The Chemistry of Caterpillar Guts

Nancy Marie Brown
September 01, 1995

Gypsy moth guts are green. Of a shade between pond-scum and kiwi, as I know well, having seen hundreds, perhaps thousands, of these slimy green guts the summer the worm invaded our mountainside. In those first May days, while the dogwoods bloomed and the green briar wrote its name through the underbrush, the worms wriggled from their eggs. By the time the oaks had pushed out leaves, the worms were ravenous. By June, they were finger thick, the trees bare as Christmas. From my window I saw caterpillars descend on their silken strings like circus acrobats, one, ten, fifteen at a time, swaying in the breeze. To lock the house door I had to brush them from the keyhole. I crushed dozens on the walk to the car. I left the country for the month to avoid them.

Heidi Appel has also seen thousands of caterpillar guts, if under rather more controlled conditions. "I chill them first," she told me when I visited her laboratory in Penn State's Pesticide Research facility. "So I don't feel guilty."

I raised an eyebrow, thinking of all the caterpillars I had deliciously squashed, and she laughed an infectious laugh. "And so they don't run away," she added. "Then I pin a caterpillar out and cut it open lengthwise. What's exposed is a long tube—the inside of a caterpillar is almost all guts. I put a platinum probe into the gut wall and measure the flow of electrons."

Appel is a research associate working with—and married to —Jack Schultz, a professor of entomology at Penn State and a 15-year veteran of the gypsy moth wars. Smartly dressed under her lab coat, she took a wry, self-deprecating delight in introducing herself to me as "one of the world's authorities on caterpillar guts." She currently co-directs (with Ken Feldman, an associate professor of chemistry) a $300,000 National Science Foundation project to learn how gypsy moths use tannins, a family of toxic plant chemicals, to protect themselves against viral infections.

"You're looking at an ancient history and music major for my first four years of college," Appel said, when asked to explain her attraction to gypsy moth guts. "Then I discovered I had no musical talent." A love of biology headed her toward the challenge of medical school. "Then I spent a night in the hospital with my dad, when he was passing a kidney stone. It was a very rude, necessary shock that this wasn't the career for me." A chance course in plant morphology and evolution ("I got closed out of human anatomy and had to take this. I thought it would be awful. I loved it.") led her, since plants make chemicals to keep insects from eating them, into caterpillar guts.

By measuring the electron flow, Appel found that tobacco hornworm guts are what's called a reducing environment—which means there are a lot of electron-rich molecules floating about in it, as in a cow's fermenting stomach; but with this difference, that the caterpillar's guts do not, like the cow's, keep out oxygen. "No one had ever dreamed you'd find a reducing environment under aerobic conditions," Appel said. "That was a big surprise."

Appel was at the time a graduate student working with University of Michigan biologist Michael Martin. "We wanted to make a model gut, a test-tube gut, in which to test certain plant chemicals, so I was measuring everything I could," she recalled. "The reducing environment was the final piece of the puzzle. We assumed all caterpillars would have reducing guts."

Yet when Appel mentioned this theory at a scientific meeting to Schultz, whom she had just met, "We got into this heated argument," she told me. "He kept saying, 'I'm sure gypsy moth guts aren't like your guts.'

"When the meeting was over, it still wasn't resolved. Two weeks later, Jack called from Penn State and said, 'Buy a ticket. We'll pay you to come here and measure gypsy moth guts.' So that was the start of both our personal and our professional relationships."

For according to Schultz, gypsy moth guts are not green. Not, at least, their midguts, where the digesting goes on. "Jack said that when he opened up his gypsy moths," Appel recalled, "they were black. It was on this basis that he made the argument to me. If they'd swallowed food and been digesting it for a while, the midgut was dark brown or black.

"When I cut up a hornworm, the gut's green all through."

Chewed up leaves left out in the air also turn dark brown or black. And the color change there has a well-known cause: oxidation, which is the opposite of reduction.

Oxidizing agents—like oxygen, which gives the phenomenon its name—are particularly good at stripping other molecules' electrons away, with possibly disastrous results. Bleach, for instance, is an oxidizing agent. When it removes electrons from a bloodstain, the blood proteins fall apart and the stain washes out of your clothes. In the gut of a caterpillar, Appel suspected, oxidized plant chemicals could "do all sorts of nasty things, like punching holes in membranes, inhibiting enzymes, messing up DNA, or binding to other compounds important to nutrition or disease resistance."

It seemed an obvious survival tactic for a caterpillar to block oxidation in its own gut. But if color was any cue, the tobacco hornworm did and the gypsy moth did not. Said Appel, "I thought that was an awfully stupid thing for a gypsy moth to do."

Chewed on by the worm, an oak tree cranks up production of the toxic tannins in its leaves.

"The tree is not defenseless," Appel said, beginning to spin out a new story.

Yet the tree may be ill-advised.

Schultz had shown that red-oak tannins depressed the gypsy moth's growth and reproduction; he expected, then, that as tannin levels climbed high, worm numbers would correspondingly decline. But in the woods he saw the opposite occur: more tannins, more worms. "Jack began fishing around in the biomedical literature," said Appel, "and found that tannins can have antiviral properties. It's a hot, hot topic of research in medicine right now.

