood area, queen-right status and had 300 bees collected into alcohol for Varroa sampling. On September 11th the remaining nucs were randomly assigned to one of four treatment groups: non treatment control, Apivar treated, formic acid treated and oxalic acid treated colonies. All treatments were applied to label direction or to current best management practice. Adult population was assessed by counting the number of bee spaces filled. Colonies were sampled again for Varroa on October 10th, and weighted on October 24th before being placed indoors for winter. Throughout the experiment the colonies were managed as commercially operated nucs would be. This included moving the nucs to 5 or 10 frame boxes as they expanded, and feeding them sugar syrup in preparation for winter.

Alberta Replicate

On June 6- 13, 2014 donor colonies for the project were collected from beekeepers in Alberta, British Colombia and Saskatchewan. The collected colonies were placed in one apiary in Edmonton, Alberta. Bee colonies were examined for diseases, mite levels and Nosema infection rates. On June 16th and 17th the nucs were created from the donor colonies using the same procedure as described in Saskatchewan trial with a small modification. For nucs established with a single brood comb, 1 frame of bees with brood, 1 frame of bees with honey and 3 empty drawn combs were combined in a 5- frame nuc box. For nucs established with 2 frames of brood, in the 5- frame nucs 2 frames of bees with brood, one frame of bees and honey were combined with 2 drawn combs. All newly established nucs were moved to a new apiary. The entrance was screened for 3 days to stop bees from drifting. A newly mated queen tagged with a colored numbered plastic tag was introduced to each nuc on June 20th.

The established nucs were then assessed every two weeks to monitor the colony population build up. The brood areas were determined using a frame with square inches placed on the comb to measure capped brood area. The portion of frames covered with bees and honey were determined to the closest ¼ of the frame. For Varroa mites, the nucs were monitored for Varroa mite infestation in 300 bees collected from brood areas in July 7th, 21st and September 8th. Surplus honey produced was harvested and determined for each colony. On September 11th the remaining nucs were randomly assigned to one of four treatment groups: non treatment control, Apivar treated, formic acid treated and oxalic acid treated colonies. All treatments were applied to label direction or to current best management practice. Colonies were sampled again for Varroa on October 20th. All bee colonies were wintered outdoors. Throughout the experiment the colonies were managed as commercially operated bee colonies including feeding them sugar syrup in preparation for winter. Colonies were not treated with any antibiotics for American Foul Brood but only treated with Fumagillin for Nosema spp.

B) Research accomplishments in the reporting period - describe progress towards meeting objectives. Please use revised objectives if Ministry-approved revisions have been made to original objectives.


This objective is progressing according to plan. Testing Hopguard II as a potential acaricide for Varroa treatment has advanced this objective’s accomplishment. Results showed that there was dose response in mite mortality ranged from 38-54% based on applied dose (Fig 1 & 2). When Hopguard doses of 0.5, 1 and 1.5 strips/ 5 frames of bees applied two consecutive times, the mortality of mites increased to 75%, 87%, and 86%, respectively (Fig 3 & 4).

The bee population and brood areas were not significantly affected. However during the first 1-4 days after applying the Hopguard II strips, almost all bees from one colony were killed in one of the treatments. Additional bees died in some cases that triggered further investigation needed to ensure applied doses are consistent. Data reported to the company and further improvement to strips are considered based on our recommendation to supply consistent dose through the application period. A dose that is safe for the bees and effective against Varroa mites.

A preparation is underway to start spring testing for 2015. In addition the lab screening test will be commenced on 4 new active ingredients for Varroa mite control as a research project for a graduate student at U of Alberta.


As previously mentioned, the source of adequate Varroa free bees for the project no longer exists. As a result this objective was put on hold during this reporting period and could only be continued on a modified basis. If it were to continue only Varroa infested-winterkilled and Varroa infested-not winterkilled colonies could be used, or colonies newly infested with Varroa. Samples currently collected for Objective 3 will fill this criteria, but Varroa free honey bee samples can no longer be found. A subsample of bees used for viral quantification in the experiment in Objective 3, will be retained to test viral strains in case this objective is retained.

Consultation with the project co-investigators and ADF staff will likely be required to determine the future course for this objective.


This objective is approximately one third done and has proceeded mostly to schedule. As mentioned the treatment group without Varroa had to be dropped due to lack of source colonies. Aside from that setback this component has continued according to plan. Colonies previously set up for this experiment will continue to be monitored as well as a new replicate started in the 2015 field season.



Approximately one third of this research objective is done. The first field summer of this field program is complete, with the continued monitoring of the colonies productivity through the next season yet to be done. During the summer of 2015 following of the first nucs established will continue as well as establishment of another replicate of the experiment. Once field work and data analysis are complete, management recommendations from the project will be created.

