LETTER

Rapid Invasive Species Detection by Combining Environmental

DNA with Light Transmission Spectroscopy
Scott P. Egan1,2,3 , Matthew A. Barnes1 , Ching-Ting Hwang1,4 , Andrew R. Mahon1,5 , Jeffery L. Feder1,2,3 ,
Steven T. Ruggiero3,4 , Carol E. Tanner3,4 , & David M. Lodge1,2,3
1
Department of Biological Sciences, University of Notre Dame, Notre Dame, IN 46556, USA
2
Advanced Diagnostics and Therapeutics Initiative, University of Notre Dame, Notre Dame, IN 46556, USA
3
Environmental Change Initiative, University of Notre Dame, Notre Dame, IN 46556, USA
4
Department of Physics, University of Notre Dame, Notre Dame, IN 46556, USA
5
Department of Biology and Institute for Great Lakes Research, Central Michigan University, Mount Pleasant, MI 48859, USA

Keywords Abstract
Carcinus maenas; Dreissena; zebra mussel;
quagga mussel; Eriocheir sinensis; Limnoperna Invasive aquatic species introductions cause tremendous environmental and
fortunei. economic damage. Conservation and management efforts will benefit from
rapid, inexpensive, and accurate on-site methods to detect harmful aquatic
Correspondence species to prevent their introduction and spread. Here, two technologies, envi-
Scott Egan, Department of Biological Sciences, ronmental DNA (eDNA) sampling and Light Transmission Spectroscopy (LTS),
University of Notre Dame, Notre Dame, IN
were combined to address this need. Specifically, eDNA filtering and extrac-
46556, USA.
Tel: 615-618-6601;
tion methods were used to isolate DNA from: (1) lake water samples that were
Fax: 574-631-7413. seeded with a microscopic fragment of five high-risk invasive species and (2)
E-mail: scott.p.egan@nd.edu untreated samples from lakes infested with the invasive zebra mussel, Dreis-
sena polymorpha, followed by polymerase chain reaction (PCR) amplification.
Received LTS was then used to detect size shifts resulting from hybridization of PCR
1 November 2012
products with nanobeads covered with species-specific oligonucleotide probes.
Accepted
The results demonstrate that coupling eDNA sampling with LTS species de-
20 February 2013
tection can provide a sensitive and real-time solution for screening real-world
Editor water samples for invasive species.
Dr. Julie Lockwood

doi: 10.1111/conl.12017

effective surveillance and management, the danger posed

Introduction
Invasive species threaten biodiversity and the function-
ing of ecosystems and economies worldwide (Pejchar &
Mooney 2009). In the U.S. alone, invasive species cause
damage in excess of $120 billion annually (Pimentel et al.
2005). Perhaps nowhere have the harmful ecological and
economic effects of aquatic invasive species been better
documented than in the Great Lakes (Keller et al. 2009).
Over 180 nonindigenous species have become established
in the Great Lakes since European settlement, with about
70% arriving through the ballast water of transoceanic
ships (Drake & Lodge 2007; Bailey et al. 2011). A subset
of those species cause $138 million to $800 million in an-
nual damages in the U.S. (Rothlisberger et al. 2012). With
ever increasing global commerce, and in the absence of
by invasive species introduced by a variety of vectors to
aquatic ecosystems worldwide will only increase (Lodge
et al. 2006; Keller et al. 2010).
Early detection is critical for conservation and man-
agement efforts aimed at mitigating the threat posed by
aquatic invasive species. Rapid and sensitive on-site de-
tection can: (1) prevent invasions through the intercep-
tion of harmful taxa at points of entry before they be-
come established and, once present, (2) help curtail their
further spread through the identification and monitoring
of affected areas. Here, we focus on the latter need by
combining advances in environmental DNA (eDNA) sam-
pling (Jerde et al. 2011) with a new Light Transmission
Spectroscopy (LTS) DNA detection technique (Li et al.
2010, 2011; Mahon et al. 2013) to screen natural lake

