coevolution along the parasitism-mutualism continuum breum andersen.pdf · the disease pressure of...

F A C U L T Y O F S C I E N C E U N I V E R S I T Y O F C O P E N H A G E N F A C U L T Y O F S C I E N C E U N I V E R S I T Y O F C O P E N H A G E N F A C U L T Y O F S C I E N C E U N I V E R S I T Y O F C O P E N H A G E N

F A C U L T Y O F S C I E N C E U N I V E R S I T Y O F C O P E N H A G E N

Dynamics of ant-microbial interactions Coevolution along the parasitism-mutualism continuum

A dissertation submitted to the University of Copenhagen in accordance with the requirements for the degree of the PhD at the Graduate School of Science, Faculty of Science, University of Copenhagen, Denmark to be defended publicly before a panel of examiners

Sandra Breum Andersen December 2011

Academic advisors: David P. Hughes & Jacobus J. Boomsma

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Preface

This PhD thesis is the result of three years of work at the Centre for Social

EvolutionatUniversityofCopenhagen,pleasantlyinterruptedbyshorterperiods

offieldworkinPanamaandBrazilandastayatPennStateUniversityUSA,under

supervisionofDavidHughes and Jacobus (Koos)Boomsma. Iwas fundedby a

grantfromtheFacultyofScience,UniversityofCopenhagen.

My project proposal was originally aimed at elucidating the role of

Ophiocordyceps fungal symbionts in leaf‐cutting ants, however I gradually

became less and less convinced that these fungi actually had a role to be

elucidatedandwedecidedtomakethescopeofthethesisbroader, includinga

varietyofdifferentmicrobialsymbiontsofants.Iamthusverygratefultobeable

to present a thesis including work on three exciting systems of microbial

symbioseswithants.

Thethesisiscomprisedofasynopsisofthecurrentunderstandingofsymbiotic

interactions, which provides the theoretical framework for the following four

chapters of original empirical works, prepared for publication. A short

concluding section aims at putting the obtained results into a broader

perspective.

SandraBreumAndersen

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Table of Contents

SUMMARIES.....................................................................................................................................7SYNOPSIS ...................................................................................................................................... 11

INTRODUCTION TO SYMBIOTIC INTERACTIONS ............................................................. 13Evolution of interactions –cooperation and conflict .............................................................13Adaptations to symbiotic life ..............................................................................................................15Complexity of multi-species interactions ....................................................................................17

THESIS OBJECTIVES ........................................................................................................................... 20The social insect as hosts and symbiotic partners...............................................................20The model systems ................................................................................................................................22Techniques and fieldwork ...................................................................................................................23Chapter outlines .......................................................................................................................................25

REFERENCES........................................................................................................................................... 26CHAPTER 1.................................................................................................................................... 31

DECONSTRUCTING A DISEASE-DEFENCE SYMBIOSIS: SPECIFICITY AND STABILITY OF ACROMYRMEX-PSEUDONOCARDIA ASSOCIATIONS IN CHANGING ENVIRONMENTS ......................................................................................................... 31

CHAPTER 2.................................................................................................................................... 65DYNAMIC WOLBACHIA PREVALENCE IN ACROMYRMEX LEAF-CUTTER ANTS: POTENTIAL FOR A NUTRITIONAL SYMBIOSIS .................................................... 65

CHAPTER 3 .......................................................................................................................... 97DISEASE DYNAMICS IN A SPECIALIZED PARASITE OF ANT SOCIETIES .......... 97

CHAPTER 4..................................................................................................................................127 ........................................................................................................................................................................127HOST SPECIFICITY OF PARASITE MANIPULATION –ZOMBIE ANT DEATH LOCATION IN THAILAND VS. BRAZIL.......................................................................................127

CONCLUSIONS AND PERSPECTIVES .............................................................................137PICTURES ....................................................................................................................................145ACKNOWLEDGEMENTS ........................................................................................................147CURRICULUM VITAE ...............................................................................................................149

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SUMMARIES ENGLISH

DANSK

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Summary Thelifehistoryofsocialinsects,withdivisionoflabour,cooperativebroodcareandoverlappinggenerations,affectsthestrategiesoftheassociatedsymbionts.Ahigh density of related individuals could be an open invitation to recurrentdisease epidemics, but the complementary layers of social and individualimmunity efficiently protect the society. In this thesis the interaction betweenants and three different microbial symbionts are dealt with, covering thespectrumfromparasitismtomutualismandsomethinginbetween.Twochaptersfocusontheleaf‐cuttingantsnativetoSouthandCentralAmericathat are (in)famous for their ability to defoliate vegetation surrounding theircolonies, making them the dominant herbivores of the region. The leaf‐cutterantsare themostadvancedof the fungus‐growingantsandthis iswhatmakesthemcapableoflivingonleaves:inundergroundchamberstheantsfarmfungusthatdegradethesubstratetheantsbringit,inreturnlettingtheantsfeedonthefungus.Theassociationbetweentheantsandthefungusarethecornerstonesinanintriguingmulti‐trophicinteractioninvolvingalsootherfungiandbacteria.

OneofthesebacterialpartnerscalledPseudonocardiagrowonthecuticleof some leaf‐cutter ants, visible to thenakedeyeas awhitepatchon theants’chest,andusedbytheantsasantibioticfactoriesemployedagainstaparasiteofthefungusgarden.Thediversityofthebacteriaontheantshasbeenthesubjectofsomecontroversy.Wefounda lowdiversitywithonlyonestrainofbacteriadominatingon theants.Bycomparingantscollected in the fieldand in the labfromthesamecoloniesweshowthatthisassociationishighlystable,evenafter10yearsinthelabandexposuretomanyotherbacteria.

Another bacterial partner is of the genusWolbachia and these are liveinside the ants’ tissues.Wolbachia are found associated with a wide range ofinsectsandtypicallyasareproductiveparasite,yetwhattheymaydointheleaf‐cutter ants is notwell understood. Ourwork show that the ants are found ingreatnumbersinsterileworkersandsurprisinglyalsoextracellularlyinthegut,suggestinganewpotentialroleintheant’snutritionalsystem.

In contrast to these likely helpful bacteria carpenter ants in tropicalregionsoftheworldareattackedbyparasiticfungiofthegenusOphiocordyceps.When infected, the ants are manipulated into leaving their colony and die in‘graveyards’,bitingunder leaves.The last twochaptersof thethesisdealswiththe disease pressure of this parasite experiencedby the ant colonies,which isfoundtobesurprisinglylow,andhowthesocialstructureofthehostapparentlyhas shaped the life‐strategy of the parasite into iteroparity. In addition,differences in the manipulation was found between species in Thailand andBrazil, likely reflecting variation in host behaviour and environmentalparameters.Together,thefourchaptershighlightsdifferentwaysinwhichthesymbiontsofantshaveadaptedtothesocialstructureofthehost.

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Resumé Desocialeinsekterslivshistorie,medopdelingafarbejdsopgaver,fællesplejeafafkom og overlappende generationer, påvirker strategierne hos de forskelligesymbionter der associerer med kolonien. En høj tæthed af nært beslægtedeindivider kan synes at være en åben invitation til gentagne epidemiskesygdomsudbrud, men de kompletterende lag af hhv. individuel og socialimmunitet beskytter effektivt kolonien. Denne afhandling ser på hvordan treforskellige symbionter interagerer med myrer, dækkende hele spektret framutualismetilparasitismeognogetindimellem.TokapitlerfokusererpåbladskærermyrernefraSydogCentralAmerika,somerberømteogberygtede for deres evnerudi defoliering af vegetationenomkringdereskolonier,medensådaneffektivitetatdeerdedominerendeherbivorer iregionen. Bladskærermyrerne er de højst udviklede af de svampedyrkendemyrer, og heri ligger forklaringen på hvordan de kan leve af blade: Iunderjordiskekamredyrkermyrerneensvampogdennenedbryderbladeneformyrerne der til gengæld for lov at spise af svampen. Mutualismen mellemmyrerne og svampen er grundlaget for en avanceret fler‐laget symbiose derinvolvererflereforskelligesvampeogbakterier. ÉnafdissebakteriellepartnerekaldetPseudonocardiagrorpåmyrernesexoskelet og kan ses med det blotte øje som en hvid plet på myrernes bryst.Bakterierne bruges af myrerne som mobile antibiotikafabrikker som kanaktivereshvisenparasitisksvampinvadererderessvampehave.Diversitetenafdissebakterierharlængeværetnogetkontroversiel.Voresstudiumfandtenlavdiversitethvorkunénslagsbakteriedominerer.Vedatsammenlignemyrerdervar indsamlet i felten og i laboratoriet fra de samme kolonier viser vi atassociationen er meget stabil, selv efter op til 10 år i laboratoriet omgivet afkoloniermedandrebakterierharmyrernedesammesomifelten. EnandenslagsbakterierafslægtenWolbachialeverindeimyrernesvæv.Wolbachia findes i et bredt udvalg af insekter, oftest som reproduktiveparasitter, men hvordan de påvirker bladskærer myrerne er ikke klart. Voresstudiumviseratbakteriernefindesivævetihøjtantalideellerssterilearbejdermyrerogoverraskendenokogsåekstracellulært ideleaf tarmen.Dettekunnetydepåatbakteriernespillerenrolleimyrernesfordøjelsessystem.

I modsætning til de tilsyneladende hjælpsomme bakterier angribes’tømrer’ myrer i verdens tropiske egne af parasitiske svampe af slægtenOphiocordyceps.Inficeredemyrermanipulerestilatforladedereskoloniogdøi’kirkegårde’ fastbidt i blade. De sidste to kapitler i afhandlingen fokuserer påhvilken effekt svampen har påmyrekoloniens tilstand, hvilken konkluderes atvære overraskede lav, og hvordan myreværtens sociale struktur har påvirketsvampen livsstrategi i retningaf iteroparitet.Dertilkommerensammenligningaf selve myremanipulationen mellem arter fra Brasilien og Thailand, hvor enforskel blev fundet, formentlig afhængig af forskelle i myrernes adfærd ogmiljømæssigevariabler.Tilsammen afdækker de fire kapitler forskellige aspekter af symbionterstilpasningtilensocialmyrevært.

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SYNOPSIS

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INTRODUCTION TO SYMBIOTIC INTERACTIONS Thetermsymbiosisisdefinedasacloseassociationoftwodifferentspeciesover

anextendedperiodoftime,andcoversacontinuousspectrumfromparasitism

to mutualism, as popularized in 1879 by the German scientist De Bary (Sapp

1994).Theimportanceofsymbiosescannotbeoverestimatedfromanecological,

evolutionary or economical perspective. The inter‐species cooperation of

mutualisms represents major evolutionary transitions, allowing some

associations to achieve ecological dominance. Prime examples are the role of

mitochondria in the evolution of the eukaryotic cell following bacterial

endosymbiosis(Margulis1993;Grayetal.2001),themutualismbetweencorals

andzooxanthellaealgaeasthefoundationoftheproductiveanddiversetropical

reefs (Muscatine 1990), and the leafcutter ants farming fungus, the primary

herbivoresinSouth‐andCentralAmerica(Schultz&Brady2008).Similarly,the

majorityofplantsaredependentonmycorrhizalfungi(Smith&Read2008),and

efficient insect pollination is crucial tomany types of plants, including human

crops (Losey& Vaughan 2006). The impact of parasites is no less impressive,

with a cautious estimate of 20‐50% of all extant species employing this life

strategy(Poulin&Morand2000).Contrarytomutualists,parasitesdecreasethe

fitness of their host, making them capable of controlling host population

densities,andtherebyindirectlyincreasingbiodiversityandspeciescoexistence

(Hudsonetal.2006).Howeverwhentargetingagricultureandhumansdirectly

parasitesareamajorcostandburden,asillustratedbytheestimated247million

annualcasesofmalariainducedfever,causedbythemosquitovectoredparasite,

resulting in 881.000 human deaths a year (WHO world malaria report 2008,

www.who.int/malaria).

Evolution of interactions –cooperation and conflict Thesymbiosisspectrumthusrangesfromconflicttocooperationbetweenhost

and symbiont, involving the exploitation of resources and services, such as

protection or transport. The cost‐benefit ratio for each partnerwill determine

the outcome on the parasitism‐mutualism scale, but quantifying this ratio is

difficultasthecurrenciesandexchangeratesmaybefarfromobvious(Herreet

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al.1999).Thisisfurthercomplicatedbyitscontextdependentnature,ase.g.the

valueofagivensymbiont‐providedresource isdependenton itsavailability in

theenvironment.

A useful framework to understand these dynamics, and predict the

outcomeofaninteraction,istheseparationofcooperationandconflictintotwo

parametersinsteadofsimplyviewingthemasopposites(Queller&Strassmann

2009). Intuitively, when cooperation is high and conflict low a mutualistic

relationship is expectedwhile the opposite results in a parasitism. Theremay

however be great differences between symbioses in the levels of both conflict

and cooperation. Both the yucca‐yucca moth and the fig tree‐fig wasp

interactions are highly evolved pollination mutualisms, yet the experienced

conflict is expected to be lower in the latter, as the transfer of host pollen

dependentonmaturewaspoffspringalignhost‐symbiont intereststoagreater

degree. Also, a study of populations of Polistes wasps differing in levels of

predation and social parasitism showed thatwhile conflict between hosts and

parasiteswerealwayshigh,cooperationbynestdefencebetweentheminhigh

predation areas lowered the overall cost of parasitism for the host (Lorenzi&

Thompson 2011). Separating cooperation and conflict also allows for a better

understandingofspecificadaptationsoftheinteractors.Adetailedcomparative

studyofrelatedspeciesoftheabovementionedfigwasp‐figtreemutualismsfor

example revealeddifferent degrees of conflict across the systems, by finding a

correlationbetweenthepresenceofwaspcheatingandhostsanctionswiththe

evolutionofactive,insteadofpassive,pollinationbythewasps(Jandér&Herre

2010).Thisframeworkmaythusmakeiteasiertoexplainhowandwhywee.g.

sometimesseehighlevelsofcooperationinspiteofextendedconflict(Queller&

Strassmann 2009). In addition, the emerging field of synthetic mutualisms,

which employs genetically engineered microbes, provides a novel way of

studying the evolution of mutualisms by varying the costs and benefits

(reviewedbyXavier2011).

Animportantfactorindeterminingtheoutcomeofaninteractionisthemodeof

symbionttransmission(Bull1994).Ifverticallytransmitted,theinterestsofthe

hostandsymbiontarealignedtosomeextent,assymbiontpropagationdepends

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onthehostreproducingsuccessfully.ThisiswellrepresentedbytheAttineant

symbiosis,wherefoundingqueensverticallytransmitthefungalcultivar,andthe

antsrestricthorizontaltransmissionbysuppressingfungalsporulation(Mueller

etal.2004).Incontrast,horizontalsymbionttransmissionmaycreateaconflict

of interests with the host, making a parasitic relationship more likely.

Ophiocordyceps fungi thatmanipulate their ant hosts are extreme examples of

this,asthehostnotonlyneedstodieinorderfortheparasitetoreproducebut

also at the right location (Andersen et al. 2009).However, transmissionmode

doesnot alwayspredict theoutcome, as illustratedby themutualismbetween

termites and their horizontally transmitted fungal cultivar (Aanen et al. 2009)

andtheverticallytransmittedWolbachiabacteriathatparasitesmanyinsecttaxa

(Werrenetal.2008).

Even inmutualistic relationshipswhere the twopartnersachieveanetbenefit

from the interaction, costs are paid and gettingmore for lesswill always be a

desirable strategy (Herreet al.1999).Explaining the stabilityofmutualisms is

thus considered one of the current major challenges of evolutionary biology.

Conditions mediating such stability are suggested to be 1) partner fidelity

ensuring consistency in the association over evolutionary time and limiting

interactions to genetically similar symbionts (Herre et al. 1999), 2) active

partner choice (Sachs et al. 2004) and 3) sanctions against non‐cooperatives

(Kiersetal.2003).

Adaptations to symbiotic life When previously free‐living species come to interact closely in a symbiosis, it

will have evolutionary consequences atmultiple levels. Interactions can range

from loose and facultative to intimate and obligate, with close coevolution

expected among partners partaking in obligate associations. The nature of the

interaction has long been thought to be important for the type of selection

experienced, where parasitism should tend to cause negative frequency‐

dependent selection of host‐symbiont genotypes, favouring diversity and

recombination, while the opposite should serve to stabilize mutualisms by

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maintainingtraitsbeneficialtotheinteraction(Sachsetal.2011).Accumulating

moleculardatahowevershowfrequentexceptionstothispattern,withapparent

similar rates of evolution in parasitic and mutualistic species and e.g.

homologous genes used for establishing host interactions between related

mutualistic and parasitic species (Sachs et al. 2011). The similarities between

mutualisticandparasiticrelationshipswerealsohighlightedinarecentstudyof

bacterialendosymbiontsofweevilbeetles,showinghowthegrowthandspread

of themutualistic bacteriawere strictly controlledbyhost immune genes also

employedaspartofthegeneralimmunedefence(Loginetal.2011).

