Reliable eDNA detection and quantification of the European weather loach (Misgurnus fossilis)

Abstract

The European weather loach (Misgurnus fossilis) is a cryptic and poorly known fish species of high conservation concern. The species is experiencing dramatic population collapses across its native range to the point of regional extinction. Although environmental DNA (eDNA)‐based approaches offer clear advantages over conventional field methods for monitoring rare and endangered species, accurate detection and quantification remain difficult and quality assessment is often poorly incorporated. In this study, we developed and validated a novel digital droplet PCR (ddPCR) eDNA‐based method for reliable detection and quantification, which allows accurate monitoring of M. fossilis across a number of habitat types. A dilution experiment under laboratory conditions allowed the definition of the limit of detection (LOD) and the limit of quantification (LOQ), which were set at concentrations of 0.07 and 0.14 copies μl–1, respectively. A series of aquarium experiments revealed a significant and positive relationship between the number of individuals and the eDNA concentration measured. During a 3 year survey (2017–2019), we assessed 96 locations for the presence of M. fossilis in Flanders (Belgium). eDNA analyses on these samples highlighted 45% positive detections of the species. On the basis of the eDNA concentration per litre of water, only 12 sites appeared to harbour relatively dense populations. The other 31 sites gave a relatively weak positive signal that was typically situated below the LOQ. Combining sample‐specific estimates of effective DNA quantity (Qe) and conventional field sampling, we concluded that each of these weak positive sites still likely harboured the species and therefore they do not represent false positives. Further, only seven of the classified negative samples warrant additional sampling as our analyses identified a substantial risk of false‐negative detections (i.e., type II errors) at these locations. Finally, we illustrated that ddPCR outcompetes conventional qPCR analyses, especially when target DNA concentrations are critically low, which could be attributed to a reduced sensitivity of ddPCR to inhibition effects, higher sample concentrations being accommodated and higher sensitivity obtained.

Environmental DNA (eDNA) Monitoring of Noble Crayfish Astacus astacus in Lentic Environments Offers Reliable Presence-Absence Surveillance – But Fails to Predict Population Density

“…While we observed similar detection sensitivity for qPCR and ddPCR on DNA extracted from crayfish tissue, we had lower detection sensitivity for ddPCR compared to qPCR for the eDNA samples. This contrasts other studies where ddPCR appears to be more sensitive than qPCR for eDNA detection (e.g., Doi et al, 2015b;Hamaguchi et al, 2018;Mauvisseau et al, 2019a;Wood et al, 2019;Brys et al, 2020). In some eDNA samples we observed a reduced amplitude for the ddPCR indicating some inhibition.…”

“…It is commonly reported that ddPCR overcomes the challenges connected to inhibitory substances to a higher extent than qPCR, and displays both higher sensitivity and higher quantification accuracy (Dingle et al, 2013;Rački et al, 2014;Zhao et al, 2016), and several studies have highlighted the benefits of ddPCR for eDNA detection (e.g., Doi et al, 2015a;Hunter et al, 2017;Hamaguchi et al, 2018;Mauvisseau et al, 2019a;Brys et al, 2020). However, while ddPCR offers absolute quantification, the variation in estimated copy numbers increases at low numbers, therefore the LOQ of ddPCR is often within the same range as LOQ for qPCR (Dobnik et al, 2015).…”

“…Quantitative real-time PCR (qPCR) or digital droplet PCR (ddPCR) are commonly used for species-specific detection (Rusch et al, 2018;Capo et al, 2019;Mauvisseau et al, 2019a;, and for relative or absolute quantification of target DNA, respectively (Demeke and Dobnik, 2018;Quan et al, 2018), while high-throughput sequencing and metagenomics is used to study whole communities (Thomsen et al, 2012;Hänfling et al, 2016;McElroy et al, 2020). While qPCR is currently the most common platform to analyze eDNA samples using species-specific assays, recent studies suggest that the detection rate of eDNA in environmental samples is higher when using ddPCR compared to qPCR technology (Doi et al, 2015a;Mauvisseau et al, 2019a;Wood et al, 2019;Brys et al, 2020). With ddPCR there is no need for standard curves and ddPCR allows for absolute quantification, even at low levels of DNA copies.…”

“…With ddPCR there is no need for standard curves and ddPCR allows for absolute quantification, even at low levels of DNA copies. Additionally, ddPCR appears to be more robust against PCR inhibition (Doi et al, 2015a;McKee et al, 2015;Mauvisseau et al, 2019a;Brys et al, 2020).…”

Invasieve Exoten in Vlaanderen: toestand en beleidsaanbevelingen: achtergrondrapport bij het Natuurrapport 2020

“…Voor een algemene opvolging van heel wat bekende invasieve probleemsoorten is doelgerichte monitoring nodig op risicolocaties voor introductie. Vaak zijn hiervoor gespecialiseerde methoden vereist, zoals milieu-DNA (stierkikkers, rivierkreeften) (Brys et al 2019(Brys et al , 2020, wildcamera's (wasberen, muntjakken) of specifieke vangtuigen. Er zou een meer actief, officieel surveillancesysteem opgezet moeten worden dat in de professionele monitoring netwerken (zoals de programma's in het kader van Natura 2000 of de KRW) verankerd is.…”