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Rapid molecular detection of invasive species in ballast and harbor water by integrating environmental DNA and Light Transmission Spectroscopy Scott Patrick Egan, Erin Grey, Brett Olds, Jeff Feder, Steve Ruggiero, Carol E. Tanner, and David Lodge Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/es5058659 • Publication Date (Web): 16 Feb 2015 Downloaded from http://pubs.acs.org on February 18, 2015

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Environmental Science & Technology

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TITLE: Rapid molecular detection of invasive species in ballast and harbor water by integrating

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environmental DNA and Light Transmission Spectroscopy

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Scott P. Egan1,2,3,*, Erin Grey3,4, Brett Olds3, Jeffery L. Feder2,3, Steven T. Ruggiero3,5, Carol E.

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Tanner3,5, & David M. Lodge2,3

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Department of BioSciences, Rice University, Houston, TX 77005

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Department of Biological Sciences, University of Notre Dame, Notre Dame, Indiana 46556

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Environmental Change Initiative, University of Notre Dame, Notre Dame, Indiana 46556

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College of Science, Governors State University, University Park, IL 60484

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Department of Physics, University of Notre Dame, Notre Dame, Indiana 46556

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*Corresponding author: Scott P. Egan, Department of BioSciences, Rice University, Houston,

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TX 77005; [email protected], phone: 615-618-6601, fax: 713-348-5232

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Running title: Aquatic invasive species detection using eDNA

ACS Paragon Plus Environment


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ABSTRACT

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Invasive species introduced via the ballast water of commercial ships cause enormous

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environmental and economic damage worldwide. Accurate monitoring for these often

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microscopic and morphologically indistinguishable species is challenging, but critical for

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mitigating damages. We apply eDNA sampling, which involves the filtering and subsequent

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DNA extraction of microscopic bits of tissue suspended in water, to ballast and harbor water

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sampled during a commercial ship’s 1,400km voyage through the North American Great Lakes.

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Using a lab-based gel electrophoresis assay and a rapid, field-ready Light Transmission

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Spectroscopy (LTS) assay, we test for the presence of two invasive species: quagga (Dreissena

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bugensis) and zebra (D. polymorpha) mussels. Furthermore, we spiked a set of uninfested ballast

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and harbor samples with zebra mussel tissue to further test each assay’s detection capabilities. In

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unmanipulated samples, zebra mussel was not detected, while quagga mussel was detected in all

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samples at a rate of 85% for the gel assay and 100% for the LTS assay. In the spiked

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experimental samples, both assays detected zebra mussel in 94% of spiked samples and 0% of

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negative controls. Overall, these results demonstrate that eDNA sampling is effective for

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monitoring ballast-mediated invasions and that LTS has the potential for rapid, field-based

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detection.

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Key Words: conservation, environmental DNA, invasive species, light transmission spectroscopy


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INTRODUCTION

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Invasive species have had a globally damaging effect on freshwater and marine ecosystems,

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adversely impacting biodiversity and human commerce.1 In the United States alone, invasive

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species cause environmental damage and economic losses in excess of US $137 billion

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annually2,3 and are now recognized as one of the leading global threats to native biodiversity and

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ecosystem functions.4,5 Within the North American Great Lakes, there are over 180 aquatic

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invasive species that have significantly altered the ecosystem and are estimated to cost up to

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$800 million annually.6-10 The majority of these aquatic invasive species (>70%) entered through

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transoceanic shipping routes, including the ballast water discharge of incoming ships.11-14 A

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general lack of sensitive technologies for early detection can allow harmful invasions to establish

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to the point where it is too late to intercede for an increasing number of highly damaging

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species.15

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The ecological and economic threats of aquatic invasions therefore require improved monitoring

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of the various anthropogenic pathways, such as ships’ ballast water. Unfortunately, ballast water

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treatment technologies are still mainly in the research and development phase.16,17 Consequently,

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there is an urgent need for rapid detection methods to identify harmful invasive species in ships’

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ballast prior to port entry to provide adequate time for initiating appropriate control actions. Even

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if ballast water treatment systems become widely adopted, the need will still exist for rapid

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inspection and enforcement tools, as a monitoring firewall against harmful invasives evading the

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first line of control.

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Traditional microscope-based methods have been used to examine ballast samples for target

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organisms, but are expensive and slow.18 By the time results from microscopic examination are

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available, ships have long since reached their destinations, making it too late to intervene.14 In

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addition, the most often encountered life stage in ballast tanks, larvae, are often not identifiable

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to the species level using morphological keys.19 Accordingly, a rapid and sensitive on-board

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detection platform that can identify target species in large volume ballast samples requiring

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minimal user expertise would greatly facilitate inspection (voluntary or regulatory), enforcement,

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and control efforts.

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Species detection also represents a fundamental tool for effective conservation and management.