Ultimatelysymbioticrelationshipscancausegene loss, if the interaction

providesapredictablelevelofservicesthatleavesthehost’sorsymbiont’sown

provisionofthesesuperfluous(Moran2007;McCutcheon&Moran2012).Thisis

true for both parasitic and mutualistic relationships. Examples include the

extensivereductionsingenomesizeandoccurrenceofpseudogenesinparasitic,

intracellularbacterialikeRickettsiaandMycobaterium,thatappeartodependon

the host’s metabolism for essential biosynthesis pathways (Andersson &

Andersson1999).TheRussiandoll‐likemutualismsbetweenabacteriumwithin

abacteriumwithinamealybugisanotherexampleofgenomereduction;neither

bacterial endosymbiont harbours an intact pathway for the biosynthesis of

essential amino acids and are thus completely dependent on each other and

potentiallyalsothehostforcompletionofthenecessarystepsintheproduction

ofthese(McCutcheon&vonDohlen2011).

Inmutualistic relationships the interests of the partners are to a large extent

aligned. Often observed adaptations are the evolution of housing structures in

thehostforthesymbionts,suchasmycangiainbarkbeetles(Six&Klepzig2004)

or bacterial pouches in ants (Billen & Buschinger 2000). Evolution of novel

behavioursarealsocommon,suchasweedingandgroomingoffungalgardensin

leafcutter ants (Currie& Stuart 2001).Above the species level, themutualistic

unionoforganismswithcomplementarytraitsmaycreateacompoundorganism

with a novel,more complexphenotype for selection toworkon (Moran2007;

Zilber‐Rosenberg&Rosenberg2008).Assuch,symbiosiscanbeahighlyefficient

way to acquire novel metabolic capabilities and allow for new niches to be

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exploitedandpotentiallydominated.Lichensillustratethiswell,asthesymbiotic

unionof fungi and cyanobacteriahave allowed them to thrive inhabitatswith

extreme conditions where few other organisms can survive (e.g. Ruibal et al.

2009).Inparasiticrelationshipsthepartners’interestshaveoppositedirection;

while the host attempt to avoid infection the parasite wants to exploit its

resources. This may lead to an arms race, as described by the ‘Red Queen

hypothesis’ (VanValen1973).Thehostandparasite co‐evolve,by respectively

improvingdefencemechanismsversusincreasinginfectivityandtransmissibility,

but the net outcome is effectively the same. An interesting consequence of

parasiteadaptationmaybehostmanipulation(Thomasetal.2005),whichwill

bedealtwithinmoredetaillater(Chapter3&4).

Complexity of multi-species interactions While much of the early literature on symbioses has focused on interactions

betweentwodifferentspeciesinisolation,itisbecomingincreasinglyclearthat

thisisapracticalyetunfortunateoversimplification.Theenvironmentinwhich

the interaction takes place is comprised of both abiotic parameters, such as

temperatureandprecipitation,andtheotherspecieslivingthere,e.g.providing

foodorpredationoradditionalsymbioticcontributions.Theseparametersmay

varyacrossaspeciesrange,potentiallycreatingdifferentselectivepressureson

the symbiotic interaction (Thompson2005;Thompson2010), as in the above‐

mentionedexampleofsocialparasitesofwaspscooperativelydefendingthenest

of their host in the presence of predators (Lorenzi & Thompson 2011). More

studies are emerging that take thisdiversity into account.An impressive long‐

term study by Palmer et al. (2010) on the ant‐acacia symbiosis highlights the

importanceofthesynergisticeffectsofamulti‐species interaction.Acaciatrees

associatewithdifferentantspeciesovertheirlifetime,andtheassociationswith

agivenantspecieshaspreviouslybeencharacterizedonascalefromparasitism

tomutualism.However, by following the assemblageof symbiontsduringhost

ontogeny a more nuanced and surprising result was found. For example,

associatingwith–whatwasconsideredtobe–acastratingparasiteatanearly,

premature,lifestagewasmorebeneficialthanbeingwithoutanyantsatall.The

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succession of symbionts over the entire lifetime of a long‐lived host thus

revealed dynamics not detectable by short‐term observations, a phenomenon

likelytoberelevantinothersystemsaswell.

Also in clearly parasitic relationships the composition of the symbionts

matters. The twomain types of interactions are at the level of exploitation of

hostresourcesandcircumventionofthehostimmunesystem.Multipleparasite

speciesmayeithercompeteforthesamelimitedresource,potentiallyincreasing

virulence,orfacilitateeachotherbyhavingcomplementaryneeds.Likewisecan

infectionbyoneparasiteimpedetheco‐infectionofanotherbyupregulatingthe

host immune response, or facilitatemultiple infections byweakening the host

(Pedersen & Fenton 2007). Such dynamics were observed in a study of four

different parasites in natural populations of voles (Telfer et al. 2010). Strong

correlations were found between the different parasites’ prevalence, where

some facilitated infection by another species, whereas others lowered host

susceptibility to a specific parasite, resulting in an intricate web of parasite

interactions.

Not onlywill a symbiotic association often be affected by the actions of other

species, diversity of the symbionts below species level may also be of great

importance. A host can associate with different genotypes or strains of

symbiontsonatemporalandspatialscale.Inmutualismsit isbelievedtobein

theinterestofthehost,and/orsymbiont, tokeepsymbiontdiversity low,orat

leastkeepdifferentgenotypesseparatedintimeorspace.Thiswilltheoretically

allow for kin selection to increase cooperation among symbionts and limit

unproductivecompetition.Intheattineantsystem,thefungalcultivariskeptas

amonoculture(at thefungus level,notmentioningthevariousmicrobes inthe

garden (Pinto‐Tomás et al. 2009)) and the fungus actively competes with

unrelated fungi by suppressing their growth and affecting the ants’ abilities to

utilizethem(Poulsen&Boomsma2005).

Whenahostisinfectedbymultiplespeciesorstrainsitissuggestedthat

competitionamongthemlikelywillincreasevirulence(Bull1994),ase.g.found

indoubleinfectionsbythetrematodeSchistosomamansonii insnails(Davieset

al.2002).Thisneednotbe thecasehowever,ase.g.observedbyMasseyetal.

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(2004) in double bacterial infections of caterpillars. Here bacterial species

interacting in the host lowers the overall virulence by killing each other with

bacteriocins,withoutharmingthehost.Thetwospeciesarecapableofcoexisting

in the host because of a spatially structured environment, creating distinct

niches for each species. Applying social evolution theory to such microbial

interactions gives very interesting results and predictions, by linking the

necessity for cooperation among bacteria to the production ofmany virulence

factors.Cooperative individualsarepronetoexploitationbyso‐calledcheaters,

that free‐load on the public goods made available by the cooperative effort.

Artificial introductionofsuchcheaters intoapopulationofpathogenicbacteria

may thus lower virulence (Brown et al. 2009; Rumbaugh et al. 2009). With

improved molecular tools, such as next generation sequencing, we are only

beginning to appreciate the true with‐in host parasite diversity. Using this

technique an extended parasite diversity withinmalaria‐infected humans was

thus discovered recently, with unknown implications for the disease

development(Julianoetal.2010).

When studying symbioses it is thus of outmost importance to recognize the

different types of interactions, i.e. at the level of the symbiont (inter‐ and

intraspecificinteractions),thehost‐symbiontassociation,andamonghosts.Add

to this the influenceof theabioticenvironment.Thispotentially leavesuswith

theungratefuljobofattemptingtodisentanglehighlycomplexsystems,butonly

byappreciatingthesedynamicsmaythetruenatureofsymbioticassociationsbe

understood.

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THESIS OBJECTIVES

The social insect as hosts and symbiotic partners Thefocusofthisthesisissymbiosesbetweenantsandmicrobes.Antsbelongto

the social insects, a group dominated by the social Hymenoptera and the

termites,anddefinedbygrouplivingwithoverlappinggenerations,cooperative

brood care by relatives and division of labour with reproductive and non‐

reproductiveindividuals(Crozier&Pamilo1996).Theantsandthetermites in

particularhavesucceededincreatingenormousandlong‐livedsocietiesplaying

alargefunctionalroleintheirgivenecosystems,withtheantsestimatedtomake

up 15‐20% of the terrestrial animal biomass (Schultz 2000). The stable, clean

andprotectedenvironmentofthecolonymakesitanattractivehost,howeverit

alsoprovidesachallengeaffectingthestrategiesoftheassociatedsymbionts.

The high density of related individuals within the colony makes it

particularlypronetoparasiteattack,wasitnotfortheextendedsocialimmune

system that complements the individual immunity. The social immunity of the

colony consists of several lines of defence, firstly avoiding encounters with

parasites, and secondly limiting parasite intake, establishment, and spread

within the colony. This is achieved through behaviours, such as grooming,

compartmentalization of the nest and labour division (Cremer et al. 2007). In

additionithasbeensuggestedthatthefeaturesofthesocietyasahostwillselect

for less virulent parasites, but also less beneficial mutualists (Boomsma et al.

2005;Hughesetal.2008).Thehomeostaticcolonycanbeviewedasanalogous

toatree, long‐livedandmodular,withindividualworkersbeingdispensableas

the leaves of the tree. Thus, while some diseases may be detrimental to the

individual ant only few will kill off an entire colony. On the other hand, the

mutualisticassociatesmaybelessefficient,e.g.byattractingtheirownparasites

andmaintainingselfishinterests.

The list of known ant symbionts is extensive and covers the whole spectrum

fromparasitestomutualistswithobligatetofacultativeassociations(Kronauer

& Pierce 2011). Of the most conspicuous interactions is the tending of

hemipterans forsugarexudates(Styrsky&Eubanks2007), themutualisticand

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parasitic relationships with caterpillars (Pierce et al. 2002) and the fungus

farmingofAttineants(Schultz&Brady2008).Inaddition,thereexistsnumerous

ant‐plantsymbioses(e.g.Oliveira&Freitas2004)suchastheabove‐mentioned

interactions with Acacia trees (Palmer et al. 2010) and the creation of

monoculture ‘devil gardens’, where ants protect the resources of their host

plantsbytheapplicationofherbicidesagainstnon‐hostinvaders(Frederickson

etal.2005).Extensivemolecularscreeningsisalsorevealingagreatdiversityof

microbial symbionts (reviewed by Zientz et al. 2005). Among the first to be

discoveredwhereBlochmannia bacteria inCamponotus ants,whichmost likely

playanutritionalrole.Subsequentlyarangeofotherbacterialspecieshasbeen

foundtobenutritionalmutualistsofants, therebyallowingthemtosurviveon

nutritionallyunbalanceddiets (Cook&Davidson2006). The functionof other

bacterial groups is less well understood such as the newly discovered

Entomoplasmatales bacteria in army ants (Funaro et al. 2011) and the

intracellularbacteriaWolbachiathatare,inadditiontobeingcommonininsects

ingeneral,foundinabroadrangeofantspecies(Wenseleersetal.1998;Russell

etal.2009;Russell2012).

The overall objective of this thesis is to elucidate the implications of

heterogeneity on the interactions between a social host and its microbial

symbionts. Variation in inherited traits is one of the required cornerstones of

evolution, and in symbiotic systems variation may occur between hosts and

among symbionts within and between hosts. In addition, environmental

heterogeneity influence the outcome of an interaction and for symbionts the

environment is comprised of the host and in some cases the external

environment.Inchapter1and2thefocuswillbeonheterogeneityintheshape

of symbiont diversity with‐in and between hosts. If multiple symbiont strains

inhabit the same host there is the potential for both conflict and cooperation

among strains, provided that the symbionts overlap temporally and spatially.

Such interactionsat thesymbiont levelwill likelyaffect thenatureof thehost‐

symbiontrelationship.Incontrast,chapter3and4dealwithhowheterogeneity

inandbetweenhostsaffects symbiont strategy.Thebehaviourof theanthost,

withwell‐protected brood and efficient grooming, in addition to temporal and

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spatial structure in the ants movement, results in a heterogeneous host

population where only a proportion of the colony members are available for

certainsymbionts.Inadditiontothefocusonthesedifferentaspectsofhostand

symbiontheterogeneity,thesystemsstudiedalsospantheparasitism‐mutualism

continuum,allowing theexplorationofhowdifferent symbiontsdealswith the

challengesofasocialanthost.

The model systems Chapter1and2inthisthesisconcernthemonophyleticgroupoffungusfarming

Attineants,whicharefoundinSouthandCentralAmericaandextendingintothe

SouthernUS.ThesymbiosisbetweentheAttineantsandtheirfungalcultivaris

toalargedegreeobligate(alwaysfortheantsbutonlyforthefungalsymbiontof

higher attines), and believed to have originatedmore than 50mill years ago.

Todaymore than230 fungus growing ant species are recognized, divided into

five major agricultural systems. The phylogeny of the Attine ants corroborate

withthesubstrateutilizedbythefungalcultivar,fromlowerattinesthatusedry

organicmattertotheleaf‐cuttingantsofAttaandAcromyrmex,theonlygenera

relying solely on fresh plant material (Schultz & Brady 2008). The fungal

monoculturefarmedbytheantsishoweverchallengedbyparasites,inparticular

the fungusEscovopsis(Currieetal.1999a).Themajorityof theAttineantsuse

antibioticsecretionsfrombacteriagrowingontheircuticletoprotecttheircrop

(Fernandez‐Marin et al. 2009). These bacteria were first believed to be waxy

exudates of the ants’ cuticle butwere subsequently identified as actinomycete

bacteria (Currie et al. 1999b). They are housed in special structures on the

cuticle and apparently supported by gland secretions by the ant (Currie et al.

2006).Thebacterialdiversityandcolonyspecificityof thecuticularbacteriaof

Acromyrmex echinatior is the focus of chapter 1. In addition to the complex

diversityofsymbiontsmentionedabove,theAcromyrmexleafcutterantsharbour

avarietyofWolbachiaendosymbioticbacteria(VanBormetal.2002;VanBorm

et al. 2003; Frost et al. 2010). While Wolbachia are often found to be

reproductive parasites, their effects in ants are not well understood andmay

likely vary between species (Wenseleers et al. 1998;Wenseleers et al. 2002).

23

Chapter2focusesonthedynamicsofmultiplestraininfectionsofWolbachia in

Acromyrmexoctospinosus.

In contrast to the likely mutualistic associates of leafcutter ants covered in

chapter 1 and 2, chapter 3 and 4 deals with a lethal parasite, the fungus

Ophiocordycepscamponotirufipedisthatinfectsandmanipulatethebehaviourof

Camponotus rufipes ants in Brazil. Ants infected with Ophiocordyceps fungi

exhibit a remarkable extended phenotype of the parasite in that they are

manipulatedtobehaveinconcordancewiththeinterestsofthefungus.Infected

antswill thus leave their colonyandbiteontovegetation to allow forparasite

development and transmission. The phenomenon arisedmore than 48million

years ago, as documented by bitemarks on fossil leaves (Hughes et al. 2011).

These Ophiocordyceps parasites have been recognized for a long time since

descriptionsbyWallace in1859(referred inHughesetal2011)andalso form

the basis of myths by indigenous Amazonian people. The occurrence of

‘graveyards’ with particularly high densities of dead infected ants was first

described from Brazil (Evans & Samson 1982) and more recently Thailand

(Pontoppidanetal.2009).Inchapter3theconsequencesofsuchantgraveyards

inregardstotheexperienceddiseasepressureontheantcolonyandthetrade‐

offsbetweenparasitetransmissionandsurvivalisinvestigated,whilechapter4

comparestheparasitcmanipulationintworelatedhost‐parasitesystems.

Techniques and fieldwork Ihaveusedavarietyoftechniquestoobtaintheresultspresentedinthisthesis.

Inchapter1Istudiedthebacterialdiversityonantcuticlesby454sequencingin

the lab of Dr. Lars Hestbjerg KU. The main challenge was obtaining an ideal

extraction of bacterial DNA, representing the natural diversity but avoiding

‘contamination’byintracellularbacteria.Iattemptedblottingthebacteriaofthe

antcuticlewithmoistcottonbutlimitedmaterialobtainedandtheriskofcotton

contamination made this unpractical. In stead, whole cuticle plates were

dissected fromtheantsandattached tissuecarefullyremoved.Thesequencing

24

was performed by Karin Vestberg and analyzed with technical assistance by

SanneNygaard.

Inchapter2,Iusedreal‐timequantitativePCR(RT‐qPCR)tomeasurethe

density of bacterial symbionts across ant life‐stages by targeting a highly

variablegeneandstandardizingforhostcellnumberbyamplificationofasingle‐

copyhostgene.RT‐qPCRisapowerfultechniqueforaccuratelyquantifyinggene

copynumber,andwasperformedinthe labofProf.CornelisGrimmelikhuijzen

KUwiththeadviceofDr.TomGilbertandMichaelWilliamson.Tovisualizethe

bacteria inside the ants I usedhistology and fluorescence in situhybridization

(FISH), which was challenging because of the hard cuticle of the adult ants.

