In a new study, Vanderbilt pharmacologist Jerod Denton, Ph.D., Ohio State entomologist Peter Piermarini, Ph.D., and colleagues report an experimental molecule that inhibits kidney function in mosquitoes and thus might provide a new way to control the deadliest animal on Earth.
The investigators aim their inhibitor, named VU041, at the mosquito Anopheles gambiae, the leading vector for malaria, and Aedes aegypti, a mosquito that transmits Zika virus and other pathogens. The study appears in the journal Scientific Reports.
Over several decades of exposure, mosquitoes have evolved genetic resistance to various insecticides that attack their nervous system. The new study shows for the first time that inducing kidney failure — or, more correctly, Malpighian tubule failure — in mosquitoes can circumvent resistance to conventional insecticides.
“We’re essentially preventing mosquitoes from producing urine after they take a blood meal,” said Denton, associate professor of Anesthesiology and Pharmacology.
According to Denton, in taking a blood meal mosquitoes can double or even triple their body weight.
Besides providing nutrients, blood meals carry toxic salts; the potassium chloride lurking in red blood cells, if not quickly voided, can depolarize cell membrane potentials and kill straightaway.
“So they’ve evolved a rapid diuretic process to very quickly separate the salt water from all the nutrients that they need for egg development. A lot of people don’t realize that mosquitoes have kidneys, and when they take a blood meal from you they also urinate on you almost simultaneously.
“What our compounds do is stop urine production, so they swell up and can’t volume regulate, and in some cases they just pop,” he said.
Conventional mosquitocides cause death of males and females at all stages of mosquito development, and in doing so exert considerable selective pressure for the development of genetic resistance.
“By targeting blood feeding female mosquitoes, we predict that there will be less selective pressure for the emergence of resistant mutations,” Denton said.
The investigators show VU041 to be effective when applied topically, which indicates that it potentially could be adapted as a sprayed insecticide. They also show that it doesn’t harm honeybees.
Arrangements are underway to test VU041 in a spray formulation. If that’s successful, additional safety testing would be needed before deciding about commercial development, Denton said.
Novel study identifies an area of the mosquito brain that mixes taste and smell
A new study by Johns Hopkins researchers suggests that a specialized area of the mosquito brain mixes tastes with smells to create unique and preferred flavors. The findings advance the possibility, they say, of identifying a substance that makes “human flavor” repulsive to the malaria-bearing species of the mosquitoes, so instead of feasting on us, they keep the disease to themselves, potentially saving an estimated 450,000 lives a year worldwide.
A report on the research appeared online on Oct. 3 in the journal Nature Communications. Malaria is an infectious parasite disease of humans and animals transmitted by the bite of the female Anopheles gambiaemosquito. In 2015, experts estimate it affected 214 million people, mostly in Africa, despite decades of mosquito eradication and control efforts. There is no malaria vaccine, and although the disease is curable in early stages, treatment is costly and difficult to deliver in places where it is endemic.
“All mosquitoes, including the one that transmits malaria, use their sense of smell to find a host for a blood meal. Our goal is to let the mosquitoes tell us what smells they find repulsive and use those to keep them from biting us,” says Christopher Potter, Ph.D., assistant professor of neuroscience at the Johns Hopkins University School of Medicine.
Because smell is essential to mosquito survival, each mosquito has three pairs of “noses” for sensing odors: two antennae, two maxillary palps and two labella. The maxillary palps are thick, fuzzy appendages that protrude from the lower region of the mosquito’s head, more or less parallel to its proboscis, the long, flexible sheath that keeps its “feeding needle” under wraps until needed. At the very tip of the proboscis are the labella, two small regions that contain both “gustatory” neurons that pick up tastes and olfactory neurons for recognizing odorants.
To better understand how An. gambiae mosquitoes that cause malaria receive and process olfactory information from so many sensory regions, Potter’s team wanted to see where olfactory neurons from those regions go to in the brain.
