*Present address:NongLamUniversity, HoChiMinhCity, Vietnam. UK. Lancaster Environment Centre, LancasterUniversity, Bailrigg, Lancaster LA14YQ, threshold (0.031 even seeninwaspsthathadfedonglucoseatthelowestacceptance linked withlowernutritionalperformance.Thisfeedinginhibitionwas inhibition seenforfructosefollowingexposuretoothersugarsisnot quality ofthesugarinlongevitytrial.Thepronouncedfeeding maltose appearsadaptiveinlightoftherelativelypoornutritional higher acceptancethresholdandshortfeedingtimeinthecaseof distinguish maltoseandfructosefromtheothersugarstested.The following abriefexposuretoothersugars,suggestingthatwaspscan showed astrongdecreaseinfeedingtimeformaltoseorfructose fructose, whichwassignificantlylowerthanformaltose.Wasps University, Fukuoka812-8581,Japan. 1692 Received 5June2013; Accepted22January2014 ‡ 1 sugars canvarywidely;whileseveralfailtoelicitanyfeeding al., 2005;WäckersandvanRijn,2012). on otherfactors,suchasflowerarchitecture(Jervis,1998;Olson et directly drivenbynectarsugarpreferences,orwhetherthisis based remains unclearwhethertheobservedinsect–flowerassociations are and Baker, 1982).However, inmanygroupsofnectarfeeders,it tendency tovisitnectarsofacertaincarbohydratecomposition(Baker rich (BakerandBaker, 1983),andinsect groupsoftenshowa their nectarcompositionassucrose-richorhexose(glucose/fructose)- Wäckers, 2005).Floralnectarshavebeencategorizedaccordingto Kloft, 1977;BakerandBaker, 1983;NemecandStarý,1990; sources, suchasnectarandhoneydew(Percival,1961;Kunkel There isalarge variationinsugarcompositionamongnatural feeding KEY WORDS:Feedinginhibition,Gustatoryresponses,Sugar glucose, fructoseandmaltoseatconcentrationsof0.008–2.0 found inplants.Whengivensinglesugarsolutionsofsucrose, common sugars,includingfructose,glucose,maltoseandsucrose, croceipes We examinedgustatoryresponsesofthelarvalparasitoid J. K.Makatiani prior sugarexposure croceipes An acquireddistaste:sugardiscriminationbythelarvalparasitoid RESEARCH ARTICLE © 2014.PublishedbyTheCompanyofBiologistsLtd|JournalExperimentalBiology(2014)217,1692-1700doi:10.1242/jeb.091843 USDA-ARS, Tifton,GA31793-0748, USA. INTRODUCTION ABSTRACT ranged between0.054and0.085 the estimatedconcentrationsatwhich50%ofwaspsinitiatedfeeding as maltoseandfructoseaftergustatorystimulationonothersugars. to showfeedinginhibitionofotherwisephagostimulantsugarssuch Author ([email protected]) for correspondence BioresourcesandBioenvironmental Sciences, Kyushu Graduate School of Parasitoid feedingresponsestodifferent nectarandhoneydew 4 Faculty of Agriculture, Kyushu University,Faculty of Fukuoka812-8581,Japan. to determinewhethertheadultsdiscriminateamong

mol 1 , H.K.Le l − 1 ) andpersistedfor24 2 1, Crop ProtectionandManagementUnit, *, D.M.Olson

3 mol Centre for Sustainable Agriculture, l − 1 for sucrose,glucoseand

(: )isaffected by h. Thisstudyisthefirst 2 , F. L.Wäckers Microplitis

mol l − 1 , 3 at concentrationsasloworlowerthan0.016 response infood-deprivedparasitoids,othersugarsstimulatefeeding social Hymenoptera, theyareunsuitablefor thestudyoffood Lanza, 1988;Lanzaetal.,1993; Völkletal.,1999). visiting thedifferent foodalternatives(LanzaandKrauss, 1984; experiments. Thesestudiestypically countthenumberofants also usedasamethodto establish preferencesinchoice intensity withwhichscoutsrecruit nestmatestofoodalternativesis Bachman, 1981;Barronetal.,2009).Inthecaseofants,relative how beesvaluateapreviouslyvisitedfoodsource(Waller and turning frequency, thisparametercanalsobeusedasanindicatorof Frisch, 1934).Asbeescommunicatefoodsourcequalitythrough the bees exhibitatypical‘beedance’ torecruitadditionalforagers(von Alm etal.,1990).Analternativemethodmakesuseofthefact that experiments (Wykes, 1952;Waller, 1972;InouyeandWaller, 1984; honeybees typicallyassesstherelativeconsumptioninchoice honeybees. Thetestsusedtodeterminefeedingpreferences in This hasfacilitatedthestudyoffoodpreferencesinants and continuously collectfoodtocovertheneedsofentirecolony. Hymenoptera, bothoftheseconditionsaremet,astheforagers inclination tosamplethefoodsource.Incaseofsocial require thatthetestorganism showsrepeatedfeedingbouts,andan measure thequantityoffoodeaten(Dethier, 1976).Thesemethods more typesoffoodsimultaneously, observeitsbehaviorand/or todiscriminateandselectfoodsispresentitwithtwoor most directwaytoinvestigatetheabilityandpropensityofan methods usedtoassesssuchpreferencesvaryamongstudies.The has beeninvestigatedinseveralvertebratesandinvertebrates.The relative preferenceforindividualsugarsremainsunknown. sugars havebeentested,theimpactofpriorexperienceand experiences. Whileparasitoidgustatoryresponsestoindividual sugars andadapttheirpreferenceinaccordancewithfeeding parasitoids canbeexpectedtohaveapreferenceforhighqualityof As naturalsugarsourcesarevitalfortheirsurvivalandreproduction, rates andtheirownphysiologicalstates(vanAlphenVet, 1986). for hostsofdifferent qualityinaccordancewiththeirhostencounter Christensen, 2001).Parasitoidsareknowntoadapttheirpreference fitness benefits(Charnov, 1976;StephensandKrebs,1986;Sih should choosedietitemstomaximizetheirnetenergy intakeornet explained byoptimalforagingtheory, whichpredictsthatforagers of parasitoidfeedingresponsestodifferent sugarscouldbe suitability ofthecarbohydratestested(Wäckers,2001),variation parasitoid feedingresponseisoftencorrelatedwiththenutritional 2001; Beachetal.,2003;Williams andRoane,2007).Becausethe fructose andsucroseascomparedwithlow-qualitysugars(Wäckers, consume larger quantitiesofhigh-qualitysugarssuchasglucose, Beach etal.,2003).Atequalconcentrations,adultparasitoids and K.Takasu While thesemethodsareeffective inestablishingpreferences in The preferenceofnectarfeedersforvariouscomponents 4,‡

