Photo copyright © 1998 Maria Eisner.

NO FREE LUNCH. Repulsed by the noxious chemicals on the squash beetle pupa, at left, a hungry ant retreats to clean its antennae.

Naked, immobile and conspicuously colored, the squash beetle pupae would be easy picking for insect predators if they hadn't long ago perfected a science called combinatorial chemistry. In the human world it is a chemical skill that pharmaceutical researchers are still learning.

By variously combining three simple molecules into a veritable arsenal of complex defensive compounds and secreting them through microscopic body hairs, Epilachna borealis pupae can thwart just about anything that would eat them, Cornell University researchers report in the July 17 issue of the journal Science.

"Industrial chemists have only begun practicing combinatorial chemistry in the last five years. They generate a very large number of variations on one architectural theme and test a library of compounds very rapidly for pharmaceutical activity with high-throughput screening," explains Jerrold Meinwald, the Goldwin Smith Professor of Chemistry at Cornell who is one of six authors of the Science article.

"This beetle pupa does the same thing, creating hundreds of deterrent compounds from three simple precursors," Meinwald adds. "Then it skips the screening process in the laboratory and goes straight to the field where the ultimate test is its survival in a bug-eat-bug world."

Photo copyright © 1998 Maria Eisner.

Scanning electron micrograph shows glistening chemicals produced by body hairs of Epilachna borealis.

One of the hundreds of different chemicals produced by E. borealis is a necklace-like structure of 280 atoms forming one single large ring.

With no other defenses than this, the beetle pupae are rarely disturbed. Hungry ants, for example, are quickly repulsed by the pupal chemicals and frantically clean the noxious substances from their antennae with special brushes on their forelegs. Within a few days, the pupae metamorphose into adult squash beetles -- and in the process become an agricultural nuisance.

"How this creature survived had been a real mystery," says Thomas Eisner, the Schurman Professor of Chemical Ecology at Cornell, and an author of the journal article. "It is bright yellow on the background of green leaves, it has no mechanical defenses, and it is exposed to anything that comes around, particularly aggressive ants."

The pupae of some beetle species defend themselves, Eisner explains, with mandible-like contraptions on their abdomens. By wiggling their abdomens, the pupae usually manage to pinch the legs or antennae of attacking insects, and thus survive into adulthood. But E. borealispupae have no such mechanical defenses, so Eisner looked a little closer. Viewed through the microscope, the pupae are seen to have fine body hairs topped by glistening droplets of chemicals.

A series of analytical investigations (including nuclear magnetic resonance spectroscopy and high-pressure liquid chromatography) performed by Frank C. Schröder, a postdoctoral researcher at Meinwald's laboratory in the Cornell Department of Chemistry and Chemical Biology, revealed the unprecedented complexity of the beetle's defenses. Jay J. Farmer, a graduate student, confirmed Schröder's structural assignments by synthesizing one of the most important of the large-ring compounds.

"One of the most exciting features of the beetle pupae's defensive chemicals is the sheer magnitude of the rings," Schröder says. "Ordinary cyclic natural products often have rings consisting of five, six, or even seven atoms. Compounds with rings of 30 or 40 atoms can already be considered very rare. The discovery of a whole library of novel compounds with ring sizes from about 30 to well over 200 atoms was therefore entirely unexpected."

And yet the beetle pupae use very simple chemistry to produce these unusual compounds. Three different small building blocks related to ordinary fatty acids are used to assemble the rings. Many different combinations of the building blocks then create a highly complex mixture, a library of large rings.

The defensive chemicals can be seen under high magnification, as shown in Maria Eisner's SEM (scanning electron microscopy) picture of E. borealis pupal hairs on the cover of Science. . The tiny insect pioneered an endeavor in which human chemists, with their costly and sophisticated machines, only now hope to succeed. Combinatorial chemistry has yielded several drugs that are expected to reach the marketplace in the near future. An estimated 1/10th of the approximately 1,900 biotechnology companies worldwide are believed to be using the approach in their research-and-development programs.

"Each time we find a new talent in the insects we study, we are brought to wonder about treasures that remain unknown. After all, most insects remain yet to be discovered," Eisner says.

The E. borealis studies by Meinwald, Eisner, Farmer and Schröder, who worked with Scott Smedley, and Athula Attygalle, Cornell postdoctoral associate, and senior research associate respectively, were supported by grants from the National Institutes of Health and the National Science Foundation.