NASHVILLE, Tenn. – 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.
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.
Honegger expects this research and ongoing 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.
"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.
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 composed.
Using this sequence, Dewey, a post-doctoral researcher in Honegger’s
laboratory, ran searches on the genome of the fruit fly and found that
three of the four sequences matched 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.
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."
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.
"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.
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 bursicon.
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
darken after the injection as they would have if they had been injected
with central nervous system samples from normal flies. These results
were consistent with the theory that the lack of bursicon in the fruit
fly mutants’ central nervous systems was responsible for their defects.
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
National Institutes of Health, the U.S. Department of Agriculture and a
Mary Gates Undergraduate Research Fellowship.
Media contact: Melanie Catania, (615) 322-NEWS
Melanie.catania@vanderbilt.edu