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Story Publication logo October 18, 2017

Outwitting Nature’s Greatest Killer

Mahal, an orangutan who was rejected by his mother at a Colorado zoo, at the Milwaukee County Zoo. Image by Mark Hoffman for the Milwaukee Journal Sentinel. United States, 2017.

The closer the contact the greater the risk humans and animals will pass devastating diseases to...

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Jimmy Mains holds an Aedes aegypti mosquito at MosquitoMate in Lexington, Ky. The small company uses Wolbachia bacterium-infected mosquitoes to reduce their numbers.

LEXINGTON, Ky. — For the second year in a row, molecular biology major Walker Vickery spent his summer internship engaged in the care and feeding of nature’s most prolific killer.

He sprinkled powdered beef liver into trays of water filled with the wriggling, swimming larvae, known as “immatures.” He made sure the adult cages had a 50-50 ratio of males to females. And in order to meet the dietary preferences of the females, he warmed cow’s blood from a nearby slaughterhouse and placed it in cups covered with sausage casings.

Much of the time a hush enveloped the humid white room where he worked, broken only by the barely audible fluttering of a million tiny wings.

In that room, in a nondescript suite of offices at the back of a quiet Lexington strip mall, one of the great battles in modern science is playing out.

Man vs. mosquito.

The mosquito, an age-old enemy, carries diseases that now kill more than 1 million people a year worldwide. To fight back, scientists have been exploring potent new weapons with the potential to replace pesticides that have harmed humans and the environment.

The scientists that Vickery has worked with during his summers at MosquitoMate hope to defeat the insect with an oddly-paradoxical idea:

They are raising mosquitoes in order to reduce the mosquito population.

The company hopes to do this by rearing male mosquitoes infected with Wolbachia, a bacterium that renders them unable to produce offspring. When released, the Wolbachia mosquitoes will compete with wild males for mates. Each time an infected male wins, procreation will fail.

The strategy, which requires annual releases, is being tested in pilot programs on opposite ends of the country — in Fresno, Calif. and the Florida Keys.

“All told we’re probably producing 500,000 mosquitoes per week,” said Stephen L. Dobson, founder and CEO of MosquitoMate, which is supplying the two pilot studies.

An international project called Eliminate Dengue is much further along than the American programs, focusing on a disease believed to infect almost 400 million people a year. Launched more than six years ago, the project has now released Wolbachia-infected mosquitoes, both male and female, in five countries, including Australia, Indonesia and Brazil, with five more soon to follow.

By infecting females, the only mosquitoes that bite and transmit disease, the project hopes to purge the insects of the illnesses they can carry: dengue, yellow fever, chikungunya and Zika. Inside the mosquitoes, Wolbachia prevents the viruses from growing and replicating.

Scientists and others are also debating more controversial methods that would alter a mosquito’s DNA, offering mankind a compelling and potentially troubling bargain.

Using genetic strategies, humans could conceivably wipe out entire species — for example, the species of Anopheles mosquitoes that carry malaria, which still kills half a million people each year.

But to save those lives, we must make peace with the uneasy fear that meddling with the blueprint for life amounts to “playing God” and risks unintended consequences.

The prospect of ending a disease as ancient and deadly as malaria is not mere wishful thinking or science fiction, according to Richard H. Ebright, professor of chemistry and chemical biology at Rutgers University’s Waksman Institute of Microbiology.

“This is something that would take a matter of years and not a matter of decades or centuries,” he said. “But you would have to have public buy-in. I would strongly support the eradication of the Anopheles mosquito … Society has to decide whether it wants to go there.”

“With malaria, the potential benefit is extraordinary,” agreed Greg Kaebnick, a research scholar at the Hastings Center, a bioethics research institute. “But there are other considerations that have to be put on the table. The environment is one. And the extent of human involvement in the natural world, the extent to which we’re remaking natural systems.”

The Florida Keys have already experienced a variation of this debate, with residents in one community rejecting a plan to test genetically-modified mosquitoes designed to control the species Aedes aegypti, carrier of Zika, dengue and yellow fever.

