implications fordiseasespreadinage-structuredpopulations epidemic spreadofinfection. consideration withrespecttoboththeirimmunesystemsandthe that invertebratedefencecapabilitieshaveanontogenymerits change overtime.Together, ourexperimentsleadustoconclude observations thatreinforcetheideaimmunityin © 2014.PublishedbyTheCompanyofBiologistsLtd|JournalExperimentalBiology(2014)217,3929-3934doi:10.1242/jeb.111260 Received 28July 2014; Accepted28August2014 *Author ([email protected]) for correspondence EH9 3JT, UK. Laboratories,Edinburgh, KingsBuildings, West Ashworth MainsRoad, Edinburgh Biology, Evolutionary BiologicalSciences, University of Institute of School of considered tobeapreciseadaptation. et al.,2003;Solomon,1971),andsothephenomenoniswidely just asoffspring antibodyproduction beginstoincrease(Grindstaff fish), butinmostcasesitappearsthatmaternalantibodiesdisappear varying degrees(forexample,formonthsinhumansanddays in (Grindstaff etal.,2003).Maternal antibodiespersistinoffspring to defence againstpathogensandtoconditiontheirimmunesystem 1971), andthereforerelyuponmaternallyderivedfactorsboth for (Brambell, 1970;Lawrenceetal.,1981;Roitt1998;Solomon, appear tohavelittleabilitysynthesiseantibodiesendogenously is muchlesscapablethanthatofadults.Specifically, neonates It iswellestablishedthattheimmunesystemofnewbornvertebrates Invertebrate KEY WORDS:Ontogeny, Immunity, Age,Epidemiology, ability of host–pathogen system.Inaseriesofexperiments,weshowthatthe hosts ofdifferent agesinanexperimentalinvertebrate disease spread.Thus,wesoughttodeterminethesusceptibilityof with agestructureinpopulations,haswidespreadimplicationsfor Yet, age-specificcapacityfordefenceagainstpathogens,coupled the ontogenyofdefenceininvertebratesismarkedlylessstudied. Immunity invertebratesiswellestablishedtodevelopwithtime,but Jennie S.Garbutt*,AnnaJ.P. O’Donoghue,SeannaJ.McTaggart, PhilipJ.Wilson andTom J.Little The developmentofpathogenresistancein RESEARCH ARTICLE a smalldoseofthepathogen(‘priming’)persistsfor7 juvenile period,andthattheprotectiveeffect ofpriortreatmentwith effect ofrestrictedmaternalfoodpersiststhroughouttheentire epidemiology. We alsoshowthatapreviously observed protective mechanism bywhichecologicalcontextcanaffect disease structure ofDaphnia tends todecline.Becausemanyecologicalfactorsinfluencetheage daphnid wouldbemostheavilyinvestinginreproduction,resistance with time.Immediatelyfollowingthis,ataboutthetimewhena magna observations indicatethatresistanceincreasesearlyinthelifeof it difficult todrawgeneralconclusions,butthemajorityof ramosa INTRODUCTION ABSTRACT , consistentwiththeideathatdefencesystemdevelops magna changes withhostage.Clonaldifferences make populations, ourresultshighlightabroad to resistitsnaturalbacterialpathogen D. magna Daphnia,

