*Author ([email protected]) for correspondence University, Halifax,NS, CanadaB3H4R2. Canada B3H4R2. 1970 Received 3February 2014;Accepted5March2014 1 One mechanismthatappearstobeacommonrouteleading to opposed torelyingona‘magicbullet’ approach(Adamo,2013). multiple mechanismssimultaneouslytoalterhostbehaviour, as Adamo, 2012;Lefèvreetal.,2009).Mostpathogensappearto use remains poorlyunderstood(Adamo,2013;vanHouteetal., a limitednumberofpotentialmoleculartools. context ofvirusesastheytendtohavesmallgenomes,andso have behavioural control.Thisproblemisespeciallyinterestingin the how evolutionhassolvedtheneurobiologicalproblem of evolutionary pressureprovidesuswiththeopportunitytodiscover pressure onthevirustomanipulateitshost.Theexistenceofthis Burand etal.,2012).Inthesesystems,therewillbeintenseselection to beanimportantelementinsuccessfulviraltransmission(e.g. 2013). Forsomeviruses,certainchangesinhostbehaviourappear Viruses areknownmanipulatorsofhostbehaviour(vanHouteetal., behaviour, STI,Sexuallytransmittedinfection KEY WORDS:Parasiticmanipulation,Sicknessbehaviour, Sexual We identifiedtheinsectiridovirusIIV-6/CrIV asapathogenofthe Shelley A.Adamo A viralaphrodisiacinthecricket RESEARCH ARTICLE © 2014.PublishedbyTheCompanyofBiologistsLtd|JournalExperimentalBiology(2014)217,1970-1976doi:10.1242/jeb.103408 behaviour. be necessaryforanypathogenthatisspreadbyhostsexual behaviour isabsent.Curtailmentofahost’s sicknessbehaviourmay IIV-6/CrIV infection,theimmunesignal(s)thatinducessickness protein productionbythefatbody. Theevidencesuggeststhatduring as wasphenoloxidaseactivity, suggestingareductioninimmune absent ininfectedcrickets.Total haemolymphproteinwasreduced, behaviour thatisinducedbyanactivatedimmunesystem,was Sickness behaviour, theadaptivereductionoffeedingandsexual studies showthattheviruscanbespreadthroughsexualcontact. benefits fromthecontinuedsexualbehaviourofitshost;transmission were quickertocourtfemalesthanuninfectedcontrols.Thevirus females continuedtomatewheninfected.Infact,infectedmales infected malesshowedlittleornomotility. Nevertheless,malesand by thevirus,althoughspermtakenfromspermatophoresof the testisofinfectedmalessuggeststhatwasnotinvaded oviducts emptyofeggs;thefemaleswereeffectively sterile.EMof hypertrophied, buteggproductionwithered,leavingthelateral immune functionandlipidstorage.Duringinfectionthefatbody attacks thefatbody, anorganimportantforproteinproduction, polymerase chainreaction(PCR)analysis.EMshowedthatthevirus INTRODUCTION ABSTRACT Department of Psychology andNeuroscience, DalhousieUniversity, of Department Halifax,NS, How viruses,oranyotherpathogen/parasite,alterhostbehaviour texensis 2 Department of MicrobiologyandImmunology, of Dalhousie Department 1 , IlyaKovalko using electronmicroscopy(EM)and 1 , RussellH.Easy 1 and DonStoltz Gryllus texensis suppress theexpression ofthehost’s sicknessbehaviour. example, adecreaseintheproduction ofhostimmuneproteinscould placed toalterimmune/neural communicationinitshost.For all majorimmunetissuesofthe cricket.Therefore,thisvirusiswell also infected(Kleespiesetal.,1999); inotherwords,thisvirusinvades (Chapman, 1998).Thehaemocytes, theimmunecellsofblood,are resistance becauseitproduces proteinsvitalforimmunefunction storage organ forlipid(Chapman, 1998).Itisalsocriticalfordisease body isthemajorsiteofproteinproductionininsects,aswell asa are arrangedinaparacrystallinearray(Williams etal.,2005).Thefat iridescent bluesheen,duetothelarge numberofviralparticlesthat body istheprimarytarget (Just andEssbauer, 2001).Ittakesonan iridovirus isolate(CrIV)(Jakobetal.,2002)]attackscrickets.The fat (Williams etal.,2005).Onetypeofiridovirus [IIV-6; cricket probably commoninfieldpopulations,andcancauseepizootics reptiles andamphibians(Williams etal., 2005).Thevirusesare icosahedral, cytoplasmicDNA virusesthatattackinvertebrates, Gryllus texensis behaviour intheirhost. host sicknessbehaviourinordertomaintainorincreasesexual transmitted pathogensprobablyneedtosuppresstheexpressionof behaviour [e.g.crickets(Jacotetal.,2004)].Therefore,allsexually , animmunechallengeresultsinareductionsexual system istheeffect ofthevirusonhostsicknessbehaviour. Inmost Burand andTan, 2006).Onemechanismnotyetexploredinthis methods tomanipulatehostsexualbehaviour(Burandetal.,2005; details remaintobedetermined,thevirusappearsusemultiple viral transmission(Burandetal.,2005).Althoughthemechanistic nudivirus 2showincreasedsexualbehaviour, leadingtoenhanced (Burand etal.,2004).Femalesinfectedwith not increasehostfitness.Itis,however, vitalforviraltransmission (Burand etal.,2004).Therefore,continuedsexualbehaviourdoes Helicoverpa zea 2012). Someofthesevirusescausehoststerility;forexample,the manipulation ofbehaviour. behaviours mayreduceviraltransmissionand/orinterferewith behavioural responsestoinfection(i.e.sicknessbehaviours)asthese host–pathogen systemsitmaybeimportanttopreventhost pathways maybeasmallevolutionarystep(Adamo,2013).Insome cytokine production).Hijackingimmune/neuralcommunication selection tointerferewithhostimmunefunction(e.g.byaltering by parasiticmanipulators,becauseallparasitesareunderheavy immune/neural connectionsmaybeespeciallypronetodisruption help animalsrecoverfrominfection(Hart,1988).Such behaviour areknowncollectivelyas‘sicknessbehaviours’,andthey invertebrates (Adamo,2008).Theseimmune-inducedchangesin behaviours inbothvertebrates(Dantzeretal.,2008)and cytokines) caninduceand/orsuppressawiderangeofhost communication signalsinthehost(Adamo,2013). altered hostbehaviouristheinterruptionofimmune/neural In thispaperweexaminedtheeffect ofaniridovirusonthecricket Some virusesaretransmittedbysexualcontact(e.g.Burandetal., Signalling moleculesproducedbytheimmunesystem(e.g. 2 nudivirus 2sterilizesitsmothhost, (Cade andOtte,2000).Iridovirusesarelarge Helicoverpa zea Helicoverpa zea

