u n i ve r s i t y o f co pe n h ag e n

Monitoring a Norwegian freshwater crayfish tragedy: eDNA snapshots of invasion,infection and extinction

Strand, David A.; Johnsen, Stein Ivar; Rusch, Johannes C.; Agersnap, Sune; Larsen, WilliamBrenner; Knudsen, Steen Wilhelm; Møller, Peter Rask; Vralstad, Trude

Published in:Journal of Applied Ecology

DOI:10.1111/1365-2664.13404

Publication date:2019

Document versionPublisher's PDF, also known as Version of record

Document license:CC BY

Citation for published version (APA):Strand, D. A., Johnsen, S. I., Rusch, J. C., Agersnap, S., Larsen, W. B., Knudsen, S. W., ... Vralstad, T. (2019).Monitoring a Norwegian freshwater crayfish tragedy: eDNA snapshots of invasion, infection and extinction.Journal of Applied Ecology, 56(7), 1661-1673. https://doi.org/10.1111/1365-2664.13404

Download date: 07. mar.. 2021

J Appl Ecol. 2019;56:1661–1673.  | 1661wileyonlinelibrary.com/journal/jpe

Received:29November2018  |  Accepted:10March2019DOI: 10.1111/1365-2664.13404

R E S E A R C H A R T I C L E

Monitoring a Norwegian freshwater crayfish tragedy: eDNA snapshots of invasion, infection and extinction

David A. Strand1,2  | Stein Ivar Johnsen3 | Johannes C. Rusch1,4  | Sune Agersnap5,6  | William Brenner Larsen5 | Steen Wilhelm Knudsen5  | Peter Rask Møller5  | Trude Vrålstad1

ThisisanopenaccessarticleunderthetermsoftheCreativeCommonsAttributionLicense,whichpermitsuse,distributionandreproductioninanymedium,providedtheoriginalworkisproperlycited.©2019TheAuthors.Journal of Applied EcologypublishedbyJohnWiley&SonsLtdonbehalfofBritishEcologicalSociety.

1NorwegianVeterinaryInstitute,Oslo,Norway;2NorwegianInstituteforWaterResearch,Oslo,Norway;3NorwegianInstituteforNatureResearch,Lillehammer,Norway;4DepartmentofBiosciences,UniversityofOslo,Oslo,Norway; 5NaturalHistoryMuseumofDenmark,UniversityofCopenhagen,Copenhagen,Denmarkand6DepartmentofBioscience,AarhusUniversity,Aarhus,Denmark

CorrespondenceDavidA.StrandEmail:david.strand@vetinst.no

Funding informationNorgesForskningsråd,Grant/AwardNumber: 243907

HandlingEditor:AyeshaTulloch

Abstract1. The European noble crayfishAstacus astacus is threatened by crayfish plaguecaused by the oomycete Aphanomyces astaci, which is spread by the invasiveNorthAmericancrayfish(e.g.signalcrayfishPacifastacus leniusculus).Surveillanceof crayfish plague status in Norway has traditionally relied on themonitoringsurvival of cage‐heldnoble crayfish, amethodof ethical concern.Additionally,trapping isused in crayfishpopulation surveillance.Here,we testwhetheren-vironmentalDNA(eDNA)monitoringcouldprovideasuitablealternativetothecagemethod,andasupplementtotrapping.

2. WetookadvantageofanemergingcrayfishplagueoutbreakinaNorwegianwa-tercoursefollowingillegalintroductionofdisease‐carryingsignalcrayfish,andini-tiatedsimultaneouseDNAmonitoringandcage‐basedsurveillance,supplementedwithtrapping.Atotalof304watersampleswerefilteredfromseveralsamplingstationsovera4‐yearperiod.eDNAdata(species‐specificquantitativereal‐timePCR [qPCR]) for thepresenceofA. astaci, noble and signal crayfishwithin thewatersampleswerecomparedtocagemortalityandtrapping.

3. ThisisthefirststudycomparingeDNAmonitoringandcagesurveillanceduringanaturalcrayfishplagueoutbreak.WeshowthateDNAmonitoringcorrespondswellwiththebiologicalstatusmeasuredintermsofcrayfishmortalityandtrap-pingresults.eDNAanalysisalsorevealsthepresenceofA. astaciinthewaterupto2.5weeksinadvanceofthecagemethod.EstimatesofA. astaciandnoblecrayfisheDNAconcentrationsincreasedmarkedlyduringmortalityandvanishedquicklythereafter.eDNAprovidesasnapshotofthepresence,absenceordisappearanceof crayfish regardless of season, and constitutes a valuable supplement to thetrappingmethodthatreliesonseasonandlegislation.

4. Synthesis and applications.SimultaneouseDNAmonitoringofAphanomyces astaci (crayfish plague) and relevant native and invasive freshwater crayfish speciesiswell‐suited forearlywarningof invasionor infection, riskassessments,habi-tatevaluationandsurveillance regardingpathogenand invasive/nativecrayfish

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1  | INTRODUC TION

EnvironmentalDNA(eDNA)monitoringofaquaticsystemsisarap-idlyadvancingresearchfieldthatpromisesimprovements,notonlytoaquaticspeciesconservation,butalsoforearlydetectionof in-vasive species andharmful pathogens at lowdensities and at anylifestageorseason(Bohmannetal.,2014;Kellyetal.,2014;Strandetal.,2014).Watercanbescreenedforthepresenceofmicro‐andmacroorganismsbyeitherabroadapproachsuchasmetabarcoding(Shawetal.,2016;Valentinietal.,2016),oratargetedapproachusingspecies‐specificquantitativereal‐timePCR(qPCR)ordropletdigitalPCR (ddPCR) (Doi, Takahara, et al., 2015;Doi, Uchii, et al., 2015;Strandetal.,2014;Thomsen&Willerslev,2015).eDNAstudieshavebeenappliedfordetectionofawiderangeofaquaticmacroorgan-ismsincludingfreshwatercrayfish(Agersnapetal.,2017;Doughertyetal.,2016;Tréguieretal.,2014).Moleculardetectionandquantifi-cationofwaterbornepathogensinenvironmentalsampleshasbeenwidelyutilisedfordecades(Ramirez‐Castilloetal.,2015).

The oomyceteAphanomyces astaci is native toNorth Americaand is an obligate parasite on American freshwater crayfish(Söderhäll & Cerenius, 1999). It is the causative agent of crayfishplague in susceptible European freshwater crayfish (Alderman,Polglase,&Frayling,1987),andislistedamongtheworld's100worstinvasive species (Lowe,Browne,Buoudjelas,&DePoorter,2004).Aphanomyces astaci infectionisanotifiablediseasebothnationallyinNorway(list3,nationaldisease;Vrålstadetal.,2017)and inter-nationally(OiE,2017).ItcausesarapiddeclineinEuropeancrayfishpopulations, and is spreadandmaintainedby invasivenon‐indige-nousNorthAmericancarriercrayfishthathaverapidlyestablishedthemselves inEurope (Holdich,Reynolds,Souty‐Grosset,&Sibley,2009).Thepathogeninvadesthecuticleofallfreshwatercrayfish,but hyphal growth is inhibited bymelanisation in resistant NorthAmericancrayfish.Insusceptiblecrayfishspecies,thehyphaegrowdeeperintotissuesandorgans,causingrapiddeath.Theoomycetereproduces asexually via clonal flagellated zoospores that locatenewcrayfishhoststhroughweakchemotaxis.Zoosporescanencystandre‐emergeseveraltimes,butbothzoosporesandcystshavearelativelyshortlifespan(2–8weeks)dependentonwatertempera-ture(Söderhäll&Cerenius,1999).

AnA. astaci species‐specific qPCRmethod is widely used forcrayfishplaguediagnostics and carrier status testing (Kozubikova,Vrålstad,Filipova,&Petrusek,2011;OiE,2017;Vrålstad,Knutsen,Tengs, &Holst‐Jensen, 2009). The samemethod,which has beenthoroughlytestedandfurtherdeveloped(Makkonen,Strand,Kokko,Vrålstad,&Jussila,2013;Strandetal.,2012),isusedforeDNAmon-itoring for the presence ofA. astaci zoospores and cysts in bothsmall(Strandetal.,2011)andlargewaterbodies(Strandetal.,2014;Wittweretal.,2018).ThesestudieshaveestablishedthatclinicallyhealthyAmericancrayfishemitalownumberofA. astacizoosporestothewaterregardlessofseason(Strandetal.,2012,2014;Wittweretal.,2018),whilemoribundinfectedsusceptiblecrayfishemithugenumbersofinfectivezoospores(Makkonenetal.,2013).