402 Conservation Letters 6:6 November/December (2013) 402–409 Copyright and Photocopying: 
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S.P. Egan et al.
water samples for target invasive species (Figure S1; we
hereafter use the abbreviation LTS to refer to both Laser
and Light Transmission Spectroscopy, with the latter
based on the same physical principles as the former but
implemented as a transportable instrument with a mod-
ified optical package). LTS works by measuring shifts in
the size of carboxylated nanobeads of a known size (typ-
ically 200 nm in diameter) that are functionalized with
18–26 bp long oligonucleotide tags encoding a species
specific diagnostic sequence. Beads in solution increase
in size when DNA from the target organism anneals to
the oligonucleotide tags, which can be detected by LTS.
Traditional surveillance methods (e.g., nets, for aquatic
taxa) usually have low capture probabilities, making
them reliable only for species that are moderately to
highly abundant (Jerde et al. 2011). Thus, traditional
methods produce many false negative inferences (Gu &
Swihart 2004). Genetic monitoring continues to grow in
importance for a variety of conservation and manage-
ment applications (Schwartz et al. 2006), and advance-
ments in noninvasive genetic sampling (Beja-Pereira et al.
2009) represent particularly important strides in the
management of rare species. For example, screening wa-
ter samples for rare invasive species can be a needle in
a haystack problem, but the eDNA approach reduces the
severity of that challenge (Lodge et al. 2012).
Species surveillance using eDNA exploits an advantage
of aquatic sampling that can aid in detection: the aque-
ous environment often contains microscopic bits of tis-
sue in suspension (e.g., sloughed tissues or cells, larvae or
adults that are microscopic, milt, eggs, extracellular DNA
from degraded tissues, scales, and invertebrate exoskele-
tons). As a result, it is possible to sample DNA from even
rare taxa that are present, but not detectable by tradi-
tional means. The eDNA method has been successfully
developed and applied to Asian carp invasions (Jerde
et al. 2011) and for other species surveillance questions
(Amos et al. 1992; Ficetola et al. 2008; Dejean et al. 2011;
Goldberg et al. 2011; Thomsen et al. 2012). Here, we use
eDNA filtering and extraction methods to isolate DNA for
PCR and LTS analysis to attempt to detect five invasive
target organisms in two different types of water samples.
The first sample type consisted of lake water collected
from the field and seeded with microscopic tissue samples
from five high-risk invasive species. The second type was
water collected from two different lakes where the ze-
bra mussel, D. polymorpha, was known to be present and
absent, respectively, to test the effectiveness of LTS on
unseeded samples. Our work expands upon previous
work (Mahon et al. 2011; Mahon et al. 2013) by demon-
strating that combining eDNA with LTS can provide a
sensitive and real-time solution for screening real-world
water samples for multiple high-risk invasive species. This
Conservation Letters 6:6 November/December (2013) 402–409 Copyright and Photocopying: 
eDNA-LTS species detection
approach will be useful for future research as well as con-
servation and management efforts involving detection of
rare species such as incipient invasions or remnant popu-
lations of imperiled species. Our approach will also ben-
efit efforts by the private sector or regulatory agencies
to detect and minimize contamination of ballast water or
live organism shipments with harmful species.
Methods
Target species, water sources, and eDNA
sampling
For the experiment in which we seeded lake water sam-
ples with DNA from target organisms, water was collected
from St. Joseph Lake, Notre Dame, IN (41◦ 42’ 21.55” N,
86◦ 14’ 20.65” W) in February 2012. The water samples
were seeded with tissue from one of five different species
that have either invaded or pose a high-risk of invading
the Great Lakes and/or estuaries in North America via
ships’ ballast waters: (1) Dreissena polymorpha (zebra mus-
sel) and (2) D. bugensis (quagga mussel), which have vi-
able populations in the Great Lakes basin, (3) Eriocheir
sinensis (Chinese mitten crab), which has been reported
in the Great Lakes and is likely able to establish (Herborg
et al. 2007), but is not yet reproducing (Tepolt et al. 2007),
(4) Limnoperna fortunei (golden mussel), a native to cen-
tral and southeastern Asia, which has invaded multiple
river drainages in South America (Pie et al. 2006), and
is likely to establish and outcompete zebra and quagga
mussels in the Great Lakes if introduced (Boltovskoy et al.
2009), and (5) Carcinus maenas (green crab), a marine crab
that has often been economically and environmentally
damaging where it has been introduced, but is not re-
ported in the Great Lakes (Deagle et al. 2003; Patil et al.
2005). None of these five species have been detected in
St. Joseph Lake.
To isolate eDNA from samples, 500 ml of lake water
was filtered through a 20 micron polycarbonate mem-
brane filter (GE Water & Process Technologies, Trevose,
PA, USA). For the seeded samples, a microscopic frag-
ment of tissue was removed from an adult specimen of
a target species (previously preserved in 95% EtOH and
frozen at –20◦ C) using fine-tipped dissecting forceps and
added to the water sample prior to filtering (see Table 1
for tissue masses). Adding tissue prior to filtering simu-
lated the normal sampling process of the eDNA approach.
We generated four replicate seeded eDNA samples for
each of the five target species from four different indi-
viduals. In addition, one control eDNA sample with no
target tissue was analyzed in parallel with each species,
resulting in a total of 25 eDNA samples for the seed
experiment.
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eDNA-LTS species detection S.P. Egan et al.

