Detecting Aquatic Invasive Species in Bait and Pond Stores With

Biological Conservation 245 (2020) 108430

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Biological Conservation

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Detecting aquatic invasive species in bait and pond stores with targeted T environmental (e)DNA high-throughput sequencing metabarcode assays: Angler, retailer, and manager implications ⁎ Matthew R. Snydera,b,c,1, Carol A. Stepiena, , Nathaniel T. Marshalla,b,1, Hannah B. Schepplerb, Christopher L. Blackd, Kevin P. Czajkowskid a Genetics and Genomics Group at NOAA Pacific Marine Environmental Laboratory, 7600 Sand Point Way NE, Seattle, WA 98115, USA b Genetics and Genomics Group, University of Toledo Department of Environmental Sciences, 2801 West Bancroft St., Toledo, OH 43606, USA c Joint Institute for the Study of the Atmosphere and Ocean, University of Washington, Seattle, WA 98195, USA d University of Toledo Department of Geography and Planning, 2801 West Bancroft St., Toledo, OH 43606, USA

ARTICLE INFO ABSTRACT

Keywords: Bait and pond stores comprise potential, yet poorly understood, vectors for aquatic invasive species (AIS). We Bait & pond stores tested for AIS and illegal native species in 51 bait and 21 pond stores from the central Great Lakes (Lake Erie, Metabarcoding Ohio and Lake St. Clair, Michigan) and the adjacent Wabash River (Indiana) using environmental DNA (eDNA) eDNA metabarcode assays of water samples and morphological identifications. Retailers were questioned about supply Invasive species chains, and anglers surveyed about baitfish use and disposal. Assays revealed unadvertised species eDNAin Anglers 100% of bait stores, with 61% containing illegal native non-bait (totaling 13 species) and 88% having AIS (11 Great Lakes species). Illegal native non-bait species included juvenile walleye, yellow perch, and white sucker eDNA. AIS eDNA included Eurasian ruffe in seven stores (all states), silver carp in five (including a Lake Erie storeintwo separate years), and bighead carp in two Lake Erie stores that also had silver carp. Among pond stores, two in Lake St. Clair had bighead carp eDNA, one also contained silver carp, and a Wabash River location showed European ide. Unadvertised invasive snails were discerned in 55% of pond stores. Four contained zebra mussel eDNA and two had invasive bryozoans. Illegal native species and AIS were widespread, but showed little relationship to the retailers' variable and extensive supply chains. Live baitfish releases were reported by 50%of Lake Erie anglers and 35% in Lake St. Clair. Consumer behavior and AIS prevalence in the bait and pond trades thus pose serious risks for introductions and spread.

1. Introduction regarded as helpful by increasing forage for fisheries and in not killing animals. However, such releases can spread AIS (Janssen and Jude, 1.1. Invasive species in the retail bait and pond trades 2001; Winfield et al., 1996) and parasites/diseases (Heckman et al., 1993; Pierce and Stepien, 2012). Moreover, intentional and/or acci- Retail trade has been implicated in introducing >150 aquatic in- dental releases can supplement genetic diversity of established AIS vasive species (AIS) in the USA, and accounts for 1/3 of the species populations, potentially increasing success and spread (Baker and listed as the world's 100 worst AIS (Padilla and Williams, 2004). Stebbins, 1965). However, finite resources and lack of cost-effective and efficient iden- AIS frequently exert severe economic and ecological effects. For tifications have limited their detections to date. For example, mor- example, 188 AIS species are listed in the NOAA Great Lakes Aquatic phological species identifications have found unadvertised native, Nonindigenous Species Information System (GLANSIS; NOAA, 2019). game, imperiled, and invasive species in bait stores (Drake and AIS cost Great Lakes' area taxpayers and businesses >$200 million Mandrak, 2014; Litvak and Mandrak, 1993), but are time consuming annually (Lodge and Finnoff, 2008; USFWS, 2012), and threaten a $7 and require taxonomic expertise. Releases of unused live bait, aqua- billion/year commercial and recreational fishing industry (GLFC, rium, and pond store species into local waterways often are mistakenly 2019). There is concern that the AIS bighead (Hypophthalmichthys

⁎ Corresponding author. Co-first author. E-mail address: [email protected] (C.A. Stepien). 1 Stepien's Genetics and Genomics Group lab Relocated to a in 2016. https://doi.org/10.1016/j.biocon.2020.108430 Received 1 September 2019; Received in revised form 4 January 2020; Accepted 20 January 2020 Available online 17 April 2020 0006-3207/ Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/). M.R. Snyder, et al. Biological Conservation 245 (2020) 108430 nobilis) and silver (H. molitrix) carps, now at the gateways to the Great “pond shop”, “pond store”, “pond fish”, “koi”, “pond snails”), con- Lakes, might become established (GLFC, 2018; Kolar et al., 2005; Stern versations with anglers and fishery managers, and roadside observa- et al., 2014). These are projected to significantly impact fisheries due to tions. We sampled stores from the Wabash River (a Mississippi River filter feeding low on the food web(Irons et al., 2007; Sass et al., 2014), tributary) in northeastern Indiana (IN), which has a history of frequent high fecundity, rapid growth, and large sizes (Cuddington et al., 2014; flooding into Lake Erie through the Wabash and Maumee rivers' con- Kolar et al., 2005). When small, these carps may accidentally be in- nection (Stepien et al., 2019), the Lake St. Clair area of southeast Mi- cluded with bait by retailers, since they resemble other “minnows” chigan (MI), and the Lake Erie Ohio (OH) shoreline (Fig. 1, Table A1). (Kolar et al., 2005). Retailers were telephoned to confirm that live baitfish or pond species The 1990 USA Lacey Act (16 U.S.C.§§3371–3378) and 1992 Alien were for sale. We sampled 51 bait stores, including 48 in 2016 and 49 in Species Prevention and Enforcement Act 2 (P.L. 102–393) contain 2017 (with 46 in common for both years) during June–August. Ac- regulations for retail bait and pond trades. They ban importation, cording to anglers, diversity of bait fishes for sale often increased later shipment, or sale of “injurious” species, and render violation of state in the season. Thus, 19 previously-sampled stores were re-sampled in wildlife regulations a federal crime (Nathan et al., 2014; U.S. 102nd September–November 2017. A bait store that had genetic evidence of Congress, 1992; USFWS, 2006). State regulations additionally govern silver carp in 2016 (see Results) was re-sampled twice. In addition, 21 transport, possession, and/or sale of invasive species (e.g., Indiana, pond supply stores were sampled in May–June 2017 (Fig. 1, Table A1). 2014; Michigan, 1994; Ohio, 2016a). At state and regional levels, sur- Collections were made under University of Toledo's Toledo veillance of bait and pond stores by fishery management agencies may Institutional Animal Care and Use Committee (IACUC) protocol include random and/or yearly inspections (AMFGLEO, 2016; LED #205400, encompassing the research team. All sampling equipment MDNR, 2018). However, morphological (MORPH) surveys rarely first was decontaminated for >10 min in 10% bleach and thoroughly identify AIS, which frequently are relatively rare in store tanks and rinsed in ddH2O between uses. We purchased >24 baitfish individuals often resemble legal bait species (e.g., many young minnow-like fishes) per bait store, and one pond fish and/or three snails from each pond (AMFGLEO, 2016; Keller and Lodge, 2007; LED MDNR, 2018; Kevin store tank. For those with a flow-through water system that supplied Kayle, ODNR Fisheries Biologist, pers. comm.). Our aim was to develop tanks in series, ≥two samples were obtained. Advertised species were and ground-truth diagnostic high-throughput sequencing (HTS) meta- recorded. In the store parking lots, specimens immediately were filtered barcode (META) assays, with an associated bioinformatic pipeline, to from the water using sterile colanders, moved to clean buckets of de- evaluate species in bait and pond store retail. chlorinated water, immediately sacrificed with an overdose of 250 mg/ mL tricaine methane sulfonate (MS-222; Argent Chemical Laboratories, 1.2. Environmental DNA detection Redmond, WA) following our IACUC protocol, placed in labeled plastic bags, and stored in a designated cooler on ice for rapid transport to the Organisms regularly shed environmental (e)DNA in mucus, skin lab. Each sample's water (containing the eDNA) was stored in a labeled cells, and waste products, which may persist for hours to days in sterile 700 mL plastic jar on ice in a separate cooler for transport, and aquatic systems (Barnes and Turner, 2016; Ficetola et al., 2008). Water soon frozen at −80 °C. In the lab, fishes were identified to species using samples containing eDNA can be genetically analyzed to determine a taxonomic keys (Hubbs and Lagler, 2007; Trautman, 1981), blotted dry, species' presence or absence (Rees et al., 2014). eDNA analyses are weighed (g), and measured to total length (mm). Taxonomy and no- more sensitive than traditional MORPH sampling for identifying rare menclature for all species followed http://www.fishbase.org for fishes taxa, with most studies to date using PCR or quantitative (q)PCR to or the World Registry of Marine Species (http://www.marinespecies. detect single species (Adrian-Kalchhauser and Burkhardt-Holm, 2016; org) for invertebrates. Erickson et al., 2016; Zaiko et al., 2018). More recently, META analyses allow simultaneous PCR amplification of multiple species to create an 2.2. Genetic detection of species amplicon library for high-throughput sequencing (HTS) (Shokralla et al., 2012). The resultant millions of sequence reads are compared to a 2.2.1. eDNA META assay design reference database to delineate component taxa. Multiple indexed Retailers' tanks typically contained hundreds to thousands of in- samples (here up to 96) can be run in a single lane on a HTS platform, dividuals in bait stores, and dozens for pond stores, and were unlikely such as Illumina MiSeq®, and then identified to species or nearest to be well characterized by visual surveys and purchased samples. We possible taxon using bioinformatic processing (Illumina, 2019; Stepien thus evaluated eDNA META assays on water samples from retail pur- et al., 2019). chases. Mitochondrial (mt) DNA cytochrome (cyt)b sequences >1000 nucleotides (NTs) were downloaded from GenBank (www.ncbi.nlm.nih. 1.3. Objectives gov/genbank) for all Great Lakes fishes (Hubbs and Lagler, 2007), all established invasive species in the USGS Nonindigenous Aquatic Spe- Our research objectives were to: (1) determine whether and to what cies database (USGS, 2019), and all present and predicted future AIS degree illegal native species and AIS were present in bait and pond from the NOAA Great Lakes Aquatic Nonindigenous Species Informa- stores from and near the central Great Lakes' region, (2) identify which tion System (GLANSIS) (NOAA, 2019; Table A2). Sequences for two species were involved and whether, and to what degree, MORPH ob- native catostomid fishes (Erimyzon claviformis and Moxostoma lacerum), servations and eDNA META identifications matched, and (3) evaluate three Coregonus spp. (Coregonus hubbsi, C. nipigon, and C. reighardi) and potential predictor variables (i.e., location, sampling season, and/or a believed-to-be extinct species (C. johannae), two cyprinids (Margar- year). We also (4) investigated store supply chains to elucidate sources iscus natchtriebi, Semotilus corporalis), and troutperch (Percopsis omisco- of illegal species, and (5) surveyed anglers to evaluate bait release maycus) were absent from GenBank at the time of our study (accessions practices, and related these to 1–3. Our eDNA META protocol was de- listed as “No records” in Table A2). The final reference dataset for designed to be adaptable for management and conservation surveys. sign of the fish META assays included >95% of Great Lakes fishes and 100% of the AIS. 2. Materials and methods Three cytb META assays were designed by our laboratory to target native and AIS (existing and those projected by GLANSIS) Great Lakes' 2.1. Bait and pond store sampling fish species, including FishCytb, CarpCytb for cyprinid (carp) AIS (Stepien et al., 2019), and GobyCytb for goby AIS. Consensus sequences Regional bait and pond stores were identified from Google Maps™ were aligned visually (Table A3) and used to design primers that sur- searches (e.g., “bait shop”, “bait store”, “bait fish”, “emerald shiners”, rounded variable sequence regions <250NTs.

