Join us for a wild ride with Dr. Brian Bahder, who’s applying digital PCR to track invasive snakes and megafauna across South Florida. From childhood bug collections to bushmaster selfies, he blends passion, fieldwork, and scientific rigor in one unforgettable episode.
This episode of Absolute Gene-ius slithers into the surprising science of invasive species monitoring with Dr. Brian Bahder. A childhood love of bugs led Brian to a dynamic career in entomology and plant pathology—and eventually to tracking large reptiles in the swamps of Florida.
We dive deep into Brian’s work developing multiplex digital PCR assays to detect DNA from snakes, caimans, and other invasive species using environmental samples like soil and water. He explains how this technology enables detection even after the animals are gone, and how sampling strategy, environmental variables, and experimental design are critical to getting reliable data. He also compares qPCR and digital PCR, emphasizing how each has its place depending on sensitivity, speed, and sample complexity.
In the career corner, Brian shares how his academic journey was shaped by travel, risk-taking, and a healthy dose of failure. From surfing and skateboarding to discovering new species and running a diagnostic clinic, his path reminds us that science thrives on curiosity—and that even mistakenly detecting your own DNA can teach you something.
Visit the Absolute Gene-ius pageto learn more about the guests, the hosts, and the Applied Biosystems QuantStudio Absolute Q Digital PCR System.
Jordan Ruggieri00:00
I think, think we need to rename our podcast Christina to Schrödinger's Cat. You won't know it's great until you listen.
Christina Bouwens 00:20
Welcome to Absolute Gene-ius, a podcast series from Thermo Fisher Scientific. I'm Christina Bouwens
Jordan Ruggieri00:25
And I'm Jordan Ruggieri, and we have an absolutely hysterical podcast for you today. If you are a fan of snakes, you're going to need to coil up with a blanket and listen to our conversation with Dr. Brian Bahder.
Christina Bouwens 00:39
Brian is an associate professor of insect vector ecology at the University of Florida. His current work uses PCR tools to track invasive snakes, caimans and lizards in South Florida and Latin America. And he's discovered over 200 species of insects as well. We loved learning from him about all these animals, and hope you enjoy our conversation.
Jordan Ruggieri00:59
Christina, Brian is searching for the truth.
Christina Bouwens 01:04
Jordan, I don't get that one.
Jordan Ruggieri01:06
Hiss searching like...
Christina Bouwens 01:09
Oh, you can go save him. The truth is out there.
Brian Bahder, PhD 01:19
I'm an associate professor of entomology at the University of Florida. I'm actually not in Gainesville. We have research centers around the state, and I'm situated at the one in Davie, which is functionally, it's Fort Lauderdale area. I've been here since 2016. All my background and formal training has been in entomology. You know, from a very early age, I was collecting bugs. And I recall my mom giving me her sewing needle box with her sewing needles and cutting foam so I could collect bugs and pin them in there. And I was about 10 when a state entomologist came to my home economics class, to date myself. They don't have those anymore, but he came in and showed us the collection and talked about his career. And I realized that you could be somebody who collected bugs and get paid for it. I went to the University of Delaware for my Bachelor's, and I studied termites early on, and then did my master’s at the University of Florida in termites. And then for my PhD, I switched over to Ag pests and plant pathogens affecting agricultural crops, and my focus was grapes up in Washington State, where I did my PhD. And then I did my postdoc training at UC Davis, working on an emerging virus in wine grapes in Napa Valley. And that was kind of where I got introduced to digital PCR. And then around 2016 I was hired at UF and here we are.
Christina Bouwens 02:57
This topic of eDNA is one that was fairly new to me, you know, getting started with dPCR, but I know there's so much more to it. Can you talk a little bit about how environmental DNA plays like an impact in your research?
