© 2014.PublishedbyTheCompanyofBiologistsLtd|JournalExperimentalBiology(2014)217,3263-3273doi:10.1242/jeb.108076 Received 15May2014; Accepted23June2014 *Author ([email protected]) for correspondence 21250, USA. Baltimore County, Maryland 1000HilltopCircle,University of Baltimore, MD are theonlyfeaturescontaining opaquepigmentsintheseanimals. transparent theyareseeminglymadeofglass.Thecompound examples ofthelattermechanism,possessingbodiesthatare so 1995; Johnsen,2014)].Manycrustaceanlarvaeprovideexquisite reviews ofgeneralpelagiccamouflagingmechanisms(Nilsson, transparency (Breder, 1962;McFall-Ngai, 1990;Johnsen,2001); Johnsen etal.,2004),andtransparentbodytissue[reviews of 1972; JohnsenandSosik,2003),counterillumination(Clarke, 1963; surroundings, themostnotablebeingmirroredsides(Denton et al., that facilitatethevisualmeldingofananimal’s bodyintoits , multiplemechanismshaveevolvedinopen-waterhabitats on theabilitytoavoidbeingseen.Giventhisstrongselection for crustacean larvae,survivalinsuchafeaturelessworldoftendepends For smallanimalsinthepelagicenvironment,suchasmarine KEY WORDS:,,Stomatopod,Visual ecology spectrum andradiancetothepelagicbackgroundlightenvironment. demonstrate anoperativemirrorcamouflagematchedinboth of eachphotographicseries.Theseresultsarethefirstto performance usingradiometricmeasurementscollectedatthetime characterized thespectrumofeyeshineandcalculatedits larval eyeshineandthebackgroundlightenvironment,we background light.To testforspectralmatchingbetweenstomatopod light environmentandanalysingthecontrastofeyeswith camouflaging mechanismbyphotographinglarvaeintheirnatural efficacy ofstomatopodcrustaceanlarvaleyeshineasanocular environment againstwhichthelarvaleyesareviewed.We testedthe eyeshine, suggestingaputativespectralmatchingtothelight wavelengths oflightaremoststronglyreflectedinstomatopodlarval to furtherreducethevisibilityofopaqueretinas.Blueorgreen crustacean larvaleyes,referredtohereaseyeshine,ishypothesized light reflectedfromstructuresoverlyingtheretinasofstomatopod are reduced,theiropacityremainsconspicuoustoanobserver. The volume oftheopaqueretina.Thoughvolumestheseretinas possess specializedopticsintheircompoundeyesthatminimizethe see them.Stomatopodlarvae,likemanytransparentcrustaceans, trade-off betweentheabilitytoseeandofotheranimals column. Transparent, pelagicanimalsmust thereforedealwiththe undermine theabilityofthatanimaltobeinvisibleinwater of image-formingeyeinatransparent,pelagicanimalwillthus preserving resolutioninimage-formingeyes.Possessionofanytype Opaque screeningpigmentsareafundamentalrequisitefor K. D.Feller*andT. W. Cronin larval eyeshineinstomatopodcrustaceans Hiding opaqueeyesintransparentorganisms:apotentialrolefor RESEARCH ARTICLE INTRODUCTION ABSTRACT camouflaging mechanism. hypothesis thatstomatopodlarval eyeshinefunctionsasanocular and periodsoftheday. Thesedataprovidesoundsupportfor the inherent contrastofthelarvalretina atdifferent depths,orientations light environment,wetestedthe efficacy ofeyeshineatreducing the stomatopod larvaleyeshineboth inthelaboratoryandnatural larvae. Throughimagingand analysis ofspectralreflectance of eyeshineinthenaturalviewingenvironmentstomatopod eyeshine camouflagehypothesisbyanalysingseveralcomponents hypothesis hasneverbeenformallytested.We soughttotestthe eyeshine hasbeenpositedasacamouflagemanytimes, the Cronin andJinks,2001).Thoughtheroleofcrustaceanlarval Douglass andForward,1989;Croninetal.,1995;Jutte 1998; retina overwhichitisproduced(NilssonandNilsson,1983; hypothesized toserveasapelagiccamouflagefortheconspicuous With noapparentroleinphotosensitivity, eyeshineisinstead the adultisopod American lobster[ caridean shrimp[ observation) (CroninandJinks,2001),embryoniclarval been observedinbrachyurancrablarvae(authors’ personal 2001). Outsideofstomatopodlarvae,thepresenceeyeshinehas et al.,1985;Cronin1995;Jutte1998;andJinks, of speciesorstage(authors’ personalobservation)(seealsoWilliams eyeshine inallstomatopodlarvaleyesexaminedtodate,regardless photoreception intheseeyes.We haveobservedstomatopod visual filteringmechanismorotherwiseacttoinfluence robust )suggeststhateyeshinedoesnotfunctionasa absorbed byphotoreceptors(asevidencedthepresenceofa (Fig. photoreceptor layersoftheeye,butabsentfromopticalpathway includes aphotonicstructurefoundbetweentheopticaland material responsibleforstomatopodlarvaleyeshinesuggeststhatit norvegicus) (Loew, 1976).Carefulobservationofthereflecting the rhabdomsofsomecrustaceaneyes(suchasthose with thatproducedbythelight-reflectingtapetumobservedbeneath 1).Theeyeshinediscussedhere isnottobeconfused eyeshine (Fig. reflect blueorgreenwavelengthsoflight,referredtohereas animal pirouettesthroughspace. only appearasmovingpairsofblackdotsfixedside-by-sidethe human peeringintoabucketofplankton,stomatopodlarvaemay species, canbeupto40 features onastomatopodlarvalbody, whichdependingonthe at maximizingtheirtransparency. Often,theeyesareonlyvisible Among crustaceanlarvae,thestomatopodsareparticularlyeffective photoreceptors inordertopreserveresolutionofthevisualscene. (Nilsson, 1983),opaquescreeningpigmentsmustremainbetween transparency withelongatedopticalelementsandcondensedretinas Though crustaceanlarvaleyeshaveevolvedtoincreasetheir 1989); Undescribed structureswithinstomatopodlarvaleyesstrongly 1C). Theabsenceofeyeshinefromthepathwaylight P. argentinus Astacilla longicornis Homarus americanus Palaemonetes pugio (Harzsch etal.,1999)],embryosofthe

mm long(Felleretal.,2013).Thustoa (Nilsson andNilsson,1983). (Harzsch etal.,1998)],and (Douglass andForward, Nephrops 3263

The Journal of Experimental individuals weremostlikelythesamedevelopmentalstage. the similaritiesinsizeandmorphology, thesixsampled either oftheidentifiedspecies.Itcanbeinferred,however, thatgiven there arenopublisheddescriptionsofthelarvalstagesavailablefor could notassignindividualstoaspecificdevelopmentalstagesince that didnotmoltbetweennocturnalcaptureanddaytimeimaging.We ( Pseudosquilloidea), andanunknownspeciesofHarpiosquilla 3264 the upperhemisphereofthree-dimensionalspacefroma fixed conditions selectedtomostbroadlysampleviewsofananimalin environment atavarietyofpositions,depths,andillumination Animals werephotographedunderwater, intheirnatural Lysiosquilloidea), in Stomatopoda: representing threeofthesevensuperfamiliespresentlydescribed Three speciesofstomatopodlarvaeweresampledinthisstudy, RESEARCH ARTICLE Image contrast analysis contrast Image hoc Post RESULTS B d A N oslVentral Dorsal =1; Squilloidea).Eachsampledindividualwasanearlystagelarva θ s animal identification Into sun (0 deg) P. thomassini Lateral P. richeri Pseudosquillana richeri Pullosquilla thomassini Dorsal Left sun(270deg) 0 deg C