LakeØymarksjøen in theHaldenwatercourse is one of a fewlakes inNorwayhostingapopulationofthenon‐indigenoussignalcrayfish Pacifastacus leniusculus, which were introduced illegallyaround twodecadesago,butnotdiscovereduntil2008 (Vrålstad,Johnsen,Fristad,Edsman,&Strand,2011).Theunknownpresenceof signal crayfish partly ruined long‐term attempts to restock thelakewithindigenousnoblecrayfish(Astacus astacus),followingthefirstoutbreakofcrayfishplaguein1989(Taugbøl,2004).Whentherestocked population increased in number, a new large outbreakof crayfish plague occurred in 2005 (Vrålstad et al., 2009). TheNorwegianFoodSafetyAuthorities (NFSA)enforcedapermanentclosureoftheØrjewaterlocksbetweenLakeØymarksjøenandLakeRødenessjøeninanattempttopreventupstreamspreadofA. astaci andsignalcrayfish(Vrålstadetal.,2011).

The noble crayfish population in Lake Rødenessjøen hasbeenmonitoredevery year since2009as a part of thenationalsurveillance programme, using baited traps set at eight stationsthroughout the lake. During this period, the relative density ofnoblecrayfishincreased,andCPUEin2014rangedbetween0.15and1.80(Johnsen,Strand,&Vrålstad,2017).InSeptember2014,bothsignalcrayfishandnoblecrayfishwerecaughtinthesouth-ernpartofLakeRødenessjøenjustabovetheclosedwaterlocks.TheNorwegianEnvironmentalAgency(NEA)regardedtheeventas another illegal introduction of signal crayfish, since long‐dis-tancemigrationoverlandorthroughtheclosedlockswashighlyunlikely (Norwegian Environmental Agency, 2014). The illegally

status.Thisnon‐invasive,animalwelfare friendlymethodexcludestheneedforcage‐heldsusceptiblecrayfish indiseasemonitoring.Furthermore,eDNAmoni-toringislesslikelytospreadA. astacithantraditionalmethods.Thisstudyresultedin the implementation of eDNAmonitoring forNorwegian crayfish plague andcrayfishsurveillanceprogrammes,andwebelieveothercountriescouldimprovemanagementstrategiesforfreshwatercrayfishusingasimilarapproach.

K E Y W O R D S

crayfishplague,diseasesurveillance,environmentalDNA,host–pathogen,invasivespecies,noblecrayfish,signalcrayfish,species‐specificdetection

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introducedsignalcrayfishwereconfirmedA. astacicarriers,indi-cating the probable onset of a new crayfish plague outbreak inthelocalnoblecrayfishpopulation.AcrayfishplaguesurveillanceprogrammecommissionedbytheNFSAwasthereforeconductedusing live noble crayfish in cages to monitor the spread of thedisease.Traditionalcageexperimentsusingnoblecrayfishas‘ca-naries ina coalmine’hadbeen the solemethodutilised for fieldmonitoringof crayfishplague since it is introduction toNorwayin the 1970s (Håstein & Unestam, 1972; Vrålstad et al., 2014).DecapodcrustaceansarenowcoveredbytheAnimalWelfareActinEuropeand theLawonAnimalWelfare (LOV‐2009‐06‐19‐97)inNorway.Thus, theuseof live crayfish formonitoring a lethaldiseaseisofstrongethicalconcern.Inadditiontofatal infectionwithcrayfishplague,cage‐heldcrayfisharealsosubjecttoothercauses of mortality such as moulting‐associated cannibalism.Furthermore, cage‐held crayfish commonlyescapedue to illegalhumaninterference(Vrålstadetal.,2017).PreviousstudieshaveshownthateDNAmonitoringofcrayfishplagueinlargewatersys-temsispossible(Strandetal.,2014),butadirectcomparisonwithtraditionalcagesurveillancehasnotyetbeenperformed.

Inthepresentstudy,wetookadvantageofanemergingcrayfishplague outbreak and compared traditional cage surveillance witheDNAmonitoring using species‐specific qPCR assays for targeteddetectionandquantificationofA. astaci(Strandetal.,2014),noblecrayfishandsignalcrayfish (Agersnapetal.,2017), fromthesamewater samples. In addition,weused trappingdata from2014and2015tocompareandverifycrayfishpresence.WeshowthateDNAmonitoringcanrevealthepresenceofA. astaciinthewaterearlierthancageswithlivecrayfish,andthatthesimultaneousmonitoringofnoble‐andsignalcrayfisheDNAprovidesadditionalinformationon habitat status that otherwisemust be obtained from separateCPUEsurveys.Consequently,weproposethateDNAmonitoringofthethreespecieswillproveasuitable,non‐invasiveandanimalwel-farefriendlyalternativetothetraditionalcagemethod.

2  | MATERIAL S AND METHODS

2.1 | Study site

The study site (Figure 1) is part of the largeHaldenwatercourse,which is149.5km longandconsistsof several lakesandconnect-ingriversandchannels.Thewatershedcovers1,584km2 and con-sistsofforestsandfarmland.TheRiverHølandselvaflowsintoLakeSkulerudsjøen(surfacearea1.7km2,retentiontime0.05year)whichconnectsandflowsintoLakeRødenessjøen(surfacearea15.3km2,retention time 0.7 year). Ørje locks are located at the outlet andsouthernendofLakeRødenessjøen(Figure1).AfterthediscoveryofA. astaci‐positivesignalcrayfishandinfectednoblecrayfishclosetoØrje locks(c.f.Table2),theNFSAextendedthecrayfishplaguecontrol zone border in theHaldenwatercourse upstream ofØrjelocks. The physicalmigration barriers (dams) in RiverHølandselva(Figure1)define thenewboarderof thecontrol zone. In thepre-sentstudy,thecontrolzoneofthewatercourseisreferredtoasthe

‘infectionzone’whilethe‘riskzone’referstotheremainingpartofthewatercourseaswellaslakesandriverswithnoblecrayfishpopu-lationsincloseproximitytotheinfectionzone(Figure1).Severalsta-tionsforcagesurveillanceandeDNAmonitoringwereestablishedandmonitoredduringsubsequentyears(2014–2017),coveringtheongoingoutbreakwithintheinfectionzone,andalsomonitoringse-lectedsitesoftheriskzone(Figure1).Trappingsurveyswereper-formedinLakeRødenessjøenin2014and2015,andcatchperuniteffort(CPUE;crayfishpertrapnight)dataforsignal‐andnoblecray-fishwereobtained.Figure2summarisesthetimelineandfrequencyofthedifferentmonitoringmethods.

2.2 | Traditional cage surveillance of crayfish plague

Fourcagestations(1–4)wereestablishedon1October2014fromupstreamofØrjelocksinthesouthtoKroksundinthenorthofLakeRødenessjøen.Eachcage (onecageperstation)containing10 livenoblecrayfishwassubmergedafewmetresfromthelake‐orrivershore.Thecagestationswerelocatedatsiteswithknowncrayfishpresenceandwerereadilyaccessibleforfrequentmonitoring.Twoadditionalcagestations(5and6)wereestablishedfurtherupstreamin thewatercourseon24April2015 (Figure1).Crayfishwereob-tainedfromalocalnoblecrayfishfarmer.Thecaptivecrayfishwereprovidedwithshelterandwerefedregularlywithbirchleavesandfish.Eachcagewasvisuallyinspectedtwiceweeklybylocallandown-erswhomanuallycountedremaininglivenoblecrayfish.Mortalityinthecageswasrecordedanddeadcrayfishcollected,frozenat−20°Cand transported to the laboratory for crayfish plaguediagnostics.Frozencrayfishwerethawed,andtissuesamplesofeye,tailmuscleandcuticleweresubjectedtoDNAextractionusingtheQIAamp® DNA mini kit on a QIAcube automated DNA extractor (Qiagen)following themanufacturers protocol.Crayfish plaguediagnosticswere performed using an A. astaci‐specific qPCR (Vrålstad et al.,2009),withmodificationsintheannealingtemperature(Kozubikovaet al., 2011). If crayfish plaguewas confirmed, the correspondingcagewasremovedfromthewatercourse.Cagesurveillance lastedfromSeptember2014toOctober2015.

