. scales togetherdeterminethewingcolorationofnymphaline stacking. Thinfilms,tunedpigmentsandcombinationsofstacked observed locallyonthewingarealsoduetodegreeofscale be welltunedtothescales’ pigmentaryspectrum.Thecolours spectrum, cruciallydeterminedbythelaminathickness,appearsto lower lamina,whichactsasanopticalthinfilm.Itsreflectance orange forscales.Thestructuralcoloursarecreatedbythe colour, whichisblueinthecaseofblue,redandblackscales,but scales fromtheiradwing(lower)sidealwaysrevealedastructural scales haveaminoramountofmelaninpigment.Observingthe pigment, blackscalescontainahighdensityofmelanin,andblue contain variousamountsof3-OH-kynurenineandommochrome depending ontheirpigmentation.Theyellow, orangeandredscales the abwing(upper)sideareblue,yellow, orange,red,brownorblack, parallel ridgesandtransversalcrossribs.Isolatedscalesobservedat structured upperlamina,whichconsistsofanarraylongitudinal, approximately flatlowerlaminaconnectedbytrabeculaetoahighly of thewingscalesappearstobebasicallyidentical,withan (micro)spectrophotometry andimagingscatterometry. Theanatomy properties byapplyingscanningelectronmicroscopy, wings. We investigatedthescales’ structuralandpigmentary species-specific patterningofdifferently colouredscalesontheir belonging tothebutterflysubfamilyNymphalinae,isdue © 2014.PublishedbyTheCompanyofBiologistsLtd|JournalExperimentalBiology(2014)217,2171-2180doi:10.1242/jeb.098673 Received 18October 2013;Accepted19March2014 ‡ Cambridge, Cambridge CB30HE,UK. Physics,*Present address:Cavendish University of Laboratory, of Department Groningen, NL-9747AG Groningen,The Netherlands. Computational Physics, Institutefor Zernike Advanced Materials, University of (Srinivasarao, 1999;Kinoshitaetal.,2008;Michielsen2010). papilionids featureiridescentcoloursduetointricatephotonicsystems being themostfamousexamples,butalsomanylycaenids and colours arewidespreadamongbutterflies,with pigmentation (KinoshitaandYoshioka, 2005).Strikingstructural pigmentary origin,dependingonthescaleanatomyand its (Nijhout, 1991).Thescalecolourscanhaveastructuraland/or a create thespecies-characteristicappearanceasinpointillistpaintings numerous smallscales,eachwithadistinctcolour, whichtogether coloration patterns.Thecolourpatternsareduetoatapestry of Butterflies areuniversallyconsideredattractivebecauseoftheirbright coloration, PigmentaryScattering KEY WORDS:Xanthommatin,3-OH-kynurenine,Structural tortoiseshell), The colorationofthecommonbutterfliesAglaisurticae Doekele G.Stavenga melanin, ommochromesandwingscalestacking Coloration principlesofnymphalinebutterflies–thinfilms, RESEARCH ARTICLE INTRODUCTION ABSTRACT Author ([email protected]) for correspondence io (peacock) and ‡ , HeinL.LeertouwerandBodoD.Wilts* Vanessa atalanta Morpho (red admiral), butterflies (small the encountered simultaneously. Forinstance,thewingsofbutterflies of Nagy, 2005;Reedetal.,2008). fritillary ( erato), thezebralongwing( the paintedlady( tortoiseshell ( peacock ( nymphalid wingscaleshasbeenstudiedinconsiderabledetail in the 1997). Thesynthesisofommochromesandexpressionpatterns in determined byextractionofthepigmentswithmethanol(Nijhout, belongs tothenymphalidsubfamilyNymphalinae,have been present inthewingscalesofbuckeye, absorbance spectra.Absorbancespectraofvariousommochromes reflectance spectraaresomewhatcomplementarytothepigment’s pigment determinesthescalecolour. Consequently, measured together withthewavelength-selectiveabsorptionbyscale’s 1998; Ghiradella,2010).Lightreflectedbythesecomponents upper lamina,whichconsistsoftheridgesandcrossribs(Ghiradella, substrate) isconnectedbyaseriesofpillars(thetrabeculae)tothe in each,thelowerlamina(theadwingside,i.e.facingwing classes arediffused throughoutthescalecomponents. Ghiradella etal.,1972;Stavenga2004),buttheotherpigment are concentratedinstronglyscatteringgranules(Yagi, 1954; (Barbier, 1981;Stavengaetal.,2010).Thepterinpigmentsofpierids a fewcases,bilepigmentsandcarotenoidscolourbutterflywings coloration (Nijhout,1997;Takeuchi etal.,2005;Nijhout,2010).In ommochromes, pigmentsthatpredominantlydeterminetheirwing produce 3-hydroxy-kynurenine(3-OH-kynurenine)andvarious 1985; Wilts etal.,2012b),whichisalsousedbynymphalidsto papiliochromes, derivedfromtheprecursorkynurenine(Umebachi, the colouredscalesofpapilionidscontainanotherpigmentclass, erythropterin (Kayser, 1985;Wijnen etal.,2007).Thepigmentsof contain theviolet-andblue-absorbingxanthopterinand/or pigment, whereastheyellow, orangeandredscalesofpierids scales ofpieridscontainleucopterin,apurelyUV-absorbing White scalesmaybepresumedtounpigmented,butthewhite families. Theblackscalesofallbutterflyfamiliescontainmelanin. Wilts etal.,2012a). Michielsen etal.,2010)andpapilionids(Saranathan2010; lycaenids (Ghiradella,1998;MichielsenandStavenga,2008; 2002), orcomplexthree-dimensionalphotoniccrystals,asinsome al., 1972;Ghiradella,1989;Vukusic etal.,1999;Kinoshita in of lycaenids(Wilts etal.,2009),multilayersinthescaleridges,as (Stavenga etal.,2012),(perforated)multilayersinthescalelumen perfect thinfilms,asinthepapilionidbutterfly Structural andpigmentarycoloration mechanismsareoften The wingscalesofmostnymphalidsareessentiallyflattenedsacs; The pigmentscontributingtocolorationvaryamongthebutterfly The scaleelementscreatingstructuralcolourscanbealmost Morpho Papilio nireus Agraulis vanillae butterflies, manypieridsandnymphalids(Ghiradellaet Aglais urticae Vanessa cardui), theredpostman( , previouslycalled group haveconspicuous blackmargins ), themapbutterfly( ) (NijhoutandKoch,1991;Reed and Heliconius charitonia Inachis io),thesmall Precis coenia Graphium sarpedon Araschnia levana), ) andthegulf Heliconius , which 2171

