Qualitative and quantitative detection using eDNA technology:A case study of Fenneropenaeus chinensis in the Bohai Sea

Mio Li,Xiujun Shn,b,*,Weiji Wng,b,Xiosong Ding,Fngqun Di,Ding Lv,Hunhun Wu

aKey Laboratory of Sustainable Development of Ma Fisheries,Ministry of Agriculture and Rural Affairs,Shandong Provincial Key Laboratory of Fishery Resources and Ecological Environment,Yellow Sea Fisheries Research Institute,Chinese Academy of Fishery Sciences,Qingdao,266071,China

bLaboratory for Marine Fisheries Science and Food Production Processes,Pilot National Laboratory for Marine Science and Technology(Qingdao),Qingdao,266237,China

ABSTRACT

Keywords:

Bohai Sea

eDNA

Fenneropenaeus chinensis

Spatial and temporal distribution

Biomass

1.Introduction

Global fisheries decline is one of the major challenges of the 21st century(Dudgeon,2010;Jin et al.,2015;Evans&Lamberti,2018).To achieve the sustainable development of marine fisheries,the development of sustainable fisheries management measures is necessary(FAO,2010).However,it is extremely difficult for researchers and policy makers to collect accurate data on the distributions of aquatic populations and population dynamics(Thomsen&Willerslev,2015),especially for certain populations with very low densities and unique life histories.At the same time,the distinct living environments of these species have increased the difficulty of research(Dejean et al.,2011;Mackenzie,Nichols,Sutton,Kawanishi,&Bailey,2005;Vermeulen and Koziell,2002).Therefore,exploring new approaches for surveys of aquatic life is necessary.

Recently,with the rapid development of molecular biology,environmental DNA(eDNA)technology has been successfully applied as a new aquatic organism survey method for research in aquatic ecosystems(Shan,Li,&Wang,2018).Environmental DNA refers to free DNA molecules released from the skin,mucus,saliva,sperm,secretions,eggs,feces,urine,blood,roots,leaves,fruits,pollen and rotten bodies(Bohmann et al.,2014;Shan et al.,2018).There are four main steps in the analysis of eDNA in water samples:collection and preservation of water samples,extraction of DNA in water samples,detection of eDNA and analysis of results(Shan et al.,2018).Currently,this technology has been successfully applied to mammals(Foote et al.,2012;Thomsenet al.,2012;Williams,Huyvaert,Vercauteren,Davis,&Piaggio,2018),amphibians(Evans&Lamberti,2018;Goldberg,Strickler,&Fremier,2018;Hall,Goldberg,Brunner,&Crespi,2018),fish(Mélanie,Valérie,Mandrak,&Nellie,2017;Rodgers et al.,2018;Yamanaka,Takao,Maruyama,&Imamura,2018),invertebrates(Deiner&Altermatt,2014;Thomsen et al.,2012;Tréguier et al.,2014)and other aquatic organisms in biological studies.At the same time,the habitat types that are applicable to eDNA technology are diverse,including ponds(Dejean et al.,2012;Takahara,Minamoto,&Doi,2013),rivers(Deiner,Fronhofer,Machler,Walser,&Altermatt,2016;Shaw et al.,2016),lakes(Balasingham,Walter,Mandrak,&Heath,2017;Larson et al.,2017),and oceans(Sigsgaard et al.,2017;Weltz,Lyle,Ovenden,Morgan,&Semmens,2017).The application of eDNA technology infishery resource surveys can detect species,evaluate biodiversity and assess population abundance.Additionally,eDNA has the advantages of convenient sampling,high sensitivity,and economic efficiency,and compared with traditional resource survey methods,this technology causes no harm to ecosystems(Jerde,Mahon,Chadderton,&Lodge,2011;Dejean et al.,2012;Xiong,Li,&Zhan,2016).However,there are still some problems in the application of eDNA in the study of aquatic systems,such as the mechanism that affects the release and degradation of eDNA,and the distance that eDNA can migrate with water in the sea.In addition,previous studies on the application of eDNA technology in aquatic ecosystems have mainly focused on freshwater ecosystems,and,to some extent,the larger size and more complex hydrological conditions of marine ecosystems increase the difficulty of using this method.Moreover,few researchers have studied crustaceans with exoskeletons(Dunn,Priestley,Herraiz,Arnold,&Savolainen,2017;Forsstrom&Vasemagi,2016;Rice,Larson,&Taylor,2018)as the research subject.Therefore,the applicability and sensitivity of eDNA technology in marine crustaceans need further study.

