Korean Journal of Ecology and Environment (생태와환경)

The Korean Society of Limnology (한국수생태학회)
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Efficiency of Density Gradient Centrifugation Method (Ludox method) Based on eDNA for the Analysis of Harmful Algal Bloom Potential

유해남조류 발생 잠재성 분석을 위한 eDNA 기반의 퇴적물 전처리 방법: 밀도 구배 원심분리법(Ludox method)

  • Kyeong-Eun Yoo (Department of Environmental Health Science, Sanghuh Life Science College, Konkuk University) ;
  • Hye-In Ho (Department of Environmental Health Science, Sanghuh Life Science College, Konkuk University) ;
  • Hyunjin Kim (Pyunghwa Engineering Consultants) ;
  • Keonhee Kim (Human & Eco Care Center, Konkuk University) ;
  • Soon-Jin Hwang (Department of Environmental Health Science, Sanghuh Life Science College, Konkuk University)
  • 유경은 (건국대학교 환경보건과학과) ;
  • 호혜인 (건국대학교 환경보건과학과) ;
  • 김현진 (주식회사 평화엔지니어링) ;
  • 김건희 (건국대학교 휴먼앤에코케어센터) ;
  • 황순진 (건국대학교 환경보건과학과)
  • Received : 2023.03.07
  • Accepted : 2023.03.20
  • Published : 2023.03.31
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Abstract

Environmental DNA (eDNA) can exist in both intracellular and extracellular forms in natural ecosystems. When targeting harmful cyanobacteria, extracellular eDNA indicates the presence of traces of cyanobacteria, while intracellular eDNA indicates the potential for cyanobacteria to occur. However, identifying the "actual" potential for harmful cyanobacteria to occur is difficult using the existing sediment eDNA analysis method, which uses silica beads and cannot distinguish between these two forms of eDNA. This study analyzes the applicability of a density gradient centrifugation method (Ludox method) that can selectively analyze intracellular eDNA in sediment to overcome the limitations of conventional sediment eDNA analysis. PCR was used to amplify the extracted eDNA based on the two different methods, and the relative amount of gene amplification was compared using electrophoresis and Image J application. While the conventional bead beating method uses sediment as it is to extract eDNA, it is unknown whether the mic gene amplified from eDNA exists in the cyanobacterial cell or only outside of the cell. However, since the Ludox method concentrates the intracellular eDNA of the sediment through filtration and density gradient, only the mic gene present in the cyanobacteria cells could be amplified. Furthermore, the bead beating method can analyze up to 1 g of sediment at a time, whereas the Ludox method can analyze 5 g to 30 g at a time. This gram of sediments makes it possible to search for even a small amount of mic gene that cannot be searched by conventional bead beating method. In this study, the Ludox method secured sufficient intracellular gene concentration and clearly distinguished intracellular and extracellular eDNA, enabling more accurate and detailed potential analysis. By using the Ludox method for environmental RNA expression and next-generation sequencing (NGS) of harmful cyanobacteria in the sediment, it will be possible to analyze the potential more realistically.

자연생태계에서 환경유전자 (eDNA)는 세포의 내부(intracellular)와 외부(extracellular) 형태로 존재할 수 있다. 유해남조류를 대상으로 할 때, 세포 외부 eDNA는 남조류의 흔적, 세포 내부 eDNA는 남조류의 발생 잠재성을 의미한다. 하지만 기존의 퇴적물 eDNA 분석법인 silica bead를 이용한 파쇄법으로는 존재 형태를 구분할 수 없기 때문에 실질적인 유해남조류 발생 잠재성을 파악하기 어렵다. 본 연구는 기존의 파쇄법의 한계를 극복하고자 퇴적물 내 세포 내 eDNA를 선택적으로 분석할 수 있는 퇴적물 전처리 방법인 밀도구배 원심분리법(Ludox method)의 적용성을 분석하였다. 그 결과, 기존의 파쇄법은 퇴적물을 그대로 사용하여 eDNA를 추출하기 때문에 eDNA에서 증폭한 mic 유전자가 세포 내 존재하는지 혹은 세포 외 DNA로만 존재하는지 알 수 없었다. 하지만 Ludox method는 여과 및 밀도 구배를 통해 퇴적물의 세포 내 eDNA를 농축하므로 남조류 세포 내부에 존재하는 mic 유전자만을 증폭할 수 있었다. 결론적으로 Ludox method는 충분한 세포내부 유전자 농도를 확보하고 세포 내부와 외부의 eDNA를 명확히 구분함으로써 보다 정확하고 세밀한 잠재성 분석이 가능하였다. 이는 퇴적물 유해남조류의 유전자 활성을 확인할 수 있는 eRNA 분석과 차세대염기서열분석(next generation sequencing; NGS)을 이용한 meta-barcoding에 Ludox method를 활용함으로써 보다 현실적인 잠재성을 분석할 수 있을 것으로 판단된다.