The Journal of Experimental Biology 2172 The wingsofthesmalltortoiseshell[ neighbouring scales. found thatthewingcolorationdependsonstackingof appears tobewelltunedthescale’s pigmentation.We furthermore the scale’s colour. Thereflectancespectrumofthelowerlamina properties ofthelowerlaminaareadominantfactorindetermining various individualwingscales.We demonstratethatthethinfilm optical propertiesandpigmentationthatdeterminethecoloursof nymphaline butterflyspecies.We specificallyinvestigatedthe (Kinoshita etal.,2008). structures andthusresultinadesaturatingbackgroundsignal potentially couldbescatteredbackbythewingorotherscale multilayered ridgeseffectively absorbstransmittedlight,which example, in cases, melaninenhancesthesaturationofcoloursignal.For reflectance (Rutowskietal.,2005;Stavenga2006).Inmany strongly absorb,ridgeinterferencereflectorscontributetothe butterflies, intheshort-wavelengthrange,wherewingpigments 2012; Wilts etal.,2012b).Intheoppositeway, inseveralpierid reflected lighttotheblue–greenwavelengthrange(Trzeciak etal., papiliochrome II,andthusactsasanopticalband-filter, limitingthe light. Thethickupperlaminacontainstheviolet-absorbingpigment blue–green bandsactsasathinfilm,reflectingbroad-bandblue surrounding blue–greenbands.Thelowerlaminaofthescalesin RESEARCH ARTICLE brown–black colour, exceptfortheredadmiralwhereventral ventral (lower)sidesofthewingshaveaveryinconspicuousdull 1A–C).Incontrast,the strikingly colourfuldorsal(upper)sides(Fig. atalanta peacock [ Wing coloration and reflectance spectra colorationandreflectance Wing RESULTS

Reflectance A Here, westudiedthewingcolorationofafewcommon 0.2 0.4 0.6 0.8 0.2 0.4 0.6 0 0 300 300 G E 2 (Linnaeus 1758)],allnymphalinebutterflies,have Aglais io 1 A. urticae V. atalanta(dorsal) Morpho 3 4

400 500 400 500 B (Linnaeus 1758)]andredadmiral[ wing scales,themelanindepositedbelow 5

6 0 0 0 300 600 700800 600 700800 12 10 11 1 3 4 2 9 8 7 Wavelength (nm) A. urticae 0.2 0.4 0.6 0.8 0.2 0.4 0.6 0 0 C 300 (Linnaeus 1758)], H F A. io V. atalanta(ventral)

400 500 400 500 11 12 Vanessa 9 10

600 700800 600 700800 D approximately flatlowerlamina. crossribs areconnectedbytrabeculae(Fig. adjacent ridges,thusmarkingopenwindows(Fig. continuous withcrossribs(Fig. microribs (Fig. parallel rowsofridges(Fig. 1998; Ghiradella,2010).Theabwingsideofthescalesconsists basic bauplan,asshowninthediagramofFig. (SEM, Fig. which areprominentlyrevealedbyscanningelectronmicroscopy show numerouslongitudinallines.Theseareduetotheridges, 2A,B),both peacock, immersedinarefractiveindexfluid(Fig. wing areas.A redscaleofaadmiralandblack we investigatedsinglescalestakenfromthedifferently coloured The wingcoloursresideinthetapestryofscales,andtherefore very lowreflectance(Fig. Fig. have asimilarbiphasicshaperidingondistinctplateau(e.g. Interestingly, reflectancespectra obtainedfromwhitewingareas 420 spectra measuredfromblueareasrevealadistinctpeakaround spectra weremeasured(indicatedinFig. spectra correspondswiththenumberoflocationwhere (Fig. wavelengths andahighreflectanceinthelong-wavelengthrange characteristic long-passfeatures,i.e.alowreflectanceatshort reflectance spectraoftheyellowtored–brownwingareasshow measured thelocalspectralreflectancewithabifurcatedprobe.The optical mechanismsunderlyingthedifferent colours,wefirst forewings featuresomecoloration(Fig. Scale anatomy 13 15 14 All nymphalinescaletypesappearedtohaveessentiallythesame nm andatroughnear620 1G,H, nos10and14).Theblackareas,notsurprisingly, havea 1E–H, nos2,3,5,6,9and13);thenumbernearreflectance 16 15 14 13 5 8 7 6 16 The JournalofExperimentalBiology(2014)doi:10.1242/jeb.098673 2C).

2D, labelledm).Someofthemicroribsare correspond. ThearrowinBcorrespondstoFig. The numbersinthephotographsandspectra surfaces; and(D,H) Vanessa atalanta tortoiseshell; (B,F) bifurcated probe. wing reflectancespectrameasuredwitha Fig.

.Threecommonnymphalinebutterflies and 1.

1F–H, nos8,12and16).

2D, labelledr)withslopesfeaturing

nm (Fig.

, theredadmiral,wingdorsal 2D, labelledc),whichconnect Aglais io (A,E) Aglaisurticae V. atalanta

1E–H, nos4,7,11 and15). 1D). To unravelthevarious

, thepeacock;(C,G) 1A–D). Thereflectance

2D, labelledt)withthe , wingventralsurfaces.

2D (Ghiradella, , thesmall

2C,D). The

6C.