Table 1Primer pairs for the mtDNA COI of F.chinensis.

Fenneropenaeus chinensis(Chinese shrimp)is an important fishery resource in China.Due to the impact of human activities,this resource has seriously declined,and wild resources are nearly exhausted.Since the 1980s,stock enhancement have occurred to restore the number of F.chinensis.By the end of the 1990s,the F.chinensis in China were almost completely dependent on stock enhancement(Liu,Li,He,Kong,&Wang,2004).Therefore,accurately evaluating the effect of stock enhancement of F.chinensis in the Bohai Sea is very important for formulating appropriate release strategies.However,evaluating the resource status of F.chinensis is the basis for accurately evaluating the effects of stock enhancement of F.chinensis to understand the spatial and temporal distribution of F.chinensis in the Bohai Sea and the dynamic change in the population abundance.However,the existingfishery resource survey methods,which are time consuming and laborious,are not effective for assessing F.chinensis populations,and these methods often cannot accurately reflect the population dynamics and spatial and temporal distribution of F.chinensis(Wang et al.,2016).Therefore,it is important to explore a method that is more suitable for the investigation of F.chinensis.

For the above reasons,the main purpose of this study is to explore the applicability of eDNA technology in the investigation of Chinese shrimp resources.In this study,F.chinensis was studied in the Bohai Sea.The applicability and sensitivity of e DNA technology in marine crustacean research were explored.Specific primers and probes for the mitochondrial DNA COI gene of F.chinensis were designed.Real-time quantitative PCR was applied.A qualitative and quantitative analysis of F.chinensis was conducted in the Bohai Sea.(1)Using eDNA technology,the spatial and temporal distribution of F.chinensis was detected in the Bohai Sea in June and August 2017.(2)The relationship between the eDNA copy number and biomass of F.chinensis caught by trawling was investigated.Through this research,the scientific problem of lack of spatial distribution data of Chinese shrimp in the early stage of value-added release was solved.In addition,this study explored the applicability of eDNA technology in the assessment of Chinese shrimp biomass for the first time.This study is intended to provide technical support for the rapid and accurate assessment of aquatic animal resources.

2.Materials and methods

2.1.Design of the specific primers and probes

Specific primers were designed for the mitochondrial cytochrome oxidative subunit I(COI)gene of F.chinensis.The COI gene sequences of F.chinensis were retrieved from the GenBank database(GenBank Accession number:gb|HQ700930.1),sequence alignment was performed using Bio Edit(Hall,1999)and MEGA6(Tamura,2013)software,primers and probes were designed using Primer Premier 6 and Beacon Designer 8 software,and primer specificity was tested on the National Center for Biotechnology Information(NCBI)website.The primers were synthesized by Sangon Bioengineering(Shanghai)Co.,Ltd.The relevant details are shown in Table 1.

2.2.DNA extraction,PCR amplification and product sequencing of muscle tissue of F.chinensis

F.chinensis muscle tissue culture was taken from F.chinensis collected from the Bohai Sea fishery resources survey in July 2016 and stored at-20°C.DNA extraction was carried out by the traditional phenol-chloroform-isoamyl alcohol method.The purity and concentration of the extracted genomic DNA solution of Chinese shrimp were detected by an ultramicro UV spectrophotometer.Finally,the DNA stock solution was diluted to 50 ng/μL and stored in a 1.5 ml centrifuge tube at-20°C.

