Detection of the endangered European weather loach (Misgurnus fossilis) via water and sediment samples: Testing multiple eDNA workflows

Abstract

The European weather loach ( Misgurnus fossilis ) is classified as highly endangered in several countries of Central Europe. Populations of M. fossilis are predominantly found in ditches with low water levels and thick sludge layers and are thus hard to detect using conventional fishing methods. Therefore, environmental DNA (eDNA) monitoring appears particularly relevant for this species. In previous studies, M. fossilis was surveyed following eDNA water sampling protocols, which were not optimized for this species. Therefore, we created two full factorial study designs to test six different eDNA workflows for sediment samples and twelve different workflows for water samples. We used qPCR to compare the threshold cycle ( C t ) values of the different workflows, which indicate the target DNA amount in the sample, and spectrophotometry to quantify and compare the total DNA amount inside the samples. We analyzed 96 water samples and 48 sediment samples from a pond with a known population of M. fossilis . We tested several method combinations for long‐term sample preservation, DNA capture, and DNA extraction. Additionally, we analyzed the DNA yield of samples from a ditch with a natural M. fossilis population monthly over one year to determine the optimal sampling period. Our results showed that the long‐term water preservation method commonly used for eDNA surveys of M. fossilis did not lead to optimal DNA yields, and we present a valid long‐term sample preservation alternative. A cost‐efficient high salt DNA extraction led to the highest target DNA yields and can be used for sediment and water samples. Furthermore, we were able to show that in a natural habitat of M. fossilis , total and target eDNA were higher between June and September, which implies that this period is favorable for eDNA sampling. Our results will help to improve the reliability of future eDNA surveys of M. fossilis .

Optimised protocol for the extraction of fish DNA from freshwater sediments

“…Furthermore, the widely used saturated phosphate buffer method for extracting only extracellular DNA (Taberlet et al, 2012) was the worst performing extraction method, resulting in low recovery of artificially spiked DNA compared to other methods tested in this study. Poor extraction efficiency using a phosphate buffer has been highlighted previously by Kusanke et al (2020), who found that its use resulted in significantly lower DNA yields of the European loach ( Misgurnus fossilis ) in sediment. A case study on historical lake sediments found that phosphate buffer extractions were consistently outperformed by the unmodified PowerSoil kit protocol when assessing sedDNA yields of various terrestrial animals including arthropods, eukaryotes and vertebrates (Capo et al, 2021).…”

“…Some previous studies have used similar sediment masses (3–10 g) to target fish DNA in both contemporary and historical sediments, although their rationale for choosing sediment mass in this range was not specifically discussed (Kuwae et al, 2020; Ogata et al, 2021; Sakata et al, 2020a, 2020b; Sales et al, 2019, 2021; Turner et al, 2015; Valdez‐Moreno et al, 2019). By contrast, other studies have used smaller amounts of sediment (<0.5 g) and successfully detected fish with either targeted or community approaches, also in a range of contemporary and historical sediments (Baldigo et al, 2017; Eichmiller et al, 2014; Kusanke et al, 2020; Matisoo‐Smith et al, 2008; Nelson‐Chorney et al, 2019; Olajos et al, 2018; Shaw et al, 2016; Stager et al, 2015). The reasons for the successful detection of fish sedDNA in smaller sediment masses remain unclear, yet there may be other factors (such as those discussed below) that influence the ability to extract and detect fish sedDNA.…”

“…By contrast, other studies have used smaller amounts of sediment (<0.5 g) and successfully detected fish with either targeted or community approaches, also in a range of contemporary and historical sediments (Baldigo et al, 2017;Eichmiller et al, 2014;Kusanke et al, 2020;Matisoo-Smith et al, 2008;Nelson-Chorney et al, 2019;Olajos et al, 2018;Shaw et al, 2016;Stager et al, 2015). The reasons for the successful detection of fish sedDNA in smaller sediment masses remain unclear, yet there may be other factors (such as those discussed below) that influence the ability to extract and detect fish sedDNA.…”

A Review of Environmental DNA Field and Laboratory Protocols Applied in Fish Ecology and Environmental Health

“…Sampling from the field is the experimental step most at risk of cross-contamination. According to previous studies, 10% bleach can be used to clean sampling appliances, including the ship or boat used, and mouth masks and disposable latex gloves should be worn by samplers to prevent DNA contamination (Sigsgaard et al, 2017;Muha et al, 2019;Kusanke et al, 2020;Riaz et al, 2020). Unmanned aerial vehicles (UAVs) have been used as a new technique to collect environmental samples for eDNA studies and avoid contamination (Doi et al, 2017).…”

Assessing Metropolitan Biodiversity Using Aquatic Environmental DNA Metabarcoding

“…In all of the cases mentioned, eDNA-based identification outperformed traditional detection, revealing targeted species' presence even at low eDNA molecule concentrations. Further development and validation of eDNA protocols has been conducted in recent years to further enhance the detection reliability of endangered taxa, additionally aiming to also provide information on quantities and abundances (Thomsen et al 2012;Harper et al 2018;Kusanke et al 2020). The further development of eDNA methodology has led to the implementation of eDNA-based biomonitoring of endangered taxa in standard national legislation, similarly to routine monitoring of great crested newts in the United Kingdom.…”