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Without accurate, up-to-date knowledge of where rare endangered species are and where

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invasive propagules reside, cost effective management is impossible. Environmental DNA

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(eDNA) sampling has become increasingly prevalent in such conservation and management

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efforts20 and has recently experienced rapid development in their application to monitoring

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biodiversity.21,22 In a management context, there are at least two major advantages of eDNA

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methods over traditional methods: (1) increased sensitivity22,23 (i.e., increased probability of

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detecting a species if it is present) and (2) potentially lower costs, especially as eDNA methods

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increase in portability and automation.24,25

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The overarching goal of our work is to enable accurate, sensitive, timely, and cost effective

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detection of target species. In the present study, we merge two new, but proven technologies for

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the purpose of quickly and accurately detecting target species in ship’s ballast and surrounding

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harbor waters. First, we use DNA filtering methods to isolate and purify field samples of


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environmental DNA (eDNA) from a ship’s ballast water and the surrounding harbor area. In this

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regard, analysis of aquatic samples with traditional methods, such as microscopy and

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morphology, often poses insurmountable logistical challenges for detecting rare and

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taxonomically-challenging organisms. However, species surveillance using eDNA exploits the

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fact that the aqueous environment often keeps microscopic bits of tissue from target invasive

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species in suspension in the water column, making it easier to sample DNA from even rare

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organisms that are present but invisible to traditional tools. We define eDNA here as all types of

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tissue samples collected by filtering water from an aquatic environment. These samples can

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include, but are not limited to: sloughed tissues or cells, larvae or adult organisms that are

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microscopic, milt, eggs, extracellular DNA from degraded tissues, scales, and invertebrate

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exoskeletons. The eDNA method has been successfully developed and applied to possible Asian

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carp invasions into the Great Lakes23,26 and for other species surveillance questions.22,27-31 The

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eDNA approach to species surveillance is rapidly developing into a burgeoning field of research

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in molecular ecology and application in conservation and invasive species management.21,32

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Second, we combine the efficacy of eDNA sampling with a new field-based tool for target DNA

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detection, Light Transmission Spectroscopy (LTS).33,34 Typically, eDNA detection methods have

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relied on PCR or qPCR to amplify target DNA present in a sample, where amplification is

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equivalent to detection.35 However, the LTS platform offers the possibility of field-based species

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detection because it is portable, rapid, and requires low electrical power to operate.24 LTS

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determines the size, shape, and number of nanoparticles in suspension by measuring wavelength-

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dependent light transmittance through a sample containing nanoparticles in suspension. 33,34 LTS

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is applicable for species detection by measuring nanoparticles that specifically bind to target


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species DNA and therefore grow in size.24,25,34 Specifically, carboxylated nanoparticles of a

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known size (typically around 200nm in diameter) are bound with species-specific tags

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representing ~18-26 base pair oligonucleotides that bind to a unique section of target DNA.

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When the tagged nanoparticles are in solution with the DNA of a target species, the tagged

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nanoparticles grow in diameter because the tags bind to PCR amplified DNA fragments. LTS

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can detect the size shift in particle size within seconds (Supplemental Figure 1). The LTS device

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is portable and can run off a car’s battery. Thus, LTS technology has promise for uses where

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speed and portability are paramount (e.g. real-time field surveys of ballast water, law

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enforcement inspections).

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In the present study, we test the effectiveness of merging eDNA sampling with LTS for invasive

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species detection using two test species that are currently found in the ballast waters of ships

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from foreign ports entering the Great Lakes: (1.) Dreissena polymorpha (zebra mussel) and (2.)

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Dreissena bugensis (quagga mussel). Both invasive species are known to be in the Great Lakes

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with viable populations. We report that combining eDNA sampling with LTS can rapidly and

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accurately detect target invasive species in two types of environmental samples where the threat

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of aquatic invasion is the highest, within the ballast water of a large commercial shipping vessel

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and in the surrounding harbor where these ship-borne aquatic invasions usually begin. Moreover,

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these data support the continued development of the LTS portable real-time DNA detection

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system that could be used for onboard monitoring during a ship’s passage prior to ballast

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discharge to dramatically reduce the impacts of future invasions.12,14

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MATERIALS and METHODS


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Ballast and harbor sampling and eDNA filtration

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Ballast water was collected directly from the intake and discharge of a 1,000-ft. shipping vessel,

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the M/V Indiana Harbor, during its five-day, 1,400-km voyage from the Indiana Harbor (41° 39'

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57.1788" N, 87° 26' 43.9038" W) near Gary, Indiana, on Lake Michigan, to the Duluth-Superior

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Harbor near Duluth, MN (46° 45' 14.3136" N, 92° 5' 59.1426" W), on Lake Superior during mid-

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October 2012 (Supplemental Figure 2). In parallel, we also collected water from the ballast

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intake source, Indiana Harbor, IN and the destination port receiving the ballast water discharge,

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the Duluth-Superior Harbor, MN. All water samples were collected in a two-liter sterile Nalgene

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bottle as follows: Ballast intake samples (n = 20) were collected during ballast intake from a

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direct tap connected to the pumping system on October 17th, 2012. Source harbor samples (n = 5)

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were collected from the surface of the water directly next to the ship during ballast intake.

