The Case of the Missing Bees Bees wax

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					     The Case of the
      Missing Bees
        How scientific sleuths at Penn State
         are helping to solve the mystery
          Story and Photos by Steve Williams

                                                                        PHOTO: ISTOCK PHOTO

18                                             Penn State Agriculture
                                                                                            Dennis vanEngelsdorp

              “The bees are a little defensive today. I think you might need some protective gear,”
              Dennis vanEngelsdorp tells an apprehensive writer making his first visit to a honey bee apiary. Van-
              Engelsdorp, the Pennsylvania Department of Agriculture’s acting state apiarist, was inspecting commer-
              cial hives along the Susquehanna River as part of an effort to assess the spread of a new, potentially
              disastrous ailment, Colony Collapse Disorder (CCD). This was the latest stop in what promises to be
              a long journey—with Penn State researchers in the lead—through a complicated scientific puzzle.

                  VanEngelsdorp and his team were count-                   This apiary belongs to beekeeper David Hacken-
              ing bees and developing brood (larvae) and look-        berg, who runs a large, migratory operation. In late
              ing for signs of brood disease. Samples were taken      fall 2006, Hackenberg transported a tractor-trail-
              to be examined for mite infestations and nosema,        er load of 400 hives to a pepper grower in Florida.
              a known disease of bees. Frozen samples would           Just another pollination job—or so he thought—for
              be analyzed for viruses and other organisms, and        some of his nearly 3,000 hives. Hackenberg’s hon-
              comb and pollen checked for nutritional quality         ey bees travel year-round, producing honey in New
              and pesticide levels.                                   York and pollinating apples and pumpkins in Penn-
                  “USDA will do the varroa mite analysis, and         sylvania, blueberries in Maine, and vegetables and
              David Tarpy at North Carolina State is getting          fruit in Florida.
              bees for genetic and protein analysis,” vanEngels-           He returned a few weeks later to check on the
              dorp says. “Diana Cox-Foster at Penn State will         hives. As he pumped smoke into the hives to calm
              get frozen bees to check for pathogens, and Penn        the bees—a common practice among beekeepers—
              State’s Maryann Frazier will analyze wax and pol-       Hackenberg became aware of a strange, dead si-
              len for pesticides. It’s a collaboration of experts.”   lence. Anyone who’s ever been around hundreds

Winter/Spring 2008                                                                                                           19
            Researchers in the
            college and across
             the country would
             begin focusing on
                 three potential
           culprits: pathogens,
                chemicals, and
          nutritional stressors.
of beehives knows how loud the buzz-
ing can be. He opened the first hive,
then the others. In every one, the adult
bees were gone. Vanished. Their newly
hatched brood abandoned.
     Hackenberg’s first thought was that
he’d done something wrong. The same
line of thinking initially kept other bee-
keepers from coming forward with simi-
lar experiences. But something felt dif-
ferent. Not only were the bees gone, no
dead bees could be found anywhere. In           the College of Agricultural Science’s Di-    on three potential culprits: pathogens,
40 years of beekeeping, he’d never seen         ana Cox-Foster in November 2006.             environmental chemicals, and nutrition-
anything like it.                                   “It would have been easy to assume       al stressors.
     “I got on the phone and started ask-       the situation was a result of a pesticide-
ing questions,” he says. “I called beekeep-     application error, a heavy infestation of    Identifying Pathogens
ers, inspectors, and scientists all over the    mites, or some other stressor common         Cox-Foster began discussing possible
country. I made so many calls that our          to bees,” entomologist Cox-Foster says.      scenarios with vanEngelsdorp, who is
cell service provider called to apologize for   “But it was hard to dismiss Dave’s insis-    also a Penn State extension entomolo-
a billing error. They told my wife it had to    tence that something different was hap-      gist. “We received samples from failing
be an error; I’d surpassed the 5,000-min-       pening. He’s a respected and responsible     colonies all over the country and tested
ute monthly limit—that’s 83 hours in one        beekeeper.”                                  them for all known viruses and bee dis-
month, talking on the phone.”                       Fingers were being pointed at every-     eases,” she says. “This little handful of
     But many credit Hackenberg’s per-          thing from cell-phone radiation to pes-      bees had almost every bee virus, often-
sistence, as well as his stature in the bee-    ticides to divine rapture. Within several    times bacteria, and fungi living in them.
keeping industry, for getting things mov-       months, researchers in the college and       But we couldn’t say that any in particu-
ing. His calls eventually brought him to        across the country would begin focusing      lar was the culprit, because we could find