Table 1 Five target species analyzed in the study (four specimens of each and 20 µl of Proteinase K, vortexed for 15 seconds, and
taxon). Also given are the mass of tissue from the specimen used to seed incubated at 63◦ C for 1 hour. After incubation, 700 µl
a water sample, the concentration of eDNA extracted from the sample,
of a 24:1 chloroform : isoamylalcohol solution was added
and the concentration of the PCR amplification product generated using
and vortexed for 5 seconds, centrifuged at 14,000 RPM
universal mtDNA invertebrate primers
for 10 minutes, and the supernatant transferred to a new
Target high- Tissue eDNA [C] PCR [C] tube. Next, 1 ml of isopropanol was added, the sam-
risk species Ind. ID mass (µg) (ng/µl) (ng/µl) ple inverted gently to mix, and incubated at –20◦ C for
Limnoperna fortunei L1 128 101.7 118.6 ≥4 hours. After this, the samples were centrifuged at
(golden mussel) L2 148 84.0 117.9 14,000 rpm for 20 minutes to form pellets that were
L3 404 136.5 114.5 washed twice with 500 µl 70% ethanol. Pellets were
L4 122 68.1 112.8 dried for 10 minutes at 45◦ C in a vacuum centrifuge
L-control 0 120.8 120.6
and then resuspended in 50 µl of TE buffer and stored
Dreissena polymorpha Z1 66 323.8 124.0
at 4◦ C. The DNA concentration of each extraction was
(zebra mussel) Z2 530 117.5 114.7
Z3 466 418.0 120.8 measured on a Nanodrop 2,000 per the manufacturer’s
Z4 284 209.3 118.0 instructions (Thermo Scientific, Waltham, MA, USA)
Z-control 0 112.6 120.5 (Table 1).
Dreissena bugensis Q1 320 50.8 118.2 Each eDNA sample was first screened using a species-
(quagga mussel) Q2 420 56.5 115.6 specific set of primers to confirm the presence of the tar-
Q3 212 51.6 122.5
get species in each of the four seeded samples for the
Q4 128 26.4 117.3
five high risk species. PCR was then performed on eDNA
Q-control 0 117.8 115.9
Eriocheir sinensis M1 528 96.1 122.9 samples using universal invertebrate mtDNA primers that
(Chinese mitten M2 100 3.8 122.2 amplify a ∼600 bp fragment of the COI gene (Table 2).
crab) M3 174 15.6 116.5 The total PCR reaction volume was 25 µl and included:
M4 56 11.8 117.6 1 µl DNA template, 2.5 µl 10X buffer, 2.5 µl MgCl2 ,
M-control 0 106.7 117.6 0.5 µl universal invertebrate forward primer (LCO-1490),
Carcinus maenus G1 156 75.3 120.4
0.5 µl universal invertebrate reverse primer (HCO-2198),
(green crab) G2 222 85.2 117.4
0.5 µl dNTPs, 0.15 µl Taq polymerase, and 17.35 µl
G3 138 64.8 117.5
G4 156 70.6 112.9 ddH2 O. PCR reaction conditions were: (1) 94◦ C for 1
G-control 0 119.8 116.5 minute, (2) 94◦ C for 30 seconds, (3) 41◦ C for 45 sec-
onds, (4) 72◦ C for 1 minutes, (5) repeat steps 2–4 for
30 cycles, and (6) 72◦ C for 8 minutes (Folmer et al.
1994). To alleviate possible stochastic effects in PCR, we
For the unseeded experiment, water was sampled in ran eight replicate 25 µl reactions per eDNA sample,
June 2012 from Eagle Lake, MI (41◦ 48’ 22.0314” N, pooling the amplification products in a total volume of
86◦ 1’ 31.2666” W), where D. polymorpha is known 200 µl. A 10 µl subsample of the pooled product was
to occur (Perry et al. 1997) and in December 2011 electrophoretically run on a 1% agarose gel to confirm
from an unnamed lake at the USGS research station in amplification.
Columbia, MO (38◦ 54’ 49.4742” N, 92◦ 16’ 22.3746”
W), which has no zebra mussels. A total of six different
500 ml water samples were analyzed for each of the two
Nanoparticle preparation and hybridization
lakes.
to PCR products
All water samples were put on ice immediately after
collection and filtered with in 24–48 hours. All filters Polystyrene nanoparticle beads were functionalized with
were frozen at –20◦ C and extracted within 2 weeks. We species-specific oligonucleotide tags as described in Li
have found that these time windows provide high qual- et al. (2011) and Mahon et al. (2013). Table 2 lists
ity samples for screening (Jerde et al. 2011; Mahon et al. the species specific tags used in the study. To de-
2013). tect target species, double-stranded PCR amplified DNA
was denatured by heating to 95◦ C for 2 minutes,
then immediately chilled on ice for 2 minutes. Af-
ter chilling, 10 µl of denatured PCR product was
eDNA extraction and PCR
combined with 20 µl of functionalized bead solution
To isolate and purify eDNA, the filter paper for each wa- and incubated at 48◦ C for 1 minute prior to LTS
ter sample was combined with 700 µl of CTAB Buffer measurement.