Even the use of Wolbachia, which involves no genetic manipulation, is treated with caution. When he finishes work each day, Vickery leaves the rooms that hold the experimental mosquitoes and ventures into the outside world where, with few exceptions, they are not allowed.

At the last door, a sign reminds all staff: “Stop. Check for mosquitoes.”

(above: Video courtesy Milwaukee Journal Sentinel)

For as long as there have been humans, there have been mosquitoes biting them.

“We know mosquitoes, or a similar insect, were around and fed on dinosaurs,” said William K. Reisen, editor of the Journal of Medical Entomology and an emeritus professor at the University of California, Davis. “Certainly they predate humans and most likely have fed on our ancestors.”

The diseases they carry are ancient, too.

Scientists have found the malaria antigen in the dessicated remains of Egyptian mummies 5,200 years old, according to a paper in the journal Transactions of the Royal Society of Tropical Medicine Hygiene.

Yellow fever is believed to have emerged 5,000 years ago in Central or East Africa. And a 2,000-year-old Chinese medical encyclopedia describes what scientists believe to be a case of dengue.

But the discovery that mosquitoes transmitted these diseases has only came in the last century or so. It was not until 1897 that a British officer working in India demonstrated that the malaria parasite could be transmitted from infected humans to mosquitoes -- then spread by the mosquitoes to more humans.

Still, long before scientists established the link between mosquitoes and diseases, people were trying to protect themselves from the insects. During summers, some Native American tribes moved from the coast to the mountains to escape mosquitoes and rubbed themselves with bear fat to protect their skin.

Mosquito nets have been used for at least several hundred years; Cleopatra was said to have slept beneath one 2,000 years ago in Egypt.

A spectacular, if far-fetched, variation was suggested in 1882 by the American doctor Albert Freeman Africanus King. He proposed protecting Washington, D.C., from malaria by constructing a wire screen net as tall as the Washington Monument and draping it over the entire city (the suggestion generally met with ridicule; the link between mosquitoes and the disease had yet to be established).

Over the years humans have drained swamps, installed screen windows, sprayed pesticides and irradiated mosquitoes — all in an effort to fend off the insects and reduce disease.

Most methods altered the natural world; none proved ideal. Pesticides poisoned wildlife and lost their effectiveness as mosquitoes built up resistance. Screen windows weren’t affordable in the poorest, most densely populated parts of the world. And irradiating mosquitoes to render them impotent proved difficult (the treatment did leave males impotent, but also made some too weak to compete for mates).

“Humans think we’ve got the answer to everything, but there’s always a hidden flaw,” explained Joseph Conlon, technical advisor to the American Mosquito Control Association, a not-for-profit professional association.

“Mosquitoes are a natural part of the environment, and to get rid of them you’re going to have to do something decidedly unnatural.”

So far mosquitoes have proved themselves among the hardiest creatures on Earth. Species have been found breeding 14,000 feet up in the Himalayas, and 5,000 feet underground in mine shafts.

“They are extraordinarily adaptable,” Conlon said.

In recent times, scientists have been exploring new strategies, seeking to kill mosquitoes not by changing their environment, but by changing their bodies at the molecular level.

Wolbachia occurs naturally in some insects; the bacterium was discovered in common house mosquitoes, Culex pipiens, in 1924. But it took decades before scientists realized its potential to control mosquitoes.

In experiments in Burma in 1967, the German scientist Hans Laven documented a promising method of mosquito control called cytoplasmic incompatibility, an inherited condition in which the male chromosomes fail to develop and the offspring die. What Laven didn’t know at the time was what triggered the condition.

That mystery was solved four years later by researchers at the University of California, Los Angeles who reported that Wolbachia appeared to cause cytoplasmic incompatibility in the common house mosquito, carrier of meningitis, Japanese Encephalitis and West Nile Virus.

The bacterium works like a lock and key for reproduction. In the male, the bacterium essentially locks the paternal chromosomes. They remain in a tightly packed ball, unable to unpack properly and pair up with the female chromosomes.

However, if the female mosquito has the same strain of Wolbachia, that releases the paternal chromosomes, allowing them to match up with their female counterparts and begin the process of cell division.