20                                                                                                                  Penn State Agriculture
Facing page (top left): Beekeeper
David Hackenberg dons a bee veil.
Above: At Hackenburg’s apiary along
the Susquehanna River, a survey team
led by Dennis vanEngelsdorp inspects
colonies awaiting transport to their
next pollination job. Right: Due to the
lack of flowering plants nearby, Hack-
enburg’s nearly 14 million bees must
be fed a dietary supplement of sugary
liquid, placed in 55-gallon drums.

the same organisms in seemingly healthy
bees. So we wondered if there was some-
thing new, something unknown to us,
that was affecting them.”
    Around that time, Cox-Foster heard

Winter/Spring 2008                        21
                                                                                                                 the technology….” She trails off with a
                                                                                                                 laugh, no doubt recalling that—at that
     Why It Matters                                                                                              time—no funds had been allocated for
                                                                                                                 working on CCD.
     Beekeepers and crop growers have been stung by the loss of honey bees to
                                                                                                                      Time passed. The problem worsened.
     Colony Collapse Disorder and other ailments. But why should the rest of us
                                                                                                                 In January 2007, during the nation-
     care? Consider these facts:
                                                                                                                 al meeting of the American Beekeeping
     • Honey bee pollination is credited with helping to produce a third of the                                  Federation in Austin, Texas, a group of
       nation’s diet.                                                                                            researchers discussed the collapse of colo-
     • More than 3.5 million acres of crops in the United States depend on honey                                 nies happening nationwide. To systemat-
       bees for pollination.                                                                                     ically gather samples and bring the right
                                                                                                                 expertise together, they formed a work-
     • Crops that require or benefit from honey bee pollination include apples,
                                                                                                                 ing group, eventually co-chaired by Cox-
       peaches, pears, pumpkins, squash, cucumbers, cherries, blueberries,
                                                                                                                 Foster and USDA’s Jeff Pettis. “The Na-
       raspberries, strawberries, peppers, squash, soybeans, almonds, cashews,
                                                                                                                 tional Honey Board agreed to pay for 10
       and sunflowers.
                                                                                                                 of the analyses using Lipkin’s methods,”
     • Nationwide, honey bee pollination is worth about $15 billion to the food                                  she recalls. “I e-mailed him, explaining
       supply.                                                                                                   that it was a potentially important eco-
     • Honey bee pollination contributes about $65 million to the value of crops in                              nomic problem, and asked if he’d consid-
       Pennsylvania.                                                                                             er doing it.”
                                                                                                                      Lipkin agreed.
     • Pennsylvania’s $51 million apple crop—the fourth largest in the country—
                                                                                                                      Fate seemed to have intervened as
       is completely dependent on insects for pollination, and 90 percent of that
                                                                                                                 well. The Honey Bee Genome Proj-
       pollination comes from honey bees. The value of honey bee pollination to
                                                                                                                 ect had just finished sequencing the ge-
       the state’s apple crop is nearly $46 million.
                                                                                                                 nome—the complete genetic composi-
     • Honey bees also pollinate many native plants in the ecosystem.                                            tion—of the honey bee and published
     • More than 700 tons of honey is produced in Pennsylvania annually.                                         the data in October 2006. Using bioin-
                                                                                                                 formatics on powerful computers, Lip-
     Source: Mid-Atlantic Apiculture Research and Extension Consortium
                                                                                                                 kin compared raw data from the samples
                                                                                                                 to the honey bee genome data. RNA
                                                                                                                 strands that were classified as part of a
                                                                                                                 bee could be eliminated, and what was
                                                                                                                 left were things that should not be pres-
                                                                                                                 ent. A bioinformatics tool known as
                                                                                                                 BLAST (Basic Local Alignment Search
                                                                                                                 Tool) made it possible to rapidly search
                                                                                                                 and compare nucleotide and protein da-
                                                                                                                 tabases and eventually identify the for-
                                                                                                                 eign organisms present in the affect-
                                                                                                                 ed colonies. By the time this work was
                                                                                           PHOTO: ISTOCK PHOTO

