Confocal image of a male mosquito antenna, used to validate a unique chemoreceptor co-expression pattern identified in the mosquito atlas. (Credit: Alexandra DeFoe)
The most dangerous animal in the world just got easier to study-and perhaps defeat one day.
Researchers from Rockefeller University's Laboratory of Neurogenetics and Behavior, in collaboration with mosquito experts around the globe, have created the first-ever cellular atlas of the Aedes aegypti mosquito, which transmits more diseases than any other species of its kind. The Mosquito Cell Atlas provides cellular-level resolution of gene expression in every mosquito tissue, from the antennae down to the legs. The dataset is freely available to all researchers (and curious members of the public). They recently published the atlas in Cell.
"This is a comprehensive snapshot of what every cell in the mosquito is doing as far as expressing genes," says lab head Leslie Vosshall, who has studied Aedes aegypti, aka the yellow fever mosquito, for nearly two decades. "It's a real achievement because we profiled so many different types of tissues in both males and females."
The atlas has already yielded new insights into the genetic secrets of Aedes aegypti, including novel cell types, subtle differences-and unexpected similarities-between male and female mosquitoes, and the dramatic changes in genetic expression that the female mosquito brain undergoes after a blood feeding.
Senior author Nadav Shai, a senior scientist in both Vosshall's lab and at the Howard Hughes Medical Institute, anticipates that by using the atlas as a starting point, many researchers will make new discoveries. "We believe this enormous data set will really move mosquito biology forward," he says. "It's a great tool for vector biologists to take whatever interests them and just run with their own line of research."
Organ by organ
In the past several years, scientists have used single-cell sequencing to identify cell types and illuminate gene expression patterns in model organisms such as Drosophila melanogaster (fruit fly), Caenorhabditis elegans (nematode), and Mus musculus (mouse), resulting in a whole organism, single-cell atlas of each species.
Mosquito researchers have been following suit but in a piecemeal way: organ by organ, tissue by tissue, all in different studies. Some of that prior work was done by Vosshall's team and fellow members of the Aedes aegypti Mosquito Cell Atlas Consortium, a global collaboration of scientists that was assembled for this project.
Most prior studies had focused on female mosquitoes, leaving out males. "Both females and males feed on nectar in their day-to-day lives, but females need blood for protein to develop their eggs and produce a new generation of mosquitoes," says first author Olivia Goldman.
"Because the female is the one that's spreading all the pathogens, there is an enormous bias toward looking at the biology of the female and very little information about the male," Vosshall says. "So we wanted to be inclusive and fill in the gap."
"We also wanted to bring the mosquito cell biology up to date in a single resource using advanced and uniform sequencing technology," Shai says.
To that end, the team used single-nucleus RNA sequencing (snRNA-seq)-which excels at capturing the biology of all insect cell types compared to single cell approaches-to create a large dataset of more than 367,000 nuclei from 19 types of mosquito tissues selected across five biological themes: major body segments; sensation and host seeking; viral infection; reproduction; and the central nervous system.
Tasting sweetness with their legs
They found 69 cell types grouped into 14 major cell categories, many of which had never been seen before.
Among the most striking findings was the pervasiveness of polymodal sensory neurons-supercharged cells that can pick up a wide variety of environmental cues, including temperature and taste. Previous research from the Vosshall lab had found that the antenna and maxillary palps were packed with these neurons, but now that they were able to look organism-wide, they found them everywhere, including the nose, tongue, and legs.
"Just like the antennae and maxillary palps, the legs and mouth parts have really powerful tools for sensing the world," Shai says. "Together they enable mosquitoes to be really good at what they do-seek hosts, feed on them, and reproduce."
Those multifunctional chemoreceptors allow them to, among other things, detect sweetness and fresh water.
"Being able to taste sweetness with their legs may be useful for detecting sugars, which both females and males need to live," Shai says. "But it's just one part of a combination of tastes that clues them into what's around them-a human to bite, a flower for sugar source or a good water source to lay eggs. We believe that the combination of a lot of sensors is important for their survival."
Brain changes that accompany behavior shifts
After feeding, a female mosquito loses all interest in humans and other hosts; her focus becomes developing and laying eggs.
"How does this incredibly strong drive to bite people get turned off?" Vosshall says.
"We knew from previous research from our lab and others that the brain transcripts change after blood feeding, and our assumption was that maybe we would find different subtypes of neurons that down- or upregulated their transcripts," Shai says.
To find out, they examined the gene expression of female mosquito brains at 3, 12, 24, and 48 hours after a blood feeding. They found dramatic changes in gene expression in all time periods, which peaked after the first few hours and gradually abated. Most of the early expression changes were of genes being upregulated, while later time periods showed a mix of both up- and downregulation.
Strikingly, these changes occurred in a completely unexpected way. Neurons account for roughly 90% of mosquito brain cells, but it was the glia-support cells that account for less than 10%-that underwent large shifts in gene expression.
"The glia are completely rewired during this time when the females lose interest in people," Vosshall says.
"That was a big surprise," Shai says. "It's evidence that glia are super important for not only supporting brain cells and function but also are physiologically relevant to behavior."
Limited sexual dimorphism
Another illuminating finding is that for all the documented morphological and behavioral differences between female and male mosquitoes, their cellular makeup is largely identical, aside from small clusters of sex-specific cells and reproductive organs.
"We were kind of expecting it to be a tale of two genomes, but that's not what we found," Vosshall says.
"In general, most cells look the same, and the transcripts they express are similar," Shai notes. "However, that doesn't mean that the regulation and level of expression are the same, and those probably drive the differences. Another factor could be how the different gene expressions work together to create new functions."
One exception was found exclusively in the male antenna, which is largely unexplored. "A small group of cells is marked by the expression of a single gene that's not expressed in any female tissue," Vosshall says. "If we hadn't compared male and female gene expression, we never would've spotted them." Their function remains to be determined.
Future directions
The Vosshall lab will mine the mosquito single-cell atlas to further its investigations into behavior such as host seeking and sensing the environment through Aedes aegypti's remarkable, widely dispersed set of multifunctional sensory neurons.
"Different people in the lab are going to take it to different places," Shai says.
They hope that researchers everywhere will find it equally inspiring. "The sheer size of the dataset opens up many new avenues of research that people couldn't study before because they didn't have this tool," Shai says.
"This is a global resource that has been open to everyone since the very inception of the project in 2021, so many people are already using it," Vosshall adds. "We're excited to see the discoveries that will come from it."