Moreover, the resinusually used for embedding antswasnot compatiblewith

FISHbutanothermaterialproveduseful.Embeddingandsectioningwasdonein

the lab of Aase Jespersen with assistance from Lisbeth Haugkrogh. FISH is

typically performed with short oligonucleotide probes that, in the case of

bacteria, target the gene 16S because of its high copy number in the cell. I

howeverwished to visualize the location of two bacterial strains that did not

differ in their 16S sequence. Using in vitro transcription I thus generated

fluorescently tagged RNA probes that targeted a single‐copy variable gene

(RING‐FISH,Zwirglmaieretal.2004),butunfortunatelytheprobesdidnotwork

successfully. 16S targeted FISH was performed at the veterinary lab of the

TechnicalUniversityofCopenhagenofMetteBoyewithadvicefromMetteBoye,

MarianneRasmussen,JoannaAmenuvorandAnnieRavnPedersen.

During my PhD I have been fortunate to do fieldwork in some extraordinary

places. Field collection ofAcromyrmex leaf‐cutter ants for chapter 1 & 2were

done in Gamboa, Panama in collaboration with the Smithsonian Tropical

Research Institute in2010.CollectionofCamponotus rufipes ants infectedwith

Ophiocordyceps camponotirufipedis for chapter 3 & 4 took place in Mata do

Paraíso, Brazil in collaboration with the Federal University of Viçosa in 2011

with visiting professor Harry Evans and co‐supervisor Dr. David Hughes.

Subsequent data analyses were done at Penn State University USA, in

collaborationwithDr.MattFerrari,hostedbyDavidHughes.

25

Chapter outlines Chapter1:Themajorityofattineantsharbouractinomycetebacteria incryptson their cuticle. The antibiotic production of these is believed to be themaindefenceoftheantsagainstthefungusEscovopsis,aspecializedparasiteoftheircrop. The first analyses suggested that there was only one strain of bacteriawithin each colony. This view has been challenged by other studies. Weaddressedthequestionofbacterialcommunitydiversityandhostspecificityby454sequencinganalysesofcuticularbacteriaofantskeptinthelabforupto10years,andsamples fromthesamecoloniescollected in the fieldover17years.We find that the cuticular diversity is dominated by Pseudonocardia bacteriawithonlyonestrainpr.colonyandtwostrainsinthepopulation.

Chapter 2:Wolbachia bacteria are found as intracellular symbionts in manyinsects,oftenasreproductiveparasitesbutsometimesasmutualists.Manyantsalso harbourWolbachia, but the consequences of these infections are notwellunderstood.WeusedquantitativePCRandfluorescence insituhybridizationtostudy the dynamics of Wolbachia infections across different life stages inworkers of Acromyrmex octospinosus leaf‐cutter ants, which harbour multipleWolbachiastrains.Thenon‐reproducingworkersofAcromyrmexwerefoundtocontain high densities ofWolbachia, and our data suggest that the differentWolbachia strains interact, competing in the immature stages.Wehypothesizethatthisisbecausethedifferentstrainsoccupythesametissuesearlyinhostlife,whiletheyspecializeondifferenttissuetypesintheadultworkers.Thepresenceof largeamountsofextracellularbacteriainthecropofthegutandinthefecaldroplets suggests that Wolbachia in Acromyrmex potentially function as anutritionalmutualist.

Chapter 3: Fungal ant parasites of the genusOphiocordyceps manipulate hostbehaviour to ensure that the host die in an appropriate location for parasitegrowth and reproduction. The existence of graveyards with a high density ofdeadantssuggeststhattheparasiteishighlyvirulent.Wemeasuredtheeffectiveparasite pressure at the ant colony level by studying parasite life‐stagedistributionwithingraveyards.Thisdatawasusedtomodelandexplorethelife‐history trade‐offs experienced by the parasite in the challenge of targeting asocialhost.We conclude that fewparasites are in an infective stage.Thewell‐defendedhostrequireslong‐termpersistenceintheenvironmentoftheparasite,resultinginaniteroparousstrategy,butthecorrespondinglyslowdevelopmentattractsanarrayofhyperparasites,resultinginlowparasitepressure.

Chapter4: Parasitemanipulationofhostbehaviour is expected tobemoreorless fine‐tuneddependingon thesystemscharacteristics.Wecompareparasiteextended phenotypes in two systems of Ophiocordceps fungi infectingCamponotusants. Infectedants inThailandhavebeenobservedtodiewithinanarrow spatial range and experimental manipulation suggested that this iswhere parasite growth is optimal, even though it comes at a trade‐off withtransmissionofpropagules.Wecompare thiswithnewlyobtaineddata fromarelatedsystem inBrazil,wheredead infectedantsare found inawiderspatialrange. We suggest that this is the result of both host and environmentaldifferencesbetweenthesystems.

26

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31

CHAPTER 1

DECONSTRUCTING A DISEASE-

DEFENCE SYMBIOSIS: SPECIFICITY AND STABILITY OF

ACROMYRMEX-PSEUDONOCARDIA

ASSOCIATIONS IN CHANGING ENVIRONMENTS

33

SandraB.Andersen1,LarsH.Hansen2andJacobusJ.Boomsma1

1Centre for SocialEvolution,DepartmentofBiology,UniversityofCopenhagen,Universitetsparken15,2100Copenhagen,Denmark

2MolecularMicrobialEvolution,DepartmentofBiology,Universityof

Copenhagen,Sølvgade83H,1307Copenhagen,Denmark

Correspondingauthors:SandraB.Andersen([email protected])and

JacobusJ.Boomsma([email protected])

Keywords: attine ant mutualism, Pseudonocardia, bacterial community, 454

pyrosequencing

34

Abstract

Fungus‐growing (attine) ants live in a complex multi‐trophic symbiosis that

involvesboth fungalandbacterialpartners.Among theseareActinobacteriaof

the genus Pseudonocardia that are maintained on the ant cuticle to produce

antibiotics, primarily against a parasitic fungus of their garden symbiont. This

associationhasbeenassumedtobeahallmarkofevolutionarystability,butthis

notionhasrecentlybeenchallengedbyculturingandsequencingdataindicating

thattheactinobacterialculturescanberelativelydiverseandvariable.Weused

454pyrosequencingofaregionofthe16SrDNAgenetoestimatethediversityof

the bacterial community on the cuticle of the leaf‐cutting ant Acromyrmex

echinatior and some other sympatric fungus‐growing ants from Gamboa,

Panama.Cuticularbacterialculturestendtobeconcentratedontheventralside

oftheantthorax,sowerestrictedoursamplingtoincludeonlythelaterocervical

platesandpronotumwithabundantbacterialcover.Weusedbothfieldandlab

samples of the same colonies, the latter after colonies had been kept under

laboratory conditions for up to 10 years.We show that the cuticular bacterial

communities are highly colony‐specific and stable over time. Colonies always

had a single dominant Pseudonocardia strain and only two such strains were

found in the Gamboa population across 17 years of sampling, confirming an

earlier study using elongation factor 1α (EF-1α). A number of other

Actinobacteria were identified at low densities in some samples, but no

consistent patterns were observed. We suggest several directions in which

future studies may shed light on the interaction‐specificity of this symbiosis,

whichapparentlycanbeverydifferentacrossspeciesandallopatricpopulations

offungus‐growingants.

35

Introduction

Questions of conflict and cooperation are fundamental for understanding the

evolutionary stability of genomes, societies and interspecific mutualisms

(Bourke, 2011;Burt andTrivers, 2006;Herre et al., 1999). This is particularly

apparentwhen consideringmulti‐species symbioses consisting of amixture of

mutualistic and parasitic partners with potentially diverging fitness interests,

suchasmicrobialgut‐communities(Qinetal.,2010),nitrogenfixingbacteriaof

legumes(Kiersetal.,2003)andmicroparasitesofwildvoles(Telferetal.,2010).

Theprevailingviewof the lastdecadehasbeenthatparasitismandmutualism

are opposites of a continuum of reciprocal exploitation (Herre et al., 1999) as

phylogeniesoftenshowtransitionsinbothdirections(SachsandSimms,2006)

andevenlocalpopulationsofthesamemetapopulationmayrepresentexamples

of net win‐win and win‐loose interactions (Hochberg et al., 2000; Thompson,

1999).

While there is now considerable consensus about the evolutionary

phenomenologyoftheparasite‐mutualistspectrumofsymbioses,thedebateon

therelative importanceofgeneticandphenotypicmechanisms formaintaining

evolutionary stability of mutualistic interactions is ongoing. Mutualisms that

involvelife‐timecommitmentsbetweenasinglehostandsymbiontstraintendto

have efficient competitive exclusion mechanisms to maintain symbionts in

monoculture (Aanen et al., 2009; Poulsen and Boomsma, 2005), so that

symbionts can remainmaximally cooperative (Frank,1996).However, a larger

rangeofmutualismshavemultiplesymbiontstrainsandcontinuetoacquirenew

ones throughout life. Suchdynamicsmayeitherbe anunavoidable liability for

thehostorapotentialassetinallowingamoreflexiblecommunitycomposition

of symbionts.Twomainmechanismshavebeensuggested to secure long term

evolutionarystabilityoftheseinteractions:hostscreeningofsymbiontsfortheir

performance before they are admitted (Archetti, 2010;Weyl et al., 2010), and

host sanctions against underperforming symbionts (Kiers and Denison, 2008;

Kiers et al., 2003). These mechanisms differ in relative importance between

mutualisms, and they make different predictions as to what kind of host

adaptationsforcontrollingsymbiontdiversityweshouldexpecttofind.

36

Thesymbiosisbetweenfungus‐growing(attine)antsandtheirmicrobial

symbiontcommunityisanintriguingexampleofacomplexmutualisminvolving

multiple partners. The ants farm basidiomycete fungi in underground nest

chambers,andarecompletelydependentonthislife‐style,whichevolvedca.50

millionyears(SchultzandBrady,2008).Thecultivarisrearedasamonoculture,

partly controlled by antagonistic behaviour of the ants and resident fungus

towardsunrelatedstrains(Botetal.,2001;PoulsenandBoomsma,2005;Ivens

etal.,2008).Incontrasttotheseeffectivemeasurestopreventcompetitionwith

related, but genetically different symbionts, the fungus garden is relatively

vulnerable to infections by the specialized parasitic ascomycete fungus

Escovopsis (Currie et al., 1999a). Tomeet these challenges, the ants employ a

rangeofbehavioural(CurrieandStuart,2001),chemical(Fernández‐Marínetal.,

2009)andbiological(Currieetal.,1999b)controlmeasures.Thelatterareoften

achievedbytheuseofantibioticcompoundsfromactinomycetebacteria,housed

inspecializedstructuresontheantcuticle(Currieetal.,2006).

Similar to the symbiosis between the ants and their crop fungus, the

associationwiththecuticularactinomyceteshasalsobeenthoughttorepresent

anancientco‐evolvedmutualismcharacterizedbyasinglestrainofbacteria in

each ant colony (Cafaro and Currie, 2005; Poulsen et al., 2005), vertical

transmission with newly eclosed workers and virgin queens obtaining the

bacteria from their sisters and the fungus garden (Poulsen et al., 2003b), and

coevolution between the ant hosts and the bacteria (Cafaro et al. 2005).

However,controversyhasariseninrecentyearsovertheextenttowhich:1.ants

andbacteriaaresufficientlyfaithfultoeachothertomakecoevolutionlikely,2.

theactinobacterialantibioticsarespecificallytargetedtowardsEscovopsisand3.

the growth‐form of a colony’s bacteria is indeed monocultural (Barke et al.,

2010; Cafaro et al., 2011;Mueller et al., 2008; Sen et al., 2009). These studies

showedthatmultipleactinomycetebacteriacanbeisolatedfromsinglefungus‐

growing ants, but also that specific Pseudonocardia lineages are consistently

associatedwithgeneraofattineantsinspiteofhavingfree‐livingcloserelatives

(Cafaroetal.,2011).

The initialstudiesof thediversityof thecuticularbacteriaofattineants

weredonebyculturingandsequencingisolates,whichproducedmonocultures

37

ofwhatwasultimatelyidentifiedasPseudonocardiabacteria(CafaroandCurrie,

2005; Currie et al., 2003). This approachwas criticized for its use of selective

media, limitingthegrowthofotherbacterialspecies thatmightalsohavebeen

present (Mueller et al., 2008). Later culturing studies indeed found a greater

diversity,includingotheractinomycetessuchasStreptomycesandAmycolatopsis

(Barke et al., 2010; Haeder et al., 2009; Kost et al., 2007; Sen et al., 2009).

However, themajoroverall limitationofculturingmethods to inferdiversity is

thatonlyasub‐sampleof the truediversitymaybeable togrowin thechosen

agar‐plateconditions,andthatnomeasureoftherelativeabundanceofcuticular

bacteriaundernaturalconditionsisobtained.

Theseissuescanbeaddressedwithnextgenerationsequencing,allowing

for a potentially unbiased picture of true diversity and species richness of

bacterial symbionts. The first surveys with 454 sequencing included three

fungus‐growing ant genera, Trachymyrmex (4 ants from one lab nest),

Cyphomyrmex (4 ants from one lab nest) andMycocepurus (4 ants from 2 lab

nests) and showed that the ants carried multiple Pseudonocardia species in

addition toawiderangeofotherbacteria (Senetal.,2009).However,a result

likethisisnotnecessarilysurprisingwhenextractsofwholeantsaresequenced,

so that also bacteria living in or passing through the gut become included. As

actinomycetes and other bacteria occur in great diversity in soil and plant

material, itmaybethatsuchsurveyshave limitedrelevanceforaddressingthe

specificity and function of cuticular bacteria in attine ants. Another potential

problemisthatantsthathavebeenkeptinthelaboratorymayhavesecondarily

acquiredbacteriathattheywouldnotassociatewithinthefield.

Inthepresentstudywerevisitthequestionsofdiversity,specificityand

stabilityof ant cuticularbacterial communities (point1and3above), focusing

ontheleaf‐cuttingantAcromyrmexechinatiorthatwasnotincludedinthestudy

by Sen et al. (2009). This species has been relatively well studied for its

phenotypic associations with actinomycete bacteria, which revealed that the

growth pattern of the bacteria on the cuticle of largeworkers of these ants is

verypredictable:Followingeclosurethelaterocervicalplates(theventralthorax

area) are quickly colonized to produce a characteristic bacterial bloom and

entire ants tend to become almost completely covered over the two following

38

weeks (Poulsen et al., 2003a). After this peak density, the bacterial bloom

graduallydisappears,untilagainonlythelaterocervicalplatesarecoveredca.a

month later (Poulsen et al., 2003a). This location appears to be particularly

adaptedtoharbouringthebacterialgrowthbecauseitisspeckledwithcuticular

tubercles that each are supplied with secretions of tiny subcuticular glands

(Currieetal.,2006).

While detailed morphological adaptations such as tubercles to feed

actinomycetesareconsistentwithalonghistoryofbacterialdomesticationand

coevolution (Currie et al. 2006), thisdoesnotnecessarily imply that therehas

been strict co‐cladogenesis. On one hand, the open ‘external’ location of the

cuticular crypts should make it relatively easy for environmental bacteria to

invade, which could lead to considerable symbiont diversity, as e.g. in the

zooxanthellae of corals (Knowlton & Rohwer, 2003) and rhizobial bacteria of

legumes (Kiers et al., 2008).On the other hand, the ants should remain under

strong selection to make their glandular secretions so specific that they

preferentially enhance the growth of bacterial cultures that produce useful

antibiotics,beitPseudonocardiaorotherlineages(BoomsmaandAanen,2009).

Toadvanceourunderstandingof the truenatureanddiversityof thebacterial

cultures on the cuticle of advanced leaf‐cutting ants, we set out to obtain a

precise culture‐independent estimate of the bacterial diversity on the

laterocervicalplatesandadjacentpartsof thepronotumofA.echinatiorandto

comparetheseestimatesacrossfieldandlabsamplesofthesamecolonies.

Materials & Methods

Antsampling

To assess the cuticular bacterial diversity, large worker ants of two age

categoriesweresampled from labcoloniesofAcromyrmexechinatior.First, the

laterocervical plates and pronotum of callow nurse workers were dissected.

Theseantshadrelativelypalecuticlesthatwerecompletelycoveredinbacteria

asistypicalforlargeworkersofA.echinatiorca.2weeksaftereclosing(bacterial

coverscales10‐12,Poulsenetal.,2003a).Theattachedsofttissuewascarefully

39

removed from the internal sides of these cuticular fragments tominimize the

presenceofWolbachiaendosymbioticbacteriathatareabundantinthethoracic

muscles(Andersenetal.,submitted).Fromthesamecoloniesandtwoadditional

ones,wealsosampledanolderantthatonlyhadvisiblebacterialgrowthonthe

laterocervicalplates.Thesewereantswithdarkercuticles,representingscales1‐

3(Poulsenetal.,2003a), i.e. the finalstageofbacterialcoverthat is typical for

foragers. The first set of samples will be referred to as category C (callow)

samples (n = 17, one ant each from 17 different colonies) and the second as

categoryM(mature)samples(n=19;Table1).Thetwocategorieswerechosen

toassesswhetherbacterialdiversitychangeswithantage,i.e.whetherbacterial

diversitywouldbehigheroncallowworkersthatareentirelycoveredinbacteria

comparedtomatureworkersortheotherwayaround.Inaddition,thecollection

ofmultiplesamplesfrom17colonies(and19coloniesoverall),allowedustoask

whethertherearewithinandbetweencolonydifferencesinbacterialcommunity

composition.All antswere sampled fromwithin the fungus gardenof colonies

that had been collected in Gamboa, Panama between 2001 and 2011 and

subsequently kept in four culture rooms in Copenhagen at ca. 25 ˚C and 70%

relative humidity, each containing multiple colonies of different attine ants.

