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Selecting for Hygienic Behaviour
in Honey Bees

See also Hygienic Behavior by Honey Bees from Far-Eastern Russia

Although the techniques described here can be safe and routine, Liquid Nitrogen is a dangerous substance if handled with less than the greatest of respect.  Please read the entire section on safety, including the links to product sheets.  If in doubt, be sure to get qualified help.

These bees pass the test and are hygienic. Click to enlarge.The brood in the circle was killed two days ago with liquid nitrogen. Not much brood has been removed after 48 hours. These bees fail the test. Don't breed from them. Click to enlarge.The term, 'hygienic', when used to describe honey bees refers to the tendency of some families of bees to identify and remove diseased and dead larvae and pupae from the brood comb and the hive much more accurately and promptly than the norm.  Hygienic behaviour is an easily identified characteristic that can be easily found and quickly reinforced in any strain of bee stock by selective breeding.  Although the expression of this  characteristic by any given hive of bees varies somewhat with time and circumstances, there is a standard quantitative test which is now routinely used to determine whether a specific hive of bees is deemed 'hygienic' -- or not.
Hygienic behaviour is assuming increased importance as its potential to reduce economic losses and to reduce the use of  chemicals in bee management is increasing appreciated.  Indeed, at present, hygienic behaviour is the best defense against the resistant AFB that was first discovered in South America and is now being discovered at many diverse locations in North America.

Freezing brood for the test. Protective clothing and common sense must be used when handling liquid nitrogen. Click to enlargeAnyone raising any queens for any purpose should be subjecting the parental stock to this test, since it is simple to do and the penalty for not doing it could be seriously reduced brood rearing, loss of bees, hives and production.  For queen breeders, lack of a hygienic testing program could result in a serious loss of customers as the beekeeping public catches on to the importance of this trait.  

Steve Taber has been advocating the selection of hygienic stock for as many years as I have read his columns.  He was ignored and even ridiculed for many years until some of the younger researchers took him seriously, picked up on the idea, brought it into the mainstream, and found that it can be a simple, reliable, cheap solution to many of our perennial disease problems.  

Jerry Bromenshenk of UMT liked Steve's idea, but found his method of cutting out a chunk of sealed brood comb, freezing it and re-inserting it too laborious and disruptive.  Moreover he says that any hive that could not notice something wrong after that was really deficient.  He developed a less invasive method using Liquid Nitrogen that soon was adopted and promoted by Marla Spivak in her important work with hygienic bees and which is now the standard method.
If everyone insists on hygienic traits when breeding and buying queens, within a few years diseases like AFB (including SAFB), chalkbrood and sacbrood should be reduced to minor nuisances, but it will take co-operation by all beekeepers.  Here is how everyone can help: 
  • DON'T BUY QUEENS UNLESS THE SUPPLIER CAN TELL YOU CONVINCINGLY HOW THEY STACK UP IN HYGIENIC TESTS.  Consider testing the queens you buy and let it be publicly known if the queens do not measure up when fairly and honestly compared to others.
  • DON'T BREED QUEENS UNLESS THE PARENT STOCK IS PROVEN TO BE HYGIENIC.  Also, test at least representative samples of the offspring periodically.  The genetics of eggs queens lay vary over time..

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What all Beekeepers Need to Know
about Hygienic Queens

We consider a queen hygienic only if her offspring pass the test described below on at least two occasions.  There are various degrees of hygienic behaviour.  Here are factors to consider when selecting a queen supplier:

  1. How hygienic are typical queens from the supplier?
  2. Quality control: 
    1. How seriously does the supplier take hygienic stock selection?
    2. Do they regularly test samples of the queens produced , or just the parent stock? 
    3. How often are the queens tested?  
    4. By whom?  
    5. Using what methods and criteria?
    6. How consistent is the hygienic trait in the producer's queens
    7. How do you know?
    8. Can they produce reports from independent testing by customers, gov't agencies, etc.?

If all the queens in your hives are not significantly hygienic, then you cannot rely on hygienic behaviour to protect you from disease.  Some hives will break down if the disease is in the locale.  Your defense is like a fence with boards missing if any queens do not measure up.  Managing bees with poor hygienic qualities in an environment where diseases are present is like trying to carry water in a bucket with holes.
There are at least three components to hygienic behaviour, and these three components are not all necessarily manifested to any degree in any one hive of bees, or necessarily present in equal measure if they are present.  Bees can be partially hygienic, and variable in how they perform in each category.  The three abilities are 
  1. Finding dead or damaged brood quickly
  2. Uncapping dead or damaged brood
  3. Removing dead or damaged brood from the cells

In addition, bees vary in associated behaviours such as how well they polish cells, clean bottom boards and frames, and groom one another.  Some bees are reputed to even scrub off the paint dots on queens that beekeepers have applied to mark them for easy recognition.  I don't know if this latter conduct is related to the characteristics that result in disease suppression.