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S.P. Egan et al.
Table 2 Universal mtDNA PCR primers and species-specific tags used in this study
Oligonucleotide
Universal PCR primers
Universal invertebrate forward (LCO-1490)
Universal invertebrate reverse (HCO-2198)
Species-specific tag for LTS
Limnoperna fortunei (golden mussel)
Dreissena polymorpha (zebra mussel)
Dreissena bugensis (quagga mussel)
Eriocheir sinensis (Chinese mitten crab)
Carcinus maenus (green crab)
LTS and nanoparticle measurement
LTS measures the size increase of functionalized nanopar-
ticles in suspension when the oligonucleotide tags on
their surface hybridize with the complementary strand
of a target species in the PCR amplification product. LTS
is based on measuring the wavelength-dependent light
transmittance through a sample cell containing nanopar-
ticles plus suspension fluid compared with that of a sim-
ilar cell containing only the suspension fluid (see Li et al.
2010; Li et al. 2011 for details). For each sample, the di-
ameter of the hybridized nanobeads was measured four
times, twice at each of two different concentrations. The
first concentration was ∼50 ng DNA/µl (range: 48–53
ng/µl) and was achieved by mixing 150 µl of pooled
PCR product with 700 µl of ddH2 O. Previous studies have
shown this to be an effective concentration for LTS mea-
surement (Mahon et al. 2013). The second concentration
of ∼25 ng/µl was half of the first (75 µl of pooled PCR
product with 775 µl of ddH2 O). LTS measurements were
performed for functionalized nanoparticles both prior to
and following hybridization to PCR products.
Results
Seeded water samples
The concentration of DNA extractions and PCR amplifi-
cations did not differ between seeded versus control sam-
ples from St. Joseph Lake (mean ± SE presented through-
out; DNA extractions: seeded = 103 ± 23 ng/µl, n = 20;
control = 116 ± 2.6 ng/µl, n = 5; tdf = 23 = 0.26, P = 0.80;
PCR product: seeded = 118 ± 0.7 ng/µl, n = 20; control =
118 ± 1.0 ng/µl, n = 5; tdf = 23 = 0.07, P = 0.95; Table 1).
These results imply that the samples from St. Joseph Lake
contained eDNA from other organisms that was PCR am-
plified by the universal mtDNA primers along with the
added DNA from the target species to similar total con-
centrations.
Conservation Letters 6:6 November/December (2013) 402–409 Copyright and Photocopying: 
eDNA-LTS species detection
Sequence Reference
5 -GGTCAACAAATCATAAAGATATTG-3 Folmer et al. 1994
5 -TAAACTTCAGGGTGACCAAAAAATCA-3 Folmer et al. 1994
5 -TCCAACCAGTCCCTACTCCACCCTCT-3 Pie et al. 2006
5 -GAATCTGGTCACACCAATAGATGTGC-3 Mahon et al. 2011
5 -TGTTCAACCCCCACCAAATCCGCCCT-3 Mahon et al. 2011
5 -AGGTGGGTAGACAGTCCACCCAGTA-3 Mahon et al. 2011
5 -GGCAAYGATAATAATAAAAGGAT-3 Senapati et al. 2009
LTS successfully distinguished seeded versus control
samples for all five target species. Figure 1 illustrates one
individual LTS measurement for (A) the control with no
target DNA present and (B–F) one seeded sample per tar-
get species. Figure 2 shows the mean peak shift across all
replicates per species. The baseline diameter of function-
alized nanobeads prior to hybridization with PCR prod-
ucts was 230 ± 0.4 nm. There was no significant shift
observed from this baseline in the control lake water
samples lacking target species tissue (229 ± 0.9 nm; tdf = 38
= 0.95, P = 0.35, n = 5) (Figures 1A and 2). Thus,
the presence of PCR product from nontarget species in
the control samples did not affect nanoparticle size, pre-
sumably because the PCR product from these organ-
isms did not anneal with the species-specific tags on the
nanobeads. In contrast, significant shifts averaging 146 ±
1.8 nm were observed for all 80 of the measured seeded
samples (Figures 1B–F and 2; Table S1). These shifts were
evident as secondary peaks of nanobeads that were dis-
tinct from the control peaks (∼229 nm); secondary peaks
resulted from annealing between amplified target species
sequences and the species-specific oligonucleotide tags.
The size shift of nanobeads was not affected by the
amount of target tissue initially added to the seeded sam-
ples, but there was a subtle (∼9 nm) increase in the mean
size shift detected for LTS conducted at the higher 50
ng/µl DNA concentration (151 ± 2.4 nm, n = 40) com-
pared to the lower 25 ng/µl concentrations (142 ± 2.4
nm, n = 40; tdf = 78 = –2.57, P = 0.01) consistent with pre-
vious studies (Mahon et al. 2013). As concentration was
not the focus of the present study, all means presented
throughout are least-square means for all four replicates
per sample adjusted for the small effect of concentration.
The size shift was not different among the four repli-
cate individuals of a given target species (ANOVA: all P-
values >> 0.05). However, the size shift did vary signifi-
cantly among target species, ranging from 132 (±1.3) nm
for Carcinus maenus up to 168 (±1.2) nm for E. sinensis
(ANOVA: F4,75 = 39.9, P < 0.0001; Figure 2; Table S1).
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eDNA-LTS species detection S.P. Egan et al.