"A light bulb went on. There's a naturally occurring virus that lives on leaves, called LdNPV." (The acronym stands for Lymantria dispar, the scientific name of the gypsy moth, Nuclear Polyhedrosis Virus.) "If a gypsy moth eats it," continued Appel, "the gypsy moth dies. The theory is that this virus could regulate the gypsy moth population and that it could be an ecologically sound microbial pesticide.

"What Jack and Steve Keating, a graduate student, found out, was that tannins inhibit this virus. It's like the tree's giving the gypsy moth an antibiotic. The tree's shooting itself in the foot.

"That was the scene when I arrived in 1989," Appel said, returning to her own story. "I was supposed to understand the chemistry and physiology of how tannins do what they do in caterpillar guts to make the gypsy moth resistant to the virus." She paused, gave a hearty laugh. "You're looking at someone who got a C in organic chemistry."

"So how did you do it?" I asked.

She shrugged. "You learn what you need to learn in the service of desire. I spent a lot of time reading and researching."

The first thing she learned was that gypsy moth caterpillar guts are not, indeed, like tobacco hornworm caterpillar guts (that is, Jack was right): Gypsy moth guts are an oxidizing environment, not a reducing one. The tannins, chewed and swallowed with the rest of the leaf, lost electrons and became unstable.

"This is peculiar," Appel remembered thinking. "How common is this?"

Investigating further, she discovered that the oxidation of phenolics, the class of plant chemicals to which the tannins belong, was quite common indeed: "When a cut apple turns brown? That's a phenolic oxidation product. The gunk in the bottom of a bottle of old wine? Phenolic oxidation. It's even why soil is brown."

But as for tannins, "people had assumed other modes of action were more important."

"So the tannins in the guts are oxidized. What happens then?" I asked.

"How do you describe the chemistry between tannins and the proteins in the gut?" Appel refocused my question. "I looked in the literature. No one's done this? Why not? I found out why: When they try to do the chemical reactions in the lab, they end up with sticky brown goo in the bottom of the test tube. It's so sticky it sticks to everything. Its structure is so complex and dense even most chemists say, No way I'm gonna work on this! And I'm going to characterize it?"

She shrugged, smiled, prolonged the suspense. "Jack had a graduate student visiting from Finland, Kari Saikkonen, who rented a room in a house with another graduate student, Susan Ensel, whose adviser was a chemist whose research specialty was —get this—Synthesis of tannins and interaction of tannins with proteins." She sucked in a breath, let it out in a whoop. "And he was here! He was at Penn State!

"And he had just published the synthesis of one of the major tannins in oak trees.

"Well, we had a momentous meeting a year and a half ago. We were jumping up and down, writing things on the board. It was like—It was like—" She smiled, daring me to guess. "You can't print what it was like.

"It turned out he wasn't working in biological systems," she hurried on. "He was interested in a purely chemical point of view. But he was interested enough in the biology. He was amused, I think, by the thought of placing his chemistry into a biological context. He'd been studying tannins in pristine clean conditions where oxidation was not occurring.

"I had to—we both had to—learn a foreign language. He's a much more reserved and formal person—but he doesn't seem to mind my lack thereof."

"Sure—" said chemist Ken Feldman, when I called for an interview, "but I have to tell you Heidi's the brains behind the project. I'm really just the technical support."

In his office, in oxford shirt and khakis, his feet up on the desk and his hands linked behind his head, he was indeed the very image of a reserved, formal, professorial chemist—except that he spoke extremely fast. "There are several different reasons why I'm interested in tannins," he began. "First, their chemical structures have features that are at the forefront of the challenges of organic synthesis. They contain pieces that chemists hadn't put together before. The formation of particular bonds had not been explained before. There was no baseline of how to do it. There are over 500 known structures of tannins. We've been able to work out the details on how to synthesize a few of them.

"Second, a few of these molecules exhibit anticancer activity. They're not cytotoxic—not cell-killing—not poisonous. They turn out to be immuno-stimulants. They appear to stimulate the immune system to generate chemical species which combat tumors, and we have some evidence, as of this morning, as to which chemical species might be involved. These compounds invoke the secretion of tumor necrosis factor alpha.

"Third, with Heidi, we're investigating how these compounds have antiviral properties."

"Does it work the same way, the anticancer and the antiviral activity?" I asked.

Feldman put his feet onto the floor, rocked forward. "It would surprise me if it did," he said. He paused a moment, as if he had lost his place in his mental outline.

"There's this professional tension between us," he mused, half his attention still clicking through mental notes. "I work small. I don't understand what I'm looking at when I work with something that big—"

"As big as a caterpillar's guts?"

"As big as a caterpillar's guts," he said.

The conversation stopped. With difficulty I abandoned the oxymoron of "big" caterpillar guts to ask a more technical question. "Why do tannins, when they're oxidized, turn into brown goo?"

