methods and software · 2020-02-04 · 1 | introduction elasmobranchs (sharks, skates, and rays)...
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Environmental DNA. 2019;00:1–10. | 1wileyonlinelibrary.com/journal/edn3
Received:24February2019 | Revised:15August2019 | Accepted:12September2019DOI: 10.1002/edn3.39
M E T H O D S A N D S O F T W A R E
Development of highly sensitive environmental DNA methods for the detection of Bull Sharks, Carcharhinus leucas (Müller and Henle, 1839), using Droplet Digital™ PCR
Katherine E. Schweiss1 | Ryan N. Lehman1 | J. Marcus Drymon2 | Nicole M. Phillips1
ThisisanopenaccessarticleunderthetermsoftheCreativeCommonsAttributionLicense,whichpermitsuse,distributionandreproductioninanymedium,providedtheoriginalworkisproperlycited.©2019TheAuthors.Environmental DNApublishedbyJohnWiley&SonsLtd
1SchoolofBiological,Environmental,andEarthSciences,TheUniversityofSouthernMississippi,Hattiesburg,Mississippi2CoastalResearchandExtensionCenter,MississippiStateUniversity,Biloxi,Mississippi
CorrespondenceKatherineE.Schweiss,SchoolofBiological,Environmental,andEarthSciences,TheUniversityofSouthernMississippi,Hattiesburg,Mississippi39406.Email:[email protected]
Funding informationUniversityofSouthernMississippi;InstitutionalDevelopmentAwardNationalInstitutesofGeneralMedicalSciencesoftheNIH,Grant/AwardNumber:P20‐GM103476
AbstractBackground: Asapexandmesopredators,elasmobranchsplayacrucialroleinmain‐tainingecosystemfunctionandbalanceinmarinesystems.Elasmobranchpopulationsworldwideareindeclineasaresultofexploitationviadirectandindirectfisheriesmortalitiesandhabitatdegradation;however,alackofinformationondistribution,abundance,andpopulationbiologyformostspecieshinderstheireffectivemanage‐ment.EnvironmentalDNAanalysishasemergedasacost‐effectiveandnon‐invasivetechniquetofillsomeofthesedatagaps,butoftenrequiresthedevelopmentofspe‐cies‐specificmethodologies.Aims: Here,weestablishedeDNAmethodologyappropriatefortargetedspeciesde‐tectionsofBullSharks,Carcharhinus leucas,inestuarinewatersinthenorthernGulfofMexico.Materials and Methods: WecompareddifferentQIAGEN®DNeasy® extractionkitprotocolsanddevelopedaspecies‐specificDropletDigital™PCR(ddPCR)assaybydesigningprimersandaninternalprobetoamplifya237basepairportionoftheND2geneinthemitochondrialgenomeofC. leucas.Tovalidatethedevelopedmethods,watersampleswerecollectedfromknownC. leucashabitatandfromanexsituclosedenvironmentcontainingasingleC. leucasindividual.TheeffectivenessoftheassayinanopenenvironmentwasthenassessedbyplacingoneC. leucasintoaflow‐throughmesocosmsystemandwatersampleswerecollectedevery30minfor3hr.Results: ThedevelopedC. leucas‐specificassayhastheabilitytodetecttargetDNAconcentrationsinareactionaslowas0.6copies/μl.DdPCRreactionsperformedonwatersamplesfromknownhabitatand30minafterasharkwasaddedtotheclosedenvironmentcontained1.62copies/μland166.6copies/μloftargetC. leucas eDNA,respectively.Carcharhinus leucas eDNAwas detected in the flow‐through systemwithin30min,butconcentrationsremainedlowandvariablethroughoutthedura‐tionoftheexperiment.
K E Y W O R D S
elasmobranch,GulfofMexico,habitatuse,mitochondrialgenome,threatenedspecies,watersample
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1 | INTRODUC TION
Elasmobranchs (sharks, skates, and rays) play a crucial role inma‐rineecosystemsasapexandmesopredators,influencingpreyabun‐dance, behavior, and trophic interactions across multiple trophiclevels inmarinefoodwebs (Ferretti,Worm,Britten,Heithaus,andLotze2010;Ritchieetal.2012).Healthyelasmobranchpopulationshelptomaintainecosystemfunction,increasebiodiversity,andbuf‐feragainstinvasivespeciesandtransmissionofdiseases(Heithaus,Frid,Wirsing,andWorm2008;Ritchieetal.2012).However,manyelasmobranchpopulationsare indeclineasaresultofexploitationviadirectandindirectfisheriesmortalitiesandhabitatdegradation(Dulvyetal.2014).Thelifehistorystrategiesofmanyelasmobranchsare characterized by late maturity, longevity, and low fecundity,making the recovery of exploited populations a biologically slowprocess (Garcia et al., 2008;Hoenig andGruber1990).Accordingto the InternationalUnion forConservationofNature (IUCN)RedListofThreatenedSpecies,one‐quarterofelasmobranchspeciesareestimatedtobethreatenedwithextinctionandalmostone‐halfarecategorizedasDataDeficient,meaningthereareinsufficientdatatoproperlyassesstheirconservationstatus(Dulvyetal.2014).Robustdata on species distribution, abundance, biology, and populationbiologyarenecessarytoenactappropriateconservationstrategiesforthemaintenanceofhealthyelasmobranchpopulations;unfortu‐nately,suchdataareoften incompleteor lackingformanyspecies(Dulvyetal.2014).
