K-State Professors Discover Enzyme Responsible For Creation Of A Beetle's Hard Shell

After a beetle first molts, its exoskeleton is soft and hydrated.Somehow, it dries out and forms a hard, stiff exoskeleton. Since the1940s, scientists have wondered which enzyme among several possiblecandidates was involved in the hardening process.

The K-State researchers have found that by knocking out anenzyme called laccase-2, cuticle tanning, the process of hardening andpigmentation, can be prevented in the red flour beetle, Triboliumcastaneum.

A paper, to be released this week in the Proceedings of theNational Academy of Sciences, presents the research results. TheK-State researchers are Yasuyuki Arakane, research associate inbiochemistry; Subbaratnam Muthukrishnan, professor of biochemistry;Richard Beeman, adjunct professor of entomology; Michael Kanost,professor and head of the department of biochemistry; and Karl Kramer,adjunct professor emeritus of biochemistry.

Kramer said K-State researchers wanted to find out what happensbetween the times when the cuticle is soft and when it is hard. Theystudied the cuticle's composition and how the components interacted togive it stiffness, flexibility and lightness. The main components inthe cuticle are proteins and chitin, which also are found incrustaceans and other invertebrates.

The researchers knew one of two classes of oxidative enzymes,tyrosinases or laccases, is likely responsible for catalyzing theexoskeleton's hardening by cross-linking cuticular proteins, Kanostsaid.

"When we knocked out tyrosinase, everything was normal," Kramersaid. "When we knocked out laccase-2, we prevented tanning from takingplace."

When the laccase-2 gene was not expressed, the newly formedcuticle remained soft and white instead of becoming hard anddark-colored. These results indicated which protein was responsible forthe hard shell's formation, Kanost said.

The identification of laccase-2 as the catalyst for cuticletanning opens up possibilities of targeting this protein as a way ofweakening the beetle's physical defenses against mechanical, chemicaland biological injuries, Muthukrishnan said. Better insecticides couldbe developed as a result of having a more insect-specific target likelaccase-2, Kramer said.

"Gaining knowledge about a molecular process required forinsect development, but absent from humans and other vertebrateanimals, such as cuticle tanning, may be useful for developing new,bio-rational methods for controlling pest insect populations," Kanostsaid.

Armed with this new information, a number of practicalapplications are possible. Materials based on the chemistry of theinsect exoskeleton could be developed to make lightweight materials foraircraft and military armor, Kramer said.

"I sometimes speculate that we might help K-State coach BillSnyder develop better football helmets and shoulder pads for hisplayers," he said.

Collaborative research with scientists at the University ofKansas is in the beginning stages to analyze quantitatively themechanical properties of insect cuticles and to perform cuticle proteincross-linking experiments that are catalyzed by insect laccase, Kramersaid. KU scientists will test the strength of the syntheticcross-linked biopolymers that are created. This could be used for thedevelopment of strong, lightweight materials.

Both Beeman and Kramer also work at the Grain Marketing andProduction Research Center, Agricultural Research Service, UnitedStates Department of Agriculture, in Manhattan.

This research has been supported by a grant from the National Science Foundation.