Mosquitoes Remade

Scientists reinvent agents of illness to become allies in fight against disease

It’s a bit unnerving that Scott O’Neill bursts out laughing at the basic premise behind the story you are beginning to read.

IN THE HOT ZONE | More than 40 percent of the world’s population lives with the risk of dengue, which is transmitted via mosquitoes. Some scientists believe they can build a better mosquito that fights dengue rather than spreading it. Two kinds of reinvented mosquitoes have been released in field tests, one in the Caribbean and the other in Australia. Future test sites are under consideration. Source: World Health Organization Geoatlas/Graphi-Ogre, adapted by Stephen Egts

James Gathany/CDC

GENETIC TAKEDOWN A field test in 2009 showed that released mosquitoes carrying lethal genes would mate and pass on the genes to offspring. Following a November release, researchers collected eggs to see how many hatched into larvae with the genes (top), marked by fluorescence (bottom). Source: A. Harris et al/Nature Biotech. 2011 Courtesy of OXITEC

BLOCK BREAKDOWN | After releasing bacteria-infected mosquitoes into an Australian town last year, researchers tested sections of the town to see how prevalent the bacteria became. Most sections (shown above) saw a big jump in the percent of infected mosquitoes during a roughly two-month period. A. A. Hoffmann et al/Nature 2011

Scott O’Neill and his Australian team have raised hundreds of thousands of bacteria-infected mosquitoes. The mosquitoes may one day help fight dengue. Colyn Huber/Eliminate Dengue Program

MICROBE PUMP-UP Researchers are testing a mosquito intended to fight dengue by passing along the disruptive Wolbachia bacteria to other mosquitoes. The bacteria successfully spread through a population after mosquito releases (bars) in a town in Australia last year. Source: A.A. Hoffmann et al/Nature 2011

He is dean of the science faculty at Monash University in Australia and lead scientist for research on developing bacteria-infected mosquitoes as a public health tool. The premise put forth was that scientists suddenly have made visible progress on a daydream that has been around for at least 50 years. Apparently, though, O’Neill thinks the “suddenly” is funny.

To the general insect-bitten public, a mosquito that fights disease instead of spreading it is the flying car of public health. Twentieth century science was supposed to create all kinds of marvels. But it’s a new millennium, cars are still grounded and mosquitoes are still dangerous. They pass along maladies, including malaria, yellow fever and dengue, that together kill hundreds of thousands of people each year, and often stump vaccine makers and drug developers. Small, frail-bodied creatures, easily knocked out of the air with the slap of a rolled-up science magazine, rank among the deadliest animals on Earth.

But now, retrofitted Aedes aegypti mosquitoes that might interrupt disease transmission are flying around freely in a wave of real-life tests.

In 2010, the British company Oxitec announced results of the first known tests of free-flying transgenic mosquitoes, a milestone for both friends and foes of genetic engineering. The mosquitoes — 3 million of them — were engineered to start a population crash after infiltrating wild, potentially disease-spreading A. aegypti swarms in a village on the Caribbean island of Grand Cayman. And last year, O’Neill and his colleagues reported results of field trials using a completely different strategy. His team’s mosquitoes, several hundred thousand of which were released into towns in Queensland, are not transgenic and are not intended to reduce the overall number of mosquitoes. Instead, regular mosquitoes infected with a bacterial disease that makes them far less likely to spread dengue were supposed to replace the usual swarms. Both research groups are continuing their tests this year, and both are discussing expansions (Vietnam for O’Neill’s group and Key West, among other places, for Oxitec).

Yes, these are just tests. The basic strategies have their pluses and minuses. And who knows what chances the saboteur mosquitoes have for ultimate success in fighting disease. But the big news is that the insects are actually out of the lab. The last widely reported releases for mosquito control that made it this far ended in 1981. Public health at least has a flying tricycle, soaring in from out of the blue.

O’Neill doesn’t see it that way. With a note of cheerful teasing, he says: “Well, I’d say you took your eyes off the ball.”

From inside the mosquito effort, he wouldn’t describe the outdoor tests as a pop-up surprise, but more as a matter of researchers continuing to attack the problems at hand. Presented with the same premise of sudden visible progress, Oxitec’s chief scientific officer, Luke Alphey, merely says that progress toward a better mosquito “took longer than we expected.”

