Dive into the World of Zebrafish: Studying eDNA with dPCR

Q&A with Dr. Zoltan Varga and Dr. Corbin Schuster from the Zebrafish International Resource Center at the University of Oregon

Introduction to Zebrafish Research

The use of model organisms in science dates to ancient Greece and represents an important means by which humans have progressed our collective understanding of biology and disease. We’ve probably all heard of using mice, rats, or even Drosophila melanogaster (i.e., fruit flies) in the lab, but zebrafish have surged in use as a model organism.

Zebrafish are small, more cost-effective to maintain, have a fast generational lifecycle, and have clear embryos that enable direct observation of their development. As is the case with any model system used across the globe, standards matter and help make results transferrable to other studies.  This is where our guests come into the picture.

For this unique episode of Absolute Gene-ius, we’re joined by both Corbin Schuster and Zoltan Varga of Zebrafish International Resource Center (ZIRC) at the University of Oregon. In their roles, they help raise and maintain over 12,000 genetically unique zebrafish lines for use in studies across the globe.

We learn about the health monitoring they conduct on a regular basis to maintain their colony and support their own and partner studies using their zebrafish.

The conversation touches on environmental DNA (eDNA) methods they employ, assay development and method selection based on their need for sensitivity and specificity, the use of qPCR and dPCR in their work, studies and monitoring of parasites and pathogens that affect their colony, and how zebrafish have helped shift research from forward genetic studies to reverse genetic studies.

To learn more, listen to the Absolute Gene-ius dPCR Podcast Episode at www.thermofisher.com/absolutegeneius

Transcript from Absolute Gene-ius episode four “Raising the fish that spawn our insights”. This transcript has been edited for clarity and brevity.

Meet the Experts: Zoltan Varga and Corbin Schuster

Interviewer: Zoltan and Corbin, thank you so much for being our Absolute Gene-iuses today and joining the podcast. We’re really, really thrilled to have you here. We were talking before about zebrafish and the facility that you have set up for zebrafish.  Can you talk a little bit about what you actually do at this facility when it comes to zebrafish? And now how’s everything set up?

Zoltan Varga, PhD: The Zebrafish International Resource Center was built in 20, well, in 1999 it started and was finished in 2000. And we have recently expanded the footprint in 2020 to 2022.

The purpose of the building was to house zebrafish from mutagenic screens that were done in 1995. We were built to house about 500 fish lines. In the meantime, we have expanded that collection to over 12,600. We have 46,000 zebrafish that we use, roughly. That is approximately 55% coverage of the zebrafish genome with on average two to four alleles per locus.

And then the other leg on which the Zebrafish International Resource Center stands is that we have health services. The large colony that we have also requires extensive health monitoring. This is where the DNA sampling comes in, which Corbin established for us last year. He was a postdoc at ZIRC and he brought environmental DNA sampling to the portfolio of our health monitoring.

There are so many lines, we can’t keep them all alive. A lot of them are imported into the quarantine room and they’re cryopreserved right away. We can’t do health checks on them until we recover them. We have to either raise them in the quarantine room, or we have to raise them in the facility.

Corbin developed our environmental sampling process to ensure that at least one of the pathogens is currently out of the system or not introduced. This makes the health monitoring approach faster, and we can be more proactive.

Interviewer: That’s a huge number of lines. Corbin, what are you what are you doing on your end? Do you house lines as well?

Corbin Schuster, PhD: It’s nowhere near the capacity that we have there at the Zebrafish International Resource Center. I think our max capacity is about 1,200.

I focus on assay development, as Zoltan mentioned, trying to develop environmental assays for zebrafish pathogens.  I don’t need necessarily a big capacity enabled to do the validation and the development of these assays, so having a smaller system really works to really focus in on culturing some of these pathogens in vivo and then being able to develop these assays.

The Role of Zebrafish as Model Organisms

Interviewer: Why are zebrafish so important to the scientific community?

Zoltan Varga, PhD: Zebrafish are essentially an almost entirely Eugene, Oregon invention by the virtue of George Streisinger who was a virologist here at The U of O.

He wanted to expand his research into vertebrates, and he was looking for a vertebrate model that lent itself as easily to genetics as viruses did. He was also hobby aquarist and he knew about zebrafish from the pet store trade, and he established them in the 70s in his lab.

