SOMETHING IN THE WATER
EDNA metabarcoding technology offers the chance for wide-scale species scanning with just a water bottle and a sense of adventure, as LAUREN FUGE reports.
A team of experienced cave explorers and a biologist armed with a new DNA survey method go deep into Christmas Island’s karst landscape in search of life. LAUREN FUGE reports.
Early one October morning in 2018, a small boat dropped 10 cavers and one scientist into the ocean off the northern coast of Christmas Island. With food, cameras, maps and scientific gear stashed in drybags, the team began a three-hour swim into the maw of the island’s most extensive cave system: Lost Lake.
It was 8:00 am, and they were on a tight deadline. “It was a race against the tide to get to the back of the cave before the water would hit the roof in the lower chambers,” explains Katrina West, the expedition’s researcher, from Curtin University in Perth.
Snorkels in mouths and wetsuit-clad, West and the cavers – all from the Western Australian Speleological Group (WASG), including trip leader Rob Susac – managed to reach the back of the cave where the chambers sloped upwards. This gifted them a precious few hours to explore the cave system before the tide began to fall again and they had to embark on another three-hour swim back to the boat.
The Lost Lake system was like a descent into another world, with vast chambers full of crystals taller than humans and massive flowstones formed over years as water seeps down walls.
“To get into a place like that was insane,” West says. “It’s just amazing that these big systems can develop for thousands of years in darkness and no one really knows about them.”
But to reach such places was dangerous work. Swimming through an ocean entrance was just one challenge – abseiling down into a limestone sinkhole
after an hour hiking in through the rainforest was another. Many caves are also flooded by the tide, and with every trip comes the risk of collapse or injury. That’s why the expedition was the culmination of a year of extensive planning – not only organising permits, equipment and travel, but also intensive caving training for West.
She was instructed by WASG in abseiling, underground navigation, cave conservation and safety drills – which came in handy when facing difficult conditions. While WASG was keen to explore and map the cave systems, West was interested in understanding how the underground ecosystems are linked to each other, using the DNA of elusive underwater critters.
“Christmas Island is like a honeycomb,” she explains. “It’s full of these caves that intersect at different points – we just don’t know where.”
Once the pinnacle of a sunken seamount, the island re-emerged from the ocean around five million years ago. It’s a karst landscape, primarily made of limestone that has dissolved to form caves, passages, chambers and whole subterranean networks, and home to animals that have been adapting and evolving in relative isolation.
These underworldly creatures live their lives in shadow. To us surface-dwellers, many of them seem ghostly – evolution has stripped them of unnecessary eyes or pigmentation, transforming them into pale spectres floating through the darkness.
They are also quite difficult to find. According to estimates, more than 80% of Australia’s subterranean fauna are yet to be discovered.
In the past, surveys of Christmas Island’s underworld have used baited traps, nets and visual surveillance to capture species. One 2010–12 study identified 54 species across 11 coastal caves, while a three-week survey in 1998 turned up 13 aquatic and 17 terrestrial taxa.
But West was trying something new: a powerful survey technique called EDNA metabarcoding, that picks up the DNA shed by an animal into the environment – for example, skin cells or mucus.
While traps and nets need to be left for days or weeks at a time, this technique requires just a single sample.
West was trying something new: a powerful survey technique that picks up the DNA shed by an animal into the environment.
At each site, West rushed to be the first at a pristine body of water not yet contaminated by her fellow cavers. She took six samples of water using hightech, sophisticated equipment – one-litre screw-top camping waterbottles, sterilised with bleach – which were then passed through a filter membrane to trap all the Dna-containing cells.
These were immediately frozen and stored at -20°C to be shipped back to the Trace and Environmental DNA (TREND) laboratory in Perth for analysis.
Over the course of two weeks, the team journeyed into 23 caves and springs scattered across Christmas Island, most of which are tricky to get in and out of. But managing these risks was well worth it for what they found down in the depths.
TRACING AND TESTING
Back in the lab, West and her colleagues at TREND used automated machines to break open the cell to extract the DNA, then heat it up to denature it – that is, peel the two strips of the DNA helix apart. They could then isolate a gene region specific to the taxa they were interested in.
“The extract would contain DNA from animals and plants in the environment,” West explains. “But say I’m only interested in looking at fish species – I then have to use what we call a primer.”
Primers are short strips of synthetic DNA, made up of 20 base pairs that are complementary to a section of the sample DNA. For each sample, two primers are attached like bookends between specific sections of the DNA the researchers are interested in, as it’s unique to a certain taxonomic group. The enzyme polymerase is then used to fill in the rest.
“The polymerase will add in the DNA from that first primer all the way down to the second primer,” West explains. “So we’re creating a synthetic DNA strand that’s about 300 base pairs long, which is complementary to the natural DNA strand.”
This technique – polymerase chain reaction (PCR; see illustration below) – essentially allows researchers to photocopy the DNA so they can sequence it. This is necessary because DNA degrades quickly in water, so it’s in very low concentrations in the samples. Making tonnes of copies means they can amplify very small samples to create enough to study in detail.
The primers, West says, can be made very specific. “If you only ever want to target one species, you can make primers that will bind to an area of DNA that has a lot of mutations,” she says – like choosing a bait that is only attractive to one type of animal.
But they can also make primers that are very relaxed, which is more like throwing out a net and seeing what it gets.
“That’s called metabarcoding – multiple species are amplified at the same time,” she says.
From there, the researchers can process the DNA again with stricter parameters, narrowing it down from genus to family group to species.
For the samples from Christmas Island, the TREND lab used three kinds of primers: fish, crustacean, and a more universal one in order to discover what other creatures were there. The resulting DNA sequence is then fed into a supercomputer and compared to a reference database to find a match. This study used National Center for Biotechnology Information’s Genbank nucleotide database, which contains hundreds of thousands of species and is being added to all the time.
