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 Elisabeth 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.
Journal
Current Biology