days, can D. 2011). Consequently, bodysizeisthoughttobe closely tiedto individuals tobeginaninfection inthehaemolymph(Duneauetal., orally, afterwhichitpenetratesthegutoroesophagusofsusceptible infection inthissystemisacommon one–thepathogenisingested to belinkedbodysizeinthe earlystagesoflife.Theroute off afterthefirstweekoflife),developmentresistance islikely larger ( resistance willthendecline.(2)Asolderindividualsinevitably be conceivably showimmunosenescence,leadingtotheprediction that at leastintheearlystagesoflife.Laterlife,however, to thepredictionthat hypothesised thattheimmuneresponsewilldevelopwithage,leading could modulatediseaseresistanceandspread. provide insightintohowecologicalfactorsthataffect agestructure Empirical dataonthesusceptibilityofdifferent ageclassescould epidemic spread(Castillo-Chavezetal.,1989;Duffy etal.,2012). 1998), andtheimpactofagestructureonvirulenceevolution and age structure(Charlesworth,1994;DeRoosetal.,2003;Sterner, has beencarriedoutonthecausesandconsequencesofvariationin specific susceptibilityfordiseasespreadinpopulations.Muchwork opportunity toshedlightonthepotentialconsequencesofage- the immuneresponse. .We assumedthatpathogenresistanceislinkedto rather wemeasuredtheirresistancetoapathogen,thebacterium control. Thetraitwemeasuredisnotanimmuneresponseperse,but the light onthedevelopmentofdefencesystemaninvertebrate, an invertebrate’s life.Themainaim ofthepresentstudywastoshed Zerofsky etal.,2005),butfewstudieshaveincludedthefirstdaysof Karlsson, 2012;Sorrentinoetal.,2002;Whitehorn2011; Laws etal.,2004;Lesser2006;Piñera2013;Prasaiand al., 2002;Eleftherianoset2008;Felix2012;Kurtz, have receivedconsiderableattention(Adamoetal.,2001;Doums the immunesystemlaterinlife(andimmunosenescenceparticular) ontogeny ofimmunityearlyinthelifeinvertebrates.Changes Little, 2011). Whathasbeenlittlestudied,however, isthebasic 2014; Littleetal.,2003;MitchellandRead,2005;Stjernman modified viamaternaleffects (Ben-Amietal.,2010;Garbutt challenges frompathogens(McTaggart etal.,2012),orcanbe there isevenevidencethatitdevelopsthroughtimeinresponseto innate immunesystemofinvertebratesissurprisinglycapable,and ontogeny similartothatofyoungvertebrates.Yet, ingeneral,the immune system,wouldnotshowansystemordefence tempting toassumethatinvertebrates,whichpossessonlyaninnate full useoftheirinnateimmunesystem.With thisinmind,itis remains undevelopedorunder-developed, youngindividualswillhave system. Itisgenerallyacceptedthat,evenasacquiredimmunity in earlylifeiscenteredonthedevelopmentofacquiredimmune We testedseveralindependent hypotheses.(1)Mostgenerally, we Additionally, bymeasuringpathogenresistance,wegaingreater Understanding ofthedevelopmentvertebrateimmunesystem Daphnia Daphnia magnaStraus,undertightexperimental have indeterminategrowth,although growthtapers Daphnia will becomemoreresistantwithtime, Daphnia : could 3929

The Journal of Experimental Biology supplementary materialFig. genotypes, susceptibilitydeclinedearlyinlife(days0–5;Fig. × ageeffect (Table clones didnotrespondinthesameway, leadingtoasignificantclone these experimentsareavailableinsupplementarymaterialTable S1. development ofresistancewithlongerexposuretimes).Datafrom assume morenatural,exposureregime(experiment4:the resistance). Finally, werepeatedexperiment1withalonger, andwe pathogen (experiment3:primingandtheearlydevelopmentof resistance), andwithvariationinexperienceofexposuretothe (experiment 2:maternaleffects ontheearlydevelopmentof Daphnia environmental contexts,i.e.howresistanceintheearlystagesof age observedinthefirstexperimentwasconsistentdifferent sought toestablishwhetherthepatternofchangeinresistancewith levels ofresistancemightdeclinewithagelaterinlife.Next,we evaluate boththeearly-lifedevelopmentofresistanceandhow of itsadulthood(Ebert,2005a).Thus,wewereabletopotentially history, includingtheentirejuvenile periodandasubstantial spread over40 follows the 3930 generalised linear models.L–R exposure) inadditiontostudyinginfection atdifferent ages.Output isfrom included additionaltreatments(maternal foodandprevious‘primary’ All experimentsusedmultiplehostgenotypes (clones).Experiments2and3 marginally non-significant( (designated FS24andGG4;seeMaterialsmethods)infectedwas The changewithageintheproportionofhostsfromtwoclones resistance) exposed defence in We performedaseriesofexperimentstoevaluatetheontogeny older individualswillbelesssusceptibletoinfection. thus couldlaunchmorerobustimmuneresponses,whichpredictsthat however, thatolder, larger individualswillhavemoreresources,and substantial, willbemoresusceptibletoinfection.(3)Itisplausible, that olderindividuals,atleastinthefirstweekoflifewhengrowthis quantities offood,andthuspathogenspores.Thishypothesispredicts resistance inthissystembecauselarger individualstakeuplarger RESEARCH ARTICLE exposure ofhosts proportion of Table xeiet4Coe23. <0.0001 35.3 2 <0.0001 38.7 <0.0001 3 107.1 <0.0001 Clone 2 29.5 Experiment 4 1 Clone Experiment 3 Clone Experiment 2 Clone Experiment 1 Experiment 1: the development of resistance Experiment 1:thedevelopment of Experimental overview RESULTS 1. Statisticaldetailsoffourexperimentstestinghowthe χ life developedundervariationinmaternalnutrition 2 Daphnia. Thefirst(experiment1:thedevelopmentof distribution. Daphnia days, whichrepresentsmuchofthiscrustacean’s life Effect g 22<0.0001 0.073 32.2 22.3 7 14 0.05 0.003 7.81 <0.0001 0.0016 <0.0001 <0.0001 13.8 37.3 21.4 0.07 3 21.4 35.4 0.035 3 Clone ×age 2 0.006 Age 4.42 13.0 6 1 6 Clone ×dayofsecondary 19.8 7 Clone ×primary 1 Day ofsecondary 7 Primary exposure Clone ×age Age Maternal food Clone ×age Age