Scott O'Neill, director of the Institute of Vector-Borne Disease at Monash University in Australia, has studied the transmission of the dengue virus to humans by Aedes aegypti mosquitoes. Image courtesy Monash University.
Scott O'Neill, director of the Institute of Vector-Borne Disease at Monash University in Australia, has studied the transmission of the dengue virus to humans by Aedes aegypti mosquitoes. Image courtesy Monash University.

The discovery of the bacterium’s effect on mosquitoes caught the attention of scientists Scott O’Neill in Australia and Stephen Dobson in the U.S., whose careers would intersect.

O’Neill, who has been studying Wolbachia for his entire 31-year career, now directs the not-for-profit Eliminate Dengue project, which has an international staff of about 350.

Dengue has been a problem in Northern Australia for the last 20 years; in 2009, the city of Cairns (population: 160,000) had roughly 1,000 cases of the virus.

In 2011, Eliminate Dengue began releasing Wolbachia-infected mosquitoes with the support of local residents. Unlike the U.S. studies, the international project focused on ridding the mosquitoes of dengue and other viruses they might carry, rather than reducing their population.

In 2014, O’Neill’s team conducted a major field trial in Townsville, an Australian city of 180,000 people. For each of the previous 15 years, Townsville had recorded a dengue outbreak. But in the three years since the release of the Wolbachia-infected mosquitoes, the city has not experienced an outbreak.

“We think that’s quite compelling,” said O’Neill.

In 2014, Eliminate Dengue also began a much larger study, involving Yogyakarta, an Indonesian city of close to half a million people. The city has been divided into 24 areas; half will get Wolbachia mosquitoes, half won’t.

The study won't conclude until 2019.

Dobson’s interest in mosquitoes went back to his childhood in rural Tennessee. He loved animals, but there were no zoos nearby.

“The most diversity I had near me was insects,” he said. “That’s how I got started.”

In 1996, Dobson first encountered Wolbachia while writing his doctoral thesis on parasitoid wasps, gruesome insects that inject their eggs into the bodies of their hosts. Interested in the properties of the bacterium, he went to work for the scientist who had studied it by then for more than a decade — O’Neill.

Later, after moving to the University of Kentucky to teach, Dobson collaborated with O’Neill on the grant that would evolve into the Eliminate Dengue project.

In 2001, Dobson and two colleagues published a paper showing that Wolbachia could be used to block Aedes albopictus, the Asian tiger mosquito, from having offspring.

Then, a decade ago, Dobson invented a way to move Wolbachia from mosquito to mosquito. The technique was patented by the University of Kentucky and became the basis for Dobson’s business, MosquitoMate.

Despite the spread of both Aedes aegypti and Aedes albopictusthrough the U.S., Wolbachia had never been tested against them in an American city.

However, in 2013 an opportunity presented itself.

Steve Mulligan was in his 23rd year of managing the Consolidated Mosquito Abatement District, which covers nine cities including Fresno, Calif. — 1,058 square miles of hot, dry land containing close to 1 million people.

In 2013, the job changed considerably. That’s when Aedes aegypti, a disease-carrying invasive species, arrived in the city of Madera, 25 miles northwest of Fresno.

The first of the new mosquitoes was caught in June and a week later, the invader was discovered in Clovis, just 9 miles from Fresno. Mulligan said no one knows for sure how Aedes aegypti wound up in Fresno County. Maybe the eggs were in a container that arrived by car or truck from one of the states where the mosquito was already established. Or maybe, the eggs or mosquitoes hitched a ride in a shipping container from another country, then were trucked inland to Fresno County.

Once in Fresno, the mosquitoes found their way into just about anything that held water, including the little plates placed under flower pots, which they found ideal for laying eggs.

“We were hoping we could eradicate it,” Mulligan said of the new mosquito. “We went door to door in the areas it was first found looking for small containers that could hold water.”

Mosquito control teams persuaded property owners to get rid of such containers, but Mulligan said, “We were not able to eliminate the mosquito. In fact, it expanded.”

The mosquitoes found their way into the underground yard drainage systems, which provided a suitable location for egg-laying.

The new invader stood out in another respect. While the native mosquitoes did most of their biting at night, Aedes aegypti hunted blood day and night.

