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Comparison of capture and storage methods for aqueous macrobial eDNA using an optimized extraction protocol: Advantage of enclosed filter

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1. Aqueous environmental DNA (eDNA) is an emerging efficient non-invasive tool for species inventory studies. To maximize performance of downstream quantitative PCR (qPCR) and next-generation sequencing (NGS) applications, quality and quantity of the starting material is crucial, calling for optimized capture, storage and extraction techniques of eDNA. Previous comparative studies for eDNA capture/storage have tested precipitation and 'open' filters. However, practical 'enclosed' filters which reduce unnecessary handling have not been included. Here, we fill this gap by comparing a filter capsule (Sterivex-GP polyethersulfone, pore size 0Á22 lm, hereafter called SX) with commonly used methods. 2. Our experimental setup , covering altogether 41 treatments combining capture by precipitation or filtration with different preservation techniques and storage times, sampled one single lake (and a fish-free control pond). We selected documented capture methods that have successfully targeted a wide range of fauna. The eDNA was extracted using an optimized protocol modified from the DNeasy Ò Blood & Tissue kit (Qiagen). We measured total eDNA concentrations and Cq-values (cycles used for DNA quantification by qPCR) to target specific mtDNA cytochrome b (cyt b) sequences in two local keystone fish species. 3. SX yielded higher amounts of total eDNA along with lower Cq-values than polycarbonate track-etched filters (PCTE), glass fibre filters (GF) or ethanol precipitation (EP). SX also generated lower Cq-values than cellulose nitrate filters (CN) for one of the target species. DNA integrity of SX samples did not decrease significantly after 2 weeks of storage in contrast to GF and PCTE. Adding preservative before storage improved SX results. 4. In conclusion, we recommend SX filters (originally designed for filtering microorganisms) as an efficient capture method for sampling macrobial eDNA. Ethanol or Longmire's buffer preservation of SX immediately after filtration is recommended. Preserved SX capsules may be stored at room temperature for at least 2 weeks without significant degradation. Reduced handling and less exposure to outside stress compared with other filters may contribute to better eDNA results. SX capsules are easily transported and enable eDNA sampling in remote and harsh field conditions as samples can be filtered/preserved on site.
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Comparison of capture and storage methods for aqueous
macrobial eDNA using an optimized extraction protocol:
advantage of enclosed filter
Johan Spens
1,2
, Alice R. Evans
1
, David Halfmaerten
3
, Steen W. Knudsen
1
, Mita E. Sengupta
4
,
SarahS.T.Mak
1
, Eva E. Sigsgaard
1
and Micaela Hellstr
om
1,5
*
1
Centre for GeoGenetics, Natural History Museum of Denmark, Øster Voldgade 5-7, 1350 Copenhagen K, Denmark;
2
Wildlife,
Fish and Environmental Studies, Swedish University of Agricultural Sciences, Skogsmarksgra
¨nd, 90183 Ume
a, Sweden;
3
Research Institute for Nature and Forest, Gaverstraat 4, 9500 Geraardsbergen, Belgium;
4
Department of Veterinary Disease
Biology Parasitology and Aquatic Diseases, Dyrlægevej 100, 1870 Frederiksberg C, Copenhagen, Denmark; and
5
Department
of Ecology, Environment and Plant Sciences, Stockholm University, 10691 Stockholm, Sweden
Summary
1. Aqueous environmental DNA (eDNA) is an emerging efficient non-invasive tool for species inventory studies.
To maximize performance of downstream quantitative PCR (qPCR) and next-generation sequencing (NGS)
applications, quality and quantity of the starting material is crucial, calling for optimized capture, storage and
extraction techniques of eDNA. Previous comparative studies for eDNA capture/storage have tested precipita-
tion and ‘open’ filters. However, practical ‘enclosed’ filters which reduce unnecessary handling have not been
included. Here, we fill this gap by comparing a filter capsule (Sterivex-GP polyethersulfone, pore size 022 lm,
hereafter called SX) with commonly used methods.
2. Our experimental set-up, covering altogether 41 treatments combining capture by precipitation or filtration
with different preservation techniques and storage times, sampled one single lake (and a fish-free control pond).
We selected documented capture methods that have successfully targeted a wide range of fauna. The eDNA was
extracted using an optimized protocol modified from the DNeasy
Ò
Blood & Tissue kit (Qiagen). We measured
total eDNA concentrations and Cq-values (cycles used for DNA quantification by qPCR) to target specific
mtDNA cytochrome b(cyt b) sequences in two local keystone fish species.
3. SX yielded higher amounts of total eDNA along with lower Cq-values than polycarbonate track-etched filters
(PCTE), glass fibre filters (GF) or ethanol precipitation (EP). SX also generated lower Cq-values than cellulose
nitrate filters (CN) for one of the target species. DNA integrity of SX samples did not decrease significantly after
2 weeks of storage in contrast to GF and PCTE. Adding preservative before storage improved SX results.
4. In conclusion, we recommend SX filters (originally designed for filtering micro-organisms) as an efficient cap-
ture method for sampling macrobial eDNA. Ethanol or Longmire’s buffer preservation of SX immediately after
filtration is recommended. Preserved SX capsules may be stored at room temperature for at least 2 weeks with-
out significant degradation. Reduced handling and less exposure to outside stress compared with other filters
may contribute to better eDNA results. SX capsules are easily transported and enable eDNA sampling in remote
and harsh field conditions as samples can be filtered/preserved on site.
Key-words: capsule, eDNA capture, environmental DNA, extraction, filter, monitoring, quantita-
tive PCR, species-specific detection, water sampling method
Introduction
The realization that DNA from macrobiota can be obtained
from environmental samples (environmental DNA, eDNA)
started with excrements (H
oss et al. 1992) and sediments
(Willerslev et al. 2003). Over the last decade, the potential of
aqueous eDNA to identify a wide range of plants and animals
from a small volume of water has been realized (Martellini,
Payment & Villemur 2005; Thomsen et al. 2012; Rees et al.
2014). Aqueous eDNA is an emerging increasingly sensitive
technique for revealing species distributions (e.g. Jane et al.
2015; Valentini et al. 2016), early detection of invasive species
(e.g. Smart et al. 2015; Simmons et al. 2016) and monitoring
rare and/or threatened species for conservation (e.g. Zhan
et al. 2013; McKee et al. 2015). Aqueous eDNA monitoring
provides possibilities to upscale species distribution surveys
considerably, because much less effort in time and resources
are required compared to conventional methods (Dejean et al.
2012; Davy, Kidd & Wilson 2015). Based on literature
*Correspondence author. E-mail: micaela.hellstrom@su.se
Joint first authors.
©2016 The Authors. Methods in Ecology and Evolution ©2016 British Ecological Society
Methods in Ecology and Evolution 2016 doi: 10.1111/2041-210X.12683
searches, we catalogue 49 studies successfully applying eDNA
from water samples to detect macro-organisms in aquatic
ecosystems, published between January 2005 and March 2015
(when this study was initiated; Table S1, Supporting Informa-
tion). To our knowledge, 39 additional empirical studies were
published since then, indicating a rapid rise of interest in this
research area (Table S2).
The field of eDNA is still evolving, and a consensus of cap-
ture, storage and extraction methods has not yet been reached
(Goldberg, Strickler & Pilliod 2015; Tables S1 and S2). In fact,
thediversityofmethodsisalmostashighasthenumberof
research groups investigating this fairly new field of research.
To ensure reliable results of downstream applications such as
quantitative PCR (qPCR) and next-generation sequencing
(NGS), the quantity and quality of the starting material is cru-
cial. From our eDNA laboratory experience, we find that a
modified easy-to-follow extraction protocol resulting in high
yields is needed. Based on eDNA studies published so far
(Tables S1 and S2), we identify three pre-PCR key issues that
hold opportunities for improvement: (i) capturing sufficient
quantities of eDNA as quite a few studies report low amounts
of captured total eDNA, (ii) effectively preserving eDNA sam-
ples before extraction and (iii) lowering contamination risks
from collection to extraction of eDNA.
Comparative studies on aqueous eDNA capture and storage
techniques (i.e. optimal ways of preserving the eDNA captured
on the filters until extraction; e.g. Renshaw et al. 2015) were
basedontheso-called‘openfilters’ (requiring handling, a filter
funnel and a vacuum pump; e.g. Liang & Keeley 2013; Turner
et al. 2014b) and ethanol precipitation (EP; e.g. Piaggio et al.
2014; Deiner et al. 2015). However, no enclosed filters were
included in previous comparative assays.
The Sterivex-GP capsule filter (SX), with a polyethersulfone
membrane, is a standard method for characterizing microbial
communities (Chestnut et al. 2014) and for removing patho-
gens from water as the organisms are captured on the filter
membranes. To our knowledge, only two published aqueous
eDNA studies have used this filter to detect aquatic macro-
organisms (fish detection: Keskin 2014; Bergman et al. 2016),
and the technique has been successful to detect a wide range of
aquatic macro-organisms in Denmark and Belgium (M.
