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Title: Environmental (e)DNA detection of the invasive pink salmon Oncorhynchus gorbuscha during
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the 2017 Norwegian invasion
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Authors: Laura M. Gargan1, Frode Fossøy2, Tor A. Mo2, Jeannette E. L. Carlsson1, Bernard Ball1,
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Jens Carlsson1
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1Area 52 Research Group, Earth Institute/School of Biology and Environmental Science, University
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College Dublin, Ireland
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2Norwegian Institute for Nature Research (NINA), Trondheim, Norway
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Corresponding author: Laura M. Gargan, E-mail: laura.gargan@ucd.ie
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Keywords: Oncorhynchus gorbuscha; invasive species; digital droplet PCR; environmental DNA;
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Norway
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ABSTRACT
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The pink salmon Oncorhynchus gorbuscha was introduced from its native range in the Pacific to
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Northwest Russia several times since the 1950’s. While this species has been regularly observed in
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rivers in Northern Norway since that time, there has been an upsurge in the numbers of odd-year O.
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gorbuscha individuals observed in rivers in southern Norway in recent years, and particularly in 2017.
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Although the wide-scale effects of this species presence are currently uncertain, there are concerns
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regarding potential competition between O. gorbuscha and native species – most notably the Atlantic
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salmon Salmo salar. Environmental (e)DNA is becoming a widely used tool to monitor rare and
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invasive species in aquatic environments. In the present pilot study, primers and a probe were
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(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
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developed to detect O. gorbuscha from eDNA samples taken from a Norwegian river system where
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the species was observed. Water samples were taken at both upstream and downstream locations of
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the Lysakerelva river during Autumn 2017 (to coincide with spawning) and during late Spring 2018.
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Autumn samples were positive for O. gorbuscha at both sampling locations, whereas Spring samples
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showed positive detection of this species in the upstream region of the river, when smolt should have
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left, or be in the process of leaving the river. These findings reveal that eDNA-based methods can be
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used detect the presence of O. gorbuscha during their spawning season. This suggests that odd-year
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populations have the potential to become established in the studied river system. We recommend that
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eDNA sampling is repeated to determine whether individuals of this odd-year population have
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survived at sea and return to spawn. Our assay specificity tests indicate that the tools developed in the
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present study can be used for detection of O. gorbuscha in both Norwegian and other European river
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systems where presence/absence data is required. We also suggest some modifications to our
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methodology that may improve upon the detection capabilities of O. gorbuscha using eDNA.
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INTRODUCTION
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Pink salmon Oncorhynchus gorbuscha (Walbaum, 1792) are native to the Pacific Ocean,
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where they typically spawn in the freshwater ecosystems of bordering countries between the latitudes
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of 40o and 70o. This species follows a strict two-year life cycle (Figure 1). Adult O. gorbuscha
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migrate from the open sea and up-river to spawn in the autumn, after which all spawning individuals
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die. The juveniles emerge the following spring, ready to migrate down-river and out into the open sea
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to mature for one winter. They return as adults to freshwater in the next autumn to spawn, thus
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completing the life cycle (Heard 1991).
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O. gorbuscha was originally introduced from their native Pacific range to North Western
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Russia several times since the 1950’s, when fry were stocked in several rivers that drain into the
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White Sea and the Barents Sea (Bakshtansky 1980). While O. gorbuscha has been regularly found in
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rivers in Northern Norway since 1960 (Berg 1961), there has been an upsurge in the observations of
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(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
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odd-year O. gorbuscha in Norwegian rivers in recent years (Mo et al., 2018), as well as in rivers in
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the UK and Ireland (Armstrong et al., 2018, Whelan 2017). However, self-reproducing populations
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have not yet appeared to become established, most likely because they have not adapted time of
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spawning to match local conditions outside of their native range (Mo et al., 2018).
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Established populations of O. gorbuscha in rivers in North Western Russia are dominated by
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odd-year individuals (Gordeeva & Salmenkova, 2011). Thus far, odd-year individuals have been
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noticeably more abundant throughout their invasive range, with a particularly large number of
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spawning O. gorbuscha recorded in 2017 (Armstrong et al., 2018, Mo et al., 2018). Therefore, it is
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these odd-year stocks that are most likely to become established as populations and the next spawning
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season (which should take place during Autumn of 2019) will be an important time for research
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scientists to determine the extent of this invasion and its potential ecological effects.
