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Serial Killer
To stop a global predator, zero in on its sense of smell
by Cathy Shufro
March/April 2006

Cathy Shufro is a writing tutor at Yale.

If you would see all of Nature gathered up at one point, in all her loveliness, and her skill, and her deadliness, and her sex, where would you find a more exquisite symbol than the Mosquito?
—Havelock Ellis, 1920

The home page of biologist Laurence J. Zwiebel’s laboratory at Vanderbilt University features a large photograph of “the most dangerous animal on the planet”: the adult female Anopheles gambiae. Most North Americans don’t think of a mosquito (or a female) as the most dangerous animal on earth. But female mosquitoes carry the malaria parasite, and malaria kills more than a million people each year. Most are African children.

This bloodthirsty animal locates her human prey with her exquisite sense of smell. She can discern human sweat from 50 meters away. But that sensitivity will be turned against her if Zwiebel and Yale molecular biologist John Carlson succeed in their new project. The two scientists have joined with three other research groups in Europe and Africa in an ambitious program to manipulate mosquitoes by using odors.

In a crude way, people have been doing that for years: repellents like Off! may deter mosquitoes by incorporating odors they dislike. The international team of two dozen investigators is taking the strategy a step further, harnessing new discoveries about how the mosquito perceives odors. On the 11th floor of Kline Biology Tower, Carlson’s group is systematically identifying odors that evoke a keen response in the insect. In collaboration with Zwiebel, Carlson hopes to develop three kinds of odor blends: odors that will lure mosquitoes to poison-laden traps, odors that will repel mosquitoes, and odors that will mask the human scents that attract them. The goal is to come up with blends that even the poorest Africans can afford.

This is how the five-year collaboration on three continents is working: when researchers in New Haven or Nashville find an odor that evokes a strong reaction in the mosquito olfactory system, they send the odor to Wageningen University in The Netherlands, where entomologist Willem Takken studies insect behavior. Takken’s lab team pumps the odor into a two-meter-long wind tunnel, lets loose a mosquito, and observes whether the mosquito seeks or shuns the odor. (In some trials, the mosquito gets to choose between an odor blend and the sweaty hand of a self-sacrificing researcher.) In six months or so, when the Dutch researchers have figured out which odors work best, Gerry Killeen and colleagues at the Ifakara Health Research and Development Centre in Tanzania will begin to test the compounds on mosquitoes in “semi-field conditions”: a simulated African settlement, complete with banana trees and a thatch-roofed house, all under a huge mesh dome. Finally, in the West African country of The Gambia, geneticist David Conway and staff at the Medical Research Council will introduce the odor blends to rural villagers and gauge how well they work.

For Carlson, the “real world” is relatively new territory for research. “I’ve always been intrigued by how biological systems work for their own inner logic and beauty,” he says. “But now there’s another dimension: how the work can be used to alleviate an enormous human health problem.”

Carlson got interested in insect olfaction as a Stanford graduate student in the early 1980s partly because of its novelty. He uses fruit flies, Drosophila melanogaster, to explore how insects sense odors. (“After you’ve worked with them for a while, they start to look beautiful,” he admits, somewhat embarrassed.) He found dozens of labs investigating fruit fly vision, but only a single scientist, in India, studying olfaction. “The problems are fascinating. How is it that animals can distinguish among different odors? Why does a peach smell different from a pineapple? It’s amazing how olfactory systems can be so exquisitely sensitive. A male moth can detect just a few molecules of a female moth’s pheromones and start flying toward her.”

He only began studying insect olfaction when Yale hired him in 1986. His new project was a switch from his postdoctoral work on genetically engineering yeast cells. “Most people start their faculty appointments with an experimental plan that’s sort of a linear extrapolation of what they did as a postdoc,” says Carlson, now the Eugene Higgins Professor of Molecular, Cellular, and Developmental Biology. “But I completely changed directions. I didn’t realize you weren’t supposed to do that. I am quite grateful to Yale for taking a risk with me.”

In 1986, he established what was probably the only fruit fly olfaction lab in the country and only the third he knows of in the world. Twenty years later, there are more than 50, and he is recognized as a leader in the field. “I don’t think anyone has put together the whole story in the way that his lab has over the last couple of decades,” says Vanderbilt’s Zwiebel, who has studied Anopheles olfaction since 1994.