Analysis of environmental DNA (eDNA) has recently emergedasanalternative,powerfulapproachtofilldatagapsonthedistri‐bution, habitat use, abundance, andpopulationbiologyof aquaticspecies (Ficetola,Miaud,Pompanon,andTaberlet2008), includingelasmobranchs(Sigsgaardetal.2016).AllorganismsleavetracesofDNA in the environment through sheddingof cellular debris, skincells, blood, and biologicalwaste, all ofwhich can be collected inwatersamples (Rees,Maddison,Middleditch,Patmore,andGough2014); however, differences in how organisms shed DNA (i.e.,mucus, scales, feces) suggest that eDNA accumulationmay differacross species (Le Port, Bakker, Cooper, Huerlimann, andMariani2018), requiring taxon‐specific research. In targeted species de‐tections,watersamplesaretypically filtered,DNAextractionsareperformedontheresultingparticulatematerial,andextractedDNAsamples are analyzed using a quantitative real‐time polymerasechain reaction (qRT‐PCR) platform with species‐specific primers,developed to amplify a smallDNA fragment in the target species(Foote et al. 2012; Taberlet, Coissac, Hajibabaei, and Rieseberg2012). The collection ofwater samples is a cost‐effective and ef‐ficientmethodofsurveyingelasmobranchpopulationswhencom‐paredtotraditionalsurveymethodsinvolvingsettingnetsorlines,whichcanhavehighincidenceofbycatchandinflictvaryingdegreesofstress tobothtargetandnontargetspecies (Larsonetal.2017;Lewison,Crowder,Read,andFreeman2004).Post‐releaserecoveryandsurvivaltendstovarywidelyacrossspecies,withsomespeciesbeingparticularlysensitivetonetcaptureandhandling(Stobutzki,Millter,Heales, andBrewer2002).With awell‐designed sampling
scheme,eDNAmethodologiesofferincreasedsensitivityfordetect‐ingthepresenceofrarespecieswhilenegatingtheneedtocapture,handle,orevenobservethetargetspecies(Portetal.2016;Reesetal.2014).Inelasmobranchs,eDNAmethodshavebeenusedintar‐geted speciesdetections for theCriticallyEndangeredLargetoothSawfish,Pristis pristis (Simpfendorfer et al. 2016), theEndangeredMaugeanSkate,Zearaja maugeana(Weltzetal.2017),theVulnerableChileanDevilRay,Mobula tarapacana (Garganetal.2017),andtheVulnerable Great White Shark, Carcharodon carcharias (Lafferty,Benesh,Mahon, Jerde, and Lowe 2018). Furthermore, eDNA hasbeen used to assess population characteristics in the EndangeredWhaleshark,Rhincodon typus(Sigsgaardetal.2016)andtoestimatesharkdiversityintropicalhabitatsusingmetabarcoding(Bakkeretal.2017;Boussarieetal.2018).
Bull Sharks,Carcharhinus leucas (Müller and Henle, 1839), arefoundintemperate,subtropical,andtropical latitudesgloballyandaredistinctiveasoneofonlyafewsharksthatcanusefreshwaterforextendedperiodsof time (Thorson1962;Thorson1971;Thorson,Cowan,andWatson1973).Asupper trophic levelpredators, theyplayacrucialroleinmaintainingecosystemhealthacrossbothma‐rineandfreshwaterhabitats(Every,Pethybridge,Fulton,Kyne,andCrook 2017; Polovina, Abecassis, Howell, andWoodworth 2009;Ritchieetal.2012).Usingacoustic telemetrydata toexamine thehabitatuseofC. leucasinnorthernGulfofMexicowaters,Drymonet al. (2014) foundC. leucasmaypreferentially selecthigher‐qual‐ity,less‐urbanizedrivers,althoughaspatiallylimitedacousticarrayhinderedafullevaluationofthispattern.TargetedeDNAsurveysofC. leucascouldprovideacost‐effective,sensitivemethodtoexam‐inethispatternmorewidely,astherecouldbesubstantialecologicalimplicationsofsuchhabitatpreference.Here,weestablishaneDNAmethodologyappropriatefortargetedspeciesdetectionsofC. leu‐casinestuarinewatersinthenorthernGulfofMexico.Specifically,we compare total eDNA yields for different QIAGEN® DNeasy® DNAextractionkitprotocolsanddevelopaspecies‐specificC. leu‐caseDNAassayusingarelativelynovel,Bio‐Rad®DropletDigital™PCR (ddPCR), platform to detect low quantities of target DNA.Finally,weapplythesemethodsto investigatethedetectabilityofC. leucaseDNAinknownhabitatinthenorthernGulfofMexicoandinexsituclosedandflow‐throughenvironmentscontainingasingleC. leucas individual.
2 | MATERIAL S AND METHODS
2.1 | Laboratory controls
Strictlaboratorycontrolswereimplementedthroughoutthisstudytoreducetheriskofcross‐contaminationandcontaminationbyex‐ogenous DNA (see Deiner,Walser, Mächler, and Altermatt 2015;Goldberg et al. 2016). Water processing, DNA extractions, andPCR amplifications were conducted in physically separated labo‐ratory spaces to prevent cross‐contamination between stages.Negative controls were incorporated into every stage of sampleprocessing,andPCRwasperformedonthemtocheckforpotential
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contamination. Filter negatives contained target‐free, autoclaveddeionized water, DNA extraction negatives contained no filteredparticulatematerial,andPCRamplificationnegativescontainednoDNA;allnegativecontrolsproducednegativeresults,indicatingnocontaminationhadoccurred.TheddPCRassay conditionsused tocarryoutthesenegativecontroltestsaredescribedbelow.
