Honey Bee Breeding Program Design: Testing for Hygienic Behavior

[Music] Hygienic behavior describes the ability of bees to identify and remove sick, damaged, or dead brood from the brood nest. This behavior is tested using a freeze kill brood asset. To perform this test, a section of brood is frozen, killing the brood. The percentage of dead brood removed after 24 hours will determine if the bees demonstrated a hygienic response. A colony will qualify as hygienic if it removes at least 95% of the dead brood within 24 hours in two separate tests. The procedure we are reviewing today requires liquid nitrogen. Liquid nitrogen is stored and transported in a tank called a doer. Doers and liquid nitrogen can be purchased from gas and welding supply stores. Liquid nitrogen is -196° or 321° F. Contact with the skin can cause severe burns. So, safe handling practices are of the utmost importance. Cryogenic gloves, safety glasses, and covering any exposed skin is required for safe handling. Other supplies which are easily obtained include two 2-in metal pastry rings, a tub of 5% bleach solution, a notebook or data sheet, a pen, and a marker. Optional supplies include a camera, insulated cup, two Now we will demonstrate a freeze kill method based on the one used by researchers at the Giovanazo lab at Laval University and Desimal Animal Sciences Research Center in Quebec, Canada. To begin, locate a frame of brood from the colony you wish to evaluate. The ideal frame will have a large area of capp brood with few uncapped cells. For higher resolution testing, uncap a few cells and identify pink or light purpleeyed pupa. Repeat this in a second location on the frame. The rings will be placed over these cells. This ensures that a similar age brood is tested in each colony. Shake or brush the bees from the frame and lay it on top of the inner cover of the colony being tested. Before inserting the rings, it is helpful to dip the bottom of the ring in honey. When the liquid nitrogen is added, it will freeze and form a seal. This keeps the liquid nitrogen contained in the ring during the test. One benefit of thin rimmed pastry rings is that they easily slide through the comb. Position the rings over the area of interest centered over the uncap cells and press firmly. If you are using plastic foundation, you will meet resistance when the ring is in contact with the foundation. When both rings are in place and secure, it is nearly time to add the liquid nitrogen. But first, mark the frame in the location of the test. This makes it easy to find the frame when it is time to evaluate the test. Now, it is time to add the liquid nitrogen. Depending on the type of doer purchased, it may come with dippers that are used to hold samples. The dipper here has been modified to hold a 50ml tube, which makes measuring the liquid nitrogen easier. If you do not have a dipper, liquid nitrogen can be carefully poured into an insulated cup. Adding liquid nitrogen happens in two phases. First, about 35 ml of liquid nitrogen is poured into each of the rings and allowed to evaporate to cool the testing area. This step is critical. If it is skipped, some of the brood will not be frozen and killed. After the first addition of liquid nitrogen has evaporated, pour an additional 40 milliliters of liquid nitrogen into each ring. If pouring is difficult, funnels can be used to control the flow of liquid nitrogen. And push fins can be used to mark the testing locations. Allow the testing area and honey to thaw before removing the rings. Otherwise, the comb and brood will be removed along with the ring. Now return the frame to the brood chamber and document the date, time, and colony number of the test in your notebook. After removing the rings, sanitize them by submerging them in a dilute bleach solution for a few minutes. This will ensure that you are not inadvertently introducing bacteria colonies. Results are interpreted after the frame has been returned to the colony for 24 hours. The 2-in rings make calculations easy as this is about the size of 100 cells per ring. Since two rings are being used, there is a total of 200 cells being tested. Colonies will qualify as hygienic if 10 or fewer cells remain in the two testing areas after 24 hours. Whatever ring size you choose to use, it is important to determine the number of cells inside the testing area so that accurate results can be calculated. This colony removed all of the frozen brood. So, the hygienic score would be 100% for the first test. This colony would need to be tested again at another date and receive another score above 95% to be considered hygienic. This colony has 13 cells in one ring and seven in the other. The score is 90% hygienic response and does not qualify as hygienic behavior. It is important to note that when an operation first starts selecting for hygienic behavior, there may be few colonies that exhibit a response above 90%. This colony demonstrated a very low hygienic response. Less than half of the cells have been removed. Recaning this colony with hygienic stock would be an excellent choice to improve genetics in the apiary. Freezekill brood testing is a useful way to select colonies for breeding hygienic behavior. Hygienic traits are linked to healthier and more [Music]