Brian Bahder, PhD 03:10
Broadly, eDNA work is focused on detecting and analyzing trace amounts of DNA that are from a specific organism that are out in the environment that you know theoretically shouldn't be there. So like you as a person, you have all your DNA is within your cells and contained within you. But if you sneeze or cough and those tiny epithelial cells go out into the environment that is now eDNA. From the eDNA perspective, I wasn't doing anything with it relative to insect work, it stemmed from some colleagues of mine in the crock dock program at UF who study invasive endangered, and endangered megafauna like alligators, crocodiles, the Burmese pythons, tegus, monitors, all that stuff. So the tool that they had an interest in using, it was digital PCR-based. I was able to take my expertise in molecular diagnostics from plant pathogens and basically convert it into molecular assays that would detect these specific invasive species. So we kind of combined our fields of study and expertise to develop this project to design these multiplex digital PCR assays and start sampling water and soil samples around the urban environment here in South Florida, but also in the Everglades, to look for these invasive species.
Jordan Ruggieri04:49
Why are invasive species important to even study?
Brian Bahder, PhD 04:53
The invasive species that we're covering, our first group we worked on was the constrictor snakes. Now there's actually four different constrictor species that are established in breeding. The Burmese python is one. The North African python, which is a snake similar in size to the Burmese python. And then we have boa constrictor and rainbow boa. You know, and a lot of them, depending on the species and where it's at, has different impacts. You know, your Burmese there. There's a lot of them, and they're out in the Everglades, competing with food for with alligators and whatnot. So there's that element. But also they're quite a, you know, aggressive, and they eat a lot, so they'll another threat to some of the native species that are already stressed from, you know, habitat loss or whatever. So, they can affect the local communities the fish species, you know, they prey on, again, native species in the canals, and can, ah. One of them, the Asian swamp eel is a burrower. So it makes, goes underground and I think there's concerns about it, like potentially draining certain areas that generally always have water, which could, you know, disrupt the ecosystem function.
Jordan Ruggieri06:12
The names are all scary, crazy names, sometimes like you're out there looking for these things that are going to eat you.
Brian Bahder, PhD 06:19
I used to do that personally. These days, I don't get out much. I have a picture from when I lived in Ecuador, I caught a bushmaster, which is a rather venomous viper. And I've got a picture somewhere with the head right up next to my face, so.
Christina Bouwens 06:34
Incredible. I wanted to kind of see if I'm catching on to the right thing. So it's more than just about researching what's there, right? So we can do all this scanning to see what types of invasive species are there. But would you say that one of the goals of this is to develop tests that you can actually take action on to help improve the invasive species situation? Can you talk a little bit about that?
Brian Bahder, PhD 06:53
Sure. So we've developed all of the multiplex assays for the digital PCR system. We've tested them against control material. They work. They're efficient. They don't cross amplify. And now we're kind of in the stage of testing them on field, positive controls, to get a sense of you know, if we put a cayman or a python in the water on this side of the pond. How long after that are we going to detect DNA on the other side of the pond, and how long will that DNA stay present in the area? And we've done some control experiments where we've had the animals in tubs with water, and we take data at like, five minutes, 30 minutes, an hour, two hours, three hours. And pretty much with all of them, even at five minutes, you can get a good hit with the reactions. But that doesn't obviously translate to the real world. So now we're in the process of doing some of these real-world experiments to get a sense of how often and where you need to sample to get reliable results. And one of the, I think, misconceptions with a lot of people, especially this day and age, there's very short attention spans, everybody wants things done like this, and they want something that it's one and done. We take a sample, we test it. Okay, it's there, it's not there. And it doesn't work like that, because you have temperature that affects dispersal of these minute particles, sunlight that can cause degradation, water flow. So doing these controlled experiments are critical to get an idea for what we need to do in terms of a sampling strategy to generate reliable results, because once we have more of this, then we can take this resource to the agencies that do the monitoring and tell them, “Okay, if you want this to sample for this species, you're going to need to take X amount of samples over this amount of distance for the summer, to have some reliable data.” And really, the way I see this being usable and practical, I kind of picture like if you're playing the game Battleship, where you have a grid and you take a sample, or where you call out your hit, and then you get negative, negative, negative, negative. And then when you hit something, finally, that's when you zoom in on the area, and you either do more sampling for eDNA testing, or, if you're lucky, you find the organism right away.
Jordan Ruggieri09:37
Interesting. Yeah, I didn't think about the sampling parameters around some of that it sounds like there's, there's a whole lot of experimental design around how sensitive it needs to be. What you're actually sampling, is it water? Is it soil? And even time of year. How those parameters, you know, work for actually detecting these, these species.
