Given the questions, I've pulled the following description of our assays
that have been published and available on our websites since 1995. Since I
retired from UM, we're shifting things over to our new company web site,
which is almost ready for launch.
As most of you know, from the early 1970s through the early 1990s, I did a
considerable amount of work for EPA regarding the use of bees as
environmental monitors and for ecological assessments of potential hazards at EPA
hazardous waste sites (i.e., EPA authorized use of bees for this purpose in
1989). One of our tasks was to identify measurement end points that could
provide data for assessments. Here are two of the methods that we produced.
FYI - I realized after posting that the hygienic assay had been
published, that the description in the book had a major error - we do NOT push the
tube into the comb, in fact, we take care NOT to damage the cells. Our
tube for the liquid nitrogen makes a SLIGHT impression in the caps, but does
not break them.
For the following tests - the Brood Survival Test is one of our go to
methods for assessing presence of materials that may poison the brood,
whether a pollutant, natural product from plants bees are foraging, or pesticides.
We will post these to our new web site, with photos. We're still
checking that everything on the site works - I'll send the URL when we're ready to
go. In the meantime, this will provide a summary of our protocols.
Brood Survival Test
For many years, we have used a simple yet effective method to assess brood
survival. We first find a comb where the queen has laid eggs in a patch
that is at least 20 cells wide by 12 cells deep. We mark six rows with
dressmaker pins. For each row, a pin is inserted into a cell on the left side of
the patch and another pin is inserted into the 25th cell to the right of
the first pin. We randomize the colors of the pins marking each row and
record the colors on a data sheet.
The test area consists of 120 cells (six rows of 20). We record the
contents of each cell-empty, honey, nectar, pollen, young larva, old larva, or
pupa. The comb is then returned to the center of the brood nest. Two weeks
later we take the comb out and again record the contents of each cell.
Assuming that a cell started with an egg or young larva, we should find a
pupa in the cell. If not, the pupa either died or was removed by the bees.
Because the bees often try to remove the pins, we mix the colors so that
we can find the original area, even if some of the pins have been pulled out
of the comb. We use a toothpick to remove the caps from any capped cells.
We also record the age of any pupae. We sometimes find pupae that are
underage. In other words, the original egg (or larva) did not survive, but the
queen replaced it soon enough for the replacement to reach the pupal stage.
We have learned never to assume that all of the initial eggs survived.
Capped brood does not guarantee survival. In many industrial areas, we find
that brood losses of up to 80% can occur, even though all of the cells may be
capped.
The empty row (left) is a positive control. At the time of the initial
inspection, we swirl a round dowel in each of the 20 cells of the sixth
(bottom) row. This provides another mark to identify the test area, tests the
ability of the queen to replace lost eggs and serves as a reference for brood
mortality. If no mortality has occurred, all of the pupae should be healthy
and similar in age (right).
Hygienic Behavior
The hygienic behavior of a colony influences its susceptibility to
microbial pathogens, including natural pathogens such as chalk brood or foul brood
and presumably microbial insecticides. Several bee researchers employ a
method advocated by Steve Taber to rank colonies according to speed of
uncapping and removal of freeze-killed pupae. His method consists of cutting out
a piece of brood comb, freezing it overnight in a freezer, and carefully
placing the brood back into the comb.
We acknowledge the usefulness of this approach but found that it takes too
much time to assess large numbers of colonies. We also had problems
getting reproducible results. The act of cutting the comb seems to be sufficient
to stimulate our colonies to remove brood. We’ve had the same problem
using the pin prick method.
Therefore, we developed a modification of the Taber technique. We use
liquid nitrogen to kill the pupae. 80 ml of liquid nitrogen is poured into a
thin-walled metal tube that is lightly pressed against the comb surface. A
cloth wrapped around the base of the tube prevents the nitrogen from leaking
out and freezing additional parts of the comb. We then have to wait about
two minutes for the nitrogen to evaporate and the metal tube to release from
the comb. We then place an acetate sheet over the comb and use a permanent
marker to mark the positions of groups of 20 undamaged cells (cells within
the circle with intact caps). The comb is then placed in the center of the
brood nest. We mark the frame with a thumb tack so we can quickly find it.
Done correctly, this procedure leaves no visible signs of having been
applied other than the slight scoring of the caps caused by the end of the
metal tube.
Twenty-four hours later, we pull out the frame with the tack, index the
acetate on the frame, and count the number of uncapped as well as the number
of cells from which the bees have removed pupae have been removed by bees.
Additional tests conducted in spring 1996 established our recommendation
in our technical report to EPA to freeze several small patches, rather than
one large patch of capped brood to improve the accuracy of the assay. We
suggest a minimum of three small frozen patches, each on a different frame
face (requires 1-3 combs). Hygienic behavior is thought to be controlled by
two recessive genes, one for uncapping, the other for removal of dead or
sick brood. Colony populations are comprised of bees from several
sub-families – they all share the same mother, the queen; but may have different
fathers (drones). Whether these sub-families are intermixed on the frames, or
occur in groups was unknown to us.
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