The barcode region of the mitochondrial COI gene was amplified using the F.chinensis-specific primer pairs COI PF/COI PR and COI DF/COI DR.A 25 μL PCR system was used:10 × Taq buffer 2.5 μL,dNTPs(2.5 mM each)0.5 μL,positive and negative primers(COI PF/COI PR)(10 mM)0.5 μL each,Taq DNA polymerase(5 U)/μL)0.5 μL,template DNA(50 ng/μL)1 μL,MgCl2(25 mM)1.5 μL,and ddH2O 18 μL.The PCR procedure was as follows:predenaturation at 94°C for 3 min;denaturation at 94 °C for 30 s,annealing at 60 °C for 30 s,and extension at 72 °C for 1 min,35 cycles;and 72 °C complex extension for 10 min.The PCR product was electrophoretically separated by a 2%agarose gel stained with a gene finder nucleic acid dye,and the fragment size of the PCR product was determined using a 2000 bp DNA ladder.

Six positive amplification products(including three pairs of COI PF/COI PR primers and three pairs of COI DF/COI DR primers)were randomly selected and sent to Sangon Biological Engineering(Shanghai)Co.,Ltd.For sequencing to obtain the mtDNA nucleotide sequences of F.chinensis.

2.3.Preparation of the recombinant plasmid DNA standards

The PCR product in 2.2 was purified,ligated into a pMD-18-T plasmid vector,transformed into E.coli competent cells,and cultured overnight on an LB solid plate medium,and a single colony expanded culture was chosen for plasmid DNA extraction.The plasmid DNA was diluted to a specific concentration and stored at-80°C until use.

2.3.1.Purification of the PCR products

The PCR product in 2.1 was detected by 2%agarose gel electrophoresis,and the single and bright product of the electrophoresis strip was selected for gel extraction using a gel extraction kit(OMEGA).The purified PCR product was stored at-20°C.The specific experimental steps refer to the instructions.

2.3.2.Plasmid ligation transformation

The purified PCR product was ligated into the pMD-18-T plasmid vector(TaKaRa).Ten microliters of the ligation product was added to 100 μL of DH5α competent cells(TaKaRa)and placed in ice for 30 min.The recombinant plasmid was transformed into competent cells by heat shock at 42°C for 1 min.One milliliter of SOC medium was added and shaken at 37 °C for 150 h at 150 rpm.One hundred and 50 μL of the bacterial solution was applied to LB solid medium(containing Amp,X-gal,and IPTG)and inverted overnight at 37°C.Six white single colonies were chosen and placed in 1.5 mL centrifuge tubes,and the bacteria were sent to Bioengineering(Shanghai)Co.,Ltd.For sequencing.The successful bacterial culture was expanded.

2.3.3.Preparation of the plasmid standard DNA

A plasmid mini kit(OMEGA)was used for the plasmid DNA extraction.After extraction,the DNA concentration was measured by an ultramicro UV spectrophotometer,diluted to 108copies/μL and stored at-80°C.

2.4.Study area and water sample collection

The Bohai Sea is the spawning and feeding grounds for F.chinensis,and the area is also important for their stock enhancement.Since the 1980s,wild F.chinensis have been on the verge of extinction.To restore F.chinensis,China has been conducting stock enhancement of F.chinensis since 1984,and the number released is substantial.In 1993,a large number of cases of white spot disease in F.chinensis occurred.Since then,the release of F.chinensis has been decreasing,and in recent years,the release of F.chinensis has increased once again(Ge et al.,2018;Zhang et al.,2009).

