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In this episode of The Highlights, we're joined by Zhilei Zhao, a former graduate student in the McBride Lab of the Department of Ecology and Evolutionary Biology and the Princeton Neuroscience Institute. We discuss his experiences working in the lab during the COVID-19 pandemic, as well as his study of the delicate neuroscience of mosquitoes and its potential impact on the fight against malaria and other insect-borne illnesses.
Brains, Black Holes, and Beyond (B Cubed) is a collaborative project between The Daily Princetonian and Princeton Insights. The show releases 3 episodes monthly: one longer episode as part of the Insights partnership, and two shorter episodes independently created by the 'Prince.' This show is produced by Senna Aldoubosh '25 under the 147th Board of the 'Prince.' Insights producers are Crystal Lee, Addie Minerva, and Thiago Tarraf Varella. This show is a reimagined version of the show formerly produced as Princeton Insights: The Highlights under the 145th Board of the 'Prince.'
Please direct pitches and questions to podcast@dailyprincetonian.com, and any corrections to corrections@dailyprincetonian.com.
[Content Warning]
Hey everyone! Welcome to episode three of the Highlights. Just a little heads up before we play this week's episode: after our theme music plays for the second time, there will be a short discussion about a research method involving mosquitoes. If that's not your cup of tea, skip ahead about 1 minute. We hope you enjoy this episode, we had a lot of fun making it!
[Theme music plays]
[Intro]
Thiago: Hi everyone! My name is Thiago, I am a graduate student at Princeton University and I'm your host. The Highlights is a sister podcast to Princeton Insights in collaboration with The Daily Princetonian. Insights is a newsletter written by Princeton undergrads, grad students, and post-docs. We write about the most exciting and groundbreaking research being conducted here at Princeton, in the form of short, fun, easy-to-read reviews. We cover a range of topics including psychology, neuroscience, biology, computer science, and physics, to name a few. Make sure to check out our website at insights.princeton.edu. Right now, I'll have the pleasure to receive my fellow graduate student Olivia Duddy as a co-host. Say hi Olivia!
Olivia: Hi everyone! I’m so excited to co-host this episode of The Highlights with Thiago. I recently wrote a review for Princeton Insights about the paper we’re discussing today, written by graduate student Zhilei Zhao and others from the McBride lab. Before we hear from Zhilei, Thiago will introduce him to you.
T: Thank you Olivia. Zhilei is currently a 6th-year graduate student in the McBride Lab, but also pursuing a joint graduate degree in neuroscience. He grew up in southwest China and went to college at Peking University in Beijing, majoring in life sciences. His research focuses on the intersection of neurobiology and evolution. Outside the lab, he enjoys jogging and reading about histories.
T: Thanks for coming, Zhilei, such a pleasure to have you here!
Zhilei: Thank you for the introduction.
O: So let's start off a little bit hearing more about you. So can you describe your journey to becoming a grad student here at Princeton?
Z: I think it's pretty straight forward — I majored in biology, life sciences, in college. I've been always interested in science, and especially for biology because I realized that there are just so many interesting and unanswered questions in biology. But in college, we study, like the major is very broad, we study a lot of different stuff. And my interest at that time was mostly on evolutionary biology and also a little bit on neuroscience. So I took one year gap in the same research lab where I did my thesis work. Then, after gap year, I applied for graduate school. I think Princeton was my best choice because of my advisor, Lindy McBride. She is doing both evolutionary biology and neuroscience, like the intersection of those two fields, so that's really ideal for me.
T: Nice! That was very useful then.
Z: Uh-huh [laughs]
T: So since you're now a graduate student, what are the best and worst parts of being a graduate student for you?
Z: The best part is, I feel like the freedom, especially in our lab, intellectual and also financial. Intellectual means my advisor, Lindy, really encourages us to explore different frontiers, even when those frontiers are kinda risky — like maybe just too ambitious. So we can really explore those frontiers, and we can dive into the unanswered questions. Like, if you read our paper, we develop a lot of new methods, and those new methods really take time to develop, and without the intellectual and financial freedom I feel like it's not possible to do that.
T: What about the worst part?
Z: The worst part, I feel like sometimes you just get too focused on the research. Like you keep thinking about it, right, even after work. Like when you're trying to sleep, you're still trying to figure out the problem, still trying to troubleshoot. I feel like that's the worst part.
