Trash to treasure: assessing viability of wing biopsies for use in bat genetic research

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Trash to treasure: assessing viability of wing biopsies for use in bat genetic research

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Title:
Trash to treasure: assessing viability of wing biopsies for use in bat genetic research
Series Title:
Conservation Genetics Resources
Creator:
Manjerovic, Mary Beth
Green, Michelle L.
Miller, Andrew N.
Novakofski, Jan
Mateus-Pinilla, Nohra E.
Publisher:
Springer
Publication Date:
Language:
English
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1 online resource

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Subjects / Keywords:
White-nose syndrome ( lcsh )
Bats ( lcsh )
Fungi -- Cultures and culture media ( lcsh )
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serial ( sobekcm )

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Abstract:
The outbreak of white-nose syndrome in North American bats has resulted in massive data collection efforts to characterize the fungus, Pseudogymnoascus destructans. Wing biopsies routinely are collected from live bats, placed in agar media to culture the fungus, and ultimately discarded. We tested whether these discarded tissues represent a viable source of host bat DNA. We found no difference in DNA concentration and no reduction of DNA quality between samples that were extracted immediately compared to samples placed in agar for fungal culture. Although recovered quantities were low, concentrations increased using a cleanup kit. Our study suggests samples collected from live bats can be leveraged across disciplines to further our understanding of bat genetics and the impact of white-nose syndrome.

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University of South Florida Library
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University of South Florida
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All applicable rights reserved by the source institution and holding location.
Resource Identifier:
K26-05074 ( USFLDC DOI )
K26.5074 ( USFLDC handle )

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TECHNICALNOTE Trashtotreasure:assessingviabilityofwingbiopsiesforuse inbatgeneticresearch MaryBethManjerovic € MichelleL.Green € AndrewN.Miller € JanNovakofski € NohraE.Mateus-Pinilla Received:18February2014/Accepted:27December2014/Publishedonline:13January2015 TheAuthor(s)2015.ThisarticleispublishedwithopenaccessatSpringerlink.com Abstract Theoutbreakofwhite-nosesyndromeinNorth Americanbatshasresultedinmassivedatacollection effortstocharacterizethefungus, Pseudogymnoascusdestructans .Wingbiopsiesroutinelyarecollectedfromlive bats,placedinagarmediatoculturethefungus,andultimatelydiscarded.WetestedwhetherthesediscardedtissuesrepresentaviablesourceofhostbatDNA.Wefound nodifferenceinDNAconcentrationandnoreductionof DNAqualitybetweensamplesthatwereextractedimmediatelycomparedtosamplesplacedinagarforfungal culture.Althoughrecoveredquantitieswerelow,concentrationsincreasedusingacleanupkit.Ourstudysuggests samplescollectedfromlivebatscanbeleveragedacross disciplinestofurtherourunderstandingofbatgeneticsand theimpactofwhite-nosesyndrome. Keywords White-nosesyndrome Genetics Bats Biopsy Fungalculture DNA White-nosesyndrome(WNS)haskilledmillionsofbats sinceitwasrstdetectedin2006(USFishandWildlife Service 2012 ),promptingeffortstocollectsamplesto betterunderstandthisfungaloutbreak.Samplinghibernatingbatspresentsinterestingchallenges.Animalsare slowmovingandaggregatedallowingformultiplesamples fromasmallarea.However,unnecessaryarousalsresultin signicantenergyexpenditures(Thomasetal. 1990 ; Speakmanetal. 1991 )sonumberofsamplingevents shouldbeminimized.Samplinglogisticsarefurthercomplicatedbecausehumanscanspreadfungalsporesandcave accessoftenisrestricted(USFishandWildlifeService 2011 ).Therefore,researcheffortsareconstrainedinthe numberofanimalsandlocationsthatcanbesampledand achievingrequiredsamplesizesisdifcult. Thesmallsizeofbatsmustbeconsideredwhencollecting tissue.Wingbiopsiesareroutinelyusedfordiagnosticsand conrmationofWNS.Federalguidelinesrecommendnomore thantwo,35mmwingpunchesshouldbetakenfromeach bat( http://www.nwhc.usgs.gov/disease_information/whitenose_syndrome/USGS_NWHC_Bat_WNS_submission_ protocol.pdf ).TheUSGSNationalWildlifeHealthCenter recommendsusingonebiopsyfromeachwingtodetect fungalpresenceeitherthroughdirectcultureorPCRmethods,precludingadditionalgeneticsampling. Tomaximizetissuescollectedfromlivebats,we determinedifsamplescollectedfor P.destructans research canbeutilizedforadditionalresearchprojectsonbat genetics.Tissuesareplacedinagarmediumtodetermine fungaldiversityviaculturingandtraditionallydiscarded onceculturesareidentied.HerewemeasuredDNA quantityandqualityoftissuesamplesthatunderwent fungalculturecomparedtofreshsamplestodeterminetheir utilityforgeneticsresearch. Weobtainedsamplesfrom15batscollectedforthe IllinoisDepartmentofPublicHealthrabiessurveillance program.Wereplicatedbiopsyprocedures,collectingtwo 3mmwingbiopsies(Fig. 1 a).DNAfromonewingpunch M.B.Manjerovic M.L.Green J.Novakofski DepartmentofAnimalSciences,UniversityofIllinoisUrbanaChampaign,1503S.MarylandDrive,Urbana,IL61801,USA M.B.Manjerovic M.L.Green A.N.Miller N.E.Mateus-Pinilla( & ) IllinoisNaturalHistorySurvey,UniversityofIllinoisUrbanaChampaign,1816S.OakStreet,Champaign,IL61820,USA e-mail:nohram@illinois.edu PresentAddress: M.B.Manjerovic LincolnParkZoo,2001NorthClarkStreet,Chicago,IL60614, USA 123 ConservationGenetResour(2015)7:325327 DOI10.1007/s12686-014-0417-z

