Research

Biting the Plant that Feeds You

She's part pilgrim and part vampire, purplish black and pear-shaped. Moments after her April birth, the aphid creeps along the smooth witch hazel leaf until she finds a suitable intersection of veins. She thrusts her needle mouth into the junction and begins to feed, sucking the sweet sap from the plant.

After she drinks, the aphid jabs the leaf with her beak. In less than a day, a swelling wall of tissue begins to grow from these nicks. "And after about a week or so of repeated feeding and stabbing," says Penn State entomologist Jack Schultz, "you can actually note the stab marks in an almost perfect circle around her." The wall diabolically rises up, enveloping her in a blood-red, Hershey's Kiss-shaped house made solely from plant material.

The aphid's house is a gall, a kind of plant tumor. To the dismay of farmers and crop managers, galls sap resources like sugars and nutrients from the plant and donate them to the feeding insect. Though scientists know the aphid induces the plant to form the gall, they don't know how she does it. Schultz's student Moriah Szpara, an undergraduate majoring in biology, thinks that the aphid affects the genes of the witch hazel plant in some way.

Szpara's interest in insect-plant interactions began in her sophomore year at Penn State, when she tried to determine whether beetle spit alerts a plant to attackers. "There's increasing evidence that plants can tell insects from scissors," Schultz says. "For example, a cucumber has some well defined defense responses that it uses against things that bite it."

four leaves

But imagine a beetle so tiny that its head is the size of hyphen (-). Then imagine repeatedly poking the beetle's mouth with a needle, and collecting enough liquid to conduct an experiment. You might need several hundred beetles, and they don't drool at the smell of pizza. It's like trying to fill a bathtub using an eyedropper. Szpara's long, diligent hours in lab didn't result in a thesis, but she remained interested in how insects manipulate plants to their advantage. She would return to Schultz's lab a year later, to begin her exploration of aphids and witch hazel.

But meanwhile, Szpara wanted to try something new. "One of the things Jack instilled in me is the need to educate the public about science," she says. "So I designed this project where I would study a zoo in Australia, then come back and study one here. This seemed like a neat way to study conservation programs and biology education." Szpara left for Melbourne, Australia in the spring of 1996. The ruddy span of land, the wildlife, and the accents all drew her to the University of Melbourne; not coincidentally the Royal Melbourne Zoological Gardens was just across the street.

Through Penn State's study-abroad program, Szpara took classes like animal behavior and marine ecology at the University of Melbourne. On her own time, she interviewed zoo employees and educators, observed student and adult classes, and analyzed the teaching and demonstration of conservation issues. In the fall of 1996, she repeated her project at the Albuquerque Biological Park in New Mexico. She presented her research at two U.S. conferences—the 1997 Ethnography in Education Research Forum at the University of Pennsylvania and the 1997 National Conference on Undergraduate Research at the University of Texas at Austin—and at a third held at the Taronga Zoo in Sydney, Australia, in March 1998.

Though she had one honors thesis under her belt, Szpara was far from finished. "I was looking at what I wanted to do for the rest of my life, as we all are," she says, "and I really always wanted to learn about genetic techniques." She returned to Penn State in the spring of 1997. Instead of renewing her relationship with the dry mouthed cucumber beetles, she turned to the aphid.

First Szpara had to discover how the gall affects the plant. Surprisingly, the green and healthy looking leaf doesn't seem to suffer from the aphid's presence. "You can have one gall on a leaf or ten," says Szpara, "and the leaf looks perfectly normal. Obviously, the gall is drawing resources from somewhere else."

Gall formation closely resembles the development of a new fruit or leaf on a plant. Fruits and leaves demand sugars and nutrients, so the plant sends these necessary materials to them through phloem, the circulating system of the plant. Such a "material-attractor," Shultz explains, is known as a sink. A gall also creates a sink, tricking the plant into thinking it's a new leaf.

"Sink formation appears to be regulated by an enzyme called invertase," Schultz explains.

"Invertase takes sucrose from phloem, breaks it into two subunits, and feeds it into cells." Think of a factory—invertase stands at the conveyer belt, taking a sucrose every now and then from the dozens floating past, and changing it into the simple sugars fructose and glucose, which are forms that cells can use, and so creating a demand for more sucrose.

Fully grown adult leaves normally photosynthesize to create sucrose. That' s their job, the creation of energy for the plants, its fruits and budding leaves. Since adult leaves have stopped growing, they export their sucrose and retire the hardworking invertases, who spend their days sleeping. But fruits and new leaves—or any sink—need large amounts of fructose and glucose to grow. So invertase activity increases. Using this as her first clue, Szpara wondered if invertase activity increased in galls.

Scientists know that like human tumors, many galls arise from uncontrolled cell division—too many cells reproducing when they should be resting. Galls also result from altered plant development. This means that the aphid has coerced the witch hazel plant into using its cells for purposes other than for forming a normal leaf. A variety of pests induce gall formation: viruses, bacteria, fungi, and other insects. Scientists have only discovered how one of them, a bacterium called Agrobacterium tumifaciens, forms galls.

