Environmental DNA (eDNA) metabarcoding is revolutionizing the monitoring of aquatic biodiversity. The use of eDNA has the potential to enable non-invasive, cost-effective, time-efficient and ...high-sensitivity monitoring of fish assemblages. Although the capacity of eDNA metabarcoding to describe fish assemblages is recognised, research efforts are still needed to better assess the spatial and temporal variability of the eDNA signal and to ultimately design an optimal sampling strategy for eDNA monitoring. In this context, we sampled three different lakes (a dam reservoir, a shallow eutrophic lake and a deep oligotrophic lake) every 6 weeks for 1 year. We performed four types of sampling for each lake (integrative sampling of sub-surface water along transects on the left shore, the right shore and above the deepest zone, and point sampling in deeper layers near the lake bottom) to explore the spatial variability of the eDNA signal at the lake scale over a period of 1 year. A metabarcoding approach was applied to analyse the 92 eDNA samples in order to obtain fish species inventories which were compared with traditional fish monitoring methods (standardized gillnet samplings). Several species known to be present in these lakes were only detected by eDNA, confirming the higher sensitivity of this technique in comparison with gillnetting. The eDNA signal varied spatially, with shoreline samples being richer in species than the other samples. Furthermore, deep-water samplings appeared to be non-relevant for regularly mixed lakes, where the eDNA signal was homogeneously distributed. These results also demonstrate a clear temporal variability of the eDNA signal that seems to be related to species phenology, with most of the species detected in spring during the spawning period on shores, but also a peak of detection in winter for salmonid and coregonid species during their reproduction period. These results contribute to our understanding of the spatio-temporal distribution of eDNA in lakes and allow us to provide methodological recommendations regarding where and when to sample eDNA for fish monitoring in lakes.
There is an urgent need to evaluate the effects of anthropogenic pressures and climatic change on fish populations’ dynamics. When monitored in lakes, fish spawning is generally assessed using ...traditional, mostly destructive or damaging, methods as gillnetting and collection of fertilized eggs. Over the last decade, environmental DNA (eDNA) based methods have been widely developed and now offer a non-invasive alternative method to these traditional biomonitoring tools. In particular, the emergence of new methods as the droplet digital PCR (ddPCR) offers the possibility to quantify an absolute eDNA signal in a very sensitive way and at a low cost. Here, we developed and implemented a quantitative eDNA approach to monitor the spawning activity in a lentic environment for two non-migratory fish species, European perch and whitefish. The ddPCR protocols were formalized based on existing and newly designed COI primers, and were applied during four spawning periods in Lake Geneva. The results demonstrate the effectiveness of weekly eDNA sampling coupled with ddPCR to identify the timing and duration of the spawning periods, as well as the peak of the spawning activity for the target species. In addition, we highlight that the use of a control species (i.e., quantification of the eDNA signal of a fish that does not reproduce during the monitoring period) helps to clearly discriminate the eDNA signal associated to the spawning activity. The filtration of 2 L of subsurface water was shown to be effective for the monitoring of spawning activity; however, using discrete sampling strategy we observed a spatial variability in the eDNA signal. For future implementation, we recommend (i) using an integrative sampling strategy to smooth the local variability of the eDNA signal, and (ii) to cover a sampling period long enough to clearly distinguish the spawning signal from the basal signal of the target species. These results show that we reached an operational level to use these non-invasive eDNA approaches to monitor the spawning periods of these two fish species in large lakes.