“They’ll follow people indoors and essentially take up residence in the house and bite people,” Mulligan said.

Of greater concern were the diseases carried by the new mosquito: yellow fever, dengue, chikungunya and a then-little known disease that would soon become front-page news, Zika.

Fresno County had experienced cases of some of these diseases in travelers who’d recently returned from countries where the diseases are common. But there had never been a local mosquito that could bite those patients and turn a single case of disease into an outbreak.

Until now.

With Aedes aegypti at large, previously unfamiliar diseases could gain a foothold in Fresno.

Mulligan had been keeping up with the latest research on mosquito control and was aware of Dobson’s work on Wolbachia.

The year Aedes aegypti arrived in Fresno, the two men met at a conference of mosquito experts, setting in motion work that would lead to the pilot study in Fresno, launched in the Spring of 2016.

The project involved MosquitoMate shipping thousands of its infected males overnight to Fresno, in containers resembling the cardboard tube inside a roll of paper towels. Local workers released 40,000 mosquitoes a week for five months in a small test area, just 120 to 130 acres — roughly 100 football fields.

Mulligan said it’s difficult to tell so far whether the number of mosquitoes is declining. Dobson said he could not release results of the trial until they have been published in a journal.

In 2017, the district broadened the study. In June, workers began releasing the Wolbachia males in two residential areas totalling 500 acres and containing about 700 households. The district and MosquitoMate also joined forces with a new collaborator, Verily, a sister company to Google that blends science, engineering and medicine.

Verily took the Wolbachia mosquitoes developed by Dobson’s company and automated the rearing process. The California company sped up the cumbersome work of separating males from females by using cameras, machine learning and algorithms.

As a result, the company is able to rear and release the insects in much greater numbers. Since July 17, Verily has been releasing roughly 1 million mosquitoes a week in the Fresno district. The company has also designed special trucks that can release mosquitoes as they cruise through the test areas, rather than having to stop at pre-set locations.

“Ultimately we’re aiming for elimination (of Aedes aegypti from the test area),” said Jacob Crawford, a senior scientist for Verily on the “Debug Fresno” project. “We’d love to be able to show elimination at the end of this year.”

So far, in the four years since it arrived in the region, Aedes aegypti has not resulted in any local transmission of Zika, dengue, chikungunya or yellow fever, said Joe Prado, division manager of community health for Fresno County.

This year, Key West became the second community in the U.S. to release the Wolbachia mosquitoes raised in Kentucky. The Florida trial ended as scheduled in August, before Hurricane Irma struck the Keys.

A tube containing bacteria-infected male mosquitoes is shown at the Florida Keys Mosquito Control District, where officials this summer tested a new way to kill mosquitoes that carry Zika and other viruses. Image by Beth Ranson / Florida Keys Mosquito Control District. United States.
A tube containing bacteria-infected male mosquitoes is shown at the Florida Keys Mosquito Control District, where officials this summer tested a new way to kill mosquitoes that carry Zika and other viruses. Image by Beth Ranson / Florida Keys Mosquito Control District. United States.

But the Florida Keys Mosquito Control District has also gone further, proposing the first U.S. use of genetic engineering to curb the mosquito population.

Since 2010, the Florida district and the English biotechnology company Oxitec had been discussing a trial that would use the biotech company’s genetically altered Aedes aegypti. The male mosquitoes Oxitec planned to release would carry a lethal gene into the wild, causing all offspring they produced to die before reaching full development.

The plan ran into strong opposition from a group of Florida Keys residents. When the public demanded a say in the decision, the district’s five-member board voted to put the issue to a public referendum on Election Day 2016.

Key Haven, the small island selected for the trial, voted in one referendum; the county as a whole in another.

The referendum passed in the county, but failed in Key Haven.

Opponents claimed the risks and uncertainty simply outweighed the benefits. The U.S. Food and Drug Administration had published a “Finding of No Significant Impact” to humans. On the other hand, there seemed little urgency to the trial, since the Keys hadn’t experienced a mosquito-borne outbreak since dengue in 2009.

