中国对虾生物量评估的环境DNA检测技术的建立及优化
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摘要
近年来,环境DNA(Environmental DNA, eDNA)技术作为一种新的水生生物调查方法发展迅速,在水生生态系统的研究领域被广泛应用到物种检测、生物多样性评价、生物量评估等方面。然而,很少有研究专门评价e DNA技术操作流程中不同的eDNA富集方法与提取方法对研究结果的影响,从而针对具体研究对象建立一套最佳的eDNA技术操作流程。此外,由于物种间生活习性的差异,不同物种释放到环境中的DNA量及DNA片段大小不同,因而,针对不同研究对象需采用不同的eDNA富集与提取方法。本研究以中国对虾(Fenneropenaeus chinensis)为研究对象,采用滤膜法富集eDNA,结合血液与组织DNA提取试剂盒提取e DNA。选取直径为47 mm的玻璃纤维膜、硝酸纤维膜、聚碳酸酯膜、尼龙膜共4种材质的滤膜,每种滤膜根据其孔径大小设置0.45、0.8、1.2、5μm共4个梯度,取样水量设置500 ml、1 L、2 L共3个梯度。结果显示,滤膜材质、滤膜孔径大小及取样水体体积均对中国对虾的定性与定量分析具有一定的影响,其中,0.45μm的玻璃纤维滤膜过滤2 L水样能够检测到的DNA拷贝数最多,并依据此建立了一套中国对虾e DNA技术的操作流程,提高了中国对虾的检出率,为后续中国对虾的分布监测及生物量评估提供了基础。
In recent years, environmental DNA(eDNA) technology has developed rapidly as a new method for investigation of aquatic organisms and has been widely used in the field of aquatic ecosystem research for species detection, biodiversity evaluation, and biomass assessment. However, few studies have specifically evaluated the effects of different e DNA enrichment and extraction methods on the results of the eDNA technology operation process to establish a set of optimal eDNA technology operation procedures for specific research subjects. In addition, because of differences in living habits between species, the amount of DNA released from different species and the size of DNA fragments are different. Therefore, different eDNA enrichment and extraction methods should be adopted for different research subjects. In this study, Fenneropenaeus chinensis was used as the research object, and eDNA was enriched by the membrane method, and eDNA was extracted by combining a blood and tissue DNA extraction kit. A filter membrane with a diameter of 47 mm, a nitrocellulose membrane, a polycarbonate membrane, and a nylon membrane were used, and each membrane was set to 0.45 μm, 0.8 μm, 1.2 μm, and 5 μm according to pore size. With 4 gradients, the sampling water volume was set to three gradients of 500 ml, 1 L, and 2 L. The experimental results showed that the membrane material size, pore size of the membrane, and the volume of the sampled water had certain influences on the qualitative and quantitative analysis of F. chinensis. The 0.45 μm glass fiber membrane filter and 2 L water sample detected the greatest DNA copy number. Based on this, a set of operational procedures for F. chinensis prawn eDNA technology was established to improve the detection rate of F. chinensis, which provided the basis for subsequent distribution monitoring and biomass assessment of F. chinensis.
In recent years, environmental DNA(eDNA) technology has developed rapidly as a new method for investigation of aquatic organisms and has been widely used in the field of aquatic ecosystem research for species detection, biodiversity evaluation, and biomass assessment. However, few studies have specifically evaluated the effects of different e DNA enrichment and extraction methods on the results of the eDNA technology operation process to establish a set of optimal eDNA technology operation procedures for specific research subjects. In addition, because of differences in living habits between species, the amount of DNA released from different species and the size of DNA fragments are different. Therefore, different eDNA enrichment and extraction methods should be adopted for different research subjects. In this study, Fenneropenaeus chinensis was used as the research object, and eDNA was enriched by the membrane method, and eDNA was extracted by combining a blood and tissue DNA extraction kit. A filter membrane with a diameter of 47 mm, a nitrocellulose membrane, a polycarbonate membrane, and a nylon membrane were used, and each membrane was set to 0.45 μm, 0.8 μm, 1.2 μm, and 5 μm according to pore size. With 4 gradients, the sampling water volume was set to three gradients of 500 ml, 1 L, and 2 L. The experimental results showed that the membrane material size, pore size of the membrane, and the volume of the sampled water had certain influences on the qualitative and quantitative analysis of F. chinensis. The 0.45 μm glass fiber membrane filter and 2 L water sample detected the greatest DNA copy number. Based on this, a set of operational procedures for F. chinensis prawn eDNA technology was established to improve the detection rate of F. chinensis, which provided the basis for subsequent distribution monitoring and biomass assessment of F. chinensis.
