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Environmental DNA from Marine Waters and Substrates: Protocols for Sampling and eDNA Extraction

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Marine Genomics

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2498))

Abstract

Environmental DNA (eDNA) analysis has emerged in recent years as a powerful tool for the detection, monitoring, and characterization of aquatic metazoan communities, including vulnerable species. The rapid rate of adopting the eDNA approach across diverse habitats and taxonomic groups attests to its value for a wide array of investigative goals, from understanding natural or changing biodiversity to informing on conservation efforts at local and global scales. Regardless of research objectives, eDNA workflows commonly include the following essential steps: environmental sample acquisition, processing and preservation of samples, and eDNA extraction, followed by eDNA sequencing library preparation, high-capacity sequencing and sequence data analysis, or other methods of genetic detection. In this chapter, we supply instructional details for the early steps in the workflow to facilitate researchers considering adopting eDNA analysis to address questions in marine environments. Specifically, we detail sampling, preservation, extraction, and quantification protocols for eDNA originating from marine water, shallow substrates, and deeper sediments. eDNA is prone to degradation and loss, and to contamination through improper handling; these factors crucially influence the outcome and validity of an eDNA study. Thus, we also provide guidance on avoiding these pitfalls. Following extraction, purified eDNA is often sequenced on massively parallel sequencing platforms for comprehensive faunal diversity assessment using a metabarcoding or metagenomic approach, or for the detection and quantification of specific taxa by qPCR methods. These components of the workflow are project-specific and thus not included in this chapter. Instead, we briefly touch on the preparation of eDNA libraries and discuss comparisons between sequencing approaches to aid considerations in project design.

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References

  1. Taberlet P, Coissac E, Hajibabaei M et al (2012) Environmental DNA. Mol Ecol 21:1789–1793

    Article  CAS  PubMed  Google Scholar 

  2. Rees HC, Maddison BC, Middleditch DJ et al (2014) The detection of aquatic animal species using environmental DNA – a review of eDNA as a survey tool in ecology. J Appl Ecol 51:1450–1459

    Article  CAS  Google Scholar 

  3. Sassoubre LM, Yamahara KM, Gardner LD et al (2016) Quantification of environmental DNA (eDNA) shedding and decay rates for three marine fish. Environ Sci Technol 50:10456–10464

    Article  CAS  PubMed  Google Scholar 

  4. Seymour M (2019) Rapid progression and future of environmental DNA research. Commun Biol 80:1–3

    Google Scholar 

  5. Thomsen PF, Willerslev E (2015) Environmental DNA – an emerging tool in conservation for monitoring past and present biodiversity. Biol Conserv 183:4–8

    Article  Google Scholar 

  6. Foote AD, Thomsen PF, Sveegaard S (2012) Investigating the potential use of environmental DNA (eDNA) for genetic monitoring of marine mammals. PLoS One 7:e41781

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Stoeckle MY, Soboleva L, Charlop-Powers Z (2017) Aquatic environmental DNA detects seasonal fish abundance and habitat preference in an urban estuary. PLoS One 12:e0175186

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  8. Knudsen SW, Ebert RB, Hesselsøe M et al (2019) Species-specific detection and quantification of environmental DNA from marine fishes in the Baltic Sea. J Exp Mar Biol Ecol 510:31–45

    Article  CAS  Google Scholar 

  9. Kelly RP, Port JA, Yamahara KM et al (2014) Using environmental DNA to census marine fishes in a large mesocosm. PLoS One 9:e86175

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  10. Sawaya NA, Djuhuus A, Closek CJ et al (2018) Assessing eukaryotic biodiversity in the Florida Keys National Marine Sanctuary through environmental DNA metabarcoding. Ecol Evol 9:1029–1040

    Article  Google Scholar 

  11. Harper KJ, Goodwin KD, Harper LR et al (2020) Finding crush: environmental DNA analysis as a tool for tracking the Green Sea turtle Chelonia mydas in a marine estuary. Front Mar Sci 6:810

    Article  Google Scholar 

  12. Laroche O, Kersten O, Smith CR et al (2020) Environmental DNA surveys detect distinct metazoan communities across abyssal plains and seamounts in the western Clarion Clipperton Zone. Mol Ecol 29:4588–4604

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Djurhuus A, Pitz K, Sawaya NA et al (2018) Evaluation of marine zooplankton community structure through environmental DNA metabarcoding. Limnol Oceanogr Methods 16:209–221