O: I absolutely understand that, it's hard to step away —
Z: Uh-huh
O: — and you know, take a step back and go home and not be still thinking about work. And so, how has your work life changed during COVID? You know, being in a hands-on type of research, I imagine things have been different for this past year.
Z: I'm lucky because most of the lab work was done before COVID. So, during COVID, I mostly focused on analysis and also writing the paper. By the way, I'm still doing a little bit of experimenting, but the efficiency is much lower than it was before. But after the vaccine, I feel it's better because right now we can really do more experiments.
T: Cool! That's great — I'm glad it didn't have too much of a negative impact.
T: So let’s get into your research. Aedes aegypti is a species of mosquito common to Brazil. So, that's something that is very interesting to me to learn more about because I'm Brazilian. So I would like to know from you — why did you choose this mosquito for your study?
Z: Okay, so let me first ask you one question.
T: Yeah!
Z: In your opinion, what's the most dangerous animal in the world?
T: Well I read your paper so...
[All laugh]
T: So should I answer from what I saw in your paper or what I might guess?
Z: [laughing] Yeah, I feel like for most people they won't think about mosquitoes because they're just tiny insects. But the real numbers, the statistics, are very striking, because nearly 1 million people die because of a mosquito-borne disease every year. It's just a crazy number in a modern society. So part of the reason is there are some species of mosquito that really just target humans. For example, the species we study, Aedes aegypti, it's called the Yellow Fever mosquito, or Dingy mosquito, because they transmit Yellow Fever, Dingy, and recently Zika. So it's a very dangerous mosquito. And this species is interesting because it originated in Africa, but in Africa we noticed the mosquito bites not only humans, they also bite animals. But, with global trade in the last 1500 years, the mosquito has spread over the world in the tropical and subtropical regions. But, outside Africa, those mosquitoes really target humans. So imagine if you're walking your dog, those mosquitoes will just target you instead of your dogs. That's why they are just so dangerous. So we are very interested in why they can do this, they can distinguish human from animal.
O: So is this, for this specific mosquito, is this fairly rare? For it to prefer humans?
Z: So most mosquitoes are generalists. There are over 3000 species of mosquito, only four or five are human specialists.
O: Wow. So, kind of getting into your paper now, what exactly is it about us that makes us a desirable target for the mosquito?
Z: Yeah, so that's the central question we want to answer in this paper because from previous research, we know mosquitoes, they really rely on olfaction, the sense of smell, to detect the hosts. So we decided to look at the olfactory system of Aedes aegypti. The way we do it is through neuroimaging. So the idea is, if we can image the neural response of this mosquito when we present the odor of human and animal, and if we can compare the neural response, we might be able to understand why they really like human odor.
O: And so when you first started this study, was there a lot already known about how mosquitoes smell? Or was it sort of a big mystery?
Z: It's still a big mystery because a mosquito is not a traditional model species. For example, in drosophila, we know a lot of the olfactory systems of the drosophila, the fruit flies. But in mosquitoes we know very little because we don't have the genetic tools to study mosquitoes.
T: That's interesting — and do you expect them to be very different? Drosophila and mosquitoes?
Z: So after we began to look at the olfactory system we realized it's not so different — the basic architecture is the same, it's conserved. Just the receptor they express might be different. So they are sensitive to different odors, but the basic architecture is the same.
T: I see… well, I'm glad!
[Theme music plays]
[Methods — CW]
T: So going to the methods then, from your paper. One thing that is kind of funny when reading your paper and looking at the first figure it looks like there are people putting their hands on a mosquito cage? So, do people have to actually sacrifice themselves to test the mosquito preference? How does that work?
Z: So in that assay the mosquitoes, they can smell the odor of the arm, but they cannot reach the hand because there is a screen. So they will try to pinch through the screen. But in a different assay, like when we need to blood feed the mosquitoes, like if we want to produce the next generation, we need to blood-feed the mosquitoes, the females. So we usually use our arm. For example, there is one cage of 500 mosquitoes. Females, they are very eager to bite you, so we insert our arm into the cage and all the mosquitoes will bite you for the blood.
T: Like, your own arm?
Z: My own arm.
T: The experimenter's arm?
Z: Yup.