45 deg 45 90 deg Right sun(90deg) Optical pathoflight Manning (N Moosa (N P. thomassini Dark Rh Down sun (180 deg) =1; =6; Ventral Dorsal CC L H V as wellthecorrespondingdarkstandard(depictedinFig. from fourindividualsthatrepresentallstudiedspeciesanddepths, above thehorizon,axialorientation,androtationmeasured individual contrastvaluesfromeachazimuthangle,viewingangle Fig. angles, rotations,andrefractedsolarelevations species measuredandatalldepths,azimuthdirections,elevation of nocontrast).Theseresultsareconsistentamongthethree background lightenvironmentarereasonablycloseto0.0(value 1 withavalueofzeroequaltonocontrast. contrast (seeMaterialsandmethods),valuesrangebetween an objectagainstabackground.PerthedefinitionofMichelson contains informationregardingtherelativebrightnessordarknessof control) andthebackground.Michelsoncontrastisametricthat contrast valueswerecalculatedbetweenthelarvaleyespot(or imaged asacontrol.Fromeachofthesephotographs,Michelson standard and/oradeadanimalwithdegradedeyeshinewasalso and methods).Within eachphotographasmall,opaquedark 2,see‘Insitu observer onthebenthos(Fig. All Michelsoncontrastvaluesoflarvaleyeshinewiththe Right PRL Left 3 arepresentedusingagrayscaletovisuallyrepresent Eyeshine The JournalofExperimentalBiology(2014)doi:10.1242/jeb.108076 data collection.Datawerecollectedatdifferent depths( for underwaterphotographicandenvironmentalradiometric design. Depictedherearealldirectionalvariablesincluded Fig. directions relativetothesun(Into0 and radiometricprobedirectionswereorientedinfour at different timesofdayor solarelevations( 90 (Dark), andHversusVbodyaxes. depict thesefourrotationalpositions, thedarkstandard positions (Ventral, Left,DorsalandRight).Insetphotographs each positioninspace,animal wasrotatedinfour three anglesrelativetothehorizon(0,45and90 positioned withitsbodyaxishorizontal(H)orvertical(V)in direction, measurementsweremadewiththeanimal OL deg, Downsun180

2. In situ 500 crystalline cones;OL,opticallayer. Scalebars, PRL, photoreceptorlayer;Rh,rhabdom;L,lens;CC, responsible forproducingstomatopodlarvaleyeshine. depicting theputativelocationofphotonicstructures the eye,acharacteristicofthisspecies.(C)Diagram the changeineyeshinebrightnessandcoloracross dorsal andventralimagesof richeri (A) Pullosquillathomassini Fig. μm.

.Stomatopodlarvaleyeshine. 1. imaging andradiometricexperimental individuals usedintheseexperiments.Lateral,

deg, Leftsun270 and (B)Pseudosquillana imaging’ inMaterials P. thomassini

deg, Rightsun

deg). Ateachsun Fg 3 (Fig. θ s ). Photographs Photographs of

deg). At ). Datain in A show − d 1 and ) and

2).

The Journal of Experimental Biology Wilcoxon signedrank Wilcoxon rank-sum RESEARCH ARTICLE Uncorrected significance thresholdwas0.05. number ofcontrastvaluesindataset; s.d.,standarddeviation; Different datafromasingleindividual. datasetcomparisonsvisualizedinFig.4.Depthand solarelevationinformationgivenasdatasetidentifiersforpaired Test background andcontrastvaluesaboveorbelow0.0arelighter value of0.0(thenocontrast)equalsthegraycircular Data points,ordots,inFig. Table Depth 17 m 6 m 5 m 1 m

1. Resultsfrompair-wisestatisticalanalysesofMichelsoncontrastdatasets Left sun Left sun Left sun x 0 deg 0 deg 0 deg 45 deg Pullosquilla thomassini 45 deg Pullosquilla thomassini x Pullosquilla thomassini* 45 deg x x Downsun 90 deg x Into sun Downsun x Downsun Into sun x Into sun x 90 deg x x 90 deg x x Pu th1 Pu th2(17 Pu th2 Pu th4(17 l 8 avlidvdas803 All (8)larvalindividuals Pu th6 All Puth6 All deadeyes All darkstandards Contrast dataset 1 1.5 11 6 17 6 * 3 areshadedsuchthatthecontrast

m/47 m/49 m/80

m/61 m/60 m/52 Right sun Left sun Right sun Right sun x

deg deg deg deg m) m) deg deg 0 deg x x 45 deg Unknown Downsun Into sun 90 deg x xx x Harpiosquilla x H α V , significancethresholdafterBonferroni correction; Orientation key 55 69 55 128 205 69 150 74 76 N x Left sun

Right sun Ventral

= nodata Left eye Dorsal x 0 deg

xx Right eye

xx Dark Pseudosquillana richeri 45 deg − − − − − − − Mean − − − − − scale (foreachdot) on theplot. 0.0 atagivenviewingco-ordinate,thelessdiscernabledotis darker gray, respectively. Thusthecloseravalueofcontrastisto 0.11 0.075 0.25 0.05 0.0098 0.11 0.12 0.25 0.6 0.09 0.1 0.099 Downsun Into sun –0.94 0.37 Contrast ratio 90 deg 1 0 The JournalofExperimentalBiology(2014)doi:10.1242/jeb.108076 Right sun − − − − − eins.d. Median − − − − − − − 0.11 .7 0.05 0.072 0.24 .1 0.14 0.06 0.013 0.11 0.13 0.24 0.61 0.09 0.11 .9 0.06 0.096 at twodifferent depths. sample photograph.*Indicatesthesameindividual contrast valuescouldnotbeextractedfromthe positions Dotswithan‘X’ denotepointswhere background andthusappeartobemissingatthese of nocontrast,0.0,areshadedthesameas inset contrastratioscale).Dotsrepresentingvalues values fromnegativetopositive,respectively(see correspond totherangeofMichelsoncontrast are shadedinascalefromblacktowhitethat solar azimuthdirectionatthisviewingangle.Dots direction, whichproducedonlyfourdatapointsper data werecollectedforeachsolarazimuth 90 images betweenHandVaxisorientationsatthe from thedarkstandard.Duetoredundancyof each setrepresentsthecontrastvaluesmeasured sun, RightDownLeftsun).Thelastdotof (0, 45,90 horizontal; V, vertical),viewingangletothehorizon Left eye,Dorsal,Righteye),axisorientation(H, exposures takenateachanimalrotation(Ventral, average Michelsoncontrastvalueofthreedifferent (dotted line).Dotswithineachplotrepresentthe individual ofagivenspeciesattheindicateddepth larvae. Fig. deg overheadviewingangle,onlytheHseriesof

3. In situ Each circleplotrepresentsdatafroman P , probabilityvalue;n.s.,notsignificant. 0.05 0.08 0.05 0.05 0.08 0.08 0.12 0.07 0.04 deg), andsolarazimuthdirection(Into contrast valuesfromstomatopod .2 0.0025 0.025 .2 <0.001 0.025 .1 <0.001 0.017 α .2 <0.001 <0.001 0.025 0.025 .1 0.029n.s. 0.017 P 3265 N ,

The Journal of Experimental Biology Fig. 3266 RESEARCH ARTICLE depth 11− variation incontrastvalues thefirstphotographicseries[deep in contrastvalues(observed in both depths andsolarelevationsproduced different ranges,orvariations photographic seriesofthesameindividualimagedattwodifferent two ofthemeasuredvariables:solarelevationanddepth.Sequential differences inthevariationof eyeshinecontrastrelatedprimarilyto Fig. with whichitwasimagedatdifferent solarelevations(Table individual (Puth6)andthecontrastvaluesofdeadeyecontrol a significantdifference between thecontrastvaluesofliving from thoseofalldarkstandards(Table values measuredfromeightindividualsweresignificantlydifferent range of− 1 was significantlydifferent fromthesecondseries(shallow depth θ Depth (m) −

RS Contrast ratio Though alleyeshinecontrastvaluesfallnear0.0,there are Overall, 95%oftotaleyeshinecontrastvaluesoccurredwithin a Species 1.5 –0.8 –0.6 –0.4 –0.2 (deg) 0.2 4B). 4B). Unfortunately, thecontributionsofdepth and/orsolar 0 m, B 17 θ .4 o025(al ,Fg 4).Alleyeshinecontrast 1,Fig. 0.346 to0.225(Table

RS Counts of contrast measurements Ps ri ri Ps 100 150 200 48 6 m, refractedsolarelevationofthe sun( 50 47− 0