    Article  PubMed  PubMed Central  Google Scholar 

  14. Berry TE, Saunders BJ, Coghlan ML et al (2019) Marine environmental DNA biomonitoring reveals seasonal patterns in biodiversity and identifies ecosystem responses to anomalous climatic events. PLoS Genet 15:e1007943

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Djurhuus A, Closek CJ, Kelly RP et al (2020) Environmental DNA reveals seasonal shifts and potential interactions in a marine community. Nat Commun 11:1–9

    Article  CAS  Google Scholar 

  16. Weltz K, Lyle JM, Ovenden J, Morgan JAT et al (2017) Application of environmental DNA to detect an endangered marine skate species in the wild. PLoS One 12:e0178124

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  17. Kim P, Kim D, Yoon TJ et al (2018) Early detection of marine invasive species, Bugula neritina (Bryozoa: Cheilostomatida), using species-specific primers and environmental DNA analysis in Korea. Mar Environ Res 139:1–10

    Article  CAS  PubMed  Google Scholar 

  18. von Ammon U, Wood SA, Laroche O et al (2019) Linking environmental DNA and RNA for improved detection of the marine invasive fanworm Sabella spallanzanii. Front Mar Sci 6:621

    Article  Google Scholar 

  19. Sigsgaard EE, Torquato F, Frøslev TG et al (2019) Using vertebrate environmental DNA from seawater in biomonitoring of marine habitats. Conserv Biol 34:697–710

    Article  PubMed  PubMed Central  Google Scholar 

  20. Sigsgaard EE, Nielsen IB, Bach SS et al (2016) Population characteristics of a large whale shark aggregation inferred from seawater environmental DNA. Nat Ecol Evol 1:0004

    Article  Google Scholar 

  21. Parsons KM, Everett M, Dahlheim M et al (2018) Water, water everywhere: environmental DNA can unlock population structure in elusive marine species. R Soc Open Sci 5:180537

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  22. Bohmann K, Evans A, Gilbert MTP et al (2014) Environmental DNA for wildlife biology and biodiversity monitoring. Trends Ecol Evol 29:358–367

    Article  PubMed  Google Scholar 

  23. Goldberg CS, Turner CR, Deiner K et al (2016) Critical considerations for the application of environmental DNA methods to detect aquatic species. Methods Ecol Evol 7:1299–1307

    Article  Google Scholar 

  24. Tsuji S, Takahara T, Hideyuki D et al (2019) The detection of aquatic macroorganisms using environmental DNA analysis—a review of methods for collection, extraction, and detection. Environ DNA 1:99–108

    Article  Google Scholar 

  25. Jeunen G-J, Knapp M, Spencer HG et al (2018) Species-level biodiversity assessment using marine environmental DNA metabarcoding requires protocol optimization and standardization. Ecol Evol 9:1323–1335

    Article  Google Scholar 

  26. Lear G, Dickie I, Banks J et al (2018) Methods for the extraction, storage, amplification and sequencing of DNA from environmental samples. N Z J Ecol 4:1–51

    Google Scholar 

  27. Kusanke LM, Panteleit J, Stoll S et al (2020) Detection of the endangered European weather loach (Misgurnus fossilis) via water and sediment samples: testing multiple eDNA workflows. Ecol Evol 10:8331–8344

    Article  PubMed  PubMed Central  Google Scholar 

  28. McGee KM, Robinson CV, Hajibabaei M (2019) Gaps in DNA-based biomonitoring across the globe. Front Ecol Evol 7:337

    Article  Google Scholar 

  29. Andruszkiewicz EA, Starks HA, Chavez FP et al (2017) Biomonitoring of marine vertebrates in Monterey Bay using eDNA metabarcoding. PLoS One 12:e0176343

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  30. Bakker J, Wangensteen OS, Chapman DD et al (2017) Environmental DNA reveals tropical shark diversity in contrasting levels of anthropogenic impact. Sci Rep 7:16886

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  31. Cowart DA, Matabos M, Brandt MI et al (2020) Exploring environmental DNA (eDNA) to assess biodiversity of hard substratum faunal communities on the lucky strike vent field (mid-Atlantic ridge) and investigate recolonization dynamics after an induced disturbance. Front Mar Sci 6:783