Listening to their accounts of what happened behind the scenes, however, makes the progress sound more, rather than less, surprising. It’s easy to understand why outsiders took their eyes off the ball. The effort stalled for years at a time, careened sideways and rolled backward as much as forward.

Death by mozzie

The perils of mosquitoes can set anyone dreaming. Female mosquitoes have evolved marvels of stealth hypodermics, drawing out blood in search of proper prenatal nutrition. So living amid the surreptitious blood-seeking moms is the disease-risk equivalent of sharing needles with anybody nearby who has some uncovered skin and a pulse.

These flying syringes spread quite a lot of unpleasantness: Beyond malaria, dengue and yellow fever, there’s also West Nile virus; lymphatic filariasis, with its elephant-scale swellings; St. Louis encephalitis; La Crosse encephalitis; eastern and western equine encephalitis; and plenty more.

Malaria is easily one of the biggest killers. Several mosquito species inject malaria parasites into the human bloodstream in the course of sucking up a meal. The World Health Organization estimates that 655,000 people died from the disease in 2010, many were children in Africa, and many more get very sick.

Almost half of humankind now lives in areas at risk for malaria, but decades of medical research have yet to yield an effective vaccine. Malaria parasites have evolved resistance to older drugs designed to treat or prevent the disease and are showing signs of getting around the current drug generation. The mosquitoes carrying the parasite have also evolved resistance to several waves of pesticides deployed against them. Particularly worrying are signs of emerging resistance to the pyrethroids. Bed nets treated with these insecticides have become the mainstay of campaigns proven to reduce malaria risk by keeping night-flying species away from still flesh.

To fight malaria and the pathogens that mosquitoes spread, “you have to be smart — it’s a very smart system,” says Raymond St. Leger of the University of Maryland in College Park. Research has therefore expanded out of the box, out of mosquito-stopping bed nets and definitely out of the insecticide canister.

St. Leger and his colleagues, for instance, are genetically engineering a fungus that targets the young malaria pathogen growing inside the mosquito. When enhanced with genes for producing the toxin scorpine, as well as some other parasite-unfriendly substances, the Metarhizium anisopliae fungus can cut a mosquito’s parasite load by 98 percent, the team reported last year in Science.

The researchers have applied for regulatory permission to test how well mosquitoes pick up the souped-up fungi in a giant enclosure in a part of Burkina Faso that St. Leger describes as squarely in Malaria Central. During mosquito season, residents there can expect some 200 mosquito bites a day, many from Anopheles gambiae, a major malaria spreader.

St. Leger’s approach lets each and every mosquito pick up the disease-fighting fungus from the environment, like a living insecticide. But a burst of new field trials for fighting another disease rely on mosquito inheritance.

Within the sweep of ideas for defending humankind, directly monkey-wrenching the mosquito so it no longer spreads a parasite or virus is “conceptually beautiful,” St. Leger says. But for malaria, it’s not exactly easy. Civilians in the war against insectborne diseases may refer to “the mosquito,” but entomologists have named roughly 3,000 species. Twenty are important malaria spreaders, he says, and more species can carry the disease. A safer mosquito for malaria zones would need to be a swarm of simultaneously modified species.

Though not as famous as malaria outside the tropics, dengue makes a worthy target. It’s the forgotten pandemic, says Scott Ritchie, a medical entomologist at the James Cook University in Cairns, Australia, who works with O’Neill. A potentially lethal viral disease, dengue has been spreading rapidly since the 1970s. Now more than 40 percent of the world’s population lives at risk. A first round of the disease brings such miseries as a fever of around 104° Fahrenheit (40° Celsius), a sharp headache behind the eyes and intense skeletal pain, inspiring the name “breakbone fever.”

“Most people remember it as one of the most miserable weeks of their lives,” says Cameron Simmons. Although he is a University of Oxford professor of medicine, Simmons lives in Ho Chi Minh City, Vietnam, in a zone at high risk for dengue. And yes, he’s had the disease once, so he is speaking from experience.