                                                                                                                 finished, researchers had identified 18
                                                                                                                 pathogens in bees from CCD colonies.
                                                                                                                      As Cox-Foster and her collaborators
                                                                                                                 reported in the October 12, 2007, issue of
                                                                                                                 Science, one pathogen in particular, Israe-
                                                                                                                 li acute paralysis virus (IAPV), appeared
about a National Academy of Medi-                     health professionals and the Department                    in almost every case, making it a prime
cine meeting in Washington, D.C., on                  of Defense.                                                suspect as the cause of the collapse, as a
emerging infectious diseases. She and                     “He introduced a new method his                        marker for some other cause, or perhaps
vanEngelsdorp attended a presentation                 lab developed for detecting unknown                        as the last straw that broke the back of al-
on new methods used to detect disease.                pathogens,” Cox-Foster says. “The tech-                    ready highly stressed colonies.
The talk, “Emerging Tools for Pathogen                nique allowed for fast sequencing of en-                        And bees are carrying a lot on their
Surveillance and Discovery,” given by Ian             tire organisms. We thought maybe we                        backs. “This is a complicated problem
Lipkin, professor of epidemiology at Co-              could figure out if there was indeed an                    with so many variables,” says Cox-Foster,
lumbia University’s Mailman School of                 unknown pathogen, using his methods.                       “and we need to quantify what is hap-
Public Health, was geared toward public               Then he mentioned the cost for using                       pening. IAPV may be a factor because

22                                                                                                                                       Penn State Agriculture
Entomologist Diana Cox-Foster (right)
and doctoral student Rob Anderson
inspect newly placed colonies in a
greenhouse at University Park. Re-
searchers are hoping to induce a col-
ony collapse in a controlled environ-
ment to help unravel how it happens.

mites suppress the bees’ immune system
and make them more susceptible to dis-
ease. Worldwide, 19 bee diseases have
been described, and we don’t know how
many others might be out there.”
    To determine if IAPV is the cause or
one of many stressors working together,
researchers will try to re-create a collapse
in a controlled environment. “We’re us-
ing healthy bees from Hawaii produced
especially for this research,” says Cox-
Foster. “We have to be certain they are
‘clean’ bees, don’t have varroa mites, and
have not been treated with miticides.”
Scientists are exposing these test colonies
to IAPV in controlled greenhouse envi-
ronments to study the virus’ effect.             for young, developing bees—and found a        dues from products that have been can-
    Fungi are being looked at as well.           pretty broad range of substances, including   celed. We’re screening for at least 175
Since early samples showed high levels           insecticides, fungicides, and herbicides,”    active ingredients.”
of fungi in CCD colonies, the research           says Maryann Frazier.                              Analyzing samples for such a broad
team asked Penn State mycologist Da-                  One class of pesticides under close      range of chemicals posed technical and
vid Geiser to join the investigation. “You       scrutiny by beekeepers and the press are      logistical challenges. The sophisticated
could see that there were a lot of fungi         neonicotinoids, which are known to be         analytical equipment that could handle
on the bees,” says Geiser. “The big ques-        quite toxic to honey bees. Mullin be-         the large number of samples did not ex-
tion was whether fungi are the ultimate          lieves a broader look is needed. “We’re       ist at Penn State or even in Pennsylvania.
cause or playing a significant role in           seeing a large sweep of active pesticides     After unsuccessful inquiries to commer-
CCD.” Right now, researchers don’t               in pollen,” he says. “Some are ingredients    cial testing labs, an agreement to examine
believe fungi are the cause of CCD but           still in commercial use, others are resi-     samples eventually was reached with the
instead are opportunistic threats that
appear in already weak colonies.

Environmental chemicals
and pesticides
On pollination jobs, bees come into con-
tact with a variety of insecticides, fungi-
cides, and herbicides used on crops. While
scientists don’t think pesticides are the sole
cause of CCD, they believe chemicals con-
tribute to the problem. Extension bee spe-
cialist Maryann Frazier is collaborating
with pesticide toxicologist Chris Mullin
and insect physiologist Jim Frazier to inves-
tigate the link between pesticides and the
general decline in honey bees. “We tested
samples of honey bee pollen—the food

Toxicologist Christopher Mullin is
looking at potential links between
CCD and the presence of pesticides
in bee pollen.