2.2 | Water sample collection and filtration
Water samples throughout this studywerecollected justbelow thesurfaceofthewater in1Lhigh‐densitypolyethyleneNalgene®bot‐tlesprecleanedina10%bleachsolutionandsanitizedunderultravio‐let(UV)lightfor20min.Newgloveswereusedtocollecteachwatersampleandsampleswerestoredoniceinacooleruntilfiltrationusingavacuumpumpcouldtakeplace,whichoccurredwithin24hrofcol‐lection(seePilliodetal.2013),exceptwhereotherwisenoted.Watersampleswerefilteredinadedicated,precleanedlaboratoryspacethathad never had C. leucastissueortotalgenomicDNA(gDNA)present.Each1Lwatersamplewasinvertedatleastthreetimestoensureho‐mogenizationofparticulatematterandwasthenvacuum‐filteredusing47‐mm‐diameter, 0.8‐μm nylon filters, which were replaced whencloggingoccurredevery~350ml (e.g., threefiltersper1L)andpre‐servedin95%ethanolatroomtemperature,unlessnotedotherwise(seeAppendixS1).Duringallwaterfiltration,filterswerehandledwith
designatedsterileforcepsforeachsampleandgloveswerechangedinbetweensamplestoavoidcross‐contamination.
2.3 | DNA extraction methods
Due to thewidevarietyofDNAextractionmethodsused ineDNAliterature(Renshaw,Olds,Jerde,McVeigh,andLodge2015),wecom‐paredeDNAextractionkitstoestablishanappropriatemethodforthenylonfiltersusedtofilterwatersamplesinthisstudy.TheQIAGEN® DNeasy®Blood&TissueKitisafrequentchoiceforDNAextractionsfromfiltersineDNAstudies,butwithnumerousvariations(seeReesetal.2014).TheperformanceofthiskitusingtheGoldbergetal.(2011)variationincorporatingQIAshredder™spincolumnswascomparedtothatofanextractionkitdesignedspecificallyforwatersamples,theQIAGEN®DNeasy®PowerWater®Kit.TheGoldbergetal.(2011)pro‐tocolincorporatingQIAshredder™spincolumnswasselectedbecauseinpreliminarytrials,ityieldedhigherrelativequantitiesofDNAcom‐paredtosomeothervariations(AppendixS2).Additionally,fourvaria‐tionsofphysicaldisruptionmethodstodislodgetheparticulatematterfrom the filters prior to digestionwere testedwith each extractionmethod:(a)nophysicaldisruption,(b)beadbeating,(c)filterscraping,and(d)freezingfilterswithliquidnitrogenandcrushingthemusinganautoclavedmortarandpestle.TheQIAGEN®DNeasy®PowerWater® Kitcontainedbeadbeatingaspartofthestandardmanufacturer'spro‐tocol,sothisstepwaseliminatedforthenophysicaldisruptionvaria‐tiontodetermineifthisstepwasacriticalfactorinDNAyields.Three×1Lwatersamplereplicateswereused ineachextraction/physicaldisruption treatment, collected fromMobileBay,Alabamausing thewatercollectionandfiltrationprotocolsdescribed.Toeliminatethefil‐terpreservationstep,thefiltersforeach1Lsamplewereimmediatelyplaced into theappropriate lysisbuffers (seeHinloetal.2017).TheDNAextractsforeach1LwatersamplewerecombinedandtheDNAqualitieswereassessedusing2%agarosegelandtherelativequanti‐tiesweremeasuredusingThermoFisherScientificNanoDrop™spec‐trophotometertechnology,witheachextractmeasuredfourtimes.
2.4 | Development of a species‐specific assay
Todevelopaspecies‐specificassay,primersandan internalprobeweremanuallydesignedinconservedregionsofthemitochondrial(mtDNA)NADHdehydrogenase2(ND2)genewithinC. leucas,butvariableregionsacross23geneticallysimilar,exclusionelasmobranchspecies, using sequences available fromGenBank and aligned viaCodonCodeAlignerv.7.0(seeAppendixS3).Forward(BULLND2F6:5′‐TCCGGGTTTATACCCAAATG‐3′) and reverse (BULLND2R5: 5′‐GAAGGAGGATGGATAAGATTG‐3′) primers were designed firstto PCR‐amplify a 237base pair portion of themtDNAND2genein C. leucas. The primers were first tested using gDNA extractedfromfiveC. leucasindividualsfromnorthernGulfofMexicowatersusingconventionalPCR.EachPCRconsistedof10mMTAQbuffer,1.5mMMgCl2,0.3μMofeachprimer,0.1mMdNTPs,1UofTaqpol‐ymerase,~25ng/μlofeachDNAextract,andPCR‐gradewaterforafinalreactionvolumeof25μl.PCRcyclingconditionsbeganwith
TA B L E 1 EighteengeneticallysimilarexclusionelasmobranchspeciesfoundintheGulfofMexico
Common name Species name
NurseShark Ginglymostoma cirratum
ShortfinMako Isurus oxyrinchus
DuskySmoothhound Mustelus canis
TigerShark Galeocerdo cuvier
GreatHammerhead Sphyrna mokarran
ScallopedHammerhead Sphyrna lewini
Bonnethead Sphyrna tiburo
AtlanticSharpnoseShark Rhizoprionodon terraenovae
LemonShark Negaprion brevirostris
FinetoothShark Carcharhinus isodon
BlacknoseShark Carcharhinus acronotus
SandbarShark Carcharhinus plumbeus
SpinnerShark Carcharhinus brevipinna
DuskyShark Carcharhinus obscurus
SilkyShark Carcharhinus falciformis
BlacktipShark Carcharhinus limbatus
CownoseRay Rhinoptera bonasus
AtlanticStingray Hypanus sabina
Note: These18geneticallysimilarexclusionspecies,andCarcharhinus leucas,weretestedforspecies‐specificityofthedevelopedprimersandinternalprobeontheBio‐Rad®QX200™DropletDigital™PCRplat‐form.AlltissuesampleswerecollectedfromtheGulfofMexico.