2.3 | eDNA water sampling

Sixstationsforwaterfiltration(eDNAstations)wereestablishedinconjunctionwiththecagemonitoring(Figures1and2)in2014–2015.Ateachstation,threereplicatewatersampleswerefilteredon‐site,withtheexceptionofstation1in2014(thesignalcrayfishinvasionsite)whereextrawatersamples(3×3)werefilteredfromthreesitesincloseproximity.Watersampleswerecollectedat7‐to10‐dayin-tervals inOctober toNovember 2014 (Figure 2) to closely followthe initial phaseof theoutbreak. In total, 72water sampleswerecollectedatstations1–3withanaverageof6.9L/filter.NoeDNAsampleswerecollectedduringwinterduetoicecoverage.In2015,water sampleswere collected every second or fourthweek fromApriltoSeptember(Figure2)tofollowupstreammovementoftheoutbreak.Intotal,120watersampleswerecollectedatfivestations

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(stations1,3,4,5and6)fromApriltoSeptemberwithanaverageof6.0L/filter.Asincreasingfocuswasplacedonupstreammovement,station 2 was excluded after 2014. Additional stations upstreamwereestablishedandsampledinJuneandAugustof2016and2017as part of a new crayfish plaguemonitoring programme (Figure1and2).Forcost‐efficiencyreasons,onlytworeplicatewatersampleswerecollectedperstation.Intotal,55and57watersampleswerecollectedwithanaverageof3.3and4.0L/filterin2016and2017re-spectively.Generally,forallstations,thewatersamplesweretakenupstreamandatsomedistance(>20mintheriverand>200minthelake)tothenearestcagednoblecrayfishtoavoiddetectionofeDNAfromthosecrayfish.Between1and10Lwerefilteredpersampledependingon the turbidityof thewater.Thewater sampleswerecollectedabovethebed(~7cm),2–5mfromtheshore,andfiltered

directlyontoglassfibrefilters(47mm,2μmporesize,AP2504700Millipore,Billerica,MA,USA)using aperistaltic pump (MasterflexL/SorE/S,Cole‐Parmer,VermonHills, IL,USA)withTygontubing(Cole‐Parmer) and an in‐line filter holder (47mm,Millipore). Eachfilterwastransferredtoa15‐mlsterilefalcontube,storedoniceinacoolingboxuntiltransportedtothe laboratorywithin12hr,andfrozenat−20°C.Thevolumeofthefilteredwaterwasmeasuredanddiscardedontheshoreateachsite.Watersampleswerealwayscol-lectedinanupstreamtodownstreamdirectiontoavoidtransferringA. astacisporesupstream.Also,stationsoutsidetheinfectionzone(riskzone)werealwayssampledbeforestationswithintheinfectionzone(Figure1).Beforefiltrationateachstation,waterwaspumpedthroughthehoseandfilterholderforafewminutestorinseawayremainsofsporesoreDNAfromthepreviousupstreamstation,and

F I G U R E 1  ThestudysiteincludespartsofthelargeHaldenwatercourseinNorwaywithnamesforinvolvedlakes,channelsandrivers.Cagestations(greensquares)andenvironmentalDNA(eDNA)stations(bluecircles)wereestablishedsuccessivelyfrom2014to2016inasouth–northdirection,startingatthesignalcrayfishinvasionsiteatØrjelocks(boldblackline;station1).Cagestations1–6andeDNAstations1–7and12arewithintheregulatedinfectionzone,whiletheeDNAstations8–11and13–15arelocatedintheriskzone,separatedfromtheinfectionzonebymigrationbarriers(boldblacklines)suchasdamsandwaterfalls

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toavoidfilteringanydisturbedsedimentsfromthecurrentstation.After samplingof all stationswithin a zone (risk zoneor infectionzone),thetubingandfilterholderweredisinfectedwith10%bleachfor30min,followedbyrinsingwith10%sodiumthiosulfate,tore-moveDNAtraces.

2.4 | Crayfish trapping—Catch per unit effort

Twoextended surveyswith baited trapswere conducted in 2015with thesamemethodsas in thenational surveillanceprogrammeofnoblecrayfish(Johnsenetal.,2017),usingconventionaltwo‐fun-neltraps(meshsize12mm)baitedwithrawchicken(Figure2).ThefirstsurveyinAugust,comprisedof1,880trapnightswheretrapsweredistributedatdifferentsites(approximately10trapspersite)coveringmostoftheshorelineofLakeRødenessjøen.Thesecond,including960trapnightsinAugustandSeptember,coveredthesus-pectedsignalcrayfishinvasionarea.Allequipmentwasdisinfectedaftereachsamplingevent.PermissionsfortrappingA. astaci-carry-ingsignalcrayfishwereobtainedfromNEAandNFSA.

2.5 | eDNA analyses

DNAwasextractedfromfiltersusingtheCTAB(cetyltrimethylam-monium bromide) extraction protocol described by Strand et al.(2014) with minor modifications (full protocol in Appendix S1).Briefly,thefilterswerefreeze‐dried,4mlofCTABbufferwasaddedand the filterswere then fragmentedusing a pestle. The sampleswerefrozen(−80°C)andthawed(65°),followedbyadditionofpro-teinaseKandincubatedat65°Cfor60min.Chloroformwasadded,thesamplewascentrifugedandthesupernatant(3mllysate)fromeachsamplewasdividedintotwo2‐mlEppendorftubesforeasierworkflow resulting in two subsamples per filter (A& B; technicalreplicates).Anadditionalchloroformstepwasperformed,followedby isopropanolprecipitationofDNA.TheDNApelletwaswashedwithethanolbeforeresuspensionin100μlTEbuffer.DuringDNAextraction,anopentubewith200μlofMilliQwaterplacedonthelaboratory work benchwas used as a laboratory work control. AtubewithCTABbuffer (extractionblankcontrol) followed theex-traction protocol alongside the real samples. Separate laboratory

roomswereusedforpre‐andpost‐PCRprocedures(Agersnapetal.,2017)tominimiseriskoflaboratory‐inducedcontamination.

TheDNA sampleswere analysed using three different probe‐based singleplex qPCR assays referred to as Aphast, Astast and Paclen(seeTable1foraqPCRassayspecifics).AphastistheA. astaci qPCR assay adapted for detection and quantification in water(Strandetal.,2014),whileAstast and PaclenrepresentqPCRassaysforeDNAdetectionandquantificationofnobleandsignalcrayfishrespectively(Agersnapetal.,2017).AllqPCRanalyseswererunonanMx3005PqPCRsystem(Stratagene);theAphastsetupfollowedStrandetal.(2014),whileAstast and PaclenfollowedAgersnapetal.(2017)withthefollowingmodifications:weused500nMprimerand250nMprobeconcentrationand60sat56°Cforannealing/exten-sionforbothassays.

StandarddilutionseriesforA. astaci,noblecrayfishandsignalcrayfishwerepreparedusinggenomicDNA,accordingtoVrålstadet al. (2009) andAgersnap et al. (2017) (i.e. ‘theNorwegian ap-proach’).Fourcalibrationpoints (standarddilutionsrangingfrom~20pg/μlto~3pg/μlgDNAofA. astaci,and~781pg/μlto~12pg/μlgDNAofbothcrayfishspecies)wereincludedineachqPCRruntogenerateastandardcurveforquantificationofeDNAinsam-ples.FourtechnicalqPCRreplicates(i.e.twopersubsampleAandB)wereanalysedperwatersample,twoundilutedandtwo10‐folddiluted replicates. The presence or absence of qPCR inhibitionwascontrolledbycalculatingthedifferenceincyclethreshold(Ct)values (ΔCt) between the undiluted and corresponding 10‐folddilutedDNAreplicates,aspreviouslydescribed (Agersnapetal.,2017;Kozubikovaetal.,2011).Briefly, thetheoreticalΔCtvalueequals3.32intheabsenceofinhibition,butvariationisexpecteddue tominor inaccuracies in amplification efficiency,manual pi-pettingandotherstochasticfactors.Weacceptedavariancelevelof 15%, allowing for quantification in sampleswhere theΔCt is3.32±0.5(range=2.82–3.82)betweentheundilutedand10‐folddilutedreplicates.IfΔCtwaswithinthisrange,DNAcopynumberswerecalculatedas themeanof theundiluted replicatesand the10‐fold diluted replicates, the lattermultiplied by10. In caseofinhibition (ifΔCt <2.82) the estimated eDNA copy number wasbasedonlyonthe10‐folddilutedDNAreplicates,while ifΔCt>3.82 (i.e. 10‐fold dilutionout of range), the estimationof eDNA