A Caveat:

The expression of these characteristics can be affected by time of year, size of the hive, numbers of bees of various ages, queen performance, flow conditions, presence of disease and predators, etc.  

Testing for these behaviours is comparative and it is therefore important that conditions in the hives under test are comparable, or consideration must be made for any differences observed.  

Periodic repetition of the tests is recommended, particularly if many daughters are to be raised from a breeder queen over time.
There are other characteristics besides hygienic behaviour that can be important for disease and pest control.  
  • The larvae of some bees are harder to infect with AFB than others.  
  • Harbo has recently shown that bees can be easily selected  that demonstrate SMR characteristics.  The characteristic seems to be naturally present to some extent in any stock of bees, and can be discovered and selected by relatively simple tests and procedures. SMR bees (Suppressing Mite Reproduction) produce pupae on which -- for some unknown reason -- foundress varroa mites are unable or reluctant to reproduce.  The mother mite enters the cell, but when the bee emerges, the mother emerges too -- sans offspring.
  • Some bee stocks groom one another to remove varroa more thoroughly than other bees.
  • Some bees chew up any varroa they find, thus ensuring the mite cannot climb onto another passing bee.
    •
    For those who want to test their bees for hygienic conduct and cannot justify the cost of a dewar:  it is possible to carry brood frames to an agricultural AI (artificial insemination) station in a nuc, brush off the bees outside (they will likely not like to have bees in their place), do the freezing there, then quickly return the frames to the nuc and take them back to the hives being tested.  As long as the brood is kept warm and at the correct humidity while in transit, the rest of the brood should be okay.  Dairy farms often have liquid nitrogen on hand too, if they are large and advanced enough to do their own AI.  (See also the second article under  Comments)

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    Testing for Hygienic Behaviour
    in Honey Bees

     by Adony Melathopoulos

    Research Update: AFB Resistant Bees
    By Adony Melathopoulos

    Background: The majority of beekeepers in Alberta have bees with genetic traits that make them resistant to American foulbrood (AFB). The problem is that the number of colonies with resistance traits is at too low a level to provide significant protection. How can AFB resistance be brought to levels high enough that beekeepers can reduce their use of antibiotic? Addressing this question is the focus of a co-operative study between Alberta beekeepers and Agriculture and Agri-Food Canada.

    Hygienic behaviour is the most studied genetically determined character honey bees carry that make them resistant to AFB. Colonies carrying the traits that make up hygienic behaviour detect early AFB infections, uncap the cells and then eat up the larvae before the disease has a chance to produce spores. Hygienic behaviour stops AFB in the same way that ploughing a field stops weeds; it stops the AFB before it can form seeds, or more precisely, spores.

    The objective of the project underway in Alberta is to determine the best ways to establish hygienic behaviour among beekeeping operations. The study will compare establishing the character by two methods; 1) introducing queen cells from hygienic selected stock into Peace River nucs for open-mating or 2) introducing mated selected queens from isolated-mated with hygienic selected drones. The change in hygienic behaviour among successive generations will be compared between open-mated queens and close-mated queens. The project will provide beekeepers with guidelines on how to best establish the character within their operations. Early results suggest the character is present at variable levels among Alberta beekeepers and may be present among anywhere from 5-50% of breeders.

    Although the results of the experiment are still in the early stages, most beekeepers can begin testing their breeder stock on their own.  

    The Method: Testing for hygienic behaviour involves freezing a patch of pupated sealed brood with liquid nitrogen. Liquid nitrogen is poured onto frames, freezing and killing the brood, and the amount of brood removed in a 48-hour period is recorded (Figure 1). The liquid nitrogen is confined to a specific spot on the brood using a tin soup can opened on both ends [2-3" diameter ´ 8-15" tall] (Figure 2). The can is driven through the brood into the mid-rib of the frame. The rim of the can must be sawed off and filed to provide a good seal between the frame and the can. A good seal is essential in preventing the leakage of liquid nitrogen from the selected area. The patches of brood selected for the test should not contain more than 10-12 empty cells. Approximately 250-300ml (9-10 ounces) of liquid nitrogen are poured on the brood. The can will require approximately 5 minutes to thaw before it can be removed and the frame replaced to the colony.