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initialdenaturationat94°Cfor5min,followedby35cyclesof94°Cfor30s,59°Cfor30s,and72°Cfor30s,finalextensionat72°Cfor7min,andafinalholdat4°C.Primerswerealsotestedagainstoneindividualofeachof18othergeneticallysimilar,localexclusionspecies,collectedfromtheGulfofMexico(Table1)toassessspeci‐ficity.TheprimersamplifiedDNAinthetargetspecies,C. leucas,butalsoamplifiedDNAfromsomeofthenontargetspeciestested.Toincreasethespeciesspecificityoftheassay,aninternalPrimeTime® double‐quenched ZEN™/IOWA Black™ FQ probe labeled with 6‐FAM(BULL_IBFQ:5’‐CAACACTAACTATAAGTCCTAACCCAATC‐3’)wasdesignedtoamplifythetargetgeneinonlyC. leucas.
DdPCR reaction mixtures and cycling conditions were opti‐mized forC. leucas by systematically adjusting the concentrationsofprimers(300–1,000nM)andinternalprobe(100–250nM),cyclenumber (30–40 cycles), ramp rate (0.5–2.0°C/s), annealing tem‐perature (54–66°C), elongation time (1–2min), and the amountofgDNA(0.2–25.0ng/μl).TheoptimizedddPCRmixturecontained1XBio‐Rad®ddPCRsupermixforprobes(nodeoxyuridinetriphosphate(dUTP)),750nMofeachprimer,and250nMofprobe,and1.1μlofextractedDNA,adjustedtoafinalvolumeof22μlwithPCR‐gradewater.DdPCRdropletsweregeneratedforeach22μlreactionusingthe Bio‐Rad® QX200™ AutoDG™ Droplet Digital™ PCR System(Instrumentno.773BR1456)andthermalcyclingconditionswereasfollows,usingaramprateof1°C/s:initialdenaturationat95°Cfor10min,followedby35cyclesof94°Cfor30sand56°Cfor2min,followedbyenzymedeactivationat98°Cfor10min,andafinalholdat4°C.Toensuretheoptimizedassaywasspecies‐specificforC. leu‐casusing theddPCRplatform, theprimersandprobewere testedusingtheseddPCRreactionandcyclingconditions,inreplicatesofthree,with0.2ng/μlofgDNAextractedfromfiveC. leucas individ‐ualsandoneindividualofeachof18othergeneticallysimilar,localexclusionspecies,collectedfromtheGulfofMexico(Table1).
All ddPCR data were analyzed with the Bio‐Rad® QX200™Droplet Reader and QuantaSoft™ software using the Rare EventDetection (RED) analysis, a manual detection threshold of 3,000amplitude(Figure1),andalimitofdetection(LoD)ofthedevelopedassay.TheLoDisconsideredthelowestconcentrationofC. leucas DNAthatcanreliablybedetectedusingtheoptimizedassaycondi‐tions.The lowerLoDwasdeterminedbyconductingddPCRswithgDNA from twoC. leucas individualsusing a sixfold seriesof10Xdilutions(e.g.,1:10to1:1,000,000),fromastartingconcentrationof25.0ng/μl.MeansandstandarderrorsofdetectedDNAconcentra‐tion(copies/μl)werecalculatedforeachindividual,acrossthethreeddPCRreplicatesforeachdilution.
2.5 | Collection of positive water samples
Carcharhinus leucaseDNAsampleswereobtainedviathecollec‐tion ofwater samples from knownC. leucas habitat and ex situexperiments. These experimentswere conducted in accordancewiththelawsofthestateofAlabamaandundertheIACUCproto‐cols(IACUCProtocolNumber974304).Allmeasuresweretakentoreducethepainorstresstheanimalunderwentduringtesting;
therefore, thewaterused in theex situexperimentswere fromnaturalsharkhabitat.WaterwascollectedfromthecoastalwatersofMobileBay,Alabama,knownC. leucashabitat,inApril2017andplaced into aprecleaned, circular fiberglass, closed‐system tank(~120cmwideandheldavolumeof~711L),andsix×1LwatersampleswereimmediatelycollectedfromthistanktodeterminewhethertargeteDNAwaspresent in theambientwater.Abub‐blerwasadded to the tank tokeep the systemoxygenatedandone wild‐caught juvenile maleC. leucas, ~930mm total length,wasaddedtothetank.ToacquireaconfirmedpositiveC. leucas eDNAsample,after30min, six×1Lwatersampleswereagaincollected from the tank. Thesewater sampleswere used in as‐pectsofmethoddevelopment (seeAppendixS1)andtovalidatethedevelopedgeneticassay.
