Advertisement

Limnology

, Volume 17, Issue 1, pp 23–32 | Cite as

Techniques for the practical collection of environmental DNA: filter selection, preservation, and extraction

  • Toshifumi MinamotoEmail author
  • Takafumi Naka
  • Kazuhiko Moji
  • Atsushi Maruyama
Research paper

Abstract

Environmental DNA (eDNA) analysis has recently been used for detection of aquatic macro-organisms; however, the analytical procedures used in previous studies have not been optimized for practical use. Here, we compared several methods for DNA enrichment and extraction from water samples to establish widely applicable techniques for eDNA analysis using common carp as the model species. First, several types of filters were compared to identify the optimal filter type. Second, the eDNA yield was compared after a variety of extraction and isolation steps, including a combination of phenol extraction, ethanol precipitation (phenol treatment), and ultrafiltration. Third, DNA fixation with ethanol was tested for the preservation of eDNA on filters. Ethanol precipitation yielded the largest number of eDNA copies, followed by filtering using a 0.2-μm polycarbonate filter and a 0.7-μm glass fiber filter. Phenol treatment resulted in collection of a higher number of eDNA copies than that collected using ultrafiltration. DNA fixation with 15 ml ethanol enabled eDNA preservation on the filters at ambient temperatures for at least 6 days. Finally, combinations of different filter types and DNA enrichment procedures were compared using field water samples. From these results, we propose that the appropriate selection method for eDNA analysis should be chosen based on context. For example, when a high concentration of the target DNA is expected, such as in an aquarium experiment, ethanol precipitation is advantageous. However, when the target DNA is rare, which is the case in most field studies, filtration followed by freezing or DNA fixation by ethanol and phenol treatment are recommended. The filter type should be decided prior to the survey based on the characteristics of the water of interest. Thus, eDNA analysis could be applied to various situations using adaptive combinations of these techniques.

Keywords

DNA extraction Environmental DNA Fish Quantitative PCR 

Notes

Acknowledgments

The authors sincerely thank Dr. M. Kondoh, Dr. H. Yamanaka, and the students in the Maruyama laboratory at Ryukoku University for discussions and comments on this study. This study was conducted as a part of the MEXT GRENEei Ecohealth Project (Project Leader: Prof. Chiho Watanabe, the University of Tokyo, Japan). This study was partly funded by the “Environmental Change and Infection Diseases in Tropical Asia” project (R-04) of the Research Institute for Humanity and Nature led by KM and by grants from JSPS KAKENHI (grant numbers 24657020 to TM and AM, and 26440238 to TM).