Winter/Spring 2008                                                                                                                     23
USDA National Science Laboratory in              A technician from Mullin’s lab moni-     habitat changes, they aren’t eating as well
Gastonia, North Carolina, under the di-      tors small cages, each containing 10 bees,   as they once did. An agricultural and
rection of chemist Roger Simonds.            and records observations every 24 hours.     suburban landscape ethos focusing on
    “The lab normally tests food products,   Each group has been exposed to a differ-     the removal of weeds as well as fencerows
such as cream, pork, and oranges, which      ent chemical. Mullin is looking at main      and other areas that once offered diverse
are fairly easy to squash up and analyze,”   pesticide ingredients found in hive sam-     and continual pollen sources has almost
Mullin says. “Pollen is more of a chal-      ples to quantify the effect they have on     eliminated natural food for bees. As a
lenge. Bee pollen is a mix of all kinds of   bees, especially when the substances in-     result, beekeepers must artificially feed
hard, colored particles containing pig-      teract with each other.                      their bees between pollination jobs.
ments and other ingredients—including                                                         “A beautiful green lawn is a desert to
bee saliva. Just figuring out how to pre-    Nutritional Stressors                        a bee,” Frazier points out. “So are farm-
pare the samples was a challenge. It was a   Contaminated or not, pollen is the nu-       lands without weeded hedgerows and fal-
blessing to be associated with that lab.”    tritional lifeblood of bees, but thanks to   low fields. This affects wild pollinators as

                                                                                          The rapid response and
                                                                                          quality of research is a
                                                                                          testament to the built-in
                                                                                          capacity of our research
                                                                                          well; butterflies, moths, bumblebees, and
                                                                                          other insects that pollinate also are in
                                                                                               To keep their colonies strong and
                                                                                          well-nourished, beekeepers are experi-
                                                                                          menting with changes in the artificial di-
                                                                                          ets they feed their bees. “Bees are gener-
                                                                                          alist pollinators and benefit from a varied
                                                                                          diet of pollen and nectar to provide di-
                                                                                          verse amino acids, which are the building
                                                                                          blocks needed for colony growth and re-
                                                                                          production,” explains Frazier. “Research
                                                                                          enables us to look at new diets that can
                                                                                          improve bee nutrition.”

                                                                                          Managing hives for disease
                                                                                          Frazier also has begun to integrate emerg-
                                                                                          ing knowledge about CCD into her ex-
                                                                                          tension programs and classroom teaching
                                                                                          to make sure the industry and the public
                                                                                          have as much current and usable informa-
                                                                                          tion as possible. One new recommenda-
                                                                                          tion is changing how beekeepers use and
                                                                                          reuse combs in their hives.
                                                                                              Early surveys of collapsed colonies
                                                                                          revealed that hives were heavily laden
                                                                                          with pathogens, which could potential-
                                                                                          ly re-infect new replacement colonies.

                                                                                          Extension bee specialist Maryann Fra-
                                                                                          zier talks to students in an apiculture
                                                                                          class about colony management. Stu-
                                                                                          dents have access to the latest emerg-
                                                                                          ing information about CCD.

