海洋沉积物病毒荧光计数法的优化
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摘要
病毒在海洋生态系统的物质循环和能流中起着重要作用,对其进行有效定量是研究其数量变动和生态功能的前提。现行的海洋沉积物病毒定量主要采用荧光染色计数法,但对技术流程中一些处理方法的定量效能尚待验证。本研究利用不同类型海洋沉积物对荧光计数法进行了效能评估和条件优化。结果表明:将沉积物采样后染色封片直接计数或-20°C保存为最佳,保存3个月后未见丰度降低。而不同类型的沉积物经液氮闪冻后于-80°C冷冻保存后的定量效能不同:沙质沉积物保存1个月后病毒丰度下降1个数量级;泥沙质沉积物保存1个月后丰度降低了约27%,2个月后则下降了60%。对焦磷酸钠最优终浓度实验结果显示,使用3mmol/L终浓度获得的病毒计数定量效能优于目前普遍使用的5mmol/L终浓度。水浴超声分离在常温下的定量效果更优,在水浴中添加冰块导致获取的沙质沉积物中的病毒丰度下降约37%。研究发现:处理中用稀释替代离心不仅可降低沉积物颗粒的干扰,而且定量效能更高;离心处理获取的沙质和泥沙质沉积物中的病毒丰度较稀释处理低约40%。对海洋沉积物中原核生物的定量效能与病毒有相似的结果。据此,本文提出了改进的沉积物病毒荧光计数法的操作流程。
Viruses play key roles in material cycles and energy flows of marine ecosystems. Effective enumeration of viruses is critical for understanding their ecological roles and dynamics. Epifluorescence microscopy has been used widely to enumerate viruses in marine sediments, but the efficiency in some procedures remains unknown. We evaluated and optimized the procedures of viral enumeration based on three types of marine sediments using epifluorescence microscopy. The results suggest that the preparation of dye-stained slides immediately after sampling and storage at-20°C yielded the greatest counting efficiency, and no decrease was observed after three months. By contrast, the counting efficiency of snap freezing in liquid nitrogen and storage at-80°C varied with different types of sediments: declined an order of magnitude in sandy sediment after one month; and decreased by about 27% after one month, and about 60% after two months in muddy-sand sediment. The results show that for the optimal performance, the final concentration of sodium pyrophosphate shall be set at 3mmol/L rather than at 5mmol/L that commonly used at present, sonication shall be placed in water at ambient temperature rather than a mixture of ice-water, with which a loss of 37% might be reached for enumeration of viruses in the sandy sediment; and dilution instead of centrifugation resulted in an increase of the viral counts by about 40% for the sandy and muddy-sand sediments. Similar efficiencies were reached for prokaryotes in all tests. Therefore, this protocol of viral counting for marine sediments in epifluorescence microscopy is improved and thus shall be recommended.
Viruses play key roles in material cycles and energy flows of marine ecosystems. Effective enumeration of viruses is critical for understanding their ecological roles and dynamics. Epifluorescence microscopy has been used widely to enumerate viruses in marine sediments, but the efficiency in some procedures remains unknown. We evaluated and optimized the procedures of viral enumeration based on three types of marine sediments using epifluorescence microscopy. The results suggest that the preparation of dye-stained slides immediately after sampling and storage at-20°C yielded the greatest counting efficiency, and no decrease was observed after three months. By contrast, the counting efficiency of snap freezing in liquid nitrogen and storage at-80°C varied with different types of sediments: declined an order of magnitude in sandy sediment after one month; and decreased by about 27% after one month, and about 60% after two months in muddy-sand sediment. The results show that for the optimal performance, the final concentration of sodium pyrophosphate shall be set at 3mmol/L rather than at 5mmol/L that commonly used at present, sonication shall be placed in water at ambient temperature rather than a mixture of ice-water, with which a loss of 37% might be reached for enumeration of viruses in the sandy sediment; and dilution instead of centrifugation resulted in an increase of the viral counts by about 40% for the sandy and muddy-sand sediments. Similar efficiencies were reached for prokaryotes in all tests. Therefore, this protocol of viral counting for marine sediments in epifluorescence microscopy is improved and thus shall be recommended.
