© 2014.PublishedbyTheCompanyofBiologistsLtd|JournalExperimentalBiology(2014)217,4213-4220doi:10.1242/jeb.108902 Received 9July2014; Accepted9October2014 *Author ([email protected]) for correspondence Kiel University, 9,24118Kiel,Germany. AmBotanischen Garten andBiomechanics, FunctionalMorphology Zoological Institute, of Department the surfaceofhostleavesduring variousenvironmentaldisturbances 1985). Stenberg andStenberg, 2012;Wahl, 2008;Wallace andO’Hop, light transmissionintothewater(Kouki,1991a;Kouki,1993; G. nymphaeae year (Wesenberg-Lund, 1943).Alongwithotherecologicalimpacts, as adults,feedontheleaves.Atleastthreegenerationsoccur per All stagesliveontheuppersurfaceofleavesandlarvae,as well surface ofleaves.Thelarvaeundergo threelarvalstagesandpupate. spring. Aftermating,femaleslayclutchesofeggsontheupper Adult beetlesoverwinterintheriparianareasandcoloniseleaves in terrestrial leafbeetle for terrestrialbeetlesthatareassociatedwithwater-plants. The necessity tostronglyattachtheirhostplantholdstrueespecially attach totheirhostsorholdontocopulationpartners.The example, theyneedtoadheretheplantsliveandfeedon, The attachmentofinsectstosurfacesiscrucialfortheirsurvival.For Biomechanics, Locomotion KEY WORDS:Adhesion,Friction,Contactangle,, contact angleof83 highest frictionforcesandsafetyfactorsonsurfaceswithawater tarsomere. Inacentrifugalforcetester, larvaeandadultsattainedthe tarsomeres anddenselyarrangedspatula-shapedonesontheirthird have pointedsetaeontheventralsideoftwoproximal structures attheirlegsandapygopodiumtheabdomentip.Adults The larvaebearasmoothattachmentsystemwitharolium-like contact anglesthataresimilartothoseofleavestheirhostplants. adult stagesgeneratethestrongestattachmentonsurfaceswith nymphaeae describe theattachmentstructuresofwater-lilyleafbeetle onto surfacesthathavedifferent chemistry. Inthepresentstudy, we different roughness;however, littleisknownaboutinsectattachment Numerous studiesdealwithinsectattachmentontosurfaces Constanze Grohmann*,AndreasBlankenstein,SvenKoopsandStanislavN.Gorb Chrysomelidae) tosurfaceswithdifferent surfaceenergy Attachment ofGalerucellanymphaeae RESEARCH ARTICLE generateonsurfaceswithdifferent surfaceenergies. We compareourfindingswithpreviousstudiesontheforcesthat result ofthesimilarchemicalcompositiontheirattachmentfluid. (86 comes closetothecontactangleoftheirhostplant INTRODUCTION ABSTRACT leaves ofthewater-lilies example, livesandforagesmainlyontheuppersurfaceoffloating For larvae,aswellforadults,itisessentialtostayattached to deg). Thesimilarityinlarvalandadultperformancesmightbea strongly reduces

deg comparedtothoseof6,26and109 and testthehypothesisthatlarval Nuphar Nuphar and Nymphaea stands andthusincreases (Linnaeus 1758),for (Smirnov, 1960).

deg. This specialisation to variousnaturalsubstrates,itmight bepossiblethat attachment padstructures,but alsohavedifferent degrees of Because different insectspeciesdonotonlyhavedifferent energy ontheadhesionabilityofinsectsremains unclear. surfaces withhighersurface energies. Thus,theroleof surface 2009; Gorbetal.,2010),theinsects performed,byfar, betteron angles weredetected,whereas indifferent studies(Lüken etal., the abilityofbeetlestoattachontosurfaceswithdifferent contact studies (AlBitaretal.,2009;Prüm2013),nodifferences in results ofthesepapersarenotconsistent.Forexample,in two Bitar etal.,2009)andsawflylarvae(Voigt andGorb,2012).The species, othersweredoneonsmoothattachmentorgans ofmoths(Al focus waslaidonhairyattachmentsystemsofdifferent Hosoda andGorb,2012;Lükenetal.,2009;Prüm2013) the Although insomestudies(GorbandGorb,2009;Gorbetal., 2010; the smallercontactangle,higherfreeenergy ofasurface. of wateronthesurfacewasmeasured.Ingeneral,onecansay that In mostofthem,insteadthefreesurfaceenergy, thecontact angle insects’ attachmentabilityhasbeenanalysedinafewstudiesonly. Voigt etal.,2008).However, theimpactofsurfaceenergy onthe Bullock andFederle,2011; Daietal.,2002;GorbandGorb,2009; to surfacesofdifferent roughness(e.g.BeutelandGorb,2001; substrate (e.g.DeSouzaetal.,2008a). on thecontactanglebetweenfluidandsurfaceofparticular (Langer etal.,2004).Thecapillaryforces,inturn,stronglydepend adhesion, capillaryforcesaccountlargely fortheattachmentability Geiselhardt etal.,2010;Ishii,1987).Inthisso-calledwet composition oftheircuticularlipids(Geiselhardtetal.,2009; of hydrocarbons,fattyacidsandalcohols,similartothe to theattachmentperformance.Inbeetles,thisfluidisamixture that issecretedintothecontactzonewithsubstratecontributes (Federle etal.,2001).Inbothtypesofattachmentsystems,afluid representatives ofOrthoptera(Gorbetal.,2000)andHymenoptera in amongstothertaxa– smooth adhesivepadsarefound– and flies(Gorbetal.,2001;Niederegger2002),whereas in adultbeetles(BullockandFederle,2011; GorbandGorb,2002) and smooth.Hairyattachmentorgans canbefound,forexample, many timesindependentlybasedontwomainprinciples–hairy Gorb, 2001;Scherge andGorb,2001),whichevolved underwater (A.B.andS.K.,personalobservations). (Balmert etal.,2011). Larvae,aswelladults,areabletomove persists for2 an airfilmrendersthebeetlewaterrepellentforsometimeand is almostcompletelycoveredwithlongsetae.Betweenthesesetae, below thewatersurface(Kouki,1991b).Thebodyofadultbeetles visible ontheircuticle,buttheysinkimmediatelywhendropped 1986), thelarvaesurviveunderwatertemporarily, havinganairfilm water. AccordingtoHippaandKoponen(HippaKoponen, (wind, waves,rain,etc.),whichcancauseleavestobecoveredby There areanumberofpapersconcerningtheattachmentinsects A varietyofattachmentorgans occurininsects(Beuteland (Coleoptera, days whensubmerging afreshlykilledspecimen 4213