2 M.R. Snyder, et al. Biological Conservation 245 (2020) 108430

Fig. 1. Map of bait and pond stores showing eDNA metabarcode assay results.

The GobyCytb and CarpCytb assays respectively amplified 167 and primer. This longer assay was substituted for the 2017 bait and pond 136NTs, beginning at base 42 or 114 in the cytb gene. An earlier shorter store samples, and all re-runs. FishCytb (-S) assay targeted a 55NT region beginning at base 954, A META assay targeting the mt 16S rRNA gene (Mollusk16S) de- which originally was thought to be possibly less susceptible to DNA signed for mollusks and other invertebrates by the Stepien lab (Klymus degradation (Table 1), but was too similar in length with primer dimers. et al., 2017) was used for pond stores selling snails. For all assays, Thus, to increase taxonomic fidelity and alleviate issues with sequen- primer sets included the Illumina® sequencing primer and four different cing very short amplicons, we designed a longer 154NT FishCytb (-L) spacer inserts, labeled e–h (7–14NT) at the 5′ end (Table A3), to in- assay beginning at base 855 (Table 1), which shared the same reverse crease library diversity and improve HTS data quality (Fadrosh et al.,

Table 1 Bait store (A) and pond store (B) morphological and eDNA HTS metabarcode assay results, by state and totals, including Unadvertised Legal for sale, Unadvertised Native Species, Aquatic Invasive Species (AIS), and Hypophthalmichthys (Hypop.) spp. (invasive silver or bighead carps). All morphological samples from pond fish retailers were found to be the advertised species, and thus just eDNA HTS metabarcode assay results are shown (B and C).

A. Bait stores State N shops Unadvertised

All Legal Native AIS Hypop. spp.

Morphology (MORPH) IN 4 2 (0.50) 2 (0.50) – – – MI 14 11 (0.79) 11 (0.79) 3 (0.21) 0 (0.00) – OH 33 23 (0.70) 21 (0.64) 1 (0.03) 4 (0.12) – Total 51 36 (0.71) 34 (0.67) 4 (0.08) 4 (0.08) – eDNA (META) IN 4 4 (1.00) 4 (1.00) 3 (0.75) 4 (1.00) 1 (0.25) MI 14 14 (1.00) 14 (1.00) 11 (0.79) 14 (1.00) 1 (0.07) OH 33 33 (1.00) 33 (1.00) 17 (0.52) 27 (0.82) 3 (0.09) Total 51 51 (1.00) 51 (1.00) 31 (0.61) 45 (0.88) 5 (0.10) B. Pond store fishes IN 1 1 (1.00) 1 (1.00) – 1 (1.00) – MI 9 5 (0.56) – 3 (0.33) 2 (0.33) 2 (0.22) OH 10 6 (0.60) 2 (0.20) 2 (0.20) 2 (0.20) – Total 20 12 (0.60) 3 (0.15) 5 (0.25) 5 (0.25) 2 (0.10) C. Pond store mollusks MI 5 – – – 3 (0.60) – OH 6 – – – 3 (0.50) – Total 11 – – – 6 (0.55) –

3 M.R. Snyder, et al. Biological Conservation 245 (2020) 108430

2014; Wu et al., 2015). We avoided sharing 3' or 5′ indices for single sold ornamental species and/or recreational fishing species for use in spacer and primer combinations. private ponds/lakes. Our pond store detections thus were categorized based on their respective state and retailer type, with non-ornamental 2.2.2. DNA processing, controls, library prep, and HTS species classified as either: legal advertised, native non-pond species Genetic material was centrifuged, extracted, and subjected to a two- (including native species sold by ornamental retailers, and illegal native step library preparation method for HTS using positive controls (see species sold for recreational fishing), or unadvertised AIS. Deiner et al., 2017) to calculate sequencing error following Stepien To our knowledge, no laws existed in the sampled states against et al. (2019). Spacer inserts offset sequence positions in the libraries, to rearing or selling native invertebrates. Most invertebrates sold by or- improve data quality (Fadrosh et al., 2014; Klymus et al., 2017; Wu namental pond stores were termed “mystery snails” (Cipangopaludina et al., 2015) and facilitate detection and removal of cross-contamina- spp.), often advertised as “trapdoor snails”. Although these are AIS in tion and/or index-hopping (see Appendix A2). No-template negative North American freshwaters, no laws prohibited them from being controls were processed alongside all reactions. Amplifications were stocked in private ponds. Thus, invertebrates in the present study were attempted on negative controls (three per primer set) from all cen- categorized either as advertised or as unadvertised AIS. trifugations (RO/DI H2O) and extractions (reagent only), and assessed with gel electrophoresis and NanoDrop (ThermoFisher Scientific, Wal- 2.4.2. META assay bioinformatic pipeline tham, MA) readings of column-cleaned reactions. After all samples were HTS data analyses were modified from Stepien et al. (2019) (Ap- processed for each marker and year, negative cleanup controls were pendix A3; Table A3). A custom PYTHON v3.7.1 script (available at created by column-cleaning the reagents alone, on which indexing was https://doi.org/10.5061/dryad.4tmpg4f5x) trimmed primers from raw attempted. We exclusively sequenced positive samples whose negative sequence reads and removed index-hops and/or possible cross-con- controls (reactions, extractions, centrifugations, and cleanups) did not tamination (MacConaill et al., 2018; Xiong et al., 2016), in reference to amplify (determined with gel electrophoresis). the correct spacer inserts and primer sets. Assay reads and positive Positive controls were constructed by mixing equal mass (μg) of control sequences were subjected to BLAST (Basic Local Alignment genomic DNA from 10 marine fish species that did not occur in the Search Tool; https://blast.ncbi.nlm.nih.gov/Blast.cgi) against the North American freshwaters. Each first was Sanger sequenced for the custom fish cytb database used for primer design and with all cytb se- cytb region of our eDNA META assays to ensure primer matches, and quences from all Actinopterygii on GenBank, and to the entirety of then were tested using META (protocols in Appendices 1–2; GenBank GenBank for the Mollusk16S META assay. AIS commonly used in fish accessions in Table A4). food (e.g., Alosa spp.) were disregarded (Frimodt and Dore, 1995; Miles and Chapman, 2006). Possible error was calculated from the fre- 2.3. Retailer and angler surveys quencies of unexpected amplicon sequence variants (ASVs) in positive controls. These can result from incorrect base calls on sequencing Sampled stores were asked to complete a survey about their supply platforms, index-hopping (when the wrong index is incorporated into a chains, over the phone or at a later visit (so as not to influence the HTS library, causing mis-assignment), or cross-contamination during species sold during sampling). Angler surveys were distributed win- library prep (MacConaill et al., 2018). Error frequencies <0.1% were ter–spring 2017 online through the Great Lakes' Sea Grant network rounded up to 0.1%, which is the observed rate of index-hopping on (https://greatlakesseagrant.com/), Ohio Sea Grant (https:// Illumina MiSeq (MacConaill et al., 2018). ohioseagrant.osu.edu), and the University of Toledo Lake Erie A species detection was ranked as valid if its frequency was above Center's public lecture series mailing list (through the Center), and at the error determined from positive controls for that marker in that run, local boat launches and marinas. Questions included fishing experience, or if present in multiple assays, since individual primer sets each might bait use and disposal, and AIS awareness (Table A4). The University of have some bias (Bylemans et al., 2018; Evans et al., 2017). META re- Toledo Institutional Review Board (IRB) reviewed this proposal and sults were combined (as above) and compared to MORPH data using a project surveys, and ruled them exempt. custom PYTHON script, which removed possible false positives using the error cutoff from positive controls, and compared BLAST hitsto 2.4. Data analyses advertised species and state regulations (including false negatives in META and MORPH results). Samples indicating AIS not known to occur 2.4.1. Categorizing detections: legal bait and pond species in the sampling area were re-run for all assays to confirm presence. All We categorized MORPH and META detections in bait stores as either custom scripts are deposited in Dryad (https://doi.org/10.5061/dryad. advertised legal bait, unadvertised legal bait, native non-bait (often 4tmpg4f5x]). All FASTQ files are in NCBI Sequence Read Archive Bio- undersized fishes), or AIS based on the respective state laws. Laws project #PRJNA548536. regulating bait species for sale in MI (MDNR, 2016) and IN (IDNR, 2019a) during this study were more restrictive than in OH (Ohio, 2.4.3. Modeling relationships among MORPH, META, and survey results 2016b), with MI banning commercial sale of all AIS (Table A5). In- Generalized linear models in R evaluated potential predictor and vasive alewife (Alosa pseudoharengus), threadfin shad (Dorosoma pete- response variables. Those included presence of advertised or un- nense), and goldfish (Carassius auratus) were legal IN bait, but only advertised bait or pond store species, illegal-to-be-sold native species, when caught and used in the same water body (making them illegal for AIS, or non-native species (e.g., mosquitofish, round goby Neogobius sale in most cases). Three AIS were legal OH bait: common carp (Cy- melanostomu), silver or bighead carps) found with eDNA META or prinus carpio), rainbow smelt (Osmerus mordax), and skipjack herring MORPH. Predictor variables examined included species advertised for (Alosa chrysochloris). sale, presence of illegal native or AIS determined with MORPH and/or No laws in the three states governed species stocked in private META, the state where the store was located (since they varied in laws ponds, provided that they were obtained in-state and that there was no and regulations), the store's supplier, and the day/month of sampling connection to a local stream or larger waterbody. Regulations specified (i.e., the potential effect of summer versus late season; see §2.1). Models which species were legal to rear and sell by aquaculture facilities. were constructed separately on MORPH and META results, for in- During our study, IN listed 36 legal species (Indiana, 2014), MI 55 dividual samplings and the combined data per store. (Michigan, 1996), and OH 94 (ODNR, 2019a) for aquaculture, with Linear models evaluated angler survey results, comparing reported endangered species and some AIS being prohibited. All ornamental bait release behavior with possible predictive factors, including years of pond species (which all are AIS) were legal to be aquacultured in these fishing experience, areas most often fished, AIS awareness, andbait three states (see Results; Table A5). The pond stores we sampled either type (live fish, live non-fish, or artificial). Separate models were