F I G U R E 2  Timelineofthesamplingmethodsandsamplingfrequency/effort.Involvedstations(environmentalDNA[eDNA]andcage)areindicatedfordifferentperiods.eDNAwassampledat10‐dayintervalsin2014andat2‐to4‐weekintervalsin2015.Cageswithlivenoblecrayfishwerecheckedtwiceaweekbylocallandowners.TrappingwasconductedateightlocationsinLakeRødenessjøenin2014aspartofthenationalsurveillanceofAstacus astacusandin2015extendedtrappingwasconductedthroughouttheentirelake

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copynumberwasbasedon theundilutedDNA replicates alone.Ifnoneoronlyoneofthereplicateswasdetectedabovelimitofquantification (LOQ), further quantification was not performedandtheresultfortheeDNAsamplewasreportedasbelowLOQ(<LOQ)(seeTable1for limitofdetection(LOD)andLOQspecif-ics).A sample resultwasonly regardedaspositive if theoveralldetection(meanforallPCRreplicates)wasaboveLOD(Table1).FollowingKozubikovaet al. (2011) andAgersnapet al. (2017), acut‐offwassetatCt41,definingpositivesignalswithaCtvalue≥41 negative (i.e. not detected). EnvironmentalDNA copy num-bers per litrewaterwere calculated from the eDNA copy num-berquantifiedintheqPCRreactionsaccordingtoAgersnapetal.(2017)using theequation:CL= (CrAB * (Ve/Vr))/Vw.Here,CL rep-resents thecopiesofeDNAper litre lakewater,CrAB representsthecopiesofeDNAinreactionvolumesummarisedforsubsampleAandB,Verepresentsthetotalelutionvolumeafterextraction,Vr representsthevolumeofeludedextractusedintheqPCRreactionand Vw represents thevolumeof filtered lakewater.TheAphast qPCRassaytargetsthemulticopyITSnrDNA‐region(seeTable1).ThesporeconcentrationsforA. astaci (spores/L)wereestimatedaccordingtoStrandetal.(2011,2014)usingtheequation:CL/138,basedon theestimation thatonesporecontains~138copiesofthetargetDNA.

2.6 | Statistics

Estimated eDNA concentrations (CL) from station 1, 3–6 in 2015werelog10transformedandconvertedtofirst‐orderdifferencese-riestotestforcorrelationbetweeneDNAconcentrationsfromthedifferentspecies.SignalcrayfisheDNAresultswereexcludedfromthecorrelationtest,sincesignalcrayfisheDNAwasonlydetectedatstation1andatlowconcentrationandfrequency.Correlationwastestedonthefirst‐orderdifferenceseriesofeDNAconcentrations(CL)fromnoblecrayfishandA. astaciusingspearmanrankcorrela-tion.ThestatisticaltestswereruninthesoftwareRStudiov.1.1.456(RStudio team, 2016) using r v 3.5.1 (RDevelopment Core Team,2018).

3  | RESULTS

3.1 | Cage surveillance versus eDNA monitoring

eDNA monitoring revealed the crayfish plague pathogen in thewaterearlierthanthecagemethod.Allthreetargets(A. astaci,noblecrayfishandsignalcrayfish)weredetectedatloweDNAconcentra-tionsatstation1onthefirsteDNAsamplingdate(3October2014;Figure3),while8weekspassedbeforeallnoblecrayfishwerefounddeadincagestation1(A. astaciinfectionconfirmed,Table2).On22December2014,allcagedcrayfishweredeadduetocrayfishplagueatstation2(Figure3a,Table2).TableS1providesdetailsforeDNAcopynumbersforalltargets,andA. astacisporeestimates.

We observed that presence/absence data, as well as fluc-tuation ineDNAconcentrations,depictedtoa largeextent theTA

BLE

1 Overviewofthethreespecies‐specificassaysusedinthestudy,targeting

Asta

cus a

stac

us, P

acifa

stac

us le

nius

culu

s(Agersnapetal.,2017)andthecrayfishplagueagentA

phan

omyc

es

asta

ci(Vrålstadetal.,2009).Thetargetgeneregionsaremitochondrialgenomiccytochromeoxidase1(CO1)andthenucleargenomicinternaltranscribedspacer(ITS)

Spec

ies

Ass

ayTa

rget

Am

plic

onFo

rwar

d pr

imer

(5′–

3′)

Prob

e (5

′–3′

)Re

vers

e pr

imer

(5′–

3′)

Asta

cus a

stac

usAs

tast

COI

65 b

pGATTAGAGGAATAGTAGAGAG

FAM‐AGGAGTAGGGACAGGATGAACT‐

BHQ1

CTGATGCTAAAGGGGGATAA

Paci

fast

acus

leni

uscu

lus

Pacl

enCOI

65 b

pAACTAGAGGAATAGTTGAAAG

FAM‐AGGAGTGGGTACTGGATGAACT‐

BHQ1

CCGCTGCTAGAGGAGGATAA

Apha

nom

yces

ast

aci

Apha

stITS

58 b

pAAGGCTTGTGCTGGGATGTT

FAM‐TTCGGGACGACCC‐MGBNFQ

CTTCTTGCGAAACCTTCTGCTA

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biologicalstatusofthecrayfishandhabitatintermsoffreedomfrom disease, early infection, mortality and extinction. Whenthe ice cover thawed in 2015, plague‐inducedmortality in thecagewasobservedatstation33weekspriortoourfirsteDNAsampling event (24 April, Figure 3a, Table 2). Here, high lev-els of eDNA fromA. astaci and noble crayfish were detected,with a further increase 2 weeks later, followed by a declineto trace amounts in the followingweekswith no detection byAugust (Figure 3c).At station4, only low levels of noble cray-fisheDNAweredetectedon24April,whilebothnoblecrayfishand A. astaciweredetected2weeks later (May8th,Figure3c).One week later, crayfish plague‐induced mortality was ob-servedinthecage(Figure3a,Table2).ConcentrationsofeDNA

for both targets continued to increase andpeakedon22May.Again,arapiddecreasefollowed,andbytheendofJune2015,noble crayfisheDNAwasdetectedonlyat lowconcentrations,whileA. astaciwasnolongerdetected(Figure3c).FromJulytoSeptember 2015, noble crayfish eDNAwas also undetectable.Atstation5,onlyeDNAfromnoblecrayfishcouldbedetectedin April and May, while A. astaci eDNA was also detected on26 June. Noble crayfish mortalities in the cage were first ob-served18days later (Figure3a,Table2).Again,concentrationsof eDNA from noble crayfish increased in parallel with eDNAfromA. astaciduringtheoutbreakperiod(Figure3c).FromJulyto August 2015, concentrations of eDNA from A. astaci de-creased,whilenoblecrayfishcouldstillbedetected.Atstation6,

F I G U R E 3  ComparisonofenvironmentalDNA(eDNA)presence/absenceofAstacus astacus, Aphanomyces astaci and Pacifastacus leniusculusandmortalityofcagedAstacus astacus(a)atthecageandeDNAstations1–6intheHaldenwatercourse(b),withdetailsfortheeDNAconcentrationdynamicsinthewaterquantifiedforAstacus astacus(greencircles),P. leniusculus(yellowsquares)andAphanomyces astaci(redtriangles)byqPCR(c).Triangularsplitcircles(a)indicatedetectionofeDNAfromAstacus astacus(green),P. leniusculus(yellow)and Aphanomyces astaci(red)perstationin2014and2015;thesearenottobeinterpretedaspiecharts.Nodetectionisindicatedwithnocolouring.Thenumbers(a)overlayingthecirclesindicatethedatewhenAphanomyces astaciwasdetectedbyeDNA,whilethenumbersoverlayingthesquaresindicatethedateformortalityinthecagescausedbycrayfishplague(i.e.Aphanomyces astaciinfection).Thecirclesandsquares(a)depictthepooledresultsfortherespectivemonth.LOQ,limitofquantification.*Sixsamplesfrom2014andanothersixfrom2015wereexcludedduetominorcontaminationinthecontrols

1668  |    Journal of Applied Ecology STRAND eT Al.