Hellstr
om, M.E. Sengupta, S.W. Knudsen, D. Halfmarten.
unpublished, S1). The SX filter is enclosed in a capsule, which
reduces handling. A water sample can easily be filtered in the
field, saving time and facilitating fixation of the eDNA imme-
diately after capture. Additionally, downstream DNA extrac-
tion takes place within the filter capsules with no need for the
membrane to be removed or handled. We therefore test the
performance of SX compared to other more frequently used
eDNA capture methods (Table S1), under different storage
conditions, in an effort to address issues 13 above. To date,
there are no studies comparing SX to other capture methods
and multiple storage treatments. We aim to fill this gap, with
an experimental study comparing SX with four other capture
methods in a set-up with five typical storage treatments and
three different storage times (up to 2 weeks). The tested open
filter materials polycarbonate, cellulose nitrate and glass fibre
(GF) and the range of tested pore sizes (0206lm) are typical
of previous studies (Tables S1 and S2). We used an optimized
extraction protocol based on a commercial kit to increase
eDNA yields. To evaluate the usefulness of the SX and preser-
vation buffers in comparison with typically used methods
(Tables S1 and S2), we test the following H
0
hypotheses:
H
0
1. CAPTURE METHOD: SX is equally effective as
other tested eDNA capturing techniques in regard to DNA
quantity and quality measured as the total extracted eDNA
concentration [eDNA
tot
] and as Cq-values (quantification
cycles, sensu Bustin et al. 2009) from two species-specific
qPCR assays.
H
0
2a. STORAGE PRESERVATIVE: Storing filters with
a preservation buffer does not affect qPCR amplification
compared to immediate extraction or freezing at 20 °C
(no buffer added).
H
0
2b. STORAGE TIME: There is no significant difference
in eDNA quality over time between SX and the other tested
capturing techniques.
H
0
3. CONTAMINATION: There is no significant differ-
ence between SX and the other tested capture techniques in
occurence of false positives.
To test these hypotheses, we use an experimental set-up with
subsampling a single large homogenous sample of water from
a Danish lake. Subsamples are subjected to different eDNA
capture methods within the same day followed by different
storage treatments. A control site (fish-free pond) is sampled
using the same set-up. Each capture and storage treatment is
assessed using concentration of total eDNA as well as species-
specific qPCR assays targeting pike Esox lucius L. and perch
Perca fluviatilis L. By testing H
0
hypotheses (13), the multiple
opportunities for optimization of eDNA surveys held by the
use of SX may be empirically evaluated. Based on the results,
we suggest recommendations for improved capture, storage
and extraction to use for aqueous eDNA, taking remote and
harsh field conditions into consideration.
Materials and methods
STUDY SITES
We chose Gentofte Lake, Denmark (N557435°,E125348°), as the
study site and a fish-free pond in Copenhagen botanical garden as a
negative field control (N556875°,E125746°). Gentofte Lake (26 ha) is
an alkaline clear water (Appendix S2) harbouring a wide range of fish
species, including pike and perch.
WATER COLLECTION
We retrieved 130 L of water from Gentofte Lake on 17 March 2015.
The water (4 °C) was collected at c. 30 points along c. 100 m of shore-
line close to the outlet of the lake. Additionally, we collected 40 L of
water from the control pond on 21 March 2015. The water was
©2016 The Authors. Methods in Ecology and Evolution ©2016 British Ecological Society, Methods in Ecology and Evolution
2J. Spens et al.
collected in sterilized 5-L buckets which prior to sampling were soaked
in bleach (5%) for 10 min, and then rinsed with laboratory-grade etha-
nol (70%). The containers were soaked repeatedly in lake water at a
location away from the collection point. Nitrile gloves were used during
cleaning, collection and filtration.
CAPTURE AND STORAGE
We carried out 41 different treatment combinations of the water sample
intotal(Table 1,Fig.S1).Weusedfivecapturetechniques,fivestorage
methods and three time regimes. All treatments were performed in trip-
licate. Apart from an in-house modified SX procedure (see Fig. 1), the
capture and storage methods were based on published sources
(Table S1). The capture methods (hereafter referred to with their abbre-
viations in square brackets) were as follows: (i) ethanol precipitation
[EP] (Ficetola et al. 2008), (ii) mixed cellulose esters membrane filters
including cellulose nitrate and cellulose acetate [CN]; Advantec 47 mm
diameter 045 lm pore size (Toyo Roshi Kaisha, Ltd., Tokyo, Japan),
(iii) polycarbonate track-etched filters [PCTE]; Whatman Nucleopore
Membrane 47 mm diameter 02lmporesize(MerckKGaA,Darm-
stadt, Germany)], (iv) glass fibre [GF] membrane filters; Advantec
GA-55 47 mm diameter 06lm pore size (Toyo Roshi Kaisha, Ltd.,
Tokyo, Japan) and (v) sterivex-GP capsule filters [SX]; polyethersul-
fone 022 lm pore size with luer-lock outlet (Merck KGaA)]. Further
downstream, SX was divided into an extraction from the filter within
the capsule (SX
CAPSULE
), after removal of the storage buffer, and an
extraction from the removed preservation buffer within a centrifuge
tube (SX
TUBE
; see DNA extraction section below). The different stor-
age methods were as follows: (i) ethanol 99% 200 proof at room tem-
perature (RT), Molecular Biology Grade (Thermo Fisher Scientific
Inc.,Waltham,MA,USA);(ii)Longmire’s buffer at RT (Longmire’s;
Longmire, Maltbie & Baker 1997); (iii) RNAlater at RT (RNA Stabi-
lization Reagent; QIAGEN, Stockach, Germany); (iv) no buffer, fro-
zen at 20 °C; and (v) no buffer, refrigerated at 810 °C. The three
time regimes between filtration and extractions were (i) within 5 hours
(5 h), (ii) within 24 h and (iii) after 2 weeks. Each treatment (n=41)
was performed in triplicate. For each filter replicate, 1 L of lake water
was processed (0015 L for EP). For each capturestorage treatment,
we included one negative control without lake water. Additionally, 1 L
tapwaterwasrunthrougheachfilter(0015 L for EP) as a control to
detect potential contamination from the filtration facilities. For the
control pond, one sample per capturestorage treatment was processed
(n=23). We captured eDNA from 155 subsamples and negative con-
trols altogether. The water samples were filtered or ethanol-precipitated
by a team of 10 researchers and the replicates of each treatment started
at different times to avoid temporal bias of filtrations. Prior to DNA
capture, bench surfaces and all equipment were wiped with bleach
(5%) and laboratory-grade ethanol (70%). Prior to each collection of
subsamples, the water was mixed thoroughly in the 130-L container.
For the open membrane filter (GF, CN and PCTE), 1 L water samples
were vacuum-filtered (c. 1530 min) using Nalgene 250-mL sterile dis-
posable test filter funnels (Thermo Fisher Scientific Inc. USA). The fil-
ters were removed from the funnel with forceps and then placed in 5-
mL DNA LoBind
Ò
centrifuge tubes (Eppendorf AG, Hamburg, Ger-
many) that were either empty (if the time regime was 5 h or the storage
method was freezing) or contained preservation buffer. For all treat-
ments and downstream applications, Eppendorf DNA LoBind
Ò
tubes
were used in order to avoid up to 50% retention of DNA by the plastic,
which is a documented problem especially for short DNA fragments
(Gaillard & Strauss 1998; Ellison et al. 2006). For the SX filters, 1 L of
water was slowly (c. 10 min to avoid tearing of filters, following manu-
facturer’s recommendations) pushed through each filter capsule using a
prepacked sterile 50-mL luer-lock syringe. Remaining water in the SX
was removed by pushing air through the filter until dry, also using the
syringe. The outlet ends of the filters were closed with MoBio outlet
caps (MOBIO Laboratories, QIAGEN) and 2 mL preservation buffer
was pipetted to the inlet end using filter tips. The inlet ends were closed
with inlet caps (MOBIO Laboratories, QIAGEN) and both ends were
sealed with parafilm whereafter the capsules were inverted vigorously.
The frozen samples and the (5 h) and (24 h) EP samples were placed at
20 °C until extraction, while the non-treated samples (5 h) were
placed in a refrigerator and extracted directly after the filtering session.
Samples containing buffers were stored at RT until processed. The
(2 weeks) EP samples were frozen for 24 h prior to extraction to allow
for precipitation. In total, we processed 96135 L of water from the lake
(32 treatments 93replicates91L+3EPtreatments93 repli-
cates 90015 L) and 20045 L of water from the control pond (20
treatments 91 replicate 91L+3EPtreatments91 repli-
cates 90015 L; Table 1).
MOLECULAR LABORATORY CONDITIONS
DNA extractions and qPCR assays took place in the laboratories at
the Centre for GeoGenetics, University of Copenhagen, Denmark.
The facilities are designed for handling environmental samples requir-
ing the most stringent precautions to avoid contamination. Pre-PCR,
extraction and PCR facilities are located in separate designated rooms
with positive air pressure. Laboratory coats are changed between
rooms. Prior to any work in the laboratory, all surfaces are washed
with 5% bleach and 70% ethanol. After completing extractions
Table 1. Outline of the number of samples processed per capture and storage treatment (negative control pond in parentheses)
Capture Sum
Storage
Refrigerated
Frozen Ethanol Longmire’s RNAlater Frozen Ethanol Longmire’s RNAlater
5h 24h 2weeks
SX
CAPSULE
27 (5) 3 (1) 3 (1) 3 (1) 3 (1) 3 (1) 3 3 3 3
SX
TUBE
18 (3) 3 (1) 3 (1) 3 (1) 3 3 3
Cellulose nitrate 15 (5) 3 (1) (1) (1) (1) (1) 3 3 3 3
Glass fibre 27 (5) 3 (1) 3 (1) 3 (1) 3 (1) 3 (1) 3 3 3 3
Polycarbonate 27 (5) 3 (1) 3 (1) 3 (1) 3 (1) 3 (1) 3 3 3 3
Precipitation 9 (3) 3 3 (3) 3
Total 123 (26)
Sterivex, eDNA extraction within capsule (SX
CAPSULE
); Sterivex, eDNA extraction from buffer in tube outside capsule (SX
TUBE
).