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wasextractedimmediately,whiletheotherwasplacedin eitherSabouraudsorPotatoDextroseAgarandstoredat 7 Cfor2weeks,replicatingthecultureprotocolusedfor WNSdetection(Fig. 1 b).After2weeks,tissueswere removedfromagarandplacedin95%ethanol.Samples arehereafterreferredtoasfresh'andagar'. WeextractedDNAusingaQuickgDNAminiprepkit (ZymoResearchCorp.,Irvine,CA,USA)followingmanufacturer'sprotocol.Weelutedsamplesin50 l Lvolumes andestimatedtotalDNAquantityandqualityusinga Nanodrop2000(NanodropTechnologies,Wilmington, DE,USA).WemeasuredtotalDNAyieldandpurityfollowingmethodsofGreenetal.( 2013 ).WeusedZymo Clean&Concentratorkit(e.g.cleanupkit')topurify DNAandrepeatedthemeasurementsandcalculationsfor DNAquantityandquality.Weveriedrecoveryofhost DNAbyamplifyingPCRproductsusingmicrosatelliteloci EF5,EF6,EF21(Vonhofetal. 2002 ),B22,E24,andF19 (CastellaandRuedi 2000 );productsfromfreshandagar tissueswerewithintheexpectedsizeranges. Wetestedwhetherdatametnormalityassumptions usingaKolmogorovSmirnovtestbeforecomparing quantityandqualityofDNAfromfreshandagarsamples, beforeandaftercleanup.WetestedfordifferencesinDNA quantityandqualityusinga t testandcomparedaverage concentrationsandabsorbanceratiosbetweensamples beforeandaftercleanupusinganANOVA.Allstatistical analyseswerecompletedinSASv.9.3(Cary,NC,USA). Alldatametnormalityassumptions.WefoundnodifferenceinDNAquantitybetweenfreshandagarsamples whetherusingDNApriortocleanup(t28= 0.49; P = 0.63)orpostcleanup(t28= 1.12; P = 0.27).Wealso foundnodifferenceinDNAqualitybetweenfreshandagar samplespriortocleanup(t28=1.20; P = 0.24)orpost cleanup(t28= 1.56; P = 0.13).AverageDNAconcentrationsofbothfreshandagarsamplesincreasedaftercleanup kitprocessing(Table 1 ;F = 8.3, P \ 0.01)independentof whethertheywerefreshoragar.Purityratiosdidnot changeafterusingthecleanupkit(F = 0.72, P = 0.49). HostDNAcanberecoveredfromwingbiopsiesplaced inagarfor P.destructans detectionwithnoreductionin quantityorquality,openingresearchpossibilitiesutilizing samplesthatwouldotherwisebediscarded.Thequantityof recoveredDNAwassuitablefordownstreamapplications, includingmicrosatellites,whichhavebeenampliedfrom batsusing1025ngofgenomicDNA(Trujilloand Amelon 2009 ;Burnsetal. 2012 ).DNArecoveryoffresh andagarsamplesfallswithinthesuitablerangefor microsatelliteamplicationevenwithoutpost-extraction cleanup.Thecleanupkitconcentratedsamplesandshould beusedwhenworkingwithsmalltissuesamplesbecause higherconcentrationsprovidemorecontrolduringdownstreamoptimization. Fig.1a Wingbiopsiestakenfromarchivedbatspecimen. b Bat biopsyfungalcultures Table1 TotalamountofDNAandpurityratiosrecoveredfromfreshandagartissues FreshAgar NDNA(ng/ l L)SERangeRatioSERangeNDNA(ng/ l L)SERangeRatioSERange Pre-cleanup1521.52.38.835.91.70.031.41.91519.92.37.738.51.80.041.62.0 Post-cleanup1543.46.315.2102.51.80.041.52.11533.75.96.773.81.70.061.42.0 DNA(ng/ l L)andpurityratios(ratio)aremeanvaluesoftriplicatemeasurementswithstandarderror(SE) 326 ConservationGenetResour(2015)7:325327123