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Presently, the spawning time of O. gorbuscha in Norway does not appear to overlap with
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native salmonids (e.g. Atlantic salmon Salmo salar and brown trout S. trutta). However, O. gorbuscha
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and S. salar have similar preferences for spawning habitats and so there is a risk of competition for
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optimal spawning sites (Sandlund et al., 2018). While O. gorbuscha juveniles emerge ready to
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migrate to sea, observations in Norwegian rivers suggest that they spend some time feeding in
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freshwater (from weeks to months) and during this time there may be interactions between juveniles
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of native salmonids (Sandlund et al., 2018). However, it is also possible that the eggs and fry of O.
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gorbuscha can provide a source of food for other native salmonid species (Rasputina et al., 2016). In
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order to fully assess the impacts of the presence of O. gorbuscha in Norwegian (and other European)
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rivers, it is first necessary to determine the spatial, as well as the temporal (e.g. time of spawning and
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migration) distribution of this species.
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Environmental DNA (eDNA) is a genetic survey method that relies on the detection of taxa
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from extracellular and intracellular material that is deposited into the environment. Subsequently, this
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material can be isolated from the environmental sample (such as water, air or soil; Taberlet et al.,
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2012) and interrogated using genetic markers for multi-species (Thomsen et al., 2012, Hänfling et al.,
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2016) or targeted species (Ficetola et al., 2008, Jerde et al., 2011, Gustavson et al., 2015) detection.
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(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
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While many studies have used quantitative PCR (qPCR) for targeted detection of species in
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an eDNA sample (Thomsen et al., 2012, Wilcox et al., 2013, Atkinson et al., 2018), more recently the
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approach of using digital droplet PCR (ddPCR) has been adopted by some researchers (Doi et al.,
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2015, Nathan et al., 2015, Evans et al., 2017, Baker et al., 2018), with either similar (Nathan et al.,
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2015) or increased (Doi et al., 2015, Hunter et al., 2017) sensitivity reported for ddPCR platforms
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compared to qPCR for the analysis of eDNA. ddPCR is a relatively recent technological
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advancement, allowing for accurate estimation of low copy DNA number (Hindson et al., 2011). It is
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an absolute quantification method which, unlike qPCR, does not rely on standard curves to estimate
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target DNA concentration. For DNA samples extracted from environmental water and containing
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potentially very low copy number of target DNA, the ability of ddPCR to detect rare events in a
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reaction is of particular interest, especially for invasive species in aquatic ecosystems where they may
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exist in low abundance and may be difficult to detect using conventional survey methods (e.g. netting
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and electrofishing).
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Whether analysis is being carried out using qPCR or ddPCR, eDNA-based detection is
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becoming increasingly used in aquatic freshwater environments for a range of low abundance or
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invasive taxa, such as amphibians (Pilliod et al., 2013, Spear et al., 2014), molluscs (Goldberg et al.,
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2013, Peñarrubia et al., 2016, Carlsson et al., 2017) and crustaceans (Tréguier et al., 2014, Harper et
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al., 2018), as well as fish (Takahara et al., 2013, Klymus et al., 2015, Davison et al., 2016) –
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including salmonid species (Gustavson et al., 2015, Atkinson et al., 2018, Rusch et al., 2018). To
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date, there has been no published studies for implementing eDNA for the detection of O. gorbuscha.
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We hypothesise that eDNA can be used for detection of non-native O. gorbuscha in running water. In
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addition, should any eradication measures be employed in the future, eDNA methods could be used to
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determine the efficacy of such efforts (Banks et al., 2015).
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This study aimed to address questions relating to the presence and distribution of O.
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gorbuscha in a Norwegian river, as part of a pilot study employing the non-intrusive and genetic
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survey method of eDNA collection and analysis. A species-specific probe-based assay was developed
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for this species and deployed in an urban river system where O. gorbuscha had been previously
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(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
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observed. We also developed this assay with the intention that it can be deployed in other European
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freshwater or marine ecosystems where this species may currently, or potentially, invade. This is of
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particular relevance for detection of the species during spawning season of this year (Autumn 2019),
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where it is unknown if the putative increased mortality of O. gorbuscha at sea (resulting from a
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mismatch between emigration time and food availability; Armstrong et al., 2018) will result in a less
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significant invasion.