mol l − 1 (Wäckers, 1999;

The Journal of Experimental Biology nutritional suitability, i.e.theirabilitytosustain adultlongevity. changes offeedingresponse arediscussedintermsofsugar stock culturefromtheUSA (Tifton strain)were alsotested.The maintained formorethan10 sugar feedingresponsesbetween ourlaboratorystrainthathadbeen feeding experiencewiththesame orothersugars.Differences in examined whetherfeedingresponses tosugarschangedaftera are abletodiscriminatebetweenindividualsugars.Subsequently, we fructose andmaltose.Thiswastodeterminewhethertheparasitoids croceipes of food-naïveandfood-experiencedmalefemaleadults of (Wäckers etal.,2006).Hereweexaminedinnatefeedingresponses fructose andmelezitoseareeffective asunconditionalstimuli conditioning offoododorsby 1997; Wäckersetal.,2002;2006).Inassociative Takasu andLewis,1995; Takasu andLewis,1996;Stapeletal., subsequently respondtothelearnedodors(LewisandTakasu, 1990; adults canalsolearntoassociateodorswithvarioussugars and 2002; NafzigerandFadamiro,2011), andbothmalefemale had beenusedinprevioussugarfeedingstudies(Wäckersetal., Wäckers etal.,2006;Zhou2012).Additionally, thisparasitoid 1993; Takasu andLewis,1996;Stapeletal.,1997;Röse2006; sugar sources,suchasnectarandhoneydew(Takasu andLewis, represents thelarge groupofparasitoidswhosedietisrestrictedto Lewis, 1993)orfeedonpollen(Jervis,1998),andthereforeit was chosenforthisstudybecauseitdoesnothostfeed(Takasu and Stadelbacher, 1983;HoangandTakasu, 2005). (Lewis andBurton,1970;KingColeman,1989;Knipling is anendo-larvalparasitoidof provide amoreappropriateassessmentofpreference. intake basedoncompleteinformationbothalternatives,itcould method allowsindividualsfromthefirstgrouptomodifytheirsugar same sugarastheoneoffered inthe with thatofacontrolgroup,whoseinitialexperienceinvolvedthe on thealternative.Thetotalamountofconsumptionisthencompared exposed toonefoodsource,beforebeingallowedfeed solitary organisms. Inthismethod,anindividualwaspisfirstbriefly solution. only whenbeesacceptedonesolutionafterrejectingtheother small amountsoftwosugarsolutions.A preferencewasobserved similar testforindividualhoneybees.Theyalternatelyoffered bees Bachman andWaller (BachmanandWaller, 1977)conducteda experienced alternative(Erhardt,1992;RomeisandWäckers,2000). butterflies imbibeonesolutionafterrejectingthepreviously drops ofalternativetestsolutions.A preferencecanbeinferredif investigated bydippingthetipofproboscisalternatelyintotwo In severalbutterflies,preferencesfornectarconstituentshavebeen measure ofpreference,asinsectsarenotexperiencingalternatives. Wäckers, 2003).However, thismethodonlyprovidesanindirect to oneparticularsugarsource(BarkerandLehner, 1974;Boevéand preferences onthebasisofsugarconsumptionwhenprovidedaccess individuals ofsocialspecies.Somestudieshaveinferredsugar developed toinvestigatesugarpreferencesforsolitaryarthropodsor foraging foralternativesugarsources.Fewmethodshavebeen typically fillitsgut,ratherthansamplethefoodsiteandcontinue of sufficient quantityandquality, afood-deprivedparasitoidwill the numberoftheirfeedingevents.Uponencounteringafoodsource their bodymassatatimeduring Moreover, parasitoidscanconsumesugarmealsofuptoathird preferences insolitaryorganisms, suchasadultparasitoids. RESEARCH ARTICLE Here weuseadifferent methodtodeterminefeedingpreferencesin to fourmajorsugarsfoundinplants:sucrose,glucose, (Cresson 1872)(Hymenoptera:Braconidae)

years inJapan(Kyushustrain) and its Helicoverpa ad libitum M. croceipes ad libitum feeding, whichrestricts and Microplitis croceipes , glucose,sucrose, Heliothis feeding. Asthis ad libitum species M. F (ANCOVA: interactionbetweenfeedingtimeandsugartype, there wasasignificantdifference amongslopesforthefoursugars y P regression resultsareasfollows:sucrose sugar solutionconsumedbyMicroplitiscroceipes Fig. sugar solutionsat ninedifferentconcentrations. Fig. (Fig. the waspstestedrejectedglucoseat0.016 2).Allof of waspsthatacceptedthesugarsolutiondecreased(Fig. As theconcentrationofsugarsolutiondecreased,percentage amount (μ Linear relationshipswerefoundbetweenfeedingtime(s)andthe As feedingtimeincreased,theamountofsugarconsumedincreased. as thegroupvariableandlogconcentrationacovariateshowed 1).Analysisofcovarianceusingsugar glucose andfructose(Table sugar) wassignificantlyhigherformaltosecomparedwithsucrose, concentration atwhichleast50%ofthewaspstestedaccepteda Bonferroni procedureformultiplecomparisons, P 0.063 glucose andfructosewerenotsignificantlydifferent ( each wassignificantlydifferent fromthatformaltose( Effect of sugars onfeeding timeand sugarintake sugars of Effect RESULTS Innate sugars feeding tosingle response =–77+8.1 <0.0001; glucose, <0.0001. 3,220

