E-DNA: THE TECHNOLOGY TRANSFORMING FISH CONSERVATION

This molecular technique is set to revolutionise freshwater biological monitoring in the 2020s, supercharging our ability to detect and identify the planet’s fish and other aquatic species. By Dr Joseph Huddart, Post-Doctoral Researcher at the Earlham Institute.

6 July 2020

Fresh water is life. The lakes and rivers that make up our surface freshwaters contain 10% of all described species and one third of all vertebrates, with more being discovered on an almost daily basis. Of the groups of animals that dwell in freshwater—including reptiles, amphibians and insects—fish are among the most recognisable and charismatic. There are over 10,000 species of freshwater fish, approximately 40% of global fish diversity; impressive considering lakes and rivers occupy less than 1% of the world’s surface, compared to the 70% covered by oceans. 

Freshwater fish species (or fishes) are vital components to food webs, sustaining both other species and millions of human communities with protein, especially in poorer parts of the world. They are, however, disproportionately threatened compared to their cousins in the marine realm, with larger species at particular risk. With just 0.01% of the world’s total water volume present as surface freshwaters (once saline, frozen and groundwaters are removed), these ecosystems are comparatively rare.

But as we scramble to exploit fresh water for our own ‘goods and services’ and alter water bodies directly through overfishing, damming and channelisation—and indirectly through pollution, climate change and invasive species introductions—we’re likely to lose many fish species before they have been formally recognised by science. 

With the Sixth Mass Extinction event well underway in freshwaters, there is real urgency to map out biodiversity hotspots and ensure they receive the necessary protections before species are lost forever.

Traditional monitoring

Getting to grips with freshwater fishes is inherently challenging. Generating the data to map species population ranges is almost entirely dependent on direct observation using capture-based methods, such as logging inland fisheries catches or actively catching fish for monitoring purposes using nets, traps or electrofishing. While fishes can be identified in the field, and subsequently returned to the water alive (albeit shaken), many are killed and then logged in fish markets or euthanised and identified in the laboratory later. The process is expensive, requiring a trained team able to operate in challenging conditions and skilled taxonomists to identify fish species based on their distinguishing physical characteristics (morphology). Even still, it’s likely that some of the more elusive or rarer species will slip through the net and remain undetected; or cryptic species, that look very similar to other species, will be misidentified. 

Moreover, the animals that make up ecological communities are not static but constantly moving and responding to environmental cues. The ability to detect, monitor and track species is what we call the ‘moving target’ of biodiversity monitoring. Each survey provides a snapshot of diversity at any one moment in time, and it’s these snapshots that are stitched together to create a time-series that are then used to look for trends and patterns. These can then be linked to environmental factors and also policy. For instance, a recent study in the UK found that while terrestrial insects have declined, pollution controls and chemical water recovery under the extensive EU Water Framework Directive have led to a measurable recovery of aquatic insect populations.  

The stats don’t lie, providing much needed quantitative evidence that policy is working and ecological recovery is possible; however, stats take time and money to acquire through surveying and always lag behind real-time, and this routine and expensive monitoring is rarely an option in poorer countries. Until now.

E-DNA metabarcoding: The game changer 

A new tool for fish detection and assessment has emerged, bearing more resemblance to a CSI investigation than to ecologists armed with waders and nets. Environmental DNA (eDNA) refers to the traces of DNA that animals lose to their surrounding environment as they move through it: faeces, skin cells, hair, slime, saliva, even their decomposing carcasses. While in land-based ecosystems DNA breaks down rapidly and is much harder to isolate from the environment, freshwater slows this process, in effect becoming a very diluted eDNA soup of the various species that call it home. However, even at this very low concentration, the eDNA present in a litre of water from a lake or river can reveal its entire fish community. The technology is of similar use for marine conservation but, due to uncertainties surrounding how far eDNA can travel before it degrades, defining the location of the source animals is much harder.

E-DNA is often used to detect the presence of specific species of interest, such as invasive crayfish or species of conservation concern like great crested newts, but the technology has developed rapidly. We’re now at a point where it’s possible to detect signals from nearly all the species making up the ecological groups in an aquatic community, from fishes to mammals to water birds and invertebrates. 

This is done through ‘eDNA metabarcoding’, in which a DNA sequence unique to a species provides a ‘barcode’ to distinguish it from other species, most commonly derived from mitochondrial DNA. These barcodes are stored in online open-source repositories, creating a genetic reference library for species (BOLD, for example). The eDNA extracted from your water sample is replicated using a process called polymerase chain reaction (PCR), and then sequenced. The barcodes are then used to match the eDNA in your sample to their species of origin. Even if an eDNA fragment doesn’t match a species barcode, it’s often possible to infer from which family the species belongs.

This is pretty amazing news for ecologists, who for the last 100 years have spent days in the field and years in the lab identifying species using taxonomic keys (written tools and diagrams used to determine species from their physical characteristics); knowing that by missing species in the environment and maybe misidentifying them in the lab, their efforts would have most likely under-represented the true ecological diversity of the area. 