O: Is that really uncomfortable?
T: [laughs]
Z: In the beginning, when I first joined the lab, it seemed scary because after feeding you see a lot of swelling on your skin. But right now I don't feel anything, like it doesn't really itch, it doesn't really hurt. The immune system can adapt to the mosquito bite.
T: Wow!
O: Wow, that's crazy!
T: You're literally sacrificing yourself to the most dangerous animal on Earth.
O: [laughs] That's true — how often do you have to do that?
Z: It depends, like for example, if you have three strains, you might need to do it once every two months because we usually breed the mosquitoes every six months. Every six months we need to produce the next generation.
O: Very fun, very cool! Alright, so now getting back to the specifics of your work and focusing on how mosquitoes smell, what's going on in their brains. You talked a little bit about how the genetic tools are very limited for mosquitoes. But I know from your work that you were able to engineer some mosquitoes to allow you to visualize what's going on in their brains. Can you talk a little bit about how that worked and how you did that?
Z: Yes. So one of the limits of working on mosquitoes is the lack of the genetic tools. We are lucky because, I don't know if you've heard of the technology CRISPR-Cas9, it's a genome editing technology. So before, you cannot really do transgenics on mosquitoes easily but with CRISPR-Cas9 it enabled us to do very complex genetic manipulation in mosquitoes.
Z: So one of the ideas is, for example, if we want to do neuroimaging, we need a reporter, it's called GCaMP. This protein is sensitive to the calcium concentration and when neurons are active, the calcium concentration will increase in the neurons and the fluorescence will increase. That's how we read out the neural activity. But in order to do that in mosquitoes, we need to put the GCaMP into the mosquito genome and we use CRISPR-Cas9 for that. So what we do is we inject CRISPR-Cas9 protein, along with GCaMP and another RNA into the embryos, into the egg of the mosquitoes, and a very small percentage of eggs will have the gene integrated into the mosquito genome.
O: Very cool, and so when you hit the mosquitoes with all these different odors, you can just look at fluorescence in the brain, or parts of the brain, to map what's exactly going on.
Z: Yes, yes exactly.
T: And are they alive while you see this happening?
Z: Yes, they are alive. So what we do is we have a mosquito holder. In the holder there is a very small hole, the size of a mosquito head.
T: Oh wow!
Z: So what we do is we push the head of the mosquito into the small hole and use some glue to fix the head so the head is not moving. But the body is moving, like the mosquito is trying to escape, flapping the wings and kicking the legs. So then we open the cuticle, basically the skin of the head, to expose the olfactory region of the brain. Then we do the imaging.
T: I imagine it must be very hard to focus on such a small head!
Z: Yes, you will need practice.
T: Could you tell us about a time when something went wrong in these experiments? What was the most difficult part about working with mosquitoes?
Z: Yeah, I feel like the most difficult part actually is not on the mosquito, it’s on the odor delivery, because you can imagine what we want to do is to deliver the real odor of a human and animal. But the thing is, the odor of us or animals is very complex. People think there are hundreds of compounds in our smell. And how to deliver the natural odor of the human or animal is actually very challenging; we spent a lot of time to do that.
T: Mhm.
Z: Traditionally, what people do is, most people in olfaction they focus on very simple stimuli, for example just one compound.
T: I see.
Z: So what they can do is they dilute the compound in solvent, then put the solution in a vial, a glass vial, then they can puff the odor to the insect. But for us, we cannot put a human in a vial —
T: [laughs]
Z: — or an animal in a vial, so we cannot do that, we need to develop new methods.
T: Unless it's the only experimenter, then it's fine! [laughing]
[both laughing]
[Theme music plays]
O: Alright, so we want to now get into the findings of your work. And so, what's the answer here? What distinguishes humans from other animals in terms of smell?
Z: What we found based on both neuroimaging and analysis on the odor profiles is we think the long chain aldehydes are very important. So this is interesting because both humans and animals have aldehydes, aldehydes are just the major component in the vertebrae odor. But what's different between humans and animals is we have different aldehydes. Humans have more of the long-chain aldehydes, but animals have the short-chain aldehydes. What we found is there is a region in the mosquito brain that's only sensitive to the long-chain aldehydes, but not to the short-chain aldehydes. So when we puff the odor of humans and animals, we only see activation with human odor, but not animal odor.
