One water bucket to find them all: Detecting fish, mammals, and birds from a single sample

Revolutionary environmental DNA analysis holds great potential for the future of biodiversity monitoring, concludes a new study.

Revolutionary environmental DNA analysis holds great potential for the future of biodiversity monitoring, concludes a new study

Collection of water samples for eDNA metabarcoding bioassessment.
Photo by Till-Hendrik Macher.

In times of exacerbating biodiversity loss, reliable data on species occurrence are essential, in order for prompt and adequate conservation actions to be initiated. This is especially true for freshwater ecosystems, which are particularly vulnerable and threatened by anthropogenic impacts. Their ecological status has already been highlighted as a top priority by multiple national and international directives, such as the European Water Framework Directive.

However, traditional monitoring methods, such as electrofishing, trapping methods, or observation-based assessments, which are the current status-quo in fish monitoring, are often time- and cost-consuming. As a result, over the last decade, scientists progressively agree that we need a more comprehensive and holistic method to assess freshwater biodiversity.

Meanwhile, recent studies have continuously been demonstrating that eDNA metabarcoding analyses, where DNA traces found in the water are used to identify what organisms live there, is an efficient method to capture aquatic biodiversity in a fast, reliable, non-invasive and relatively low-cost manner. In such metabarcoding studies, scientists sample, collect and sequence DNA, so that they can compare it with existing databases and identify the source organisms.

Furthermore, as eDNA metabarcoding assessments use samples from water, often streams, located at the lowest point, one such sample usually contains not only traces of specimens that come into direct contact with water, for example, by swimming or drinking, but also collects traces of terrestrial species indirectly via rainfalls, snowmelt, groundwaters etc. 

In standard fish eDNA metabarcoding assessments, these ‘bycatch data’ are typically left aside. Yet, from a viewpoint of a more holistic biodiversity monitoring, they hold immense potential to also detect the presence of terrestrial and semi-terrestrial species in the catchment.

In their new study, reported in the open-access scholarly journal Metabarcoding and MetagenomicsGerman researchers from the University of Duisburg-Essen and the German Environment Agency successfully detected an astonishing quantity of the local mammals and birds native to the Saxony-Anhalt state by collecting as much as 18 litres of water from across a two-kilometre stretch along the river Mulde.

After water filtration the eDNA filter is preserved in ethanol until further processing in the lab.
Photo by Till-Hendrik Macher.

In fact, it took only one day for the team, led by Till-Hendrik Macher, PhD student in the German Federal Environmental Agency-funded GeDNA project, to collect the samples. Using metabarcoding to analyse the DNA from the samples, the researchers identified as much as 50% of the fishes, 22% of the mammal species, and 7.4% of the breeding bird species in the region. 

However, the team also concluded that while it would normally take only 10 litres of water to assess the aquatic and semi-terrestrial fauna, terrestrial species required significantly more sampling.

Unlocking data from the increasingly available fish eDNA metabarcoding information enables synergies among terrestrial and aquatic biodiversity monitoring programs, adding further important information on species diversity in space and time. 

“We thus encourage to exploit fish eDNA metabarcoding biodiversity monitoring data to inform other conservation programs,”

says lead author Till-Hendrik Macher. 

“For that purpose, however, it is essential that eDNA data is jointly stored and accessible for different biodiversity monitoring and biodiversity assessment campaigns, either at state, federal, or international level,”

concludes Florian Leese, who coordinates the project.

Original source:

Macher T-H, Schütz R, Arle J, Beermann AJ, Koschorreck J, Leese F (2021) Beyond fish eDNA metabarcoding: Field replicates disproportionately improve the detection of stream associated vertebrate species. Metabarcoding and Metagenomics 5: e66557.

Scientists took a rare chance to prove we can quantify biodiversity by ‘testing the water’

Recent study conducted at a UK fishery farm provides new evidence that DNA from water samples can accurately determine fish abundance and biomass

Organisms excrete DNA in their surroundings through metabolic waste, sloughed skin cells or gametes, and this genetic material is referred to as environmental DNA (eDNA).

As eDNA can be collected directly from water, soil or air, and analysed using molecular tools with no need to capture the organisms themselves, this genetic information can be used to report biodiversity in bulk. For instance, the presence of many fish species can be identified simultaneously by sampling and sequencing eDNA from water, while avoiding harmful capture methods, such as netting, trapping or electrofishing, currently used for fish monitoring.