TotesttheeffectivenessofthedevelopedC. leucasassayinanopensystemwithasingletargetspeciespresent,aflow‐throughmesocosm (~365 cmwide containing a volume of ~14,500 L) atDauphin Island Sea Lab, Alabamawasmaintained inApril 2017.The flow rate of the mesocosm was designed to mimic flow ina coastal system at ~30 cm3/hr,with complete system turnoverat approximately 2 hr. One wild‐caught juvenile male C. leucas,~930mmtotal length,was introduced to this systemand five×
F I G U R E 1 RawoutputoftheoptimizedDropletDigital™PCR(ddPCR)forthedesignedCarcharhinus leucasspecificassayshowingoneddPCRreplicateforoneindividual(0.2ng/μlofgenomicDNA)andonereplicatefortheddPCRnegativefromtheBio‐Rad® QX200™DropletReader.Eachdropletineachwellwasclassifiedaseitherpositive(bluedroplets)ornegative(graydroplets)fortargetDNA,basedonamanualdetectionthresholdsetto3,000amplitude(thehorizontalpinkline)usingtheQuantaSoft™RareEventDetectionanalysis.Eventnumberreferstothenumberofdropleteventsgeneratedforagivenwellorsample;Ch1amplitudemeasurementreferstotheleveloffluorescenceemittedbyadropletevent;andeachcolumnisasinglewell
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1Lwatersampleswerecollectedimmediately(time0.0),spanningthediameterofthemesocosm;thissamplingregimewasrepeatedevery0.5hrfor3hr,allowingforcompleteturnoverofthesystem.Watersampleswerestoredina−20°Cfreezerfor1month,duetolaboratoryequipmentconstraints, similar toBakkeret al. (2017)andGarganetal. (2017),andwerethawedat roomtemperaturepriortofiltration.
Water samples from these experiments were vacuum‐filteredusing 47‐mm‐diameter nylon 0.8‐μm filters (three per 1 L), whichwere preserved in 95% ethanol at room temperature (AppendixS1) andDNAextractions followed theGoldberget al. (2011)pro‐tocolincorporatingtheQIAshredder™spincolumns(AppendixS2).DdPCR amplificationswere carried out in replicates of five, usingtheoptimizedC. leucasassaypreviouslydescribedinthisstudy.AllddPCRreactionsweresetupusingaerosolbarrierfilterpipettetipsanddesignatedpipettes,separatefromthoseusedinsettingupPCRreactions,wereusedtoaddeDNAextractstothereactions.DdPCRresultswereanalyzedusingtheBio‐Rad®QX200™DropletReaderand QuantaSoft™ RED analysis, a manual detection threshold of3,000amplitude,andtheLoD.
3 | RESULTS
3.1 | Optimal eDNA methods
TheGoldbergetal. (2011)protocolusingtheQIAGEN®DNeasy® Blood&TissueKitandQIAshredder™spincolumnsyieldedhigherrelative quantities of total eDNA from filters compared to theQIAGEN®DNeasy®PowerWater®Kitprotocol,acrossallvariationsinphysicaldisruptionmethods(Figure2).TheDNAyieldsfromthefour physical disruption methods used with the Goldberg et al.(2011)protocolweresimilar:NophysicaldisruptionyieldedatotalDNA average of 61.19 ng/μl (SE = 1.65), bead beating the filtersyielded56.83ng/μl (SE = 6.75), filter scraping yielded56.78ng/μl (SE=1.77),and freezing filterswith liquidnitrogenandcrush‐ingyielded64.93ng/μl(SE=2.36)(Figure2).SincethetotalDNAyieldsweresimilaracrossthesemethodsandbecausetheadditionofaphysicaldisruptionstep is time‐consumingandallowsforanadditionalopportunity forcontaminationbyexogenousDNA,wedeterminedtheoptimalDNAextractionmethodforourpurposestobetheGoldbergetal. (2011)protocolwithnophysicaldisrup‐tionmethod.
Thecombinationofprimersandprobedesignedinthisstudyweredemonstratedtobespecies‐specificforC. leucasinourstudyareabysuccessfullyamplifyingtargetDNAinallddPCRreplicatesfor the fiveC. leucas individuals andnot amplifyingDNA in anyoftheddPCRreplicatesofthe18localexclusionspeciesorPCRnegative controls. The LoD, as determined using the Bio‐Rad® QX200™ Droplet Reader and QuantaSoft™, was the 1:10,000dilution, corresponding to 2.5 pg of targetDNA in the reaction(Figure3).Therewereseveralpositivedropletspresentabovethemanual threshold in the1:10,000dilutions,andthestandarder‐rorsdidnot includezerooroverlapwiththoseofthe1:100,000
dilutions. In contrast, there were no positive droplets in the1:100,000dilutionsandthestandarderrorsoverlappedwithzero,suggestingC. leucas DNA could not be reliably detected at thisdilution (Figure3).Usingthenumberofcopiesof targetDNA/μl in the 1:10,000 dilutions and applying the lower standard errorastherelaxeddetectionthresholdforeachofthetwoindividuals(seeBakeretal.2018),theaverageLoDthresholdwasdeterminedtobe0.6copies/μlinareaction.
3.2 | Analysis of water samples
UsingthedevelopedddPCRassayandtheQuantaSoft™REDanaly‐siswithamanualdetectionthresholdof3,000amplitude,anaverageof1.62copies/μl(SE=0.12)ofC. leucasDNAwasdetectableintheddPCRreactionsfromwatersamplescollectedfromknownhabitat,Mobile Bay,without visually confirming the presence ofC. leucas (Figure4).IntheexsitupositiveeDNAexperiment,30minafteraC. leucaswasaddedtotheclosedtankcontainingthiswater, largeamountsoftargeteDNAwerepresent,withanaverageconcentra‐tionof166.6copies/μl(SE=3.01)intheddPCRreactions(Figure4).Intheflow‐throughmesocosmexperiment,whenapplyingalowerLoDof0.6copies/μltothedataanalysis,targetC. leucasDNAwasnotdetectable inanyof theddPCRreplicatesat time0.0butwasdetectableinallddPCRreplicates0.5hrafterthesharkwasadded(Figure 5). Average target eDNA concentration peaked by 1.0 hr,withanaverageof5.8copies/μl (SE=0.27)acrossallddPCRrep‐licates,andthendeclinedoverthenexthour (Figure5).By2.0hr,theaverageconcentrationofC. leucaseDNAdippedbelowtheLoD,withpositivedetectionsinonlytwoofthefiveddPCRreplicatesforthissample(Figure5).Therewasasecond,smallerspikeinC. leucas eDNAby2.5hr,thatagaindecreased,buttheaverageconcentration
F I G U R E 2 ConcentrationsofDNAextractsfromwatersamplesusingtheQIAGEN®DNeasy®Blood&TissueKitwiththeGoldbergetal.(2011)protocolandtheQIAGEN®DNeasy® PowerWater®Kit,incombinationwithadditionalphysicaldisruptionmethods.SEbarswereusedtoshowtheerrorinmeanDNAconcentrationsbetweencategories,usingfourThermoFisherScientificNanoDrop™spectrophotometerreadingspersample.TheDNAextractsforeach1Lwatersamplewerecombinedandeachcategorycontainedthree×1Lwatersamplereplicates
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oftargetDNAremaineddetectableat3.0hr,althoughonlytwoofthefiveddPCRreplicatesforthissamplehadconcentrationsabovetheLoD(Figure5).