Brian Bahder, PhD 10:02
Another critical element is the language used for describing some of these results. An example, when we caught a caiman the other day, they took a water sample right next to where they got it, and it tested negative. We hear some people say that's a false negative, and it's not a false negative. That's a negative sample, but a false negative is if that caiman in DNA was in the sample and it failed to amplify. So how people term this stuff can have a big influence. And when people say a false positive, a false positive truly is if there's no DNA and you're getting amplified pixels on your reaction, that's a false positive. If you say “There's none of this organism in this pond, and we removed it,” and you know that species isn't there, but you're still detecting DNA. That's still a positive reaction. That means the DNA is still there, even though the organism is gone.
Christina Bouwens 11:03
I think this is actually something that is super common, with dPCR right, because it's touted for its sensitivity and its precision and its accuracy. But across applications, we also see this. It's how to term the phrases appropriately, false negatives, false positives. So I really like your overlay of the sampling strategy being such a fundamental point. I think it's applicable for not just this application, but honestly, for anything that's looking for rare variants or rare targets.
Jordan Ruggieri11:27
Do you ever run into, with these samples as well, any type of inhibitors? I mean, I'm assuming with dirt and water or anything like that. Polyphenols, or anything that might actually impact reaction efficiency?
Brian Bahder, PhD 11:41
We haven't noticed any significant influence of inhibitors. I mean, we've done soil samples. Some of the fish stuff was pretty nasty from the water because it, like they get that mucus membrane that clogs up the filter. We do have an experiment plan. It's, it's not a critical one, simply because we haven't had any issues, but it's one that I'm curious about, where, basically, we buy liquid tannins, like from oaks, which are, you know, commonly, a big inhibitor, and basically spiking our extract, the template, with different concentrations to see how much we can get away with having in those samples. Now, for us, with a lot of samples, probably the biggest influencer of inhibition is going to be ethanol. But again, with a lot of the kits and cleanup, it's we really haven't seen any negative effects. And that's one of the common comments I get from reviewers on manuscripts, is like, “Well, how do you know inhibition isn't doing this?” I'm like, like, “Well, that's why we have a spectrophotometer to measure how good the sample is, so we know there's not a problem. And even if there is, the system partitions this thing out 20,000 times or more. And the idea is some wells get inhibitors and may shut it down.” And they’re like, “Well, what if your two positives are in those wells?” I'm like, “Well, trying to give me a hard time rather than think about it practically.”
Christina Bouwens 13:15
I actually want to step back a little bit, because I know, I know you've been alluding a lot to your paper, and I want to give us a chance to shout it out officially. It's been a really fascinating read for me. So could you give us a little bit of background on the goals of the paper and just give everybody a shout out?
Brian Bahder, PhD 13:28
So yeah, the paper we studied published it in Ecology and Evolution, and it was our first paper from the project that we got funded from the South Florida Water Management District to design. And this project was specifically for designing these assays, making sure they work, and then applying them to some field samples to make sure, yes, they work on actual eDNA samples. So this first paper was the process of designing the multiplex, designing it so that each separate assay within it does not cross amplify the other snake species. And we did kind of a theoretical analysis of aligning COI sequences with native species to show that the binding regions for the primers and probes had X number of SNPs. So we know that, you know, generally above four, you're not going to have issues. And similar to what I mentioned about lowering your cycle number, if it does amplify, if there's only, like, one or two SNPs, that's going to reduce the efficiency. And then if you lower your cycle number, then you weed out those, those cross amplifications, if they're going to be present. So with this study, we designed the tetraplex, and we got the four snake species into one. We purchased these assays separately. Determine the optimal annealing temperature for the assays to make sure they all operated at the same temperature. Then we screen them with qPCR to make sure that each species does not cross amplify one of the other four species. And then once we got that, then we purchased the multiplex assay that had all of them in there. And I know that has to go undergo a quality check, and if it passes that at the company, then then we get it and it should work. And once we got the multiplex assay, then we had to validate that on the digital PCR system, so that's where we really showed the functionality of the multiplex. So we screened it on samples individually and showed no cross amplification. One of the figures in that it kind of looks like a, almost like a Rubik's cube or something, where we have it lined up so you can see the assays not amplifying species A, B and C, but it does the last one. So basically, once we validated that, then we did mixed populations where, like we mixed different combinations of the snake DNA in, in the lab, and run it to show what kind of reactions you can expect if you're an environment where both snakes or multiple snake species are present. And then once all that was done, then we did our kind of our limits of detection, to say we put different amounts of tissue in water and filtered it to see how low we could go and get detection. We did our plasmids, serial diluted, the final eluate from the extraction, diluted that just to see how, how sensitive it would be. And that was basically to validate that the assay itself was fully functional. Then we had to do some control experiments to show that these things actually work on water or soil samples.
