01&-(+' 2*&3**+'4(5&5'$+6'#$.$5-&*5 ' · Faculté des sciences Secrétariat-décanat de Faculté Rue Emile-Argand 11 2000 Neuchâtel - Suisse Tél: + 41 (0)32 718 2100 E-mail: [email protected]

Impactofenvironmentonco-evolution

betweenhostsandparasites

THÈSEPRÉSENTÉEÀLAFACULTÉDESSCIENCES

UNIVERSITÉDENEUCHÂTEL

POURL’OBTENTIONDUGRADEDEDOCTEURÈSSCIENCES

PAR

ZellerMICHAEL

AcceptéesurpropositionduJury:

Prof.KoellaJacob,directeurdethèse,UniversitéNeuchâtel

Prof.FlattThomas,rapporteur,UniversitéLausanneProf.HelfensteinFabrice,rapporteur,UniversitéNeuchâtel

Soutenuele24juin2016

UniversitédeNeuchâtel

2016

Faculté des sciences

Secrétariat-décanat de Faculté Rue Emile-Argand 11

2000 Neuchâtel - Suisse Tél: + 41 (0)32 718 2100

E-mail: [email protected]

Imprimatur pour thèse de doctorat www.unine.ch/sciences

IMPRIMATUR POUR THESE DE DOCTORAT

La Faculté des sciences de l'Université de Neuchâtel autorise l'impression de la présente thèse soutenue par

Monsieur Michael ZELLER

Titre:

“Impact of environment on co-evolution between hosts and parasites”

sur le rapport des membres du jury composé comme suit :

- Prof. Jacob Koella, directeur de thèse, Université de Neuchâtel, Suisse - Prof. ass. Fabrice Helfenstein, Université de Neuchâtel, Suisse - Prof. Thomas Flatt, Université de Lausanne, Suisse

Neuchâtel, le 4 juillet 2016 Le Doyen, Prof. B. Colbois

5

Abstract

Manyparasitescausepathologyandmortalityandcanbeamajorsourceofselectionontheirhosts,creatingstrongselectionpressuresontheevolutionofhostsdefensestrategies.Changesinthehost’stoleranceandresistancetopathogencanstrongly influencethespreadofadiseaseandhenceinfluenceselectiononvirulence.Understandinghowecologicalconditionsinfluencethehost’slifehis-toryanddefensemechanisms,howtheyalterinfectiondynamicsandcontributetotheparasitesviru-lenceandtransmissionandhowtheyshapetheco-evolutionarydynamics isessential forgaining fur-therinsightsintohost-parasiteinteraction.

Inthisthesis,singlegenerationexperimentsandexperimentalevolutionwereusedtoexploretheim-pactof theenvironmentonhost-parasite interactionand theirevolution.Firstly, the roleof resourcevariabilityonthelifehistoryofthemosquitohostAe.aegyptiwasinvestigated.Secondly,therelation-ship between the growthof theparasiteV. culicisand thehealthof itsmosquito hostwas exploredacross ecological variables. The parasites growth rate and asymptotic load was estimated and com-pared in living and naturally dying hosts. Thirdly, theoretical predictions about the evolution of hostdefenseagainstparasitesweretested.Inparticular,theroleofresourceavailabilityontheevolutionofthehost’stoleranceandresistancetopathogenwasinvestigated.Finally,thisthesisexaminestheim-pactoftheenvironmentandco-evolvingparasitesonthehost’slifehistoryevolution.Theexperimentsintroduced here show that variable environmental conditions can influencemany central aspects ofhost-parasiteinteractions,onesthatplayimportantrolesinshapingevolutionarydynamics.Inthisthe-sis,theresultsofthoseexperimentsaredescribedindetailandtheirimplicationsforhost-parasiteco-evolutionarediscussed.

Overall,thisthesisemphasizesthecomplexityanddependencefromenvironmentalconditionsofhost-parasite interactionand theirevolution.Whenconsidering the spatialand temporalecologicaldiffer-encesofnaturalhabitats,theresultspresentedheremayhelptoleadtoamoreprofoundknowledgeofhost-parasiteinteractions.

Keywords

Aedesaegypti,co-evolution,epidemiology,experimentalevolution,host-parasite interactions,life-historyevolution,resourcevariability,Vavraiaculicis,virulence

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Résumé

Beaucoup de parasites provoquent des pathologies et de la mortalité et peuvent être unesourcemajeuredesélectionsurleurhôte,enappliquantunefortepressiondesélectionsurl’évolutionde leurs stratégies de défense. Les changements de tolérance et de résistance de l’hôte face aupathogènepeuventfortementinfluencerlapropagationd’unemaladie,etdoncinfluencerlasélectionde la virulence. Comprendre comment les conditions écologiques influencent l’histoire de vie et lesmécanismesdedéfensedel’hôte,commentellesaltèrentladynamiqued’infectionetcontribuentàlavirulenceetlatransmissionduparasite,etcommentellesfaçonnentladynamiquecoévolutiveestes-sentielpouravoirunemeilleurecompréhensiondesinteractionshôte-parasite.

Dans cette thèse, des expériences sur une seule génération et des expériences d’évolution expéri-mentaleontétéutiliséespourexplorer l’impactde l’environnementsur les interactionshôte-parasiteetleurévolution.Premièrement,lerôledelavariabilitédesressourcessurl’histoiredeviedel’hôte,lemoustiqueAe.aegypti,aétéinvestigué.Deuxièmement,lelienentrelacroissanceduparasiteV.culiciset la santée de son hôte moustique a été exploré sous différentes conditions écologiques.Troisièmement, le rôlede ladisponibilitédesressourcessur l’évolutionde la toléranceetde la résis-tancede l’hôteaupathogèneaétéexploré.Finalement, l’impactde l’environnementetdesparasitessur l’évolution des traits d’histoire de vie de l’hôte a été étudié. Les expériences introduites ici ontmontréquedes conditions environnementales variablespeuvent influencerbeaucoupd’aspects cen-traux des interactions hôte-parasite, en particulier ceux façonnant de manière importante la dy-namiqueévolutive.Danscettethèse,lesrésultatsdecesexpériencessontdécritsendétailetleursim-plicationspourlacoévolutionhôte-parasitesontdiscutées.

Plusglobalement,cettethèsesouligne lacomplexitéet ladépendanceauxconditionsenvironnemen-talesdesinteractionshôte-parasiteetleurévolution.Enconsidérantlesdifférencesspatialesettempo-rellesdeshabitatsnaturels,lesrésultatsprésentésicipeuventaideràconduireàuneconnaissanceplusprofondedesinteractionshôte-parasite.

Mots-clés

Aedesaegypti,évolutiondel’histoiredevie,co-évolution,épidemiologie,évolutionexperimen-telle,interactionshôte-parasite,variabilitéderesources,Vavraiaculicis,virulence

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Contents

Abstract..................................................................................................................................................5

Résumé...................................................................................................................................................7

ListofFigures........................................................................................................................................10

ListofTables.........................................................................................................................................11

Generalintroduction........................................................................................13Chapter1

EffectsoffoodvariabilityongrowthandreproductionofAedesaegypti.........25Chapter2

Context-dependentrelationshipbetweenparasitegrowthandChapter3

virulenceinamicrosporidia-mosquitointeraction...........................................39

TheroleoftheenvironmentontheevolutionoftoleranceandChapter4

resistancetoapathogen..................................................................................53

Antagonisticcoevolutionandresourcesalterthehost’slifehistoryChapter5

evolution.........................................................................................................65

Synthesisandfutureresearch..........................................................................79Chapter6

Acknowledgements..............................................................................................................................83

References............................................................................................................................................85

10

ListofFigures

Figure1:1Resourcesinhost-parasiteinteraction.................................................16

Figure1:2Mosquitolifecycle................................................................................20

Figure1:3LifecycleofVavraiaculicis....................................................................21

Figure2:1Mosquitogrowth..................................................................................31

Figure3:1:Sporeloadandprobabilityofinfection...............................................45

Figure3:2Parasitegrowthparameters.................................................................47

Figure3:3Longevityofinfectedmosquitoes.........................................................49

Figure4:1Toleranceandresistance......................................................................61

Figure4:2Tolerancevs.resistance........................................................................62

Figure5:1Ageandsizeatmaturity........................................................................72

Figure5:2Phenotypicplasticity.............................................................................73

Figure5:3Probabilityofemergenceandsporeload.............................................75

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ListofTables

Table2:1Statisticalsummaryforjuveniletraits……….……......................................31

Table2:2Statisticalsummaryforadulttraits………………………………………………..……33

Table2:3Repeatedmeasuresanalysisofclutchsizes.…………………………….………….35

Table3:1Sporeloadandprobabilityofinfection……..……………………………………..…46

Table3:2Parasitegrowthparameters……..…………………………………………………………48

Table3:3Statisticalsummaryofsurvivalanalyisis….………………………………………..…49

Table4:1Statisticalsummaryforresistance…………….………..……………………………….59

Table4:2Statisticalsummaryfortolerance………….…………………………………..………..60

Table5:1Statisticalsummaryoflife-historytraits……………..……………………………….76

Impactofenvironmentonco-evolutionbetweenhostsandparasites

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GeneralintroductionChapter1

1.1 Background

Parasites1areomnipresentinnatureancanbeasubstantialsourceofselectionontheirhosts.Parasites

areinvolvedinmanyecologicalandevolutionaryprocessesincludingtheevolutionofsex,socialbehav-

ior,maintainingorincreasinggeneticdiversityofhosts,alteringpopulationstructuresandeveninthe

diversificationofspecies.Someparasitesareknowntobehighlyvirulentbycausingseriousharmand

deathintheirhostpopulations.Forexample,ithasbeenestimatedthat20%ofthemarinebiomassis

killedbyviruseseverydayand that in theocean1023newviral infectionsoccurevery second (Suttle

2007).Therearemorethan1,400describedparasitespeciesthatcan infecthumans(Woolhouseand

Gowtage-Sequeria 2005) and approximately 15% of human death is caused by pathogens. Malaria

aloneaccounts for20%ofchildhoodmortalityand in2015therewereanestimated214millionnew

caseswith440,000deaths (WorldHealthOrganization2015).Otherparasitesare less severebutstill

causedebilitatingsymptoms,whichcanreduceorpreventreproductionanddecreasecompetitiveabili-

ties.

Whysomeparasitescauseveryseverepathologyandmortalitywhileothersarerelativelybenign,and

whysomeparasitesareveryefficientinspreadinginapopulationandbecomeepidemicwhileothers

stay in small frequencies, has fascinated the scientific community for decades. Such diverse disease

characteristicsaretheresultofcomplexco-evolutionarydynamicsbetweenhostsandparasites.Para-

sites thatcausepathologyandmortalitycanbeamajorsourceofselectionontheirhosts.Thehosts

haveevolveddiversemechanismsofdefense to reduce the successof infection, increase the rateof

clearance,orat leastby reducing thedetrimentaleffectsof theparasite. Thesedefensemechanisms

rangefromverycompleximmunesystemprocesses,modificationofcellsurfacestopreventinfection,

tochangesinhostbehaviorandlife-historystrategiesinordertoresistortolerateparasites.Thepara-

sitesontheotherhand,aredependentonthehost’sresourcesandaimtoreachahighdensity,within

1 Parasites in thesis are defined broadly as infecitous agents that cause disease (including virus, bacteria, fungi, protozoa,helminths).


14

thehost,inordertobetransmittedefficientlyinapopulation.Howeverbecauseahighparasitedensity

isexpectedtoreducethehost’ssurvivalandthusthedurationofthetransmissionperiod,theparasites

virulence2istraded-offwithitstransmission(AndersonandMay1982;Alizonetal.2009).

Accordingly,thehost’sabilitytoresistparasites is incontinuousconflictwiththeparasites infectivity,

thegrowthanditstransmissibility.Anevolutionaryresponsetoparasitismofthehostmayagainalter

theselectionpressureoftheparasite.Thisreciprocalselectioncanresultinco-evolution,withcontinu-

ouschangesofallelefrequenciesinhostsandparasites(Gandonetal.2008;GabaandEbert2009).This

canbedrivenbyparasite-mediatedselectionagainstcommonallelesorbydirectionalselectionthrough

thesuccessivefixationofadvantagesmutations.Accordingtothered-queenhypothesistheevolution-

ary ratesofchangeshouldbeacceleratedwithcoevolution. Indeedgenomesofcoevolvinghostscan

evolvefastercomparedtopopulationsevolvingagainstaconstantparasitepopulation(Patersonetal.

2010;KashiwagiandYomo2011).Howeverinmanysituationssuchpredictionsaretoosimple,especial-

ly when hosts develop tolerance instead of resistance, when there is enhanced competition for re-

sourcesorwhenimmunopathologycausesabigpartofthedamage(RestifandGraham2015).Forex-

ample it has been shown that co-evolutionary dynamics canbe alteredby resource availability; high

resource levels leads todecreased fluctuatingselection in resistance (LopezPascuaetal.2014).Such

studiesillustratetheneedtounderstandhost-parasiteinteractionsinan“eco-co-evolutionary”context

because of the presence of genotype-by-genotype-by-environment interactions that influence their

evolution.

Hostsresourcesaffectmanyaspectsofhostparasiteinteractionandhaveshowntodrasticallychange

theoutcomeofinfection(Shetty2010).However,inmosttheoreticalstudiesthatpredicttheevolution

ofhost-parasite interactions,one fundamentalaspectofparasites isgenerally ignored: thatparasites

steal resources fromtheirhost to support theirowndevelopment (see (Smith1993;Halletal.2007;

Halletal.2009a;Halletal.2010)forexceptions).Inadditiontogeneticfactorsofhostsandparasites,

resourceavailabilityhasbeenrecognizedasakeyaspect inthedynamicsof infectiousdiseases, influ-

encinghostdefense,parasite transmissionandvirulence (Lazzaroand Little2009;WolinskaandKing

2009).Severalstudiesshowhowresourcequality,aswellasquantity,shapesparasitevirulence(Jokela

etal.1999;Brownetal.2000;FergusonandRead2002)andalsodirectlyinfluencestheproductionof

parasites(Bedhommeetal.2004;Johnsonetal.2007;DeRoodeetal.2008;Halletal.2009b).

2Virulence in this thesis isdefindedas theparasite inducedhostmortalityand fecundity loss fromtheageof infectionon-wards.


15

There are twogeneralways inwhich resource availability in thehost’s environment influenceshost-

parasiteinteractions.Ontheonehand,highlevelsofresourcescanpositivelyinfluencethehost’s im-

muneresponseandthereforeincreaseitsresistancetoparasiteinfection(KoellaandL.2002;Ayresand

Schneider2009).Malnourishedhostsmaybeweakerandmoresusceptibletoinfectiousdisease(Moret

2000) so that the lower the food availability, the higher the costs of parasitism (Ferguson and Read

2002).Forexample,thefleaXenopsyllaramensisproducesmoreeggswhenfeedingonhostsexperienc-

ing diet restriction (Krasnov et al. 2005). On the other hand, a parasite uses the host’s internal re-

sourcesforitsowndevelopment.Accordingly,hostsrearedonhighlevelsoffoodmaystoremoreen-

ergy reserves and therefore present a better environment for the parasite to develop. Indeed, en-

hancedqualitiesandquantitiesof resources increase theproductionofeffectivepropagulesofpara-

sites(e.g.ascogregarines(Tseng2006)ormicrosporidians(AgnewandKoella1999b;Bedhommeetal.

2004)) inmosquitoes, and trypanosomes in bumble-bees (Brown et al. 2000), but also increase host

survival(asin(Jokelaetal.1999)),andcannegativelyaffecthostreproductivesuccess.Well-feddaph-

nia hosts infectedwithCaulleryamesnili, for example, showed, in regard to fecundity,more severe

parasiticeffects(Bittneretal.2002).Itfurtherhasbeenshownthatincreasedfoodavailabilityincreas-

esparasitetransmissionrate(Ebertetal.2000;J.Ryderetal.2007;Valeetal.2013).

Becauseparasitegrowth,virulenceandtransmissionareprincipalparametersinepidemiologyandthe

evolution of host-parasite interactions, ignoring the impact of resource availability limits our under-

standingofhost-parasiteinteractionsandevolution.Studyingthesetraitsunderdifferentresourcelev-

els is thereforevital tounderstand theparasiteswithinhost-dynamics (e.g. the relationshipbetween

parasiteburdenandhosthealth),whichplaysanimportantroleintheevolutionofparasitesandhosts

(Antiaetal.1994;AlizonandvanBaalen2005a).Byconsideringthatthehost’snaturalhabitatcanvary

drastically in space and time, our understandingof host-parasite interactions, their co-evolution, the

predictions of parasite evolution and especially the management of parasites must incorporate

knowledgeofhowenvironmentalvariationsinfluenceparasite-hostinteractions.Itisthereforeofgreat

importancetoscienceandsocietytodeepenourknowledgeinthisfield.


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Figure1:1Resourcesinhost-parasiteinteraction

Resourceavailabilityasakeyfactorinhost-parasiteinteractions:Resourceavailabilitycaninfluencethehost’sgrowthrate,

ageandsizeatmaturity,longevityandfecundity.Resourcescanalsobeimportantforthedevelopmentoftheparasitewithin

itshostbecauseofadirectinfluenceoftheresourcesavailabletotheparasite,andviathehostslifehistory,inparticularbody

sizethatoftenconstrainstheparasitedevelopment.Theamountofresourcescanalsoinfluencethehost’sresistance(limit

parasitedevelopment)andtolerance(reducedetrimentaleffectsofparasiteswithoutaffectingtheparasitesdevelopment)to

pathogens.Thedevelopmentoftheparasitecanfeedbackontothehost’slifehistory,especiallybyreducingitslongevity.The

transmissionoftheparasiteisdependentonparasitegrowthandthelongevityofthehost,andindirectlyonthehost’sre-

sources.

1.2 Thesisintroduction

InthisthesisIstudytheimpactoftheenvironmentonhost-parasiteinteractions(summarizedinFigure

1.1)andhowthis influencestheirevolution. IusethemosquitoAedesaegyptiand itsmicrosporidian

parasiteVavraia culicis. I aim toempirically test certainaspectsofmathematicalmodels thatpredict

theevolutionofthehostandparasiteslife-history,virulenceandtheparasitesdefensestrategies.The-

seresultswillhopefullyenlargeourknowledgeabouttheevolutionofhostparasiteinteractionsingen-

eral,andcontributetothedevelopmentofmorepowerfulandrealisticlife-historyandepidemiological

models.Furthermore,thedatamightbedirectlyrelevantforpublichealthbecauseoftheroleofmos-

quitosasavectorforseveralinfectiousdiseases.Theresultsmightalsobeusefulforpeopleworkingon

biologicalcontrolasmicrosporidianshavebeenproposedforthecontrolofmosquitoesandtheirvec-

tor-bornediseases(SweeneyandBecnel1991;Koellaetal.2009;LorenzandKoella2011).