The Journal of Experimental Biology an experiment,wetestedthehypothesisthatin 4214 The thirdinstarlarvaewereroughly7 surfaces withsurfaceenergies similartothatoftheirhostplant. Nuphar lutea insect speciesthathasspecialisedtoliveonandattachmainly attachment structuresoflarvalandadult ‘generalist’ ones. be exhibitedtoagreaterextentin‘specialist’ speciescomparedto adaptation tothesubstratechemistryisspeciesspecificandmight RESEARCH ARTICLE Larvae Morphology RESULTS otc ihtesrae(i.1E–J). contact withthesurface(Fig. ventral partsoftheabdominalsegmentsandpygopodiummake observations). Whilethelarvaeattachtoasurface,thoraciclegs, total adhesionforceofthelarvae(C.G.,A.B.andS.K.,personal 1C–F),whichstronglycontributestothe a fleshypygopodium(Fig. 1A,B,G).Theterminalabdominalsegmentischaracterisedby (Fig. legs bearanarolium-likestructureandaclawattheirdistalend In thispaper, westudythemorphologyofsmoothandhairy leaves, thehighestattachmentforcesarereachedon

mm long( G. nymphaeae N G. nymphaeae,an =20). Allthoracic . In However, femalebeetlespossessedmoresetaecomparedtomales 2E,F). between theshapeoffemaleandmaleadhesivesetae(Fig. 10.4±1.1 (N to thedistalendofthirdtarsalsegment. the setae’s endsandthenumberofnubsincreasefromproximal that areupto2 The uppersideofthesespatula-shapedsetaeiscoveredwithnubs widen towardstheirtips,reachingupto6 3 shaped setaearecylindricalattheirbasewithadiameterof1to 2D).Thesespatula- spatula-shaped setaeatitstheventralside(Fig. however, onlythethirdtarsalsegmenthasmoredenselypacked but theforthtarsalsegmentarecoveredwithlongandpointedsetae; strong tipattheoutersideandahalfaslonginnerside.All It iscomposedoftwohalvessimilarinshape,eachwithalonger 2A–C,E,F).A clawissituatedatthedistalendofeachtarsus. (Fig. consists oftwolobesandthereducedforthoneishardlyvisible bears fivetarsalsegmentsandaclaw. Thethirdtarsalsegment Adult beetleswere~6 Adults μm andalengthof40to62 Females outweighedmales[12.3±1.7(mean±s.d., The JournalofExperimentalBiology(2014)doi:10.1242/jeb.108902 =5), respectively].We didnotfinddistinctdifferences μm inlength.Thereisatendencythatthewidthof

mm longand3 (A,C,E); 50 arolium-like structure;cl,claw. Scalebars:1 to asmoothglasssurface,ventralview. ar, at coaxialillumination.(J)Pygopodiumattaching surface. Areasincontactwithglassappeardark of alarvaattachingupsidedowntosmooth abdominal segments(H)andthepygopodium(I) attaching toasurface.(H,I)Contactareaof (G) Arolium-likestructureofathoracicleg specimen. (F)Detailofthepygopodium. attached toasmoothsurface.(E)Whole (E,F) Ventral viewofaspecimenfrozenwhileit abdominal appendage(pygopodium). forefoot. (C)Ventral view. (D)Detailofthe microscopy images.(B)Thoracicleg,right (A) Lateralview. (B–F)Cryo-scanningelectron Galerucella nymphaeae Fig.

