Hygienic Behaviour Testing

The hygienic behaviour test is a simple field test that determines a bee colony's inherited defence against disease by measuring how quickly it finds killed sealed brood and cleans it out of the cells. Its most common forms are the pin-killed brood test and the freeze-killed brood test. Colonies with high scores are more resistant to chalkbrood and foulbrood; that is why hygienic behaviour is one of the most valuable indicators in breeding.

What is hygienic behaviour?

Hygienic behaviour is when young worker bees recognise dead, diseased or parasitised brood in a sealed cell by its smell, uncap the cell and throw the brood out. It has two stages: first uncapping, then removal of the brood. In some colonies the first may be strong and the second weak; the test measures both together.

This behaviour is heritable. It was shown in classic studies in the 1960s on lines resistant and susceptible to American foulbrood, and from the 1990s onwards lines selected solely for this trait were developed. It responds well to selection; clear progress can be made within a few generations.

Which diseases do hygienic colonies have an advantage against?

• Chalkbrood (Ascosphaera apis): Because the diseased larva is removed before the fungal spores form, the disease cannot spread. This is where hygienic behaviour brings its clearest benefit.
• American foulbrood (Paenibacillus larvae): Early removal of the dead larva reduces the number of spores and increases resistance; but this does not mean it prevents the disease. This disease is notifiable; if you suspect it, isolate the hive and report it to the official veterinary service / local agriculture authority.
• Varroa (Varroa destructor): Some of the cells in which mites have reproduced are uncapped and cleaned out; mite reproduction is partly slowed. The varroa-specific form is VSH behaviour, described on the "Resistance to Diseases and Pests" page.

What methods are used for the hygienic behaviour test?

Whichever method you choose, use the same method and the same time interval in all colonies. Results from different methods cannot be compared with each other.

How do you do the pin test, step by step?

1. Choose a suitable frame: Take a frame with compact, even sealed worker brood at the pupal stage. The cappings should be light-coloured and flat, not dark.
2. Mark the area: Choose a square area of 10 × 10 cells (100 cells) or smaller. Stick coloured pins in the corners or write on the bar; take a photo.
3. Kill the brood: Pierce each sealed cell from the capping to the bottom with a fine entomological pin. Do not count empty or open cells; note them.
4. Put the frame back: Return the frame to the same place, facing the same way.
5. Count after 24 hours: Count the completely emptied cells. Note separately the cells that have been uncapped but are still full.
6. Calculate the rate: Divide the number of cleaned cells by the number of killed cells and multiply by 100.

How do you do the freeze test?

In the liquid nitrogen method, a metal or plastic cylinder open at both ends is pressed gently into an area of sealed brood and the sealed cells inside are counted. Liquid nitrogen is poured into the cylinder, you wait until the brood is frozen, the cylinder is removed and the frame is returned to the hive. Safety goggles and gloves are essential when working with liquid nitrogen; a special transport container is required.

In the freezer method, a rectangular piece is cut from the brood comb, kept in a freezer overnight, then fitted back in its place in the frame and returned to the hive. Counting is done as in the pin test.

How are the results interpreted?

A common criterion in the freeze test is that a colony that removes 95% or more of the killed brood within 48 hours is considered hygienic. Breeders selecting more strictly look for the same rate within 24 hours. In the pin test, because the brood is easier to find, rates usually come out higher; here it is more accurate to compare colonies with each other rather than use a fixed threshold.

The limits in this table are guiding examples; use them to rank the colonies in your own apiary.

Worked example: An area of 100 cells was marked, of which 4 were empty from the start; so 96 brood were killed. After 24 hours, 90 cells were completely cleaned out and 3 cells had been uncapped but not emptied. The removal rate is 90 ÷ 96 × 100 ≈ 94%. Cells uncapped but not emptied are not counted in this calculation but noted separately; they show the difference between uncapping and removing brood.

What should you watch out for in the test?

• Test at least twice: Leave a week or more between the two tests; take the average of the two results.
• Test during a strong period: Test when there is a nectar flow and the colonies are strong; results drop during a break in forage.
• Test all colonies on the same days: That way weather and forage differences affect everyone equally.
• Do not test a new queen straight away: The workers need to be the new queen's daughters; test at least 6–8 weeks after the queen starts laying.
• Do not unfairly cull a weak colony: A colony with few bees cleans slowly; this is a strength issue, not a genetic one.

How should the test result be used in breeding?

Colonies that score highly in both tests become breeder candidates if their other traits, such as gentleness and wintering, are also acceptable. Rear queens from these colonies and give them drone comb; this also improves the drone pool in your area. Requeen low-scoring colonies with queens from the selected line. Track progress by repeating the same test in the new generation.