4 | DISCUSSION
TheuseofeDNAasatooltostudythedistributionandecologyofmarinespecieshasincreasedsubstantiallyinrecentyears(Bakkeretal.2017;Footeetal.2012;Laffertyetal.2018;Portetal.2016).However,carefulconsiderationandoptimizationofthemethodsemployedinsuchstudiesarenecessary,ultimatelyallowingforanappropriateinterpretationoftheresults.Here,wefoundfilteringwaterwithnylon0.8‐μmfilters,preservingthefiltersin95%etha‐nol(AppendixS1),andthenperformingDNAextractionsusingtheGoldbergetal.(2011)protocolwiththeQIAGEN®DNeasy®Blood&TissueKitandQIAshredder™spincolumns tobeanappropri‐atemethodofisolatingtotaleDNAfromwatercollectedfromthenorthern Gulf of Mexico. Although the number of replicates intheexperimentwassmall,theGoldbergetal.(2011)protocolwasfoundtooutperformthePowerWater®kitacrossallfourphysicaldisruptionmethods,despitethelatterbeingspecificallydesignedandmarketed for eDNAextractions fromwater samples, andatahighercost.ThetotalDNAyieldsusedtoevaluate theperfor‐mancesof theseextractionmethods areunlikely tobe accurateinanabsolutesenseduetothe inabilityofNanoDrop™spectro‐photometertechnologytodecipherDNAfromotherpossiblebio‐logicalmacromolecules,buttherelativedifferencesbetweenDNAyieldsweresubstantial.Thecombinationofprimersand internalprobefor themtDNAND2genedesigned inthisstudyareopti‐mizedforC. leucasintheestuariesinthenorthernGulfofMexico;however,whethertheyareappropriate(e.g.,species‐specific)foruse in other geographic regions, such as northern Australia, orin fullymarinewaters,wheretheremaybeadditionalspeciesofcloselyrelatedcarcharhinidspresent,requiresfurthertesting.TheLoDdeterminedinthisstudyshowsthesensitivityanddetectioncapabilityofthedevelopedassayandwasdemonstratedtobesuf‐ficientforC. leucaseDNAdetectioninMobileBayandinexsitupositive samples.However, the LoDmay require further refine‐mentthroughadditionaldilutionseriesbetweenthe1:10,000and1:100,000 dilutions before being used in data analysis for largenumbers of field samples. Furthermore, due to potential differ‐encesacrossddPCRmachines,werecommendtheLoDtobere‐fined independently for eachmachine, using the LoD here as astartingreferencepointforthisassay.
The ability of ddPCR to detect low concentrations of targetDNA,forexample,2.5pgofC. leucasDNAinthisstudy,meansthisplatformmaybeless likelytoproducefalsenegativeswhenusedalongside an appropriate sampling regime and water processingmethods(e.g.,spatialanddepthcoverage,volumecollected,filterporesize).FalsenegativescanoccurwhentargetDNAiscapturedinwatersamplesbutisnotdetectedduetolimitationsofthege‐netic assays employed (Darling andMahon 2011; Ficetola et al.2015;Goldbergetal.2016;Lahoz‐Monfort,Guillera‐Arroita,andTingley2016).Todate,themajorityofstudiesthatuseeDNAintar‐getedspeciesdetectionshaveusedqRT‐PCR,butthedetectionca‐pabilitiesofthisplatformmaybelimited,whencomparedtothoseofddPCR(Doi,Takahara,etal.2015;Doi,Uchii,etal.,2015).The
FIGURE 3 Limitofdetection(LoD)testsusinga6‐fold10Xdilutionseries(1:10–1:1,000,000)oftotalgenomicDNA(gDNA)fromtwoCarcharhinus leucasindividualsfromthenorthernGulfofMexico.(a)ThemeanDNAconcentrations(copynumber/μl)andstandarderrorbarswerecalculatedfromthreeDropletDigital™PCR(ddPCR)replicatesforeachoftwoindividuals,usingamanualdetectionthresholdof3,000amplitudeandtheRareEventDetectionanalysissettingontheBio‐Rad®QX200™DropletReaderandQuantaSoft™software.The1:10and1:1,000,000werenotgraphedduetooversaturationofthePCRproduct,andthelackofDNAcopiespresentshowingnopositivedropletdetections,respectively.TheLoD(0.6copies/μl)isrepresentedbyadottedline.(b)RawdropletoutputofddPCRserialdilutionproductsfromoneddPCRreplicateofoneC. leucasindividualdetectedbytheBio‐Rad®QX200™DropletReaderandQuantaSoft™software.Eachdropletineachwellwasclassifiedaseitherpositive(bluedroplets)ornegative(graydroplets)fortargetDNA.EachwellisseparatedbyyellowbarsandcorrespondstothesamedilutionconcentrationsgraphedinFigure3a,labeledwitheachdilutionseriesitrepresents
(a)
(b)