The Journal of Experimental Biology RESEARCH ARTICLE scales fromtheeyespotofdorsal hindwing(Fig. spectrumofblue and theyellowcurveholdsforscalesfromasmallyellowishareaon ventralwingsurface;thebluecurveisaverageabsorbance tortoiseshell. Theredandyellowcurvesarefromscalesinareas3 2, respectively, ofFig. Fig. and reflectionthatunavoidablyoccuratboundariesbetweenmedia immersed thescalesinimmersionoil,soastoreducelightscattering coloured wingareasofthethreenymphalinesFig. investigated nymphalines,weisolatedscalesfromthedifferently pigments. To identifythepigmentsinwingscalesof The scalesofFig. xanthommatin (xan). 3-OH-kynurenineitselfcanbetaken updirectlyintoscales(afterReed andNagy, 2005;Reedetal.,2008). furtherinto processed byseveralenzymesincluding vermilliontokynurenine(kyn)andcinnabarinto3-OH-kynurenine (3-OHK),whichisprocessed Drosophila and Nagy, basedonanalysesof 2005)andxanthommatin(fromNijhout,1997).Inset:hypothetical modelofommochromesynthesisinbutterflywingscales kynurenine (3-OH-kynurenine),extracted fromyellowscalesof (Fig. scales fromtheorange–redbandon thedorsalforewing(Fig. Pigments of nymphalinewingscales Pigments of A

3. Absorbancespectraofisolatedyellowtored–brownwingscales inimmersionoil(refractiveindex 1D, area 13); the yellow curve is from a small yellow area in the ventral forewing. (D) Normalised absorbance spectra of the pigments 3-hydroxy- 1D, area13);theyellowcurveisfrom asmallyellowareaintheventralforewing.(D)Normalisedabsorbance spectraofthepigments colour mutants. The ommochrome precursor tryptophan (try) is thought to be transported from the haemolymph (h) into a scale (s) cell,whereitis colour mutants.Theommochromeprecursor tryptophan(try)isthoughttobetransportedfromthehaemolymph (h)intoascale(s)

2A,B evidentlycontainverydifferent absorbing

Absorbance 0.2 0.4 0.6 0.2 0.4 0.6 0 0 B CD A 400 400 V. atalanta A. urticae

500 600 500 600

1B, area7),showingsomemelanin pigmentation.(C)Redadmiral.Thebrownandredcurvesarefor

1C, area9),andtheorangecurveis forscalesfromtheorange–redbandatventralforewing Heliconius melpomene

700 700

1. We C D C Wavelength (nm) ll

800 800 l side bandnear500 scales peakedintheultraviolet,ataround380 red scalespeakedat480–500 3A–C).Theabsorbanceoftheorangeand were rathervaried(Fig. spectra thusobtainedfromtheindividualyellow, redandbluescales scale transmittancewithamicrospectrophotometer. Theabsorbance with different refractiveindexvalues,andwethenmeasuredthe 0.2 0.4 0.6 0.2 0.4 0.6 0.8 1.0 r 0 0 c with methanol(D.G.S.,H.L.L.andB.D.W., unpublished)(seealsoReed B 400 400 m V. atalanta A. io

1A. (B)Peacock.Theredcurvecorrespondstoarea6ofFig. t The JournalofExperimentalBiology(2014)doi:10.1242/jeb.098673

500 600 500 600

nm (Fig. of thescale. are connectedbytrabeculae(t)tothelowerlamina(ll) of whicharecontinuouswiththecrossribs(c), the ridges(r)withlamellae(l)andmicroribs(m),some wing scale(modifiedfromGhiradella,1998),showing in B(scalebar, 2 black scaleofapeacockcorrespondingtothesquare (C) Scanningelectronmicroscopyphotographofa in arefractiveindexfluid(scalebars,20 red admiralandablackscaleofpeacockimmersed (A,B) Transmitted lightmicroscopyofaredscale Fig.

2. Anatomyofnymphalinewingscales. Xanthommatin 3-OH-kynurenine h s kyn n try

3B,C). =1.515) andpigments.

700

700 nm, andtheabsorbanceofyellow 3-OHK 3-OHK xan μm). (D)Drawingofanymphaline

800 800

nm, butoftenhada (A) Small μm). 2173

1B,

The Journal of Experimental Biology of thelowerlaminabluescales (Fig. minima, whicharemostlikely duetoaslightlyvariablethickness from thebluewingareas(Fig. minima at~320and620 therefore wealsocalculatedthe reflectancespectrumofa200 high pigmentconcentrations (e.g. Stavengaetal.,2013),and hold forunpigmentedscales.The refractiveindexismodifiedby 200 thicknesses of125–225 below 100 2013)] havescaleswherethelowerlaminaethicknesses well a structuralorigin. conclusion thereforemustbethatthebluecolourofthesescaleshas with theabsorbancespectrumofitspigment,however. The have reflectancespectrathatcannotbestraightforwardlyreconciled black scales.Thebluescales,containingasmallamountofmelanin, range andthuswithsufficient densitywillcauselow-reflecting, strongly absorb.Melaninabsorbsthroughoutthewholewavelength range wherethe3-OH-kynurenineandxanthommatinpigments orange andredscaleshavealowreflectanceinthewavelength pigments determinethescalecolour. Forinstance,theyellow, reflectance spectraofFig. present, butthemaincomponentwasclearlymelanin. absorbance valueof1.Traces ofommochromesappearedtobeoften the amplitudewasquitevariable,frequentlyexceedingan shape, i.e.monotonicallydecreasingwithincreasingwavelength,but absorbance spectraoftheblackscales(notshown),hadasimilar indicating arathersmallamountofmelaninpigment(Fig. peacock, wasminor, decreasingmonotonicallywithwavelength, absorbance ofthebluescales,takenfromeyespot 3-OH-kynurenine withvarioustracesofxanthommatin.The 2174 thick scalecontaining thepigmentmeasured inthebluescaleof [ of Stavengaetal.(Stavengaal.,2004)]andtheangledsunbeam (Vukusic etal.,2009),andthesmall white[ of thelowerlamina rather different valuesexistin othercases;forinstance,thethickness butterflies (Trzeciak etal.,2012;Wilts etal.,2012b).However, was concludedforthelowerlaminaofgreenscales of about200 Those scalesconsistoftwocollapsedlayers,eachwithathickness papilionid butterfly dispersion datadeterminedfortheglasswingscalesof 2012). Fortherefractiveindexofthinfilm,weused formula fornormallightincidence(Yeh, 2005;Stavengaetal., of athinfilmwithvariousthicknessesusingtheclassicalAiry dielectric thinfilm.We thereforecalculatedthereflectancespectrum suggests thatthelowerlaminaoflocalscalesactsasanoptical, The shapeofthereflectancespectrabluewingareas(Fig. RESEARCH ARTICLE yellow scales(Fig. unpublished) (seealsoReedandNagy, 2005).Thespectraofthe Heliconius melpomene precursor, 3-OH-kynurenine(extractedfromyellowscalesof of thebuckeye(Nijhout,1997),togetherwithspectrumits absorbance spectrumofxanthommatinextractedfromwingscales butterflies (Nijhout,1997;ReedandNagy, 2005).Fig. reminiscent oftheommochromes,identifiedinrelatednymphalid Thin films Thin We thereforecalculated reflectancespectraforfivethinfilmswith Most oftheabsorbancespectraFig. The absorbancespectraoftheorangeandredscalesarevery nm (Fig. Curetis acuta nm. nm (Stavengaetal.,2010).Thesamethicknessvalue 4A, bluecurve),withmaximumat~420

3A–C) indicatethatthesescalescontainmainly Graphium sarpedon with methanol;D.G.S.,H.L.L.andB.D.W.,

m(i.4A).Thereflectancespectrumfor nm (Fig. Argyrophorus argenteus nm, closelyresemblesthespectrameasured ; seefig.