©2016 The Authors. Methods in Ecology and Evolution ©2016 British Ecological Society, Methods in Ecology and Evolution
Testing enclosed filter for eDNA capture and storage 3
Fig. 1.
©2016 The Authors. Methods in Ecology and Evolution ©2016 British Ecological Society, Methods in Ecology and Evolution
4J. Spens et al.
involving guanidiumthiocyanate, surfaces are washed with 70% etha-
nol (to avoid reactions between chlorine in the bleach and guanidi-
umthiocyanate in two of the buffers provided with the Qiagen kit), 5%
bleach and then 70% ethanol. All extractions of eDNA took place in
laminar flow hoods which were UV-treated before and after extrac-
tions. Every night, the entire facilities are automatically UV-
treated for a 2-h period.
DNA EXTRACTION
We extracted the eDNA using the extraction protocol outlined in
Fig. 1 and Appendix S1. The SX filters containing preservation buffers
underwent two extractions, one extraction from the buffer and one
extraction within the filter capsule after it had been emptied of buffer
(hereafter referred to as SX
TUBE
and SX
CAPSULE
). Altogether, 179 (24
SX
TUBE
+155 (see ‘Capture and storage’ section above) samples from
the study lake and the control pond were extracted. We measured
[eDNA
tot
] in each extraction using a Qubit 1.0 fluorometer (Thermo
Fisher Scientific Inc.) applying the high-sensitivity assay for dsDNA
(Life Technologies, Carlsbad, CA, USA).
QUANTITATIVE PCR
For the qPCR assays (e.g. Wilcox et al. 2013), two species-specific Taq-
Man primers/probe sets were used targeting 84 and 89 base pair frag-
ments of the mitochondrial cytochrome b(cyt b)geneinpikeand
perch, respectively (Table S3). Species specificity of the assays was
tested on extracted DNA from non-target species (Table S3) using the
qPCR set-up described below. These non-target species did not gener-
ate any amplification signals. The optimal ratio of probe: primer con-
centration was tested prior to the study. The final PCR set-up to detect
the target species was as follows: pike 5lLtemplateDNA,125lL
TaqMan Environmental Master Mix 20 (Life Technologies), 3 lLfor-
ward primer (10 lM), 2 lL reverse primer (10 lM)and3lL probe
(25lM); and perch 5lLtemplateDNA,125lL TaqMan Environ-
mental Master Mix 20 (Life Technologies), 05lLforwardprimer
(10 lM), 25lL reverse primer (10 lM), 3 lL probe (25lM)and
15lL UV-treated laboratory-grade water. The TaqMan qPCRs were
performed on a Stratagene Mx3005P (Thermo Fisher Scientific Inc.)
using thermal cycling parameters of 50 °C(5 min),95 °C(10 min)fol-
lowedby50cyclesof95 °C(30s)and60°C (1 min). For each plate,
no-template controls (NTCs) and positive/negative tissue extracts were
run alongside the samples. All filtering and extraction negatives were
included in the qPCR assays. Additional qPCR replicates were run in
order to detect effects of freezing and thawing of the samples. To check
for PCR inhibition in the lake, separate qPCR assays for both species
following the protocols above were performed in a dilution series
(1:1,1:2,1:10and1:20)ofextractedDNAonfoursamplesrepli-
cated twice plus two positive and two negative controls to determine
any deviation of the amplification curves. The dilution series did not
indicate inhibition.
DATA ANALYSIS
To compare detection probability (i.e. diagnostic sensitivity) between
eDNA capture methods, the proportion of positive qPCR replicates
was calculated for each target species. Positive samples were analysed
Fig. 1. Flow chart illustrating the modified environmental DNA (eDNA) extraction protocol based on DNeasy Blood & Tissue Kit (QIAGEN,
Carlsbad, CA, USA). *) Capture: SX, Sterivex-GP polyethersulfone capsule filters, Note that SX
CAPSULE
and SX
TUBE
are treated as separate sam-
ples from step 2. CN, cellulose nitrate; PCTE, polycarbonate track-etched; GF, glass fibre filters; EP, ethanol precipitation. Storage: Frozen at
20 °C, Refrigerated are samples stored at 810 °C and processed within 5 h. Steps 926 see Appendix S1.
©2016 The Authors. Methods in Ecology and Evolution ©2016 British Ecological Society, Methods in Ecology and Evolution
Testing enclosed filter for eDNA capture and storage 5
using multivariate decision trees and univariate tests of ‘no-effect’ null
hypotheses. To explore the effect of capture and storage on qPCR Cq-
values, Chi-square Automatic Interaction Detector (CHAID) decision
tree was used. CHAID is a nonparametric tree-building method that
can handle multivariate categorically induced quantitative responses
(IBM Corp. (2013)). It defines optimal multiway splits and adjusts for
Bonferroni. The main advantage of this approach is to analyse a data
set all-in-one (rather than manually splitting the data into user-selected
subgroups and thereafter choosing and performing multiple tests). The
approach offers a number of other advantages including its ability to
handle categorical (ordered, nominal) data types well and to model
nonlinear relationships without having to specify apriorithe form of
the interactions. A CHAID tree produces an overview, grouping or sin-
gling out the factors that predict the variation in the response variable.
Categorical variables (capture method, storage treatment and storage
time) were used as model predictors, and Cq-value from qPCR was set
as the response target. Two trees were generated: the first targeting
perch and the second pike. Tree depth, that is the maximum number of
branching levels, was set to two (realized from ten 50/50 split valida-
tions) to reduce overfitting.
For a univariate test of H
0
(12a,b), first a Wilcoxon signed-rank test
forpairedsampleswasappliedtodeterminewhether[eDNA
tot
]and
Cq-values attained using SX
CAPSULE
differ significantly, from any of
the other tested capture methods (CN, GF, PCTE, EP and SX
TUBE
).
Secondly, SX, GF and PCTE filter results were tested for signs of
eDNA degradation over time, that is detecting any significant differ-
ence in Cq-values or [eDNA
tot
] between 24 h and 2 weeks of storage.
Wilcoxon signed-rank test was used as data exhibited non-normal dis-
tributions. Thirdly, guided by results from the CHAID trees, results
from SX
CAPSULE
stored in ethanol or Longmire’s were tested (Mann
Whitney) for differences in Cq-value against SX
CAPSULE
without
preservation buffer. The CN filter group was reduced, as the planned 1-
day storage treatment was omitted due to filtering time constraints.
The mean difference in Cq-value and associated 95% CI of all qPCR
replicates was calculated. All statistical analyses were performed using
SPSS IBM Corp. (2013).
Results
SPECIES DETECTION
Altogether 713 qPCR samples, including controls, were anal-
ysed. No samples were discarded. Perch and pike were both
detected in most of the qPCR runs from the study lake (314 of
365, Fig. 2). For both species, SX
TUBE
showed the highest
overall detection rate (95% perch and 96% pike) and EP the
lowest (89% perch and 56% pike; overall difference SX
TUBE
EP: Pearson v
2
(1, n=62) =69, Fisher’s exact P=002).
CAPTURE METHOD
A CHAID tree multivariate predictive model was successfully
generated from perch Cq-values. Capture method was the best
overall predictor of Cq-values, better than storage media or
storage time. In general, the lowest Cq-values were generated
from SX
CAPSULE
samplesincomparisonwithothercapture
methods (Fig. 3a). We validated the fundamental first-level
outcome from this multivariate model for perch with new data
in the build of a second CHAID tree, modelling pike Cq-values
(Fig. 3b). In this second variant, capture was also the best
predictor of Cq-values and SX
CAPSULE
tied with the CN and
GF filters in the lowest value category.
The fundamental first-level outcome of both the CHAID
tree multivariate predictive models was supported in a one-by-
one comparison of capture methods including both species and
all treatments. Overall, SX
CAPSULE
was more efficient than the
other capture methods apart from CN. SX
CAPSULE
yielded
significantly higher [eDNA
tot
] and lower Cq-values (Table 2).
SX samples contained up to 118 ng total eDNA lL
1
and
most SX
CAPSULE
amplified before 36 cycles (Fig. 4). [eDNA
tot
]
from the fish-free control pond showed a similar pattern, being
higher for CN and SX
CAPSULE
compared with GF and PCTE
(MannWhitney U=12, n
1
=n
2
=10, Fisher’s exact
P=0003), but with no Cq-values from qPCR as target species
were not present. Overall, capture method and [eDNA
tot
]were
fundamental predictors of Cq-values (Fig. 4).
STORAGE PRESERVATIVE
SX-specific storage results are singled out and illustrated in
Fig. 5. SX
TUBE
samples treated with RNAlater, a significant
predictorofpoorerCq-valuesintheCHAIDtrees,wereleast
successful. For SX
CAPSULE
, preservation in ethanol or Long-
mire buffer improved Cq-values for perch in comparison with
frozen, 5 h and preservation in RNAlater (Figs 3a and 6).
Also for both species pooled, these two buffers (ethanol or
Detection rate
50
100
SXCAPSULE CN GF PCTE EP
SXTUBE
PIKE
PERCH
%
n = 72 n = 44 n = 48 n = 97 n = 86 n = 18
Capture method
(0·22 µm)
1
(0·45 µm) (0·6 µm) (0·2 µm)
Fig. 2. Detection rate using quantitative PCR (qPCR; study lake).