A total of 60 sampling stations were established throughout the Bohai Sea(Fig.1).Water sample collection in the field occurred during the maritime resource surveys in the Bohai Sea.The two voyages occurred June 8-June 17 and August 9-August 18,2017.The sampling method,sampling device and filtering device used during the two voyages were the same.At each sampling point,2 L of surface water(0-1 m depth)was taken from each sampling point with a glass water sampler,and 3 technical repeats were made at each sampling stations;negative controls were made at every 10 sampling stations(2 L of distilled water was filtered for each sample,and 3 technical repeats were made).Immediately after the water sample collection,the samples were filtered with a vacuum pump and a GF/F glass fiber filter with a diameter of 47 mm and a pore size of 0.45 μm(Li et al.,2019).After thefiltration was completed,each filter was individually folded and wrapped in aluminum foil(Takahara,Minamoto,Yamanaka,Doi,&Kawabata,2012)and stored at-20°C in the dark until the DNA was extracted in the laboratory.The glass water collector and all filtration devices were bleached with 10%bleach before sampling and washed with distilled water(ultrapurified deionized filtration to reduce the DNA),and all devices were sterilized with UV light for 30 min before use.To preventcross-contamination,allfiltration deviceswere carefully rinsed with distilled water after each filtration,and the eDNA extraction and PCR amplification were performed in different laboratories during laboratory experiments.

2.5.Extraction of eDNA

The extraction of eDNA was carried out using the DNeasy Blood and Tissue kit(Qiagen GmbH,Hilden,Germany)with reference to the method of Renshaw et al.(Renshaw,Olds,Jerde,Mcveigh,&Lodge,2015).The specific steps were as follows.(1)After the filter was removed and thawed,the filter was cut into thin strips with a small pair of scissors and placed in a 2 mL centrifuge tube;570 μL of ATL buffer and 60 μL of proteinase K were added,vortexed and mixed,and heated at 65 °C for 3 h(2)The sample was vortexed for 15 s,and 630 μL of AL buffer was added to the sample and mixed thoroughly by vortexing.Then,630 μl of ethanol(96%-100%)was added,and the mixture was mixed thoroughly by vortexing.(3)The mixture from step 2(including any precipitate)was pipetted into the DNeasy mini spin column and placed in a 2 mL collection tube(provided).The sample was centrifuged at≥6000 g(8000 rpm)for 1 min.The flow-through and collection tubes were discarded.(4)The spin column was placed into a new collection tube,and 500 μL of AW1 buffer was added.The sample was centrifuged at 8000 g for 1 min at room temperature,and the filtrate and collection tube were discarded.(5)The spin column was placed into a new collection tube,and 500 μL of AW2 buffer was added.The sample was centrifuged at 12,000 g for 3 min at room temperature,and the filtrate and collection tube were discarded.(6)The spin column was placed in a new 1.5 mL centrifuge tube,and 30 μL of TE buffer was added in the middle of the spin column(instead of the AE buffer in the kit).The mixture was incubated for 1 min at room temperature and then centrifuged at 8000 g for 1 min at room temperature.(7)Step(6)was repeated once to increase the DNA yield.Three negative controls(extraction blanks)were set up each time the eDNA was extracted,and the extraction method was consistent with the field environment samples.

2.6.Real-time fluorescence quantitative PCR

Quantitative analysis was performed using a 2×TaqMan Fast qPCR Master Mix(Low Rox)real-time PCR kit from BBI Life Sciences.An Applied Biosystems model 7500 quantitative PCR machine and a 96-well plate(ThermoFisher)were used.Three positive controls(genomic DNA of F.chinensis)and three negative controls(no template control:ultrapure water)were placed in each 96-well plate.Plasmid DNA was diluted from 108copies/μL in a 10-fold concentration gradient to 102copies/μL as a standard for the quantitative PCR.A 20 μL system was used for the PCR:10 μL 2×TaqMan Fast qPCR Master Mix,0.4 μL forward primer(10 μmol/L),0.4 μL reverse primer(10 μmol/L),0.4 μL probe(10 μmol/L),2 μL of template DNA,and 6.8 μL of PCR-grade water.The amplification reaction procedure was as follows:predenaturation at 94°C for 3 min and then 40 cycles of denaturation at 94 °C for 5 s and annealing at 60 °C for 34 s.The experimental data were analyzed by the absolute quantitative method.