This Agrobacterium, a single celled organism, injects its DNA into a plant. Part of this DNA codes for cytokinin, a class of plant growth hormones. But cytokinin itself doesn't cause galls. Cytokinin acts as invertase's slavemaster, awakening the invertase in adult leaf cells and commanding it not only to get back to work, but to work harder and faster than ever before. With a sink created and localized on the adult leaf, cells again begin to grow and divide. The tumor caused by Agrobacterium is known as crown gall.

Bacteria and insects belong to entirely different taxonomic kingdoms, but some gallforming insects seem to produce cyto kinin too. So aphids and Agrobacterium may manipulate plants in some of the same ways. "In our project," says Schultz, "we're assuming that insects may use some of the same tricks."

For Szpara, a sort of Euclidian logic leads to a hypothesis: Galling insects may produce cytokinin. Cytokinin increases invertase activity. Therefore, invertase activity must be higher in galls than in the rest of the plant. Szpara set out to test this hypothesis.

Her task proved to be no easier than extracting beetle spit. Szpara first had to isolate DNA from the leaves of the witch hazel plant. "It's notoriously difficult to get DNA out of woody plants," she says. "Witch hazel is pretty well defended with phenolics and polysaccharides, chemicals that gum up the whole system." But somehow Szpara managed to sequence genes from witch hazel that code for three different forms of invertase.

With this information, Szpara can compare the levels of invertase in normal plant tissue versus the amount present in gall tissue. She hopes she'll find that gall tissue has a lot more invertase than normal tissue—at least that's what all the theoret ical evidence points to.

"The critical issue for Moriah's study," says Schultz, "is that nobody has measured any of the aspects of gall formation by insects at a level that suggests the insect is manipulating gene expression. Moriah will be a real pioneer here, as one of the first people to ask such questions about an insect gall system. It's absolutely amazing that she was able to amplify genes from something that nobody's ever worked on before, the witch hazel." He also stresses that her research spans several disciplinary boundaries including molecular biology, biochemistry, ecology, plant physiology, and entomology.

As spring moves into summer and the female aphid grows comfortable in her spacious gall, she decides to start a family. In her characteristically otherworldly manner, she emits hundreds of asexual clones of her own body; each clone grows into a new aphid. Now the gall needs to feed not only the mother aphid, but all of her babies.

Meanwhile, the aphids have started a candy factory inside the now-bustling gall. The sap in phloem, their only source of nutrients, is the ultimate junk food, teeming with sugar and containing very little protein. But aphids need protein. So instead of turning cannibalistic or leaving the nest and venturing out to forage, they merely drink more and more sap. "They pass tremendous amounts of sugar water," says Schultz, "to get a little bit of protein." What happens to all the sugar in the tiny gall?

The aphids make a waste called honeydew. They cover the sticky droplets with a dry wax produced from glands on their backs and push the coated sugarball out of the gall. Then ants, those clever scavengers, carry the balls away for their own personal use.

By the end of summer, the aphids grow restless. "As the aphids grow more and more crowded," says Schultz, "some of the babies begin to have wings; normally they don't." Instead of pushing just sugar drops out of the floor of their home, the aphids start pushing babies out as well. These babies aren't innocent like their home-raised older sisters. They change sex, mate with their siblings, and eat leaves without creating galls. The ones with wings may fly to birch trees to feed and mate, before returning home to a witch hazel plant to lay their eggs on the stems.

The aphids are a curious family, but a dangerous one as well. Though they don't kill the the plant, they do usurp its resources. Fruiting plants suffer the most from galls, since nutrients that should be creating plump produce actually go into forming colorful galls and feeding hundreds of insects. "Corn, wheat, cacao, and grapes are all plagued by some kind of galling pests," Schultz notes. "Moriah's genetic research provides the first step in unravelling the mystery of tumor induction in plants. It could lead to developing crops that are resistant or immune to this destructive and bizarre kind of attack."

Moriah Szpara received her B.S. in biology, with honors in biology and anthropology, in May 1998 from the Eberly College of Science, the College of the Liberal Arts, and the Schreyer Honors College. She received the Paul Axt Award as the outstanding Penn State honors student this year. Her thesis advisor for anthropology is Stephen J. Beckerman, Ph.D., associate professor of cultural anthropology in the College of the Liberal Arts, 514 Carpenter Building, University Park, PA 16802; 8148633869; stv@psu.edu. Her thesis advisor for biology is Jack Schultz, Ph.D., professor of entomology in the College of Agricultural Sciences, 0001 Pesticide Lab, University Park, PA 16802; 8148634438; ujq@psu.edu. Szpara also collaborated with professors Bruce McPheron, Diana CoxFoster, and Eva Pell of the College of Agricultural Sciences.

Last Updated January 10, 2014