    Liquid nitrogen has a boiling point of almost -200°C and care must be taken to prevent frost bite. Protective clothing including heavy gloves, boots and safety glasses should be worn (Figure 3). The liquid nitrogen must be stored in a special tank. Liquid nitrogen and used 20 litre tanks (holds enough liquid nitrogen to test 50-75 hives) are available for from companies supplying artificial insemination services for livestock. Two sources are Westgen in Armstrong, BC (call Roy at 604-530-1141) and Alberta Breeders Service (call Neil at 403 507 8771). Expect to pay around $300 for the tank and $45 for a filling of liquid nitrogen.

    The pictures:

    1. Nitrogen comes in a dewar.  A dewar is basically a big, strong, specially built thermos bottle. 
    2. Protective clothing and common sense must be used when handling liquid nitrogen. Click Here
    3. Modified Campbell's soup being used to isolate a patch of brood for freezing with liquid nitrogen.
    4. Two patches of frozen brood 48 hours after freezing
      1. not hygienic
      2. hygienic. 

    Freezing tests should repeated and only colonies removing 95% of frozen brood for two consecutive tests should be bred from.

    Pictures 2,3,4, &5 and text courtesy Adony Melathopoulos
    Agriculture and Agri-Food Canada, 
    Beaverlodge, Alberta, CANADA T0H 0C0
    Office: +1 780 354 5130, Lab: +1 780 354 5135, Fax: +1 780 354 8171

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    Comments:

    From: Jerry J Bromenshenk [jjbmail@SELWAY.UMT.EDU]
    Sent: May 19, 2001 3:05 PM
    To: BEE-L@listserv.albany.edu 
    Subject: Don't buy any queens...

    Allen Dick posted a nice synopsis of the fast-freeze liquid nitrogen method
    used to assay honey bee colonies for hygienic behavior.

    We developed this procedure under funding from the U.S. EPA and posted it
    to our web pages several years ago.

    Marla Spivak later wrote an article in Bee Culture describing our procedure
    and how it compared to the one described by Steve Taber.

    Steve and others cut out bits of comb, freeze them overnight in a freezer,
    and then return them to the hive. We found this to be too time consuming
    and the act of cutting the comb often induced vigorous house cleaning
    activities. We were never able to get consistent results with this method.

    With the liquid nitrogen procedure and a sharpened tube (we used metal
    flashing rather than a soup can), you get a consistent amount of damage to
    the comb; the liquid nitrogen thoroughly kills the brood, and you don't
    have to try to re-insert a piece of cut-out comb.

    Pat Heitkam, a queen producer in California, was the first to try our
    method in a commercial application mode. I told him about the method at
    breakfast at an ABF meeting in Portland. He had been working with Marla on
    breeding for this trait. Marla thought he would have trouble getting the
    nitrogen, but Pat was able to borrow a Dewar from a local veterinarian, who
    used it in his artificial insemination of livestock. Pat quickly became
    convinced that it was a fast and practical method, and he was able to get a
    Dewar and find a local supplier was happy to sell him the nitrogen (the
    same one supply the nitrogen to the vet). (In the U.S., you can usually
    buy or rent Dewars - check with the companies that sell gases for medical
    or industrial purposes).

    You don't need to freeze a very large area - in fact the test is a bit more
    sensitive if you freeze small areas of brood. But you must thoroughly
    freeze the brood. Medhat was using a double treatment with liquid nitrogen
    to be sure that the brood was frozen. We usually just checked the opposite
    side of the comb. If the brood is frozen solid on the underside of the
    comb, in addition to the area where you poured the nitrogen, you can be
    sure the brood has been killed. But don't use so much nitrogen that it
    pours over the sides of the tube and kills most of the surrounding brood -
    you want a nice, clean delineation as illustrated by Allen and on our pages.

    And one more caution, if you don't think liquid nitrogen is cold, drop a
    rubber ball into it (on a string). Pull it out and toss it onto a concrete
    sidewalk. It will shatter.

    One word of caution - as stated on Allen's site, liquid nitrogen can cause
    serious burns. Protective clothing includes gloves, BUT NOT JUST ANY OLD
    GLOVES. Cloth gloves may be worse than no gloves at all, because the
    liquid will pass right through the fabric and the gloves will hold it
    against your skin. Be sure that the gloves are made of a material that
    will not pass liquids through.

