Live and dead fish shed different amounts of intact cells: Implications for advancing environmental DNA methodologies

Environmental DNA (eDNA) analysis has significantly contributed to biodiversity monitoring due to its sampling simplicity and inherent high sensitivity. While the technique has matured considerably for species detection and characterising community compositions via metabarcoding, its applications in population and landscape genetics remain underexplored, primarily because these fields require the extraction of high-quality genetic information from samples. To facilitate further technical development in this domain, we investigated the availability of intact fish cells in water — a source expected to yield high-quality DNA templates — by performing a five-day replicated tank experiment using live and dead Rainbow Trout ( Oncorhynchus mykiss ) as experimental subjects for eDNA production. Propidium monoazide (PMA), a photoreactive intercalating dye which is permeable to disrupted biological membranes and thereby inhibits PCR amplification of DNA, was employed to differentially quantify PMA-resistant eDNA as a proxy for eDNA derived from intact versus compromised cells relative to the total eDNA pool, based on whether fish were living or dead. Our results showed significantly higher concentrations of total eDNA released by dead fish compared to live fish on Day 2 (12.9×) and Day 3 (7.4×). Furthermore, the amount of PMA-resistant eDNA was significantly elevated in the dead fish treatments on Day 2 (29.3×), Day 3 (31.3×) and Day 5 (5.2×). Collectively, these findings indicate that dead fish release significantly greater amount of both total eDNA and PMA-resistant eDNA, which likely represents eDNA from intact cells, into the surrounding environment compared to live fish. Our established methodology for assessing the availability of intact cells in water and the resulting quantitative data provide crucial insight for advancing environmental genetics approaches within the eDNA framework.