8.0x1010 1.0x108
A Control blank: B Limnoperna fortunei C Dreissena polymorpha
no target present Golden mussel Zebra mussel
7
8.0x10
10
6.0x10

6.0x107
Density distribution (1/mL*nm)

10
4.0x10
7
4.0x10

2.0x1010
2.0x107

0 0
8
1.4x10
D Dreissena bugensis E Eriocheir sinensis F Carcinus maenus
Quagga mussel Chinese mitten crab Green crab
1.2x108

1.0x108

8.0x107

6.0x107

4.0x107

2.0x107

0
0 200 400 600 0 200 400 600 0 200 400 600
Diameter (nm)

Figure 1 DNA-based invasive species detection using LTS. Each panel represents the density distribution generated by LTS for species detection. (A)
LTS particle size distribution for nanobeads functionalized with the zebra mussel tag and hybridized with an eDNA extraction without the zebra mussel
tissue added (control). The other five panels (B–F) represent one of the 16 measurements made on a specimen of each target species (see Methods). In
each trial, there is a peak at ∼230 nm representing nanobeads that did not bind to the target and a second peak (between 300 and 450 nm) representing
functionalized beads bound to the target DNA fragment (positive detection).

260
Average shift in diameter (nm)

220
180
140
100
60
20
-20 _ _ _ _ _
bead + + + + +
+ L. fortunei D. polymorpha D. bugensis E. sinensis C. maenus
tag

Figure 2 Average shift (± standard deviation) in diameter between baseline beads attached with species specific oligonucleotide tags (beads + tag)
and the seeded (+) and control (–) samples for each of the five target high-risk invasive species. All means presented are least-square means of the four
replicates, which remove the significant, but small effect of concentration. All seeded and control samples differed significantly (P < 0.001) for all species
(see Table S1 for details).

Untreated water samples water experiment, zebra mussel was not detected in any
of the control samples from St. Joseph Lake, IN, which is
Zebra mussel was detected in all six water samples from also believed to be without zebra mussel (Figures 1a and
Eagle Lake, MI, which is known to be infested with zebra 2). Across all replicates, the mean size shift was greater
mussel (Figure 3). In contrast, no sample from the USGS for infested (Eagle Lake, 129 ± 1.3 nm, n = 24) than un-
research station lake without zebra mussel tested positive infested (0.5 ± 2.7, n = 24) lake water (ANOVA: F1,47 =
(Figure 3). In addition, as reported earlier for the seeded 1358.0, P < 0.0001; Figure 3).

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S.P. Egan et al. eDNA-LTS species detection

10 8 260
8.0x10 1.0x10
Density distribution (1/mL*nm)

Density distribution (1/mL*nm)

A D. polymorpha absent B D. polymorpha present C

Average shift in diameter (nm)

Columbia, MO Eagle Lake,MI 220
7
8.0x10
10
6.0x10 180
7
6.0x10 140
10
4.0x10
7 100
4.0x10
60
10
2.0x10
2.0x107
20

0 0 -20
0 200 400 600 0 200 400 600 Eagle Lake Columbia
+ _
Diameter (nm) Diameter (nm)
D. polymorpha

Figure 3 DNA-based zebra mussel detection using LTS from naturally infested or uninfested lake water. One of twenty-four individual LTS measurements
from (A) the USGS research station in Columbia, MO, where no D. polymorpha have been reported and (B) Eagle Lake, MI, where D. polymorpha have
been documented for over 20 years (Perry et al. 1997). In panels (A) and (B), there is a peak at ∼230 nm representing functionalized beads and, in the
Eagle Lake panel (B), a second peak at ∼340 nm representing functionalized beads that have bound to the target DNA fragment and represent a positive
detection. In panel (C) average shift (±standard deviation) in the diameter of nanoparticles relative to the baseline beads attached with species specific
oligonucleotide tags (beads + tag) for the infested (+ = Eagle Lake, MI) and uninfested (– = Columbia, MO) lake water samples. Means presented are
least-square means. Infested and uninfested samples differed significantly (P < 0.001) in panel (c) (see Results for details).