Throughouttheirlaboratory‘tenure’,coloniesreceivedthesamelocallycollected

brambleleaves,fruitfragmentsanddryrice.

For 11 of the sampled lab colonies field collected samples of mature

workers were available, stored in 96% ethanol at ‐20 °C in tubes containing

multipleworkers from the same colony.Onematureworker fromeach colony

was oven‐dried and the laterocervical plates dissected. Freezer samples from

anothersixfield‐collectedantsfromA.echinatiorcoloniesanalysedinthestudy

by Poulsen et al. (2005) were also included, to represent the two clusters of

Pseudonocardiastrainsidentifiedinthatearlystudywheretheelongationfactor

1α (EF-1α) genewas sequenced. These samples are referred to as category F

(field;Table1).InadditiontotheA.echinatiorants,sixlabsamplesrepresenting

fourotherspeciesofattineantsfromthesamestudysite,sharingtheirculture

roomswithsomeofthesampledAcromyrmexcolonies,werecollected.Thesesix

samples contained the laterocervical plates of two samples of Trachymymex

zeteki (three individuals pooled to compensate for bodymasses being smaller

40

than in A. echinatior), two samples of Cyphomyrmex costatus (five individuals

pooled in each sample), C. longiscapus (three individuals pooled) and

Acromyrmexvolcanus(Table1).

DNAextraction

Thedissectedcuticularfragmentswereplacedindividuallyinsterile2mlscrew

lidtubeswith0.1mmglassbeads(MOBIOlaboratories,Inc.)andDNAextracted

withaMasterPureDNApurificationkit(EpicentreTechnologies),whichextracts

Gram‐negative and Gram‐positive bacteria with about equal efficiency

(Rantakokko‐Jalava and Jalava, 2002). In short, 300 µl tissue lysis buffer was

addedandthebacterialmembranesdisruptedinaFastPrepmachinefor45s.at

4.5speed.ThreeµlofProteinaseK(Invitrogen)wasaddedfollowedby>25min

incubation at 65 °C with frequent vortexing. The samples were cooled and

precipitatedaccordingtothemanufacturer’sinstructionsandtheDNAelutedin

35µlTEbuffer.

Amplificationof16SrDNAbyPCRandtagencodedFLX454pyrosequencing

Bacterial DNA was amplified with the general bacterial primers 341F/806R

spanningthehypervariableregionV3(Masoudetal.2011).PCRwasperformed

in a final volumeof 20µlwith4µl 5xPhusionHFbuffer, 0.4µl 10mMdNTP

mixture, 0.2 µl Phusion Hot Start DNA Polymerase (Finnzymes), 1 µl of each

primer(10µM),1µl10xdilutedtemplateandwaterattheconditions:98˚Cfor

30s,followedby35cyclesof98˚Cfor5s,56˚Cfor20sand72˚Cfor20s,anda

finalextensionat72 ˚C for5min.Thesamplesweremoveddirectly to iceand

runona1%agarosegelcontainingEtBrfor50min.Thespecificbandswerecut

andpurifiedfromthegelusingtheMontageDNAgelextractionkit(Millipore).

To each sampleA andB adaptors for emPCRandpyrosequencingwere

added togetherwitha sample‐unique tag inanadditionalPCR.Thisprocedure

wasperformedasaboveexcept that the forwardprimerwas replacedwith59

differentlytaggedforwardprimersandwithonly15cyclesinthePCR.Thefirst

36ofthesesampleswereprovidedwithanAadaptor,LinA_341F_1–36,andthe

reverse primer with a B adaptor LinB_806R, whereas the last 23 samples

received a B adaptor, LinB_341F_58‐80 and the reverse primer with an A

41

adaptorLinA_806R(Masoudetal.2011).ThePCRproductwasrunonageland

purifiedasdescribedabove,afterwhich theDNAconcentrationwasquantified

using a Quant‐iT dsDNA High‐Sensivity Assay Kit and a Qubit fluorometer

(Invitrogen).Ampliconsweremixed to ensureanequal representationof each

sampleand twoone‐region454sequencing runswereperformedonaGSFLX

TitaniumPicoTiterPlate(70X75)usingaGSFLXTitaniumSequencingKitXLR70

according to the manufacturer's instructions (Roche). The A and B tagged

sampleswereprepared for two separate454 sequencing runs,where the first

included all the lab samples of A. echinatior and the second run all the field

samplesandtheotherspeciesofattineants.

Dataanalyses

The data of the two runs were analysed using the QIIME pipeline

(http://qiime.sourceforge.net/index.html#, Caporaso et al., 2010). The raw

sequence data of the two runs were trimmed separately using the default

settings (minimum average quality score = 25, minimum/maximum length

200/1000 bp, removal of forward primer) and sorted by sample ID. The

resulting FASTA files were compiled and operational taxonomic units (OTUs)

werepickedwiththedefaultsettingsusingthe‘uclust’algorithmbasedon97%

similarity, and a representative sequence for eachOTUwas selected using the

‘first’ algorithm. The identified OTUs were aligned with PyNAST, after which

nameswereassignedtoOTUswiththedefaultRDPclassifier.Thisallowedforan

OTUtableandOTUheatmap tobeconstructed. Inaddition, thealpha‐diversity

for each sample was computed and rarefaction curves of the diversity index

Chao‐1(Chao1984)andtheobservedspeciesnumber,asafunctionofsimulated

sequencingeffort,generatedinQiimewiththedefaultsettings.

Wolbachia isaprevalentendosymbiontofAcromyrmexants(Frostetal.,

2010;VanBormetal.,2001;Andersenetal.,submitted)andisnotexpectedtobe

relevantforcuticularbacterialdiversity,soallOTUsclassifiedasWolbachiawere

removed from the data set. As different depths of sequencing were achieved

across thesamples, thepercentprevalenceofagivenOTU ineachsamplewas

calculatedtominimizebiastowardsproportionallyrareOTUsbeingrepresented

bymanysequencesinsomedeeplysequencedsamples.Theseproportionswere

42

used to focus the further analyses on biologically relevant bacteria, by

conservativelynarrowingtheOTUcollectiontoOTUswith>5%prevalenceinat

leastonesample.The identifiedPseudonocardiaOTUsweremanuallyvalidated

against five high‐quality sequences of different Pseudonocardia species

representing the diversity of the genus with Genbank accession numbers

EF114314,AJ252833,AJ252827,AJ249206andAJ252822(Muelleretal.,2010)

in Sequencher 4.7. All gaps and ambiguous base pairs not found in these five

sequenceswereremovedfromtheOTUs.

Statisticalanalyses

Bacterial diversitywas further analysed in JMP9.0.2 forMacOSX. Community

composition was assessed by principal component analysis, using the first

principal component as the ordering variable in a subsequent two‐way

hierarchical clustering with Ward’s minimum variance method. The diversity

withineachsampleaftertheexclusionofWolbachiaOTUswasestimatedbythe

Simpson’s index1‐D calculated as 1 ‐∑(n/N)2withnbeing the frequencyof a

givenOTUinasampleandNthetotalfrequencyoftheOTUswithaprevalence>

5% in the sample. Thediversity indexwas comparedbetween sampleswith a

Two‐WayANOVAwithTukey‐KramerHSDposthoctesting.

Results

Dataquality,readdistributionandPseudonocardiadiversity

In the first runwith 36 samples 266520 sequences out of 446202 passed the

qualitycontrols.Thedistributionofreadspersamplewashighlyskewed,asone

sample contributed 20.5% of the sequences (sample Ae.480M, Fig.1A). The

remaining samples contributed on average 6052 ± 2635 SD sequences. In the

secondrunwith23samples202375sequencesoutof504794passedthequality

controlwithamoreevendistributionof8796±3093SDsequencespersample

(Fig.1A).SomeofthesequencescontainedahighproportionofWolbachiaOTUs,

whichwere removed to focus on the cuticular bacterial diversity (Fig. 1A, red

bars). Rarefaction curves showed that a deeper sequencing likely would have

43

revealedmorerarespecies(Fig.1B,includingWolbachiaOTUs),butthatthedata

provideareliablepictureofthetotaldiversityasestimatedbytheChao‐1index,

astheyincludeallcommonOTUs(Fig.1C,includingWolbachiaOTUs).

After removal of the Wolbachia

sequencesthe2678identifiedOTUsamountedto2491.Whenincludingonlythe

OTUs that contributed > 5% of the total sequences in at least one sample the

diversitywasnarroweddownto35OTUs,comprisingonaverage84±6%SDof

thesequencesofeachsample.AtotalofeightPseudonocardiaOTUsdominated

thebacterial communities,with ameanoverall prevalenceof 70%of the total

sequences in theA. echinatior samples. As the diversity of this genuswas the

focus of the study, these eight OTUs were validated against high‐quality

Pseudonocardia sequences to identify potential errors introduced in the

sequencingorsubsequentanalysis.Themajorityoferrorswerefoundinhighly

conserved regions andvalidation revealed that the eightPseudonocardiaOTUs

identified in the analysis in reality only represented four different

Pseudonocardiastrains,twofoundonAcromyrmexandCyphomyrexants,oneon

A. volcanus and one on C. longiscapus. On A. echinatior, the two strains were

generallynotfoundtoco‐occuronindividualantsorinsinglecolonies.ABLAST

search suggested that also the two Amycolatopsis OTUs found on the

Trachymyrmexantsonlyrepresentedoneactualstrain.TheidenticalOTUswere

thuscollapsedandthedistributionrecalculated.

Otherbacteria

Three samples stood out by containing a large proportion of Archaea bacteria

(26%, Ae.153M), Cyanobacteria/chloroplast DNA (41%, Ae.480M) and

Enterobacteriaceae (Ae.342F), which likely reflects contamination of an

unknownorigin. Someof the samples containedPseudomonas OTUs, but these

wereonly identified in fieldsamplesand labsamplesof lowerattines fromthe

second454 run, suggesting that theymay represent contaminants fromeither

theDNAextractionoraPCRstep.

In addition to the removedWolbachia bacteria, also some other OTUs

weresuspectedtobeofantsoft‐tissueorigin.ThereweretwoRhizobialesOTUs

reachingfrequenciesof35and53%inoneoftheT.zetekiandtheC.longiscapus

44

samples,respectively,witha98‐99%sequencesimilaritytobacteriapreviously

identified as gut symbionts of various ant species (e.g. GenBank acc.no.

FJ477647; Russell et al. 2009), three other Rhizobiales OTUs with closest

matches to environmental samples, and an Entomoplasmataceae OTU that

closelymatchedbacteriafoundinassociationwithantguts(93‐94%similarity,

e.g.GenBankacc.no.HM996870;Funaroetal.,2011;seealsoRusselletal.,2009;

Stoll et al., 2007). In addition, OTUs from the Burkholderiales and

Xanthomonadaleswereidentified,orderswhichalsohavebeenfoundpreviously

inassociationwithants(Russelletal.2009).TheseOTUswereonlyidentifiedin

a minority of the samples and may result from contamination from the

oesophagusduringdissection.However,astheiroriginwasnotconfirmedthey

were included in the analysis. Other OTUs represented Chitinophaga

(Crenotrichaceae,Sphingobacteria)andActinobacteriainRubrobacteraceae(not

identifiedtogenuslevel),Aeromicrobium(Nocardioidaceae),Intrasporangiaceae

(notidentifiedtogenuslevel),Microbacteriaceaea(notidentifiedtogenuslevel)

and Amycolatopsis (Pseudonocardiaceae). None of the Actinobacteria were

identifiedasbelonging to thegenusStreptomyces(Pseudonocardiaceae),which

haspreviouslybeenfoundtobeassociatedwithattineants(Haederetal.,2009;

Kostetal.,2007;Barkeetal.,2010).

Multivariateanalysesofcommunitycomposition

PrincipalcomponentanalysiswasperformedonthefourPseudonocardiaandthe

singleAmycolatopsisOTU.Thefirstprincipalcomponent,explaining53.1%ofthe

variation between samples, was saved and used to subsequently order the

samples in a hierarchical clustering analysis using Ward’s minimum variance

method. The analysis visualizes the clusters of samples with similar bacterial

communities in a heatmap (Fig. 2). Five clusters of attine ant samples were

identified,twowithA.echinatiorsamplesandasingleC.costatussample(cluster

1and2),onewithTrachymyrmex samples(cluster3),onewithamixtureofA.

echinatior,CyphomyrmexandA.volcanussamples(cluster4),andonecomprised

of a single A. echinatior sample (cluster 5, Fig.2). Cluster 1 and 2 were each

dominated by a single Pseudonocardia OTU. Cluster 1 was comprised of all

samples from 10 different colonies (C, M and/or F) and the field‐collected

45

sample from Ae.263. Sample Ae.153Mwas, however, placed as its own group

next to cluster1becauseof thehighproportionofArchaeabacteria.Cluster2

wascomprisedofall samples from fivedifferentcoloniesand the labcollected

sample(s)fromAe.342andAe.356,inadditiontoAe.263C,Ae.280CandAe.480C.

ThefieldsampleofAe.342formeditsowncluster(5,Fig.2)asthissamplewas

dominated by Enterobacteriaceae bacteria, likely of soft ant tissue origin. The

only other bacteria present were the Pseudonocardia OTU from cluster 2,

confirmingthatbothfieldandlabsamplesofthiscolonystillharbouredthesame

cuticularcommunity.SampleAe.263FandAe.263Cclusteredineachofthetwo

main clusters, suggesting a complete change in bacterial community from the

fieldtothelab.However,whetherthesampledantsactuallycamefromthesame

colonyremainstobecheckedwithmicrosatteliteanalysisofthehostDNA.

Thefifthclustercontainedamixtureofsamples,whereAe.26F,Ae.263M,

Ae.280F and Ae.356F were apparently grouped together because of slightly

higherlevelsofsuspectedcontaminantsfromRhizobialesandPseudomonadales,

whilethesesamplesotherwiseclearlybelongedincluster2.Ae.480Mwasplaced

on its own within cluster 4 because of the high proportion of the

Cyanobacteria/chloroplastOTUbutthePseudonocardiaOTUclearlyplaceditin

cluster2.ThefieldsamplefromAe.480andsevenothersamplesfromcluster5

stoodoutbybeingunusuallydiverse.Ae.480FhadlowprevalencesforallOTUs,

withthe30mostprevalentOTUsonlycontributing62%ofthesequencesofthis

sample, potentially indicating low sequence quality. The only Pseudonocardia

OTUidentifiedwasthatofcluster2,placingthesampleinthesameclusterasthe

labsamplesofthesamecolony,eventhoughotherActinomycetalesbacteriaalso

contributed to the diversity as did some Betaproteobacteria from

Burkholderiales.

The bacterial communities of the two

samples from Ae.406 were more complex as they were comprised of

Pseudonocardia primarily belonging to cluster 1 but having also some

representatives of cluster 2, in addition to significant amounts of

Actinomycetales bacteria from Intrasporangiaceae and Nocardioidaceae

respectively. The Nocardiaceae OTU(s), identified as Aeromicrobium, was also

found in Ae.24F and Ae.220M, which were otherwise dominated by the

46

Pseudonocardiafromcluster1and2respectively.Aslightlyhigherproportionof

potentiallynon‐cuticularbacteriafromRhizobialesmayagainpartlyexplainthe

placementoftheC.longiscapusandC.costatussamplesinthemixedcluster4,in

addition to various Gammaproteobacteria and Sphingobacteria of unknown

functionandimportance.ThemostprevalentPseudonocardiaOTUonC.costatus

belonged to cluster 2, while the C. longiscapus sample carried another unique

Pseudonocardia and the Amycolatopsis from the Trachymyrmex colonies in

smalleramounts.TheA.volcanussamplewasinterestingasitharbouredthetwo,

otherwise not co‐occuring, main Pseudonocardia OTUs, in addition to an OTU

specifictothissample,allinaboutequalamounts.

Samples from six field colonies previously analysed by Poulsen et al. (2005)

wereincludedintheanalyses.Oneofthesewasassignedtoclusterone(Ae.33F),

which is likely also where Ae.24F belonged, three were assigned to cluster 2

(Ae.47F, Ae.26F, Ae.112F), while the last (Ae.44F) could not be confidently

placedineitheroftheclusters.ThefindingoftwoPseudonocardiaOTUsandthe

clustering of Ae.33F with Ae.24F, and Ae.47F with Ae.26F and Ae.112F thus

replicatedtheresultsofPoulsenetal.(2005),astudythatonlyusedtheEF-1α

gene,furthercorroboratingthevalidityofthetwomainPseudonocardiaclusters

inourGamboapopulation.