Blue bars and clear bars show positive detections of perch and pike,
respectively. Pore size of filters within parentheses. SX
CAPSULE
,Steri-
vex, extraction within filter capsule; SX
TUBE
, Sterivex, extraction in
tube outside capsule from removed preservation buffer; CN, cellulose
nitrate; PCTE, polycarbonate track-etched; GF, glass fibre; EP, etha-
nol precipitation. Error bars represent standard errors; nindicates
number of trials pooling all replicatesforeachmethodandbothspecies
combined.
1
Deviating from protocol, 12 SX
CAPSULE
replicates were
over-vortexed and tested mainly negative. If these 12 over-vortexed
samples are omitted, the detection rate estimate for SX
CAPSULE
increases to 100% for perch and to 91% for pike.
©2016 The Authors. Methods in Ecology and Evolution ©2016 British Ecological Society, Methods in Ecology and Evolution
6J. Spens et al.
Longmire) in SX
CAPSULE
resulted in lower Cq-values com-
paredwithfrozenor5 h(MannWhitney Test U:35,n
1
=23,
n
2
=15, Z=41; P=4910
5
).
STORAGE TIME
Storage time in the second-level outcome from the first
CHAID tree was classified as a positively correlated predictor
of Cq-values for all capture methods apart from SX (Fig. 3a).
This was supported in a one-by-one comparison of capture
methods including both species and 24 h to 2 weeks
treatments (Table 3). Cq-values did not increase significantly
with time using SX, but did with GF and PCTE.
The mean difference between Cq-values of paired qPCR
replicates run within the same day was +0302SE.This
difference increased to +1302 SE when replicates run on
different days were included, indicating that freezing and thaw-
ing of eDNA once or twice between measurements decreased
DNA quality [Welch’s test t(1, 68) =71, n
1
=20, n
2
=80,
P=9910
10
]. To avoid introducing this error, only DNA
templates thawed for the first time were included when calcu-
lating average Cq-values for the samples.
SXCAPSULE PCTE
SXTUBE, CN, GF, EP
P = 0·02 F = 13·5 P = 0·002 F = 12·3 P = 0·001 F = 15·0
P<0·001 F = 20·2
CAPTURE METHOD
38·3
±0
·
7
35·2
±0
·
4
n = 11
n = 15
n = 35
n = 32
n = 13
n = 12
39·5
±0
·
3
36·8
+0·5
n = 67 n = 26
n = 25
n = 118
TIME
38·4
±0
·
4
41·2
±0
·
4
40·4
±0
·
4
40·1
±0
·
4
42·7
±0
·
6
39·3
±
0·3
Ethanol
or
Longmire
2 w
2 w
<24 h<24 h
TIME
Refrigerated,
Frozen or
RNAlater
STORAGE
SXCAPSULE, CN, GF SXTUBE, PCTE
Refrigerated, Frozen,
Ethanol or Longmire RNAlater
EP
P<0·001 F = 31·6
CAPTURE METHOD
n = 11
n = 26
38·1
±0
·
4
35·6
+0·2
n = 37 n = 5
n = 67
n = 109
37·2
±0
·
3
41·1
±0
·
8
40·1
±0
·
5
36·7
±
0·2
P = 0·001 F = 21·2
STORAGE
Perch
(a)
Pike
(b)
Fig. 3. Chi-square Automatic Interaction Detector decision trees relating three categorical variables (capture method, storage treatment and storage
time) as model predictors for Cq-values as response target (study lake). (a) Perch. Best predictor was capture method, followed by storage time, and
finally, storage treatment. (b) Pike. Best predictor was capture method followed by storage treatment. SX
CAPSULE
, Sterivex, extracted within capsule;
SX
TUBE
, Sterivex, extraction in tube outside capsule; CN, cellulose nitrate; GF, glass fibre; PCTE, polycarbonate track-etched fibre; EP, ethanol pre-
cipitation; h, hours; w, weeks. Blue bar charts indicate relative size distribution of Cq-values within each category before split. Number under bar
charts indicate mean Cq-value for the given category SE.
Table 2. SX
CAPSULE
in comparison with other eDNA capture methods
SX
CAPSULE
comparison of Cq-values (SX
CAPSULE
comparison of [eDNA
tot
])
Capture Pairs of nP Significance* ZRank
SX
TUBE
33 (18) 1910
5
(5 910
4
)*** (**)44(35) SX
CAPSULE
<SX
TUBE
(>SX
TUBE
)
GF 50 (27) 7910
3
(2 910
5
)*(***)27(43) SX
CAPSULE
<GF (>GF)
PCTE 44 (27) 1910
5
(6 910
6
)*** (***)44(45) SX
CAPSULE
<PCTE (>PCTE)
EP 13 (9) 1910
3
(8910
3
)** (*)32(27) SX
CAPSULE
<EP (>EP)
CN
29 (15) 032 (055) N.S. (N.S.) 10(06)
Wilcoxon matched-pair signed-rank test of both Cq-values from qPCR and [eDNA
tot
] (denoted in parentheses). Significant P-values are in bold and
non-significant P-values are denoted as N.S.
SX
CAPSULE
, Sterivex, extracted within capsule; SX
TUBE
, Sterivex, extraction in tube outside capsule; GF, glass fibre; PCTE, polycarbonate track-
etched filter; CN, cellulose nitrate; EP, ethanol precipitation; [eDNA
tot
], total eDNA concentration.
*Bonferroni corrected (5 tests): a=005 lowered to 001, a=001 lowered to 0002 and a=0001 lowered to 00002.
Due to time constraints, CN (24 h) were cancelled reducing sample size and statistical power for CN in comparison.
©2016 The Authors. Methods in Ecology and Evolution ©2016 British Ecological Society, Methods in Ecology and Evolution
Testing enclosed filter for eDNA capture and storage 7
CONTAMINATION
One false-positive signal for perch was detected at 42 cycles in
an EP ‘no-water’ negative control. Remaining negative con-
trols for capture/storage treatments (n=80) and negative
pond water (n=85), NTCs (n=64) and 37/40 tissue negative
controls for species specificity did not amplify. The contami-
nated tissue control was replaced and showed no amplification.
One extraction blank came up positive in one of the seven runs,
but at a very high Cq of 462.
Discussion
To our knowledge, this is the first study comparing enclosed fil-
ters (SX) with commonly used eDNA capture and storage
techniques. Similarly to other capture methods, SX can be used
to target a wide range of macro-organisms successfully (using
PCR, qPCR or NGS; Table S1), ensuring the generality of SX
for surveys of aquatic biodiversity.
Specifically, SX with added preservation buffer (ethanol or
Longmire’s) is the optimal approach of the tested treatments in
regard to [eDNA
tot
] yield and detection sensitivity for target
species. Other eDNA studies of macrobiota using SX (Keskin
2014; Bergman et al. 2016) did not apply preservation buffers.
Although our study set-up was different, the lake sample
results are consistent with the mesocosm experiment of Ren-
shaw et al. (2015), showing that open CN filter and polyether-
sulfone filters (same material as SX in this study) were more
effective than PCTE and GF. Additionally, we demonstrate
that SX eDNA retains integrity over time, whereas eDNA
from the open filters degrades significantly. These results sug-
gest that SX eDNA is more effectively preserved, possibly due
to the fact that it is considerably less handled by the user. The
capsule may reduce risks of exposure to physical and biogenic
stress as well as contamination, because capture, storage and
extraction take place within the filter capsule. This, together
with extended field usage possibilities, and higher eDNA
yields, constitutes reasons to recommend enclosed filters before
other capture methods.
CAPTURE METHOD
Based on our results, we reject H
0
hypothesis 1 stating that SX
and commonly used techniques in our study are equally
50 100 (ng µL–1) 100 (ng µL–1)
34
36
38
40
42
Perch
44
Cq
46
32
(a)
0
CN
GF
PCTE
EP
SXCAPSULE
SXTUBE
50
34
36
38
40
42
Pike
44
[eDNAtot]
32
(b)
0
Cq
Fig. 4. Environmental DNA (eDNA) capture methods: relationship between total eDNA concentration ([eDNA
tot
]) and quantification cycles in
qPCR (Cq-value) in study lake. Line represents best-fit power function where Cq decreased as a function of [eDNA
tot
]. (a) Perch.
Cq =4189[eDNA
tot
]
0024
;P<0001, R
2
=023. (b) Pike: Cq =4009[eDNA
tot
]
0031
;P<0001, R
2
=042. Dotted lines represent lower or
upper limits of 95% CI for slope of regression. SX
CAPSULE
, Sterivex, extracted within capsule; SX
TUBE
, Sterivex, extracted from buffer in tube out-
side capsule; CN, cellulose nitrate; GF, glass fibre; PCTE, polycarbonate track-etched fibre; EP, ethanol precipitation.
50 100 (ng µL–1)100 (ng µL–1)
44
Cq
Perch
[eDNAtot]
(a)
42
40
38
36
34
32
0
SXTUBE
24 h 2 w
Frozen
2 w
BUFFER NO BUFFER
Ethanol
Longmire's
RNAlater
24 h
5 h
Refrigerated
SXCAPSULE
Time:
50
42
40
38
36
34
32
(b)
Pike
0
Cq Fig. 5. Environmental DNA (eDNA) storage
treatment using SX: relationship between
total eDNA concentration ([eDNA
tot
]) and
quantification cycles in qPCR (Cq-value) in
study lake. Line represents best-fit power
function of the negative correlation between
Cq and [eDNA
tot
]. (a) Perch: Cq =4099
[eDNA
tot
]
0026
;P<0001, R
2
=028. (b)
Pike: Cq =4089[eDNA
tot
]
0030
;P<0001,
R
2
=045. Dotted lines represent lower or
upper limits of 95% CI for slope of regres-
sion. Sterivex, extracted within capsule
(SX
CAPSULE
) and from buffer in tube outside
capsule (SX
TUBE
) shown in black and blue
symbols, respectively. h, hours; w, weeks.
