Once Bitten…

Not shy: How UTMB researchers are fighting the pesky mosquitoes that transmit deadly pestilences including malaria and West Nile and yellow fever viruses

Graduate student Ivy Greene sits in a small, white, uncomfortably warm room in UTMB's Keiller Building, using tweezers to separate mosquitoes into two piles. The insects are still alive, but unlike the thousands of others that occupy the clear-plastic-and-mesh cages on shelves nearby, they aren't moving; they lie in Petri dishes embedded in crushed ice, and the cold keeps them immobilized.

Mostly immobilized, that is.

“Oh, you're way too awake,” Greene says, noticing a mosquito beginning to twitch its legs. “You're going to make me kill you.” She picks it up with her tweezers and buries it in the ice. “Bye-bye,” she says, and returns to her sorting without missing a beat, carefully picking up chilled mosquitoes by their legs, peering at the antennae on their tiny heads, and then putting them into one dish or the other depending on whether they're male or female. She wants only the females, because only females feed on blood and transmit the virus she's studying.

“The males have—I call them bushy eyebrows.” She holds one up to demonstrate. “The females are like this, theirs are straight.”

K nowing how to tell the sex of a mosquito is just one trick of the trade for the fifteen or so UTMB scientists, students, and lab technicians whose lab and fieldwork explores the complex relationship between mosquitoes and human and animal diseases. Their ultimate goal is to fight mosquito-borne plagues like yellow fever, dengue, malaria, West Nile virus, and Venezuelan equine encephalitis—diseases that sicken and kill millions of people worldwide every year.

To do so, the UTMB researchers—concentrated in two groups led by vector biologist Stephen Higgs and virologist Scott Weaver—need to know, inside and out, the mosquito species that carry those diseases. They need to know where mosquitoes get viruses and other pathogens—what animals they feed on, and under what environmental conditions. They need to know what genetic, physiological, and behavioral factors determine whether a particular mosquito is likely to acquire an infection and pass it along to humans. Finally, they need to know how ecological change—the felling of forests for farms, and the growth of cities and suburbs, for example—alters the dynamics of both mosquito and human populations, and brings people and disease-causing agents into contact in new and dangerous ways.

That kind of knowledge has allowed UTMB scientists to make substantial contributions to public health both at home and abroad, and to lay the basic-science groundwork for new and perhaps crucial efforts to control mosquito-borne disease. In Venezuela, UTMB researchers have used satellite imagery and ground-level investigations to produce Venezuelan equine encephalitis “risk maps” that could enable authorities to target severely limited vaccination and control resources at the areas where they will do the most good—protecting children and valuable horses. In Mexico, they're keeping an eye on the states of Oaxaca and Chiapas, where the disease has flared twice in the last ten years—and through which it passed in 1969 on its way from Guatemala to the United States, in an outbreak that killed 1,500 horses and sickened 300 people in Texas during 1971. In the cities of Bolivia, they've captured yellow fever mosquitoes in an attempt to understand whether that deadly virus may be poised to make the jump from rural to urban environments. And in Harris County, they've been working to determine whether the West Nile virus—first detected in Texas by UTMB's Robert Tesh last year—will make the jump into new species of mosquitoes far more likely to spread it to humans.

Getting that knowledge means getting up close and personal with creatures that most of us go out of our way to avoid. It also requires adaptability and endurance. To catch wild mosquitoes for disease surveillance and infection experiments, you have to go where they live, whether that's a tire dump in Houston or a tropical rain forest in Peru. To keep mosquitoes alive and happy enough to reproduce in the lab, you have to work with them under artificially hot, humid conditions, coaxing them to feed and breed with specially warmed animal blood and cages designed to accommodate their mating preferences. And to unravel the connections that link them with disease, and do it safely, you have to infect mosquitoes with dangerous pathogens and study them without allowing the mosquitoes—perhaps the most efficient means of infection ever devised by nature—to infect you.