2.7.Statistical analysis

All data were analyzed using R3.5.0,and the error was controlled within a 95%confidence interval.

3.Results

3.1.Specificity of the primers and probes

Fig.1.Map of the sampling stations.The sampling stations in June 2017 were the same as those in August 2017.

Fig.2.Amplification of the primers COI PF/PR and COI DF/DR.M stands for DNA marker DL 2000,lanes 1-3 were the PCR products of COI PF/PR,and lanes 4-6 were the PCR products of COI DF/DR.

The PCR product was detected by 2%agarose gel electrophoresis.The results showed that the primers designed in this experiment successfully amplified the specific target fragments of 597 bp and 106 bp,which were completely consistent with the expected results.Primer specificity was verified by PCR amplification and agarose gel electrophoresis of the muscle tissue DNA of F.chinensis with single and bright target bands(Fig.2).At the same time,with reference to the NCBI database,the sequencing results were aligned using Blast(http://blast.ncbi.nlm.nih.gov/Blast);the results showed that the sequence of PCR amplification product had 100%homology with the mtDNA COI gene(GenBank Accession number:gb|HQ700930.1)and indicated that the designed primer had strong specificity.

3.2.Establishment of the standard curve of qPCR amplification of the COI gene of F.chinensis and determining the regression equation

Through real-time quantitative PCR amplification,the system automatically generated a standard curve and amplification curve of the mtDNA COI gene of F.chinensis according to a change in the fluorescence value(Fig.3).The correlation coefficient,R2,of the curve was 0.999,and the regression equation was y=-3.4 x+38.This result shows that there is a good linear relationship within the range of diluted plasmid standards.The established standard curve correctly reflected the amplification of the mtDNA COI gene of F.chinensis.

Fig.3.The standard curve of qPCR of the F.chinensis COI gene.The x axis represents the logarithm of the DNA concentration of the plasmid,and the y axis represents the threshold cycle value of the qPCR.

3.3.Detection of eDNA

The purity and concentration of the extracted genomic DNA of F.chinensis were high,and the target strips of the PCR products were single(Fig.2).

Fig.4.Detection of eDNA by agarose gel electrophoresis.The agarose gel electrophoresis results of the eDNA samples from June 2017 are on the left,and the agarose gel electrophoresis results of the eDNA samples from August 2017 are on the right;M stands for marker DL 2000.

After the water sample eDNA extraction was completed,the eDNA was detected by an ultramicro UV spectrophotometer.The results showed the following.(1)The minimum eDNA concentration in June was 4.5 ng/μL,and the maximum value was 351.75 ng/μL(2)The minimum eDNA concentration in August was 1.3 ng/μL,and the maximum value was 359.5 ng/μL(3)The A260/A280 values of most eDNA samples in the two months ranged from 1.8 to 2.0,indicating that the extracted eDNA was of a relatively higher quality,while only a small portion of the extracted eDNA samples were of a relatively lower quality;this result was mainly due to the low concentration of DNA in the water around the sampling site and the inclusion of various impurities,such as humus.(4)The eDNA was a mixture of the DNA from all the organisms around the sampling site.Due to the inconsistent sizes of DNA fragments of different organisms,the bands of eDNA will show a serious towing phenomenon when agarose gel electrophoresis is used to detect the eDNA(Fig.4).

3.4.Comparison between eDNA technology and traditional resource survey methods

According to the results of real-time quantitative PCR(Fig.5),in June 2017,54 of the sites sampled were able to successfully amplify the mtDNA COI gene of F.chinensis,and the detection rate reached 100%.However,only approximately 23 of the 60 sites sampled in August 2017 were able to detect F.chinensis in the waters around the site,and the detection rate was only 38%.According to the results of the bottom trawl surveys(Fig.5),no Chinese shrimp was captured in June 2017,and only 11 sampling stations were able to capture Chinese shrimp in August 2017.Compared with traditional bottom trawl surveys,eDNA technology has a very high sensitivity and can be used to study the temporal and spatial distribution of F.chinensis.

