Discussion
eDNA detection
Prevention of false negatives is an issue that receives much attention
in monitoring freshwater biodiversity using environmental DNA (e.g.
Darling & Mahon 2011; Buxton et al., 2017). Therefore, a better
understanding of the limits of eDNA detection is essential. This study
shows that eDNA of live shrimps degrades faster in the presence of OM,
resulting in reduced amounts of detectable eDNA, especially when pH is
low, as might be found in peat bogs. We found the level of PCR
inhibition to be unaffected by pH or the presence of OM. Therefore,
detection of reduced amounts of eDNA when OM was present must be
explained by a decline in rate of decay and by a failure to sample eDNA
instead of by PCR inhibition. As spiked DNA degraded significantly
faster than eDNA we believe most eDNA detected in natural systems must
be contained inside cells or mitochondria. This is in line with findings
of Turner et al., (2015) who found that only a minor fraction of
carp eDNA to be extra-cellular. Dupray et al., (1997) reports
that heat-killed cells of Salmonella typhimurium persist in
seawater longer than purified DNA. Nielsen et al., (2007) show
that the residence time of bacterial DNA in soil is generally longer
when dead cells are used as DNA source compared to purified DNA.
In aquatic environments, DNA is known to degrade faster in the presence
of DNA-consuming microorganisms (Alvarez et al., 1996; Duprayet al., 1997). The longer persistence of cellular DNA can be
explained by the presence of cellular compounds such as cell membranes
that form a barrier against DNA consuming microorganisms and nucleases
in the environment (Dupray et al., 1997).
Humic acids can strongly adsorb DNA, probably by ligand binding,
hydrophobic interaction, aggregation or precipitation (e.g. Saekiet al., 2011), and eDNA therefore might have been adsorbed to
organic particles that were deposited at the bottom of the aquariums.
Stotzky (2000) found that DNA bound to humic acids and clay-humic acid
complexes becomes more resistant to degradation by DNases. However, as
we infer that most eDNA is cellular, these processes might have a minor
effect on eDNA contained in cells or mitochondria. Sampling of organic
material or sediments might increase the yield of target eDNA, though
PCR might be inhibited by organic acids in such cases. However, sampling
of organic material or sediments might result in detection of historical
eDNA, not representing the actual presence of target species (Olajoset al., 2018).
In a comparable experimental set-up to ours, using tanks, Buxtonet al., (2017) found the effect of pH on eDNA survival to be
insignificant (which is in line with our findings), but that sediment
has a strong effect. The authors conclude that especially “ponds with
organic sediment types—or sediments that become suspended easily—can
be a source of false negative results” (Buxton et al., 2017).
Remarkably, in our aquarium treatment B (high pH and no added OM), eDNA
could be detected more than six weeks later, whereas other studies found
that eDNA degrades beyond detection ability within two weeks (Dejeanet al., 2011; Thomsen et al., 2012a; Thomsen et
al., 2012b; Strickler et al., 2015; Eichmiller et al., (2016).
However, the eDNA concentrations in these aquariums were unnaturally
high, thus not reflecting a natural situation. This might have resulted
in relatively high amounts of detectable eDNA and probably lengthened
eDNA survival.
Several studies show a correlation between eDNA concentration and
population density (Maruyama et al., 2015; Wilcox et al.,2016; Baldigo et al., 2017). This study, as well as previous
studies (Strickler et al., 2015; Echmiller et al., 2016)
show that environmental conditions strongly affect eDNA concentration.
We therefore believe caution is warranted when using eDNA concentrations
as proxy for population density. Environmental conditions might
specifically affect eDNA concentrations on the sampling site. Therefore,
it is necessary to correct measured eDNA concentrations for local
environmental conditions such as pH and amount of OM.
Our study, as well as previous studies, focused on selected
environmental factors only and was conducted in an artificial ecosystem
(i.e. an aquarium) (Nielsen et al., 2007). Complex interactions
between eDNA degradation and additional factors such as the presence of
DNA-consuming microorganisms remain largely unknown, and future studies
should therefore include microbial activity as well. In addition,
species that occur in a wide range of habitats should be used to
investigate the relation between amount of detectable eDNA and other
environmental conditions in the field such as seasonality (de Souzaet al., 2016) or soil type (Buxton et al., 2017).