.Habitusandattachmentorgansof 1. μm. Allbutthemostproximalsetae μm (B);250 mm wide(N μm inwidthattheirend. μm (D,F–H);100 at the3rdlarvalstage. =26). Eachleg N =4) and μm (I,J).

mm

The Journal of Experimental Biology RESEARCH ARTICLE individuals, is giveninbrackets; (3.67–14.13 all surfaceswas1.50 The medianfrictionforcecalculated foralldatapooledtogether ( The surfaceroughnessof (Student’s ANOVA, hairs onthefore-,middleandhindleg(one-wayrepeatedmeasures values of6.729±1.493 μm(mean±s.d., for middleversushindlegs). fore- andmiddlelegs, comparisons withHolm–Sidakmethod, (1285±150) andleasthairsontheforeleg(1242±139;pairwise (1320±134, mean±s.d., t differences betweensexeswerenolongerdetected(Student’s were normalisedtothebodymassofbeetles,significant Friction force of Surface roughness 7 R = a − ) valuesof5.143±1.413 0.171, F t P 2 -test,

mN) foradults(medianvalues, theinterquartilerange =11.517, n =0.869). We founddifferent numbersofattachment =5 repetitionsfor eachindividualonsurface). t 7 =2.521, mN (0.78–2.63 P P <0.001) withmosthairsonthemiddleleg =0.031 forfore-versushindlegs,P N. lutea N. N. lutea N =18 larvalindividuals, P μm androotmeansquare(rms,R N =0.040). Whenthenumberofsetae =12), followedbythehindleg leaves leaves yieldedroughnessaverage mN) forlarvaeand6.97 P n <0.001 forcomparisonof =7). N =30 adult =0.041 t -test,

mN q ) i.4. Fig. adjusted pairwisecomparisonsofthesafetyfactorspleaserefer to 3).ForTukey’s multiplicity- both larvalandadultindividuals(Fig. factors werehighestonsurfaceswithacontactangleof83 angle (N structures between theclaws(e.g.Gannonet al., 1994;Nieet Chrysomelidae (e.g.Baselga,2007; Huaetal.,2013).Arolium-like resembles thoseofother representatives ofthefamily The generalhabitusofthird larvalinstarsof ( contact angles[ individuals differed significantly betweensurfacesofdifferent weight. and thatadultsresistedforces63(34–109)timeshigherthan their forces, onaverage,15(9–27)timeshigherthantheirbodyweight individuals. Itsvaluesrevealedthatlarvaewereabletowithstand weight) wascalculatedtoaccountforweightdifferences between The dimensionlesssafetyfactor(frictionforcedividedbybody Larvae Morphology DISCUSSION N Both frictionforcesandsafetyfactorsoflarvalaswelladult =30 adults, =4) asthefixedfactor].Thefrictionforcesandsafety The JournalofExperimentalBiology(2014)doi:10.1242/jeb.108902 N P =18 larvae, <0.001, linearmixedeffects model withindividual substrate, afloatingleafof G. nymphaeae. Fig. segments; T4isreduced.Scalebars:1 male (F)beetle,ventralview. T1–T5,tarsal segment ofarightmiddlelegfemale(E)and a male,disto–lateralview. (E,F)Thirdtarsal the thirdtarsalsegmentindetail,righthindlegof ventral view. (C)Tarsus, lateralview. (D)Setaeof 100 μm (B,C);10(D);50(E,F).

.Habitusandattachmentorgansofadult 2. n =5) astherandomfactorandcontact (A) Beetleonitstypical N. lutea G. nymphaeae . (B)Tarsus,

mm (A);

deg for 4215

The Journal of Experimental Biology 4216 covered withspatula-shapedsetae.Inmanychrysomelids,allthree studied arecoveredwithpointedsetae,thethirdoneisdensely The ventralsidesofthetwoproximaltarsalsegmentsspecies pads ofthoraciclegs. segments seemtocontributeevenlessoveralladhesionthanthe forces thantheattachmentdevicesassociatedwithlegs.Abdominal we assumethatthepygopodiumcontributesmoretoattachment demonstrated hereforthefirsttime.Basedonourownobservations, segments totheattachmentperformanceofleafbeetlelarvaewas nymphaeae. Thecontributionoftheventralsurfaceabdominal described forotherchrysomelidspecies,butnotyet 2012) andthepygopodium(Nieetal.,havebeenpreviously RESEARCH ARTICLE Adults Contact angle (deg) Safety factor Friction force (mN) 109 109 109 109 100 120 140 160 83 109 83 109 10 20 40 60 80 83 83 26 26 0 2 4 6 8 0

– – – – – – B A

– – – – – – 04 08 0 120 100 80 60 40 20 0 204060801001200 83 26 26 83 26 26 a,b a,b

6 6 6 6 6 6 BD –0.4 –0.4 AC ( (

b c b ( (

–0.2 –0.2 ) ) ( ( ) ) ( (

0 0 ) ) ( (

c

( ( 0.2 0.4 0.2 0.4 ) )

) )

