Boulder, CO80309,USA. 2250 Received 8July2013; Accepted27March2014 ‡ 1 Hempel, 2005;Schulenburg etal.,2009). Rolff andSiva-Jothy, 2003;Schmid-Hempel, Schmid- on timescalesrelevanttotheindividual’s fitness(Bestetal.,2013; in insectsrespondtovariousecologicalfactors,andmaybeinduced field ofecologicalimmunologyhasshownthatimmuneparameters Janeway, 1998).However, agrowingbodyofempiricalworkinthe as havingbothinnateandacquiredcomponents(Medzhitov and in contrasttoimmunityvertebrates,whichhasbeenrecognized Immunity ininsectshastraditionallybeencharacterizedasinnate, Bead injection,Illness-inducedanorexia KEY WORDS:Ecologicalimmunity, Macronutrient,Parasitoid, self-medicationresponse. nutritional interactionswiththecaterpillar’s could negativelyorpositivelyaffect parasitizedcaterpillars,including discuss alternativemechanismsbywhichvariationinproteinintake melanization, suggestingaprophylacticroleforcarbohydrates.We melanization response,increasedcarbohydrateintakedidincrease Although variationinproteinintakedidnotchangethecaterpillars’ immune challenge,whereasthoseoffered alow-proteinfooddidnot. concentration thatisoptimalforgrowthreducedfeedingfollowing no-choice experiment,caterpillarsoffered foodwithaprotein foods reducedtheirintakeofthehigh-proteinfood.Furthermore,ina or injectedcaterpillarsgivenachoicebetweenhigh-andlow-protein feeding assays.Contrarytoourprediction,wefoundthatparasitized of dietonmelanization,wequantifiedmelanizationbeadsfollowing on dietsvaryingintheirmacronutrientcontent.To evaluatetheeffects parasitism, andmeasuredfeedingbeforeafterimmunechallenge a beadinjectiontechniquethathasbeenusedinstudiestosimulate challenged theimmunesystemusingeithertachinidflyparasitoidsor component oftheimmunesystemthatactsagainstparasitoids.We that thisresponsewouldenhancethemelanizationresponse,a would increasetheirintakeofdietaryprotein.We furtherpredicted behavior inresponsetoimmunechallenge,predictingthatcaterpillars Edwards) (Family:)wouldadaptivelychangeitsfeeding hypothesis thatthepolyphagouscaterpillar intake inmountinganimmuneresponse.Inthisstudy, wetestedthe particular, recentstudieshavepointedtotheimportanceofprotein changes infeedingbehaviorthataidtheimmunesystem.In vertebrates, insectsexhibitadaptiveimmunity, includinginduced Advances inecologicalimmunityhaveillustratedthat,like Peri A.Mason challenge inapolyphagouscaterpillar Reduced consumptionofprotein-richfoodsfollowsimmune RESEARCH ARTICLE © 2014.PublishedbyTheCompanyofBiologistsLtd|JournalExperimentalBiology(2014)217,2250-2260doi:10.1242/jeb.093716 *These authors contributed*These equally tothiswork. authors CT 06457,USA. Reno, NV89557,USA. INTRODUCTION ABSTRACT Author (peri.mason@.edu) for correspondence Biology, EcologyandEvolutionary Colorado Boulder, of University of Department 1, 3 * Department of Biology, of WesleyanDepartment University, Middletown, ,‡ 2 , AngelaM.Smilanich Department of Biology, Nevada Reno, of University of Department incorrupta 2, * andMichaelS.Singer (H. and Joern,2008; Cotteretal.,2011; Poveyetal.,2009; Simpsonand ’s sex,geneticlineandphysiologicalcondition(e.g.Behmer intra-specific variationinthese nutritionaloptima,basedonthe overall physiologicalrequirements ofagivenspecies,thereisalso 1993). Althoughproteinandcarbohydrate intaketargets reflect the Raubenheimer andSimpson, 2003; SimpsonandRaubenheimer, 1981) andmaybetightlyregulated asinsectsforage(Behmer, 2009; normal physiologicalfunctioningininsects(ScriberandSlansky, Povey etal.,2009;Srygley2009).Macronutrientsmediate carbohydrate (Cotteretal.,2011; Leeetal.,2006b;2008; typically studiedintermsofthemacronutrientsprotein and Thompson, 2002). contributing toimmuneefficiency (Leeetal.,2008;Siva-Jothyand (Schmid-Hempel, 2003),anddietcompositionisanimportantfactor These responsesrequiresignificantamountsofnutrientsandenergy melanization cytotoxicity(KanostandGorman,2008;Strand,2008). invaders andneutralizethemviahemocyteasphyxiationand/or signaling cascadesinitiatedbythehumoralresponsetoisolate (Beckage, 2008).Specializedimmunecells(hemocytes)respondto responses thatworkinconcerttodefendagainstinternalenemies Lee etal.,2008;Povey2009;Srygley2009). mediating theseinteractions(Adamoetal.,2010;Cotter2011; nutritional studiesfocusontheroleofprimaryplantmetabolitesin 2010; Simone-FinstromandSpivak,2012;Singeretal.,2009), individuals infectedwithparasitesorpathogens(Lefèvreetal., on thetherapeuticeffects ofplantsecondarymetaboliteson in offspring (Lefèvreetal.,2010).Whereasmedicationstudiesfocus protozoan parasiteadaptivelyselecthostplantsthatreduceinfection transgenerational example,monarchbutterfliesinfectedwitha Jothy andThompson(Siva-JothyThompson,2002)].Ina aspects arereviewedinPontonetal.(Pontonal.,2013)andSiva- et al.,2011; Leeetal.,2006b;Lefèvre2010)[nutritional the system(Adamoetal.,2010;Ayres andSchneider, 2009;Cotter also highlightstheimportanceofchemicalandnutritionalinputsto Elliot etal.,2002;Inglis1996),ecologicalimmunityresearch metabolic processescanaffect immunity(Andersonetal.,2013; to illustratingthatbehavioralalterationofthethermalcontextfor thermoregulate tofevertemperatures(Elliotetal.,2002).Inaddition able toproduceviableoffspring whentheywerepermittedto ( in behavior(Adamo,2004).Forexample,desertlocusts inoculation effects, andmayactthroughpathogen-inducedchanges induced immunologicaldefenseininsectsisnotlimitedto Moret andSiva-Jothy, 2003; Tidbury etal.,2011). However, offspring (KurtzandFranz,2003;MoretSchmid-Hempel,2001; regard tofutureexposuresineitherthetreatedindividualortheir whereby exposuretoapathogenactsinaninoculativemannerwith vertebrates (i.e.immunologicalmemory)ispriming, Schistocerca gregaria The effects ofdietarynutrients ontheinsectimmunesystemare The insectimmuneresponseiscomposedofcellularandhumoral In ,thestrongestparalleltoadaptiveimmunityin 3 ) infectedbyafungalpathogenwereonly

The Journal of Experimental Biology It thusprovided ararecomparativetestofthe effects ofparasitism endoparasites asimmunechallenges totestbehavioralpredictions. This experimentisunusualin usingbothbeadinjectionandlive 1996) withthatofindividuals wereparasitizedbytachinidflies. challenged byinjectionwithSephadex beads(LavineandBeckage, compared macronutrientregulation inindividualsthatwere proportion ofproteininthe diet. Inthefirstexperiment,we prediction thatimmune-challengedcaterpillarswouldincrease the et al.,2006b),wedesignedexperimentstoaddressthespecific Povey etal.,2009),andthemelanizationresponseinparticular (Lee immune responseingeneral(Leeetal.,2006b;Lee2008; results showingtheimportanceofdietaryproteininmounting an change affects thecaterpillars’ melanization response.Basedon following immunechallengein there isachangeinrelativeintakeofproteinandcarbohydrate defense fails(Smilanichetal.,2011a). medication behaviorensuesifthisrelativelylowcost,firstline of nutritional intaketobolstertheimmunesystem,andthat self- hypothesis thatduringtheearlystage,caterpillarsaltertheir occurred duringthelatestageofparasitoidinfectionledto mortality (Singeretal.,2009).Theobservationthatself-medication pyrrolizidine alkaloidsintheabsenceofparasitismcanresult this defensivestrategyiscostly:ingestinglarge quantitiesof species (BernaysandSinger, 2005;Singeretal.,2009).However, medicates usingpyrrolizidinealkaloidsfoundinsomehost-plant When infectedwiththelarvaeoftachinidflies,speciesself- Edwards) [formerly hypothesis inthegrazingcaterpillar parasitoids (LavineandStrand,2002;2008).We testedthis a componentoftheinsectimmunesystemthatactsagainst predicting thatthealtereddietincreasesmelanizationofhemocytes, alter macronutrientintakeinresponsetoimmunechallenge, Raubenheimer andSimpson,2003). 2006a; Leeetal.,2003;RaubenheimerandSimpson,1999; capitalize onbothintra-andinter-specific plantvariation(Leeetal., grazing herbivoreswouldbepositionedparticularlywellto attributes encounteredbyindividualsintheirenvironments.Ifso, this adaptivestrategywouldseemtodependonthevariationinfood monophagous oroligophagousspecies.However, theplausibilityof of macronutrientstoenhanceimmunityisavailableeven Baldwin, 1997;Mattson,1980),suggeststhattheadaptiveregulation can existwithinplantpopulations,andevenindividuals(Karban their study, thevariationinchemical andnutritionalattributesthat of proteintocarbohydratethancontrols(Leeetal.,2006b). intake madetheadaptivedietarychange,consumingagreaterratio Caterpillars thatwereallowedtoself-regulatetheirmacronutrient costs ofresistanceweregreaterthanenergy costs(Leeetal.,2006b). fed carbohydrate-biaseddiets.Thisledtotheconclusionthatprotein stronger constitutiveimmunefunctioncomparedwithindividuals shown tohavebothincreasedresistancethepathogenand individuals feddietswithhighproteintocarbohydrateratioswere Spodoptera littoralis al., 2009).Forexample,inastudywhichthegeneralistcaterpillar enhancement oftheimmuneresponse(Leeetal.,2006a;Povey case, plasticityinmacronutrientregulationmayfacilitate 2006b; Poveyetal.,2009;Srygley2009).Whenthisisthe carbohydrate intensivethanothers(Cotteretal.,2011; Leeetal., processes involvedininsectimmunity, maybemoreproteinor Raubenheimer, 1993).Somephysiologicalfunctions,including RESEARCH ARTICLE The particularquestionsaddressedinthisstudyare(a)whether In thisstudy, wetestedthehypothesisthatherbivoresadaptively Although Leeandcolleaguesusedageneralistinsectherbivorein G. geneura was exposedtoanucleopolyhedrovirus, G. incorrupta,and(b)whetherthat (Strecker)] (Family:Erebidae). Grammia incorrupta (H. principally duetoreductionsinintakeofthehigh-proteinfood.In Contrary toourprediction,reductionsinfoodconsumptionwere 1). injected andparasitizedindividualscomparedwithcontrols(Fig. P (ANCOVA day1:F amount offoodeatenoneachdayfollowingparasitismorinjection challenge. Therewasasignificanttreatmenteffect onthetotal significantly fromthatofcontrolsforthe2 The feedingbehaviorofimmune-challengedcaterpillarsdiffered demands oftheimmuneresponse. ingest agreateramountoffoodinordertoanswertheprotein injection, anticipatingthatcaterpillarsfedthelow-proteindietwould for growthoralow-proteindiet,priortoandsubsequentbead caterpillars eitheradietwithproteinconcentrationthatisoptimal more protein-biaseddiet.Inthethirdexperiment,weoffered injection, predictingthatbead-injectedcaterpillarswouldchoosea self-regulate theirintakeofmacronutrientsbeforeandafterbead other studies.Inthesecondexperiment,weallowedcaterpillarsto and beadinjection,apresumedsurrogateofparasitismusedinmany standard errors,and numbersatthebaseofcolumns indicatesamplesize. letter ofthesame case orstylearestatisticallydistinct. Errorbarsshow significantly acrosstreatments.Columns orpairsofcolumnsnotsharinga bars inday1databecauseamounts ofindividualfoodsdidnotdiffer of low-proteinfood(lowercase)eaten. Lettersareabsentaboveindividual amount offood(italics),high-protein food(uppercase)andamount Table the repeatedmeasuresANCOVA, detailedinsupplementarymaterial parasitism/injection experiment. daysfollowingtheimmune challengeinthe caterpillars forthe2 Fig. Parasitism/injection experiment RESULTS =0.0001). TheTukey testshowsthatfeedingwasreducedinboth