One warm, humid morning a few weeks before the first big rain of summer, Stephen Higgs and his lab assistant Kim Klingler head north on I-45 to gather mosquitoes for West Nile research. Higgs, an Englishman in his mid-40s, is dressed as he almost always is, in a short-sleeved shirt and khaki pants. Klingler wears her usual field gear: blue jeans and a white, long-sleeved T-shirt that completely covers her arms. Their destination is a place called Flickinger's Farm, just inside Beltway 8 in far southeast Houston. It's one of those odd rural enclaves that still survives inside the rim of the expanding city—cattle graze behind barbed wire on the other side of Old Genoa Red Bluff Road, and baby chicks send up a cheeping chorus from a cage outside owner David Flickinger's machine shop. But what brings Klingler here every week is a less idyllic feature of the landscape: the three-acre tire dump that abuts Flickinger's property and spawns clouds of bloodthirsty mosquitoes from May to November. Tires catch rainwater, which turns them into the perfect place for mosquitoes to lay eggs and mosquito larvae to develop into adults. All over the world, tire dumps are notorious as mosquito breeding sites. (Last year, when Higgs, UTMB virologist Alan Barrett, and postdoctoral fellow John-Paul Mutebi visited La Paz, Bolivia, to study the potential for urban yellow fever transmission, they initially had trouble finding enough mosquitoes. Local mosquito-control workers took the visitors to a back-street tire dump). Tires have also introduced at least one exotic mosquito species into the United States—the infamous, highly aggressive Asian tiger mosquito, Aedes albopictus , which arrived in Houston in the early 1980s in used tires being brought over from Japan and Taiwan for recapping.

Although it is only early June, there are plenty of Aedes albopictus adults around these tires, piled about 15 feet high along the western edge of Flickinger's land. There are also Culex quinquefasciatus and probably Aedes aegypti , thought to have come to the New World from Africa in the water casks and bilges of slave ships. ( Aedes aegypti has become rarer in recent years, under pressure from albopictus , which occupies similar territory.) Flickinger has been suffering their bites for decades, but he's managed to keep a sense of humor about it. “Don't take too many of my mosquitoes away,” he says, chuckling. “You can feed 'em while you're here, though.”

“The more of our blood they get, the less of yours they want,” Higgs rejoins, watching Klingler remove captured mosquitoes from an odd-looking contraption hanging from a Chinese tallow tree. It's a CDC light trap, a device that uses light bulbs, carbon-dioxide-producing dry ice, and a fan to lure mosquitoes close and then suck them down a tube into a mesh bag. Nearby is another trap, a propane-burning machine its manufacturer calls a Mosquito Magnet; it also lures mosquitoes by generating carbon dioxide, which they mistake for the exhalations of their animal prey.

“There you go—we've got a couple here,” Klingler says, opening a panel in the side of the Mosquito Magnet. “We're capturing Culex quinquefasciatus , Aedes albopictus , and Aedes aegypti , to test the last two for West Nile.” She transfers the captives into a net-and-metal cage, which will give the delicate insects some protection from dehydration on the ride down to Galveston. There, sensitive molecular biological techniques will be used to test them for West Nile virus.

“ Culex quinquefasciatus is the primary species for transmitting West Nile, but feeds primarily on birds,” Higgs explains. “Now albopictus and aegypti tend to live very close to people's houses and feed very readily on humans. So if West Nile is infecting those species, then transmission to humans is more likely.” He walks over to where Klingler is checking a red plastic cup under an old Ford pickup—one of several placed in sheltered locations in Flickinger's yard. Each contains water and a brown paper towel, meant to collect eggs laid at the water's edge by albopictus and aegypti . Klingler removes the paper towels and places them into Tupperware containers to keep them from drying out. The eggs form a tiny line on the towel's surface. A few days after they're laid, Klingler says, the eggs develop to the point where they can survive if the water in the tree hole or tire they're in dries up. They can hold out for months in this desiccated state, waiting for the water level to be raised by a rainstorm—or, in the case of these eggs being taken back to the Keiller Building, by a researcher who needs more mosquitoes for experiments.

When that happens, the result is mosquito larvae. Higgs' backyard next to Dickinson Bayou is a prime location for collecting mosquitoes in their aquatic larval stage; one day last summer, he recovered five different species from his four-year-old son's play pool. (Naturally, Higgs' yard is also a prime spot for adult mosquitoes. To take advantage of this resource, he keeps a Mosquito Magnet of his own just off his back patio.) Even this early in the season, Klingler has no trouble capturing multitudes of future bloodsuckers from the large water-filled pots on Higgs' patio. “Wow, Steve, you've been doing a good job for me here,” she says, holding up a clear plastic syringe full of wriggling larvae just sucked up from their nursery. “I think you've got some Aedes in here—I can see some siphons.”