1, seemtoconfirmthecommonviewthat

1) exceptforthelatter’s non-zero 4c ofWilts etal.(Wilts etal.,

3, whencomparedwiththe (Leertouwer etal.,2011).

4B). ThespectraofFig. Pieris rapae;seefig. scales is120

3D showsthe

3B). The P. nireus nm and

nm nm

4a 1)

4 wrinkled adwing surface.Incontrast,theabwing scatterogramshows the scatterogramisslightlylarger, obviously duetothesomewhat microspectrophotometer (Fig. column) andwealsomeasuredthereflectancespectrawith a of theilluminationbeamwasabout 5 of theadwingsideisalocalised spot(Fig. which ishighlystructuredwithridges andcrossribs.Thescatterogram smooth, createsamuchglossier photographthantheabwingside, on bothsides(Fig. sides withanarrowaperturewhitelightbeam(Fig. microscope ( side andadwing(lower)withanepi-illumination light micropipette andobservedthescalesatbothabwing(upper) single scales.We glued singlescalestothethintipofaglass investigated thespectralandspatialreflectioncharacteristics of To understandhowthe local wingcoloursarecreated,we scales ofthenymphalinesbyinspectingbothsidesscale. detailed studyofthereflectionpropertiesvariouslycoloured to thecolourofotherscaletypes.We thereforeperformeda the bluescalessuggeststhatthinfilmreflectioncanalsocontribute unpigmented scale(notshown). Fig. thin filmswiththicknessesof180,200and220 of butterflywingscales(Leertouweretal.,2011). (B)Reflectancespectraof spectra ofthinfilmswiththicknesses125–225 Fig. scales single characteristicsof Spatial reflection andspectral A bluescale(fromapeacock’s wingeyespot;Fig. The conclusionthatthinfilmreflectiondeterminesthecolourof

.Reflectancespectraofcuticlethinfilms inair. 4. 3B; thisyieldedavirtuallyidenticalspectrumasthatforthe Reflectance 0.1 0.2 0.1 0.2 0 0 The JournalofExperimentalBiology(2014)doi:10.1242/jeb.098673 i.5, Fig. 300 B A 300 5A, left).Theadwingside,which ismoreorless left). We measuredscatterograms ofbothscale

400 500

400 500 Wavelength (nm)

5, right).

deg, butthereflectionspotin Mean 220 nm 200 nm 180 nm

225 nm 200 nm 175 nm 150 nm 125 nm 600

600 700 nm andtheirmean.

5A, middle).Theaperture

nm andtherefractiveindex

700 (A) Reflectance

1B, no.7)isblue

5, middle 800 800

The Journal of Experimental Biology (Fig. 3B), butthemain effect mustbeattributed tothelimited in amplitude,whichispartlydue totheminoramountofpigment the scalecolour. Thetworeflectancespectraofthebluescalediffer properties ofthelowerlamina thebluescaledominantlydetermine RESEARCH ARTICLE thickness of200 calculated forathinfilmwithslightly varyingthicknessandamean very similarshape(Fig. (Fig. with adiffuse pattern,presumably duetoscatteringbythecrossribs a diffraction pattern,created bythearrayofparallelridges,together A B D C The reflectancespectraofthetwosidesbluescalehave a 5A, middle)(seealsoStavengaetal.,2009). nm (Fig.

5A, right)andcloselyresemble the spectrum 4B, mean).Thissuggeststhatthe thinfilm ad ad ad ad ab ab ab ab

Reflectance 0.05 0.10 0.15 0.05 0.10 0.15 0.05 0.10 0.15 0.05 0.10 0.15 0 0 0 0 400 400 400 400

500 500 500 500 Wavelength (nm) Orange Blue Red Black

600 600 600 600 ad ab ad ab ad ab ad ab

700 700 700 700 spectrum, which hasadistincttroughat~460 diffuse backgroundofthesamecolour. Theadwingreflectance abwing scatterogramshowsa bright orangelinetogetherwitha a spatiallyrestrictedreflection pattern (Fig. a metallicreflection.Alsosimilarly, theadwingscatterogram reveals scale, theabwingsidehasamatte colour, andtheadwingsidefeatures on boththeabwingandadwing side (Fig. the directionaladwingreflection. captures amuchsmallerpartofthediffuse abwingreflectionthanof aperture oftheobjectivemicrospectrophotometer, becauseit An orangescaleofthesmalltortoiseshell(Fig.

800 800 800 800 The JournalofExperimentalBiology(2014)doi:10.1242/jeb.098673 supplementary materialFig. indicate scatteringanglesof5,30,60and90 column) withanarrowaperturelightbeam.Theredcircles (ad) sides(dashedcirclesinthephotographsofleft illuminating asmallareaintheabwing(ab)andadwing column: scatterogramsofthefourscales,obtainedby micropipette (shinyand/orcolouredhorizontalbars).Middle scale bars,50 red admiral(C)andablackscaleof(D); orange scaleofasmalltortoiseshell(B),red adwing (bottom)sidesofabluescalepeacock(A),an scales. Fig. spectrum ×10. in D(rightcolumn)istheblackscale’s abwingreflectance circle inthephotographsofleftcolumn).Thedottedline spectra measuredfromasmallarea(similartothedashed

.Reflectionpropertiesofsinglenymphalinewing 5. Left column:photographsofabwing(top)and μm. Thewingscalesweregluedtoaglass

