Environmental DNA (eDNA) survey of tilapia in the Fitzroy Basin.

ENVIRONMENTALDNA(eDNA)SURVEYOFTILAPIAINTHEFITZROY

BASIN

TansynNoble,HeatherRobson,DamienBurrows,DeanJerry

ReportNo.14/61

August2014

ENVIRONMENTALDNA(eDNA)SURVEYOFTILAPIAINTHEFITZROYBASIN

AReportforFitzroyBasinAssociation,Rockhampton

ReportNo.14/61

August2014

PreparedbyTansynNoble,HeatherRobson,DamienBurrowsandDeanJerry

CentreforTropicalWater&AquaticEcosystemResearch(TropWATER)

JamesCookUniversityTownsville

Phone:(07)47814262Email:[email protected]

Web:www.jcu.edu.au/tropwater/

Informationshouldbecitedas:Noble, T, Robson,H, Burrows,D& Jerry,DR 2014, ‘EnvironmentalDNA (eDNA) Survey of Tilapia in theFitzroyBasin’,Centre forTropicalWater&AquaticEcosystemResearch (TropWATER)Publication14/61,JamesCookUniversity,Cairns,11pp.Forfurtherinformationcontact:CentreforTropicalWater&AquaticEcosystemResearch(TropWATER)JamesCookUniversityATSIPBuilding145TownsvilleQLD4811This publication has been compiled by the Centre for Tropical Water & Aquatic Ecosystem Research(TropWATER),JamesCookUniversity.©JamesCookUniversity,2014.ExceptaspermittedbytheCopyrightAct1968,nopartoftheworkmayinanyformorbyanyelectronic,mechanical,photocopying,recording,oranyothermeansbereproduced,stored inaretrievalsystemorbe broadcast or transmitted without the prior written permission of TropWATER. The informationcontainedhereinissubjecttochangewithoutnotice.Thecopyrightownershallnotbeliablefortechnicalor other errors or omissions contained herein. The reader/user accepts all risks and responsibility forlosses,damages,costsandotherconsequencesresultingdirectlyorindirectlyfromusingthisinformation.Enquiries about reproduction, includingdownloadingorprinting thewebversion, shouldbedirected [email protected]:This investigation was funded by the Fitzroy Basin Association as part of the QLD Regional NRM Program (FB QNRM 02) for Fitzroy Pest and Weed Management, 2013-2016.

TilapiainFitzroyBasin–TropWATERReportno.14/612014

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TABLEOFCONTENTS1 OBJECTIVES..........................................................................................................................................2

2 METHODOLOGY...................................................................................................................................2

3 RESULTS...............................................................................................................................................4

3.1 NankinCreekLagoon....................................................................................................................43.2 MooresCreek...............................................................................................................................43.3 RiverRoadLagoon1.....................................................................................................................43.4 RaglanCreek.................................................................................................................................43.5 BajoolWeir...................................................................................................................................43.6 GracemereLagoon........................................................................................................................43.7 YeppenLagoon.............................................................................................................................43.8 LionCreek.....................................................................................................................................53.9 SplittersCreek..............................................................................................................................53.10BelmontCreek..............................................................................................................................53.11AlligatorCreek..............................................................................................................................53.12RossmoyaRoad............................................................................................................................53.13HedlowCreek...............................................................................................................................53.14EdenBannWeir............................................................................................................................5

4 DISCUSSION&RECOMMENDATIONS...................................................................................................7

5 REFERENCES.........................................................................................................................................8


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1 OBJECTIVESThis report presents the results of an environmental DNA (eDNA) survey of Tilapia (Oreochromismossambicus)intheFitzroyRiverBasin,QueenslandinAugust2014.Theobjectivesofthissurveywereto:

i) UsingeDNAasthedetectionmethod,determinethepresence/absenceoftilapiainvariouswaterbodieswithintheFitzroyRiverBasin.

ii) ComparetheresultsfromtheeDNAsurveywiththeresultsfromelectrofishingconductedatthesamesites.