1.2.1 Foodvariability,growthandlaterlifeconsequences

Resource availability is as key component of life history theory because of its role in determining

growth,survivaland fecundityof individuals (Stearns1992).Diet restrictionhasbeenassociatedwith


17

slowergrowth,smalleradultsize,delayedmaturityandlowerfecundity,butwithalongerandhealthier

life(StearnsandKoella1986).Becauseadultsizeisaveryimportantdeterminantoffitness,adaptations

tohandleperiodsoffoodrestrictionsareveryimportant.Onepossibility,whichisreferredtoascom-

pensatorygrowth,istogrowatacceleratedratesafteraperiodofundernourishmentinordertocatch

up in size. Rapidly growing individualsmight respond to food stress by decelerating growth rates in

ordertousetheavailableresourcesformaintenanceandreproduction.Compensatorygrowth,aswell

asdeceleratinggrowth,aregenerallythoughtofasadaptiveresponses,buttheirconsequenceslaterin

liferemainlargelyunexplored. Inthesecondchapter, Iexperimentallytesttheeffectofvariablefood

availability during the development of the mosquito host Aedes aegypti (leading to compensatory

growthordeceleratedgrowth),andtheassociatedchangesinlongevityandreproductivesuccess.Such

dataisrelevantforlife-historytheoryandisalsodirectlyrelevantforpublichealthduetothemosqui-

to’sroleasvectorforseveralinfectiousdiseases.

1.2.2 Within-hostgrowthandvirulence

Mostmodelsfortheevolutionofhostparasiteinteractionsarebasedontheassumptionthatthereis

trade-off between virulence and the rate of transmission (Anderson andMay 1979; Alizon and Lion

2011,Gandonetal.2001;Dieckmannetal.2002).Underlyingthisassumptionistheideathatparasites

mustgrowrapidly toahigh load inorder tobe transmittedefficiently,but thathighparasitedensity

alsoreducesthehost’ssurvivalandthusthedurationofthetransmissionperiod.Thistrade-offimplies

thatmaximaltransmissionisoftenhighestatintermediatelevelsofvirulence.Howeveroptimallevels

ofvirulencedependontheshapeofthetrade-off(AndersonandMay1982;BremermannandPickering

1983).Thegeneralassumptionthatparasiteburden ispositivelycorrelatedwith itsvirulenceholds in

severalsystems(Mellorsetal.1996;MackinnonandRead1999;DeRoodeetal.2008;DeRoodeand

Altizer 2010). However, because resource availability has been shown to influence parasite growth

(Johnson et al. 2007; De Roode et al. 2008;Michalakis et al. 2008; Hall et al. 2009b) and virulence

(Jokelaetal.1999;Brownetal.2000;FergusonandRead2002),itislikelytoaffecttheassociationbe-

tweenthetwo,sothatweonlyseeanegativerelationshipbetweenparasitedevelopmentandlongevi-

tyinsomeenvironments.InthethirdchapterIinvestigateexperimentallyhowaspectsoftheenviron-

ment (the amountof food available to larvae and to adults, and the age at infection) influences the

growthoftheparasite,thelongevityofthehostandtherelationshipbetweenthetwo.Studyingfactors

thatinfluencetherelationshipbetweenthehost’sgrowthanditsvirulenceisimportant,becausethey

mayaltertherelativecostsandbenefitsofrapidparasitereplicationandthereforedetermineadaptive

levelsofvirulence.


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1.2.3 Toleranceandresistance:twofundamentallydifferentdefensetraits

Adaptationsofthehosttoreducenegativeeffectsofparasitescanbeclassifiedintotwobroadcatego-

ries:Resistanceandtolerancetoparasites(Readetal.2008;Råbergetal.2009;Littleetal.2010).Re-

sistancereducesthesuccessofinfectionorincreasestherateofclearance,whereastolerancereduces

thedetrimentaleffectsoftheparasitewithoutaffectingthepathogendirectly.Itisimportanttodistin-

guishbetweenthetwotraitsbecausetheyhaveaverydifferenteffectontheevolutionofhost-parasite

interactions(RoyandKirchner2000;RestifandKoella2003;Boots2008).Tolerancegenesarepredicted

tobecomefixed,becausetheyarebeneficialforthehostandparasite,whileresistancegenesarepre-

dictedtorapidlychangebecausetheywouldprovokecounter-adaptationoftheparasitetoovercome

resistance (Roy and Kirchner 2000;Miller et al. 2006).One example that illustrates the evolutionary

significanceof tolerance is thecaseofsimian immunodeficiencyvirus (SIV) inmonkeys. Inmacaques,

whicharenon-naturalhosts,thevirusreplicatesrapidlyandinfectedanimalsdevelopAIDS.Incontrast,

insootymangabeys,whicharenaturalhostsofSIV,infectedindividualsdonotshowdiseasepreogres-

sionandshownodevelopmentofAIDS,evenwhencarryinghighvirusloads(Chakrabarti2004).Sooty

mangabeys,whichhavealongevolutionaryhistorywithSIV,thereforereducedthedamageofparasite

infectionandevolvedhighlevelsoftoleranceinsteadofresistance.Tolerancehasalsobeendiscussed

tobeclinicallyrelevant(Medzhitovetal.2012).Incontrasttoresistant-basedtherapy,tolerance-based

therapyaimstoimprovethehealthofthehostatagivenparasiteloadinsteadofreducingtheparasite

burden.Thismaynotleadtoselectionforresistantparasitesandwasconsideredas“evolution-proof”

(Readetal.2008;SchneiderandAyres2008).

Differentiatingbetweenresistanceandtolerance,studyingtheassociationbetweenthetwo,andpre-

dictinghowthetwoevolvehavebecomeimportanttopicsofevolutionaryparasitology.Whatismissing

areexperimentalstudiesontheextenttowhichevolutionfavorstoleranceorresistanceunderdiffer-

ent ecological settings. Using experimental evolution, I aim to test theoretical predictions about the

evolutionofhostdefenseagainstparasitesandinvestigatetheroleofresourceavailabilityonthecon-

currentevolutionoftoleranceandresistance.

1.2.4 Changeinlifehistorytoreducethecostsofparasitism

Achangeinhost’slifehistorycanbeanotherformtoreducethecostofparasitism.Lifehistorytheory

predictsthatearlymaturinghostsmayhaveaselectiveadvantagebecausetheycanevadeparasitismin

timeandwhenparasitizedreducedetrimentaleffectsonreproductionand longevity (Hochbergetal.

1992;Forbes1993;PerrinandChriste1996).Howeversuchadaptationscanbeassociatedwithcosts

laterinlife(reducedlate-lifefecundityandlongevity).Astunningexampleisthespreadofaninfectious


19

cancerinTasmaniandevilsthatcausesalmostcompletemortalityafterthefirstyearofadulthood.This

facialtumorcausesanabruptshiftinthehost’slifehistoryfrommultibreedingtowardsinglebreeding.

The Tasmanian devils responds to this strong selection pressure, by a 16-fold increase in precocious

sexualmaturity(Jonesetal.2008).Suchanalterationinlifehistorycanbeseenasaformofresistance,

whichmight leadtocomplexco-evolutionarydynamicsbetweenthe lifehistoriesofthehostandthe

parasites. In the fifthchapter Iaimtostudyhowcoevolvingandconstantparasitescan influencethe

hosts’lifehistoryunderdifferentresourcelevels.Studyingtheroleofresourceavailabilityispotentially

relevant because trade-offs between different life history traits might only be detectable when re-

sourcesarescarce.Accordingly,variableenvironmentsmightinfluencethelong-termhostevolution.

1.3 Experimentalsystem

In this thesis themosquitospeciesAedesaegyptiand itsmicrosporidianparasiteVavraiaculiciswere

usedasmodelorganismsto investigatethe impactof thehostenvironmentonhost-parasite interac-

tion.

1.3.1 ThemosquitoAedesaegyti

TheUGALstrainofthemosquitospeciesAe.aegypti(obtainedfromPatrickGuérin,UniversityofNeu-

châtel)wasusedforalloftheexperimentspresentedinthisthesis.ThemosquitospeciesAedesaegypti

is the principal vector of yellow-fever (Tomori 2004), dengue (World Health Organisation 2002) and

Zika-virus(Petersenetal.2016),growsinavarietyofdifferentnaturalandartificialcontainersholding

cleanfreshwater(Southwoodetal.1972)andoccursthroughoutthetropicsandsubtropics.Itisavery

wellstudiedspecies;itsecologyisknownindetail(Christophers1960),ithasbeenamodelorganismin

insectphysiologystudies(Clements1999),anditsfullgenomehasbeenpublished(Neneetal.2007).

Aedesaegyptiarehighlysusceptibletoawholerangeofenvironmentalconditions(Christophers1960).

Forexampleduringtheaquaticlarvalstages,wildmosquitoesareoftenundergoingperiodsofnutrient

restrictionandcompetitionforresourceslikebacteria,algaeandorganicmatter(ReiskindandLounibos

2009).


20

Figure1:2Mosquitolifecycle.

Generalizedlifecycleofmosquitoes:Aedesaegyptimosquitoesareholometabolousorganisms.Eachmosquitogoesthrough

fourdistinctstagesofitslifecycle:Egg,larva,pupaeandimago.Theadulthoodistheonlynon-aquaticstage.

1.3.2 Vavraiaculicis

Microsporidiaconstitutealarge,verydiversegroupofsingle-cell,endocellularparasites.Theybelongto

the kingdomof fungus, arewidespread in nature andhave a broad rangeof hosts (fromhumans to

invertebrates).Microsporidiaarespeciallycommoninarthropods;closetohalfofthedescribedgenera

haveinsectsastheirhosts(BecnelandAndreadis1999).ForthisthesisIusethemicrosporidanparasite

VavraiaculicisthatwasoriginallyderivedfromAe.albopictusinFloridaandkindlyprovidedbyJ.JBecnel

(USDA,Gainesville,USA). It is anaturalparasiteof several generaofmosquitoes includingAedesae-

gypti(WeiserandColuzzi1972).Naturalprevalenceratesofinfectionrangingfromlessthan1%upto

54%differing frommosquitospeciesandgeographical location (Andreadis2007). It isanobligateen-

docellularparasite,orally infectiousandistransmittedbetweenhostsbyinfectivespores(Figure1.3).

Thesporesaretheyonlystageoftheparasitethatcanpersistintheenvironmentoutsidethehostcell.

ThehostlarvaeingestthesporesofVavraiaculiciswiththeirfood,resultingininfectionofgutcellsand

epithelialcells.Afterpassingaseriesofdevelopmentalstageswithinthelarvae,theparasitebeginsto

produceitsinfectiousspores.Theinfectionthenspreadstogutandfatbodycells.Nocomponentofan

insect’simmunesystemhasbeendescribedwhichcanneutralizethisintracellularparasite(Bironetal.


21

2005).Theparasite is transmitted in twoways.First, transmissioncanoccur from larva to larvaafter

larvaldeath.Inparticularcasesoffoodstressorstronginfection(Bedhommeetal.2004)larvaldeathis

enhanced,which initiatesanother roundofhorizontal transmission. Second, larvae survive the infec-

tion,withoutclearingitanddevelopintoadults.Thesporeswithinthehostcontributestotheparasites

fitnesseitherbythereleaseofsporeswhendying intheaquaticenvironmentor,becausesporescan

adheretothesurfaceoftheeggsandinfectthenewlyhatchedlarvae(Andreadis2007).

Figure1:3LifecycleofVavraiaculicis

LifecycleofVavraiaculicis:Sporesarereleasedintotheaquaticenvironmentwhenjuvenileoradultmosquitoescarrying

sporesdieandareorallyingestedbylarvae.Transmissioncanoccurfromlarvatolarvawhensporesarereleasedafterthe

larvadies(solidlines)oriflarvaesurvivetheinfectionanddevelopintoadults;thesporescanbereleasedwhenthemosquito

diesintheaquaticenvironmentortheycanadheretothesurfaceoftheeggsandinfectthenewlyhatchedlarvae(dashed

lines).

1.3.3 Resourcesaffectlife-historiesofhostandparasite

Resourceavailabilityplaysafundamentalroleintheinteractionbetweenmosquitoesandmicrosporidi-

anparasites.Larvalresourceavailabilityofthehostinfluencesthemosquito’sgrowthrate,ageatma-

Like all microsporidia, Vavraia culicis is an obligate endocellular parasite of several mosquito genera, including Aedes, Culex and Anopheles. Natural prevalence rates of infection range between 1% and 54% depending on the mosquito species and geographical location [69]. Vavraia culicis is horizontally transmitted when mosquito larvae ingest its spores. The spores first infect the mosquito gut epithelial cells, and the infection then spreads to other gut and fat body cells. After several rounds of replication within a larva, the parasite begins to produce its infectious spores. In some cases, in particular in conditions of food stress or intense infection [15], these kill the larva or pupa and are released into the breeding site, thus initiating another round of horizontal transmission. In other cases, larvae juveniles the infection and develop into adults (without clearing the infection). There is no transovarial transmission (i.e. the parasite does not penetrate the eggs of infected females), but spores can adhere to the surface of the eggs and infect the newly hatched larvae [69].

Resources affect life-histories of the host and parasite of this project

Resource availability is a key component of the interaction between mosquitoes and microsporidians. Larval food influences, for example, the mosquito’s growth rate and age at metamorphosis [15,70,71]). The resources available to the host are also important for the dynamics of the parasite within its hosts. Increasing larval food increases the production of spores [15], probably because of a direct influence of the energy available to the parasite (as in [8]) and an indirect influence via the host’s life-history, in particular body size, that constrains the parasite’s development (as in [70]). Larval food also influences the parasite’s virulence: less food increases the probability that infected mosquitoes die before their emergence [15,71] and decreases the longevity of infected adults [71]. It thus largely affects the parasite’s transmission route, either horizontally from dead juveniles or vertically from females as they lay their eggs. Although the mechanisms underlying virulence are not known, they may include a direct effect of the parasite (as assumed in many models, e.g. [46-48]) or the depletion of energy below a threshold necessary for the host’s survival [8,72]}.

Choice of experimental system

The mosquito Aedes aegypti is the major vector of dengue and yellow fever. It is a subtropical mosquito, whose larvae grow in natural or artificial containers [73]. It is a very well-studied organism: its ecology is known in detail [74], it has been a model organism in insect physiology [75], and its full genome has been published [76]. Its eggs can be hatched synchronously, and it can be reared easily in the laboratory.

The Vavraia-Aedes system is well suited for this research for many reasons. (i) The host and parasite have short generations, enabling rapid single-generation experiments and feasible experimental evolution. (ii) The mosquito’s eggs and the parasite’s spores can be stored for several months, which simplifies the logistics of experiments and enables to compare the evolved host and parasite with the original populations. (iii) The resource availability and the exposure of the host to the parasite are easy to control. (iv) The influence of the

Resource ecology of parasite evolution! 7/22


22

turity,reproductionandlongevity(ZellerandKoella2016)aswellas it influencesthedevelopmentof

theparasiteby increasing theproductionof infectious sporesofdeadmosquitoes (Bedhommeet al.

2004).Thewithingrowthoftheparasitemaythereforebelimitedbytheconditionofthehost,proba-

blybecauseofadirectinfluenceoftheenergyavailablefortheparasite(Halletal.2009b)andanindi-

rectimpactviathehost’slifehistory,speciallybodysize,thatconstrainsthedevelopmentofthepara-

site(AgnewandKoella1999b).Larvalfoodalsodeterminesthevirulenceoftheparasite:lessfooden-

hancesjuvenilemortality(Bedhommeetal.2004;LorenzandKoella2011)anddecreasesadultlifespan

ofinfectedmosquitoes(LorenzandKoella2011).Italsoconsiderablyaffectstheparasite’stransmission

route,eitherhorizontallyfromdeadjuvenilesorverticallyfromfemaleswhenthelaytheireggs.


23

1.4 Researchaims

TheoverallobjectofthisPhDthesis istounderstandempiricallyhowtheenvironment influencesthe

co-evolutionarydynamicsbetweenthemosquitoAedesaegyptianditsmicrosporidianparasiteVavraia

culicis.Iaimtoempiricallytestassumptionsandpredictionsofmodelsofhostandparasiteslife-history

andepidemiology.Iusesinglegenerationexperimentsaswellasexperimentalevolutiontoaddressthe

openquestions.

Theprojecthasfourmajorgoals:

I. Experimentalexaminationofresourcevariabilityongrowth,reproductionandlongevityinthe

mosquitoAedesaegypti

II. Description of the parasites within-host dynamics; investigation of the relationship between

parasitedevelopmentandhost-longevityasafunctionofthehostsresourcesandageatinfec-

tion

III. Testtheoreticalpredictionsabouttheevolutionofhostdefenseagainstparasites.Investigation

of the roleof resourceavailabilityon theevolutionof thehost’sability to tolerateand resist

parasites.

IV. Examinationofresourceavailabilityandco-evolutionarydynamicsonthehostlife-historyevo-

lution.


25

Effects of food variability onChapter2

growthandreproductionofAedesaegypti

MICHAELZELLER1,JACOBC.KOELLA1

1LaboratoryofEcologyandEpidemiologyofParasites,InstituteofBiology,UniversityofNeuchâtel,Rue

Emile-Argand11,2000Neuchâtel,Switzerland

PublishedinEcologyandEvolution:

Zeller,M.andKoella, J.C. (2016),Effectsof foodvariabilityongrowthandreproductionofAedesaegypti.Ecol

Evol,6:552–559.doi:10.1002/ece3.1888


26

Abstract

Despitea largebodyof knowledgeabout theevolutionof life-histories,weknow little about

howvariable foodavailabilityduringan individual’sdevelopmentaffect its life-history.Wemeasured

theeffectsofmanipulatingfoodlevelsduringearlyandlatelarvaldevelopmentofthemosquitoAedes

aegyptionitsgrowthrate,life-historyandreproductivesuccess.Switchingfromlowtohighfoodledto

compensatorygrowth:individualsgrewmorerapidlyduringlatelarvaldevelopmentandemergedata

sizeclosetothatofmosquitoesconsistentlyrearedathighfood.However,switchingtohighfoodhad

verylittleeffectonlongevity,andfecundityandreproductivesuccesswereconsiderablylowerthanin

consistentlywell fedmosquitoes.Changing fromhighto lowfood ledtoadultswithsimilarsizeas in

consistentlybadlynourishedmosquitoes,buteven lower fecundityandreproductivesuccess.Arapidresponseofgrowthtochangingresourcescanthushaveunexpectedeffectsinlaterlifeandinlifetime

reproductivesuccess.Moregenerally,ourstudyemphasizestheimportanceofvaryingdevelopmental

conditionsfortheevolutionarypressuresunderlyinglife-historyevolution.


27

2.1 Introduction

Howlife-historiesrespondtovariationinfoodavailabilityisacentralquestionofevolutionaryecology.

Considerableeffort,bothwiththeoreticalandempiricalapproaches,hasbeenspentonansweringthe

questionforenvironmentsthatvaryspatially(KaweckiandStearns1993;Ernandeetal.2004)andfrom

onegenerationtothenext(Bashey2006)inresourceavailability.Yet,animportantaspectofvariability

has receivedconsiderably lessattention: that resource levelscanvaryduringan individual’sdevelop-

ment.

Eventhoughthere issubstantialevidencethatvariation in food levelsduringdevelopmentcanaffect

ageandsizeatmaturity(e.g.Leips&Travis1994;Hentschel&Emlet2000),weknowlittleabouthow

this variation affects reproductive success andadult survival.As food restriction severely affects life-

historyparameters - itgenerallyslowsgrowth,delaysmaturityand leads tosmalladultswith lowfe-

cundity (Stearns and Koella 1986) - it seems plausible that individuals that grow slowly early in life

shouldtrytomakeuptheirsizedeficitwithcompensatorygrowth,i.e.bygrowingmorerapidlyorfora

longerperiodoncetheyobtainmorefood (Dmitriew2011).Rapidlygrowing individuals,ontheother

hand,mightrespondtofoodstressbydeceleratinggrowthratesinordertousetheavailableresources

formaintenance and reproduction. Slow growth has shown to be adaptive for dealingwith nutrient

stress(Arendt1997).Compensatorygrowthfollowingaperiodofunfavorableenvironmentalconditions

hasbeendescribedformanyvertebratesandinvertebrates(Dmitriew2011).However,compensatory

growthneednotbeevolutionarilybeneficial.Indeed,thepresumedbenefitofcompensatorygrowth-

largerindividualshavegreaterfecundity-isnotalwaysobserved.InTrinidadianguppies,forexample,

compensatorygrowthisnotassociatedwithincreased,butwithdecreasedfecundity(Aueretal.2010).