Residents feared the effect an “immature” technology could have on people and the environment. Female mosquitoes weren’t supposed to be released, but what if one was and happened to bite someone? What if the mutation somehow persisted in the wild? And what if the mosquito harmed the ecosystem?

Although these concerns were all addressed in the FDA’s final environmental assessment, opponents cited a scarcity of peer-reviewed research by scientists not involved in the Oxitec project.

“You’re constantly trying to keep up with what the fox is doing in the hen house and it’s been a chase,” said Meagan Hull, a Keys resident of 14 years. Her view is echoed by others who opposed the trial.

Meanwhile, Oxitec mosquitoes have been released in trials in Brazil, the Cayman Islands and Panama; all three tests have reported reductions of over 90% in the mosquito populations. But the Keys trial remains on hold until the Environmental Protection Agency approves a new site.

Last year’s referendum in Florida may signal more intense debates likely to play out in the coming years. Scientists have been discussing a controversial method with the potential to wipe out a species over a much larger geographical area -- possibly even across the planet.

In 2003, a British biologist named Austin Burt wrote a paper explaining how insects might be controlled by exploiting a quirk of genetics called “selfish genes.” Selfish genes turn genetics into an uneven playing field by increasing their own transmission while reducing the transmission of other genes.

A trait encoded by a selfish gene has a better-than-average chance of being inherited and passed down through generations. Moreover, selfish genes have the special ability to be passed down even if their trait may ultimately doom the species. For example, a gene could be engineered to impair fertility or cause the early death of offspring.

The idea of driving such a gene from one generation to the next is what scientists mean when they speak of a “gene drive.” The technology represents a new kind of power, the ability to steer evolution.

So far, humans have never released a gene drive into the natural world.

Two years after writing the paper in the Proceedings of the Royal Society B, Burt and colleagues at Imperial College London received funding for a project that would come to be known as Target Malaria.

Today the project includes 13 institutions across the globe including the U.S. Centers for Disease Control and Prevention, the universities of Oxford and Cambridge in England, two centers in Italy and three in Africa.

So far, the specific genetic method hasn’t been decided, though lab work on two possibilities is “well advanced,” said Delphine Thizy at Imperial College London, who works for Target Malaria. “We hope to make an application in 2023.” The application would request permission to import gene drive mosquitoes for testing, most likely into the African nation of Burkina Faso.

Thizy said the first method under development attacks the so-called sex chromosomes that determine whether offspring are male or female. Using a special enzyme that destroys the X chromosome the moment sperm is produced, scientists hope to insure that a mosquito can only generate male offspring.

“We’ve managed to do that in the lab,” Thizy said. “Now we’re trying to find a way to drive that modification so that all of males will carry the modification.”

They are testing the technique on Anopheles gambiae, one of 30 to 40 mosquito species responsible for transmitting malaria.

The second method under investigation involves targeting a key gene necessary for female fertility and knocking it out so that female mosquitoes would no longer be able to have offspring.

Thizy stressed that Target Malaria is moving gradually, conducting rigorous testing and seeking public input throughout the process.

Each of the two methods is tested first in small cage in London, then in much larger, room-sized cages in Italy designed to mimic the temperature, light and humidity of the natural environment. Testing would then take place in labs in Africa. Only after passing through these stages would scientists apply for permission to test a controlled release of mosquitoes in a small geographic area in Africa.

Target Malaria is investing considerable effort in engaging interested groups throughout the process, from villagers to national authorities and policy makers.

“It’s still early days and it’s important that people realize they have a voice and can express their hopes and concerns,” Thizy said.

The new methods of mosquito control have raised the possibility of species eradication and with it two questions fundamental to science:

Can we?

And if so, should we?

To the first question, it won’t be easy to defeat mosquitoes or the diseases they carry.

“Wolbachia and gene drives might be able to provide permanent replacements and reduce pathogen transmission, but to expect nature not to find a way to prevent extinction may be optimistic,” said Reisen.

He speaks from experience, having worked in Pakistan from 1975 to 1980, on an effort to develop strains of sterilized Culex and Anopheles mosquitoes that could be released to control the population in the wild.