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MATERIALS AND METHODS
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Water sampling and eDNA extraction
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For this study, water samples were collected from the Lysakerelva river in the south-east
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region of Norway (Table 1) during Autumn of 2017 and Spring/early Summer of 2018. This river
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system runs through a large urban area outside of Oslo and is characterised by a number of natural
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migration barriers, including waterfalls. The smallest of these waterfalls (Møllefoss) is a few metres
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in height and is fitted with fish ladders to enable upstream migration. The next waterfall, Granfoss, is
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sufficiently high (~12-15 metres) as to completely prevent upstream fish migration. In terms of the
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diversity of fish found in the study area, the Lysakerelva river fish fauna is dominated by S. salar, S.
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trutta and the European minnow Phoxinus phoxinus (Saltveit et al., 2013). Several O. gorbuscha have
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previously been caught in the Lysakerelva river (>20 fish; Sandlund et al., 2018).
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During September 2017, two water samples were taken below Møllefoss and just above upper
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high tide close to the mouth of the Lysakerelva river, and two water samples were taken immediately
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below the Granfoss waterfall (~800 metres upstream from the river mouth; Table 1). These samples
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were taken to coincide with the spawning season of O. gorbuscha. This sampling was repeated the
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following May, the time period when it is expected that juvenile O. gorbuscha would be undertaking
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seaward migration (Table 1). In June 2018, two water samples were also taken from the estuary as it
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was considered possible that O. gorbuscha may also be found in the coastal area after migrating
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downriver (Heard 1991; Moore et al., 2016). Additional samples (n=4; Table 1) were taken from
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above the Granfoss waterfall in June 2018 as negative eDNA field control samples, as it was expected
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that no O. gorbuscha would be present at this location due to the barrier of the Granfoss waterfall.
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For each locality, we filtered two replicate samples of either c. 1 L water on a 0.45 µm
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cellulose filter (Pall MicroFunnel 300 ST; Pall Corporation, New York, USA; Table 1) or two
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replicate samples of c. 10 L water on a 2.0 µm glassfiber filter (Merck Millipore, Burlington,
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Massachusetts, USA; Table 1) using a peristaltic pump (Vampire sampler, Bürkle, Bad Bellingen,
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Germany). The 0.45 µm cellulose filters were immediately placed in 2 mL tubes with 1440 µL ATL-
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buffer (Qiagen, Hilden, Germany), whereas the 2.0 µm glassfiber filters were placed in 5 mL tubes
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with 4050 µL ATL-buffer. All samples were stored at room temperature until further processing in the
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genetics laboratory. All field equipment (e.g. filtering tubes and collection bottles) was sterilised
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between collection of each sample using 10% bleach solution for approximately 60 minutes.
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In the laboratory, 160 µL or 450 µL Proteinase-K (Qiagen) was added to the 2 mL and 5 mL
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sampling tubes, respectively. All samples were incubated overnight at 56°C. DNA was isolated from
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0.45 µm cellulose filters using DNeasy DNA Blood & Tissue kit (Qiagen), and from the 2.0 µm
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glassfiber filters using NucleoSpin Plant II Midi kit (Macherey-Nagel, Düren, Germany), following
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the manufacturers protocol except that Qiagen buffers were used instead of those supplied with the
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kit. DNA extracted from the 0.45 µm cellulose filters was eluted in 100 µL AE buffer, whereas DNA
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extracted from the 2.0 µm glassfiber filters was eluted in 200 µL AE buffer. All samples were re-
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eluted in order to maximise the output of DNA. Final concentrations of the eluted DNA samples
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(Supplementary Table 1) were determined using a Nanodrop 1000 Spectrophotometer (Thermo Fisher
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Scientific, Waltham, Massachusetts, USA).
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Molecular assay development and specificity testing
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An assay was designed to amplify a 98 bp region of the mitochondrial COI gene of O.