Christina Bouwens 16:58
I told Jordan before we started this call, I was like, “I did not know I had so many questions about this, but I have so many questions about this.” So I said, “Jordan, you're going to have to cut me off.” What is the unit of measure that you're using? And what is your LOD that would be, you know, a really good improvement on what's currently available?
Brian Bahder, PhD 17:17
That's a, it's a good question because it highlights the strategy needed is going to be very different for soil versus water. If somebody finds a burrow and they say, “We saw this snake hanging out here for a day,” how do we go about sampling that? And it seems like, if somebody says no, it is like if they take a picture of it and we can see, okay, it was sitting right here, then it's easy. We can just go in and scoop the dirt. If it's more ambiguous, some people have talked about taking like paint rollers with like tape and running it over to collect larger areas. But the logistics of doing extractions from, forget it. I think it's probably just easier to do more soil scrapes than the headache of dealing with paint rollers. And water sampling is pretty straightforward. I have a picture somewhere. We were collecting from a canal close to my house, where I've seen these big Bullseye snakehead fish. So, we were collecting samples from this canal to validate as a positive control on our fish assay. We were at the park, and there's a bridge going over the canal, so I had my kids dumpster dive for a piece of long like plastic, and I pulled them out of the dumpster, and I tied the plastic around the Nalgene bottle, and I have them, like I took a picture of them on top of the bridge, like lowering this piece of plastic with the bottle to get the water sample.
Christina Bouwens 18:50
I love it.
Jordan Ruggieri18:52
This is why you have kids everybody.
Brian Bahder, PhD 18:53
Absolutely.
Christina Bouwens 18:55
My kids would sign up 100%. They'd think that was the coolest thing ever.
Jordan Ruggieri18:57
I picked up on even just something you're saying about how you're using qPCR and dPCR together, right. And it's a common theme I think Christina and I have run across for many of these interviews now is, you know, it's not one or the other, it's how you can use all the different tools in your toolbox to make sure that you get reliable results and answer the questions that you are trying to answer as part of your experimental design. Is that something you notice as well?
Brian Bahder, PhD 19:26
It was until recently. I think I mentioned in our first call that I was introduced to digital PCR with the first chip-based system. And when I was at Davis, my first experience was loading those chips by hand with the blade, smearing it. And then we got the chip loader, which was a huge plus to do, but then we still had to do that, and then it had the glue, that little light that you put in. So the just the time to set up 24 reactions would take me three hours. Early on, I see that using qPCR with digital, depending on what you need, like, if you're dealing with research questions or you need the high sensitivity, yeah, the digital PCR it's worth taking the time to do that. But if you're running like a diagnostic service, where you need to get results, like quickly, you can somewhat sacrifice the sensitivity because you need, and qPCR is, for a lot of stuff, the sensitivity is just fine. There's no problem with it. And it's faster just because of the setup. With this new system, as long as you have the, I guess, the funding and support and staff, the workflow of setting up the digital PCR plate, with how it's currently designed, is the difference with q, it still might be a little slower than qPCR, but it's so close that, in my mind, with this system, you could do away with qPCR pretty much for any research purposes. The only scenario, you know, and for my diagnostic clinic, just because of the resources, we still run qPCR on our assays, because it's cheaper and people have to pay for the service and for the samples you don't, they always send in palm samples where it's symptomatic. And when the palm is symptomatic, the phytoplasma is fully systemic, so you don't need that level of sensitivity that you get with the digital PCR. So it's not cost effective to use it. So we stick with qPCR for that. But from a research perspective, there is a very specific niche that I use qPCR for, and that's with HRM analysis. If you're looking at a single SNP within a region or a few, you can't design assays that are specific for those and use it on the digital PCR to be exclusive. It's just not, not possible with every scenario. So that's a case where, like the insect species I work with that transmits the pathogen. We did a population genetic study in Florida and found four distinct haplotypes that differ by one to two nucleotides, like really, really being stringent, you might be able to get it to where the probe doesn't work, or one of the primers doesn't, but it's it. I, you just can't get that specificity with some things that are so closely related. So with HRM, with the qPCR, basically we have that app that gives you really high resolution. So, you know, we run the qPCR, we amplify the COI gene for these bugs, and then eventually we want to use HRM to be a cost-effective way to screen these genetic variants that only differ by one nucleotide.
