Last night we had our May meeting for the Minnesota Hobby Bee Keepers
Association (MHBKA)and a few guys were talking about this article written by
two of our well known leaders.  I looked it up today and thought I would
pass it on in light of all the missives about hygenic queens lately...

One more thing, Gary and Marla teach a great 2-3 day short course for
Northern Bee keepers every March (Friday is on Bee Biology).  It was a very
enjoyable weekend, I almost learned more during breaks hanging around and
talking / listening to them than I did in the class...and I see them quite
often at the monthly MHBKA meetings. If you can make it to Minnesota in
March it is well worth the time.


THE HYGIENE QUEEN
by Marla Spivak & Gary Reuter

Hygienic behavior of honey bees is the primary natural defense against
American foulbrood (Park et al., 1937; Woodrow and Holst, 1942;
Rothenbuhler, 1964) and chalkbrood (Gilliam et al., 1983). Hygienic bees
detect, uncap, and remove diseased brood from the combs before the disease
becomes infectious. Hygienic behavior also is one defense against Varroa
mites (Peng et al., 1987), and although it is not the main mechanism of
resistance to the mites (Harbo and Hoopingarner, 1997), it appears to limit
their reproduction and population growth to some degree. Our studies have
shown that it is possible to select for hygienic behavior without
compromising honey production or gentleness (Spivak, 1996; Spivak and
Reuter, in press). The trait can be found in approximately 10 percent of the
managed colonies found in the United States, in any race or stock of bees.
We feel it would benefit the beekeeping industry to have hygienic lines of
bees commercially available.

In this article, we present a simple way of screening colonies for hygienic
behavior. We also discuss some frequently asked questions about the
behavior, and how to breed hygienic colonies.


Using Liquid Nitrogen

For years, we screened colonies for hygienic behavior by cutting out a
section of comb (2 x 2.5 inches) containing sealed brood, freezing it for 24
hours, then placing the frozen comb section in the colony to be tested. If
the test colony was hygienic, the bees would uncap and remove the
freeze-killed brood within 48 hours when tested repeatedly (Taber, 1982;
Spivak and Downey, 1998). Cutting comb sections out of frames is relatively
messy and damages the combs, so we sought a better way of killing brood
without having to handle the combs.

Dr. Jerry Bromenshank at the University of Montana was the first to suggest
using liquid nitrogen (N2) to freeze a section of sealed brood within the
frame. He found that freezing the brood this way was more efficient than
cutting, freezing, and replacing comb inserts. Based on his suggestions, we
conducted several tests to determine how much liquid N2 was necessary to
completely kill the brood, and whether the test yielded the same results as
cutting and freezing comb sections. We are now convinced that freezing brood
with liquid N2 is the best screening procedure found to date for assaying
hygienic behavior.

Liquid N2 is relatively inexpensive and easy to obtain; check with your
local veterinarian or livestock artificial inseminator. It must be kept in
an appropriate tank and securely fastened to the truck during travel to
avoid spillage.

Common sense and several precautions must be used when handling liquid
nitrogen. It has a boiling temperature of -320F (-195.8C)
which means that it is extremely cold and will kill skin (causing severe
frostbite) on contact. Protective clothing including heavy gloves, boots, a
face shield and safety glasses should be worn. The boots should be
sufficiently secure so that the liquid N2 can not be spilled into them.

You will need to construct (or find) a hollow cylinder into which you will
pour the liquid N2 to freeze a circular section of sealed brood. We have
been using a 3-inch diameter cylinder, cut from galvanized clothes-dryer
vent. The cylinder must be at least 4 inches long because the nitrogen will
boil on contact with the brood. The thinner the walls of the cylinder, the
easier it is to press into the comb assuring a good seal.

Nine to 10 ounces (250-300 ml) of liquid N2 is needed to freeze-kill all the
brood (approximately 160 cells) within a 3-inch diameter cylinder. A smaller
amount will not kill all of the brood, leading to erroneous results. Use a
10-ounce or larger Styrofoam coffee cup for measuring and pouring. Other
materials will shatter on contact with the liquid N2.

Select a frame with at least a 3-inch diameter circle of sealed brood
containing fewer than 30 unsealed cells within the circle. Lay the frame
horizontally across a support (i.e. an empty super). Twist the metal
cylinder into the sealed brood until it reaches the midrib. Record the
number of unsealed cells inside the cylinder. Pour a couple ounces of the
liquid N2 into the cylinder and wait for it to freeze the edges or
evaporate. Then pour the remainder of the liquid N2 into the cylinder. Wait
to remove the cylinder until it thaws, which may take three to five minutes.
If you have additional cylinders, you can start the next test while you are
waiting for previous ones to thaw. We put a thumbtack in the top of the
frame to mark the frame and the location of the test on the frame. Some
hygienic colonies clean and repair the comb so quickly that it is hard to
locate the test when you return. Place the frame in the center of the brood
nest.

Remove the frame containing the frozen brood 48 hours later, and record the
number of sealed cells remaining within the circle. When testing a colony
that has been requeened, six to eight weeks must elapse after requeening for
the bees in the colony to be daughters of the new queen.