S1). Rightcolumn:reflectance 5B, left).Similartotheblue

5B, middle),whilethe

nm (Fig. 1A, no.3,)isorange

deg (see 5B, right), 2175

The Journal of Experimental Biology with increasing wavelength. understood fromthemelaninabsorbance spectrum,whichdecreases in thefar-red oftheabwing reflectancespectrumcanbeimmediately melanin suppressesthelowerlamina’s contribution,buttheupswing contributes totheabwingreflectance spectrum.Thehighlyabsorbing curve). Thisconfirmstheinterpretation thatthelowerlamina reminiscent oftheadwingreflectance spectrum(Fig. the abwingspectrumbyafactorof10revealsspectral shape blocked thereflectanceoflowerlamina.Interestingly, multiplying reflectance wasminimal,clearlybecausethescale’s melaninpigment with arelativelylongexposuretime,andaccordinglytheabwing distinct diffraction patternin theabwingscatterogramwasobtained spectrum indicatesathicknessof~200 reflecting thinfilm(Fig. scatterogram showsalocalisedspot,asexpectedfromdirectionally metallic navy-bluecolouredadwingside(Fig. wavelength range(Fig. unimpeded, thusyieldingasubstantialreflectanceonlyinthe red by thelowerlamina,whileleavinglong-wavelengthpart pigment inthescalewillstronglysuppressbluelightreflected right). With abwingillumination,thelarge amountofxanthommatin unequivocally indicatesathinfilmwiththickness~190 pattern (Fig. local, bluespot,documentingaverydirectional,specularreflection diffuse redbackground,whiletheadwingscatterogramshowsa diffraction pattern,perpendiculartotheridgearray, togetherwitha blue (Fig. the abwingsideisredcoloured,whileadwingmetallic increasing wavelength(Fig. thus causingthealmostmonotonicincreaseofreflectancewith the scale’s mainpigment,xanthommatin, issubstantial(Fig. be suppressedintheshort-wavelengthrange,whereabsorbanceof less diffusely scatteredbythecrossribs.Theabwingreflectancewill by thelowerlamina’s thinfilmisdiffracted bytheridgesandmoreor As withthebluescale,abwingillumination,lightreflected of theorangescaleactsasathinfilmwithmeanthickness~150 2176 compared withthespectraofFig. RESEARCH ARTICLE C AB An extremecaseisablackscaleofredadmiral,whichhas a A redscaleoftheadmiral(Fig.

5C, left).Theabwingscatterogramfeaturesaclear

5C, middle).Theadwingreflectancespectrum

5C, right).

5D, middle).Theadwingreflectance 5B, right).

4A, suggeststhatthelowerlamina

1C, no.9),isverydifferent as

nm (Fig. D

5D, left).Theadwing

5D, right).The 5D, right,dotted

nm (Fig.

3A,D),

nm. 5C, reflectance spectrum, at thesamelocationyieldsidenticaloscillationstothoseseen in the The transmittancespectrum, [following apreviouslypublishedmethod(Stavengaetal.,2011)]. oscillations, alocalwingthicknessof~1.1 substrate hasthinfilmproperties.Fromtheperiodicityof the reflectance spectrumfeaturesoscillations,indicatingthatthewing 7).The reflectance overthewholewavelengthrange(Fig. wing (Fig. shorter wavelengths)whenthefilmplaneistilted(Fig. films thatthereflectancespectrumshiftshypsochromically(towards show avioletcolour(Fig. A transmittance spectrashowsthatthewingabsorptance, purplish (Fig. negligible (Fig. scales showsthatreflectanceofthewingsubstrateiscertainlynot contribute tothereflection(Stavengaetal.,2006),asremovalofits one. Inthecaseofstackedbluescales,wingsubstratecanalso has passedonebluescaleandisthenreflectedbytheunderlying related mapbutterfly ( pigment-based coloration.Following previousworkontheclosely The wingscalesofnymphaline butterflieshavestructure-aswell a whitishcolour(Fig. by blackscales,butstackedaboveoneanother, thebluescaleshave (Fig. peacock, bluecoverscalesarebackedbyblackground 6).Intheeyespotsofdorsalhindwings rows (Fig. Butterfly wingscalesarearrangedinregular, usuallyoverlapping Scale stacking Pigments in the wing scales of nymphalinebutterflies Pigments inthewingscales of DISCUSSION kynurenine istakenupfromthewinghaemolymph(Fig. (Fig. kynurenine andxanthommatin,similartotheyellowishscales not negligible.Presumablythewingcontainstracesof3-OH- (see Koch,1991;ReedandNagy, 2005;Reedetal.,2008). ( Measurements onabare,descaledareaofsmalltortoiseshell λ )=1–T 6A), whichthusensurethebluecoloration.Whennotbacked 3). Thismaynotbesurprising,asduringdevelopment3-OH- (

λ 6D) showthatthewingsubstratehasaratherconstant The JournalofExperimentalBiology(2014)doi:10.1242/jeb.098673 )–R hindwing ofapeacockbutterfly(Fig. butterfly wings. Fig. peacock (indicatedbythearrowinFig. (C) Borderofthewhitespotindorsalforewinga scales aboveblackgroundappearblue(arrowhead). colour (areawithdottedwhitecircle),butunpigmentedcover ground scales,togetherwiththewingsubstrate,yieldawhite (Fig. band ontheventralforewingofaredadmiralbutterfly (see Results,Scalestacking).(B)Borderareaofthewhite of thebluescalesarecurvedandthusshowavioletcolour there existblackgroundscales(smallarrowheads).Thetips blue coverscales(arrows)andsomeblack removed scales(arrowhead).ScalebarforA–D,200 showing thebarewingsubstratewithsocketsof scales onthedorsalforewingofasmalltortoiseshell, appear pink(arrowhead).(D)Damagedareawithblack unpigmented coverscalesaboveorangeground membrane yieldwhite(areawithdottedcircle),but unpigmented coverscales,groundscalesandwing 6C). Thetipsofthebluescalesarecurvedandthus (

6D). Bluescalesaboveorange–redbecome λ

) (theareabetween1– .Packingandstackingofscalesonnymphaline 6. 1D, no.14).Stackedunpigmentedcoverscalesand

6B,C). Thecolourchangeisduetolightthat R A. levana ( λ

) (Fig. 6A) becauseofthebasicpropertythin T (A) Bluescalesintheeyespotofadorsal ( λ (where ) ) (Koch,1991) and paintedlady( 7). Combiningthereflectanceand λ T iswavelength),measured ( λ ) andR

1B, no.7).Belowthe μm canbederived

1B). Stacked ( λ ) inFig.