PhylogeneticplacementoftheAmycolatopsisandPseudonocardiaOTUs

ThetwosampledcoloniesofTrachymyrmexantsprimarilycarriedAmycolatopsis

Actinobacteria with a 100% identity to various environmental samples,

representing91%and52%oftherespectivesequences.ThesameAmycolatopsis

wasalso found toaminordegreeonC. longiscapus (3%).Oneof theT. zeteki

samplescontained37%Rhizobialesbacteria,whicharelikelycontaminantsfrom

the ant tissues not properly removed during dissection of these small ants,

suggesting that theAmycolaptopsisbacteriadominateon thecuticle. Itwasnot

possibletocomparetheentireAmycolatopsissequencetothoseobtainedbySen

etal.(2009)fromMycocephorusattineants,asadifferentregionofthe16SrDNA

wastargeted.Howevera166bpoverlapbetweentheOTUandGenBankacc.no.

FJ948128showedonly93.4%similarity.NoPseudonocardiaOTUswerefoundat

47

a prevalence > 5% in the Trachymyrmex samples. Cafaro et al. (2011) found

Pseudonocardia onT. cf. zeteki but it is not clearwhether this is the same ant

speciesasthatofthepresentstudy.

The four Pseudonocardia OTUs were

tentatively assigned to the six known Pseudonocardia clades associated with

attine ants described by Cafaro et al. (2011). The OTU from cluster 1 was

identical to Pseudonocardia strains from Cafaro’s clade VI isolated from

AcromymexandafewTrachymyrmexspecies,whiletheOTUfromcluster2was

most similar (99%) to strains from Cafaro’s clade IV and V isolated from

primarilyApterostigma,Trachymyrmex andAcromyrmex ants.TheOTU fromA.

volcanuswasalsomostsimilar(99%)tostrainsfoundincladeIVwhiletheOTU

from Cyphomyrmex was most similar (99%) to strains in Cafaro’s clade III

isolatedfromCyphomyrmex,TrachymyrmexandMycetarotes.Thetwodominant

Pseudonocardia OTUs fromA. echinatior had 98% sequence similarity but the

alignmentintroducedfourgaps,makingthetwoOTUsclearlydistinguishable.

Comparingdiversitybetweensampletypesandclusters

ThebacterialdiversityonA.echinatiorcuticlesasestimatedwiththeSimpson’s

indexwascomparedbetweencallowworkersfromthelabandmatureworkers

from the lab and the field and between the twomain clusters by a Two‐Way

ANOVA. Only samples that had been placed in either cluster 1 or 2 by the

hierarchicalclusteringanalysis(Fig.1)wereincluded.Therewasnosignificant

difference in the diversity index between the callow and mature individuals

sampled from the same cluster, but the diversity of lab callow workers from

cluster 1 was significantly lower than that of mature and field samples from

cluster2(F5,32=5.09,p≤0.05;Table3).AOne‐WayANOVAwithTukey‐Kramer

post‐hoc testing including all A. echinatior samples found an overall lower

diversity inthesamplesofcallowworkers(F2,50=10.12,p≤0.05). Whenonly

lookingatthelabcollectedsamplestherewasnocorrelationbetweenhowlong

theantshadbeenkeptinthelab(measuredasyearssincecollection)andtheir

cuticularbacterialdiversity(Lin.reg.cluster1:R2=0.057,p=0.92;cluster2:R2

=0.11,p=0.17;allsamples:R2=0.0087,p=0.59).

48

Of the Acromyrmex colonies from the

lab thatwere assigned to either cluster 1 or 2, nine had been kept in climate

roomsprimarilywithleaf‐cutterants(room‘Acro3’androom‘Atta’)andeight

together with a variety of lower attines and non‐leafcutter attine ants (room

‘Acro1’androom‘Q’;Table4).Therewasnostatisticallysignificantcorrelation

betweenwhichroomthecolonyhadbeenkeptinandwhichclusteritsbacterial

community belonged to (Likelihood ratio test, χ21,17 = 1.55, p = 0.08). A

suggestivetendencytowardscolonieswithacluster1communitytohaveshared

rearingroomswithonlyleaf‐cutterantsandcolonieswithacluster2community

to have shared rooms with other attine ants is expected to be merely

coincidental,as thecolonieswereplaced in theserooms innoparticularorder

andallretainedthebacteriathattheyhadinthefield.

Discussion

Bacterialdiversityontheantcuticle

Multivariate analyses of the bacterial communities identified two

Pseudonocardia OTUs that dominated the cuticular diversity of Acromyrmex

echinatiorbutalmostneverco‐occuredinthesamecoloniesoronthesameants

(Theexceptionbeing the fieldand labsamples fromAe.263placed indifferent

clusters and sample Ae.406M and F, see Results; Fig. 2). This confirms the

conclusionsdrawnfromstudiesthatemployedculturinganddirectsequencing

of only one Pseudonocardia strain pr. colony (e.g. Poulsen et al., 2005). The

associationbetweenA.echinatior coloniesand theirPseudonocardia strainwas

apparentlyverystable,withcoloniesthathadbeenkeptinthelabforuptoten

yearsineachother’scloseproximity,retainingtheiroriginalstrainsinallcases

butone, inspiteofampleopportunities forhorizontal transmission.Thesingle

exceptionwill need to be checked to seewhether it indeedwere ant samples

fromthesamecolonysampledinthefieldandthelab.

Thecoloniesusedinthepresentstudyhadbeencollectedfromthesame

fieldsiteoveraperiodof17yearswithbothstrainsbeingsampledacrossthat

period, confirming the stable co‐occurrence of both strains in the Gamboa

49

Acromyrmex population. Analysis of less conserved genes than the 16S rDNA

region analyzed here may reveal a greater diversity within and between

colonies, but sequencing of themore variable geneEF-1α also found just two

Pseudonocardia strainsonA. echinatior and its sister speciesA.octospinosus in

the study area (Poulsen et al. 2005), so we consider the results of the two

combinedstudiestoberobust.

Other Actinomycetales bacteria were found to be inconsistently and

rathersparselyassociatedwiththeattineantsthatweanalysed.Infoursamples

(three from the fieldandone froma labcolony)OTUs fromMicrobacteriaceae

and Nocardiaceae reached densities of 12‐23%. The closest BLAST search

matches to these OTUs were different environmental isolates. Relatively low

densitiesofPseudonocardiabacteriawere found inthesesamples,butwhether

thegreateroccurrenceoftheotheractinomycetesisthecauseortheeffectofa

limitedPseudonocardia coveron these respective ants remains tobe explored.

Thepossibleecologicalimportanceoftheseandothernon‐PseudonocardiaOTUs

remainsunknown,butco‐occurrenceofbacterialspeciesontheantcuticlehas

been hypothesized to be facilitated by the sharing of resistance genes against

bacteriocins(Barkeetal.,2011).

No Streptomyces bacteria were observed in any of our lab or field

samples. This contrastswithwhat has been found onA. octospinosus workers

fromTrinidadandTobagoafter culturingwashes from field‐samples (Barkeet

al.,2010),fromthesamespeciesinFrenchGuianabyrubbingantcuticlesagainst

agar plates (Kost et al., 2007), from fungus gardens of A. echinatior and A.

octospinosus,andfromtoA.volcanusworkersfromthesamesiteasthesamples

ofourpresentstudy,GamboainPanama(Haederetal.,2009).Thismayreflect

geographicaldifferencesorthatthesebacteriawerelocatedonotherpartsofthe

cuticle(thanthelaterocervicalplatesandpronotum),orinthefungusgardenas

suggestedbyHaederetal.(2009).Streptomycesmaythereforenotbeassociated

withA.echinatioror, ifonly foundat lowfrequency inthe field,havebeen lost

from the cuticle during storage in ethanol. The Streptomyces previously found

associatedwith antswere hypothesized to be regularly acquired from the soil

(Barkeetal.,2010),suggestingthattheymayalsobelostmoreeasilywithoutthe

possibilityofreacquisitioninthelab.

50

Communitycompositionacrosslifestagesandenvironments

Nodifferencewas found in thediversity indexbetweenmatureworkers in the

lab and field, suggesting that the transition does not drastically affect the

diversityofcuticularsymbionts.Theslightlylowerdiversityfoundonthecallow

workers fromcluster1 is likely the resultof ahigherproportionofWolbachia

sequences in these samples, as a larger surface of cuticle and thus potentially

more soft tissue was included in the samples of callow workers. Fewer

sequencesoverallwouldthuslimittheprobabilityofsequencingrarerOTUs.

Horizontal transmission between colonies and species under lab

conditions could not clearly be inferred from our present data. A. echinatior

colonies with communities from each Pseudonocardia cluster are apparently

capableofmaintaining theiroriginalPseudonocardia symbiontevenwhenkept

in close proximity (see above). The Trachymyrmex species and C. longiscapus

carriedanentirelydifferentgenusofActinobacteria,Amycolatopsis, inaddition

to a specific Pseudonocardia OTU in the case of C. longiscapus. There was

extensive overlap in the actinomycete communities of A. echinatior and C.

costatusbutitwasnotpossibletoevaluatewhetherthishadalsobeenthecase

inthefield.A.volcanuswasinterestinginapparentlyharbouringthreedifferent

Pseudonocardia OTUs in similar prevalences. A. volcanus has only rarely been

encounteredatthestudysiteandistypicallyfoundnestinginwoodcavitiesthat

areusuallyinthecanopy.Theforagingworkersareclearlydistinctfromthoseof

A. echinatior and A. octospinosus by being almost black and covered in white

bacterial growth throughout the mature stage. How and why this growth is

sustained remains unclear, but maintaining high degrees of bacterial bloom

throughoutolderworkersmaywellchangethebacterialinteractionsandonthe

antcuticleandpotentiallyenableco‐occurrenceofotherwisesegregatingOTUs.

The potential for colony‐level

adaptations to thecuticularbacterialcommunitywasnotdirectlyaddressed in

this study, but our results indicate that this is a possibility. The ca. 50:50

distribution of the two Pseudonocardia strains in the population across time

suggests that some formof balancing selectionmayact on thePseudonocardia

communitycomposition.Cross‐fosteringexperimentshaverecentlyshownthat

51

thebacteriagrowlesswellonnewlyeclosedantsdevelopinginanothercolony

than theirown,although thePseudonocardiaof thedifferentcolonieswerenot

identified (Armitage et al. 2011). Such an approach in colonies with known

bacterialcompositioncouldbeusedinfurtherattemptstoelucidatewhetherand

how the ants manage and control their interactions with Pseudonocardia and

otheractinomycetebacteria.

Conclusions

Inthepresentstudyweexploitedthepotentialofnextgenerationsequencingto

obtainpreciseestimatesofthediversityofbacterialcommunitiesonthecuticle

ofPanamanianAcromyrmexleaf‐cuttingants.Weshowthatdiversitypercolony

is relatively low and always revolves around a single dominant strain of

Pseudonocardia, consistent with earlier findings by Poulsen et al. (2005). The

relativelylowdiversityofactinomycetes,incomparisonwithestimatesbySenet

al. (2009),Kostetal. (2007)andBarkeetal. (2010),maybeduetoourrather

precisetargetingofthecuticularregionwiththemostexplicitconcentrationof

glands feeding the actinomycete symbionts. However, even after taking

substantial care to include only cuticular bacteria it was difficult to avoid

contaminations with other bacteria such as thorax muscleWolbachia and gut

bacteria.ThissuggeststhattheuseofextractsofwholeantsasemployedbySen

et al. (2009) may not tell us much about symbiotically relevant diversity of

bacteriaonthecuticle. Inaddition,wediscoveredthattheslightlyhighererror

rates in pyrosequencing, compared to Sanger sequencing, offered considerable

challengesindataanalysis,whichcouldonlyberesolvedbymanuallyseparating

very closely related strains. These manual validations of OTUs produced

considerably lowerdiversityestimates thanthoseobtained fromtheautomatic

analysesoftherawsequencedata.Weconclude,therefore,thattheAcromyrmex

Pseudonocardia symbiosis in our study population combines intriguing

characteristics of long‐term interaction‐ specificity with indications that

horizontal acquisition of other Actinobacteria may indeed happen at low

frequencies.Itisthustooearlytodismissthatco‐evolutionaryinteractionsmay

be frequent, even though they will only broadly result in co‐cladogenesis,

consistentwithfindingsbyCafaroetal.(2010).

52

Acknowledgements

We would like to thank Karin Vestbjerg for assistance in the lab, Panagiotis

Sapountzisforcomputerassistance,SanneNygaardandMartinAsserHansenfor

bioinformaticadviceandSørenSørensen foraccess to the454pyrosequencing

facilities.SBAwasfundedbyaPhDScholarshipfromtheScienceFacultyofthe

University of Copenhagen. SBA and JJBwere further supported by the Danish

NationalResearchFoundation.

53

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57

Figure legends Figure1

Data obtained by 454 sequencing. (A): Histograms showing the number of

sequencesobtainedforeachsampleafterqualitycontrolforallsequences(blue

bars)andafterWolbachiaOTUshadbeen removed (redbars).The sampledA.

echinatior colonies are arranged along the x‐axis in chronological order of

collection,spanningaperiodof17years(1994forAe.24Fto2011forAe.529),

with the other attine ant species added towards the right. The name tags

representingthesamplesfromthefirstrunareinfullcolourwhilethesamples

from the second run are in lighter shades. (B, C): Rarefaction curves of

sequencing depth (includingWolbachia OTUs). The curves each represent an

individual sampleandshow theobservednumberofOTUs (B)and theChao‐1

indexofdiversity(C)asafunctionofsimulatedsequencingeffort.

Figure2

Hierarchical clustering of the bacterial communities on the cuticle of

Acromyrmex echinatior and other attine ants. On the x‐axis are the 30 most

common OTUs, with the different taxonomic groups highlighted. The

dendrograms illustrate clustering in the samples and not phylogenetic

relationship between the OTUs. On the y‐axis are the sampled ants, with the

frequency of each OTU illustrated by a color scale from blue (= zero to low

frequency) over gray to red (= high frequency to complete dominance). The

colourof the sample IDs towards the left and thebranchesof thedendrogram

indicatetheannotationobtainedbyhierarchicalclusteringanalysis;theclusters

arenumberedasmarkedontheleftsideofthesamplenames.Fiveclusterswere

identified;cluster1and2wereeachdominatedbyonePseudonocardiaoutthat

wasnotfoundintheothercluster(thetentativeplacementofthesetwoOTUsin

thecladesofthephylogenybyCafaroetal.(2011)ismarkedbeneaththecluster

number). Cluster 3 contained the Trachymymex samples, which harboured

Amycolatopsisbacteria,whilecluster4containedamixtureofsamplesgrouped

together because of a higher non‐Pseudonocardia diversity. Some of this

diversity could be explained by the presence of OTUs suspected to reflect

contaminations rather than cuticular diversity. For these nine samples the

58

suggestedplacementineithercluster1or2ismarkedbymatchingthecolourof

the sample name tag to that of cluster 1 (green) or 2 (blue). Cluster 5 was

comprised of a single sample, Ae.342F, with a high prevalence of

Enterobacteriaceae; the Pseudonocardia OTU of this sample indicated that it

shouldbeplacedincluster2.

FigureS1(onlyaccessibleviathepdfversionofthisfile)

TheOTUheatmapshowingthedistributionofthe2678OTUsobtainedfromthe

antcuticlesamplesandthenumberofsequencesthateachsamplecontributed

toeachOTU.

59

Figures

Figure1

60

Figure2

61

Tables Table1

Colony ID, sample tag (C = callow workers for which both the laterocervical

platesandthepronotumweredissected;M=matureworkersforwhichonlythe

laterocervical plates were dissected; F =matureworkers from field colonies),

collection year (Gamboa, Panama), and the name of the ant rearing room in

which the sampled colony had been kept (Atta, Acro 1, Acro 3, Q). Ae. = A.

echinatior,Av.=A.volcanus,Tz=T.zeteki,Cc.=C.costatus,Cl.=C.longiscapus

ColonyID Collectionyear Categories

sampledNameofantroominthelab

Ae.24 1994 F Ae.26 1994 F Ae.33 1996 F Ae.44 1996 F Ae.47 1996 F Ae.112 2000 F Ae.150 2001 C,M,F AttaAe.153 2001 C,M AttaAe.160 2002 C,M,F Acro1Ae.220 2004 M Acro3Ae.263 2004 C,M,F Acro3Ae.280 2004 C,M,F Acro3Ae.282 2004 C,M,F Acro1Ae.322 2006 C,M,F Acro3Ae.331 2007 C,M Acro1Ae.335 2007 C,M AttaAe.342 2007 C,M,F Acro3Ae.356 2008 C,M,F Acro3Ae.406 2009 M,F Acro3Ae.420 2009 C,M Acro3Ae.480 2010 C,M,F Acro1Ae.488 2010 C,M Acro1Ae.505 2011 C,M QAe.528 2011 C,M,F QAe.529 2011 C,M QAv.520 2011 M QTz.15‐022010‐2 2010 3xM QTz.022‐0509 2009 3xM Acro1Cc.RMMA100603‐04 2004 5xM QCc.011‐170507 2007 5xM Acro1Cl 3xM Acro1

62

Table2

Phylogeneticdistributionofthe35OTUsthathadprevalencesof>5%inatleast

onesampleafterexclusionofWolbachiaOTUs.TheeightPseudonocardiaOTUs

and two Amycolatopsis OTUs were subsequently collapsed into four and one,

respectively, followingmanualvalidationagainstreferencesequences.Foreach

group, themeancontributionstothe59samplesaregivenaspercentages.The

Amycolatopsis OTUs were only identified in Trachymyrmex and C. longiscapus

samples.