2 METHODOLOGY

Theoverallexperimentalplanforthissurveyinvolvedsampling14siteswithintheFitzroyRiverBasinusingeDNA as the detection tool (Figure 1). Each sample collectedwas analysed for presence or absence ofTilapiaDNAusingtilapinespecificDNAmarkers.Resultswereusedtoestablishlikelihoodofthepresenceof Tilapia at each site, as well as make a quantitative comparison with the results collected viaelectrofishing.Atotalof70x2Lwatersampleswerecollectedfrom14siteswithintheFitzroyRiverbasin(seeTable1forlistofsites).Ateachsite,fiveindependent2Lwatersampleswerecollectedatapproximately20mto50mapart to ensure the site locationwas adequately covered and to increase the likelihoodof capturingTilapia DNA. Sites included lagoons, creeks and weir pools. Samples were either filtered straight aftercollection in the field, or kept in the dark and cool until processing could take place; all sampleswerefilteredwithin9hrsofcollection.ThewatersamplefiltrationprotocolusedtoconducttheeDNAsurveywasmodifiedfromJerdeetal(2011).Watersamples(2L)werevacuumfilteredthrougha20µmnylonnetfilter (Merck NY2004700), filters were then stored in the freezer until further processing back in thelaboratoryatJamesCookUniversity(JCU),Townsville.To ensure quality control of the eDNA survey all filtration equipment was sterilised with 10% bleachsolution before use. A negative sampling control was included for each site; this was a 2 L sample ofdeionisedwatertakenintothefield,storedwiththesamplesandprocessedinthesamewayasabovetotestforfieldcontamination(i.e.dunkingthefilledandcappedbottleintothewater).Furthermore,priortofiltrationofeverysample,500mlofdeionisedwaterwasfilteredthroughthefiltrationapparatus,thefilterpaperwasthenstoredandkeptforfurtheranalysis.Thisiscalledtheequipmentcontrolandisusedtotestforequipmentcontaminationforcorrespondingpositivesamples.All further processing of samples took place in theMolecular Ecology and Evolution Laboratory at JCU.DNAon the filterswasextractedand isolatedusing theBioline ISOLATE IIGenomicDNAkit (BIO-52067)followingthemanufacturersstandardprotocol.AthreephaseqPCRanalysiswasthencarriedoutforeachsample. All three phases followed the same qPCR protocol which was a 40 cycle quantitation analysisfollowedbyameltanalysis.Thequantitationanalysisidentifieswhetherasamplehasamplifiedornotandcan be compared to standard controls (samples spiked with known DNA quantities) to estimateconcentrationofthetargetspeciesDNAinthewatersample.ThemeltanalysisisusedtoconfirmthePCRproductformedisfromtilapiaDNA.ForeachrunofqPCRanalysis,positiveandnegativePCRcontrolswereruntoensurequalitycontrol.Phase1oftheqPCRanalysiswastotesteachsampleforPCRinhibitionfromcontaminantsinthewaterbyrunningsampleswithaninternalpositivecontrolincluded(QiagenRT2qPCRPrimergDNAcontrol,cat.no.330011).SamplesthatdisplayedsignsofPCRinhibition(e.g.failedtoamplify


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thespikedcontrolDNA)werethenfurthertreatedbyrunningthesamplethroughaDNAcleanupcolumn(BiolineISOLATEFecalDNAkit,BIO52082).Intotal27samplescollectedfromtheFitzroyRiverBasinrequiredfurthertreatmentduetosignsofPCRinhibition. Next, during phase 2 samples were analysed for presence or absence of tilapia eDNA byconductingaqPCRusingtilapinespecies-specificDNAprimers.EachwatersamplecollectedwasanalysedusingfivereplicateqPCRreactions.PositivereactionswereidentifiedfortilapiaDNAwhentherewasthepresenceofanamplificationcurveinthequantitationanalysis,aswellasadistinctpeakoftherightsizecomparedwiththepositivecontrolofknowntilapiaDNAinthemeltanalysis.Forallwatersampleswhichtested positive for tilapia DNA the corresponding equipment control was then analysed to test forcontamination(phase3).IftheequipmentcontrolwasclearofcontaminationthewatersamplewasthenclassifiedaspositivefortilapiaDNA.Failuretomeetanyofthesequalitycontrolsresultedinthesamplebeingclassifiedasnegative(orstatusuncertain)duetoDNAcontamination.