The Journal of Experimental Biology suggested that there wasnogrossdecrease insize,although ovaries containedfewornodeveloping follicles. lateral oviductshold~120eggs (AdamoandBaker, 2011). The females hadfewerthan10eggs perlateraloviduct).Normally, oviducts typicallyhadfeworno eggs(N females weresexuallyactive( The spermathecaewerefullofsperm,demonstratingthatinfected haemolymph frominfectedcrickets( particles werenotfoundinthecellsoftestis. In somecells,virionsformedsmallparacrystallinearrays.Viral particles werealsofoundinthetrachealepithelialcellsandmuscle. 1).Thefatbodycellswereheavily packedwithvirions.Viral (Fig. confirmed thepresenceofalarge (~150 iridovirus (Williams etal.,2005).Electronmicroscopy(EM) The bluesheenofthefatbodysuggestedpresencean RESEARCH ARTICLE Identity of thevirus Identity of RESULTS Viral effects on reproductive tissuein onreproductive effects Viral viral proteins,includingthemajorcapsidproteinofIIV-6/CrIV. protein (E confirmed itsidentityas DNA extractedfromthefatbody. SequencingofthePCRamplicon major capsidproteinofthisvirusproducedasingleamplicon from with CrIV avariantofIIV-6 (Jakobetal.,2002)].Primersforthe type 6(IIV-6) [theyareconsideredtobe the samespeciesofvirus, tentatively identifiedascricketiridovirus(CrIV)orinsect Kleespies etal.,1999;Williams etal., 2005),theviruswas were nocommonbacterialorfungalpathogenspresent. produced nogrowthonnutrientagarplates,suggestingthatthere In contrast,observationsofthe testesofinfectedmalecrickets No otherpathogenswerefound.Culturingoffatbodyor Given theEMresults,andfromdescriptionsinliterature(e.g. value=0.0). Crickethaemolymphwasfoundtocontain Gryllus bimaculatus N =18/18 infectedfemales).Thelateral N =6 femalesand =18 infectedfemales;all G. texensis G.

nm) icosahedralvirus iridovirus majorcapsid N =4 males) spermatophores ofcontrolmales( observation period.Incontrast,spermliberatedfromthe ( from theremainsofspermatophoreduringsametimeperiod. females) crickets(two-wayANOVA, F to, theremainsofcrushedspermatophoreduring10 F F control cricketsasestimatedbyfemurlength(two-wayANOVA, P P P between infectedanduninfected crickets(Mann–Whitney, There wasnosignificantdifference inanti-predatorbehaviour with aninfectedfemale. However, one maleremaineduninfectedevenafterhecopulated females, twobecameinfectedaftermatingwithfemales. contact). Ofthefouruninfectedmalesthatwereplacedwithinfected merely courtedbythatmale(courtingincludesclosephysical including onethatdidnotcopulatewithaninfectedmale,but was infected afterphysicalcontactwithmalesduringsexualbehaviour, six uninfectedfemalescourtedbyinfectedmales,threebecame The fatbodyappearednormalandwasdevoidofviralDNA.Of the None ofthesentinelanimals( little ornomotility( infected malesheldsperm(4/4males).However, thespermshowed testicular sizewasnotquantified( females ( Behavioural effects of thevirus of Behavioural effects Evidence for sexual transmission N =0.67). Therewasalsonodifference insizebetweeninfectedand =0.38; samplesizesasshownabove). 1,45 1,45 =0.5; control There wasnodifference inlivemassbetweeninfectedcrickets =13 males, =0.10, =22.8, N =28) wereheavierthanmales( The JournalofExperimentalBiology(2014)doi:10.1242/jeb.103408 P P =0.38; sexF virus. Fig. <0.0001; nosignificantinteraction, N