6A).

3D, inset) μm. 7) is V.

The Journal of Experimental Biology RESEARCH ARTICLE synthesis intheascidian contain tracesofmelanin. pigmented (coloured)scalessuggestthatmanyofthese measurements ontransparent(blue)aswellommochrome- The situationmaybeevenmorecomplicated,astheabsorbance kynurenine andxanthommatinbutalsoaddingotherommochromes. scale colourscanbefinelytuned,notonlybymixing3-OH- by (Riou andChristidès,2010)thuscannotbeeasilydistinguished absorbance spectrathatallpeakintheblue–greenwavelengthrange expression (Nijhout,1997).Thedifferent ommochromeshave to eitherxanthommatinordihydro-xanthommatinandommatin-D develops eitherapaletanordarkreddish-brownpigmentation,due depending onthedurationofrearingconditions,buckeye the wingcolorationofnymphalinebutterflies.Forinstance, However, itisnotunlikelythatseveralommochromesparticipatein pigments xanthommatinanditsprecursor3-OH-kynurenine. cardui) (ReedandNagy, 2005),weidentifiedastheimportant Fig. indicates thatthewingcontains3-OH-kynurenineaswellxanthommatin. absorptance chordate, demonstratedthatthegenomecontainedawidesetof C A A genome-widesurveyofgenesfor enzymesinvolvedinpigment in situ

.Thinfilmpropertiesoftheclearwingasmalltortoiseshell. 7. Reflectance/(1–transmittance) 0.1 0.2 0.3 0 measurements. Presumably, therefore,theyellowtored A =1–T 400 – R (where

Ciona intestinales 500 600 1–T T R Wavelength (nm) is transmittanceand , amarineinvertebrate B R

700 800 is reflectance) The D resulting inaredcolourwithslightpurplishhue(Fig. the thinfilmreflectioninredwavelengthrangeisleftunaffected, reflection intheshort-wavelengthrangeislargely suppressed,but blue-absorbing xanthommatin,whichisthecaseinredscales, fully suppressed(Fig. visible aswellultravioletwavelengthrange,abwingreflectionis concentration ofmelanin,whichabsorbsthroughoutthewhole This isthecaseofbluescales(Fig. spectrum willbeobtainedforbothabwingandadwingillumination. a scalehaslittleornopigment,andthenthesamereflectance transmittance spectrumofthescale’s pigment. spectrum ofthelowerlamina’s thin filmmultipliedbytheeffective spectrum measuredwithabwingilluminationisthereflectance shown inFig. reflectance spectrum.Inasimpleinterpretation,diagrammatically will actasaspectralfilterandthusmodifythelowerlamina’s the ridgesandcrossribs.Pigmentpresentinscale’s components the lowerlaminaispartlyreflectedthereandsubsequentlypasses windows formedbytheridgesandcrossribs.Thelightfluxreaching crossribs, whereitisscattered,butalsoentersthroughthelarge incident lightattheupper, abwingsidepartlyhitstheridgesand i.8A foraredscaleoftheadmiral(Fig. Fig. illumination fromtheadwingsideanddiagrammaticallyshownin lamina actsasathinfilm,isimmediatelydemonstratedwith A centralfindingofthepresentstudyisthatinallcaseslower scales insomeheliconiinebutterflies(Simonsen,2007). species (Kinoshitaetal.,2008),andclosedwindowsresultinsilvery multilayered ridgescausethebrightblueofwings creating muchmoreintensestructuralcolours.Forinstance, patterns. Othernymphalidshavediversifiedscalestructures,thus in different combinations,thuscreatingcomplexandstriking pigments inrathersimplystructuredscales,variableamountsand their wingcoloration.Thenymphalineshaveexpressedthese butterflies haveclearlyfavouredommochromesandmelaninfor papiliochromes colourthewingsofpapilionids,nymphalid Whereas pterinsarethewingpigmentsofpierids,and papiliochromes, pterinsaswellhaemes(Takeuchi etal.,2005). enzymes involvedinthesynthesisofmelanin,ommochromes, Structural and pigmentary coloration of nymphalinescales colorationof andpigmentary Structural The spectralfilteringofthethinfilmreflectionisnegligiblewhen The JournalofExperimentalBiology(2014)doi:10.1242/jeb.098673 8B fortheredadmiral’s redscale, thereflectance wing substrate. reflection andscatteringbythestack ofscalesandthe the observedscalecolouriscumulative resultof with stackedredscales.(D)Detailof Ctoillustratethat backscatters redlight,whichbecomes moresaturated reflectance. A red-pigmentedscalereflectsand melanin-pigmented scaleisblackbecause ofitslow melanin-pigmented, blackscaleyieldsabluecolour. A white–bluish colour. Anunpigmentedscale ontopofa scale ontopofanotherunpigmentedyieldsa on thelocalstackingofscales.Anunpigmented of thescalesonwingresultsincolorationdepending absorption bythepigmentinscale.(C)Illumination ridges andcrossribs,thuscausingconsiderable at theabwingsideisdominatedbyscattering (B) Lightreflectionofaredpigmentedscaleilluminated the lowerlamina,yieldingabluedirectionalreflection. adwing sideisdominatedbythethinfilmpropertiesof reflection ofaredpigmentedscaleilluminatedatthe the wingscalesofnymphalinebutterflies. Fig.