While the eDNA approach has already been applied in a number of studies concerning fish diversity in different types of aquatic habitats: rivers, lakes and marine systems, its efficiency in quantifying species abundance (number of individuals per species) is yet to be determined. Even though previous studies, conducted in controlled aquatic systems, such as aquaria, experimental tanks and artificial ponds, have reported positive correlation between the DNA quantity found in the water and the species abundance, it remains unclear how the results would fare in natural environments.

However, a research team from the University of Hull together with the Environment Agency (United Kingdom), took the rare opportunity to use an invasive species eradication programme carried out in a UK fishery farm as the ultimate case study to evaluate the success rate of eDNA sampling in identifying species abundance in natural aquatic habitats. Their findings were published in the open-access, peer-reviewed journal Metabarcoding and Metagenomics.

“Investigating the quantitative power of eDNA in natural aquatic habitats is difficult, as there is no way to ascertain the real species abundance and biomass (weight) in aquatic systems, unless catching all target organisms out of water and counting/measuring them all,”

explains Cristina Di Muri, PhD student at the University of Hull.
Drained pond after fish translocation.
Photo by Dr. Watson H.V.

During the eradication, the original fish ponds were drained and all fish, except the problematic invasive species: the topmouth gudgeon, were placed in a new pond, while the original ponds were treated with a piscicide to remove the invasive fish. After the eradication, the fish were returned to their original ponds. In the meantime, all individuals were counted, identified and weighed from experts, allowing for the precise estimation of fish abundance and biomass.

“We then carried out our water sampling and ran genetic analysis to assess the diversity and abundance of fish genetic sequences, and compared the results with the manually collected data. We found strong positive correlations between the amount of fish eDNA and the actual fish species biomass and abundance, demonstrating the existence of a strong association between the amount of fish DNA sequences in water and the actual fish abundance in natural aquatic environments,”

reports Di Muri.
Environmental DNA sampling using water collection bottles
Photo by Dr. Peirson G.

The scientists successfully identified all fish species in the ponds: from the most abundant (i.e. 293 carps of 852 kg total weight) to the least abundant ones (i.e. one chub of 0.7 kg), indicating the high accuracy of the non-invasive approach.

“Furthermore, we used different methods of eDNA capture and eDNA storage, and found that results of the genetic analysis were comparable across different eDNA approaches. This consistency allows for a certain flexibility of eDNA protocols, which is fundamental to maintain results comparable across studies and, at the same time, choose the most suitable strategy, based on location surveyed or resources available,”

elaborates Di Muri.

“The opportunity of using eDNA analysis to accurately assess species diversity and abundance in natural environments will drive a step change in future species monitoring programmes, as this non-invasive, flexible tool is adaptable to all aquatic environments and it allows quantitative biodiversity surveillance without hampering the organisms’ welfare.”


Original publication:

Di Muri C, Lawson Handley L, Bean CW, Li J, Peirson G, Sellers GS, Walsh K, Watson HV, Winfield IJ, Hänfling B (2020) Read counts from environmental DNA (eDNA) metabarcoding reflect fish abundance and biomass in drained ponds. Metabarcoding and Metagenomics 4: e56959.

Nature Conservation Special: Guidelines for the monitoring of beetles protected in Europe

In a follow-up to a recent special issue, 8 research articles outline a set of verified guidelines for the monitoring of 5 saproxylic beetle species listed in the Habitats Directive

In a set of eight research publications, scientists tested various methods for the monitoring of five European saproxylic (i.e. dependent on dead wood) beetle species protected by the Habitats Directive. The aim of their work was to test and propose a standard method for each species. A key role in this conservation initiative was played by citizen scientists who made it possible for sufficient data to be collected within a significantly shorter time frame.

The special issue “Guidelines for the monitoring of the saproxylic beetles protected in Europe” is the second in succession published in the open access journal Nature Conservation. Both are produced within the framework of the European Union’s LIFE Programme Project “Monitoring of insects with public participation” (LIFE11 NAT/IT/000252 MIPP) and were presented at the European Workshop held in Mantova in May 2017. Colonel Franco Mason, project manager of the MIPP project, notes that the workshop was aimed primarily at monitoring of saproxylic beetles.

While the first article collection focused on reporting recent findings derived from monitoring surveys across the European Union, the papers in the latest issue are devoted to testing various methods for the monitoring of five selected species of protected beetles, in order to determine the most efficient methods and, subsequently, to propose them as standard methods.

12761_Public participation 2nd tweetCuriously, the public participation in the project was not limited to ecology and entomology semi-experts and aficionados. The team specifically targeted children when recruiting volunteers. One of the dissemination activities of the MIPP project was the “MIPP-iacciono gli insetti” (translated to “I like insects” from Italian), where 3000 students from primary to high school undertook 60 activities per year in order to learn how to locate and identify the target insects.