–0.80

UK UK Ha 56 5 A 52 47 deg) foreachoftheseindividuals (Table

1

h1 t Pu –0.60 Eyeshine Dark standard Individual photographseries 61 11

Binned contrast values 1.5

52

2 th Pu –0.40

60 17

h3 t Pu

50 –0.20 1, Fig. 6

Pu th1

h4 t Pu 57 17

4A). Therewasalso

th5 Pu 80 and 6

0 ‡ θ RS 49 6

Pu th2).The

6 th Pu ) 60− 0.20

80 ‡ 6 61 49 6

deg]

ead D

1, 1, ‡ 80 6 during ourcontrast analysisexperiments,these data(collectedJune Gonodactylaceus falcatus with angleofilluminationand polarizationin These dataprovideageneralcharacterization ofhoweyeshinevaries 5). independent setofexperiments conductedinJune2010(Fig. properties ofstomatopodlarval eyeshinereflectanceinan natural environment,wecharacterized someofthefundamental Prior toanalysingtheinherentcontrastoflarvaleyeshinein the same depth(6 2014, definitively. elevation tovariationsincontrastwereunablebedeciphered significant afterBonferronicorrection(Table in contrastvaluesbetweenthetwosolarelevationswas not elevation oncontrastperformanceatagivendepth.Thedifference 80 Spectral properties of larval eyeshine larval of properties Spectral In aseparatesetofimagingexperimentsperformedinMarch deg). Thisexperimentspecificallytestedtheeffects ofsolar twice underdifferent illuminationconditions(different depthsor series. Threeindividuals( background visuallyseparateboxplotsbyindividualphotographic overlap withdarkstandarddata.Grayandwhiteverticalbarsin stippled, deadeyecontrast.Notethatsomeexperimentaloutliers white, liveanimaleyeshinecontrast;solidgray, darkstandardcontrast; horizontal lineineachbox.Boxesandoutliersareshadedasfollows: range; circulardots,outliers.Medianisdenotedbyacontinuous,black photographic series.Box,interquartilerange;whiskers,1.5 all measuredeyeshineandcontrolcontrastvaluesbyindividual significantly different fromthoseofeyeshine(Table values astheeyeshinedata.Darkstandardcontrastwere rotations), thedarkstandarddatasetcontainsone-quarterasmany solar azimuthandhorizontalelevationdirections(notforanimal space. Sincedarkstandardswereonlymeasuredforaxisorientation, different imageexposuresforagivenorientationorrotationanglein value representstheaverageofthreerawcontrastvaluesmeasuredat Harpiosquilla th1 in theboxplotofeachdatasetareasfollows: shading; includes: contrast values(binsize,10 Fig. the sun. contrast withthenaturalenvironment. thomassini Ha UK Pullosquilla thomassini separate pair-wisestatisticaltestssummarizedinTable Brackets atthetopofboxplotindicatedatasetsincludedin 74; Dead49 Pu th3,80;th4th560;th6 P. thomassini 47

.Summaryofimagecontrastanalysis. 4. , unknown deg, 58;Puth1 The JournalofExperimentalBiology(2014)doi:10.1242/jeb.108076 ‡ March 2014samples.Numberofcontrastvaluesrepresented ) imagedwith m) andlocationatdifferent times ofday(

deg, 65;Dead80 , N =1) anddarkstandards(blackshading).Eachcontrast Harpiosquilla individual 6(Puth6)wasphotographedatthe P. thomassini 61 (individuals 1 Pu th6. . ThoughG.falcatus deg, 77;Puth2 Pu th1,th2

deg) fromallsampledindividuals(gray

deg, 63. ; Dead,deadeyeanimalcontrol(likely , N Dashed linerepresents0.0valueofno =4; P. richeri,N − 6); Psri, θ RS and Puth6)wereimaged 52 , refractedsolarelevationof 49 deg, 69;Puth2 deg, 76;Puth6

KHa Ps ri,76;UK 1). Pseudosquillana richeri (A) Histogramofbinned =1; unknown P. thomassini

1). (B)Boxplotsof was notsampled

1. Puth, θ 60 80 RS , 73;Pu θ deg, 80; deg, 49 and RS ). and P. ;

The Journal of Experimental Biology wavelengths below410 small, earlystageeyesresults inthespectralnoiseobservedat are fromearlystagelarvae.The lowlevelsoflightreflectedfrom individual measuredinFig. the spectrometer. With theexceptionoflast stage retinal areathatisroughlytwice thatandthusreflectmorelightto is ~500 μm.A laststagelarvaofeitherthesespecieswillhavea an earlystagelarvainspeciessuchas with thestage,andthuseye-sizeofanimal.Theeyediameter of of theretina(Marshalletal.,1991). ommochrome screeningpigmentsexpressedintheopaqueportion noted thatwhile theamountoflightreflectedchanges withontogeny polarized atmost(Fig. wavelength peakinaneyeshinereflectancecurveisweakly spectrum (G.falcatus structures moststronglyreflectinthisregionofthereflectance reflectance curve,whichsuggeststhateyeshine-producing changes inpolarizationtheshort-wavelengthregionof the structures torespondpolarizationfiltering.We observedstrong only theportionsofreflectancecurvecontributedbyeyeshine Stavenga etal.,2010;2011); thusweexpected linearly polarized(Chiouetal.,2007;Douglas established thatreflectivestructuresoftenproducespectraare properties ofstomatopodlarvaleyeshinereflectance.Itiswell reflectance spectra,wechosetoexaminethelinearpolarization In ordertoresolvethecontributionofeyeshineobserved RESEARCH ARTICLE (450–550 two characteristicpeaksinreflectivity:ashort-wavelengthpeak a widerrangeofspecies. properties oflarvaleyeshinenecessaryforfurtheranalysesamong 2010) provideafundamentalunderstandingofthespectral A C The amountoflightreflectedfromeyeshineisstronglyassociated Most eyeshinereflectancespectra,regardlessofspecies,contain

% Reflectance source light White 35 10 15 20 25 30 0 0 0 0 800 700 600 500 400 Microscope 0 5 eyepieces nm) andalong-wavelengthpeak(>800 Optical fiber Microscope deg objective 45 10×

nm inFig. data inFig.

5B), suggestingthatitarisesfrom Pullosquilla thomassini

5C, alleyeshinereflectancespresented To spectrometer P and computer 5D andFigs 90 deg 75 deg 60 deg 45 deg P. thomassini

Wavelength (nm) 5B). Thesecond,long-

% Reflectance B 10 15 20 10 D 5 0 0 500 400 0 2 4 6 8 6–8. Itshouldbe

nm; Fig. 0 0 0 0 800 700 600 500 400 and P. thomassini G. falcatus

Horizontal polarizedlight 5B–D). Wavelength (nm) Vertical polarizedlight 0 700 600 Gonodactylaceus falcatus the eyein15 Pu th1whileholdingtheilluminationangleat45 demonstrates howreflectancechangesacrosstheeyeofindividual band longerwavelengthreflectance(470–600 whereas thegreendorsalregionproducesahigherintensity, broad- reflects anarrow-bandshortwavelengthspectrum(~445 eye region(Fig. color transitionstogreenasonetravelsacrosstheretinadorsal stage larvae,theventralregionofeacheyeappearsblue,and unpublished dataandpersonalobservations).Inbothearlylate all crustaceanlarvalspeciesexaminedtodate(K.D.Feller, stomatopod larvalspecies,bothexaminedinthisstudyandamong Since reflectances: situ and methods).Thespectralproperties ofeyeshinewereanalysed in situ performed usingradiometricdatacollectedconcurrentlywith each The analysisofeyeshinespectrainthenaturalenvironment was 5C,D). apparatus (Fig. which wasalsotheminimumilluminationanglepossiblewithinour illumination angleof45 10× objective).Maximumeyeshinereflectancewasmeasuredatan from fourilluminationangles(45,60,75and90 maximum eyeshinereflectance,wemeasuredreflectance constant acrossdevelopmentalstages. of theeye,spectralshapeeyeshinereflectanceremains measurement in ourspectralanalysis.To examine furtherwhether retina (Fig. In situ In Gonodactylaceus falcatus Pullosquilla thomassini To determinetheilluminationanglethatwouldproduce from twoindividualsrepresenting different speciesandeyeshine P. thomassini photographic series(see‘ spectral analysis of larval eyeshine larval of analysis spectral 90 deg 75 deg 60 deg 45 deg 800 The JournalofExperimentalBiology(2014)doi:10.1242/jeb.108076 6), weincludedbothadorsal and ventralreflectance

Pullosquilla thomassini deg stepsfromventraltodorsal.