Figure1:LocationofsitessampledwithintheFitzroyRiverBasinduringtheeDNAsurveyoftilapia.Resultsof the eDNA surveywhich returned the positive presence of tilapia DNA are represented by images oftilapia.


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3 RESULTSOf the 14 sites sampled eight sites resulted in positive detection of tilapia eDNA, two sites had nodetectableDNAandfoursitesfailedtomeetthenecessaryqualitycontrolsandthereforeresultsfromthesesites could not be confirmed (Table 1). In all instances where tilapia were caught using electrofishing(YeppenLagoon,LionCreekandSplittersCreek)eDNAalsosuccessfullydetectedtilapiaDNAinthesewaterbodies.ResultsoftheeDNAsurveyateachsiteareasfollows:3.1 NankinCreekLagoon

Tilapia eDNA was detected in one out of the five samples collected at Nankin Creek Lagoon, all othersampleswerenegative.AllfivereplicateqPCRreactionsofthepositivesamplewereconsistentwithtilapiaDNA. Although, tilapia were not detected by electrofishing and only one eDNA water sample returnedpositive for tilapia at this site, the eDNA samplewas truly positive due to the high consistency betweenqPCR replicate reactions within the sample and that all quality controls were checked and clear ofcontamination. This resultmeans tilapia eDNAwas present at detectable levels at one sampling locationwithinNankinCreekLagoon,butnottheothersandamorethoroughelectrofishingresamplingeffortatthissiteshouldconfirmthepresenceoftilapia.3.2 MooresCreek

Tilapia were detected in one of the five eDNAwater samples collected atMoores Creek. However, thissampleconsistedofonlyonepositiveqPCRreactionoutoffivereplicatereactions.Thelowrateofdetectionat Moores creek indicates that tilapia are likely to be present, but in low abundances. Due to the lowdetection rate it is recommended this site is re-surveyed via electrofishing or eDNA analyses in order toconfirmthepositiveeDNAresult.3.3 RiverRoadLagoon1

TilapiawerenotdetectedatthissitebyeDNAorelectrofishing.3.4 RaglanCreek

Tilapia were detected in one of the five eDNA samples collected at Raglan Creek. The positive sampleconsistedofonlyonepositiveqPCRreactionoutoffivereactionsforthissample.ThelowrateofdetectioninRaglanCreekmaymeantilapiaarepresent,butinlowabundance.Becauseofthelowlevelofdetectionwerecommendthissiteisre-surveyedtoconfirmthepositiveeDNAresult.3.5 BajoolWeir

TilapiaeDNAwasdetectedintwoofthefive2LwatersamplescollectedatBajoolWeirandwithinthetwopositivewater samples, fouroutof five replicateqPCR reactionswerepositive.All quality control checkswereclearofcontamination.TilapiawerenotdetectedatBajoolWeirusingelectrofishing,however,giventhepositiveresultsoftwosamplesintheeDNAsurveywerecommendre-surveyingthissitetoconfirmthepresenceoftilapiaaseDNAanalysesindicatethespeciesbeingpresent.3.6 GracemereLagoon

TilapiawerenotdetectedatthissitebyeDNAorelectrofishing.3.7 YeppenLagoon


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Tilapia eDNA was found in three out of the five 2 L water samples collected.Within the three positivesamples found, thedetection rateof theqPCR replicate reactionswasquitehigh; all fiveqPCR reactionswerepositiveintwosamplesandanothertwoqPCRreactionswerepositiveinthethirdsample.Allqualitycontrol checks for Yeppen Lagoon were clear. Tilapia presence was also confirmed in Yeppen Lagoonthroughelectrofishing.3.8 LionCreek

All five samples returnedpositive results for tilapiaeDNA in LionCreek. ThepercentageofpositiveqPCRreactionsoverallfivesamplescombinedwas44%.ThismeansnotallqPCRreactionswerepositiveandthatsome failed to detect DNA. However, at least one ormore reactionswere positive in each of thewatersamplescollected.TilapiapresencewasalsoconfirmedinLionCreekthroughelectrofishing.3.9 SplittersCreek

