eRNA metabarcoding:

• Discovered recently, metabarcoding is a powerful tool for assessing species composition by scanning the ‘barcode’ of DNA or RNA to identify plants and animals.
• However, DNA metabarcoding analysis can provide false positive results.
• Dr Kaede Miyata, Mr Yasuaki Inoue, and colleagues at Kao Corporation, Japan have developed an innovative approach – metabarcoding environmental RNA (eRNA).
• Importantly, eRNA analysis could serve as an indicator of the living biotic assemblages actually inhabiting an environment, improving the evaluation of biodiversity and water quality and mitigating the effect of wastewater contamination in analysis.

Population growth and rapid climate change have put immense pressure on Earth’s ecosystems. At the UN COP15 international conference on biodiversity in 2022, a new framework to halt and reverse nature loss was agreed upon. This nature-positive pledge aims to achieve a world where nature is restored and regenerated above and beyond baseline levels recorded in 2020.

Assessing biodiversity – tracking the rise and fall of populations over time – is crucial for achieving nature-positive goals. However, accurately measuring a habitat’s biodiversity and species composition is a real challenge. Fortunately, a team of researchers in Japan, led by Dr Kaede Miyata of Kao Corporation and including Mr Yasuaki Inoue, have a solution that could help turn the tide in the fight to save our precious natural world. Using a tool called metabarcoding, they are assessing the potential usefulness of environmental RNA (eRNA).

Capturing biodiversity

Traditional methods for assessing biodiversity involve capturing organisms and visually classifying them via taxonomic features or unique physical attributes of a species. This method of identification can be tricky, even for experts. Key identification features for some species are not always present or are difficult to distinguish; some species can only be identified genetically, and capturing an accurate number of samples for analysis can be time-consuming or even impossible. To assess the species composition of entire aquatic ecosystems, researchers have – up until now – turned to DNA for the answers.

Metabarcoding is a recent breakthrough that enables researchers to conduct large-scale ecological surveys using many genetic samples.

Metabarcoding: scanning the barcode

To assess biodiversity, ecologists often measure DNA – the molecule that carries an organism’s genetic information. However, living organisms contain vast amounts of DNA so measuring the entire DNA sequence to identify an organism is expensive and time-consuming. To more quickly assess an organism’s DNA, researchers sequence only a small fragment unique to the organism – a short sequence, known as a ‘barcode’. The technique, called DNA metabarcoding, is a recent breakthrough that combines the accuracy of genetic sequencing with the low cost and speed of traditional DNA identification. Using barcoding, a high number of DNA molecules can be sequenced at the same time in a mixed sample. However, DNA metabarcoding does have limitations. DNA persists in the environment for extended periods of time, which may generate false positive results.

Environmental RNA metabarcoding

Before now, little had been known about how useful eRNA could be for metabarcoding in ecological surveys. But Miyata, Inoue and colleagues hypothesised that RNA could offer a better template for metabarcoding than DNA. Because DNA is stable, it causes false positives for species not living in the ecosystem being surveyed. As RNA is less stable than DNA, it disintegrates faster once outside the organism, offering a more accurate real-time environmental indicator.

The researchers tested the feasibility of environmental RNA as a barcode template by comparing it to environmental DNA barcodes of fish from a freshwater ecosystem. They were looking to see the difference in accuracy between eDNA and eRNA metabarcoding analyses by measuring each technique’s false positives (samples containing genetic material from species not present in the observed habitat or ecosystem), which the researchers thought were likely to be caused by wastewater contaminants containing fish that were not living in the aquatic ecosystems.

Observing a wide range of aquatic species, the researchers detected more false positives (eg, fish DNA or RNA from wastewater) using eDNA metabarcoding compared to eRNA metabarcoding.

Fishing out false positives

To see if eRNA metabarcoding analysis was as accurate as eDNA, Miyata’s team investigated the fish populations of several Japanese rivers. In 2021, their study uncovered that there were sufficient levels of eRNA present in the water for testing using metabarcoding. The team found that eRNA testing using metabarcoding was equally as sensitive as eDNA analysis, but was superior in terms of positive predictability. eRNA also more accurately distinguished false positives by identifying DNA or RNA from saltwater and brackish water fish. These would not have been native to the freshwater river ecosystems, and likely originated from domestic wastewater. These findings represent the first evidence of eRNA’s potential as an indicator of the biotic assemblages inhabiting an ecosystem.

Subsequently in 2022, the researchers performed a comparative eDNA/eRNA analysis of algae and arthropods (insects) in a Japanese river ecosystem to evaluate and compare their potential for biological monitoring and assessing water quality. This ecological survey revealed a high number of species, but the composition – or ratios of species to each other – was very different from the traditional field survey. For algae, eDNA and eRNA metabarcoding produced comparable results and, compared to traditional field surveys, which focus on attached algae, metabarcoding analysis was able to detect the free-floating planktonic algae and attached algae. Meanwhile, for arthropods, eRNA distinguished false positives by identifying DNA or RNA from terrestrial arthropods. The results indicated that eRNA was an effective indicator of false positives. The researchers showed that algae and arthropod eDNA/eRNA metabarcoding analysis enables water quality estimation and that eRNA can be used to evaluate biodiversity and water quality and also identify false positives.

This year, the researchers showed that both environmental DNA and RNA analysis can produce false positive results for species identification, especially in aquatic environments contaminated with domestic wastewater. A metabarcoding analysis of influent and effluent of a wastewater treatment plant revealed that wastewater was contaminated with fish DNA and RNA, most of this constituting consumed fish. The levels of these nucleic acids were extremely high at the influent of the treatment plant, meaning areas with poor waste treatment infrastructure may be subject to higher contamination. However, the influent wastewater had less RNA than DNA, meaning the number of false positives (food fish contamination) was reduced in the eRNA metabarcoding analysis compared to eDNA, supporting its potential to mitigate errors in identification, particularly in areas receiving wastewater. Therefore, analysing wastewater near sampling locations and the sewerage coverage rate will allow for the selection of the appropriate nucleic acid (DNA or RNA) to be used in metabarcoding analysis, giving a more accurate evaluation of the fish species present in the sample environment.

Assessing the environmental RNA in a sample should give a more accurate reading of the species currently in the environment.

Ushering in sea of change

Miyata’s new eRNA metabarcoding tool could be used for a range of applications in environmental studies, such as water quality estimations. In their most recent study, the researchers focused on domestic wastewater as a possible source of false positives and characterised fish nucleic acid in wastewater. As they found that a certain amount of DNA/RNA derived from mainly consumed fish, these DNA/RNA contained in wastewater is an important source of false positives in ecological survey using eDNA/eRNA.

Comparing the RNA and DNA analysis helped scientists distinguish whether fish were living in the environment. In addition, analysis of wastewater near sampling locations is essential to accurately understand the survey results. This innovative application of environmental nucleic acid analysis will help environmentalists obtain more accurate results and better protect our fragile ecosystems.

Through their research, Miyata and colleagues have shown the many ways that the eRNA metabarcoding technique they developed may be used in the field of ecology, including being used to evaluate biodiversity and water quality, improve species detection, and mitigate wastewater contamination. This novel tool offers potential for the improvement of monitoring and evaluation of aquatic ecosystems in the future.