Keywords

Acknowledgement

본 연구는 한국환경산업기술원의 '수생태계 건강성 확보 기술개발사업'인 '최첨단 위치기반 USBL ROV로봇을 탑재한 휴면포자 함유 퇴적물 준설 기술 개발' 과제에 의해 수행되었습니다 (2022003040003).

References

  1. Alaeddini, R. 2012. Forensic implications of PCR inhibition-a review, Forensic Science International: Genetics 6(3): 297-305.  https://doi.org/10.1016/j.fsigen.2011.08.006
  2. Beng, K.C. and R.T. Corlett. 2020. Applications of environmental DNA (eDNA) in ecology and conservation: opportunities, challenges and prospects. Biodiversity and Conservation 29: 2089-2121.  https://doi.org/10.1007/s10531-020-01980-0
  3. Bohmann, K., A. Evans, M.T.P. Gilbert, G.R. Carvalho, S. Creer, M. Knapp, W.Y. Douglas and M. De Bruyn. 2014. Environmental DNA for wildlife biology and biodiversity monitoring. Trends in Ecology & Evolution 29(6): 358-367.  https://doi.org/10.1016/j.tree.2014.04.003
  4. Ceccherini, M.T., J. Ascher, G. Pietramellara, T.M. Vogel and P. Nannipieri. 2007. Vertical advection of extracellular DNA by water capillarity in soil columns. Soil Biology and Biochemistry 39(1): 158-163.  https://doi.org/10.1016/j.soilbio.2006.07.006
  5. Corinaldesi, C., R. Danovaro and A. Dell̓Anno. 2005. Simultaneous recovery of extracellular and intracellular DNA suitable for molecular studies from marine sediments. Applied and Environmental Microbiology 71(1): 46-50.  https://doi.org/10.1128/AEM.71.1.46-50.2005
  6. Creer, S., K. Deiner, S. Frey, D. Porazinska, P. Taberlet, W.K. Thomas, C. Potter and H.M. Bik. 2016. The ecologist̓s field guide to sequence based identification of biodiversity. Methods in Ecology and Evolution 7(9): 1008-1018.  https://doi.org/10.1111/2041-210X.12574
  7. Damerval, T., A.-M. Castets, G. Guglielmi, J. Houmard and N. Tandeau de Marsac. 1989. Occurrence and distribution of gas vesicle genes among cyanobacteria. Journal of Bacteriology 171(3): 1445-1452.  https://doi.org/10.1128/jb.171.3.1445-1452.1989
  8. Dineen, S., R. Aranda IV, D. Anders and J. Robertson. 2010. An evaluation of commercial DNA extraction kits for the isolation of bacterial spore DNA from soil. Journal of Applied Microbiology 109(6): 1886-1896.  https://doi.org/10.1111/j.1365-2672.2010.04816.x
  9. Ficetola, G.F., C. Miaud, F. Pompanon and P. Taberlet. 2008. Species detection using environmental DNA from water samples. Biology Letters 4(4): 423-425.  https://doi.org/10.1098/rsbl.2008.0118
  10. Harper, L.R., A.S. Buxton, H.C. Rees, K. Bruce, R. Brys, D. Halfmaerten, D.S. Read, H.V. Watson, C.D. Sayer and E.P. Jones. 2019. Prospects and challenges of environmental DNA (eDNA) monitoring in freshwater ponds. Hydrobiologia 826: 25-41.  https://doi.org/10.1007/s10750-018-3750-5
  11. Hilton, J.A., J.C. Meeks and J.P. Zehr. 2016. Surveying DNA elements within functional genes of heterocyst-forming cyanobacteria. PLoS One 11(5): e0156034. 
  12. Kim, K., S. Lee, K. Seo and S.-J. Hwang. 2020a. Molecular Identification of Pseudanabaena Strains and Analysis of 2-MIB Production Potential in the North Han River System. Korean Journal of Ecology and Environment 53(4): 344-354.  https://doi.org/10.11614/KSL.2020.53.4.344
  13. Kim, K., Y. Yoon, H. Cho and S.-J. Hwang. 2020b. Molecular probes to evaluate the synthesis and production potential of an odorous compound (2-methylisoborneol) in cyanobacteria. International Journal of Environmental Research and Public Health 17(6): 1933. 
  14. Kosowski, R., N.Y. Naik and M. Teo. 2007. Do hedge funds deliver alpha? A Bayesian and bootstrap analysis. Journal of Financial Economics 84(1): 229-264.  https://doi.org/10.1016/j.jfineco.2005.12.009