It’s a great model for doing genetics. George Streisinger was interested in, as far as I understand, in the genetics on one side, but also the neuroscience. And then so at the Institute of Neuroscience, several researchers picked up the baton and continued zebrafish.

And they exploited zebrafish for its genetic amenability, but also because as an embryo, it is transparent. It grows fast and you can see single cells forming tissues, tissue layers, and the organs. The research community has expanded zebrafish into virtually any biomedical research field. Now, there’s hardly any university that doesn’t use zebrafish in some biomedical application.

Interviewer: It’s crazy to me how we’ve gone from somebody being interested in this stuff and looking at zebrafish in a pet store, to now where we’re at. It’s such a huge leap in a relatively short amount of time, when you think about it, pretty wild.

Zoltan Varga, PhD: It is. We’re talking about 40 years, maximum 50 years.

Interviewer: It’s awesome. So, I know you had mentioned that one of the benefits is the transparency, especially in early development. What are some other benefits that zebrafish have as a model organism?

Zoltan Varga, PhD: They have a relatively short generation time. In about three to four months, you can breed a new generation, and then one litter one batch of eggs, has enough embryos that you can do genetics very easily. Typically, it’s at least 100 eggs.It’s so much easier if you have a batch and you have 25% mutations right in front of you.

And then the other part of it is that very recently with CRISPR Cas mutagenesis, we came away from regular forward genetics, to reverse genetics, where we can target specific genes and ask what the phenotypes are. With CRISPR Cas genetic editing, this became very, very straightforward.

Previously, it was a random approach, and you could only identify mutants that actually had a phenotype. Now, if there is a mutated gene, you can now look thoroughly with the help of other model organisms, make predictions about the phenotype, investigate particular cell types and gene expression patterns, and really hone in on a specific gene much better than that used to be possible with forward genetics.

Interviewer: Absolutely incredible. Corbin, I think this might be more on your end. But I want to talk a little bit about how you maintain the zebrafish as well and what that entails.

You mentioned something about looking at the different pathogens that they might be exposed to. What exactly do you look for there? Why is it important that you understand some of the pathogens they may carry?

Corbin Schuster, PhD: Well, one of the main pathogens that I worked with, there at ZIRC and as well as in my doctoral training, was an opportunistic pathogen. It’s Pseudoloma neurophilia, a microsporidian parasite.

This pathogen is largely asymptomatic until it reaches high prevalence, so you wouldn’t necessarily know that your population is infected until 10-11 months down the road. When that prevalence has peaked, it’s pretty obvious that your fish are pretty sick.

So, being able to be proactive about detecting these pathogens is pretty important, especially when we’re talking about reproducibility in different research studies. These pathogens can also impact the behavior of these fish.

Infections like these could be really confounding factors, or what we like to call non-protocol induced variation, in these research studies. Understanding how these pathogens interact with the host and interact on the population scale, is very important for maintaining rigor and reproducibility in research studies.

One of the obvious things that we do is, we obviously look for clinical manifestations of these different pathogens that we study. I also worked very closely with Dr. Katie Murray, who was the attending veterinarian there at the Zebrafish International Resource Center, who showed me that it’s very important to be able to bring in traditional surveillance efforts such as histopathology and other types of efforts to really be proactive in trying to eliminate these pathogens from zebrafish facilities.

I believe the pathogen that I’ve worked with, Pseudoloma neurophilia, may have decreased now, but a few years ago, it was reported that this pathogen impacted about 50% of all research laboratories that reported to ZIRC. Understanding the origin of these pathogens is important.

Also, understanding what the genetic variation of these pathogens is across zebrafish laboratories, is also something that I’ve been particularly interested in, especially as we find more microsporidia species can actually infect other types of fishes. We recently found a microsporidian that is, after doing some gene sequencing and alignments, actually relatively closely related to the Pseudoloma neurophilia that I’ve been studying.

There have been some reports, in labs that I’ve talked to, where they had it in the microscope and they’re saying, I’m pretty sure it’s Pseudoloma, but it’s not being detected by our qPCR assays, which are by design very specific. Sometimes it can be 113 base pairs.

And so that’s a very small sequence and very specific to be looking at in regard to pathogens, and if it’s not being detected, why is that so? So that’s what had me thinking about the genetic variation in these different laboratories.

Digital PCR and qPCR in Zebrafish Research

Interviewer: Is there a way that you implement qPCR versus digital PCR? We love digital PCR here at Absolute Gene-ius. So how does digital PCR and qPCR kind of fit into your workflow as you’re looking at these pathogens?