If an exact match isn’t found in the database, a species can’t be assigned – though often a sample can still be narrowed down to within a genus or family group. “Say I have a DNA sequence that only gets a 95% match,” West says. “It probably isn’t that exact species, but it’s perhaps something closely related.
“That’s quite useful for directing certain researchers – taxonomists for example – to specific spots where we know we have an animal that is potentially undescribed.”
Of course, the species might be known, it just may not have been barcoded before and so isn’t in the database – only when a taxonomist has seen, identified and collected a specimen can it be added.
“The main benefit of this technique is that it doesn’t require taxonomic expertise,” West adds – at least, not in the field. “If we catch something by trapping and have crustacean species, fish species, molluscs and so on, we would need to have a specialist taxonomist for every single one of those taxonomy groups to be able to classify exactly what it is.”
This simplifies planning. An already arduous caving expedition would be much trickier if a cluster of taxonomists also needed to be lowered through a sinkhole and into the karst.
INTO THE UNKNOWN
One thorny problem remains: even if a DNA sample leads to a 100% species match, how do the researchers know that critter is really there without spotting it? Could the DNA have been swept in from somewhere else, especially in environments with tidal movements?
West explored this problem in 2017 in the neighbouring Cocos Keeling Islands, which are home to many different marine habitats very close to each other. Alongside EDNA metabarcoding, her team deployed baited remote underwater video stations (BRUVS) to provide independent data.
Despite the massive tidal movements through these diverse habitats, West and her colleagues found they were still able to distinguish between areas.
“The DNA gets diluted out very quickly,” she explains. “We weren’t going to pick it up in another location because there isn’t enough of it.”
Interestingly, the EDNA technique was also able to pick up many smaller fish species the camera didn’t have the resolution to detect.
An already arduous caving expedition would be much trickier if a cluster of taxonomists also needed to be lowered through a sinkhole.
Yet in still underwater environments, other challenges arise. “It was the first time this technology had been used in cave ecosystems,” West says. “My main concern was I wouldn’t actually pick up much DNA at all.”
Plus, because cave systems are notoriously underresearched, the reference databases for these types of species are limited. “That was the biggest challenge – can we actually detect species in cave systems? Are reference databases adequate enough to actually distinguish between different species?”
Yes. West and her team found 115 distinct species in the Christmas Island cave ecosystem, though some could only be narrowed down to the genus or family level.
“Of those, 21 were actually new-occurrence records for Christmas Island,” she says – meaning they had been found elsewhere but never here.
Small arthropods called springtails, for example, had previously been discovered in a big karst system in Cape Range, in northern WA. They must have somehow found their way to Christmas Island before the landscape became isolated and have been stuck there for millions of years.
“My favourite finding was the Indonesian shortfin eel – a super-elusive species,” West says. “There was only one that was found in a cave previously, and that was a juvenile. Then the public spotted it previously in one part of Christmas Island that’s a rainforest wetland area and it hasn’t been seen anywhere else.”
Finding the eel in a cave on the other corner of the island is exciting, she says, because it indicates the species is more widespread than anyone imagined. They also identified a freshwater jellyfish, typically found in calm lakes and reservoirs – but never before on Christmas Island. “The freshwater jellyfish was probably the most surprising,” West says. “I had no idea that they even existed before that finding.”
EDNA metabarcoding allowed the team to pick up more species than any previous study. “That’s the power of using this technique,” West says. “Picking up over 100 different species within a two-week period was really quite extraordinary compared to previous survey efforts.”
“Picking up over 100 different species within a two-week period was really quite extraordinary compared to previous survey efforts.”
West isn’t just focused on biodiversity; she is attempting to use the species to figure out how the caves are interconnected. “If we can detect the same sort of composition of subterranean species in caves that are closely located to each other and that these caves have the same types of environmental variables, then we could actually predict which caves are connected under the ground,” she explains.
By targeting many different taxonomic groups in one go, EDNA metabarcoding creates the equivalent of a whole ecosystem survey. West’s team was able to pair this with other measurements taken in each cave system – like salinity, dissolved oxygen, acidity and temperature – to begin to map the interconnectivity of the cave systems. They identified three cave and spring groups that shared the same types of species and water quality readings.
“To confirm interconnectivity, we would need to get our cavers back down there and potentially conduct a salt test, whereby we drop salt in the water and see if we can detect an increase in salinity in the next cave over,” West says.
She is soon moving to a new position at CSIRO to tackle the next challenge in her EDNA metabarcoding work – using DNA samples to determine not only the species an animal belongs to, but which specific population within a species. A simple water sample could tell biologists whether populations are migrating or interbreeding or declining – or even when they diverged from other species. “It’s definitely a possibility because we know there’s intact DNA in the environment,” West says. “It’s just a matter of being able to sequence longer DNA strands.”
For this, she’ll likely head to the Kimberley to study the rapidly declining populations of sawfish – a tricky thing to do with regular surveying techniques, because the water is turbulent and chock full of crocs. But with EDNA metabarcoding, she hopes to learn more about their movements and breeding and thus inform management decisions about water use in the region – including for proposed dams and mines.
“I’ve always been trying to pivot the science research I do to have immediate applications,” West says. But she also wants to keep caving.
“My biggest dream would be going to the Son Doong cave in Vietnam,” she says. “That’s one of the largest cave systems in the world and it has its own underground rainforest and underground rivers. If we could apply this technology in a place like that, it would probably reveal a lot of interesting things.”
LAUREN FUGE is a journalist at Cosmos, writing regularly on cosmosmagazine.com. Her last story, about human-wolf interaction, appeared in Issue 84.