Contact angle (deg) angle Contact a a ) ) P<0.001 P=0.003 P=0.002 P=0.326 P<0.001 P=0.300 P<0.001 P=0.003 P=0.002 P=0.326 P<0.001 P=0.300 Linear function 100 200 300 400 500 10 20 30 40 50 60 70 0 0 109 109 109 109 C 83 109 83 109 D 83 83 26 26 04 08 0 120 100 80 60 40 20 0 04 08 0 120 100 80 60 40 20 0

a – – – – – – a

– – – – – – 83 26 26 83 26 26

6 6 6 6 6 6 a b a ( ( –0.1 0 –0.1 0 ( ( G. ( ( ( ( nymphaeae the surfacemorphologyoffemales’ elytra,astheelytraof assume thatthelackofdiscoidsetaein during copulation(Stork,1980a;Stork,1980b;1983).We probably adaptedforattachmenttothesmoothelytraoffemales species withratherglaborouselytrainthefemales.Suchsetaeare found infemales(Stork,1980a).However, theyoccuronlyin Cerambycidae andCoccinellidaebeardiscoidsetaethatcannotbe beetle species.MostmaleindividualsintheChrysomelidae, nymphaeae. Thisisdifferent tothepadmorphologyofmanyother differences intheshapeofadhesivehairsmaleandfemale 2002; Stork,1980a;Voigt etal.,2008).We couldnotfindany shaped ordiscoidal(BullockandFederle,2009;GorbGorb, visible tarsalsegmentshavesetaethatcanbepointed,spatula ) ) ( ( ( ( b ) )

0.1 0.1 a,b a,b ) ) The JournalofExperimentalBiology(2014)doi:10.1242/jeb.108902 are denselycoveredwithwater-repellent setae.Owing ) )

0.2 0.2 ) ) ) ) P=0.295 P=0.112 P<0.001 P=0.325 P=0.003 P=0.948 P=0.292 P=0.114 P<0.001 P=0.331 P=0.003 P=0.947 Fig. water contactangles. nymphaeae significantly iftheydonotsharethesameletter. Pairwise comparisonswithinasingleplotdiffer percentiles (errorbars)andoutlyingvalues(circles). 75th percentiles(endsofboxes),10thand90th show themedians(lineswithinboxes),25thand safety factors,(A,B)larvae,(C,D)adults.Theplots

.Frictionforcesandsafetyfactorsof 3. given second. (larger) comparedtothecontactangle angles givenfirstaresignificantlysmaller zero line,thesafetyfactorofcontact completely totheleft(right)sideof differences. Iftheconfidence intervallies plot. P displayed ontheright-handsideofeach given. Thecorresponding of Tukey’s all-paircomparisonsare estimators andtheconfidenceintervals log adults. Values ofthesafetyfactorwere (B,D) safetyfactors,(A,B)larvae,(C,D) contact angles. on glasssurfaceswithdifferent and safetyfactorsof linear mixedmodeloffrictionforces Fig. on glasssurfaceswithdifferent 10

4. -transformed beforeanalysis.The -values inboldindicatesignificant Post hoc G. nymphaeae (A,C) Frictionforces,(B,D) (A,C) Frictionforces, performance ofthe G. nymphaeae P -values are is owingto G. G. G.

The Journal of Experimental Biology highest forceson thesurfaceof83 contact angles.Transferred toourresults,wewouldexpectthe constant, theforcesdecreasewith increasingasymmetryinthetwo contact anglesandstatethatif the sumoftwocontactanglesis strength ofcapillaryforcesbetween twosurfacesthathavedifferent principle (DeSouzaetal.,2008b). DeSouzaetal.calculatedthe regardless ofthenumbercontacts mightbeevokedbyaphysical possible inanyspecies. specialist species;or(iii)whethermodificationsofthefluidare not properties ofthefluidareconstant,whichmightbecase in which mightbethecaseingeneralistspecies;(ii)whether the attachment fluidtothesurfacechemistryinashort-termrange, studies shouldhenceconsider(i)whetherinsectscanadapt their components areproducedbydifferent glands(Betz,2003).Further might beadaptedrapidlytothecontactsurfacebecause these Stenus the productionofanon-lipoidfractionintarsalsecretion of (Federle etal.,2002;Gorb,2001;Vötsch2002).Forinstance, allows themtoattachsurfaceswithdifferent surfaceenergies has beenpreviouslydiscussedthattheoil-watermixtureoffluid adaptation. Forotherinsecttaxa,suchasants,fliesandlocusts,it attachment abilitiesmightbetheresultofalong-termevolutionary exhibited watercontactanglesofaround86 the liquiddeliveredintocontactzone.Because explained byapossiblesimilarityinthechemicalcompositionof similarity ofthedataobtainedforlarvaeandadultsmightbe RESEARCH ARTICLE (6 contact angleof83 nymphaeae et al.,2000;Voigt andGorb,2012). beetles. Forsafetyfactorsofothertaxa,please,seereviews(Federle latter studydealtwithtractionforcesgeneratedbywalkingtethered forces weremeasuredusingacentrifugalforcetester, whereasthe female onglass)(Stork,1980b).Inthefirsttwostudies,friction al., 2009)and39–70( Brentidae, maleandfemaleonglasssilanisedglass)(Lükenet male onglass)(Voigt etal.,2008),45–268( are 60–71(Leptinotarsadecemlineata studies usingvariousinsecttaxa.Previouslypublishedsafetyfactors nymphaeae values calculatedfrommeasuredfrictionforcesofadult completely lackingintheliterature.However, thesafetyfactor (34–109) foradults.Safetyfactordataonlarvalcoleopteransare the interquartilerangeisgiveninbrackets)forlarvaeand63 weight ofanimalsandyieldedvalues15(9–27)(medianvalue, device, tractionforcemeasurementsintetheredwalkinganimals). individual andthemethodusedtomeasureforces(centrifugalforce not onlyontheinsectspecies,butalsoweightofan data fromotherstudiesrevealsgreatdiversity, astheforcedepends The comparisonofthefrictionforcemeasuredinthisstudywith structures preventconglutinationofneighbouringsetae. 2006; HaasandGorb,2004).Itwasassumedthatsuchsurface spatula-shaped insectsetaehasbeendescribedpreviously(Federle, surface oftheelytra. the properadhesivecontactbetweenitshairsand to thesehairystructures,itmightbeimpossibleforamalemake Friction force The similarityofthetrendinlarvalandadult Friction forcesandsafetyfactorsofbothlarvaladult We computedthesafetyfactortoaccountfordifferences inthe The presenceofsmallnubsorcorrugationsontheupperside deg), untreatedglass(26 beetle speciesshowsthatthecompositionoftarsalliquid lie withinthesamerangeaspreviouslyshowninsimilar were thehighestonsilanisedglasssurfacewitha