“Participation by children in environmental education programmes seems to have a great impact on their attitude and behaviour,” notes Giuseppe Carpaneto, Roma Tre University and his co-authors in their introductory article.

“Some studies have shown that children who participate in such programmes are more concerned about nature, want to learn more about environmental issues and are more prone to follow pro-environmental behaviour (e.g. waste recycling) than children who did not participate”.

In another article, included in the special issue, Fabio Mosconi of the Italian Agricultural Research Council and Sapienza University of Rome and his co-authors tested whether a specially trained Golden Retriever could locate the threatened hermit beetle faster and more efficiently than scientists using the standard “wood mould sampling” method.


Additional information:

About the Life project MIPP

The main objective of the project MIPP is to develop and test methods for the monitoring of five beetle species listed in Annexes II and IV of the Habitats Directive (Osmoderma eremitaLucanus cervusCerambyx cerdoRosalia alpinaMorimus funereus).

Efficiency of insect biodiversity monitoring via Malaise trap samples and DNA barcoding

The massive decline of over 75% insect biomass reported from Germany between 1989 and 2013 by expert citizen scientists proves the urgent need for new methods and standards for fast and wide-scale biodiversity assessments. If we cannot understand species composition, as well as their diversity patterns and reasons behind them, we will fail not only to predict changes, but also to take timely and adequate measures before species go extinct.

An international team of scientists belonging to the largest and connected DNA barcoding initiatives (iBOL, GBOL, BFB), evaluated the use of DNA barcode analysis applied to large samples collected with Malaise traps as a method to rapidly assess the arthropod fauna at two sites in Germany between May and September.

One Malaise trap (tent-like structure designed to catch flying insects by attracting them to its walls and then funneling them into a collecting bottle) was set in Germany’s largest terrestrial protected natural reserve Nationalpark Bayerischer Wald in Bavaria. Located in southeast Germany, from a habitat perspective, the park is basically a natural forest. The second trap was set up in western Germany adjacent to the Middle River Rhine Valley, located some 485 kilometers away from the first location. Here, the vegetation is eradicated annually due to St. Martin’s fires, which occur every November. Their findings are published in the open access Biodiversity Data Journal.

DNA barcoding enables the identification of a collected specimen by comparing its BIN (Barcode Index Number) against the BOLD database. In contrast to evaluation using traditional morphological approaches, this method takes significantly less experience, time and effort, so that science can easily save up on decades of professional work.

However, having analyzed DNA barcodes for 37,274 specimens equal to 5,301 different BINs (i.e., species hypotheses), the entomologists managed to assign unambiguous species names to 35% of the BINs, which pointed to the biggest problem with DNA barcoding for large-scale insect inventories today, namely insufficient coverage of DNA barcodes for Diptera (flies and gnats) and Hymenoptera (bees and wasps) and allied groups. As the coverage of the reference database for butterflies and beetles is good, the authors showcase how efficient the workflow for the semi-automated identification of large sample sizes to species and genus level could be.

In conclusion, the scientists note that DNA barcoding approaches applied to large-scale samplings collected with Malaise traps could help in providing crucial knowledge of the insect biodiversity and its dynamics. They also invite their fellow entomologists to take part and help filling the gaps in the reference library. The authors also welcome taxonomic experts to make use of the unidentified specimens they collected in the study, but also point out that taxonomic decisions based on BIN membership need to be made within a comparative context, “ideally including morphological data and also additional, independent genetic markers”. Otherwise, the grounds for the decision have to be clearly indicated.

The study is conducted as part of the collaborative Global Malaise Trap Program (GMTP), which involves more than 30 international partners. The aim is to provide an overview of arthropod diversity by coupling the large-scale deployment of Malaise traps with the use of specimen-based DNA barcoding to assess species diversity.

Sequence analyses were partially defrayed by funding from the government of Canada through Genome Canada and the Ontario Genomics Institute in support of the International Barcode of Life project. The German Barcode of Life project (GBOL) is generously supported by a grant from the German Federal Ministry of Education and Research (FKZ 01LI1101 and 01LI1501) and the Barcoding Fauna Bavarica project (BFB) was supported by a 10-year grant from the Bavarian Ministry of Education, Culture, Research and Art.



Original source:

Geiger M, Moriniere J, Hausmann A, Haszprunar G, Wägele W, Hebert P, Rulik B (2016) Testing the Global Malaise Trap Program – How well does the current barcode reference library identify flying insects in Germany? Biodiversity Data Journal 4: e10671.