.Rltosi ewe edn ieadaon fa1mll mol Relationshipbetweenfeedingtimeandamountofa1 1. .Feedingacceptance by 2. Amount of sugar consumed (μg) =5.15, 2). TheProbitanalysisestimatedthattheEC 1000 1200 1400

% Acceptance mol 200 400 600 800 100 20 40 60 80 0 .0 .1 .3 .6 .2 .50512 1 0.5 0.25 0.125 0.063 0.031 0.016 0.008 x 0 g) ofsugarconsumedforthefoursugars( 06 010150 120 90 60 30 l , R − The JournalofExperimentalBiology(2014)doi:10.1242/jeb.091843 1 P , andthetwoothersugarswererejectedat0.008 2 =0.8621, <0.0001). Theslopesoflinearregressionsforsucrose, ■ ○ ● y =–166+10.8 Maltose Fructose Sucrose Glucose Sucrose Maltose Glucose Fructose P <0.0001; maltose, Sugar concentration(moll Microplitis croceipes x Feeding time(s) , R 2 =0.6225, y =–183+10.2 y =66+3.9 P <0.0001; fructose,

mol females. P x x females towardfour , R –1 l <0.05). , R − ) Fig. 1). 1). Fig. 1 2 50 F 2 =0.6170, and maltoseat =0.7272, 2,165 (the effective F Linear 1,230 =1.15), but However, =11.89;

mol − 1 1693 l − 1

The Journal of Experimental Biology H Sucrose H P tests: female,d.f.=3, d.f.=3, (Steel–Dwass tests:female,d.f.=3, significantly reducedafterwaspshadfedontheglucosesolution 1694 RESEARCH ARTICLE (control) (Steel–Dwasstest:d.f.=3, significantly lowerthanfeedingtimeonthefructosesolution alone feeding timeonthemaltosesolution(control)alone,but was females afterfeedingonthemaltosesolutionwasnotdifferent from Fig. fructose solution(Steel–Dwasstest:d.f.=3, solution significantlyreducedsubsequentfeedingtimeonthe solution, butpriorfeedingby fructose solutiondidnotaffect subsequentfeedingonthemaltose 1 Prior feedingbymaleandfemale F percentage ofwaspsthatacceptedthesugars(ANCOVA: that therewasasignificantdifference amongthesugarsin y Maltose Glucose Fructose Sugar Values withthesamesuperscripted letterswithintheEC (Steel–Dwass tests:glucose,female,d.f.=3, significantly reducedsubsequentfeedingtimeonthemaltosesolution 4A).However, priorfeedingontheglucoseorsucrosesolutions Fig. female, d.f.=3, feeding timeoneitherofthosesugarsolutions(Steel–Dwasstests: H Table Effect of previous experience onfeeding tosugars response of Effect than onothersugarsat0.5 3).Feedingtimeonmaltosewassignificantlylower decreased (Fig. P male, d.f.=3,H , probitacceptance;

<0.0001; male,d.f.=3, 3,31 <0.0001; Fig. mol =18.23, =28.71, =36.50, Prior feedingbymaleandfemale Feeding timeforallsugarsdecreasedassugarconcentration Mean feeding time (min) Similarly, feedingtimeonthe1 00 11 22 33 44 55 66 =68.06, 4F). Feedingtimeonthefructosesolutionby 1. ProbitanalysesofinnatefeedingresponsestofoursugarsolutionsbyMicroplitiscroceipes l − 1 H .0 .1 .3 .6 .2 .50.5 0.25 0.125 0.063 0.031 0.016 0.008 solutions ofglucoseorsucrosedidnotaffect theirsubsequent =71.06, P P P =0.0004; Fig. <0.0001) and0.125 P <0.0001), 0.25 <0.0001). =48.15, Maltose Fructose Sucrose Glucose 4E). H a =4.73, b P x, b <0.0001; Fig. natural logofsugarconcentration. Regression y y y y a H =0.858 =1.173 =1.019 =1.196 P =58.91, H <0.0001; sucrose,female,d.f.=3,H a P 3). =71.49, a =0.193; male,d.f.=3, a x x x x 75171(.1)0.054(0.039–0.073) 7.1(0.418) +7.501 +6.486 +7.505 +8.404 mol bb

mol M. croceipes P mol a <0.0001; male,d.f.=3, P 4D) oronsucrose(Steel–Dwass l Concentration (mol l M. croceipes − a <0.0001; Fig. l H 1 −