deg comparedwiththoseonplasma-treatedglass Chrysolina polita

deg) andsilanisedglass(109

deg ifthecontact angleofwater , Chrysomelidae,femaleand , Chrysomelidae,maleand Cylas puncticollis

deg, thebeetles’ N. lutea G. nymphaeae

deg). The leaves G. G. , possess suchafineroughness( taken intoaccount. species, developmentalstages,sexes andexperimentaldesignswere friction forceswithincreasing contactangle,althoughdifferent studies thatdetectedsignificant differences showeddecreasing than thosewithcontactangles below40 on surfaceswithlowroughnessvalues(R roughness (Gorbetal.,2010;Kovalev2012). higher amountofthefluidowingtoitsstrongabsorptionbyfine with theterminalcontactelements):thesespeciesmightneeda surfaces withfineroughness(ofmuchsmallerdimensionscompared setae. Thelattercasemighthaveevolvedinspecieswalkingon might berelativelyhightopreventextensivefluidlossfromthe angles betweenthesetae–liquidandliquid–surfaceinterfaceorit attachment hairsmighteitherbeadaptedtoachievesimilarcontact (Kovalev etal.,2012).Hence,thesurfaceenergy ofinsect attachment hairswouldpreventinsectsfromahighrateoffluidloss fluid. Iftheproductionislimited,ahighsurfaceenergy ofthe attachment abilitiesofinsectsarelimitedbytheproduction surface afterfootdetachment.Itisnotsolvedyetwhetherthe attachment fluidbecauseatleastpartofitremainsattachedtothe the insects’ host plant.Whilewalking,insectsmustrenewtheir in ordertodrawconclusionsonapossibleadaptationofthefluid insects ontheirhostplantsandvariousfurthersubstrataareneeded experimental studiesonthepropertiesofattachmentfluid surfaces (R compared withthosehigherthan953 lower frictionforcesonsurfaces withcontactanglesabove100 from fivedifferent studies(excludingthepresentone)revealed contact anglesweredetected.However, six experimentalset-ups different studies,nosignificant dependenciesoffrictionforceson 5).Infiveexperimentsfromfour 1;Fig. surface energies (Table with forcemeasurementsofinsectsonsurfacesdifferent matter areratherheterogeneous.We analysedliteraturedatadealing in insectattachment.However, theresults of previousstudiesonthis apply toG.nymphaeae. of fluidproductiontofillthefinesurfaceirregularitiesshould not between thebeetles’ fluidand remains uncleartowhatextentthesemodelsfitthecontactangle contact elementsunderlieevolutionarypressure.However, it angles of90 achieve thehighestattachmentforcesonsurfaceswithcontact number orareaofcontactelements,insectsingeneralshould from thisoutcomethat,ifthereisnoevolutionarypressureonthe 90 favourable ifahightotalcapillaryforceistobeachieved.Anglesof liquid bridges,contactanglesofapproximately70 angle asvariablefactors.Theirresultsshowedthat,atnumerous between themandusedthenumberofliquidbridgescontact between twohomogeneoussurfaceswithaconstantliquidvolume Souza etal.,2008a).Theauthorsanalysedtheseparationforce contact angleofthesurfacehasalsobeensubjectmodels(De angles ofaround80 for thegenerationofstrongfrictionforcesonsurfaceswithcontact data onthecontactanglesofattachmentsetaeinsects. to 166 the setaetipsis57 on thesurfacewitha109 on thesetaetipsalsoliesinmagnitudeof83 In experiments,beetlesshowedstronglyreducedfrictionforces Our hypothesisisthatsurfaceenergy doesplayanimportantrole However, itcannotbeexcludedthatotherfactorsareresponsible deg optimisethecapillaryforcepercontactarea.We conclude deg. Unfortunately, tothebest ofourknowledge,thereisno a The JournalofExperimentalBiology(2014)doi:10.1242/jeb.108902 =30 deg andonthoseof70 nm) (Voigt etal.,2008).Because