Discussion tions in the eDNA samples. It may therefore be possi-

ble to design species specific PCR primers to improve the
Previous research suggested that the detection sensitivity specificity and quantitative aspects of target taxa detec-
of LTS was orders of magnitude greater than traditional tion. Second, there was variation in the peak shift asso-
surveillance methods (Mahon et al. 2013). Specifically, ciated with the concentration of DNA measured. Further
LTS successfully detected PCR amplified DNA isolated analysis is therefore warranted to determine the minimal
directly from target species tissue under laboratory con- threshold of DNA needed to positively confirm the pres-
ditions for samples containing: (1) only the target species, ence of a target taxa, potentially reducing the number of
even at low DNA concentrations (Li et al. 2011); and PCR cycles needed for LTS, and possibly eliminating PCR
(2) target species along with DNA from closely related altogether in some applications.
species (Li et al. 2011; Mahon et al. 2013). Here, we Species detection, including screening pathways for in-
present results demonstrating that a combined eDNA iso- vasive propagules and monitoring habitats for newly in-
lation and LTS approach (Figure 1) will work directly for troduced species (Lodge et al. 2006) as well as monitoring
real-world applications on field collected water samples the distribution and abundance of threatened and en-
from lakes typically containing large numbers of back- dangered populations (Joseph et al. 2006), represents a
ground species of plants, animals, microbes, etc. LTS was fundamental tool for effective conservation and manage-
100% accurate in all cases for: (1) seeded versus control ment. Without accurate, up-to-date knowledge of where
natural water samples containing or lacking five high-risk rare species are, cost effective management is impossible.
invasive species; and (2) for all untreated samples directly Genetic tools such as eDNA sampling and LTS have be-
surveyed from the field for presence or absence of zebra come increasingly prevalent in conservation and man-
mussels. Thus, the eDNA-LTS approach delivered zero agement efforts (Schwartz et al. 2007), and such tools
false positive and zero false negative rates for species have recently experienced rapid development in their ap-
detection. plication to monitoring biodiversity (Lodge et al. 2012;
Our findings highlight two areas for additional research Thomsen et al. 2012). In a management context, there
to further optimize the application of LTS to species de- are at least two major advantages of eDNA methods
tection. First, while LTS was effective in all tests where over traditional methods: (i) increased sensitivity, that
target species tissue was added, there was variation in is, increased probability of detecting a species if it is
the peak shift among taxa. This may have been due to present; and (ii) potentially lower costs, especially as
using universal mtDNA primers that PCR amplified the eDNA methods increase in portability and automation.
five different species to different final DNA concentra- The overarching goal of our work is to enable accurate,