a mol 1 l =27.58, − (Steel–Dwass test:d.f.=3, M. croceipes H 1 (Steel–Dwass test:d.f.=3, =61.69, (Steel–Dwass test:d.f.=3, a,b l − 1 b fructose solutionwas a males onthemaltose P H H c <0.0001). =21.34, Chi-square (d.f.=7)( 5.4 (0.617) 5.5 (0.598) 13.8 (0.054) =56.11, (Kyushu strain)on 50 H 4B,C). P a =6.50, column arenotsignificantlydifferent (likelihoodratiotest: <0.0001; male, b on a1 –1 a M. croceipes ) c P P H P <0.0001; <0.0001; a =63.97, =0.089; =61.13, mol a,b b c l − P 1 -value) a b 12 b,c Fig. control treatments(Steel–Dwasstest:d.f.=3, the glucosesolutionfor50 Similarly, feedingtimeonthefructosesolution18 H H P (Steel–Dwass tests:0.5 significantly reducedatallconcentrationsofglucosetested glucose, d.f.=3,H solution (Steel–Dwasstests:sucrose,d.f.=3, significantly reducedsubsequentfeedingtimeonthefructose 0.25 (Steel–Dwass tests:30 reduced relativetothecontroltreatmentsforalltimeintervals following 5 6A).Nevertheless,feedingtimeonthefructosesolution (Fig. as theintervalbetweenexposurestothosesugarsincreased solutions insequence,feedingtimeonfructoseincreasedgradually When waspswereprovidedwith1 2 Survival curvesforbothsexesdiffered significantlyamongthefour glucose, theirsubsequentfeedingtimeon1 When femalewaspswereprovidedwithvariousconcentrationsof 0.031 prior feedingona1 H experience feeding inhibitionfollowing fructose glucose Persistence of Effect of sugars onlongevity sugars of Effect feeding inhibition concentrationonsubsequentfructose glucose of Effect

c 000;Fg 5). <0.0001; Fig. mol =49.02, =46.08, =45.02, The Tifton strainshowedsimilarresults.Hereaswellweseethat 6B). mol a mol l a − 1 b l sugar solutions(female:d.f.=4, b − P l P P 1 − The JournalofExperimentalBiology(2014)doi:10.1242/jeb.091843 , d.f.=3,H <0.0001; 1 1 <0.0001; 0.063 <0.0001; 12 s feedingontheglucosesolutionwassignificantly , d.f.=3,H 0.282 (0.217–0.366) 0.085 (0.065–0.113) 0.058 (0.045–0.075) EC Fig. (Steel–Dwass test: same concentration(mol bars represent±s.e.m.Barswiththesameletterwithin on foursugarsolutionsatnineconcentrations. 50

=47.24, .FeedingtimebyMicroplitiscroceipes 3. (mol

mol s, d.f.=3,H =36.33, h, d.f.=3,H =55.00, l –1 h, d.f.=3,H l ) (95%CI) s wassignificantlyreducedrelativetothe − mol 1 P mol sucrose, glucoseormaltosesolution P <0.0001; maltose,d.f.=3, females =0.05). l P − P 1 a 000;Fg 7). <0.0001; Fig. P b a a l =0.05). =46.02, , d.f.=3,H − <0.0001; 0.125 1 =45.71, , d.f.=3,H

l mol − 1 =39.14, ) arenotsignificantlydifferent P l − <0.0001; 30 1 χ P 2 glucose andfructose

<0.0001; 6 H H mol =126.02, =41.90, =41.24, P =28.15, =46.69,

<0.0001; Fig. h afterfeedingon l mol − 1 fructose was min, d.f.=3, l P P P P P − females H 1 h, d.f.=3, <0.0001; <0.0001; <0.0001; <0.0001; <0.0001; , d.f.=3, =21.28, Error 6).

The Journal of Experimental Biology sugar combinationarenotsignificantlydifferent (Steel-Dwasstest: and fructose(F)maltose.S,sucrose;F, thesameletterwithin a fructose;G,glucose;M,maltose.Errorbarsrepresent±s.e.m.Bars with andfructose,(E)sucrose two sugarsgiventoindividualwaspswere:(A)glucoseandsucrose,(B)maltose,(C)sucrose(D) (Siekmann etal., 2001;Wäckers,Wäckers etal.,2006;Faria of different feedingspeedsduetodifferent viscositiesofthesugars solution maynotalwaysreflect consumption ofthesolutionbecause feeding timeandconsumption. However, feedingtimeonasugar assumption thatthereisapositive relationshipbetweensugar response andfoodconsumption (Wäckersetal.,2006),withthe Feeding timeisoftenrecorded asameasureofinsectgustatory male: d.f.=4, Fig. RESEARCH ARTICLE significantly higherthanthatofmales(d.f.=1, Longevity offemalesgivendifferent sugarsolutionswas shorter thanthatofthosefedontheothersugarsolutions. longevity ofwaspsthatfedonthemaltosesolutionwassignificantly Relationship between sugarfeeding timeandconsumption DISCUSSION

.Efc fpeiu ua edn nfeigtm nfu o l mol Effectofprevioussugarfeedingon feedingtimeonfour1 4.

χ Mean feeding time (min) 2 00 11 22 33 44 =172.39, 00 11 22 33 44 00 11 22 33 44 A C E a a aa Female S–S eaeMale Female Female P S–S b a b G–G 000;Fg 8).Forbothsexes, <0.0001; Fig. a,b c a a a M–M c S–S F–F χ a 2 a S–M G–S =24.36, F–S a c Male b Male a d a P S–G M–S <0.0001). P S–F a =0.05). b c − 1 sugar solutionsin for 1 positive linearrelationshipsbetweenfeedingtimeandconsumption et al.,2008;Wyckhuys etal.,2008). Inthepresentstudy, we found what properties ofmaltosereducethefeeding time in equal viscosityofsucroseand maltose.Thus,itremainsunclear 2013) andChirifeBuera (Chirife andBuera,1997)reported Nithiyanantham andPalaniappan (NithiyananthamandPalaniappan, maltose mayreducefeedingspeed relativetotheothersugars,but al. (Wyckhuys etal.,2008)suggestthatthehigherviscosity of solutions. Siekmannetal.(Siekmann etal.,2001)andWyckhuys et feeding speedislowerformaltosesolutionsthantheother sugar steeper thanthoseoftheotherthreesugars.Thissuggests that consumption inallsugarstested. feeding timeisagoodindicatorofgustatoryresponses and However, theslopeofregressionformaltose wassignificantly 0 1 2 3 4 00 11 22 33 44 00 11 22 33 44 mol D F B l a a a − 1 The JournalofExperimentalBiology(2014)doi:10.1242/jeb.091843 in allfoursugars,suggestingthatatthisconcentration, G–G b,c Female G–G Microplitis croceipes Female Female F–F b b a,b a c c c a M–M M–M F–F (Kyushu strain). a F–M a F–G a G–M a b b Male Male Male a a c M–F G–F a,b M–G b c The combinationsof M. croceipes. 1695