=18 females)anduninfected( N .CytoplasmoffatbodycellinfectedwithIIV-6/CrIV 1. =12, infected The scalebarrepresents500 N =4), withthespermremaininginside,orclose 1,45 =0.78, N =4) showedevidenceofinfection. N N =29). Examining locomotion P N =0.38; interaction =3) movedvigorouslyaway =7). Thespermatophoreof 1,45 N =0.06, =21; two-wayANOVA,

nm. N P =8 males, =0.80), although F F 1,45 1,45 U =0.19, =0.77, =150, N 1971

=10 min

The Journal of Experimental Biology Fg 3; (Fig. (Kruskall–Wallis=0.65, infected females( injected crickets comparison, nosignificantdifference betweencontrolandsham- 1972 RESEARCH ARTICLE t immune challengedwithheat-killed infection ofthecolony( controls haemolymph (349.8±12.02 Infected cricketshadsignificantly lesstotalproteinintheir Total haemocytecountdidnotdiffer betweeninfected( crickets (N There wasnoevidenceofillness-inducedanorexiaininfected N during theirinitialpresentationaswereuninfectedcrickets[50%, Infected malecricketswereaslikelytocourtfemales(43.8%, H the samecolonyrun6 significantly whencomparedwithcontrol,uninfectedcricketsfrom N and uninfectedcrickets(Mann–Whitney, separately, wefoundnodifference inlocomotionbetweeninfected challenged cricketstestedpriortotheinfectionenteringcolony. infection enteringthecolony. Pre-con+Bac wereuninfected,immune- con+Sh wereuninfectedsham-challengedcricketstestedpriortothe uninfected cricketstestedpriortotheinfectionenteringcolony. Pre- bars denotemeans,andtheerrorrepresents.e.m.Pre-conwere Fig. unchallenged controls(N longer tocourtfemalescomparedwithsham-injected( males challengedwithheat-killed Mann–Whitney producing courtshipsong)thanuninfectedmales( three trials,infectedmales( Total haemolymphproteinandphenoloxidaseactivity Haemocyte count Physiological effects anorexia) infection onsickness behaviour (illness-induced of Effect infection onsexual behaviour of Effect uninfected ( 30 =12, infected =6; =0.57, =3.96, There wasnosignificantdifference inthetimerequiredfor

.Infectedmaleshaveashorterlatencytosingthancontrols. 2. G F -test (1d.f.)=0.07,P Latency to sing (s) 1000 P 3,52 P N 200 400 600 800 =0.75 (Adamoetal.,2013)]. =0.0004). =21) evenafterinjectionofheat-killedbacteria( =11). Healthy cricketsthatweretestedpriortothe N =0.12, 0 =8) crickets( N Control U=0.001, =29). Infectedanduninfectedcricketsdidnotdiffer q =0.25, P =0.95, sham-injectedcrickets

N months previously[N P N =12) (F Infected =5) tomountacourtingmale q =0.73) comparedwithcontrols( =16) significantlyreducedfeedingwhen =4.6, 002,Fg 2).However, uninfected P=0.0025, Fig. t 13 N

mg =1.13, =7) hadashorterlatencytocourt(i.e. =0.79]. Taking theaveragevalueof 2,33 P ml >0.05) (Fig. ertamarcescens Serratia r-o Pre-con Pre-con =7.6, − P 1 S. marcescens , =0.28). N P =46) thandid controls =0.002; Tukey’s multiple U =160, +Sh =23; Kruskal–Wallis 2). N P =11, uninfected ( =0.70; control Pre-con N +Bac =16) (Fig. ( N =12) took N N N =7) and =12) or =8). N N The N =11) =16) =5, 3; challenged cricketstestedpriortotheinfectionenteringcolony. infection enteringthecolony. Pre-con+Bacwereuninfected,immune- uninfected; inf,infected).Pre-conwereuninfectedcricketstestedpriortothe against theaveragetimeuninfectedcricketsspentfeeding(notinf, Bars denotemeansanderrorbarss.e.m.Allvalueswerenormalized Fig. denote meansand errorbarsrepresents.e.m. Fig. activity wassignificantlylowerininfectedcricketsevenwhen (570.5±21.33 significantly lowerininfectedcrickets( (Chapman, 1998).Total phenoloxidaseactivitywasalso phenoloxidase activity, anenzymeimportantformelanization wounds thancontrols.Thisobservationledustomeasure anecdotally thatinfectedcricketstooklongertomelanizetheir crickets (

.Infectedcricketsshownoevidence of illness-inducedanorexia. 3. .Infectedcricketshave lowertotalphenoloxidaseactivity. 4.

Control (not inf) Normalized time spent feeding N 0.5 1.0 1.5 =55; 0 The JournalofExperimentalBiology(2014)doi:10.1242/jeb.103408

Tyrosinase equivalents (µg) mg 0.2 0.4 0.6 0.8 t 0 108 ml Control (inf) =2.44, − 1 , N Control =67) (t Sham (inf) P 006 i.4).Total phenoloxidase =0.016; Fig. 111