It wasn’t until 1995, when Carlson attended a conference in London on mosquito olfaction, that the enormity of the malaria problem became clear to him. In London, he heard from scientists about the suffering caused by the Plasmodium falciparum parasite. “That woke me up. I was fairly naïve. I hadn’t realized the immensity of the scope of malaria, the dimensions of the havoc it causes in the developing world. I had no idea that 10 percent of the world’s population gets it every year. I hadn’t a clue. We don’t think about it in North America.”

If you visit an elementary school near the Ifakara Health Research and Development Centre in Tanzania, you’ll find that even among students healthy enough to attend school, half are carrying the parasites that cause malaria. Ten percent of these children of subsistence rice farmers are so anemic that “they'd get a blood transfusion if they turned up in a hospital near you [in the United States] or where I come from in Ireland,” says disease ecologist Gerry Killeen. Killeen is Carlson’s counterpart in East Africa and a Wellcome Trust international research fellow. “Anemia has big effects on intellectual development, on size, on energy levels.” In many parts of rural Tanzania, getting any kind of health care—much less a blood transfusion—is not an option. “You have to go a long, long way in Tanzania to find a place where you can’t buy a Coke, but it’s very easy to find a place where you can’t find a doctor,” says Killeen.

The Ifakara research center has made inroads into malaria rates with insecticide-treated bed nets. Three out of four people in the area now hang nets around their beds to reduce the number of bites inflicted while they sleep. Still, Killeen says, most area residents get bitten several times each night by an infected mosquito.

Those biting mosquitoes are females. Males don’t bite. (Male Anopheles, for instance, drink nectar.) But for the female mosquito, finding a post-coital blood meal is essential for egg production, and the female Anopheles gambiae prefers human blood above all. When she sucks blood from a person infected with Plasmodium, she ingests some of the parasites. They live in her gut for about ten days while they mature. Then they infiltrate her saliva.

For the rest of her three-week-long life, whenever the mosquito bites a human being, she passes on malaria parasites. Within minutes, the parasites travel to the person’s liver and begin to multiply there. They move to the human bloodstream about a week later, where they proliferate and damage red blood cells. A few days later, the infected person generally begins to suffer waves of fever, chills, and drenching sweats. “You feel totally miserable,” says Takken, the Dutch researcher. He’s had malaria several times while working in Africa.

Young children, pregnant women, and newcomers to Africa are most vulnerable to malaria, as they have little immunity. Most who die are under five. Many adults become highly immune, but there are so many strains of Plasmodium, Killeen says, that “there’s a decent chance that if you get bitten by an infected insect, you’ll get sick.” Moreover, “it’s common for an individual to host a handful of strains at any one time. Most people are exposed to dozens, hundreds, and even thousands of infectious bites per year, so such multiple infection is common.” In a small village just outside of Ifakara, Killeen’s center recorded exposure rates of 2,700 infectious bites per person per year.

Even people with some immunity are likely to get at least mildly ill with malaria a couple of times a year. Without effective treatment, damage to red blood cells will cause anemia. Anemia, in turn, weakens defenses against other common illnesses like diarrhea, which can kill young children. Infected blood cells can clog vessels, starving vital organs of oxygen. Cerebral malaria may cause seizures and coma and, unless it’s treated quickly, often proves fatal.

Malaria deaths in sub-Saharan Africa are increasing. Contributing factors include drug and insecticide resistance, population growth without adequate health care, changes in weather patterns, and wars. Malaria saps economies; poverty and malaria sustain one another. Rich and poor alike get malaria, notes epidemiologist Hassan Mshinda, director of the Ifakara center; his wife became ill in January. But because Mshinda lives in a town and has money in his pocket, “I can rush [out] and get the most effective drug, which is expensive.”

Many Africans can neither find nor afford such a drug. The disease kills an African child every 30 seconds, according to the World Health Organization. It’s as if six 747 jets filled with children crashed in Africa daily, killing all aboard.

You won’t find a single mosquito in Carlson’s mosquito olfaction lab. Mosquitoes are notoriously difficult to manage in the lab—for starters, they need human blood to thrive—so, through ingenuity, patience, and one stroke of particularly good luck, Carlson and his team developed a clever work-around. They adapted the tractable, familiar, and less-finicky fruit fly into a model for the mosquito. Their fruit fly looks like a fruit fly and generally acts like a fruit fly for its few weeks on earth. But when it perceives odors, it’s partly mosquito.