Every mosquito larva, she explains, breathes through a tube that extends from its rear end to the surface. The larva hangs from this tube, feeding on bacteria and detritus scooped toward its mouth by tiny, constantly moving bristles. “A lot of these are probably Culex , they have really long siphons. But there are a few that have kind of shorter, stouter siphons, and those are Aedes mosquitoes.” Klingler and Higgs move from pot to pot, exclaiming over their finds. “There's some pupae, those little dark commas,” Higgs points out, indicating larvae that have reached the final stage before becoming adults—plumper and only moving if they have to. “Now look at that one—that's a rich one. They like to get into those dark corners.” Klingler adds each syringeful of water and larvae to a clear plastic container with air at the top so the larvae can breathe. They, too, will go back to the Keiller Building, to be raised to adulthood in captivity. “We just wait for them to become adults, and when they're adults we see if we have anything interesting,” Klingler says. “This is really very exciting for me. Not many people get this excited about this, but I have all these mosquitoes to go back and identify. That makes me happy.”

Klingler isn't the only person happy to be working with Higgs and UTMB on mosquitoes; Ray Parsons, the head of the Harris County Mosquito Control District, seems unable to find enough good things to say about the help UTMB has given his organization. In addition to Tesh's ongoing efforts to identify West Nile-infected birds (in the last two years, the virologist has processed more than a thousand birds sent to Galveston by Parsons' organization), Parsons singles out Higgs' lab for praise. Last summer, he notes, Higgs' group used advanced molecular biology techniques to double-check Harris County Mosquito Control's identifications of mosquitoes infected by West Nile virus, and the Higgs team's ongoing “vector competence” work—checking to see which of the eighty-four species of mosquito found in Harris and Galveston counties can become infected by and transmit West Nile—provides critical intelligence to mosquito control authorities. “It's really been a wonderful, unique opportunity for us, to have them available to do laboratory work,” Parsons says. “I've never seen a situation anyplace that I think has worked better than this.”

A few days later, with Diana Ortiz, a postdoctoral fellow in Scott Weaver's lab, I visit Klingler and Higgs' captives in their new home. This “insectary” contains no lab-infected mosquitoes—experimental infections are done in another facility, but nevertheless, full precautions are taken to make sure none escape. After entering an outer door we don white lab coats—to make any hitchiking mosquitoes easier to spot—and then pass through two more doors, one of which must be closed before the other can be opened. The mosquito-friendly 80-degree heat and 80 percent humidity of the room hit us immediately, along with the sharp odor of dry fish food. It's part of the diet given to the larvae, which live in water-filled Tupperware-like containers along the back wall. On our right as we enter is an refrigerator-shaped incubator, for storing eggs; on our left is a coffee urn, for sterilizing water before it's poured down the drain, thus ensuring that no larvae or eggs escape.

In the cages that line the room's two other walls are more mosquitoes per square foot than anywhere else on Galveston Island. Some rest placidly, like tiny specks of bark on the floors and walls of their cages, while others whirl in agitation, or suck on thick, sugar-water-soaked cotton wicks. (Male mosquitoes never feed on blood, and females do so only to produce eggs; most species draw the bulk of their sustenance from sugars made by plants, sucking nectar from flowers or sucrose from rotting fruit.) Labels on the cages indicate the species inside: Aedes albopictus and Culex quinquefasciatus from Flickinger's Farm, Ochlerotatus taeniorhynchus from the marshes near Weaver's house on Galveston's West End, Anopheles gambiae from West Africa, and Aedes aegypti from Puerto Rico and Iquitos, Peru.

Ortiz herself is about to leave for Iquitos, a city of 300,000 people on the Amazon River. Her goal is to spend a month collecting mosquitoes that spread Venezuelan equine encephalitis, called VEE by scientists, which is one of Weaver's primary interests. The virus, which can cause death and serious neurological damage, strikes human beings and horses in South America and Mexico, and in 1971 broke out in South Texas. (During the Cold War, it was also mass-produced as a potential weapon by Soviet and U.S. scientists.) In humans, VEE hits children hardest. Weaver, the father of two young children, says, “It's a really scary thing, if you're a parent, to see your kids getting neurological disease and there's nothing you can do.”

