Quantitative monitoring of diverse fish communities on a large scale combining eDNA metabarcoding and qPCR

Abstract

eDNA metabarcoding is an effective method for studying fish communities but allows only an estimation of relative species abundance (density / biomass). Here, we combine metabarcoding with an estimation of the total abundance of eDNA amplified by our universal marker (teleo) using a qPCR approach to infer the absolute abundance of fish species. We carried out a 2850 km eDNA survey within the Danube catchment using a spatial integrative sampling protocol coupled with traditional electrofishing for fish biomass and density estimation. Total fish eDNA concentrations and total fish abundance were highly correlated. The correlation between eDNA concentrations per taxon and absolute specific abundance was of comparable strength when all sites were pooled and remained significant when the sites were considered separately. Furthermore, a non-linear mixed model showed that species richness was underestimated when the amount of teleo-DNA extracted from a sample was below a threshold of 0.65.106 copies of eDNA. This result, combined with the decrease in teleo-DNA concentration by several orders of magnitude with river size, highlights the need to increase sampling effort in large rivers. Our results show a comprehensive description of longitudinal changes in fish communities and underline our combined metabarcoding/qPCR approach for biomonitoring and bioassessment surveys when a rough estimate of absolute species abundance is sufficient.

Sturgeons in large rivers: detecting the near-extinct needles in a haystack via eDNA metabarcoding from water samples

“…1 in Pont et al (2021); and in Suppl. Figure 1 in Pont et al (2022). This value was comparable to the total number of 71 species captured in the TEF survey conducted during the same period (Bammer et al 2021).…”

“…Sequences shorter than 20 bp, or with fewer than 10 (or labeled "internal" by the OBICLEAN program) occurrences were excluded. The taxonomic assignment of molecular operational taxonomical units was performed using the ECOTAG program, based on a local "Sturgeon" reference database (see later), the "Danubian" database, obtained from (Pont et al 2022), the database obtained from Valentini et al (2016), and the sequences extracted from the release 142 (standard sequences) of the ENA database (http:// www. ebi.…”

Temperature moderates eDNA-biomass relationships in northern pike

“…Since the proportion of preferred habitat scales disproportionately with the square of bay area, and given that the eDNA is thoroughly mixed within the bay, this results in a dilution effect. Similar patterns of DNA dilution have been observed in rivers with elevated water discharges (Pont et al 2022).…”

Novel assay for endangered Hong Kong grouper (Epinephelus akaara) to assess eDNA shedding, decay, and population status

“…Specifically, the assay showed an amplification efficiency of 95.7%, which falls within the acceptable criteria range (90%-110%) of qPCR validation guideline set by Broeders et al (2014), and indicates that a high fraction of target DNAs are copied in each cycle. The curve-fitted model determined limit of detection (LOD) of this assay to be at 3.71 copies per reaction, which is at similar levels to other established eDNA assays on fish species, ranging between 2.19 to 22.1 copies per reaction (Kirtane et al, 2021;Klymus et al, 2020;Pont et al, 2023). On the other hand, the determined LOQ was 30 copies per reaction, which is an order of magnitude lower than some of the established fish eDNA assays at a range of 10 to 1000 copies per reaction (Kirtane et al, 2021), demonstrating the ability of the developed assay to detect target eDNA even at low concentration levels.…”