Phylum Order/Family/Genus Numberof

OTUsMean%contributionpersample±SD

Actinobacteria Pseudonocardia 8 66±26% Intrasporangiaceae 2 1±3% Microbacteriaceae 1 2±3% Nocardioidaceae 3 3±5% Amycolatopsis 2 2±14% Rubrobacteracea

Unknown11

0±1%0±1%

Proteobacteria Rhizobiales 5 2±10% Pseudomonadales 2 1±3% Xanthomonadaceae 2 1±5% Rhodospirillales

BurkholderialesEnterobacteriaceae

111

0±2%0±2%1±8%

Bacteroidetes Chitinophagaceae 2 0±2%Tenericutes Entomoplasmataceae 1 0±3%Euryarchaeota(Archaea)

Methanosaetaceae 1 0±3%

Cyanobacteria/cloroplast

1 2±6%

63

Table3

Results of a Two‐Way ANOVA with the Simpson diversity index of cuticular

bacteriainA.echinatiorsamplesfromcluster1and2asdependentvariableand

callowworkersfromthelab,matureworkersfromthelab,andmatureworkers

fromthefieldascategories.

Dependent

variable

Source F d.f. P

Diversity

index1‐D

Cluster(1or2)

Antcategory(C,MorF)

Antcategory*cluster

2.77

4.82

3.46

2,32

1,32

2,32

0.106

0.0148

0.0437

Table4

The species distribution of attine ants across the culture rooms in which the

colonies had been kept for 1‐10 years before their cuticular bacterial

communitiesweresampled.

Species/rearingroom Acro1 Acro3 Q AttaOnlyleaf‐cuttingants(3Atta*species,4Acromyrmexspecies)

17 21 19 24

Highernon‐leaf‐cuttingattineants(2Sericomyrmexspecies,3Trachymyrmexspecies)

Ca.50 0 Ca.35 0

Lowerattineants(4Cyphomyrmexspecies,2Apterostigmaspecies)

Ca.50 Ca. 20 (colonyboxes covered inplastic)

Ca.20 0

* Atta species do not rear actinomycete bacteria on their cuticle (Fernández‐

Marínetal.2009)

65

CHAPTER 2

DYNAMIC WOLBACHIA PREVALENCE IN

ACROMYRMEX LEAF-CUTTER ANTS:

POTENTIAL FOR A NUTRITIONAL SYMBIOSIS

67

Dynamic Wolbachia prevalence in Acromyrmex leaf-cutting ants: potential for a nutritional symbiosis

SandraB.Andersen1*,MetteBoye2,DavidR.Nash1&JacobusJ.

Boomsma1

1:CentreforSocialEvolution,DepartmentofBiology,UniversityofCopenhagen

Universitetsparken15,DK‐2100Copenhagen,Denmark

2:NationalVeterinaryInstitute,TechnicalUniversityofDenmark,Bülowsvej27,

DK‐1790Copenhagen,Denmark

*:Correspondingauthor:[email protected]/+4526209197

Runningtitle:WolbachiainAcromyrmexants

INREVIEW

68

Abstract

Wolbachiaarerenownedasreproductiveparasites,buttheirphenotypiceffects

ineusocial insectsarenotwellunderstood.Weusedacombinationofqrt‐PCR,

fluorescence in situ hybridisation, and laser scanning confocal microscopy to

evaluate the dynamics of Wolbachia infections in the leaf‐cutting ant

Acromyrmex octospinosus across developmental stages of sterile workers. We

confirm thatworkers are infectedwith one or twowidespreadwsp strains of

Wolbachia,showthatcolonyprevalencesarealways100%,andcharacterizetwo

rare recombinant strains. One dominant strain is always present and most

abundant while another strain only proliferates in adult workers of some

colonies and is barely detectable in larvae and pupae. An explanationmay be

thatWolbachia strains compete for host resources in immature stages while

adulttissuesprovidesubstantiallymorenichespace.Tissue‐specificprevalences

ofthetwostrainsdiffer,withtherarerstrainbeingoverrepresentedintheadult

foregut and thorax muscles. Both strains occur extracellularly in the foregut,

suggesting an unknown mutualistic function in worker ant nutrition. Both

strains of bacteria are also abundant in the faecal fluid of the ants, suggesting

thattheymayfurtherhaveextendedfunctionalphenotypesinthefungusgarden,

whichtheantsmanurewiththeirownfaeces.

Keywords:Wolbachia,Acromyrmexants,symbiosis,gutbacteria,fluorescencein

situhybridization

69

Introduction

Symbiotic interactions span the entire spectrum between mutualism and

parasitism, because the respective costs and benefits for hosts and symbionts

ultimately determine whether interactions become “win‐win” or “win‐lose”

(Bull, 1994; Herre et al., 1999). Vertical transmission typically aligns the

reproductive interests of host and symbiont but this transmission mode is

neithernecessarynorsufficienttokeepasymbioticinteractionmutualistic.For

example,Wolbachia is usually a vertically transmitted parasite with relatively

highvirulence(Werrenetal.,2008),whileTermitomyces,thegardensymbiontof

fungus‐growing termites, is a horizontally transmitted mutualist with an

unusuallystablecommitmenttoitshosts(Aanenetal.,2009).Inaddition,even

vertically transmittedmutualists are not permanently evolutionarily stable, as

someareknowntohavebeenlostovertime(Sachs&Simms,2006).

Symbioses are increasingly known to involve more than two partners (e.g.

Palmeretal.,2010).Thisfurthercomplicatesthedynamicsandselectiveforces

that shape the ultimate nature of these interactions, because cooperation and

conflict in such multiple partnerships depend on the interactions between

symbionts in addition to those betweenhost and symbionts (Vautrin&Vavre,

2009;Telferetal.,2010).Suchinteractionscaneitherhavepositiveornegative

effects on the host, but typically require that symbionts have spatially and

temporallyoverlappingnicheswithinhosts.

Communities of bacterial symbionts with complementary roles may produce

stablemutualismswhenconfinedtospecifichostorgansortissues.Examplesare

the gut pouches of Tetraponera ants that contain multiple highly divergent

speciesofnitrogenfixingbacteria(VanBormetal.,2002)andthebacteriomesof

hemipteran sharpshooters (Homalodisca coagulata) that contain two bacterial

species supplying amino acids and vitamins to the host (Wu et al., 2006).

However, when it comes to genetic variation among symbionts with similar

roles,diversitymaybecostlyforhosts,aswithin‐hostcompetitionoftenselects

70

formorevirulentparasites(Frank,1996;Daviesetal.,2002)orlesscooperative

mutualists(Herreetal.,1999;Poulsen&Boomsma,2005).

Wolbachiaα‐proteobacteriaare intracellularsymbionts inmany insects,mites

and some nematodes and crustaceans. They often affect host fitness as

reproductiveparasitesbycausingcytoplasmic incompatibility, as inDrosophila

flies (Bourtzis et al., 1996), Ephestia moths (Lewis et al., 2011) and Nasonia

wasps(Tram&Sullivan,2002).OtherWolbachiacausehostparthenogenesisas

in Bryobia mites (Weeks & Breeuwer, 2001), male‐killing as in ladybirds and

butterflies(Hurstetal.,1999),orfeminizationasinvariousisopods(Bouchonet

al., 1998). However, in other associations the host has become dependent on

these bacteria as nutritional mutualists or reproduction facilitators

(Pannebakker et al., 2007: Hosokawa et al., 2010). The default Wolbachia

transmission‐modeisvertical, frommothertooffspring,buthostandsymbiont

phylogenies often indicate that horizontal transmission occurs frequently

enoughoverevolutionarytimetopreventco‐cladogenesis(Werrenetal.,2008).

ManyhostspecieshavealsobeenfoundtocarrymultipleWolbachiastrains,and

insomecasesthesestrainsresideindifferenttissues(e.g.Ijichietal.,2002).

Whileanumberofthoroughcasestudieshaveclarifiedthephenotypiceffectsof

Wolbachia infections inmodels of solitary invertebrates, rather little progress

hasbeenmade inunderstanding thephenotypiceffectsof similar infections in

eusocialinsects.Surveyshaveshownthatawiderangeoftermitesareinfected,

but that eusocialwasps and bees are rarely hosts (Lo& Evans, 2007; Russell,

2012).ManyantsarealsoknowntoharbourWolbachia(Wenseleersetal.,1998;

Russelletal.,2009;Russell,2012),butprevalencesvaryconsiderablybetween

species, between colonies in populations, and between castes within colonies.

Some correlation between mode of colony founding and the likelihood of

infectionhasbeensuggested,asspeciesthatfoundcoloniesaidedbyworkersof

the parental colony have slightly higher prevalences than species that found

coloniesbysinglenewly‐matedqueens(Wenseleersetal.,1998).Otherstudies

haveshownthatWolbachia infectionsarefrequentlylostininvasiveantswhen

these populations are compared to their native sister populations or species

71

(Shoemakeretal.,2000;Reuteretal.,2004;Cremeretal.,2008).Female‐biased

sex ratios in coloniesof eusocialHymenopterahavealsobeen suggested tobe

influencedbyWolbachia,butnoevidenceforthiswasfoundinFormicaexsecta

(Keller et al., 2001) and Formica truncorum (Wenseleers et al., 2002). In the

latter case, and in the fire ant Solenopsis invicta, Wolbachia infections may

potentially reduce host fitness (Shoemaker et al., 2000), but otherwise the

phenotypic consequences ofWolbachia infections for ant hosts have remained

enigmatic.

In the present studywe use a novel combination of techniques to assess how

strain‐specificWolbachiaprevalencevariesacrossdifferentlifestagesofsterile

workersof the fungus‐growingantAcromyrmexoctospinosus.Earlier studiesof

this anthave indicated thatmostworkersare infected (VanBormetal., 2001;

Frost et al., 2010) and often by multiple strains (Van Borm et al., 2003). By

measuringthediversityandtissuedistributionofthesestrainswithinindividual

ants we aimed to elucidate the potential for interaction betweenWolbachia

strainsandtoevaluatethephenotypiceffectsoftheseinfectionsonhostfitness.

We used quantitative real time PCR (qrt‐PCR) and fluorescence in situ

hybridization (FISH) to measure the density ofWolbachia symbionts and the

distributionofbacteriaamonghosttissues.Afterestablishingthatconsiderable

concentrations ofWolbachia are associated with the ant gut, we used laser

scanningconfocalmicroscopytodocumentthisinmoredetail.Ourvisualizations

of bacteria in ant tissues revealed an unexpected extracellular presence of

Wolbachiaintheantgut,whichsuggestsanovelroleofWolbachiainthefungus‐

growingantsymbiosis.

Methods

DNAextraction,sequencingandquantitativePCR

Acromyrmex octospinosus colonies were collected in Gamboa, Panama in the

period 2004‐2010 (Table 1). DNAwas extracted fromwhole individuals after

crushing themwith a plastic pestle, and fromdissected tissues (DNeasy blood

72

and tissue kit, Qiagen). The wsp primers from Zhou et al. (1998), targeting a

surface protein, were used to amplify ca. 560 bp ofWolbachia DNA. The PCR

productwascloned(TOPOTAcloningkit, Invitrogen)and10‐23clones froma

singleadultworker fromsix field collected colonies (102clones in total)were

sequenced by Eurofins MWG Operons (Ebersberg, Germany). Two dominant

strainswhere identified,whichwere identical to strains previously sequenced

fromants(Shoemakeretal.,2000;VanBormetal.,2001;VanBormetal.,2003).

The sequences were translated to amino acids

(http://expasy.org/tools/dna.html) and the four hypervariable regions (HVRs)

ofwsp (Baldo et al., 2005)were identified using theWolbachiaMLSTwebsite

(http://pubmlst.org/wolbachia/;Jolleyetal.,2004).FollowingShoemakeretal.,

(2000) we called the two dominant strains “WSinvictaA” and “WSinvictaB”.

Specific primers for these strains were designed (wspa F: 5’‐

GAAAACTGCTGTGAATGGTC‐3’, wspa R: 5’‐TCCTCCTTTGTCTTTCTC‐3’; wspb F:

5’‐GAAAACTGCTGTGAATGGTC‐3’,wspbR:5’‐ATTKCAGCATCGTCTTTARCT‐3’)to

amplify167‐170bp,andthespecificityof theprimerswascheckedwithdirect

sequencing. The primers amplified a region where theWSinvictaB strain was

100%identical to theothernon‐dominantstrains(seeresults)and itwasthus

notpossibletoquantifythepresenceoftheserareadditionalstrainsanyfurther.

ForanalysisofthedistributionofWolbachiastrainsacrossdifferentindividuals,

castesandcoloniesDNAwasextractedfromeightcoloniessampledinthefield

and from six colonies reared under lab conditions for >7months (no colonies

were sampled both in the field and in the lab). From each colony eight entire

largelarvae,pupaeandadultworkersweresampled(seeTable1forcolonyID

andexactsamplenumber).Fieldcoloniesweresampledaftertheannualmating

flight, when they were not producing sexuals, to ensure that the large larvae

were immature large workers. For analysis ofWolbachia strain distributions

across worker tissues, DNA was extracted from dissected thoracic muscle

tissues, from three different parts of the gut, and faecal droplets of eight ants

from a single lab reared colony (Ao492). Absolute wsp copy numbers were

quantified by quantitative real time PCR (qrt‐PCR) using SYBR Premix Ex Taq

(TakaraBioInc.)ontheMx3000Psystem(Stratagene).Reactionstookplaceina

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finalvolumeof20.5µlcontaining10µlbuffer,8.8µlddH2O,0.4µlofeachprimer

(10µM),0.4µlROXstandardand0.5µltemplateDNA.Bacterialmeasurements

werestandardisedwithqrt‐PCRofthesinglecopyantgene,elongationfactor1α

(primers EF‐1α f: 5’ ACGGAAGCTCTGCCCGGTGA‐3’ EF‐1α r: 5’‐

TGGCAGTCAAGCACTGGCGT‐3’), providing an estimate of host cell number,

under the assumption that bacterial and antDNAarepreserved and extracted

equallywellbetweencastesandindependentofstoragemethod(inethanolat‐

20°Cvs. freshlycollected).AllPCRreactionsconsistedofa2mindenaturation

stepat95°C,35cyclesof95°Cfor30s,52°Cfor30sand72°Cfor30s,followed

byadissociationcurveanalysis.Allsampleswerereplicatedinthesamerunand

themeanwasusedforanalysis.Eachrunalso includedthreenegativecontrols

withnoaddedtemplate.Theinitialtemplateconcentrationwascalculatedfroma

standardcurvewithPCRproductintenfolddilutionsofknownconcentration,as

quantifiedbynanodrop.

Crosssectioningandembedding

Larvae(n=4),pupae(n=2)andworkers(n=8)fromcoloniesAo49a,Ao491

andAo496werefixedandembeddedfollowingtheprotocolofKulzerTechnovit

8100 (HeraeusKulzer,Germany).Tissueswere cut to allowpenetrationof the

fixative (2%paraformaldehyde inphosphate‐buffer,pH7.4) for<4h followed

byovernightwashinginPBSpH7.4at4°C.Thetissuesweredehydratedin100%

acetone for 1 h at 4°C and infiltrated with Technovit 8100 solution for 6‐10

hoursat4°C,followedbytransfertotheembeddingsolution,agitationfor5min,

andtransfertoaplasticmould.Mouldsweresealedwithplastic foilandleft to

hardenoniceat4°Covernight.Thetissueblockswerecutwithaglassknifeand

sectionsattachedtosuperfrostplusslides(Menzel‐Gläser,Germany)byheating

for 15 min. For whole‐mount laser scanning confocal microscopy, eggs were

collectedfromthefungusgardenofanisolatedlayingqueen(n=5,Ao492)and

antgutsweredissectedoutinfixative,fixedfor>4handwashedinPBS(Ao492,

n=10).

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Fluorescenceinsituhybridization(FISH)

Tissue sections were treatedwith lysozyme (5mg/ml) for 30min at 37°C to

increasecellpermeabilization(Moter&Göbel,2000)anddehydratedfor3min

each in 50%, 70% and 100% ethanol prior to hybridization. Slides were

hybridizedwitha16SrRNAtargetedprobespecific forWolbachiaand labelled

with Cy3 (Wol: 5’‐ CTAACCCGCCTACGCGCC‐3’, from Eurofins MWG Operons,

Germany)overnightat46°C.Thiswasdonein100µlhybridizationbuffer(100

mMTrispH7.2,0.9MNaCl,0.1%sodiumdodecylsulphate)with5ng/μlprobe

in a Sequenza slide rack (Thermo Shandon, Cheshire, United Kingdom). As a

negative control a Cy3 labelled probe targeting the spirochaete bacteria

Treponema sp. was used (S‐S‐ TrepDDKL 12‐432: 5’‐CATCTCAAGGTCATTCCC‐

3’).Slideswerethenwashedwithpreheated(46°C)hybridisationbufferfor3x3

min followedbywashwithpreheated(46°C)washingbuffer (100mMTrispH

7.2,0.9MNaCl)for3x3min.Finallytheslideswererinsedinwater,airdried

and mounted with Vectashield (Vector Laboratories Inc., Burlingame, CA) for

epifluorescence microscopy using an Axioimager M1 epifluorescence

microscope. ImageswereobtainedusinganAxioCAMMRmversion3FireWire

monochromecamera(CarlZeiss,Oberkochen,Germany).

