Cooperative Fish and Wildlife Research Units Program: all
Education, Research and Technical Assistance for Managing Our Natural Resources

Environmental DNA (eDNA) analysis has enabled more sensitive and efficient biologicalmonitoring than traditional methods. However, since the target species is not directlyobserved, interpretation of results cannot preclude process Type I errors. Specifically,there may be a spatial or temporal gap between the target eDNA and the eDNAsource in the sampled area. Moreover, eDNA surveillance lacks the ability to distinguishwhether eDNA originated from a living or non-living source. This kind of Type I error isdifficult 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 appliedPMA (Propidium monoazide) to eDNA analysis which enabled us to differentiate “intactcells” from “disrupted cells.” PMA is a dye that has a high affinity for double-strandedDNA and forms a covalent bond with double-stranded DNA and inhibits amplification ofthe 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 ofboth intracellular and extracellular eDNA.We developed ten species-specific quantitativePCR assays for D. rerio with different amplification lengths that enabled independentquantification of total eDNA (sum of intracellular and extracellular eDNA, commonlymeasured in other studies) and intracellular eDNA (DNA in intact cells) and allow foranalyses 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 effortsusing eDNA analyses, it is necessary to increase the reliability of the analysis results. Theresearch presented here provides new analysis tools that expand our understanding ofthe ecology of eDNA, so that more accurate and reliable conclusions can be determined.