by: Nancy Marie Brown
From Research/Penn State, Vol. 16, no. 3 (September, 1995))
ypsy 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?"
nvestigating 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."
n 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."
e 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."
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.