Corbin Schuster, PhD: The nice thing with qPCR is it’s essentially, we will build our qPCR assays and that’s what we will use to do detection of these pathogens for tissues and stuff, and in different sorts of tissues.

Pseudoloma neurophilia specifically targets the central nervous system, so we look at the hindbrain and spinal cord tissue, or in the spinal column. It’s really nice. That’s sort of where we start.

From there, it’s a very easy transition to take our qPCR assay and take it to the digital PCR platform. It’s essentially the same primers, a little different master mix, but we can take our primers and our probes and directly translate that over to digital PCR.

Where I think the digital PCR has really complimented our qPCR efforts has been in the fact that we can detect the pathogen at much lower concentrations than what we would have with the qPCR. This is especially what we’ve seen with environmental sampling. We’ve used our qPCR assay for environmental samples, whether that be tank detritus or biofilms, or straight tank water, and it was very inconsistent.

We could detect it if the pathogen had a really high concentration in an environment, but Pseudoloma neurophilia is an intracellular parasite, so there’s also an intracellular phase that moves essentially through every bodily organ system until it finally reaches its, quote, final destination in the spinal column. Regarding that movement, we’ve done transmission studies looking at what does that look like, actually, for the detecting the parasite in the environment.

And what we found was really, during those initial intracellular stages, it’s not really detectable as it’s moving through those organ systems within the host. There’s some gaps in detection and that gap in detection is very, it’s varied. It varies by how big your population is and how big of an inoculum or a dose that the actual fish are exposed to.

So being able to detect it at very low concentrations was very important. And the only way we were able to truly achieve that was with the digital PCR.

Developing Assays for Pathogen Detection

Interviewer: When you’re actually developing assays, what exactly are you looking for? Are you creating primers and probes against these different pathogens? Are you actually doing any type of sequencing to look at all pathogens or all organisms in the water?

Corbin Schuster, PhD: Certainly, certainly. So, the primary thing, at least for the assays that I have worked with, has been to make them very specific and make them very sensitive.

We do look at other pathogens that may be present within a certain system, but we want that sequence that we are amplifying to be very specific to the type of pathogen that we’re looking for. We don’t want that any cross-reactants or any sort of things like that going on.

So I would say that the primary thing that we’re trying to make sure is that our assay is one, sensitive enough to detect a pathogen and then two, to make sure that it’s specific enough that it’s not cross-reacting with any sort of other species that may be present in in the zebrafish population or in a different fish species population.

This can be challenging however, especially if we’re looking for, say,  Mycobacterium, which there’s several different strains that may impact the zebrafish populations. Making assay that are very specific in regard to that without cross-reactivity can be a bit of a challenge.

Digital PCR for Pathogen Surveillance and Environmental Sampling

Interviewer: Zoltan, are you using digital PCR on your end as well, or any comments on how digital PCR is used in your space?

Zoltan Varga, PhD: So, Corbin taught us digital PCR, right? He, he helped us adopt the methodology. And now Katie Murray and Evan Loucks, are doing routine screens. And in the same way, it is of interest to do the environmental sampling. We know that the UV sterilization, the last step in the filtration system, is a very effective method to knock down microsporidia, Pseudoloma neurophilia specifically, but we can’t be sure if it’s 99% or 99.9%.

Previously, we thought, “Well, this is endemic to zebrafish, there’s nothing we can do about it.” Then we said, “Well, this is in the water system. Even if we clean up the fish population, there are niches in our water system like piping, like pumps, aquarium maybe or fish populations that shed the pathogen, we will never get rid of it.”

With the environmental DNA sampling, now we can go in and go on a tank level and say, well, “We screened these in these tanks, the overall prevalence has been reduced from let’s say, I don’t know 10% to 1%.” But in addition to that, we can also go, and we can say, “Well, these are really old pipes. Let’s sample them,” for example, biofilm that is something we still need to develop as a technology and see if we have any significant reservoirs of microsporidia spores in our system that we need to clean out.

We can go step by step through our filtration system in addition to the fish population, and really try and eliminate the pathogen altogether.

End of Transcript.

To get more information and hear career advice, listen to the Absolute Gene-ius dPCR Podcast Episode at www.thermofisher.com/absolutegeneius

Learn more about digital PCR at www.thermofisher.com/absoluteq