引文
Bohmann K,Evans A,Gilbert MTP,et al.Environmental DNAfor wildlife biology and biodiversity monitoring.Trends in Ecology&Evolution,2014,29(6):358-367
Costas BA,Mc Manus G,Doherty M,et al.Use of speciesspecific primers and PCR to measure the distributions of planktonic ciliates in coastal waters.Limnology&Oceanography Methods,2007,5(6):163-173
Deiner K,Altermatt F.Transport distance of invertebrate environmental DNA in a natural river.PLoS One,2014,9(2):e88786
Deiner K,Walser JC,M?chler E,et al.Choice of capture and extraction methods affect detection of freshwater biodiversity from environmental DNA.Biological Conservation,2015,183(9):53-63
Eichmiller JJ,Miller LM,Sorensen PW.Optimizing techniques to capture and extract environmental DNA for detection and quantification of fish.Molecular Ecology Resources,2016,16(1):56-68
Evans NT,Lamberti GA.Freshwater fisheries assessment using environmental DNA:A primer on the method,its potential,and shortcomings as a conservation tool.Fisheries Research,2017
Ficetola GF,Miaud C,Pompanon F,et al.Species detection using environmental DNA from water samples.Biology Letters,2008,4(4):423-425
Geerts AN,Boets P,Heede SVD,et al.A search for standardized protocols to detect alien invasive crayfish based on environmental DNA(eDNA):A lab and field evaluation.Ecological Indicators,2018,84(84):564-572
Goldberg CS,Pilliod DS,Arkle RS,et al.Molecular detection of vertebrates in stream water:A demonstration using rocky mountain tailed frogs and Idaho giant salamanders.PLoSOne,2011,6(7):e22746
Haile J,Froese DG,Macphee RDE,et al.Ancient DNA reveals late survival of mammoth and horse in interior Alaska.Proceedings of the National Academy of Sciences of USA,2009,106(52):22352-22357
Hebert PD,Gregory TR.The promise of DNA barcoding for taxonomy.Systematic Biology,2005,54(5):852-859
Jerde CL,Mahon AR,Chadderton WL,et al.“Sight-unseen”detection of rare aquatic species using environmental DNA.Conservation Letters,2011,4(2):150-157
Jiao MC,Zhao DX,Ouyang S,et al.Application prospect of DNA barcode technology in taxonomy.Hubei Agricultural Sciences,2011,50(5):886-890[焦明超,赵大显,欧阳珊,等.DNA条形码技术在生物分类学中的应用前景.湖北农业科学,2011,50(5):886-890]
Klymus KE,Richter CA,Chapman DC,et al.Quantification of e DNA shedding rates from invasive bighead carp Hypophthalmichthys nobilis and silver carp Hypophthalmichthys molitrix.Biological Conservation,2015,183:77-84
Minamoto T,Yamanaka H,Takahara T,et al.Surveillance of fish species composition using environmental DNA.Limnology,2012,13(2):193-197
Minamoto T,Naka T,Moji K,et al.Techniques for the practical collection of environmental DNA:Filter selection,preservation,and extraction.Limnology,2016,17(1):23-32
Moyer GR,Díaz-Ferguson E,Hill JE,et al.Assessing environmental DNA detection in controlled lentic systems.PLoS One,2014,9(7):e103767
Rees HC,Maddison BC,Middleditch DJ,et al.The detection of aquatic animal species using environmental DNA-Areview of eDNA as a survey tool in ecology//Dynamic 3DImaging,DAGM 2009 Workshop,Dyn3D 2009,Jena,Germany,September 9,2009.Proceedings.DBLP,2014,110-125