1A). Under45 displays different reflectancespectraacrossthe (B) Horizontal(0 were placedatlocationofdashedline,P. polarization measurements,linearpolarizers head viaa1000 and computersystemattachedtothetrinocular reflectance wasmeasuredviaaspectrometer imaged throughamicroscope,andthe seawater. Theilluminatedlarvaleyewas fixed at45 illuminated byasubmergedwhitelightsource Larvae werefixedtoathinplasticrodand eyeshine reflectance(notdrawntoscale). experimental set-upformeasurementof eyeshine reflectance Fig. observed below410 to eyearea)isresponsibleforthenoise the relativelyweakreflectanceofeye(due available fromthelightsourcecombinedwith The lowlevelsofshort-wavelengthlight thomassini 10× objectiveaxismeasuredfrom from different illuminationanglesrelativetothe illumination. (C)Larvaleyeshinereflectance eyeshine reflectancein vector polarizationreflectancemeasurementsof

deg ofadorsal-laterallypositionedeye, eyeshine isunlikethatofotherexamined

.Characterizationofstomatopodlarval 5. deg illumination,theblueventraleye In situ

deg toasubmerged10×objectivein and (D) and spectral analysis’ inMaterials μm opticalfiber. For

deg) andvertical(90 Gonodactylaceus mutatus Pseudosquillana richeri.

nm inD. . (A)Diagramof G. mutatus

nm flatpeak).Fig.

deg relativetothe

deg androtating Pullosquilla from 45

nm peak),

deg) e- 3267 deg . in 6

The Journal of Experimental Biology matched tothe long-wavelength shiftedradiance ofitsnativelight environment (Fig.8). reflectance estimatesarepoorly matchedtotheLizardIslandlight spectrum ismuchbroader(Fig. measured atshallowdepths(1 appears toprovideanadequatematchthepeakwavelengths wavelength shifted,broadspectralreflectanceof 8A,B,D).Thelong- most LizardIslandspectralenvironments(Fig. S. empusa comparison, thecalculatedreflectancespectrumfromlarvaleyesof matched tothebackgroundradianceatthisdepth(Fig. shallow, broad-spectrumwater, thegreendorsaleyeshineiswell eyeshine ofP. thomassini occurring species.Itshouldbenotedthatwhiletheblue,ventral time ofday, ordepth(Fig. environment. Theseresultsareconsistentregardlessofsunposition, reflection andtheradiancespectraofbackgroundlight eyeshine demonstratesaclosematchbetweenitswavelengthofpeak spectrum measuredduringeachphotographicseries,calculated richeri 3268 environmental radiance,wefound that might suspectthatanyeyeshine reflectorcouldmatchthe matching isadaptivetoparticular habitats(Figs calculated eyeshinefromendemic speciessuggestthatspectral at depth.Matchesbetweenspectraofbackgroundradiances and retinal contrastandisspectrallymatchedtotheviewingbackground we demonstratedthatstomatopodlarvaleyeshineeffectively reduces By systematicallytestinganimalsintheirnaturallightenvironments, USA; July2010). the DukeUniversityMarineLaboratory(Beaufort,NorthCarolina, P. thomassini RESEARCH ARTICLE General DISCUSSION same methodsas empusa eyeshine reflectancebyLizardIslandirradiancespectra. in theLizardIslandlightenvironmentbymultiplying empusa calculated whattheeyeshineofanon-endemicspecies, AB White Normalized, raweyeshinereflectancesfrom light source r rsne nFg 7A.Whenmultiplied bytheirradiance are presentedinFig. from theWestern AtlanticOcean,wouldproduceifitwere larval eyeshinereflectancedatawerecollectedusingthe demonstrates apoormatchtobackgroundradiancesfor and G. falcatus objective P. richeri deg 45 D 10× V Squilla empusa M may notseemtobewellmatchedin

7B–E) forbothoftheLizardIsland- during anindependentfieldstudyat eyeshine spectraareadaptive,we

8C).

% Reflectance 10

0 2 4 6 8 m), wheretheenvironmental 0 0 0 0 800 700 600 500 400 eyeshine reflectanceislikely Squilla empusa

P. thomassini 7, 8).Although,one S. empusa Wavelength (nm) S. empusa

eyeshine 7C). By Squilla Squilla and only P. Pullosquilla thomassini illumination conditions( where asingleanimalwasphotographedintwodifferent values withinasinglephotographicserieswas− of contrastvaluesamongdatasets.Themaximumrange close to0.0,thevalueofnocontrast,therewasvariationinrange stomatopod larvaleyes. that eyeshineservesasacamouflagefortheopaqueretinain a livinganimal(Fig. in thecontrastofeyewhencomparedtoeyeshine eyeshine reflectivityindeadlarvaeproducesasignificantincrease the variationof eyecontrast.Whiletheseresults maysuggestthat elevations, controllingfordepth, andfoundnosignificantchangein successfully imagedanindividual ( ascertained. We revisitedthisexperimentinMarch 2014and Given thesedata,theinfluence ofeachvariablecouldnotbe th2) wereeachphotographedatdifferent depthsandsolarelevations. photographed intwodifferent seriesinJune2012( reconcile thetwofactorsfromoneanother. Theindividuals factors withinourdatasettoaffect eyeshinecontrast,wecouldnot reflecting propertiesofthereflector. (such asdepthandsolarelevation)ratherthanthephysical light- that eyeshinecontrastisinfluencedbytheilluminationconditions rotation oftheanimalorviewingangle,leadingtoconclusion P measured betweenthetwoconditions( demonstrated asignificantchangeintherangesofcontrastvalues varied from th1, 11 depths, androtations(Figs examined viewingangles,solarazimuthsandelevations, background environmentthanadarkobjectcontrolfromall 2003). Eyeshineproducessignificantlylesscontrastwiththe as predictedbytheoreticalmodelsofcrypsis(JohnsenandSosik, viewing background. example ofapelagicreflectivecamouflagespectrallymatchedtothe Lizard Islandstomatopodlarvaepresentthefirstdocumented data necessarytotestthisareunavailable.Regardless,from environment intheWestern AtlanticOcean,thoughtheradiometric =4.51e Though alleyeshinemeasurementsofMichelsoncontrastwere Though solarelevationanddepthweretheonlyillumination In general,eyeshineperformslikeotherpelagicmirrorreflectors, m depth),whiletheminimumrangeofcontrastvaluesonly − 7 ). Changesincontrastdidnotvarysignificantlywiththe The JournalofExperimentalBiology(2014)doi:10.1242/jeb.108076 − 0.16 to−

4B). Thesedatastronglysupportthehypothesis collected in15 locations ofreflectancespectralmeasurements reflectance acrosstheeye. Fig. plot below410 responsible forthenoiseobservedinspectral weak reflectanceoftheeye(duetoarea)is from thelightsourcecombinedwithrelatively A. Thelowlevelsofshort-wavelengthlightavailable by thecoloredbarsonlateraledgeofeyein location ofeachmeasurementontheeye,depicted eye. Thecolorofeachspectrumrepresentsthe wavelength reflectanceandintensityacrossthe (B) Reflectancespectrachangesinboth D, dorsaleyeregion;M,medialedgeofeye. illumination ofasingleeye.V, ventraleyeregion; 0.03 (Puth4,depth17

6. Pu th1 Pullosquilla thomassini

3, 4).Additionally, thereductionof

deg steprotationsunder45 nm. and Pu th6)atdifferent solar Pu th1, Pu th2),theanimal m; Fig. (A) Diagramdepicting P 0.28 to+0.22(Pu =0.0025; eyeshine 4). Fordatasets Pu th1