Gutdissectionsandanteggsweretreatedwithlysozyme,dehydrated,hybridized

and washed as above in an Eppendorf tube and mounted on slides with

VectashieldcontainingDAPI(DAPIstainshostnucleiblueanditisthuspossible

toinferwhetherbacteriaareintra‐orextracellularlylocated).Theseslideswere

observed and photographed with a Zeiss LSM 710 laser scanning confocal

microscope equippedwith Zen 2009 software. After some editing, the images

were further processed to adjust contrast and crop irrelevant parts using

PhotoshopCS3forMac.

Live/deadbacterialstaining

Toevaluatetheoccurrenceofbacteriainthefaecalfluidoftheants,adropletof

ca.0.5µlwasdepositedonamicroscopeslidebysqueezingtheantgasterwith

forceps(asdescribedinSchiøttetal.,2010).Thebacteriawerestainedwiththe

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BacLightL13152live/deadstain(MolecularProbesInc.),staining livebacteria

green (Syto‐9 probe) and dead bacteria (i.e. cells with a compromised

membrane)red(propidiumiodide).0.5µlofeachstainwasaddedtoeachfresh

faecaldropletandslidesweresealedwithacoverslideandincubatedinthedark

for 15 min, after which slides were analysed using the Axioimager M1

epifluorescencemicroscope(n=5).

Results

IdentificationofWspstrainsusingtheHVRtypingsystem

Previously,Wolbachiaphylogenieswereprimarilybasedonthehighlyvariable

Wsp gene, but this gene later turned out to be unsuitable for inferring

phylogenetic relationships, because of its high divergence and recombination

rate (Baldo et al., 2005; Baldo et al., 2010). However, Wsp remains a useful

markerforidentifyingdifferentstrains,andallowedustoidentifyfourdifferent

Wolbachiawspstrainsfromthesixscreenedcolonies.Twowereidenticaltothe

WSinvictaAandWSinvictaBstrainsfoundinSolenopsisinvicta(GenBankacc.no.

AF243435 and AF243436, Shoemaker et al., 2000) and in three Panamanian

Acromyrmexspecies(VanBormetal.,2003).AllcoloniescarriedtheWSinvictaB

strain,whileonlysomehadtheWSinvictaAstrain.3.9%ofthesequenceswere

different with colonies Ao483 and Ao493 each yielding an additional strain

(GenBankacc.no.tobeadded)thatwas98‐99%similartoapreviouslyidentified

strain in A. octospinosus (Genbank acc. no AF472561.1). A new strain was

obtained from colony Ao496 (GenBank acc. no. to be added), showing 90%

similaritytoothersequencesinGenBank.

TheWspgeneconsistsoffourhypervariableregions(HVRs),eachwithmultiple

alleles that have been numbered, alternating with conserved sequences.

Recombinationtypicallytakesplacebetweenthefourregions,andHVRtypingis

ausefulwayofidentifyingrecombinationpoints(Baldoetal.,2005).Allstrains

werethusfurthercharacterizedwiththeHVRsystem.TheWSinvictaAstrainof

Acromyrmex octospinosus contained the elements 42‐43‐198‐25 and the

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WSinvictaBstrainhadtheelements21‐21‐25‐21.Theotherthreestrainsturned

outtobechimerasofthedominantstrains,andhadHVRs42‐43‐25‐21(foundin

colony Ao496) and 42‐21‐25‐21 (found in colony Ao493 and Ao492, the

sequences from each colonywere slightly different but translated to the same

protein sequence). The fact that recombination was localized between the

hypervariable regions, as previously reported for other strains, confirms that

thesestrainsaretruechimerasandnotsimplytheresultofsequencingerrors.

qrtPCR

All individuals fromallcolonieswerefoundtobe infectedwithWolbachia.qrt‐

PCRshowedthattheWSinvictaBstrainwasdominantinallindividualsatalllife

stages(Fig.1,Table2).Inthreeofthefield‐collectedcoloniesthiswastheonly

strain found in measurable amounts, except for two adult workers from one

colony that also carried theWSinvictaA strain. This colony (Ao471) had been

kept in the lab at the field site in Gamboa, Panama for > 1month,whichmay

have enhanced the expression of the WSinvictaA strain (see below). In the

remainingcoloniesalladultindividualscarriedbothstrains.

Basedon theprevalencedifferencesof theWSinvictaAandWSinvictaBstrains,

coloniesweredividedintothreecategories:fieldcollectedsingleinfected(FS,n

=3), fieldcollecteddouble infected(FD,n=5),and labreareddouble infected

(LD, n = 6).No lab reared colonies showed single infection. Thedifferences in

bacterial densities were analysed in JMP 9.0.2 for Mac OSX using a repeated‐

measures ANOVA, as individuals collected from the same colony could not be

regarded as independent. Therewas considerable between‐colony variation in

standardised bacterial densities within colonies and castes, with outliers

apparent inmany combinations, so the geometricmean density per caste per

colony was analyzed, as this showed the most homogenous variance of all

measures examined. Categorywas included as the between‐subject effect, and

caste and the category by caste interaction included as within‐subject effects.

Post‐hoc testing was by paired or unpaired t‐tests for within‐ and between‐

subject effects respectively, with Bonferroni correction based on the total

77

number of tests carried out. Overall there was an increase in total bacterial

numberwithdevelopmentalstage,withthebacterialdensitybeingsignificantly

higher in pupae than larvae, and significantly higher in workers than pupae.

There was also a significant caste by category interaction, due to somewhat

different development of bacteria in the different categories. In single infected

fieldcolonies,thebacterialdensitydidnotvarysignificantlybetweencastes.In

double infected field colonies, the increase was significant between all castes,

whileitwasonlysignificantbetweenlarvaeandpupaeandlarvaeandadultsin

labreareddoubleinfectedcolonies.Therewasasignificantdifferenceinthetotal

number of bacteria between categories, with lab colonies contained slightly

higherdensitiesatalllifestages(Table2and3,Fig.1).

LookingattheWSinvictaBstrainonly,theoverallpatternwasbacterialdensity

increasingfromthelarvaltothepupalstageandremainingatthishighlevelin

theadults.Therewasnosignificantcastebycategoryinteraction,showingthat

this pattern was the same in each category, and the difference between

categoriesdidnotquitereachsignificance(Table2and3,Fig.1).

The highest prevalence of the (non‐dominant)WSinvictaA strainwas found in

adult workers of FD and LD colonies, where they reached a mean of 29% (±

0.015SE)ofthetotalbacteria.IntheFScolonies,theWSinvictaAstrainwasnot

present in measureable amounts, and abundances in the immatures of FD

colonies were only slightly (not significantly) higher. The LD colonies carried

significantlyhigheramountsoftheWSinvictaAstraininthepupalstages(Table

2&3,Fig.1).

Thebacterialestimatesobtainedfromdissectionsofdifferenttissuetypeswere

not standardizedwith host gene copy number, as themajority of the bacteria

were foundtobeextracellular(seebelow).As thevariance inproportionswas

very different across tissues, with the faecal droplet material in particular

containingeitherhighorlowproportionsoftheWSinvictaAstrain(Fig.2)they

were compared pair wise using the Steel‐Dwass non‐parametric test. The

proportions of theWSinvictaA strainwere significantly higher in the crop and

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the muscle tissues (44%) compared to the rest, while the rectum (37%)

containedsignificantlymore than thewholeant (26%),and themidgut(24%).

BecauseofthehighvarianceintheWSinvictaAproportioninthefaecaldroplets,

the mean proportion in these (29%) was not significantly different from the

othertissues.

FISH

TheFISHanalysesshowedbacterialcolonizationofmultipletissuetypes.Inthe

anteggs,thebacterialdensitywashighestaroundonepole(SIFig.1).Inlarvae,

thedominantfatbodycellswerecarryingmanybacteriaandtheguttissuealso

housedsome (Fig.3A,B). In thepupae theant cellsarediversifying intomore

tissuetypes,whichwerewidelyinfected(e.g.musclefibresandfatcells,datanot

shown).Thiswasalsothecaseintheadults,whereparticularlythemusclecells,

fatbodyandguttissueharbouredmanybacteria(Fig.3C,D).Histologyshoweda

largeamountofWolbachiaoccurringextracellularly in thecroppartof thegut

(in 6 out of 8 individuals, Fig. 3C), and this was confirmed by confocal

microscopyofwholeguts(in10outof10individuals,Fig.4).Theseextracellular

bacteria were to a lesser extent also seen in the midgut (SI Fig. 2). No clear

identification of Wolbachia in the rectum could be made because of strong

autofluorescenceofthetissues.Asanegativecontrolforunspecifichybridization

a probe specific to the bacteriumTreponema sp. was used. This showed some

unspecifichybridizationtothepartofthegutleadingtothecropandtheileum

connecting the midgut and rectum, so hybridization to these tissues by the

Wolbachiaprobewasignored,asitwaspossiblyunspecific.

Thelive/deadbacterialstainingoffaecaldropletsshowedahighdensityofliving

bacteria,butitwasnotpossiblewiththeappliedmethodstoconfirmhowmany

ofthesewereWolbachia.

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Discussion

Wolbachiaprevalenceanddiversity

Wefoundthatallindividualsandalllifestagesandcolonieswereinfectedwith

Wolbachia,andverticalsymbionttransmissionwasconfirmedbyvisualizationof

Wolbachiainanteggs(SIFig.1).Thishighprevalencecontrastssomewhatwith

previousstudies,onthesameantspeciesfromthesamearea,whereallcolonies

were also found to be infected, but individual infection rateswere lower (Van

Bormetal.,2001,mean infectionrateof individualsof40%;Frostetal.,2010,

81%).Theexplanationforthismaybetechnicalratherthanbiological,because

qrt‐PCRallowsforahigherlevelofdetectionandtheamplificationofashorter

DNA fragment (ca. 170bp in thepresent studyvs. 783bpbyVanBormet al.,

2001),whichensuresthatevenslightlyfragmentedDNAisamplified.

ThetwodominantWolbachiawspstrainsthatwefoundhavebeenobservedin

otherantsaswell(Shoemakeretal.,2000;VanBormetal.,2003)andarealso

verysimilartostrainsinbeetlesandspiders(e.g.Sintupacheeetal.,2006).The

WSinvictaB strain was found in all colonies of A. octospinosus while the

WSinvictaAstrainonlyoccurred insomecoloniesandneveralone.Therewere

thus colony level differences in strain diversity, as either all or no individuals

withina colony carried theWSinvictaA strain.Thepresenceof theWSinvictaA

strain was not correlated with sampling site, indicating that geographic

clustering in the sample population is unlikely (data not shown).However, no

goodestimatesofcolonyageandcolonysizeuponcollectionwereavailable,so

wecannotdirectlyevaluatewhetherthesevariables,whichmaybeimportantfor

thedevelopmentofbacterialinfections,hadanyeffect.

In the coloniesharbouringboth strainswe identified two recombinant strains.

Althoughtheprevalencesof thesestrainswerenotassessedbyqrt–PCRforall

life stages, the low frequencies in three adult ants forwhich cloning estimates

were available suggest that they were rare. Our HVR typing further indicated

that these chimera strains arose by recombination of the WSinvictaA and

WSinvictaBstrainsbetweenthefirstandsecondHVR,andthesecondandthird

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HVR, respectively.Recombinationmay thusbe rather frequentbutwhile these

recombinant strains may persist in the population they do not appear to be

particularlysuccessful.Theveryoccurrenceofwithinhostrecombinationshows

thatsomedegreeofinteractionbetweentheWSinvictaAandWSinvictaBstrains

occurs. Wsp is a major outer surface protein of Wolbachia and has been

suggested to mediate contact with the host cells via its two transmembrane

regions that likely interact with the host immune system (Braig et al., 1998;

Bazzocchi et al., 2007), so that recombination may affect these recognition

processes.RecombinationbetweenWolbachiastrainshaspreviouslybeenfound

inotherhostspecies,includingants(Reuter&Keller,2003).

Bacterialdensityincreaseswithhostage

Wefoundan increase in thebacterial loadwithage,suggestingthatWolbachia

thrive in the mature workers. Adult worker ants of the species Formica

truncorumwerepreviouslyfoundtohavelowerinfectionratesthanimmatures

(Wenseleersetal.,2002),whichgeneratedthehypothesisthatworkersmaylose

infection for reasons that are adaptive for the bacteria, because they are

evolutionarydeadends fora reproductiveparasite.Thisappearsnot tobe the

case for A. octospinosus. For the dominant WSinvictaB strain, the increase in

densityoccursbetween the larval and thepupal stage andprevalence stays at

thislevelinadults,equivalenttowhathasbeenfoundintheAdzukibeanbeetle,

whereWolbachiaisaconfirmedreproductiveparasite(Ijichietal.,2002).

The increase inbacterial loadcouldreflect theappearanceofnew tissue types

that the bacteria are able to invade aftermetamorphosis (see below). Inmost

host‐symbiontinteractions,whetherparasiticormutualistic,thehosthasaclear

interestincontrollingbacterialgrowthanddispersal.InDrosophila,theabilityto

do so appears connected to life‐stage‐specific expression of immunity genes

(Samakovlisetal.,1990).IntheeusocialhoneybeeApismellifera,phenoloxidase

activity(ameasureofimmunedefence)waslowinbothlarvaeandpupae,most

likely because alternative social immunity mechanisms provide efficient

protection of brood (Wilson‐Rich et al., 2008). Such a down‐regulation of the

individual immune defence could be of importance for the ability of vertically

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transmittedsymbiontstogrowintheimmaturestagesofsocialinsects.Ifthisis

also the case forAcromyrmexoctospinosis itmaypartly explain the increase in

Wolbachiadensitywithhostage, suggesting that thebacteriamostlygrowand

dispersewhenhostcontrolmechanismsareconstrained.

Nichesegregationofbacterialsymbionts

Our results (Fig. 1) indicate that theWSinvictaA strain proliferates mainly in

adultindividuals.However,whencomparingthesingleanddoubleinfectedfield‐

coloniesthereisasuggestionofWSinvictaAstrainproliferationinadultworkers

being associated with lower WSinvictaB strain prevalences in the larval and

pupal stages. This could reflect some form of scramble competition between

WSinvictaA and WSinvictaB strain bacteria in the immature developmental

stages. In this hypothetical scenario, the initial degree of dominance of the

WSinvictaBstrainwouldthendeterminetheavailablenichespacefortheother

strain, so that individualswhereWSinvictaA strain bacteria remain under the

detectionlimitintheimmaturestageswillonlybeabletogrowveryfewofthem

asadults(theobservedpatternofsinglyinfectedfieldcolonies;Fig.1).However,

when theWSinvictaA strain bacteria for some reason becomemore abundant

alreadyintheimmaturestages(sothatimmaturefieldindividualsarescoredas

double infected), they are much more likely to proliferate further in adult

workers.

A competitive scenario as outlined abovewould bemost likelywhenbacterial

strainsinteractinthesamehosttissuesduringthelarvalandpupalstagesbut,at

least partly, segregate into different tissue types in adultworkers. Such tissue

tropism of Wolbachia strains has previously been observed in Adzuki bean

beetles(Ijichietal.,2002).AsourFISHresultsshowedahighdensityofbacteria

in the muscle fibres and the gut we further measured the distribution of

Wolbachiastrainsinthesetissues.Theqrt‐PCRsofspecifictissuetypesshowed

thattheWSinvictaAstrainwassignificantlymoreabundantinmusclefibresand

inthecropofthegut,relativetolaterstagesinthedigestiveprocess(midgutand

rectum)andthewholeant(Fig.2).Themuscletissueisonlyfullydevelopedin

theadultantsandtheadultgut isverydistinctfromthelarvalgut,beingmore

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complex anddivided into sections varyingmorphologically and in pH, enzyme

activityandretentiontimeofcontents(ErthalJr.etal.,2004).Thiscorroborates

thenotionthat,althoughoverlapping,theadultWolbachianichesaresomewhat

distinct and that they are unlikely to be differentiated earlier in development.

However, the substantial overlap inWolbachia nicheswithin hosts also raises

thepossibilitythatthesestrainsmayhavedifferentfunctionalrolesinadultants.