.Amountofhigh-proteinandlow-proteinfoodsconsumedby 1.

S1. Lettersabovebarscorrespondto Tukey testsperformed ontotal

Amount eaten (mg) The JournalofExperimentalBiology(2014)doi:10.1242/jeb.093716 20 40 60 20 40 60 0 0 Day 2 Day 1 A oto netdParasitized Injected Control A A 2,79 29 a =9.45, Least squaremeanswerederivedfrom Treatment A,B P B B =0.0002; day2: 24 a

Low proteinfood High proteinfood days followingimmune B B B 27 a F 2,74 =9.94, 2251

The Journal of Experimental Biology bivariate analysis revealedamarginally significant changein P during thefirstday(ANCOVA protein: associated withareductioninbothproteinandcarbohydrateintake bivariate responsebetweenparasitizedandcontrolindividualswere treatment ×timeinteraction;supplementary materialTable after thetimeofinjection(reflected inthelackofasignificant treatment onthechangeinprotein tocarbohydrateratiobeforeand macronutrient ratioconsumed, nordidwedetectaneffect of Table self-chosen dietsobscuredthese differences (supplementary material analyzing dataintermsoftheratioproteintocarbohydrate in 3).Alsoinkeepingwiththeparasitism/injection experiment, (Fig. consumption ofthehigh-proteinfoodfollowingimmunechallenge than controls,andreducedfeedingwasunderlainbyreductions in material Table d.f.=26, second day(t ate similaramountsoflow-proteinandhigh-proteinfoodonthe high-protein foodonthefirstdayfollowingimmunechallenge,but than low-proteinfoodonbothdays( second dayfollowinginfection.Controlsatemorehigh-proteinfood preference forhigh-proteinfoodinparasitizedindividualsonthe t 1: only significantontheseconddayfollowinginfection(t-tests P Table 2252 RESEARCH ARTICLE experiment (ANCOVA F caterpillars inthisexperiment,whichwewillrefertoasthechoice treatment effect onthetotal amountoffoodconsumedby Consistent withtheparasitism/injectionexperiment,therewas a the seconddayfollowinginfection(ANCOVA day1: protein dietthancontrols,adifference thatwashighlysignificanton particular, parasitizedindividualsatesignificantlylessofthehigh- (ANCOVA day1: the ratioofproteintocarbohydratechosenbycaterpillars effect oftheinteractionbetweencaterpillarfamilyandtreatmenton (supplementary materialTable P F and controlsdidnotdiffer significantly(MANCOVA day1: P immune challengediddiffer (MANCOVA day 1: protein andcarbohydrateconsumedbycaterpillarsfollowing ratios ofproteintocarbohydrateconsumed,therawamounts P P carbohydrate: infection (ANCOVA protein:F F Feeding behavior before andafterimmunechallenge (ANCOVA day1:F protein tocarbohydrateconsumedbydifferent treatmentgroups challenge, wedidnotfindasignificantdifference intheratioof driven byreducedintakeofthehigh-proteinfoodfollowingimmune (MANCOVA day1: significantly fromcontrolsonbothdaysfollowinginfection the macronutrientintakeofparasitizedindividualsdiffered Table =2.23, d.f.=21,P 1,53 =0.0049). Injectedcaterpillarsshowedthesametrend,butitwas 2,52 =0.0043; day2: =0.016; day2: =0.35; day2: =0.0001), whereasmacronutrientintakebyinjectedindividuals =0.016). AlthoughtheANCOVA didnotdetectdifferences in Although theconsumptiondatashowthatreducedfeedingis t =1.81, d.f.=26,P =8.43, =2.39,

3 Fg 2).Inparticular, plannedcomparisonsshowedthat S3) (Fig.

S5). Treatment itselfwasnotasignificantdeterminantofthe S1) (Fig. P =0.54; total: P P =0.0054). -tests day1: =0.10; day2:

F S4). Immune-challengedindividualsatelessfood 1). Thisresultisalsoreflectedinthediminished 1,55 F t =0.037), whereasparasitizedcaterpillarsatemore =3.39, d.f.=57,P 2,76 F =19.43, 4,146 =0.083; day2:t 2,75 =10.84, F F t 4,75 =2.24, d.f.=26,P =1.01, 2,54 =4.04, t =3.036, d.f.=28,P =6.05, =10.70, F P 2,42 2,49 <0.0001) andseconddayfollowing

P S2). However, therewasasignificant P P 1,53 =3.88, <0.0001) (supplementarymaterial =1.96, =0.0039) (supplementarymaterial =0.44; day2:F =21.57, P =0.0021; total: P =0.0003; day2:F =2.55, d.f.=23,P =0.0001; day2: t -tests day1: P P =0.034). =0.15). Differences inthe =0.028) (supplementary P F <0.0001; carbohydrate: =0.0051; day2:t 1,55 =10.43, 2,75 t t =0.31, =3.07, d.f.=27, =2.55, d.f.=28, =0.018; total: F F F 2,52 P 4,162 2,76 4,75 =0.0021;

S5). The P =10.80, =0.75) =1.36, =3.27, =3.96, =0.62, (ANCOVA intake wassignificantlyreducedinsham-injectedindividuals amounts ofprotein andcarbohydrateconsumed werehighly weakly) associatedwithbead melanization (Fig. consumed bycaterpillarsfollowing injectionwaspositively(though 5).Theamountoffood not improvemelanizationcapability (Fig. macronutrients, thetendencyto eatlessfoodfollowinginjectiondid immune challenge(Fig. experiment, whichpreferredthehigh-proteinfoodinabsence of protein foodincontrasttothosetheparasitism/injection choice experimentalsoshowedamarkedpreferenceforthe low- P before: nutrient regulationfollowingimmunechallenge(MANCOVA intake points,seesupplementarymaterialTable equal amountsofproteinandcarbohydrate.Forstatisticalcomparison challenge. Thedashedlineindicatesthetrajectoryifcaterpillarshadeaten the intakepoints(non-cumulative)reachedeachdayfollowingimmune initial massesofcaterpillars.Symbolswheretrajectoriesterminaterepresent means accountforvariationinfamily, itsinteractionwithtreatment,andthe daysfollowingimmunechallenge.Leastsquare experiment inthe2 carbohydrate intakeforcaterpillarsintheparasitism/injection Fig. carbohydrate followinginjection(ANCOVA protein: individuals wereduetoreducedintakeofbothproteinand following infection.Differences betweeninjectedandcontrol differed significantlyfromcontrols(MANCOVA before: comparisons showedthatcaterpillarsintheinjectedtreatment P (caterpillar mass): 30% larger thanthoseinthe parasitism/injectionexperiment[ their greatersize:caterpillarsusedinthechoiceexperiments were in theparasitism/injectionexperiment,whichcanbeexplained by (MANCOVA before:F sham-injected individualsandcontrolsweremarginally significant =0.0012; carbohydrate: =0.45; after: When caterpillarswereallowed toself-regulatetheirintakeof Caterpillars inthisexperimentatesignificantlymorethanthose