Using the biomass data of F.chinensis collected from the bottom trawling survey in August 2017 and the DNA copy number obtained by real-time fluorescence quantitative PCR of the eDNA samples in August 2017,there was no significant linear correlation(R2=0.26)between the copy number of eDNA and the biomass obtained from bottom trawling(Fig.6).

In addition,the two fishery resources survey lasted 10 days at sea,costing at least 10,000 yuan per day,and required at least 5 staffmembers.After returning to land,it took about 1 week for sample processing.The collection of eDNA samples requires only one staffmember,and the sample processing for two months costs less than 10,000 yuan,and the time required to process the samples also takes only three days.From this point of view,eDNA technology saves time and effort and costs compared to traditional survey methods.

4.Discussion

4.1.Applicability of eDNA technology in studies with different species and the factors affecting the detection rate of eDNA

Different species have different life history characteristics,and therefore,the amount of DNA released by different species into the environment and the eDNA released by them are different in water(Geerts,Boets,Stef,Goethals,&Christine,2018).Therefore,using eDNA technology,when different species are tested,the detection rates of the species differ.When biomonitoring using eDNA technology,a biological group that is in direct contact with a water body will have a higher detection rate than a biological group with an exoskeleton(Forsstrom&Vasemagi,2016).Studies have shown that the detection rate of fish and amphibians using eDNA technology was almost 100%;the detection rate was only 82%for dragonflies(Thomsen et al.,2012)and was even lower for crustaceans(59%in crayfish and 57%in crabs)(Tréguier et al.,2014;Forsstrom&Vasemagi,2016).In addition,Machler,Deiner,Steinmann,&Altermatt(2014)found that in the detection of Asellus aquaticus,Crangonyx pseudogracilis,Gammarus pulex,Tinodes waeneri,etc.,the traditional survey method was better than the use of eDNA technology(Machler et al.,2014).The differences in the detection rates of eDNA mentioned above are mainly due to species differences(such as differences in life history type,target species resources,etc.)and environmental factors,such as temperature,pH,ultraviolet light,and hydrological conditions(Atsushi et al.,2014;Kelly,Port,Yamahara,&Crowder,2014;Pilliod,Goldberg,Arkle,&Waits,2014).In addition,the detection rate of eDNA is also affected by subjective factors,including the collection of eDNA water samples and the enrichment,extraction,and analysis of eDNA(Deiner,Walser,Machler,&Altermatt,2015).

Fig.5.Detection of the distribution of F.chinensis in the Bohai Sea based on eDNA technology and bottom trawl surveys.The detection of the spatiotemporal distribution of F.chinensis in the Bohai Sea in June 2017 by eDNA technology is on the left,and the detection in August 2017 is on the right.The circles in the figure represent the sampling points with positive qPCR results.

Fig.6.The relationship between the copy number of eDNA and the biomass from trawl survey.

