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BIBD STFIIKECOMMITTEEEUROPE

KERATINPROTEIN LECTROPHORESISND THEIDENTIFICATIONF FEATHERREMAINS:NEWDEVELOPMENTSNDUPDATE

BSCE2aMP90vienn.,29 August o z september s94

HenriOuelieloepartenent de scences b ologiqlesUnive.silb e Montrealc6e patde 6123,succusaie "4"Monrrea Qu6bec), anadaH3C3J7

CanadianMlseum ot NalurePO Box 34.13, tation D"oiawa onra o, canada KlP 6P4

ABSTRACTFeather deftinlon by visuaimeans eavesa percenlage iunidenlitied ahplesallrre rolevels genusand species, ancu|any ingjeleatheEors.rial lragments.Aier an inital periodol technicaldevelopmentand esearch, ldrcphoresrsl proteins ltracted dm leathersha beenused orthe laslsi xyearswth a high relab ity and repalabiityn ealher dentincationegardless l tlieir origin. Prolenextraclionrom keratin 6 been refnedand standddsed @curcntly wnh he methods sed orelectroporz g protein oncenrales cunert resuLls llowaccuratedentiiications l unk.ow. sao ples othe specesleve proMdedhe sampescontainenooghpluhaceousorpennaceousleatlier lemeds oenraci abaLt10 !loi protlns.comparsonsol keralinproliless now easierbecaus the colleclonol gelsand keratinprolilesusedas.eierenceconprisesovef 300 Nodh Ameicanspeces represented v morethan one sanple in mostcases, or a tota of neany3,s00keralinpoiles. Results how hat here s ltleindivdual aiation and no sexualvaialron. Dilterences etween pecies l a sinoeqenus ca. be imponanland can sofrelimesbe assessed isua v but gelsare roul ney scannedwiih a lasetdenslometertotdetecting nd measuing difie.encs nveen udes Thevaluesandpatter.s husoblained an be usedlor separanng losely elaledspecLes.DiJJerenceserween pecles an be sma butare sulricientodenlt sahples Th s nelhodoLogD,s re able or eathersamplesor nore than 10 mg nol alteredbv hightemperatures r bychem calprodlcG. o!r resu s indLcate hrgrr uccess aie andprecFronnLdentlicatioFs,xceedinghe resuhs btanedeader uslngothermeansoi identitcalLon