AtthissitetilapiaeDNAwasdetectedinfourofthefivewatersamples.TheoveralldetectionrateoftilapiaeDNAwithinqPCRreactionswas32%,meaningnearlyathirdofqPCRreactionsweresuccessfulindetectingDNA.Tilapiawerealsoconfirmedtobepresentinthiscreekthroughelectrofishing.3.10 BelmontCreek

Contaminationof thenegative sampling control occurredat this site.All sampleswerealsopositivewith40% of the qPCR reaction replicates indicating presence of tilapia DNA. However, due to the negativesamplingcontrolbeingcontaminatedthepositiveresultsofthesamplescannotbetrusted(seediscussionin“Outcome” for an explanation of the contamination). It is recommended that this site is re-surveyed fortilapia.3.11 AlligatorCreek

Contamination of the negative sampling control also occurred at this site. Additionally, four of the fivesamplescollectedwerealsofoundtobepositivebutduetocontaminationofthenegativesamplingcontrol,thepositiveresultsfromfoursamplescannotbetrustedanditisrecommendedthissiteisre-surveyed.3.12 RossmoyaRoad

TilapiaeDNAwasdetectedinonewatersamplecollectedatthissite.ThepositivesampleconsistedoftworeplicateqPCRreactionsoutoffivebeingpositiveresultingin8%detectionrateofqPCRreactionsforthissite.Thenegativesamplingcontrolforthissitewasclearofcontamination.Giventhelowrateofdetectionaswell asother sites sampledon the samedayhaving contamination issueswe recommend re-samplingthissitetoconfirmthepositiveeDNAresults.3.13 HedlowCreek

Contaminationofthenegativesamplingcontroloccurredatthissiteandafurtheroneofthefivesampleswasalsopositive.Duetofailuretomeetqualitycontrolstheresultsof fromthissitecannotbeused.Wesuggestthissiteisre-surveyedtoconfirmpresence/absenceoftilapia.3.14 EdenBannWeir

Contaminationofthenegativesamplingcontrolalsooccurredatthissite,aswellasoneeDNAwatersamplebeingpositive.However,thispositiveresultfromtheeDNAcannotbeusedtoindicatepresenceoftilapiaduetocontaminationofthenegativecontrolatthissite.Re-samplingthissiteisrecommended.


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Table1:SitessurveyedforthepresenceoftilapiausingbothelectrofishingandeDNAsamplingapproaches.eDNA test results are presented as both an overall positive or negative assay for tilapia eDNA in eachsample(totalfivesamplespersite)andpositiveornegativeforeachqPCRreaction(total25qPCRreactionspersite).

