Profile of David L. Denlinger

David L. Denlinger moonlights as a surgeon, performing brain transplants. “It's not as hard as it sounds,” he says. “You don't have to bother reconnecting things.” His attitude might seem cavalier, but Denlinger is describing experiments to explore the effect of hormones on insect hibernation, known as “diapause.” “We just take the brains out, we can even plop them back into the abdomen of another fly, we can put them almost anywhere, and they can still retain their function of releasing hormones,” he says. The hormones, depending on the species, can either induce diapause or cause a dormant insect to wake up. Denlinger's career has centered on diapause, beginning with the study of whole insects and then moving on to hormones and molecular mechanisms and currently to data-intense genomic approaches. For his contributions to entomology and their application to agriculture and pest control, Denlinger was elected to the National Academy of Sciences in 2004.

David L. Denlinger (Right), Rick Lee (Left), and feathered friends in Antarctica.

He grew up on the family farm near Lancaster, PA. “I came up the route of the gung-ho kid bug collector,” he says. “Once the chores were over, I'd be heading out to the fields and the fence-rows with my insect net and trying to see what I could find out there, turning over logs in the orchard and looking for bugs.” As a member of a local 4-H club, the youth agriculture program, he was required to choose a project. Denlinger decided he would work on insects. Once a year, a professor from Pennsylvania State University, John Pepper, came to the 4-H club to talk to the children who had entomology projects. “He would give us real insect pins and tell us about the insects we had collected,” Denlinger recalls. “Here I met someone who was doing something that I assumed people just did as a hobby, and this guy was actually getting paid for it. How cool was that! So that was an ‘aha’ moment. I realized you can do something you enjoy and do it as a career.”

Denlinger soon moved beyond collecting insects and began to wonder how they reproduced and survived. He knew they were an integral part of the environment on the farm. “You first pick up on the destructive aspects of insects from just observing what needs to be done to protect crops, but I certainly remember from an early age being a strong advocate for the idea that insects are our friends too,” he says. “They're doing a lot of really useful things for us. It's just a few of them that are out there being the bad guys and giving the rest of insects a bad name.” In 1964, he began a B.S. in zoology at Penn State. He knew that he wanted to be an entomologist, but his professors advised him that he could wait until graduate school to specialize and that he would be better off building a solid foundation in zoology. “That was very good advice, because I could have very well narrowed myself much too early,” he says.

Meet the Flesh Flies

Early in the summer of 1967, he set off for the University of Illinois at Urbana–Champaign, which had a strong program in insect physiology. Before the summer was over, he had found a Ph.D. supervisor. Two supervisors, in fact: Gottfried Frankel and Judy Willis. “Frankel had just been to Europe, and he brought back a colony of flesh flies from France. He was trying to slow down their development because he still had some more traveling to do, and he put them on the windowsills of the pensiones (guest houses) where he was staying in Europe. When he got back to Urbana, some of these pupae were in a developmentally arrested state, and at that point, no one knew really much about fly diapause.” The phenomenon had been studied in moths and butterflies but not in flies. “[Frankel] pointed me in that direction, and Judy Willis showed me how to use her respirometer, so I started measuring oxygen consumption in the pupae right away. And I was hooked on it.” In the laboratory at the University of Illinois, Denlinger became friends with a Czechoslovakian postdoc, Jan Zdarek, with whom he has had a strong research partnership throughout his career.

To this day, Denlinger continues to work on flesh flies. “These are not fruit flies; these are flesh flies, much more macho beasts,” he says. “As the name implies, they do feed on carrion, so the smell is a bit gross. You have to feed them on rotting liver.” But the flies are ideal for experiments. “They're nice big flies; it's relatively easy to do surgery on them. You can produce lots of them without too much effort. They make a great model system for understanding how diapause works.”

In his Inaugural Article in this issue of PNAS (1), Denlinger explores how insects up-regulate heat shock proteins (Hsps) to protect themselves from freezing as they hibernate through the winter. Hsps are molecular chaperones that ensure proper protein folding under stressful conditions such as temperature extremes. During diapause, flesh flies express most of the Hsps in their genome (but also continue to express other proteins, which does not happen during the stress response). Denlinger and colleagues used hybridization analysis to catalog all of the Hsps that flesh flies up-regulate during diapause. The authors found several new Hsps, including a member of a new family, Hsp60. Using RNA interference, the researchers shut down transcription of Hsp23 and Hsp70, the Hsps expressed at highest levels during diapause. Flesh fly pupae unable to produce Hsp23 and Hsp70 could not survive freezing temperatures. Because the profile of Hsp expression is similar across several species, Denlinger concludes that these chaperones are most likely critical to cold tolerance in many insects.

A Pennsylvanian Dutchman in Africa

A missionary aunt had regaled Denlinger with stories of eastern Africa during his childhood. “I knew I needed to get there sometime myself,” he says. A new research center, the International Centre of Insect Physiology and Ecology, was opening in Nairobi, Kenya, just as Denlinger was finishing his Ph.D. Unfortunately, several American scientists were ahead of him in the queue. Jan de Wilde, a Dutch specialist in diapause, was one of the directors of the center. In 1971 he invited Denlinger to work in his laboratory in Wageningen, The Netherlands, while Denlinger was awaiting a turn in Nairobi. “He was a really wonderful person, a pioneer in insect endocrinology,” Denlinger recalls. After about a year, a position opened in Nairobi for a citizen of The Netherlands. “[de Wilde] kindly decided that a Pennsylvanian Dutchman would be just as good,” Denlinger says, “so he sent me down to Kenya as a Dutch representative. I've always appreciated his doing that for me.” Denlinger's first job in Nairobi was to supervise construction of his research building. “It was just a great environment. The center at that point was just starting, and it was a very exciting place to be. There were postdocs from around the world and great camaraderie.”