Furthermore, anybenefit of compensatory growthwith regard to fecunditymaybe counteractedby

costswithregardtootherpartsofthelife-history.Longergrowthandthusdelayedmaturity,forexam-

ple,canbeassociatedwithagreaterriskofdyingbeforematurity(AbramsandRowe1996).Evenfor

purecompensatorygrowth,i.e.whenmaturityisnotdelayed,thegreatergrowthratemayhavecosts

(physiological/cellular level),whichareoftenonlyevidentmuch later in life (MetcalfeandMonaghan

2001;Alonso-Alvarezetal.2007;DeBlockandStoks2008).Indeed,dietrestrictionisoftenassociated

with a longer and healthier life (Chippindale et al. 1993; Masoro 2005). Accordingly compensatory

growthinfishreducedlifespanwhereasdeceleratedgrowthextendedit(Leeetal.2013).Thismay,in

part,beduetodevelopmentalerrorsandstructuralinstabilityasaresultofincreasedgrowth(Mangel

andMunch2005).Thus,althoughtheroleofre-feedingafteraperiodofdietaryrestriction(and,more

generally,theroleofchangingresourceavailabilityduringanindividuals’development)ontraitssuch


28

asgrowthrate, longevityandageatmaturityhaveacquiredsomeattention, little isknownabout its

roleonreproductivesuccess.

Inthisstudy,weprovidedataontheeffectofvariabilityindevelopmentalfoodconditions(leadingto

compensatory growth or decelerated growth) and associated changes in longevity and reproductive

successofthemosquitoAedesaegypti.Suchdatanotonlyformthebasisforourunderstandingoflife-

historyevolution,butarealsodirectlyrelevantforpublichealthduetomosquito’sroleasavectorof

severalinfectiousdiseases.

2.2 Materialandmethods

2.2.1 Experimentalsystem

WeusedtheUGALstrainofthemosquitoAe.aegypti(obtainedfromPatrickGuérin,UniversityofNeu-

châtel).Aedesaegyptioccurs throughout the tropicsandsubtropics.During theaquatic larval stages,

mosquitoesinnaturecanexperienceperiodsofnutrientrestrictionandcompetitionforresourceslike

bacteria,algaeandorganicmatter(ReiskindandLounibos2009).

2.2.2 Experimentaldesign

Theexperimentwasruninaclimatechambersetto26°C,70%relativehumidityandat12hlightand

12hdarkregime.Weuseda2x2factorialdesign,wherelarvaewerefedeitherwithastandardamount

of food (Day 1: 0.06mg of tetramin fish food, day 2: 0.08mg, day 3: 0.16mg, day4: 0.32mg, day 5:

0.64mg,day6orlater:0.32mg)orwithhalfofthestandarddietduringeitherearly(0to3daysafter

hatching)or latedevelopment (4ormoredaysafterhatching).The four treatmentsarehereafter re-

ferredtoasLL,LH,HHandHL,withthefirstletterreferringtotheamountoffoodduringearlydevel-

opment(LoworHigh)andthesecondlettertotheamountoffoodduringlatedevelopment.Eggswere

hatchedindeionizedwater.Fourhoursafterhatching,384first-instarlarvaeweremovedinto12-well

platesandkeptindividuallyin3mlofdeionizedwater.Eachlarvawashaphazardlyassignedtooneof

the four feeding regimes and fed every 24 hourswith the appropriate amount of food. Pupaewere

moved to300mlplastic cupscontainingdeionizedwaterandapieceof filterpaperasanoviposition

substrate.Thecupswerecoveredwithmosquitonetting,andcottonwoolmoistenedwith10%sugar

solutionwas placed onto the netting and changed every 48 hours.One day after emergence,males

werediscardedandeachfemalewasgivenamalechosenhaphazardlyfromourcolony.Thenextday

andeverytendaysthereafter, thefemalesweregiventheopportunitytotakeabloodmealonMZ’s


29

armforfiveminutes.Thefemaleswherecheckedeverydayforsurvival.Ninedaysafterbloodfeeding,

thefemaleswereplacedintofreshlypreparedplasticcupsandtheireggswereremovedandcounted.

Fecunditywasdefinedasthenumberofmelanisedeggs laiduptoninedaysafterbloodfeeding.The

experimentwas stoppedafter six roundsof egg-laying, atwhich time85.4%of themosquitoeshad

died.

2.2.3 Traitmeasurement

Weestimated larvalbodysizeby takingstandardizeddigitalpicturesofall individualsevery24hours

starting on the day of hatching (age 0) andmeasuring the length of the larvawith the open-access

softwareIMAGEJ.Whenphotosoflarvaewereconsideredtoolowinqualityforanaccuratemeasure-

menttobetaken,theindividualswerenotincludedintheanalyses.Larvalgrowthwasmeasuredasthe

differenceinsizebetweenage0andage4(earlygrowth)andbetweenage4andage6(lategrowth)

forallindividuals.Thesizeofadultswasassayedasthemeanoftheirwinglength,whichstronglycorre-

lateswiththeweightofmosquitoes(KoellaandLyimo1996)andiswidelyusedasanapproximationfor

adult size. The wings were removed and mounted on microscope slides. The slides were digitally

scannedandthewingsweremeasuredwithIMAGEJ

2.2.4 Statisticalanalysis

Weconsideredonly females, and ignored the growthof the6 (out of 384) individuals that haddied

beforepupation.Weassayed185femalemosquitoes,between43and49ineachfoodtreatment.The

difference insizebetweenage0andage4 (earlygrowth)wasevaluatedwithananalysisofvariance

(ANOVA) that includedthe levelofearly foodasa fixedbinomial factor.Becausethesizedifferences

betweentheages4and6(lategrowth)wereclosetolinearandindividualsnotyetreachedasymptotic

sizetheywereevaluatedwithananalysisofcovariance(ANCOVA)thatincludedearlyandlatefood,the

interactionbetweenthetwoasfixedfactors,andthesizeatagefourasacovariate.Assizeatagefour

didnot interactwithearlyor latefood,weomittedthese interactionsfromtheanalysis.Additionally,

becausewemeasuredindividualsrepeatedly,wecheckedthattheresultsweresimilar,whenwecor-

rectedforregressiontothemean(analysisnotshown).Forbothanalyses (earlyand lategrowth)we

verifiedthattheassumptionsofANOVAandrespectivelyANCOVAwerenotviolated.Ageatemergence

andlongevitywereanalyzedwithsurvivalanalysesthatincludedearlyandlatefoodandtheirinterac-

tionasfixedfactors. Intheanalysisof longevityweaddedwinglengthasapotentialconfounder.We

usedthedistributionsthatgavethebestfit,solog-logisticforageatemergenceandWeibullforlongev-

ity; using proportional hazards gave similar results (not shown). Wing length was analyzed with an

ANOVAthat includedearlyfoodand latefoodandtheir interactionasfixedfactors.Thewing lengths


30

wereBox-CoxtransformedtomeetANOVArequirements.Weanalyzedfecundity inthreeways.First,

weanalyzedtheproportionofblood-feedsthatledtoatleastoneeggwithaGLM(binomialdistribu-

tion).Second,weanalyzedthetotalnumberofeggslaidthroughouttheexperimentwithaGLMwith

quasi-Poissondistribution (corrected foroverdispersion). Inbothanalyses,we includedearlyand late

foodand their interactionas fixed factorsandwing lengthasapotential confounder.Third,weana-

lyzedtheage-specificclutchsizes(consideringonlythoseblood-feedsafterwhichatleastoneegghad

been laid)withamixedeffectANOVA,usingearly food, late food, clutchnumber (i.e.age)and their

interactionsasfixedfactors,winglengthasapotentialconfounder,andmosquitoasarandomeffect.

Wepresent theanalysis using all clutches.As thenumberofmosquitoes surviving to theendof the

experimentwaslow,weverifiedthattheresultsweresimilarifweconsideredonlythefirstthreeorthe

firstfourclutches(analysesnotshown).Themixed-effectANOVAwasdonewithRv.0.98.1056(RDe-

velopmentCoreTeam,2015)usingthelme4package;theotheranalysesweredonewithJMP12.0.0.

2.3 Results

2.3.1 Developmentaltraits

ThegrowthdataaresummarizedinFig.2.1.Larvaerearedonhighfoodgrewmorebetweenage0and

age4(mean=2.64mm,standarderror=0.072)thanthoserearedonlowfood(mean=1.86mm,se=

0.063)(F=66.86,p<0.001)(Fig.2.1).Growthafterage4decreasedwithincreasingsizeatday4(Table

2.1).Itwasgreatestformosquitoesthatswitchedfromlowtohighfoodatage4(2.21mm,se=0.120),

lowestformosquitoesthathadswitchedfromhightolowfood(1.57mm,se=0.118)andintermediate

formosquitoeswiththesamefoodlevelthroughouttheirdevelopment(Fig.2.2b).Theeffectsofearly

andoflatefood,butnottheinteractionbetweenthetwo,werestatisticallysignificant(Table2.1).


31

Table2:1Statisticalsummaryforjuveniletraits

ANCOVAfordifferencesinlategrowth,survivalanalysis(logLogisticdistribution)forageatemergenceandANOVAfordiffer-

encesinwinglength.

Lategrowth Ageatemergence Winglength

Factor df F SS p df χ2 p df F SS p

Earlyfood 1 5.07 1.77 0.026 1 173.6 <0.001 1 2.84 0.08 0.094

Latefood 1 14.87 5.18 <0.001 1 25.5 <0.001 1 41.21 0.02 <0.001

Earlyfood*latefood 1 0.24 0.08 0.63 1 6.6 0.01 1 0.13 <0.01 0.721

Sizeatage4 1 90.62 31.57 <0.001

Error 155 52.61 166 4.64

Figure2:1Mosquitogrowth

Bodylengthformosquitolarvaeasafunctionofage.Symbolsrepresentthemeanswitheachfoodtreatment,verticallinesthe

standarderrors.Trianglesrepresenttreatmentswithlowfoodavailabilityduringearlydevelopment;circlesrepresenttreat-

mentswithhighfoodavailabilityinearlydevelopment.Opensymbolsrepresenttreatmentswithlowfoodduringlatedevel-

opment;solidsymbolsrepresenthighfoodduringlatedevelopment.

12

34

5

Age of mosquito (days)

Larv

al g

row

th (m

m)

0 1 2 3 4 5 6 7 8 9 10

HHHLLHLL


32

Ageat emergence increased from9.9days (se=0.11) formosquitoes consistently fed thehigh food

levelto11.9days(se=0.09)formosquitoesconsistentlyfedthelowfoodlevel(Fig.2.2d).Mosquitoes

thathadswitched fromhigh to low foodemergedearlier (10.1±0.09) than those thathadswitched

fromlowtohighfood(11.2±0.10);theinteractionbetweenearlyandlatefoodlevelswasstatistically

significant(Table1,Fig.2.2c).

Winglengthincreasedfromameanof2.35mm(se=0.019)formosquitoesthathadbeenconsistently

rearedonlowfoodto2.55mm(se=0.027)formosquitoesthathadbeenconsistentlyrearedonhigh

food.Wing lengthwas influencedsignificantlybytheavailabilityof foodafterage4,whileearly food

andtheinteractionbetweenearlyandlatefoodhadnosignificanteffects(Table1,Fig.2.2d).

Figure2:2Juveniletraits

Theeffectoflarvalfoodduringearlyandlatestagesofdevelopmentfor(a)Earlygrowth(sizedifferencebetweenage0and

age4),(b)Meanlategrowth(sizedifferencebetweenday4andday6),(c)Ageatemergence±SE,(d)Adultsize(winglength).

Thedataforearlygrowth(a)waspooledforlatefoodtreatment.Symbolsrepresentthemeanswithintreatments;thevertical

linestheirstandarderrors.Opensymbolsrepresenttreatmentswithlowfoodduringlatedevelopment;solidsymbolsrepre-

senthighfoodduringlatedevelopment.

(a)

Early food

Early

gro

wth

(mm

)

Low High

1.5

2.0

2.5

3.0

(b)

Early food

Late

gro

wth

(mm

)

Low High

1.5

2.0

2.5

3.0

Late foodHighLow

(c)

Early food

Age

at e

mer

genc

e (d

ays)

Low High

1011

12

(d)

Early food

Win

g le

ngth

(mm

)

Low High

2.3

2.4

2.5

2.6


33

Table2:2Statisticalsummaryforadulttraits

Survivalanalyses(Weibulldistribution)forlongevity,binomialGLMfortheproportionofblood-fedsandGLM(quasi-Poisson

distribution)forthetotalnumberofeggs.

2.3.2 Adulttraits

Adult mosquitoes lived longest if they had been reared on low food throughout their development

(39.1days±1.97;thisandotheraveragesarebiased,fortheexperimentwasstoppedwhen14.6%of

themosquitoeswerestillalive),followedbythosethathadswitchedfromlowfoodtohighfoodwhen

theywerefourdaysold (36.8days±2.44). Incontrast tothesizeofadultmosquitoes, longevitywas

significantly affected by early food (Table 2.2),while late food and the interaction between the two

foodlevelshadnosignificanteffects.Winglengthhadnosignificanteffectonlongevity(Table2.2).

Thepercentageofthesixblood-feedsthatwerefollowedbylayingatleastoneeggrangedfrom0%to

100%;theaveragepercentagerangedfrom50%forthemosquitoesthathadbeenrearedonhighfood

throughouttheirdevelopmentto28%ifthemosquitoeshadswitchedfromhighfoodtolowfoodwhen

theywerefourdaysold(Fig.2.3B).About35%oftheblood-feedsledtoegg-laying,ifmosquitoeshad

initiallybeenrearedonlowfood,independentlyofthefoodavailabletothemduringtheirlatedevel-

opment (Table 2.2). Similarly, the total number of eggs was highest for mosquitoes that had been

rearedonhighfoodthroughouttheirdevelopment(67±8.1),lowestformosquitoesthathadswitched

food fromhigh to low(31±5.2)and intermediate formosquitoes thathadbeenrearedon lowfood

earlyintheirdevelopment(forLL:38±4.7;forLH:48±6.9)(Table2,Fig.2.3C).Latefoodenvironments

hadsignificanteffects indeterminingtheprobabilityof layingeggsandthetotalnumberofeggs.The

interaction between early food and late food had marginally significant effects in determining egg-

layingsuccessandmarginallynon-significanteffects indeterminingthetotalamountofeggs.Neither

theegg-layingsuccessnorthenumberofeggsweresignificantlyinfluencedbywinglength(Table2.2).

LongevityEgg-layingafterblood

feedingTotalnumberofeggs

Factor df χ2 p χ2 p χ2 p

Earlyfood 1 3.87 0.049 0.74 0.39 0.03 0.857

Latefood 1 0.22 0.636 4.58 0.032 11.79 <0.001

Earlyfood*latefood 1 <0.01 0.969 4.00 0.046 3.66 0.055

Winglength 1 1.17 0.279 0.14 0.712 0.06 0.803


34

Figure2:3Adulttraits

(a)Theeffectoflarvalfoodduringearlyandlatestagesofdevelopmentfor(a)Longevityofadultfemalemosquitoes(age0is

ageafteremergence).(b)Proportionofblood-fedsthatledtoegg-laying,(c)Totalnumberofeggs±SE.LLstandsforlowfood

availabilityduringthewholelarvaldevelopment,LHforlowfoodduringearlydevelopment,highfoodduringlatedevelop-

ment,HHforhighduringthewholedevelopmentandHLforhighfoodduringearlydevelopment,lowfoodduringlatedevel-

opment.In(b)and(c),opensymbolsrepresenttreatmentswithlowfoodduringlatedevelopment;solidsymbolsrepresent

highfoodduringlatedevelopment.

The clutch size (considering only those blood-feeds after which at least one egg had been laid) de-

creasedwiththeageofadultmosquitoes(Fig.2.4).Foodlevelduringlatelarvallifeaffectedthenum-

berofeggsinthefirstclutchandtherateatwhichfecunditydecreasedwithagewasinfluencedbythe

interactionbetweenearlyand late food treatment (Table2.3). Switching from low food tohigh food

four days after hatch led to themost eggs in the first clutch, but then to the greatest decline over

clutches(Fig.2.4).Therateofthedecreasewasmostlyinfluencedbytheinteractionbetweenearlyand

latefoodtreatments(Table2.3).

(a)

Age after emergence [days]

Prop

ortio

n su

rviv

ing

HHHLLHLL

0 10 20 30 40 50 60

0.0

0.2

0.4

0.6

0.8

1.0

(b)

Early food

Egg−

layi

ng a

fter b

lood−f

eedi

ngLow High

0.2

0.3

0.4

0.5

0.6

(c)

Early food

Tota

l num

ber o

f egg

s

Low High

2030

4050

6070

80 Late foodHighLow


35

Figure2:4Clutchsize

Relationshipbetweennumberofeggsperclutchandclutchnumber(i.e.,age).Circlesrepresenttreatmentswithhighfood

availabilityduringearlydevelopment;trianglesrepresenttreatmentswithlowfoodavailabilityinearlydevelopment.Open

symbolsrepresenttreatmentswithlowfoodduringlatedevelopment;solidsymbolsrepresenthighfoodduringlatedevelop-

ment.

Table2:3Repeatedmeasuresanalysisofclutchsizes

Onlyblood-feedingattempts,whichledtoatleastoneegg,wereconsidered.

Numberofeggs

Factor df F SS p

Earlyfood 1 0.02 2.8 0.885

Latefood 1 15.00 2005.0 <0.001

Earlyfood*latefood 1 3.67 490.6 0.056

Winglength 1 0.15 19.8 0.700

Clutchnumber 1 33.81 4518.5 <0.001

Clutchnumber*earlyfood 1 0.29 38.3 0.593

Clutchnumber*latefood 1 0.61 81.8 0.435

Clutch number * early food * late

food1 5.52 737.6 0.020

Error 267 133.6

Clutch number

Num

ber o

f egg

s

1 2 3 4 5 6

1020

3040

50

HHHLLHLL


36

2.4 Discussion

VariabilityindevelopmentalfoodconditionsinAedesaegyptihadqualitativelydifferenteffectsonthe

life-history traits we investigated: adult size, fecundity, survival and reproductive success. Thus, for

example,wing lengthwasdeterminedmainlyby foodavailabilityduring late larvaldevelopment, sur-

vivalbyfoodavailabilityduringearlydevelopment,andtotalnumberofeggsbyacombinationofthe

two.

When food availabilitywas held constant during themosquitoes’ development, their life-history fol-

lowedthegeneralpredictionsoflife-historytheory(e.g.StearnsandKoella,1986):lowfoodthusledto

slowgrowth, latepupation, smalladults,and low fecundity. Italsocorroboratesmanystudieswhere

food restriction increased longevity (Weindruch 1996; Shanley and Kirkwood 2000;Mair et al. 2003;

KirkwoodandShanley2005;Masoro2005).