引文
马玉,汪岷,夏骏等,2016.秦皇岛褐潮期超微型浮游生物丰度及多样性研究.中国海洋大学学报(自然科学版),46(6):142-150
中国科学院海洋研究所,2011.HY/T 140-2011海洋微型底栖生物调查规范.北京:中国标准出版社,1-30
王海丽,杨季芳,屠霄霞等,2011.象山港海洋病毒时空分布特征及其环境影响因素.中国环境科学,31(5):834-844
卢龙飞,汪岷,梁彦韬等,2013.东海、黄海浮游病毒及异养细菌的分布研究.海洋与湖沼,44(5):1339-1346
白晓歌,汪岷,梁彦韬等,2008.运用荧光显微技术分析北黄海夏季浮游病毒的分布.中国海洋大学学报(自然科学版),38(4):609-613
刘晶晶,曾江宁,杜萍等,2011.长江口及邻近海域夏、冬季浮游病毒丰度分布.应用生态学报,22(3):793-799
孙辉,汪岷,汪俭等,2014.养殖活动对超微型浮游生物分布影响的研究.海洋与湖沼,45(6):1272-1279
孙小磊,赵以军,刘妮等,2009.淡水湿地浮游病毒的空间分布.生态学报,29(2):1048-1054
张喆,王晓红,巩秀玉等.2017.南海北部海域春季浮游细菌和病毒空间分布及其影响因素.生态学报,37(5):1639-1649,doi:10.5846/stxb201509231958
张喆,孟祥红,肖慧等,2008.青岛近岸水体夏冬季浮游病毒、细菌分布特征及其与环境因子的关系.武汉大学学报(理学版),54(2):209-214
张永雨,黄春晓,杨军等,2011.海洋微生物与噬菌体间的相互关系.科学通报,56(14):1071-1079
李清,闫启仑,李洪波等,2016.大连湾和大窑湾表层沉积物病毒分布特点.海洋环境科学,35(2):184-189
李洪波,肖天,林凤翱,2010.海洋浮游病毒的研究方法.海洋科学,34(9):97-101
李洪波,崔向阳,林凤翱等,2010.河北近岸海域浮游细菌与病毒的关系.海洋环境科学,29(2):187-190
李胜男,王秀娟,周建等,2015.利用流式细胞仪计数微型浮游生物的方法.湖泊科学,27(5):757-766
国家海洋局,2007.GB/T 12763.6-2007海洋调查规范第6部分:海洋生物调查.北京:中国标准出版社,159
赵苑,武洪庆,李洪波等,2010.胶州湾浮游病毒的分布研究.海洋科学,34(7):86-92
李娟,2005.病毒:海洋生态动力学和疾病学研究的创新点.海洋湖沼通报,2:79-87
潘洛安,张利华,张经,2006.应用流式细胞术检测水体病毒.海洋环境科学,25(3):78-85
Brussaard C P D,2004.Optimization of procedures for counting viruses by flow cytometry.Applied and Environmental Microbiology,70(3):1506-1513
Danovaro R,Dell’Anno A,Trucco A et al,2001.Determination of virus abundance in marine sediments.Applied and Environmental Microbiology,67(3):1384-1387
Danovaro R,Corinaldesi C,Filippini M et al,2008a.Viriobenthos in freshwater and marine sediments:A review.Freshwater Biology,53(6):1186-1213
Danovaro R,Dell’Anno A,Corinaldesi C et al,2008b.Major viral impact on the functioning of benthic deep-sea ecosystems.Nature,454(7208):1084-1087
Danovaro R,Middelboe M,2010.Separation of free virus particles from sediments in aquatic systems.Manual of Aquatic Viral Ecology,8:74-81
Dell’Anno A,Corinaldesi C,Magagnini M et al,2009.Determination of viral production in aquatic sediments using the dilution-based approach.Nature Protocols,4(7):1013-1022
Duhamel S,Jacquet S,2006.Flow cytometric analysis of bacteria-and virus-like particles in lake sediments.Journal of Microbiological Methods,64(3):316-332
Ellery W,Schleyer M,1984.Comparison of homogenization and ultrasonication as techniques in extracting attached sedimentary bacteria.Marine Ecology Progress Series,15(3):247-250
Fischer U R,Kirschner A K T,Velimirov B,2005.Optimization of extraction and estimation of viruses in silty freshwater sediments.Aquatic Microbial Ecology,40(3):207-216
Helton R R,Liu L,Wommack K E,2006.Assessment of factors influencing direct enumeration of viruses within estuarine sediments.Applied and Environmental Microbiology,72(7):4767-4774