©2016 The Authors. Methods in Ecology and Evolution ©2016 British Ecological Society, Methods in Ecology and Evolution
8J. Spens et al.
effective, because SX
CAPSULE
yields the lowest Cq-values for
perch (Fig. 3a). However, this is only partially validated in the
case of pike (Fig. 3b), where SX
CAPSULE
,GFandCNgroup
together for the lowest Cq-values. Overall, SX
CAPSULE
yields
higher [eDNA
tot
] and generates better qPCR results than other
capture methods, with the exception of CN. Our CN/SX com-
parisons are not as extensive as the SX/GF and SX/PCTE
comparisons (Table 2). We show that higher levels of
[eDNA
tot
] are related to lower Cq-values of target species
DNA (R
2
=023045, Figs 4 and 5) and therefore suggest
measurements of [eDNA
tot
] for approximate indications of
eDNA capture efficiency.
The comparison in this study of SX
TUBE
to SX
CAPSULE
demonstrates that utilizing both these sources of eDNA should
be useful. Pooling of these in the final elution step would be
advisable for gaining even higher final yields of eDNA.
SX
TUBE
exhibits the highest overall detection rate for both spe-
cies (9596%) in our study, significantly higher than EP results.
Higher amounts of false negatives from EP field samples may
be due to DNA retention in the falcon tubes (Gaillard &
Strauss 1998) and/or to the low water volume processed
(0015 L; Deiner et al. 2015; Eichmiller, Miller & Sorensen
2016; Minamoto et al. 2016).
STORAGE PRESERVATIVE
We reject H
0
hypothesis 2a stating that preservation buffers for
storage of SX do not affect qPCR amplification in comparison
with extraction within 5 h or freezing at 20 °C. Two-thirds
of published aqueous eDNA surveys reporting storage details
apply freezing of filters as a preservation method (Table S1
and S2), while less than one-third of surveys use buffer storage.
Our results indicate that addition of ethanol or Longmire’s
immediately after SX filtration provides the lowest Cq-values,
and is significantly better than freeze storage or extraction
within 5 h. Based on our results as well as the results of three
previous studies (Renshaw et al. 2015; Wegleitner et al. 2015;
Minamoto et al. 2016), we recommend addition of preserva-
tion immediately after filtration.
STORAGE TIME
We reject H
0
hypothesis 2b that degradation of captured
eDNA is the same in SX filters and the other capture
techniques tested in this study. Cq-values increase significantly
with storage time for GF and PCTE samples, indicating degra-
dation of eDNA. In contrast, Cq-values for SX samples
(SX
CAPSULE
or SX
TUBE
) do not differ significantly after
2 weeks of storage at RT.
We note that repeated use of the same extracted eDNA
sample (eluted in TE-buffer) for qPCR on different days,
entailing repeated freezing and thawing, resulted in higher
Cq-values. Freezethaw-induced degradation and/or inhibi-
tion of DNA is previously acknowledged (e.g. Ross, Haites
40
Cq
38
36
34
Buffer (Ethanol or Longmire's)
No Buffer (Refrigerated or Frozen)
42
44
PikePerch
32
Fig. 6. Boxplots of Cq-values showing SX
CAPSULE
(extraction within
Sterivex capsule) filter storage with and without preservation buffer
(ethanol or Longmire’s).
Table 3. Effect of storage time for eDNA results with different capture methods
Paired test of Cq-values
Storage Pairs of nP Significance* ZRank
SX
CAPSULE
20 015 N.S. 15
SX
TUBE
16 018 N.S. 13
PCTE 16 0002 ** 31PCTE24h<PCTE 2 weeks
Glass fibre (GF) 24 0002 ** 31GF24h<GF 2 weeks
Wilcoxon matched-pair signed-rank test of Cq-values from qPCR. Storage 24 h paired with storage 2 weeks. Significant P-values are in bold and
non-significant P-values are denoted as N.S.
Due to time constraints, cellulose nitrate treatments (24 h) were cancelled.
SX
CAPSULE
, Sterivex, extracted within capsule; SX
TUBE
, Sterivex, extraction in tube outside capsule; PCTE, polycarbonate track-etched filter.
*Bonferroni corrected (4 tests): a=005 lowered to 00125, a=001 lowered to 00025.
©2016 The Authors. Methods in Ecology and Evolution ©2016 British Ecological Society, Methods in Ecology and Evolution
Testing enclosed filter for eDNA capture and storage 9
& Kelly 1990; Takahara, Minamoto & Doi 2015). We there-
fore recommend that extracted eDNA samples are divided
into many aliquots immediately after extraction, in order to
avoid compromising eDNA quality by repeated freezing and
thawing.
CONTAMINATION
We cannot yet reject H
0
hypothesis 3 stating that SX leads to
as many false positives as typically used methods. We only pro-
duced one false positive (EP) which is insufficient for any statis-
tical inference. The SX approach using sealed pre-sterilized
equipment until sampling, and capping filter immediately after
filtration, should reduce contamination risk. The contamina-
tion variance between these capture methods remains to be
tested using more observations and possibly synthetic controls
(Wilson, Wozney & Smith 2016).
LIMITATIONS
The hand-held syringe used with SX filter units is convenient
but turns into a labour-intensive bottleneck when processing
many samples. This can be alleviated by switching to battery-
powered pumps (Sterivex
TM
2013). In ‘algal soup’ or turbid
waters, 02lm pore size may pose a problem as the filters clog
easily and less water can be processed (Turner et al. 2014a).
This can be overcome by pre-filtering (Robson et al. 2016)
and/or increasing the number of filter replicates. Future
research is needed to identify optimal procedures for highly
productive and/or turbid waters.
Conclusion
In conclusion, we recommend SX filters as an efficient capture
method for aqueous eDNA sampling of macro-organisms.
Preservation of SX in ethanol or Longmire’s buffer immedi-
ately after filtration is recommended. Preserved SX capsules
may be stored at RT for at least 2 weeks without significant
degradation. Water samples can be quickly filtered and pre-
served on site requiring less equipment, easing transport.
Therefore, SX capsules are logistically compatible with remote
and harsh field conditions.
Authors’ contributions
M.H. and J.S. conceived and designed initial experiment. All authors (except
D.H.) contributed to final design and participated in ‘sample collection/filtration
day’. J.S. analysed data and drafted the manuscript. M.H. developed protoco l for
eDNA capture/extraction. J.S., M.H. and A.E. wrote the manuscript. A.E. and
S.S.T.M. coordinated field experiment and contributed to extraction protocol.
A.E., M.H., S.W.K., S.S.T.M., E.E.S. and M.S. extracted DNA. S.W.K. opti-
mized qPCR protocol. S.W.K., M.H. and M.S. performed qPCR assays. All
authors revised the manuscript. No conflict of interest exists.
Acknowledgements
We thank Philip Francis Thomsen (PFT), Ian Eirød and Peter Rask Møller for
participating in ‘experimental planning’ and ‘collection/filtration’ day; PFT for
providing qPCR primers/probes and collaborating on earlier SX extraction
protocols; Eske Willerslev for laboratory facilities, funding and comments on ear-
lier drafts.
Data accessibility
Data are deposited in the Dryad Data Repository http://dx.doi.org/10.5061/
dryad.p2q4r (Spens et al. 2016).
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Handling Editor: Douglas Yu
Supporting Information
Additional Supporting Information may be found online in the support-
ing information tab for this article:
Fig. S1. Flow chart illustrating the different capture and storage treat-
ments.
Appendix S1. eDNA extraction protocol.
Appendix S2. Water quality in Gentofte lake.
Table S1. Empirical field-studies targeting macrobial eDNA in aquatic
ecosystems with water sampling, January 2005 to March 2015.
Table S2. Empirical field-studies targeting macrobial eDNA in aquatic
ecosystems with water sampling, published after the current study was
initiated in March 2015.
Table S3. Primers and probes used in this study.
©2016 The Authors. Methods in Ecology and Evolution ©2016 British Ecological Society, Methods in Ecology and Evolution
Testing enclosed filter for eDNA capture and storage 11
... Polyethersulfone (PES), cellulose nitrate, and glass fiber are the most commonly used types of filters in DNA research. Glass fibre filters are commonly suggested due to their higher capability to absorb DNA (Muha et al., 2019;Spens et al., 2017;Tsuji et al., 2019). ...
... The type of filter is one of the most important decisions to be made when designing the sampling strategy. Filters can be classified as open or encapsulated/cartridge filters (Spens et al., 2017;Tsuji et al., 2019). Open filters are tiny membranes that are usually fixed on an immobilized manifold system that is connected to a vacuum pump for filtering the water. ...
... This reduces the effort and time required for field sampling. As contamination can also be prevented immediately after water filtering, encapsulated filters offer many advantages over filtering on-site (Spens et al., 2017;Thomas et al., 2019). A key drawback of encapsulated filters is cost: encapsulated filters generally cannot be used more than once, so the total cost of filters needs to be considered, in particular for large-scale projects. ...