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Receiving harbor surface water samples (n = 3) were collected five days later (October 22nd,

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2012) in the same manner just prior to ballast discharge. Ballast discharge samples (n = 13) were

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collected from the direct line to the pumping system from three separate tanks (designated tank

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#3 = 3 samples, tank #678 = 3 samples, and tank #5 = 5 samples). Each ballast tank is

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independently sealed from the other and thus, can potentially harbor different contaminants

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(biotic or abiotic) in each.

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For each of the ballast and harbor samples, a 250mL aliquot was filtered through a 1.2-micron

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polycarbonate membrane filter (GE Water & Process Technologies) to screen for the aquatic

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invasive species, quagga mussel (Dreissena bugensis), expected to be present and active in the


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Great Lakes region at the time of sampling36-38 and zebra mussel (Dreissena polymorpha), also

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present but not expected to be as active as quagga mussels at this time of year. We will refer to

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these samples throughout as the ‘standard’ ballast and harbor samples.

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Spiked experimental ballast and harbor samples

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To further test the effectiveness of eDNA sampling, we spiked a second 250mL aliquot of ballast

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water samples with a small fragment of tissue (range in tissue amount: 66 – 520 µg) from the

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aquatic invasive species zebra mussel (Dreissena polymorpha). As discussed above, zebra

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mussels are present in the Great Lakes region, but not present in the water column as

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temperatures cool during the fall, such as when we sampled in October of 2012.37,38 Thus, the

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prediction is that the spiked samples should serve as a positive control for the negative result

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expected for zebra mussel from the field samples. All samples (N = 41) were screened for zebra

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mussels using eDNA, species-specific mitochondrial DNA (mtDNA) markers (Table 1) and gel

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electrophoresis prior to the spiking experiment, with 0% detection of zebra mussels. Each tissue

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sample was removed from a preserved adult using fine-tipped dissecting forceps and added to the

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eDNA filter to imitate the event that this target invasive species was collected during ballast

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water screening. We spiked sixteen samples distributed equally across the four sampling

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categories. In addition, we generated eight control eDNA samples where no target zebra mussel

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tissue was added. The control eDNA extractions were done in parallel with the spiked samples.

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We will refer to these samples henceforth as the ‘experimental’ ballast samples. All experimental

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ballast samples were filtered through a 1.2-micron polycarbonate membrane filter (GE Water &

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Process Technologies).


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DNA extraction from filter and Polymerase Chain Reaction

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eDNA was isolated from filters following the protocol described in of Jerde et al.23 and Egan et

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al.24. In brief, the polycarbonate membrane filter from standard and experimental ballast and

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harbor samples was combined with 700 µL of CTAB Buffer and 20 µL of Protinase K, vortexed

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for 15 seconds, and incubated at 63ºC for 1hour. After incubation, 700 µL of a 24:1 chloroform :

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isoamylalcohol solution was added to the solution and vortexed for 5 seconds, centrifuged at

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14000 RPM for 10 minutes, and the supernatent was transferred to a new tube. Next, 1mL of

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isopropanol was added, the sample was inverted gently to mix, and incubated at -20ºC for ≥4

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hours. Samples were removed from -20ºC, centrifuged at 14000 RPM for 20 minutes to form a

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pellet, the supernatent was poured off, and washed twice with 500µL 70% ethanol, pouring off

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ethanol and maintaining the pellet each time. Pellets were dried for 10 minutes at 45ºC in a

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vacuum centrifuge and then resuspended in 50µL TE Buffer overnight at 4ºC in a refrigerator.

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We measured the DNA concentration of each sample extracted using a Qubit 2.0 fluorometer

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(Life Technologies) with either the dsDNA Broad Range or High Sensitivity assay kits

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(Invitrogen). Sample DNA concentration was first measured with the Broad Range kit, and

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samples with undetectable DNA concentration were then measured again with the High

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Sensitivity kit. Two PCRs were performed on each sample. First, a species-specific primer was

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used in concert with gel-electrophoresis for a standard detection. Second, a universal primer,

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which amplifies the same section of the mtDNA in most invertebrates (Folmer et al. 1994), was

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used in concert with the LTS device to test this new, field-ready device.


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The first PCRs were performed using species-specific primers listed in Table 1 that would

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amplify a section of the cytochrome c oxidase subunit I (COI) gene in the mtDNA for one of the

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target species only (quagga or zebra mussel). The PCRs were used to confirm the success of

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eDNA sampling from ballast and harbor water for species-specific detection of the two taxa. The

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species-specific mtDNA markers were developed for previous studies where they were tested for

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specificity, even against their closest related sympatric relative (i.e., quagga versus zebra and

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vice versa), and some fragments were sequence verified for additional verification (Li et al.

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2011, Mahon et al. 2011, Mahon et al. 2013, Egan et al. 2013). Species-specific PCR was

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followed by standard gel electrophoresis detection analysis and included positive and negative

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controls. The lab-based gel detection offered a baseline for detection to which we could compare

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results from LTS detection. For each sample, nine replicate PCRs were run.

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The second set of PCRs used universal invertebrate primers (Table 1), which amplify a ~600

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base pair section of the mtDNA COI gene for both target and non-target invertebrate species.