Frequently Asked Questions

Often we are asked if hygienic colonies tend to have clean bottomboards, or
if they tend to remove debris (such as wax paper, newspaper or cardboard)
from the colony more quickly than other colonies. Mayer (1996) suggested
that if colonies eat grease patties quickly, they might be hygienic.
Removing debris from the hive is a form of cleanliness, but it is not
necessarily a sign that the bees carry the hygienic trait.

Although the common usage of the word hygienic denotes cleanliness, hygienic
behavior is a specific response by the bees to diseased and parasitized
brood. A colony that keeps its hive clean does not imply that it will be
resistant to diseases. Colonies must be screened for hygienic behavior using
an assay such as the one described above. If a colony removes all of the
freeze-killed brood within 48 hours, the colony will probably be resistant
to diseases and will tend to remove mite-infested pupae. To determine
whether they can actually resist the diseases or mites, the colony would
have to be challenged with American foulbrood, chalkbrood or mites.

Another question we encounter concerns the difference between hygienic and
grooming behaviors. Grooming behavior involves an interaction between adult
bees; one bee removes mites or debris from the body of another bee.
Alternatively, a bee may groom herself. Grooming and hygienic behaviors are
different traits, and selecting for one does not imply selection for the
other.

It is assumed by some beekeepers that hygienic behavior is associated with a
high degree of defensive (stinging) behavior. This assumption stems from the
reputation of the Brown line of hygienic bees studied by Rothenbuhler.
Rothenbuhler (1964) showed that stinging behavior and hygienic behavior are
inherited separately. Our experience has shown that hygienic colonies are as
gentle as the stock from which they were bred.


Propagating Hygienic Colonies

Any race or line of bees can be bred for hygienic behavior. We recommend
that bee breeders select for hygienic behavior from among their best breeder
colonies; i.e., from those that have proven to be productive, gentle, and
that display all the characteristics desired by the breeder. A breeder can
get a head start on selecting for hygienic behavior simply by rearing queens
from colonies that do not have chalkbrood.

When colonies are first screened for hygienic behavior using liquid N2, they
may not remove all of the frozen brood within 48 hours. The colonies that
remove the most freeze-killed brood within 48 hours should be propagated by
rearing queens from them. Subsequent generations will remove the brood more
quickly, because hygienic queens from the first generation will produce
drones for the second generation. If the hygienic queens are instrumentally
inseminated with semen collected from drones from hygienic colonies, or are
mated naturally in an isolated area, where all the surrounding drones are
from hygienic colonies, it will be easier to fix the trait in your line of
bees.

Beekeepers should rear queens from unrelated hygienic colonies each year to
avoid the negative effects of inbreeding. In time, if many bee breeders
select for hygienic behavior, the frequency of the trait should increase in
the general population of bees, which will increase the chances that any
queen will encounter drones that carry the trait.

The effects of American foulbrood, chalkbrood and Varroa mites can be
alleviated if queen producers select for hygienic behavior from their own
lines of bees. Because a small percentage of the managed colonies today
express hygienic behavior, it is important for many bee breeders to select
for the behavior to maintain genetic variability within and among bee lines.
Our experience has shown there are no apparent negative characteristics that
accompany the trait. Years of research experience have shown it would
greatly benefit the beekeeping industry if productive, hygienic lines were
available commercially.


Marla Spivak is Professor and Extension Specialist in Apiculture, University
of MN. Gary Reuter is a Research Technician working with Marla.


References

Gilliam, M., S. Taber III and G. V. Richardson. Hygienic behavior of honey
bees in relation to chalkbrood disease. Apidologie 14: 29-39. 1983.

Harbo, J.R., Hoopingarner, R.A. Honey bees (Hymenoptera: Apidae) in the
United States that express resistance to Varroa jacobsoni (Mesostigmata:
Varroidae). J. Econ. Ent. 90: 893-898. 1997.

Mayer, M. Testing for super hygienic bees. Bee Culture 124: 517-519. 1996.

Park, O.W., Pellett, F.C., Paddock, F.B. Disease resistance and American
foulbrood. Amer. Bee J. 77: 20-25. 1937.

Peng, Y.S., Fang, Y., Xu, S., Ge, L., Nasr, M.E. Response of foster Asian
Honey bee (Apis cerana Fabr.) colonies to the brood of European honey bee
(Apis mellifera L.) infested with parasitic mite Varroa jacobsoni Oudemanns.
J. Invertebr. Pathol. 49: 259-264 1987.

Rothenbuhler, W.C. Behaviour genetics of nest cleaning in honey bees. IV.
Responses of F1 and backcross generations to disease-killed brood. Am. Zool.
4: 111-123. 1964.

Spivak, M. Honey bee hygienic behavior and defense against Varroa jacobsoni.
Apidologie 27: 245-260. 1996.

Spivak, M., Downey, D. Field Assays for Hygienic Behavior in Honey Bees
(Apidae: Hymenoptera). J. Econ. Entomol. 91(1): In press. 1998.

Spivak, M., Reuter, G.A. In press. Performance of Hygienic Colonies in a
Commercial Apiary. Apidologie.

Taber, S. III. 1982. Determining resistance to brood diseases. Am. Bee J.
122: 422-425. 1982.

Woodrow, A.W., Holst, E.C. 1942. The mechanism of colony resistance to
American foulbrood. J. Econ. Entomol. 35(3): 327-330.



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