    Article  Google Scholar 

  32. Brandt MI, Pradillon F, Trouche B et al (2021) Evaluating sediment and water sampling methods for the estimation of deep-sea biodiversity using environmental DNA. Sci Rep 11:7856

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Herbold CW, Pelikan C, Kuzyk O et al (2015) A flexible and economical barcoding approach for highly multiplexed amplicon sequencing of diverse target genes. Front Microbiol 6:731

    Article  PubMed  PubMed Central  Google Scholar 

  34. Hebert PDN, Braukmann TWA, Prosser SWJ et al (2018) A sequel to sanger: amplicon sequencing that scales. BMC Genomics 19:219

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  35. Einsen JA (2007) Environmental shotgun sequencing: its potential and challenges for studying the hidden world of microbes. PLoS Biol 5:e82

    Article  CAS  Google Scholar 

  36. Stat M, Huggett MJ, Bernasconi R et al (2017) Ecosystem biomonitoring with eDNA: metabarcoding across the tree of life in a tropical marine environment. Sci Rep 7:12240

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  37. Coissac E, Riaz T, Puillandre N (2012) Bioinformatic challenges for DNA metabarcoding of plants and animals. Mol Ecol 21:1834–1847

    Article  CAS  PubMed  Google Scholar 

  38. Hajibabaei M, Porter TM, Wright M et al (2019) COI metabarcoding primer choice affects richness and recovery of indicator taxa in freshwater systems. PLoS One 14:e0220953

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Singer GAC, Shekarriz S, McCarthy A et al (2020) The utility of a metagenomics approach for marine biomonitoring. BioRxiv

    Google Scholar 

  40. Miya M, Sato Y, Fukunaga T et al (2015) MiFish, a set of universal PCR primers for metabarcoding environmental DNA from fishes: detection of more than 230 subtropical marine species. R Soc Open Sci 2:150088

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Valentini A, Taberlet P, Miaud C et al (2016) Next-generation monitoring of aquatic biodiversity using environmental DNA metabarcoding. Mol Ecol 25:929–942

    Article  CAS  PubMed  Google Scholar 

  42. DiBattista JD, Coker DJ, Sinclair-Taylor TH (2017) Assessing the utility of eDNA as a tool to survey reef-fish communities in the Red Sea. Coral Reefs 36:1245–1252

    Article  Google Scholar 

  43. Cowart DA, Murphy KR, Cheng CH (2018) Metagenomic sequencing of environmental DNA reveals marine faunal assemblages from the West Antarctic peninsula. Mar Genomics 37:148–160

    Article  PubMed  Google Scholar 

  44. Zilius M, Samuiloviene A, Stanislauskienė et al (2021) Depicting temporal, functional, and phylogenetic patterns in estuarine diazotrophic communities from environmental DNA and RNA. Microb Ecol 81:36–51

    Article  CAS  PubMed  Google Scholar 

  45. Lafferty KD, Garcia-Vedrenne AE, McLaughlin JP et al (2020) At Palmyra atoll, the fish-community environmental DNA signal changes across habitats but not with tides. J Fish Biol 98:415–425

    Article  PubMed  CAS  Google Scholar 

  46. Jamy M, Foster R, Barbera P et al (2019) Long-read metabarcoding of the eukaryotic rDNA operon to phylogenetically and taxonomically resolve environmental diversity. Mol Ecol Resour 20:429–443

    Article  PubMed  CAS  Google Scholar 

  47. van der Loos LM, Nijland R (2020) Biases in bulk: DNA metabarcoding of marine communities and the methodology involved. Mol Ecol 00:1–19

    Google Scholar 

  48. Pedersen MW, Overballe-Petersen S, Ermini L et al (2015) Ancient and modern environmental DNA. Philos Trans R Soc Lond B Biol Sci 370:20130383

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  49. Wilcox TM, McKelvey KS, Young MK et al (2013) Robust detection of rare species using environmental DNA: the importance of primer specificity. PLoS One 8:e59520

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Stewart K, Ma H, Zheng J, Zhao J (2017) Using environmental DNA to assess population-wide spatiotemporal reserve use. Conserv Biol 31:1173–1182

    Article  PubMed  Google Scholar 

  51. Baker CS, Steel D, Nieukirk S et al (2018) Environmental DNA (eDNA) from the wake of the whales: droplet digital PCR for detection and species identification. Front Mar Sci 5:133