TAB

LE 2

 OverviewofnobleandsignalcrayfishanalysedwithA

stac

us a

stac

iquantitativereal‐timePCR(qPCR).Threetissueswerescreenedpercrayfish.InfectionofA

. ast

aciisreported

atagentlevelsaccordingtoVrålstadetal.(2009),whichreflectsincreasing,semi‐quantitativeintervalsofDNAconcentrationsfoundintheinfectedcrayfishtissues.Theshadednumbers

indicatethenumbersofcrayfishwithpositivedetectionofA

. ast

acicolouredaccordingtotheagentlevel.Onlythehighestobservedagentlevelisincludedinthetableregardlessoftissue

Orig

inLo

catio

nD

ate

NV

I ref

.Cr

ayfis

h sp

ecie

s#

Ana

lyse

dA

. ast

aci

Prev

alen

ce

Age

nt le

vels

eDN

A d

etec

tion

A. a

stac

i

Neg

ativ

ePo

sitiv

e

A0

A1

A2

A3

A4

A5

A6

A7

Trapping

Rødenessjøensouth

12.0

9.20

1420

14-2

3-23

7Noblecrayfish

557%

30

11

00

00

Trapping

Rødenessjøensouth

12.0

9.20

1420

14-2

3-23

7Signalcrayfish

2100%

00

11

00

00

Cage

Rødenessjøensouth

12.0

9.20

1420

14-2

3-23

7Noblecrayfish

2100%

00

00

01

10

Ashore/Dead

Rødenessjøensouth

29.0

9.20

1420

14-2

3-26

5Noblecrayfish

2100%

00

00

10

10

Unknown

Rødenessjøensouth

29.0

9.20

1420

14-2

3-26

5Signalcrayfish

3100%

00

11

10

00

Cagemonitoring

St.1

27.1

1.20

14a

2015

-23-

46Noblecrayfish

3100%

00

00

11

10

03.1

0.20

14

Cagemonitoring

St.2

22.1

2.20

14a

2015

-23-

44Noblecrayfish

5100%

00

00

00

23

-

Cagemonitoring

St.3

03.0

4.20

15a

2015

-23-

88Noblecrayfish

3100%

00

00

00

21

24.0

4.20

15

Cagemonitoring

St.4

12.0

5.20

15a

2015

-23-

121

Noblecrayfish

3100%

00

00

21

00

08.0

5.20

15

Cagemonitoring

St.5

13.0

7.20

15a

2016

-23-

8Noblecrayfish

5100%

00

00

13

10

26.0

6.20

15

Cagemonitoring

St.6

01.0

7.20

15b

2016

-23-

7Noblecrayfish

30

30

00

00

00

04.0

9.20

15

Not

e: a Mortalitydateofcagedcrayfish.

b Threecrayfishdied,butwerenegativeforcrayfishplague.

     |  1669Journal of Applied EcologySTRAND eT Al.

eDNAfromnoblecrayfishwasdetectedfromApriltoSeptember2015 (Figure3a,c),while eDNA fromA. astaciwasdetected atlow concentration in September samples. No crayfish plague‐inducedmortality of noble crayfish was observed in this cage(Table 2), and the eDNA concentrations of noble crayfish re-mained stable throughout the sample period. No eDNA fromsignal crayfish was detected at any station other than station1 (Figure 3). The parallel increase and subsequent decrease ineDNAconcentrationsofA. astaciandnoblecrayfishcorrelatedsignificantly (rho=0.485;p=0.0043,Figure3c).TableS2pro-videseDNAcopynumbersforalltargets,andsporeestimatesofA. astaci for2015.Sixsamplesfrom2014andanothersixfrom2015wereexcludedduetominorcontaminationdetectedinthelaboratoryworkcontrolorDNAblankcontrolforthesesamplesrespectively(c.f.Figure3).

3.2 | Trapping data versus eDNA

WefoundthattrappingdataandeDNAdataareinagreementwithregardtopresence/absenceresults.Atstations2and3,noblecray-fisheDNAwasdetectedin2014(TableS1),correspondingwellwiththetrappingof135noblecrayfish(CPUE=0.86)duringthenationalsurveillance programme the same year. In Lake Rødenessjøen, notracesofeDNAfromnoblecrayfishweredetectedafterJuly2014(Tables S1–S3).No noble crayfishwere caught duringAugust andSeptember2015,despite2,840trapnights,suggestinglocalextinc-tion.Attheinvasionsite(station1),only11%ofthewatersamplesanalysedfrom2014to2015werepositiveforsignalcrayfisheDNA(TablesS1 andS2). The trapping surveys suggest that signal cray-fishwererestrictedtothesouthernpartofthelakeatlowdensity.Here,110signalcrayfishwerecaughtin2015using960trapnights

F I G U R E 4  TriangularsplitcirclesindicatedetectionofeDNAfromAstacus astacus(green),Pacifastacus leniusculus (yellow)andAphanomyces astaci(red)perstationin2016and2017;thesearenottobeinterpretedaspiecharts.Nodetectionisindicatedwithnocolouring.Stations1,4,6–7and12arewithintheinfectionzone,whilethestations8–11and13–15arelocatedintheriskzone,separatedfromtheinfectionzonebymigrationbarriers(boldblacklines)suchasdamsandwaterfalls.Theonlychangefrom2016to2017isfoundatstation6,whereeDNAfromAphanomyces astaci wasdetectedonlyin2016

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(CPUE = 0.12), and only large individuals were trapped (average118.2mm,N=91),suggestingtheirrecentrelease.

3.3 | Implementing eDNA monitoring

The comparative data obtainedwith eDNAmonitoring and tradi-tionalmethods(cagesandtrapping)convincedtheauthoritiestoof-ficiallyincludeeDNAasamonitoringmethod.Thus,in2016,eDNAwasofficially integrated into thenationalcrayfishplaguemonitor-ing programme commissioned byNFSA. Cageswere only used inthe risk zone (data not shown), and cage surveillancewas discon-tinuedfrom2017.TheeDNAmonitoringfocusshiftedtotheRiverHølandselva(station6–7),andupstreamlocations(station8–15)inadditiontostations1and4(Figure1).Severalnewstations(8–10,13–15)wereestablishedintheriskzonetomonitorpotentialspread.NoblecrayfisheDNAwasdetectedatall stations in the risk zone(Figure4,TableS3),whilenosignalcrayfishorA. astacieDNAwasdetectedhere. In theRiverHølandselva,eDNAfromA. astaci and noblecrayfishwasdetectedattheoutletoftheriverin2016(sta-tion6),whileonlyeDNAfromnoblecrayfishwasdetectedfurtherupstream in the river (station7) (Figure4).At station4, eDNAofA. astaciandnoblecrayfishwasnolongerdetected,andin2017,allsignsofA. astacihaddisappearedfromallstationswiththeexcep-tionofstation1(Figure4).Atstation1,eDNAfromsignalcrayfishand A. astaciwasstilldetected(Figure4).TableS3providesdetailsforeDNAdetectionfrequencyforalltargetsfor2016–2017.

4  | DISCUSSION

eDNAmonitoringprovidesareliable,non‐invasive,ethicalandani-malwelfarefriendlyalternativetocagemonitoringforearlydetec-tion of crayfish plague. During the predicted freshwater crayfishdisaster in theNorwegianHaldenwatercourse, we demonstratedthateDNAmonitoringcanrevealtheinvasionofsignalcrayfishatlowdensities,aswellaslownumbersofwaterborneinfectiousA. astaci spores 2–3 weeks prior to observation of mortality in cage‐heldsusceptiblecrayfish.Furthermore,eDNAmonitoringislesslikelytospreadA. astacithantraditionalmethods.Asadirectconsequenceofthepresentstudy,eDNAmonitoringhasbeenadoptedincrayfishplaguediseasemanagement inNorway (Vrålstad,Rusch, Johnsen,Tarpai,&Strand,2018;Vrålstadetal.,2017).Wealsoconfirmedtheefficacy of simultaneous eDNAmonitoring of three target organ-isms,representedinthisstudybyaRedlistspecies,aninvasivespe-ciesandaharmfulpathogen,whichhasrecentlybeendemonstratedfor invasivesignalcrayfish,endangeredwhite‐clawedcrayfishandthecrayfishplaguepathogenintheUK(Robinson,Webster,Cable,James,&Consuegra,2018).