Varyingfoodavailabilityledtolife-historiesthataremoredifficulttoexplainwithlife-history,similarly

tothestudyofYearsley,Kyriazakis&Gordon(2004).Increasingfromlowtohighfoodled,asfrequently

observed (Metcalfe andMonaghan 2001), to compensatory growth: at emergence,mosquitoes that

hadbeenfirstbadlyandthenwellnourishedcaughtupinsizebygrowingmorerapidlyandbydelaying

pupation,andtherebybecamealmostaslargeasmosquitoesthathadbeenfedwellthroughouttheir

development.However,althoughsizecaughtup,weobservednotoverylittlecatchingupoffecundity,

longevity,orlife-timereproductivesuccess.Togetherwiththeobservationthatthenumberofeggsper

clutchdeclinedstrongestwithagefor individualsthathadswitchedfromlowtohighfoodduringde-

velopment(Figure2.4), theseresultscouldmeanthatcompensatorygrowthearly in life isassociated

withreproductivecosts later in life,whichleadto, inour laboratoryconditions, lowerlife-timerepro-

ductivesuccess.Inadditiontoconsiderableevidencefortrade-offsbetweenlife-historytraitsearlyand

late in life,bothfromlaboratorysituations (e.g.Rose1984)and,morerecently, fromnaturalpopula-

tions (Lemaîtreetal.2015),our resultssupport the findingsofAueretal. (2010),whichsuggest that

thereare reproductivecostsassociatedwithcompensatorygrowth.The trade-offweobservedraises

thequestionabouttheadaptivenatureofcompensatorygrowth.However,althoughinourlaboratory

conditions,compensatorygrowthhadanegativeconsequence for reproductivesuccess, thesituation

maychangeinnaturalconditions.Bothjuvenileandadultmortalityratesmaybesubstantiallyhigherin

thefieldthaninthelaboratory.Accordinglythebenefitsof largersizeandearliermaturityassociated

withcompensatorygrowthmayoutweighitsreproductivecostsinoldmosquitoes.

Whenmosquitoesstartedoutatgoodfoodconditionsandthenswitchedtolowfood,theirgrowthand

adultsizedecreasedasexpected.Whatwasmoresurprisingwasthattheindividualswithdecelerated


37

growthhave lowerreproductivesuccessthanthosethathadexperiencedfoodrestrictionthroughout

theirdevelopment.However,becausetheinteractionbetweenearlyandlatefoodwasmarginallynot

significant,wecannotdrawstrongconclusions.Neverthelessthistrendcouldbetheresultofphysiolog-

icalresponsestothefoodenvironment inearlydevelopmentthatpreparethe individual forasimilar

environmentlaterinlife(GluckmanandHanson2004).Therefore,mosquitoesthatareundernourished

earlyinlifecancopewithfoodrestrictionlaterinlifebetterthanthosethathavebeenpreparedforan

environmentwithplentifulfood.

Astrikingresultwasthatwinglengthhadverylittleeffectonreproductionorlongevity,althoughasso-

ciations of life-history traitswith size are central tomany ideas in life-history theory (e.g. Stearns&

Koella1986;Rowe&Ludwig1991;Abrams&Rowe1996).Forexample,mostmodelsthatpredictthe

evolutionarilyoptimalageatmaturityassumethatfecundity increaseswithbodysize (e.g.Roff1984;

Stearns&Koella1986;Berrigan&Koella1994).Suchassociationsareoftenfoundwhenfoodavailabil-

ityisheldconstant(LyimoandTakken1993;McCannetal.2009).However,inourexperiment,where

food availability varies during themosquito’s development, the environmental factor over two time-

periods thatdeterminedbodysize (foodavailabilityduringearlyandduring latedevelopment)affect

the life-history traits rather than body size itself. If this is generally the case, it would imply major

changesinthewaywethinkaboutlife-historyevolution.Thetimingofresourcerestrictionduringde-

velopmentalsoaffecteditseffectonlongevity.Weobservedonlyaneffectiftherestrictionwasduring

earlydevelopment.Thisisconsistentwiththecommonfindingthatfoodrestrictioncanslowtheageing

process(Weindruch1996;ShanleyandKirkwood2000;Mairetal.2003;KirkwoodandShanley2005;

Masoro2005).However,thatchangingfromlowtohighorfromhightolowfoodhadnegligibleeffects

onlongevitycontradictsotherstudiesshowingthatcompensatorygrowthassociatedwithbetterfood

conditionsreversestheeffectofearlyresourcerestrictiononlongevity(Merry2002;Dhahbietal.2004;

Spindler2005).Wehavenoexplanationforthedifferenceoftheseresults.

2.4.1 Conclusion

Inconclusion,weshowedthatvariabilityofdevelopmental foodconditions inAedesaegyptimosqui-

toeshas strongeffectsonadult size, reproductivesuccessandmortalityofadult females,withsome

traitsbeingmostlyaffectedbythefoodavailability inearlydevelopmentandotherbeingaffectedby

late foodavailability.Sucheffectsmayhave importantconsequences forenergyallocationstrategies,

butaregenerallynotconsideredinmodeloflife-historyevolution.Wefurthershowedthatcompensa-

torygrowth,whichisgenerallyconsideredanadaptivestrategy,doesnotincreaseitsreproductivesuc-

cess,atleastforAedesaegyptiinourlaboratoryconditions.Thereproductiveburdensassociatedwith

compensatory growthmay play an important and limiting role in the evolution of growth and other


38

relatedtraits.Finally,thatthemosquitoes’reproductivesuccesswasnotdirectlyconnectedwithadult

size,butwas,rather,influencedbythefoodconditionsthattheyexperiencedduringdevelopmentcon-

trastsacentralassumptionofmanyideasinlifehistorytheory.Thus,wesuggestthatourunderstand-

ingof theevolutionof life-historieswillbegreatlyenhanced ifweconsider theeffectsofvaryingthe

environmentalconditionsduring juveniledevelopment.Such information is important inorder tode-

velopeffectivepredictionsofdiseasetransmissionandstrategiesofmosquitocontrol.

39

Context-dependent relationshipChapter3

between parasite growth and virulence in a

microsporidia-mosquitointeraction






40

Abstract

Manyideasabouttheevolutionofparasitesrelyontheassumptionthattheirhostsincreasingly

suffer as parasite load increases, and that the relationship between growth and virulence is similar

among environments.We investigatedwhether the interaction between the parasiteVavraia culicis

anditshost,themosquitoAedesaegypti,isaffectedbythreeaspectsoftheenvironment:theamount

of foodavailableto larvaeandtoadults,andtheageat infection.Wemeasuredspore loadandesti-

matedtheparasite’sgrowthrateandasymptotic load inmosquitoesthatdiedduringtheexperiment

and in haphazardly selected, living mosquitoes. In most environments, the probability of infection,

spore load, bothmeasures of the parasite’s developmentwere higher in dead than in age-matched

living mosquitoes, corroborating the idea that virulence increases with the parasite’s development.

However, these relationshipsdependedon the foodavailability andage at infection, suggesting that

thetrade-offsunderlyingtheevolutionofvirulencedependontheenvironment.Ideasabouttheevolu-

tionofvirulencemustthereforeconsidernotonlyhowtheenvironmentaffectsepidemiologicallyrele-

vantparameters,butalsohowitaffectstherelationshipsbetweenthem.


41

3.1 Introduction

Whydosomeparasitescauseseverepathologyandmortalitywhileothersarerelativelybenign?Many

theoreticalattempts tounderstand thisvariation relyon theassumption that there isa trade-offbe-

tweentherateoftransmissionandthedurationofthetransmissionperiod(AndersonandMay1979;

Alizonand Lion2011,Gandonet al. 2001;Dieckmannet al. 2002).Underlying this assumption is the

ideathatparasitesmustreplicaterapidlytoahighloadinordertobetransmittedefficiently,butthat

highparasite load(orrapidparasitedevelopment)alsoreducesthehost’ssurvivalandthusthedura-

tionofthetransmissionperiod.

Manyaspects,however,of the relationshipbetweenparasite loadand thehost’sdeathareobscure.

Whileseveralexamples indeedshowthatgreaterparasitemiacan increasetheriskofdeath(e.g.HIV

(Mellors et al. 1996), rodentmalaria (Mackinnon and Read 1999;Mackinnon and Read 2004) and a

gregarine parasite ofmonarch butterflies (De Roode et al. 2008; De Roode and Altizer 2010),many

shownorelationship (Salvaudonetal.2007;Littleetal.2008). Indeed,wemightexpect that thebe-

tweenparasiteburdenandhostmortalityisweakforparasiteswhosesymptomsaremainlyduetothe

host’simmuneresponse,sothatlowparasiteloadsmaybeasharmfulashighloads.Forexample,clini-

calmalariaand,inparticularcerebralmalaria,appeartosteminlargepartfromanoveractiveimmune

system(Artavanis-Tsakonasetal.2003).

Another,lessexploredreasonforthevariabilityintheassociationbetweenparasitedevelopmentand

hostmortality is the role of the environment on the association. The environment, in particular re-

sourcescanaffectvirulence(Jokelaetal.1999;Brownetal.2000;FergusonandRead2002);resources

canalsoaffectparasitegrowth(Bedhommeetal.2004;Johnsonetal.2007;deRoodeetal.2008;Hall

etal.2009a).Ifvirulenceandgrowthareaffecteddifferently,wemightseeanegativerelationshipbe-

tweenparasitedevelopmentandsurvivalinsomeenvironments,butnotinothers.Indeed,inanexper-

iment withDaphnia magna and its intestinal, castrating parasite Pasteuria ramose, the relationship

betweenparasiteloadandmortalityrateswitchedfrompositiveatlowfoodconditionstonegativeat

highones(Valeetal.2011).Suchstudiescanbe,however,difficulttointerpret,astheymeasurepara-

siteloadatthetimeofthehost’sdeath.Asparasiteloadisexpectedtoincreasewithtimeafterinfec-

tion,itisintrinsicallyrelatedtolongevityandthusmortalityrate.Inaddition,parasiteloadinsurviving

individuals is often notmeasured, so thatwe do not knowwhether dead individuals indeed harbor

moreparasites than survivingones. Yet this is a critical assumption ifwewant to conclude anything

aboutarelationshipbetweenparasiteloadanddeath.Iftheenvironmentindeedchangestherelation-

ship between the parasite’s growth and virulence, testing ideas about the evolution of virulence by

changing environmental parameters (as, for example, (Ebert andMangin 1997; Nidelet et al. 2009))


42

wouldbeproblematic,asthedifferenttreatmentswoulddifferinthetrade-offsunderlyingtheevolu-

tionarypredictions.

Here,westudywhethertheenvironment(resourceavailabilityandageatinfection)affects,inaddition

tohosthealthandparasitedevelopment(growthandparasiteload),therelationshipbetweenthetwo,

usingexperimentalinfectionsofthemosquito,AedesaegyptiwiththemicrosporidianparasiteVavraia

culicis.Wecomparethenumberofspores indeadmosquitoesand inasampleofage-matched living

mosquitoes.Thisallowsustodecoupletheeffectofparasite loadfromthe longevityofthehost.We

furthermodelthedevelopmentoftheparasitewithinthelivingandthedeadmosquitoesandcompare

the growth parameters between the two groups. Finally, bymanipulate age at infection, larval and

adultresources,wefindtheroleoftheenvironmentontherelationshipbetweenparasitegrowthand

hostmortality.



ThemicrosporidianparasiteVavraiaculiciswasprovidedbyJ.JBecnel(USDA,Gainesville,USA).Itisan

obligate,intracellularparasitethathasbeenreportedinnaturalpopulationsofseveralgeneraofmos-

quitoesincludingAedes(WeiserandColuzzi1972).ThehostlarvaeingestthesporesofVavraiaculicis

withtheirfood,resultingininfectionofgutcellsandepithelialcells.Afterseveralroundsofreplication

withinthelarvaetheparasitebeginstoproduceitsinfectiousspores.Theparasiteistransmittedintwo

ways.First,transmissioncanoccurhorizontallyfromlarvatolarva,eitherbysporesreleasedinfaeces

orafter larvaldeath. Insomecases,e.g.with foodstressorheavy infection(Bedhommeetal.2004),

larvaldeath isenhanced.Second, if juveniles survive the infection, theydevelop into infectedadults.

Thesporesaretransmittedfromfemalestothenextgenerationofmosquitoesbyarrivinginabreeding

siteeitherwhenamosquitodiesonabreedingsiteoronthesurfaceoftheeggs(Andreadis2007).We

usedtheUGALstrainofthemosquitoAe.aegypti(obtainedfromPatrickGuérin,UniversityofNeuchâ-

tel).Aedesaegyptioccursthroughoutthetropicsandsubtropics,isanimportantvectorfor,e.g.dengue

andZikaviruses.

3.2.2 Experimentaldesign

Theexperimentwasruninaclimatechambersetto26°C,70%relativehumidityandat12hlightand

12hdarkregime.Weusedafullfactorialdesign,withageatinfection(twoorfivedaysafterhatching),


43

larval food (the standarddietofour lab (Day1: 0.06mgof tetramin fish food,day2: 0.08mg,day3:

0.16mg,day4:0.32mg,day5:0.64mg,day6orlater:0.32mg)or40%ofthestandarddiet)andadult

food(10%or2%sucrose-solution)asexperimentalfactors.

Eggsweresoakedindeionizedwaterandsimultaneouslyhatchedunderreducedatmosphericpressure.

2010first-instarlarvaeweremovedinto12-wellplatesandkeptindividuallyin3mlofdeionizedwater.

Each larvawashaphazardlyassignedtooneof theeight treatmentsand fedevery24hourswith the

appropriateamountoffood.Weexposedlarvaetoinfectionbyadding2.0x105sporesin100µldeion-

izedwaterper individual.Pupaeweremoved to50mlFalcon tubecontainingdeionizedwateranda

pieceoffilterpaper.Thecupswerecoveredwithmosquitonetting,andmosquitoesweregivenaccess

tocottonwoolmoistenedwitheither10%or2%sugarsolution.Toestimatetheparasite’sgrowth,we

countedsporesfromall thedead individualsand,startingelevendaysafterhatching, fromahaphaz-

ardly selected sample (8-12 individuals) of livingmosquitoesof each treatment. Theexperimentwas

stoppedwhenallofthemosquitoeshaddied(32daysafterhatching).


The size of adultswas assayed as themean of theirwing length,which strongly correlateswith the

weightofmosquitoes (KoellaandLyimo1996)and iswidelyusedasanapproximation foradult size.

Thewingswereremovedandmountedonmicroscopeslides.Theslidesweredigitallyscannedandthe

wingsweremeasuredwiththeopen-accesssoftwareIMAGEJ.Tocounttheparasite’ssporeswerewe

placedeachmosquito intoa2mlEppendorf tubecontaining180µldeionizedwateranda5mmsteal

bead.We crushed themosquitoesby shaking the tube for4minutes at 35Hz (Tissue Lyser,Qiagen,

Valancia,California).Thestealbeadwasremovedandthesporeswerecountedinasampleofthesolu-

tionwithahaemocytometer(Neubauerimproved).


Weassayed2014mosquitoes (between248 and253 in each treatment) ofwhich925becameadult

females (between 61 and 131 in each treatment). For the analysis of the juvenilemortality the two

adult food treatmentswerepooled.Ageneralized linearmodel (GLM) fittedwithabinary logistic re-

gressionwasperformedtodeterminetheeffectsoflarvalfood,ageatinfectionandtheirinteractionon

the likelihoodthat larvaesurvivedtoemergeasadults.Weanalyzedtheproportionsof individuals in

whichwefoundatleastonesporewithaGLM(binomialdistribution),andthenumberofsporeswitha

GLMwithquasi-Poissondistribution(correctedforoverdispersion).Forbothanalysesweincludedlar-

valfood,adultfood,ageatinfectionandsurvival(deadoraliveattimeofsampling)andtheirinterac-

tionasfixedfactorsandthetimeafterinfectionasanominalconfounder.Notethatwedonotconsider


44

timeafterinfectioncontinuousbecauseofitsstronglynonlinearrelationshipwithsporeload.Parasite

sporeloadswerelog10-transfromed.

Wefittedthenumberofsporesusinggeneralizedleastsquareswiththefollowingequation

! = ! ∗ (1 − !!!∗!)

Equation3:1-Parasitegrowthequation

wheren represents the log10-transformednumberof spores,c theasymptoticnumberof spores,k a

parameterrelatedtothegrowthrateatlowsporeloadsandtthetimeafterinfection.Wecomparedc

andkbetweenexperimentalgroupswiththegnlsfunctionfromthenlmepackageincludinglarvalfood,

adultfood,ageatinfection,survivalandalltheirinteractionsasfixedfactors.Thelongevityofthemos-

quitoeswasanalyzedwithasurvivalanalysisthatincludedlarvalfood,adultfood,infectiontime-point

andallpossibleinteractionsandaddedwinglengthasapotentialconfounder.WhileweusedWeibull

distributions because it gave the best fit, Cox proportional hazard gave similar results. All statistical

analyseswereperformedwithRversion3.2.3(RDevelopmentCoreTeam,2015).

3.3 Results

3.3.1 Juvenilemortality:

Theprobabilitythata juvenilediedbeforeemergencerangedfrom1.8%(confidence intervalofpro-

portion:lowerlimit=0.95%,upperlimit=3.4%)formosquitoesthathadbeeninfectedfivedaysafter

hatchingandrearedonhighlevelsoffoodto33.2%(CI:lowerlimit=29.1%,upperlimit=37.6%)for

mosquitoesthathadbeeninfectedatagetwoandrearedonlowfood.Mosquitoesthathadbeenin-

fectedatagefiveandrearedonlowfood(2.6%;CI:lowerlimit=1.5%,upperlimit=4.4%)andatage

twoandrearedonhighfood(13.5%CI: lower limit=10.7%,upper limit=16.7%)had intermediate

risksofjuveniledeath.Larvalfood(χ2=17.9,p<0.001)andageatinfection(χ2=15.9,p<0.001),but

nottheirinteraction(χ2=3.02,p=0.082),hadsignificanteffectsinfluencingjuvenilemortality.

3.3.2 Probabilityofinfection,sporeloadandparasitegrowth:

The probability of harboring spores significantly increased with infection period and was higher for

mosquitoeswithayoungageat infection(Table3.1,Figure3.1A).Theanalysisofsporeloadrevealed

statisticallysignificanteffectsoftimeafterinfection,survival,larvalfood,ageatinfectionandtheinter-

actionbetweenlarvalfoodandageatinfection(Table3.1,Figure3.2B).Whenmosquitoesdiednatural-


45

lytheygenerallyhadmoresporesthanlivingonesatthesameage.Thesporeloadofmosquitoessignif-

icantlyincreasedwithtimeafterinfection,andwashigherforindividualswithhighlarvalfoodandwith

ayoungageatinfection.

Figure3:1:Sporeloadandprobabilityofinfection

Theeffectoflarvalfoodavailabilityandageatinfectionfor(A)theprobabilitytofindspores(mean±confidenceintervalof

proportion),for(B)thelognumberofspores(mean±se)andfor(C)themeandifferenceofsporeloadbetweennaturally

deadandlivingmosquitoes.Plottedare,forcolumnIindividualswithhighadultfoodavailabilityandforcolumnIIindividuals

withlowfoodadultavailability.Solidsymbolsrepresenttreatmentsalowageatinfection(agetwo);opensymbolsrepresent

treatmentswithanageatinfectionoffive.Circlesrepresenttreatmentswithhighfoodavailabilityduringdevelopment;

squaresrepresenttreatmentswithlowfoodavailabilityduringdevelopment.ThedashedlinesinfiguresCindicatetheexpec-

tationifsporeloadwouldbeequalfornaturallyandartificiallydyingmosquitoes.