    Article  Google Scholar 

  52. Ficetola GF, Miaud C, Pompanon F et al (2008) Species detection using environmental DNA from water samples. Biol Lett 4:423–425

    Article  PubMed  PubMed Central  Google Scholar 

  53. Jerde CL, Mahon AR, Chadderton WL et al (2010) “Sight-unseen” detection of rare aquatic species using environmental DNA. Conserv Lett 4:150–157

    Article  Google Scholar 

  54. Ma H, Stewart K, Lougheed S (2016) Characterization, optimization, and validation of environmental DNA (eDNA) markers to detect an endangered aquatic mammal. Conserv Genet Resour 8:561–568

    Article  Google Scholar 

  55. LeBlanc F, Belliveau V, Watson E et al (2020) Environmental DNA (eDNA) detection of marine aquatic invasive species (AIS) in Eastern Canada using a targeted species-specific qPCR approach. Manag Biol Invasion 11:201–217

    Article  Google Scholar 

  56. Simpson TJS, Dias PJ, Snow M et al (2017) Real-time PCR detection of Didemnum perlucidum (Monniot, 1983) and Didemnum vexillum (Kott, 2002) in an applied routine marine biosecurity context. Mol Ecol Resour 17:433–453

    Article  CAS  Google Scholar 

  57. Edgar RC (2010) Search and clustering orders of magnitude faster than BLAST. Bioinformatics 26:2460–2461

    Article  CAS  PubMed  Google Scholar 

  58. Caporaso JG, Kuczynski J, Stombaugh J et al (2010) QIIME allows analysis of high- throughput community sequencing data. Nat Methods 7:335–336

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Rognes T, Flouri T, Nichols B et al (2016) VSEARCH: a versatile open source tool for metagenomics. PeerJ 4:e2584

    Article  PubMed  PubMed Central  Google Scholar 

  60. Callahan BJ, McMurdie PJ, Rosen MJ et al (2016) DADA2: high-resolution sample inference from Illumina amplicon data. Nat Methods 13:581–583

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  61. Escudié F, Auer L, Bernard M et al (2018) FROGS: find, rapidly, OTUs with galaxy solution. Bioinformatics 34:1287–1294

    Article  PubMed  CAS  Google Scholar 

  62. R Core Team (2021) R: a language and environment for statistical computing. R Foundation for Statistical Computing, Vienna. https://www.R-project.org/

    Google Scholar 

  63. Turner CR, Uy KL, Everhart RC (2015) Fish environmental DNA is more concentrated in aquatic sediments than surface water. Biol Conserv 183:93–102

    Article  Google Scholar 

  64. Buxton AS, Groombridge JJ, Griffiths RA (2018) Seasonal variation in environmental DNA detection in sediment and water samples. PLoS One 13:e0191737

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  65. Holman LE, de Bruyn M, Créer S et al (2019) Detection of introduced and resident marine species using environmental DNA metabarcoding of sediment and water. Sci Rep 9:11559

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  66. Brandt MI, Trouche B, Henry N et al (2020) An assessment of environmental metabarcoding protocols aiming at favoring contemporary biodiversity in inventories of deep-sea communities. Front Mar Sci 7:234

    Article  Google Scholar 

  67. Deiner K, Lopez J, Bourne S et al (2018) Optimising the detection of marine taxonomic richness using environmental DNA metabarcoding: the effects of filter material, pore size and extraction method. Metabarcoding Metagenom 2:1–15

    Article  Google Scholar 

  68. Robson HLA, Noble TH, Saunders RJ et al (2016) Fine-tuning for the tropics: application of eDNA technology for invasive fish detection in tropical freshwater ecosystems. Mol Ecol Resour 16:922–932

    Article  CAS  PubMed  Google Scholar 

  69. Turner CR, Barnes MA, Charles CYX et al (2014) Particle size distribution and optimal capture of aqueous macrobial eDNA. Methods Ecol Evol 5:676–684

    Article  Google Scholar 

  70. Eichmiller JJ, Miller LM, Sorensen PW (2016) Optimizing techniques to capture and extract environmental DNA for detection and quantification of fish. Mol Ecol Resour 16:56–68

    Article  CAS  PubMed  Google Scholar 

  71. Renshaw MA, Olds BP, Jerde CL et al (2015) The room temperature preservation of filtered environmental DNA samples and assimilation into a phenol-chloroform- isoamyl alcohol DNA extraction. Mol Ecol Resour 15:168–176