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1. INTRODUCTIONThe dentiticationl tealhef emains anbe a difiicuh6k oanrcularlvhen he remnantseceivedare very small and badly mangrdwhen rhe sanple are large enough,direci compaison w hspclmensn referenceolltions oupled lth e macroeopic xamrnaionI vlsualclus slsuallysulficienro insure ccuratedeftilications.sing lraiqht isualcompasons rtheexternalmorphology nd disiinclive eaiur* ot fealhels,wirh or wihotr lhe use ol low optical magnificalion,no less han 500,tol the smd6 cd be identifiedby a expeaencd rnnhologisl.t is necessaryoemploydillerenr methods o idermty he remaining @/0. numberol identificationchniqueshavebeendescribed Robensonel a/. 1gaq and havebeen usedby various denliters and rhe thniqusof iderniicationol unknownsarnpiesusedby myselfor my Naiares include he examination f themicroscopicrruclure f eathers,ncluding owny arbules8rom1986, 991), sing isselio,conpound. ndscanning lectrcnmicroscopeschandler,916 Davis, 1970i ick,1937iVonkevich,1966).A cririczlevaluarion t the resulrsobtainedusing hese rechniqus,ncludingseri6 ot esls lovetily rheir accuracy,has shown hal rhe delailedand prsise identificalionof nondscripteatherscan be achievedo.ly lor an additional25qool fte sampls.hus lavingaboul25% of lhe samplBsubmfled nidentiriederos heFamilyevel. s@med ertinento explore ther echn.quesoreduce he percentageof samplesunidentifted elow he highercareqories.Alier a sei. ol lesis andexpefimeds n the lale 1980s,eleclrophoresis l reatherkeratinproteins proved o be reliable nprosding n accuratemeans l idemiti.qlhGe sample whch couldnotbe denilied lheMise.The r6ulls obtainedproved o b beyondour mo$ ambftio{]s xp*lalions and ft was decided opursue ith his echnique henhe othermeans annor rovidehe inlormationequ6led.Electophoresisoi feaiher keraln is now usedwhen t rsnol possible o idenlily otheruisemaleia al2. ME'}ODSEleclroohoresis f proteinsobtained rom the kerarinol leathershas been usd n a limitedway inlaonomicworkdd indicalesha these roleins ave imileitis amonghemselves,ave malmolecularwegtrrs,and vary rom species10species Br|.ish 976;Erushandwrtt 1983i BuschandErush 979iKnox1980a, 980br oonnell 973; lsenetal. 9a9).These esLrltshow ha| hetshnique can becomea valuablalternaive in rhe rdentificalion ! tearhersamples hat cannolbeidentifiedy othermeans.3. PROIEINX.rRACTIONwhole tealhers,or partsol teahe.s, are cleanedn barche by wdhing m hot detergentand I nsinqin hor rap water.when dry, rhey are nnsed n l{o charges ot naphtha hexanes,Fisheq,and once ndistilled atr. inally,heyarewashed qain n twochanges t denaturedlcohol. ner ie tinaldryngeach amples packagedndstoredor dure use Knox 9a0aJ.eralins extracledfom 10mgol inelycut earher md6lrom wtich shalts r calami avebeenexcludd.lo achof hesesarnples 0 ml ol the xtracrinqolutiononsistingt 0 05M iHAM (Flsher -370), Murea Fisher4204-1),o which .05M iihiothrcitolDrrJ eharmacia)sadded r he aslmomemMarsialler l1986t.The sampiesare $nred overnrgh ar room empraiure nderan amcpheie ot nilroqen.Eachreaction i{ure is ceftrituged 12,000pm,10 min;Eppendorl5415icrotugeltodimenlhefeidua fealher ragments.The supernalan s removed ndslord d 20 C.To 25lL ol eachenradedsamole, tL of 0.1MDTT s added l east10minbeiore ypinq caracdo al .198).The samplesare added o Phaimaciapolyacryloide Pha$get EF3-9,presoakedor 15 min in asoluriononsislingf 1.011 harmaciaharmalyler0, 25011 0ohNP-40, 2016ucrose, nds.Oml4. PROIEINELECTFOPHOEESISsoelecric ocusingprocedsai 2ooov, 25mA,4w. 20' C ior 400vh wilh a prefocusing haseal

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2ooov 25mA, w, 20' C lor5OVh singa PhemaciaPhast vstem eparalionndCodrolUnit'rheoelsare ained rna Phdmacia hasl v$emDeveLopnrntnit lsinq theprotaoltor PhastGelIEFsi verstarninaehrroues Pnsl Svsler Owner Manua modilredo addan e{ra s|ep usingo o r 2 5 q o D T T { P h * m a c i a ) l o r t o r r . a ' , r o ' c w l r l s d d o 6 o F f w e e . s l e p s N o 6 S d d 9 0 f l t eprotocoluppliedy he nanuladurer5 GELANALYSISAner drying he gels are examined islallv and ditterencesbetweenhe bandsoi the tracls are noledand c;pa;ed. Then each ck, I on eachgel, is processed n a laser densitomeler!KB U[ro ScanxL) in or;er ro obtainquantitativlu6 tor eachof the bandsshownon $e tracks as wellas aoraohc eoresenrationl 16e valu6. someor le bandsmav90 undetededl'rough isual;.;mnarionbeause heyappea o be used o he aoorning nesbul ce besepaaldbvlhebeamoi the densiomet;. l; ihis manner, t is possibleo oblaindin'ibution cuwes ot the valu6recordedor each samplandcompareh6e withcurvesoi unknownsamplesThe curves husobrainedavebeend;ignaed as KEFATINPFOFILES' nd ie comp:fison f unknoMkeratinprofilesaqaing prol5les btainedrom loown sampls permitshe identiiicationot unknowneathersln exact;arch cd be obtarndn mostc66 ahhough lightvariarionshaveben observedand areconsidered 0be the reuh or individual arialionor irom iation due lo the instrumemanonnd/or