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The long term strategy for using universal primers is to develop a single PCR from which it

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would be possible to use LTS to detect the presence or absence of multiple species of interest

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using nanoparticles functionalized with different species-specific tags (i.e., single stranded

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oligonucleotides). The current LTS sequence tag is ~340bp downstream of the 5’ end of the

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~600bp mtDNA fragment amplified by the universal primers. In effect, the universal primers

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would amplify all invertebrates present in a sample. Then, separate LTS analyses could be

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conducted on the single PCR reaction using a different species specific set of functionalized


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nanoparticles for each LTS trial to determine if a particular target species of invertebrate was

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present in the sample.24,25,34

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PCRs with both primer sets were performed as follows: (i) 94ºC for 1 minute, (ii) 94ºC for 30

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seconds, (iii) 41ºC for 45 seconds, (iv) 72ºC for 1 minute, (v) go to step 2 and repeat 30 times,

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and (vi) 72ºC for 8 minutes.39 The total reaction volume was 25 µL and included: 1µL DNA

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template, 2.5µL 10X buffer, 2.5µL MgCl2 (25mM), 0.5µL forward primer (10µM), 0.5µL

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reverse primer (10µM), 0.5µL dNTPs (2.5mM each; Invitrogen), 0.15µL Taq polymerase

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(5Prime), and 17.35µL ddH2O.

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Test of PCR inhibition

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We also tested for PCR inhibition in each sample. Abiotic substances, such as humic and fulvic

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acids, are known PCR inhibitors that are likely to be suspended or dissolved in ballast and port

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waters, where they are found in soils and sediments and get turned up a lot due to boat traffic and

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repeated dredging within the ports.40 To test for PCR inhibition, we compared the observed and

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expected concentration of a positive control DNA sequence (Eurogentec Universal Exogenous

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qPCR Positive Control) using quantitative real-time PCR on an Eppendorf MasterCycler

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realplex machine. The master mix for each sample included 3.6 µL H2O, 10.0 µL ABI TaqMan

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Environmental, 2.0 µL positive control mix (Eurogentec), 0.4 µL positive control DNA

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(Eurogentec), and 4.0 µL template DNA.

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Nanoparticle preparation and hybridization to eDNA samples for LTS

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To detect target species using LTS,25,34 we first bound polystyrene beads with a carboxylated

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surface to a species-specific oligonucleotide tag that will only bind to DNA fragment amplified

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in the target species (see Table 1 for species specific tags). To generate these ‘functionalized’

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nanoparticles for species detection we followed published protocols.24,25,34 Briefly, we combined

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10 µL of Polybead Carboxylate Microspheres (diameter = 209 nm; [C] = 5.68x1012 beads/mL;

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Polysciences, Inc.) with 1 mL of ddH2O, 50 µL of Bead Coupling Buffer (Polysciences, Inc.),

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and 25 µL of an EDC coupling activating solution [EDC = 1-Ethyl-3-(3-dimethylaminopropyl)

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carbodiimide HCl at 192 g/mol; solution = 1mg EDC in 5mL of ddH2O]. Bead solution was

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mixed at 300 rpm on an orbital shaker for 15 minutes. Separately, 10 µL of each species-specific

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oligonucleotide tag ([C] = 100 ng/µL) was combined with 1 mL of ddH2O and 50 µL of Bead

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Coupling Buffer (Polysciences, Inc.) and mixed for 1 minute at 300 rpm on an orbital shaker.

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The bead solution and the tag solution were then combined and mixed constantly at 300 rpm for

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two to four hours. For downstream LTS measurement, a ‘reference’ solution containing all

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reagents except the beads and tags was also made, where bead and tag volume was replaced by

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equal amounts of ddH2O.

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PCR products (double stranded DNA from the amplified mtDNA COI region) from each sample

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was denatured by heating to 95ºC for 2 minutes, then immediately chilled on ice for 2 minutes.

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PCR product was combined with functionalized bead solution ([C] = 1.046109 x 109 beads/mL)

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in a 1:2 ratio at 48ºC for one minute.34 If target DNA was present, it would anneal (or bind) to

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the complementary species-specific tag and the nanoparticle would grow in diameter. We have

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previously shown that it does not grow when combined with non-target DNA.24,25,33,34 Each

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PCR-bead hybridization reaction was then placed on ice until LTS measurement.


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Laser Transmission Spectroscopy (LTS) and nanoparticle measurement

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LTS is used in the context of species detection to measure the size of nanoparticles in

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suspension. For the purpose of DNA diagnostics, LTS can detect the increase in size of the

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functionalized nanoparticles if the species specific tag has bound to the target species PCR

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product causing an increase in the diameter of the bead. LTS is based on measuring wavelength-

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dependent light transmittance through a sample containing nanoparticles in suspension. Briefly,

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the transmission of light through a sample cell containing particles plus suspension fluid is

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recorded along with that of a similar cell containing only the suspension fluid. The fundamental

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data-acquisition process involves measuring the wavelength-dependent transmission of light

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(quantified as extinction) through an aqueous suspension of nanoparticles. Here, the pertinent

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wavelength range is from ~300 to 1000 nm. Given the extinction information, and the known

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wavelength-dependent properties of the beads, Mie theory 33 can be used to accurately determine