.Bivariateleastsquaremeans(±1s.e.) ofproteinand 2. F

4,132 Carbohydrate eaten (mg) The JournalofExperimentalBiology(2014)doi:10.1242/jeb.093716 F 10 12 10 12 1,43 0 2 4 6 8 0 2 4 6 8 =1.08, F 01 Day 2 =5.92, Day 1 2,44 t =–11.03, d.f.=168,P =6.39, P =0.37; after: 2,42 5 P

1). 009 Fg 4). =0.019) (Fig. F =2.05, P 1,45 Protein eaten(mg) =0.0037), whereasdifferences between =5.76, 01 P =0.14; after: F P 4,90 =0.021), whereasonlyprotein <0.0001]. Caterpillarsinthe 52 =2.29,

S3. F Parasitized Injected Control P 2,42 02 =0.066). Planned

5). Becausethe =3.02, F 2,45 F 5 P 2,44 =12.03, =0.059) =0.82, t -test

The Journal of Experimental Biology challenge anddiet( the three-wayinteractionbetweentimepoint,levelofimmune immune challengetreatment(ANCOVA: The amountoffoodthatcaterpillarsconsumeddependedon the ρ melanization, ityieldedamarginally significantresult(Spearman’s relationship. However, whenprotein wascorrelatedwith of thesemacronutrientsortheotherisresponsibleforthis correlated ( indicate samplesize. Error barsshowstandarderrors,andnumbersatthebaseofcolumns columns notsharingaletterofthesamecaseorstylearestatisticallydistinct. foods didnotdiffer significantlyacrosstreatments.Columnsorpairsof absent aboveindividualbarsinday1databecauseamountsof (uppercase) andamountoflow-proteinfood(lowercase)eaten.Lettersare tests performedontotalamountoffood(italics),high-protein in supplementarymaterialTable square meanswerederivedfromtherepeatedmeasuresANCOVA, detailed before andafterimmunechallengeinthechoiceexperiment. h caterpillars incontrol,sham-injectedandinjectedtreatmentsthe24 Fig. RESEARCH ARTICLE optimal protein foodthancontrols,withthesize ofthereduction injected andsham-injectedcaterpillars consumedsignificantlyless 6).However, bothbead- of low-proteinfoodafterinjection (Fig. caterpillars inallthreetreatment groupsconsumedthesameamount their intakeoflow-proteinfood inresponsetoanimmunechallenge, Table interaction betweentimeandtreatment( the macronutrientcontentofdiet( No-choice experiment ρ were correlated,asignificantresultwasobtained(Spearman’s =0.50, =0.45,

.Amountofhigh-proteinandlow-protein foodconsumedby 3.

S6). Contrarytothepredictionthat caterpillarswouldincrease P P =0.047). =0.074), whereaswhencarbohydrateandmelanization Amount eaten (mg) r 100 150 200 100 150 200 =0.95, 50 50 0 0 After immune Before immune challenge P F A oto hmInjected Sham Control challenge <0.0001), itisdifficult todiscernwhetherone 2,105 A A 16 =4.16, a

S4. LettersabovebarscorrespondtoTukey P Treatment A,B =0.018) (supplementarymaterial A,B A F 16 F a 1,105 2,105 F =13.03, 2,105 =5.69, Low proteinfood High proteinfood B =5.98, A B P P 17 =0.0045), and a =0.0007), the P Least =0.0045), were collectedfrom thewild,whereasthoseused inthechoiceand as theparentsofcaterpillarsin theparasitism/injectionexperiment genetic, orcouldstemfromtransgenerational environmentaleffects, of differences incaterpillarstockused.Thesedifferences couldbe this difference inpreferencebutspeculatethatitmaybethe result low-protein food(Figs high-protein food,whereasthose inthechoiceexperimentpreferred control caterpillarsintheparasitism/injectionexperimentpreferred the high-proteinfoodthantolow-proteinfood.Interestingly, immune challenge,andthisreductionwasstrongerwithregard to dietary protein.Infact,caterpillarsreducedfeedinginresponse to immune-challenged caterpillarsdidnotincreasetheirintake of response toimmunechallenge.However, contrary toourprediction, herbivore Our findingssupportthehypothesisthatdietarygeneralist in howmuchfoodtheyconsumed( controls (Fig. injected individualsingested58.3%lessoptimalproteinfoodthan caterpillars ingested26.9%lessoptimalproteinfood,andbead- tracking theseverityofimmunechallenge;sham-injected intake points,seesupplementarymaterialTable equal amountsofproteinandcarbohydrate.Forstatisticalcomparison challenge. Thedashedlineindicatesthetrajectoryifcaterpillarshadeaten (non-cumulative) reachedforthe24 caterpillars. Symbolswheretrajectoriesterminaterepresenttheintakepoints variation infamily, itsinteractionwithtreatment,andtheinitialmassesof hbeforeandafterimmunechallenge. 24 carbohydrate intakeforcaterpillarsinthechoiceexperiment Fig. beads weremelanized(Fig. was significantlyandpositivelyassociatedwiththedegreetowhich Instead, theamountoflow-proteindietconsumedpriortoinjection of foodeatenandmelanizationbutitwasnon-significant(Fig. melanization. We didseeanegativecorrelationbetweentheamount immune-challenged individualsdidnotadaptivelyaffect DISCUSSION The observedreductioninoptimalproteinfoodintakeamong

.Bivariateleastsquaremeans(±1s.e.) ofproteinand 4.

G. incorrupta Carbohydrate eaten (mg) The JournalofExperimentalBiology(2014)doi:10.1242/jeb.093716 100 120 100 120 20 40 60 80 20 40 60 80 6). Inaddition,caterpillarfamiliesvariedsignificantly 0 0 04 08 0 120 100 80 60 40 20 0 Before immune After immune challenge challenge 1, 3).We areuncertainastowhatunderlies modifies itsmacronutrientintakein

5). Protein eaten(mg)

h periodbeforeandafterimmune F 3,105 Least squaremeansaccountfor =6.57,

S3. P Injected Sham Control =0.0006). 2253 5).

The Journal of Experimental Biology − 2254 RESEARCH ARTICLE However, thisisapossibility associatedwiththeexperimental diets significantly changingtheratio ofmacronutrientsinthediet. reduce theirintakeofhigh-protein foodfollowingparasitismwithout background, hormonalmilieu) vary. response maybeadaptivewhen circumstances(e.g.genetic protein-rich foodfollowingimmune challengesuggeststhatthis in thetwoexperimentsconverged onthetendencytoeatless and M.S.S.,personalobservation).Theobservationthatcaterpillars individuals duringbothstagesoftheirlifehistory(P.A.M., A.M.S. tachinid fliesreadilyattackandaresuccessfulon If so,itwouldnotchangetherelevanceofthisstudy, giventhat be surewhethertheywouldhaveundergone anadditionalstadium. caterpillars werefreeze-killedfollowingfeedingassays,wecannot a highdegreeofbodysizevariationwithineachstadium. As history plasticityinthenumberofstadiaitundergoes, andthereis rather thanfinallarvalstadium. parasitism/injection experimentcouldhavebeenintheirpenultimate laboratory conditions.Alternatively, somecaterpillarsusedinthe no-choice experimentshadbeenbredforseveralgenerationsunder omitted fromsomepanelsforclarity, butineachcase,the only whentheSpearman’s rankcorrelationwassignificant.Scale high- andlow-proteinfoods.Trendlines aredrawn,andstatisticsprovided, size. LP, lowprotein;OP, optimalprotein;Choice,self-regulatedbetween three experimentaldiets. beads inG.incorrupta Fig. 200 to200,andthe It mayseemcounterintuitivethat caterpillarscouldspecifically

% Melanization .Correlationsbetweenfoodconsumption andmelanizationof 5. 20 40 60 80 20 40 60 80 20 40 60 80 0 0 0 20–0 0 200 100 0 –100 –200 P=0.036 ρ=0.50 Before y -axis rangesfrom0to80%. before andafterimmunechallengewhenfed Food consumptionwascorrectedforcaterpillar hieChoice Choice Residual amounteaten OP LP Grammia incorrupta P=0.055 ρ=0.49 10010200 100 0 –100 After x -axis rangesfrom G. incorrupta exhibits life- OP LP column. bars showstandarderrorsandsamplesizesappearatthebaseofeach Columns notsharingaletterofthesamecasearestatisticallydistinct.Error performed ondatafromhigh-proteinandlow-proteingroupsseparately. Table from therepeatedmeasuresANCOVA, detailedinsupplementarymaterial challenge intheno-choiceexperiment. hbeforeandafterimmune injected andtreatmentsinthe24 Fig. nutrients interactwiththemelanization responsein with melanization,asanticipated, ourresultsdosuggestthatdietary regulated diet. to ahigh-proteindiet,ratherthananoptimal-proteinor self- hypothesis wouldrequiremeasuringimmuneattributesinresponse the choiceexperiment(Fig. bead melanization,anexpectationthatwasnotmetbytheresults of protein consumptionafterinjectiontobenegativelycorrelated with protein onthemelanizationresponse,wewouldhaveexpected of consumingahigh-proteindietstemmedfromnegativeeffect of be detrimentaltoimmunityin (Lee etal.,2006b;Povey2009),excessdietaryprotein may bolstering theimmuneresponseasshownin aversion tohigh-proteinfoodsseenheresuggeststhat,ratherthan swamping outvariationinproportionalconsumption.Therelative necessarily ingestsomecarbohydrateandviceversa,perhaps used here.Eachtimecaterpillarsingestedsomeprotein,theywould carbohydrate were inverselycorrelatedinexperimental diets,this caterpillars fedtheoptimalprotein diet(Fig. with beadmelanization.However, thesamewasnottrueof amount offoodconsumedbefore injectionwaspositivelycorrelated When caterpillarswerefedalow-protein, carbohydrate-richdiet,the Although proteincontentofthedietwasnotpositivelycorrelated

.Amountoffoodconsumedbycaterpillars incontrol,sham- 6.