deg Pu th2, and Pu

The Journal of Experimental Biology A RESEARCH ARTICLE analogous tothose withtransparentcladocerans willprovidean hypothesis remainsuntested. Futurebehavioralexperiments the evolutionofstomatopod larvaleyeshineevolution,this India ink(Zaret,1972).Though predationisalikelyforcedriving and pigmentationoftheeyespot isincreasedviadietaryuptakeof of fishpredationontransparent cladoceransincreasewhenthesize stomatopod larvaleyeshine.Previous workdemonstratesthatrates isastrongselectiveforcethatmaydrivetheevolution of of reducingcontrastinanynaturalilluminationconditions. evidence suggeststhatoveralleyeshineisaneffective mechanism which cannotberesolvedwithoutfurtherstudies.Regardless, the elevation anddepthmayalsobeanartifactofindividualvariation, sample size,ourobservationsofchangestoeyecontrastwith solar influence oneyecontrastwiththebackground.Duetoour small for depthandsolarelevationarenecessaryresolving the to interpretwithoutfurtheranalysis.Futureexperimentsthatcontrol depth isaprimaryinfluenceineyecontrastvariation,itdifficult B Ecological significance Normalized units of light Normalized % reflectance 0.2 0.4 0.6 0.8 1.0 0.2 0.4 0.6 0.8 1.0 0 0 0 0 0 0 800 700 600 500 400 0 0 0 0 800 700 600 500 400 5 5 5 750 650 550 450 5 5 5 750 650 550 450 Wavelength (nm) 45 deghorizontalelevation 90 deghorizontalelevation PsRi eyeshine 0 deghorizontalelevation PsRi PuTh ventral PuTh dorsal E D C Normalized units of light Normalized units of light Normalized units of light 0.2 0.4 0.6 0.8 1.0 0.2 0.4 0.6 0.8 1.0 0.2 0.4 0.6 0.8 1.0 0 0 0 0 0 0 0 800 700 600 500 400 0 0 0 0 800 700 600 500 400 0 0 0 0 800 700 600 500 400 green eyeshine in P. thomassini,however, suggeststhatthis species of spectralmatchingbetweenshallow (1 in nature,sincelarvaewouldrarely occurinsuchahabitat.Evidence radiance atshallow, 1 matching betweenlarvaleyeshine andbroad-spectrumenvironmental be foundawayfromthesurface atdepthduringtheday. Poor this aspectofstomatopodlarval ecology, sincelarvaeareexpected to are wellmatchedtolightenvironmentsatdeeperdepthsagrees with feed (Ohtomietal.,2005).Ourfindingsthatmosteyeshinespectra are foundatdepthduringthedayandrisetosurfacenight to modeling experiments. detection forapredatormustbedeterminedthroughsensitivity conditions (mostcontrast),thelowerthresholdlimitsofcontrast contrast isecologicallysignificantinitsworstillumination larval eyesaredetectablebypredators.To determineifeyeshine whether therangeofcontrastvaluesobservedfromstomatopod predation rates.Suchexperiments,however, wouldnotaddress interesting testoftherelationshipbetweeneyeshinecontrast and Wavelength (nm)

Stomatopod larvaeperformdielverticalmigrations,wherebythey

v PuTh dorsaleye PuTh 90 degradianceoverhead 45 degradiancedownsun 45 degradianceintosun PuTh dorsal PuTh ventral 0 degradiancedownsun 0 degradianceintosun

The JournalofExperimentalBiology(2014)doi:10.1242/jeb.108076

e entral

PuTh dorsal PuTh ventral down sun 0 degradiance into sun 0 degradiance ye

m daytimedepthsmaybeoflittle consequence observed below410 natural lightingconditions. Fig. depth versus0 thomassini from Downsunsolarazimuth.(C) 90 (B) P. richeri represent calculatedeyeshinereflectance. background radiances;bluetraces All blackandgraycurvesrepresent different viewinganglesabovethehorizon. reflectances versusselectedradiancesof analyses. (B regions ofPuTheyeswereusedinour curves fromboththeventralanddorsal eyeshine reflectancecurves.Reflectance Pseudosquillana richeri (A) Pullosquillathomassini reflectance oftheeye(duetoarea). source combinedwiththerelativelyweak wavelength lightavailablefromthe reflectances isduetolowlevelsofshort- radiance. (D) eyeshine at11 calculated eyeshineat11 viewing angleradiance.(E) 45 and90 indicated inthekeys. Radiance solarazimuthsforC deg viewingangleradiancescollected

.Calculatedeyeshinespectrain 7.

deg viewingangleradiances. calculated eyeshineat1

m) radiancesandthedorsal − at 6 P. thomassini E) Calculatedeyeshine

m depthversus0 deg viewingangle m depthversus0,45and

nm ineyeshine (PsRi) larval

m depthversus (PuTh) and calculated P. thomassini Noise − E are

deg P.

m 3269

The Journal of Experimental Biology eyeshine solution. the particularevolutionarypressures thatledtosuchanovel the dielverticalmigrationrange ofthisspeciesmayhelptoidentify Studies thattarget theswimming postureof ecology andisnotrelatedtodevelopmentalchangesthe eye. we canassumethisnovelstructureplaysaspecificrolein the larval stages(authors’ personalobservationsinearlyandlaststages), eyeshine of eyeshine. However, giventhattheblue-ventral, green-dorsal we cannotevaluatethefunctionalsignificanceofthisregionalized regarding thedaytimebehaviorandlocationof for camouflageinashallowerhabitat.Untilmoreisknown shallow, backgroundradiances,suggestingthatitmaybeadaptive The greenregionofeyeshineshowsbetterspectralmatchingagainst and blue,ventralhemisphere)areamechanismforcounter-shading. two spectrallydifferent regionsoftheeye(green,dorsalhemisphere regarding itsfunctioninthisparticularspecies.Itispossiblethatthe thomassini 3270 are notcurrently understood,thoughphotonic structures arewidely The structuresresponsibleforproducing stomatopodlarvaleyeshine behavioral ecologythatremainstobecharacterized. may becamouflagingatshallowerdepthsperanaspectoftheir Fig. RESEARCH ARTICLE radiances; green( horizontal elevation;(C)1m,45degand(D)11 measuredbackground m,45deghorizontalelevation.Blackandgraytracesrepresent solar azimuthsandviewinganglesabovethehorizon.Depthdirectionareasfollows:(A)1m,0deghorizontalelevation;(B)11 m,0deg Eyeshine-producing structures The unusualblueandgreenregionalizedeyeshineseenin

.Calculatedeyeshinereflectancefrom 8. P. thomassini larval eyessuggestssomeinterestinghypotheses 11 mdepth 1 mdepth S. empusa)andbluetraces(

Normalized units of light is presentthroughouttheentirerangeof 0.2 0.4 0.6 0.8 1.0 0.2 0.4 0.6 0.8 1.0 0 0 B A 0 0 0 0 800 700 600 500 400 800 700 600 500 400 P. thomasini Pullosquilla thomassini 0 deghorizontalelevation P. thomassini P. thomassini ) representcalculatedreflectancefromeyeshinefortheindicatedenvironment. larvae and larvae, and Squillaempusa P. Wavelength (nm) Transmission electronmicroscopic imagesofeyestructures in may becomposedofthree-dimensionally arrangedvesicles. reflectance spectra. crystal mayberesponsible for producingsuchfinelytuned in stomatopodlarvaleyes,acomparable large-domain-type photonic While itisunlikelythisspecific typeofphotonicstructureisfound crystals forcamouflageinforestedenvironments(Wilts etal.,2012). use spectrallymatchedreflectionsfromdiamond-typephotonic terrestrial diamondweevil, a relativelythincoherentscatteringphotonicstructure. The produced byasimplequarter-wavelength multilayer reflectororby (Denton andLand,1971);therefore,eyeshinemusteither be stomatopod eye,whichisonly~500 required foratypicalsilverreflectorisperhapsnotavailable ina quarter-wavelength thicknesses(DentonandLand,1971). Thespace reflection viamultiplelayersofguaninecrystalswithvarying flanks ofasilveryfish,whichareunderstoodtoproducebroad-band seem prominent,theyareinfactquitesmallbycomparisonwith the their opaqueretina.Thoughthestalkedeyesonalarvalstomatopod as towhystomatopodlarvaedonotuseasilverreflectorhide 2003). Thesizeoftheobjectbeingcamouflagedmayprovideinsight to camouflaginginthepelagicenvironment(JohnsenandSosik, reflectors, orsilverymirrors,areacommonandeffective solution used forcamouflageintheaquaticenvironment.Broad-band Existing evidencesuggeststhat eyeshine-producingstructures 0.2 0.4 0.6 0.8 1.0 0.2 0.4 0.6 0.8 1.0 0 0 D C 0 0 0 0 800 700 600 500 400 0 0 0 0 800 700 600 500 400 The JournalofExperimentalBiology(2014)doi:10.1242/jeb.108076 larvae versusradiancemeasurementsatdifferentdepths, 45 deghorizontalelevation Entimus imperialis SqEm PuTh dorsal PuTh ventral Radiance downsun Radiance intosun μm inmaximumdiameter , hasbeenshownto