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the bead diameter. The extinction data are analyzed and inverted by a computer algorithm that

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outputs the particle size distribution. The sensitivity limit of LTS reported in Li et al.33 for 1025

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nm polystyrene spheres is ~3580 particles/mL (i.e. 3.5x10-17 molar). The precision, accuracy,

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sensitivity, and resolution of LTS using NIST traceable polystyrene particles are detailed in Li et

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al.33, where these properties are quantified for the size range important for DNA detection (~50–

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1000 nm). Further details of LTS theory and operation are detailed in Li et al.33 and Li et al.34

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RESULTS & DISCUSSION

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Species detection probabilities are typically low in aquatic environments, especially for the

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ballast of large commercial shipping vessels.11-14 Yet, effective management of high-risk

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invasive species requires the early detection of an incipient invasion, which increases the

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feasibility of rapid responses to eradicate the species or at least contain its spread.15,41 Here, we

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tested whether eDNA sampling can be an effective tool for species detection in heterogeneous

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ballast and harbor water samples, which contain a variety of aquatic species, their eDNA filtered

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tissues and cells, and a mixture of biotic and abiotic debris.

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Amounts of eDNA isolated from harbor and ballast samples

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First, the concentration of DNA isolated from ballast and harbor water samples was quantified

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using a Qubit 2.0 flourometer (Life Technologies). Ballast intake and discharge were slightly

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less than the harbor samples, which is consistent with ballast representing a subset of available

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eDNA content in each harbor (mean ± SE: Indiana and Duluth harbors = 5.54 ± 1.32 µg/mL,

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Ballast intake and discharge = 3.28 ± 0.65 µg/mL; t-test assuming unequal sample sizes: tdf=19.45

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= 2.16, P = 0.04). However, ballast intake and discharge did not differ significantly (Ballast

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intake = 3.67 ± 0.84 µg/mL, Ballast discharge = 2.68 ± 1.04 µg/mL; t-test assuming unequal

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sample sizes: tdf=27.81 = 0.81, P = 0.43), nor did samples from the two harbors (Indiana source

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harbor = 6.42 ± 0.95 µg/mL, Duluth destination harbor = 4.09 ± 1.17 µg/mL; t-test assuming

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unequal sample sizes: tdf=2.29 = 1.24, P = 0.32). In addition, the DNA concentrations did not differ

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between the spiked and control samples in our experiment (DNA [C] ± SE: spiked = 1.68 ± 0.21

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µg/mL, control = 1.26 ± 0.29 µg/mL; t df=22 = 1.21, P = 0.24). These results are consistent with an


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expectation from real-world ballast and harbor water samples, where the DNA from many

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different organisms would be filtered and extracted from a sample.

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Tests for PCR inhibition

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Prior to screening eDNA samples for invasive species with each technology, we tested for PCR

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inhibition since PCR inhibitors are likely present in ballast and harbor waters.40 There was no

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evidence for systematic biotic or abiotic inhibition of PCRs in the study. We compared the

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observed and expected concentration of a positive control DNA sequence added to each sample

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using quantitative real-time PCR. The greater the difference generated between observed from

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expected concentrations suggests an increased chance of PCR inhibition. None of the 41 standard

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samples (ballast or harbor) or the 24 experimentally spiked samples (zebra or controls) showed

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any evidence of PCR inhibition (all values were less than 1.4; mean ± SE: 0.64 ± 0.01).

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Standard lab-based eDNA detection using gel electrophoresis

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Each standard ballast water sample from the field was screened for quagga and zebra mussel

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eDNA using species specific primers (Table 1) under the prediction that we should detect quagga

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mussels at a higher rate than zebra mussels in the Great Lakes during October because they are

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known to be more cold-tolerant.36-38 Based on gel electrophoresis, quagga mussel was detected in

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35/41 (85%) of the field samples, representing 17/20 of the ballast intake samples, 5/5 of the

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source harbor samples, 12/13 of the ballast discharge samples and 1/3 of the destination harbor

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samples (Table 2). The six samples where quagga was not detected may represent: (1) a natural


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failure to capture quagga mussel in the samples, (2) a failure to isolate quagga DNA in samples

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in which it was present, but perhaps in low frequency, (3) PCR failure, or (4) a failure to detect

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DNA by gel electrophoresis in PCRs in which quagga amplification was low. The overall DNA

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concentration in six negative quagga samples did not, however, differ from the overall mean

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(failed quagga detection samples: 2.36 ± 1.12 µg/mL; one-sample t-test: tdf=5 = -0.88, P = 0.42).

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Thus, it was not the case that the negative quagga results for these six samples was due to little or

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no DNA captured and extracted from the filters.

339 340

Zebra mussel eDNA was not detected in any standard field sample using gel electrophoresis

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(0/41), as expected based on their natural history. However, zebra mussels were detected in

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15/16 of the spiked environmental samples compared to 0/8 control samples (Table 2). The one

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failed zebra detection had undetectable amounts of DNA present based on the Qubit analysis,

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suggesting that zebra mussel tissue was not captured on the filter in this eDNA sample or that the

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DNA extraction itself failed.

