How mosquitoes time their bites

[Music] Welcome to Science Sessions, the podcast of the proceedings of the National Academy of Sciences, where we connect you with Academy members, researchers, and policymakers. Join us as we explore the stories behind the science. I'm Paul Gabrielson. Fighting mosquitoes are more than a nuisance. They transmit diseases like yellow fever and malaria posing a serious health risk, so understanding the molecular underpinnings of biting behavior may be key to mitigating mosquito risks. In a recent PNAS study, Laura DuVal of Columbia University and colleagues identified a peptide in the circadian clock system of 80s-adjipti mosquitoes that may regulate their dawn and dusk biting pattern. Understanding what drives mosquitoes to feed the author's right is an early step towards disrupting their feeding patterns to protect human health. Laura introduced us to normal biting behavior of mosquitoes. How do they find their targets and at what times of day? One of the things that we have known probably for millennia is that different species of mosquitoes tend to bite at different times of day. So in fact, there are writings from the Roman era where people talk about trying to attack at particular times of day to avoid the mosquitoes that were found in these swamps. So some species of mosquito bite primarily at nighttime. This is when the malaria mosquito caught an athlete's gambiae often bites. But the species that we studied here called 80s-adjipti mostly bites at dawn and at dusk. And this has been really well established in decades of field studies. So the way that you normally carry out these field studies is what is called the land and catch technique. You send someone, maybe one of your students or your postdocs out to go and sit in a field or in a hut and expose part of their body and count the number of mosquitoes that land on them and try and bite them. A lot of labs have worked on trying to understand what cues mosquitoes are paying attention to when they're trying to bite us. We know that they are really attracted to things like visual contrast to a human body odor. Once they get close enough to us, even human body temperature. But they are really exquisitely sensitive to detect even small changes in carbon dioxide. So this is a really potent cue to tell them that something alive is nearby. And if you give them even just a small pulse of carbon dioxide like what we exhale, this will drive a lot of flight activities. You can sort of think of this as like starting the hunt for a human. Why is the biting behavior of the 80s agypti mosquito of particular interest? 80s agypti mosquitoes can transmit the pathogens that cause diseases like yellow fever, dengue, chikengunya, and zika. So they transmit those pathogens to humans when they find us and bite us. And this species really strongly prefers to bite humans. So what this means is that if you were out in the evening walking your dog, an 80s agypti female would really strongly prefer to bite you. And that's part of the reason that this species is so effective at contributing to the spread of diseases that affect humans. What did you find about how mosquitoes response to CO2 varied over the day? Based on these field studies, mosquitoes are mostly biting at dawn and at dusk. But because so many things might be varying in those field studies, one of the things that Lynn Han Dong, who is the first author of this study really wanted to do is to bring this into the lab and an environment that we could really carefully control. Because not many people have studied circadian rhythms in mosquitoes, we had to invent a lot of the tools from the ground up. We built a mosquito arena with a pretty fancy camera setup so that we could puff in carbon dioxide at different times of day and track the mosquitoes flight activity. That is something that Lynn Han actually constructed himself by screwing together pieces of plastic, gluing them together, screwing the camera and writing all the code that we use to analyze this data. And so because we could track their flight activity for tens of minutes, this meant that we could go beyond just asking whether or not they responded to the carbon dioxide or not, but we could measure different parts of their responses. And one really important thing that we were able to measure here was how long that response lasted. So we can call this either the duration of the response or the response persistence. One of the first things that we noticed is that if you pulse carbon dioxide in the middle of the night, the mosquitoes basically don't respond at all. And that was interesting because it tells us that if you give this normally very potent queue associated with finding humans at the wrong time of day, the mosquitoes don't seem to be responding to it. Even though the mosquitoes responded to carbon dioxide throughout the light part of the day, we noticed that the responses were most persistent exactly at dawn and dusk in the middle of the day, the mosquitoes are relatively inactive. And even though they will initiate this flight activity, if you give them a burst of carbon dioxide, they sort of give up more quickly than they do at dawn and at dusk. And I think it's pretty intuitive to imagine why a mosquito that has a more persistent response might be able to be a more effective hunter of humans at those particular times of day. What peptide did you find that might be regulating this behavior and how does losing this peptide change mosquitoes behavior? In many insects, these behavioral patterns of activity are driven by circadian clock cells in the brain. And there's not just one clock in the brain. There are dozens or hundreds of individual neurons that need to work together to allow the animal to produce these coherent behavioral rhythms. Now, there's a gene that you can find in many insects called PDF. This stands for pigment dispersing factor. That's an important neuro peptide that is made by some of those clock cells and that works to keep the whole system synchronized and organized together. So based on what we knew in other insects, we thought that if we took away this important neuro peptide, we would desynchronize the clock cells and the mosquito brain. We wanted to ask how this might break or disrupt those rhythms that we saw in carbon dioxide responses in the mosquitoes that we could actually go in and genetically disrupt things. That's something that was impossible or at least very hard to do with mosquitoes for a long time. The advent of a lot of these CRISPR-CAS-based technologies really means that we can ask questions about the regulation of mosquito behavior in a way where we can make genetic interventions and go beyond just observation of these patterns. We actually looked at the clock cells in the brain to make sure that when we disrupted this gene in mosquitoes, we also saw that the clock cells were desynchronized when we mutated or removed this gene. These animals also had behavioral consequences as well. So the animals that lost PDF were not able to maintain their normal daily rhythms and activity. When we looked at their carbon dioxide responses, they no longer showed those normal rhythms in persistence. And the most dramatic change that we saw was that there was a loss of mourning persistence when these animals lost the important synchronizing factor. Then when we actually tested these animals in a laboratory assay where they actually get to find and take a blood meal, but we give them access to what we call an artificial membrane feeder. Normally mosquitoes are really good at finding this artificial feeder in the morning in the evening and taking a blood meal, but the PDF mutants were much less effective at blood feeding specifically in the morning, the same time of day when they had reduced carbon dioxide persistence. What are the takeaways for control of mosquito-borne illnesses? So we are a basic or discovery research lab. One of our main goals was to understand more about how the system works. This isn't something that we're immediately commercializing, but we were interested in understanding why mosquitoes are such good predators of humans at specific times of day. The other side of this is that that means there are certain other times of day and mosquitoes are not particularly responsive to human host associated cues or not particularly efficient predators. And so if we can find ways to mimic that internal state, then we might be able to find new approaches to prevent mosquitoes from finding us and biting us as effectively. What are the caveats or limitations of the study? We chose to focus specifically on carbon dioxide, because we know it's really critical for mosquito host seeking, but also because we can deliver it in a very standardized and reliable way. However, mosquitoes are normally using multiple cues to find us at once, things like human body odor and body temperature. And so one important area for future study will be to ask whether or not responses to those cues or to multiple cues in combination are regulated in the same way. Thanks for tuning into science sessions. 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