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There’s a new detective in town: SHERLOCK technology is reimagining species detection and identification in fish

Longfin smelt being swabbed to collect mucus samples for SHERLOCK validation.
Longfin smelt being swabbed to collect mucus samples for SHERLOCK validation. Photo by: Alisha Goodbla
Written by: Jenna Quan

There’s a new detective in town: SHERLOCK technology is reimagining species detection and identification in fish.

Understanding species interactions and population dynamics are important for tracking the success and spread of threatened and endangered species. But how can scientists accurately track these data for species that look the same and cannot be identified via visual comparisons of two individuals? The answer may lie in the realm of conservation genetics and genomics. In addition to being able to provide species-level identification (and even individual-level identification), this field obtains and analyzes organisms’ genetic material to gain insight into population functions. Data obtained from genetic material can shed light on how isolated populations are from one another, how large populations are, and how populations are related to each other in time and space.

The Genomic Variation Lab at UC Davis, directed by Dr. Andrea Schreier, utilizes genetic and genomic methods in the context of fish and wildlife conservation. Their work is motivated by a desire to protect plant and animal populations by providing knowledge that can help wildlife and aquaculture managers preserve populations. Dr. Schreier’s lab has many areas of focus in which they work to achieve these goals, but two main ones are in tool development and sustainable aquaculture. 

Dr. Andrea Schreier, Director of the Genomic Variation Laboratory at UC Davis.
Dr. Andrea Schreier, Director of the Genomic Variation Laboratory at UC Davis.

Tool Development: Making research safer and more accessible

One problem faced by scientists studying endangered species is that there are often restrictions that limit the organisms they are allowed to capture, handle, or take samples from for research purposes. Although taking a fin clipping from a fish doesn’t actually hurt the fish, it is often against the law to take tissue from an endangered species without proper permits and often the number of individuals that can be sampled under these permits is highly restricted by management agencies. So the question becomes, how can scientists accurately identify species if they can’t tell them apart visually and can’t collect tissue samples for genetic analysis?

Dr. Schreier’s lab has recently tackled this problem head-on after hearing about a lab at MIT that used CRISPR-Cas13 technology for identifying pathogens in small biological samples. Although many people hear “CRISPR” and immediately think of the CRISPR-Cas9 technology that functions in gene editing, CRISPR-Cas13 is a different technology that can be used to identify species presence. Dr. Schreier and her lab, in collaboration with Dr. Melinda Baerwald of the California Department of Water Resources, are pioneering the use of CRISPR-Cas13 for this purpose by developing a tool called SHERLOCK (Specific High-sensitivity Enzymatic Reporter unLOCKing), a non-invasive method of genetic analysis that can yield genetic sequence information from a swab of mucus off the side of a fish’s body in only ~30 minutes at room temperature. This is a huge improvement from other methods that involve obtaining a genetic sample (such as a fin clipping), extracting DNA from it, performing PCR (which takes a long time and requires many different temperatures for different steps). This process can take days to complete in the lab, depending on the DNA extraction protocol used, whereas this CRISPR-based SHERLOCK technology can be done in the field almost immediately.

Graduate students Grace Auringer (left) and Aviva Fiske (right) collect tissue samples from white sturgeon at the UCD Center for Aquatic Biology and Aquaculture for ploidy analysis and transcriptome sequencing.
Graduate students Grace Auringer (left) and Aviva Fiske (right) collect tissue samples from white sturgeon at the UCD Center for Aquatic Biology and Aquaculture for ploidy analysis and transcriptome sequencing.
Photo credit: Fred Conte

Once a researcher collects mucus from a fish’s scales, the DNA from the mucus is amplified in a process called recombinase polymerase amplification (RPA). This step replaces the time-consuming and multi temperature-dependent process of PCR in other methods, which requires expensive lab equipment to perform. Next, the amplified DNA created during RPA is transcribed into RNA, then SHERLOCK technology deploys a guide RNA that identifies a species specific RNA sequence that CRISPR-Cas13 will act on. Once it identifies the correct portion of fluorescent-tagged RNA, it cuts it up into multiple segments, freeing the fluorescent probe and creating a signal. Researchers can then use a hand-held fluorescent reader to measure the fluorescent signal from this process and determine whether or not a species’ DNA is present in the sample. In addition to the increased ease and speed of result turnaround, this technology decreases organism handling time and risk, thus providing a safer way to obtain genetic material for analysis. This technology allows scientists to learn valuable information about wild populations while vastly reducing human impact.

Sustainable Aquaculture: Combatting spontaneous autopolyploidy in sturgeon

Another area that Dr. Schreier’s lab focuses on is sustainable aquaculture - the process of culturing and raising fish and other aquatic organisms in captivity for caviar and meat harvest so as to reduce the need for fishing wild populations of these species. Earlier on in her career, while obtaining her PhD, Dr. Schreier made a serendipitous discovery while working in white sturgeon aquaculture - she found that an unprecedented number of individuals exhibited a strange phenomenon: they had more chromosome copies than normal. 