Thesis
Full-text available
Diet information is key when implementing conservation strategies for species habitat protection. Non-molecular methods used to attain herbivore diet information are time-consuming and generally have low taxonomic resolutions. Hence, researchers are limited by the number of individuals that they can study within a short time frame. Advances in high-throughput sequencing technologies overcome these limitations as they allow rapid sequencing of multiple samples in parallel. The species of interest in my thesis is the herbivorous wood grouse, the western capercaillies (Tetrao urogallus). The western capercaillie is a targeted species when carrying out forest protection due to its unique habitat requirements. As this bird is particularly susceptible to anthropogenic disturbances, I used non-invasive environmental DNA analyses of its faeces to study its diet. The first three chapters in my thesis are educational book chapters describing the use of faecal samples in molecular diet studies (Chapter 1), and two high-throughput sequencing techniques used in plant identification; metabarcoding (Chapter 2) and metagenomics (Chapter 3). I explored the advantages and challenges of using these two techniques to reconstruct the western capercaillie’s diet in Chapters 4 and 5. In Chapter 4, I developed an in silico approach to validate metagenomics taxonomic assignment steps for the identification of plant taxa. In Chapter 5, I present the first large-scale capercaillie molecular diet study carried out using metabarcoding. A common issue of these two high-throughput sequencing techniques is the lack of a curated and comprehensive DNA reference database used in the taxonomic identification of sequenced data. As a small but important step forward to overcome this limitation for future plant biodiversity monitoring studies in Denmark, in Chapter 6, I generated plant DNA reference data using genome skimming. These reference data consist of the chloroplast and nuclear DNA sequences of 184 Danish plant species.
... Sampling occurred from 30 May until 10 June 2018. Environmental DNA filtration followed recommendations from (Spens et al. 2016). Briefly, pre-packed sterile 50 ml luer-lock syringes were used to push the sampled water through the Sterivex (Millipore, Merck KGaA, Darmstadt, Germany) column until clogging. ...
... Sample processing followed the recommendations in Spens et al. (2016) with slight modifications. Briefly, DNA was extracted solely from the Longmire's Buffer, since DNA extracts from filter capsules did not show amplification success during initial testing, most likely due to the lysis and leaching of DNA into the Longmire's Buffer (Williams et al. 2016;David et al. 2021). ...
... Finally, false-negative fish NIS detections could also be a consequence of our experimental design. For eDNA capture, we opted to employ the frequently used Sterivex filters with a pore size of 0.22 µm (Spens et al. 2016). However, the high turbidity at our sampling sites limited the volume processed until the filter clogged. ...
Article
Full-text available
Active environmental DNA (eDNA) surveillance through species-specific amplification has shown increased sensitivity in the detection of non-indigenous species (NIS) compared to traditional approaches. When many NIS are of interest, however, active surveillance decreases in cost- and time-efficiency. Passive surveillance through eDNA metabarcoding takes advantage of the complex DNA signal in environmental samples and facilitates the simultaneous detection of multiple species. While passive eDNA surveillance has previously detected NIS, comparative studies are essential to determine the ability of eDNA metabarcoding to accurately describe the range of invasion for multiple NIS versus alternative approaches. Here, we surveyed twelve sites, covering nine rivers across Belarus for NIS with three different techniques, i.e. an ichthyological, hydrobiological and eDNA survey, whereby DNA was extracted from 500 ml surface water samples and amplified with two 16S rDNA primer assays targeting the fish and macroinvertebrate biodiversity. Nine non-indigenous fish and ten non-indigenous benthic macroinvertebrates were detected by traditional surveys, while seven NISeDNA signals were picked up, including four fish, one aquatic and two benthic macroinvertebrates. Passive eDNA surveillance extended the range of invasion further north for two invasive fish and identified a new NIS for Belarus, the freshwater jellyfish Craspedacusta sowerbii . False-negative detections for the eDNA survey might be attributed to: (i) preferential amplification of aquatic over benthic macroinvertebrates from surface water samples and (ii) an incomplete reference database. The evidence provided in this study recommends the implementation of both molecular-based and traditional approaches to maximise the probability of early detection of non-native organisms.
... Sampling occurred from 30 May until 10 June 2018. Environmental DNA filtration followed recommendations from (Spens et al. 2016). Briefly, pre-packed sterile 50 ml luer-lock syringes were used to push the sampled water through the Sterivex (Millipore, Merck KGaA, Darmstadt, Germany) column until clogging. ...
... Sample processing followed the recommendations in Spens et al. (2016) with slight modifications. Briefly, DNA was extracted solely from the Longmire's Buffer, since DNA extracts from filter capsules did not show amplification success during initial testing, most likely due to the lysis and leaching of DNA into the Longmire's Buffer (Williams et al. 2016;David et al. 2021). ...
... Finally, false-negative fish NIS detections could also be a consequence of our experimental design. For eDNA capture, we opted to employ the frequently used Sterivex filters with a pore size of 0.22 µm (Spens et al. 2016). However, the high turbidity at our sampling sites limited the volume processed until the filter clogged. ...
Article
Full-text available
Active environmental DNA (eDNA) surveillance through species-specific amplification has shown increased sensitivity in the detection of non-indigenous species (NIS) compared to traditional approaches. When many NIS are of interest, however, active surveillance decreases in cost-and time-efficiency. Passive surveillance through eDNA metabarcoding takes advantage of the complex DNA signal in environmental samples and facilitates the simultaneous detection of multiple species. While passive eDNA surveillance has previously detected NIS, comparative studies are essential to determine the ability of eDNA metabarcoding to accurately describe the range of invasion for multiple NIS versus alternative approaches. Here, we surveyed twelve sites, covering nine rivers across Belarus for NIS with three different techniques, i.e. an ichthyological, hydrobiological and eDNA survey, whereby DNA was extracted from 500 ml surface water samples and amplified with two 16S rDNA primer assays targeting the fish and macroin-vertebrate biodiversity. Nine non-indigenous fish and ten non-indigenous benthic macroinvertebrates were detected by traditional surveys, while seven NIS eDNA signals were picked up, including four fish, one aquatic and two benthic macroinvertebrates. Passive eDNA surveillance extended the range of invasion further north for two invasive fish and identified a new NIS for Belarus, the freshwater jellyfish Craspedacusta sowerbii. False-negative detections for the eDNA survey might be attributed to: (i) preferential amplification of aquatic over benthic macroinvertebrates from surface water samples and (ii) an incomplete reference database. The evidence provided in this study recommends the implementation of both molecular-based and traditional approaches to maximise the probability of early detection of non-native organisms.
... Thus, our first experiment explored the efficacy of storing rollers within sterile bags in conditions that will slow the rate of DNA degradation ( Figure 1). We selected two methods of preservation, ethanol, and cold storage, as they are practical to use in the field and are used in other eDNA survey protocols (Goldberg, Sepulveda, Ray, Baumgardt, & Waits, 2013;Minamoto, Naka, Moji, & Maruyama, 2016;Rees, Maddison, Middleditch, Patmore, & Gough, 2014;Renshaw et al., 2015;Spens et al., 2017;Strickler et al., 2015). Cold temperature storage was achieved using a commercially available cooler filled with a bottom layer of ice. ...
... Letters indicate statistical significance in difference in eDNA recovered among number of trees sampled within the forward experiment (uppercase letters) and the reverse experiment (lowercase) and can be an important limiting step in the detection of rare species (Goldberg et al., 2016(Goldberg et al., , 2018Pilliod et al., 2014;Strickler et al., 2015). Cold storage of water samples in the field is a common method of preserving eDNA within samples for longer timeframes before filtration and extraction (Strickler et al., 2015;Tsuji et al., 2017), while ethanol and other chemical preservatives have also been used effectively to slow degradation rates (Minamoto et al., 2016;Renshaw et al., 2015;Spens et al., 2017). Although our study involved the collection of eDNA from dry surfaces, once the DNA is transferred onto damp rollers, many of the same principles of DNA degradation in aquatic systems likely apply (Barnes et al., 2014). ...
Article
Full-text available
Environmental DNA surveys have revolutionized monitoring of rare or cryptic species and species inhabiting areas where conventional sampling is difficult or dangerous. Recent advancements within terrestrial environments include the capture of eDNA deposited by animals on surfaces such as tree bark and foliage, hereafter “surface eDNA.” Notably, a technique which uses commercial paint rollers to aggregate surface eDNA has been deployed with success to detect the presence of forest insect pests providing a potentially powerful new management tool. However, before widespread adoption is feasible, the efficiency and logistics of roller sample collection and study design, especially relative to realistic survey conditions, must be evaluated. We compared the performance of two DNA preservation treatments—cold and ethanol—on their ability to reduce the loss of captured eDNA on rollers over time. Additionally, we evaluated how the detection probability of our target species, the spotted lanternfly (Lycorma delicatula), varied with sampling effort (time spent rolling per sample) and the initial quantity of eDNA present. Finally, we evaluated how the number of trees sampled per roller influenced the final concentrations of lanternfly eDNA remaining on the roller. We found storing rollers with ethanol or cold temperatures resulted in 3–10‐fold greater concentrations of experimentally controlled eDNA relative to no treatment after 24 h. Detection probability declined as the amount of lanternfly eDNA decreased, but did not change in response to sampling effort over sample time (10–80 s/tree). Finally, recovered lanternfly eDNA decreased as more trees were sampled by a single roller—a 91% reduction after 7 trees—potentially due to captured DNA being transferred back from the roller onto the bark. Our results provide improved guidance for deploying roller surface eDNA methods for spotted lanternfly surveys, and for invasive insect pest surveillance and monitoring programs generally. Surveying plant surfaces for eDNA is relatively novel. We present several experiments to improve on a recent technique used for surveying bark and branches of trees for insect DNA. Cold temperatures can preserve DNA on paint rollers, used to sample trees, while sampling more than a few trees can lead to DNA loss.