1; supplementarymaterialFig. Daphnia hosts infectedisdependentupontheageat P χ =0.07; T 2

is thelikelihood-ratio teststatistic,which S1), butafterday5,changesof to theparasiteateighttimepoints be1 i.1 1,Fig. able ..L–Rχ d.f. ); however, thetwo 2

S1). Inboth Probability

1; as the0and10dayolds. one offourages(indays)earlyinlife. Themothersofthese and arethestandard errorsofthepredictedvaluesfor each clone. the generalisedlinear model.Errorbarsshowthevariation betweenclones, the meanproportionofinfected raised undereitherhighfood(HF)or lowfood(LF).Eachdatapointrepresents predicted proportionof clone. Notethat30and40 between clones,andarethestandarderrorsofpredictedvaluesforeach predicted bythegeneralisedlinearmodel.Errorbarsshowvariation represents themeanproportionofinfected pathogen predicted proportionof Fig. Fig. treatments (i.e.underbothhighandlowmaternalfood;Table in adaphnid’s life,andthispattern wasconsistentacrossmaternal Consistent withexperiment1,susceptibilitydeclinedageearly to fallduringthesameperiod(supplementarymaterialFig. 5 today40whilsttheproportionofcloneGG4hostsinfectedappears proportion ofcloneFS24hostsinfectedappearstoincreasefromday susceptibility withagediffered betweenthetwogenotypes: resistance development ontheearly of effects Experiment 2:maternal

.Maternaleffectsontheearlydevelopmentofresistance. 2. .Thedevelopmentofresistancein 1. Mean predicted proportion infected Mean predicted proportion infected Pasteuria ramosa 0.35 0.40 0.45 0.50 0.55 0.60 0.65 0.70 0.30 0.40 0.50 0.60 0.70 The JournalofExperimentalBiology(2014)doi:10.1242/jeb.111260 HF LF 0 1 D. magna 0 1 D. magna

day old at oneofeightages(indays).Eachdatapoint

Daphnia HF LF

infected followingexposureto 3 infected followinga4 Day ofexposure Day ofexposure Daphnia

5 10 across threeclones,aspredictedby Daphnia Daphnia magna. were exposedonthesameday