Feldman seemed to relax, as if I'd given him his cue. "These compounds seem to have been evolved by nature," he began, "to act as a molecular glue. Their role is to attach big molecules to each other in plants and insects. Once they're oxidized, they are very chemically reactive. Here—Let me show you."

Leaving his desk, he crossed the room to a PC, called up a series of chemical diagrams: molecules stretching across the screen in long, dragonish chains studded with the familiar hexagonal benzene rings. As he scrolled through the screens he stopped, pointed out two short lines of the dozens in the diagram. "This bond here and this bond here," he said, confidingly, "are what make this molecule interesting to a chemist. These two bonds are very hard to make."

He clicked on through the screens until he found the diagram of the oxidizing reaction. The original tannin is rather small, just one ring with a few oxygen and hydrogen atoms sticking off. Exposed to an oxidant, it appears to lose two hydrogen atoms. ("Why is it called oxidation when it has to do with hydrogen atoms?" I asked. Feldman nodded. "It's a historically poor name. It has to do with the gain or loss of electrons.") Once oxidized, the tannin becomes active: any protein in the area will stick to it, making those two hydrogen atoms reappear in the diagram, and making it easy to oxidize again. After the second oxidation, explained Feldman, "it traps another protein. Once you have two proteins stuck on, you can't work with these things." You've got Appel's brown goo.

"The chemistry I've been working on," Feldman continued, "is to trap this molecule"—he taps the image of the first tannin-protein cluster—"before it turns to crap.

"I can't say it works yet. In a model system it works. I can make a protected form"—a tannin-like molecule that can only oxidize once—"and it's stable. I can keep it in a bottle. It stays a nice purple color."

But from this model system to a red oak tannin and a gypsy moth viral protein, Feldman conceded, is a large step. "I was rather surprised that the NSF bought it," he said. He and his postdoctoral assistant, Stephane Quideau, "have to build a piece of apparatus—build the molecules themselves—build how the molecules will stick to the apparatus—

"There's slightly more to it than throwing things in a flask. It'll take us six months to put it together."

The trick is to keep the tannins apart from each other and to drag the reactions out in time so that what seems an immediate clumping to brown goo can be separated into an orderly series of chemical steps.

Feldman went to the blackboard. He plans, he said, drawing it while he explained, to attach each individual tannin molecule to a tiny (but still visible) plastic pole with an invisible chemical linker.

"Like a plastic hook?" I asked.

"Like a chemical hook," he agreed. "The tannins can't wave around too much because of the linker—the hook," he said, "and if they can't touch each other, they can't react with each other." Once the tannins are hooked to the pole, he'll expose them to an oxidant, wash it away, then send down a gypsy moth viral protein. "The protein hopefully will do the chemistry I just showed you"—he gestured toward the PC—"and stick onto the tannin molecule."

Feldman will then "unhook" the tannin-protein complex from the plastic pole—"We have to be clever as chemists to design this linker to dissolve when we want it to, without destroying the molecules we're interested in," he noted—and determine where and how the two molecules stick together.

Or, as Appel puts it, "The rest is just fancy chemistry. Fairly routine for someone with his skills."

In approximately two years, Appel and Feldman should know which atom on which amino acid of the protein sticks to the oxidized tannin molecule (Feldman thinks it's a sulfur) in the gypsy moth caterpillar's gut.

"That's important," Appel explained, "because it will be the first demonstration ever of tannins binding to proteins in insect guts under oxidizing conditions—which is probably the dominant mechanism for tannin action. And if this is the mechanism—if the tannin oxidizes first, then binds to the protein—we can go about uncoupling it."

A chemical that blocks one crucial step along the brown-goo pathway could turn oak-leaf tannins back into harmless glue, making the worms once again susceptible to LdNPV and the woods safe for spring leaves. Alternately, using Feldman's apparatus as a screen test, Appel might be able to identify a strain of LdNPV to which an oxidized tannin cannot stick, making the virus a sure worm-killer.

"And there's another curlicue I've left out," Appel added. "Plant phenols—like tannins—are ubiquitous. All microbes, all insects have to deal with them." As, she has written in the Journal of Chemical Ecology, do isopods, gastropods, sea urchins, snails, earthworms, nematodes, amphibians, mites, crayfish, true fish, all birds and mammals—even humans: What makes the itch of poison ivy? An oxidized phenol sticking to the proteins of your skin. Even dirt depends on phenols: As a main ingredient in humus, they determine the fertility of soils, sediments, lakes, streams, estuaries, and oceans.

"So oxidation of tannins has ecosystem-level effects," concluded Appel. "The same chemical principles apply to much larger processes than caterpillar guts."

Heidi M. Appel, Ph.D., is research associate in the entomology in the College of Agricultural Sciences, 122 Pesticide Research Laboratory, University Park, PA 16802; (814) 863-3380. Her article, "Phenolics in Ecological Interactions: The Importance of Oxidation," appeared in the Journal of Chemical Ecology, Vol. 19, No. 7 (1993). Ken S. Feldman, Ph.D., is associate professor of chemistry in the Eberly College of Science, 122 Chandlee Laboratory; 863-4654. This research was funded by the National Science Foundation.

Last Updated September 01, 1995