Hygienic Testing
November 2024
By Leilani Pulsifer
Hygienic behavior in honey bees corresponds to a colony’s ability to detect and remove diseased brood before it reaches an infectious stage (Frost, 2014). This is extremely beneficial when trying to minimize the effects of diseases such as American foulbrood, European foulbrood, and Chalkbrood. Genetic traits, like hygienic behavior, have different levels of heritability - the transmissibility of a trait between parent and offspring. The test demonstrated below is a great way to identify whether your honey bees have hygienic traits that can be passed along to the next generation.
How to: Hygienic testing in Honey Bees | BC Technology Transfer Program: A video tutorial on how to test honey bees for hygienic traits.
Hygienic Testing | Written instructions:
Considerations:
1. Hygienic behaviour testing should be done when there is no nectar flow.
2. If there are wasps or robbing bees, a canopy with a mosquito screen will be necessary to protect the frames.
3. It is possible to work on a number of hives simultaneously.
4. There are approximately 73 cells in a 6 mm diameter PVC tube.
Reagents and supplies (as described or the appropriate equivalent):
1. Liquid nitrogen (purchase from Linde Canada)
Equipment (as described or the appropriate equivalent:
1. Liquid nitrogen dewar 20 to 35 L (Fisher scientific NC1628021). A 20 L dewar will test approximately 40 colonies.
2. Ratchet straps (Stanley S9500 1”x10’ ratchet straps)
3. Cryo-gloves (Unline S-21621)
4. Safety glasses (splash-proof; Fisher scientific 19-151-423)
5. Safety boots (waterproof)
6. Liquid nitrogen dipper (Sigma Aldrich Z150851); 500 ml per colony.
7. POSCA marker
8. Index cards (3x4”)
9. Permanent markers (Sharpie fine point)
10. Camera (phone camera or other)
11. Push pins (Staples; assorted colour push pins)
12. PVC tubes: Pipe PVC outer rim diameter of 6 cm (2 3/8”) and inner rim diameter of 5.5 cm (2 3/16”) and 15 cm (6”) tall. Groove on one side is optional.
13. Foldable table (optional)
14. Newspaper (optional)
15. Watch
16. Field notes
17. Beekeeping equipment:
a. Coverall e. Smoker
b. Veil f. Fuel for smoker
c. Hive tool g. Bee Brush
d. Torch h. Fist aid kit
Procedure
1. Set up your working station: assemble the foldable table, place newspaper on the table, have blankets prepared, and if necessary, a canopy with a screen for protection against sun, wasps, and robbing.
2. Open the hive, locate the queen, and cage her.
3. Select a frame with capped brood. The best frames are the ones with a solid brood pattern. Avoid frames with many empty cells, uncapped brood, or emerging brood.
4. Uncap a few cells to find brood in the pupal stage (white to purple-eyed pupa).
5. Shake the adult bees off the frame. You can use the brush to remove the bees.
6. Label the top bar of the frame with the Posca marker to indicate the colony number and draw an arrow to indicate the side of the frame that will be tested.
7. Place the frame on the table or on a flat surface (like an outer cover on a wooden pallet).
8. Place two PVC tubes on the frame, press and twist the PVC tubes into the selected brood areas.
9. Pour a small amount of liquid nitrogen (approximately 50 ml) into the PVC tube using the liquid nitrogen scoop, this will help to “seal” the PVC tube to the comb. Wait until the initial pour of liquid nitrogen evaporates.
10. After, pour 250 ml of liquid nitrogen into the PVC tube and wait until it evaporates.
11. It takes approximately 15 minutes for the comb to thaw after pouring liquid nitrogen, depending on the ambient temperature.
12. Wait until the comb thaws, and until you can easily remove the PVC tube. Gently twist the tube to remove, do NOT pull on it as this could break cappings.
13. Count the number of open and capped cells and record the numbers in your field sheet.
14. Use the index card to write the identification number of the colony, date and time. Take a photo of the frame with the index card on the side of the tested areas.
15. Return the frame to its respective hive in the center of the brood chamber and record the time in your field sheet.
16. Release the queen and close the hive.
17. Exactly 24 hours later, open the hive, and cage the queen.
18. Remove the marked frame, brush off bees and place frame on the table (or flat surface).
19. Record the number of uncapped, capped, and partially capped cells.
20. Take a photo of the frame with the index card, adding the date and time of its removal from the hive.
21. Return the frame, release the queen, and close the hive.
22. Calculate the percentage of the colony’s hygienic behaviour following the example below for PVC tube 1:
Average number of cells in 6mm diameter PVC: 73
Number of uncapped cells on day one: 3
Total number of capped cells: 73-3 = 70
Number of open cells and partially open cells after 24 hours (not counting the 3 cells that were already open) = 69
Percentage of uncapped cells: (100*69)/70 = 98%
Repeat the same calculations for the second PVC tube and calculate the average. For example, if for the second tube the percentage of uncapped cells was 95%, then the average for this colony will be: (98+95)/2 = 96.5%.
The percentage of uncapped cells (aka removed brood) corresponds to how hygienic your bees are. A colony is considered hygienic if the worker bees removed at least 95% of the freeze-killed brood. In a selective breeding program, it is important to find variability. Beekeepers can start breeding for hygienic behavior by selecting colonies with the highest hygienic performance.
References:
Reuter, GS & Spivak, M. Testing for hygienic behaviour. University of Minnesota Instructional poster #162. Department of Entomology. Available at https://bees.caes.uga.edu/content/dam/caes-subsite/bee-program/images/bees%2C-beekeeping-%26-pollination/other-topics/Poster162.pdf
Frost, E. Testing for hygienic behaviour. Department of Primary industries. November 2014, primefact 1378 first edition. Available at https://www.dpi.nsw.gov.au/__data/assets/pdf_file/0005/535604/Testing-for-hygienic-behaviour.pdf
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We hope this tutorial on testing honey bees for hygienic behaviour has been helpful. If you have any questions, don’t hesitate to reach out to us by emailing info@ttp-bchpa.ca or by sending us a direct message on Facebook (@British Columbia Tech Transfer Program) or Instagram (@BC_TTP).