4 Influential factors of eDNA detection

Lindahl showed that the detection and quantification of DNA in specific freshwater animal species depends on DNA release and degradation rates. eDNA release is related to population density and organism size, and DNA degradation is determined by a simple relationship between microbial or enzymatic attack and spontaneous chemical reactions such as hydrolysis and oxidation (Lindahl, 1993). Conceptually, three processes determine the detection of DNA in environmental samples: production, dissipation and degradation.

4.1 Production of eDNA

For aquatic eDNA, production, the rate at which DNA is released in water, is a highly variable function, influenced by population density and species-specific characteristics, such as individual size and metabolic rate (Strickler et al., 2015). Thomsen conducted Pearson correlation analysis of population size and the mean number of DNA molecules in ponds for two amphibian species (Pelobates fuscusand Triturus cristatus ) and found that DNA release increased with population density and animal size (P. F. Thomsen, J. Kielgast, L. L. Iversen, C. Wiuf, et al., 2012). In order to examine the effect of different growth stages of amphibians on the efficiency of eDNA detection, Everts et al. compared the eDNA emission rates of tadpoles and juveniles of American bullfrogs (Rana catesbeiana ) and found that the average individual eDNA emission rate of tadpoles was not significantly different from that of juveniles, while the average emission rate per unit biomass of tadpoles was significantly lower than that of juveniles (Teun Everts et al., 2021).

4.2 Dissipation of eDNA

The dissipation pattern of eDNA was shown to vary between species and experimental conditions. In order to obtain more accurate results for amphibian detection by eDNA methods, Brys et al. estimated the temporal and spatial dissipation of eDNA by placing a group of different fish and amphibians in a cage on one side of a pond (thereby creating eDNA emission sites) and assessing the dissipation of eDNA with distance by collecting water samples at different distances from the cage. Detection rate of the locally released eDNA and the standardized index of relative eDNA abundance of the cage species decreased significantly with distance within a radius of 5–10 m from the cage. After removing the cage community for one week, no DNA of any species could be detected in the study system, indicating that the eDNA had degraded below the detection threshold within that period (Brys et al., 2021). Dejean et al.’s study found that eDNA from American bullfrog (Rana catesbeiana ) tadpoles was able to persist for 25 days under a laboratory microscope (Dejean et al., 2011). Thomsen et al. found that in experiments conducted in outdoor ponds, eDNA from larvae of the amphibiansPelobates fuscus and Triturus cristatus could be detected for 7–14 days (P. F. Thomsen, J. Kielgast, L. L. Iversen, C. Wiuf, et al., 2012).

4.3 Degradation of eDNA

Studies have shown that degradation of DNA in water is one of the main factors that reduce detectability (Barnes et al., 2014; Dejean et al., 2012; Pilliod et al., 2014). An understanding of the relevant environmental factors is therefore essential for controlling degradation rate and optimizing sampling strategy when using eDNA methods. DNA in water is broken down by chemical hydrolysis, primarily by exposure to acids or by enzymatic action. Microbial activities in water directly promote enzymatic hydrolysis by producing exogenous nucleases, which break down DNA into its components (Lindahl, 1993).
High temperatures accelerate the degradation of DNA (Corinaldesi et al., 2008; Okabe & Shimazu, 2007). When temperatures are very high (>50°C), direct denaturation can occur; however, most of the effects of high temperatures on DNA degradation arise indirectly, as moderately high temperatures stimulate microbial metabolism and exonuclease activity (Corinaldesi et al., 2008; Fu et al., 2012; Hofreiter et al., 2001; Pote et al., 2009; Zhu, 2006). Strong ultraviolet radiation can also enhance DNA degradation. This particularly applies to ultraviolet B (UV-B) light, as UV-B wavelengths are the most harmful to aquatic organisms among wavelengths most likely to occur in terrestrial sunlight (Diffey, 2002), causing photochemical damage to DNA and thus inhibiting DNA amplification (Hader et al., 2003; Ravanat et al., 2001). Acidic conditions catalyze the hydrolysis process that degrades DNA (Alaeddini et al., 2010; Lindahl, 1993), and thus eDNA is present for longer in samples with neutral or slightly alkaline pH (Lindahl, 1993).
A study by Pilliod et al. clearly quantified the influence of specific environmental factors on eDNA degradation. eDNA of Dicamptodon aterrimus can persist for 8 days in an outdoor container under ambient light and temperature conditions, at least 11 days under ambient temperature and low light conditions, and for at least 18 days under refrigeration without light (Pilliod et al., 2014). Similarly, to quantify the independent and interactive effects of environmental factors on eDNA persistence, Strickler et al. measured eDNA degradation rates at different levels of UV-B, pH, and temperature in a laboratory setting. The results showed that temperature had a strong effect on the amount of eDNA detected over time; the degradation rate at 5°C was significantly lower than that at 20°C and 35°C, indicating slower microbial growth at low temperatures, which may delay the microbial degradation process of eDNA. There was no significant difference in degradation rate between pH 4 and pH 7, but rates at both pH levels were higher than that at pH 10. There was no significant difference in the degradation rate of UV-B at 2, 25 and 50 KJ/m2/day, however, the degradation rate was faster at higher UV-B levels when the level was between 5 and 25 KJ/m2/day and the amount of residual eDNA was lower than 5%. This indicates that there is a nonlinear correlation between UV-B intensity and eDNA degradation (Strickler et al., 2015). Since geographical features such as latitude and altitude are the main determining factors of UV-B radiation (Godar, 2005), radiation effect on eDNA degradation is more significant in aquatic environments at high altitudes or near the equator. Although studies have shown that higher acidity (pH 4) accelerates the degradation of eDNA, McKee et al. successfully detected four endangered amphibian species (Ambystoma cingulatum , Ambystoma bishopi , Notophthalmus perstriatus and Lithobates capito ) in natural acidic wetlands (pH close to 4) of longleaf pine in the southeastern United States (A. M. McKee et al., 2015). Goldberg et al. also detected the endangered species Ambystoma bishopi andPseudacris ornata at the Eglin Air Force Base and demonstrated that a pH value as low as 4 would not degrade eDNA to an undetectable state (Goldberg et al., 2018).