... All DNA extractions and setup of quantitative PCR (qPCR) reactions were conducted in a dedicated clean laboratory facility at DTU Aqua (Technical University of Denmark, Silkeborg, Denmark). DNA extraction was based on a modified version of a previously published eDNA extraction protocol (Spens et al., 2017). ...
... We note that prior to potentially realizing any of the promising developments above, eDNA and video sampling can already be combined with relative ease in the field by bringing low-cost enclosed Sterivex filters (Spens et al., 2017) on field expeditions to filter water samples prior to camera deployments. The filtered samples can afterwards be stored at freezing temperature for later use, in case further eDNA analysis is not immediately feasible. ...
... All DNA extractions and setup of quantitative PCRs (qPCRs) were conducted in a dedicated clean laboratory facility. DNA extraction was based on a modified version of a previously published eDNA extraction protocol (Spens et al., 2017). This protocol uses the DNeasy blood and tissue kit (Qiagen, Hilden, Germany) to extract eDNA directly from the Sterivex filters (Supporting information, Note S1). ...
Article
Full-text available
Accurate knowledge on spatiotemporal distributions of marine species and their association with surrounding habitats is crucial to inform adaptive management actions responding to coastal degradation across the globe. Here, we investigate the potential use of environmental DNA (eDNA) to detect species–habitat associations in a patchy coastal area of the Baltic Sea. We directly compare species‐specific qPCR analysis of eDNA with baited remote underwater video systems (BRUVS), two non‐invasive methods widely used to monitor marine habitats. Four focal species (cod Gadus morhua, flounder Platichthys flesus, plaice Pleuronectes platessa, and goldsinny wrasse Ctenolabrus rupestris) were selected based on contrasting habitat associations (reef‐ vs. sand‐associated species), as well as differential levels of mobility and residency, to investigate whether these factors affected the detection of species–habitat associations from eDNA. To this end, a species‐specific qPCR assay for goldsinny wrasse is developed and made available herein. In addition, potential correlations between eDNA signals and abundance counts (MaxN) from videos were assessed. Results from Bayesian multilevel models revealed strong evidence for a sand association for sedentary flounder (98% posterior probability) and a reef association for highly resident wrasse (99% posterior probability) using eDNA, in agreement with BRUVS. However, contrary to BRUVS, eDNA sampling did not detect habitat associations for cod or plaice. We found a positive correlation between eDNA detection and MaxN for wrasse (posterior probability 95%), but not for the remaining species and explanatory power of all relationships was generally limited. Our results indicate that eDNA sampling can detect species–habitat associations on a fine spatial scale, yet this ability likely depends on the mobility and residency of the target organism, with associations for sedentary or resident species most likely to be detected. Combined sampling with conventional non‐invasive methods is advised to improve detection of habitat associations for mobile and transient species, or for species with low eDNA concentrations. There is a growing demand for low‐cost marine monitoring techniques capable of documenting species‐habitat associations (SHAs). In this comparative assessment with baited remote video, we show that eDNA can detect SHAs on a fine spatial scale, yet this ability likely depends on the mobility and residency levels of target organisms.
... The Sterivex capsules preserved in Longmire's buffer were stored at room temperature for seven weeks prior to DNA extraction following the protocol described by Spens et al. (2017) using the DNeasy Blood & Tissue Kit (Qiagen). The lysis solutions from the capsule and buffer components were combined before the addition of buffer AL and the continuation of the Blood & Tissue Kit protocol. ...
Article
Full-text available
As elasmobranchs are becoming increasingly threatened, efficient methods for monitoring the distribution and diversity of elasmobranch populations are required. Environmental DNA (eDNA) metabarcoding is a progressively applied technique that enables mass identification of entire communities and is an effective method for the detection of rare and elusive species. We performed an eDNA metabarcoding survey for fish communities around a coral reef atoll in the Chagos Archipelago (Central Indian Ocean) and assessed the diversity and distribution of elasmobranch species detected within these communities. Our eDNA survey detected 353 amplicon sequence variants (ASVs) attributed to fishes, 12 of which were elasmobranchs. There were no differences in fish communities based on the presence and absence of ASVs between sample depth (surface and 40 m) or sampling habitat, but communities based on read abundance were significantly different between habitats. The dominant elasmobranch species were grey reef (Carcharhinus amblyrhynchos) and silvertip (C. albimarginatus) sharks, and elasmobranch communities were significantly different between sampling depth and habitat. Overall, we find that eDNA metabarcoding can be used to reveal the diversity of elasmobranchs within broader taxonomic assays, but further research and development of targeted metabarcoding primers may be required before it can be integrated into a toolkit for monitoring these species. ADDITIONAL KEYWORDS: barcoding-conservation genetics-eDNA metabarcoding-fish-marine-sharks.
Article
Environmental DNA (eDNA) technology has revolutionized biomonitoring in recent years; however, eDNA collection from aquatic environments generally relies on the time-consuming and equipment-dependent process of water filtration. Passive eDNA sampling deploys sorbent materials to capture eDNA from water, circumventing many problems associated with active filtration; yet, very few candidate materials have been systematically evaluated for this purpose. Here, we evaluated the ability of 12 different types of common loose sorbents and filter membranes to capture eDNA in laboratory and field experiments compared with conventional water filtration. Glass fiber filters (GF) outperformed all other materials in laboratory experiments with respect to their quantitative capacity to recover amphibian eDNA, with the eDNA yield increasing linearly with submersion time up to 72 h. Furthermore, GF rapidly (within 0.5 h) captured the eDNA of up to 71% of the total fish species in a lake, in addition to detecting the entire fish community by 8 h, as assessed by metabarcoding analysis. Our results demonstrate that GF could passively capture aqueous eDNA with a similar or greater efficiency than conventional methods, thus paving the way for convenient, effective, and eco-friendly eDNA sampling in aquatic environments.
Chapter
Environmental DNA (eDNA) analysis has emerged in recent years as a powerful tool for the detection, monitoring, and characterization of aquatic metazoan communities, including vulnerable species. The rapid rate of adopting the eDNA approach across diverse habitats and taxonomic groups attests to its value for a wide array of investigative goals, from understanding natural or changing biodiversity to informing on conservation efforts at local and global scales. Regardless of research objectives, eDNA workflows commonly include the following essential steps: environmental sample acquisition, processing and preservation of samples, and eDNA extraction, followed by eDNA sequencing library preparation, high-capacity sequencing and sequence data analysis, or other methods of genetic detection. In this chapter, we supply instructional details for the early steps in the workflow to facilitate researchers considering adopting eDNA analysis to address questions in marine environments. Specifically, we detail sampling, preservation, extraction, and quantification protocols for eDNA originating from marine water, shallow substrates, and deeper sediments. eDNA is prone to degradation and loss, and to contamination through improper handling; these factors crucially influence the outcome and validity of an eDNA study. Thus, we also provide guidance on avoiding these pitfalls. Following extraction, purified eDNA is often sequenced on massively parallel sequencing platforms for comprehensive faunal diversity assessment using a metabarcoding or metagenomic approach, or for the detection and quantification of specific taxa by qPCR methods. These components of the workflow are project-specific and thus not included in this chapter. Instead, we briefly touch on the preparation of eDNA libraries and discuss comparisons between sequencing approaches to aid considerations in project design.
Article
Environmental DNA (eDNA) quantification and sequencing are emerging techniques for assessing biodiversity in marine ecosystems. Environmental DNA can be transported by ocean currents and may remain at detectable concentrations far from its source depending on how long it persist. Thus, predicting the persistence time of eDNA is crucial to defining the spatial context of the information derived from it. To investigate the physicochemical controls of eDNA persistence, we performed degradation experiments at temperature, pH, and oxygen conditions relevant to the open ocean and the deep sea. The eDNA degradation process was best explained by a model with two phases with different decay rate constants. During the initial phase, eDNA degraded rapidly, and the rate was independent of physicochemical factors. During the second phase, eDNA degraded slowly, and the rate was strongly controlled by temperature, weakly controlled by pH, and not controlled by dissolved oxygen concentration. We demonstrate that marine eDNA can persist at quantifiable concentrations for over 2 weeks at low temperatures (≤10 °C) but for a week or less at ≥20 °C. The relationship between temperature and eDNA persistence is independent of the source species. We propose a general temperature-dependent model to predict the maximum persistence time of eDNA detectable through single-species eDNA quantification methods.
Article
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Environmental DNA (eDNA) is an emerging sampling method that has been used successfully for detection of rare aquatic species. The Identification of sampling tools that are less stressful for target organisms has become increasingly important for rare and endangered species. A decline in abundance of the Southern Distinct Population Segment (DPS) of North American Green Sturgeon located in California's Central Valley has led to its listing as Threatened under the Federal Endangered Species Act in 2006. While visual surveys of spawning Green Sturgeon in the Central Valley are effective at monitoring fish densities in concentrated pool habitats, results do not scale well to the watershed level, providing limited spatial and temporal context. Unlike most traditional survey methods, environmental DNA analysis provides a relatively quick, inexpensive tool that could efficiently monitor the presence and distribution of aquatic species. We positively identified Green Sturgeon DNA at two locations of known presence in the Sacramento River, proving that eDNA can be effective for monitoring the presence of adult sturgeon. While further study is needed to understand uncertainties of the sampling method, our study represents the first documented detection of Green Sturgeon eDNA, indicating that eDNA analysis could provide a new tool for monitoring Green Sturgeon distribution in the Central Valley, complimenting traditional on-going survey methods.