eDNAmonitoringprovidesasnapshotofthecrayfishandhabitatstatus,suchasinvasion,infectionandextinction.AfterthediscoveryoflowsignalcrayfisheDNAlevels(earlyinvasionstate),therepeat-edlyobservedandsignificantlycorrelatedincreaseandsubsequentdeclineofeDNAfromA. astaciandnoblecrayfishspanningonlyafew

weeksateachstationdepict theacutediseasesituation (infectionoutbreak)followedbylocalnoblecrayfishextinction.IncreasedlevelsofnoblecrayfisheDNAduringthecrayfishplagueoutbreakcouldbecausedbydecayofdeadnoblecrayfish,resultinginincreasedeDNAreleasetotheambientwater.However,behaviouralchanges,suchasuncoordinatedspasmodiclimbtremors(Aldermanetal.,1987),lossofnocturnality (Westman,Ackefors,&Nylund,1992), reducedes-capereflexandprogressiveparalysis(OiE,2017)makenoblecrayfisheasierprey.Increasedfeedingoncrayfishbypredatorsmayalsocon-tributetoincreasedeDNAshedding.Therapiddeclineanddisappear-anceofA. astacieDNAalsosupportspreviousstudiesshowingthatA. astacihasashortlifespanoutsideitshost(Svensson&Unestam,1975;Unestam,1966).TherapidtransmissionofcrayfishplagueandthesubsequentlossofnoblecrayfishthroughoutLakeRødenessjøen(15.95 km2), Lake Skulerudsjøen (1.82 km2) andRiverHølandselvafromSeptember2014toAugust2015,demonstratesthedevastat-ingeffectofcrayfishplagueonindigenousEuropeancrayfishpopu-lations (Holdichetal.,2009;Söderhäll&Cerenius,1999;Svoboda,Mrugala,Kozubikova‐Balcarova,&Petrusek,2017).TherapidspreadofA. astacithroughoutthelakescanbefacilitatedbyseveralfactors,includinganenormousbloomofinfectiousswimmingzoosporespro-ducedfromeachdyingcrayfish individual (Makkonenetal.,2013),andwind driven currents leading to rapid spread from crayfish tocrayfishinthepopulation.Furthermore,fishfeedingondiseasedanddyingcrayfishactaslong‐distancevectorssinceA. astacisurvivethepassagethroughthefishgut(Oidtmann,Heitz,Rogers,&Hoffmann,2002).However,despitetherapidspreadthroughoutthetwolakes,theoutbreakwasstillactiveinRiverHølandselva1yearafterinitialinfection.Advancementof spread then slowed,most likelydue toslowerupstreamspreadinaflowingrivercombinedwiththeabsenceorverylowdensityofnoblecrayfish,workingasbarriersforfurtherspread. Infact, thecrayfishplagueseeminglyburntout,as it isnolongerdetectableintermsofeDNAin2017.

OurstudyindicatesthattrappingdataandeDNAdataarecom-parablewhen used tomeasure the presence/absence, but do notalways agree for measuring biomass. Relatively low CPUE mea-surements (0.15–1.8; Johnsen et al., 2017) correlatedwith a highfrequencyofpositiveeDNAsamplesfornoblecrayfish,whilenega-tivetrappingresults(2,840trapnights)thefollowingautumnwereconfirmed by negative noble crayfish eDNA results. These twofactors togetherprovidedstrongevidence for localnoblecrayfishextinction.Lowdensitiesofsignalcrayfishonlyattheinvasionsite(CPUE=0.12)correlatedwithinfrequenteDNAdetectionofsignalcrayfish in11%ofthesamples,whichdemonstratesthat it ispos-sible todetect freshwatercrayfishatvery lowdensities ina largelakebymeansofeDNA.These results are similar to the studybyDoughertyetal.(2016),where10%oftheeDNAsampleswerepos-itivefortheinvasivefreshwatercrayfishFaxonius rusticus ina lakewithaCPUEvalueof0.17.Ourresultssupport theconclusionsofRobinsonetal.(2018)whodetectedendangerednativecrayfishinareasinwhichtrappingfailed,andsuggestedeDNAassuitablefordetectionofnativeandinvasivecrayfishandtheirinfectionstatusinarapid,costeffectiveandhighlysensitiveway.

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FalsenegativesresultingfromPCRinhibitionarealwaysariskwithenvironmentalsamples.ThewaterinHaldenwatercourseisrelativelyturbid(e.g.LakeSkulerudsjøenandLakeRødenessjøenhadaverageseccidepthsof1.2and1.6m,respectively,in2016).FilteringlargervolumesofwatermightincreasetheriskofinhibitionduringPCR,duetothepresenceofPCRinhibitorssuchashumicacids.Alloursampleswererunbothundilutedand10‐folddilutedinordertoaccountforPCR inhibition,andseveralsamplesshowedsignsof inhibition (dif-ference inCtvaluesof<2.85).Thismay insomecaseshave led tounderestimationoftheactualeDNAconcentrationofsomesamplesinthisstudy.Additionally,thepresenceoflowlevelsofeDNAfromcrayfishmaybemaskedinsomesamplesduetoinhibitionofthePCRreaction.RecentstudiessuggestthattheuseofddPCRincreasesthedetectionrateofeDNAcomparedtoqPCR,especiallyat lowDNAconcentrations,andismorerobustagainstinhibition(Doi,Takahara,etal.,2015;Doi,Uchii,etal.,2015).ddPCRalsooffersabsolutequan-tificationandprecisemultiplexing(twoormoretargetsinthesamereaction) (Whale, Huggett, & Tzonev, 2016). Adopting the existingassaystodevelopamultiplexassayforeDNAdetectionofallthreespeciesinasinglereactionwouldthusbebeneficial.Additionally,fu-tureeDNAstudiesshouldalsobedesignedtoincorporateoccupancymodellingtoestimatethedetectionsensitivityusingtraditionalsur-veillanceandeDNAmonitoring(Schmelzle&Kinziger,2016).

An important goal of this studywas to contribute to the re-ductionorreplacementoflivecrayfishincrayfishplaguemonitor-ing.Asadirectresult,NFSAreplacedcagesurveillanceofcrayfishplaguewith eDNAmonitoring, contributing to the3Rs (replace-ment, reduction, refinement; https://www.nc3rs.org.uk/the‐3rs)and improved animal welfare. From 2018, NEA has also imple-mentedeDNAmonitoringofnoblecrayfishandsignalcrayfishasasupplementtothetraditionalCPUEsurveillance,whichalsoin-creasesthenumberofsurveyedwatercourses.Asthereisnocureforcrayfishplague,itisessentialtominimisetheriskofspreadingthepathogentonewareas.SinceA. astaci isanotifiablediseaseinNorway,nationallegislationdemandsmonitoringmeasuresandcontrolstrategiestoreducetheriskoffurtherspread.Othercoun-triesinEuropemayalsochoosetomonitorcrayfishplague,sincethisisalsoanOiE‐listed,notifiabledisease(OiE,2017).MitigationstrategiesinNorwayincludearearestrictions,prohibitingcrayfishtrapping,increasingpublicawarenessandmandatorydisinfectionof equipment.We advocate the use of the presented approachfor early warning and targeted surveillance of non‐indigenouscrayfish species and crayfish plague in natural habitats, and fordeterminationofthemagnitudeofanoutbreak.Itcanalsobeusedforimprovedconservationofindigenouscrayfish,forexampleforassessinghabitatstatusforcrayfishrestockingpurposesorselec-tionofArksites(Nightingaleetal.,2017).

OneoftheprimarybenefitsofeDNAmonitoringinaquaticen-vironmentsisthepossibilityfortemporalandspatialmonitoringofseveralorganisms fromthesameeDNAsamples.Thisapproach ishighlyrelevantforthestudyofotherhost–carrier–pathogengroupsinmarineandfreshwaterenvironments(Bass,Stentiford,Littlewood,& Hartikainen, 2015; Rusch et al., 2018). Additionally, recurrent

samplingandlong‐timestorage(e.g.biobank)ofeDNAsamplesgivesthepossibilityforretrospectiveanalysisforotherspeciesof inter-est or evenwhole communities using environmentalmetabarcod-ing(Deineretal.,2017).Environmentalmetabarcodingmightevenreveal emerging pathogens and/or invasive species thatwould goundetectedunlessspecificallyscreenedfor,andcould identifythecausativeagentsfordeclinesinotherindigenousspecies.Inthenearfuture, technological advances will propel the eDNA monitoringconceptforward,maturingfrommanuallysampledeDNAsnapshotstoautomatedandcontinuouseDNAmonitoringinrealtime.

