Development of a Quantitative PCR Assay for Detecting Egeria Densa in Environmental DNA Samples

Conservation Genetics Resources https://doi.org/10.1007/s12686-020-01152-w

TECHNICAL NOTE

Development of a quantitative PCR assay for detecting Egeria densa in environmental DNA samples

Dorothy M. Chase1 · Lauren M. Kuehne2 · Julian D. Olden2 · Carl O. Ostberg1

Received: 26 September 2019 / Accepted: 14 May 2020 © This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply 2020

Abstract Brazilian elodea (Egeria densa) is an invasive freshwater plant that demonstrates widespread ecological impacts in freshwater ecosystems and causes substantial economic damage. Here, we developed an environmental DNA assay for detection of E. densa to provide resource managers with a tool for early detection, identifcation, and monitoring of invasive populations.

Keywords Egeria densa · eDNA · Environmental DNA · Invasive aquatic plants · Quantitative PCR · qPCR

Brazilian elodea (Egeria densa) is a macrophyte native to To develop the E. densa assay, we retrieved DNA South America. Their introduction to numerous regions of sequences for several genes from E. densa and Elodea native the world has led to infestations that negatively impacted to North America (Elodea canadensis, Elodea nuttallii, aquatic ecosystems (de Winton and Clayton 1996; Roberts and Elodea bifoliate) from GenBank (Supplemental File). et al. 1999; Lund et al. 2007; Santos et al. 2011) and dis- Sequences were aligned using MEGA 7.0.21 (Kumar et al. rupted ecosystem function and services, causing economic 2016) and surveyed for sequence variation. The ITS1-5.8s- impacts (Hussner et al. 2017; Yarrow et al. 2009). Early ITS2 region was selected for assay development because it detection of invasive species and understanding of their dis- contained many candidates for comparison and many inter- tribution is essential to preventing further spread (Vander specifc, single nucleotide polymorphisms (SNPs). The E. Zanden and Olden 2008; Hussner et al. 2017). Traditional densa assay (EdITS1) was developed using the PrimerQuest methods for surveying invasive aquatic plants can be time Tool (Integrated DNA Technologies). The assay amplifes consuming and costly. Surveys of aquatic environmental 79 bases of the internal transcribed spacer-1 (ITS1) using DNA (eDNA) are an alternative that can provide improved primers (F: 5′-GGTCAATGGCAATTCCTTCTTG-3′; R: detectability relative to traditional feld survey methods 5′-GCGCACCACCCAAATAGA-3′) and a MGB non-fuo- (Ficetola et al. 2008; Jerde et al. 2011; Larson et al. 2020) rescent quencher probe (6FAM-5′-CCATGC CCA ATG AGA while requiring less efort (Biggs et al.; 2015; Sigsgaard GTCGCG TAT -3 ′). Assay specifcity was confrmed in silico et al. 2015; Evans et al. 2017). Here, we developed and vali- using primer-BLAST and BLAST. The closest non-E. densa dated an eDNA assay as a tool for early detection of E. densa match (Elodea canadensis and Elodea nuttallii) had 22% in aquatic environments. mismatch across primer and probe sequences. Tissue samples from E. densa, closely related species in the Hydrocharitaceae family and other aquatic plants were collected for testing of EdITS1 (Table 1). Genomic DNA Electronic supplementary material The online version of this was extracted and purifed from plant tissue samples using article (https://doi.org/10.1007/s12686-020-01152-w) contains DNeasy PowerPlant Pro Kit (Qiagen) and OneStep PCR supplementary material, which is available to authorized users. Inhibitors Removal kit (Zymo Research). To confrm that * Dorothy M. Chase the DNA quality was sufcient for PCR amplifcation, the [email protected] DNA was amplifed by using universal chloroplast primers C and D as described by Taberlet et al. (1991). 1 U.S. Geological Survey, Western Fisheries Research Center, In vitro testing of EdITS1 was accomplished using 60 Seattle, WA 98115, USA pg of genomic DNA, and each sample was tested in trip- 2 School of Aquatic and Fishery Sciences, University licate. Quantitative PCR (qPCR) assays were performed of Washington, Seattle, WA 98195, USA

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Table 1 Mean eDNA copy number/qPCR following testing of the EdITS1 assay against 60 pg genomic DNA from aquatic plants sampled in USA (unless indicated otherwise) Species (herbarium catalogue number) Mean DNA copy number Sample source location (county, state)