    Jerry
    Jerry J. Bromenshenk, Ph.D.
    Director, DOE/EPSCoR & Montana Organization for Research in Energy
    The University of Montana-Missoula
    Missoula, MT 59812-1002
    E-Mail: jjbmail@selway.umt.edu
    Tel: 406-243-5648
    Fax: 406-243-4184
    http://www.umt.edu/biology/more
    http://www.umt.edu/biology/bees

    And from a private communication discussing this liquid nitrogen
    technique which he and his group developed over a two year period
    as an alternate to the previous methods which involving cutting chunks
    of  comb or pin pricks.  (His LN technique was subsequently adopted
    and popularized by Marla Spivak.):

    ..I am glad people are using the procedure that we developed...

    ...The queen breeders have simplified it - all or nothing. Actually, the
    original test scores the percentage of cells 1) opened, and 2) emptied.
    There are two recessive genes working here (if we believe the geneticists).
    One for uncapping, the other for removal. Real colonies vary along a
    graded response from 0-100%. However, Pat Heitkham, as a practical
    breeder, had to find a quicker metric, so he went with all or none...

    Jerry
    Jerry J. Bromenshenk, Ph.D.
    Director, DOE/EPSCoR & Montana Organization for Research in Energy
    The University of Montana-Missoula
    Missoula, MT 59812-1002

    From: Informed Discussion of Beekeeping Issues and Bee Biology
    Sent: June 16, 2001 3:08 AM
    To: BEE-L@listserv.albany.edu 
    Subject: Re: Don't buy and queens...

    > ...Bob commented about the issue of open
    > mated production queens resulting in a lower level of hygenic
    > behavior or SMR trait. In both cases researchers have done tests and
    > have shown that the selected X nonselected cross shows intermediate
    > levels of the selected trait i.e. not as good as the selected parent
    > but considerably better than nonselected stock. Both were shown by
    > this testing to have much better disease ( hygenic ) or mite ( SMR )
    > resistance than the nonselected stock they were crossed with. Bottom
    > line: even if you don't control the mating you will get some
    > improvement in the first year.

    That is encouraging.

    I would also like to add that the belief that perfection may not be achieved
    immediately -- or ever -- is no reason not to start in that direction.

    For that matter, no one knows exactly what degree of hygienic behaviour (HB) is
    desirable, and if it can be overdone. Maybe, in the extreme, it has a downside.
    Maybe a consistent 50% is all we need. Maybe even 20% across the board (or even
    just eliminating those that score near zero) can make a huge difference -- or
    mean the difference between never seeing any AFB and having a bonfire.

    People are assuming that scoring 100% on the HB test is desirable -- and
    necessary. It is not. I think we must remember that the HB tests are extremely
    rigourous artificial benchmarks, and no one knows what level of HB test
    performance is necessary to get some significant improvement in AFB resistance
    in real life.

    Apparently it does not take much HB to knock out all signs of chalkbrood. It is
    not hard to visualize how *even a little resistance to AFB*, and the resulting
    early cleanup of diseased pupae, could prevent spore formation in hives that
    have only an occasional diseased cell. This could ultimately protect the
    hive -- and perhaps subsequently the whole operation or neighbourhood -- from
    avalanching into AFB breakdown. HB is a finger in the dyke.

    As it stands now, some bees in circulation are very susceptible to AFB. Once
    the hives they occupy break down with a few cells, then the rest of the hive
    gets contaminated and there is a serious risk to surrounding bees, even those
    that do have a higher breakdown threshold. This is due to the high levels of
    AFB spores that have been incubated in the susceptible hives and which get
    distributed in the normal course of bee and human activity. Eliminating
    extremely susceptible bees from the general population by flooding
    neighbourhoods with increased levels of HB will make it much harder for AFB to
    get a foothold.

    AFB starts with one or two hives in a yard breaking down, then that disease is
    spread through the other hives by the beekeeper and the bees. If that initial
    breakdown never happens, then we will never know how we have been saved from
    disaster.