S6. UppercaseandlowercaseletterscorrespondtoTukey tests

Amount eaten (mg) The JournalofExperimentalBiology(2014)doi:10.1242/jeb.093716 100 150 200 250 100 150 200 250 50 50 0 0 24 A A oto hmInjected Sham Control Before immune After immune challenge challenge 23 a a

5). A stringenttestofa‘costlyprotein’ G. incorrupta.However, ifthecost Treatment 20 B B Least squaremeanswerederived 22 a a

5). Becauseproteinand Low protein Optimal protein A,B Spodoptera 425 24 C G. incorrupta a a species .

The Journal of Experimental Biology work. Oneisthat parasitoidsinducereductions tofeedingfortheir 1998), andofthese,fourcanbe addressedtosomeextentbythis behavior inresponsetodisease (Adamo,2006;Kyriazakisetal., al., 2010). preference forfoodswithlow-lipid contentatthattime(Adamoet feeding followingimmunechallenge, theyexhibitedanadaptive crickets werefedlipid-richfoods,andalthoughreduced al., 2010).Resistancetobacterialinfectionwasreduced when immunity anddigestioninthecricket illness-induced anorexiamightreducecompetingdemands of incorrupta. A similareffect wasseeninaninvestigationofhow quantity offoodsmayinteracttoaffect immuneparametersin caloric densityoffood.Instead,itsuggeststhatthequality and ratio inthedietaffects immune attributesmorestronglythanthe finding byCotteretal.(Cotteral.,2011) thatthemacronutrient self-regulate dietarymacronutrients(Fig. were onlypositivelycorrelatedwhencaterpillarsallowed to 2002; SlanskyandWheeler, 1992). preference andtheperformanceconsequencesthereof(Behmeretal., conjunction withsecondarymetabolitescanindeedaffect dietary sub-optimal melanizationresponse,asseenhere.Macronutrientsin to mixfoodsonshorttimescales,evenifdoingsowouldleada enhancements totheimmunesystem,caterpillarswouldbeexpected absence ofparasitism)isstrongerthanthebenefitprophylactic unpublished). Ifthedefensivebenefitofmixinghostplants(in generalist predator(P.A.M., M.A.BernardoandM.S.S., defensive chemicalsactstodefend in theinsect’s diet.Eatingamixture ofplantscontainingdifferent of nutrientsandthebalancebeneficialplantsecondarymetabolites particular relevancetothissystemmightbethatbetweenthebalance Cotter etal.,2011; Poveyetal.,2009).A potentialtrade-off with within theimmunesystem,arewelldocumented(Cotteretal.,2004; Zuk andStoehr, 2002),aswellthosebetweendifferent parameters 2010; Cotteretal.,2008;Fedorka2004;Ponton2011; between theimmunesystemandlife-historytraits(Adamoetal., with theproteinrequirementofgrowthandreproduction.Trade-offs the carbohydraterequirementofmelanizationresponseconflicted compared with15P:25C](Fig. system [21protein(P):19carbohydrate(C)whenself-regulated, intake target thanwouldprovideaprophylacticbenefittotheimmune immune challenge,caterpillarsself-regulatetoalowercarbohydrate (choice experiment,Fig. allowed toself-regulatetheirmacronutrientintakepriorinjection eaten andthedegreeofbeadmelanizationwhencaterpillarswere melanization capabilitymayhavebeenenhanced. the massoffatbody, thisisonemechanismbywhich precursors (Beckage,2008).Ifcarbohydrateconsumptionincreases greater massofthefatbody, the siteofproductionformanyimmune 2002). Increasedfeedingoncarbohydrate-richfoodsmayleadto degree whenrearedondietsrichinglucose(SchwartzandKoella, mosquito of nutrientstowardimmunity. A similarresultwasfoundinthe carbohydrate, ratherthanprotein,maylimittheprophylacticaction melanization, whichwasnotthecase(Fig. consumed priortoinjectionbenegativelycorrelatedwith we wouldhaveexpectedtheamountofoptimalproteindiet challenge. Ifproteinweredetrimentaltothemelanizationresponse, carbohydrate orfromreducedproteininfoodpriortoimmune could meanthatcaterpillarsbenefitedeitherfromincreased RESEARCH ARTICLE A number ofhypotheseshavebeenputforthtoexplainanorexic The observationthattheamountoffoodeatenandmelanization Interestingly, therewasnocorrelation betweentheamountoffood Anopheles gambiae,whichmelanizedbeadstoagreater

5). Thissuggeststhat,intheabsenceof

5). Thiscouldresult,forexample,if G. incorrupta Gryllus texensis

5) contrastswiththe

5). Thissuggeststhat against atleastone (Adamo et G. parasitism/injection experiment (Fig. nutrient intakebetweenparasitized andinjectedcaterpillarsinthe caterpillars exhibitedanorexia, wealsoobservedadifference in Raubenheimer andSimpson,2003). 2006a; Leeetal.,2003;Raubenheimer andSimpson,1999; complex, immunity-enhancingbehavioralstrategies(Leeet al., herbivores shouldbepositionedparticularlywelltoemploy hypothesis isconsistentwiththeexpectationthatgeneralist harness bothmacronutrientandchemicalvariationinplants. This consequences undernaturalcircumstances,whencaterpillars can caterpillar dietsonparasitoiddevelopmentcouldhavemajorfitness condition (e.g.size)ofparasitoidsatthattimepoint,theeffects of et al.,2011a). Iftheefficacy ofself-medicationiscontingentonthe oviposition), enhancingtheirsurvival(Singeretal.,2009;Smilanich alkaloids duringthelatestageofparasitoidinfection(~96 Grammia incorrupta medication todefendcaterpillarsagainstparasitoidinfection. that anorexiaactsinconjunctionwithmelanizationand/orself- alone isinsufficient toovercomeparasitoids.However, itispossible parasitoid infection(datanotshown),weconcludethatanorexia that parasitoids hadtakencontrolofhostnutrient intakefortheir (see supplementarymaterialFig. comparable growthbenefittotheoptimalproteinfoodusedhere cost, giventhatalowerproteindietcanafford personal observation).Moreover, suchastrategymayincurlittle melanization responserelativetoothercaterpillarspecies(A.M.S., nutritional variation,and(b)theypossessaparticularlystrong grazing feedingstrategyallowsthemtoaccessthenecessary could beparticularlyeffective in parasitoids bynutritionalmeanswiththemelanizationresponse immunological strategythatcombinesslowingthegrowthof to bemostvulnerablethehost’s melanizationresponse.An during theearlystageofinfection,whenparasitoidlarvaearelikely in thehemolymphcouldtranslatetoslowergrowthofparasitoids (Thompson etal.,2005).Ifthisisthecasehere,lowerproteintiters Thompson etal.,2005),andcanaffect parasitoiddevelopment respond todietaryprotein(Leeetal.,2008;Povey2009; development ofparasitoids.Proteinlevelsinthehemolymph with regardtoprotein-richfoods(seealsoAdamoetal.,2010). meet thisexpectation;caterpillarsexhibitedanorexiaparticularly low-quality foods(Kyriazakisetal.,1998).However, ourresultsdo should bemorepronouncedwithregardtohigh-qualityfoodsthan 1996; Kyriazakisetal.,1994),namelythattheanorexicresponse the hypothesisisnotgenerallymetinmammals(Kyriazakisetal., discounted tosomeextentonthebasisthatmainpredictionof types offoods,supportingtheformerhypothesis.Thelatterhasbeen exhibited ananorexicresponsethatdiffered withrespecttodifferent that anorexiaservestostarveparasites.Inthisstudy, caterpillars individuals tobemoreselectiveinthefoodsthattheyeat,and(2) seem tobesupportedbythisstudy:(1)thatanorexiaallows many immuneparametersthatwedidnotmeasurehere. is notsupportedbyourmelanizationresults;however, thereare Another hypothesis,thatanorexiaenhancestheimmuneresponse, that waslesspronouncedthanofparasitizedindividuals). injected individualsalsoexhibitedananorexicresponse(thoughone 2012; Moore,2002),thisexplanationisunlikelygiventhatbead- feeding behaviorhasbeenshowninsomesystems(Hughesetal., own benefit.Althoughadaptiveparasitemanipulationofhost Although bothparasitizedandunparasitized, immune-challenged Because parasitizedcaterpillarsinthisstudysuccumbedto Perhaps reducedingestionofhigh-proteinfoodsactstoretardthe Two relatedhypothesesregardingdisease-inducedanorexiado The JournalofExperimentalBiology(2014)doi:10.1242/jeb.093716 caterpillars self-medicateusingpyrrolizidine