3 5

20 3040 across bothclones,as

h exposuretothe

HF Daphnia The mean P. ramosa

LF The mean

S1). were at

1,

The Journal of Experimental Biology RESEARCH ARTICLE The abilityof infected significantlychangedwithage(Table In thisexperiment,weagainfoundthattheproportionof hosts control andprimedindividualsexposedatthesameage). ‘primed’ treatmentwerelesslikelytobecomeinfected(comparing al., 2012),wasalsoseenhere(Table pathogen (i.e.priming)improvesresistance(McTaggart et A patterndocumentedinanotherstudy, thatpreviousexposuretothe Kc200a andKc49a),butthe‘humped’ relationshipincloneFS24. decrease insusceptibilitywithagethreeoutoffourclones(GG4, supplementary materialFig. a clone×ageofsecondaryexposureinteraction(Table of theparasite;Table treatments (i.e.withandwithoutpreviousprimingalowdose declined, andthispatternwasconsistentacrossprimaryexposure Consistent withtheotherexperiments,susceptibilityinitially 2005; StjernmanandLittle,2011), wasalsoobservedhere. (Ben-Ami etal.,2010;Garbutt2014;MitchellandRead, pattern, thatlowmaternalfoodimpartshighparasiteresistance appears absentfromathirdclone,KB5A.A previouslyobserved from day0to5inclonesFS24andGG4,whilethispattern (Table 2).Inthisexperiment,therewas alsoaclone×ageinteraction Fig. and arethestandarderrorsofpredictedvaluesforeachclone. the generalisedlinearmodel.Errorbarsshowvariationbetweenclones, the meanproportionofinfectedDaphnia (filled circles)oracontrolexposure(opencircles).Eachdatapointrepresents time pointsfollowingaprimary(andnon-infective)exposuretothepathogen proportion ofD.magna Fig. substantially with hostage,asindicatedby the significantmain DISCUSSION exposure times withlonger resistance Experiment 4:thedevelopment of resistance Experiment 3:primingandthedevelopment of susceptibility appearstodecline again. clones showanincreaseinsusceptibility onday5,afterwhich early inlifeclonesFS24and Kc49a,butnotcloneKc200a.All supplementary materialFig. found anear-significant (

.Primingand thedevelopmentofresistance. 3.

1; supplementarymaterialFig. Mean predicted proportion infected 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 0.50 0.55 2 D. magna infected afterexposuretoP. ramosa

,Fg 3).Inthisexperiment,therewasalso 1, Fig. Day ofsecondaryexposure

4 P

S3), probablyreflectingtheverystrong =0.073) clone×ageeffect (Table

S4). Susceptibilityappearstodecline to resistP. ramosa

7 across fourclones,aspredictedby

1, Fig. S2). Infectionlevelsdeclined

2 3) –individualsinthe The meanpredicted

,Fg 4),andalso 1, Fig.

4 7 at threedifferent can change

1; 1; the experiment thatmirrorsexperiment4,aside fromexposure declining afteraninitialrise,as itdoesinexperiment1(whichis include days5to10,when we mightexpectresistancetobe other experiments)andthus,for example,the‘day5’ exposures used longerexposures(5 (Fig. week; Figs P. ramosa observations consistentlyindicatethatresistancetothepathogen many genotypes. a completecharacterisationofthiswillrequirethestudy great revealed that,althoughresistanceclearlychangeswithage,gaining we emphasisethatitisastrengthofthecurrentreportto have require theextensivecollectionoffurtherdata,butformoment immunocompetence laterinlife)?Answerstosuchquestions reproduction (andhavethus‘traded-off’ earlylifetraitswith have exceptionallystronginvestmentinearly-liferesistance or clone GG4inexperiment1;seesupplementarymaterialFig. do clonesthatshowpatternsofapparentimmunosenescence(e.g. strikingly different patternsofage-relatedresistance.Forexample, to gatherfurtherdataonthedifferent genotypesthatfollowsuch may bemaintainedinnaturalpopulations.Itwouldintriguing × ageinteractionindicatesamechanismbywhichgeneticvariation favoured atdifferent times,orindifferent locations,this genotype 2006). Assumingthat,inthewild,different strategiesmightbe with age(Felixetal.,2012;KhanandPrasad,2013;Lesser for immunosenescenceorwhereimmunocompetenceincreases the currentresults,therealsoexistexamplesofgeneticvariation 2012; Whitehornetal.,2011; Zerofskyetal.,2005),but,aswith al., 2002;Kurtz,Lawset2004;PrasaiandKarlsson, with advancedageininvertebrates(Adamoetal.,2001;Doums supported thetheorythatimmunocompetencegenerallydecreases might reflectimmunosenescence.Studiesonotherorganisms have genetic variationforalate-lifedeclineindefensiveability, which conclusions. Forexample,ourdatahighlightthatthereisextensive variation weobservedmakesitdifficult todrawgeneral × ageinteractions(Table effect ofageinthreefourexperiments,andtheprevalentclone Fig. and thusthedayshownisfirstofarelativelylongexposureperiod. ramosa proportion of Despite thestrongclone-specificeffects, aslightmajorityof

