Droplet digital PCR assays for the quantification of brown trout (Salmo trutta) and Arctic char (Salvelinus alpinus) from environmental DNA collected in the water of mountain lakes

Abstract

Classical methods for estimating the abundance of fish populations are often both expensive, time-consuming and destructive. Analyses of the environmental DNA (eDNA) present in water samples could alleviate such constraints. Here, we developed protocols to detect and quantify brown trout (Salmo trutta) and Arctic char (Salvelinus alpinus) populations by applying the droplet digital PCR (ddPCR) method to eDNA molecules extracted from water samples collected in 28 Swedish mountain lakes. Overall, contemporary fish CPUE (catch per unit effort) estimates from standardized survey gill nettings were not correlated to eDNA concentrations for either of the species. In addition, the measured environmental variables (e.g. dissolved organic carbon concentrations, temperature, and pH) appear to not influence water eDNA concentrations of the studied fish species. Detection probabilities via eDNA analysis showed moderate success (less than 70% for both species) while the presence of eDNA from Arctic char (in six lakes) and brown trout (in one lake) was also indicated in lakes where the species were not detected with the gillnetting method. Such findings highlight the limits of one or both methods to reliably detect fish species presence in natural systems. Additional analysis showed that the filtration of water samples through 1.2 μm glass fiber filters and 0.45 μm mixed cellulose ester filters was more efficient in recovering DNA than using 0.22 μm enclosed polyethersulfone filters, probably due to differential efficiencies of DNA extraction. Altogether, this work showed the potentials and limits of the approach for the detection and the quantification of fish abundance in natural systems while providing new insights in the application of the ddPCR method applied to environmental DNA.

Lake Sedimentary DNA Research on Past Terrestrial and Aquatic Biodiversity: Overview and Recommendations

“…Alternatively, this bias can be utilized to quantify inhibition (e.g., [189]). Unlike qPCR, the recently developed ddPCR does not require standard curves and inhibition assays, due to pre-amplification partitioning of target templates into thousands of droplets of defined minute volumes where individual PCR reactions will take place (see [196,197] for application to modern environmental samples). For this method, the detection limit is very low, which may be advantageous given the issues that can be present in sedaDNA extracts.…”

Droplet digital PCR applied to environmental DNA, a promising method to estimate fish population abundance from humic‐rich aquatic ecosystems

“…Numerous studies have applied molecular methods to natural water samples to estimate the abundance, species composition, and diversity of fish communities (Evans & Lamberti, 2017; Hansen, Bekkevold, Clausen, & Nielsen, 2018; Wilcox, Carim, et al, 2018). However, results are not always straightforward and current knowledge highlights both the potentials and limits of eDNA methods to quantify the abundance of fish populations (e.g., Capo, Spong, Norman, et al, 2019; Levi et al., 2019; Nevers et al., 2018; Wilcox et al., 2016; Wilcox, Young, et al, 2018; Yates, Fraser, & Derry, 2019).…”

“…While many studies used the quantitative PCR method (qPCR) to quantify fish population abundance, the droplet digital PCR (ddPCR) method may be even more suitable for fish eDNA abundance estimation. This method has proved to be powerful for quantifying absolute numbers of DNA sequences, even at very low concentrations such as in aquatic systems with low population abundance (e.g., Capo, Spong, Norman, et al, 2019; Doi, Takahara, et al, 2015; Doi, Uchii, et al, 2015; Hunter et al., 2017). Moreover, the effect of humic substances acting as PCR inhibitors may be reduced by the specific procedure of partitioning a high number of target droplets—because of lowered co‐occurrence of humic compounds and DNA molecules in droplets—thereby reducing biases related to PCR inhibition (Hoshino & Inagaki, 2012).…”

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

“…Thus, from a water sample, it is possible to detect specific species or even whole communities (Deiner et al, 2017;Bylemans et al, 2019;McElroy et al, 2020). 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).…”