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Experimental field-based eDNA detection using Light Transmission Spectroscopy

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Prior to LTS species detection tests, nanoparticles were attached with species-specific

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oligonucleotide tags and measured to be 230 (± 0.4) nm in diameter (Fig. 1A). This LTS size

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distribution is larger than the original diameter of 209-nm for the carboxylated beads and

352

represents the binding of the oligonucleotide tags to the beads.24,34 Thus, 230 nm is the baseline

353

diameter of beads used to test for size shifts associated with positive detection of target quagga

354

and zebra mussel DNA.


16

Page 17 of 38


355 356

LTS successfully detected quagga mussel eDNA in all 41 standard field samples, including the

357

six samples where quagga mussel was not detected using gel electrophoresis. The LTS result

358

suggests that the failure to detect quagga in the six cases by gel electrophoresis was due to a

359

reduced sensitivity of electrophoresis versus LTS to detect target DNA at low concentration. We

360

present LTS measurements from four representative positive quagga mussel detections from the

361

source harbor, ballast intake, ballast discharge, and receiving harbor in Figs. 1B-E, respectively.

362

Positive detection is indicated by two peaks in the size distribution of nanoparticles. The peak

363

having smaller diameter size represents functionalized nanoparticles that did not hybridize with

364

target DNA in the reaction, while the peak of larger-size diameter represents instances when the

365

oligonucleotide tag on the nanobead hybridized with target species DNA. Note, that in Figures

366

1B, 1C, and 1E, the estimated diameters of the nanoparticles measured by LTS matched the

367

predicted sizes of functionalized, non-hybridized nanoparticles (~230 nm) determined in control

368

experiments, with positive detection causing a size shift of from 140 to 160 nm for a subset of

369

nanoparticles that bound target species DNA to mean diameters of 370 to 390 nm (see Fig. 1F

370

for summary statistics for all 41 positive quagga mussel results). In comparison, in Figure 1D,

371

the two peaks were also present indicating a positive detection. However, both peaks were

372

shifted larger than the mean diameters expected for non-hybridizing (230 nm) and hybrizing

373

(~380 nm) nanoparticles. Variation from anticipated peak shifts was observed in 5 of the 57

374

positive results (8.8%) in the current study. The presence of the two peaks represented a positive

375

detection in these 5 cases; since we have never observed a double peak in any negative control in

376

this or any previous study.24,25 However, it is possible that interactions between bound and non-

377

bound nanoparticles may occasionally occur in the hybridization reaction to generate the greater


17


Page 18 of 38

378

than expected shifting of peaks in positive detections. Further work is needed to test this

379

hypothesis. The average size shift (± standard deviation) in the diameter of nanoparticles relative

380

to the baseline beads attached with quagga-specific tags for all 41 standard samples (ballast and

381

harbor) was 139 ± 4.18 nm (Figure 1.F).

382 383

In the experimentally manipulated ballast and harbor water samples, LTS successfully detected

384

zebra mussel eDNA in 15/16 of the spiked samples (94%). The one sample where zebra mussel

385

was not detected by gel electrophoresis coincided with the sample that zebra mussel was not

386

detected by LTS. Thus, either DNA was not successfully isolated from this sample or the PCR

387

failed, which is to be expected with real-world sampling of eDNA. As was the case for gel

388

electrophoresis, all eight control samples were also negative for zebra mussel in LTS tests.

389

Figure 2 shows in panel (A) the LTS measurement of one control sample where no target zebra

390

mussel tissue was added, (B) the positive LTS result for one individual sample spiked with zebra

391

mussel tissue, and (C) the average size shift of 141 ± 6.33 nm standard deviation observed in the

392

diameter of nanoparticles relative to the baseline beads for the 15 cases of positive LTS

393

detection.

394 395

eDNA as an effective tool for species detection in ballast and habor waters

396 397

In unmanipulated field samples, eDNA species detection matched expectations from the natural

398

history of each species.36-38 Zebra mussel, which are known to be less cold-tolerant that quagga

399

mussels, was not detected, while quagga mussel was detected in most samples at a rate of 85%

400

for the gel assay and 100% for the LTS assay. In the spiked experimental samples, both assays


18

Page 19 of 38


401

detected zebra mussel in 94% of spiked samples and 0% of negative control samples. Overall,

402

these results demonstrate that eDNA sampling is effective for monitoring ballast-mediated

403

invasions and that LTS is a sensitive species-specific assay with the potential for rapid, field-

404

based detection. Moreover, using our eDNA sampling and species detection methods, we found

405

clear evidence of naturally occurring quagga mussel DNA in all four sampling localities (ballast

406

and harbor water from the Indiana and Duluth harbors). Using traditional methods, it is common

407

to find the dreissenid veligers (i.e., planktonic larval forms) in the ballast tanks of large ships,

408

however they are nearly impossible to determine morphologically to species based on standard

409

microscopy.42 Thus, in addition to detection, eDNA can offer greater taxonomic clarity.

410 411

One previous study has applied species-specific PCR to ballast water surveillance43 in which

412

standard PCR was used to screen for starfish larvae from bulk plankton samples, not eDNA.