White sturgeon, which are highly prized for their caviar, typically have 8 sets of chromosomes (denoted as “8N”), which is different from humans and most mammals, who have 2 sets of chromosomes (2N). When white sturgeon spawn, the mother and the father each pass on half of their genes to their progeny, resulting in the creation of normal 8N offspring. However, Dr. Schreier found many individuals in multiple aquaculture farms that did not exhibit this 8N ploidy level. These differences in chromosome numbers were a result of spontaneous autopolyploidy, a phenomenon in which the mother passes on all of her genes to her offspring. In the case of white sturgeon, this means that the mother’s 8N egg combines with the father’s 4N sperm to create an offspring with a 12N ploidy.

Upon further investigation, Dr. Schreier realized that these strange 12N individuals were occurring at an alarmingly high frequency in aquaculture farms (~10%) compared to their frequency in wild populations (<1%). These observations led her to the questions: why is the frequency of spontaneous autopolyploids so much higher in captivity than in the wild, and what are the effects of this change in ploidy on the fitness and performance of individuals exhibiting it?

After taking a variety of data on ploidy levels of white sturgeon in captivity and in the wild, Dr. Schreier and her team were able to get to the bottom of these questions. They found that the vigorous stirring of fish eggs during the artificial fertilization process used by farmers was actually the cause of the increased frequency of spontaneous autopolyploidy in captivity. They also found that although 12N fish (the product of spontaneous autopolyploidy) had no noticeable fitness or productivity differences compared to 8N fish, individuals with an intermediate ploidy of 10N (the progeny of 8N and 12N parents) did have fitness issues. Specifically, these intermediate 10N individuals often take much longer to  produce eggs than most 8N and 12N females, which has a substantial effect on the success of aquaculture farms whose main product from white sturgeon is caviar, not meat. 

Thanks to the work of Dr. Schreier and her lab, aquaculture farms now know how to go through the process of artificial fertilization while minimizing cases of spontaneous autopolyploidy. Dr. Schreier has also worked to sample fish from virtually every white sturgeon aquaculture farm and has successfully identified all of the 12N and 10N individuals in the population. Now, farms can harvest those individuals early and sell them for meat instead of spending additional resources on them in hopes that they will produce profitable caviar. 

Future Endeavors in the Genomic Variation Lab

Dr. Schreier and her team are actively working to improve the CRISPR-based SHERLOCK technology. They are also developing new tools to use in eDNA (DNA from environmental samples like water) analysis, such as developing eDNA metabarcoding methods for monitoring of fish and macroinvertebrate species in the San Francisco Estuary.. Other projects in sustainable aquaculture of white sturgeon, as well as population monitoring of wild populations of sturgeon, are also underway. Dr. Schreier hopes to continue using genetic and genomic methods for fish and wildlife conservation efforts, as well as developing new tools to make doing so easier and safer.


Meet the Author: Jenna Quan

Jenna Quan is a fourth-year undergraduate student majoring in evolution, ecology, and biodiversity and minoring in education. She has a passion for ecology and biology, especially in marine systems. Upon graduation, she hopes to pursue a PhD in ecology and continue on in academia. When Jenna is not working on research projects at BML or in a genetics lab, she is co-captaining the UC Davis Dance Team and working on her knitting projects!

 

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Demystifying Undergraduate Research Experiences

Priya Shukla and a classmate working on a research project

by MCS Lead Mentor Priya Shukla

For many undergraduate students interested in pursuing marine science as a career, getting research experience (even if its research units required for your degree) is a necessary, and sometimes intimidating, process. 

Below, CMSI Lead Mentor Priya Shukla shares some of her thoughts on finding and learning from her undergraduate research experiences.

How did you get into doing research?

After years of learning about scientific discoveries in grade school, I was craving my own ah-hah! Moment. Like many of you, I did my undergraduate degree (in Environmental Science & Management) at UC Davis, and there was a high volume of research happening on campus. So, opportunities abounded for me to get some experience - I just needed to know where to dig in. Because I wanted to earn some extra income*, I prioritized paid research assistant positions over volunteer work and unpaid internships. I went onto have several positions that taught me what I didn’t want to do, before I realized what sort of work not only helped me pay the bills, but piqued my curiosity even while doing the most mundane tasks. 

Priya ShuklaMy first job on campus involved doing air quality research. I then worked at greenhouses located in west campus and an agricultural pest lab in the basement of Briggs Hall. In my last two years of undergrad, I worked in a fish conservation and at a paleoceanography lab (Tessa Hill’s lab) out at the Bodega Marine Lab.

Each of these jobs taught me what sort of tasks I did and didn’t enjoy doing, but it was through the later two jobs that I learned how much I loved doing work on, in, near, about water and climate change -- specifically the ocean!