.Diagramsoflightreflectionandscatteringby 8. 5D). With aconsiderableconcentrationof

5A). With ahigh

5C). Incontrast,

5C). (A) Light Morpho 2177

The Journal of Experimental Biology the enhancedreflectance inthelongerwavelength range.Thisisthe the wingsubstratewillresultin amoresaturatedredcolour, dueto thin films.Similarly, aredscaleontopofanotherand/or (Fig. areas neverthelesshavepeaks inthebluewavelengthrange the resultisawhitishcolour. Thereflectancespectraofthewhitish desaturated bluishcolour. Whenthewingsubstratealsocontributes, blue wingcolour, butbluescalesontop ofeachotheryielda is negligible,bluescalesoverlappingblackgroundcreate a 2178 the wingreflectance(Fig. overlapped groundscales.Thesescalesthuscanalsocontribute to fraction oftheincidentlightpassescoverscalesandreaches the transmitted. Scalesonthewingarestacked,sothataconsiderable rather moderate,oftheorderatmost20%,sothat>80% is The reflectanceofthescalesforaboutnormalilluminationisusually RESEARCH ARTICLE with theredscales(Fig. reflectance attheredwavelengthsremains.Thisisinfactcase in thescale’s (abwing)reflectancespectrumsothatonlythe lamina asthatofthebluescale(Fig. of xanthommatininascalewithreflectancespectrumthelower transmittance spectrum)ofthescale’s pigment.A highconcentration that itistunedtotheabsorbancespectrum(orrather coloration effect. orange colourhenceisacombinedstructuralandpigmentary monotonically increasingreflectancespectrumresults(Fig. wavelength reflectionofthelowerlamina,andthusanalmost well assome3-OH-kynurenine,whichtogethersuppresstheshort- The orangescalescontainamodestamountofxanthommatinas spectrum, however, indicatingamuchsmallerthickness(Fig. film thickness.Theorangescaleshaveaverydifferent reflectance scales allhaveablue-peakingreflectance,indicatingsimilarthin always thesame.Thelowerlaminaeofblue,blackandred al., 2012b). narrow-band blue–greenreflection(Trzeciak etal.,2012;Wilts et abwing side,whichisreflectedbythelowerlamina,thuscausinga spectral filterforabroad-bandwhitelightbeam,incidentfromthe papiliochrome II.Theupperlaminaactstwiceasaneffective upper lamina,whichcontainstheviolet-absorbingpigment a blue-reflectingthinfilm.Thecrossribsofthesescalesformthick scales ofbutterfliesthe blue groundscales(Yoshioka andKinoshita,2004).Also,thegreen wings, thesescalesfunctionasdiffusers forthehighlyreflective, (Yoshioka andKinoshita,2004).However, ratherthan colouringthe previously noticedforthebluecoverscalesof pigment absorbancetogetherdeterminethescalecolour. scales. Thegeneralconclusionisthatthinfilmreflectanceand pigment concentration,especiallyinthecaseofredandblack colour, whilethepigmentbecomesadominantfactorathigh negligible pigmentationthelowerlaminadeterminesscale nymphalines leadstoanimportantconclusion,namelythatwith enhanced hueofthescale. wavelength reflectionrelativelyunhampered,resultinginan suppress thereflectionatshortwavelengthsandleavelong- range, whichactaslong-passspectralfilters,cantheneffectively shorter wavelengths.Pigmentsabsorbingintheshort-wavelength is essentialtoshiftthethinfilmreflectanceminimumtowardsmuch Wing colorationandscalestacking Wing The reflectancespectrumoftheorangescale’s thinfilmsuggests The thicknessofthelowerlaminavariousscalesisnot The importanceofthelowerlamina’s thinfilmhasbeen The surveyoftheopticalpropertiesvariousscales 1G,H, nos10and14),betraying thepresenceofblue-reflecting

5C). Forayellowororangescalecolourit

6). Becausethereflectanceofblackscales P. nireus group havealowerlaminawith

5A) willsuppressthebluepeak Morpho didius

5B). The 5B). wavelength receptoridentifiedhasapeakaround530 absorbing rhodopsins(BriscoeandBernard,2005).Thelongest- been characterisedinnymphalines;ultraviolet-,blue-andgreen- studied nymphalinebutterflies.Threedifferent visualpigmentshave A specialpointtoconsideristheprominentredcolorationof appreciable reflectancevalues(Fig. moderate; bystackingafewscales,theadditiveeffect yieldsquite basis. Furthermore,thereflectanceofasinglescalemaybe diffusive upperlamina,mayinfacthaveastraightforwardfunctional butterfly scales,withthewell-reflectinglowerlaminaand a spectralfilter. Inotherwords,theasymmetricalanatomyof as adiffuser. Whenpigmented,theupperlaminaadditionallyactsas therefore theupperlamina,consistingofridgesandcrossribs,acts reflector. Itsreflectivityishighlydirectional,andpresumably lower laminaisanextremelylightweight,reasonablyeffective bright coloration.Atthelevelofasinglescale,~200 material Fig. diagrammatically showninFig. (Fig. case forthepeacockwingareaswithdenselypackedredscales the individualscalestoaglassmicropipette (Fig. gently pressingthewingsontoamicroscope slideandsubsequentlygluing adwing (lower)sideandabwing(upper) sideofsinglescales,obtainedby DP70 digitalcamera(Olympus,Tokyo, Japan).Photographs of boththe taken withanOlympusSZX16stereomicroscopeequipped Photographs ofthetapestryscalesdifferent wingparts(Fig. Netherlands, werecapturedlocallyinthesummerof2013. the mostcommonnymphalidbutterfliesinnorthernpartof The previously Specimens ofthesmalltortoiseshell( substrate finetunethewingcolour. colour. Thelocalscalestacking as wellthereflectingwing of thelightreflectedbylowerlaminadeterminesscale pigmented, however, spectrallyselectiveabsorptionbythepigment to thelight-scatteringridgesandcrossribs.Whenscalesare Blue scalesarevirtuallyunpigmentedandhaveadiffuse colourdue wings. Thelowerlaminaofthescalesactsasathinfilmreflector. A fewlevelsofcomplexitydetermine thecolorationofnymphaline Wing colorationanddisplay Wing Photography MATERIALS ANDMETHODS Conclusions scales inimmersionfluid(refractive index1.60;Fig. 16×/0.35 objective(Zeiss,Oberkochen, Germany).Photographsofsingle Universal Microscope,usingepi-illumination throughaZeissEpiplan wing patternshaveavisuallydisruptivefunction. the wingsofsmalltortoiseshellandredadmiral.Possibly, their conceive thatasimilarmimicryorthreateningdisplayisrealisedin warning signalofthewings’ eyespots(Blest,1957),itishardto Whereas inthecaseofpeacock,redcolorationmayaddto photoreceptors thatarehighlysensitiveinthered(e.g.Hart,2001). conspecific butterfliesbut,rather, forpredatorybirds,whichhave 1997). Theredwingpartshencemaybebrightsignallersnotfor and theredreflectancespectraofwing(seealsoKinoshitaetal., meaning averylimitedoverlapofthereceptor’s spectralsensitivity camera wasaKappa DX-40(KappaOptronicsGmbH, Gleichen,Germany). Nikon Fluor40×/1.30oilobjective (Nikon,Tokyo, Japan).Thedigital Wing scalestackingappearstobeaneffective methodtoachieve 1B, Fig. Inachis io)andtheredadmiral( The JournalofExperimentalBiology(2014)doi:10.1242/jeb.098673