Jordan Ruggieri23:26
I mean, even just you walking through those different case scenarios, I think shows right like there's different, different ways and different techniques and different, you know, features and benefits of each of the technologies that, again, just depending on what you're looking to do, you can use both, or one over the other, or...
Brian Bahder, PhD 23:24
And I would like, I said, with the diagnostic, if you have a diagnostic clinic where you're processing samples for the public, it's you're probably going to want to have a qPCR. I think that's always going to be the case. If it gets to the point where the cost of running the digital PCR is comparable, or the same as qPCR, then yeah, why not run the higher sensitivity? Because, like, let's say you're dealing with a latent infection before symptoms and the titer is lower. That's where digital PCR would be more valuable.
Steve Lewis24:02
Hey there Absolute Gene-ius listeners, if you appreciate molecular biology and like learning about its use in trending applications in life sciences, I've got something for you. I'm Steve Lewis, the host of Speaking of Mol Bio, the podcast series that covers these topics. In each episode, I sit down with top scientists, industry experts and innovators to explore breakthroughs shaping our molecular biology world, from gene editing to synthetic biology to biosecurity and single cell. Many of these applications we discuss are actually great applications for digital PCR as well, which I know you obviously care about if you're listening to me right now.Whether you're at the bench, in the office, or just geeking out on science in general, Speaking of Mol Bio is your go-to for insightful conversations and real-world tips and tricks to help drive your molecular biology research. So, after this episode, why not give us a listen? Find Speaking of Mol Bio wherever you get your podcast today or visit thermofisher.com/molbiopodcast, that's thermofisher.com/molbiopodcast. Hope to see you over there. Cheers and good science.
Jordan Ruggieri25:25
Welcome to the career journey element of the podcast. You talked about collecting bugs as a kid and some of your educational journey. But are there any critical steps along that journey that stand out in your mind is really having kind of a defining moment in setting your career?
Brian Bahder, PhD 25:44
There's a few. As an undergrad and even as a kid, and I hope my kids don't hear this, but I was a terrible student. I hated school. I did not like the classroom. I wanted to be outside seeing things, experiencing things. So my first few years in undergrad were rough. I was more into skateboarding and surfing and playing guitar than studies. And then, obviously, grades weren't good. But then I got a intro biology teacher my sophomore year that was very hands on and very passionate, and how he taught and explained, and it kind of sparked and reconnected the little kid who likes collecting stuff now to an actual career and taking something a little more seriously. So that was kind of the first catalyst to spark, you know, the innate interest that I had. And then, once I got into the main campus at my university, you know, I started working in the department and doing some undergrad research. And when I started getting more involved in the department, probably the next big change for me was I did a study abroad. I did a two-week trip to the Galapagos and that was kind of an eye-opening experience. And I caught the travel bug after that. So then, when I got back, one of the professors from that trip approached me about doing a semester in Ecuador, in the Amazon, and that opened all sorts of doors for me for graduate school and now it's come back full circle and benefiting my career. Those were kind of, laid the foundation for my desire to travel and explore and take risks, because if you don't take risks, you're going to be you're going to be bored, and you can have a good career doing that but I found with taking risks and exploring every possible new opportunity, you're going to get into all sorts of exciting new things.
Jordan Ruggieri27:59
It's great advice. I 100% agree. I think, I think taking risks is really important, especially early on in your career development, educational journey. It's cool to hear you say that. And let me tell you, the Galapagos is a bucket list of mine. I have always wanted to go. I wrote a report about the Galapagos tortoises. And I am super jealous, so one day I will get there, but...