Egeria densa (not herbarium sample) 3491 King, Washington Egeria densa (WTU373946a) 2313 Pacifc, Washington Egeria densa (WTU409813a) 11231 King, Washington Egeria densa (WTU420154a) (GB: MT276593) 3017 Snohomish, Washington Egeria densa (WTU418935a) 4513 King, Washington Egeria densa (WTU412136a) (GB: MT276594) 7031 Clatsop, Oregon Egeria densa (WTU396706a) 5374 Benton, Oregon Egeria densa (WTU364520a) (GB: MT276595) 9364 Island, Washington Egeria densa (WTU342085a) 8543 San Juan, Washington Egeria densa (BRYV0089283b) (GB: MT276588) 3539 Sierra, New Mexico Egeria densa (BRYV0089130b) 3206 Somerset, New Jersey Egeria densa (CIC044457c) (GB: MT276590) 9059 Blaine, Idaho Egeria densa (CM 529914d) (GB: MT276589) 3455 Allegheny, Pennsylvania Egeria densa (CM537391d) 1041 Allegheny, Pennsylvania Egeria densa (UOS07132e) 2164 Franklin, Tennessee Egeria densa (LSU001115041f) (GB: MT276591) 3171 East Baton Rouge, Louisiana Egeria densa (LSU00139347f) (GB: MT276592) 1398 Calcasieuo, Louisiana Blyxa aubertii (LSU00031902f) – Evangelline, Louisiana Elodea bifoliate (CIC45326c) – Owyhee, Idaho Elodea canadensis (not herbarium sample) – Lewis, Washington Elodea canadensis (WTU412207a) – British Columbia, Canada Elodea canadensis (WTU410341a) – Deschutes, Oregon Elodea canadensis (WTU416493a) – Gilliam, Oregon Elodea nuttallii (WTU418682a) – Berks, Pennsylvania Elodea nuttallii (WTU416900a) – Thurston, Washington Elodea nuttallii (WTU412140a) – Whatcom, Washington Ceratophyllym demersum (not herbarium sample) – Thurston, Washington Hydrilla verticillata (WTU350381a) – King, Washington Hydrocharis morsus-ranae (WTU409810a) – Snohomish, Washington Hydrocharis morsus-ranae (WTU417097a) – Wayne, New York Limnobium laevigatum (WTU419663a) – Pacifc, Washington Limnobium laevigatum (WTU412560a) – Pacifc, Washington Limnobium spongia (LSU00118650f) – Lafourche, Louisiana Limnobium spongia (LSU00067022f) – Saint John the Baptist, Louisiana Myriophyllum aquaticum (WTU412475a) – Wahkiakum, Washington Myriophyllum heterophyllum (not herbarium sample) – Pierce, Washington Myriophyllum hippuroides (WTU350337a) – Whatcom, Washington Myriophyllum sibiricum (not herbarium sample) – Pierce, Washington Myriophyllum spicatum (WTU409806a) – King, Washington Myriophyllum verticillatum (WTU 412160a) – Pierce, Washington Najas canadensis (WTU409765a) – Pierce, Washington Najas fexilis (WTU409674a) – Grant, Washington Najas fexilis (not herbarium sample) – King, Washington Najas gracillima (MIN1145338g) – Becker, Minnesota Najas gracillima (MIN1145343g) – Clearwater, Minnesota Najas guadalupensis (WTU370895a) – Wahkiakum, Washington Najas marina (MIN114546g) – Polk, Minnesota Najas marina (MIN1145473g) – Grant, Minnesota Najas minor (LSU00132926f) – Plaquemines, Louisiana

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Table 1 (continued) Species (herbarium catalogue number) Mean DNA copy number Sample source location (county, state)

Najas minor (LSU00198283f) – Cado, Louisiana Ottelia alismoides (LSU005755544f) – Livingston, Louisiana Ottelia alismoides (LSU00575735f) – Cameron, Louisiana Potamogeton amplifolius (WTU359714a) – Jeferson, Washington Potamogeton crispus (not herbarium sample) – King, Washington Potamogeton pusillus (not herbarium sample) – King, Washington Potmaogeton robbinsi (not herbarium sample) – Thurston, Washington Potomogeton richarsonii (not herbarium sample) – Chelan, Washington Stuckenia pectinata (WTU410495a) – San Juan, Washington Utricularia vulgaris (not herbarium sample) – King, Washington Vallisneria americana (WTU361075a) – Mason, Washington Vallisneria americana (WTU412779a) – Josephine, Oregon