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eDNA-LTS species detection
sensitive, timely, and cost effective detection of target
species.
We now have demonstrated the efficacy of eDNA sam-
pling and LTS measurement as a laboratory-based detec-
tion method. Our ultimate objective is to apply the tech-
nology for rapid point of sampling detection in nature.
To this end, LTS technology is portable, fast, and rela-
tively inexpensive; we have recently developed a beta-
model LTS device that together with a field-based PCR
unit fit in a “carry-on” sized suitcase, run for a day from
a rechargeable 12-volt battery, and following amplifica-
tion can measure a sample in less than 10 seconds. The
next major phase of research is therefore to conduct LTS
testing on site and eventually to transfer the technology
to personnel on the front line of invasive species detec-
tion in the field. In this regard, one extremely valuable
application of LTS would be to rapidly screen the bal-
last tanks of transoceanic ships for invasive species. Cur-
rently, the most common management strategy, ballast
water exchange, is of unknown effectiveness (Costello
et al. 2007; cf. Bailey et al. 2011). Moreover, ballast wa-
ter treatment technologies are still in the research and
development phase (Tsolaki & Diamadopoulos 2010),
and are many years from implementation for most ships
worldwide. An urgent need therefore exists in the mean-
time for rapid detection methods like LTS to identify
harmful invasive species in ballast prior to port entry
to provide adequate time for implementing appropriate
management actions.
In conclusion, our work implies that eDNA sampling
and LTS could enable rapid species detection in the
field in the context of research, voluntary or regulatory
surveillance, and management actions to lower the risk
of the introduction or spread of harmful species. Ballast
water monitoring is one of many potential applications
for LTS with ramifications for environmental protection,
public health, and economic health. Although we have
focused on aquatic habitats, the technology could be used
to test for any organism in any habitat. Future research
will expand analyses to include threatened or endangered
species, pest species of agricultural concern, infectious
species of health concern (e.g., pathogens and parasites),
and species that pose biosecurity risks.
Acknowledgments
We would like to thank the Great Lakes Protection Fund,
the U.S. Environmental Protection Agency, and the Uni-
versity of Notre Dame’s Environmental Change Initiative
and its Advanced Diagnostics & Therapeutics Initiative for
providing funding for this research.
408 Conservation Letters 6:6 November/December (2013) 402–409
S.P. Egan et al.
Supporting Information
Additional Supporting Information may be found in the
online version of this article at the publisher’s web site:
Figure S1. General schematic of the eDNA – LTS
species detection process. (1) Water samples are taken
from aquatic environments (photo credit: Frank Oliver,
Indiana DNR), (2) water sample is run through a filter,
which captures eDNA fragments (photo credit: Univ. of
Notre Dame), (3) eDNA is extracted directly from the
filter paper, (4) PCR amplification of mtDNA fragment
containing species diagnostic differences using universal
primers, (5) hybridization of denatured, single-stranded
PCR product with nanobeads covered with a short ∼30 bp
oligonucleotide that will bind only to the target species,
and (6) the diameter of the nanobeads in solution are
measured by LTS platform, where a single peak around
the original diameter of the beads will appear when no
target species DNA is present and two peaks, one of the
original size and one ∼150 bp larger, will appear when
the target species is present. See Methods section for
detailed descriptions of each step. Depicted in step 6 is
the original (bench-top) implementation of LTS (Li et al.
2010, 2011), which has been modified in part to create a
portable instrument, on which the data for these experi-
ments were taken.
Table S1. Mean peak shift detected by LTS follow-
ing hybridization for individual specimens and for the
pooled total across all four specimens (all) for each of
the five tested target species. Significance determined by
one-sample t-tests to null hypothesis of no peak shift (= 0
nm). All means presented are least-square means of the
four replicates, which remove the significant, but small
effect of concentration (see Results).
References
Amos, W., Whitehead, H., Ferrari, M. J., Glockner-Ferrari,
D.A., Payne, R. & Gordon, J. (1992). Restrictable DNA
from sloughed cetacean skin: its potential for use in
population analysis. Mar. Mam. Sci., 8, 275–283.
Bailey, S.A., Deneau, M.G., Jean L, Wiley, C.J., Leung, B. &
MacIsaac, H.J. (2011). Evaluating efficacy of an
environmental policy to prevent biological invasions.
Environ. Sci. Technol., 45, 2554–2561.
Beja-Pereira, A., Oliveira, R., Alves, P.C., Schwartz, M.K. &
Luikart, G. (2009). Advancing ecological understandings
through technological transformations in noninvasive
genetics. Mol. Ecol. Res., 9, 1279–1301.
Boltovskoy, D., Karatayev, A., Burlakova, L., et al. (2009)
Significant ecosystem-wide effects of the swiftly spreading
invasive freshwater bivalve Limnoperna fortunei.
Hydrobiologia, 636, 271–284.
Copyright and Photocopying: 
