Materials and method
Environmental conditions
Our experimental design is summarized in Table 1 and encompassed eight
treatments A-H. We filled each of 28 aquariums with 3.7 L water obtained
from a natural water system in The Netherlands (GPS: N 52 10.056, E 4
28.086). We kept the aquariums under controlled conditions in the
laboratory facility of Naturalis Biodiversity Center (Leiden, The
Netherlands). There was no gravel or substrate inside and the aquariums
were not aerated. The aquariums were placed on a laboratory bench and
the treatments were equally distributed over the space. We varied the pH
in the aquariums to either ‘high’ (above 8) or ‘low’ (below 5.7) and the
amount of OM to either 10 grams added, or none at all, resulting in four
treatments (see Table 1). OM content of the water was increased in the
following way: five grams of decaying leaf material of locally growing
plane trees (Platanus hispanica ) and five grams of leaf material
of locally growing European beech (Fagus sylvatica ) was added to
the water after sterilizing the leaves for 1 hour at 120 ºC to prevent
introduction of microorganisms that degrade eDNA. To lower the pH we
acidified the water using 3.7% HCl to a pH of 5.
During
the experiments we monitored the pH of the water (17 measurements,
Supplemental Table 1) and we added additional HCl if the pH exceeded
5.7. We refilled the aquariums to the original level, each time that
samples were collected for eDNA sampling of pH monitoring. The water in
the aquariums was kept at room temperature.
Inoculation of living
shrimps
Prior to inoculation with DNA sources, we took samples from all 28
aquariums to estimate the level of background DNA of Gammarus
pulex present. Four aquariums were not inoculated with any DNA source
and served as control.
In twelve aquariums, we added eight live shrimps (G. pulex ) in
the final stages of their development. Last instars were chosen to avoid
differences in molting and propagation between the aquariums. All
individuals used in this study were collected in Wageningen, the
Netherlands, from a single population in the wild (GPS: N 51 58.500, E 5
38.820). We removed dead shrimps and replaced them with live ones, and
we also removed newborn shrimps (for details see supplemental material:
STable2).
Spiking DNA
On the date that we removed the shrimps from the aquariums, we spiked
another twelve aquariums with 4.99 μg tissue-derived extracellular
genomic DNA of G. pulex . We measured DNA degradation in these
aquariums from two hours after spiking, measuring every 60 minutes. The
DNA used for spiking was extracted from tissue of G. pulex using
the Qiagen DNeasy Blood & Tissue following the Spin-column protocol. We
measured DNA concentration in the extracts using a Qubit 2.0 fluorometer
(Life Technologies).
Real-time quantitative
PCR
eDNA degradation was monitored using a CFX96TMReal-Time PCR System. We developed a species-specific qPCR primer set
using Geneious (PulexF1: ACGTAGACCTGGTATATCTATAGACC & PulexR1
CCGGCTAAAACAGGTAAGGA) to amplify a 98bp fragment of COI; we developed
another primer set using primer-BLAST of NCBI ((Ye et al., 2012)
(PulexF2: GGAGCTTGGGCTAGTGTTGT and PulexR2: CGTGAGCGGTGACTAATGACG) to
amplify an 118bp fragment of COI. Both primer combinations worked well,
but we selected primer pair PulexF1 & PulexR1 to do the experiment. We
checked the specificity of both primers in silico using
primer-BLAST (2013/02/28) with the setting that unintended targets
should have at least two mismatches within the last five base pairs at
the 3’ end for one of the primers. Primer-BLAST only showed hits of
indigenous organisms except for Gammarus duebeni . However, in the
case of the primer pair PulexF1 & PulexR1 a total of seven mismatches
was found. Furthermore, G. duebeni does not occur in the region
and occurs in a habitat type different from that at the location where
we obtained aquarium water.
eDNA extraction For extracting eDNA, we added 15 mL of water samples to 1.5 mL of 3M
sodium acetate and 33mL absolute ethanol and stored it at -20 ºC
(following Ficetola et al., (2008)). We centrifuged the mixture
(9400g, 35 min, 6ºC) and discarded the supernatant. To extract DNA from
the pellets, we used the Qiagen DNeasy Blood & Tissue kit (spin-column
protocol) after Thomsen et al., (2012a); Thomsen et al.,(2012b). Quantitative real-time PCR (qPCR) was performed in a total
volume of 20 µL using 10 µL GoTaq PCR Master Mix 2X (Promega), 0.4 µL of
both primers, 5.2 µL nuclease-free water and 4 µL template. We performed
PCRs in 96-well plates and included in each plate at least one negative
and one positive PCR control reaction (both in triplicate).
eDNA samplingWe sampled eDNA in the aquariums 28 days after they had been inoculated
with live shrimps to estimate the amount of eDNA that had been
accumulated. Thereafter, the shrimps were removed. To estimate the
survival of eDNA, samples were collected after 12, 24, 36, 48, 60, 72,
96, 168, 288, 504, 1008 and 1680 hours. We stopped sampling when the
average Ct-value of a sample exceeded 47 (see below).