Fish eDNA metabarcoding is usually performed from filtered water samples. The volume of filtered water depends on the study scope and can rapidly become time consuming according to the number of ...samples that have to be processed. To avoid time allocated to filtration, passive DNA samplers have been used to recover fish eDNA from marine environments faster. In freshwater ecosystems, aquatic biofilms were used to catch eDNA from macroinvertebrates. Here, we test the capacity of aquatic biofilms to entrap fish eDNA in a large lake and, therefore, the possibility to perform fish eDNA metabarcoding from this matrix compared to the traditional fish eDNA approach from filtered water samples. Methodological aspects of the use of aquatic biofilms for fish eDNA metabarcoding (e.g. PCR replicates, biological replicates, bioinformatics pipeline, reference database and taxonomic assignment) were validated against a mock community. When using biofilms from habitats sheltered from wind and waves, biofilm and water approach provided similar inventories. Richness and diversity were comparable between both approaches. Approaches differed only for rare taxa. Our results illustrate the capacity of aquatic biofilms to act as passive eDNA samplers of fish eDNA and, therefore, the possibility to use biofilms to monitor fish communities efficiently from biofilms. Furthermore, our results open up avenues of research to study a diversity of biological groups (among which bioindicators as diatoms, macroinvertebrates and fish) from eDNA isolated from a single environmental matrix reducing sampling efforts, analysis time and costs.
The taxonomic identification of organisms based on the amplification of specific genetic markers (metabarcoding) implicitly requires adequate discriminatory information and taxonomic coverage of ...environmental DNA sequences in taxonomic databases. These requirements were quantitatively examined by comparing the determination of cyanobacteria and microalgae obtained by metabarcoding and light microscopy. We used planktic and biofilm samples collected in 37 lakes and 22 rivers across the Alpine region. We focused on two of the most used and best represented genetic markers in the reference databases, namely the 16S rRNA and 18S rRNA genes. A sequence gap analysis using blastn showed that, in the identity range of 99–100%, approximately 30% (plankton) and 60% (biofilm) of the sequences did not find any close counterpart in the reference databases (NCBI GenBank). Similarly, a taxonomic gap analysis showed that approximately 50% of the cyanobacterial and eukaryotic microalgal species identified by light microscopy were not represented in the reference databases. In both cases, the magnitude of the gaps differed between the major taxonomic groups. Even considering the species determined under the microscope and represented in the reference databases, 22% and 26% were still not included in the results obtained by the blastn at percentage levels of identity ≥95% and ≥97%, respectively. The main causes were the absence of matching sequences due to amplification and/or sequencing failure and potential misidentification in the microscopy step. Our results quantitatively demonstrated that in metabarcoding the main obstacles in the classification of 16S rRNA and 18S rRNA sequences and interpretation of high-throughput sequencing biomonitoring data were due to the existence of important gaps in the taxonomic completeness of the reference databases and the short length of reads. The study focused on the Alpine region, but the extent of the gaps could be much greater in other less investigated geographic areas.
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•Metabarcoding needs discriminating information in sequences and database coverage.•Requirements were tested by analyzing 16S-18S rRNA genes in 59 Alpine water bodies.•A high % of sequences and species is not included in the molecular databases.•The extent of sequence and taxonomic gaps differed in the major taxonomic groups.•Incomplete coverage of taxonomic databases hinders the application of metabarcoding.
Anthropogenic pressures and more recently climatic change have increased the interest to study the impact of environmental changes on the key stages of fish life cycle. In lake Geneva, a deep ...peri-alpine lake, climate change and phosphorous level are known to have consequences on salmonid and percid populations, including key species for recreational and commercial fisheries, whose stocks are subject to significant fluctuations. To follow these stock variations, the spawning activity of European perch (
Perca fluviatilis
) and whitefish (
Coregonus lavaretus
) is monitored in this lake since several years using traditional methods, unfortunately mostly destructive or damaging (e.g. gillnetting and collection of fertilized eggs).
DNA isolated from the environment (eDNA) has been widely developed for the detection of specific species or whole biological communities, and this non-invasive method offers an alternative to conventional surveying tools. Until recently, the methods used for eDNA analysis (e.g. qPCR, metabarcoding) could be limited by their sensitivity, quantification limit or price, but the emergence of new methods, such as the droplet digital PCR (ddPCR), offers the possibility to quantify an absolute eDNA signal in a very sensitive way and at a lower cost.
Here, we show for the first time the applicability of an eDNA method to monitor the spawning activity of two fish species in a lake by using ddPCR.