What he explains, with remarkable matter-of-factness, is that enduring one round gives long-lasting immunity to that particular group of dengue viruses — but not to the three others. The first bout actually increases the risk that infection with a second kind will turn into what’s appropriately called severe dengue. Symptoms include bleeding gums, plasma leaking out of blood vessels, difficulty breathing and vomiting up blood. In 2010, 49,000 people in the Americas alone developed severe dengue.

No drugs or vaccines for dengue have made it to the market yet, although trials are under way. Even if the leading vaccine candidate aces its tests, it requires three doses spaced six months apart, Simmons points out. For many high-risk dengue zones, that’s not an easy vaccine to spread around.

But, when it comes to a mosquito-based solution, targeting dengue has an advantage over malaria. One species, A. aegypti, is mostly responsible for spreading dengue, making the problem more manageable than malaria for the Oxitec and Australian teams. St. Leger, with ironic undertones, says, “Dengue is the simple one.”

Sterile strategy

Even with the aid of the 20th century’s scientific advances, not much has been simple about controlling mosquitoes. But in the 1990s, Oxitec’s Luke Alphey took a new look at some early failures. One was the sterilization technique.

In the 1950s, U.S. Department of Agriculture researchers developed a way to irradiate the pupal resting stage of screwworms just enough to let them develop into adults that can fly around and mate but can’t sire viable young. Releasing clouds of these sterile male adults lured natural females into dead-end relations. By 1966, waves of releases had eliminated the screwworm from the southern United States. Rare among insects as a dedicated eater of the living flesh of warm-blooded mammals, its disappearance was not mourned.

Trying to radiate mosquitoes by just the right amount proved difficult. In spite of the death they deal to humankind, mosquitoes are fragile. Among other problems, too much radiation kept males from competing well for wild females.

Alphey says he heard about the screwworm eradication in the 1990s from a colleague at the University of Oxford. It was hardly a secret among pest specialists, but, he deadpans, “I’m a geneticist.”

Even though he came to the concept late, his years of research on fruit fly genetics gave him an unusual take on irradiating mosquitoes. Why bother, he wondered. Considering what geneticists can do as a matter of routine, it might be possible to sterilize male mosquitoes genetically. Alphey says his inspiration was “an idea at the right time.”

In 1982 geneticists had reported how to insert new genes into the natural complement of DNA in the Drosophila melanogaster fruit fly. Hopes rose among entomologists that some kind of genetic redesign of the mosquito would render disease-carrying killers harmless nourishment for birds and bats. This feat proved much harder than expected. The bit of DNA used to insinuate a gene into the fruit fly refused to cooperate in many other insects, mosquitoes included. “A lot of time was wasted and postdocs burned up — though not by me,” Alphey says.

Fortunately, he had his inspiration for genetic tinkering after this long, dark period. Sixteen years after the announcement of the genetically engineered fruit fly, researchers reported coaxing a gene into a mosquito’s DNA. Thus began an ongoing effort to change insect biology to render a mosquito less likely to pick up, harbor or transmit a pathogen.

When Alphey learned about the screwworms, he realized he just needed to make the mosquito sterile. That’s not a small “just,” but he and his colleagues did it by refining a genetic system they call RIDL. Pronounced “riddle,” it stands for Release of Insects carrying a Dominant Lethal genetic system. The fatal genes can be rendered harmless in the lab. But outdoors, RIDL males pass along the genetic booby trap to their offspring, so the youngsters die. In 2002, Oxitec was formed to commercialize the technique.

A small test on Grand Cayman in 2009 confirmed that RIDL lab mosquitoes could survive the shock of the wild. “They hadn’t seen a predator in 100 generations,” Alphey says. “They hadn’t seen rain. But they did OK.” And a bigger test of free-flying mosquitoes on the island in 2010 demonstrated that they could fool the local females into mating, thus reducing the population by 80 percent.

Bacterial intervention

O’Neill’s reinvention of the mosquito takes a very different approach. He doesn’t want to wipe out a population. He wants A. aegypti still flying around — just not spreading dengue.

He started thinking about disease-fighting mosquitoes during the 1980s, when he was in grad school working on remarkable bacteria in the group Wolbachia. Various species in this group naturally infect thousands upon thousands of different insects, perhaps three out of four of all kinds in existence. The bacteria often don’t kill their insect host, instead transforming it into a lean, mean, baby-bacteria machine. Wolbachia infections spread from mother to offspring. Fathers aren’t much use as far as the bacteria are concerned, and Wolbachia do have tricks for this, such as turning genetic males into bacteria-spreading females.