4 . Thedevelopmentofresistancewithlongerexposuretimes. 4), butitisimportanttobear inmindthatthisexperiment

at oneofeightages(indays).Eachexposureperiodlasted5 Mean predicted proportion infected 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 0.50 increases intheearlieststagesoflife(roughlyfirst

D. magna The JournalofExperimentalBiology(2014)doi:10.1242/jeb.111260 1–4). Thispatternwaslessclear inourexperiment4 0 1 infected followingexposuretothepathogen days, comparedwithjusthours forthe

1). However, theamountofgenetic

3 Day ofexposure

5 10

20 3040 P.

days, 3931 The

S1)

The Journal of Experimental Biology clones varyindefence abilitylaterinlife. in theirinter-clutch interval, andthisvariationmayexplainwhy Again, clonestendtovaryinhow quicklytheyreachmaturity, and understanding oftheinterplaybetween immunityandreproduction. age-specific virulence,and an importantcontributiontoour system isfecundityreduction, this wouldessentiallybeastudyof age isstillrequired.Giventhat themaineffect ofinfection inthis designed tocorrelatereproductionandimmunityacrosstime and 2013; StjernmanandLittle,2011), thoughastudyexplicitly between reproductionandresistanceinthissystem(Schlotz et al., lines ofevidencesupportaconnection(or, perhaps,atrade-off) reproduction isapproximatelywhensusceptibilityhighest. Other clutch intervalgraduallyincreases.Thus,themostintenseperiod of are released2–3 conditions) around3dayslater(Ebert,2005a).Subsequentclutches and theirfirstclutchisreleased(again,dependingon food chamber (dependingonfoodconditions)arounddays5to10of life, organisms. corresponds withthepeakofreproductiveactivityin these clones, sowegeneralisewithcaution.Thisriseapproximately Fig. and thetimingofrisevariedfromaround5 starting pointforfurtherexplorationofthesefactors. observations ofchangedresistancewithagerepresentanimportant the proportionofadults(vanLeeuwenetal.,1986).Our food availabilityaltersagestructure,withdecliningincreasing are likelytoaffect diseasedynamics;thereissomeevidencethat 3932 ecological factorsthataltertheagestructureof in populationsdominatedbyolder (Riessen andYoung, 2005)]willhavetheoppositeeffect, resulting presumably youngest,individuals[liketheinvertebrate population. Ofcourse,predatorsthatselectthesmallest,and changes intheagestructure,andthusresistancecompetence,of Duffy etal.,2005),fishpredationalsoaltersepidemicsizethrough Daphnia possible thatinadditiontotheselectivecullingbyfishofparasitised parasite predation hasbeenshowntolimitthesizeofepidemicsyeast Daphnia, whichtendtobemoresusceptibleinfection.Fish predation couldresultinpopulationsdominatedbyjuvenile individuals (Galbraith,1967;Gibson,1980).Highlevelsoffish fish, whichselectivelyremovesthelargest, andpresumablyoldest, prominent influenceonagestructureapparentlybeingpredationby age structureofdaphnidpopulationsiswellstudied,withthemost dependent ontheageandgeneticstructureofpopulation.The parameters, forinstancepathogenprevalence,willbestrongly low doseofthepathogen. development ofimprovedimmunityfollowingpriorexposuretoa defences candevelopinthisorganism, showingasitdoesthe The primingeffect observedinexperiment3reinforcesthat have amoredevelopedimmunesystem,thebestdefences. perhaps becausetheyhavemoreresourcesattheirdisposaland/or observations supporttheideathatolderandthereforelarger hosts, of thechangesinresistanceearlylife,butformomentour and feedingratewillberequiredtofullydisentanglethecause(s) because theyfeedmore.Simultaneousmeasurementofage,size Daphnia develops withtime,anddoesnotfittheideathatlarger compatible withtheideathatdefencesystemof time). Theobservationofincreasingresistanceearlyinlifeis RESEARCH ARTICLE Early declinesinsusceptibilityweregenerallyfollowedbyarise, The clearimplicationofourresultsisthatepidemiological 4) toaround10 Metschnikowia bicuspidata or reductionsinoverallhostdensity(Duffy andHall,2008; will bemoresusceptibletoanorallyacquiredpathogen Daphnia