They used a powerful genetic technique — never before accomplished in mosquitoes, according to Potter — to make certain neurons “glow” green. The green glowing label was designed to appear specifically in neurons that receive complex odors through proteins called odorant receptors (ORs), since OR neurons are known to help distinguish humans from other warm-blooded animals in Aedes aegypti mosquitoes, which carry the Zika virus.
“This is the first time researchers managed to specifically target sensory neurons in mosquitoes. Previously, we had to use flies as a proxy for all insects, but now we can directly study the sense of smell in the insects that spread malaria,” says Olena Riabinina, Ph.D., the lead author of the study and a postdoctoral fellow now at the Imperial College London. “We were pleasantly surprised by how well our genetic technique worked and how easy it is now to see the smell-detecting neurons. The ease of identification will definitely simplify our task of studying these neurons in the future.”
As expected, Potter says, the OR neurons from the antennae and maxillary palps went to symmetrical areas of the brain called antennal lobes, just as they do in flies. But Potter was quite surprised when he saw that the OR neurons from the labella went to the so-called subesophageal zone, which, he says, had never before been associated with the sense of smell in any fly or insect; it had only been associated with the sense of taste.
“That finding suggests that perhaps mosquitoes don’t just like our smell, but also our flavor,” says Potter. “It’s likely that the odorants coming off our skin are picked up by the labella and influence the preferred taste of our skin, especially when the mosquito is looking for a place to bite.”
Potter says the finding potentially offers researchers one more way to repel mosquitoes. The antennae and maxillary palps are more specialized for picking up long-range signals, while the labella come into direct contact with our skin. In fact, Potter says, before injecting their needlelike proboscis, mosquitoes use the labella to probe about on a victim’s skin. “We don’t really know why they do that, but we suspect that they’re looking for sensory cues that hint at easy access to a blood vessel,” he says. “This suggests that a combination of repellants could keep mosquitoes from biting us in two ways. One could target the antennal neurons and reduce the likelihood that they come too close, while another could target the labellar neurons and make the mosquitoes turn away in disgust — before sucking our blood — if they got close enough to land on us.”
The two-part genetic system Potter devised to generate the glowing neurons will make it much easier for his and other laboratories to mix and match genetically altered mosquitoes to generate new traits, he says. His group has already created a strain of An. gambiaemosquitoes whose OR neurons glow green upon activation. Scientists can thus see which neurons light up in response to a specific smell.
“Using this method, we hope to find an odorant that is safe and pleasant-smelling for us but strongly repellant to mosquitoes at very low concentrations,” says Potter.
His group was also able to compare the brains of male and female mosquitoes. Since only females use their sense of smell to find humans and males feed only on nectar, it was previously thought that males had just a rudimentary sense of smell. The Potter group found instead that males have the same level of complexity in their brains to detect odors as females but have fewer olfactory neurons. “It appears that males might just have a scaled-down version of a female’s sense of smell. So they can still smell everything a female smells, just not as well,” Potter says.
His group plans to study other types of neurons to better understand how signals from the mosquitoes’ three types of olfactory receptors interact to influence their behavior. For example, why is lactic acid not attractive on its own but highly attractive when mixed with carbon dioxide?
“We’d like to figure out what regions and neurons in the brain lead to this combined effect,” says Potter. “If we can identify them, perhaps we could also stop them from working.”
South America is fighting a battle against tiger mosquitoes that transmit yellow fever, dengue fever and the Zika virus. In Central Europe, wine and fruit growers fear another year of massive crop failures due to a type of vinegar fly known as the spotted-wing drosophila. Since the success rate of conventional methods continues to fall, researchers from Fraunhofer and the Justus-Liebig-University Gießen are developing new species-specific strategies that keep pests under control without resorting to environmental toxins.