S1; Appendix2). G. incorrupta

1). Onepossibleexplanationis because (a)their G. incorrupta

h after 2255 a

The Journal of Experimental Biology 2256 RESEARCH ARTICLE Singer andStireman,2003).Onaverage,15%of as wellprovidingdefenseagainstnaturalenemies(Singeretal., 2004; physiological efficiency (P.A.M., M.A.BernardoandM.S.S.,unpublished), 2002). Thisgrazingdietarystrategybenefitsthespeciesbyimproving its host plantsoverthecourseofadayisregularoccurrence(Singer et al., Host-plant switchingisacommonbehaviorandmovingbetweenindividual southwestern USA andnorthwesternMexico(SchmidtSperling,2008). Stireman, 2001).Thisspeciesinhabitsaridgrasslandsandwoodlands of on over80speciesofplantsin50different plantfamilies(Singerand Grammia incorrupta considerations. host, thepathogenorparasite,andnumerousotherecological behavioral components,canbeexpectedtodiffer dependingonthe findings reinforcethenotionthatimmuneresponse,includingits melanization responseandself-medicationbytheirhosts.These their susceptibilitytoanti-parasitoidresistancefromboththe dietary attributesmayalsointeractwithdevelopingparasitoids,and ratio) andquantityin affected bytheinteractionbetweenfoodquality(macronutrient macronutrient ratiosdidnot.Thissuggeststhatimmunefunctionis improved melanization,whereaseatingmoreofdietswithfixed increased feedingondietswithself-selectedmacronutrientratios improved themelanizationresponse.Afterimmunechallenge, immune challenge,greaterintakeofcarbohydrate-biaseddiets caterpillars reducedtheirintakeofhigh-proteinfood.Priorto Contrary tofindingsfromsimilarstudies,immune-challenged that variancesdidnotdiffer amongtreatments(day1: high-protein foodeateneachdayfollowingparasitism,andfound Brown–Forsythe testsforunequalvariancestotheproportionof parasitized andcontrolindividuals.To testthis,weapplied would expectgreatervarianceintheamountofeachfoodeatenby nutrient regulationhadbrokendowninresponsetoparasitism,we precludes usingthismethodtodrawsuchaconclusion;however, if offered (ThompsonandRedak,2005).Ourexperimentaldesign target regardlessofthemacronutrientratiosinpairsfoods indiscriminately, whereascontrols maintainedamacronutrientintake design theywereabletoconcludethatparasitizedindividualsfed macronutrient content(ThompsonandRedak,2005).Usingthis employing multiplepairsoffoodsthatdiffered intheir in responsetowaspparasitismbyusingchoiceexperiments and Redakshowedsuchabreakdownin disrupted thecaterpillar’s regulationofnutrientintake.Thompson is difficult toevaluate.Anotherpossibilityisthatparasitism measure theeffects ofdietonparasitoidfitnesshere,thishypothesis own benefit(Hughesetal.,2012;Moore,2002).Aswedidnot P change isbiotic. individuals illustratesthatatleastsomepartofthecueinducingthis extent towhichfeedingwasaffected inparasitizedandinjected Study system MATERIALS ANDMETHODS Conclusions The choiceandno-choice experimentswereperformed intheautumnof their diettosupporttheimmunesystem. plants, itseemslikelythatgrazing individualscouldalsoadaptivelyalter individuals arelikelytoencounter byusingsuchabroadrangeofhost parasitoids (StiremanandSinger, 2002).Giventhe nutritionalvariationthat mella majority ofparasitismcomingfrom tachinidflyspecies,including in naturalpopulationsexperiencemortalityfromparasitoids,with the =0.20; day2:F These experiments tookplaceintheSingerlabatWesleyan University. and Chetogena 2,76 caterpillars aregrazinggeneralistherbivores,feeding =2.21, species, andtoalesserextentfrom hymenopteran G. incorrupta.We hypothesizethatthese P =0.12). Nonetheless,differences inthe Manduca sexta G. incorrupta F caterpillars 2,82 caterpillars =1.166, Exorista either caterpillars toself-regulateatarget ratio. Simpson etal.,2004).Presentingfoodtocaterpillarsinthismannerallowed Raubenheimer andSimpson,2004;SimpsonRaubenheimer, 1993; herbivores non-independent(RaubenheimerandSimpson,1999; correlated (BernaysandChapman,1994)theirconsumptionbyinsect because proteinandcarbohydrateconcentrationsinplantsareofteninversely ingredients). We variedmacronutrientratios,ratherthanrawamounts, carbohydrate, bydrymass(seeAppendix1foracompletelistof mass, andthehigh-proteinfoodcontained35%protein5% low-protein foodcontained15%proteinand25%carbohydrate,bydry be injectedwithbeadsandthosethatwouldreceiveparasitoideggs.The among threetreatments:thosethatwouldactascontrols, caterpillars intheirfinalstadiumuponwhichflyeggswerevisible. obtained fliesforthelaboratorycolonybycollecting G. incorrupta food thancontrols.We areconfident thattheflyspeciesusedhereattacks parasitized andinjectedcaterpillarswouldconsumemoreofthehigh-protein a tachinidfly. We predictedthat,whenallowedtoself-regulate,both studies tomimicparasitoidinfectionin bead injectiontechnique(describedbelow),whichhasbeenusedinprior behavior inresponsetoimmunechallenge,and(b)whethertheSephadex The purposeofthisexperimentwastotest(a)forchangesinfeeding in experimentswerehoused167.2 in experimentspriortofeedingonexperimentaldiets.Allcaterpillarsused wheatgerm-based rearingdiet(Yamamoto, 1969),aswereindividualsused southeastern ,USA.Colonyindividualswererearedonanutritious, laboratory breedingcolony, initiatedfromcaterpillarsoriginallycollectedin summer of2009.Caterpillarsusedfortheexperimentsweretakenfroma 2008, andtheparasitism/injectionexperimentwasperformedduring injection byingesting agreateramountofthehigh-protein foodthan caterpillars would biasmacronutrientintaketowards proteinfollowing intake differently beforeandafterimmunechallenge.We predictedthat In thisexperimentwetestedwhether caterpillarsregulatemacronutrient mass offoodeateneachday. feeding days)wasthensubtractedfrom initialdrymasstodeterminethe food thatremainedafter24 these valuestodrymassusingawet–dryconversioncurve.Dry of of thefoodsprovidedtocaterpillarsonbothfeedingdaysandconverted to carbohydratethattheyconsumed.To dothis,weweighedinitial amounts controls orparasitizedcaterpillarsinhowmuchfoodandtheratioofprotein challenge inordertoassesswhetherinjectedcaterpillarsgrouped with caterpillars inallthreetreatmentsfor2 assay’ below).We measuredtheamountofeachfoodblock eatenby that parasitoidsenteredcaterpillars(forinjectiontechnique,see‘Immune larval molt,sothatthemomentofinjectionwouldapproximate caterpillars intheinjectiontreatmentwereinjected2 hatch fromeggsandburrowthroughthecuticle(Smilanichetal.,2011a), at leastoneeggwaspresent.Asittakes48–60 caterpillars wereinspectedmorecloselyinaclearplasticvialtoensurethat they hadreceivedonetothreeeggs.Threeattemptswerepermitted,andthen experimentally. Caterpillarswereexposedtofliesforseveralminutesuntil different feedingresponsesin saved fromtheexperiment.Althoughitispossiblethatthesecongenerselicit Stireman III,Wright StateUniversity)ontheidentitiesoftachinidspecimens experiment, wereceivedconfirmationfromataxonomicexpert(J.O. not reliablydistinguishthetwospeciesduringexperiments.After because bothwerepresentinourtachinidcolonyatthetimeandwecould larval molt.We usedtwocloselyrelatedflyspeciesintheseexperiments 2011b), elicitsthesamefeedingbehaviorin (Lavine andBeckage,1996;Smilanichetal.,2011a; Smilanichetal., Meriden, CT, USA). Parasitism/injection experiment Feeding behavior before andafterimmunechallenge Caterpillars intheparasitismtreatmentwereexposedtotachinidflies, After thefinallarvalmolt,weweighedcaterpillarsanddistributedthem Chetogena edwardsi The JournalofExperimentalBiology(2014)doi:10.1242/jeb.093716 during thefinallarvalstadiuminwildbecausewe or

h (foodwasremovedaftereachofthetwo C. tachinomoides G. incorrupta,wedidnottestthat

ml clearplasticcups(RussellHallCo.,

days followingthetimeofimmune G. incorrupta G. incorrupta h forChetogena , onthedayoftheirfinal

days afterthefinal and otherspecies as parasitismby G. incorrupta larvae to

The Journal of Experimental Biology the feedingtrialanddissectedlatertodeterminebeadmelanization. as describedabove,andinjectedindividualswerefreeze-killedattheendof receiving theimmunechallenge.Amountsoffoodeatenweredetermined day numbertovaryensurethatcaterpillarshadinitiatedfeedingbefore some caterpillarsdidnoteatforseveraldaysaftermolting,weallowedthe later inthisexperimentthantheparasitism/injectionexperiment.Because an additional24 challenge, caterpillarsweregivenfreshfoodblocksandallowedtofeedfor injection, shaminjectionandcontroltreatments.Afterthetimeofimmune observation). Forcomparison,thetimingofimmunechallengewas1 when caterpillarsfeedmost(P.A.M., A.M.S.andM.S.S.,personal feeding assaysbecausetheyrepresentthemiddleoffinallarvalstadium, suspended inRinger’s solutionsothat5–10beads couldbeinjectedintothe red using0.1%Congo Red(dyecontent35%;Sigma-Aldrich) andwere optimal ratioofdietaryproteintocarbohydrate. Sephadexbeadsweredyed predicted anincreaseinthemelanization responseinindividualswithan Beckage, 1996;Smilanichetal., 2009a;Smilanichetal.,2009b).We Sigma-Aldrich, StLouis,MO,USA) asaproxyforparasitism(Lavineand caterpillars wereinjectedwithSephadex beads(SephadexA25,40–120 To measurethemelanization responsetodietarynutrition, (see ‘Immuneassay’ below). killed attheendoffeedingtrialanddissectedlaterforretrieval beads day usingthemethoddescribedabove.Injectedindividualswerefreeze- feeding foranother24 beads orshaminjected,thenreturnedtotheirrespectivedietscontinue individuals. After24 protein dietsorlow-proteindiets.Eachtreatmentlevelreceived 20 assigned toinjection,shaminjectionorcontrolgroupsaswelloptimal Fig. allowed toself-selectamacronutrientintaketarget (supplementarymaterial material Fig. experimental dietsthatvariedintheirmacronutrientratios(supplementary it afforded 25P:15C anoptimalratiobecausepreliminaryexperimentsshowedthat(a) mass, respectively),sothatmixingfoodswasnotapossibility. We consider either alow-proteinoranoptimalproteinfood(15P:25Cand25P:15Cdry to, and24 alternative. differentiate betweenpreferenceforagivenfoodtypeandaversiontothe conjunction withthechoicetestdescribedabovewouldalsopermitusto sham-injected andcontrolgroups.Conductingano-choicetestin stadium, andcomparedfeedingresponsesbetweeninjectedindividuals, immune systemusingbeadinjectionduringthefourthdayoffinallarval individuals. Asinthechoiceexperimentdescribedabove,wechallenged low-protein dietthanthehigh-proteinamongimmune-challenged and Wheeler, 1992). Therefore,weexpectedgreaterconsumptionofthe feeding onthelow-proteindiet(RaubenheimerandSimpson,1993;Slansky caterpillars wouldincreaseproteinconsumptionthroughcompensatory ratio afterimmunechallenge.We predictedthatimmune-challenged caterpillars’ consumptionoftwodietsthatdiffered in theirmacronutrient In thisexperiment,wetestedwhethertherewouldbedifferences inthe allowed toself-regulatetheirmacronutrientintakefor24 high-protein foods(15P:25Cand35P:5Cdrymass,respectively), larval stadium,caterpillarswereoffered blocksofbothlow-proteinand intake inresponsetotheimmunechallenge.Onthirddayoffinal individuals wouldregulatetheirmacronutrientratiotowardahigherprotein injection (Smilanichetal.,2011a). We predictedthatimmune-challenged Ringer’s solutionandnobeadstocontrolforthewoundresponse injection groupinwhichindividualswereinjectedwithonlyisotonic 1996). Unlikeintheparasitism/injectionexperiment,weincludedasham beads representedthechallengetoimmunesystem(LavineandBeckage, controls. Asintheparasitism/injectionexperiment,injectionwithSephadex RESEARCH ARTICLE No-choice experiment Immune assay On thethirddayofseventhlarvalstadium,individualswererandomly All individualsweresubjectedtoano-choicefeedingassayfor24 S2; Appendix2). h subsequentto,thetimeofinjection.Caterpillarswereoffered G. incorrupta

