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Researchers determine structure of secret hormone that hardens insect by krx14451


									                                                                              Photo by Neil Brake

       Hans-Willi Honegger used the American cockroach, P. americana, to collect the secret hormone
       that insects need to develop hard outer shells

Researchers determine structure of secret hormone
that hardens insect’s outer shells

By Melanie Catania
Published: August 12, 2004

A team of biologists has discovered the structure and genetic sequence of the hormone that
makes insects develop their hard outer shells and allows them to spread their wings. The findings
answer more than 40 years of questions about insect development.

Researchers determine structure of secret enzyme
that hardens insect’s outer shells
                                                           Using the fruit fly, the researchers determined
                                                           the genetic sequence of the hormone
                                                           bursicon, confirmed that it is responsible for
                                                           the hardening of the soft exoskeleton after
                                                           each molt of an insect as it grows into
                                                           adulthood, and discovered that it is also
                                                           responsible for enabling developing insects to
                                                           spread their wings. The research was
                                                           published July 13 in the journal Current
                                                           Biology by Vanderbilt University biologists
                                                           Hans-Willi Honegger and Elizabeth Dewey
                                                           and researchers at Cornell University and the
                                                           University of Washington, Seattle.
                                   Photo by Neil Brake
                                                           Honegger expects this research and ongoing
Elizabeth Dewey and Hans-Willi Honegger in the
                                                           studies to identify the receptor for bursicon to
                                                           open new doors for pest control.

“Bursicon is absolutely necessary for insect survival. When you know the receptor and you know
the hormone, you can produce an inhibitor which fits to the receptor,” he explained. “It would act
only on insects that are in the process of molting, so you could time it precisely to the time that
specific pest insects are molting. This is especially applicable to epidemic outbreaks of pest
insects like migratory locusts which molt synchronously by
the thousands.”

The unassuming fruit fly, Drosophila melanogaster, has
long been a critical player in biological research. The same
characteristics that make it maddening in your kitchen—
small size, prolific reproduction and rapid growth—make it
a perfect model for studying genetics and development. It
has been the focus of research by thousands of scientists
for more than 100 years.

Despite such rigorous study, the genetic structure of one of
the key hormones involved in the fruit fly's development,
bursicon, remained unknown.
                                                                       Drosophila melanogaster
“Bursicon was first discovered in 1935. A study by
Gottfried Fraenkel in 1962 showed its role in cuticle
hardening and darkening,” Honegger said. “We now have the first real information about it,
information that people had about other insect hormones 15 years ago, so we are quite excited.”

All insects must shed their old outer skin or cuticle periodically in order to grow. The new outer
shell then hardens and its color darkens. Both processes take place through the activation of a
series of five hormones. The structure, genetic sequence and biochemical properties of four of
these hormones were known since 1990; that of the fifth, bursicon, was not.

Researchers determine structure of secret enzyme
that hardens insect’s outer shells
                                                                 Using biochemical methods, the
                                                                 researchers set out to determine
                                                                 bursicon's genetic sequence and
                                                                 molecular structure and also to
                                                                 confirm that it indeed triggered the
                                                                 hardening process.

                                                                 In the first phase of the work, the team
                                                                 went to work to determine the genetic
                                                                 sequence of bursicon. Using
                                                                 cockroaches, Honegger's students
                                                                 were able to collect and purify a small
                                                                 sample of the hormone. They sent this
                                                                 sample to a laboratory at Harvard
                                                                 University that chemically sequenced
                                                                 it and sent back four short amino acid
                                                                 sequences of which the sample was

                                                                       Using this sequence, Dewey, a post-
                                                                       doctoral researcher in Honegger's
                                By Roger Hangarter, Indiana University laboratory, ran searches on the
                                                                       genome of the fruit fly and found that
 View a time-lapse series of cicada nymphs going through the           three of the four sequences matched
 molting process
                                                                       the sequence of the fruit fly gene
                                                                       CG13419. She subsequently
compared the sequence to known genomes for other insects and also found matches, leading the
team to determine that bursicon has the same genetic sequence across species.