24                                                                                                                Penn State Agriculture
“Beekeepers used to take pride in saying         economic viability, we don’t see a crowd      collaboration and speed in which a sci-
they’d had a comb for 25 years,” says Fra-       of people lining up to replace them.”         entific paper was published outlining the
zier. “But we have found those combs to              A bright side to this story is how rap-   metagenomic survey was impressive.”
be a reservoir of disease and possibly pes-      idly beekeepers and researchers have re-          Bruce McPheron, director of Penn
ticides. We’re encouraging people to not         sponded. A little more than a year has        State’s Agricultural Experiment Station,
reuse comb materials over long periods.”         elapsed since beekeeper David Hack-           also was encouraged. “The rapid response
    On a related front, college researchers      enberg started making phone calls, and        and quality of research is a testament to
are collaborating with Penn State’s Ra-          much has been accomplished. As the cri-       the built-in capacity of our research in-
diation Science and Engineering Center           sis unfolded, scientists across the country   stitutions,” he says. “We hire creative
to determine if and how radiation works          in government, industry, and land-grant       people who are prepared to tackle un-
to sanitize a hive and disrupt the collapse      universities mobilized. Interdisciplinary     expected problems. We began here with
cycle. Preliminary results are promising.        teams collaborated. The system worked.        just one person focused on bees, but that
                                                     “This was an unusual case,” mycolo-       did not hinder our ability to respond.”
What the future holds                                                                              At Penn State, scientists are still look-
With the onset of colder weather, reports                                                      ing, still responding, still working to un-
of collapses are on the rise. Until more                                                       ravel the mystery of the missing bees.
is known about the CCD phenomenon,
researchers can’t predict what will hap-                                                       Faculty and staff referenced in this article
pen in the coming months. With the                                                             are Diana Cox-Foster, professor of entomol-
potential for a continuing and exponen-                                                        ogy; Maryann Frazier, senior extension
tial decline in bees, beekeepers are                                                           associate in entomology; David Geiser,
struggling and growers are worried.                                                            associate professor of plant pathology and
     VanEngelsdorp suggests caution in                                                         director of the Fusarium Research Center;
affixing blame to any one cause. “One op-                                                      Christopher Mullin, professor of entomol-
eration we are monitoring has already lost                                                     ogy; Bruce McPheron, associate dean
30 percent of its bees, which mirrors what                                                     for research and graduate education and
happened last year,” he says. “Whether it’s                                                    director of the Pennsylvania Agricultural
CCD or other known problems is still a                                                         Experiment Station; and Dennis vanEn-
question. People are quick to jump on the                                                      gelsdorp, senior extension associate in
CCD bandwagon. We are working with                                                             entomology and acting state apiarist for the
USDA to develop a systematic protocol                                                          Pennsylvania Department of Agriculture.
for sample collection so we know exactly                                                            Other Penn State researchers actively
what we are looking at and can rule out                                                        studying CCD and/or bee health include
collapses from known causes.”                                                                  Liwang Cui, associate professor of entomol-
     Frazier already has heard from several                                                    ogy; James Frazier, professor of entomology;
large beekeepers who had significant col-                                                      Edward Holmes, professor of biology and
lapses in the fall. “We expect things to                                                       Eberly College of Science Distinguished Se-
worsen over the winter,” she says. “Large                                                      nior Scholar; and Nancy Ostiguy, associate
beekeepers are going out of business over                                                      professor of entomology.
this. And since this is a small industry to                                                         This research is being supported by
start with, the impact of even a few clo-                                                      Hatch Act research funds from the federal
sures would be heavy. My sense is that                                                         government, in addition to funds from the
                                                 Research technician Sara Ashcraft
this is going to be a very, very big prob-                                                     Pennsylvania Department of Agriculture,
                                                 works in Chris Mullin’s lab inoculating
lem this winter, and we are going to lose        bees with a wide range of pesticide           the U.S. Department of Agriculture, the
beekeepers. They just can’t sustain these        ingredients to help determine wheth-          National Honey Board, and a gift from
kinds of dramatic losses.”                       er they play a role in CCD. Bees are          Häagen-Dazs in support of pollinator
     Adds Cox-Foster: “We think IAPV             kept in groups of ten in small cages          health and research.
is here to stay. If it’s extremely virulent it   and are monitored daily.                           Other institutions and agencies address-
could burn itself out, but that could be                                                       ing or collaborating on various aspects of
bad news for the beekeeping industry if          gist Geiser says. “Priorities were shifted    Colony Collapse Disorder include Colum-
colony losses cause bee populations to           and work undertaken long before any           bia University, the University of Arizona,
drop below a level of economic viability.        formal structure or system of grant-fund-     the University of Illinois, the University of
Pollination services for crops such as al-       ed research could be put in place. The        Delaware, North Carolina State Univer-
monds, blueberries, and apples are com-          collapse of bee colonies across the coun-     sity, the University of Montana, and the
ing from a very small number of opera-           try was a big, potential crisis and needed    United States, Pennsylvania, and Florida
tions. If those outfits can’t maintain their     immediate attention. The scope of the         departments of agriculture.

Winter/Spring 2008                                                                                                                       25

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