413

Deagle et al.43 showed it worked with no false positives detected and could detect 1

414

larvae/200mg plankton. In general, the eDNA approach has been applied successfully for species

415

detection to multiple aquatic organisms (fish, amphibians, and invertebrates) worldwide22,23,26,29-

416

31

417

application in conservation and invasive species management.21,32 Our results suggest that eDNA

418

species detection from the ballast water of large transoceanic ships is a natural extension of the

419

eDNA approach.

and is rapidly developing into a burgeoning field of research in molecular ecology and

420 421

Next steps: Moving into rapid field-based detection with LTS

422 423

We also demonstrate that the field-ready LTS is a very sensitive technique for rapid target


19


Page 20 of 38

424

species detection from ballast and harbor waters. Previous research regarding LTS as a DNA

425

detection tool has shown that it can work effectively in species detection using pure lab samples

426

with one species present,34 when in the presence of the DNA of other closely related species,25,33

427

at low concentrations, 25 and for real-world samples, where there are a variety of species present

428

and the target DNA fragment is amplified with universal invertebrate primers and species

429

specific LTS tagged nano beads.24 In fact, detection capabilities of this LTS system suggest

430

potential detection to the picomolar (10−12) range.25 The LTS device fits into a large briefcase

431

and could run on a car battery in the field, thus LTS offers the opportunity to become a rapid

432

DNA detection method for real-world applications.33,34 Our ultimate objective is to apply the

433

technology for rapid point of sampling detection in nature. To this end, LTS technology is

434

portable, fast, and relatively inexpensive; we have recently developed a beta-model LTS device

435

that together with a field-based PCR unit fit in a “carry-on” sized suitcase, it can run for a day

436

from a rechargeable 12-volt battery, and following PCR amplification can measure a sample in

437

less than 10 seconds. The next major phase of research is two fold: (1.) integrate already

438

developed field DNA extraction techniques 44,45 and field PCR technologies 46,47 to be fully field-

439

ready and (2.) conduct LTS testing on site and eventually to transfer the technology to personnel

440

on the front line of invasive species detection in the field. In this regard, one extremely valuable

441

application of LTS would be to begin rapidly screening the ballast tanks of transoceanic ships

442

coming into the U.S. for invasive species. An urgent need exists for rapid detection methods like

443

LTS to identify harmful invasive species in ballast prior to port entry to provide adequate time

444

for implementing appropriate management actions.

445


20

Page 21 of 38


446

The general purpose of our work is to enable improved environmental protection, which requires

447

timely data to determine if potentially invasive species are contained in particular ships entering

448

the Great Lakes and other coastal regions of the world. Only then can the risks be understood at

449

the spatial and temporal scales needed to make appropriate decisions to reduce the potential for

450

harm. We feel that the further development and implementation of the technology tested here

451

could provide important information to guide decisions in the public and private sector, including

452

those by government agencies, the shipping industry, insurers, and other stakeholders, as it will

453

become possible to rapidly test for the presence of target organisms in specific ships. For

454

example, the tools described here could enable regulators to ensure compliance in actionable

455

time frames, and could be used by ship operators to document compliance with various enacted

456

and proposed ballast water concentration standards.48 Finally, ballast and harbor water

457

monitoring is only one of many potential applications for the tested technology, including

458

detection of invasive species in ports or other environments, threatened or endangered species,

459

species of public health concern (e.g., pathogens, parasites, or indicator species), and species of

460

concern for biosecurity.

461 462

Acknowledgements

463 464

For providing funding for this research, we would like to thank the U.S. Environmental

465

Protection Agency’s Great Lakes Restoration Initiative, and two interdisciplinary research

466

initiatives funded by the University of Notre Dame, the Environmental Change Initiative and the

467

Advanced Diagnostics and Therapeutics Initiative. We would also like to thank the American

468

Steamship Company and the captain and crew of the M/V Indiana Harbor for access to ballast


21


469

Page 22 of 38

and harbor water.

470 471


22

Page 23 of 38

472


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631

48. IMO. International Convention for the Control and Management of Ships’ Ballast Water and

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49. Mahon, A.R.; Barnes, M.A.; Senapati, S.; Feder, J.L.; Darling, J.A. et al. Molecular detection

635

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30

Page 31 of 38

637


Data Accessibility

638 639

All data presented in this paper will be publicly available on the Dryad Digital Repository.


31


640

Page 32 of 38

TABLE 1. List of species-specific and universal PCR primers and species-specific tags for LTS used in the present study,

641

Oligonucleotide

Sequence

Ref.