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gorbuscha (Table 2). This assay consisted of primers and a 5’ VIC labelled TaqMan® minor groove
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binding (MGB) probe. The primers and probes were designed using Primer3 software (Rozen and
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Skaletsky 2000). Specificity of the assay was checked in-silico, by aligning the primers and probe
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with the consensus sequence generated from publicly available O. gorbuscha sequences as well as
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those from other salmonids commonly occurring in the study area (e.g. S. salar and S. trutta). The
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primer and probe sequences were also checked against the NCBI database to ensure specificity to the
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target organism. Furthermore, specificity of the assay was checked using qPCR, with tissue-extracted
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DNA from O. gorbuscha, as well as from S. salar and S. trutta. Tissue-extracted DNA was also
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acquired and tested by qPCR for rainbow trout (O. mykiss) which, while not a native salmonid, has
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been widely introduced throughout Europe (Stanković et al., 2015). In addition, DNA extracted from
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the closely-related chum salmon O. keta tested to check the specificity of the assay. The latter is not
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currently found in Europe but this species overlaps with O. gorbuscha in its native range.
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All qPCR reactions took place in a 20 µl reaction volume, containing 10 µl of TaqMan™
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Environmental Master Mix 2.0 (Thermofisher), 2 µl of each primer (2 µM), 2 µl of probe (2 µM;
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Applied Biosystems) and 2 µl of template DNA (where extracts were normalised to 34 ng/µl). The
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PCR program consisted of 50oC for 2 min, 95oC for 10 min, followed by 40 cycles of 95oC for 15 s
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and 60oC for 1 min. All qPCR reactions were carried out using QuantStudio™ 7 Flex Real-Time PCR
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System (Applied Biosystems). All qPCR analysis took place in University College Dublin.
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Potential cross-amplification of other salmonids (O. mykiss, S. salar, S. trutta, Salvelinus
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alpinus, Salvelinus fontinalis, Salvelinus namaycush and Thymallus thymallus) using the O.
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gorbuscha assay was also tested using ddPCR at NINA using the same PCR conditions as those
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detailed in the next section.
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Digital droplet (dd)PCR analysis of eDNA samples
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Sample collection and eDNA extraction resulted in a total of 14 samples originating from the
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Lysakerelva river (Table 1). Detection and concentration of target-DNA was assessed using droplet-
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digital-PCR (QX200 Droplet Digital PCR system with AutoDG, Bio-Rad Laboratories, Hercules,
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USA). A tissue-extracted DNA sample of O. gorbuscha was included in the analysis, as a positive
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control. A no-template control was also included in the analysis. The eDNA samples, along with
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positive, negative and no-template controls, were analysed in triplicate with the exception of the
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samples from 2017 (Table 1), which were analysedsingularly. The ddPCR reactions consisted of 0.9
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µM forward and reverse primers, 0.25 µM of the probe, ddPCR™ Supermix for Probes (No dUTP)
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(Bio-Rad Laboratories), dH2O and 5 µL template. In order to assess potential problems with inhibition
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in our samples we also reran the samples with only 1 µL template. However, we found no indications
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of inhibition among our samples. To generate droplets, an AutoDG Instrument (Bio-Rad
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Laboratories) was used, with subsequent PCR amplification in a Veriti96-Well Thermal Cycler
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(Applied Biosystems). The following thermal cycling conditions were used: an initial denaturation
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step at 95°C for 10 min, 40 cycles of denaturation at 95°C for 30 sec, annealing and extension at 60°C
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for 1 min, a final step of denaturation at 98°C for 10 min, and a final hold at 4°C. PCR plates were
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transferred to a QX200 Droplet Reader (Bio-Rad Laboratories) to automatically detect the fluorescent
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signal in the droplets. QuantaSoft software v.1.7.4 (Bio-Rad Laboratories) was used to separate
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positive from negative droplets according to manufacturer’s instructions. A threshold of minimum 3
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positive droplets were used as a criterion for positive detection Wacker et al. 2019). All ddPCR-
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analyses took place at the genetic lab at NINA in Trondheim, Norway.
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RESULTS
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Molecular assay development
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Testing of the O. gorbuscha assay with non-target salmonids using tissue-extracted DNA and
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qPCR, revealed that the assay did not cross-amplify S. salar, S. trutta, or O. mykiss. ddPCR testing of
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non-target salmonid species (O. mykiss, S. salar, S. trutta, S. alpinus, S. fontinalis, S. namaycush, T
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thymallus) did not produce any detectable amplification. However, amplification was observed for O.
