| Fenneropenaeus chinensis is one of the important fishery resources in the Yellow Sea and Bohai Sea.In the impact of human activities,the resource decline is very serious and the wild resources are almost exhausted.Since the end of the 1990s,the marine fishing production of Fenneropenaeus chinensis has almost completely relied on artificial discharge,but the research on the evaluation of the effects of proliferation and release has not yet matured.Therefore,accurately evaluating the effect of release is the fundamental premise for the government to formulate relevant release strategies.However,due to the special life history type of Fenneropenaeus chinensis,the traditional fishery resources survey method can not correctly reflect the temporal and spatial distribution of Fenneropenaeus chinensis in the Bohai Sea and the dynamic changes of population resources.At the same time,traditional fishery resources survey methods often consume a large amount of money.Time and money,and the results are not satisfactory.In recent years,environmental DNA(eDNA)technology has been applied to the research field of aquatic ecosystems as a new aquatic organism survey method.However,due to the difference between species,the amount of eDNA released is different from the size of the eDNA fragment,and the eDNA released by different species has different retention time in the water.Therefore,before applying the eDNA technology to the investigation of the resources of the Fenneropenaeus chinensis in the Bohai Sea,it is essential to explore the retention time of the eDNA released by Fenneropenaeus chinensis in the water and to establish an operational procedure that is most suitable for the Fenneropenaeus chinensis DNA technology.In this study,Fenneropenaeus chinensis was used as the research object,and the following four aspects were studied:(1)Real-time quantitative PCR was used to quantitatively analyze the degradation of eDNA released by Fenneropenaeus chinensis in water environment.Based on Akaike Information Criterion(AIC),the most suitable statistical model for eDNA degradation over time was selected.(2)Enriching eDNA by filter method,extracting eDNA by DNeasy Blood and Tissue kit,selecting a glass fiber membrane with a diameter of 47 mm,a nitrocellulose membrane,a polycarbonate membrane,and a nylon membrane.Membrane,each filter has 4 gradients of 0.45μm,0.8μm,1.2μm and5μm according to its pore size.The sampling water volume is set to three gradients of500 mL,1 L and 2 L.Based on this,the most suitable operation procedure for eDNA technology of Fenneropenaeus chinensis was established.(3)According to the size of biomass set up three different Fenneropenaeus chinensis breeding density gradient,150/m~3,respectively 300/m~3,666/m~3,on May 3,2018,at the same time,May 21,June 7,respectively,using the membrane filter technique enrichment eDNA,using real-time fluorescent quantitative PCR detection of the Fenneropenaeus chinensis released into the environment in different growth periods of the relationship between DNA content and biomass.(4)Based on water samples from 54 stations in June and 60 stations in August in Bohai Sea in 2017,2L sea water was filtered with 0.45 m glass fiber filter membrane combined with vacuum filter device for eDNA enrichment,and DNeasy Blood and Tissue kit was used for eDNA extraction.DNA extraction design after the completion of the Fenneropenaeus chinensis mitochondrial DNA COI gene specific primers and probes,application of real-time fluorescent quantitative PCR for the Bohai Sea eDNA do the qualitative and quantitative analysis,eDNA technology were used to detect the space-time distribution of Fenneropenaeus chinensis,explores the eDNA copy number and trawl survey of Fenneropenaeus chinensis,the relationship between the biomass of eDNA is verified the applicability of the technology in Marine crustaceans,and sensitivity.Experimental results show that:(1)After the release source of eDNA is removed,the copy number of eDNA of Fenneropenaeus chinensis in water is negatively correlated with time,and the retention time in water is about one month.(2)The material of the membrane,the pore size of the membrane and the volume of the sampled water has certain influence on the qualitative and quantitative analysis of Fenneropenaeus chinensis.The 0.45μm glass fiber membrane filtration 2L water sample can detect the highest number of DNA copies.Based on this,a set of operational procedures for Fenneropenaeus chinensis prion eDNA technology was established.(3)The biomass of Fenneropenaeus chinensis is positively correlated with the eDNA released into the water environment,and the amount of eDNA released into the water environment is different during different growth periods.(4)The mtDNA COI gene of Fenneropenaeus chinensis was successfully amplified in 54 water samples sampled in June,and the detection rate was 100%.Only 23 of the 60 stations sampled in August could detect Fenneropenaeus chinensis.The detection rate is only 38%.The copy number of eDNA obtained by real-time PCR was fitted to the density of Fenneropenaeus chinensis resources investigated by bottom trawl.There was no significant positive correlation between the two.In summary,eDNA technology is currently used as an aid to fishery resource surveys and can be used for qualitative research in marine ecosystems,but quantitative studies require further investigation due to some uncertainties.Through this study,we aim to explore a new Fenneropenaeus chinensis resource survey method,which provides a strong scientific basis for rational use and scientific release of Fenneropenaeus chinensis. |
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