Avoiding false positives
In this study, we took several measures to avoid false positives (i.e.
detecting eDNA when no animals were around). For detection of
invertebrates in field samples, the use of specific-binding probes is
paramount for reliably detecting target organisms. Even when the
concentration is extremely low, this approach can result in more
sensitive and specific detection of target DNA (Schultz and Lanze, 2015;
Goldberg et al., 2016). However, because the concentration of
eDNA in our controlled aquariums was relatively high we were able to use
a less sensitive, low-cost approach including GoTaq qPCR 2X Master Mix
in a real-time quantitative PCR assay, which contained BRYT Green, a
fluorescent dye that binds to double-stranded DNA. Since BRYT Green dye
binds to all double-stranded DNA, the presence of double stranded
non-target DNA, such as primer dimers, can also result in a fluorescent
signal. Ct-values were converted to numbers of molecules based on the
principle that 2[CtStandard –CtSample] is the fold difference in concentration of
sample and standard used. Standards (i.e. series of increasing known
concentrations) were made for each PCR plate and resulting Ct-values
plotted against the 10log(number of molecules). Linear regression
analysis of the average across plates then enabled calibrating the
standards and calculating numbers of molecules in the aquarium samples.
The detection limit was thereafter determined based on sample
concentrations collected from control aquariums, and the from all other
aquariums prior to inoculation (see also supplementary figure S1).
Defining the amount of detectable eDNAEach aquarium was sampled twice at each sampling time. Three water
samples were collected from the aquariums with live shrimps just before
they were removed from the aquariums, to be able to accurately determine
the accumulation of eDNA in de aquariums. In 12 samples the DNA pellet
did not form properly during extraction, in which cases only one sample
was analyzed.
Quantifying qPCR
inhibition
We quantified the amount of PCR inhibition in the samples (N=36) that
were collected from the aquariums containing shrimps just before they
were removed from aquariums. We did this by performing an inhibition
qPCR test (see details below). We repeated this in the samples obtained
from the spiked aquariums just after spiking (N=23) and in the samples
collected from the control aquariums that were obtained at the same time
(N=8). The qPCR reactions were spiked with an artificial fragment of DNA
(CGGAGGTGCACTTACAGATAGAGTCACATGTCGTGTCTAACGCGCAGCAGTAGTGTCTGAACACGAGTCCTTCC)
cloned into an pUC57 plasmid. The primers ART3-F
(CGGAGGTGCACTTACAGATAGAG) and ART3-R (GGAAGGACTCGTGTTCAGACA) were used
to amplify the fragment. For each sample, three qPCR reactions were
performed containing 33, 333 and 3333 molecules of the artificial DNA
fragment.
We performed the inhibition qPCRs in a total volume of 20 µL using 10 µL
GoTaq PCR Master Mix 2X (Promega), 0.4 µL of both primers, 4.2 µL
nuclease-free water, 4 µL template (either aquarium water or
nuclease-free distilled water) and 1 µL containing the artificial DNA
molecules. The cycling conditions were identical to those used for
detection of the shrimp DNA. A standard curve was generated using each
DNA concentration in triplicate, in which nuclease-free distilled water
was added instead of aquarium sample. We assessed response variable
CT-values of the control, shrimp and spiked data and explanatory
variable CT-values of the standard deviated from slope 1 in order to
assess the presence of inhibition. Therefore, linear mixed effect models
were used (see below under Controls and limit of detection ). The
R2 and efficiency of the qPCR assay were calculated
based on a standard containing 10, 100, 1000, 10000 target-molecules
(results not shown).
Statistics
In general, best-fitting models were selected with Akaike’s Information
Criterion corrected for small sample sizes (AICc, see eqn. 1):
AICc=2k −2Log(L )+(2k (k +1))/(n −k −1)
(eqn.1)
with Log denoting the natural logarithm, L the likelihood of the
model, k the number of estimated parameters in the model, andn the sample size (Bolker 2008). The minimum AICc value indicates
the best-fitting model. Model fits are evaluated with respect to the
AICc-difference (ΔAICc) between the considered model and the best model.
Models within the interval ΔAICc < 2 are considered equivalent
(Bolker 2008). In this set of models, Ockham’s razor (parsimony
criterion) was used to choose the best model, containing the smallest
number of parameters. We used Fisher’s least square difference (LSD)
test with Bonferroni correction from the agricolae R library (Mendiburu,
2016) to test for differences in the amount of eDNA accumulated in the
aquariums with live shrimps.