References

  1. Brook BW, O’Grady JJ, Chapman AP, Burgman MA, Akçakaya HR, Frankham R (2000) Predictive accuracy of population viability analysis in conservation biology. Nature 404(6776):385–387. doi: 10.1038/35006050 CrossRefPubMedGoogle Scholar
  2. Deiner K, Altermatt F (2014) Transport distance of invertebrate environmental DNA in a natural river. PLoS One 9(2):e88786. doi: 10.1371/journal.pone.0088786 PubMedCentralCrossRefPubMedGoogle Scholar
  3. Deiner K, Walser J-C, Mächler E, Altermatt F (2015) Choice of capture and extraction methods affect detection of freshwater biodiversity from environmental DNA. Biol Conserv 183:53–63. doi: 10.1016/j.biocon.2014.11.018 CrossRefGoogle Scholar
  4. Dejean T, Valentini A, Duparc A, Pellier-Cuit S, Pompanon F, Taberlet P, Miaud C (2011) Persistence of environmental DNA in freshwater ecosystems. PLoS One 6(8):e23398. doi: 10.1371/journal.pone.0023398 PubMedCentralCrossRefPubMedGoogle Scholar
  5. Dejean T, Valentini A, Miquel C, Taberlet P, Bellemain E, Miaud C (2012) Improved detection of an alien invasive species through environmental DNA barcoding: the example of the American bullfrog Lithobates catesbeianus. J Appl Ecol 49(4):953–959. doi: 10.1111/j.1365-2664.2012.02171.x CrossRefGoogle Scholar
  6. Doi H, Takahara T, Minamoto T, Matsuhashi S, Uchii K, Yamanaka H (2015a) Droplet digital polymerase chain reaction (PCR) outperforms real-time PCR in the detection of environmental DNA from an invasive fish species. Environ Sci Technol 49(9):5601–5608. doi: 10.1021/acs.est.5b00253 CrossRefPubMedGoogle Scholar
  7. Doi H, Uchii K, Takahara T, Matsuhashi S, Yamanaka H, Minamoto T (2015b) Use of droplet digital PCR for estimation of fish abundance and biomass in environmental DNA surveys. PLoS One 10(3):e0122763. doi: 10.1371/journal.pone.0122763 PubMedCentralCrossRefPubMedGoogle Scholar
  8. Dunnett CW (1964) New tables for multiple comparisons with a control. Biometrics 20(3):482–491CrossRefGoogle Scholar
  9. Eichmiller JJ, Bajer PG, Sorensen PW (2014) The relationship between the distribution of common carp and their environmental DNA in a small lake. PLoS One 9(11):e112611. doi: 10.1371/journal.pone.0112611 PubMedCentralCrossRefPubMedGoogle Scholar
  10. Ficetola GF, Miaud C, Pompanon F, Taberlet P (2008) Species detection using environmental DNA from water samples. Biol Lett 4(4):423–425. doi: 10.1098/Rsbl.2008.0118 PubMedCentralCrossRefPubMedGoogle Scholar
  11. Foote AD, Thomsen PF, Sveegaard S, Wahlberg M, Kielgast J, Kyhn LA, Salling AB, Galatius A, Orlando L, Gilbert MTP (2012) Investigating the potential use of environmental DNA (eDNA) for genetic monitoring of marine mammals. PLoS One 7(8):e41781. doi: 10.1371/journal.pone.0041781 PubMedCentralCrossRefPubMedGoogle Scholar
  12. Fukumoto S, Ushimaru A, Minamoto T (2015) A basin-scale application of environmental DNA assessment for rare endemic species and closely related exotic species in rivers: a case study of giant salamanders in Japan. J Appl Ecol 52(2):358–365. doi: 10.1111/1365-2664.12392 CrossRefGoogle Scholar
  13. Goldberg CS, Pilliod DS, Arkle RS, Waits LP (2011) Molecular detection of vertebrates in stream water: a demonstration using Rocky Mountain tailed frogs and Idaho giant salamanders. PLoS One 6(7):e22746. doi: 10.1371/journal.pone.0022746 PubMedCentralCrossRefPubMedGoogle Scholar
  14. Hajibabaei M, Spall J, Shokralla S, van Konynenburg S (2012) Assessing biodiversity of a freshwater benthic macroinvertebrate community through non-destructive environmental barcoding of DNA from preservative ethanol. BMC Ecol 12(1):28. doi: 10.1186/1472-6785-12-28 PubMedCentralCrossRefPubMedGoogle Scholar
  15. Honjo MN, Minamoto T, Matsui K, Uchii K, Yamanaka H, Suzuki AA, Kohmatsu Y, Iida T, Kawabata Z (2010) Quantification of cyprinid herpesvirus-3 in environmental water by using an external standard virus. Appl Environ Microb 76:161–168. doi: 10.1128/AEM.02011-09 CrossRefGoogle Scholar
  16. Janosik AM, Johnston CE (2015) Environmental DNA as an effective tool for detection of imperiled fishes. Environ Biol Fishes. doi: 10.1007/s10641-015-0405-5 Google Scholar
  17. Jerde CL, Mahon AR, Chadderton WL, Lodge DM (2011) “Sight-unseen” detection of rare aquatic species using environmental DNA. Conserv Lett 4(2):150–157. doi: 10.1111/J.1755-263x.2010.00158.X CrossRefGoogle Scholar
  18. Krebs C (1978) Ecology: The Experimental analysis of distribution and abundance, 2nd Edn. Harper and Row, New YorkGoogle Scholar
  19. Lowe S, Browne M, Boudjelas S, DePoorter M (2000) 100 of the world’s worst invasive alien species: a selection from the global invasive species database. 1–12Google Scholar
  20. Lydolph MC, Jacobsen J, Arctander P, Gilbert MTP, Gilichinsky DA, Hansen AJ, Willerslev E, Lange L (2005) Beringian paleoecology inferred from permafrost-preserved fungal DNA. Appl Environ Microb 71(2):1012–1017. doi: 10.1128/Aem.71.2.1012-1017.2005 CrossRefGoogle Scholar