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differenceindetectionabilitiesbetweenthetwoPCRplatformsislikelyduetofundamentaldifferencesinhowtheyquantifytargetDNA.DdPCRquantifiesthestartingDNAcopynumberpresentinasampleusingend‐pointPCRwithoutreferencetoastandard(abso‐lutequantification)(Whaleetal.2012),makingitamoresensitiveand precise assay, ideal for eDNA applications targeting a singletargetspecies.Additionally,theREDanalysissettingusingtheBio‐Rad®QuantaSoft™softwareisdesignedtoidentifylowcopynum‐bersoftargetDNAinabackgroundlargelycomposedofnontargetDNAcopies (Bio‐Rad®DropletDigital™PCRApplicationsGuide).GiventheabilityofddPCRtodetectsuchlowquantitiesofDNA,itmayreplaceqRT‐PCRineDNAresearch(Doi,Uchii,etal.,2015;Nathan,Simmons,Wegleitner, Jerde,andMahon2014)assessingthedistribution,habitatuse,andabundanceofspeciesfoundinlowabundanceand/orareofconservationconcern(Bakeretal.2018;
Hunteretal.2018;Tréguieretal.2014), includingelasmobranchs(Bohmannetal.2014;Laffertyetal.2018).However,wecautionthattheabilitytodetectsuchlowquantitiesofDNAalsoincreasesthe potential for false positives (Goldberg et al. 2016; Huggett,Cowen,andFoy2015).AlleDNAstudies,butespeciallythoseusingddPCR,requirestrictfieldandlaboratorycontrolsandproceduresbeinplacetoreducethepotentialforfalsepositives,typicallytheresultofcontaminationbyexogenousDNAorcross‐contaminationof samples (seeFicetola,Taberlet, andCoissac2016). InadditiontothecontaminationcontrolsdescribedbyGoldbergetal.(2016),Deineretal.(2015),andPortetal.(2016),whenusingddPCR,wealsosuggest: (a)usingtwocleaningmethodsfordecontaminationof all field and water filtration equipment (e.g., a bleach wash,plus autoclaving, and/orUV light exposure), (b) thatwater filtra‐tion isconducted ina laboratoryspacethathasneverhadtissueorgDNAfromthetargetspeciespresent, (c) thatglovesandanytoolsarechangedinbetweensamplesduringwaterfiltration(seePilliod,Goldberg,Arkle,andWaits2013),(d)thatnegativesbein‐corporated into field collection,water filtration,DNAextraction,andPCR,witheachnegativerunthroughtoPCR(seeBakkeretal.2017;Jerde,Mahon,Chadderton,andLodge2011),(e)thatadesig‐natedpipette,separatefromthatusedtosetupreactions,beusedtoaddDNAextractstoddPCRreactions,and(f)thatmultiplerep‐licatesforeachsamplearerunduringddPCR(seeReesetal.2014).Strictfieldandlaboratorycontrolswillensuretheauthenticityandreliabilityof eDNA results,which is increasingly critical in eDNAresearchusinghighlysensitivetechnologies,suchasddPCR,espe‐ciallywhentheresultsofsuchstudieswillbeusedtoinformcon‐servationandmanagement initiatives (Hunteretal.2017;Hunteretal.2018).
F I G U R E 4 RawDropletDigital™PCR(ddPCR)outputfromtheambientwatersampleinMobileBay,theCarcharhinus leucaseDNApositivewatersampletakenfromaclosedsystem30minafteraddingtheshark,andeachnegativecontrolfromtheBio‐Rad® QX200™DropletReader.Eachdropletineachwellwasclassifiedaseitherpositive(bluedroplets)ornegative(graydroplets)fortargetDNAbasedonamanualdetectionthresholdsetto3,000amplitude(thehorizontalpinkline)usingtheQuantaSoft™RareEventDetectionanalysis.Eventnumberreferstothenumberofdropleteventsgeneratedforagivenwellorsample;Ch1amplitudemeasurementreferstotheleveloffluorescenceemittedbyadropletevent;andeachcolumnisasinglewell.Columns,orwells,areseparatedbyyellowbars;ColumnD01correspondstooneddPCRreplicatefromtheambientMobileBaywatersampleandF01correspondstooneddPCRreplicatefromtheC. leucaseDNApositivewatersample.ColumnsB11,A12,andB12correspondtooneddPCRreplicatefromeachnegativecontrolincorporatedandshowsnocontaminationoccurredduringanystageofthisexperiment
F I G U R E 5 Carcharhinus leucasmeaneDNAconcentrations(unitofmeasure)inaflow‐throughmesocosmdetectedusingtheBio‐Rad®QX200™DropletReaderandQuantaSoft™usingamanualdetectionthresholdof3,000amplitudewiththeRareEventDetectionanalysissetting.EachtimepointsamplewasruninDropletDigital™PCR(ddPCR)replicatesoffive,andstandarderrorbarswereusedtoshowthevariationinconcentrationestimatesacrossthefiveddPCRreplicatesforeachsample.Thelowerlimitofdetection,foundtobeatleast0.6copies/μlinthisstudy,isindicatedbyadottedline
