【作者】 黄磊；

【导师】 王玉蓉；

【作者基本信息】 四川大学 ， 水力学及河流动力学， 2023， 硕士

【摘要】 环境DNA（environmental DNA,eDNA）技术利用了生物体向环境中释放的遗传物质实现对水生物种的监测。对于河流这种开放、动态的生态系统,繁冗庞杂的生境因素可以直接或间接影响生物eDNA在水中的持久性,进而影响eDNA技术的应用。山区河流中的水电开发会造成河床沉积物的组成发生改变,而河床沉积物的稳定性以及沉积物表层生物膜的定植会影响水生生物的活动轨迹,继而会影响生物eDNA在河流中的降解、沉降、输移和再悬浮过程。因此,探究不同水流条件下,沉积物和生物膜条件变化对于eDNA持久性的影响特性十分必要,研究结果可以为eDNA技术在水生环境中的应用提供理论支撑。本文以草鱼为研究对象,在标准化的实验设置下,对3种沉积物类型（0.5-1mm沙粒、4-6mm砾石和20-40mm卵石）、4种生物膜发育水平（生物膜培养时间分别为0天、10天、18天和25天）以及3种水流条件（静水条件、动水条件和动静水组合条件）进行组合实验,利用荧光定量PCR技术检测不同工况中草鱼eDNA浓度的变化情况,得到的主要实验成果和结论如下:（1）实验中草鱼eDNA浓度随时间呈现指数降解模式。沉积物类型、生物膜发育水平以及水流条件的变化不会影响eDNA的降解模式,但会影响eDNA的降解速率,这种影响不仅来自于单因素作用,同时还存在多因素的耦合作用。此外,在降解初期,草鱼eDNA拥有着较大的降解速率,但是随着时间的推移,降解速率逐渐减小。（2）生物膜存在会降低eDNA在水中的持久性,并且这种影响会随着生物膜发育水平的提升而加大。在生物膜的作用下,eDNA的降解速率常数（K₁）增大了9.13倍（最大幅度）,T₅₀和T₉₀相应缩短了7.43小时和24.67小时。（3）沉积物作用会因为表层生物膜的定植而发生变化。当生物膜未存在时,细颗粒沙粒可以直接影响水中eDNA的存在;当生物膜存在时,沉积物会与生物膜产生耦合作用,细颗粒沙粒凭借其相对较大的比表面积和平整的固液交界面使其表层生物膜中的细菌含量相对较高,从而促进草鱼eDNA降解。沙粒工况中eDNA的降解时间较砾石和卵石工况分别缩短了0.74～1.06小时(T₅₀)和2.46～3.52小时(T₉₀)。但是当生物膜发育到一定程度时,细颗粒沙粒中eDNA的降解时间与其他类型沉积物相比无明显差异。（4）水流条件会与生物膜产生显著的耦合作用。动水条件下水流的扰动作用可以使生物膜中的细菌进入水体,增大细菌的影响范围,进而显著加快了eDNA的降解速率。同时这种作用还会随着生物膜发育水平的提高而增大。动水条件下草鱼eDNA的降解速率常数K₁比静水条件和动静水组合条件增长了1.24～4.98倍,T₅₀和T₉₀较静水条件和动静水组合条件分别减少了3.13～4.64小时、10.4～15.4小时。此外,当水流条件在实验过程中发生变化后,草鱼eDNA在水中的再悬浮会受到eDNA的异质性分布与快速降解,eDNA和沉积物、生物膜之间的静电相互作用以及水中缺乏大颗粒的eDNA等诸多因素的影响,从而造成本研究未发现明显的eDNA再悬浮现象。更多还原

【Abstract】 The environmental DNA（eDNA）technology utilizes the genetic material released by organisms into the environment for monitoring aquatic species.For open,dynamic ecosystems such as rivers,a wide range of habitat factors can directly or indirectly affect the persistence of biological eDNA in water,thus affecting the application of eDNA technology.Hydropower development in mountainous rivers will cause changes in the composition of riverbed sediments,and the stability of riverbed sediments and the colonization of the biofilm on the surface of sediments will affect the activity track of aquatic organisms,and then affect the degradation,sedimentation,transport and re-suspension processes of biological eDNA in the river.Therefore,it is necessary to explore the influence characteristics of sediment and biofilm condition changes on eDNA persistence under different flow conditions,and the research results can provide theoretical support for the application of eDNA technology in aquatic environments.Grass carp were used as the target species in this study,and an established experimental design was applied.Three types of sediment（0.5-1 mm sand,4-6 mm gravel,and 20-40 mm pebble）,four biofilm growth stages（0,10,18,and 25 days of biofilm incubation）,three water flow conditions（static water condition,dynamic water condition,and combined dynamic and static water condition）were combined.Fluorescence quantitative PCR was utilized to identify variations in eDNA concentration of grass carp under various working conditions,and the main experimental findings and conclusions were as follows:（1）In the experiment,eDNA concentration of grass carp showed an exponential degradation pattern with time.The changes of sediment type,biofilm development level and water flow conditions did not affect the degradation mode of eDNA,but affected the degradation rate of eDNA.This effect was not only due to the single factor,but also the coupling effect of multiple factors.In addition,at the initial stage of degradation,eDNA of grass carp had a relatively high degradation rate,but with the passage of time,the degradation rate decreased gradually.（2）The presence of biofilm significantly decreased the persistence of eDNA in water,and this effect was enhanced by the increase in the biofilm development level.In the presence of biofilm,the degradation rate constant（K₁）was increased by a maximum of 9.13 times,while the T₅₀ and T₉₀ were shortened by 7.43 h and 24.67 h respectively.（3）The role of sediment was changed by the colonization of surface biofilm.When the biofilm did not exist,the fine-grained sediment could directly affect the presence of eDNA in water.When the biofilm existed,the sediment had a coupling effect with the biofilm.With the relatively large specific surface area and the flat solid-liquid interfacial interface of the small particle size sand,the bacteria content in biofilm was relatively high,thus promoting eDNA degradation.The corresponding eDNA degradation time in the sand condition was shortened by 0.74～1.06 h(T₅₀)and2.46～3.52 h(T₉₀)compared with the gravel and pebble condition,respectively.However,when the biofilm development level reached a certain level,the degradation time of eDNA in fine-grained sand particles was not significantly different compared with other sediment types.（4）The dynamic water condition had a significant coupling effect with biofilm.The disruption caused by flowing water could allow bacteria in biofilm to penetrate the water column and expand their influence,which in turn significantly accelerated the degradation rate of eDNA.Additionally,as the biofilm development level improved,this effect grew as well.When compared to static water conditions and combined static and dynamic water conditions,the degradation rate constants（K₁）under dynamic water condition increase by 1.24 to 4.98 times,T₅₀ and T₉₀ were reduced by 3.13～4.64 hours and 10.4～15.4 hours,respectively.In addition,when the water flow condition was changed during the experiment,the resuspension of grass carp eDNA in water would be affected by many factors,heterogeneous distribution and rapid degradation of eDNA,electrostatic interaction between eDNA and sediment and biofilm,and the lack of large particles of eDNA in water,which caused no significant eDNA resuspension in this study.更多还原