deg. Inbothcases,thetwocontactanglessumup deg. Thedependenceofcapillaryforcesonthe

deg contactangleiftheon Nuphar R a =5143 deg ifthenumberorareaof or Nymphaea

nm), theneedforahighrate nm roughnessandsmooth a ) between74and198

deg. Interestingly, all

deg, butlessforces N. lutea leaves. Further

deg are does not 4217

deg nm

The Journal of Experimental Biology 4218 RESEARCH ARTICLE

Table 1. Literature in insect adhesion on surfaces with different but defined surface energies Source Species Systematic position Force measured (method) Surfaces (contact angles, deg) Comments 1 Al Bitar et al., 2009 Cydia pomonella Lepidoptera, Tortricidae Friction force (centrifuge) Glass (39), Plexiglas (74, females Adults (females and males) only), silanised glass (109) 2 Gorb and Gorb, 2009 Gastrophysa viridula Coleoptera, Chrysomelidae Friction force (centrifuge) Glass (30), silanised glass (102) Adults (not differentiated between sexes) 3 Lüken et al., 2009 Cylas puncticollis Coleoptera, Brentidae Friction force (centrifuge) Glass (39), silanised glass (110) Adults (females and males) 4 Gorb et al., 2010 Coccinella septempunctata Coleoptera, Chrysomelidae Traction force (force Glass (30), sapphire samples (85) Adults (females and males) transductor) 5 Hosoda and Gorb, 2012 Gastrophysa viridula Coleoptera, Chrysomelidae Traction force (force Soda-lime glass (43), smooth Adults (females) transductor) polycarbonate (59), modified spurr (104), smooth polycarbonate (108) 6 Voigt and Gorb, 2012 Rhadinoceraea micans Hymenoptera, Tenthredinidae Friction force (centrifuge) Glass (39), silanised glass (109) Larvae (in longitudinal and transverse orientation) 7 Prüm et al., 2013 Leptinotarsa decemlineata Coleoptera, Chrysomelidae Traction force (force Glass (20), epoxy resin replica Adults (males) transductor) untreated (83) and hydrophobised with antispread (105) 8 Present paper Galerucella nymphaeae Coleoptera, Chrysomelidae Friction force (centrifuge) Plasma treated glass (6), glass (26), Larvae, adults (no differences between 2× silanised glass (83, 109) sexes) References are numbered according to Fig. 5. viridula study number5(HosodaandGorb,2012)workedonGastrophysa accompanied byanincreaseinattachmentforces.Forexample, angle uptothecontactofhostplantwouldbe similar tothehostplants,wereincluded,anincreaseincontact other studies.We assumethat,ifsurfaceswithcontactangles, Similarly, thecontactanglesofhostplantswereseldomgivenin 4). with anincreasingcontactangleforlarvae(oradults;seeFig. we likewisewoulddetectdecreasing(orthesame)frictionforces included inotherstudies.Ifweomitthissurfacefromtheanalysis, Surfaces withcontactanglesofapproximately83 might beowingtoparticularsurfacesusedintheexperiments. with anincreasingcontactangle.Thereasonforthesedifferences remained thesame,whereasinourstudy, frictionforceincreased with anincreasingcontactangle(butseeanglesabove100 these twocontactanglesdiffered by4 that inturnhasacontactangleof63 on surfacesof108 that witha59 partly bredfromtheselarvaeandcollectedthefield. studied larvaeofthethirdlarvalstage.Adultsusedinexperiments were which wasreplacedeverythirduptofifthday. Forhandlingreasons,we were supplementedwithmoisttissueunderneathadiscof on surfaceswithcontactanglesabove100 different surfaces. the underlyingfactorsrulingattachmentbehaviourofspecieson angle(s) oftheirhostplant(s)torevealadaptations,whichmightbe representatives ofasingleinsectorderand(iii)providethecontact chemistry evenatthesamecontactangles,(ii)concentrateon of surfaceswithdifferent contactanglesanddifferent individual hence proposethatfurtherstudiesshould(i)coveralarger variety contact angleof43 significant, increaseinthefrictionforcesfromsurfacewitha of Hosoda andGorbtestedasurfacewithcontactanglesimilartothat were loweronthesurfacewitha104 that hadcontactangleshigherthan100 study (HosodaandGorb,2012)differentiated betweentwosurfaces on surfaceswithcontactanglesbetween40and100 angle mightinfluencefrictionforcesinatotallydifferent mannerthan 45–55% relativehumidity)inPetridishesof35 werekeptindividuallyunderlaboratoryconditions(22–24°C, floating leavesof First, weletlarvae attachthemselvestothereverse side ofaPetridishand We usedtwodifferent techniquestostudythecontactareasof thelarvae. The samplesweresputteredwithalayerof~20 Eggs, larvaeandadultindividualsof angles ofroughly40 Visualisation of contact areas Visualisation of microscopy Scanning electron MATERIALS ANDMETHODS at 3 with aGatanALTO 2500cryo-preparationsystem(Gatan,Abingdon,UK) Technologies, Tokyo, Japan)scanningelectronmicroscope (SEM)equipped Images ofthelarvaeweretakenwithaHitachiS4800(Hitachi High- device at3 images ofthesetaeweretakenwith theabovementionedHitachiS4800 TM-3000 (HitachiHigh-Technologies, Tokyo, Japan)at5 chamber. Theattachmentsystemofadult beetleswasstudiedwithaHitachi In allthestudiesweanalysed,frictionforceeitherdecreased Most studiescomparedperformancesonsurfaceswithcontact R. obtusifolius kV acceleratingvoltageand atemperatureof , aspeciesthatalmostentirelyliveson

