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).