                           Common Insect Pests That Rely On Bursicon

                         Mosquito                                       Cockroach

                          Locust                                           Aphid

Researchers determine structure of secret enzyme
that hardens insect’s outer shells

                           Ant                                              Moth

                        Termite                                          House Fly
                                                       Images Courtesy of the Agricultural Research Service

The researchers then used the sequencing information to determine the structure of the bursicon
molecule. They found that bursicon's structure makes it a member of a group of molecules known
as the cystine knot proteins. Cystine knot proteins are so known due to their molecular structure,
repeated across mammalian species, of three loops of amino acids linked together in a specific,
unique configuration. Proteins such as growth factors have the cystine knot configuration.

“The exciting thing is that this is the first cystine knot protein with a function that has been found
in insects,” Honegger said. “What you can gather from that is that nature is really very
conservative. It creates the same structure but uses it for different functions.”

Researchers determine structure of secret enzyme
that hardens insect’s outer shells
Honegger and his colleagues then wanted to take their findings to the next level and determine
that the genetic sequence they had found was in fact coding for bursicon.

                                                                                     Courtesy of Willi Honegger

      Identification of the nerve cells in the abdominal ganglion of cockroaches using an antibody
      against bursicon. The nerve cells which contain bursicon also produce another hormone, called
      crustacean cardioactive peptide or CCAP. CCAP is involved in triggering the motor activity that
      allows the animal to crawl out of its old cuticle. Bursicon is labeled in red and CCAP is labeled in
      green. The two figures are overlaid to show that CCAP and bursicon are both in the same nerve

“Based on previous research, we knew that certain nerve cells produce bursicon and that the very
same cells produce another protein, crustacean cardioactive peptide (CCAP),” Honegger said.
“We used a molecular probe that would attach to bursicon messenger RNA and an antibody that
would work against CCAP. From the reaction, we saw that the same cell was producing both. The
molecular probe showed us that we really had the right stuff.”

Honegger's colleague at Cornell, John Ewer, then made transgenic fruit flies by using a “death
gene” that targeted CCAP cells. The cells disappeared, prohibiting the production of bursicon and
confirming that the genetic sequence the researchers had for the hormone was correct.

In the final test, Susan McNabb from the University of Washington looked at mutant fruit flies
whose outer shells showed defects or did not harden completely. She found that all of the
mutants had mutations in the gene they had identified for bursicon.

Researchers determine structure of secret enzyme
that hardens insect’s outer shells
To determine that decreased levels of
bursicon were responsible for the defects
to the mutants' shells, the researchers
used a test previously used to
demonstrate that bursicon levels in the
central nervous system are responsible
for shell hardening and pigmentation. The
shells of blow flies that are treated shortly
after they leave their pupae to prevent
them from releasing their own bursicon
will harden and darken if they are injected
with central nervous system samples from
other flies or insects which are producing

The researchers injected samples of
central nervous systems from the fruit fly
mutants into blow flies that had been
treated to prevent bursicon release. The
shells of the blow flies did not harden nor
                                                              Courtesy of Susan McNabb, University of Washington
darken after the injection as they would
have if they had been injected with central     Three fruit flies shortly after molting. The fly on the left (A)
nervous system samples from normal              is a normal, “wild type.” The other two (B,C) are mutant
flies. These results were consistent with       strains that cannot make bursicon. Note the white color on
                                                the abdomens of the mutants: That indicates their
the theory that the lack of bursicon in the
                                                exoskeletons have not hardened properly. Also note the
fruit fly mutants' central nervous systems      shriveled, deformed wings. The bottom row of photos (D,
was responsible for their defects.              E, F) point out the malformation of bristles on the mutants
                                                (E,F) that indicate their thorax did not expand properly.

The mutants also revealed a surprise:
Not only were their shells not properly formed, but they could not expand their wings.

                                                  “This means that bursicon has a second
                                                 function—not just for hardening of the
                                                 exoskeleton, but also for wing expansion,”
                                                 Honegger said.

                                                 The research was conducted by Honegger and
                                                 Dewey at Vanderbilt; Susan McNabb, Gloria Kuo,
                                                 Christina Takanishi and James Truman at the
                                                 University of Washington, Seattle; and John Ewer
                                                 at Cornell University. It was supported by grants
                                                 from the National Science Foundation, the
Hans-Willi Honegger, Elizabeth Dewey and lab     National Institutes of Health, the U.S. Department
technician Daniel Market                         of Agriculture and a Mary Gates Undergraduate
                                                 Research Fellowship.

                                                - VU -


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