This transformation began a few years ago, when Carlson was doing basic research on odor perception in Drosophila. He and other scientists had already worked out how fruit flies perceive odors via 800 sensory hairs, or sensilla, on their two antennae (see graph, next page). Each sensory hair contains nerve cells; each of these, in turn, has particular “receptor” proteins on its surface that are capable of binding to particular odor molecules. When one of these molecules floats by in the air, the receptor protein binds to it and sends a signal through the neuron in the sensillum. The tiny fruit fly brain perceives the molecule as an odor.

Carlson’s daunting first task was to identify the genes governing insect olfaction. “People were hitting their heads against the wall on this for years,” says Zwiebel. “Postdoc after postdoc bit the dust on this topic.” But in the late 1990s, Carlson and evolutionary biologist Junhyong Kim (then a Yale faculty member and now at the University of Pennsylvania), created a novel computer program to search the fruit fly genome database for olfactory genes. The program was able to find patterns of genetic building blocks (or nucleotides) that resembled—very faintly—the olfactory receptor genes already identified in mice. In 1999, they identified the first odor receptor genes in the fruit fly. It was for that work, says Zwiebel, that Carlson earned a reputation as “the real deal in Drosophila olfaction.”

Key questions remained. When a fruit fly responded to a particular odor, Carlson did not know which of the many receptor proteins on its antennae had bound the odor molecule. Given the fly’s 1,000 sensory hairs and a potentially infinite number of odor molecules, the possibilities were overwhelming.

Then one day in November 2001, a stranger unexpectedly presented Carlson with a gift. Carlson had just given a talk at Brandeis University when a Brandeis biologist, Michael A. Welte, approached him. Welte asked if Carlson was interested in using a mutant strain of fruit fly that he had developed for his own work. This fly was missing one of the genes for odor perception.

With a vial containing 50 mutant flies in his backpack, Carlson headed south on the highway to his lab. When he arrived, he handed the vial to doctoral student Anna Dobritsa '03PhD. “Here, Anna,” he recalls saying. “We can do wonderful things with these.”

At first, Carlson simply envisioned exploring the effects of deleting that missing gene. But then he and Dobritsa realized they could use the fly as a living test tube—a vehicle for learning about all the olfactory genes. That strain of mutant flies, he says, “changed our lives.” The crucial element was this: although one olfactory gene was missing, the corresponding odor-sensing neuron was intact. They called it an “empty neuron,” because, without its gene, the sensillum could not produce a receptor protein. And so Carlson’s team filled the empty neuron.

Dobritsa and postdoc Wynand van der Goes van Naters inserted one of the genes for fruit fly odor reception into mutant fruit flies. Then Elissa Hallem '05PhD, in what Carlson calls a “heroic” effort, inserted the remaining 31 genes, one by one, into mutant flies. Each gene produced its specialized protein in the (formerly) empty neuron.

The system was “elegant,” says Stanford neurobiologist Liqun Luo, because introducing a gene into a living fly “closely mimics the native environment of that neuron.” It enabled Carlson’s team to start developing a response profile for the fruit fly olfaction genes. And then Hallem and Carlson made another leap. They decided to try inserting mosquito DNA into Drosophila: an in vivo experiment that would allow them to use fruit flies to examine mosquito olfaction gene by gene.

Carlson got female mosquito DNA from the Zwiebel lab, and Hallem injected it into embryonic fruit flies. Each injection contained a single mosquito olfaction gene, along with “guiding” DNA to ensure that the odor receptor was produced in the empty neuron. The injection also included the fruit fly DNA for red eyes. Every member of the next generation that picked up the mosquito gene would be instantly recognizable by its eyes. The introduced gene was incorporated in about one in ten fly offspring. (The rest were consigned to a bottle of ethanol labeled R.I.P.)

One afternoon in late 2003, Hallem tested a series of odors on the transgenic fly to discover whether the introduced mosquito odor receptors would work. She suctioned a fruit fly into a pencil-shaped plastic pipette and jiggled the pipette until the fly lodged in its narrow tip. Only its head and antennae protruded from the tiny opening. Then, looking through a microscope, Hallem inserted a tiny electrode into the cuticle of the sensillum containing the formerly “empty neuron.” She blew a puff of air containing an odor sample across the antenna, and she waited. If the odor molecule bound to the receptor, the neuron would fire, and the electrode would register the resulting changes in voltage within the neuron. Hallem would see sharp spikes on the computer screen and hear buzzing from a sound box hooked to the electrode.