4 M.R. Snyder, et al. Biological Conservation 245 (2020) 108430 constructed for all anglers and for those using live fish bait. AIS 2016 and one from 2017), likely due to inhibition or lack of eDNA (see awareness and eDNA results, supply chains, and angler survey data Appendix A2). were mapped in ESRI ArcGIS v10.7 (Redmond, CA). To depict species Among 113 samples from bait stores, at least one from every store detections from MORPH and META, we constructed phylogenetic trees was successfully sequenced for at least one assay and 81% were se- with Bayesian analyses in MrBayes 3.2 (Ronquist et al., 2012). That quenced with all three fish assays (Mean = 2.7 ± 0.07; Table A8). Of dataset included 1033NT of cytb and 608NT of cytochrome oxidase c 19 late season bait store samples, 94.7% (18) successfully sequenced subunit I (COI) for all fishes detected in our study (Table A2). The with the three assays, and one with just two. model was run using GTR substitution with gamma-distributed rate Twenty (95.2%) of the 21 pond fish retailer samples were success- variation across sites and a proportion of invariable sites (lset nst = 6 fully sequenced with all three fish assays. One pond sample did not rates = invgamma). Analyses ran for 300,000 generations, with sam- yield successful libraries. All (100%) of the 13 pond stores that sold pling every 1000th generation and the first 25% generations discarded mollusks were successfully sequenced with the Mollusk16S assay, but as burn-in. only 85% (11) contained invertebrate sequences. Of 5569 ASVs with a BLAST hit, 94% were identified to the species level (Appendix A3). 3. Results 3.2.2. Detections from META assays versus MORPH 3.1. Store advertisement, MORPH sampling, and identifications Just 32.6% (15) of the 46 fish species' eDNA detected in bait stores using META also were found by MORPH observations (Table A9). Of Five legal bait fish species (all cyprinids) were advertised during the 215 total MORPH fish detections (from all bait stores and sam- visits to bait stores (Table A6), and 39% (18) of 46 stores that were plings), META discerned 96% (206) of them. An initial potential dis- sampled two or more times did not advertise any of the same species in crepancy was that creek chub (Semotilus atromaculatus) was not verified repeat visits. Some advertised ambiguously (e.g., “minnows” or “shi- by META in three shop samples in which it was tentatively observed ners”, in 14% of 113 sampling events). Two (14.2%) of the 14 MI stores using MORPH by the student researcher. Notably, creek chub fre- advertised mud minnows (Umbridae), yet none were detected with quently was advertised by many of the bait stores. However, our META MORPH or META. One or more unadvertised species was/were for sale analyses identified those individuals as being white sucker (Catostomus in 71% (36) of the 51 stores during visits (Table 1A), and 67% (34) sold commersoni). This initial discrepancy was due to MORPH mis- unadvertised legal bait species. Five (10.4%) of 48 stores sampled in identification, which then was acknowledged by the student researcher 2016 and two (4.1%) of 49 stores in 2017 exclusively sold unadvertised and confirmed by the research team. With that correction, 210ofthe legal bait species (Table A6). In 2016, all unadvertised species observed 215 (98%) individual MORPH findings were confirmed by META, with with MORPH were legal bait. In 2017, both native non-bait and AIS just 2.3% (five) missing. These were a small proportion of grams ofbait were for sale in 8% (four) of the stores. Bait store advertisements thus (mean = 5.5% ± 3.59%). Four of the five individuals missing from often were inconsistent, inaccurate, and misleading. META were identified by MORPH as unadvertised golden shiner States differed in regulations and species for sale. For example, (Notemigonus crysoleucas) or common shiner (Luxilus cornutus); both brook stickleback (Culaea inconstans), an unadvertised legal bait sold by were legal bait species. The fifth individual missing from META wasa 6.1% (two) of the 33 OH stores also occurred in 7.1% (one) MI store, goldfish in a single sample, which was amplified by just one fishassay where it belonged to the native non-bait category (Table A6). Yellow (FishCytb-L), potentially indicating PCR inhibition. perch (Perca flavescens), a native non-bait species in all states, occurred META eDNA assays of bait stores yielded many more individual in 6.1% (two) of the OH stores, and the AIS western mosquitofish detections (totaling 619), of which 65% (402) were not observed with (Gambusia affinis) was sold by 12.2% (four). Five (9.8%) of 51 total MORPH. META discerned 31% (15) more bait stores positive for un- stores advertised and sold AIS goldfish as bait, including 50% ofthe advertised species (legal bait, native non-bait, and/or AIS; p < 0.001 four IN stores (where it was illegal) and 9.1% of the OH stores (where it for each) than MORPH. All 23 unadvertised species identified with was prohibited for sale as bait) (Table A5). Models showed that neither META in pond stores were undetected by MORPH. state nor supplier significantly explained occurrences of unadvertised bait, native non-bait, or AIS in stores, and year was not a predictor for 3.2.3. Species, categories, and model predictors in bait stores any type of unadvertised bait. Since META eDNA results did not significantly differ between Of 21 pond store retailers sampled, 90.4% (19) solely sold ad- sampling years and/or seasons, temporal results from individual shops vertised ornamental species, including 95% with koi (a cultivar of were combined for further analyses. One or more unadvertised species common carp), 76% having goldfish, and 71% with mystery snails. were discerned in all (100%) of the bait stores during at least one visit Additionally, 24% sold mosquitofish for insect control. Two stores (Table 1A), which included one or more legal bait species (Table A10). (9.5%) also sold recreational fish species. All species were confirmed Five stores (10%) solely contained unadvertised legal bait (no native with both MORPH and META. All native species sold by recreational non-bait or AIS), according to META results (Fig. 1, Table A10). Just 5% pond fish retailers were legal for sale, except for striped bass(Morone (five) of 106 total bait store samples exclusively contained bait species saxatilis), which was an AIS sold in OH. Most pond supply stores pro- advertised for sale. Twenty-four (23%) of the samples indicated un- viding recreational fish species delivered them directly to the custo- advertised legal bait alone (no native non-bait or AIS). mer's pond. Proportions of stores containing eDNA of illegal-to-be-sold native fish species or AIS did not statistically differ among the three states. 3.2. Genetic detection of species Three legal-to-be-sold bait fish species were found in all states: golden shiner, emerald shiner (Notropis atherinoides), and bluntnose minnow 3.2.1. META HTS metrics (Pimephales spp.). Some species findings varied among the three states. Sequence reads from bait and pond store HTS libraries totaled Native species that were illegal-to-be-sold as bait, detected in just a 49,275,676 (mean/sample/assay±SE = 113,801 ± 3624; Table A7). single state included: northern pike (Esox lucius) and roughbelly darter DADA2 merged an average proportion of 0.79 ± 0.01 trimmed reads/ (Percina spp.) in MI, and rock bass (Ambloplites rupestris), white bass sample/assay (mean = 64,385 ± 1400), averaging 18 ± 0.6 ASVs/ (Morone chrysops), and whitefish (Coregonus spp.) in OH (Fig. 2A). sample/assay. Error in the positive controls was low, ranging from Mosquitofish and white perch (Morone americana) were AIS solely 0.07%–0.29% (mean = 0.19 ± 0.02%). Amplification was not dis- identified in OH stores. cerned in any negative centrifugation, extraction, or cleanup control. Native non-bait species were uncovered in 61% (37) of the 51 bait Six bait store samples did not produce successful libraries (five from stores (Fig. 1, Table 1A), which most frequently were walleye (Sander

