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eDNA Tracking Method

DNA tracking method - a new way to save endangered turtles

Nature pays the price for continuous human development. According to the World Wide Fund for Nature, global animal populations have dropped by nearly 70% over the past 50 years. Hence, conservation work is urgently needed to make up for the loss.

Although Hong Kong is a developed city, rare and endangered animals persist here. Lingnan University Science Unit Associate Professor Jonathan Fong and his team have introduced DNA-based methods to study and conserve endangered animals.

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The crisis turtles facing

In Asian societies, tortoises and freshwater turtles are often used as food, traditional medicine, decorations, and pets . Since turtles have a relatively low reproduction rate , intense hunting for these uses threatens them with extinction.

Moreover, turtles have also become the victims in Asia’s illegal markets. In the pet market, wild individuals of rare species can be worth up to HKD$10,000. Some people use turtles as their wealth-flaunting tools, while some animal traders breed turtles for great profits.

Asian turtle species are in danger of going extinct. We should act now to save them.

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Conservation of Hong Kong’s native freshwater turtles

Although Hong Kong is sometimes referred to as a concrete jungle, it is also blessed with a large area of protected country parks. The natural features, such as the presence and abundance of rivers, swamps, and islands, facilitate the high diversity of living organisms.

In Hong Kong, there are five native turtle species, namely the Reeves’ turtle, Chinese three-lined box turtle, Beale’s-eyed turtle, Chinese softshell turtle, and big-headed turtle.

Let’s find out more about them through the pictures below.

In addition to hunting and trafficking, Hong Kong turtles are also threatened by exotic turtle species (e.g., red-eared slider; Trachemys scripta elegans). These exotic turtles are released into nature usually from Buddhist release or discarding of unwanted pets. They compete with local species for resources and damages the ecosystem. As a result, the populations of local turtle species continue to shrink.

Beale’s turtle and big-headed turtle are relatively long lived. However, due to their long reproductive cycles and the abovementioned threats, they are listed as endangered and critically endangered in the Red List of Threatened Species by the International Union for Conservation of Nature. This highlights their risk of extinction. Despite their rarity, both species can be found in Hong Kong, which provides Professor Fong and his research team a special opportunity to study and protect them.

Traditional Methods

The first step to studying freshwater turtles is to locate them. Traditionally, this is done by trapping, which is time-consuming, exhausting, and sometimes dangerous for researchers. Trapping is a two-day event that takes advantage of turtles being more active at night, coming out to look for food and mates. On the first day, researchers hike along streams and place baited traps into the water, then return the next day to collect traps and any turtles. Due to the long and bumpy routes, they only lay traps in 1–2 locations each trip. All this hard work does not guarantee success in trapping turtles. While searching for turtles in Guangdong Province, Professor Fong and colleagues spent one month putting out traps and only caught four turtles.

To more effectively find and study Hong Kong’s wild freshwater turtles, Professor Fong’s research team developed and tested a new method to aid in finding turtles that centers around environmental DNA (eDNA).

eDNA as evidence for the survival of Hong Kong turtles

Crime movies often show how even when a criminal is not captured at the crime scene, circumstantial evidence can help solve the crime. The same applies to the research on Hong Kong’s wild turtles. Organisms leave evidence of their presence in the environment through the DNA in shed cells, such as skin, gametes, feces, and blood. This DNA is called Environmental DNA.

Environmental DNA is also called eDNA. Living organisms release cells that contain DNA into our environment, such as air, water and soil. Through eDNA, we can extract DNA from environmental samples such as soil, water and air instead of extracting DNA directly from the body. Foreign studies have also proved that eDNA helps to find a variety of aquatic vertebrates, including turtles.

When the research team arrives at a site, they collect 1 L of water in a sterile container, which is filtered in the field or back at the laboratory. Then, the eDNA is amplified using a quantitative PCR (qPCR) technique. This eDNA technique can be used to identify the diversity of organisms (such as plants, fungi, animals, and bacteria) in the water. In the right conditions, even the number of individuals can be estimated.

eDNA methods enable us to study organisms without seeing them. This is important for rare and secretive species. However, eDNA methods do not solve everything. Many research projects still require organisms to be captured to study. eDNA methods can be used to quickly and efficiently evaluate the probability of organisms existing at a site, increasing the chances of traditional trapping methods.

Turtle evolution and genetics

Another important part of conservation is to identify and delineate each species. We can use DNA to estimate how closely related two individuals are, and whether they are different species. Another project of Professor Fong and his team is to study the genetics of Hong Kong species, especially amphibians and reptiles. For example, the big-headed turtle is found in China, Vietnam, Laos, Cambodia, Thailand, and Myanmar. By using DNA data, we can see how similar populations are from the different countries. If different, we will need to work hard to conserve all populations.