  15. Larkin, M.A., G. Blackshields, N.P. Brown, R. Chenna, P.A. McGettigan, H. McWilliam, F. Valentin, I.M. Wallace, A. Wilm and R. Lopez. 2007. Clustal W and Clustal X version 2.0. Bioinformatics 23(21): 2947-2948.  https://doi.org/10.1093/bioinformatics/btm404
  16. Mustafa, I., Hadiatullah and Sustiyah. 2017. Removal of humic acid from peat soils by using AlCl3 prior to DNA extraction, AIP Conference Proceedings. 
  17. Miya, M. and T. Sado. 2019. Environmental DNA sampling and experimental manual version 2.1. Ed. by eDNA Methods Standardization Committee, The eDNA Society. 
  18. Nubel, U., F. Garcia-Pichel and G. Muyzer. 1997. PCR primers to amplify 16S rRNA genes from cyanobacteria. Applied and Environmental Microbiology 63(8): 3327-3332.  https://doi.org/10.1128/aem.63.8.3327-3332.1997
  19. Portt, C.B., G. Coker, D. Ming and R.G. Randall. 2006. A review of fish sampling methods commonly used in Canadian freshwater habitats. Fisheries and Oceans Canada, Ontario, p. 1-50. 
  20. Roose-Amsaleg, C., E. Garnier-Sillam and M. Harry. 2001. Extraction and purification of microbial DNA from soil and sediment samples. Applied Soil Ecology 18(1): 47-60.  https://doi.org/10.1016/S0929-1393(01)00149-4
  21. Santoso, F., B.P. Sampurna, Y.-H. Lai, S.-T. Liang, E. Hao, J.-R. Chen and C.-D. Hsiao. 2019. Development of a simple ImageJ-based method for dynamic blood flow tracking in zebrafish embryos and its application in drug toxicity evaluation. Inventions 4(4): 65. 
  22. Sassoubre, L.M., K.M. Yamahara, L.D. Gardner, B.A. Block and A.B. Boehm. 2016. Quantification of environmental DNA (eDNA) shedding and decay rates for three marine fish. Environmental Science & Technology 50(19): 10456-10464.  https://doi.org/10.1021/acs.est.6b03114
  23. Schrader, C., A. Schielke, L. Ellerbroek and R. Johne. 2012. PCR inhibitors-occurrence, properties and removal. Journal of Applied Microbiology 113(5): 1014-1026.  https://doi.org/10.1111/j.1365-2672.2012.05384.x
  24. Shin, J. and K. Cho. 1997. Distribution and population dynamics of Microcystis (Cyanophyta) in the Naktong River. Algae 12(4): 283-2903. 
  25. Sigsgaard, E.E., H. Carl, P.R. Moller and P.F. Thomsen. 2015. Monitoring the near-extinct European weather loach in Denmark based on environmental DNA from water samples. Biological Conservation 183: 46-52.  https://doi.org/10.1016/j.biocon.2014.11.023
  26. Tamura, K., G. Stecher and S. Kumar. 2021. MEGA11: molecular evolutionary genetics analysis version 11. Molecular Biology and Evolution 38(7): 3022-3027.  https://doi.org/10.1093/molbev/msab120
  27. Torti, A., M.A. Lever and B.B. Jorgensen. 2015. Origin, dynamics, and implications of extracellular DNA pools in marine sediments. Marine Genomics 24: 185-196.  https://doi.org/10.1016/j.margen.2015.08.007
  28. Tsao, H.-W., A. Michinaka, H.-K. Yen, S. Giglio, P. Hobson, P. Monis and T.-F. Lin. 2014. Monitoring of geosmin producing Anabaena circinalis using quantitative PCR. Water Research 49: 416-425.  https://doi.org/10.1016/j.watres.2013.10.028
  29. Turner, C.R., D.J. Miller, K.J. Coyne and J. Corush. 2014. Improved methods for capture, extraction, and quantitative assay of environmental DNA from Asian bigheaded carp (Hypophthalmichthys spp.). PLoS One 9(12): e114329. 
  30. Vuillemin, A., F. Horn, M. Alawi, C. Henny, D. Wagner, S.A. Crowe and J. Kallmeyer. 2017. Preservation and significance of extracellular DNA in ferruginous sediments from Lake Towuti, Indonesia. Frontiers in Microbiology 8: 1440. 
  31. Zhang, L., J. Yang, Y. Zhang, J. Shi, H. Yu and X. Zhang. 2022. eDNA biomonitoring revealed the ecological effects of water diversion projects between Yangtze River and Tai Lake. Water Research 210: 117994.