The Journal of Experimental Biology 90, V])onlyHimages werecollectedforall90 between HandV animals(e.g.[Intosun,90, H]isthesameas[Rightsun, horizontally (H).Asthe90 90 animal wasviewedatthreeviewing anglesrelativetothehorizon(0,45and left ofthesun(‘Leftsun’,270 RESEARCH ARTICLE sun (‘Rightsun’,90 directions included:intothesun’s azimuth (‘Intosun’,0 long axisorientation,andlarvalrotationalangle(Fig. four components:solarazimuth,viewingangleabovethehorizon, larval Each selectedviewingangleandlarvalposturecanbebrokendown into from afixed,benthicpositionthatprovidedviewsoftheanimalbelow. of viewforanobserverpositionedunderwater(Fig. of viewinganglesanddirectionsthatbestrepresentedtheupperhemisphere illumination angle.Consequently, wetestedeyespotcontrastfromavariety established thatthespectralpropertiesoflarvaleyeshinechange with a calibratedrotationalchuckmountedontripod.Priortoimaging, we situ) usingaCanonG12camera(LakeSuccess,NewYork, NY, USA)and Live-mounted animalswerephotographedintheirnaturalenvironment ( 20:00 and22:00 (~1 sunset andhadamaximumdurationof40 either whilewadingorscubadiving.Duringnightdivesatdepthsof6 at thesamelocationinMarch2014.Allcollectionswereperformednight Station (Queensland,Australia),withasmallersubsetofsamplescollected The majorityoflarvaewerecapturedinJune2012atLizardIslandResearch among stomatopods. as toexaminehowthesenovellight-reflectingstructuresvary eyeshine shouldincludeadiversetaxonomicsamplingofspeciesso characterize thephotonicstructuresresponsibleforproducing eyeshine reflectancespectraexist.Futureinvestigationsthat used toproduceeyeshineisidenticalacrossspecies,sincedifferent stomatopod larvalspecies,itisunclearastowhetherthemechanism camouflage. Thougheyeshinehasbeenobservedinallexamined widespread withinStomatopodaasamechanismoflarvalocular eyeshine arecurrentlyunderway. into theultrastructurethatissourceofstomatopodlarval structures mayexistintheeyesofstomatopodlarvae.Investigations amorphous solidwithshort-rangeorder. We hypothesizethatsimilar these structuresarecomposedofsphericalvesicles,indicativean retina (Marshalletal.,1991).Aswiththe possess greenlight-reflectingpigmentsontheoutermargin ofthe species ofadultSquilloidandLysiosquilloid stomatopodsalso structures (Douglass,1986;DouglassandForward,1989).Some the hypothesizedlocationofcrustaceaneyeshine-producing vesicles atthejunctionofopticsandphotoreceptorlayer gold reflectingeyeshine,showlayersoftightlypacked,spherical caridean shrimp(Palaemonetes)larvae,whichpossessagreenish- USA) (seeFelleretal.,2013). barcoding attheUniversityofMaryland,BaltimoreCounty(Baltimore,MD, conducted within48 temperatures (~26°C)inseawaterwithoutfood.Allexperimentswere maintained individuallyinplasticcupsthelaboratoryatambient same lightsourcewasusedduringcollectionsatdepth.Larvaewere in thelaboratory. Alldeep-watercollectionperiodsbeganwithin30 USA). Larvaewerecollectedingallon-size,resealablebagsandlatersorted underwater lightsource(UnderwaterKineticsAquaSuneLED;Poway, CA, pelagic stagestomatopodlarvae(Dingle,1969)andattractedtoan we tookadvantageoftheinnate,positivephototacticbehaviorearly In situ In Animal collection MATERIALS ANDMETHODS Our diversetaxonomicsamplingsuggeststhateyeshineis deg) whilethelongaxisofanimal washeldeithervertically(V)or m depth)wereroutinelyperformedwhilewadingnearshorebetween imaging

h. Animalswerecapturedusinghand-helddipnetsandthe

h ofcapture.Larvaewereidentified deg), awayfromthesun(‘Downsun’, 180

deg viewinganglepositionproduces redundancy

deg). Ineachdirectionrelativetothe sun,the

min. Collectionsinshallowwater

deg viewsrelative tothe Palaemonetes

2). We imagedtargets

2). Thefoursolar post hoc

deg), rightofthe deg), and via DNA larvae,

min of − 18 m in − elevations werecollectedusingthedeadeyecontrol. centimeter ofthelivingspecimen.Two photographicseriesatdifferent solar prior toexperimentation)andadarkstandardweremountedwithin Pu th6 in eyeshinepostmortemasasecondcontrolbyincludingdeadlarvathe procedure wereusedfortheexperimentalanalysis.We utilizedthereduction often disappearswhenalarvadies,onlyanimalsthatsurvivedtothefixation KJ828807, KJ828808,KJ828809,KJ828810,KJ828811). Sinceeyeshine gene weredepositedinGenBank(GB-KJ828804,KJ828805,KJ828806, true identityofthisindividualwillremainunclear. barcodes aredocumentedforagreaterselectionofstomatopodspecies,the passing throughfromadistantlocationonlocalcurrent.UntilDNA from thisregion.Alternatively, theindividualcouldrepresentalarvathatis Island, itislikelythatthislarvarepresentsanundescribedsquilloidspecies barcoding andidentified After photographing,eachanimalwasfixedinabsoluteethanolforDNA photographic measurement,placedwithinacentimeterofthelarvaltarget. opaque darkstandard.This1-mmdiameterstandardwasincludedineach final totalof240imagesperserieswascollectedforimagecontrastanalysis. and anoverexposedimageonestopabovetheautomaticexposure.Thusa the automaticallyselectedexposure,anunderexposedimageonestopbelow, generated threesuccessiveexposuresforeveryrawformatimagecaptured: shutter speedselection.UsingafixedF-stopof8,theautomaticcamera and directionsofourimagesnecessitatedtheusecamera’s automatic from thedescribedcomponents. each series,atotalof80uniqueviewsanimalwerethusproduced from theventral,left,dorsalandrightaspects(Fig. position theanimalwasrotatedinfour90 horizon. To furthervieweyeshinereflectanceatvariousorientations,each formula: green channelmeasurementsandusedtoproducethefollowingcorrection A linearregressionofthisplotwasthencalculatedfromboththeblueand determine howpixelvaluechangeswithexposureusingourcamerasystem. between neighboringgraystandardsandplottedversuspixelvalue to 8-bit pixelchannel.Foreveryexposure,fiveerrorratioswerecalculated series. Thisproducedvaluesbetween0and255,themaximumrangefor any values weremeasuredfromeachgraystandardinexposure in the sunlight. InAdobePhotoshop,boththeaveragegreenandbluechannel pixel Colorchecker; Baltimore,MD,USA)illuminatedunderdiffuse, natural standards (white,gray8,6.5,5,3.5andblack;Macbeth (G) orblue(B)pixel.To doso,wephotographedaseriesofsixgray brightness sensitivityacrossthe8-bitdynamicrangeofeachred(R),green Before analysingthe barcode recordofanyadult to thatof because thesmallestsequencedistancetoaCOIgenereference(10%)was specimen inthisstudywastentativelyassignedtothegenus I (COI)genewas≤ genetic distancerelativetoareferencesequenceofthecytochromeoxidase (Feller etal.,2013).Specimenswereconsideredpositivelyidentifiedifthe wavelengths attenuate ataslightlyfasterratethan blue,weselectedthe since therewas no difference inthecorrectionformula.Because green Measurements fromeitherofthese channelsweresufficient for ouranalysis reliable datafortherangeofdepths measuredintheseexperiments. light underwaterwithdepth,onlythe blueandgreen8-bitchannelsprovide same correctionformula.Duetothe attenuationofdifferent wavelengths of Calibration measurementsfromthe blueandgreenchannelsproducedthe could becalculatedforeachraw8-bit measurement( Image analysis Image All barcodesequencesofthecytochromeoxidasesubunitImitochondrial As acontrol,small,black-paintedsphericalobjectwasusedasan The changesinlightingconditionsthataccompaniedtheviewingangles Using thisformula,acorrectedpixelvalue( photographic series(March2014).Boththedeadlarva(whichdied Harpiosquilla harpax.Sincenomorphologicalormolecular The JournalofExperimentalBiology(2014)doi:10.1242/jeb.108076 3% (BarberandBoyce,2006).Theonlyunknownlarval in situ post hoc Harpiosquilla images, wecalibratedthedigitalcameraforits y = − .040.8 0.0004 per themethodsdescribedinFelleretal. x x

deg turnssoastoimageeyeshine + species presentlyexistsatLizard y ) ofeachexperimentalimage .(

2, insetphotographs).In x ) fromaphotograph. Harpiosquilla 3271 1 )