ACKNOWLEDG EMENTS

ThisworkwasfundedbytheNorwegianResearchCouncilthroughtheproject‘Targetedstrategiesforsafeguardingthenoblecrayfishagainstalien and emerging threats’ (TARGET; NFR‐243907), the NorwegianVeterinary Institute, theNorwegian Institute ofNatureResearch andtheNorwegianFoodSafetyAuthority.Thecageexperiments, fundedbyNFSAandpartlybyTARGET,wereconductedbyØysteinToverud(AgencyofOutlayingFields,AOF).CrayfishtrappingwasconductedbyAOFandfundedbytheNorwegianEnvironmentalAgency.SpecialthanksgotoAttilaTarpaifordesigningthemaps,andHildegunnViljugreinforsupportwiththestatistics.DevelopmentofthecrayfishprimersweresupportedbytheDanishInnovationFoundation104‐2012‐1.

AUTHORS’ CONTRIBUTIONS

D.A.S., S.I.J. and T.V. designed the study; D.A.S., J.C.R., S.A.,W.B.L.,S.W.K.andP.R.M.contributedtothemethoddevelopment;D.A.S.,J.C.R.,S.I.J.andT.V.carriedoutthefieldwork;D.A.S.andJ.C.R.performedthemolecularanalyses;D.A.S.,S.I.J.andT.V.draftedthemanuscript,whileallotherauthorscontributedtoandapprovedthefinalversion.

DATA ACCE SSIBILIT Y

Data available via the Dryad Digital Repository https://doi.org/10.5061/dryad.vf86jb2(Strandetal.,2019).

ORCID

David A. Strand https://orcid.org/0000‐0001‐8804‐7994

Johannes C. Rusch https://orcid.org/0000‐0003‐3935‐3009

Sune Agersnap https://orcid.org/0000‐0001‐9193‐8069

Steen Wilhelm Knudsen https://orcid.org/0000‐0003‐0428‐9940

Peter Rask Møller https://orcid.org/0000‐0002‐0177‐0977

TrudeVrålstad https://orcid.org/0000‐0002‐5859‐8039

R E FE R E N C E S

Agersnap,S.,Larsen,W.B.,Knudsen,S.W.,Strand,D.,Thomsen,P.F.,Hesselsoe,M.,…Moller,P.R.(2017).Monitoringofnoble,signalandnarrow‐clawedcrayfishusingenvironmentalDNAfromfreshwater

1672  |    Journal of Applied Ecology STRAND eT Al.

samples. PLoS ONE, 12, e0179261. https://doi.org/10.1371/journal.pone.0179261

Alderman,D.J.,Polglase,J.L.,&Frayling,M.(1987).Apahanomyces astaci pathogenicityunder laboratoryandfieldconditions.Journal of Fish Diseases, 10, 385–393. https://doi.org/10.1111/j.1365‐2761.1987.tb01086.x

Bass,D.,Stentiford,G.D.,Littlewood,D.T.J.,&Hartikainen,H.(2015).Diverse applications of environmental DNAmethods in parasitol-ogy. Trends in Parasitology, 31, 499–513. https://doi.org/10.1016/j.pt.2015.06.013

Bohmann,K.,Evans,A.,Gilbert,M.T.P.,Carvalho,G.R.,Creer,S.,Knapp,M.,…deBruyn,M.(2014).EnvironmentalDNAforwildlifebiologyandbiodiversitymonitoring.Trends in Ecology & Evolution,29,358–367.https://doi.org/10.1016/j.tree.2014.04.003

Deiner, K., Bik, H.M.,Machler, E., Seymour,M., Lacoursiere‐Roussel,A.,Altermatt,F.,…Bernatchez,L. (2017).EnvironmentalDNAme-tabarcoding:Transforminghowwesurveyanimalandplantcommu-nities.Molecular Ecology, 26, 5872–5895. https://doi.org/10.1111/mec.14350

Doi, H., Takahara, T., Minamoto, T., Matsuhashi, S., Uchii, K., &Yamanaka, H. (2015). Droplet digital polymerase chain reaction(PCR) outperforms real‐time PCR in the detection of environ-mentalDNA froman invasive fish species.Environmental Science & Technology, 49, 5601–5608. https://doi.org/10.1021/acs.est.5b00253

Doi, H., Uchii, K., Takahara, T., Matsuhashi, S., Yamanaka, H., &Minamoto, T. (2015). Use of droplet digital PCR for estima-tion of fish abundance and biomass in environmental DNA sur-veys. PLoS ONE, 10, e0122763. https://doi.org/10.1371/journal.pone.0122763

Dougherty,M.M., Larson, E. R., Renshaw,M. A., Gantz, C. A., Egan,S. P., Erickson,D.M., & Lodge,D.M. (2016). EnvironmentalDNA(eDNA) detects the invasive rusty crayfish Orconectes rusticus atlowabundances.Journal of Applied Ecology,53,722–732.https://doi.org/10.1111/1365‐2664.12621

Håstein, T., & Unestam, T. (1972). Krepsepest nå i Norge. Fauna, 25,19–22.

Holdich,D.M.,Reynolds,J.D.,Souty‐Grosset,C.,&Sibley,P.J.(2009).Areviewoftheever increasingthreattoEuropeancrayfishfromnon‐indigenous crayfish species. Knowledge and Management of Aquatic Ecosystems, 394–395, 11. https://doi.org/10.1051/kmae/2009025

Johnsen,S. I.,Strand,D.,&Vrålstad,T. (2017).Nasjonalovervåkingavedelkreps ‐ presentasjon av overvåkingsdata og bestandsstatus.NINA Rapport,pp.100.(InNorwegian).

Kelly, R. P., Port, J. A., Yamahara, K. M., Martone, R. G., Lowell, N.,Thomsen,P.F.,…Crowder,L.B.(2014).HarnessingDNAtoimproveenvironmental management. Science, 344, 1455–1456. https://doi.org/10.1126/science.1251156

Kozubikova,E.,Vrålstad,T.,Filipova,L.,&Petrusek,A.(2011).Re‐exam-inationoftheprevalenceofAphanomyces astaciinNorthAmericancrayfish populations in Central Europe by TaqManMGB real‐timePCR. Diseases of Aquatic Organisms, 97, 113–125. https://doi.org/10.3354/dao02411

Lowe,S.,Browne,M.,Buoudjelas,S.,&DePoorter,M.(2004).100 of the world's worst invasive alien species a selection from the global invasive species database.Auckland,NewZealand:ISSGandSSCofIUCN.

Makkonen, J., Strand, D. A., Kokko, H., Vrålstad, T., & Jussila, J.(2013). Timing and quantifying Aphanomyces astaci sporula-tion from the noble crayfish suffering from the crayfish plague.Veterinary Microbiology, 162, 750–755. https://doi.org/10.1016/j.vetmic.2012.09.027

Nightingale, J., Stebbing, P., Sibley, P. S., Brown, O., Rushbrook, B.,& Jones, G. (2017). A review of the use of ark sites and associ-ated conservation measures to secure the long‐term survival of

White‐clawed crayfish Austropotamobius pallipes in the UnitedKingdomandIreland.International Zoo Yearbook,51,50–68.https://doi.org/10.1111/izy.12161

Norwegian Environmental Agency. (2014). Krepsepest påvist iRødenessjøen.Newspage19.09.2014.Retrievedfromhttp://www.miljodirektoratet.no/no/Nyheter/Nyheter/2014/September‐2014/Krepsepest‐pavist‐i‐Rodenessjoen/(InNorwegian).