She tried one odor after another. A few random spikes showed on the screen, and the sound box occasionally buzzed, but the haphazard signals marked only spontaneous activity in the neuron. Hallem was discouraged. And then, as Carlson recalls, “Elissa was hunched over her electrophysiology setup and tried 4-methyl phenol”—a chemical in human sweat. “She got a screaming response.”

Carlson was elated. (Hallem was frustrated: the response was so robust she had trouble counting the number of spikes for her records.) Finding an odor sample that triggered a reaction was partly luck, says Carlson: “We could have tried a thousand odors and not gotten a response.” But the experiment had worked. The fruit fly was detecting odors with the sensibilities of a mosquito. Hallem’s doctoral dissertation describing this work won three prizes, including last year’s Distinguished Dissertation Award from the Council of Graduate Schools, given to only two students nationwide.

Hallem has moved on to do postdoctoral work at Caltech, but Allison Carey '09MD/PhD has picked up where she left off. Carey has so far tested 60 different odors on five lines of transgenic flies, each carrying one of the mosquito’s 79 odor receptor genes. Carlson and Carey plan to assemble a gene-to-odor list—matching every Anopheles gambiae receptor with an odor or odors it responds to. With that detailed map of the mosquito’s sensory world in hand, researchers may be able to create more refined and effective attractants and repellants than have ever before been possible.

Among the photographs that Carlson snapped during a family jaunt to the Bronx Zoo last October for his 50th birthday, there are no pictures of the wedge-capped capuchins in the monkey house. While five-year-old Eliot and nine-year-old Evan watched the little “organ grinder” monkeys swinging on the vines, their father occupied himself in photographing the sign outside the cage.

“Some New World monkeys,” the sign read, “know how to fight off mosquitoes: they rub themselves with a millipede that oozes chemicals mosquitoes hate.” As it turns out, millipedes contain chemicals from the benzoquinones family. They might be worth testing on transgenic fruit flies.

As word of Carlson’s research has circulated, he has received ideas for other chemicals worth testing. An East African scientist e-mailed him that she had collected dozens of local plants that might deter mosquitoes. “There’s a huge lore of products from the real world, plants and animals that repel mosquitoes,” says Carlson. He tells of a Russian scientist working in mosquito-rich Siberia, where people who step outside can find their skin blackened with mosquitoes. The scientist encountered a man who could go outdoors stark naked without suffering a single bite. In Carlson’s world, that man’s sweat might be the elixir in the Grail.

But there can be no true cure-all. “The people in Europe and America do not understand some of the complexities of these issues,” says epidemiologist Mshinda. He gives an example from Tanzania. When the Ifakara health center promoted insecticide-impregnated bed nets in the late 1990s, the death rate for children under five dropped 27 percent—temporarily. The nets must be re-treated with insecticide every few months. Human nature and household economics being what they are, only one in ten households re-treats its nets.

Tanzania is developing nets that work longer. But such complications abound. Some mosquitoes have developed resistance to pyrethroid, the insecticide in bed nets. The longtime standard for treating malaria, chloroquine, no longer works in some regions because the Plasmodium parasite has become resistant. Eight out of ten Tanzanians live so far from clinics that, even if they do come up with the money for medicine, they buy from local shops whose drugs may be ineffective.

Because the problems are so complex, scientists and policy makers agree that malaria can only be drastically reduced by using a combination of tools: vaccines, drugs, and mosquito control. The funder of the insect olfaction project—the Grand Challenges in Global Health Initiative, primarily financed by Bill and Melinda Gates—is backing this idea. When the Global Health Initiative gave the olfaction project an $8.5 million five-year grant, it also funded six other anti-malaria projects, which take other approaches: developing vaccines, studying immunity to malaria, and testing ways to control insects with insecticides or through genetic engineering. The olfaction research project was one of 43 chosen from among more than 1,500 proposals submitted to the Global Health Initiative.

In Tanzania, Killeen, surrounded by the suffering of malaria, says, “We’re pressured. We should be.”

Takken appreciates Zwiebel andCarlson for feeling that pressure in remote North America. “John Carlson doesn’t have to worry about the African child,” he says. “His scientific achievements are sufficiently great that he can get funding for 10 or 15 years based on current achievements. He could close his office door and be fine.”

Carlson believes that even limited success could be significant. “I don’t know how well this project is going to work,” he says, “but I say to myself, with 500 million people in the world suffering from malaria each year, if we can just reduce the incidence by 0.1 percent, that’s 500,000 people we could help each year. That’s a lot of people.”

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