5 M.R. Snyder, et al. Biological Conservation 245 (2020) 108430

Fig. 2. eDNA metabarcode assay detections of fish species in (A) bait and (B) pond stores, and (C) invertebrates from the latter. Bar plots show % ofdetectionsfrom stores in IN, MI, and OH (for sample sizes see Table A1). Shades denote legal bait or pond species, native non-pond/bait species, or AIS. Some native species in OH were sold by pond stores that advertised them for sale and others that did not. Tree for fishes was constructed using representative cytb and COI sequences for each species (Table A2). Not all invertebrate species had representative sequences for these two genes available, and thus we did not illustrate their relationships with a tree. vitreus) (22% of stores) or yellow perch (33%) (Fig. 2A). We also de- our re–runs (see Methods; Fig. 2A), including Eurasian ruffe (Gymno- tected river herring (Alosa spp.), which are AIS often used in fish food; cephalus cernua) in 14% (seven), bighead carp in 4% (two), and silver these were disregarded (see Methods). META assays identified un- carp in 10% (five) of the stores; one OH store contained the latterin advertised AIS in 88% of stores (Table 1A), which were predominantly both sampling years (Fig. 1, Tables 1A, A8). All bighead carp eDNA goldfish (25% of stores), mosquitofish (31%), round goby (55%), tu- occurred together with silver carp. benose goby (Proterorhinus semilunaris; 10%), and grass carp (Cteno- In 2017, AIS detections increased by 25% (from 22 to 37 stores) and pharyngodon idella; 8%) (Fig. 2A). Three species that were unreported native non-bait by 10% (from 14 to 22 stores). Despite these apparent and unknown from the central–lower Great Lakes were confirmed by increases, year did not appear to significantly predict AIS or native non-

6 M.R. Snyder, et al. Biological Conservation 245 (2020) 108430 bait. When comparing the 19 late season (September–November) adjacent U.S. states, except Illinois. Anglers reported having 0–70 samples to the 93 from June–August, season also was not significant. (mean = 36.4) years of fishing experience, with 96.7% (173) aware of However, presence of native non-bait species significantly predicted at least one AIS, some familiar with all 10 species listed, and several AIS (p < 0.001), increasing the likelihood of AIS by 28%. Stores that naming others (e.g., dreissenid mussel, alewife, Atlantic salmon). Live advertised ambiguously (i.e., minnows or shiners), constituting 16% bait use was reported by 53% of all anglers (Fig. 3), and was sig- (17) of the 106 samples successfully sequenced, were 38% more likely nificantly greater for Lake Erie, in 61.6% (40) of the 61reports to have AIS eDNA (p = 0.032). Other possible predictors were insig- (p < 0.001). Bait dumping into waterways was reported by 23% of all nificant (i.e., presence of AIS, native non-bait, and/or specific non-na- anglers and 44% of those using live fish bait (Table A13). Bait release tive species), for analyses of the individual store samplings. practices significantly varied by region (Fig. 4; Table A13), being re- When data were combined for all samples per bait store, no vari- ported by 80% using live bait into the Lake St. Clair region (p = 0.016), ables significantly predicted overall native non-bait or AIS detections. 60% into OH inland lakes and streams (p = 0.037), and 50% into Lake However, round goby (slope = 0.34, p < 0.040) and Eurasian ruffe Erie (p = 0.042). (slope = 0.30, p < 0.032) eDNA each occurred more frequently in the MI stores than in OH. IN stores contained significantly more eDNA 4. Discussion positives for Eurasian ruffe, in 50% (two) of the four stores sampled (slope = 0.81, p < 0.003), than were found in OH. 4.1. Unadvertised species detection implications

3.2.4. Species, categories, and model predictors in pond store retailers Most MORPH and META detections of unadvertised species in bait For pond stores, 15% (three) of the 20 with successful META results and pond stores were legal for sale as bait or by aquaculture facilities. contained eDNA of unadvertised legal koi, goldfish, or mosquitofish Releases of legal species into a different geographic region or from (Fig. 2B). Native non-pond fishes (excluding those advertised for sale by aquaculture could transmit parasites or diseases into wild populations recreational pond fish suppliers) were identified with META in25% (Goodwin et al., 2004; Meyer, 1991; Walker and Winton, 2010). For (five) of the stores (Fig. 1, Table 1B), including single store occurrences example, a non-native tapeworm parasite (Schyzocotyle acheilognathi c.f. of gizzard shad (Dorosoma cepedianum), largemouth bass (Micropterus Bothriocephalus acheilognathi) was introduced into Lake Mead, NV, salmoides), walleye, white sucker, and yellow perch (Fig. 2B). State or through bait releases of infected invasive grass carp (Heckman et al., supplier did not significantly predict native non-pond species. 1993). Viral hemorrhagic septicemia virus (VHSV) is a swiftly evolving Pond store META identified three Alosa spp. commonly used in fish finfish disease known to occur in aquaculture operations, whichhas food, which were disregarded (as for bait stores). Grass carp eDNA caused large fish kills in the Great Lakes and beyond (Pierce and occurred in 5% (one store), for an ornamental pond fish supplier in OH, Stepien, 2012; Stepien et al., 2015; Walker and Winton, 2010). where it is a legal AIS to aquaculture (if genetically triploid and unable Although not statistically significant, the increase in native non-bait to reproduce; Ohio, 2016a). Round goby eDNA was in 20% (four) of the and AIS observed in our 2017 results (both META and MORPH) may stores (Fig. 2B, Table A11). Three fish species that have not established have reflected declining availability of local bait species (Copper, 2017; in the Great Lakes were detected and confirmed with our pond store Egan, 2017) or variations in suppliers. Additional declines in bait fish runs and re-runs, including Eurasian ide (also called orfe) (Leuciscus availability in the face of silver and bighead carp invasions are pre- idus) in one store and bighead carp in two, one of which also was po- dicted (Zhang et al., 2016). It may become more difficult to collect legal sitive for silver carp (Figs. 1, 2B, Table A11). bait, necessitating that retailers source from out of state, further in- Eleven of the 13 (85%) pond stores amplified with the Mollusk16S creasing AIS risk. The two most frequently found AIS with our META primer, yielding positives for eight different snail species (Fig. 2C). Four assays (round goby and western mosquitofish) occupy the same habitats stores solely contained Cipangopaludina chinensis, four had C. japonica as legal bait species, implicating collections from shared waterways as alone, and three possessed both. All advertised snails were AIS, and their sources. If native bait species continue to decline, more illegal 55% (six) of the 11 pond stores that sold mollusks contained genetic species and AIS may appear in retail supply chains. evidence of unadvertised AIS snails (Table 1C). These included Gyraulus Bait releases are a known vector for introductions and secondary parvus, Melanoides tuberculata, Planorbarius corneus, Physella acuta, and spread of AIS, including Eurasian ruffe (Winfield et al., 1996), round Planorbella trivolvis, with the latter two found in multiple stores goby (Janssen and Jude, 2001), and smallmouth bass (Micropterus do- (Fig. 2C). META identified zebra mussel Dreissena polymorpha in 36% lomieu; native in the Great Lakes but invasive in the western U.S.) (four) of the stores and invasive Bryozoa in 18% (two), including Fre- (Jackson, 2002). Many AIS that we detected are well-established in the dericella indica and Lophopodella carteri (Fig. 2B, Table A12). No vari- central Great Lakes region (i.e., goldfish, grass carp, mosquitofish, and ables (state, supplier, species sold, or native non-ornamental detections) round goby). However, releases can supplement the genetic variation of significantly predicted these AIS. an established AIS population, furthering success (Baker and Stebbins, 1965), as well as spread them into new areas. Reproductive grass carp 3.3. Retailer suppliers and angler survey results are locally established in the Sandusky River, a western Lake Erie tributary (Embke et al., 2016), nearby where many stores and suppliers Among bait stores, 50% (24) of 48 in 2016 and 61% (30) of 49 in reported collecting their bait in our study. Our META assays cannot 2017 divulged their suppliers, with most sourcing from a variety to discriminate whether the eDNA originated from triploid (infertile) in- ensure that bait remained in stock all season. Ten (19.6%) of the 51 bait dividuals that are legal in IN and OH aquaculture, or from diploid ju- stores named the same supplier and 11 specified that they sourced veniles captured locally. within their state (OH) (Fig. 3). Seven bait stores that sourced from a Several AIS that currently are not known in the central–lower Great single supplier (located along the southwestern Lake Erie shore (OH)) Lakes region were discerned by our investigation. Predicted impacts of were 43% (slope = 0.43) more likely to contain native non-bait species Eurasian ruffe if it reaches Lake Erie include competition with the eDNA (p = 0.043). That supplier declined to divulge its source(s). economically valuable native yellow perch fishery (Ogle, 1998). The Just two pond stores had the same supplier (Fig. A2), who did not ruffe has been established in the upper Great Lakes for ~30 years,yet appear correlated to presence of unadvertised legal bait or AIS. No has not been reported from Lake Erie, despite ~25 years of predictions significant relationship was found between unadvertised species (ofany that it would soon arrive (Stepien et al., 2018). Silver and bighead carps type) and supply chains for the pond stores. are predicted to significantly alter food webs and threaten native fish- We received 179 completed angler surveys (Table A13), who re- eries if they become established (Irons et al., 2007; Kolar et al., 2005; ported fishing in all five Great Lakes and inland lakes and streams inall Sass et al., 2014). Potential impacts of Eurasian ide invasion are