days later, andafterthefirstfewclutches the inter- days (experiment1;Fig. typically begintodepositeggsinthebrood (Duffy etal.,2012);itisthus Daphnia. Likewise,other 1) and,again,between Daphnia

days (experiment4; populations Chaoborus D. magna design, butwith animportantextrasteptoaccount fortimeeffects. 30 and40 experimental animalsandwereexposed tothepathogenat0,1,3,5,10,20, ensure thateachreplicateisindependent (seeEbertetal.,1998). day. Acclimating allreplicatesforthreegenerationsisaprocessdesignedto when offspring wereobservedinthejaror, ifnonewerepresent,everythird culture, whichrepresentsabout5×10 cycle incontrolledclimatechambersat20°C. for threegenerationsunderstandardisedconditionsona12 population). Initially, 24replicatesofeachhostclonewereacclimatised FS24 fromtheKaimespopulationinScotland,andGG4German This experimentwasconductedontwo experimental infectionswiththissystem,infected Daphnia when sporesarereleasedfromdeadhostsandsubsequentlypickedupby is horizontal(transovarialtransmissionhasneverbeenobserved),achieved reproduction ininfectedhosts(Ebertetal.,1996).Transmission of The pathogen susceptible to. were exposedtoparasitesfromtheirownpopulationthatthey Gaarzerfeld populationfromGermany(Cariusetal.,2001).Allgenotypes (Auld etal.,2012;Auld2014)intheScottishBorders,and magna (Ebert, 2005b)(supplementarymaterialFig. through thecarapace,andbecauseinfected are easytodiagnosewiththenakedeyebecauseredsporegrowthisvisible found insmallfreshwaterponds. D. magna a countingchamber(Neubauer-improved, 0.1 and dilutedforuseinexposuresaccordingtodeterminationofdensityusing form asporesuspension.Sporesuspensionswerestoredfrozenat of primingafter8 (McTaggart etal.,2012)].Thisiscomparablewiththeobservations immediate effects andthose2 7 treatment withasmalldoseofthepathogen(‘priming’)extendsfor to granddaughters).We alsofoundthataprotectiveeffect ofprior potential fortheeffect tobetransmittedonegenerationfurther(i.e. maturity (thetimethateggsbegintodevelop)highlightsthe persists untilthetimethatdaphnidnearlyreachesreproductive through muchoftheirjuvenileperiod.Thatthematernaleffect 2014; MitchellandRead,2005;StjernmanLittle,2011) persists of food-restrictedmothers(Ben-Amietal.,2010;Garbutt particular, weshowthattheprotectiveeffect evidentintheoffspring experience ofpreviousexposureandtomaternalnutritionalstate.In spread andhost–pathogenevolutioninthisothersystems. effects andpriming–meritfurtherstudyasmajordriversofdisease effects ofthephenomenareportedhere–ageeffects, maternal and destabilisehost–pathogendynamics.We suggestthatthelinked that immuneprimingmayincreasethepersistenceofpathogen priming. Forexample,Tidbury etal.(Tidbury etal.,2012)showed implications forunderstandingtheepidemiologicalconsequencesof system isrelativelypersistent.Thispersistentprimingalsohasbroad moths (Tidbury etal.,2011) andshowsthatprimingoftheinnate Experiment 1: the development of resistance Experiment 1:thedevelopment of Organisms MATERIALS ANDMETHODS absorbance unitistheopticalof650 quantity duringthisperiodwas1absorbanceunitperjarday (1 spp., agreenalgaeculturedinchemostatswithChuBmedium. Food synthetic pondmedium(Klüttgenetal.,1994),andwerefedon

days [previousexperimentsinthissystemhadonlylookedfor Second-clutch offspring fromthethird-generation Our resultsalsoextendstudiesofhowresistancerelatesto genotypes fromtwopopulations:theKaimespondnearLeitholm during filterfeeding(Ebertetal.,1996).Thus,toperform days old.Theexperimentalset-up wasbasedonasplit-clutch (Crustacea: )isaplanktoniccrustaceancommonly The JournalofExperimentalBiology(2014)doi:10.1242/jeb.111260 P. ramosa

days inbeetles(Rothetal.,2009)and6 is aspore-formingbacteriumthatgreatlyreduces Pasteuria ramosa