kV. The JournalofExperimentalBiology(2014)doi:10.1242/jeb.108902

deg contactangle,higherfrictionforcesweredetected Nuphar lutea (59 deg comparedwiththoseof104

deg tothatwithacontactangleof59 deg withthoseonsurfacesof105 deg) andindeeddetectedaslight,althoughnot in theBotanicalGarden,Kiel,Germany. G. nymphaeae

deg contactanglecomparedto deg only.

deg (GorbandGorb,2009). deg. Althoughfrictionforces

deg, changesinthecontact

mm indiameter. Petridishes

nm thickgold–palladium. − 140°C inthepreparation Rumex obtusifolius were collectedfrom

deg wererarely

deg. However, N. lutea

deg, although deg. A single

kV. Detailed

deg. We

deg) or leaves,

The Journal of Experimental Biology measurement deviceOCA 20(Dataphysics,Filderstadt,Germany). measured at10randomlychosenspotsoneachdiscusingthecontactangle the higherfreeenergy ofasurface.Contactanglesthesurfaceswere representative parameterofthesurfaceenergy: thelowercontactangle, n angles, twoglassplatesweresilanisedtocontactanglesof83±3 contact angleof26±2 N Discs treatedinthiswayobtainedacontactangleof6±5 Ebhausen, Germany)withoxygenasthecarriergasfor~2 treated intheplasmadeviceZEPTO B(DienerelectronicGmbH+Co.KG, surface energies. To obtaindiscswithhighsurfaceenergies, theywere RESEARCH ARTICLE diameter wasrotatedwithaspeedof50 adult beetlesondifferent surfaces (Gorbetal.,2001).A drumof10 (Tetra GmbH,Ilmenau,Germany)toassessattachmentforces of larvaland We usedthecomputer-controlled centrifugal forcetesterTetra Zentri-01-P Leaves ofN The experimentswererunonglassdiscsof10 glass appeareddarkincoaxialillumination,whereasothersbright. inverted sothattheattachingorgans facedupwards.Surfacesincontactwith For thispurpose,larvaewereplacedinPetridishesthatsubsequently incident illuminator(LeicaGmbH,Wetzlar, Germany) andavideocamera. a LeicaMZ205A stereomicroscopeequippedwithanintegratedcoaxial dorsal sideontoametalholder. Second,wevisualisedthecontactareaswith deep-frozen larvaewereremovedfromthePetridishandgluedwiththeir filled itwithliquidnitrogen.Thecontactareaswerefrozenimmediately. The individuals facing inwardsversusoutwards)atthemoment ofdetachment. not possibletoconsider theorientationofbeetles inthedrum(e.g. (Gorb andGorb,2004).Owingtothe highmobilityoftheindividuals,itwas factors bydividingthefrictionforce bythebodyweightofeachindividual account fordifferences inweights,weobtainedthedimensionless safety with otherstudiesthatdidnotcompute thesafetyfactors.However, to was droppedandthemassofindividual (Gorbetal.,2001). of rotation,therotationalspeeddrumatmomentindividual in eachsinglerunwascalculatedfromthedistanceofinsectto centre on thedrumwaslocatedusingalightbarrier. Themaximumfrictionforce a maximumof3000 Middlefield, USA)usingthe×5lens. with thewhitelightinterferometerZygoNewView 5000(ZygoCorporation, leaves measured, Friction force Surfaces of Surfaces usedfor experiments =10) and109±3 =6 discs,n We measuredthecontactanglesofbi-distilledwateronsurfacesasa We usedfrictionforcestoallowforcomparisonsoftheobservedtrend Force (mN) 10 15 20 25 30 35 40 0 5 04 08 0 120 100 80 60 40 20 0 AB 7m 4m * . =10 measurementsoneachdisc).Thenon-treateddischada 1m N. lutea N. lutea 6l 1f t n deg (N =10 timeseach).Theirsurfaceroughnesswasmeasured