7 M.R. Snyder, et al. Biological Conservation 245 (2020) 108430

Fig. 3. Map of bait fish retailer supply chains across the sampling region, with inset of the Sandusky Bay region of western LakeErie,OH. understudied, but include competition with native species (USGS, bait and one AIS (common carp) in their MORPH survey of just four 2018). A cultivar of this fish, the golden orfe, whose coloration renders baitfish dealers in Ontario, Canada. Drake and Mandrak (2014) used it a popular ornamental species, is widely sold in many areas of the MORPH to identify eight unadvertised native non-bait species, in- world, including the U.S. (USGS, 2018), and thus might become es- cluding the imperiled river redhorse (Moxostoma carinatum) and three tablished. AIS from 50 Ontario, Canada bait retailers, who might have sourced Gastropod mollusks (snails), including cryptic AIS, frequently are from Lake Erie. Nathan et al. (2015) detected bighead and silver carps, introduced into new ecosystems via pond and home aquarium releases goldfish, rudd (Scardinius erythrophthalmus), and round and tubenose (Keller and Lodge, 2007). Species in the families Physidae, Thiaridae, gobies in single-species qPCR surveys of 576 bait stores across the eight Planorbidae, and Lymnaeidae have established populations globally U.S. Great Lakes states. Three of our five bait stores containing silver (Cowie and Robinson, 2003; Pointier and Augustin, 1999) and were carp eDNA also were sampled in 2012–13 by Nathan et al. (2015), who found in the pond stores here. Duggan (2010) identified similar snail did not then find it or other AIS (L. Nathan pers. comm.). However, AIS from New Zealand home aquaria. Pond and aquarium snails, in- those stores were located relatively near (<120 km) three other stores cluding Ph. acuta and Me. tuberculata, typically live in association with where their qPCR found silver carp eDNA (L. Nathan pers. comm.). aquatic plants and often are cryptic (Cowie and Robinson, 2003; Using multiple META assays, we discovered six AIS that Nathan et al. Duggan, 2010). Many of these snails have histories of accidental re- (2014) did not test for or find: Eurasian ruffe, common carp, grass carp, leases leading to established AIS populations, raising ecological con- mosquitofish, rainbow smelt, and white perch. Nathan et al. (2015) also cerns (Duggan, 2010). Introductions can lead to negative ecological did not attempt to detect illegal native taxa, whereas we identified interactions with related native species (Zukowski and Walker, 2009). eDNA from 13 illegal native species. For example, continued releases and range expansions of invasive Ci- Mahon et al. (2014) applied a single META assay to six bait stores in pangopaludina spp. (the Chinese mystery snail group) pose significant the Great Lakes, finding a maximum of three unadvertised legal bait threats to native snails (Van Bocxlaer and Strong, 2019). Moreover, species per retailer. They also found two unadvertised native non-bait invasive Me. tuberculata found here are intermediate hosts to several species and the AIS white perch in MI, where it already was established. human pathogenic trematode flukes, constituting human health risks In comparison, we discerned 13 native non-bait species and 11 AIS with (Pinto and Melo, 2011). Stricter laws and policies are needed to reg- our multiple META analyses of 51 bait stores, in a more comprehensive ulate sale of potential and already-established AIS (Keller and Lodge, investigation. Most sequence reads from the stores in Mahon et al. 2007), since all advertised pond snails in our investigation were AIS. (2014) were classified as “unknown fish”. It is unclear if this lower In comparison, Litvak and Mandrak (1993) found five native non- resolution was due to their marker selection or their bioinformatic

8 M.R. Snyder, et al. Biological Conservation 245 (2020) 108430

Fig. 4. Angler survey results for bait use and reported release (=dumping) in regions where three or more respondents reported fishing. pipeline. Almost all sequences here were resolved to species, with high (Kelly et al., 2014; Miya et al., 2015). This can be alleviated with degree of confidence due to our bioinformatic filtering criteria. multiple primer sets (Evans et al., 2017; Thomsen et al., 2012), con- Our approach, with multiple META assays, identified a wide range trols, and a stringent bioinformatic pipeline, as accomplished here. Our of species, significantly extending and enhancing prior studies ofAIS approach, which employed three META assays, indicated that multiple and non-legal species for sale in bait stores. Moreover, we conducted a assays markedly improved resolution over MORPH surveys. novel investigation of pond stores, discerning many AIS snails. If ap- In this study, most unadvertised species for sale in bait and pond plied more broadly, these protocols likely would uncover additional AIS stores were legal. It is unlikely that each shipment to a store would and illegal species occurrences in the retail vector. For example, contain exactly the same species over the fishing season. Although some Trujillo-González et al. (2019) showed use of eDNA as a biosecurity tool species might no longer be present in a store, the eDNA from those to screen pathogens in the aquarium trade, indicating potential of this individuals might remain for a few days (Zaiko et al., 2018). With large biotechnology. numbers of individuals in their tanks, retailers may be incapable or unmotivated to determine exactly which species they have for sale. Improved identifications of AIS and illegal native species are necessary 4.2. MORPH versus META analyses for their removal from shops (Litvak and Mandrak, 1993; Nathan et al., 2014), and may be significantly improved with META and bioinfor- Our eDNA META results revealed relatively few false negatives and matics. more detections of unadvertised species than MORPH. This is expected as retailer tanks typically contain dozens (pond stores) or hundreds to thousands (bait) of individuals, and thus are unlikely to be accurately 4.3. Sources of eDNA in tanks represented in relatively small samples. Confidence levels in genetic results are much higher than for MORPH (Imtiaz et al., 2017), corre- Cells and free DNA can adhere to nets, traps, or other species during sponding to our findings. MORPH provides counts of species in tanks, collection, persist in water from which species were harvested/reared, but results in many more false negatives. Our META assays are re- or be accidentally introduced to tanks (such as via filleting of game fish stricted to relative abundances of eDNA reads, but sampled genetic in a bait store). If any were the case here, high numbers of eDNA se- material from all individuals and thus found many more unadvertised quences for native non-bait species would be expected in stores that species. We employed a pipeline that removed false positives. filleted game fish, and/or for native non-bait and established AISin Research comparing META with MORPH sampling in the environ- stores sourcing from the environment. None of those trends occurred ment showed that it was at least as good or provided a valuable com- here. We did not collect data on water sources for tanks in our retailer plement (Deiner et al., 2017). Investigations employing just a single survey, but observed that most were filled from tap water. eDNA META assay often had some false negatives due to primer bias Persistence of eDNA in store tanks might be influenced by water

9 M.R. Snyder, et al. Biological Conservation 245 (2020) 108430 chemistry, temperature, microbial communities, total biomass present, behavior, and presence of AIS in bait stocks and pond stores, hundreds and/or UV radiation (Barnes et al., 2014). Studies of eDNA degradation to possibly thousands of introductions likely occur every year due to in tanks or mesocosms estimated that eDNA is undetectable after these vectors. Those other studies, and our findings, illustrate the im- <14 days (Barnes et al., 2014; Jo et al., 2019; Strickler et al., 2015). portance of further AIS education, including against releases of bait, Moreover, almost every bait and pond store sampled here had filters aquacultured species, aquarium pets, and pond species. that actively removed waste from tanks, and the tanks contained very large numbers of live individuals. Thus, it is most likely that our eDNA 5. Conclusions META results reflected the actual present-day or very recent physical compositions of the species in tanks. The addition of eRNA analyses (see Our analyses of bait and pond stores discerned prevalence of eDNA Pochon et al., 2017) could differentiate live metabolizing species in the from unadvertised species, illegal native species, and AIS, including tanks, if applied to our study design. silver and bighead carps. Retailers appear to do little to prevent accidental and intentional releases, with the latter being a common practice 4.4. Retailers and angler behavior among surveyed anglers. AIS presence appeared unrelated to specific retailer supply chains, highlighting an industry-wide lack of will- Our investigation showed that retailers obtained their inventories ingness, motivation, and/or ability to identify species for sale and re- from a wide diversity of sources, with few sharing common suppliers. move those that are illegal. Pond stores communicated little to no in- This appeared especially true for pond stores. Survey response fre- formation about potential escape of species, or proper pond quencies resembled those reported in other studies (e.g., Kilian et al., construction and management. Thus, there is high risk of further AIS 2012; Litvak and Mandrak, 1993). One OH bait supplier was sig- introductions and spread via these vectors. A genetic surveillance pro- nificantly associated with native non-bait species presence, whose gram, such as employing our META assays, could help fishery and proximity to Lake Erie indicated that their bait was obtained from local conservation managers to accurately screen for illegal species and AIS waters. The supplier and stores were unwilling or unable to identify the in retail. species present. The significant relationship between native non-bait and AIS in retailers implies that sourcing from the environment, or from Funding a supplier that does, increases the likelihood of AIS spread. Present results indicated that 20% of bait stores sourced bait from out of state This study was funded by USEPA GLRI grants GL-00E01149-0 and (similar to numbers from Kilian et al. (2012)), posing risk of AIS in- GL-00E01289 (to CAS) and GL-00E01898 (to CAS and KPC); the latter troductions and spread. Additional regulations requiring stores and two were partially subawarded to the University of Washington Joint suppliers to list bait source(s) likely would help alleviate potential AIS Institute for the Study of the Atmosphere and Ocean (JISAO). Summer spread and illegal sales of young native species. students (HPS, MRS) were supported by NSF Research Experiences for Few bait stores (<10) sampled here posted information about Undergraduates (REU) DBI-1461124 (to CAS and KPC). MRS was par- proper disposal of unused bait or AIS. One store in northwest OH dis- tially supported by a graduate student research fellowship from NSF played a “best practices” approach, providing pamphlets with pictures DGE-0742395 (to CAS and KPC) and by a fellowship from the of legal bait and AIS (including bighead and silver carps), encouraging University of Toledo, and by JISAO in Summer 2019. anglers to discard unused bait in the trash. In a mail survey, 49–84% of Great Lakes' bait stores claimed to communicate AIS information to Role of funding customers either with signage, printed information, or in conversations (Connelly et al., 2018), but were not verified with actual visits to re- The funding sources had no role in the study design; in the collec- tailers, and were not the case in our study. Nathan et al. (2014) dis- tion, analysis and interpretation of data; in the writing of the report; or covered that just 22% of 525 bait stores in the Great Lakes region in the decision to submit the article for publication. displayed signage or other materials about AIS. When provided with materials and signage, only 54% of those stores continued to display Credit authorship contribution statement them one year later (Nathan et al., 2014). We found three stores selling goldfish for bait in OH, despite not being legal for that purpose. Overall, Matthew R. Snyder: Data curation, Formal analysis, Writing - most retailers appear to be unaware of regulations. draft, Writing - review & editing. Carol A. Stepien: Project Lead, All three states provided online resources on best practices for pond Supervision, Conceptualization, Funding acquisition, Writing - review & construction and management to ensure that there were no connections editing, Methodology. Nathaniel T. Marshall: Data curation, Formal to other water bodies (IDNR, 2019b; MDEQ, 2017; MDNR, 1999; analysis, Writing - review & editing. Hannah B. Scheppler: Formal ODNR, 2019b). However, we observed no information posted in the analysis, Writing - review & editing. Christopher L. Black: Formal pond stores about this topic or AIS. To our knowledge, ours appears to analysis, Writing - review & editing. Kevin P. Czajkowski: be the first study to investigate and identify unadvertised species for Conceptualization, Supervision, Formal analysis, Writing - review & sale in regional pond stores or to examine their communications with editing. customers. Most anglers surveyed were aware of AIS, which did not necessarily result in proper bait disposal behavior. Some survey respondents ob- Declaration of competing interest tained from Sea Grant programs likely were more informed about AIS issues, thereby underestimating the true prevalence of bait and AIS The authors declare no financial or other conflict of interests. releases into waterways by the general population. Surveys conducted in the Canadian Great Lakes' provinces indicated that >50% of anglers Acknowledgements were unfamiliar with bait fish disposal regulations and 41% released unused bait (Litvak and Mandrak, 1993). Nearly all of their respondents This is contribution #4940 from NOAA PMEL and JISAO #2020- reportedly regarded bait releases as a service to the environment and 1060. This study was funded by USEPA GLRI grants GL-00E01149-0 the fishery (Litvak and Mandrak, 1993). Another study found that 65% and GL-00E01289 (to CAS) and GL-00E01898 (to CAS and KPC), sub- of Maryland anglers using live fish bait reported releasing it(Kilian awarded to the University of Washington Joint Institute for the Study of et al., 2012). Given the >13 million yearly angler fishing days in the the Atmosphere and Ocean (JISAO). Summer students (HPS, MRS) were Great Lakes (USDI et al., 2016), the frequency of reported release supported by NSF Research Experiences for Undergraduates (REU) DBI-