days followingprimingexposure 6 D. magna algal cells).Mediumwaschanged

S5). Inthisstudy, weusedD. Daphnia mm, Marienfield).Thehost

nm lightbythe clones (aclonedesignated Daphnia Daphnia infections inDaphnia cease toreproduce Daphnia h:12 are crushedto were keptin h light:dark P. ramosa Chlorella Chlorella were the

days in −20°C,

The Journal of Experimental Biology 25 Daphnia in theoffspring generation. thus, thevariationinfoodleveloccurredmaternalgenerationand not at 1.0absorbanceunitsoffoodperdayfortheremainderexperiment; plate (CostarCorning,NY, USA),one acclimation perioddescribedabovefor25 jars andmaintainedunderthesameconditionsexperiencedduring measures onthesameindividuals. individuals (inasplit-clutchdesign)andthereforetherewerenorepeated offspring fromeachreplicate jartosetuptheoffspring orF day. Atthisstageoftheexperiment,therewere144 was changedwhenoffspring wereobservedor, alternatively, everythird to ahighfoodtreatment(1.5absorbanceunitsperjarday).Medium to alowfoodtreatment(0.3absorbanceunitsperjarday)andtheother from eachofthe24acclimatisedreplicatesforcloneandassignedone medium everyotherdayorwhenclutcheswerepresent. diagnosed. We recordeddailywhetheraclutchwaspresentandchanged hosts weremaintainedin60 which wereexposedtothepathogen.Thus,there576F RESEARCH ARTICLE added twofurther clones,designatedKc200aandKc49a, fromtheKaimes This experiment used thetwoclonesstudiedin firstexperiment,but their interactions. factor), ‘clone’ (athree-levelfactor),‘maternalfood’ (atwo-levelfactor)and (link=logit, dist=bin)andtheexplanatory variables‘age’ (afour-level We analysedtheproportionofhostsinfectedwithageneralisedlinear model generation) ofthefocalanimals(calledF design featureofthisexperimentwastoraisethemothers(calledF three generationsunderstandardisedconditionsasabove.Theessential studied. Again,24replicatesofeachhostclonewereacclimatisedfor but anadditionalclone,KB5A,fromtheKaimespopulation,wasalso This experimentwasconductedonthesametwo effects orsignificantinteractiontermsremained. were reducedbyremovingnon-significantinteractiontermsuntilonlymain factor), ‘clone’ (atwo-levelfactor)andtheirinteraction.Thisallmodels (link=logit, dist=bin)andtheexplanatoryvariables‘age’ (aneight-level We analysedtheproportionofhosts infectedwithageneralisedlinearmodel times). were 384Daphnia into different treatments(i.e.wereinfectedatdifferent ages).Intotal,there brood designmeantthatindividualssharedamotherwerealwaysplaced There werenorepeatedmeasuresonthesameindividuals,andsplit- allowed us,tosomedegree,disentangledayeffects fromageeffects. day 40exposuresweresetuponthesameas10exposures.This the day0exposuresand30onsameday, whilethe (though runthroughthesameacclimationprocess),suchthatwecarriedout exposures doneonday30or40weresetuppriortotheothermothers first sixageclasses,butmothersdestinedtocontributeoffspring tothe Specifically, eachreplicatemothercontributedoneoffspring toeachofthe received 7.5×10 (144×4 exposuredays).Forpathogenexposure,F or lowfoodconditionsandthenexposeF Corning) containing1×10 individually in2 5 these F × twofoodlevelsthreeclones=144 Experiment 3: priming and the development of resistance Experiment 3:primingandthe development of Analysis resistance development ontheearly of effects Experiment 2:maternal Analysis

days tothepathogen.Again,eachagetreatmentwascomposedofdifferent To setupthematernal(F Exposures werecarriedoutin2 day exposureperiodwerescoredaseither‘infected’ or‘uninfected’. 0 females, wetookfour(oneforeachexposureday:0,1,3,5) were checkedforsignsofinfectiondailyandattheend 5 spores for4

ml ofmediuminthewellsa24wellplate(Costar in thisexperiment(24replicates×2clones×8exposure 5 0 ) generation,wetooktwothird-clutchoffspring P. ramosa h, afterwhichtimetheywerereturnedto60