S1; Appendix2),and(b)caterpillarschoseasimilarratiowhen

h. Thethirdandfourthdayofthestadiumwerechosenfor

h offeeding,individualswereinjectedwithSephadex

h. We measuredtheamountoffoodeatenoneach the greatestgrowthonaverageamongfive

h priortobead G. incorrupta

h prior μm;

day differences betweentreatments. whether intakeofprotein,carbohydrateorbothwasresponsibleforsignificant the control.We alsoperformedunivariateplannedcomparisonstoidentify performed plannedcomparisonstodiscernwhichtreatment(s)differed from to identifytreatmentdifferences inself-regulatedmacronutrientintake,and consumed usingMANCOVA (maineffect: treatment;covariate: initialmass) the consumptiondata.We analyzedamountsofproteinandcarbohydrate analyzed usingANCOVA withthesamefactorsinmodelsasweusedfor The ratiosofproteintocarbohydrateconsumedwerelogtransformedand and thebivariateresponse,amountsofproteincarbohydrateconsumed. immune challenge,weanalyzedtheratioofproteintocarbohydrateconsumed, preference associatedwithimmunechallenge,weusedpaired food eatenbycaterpillarsindifferent treatments.To assesschangesin interactions. We usedTukey teststoidentifydifferences in theamountof family (treatedasarandomeffect), initialmassandsignificanttwo-way high-protein andlow-proteinfoodsseparately. Modelsincludedtreatment, following immunechallenge.Thiswasdonefortotalfoodeaten,and We usedANCOVA toassessdifferences intheamountoffoodeaten and viceversa(Smilanichetal.,2009a;Smilanich2009b). interpretation, sothathighvaluesindicateagreaterdegreeofmelanization a percentageofmelanization[1–( score foreachindividualcaterpillar. Themean bead withinacaterpillarandthesevaluesaveragedtoprovidean Using AdobePhotoshop(version6.0),ther r to 255,where0=puregrayand255=purered,foreachbead.Thelowerthe quantified melanizationbymeasuringtheredvalue,ascalerangingfrom0 As thebeadsweredyedredbeforeinjectingthemintocaterpillars,we AxioVision software;CarlZeissMicroscopy, LLC,Thornton,NY, USA). dissection microscopefocusedat80×magnification(DiscoveryV.8, methanol andbeadswerephotographedusingacameramountedon additional 24 to theirtestdietsandfreeze-killedattheendoffeedingtrial(afteran glass needles(LavineandBeckage,1996).Caterpillarswerethenreturned (Sigma-Aldrich) thatwehadstretchedunderheatinordertocreatetiny base ofthethirdproleg.InjectionswerecarriedoutusingPasteurpipettes each macronutrient affected melanization. with theamount of foodconsumed,precludingseparate analysesofhow in thisexperiment,proteinandcarbohydrate intakewereperfectlycorrelated differences inamountoffoodeaten.Becausecaterpillarsate only onediet (low-protein, optimalprotein).Tukey testswereusedtoidentifytreatment indicated forthechoiceexperiment, withtheadditionofvariablediet injection usingrepeatedmeasures ANCOVA, withthesame variables We analyzedtheamountoffoodingestedbycaterpillarsbeforeand after interpretation offamilyeffects. We didnotuse individuals ofthesamefamilyusedinexperimentsformeaningful models, orinthosefortheno-choiceexperimentbecausethereweretoo few treatment interaction.We didnotincludefamilyasacovariateinthese injected, control),time(beforeinjection,afterinjection)and × included theindependentvariablesimmunechallenge(beadinjected, sham protein tocarbohydrateratios.RepeatedmeasuresANCOVA models used repeatedmeasuresANCOVA toanalyzebothconsumptiondataand case, wemeasuredconsumptionbeforeandafterimmunechallenge, parasitism/injection experiment,withafewmodifications.Because,inthis We usedthesameanalyticalproceduresinchoiceexperimentas data. this preferencearereflectedinresultsofTukey testsappliedtoconsumption protein foodregardlessofdietarytreatment.Differences inthestrengthof caterpillars usedinthisexperimentexhibitedaclearpreferenceforthe low- food preferenceassociatedwithimmunechallengebecausethe set of Parasitism/injection experiment Statistical analysis No-choice experiment Choice experiment -value, theblackerbead,indicatingincreasinglevelsofmelanization. To identifydifferences innutrientregulationthe2 The JournalofExperimentalBiology(2014)doi:10.1242/jeb.093716

h). To retrievebeads,caterpillarsweredissectedin95% r -value/maximum -value wasobtainedforeach r t -tests toevaluatechangesin -value wastransformedinto r -value)] foreaseof

days following t -tests. r -value 2257

The Journal of Experimental Biology The remainingfoodwasremovedeachday, driedandweighed, blocks werecalculatedusingawettodrymassconversioncurve. cups wherecaterpillarswerehoused,andthedrymassesof food Each foodblockwasweighedbeforebeingplacedintheplastic food (P:Cratioof35:5)andalow-protein15:25). families represented)wasoffered thechoicebetweenahigh-protein two larvalstadia.A groupofcaterpillars ( and carbohydrate(accordingtothefirstexperiment)duringfinal caterpillars wouldadaptivelyself-selectanoptimalratioofprotein dietary treatmentswerestatisticallydifferent. normalize residuals,andaTukey testwas used todiscernwhich dry massanddurationofdevelopmentwerelogtransformedto of sexualdimorphisminG.incorrupta.Theresponsevariablesadult diet asthemaineffect andsexasacovariatetocorrectfortheeffects models. We analyzedthesedatausingANCOVA withexperimental considered indicesofgrowthandusedasresponsevariablesinour and weighed.Adultdrymassdevelopmentaldurationwere caterpillar, andadultcaterpillarsweredriedatapproximately60°C The durationofthefinaltwolarvalstadiawasnotedforeach food wasprovided 2258 RESEARCH ARTICLE Water Agar Choline chloride Vitamin mix Methyl paraben Ascorbic acid Cholesterol Linoleic acid Wesson’s saltmixture Cellulose Sucrose Casein Ingredient in 167.2 30:10 and35:5(seeAppendix1fordetails).Caterpillarswerekept in theirproteintocarbohydrate(P:C)ratios:15:25,20:20,25:15, treatments) werefedoneoffiveexperimentaldietsthatvariedonly nine full-siblingfamilies(distributedinabalancedmanneramong incorrupta of proteintocarbohydrateratiosismostbeneficialgrowthin The purposeofthefirstexperimentwastoidentifywhicharange macronutrients varied(Table totaled 16 carbohydrate sources(caseinandsucrose,respectively)always et al.(Singeral.,2002)(Table The experimentaldietsusedinthisstudyweremodifiedfromSinger their resultscanbefoundinsupplementarymaterialTables S1–S6. ver. 7,1989–2007,SASInstituteInc.,Cary, NC,USA).Fullmodelsand with melanizationdata. over caterpillarmass,andusedresidualsinSpearman’s rankcorrelations caterpillar bodysizewasaccountedfor, weregressedamountoffoodeaten To assesstheeffect oftheamountfoodeatenonmelanizationwhen Table A1.Experimental dietingredients Injection assay Methods APPENDIX 2:PRELIMINARY NUTRITIONAL EXPERIMENTS APPENDIX 1:EXPERIMENTAL DIETS All statisticswerecalculatedusingJMP statistical software(JMP 2007, The purposeofthesecondexperimentwastotestwhether

ml clearplasticcups(seeMaterialsandmethods),inwhich

g (40%ofthedrymassfood);onlyratio in itsfinaltwolarvalstadia.Groupsofcaterpillarsfrom dlibitum ad

A2). and wasreplacedeverysecondday. 0.3 0.25 0.2 0.2 22.27 0.21 0.12 0.96 Varied (seeTable A2) Varied (seeTable A2) Quantity 160 5.12