=7.99, Bac (inf) Infected N

P Pre-con =55) thanincontrol <0.0001). We noted

Pre-con+Bac Bars

The Journal of Experimental Biology haemolymph suggests thattheamountofvitellogenin availableto fat body(Chapman,1998).The steepdeclineintotalproteinthe haemolymph offemalesisvitellogenin, ayolkproteinmadebythe making hostproteins.Oneof themostabundantproteinsin the hostfatbodytowardsmaking viralparticlesandawayfrom this resultisconsistentwiththe hypothesisthatthevirusredirects haemolymph aremadebythefat body(Chapman,1998).Therefore, infected animalsthanincontrols.Mostoftheproteinsinsect under naturalconditions. Therefore, hostsexualbehaviourmaybeimportantfortransmission cricket ( 2001) foundnosignofinfectionintheovariesandtestesarelated et al.,2005).Furthermore,JustandEssbauer(JustEssbauer, the hypothesisthatthisvirusisnotverticallytransmitted(Williams corpses. We foundno evidence ofinfectionthetestes,supporting the fieldbecauseofcompetitionfromotherscavengersforcricket other (Adamoetal.,2010),butthismaybeanuncommonevent in (Williams andHernandez,2006).Crickets willcannibalizeeach and Dambach,1989).Iridovirusescanalsobespreadbycannibalism behaviour onlyoccurswithinthecontextofsexual(Loher other duringaggressivebehaviour(Williams etal.,2005),butthis 1992), andthisspeciesisasocial.Malesmaybeabletoinfecteach 2005). Cricketdensitiesareusuallylowinthefield(CadeandCade, and theseparticlesloseactivityrapidlyondrysoil(Williams etal., Humidity levelsareoftenlownearAustin(TX,USA)(Ward, 2010), from viralparticlesshedintotheenvironmentmaybeuncommon. viral transmissionmayberareinthiscricket.Forexample,infection cricket hosts(e.g.Kleepsiesetal.,1999).Otherpossiblemodesof for infection.ThisisconsistentwithotherpapersonIIV-6 and merely beinginthesameroomasinfectedanimalsisnotsufficient with noevidenceofviralDNA in theirfatbody, suggestingthat for thisvirus(Williams etal.,2005).Oursentinelsremainedhealthy, mouth. 1994), andthiscouldtransferparticlesfromtheantennaeinto antennae withtheirmouthpartsduringcourtship(AdamoandHoy, other’s cuticle(AdamoandHoy, 1994).Cricketsgroomtheir example, bothmalesandfemalesbrushtheirantennaeovereach 2003). Cricketscontacteachotherfrequentlyduringcourtship;for behaviour, iscapableoftransmittingthevirus(alsoseeHunteretal., phenotype islikelytoenhanceviraltransmission. indistinguishable fromthatofcontrols.Thisnewhostbehavioural behaviour criticalforhostsurvival(Adamoetal.,2013),was infected animals.However, hostanti-predatorbehaviour, a produce asimilarresult–continuationofsexualbehaviourin behaviour wascurtailed(Fig. courtship singingsoonerthancontrols(Fig. 2013), iscapableofalteringhostbehaviour. Infectedcricketsbegan showed thatIIV-6, likesomeotherinsectviruses(vanHouteetal., iridescent virus,type6(IIV-6, cricketvariantCrIV).We also able toidentifythepathogeninourcricketcolonyasinsect Using bothmolecularmethodsandelectronmicroscopy, wewere corrected fortotalproteincontentintheblood( RESEARCH ARTICLE U baseline valuesthandidinfectedcrickets( had greaterinducible N DISCUSSION animals (Pearson’s r between totalproteincontentandphenoloxidaseactivityininfected =58 infected, =28.5, Total proteinconcentration inthehaemolymphwaslower Our resultssuggestthatphysicalcontact,akeyfeatureofsexual Per os P Gryllus bimaculatus)infectedwiththesamevirus. =0.005). infection isthoughttobethemainrouteof N =30 control).Therewasasignificantcorrelation 2 =0.40, in vitro P

=0.001, 3). Thesetwochangeswouldtendto phenoloxidase activityrelativeto N =58). Controls(N N