The trick that intrigued O’Neill gives infected females an unfair advantage in the mating scene. Bacteria-carrying females produce viable bacteria-carrying youngsters regardless of whether their dad is infected or not. But an uninfected female won’t have viable young if she mates with an infected male. So that male, who wouldn’t have passed on his infection directly, still does his bacteria-spreading part by taking a healthy female out of the gene pool.

Wolbachia infections sweep through populations at great speeds, and O’Neill began to wonder if this spreading power could be harnessed to drive some yet-to-be-invented disease-fighting trait through a mosquito population. Instead of a eureka moment, he says, “It crept up on me.”

Even if safer mosquitoes can be made, a further challenge is making them numerous. The best, safest, most cleverly engineered mosquito does nothing for public health if wild females don’t mate with it or if helpful traits get swamped by wild genes. Wolbachia might give some disease-fighter a chance to spread its genes far and wide.

O’Neill’s big problem was that no natural Wolbachia infected A. aegypti. He had to find a way to coax a mosquito to catch the insect disease. “Everybody said it was a great idea,” he says. “But we couldn’t get it to work.”

For years he and his colleagues tried to persuade Wolbachia from fruit flies to colonize the mosquito. “It was a big jump,” he says. “They would get it, and then it would fall out of the population.”

He did succeed in coaxing the bacteria to grow in lab dishes of cells from mosquitoes, and, although he moved on to other projects and also moved back to his native Australia, he kept those cultures going. When the Bill & Melinda Gates Foundation announced their Grand Challenges funds for edgy science on intractable problems in 2003, O’Neill applied. The foundation also thought the approach was a “great idea,” he says.

Receiving Gates money brought on a chest-tightening moment for O’Neill. He was delighted to have another chance to try his Wolbachia scheme, but its track record was terrible.

This time, he got his bacteria to infect the mosquitoes and travel from mother to offspring for generations. What made the difference, he speculates, is the four years of growing in mosquito cells.

But the project strategy took a sharp left turn when research showed that Wolbachia could do more than accelerate the spread of some other disease-stopping trait. A strain also shortened the life of its mosquito hosts. Because the dengue pathogen takes time to multiply and render mosquito spit truly dangerous, any shortening of life reduces mosquitoes’ abilities to pass along the infection to humans. This Wolbachia strain alone, then, might have a big effect on disease transmission.

Then came another scientific twist from research on fruit flies. A version of the same Wolbachia strain interfered with pathogens multiplying inside the flies. Research confirmed a similar interference in mosquitoes with dengue. So, in the best news for the project yet, a Wolbachia strain not only shortens the life of a mosquito, but also reduces the amount of virus it develops. Wolbachia infection may have two ways to make mosquitoes less likely to transmit dengue — at least that’s how it plays out in the lab. But the idea needed testing in the field.

To make sure captive-reared Wolbachia carriers have what it takes to go wild, hundreds of thousands of test mosquitoes grow up biting real people. “We need very, very high-quality mosquitoes,” says geneticist Ary Hoffmann, a collaborator at the University of Melbourne.

Releases in Queensland last year showed that Wolbachia could spread through a wild population. And O’Neill, Hoffmann and their colleagues spent the early months of this year testing the spreading power of a more effective disease-fighting Wolbachia.

As exciting as the Australian and the Oxitec tests are for anyone dreaming of a better mosquito, the efforts are far from any real-world disease control. So far no tests have looked at whether the efforts actually whittle away at dengue.

Neither O’Neill nor Alphey sounds particularly dismayed about how far their projects still have to go. Maybe — at the risk of speculating too much — their perseverance comes along with the special point of view it takes to get even this far. Steps forward can’t seem too astonishing or too sudden. The way onward can’t seem overly twisted or steep. Saying somebody has to be a special kind of crazy sounds too rude, but to keep fiddling with tricycles does take openness to the possibility that, despite half a century on the ground, cars really could fly.

Susan Milius is the life sciences writer, covering organismal biology and evolution, and has a special passion for plants, fungi and invertebrates. She studied biology and English literature.

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