ml jarsfor30

ml ofmediuminthewellsa24well spores for4 Daphnia). Fromthethirdclutchof 1

days toallowinfectionsbe 1 Daphnia generation) undereitherhigh Daphnia

days followingexposure. D. magna 1 h. Followingexposure, Daphnia offspring wereplaced of ages0,1,3and Daphnia per well.Eachhost clones asabove, (24 replicates 1 generation, 1 were kept Daphnia

ml 0 (a three-levelfactor)andtheirinteractions. two-level factor),‘clone’ (afour-level factor),‘dayofsecondaryexposure’ (link=logit, dist=bin)andtheexplanatoryvariables‘primaryexposure’ (a We analysedtheproportionofhostsinfectedwithageneralisedlinearmodel secondary exposures,hostswereexposedto5×10 solution. After4 half ofthewells.Theotherreceivedsamevolumecontrol individual 2 Daphnia. Onthedayofprimaryexposure,each infected solution. Thesporesolutionwaspreparedasabove(i.e.fromcrushed infectious doseof500spores,withtheotherhalfbeingexposedtoacontrol were 7,9or12 occurred 2,4or7 primary exposurethatoccurredonday5,andalatersecondary studied thedevelopmentofresistance,butalsoincludedtwoexposures:a day ofbirthandassignedtoonesixtreatmentpaths.Thisexperiment of thethirdgenerationwereselectedatrandomfromeach acclimatised forthreegenerations.Sixoftheoffspring fromthethirdclutch population. Asabove,initially, 24replicatesofeachhostclonewere http://jeb.biologists.org/lookup/suppl/doi:10.1242/jeb.111260/-/DC1 Supplementary materialavailableonline at 095831). Trust totheCentreforImmunity, InfectionandEvolution(grantreferenceno. (NE/I026405/1). S.J.M.wassupportedbyastrategicawardfromtheWellcome This workwassupportedbytheNaturalEnvironmentResearchCouncil J.S.G. analysedthedataandwrotepaper. conceived ofthestudyanddesignedittogetherwithP.J.W. andA.J.P.O. T.J.L. and P.J.W., A.J.P.O. andJ.S.G.carriedouttheexperiments.T.J.L., S.J.M.andJ.S.G. The authorsdeclarenocompetingfinancialinterests. We thanktwoanonymous refereesfortheirhelpfulcommentsonthemanuscript. factor), ‘clone’ (athree-levelfactor)andtheirinteraction. (link=logit, dist=bin)andtheexplanatoryvariables‘age’ (aneight-level We analysedtheproportionofhostsinfectedwithageneralisedlinearmodel This experimentwasconductedonthree period andobservedforinfection28 exposure infectious dose,2,4or7 following exposure. checked dailytodetermineinfectionstatus(infectedornot)for25 maintained astheywereduringtheacclimationperiod.All ended onday5,etc.Followingexposure,hostsweremovedtofreshjarsand exposure lastedfromday0to4,the1beganonand dm,S . esn .adYugr M. Younger, and M. Jensen, S.A., Adamo, jars with5×10 identical toexperiment1,exceptthatexposureswerecarriedoutin60 Kc200a andKc49a)fromtheKaimespopulationinScotland.Methodswere Analysis References Supplementary material Funding Author contributions Competing interests Acknowledgements Analysis exposure times withlonger resistance Experiment 4:thedevelopment of 576 or control)×3secondaryexposuretimes]. offs between immunityandreproduction. immunocompetence inmaleandfemale For theprimaryexposure,halfof Daphnia Daphnia), whilethecontrolsolutionwascrusheduninfected Daphnia

ml well,andanon-infectiousdoseof500sporeswasaddedto The JournalofExperimentalBiology(2014)doi:10.1242/jeb.111260 [24 replicates×4clones2primaryexposurelevels(exposed 5 days old). spores andlastedfor5 h, theDaphnia days aftertheprimaryexposure(i.e.when were maintainedastheyduringtheacclimation

days aftertheprimaryexposure.Post-secondary were returnedtotheiroriginaljars.Forthe Gryllus texensis Anim. Behav.

days. Thus,forexample,theday0 days. Inthisexperimenttherewere D. magna Daphnia (2001). Changesinlifetime Daphnia 62, 417-425. clones (designatedFS24, were exposedtoanon- (formerly 5 spores, whichisan was placedinan Daphnia G. integer):trade- Daphnia Daphnia on the 3933

were days ml

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