A1). Thedrymassofproteinand

ml g g

ml

ml g g g g

g N =30, fivefull-sibling G. 35P:5C 30P:10C 25P:15C 20P:20C the manuscript. also thankananonymousreviewerandSpenceBehmerforvaluablecomments on taken fortheseexperiments,andLucySprungassistanceinbeadretrieval. We F treatments). Itdid,however, significantlyaffect insectfinalmass(diet: experiment failedtoeclose(onefromeachofthetwohighestprotein caterpillars’ abilitytoreachadulthood;onlytwocaterpillarsinthe The macronutrientcontentoffooddidnotsignificantlyaffect the developmental stadia. each day. We didthisforthedurationofpenultimateandfinal raw amountsofproteinandcarbohydrateingestedbycaterpillars allowing ustocalculatehowmuchofeachfoodwaseaten,andthe P, protein;C,carbohydrate. 15P:25C Experimental diet and NSFno.0744676 toM.S.S.]. This workwassupported byTheNationalScienceFoundation [DDIGno.1011503 M.S.S. andA.M.S.revisedit. were involvedintheinterpretationofresults. P.A.M. wrotethemanuscript, and experimental design,P.A.M. andA.M.S.executedtheexperiments, and allauthors M.S.S. wasresponsibleforconception oftheproject.Allauthorswereinvolvedin The authorsdeclarenocompetingfinancialinterests. help maintainingthebreedingcolonyof and CollinMcMichaeloftheSingerLaboratoryatWesleyan Universityfortheir We thankNickolasFesenko,AaronGreenberg,Yumin He,IsaacLichter-Marck F significantly affect thedurationoffinaltwolarvalstadia(diet: significantly less(Fig.S1).Themacronutrientcontentoffooddidnot However, aboveagiventhresholdofproteinbias,adultsweighed toward eithermacronutrientwasslight(15P:25C,25P:15C)(Fig.A1). carbohydrate werebalancedinthediet(20P:20C)orwhenbias attained equallylarge adultmasses,onaverage,whenproteinand Table A2.Proteinandcarbohydratecontentofthediet macronutrients. individuals forageinanoptimalmannerwithregardtotheirintakeof containing aratioof25P:15C,andillustratesthat incorrupta consistent withthefindingfromfirstexperimentthat 25.16% proteinbydrymass(range21.27–29.58%).Thisresultis 19.78–29.22%). Theoverallmeanforthetwofinallarvalstadiawas selected dietscontaining24.26%proteinbydrymass(range average (range21.62–34.45%).Duringthefinalstadium,caterpillars caterpillars selecteddietscontaining28.43%proteinbydrymasson (supplementary materialFig. in proteinintakeatthebeginningandendofeachstadium intake overthecourse consumed (supplementarymaterialFig. to carbohydrate,theyvaried Results Funding Author contributions Competing interests Acknowledgements 4,113 4,95 When caterpillarswereabletoself-selecttheratioofdietaryprotein =9.82, =1.20, attains thegreatestadultbodymasswheniteatsadiet The JournalofExperimentalBiology(2014)doi:10.1242/jeb.093716 P P =0.31; sex:F <0.0001; sex:F fthefinaltwolarvalstadia,showingspikes of 1,113 14.0 12.0 10.0 8.0 6.0 Casein (g) both theamountsoffoodsthatthey

S3). Duringthepenultimatestadium, =1.12, 1,95 G. incorrupta =54.91, P =0.29).

S2) andtheirmacronutrient P from whichindividualswere <0.0001). Caterpillars 2.0 4.0 6.0 Sucrose (g) 8.0 10.0 G. incorrupta G.

