The Application of PMA (Propidium Monoazide) to Different Target Sequence Lengths of Zebrafish eDNA: A New Approach Aimed Toward Improving Environmental DNA Ecology and Biological Surveillance

Abstract

Environmental DNA (eDNA) analysis has enabled more sensitive and efficient biological monitoring than traditional methods. However, since the target species is not directly observed, interpretation of results cannot preclude process Type I errors. Specifically, there may be a spatial or temporal gap between the target eDNA and the eDNA source in the sampled area. Moreover, eDNA surveillance lacks the ability to distinguish whether eDNA originated from a living or non-living source. This kind of Type I error is difficult to control for, in part, because the relationship between the state of eDNA (i.e., intracellular or extracellular) and the degradation rate is still unclear. Here, we applied PMA (Propidium monoazide) to eDNA analysis which enabled us to differentiate “intact cells” from “disrupted cells.” PMA is a dye that has a high affinity for double-stranded DNA and forms a covalent bond with double-stranded DNA and inhibits amplification of the bonded DNA molecules by PCR. Since PMA is impermeable to the cell membrane, DNA protected by an intact cell membrane can be selectively detected. In this study, we investigated the workability of PMA on vertebrate eDNA using zebrafish, Danio rerio. Aquarium water was incubated for 1 week to monitor the eDNA degradation process of both intracellular and extracellular eDNA. We developed ten species-specific quantitative PCR assays for D. rerio with different amplification lengths that enabled independent quantification of total eDNA (sum of intracellular and extracellular eDNA, commonly measured in other studies) and intracellular eDNA (DNA in intact cells) and allow for analyses of sequence length-dependent eDNA degradation in combination with PMA. We confirmed that PMA is effective at differentiating “intact” and “disrupted” fish cells. We found that total eDNA and intracellular eDNA have different degradation processes that are dependent on the length of target sequence. For future conservation efforts using eDNA analyses, it is necessary to increase the reliability of the analysis results. The research presented here provides new analysis tools that expand our understanding of the ecology of eDNA, so that more accurate and reliable conclusions can be determined.

Linking the state of environmental DNA to its application for biomonitoring and stock assessment: Targeting mitochondrial/nuclear genes, and different DNA fragment lengths and particle sizes

“…For example, viability PCR, using a fluorescence dye that does not penetrate intact cell membranes, can selectively detect viable cells (Carini et al, 2016). Hirohara et al (2021) recently applied viability PCR using propidium monoazide (PMA) for zebrafish eDNA detection and suggested that the degradation processes might be different between total eDNA (measured by qPCR) and intra‐cellular eDNA (measured by qPCR following PMA treatment). Alternatively, environmental RNA (eRNA), which is considered to be less stable than eDNA, might provide finer spatiotemporal and fresher inferences of target organisms (Cristescu, 2019; Marshall et al, 2021; Tsuri et al, 2021).…”

Fine‐tuning the performance of abundance estimation based on environmental DNA (eDNA) focusing on eDNA particle size and marker length

“…The zebrafish eDNA concentration in the water sample was estimated by quantifying the copy number of mitochondrial genes using the StepOnePlus Real-Time PCR system (Thermo Fisher Scientific). We used the primer/probe sets to amplify the different lengths of mitochondrial genes, including cytochrome b, tRNA-Glu, and ND6 regions of the target species (132, 430, 715, and 1021 bp) developed by Hirohara et al (2021). Each 15 μl of TaqMan reaction mixture contained a 2 μl DNA template, a final 900 nM concentration of each forward and reverse primer, and 125 nM of TaqMan probe in 1 × TaqPath qPCR Master Mix, CG (Thermo Fisher Scientific).…”

Using eDNA metabarcoding to establish targets for freshwater fish composition following river restoration

“…DNA extraction was performed using the DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany), basically following Miya et al (2016), with some modifications following Hirohara et al (2021).…”