Hennes K P,Suttle C A,1995.Direct counts of viruses in natural waters and laboratory cultures by epifluorescence microscopy.Limnology and Oceanography,40(6):1050-1055
Hewson I,Fuhrman J A,2003.Viriobenthos production and virioplankton sorptive scavenging by suspended sediment particles in coastal and pelagic waters.Microbial Ecology,46(3):337-347
Noble R T,Fuhrman J A,1998.Use of SYBR Green I for rapid epifluorescence counts of marine viruses and bacteria.Aquat Microb Ecol,14:113-118
Patel A,Noble R T,Steele J A et al,2007.Virus and prokaryote enumeration from planktonic aquatic environments by epifluorescence microscopy with SYBR Green I.Nature Protocols,2(2):269-276
Siem-J?rgensen M,Glud R N,Middelboe M,2008.Viral dynamics in a coastal sediment:Seasonal pattern,controlling factors and relations to the pelagic-benthic coupling.Marine Biology Research,4(3):165-179
Suttle C A,2005.Review Viruses in the sea.Nature,437(7057):356-361
Suttle C A,Fuhrman J A,2010.Enumeration of virus particles in aquatic or sediment samples by epifluorescence microscopy.Manual of Aquatic Viral Ecology,15:145-153
Wen K,Ortmann A C,Suttle C A,2004.Accurate estimation of viral abundance by epifluorescence microscopy.Applied and Environmental Microbiology,70(7):3862-3867
Wilhelm S W,Suttle C A,1999.Viruses and nutrient cycles in the sea-viruses play critical roles in the structure and function of aquatic food webs.Bioscience,49:781-788
中国科学院海洋研究所,2011.HY/T 140-2011海洋微型底栖生物调查规范.北京:中国标准出版社,1-30
王海丽,杨季芳,屠霄霞等,2011.象山港海洋病毒时空分布特征及其环境影响因素.中国环境科学,31(5):834-844
卢龙飞,汪岷,梁彦韬等,2013.东海、黄海浮游病毒及异养细菌的分布研究.海洋与湖沼,44(5):1339-1346
白晓歌,汪岷,梁彦韬等,2008.运用荧光显微技术分析北黄海夏季浮游病毒的分布.中国海洋大学学报(自然科学版),38(4):609-613
刘晶晶,曾江宁,杜萍等,2011.长江口及邻近海域夏、冬季浮游病毒丰度分布.应用生态学报,22(3):793-799
孙辉,汪岷,汪俭等,2014.养殖活动对超微型浮游生物分布影响的研究.海洋与湖沼,45(6):1272-1279
孙小磊,赵以军,刘妮等,2009.淡水湿地浮游病毒的空间分布.生态学报,29(2):1048-1054
张喆,王晓红,巩秀玉等.2017.南海北部海域春季浮游细菌和病毒空间分布及其影响因素.生态学报,37(5):1639-1649,doi:10.5846/stxb201509231958
张喆,孟祥红,肖慧等,2008.青岛近岸水体夏冬季浮游病毒、细菌分布特征及其与环境因子的关系.武汉大学学报(理学版),54(2):209-214
张永雨,黄春晓,杨军等,2011.海洋微生物与噬菌体间的相互关系.科学通报,56(14):1071-1079
李清,闫启仑,李洪波等,2016.大连湾和大窑湾表层沉积物病毒分布特点.海洋环境科学,35(2):184-189
李洪波,肖天,林凤翱,2010.海洋浮游病毒的研究方法.海洋科学,34(9):97-101
李洪波,崔向阳,林凤翱等,2010.河北近岸海域浮游细菌与病毒的关系.海洋环境科学,29(2):187-190
李胜男,王秀娟,周建等,2015.利用流式细胞仪计数微型浮游生物的方法.湖泊科学,27(5):757-766
国家海洋局,2007.GB/T 12763.6-2007海洋调查规范第6部分:海洋生物调查.北京:中国标准出版社,159
赵苑,武洪庆,李洪波等,2010.胶州湾浮游病毒的分布研究.海洋科学,34(7):86-92
李娟,2005.病毒:海洋生态动力学和疾病学研究的创新点.海洋湖沼通报,2:79-87
潘洛安,张利华,张经,2006.应用流式细胞术检测水体病毒.海洋环境科学,25(3):78-85
Brussaard C P D,2004.Optimization of procedures for counting viruses by flow cytometry.Applied and Environmental Microbiology,70(3):1506-1513
Danovaro R,Dell’Anno A,Trucco A et al,2001.Determination of virus abundance in marine sediments.Applied and Environmental Microbiology,67(3):1384-1387
