Results
Controls and limit of
detection
The qPCR assay had typically a R2 of over 0.99 and an
efficiency of 70%. Although PCR efficiency was quite low we could
amplify even single molecules, indicating that the assay was rather
sensitive. The negative PCR control reactions did not result in any
amplification. However, we amplified low levels of eDNA from the samples
collected prior to inoculation with DNA sources and in the samples
collected from the control aquariums. For all samples analyzed of the
control aquariums and samples collected prior to inoculation, we plotted
a cumulative density function in R (R core team, 2014) on the CT-values.
From this we estimated the 5% percentile to be 45.5 (Figure S1). Based
on this result, we estimated our detection limit to be 45 cycles, which,
given our standards corresponds to 8221 molecules of DNA.
CT-values
exceeding 45 in the non-control measurements were subsequently set to 45
as such values are likely to be caused by low levels of contamination or
by double-stranded non-target DNA, such as primer dimers. Based on the
melting curve of the positive controls, the typical melting temperature
of target DNA (e.g. the temperature that the highest amount of DNA
products dissociates and becomes single-stranded) was inferred to be in
the range of 75.5 and 77.5 ºC. We assumed reactions showing a melting
temperature outside this range to be non-target DNA such as primer
dimers.
Therefore,
we set their CT-values to 45.
eDNA accumulation in aquariums with live shrimps
Significantly less eDNA was accumulated in the aquariums to which
additional organic matter was added (P<0.05, Figures 1-2).
CT-values were on average 5.4 higher in the aquariums to which organic
matter was added, and on average 1.3 higher in the aquariums with low
pH. However, the effect of pH on eDNA accumulation was not significant
at the 5% level.
eDNA survival over time
For this analysis, we only used the aquariums that were monitored over
time until the eDNA concentration dropped below the limit of detection.
The spiked DNA was degraded beyond detectability within 2 to 60 hours,
whereas the eDNA from the live shrimps was degraded in 0 to 1680 hours.
We performed a survival analysis with the Cox’s proportional hazards
model to estimate how the treatments affected the time needed for eDNA
to degrade beyond the detection limit (45 cycles) (Therneau, 2014;
Therneau et al., 2000). We used OM, pH and DNA source as
treatment groups to estimate the effect of the treatments. eDNA degraded
significantly faster in the treatments in which OM was added (p=0.003)
whereas the pH did not significantly affect eDNA degradation (p=0.360).
The survival analysis shows that spiked DNA was degraded significantly
faster than the eDNA released by the living shrimps (p=0.023). The
largest difference in eDNA survival versus survival of the spiked DNA
was found in the treatment with high pH and no OM. The spiked DNA
(treatment F) was degraded between 0 and 12 hours whereas it took
between 1008 and 1680 hour for the eDNA to degrade (treatment B).