Dr Jessica Tout-Lyon (pictured above) Lecturer and early career researcher in the Charles Sturt School of Agricultural, Environmental and Veterinary Sciences in Port Macquarie and the Charles Sturt  Gulbali Research Institute of Agriculture, Water and Environment said anything that has DNA or leaves DNA in the environment can be studied.

Her previous research has focused on using eDNA to study the ecology of coral reefs and potential pathogenic bacteria during coral bleaching events.

Dr Tout-Lyon said over 50 per cent of turtle species are at risk of extinction, making them one of the most threatened vertebrate groups globally.

“The only two freshwater species  ̶  the Manning River Turtle (Myuchelys Purvisi) (pictured left) and the Bellinger River Turtle (Myuchelys georgesi)  ̶  that are endemic to NSW (that is, found nowhere else in the world) are also classified as being endangered,” she said.

“The ability to apply emerging and citizen science-friendly technologies such as eDNA has the potential to revolutionise the conservation and management of turtles.”

She said the Manning River Turtles’ distribution in the environment is greatly reduced due to habitat loss and feral animal predation, and the increasing time and high costs (up to six-times more) associated with their detection using traditional ecological surveys, such as snorkelling and trapping, limits research efforts.

Dr Tout-Lyon and Dr Elise Furlan from the University of Canberra, and their other collaborating colleagues, are designing and developing species-specific molecular tools, known as primers, to be able to identify and detect the DNA of endemic and endangered freshwater turtle species in the wild.

“These tools will allow us to detect the DNA turtles leave behind in their environment, which are predominantly river systems,” Dr Tout-Lyon said.

Dr Tout-Lyon explained that before COVID not many people would understand how the team uses the technology, but thanks to COVID, the technology and methods the team uses are now a household name.

“Once the water samples are collected from the environment - in this case where we think or know these turtles exist - we conduct similar lab work to the process used to test for COVID in wastewater or to confirm its presence in an infected person using PCR,” she said.

“We firstly filter the water sample to capture the DNA in the water; this can be done in-situ in the field, which is what I have done for the current field work, with my five-month-old baby boy (pictured) in tow.

“Then we extract the DNA from the filter and target the DNA we are interested in (the turtles) by using the species-specific primers we are developing.

“PCR is then used to amplify the amount of DNA we have in our sample, which is then sent to be sequenced to confirm that what we have detected is in fact our target species of interest.”

The focal species for the research include the local (Manning River catchment) endemic and endangered Manning River Turtle (Myuchelys purvisi), the Bell’s Turtle ( Myuchelys belli) and, a native but introduced species (Emydura macquarii) that is now thought to be hybridizing with the two endemic Myuchelys species.

“When compared to traditional ecological surveys, the use of eDNA in aquatic ecology as a sampling tool for assessment and monitoring is becoming a favoured technique,” Dr Tout-Lyon said.

“It is non-invasive, highly sensitive, reduces the sampling effort, time and costs, and has been shown to detect cryptic species or those in remote and otherwise difficult to survey locations.

“It allows researchers to be able to detect the DNA of endemic and endangered turtle species by taking a water sample from a river system.

“But the beauty of this technology is that the water samples do not have to be collected only by researchers, and this is where it is really beneficial,” she said.

Dr Tout-Lyon explained that the water samples can be collected by anyone provided a few guidelines are followed about the type of container the water is collected in and how it is stored.

“This is a ‘citizen science’-friendly approach to being able to detect endangered species on a more frequent basis with a broader scale approach than if we solely rely on scientists being deployed in the field a few times a year,” she said.

“Once we have validated the use of the species-specific primers, we will be able to work together with citizen-scientists to take water samples from their local river systems on a more regular basis.

“This will help to strengthen the collection of long-term spatio-temporal baseline data that is currently reliant upon scientists conducting time-intensive and expensive field surveys.

“If you think about the ability of land holders and community members to regularly and frequently sample their section of water way which they live either next to or nearby, compared to a scientist who lives a two-hour drive away, or in some instances, inter-state, we are able to open up the frequency of water sampling events as well as the number of sites sampled.

“This is crucial to building that long-term spatio-temporal data set that is so important to getting a better understanding of what is happening to these endangered species in the wild.

“We as researchers, and the future of these species, will be relying on people who live along these river systems to conduct regular water sampling to build this data set.”

Dr Tout-Lyon said the turtle species are threatened by invasive animals such as feral pigs and foxes that destroy critical habitat important to nesting, as well as the nests themselves.

“If we have a good indication of where these turtles are in the river, given that some species tend to favour particular stretches, we can potentially link this data to informing on-ground conservation and management practises. This includes the creation of predator-proof exclusion zones so that habitat and nests are not destroyed and thus give the species every chance of making a comeback from the endangered listing,” she said.

“It will also allow us to track the species throughout environmental disturbances, such as the recent trifecta of extreme weather events  ̶  the 2018-19 drought which dried up a significant proportion of the river, leaving only deep refuge pools for them to survive in; the ‘Black Summer’ mega fires of 2019-20 destroying much of the riparian habitat; and the 2021-22 floods, resulting in the destruction of riparian habitats and the potential relocation of turtles throughout the river.

“We may also be able to start investigating where in a river system the potential hybridisation with a native but introduced species of turtle (Emydura macquarii) is occurring, if we are able to detect its presence at a site.”

Dr Tout-Lyon and Dr Furlan are currently working on developing the species-specific primers in collaboration with project partners and the project funders, Mr Mat Bell from MidCoast Council and Dr Deb Bower at the University of New England (UNE). (See Media Note at the end for the full list of research collaborators.)

The results of the research are expected towards the end of 2023, and the technology will be used by MidCoast Council and UNE to conduct regular research into the presence and distribution of the two endemic species and to inform on-ground conservation and management practices.

Dr Tout-Lyon will also lead an all-female research team to explore the use of eDNA in detecting estuarine fish populations on the Mid North Coast under extreme weather events such as drought, fire and flood, and has recently applied for a L’Oreal UNESCO Women in Science Fellowship to explore the way in which turtles shed DNA.

“Unlike other vertebrate phyla, turtles’ hard keratinised outer surface is thought to inhibit their ability to leave traces of DNA in the surrounding environment compared to soft-bodied organisms, such as fish or mammals, and is termed by Adams et al., (2019) as the ‘Shedding Hypothesis’,” she said.

“We know the team of researchers at James Cook University used eDNA in 2022 to successfully re-discover the endemic Australian Irwin’s turtle (Elseya irwini) in Queensland, so there’s more to explore here.”