    Article  CAS  PubMed  Google Scholar 

  72. Wegleitner B, Jerde C, Tucker A et al (2015) Long duration, room temperature preservation of filtered eDNA samples. Conserv Genet Resour 7:789–791

    Article  Google Scholar 

  73. Thomsen PF, Kielgast J, Iversen LL et al (2014) Detection of a diverse marine fish fauna using environmental DNA from seawater samples. PLoS One 7(8):e41732

    Article  CAS  Google Scholar 

  74. Hinlo R, Gleeson D, Lintermans M et al (2017) Methods to maximise recovery of environmental DNA from water samples. PLoS One 12:e0179251

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  75. Guardiola M, Wangensteen OS, Taberlet P et al (2016) Spatio-temporal monitoring of deep-sea communities using metabarcoding of sediment DNA and RNA. PeerJ 4:e2807

    Article  PubMed  PubMed Central  Google Scholar 

  76. Huber DH, Ugwuanyi IR, Malkaram SA et al (2018) Metagenome sequences of sediment from a recovering industrialized Appalachian River in West Virginia. Genome Announc 6:e00350–e00318

    PubMed  PubMed Central  Google Scholar 

  77. Sellers GS, Di Muri C, Gómez A et al (2018) Mu-DNA: a modular universal DNA extraction method adaptable for a wide range of sample types. Metabarcoding Metagenom 2:1–11

    Article  Google Scholar 

  78. Giguet-Covex C, Ficetola GF, Walsh K et al (2019) New insights on lake sediment DNA from the catchment: importance of taphonomic and analytical issues on the record quality. Sci Rep 9:14676

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  79. Wei N, Nakajima F, Tobino T (2018) Effects of treated sample weight and DNA marker length on sediment eDNA based detection of a benthic invertebrate. Ecol Indic 93:267–273

    Article  CAS  Google Scholar 

  80. Spens J, Evans AR, Halfmaerten D et al (2017) Comparison of capture and storage methods for aqueous macrobial eDNA using an optimized extraction protocol: advantage of enclosed filter. Methods Ecol Evol 8:635–645

    Google Scholar 

  81. Majaneva M, Diserud OH, Eagle SHC et al (2018) Environmental DNA filtration techniques affect recovered biodiversity. Sci Rep 8:4682

    Google Scholar 

  82. Wacker S, Fossøy F, Larsen BM (2019) Downstream transport and seasonal variation in freshwater pearl mussel (Margaritifera margaritifera) eDNA concentration. Environ DNA 1:64–73

    Google Scholar 

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Acknowledgments

We thank Cinzia Verde for inviting this submission. We are grateful to colleagues at Ifremer for their advice with sampling substrates, as well as to the Laboratories of Analytical Biology at the National Museum of Natural History, Smithsonian Institution, for their support in marine water protocol development. We thank Elliot DeVries for his artwork used in the figure. CHCC acknowledges U.S. NSF Polar Programs grant support (ANT1142158, OPP1645087) that enabled sampling of Southern Ocean waters and development of some of the described protocols.

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Authors and Affiliations

  1. Company for Open Ocean Observations and Logging (COOOL), Saint Leu, La Réunion, France

    Dominique A. Cowart

  2. Laboratories of Analytical Biology, National Museum of Natural History, Smithsonian Institution, Washington, DC, USA

    Katherine R. Murphy

  3. Department of Evolution, Ecology, and Behavior, University of Illinois at Urbana – Champaign, Urbana, IL, USA

    C.-H. Christina Cheng

Authors
  1. Dominique A. Cowart
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  2. Katherine R. Murphy
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  3. C.-H. Christina Cheng
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Corresponding author

Correspondence to C.-H. Christina Cheng .

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Editors and Affiliations

  1. Institute of Biosciences and BioResources (IBBR), National Research Council, Naples, Italy

    Cinzia Verde

  2. Institute of Biosciences and BioResources (IBBR), National Research Council, Naples, Italy

    Daniela Giordano

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Cowart, D.A., Murphy, K.R., Cheng, CH.C. (2022). Environmental DNA from Marine Waters and Substrates: Protocols for Sampling and eDNA Extraction. In: Verde, C., Giordano, D. (eds) Marine Genomics. Methods in Molecular Biology, vol 2498. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2313-8_11

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  • DOI: https://doi.org/10.1007/978-1-0716-2313-8_11

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