The Journal of Experimental Biology a drawingtablet(Wacom Intuos; Vancouver, WA, USA).The15 selecting theaveragedareaofscatteredpelagiclightwithincircleusing the larvalmountorreefsubstrate,wereexcludedfromthismeasurementby circle wastakentobethebackgroundradiance.Near-field objects,suchas Photoshop. Thecorrectedaveragegreenchannelpixelvaluewithinthis centered onthemountedlarvaineachrawformatimageusingAdobe 145.454°E). each photographicseriesattheLizardIslandco-ordinates( gmd/grad/solcalc/; accessed21April2014)forthespecificdateandtimeof acquired fromtheNOAA SolarPositionCalculator(www.esrl.noaa.gov/ sun andthezenith(θ underwater viewer(Lythgoe, 1979;Johnsen,2012). through whichlightintheairisrefractedandcompressedabove an provide thepositionofsunwithinSnell’s window, the96 3272 corrected significance thresholds,aresummarizedin Table given analysiswas either0.025or0.017.Allstatistical results,aswell were comparedtwotothreetimes, thusthesignificancethreshold( multiple comparisonsofspecific data. Inouranalyses,specificdatasets and/or timesofday. A Bonferronicorrectionmethodwasemployedduring contrast datasetsmeasuredfrom asingleindividualatdifferent depths Wilcoxon signedranktestwasusedtofordifferences betweeneyeshine known asaMann–Whitney from animaleyeshineandthedarkstandards,aWilcoxon ranksumtest(also normally distributed.To testfordifferences betweencontrastdatameasured normal distributionusingaShapiro–Wilk normalitytest.Alldatasetswere All statisticalanalyseswereperformedinR.Eachdatasetwastested for of pixelvaluesatshallowdepths. green 8-bitchannelforouranalysissoastominimizepotentialsaturation radiometric datacollection(see‘ was selectedtoduplicatetheacceptanceangleofprobeusedfor where background: ( a different refractiveindex( Snell’s lawdescribestheanglelightisrefractedasitentersamediumwith series wascalculatedusingSnell’s law: elevation, therefractedsolarelevationattimeofeachphotographic contrast equaltozero. produced fromthisformularangebetween this way, wewerebetterabletoanalyseinherentcontrast,sincethevalues RESEARCH ARTICLE Statistical analyses chose tocalculatetheMichelsoncontrast( contrast ofstomatopodlarvaleyeswiththebackgroundenvironment,we visibility oflarvaleyestohypotheticalviewers,butrathertesttheinherent Wiederman, 2014). Sinceourobjectivewasnotdesignedtomodelthe sensitivity modelingofanimalsviewingeyeshine(O’Carrolland attenuation ofcontrastwithdistancesmallobjects(Johnsen,2012)orfor may beused(Johnsen,2012).Weber contrastisoftenpreferredtocalculate was thecasewiththisstudy, eitherMichelsonorWeber contrastformulae addressing questionsofinherentcontrastanobjectatafixeddistance,as Michelson ortheWeber formula forcontrast(Johnsen,2012).When relative toagivenbackgroundandcanbemeasuredusingeitherthe in thefinalanalysis. standard. Photographsinwhichtheretinawasnotfocuswereincluded same procedurewasperformedforradiancemeasurementsofthedark radiance wasdeterminedbyaveragingallpixelswithinthelarvalretina.The ( θ n RS 1 For analysisofexperimentalimages,a15 To examinechangesineyeshinecontrastwithtimeofday, orsolar Contrast isaquantitativemeasurementofhowbrightordarkanobject =1.0) andthen ) wasdeterminedbysubtracting L eye is theeyeradianceand θ S was thenusedtodetermine 1 of water(n =90 C deg M θ 2 n = − = ). Forourcalculations,weusedthe () ()

U θ sin LL LL S 2 y background backgroundeye eye -test) wasperformed.Alternatively, a =1.33). Solarelevationdata( ). Therefractedsolarelevationofthesun L − In situ background 1 + − ⎝ ⎜ ⎛ θ n 2 11 sin n from 90 2 spectral analysis’ below).Eye − θ θ is thebackgroundradiance.In 1 and1,withthevalueofno 1 C , theanglesubtendedby ⎠ ⎟ ⎞

M deg circulartemplatewas .( ) ofoursubjectwiththe deg. Thesecalculations ,(2)

1. −

deg region 14.673°S,

deg cone θ S n α ) were ) fora of air 3 ) background, orifinsteaditisadaptivetospecificenvironments. Western Atlanticocean)canprovideanadequatespectralmatchtoany eyeshine fromaspeciesinhabitingdifferent lightenvironment(suchasthe stomatopod species,intheLizardIslandlightenvironmenttotestwhether eyeshine reflectancefromlarvaeof maxima forcomparison.Usingthesesamemethods,wealsocomputedthe radiance andreflectancespectraldata,allspectrawerenormalizedtotheir image capture.Duetodifferent valuesofintensitiesamongirradiance, series, thisprovidedreal-timeenvironmentalspectrapresentduringeach condition. Sinceradiancewascollectedintandemwiththephotographic radiance spectracollectedfromallviewingdirectionsforagiven for aninsitu for eachindividualwasmultipliedbythedownwellingirradiancemeasured angle fromwhichmaximumreflectancewasdetermined,Fig. eye reflectancespectrumfroma45 specimens. reflected fromtheretina.Allmeasurementswereacquiredlive 90 were notincludedinouranalyses.A linearpolarizerwasrotatedto0and taken wasmeasuredusinga15 downwelling irradiance.Radianceforallanglesatwhichphotographswere 100 μm diameteropticalfiber(UV-Vis; OceanOptics)tocollect (CC-3, OceanOptics,Dunedin,FL,USA)wasattachedtoa40 Brisbane, Queensland).Atthebeginningofeachseriesacosinecorrector a calibratedunderwaterspectrometer, Subspec(AndorTechnology/Oriel, Environmental spectraweremeasuredduringeachphotographicseriesusing Scientific Research (FA9550-12-1-0321). This workwassupported byagrantfromtheUnitedStates AirForceOffice of The manuscriptwascomposedinitsentirety byK.D.F., withrevisionsbyT.W.C. methods andanalyseswereperformed byK.D.F. underthementorshipofT.W.C. The initialconceptforthisstudywasproposed byT.W.C. Allfurtherdesigns, The authorsdeclarenocompetingfinancialinterests. aspects ofthisproject. Justin Marshall(UniversityofQueensland)fortheirexpertcounselonvarious Johnsen (DukeUniversity),DrNicholasRoberts(UniversityofBristol)and N. Station staff fortechnicalandlogisticalsupportinthefield;aswellDrSönke Partridge (UniversityofBristol)forspectralanalyses;LizardIslandResearch Baltimore County)forconstructionofcustomexperimentalapparatuses;Julian assistance withSubspecmeasurements;JohnCataldi(UniversityofMaryland, The authorsaregratefultoDrMeganPorter(UniversityofSouthDakota)for ultraviolet opacityofthemicroscopesystem,wavelengthslessthan400 diffuse whitestandard(Labsphere;NorthSutton,NH,USA).Duetothe obtained atthebeginningofeachdatacollectionsessionusingaSpectralon (Apple; Cupertino,CA,USA).A measurementof100%reflectancewas using SpectraSuitesoftware(OceanOptics)operatedonaMacBookPro of theactualeyepiece;Fig. microscope adaptorattachedtotheepi-eyepieceof(inplace a 1-μ 45 prevented reflectancemeasurementsfromilluminationanglessmallerthan 45 fixed anglesrelativetotheopticalaxisof10×objective:90,75,60or fixed onthecalibratedanglemounttoilluminatespecimenatoneoffour optical fiber(UV-Vis; OceanOptics).Theopticalfiberwassubmerged and white-light, LS-1tungstenhalogenlamp(OceanOptics)connectedtoa1 seawater bywrappingitinaplasticfilm.Larvaewereilluminated Germany) (Fig. via asubmerged 10×microscopeobjective(LeitzDialux22;Wetzlar, to acalibratedanglemount,whichwassuspendedunderwaterandimaged In situ In Funding Author contributions Competing interests Acknowledgements To calculatetheeyeshinereflectancespectrainnaturalenvironment, Prior tounderwaterphotography, larvaeweregluedtosticksandaffixed deg. Lightwasguidedtoaspectrometer(QE65000;OceanOptics)using deg inthepathofreflectedlighttomeasuredegreepolarized deg. Physicalinterferencebetweenthelightsourceandobjective m diameteropticalfiber(UV-Vis; OceanOptics)andacustomprobe- spectral analysis spectral The JournalofExperimentalBiology(2014)doi:10.1242/jeb.108076 condition ofinterest.Computedeyeshinewascomparedto 5A). Thefrontfaceofthe10×objectivewasprotectedfrom

5A). Reflectancemeasurementswerecollected

deg acceptanceangleprobe.