Days after infection

Spor

e pr

obab

ility

6 9 12 15 18 21 24

0.0

0.2

0.4

0.6

0.8

1.0A

I


Spor

e pr

obab

ility

6 9 12 15 18 21 240.0

0.2

0.4

0.6

0.8

1.0

II


Spor

e lo

ad

6 9 12 15 18 21 24

3.5

4.0

4.5

5.0B


Spor

e lo

ad

6 9 12 15 18 21 24

3.5

4.0

4.5

5.0


Diff

. in

spor

e lo

ad

6 9 12 15 18 21 24

−1.0

−0.5

0.0

0.5

1.0

(d)

C

Days after infection6 9 12 15 18 21 24

−1.0

−0.5

0.0

0.5

1.0


46

Table3:1Sporeloadandprobabilityofinfection

Statisticalsummaryfortheprobabilityofinfection(GLMwithbinomialdistribution)andsporeload(GLMwithPoissondistri-

butioncorrectedforoverdispersion).Statisticallysignificantp-valuesaregiveninbold.

Sporeprobability Sporeload

Factor df χ2 p χ2 p

Survival 1 2.40 0.121 4.89 0.027

Larvalfood 1 1.49 0.223 7.34 0.006

Adultfood 1 1.39 0.239 1.35 0.245

Ageatinfection 1 7.51 0.006 12.42 <0.001

Survival*larvalfood 1 0.86 0.354 0.23 0.635

Survival*adultfood 1 0.58 0.446 1.19 0.276

Larvalfood*adultfood 1 2.44 0.118 0.08 0.779

Survival*ageatinfection 1 0.73 0.393 1.08 0.298

Larvalfood*ageatinfection 1 1.92 0.166 10.17 0.001

Adultfood*ageatinfection 1 1.00 0.318 0.61 0.437

Survival*larvalfood*adultfood 1 0.51 0.477 0.29 0.591

Survival*larvalfood*ageatinfection 1 0.62 0.431 0.61 0.433

Survival*adultfood*ageatinfection 1 1.32 0.250 0.78 0.378

Larvalfood*adultfood*ageatinfection 1 3.04 0.081 3.02 0.082

Timeafterinfection 1 54.64 <0.001 60.71 <0.001


47

Thegrowthparameterkandtheasymptoticsporenumbercweresignificantlyhigherfornaturallydy-

ingmosquitoesthanfor livingones(Figure3.2).Whilenoneoftheenvironmentalfactorssignificantly

influencedk,theasymptoticnumberofsporescwassignificantlyaffectedbylarvalfood,ageatinfec-

tion,adultfoodandbysomeinteractionsbetweenthefactors(summarizedinTable3.2).Inparticular,

theasymptoticsporenumberwashigherformosquitoeswithalowageatinfectionandformosquitoes

rearedonhighlevelsoffoodaslarvae.

Figure3:2Parasitegrowthparameters

Theeffectoflarvalfoodavailabilityandageatinfectionfortheasymptoticsporeloadc(A)andthegrowthparameterk(B)±

standarderrorfor(I)individualswithhighadultfoodavailabilityandfor(II)individualswithlowadultfoodavailability.Solid

symbolsrepresenttreatmentswithalowageatinfection(agetwo);opensymbolsrepresenttreatmentswithanageatinfec-

tionoffive.Circlesrepresenttreatmentswithhighfoodavailabilityaslarvae;squaresrepresenttreatmentswithlowlarval

foodavailability.

cause of death

c3.5

4.0

4.5

5.0

5.5

AI

cause of death

c3.5

4.0

4.5

5.0

5.5 II

cause of death

k

artififcial natural

0.0

0.1

0.2

0.3

0.4

0.5B

cause of death

k

artififcial natural

0.0

0.1

0.2

0.3

0.4

0.5


48

Table3:2Parasitegrowthparameters

Statisticalsummaryforthecomparisonofk(growthparameter)andc(asymptoticsporeload)betweenexperimentalgroups.

Statisticallysignificantp-valuesaregiveninbold.

Growthparameter Asymptoticsporeload

Factor df F p F p

Survival 1 68.533 <0.001 11260.34 <0.001

Larvalfood 1 0.059 0.8086 12.745 <0.001

Adultfood 1 0.110 0.7398 4.325 0.0382

Ageatinfection 1 0.384 0.5356 16.900 <0.001

Survival*larvalfood 1 <0.01 0.9894 2.136 0.1447

Survival*adultfood 1 0.128 0.7203 9.670 0.002

Larvalfood*adultfood 1 0.071 0.7904 0.012 0.9115

Survival*ageatinfection 1 0.251 0.6163 13.574 <0.001

Larvalfood*ageatinfection 1 0.039 0.8434 26.167 <0.001

Adultfood*ageatinfection 1 0.381 0.5376 2.503 0.1143

Survival*larvalfood*adultfood 1 0.063 0.8020 0.053 0.8173

Survival*larvalfood*ageatinfection 1 0.450 0.5026 3.499 0.0621

Survival*adultfood*ageatinfection 1 0.086 0.7689 14.462 <0.001

Larvalfood*adultfood*ageatinfection 1 0.805 0.3702 7.070 0.0081

Survival*Larvalfood*adultfood*ageatinfection 1 0.073 0.7870 14.819 <0.001

3.3.3 Longevity:

Adultmosquitoeslivedlongestiftheyhadbeeninfectedatagefive,rearedaslarvaeonhighfoodand

asadultsonhighfood(13.8days;se=0.70),andtheylivedshortestiftheyhadbeeninfectedatage2

andrearedonlowfoodaslarvaeandasadults(3.4days;se=0.60).Theeffectsofageatinfection,the

interaction between larval and adult food, and the three-way interaction between larval food, adult

foodandageat infectionwerestatistically significant (Table3.3).Completedataon longevitycanbe

obtainedfromFigure3.3.


49

Figure3:3Longevityofinfectedmosquitoes

TheeffectsoflarvalfoodandageatinfectionforlongevityofadultfemalesforI)individualswithalowadultfoodavailability

andII)forindividualswithhighadultfoodavailability(age0isageafteremergence).Darkblacklinesrepresenttreatments

withhighfoodavailabilityaslarvae;greylinesrepresenttreatmentswithlowfoodavailabilityaslarvae.Solidlinesrepresent

treatmentswithalowageatinfection(age2);dashedlinesrepresentstreatmentswithhighageatinfection(age5).

Table3:3Statisticalsummaryofsurivalanalysis

Statisticalsummaryforthesurvivalanalysis(Weibulldistribution).Statisticallysignificantp-valuesaregiveninbold.

Longevity

Factor df χ2 p

Larvalfood 1 0.44 0.509

Adultfood 1 0.44 0.509

Ageatinfection 1 7.49 0.006

Larvalfood*adultfood 1 38.67 <0.001

Larvalfood*ageatinfection 1 0.09 0.769

Adultfood*Ageatinfection 1 0.34 0.561

Larvalfood*Adultfood*ageatinfection 1 20.98 <0.001

Winglength 1 0.41 0.522

3.4 Discussion

The foodofAedesaegyptiand theageat infectiongreatlyaltered thedynamicsof thehostparasite

interactionby influencing theprobabilityof infection thegrowthof theparasiteand the longevityof

thehost.Theprobabilityofinfectionwasmainlydeterminedbyageatinfection,thesporeloadbylar-

0 5 10 15 20

0.0

0.2

0.4

0.6

0.8

1.0

Days after emergence

Prop

ortio

n su

rviv

ing

I

0 5 10 15 20

0.0

0.2

0.4

0.6

0.8

1.0

Days after emergencePr

opor

tion

surv

ivin

g

II


50

valfoodandbyageatinfectionandparasitegrowthwasinfluencedbylarvalfood,adultfoodandage

atinfection.Thelongevityofmosquitoeswasmostlydeterminedbyageatinfectionandbyinteractions

betweenlarvalfoodadultfoodandhostageatinfection.Furthermore,theprobabilityofinfection,the

spore loadand the sporegrowthwas significantlyhigher fornaturallydyingmosquitos than for age-

matchedlivingones,indicatingthatthesetraitsareimportantcausesofvirulence.

Inoursystem,virulencehastwocomponents:theprobabilitythatinfectedindividualsdiebeforeemer-

genceandthelongevityofinfectedadults.Bothcomponentswerehigherifthelarvaewereyoungerat

infectionand if theyobtained less food.This isconsistentwith the literature that less foodenhances

juvenilemortality(Bedhommeetal.2004;LorenzandKoella2011)anddecreasesadultlifespanofin-

fectedmosquitoes (Lorenz andKoella 2011), and it suggests early infection amplifies thesepatterns.

Althoughthemechanismsunderlyingvirulencearenotknown,theymayincludeadirectanddensity-

dependent effect of the parasite (as assumed inmanymodels (Ganusov et al. 2002; Alizon and van

Baalen2005b))orthedepletionofenergybelowathresholdnecessaryforthehost’ssurvival(Halletal.

2009b),whichislikelytodependnotonlyonparasiteloadbutalsoontheperiodofinfection.

We were particularly interested in whether the environment affected the way that the parasite’s

growth and density influenced the severity of parasitism, in particular its host’s longevity. Naturally

dyingmosquitoesgenerally showedahigher spore load (Figure3.1C,Table3.1), ahighergrowthpa-

rameteroftheparasiteandahigherasymptoticsporeloadthanlivingonessampledatthesameage

(Figure3.2,Table3.2).Thus,ourdataagenerallypositiverelationshipbetweentheparasitesdevelop-

mentanditsvirulence, inaccordancewiththecentralassumptionofthevirulencetransmissiontrade

off(AndersonandMay1982).Howeverwefoundthattherelationshipbetweenthehost’shealthand

theparasite’sdevelopmentvariedbetweenagesatinfectionandamongfoodlevels.Thustheenviron-

mentalfactorsinfluencedtheasymptoticsporedensity(c)differentlyinlivingandnaturallydyingindi-

viduals(significantinteractionsbetweensurvivalandadultfoodandbetweensurvivalandageatinfec-

tion,Table3.2).

Ageatinfectionandfoodavailabilityalsointerfereinhowtheyexpresstheparasite’sexploitationand

virulence.Thus,whichenvironmental factorsare importantdiffer for theparasite’sdevelopmentand

forthehost’slongevity.Thus,whileyoungageatinfectionledtohighersporeloadsandalsoreduced

adult longevity, mosquitoes reared on ample larval food producedmore spores (higher growth and

parasiteload)thanmosquitoesrearedonlittlefood,buthadverysimilaradultlongevity;ortheinterac-

tionbetweenlarvalfoodandadultfoodavailabilityhadlittleinfluenceontheproductionofspores,but

butalargeeffectonlongevity.Itthereforeseemsthatwhenlarvalfoodavailabilityishightheparasite

growswithlittleimpactonthehost’slongevity,butwhenageatinfectionislowthehostismorevul-


51

nerabletohighparasiteloads.Thefactthathostsrearedonhighlevelsoffoodseemtobemoretoler-

ant tohighparasiteburdencanbe interpretedas reducedconflict for shared resourcesunderample

foodandisconsistentwiththefindingsfromValeetal.(2011)andZellerandKoella(underreview).If

weassumethattransmissionislinkedtosporeloadsimilarlyinallenvironments(whichis,ofcoursenot

necessarily the case), our results imply that the virulence-transmission trade-off differs among envi-

ronments,withimportantconsequencesforpredictionsabouttheevolutionofvirulence.

Two general mechanisms can explain the effect of resource availability on the outcome of a host-

parasite interaction.Ontheonehandtheampleresourcescan increasetheefficacyof thehost’s im-

muneresponseandtherebyincreasetheresistanceofhoststoparasite infection (KoellaandL.2002;

AyresandSchneider2009).Malnourishedhostsmaythereforebeweakerandmoresusceptibletoin-

fectiousdisease(Moret2000)sothatthelowerthefoodavailability,thehigherthecostsofparasitism

(FergusonandRead2002).Ontheotherhand,theparasitesusetheirhost’sinternalresourcesfortheir

owndevelopment; resources that arenormally allocated to thehost’s growth,maintenance, and re-

production.Accordinglyhostsrearedonhighlevelsoffoodmaypresentabetterenvironmentforthe

parasitetodevelop(AgnewandKoella1999a;Brownetal.2000;Bedhommeetal.2004;Tseng2006).

Becauseinourstudytheparasiteloadwashigherforindividualswithaccesstolotsoffoodandwhen

theywereinfectedatayoungage,itsuggeststhattheparasitewasabletobenefitfromthehost’sam-

plefoodenvironmentandprobablydirectlyaccessedthehostresourcestoproducesitsspores.

3.4.1 Conclusion

Weshowed that the susceptibility, parasite loadand theparasite’swithin-host growthandvirulence

arecomplex,ageandresource-dependenttraits.Thereforethecorrelationamongagesandfoodlevels

betweenparasitedevelopment and virulence canhave considerable impacton theevolutionaryout-

comeof infectiousdisease.Theecologicalconditionsandtheageat infectionofhostshavetherefore

thepotential to change the relative costsandbenefitsofparasite replicationandare likely todeter-

minetheadaptivelevelsofvirulence.


53

The role of the environment onChapter4

theevolutionoftoleranceandresistancetoa

pathogen





54

Abstract

Defenseagainstparasitescanbedividedintoresistance,whichlimitsparasiteburden,andtol-

erance,which reducespathogenesis at a givenparasite burden.Distinguishingbetween the twoand

understandingwhichdefenseisfavoredbyevolutionindifferentecologicalsettingsareimportant,for

theyleadtofundamentallydifferentevolutionarytrajectoriesofhost-parasiteinteractions.Weletthe

mosquitoAedesaegyptievolveunderdifferentfoodlevelsandeitherwithnoparasite,withaconstant

parasite,orwitha coevolvingparasite (themicrosporidianVavraia culicis).We then tested tolerance

and resistanceof theevolved linesat the two food levels. Exposure toparasitesduringevolution in-

creased resistance and tolerance, but therewere no differences between the lines evolvedwith co-

evolvingorconstantparasites.Mosquitoesthathadevolvedwithfoodrestrictionhadhigherresistance

thanthoseevolvedwithhighfood,butsimilartolerance.Themosquitoesthathadrestrictedfoodwhen

being tested had lower tolerance than those with normal food, but there was no difference in re-

sistance.Our results emphasize the complexity and dependence on environmental conditions of the

evolution and expression of resistance and tolerance, and help to evaluate some of the predictions

abouttheevolutionofhostdefenseagainstparasites.


55

4.1 Introduction

Defensemechanismsagainstparasitescanbedividedintotwobroadclasses:resistanceandtolerance.

Resistancereducestheharmcausedbydiseasebypreventinginfectionorlimitingtheparasite’sdevel-

opmentwithin thehost, thus leading to lowerparasite loads. Tolerance reduces theparasite’sdetri-

mentaleffectswithoutaffectingparasiteload(Readetal.2008;Råbergetal.2009;Littleetal.2010).

Whileresistanceandtolerancebothincreasethefitnessofaninfectedindividual,resistancedoessoby

reducing the parasite’s fitness, whereas tolerance does not. Whether hosts evolve tolerance or re-

sistancehas fundamentallydifferenteffects for theevolutionary trajectoryofhostpathogen interac-

tions(RoyandKirchner2000;RestifandKoella2003;Boots2008).Severaltheoreticalstudieshavepre-

dictedhowtoleranceandresistancemightevolve.Forexampleitwaspredicted(i)thathostsmaintain

geneticpolymorphismforresistance,butnotfortolerance(RoyandKirchner2000;Milleretal.2005).

Theevolutionofresistancewouldprovokecounter-adaptationoftheparasitetoovercomeresistance,

whichwould lead to co-evolutionary dynamicswith rapidly changing allele-frequencies in resistance-

genes.Incontrast,theevolutionoftolerancewouldbenefitbothhostandparasite,soenablingitsfixa-

tion.Thus,(ii)tolerancewouldincreasemoreeasilythanresistance(RoyandKirchner2000).However,

(iii) theparasites couldalso respond to theevolutionof toleranceby increasing their growth to take

advantageoftheweakerconstraint(Milleretal.2005).Thus,tolerancewouldonlybeobservedifthe

hostsareinfectedwithparasitesthathavenotco-evolvedwiththehost.Finally,(iv)parasiteswithlow

virulencecouldbemore likely toselect for theirhosts’ tolerance,whereashighvirulencecould favor

resistance(RestifandKoella2004).

While this short list emphasizes thatwe expect and can predict different evolutionary outcomes for

resistanceandfortolerance,theseideasrelyontheideathatthetwodefensestrategiesarenotlinked,

asforexampleobservedinnaturalpopulationsofmonarchbutterfliesand(Lefèvreetal.2010)anda

cyprinidfish(Mazé-Guilmoetal.2014)andtheirparasites.Inothersystems,however,evolutionofthe

twodefensestrategiesappearstobeconstrainedbyanegativegeneticcorrelation.Forexample,mice

infected with Plasmodium chabaudi show a negative genetic correlation between resistance (peak

pathogen load)and tolerance (weightand redbloodcells) (Råbergetal. 2007). This couldbedue to

linkagedisequilibrium,pleiotropiceffectsorphysiologicalconstraints.

Furthercomplicatingtheevolutionarypressuresisthefactthattheenvironment,andinparticularre-

sourceavailabilitycanaffectthepredictionsmentionedabove.First,becauseresourceavailabilitycan

influencenotonlythehost’sabilitytotolerateparasites,butalsohowthehost’sgenotypeaffects its

(Sternbergetal.2012;HowickandLazzaro2014;Mazé-Guilmoetal.2014),toleranceislikelytoevolve


56

differently under different food environments. Second, because food availability often alter virulent

parasitic effects (including theAe. aegypti - V.culicis system that we study (Bedhomme et al. 2004;

LorenzandKoella2011)),itshouldinfluencetheevolutionoftoleranceandresistance(RestifandKoella

2004).Third,theevolutionarycostofresistancedependsontheavailabilityofresources,sothatdiffer-

entenvironmentsconstrainevolutionofthetwotraitsindifferentways(HochbergandBaalen1998;D.

C.Lopez-PascuaandBuckling2008;Boots2011;Harrisonetal.2013).

WestudiedsuchaspectsoftheevolutionofresistanceandtolerancewiththemosquitoAedesaegypti

and itsmicrosporidian parasiteVavraia culicis.With an experimental evolution approachwe let the

hostsevolveinresponsetoparasitesindifferentenvironments.Ourgeneralgoalwastoevaluatesome

of the ideasmentionedabove.Wetherefore investigated (i)whetheroursystemhasenoughgenetic

variabilityandlowenoughcoststoenabletheevolutionofresistanceandtolerance,(ii)howresource

availability influencestheevolutionoftoleranceandresistance,and(iii)whethercoevolvingparasites

influencethetwodefensetraitsdifferentlythanconstantparasites.


Ourexperimentwasruninaclimatechambersetto26°C,70%relativehumidityanda12hlightand

12hdarkregime.


WeusedthemicrosporidianparasiteVavraiaculicis (providedbyJ.Becnel,USDAGainesville)andthe

UGAL strain of one itsmosquito hosts,Ae. aegypti (provided by P, Guérin, University ofNeuchâtel).

Aedesaegyptioccursthroughoutthetropicsandsubtropics,isanimportantvectorfor,e.g.dengueand

Zikaviruses.