In Australia, a Queensland scientist monitors Wolbachia-infected mosquitoes that can block dengue and other viruses including Zika. Image courtesy Eliminate Dengue Townsville.
In Australia, a Queensland scientist monitors Wolbachia-infected mosquitoes that can block dengue and other viruses including Zika. Image courtesy Eliminate Dengue Townsville.

“To my knowledge,” he said, “except for the Australian experience (with Wolbachia), we have not been successful in eliminating any of the world’s major mosquito-borne pathogens using any method.”

Humans have attacked mosquitoes with pesticides for over a century with limited success. While the U.S. managed to wipe out local outbreaks of yellow fever and malaria, the diseases continue to thrive in poorer countries.

And just as mosquitoes have developed resistance to the chemicals we’ve sprayed on them, they may also find ways to defeat a gene drive, said Edward Blumenthal, chairman of biological sciences at Marquette University.

Develop a gene drive that impairs fertility, and “there’s going to be a tremendous selective pressure,” Blumenthal said. “Any mosquito that develops a mutation that allows fertility will out-compete the rest.”

Even getting rid of the mosquito may not eliminate the disease. Kill Anopheles mosquitoes and you may only force the malaria parasite to find a different host.

The second question — whether we should use gene drives to eradicate a species -- is thornier still.

True, entomologists aren’t exactly lining up to sing the praises of mosquitoes. The insect’s main contribution to the planet seems to be as food for other species such as frogs, waterfowl and migratory songbirds.

But ending a species is no trivial decision, and the effort could backfire.

Blumenthal said a gene drive could work on the intended species, but spread to an unintended species. Also, it is hard to say how a major change in one species, including its elimination, might affect the much larger ecosystem.

“The biologist in me says, ‘Preserve everything. We don’t have the right to make the decision to get rid of an entire species,’ ” says Susan Paskewitz, a professor at the University of Wisconsin-Madison and chairman of the Department of Entomology. “The global health specialist in me says, ‘A half a million people die from malaria, most of them under the age of 5. That’s an awful lot of suffering.’ ”

Whether scientists are infecting mosquitoes with Wolbachia or tinkering with their genes, the tasks will involve a major change in our relationship with the insects.

In order to kill them, we will first have to raise them.

At MosquitoMate, it is clear that rearing mosquitoes on an industrial scale requires care, if not love.

Vickery and the other staff provide for all of the insects’ needs. While the female mosquitoes feast on blood from the slaughterhouse, the males get sucrose.

In the room where Vickery works, the temperature is kept at 80 degrees Fahrenheit, the humidity at 65%; that’s a little warm for him, but the mosquitoes prefer it.

The cages used for mating are ordinary. They include small plastic containers where the females go to lay their eggs on clumps of brown paper that resembles the paper used for seed germination.

The lifespan of a mosquito typically lasts two- to three weeks. In the first week, they pass through four stages of life, from eggs to larvae to pupa to adult. In the larval stage alone, the mosquitoes shed their skin no less than four times.

The company keeps the larvae in pans of water where they swim like tiny worms. In the pupa stage they lose their tails and develop large heads. At this point, males and females are separated, using a device with angled panes of glass that catch the larger females, while allowing the smaller males to slip to the bottom.

“Once they are adult, we feed them, they lay eggs for us and then we kill them,” said Karen Dobson, Stephen Dobson’s wife and director of mass rearing operations at MosquitoMate.

While testing of Wolbachia-infected mosquitoes has focused on communities, Stephen Dobson said his company has an additional use in mind. He hopes to get a permit to sell infected Aedes albopictus mosquitoes to individual homeowners.

Just as some homeowners treat their lawns with weedkillers, they would have the option of buying a treatment aimed at lowering the number of mosquitoes in their yards. Mosquitoes travel only a quarter of a mile in their brief lives.

Dobson isn’t worried about competition between his company and others working on genetically-modified mosquitoes.

“We need to be exploring lots of new technologies because there’s great need,” he said. “It has a lot to do with globalization. There’s a lot more transport of goods around the world, and the spread of mosquitoes is a byproduct of that.

“The world’s just becoming a very small place, and the same thing is true for pathogens.”


Devi Shastri, a Marquette University student researcher and 2017 intern at the Milwaukee Journal Sentinel, contributed to this report.


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