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Giventhecomplexitiesinvolvedwithsamplingbat populations,tissuescollectedforfungaldetectionrepresent avaluablegeneticresource.Althoughmolecularapproachesrequireintensivesamplingefforts,theyallow researcherstoasknewquestionsatresolutionspreviously unavailable(Kellyetal. 2010 ;Greenetal. 2014 ).This researchwillpavethewayforinterdisciplinarypartnershipstoincreasetheknowledgeproducedfromasingle sampleandformolecularapproachestoenhanceour understandingofWNSecology(Archieetal. 2009 ).Acknowledgments ResearchsupportedbyUSFWSFederalAidin WildlifeRestorationProject(W-146-R)andUniversityofIllinois OfceoftheViceChancellorforResearch.TheauthorsthankDaniel RaudabaughandNicoleConwayfortheirassistance. OpenAccess Thisarticleisdistributedunderthetermsofthe CreativeCommonsAttributionLicensewhichpermitsanyuse,distribution,andreproductioninanymedium,providedtheoriginal author(s)andthesourcearecredited.ReferencesArchieEA,LuikartG,EzenwaVO(2009)Infectingepidemiology withgenetics:anewfrontierindiseaseecology.TrendsEcol Evol24:2130.doi: 10.1016/j.tree.2008.08.008 BurnsLE,BrodersHG,FrasierTR(2012)Characterizationof11 tetranucleotidemicrosatellitelociforthelittlebrownbat( Myotis lucifugus )basedoninsilicagenomesequences.ConservGenet Resour4:653655.doi: 10.1007/s12686-012-9615-8 CastellaV,RuediM(2000)Characterizationofhighlyvariable microsatellitelociinthebat Myotismyotis (Chiroptera:Vespertilionidae).MolEcolNotes9:9931011 GreenML,TingT,ManjerovicMBetal(2013)Noninvasivealternatives forDNAcollectionfromthreatenedrodents.NatSci5:1826 GreenML,ManjerovicMB,Mateus-PinillaNE,NovakofskiJ(2014) Geneticassignmenttestsrevealdispersalofwhite-taileddeer: implicationsforchronicwastingdisease.JMammal95:646654 KellyAC,Mateus-PinillaNE,DouglasMetal(2010)Utilizing diseasesurveillancetoexaminegeneowanddispersalinwhitetaileddeer.JApplEcol47:11891198.doi: 10.1111/j.1365-2664. 2010.01868.x SpeakmanJR,WebbPI,RaceyPA(1991)Effectsofdisturbanceonthe energyexpenditureofhibernatingbats.JApplEcol28:10871104 ThomasDW,DoraisM,BergeronJM(1990)Winterenergybudgets andcostofarousalsforhibernatinglittlebrownbats, Myotis lucifugus .JMammal71:475479 TrujilloRG,AmelonSK(2009)Developmentofmicrosatellite markersin Myotissodalis andcross-speciesamplicationin M. gricescens M.leibii M.lucifugus ,and M.septentrionalis ConservGenet10:19651968.doi: 10.1007/s10592-009-9869-1 USFishandWildlifeService(2011)Anationalplanforassisting states,federalagencies,andtribesinmanagingwhite-nose syndromeinbats.whitenosesyndrome.org/national-plan/whitenose-syndrome-national-plan.Accessed11Dec2013 USFishandWildlifeService(2012)NorthAmericanbatdeathtoll exceeds5.5millionfromwhite-nosesyndrome. http://www. batcon.org/pdfs/USFWS_WNS_Mortality_2012_NR_FINAL.pdf Accessed23Sep2013 VonhofMJ,DavisCS,FentonMB,StrobeckC(2002)Characterizationofdinucleotidemicrosatellitelociinbigbrownbats ( Eptesicusfuscus ),andtheiruseinotherNorthAmerican vespertilionidbats.MolEcolNotes2:167169.doi: 10.1046/j. 1471-8286.2002.00189.x ConservationGenetResour(2015)7:325327 327123


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