Vavraiaculicis isanobligate,intracellularparasitethatinfectsseveralgeneraofmosquitoes,including

Aedes(WeiserandColuzzi1972).Mosquitolarvaeingesttheparasite’ssporeswiththeirfood,resulting

in the infectionof gutandepithelial cells.Afteraperiodof replicationwithin the larva theparasites

begin to produce their infectious spores,which are transmitted in twoways. First, transmission can

occurfromlarvato larvawhensporesarereleasedafterthe larvadies.Thistransmissionroute isen-

hancedbyfoodstressorstronginfection(Bedhommeetal.2004).Second, if larvaesurvivetheinfec-

tiontodevelopintoadultsthesporescanbereleasedwhenthemosquitodiesintheaquaticenviron-

ment or they can adhere to the surface of the eggs and infect the newly hatched larvae (Andreadis

2007).


57

4.2.2 Experimentalevolution

Weletthemosquitoevolvefor10generations(i)eitherwithouttheparasite,withanexternallymain-

tainedparasite,orwithaco-evolvingparasite,and(ii)eitherwithhighorwithlowfoodavailabilitydur-

ingthelarvalstage.The‘constant’parasitesweretakenfromourstandardlinemaintainedinourmos-

quitocolony.Theco-evolvingparasitesweretakenfromthepreviousgenerationoftheinfectedmos-

quitoes.Eachtreatmentwasreplicatedthreetimes.Thefirstgenerationoftheexperimentwascreated

byhaphazardlymoving200one-dayoldlarvaefromthecolonytoeachline.Torearthemosquitos,we

hatchedeggssimultaneouslyunderreducedatmosphericpressure.Forthefirstfourdayswerearedfor

eachline50larvaein4petridishes(8cmdiameter)containing30mldeionizedwater(Rearingthelar-

vaeinpetridishesratherthanalargetrayhelpstoobtainasuccessfulinfection).Every24hourswefed

thelarvaewithourstandardamountoffood(Day1:0.06mgoftetraminfishfood,day2:0.08mg,day3:

0.16mg,day4:0.32mg,day5:0.64mg,day6orlater:0.32mg)orwithhalfofthestandarddiet.Weex-

posedthelarvaetoinfectiontwodaysafterhatchingbyadding5.0x106Vavraiaculicissporesin1ml

deionized water. In the first generation constant parasite and coevolution treatments received the

same solution of spores prepared from the standard lab colony. Two days after infection the four

groupsoflarvaefromeachlineweremovedtoa200*150*50mmplastictraycontaining1.5ldeion-

izedwater.Pupaeweretransferred intocages(30x30x30cmsize)containingfilterpapersoakedwith

10%sugarsolutionasfoodandacupcontainingdeionizedwaterandapieceoffilterasanoviposition

substrate.Four,six,elevenandthirteendaysafterthedaywhen75%ofmosquitoesofagivenlinepu-

patetheyweregiventheopportunitytotakeabloodmealonMZ’sarmfor8minutes.Theeggswere

removedevery48hoursandstoredat26°Cand70%relativehumidityuntilthestartofanewgenera-

tion.Fortheco-evolvedparasitepopulation(coevolution)wecollectedthedead infectedmosquitoes

(larvaeandadults),groundtheminaneppendorftube,countedthesporesandkeptthemat5°Cuntil

the next generation of hosts was started. Before starting a new generation we eliminated Vavraia

sporesfromtheeggsbybleachingtheeggsofalllineswith1%householdbleach.

4.2.3 Measuringresultofexperimentalevolution

After 10 generation of evolutionwemeasured spore load and longevity of themosquitoes from all

evolvedlinesexposedtoV.culicissporesfromthelabcolonyandfedeitherwiththestandardamount

offoodorwithhalfofthestandarddiet.Werearedthemosquitoesasdescribedabove,withthefol-

lowingdifferences.First,werearedthelarvaeindividuallyin3mldeionizedwaterinthewellsof12-well

plates.Wehadbetween109and112first-instar larvaeper line (intotal2009 larvae).Each larvawas

haphazardlyassignedtothehighorthelowfoodtreatment(between54and56individualspertreat-

mentandline).Second,weexposedlarvaetotheparasitebyadding100µlofasolutioncontaining2.0x


58

106Vavraiaculicissporespermldeionizedwater.Third,pupaeweremovedto50mlFalcontubecon-

tainingdeionizedwaterandapieceoffilterpaper.Thecupswerecoveredwithmosquitonetting,and

cotton woolmoistenedwith 10% sugar solution was placed onto the netting remoistened every 48

hoursandchangedevery72hours.Onedayafteremergencethemaleswerediscardedandthefemales

where checked every day for survival. The experimentwas stoppedwhen all of themosquitoes had

died(57daysafterhatching).

4.2.4 Sporemeasurement

Vavraiaculicissporesweremeasuredwithahaemocytometer.Eachmosquitowasindividuallyplaced

into a 2ml Eppendorf tube containing200μl distilledwater anda5mmsteal bead.Mosquitoeswere

crushedbyshakingthetubefor3minutesat35Hz(TissueLyser,Qiagen,Valancia,California).Eightμl

of themixwere added to the haemocytometer (Neubauer improved) and the sporeswere counted

withacellcounter.

4.2.5 Measurementofhostresistanceandtolerance

Wemeasuredtwotypesofresistance:qualitativeresistanceastheproportionof individuals inwhich

wefoundspores,andquantitativeresistanceastheinverseofthesporeload.Wemeasuredtolerance

astheslopebetweenlongevityandsporeloadatthetimeofdeath(Råbergetal.2009).Notethatall

mosquitoesdied laterthan14daysafter infection(~sevendaysafteremergence),atwhichtimethe

spore loadhasgenerally reachedanasymptoticvalue (ZellerandKoella, inprep.).Therefore, it isex-

pectedthatsporeloadwouldnotfurtherincreasewiththemosquito’sageandthusthatitisnotauto-

correlatedwithageatdeath.


AllanalysesweredonewithRv.3.2.3(RDevelopmentCoreTeam,2015).Differencesintheprobabili-

tiesof infectionwereanalyzedwithageneralized linearmixedeffectmodel (GLMM;binomialerrors,

logit link, using the lme4package) that included food level, the two factors (parasite and food level)

duringtheevolutionaryhistoryandalltheirinteractionasfixedfactors,andreplicateoftheevolution

treatmentasarandomeffectnestedwithinevolutiontreatment.Sporeloadofinfectedindividualswas

analyzedwitha linearmixedeffectmodel that includedthesamefactors.Longevityofadultmosqui-

toeswasanalyzedwitha linearmixedeffectmodel that includedfood,parasiteduringevolutionand

foodduringevolutionasfixedfactors,sporeloadascontinuousvariableandreplicatenestedwithinthe

evolution treatments.A significant interactionbetween spore load andexperimental factors indicate

differences in tolerance (Simms 2000; Råberg et al. 2007). The number of spores (+ 1) was log10-


59

transfromed for all analyses. Fullmodels includedall possible interactions.Minimalmodelswerede-

rived by removing unsignificant terms followed by model comparisons with likelihood-ratio tests. If

removingatermsignificantlyreducedtheexplanatorypowerofthemodel,itwaskeptinthemodel.

Therelationshipbetweenresistance(meaninverseparasiteburden)andtolerance(slopebetweenpar-

asiteburdenand longevity)wasanalyzedwith linear regression that considered foodavailability and

theinteractionbetweenfoodavailabilityandtoleranceasfactors.

4.3 Results

Atotalof1814outof2009(90%)mosquitoessurvivedtoadulthood.881(48.6%)ofthesewerefemales

andwereanalyzed(between18and33individualsperlineandtreatment).

Qualitative resistancewas affected by none of our experimental factors (Table 4.1).Quantitative re-

sistancewasaffectedbyparasitismandfoodlevelduringevolution(Table4.1,Figure4.1).Whenmos-

quitoeswereexposedtoparasitesduringevolutiontheygenerallyshowedhigherresistances.Post-hoc

test between coevolution and constant parasite treatments showedno significant differences for re-

sistance(analysisnotshown).Mosquitoesoriginatingfromlineswithfoodrestrictionduringevolution

hadahigher resistance.Neither foodavailabilitynor interactionsamong factorshadaneffect forre-

sistance.

Table4:1Statisticalsummaryforresistance

Statisticalsummaryforquantitative(sporeload)andqualitative(probabilityofinfection)resistanceanalysis.

SporeLoad Probabilityofinfection

Factor df χ2 p χ2 p

Food 1 0.19 0.667 >0.01 0.953

Evolutionparasite 2 6.05 0.049 2.89 0.236

Evolutionfood 1 7.13 0.008 0.52 0.473

Food*Evolutionparasite 2 1.66 0.436 1.91 0.384

Food*Evolutionfood 1 1.19 0.276 0.09 0.767

Evolutionparasite*Evolutionfood 2 1.84 0.398 0.40 0.819

Food*Evolutionparasite*Evolutionfood 2 1.40 0.497 1.96 0.375

Tolerancesignificantlyvariedbetweenfoodtreatmentsandbetweenlineswithdifferenttypesofpara-

sitismduringevolution(significant interactionbetweenspore loadandfood,andbetweenspore load


60

and evolution parasite (Table 4.2, Figure 4.1)). Tolerancewas significantly higher for lines with high

foodavailability and for linesoriginating from lines thatwereexposed toparasitesduringevolution.

Post-hoctestbetweencoevolutionandconstantparasitetreatmentsshowednosignificantdifferences

(analysis not shown).We foundnodifference in tolerancebetween lines rearedat the two levels of

foodduringevolution.Thelongevityofinfectedmosquitoes(whencontrolledforparasiteinducedfit-

nessloss)wassignificantlyinfluencedbytheinteractionbetweenevolutionparasiteandevolutionfood

(Table4.2).Thelongevitywasgenerallyhigherforlinesthathadevolvedwithparasitesandhigherfood

levels.Theinteractionbetweenfoodandevolutionparasitehadaclosetosignificanteffectonthelon-

gevity.

Table4:2Statisticallysummaryfortolerance

Statisticalsummaryforlongevityanalyses.Significantinteractionsbetweenparasiteloadandexperimentalfactorsindicate

differencesintolerance.

Longevity

Factor df Chisq p

Parasiteload 1 2.53 0.111

Food 1 1.19 0.275

Evolutionparasite 2 0.70 0.706

Evolutionfood 1 2.50 0.114

Parasiteload*Food 1 18.95 <0.001

Parasiteload*Evolutionparasite 2 7.03 0.030

Food*Evolutionparasite 2 4.89 0.087

Evolutionparasite*Evolutionfood 2 8.50 0.014


61

Figure4:1Toleranceandresistance

AedesaegyptitoleranceandresistancetoVavraiaculicisformosquitoesfromdifferentevolutionarylinesandtestedatdiffer-

entfoodlevels.Box-plotsaboveandtotherighteachpanelshowthemedian,the25thand75thquantileandtherangeof

longevityandsporeload.Reddotsandbox-plotsrepresentindividualsoriginatingfromlineswithcoevolvingparasites,yellow

dotsrepresentindividualswithconstantparasitesduringevolutionandbluedotsrepresentindividualsthathadevolvedwith-

outparasites.Linesshowleast-squaresregressionsfordifferentevolutionlines.Panelsinthefirstrowrepresentmosquitoes

testedathighfood;thoseinthesecondrowrepresentmosquitoestestedatlowfood.Panelsinthefirstcolumnrepresent

linesthathadevolvedathighfoodavailability;thoseinthesecondcolumnrepresentlinesthathadevolvedatlowfoodavaila-

bility.Individualsfromdifferentreplicatesarepooled.

coevno

0 1 2 3

par.e

vo

coevno

0 1 2 3

par.e

vo

0

10

20

30

40

50

0 1 2 3

Long

evity

par.evo

0

10

20

30

40

50

0 1 2 3

Evolution LinesCoevolutionConstant parasiteNo parasite

par.evo

coevno

0 1 2 3

par.e

vo

coevno

0 1 2 3

par.e

vo

0

10

20

30

40

50

0 1 2 3Spore load

Long

evity

par.evo

0

10

20

30

40

50

0 1 2 3Spore load par.evo

Test

ed a

t hig

h fo

odTe

sted

at l

ow fo

odEvolved at high food Evolved at low food


62

Wefoundasignificantpositivecorrelationbetweenresistanceandtolerance(f=4.60p=0.040).Food

availability(f=0.07p=0.795)andtheinteractionbetweenfoodavailabilityandtolerance(f=1.93,p=

0.174)hadnon-significanteffectsindeterminingresistance(Figure4.2).

Figure4:2Tolerancevs.resistance

Therelationshipbetweensporeloadandtoleranceformosquitolineswithdifferentevolutionaryorigin.Notethatthere-

sistanceincreasesfromtoptobottom,soanegativeslopeimpliesapositiveassociationbetweenresistanceandtolerance.

Dotsrepresentmeansporeloadandtoleranceofmosquitoesthathadevolvedathighfoodavailability;trianglesrepresent

mosquitoesthathadevolvedatlowfoodavailability.Redsymbolsrepresentindividualsthathadevolvedwithcoevolving

parasites,yellowsymbolsrepresentindividualsthathadevolvedwithconstantparasitesandbluesymbolsrepresentindividu-

alsthathadevolvedwithoutparasites.Thepanelontheleftshowsmosquitoestestedathighfood;theoneontherightshows

mosquitoestestedatlowfood.Linesshowleast-squaresregressionspooledforalllines.Thep-valuesonthisfigurewerecalcu-

latedforeachfoodtreatmentseparately

4.4 Discussion

InourcolonyofAe.aegyptimosquitoes,toleranceofandresistancetothemicrosporidianparasiteV.

culicis are evolvable traits, so thatmosquito lines that were exposed to parasitism during evolution

showbothahigherresistanceandtolerancetoparasitism.Howeverthewaythesetwodefensetraits

evolve depends on the ecological settings. Thus, restricting food during evolution led to higher re-

sistance,buthadnoimpactontolerance.Furthermore,theecologicalsettingsduringthetestingofthe

−8 −6 −4 −2 0 2 4

0.8

1.0

1.2

1.4

1.6

Tested at high food

Tolerance

Mea

n lo

g−10

spo

re lo

ad

Evolution linesCoevolutionConstant parasiteNo parasite

p = 0.016

−8 −6 −4 −2 0 2 4

0.8

1.0

1.2

1.4

1.6

Tested at low food

Tolerance

Res

ista

nce

p = 0.596


63

mosquitoes also affected theobserveddefense strategies. Thus, food restrictiongenerallydecreased

tolerance,buthadnoeffectonresistance.

Themosquitoes’parasiteloadandthereductionoflongevitywithincreasingparasiteburdenwaslower

forlinesthathadexposedtoparasitesduringevolution,andindicatesageneticvariationinresistance

andtoleranceforthoselines.Thisisinconsistentwithmodelsthatpredictevolutionaryfixationoftol-

erancealleles(RoyandKirchner2000;Milleretal.2006).Atleastfiveotherstudiesfoundevidencefor

variationintolerance(Råbergetal.2007;ValeandLittle2009;HowickandLazzaro2014;Regoesetal.

2014;Loughetal.2015);twonot(Lefèvreetal.2010;Haywardetal.2014).

That tolerance and resistance both increased as an evolutionary response tomicrosporidia infection

suggests that there isnomajor internal constrain in the formofa stringnegativegenetic correlation

betweenthetwotraits.Thepositiveassociationbetweenthetwotraitsobservedafterevolutioncould

beduetoindependentevolutionofthetwotraitsorduetoapositivegeneticcorrelationbetweenthe

twotraits.Wehavenoindicationfromourstudytosuggestwhichofthetwopossibilitiesiscorrect.The

possibilityofapositivecorrelationisplausible,andsuchacorrelationwasfoundinat leastoneother

study (Howick and Lazzaro 2014), although most other studies show either no genetic correlation

(Sternberg et al. 2012;Mazé-Guilmo et al. 2014) or a negative one (Råberg et al. 2007; Vincent and

Sharp2014).However,thefactthatdifferentevolutionaryenvironmentsledtodifferentevolutionary

pathways suggests that anypossible positive genetic correlationwasnot strong enough to constrain

theevolutionofthetwodefensestrategies,sothatthetwocanevolvemoreorlessindependently.The

observedpositivecorrelationalsoemphasizesthatcorrelationsamongpopulationshouldnotbecon-

sideredasevidence for trade-offsorpositive links,as they result fromacombinationof selectionon

bothtraitsandthegeneticcorrelationbetweenthem(SimmsandTriplett1994;RestifandKoella2004).

We foundnodifferences in resistanceand tolerancebetween lines thatwereexposed to coevolving

parasitesor constantparasites.Accordinglyourdatadoesnot supportbothof the self-contradictory

predictions that coevolution either increases tolerance because of its lower impact on the parasites

fitness (Roy and Kirchner 2000) or decreases because parasites would respond to tolerant hosts by

growingfaster(Milleretal.2006).However,thedurationoftheexperimentmighthavebeentooshort

todifferentiatetolerancebetweenlineswithcoevolvingorconstantparasites.

Eventhoughfoodhadnodirectinfluenceonresistance,mosquitoesevolvedresistancemoreeasilyin

resource-poorenvironments,contrastingfindingsthathighfoodlevelstendtoresultintheevolutionof

elevated resistance (Hochberg and Baalen 1998; D. C. Lopez-Pascua and Buckling 2008; Boots 2011;

Harrison et al. 2013), probably because resistance is less costly when there resources are plentiful.


64

However,because inoursystemwell-fedmosquitoesevolvedtobetolerant to infection,mosquitoes

mighthavebenefitedonly slightly fromresistance. In resource-poorenvironments,where the fitness

losswithincreasingparasiteburdenwasmuchhigher,resistantindividualsmighthavebeenunderposi-

tiveselectionand increasedwithtimeinfrequency. Inotherwords,theevolutionarytrajectoryofre-

sistanceindifferentenvironmentscoulddependonhowtheenvironmentinfluencestolerance.These

results are inaccordancewith theprediction thathigher levelsof virulence (inour case triggeredby

resourcerestriction)resultsintheevolutionofincreasedresistance(RestifandKoella2004).Analter-

nativeexplanation is that thecostof resistance isonlyapparent ingoodenvironments.Nevertheless

thefactthatresistancecanmoreeasilyevolveinresource-poorenvironmentissurprisingbecausere-

source-harshenvironmentscanthereforeincreasethehostabilitytodealwithparasites.

Aconsiderablepartofthemosquito’slongevityisexplainedbyfactorsthatarenotrelatedwithparasite

burden.Mosquitoesthatwereexposedtoparasitesduringevolutiongenerallylivedlonger.Thiscanbe

explainedbypotentialcostsofquantitativeresistanceorbyevolvinga longeruninfectedlongevity. In

uninfectedmosquitoes this trendwassimilar (Zeller&Koella inprep.) suggesting that theconditions

duringevolutionaffectedtheevolvedlongevity(generalvigor).

4.4.1 Conclusions

Ourstudyisthefirstexperimentaltest,whichfoundenoughgeneticvariabilityandlowcostthattoler-

anceandresistanceevolvedwhenfacedtoparasitesandunderdifferentresourcelevels.Thefactthat

differentcombinationsofresistanceandtoleranceevolvedindifferentecologicalsettingsillustratesthe

importancetostudythosetraitsacrossenvironmentalvariables.Inadditionbecausemanyofthepre-

dictionsdidnotholdtrueinoursystemunderlinestheimportancetoincorporatesuchenvironmental

heterogeneity, condition-dependent evolutionary costs and non-independence between both traits

whenmodeling the evolution of resistance and tolerance.Our study provides further an example of

how resources and the ability to tolerate parasitesmight interact to determine the evolution of re-

sistance.