The Journal of Experimental Biology test: P differentfructose; G,glucose;M,maltose.Errorbarsrepresent±s.e.m.Barswiththesameletterwithinasugarcombinationarenotsignificantly (Steel–Dwass andmaltose.S,sucrose;F,combinations oftwosugarsgiventoindividualwaspswere:(A)sucroseandfructose,(B)glucose(C)fructose sugars. longevity ofparasitoidsfedthissugar, ascomparedwiththeother nutritional valueofmaltoseisexpressedinthesubstantiallyreduced Roane, 2007;Luoetal.,2013).Similarly, in utilization andfitness(Wäckers,1999;Wäckers,2001;Williams and the relativenutritionalqualityofsugarintermsmetabolic that innategustatorysugarresponsesarepositivelycorrelatedwith nutritionally lesssuitable.Previousparasitoidstudieshaveshown may reflectthefactthatthissugarislesscommoninnectarand shorter timeonmaltosewhencomparedwiththeothersugars.This The EC is lesssensitivethantheirresponsetosucrose,glucoseandfructose. and honeydews. croceipes honeydews (BakerandBaker, 1983).Thissuggeststhat concentrations atwhichthosesugarsnaturallyoccurinnectarsor parasitoids (Wäckers,1999;Beachetal.,2003)andfarbelowthe acceptance thresholdsforthesesugarsasreportedother 1696 RESEARCH ARTICLE 0.016 and0.031 which thesesugarsevokeafeedingresponse,wereintherangeof for sucrose,glucoseandfructose,i.e.thelowestconcentrationat Beach etal.,2003).Inthepresentstudy, theacceptancethresholds parasitoids areknowntovarybetweensugars(Wäckers,1999; Gustatory responsesbyfeeding-inexperiencedhymenopteran Fig. Innate gustatory response to single sugars tosingle response Innate gustatory 1

Mean feeding time (min) mol However, theinnateresponseby

0 1 2 3 4 5 6 .Efc fpeiu ua edn nfeigtm nfu o l mol Effectofprevioussugarfeedingontimefour1 5. =0.05). l − 50 A 1 a or lowerconcentrations,waspsfedforasignificantly was higherformaltosethantheotherthreesugars.At should havenoproblemdetectingthesesugarsinnectars 0s3 i 12h 6h 1h 30min 30 s a a

b Mean feeding time (min) 0 1 2 3 4 5 G–G mol a,b A a a l − b 1 S–S c (Fig. F–F a a 6). Thisiscomparabletothe F–F a M. croceipes a Time intervalbetween twosugars a b F–S F–G M. croceipes a b a S–F females tomaltose a b 0 1 2 3 4 5 G–F B a , thelower b a a G–G c M. − 1 b sugar solutionsin 0 1 2 3 4 5 6 F–F Duan andMessing,1999;Fujiwaraetal.,2000). and Vet, 1990;PoolmanSimonsetal.,1992;Geervliet1998; they changehostacceptancebasedonpreviousexperience(Papaj in andamongpatchescontainingdifferent qualitiesofhosts,and sugars. Similarly, parasitoidseffectively discriminatebetweenhosts lesser qualityfoodinfavorofforagingforthenutritionallysuperior such asglucose,sucroseorfructose,parasitoidsrapidlygiveupthe feeding inhibitionimpliesthatinapatchcontainingsuperiorsugars having experiencednutritionallysuperiorsugars.Thisacquired refraining fromfeedingonthenutritionallyinferiormaltose,when sugars couldbeexplainedintheseadaptiveterms,withparasitoids feeding inhibitiontomaltosefollowingpreviousonother Charnov, 1976;SihandChristensen,2001).Inthepresentstudy, reduce consumptionofthelowerqualityfood(Pulliam,1974; that differ inquality, theyshouldchoosethehigherqualityfood,and when anorganism isprovidedachoicebetweentwotypesoffood feeding responsestomaltose.Optimalforagingtheorypredictsthat other sugars,priorfeedingonglucoseorsucrosefurtherinhibited ability ofparasiticwaspstodiscriminatebetweensinglesugars. 2003; Vollhardt etal.,2010),thisisthefirstdemonstrationof sugars isknowninafewparasitoids(Wäckers,1999;Beachetal., three sugars.Althoughfeedingdiscriminationagainstamixtureof croceipes time inthecaseofmaltoseorfructose.Thissuggeststhat A pre-exposuretosucroseandglucosesignificantlyreducedfeeding Gustatory discrimination between sugars Gustatory B While innatefeedingresponseswereweakerformaltosethanthe a,b a F–G 8h24h 18 h b a adults candiscriminatemaltoseorfructosefromtheother c c The JournalofExperimentalBiology(2014)doi:10.1242/jeb.091843 G–F Microplitis croceipes a 0 1 2 3 4 5 b C a a b F–F b test: P significantly different (Steel–Dwass combination oftwosugarsarenot Bars withthesameletterwithina maltose. Errorbarsrepresent±s.e.m. S, sucrose;F, fructose;G,glucose;M, the first1 solution for5 1 (A) Wasps weregiventhesecond solution by on feedingtimethesecondsugar feeding onthefirstsugarsolution Fig. after feedingonthefirst1 females (Tifton strain).The M–M

mol

.Effectoftimelapseafter 6. l − =0.05). 1 b sugar solution30 mol F–M Microplitis croceipes. s. (B)Wasps weregiven l − 1 sugar solutionfor50 c M–F

mol s to12 l − 1 sugar h M.

s.