* I mentioned that I only accepted positions that would pay me, but there are several different ways to do research: you can get paid like I did, get compensated in the form of research units (many science majors at UC Davis require research credits to graduate), or you can volunteer your free time. If you’re interested in a research position that doesn’t necessarily come with a salary or stipend, you should talk with your research mentor about ways to apply for funding (e.g., work-study).

What is your favorite thing about doing research?

Like many people, I love that doing research - especially fieldwork - means I get to work in some breathtaking places! But, my favorite thing about doing research is the part that many people dread - writing it all up! There is so much that goes into a given project - brainstorming, finding funding, setting up an experiment, watching it fail and re-doing the work, interpreting unexpected results, and then passing it along to fellow scientists for their perspective on your work (peer review) before you share it with your colleagues. 

Don’t get me wrong - putting all of these pieces together is really challenging and fun! But, writing publications (along with presenting at conferences) is also the part of the process that helps science get out of the lab and into the world. Getting your work peer-reviewed and published is a lengthy process, but it also is what allows “sound science” to be used to make decisions. And, there’s something thrilling about seeing your name as an author!

Is there anything about doing research that you don’t like?

My least favorite thing about doing research is also one of the things that makes it really fun - early morning low tides. Low tides are beautiful and epic opportunities to see the diversity of life and interesting ecological interactions that occur in the zone where the water rises and falls every day. These low tides start at convenient times at the beginning of summer, but get progressively earlier as summer edges into fall. This means getting up at 4:00am (which is hard, but not the part I don’t like) and having to be extremely careful so that I don’t make silly mistakes in my sleep-deprived state and up feeling scornful towards my past self. Priya Shukla

For example, one of my field sites has much softer mud than the other, so I can wear some standard rainy day galoshes at one, while the other requires full on wader so that I can maneuver around in the mud. During one of my first excursions, when I was still getting comfortable at the mushier site, I forgot to put on my waders. I tromped out to my experimental baskets - or at least I tried to - in the rainboots. I had maybe taken 10 steps before my left foot sank into the mud up to the middle of my calf. I went to take another step, and my foot slipped out and I managed to catch myself before my socked foot hit the mud. I ground down with my right foot to maintain my balance and then hoved my left foot back into its boot. To make my way out of the situation, I had to pull on the lip of both boots as I took each step to prevent that from happening again. I managed to make it back to my car and change into my waders … but then managed to make the same mistake at least once more before I fully learned my lesson!

What surprised you about doing research?

I had romanticized the ah-hah! moment I was seeking! There is so much failure before you get to that point. That sometimes means you end up literally shedding blood (because you banged your knuckles against the water table), sweat (because lifting multiple buckets of water is exhausting), and tears (because sometimes no matter how much you try to get things right, nature is complicated and the species you’re working with don’t cooperate). You are asking questions and doing work that no one else has - so of course you’re going to make mistakes! 

Priya ShuklaOver time, I’ve gotten better at accepting failure, but it still bums me out when things don’t work. But, because I’m the kind of person who loves planning, I build failure into my plans. That way, when things don’t work out, I can pretend it was part of the plan all along (because it was!). Also, because there is so much failure between conceiving an idea and writing it up (because something breaks, your data don’t tell a clear story, your code won’t run, your figures don’t make sense), it is really satisfying when things begin to fall into place, and it becomes clear what your data are telling you; that ah-hah! moment is truly rewarding!

Is getting research experience worth it if you don’t want to do it for a living?

Absolutely - because it is an invaluable opportunity to push yourself out of your comfort zone while learning new skills. Yes, you might learn niche skills like counting otolith rings or measuring seawater carbonate chemistry, but you’ll also learn time management and organization skills, what working collaboratively looks like, and gain more abstract qualities like patience and perseverance -- all of which have application beyond marine science research.

How can you find research experiences?

Through the job application system (currently Handshake) and departmental emails, I found and heard about several research opportunities to work directly with graduate students, post-docs and professors. I applied to dozens of these and would hear back from ~ 20% of the people I reached out to and positions I applied for. For opportunities associated with UC Davis, you can also check out this Research Opportunities Database.

Also, if you are concerned that you won’t be competitive because you don’t have any prior research experience - you should recognize skills you’ve gained in other positions you’ve held! For example, working at a restaurant, you have to be adaptive and juggle multiple tasks at once on a strict schedule. Babysitting requires you to be extremely responsible and careful. Being an athlete means that you have grit and know how to strategize, follow complex plans, and achieve goals.

Bear in mind that you likely won’t be starting out planning your own experiments. You’ll probably be working with someone who needs help setting up and carrying out an experiment or has completed one and is inundated with samples to process. Learning these different components makes the whole process more accessible.   

If you have any questions about research that were not answered here, consider submitting them to the new Ask A Grad Student Blog!

 

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