Electrofishing eDNA Positive/Negative of WaterSamples

eDNA Positive/Negative of qPCRReactions

Site DateSampled

Tilapiapresent/absent

Number ofSamples

NumberPositive

%Positive

Number ofReactions

NumberPositive

%Positive

NankinCreekLagoon

11/08/2014 Absent 5 1 20% 25 5 20%

MooresCreek

11/08/2014 Absent 5 1 20% 25 1 4%

River RoadLagoon1

12/08/2014 Absent 5 0 0% 25 0 0%

RaglanCreek

12/08/2014 Absent 5 1 20% 25 1 4%

Bajool weirpool

12/08/2014 Absent 5 2 40% 25 8 32%

GracemereLagoon

13/08/2014 Absent 5 0 0% 25 0 0%

YeppenLagoon

13/08/2014 Present 5 3 60% 25 12 48%

LionCreek 13/08/2014 Present 5 5 100% 25 11 44%SplittersCreek

13/08/2014 Present 5 4 80% 25 8 32%

BelmontCreek

14/08/2014 Absent 5 NegativeSamplingControlContaminated

AlligatorCreek


RossmoyaRoadCreek

14/08/2014 Absent 5 1 20% 25 2 8%

HedlowCreek


Eden Bannweirpool



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4 DISCUSSION&RECOMMENDATIONSTheeDNAsurveyperformedjustaswellastheelectrofishingsurveybasedontheresultsabove.TheeDNAsurvey was able to reliably detect tilapia at the same sites where they were also caught throughelectrofishing.eDNAwasabletoalsodetecttilapiaatafurtherfivesitescomparedwhenthesesiteswereelectrofished. However, it is to be noted that some of these sites, Moores Creek, Raglan Creek andRossmoya Road, the detection rate of the eDNA was low. The low detectability at these sites may beexplained by two possibilities: a) tilapia are present in these water bodies, but in low abundances; b)alternativelylowlevelsofcontaminationmayhaveoccurredsomewhereduringcollection(seebelow).Lowrates of detection are not uncommon when working with low amounts of the target species DNA inenvironmentalwatersamples(seeFicetolaetal.2008,Jerdeetal.2011&Dejeanetal.2012).ThisiswhyhighreplicationofsamplesandPCRtestreactionsarenecessary.Thedisappointing result of four sitesBelmontCreek,Alligator Creek,HedlowCreek and EdenBannWeirPoolhavingcontaminationoftilapiaDNAinthenegativesamplingcontrolsmostlikelyoccurredfromwhentilapiawere caught in Yeppen Lagoon, Lion and Splitters Creeks. The capturing and handling of tilapia ishighlylikelytohaveleftbehindtracesofDNAonboardtheboatandthisDNAwasnoteffectivelyremovedbefore sampling took place at the next site. All four sites that were contaminated were the next sitessampledimmediatelyfollowingthecaptureoftilapia.SamplingandhandlingofwatersamplesfortheeDNAsurveyatthethesesitestookplaceonboardtheboatinthefrontdeckareawheretilapiawerepreviouslycaughtandhandled.DuetotheextremelyhighsensitivityoftheeDNAapproachanytracesoftilapiaDNAonboard the boat or released into the surroundingwater (e.g. flushed from the live fishwellwhere thetilapia were also held) are likely to be detected in the downstream qPCR tests. We suspect thatcontaminationwasoccurringon theboat immediately following captureof tilapiabyelectrofishing, as intheoneinstancefollowingelectrofishingoftilapiawherenofieldcontaminationoccurred(thatofRossmoyaRoad)eDNAsampleswerecollectedfromthebankandupstreamfromtheboatthereforeawayfromanytracesoftilapiaDNAthatmayhavebeenontheboat.ForfutureeDNAsurveys it ishighlyrecommendedthat the two sampling approaches take place separately; for example, if possible eDNA water samplingshouldtakeplacefromthebankandbecollectedpriortotheelectrofishingboatenteringthewater(suchas inTréguieretal.2014).Whensamplesareunabletobecollectedfromthebankit iscrucialtosterilizethe electrofishing boat both inside and out by washing/wiping with 10% bleach solution to avoid crosscontaminationbetweensites.Insummary,basedontheeDNAresultsandelectrofishingitappearsasiftilapiaarespreadingthroughoutthe Fitzroy River Basin. In somewater bodies they appear to be at reasonable densities as indicated byeDNA and the success of electrofishing, while in others low eDNA detection levels (but still the positivepresenceoftilapiaDNAdetected)suggestlowerabundances,ormorelocalizedinfestations.


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5 REFERENCESDejeanT,ValentiniA,MiquelC, TaberletP,BellemainE,MiaudC (2012) Improveddetectionof analieninvasivespeciesthroughenvironmentalDNAbarcoding:TheexampleoftheAmericanbullfrogLithobatescatesbeianus.JournalofAppliedEcology49:953-959Ficetola GF,Miaud C, Pompanon F, Taberlet P (2008) Species detection using environmental DNA fromwatersamples.Biologyletters4:423-425JerdeCL,MahonAR,ChaddertonWL, LodgeDM (2011) “Sight-unseen”detectionof rareaquatic speciesusingenvironmentalDNA.ConservationLetters4:150-157TréguierA,PaillissonJ-M,DejeanT,ValentiniA,SchlaepferMA,RousselJ-M,CrispoE(2014)EnvironmentalDNAsurveillanceforinvertebratespecies:AdvantagesandtechnicallimitationstodetectinvasivecrayfishProcambarusclarkiiinfreshwaterponds.JournalofAppliedEcology51:871-879

Environmental DNA (eDNA) survey of tilapia in the Fitzroy Basin.

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