Oidtmann, B., Heitz, E., Rogers, D., & Hoffmann, R. W. (2002).Transmission of crayfish plague.Diseases of Aquatic Organisms,52,159–167.https://doi.org/10.3354/dao052159

OiE. (2017). Infection with Aphanomyces astaci (Crayfish plague).Chapter2.2.2.ManualofDiagnosticTestsforAquaticAnimals2017(ed.W.O.f.A.Health),pp.1‐18.Paris:OiE (Office InternationaldesEpizooties), Office International des Epizooties. Retrieved fromhttp://www.oie.int/international‐standard‐setting/aquatic‐manual/access‐online/

RDevelopmentCoreTeam.(2018).R:Alanguageandenvironmentforstatistical computing. Vienna, Austria: R Foundation for StatisticalComputing.Retrievedfromhttp://www.R‐project.org

Ramirez‐Castillo, F. Y., Loera‐Muro, A., Jacques, M., Garneau, P.,Avelar‐Gonzalez,F.J.,Harel,J.,&Guerrero‐Barrera,A.L. (2015).Waterborne pathogens: Detection methods and challenges.Pathogens, 4, 307–334. https://doi.org/10.3390/pathogens4020307

Robinson,C.V.,Webster,T.M.U.,Cable,J.,James,J.,&Consuegra,S.(2018). Simultaneous detection of invasive signal crayfish, endan-geredwhite‐clawedcrayfishandthecrayfishplaguepathogenusingenvironmentalDNA.Biological Conservation,222,241–252.https://doi.org/10.1016/j.biocon.2018.04.009

RStudioTeam. (2016).RStudio: Integrateddevelopment forR.Boston,MA:RStudio,Inc.Retrievedfromhttp://www.rstudio.com/

Rusch, J. C.,Hansen,H., Strand,D.A.,Markussen, T.,Hytterod, S.,&Vrålstad,T. (2018).Catching the fishwith theworm:Acase studyoneDNAdetectionofthemonogeneanparasiteGyrodactylussala-ris and twoof itshosts,Atlantic salmon (Salmo salar) and rainbow trout(Oncorhynchus mykiss). Parasites & Vectors,11,333.https://doi.org/10.1186/s13071‐018‐2916‐3

Schmelzle, M. C., & Kinziger, A. P. (2016). Using occupancy model-ling to compare environmental DNA to traditional field meth-ods for regional‐scale monitoring of an endangered aquaticspecies. Molecular Ecology Resources, 16, 895–908. https://doi.org/10.1111/1755‐0998.12501

Shaw, J.L.A.,Clarke,L. J.,Wedderburn,S.D.,Barnes,T.C.,Weyrich,L.S.,&Cooper,A. (2016).ComparisonofenvironmentalDNAme-tabarcodingandconventionalfishsurveymethodsinariversystem.Biological Conservation, 197, 131–138. https://doi.org/10.1016/j.biocon.2016.03.010

Söderhäll,K.,&Cerenius,L.(1999).Thecrayfishplaguefungus:Historyandrecentadvances.Freshwater Crayfish,12,11–35.

Strand,D.A.,Holst‐Jensen,A.,Viljugrein,H.,Edvardsen,B.,Klaveness,D.,Jussila,J.,&Vrålstad,T.(2011).Detectionandquantificationofthe crayfish plague agent in naturalwaters:Directmonitoring ap-proachforaquaticenvironments.Diseases of Aquatic Organisms,95,9–17.https://doi.org/10.3354/Dao02334

Strand,D. A., Johnsen, S. I., Rusch, J. C., Agersnap, S., Larsen,W. B.,Knudsen, S. W., … Vrålstad, T. (2019). Data from: Monitoring aNorwegianfreshwatercrayfishtragedy–eDNAsnapshotsofinva-sion, infection and extinction.Dryad Digital Repository, https://doi.org/10.5061/dryad.vf86jb2

Strand, D. A., Jussila, J., Johnsen, S. I., Viljamaa‐Dirks, S., Edsman, L.,Wiik‐Nielsen,J.,…Vrålstad,T. (2014).Detectionofcrayfishplaguespores in large freshwater systems. Journal of Applied Ecology, 51,544–553.https://doi.org/10.1111/1365‐2664.12218

Strand,D.A.,Jussila,J.,Viljamaa‐Dirks,S.,Kokko,H.,Makkonen,J.,Holst‐Jensen,A.,…Vrålstad,T.(2012).Monitoringthesporedynamicsof

     |  1673Journal of Applied EcologySTRAND eT Al.

Aphanomyces astaci in theambientwaterof latentcarriercrayfish.Veterinary Microbiology, 160, 99–107. https://doi.org/10.1016/j.vetmic.2012.05.008

Svensson, E., & Unestam, T. (1975). Differential induction of zoo-spore encystment and germination in Aphanomyces astaci,Oomycetes. Physiologia Plantarum, 35, 210–216. https://doi.org/10.1111/j.1399‐3054.1975.tb03895.x

Svoboda, J., Mrugala, A., Kozubikova‐Balcarova, E., & Petrusek, A.(2017). Hosts and transmission of the crayfish plague pathogenAphanomyces astaci:Areview.Journal of Fish Diseases,40,127–140.https://doi.org/10.1111/jfd.12472

Taugbøl, T. (2004). Reintroduction of noble crayfish Astacus asta‐cus after crayfish plague in Norway. Bulletin Francais de la Peche et de la Pisciculture, 372‐373, 315–328. https://doi.org/10.1051/kmae:2004006

Thomsen,P.F.,&Willerslev,E.(2015).EnvironmentalDNA–Anemerg-ing tool in conservation formonitoring past and present biodiver-sity. Biological Conservation, 183, 4–18. https://doi.org/10.1016/j.biocon.2014.11.019

Tréguier,A.,Paillisson,J.‐M.,Dejean,T.,Valentini,A.,Schlaepfer,M.A.,&Roussel,J.‐M.(2014).EnvironmentalDNAsurveillanceforinverte-bratespecies:AdvantagesandtechnicallimitationstodetectinvasivecrayfishProcambarus clarkii in freshwaterponds.Journal of Applied Ecology,51,871–879.https://doi.org/10.1111/1365‐2664.12262

Unestam,T.(1966).Chitinolytic,cellulolytic,andpectinolyticactivityinvitroofsomeparasiticandsaprophyticOomycetes.Physiologia Plantarum,19,15–30.https://doi.org/10.1111/j.1399‐3054.1966.tb09070.x

Valentini,A.,Taberlet,P.,Miaud,C.,Civade,R.,Herder,J.,Thomsen,P.F.,…Dejean,T.(2016).Next‐generationmonitoringofaquaticbiodiver-sityusingenvironmentalDNAmetabarcoding.Molecular Ecology,25,929–942.https://doi.org/10.1111/mec.13428

Vrålstad,T.,Johnsen,S.I.,Fristad,R.F.,Edsman,L.,&Strand,D.(2011).Potent infection reservoirof crayfishplaguenowpermanently es-tablishedinNorway.Diseases of Aquatic Organisms,97,75–83.https://doi.org/10.3354/Dao02386

Vrålstad, T., Knutsen, A. K., Tengs, T., & Holst‐Jensen, A. (2009). AquantitativeTaqMan (R)MGBreal‐timepolymerasechain reactionbasedassayfordetectionofthecausativeagentofcrayfishplagueAphanomyces astaci. Veterinary Microbiology,137,146–155.https://doi.org/10.1016/j.vetmic.2008.12.022

Vrålstad,T.,Rusch,J.,Johnsen,S.I.,Tarpai,A.,&Strand,D.(2018).Thesurveillance programme for Aphanomyces astaci in Norway 2017. Annual Report.NorwegianVeterinaryInstitute

Vrålstad, T., Strand, D. A., Grandjean, F., Kvellestad, A., Håstein, T.,Knutsen, A. K., … Skaar, I. (2014).Molecular detection and geno-typing of Aphanomyces astaci directly from preserved crayfishsamples uncovers the Norwegian crayfish plague disease history.Veterinary Microbiology, 173, 66–75. https://doi.org/10.1016/j.vetmic.2014.07.008

Vrålstad,T.,Strand,D.,Rusch,J.,Toverud,Ø.,Johnsen,S.I.,Tapai,A.,…Gjevre,A.‐G.(2017).ThesurveillanceprogrammeforAphanomyces astaci in Norway 2016. Annual Report. Norwegian VeterinaryInstitute.

Westman,K.,Ackefors,H.,&Nylund,V.(1992).Kräftor – Biologi, odling og fiske.Ystad:Kiviksgårdensförlag.(InSwedish).

Whale,A.S.,Huggett,J.F.,&Tzonev,S.(2016).Fundamentalsofmulti-plexingwithdigitalPCR.Biomolecular Detection and Quantification,10,15–23.https://doi.org/10.1016/j.bdq.2016.05.002

Wittwer,C., Stoll, S., Strand,D.,Vrålstad, T.,Nowak,C.,&Thines,M.(2018). eDNA‐based crayfish plague monitoring is superior toconventional trap‐based assessments in year‐round detectionprobability. Hydrobiologia, 807, 87–97. https://doi.org/10.1007/s10750‐017‐3408‐8

SUPPORTING INFORMATION

Additional supporting information may be found online in theSupportingInformationsectionattheendofthearticle.

How to cite this article:StrandDA,JohnsenSI,RuschJC,etal.MonitoringaNorwegianfreshwatercrayfishtragedy:eDNAsnapshotsofinvasion,infectionandextinction.J Appl Ecol. 2019;56:1661–1673. https://doi.org/10.1111/1365‐2664.13404