In Venezuela and Colombia, according to Weaver, VEE epidemics probably could be prevented with a large-scale horse vaccination campaign, but long periods of dormancy between outbreaks discourage the governments of both nations from spending scarce public health money on the problem. Weaver has focused on an inquiry that could make a crucial difference: figuring out where the particular strains of the virus that cause outbreaks comes from, and in what geographic areas they pose the greatest danger. From fieldwork done throughout the 1990s, Weaver has determined that VEE outbreaks occur when virus strains normally confined to what he describes as “little islands of forest in a sea of ranches” undergo specific mutations that enable them to generate high virus concentrations in the bloodstream of horses­. The outbreak in horses, called “amplification hosts” of VEE, makes it easier for the virus to infect certain mosquito species that are likely to bite both horses and humans.

Further field studies in Venezuela (all conducted in what one of Weaver's journal articles, with characteristic understatement, calls a “politically unstable location” near the Colombian border) have yielded information that Weaver and his collaborators combined with satellite images of forest vegetation. The result is a map showing the areas of greatest danger for VEE outbreaks. Since then, the group has used satellite-based vegetation-scans of “forest islands” not previously sampled at ground level to achieve 90 percent accuracy in predicting the presence of VEE in an area—VEE that could mutate into an “outbreak-ready” strain at any time.

In southern Mexico, Weaver, UTMB Assistant Professor Jose Estrada-Franco, and their Mexican collaborators have begun a VEE study with direct implications for the United States. They are collecting mosquitoes and rodents from Oaxaca and Chiapas states, which experienced small outbreaks in 1993 and 1996 and served as way stations for the epidemic that reached Texas in 1971. Surprisingly, he says, the Mexican strains produce encephalitis in horses but only low concentrations of virus in the bloodstream, a characteristic that probably keeps the strains confined by making it harder for mosquitoes to acquire the virus. “We're going to keep monitoring that situation,” Weaver says. “If there's evidence these viruses can start amplifying better in horses, then we'll be more worried about them moving up into the U.S.”

Iquitos—Ortiz's destination—is a third theater of operations for Weaver's group, one where unique ecological circumstances make it an ideal place to study what happens when a large human community encounters mosquito-borne forest viruses. “Iquitos is right in the middle of the jungle—the jungle comes right up to the edge of the city,” Ortiz says. That means the jungle viruses come right up to the edge of the city too, with twenty different mosquito-borne viruses identified on the outskirts of Iquitos.

As the city grows and people clear nearby areas in the rain forest to grow crops, mosquitoes and the viruses they carry are forced to adapt to new conditions, undergoing genetic change at the same time they are being brought into contact with large numbers of humans. Weaver explains,“We want to know how this affects the exposure of people to these viruses, so we're studying the mosquito fauna and the reservoir hosts of the viruses, seeing how those change in response to deforestation.”

UTMB maintains a house and lab in Iquitos, which serve as a base for several insect and mammal ecologists working mainly in nearby forest and farm areas. Ortiz will work in the city itself, focusing on mosquitoes believed to be carrying VEE. Building on a study of unidentified
fever-producing illnesses that the U.S. Navy has been conducting in Peru since 1993, Ortiz wants to identify the mosquito type or types thought to be spreading VEE in Iquitos, determine what hosts those mosquitoes are feeding on, and try to find VEE's urban “reservoir host”—an animal the virus does not make sick, but from which it can cycle to mosquitoes and back indefinitely. “The idea is to collect blood-fed mosquitoes,” Ortiz says. “After mosquitoes bite, they go and rest on surfaces—walls in buildings, outside on vegetation—for a few days to digest the bloodmeal, before seeking a place to lay their eggs. So we can use what we call a backpack aspirator, kind of a vacuum cleaner, to suck them up.” The mosquitoes can then be frozen and shipped back to UTMB, where the Weaver group will attempt to isolate VEE from the blood the bugs have sucked from their victims—and, in a procedure that evokes Jurassic Park , examine that blood's DNA to try to determine just what animal the blood came from.

