Finding Frogs, Flies and Fungi: Using Environmental DNA to Survey Amphibians and Their Pathogens

A simple water sample can be used to simultaneously track wildlife, reveal biodiversity, monitor pollution, and survey pathogens. In our recent study (1), we took water samples from the Avoca River in Ireland and found not only evidence of the presence of Ireland's only frog species (the European common frog, Rana temporaria), but also Batrachochytrium dendrobatidis (Bd, chytrid fungus), which causes chytridiomycosis disease in amphibians. Bd is responsible for dramatic declines in amphibian populations around the globe, but this was the first reported detection of Bd in Ireland. We did not physically observe or capture any frogs, nor did we set out specifically to investigate this pathogen. The key to finding this information lay in environmental DNA (eDNA).

What is environmental DNA?

Organisms are constantly shedding fragments of their DNA into the environment. Things like skin cells, feces, mucus, fur, and spores all carry the unique genetic code of the organism they came from. This shed DNA is known as environmental DNA (eDNA) and can provide vast amounts of information without ever having to see or interact with the species in question. It can be used to assess biodiversity (2), to monitor the spread of invasive species (3), and to detect pathogens (4). Traditional monitoring methods, such as observation or trapping of the species of interest, can be difficult or intrusive. eDNA allows researchers to collect information about a species using just a cup of water, a few ounces of sand, or even air sucked through a filter (5). In fact, amphibian eDNA was abundant in the air at another of our team's study sites in Florida (5).

A 2019 study used aquatic eDNA to detect a Ranavirus outbreak in frogs more effectively than traditional observation methods (6). In that study, a targeted approach called qPCR was used. qPCR is also used clinically in human diagnostics and, while it is the gold standard for pathogen detection, it will only relay information about one or a few species or pathogens. For rapid ecosystem-wide biodiversity assessments of the DNA of all species in a sample, other approaches are required.

Environmental DNA biodiversity research has long relied on metabarcoding: amplifying short genetic markers to identify particular species. This approach has powered a global expansion of biodiversity monitoring, from insect populations (7) to coral reefs (8). Yet metabarcoding is inherently selective. It finds what it is designed to find, and ignores anything outside its targeted range.

Emerging technologies such as long-read shotgun metagenomic sequencing are enabling even broader simultaneous detection, from viruses to vertebrates, without requiring pre-selection of what species or pathogens to examine. Shotgun metagenomic sequencing examines all DNA present in a sample and determines the relative proportions (quantity) of DNA from each species within the sample. It is not susceptible to the primer and PCR biases of metabarcoding. Amphibians and reptiles have historically been underreported in eDNA metabarcoding studies (5) due to primer bias toward more intensively studied groups (e.g., mammals).

River eDNA

Our research involved collecting water and sand samples at various points along the Avoca River in Ireland, from one of its source streams in the mountains all the way down to the Irish Sea at Arklow Town. We used fine filters to trap tiny bits of biological material, which were then processed so that DNA could be extracted and analysed.

We wanted to find out how much information could be obtained from a simple water sample using the long-read nanopore shotgun metagenomic DNA sequencing technique. This technique reads long stretches of DNA, and sequences all the DNA in a sample at once. Large reference genome databases can then be used to determine which species the DNA came from. The result is a broad snapshot of ecological communities, including the abundance of each species' eDNA in the sample. Microbes, fungi, plants, animals, and viruses can all be detected in a single rapid assessment. Technological advances even allow researchers to recover not just species' identities but genomic structure and variation.

When we sequenced the DNA that we filtered from the water and sand samples, we found DNA from a huge range of living organisms that had been in or near the water: from otters to oysters, cows to coral, foxes to fish, eagles to echinoderms, and badgers to bacteria. We also found DNA from one of Ireland's three native amphibian species, the European common frog (Rana temporaria). Loss of habitat and poor water quality are considered to be the main threats to amphibians in Ireland (9). Meanwhile, in many other parts of the world, Batrachochytrium dendrobatidis (Bd) is causing widespread reduction and extinction of amphibian populations (10). To the best of our knowledge, Bd has not previously been found in Irish amphibian populations (11).

This broad biodiversity screening is the first reported identification of this devastating amphibian disease in Ireland. Its potential presence is cause for concern, given the impact Bd has had on amphibian populations around the globe. Now that the presence of Bd and its host species have been detected, further targeted investigation at those sampling sites is warranted. This could include using pathogen-specific qPCR approaches to confirm the presence of Bd in eDNA samples from these habitats, as well as direct capture and swabbing of frogs from highlighted locations. Airborne DNA sampling and shotgun metagenomic analysis near the upland stream site also detected both frog (5) and Bd (unpublished) eDNA.