2). Moreover, sickness =12; Mann–Whitney, t 86 =5.29, P =20) also <0.0001, significant differences betweenthetwopopulations. differ intheirmatingbehaviour, suggesting thattherewereno historical controlsandtheuninfected animalsfromOttawadidnot colony) aswellwithuninfected animalsfromOttawa.The controls (datacollectedfromanimals priortotheinfectionof behaviour, we comparedourinfectedcolonyanimalswithhistorical between thetwopopulationsof crickets.Forexample,withsexual However, wedonotbelievetherewereany significantdifferences these animalsweredescendedfromcricketsourlabcolony. group weresometimesobtainedfromadifferent colony, although an unusualsubsetofthepopulation.Similarly, cricketsinourcontrol pathological andnotlikelytobetheresultofvirusselecting for (e.g. totalphenoloxidaseactivity, Fig. values beyondtherangeoffoundwithinourcolonyanimals any directviraleffects. However, infectedanimalsoftenhadassay Such asubsetmightbedifferent fromcontrolcricketsregardlessof infected animalscouldrepresentanon-randomsubsetofourcolony. Therefore, itisunlikelythatincreasedmatingbenefitsmales. Infected maleshavelowmotilityspermandmaybeinfertile. storing spermfrompreviousmatings(LoherandDambach,1989). unlikely toincreasetheirfitness,especiallyastheyarecapableof because infectedfemaleshavefeweggs,continuedmatingis female crickets(Adamo,1999;Shoemakeretal.,2006).However, an increaseinreproductionpriortodeath,isknownoccur terminal reproductiveinvestment.Terminal reproductiveinvestment, female sexualbehaviour, isunlikelytobeahostresponse,i.e. discussion ofthisissue(Poulin,2010)]. to specificallysuppresshostsicknessbehaviour[seePoulinfora is neededtodeterminewhethertherepositiveselectionforthevirus of effects onfatbodyproteinsneededforhostsurvival.Moreresearch effects couldleadtoaugmentationofthesechanges,withadiminution effect ofthevirus’s attackonthefatbody, positiveselectionforthese However, evenifthedeclinesinsicknessbehaviourbeganasaside- pre-adapts itfortheabilitytosuppresshost’s defenceagainstit. molecules importantforimmunefunction(Chapman,1998),probably behaviours inthehost. proteins suchascytokines,thiscouldexplainthedeclineinsickness immune systemfromproducingimmune-relatedpeptidesand this stateprobablybenefitsthevirus.Ifvirusinhibitshost’s induction ofananti-viralstate(KempandImler, 2009).Preventing infection cantriggercytokinereleaseininsects,leadingtothe inducing thesamephenomenaininvertebrates(Adamo,2008).Viral vertebrates (Dantzeretal.,2008),andarethoughttobeinvolvedin Cytokines areresponsibleforinducingsicknessbehavioursin made bythefatbodyand/orhaemocytes(Kamimura,2012). cytokines (e.g.immuneactivatorslikeENFfamilypeptides)are may alsohelpexplainthelackofsicknessbehaviour. Ininsects, in theproductionofimmune-relatedcompoundsbyfatbody therefore, thisdeclinewouldprobablybenefitthevirus.The being made.Phenoloxidaseisvirucidal(ShelbyandPopham,2006); was significantlyreduced,suggestingthatlessprophenoloxidaseis during infectionisunknown,buttotalphenoloxidaseactivity(Fig. Whether theproductionofallhostproteinswasequallysuppressed results providesomeindirectevidencesuggestingamechanism. infection becausetheyrelylessonproteinsmadebythefatbody. made withouttheyolkprotein.Thetestesmaybelessaffected by reduction couldexplainthelossofeggproduction;eggscannotbe the ovariesisprobablyalsogreatlyreducedduringinfection.This Because theinfectionofourcolonywasunintentional, the The increaseinmalesexualbehaviour, andthecontinuationof The abilityofthisvirustoattackthefatbody, akeyproducerof We donotknowhowthevirusaltershostbehaviour, butour The JournalofExperimentalBiology(2014)doi:10.1242/jeb.103408

4). Thesevaluesare 1973

4)

The Journal of Experimental Biology unless otherwisenoted. during mid-photophase. performed onlastinstarnymphs.Allbehaviouraltrialswereconducted field (MurrayandCade,1995).Illness-inducedanorexiastudies were Wyatt, 1984;Solymarand Cade,1990)butstillwithintheirlifespaninthe after themoulttoadulthood.Atthisagetheyaresexuallymature(Cade and Council onAnimalCare.Adultcricketswereusedbetween8and21 Dalhousie University(no.I11-025) andareinaccordancewiththeCanadian cycle. AllstudieswereapprovedbytheAnimalCareCommittee of 1974 RESEARCH ARTICLE from thesamegenetic pool.ThecricketsfromDr Bertram’s colonywere colony toherlaboratory about2 from DrBertram’s colonyweredescended fromanimalsshippedour Bertram’s colonyatCarletonUniversity inOttawa,Canada.Theanimals colony wasquarantined.Uninfected animalswereobtainedfromDrS. females exhibitedahypertrophicfat bodywithaniridescentbluesheen.The Some femalesinourcolonystopped layingeggs.Ondissectionthese during rearing.Cricketswererearedat25±2°Cona12 generations. Pelletsofdrycatfoodandwaterwereprovided TX, USA,andhavebeenmaintainedasalaboratorycolonyformany Crickets (long-winged (Adamo, 2014). suppression ofsicknessbehaviourmaybewidespreadinSTIs and Immerman,2003;Antonovicsetal.,2011), suggestingthatthe asymptomatic thanotherdiseases(Lockhartetal.,1996;Mackey pathogen reproduction.Inmammals,STIsaremorelikelytobe STI bypreventingadeclineintheavailabilityofresourcesfor behaviour, suchasillness-inducedanorexia,mightalsobenefitan STI likeIIV-6/CrIV. Suppression ofotheraspectssickness Therefore, sicknessbehaviourswoulddecreasetransmissionofan determinant oftransmissionforSTIs(Webberley etal.,2006). opportunity tocontactanothercricket.Matingrateisakey males tocourtanyfemalethatwasattracted,furtherreducingthe results showthatimmuneactivationalsodecreasestheabilityof mating partnerstheanimalattracts(GerhardtandHuber, 2002).Our campestris). A reductionincallingeffort willreducethenumberof immune activationreducescallingeffort inmalecrickets( animals. Forexample,Jacotetal.(Jacotal.,2004)foundthat a matingpartnerbutalsoreducestheeffort ofinfected transmission ofanySTI(Adamo,2014). individuals tosecurematingpartners–anabilitythatiskeyforthe sickness behaviourmaybecriticalfortheabilityofinfected will notattractmates(KnellandWebberley, 2004).Suppressionof Knell andWebberley pointout,ahostthatbehavesasifitweresick mating withconspecificscontagiousdiseases(Able,1996).As very sensitivetothestateofaprospectivemate’s health,andavoid of matesinfectedwithSTIsissurprisingasanimalsaretypically and Martin,2012),vertebrates(Dassetal.,2011)]. Thepopularity uninfected controls[(KnellandWebberley, 2004;Goodacre infected withSTIsareabletoattractevenmorematingpartnersthan with non-STIs(deRoodeandLefèvre,2012).Infact,someanimals STIs, eventhoughsomeinsects(e.g.ants)avoidindividualsinfected evidence thatinsectsavoidmatingwithconspecificsinfected selected toavoidinfectedconspecifics.Nevertheless,thereisno fertility (KnellandWebberley, 2004);therefore,animalswillbe (Lockhart etal.,1996).STIscommonlyresultinreductions (Knell andWebberley, 2004;Burandetal.,2012)andotheranimals Source of infected animals of Source Animals MATERIALS ANDMETHODS Chemicals wereobtainedfromSigmaChemicalCo.(StLouis,MO,USA) Sickness behaviournotonlymakesanindividuallessdesirableas Sexually transmittedinfections(STIs)arewidespreadininsects G. texensis