6. PFIOTEINARIATIONN RELATIONO OBIGINOF FE{THEFSON BODYll waso'ornahv Jspecedharva|a||o.courd .cLr n 'eral'n rollesco(6pond'19lo nF nalure lIhe a|h;s or he. oigrnol lheoodv Tnispcs|orliwwa lesleownh wosetsol sample fof twod lerenl brds.Fealhea or o'tferemansol he oodvor a srnglendividJdl ere etecleo losubisled o theenireeletrophoresisrocess d lh r6uhs indicaGhat here s no siqniica'tdifi;rence r?aiarionn theproteinands tlhe gelsnor n hekerai' profiles llhe iealhersobrained rom he sme indivdual,regardlessof their originon he bodyln adifferenl xperiment,hen ealing th leathersongerthan 5lo 20 mmor witha lhickshalt{calamus id .&his), only he vanesare useObecausehe r6uhs showedmeeingtul ditlerencesiaween samptesaten rom hevan6 and hce conlarningrly shaitmalenal7. INTEFISEXUALAFIlATIONNo niersexualditlerencGin kerain prolilowere notedberweenndividualsol rhe sme spe''esuoon et(ensivecomoarisons i keratinprofilesoi over aoo sp6res'8 INDIVIOUALARIATIONThe.esults hainedndicalerhalhere s ntle ndividualvariatonn a given pciesAs an etamp|eeg dltterenl noiviouats t the Rrng{illed Gulis lrrrs delaM.ens's) Figure1) replsenli'g difte'eftsera andaqa were o-paredm eacl otnerand ale'verilied y compar4orargeamol6olk-rarnp olles romsevetaspec . Tl'e rdc\sol me gFls how greaiulilormrtYn e local|on 'thevari;usbandsmd theke;dn pronles reverysmiar, alrowrngor negligibrendividualariaion9. INTEFSPECIFICIFFERENCESInersp4.ncdrflerenceseMeen roselvelaredongelc6 ca ' bc mponani ' some ases noreoien 6e es$-aled r\ualry s ldrared In Fgures d ard b In hs cass tnege showsne prolEnetectrootrorat oatternsoiseven speciesot gulis (rlrus debwatq'sis L atg$ta|IJs L glaucdd$I^untienl L hv1ehDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDD 4a4nus L netnahnL L cal4omrc's L gbucascss' rlcanbe","'rarei u'"rlttv wnhoulhpad ol anvequDrenl lnald nJaberol bands r each tr\ naveco;;;n posirid dd that the olhersar; siiuatd n a difierentplaceon lhe rack Bands iat occupva smilar bosilion n a se s or acks can be interpreld6 characleristics ommono specresn a""ii" &6'i".t *r"g".v *ch as heGenusF$ilv or evenhorder rhe other ands ocaed n

10. oNc

12. FIEFEBrom T. G

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difterentplaceson the tack, are considefedo be Specis characteristics. hesedme.ences .emofeobvious n he keratin rotilesbkindhroughdenshmetric canninghan rom heexaminatEnfth gelsas shownof he eight pecies l gutlsol Fqures2.1to 2.9 In addnion,iediftsencs betwen each curuecan be neasufedas wellas dY oointon he curue.The valu6ohained can then be used or separaling losely elatedSpeciesor Speis tna havesmal dilrerencs such as is rh case wilh fte eioht speies ofgulls.certaln diliarencesbetween pec es aresmallbulare suffrcientroistinguishetweenpecres,aniculdlywhen alues recomputed ndave.aged or the peaksor highesrvalu6 ot he curves.similany,ma.kedd fierenceswereobsetuedn ihe keralinpronl6 of two populatons or he Gray{heked Ihfush (Carial6 n rtmus)(Figlres 31,3.2) Trresemarkddilterenca providedhe basicmaterial or an elaborate atonomicstudvof thsepopulaiionshichprovedo betuo dinerentpe4 (ouellet 993)10.coNclustoNThe merhodololydscribed aboveprovidesa tchniquehat allos the accufate dentiiicaion oileathersamplese!n ol smallsize.The amouft ol lealhersavarlableor analtis shouldbe in exc6sot I 0 mg and the learhe6 shouldnol have been aheredby excessivehed or degene.ated ychemica!roducts.orany eathermeeldghese 6ic requiiementsndunsuilableor dentficationbyvisualmethods ur esuhsndicarehata hrgb uccssate, ombined itha highdeg.e olprecEion,exceedrng y lar rhe resultsssured by any other dernilicalionechniques an be ananedthrough kerarineleclrophoi6is and subsequent nalysisof the gel paiternswth a scannrngdensitomete..rprese.! hedatabase ontainscre than800spcies. ajnlyromNonhAmericaand ahost 3,5@proliles(Figu.6 4 and 5). 11s hopdhar rhe rsuhs ot fte kerarinprofils n becompdenzed o generale apid and accurare omparisons i unknown amplesagain$ he proreinprofiles l a darabank. 11.11,ACKNOWLEDGMENTSI wishrc rhdk Transport anadafor fie su$ainedsuppo.lgiven o fiis prqect paniculariyo BrlceMcKrnnnoni theEnvndmenlalReview ervices,nd u Mrs.S. A. vanZylldeJong or havngperfo.medhe laboratory nalyses12.REFERENCESBrom,T. G. 1986.Mc.oscoprc dentiJicarionl teathersand leather ragmentsot PalarclicbirdsBijdraqn ierkunde,6(2)181204.Brom, G. 1991 Thediagno$ic ndphylogeneticignificancef eathertucturs Pho Th6is,Universltyl Amsterdam,stiluulTaronomi$he oologie,279ag6.Brush.A H 1976.WarerlowleatherDrorcinsanalysisot use n tdonomic srudiA Journalol ZoologvLondon. 79(4) 67-494.Brush, . H.,andH. H. W-dl1943. taordinal elalionshrpsfthe Pelicdilo.me6ndClculilormselectroohoresisi eather eratins.bis 125:141199Busch.N. E, andA. H. Brush. 979. vianearhr eratins: olecula spects l $ruduralhereroqeneityournal f Experimentaloology,10{1): 947.Carracedo. J. M.Prieto. . conchei.o. ndJ. Estelania.987 soelecric@using anems l somemammalianeratins ournal i Forensic ciences ociety, 2 1) :93 99Chandler. C.191 .A studyol hsttudureol feathers, jth eterenceorhe xonomrc igniiicanceUnive6iiv alifomia ublicaionsn zoology, 301): 43.446,337 pls T igsoavies. . 1970 Mic.omo.phologvf eathers singhe scatung eEcrronmicroscopeournar rForensic crenceseielv. 10:165174.Knox.A. G. 198oa. earherrolein s a source l avid taonomic nlormation omparativelehernistrvPhysiology,58:45-s4.Knox,A G 1980b.Appendix: eatherproteinevidenceof the relalionships i AlricanHalcyo, speieslbis.122:72 74.Marshall .. S. A. M. Paviour, . M.williams. 986SOSPAGE t enracted uman ndanimal aproleins. ages 50-353. M.J. Ouni(Ediror) l6trophorsis86.Proceedmgsi 5thMeetLng