When Prof. Marc F. Schetelig takes visitors through his laboratories at Justus-Liebig-University Gießen and the Fraunhofer Institute for Molecular Biology and Applied Ecology IME in Gießen, one of the things he shows them is a climate chamber that is kept at a temperature of 27 degrees Celsius. Inside are thousands of tiger mosquitoes (Aedes aegypti) in net cages. “We have to keep the females on a diet of cow’s or pig’s blood, served at 37 degrees Celsius, which we get directly from the abattoir,” says Schetelig. “This helps them lay their eggs, which we then put in bowls of water, where the larvae hatch and pupate.”
Schetelig is an expert on the sterile insect technique, or SIT, in which the mass release of sterile males decimates the population of insect pests (see box). With his team of researchers at the LOEWE Center for Insect Biotechnology & Bioresources, which is sponsored by the German state of Hesse, Schetelig is developing methods that increase the effectiveness of the sterile insect technique and allow it to be transferred to other insect pests.
A genetic on and off switch
Schetelig’s highest achievement to date has been his work on the spotted-wing drosophila vinegar fly, Drosophila suzukii, which comes from Southeast Asia and is spreading worldwide. The females lay their eggs in ripe fruit just before the harvest, when the farmers are generally no longer allowed to spray insecticide. This means that the fly can evade even efficient insecticides and at present, an effective control strategy is not on the market. In 2014, the spotted-wing drosophila caused massive crop failures to cherries, raspberries, plums and grapes in many parts of Central Europe. Mass propagation of the insects was feared for 2015, but was derailed by the hot, dry summer. This year, the wet early summer weather could once again provide the ideal conditions for a further spread.
Schetelig has designed a genetic system that causes the flies’ offspring to not survive beyond the embryonic stage. If male flies carrying this system mate with naturally occurring females, the fertilized eggs will not produce larvae. But the genetic system also includes a switch that can be used to turn the program off. This switch can be activated by feeding the flies the antibiotic tetracycline: the tetracycline containing diet allows the flies to be successfully rearing in the laboratory. “The new method doesn‘t require antibiotics for larval rearing and only a little amount of antibiotics is needed for the adult flies. So no antibiotics are transferred to the environment,” says Schetelig.
To help with mass breeding, the research team anchored yet another genetic system within the genome that can cause all females to die at the embryonic stage. “With this sexing system, we can considerably improve the effectiveness of mass breeding, because we raise only the males. Females need to be sorted out,” explains Schetelig. In the majority of conventional breeding methods, the females needed to be weeded out, sometimes manually, before a mass release.
Curbing dengue and Zika viruses
The team is currently working on transferring the genetic systems it developed for the spotted-wing drosophila to the tiger mosquito. It is hoped that this use of the sterile insect technique will help stop the tiger mosquito from continuing to spread a number of dangerous viruses. In addition to the dengue and Zika viruses, the tiger mosquito can also carry the viruses for yellow fever, Chikungunya and Rift Valley fever. Despite being indigenous to the tropics, this insect spread across the world. A related Aedes species has already established itself in Italy. And the first case of tiger mosquitoes surviving the German winter and hatching has been reported near Freiburg im Breisgau.
In addition to his high hopes in the new technology, Schetelig is also looking into the risks of releasing transgenic insects. As part of a project funded by the German Research Foundation DFG through the Emmy Noether Programme, he is examining for example how stable the integration of foreign genes into genomes really is and what happens when other animals eat the genetically modified insects.
“Since we use radioactive radiation to sterilize the males before their release, we are convinced that they will not multiply,” says Schetelig. He expects the technology will most likely find large-scale application in countries that already have regulations for the release of genetically modified organisms in place, such as in South America and to some extent in the United States. “The decisive factor in terms of acceptance is the level of threat posed by the pests,” Schetelig says. “If we had the same problems with dengue fever and Zika virus in Germany as they have in Brazil, we would also be searching for and evaluating different solutions. And the sterile insect technique with transgenic mosquitoes would definitely be part of the discussion.”