During two spawning seasons for whitefish and one spawning season for European perch, water samples were collected every week from the subsurface, simultaneously to traditional monitoring sampling, and filtered through sterile cartridges. The eDNA was then extracted and analyzed using ddPCR, targeting the mitochondrial DNA of the two fish species.
The results demonstrate the efficiency of eDNA coupled with ddPCR to identify the timing and duration of the spawning periods, as well as the peak of the spawning activity for whitefish and European perch in Lake Geneva. This study shows that we have reached an operational level to use this non-invasive eDNA monitoring of the spawning activity of these fish species in lakes.
The analysis of environmental DNA (eDNA) by high throughput sequencing (HTS) is proving to be a promising tool for freshwater fish biodiversity assessment in Europe within the Water Framework ...Directive (WFD, 2000/60/EC), especially for large rivers and lakes where current fish monitoring techniques have known shortcomings. These new biomonitoring methods based on eDNA show several advantages compared to classical morphological methods. The sampling procedures are easier and cheaper and eDNA metabarcoding is non-invasive and very sensitive, allowing for the detection of traces of DNA. However, eDNA metabarcoding methods need careful standardization to make the results of different surveys comparable. The aim of the EU project Eco-AlpsWater is to test and validate molecular biodiversity monitoring tools for aquatic ecosystems (i.e., eDNA metabarcoding) to improve the traditional WFD monitoring approaches in Alpine waterbodies. To this end, an inter-calibration test was performed using fish mock community samples containing either tissue-extracted DNA, eDNA collected from aquaculture tanks and eDNA samples collected from Lake Bourget (France). Samples were analysed using a DNA metabarcoding approach, relying on the amplification and HTS of a 12S rDNA marker, in two separate laboratories, to evaluate if different laboratory and bioinformatic protocols can provide a reliable and comparable description of the fish communities in both mock and natural samples. Our results highlight good replicability of the molecular laboratory protocols for HTS and good amplification success of selected primers, providing essential information concerning the taxonomic resolution of the 12S mitochondrial marker in describing the Alpine fish communities. Interestingly, different concentrations of species DNA in the mock samples were well represented by the relative DNA reads abundance. These tests confirm the reproducibility of eDNA metabarcoding analyses for the biomonitoring of freshwater fish inhabiting Alpine and peri-Alpine lakes and rivers.
Environmental DNA (eDNA) based methods (Fig. 1) are proving to be a promising tool for freshwater fish biodiversity assessment in Europe within the Water Framework Directive (WFD, 2000/60/EC) ...especially for large rivers and lakes where current fish monitoring techniques have known shortcomings. Freshwater fish are actively involved in aquatic ecosystems functioning and diversity, contributing to the health, well-being and economy in every geographic realm. Unfortunately, many freshwater fish are experiencing critical population decline with risk of local or global extinction because of intense anthropogenic pressure. Within the EU project Eco-AlpsWater, advanced high throughput sequencing (HTS) techniques are used to improve the traditional WFD monitoring approaches by using environmental DNA (eDNA) collected in Alpine waterbodies. To evaluate the performance of the metabarcoding approach specifically designed to measure freshwater fish biodiversity in Alpine lakes and rivers, an intercalibration test was performed. This exercise forecasted the use of mock samples containing either tissue-extracted DNA of different target species or water collected from aquaculture tanks to mimic real environmental water sampling and processing. Moreover, three water samples collected in Lake Bourget (France) were used to compare the efficiency of taxonomic assignments in natural and mock community samples. Our results highlighted a good efficiency of the molecular laboratory protocols for HTS and a good amplification success of the selected primers, providing essential information concerning the taxonomic resolution of the 12S mitochondrial marker. As further confirmation, different concentration of species DNA in the mock samples were well represented by the relative read abundance. This preliminary test confirmed the applicability of eDNA metabarcoding analyses for the biomonitoring of freshwater fish inhabiting Alpine and perialpine lakes and rivers.
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