Environmental RNA Reveals Heat Stress in Water Fleas

Every inch of the environment that surrounds us, from the ground we tread, to the water we drink and the air we breathe, potentially contains genetic information from other organisms that inhabit our world. But until recently, researchers lacked the tools needed to make use of that information, which comes in the form of environmental DNA (eDNA) or environmental RNA (eRNA). While scientists have begun to explore uses for eDNA in recent years, fewer have tackled its fickler, more elusive sister eRNA. Now, a team of researchers in Canada have used eRNA to identify signs of heat stress in water fleas, they report in a preprint posted November 18 on bioRxiv.

It’s one of the first studies to use eRNA to make inferences about organism health, says Caren Helbing, a biochemist at the University of Victoria in British Columbia, Canada, who wasn’t involved in the research. “There’s been a lot of speculation about the use of environmental RNA,” she says. “It’s really neat to see how that speculation is starting to become reality.”

All animals shed skin, excrement, and other waste throughout their lives. The eDNA within this organic detritus settles in its surroundings, where it remains intact for an average of a couple of weeks. In the mid 2010s, eDNA began to take off as a new tool for monitoring biodiversity and community makeup. By taking a sample of a river, a lake, the ocean, or even the air, scientists can pull out trace DNA and use it to figure out what species live in the area, all without ever having to see or catch an animal.

See “Scientists ID Dozens of Plants, Animals from Free-Floating DNA”

“It’s literally a game changer in how we assess our environment and the impacts that climate change and humans have on the ecosystems around us,” Helbing says.

Along with eDNA, organisms also shed eRNA. However, while eDNA has become a valuable tool for ecosystem ecologists in recent years, eRNA hasn’t received the same attention. According to Robert Masaki Hechler, an aquatic ecologist at the University of Toronto and a coauthor of the preprint research, this is because RNA is generally less stable than DNA, degrading so swiftly that many scientists assumed it would be difficult or impossible to harvest and analyze it quickly enough to get any results. However, ecologists and eDNA scientists had long speculated that, if possible, analyzing eRNA could provide insights into ecosystem health that eDNA cannot. Ironically, eRNA’s rapid degradation could allow it to provide a more accurate snapshot of what organisms are currently in a system, scientists posit. Moreover, the RNA an organism sheds can change throughout its life whereas its DNA remains fixed over time.

a daphnia, with a rounded, transparent body with green stripe running through its center, is lain on a taupe-colored slide under a microscope. a black needle points towards the front of the <em>Daphnia</em> and the image is constrained by a black circle. 

One of the Daphnia cultivated in the experiment on display under a microscope.

Robert Masaki Hechler

“The RNA that an organism produces changes in response to the environment or other stress factors,” Hechler explains, so “with RNA, we can get some insights into how [organisms] are actually doing.”

Hechler teamed up with his then-master’s advisor Melania Cristescu, an ecologist at McGill University who put forward the idea of using eRNA in an opinion piece in 2019, to determine whether it was possible not only to capture and analyze the nucleic acid fragments, but to use them to determine whether a population was suffering from heat stress. Setting up eight tanks of artificial lake water in the lab, Hechler and Cristescu along with other scientists cultivated populations of Daphnia (Daphnia pulex), a water flea that is commonly used in ecology experiments. They then set the temperature of half of the tanks at 20°C, which is normal for Daphnia, and the other half at 28°C, which is almost hot enough to kill the crustacean. The team then took tissue samples, which contain organismal RNA (oRNA), and water samples, which contain eRNA, from each tank, and immediately put them into -80°C freezers in order to prevent the RNA from degrading. oRNA samples, Hechler explains, are the gold standard for measuring an organism’s health status and response to stress. By comparing them to the eRNA samples, he and Cristescu hoped they would be able to tell whether the latter provided useful information.

“This was the right first experiment to do in order to test whether eRNA can actually do what we were hoping it could do,” Helbing says.

The researchers slightly thawed the samples so they could extract RNA and used RNA sequencing technology to look at each sample’s gene expression profile. Daphnia downregulate certain genes to cope with high levels of heat stress, and the researchers expected to be able to detect that change through RNA analysis of the Daphnia in heated tanks compared to the unstressed controls. In general, the researchers were able to identify far more genes’ transcripts in the oRNA samples than in the eRNA samples, which made sense given that oRNA comes directly from the organism and eRNA comes from the organism’s traces. However, the scientists were still able to identify 32 genes from eRNA that were differentially expressed in response to heat stress, 17 of which lined up with differentially expressed genes detected through the oRNA sampling. Hechler says this confirmed what he, Cristescu, and other scientists had hoped: that eRNA could be used to determine how an organism is responding to its environment.

Joanne Littlefair, a molecular ecologist at the Queen Mary University of London who wasn’t involved in the work, says she is excited to see eRNA research taking off. “There’s such a global attention on biodiversity at the moment,” she says. “There’s a lot of attention on biological monitoring, and the kinds of ways we can quickly and efficiently and noninvasively collect data. So I think this [innovation] comes at a good time.”

The next step, Hechler says, would be to take this research into the field, where eRNA is less predictable and harder to collect. In fine-tuning eRNA research for a larger ecosystem scale, he says he’s hopeful that it can one day be used to catch “early warning signs” of environmental stress in ecosystems, allowing for problems to be detected and remedied in time to preserve an ecosystem or save an organism from extinction. “I hope that people will start doing studies like that,” he says. “That would be really exciting to see.”

Correction (December 5): An earlier version of this article referred to Daphnia as insects. They are in fact crustaceans. The Scientist regrets the error.

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