The Journal of Experimental Biology RESEARCH ARTICLE and withbothcoloniesof this phenomenonwasconsistentlyobservedinmultipleexperiments reduction infeedingtimefollowingexposuretoglucoseorsucrose; fructose withoutpriorfeedingonanothersugar. Unliketheexample Moreover, thisbehaviorwasalsonotseeninwaspsencountering was notobservedwhenwaspsencounteredtheothersugars. quick turnswhilewalkingaroundthesugardroplet.Thisbehavior with theirmouthparts,antennaeandfrontlegs,madeseveral fructose droplet,thewaspsrepeatedlytouchedsolution suggesting thattheywerestillhungry. Uponeachcontactwitha followed byrepeatedreturnvisitstothefructosesolution, when exposedtothefructose.Theyexhibitedfrequentrejections other sugars,thewaspsshowedapronouncedchangeinbehavior Proportion surviving Mean feeding time (min) Surprisingly, alsointhecase of fructoseweseeapronounced 0.2 0.4 0.6 0.8 0 1 2 3 4 5 6 0.2 0.4 0.6 0.8 0 1 0 1 0 369121518 0 3691215 18 B A a Male Female .3 .6 .2 0.25 0.125 0.063 0.031 G–G a a b F–F a Concentration offirstsugar(moll a M. croceipes. Followingexposuretothe a F–G b Days G–F a b a,b

21 2427303336 21 242730 c a b –1 a ) c Water Maltose Sucrose Glucose Fructose a a 0.5 a fructose feedingfollowingexposuretotheothersugarstherefore natural conditions.Thepossiblefunctionfortheinhibitionof fructose onreproductiveperformanceby conditions. However, wedonotknowtheeffect offeedingononly nutritionally equivalenttoglucoseorsucroseundertheexperimental (Wäckers etal.,2006).Allofthesefactssuggestthatfructoseis unconditional stimulusforassociativelearningoffoododors Fructose, aswellglucoseandsucrose,isalsoaneffective females andglucose-orsucrose-fed(K.T., unpublished). not findanydifference inovipositionbehaviorbetweenfructose-fed survival equallyaswellglucoseandsucrose.Inaddition,wedid nutritional suitabilityoffructose.Actually, fructosesupportedadult experience withglucoseorsucroseisnotexplainedbyalower of maltose,thefeedinginhibitionincasefructosefollowing b provided with2 Fig.

.Survivorshipcurvesforbothsexesof 8. Bars withthesameletterwithinaconcentration(mol fructose; G,glucose;M,maltose.Errorbarsrepresent±s.e.m. sugar arenotsignificantlydifferent (Steel–Dwasstest: fructose feedingbyMicroplitiscroceipes Fig. The JournalofExperimentalBiology(2014)doi:10.1242/jeb.091843

.Effectofglucoseconcentrationonsubsequent 7. o l mol − 1 solutions offoursugarsandwater. M. croceipes Microplitis croceipes . S, sucrose;F, adults inthe

l − 1 P ) offirst =0.05). 1697

The Journal of Experimental Biology 1698 RESEARCH ARTICLE Individual female waspswereplacedat5°Cfor10 time andintakeof thesucrose,glucose,fructose and maltosesolutions. al., 2008).Therefore,wefirstexamined therelationshipbetweenfeeding (Siekmann etal.,2001;Wäckers, 2001; Azzouzetal.,2004;Wyckhuys et However, feedingtimeofafooditemmaynotaccurately reflectitsintake Gustatory responsesininsectsare often assessedintermsoffeedingtime. maltose, andthemonosaccharides The sugarsusedintheexperimentsweredisaccharidessucroseand 10 droplet ofwaterandkeptindividuallyinglassvials(diameter1.7 were usedinalltheexperiments.Individualwaspsprovidedwitha (Lepidoptera: Noctuidae)larvaewithhoneyasfood.Two-day-old adults that weretestedatCPMRUhadbeenrearedon width ×height)andprovidedwithdistilledwateronly. emerged adultswerekeptinaplasticrearingcage(30×30×25 photoperiod at25°C(HoangandTakasu, 2005).Fortheexperiment,newly Noctuidae) larvaewithhoneyasfoodundera16 parasitoids wererearedon University sinceJune2001,withnoinfusionferalwasps.The Unit (CPMRU)inTifton, GA,USA,andhasbeenrearedatKyushu originated fromtheUSDA-ARSCropProtectionandManagementResearch Management, KyushuUniversity, Japan.Theparasitoidstockculture Microplitis croceipes ecological implications. studies willhavetoaddressthephysiologicalmechanism(s)and an inhibitoryinteractionbetweentwophagostimulantsugars.Future However, toourknowledge,resultspresentthefirstexampleof 1973; Blaneyetal.,1986;Bernays,1995;Wäckers,2001). cells ortonutrientunsuitabilityofthosecompounds(Stoffolano, attributed todifferences inresponsebyinsectgustatoryreceptor (Hymenoptera: Mymaridae).Suchdiscriminationabilitieshavebeen inhibited acceptanceofthelatterbyparasitoid 2003) foundthatrhamnose(monosaccharide)mixedwithmaltose Braconidae) relativetopuresucrose,andBeachetal.(Beachal., feeding bytheparasiticwasp 1999) foundthatsucrosemixedwithmannoseorraffinose inhibited (monosaccharide) andraffinose (trisaccharide).Wäckers(Wäckers, inhibition ofblowflyfeedingonfructosewhenmixedwithmannose Wieczorek andWolff (Wieczorek andWolff, 1989)demonstrated an sugars inmixturescanbeinhibitedbyphagodeterrentsugars, nectars andhoneydew. and fructosebecausemultiplesugarsaretypicallypresentinplant responses tofructoseafterfeedingonamixtureofglucose,sucrose remains tobeinvestigated.Itwouldinterestingtestthewasps’ feeding. contact withthesugarsolution.Thewaspsneverpausedoncetheyinitiated time duringwhichthewasp’s mouthpartswereseentobeincontinuous the solutionwasobservedandrecorded.Feedingtime(s)recorded as increase inthetestsolutionduetoevaporation.Thewasp’s feedingtimeon the cupwasplacedupsidedownonwetfilterpapertoavoidaconcentration vial tothedropletofsolutionincup.Afterwaspmoved cup, 10 center ofatruncatedcone-shapedplasticcup(diameter:top12 Fresh sugarsolutionswerepreparedforeachoftheexperiments. Effect of sugars andfeeding timeon sugarintake sugars of Effect tested Sugars Parasitoids MATERIALS ANDMETHODS molecular weights,thesugarsweredilutedwithdistilledwaterto1 (Levulose) (allfromNacalaiTesque, Inc.,Kyoto,Japan).Basedontheir In allexperiments,a10 Although itisknownthattheacceptanceofphagostimulatory cm; height6 cm) for30 min beforeeachtest.