In this study,the mtDNA COI gene of F.chinensis was successfully amplified in all the water samples taken at 54 stations in June 2017,with a detection rate as high as 100%,while only 23 of the water samples taken at 60 stations in August 2017 could detect F.chinensis,with a detection rate of only 38%.With the growth of Chinese shrimp,the detection rate of eDNA of the Chinese shrimp was very different between the two months;this finding indicates that the detection rate of the same species was different in different growth periods when eDNA technology was used as the detection method.The difference in detection rate may be caused by the following reasons.(1)The eDNA release rate of the same species in different growth periods is not the same(Atsushi et al.,2014),and the time between molting of F.chinensis is continuously extended with increasing individual growth(Deng,Ye,&Liu,1990).As a result,F.chinensis shed their exoskeletons more often in June than in August,resulting in more DNA being released into the water environment in June than in August.Therefore,there will be different detection rates in different stages.(2)The unique life history characteristics of F.chinensis are also one of the reasons for the difference in detection rates between the two months.F.chinensis is a large migratory shrimp that has different distributions in the Bohai Sea during different growth periods(Deng et al.,1990).In June and July each year,F.chinensis feed in shallow coastal waters,and during this period,its swimming ability is weak.Under the action of currents,the shrimp will also be distributed in the sea far away from the coast,and during that time,the detection rate can be as high as 100%.At the beginning of August each year,the shrimp group and begin to move from shallow water areas to water areas approximately 10 m deep(Deng et al.,1990),and by the end of August each year,the body lengths of the shrimp can reach 80-100 mm;at this size,the shrimp are fully capable of swimming,and the possibility of movement by currents is very low.The distribution was relatively more concentrated in somefixed areas;therefore,there were no F.chinensis in the survey sites in the shallow coastal waters,and as a result,the detection rate of eDNA was much lower in August than in June.(3)The natural death of F.chinensis is also the cause of the difference in the detection rate between the two months.Currently,F.chinensis are mainly derived from stock enhancement.However,the annual release from the beginning of May to August was not supplemented by additional resources,and the densities of F.chinensis were theoretically decreasing.This decrease could have resulted in a higher detection rate in June than in August.Related research shows that the detection rate of eDNA increases with the increase of population resources(Pilliod et al.,2014).(4)Changes in environmental factors could also have led to the differences in detection rates between the two months.The temperature of the sea surface was higher in August than in June;the higher the sea temperature is,the more active the microbes in the ocean will be,and the rate of degradation of eDNA will increase(Eichmiller,Sendréa,&Sorensen,2016).As a result,the eDNA released by F.chinensis in August remained in the seawater for a shorter period of time than that in June,resulting in a difference in the detection rate.In addition to the above objective factors,human factors also affect the detection rate of eDNA.However,the sampling method,sample treatment method and subsequent analyses in the two months in this study were the same,and the eDNA processing methods used in this study were optimized.Therefore,the influence of human and subjective factors on the detection rate difference between the two months in this study can be ignored.

4.2.Application of eDNA technology to assess the uncertainty of the biomass of F.chinensis in the Bohai Sea

In this study,the relationship between the copy number of eDNA obtained by real-time fluorescence quantitative PCR and the biomass of F.chinensis obtained by bottom trawling were analyzed.There was not a positive correlation between the copy number of eDNA and the biomass,and this finding was different from the results of studies in artificial and freshwater ecosystems.When the presence of F.chinensis was detected with bottom trawling and the eDNA technique,the results of quantitative analysis in this study indicated the following three scenarios:(1)the copy number of eDNA was positively correlated with the biomass of F.chinensis;(2)the copy number of eDNA was very high,but the number of F.chinensis detected by trawling was very small;and(3)the copy number of eDNA was very low,but the number of F.chinensis detected by trawling was high.The first scenario is optimal,but in the study of marine ecosystems,the second and third scenarios are more likely.This result is mainly due to the complex environmental conditions of marine ecosystems,which are full of uncertainties.Various physical,chemical and biological processes have an impact on the production,degradation and migration of eDNA(Hansen,Bekkevold,Clausen,&Nielsen,2018),making it difficult for researchers to establish relationships between target species biomass and the copy number of the DNA these species release.

With regard to how external factors affect the production of eDNA,current research suggests that the interaction between biological and abiotic factors can have some potential effects on organisms,such as behavior,feeding,health,metabolism,and the male to female ratio(Kelly et al.,2014;Pilliod,Goldberg,Arkle,&Waits,2013),resulting in a change in the rate at which organisms release eDNA.Takahara et al.believe that temperature changes do not affect the rate at which organisms release eDNA(Takahara et al.,2012),though Lacoursière Roussel et al.(2016)suggest that temperature changes have an impact on the accuracy of species biomass assessments(Lacoursière Roussel et al.,2016).There are many disputes concerning this aspect of research that need to be resolved in the future.