But before Ortiz leaves for Peru, she has lab work to wind up. She's about to go do a job similar to that undertaken by Ivy Greene, who took her female mosquitoes up to the BSL-3 lab to infect them with VEE. Since I can't go into the Level 3 lab with her—I'm not properly trained and don't have the necessary immunizations—Ortiz has offered to show me the procedures in a Level 2 lab, in conjunction with another Weaver lab experiment involving the relatively harmless Sindbis virus. Two graduate students, Nikos Vasilakis and Shannan Rossi, are infecting Aedes aegypti and Aedes albopictus with Sindbis virus, and they stand by in lab coats and shorts while Ortiz dons the protective “bunny suit” she would normally wear in the BSL-3. “You're going to laugh,” she warns, zipping the white coverall up over her dark-blue Student Government Association Intramural Sports
T-shirt and pulling a hood with a clear face shield down on her head.

“This is for protection from flying insects and pieces of insects—and also from inhaling aerosolized droplets of VEE,” she says. It's made from tear-resistant Tyvek, which feels like a cross between paper and fabric. The suit's elastic cuffs close tightly over ankles and glove-covered wrists. The hood overlaps the coverall, and has its own elastic seal beneath the wearer's chin. To prevent asphyxiation, a black hose feeds air into the hood from a belt-mounted battery-powered turbine.

Inside a screened-in area about the size of a large walk-in closet, Vasilakis and Rossi have set up the feeding apparatus. It's a Rube Goldberg-looking contraption that passes warm water around small glass chambers containing virus-infected sheep's blood, covered with a thin membrane made from chicken skin. The feeders are placed, membrane side down, on the screen tops of four cylindrical cardboard mosquito cages, each about the size of an ice-cream carton and containing 50 mosquitoes. The idea is to provide such a good imitation of a warm-blooded animal that the hungry mosquitoes will plunge their proboscises through the screens and membranes and suck down the blood.

If this had been VEE, things would have been different. The mosquitoes would have been held in the same “ice-cream boxes,” but those boxes would have been isolated inside a glove box, a closed container with gloves into which the protective-suited lab worker could extend his or her arms. Everything involving live infected mosquitoes in the BSL-3 happens in a glove box: feedings on anesthetized hamsters, mosquito sorting, the removal of legs and wings for analysis to determine whether a virus has reached all parts of a mosquito's body, even “salivating”—a process by which mosquitoes are persuaded to spit into a tiny tube so their saliva can be tested for virus. (Higgs is also collaborating with Dr. Lynn Soong to see whether proteins in mosquito saliva make it easier for West Nile virus and dengue virus to evade immune defenses.) When work is finished, mosquito-holding ice-cream-box cages are sealed into a Tupperware container before removal from the glove box, and then transferred to an incubator. “The rule is that all the mosquitoes have to be accounted for,” Ortiz says. “If you started with 50 in a box, there must be 50 when you finish. If we ever had a missing mosquito, our safety rules say that no one can leave and no one can enter—we'd notify a supervisor and search for the mosquito until we found it and killed it. We don't want infected, hungry mosquitoes flying around.”

That prospect can sometimes play tricks on the mind of someone working the lab alone, with vision and hearing limited by the suit hood and its constantly running air system. “I've worked six or seven hours at a time in the Level 3 lab, and sometimes it's like—all of a sudden you think, I saw something flying ,” Ortiz says, her eyes widening behind the face shield. “Or maybe— I thought I saw something flying , but… ”

Usually, though, the difficulties encountered are more prosaic, like the one Vasilakis and Rossi are facing now. The aegypti are feeding enthusiastically, with seven or more at a time clustered on each feeding membrane. (“Check out the abdomens—they're really getting full,” Ortiz says, pointing to some particularly engorged specimens.) But the albopictus , so voracious when humans are the target, have turned up their proboscises at the chicken-skin membranes. “I think these membranes are too old,” Vasilakis says.

“Did you sing to the mosquitoes?” Ortiz asks.

“What?” Vasilakis responds.

“You have to sing to your mosquitoes,” Ortiz says. “You have to make them happy.”

Ortiz won't have to sing to the Iquitos mosquitoes to make them happy; the ones she'll collect will already be fat with blood. Some of it may be hers, an occupational hazard of mosquito research. But she doesn't mind. Mosquito fieldwork is “the most fun you can have studying viruses,” she says. “You put on your boots in the morning and you're thinking about what you're going to do that day in the field, where you're going to go, where you're going to put your traps—you try to think like a mosquito. It's a challenge, and it's very interesting. It gets in your blood.”