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Fundamental researchon the accumulation, persistence, anddegradationofelasmobrancheDNA isnecessary to improve theinterpretation of results in eDNA field research. Here, we haveshown that after adding a shark into closed and flow‐throughsystems,targeteDNAwasdetectablewithin30min.Intheflow‐throughsystem,theinitialspikeintargeteDNAthatoccurredbe‐tween 0.5 and 1.0 hr could be due to initial stress experiencedby the shark after being added to the mesocosm, causing it toexpelmoreDNA (e.g., Barnes et al. 2014). The overall decreaseintargeteDNAbetween1.0and2.0hrmaybetheresultoftheshark acclimating to theenvironment and releasing lessDNAorturnoverofwaterinthemesocosmifthesharkisreleasingDNAintothesysteminpulsesratherthancontinuously;however,thishasnotbeenexplicitlyexploredinelasmobranchs.TheinabilitytodetectC. leucasDNAinsomeoftheddPCRreplicatesat2.0and3.0hr,despitetheconfirmedpresenceofasharkandtheuseofahighlysensitiveddPCRassay,suggeststheremayhavebeenverylittleC. leucasDNApresentat those times,whichcouldoccur ifDNAwasshed inpulses,andthenflowedoutof themesocosm.However, thispatterncouldalsobe indicativeofsamplingerror,where C. leucasDNAwaspresent,butnotcaptured,highlightingtheneed forcarefulconsiderationofsampling regimeaswellasthe interpretation of the results of eDNA studies. Becauseme‐socosmwatersampleswere frozenaftercollection, itcannotbecompletely ruledout that theeDNAdegradedprior to filtration(Hinloetal.2017;Takahara,Minamoto,andDoi2015);however,theconcentrationsofthetotaleDNAextractsfromthesesampleswerenotunusuallylowcomparedtotheothereDNAextractsana‐lyzedforthisstudy.Furthermore,othereDNAstudieshavefrozenwatersamplespriortofiltrationwithoutapparentnegativeeffects(Bakkeretal.2017;Garganetal.2017)makingitunlikelytobethesoleexplanationfor theobservedpatternsofC. leucasDNAde‐tectedinthisexperiment.Ideally,theseexperimentsshouldhavebeenreplicatedand includedasecondtankwithoutasharkasanegative control, with water samples filtered immediately aftercollection;however,duetolimitedfacilitiesandtheconstraintsofusingliveanimals,theseimprovementstothestudydesignwerenotfeasible.Regardless,thisisthefirstelasmobrancheDNAstudythathasplacedasingletargetanimal intoclosedandthenopen,flow‐throughsystemstoquantifytargeteDNAfromasinglean‐imalovertime,creatingabaselinefor futureexsituresearch. Incomparison,othereDNAstudiesofelasmobranchshaveacquiredpositiveeDNAsamplesbycollectingwatersamplesfromaquariawith the target species present (e.g., Simpfendorfer et al. 2016)orcollectingwatersamplesfromknownhabitats,butwithoutvi‐sually confirming the presence of the target species (e.g.,Weltzetal.2017).FuturestudiesshouldassessDNAaccumulationoverdifferent timescales thanpresentedhere,aswellashowalteredflow rates, water conditions (pH, temperature), weather condi‐tions (photoperiod, cloud cover), and number and size of targetspeciesimpacttheaccumulationandpersistenceofelasmobrancheDNAinmarinesystems.
ACKNOWLEDG MENTS
ThisresearchwasfundedbyTheUniversityofSouthernMississippiand was supported by the Mississippi INBRE, funded underInstitutionalDevelopmentAward(IDeA)numberP20‐GM103476from theNational Institutes ofGeneralMedical Sciences of theNationalInstitutesofHealth.WewouldliketoshowourgratitudetoDeanGrubbs,JillHendon,andTobyDaly‐Engelforprovidinguswith elasmobranch tissue samples. Thank you to Emily Seubert,GrantLockridge,andMattJargowskyforcollectionofBullSharkspecimens andmesocosmmaintenance and to Ruth Carmichaelforaccesstofreezerspace.ThankyoutoJoshuaSpeed,LondonWilliams, and Michael Garrett for kindly accommodating us atUniversityofMississippiMedicalCenterandprovidingaccesstoallDropletDigital™PCRequipment.WewouldalsoliketothankGlenmoreShearer,LoganBlancett,andJonathanLindnerforad‐vice,assistance,andaccesstoequipmentusedinthisstudy.HelpprovidedbyElannaThompson,MadisonVitale,andArielWilliamsin collection, filtering, and measurement of water samples wasgreatlyappreciated.
CONFLIC T OF INTERE S T
Theauthorsdeclarethattheyhavenoconflictofinterest.
AUTHOR CONTRIBUTIONS
Allauthorscontributedtotheconceptionanddesignofthestudy,theacquisitionand the interpretationof thedata, andwriting themanuscript.
ORCID
Katherine E. Schweiss https://orcid.org/0000‐0002‐4795‐1933
Nicole M. Phillips https://orcid.org/0000‐0002‐4138‐4966
DATA AVAIL ABILIT Y S TATEMENT
Thedatasetsgeneratedduringand/oranalyzedduringthecurrentstudyareavailablefromN.M.Phillipsonreasonablerequest.
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SUPPORTING INFORMATION
Additional supporting information may be found online in theSupportingInformationsection.
How to cite this article:SchweissKE,LehmanRN,DrymonJM,PhillipsNM.DevelopmentofhighlysensitiveenvironmentalDNAmethodsforthedetectionofBullSharks,Carcharhinus leucas(MüllerandHenle,1839),usingDropletDigital™PCR.Environmental DNA. 2019;00:1–10. https://doi.org/10.1002/edn3.39