Alberta Replicate

The first field summer of this field program is successfully completed, with the continued monitoring of the colonies productivity through the next season yet to be done. During the summer of 2015 nucs will be prepared using queen cells vs mated queens thus we will be able to test differences between using queen cells and mated queens in building up bee colonies for wintering and increasing numbers of colonies in a bee operation. Once field work and data analysis are complete, management recommendations from the project will be created.

C) Discussion - Provide discussion necessary to the full understanding of progress made during this reporting period and the relevance of any findings. Detail any major concerns or project setbacks.


Research experiments planned for this objective are in progress as planned. We have taken a jump start to work on Hopguard II that is available in the USA for beekeepers. Modification of the delivery method based on research done in our laboratory, was tested last summer to speed up collecting supporting data for product registration. Once again data provided support to the product. However the efficacy was variable from colony to colony. This variation required to go back to the supplier to improve the dosages to be applied in bee colonies. A different preparation and packaging method is needed. The supplier accepted recommended modification and new strips delivered to be tested in 2015.


As previously mentioned, this component of the research project has to be modified based on current status of the Varroa mites in Lake Meadows. This new modification will be shared with the ADF staff to determine if the modified objective will meet our overall objectives of the project.


Progress has been good on this project objective. Despite the loss of the treatment group “Varroa Free” the remaining experiment has continued as planned. The first summer and winter of the first replicate have been completed. Another replicate in the 2015 summer is planned in addition to continued monitoring and sampling of the 2014 summer replicate. With only partial completion of a full replicate of this experiment, no conclusions can be made at this time.



With approximately one third of the experiment completed it the experiment is running to schedule, although slightly delayed by the slow spring and summer in 2014. At this time the relevance of the experiment and the information resulting from it are too early to yield much toward the final goal of the objective. When one season is complete in the summer 2015 early conclusions could be made from the experiment.

Alberta Replicate

Research experiments planned for this objective are in progress according to schedule. This type of experiment requires a long term for evaluations of the experimental colonies. Once all relevant data collected and analyzed, recommendations will be prepared for beekeepers. Presentations of the results will be shared with beekeepers during field demonstrations and extension talks.

D) List and briefly discuss any interim conclusions

The project has not completed one full field season yet, and without information on colony survival through the winter, limited or no conclusions can be made at this time. Much data has been collected on the experimental colonies, however without information on their survival the data points will provide little meaningful data until they can be put in context with survival.

3. List any technology transfer activities undertaken in relation to this project - include conference presentations, talks and paper published etc.

As the project is in early stages at this point little data that would be immediately useful to beekeepers has yet to be collected and analysed. It is expected that at the end of the first and second field seasons technology transfer and extension activities will continue to increase.

Graham Parsons Extension Activities:

1) Title: ADF Project: Honey Bee Health: Management of Varroa mites and virusesMeeting: at the Saskatchewan Beekeepers’ Association Annual Convention, Saskatoon , SKDate: November 22nd 2014Number of participants: 150 beekeepers from Sk and few from out of the province

2) Title: Beekeeping in Western Canada: Varroa Management and making nucs.Meeting: Educational Day of Canadian Association of Professional Apiculturists (CAPA) Moncton New BrunswickDate: on January 31, 2015Number of participants: 120 beekeepers mainly from Maritimes and some from across Canada

Medhat Nasr Extension Activities:

1) Title: Honey Bee Health: Alternatives for Integrated Pest Management including HopGuardMeeting: British Colombia Honey Producers Association, Vancouver, BC. Date: September 27th. 2014 Number of participants: 200 beekeepers from BC and Western Canada

2) Title: Updates on Hopguard as a potential biopesticide for Varroa controlMeeting: Alberta Beekeepers Commission. Edmonton, ABDate: November 4th. 2014 Number of participants: 90 beekeepers from Alberta and Saskatchewan.

3) Title: Honey Bee Health: Hopguard as a new tool for integrated management of Varroa mitesincluding HopGuardMeeting: Educational Day of Canadian Association of Professional Apiculturists (CAPA) Moncton New BrunswickDate: on January 31, 2015Number of participants: 120 beekeepers mainly from Maritimes and some from across Canada

4. Identify any changes to industry contributions, in-kind support, collaborations or other resources

No changes were identified to the industry contribution, or in kind support. The collaborators may have done less processing of samples and data collection to date than planned due to the changes in Objective 2 and 3 brought about by the lack of Varroa free colonies. If this Objective 2 is modified some of the contributions of time, travel and sample processing may be moved to later in the project.

5. Appendices - include any additional materials supporting the previous sections, e.g. detailed data tables, maps, graphs, specification, lit cited acknowledgments.

Objective 1.

Objective 3

Figure 1 Shaking bees into one of the large screened bulk boxes, for mixing of bees and Varroa mites

Objective 4

Figure 2 Evaluation and sampling of nucleus colonies for Objective 4

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