65

Antagonisticcoevolutionandre-Chapter5

sourcesalterthehost’slifehistoryevolution





66

Abstract

Earlyreproductionasanadaptiveconsequenceofparasitismhasbeenpredictedbylifehistorytheory.

However,empiricalvalidationofthispredictionis limitedanddirectexperimentalevidencethatpara-

sitescaninfluencethehost’sgeneticallydetermineddevelopmenttimeismissing.Wehavesetupan

evolutionexperimentbylettingAedesaegyptimosquitoevolveeitherwithnoparasite,withaconstant

parasite,orwithaco-evolvingparasite(Vavraiaculicis)witheitherhighorlowresourceavailability.We

testedthehost’slifehistorytraitsoftheevolvedlinesathighandatlowfoodavailabilitywithorwith-

outparasites.Wefoundthatwhenmosquitoeswereexposedtoparasitesduringevolutiontheyhada

shortergeneticallydetermineddevelopmenttime,anequalbodysizeandalongerlongevitycompered

tomosquitoesoriginatingfromcontrollines.Theseresultssuggestnoevidenttrade-offsamongageat

maturity and other traits. We also found no differences in age at maturity between lines with co-

evolvingorconstantparasites,but lineswithco-evolvingparasites livedsignificantly longer.Theenvi-

ronmentalconditionsinfluencedthehost’slife-historiesandthephenotypicplasticresponsestopara-

sitisminmanyways,suggestingthatvariableenvironmentsinfluencethelong-termhostevolution.


67

5.1 Introduction

Theco-evolutionaryinteractionsbetweenhostsandparasitesarerecognizedasimportantfactorsshap-

inghostslifehistoryevolution(Hochbergetal.1992;Koellaetal.1998;KoellaandRestif2001;Gandon

etal.2002;AshbyandBoots2015).Parasitesmodifytheecologicalcontextinwhichhosttraitsevolve

byaffectingcomponentsofthehost’sage-dependentfecundityandmortality.Thisselectionpressure

caninfluencethehost’soptimalpatternofresource-allocation.

Thereisincreasingevidencethathostsmayaltertheirlifehistorytraitsinawaytocompensateforthe

negativeeffectsofparasitism(Minchella1985;Koellaetal.1998;Richner1998;Agnewetal.2000).Life

history theory predicts that earlier reproducing hosts will have a selective advantage, because they

mightbeable toevadeparasitism in timeand,whenparasitized, reduce the impactofparasitismon

reproductive success and survival (Hochberg et al. 1992; Forbes 1993; Perrin and Christe 1996). Life

history traitsof thehost,whichhavebeen shown to respond toparasitism, includeearly versus late

fecundity (Minchella and Loverde 1981; Gérard and Théron 1997; Adamo 1999), reproductive effort

(Sorcietal.1996;PolakandStarmer1998;Krist2001),parentalcare(Christeetal.1996;Richnerand

Tripet1999),bodysize(Lafferty1993;Pontieretal.1998;Arnottetal.2000)anddevelopmentaltime

(Agnewetal.1999; Jonesetal.2008). Insomecasesthesemodificationshadageneticunderpinning

(Lafferty1993;KoellaandAgnew1999),inothers,theywereplasticresponses.However,anoptimiza-

tion in one trait (e.g. maturing early) might be associated with penalties in another trait, such as

maintenance functions, immune-related traits or late-life reproduction. This can be induced by re-

source-allocationtrade-offs,linkagedisequilibriumorpleiotropy.

Suchmodificationinlifehistorycanbeseenasaformofresistance,whichmightleadtocomplexco-

evolutionarydynamicsbetweenthelifehistoriesofthehostandtheparasites.Becauselifehistoriesof

hosts and parasites are at least partly determined by the genotype of the counterpart (Koella and

Agnew1999),anevolutionaryresponseofhost’slifehistorytoparasitismmayagainaltertheselection

pressureoftheparasite.Thisreciprocalselectionmightresultincoevolution,withcontinuouschanges

ofhostsandparasites life-histories (Gandonetal.2008;GabaandEbert2009).Accordingtothered-

queenhypothesistheevolutionaryratesofchangeingenesrelatedtoresistancetraitsshouldbeaccel-

eratedthroughco-evolutionarydynamics.Indeed,genomesofcoevolvinghostshaveshowntoevolve

fastercomparedtopopulationsevolvingagainstaconstantparasitepopulation (Patersonetal.2010;

Kashiwagi and Yomo2011). Theoretical studies reveal that co-evolutionary dynamics can havemany

effectsfortheevolutionofthehostlifehistory(KoellaandRestif2001;Restifetal.2001;Gandonetal.

2002;AshbyandBoots2015),howeverdirectexperimentalevidencethatparasites,and inparticular,

co-evolvingparasitescaninfluencethehost’sgeneticallydeterminedlifehistoryismissing.


68

Contrarilytotheevolvedadaptations in lifehistorytraits,manyhostsrespondtoparasitismandfood

restrictionphenotypicallyplastic (includingAe.aegypti -V.culicis systemthatwestudy).A lotofhosts

decrease their growth, mature later and with a smaller body size once infected (Bedhomme et al.

2004).Inadditiontothelifehistorytraitsthemselves,thedegreeofphenotypicplasticity,theabilityof

singlegenotypestoproducemorethanonephenotypeacrossdifferentenvironments(Pigliucci2001),

isalsogeneticallydetermined.Ifthereexistsadaptivevariationforphenotypicplasticityamonggeno-

types, it is likely to evolve differently under variable conditions. However, how phenotypic plasticity

evolvesexperimentallyhasrarelybeeninvestigated.

Weuseanexperimentalevolutionapproachwith themosquitoAedesaegypti and itsmicrosporidian

parasiteVavraiaculicistoexaminehowthelifehistoriesofthehostchange,bylettinghostsevolvein

responsetoparasitesunderdifferentresourcelevels.Studyingdifferentlevelsofresourcescanberele-

vantbecausepotential trade-offsbetweendifferent lifehistory traitsmightonlybedetectablewhen

resources are scarce. Specificallywewant to investigate: (i)whetheranexposition toparasitesover

severalgenerationleadstoearlymaturinghost’s,(ii)whetherevolvedearlymaturityisassociatedwith

costslaterinlife,(iii)whetherco-evolvingparasitesinfluencethehostslifehistoryevolutiondifferently

thanconstantparasites(iv)andwhetherourmosquitocolonyhasenoughgeneticvariabilityandadap-

tivedifferencestoenabletheevolutionofphenotypicplasticity.

5.2 Materialsandmethods

Theexperimentwasruninaclimatechambersetto26°C,70%relativehumidityanda12hlightand

12hdarkregime.


WeusedthemicrosporidianparasiteVavraiaculicis (providedbyJ.Becnel,USDAGainesville)andthe

UGALstrainoneofitsmosquitohosts,Aedesaegypti(providedbyP,Guérin,UniversityofNeuchâtel).

Vavraiaculicisisanobligate,intracellularparasitethatinfectsinnatureseveralgeneraofmosquitoes,

includingAedes(WeiserandColuzzi1972).ThemosquitolarvaeingestthesporesofVavraiaculiciswith

theirfood,resultingininfectionofgutcellsandepithelialcells.Afteraperiodofreplicationwithinthe

larvaetheparasitesbegintoproducetheirinfectiousspores,whicharetransmittedintwoways.First,

transmissioncanoccurfromlarvatolarvawhensporesarereleasedafterthelarvadies.Thistransmis-

sionrouteisenhancedbyfoodstressorstronginfection(Bedhommeetal.2004).Second,iflarvaesur-

vive the infection to develop into adults the spores can be releasedwhen themosquito dies in the


69

aquaticenvironmentortheycanadheretothesurfaceoftheeggsandinfectthenewlyhatchedlarvae

(Andreadis2007).

5.2.2 Experimentalevolution

Weletthemosquitoevolvefor10generations(i)eitherwithnoparasite,withanexternallymaintained

parasite,orwithaco-evolvingparasite,and(ii)eitherwithhighorwithlowfoodavailabilityduringthe

larval stage.Eachparasite treatmentevolved.The ‘constant’parasiteswere taken fromour standard

linemaintainedinourmosquitocolony.Theco-evolvingparasitesweretakenfromthepreviousgener-

ationoftheinfectedmosquitoes.Eachtreatmentwasreplicatedthreetimes.Thefirstgenerationofthe

experimentwascreatedbyhaphazardlymoving200one-dayoldlarvaefromthecolonytoeachline.

Torearthemosquitos,wehatchedeggssimultaneouslyunderreducedatmosphericpressure.Forthe

firstfourdayswerearedforeach line50 larvae in4petridishes(8cmdiameter)containing30mlof

deionizedwaterinordertoensureasuccessfulinfection.Every24hourswefedthelarvaeeitherwith

ourstandardamountoffood(Day1:0.06mgoftetraminfishfood,day2:0.08mg,day3:0.16mg,day4:

0.32mg,day5:0.64mg,day6orlater:0.32mg)orwithhalfofthestandarddiet.Weexposedthelarvae

toinfectiontwodaysafterhatchingbyadding5.0x106Vavraiaculicissporesin1mldeionizedwater.

In the first generation constant parasite and coevolution treatments received the same solution of

sporespreparedfromthestandardlabcolony.Twodaysafterinfectionthefourgroupsoflarvaefrom

each lineweremovedtoone200*150*50mmplastic traycontaining1.5 literofdeionizedwater.

Pupaeweretransferred intocages(30x30x30cmsize)containingsugarsolutionandacupcontaining

deionizedwater and a piece of filter as an oviposition substrate. Four, six, eleven and thirteen days

afterthedaywhen75%ofmosquitoesofagivenlinepupatedtheyweregiventheopportunitytotake

abloodmealonMZ’sarmfor8minutes.Theeggswereremovedevery48hoursandstoredat26°Cand

70%relativehumidityuntilthestartofanewgeneration.Fortheco-evolvedparasitepopulation(co-

evolution)wecollectedthedeadinfectedmosquitoes(larvaeandadults),groundtheminaneppendorf

tube,countedthesporesandkeptthemat5°Cuntilthenextgenerationofhostswasstarted.Before

startinganewgenerationweeliminatedVavraiasporesfromtheeggsbybleachingtheeggsofalllines

with1%householdbleach.

5.2.3 Measuringresultofexperimentalevolution

After10generationofevolutionwemeasuredtheprobabilityofemergence,ageatpupation,adultsize

and longevityofthemosquitoesfromtheevolved lines.Mosquitoesfromthe18selection lineswere

exposedtoV.culicissporesfromthelabcolonyandfedeitherwiththestandardamountoffoodorwith

halfofthestandarddiet.Werearedthemosquitoesasdescribedabove,withthefollowingdifferences.


70

First,we reared the larvae individually in3mldeionizedwater in thewellsof12-wellplates.Wehad

between219and224first-instar larvaeper line(intotal4005larvae).Eachlarvawashaphazardlyas-

signedtooneofthefourtreatments(between53and56individualspertreatmentandline).Second,

we exposed larvae to the parasite by adding 100µl of a solution containing 2.0 x 106Vavraia culicis

spores perml deionizedwater. Third, pupaeweremoved to 50ml Falcon tube containing deionized

waterandapieceoffilterpaper.Thecupswerecoveredwithmosquitonetting,andcottonwoolmois-

tenedwith10%sugarsolutionwasplacedontothenettingremoistenedevery48hoursandchanged

every72hours.Onedayafteremergence themaleswerediscardedand the femaleswherechecked

everydayforsurvival.Theexperimentwasstopped69daysafterhatchwhenallofthemosquitoeshad

died.


The size of adultswas assayed as themean of theirwing length,which strongly correlateswith the

weight ofmosquitoes (Koella and Lyimo 1996) and is commonly used as an approximation for adult

size.Thewingswereremovedandmountedonmicroscopeslides.Theslidesweredigitallyscannedand

thewingsweremeasuredwiththeopen-accesssoftwareIMAGEJ.


Differencesintheprobabilitiesofemergencewereanalyzedwithgeneralizedlinearmixedeffectmodel

(binomialdistribution) that includedparasite infection, food level, the two factors (parasiteand food

level)duringtheevolutionaryhistoryandalltheirinteractionasfixedfactors.Replicatewastreatedas

randomeffect,nestedwithinevolutiontreatment.Weusedtheglmerfunctionfromthelme4package

fromRv.3.2.3(RDevelopmentCoreTeam,2015).Ageatpupationandlongevitywereanalyzedwitha

mixedeffectsurvivalanalysis(cox-proportionalhazard)thatincludedparasiteinfection,foodavailabil-

ity,parasiteduringevolutionandfoodduringevolutionasfixedfactors,replicatewastreatedasaran-

domeffect,nestedwithintheevolutiontreatments.Intheanalysisoflongevity,weaddedwinglength

asapotentialconfounder.Significantinteractionsbetweenevolutionaryfactorsandtestedfactorsindi-

cate evolutionary differences in phenotypic plasticity.Wing lengthwas analyzedwith a linearmixed

effectmodel(lmerfunctionfromlme4package)thatincludedparasiteinfection,foodavailability,para-

site during evolution and foodduring evolution and all their interaction as fixed factors, replicate as

randomeffect,nestedwithinevolutiontreatment.


71

5.3 Results

Atotalof3716outof4005 (93%)mosquitoessurvivedtoadulthood.1764 (47.5%)of thesewere fe-

males andwere analyzed (between 17 and 33 individuals per line and treatment). Food variability,

parasiteinfectionandtheirinteractionstronglyinfluencedthemosquitos’lifehistory(Table5.1,Figure

5.1,5.2&5.3).Wewillfocushereonhowthefoodavailabilityandthetypeofparasitismduringevolu-

tioninfluencedthemosquito’slifehistoryandhowthesefactorsinfluencedthemosquito’sphenotypic

plasticresponsetofoodstressandparasiteinfection.Becausemanyfactorsandinteractionsinfluenced

themosquitos’lifehistorywedescribeonlytheresults,whichseemmostimportanttous.

5.3.1 Ageandsizeatmaturity

Theageatpupationwassignificantlyinfluencedbythetypeofparasitismduringevolution(Table5.1,

Figure 5.1)).Whenmosquitoeswere exposed to parasites during evolution theymatured at a lower

age.ThiswasparticularlythecasewheninfectedwithV.culicis(significantinteractionbetweenparasite

infectionandtypeofparasitismduringevolution).Tukey’sHSDposthoctestbetweencoevolutionand

constant parasite treatments showed no significant differences for age at pupation (analysis not

shown).Inadditionthetypeofparasitismandfoodavailabilityduringevolutionaffectedthephenotyp-

icplasticresponsetoparasitismandfoodrestriction(significantinteractionsbetweenevolutiontreat-

ments and “tested” treatments). For example, mosquitoes that were not exposed to parasites and

raisedonamplefoodduringevolution(controlhighfood),showedindevelopmentaltimethehighest

phenotypicplasticresponsewhenexposedtoparasitism(forhighandlowfood(Figure5.2aand5.2c).)

Contrary to that, we find the lowest phenotypic plastic response to food restriction for individuals,

whichwerenotexposedtoparasitismandhadlowfoodavailabilityduringevolution(controllowfood,

Figure5.2b).

Thewing lengthwassignificantly influencedby foodavailabilityandby the interactionbetween food

andparasiteinfection.Itwaslowestforparasitizedmosquitoeswithlowfoodavailability.Furthermore

the three-way interaction between parasite infection, type of parasitism and food availability during

evolutionaswell as the three-way interactionbetween foodavailability, typeofparasitismand food

availabilityduringevolutionhadsignificanteffectsindeterminingthewinglength(Figure5.1andTable

5.1).Thephenotypicplasticresponsetofoodrestrictionandtofoodrestrictioncombinedwithparasite

infectionwassmallestforcoevolvedmosquitoeswithhighlarvalfoodavailability(Fig.5.2B&C).Tuk-

ey’sHSDpost hoc testbetween coevolution and constant parasite treatments showedno significant

differences(analysisnotshown).


72

Figure5:1Ageandsizeatmaturity

Meanwinglength±SEagainstmeanageatpupation±SE.Redsymbolsrepresentcoevolutionlines,yellowsymbolsrepresent

lineswithconstantparasites,andbluesymbolsrepresentslineswithoutparasites.Trianglesrepresentlinesthathadlowfood

availabilityduringevolution;squaresrepresenttreatmentswithhighfoodavailabilityduringevolution.Opensymbolsrepre-

senttreatmentsthatwerenotinfected;filledsymbolsrepresenttreatmentsthatwereinfectedwithparasites.Individualsfrom

differentreplicatesarepooled.

7 8 9 10 11

0.22

50.

230

0.23

50.

240

0.24

50.

250

0.25

5

Mean age at pupation (days)

Mea

n w

ing

leng

th (c

m)

Tested at high food

Tested at low food

Evolution linesCoevolutionConstant parasiteNo parasite


73

Figure5:2Phenotypicplasticity

PhenotypicplasticresponsetoA)parasiteinfection,B)foodrestrictionandC)parasiteinfectionandfoodrestrictionforcol-

umnI)ageatpupationandcolumnII)winglength.Phenotypicplasticitywasestimatedforeachevolutiontreatment.We

calculatedthedistancebetweenthemeanvalues±SEfromhighfoodun-parasitizedtreatmentstohighfoodparasitized,low

foodun-parasitizedandlowfoodparasitizedtreatments.Individualsfromdifferentreplicatesarepooled.

5.3.2 Probabilityofemergenceandadultlongevity

Theprobabilitythatjuvenilemosquitoesemergedintoadultswassignificantlyaffectedbytheamount

offoodavailabilityandbytheinteractionbetweenfoodavailabilityandparasiteinfectioninthecurrent

condition(Table5.1,Fig.5.3a-c).Itwasgenerallylowerforparasitizedmosquitoesandunderfoodre-

striction.Parasitesandfoodduringevolutionhadnosignificanteffectsfortheprobabilityofemergence

buttheinteractionbetweenthemwasclosetostatisticallysignificant.Additionallythethree-wayinter-

Index

Control high food

Control low food

Constant parasite high food

Constant parasite low food

Coevolution high food

Coevolution low food

0 1 2 3 4

Index

Control high food

Control low food





0 1 2 3 4

Change in age at pupation (days)

Control high food

Control low food





0 1 2 3 4

Index0.00 0.02 0.04

Index0.00 0.02 0.04

Change in wing length (cm)

0.00 0.02 0.04

I IIA

B

C


74

actionbetweenparasiteinfection,foodandfoodduringevolutionwasmarginallynotsignificant.Tuk-

ey’sHSDposthocbetweencoevolutionandconstantparasitetreatmentsshowednosignificantdiffer-

ences(analysisnotshown).

Theadult longevitywassignificantly lower for infected individualsandwith low foodavailability (Fig.

5.3d-f).Parasiteinfection,foodavailabilityanditsinteractionhadhighlysignificanteffectsindetermin-

inglongevity.Parasitesduringevolutionhadaclosetosignificanteffectindetermininglongevityanda

significanteffectinaffectinglongevityincombinationwithparasiteinfectionandalsowithfoodavaila-

bility.Thefoodavailabilityduringevolutionhadnosignificanteffectsfortheadultlongevity,butitin-

fluenced the longevity in combinationwith parasites during evolution. The coevolved lines generally

lived longer compared to lines kept on constant parasites (Fig. 5.3f). Tukey’sHSDpost hoc testsbe-

tweencoevolutionandconstantparasitetreatmentsrevealedsignificantdifferencesinadultlongevity

(z=2.64,p=0.022).


