Aquaculture

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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Warming oceans could mean bad news for oyster aquaculture

Juvenile Oysters
Juvenile Pacific oysters (Crassostrea gigas) in a nursery.

Warming oceans could mean bad news for oyster aquaculture

Written by Undergraduate Intern Jane Park

Highly prized as delicacies, oysters are farmed all over the world. In the US, they account for the highest volume of marine shellfish production, amounting to a total of 36.5 million pounds in 2018. However, as seawater temperatures rise due to global warming, oyster aquaculture will face obstacles and will likely need to reshape its practices to continue its success.

An oyster’s journey to a dinner plate begins on land at a hatchery, where breeding oysters produce larvae, which are then transferred to nurseries, also on land. The larvae develop into juveniles, and once they reach about 19 to 24 millimeters, they are moved to farms in brackish or marine waters, where they grow until they’re ready for the market and then consumption.

Priya Shukla
Priya Shukla, a UC Davis graduate student. She is conducting her research at Tomales Bay, California.

Because the juveniles develop in the ocean, they are vulnerable to the effects of climate change. Priya Shukla, a Ph.D. student at UC Davis, is currently studying the Pacific oyster (Crassostrea gigas), a globally farmed species that has experienced multiple mortality events. “In the seventies, researchers started documenting summer seed mortality,” Shukla explains. Seeds are the small, juvenile oysters. “Every summer, they would perish. And more recently, in the past 20 years, scientists have noticed that the oyster seed die-off is often associated with the outbreak of certain diseases.”

The ostreid herpesvirus (OsHV-1) is a primary suspect. Researchers first documented herpes infections in 1972 in the eastern oyster (Crassostrea virginica). Since then, several cases have been observed throughout the world. In 1991 and 1992, larval Pacific oysters exhibited symptoms in New Zealand and France, respectively. Though herpesvirus can affect oysters from all life stages, it is fatal for early juveniles. This poses a problem for growers, who are left without adult oysters to give to the market.

The heating of our planet can also exacerbate the situation, as research has indicated that the herpesvirus is associated with warming waters. “As you increase temperatures, you see that the viruses can replicate and that disease outbreaks accelerate,” says Shukla. “As we see warming conditions due to the changing climate, we might see that the virus gets more virulent, and that can be devastating for the oyster industry.”

For instance, in 2008 and 2009, the herpesvirus decimated up to 100 percent of oyster stocks in France, according to Shukla. And in Tomales Bay, California, it can devastate close to 40 percent.

Through her research, Shukla is hoping to find solutions. One possible answer is to train the oysters to respond differently to higher temperatures. “Can we expose these early life stage oysters to warmer conditions early on, before we put them out into the wild, so that they’re ready for these warm temperature outbreaks and therefore, ready for the disease outbreaks?” she asks.

To answer this question, Shukla will expose groups of young oysters to a varying number of temperature spikes then return them to the temperature they were originally at. She will then observe their responses to see if they’re improving at tolerating multiple temperature spikes. Are their growth rates increasing as the number of spikes increase? Are they producing more immune cells, or hemocytes, and therefore having better immune response? “Oysters in general are hardy animals,” says Shukla. “So I’m hoping that if you stress them out a little bit that makes them really strong and able to withstand future stresses.”

However, even if this works, there are further angles to consider. “From a grower’s perspective, you want to grow oysters in the most ideal conditions for rapid, high growth,” Shukla says. “But those conditions may not be what also makes them most immune to outbreaks of disease or increases in temperature.” Therefore, as our oceans warm, growers may have to compromise efficient growth to ensure that the juveniles survive.

Regardless, the aquaculture industry will likely need to adapt to a changing environment if oysters are to remain a reliable food source. With the help of scientists like Shukla though, the industry and its oysters are in good hands.

 

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