Article
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Invasive species pose a major threat to aquatic ecosystems. Their impact can be particularly severe in tropical regions, like those in northern Australia, where >20 invasive fish species are recorded. In temperate regions, environmental DNA (eDNA) technology is gaining momentum as a tool to detect aquatic pests, but the technology's effectiveness has not been fully explored in tropical systems with their unique climatic challenges (i.e. high turbidity, temperatures and ultra-violet light). In this study, we modified conventional eDNA protocols for use in tropical environments using the invasive fish, Mozambique tilapia (Oreochromis mossambicus) as a detection model. We evaluated the effects of high water temperatures and fish density on the detection of tilapia eDNA, using filters with larger pores to facilitate filtration. Large-pore filters (20 μm) were effective in filtering turbid waters and retaining sufficient eDNA, whilst achieving filtration times of 2-3 minutes per 2-L sample. High water temperatures, often experienced in the tropics (23, 29, 35 °C), did not affect eDNA degradation rates, although high temperatures (35 °C) did significantly increase fish eDNA shedding rates. We established a minimum detection limit for tilapia (1 fish/ 0.4 megalitres/ after 4 days) and found that low water flow (3.17 L/s) into ponds with high fish density (>16 fish/ 0.4 megalitres) did not affect eDNA detection. These results demonstrate that eDNA technology can be effectively used in tropical ecosystems to detect invasive fish species. This article is protected by copyright. All rights reserved.
Article
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Environmental DNA (eDNA) is an emerging tool that allows low-impact sampling for aquatic species by isolating DNA from water samples and screening for DNA sequences specific to species of interest. However, researchers have not tested this method in naturally acidic wetlands that provide breeding habitat for a number of imperiled species, including the frosted salamander (Ambystoma cingulatum), reticulated flatwoods salamanders (Ambystoma bishopi), striped newt (Notophthalmus perstriatus), and gopher frog (Lithobates capito). Our objectives for this study were to develop and optimize eDNA survey protocols and assays to complement and enhance capture-based survey methods for these amphibian species. We collected three or more water samples, dipnetted or trapped larval and adult amphibians, and conducted visual encounter surveys for egg masses for target species at 40 sites on 12 different longleaf pine (Pinus palustris) tracts. We used quantitative PCRs to screen eDNA from each site for target species presence. We detected flatwoods salamanders at three sites with eDNA but did not detect them during physical surveys. Based on the sample location we assumed these eDNA detections to indicate the presence of frosted flatwoods salamanders. We did not detect reticulated flatwoods salamanders. We detected striped newts with physical and eDNA surveys at two wetlands. We detected gopher frogs at 12 sites total, three with eDNA alone, two with physical surveys alone, and seven with physical and eDNA surveys. We detected our target species with eDNA at 9 of 11 sites where they were present as indicated from traditional surveys and at six sites where they were not detected with traditional surveys. It was, however, critical to use at least three water samples per site for eDNA. Our results demonstrate eDNA surveys can be a useful complement to traditional survey methods for detecting imperiled pondbreeding amphibians. Environmental DNA may be particularly useful in situations where detection probability using traditional survey methods is low or access by trained personnel is limited.
Article
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Global biodiversity in freshwater and the oceans is declining at high rates. Reliable tools for assessing and monitoring aquatic biodiversity, especially for rare and secretive species, are important for efficient and timely management. Recent advances in DNA sequencing have provided a new tool for species detection from DNA present into the environment. In this study, we tested if an environmental DNA (eDNA) metabarcoding approach, using water samples, can be used for addressing significant questions in ecology and conservation. Two key aquatic vertebrate groups were targeted: amphibians and bony fish. The reliability of this method was cautiously validated in silico, in vitro, and in situ. When compared with traditional surveys or historical data, eDNA metabarcoding showed a much better detection probability overall. For amphibians, the detection probability with eDNA metabarcoding was 0.97 (CI = 0.90-0.99) versus 0.58 (CI = 0.50-0.63) for traditional surveys. For fish, in 89% of the studied sites, the number of taxa detected using the eDNA metabarcoding approach was higher or identical to the number detected using traditional methods. We argue that the proposed DNA-based approach has the potential to become the next-generation tool for ecological studies and standardized biodiversity monitoring in a wide range of aquatic ecosystems. This article is protected by copyright. All rights reserved.
Article
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Effective management of alien species requires detecting populations in the early stages of invasion. Environmental DNA (eDNA) sampling can detect aquatic species at relatively low densities, but few studies have directly compared detection probabilities of eDNA sampling with those of traditional sampling methods. We compare the ability of a traditional sampling technique (bottle trapping) and eDNA to detect a recently established invader, the smooth newt Lissotriton vulgaris vulgaris, at seven field sites in Melbourne, Australia. Over a four-month period, per-trap detection probabilities ranged from 0.01 to 0.26 among sites where L. v. vulgaris was detected, whereas per-sample eDNA estimates were much higher (0.29-1.0). Detection probabilities of both methods varied temporally (across days and months), but temporal variation appeared to be uncorrelated between methods. Only estimates of spatial variation were strongly correlated across the two sampling techniques. Environmental variables (water depth, rainfall, ambient temperature) were not clearly correlated with detection probabilities estimated via trapping, whereas eDNA detection probabilities were negatively correlated with water depth, possibly reflecting higher eDNA concentrations at lower water levels. Our findings demonstrate that eDNA sampling can be an order of magnitude more sensitive than traditional methods, and illustrate that traditional-and eDNA-based surveys can provide independent information on species distributions when occupancy surveys are conducted over short timescales.
Article
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Environmental DNA (eDNA) is useful for delimiting species ranges in aquatic systems, whereby water samples are screened for the presence of DNA from a single species. However, DNA from many species is collected in every sample, and high-throughput sequencing approaches allow for more passive surveillance where a community of species is identified. In this study, we use active (targeted) and passive molecular surveillance approaches to detect species in the Muskingum River Watershed in Ohio, USA. The presence of bighead carp (Hypophthalmichthys nobilis) eDNA in the Muskingum River Watershed was confirmed with active surveillance using digital droplet polymerase chain reaction (ddPCR). The passive surveillance method detected the presence of eDNA from northern snakehead (Channa argus), which was further confirmed with active ddPCR. Whereas active surveillance may be more sensitive to detecting rare DNA, passive surveillance has the capability of detecting unexpected invasive species. Deploying both active and passive surveillance approaches with the same eDNA samples is beneficial for invasive species management. © 2015, National Research Council of Canada. All rights reserved.
Article
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The use of environmental DNA is a rapidly evolving approach for surveillance and detection of species. Often water samples are collected in the field and then immediately cooled, filtered, and the resulting filters are stored in freezers to preserve the DNA for subsequent analyses. Recently it was shown that filtered samples could be stored at room temperature for 14 days without any discernable loss in the total DNA. However, for many conservation applications, particularly in remote settings with limited capacity to freeze samples, it would be advantageous to store samples at room temperature for longer periods of time. Here we test for significant loss of DNA yield from storage of polycarbonate track etched filters in Longmire’s lysis buffer at room temperature (20 °C) for 150 days.
Article
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Environmental DNA (eDNA) is increasingly used for surveillance and detection of species of interest in aquatic and soil samples.A significant risk associated with eDNA methods is potential false positive results due to laboratory contamination.To minimize and quantify this risk, we designed and validated a set of synthetic oligonucleotides for use as species-specific positive PCR controls for several high-profile aquatic invasive species.The controls consist of species-specific sequences for the species of interest, with the addition of a synthetic insert containing recognition sites for several restriction enzymes.Following PCR, the presence of the synthetic insert can be detected using gel electrophoresis, restriction enzyme digests, or DNA sequencing. For quantitative PCR (qPCR), false positives in environmental samples can also be detected using a fluorescent probe designed to detect the synthetic insert.The generation of synthetic controls is a cost-effective, reproducible method that increases the power and reliability of eDNA testing by eliminating misinterpretation of false positive results from laboratory contamination.This article is protected by copyright. All rights reserved.
Article
Environmental DNA (eDNA) analysis has recently been used for detection of aquatic macro-organisms; however, the analytical procedures used in previous studies have not been optimized for practical use. Here, we compared several methods for DNA enrichment and extraction from water samples to establish widely applicable techniques for eDNA analysis using common carp as the model species. First, several types of filters were compared to identify the optimal filter type. Second, the eDNA yield was compared after a variety of extraction and isolation steps, including a combination of phenol extraction, ethanol precipitation (phenol treatment), and ultrafiltration. Third, DNA fixation with ethanol was tested for the preservation of eDNA on filters. Ethanol precipitation yielded the largest number of eDNA copies, followed by filtering using a 0.2-μm polycarbonate filter and a 0.7-μm glass fiber filter. Phenol treatment resulted in collection of a higher number of eDNA copies than that collected using ultrafiltration. DNA fixation with 15 ml ethanol enabled eDNA preservation on the filters at ambient temperatures for at least 6 days. Finally, combinations of different filter types and DNA enrichment procedures were compared using field water samples. From these results, we propose that the appropriate selection method for eDNA analysis should be chosen based on context. For example, when a high concentration of the target DNA is expected, such as in an aquarium experiment, ethanol precipitation is advantageous. However, when the target DNA is rare, which is the case in most field studies, filtration followed by freezing or DNA fixation by ethanol and phenol treatment are recommended. The filter type should be decided prior to the survey based on the characteristics of the water of interest. Thus, eDNA analysis could be applied to various situations using adaptive combinations of these techniques.