E-DNA metabarcoding also enables you to identify species at all stages of life, such as sperm or barely visible fish fry, which would be near impossible to detect and distinguish from other species using traditional morphological methods. Surveying is also much less destructive (if at all): the assault on the environment with boats, nets, traps and electric fishing gear, and necessary fish mortality and disturbance—typical prerequisites of fish surveying—is replaced by filling sterilised bottles with water from sites of interest. In terms of efficacy, numerous studies using natural-study and controlled systems (even large aquariums in theme parks) have found eDNA to outperform traditional methods, and the technology is now commercially available from private companies. While the approach isn’t yet able to substitute for the data on a species’ population size collected through quantitative capture-based methods, it complements these data with an added level of detection covering a larger spatial-scale. 

And there’s also another important utility for eDNA: the detection of non-native species. Invasive species are recognised as a major threat to global freshwater biodiversity, where even the invasion of small invertebrates such as water fleas and mussels can subtly alter food webs and shift ecosystems into a new equilibrium, leading to the extinction of seemingly unaffected species. For the many species of exotic fish being reared for aquaculture, such as tilapia and catfish, chosen not just for taste but also for their voracious unfussy diets and hardiness, the impact of their release and successful colonisation on native species is far more obvious. The early detection of their presence using eDNA can lead to quick and effective management to limit the extent of their damage.

With the falling costs of sequencing and other operational functions, alongside ever more species barcodes added to the reference libraries, this molecular method is set to transform the freshwater biological monitoring landscape in the 2020s. It will turbo-charge our ability to detect and identify the species of the planet’s aquatic ecological communities, and has vast potential in remote, hard-to-access locations where traditional netting and electrofishing aren’t viable. Already, countries such as Canada are embracing eDNA metabarcoding and embarking on trans-national monitoring programs to assess their freshwater fishes.

Tropical potential

With eDNA having been pioneered in the well characterised, heavily modified and relatively depauperate freshwaters of Western Europe and North America, tropical countries offer the technology the opportunity to go off-road, into ecosystems that are yet to be studied and far more biodiverse. Take, for instance, the UK, with it’s relatively few (but still charming) 38 species of freshwater fish, and compare it to a tropical country like Colombia. Taking in the Amazon, Orinoco, Andes, Pacific and Caribbean zones, the country boasts more than 1,500 species of freshwater fish, with new species being discovered all the time.

In such countries, the level of skill required to identify species is obviously much higher, and in many cases—in the field, with live specimens under the naked eye—almost impossible. Microscopes are often required to count or look for morphological indicators, such as the number of fin rays (or even individual scales!). Expeditions and surveys typically use gill nets or other destructive means to harvest fish samples, which are preserved and identified in laboratories—a process that can take months to complete. This means our estimates of the moving target of biodiversity is lagging even further behind and rarely frequent enough to generate a time-series. And let’s not get ahead of ourselves: often megadiverse tropical countries lack the resources to conduct extensive freshwater surveys and monitoring. Cash-strapped governments are unable to pump unlimited funds into extensive field trips to remote locations, which present a logistical maelstrom of expensive equipment and treacherous conditions.

It’s here where the potential for eDNA is arguably greatest. Unleashing fish scientists from the shackles of tonnes of fishing gear, eDNA monitoring could rapidly accelerate our understanding of fish communities in the world’s biologically richest, economically poorest and most understudied countries in a matter of years rather than the decades previously assumed. We have a real opportunity to quantify the true extent of freshwater biodiversity and perform high-frequency, large-scale and long-term biological monitoring much faster and at much less cost than ever previously possible. 

Nonetheless, there remains one overriding necessity required to facilitate eDNA as a viable fish surveying method, and that is the need to first gather ‘voucher’ specimens of species from which to obtain the original barcodes for the reference libraries. This will require strong collaboration with fish taxonomists, tracking down viable specimens in museum collections and other repositories, as well as from further expeditions into the field. 

It will also require some technical fine-tuning to ensure that the genetic barcodes can differentiate what we consider different species based on morphological differences, and already there is heated debate as to whether physical or genetic differences should take precedence when delineating species. 

The 2020s is likely to be a decade where the environment, particularly our treatment of it, is by necessity put under the spotlight. Speeding up how we detect and monitor the species with which we share this planet will be vital for gathering the evidential momentum necessary to change and inform policy and prevent further damage. 

Armed with eDNA metabarcoding, we’ll be able measure diversity and track the loss of species from the environment and exotic species invasions. But perhaps more importantly, we’ll be able to identify species that could be recolonised in areas where they have been previously driven to extinction. This will help shift the dystopian narrative of biodiversity loss to one of ecological recovery.

For the world’s freshwater fishes, it marks the beginning of a period of greater visibility and recognition—and, one would hope, protection.

The author is currently working for GROW Colombia,​ an initiative​ funded by the UK Government's Global Challenges Research Fund aiming to protect, preserve and manage biodiversity in the Latin American country. He is focusing on developing eDNA as a tool for detecting, monitoring and advancing the understanding of Colombia’s freshwater fish species.