In northern climates, the yearly variation in daytime length drives insect diapause. The research question that drew Denlinger to Nairobi was what exactly the signal for diapause could be in the tropics. (Nairobi is one degree of latitude south of the equator.) “It certainly seemed like photoperiod was not going to be an important regulator when there was only a 7-minute difference between the longest and the shortest day,” he says. He established a colony of local flesh flies, conducted experiments, and found that cool daytime temperatures, which prevail during July and August, trigger diapause in Kenyan flesh flies.

In the meantime, he found himself fascinated by the work his colleagues at the center were doing on the tsetse fly. “Unlike the flesh fly, tsetse is an insect of considerable medical and veterinary importance,” he says. “There are areas of sub-Saharan Africa roughly the size of Europe where you cannot raise cattle, and of course there are lots and lots of people who get sleeping sickness [trypanosomiasis]; 20,000, 25,000 or so people a year die of sleeping sickness, which is the disease that tsetse carries.” While continuing his flesh fly diapause project, Denlinger began to investigate the tsetse's unusual pregnancy cycle. “Tsetse produces a single egg at a time. That egg is fertilized and ovulated into her uterus, where the larva hatches and is nurtured by the mother's milk gland. The larva spends its entire larval development inside the female,” he says. “After a pregnancy of 9 or 10 days, she gives birth to this full-grown larva that can actually weigh more than she does. So it's a mammalian-type reproductive strategy that very few insects use.”

Together, the tsetse and flesh flies gave Denlinger's career a powerful boost.

Together, the tsetse and flesh flies gave Denlinger's career a powerful boost. In a single year, from February 1974 to January 1975, he published three letters in Nature. The first described the function and microflora of the mother tsetse fly's milk gland, which nourishes the larva (2). The second announced the potential for diapause in tropical flesh flies (3). The third reported that insect hormones might be used to induce abortion in pregnant tsetse flies (4). “I thought, well, this is so easy, everyone should at least have one paper in Nature a year,” he says now. “I had some other things that I felt were equally good that they rejected, so I didn't bother trying anymore.” He often recounts to his students the elation and disappointment he experienced, to prepare them for similar career roller-coasters.

Return of the Native

Returning to the U.S. to seek a university position proved difficult to arrange from Africa. Fortunately one of the directors of the center in Nairobi, Carroll Williams, was an American who specialized in diapause and developmental hormones in insects. Williams, a professor at Harvard, provided Denlinger with a position in his laboratory, a stepping-stone out of Africa. In 1976, Denlinger landed a professorship at Ohio State University (Columbus, OH), where he has taught and carried out research ever since.

The trajectory of Denlinger's research over the past 30 years has taken him far from beetle and butterfly collecting. “We are very much engaged in the big-picture genomics approach,” he says, “as well as doing proteomics and metabolomics,” the computer-driven analysis of entire protein output and metabolic pathways. “Until fairly recently, we've been doing one gene at a time. But this broader approach does allow us to see some of the big pictures that we've been overlooking.” Occasionally he feels wistful about his changes in focus, but he embraces each new revolution as it happens. “We continue to work the whole gamut from the whole animal down to the molecular and metabolic.”

In recent years, he has decided to channel his efforts into research that has direct practical applications. The virus that causes West Nile disease is spread by mosquitoes that undergo diapause in the adult stage, and Denlinger is looking into what genes might be turned on or off to initiate hibernation. The results of this research might speed the development of agents to control the mosquitoes and hence the virus. Another venture of Denlinger's is an investigation of diapause hormones in common agricultural pests such as the corn earworm.

Antarctica's Biggest Tenant

Not all of Denlinger's work is aimed at immediate medical or commercial payoff. His interest in cold hardiness took him to Antarctica in 2004 on a quest for the largest animal that lives year-round on the coldest continent: the wingless midge Belgica antarctica. On this ongoing project, he collaborates with Rick Lee of Miami University (Oxford, OH). “That this organism is capable of surviving in an environment that most cannot suggests that it has some adaptations that might be rather interesting,” he says. To survive in the extreme cold, the midge activates its Hsps. However, “unlike other insects that just turn them on periodically, these larvae are making those stress proteins all the time.” The Hsps are necessary to ensure proper protein folding in the midge, which has a high concentration of salt and natural antifreeze. “The real problem that you encounter at low temperatures is ice formation,” Denlinger says. “A little crystal will form in the cell or rupture the cell, and then you're toast. But there's a way around that. And that is, get rid of your water. These midges can survive a loss of 70% of their body water. So they look like little dried-up raisins. You can't imagine they would actually still be living.” Out of the Antarctic project has come a kaleidoscope of insights into how the midges regulate the Hsps, alter their cell membranes to prevent fracture from the expansion of freezing water, and survive the assault of ultraviolet rays through the ozone hole.

One of Denlinger's many accolades, of which he is particularly proud, is the Gregor Mendel medal, presented to him by the Academy of Sciences of the Czech Republic in 2006. This award recognizes the long and fruitful research relationship Denlinger has had with Jan Zdarek, his former labmate at the University of Illinois, who is now a professor in Prague. Even when Denlinger was in Kenya in the early 1970s, he found time to travel to Prague for a research visit. Zdarek collaborated with Denlinger on his tsetse work through the 1980s, and every once in a while one of the two scientists will send a student to work in the other's laboratory to share techniques. “I remember being mesmerized by the story of Mendel and his pea patches in high school biology and certainly never dreamed that I would someday get a medal that bore his name,” Denlinger says. “And I can imagine my high school biology teacher would have been even more surprised!”