Renshaw MA,Olds BP,Jerde CL,et al.The room temperature preservation of filtered environmental DNA samples and assimilation into a phenol-chloroform-isoamyl alcohol DNAextraction.Molecular Ecology Resources,2015,15(1):168-176
Shan XJ,Li M,Wang WJ.Application of environmental DNAtechnology in aquatic ecosystem.Progress in Fishery Sciences,2018,39(3):23-29[单秀娟,李苗,王伟继.环境DNA(e DNA)技术在水生生态系统中的应用研究进展.渔业科学进展,2018,39(3):23-29]
Sigsgaard EE,Nielsen IB,Carl H,et al.Seawater environmental DNA reflects seasonality of a coastal fish community.Marine Biology,2017,164(6):128
Takahara T,Minamoto T,Yamanaka H,et al.Estimation of fish biomass using environmental DNA.PLoS One,2012,7(4):e35868
Thomsen PF,Kielgast J,Iversen LL,et al.Detection of a diverse marine fish fauna using environmental DNA from seawater samples.PLoS One,2012,7(8):e41732
Thomsen PF,Kielgast J,Iversen LL,et al.Monitoring endangered freshwater biodiversity using environmental DNA.Molecular Ecology,2012,21(11):2565
Turner CR,Barnes MA,Xu CCY,et al.Particle size distribution and optimal capture of aqueous macrobial eDNA.Methods in Ecology&Evolution,2014,5(7):676-684
Yan H,Dong J,Huang J,et al.Study on the DNA extraction from shrimp specimens by different methods.Applied Chemical Industry,2012,41(3):548-551[闫晗,董君,黄静,等.不同固定剂处理的对虾基因组DNA提取方法研究.应用化工,2012,41(3):548-551]
Yuan W,Lin Q,Wang J,et al.Assessment of enhancement effectiveness of Chinese shrimp Fenneropenaeus chinensis stock in the Laoshan Bay.Progress in Fishery Sciences,2015,36(4):27-34[袁伟,林群,王俊,等.崂山湾中国对虾(Fenneropenaeus chinensis)增殖放流的效果评价.渔业科学进展,2015,36(4):27-34]
Vermeulen S,Koziell I.Integrating local and global biodiversity values:A review of biodiversity assessment.2002-IIED,2002
Costas BA,Mc Manus G,Doherty M,et al.Use of speciesspecific primers and PCR to measure the distributions of planktonic ciliates in coastal waters.Limnology&Oceanography Methods,2007,5(6):163-173
Deiner K,Altermatt F.Transport distance of invertebrate environmental DNA in a natural river.PLoS One,2014,9(2):e88786
Deiner K,Walser JC,M?chler E,et al.Choice of capture and extraction methods affect detection of freshwater biodiversity from environmental DNA.Biological Conservation,2015,183(9):53-63
Eichmiller JJ,Miller LM,Sorensen PW.Optimizing techniques to capture and extract environmental DNA for detection and quantification of fish.Molecular Ecology Resources,2016,16(1):56-68
Evans NT,Lamberti GA.Freshwater fisheries assessment using environmental DNA:A primer on the method,its potential,and shortcomings as a conservation tool.Fisheries Research,2017
Ficetola GF,Miaud C,Pompanon F,et al.Species detection using environmental DNA from water samples.Biology Letters,2008,4(4):423-425
Geerts AN,Boets P,Heede SVD,et al.A search for standardized protocols to detect alien invasive crayfish based on environmental DNA(eDNA):A lab and field evaluation.Ecological Indicators,2018,84(84):564-572
Goldberg CS,Pilliod DS,Arkle RS,et al.Molecular detection of vertebrates in stream water:A demonstration using rocky mountain tailed frogs and Idaho giant salamanders.PLoSOne,2011,6(7):e22746
Haile J,Froese DG,Macphee RDE,et al.Ancient DNA reveals late survival of mammoth and horse in interior Alaska.Proceedings of the National Academy of Sciences of USA,2009,106(52):22352-22357