This work is being done in collaboration with his Lingnan University colleague, Assistant Professor Yik Hei Sung. Professor Sung has studied the ecology of Hong Kong turtles for almost 10 years. During this time, he has collected hundreds of tissue samples that can be used for DNA studies. The DNA is extracted from the cells and can be sequenced then compared between individuals. To get samples from other countries, Professors Fong and Sung are collaborating with other turtle researchers in Southeast Asia, as well as using samples from museum specimens in natural history collections around the world. For these studies, they have collected hundreds of samples.

This research has several of practical conservation uses. First, this work can help identify the origin of smuggled animals, so that they can be returned to the proper country. Also, many endangered species are bred in captivity, so that they can be released back into the wild in the future. DNA data can help identify which individuals to breed together to maintain a healthy population. Lastly, sometimes a population is identified as being so different that we need to recognize a new species.

Research Progress

Through their eDNA research, the team hopes to learn about the diversity of Hong Kong’s freshwater turtles as well as understand the distribution of exotic turtle populations. The team successfully developed eDNA methods to locate two local turtle species (big-headed turtle and Chinese softshell turtle) and one exotic species (red-eared slider).

The red-eared slider, a native species of North America, is no stranger to Hong Kong residents. It is an omnivorous animal with strong adaptability to new environments, and often competes with native species for resources. Due to the release of unwanted pets and Buddhist release, local Hong Kong habitats have been invaded by the red-eared slider. Local species are likely affected negatively by competition with the red-eared slider. Professor Fong and his team used eDNA to track the distribution of red-eared slider to evaluate the harm they have brought to the native turtles.

In addition, the team collected water and soil samples from more than 40 rivers and lakes in Hong Kong four times a year, for three years. By comparing the results, they hope to understand how different seasons and variables in the environment affect the preservation of eDNA in water. By 2021, more than 500 samples had been collected for this research.

Citizen science for all

The preservation of eDNA in water is affected by temperature, pH, and water velocity. For example, warmer temperatures and lower pH will cause DNA to degrade, reducing its presence in the water. Despite limitations to eDNA methods, one of its advantages is that one doesn’t need to be a researcher to collect eDNA samples. Everyone can do their part and contribute to the research. Incorporating non-scientists in the research process is called citizen science.

The team incorporated citizen science in this turtle eDNA study to aid the research and promote science research to the general public. Professor Fong invited university undergraduates and the elderly to take part in sample collection, providing them with necessary tools such as containers, thermometers, and pH detectors. The participants attended training workshops and collected water samples to be used in the study. Their contributions contributed to the project’s success.

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Using eDNA helps facilitate the tracking of turtles which is vital for future conservation efforts. Click to understand more about the significance of eDNA!

Data sharing

The research team and the Agriculture, Fisheries and Conservation Department share the location data of wild turtle populations obtained through eDNA so that authorities can better monitor the living conditions of turtle species and even prevent illegal hunting.

Saving smuggled turtles

Using DNA, the research team hopes to identify the origin of smuggled animals so they can be returned to their natural habitat.

Conservation of other species

Although the research only targeted turtles, it has helped in the understanding and promoting animal conservation.

Community education

During the research, students were trained to promote conservation. Through observing wild turtles in country parks, it helped increase their awareness of the importance of conservation.

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Research journey

Professor Fong specialises in research on the evolution and ecology of amphibians and reptiles, including the unique Romer’s tree frog in Hong Kong. He successfully built a Natural History Collection of Hong Kong’s amphibians and reptiles, which has over 550 specimens. These specimens are an important resource for research and public education. Watch the video to learn more about his ambition as a researcher.

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Encouragement to researcher

Growing up in the United States, Professor Jonathan Fong's curiosity about nature, fostered by his parents and teachers, led him to become a scientist who promotes conservation.

Research success is a team effort and depends on collaborations with government officials, NGOs, and other scientists. Additionally, the research involves hiring and training students. As other mentors did for him, Professor Fong’s hopes give opportunities to young scientists to explore their interests and pursue a career in the field. To his delight, several of his research assistants have chosen to pursue careers in science and have continued their postgraduate studies in biology.

Professor Fong believes that although enjoyable, research can be difficult, "If it were easy, someone would have done it already!” His interest and motivation to understand the natural world, as well as make an impact to society, drives him to solve these problems. For anyone who is interested in research, he said “Scientific research is never perfect, problems will always arise, and scientific research is about learning from your mistakes. The research journey may be solitary but not isolated, and generations of scientists continue to build on past research in the hope of benefiting future generations. As researchers, we should never forget to enjoy the present.”