More than a decade ago, Leslie Vosshall, then a relatively new Howard Hughes Medical Institute (HHMI) Investigator, decided to switch from studying innocuous fruit flies to a far deadlier creature—the mosquito. Perhaps her extensive knowledge of how the fruit fly sniffs out its food could be applied to mosquitoes, she wondered, uncovering new ways to blunt the blood-sucking insect’s uncanny ability to find human prey. “I wanted to do something the public could be excited about,” she says.

Her new work would indeed prove to have a major impact—just not what she’d anticipated. It’s been “a huge, staggering surprise,” she says. As she and the team she leads at the Laboratory of Neurogenetics and Behavior at The Rockefeller University now report in a paper published August 18, 2022, in Cell, her research has overturned the conventional model of the neural circuity animals use to detect—and to distinguish among—thousands of distinct smells in their olfactory systems. “This is a big deal,” says neuroscientist Christopher Potter of the Johns Hopkins University School of Medicine. “It really changes the way we think the insect olfactory system is working.” 

Moreover, the unexpected new result shows that it’s even harder than previously thought to confuse mosquitoes as they relentlessly search for human blood. In the fight to cut the enormous toll in illnesses and deaths by mosquito-borne diseases, “this is not a good news paper,” says Vosshall, now also vice president and chief scientific officer at HHMI.

When Vosshall’s HHMI lab at Rockefeller turned its attention to the mosquito, one of the initial tasks they successfully tackled was assembling the first complete genome of the insect. “No one had done genome editing before in part because the genome was so fragmented,” Vosshall explains. Then, with the genome in hand, Meg Younger a former postdoc in the lab set out to try to answer a puzzling question. Mosquitoes are attracted both to the CO2 that people breathe out and to human body odor. “But there’s something magical about adding those two ingredients together, where one plus one equals twenty,” she says. The insects get super excited, becoming very focused and very ferocious human hunters. So how are the two signals being added together and amplified so much in the olfactory system?

To try to find out, Younger figured they could identify which olfactory neurons responded to CO2 and which to body odor, and then trace the pathways of the signals to the brain. So they used the gene editing tool CRISPR to slip a fluorescent marker protein into the neurons that had receptors for CO2 and another marker into those that could sense chemicals from body odor.

That’s when the research took an unexpected turn. “It was like Alice in Wonderland—where nothing makes sense,” Vosshall says.

The scientific dogma, based on the Nobel Prize-winning research of Linda Buck (now at Fred Hutchinson Cancer Center) and Richard Axel at Columbia University in mice, was that the smell sensing systems in animals are exquisitely specialized and organized. Each olfactory neuron has just one type of receptor, which detects a specific set of chemicals and then which connects to just one structure (called a glomerulus) in the olfactory bulb. By this logic, there would be separate types of neurons that respond to strawberry smell, for example, others for peanut butter, yet others for gasoline, and so on. “We as a field were so influenced by Buck and Axel,” says Vosshall (who was a postdoc in Axel’s lab). “Those were the rules.”

By probing receptor genes with different fluorescent colors, Margaret Herre a former MD-PhD student in the lab, discovered that individual neurons were chock full of multiple types of receptors, not just one. We found that “all the Buck and Axel rules were thrown in the garbage can by mosquitoes,” says Vosshall.

The results were so startling that Vosshall’s lab has spent years painstakingly proving that they were actually real, using several additional lines of evidence. For instance, Olivia Goldman, a PhD student in the lab, harnessed a relatively new and revolutionary technique called single nucleus RNA sequencing (snRNA-seq) to probe which genes are turned on in individual neurons. The approach confirmed that each neuronal cell is indeed making many kinds of receptors. 

They also teamed up with scientists at the Swedish University of Agricultural Sciences, who had done ground-breaking work to figure out how to stick electrodes into individual mosquito olfactory neurons and measure the cells’ responses to various smells. That method also confirmed that a single mosquito neuron can detect different smells—even two different flavors of body odor, a perfumy smell and a stinky foot odor, which require two entirely different classes of receptors. Those results “were a huge relief,” Vosshall says. She anticipated widespread skepticism to her conclusions, “so the number of levels of evidence that we used to prove it was intense,” she says,

As word and preprints of Vosshall’s team’s results spread through the community, in fact “there was a lot of skepticism at first,” says Potter. But not only was the evidence overwhelming, in fact, similar findings also were emerging from Potter’s lab at Johns Hopkins. Working with both the fruit fly and a mosquito species, Potter’s team published a paper in eLife in April suggesting that “co-expression of chemosensory receptors is common in insect olfactory neurons.” In the past, the conventional wisdom of one receptor per smell and one receptor per neuron was so strong that there was no reason to probe for multiple receptors, says Potter. “Now we know to look for it.”

In retrospect, the added complexity of the insect olfactory system makes perfect evolutionary sense, especially for mosquitoes that must find humans to survive. Having multiple types of receptors in each neuron amps up the bugs’ ability to detect exhaled CO2 and the whole smorgasbord of body odors. And when people try to rebuff the biting insects by blocking some receptors, the mosquitoes can still easily home in on blood using their other receptors. “It is a really good trick,” explains Vosshall. “Mosquitoes have Plan B after Plan B after Plan B. To me the system is unbreakable.” That’s obviously not good news for the effort to reduce the toll from mosquito-borne diseases, such as malaria, yellow fever, and dengue, by trying to block receptors. But perhaps an alternative strategy might be to overwhelm the whole system with alternative smells, adds Potter. At least now “we have a more realistic view of what we are up against,” he says.

In the meantime, Vosshall aims to compare the olfactory neurons of blood-dining mosquitoes with those from purely vegetarian mosquito relatives to see if the more extreme receptor complexity is a unique adaptation for those species that only hunt humans. And as for the puzzle Vosshall first started to probe—how the combined sensing of both CO2 and body odor greatly amplifies the message to the brain? One of her former postdocs, Meg Younger, is tackling the question in her new lab at Boston University.

IMAGE CREDIT: NASA.