10 M.R. Snyder, et al. Biological Conservation 245 (2020) 108430

1461124 (to CAS and KPC). MRS was partially supported by a graduate Fadrosh, D.W., Ma, B., Gajer, P., Sengamalay, N., Ott, S., Brotman, R.M., Ravel, J., 2014. student research fellowship from NSF DGE-0742395 (to CAS and KPC) An improved dual-indexing approach for multiplexed 16S rRNA gene sequencing on the Illumina MiSeq platform. Microbiome 2 (1), 6. https://doi.org/10.1186/2049- and by a fellowship from the University of Toledo. We thank Scott 2618-2-6. McBride, David Butterfield, Fred Averick, Thomas Ackerman, and Anna Ficetola, Claude, M., Pompanon, F., Taberlet, P., 2008. Species detection using environ- Elz for logistical support. We also thank Kevin Kayle (Ohio Division of mental DNA from water samples. Biol. Lett. 4, 423–425. https://doi.org/10.1098/ rsbl.2008.0118. Wildlife) and Thomas Bacula (Indiana Department of Natural Frimodt, C., Dore, I., 1995. In: Farnham, S. (Ed.), Multilingual Illustrated Guide to the Resources) for help with understanding and locating legal documents World’s Commercial Coldwater Fish, 1st edition. Wiley-Blackwell, Hoboken, New about the bait and pond trades. Jersey. GLFC, 2018. Asian carp [WWW Document]. URL Great Lakes Fishery Commissionhttp:// www.glfc.org/asian-carp.php, Accessed date: 6 January 2020. Appendix A. Supplementary data GLFC, 2019. The Great Lakes fishery: a world-class resource! [WWW Document]. URL Great Lakes Fishery Commissionhttp://www.glfc.org/the-fishery.php, Accessed date: Supplementary data to this article can be found online at https:// 6 January 2020. Goodwin, A.E., Peterson, J.E., Meyers, T.R., Money, D.J., 2004. Transmission of exotic doi.org/10.1016/j.biocon.2020.108430. Bioinformatics scripts and pi- fish viruses. Fisheries 29, 19–23. https://doi.org/10.1577/1548-8446(2004)29[19, pelines are at https://doi.org/10.5061/dryad.4tmpg4f5x. A video TOEFV]2.0.CO;2. about the results and its implications, featuring viewpoints of invasive Heckman, R.A., Greger, P.D., Furtek, R.C., 1993. The Asian fish tapeworm, Bothriocephalus acheilognathi, in fishes from Nevada. J. Helminthol. Soc. Wash. 60, species experts, charter boat captains, and a bait store owner is avail- 127–128. able at: https://www.wgte.org/tv/programs/detecting-invasive- Hubbs, C.L., Lagler, K.F., 2007. Fishes of the Great Lakes Region, Revised Edition. species-great-lakes. University of Michigan Press, Ann Arbor, Michigan. IDNR, 2019a. Indiana bait dealer's license regulations [WWW Document]. URL Indiana Department of Natural Resourceshttps://www.in.gov/dnr/fishwild/files/fw-Bait_ References Dealer_Regs.pdf, Accessed date: 6 January 2020. IDNR, 2019b. Indiana fish pond management [WWW Document]. URL Indiana Department of Natural Resourceshttps://www.in.gov/dnr/fishwild/files/fishmgt. Adrian-Kalchhauser, I., Burkhardt-Holm, P., 2016. An eDNA assay to monitor a globally pdf, Accessed date: 6 January 2020. invasive fish species from flowing freshwater. PLoS One 11, e0147558. https://doi. Illumina, 2019. Nextera XT DNA Library Prep Kit Reference Guide. [WWW Document]. org/10.1371/journal.pone.0147558. URL. https://support.illumina.com/content/dam/illumina-support/documents/ AMFGLEO, 2016. Annual Agency Reports [WWW Document]. URL Association of documentation/chemistry_documentation/samplepreps_nextera/nextera-xt/nextera- Midwest Fish and Game Law Enforcement Officershttp://static1.squarespace.com/ xt-library-prep-reference-guide-15031942-05.pdf, Accessed date: 6 January 2020. static/53dd062ce4b0461efc568dee/t/57d811c320099e5bb1f2a9d6/ Imtiaz, A., Nor, S.A.M., Naim, D.M., 2017. Review: Progress and potential of DNA bar- 1473778201251/2016+AMFGLEO+Agency+Reports.pdf, Accessed date: 6 coding for species identification of fish species. Biodiversitas 18, 1394–1405. https:// January 2020. doi.org/10.13057/biodiv/d180415. Baker, H., Stebbins, G. (Eds.), 1965. The Genetics of Colonizing Species. Academic Press, Indiana, 2014. Article 9. Fish and Wildlife. 312 IAC [WWW Document]. Indiana New York, NY. Administrative Code URL. http://www.in.gov/legislative/iac/T03120/A00090.PDF, Barnes, M.A., Turner, C.R., 2016. The ecology of environmental DNA and implications for Accessed date: 6 January 2020. conservation genetics. Conserv. Genet. 17, 1–17. https://doi.org/10.1007/s10592- Irons, K.S., Sass, G.G., McClelland, M.A., Stafford, J.D., 2007. Reduced condition factor of 015-0775-4. two native fish species coincident with invasion of non-native Asian carps inthe Barnes, M.A., Turner, C.R., Jerde, C.L., Renshaw, M.A., Chadderton, W.L., Lodge, D.M., Illinois River, U.S.A. Is this evidence for competition and reduced fitness? J. Fish Biol. 2014. Environmental conditions influence eDNA persistence in aquatic systems. 71, 258–273. https://doi.org/10.1111/j.1095-8649.2007.01670.x. Environ. Sci. Technol. 48, 1819–1827. https://doi.org/10.1021/es404734p. Jackson, D.J., 2002. Ecological effects of Micropterus introductions: the dark side of black Bylemans, J., Gleeson, D.M., Hardy, C.M., Furlan, E., 2018. Toward an ecoregion scale bass. In: Phillip, D.P., Ridgway, M.S. (Eds.), Black Bass: Ecology, Conservation, and evaluation of eDNA metabarcoding primers: a case study for the freshwater fish Management. American Fisheries Society Symposium 31, pp. 221–232 Bethesda, biodiversity of the Murray–Darling Basin (Australia). Ecol. Evol. 8, 8697–8712. Maryland. https://doi.org/10.1002/ece3.4387. Janssen, J., Jude, D.J., 2001. Recruitment failure of mottled sculpin Cottus bairdi in Connelly, N.A., Lauber, T.B., Stedman, R.C., Knuth, B.A., 2018. Bait dealers’ roles in Calumet Harbor, southern Lake Michigan, induced by the newly introduced round preventing the spread of aquatic invasive species and fish pathogens in the Great goby Neogobius melanostomus. J. Great Lakes Res. 27, 319–328. https://doi.org/10. Lakes region. J. Great Lakes Res. 44, 514–520. https://doi.org/10.1016/j.jglr.2018. 1016/S0380-1330(01)70647-8. 04.005. Jo, T., Murakami, H., Yamamoto, S., Masuda, R., Minamoto, T., 2019. Effect of water Copper, M., 2017. Erie-area bait shops, anglers search for Lake Erie emerald shiners temperature and fish biomass on environmental DNA shedding, degradation, and size [WWW Document]. URL GoErie.comhttps://www.goerie.com/news/20170704/ distribution. Ecol. Evol. 9, 1135–1146. https://doi.org/10.1002/ece3.4802. erie-area-bait-shops-anglers-search-for-lake-erie-emerald-shiners, Accessed date: 6 Keller, R.P., Lodge, D.M., 2007. Species invasions from commerce in live aquatic or- January 2020. ganisms: problems and possible solutions. BioScience 57, 428–436. https://doi.org/ Cowie, R.H., Robinson, D.G., 2003. Pathways of introduction of nonindigenous land and 10.1641/B570509. freshwater snails and slugs. In: Carlton, J. (Ed.), Invasive Species: Vectors and Kelly, R.P., Port, J.A., Yamahara, K.M., Crowder, L.B., 2014. Using environmental DNA to Management Strategies. Island Press, pp. 93–122. census marine fishes in a large mesocosm. PLoS One 9, e86175. https://doi.org/10. Cuddington, K., Currie, W.J.S., Koops, M.A., 2014. Could an Asian carp population es- 1371/journal.pone.0086175. tablish in the Great Lakes from a small introduction? Biol. Invasions 16, 903–917. Kilian, J.V., Klauda, R.J., Widman, S., Kashiwagi, M., Bourquin, R., Weglein, S., Schuster, https://doi.org/10.1007/s10530-013-0547-3. J., 2012. An assessment of a bait industry and angler behavior as a vector of invasive Deiner, K., Bik, H.M., Mächler, E., Seymour, M., Lacoursière-Roussel, A., Altermatt, F., species. Biol. Invasions 14, 1469–1481. https://doi.org/10.1007/s10530-012- Creer, S., Bista, I., Lodge, D.M., Vere, N. de, Pfrender, M.E., Bernatchez, L., 2017. 0173-5. Environmental DNA metabarcoding: transforming how we survey animal and plant Klymus, K.E., Marshall, N.T., Stepien, C.A., 2017. Environmental DNA (eDNA) meta- communities. Mol. Ecol. 26, 5872–5895. https://doi.org/10.1111/mec.14350. barcoding assays to detect invasive invertebrate species in the Great Lakes. PLoS One Drake, D.A.R., Mandrak, N.E., 2014. Ecological risk of live bait fisheries: a new angle on 12, e0177643. https://doi.org/10.1371/journal.pone.0177643. selective fishing. Fisheries 39, 201–211. https://doi.org/10.1080/03632415.2014. Kolar, C., Chapman, D., Courtenay, W., Housel, C., Williams, J., Jennings, D., 2005. Asian 903835. carps of the genus Hypophthalmichthys (Pisces, Cyprinidae) — a biological synopsis Duggan, I.C., 2010. The freshwater aquarium trade as a vector for incidental invertebrate and environmental risk assessment [WWW Document]. URL National Invasive fauna. Biol. Invasions 12, 3757–3770. https://doi.org/10.1007/s10530-010-9768-x. Species Council materialshttps://digitalcommons.unl.edu/cgi/viewcontent.cgi? Egan, D., 2017. Shiner shortage has bait shops all in a tangle [WWW Document]. URL article=1004&context=natlinvasive, Accessed date: 6 January 2020. Crain's Cleveland Business https://www.crainscleveland.com/article/20170701/ LED MDNR, 2018. 2018 Annual Programs Report. Law Enforcement Division of the news/170709996/shiner-shortage-has-bait-shops-all-tangle, Accessed date: 6 Michigan Department of Natural Resources URL. https://www.michigan.gov/ January 2020. documents/dnr/law_division-annual_report_638555_7.pdf, Accessed date: 6 January Embke, H.S., Kocovsky, P.M., Richter, C.A., Pritt, J.J., Mayer, C.M., Qian, S.S., 2016. First 2020. direct confirmation of grass carp spawning in a Great Lakes tributary. J. GreatLakes Litvak, M.K., Mandrak, N.E., 1993. Ecology of freshwater baitfish use in Canada and the Res. 42, 899–903. https://doi.org/10.1016/j.jglr.2016.05.002. United States. Fisheries 18, 6–13. https://doi.org/10.1577/1548-8446(1993) Erickson, R.A., Rees, C.B., Coulter, A.A., Merkes, C.M., McCalla, S.G., Touzinsky, K.F., 018<0006:EOFBUI>2.0.CO;2. Walleser, L., Goforth, R.R., Amberg, J.J., 2016. Detecting the movement and Lodge, D.M., Finnoff, D., 2008. Annual losses to Great Lakes region by ship-borne invasive spawning activity of bigheaded carps with environmental DNA. Mol. Ecol. Res. 16, species at least $200 million [WWW Document]. In: Invasive Species in the Great 957–965. https://doi.org/10.1111/1755-0998.12533. Lakes: Costing Us Our Future, URL. https://www.invasive.org/gist/products/ Evans, N.T., Li, Y., Renshaw, M.A., Olds, B.P., Deiner, K., Turner, C.R., Jerde, C.L., Lodge, library/lodge_factsheet.pdf, Accessed date: 6 January 2020. D.M., Lamberti, G.A., Pfrender, M.E., 2017. Fish community assessment with eDNA MacConaill, L.E., Burns, R.T., Nag, A., Coleman, H.A., Slevin, M.K., Giorda, K., Light, M., metabarcoding: effects of sampling design and bioinformatic filtering. Can. J.Fish. Lai, K., Jarosz, M., McNeill, M.S., Ducar, M.D., Meyerson, M., Thorner, A.R., 2018. Aquat. Sci. 74, 1362–1374. https://doi.org/10.1139/cjfas-2016-0306. Unique, dual-indexed sequencing adapters with UMIs effectively eliminate index