Applications of eDNA research

Sequencing eDNA can provide valuable guidance in deciding how to allocate resources for further field research and conservation efforts. Indications of the presence of a particular species can help to determine hotspots and refine search locations of the species in question. Furthermore, eDNA sequencing can provide information about the genetic variation in a population (1, 5). Researchers can discover not just what species are present, but the origins of migratory species, pathogen strains, and patterns of spawning dispersal (1, 5).

Environmental DNA presence/absence and quantity information can be compared over time to provide ongoing indications of the wellbeing of a population, ecosystem health, prey and food availability, and can be used to determine where more focused field research and conservation work need to be undertaken. It is also key in developing environmental management policies and pollution mitigation strategies. When we know what is out there, we can be more effective in protecting it.

The ability to identify eDNA from pathogens as well as their host species, all in a single sample and analytical technique, raises the possibility of integrated surveillance systems that monitor biodiversity, human health, wildlife health, and environmental change simultaneously in a 'One Health' approach. The potential applications are wide-ranging: identifying emerging pathogens, tracking invasive species, and monitoring environmental reservoirs of disease. Reducing the amount of direct contact between researchers and their target species would also minimize the potential risk of transmitting diseases between study populations.

Our findings highlight just how powerful the application of long-read shotgun metagenomic DNA sequencing can be. Entire food webs can be captured in a water sample, providing indicators of amphibian prey and predator species, and of the beneficial aquatic flora, keystone species, and ecosystem engineers that support amphibian ecosystems.

eDNA collection and sequencing

Researchers do not necessarily need to trap amphibians to retrieve DNA, or to take swabs to monitor for Bd. The collection of water samples is straightforward and can be done by citizen scientists and field biologists with basic training. In fact, sample collection for our study (1) was a family affair, with our children helping to collect and filter samples while on a summer visit to Ireland. We have also had extensive citizen-scientist sampling networks for some of our sea turtle eDNA projects (12). In the UK, citizen scientists have used eDNA sampling very effectively to monitor the great crested newt (Triturus cristatus) (13).

The process of filtering DNA from water samples, or from sand, sediment, or soil samples, is also relatively straightforward and requires minimal field and laboratory equipment such as bottles, syringes, and filters. Extracting eDNA from the samples only requires basic laboratory equipment like a shaker and a benchtop centrifuge.

Looking for individual species or pathogens in eDNA is cost-effective with qPCR, while whole-biodiversity surveys are becoming ever cheaper. Ownership of qPCR and sequencing machines is not required; samples can be run in university core facilities or by commercial vendors. qPCR currently has an approximate price point of USD $20 per sample. Commercial metabarcoding services can charge as little as USD $100 per sample per species group (barcode), including sampling consumables, sequencing, and analysis. Shotgun metagenomic sequencing, which captures all groups simultaneously, costs approximately USD $200 per sample for short-read sequencing, and USD $500 per sample for long-read sequencing. The cost of these sequencing approaches continues to fall, while the base-calling accuracy of long-read sequencing continues to increase.

Reference databases

The information resulting from eDNA sequencing depends on the quality of the reference databases available. Databases are continually being improved and updated with additional species genomes, but there are still gaps, and not all species are included. Some eDNA sequences might be assigned as a match with the nearest related species in the database rather than the actual species in question. As large-scale efforts to populate these databases continue, they will provide increasingly reliable matches. In the meantime, it is important that species identified through eDNA sequencing are cross-checked and interpreted by researchers with expertise in the relevant field and local fauna assemblages. Additionally, taking single samples risks missing some species, especially less abundant species or species in large waterbodies. However, this can be mitigated through biological, spatial, or temporal replicates, and survey design informed by species-habitat-use expertise.

Whether from air, sediment, or water sampling, environmental DNA offers huge potential for amphibian and ecological monitoring. The contents of a single cup of water can unlock vast quantities of information: biodiversity in and around the water, the effects of pollution and recovery, the spread of disease, and the ongoing effects of global environmental changes.

Acknowledgments

Sincerest thanks to our study co-authors: Orestis Nousias, Mark McCauley, Fiona Duffy, and Isabelle Duffy. Thanks also to Paula Murphy and Rebecca Rolfe (Trinity College Dublin), and Aleksandar Krstic and Walter Kolch (University College Dublin), for facilitating some of the lab work discussed in this article. Thanks to the University of Florida for funding the project. We gratefully received sampling equipment from the Corrib Conservation Centre.

References

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