years previously, andweretherefore ) wereoriginallycollectednearAustin,

h/12 h light/dark ad libitum Gryllus

days were transferredto aclean,freshcontainer(17×15×9.5 (http://blast.ncbi.nlm.nih.gov/Blast.cgi) withoverlappingregionsremoved. Homologous sequenceswereidentifiedusingBLAST searches to editsequencedataandproducecontiguousalignments. and Bioedit(http://www.mbio.ncsu.edu/BioEdit/bioedit.html) wereused amplicon wassentforsequencing.Mega5(http://www.megasoftware.net/) were treatedwithExoSap(Affymetrix, www.affymetrix.com) anda510 following amplificationwiththeWeinmann primers.PCRproducts in 0.5×Tris base/boricacid/EDTA buffer solutionandstainedwithgelgreen (see Weinmann etal.,2007).PCRproductswererunona1.5%agarosegel for atleast1 TGATTTGG-3′ the majorcapsidproteingenewerePCRFOR:5 extension at72°Cfor10 primer, 0.2 cacodylate buffer containing250 used asatemplatein30 following themanufacturer’s instructions.Approximately300 frozen at saline (PBS)andexaminedusingphasecontrastmicroscopy. Sampleswere opened usingfinedissectingscissorsinabout200 spermatheca containedspermweredetermined.Spermatophores fat body, numberofeggsinthelateraloviducts,andwhether dissected understerileconditions.Duringthedissection,colourof Fat body, spermatheca(females),spermatophoreandtestes(males)were primary fixative(3%glutaraldehyde/1%acroleinin50 Tissues tobeprocessedforthinsectioningweredissectedoutintothe confirm theiruninfectedstatus. to preventinadvertentinfection.TheseanimalsweretestedusingPCR held undersimilarconditionstoourinfectedcolonybutinaseparateroom and uninfectedcricketswereisolated intosinglecontainers(17×15×9.5 placed infectedmaleandfemalecrickets withuninfectedcrickets.Infected To testwhethertheviruscould alsobetransmittedviasexualcontact,we NaCl, 10 pipetter andanice-coldtipflushedwithanti-coagulant[0.15 collected understerileconditionsfromcricketsusinga10 polyacrylamide gelelectrophoresis.A 4 Viral proteinswereidentified throughsodiumdodecylsulphate haemolymph andplatedtheseonnutrientagarplates. contributing totheinfection,weasepticallyharvestedfatbodyand using a12HRdigitalHamamatsuC4742-51camera. were takenusingaJeolJEM1230electronmicroscopeoperatingat80 was inepon/aralditeresin,followingdehydrationacetone.Micrographs followed byovernightstainingin0.1%aqueousuranylacetate.Embedment temperature for2 buffered saline(PBS)and frozen at supersaturated solution)].Thesampleswereplacedin12 phenothiocarbamide andproteaseinhibitorcocktail(enoughtoform a cycles of1 Cycling wasperformedunderthefollowingconditions:5 polymerase (Fermentas,http://www.thermoscientificbio.com/fermentas/). PCR formolecularidentificationofthevirus Electron microscopy virus Identification of Evidence for sexual transmission inhaemolymph proteins viral Identification of (Proteomics CoreFacility, DalhousieUniversity). band visibleinthelanesofinfectedsampleswassentoutforsequencing Proteins werevisualizedbysilverstaining(SwainandRoss,1995).A strong run inMini-PROTEANTGXPrecastGels(Bio-Rad)at150 The tubeswerelefttoincubateinboilingwaterfor3 diluted 1:2usingloadingbuffer withdithiothreitolandphenylthiocarbamide. DNA was extractedfromthawedsamplesusingaDNAeasykit(Qiagen) To determinewhetheranycommonbacterialorfungalpathogenswere − mmol

80°C untiluse. min at94°C,1 mmol The JournalofExperimentalBiology(2014)doi:10.1242/jeb.103408

day priortothemating trial.Bothmaleandfemale crickets andPCRREV: 5′

l

− l − 1

1 h. Post-fixationwasin2%OsO4thesamebuffer, EDTA, 10 dNTPs, 1.5 min. Theforwardandreverseprimersequencesof

min at50°C,and2 μl PCRreaction,including0.4 mmol mmol -TTTTGACGTGGTGCAGTTTGAAC-3

mmol

l

l − − − 1 1 80°C untiluse.Eachsamplewas glutathione andafewcrystalsof MgCl μl sampleofhaemolymphwas

l − 1

min at72°C,followedbyfinal sucrose) andleftatroom 2 , 1×PCRbuffer and2 μl ofphosphate-buffered

min. Thesampleswere ′ -CCATTACATTTAA -

cm) separatedby a

mmol μl ofphosphate- μmol

min at95°C,30

ng ofDNA was

V for90 μl automatic

l l − − 1 1 sodium of each

mol U min.