Danovaro R,Corinaldesi C,Filippini M et al,2008a.Viriobenthos in freshwater and marine sediments:A review.Freshwater Biology,53(6):1186-1213
Danovaro R,Dell’Anno A,Corinaldesi C et al,2008b.Major viral impact on the functioning of benthic deep-sea ecosystems.Nature,454(7208):1084-1087
Danovaro R,Middelboe M,2010.Separation of free virus particles from sediments in aquatic systems.Manual of Aquatic Viral Ecology,8:74-81
Dell’Anno A,Corinaldesi C,Magagnini M et al,2009.Determination of viral production in aquatic sediments using the dilution-based approach.Nature Protocols,4(7):1013-1022
Duhamel S,Jacquet S,2006.Flow cytometric analysis of bacteria-and virus-like particles in lake sediments.Journal of Microbiological Methods,64(3):316-332
Ellery W,Schleyer M,1984.Comparison of homogenization and ultrasonication as techniques in extracting attached sedimentary bacteria.Marine Ecology Progress Series,15(3):247-250
Fischer U R,Kirschner A K T,Velimirov B,2005.Optimization of extraction and estimation of viruses in silty freshwater sediments.Aquatic Microbial Ecology,40(3):207-216
Helton R R,Liu L,Wommack K E,2006.Assessment of factors influencing direct enumeration of viruses within estuarine sediments.Applied and Environmental Microbiology,72(7):4767-4774
Hennes K P,Suttle C A,1995.Direct counts of viruses in natural waters and laboratory cultures by epifluorescence microscopy.Limnology and Oceanography,40(6):1050-1055
Hewson I,Fuhrman J A,2003.Viriobenthos production and virioplankton sorptive scavenging by suspended sediment particles in coastal and pelagic waters.Microbial Ecology,46(3):337-347
Noble R T,Fuhrman J A,1998.Use of SYBR Green I for rapid epifluorescence counts of marine viruses and bacteria.Aquat Microb Ecol,14:113-118
Patel A,Noble R T,Steele J A et al,2007.Virus and prokaryote enumeration from planktonic aquatic environments by epifluorescence microscopy with SYBR Green I.Nature Protocols,2(2):269-276
Siem-J?rgensen M,Glud R N,Middelboe M,2008.Viral dynamics in a coastal sediment:Seasonal pattern,controlling factors and relations to the pelagic-benthic coupling.Marine Biology Research,4(3):165-179
Suttle C A,2005.Review Viruses in the sea.Nature,437(7057):356-361
Suttle C A,Fuhrman J A,2010.Enumeration of virus particles in aquatic or sediment samples by epifluorescence microscopy.Manual of Aquatic Viral Ecology,15:145-153
Wen K,Ortmann A C,Suttle C A,2004.Accurate estimation of viral abundance by epifluorescence microscopy.Applied and Environmental Microbiology,70(7):3862-3867
Wilhelm S W,Suttle C A,1999.Viruses and nutrient cycles in the sea-viruses play critical roles in the structure and function of aquatic food webs.Bioscience,49:781-788