Felton, working with Richard O. Musser, the lead author on this paper and Sue M. Hum-Musser, graduate students at University of Arkansas; Herb Eichenseer, University of Arkansas post doctoral fellow now at Pioneer Seed Company, Gary Ervin, postdoctoral fellow now at Mississippi State University; and J. Brad Murphy, professor of horticulture, University of Arkansas; and Michelle Peiffer, research assistant, Penn State, identified glucose oxidase as an active ingredient of caterpillar saliva. Glucose oxidase converts the simple sugar glucose into gluconic acid and hydrogen peroxide.
"While glucose oxidase is produced in many biologic systems and glucose oxidase extracted from fungi is used in diabetic test strips, this is the first time this enzyme has been found in caterpillars," Felton says Caterpillars produce saliva in two large salivary glands that excrete through the spinneret located in the lower jaw. This organ is not located in the mouth. As the caterpillar goes along, it puts down watery saliva as it feeds.
To test that the saliva actually inhibited plant defenses, the researchers cauterized the spinnerets of some caterpillars and placed them on leaves of common commercial tobacco plants, while they placed unaltered caterpillars on other leaves. After the corn earworms fed for 24 hours, the leaves continued to grow for another three days before they were harvested and tested. The leaves fed on by unaltered corn earworms had 26 percent less nicotine content than those fed on by caterpillars that did not secrete saliva. To show how increased nicotine affects the growth and health of caterpillars, pieces of the leaves fed on by intact and altered caterpillars were fed to newly hatched corn earworms. Those eating leaves from intact caterpillars fared much better than those eating the high nicotine leaves fed on by altered corn earworms.
The researchers then tested to see if glucose oxidase was the actual compound suppressing the leaf's defense mechanism. They cut holes in leaves to mimic caterpillar feeding and applied purified active glucose oxidase, unpurified salivary gland extract, inactive glucose oxidase and water. The purified glucose oxidase and the saliva extract both suppressed nicotine production to about the level of a leaf that had not been cut or chewed. To test the products produced when glucose oxidase reacts with sugar, the researchers looked at gluconic acid and hydrogen peroxide. The gluconic acid reduced nicotin production by 29 percent and the hydrogen peroxide reduced production about 44 percent. According to the researchers,"as glucose oxidase is produced by a wide variety of caterpillar species, we may have discovered a new feature of the evolutionary arms race between plants and herbivores."
Felton and collaborators have gone on to clone and sequence the gene that produces glucose oxidase in corn earworm. He found that introducing this gene into plant chromosomes, makes the plants more resistant to pathogens and temperature stress. He holds a patent on this technology filed when he was at the University of Arkansas.
"The resistance to pathogens and temperatures in plants that have the glucose oxidase gene may be because glucose oxidase serves to confuse the plant," says Felton. "The enzyme may cause the plant to react more to stresses other than insect chewing, stresses like temperature or pathogens."
Journal
Nature