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Up close and personal with one of the self-cloning marbled crayfish from Neurophysiology Professor Wolfgang Stein’s research laboratory.

Up close and personal with one of the self-cloning marbled crayfish from Neurophysiology Professor Wolfgang Stein’s research laboratory.

Decoding the mutant, all-female, self-cloning crayfish

In the early 1990s, something strange happened in the crustacean world. A pair of mating slough crayfish produced a daughter that was born with an extra set of chromosomes. Now, that’s a rare event. But what is even stranger is that this mutant offspring began reproducing asexually and went on to propagate a globetrotting line of all-female clones.

“From that one mating that went wrong, essentially, the whole species came into existence,” said Wolfgang Stein, a professor of neurophysiology in Illinois State’s School of Biological Sciences.

Scientists are unsure whether the original mating occurred in the slough crayfish’s native waters in Georgia and Florida, or in a pet owner’s tank somewhere else. Nevertheless, the new self-replicating species, known as the marbled crayfish for the patterns on its shell, has spread quickly by way of the pet trade to ecosystems around the world.

Despite a lack of genetic variability, the freshwater animal has adapted to environments as different as Germany and Madagascar, overwhelming native species due in part to its prolificacy.

Four years ago, Stein brought the marbled crayfish into his laboratory. He wanted to decode the crustacean’s genome so neuroscientists could use the unique species to unlock mysteries in the human brain.

“We can now look at this species that is brand new. It is spreading across the planet, and so far, it is genetically identical,” Stein said. “We can look how it evolves as a species. We can study the environmental impacts versus the genetic differences. To tease that out is not very simple. Before this, we didn’t have that chance. More importantly, we can now separate environmental from genetic influences on brain function.”

Assistant Professor of Molecular Neuroethology, Andres Vidal-Gadea, master’s students Casey Gährs ’17 and Abbi Benson ’15, and Professor Wolfgang Stein are hoping the mutant crayfish can help them and other scientists solve mysteries in neuroscience.

Assistant Professor of Molecular Neuroethology, Andrés Vidal-Gadea, master’s students Casey Gährs ’17 and Abbi Benson ’15, and Professor Wolfgang Stein are hoping the mutant crayfish can help them and other scientists solve mysteries in neuroscience.

Stein researches the genetic origins of the marbled crayfish with Assistant Professor of Molecular Neuroethology Andres Vidal-Gadea and master’s students Casey Gährs ’17 and Abbi Benson ’15. They successfully sequenced the crayfish’s genome through a collaboration with the German Cancer Research Center and traced the species to a single crayfish brought to Germany.

In February, the research team published its findings in the journal Nature Ecology & Evolution. News of this research, and the odd self-replicating crayfish itself, drew international media attention from prominent outlets like the BBC and NPR’s Science Friday.

“A lot of the research and work being done here in the School of Biological Sciences is world-class,” Vidal-Gadea said. “This an example of that.”

The German Cancer Research Center is hoping the marbled crayfish can provide clues about how cancerous tumors spread among a population. That can be difficult to determine due to humans’ genetic diversity. “There are many things that you inherit that might cause cancer, but then there are environmental causes for cancer, and some from the environment make it into your genetics later on,” Stein said.

“So it is very difficult to tease out what actually causes cancer. With these animals we can easily track the environmental differences.”

For the Illinois State scientists, the publication was only the beginning of their work. They used a laboratory at the University of Illinois Urbana-Champaign—which has machines that can read the DNA—to carry out the original sequencing. However, the researchers are still filling in gaps in the sequence.

“You can imagine DNA like a book you want to know every page of. But this technology doesn’t give you the book; it gives you chunks of pages,” Vidal-Gadea said. “You have to align the different pieces together. It’s like putting a puzzle together to figure out exactly where it begins and where it ends. Is this the beginning of one gene? Is this the end of the second gene? This is a gigantic puzzle—thousands and thousands of pages.”

The Illinois State students—Gährs and Benson—have been tasked with figuring out important parts of this puzzle. They spend about 60 hours per week in the lab acting as scientific sleuths, comparing the DNA sequences of previously studied animals to the sequences of the marbled crayfish in order to solve these genetic riddles.

In addition, both students are looking at the transcriptome, or the actual genes expressed. The genome contains all of the genes that can possibly be produced in this animal. The transcriptome, on the other hand, is composed of only the genes being expressed in particular cells. Comparing the two gives the researchers a much better idea of what genes marbled crayfish turn on or off when the environment changes.

Gährs is creating a transgenic marbled crayfish in order to study the function of genes by modifying them. Specifically, she is investigating actin, a protein found in animal muscles. She injects green fluorescent protein, isolated from a glowing jellyfish, into the crayfish eggs, and then raises the crayfish to see how the genes function. She can then see where actin shows up in the crayfish by identifying where the animal glows green.

Benson is using a molecular technique known as RNA interference, or RNAi, to study the expression of genes in the adult crayfish. She is researching structures called gap junctions in brain cells, which act as bridges that enable these cells to communicate with one another. Using RNAi, she can cut off these communications and see how that affects behavior.

The students are developing the genetic and molecular techniques for the marbled crayfish that scientists could use one day to answer their own research questions.

“This is something I think is important to understand: In principle, we are not that much interested in the behavior of this specific species,” Stein said. “We are interested in how the brain functions, and the molecular tools we develop here will help us do so.”

The marbled crayfish offers important advantages to researchers. The animals reproduce in large numbers, laying 200 eggs every three months, and the offspring mature within a few months. The crabs the laboratory usually studies can take several years to become adults.

Furthermore, crayfish neurons, like those in other crabs, are large enough for the scientists to study the communication between different parts of the brain, but there are not so many neurons, like in mice or rats, where it becomes impossible to study an individual cell.

Redbird Scholar cover Volume 4 Issue 1 Fall 2018 Meet the Mutant: ISU scientists are hoping an all-female, self-cloning crayfish species holds the genetic key to unlocking mysteries in the human brain. Page 20

Marbled crayfish pictured on the cover of Redbird Scholar’s fall 2018 issue

Benson said there is a lot of trial and error involved in the students’ research. Working through these problems has given her the critical-thinking skills and scientific knowledge she hopes to apply one day as a patent lawyer.

Gährs plans to pursue an M.D.-Ph.D. so she can both treat patients and conduct medical research. She began researching in Vidal-Gadea’s and Stein’s laboratories as an undergraduate student. “When I started, I really had no idea what I was doing. But in these last three years, I’ve acquired a repertoire of skills I can use to study the genes and nervous system of this animal.”

Stein hopes marbled crayfish can further his laboratory’s research into how people respond to medications. For example, when treating a patient for depression, it may take the doctor several months to determine the appropriate dosage.

“Why is that?” Stein said. “Well, the underlying assumption is always it’s the genetic differences. Your brain is slightly different from my brain, so you need a different dose than I do. That’s an assumption. No one knows if that is actually the case.

“So why do we not know this? We have no way of teasing out the genetic differences versus the life history differences versus the environmental influences.”

Due to a weird bit of reproduction 25 years ago, and the work of Illinois State researchers today, neuroscientists may be able to answer these and many other questions in the years ahead.

To read the marbled crayfish research paper, visit nature.com.

Kevin Bersett can be reached at kdberse@IllinoisState.edu.

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