A dashed line (–) indicates a product was not amplifed. GB indicates GenBank accession number for E. densa specimens that were sequenced for this project a Burke Museum Herbarium at the University of Washington b Stanley L. Welsh Herbarium at Brigham Young University c Harold M. Tucker Herbarium at the College of Idaho d Carnegie Museum of Natural History Herbarium e Sewanee Herbarium at the University of the South f Shirley C. Tucker Herbarium at Louisiana State University g Bell Museum Herbarium at the University of Minnesota using the ViiA 7 real-time PCR System (Applied Biosys- − 122.270727) and one sample where E. densa has not tems) and data were analyzed using ViiA 7 RUO 1.2.4 soft- been reported (Lake Meridian, King County, WA; latitude: ware. Assays were conducted in 14 µl volumes containing 47.362252; longitude: − 122.152337). Three 1-l water sam- 1 × Gene Expression Master Mix (Life Sciences), 0.7 µM ples were collected at four sampling locations from the sur- forward primer and 0.7 µM reverse primer, 0.2 µM probe, face of each lake in August 2018 as part of a larger project and 6 µl of DNA extract. Cycling conditions consisted of 10 assessing eDNA detection for invasive macrophytes (Kuehne min initial heat activation at 95 °C, followed by 40 cycles et al. in press). Water samples were fltered through 1.5 µm of denaturing at 95 °C for 15 s and annealing/extension at glass fber flters (GE Health Care) and flters preserved in 60 °C for 1 min. A negative template control (sterile water 95% ethanol. Two 1-L deionized water samples were fltered in place of DNA) was included on each qPCR plate. In vitro on each sampling day and used as negative controls. DNA test results showed that only E. densa DNA amplifed with was extracted from flters using the DNeasy PowerPlant the EdITS1 assay (Table 1). A subset (8/17) of E. densa kit (Qiagen) and stored at − 20 °C. A negative extraction amplicons were sequenced and the results confrmed that control was included in the extraction procedure. PCR inhi- primer and probe sequences were conserved across speci- bition testing on all eDNA extracts was performed using mens from broad geographic regions (Supplemental File). the TaqMan Exogenous Internal Positive Control Reagent To determine the sensitivity of EdITS1, we performed (Applied Biosystems). The EdITS1 assay was performed in qPCR on a dilution series of a gBlock (Integrated DNA triplicate on each DNA sample. Technologies) consisting of the E. densa amplicon. The We detected eDNA from E. densa at all four locations gBlock dilution series consisted of 1, 2, 3, 6, 60, 600, 6000 in Lake Fenwick where E. densa was known to be present and 60,000 copies/qPCR, with eight qPCR replicates per (Table 2). E. densa eDNA was not detected at Lake Merid- concentration. Using the methods of Armbuster and Pry ian where E. densa has not been observed. All lab and feld (2008), the limit of detection was estimated at 2.29 copy/ negative controls were negative for E. densa. We confrmed qPCR and the limit of quantifcation was estimated at 4.68 that the qPCR-detected DNA was from the target species copy/qPCR. The PCR efciency was 96.25% and R2 = 0.99. by sequencing a subset of amplicons (Supplemental File). We collected water samples from two lakes to feld vali- In conclusion, we developed a qPCR assay for detection date EdITS1: one sample where E. densa was present (Lake of E. densa. We confrmed assay specifcity, established Fenwick, King County, WA; latitude: 47.365161; longitude: the limits of detection and quantifcation and validated the

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Table 2 The number of qPCR technical replicates (out of 3 technical replicates performed on each feld water sample) that were positive for E. densa eDNA, mean DNA copy number/qPCR, and standard error (SE) at each of the four locations sampled in Lake Fenwick and Lake Meridian

Lake Location Water sample 1 Water sample 2 Water sample 3 Location total No. positive Mean copy no., No. positive Mean copy no., No. positive Mean copy No., No. positive SE SE SE