75

Figure5:3Probabilityofemergenceandsporeload

Meanprobabilityofemergenceof(a)coevolvedversuscontrollines(noparasite),(b)constantparasiteversuscontrollines

and(c)coevolvedversusconstantparasitelines.Meanlongevityof(d)coevolvedversuscontrollines,(e)constantparasite

versuscontrollinesand(f)coevolvedversusconstantparasitelines.Everysymbolindicatesapairwisecomparisonofasingle

treatmentbetweenlinesfromdifferentorigins.Trianglesrepresentlinesthathadlowfoodavailabilityduringevolution;

squaresrepresenttreatmentswithhighfoodavailabilityduringevolution.Redsymbolsrepresenttreatmentsthatwereinfect-

edwithV.culicis,blacksymbolsrepresentstreatmentsthatwerenotinfected.Opensymbolsrepresenttreatmentswithhigh

foodavailabilityaslarvae;filledsymbolsrepresenttreatmentswithlowfoodavailability.Thedashedlineindicatestheexpec-

tationifmortality,respectivelylongevitywouldbeequalforcontrol,coevolvedandconstantparasitelines.Individualsfrom

differentreplicatesarepooled.

Prob. of emergence control lines

Prob

. of e

mer

genc

e co

evol

ved

lines

0.75

0.80

0.85

0.90

0.95

1.00

0.75 0.80 0.85 0.90 0.95 1.00

Prob. of emergence control linesPr

ob. o

f em

erge

nce

evol

ved

lines

0.75

0.80

0.85

0.90

0.95

1.00

0.75 0.80 0.85 0.90 0.95 1.00

Prob. of emergence evolved lines

Prob

. of e

mer

genc

e co

evol

ved

lines

0.75

0.80

0.85

0.90

0.95

1.00

0.75 0.80 0.85 0.90 0.95 1.00

Longevity control lines

Long

evity

coe

volve

d lin

es

22

24

26

28

30

32

34

36

38

22 24 26 28 30 32 34 36 38

Lonegvity control lines

Long

evity

evo

lved

lines

22

24

26

28

30

32

34

36

38

22 24 26 28 30 32 34 36 38

Longevity evolved linesLo

ngev

ity c

oevo

lved

lines

22

24

26

28

30

32

34

36

38

22 24 26 28 30 32 34 36 38

a) b) c)

d) e) f)


76

Table5:1Statisticalsummarylifehistorytraits

Statisticalsummaryforthehosts’lifehistorytraits.Generalizedlinearmixedeffectmodel(binomialdistribution)fordiffer-

encesinprobabilityofemergence,mixedeffectsurvivalanalysis(coxproportionalhazard)forageatpupation,linearmixed

effectmodelforwinglengthandmixedeffectsurvivalanalysis(coxproportionalhazard)fordifferencesinlongevity.Statisti-

callysignificantvaluesaregiveninbold.

Probabilityof

emergence

Ageatpupation Winglength Longevity

Factor df χ2 p χ2 p χ2 p χ2 p

Parasite 1 0.38 0.538 133.67 <0.001 0.02 0.896 104.49 <0.001

Food 1 9.95 0.002 1446.75 <0.001 241.62 <0.001 244.14 <0.001

Evolutionparasite 2 2.91 0.233 7.94 0.019 1.46 0.483 4.88 0.087

Evolutionfood 1 1.00 0.317 2.38 0.123 0.01 0.919 0.00 0.968

Parasitexfood 2 7.34 0.007 40.09 <0.001 5.60 0.018 10.44 0.001

Parasitexevolution

parasite

2 0.12 0.943 18.52 <0.001 3.11 0.211 10.69 0.005

Foodxevolutionparasite 2 1.02 0.601 6.94 0.031 3.40 0.183 6.16 0.046

Parasitexevolutionfood 1 0.03 0.862 6.37 0.012 1.12 0.290 0.35 0.557

Foodxevolutionfood 1 0.91 0.339 8.60 0.003 0.01 0.917 2.20 0.138

Evolutionparasitex

evolutionfood

2 5.14 0.076 3.84 0.146 3.86 0.145 9.57 0.008

Parasitexfoodxevolution

parasite

2 0.11 0.949 0.26 0.878 2.36 0.308 7.73 0.021

Parasitexfoodxevolution

food

1 3.04 0.081 4.01 0.045 0.48 0.489 2.16 0.141

Parasitexevolution

parasitexevolutionfood

2 2.52 0.283 5.76 0.056 6.35 0.042 1.29 0.526

Foodxevolutionparasitex

evolutionfood

2 0.28 0.871 4.06 0.131 7.21 0.027 3.40 0.183

Winglength 1 - - - - - - 1.60 0.206


77

5.4 Discussion

Parasitismandthefoodavailabilityduringevolutionalteredthehost’slifehistoryinmanyways.Mos-

quitoesoriginating from lines thatwereexposed toparasitismduringevolutionpupatedearliercom-

pared to control lines. These results are in accordance with the prediction that earlier reproducing

hostswill evolve to reduce the impact of parasitism (Hochberg et al. 1992; Forbes 1993; Perrin and

Christe 1996). Thiswas particularly the casewhenmosquitoeswere exposed to infection and under

food restriction. In other words, the parasite-induced selection over 10 generations shortened the

mosquitosgeneticallyunderpinneddevelopmenttime.Toourknowledge,thisisthefirstexperimental

evidenceshowingparasiteinfectionleadstoearlyreproducinghosts.Theshifttowardsearliermatura-

tionweobservedhere,mightindeedreducethecostsofinfectionaswithongoingtimeV.culicisprolif-

eratesandproducesdamagingspores(reducedreproductivesuccessandreducedlongevityoffemale

mosquitoes(Reynolds1970,Zeller&Koella,inprep)).

The shift had no negative consequences for the adult body size. Accordingly,mosquitoes that were

facedtoparasitismduringevolutionareabletogrowfaster,whenparasitized.Asthelevelofparasites

virulence is at leastpartlydeterminedby thegeneticbasisofmosquitosageatpupation (Koellaand

Agnew1999),earlyemergingindividualswereunderpositiveselectionwhenexposedtoV.culicis.Simi-

larly,theprobabilitythatmosquitoesemergedintoadultswashigherformosquitoesoriginatingfrom

linesthatwerefacedtoparasitismduringevolution(whenparasitizedandunderfoodrestriction(Fig-

ure5.3a&b)).Inadditiontothatmosquitoesoriginatingfromlinesthatwerefacedtoparasitesduring

evolutiongenerally lived longer,againespeciallyunderparasiteexposureand foodrestriction (Figure

5.3d and5.3e). These results indicate, that the evolvedearlymaturation seemsnot tobe traded-off

withthemosquito’sbodysizeortheadultlongevity.Mosquitoesthatwereexposedtoparasitesduring

evolutiongenerallyshowedashorterdevelopmenttime,anequalbodysizeandalongersurvivalcom-

pared tomosquitoes originating from control lines. It therefore seems thatV.culicis exerts a general

anddirectionalselectionpressureonlifehistoriesofAedesaegypti.However,wedonotknowwhether

V.culicisdirectlyexertsselectiononthemosquitosageatmaturity, longevityorreproductivesuccess,

orwhetherthisistheresultofselectiononacorrelatedtrait.

Wefoundthatcoevolvingparasitesdidnotinfluencethehost’sdevelopmenttimedifferentlythancon-

stantparasites.However,thedurationoftheexperimentmighthavebeentooshorttofindsuchdiffer-

ences.Astrikingresultwas, that individualsoriginating frommostof thecoevolved lines lived longer

comparedtomosquitoesfromlineswithconstantparasites.Oneexplanationcouldbe,thattheevolu-

tionary rate of change in the host’s longevitywas accelerated by co-evolving parasites (evolutionary

arms race).However,when foodavailability during evolutionwas lowandwhen tested at high food


78

levels,themosquitoesfromlineswithconstantparasiteslivedlonger.Thispatternwasconsistentwith

and without parasites. Restricted resources therefore seem to impede the host’s ability to adapt

againstco-evolvingparasites.Accordingly,co-evolutionagainstV.culicisparasitesmightbecostly.

In addition to the evolved adaptations in life histories discussed above, the ecological setting during

evolutionalsoaffectedthemosquito’sphenotypicplasticity.Theenvironmentinthattheyweretested

explained the biggest part of the phenotypic plasticity (development time andwing length). Still we

foundenoughgeneticvariability inphenotypicplasticity toevolveunderdifferentecological settings.

Forexample,coevolvedmosquitoesthathadampleresourcesduringevolutionshowthesmallestplas-

ticresponseinwinglengthwhenfacedwithparasitism.Accordinglyco-evolutionmighthaveincreased

thehost’sadaptiveabilitytodealwithparasites.However,thetrendswedescribehereareverydiffi-

culttointerpretandmightbeinfluencedbyepistasisandpleiotropy(LynchandWalsh1998).Neverthe-

lesstheyillustratethecomplexityanddependencefromenvironmentalconditionofthehosts’lifehis-

toryevolution.


79

SynthesisandfutureresearchChapter6

6.1 Summaryofresults

This thesis shows that environmental variability can influencemany aspects of host-parasite interac-

tions.InthesecondchapterIdescribetheeffectofvariablefoodavailabilityduringthedevelopmentof

themosquitoAedesaegyptionitslife-history.Oneofourquestionsiswhether‘compensatorygrowth’

afteraperiodofundernourishment,whichgenerallyappearstobethoughtofasanadaptiveresponse

(Dmitriew 2011), increases reproductive success.We show, however, that compensatory growth did

notincreasereproductivesuccess.Movingfromhightolowfoodavailabilityalsohadunexpectedcon-

sequences,leadingtolowerreproductivesuccessthanconsistentlybadlynourishedindividuals.Varying

nutrition is thusclearly importanttounderstandpopulationecologyand life-historyevolution. I think

thatlife-historytheoryshouldbeextendedtoincludetheselong-termeffectsofearlynutrition.These

resultsarealso importantbecausesucheffectsofearlynutrition,thatalteradulttraits,can influence

the mosquito’s capacity to transmit vector born diseases (Sumanochitrapon et al. 1998; Alto et al.

2008).Accordingly,ourdatamaybeuseful forpredictingdiseasetransmissionanddevelopingstrate-

giesformosquitopopulationcontrol.

Inthethirdchapterwestudytheeffectoftheenvironmentontherelationshipbetweenthegrowthof

themicrosporidianparasiteVavraiaculicisandthelongevityof itshosts,themosquitoAedesaegypti.

Ourdatasuggestthat,inmostenvironmentswestudy,thereisanegativerelationshipbetweenpara-

site development and host health. Howeverwe show that food availability and age at infection can

changetheeffectofparasitegrowthonhostlongevity.Suchcontext-dependentrelationshipbetween

parasitedevelopment and virulence canhavea considerable impacton theevolutionaryoutcomeof

infectiousdisease.Accordingly,theecologicalconditionswillchangetherelativecostsandbenefitsof

parasitereplicationandarelikelytodetermineadaptivelevelsofvirulence.Therefore,theoreticalstud-


80

iesthatpredicttheevolutionofvirulenceshouldconsider,inadditiontohowtheenvironmentaffects

epidemiologicallyrelevantparameters,alsohowitaffectstherelationshipsbetweenthem.

Inafurtherstep,wetesthowtheabilityofAe.aegyptimosquitoestoresistandtoleratetheV.culicis

parasite evolves in different environments. Unsurprisingly, tolerance and resistance to disease both

increased ifmosquitoeswereexposed toparasites.However, indifferentevolutionary scenarios,dif-

ferentcombinationsofthesetwodefensestrategiesevolved,andindifferentecologicalsettingstheir

expression varied. For example, we found that lines that had evolvedwith low food had higher re-

sistancethanthoseevolvedwithhighfood,buttherewasnodifferenceintolerance.Whenwetested

theevolvedmosquitoes, those thatweregiven restricted foodhad lower tolerance than thosegiven

normalfood,buttherewasnodifferenceinresistance.Suchfindingsillustratetheimportanceofincor-

poratingenvironmentalheterogeneity,conditiondependentevolutionarycostsandnon-independence

betweenbothtraitswhenpredictingtheevolutionoftoleranceandresistance(similarlyto(Carvaland

Ferriere2010)).Ourstudyalsoprovidesanexampleofhowresourcesandtheabilitytotoleratepara-

sitesmight interact todeterminetheevolutionof resistance.Suchfindingsarealsoclinically relevant

andmighthelptoelucidatetheevolutionaryimplicationsoftoleranceandresistancebasedtherapies.

More broadly this chapter should help to increase our knowledge of the environmental and genetic

sources of variation in tolerance and resitance, and how these variations affect fitness. Considering

variableecologicalconditionsisthusclearlyimportanttoundersandhost-parasiteco-evolution.

Foodavailabilityandparasiteinfectionalsoinfluencetheevolutionofthehosts’lifehistory.Weshow

thatparasite-inducedselectionover10generationscanshortenthehostsgeneticallydeterminedde-

velopmenttime.Thisisinaccordancewithlife-historytheory,whichpredictsthatearlymaturinghosts

havefitnessbenefitswhenexposedtoparasitism(Hochbergetal.1992).Toourknowledgethisisthe

firstexperimentalevidenceshowingparasiteinfectionleadstothehostreproducingearlierthanwhen

there isno infection.Mosquitoes thatwereexposed toparasitesduringevolutionhadshorterdevel-

opment times, an equal body size and even a longer longevity compared tomosquitoes originating

fromcontrollines.Thissuggestsnoevidenttrade-offsbetweenthetraitswemeasured.Themicrospor-

idianparasite thereforeseemstoexertageneralanddirectional selectionpressureon thehost’s life

histories.However,thefactthatenvironmentalconditionsduringevolutionandco-evolutionhadmany

effects in theexpressionof thehost’s life-history traits illustrates thecomplexityofhost-parasite co-

evolution. Our results further suggest, that variable environmentsmay influence the long-term host

evolution.


81

6.2 Futuredirections

Inchaptertwowedescribethereproductiveburdenassociatedwithcompensatorygrowth.Anextstep

couldbe toquantifycostsof fastgrowthbycomparingoxidative stressandphysiologicalparameters

(carbohydrates, lipids,proteins)between fastgrowingandnormallygrowing individuals (triggeredby

foodavailabilityortemperature).Similartestscouldbeperformedtoexplorewhetherpartsofthepar-

asitesvirulencearecausedbyaparasite-inducedincreaseinthetotalmetabolicrate,whichincreases

theproductionoffreeradicals,cellulardamageandacceleratesageingprocesses(vanLeeuwenetal.

2010).

Anotheropenquestionconcernshowenvironmentalheterogeneityinfluenceshost-parasitedynamics.

Environmentalconditionscanvarystronglyacrossthehost’shabitat,andhostswithinapopulationare

unlikelytoallexperiencethesameconditions.Ithasthereforebeenarguedthatenvironmentalhetero-

geneitymayaffectthegeneticdiversityofhostsandparasites(WolinskaandKing2009),theseverityof

disease outbreaks and virulence (Duffy et al. 2012). Such expectations could be tested with experi-

mentalevolution.Diseasecharacteristicscouldbecomparedbetweenparasitesoriginatingfromeither

evolutionarylines,wheretheco-evolvedhostsreceivedastandardamountoffood,orfromlineswhere

theco-evolvedhostsreceivedvariableamountsoffood.

Inexperimentalstudiesoftolerance,themostcommonlyusedmeasurementofparasiteburdenisthe

peak parasite density. This is done because parasite load increases with time after infection and is

therefore auto-correlated with longevity and intrinsic mortality rate. However, such measurements

misstheearlyphaseofinfection.Afurtherstepcouldbetoincorporatethegrowthoftheparasiteinto

measurementoftolerance.Onepossibleapproachcouldbetoestimatehowtheprobabilityofdying(at

differenttimepointsafterinfection)isinfluencedbythedifferenceinparasiteloadbetweennaturally

dying and living hosts. Suchmeasurements are further relevant as resistance and tolerance are ex-

pectedtochangethroughoutanindividual’slife(Loughetal.2015).

Asdescribedinchapterfour,toleranceandresistancecanbecorrelatedandincreaseasanevolution-

aryresponsetoparasiteinfection.Studying,whetherthesetraitcovariancesaretheresultfromadap-

tiveresponsestophysiological,environmental,orepidemiologicalfactorsorwhethertheyresultfrom

geneticlinkage(pleiotropy,linkagedisequilibriumorepistasis)couldbeanextstep.Quantitativeanaly-

sisofsuchgeneticcovariance is,as faras Iknow,still lackingandcouldbe investigatedbyecological


82

genomics.Theoreticalmodelscouldbeappliedtoidentifysituationsinwhichgeneticcovariationshould

havestrongco-evolutionaryconsequences.

Additionally,regionsinthegenomewithecologicallyrelevantfunctions,thatplayanimportantrolein

the evolution of tolerance, could be tracked. This could illuminate the genetic architecture of evolu-

tionarytransitionsbetweenantagonism,commensalismandevenmutualism.

6.3 Conclusion

Overallthisthesishasshownthattheenvironmentcaninfluencemanyaspectsofhost-parasiteinterac-

tions,onesthatplayimportantrolesinshapingevolutionarydynamics.Thetopicsthathavebeencov-

ered areof relevance for severalmain areas in evolutionary ecology, including life history evolution,

epidemiologyandresourceecology,andhave implications for futureresearch inthesefields.Bycon-

sideringthatenvironmentalconditionscanvarydrasticallyacrossthehost’shabitat,andbecause“the

only thing that is constant is change” (Heraclitus, ~ 500 BC), the knowledge presented in this thesis

mustbeconsideredto fullyunderstandhost-parasite interactionsandtheirco-evolution. Italsomust

be incorporatedwhenpredictingparasite evolutionandespeciallywhenmanagingparasites. The re-

sults presented here contribute towards a better understanding of host-parasites interactions, but

manyquestionsremain.


83

Acknowledgements

IamverygratefultohavetheopportunitytoworkwithJacobKoella.Itwasaprivilegetowork

inthisinterestingfieldandtocollaboratewithsuchanexperiencedandpassionatescientist.Duringthe

many inspiringdiscussions I learneda lot fromhisclearanalyticalwayofthinkingandfromhisdirect

andveryefficientapproachtoaddressproblems.Ithankfortheguidance,adviceandinspiration.

AspecialthanksgoestoJonforallthecommentsonthemanuscripts,forthemanyfruitful(sometimes

controversial)discussionsaboutscienceandlifeingeneral.Thanksforthegoodtimesatunineandin

themountains. Further thanks go to Giacomo, Gael, Kevin, Antoine,Marion, Nathalie, Elise, Naiara,

Priscille,Adam,Marek,Pitou,Philip,LouisandLorenzforthehelpinthelab,discussions,commentsand

support.

IalsothankThomasFlatt,RolandRegoesandYannickMorêtfortheirvaluablecommentsonthemid-

thesisreportandespeciallyThomasFlattandFabriceHelfensteinfortheexaminationofthisthesis.

AverybigthanksgoestomyfamilyandmyfriendsfortheirsupportoverthewholeperiodofthePhD.

Miri,thanksfortheunlimitedsupportyouhaveprovidedmewithinthelaststressfulmonths,thanksfor

allthelittlethingsyouhavedoneforme,forhelpingoutinthelabduringtheverybuzzydaysandfor

allthepatienceandencouragementsduringtheseyears.Iamsohappytohavemetyou.

Thisworkwasfundedbygrant31003A_144207oftheSwissNationalScienceFoundation(SNF).

Neuchâtel,le6juillet2016


85

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