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lftemational lclrophoresisociery.WeinheimF.B.G): CHV.laggsellschaftbH. XVlll +1 765.O'Oonnell, J. 1973. seatchor a simple erarinracrionarion.d peptidemapping l proteinsomiearher eErinsAusrralian. Blologicaici., a6(2): 01,413.Olsen, D. Fl.C. Marshall,ndA. Ga"l.1989.Felalionshipshhinhe Oenus abo: compansonlrhe electrophortic anernsof feaher prolems,Emu, a9 (4):193 203oueller,H. 1993.Bicknells hrush:donomicpositionnddistriburion. lsonBu]letr,105 4):545572.Robenson... C. Barkin. ndJ. Govan. 986. he dsriricalion t bnd earhe6.Schemeor eatheridentiricaiion.ournal f Forensic ciences ociety, 4O): 5 98.Slck,H. 1937.MorphologischjunKionellentersuchengenber dieFeinstruklurerVogeliederournaliiir Ornilhologie,a5 3): 206 372.voirkevich.. A. 1966. he earhers ndpltrmagel birds.NewYork:Ocrober ousenc pp l-wl.1 335.

Figur

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+

lled Gull {larus olg/agvarcrsrs).

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Figure .Proteinrackson gelof ividua

Figure2A.Protein racks on gelof eight speciesof NonhAmericangLrlls Larussp.).

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Figure 8.Schemarized rotein racks of eight speciesof North Americangulls (Larussp

showing ocationsof bands rom originalgel (Figute2A)

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[ . . r .Figuto 2.1 R.!.billed Gu

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F ig l re23 r candGJ :LarDs gtaucatdes lkunheh,

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Figure2.4 GaLcousGd

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Fagure .5. N eerfrann Gln

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t" , ],.,,1,,'1, ', ' i*1i'"1,,-1.,,1"'f, . .1

Figu,e 2,4 - Gblcoltw.ged G! l

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Figure3.1 GraYt.heekedThrushCathatus nhtnus

i , r l __ - ' '!,r: Figure -2 Blcknells hrush

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NUMBER FSPECIES REFERENCEIBRARYFqure4.BIFD STA

BIR

NUMBER900400700ut f500400

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i993 1994 1995 1996

NUMBER

1990 1991 1W

KERATINPROFILESFigure.

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1991 1992 1993 1994 1995 1996