  21. Maruyama A, Nakamura K, Ymanaka H, Kondoh M, Minamoto T (2014) The release rate of environmental DNA from juvenile and adult fish. PLoS One 9(12):e114639. doi: 10.1371/journal.pone.0114639 PubMedCentralCrossRefPubMedGoogle Scholar
  22. Minamoto T, Honjo MN, Kawabata Z (2009) Seasonal distribution of cyprinid herpesvirus 3 in Lake Biwa. Appl Environ Microb 75:6900–6904. doi: 10.1128/AEM.01411-09 CrossRefGoogle Scholar
  23. Minamoto T, Yamanaka H, Takahara T, Honjo MN, Kawabata Z (2012) Surveillance of fish species composition using environmental DNA. Limnology 13(2):193–197. doi: 10.1007/S10201-011-0362-4 CrossRefGoogle Scholar
  24. Nathan LM, Simmons M, Wegleitner BJ, Jerde CL, Mahon AR (2014) Quantifying environmental DNA signals for aquatic invasive species across multiple detection platforms. Environ Sci Technol 48(21):12800–12806. doi: 10.1021/es5034052 CrossRefPubMedGoogle Scholar
  25. Olson ZH, Briggler JT, Williams RN (2012) An eDNA approach to detect eastern hellbenders (Cryptobranchus a. alleganiensis) using samples of water. Wildlife Research 39(7):629–636. doi: 10.1071/WR12114 CrossRefGoogle Scholar
  26. Pilliod DS, Goldberg CS, Arkle RS, Waits LP (2013) Estimating occupancy and abundance of stream amphibians using environmental DNA from filtered water samples. Can J Fish Aquat Sci 70(8):1123–1130. doi: 10.1139/cjfas-2013-0047 CrossRefGoogle Scholar
  27. R Development Core Team (2011) R: a language and environment for statistical computing. R Foundation for Statistical Computing, ViennaGoogle Scholar
  28. Renshaw MA, Olds BP, Jerde CL, McVeigh MM, Lodge DM (2015) The room temperature preservation of filtered environmental DNA samples and assimilation into a phenol-chloroform-isoamyl alcohol DNA extraction. Mol Ecol Resour 15(1):168–176. doi: 10.1111/1755-0998.12281 PubMedCentralCrossRefPubMedGoogle Scholar
  29. Sogin ML, Morrison HG, Huber JA, Welch DM, Huse SM, Neal PR, Arrieta JM, Herndl GJ (2006) Microbial diversity in the deep sea and the underexplored “rare biosphere”. Proc Natl Acad Sci USA 103(32):12115–12120. doi: 10.1073/pnas.0605127103 PubMedCentralCrossRefPubMedGoogle Scholar
  30. Taberlet P, Coissac E, Hajibabaei M, Rieseberg LH (2012) Environmental DNA. Mol Ecol 21(8):1789–1793. doi: 10.1111/J.1365-294x.2012.05542.X CrossRefPubMedGoogle Scholar
  31. Takahara T, Minamoto T, Doi H (2013) Using environmental DNA to estimate the distribution of an invasive fish species in ponds. PLoS One 8(2):e56584. doi: 10.1371/journal.pone.0056584 PubMedCentralCrossRefPubMedGoogle Scholar
  32. Takahara T, Minamoto T, Yamanaka H, Doi H, Kawabata Z (2012) Estimation of fish biomass using environmental DNA. PLoS One 7(4):e35868. doi: 10.1371/journal.pone.0035868 PubMedCentralCrossRefPubMedGoogle Scholar
  33. Takahara T, Minamoto T, Doi H (2015) Effects of sample processing on the detection rate of environmental DNA from the Common Carp (Cyprinus carpio). Biol Conserv 183:64–69. doi: 10.1016/j.biocon.2014.11.014 CrossRefGoogle Scholar
  34. Thomsen PF, Kielgast J, Iversen LL, Moller PR, Rasmussen M, Willerslev E (2012a) Detection of a diverse marine fish fauna using environmental DNA from seawater samples. PLoS One 7(8):e41732. doi: 10.1371/journal.pone.0041732 PubMedCentralCrossRefPubMedGoogle Scholar
  35. Thomsen PF, Kielgast JOS, Iversen LL, Wiuf C, Rasmussen M, Gilbert MTP, Orlando L, Willerslev E (2012b) Monitoring endangered freshwater biodiversity using environmental DNA. Mol Ecol 21(11):2565–2573CrossRefPubMedGoogle Scholar
  36. Treguier A, Paillisson J-M, Dejean T, Valentini A, Schlaepfer MA, Roussel J-M (2014) Environmental DNA surveillance for invertebrate species: advantages and technical limitations to detect invasive crayfish Procambarus clarkii in freshwater ponds. J Appl Ecol 51:871–879. doi: 10.1111/1365-2664.12262 CrossRefGoogle Scholar
  37. Turner CR, Barnes MA, Xu CCY, Jones SE, Jerde CL, Lodge DM (2014) Particle size distribution and optimal capture of aqueous macrobial eDNA. Method Ecol Evol 5:676–684. doi: 10.1111/2041-210X.12206 CrossRefGoogle Scholar
  38. Uchii K, Telschow A, Minamoto T, Yamanaka H, Honjo MN, Matsui K, Kawabata Z (2011) Transmission dynamics of an emerging infectious disease in wildlife through host reproductive cycles. The ISME Journal 5:244. doi: 10.1038/ismej.2010.123 PubMedCentralCrossRefPubMedGoogle Scholar

Copyright information

© The Japanese Society of Limnology 2015

Authors and Affiliations

  • Toshifumi Minamoto
    • 1
    • 2
    Email author
  • Takafumi Naka
    • 3
  • Kazuhiko Moji
    • 2
    • 4
  • Atsushi Maruyama
    • 3
  1. 1.Graduate School of Human Development and EnvironmentKobe UniversityKobeJapan
  2. 2.Research Institute for Humanity and NatureKyotoJapan
  3. 3.Faculty of Science and TechnologyRyukoku UniversityOtsuJapan
  4. 4.Graduate School of Tropical Medicine and Global HealthNagasaki UniversityNagasakiJapan

Personalised recommendations