The uncertainty of the mechanism by which eDNA degrades is also one of the important reasons that impedes the accurate assessment of biomass.At the moment eDNA is released into the environment by an organism,it begins to degrade,and the eDNA retention time in water is between one week and one month.Different organisms have different eDNA retention times in water(Dejean et al.,2012;Goldberg,Sepulveda,Ray,Baumgardt,&Waits,2013;Thomsen et al.,2012),and the main factors affecting eDNA degradation are environmental factors such as temperature,pH and light.Temperature is currently considered to be the most important factor affecting eDNA degradation.Strickler,Fremier,and Goldberg(2015)studied the degradation of eDNA released into the water by American bullfrog,Lithobates catesbeianus,at 5 °C,25 °C,and 30 °C.The authors found that the amount of eDNA in the environment was the same when the temperature was 5°C.The degradation of eDNA is the slowest at this temperature(Strickler et al.,2015),confirming that low temperatures can slow the rate of degradation of eDNA.There is some controversy about how pH and light are degraded by eDNA.Some studies suggest that the stronger the light is,the faster the degradation rate of eDNA.Other authors determined thatillumination had no effecton thedegradation ofeDNA(Andruszkiewicz et al.,2017;Pilliod et al.,2014);Strickler et al.,2015).Many researchers have explored how a single environmental factor affects eDNA degradation;however,in marine ecosystems,several of the environmental factors previously mentioned do not act solely on the degradation of eDNA but rather affect the eDNA itself.There is no relevant research on the antagonism or promotion of eDNA degradation by several environmental factors.Therefore,it is important to resolve the abovementioned problems before the relationship between the copy number of eDNA and biomass can be accurately established.

The migration of eDNA with water flow also has an impact on the assessment of biomass.Deiner et al.(2014)examined the eDNA released by two invertebrates,Daphnia longispina and Unio tumidus,living in lakes.In the rivers connected to the lake,sampling points were chosen based on distance,and the distance between the river and the lake was determined.The lower the copy number of eDNA detected by the macro was,the more the target eDNA was no longer available at a distance of 9.1 km from the lake(Deiner&Altermatt,2014).At the same time,Jane,Wilcox,Mckelvey,Young,and Whiteley(2014)showed that the greater the flow rate of water in a river was,the faster the rate of degradation of eDNA(Jane et al.,2014).There is no relevant research on the distance of eDNA transmission in marine ecosystems,although some scholars speculate that eDNA can move 600 km in ocean water in one week(Hansen et al.,2018);however,the marine environment is complex,and different sea states may cause eDNA to have different degradation rates.In addition,the direction of water flow in marine ecosystems is variable,and the migration of eDNA with waterflow is also multidirectional.This aspect is one of the reasons leading to the occurrence of false positive or false negative results.Therefore,future research on the distance of eDNA transmission in marine ecosystems is also worthy of attention.

5.Conclusion

Through this study,the author explored the applicability and sensitivity of eDNA technology for assessing the spatial and temporal distribution and biomass of marine crustaceans and successfully amplified the mtDNA COI gene of F.chinensis from the eDNA in water samples in the Bohai Sea for the first time.Correlation analysis was carried out on the spatial and temporal distribution and biomass assessment of F.chinensis in the Bohai Sea.The research results show that compared with the traditional trawl survey method,eDNA technology saves time and effort,is cost-effective,and requires few inputs from sampling personnel.At the same time,the method has a very high sensitivity and can be used for monitoring the temporal and spatial distribution of F.chinensis.However,there is a certain difficulty in the application of eDNA technology in the evaluation of F.chinensis biomass.There was not a good positive correlation between the copy number of eDNA and the biomass of F.chinensis obtained from the bottom trawl survey.The application of assessment also requires in-depth research.

Acknowledgments

This work was supported by the National Key R&D Program of China(2017YFE0104400),the National Basic Research Program of China(2015CB453303),Special Funds for Taishan Scholar Project of Shandong Province,and the Aoshan Talents Cultivation Program supported by the Pilot National Laboratory for Marine Science and Technology(Qingdao)(2017ASTCP-ES07).