11 M.R. Snyder, et al. Biological Conservation 245 (2020) 108430

cross-talk and significantly improve sensitivity of massively parallel sequencing. BMC 539–542. https://doi.org/10.1093/sysbio/sys029. Genomics 19, 30. https://doi.org/10.1186/s12864-017-4428-5. Sass, G.G., Hinz, C., Erickson, A.C., McClelland, N.N., McClelland, M.A., Epifanio, J.M., Mahon, A.R., Nathan, L.R., Jerde, C.L., 2014. Meta-genomic surveillance of invasive 2014. Invasive bighead and silver carp effects on zooplankton communities in the species in the bait trade. Conserv. Genet. Resour. 6, 563–567. https://doi.org/10. Illinois River, Illinois, USA. J. Great Lakes Res. 40, 911–921. https://doi.org/10. 1007/s12686-014-0213-9. 1016/j.jglr.2014.08.010. MDEQ, 2017. Building a Pond in Michigan [WWW Document]. Michigan Department of Shokralla, S., Spall, J.L., Gibson, J.F., Hajibabaei, M., 2012. Next-generation sequencing Environmental Quality URL. https://www.michigan.gov/documents/deq/wrd- technologies for environmental DNA research. Mol. Ecol. 21, 1794–1805. https://doi. permit-pond_559023_7.pdf, Accessed date: 6 January 2020. org/10.1111/j.1365-294X.2012.05538.x. MDNR, 1999. Building and Managing Ponds [WWW Document]. Michigan Department of Stepien, C.A., Pierce, L.R., Leaman, D.W., Niner, M.D., Shepherd, B.S., 2015. Gene di- Natural resources URL. https://www.michigandnr.com/publications/pdfs/ versification of an emerging pathogen: a decade of mutation in a novel fishviral huntingwildlifehabitat/landowners_guide/Habitat_Mgmt/Wetland/Building_ hemorrhagic septicemia (VHS) substrain since its first appearance in the Laurentian Managing_Ponds.htm, Accessed date: 6 January 2020. Great Lakes. PLoS One 10, e0135146. https://doi.org/10.1371/journal.pone. MDNR, 2016. Minnow, Wiggler, and Crayfish Regulations for Commercial Purposes and 0135146. Waters That Are Closed to the Personal Take of Minnows [WWW Document]. Stepien, C.A., Eddins, D.J., Snyder, M.R., Marshall, N.T., 2018. Genetic change versus Michigan Department of Natural Resources URL. https://www.michigan.gov/ stasis over the time course of invasions: trajectories of two concurrent, allopatric documents/dnr/FO-216-07_182409_7.pdf, Accessed date: 6 January 2020. introductions of the Eurasian ruffe. Aquat. Invasions 13, 537–552. https://doi.org/ Meyer, F.P., 1991. Aquaculture disease and health management. J. Anim. Sci. 69, 10.3391/ai.2018.13.4.11. 4201–4208. https://doi.org/10.2527/1991.69104201x. Stepien, C.A., Snyder, M.R., Elz, A.E., 2019. Invasion genetics of the silver carp Michigan, 1994. Natural Resources and Environmental Protection Act (Excerpt) Act 451 Hypophthalmichthys molitrix across North America: differentiation of fronts, in- of 1994. URL. http://www.legislature.mi.gov/(S(r30ikfhhyvpgirfx2zvfp2y1))/mileg. trogression, and eDNA metabarcode detection. PLoS One 14, e0203012. https://doi. aspx?page=GetObject&objectname=mcl-Act-451-of-1994, Accessed date: 9 January org/10.1371/journal.pone.0203012. 2020. Stern, C.V., Upton, H.F., Cynthia, B., 2014. Asian Carp and the Great Lakes Region (No. Michigan, 1996. Michigan Aquaculture Development Act. Act 199 of 1996. URL. http:// R41082), Prepared for Members and Committees of Congress. Congressional www.legislature.mi.gov/(S(vfrgtb55ih0jydqw2wuzj545))/documents/mcl/pdf/mcl- Research Service URL. https://archive.org/stream/ act-199-of-1996.pdf, Accessed date: 9 January 2020. R41082AsianCarpandtheGreatLakesRegion-crs/R41082%20Asian%20Carp%20and Miles, R.D., Chapman, F.A., 2006. The Benefits of Fish Meal in Aquaculture Diets [WWW %20the%20Great%20Lakes%20Region_djvu.txt, Accessed date: 9 January 2020. Document]. University of Florida Institute of Food and Agriculture Sciences URL. Strickler, K.M., Fremier, A.K., Goldberg, C.S., 2015. Quantifying effects of UV-B, tem- http://edis.ifas.ufl.edu/pdffiles/fa/fa12200.pdf, Accessed date: 6 January 2020. perature, and pH on eDNA degradation in aquatic microcosms. Biol. Conserv. 183, Miya, M., Sato, Y., Fukunaga, T., Sado, T., Poulsen, J.Y., Sato, K., Minamoto, T., 85–92. https://doi.org/10.1016/j.biocon.2014.11.038. Yamamoto, S., Yamanaka, H., Araki, H., Kondoh, M., Iwasaki, W., 2015. MiFish, a set Thomsen, P.F., Kielgast, J., Iversen, L.L., Møller, P.R., Rasmussen, M., Willerslev, E., of universal PCR primers for metabarcoding environmental DNA from fishes: detec- 2012. Detection of a diverse marine fish fauna using environmental DNA from sea- tion of more than 230 subtropical marine species. R. Soc. Open Sci. 2, 150088. water samples. PLoS One 7, e41732. https://doi.org/10.1371/journal.pone.0041732. https://doi.org/10.1098/rsos.150088. Trautman, M.B., 1981. The Fishes of Ohio, Revised Edition. Ohio State University Press, Nathan, L.R., Jerde, C.L., McVeigh, M., Mahon, A.R., 2014. An assessment of angler Columbus, Ohio. education and bait trade regulations to prevent invasive species introductions in the Trujillo-González, A., Becker, J.A., Huerlimann, R., Saunders, R.J., Hutson, K.S., 2019. Laurentian Great Lakes. Manag. Biol. Invasions 5, 319–326. https://doi.org/10. Can environmental DNA be used for aquatic biosecurity in the aquarium fish trade? 