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keta DNA analysed by qPCR (data not shown). This species is closely related to O. gorbuscha and
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there are very few nucleotide differences between these species for the COI region targeted by our
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assay (Supplementary Figure 2). However, this should not present a major concern for researchers
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9
wishing to apply this assay for O. gorbusha detection in Norwegian and other European locations, as
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neither O. keta nor other species of Pacific salmon (O. nerka, O. kisutch, O. tshawytscha and O.
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masoudo) currently occur in these regions.
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ddPCR analysis of eDNA samples
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An average number of 15,299 droplets were analysed for each sample included in the ddPCR
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analyses. Using the O. gorbuscha assay in a ddPCR analysis, there was target DNA detected in six out
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of 14 eDNA samples (Table 3).
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In Autumn 2017, O. gorbuscha DNA was detected at Granfoss but not at Møllefoss (Table 3,
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Figure 2). In Spring, target DNA was detected in both samples taken below the Granfoss waterfall,
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which indicates that there were still young O. gorbuscha in the sampling area that had not yet left the
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river. No target DNA was found in samples taken near to the mouth of the river in June 2018, which
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is perhaps unsurprising, considering that upstream and downstream samples were taken 20 days apart
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and young O. gorbuscha may have already migrated to sea.
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No detectable target DNA was found in samples taken from the estuary. It is possible that,
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similar to the samples taken in the downstream region of the Lysakerelva river, young O. gorbuscha
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may have already migrated out to sea when this sampling took place in the Summer. Alternatively, a
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lack of detection of O. gorbuscha at this location may be a false negative, as a result of the relatively
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low number of samples taken over a large sampling area (Table 1).
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Interestingly, target DNA was also detected in low copy number from one out of four of the
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control samples above the migration barrier (Figure 2) indicating alternative means of target DNA
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transport to this site, or contamination of the sample in the field (see Discussion). All no-template
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controls were negative for target DNA.
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10
DISCUSSION
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The year 2017 was unprecedented in terms of the number of O. gorbuscha that were observed
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in Norwegian rivers (Mo et al., 2018), as well as in Ireland and Scotland (Whelan 2017, Armstrong et
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al., 2018). In the present study, we were successfully able to detect O. gorbuscha from environmental
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water samples in Norway during this invasion. Target DNA was amplified from samples taken in both
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Autumn (during adult spawning) and the following Spring (during the migration of juveniles),
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indicating that spawning in the focal river system was successful. However, the survival of juvenile
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O. gorbuscha at sea is poorly understood and it is unknown whether mortality will limit the ability of
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this species to return to the river and establish self-reproducing populations.
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The assay developed and validated in this study not only has applications for monitoring
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presence of O. gorbuscha spatially and temporally in Norwegian rivers, but also in countries where it
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currently exists outside of its native range, as well as those where it has not been observed but may
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potentially occur in the future. Further, this assay can be used to monitor O. gorbuscha presence
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following any future eradication efforts that may be implemented. While the utility of the assay
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developed in this study is limited to those areas where O. gorbuscha does not overlap with O. keta,
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this should not be of concern for researchers using this assay to detect O. gorbuscha within its
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invasive range as O. keta is not currently found outside the North Pacific.
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The finding of a positive detection of O. gorbuscha above the impassable barrier of Granfoss
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waterfall demonstrates the potential drawbacks of using eDNA to infer species presence. There was
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no indication of contamination in any of the negative controls included in this study. Specificity
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testing by qPCR and ddPCR revealed that this assay does not amplify COI from other commonly co-
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occurring native or introduced salmonids. The sample location was the only site visited on the day of
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sampling, yet it is impossible to rule out contamination of field equipment from previous sampling
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events downstream of the barrier. It is also possible that an alternative vector, such as predation (by
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birds for example; Merkes et al., 2014), can explain the detection of target DNA in a location where
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the organism is not present.
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11
The results of our pilot study show that this assay can be used to detect the presence of O.
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gorbuscha in running water. However, we can make some suggestions to increase the efficacy of our
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approach for future studies. The lack of detection of O. gorbuscha in water samples taken from the
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estuary and at the mouth of the Lysakerelva river in early Summer indicated that the juveniles had
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already migrated to sea. We would recommend an increase in temporal sampling, as data derived
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from these samples may have been able to reveal the timing of juvenile migration to sea, increasing
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our knowledge about O. gorbuscha behaviour in Norwegian rivers and indicating the extent of
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potential interactions with local fauna.