cm). Anindividualwaspwasallowedtowalkfromtheglass were rearedatthelaboratoryofBioresourceand μl dropletofasugarsolutionwaspipettedatthe Helicoverpa armigera Cotesia glomerata D -(+)-glucose and

min toimmobilize them, (Hübner) (Lepidoptera: Microplitis croceipes (Hymenoptera:

h:8 Anaphes iole D

-(–)-fructose h light:dark cm, length×

cm, bottom

cm, height

mol l − 1 . EC 5 acceptance. Whenawaspstopfeedingonandleftthesugarsolutionwithin tested foreachcombinationoftreatment andtimeinterval. Feeding time(s)onthesecondsugar wasrecorded.A totalof20waspswere fructose orglucosefirst,andthenprovidedwiththesamesugarsolution. fructose, followedbyglucose.Anothercontrolwaspwasallowedtofeed on manner asdescribedaboveexceptthatherethewaspwasfirstprovided with fructose solution.Inbothexperiments,controlwaspsweretestedinasimilar access towateronlyforeither18or24 glucose solutionfor50 was carriedout,inwhichanindividualfemaleallowedtofeed on sugar couldnotsustainawaspformorethan12 accepted asugar).Foreachsugarconcentration,20waspsweretested. was providedwitha1 access towateronlyfor30 then removedfromthesolution.Thewaspwasplacedinavial with each sugarfeedingtime,16waspsweretested. measurements indicatedthequantityofsugaringestedbyeachwasp.For at 5°Cfor10 60, 90,120or150 sucrose–fructose andmaltose–fructosecombinations. for eachoftheglucose–fructosecombinations,and12 using theTifton strainof reference forcomparison,thesameexperimentwasconductedinTifton, feeding inhibitiontofructoseafterhavingfedonothersugars.To providea and 24forthefourremainingsugarcombinations. glucose–maltose combinations,25forthesucrose–maltose recorded. Thenumberofwaspstestedwas20foreachthefour second sugarsourceandthedurationof source wasremoved.Aftera30 wasp wasallowedtofeedononesolutionfor50 sugar combinations(AA,AB,BA,BB).Foreachtreatment,anindividual fructose andmaltose(sixpairs).Eachpairwastestedatthefourpossible was conductedwitheachpairofthefourfollowingsugars:sucrose,glucose, or offering adifferent sugarduringthesecondfeeding(AB orBA).Thetest feeding bouts,eitherprovidingthemwiththesamesugartwice(AA orBB), croceipes This experimentwasdesignedtodeterminewhethermaleandfemale sucrose, glucose,fructoseandmaltose.A 2 M. croceipes The effect ofsugarconcentrationontheinnatefeedingresponsefemale continuously onadropletof1 Limited, Tokyo, Japan).Thereafter, eachwaspwasallowedtofeed and thenweighedonaprecisionbalance(GR-60series,A &DCompany sequence of0.25, 0.125,0.063and0.031 glucose solutionwas preparedandserialdilutionswere madeinageometric glucose thatcouldinhibit This experimentwasdesignedto determinethelowestconcentrationof wasp wasfirstallowedtofeedona1 feeding theinhibitionoffructoseintakeismaintained.Anindividualfemale further experimentdesignedtodeterminehowlongfollowingglucose sucrose drasticallyreducedsubsequentfeedingonfructose,weconducteda As ourfirstexperimentshowedthatabriefexposuretoeitherglucoseor time (s)wasrecorded.Iffeedinglastedmorethan5 individual waspwasallowedtofeedonadropletofsolutionanditsfeeding sequence of1,0.5,0.25,0.125,0.063,0.031,0.016and0.008 was firstpreparedandthenserialdilutionsweremadeinageometric Effect of previous experience onsugarfeeding response of Effect Innate sugars feeding tosingle response Effect of glucose concentration on fructose feeding inhibition concentrationonfructose glucose of Effect inhibitionfollowing feeding fructose glucose Persistence of