Species-specific PCR primers Dreissena bugensis (quagga mussel) forward

5’-CCTTATTATCTGTTCGGCGTTTAG-3’

49

Dreissena bugensis (quagga mussel) reverse

5’-ACTTGTAACACCAATAGAAGTAC-3’

49

Dreissena polymorpha (zebra mussel) forward

5’-GGGATTCGGAAATTGATTGGTAC-3’

49

Dreissena polymorpha (zebra mussel) reverse

5’-GAATCTGGTCACACCAATAGATGTGC-3’

49

Universal invertebrate PCR primers Universal invertebrate forward (LCO-1490)

5’-GGTCAACAAATCATAAAGATATTG-3’

39

Universal invertebrate reverse (HCO-2198)

5’-TAAACTTCAGGGTGACCAAAAAATCA-3’

39

Species-specific tag for LTS Dreissena polymorpha (zebra mussel)

5’-GAATCTGGTCACACCAATAGATGTGC-3’

49

Dreissena bugensis (quagga mussel)

5’-ACAAGTTGGGGGTGGTTTAGGCGGGAGT-3’

49

642 643


32

Page 33 of 38


644

TABLE 2. Results of quagga mussel (Dreissena bugensis) screens of the standard ballast and harbor water samples for the standard

645

gel and LTS assay methods. There were no positive hits for zebra mussel (D. polymorpha) using either of the two methods. PCR

646

inhibition was also not observed in any of the samples. (Gel assay 1: % of three separate triplicate PCR reactions, where at least one

647

positive reaction occurred out of the three; Gel assay 2: % of all nine total PCR reactions that was positive; LTS assay: % of samples

648

with a clear double peak demonstrating a positive species detection) eDNA sample

Standard

Experimental

Type

DNA Concentration

(number of samples)

(mean µg/mL ± sd)

Gel assay 1

Gel assay 2

LTS assay

Source harbor (5)

6.43 ± 1.01

100% (5/5)

89% (40/45)

100% (5/5)

Ballast intake (20)

3.86 ± 4.90

85% (17/20)

63% (113/180)

100% (20/20)

Receiving harbor (3)

4.01 ± 3.14

33% (1/3)

17% (3/18)

100% (3/3)

Ballast discharge (13)

2.68 ± 2.01

92% (12/13)

54% (63/117)

100% (13/13)

Spiked (16)

1.68 ± 0.79

94% (15/16)

94% (45/48)

94% (15/16)

Control (8)

1.26 ± 1.09

0% (0/8)

0% (0/24)

0% (0/8)


33


649

Page 34 of 38

FIGURE LEGEND

650 651

Figure 1. Naturally infested ballast and harbor water samples

652 653

Environmental DNA-based quagga mussel detection using LTS from naturally infested ballast

654

water samples from the M/V Indiana Harbor and surrounding source and receiving harbor

655

waters. Density distribution for one LTS measurement of (A.) the control blank, where no eDNA

656

was added; four positive quagga mussel detections from the (B.) source harbor, (C.) ballast

657

intake, (D.) ballast discharge, (E.) receiving harbor; and (F.) the average shift (± standard

658

deviation) in the diameter of nanoparticles relative to the baseline beads attached with quagga-

659

specific tags for all 41 standard samples (ballast and harbor). Dashed line in pane (F.) marks zero

660

peak shift for nanoparticle and represents no detection of target.

661 662

Figure 2. Experimentally spiked ballast and harbor water samples

663 664

Environmental DNA-based zebra mussel detection using LTS from experimentally spiked ballast

665

water samples collected from the M/V Indiana Harbor and surrounding source and receiving

666

harbor waters. (A.) Density distribution of one LTS measurement of a control sample, where no

667

zebra mussel tissue was added, (B.) density distribution of one LTS measurement where zebra

668

mussel tissue was added, and (C.) the average shift (± standard deviation) in the diameter of

669

nanoparticles for ballast samples where zebra mussel was added (+) relative to the samples were

670

zebra mussel was not added (–). Dashed line in pane (C.) marks zero peak shift for nanoparticle

671

and represents no detection of target.


34

3x107

2x107

1x107

0x100 0

200

400

600

Quagga mussel positive Source harbor

B.

1x108

5x107

0x100 0

200

E.

1x107

Quagga mussel positive Ballast discharge

8x106 6x106 4x106 2x106 0x100 0

200

400

Diameter (nm)

600

Quagga mussel positive Ballast intake

C.

6x107

4x107

2x107

0x100 0

200

Diameter (nm)

600

4x107

Density distribution (1/mL*nm)


Diameter (nm)

400


Quagga mussel negative Control blank

8x107


Quagga mussel positive Receiving harbor

E.

3x107

2x107

.

1x107

0x100 0


200

400

Diameter (nm)

400

600

Diameter (nm)

Mean shift in diameter (nm)

A.

2x108



7 4x10 Page 35 of 38

140

F.

E

100

60

20 E -20

600

Quagga eDNA

Control

A.

Zebra mussel negative Control

6x106

2x107

Environmental Technology Zebra Science mussel&positive

Spiked sample 1x107

4x106

8x106

2x106

4x106

0x100

B.

0

200

400

0x100 600 0

Mean shift in diameter (nm)

8x106

140

Page 36 of 38

E

100

60

20 E -20

200 400Environment 600 ACS Paragon Plus

C.

_

+ Zebra mussel

Page 37 of 38


Taking an environmental DNA sample. 363x241mm (72 x 72 DPI)



Laser transmission spectroscopy read-out and briefcase size device. 404x253mm (240 x 240 DPI)


Page 38 of 38

Rapid Molecular Detection of Invasive Species in ... - ACS Publications

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