样品保存温度和时长对环境DNA鱼类监测的影响  

Effects of storage temperature and time of sampling and pre-treatment on fish biomonitoring through environmental

作　　者：母亚雯 罗仪宁 汤楠 杨江华 张咏[1] 张效伟[2] DNA Mu Yawen;Luo Yining;Tang Nan;Yang Jianghua;Zhang Yong;Zhang Xiaowei(Jiangsu Provincial Environmental Monitoring Center,Nanjing 210019,P.R.China;State Key Laboratory of Pollution Control&Resource Reuse,School of the Environment Nanjing University,Nanjing 210023,P.R.China;Shaanxi Province Environmental Monitoring Center,Xian 710054,P.R.China)

机构地区：[1]江苏省环境监测中心,南京210019 [2]南京大学环境学院,污染控制与资源化研究国家重点实验室,南京210023 [3]陕西省环境监测中心,西安710054

出　　处：《湖泊科学》2024年第6期1853-1863,共11页Journal of Lake Sciences

基　　金：江苏省卓越博士后资助项目(2022ZB811);国家自然科学基金项目(42377277);江苏省环境监测科研基金项目(2203,1802)联合资助。

摘　　要：环境DNA技术作为最具潜力的生物监测手段正逐渐应用于生物多样性调查。水样采集和前处理对环境DNA生物监测至关重要。通过实验室水箱实验分析4种温度梯度下(4、15、25和35℃)7种常见鱼类环境DNA随时间的降解规律,探究保存温度和时长等前处理条件对环境DNA鱼类监测的影响,并在25℃下测定了野外环境中鱼类环境DNA的降解速率。结果表明:整体上,温度越高,鱼类环境DNA降解越快。水箱实验中,除4℃外,各鱼类环境DNA在采集后24 h内降解超过90%,计算半衰期在0.91~5.80 h之间。野外环境样品中,鱼类环境DNA含量更低、降解更快,总鱼类环境DNA计算半衰期仅为0.15 h。环境DNA宏条形码比定量PCR检测灵敏度高,在水样采集第13天后仍能检出大量鱼类信息,但定量PCR在水样采集第2天就无有效检出。宏条形码检出各鱼类相对丰度在水样采集前5天内基本保持稳定。综上,采用定量PCR方法开展鱼类监测时,建议采样后4 h内完成过滤;采用宏条形码方法时,建议24 h内完成样品过滤,以最大程度降低环境DNA降解对监测结果的影响。As the most promising biological monitoring approach,environmental DNA technology is gradually being applied to the biodiversity survey.Water sampling and pre-treatment are crucial to the results of environmental DNA biomonitoring.Laboratory tank experiments were conducted to analyze the environmental DNA degradation rules of 7 common fish species over time under four different temperatures gradients(4℃,15℃,25℃and 35℃),to explore the effects of water samples pre-treatment conditions(temperature,storage time and so on)on environmental DNA fish monitoring,and to further test the degradation rate of environmental DNA in field assays at 25℃.The results showed that the higher the temperature,the faster the degradation of environmental DNA,as a whole.In laboratory tank experiments,fish environmental DNA degradation exceeded 90%within 24 h of water sampling except at 4℃,and calculated half-life of fluctuated between 0.91 and 5.80 h.In the field experiment,the content of fish environmental DNA was lower,the degradation was faster,and the degradation calculated half-life was only 0.15 h.The detection sensitivity of metabarcoding was higher than that of quantitative PCR.A large number of fish species eDNA information could still be detected by metabarcoding after 13 days of water sampling,while the DNA signals could not be detected almost on the second day of sampling by quantitative PCR.Metabarcoding results showed that the relative abundance of each fish remained basically stable within 5 days.In conclusion,when quantitative PCR is used for fish biomonitoring,it is recommended to filter the water samples within 4 h.When using metabarcoding method,it is recommended to carry out sample filtration within 24 h at 4℃to minimize the impact of environmental DNA degradation on monitoring results.

关 键 词：宏条形码 定量PCR 鱼类多样性 DNA降解 温度 保存时长 

分 类 号：X835[环境科学与工程—环境工程]