- Scientists sampling water for the environmental DNA of fish had the unique opportunity to test the potential of using eDNA to detect the presence of a fungus deadly to frogs while the animals are still healthy.
- The Batrachochytrium dendrobatidis (Bd, or chytrid) fungus has decimated frog populations across the world and is very difficult to detect until the frogs of a newly infected population start to die.
- The research suggests that eDNA could help managers predict which lakes and other water bodies harbor the chytrid fungus and take action to protect surviving amphibian populations.
When Colleen Kamoroff and her graduate advisor Caren Goldberg collected water samples to look for the DNA of non-native fish species, they could not have guessed the potential importance of their timing.
One month after they sampled water from 13 lakes in California’s Sequoia Kings Canyon National Park in 2015, endangered mountain yellow-legged frogs in three of the seven lakes they’d inhabited died in large numbers. Kamoroff and Goldberg, assistant professor at Washington State University, suspected the presence of the Batrachochytrium dendrobatidis (Bd) fungus. Bd, a.k.a. chytrid fungus, causes Chytridiomycosis, which has devastated populations of at least 200 species of frogs and salamanders across the globe.
The fungus feeds on keratin in a frog’s skin, causing the skin to thicken and peel. Frogs use their skin to breathe, so chytrid makes breathing difficult and causes the frog to move sluggishly and react slowly to danger. It also damages the nervous system and causes them to behave abnormally, including sitting out in the open rather than protecting themselves.
Chytrid doesn’t kill frogs immediately, so they will first hop and swim around, spreading the fungus to other ponds and streams. This also makes infection difficult to detect prior to a major outbreak that can kill whole frog populations. Moreover, once chytrid has infected a pond, the fungus may remain in the water forever.
Detecting disease through distributed DNA
Kamoroff and Goldberg analyzed the water samples for environmental DNA, or eDNA—genetic material from the skin, hair, scales, or waste products shed by living creatures into the environment and extracted from soil, water or air, rather than directly from a plant or animal. Its lower cost and noninvasive nature has increased interest in using eDNA, both for detecting the presence of particular species and for describing whole communities. To sample for the presence of the Bd fungus, for instance, would otherwise involve capturing frogs and swabbing their skin.
Armed with pre die-off water samples, they re-analyzed the samples to look specifically for the fungus that is killing frogs so relentlessly.
“I was curious if Bd would be detectable at the lakes I took samples from even though there were no sick looking frogs at the time,” Kamoroff said in a statement. “So I decided to run the eDNA samples I originally collected for the lake restoration project to test for the presence of Bd.”
The researchers did not see any sick frogs even at the three lakes where so many frogs died just a month later. Their analysis of the eDNA in water samples, however, found chytrid fungus in all three of those sites, suggesting that they detected it before it had reached a lethal threshold. They did not find the fungus in the four lakes where no die-off occurred.
The scientists published their methods and findings in the journal Diseases of Aquatic Organisms.
Kamoroff told Mongabay-Wildtech she was surprised that not only were they were able to detect Bd in the water a full month prior to the observed yellow-legged frog die-off, but also that some of the samples contained large quantities of the fungus.
Detecting Bd before its prevalence levels reach that lethal threshold (~10,000 zoospores) would enable resource managers to take action to save frog populations that have not developed a natural resistance to Bd infection. These “naive populations” are thus particularly vulnerable to infection. For example, frog researchers have discovered that physically washing frogs in an anti-fungal drug for a few minutes has cured infected frogs, reduced infection levels, or at least increased survival of animals infected with Bd. Taking such action before it’s too late could bolster the survival rate in these populations.
Applying eDNA analysis to other frog populations
Kamoroff said that detecting eDNA in filtered water samples requires equipment similar to that used for extracting and detecting DNA in traditional samples.
“However,” she added, “it is necessary to use a ‘clean room,’ without high-quality DNA samples (toe-clips, mouthswabs, blood, etc.) or qPCR product because the amount of eDNA in filtered water samples can exist in low quantities. It is possible that the high-quality DNA samples and qPCR product could contaminate the eDNA samples.”
Kamoroff also recommended that researchers or resource managers collect large water samples (2 liters per sample) and use filters sensitive to low quantities of DNA. “If managers detect Bd at naive populations,” she said, “they can move forward with management options such as anti-fungal baths or captive rearing.”
The research suggests that eDNA could allow managers to screen lakes and other bodies of water for the presence of the chytrid fungus and thus help inform efforts to protect surviving populations.
The researchers are expanding their study of Bd through analysis of eDNA from other locations. Kamoroff is working to incorporate eDNA in management, restoration, and research in Yosemite National Park. “This summer, with the help of Dr. Goldberg” she added, “I will be looking at how the quantities of Bd in eDNA filtered water samples compare to Bd zoospore load on sick frogs.”
Goldberg told Mongabay-Wildtech that eDNA is working well for detecting golden frogs and Bd in Panama. “These populations are not Bd naïve, so the use of eDNA detection in this system is not necessarily the same,” she said, “but we can use it to understand where the pathogen is persisting and how that relates to where we find golden frogs.”
“If we can predict when an outbreak is imminent, we can proceed with management actions such as anti-fungal baths that kill Bd,” Kamoroff said in the statement. “Mountain yellow-legged frogs in the Sierra Nevada have experienced a population decrease of over 90 percent in recent years. Environmental DNA could help save these frogs and other species of amphibians around the world from extinction.”
Kamoroff, C., & Goldberg, C. S. (2017). Using environmental DNA for early detection of amphibian chytrid fungus Batrachochytrium dendrobatidis prior to a ranid die-off. Diseases of aquatic organisms, 127(1), 75-79.
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