Brian Bahder, PhD 28:24
It's a cool place. Now, obviously the islands are awesome. They're exciting to see. It's interesting biology. But the part that's going to blow your mind is if you go snorkeling or scuba diving, the underwater world there is just phenomenal.
Jordan Ruggieri28:43
That's a dream. So you talk about taking risks. Is there ever a time where you took a risk and maybe failed, but you are still able to learn a little bit about that and maybe adjust some of your trajectory? Or even just discover about yourself a little bit?
Brian Bahder, PhD 29:01
Sure, yeah, for me, failure is a very important learning mechanism. You know, we had a study where we found more specimens of the vector on infected plants. So initially I thought, you know other systems, plants emit volatiles and attract the vector to increase spread. So I was like, okay, maybe that's going on. So, we invested in that, and we did all this work, and it turned out to be a dead end. It didn't, didn't work. But in the process, there was data in there that this, and this is another important thing, I think, for people to, I don't know if you can teach this, but to incentivize and tell people not to be afraid to use some creativity and imagination is when. So this one experiment failed. We ended up analyzing the volatiles emitted from these palms, and none of them were attractive to the vector at all. So it was a bust, and we spent all this time and money, but what we found was the chemicals that we did get, there was a difference in infected plants and threatened plants and threatened plants were producing a different signal. So that's when I was like, okay, my initial thought was wrong and I failed, but there's something, there's something here. So basically, we look closer at the data and found that the compound the threatened ones were producing actually had antimicrobial properties that they use in post crop harvest protection. So, looking at the distribution of infections in the environment, we realized that these plants might be responding to infections and producing their own defense. I was wrong in one regard, but if you take that data and step back, you have something it's just you may not realize.
Jordan Ruggieri31:06
I just have one more question on my list here, and we ask this of everybody. It's a two-parter. What is your proudest moment that you've had in your career so far, and then maybe what is your most embarrassing lab moment as well?
Brian Bahder, PhD 31:23
Gotcha. So, for me personally, it's our recent discovery of the vector in Madagascar. Because what started off as what my wife called a boondoggle, has turned into a project that is we're going to be applying for NSF funding, and has some real potential down the road. Because of the discovery of the vector, we have some real, solid data to continue this research, and it was kind of a grassroots like, from start to finish, like homegrown in my lab. So that was from that area of research, that's probably my most proud. The first species I discovered, I was super excited about that. That's kind of like, you know, every entomologist dreams they want to discover a new, new species. So that one that was pretty exciting. Embarrassing, probably the most embarrassing was when I was first learning some of these plant pathology techniques, I was working with a group of guys up at Washington State and they were showing me the process. And they, they, you know, turned me loose on, on my samples. And I was processing it, and you know we're looking for viruses in grapes, and I got some, some bands on, on some of the reactions from some of the plants that were supposedly recipients of the virus from my transmission experiment. So I, I'd spend months and, you know, got some bands, and I was super excited. So we, we cleaned them up and sequence it, and they were showing me how to analyze the data and assemble it. So they're with me. So we get it to the end. We plug it into GenBank and BLAST and bam, homo sapien. That's probably the one time that I'm like, ohh.
Jordan Ruggieri33:28
Oh, that's amazing.
Brian Bahder, PhD 33:29
Yeah, I've had some other knucklehead moves, but nobody was around. So, you know, it's not a big deal, but that was the one where there was witnesses. I was like, ugh.
Jordan Ruggieri33:38
Oh, that's awesome, Brian. Just thank you so much for being on the podcast. We really appreciate your time. Fascinating story and just a really, really good conversation. So thank you very much for your time. Appreciate it.
Brian Bahder, PhD 33:52
Absolutely, thank you. Thank you all for having me on. I appreciate it.
Christina Bouwens 33:59
That was Dr. Brian Bahder, associate professor of insect vector ecology at the University of Florida, based in Davie, Florida. This episode of Absolute Gene-ius was produced by Sarah Briganti, Matt Ferris and Matthew Stock, with more fascinating conversations around the corner, stay curious, and we'll see you next time.
Jordan Ruggieri34:16
I'm out of puns. They're gone.
Christina Bouwens 34:19
Should I go get the five-year-old?