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We detected a mismatch in the forward primer sequence (Supplementary Figure 1), based on
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the O. gorbuscha consensus sequence generated from publicly available COI records on GenBank.
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The source of this mismatch is a sequence from an odd-year individual (Accession No. MG951587.1)
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from the White Sea that was submitted to the NCBI database after the assay was developed and
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tested. It is therefore possible that this haplotype is found in Norwegian rivers. This mismatch is
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found on the 5’ end of the forward primer, therefore it is unlikely that our assay efficiency was
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severely compromised in the present study. However, to ensure optimal efficiency in the assay (and
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particularly to ensure sensitive detection of the low copy numbers frequently found in eDNA
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samples), we recommend that the forward primer incorporate a degenerate base at this position should
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the assay be deployed in other studies. Further, we concur with other eDNA researchers (e.g.
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Goldberg et al., 2016) that concerted efforts are made to minimise any contamination in the field,
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through the implementation of careful sterilisation and sampling techniques, as well as the use of
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strict controls (e.g. field blanks, filter blanks) to monitor for exogenous sources of DNA during the
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entire eDNA sampling and analysis workflow.
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ACKNOWLEDGMENTS
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The authors would like to thank Hege Brandsegg for running the ddPCR genetic analysis. We also
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gratefully acknowledge Sigrid Skoglund for drawing the pink salmon life cycle.
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COMPLIANCE WITH ETHICAL STANDARDS
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Conflict of interest The authors declare that they have no conflict of interest.
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Ethical approval All applicable international, national, and/or institutional guidelines for the care and
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use of animals were followed.
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TABLES AND FIGURES
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Figure 1: Life cycle of the pink salmon Oncorhynchus gorbuscha, ill.: Sigrid Skoglund, NINA.
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425
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.CC-BY-NC-ND 4.0 International licenseIt is made available under a
(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint. http://dx.doi.org/10.1101/651554doi: bioRxiv preprint first posted online May. 31, 2019;
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Table 1: Details of water sampling in Lysakerelva river.
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Date
Locality
No.
samples
Water
volume
Filter
size
Latitude, Longitude
04.09.2017
Granfoss below waterfall
2
1
0.45µm
59.917894, 10.631348
04.09.2017
Møllefoss below waterfall
2
1
0.45µm
59.914629, 10.636455
31.05.2018
Granfoss below waterfall
2
0.8
0.45µm
59.917894, 10.631348
20.06.2018
Estuary
2
10
2.0µm
59.911005, 10.643129
20.06.2018
Møllefoss below waterfall
2
10
2.0µm
59.914629, 10.636455
22.06.2018
Granfoss above waterfall
2
10
2.0µm
59.918166, 10.630236
22.06.2018
Granfoss above waterfall
2
0.8
0.45µm
59.918166, 10.630236
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430
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Table 2: Details of the assay that was designed, tested and deployed for detection of a 98bp region of
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the mitochondrial COI gene of O. gorbuscha in this study.
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Primer Type
Name
Sequence 5' -3'
Length (bp)
Forward Primer
PinkF
CACCGCCCTAAGCCTACTAA
20
Reverse Primer
PinkR
AGGCATGGGCTGTAACGATT
20
Probe *
PinkPr
CGCTCTTCTAGGGAATGACCA
21
* 5' VIC labelled reporter dye and 3’ non-fluorescent quencher
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435
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Table 3: Detection of O. gorbuscha using eDNA shown as the number of samples that were positive
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for detection out of the total number of samples analysed by ddPCR.
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Locality
Date
eDNA detection
Granfoss below waterfall
04.09.2017
1/2
Granfoss below waterfall
31.05.2018
2/2
Granfoss above waterfall
22.06.2018
1/4
Møllefoss below waterfall
04.09.2017
2/2
Møllefoss below waterfall
20.06.2018
0/2
Estuary
20.06.2018
0/2
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(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
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17
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Figure 2: Boxplot showing the number of positive droplets from ddPCR detection of O. gorbuscha in
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relation to date and locality. The horizontal dashed line indicates the lower threshold of three droplets
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for assessing a sample as positive. See Table 1 for details of each sample.
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.CC-BY-NC-ND 4.0 International licenseIt is made available under a
(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint. http://dx.doi.org/10.1101/651554doi: bioRxiv preprint first posted online May. 31, 2019;