bore acontactangleof86±17 rounds

deg (N leaves =1, min =1, n =10). –1 n =10). To preparediscswithhighercontact within 20 Contact angle (deg) angle Contact

rounds

s. Thepositionoftheindividual min

cm diameteranddifferent 10 15 20 25 30 35 40 0 5

–1 deg (mean±s.d., 204060801001200 8l 8a and acceleratedupto

2a deg (mean±s.d.;

min at0.2 4f 3m 6l 3f 5f l

deg (N

cm in mbar. N =1, =7 performed withthe package a fixedfactorandindividual( surfaces usingalinearmixedeffects model.Surface( the residuals.We comparedthevaluesindividualsyieldedondifferent log Prior totheanalyses,obtainedfrictionforcesandsafetyfactorswere Project No.C-10withinCollaborativeResearchCenter(SFB677)toS.G.]. This workwassupportedbytheGermanScienceFoundation[GO995/7-1and commented onthemanuscript. C.G., A.B.andS.K.wrotethemanuscript.S.G.interpretedresults the experiments.C.G.,A.B.andS.K.tookimagesanalysedresults. S.G. conceived,andC.G.designedtheresearch.A.B.S.K.performed The authorsdeclarenocompetingfinancialinterests. thank twoanonymousreviewerswhohelpedtoimprovethispaper. A. Kovalevhelpedwithmeasurementtechniquesandfruitfuldiscussions.We also collect beetlesandleaves.E.Appel,Gorb,L.Heepe,Kizilkan,M.Klein We wouldliketothankthe staff oftheBotanicalGarden,Kiel,forpermissionto each ofthemfivetimes. amr,A,FoinBh,H,DtceKr,P n atlt,W. Barthlott, and P. Ditsche-Kuru, H., FlorianBohn, A., Balmert, S.N. Gorb, and C.P. W. Zebitz, D., Voigt, L., Al Bitar, aeg,A. Baselga, Development CoreTeam, 2012)usingthe multiplicity-adjusted pairwisecomparisons. forces andsafetyfactorsbetweensurfaceswereanalysedusingTukey’s individual oneachsurface)asarandomfactor. Differences inthefriction 2008). Statistical analyses References Funding Author contributions Competing interests Acknowledgements ez O. Betz, etl .G n ob S.N. Gorb, and R.G. Beutel, surfaces. J.Morphol. water: comparativemorphologyandfunctionalaspectsofair-retaininginsect the larvaeofEuropeanGastrophysa. 1851) [= to smoothsurfaces. attachment abilityofthecodlingmoth 255 Staphylinidae): externalmorphology, ultrastructure, andtarsalsecretion. phylogeny. hexapods (Arthropoda): evolutionarypatternsinferredfrom arevisedordinal In total,18larvaeand30adultindividualsweretestedoneachsurface, We ranthelinearmixedeffects modelswiththesoftwareR(R 10 -transformed toachieveahomogeneousdistributionofthevariances , 24-43. G. unicolor (2003). Structureofthetarsiinsome nlme (2007). Descriptionofthematurelarva of information seeTable plots giveninthepreviousstudies(numbered1–8,formore 2010) andmedians(allotherpapers)weremeasuredfromthe different contactanglesofwater. Fig. respectively (presentpaper). adult males(Prümetal.,2013);8land8a,larvaeadults, transversal orientation,respectively(Voigt andGorb,2012);7m, (Hosoda andGorb,2012);6l and males,respectively(Gorbetal.,2010);5f,adultfemales males, respectively(Lükenetal.,2009);4fand4m,adultfemales females (GorbandGorb,2009);3f3m,adult females andmales,respectively(AlBitaretal.,2009);2a,adult droplets astheywerespreadingonthesurface.1fand1m,adult by solidlines.*Thecontactanglewasnotmeasureableformost differences betweentwoproximatecontactanglesareindicated significant differences betweencontactangles.(B)Significant J. ZoologicalSyst.Evol. Res. The JournalofExperimentalBiology(2014)doi:10.1242/jeb.108902

.Adhesionforcesyieldedonsmoothsurfaceswith 5. (Pinheiro etal.,2012). J. InsectPhysiol. auct.] (Coleoptera:Chrysomelidae:Chrysomelinae) andkeyto glht 272 , 442-451. function ofthepackage (2001). Ultrastructureofattachmentspecializations of

1). (A)Experimentsthatdidnotdetect N larva 55, 1029-1038. Cydia pomonella 39, 177-207. Zootaxa =18, l and 6l lme N Post hoc 1594 adult t The means(Gorbetal., , larvaeinlongitudinaland function implementedinthe Gastrophysa janthina (2009). Tarsal morphologyand Stenus =30, multcomp , 61-68. L. (Lepidoptera,Tortricidae) N n =4) wasincludedas comparisons were =5 repetitionsper species (Coleoptera, (Hothorn etal., (2011). Dryunder J. Morphol. (Suffrian, 4219

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