cm)

Taq kV,

bp l − 1 ′

The Journal of Experimental Biology from bothsexeswerepooled. sex differences inplusmazeperformance(Adamoetal.,2013);therefore,data scoring thetrialswasblindtoinfectionstatusofcricket.Thereareno spaces. Theapparatuswascleanedwithdisinfectantbetweentrials.person the timeitspentundercoveredarmstoassessitstendencyavoidopen cricket spentlocomotingasaproxyforbehaviouralactivity. We alsomeasured tendency toexploreitsenvironment.We measuredtheamountoftimeeach of timeseachcricketenteredanarmtheplusmazeasassessmentits 2012) includingcrickets(Niemeläetal.,2012).We alsomeasuredthenumber response associatedwithpredationavoidanceacrosstaxa(StynoskiandNoble, called thistimeperiod‘freezing’.Freezingisastereotypicanti-detection then removedandwemeasuredthetimecricketremainedmotionless.We uncovered area.Theyremainedunderthetransfercupfor1 Crickets weregentlytransferredfromtheirhomecagetothecentral, covered withheavyblackbristleboard,creatingdarkspacesunderneath. al., 2008). injection ofheat-killed deprived offood,butnotwater, for2 previously usedprocedure(Adamo etal.,2010).Infectedcricketswere activation incrickets(Adamoetal.,2010).Theexperimentfollowed a could expressillness-inducedanorexia,arobustresponsetoimmune normal behaviouruntilclosetodeath.We testedwhetherinfectedcrickets Anecdotal observationssuggestedthatvirus-infectedcricketsshowed uninfected cricketswereisolatedintosinglecontainers(17×15×9.5 behaviour inmaleandfemaleinfecteduninfectedcrickets.Infected We assessedthelatency(timetosinging)anddurationofcourtship an opencentralarea(8×8×6.5 constructed ofblackacrylic.Itconsistedfourarms(14×8×6.5 et al.,2013).Briefly, theplusmazewasshapedlikeasignand A modifiedplusmazewasusedtoassessanti-predatorbehaviour(seeAdamo above. time. TheharvestedfatbodywastestedforvirususingPCRasdescribed mating trialswithinfectedcrickets.Theirfatbodywascollectedatthesame sentinels. Theywerehousedinthesameroom,butdidnotparticipate (Kleespies etal.,1999).Fourcrickets(twomaleandtwofemale)actedas Kleespies etal.foundthatcricketsbegantodie2 the fatbodyfromcrickets10 mounting wasalsonoted,asthetimeofcopulation,ifany. We collected until themalebegantosing(i.e.latencysinging).Thetimeofeachfemale divider wasremoved.Thetimetofirstcontactrecorded,asthe infected femaleswerepairedwithuninfectedmales( divider. Infectedmaleswerepairedwithuninfectedfemales( pellet intheother, andtheamount oftimetheyspentfeedingover the placed ononeside ofanexperimentalchamber(17×15×9.5 this (Adamoetal.,2010). Onehourafterinjection,cricketswere heat-killed injection ofsterilewater, oractedasanunhandled control.Injectionsof at least1 a 2 healthy malecricketstoproducecourtshipsong.Malewere given transferred toaclean,freshcontainer(17×15×9.5 further 40 RESEARCH ARTICLE lipid release)90 time pointwaschosenbecausethereisaclearphysiologicalresponse (i.e. were givenmatingtrialswithuninfectedfemalesasdescribedabove. This sterile water, oractedasanunhandledcontrol.Ninetyminuteslater, males no matingafter40 mounting wasalsonoted,asthetimeofcopulation,ifany. Iftherewas as wasthetimeuntilmalebegantosing.Theofeachfemale After 1 Effect ofinfectiononsicknessbehaviour(illness-inducedanorexia) Effect ofinfectiononsexualbehaviour Effects ofinfectiononanti-predatorbehaviour Behavioural effects We alsotestedwhetheranimmunechallengeaffected thelatencyof μl injectionofheat-killed min thedividerwasremoved.Thetimetofirstcontactrecorded,

day priortothematingtrial.Bothmaleandfemalecricketswere min. S. marcescens

min afterinjectionofheat-killed

min, cricketswerere-testedwithafreshpartnerfor S. marcescens are knowntoinduceillness-induced anorexiain

days afterthecourtshiptrialsdescribedabove.