Lake Fenwick 1 3/3 (412.5, 159.3) 3/3 (932.2, 40.4)a 3/3 (3.4, 1.8)a 9/9 2 0/3 – 3/3 (50.2, 10.8)a 3/3 (13.3, 3.8) 6/9 3 3/3 (122.1, 31.7) 3/3 (54.6, 14.9) 0/3 - 6/9 4 3/3 (5.3, 2.1) 3/3 (100.5, 20.8) 3/3 (28.0, 5.0) 9/9 Lake Meridian 1 0/0 – 0/0 – 0/0 - 0/9 2 0/0 – 0/0 – 0/0 - 0/9 3 0/0 – 0/0 – 0/0 - 0/9 4 0/0 – 0/0 – 0/0 - 0/9 a Indicates E. densa positive detections that were confrmed by sequencing efcacy of EdITS1 for detecting E. densa in environmental Hussner A, Stiers I, Verhofstad MJJM, Bakker ES, Grutters BMC, samples. The assay has application for early detection and Haury J, van Valkenburg JLCH, Brundu J, Newman J, Clayton E. densa JS, Anderson LJW, Hofstra D (2017) Management and control monitoring of , thus providing resource managers methods of invasive alien freshwater aquatic plants: a review. the opportunity to identify and rapidly respond to new infes- Aquat Bot 136:112–137 tations that could otherwise negatively impact ecosystems Jerde CL, Mahon AR, Chadderton WL, Lodge DM (2011) “Sight- and economies. unseen” detection of rare aquatic species using environmental DNA. Conserv Lett 4:150–157 Kuehne LM, Ostberg CO, Chase DM, Duda JJ, Olden JD (In Press) Use of environmental DNA to detect the invasive aquatic plants Acknowledgements Plant specimens were provided by the Burke Myriophyllum spicatum and Egeria densa in lakes. Freshwater Sci Museum Herbarium at the University of Washington, Stanley L. Welsh Kumar S, Stecher G, Tamura K (2016) MEGA7: molecular evolution- Herbarium at Brigham Young University, Harold M. Tucker Herbarium ary genetics analysis version 7.0 for bigger datasets. Mol Biol at the College of Idaho, Carnegie Museum of Natural History Her- Evol 33:1870–1874 barium, Sewanee Herbarium at the University of the South, Shirley C. Larson ER, Graham BM, Achury R, Coon JJ, Daniels MK, Gambrell Tucker Herbarium at Louisiana State University, Bell Museum Her- DK, Jonasen KL, King GD, LaRacuente N, Perrin-Stowe TIN, barium at the University of Minnesota, and Jen Parsons (Washington Reed EM, Rice CJ, Ruzi SA, Thairu MW, Wilson JC, Suarez State Department of Ecology). Funding was provided by the Washing- AV (2020) From eDNA to citizen science: emerging tools for the ton State Department of Ecology (Aquatic Weed Management Program early detection of invasive species. Front Ecol Environ. https:// Agreement No. WQAIP-2018-UWFECL-00009). Any use of trade, doi.org/10.1002/fee.2162 frm, or product names is for descriptive purposes only and does not Lund JR, Hanak E, Fleenor W, Howitt R, Mount J, Moyle P (2007) imply endorsement by the U.S. Government. Envisioning futures for the Sacramento-San Joaquin Delta. Public Policy Institute of California, San Francisco Roberts DE, Church AG, Cummins SP (1999) Invasion of Egeria into References the Hawkesburg, Nepean River, Australia. J Aquat Plant Manage 37:31–34 Santos MJ, Anderson LW, Ustin SL (2011) Efects of invasive species Armbuster DA, Pry T (2008) Limit of blank, limit of detection and on plant communities: an example using submersed aquatic plants limit of quantitation. Clin Biochem Rev 29:49–52 at the regional scale. Biol Invas 13:443–457 Biggs J, Ewald N, Valentini A, Gaboriaud C, Dejean T, Grifths RA, Sigsgaard EE, Carl H, Moller PR, Thomsen PF (2015) Monitoring Foster J, Wilkinson JW, Arnell A, Brotherton P, Williams P, Dunn the near-extinct European weather loach in Denmark based on F (2015) Using eDNA to develop a national citizen science-based environmental DNA from water samples. Biol Conserv 183:46–52 monitoring program for the great crested newt (Triturus cristatus). Taberlet P, Gielly L, Pautou G, Bouvet J (1991) Universal primers for Biol Conserv 183:19–28 amplifcation of three non-coding regions of chloroplast DNA. de Winton MD, Clayton JS (1996) The impact invasive submerged Plant Mol Biol 17:1105–1109 weed species on seed banks in lake sediments. Aquat Bot Vander Zanden MJ, Olden JD (2008) A management framework for 53:31–45 preventing the secondary spread of aquatic invasive species. Can Evans NT, Li Y, Renshaw MA, Olds BP, Deiner K, Tuner CR, Jerde J Fish Aquat Sci 65:1512–1522 CL, Lodge DM, Lamberti GA, Pfrender ME (2017) Fish com- Yarrow M, Marin VH, Finlayson M, Tironi A, Delgado LE, Fisher F munity assessment with eDNA metabarcoding: efects of sam- (2009) The ecology of Egeria densa Planchon (Lilopsida: Alis- pling design and bioinformatic fltering. Can J Fish Aquat Sci matales): awetland ecosystem engineer? Rev Chilena de Hist 74:1362–1374 Natural 82(2):299–313 Ficetola GF, Miaud C, Pompanon F, Taberlet P (2008) Species detection using environmental DNA from water samples. Biol Lett Publisher’s Note Springer Nature remains neutral with regard to 4:423–425 jurisdictional claims in published maps and institutional afliations.

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