    Black and white thinking and an emphasis on 'perfection or nothing' can keep us
    from many worthwhile projects. In this case perfection is not necessary and
    perhaps not even desirable. A little improvement in resistance could save a lot
    of money, and constant pressure towards the goal of increased HB (together with
    determining an ideal level of the trait) will pay huge dividends over time.

    allen
    http://www.honeybeeworld.com//diary/

    PS: I wonder what those using the current hygienic stocks are experiencing in
    terms of AFB breakdowns and subsequent cleanup. I also wonder what the
    perspective on HB is from those countries which do not permit use of drugs to
    prevent AFB, and if they are using the HB test to improve their stocks. As I
    indicated in a previous post, some Australian bees I have here demonstrate truly
    amazing levels of chalkbrood, so I am wondering if HB awareness is strictly a
    North American phenomenon.

    From: Steve Moye <steve_moye@yahoo.com>
    Date: Sat, 19 May 2001 18:08:31 -0700 (PDT)
    Subject: Don't buy and queens...
    To: bee-l@listserv.albany.edu
    MIME-Version: 1.0
    Content-Type: text/plain; charset=us-ascii

    If you need liquid nitrogen to freeze brood for
    hygienic behavior determination, you can transport it
    in a coffee Thermos. Sources of nitrogen include
    industrial gas suppliers, beef and diary farmers who
    perform artificial insemination and store bull semen in
    nitrogen, dermatologists, and some family practice or
    internal medicine physicians who use it to freeze skin
    lesions, and researchers who use it for a variety of
    purposes.

    You can also transport liquid nitrogen for short
    periods in a styrofoam coffee cup. If the cup were to
    break you could put yourself in danger as the cold
    nitrogen will frostbite your skin.

    From: BOGANSKY,RONALD J. [BOGANSRJ@APCI.COM]
    Sent: May 21, 2001 12:37 PM
    To: BEE-L@listserv.albany.edu 
    Subject: Liquid Nitrogen Safety

    Hello All,

    I have been following the thread on hygienic behavior. If we follow Allen's
    advice (and Steve Taber's before him) a number of folks will be using, or
    attempting to use liquid nitrogen (LIN). I think the information on Allen's
    website is excellent. I just want to talk a little about the safe handling
    of the product.

    There are two major hazards associated with LIN. First, the obvious, cold
    temperature. LIN can freeze flesh on contact. The best protection comes
    from LOOSE FITTING leather gloves thick enough to offer cold protection.
    Leather welding gloves are excellent for this. There are also some
    leather/polyester combination gloves sold for handling cryogenic products.
    It is important that the glove is loose fitting. In the event that you
    spill (and you will) or come in direct contact with the LIN it is important
    to quickly remove the glove. You can shake off a loose fitting glove with
    one hand. If the glove is tight, the time spent trying to remove it can
    allow a burn to occur. (Cryogenic "burns" are painful). Do not use plastic
    or rubber gloves as they offer little thermal protection. When pouring the
    LIN it will spatter and splash, similar to a drop of water on a very hot
    pan. For this reason I highly recommend using safety glasses.

    The other hazard is the liquid to gas expansion ratio. One volume of LIN
    will expand to 697 equivalent volumes of gas. This is a major concern when
    working in a confined space where the oxygen can be displaced by the
    nitrogen resulting in an asphyxiation hazard. If you are working outside,
    or in areas of good ventilation this hazard is greatly reduced. However,
    this expanding gas will build pressure rapidly if confined. There was a
    suggestion that LIN can be transported in a coffee thermos. Although they
    are not sold for that purpose I suppose they work, although I would not use
    a plastic one. At cold temperatures the plastic will become brittle and
    break. Most importantly, DO NOT SEAL THE THERMOS with its cap. A piece of
    loose fitting Styrofoam with a small vent hole can be safely used as a lid.
    This will allow the expanding gas to escape without building pressure while
    keeping the liquid from spilling out. Periodically check to ensure it is
    loose and has not become a frozen plug.

    For additional safety information on liquid nitrogen you can visit the below
    website.

    http://www3.airproducts.com/productstewardship/product-safety/safetygrams/sfgrm7.pdf 

    For a liquid nitrogen material safety data sheet (in Microsoft Word format) visit:
    http://www3.airproducts.com/productstewardship/product-safety/msds/lookup.asp?letter=N 

    To get a faxed copy of the MSDS:
    http://www3.airproducts.com/productstewardship/product-safety/msds/ 

    I hope this information is helpful.

    Ron Bogansky
    Kutztown, (eastern) PA, USA

    Although I hate disclaimers:
    The above information and opinions are my own and are not associated with
    nor do they reflect that of my employer.

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