S. marcescens cm). Duringthetrial,twoopposingarmswere (approximately 1×10

days. Theywerethengivena2 (approximately 1×10

cm) separatedbyadivider. S. marcescens

weeks afterinfection N =4). After1

min. Thecupwas

4 cm) withafood cells) ora2

cm each)and (Adamo et N 4 =6), and cells) in

min the cm) for μl μl tube containing20 measure. A further15 spontaneous activity(Adamo,2004)andthistubeprovidesabaseline to atubecontaining20 challenge assesses theamountofactivationprophenoloxidaseduringanimmune added to18 potential activity. Another15 active form(Adamo,2004).Thistubeprovidesanestimateofthemaximal dissolved inwater. Thechymotrypsinconvertsprophenoloxidaseintothe mushroom tyrosinasewererunaswellonthesameplate. showed thatthiswaswithinthelinearrangeofassay. Standardsusing in absorbanceat490 1984). than thiscannot be adequatelytestedforanormal distribution(Meddis, groups hadasamplesizelessthan6; Meddisargue thatsample sizes smaller tests werealsousedforanalyses inwhichatleastoneofthetreatment analysed usingnon-parametricstatistics (Meddis,1984).Non-parametric distributed dataweretestedwithparametric statistics.Non-normaldatawere summary anti-predatorscore.Data weretestedfornormality;normally 15 removed asdescribedabovefrombothinfectedanduninfectedcrickets.A Bidochka etal.(Bidochkaal.,1989).A 4 bovine serumalbumen. absorbance wasreadat590 reagent ina96-wellplate.After15 of thehaemolymph/salinemixturewasthenaddedto180 phosphate-buffered salineandimmediatelyvortexedfor5 uninfected crickets.Thehaemolymphwasaddedto60 haemolymph wasremovedasdescribedabovefrombothinfectedand To measurethetotalproteincontentofhaemolymph,4 therefore, resultsfrommalesandfemaleswerepooled. Haemocyte numberdoesnotdiffer betweenthesexes(Adamo,2010); 1 a 10 μlsamplewasplacedonFuchs–Rosenthalcountingchamberwithin transferred to200 cocktail (enoughtoformasupersaturatedsolution)].Haemolymphwas glutathione andafewcrystalsofphenothiocarbamideproteaseinhibitor with anti-coagulant[0.15 crickets usinga10 Haemocyte countwasobtainedbyremoving4 animals. al., 2010).Theresultswerealsocomparedwithdatacollectedonuninfected induced anorexia,soinformationonthetwosexeswaspooled(Adamoet study (Adamoetal.,2010).Adamoal.foundnoeffect ofsexonillness- 30 sphericity ( (Kaiser–Meyer–Olkin=0.46) andahighlysignificantBartlett’s testof validity ofthePCA wasshownbythemeasureofsamplingadequacy (score=2.1) anditexplained70%ofthetotalvariance( 2013). OnlythefirstcomponentofPCA hadaneigenvalueabove1 the timespentincoveredarms)asdescribedpreviously(Adamo etal., measures (locomotion,timespentfreezing,thenumberofarmentries and components analysis(PCA)wasperformedontheplusmazebehavioural Prizm (v. 5.0b,GraphPadSoftware,LaJolla,CA,USA).A principal Statistics wereperformedusingIBMSPSS(v. 19)andGraphPad to 180 temperature at10,000for5 20 Total haemolymphproteinandphenoloxidaseactivity Haemocyte count function immune –evidencePhysiological effects for alterationof Statistics

min ofcollection.Cellswerecountedusingphase-contrastmicroscopy. Phenoloxidase activitywasmeasuredusingamethodmodifiedfrom μ min trialwasrecorded.Thesetimeswerechosenbasedontheearlier min. Aftertheincubationperiod,tubeswerecentrifugedatroom l sampleofthehaemolymph/salinemixture(seeabove)wastransferred μl of0.02 in vitro P μl ofwaterand2 The JournalofExperimentalBiology(2014)doi:10.1242/jeb.103408 <0.0001). Thefirstcomponentof thePCA wasusedasa mol . Thetubeswerelefttoincubateatroomtemperaturefor μl ofice-coldanti-coagulantandgentlymixed,then μl ofbovinepancreasalphachymotrypsin(1.2 μl automaticpipetterandanice-coldtipflushed

l −

μl ofthehaemolymph/salinemixturewasaddedtoa nm wasmonitoredfor20 μl ofwater. Crickethaemolymphrarelyexhibitsany 1 L -Dopa ina96-wellplate.Aftermixing,thechange

mol min. A 30 μlsampleofsupernatantwasadded

nm. Eachassaywasrunwithstandardsof μl ofthehaemolymph/salinemixturewas μl ofheat-killed l − 1 NaCl, 10

min incubationwiththereagent, μ mmol l sampleofhaemolymphwas

S. marcescens. Thistube μl ofhaemolymphfrom min. Preliminarystudies

l − 1 EDTA, 10 N =23 crickets).The

s. A 10 μl sample μl ofBradford μl ofice-cold

mmol mg μl of 1975 ml

− l − 1 1 )

The Journal of Experimental Biology hpa,R.F. Chapman, ae .H n te D. Otte, and W. H. Cade, ae .H n yt,D. Wyatt, and W. H. Cade, uad .P,Tn . i,W,Njm,S n olf,W. Roelofs, and S. Nojima, W., Kim, W., Tan, J.P., Burand, 1976 RESEARCH ARTICLE Council ofCanada)grantstoS.A.A.andD.S. This studywasfundedbyNSERC(NaturalSciencesandEngineeringResearch EM studyandhelpededitthepaper. the PCRanalysisandcriticallyreadpaper. D.S.performedandanalyzedthe PCR analysis,andcriticallyreadthepaper. R.H.E.designedandhelpedperform the paper. I.K.helpeddesignandperformthebehaviouralassays,performed S.A.A. generatedthehypothesis,designedstudy, analyzedthedataandwrote The authorsdeclarenocompetingfinancialinterests. using LC-MS/MS. behavioural trials.We thankA.Cohenforassistancewithproteinidentification We thankJ.Beach,K.Bradley, E.FairnandN.Parsonsforassistancewith ae .H n ae E.S. Cade, and W. H. Cade, Rock, and Z. Lu, G.F., Kutish, E.R., Tulman, C.L., Afonso, W., Kim, J.P., Burand, uad .P,Rli,C .adTn W. 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