deg illuminationangle(the Squilla empusa,aWestern Atlantic

5C,D) measured

cm longby in situ μm

nm

The Journal of Experimental Biology ho,T . ähe,L . aln .T n rnn T. W. Cronin, and R.T. Hanlon, L.M., Mäthger, T. H., Chiou, rdr C.M. Breder, RESEARCH ARTICLE one,S. Johnsen, rnn .W,Mrhl,N . adel .adPls D. Pales, and R. Caldwell, N.J., Marshall, T. W., Cronin, R. Jinks, and T. Cronin, W. D. Clarke, etn .J,Gli-rw,J .adWih,P. G. Wright, and J.B. Gilpin-Brown, E.J., Denton, S.L. Boyce, and P. Barber, References elr .D,Coi,T . hog .T n otr M.L. Porter, and S.T. Ahyong, T. W., Cronin, K.D., Feller, R. Forward, and J. Douglass, J.K. Douglass, oga,J . rnn .W,Ciu .H n oiy N.J. Dominy, and T. H. Chiou, T. W., Cronin, J.M., Douglas, H. Dingle, M.F. Land, and E.J. Denton, one,S. Johnsen, one,S. Johnsen, B. Beltz, and R. Dawirs, J., Benton, S., Harzsch, B. Beltz, and R. Dawirs, J., Benton, J., Miller, S., Harzsch, spatial propertiesofpolarizedlightreflectionsfromthearmssquid( larvae. 198 and cuttlefish( 2053-2061. stomatopods throughDNA barcodingofstomatopodlarvae. Princeton, NJ:PrincetonUniversityPress. countermeasures. 41, 1098-1107. (:Nymphalidae). and theroleofpolarizediridescenceinsensoryecologyneotropicalnymphalid R. Soc.B of thelightproducedbysomemesopelagicfishinrelationtotheircamouflage. Mar. Freshwat.Behav. Physiol. and ocularpigmentsofcrustaceanlarvae(StomatopodaDecapoda,Brachyura). and cephalopods. Durham, NC,USA.Accessedfromauthor. Eyes ofGrassShrimp,PalaemonetesPugio. transparency. Biol.Bull. Stomatopoda) fromLizardIsland,Australia. and moleculardescriptionofthelate-stagelarvaeAlimaLeach,1817(Crustacea: in ashrimpeye:hatchingthroughmetamorphosis. larvae. neurogenesis, andapoptoticcelldeath. development ofthevisualsystemindecapodcrustaceans:neuropilformation, development. in thethoracicneuromeresoftwocrustaceanswithdifferent typesofmetamorphic , 1244-1246. Copeia Crustaceana (1969). Ontogeneticchangesinphototaxisandthigmokinesisstomatopod 182 (2001). Hiddeninplainsight:theecologyandphysiologyoforganismal (2014). Hideandseekintheopensea:pelagiccamouflagevisual (1962). OnthesignificanceoftransparencyinOsteichthidfisheggsand (1963). Functionofbioluminescenceinmesopelagicorganisms. (2012). , 145-158. J. Exp.Biol. Sepia officinalis (1986). 1962, 561-567. Proc. R.Soc.B Ann. Rev. Mar. Sci. 16, 108-110. The OpticsofLife:A The OntogenyofLightandDarkAdaptationintheCompound (2001). Ontogenyofvisioninmarinecrustaceans. 201 201 , 301-318. (1989). Theontogenyoffacultativesuperpositionoptics (2006). EstimatingdiversityofIndo-Pacificcoralreef (1971). Mechanismofreflexioninsilverylayersfish , 2465-2479. L.). J.Exp.Biol. 26, 219-231. 178 , 43-61. 6 J. Exp.Biol. , 369-392. J. Neurobiol. Zootaxa Biologist’ 210 PhD dissertation,DukeUniversity, Cell Tissue Res. , 3624-3635. 210 (1999). A newlookatembryonic 3722, 22-32. (1972). Theangulardistribution 39, 294-306. s GuidetoLightinNature , 788-799. (1995). Compoundeyes (2013). Morphological (1998). Neurogenesis (2007). Lighthabitats Proc. Biol.Sci. (2007). Spectraland 258 Loligo pealeii , 289-300. Am. Zool. Nature Proc. 273 ) , . ae,T. M. Zaret, one,S n oi,H.M. Sosik, and S. Johnsen, one,S,Wde,E .adMbe,C.D. Mobley, and E.A. Widder, S., Johnsen, W T. Hariyama, and H.L. Leertouwer, B.D., Wilts, D.G., Stavenga, Williams, B.,Greenwood,J.andJillett, H.L. Leertouwer, and M.A. Giraldo, D.G., Stavenga, T. Furota, and H. Kawazoe, J., Ohtomi, R.L. Caldwell, and T. W. Cronin, P. A., Jutte, ’arl,D .adWeemn S.D. Wiederman, and D.C. O’Carroll, ow E.R. Loew, iso,D .adNlsn H.L. Nilsson, and D.E. Nilsson, D.-E. Nilsson, T. Cronin, and C. King, M., Land, N., Marshall, M. McFall-Ngai, J.N. Lythgoe, iso,D.E. Nilsson, ilts, Cladocera (ClassCrustacea). Proc. Biol.Sci. Brilliant camouflage:photoniccrystalsinthediamondweevil, predator avoidance. camouflage strategiesinnear-surfacepelagichabitats:implicationsforforagingand enhance blue/greenpigmentcolorationofthewingmembrane. colors: glassscalesof 207 bioluminescence: factorsaffecting counterilluminationasacrypticstrategy. fulgidissima iridescence ofthemultilayeredelytraJapanesejewelbeetle, 1731-1739. Sci. Stomatopoda) andthereplacementoflarvaleyeatmetamorphosis. developmental stagesof Japan. Crustaceana Oratosquilla oratoria morphological changeduringlarvaldevelopmentoftheJapanesemantisshrimp, of movingpatternsandfeatures. crustacean. planktonic larvaeoftwospeciespullosquilla,alysiosquilloidstomatopod Nephrops norvegicus Astacilla longicornis Purcell andD.L.Macmillan),pp.149-162.Amsterdam:GordonBreach. Zooplankton: SensoryEcologyandPhysiology in crustaceaneyes. Trans. R.Soc.B stomatopod crustaceans:Polychromaticvisionbyserialandlateralfiltering. shrimps (Crustacea,Hoplocarida,Stomatopoda).II.Colourpigmentsintheeyesof , 1-16. .D,Mcile,K,Kies . eRet .adSaeg,D.G. Stavenga, and H. DeRaedt, J., Kuipers, K., Michielsen, B. D., 36 , 104-114. (1972). Predators,invisibleprey, andthenatureofpolymorphismin . Philos.Trans. R.Soc.B (1976). Light,andphotoreceptordegenerationintheNorwaylobster, J. Exp.Biol. The JournalofExperimentalBiology(2014)doi:10.1242/jeb.108076 (1979). (1995). Eyedesign,visionandinvisibilityinplanktonicinvertebrates.In (1983). Evolutionarylinksbetweenappositionandsuperpositionoptics (1990). Crypsisinthepelagicenvironment. 279 334 , 2524-2530. Nature (Sowerby). Limnol. Oceanogr. The EcologyofVision , 57-84. 78, 1325-1337. (L.). Proc.R.Soc.B (De Haan,1844)(Stomatopoda,Squillidae)inTokyo Bay, 201 Graphium sarpedon 302 , 2481-2487. Heterosquilla tricarinata Limnol. Oceanogr. , 818-821. J. Mar. Biol.Ecol. Philos. Trans. R.Soc.B (2003). Crypticcolorationandmirroredsidesas (1983). Eyecamouflageintheisopodcrustacean 366 (2014). Contrastsensitivityandthedetection 48, 1277-1288. , 709-723. . Oxford:ClarendonPress. 193 (1985). Seasonalityanddurationofthe (2005). Larvalstagecompositionand (2004). Propagationandperceptionof , 31-44. (1991). Thecompoundeyesofmantis (ed. P. H.Lenz,D.K.Hartline,J.E. 17, 171-184. cause polarizediridescenceand 68, 105-110. (1998). Photoreceptioninthe (Claus, 1871)(Crustacea: 369 Am. Zool. , 20130043. (2010). Butterflywing Entimus imperialis J. Exp.Biol. (2011). Polarized 30, 175-188. Chrysochroa Bull. Mar. Biol. Bull. (2012). 3273 Philos. 213 . ,

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