The Journal of Experimental Biology otr .C,Smsn .J,Ruehie,D n isn K. Wilson, and D. Raubenheimer, S.J., Simpson, S.C., Cotter, otr .C,Mat .P,Bnkn .M .adWlo,K. Wilson, and C.M.H. Benskin, J.P., Myatt, S.C., Cotter, K. Wilson, and L.E.B. Kruuk, S.C., Cotter, lit .L,Bafr,S n hms M.B. Thomas, and S. Blanford, S.L., Elliot, emr .T n or,A. Joern, and S.T. Behmer, S.T. Behmer, Hughes, N.E. Beckage, http://jeb.biologists.org/lookup/suppl/doi:10.1242/jeb.093716/-/DC1 Supplementary materialavailableonlineat RESEARCH ARTICLE nls .D,Jhsn .L n ote,M.S. Goettel, and D.L. Johnson, G.D., Inglis, aie .D n tad M.R. Strand, and M.D. Lavine, N.E. Beckage, and M.D. Lavine, M.R. Hutchings, and B. J. Tolkamp, I., Kyriazakis, F. Jackson, and R.L. Coop, J.D., Oldham, D.H., Anderson, I., Kyriazakis, e,K . abnemr . emr .T n ipo,S.J. Simpson, and S.T. Behmer, D., Raubenheimer, K.P., Lee, et . ibr,H,Wie .adBos M. Boots, and A. White, H., Tidbury, A., Best, M.S. Singer, and E.A. Bernays, R.F. Chapman, and E.A. Bernays, D. Raubenheimer, and S.J. Simpson, S.T., Behmer, eok,K . u,M n oseu T. A. Mousseau, and M. Zuk, K.M., Fedorka, D.S. Schneider, and J.S. Ayres, S.A. Adamo, yizks . lhm .D,Co,R .adJcsn F. Jackson, and R.L. Coop, J.D., Oldham, I., Kyriazakis, ut,J n rn,K. Franz, and J. Kurtz, e,K . oy .S,Wlo,K,Ruehie,D n ipo,S.J. Simpson, and D. Raubenheimer, K., Wilson, J.S., Cory, K.P., Lee, S.J. Simpson, and S.T. Behmer, K.P., Lee, References Supplementary material nesn .D,Bafr,S n hms M.B. Thomas, K. and S. Sullivan, Blanford, and R.D., N. Anderson, Spencer, J., Le, A., Bartlett, S.A., Adamo, dm,S.A. Adamo, aot .R n omn M.J. Gorman, and M.R. Kanost, abn .adBlwn I.T. Baldwin, and R. Karban, eèr,T,Oie,L,Hne,M .adD od,J.C. DeRoode, and M.D. Hunter, L., Oliver, T., Lefèvre, K. Wilson, and S.J. Simpson, K.P., Lee, traits. Funct.Ecol. Macronutrient balancemediatestrade-offs betweenimmunefunctionandlifehistory littoralis. cuticular melanismrevealsimmunefunctionandlife-historytrade-offs inSpodoptera 421-429. correlations andpotentialtrade-offs inaninsectimmunesystem. 20120887. intake, dietselectionandperformanceofgrowinglambs. Long-term subclinicalinfectionwith 269 varying environment:temperature,behaviouralfeverandfitness. within-generation immuneprimingininvertebratehosts. nutritional feedingniches. 165-187. 2485. tolerance toinfectionsinDrosophila. thermoregulation onmycosisby Oxford: OxfordUniversityPress. Insect Physiol. immunosuppression in 56, 265-274. explanation fortheoccurrenceofanorexiaduringparasiticinfections. Nutr. subclinical intestinalnematodeinfectiononthedietselectionofgrowingsheep. Insect Biochem.Mol.Biol. diet breadth:acomparisonof specialist caterpillar between macronutrientbalancingandinsecthost-plantrange:evidencefrom the Insects. NewYork, NY: Chapman&Hall. Ecology chemically heterogeneousenvironments:nutrientsandsecondarymetabolites. cost ofreproductioninthegroundcricket, endoparasites. infection byfevering:atacost. Anim. Behav. induced anorexiamayreducetrade-offs betweendigestionandimmunefunction. insects. Behav. Cogn.Neurosci.Rev. immunity? 7 immunity. The UniversityofChicagoPress. 2076-2084. Biol. Sci. Flexible dietchoiceoffsets proteincostsofpathogenresistancein a caterpillar. generational medication innature. Immunology melanization andimmunefunctioninan insect. , 131-139. , 1599-1607. 72, 665-677. .P,Boer .adToa,F. Thomas, and J. Brodeur, D. P., 83, 2489-2501. J. Evol.Biol. 273 Nature Anim. Behav. (ed. N.E.Beckage),pp.69-96.Amsterdam:AcademicPress;Elsevier. (2004). Howshouldbehaviouralecologistsinterpretmeasurementsof , 823-829. (2009). Insectherbivorenutrientregulation. (2008). 79, 3-10. (2006). Comparativepsychoneuroimmunology:evidencefromthe 42, 41-51. Nature 425 25, 186-198. Spodoptera exempta , 37-38. Insect Immunology 21, 1744-1754. 436 (2003). Innatedefence:evidenceformemoryininvertebrate Manduca sexta 68, 1443-1449. Proc. Natl.Acad.Sci.USA 32, 1295-1309. , 476. (2008). Coexistinggeneralistherbivoresoccupyunique (1997). Heliothis Ecol. Entomol. (2002). Insecthemocytesandtheirroleinimmunity. (2008). Phenyloxidasesininsectimmunity. In Beauveria bassiana Ecol. Lett. (2009). Theroleofanorexiainresistanceand (1996). Temporal patternofparasitism-induced PLoS Biol. (2005). Insectdefences:tastealterationand (1994). rcotoglscolubriformis Trichostrongylus Induced ResponsestoHerbivory 5 , 128-140. sister speciesandahybrid. larvae parasitizedby . Amsterdam:AcademicPress;Elsevier. (Walker). J.InsectPhysiol. loeoissocius Allonemobius (2008). Dietaryprotein-qualityinfluences (2006a). Nutrientregulationinrelationto (2012). Host-Plant SelectionbyPhytophagous (2002). Host-pathogeninteractionsina 13, 1485-1493. 38, 1-10. (2004). Immunesuppressionandthe (2013). Theevolutionarydynamicsof Funct. Ecol. (2004). Costsofresistance:genetic 7 , e1000150. (1996). Effects oftemperature and (2013). Housefliesdelayfungal Host ManipulationbyParasites 105 in grasshoppers. (1998). Towards afunctional (2002). Herbivoreforagingin Vet. Parasitol. , 1977-1982. (2010). Evidencefor trans- Annu. Rev. Entomol. J. R.Soc.Interface 22, 1052-1061. Cotesia congregata (1994). Theeffect of . (2008). Selectionfor (2003). A correlation Evolution 49, 1161-1171. : effects onfood J. Exp.Biol. J. Evol.Biol. Proc. Biol.Sci. (2010). Illness- . Chicago.IL: Anim. Behav. 61, 297-313. Biol. Control 58, 2478- (2006b). (1996). (2011). Insect Proc. Br. J. 209 . 17, 10, 54, J. . , igr .S,Mc,K .adBras E.A. Bernays, and K.C. Mace, M.S., Singer, lnk,F n hee,G.S. Wheeler, and F. Slansky, igr .S,Crir,Y,Tern,C n atan T. Hartmann, and C. Theuring, Y., Carrière, M.S., Singer, mlnc,A . yr .A,Cabr,J .adBwr,M. D. Bowers, and J.Q. Chambers, L.A., Dyer, A.M., Smilanich, J.W. Thompson, and M.T. Siva-Jothy, oe,Y n iaJty M.T. Siva-Jothy, and Y. Moret, P. Schmid-Hempel, and Y. Moret, J. Moore, igr .S n trmn J.O. Stireman, and M.S. Singer, J.O. Stireman, and M.S. Singer, M. Spivak, and M.D. Simone-Finstrom, F. Slansky, and J.M. Scriber, igr .S,Bras .A n aree Y. Carriere, and E.A. Bernays, M.S., Singer, mlnc,A . ags . yr .A n oes M.D. Srygley Bowers, and L.A. Dyer, J., Vargas, A.M., Smilanich, M.S. Singer, and T. R. Chase, L., Sprung, P. A., Mason, A.M., Smilanich, G.L. Gentry, and L.A. Dyer, A.M., Smilanich, oe,S,Cte,S . ipo,S . e,K .adWlo,K. Wilson, and and K.P. Lee, D. S.J., Raubenheimer, Simpson, S.C., S.C., Cotter, S., Cotter, Povey, A.J., Holmes, S.J. K., Simpson, and Wilson, C. F., Behm, Ponton, K., Wilson, C., Fromont, F., Lalubin, F., Ponton, ezio,R n aea,C . Jr C.A., Janeway, and R. Medzhitov, W. J. Mattson, ipo,S . il,R . e,K . emr .T n abnemr D. Raubenheimer, and S.T. Behmer, K.P., Lee, R.M., Sibly, S.J., Simpson, D. Raubenheimer, and S.J. Simpson, cwrz .adKel,J.C. Koella, and A. Schwartz, abnemr .adSmsn S.J. Simpson, and D. Raubenheimer, abnemr .adSmsn S.J. Simpson, and D. Raubenheimer, S.J. Simpson, and D. Raubenheimer, S.J. Simpson, and D. Raubenheimer, cuebr,H,Krz . oe,Y n iaJty M.T. Siva-Jothy, and Y. Moret, J., Kurtz, H., Schulenburg, F. A.H. Sperling, and B.C. Schmidt, P. Schmid-Hempel, P. Schmid-Hempel, M.T. Siva-Jothy, and J. Rolff, Am. Nat. quality fromresistanceagainstparasitoids:dietchoicebyageneralistcaterpillar. 4 plasticity: increasedingestionofplanttoxinsbyparasitizedcaterpillars. diluted nutrientsleadstotoxicallelochemicaldose. affects immunefunction. Behav. balancing andtoxindilutioninforagingbyageneralistinsectherbivore. Ecol. Lett. Immunological costofchemicaldefenceandtheevolutionherbivoredietbreadth. prophylaxis inthemealwormbeetle, Nature Press. Curr. Opin.Immunol. protein dietaryeffects onmovementandthe generalisedimmunocompetenceof plant selectionbyapolyphagouscaterpillar? use byapolyphagouscaterpillar. 1311. Optimal foragingwhenregulatingintakeofmultiplenutrients. parasite challenge:acaseofself-medicationinhoneybees? Annu. Rev. Entomol. southern Tanzania. 25 sephadexbeadsbyfield-caught caterpillar Grammiaincorrupta. ingested secondarymetabolitesonthe immuneresponseofapolyphagous caterpillar. Complex effects ofparasitoidsonpharmacophagyanddietchoice ofapolyphagous 1440. and otherputativedefensesaseffective predictorsofparasitism. Ecol. protein costsofbacterialresistancebeoffset byalteredfeedingbehaviour? Insect Physiol. S.J. Simpson, reduction infecundity. (2011). Hostsusealteredmacronutrientintaketocircumventparasite-induced 2480. Syst. 402. behavior: thegeometryofnutritionaldecisions. feeding inthelocust. behavioural andphysiologicalcorrelatesofdietarybreadth. non-independence amongfoodcomponents. 1681. approach. 301 Ecological immunology. Syst. Entomol. variation andspeciesintegrityin Rev. Entomol. ecology. , e4796. , 472-475. .B,Lrh .D,Smsn .J n wr,G.A. Sword, and S.J. Simpson, P. D., Lorch, R.B., , 11 78, 437-446. 414 64, 629-643. , 119-161. (2002). Proc. Biol.Sci. 164, 423-429. 12, 612-621. Entomol. Exp.Appl. Oecologia , 506-506. The JournalofExperimentalBiology(2014)doi:10.1242/jeb.093716 (1980). Herbivoryinrelationtoplantnitrogencontent. 50, 529-551. 59, 130-137. 33, 613-634. Parasites andtheBehaviorofAnimals (2013). Integratingnutritionandimmunology:anewfrontier. (2003). Variation inimmunedefenceasaquestionofevolutionary (2005). Evolutionaryecologyofinsectimmunedefenses. J. Med.Entomol. 26, 183-211. Anim. Behav. 10, 12-15. 165 Int. J.Parasitol. 270 Philos. T Physiol. Entomol. , 995-1005. , 357-366. (1992). Caterpillarscompensatoryfeedingresponseto (2002). MelanizationofplasmodiumfalciparumandC- (2003). Adaptiveinnateimmunity?Responsive-mode (2003). Invertebrateecologicalimmunology. (1981). Thenutritionalecologyofimmatureinsects. 91, 67-82. J. Chem.Ecol. rans. R.Soc.B Oecologia (2003). Doesanti-parasitoiddefenseexplainhost- (2001). Immunedefenceinbumble-beeoffspring. (2001). Howforagingtacticsdeterminehost-plant 45, 953-964. Grammia 39, 84-88. Anopheles gambiae 41, 43-50. Tenebrio molitor (2004). Organismalstoichiometry:quantifying (1998). Anancientsystemofhostdefense. (2003). Nutrientbalancingingrasshoppers: (2008). WidespreaddecouplingofmtDNA (1999). Integratingnutrition:ageometrical (1993). A multilevelanalysisoffeeding- (1993). Thegeometryofcompensatory 27, 206-212. (2012). Increasedresincollectionafter 129, 98-105. Oikos (2002). Short-termnutrientdeprivation Ecology tiger (:Noctuidae). (2002). Theinterplaybetweennutrient (2009b). Theinsectimmuneresponse 37, 239-245. (2009). Self-medicationasadaptive 364 Philos. Trans. R.Soc.B Entomol. Exp.Appl. 100, 554-562. , 3-14. 85, 1203-1216. . Proc. Biol.Sci. . Oxford:OxfordUniversity (2004). Disentanglingfood (Diptera: Culicidae)from J. Exp.Biol. PLoS ONE Anim. Behav. (2009). Introduction. (2011b). Effects of (2009). Immediate Ecology Annu. Rev. Ecol. (2009). Canthe 65, 171-186. 7 PLoS ONE 270 206 , e34601. 342 90, 1434- 68, 1299- (2009a). (2011a). J. Anim. Science , 2475- , 1669- (2004). 2259 , 381- Annu. Anim. J.

The Journal of Experimental Biology hmsn .N n ea,R.A. Redak, and S.N. Thompson, M.R. Strand, trmn .O n igr M.S. Singer, and J.O. Stireman, 2260 RESEARCH ARTICLE Physiol. A in aninsect Immunology 588-600. parasitoid assemblageofanexophyticpolyphagouscaterpillar. Entomol. migrating Mormoncrickets 34, 663-668. 191 (ed. N.E.Beckage),pp.25-48.Amsterdam:Elsevier. Manduca sexta , 909-923. (2008). Insecthemocytesandtheirroleinimmunity. In Anabrus simplex L. andalterationsinducedbyparasitism. (2005). Feedingbehaviourandnutrientselection (2002). Spatialandtemporalvariationinthe (Orthoptera: Tettigoniidae). Ecol. Entomol. J. Comp. Insect Ecol. 27, aaoo R.T. Yamamoto, M. Boots, and A.B. Pedersen, H.J., Tidbury, L.W. Wang, and R.A. Redak, S.N., Thompson, u,M n ter A.M. Stoehr, and M. Zuk, Manduca sexta blood nutrientcompositionandmediatesparasitedevelopmentalsuccess: pupation. immune priminginaninsecttoaDNA virus. 635. Suppl. 4,S9-S22. J. Econ.Entomol. The JournalofExperimentalBiology(2014)doi:10.1242/jeb.093716 L. parasitizedby (1969). Massrearingoftobaccohornworm.2.Larvaland (2002). Immunedefenseandhostlifehistory. 62, 1427-1431. Cotesia congregata Proc. Biol.Sci. (2011). Withinandtransgenerational (2005). Hostnutritiondetermines (Say). 278 J. Exp.Biol. , 871-876. Am. Nat. 208 , 625- 160

The Journal of Experimental Biology