Hebert PD,Gregory TR.The promise of DNA barcoding for taxonomy.Systematic Biology,2005,54(5):852-859
Jerde CL,Mahon AR,Chadderton WL,et al.“Sight-unseen”detection of rare aquatic species using environmental DNA.Conservation Letters,2011,4(2):150-157
Jiao MC,Zhao DX,Ouyang S,et al.Application prospect of DNA barcode technology in taxonomy.Hubei Agricultural Sciences,2011,50(5):886-890[焦明超,赵大显,欧阳珊,等.DNA条形码技术在生物分类学中的应用前景.湖北农业科学,2011,50(5):886-890]
Klymus KE,Richter CA,Chapman DC,et al.Quantification of e DNA shedding rates from invasive bighead carp Hypophthalmichthys nobilis and silver carp Hypophthalmichthys molitrix.Biological Conservation,2015,183:77-84
Minamoto T,Yamanaka H,Takahara T,et al.Surveillance of fish species composition using environmental DNA.Limnology,2012,13(2):193-197
Minamoto T,Naka T,Moji K,et al.Techniques for the practical collection of environmental DNA:Filter selection,preservation,and extraction.Limnology,2016,17(1):23-32
Moyer GR,Díaz-Ferguson E,Hill JE,et al.Assessing environmental DNA detection in controlled lentic systems.PLoS One,2014,9(7):e103767
Rees HC,Maddison BC,Middleditch DJ,et al.The detection of aquatic animal species using environmental DNA-Areview of eDNA as a survey tool in ecology//Dynamic 3DImaging,DAGM 2009 Workshop,Dyn3D 2009,Jena,Germany,September 9,2009.Proceedings.DBLP,2014,110-125
Renshaw MA,Olds BP,Jerde CL,et al.The room temperature preservation of filtered environmental DNA samples and assimilation into a phenol-chloroform-isoamyl alcohol DNAextraction.Molecular Ecology Resources,2015,15(1):168-176
Shan XJ,Li M,Wang WJ.Application of environmental DNAtechnology in aquatic ecosystem.Progress in Fishery Sciences,2018,39(3):23-29[单秀娟,李苗,王伟继.环境DNA(e DNA)技术在水生生态系统中的应用研究进展.渔业科学进展,2018,39(3):23-29]
Sigsgaard EE,Nielsen IB,Carl H,et al.Seawater environmental DNA reflects seasonality of a coastal fish community.Marine Biology,2017,164(6):128
Takahara T,Minamoto T,Yamanaka H,et al.Estimation of fish biomass using environmental DNA.PLoS One,2012,7(4):e35868
Thomsen PF,Kielgast J,Iversen LL,et al.Detection of a diverse marine fish fauna using environmental DNA from seawater samples.PLoS One,2012,7(8):e41732
Thomsen PF,Kielgast J,Iversen LL,et al.Monitoring endangered freshwater biodiversity using environmental DNA.Molecular Ecology,2012,21(11):2565
Turner CR,Barnes MA,Xu CCY,et al.Particle size distribution and optimal capture of aqueous macrobial eDNA.Methods in Ecology&Evolution,2014,5(7):676-684
Yan H,Dong J,Huang J,et al.Study on the DNA extraction from shrimp specimens by different methods.Applied Chemical Industry,2012,41(3):548-551[闫晗,董君,黄静,等.不同固定剂处理的对虾基因组DNA提取方法研究.应用化工,2012,41(3):548-551]
Yuan W,Lin Q,Wang J,et al.Assessment of enhancement effectiveness of Chinese shrimp Fenneropenaeus chinensis stock in the Laoshan Bay.Progress in Fishery Sciences,2015,36(4):27-34[袁伟,林群,王俊,等.崂山湾中国对虾(Fenneropenaeus chinensis)增殖放流的效果评价.渔业科学进展,2015,36(4):27-34]
Vermeulen S,Koziell I.Integrating local and global biodiversity values:A review of biodiversity assessment.2002-IIED,2002