C 2013 Wiley Periodicals, Inc.
S.P. Egan et al.
Costello, C., Drake, J.M. & Lodge, D.M. (2007). Evaluating an
invasive species policy: ballast water exchange in the Great
lakes. Ecol. Appl., 17, 655–662.
Deagle, B.E., Bax, N., Hewitt, C. L. & Patil, J.G. (2003).
Development and evaluation of a PCR-based test for
detection of Asterias (Echinodermata: Asteroidea) larvae in
Australian plankton samples from ballast water. Mar.
Freshwater Res., 54, 709–719.
Dejean, T., Valentini, A., Duparc, A., et al. (2011). Persistence
of environmental DNA in freshwater ecosystems. PLoS
ONE, 6, e23398.
Drake, J.M. & Lodge, D.M. (2007). Rates of species
introductions in the Great Lakes via ships’ ballast water
and sediments. Can. J. Fish. Aquat. Sci., 64, 530–538.
Ficetola, G.F., Miaud, C., Pompanon, F. & Taberlet, P. (2008).
Species detection using environmental DNA from water
samples. Biol. Lett., 4, 423–425.
Folmer, O., Black, M., Hoeh, W., Lutz, R. & Vrijenhoek, R.
(1994). DNA primers for amplification of mitochondrial
cytochrome c oxidase subunit I from diverse metazoan
invertebrates. Mol. Mar. Biol. Biotechnol., 3, 294–
299.
Goldberg, C.S., Pilliod, D.S., Arkle, R.S. & Waits L.P. (2011).
Molecular detection of vertebrates in stream water: a
demonstration using Rocky Mountain Tailed Frogs and
Idaho Giant Salamanders. PLoS ONE, 6, e22746.
Gu, W. & Swihart, R.K. (2004). Absent or undetected? Effects
of non-detection of species occurrence on wildlife-habitat
models. Biol. Cons., 116, 195–203.
Herborg, L.-M., Jerde, C.L., Lodge, D.M., Ruiz, G.M. &
MacIsaac, H.J. (2007). Predicting invasion risk using
measures of introduction effort and environmental niche
models. Ecol. Appl., 17, 663–674.
Jerde, C.L., Mahon, A.R., Chadderton, W.L. & Lodge, D.M.
(2011). “Sight-unseen” detection of rare aquatic species
using environmental DNA. Conserv. Lett., 4, 150–157.
Joseph, L.N., Field, S.A., Wilcox, C. & Possingham, H.P.
(2006). Presence-absence versus abundance data for
monitoring threatened species. Conserv. Biol., 20,
1679–1687.
Keller, R.P., Lodge, D.M., Shogren, J.F. & Lewis, M. (2009).
Bioeconomics of Invasive Species: Integrating Ecology, Eco-
nomics, Policy, and Management. Oxford University Press,
Oxford.
Keller, R.P., Drake, J.M., Drew, M.B. & Lodge, D.M. (2010).
Linking environmental conditions and ship movements to
estimate invasive species transport across the global
shipping network. Divers. Distrib., 17, 93–102.
Li, F., Schafer, R., Hwang, C.-T., Tanner, C.E. & Ruggiero, S.T.
(2010). High-precision sizing of nanoparticles by laser
transmission spectroscopy. Appl. Optics., 49, 6602–
6611.
Conservation Letters 6:6 November/December (2013) 402–409 Copyright and Photocopying: 
eDNA-LTS species detection
Li, F., Mahon, A.R., Barnes, M.A., et al. (2011). Quantitative
and rapid DNA detection by laser transmission
spectroscopy. PLoS One, 6, e29224.
Lodge, D.M., Williams, S., MacIsaac, H.J., et al. (2006).
Biological invasions: recommendations for U.S. policy and
management. Ecol. Appl., 16, 2035–2054.
Lodge, D.M., Turner, C.R., Jerde, C.L., et al. (2012).
Conservation in a cup of water: estimating biodiversity and
population abundance from environmental DNA. Mol.
Ecol., 21, 2555–2558.
Mahon, A.R., Barnes, M.A., Senapati, S., et al. (2011).
Molecular detection of invasive species in heterogeneous
mixtures using a microfluidic carbon nanotube platform.
PLoS One, 6, e17280.
Mahon, A.R., Barnes, M.A., Li, F., et al. (2013). DNA-based
species detection capabilities using Laser Transmission
Spectroscopy. J. R. Soc. Interface, 10, 20120637. doi:
10.1098/ rsif.2012.0637.
Patil, J. G., Gunasekera, R. M., Deagle, B. E., Bax, N. J. &
Blackburn, S. I. (2005). Development and evaluation of a
PCR based assay for detection of the toxic dinoflagellate,
Gymnodinium catenatum (Graham) in ballast water and
environmental samples. Biol. Invasions, 7, 983–994.
Pejchar, L. & Mooney, H. (2009). Invasive species, ecosystem
services and human well-being. Trends Ecol. Evol., 24,
497–504.
Perry, W.L., Lodge, D.M. & Lamberti, G.A. (1997). Impact of
crayfish predation on exotic zebra mussels and native
invertebrates in a lake-outlet stream. Can. J. Fish. Aquat.
Sci., 54, 120–125.
Pie, M.R., Boeger, W.A., Patella, L. & Falleiros, R.M. (2006). A
fast and accurate molecular method for the detection of
larvae of the golden mussel Limnoperna fortunei (Mollusca:
Mytillidae) in plankton samples. J. Mollus. Stud., 72,
218–219.
Pimentel, D., Zuniga, R. & Morrison, D. (2005). Update on the
environmental and economic costs associated with
alien-invasive species in the United States. Ecol. Econ., 52,
273–288.
Schwartz, M.K., Luikart, G. & Waples, R.S. (2007). Genetic
monitoring as a promising tool for conservation and
management. Trends Ecol. Evol., 22, 25–33.
Tepolt, C.K., Blum, M.J., Lee, V.A. & Hanson, E.D. (2007).
Genetic analysis of the Chinese mitten crab (Eriocheir
sinensis) introduced to the North American Great Lakes and
St Lawrence Seaway. J. Great Lakes Res., 33, 658–667.
Thomsen, P.F., Kielgast, J., Iversen, L.L., et al. (2012).
Monitoring endangered freshwater biodiversity using
environmental DNA. Mol. Ecol., 21, 2565–2573.
Tsolaki, E. & Diamadopoulos, E. (2010). Technologies for
ballast water treatment: a review. J Chem. Technol.
Biotechnol., 85, 19–32.
C 2013 Wiley Periodicals, Inc. 409