S2). 6C). Theconsequencesofwingscalestackingare

8C,D (seealsosupplementary

1). A. urticae V. atalanta

5), weremadewithaZeiss ), thepeacock(

2) weremadewitha ), whichareamong

nm thick

6) were A. io;

nm,

The Journal of Experimental Biology reference object(seeStavengaetal.,2009;Wilts etal.,2009). then monitored.A flakeofmagnesiumoxideservedasawhitediffuse the wingpatch),andspatialdistributionoffar-field scatteredlightwas to asmallcirculararea(diameter~13 therefore wedividedthemeasuredreflectancespectrainFigs adwing sidesofthescalesandwingsubstrate)isaboutafactor5, tile withamirror, thatthereflectanceofspecularreflectingobjects(e.g. directionally reflecting.We estimated,bycomparingthediffuse reflectance overestimated reflectancevalueswhenthemeasuredobjectisnotdiffuse but diffuse reflectancetilewasalsousedasareference,butthiscausesseverely the reflectancemeasurementswithmicrospectrophotometer, thewhite microspectrophotometer spectrawerelimitedtowavelengths>350 was anOlympusLUCPlanFL N20×/0.45.Because oftheglassoptics, was aboutsquarewithsidestypically~15 absorption intheUV. Theareameasuredwiththemicrospectrophotometer higher refractiveindices,becausetheindexfluidshaveahigh microspectrophotometer. We usedimmersionoilinsteadoffluidswith immersion oil(refractiveindex1.515),werealsomeasuredwiththe xenon arclightsource.Absorbancespectraofsinglescales,immersedin Wetzlar, Germany)connectedtothedetectorarrayspectrometer, witha measured withamicrospectrophotometer(Ortholuxmicroscope,Leitz, RESEARCH ARTICLE http://jeb.biologists.org/lookup/suppl/doi:10.1242/jeb.098673/-/DC1 Supplementary materialavailableonline at AFOSR/EOARD [grantFA8655-08-1-3012]. Research/European Office ofAerospaceResearchandDevelopment This studywasfinanciallysupportedbytheAirForceOffice ofScientific analyses andwrotethepaper. D.G.S., H.LL.andB.D.W. performed theexperiments,andD.G.S.performed The authorsdeclarenocompetingfinancialinterests. We thankAidanVey andHelenGhiradellaforreading themanuscript. obtained byfocusingawhitelightbeamwithnarrowaperture(<5 of theellipsoidalmirrorimagingscatterometer. Thescatterogramswere Fig. An isolated,singlescale(Fig. performed imagingscatterometry(Stavengaetal.,2009;Wilts etal.,2009). For investigatingthespatialreflectioncharacteristicsofwingscales,we with palladium. Eindhoven, TheNetherlands).Priortoimaging,thesamplesweresputtered The scaleanatomywasvisualisedbySEM(XL-30ESEM,Philips, micropipette (Fig. Reflectance spectraofbothsidessinglescalesattachedtoaglass captured thelightreflectedinasmallspatialangle,aperture~20 bifurcated probeilluminatedanareawithadiameterofabout1 reference wasawhitediffuse reflectancetile(WS-2,Avantes). The source wasadeuterium–halogenlamp[AvaLight-D(H)-S, Avantes], andthe an AvaSpec 2048-2CCDdetectorarrayspectrometer(Avantes). Thelight bifurcated probe(FCR-7UV200,Avantes, Eerbeek,TheNetherlands),using Reflectance spectraofdifferent wingareas(Fig. or lessdiffusely reflectingobjects. low valuesforthespectraofabwingsidesscales,whicharemore factor. We havetonote,however, thatthiswillinevitablycauseartificially abe,M. Barbier, References Supplementary material Funding Author contributions Competing interests Acknowledgements scatterometry Imaging SEM Spectroscopy phototransformations. S2) attachedtoaglassmicropipettewaspositionedatthefirstfocalpoint (1981). Thestatus of blue-greenbilepigmentsbutterflies, andtheir

5) andofbare,descaledwingareas(Fig. Experientia

5; orawingpatch:supplementarymaterial 37, 1060-1062. μm) oftheisolatedscale(ora μm. Themicroscopeobjective

1) weremeasuredwitha

5 and7bythat

mm, and

7) were

nm. For deg) on

deg. ed .D n ay L.M. Nagy, and R.D. Reed, Stavenga, B.D. Wilts, and H.L. Leertouwer, J., Tinbergen, D.G., Stavenga, H.L. Leertouwer, and M.A. Giraldo, D.G., Stavenga, B.J. Hoenders, and M.A. Giraldo, D.G., Stavenga, tvna .G,Soe . ibe . el .adAiaa K. Arikawa, and J. Zeil, K., Siebke, S., Stowe, D.G., Stavenga, M. Srinivasarao, hrdla . nsase,D,Ese,T,Slegid .E n itn H.E. Hinton, and R.E. Silberglied, T., Eisner, D., Aneshansley, H., Ghiradella, H. Ghiradella, Nijhout, H.F. andKoch,P. B. tvna .G,Letue,H . ii,P n eln,M.F. Wehling, and P. Pirih, H.L., Leertouwer, D.G., Stavenga, L.M. Nagy, and W. O. McMillan, R.D., Reed, iosn T. J. Simonsen, A., Sandy, S., Narayanan, H., Noh, S.G., Mochrie, C.O., Osuji, V., Saranathan, L. Taylor-Taft, and N. Morehouse, J.M., Macedonia, R.L., Rutowski, J.P. Christidès, and M. Riou, ioht,S n ohoa S. Yoshioka, and S. Kinoshita, H. Kayser, at N.S. Hart, H. Ghiradella, H. Ghiradella, G.D. Bernard, and A.D. Briscoe, ls,A.D. Blest, ioht,M,St,M n rkw,K. 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