T: That's cool — so the specific molecules that would set us apart would be these aldehydes. How did you figure that specific part out, that these are the molecules?
Z: Yeah, so what we do is we first did the imaging to the complex human odor and animal odor, and we saw this unique region in the mosquito brain that's only sensitive to human. Later, we focus on that region. What we did is we puff the single component, each component individually, to the mosquito and record neural activity, and try to find out which molecule, which compound activates this region. What we found is it's the long-chain aldehydes.
O: And so, thinking again more broadly here, you're studying one specific mosquito. But, do you think what you've discovered about how this mosquito smells is applicable to all mosquitoes? Or even beyond mosquitoes, like drosophila, or fruit flies?
Z: Yeah, yeah. I think it might be applicable to the other mosquitoes, for example the malaria mosquito. Those are different kinds of mosquitoes, but they also specialize on humans. And what we found, last year there was a interesting paper on malaria mosquitoes. What they studied is a very different system, they're trying to find a blend that attract the Anopheles. What they found is a major component in the blend is actually those long-chain aldehydes. So we think what we found might be applicable to Anopheles as well.
O: That's awesome, super exciting!
T: Yeah! So are you working on these ideas as well? Is there a follow up to these results that you or your lab are working on? And also, because you're a sixth year now, what else are you working on now and what are your plans after graduation?
Z: So first on the follow up of our study, what we focus on right now is behavior, because what we saw is this neural response in the mosquito brain, but we don't know whether the mosquito really uses this neuro code. So what we're trying to do right now is to the test behavior. For example, we know there’s two regions in the brain that are activated by a mosquito brain. What we can do is use a very simple blend, just two components. If this simple blend is attractive, and evokes host-seeking behavior, that means the code might be used by the mosquito. So we are trying to do that right now. The preliminary result shows the simple blend is indeed attractive. So once mosquitoes smell the blend, the mosquito will show very typical host-seeking behavior.
Z: Another follow up, not for me because I'm moving on to a post-doc, but for maybe other future graduate students or post-docs is to try to find the receptor, the actual gene of the protein that senses the long-chain aldehydes. If we can find the receptor, what we can do is we can probably try to black out the receptor. If we black out the receptor, maybe the mosquito don't like humans any more. That would be ideal for us!
O: Yeah, it's super cool thinking about how you can inhibit it or learn so much biology from that.
Z: But my long term plan, because I plan to defend in July, so I’m going to do a post-doc but I’m going to switch to a different system. I'm going to study birds, and social behavior in birds.
O: Can I ask, why the switch?
Z: Because I've been always interested in birds. I feel they are very smart, also they are kind of understudied. There are a lot of unknowns in birds.
T: Crows are super smart, you should study crows as well!
[all laugh]
O: Alright, finishing this out — I learned recently that there are a lot of efforts to control mosquito driven diseases by use of genetically engineered mosquitoes. For example, I think they're trying to do this in Florida. I was wondering -- do you envision your work being applied in a similar context?
Z: Yes! So the commonly used method is called the gene-drive method. So what they do is they try to wipe out the mosquito population by releasing transgenic mosquitoes. But, one major hurdle in that direction is because of evolution, the system will cease to work after some time. So for us, one idea is maybe we don't want to wipe out the population, we just don't want the mosquitoes to bite us. They can bite animals, but it's okay. If we can engineer the mosquito to not like humans, that would be great!
T: I think that's about it for what we wanted to know! It was super, super nice talking to you, that's very exciting. I was very amazed to learn that we have graduate students being served as sacrifice in Princeton!
[all laugh]
T: But this is really cool, thank you so much for talking to us!
Z: Thank you for having me!
O: Yeah of course, super fun! Thanks for coming!
[Theme music plays]
[Outro]
T: This episode of the Highlights was written by Thiago Tarraf Varella and Olivia Duddy. It was produced by Isabel Rodrigues under the 145th Managing Board of The Daily Princetonian. For more podcasts and other digital media from the 'Prince', visit www.dailyprincetonian.com.
T: Many thanks to Zhilei Zhao for speaking with us! To read more about Zhilei's work, check out the Princeton Insights article covering his research, which can be found in the description of this episode.
T: Thank you for listening, and until next time!