s, thiswasrecordedasarejection.Probitanalysisusedtoestimatethe Early resultswithwaspsoftheKyushustrainshowedthattheyexhibit 50 (the effective concentrationatwhichleast50%ofthewaspstested discriminate betweentwosugars.Parasitoidsweregivensugar The JournalofExperimentalBiology(2014)doi:10.1242/jeb.091843 min andre-weighed.Thedifference betweenthetwoweight was examinedbyprovidingthemwithsinglesolutionsof s. Immediatelyafterfeeding,eachwaspwasagainplaced

mol

s. Thereafter, thewaspwasplacedinavialwith M. croceipes. Thenumberofwaspstestedwas20 M. croceipes

l s, 30 − 1 fructose solution.Because5

mol s interval,thewaspwasprovidedwith min or1,612 l − 1 moll solution ofeachsugarforeither30, feeding onfructose.A 0.5 h, beforebeingprovidedwiththe mol −1

mol l glucose solutionfor5 − ad libitum 1 . Anindividualfemale was

s. Subsequently, thesugar l − h, aseparateexperiment 1

h. Thereafter, thewasp s, itwasregardedasan solution ofeachsugar feeding (s)was

s feedingona moll mol s, and −1 . An M. l − 1

The Journal of Experimental Biology ae,H .adBkr I. Baker, and H.G. Baker, G.D. Waller, and W. W. Bachman, plastic cup(diameter:top12 Newly emerged waspswereindividuallyplacedinatruncatedcone-shaped ( correction wasusedtoaccountforeachofthepair-wise comparisons estimates ofthesurvivalfunction(Lavanderoetal.,2005).A Bonferroni moment intime)weregeneratedandcomparedusingKaplan–Meier analysis. Survivalcurves(functionofwaspssurvivingataparticular test followedbySteel–Dwasstests.Longevitywasanalyzedusingsurvival different sugarsonwasp’s feedingtimewasanalyzedbyaKruskal–Wallis zoz . iraeg,P,Wces .L n asr L. Kaiser, and F. L. Wäckers, P., Giordanengo, H., Azzouz, tested foreachtreatmentwithfirstsugarconcentration. Feeding time(s)onthesecondsugarwasrecorded.A totalof18waspswere KAKENHI [grantnumber24658052]. This workwassupportedbyJapanSocietyforthePromotionofScience(JSPS) to writingofthemanuscript. of themanuscript,K.T. conceivedanddesignedtheexperiments,contributed the manuscript,F.L.W. contributedtothedesignofexperimentsandwriting the manuscript,H.K.L.designedexperiments,D.M.O.contributedtowriting of J.K.M. performedtheexperiments,analyzeddataandcontributedtowriting of The authorsdeclarenocompetingfinancialinterests. anonymous refereesforimprovingthemanuscript. We wouldliketothankK. Urakawaforrearinginsects.We arealsogratefultotwo ( sugars andtheslopeformaltose.A Bonferronicorrectionwasused wise comparisonwasmadebetweenthecombinedslopesforthree times werenotdifferent, thedataforthesesugarswerecombinedandapair- for amountofsucrose,glucoseandfructoseconsumeddifferent feeding analysis ofcovariance(ANCOVA). Astheslopesfromlinearregressions Data ontheeffect ofsugarandfeedingtimeonconsumptionweretestedby provided with2 Longevity of for 5 control waspswasallowedtofeedonaglucoseorfructoseconcentration wasp wasprovidedwitha1 allowed tofeedononeoftheglucoseconcentrationsfor5 RESEARCH ARTICLE 0.5 for 5 individual waspwasallowedtofeedononeofthefructoseconcentrations were keptundera16 to avoidmicrobialgrowthandcrystallizationdueevaporation.Wasps l,J,Ones,T . az,J n reeg,L. Vriesenga, and J. Lanza, T. E., Ohnmeiss, J., Alm, of concentrations,EC Funding Author contributions Competing interests Acknowledgements Statistical analyses onlongevity sugars of Effect References solution. was checkedonceaday. A totalof20waspswereexaminedforeachsugar 10 μl ofdistilledwater. Sugarsolutionsorwaterwererenewedevery2 provided witha10 P P different compositions. 452. parasitoid: frequency andsugarconcentrationon behaviorandlongevityoftheadultaphid Biology pollination mechanisms andphylogeny. In 53-57. white butterfliesandhoneybeesfornectarthatcontainsaminoacids. =0.05/k =0.05/k mol s, andafter30 s andafter30 l − (ed. M.H.Nitecki),pp. 131-171.Chicago,IL:UniversityofChicago Press. 1 , wherek , wherek fructose solutionwaspreparedandseriallydilutedasabove.An Aphidius ervi M. croceipes mol =2 forallsugars).Afternaturallogarithmtransformation μl ofsugarsolution.Controlwaspswereprovidedwith =10 foralltreatmentsinalog-ranktest). s wasprovidedwith1 s wasprovidedwith1

l h:8 − J. Apic.Res. 1 50 (Haliday) (Hymenoptera:Braconidae). solutions ofsucrose,glucose,fructoseandmaltose. h light:darkphotoperiodat25°C,andtheirsurvival was estimatedbyProbitanalysis.Theeffect of (1982). Chemicalconstituentsofnectar inrelationto

cm, bottom10 adults wasexaminedformalesandfemales mol (1977). Honeybeeresponsetosugarsolutions of 16, 165-169. l − 1 fructose solution.Asacontrol, mol mol Biochemical Aspects ofEvolutionary

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The Journal of Experimental Biology