3391/mbi.2014.5.4.02. Biol. Invasions. https://doi.org/10.1007/s10530-019-02152-0. Nathan, L.R., Jerde, C.L., Budny, M.L., Mahon, A.R., 2015. The use of environmental DNA U.S. 102nd Congress, 1992. Alien Species Prevention and Enforcement Act [WWW in invasive species surveillance of the Great Lakes commercial bait trade. Conserv. Document]. Government Publishing Office URL. https://www.govinfo.gov/content/ Biol. 29, 430–439. https://doi.org/10.1111/cobi.12381. pkg/STATUTE-106/pdf/STATUTE-106-Pg1729.pdf, Accessed date: 6 January 2020. NOAA, 2019. GLANSIS – Great Lakes Aquatic Nonindigenous Species Information System USDI, USFWS, USDOC, USCB, 2016. National Survey of Fishing, Hunting, and Wildlife- [WWW Document]. URL. https://www.glerl.noaa.gov/glansis/, Accessed date: 6 Associated Recreation. U.S. Department of the Interior, U.S. Fish and Wildlife Service, January 2020. and U.S. Department of Commerce, U.S. Census Bureau. ODNR, 2019a. Aquaculture Law Digest. Ohio Department of Natural Resources Division USFWS, 2006. Lacey Act. 18 USC 42–43, 16 USC 3371–3378. U.S. Fish and Wildlife of Wildlife, Columbus, OH URL. http://wildlife.ohiodnr.gov/portals/wildlife/pdfs/ Service URL. https://www.fws.gov/international/laws-treaties-agreements/us- publications/laws%20&%20regs/pub061.pdf, Accessed date: 9 January 2020. conservation-laws/lacey-act.html, Accessed date: 9 January 2020. ODNR, 2019b. Ohio Pond Management Handbook, a Guide to Managing Ponds for USFWS, 2012. The Cost of Invasive Species [WWW Document]. U.S. Fish and Wildlife Fishing and Attracting Wildlife. Ohio Department of Natural Resources Division of Service URL. https://www.fws.gov/verobeach/pythonpdf/costofinvasivesfactsheet. Wildlife, Columbus, OH URL. http://wildlife.ohiodnr.gov/portals/wildlife/pdfs/ pdf, Accessed date: 6 January 2020. publications/fish%20management/Pub432.pdf, Accessed date: 9 January 2020. USGS, 2018. Ide (Leuciscus idus) – species profile [WWW Document]. In: USGS Ogle, D.H., 1998. A synopsis of the biology and life history of ruffe. J. Great Lakes Res. 24, Nonindigenous Aquatic Species Database, URL. https://nas.er.usgs.gov/queries/ 170–185. https://doi.org/10.1016/S0380-1330(98)70811-1. factsheet.aspx?SpeciesID=557, Accessed date: 6 January 2020. Ohio, 2016a. 1501:31-19-01 Wild Animal Importing, Exporting, Selling and Possession USGS, 2019. NAS – Nonindigenous Aquatic Species Database [WWW Document]. URL. Regulations. Columbus, OH. URL. http://codes.ohio.gov/oac/1501%3A31-19, https://nas.er.usgs.gov/, Accessed date: 6 January 2020. Accessed date: 9 January 2020. Van Bocxlaer, B., Strong, E.E., 2019. Viviparidae Gray, 1847. In: Lydeard, C., Cummings, Ohio, 2016b. 1501:31-13-04 Bait and Bait Dealers [WWW Document]. Ohio Division of C.S. (Eds.), Freshwater Mollusks of the World. A Distribution Atlas. John Hopkins Wildlife URL. http://codes.ohio.gov/oac/1501:31-13-04v1, Accessed date: 6 University Press, Baltimore, Maryland, pp. 43–50. January 2020. Walker, P.J., Winton, J.R., 2010. Emerging viral diseases of fish and shrimp. Vet. Res. 41, Padilla, D.K., Williams, S.L., 2004. Beyond ballast water: aquarium and ornamental trades 51. https://doi.org/10.1051/vetres/2010022. as sources of invasive species in aquatic ecosystems. Front. Ecol. Environ. 2, 131–138. Winfield, I.J., Adams, C.E., Fletcher, J.M., 1996. Recent introductions of theruffe https://doi.org/10.1890/1540-9295(2004)002[0131:BBWAAO]2.0.CO;2. (Gymnocephalus cernuus) to three United Kingdom lakes containing Coregonus species. Pierce, L.R., Stepien, C.A., 2012. Evolution and biogeography of an emerging quasis- Ann. Zool. Fenn. 33, 459–466. pecies: diversity patterns of the fish viral hemorrhagic septicemia virus (VHSv). Mol. Wu, L., Wen, C., Qin, Y., Yin, H., Tu, Q., Nostrand, J.D.V., Yuan, T., Yuan, M., Deng, Y., Phylogenet. Evol. 63, 327–341. https://doi.org/10.1016/j.ympev.2011.12.024. Zhou, J., 2015. Phasing amplicon sequencing on Illumina Miseq for robust environ- Pinto, H.A., Melo, A.L.D., 2011. A checklist of trematodes (Platyhelminthes) transmitted mental microbial community analysis. BMC Microbiol. 15, 125. https://doi.org/10. by Melanoides tuberculata (Mollusca: Thiaridae). Zootaxa 2799, 15–28. https://doi. 1186/s12866-015-0450-4. org/10.11646/zootaxa.2799.1.2. Xiong, W., Li, H., Zhan, A., 2016. Early detection of invasive species in marine ecosystems Pochon, X., Zaiko, A., Fletcher, L.M., Laroche, O., Wood, S.A., 2017. Wanted dead or using high-throughput sequencing: technical challenges and possible solutions. Mar. alive? Using metabarcoding of environmental DNA and RNA to distinguish living Biol. 163, 139. https://doi.org/10.1007/s00227-016-2911-1. assemblages for biosecurity applications. PLoS One 12, e0187636. https://doi.org/ Zaiko, A., Pochon, X., Garcia-Vazquez, E., Olenin, S., Wood, S.A., 2018. Advantages and 10.1371/journal.pone.0187636. limitations of environmental DNA/RNA tools for marine biosecurity: management Pointier, J.-P., Augustin, D., 1999. Biological control and invading freshwater snails. A and surveillance of non-indigenous species. Front. Mar. Sci. 5. https://doi.org/10. case study. C. R. Acad. Sci. III 322, 1093–1098. https://doi.org/10.1016/S0764- 3389/fmars.2018.00322. 4469(99)00108-0. Zhang, H., Rutherford, E.S., Mason, D.M., Breck, J.T., Wittmann, M.E., Cooke, R.M., Rees, H.C., Maddison, B.C., Middleditch, D.J., Patmore, J.R.M., Gough, K.C., 2014. Lodge, D.M., Rothlisberger, J.D., Zhu, X., Johnson, T.B., 2016. Forecasting the im- REVIEW: the detection of aquatic animal species using environmental DNA – a review pacts of silver and bighead carp on the Lake Erie food web. Trans. Am. Fish. Soc. 145, of eDNA as a survey tool in ecology. J. Appl. Ecol. 51, 1450–1459. https://doi.org/ 136–162. https://doi.org/10.1080/00028487.2015.1069211. 10.1111/1365-2664.12306. Zukowski, S., Walker, K.F., 2009. Freshwater snails in competition: alien Physa acuta Ronquist, F., Teslenko, M., van der Mark, P., Ayres, D.L., Darling, A., Höhna, S., Larget, B., (Physidae) and native Glyptophysa gibbosa (Planorbidae) in the River Murray, South Liu, L., Suchard, M.A., Huelsenbeck, J.P., 2012. MrBayes 3.2: efficient Bayesian Australia. Mar. Freshw. Res. 60, 999–1005. https://doi.org/10.1071/MF08183. phylogenetic inference and model choice across a large model space. Syst. Biol. 61,