Interpreting the infection patterns of double infected lab colonies as being

consistent with strain competition would imply that resource constraints

somehowaffect fieldcoloniesmorethanlabcolonies.This isreasonable,as lab

colonieswerebeing fed regularlywith a standard selectionofDanishbramble

leaves (Rubus sp.), experienced no predation or other hazards while foraging,

and generally had large and thriving fungus gardenswhile living under stable

humidity and temperature regimes. All of these lab colonies were double

infectedandalsoharbouredslightlyhigherdensitiesoftheWSinvictaAstrainin

the immature life stages compared to the field sampled colonies (Fig. 1). In

addition,thetotalbacterialnumberinthelab‐rearedcolonieswasslightlyhigher

thanthatfoundinthefield,suggestingthatthebacteriathrivewhentheirhosts

experience lab conditions. The finding of two double infected workers in an

otherwise single infected colony in the field seems consistent with this

interpretation, as thiswas theonly colony thathadbeenkept formore thana

monthinthefieldlabinPanamaunderadlibitumresourceconditionsbeforeant

samples were collected (Ao471). The presence of the WSinvictaA strain in

measurable quantities early on could thus be dependent on colony resource

condition,whichinthefieldmaybecorrelatedwithcolonysize.

AreWolbachianewmutualistsintheattinefungusfarmingsymbiosis?

TheFISHdatasurprisinglyshowedthatWolbachiabacteriaareabundantinthe

lumenof theadultworkergut(Fig.3&4).WhileWolbachiahasbeenfound in

gut tissue (Dobson et al., 1999; Ijichi et al., 2002) an extracellular location is

highlyunusualandhastoourknowledgenotbeendocumentedbefore(butsee

Fischer et al., 2011 showing the occasional appearance of extracellular

Wolbachia close to ovarian tissue in nematodes). However, this observation is

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consistentwithWSinvictaA andWSinvictaB strainWolbachia being present in

thefaecaldropletsofAcromyrmex(Fig.2).Thefaecaldropletscontainedalarge

amountofviablebacteriaandpositiveDNA‐levelevidencesuggeststhatatleast

partofthesebacteriawereWolbachia.Thiscombinedresultthereforeindicates

thatWolbachia cells arenotharmedbydigestivegut enzymes, consistentwith

thisenvironmentbeingtheirnaturalniche.

The faecal droplets have unique functions in the fungus‐growing ants. They

containproteinsfromthefungalgarden,whichareingestedbytheantsbutpass

undigestedthroughtheguttoassistdecompositioninnewlyestablishedfungus

garden (Schiøtt et al., 2010). They also play a role in the recognition and

elimination of genetically different fungal cultivars that workers may collect

(Poulsen & Boomsma, 2005). The various adaptative functions of the faecal

droplets to the ant‐fungal symbiosis suggest that there is strong selective

pressureon the gut environment and the compositionof faecal fluid.This and

theatypicallocationofWolbachiainthegutlumenandfaecaldroplets,suggests

thatWolbachia inA.octospinosusmayhaveamutualisticnutritionalrole inthe

ant‐funguscultivationsymbiosis.

The recent finding of a beneficial role ofWolbachia symbionts in the

Western rootworm, larvae of Diabrotica virgifera virgifera, causing the down

regulation of defence compounds in the plants that they feed on (Barr et al.,

2010), offers an intriguing possible analogue to our present results. Similar to

the herbivorous beetle larva, the alliance of leaf‐cutting ants and their fungus

garden symbionts also faces challenges from secondaryplant defences. Recent

work (Schiøttetal., 2010)has shown that the fungal symbiontsof leaf‐cutting

antshaveconvergentlyevolvedanentiresetofpectinasesthatarenormallyonly

found in pathogenic fungi that attack live plant hosts, and also these enzymes

pass the ant gut unharmed. It therefore seems highly worthwhile to further

explorethefunctionalroleofWolbachiainAcromyrmex,bothintheworkerguts

and in the faecal fluid where the bacteria interact with the multiple

microorganisms that are now known from attine ant fungus gardens (Pinto‐

Tomás et al., 2009). We note that recent work has also suggested that plant

defencesmaynotonlybechemical,butalsobiotic,asleaf‐substratechoicebythe

84

ants is affected by the endophytic community of the leaves (Bittleston et al.,

2010;VanBaeletal.,2009).Furtherstudiesalongtheselineswillalsohavethe

potential to elucidate why only some colonies carry the WSinvictaA strain in

measurableamounts.

Acknowledgements

WewouldliketothankJoannaAmenuvorandAnnieRavnPedersen(DTU),and

LisbethHaugkroghandAaseJespersen(KU),foradviceconcerninghistologyand

FISH,MortenSchiøtt,HenrikdeFineLichtandTomGilbert (KU) foradviceon

qrt‐PCR,andPanagiotisSapountzis forcommentson themanuscript. SBAwas

funded by a PhD Scholarship from the Science Faculty of the University of

Copenhagen, and SBA, DRN and JJB were supported by the Danish National

ResearchFoundation.

85

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Figure legends

Figure 1. The density ofWolbachia bacteria in three different life stages (L =

larvae,P=pupaeandW=workers) in threedifferent colonycategories (FD=

fieldcollecteddoubleinfected,FS=fieldcollectedsingleinfectedandLD=lab‐

reareddoubleinfected).EachbarrepresentsthenumberofWolbachiacellsper

hostcell,standardizedbythecopynumberofthehostgeneEF‐1αestimatingthe

total numberof host cells, asmeasuredwithqrt‐PCR (dark grey=WSinvictaA

strain; light grey = WSinvictaB strain). For each caste in each colony the

geometric mean was calculated and the depicted value is the mean for each

colonycategory.Thereisanincreaseintheamountofbacteriawithprogressing

life stages and lab‐reared colonies have slightly higher numbers than field

colonies.TheWSinvictaBstraindominates inall lifestagesandtheWSinvictaA

strainonlyproliferatesinadultsofthedoubleinfectedcolonies.

Figure 2. Box‐plots showing the proportion ofWolbachia strainWSinvictaA in

thoracicmuscletissue,threedifferentpartsoftheantgut(thecrop,midgutand

rectum) and faecal droplets in comparison to in whole ant samples. The

bacterial load of WsinvictaA and WSinvistaB was measured by qrt‐PCR of

individualsamples.Thecentrallinerepresentsthemedianproportion,whilethe

boxruns fromthe lower25%to theupper75%quartile foreachsample,with

the whiskers linking the extremes of the data. Letters indicate group‐level

differences following the pairwise Steel‐Dwass method. The insert shows the

correspondingoverviewoftheantgutanditssurroundings,withthecropbeing

connected to the midgut, where the Malphigian tubules attach, and the ileum

connectingthemidguttotherectum.

Figure 3. Fluorescence in situ hybridization ofWolbachia (stained red) in ant

tissues. (A) Larval gut showing some hybridization. (B) A larval fat bodywith

manyWolbachiaaroundcellnuclei.(C)Crop(foregut)ofanadult largeworker

ant with extracellularWolbachia in the lumen. (D) Muscle fibres of an adult

workerwithsomeintracellularconcentrationsofWolbachia.Scalebar50µm(A)

and20µm(B‐D).

90

Figure 4. ExtracellularWolbachia bacteria in the crop of the ant gut, a highly

flexible sac that is slightly deflated so it appears somewhat folded. The LSCM

imageshowsthe3DstructureofthecropcontainingWolbachiabacteria(stained

red). The central image shows a horizontal optical section while the flanking

imagesrepresenttheverticalopticalsections.Scalebar50µm.

SIFigure1.Wolbachiabacteria ineggsofAcromyrmexoctospinosus,withLSCM

images showing the3D structureof the ant egg containingWolbachia bacteria

aroundtheeggpole(stainedred).Thecentralimageshowsahorizontaloptical

sectionwhile the flanking images represent theverticaloptical sections. Scale

bar50µm.

SI Figure 2. ExtracellularWolbachia in the antmidgut. (A)Wolbachia bacteria

stainedredagainstthegreenautofluorescenttissue.(B)Thesamebacterianow

visualized with a DAPI staining. DAPI stains nuclei blue and the absence of

stainedhostnucleithusconfirmsthatbacteriaareextracellular(Insertshowsan

exampleofhostnucleistainedwithDAPI).Scalebar50µm.

91

Figures

Figure1

Figure2

92

Figure3

Figure4

93

SIFigure1

SIFigure2

94

Tables Table1

CollectiondataforthecoloniesofAcromyrmexoctospinosusthatwereusedfor

estimatingWolbachiaabundancebyqrt‐PCR.

Samplesize DateColony

ID Larvae Pupae Workers

Lab/

Field Collection Sampling

Infection

status

Ao273 8 8 8 lab May‐04 Dec‐10 Double(A/B)





Ao471 8 7 8 field Apr‐10 May‐10 Single(B)

Ao482 4 8 8 field May‐10 May‐10 Double(A/B)


Ao491 7 8 8 field May‐10 May‐10 Single(B)

Ao49a 8 8 8 field May‐10 May‐10 Single(B)




AoClay 8 8 8 field May‐10 May‐10 Double(A/B)

95

Table2

Standardizedbacterialdensities(±SE)ofstrainsWSinvictaAandWSinvictaBand

their cumulative densities estimatedbyqrt‐PCR.The geometricmean for each

colony was used to calculate the mean for each category. Superscript letters

indicate the grouping in Bonferroni‐corrected paired t‐tests within each

category, following repeated measures ANOVA analysing the differences

between castes (larvae, pupae, adults: within‐subject effect), categories (field

single infected, field double infected and lab double infected: between‐subject

effect)andtheirinteraction.ForWSinvictaAandthetotalbacteria,letterstothe

right of each category represent groupings in post‐hoc Bonferroni‐corrected

unpairedt‐testsbetweenpairsofcategories.

Category WSinvictaA WSinvictaB Totalbacteria

Fieldsingleinfectedlarvae

0.002±0.0004A 3.413±0.788A 3.415±0.788A

Fieldsingleinfectedpupae

0.002±0.0002A 6.196±1.286A 6.198±1.286A

Fieldsingleinfectedworkers 0.279±0.277A

A

6.324±1.114A 6.603±1.002A

A

Fielddoubleinfectedlarvae 0.027±0.016A 1.898±0.269A 1.925±0.281AFielddoubleinfectedpupae 0.134±0.090A 4.582±0.214B 4.716±0.272BFielddoubleinfectedworkers 2.231±0.328B

B

6.188±0.925B 8.419±0.942B

A

Labdoubleinfectedlarvae 0.338±0.061A 3.898±0.262A 4.236±0.295ALabdoubleinfectedpupae 0.780±0.058B 7.028±0.395B 7.808±0.398BLabdoubleinfectedworkers 2.607±0.107C

C

6.994±0.525B 9.600±0.571B

B

96

Table3

Theresultsoftherepeated‐measuresANOVAforthedensityofWSinvictaAand

WSinvictaB and their cumulative amounts estimated by qrt‐PCR, testing the

effectofcasteandcategoryandtheinteractionbetweenthem.Forcategory(the

between‐subject effect), the F‐test shown is exact,while for thewithin‐subject

effects (caste and the casteby category interaction), theF‐test is approximate,

andbasedonWilk’sλ.

Dependentvariable Source F d.f. P

WSinvictaA Category 30.30 2,11 <0.0001

Caste 58.49 2,10 <0.0001

Caste×Category 11.35 4,20 <0.0001

WSinvictaB Category 3.21 2,11 0.080

Caste 206.2 2,10 <0.0001

Caste×Category 1.36 4,20 0.284

TotalNo. Category 5.46 2,11 0.023

Caste 273.4 2,10 <0.0001

Caste×Category 4.68 4,20 0.008

97

CHAPTER 3

DISEASE DYNAMICS IN A SPECIALIZED PARASITE

OF ANT SOCIETIES

99

Disease dynamics in a specialized parasite of ant societies

Sandra B. Andersen1, Matt Ferrari2, Harry C. Evans3,4, Sam L. Elliot4,

JacobusJ.Boomsma1.&DavidP.Hughes5

1Centre for Social Evolution, Department of Biology, University of Copenhagen,Universitetsparken15,2100Copenhagen,Denmark

2CenterforInfectiousDiseaseDynamics,PennStateUniversity,PA16802,USA

3CABInternational,E‐UK,Egham,Surrey,TW209TY,UK4DepartmentofEntomology,FederalUniversityofViçosa,36571.000,Viçosa,Brazil

5Department of Entomology and Department of Biology, Penn State University, PA16802,USA

Authors for correspondence: Sandra Breum Andersen ([email protected]) and

DavidP.Hughes([email protected])

Co‐authors email addresses: [email protected], [email protected], [email protected] and

[email protected]

Statementofauthorship

SBAandDPHdesignedthestudy;SBA,DPH,HCEandSLEcollectedthedatainthefield;

MFbuild themodel;SBA,DPHandMFanalysed thedata;SBA,DPHand JJBwrote the

manuscriptandallauthorscontributedsubstantiallytorevisions.

Runningtitle

Diseasedynamicsofantsocieties

Keywords

Ant,parasite,diseasepressure,society,manipulation,entomopathogen

TobesubmittedJanuary2012

100

Abstract

Coevolutionbetweenantcoloniesandtheirspecializedparasitesareintriguing,

because lethal infections of workers may correspond to tolerable chronic

diseasesofcolonies,butthelife‐historyadaptationsthatallowstablecoexistence

withanthostsarevirtuallyunknown.Weexplorethetrade‐offsexperiencedby

Ophiocordycepsparasitesmanipulatingantsintodyinginnearbygraveyards.We

usedfielddatafromBrazilandThailandtoparameterizeandfitamodelforthe

growthrateofgraveyards.Weshowthatparasitepressure ismuchlowerthan

the abundanceof ant cadavers suggests and thathyperparasitesoften castrate

Ophiocordyceps. However, once fruiting bodies become sexually mature they

appearrobust.Suchparasitelife‐historytraitsareconsistentwithiteroparity–a

reproductive strategy rarely considered in fungi. We discuss how tropical

habitatswithhighbiodiversityofhyperparasitesandhighsporemortalityhave

likelybeencrucialfortheevolutionandmaintenanceofiteroparityinparasites

withlowdispersalrates.

101

Introduction

Specializedparasitesthatinteractwithasingleornarrowspectrumofhoststend

tohavefascinatinglife‐histories,becausevirulenceanddefencetraitsarelikely

to have been shaped by co‐evolutionary arms races (Poulin 2007; Schmid‐

Hempel2011).This isparticularlytrueforparasitesthathaveevolvedwaysto

manipulatehostbehaviour,sothatdyinghostsexpressextendedphenotypesfor

the benefit of parasite reproductive success (e.g. Poulin 2010; Hoover et al.

2011). The fungal hypocrealean genusOphiocordyceps (formerly Cordyceps) is

well known for attacking specific hosts fromdiverse insect orders (Sung et al.

2007). Several lineages have evolved species that attack ants (Evans 1982b)

leading to manipulative extended phenotypes, that make infected ants leave

their nests to die and disperse spores in ways that serve parasite fitness

(Andersenetal.2009).

Ant coloniesarepeculiarhosts forparasites. Followingahighmortality

rate at the founding stage, mature colonies are typically long‐lived and

experiencelowextrinsicmortality.Thehighdensityofcontinuouslyinteracting

individualswithincoloniesimpliesthatinfectionrisksarehigh(Hamilton1987;

Sherman etal.1988),butalso thatselection forefficientprophylacticdefences

hasbeenstrong(Schmid‐Hempel1998;Naug&Camazine2002;Boomsmaetal.

2005). Recent reviews (Cremer et al. 2007;Hughes et al. 2008; Cremer& Sixt

2009)haveemphasizedthatbehaviouralformsofsocialimmunityarenormally

veryefficient,sothatantparasitesposealimitedthreatforescalatingepidemics

within colonies. Thus, even though individual ants may die from infection,

disease‐inducedcolonymortalityislow(Hughesetal.2008).

Horizontal disease transmission requires the introduction of parasite

propagules to uninfected nests. This process may not be very efficient as

territoriality often limits overlap between infected and susceptible colonies

(Boomsma et al. 2005) and propagules can often only reach the modest

percentageofworkers thatareout foraging (typicallybetween10‐25%of the

workers; Mirenda & Vinson 1981; Porter & Jorgensen 1981; MacKay 1985).

However, as chronically infected ant colonies tend to be long‐lived, a

combination of frequent vertical (nestmate to nestmate) infection and rare

102

horizontal transmission across colonies appears to have secured stable host‐

parasite interactions inants(Marikovsky1962;Charney1969;Schmid‐Hempel

1998: appendix 2, p.291‐324; Yanoviak et al. 2008). Such situations of stable

coexistence between hosts and parasites are examples of colony‐level disease

toleranceratherthandiseaseresistance(Milleretal.2005,2006).

Herewesetouttoexaminethedynamicsoftheinteractionbetweenant

hostsandOphiocordycepsparasites,whichallavailableevidenceshowisahighly

specialized, in tropical forests. The fungusmanipulatesworkers to leave their

nesttodieclosetotheirhostcolonyinhigh‐densitygraveyardsthatmaypersist

onthesamelocationforyears,offeringtheadvantagethatmortalityratesdueto

chronic parasitism can be estimated (Evans 1974; Evans & Samson 1984;

Sanjuánetal.2001;Pontoppidanetal.2009).Apartfromtheintriguingextended

phenotype adaptations that allow the fungus to control ant behaviour,

Ophiocordycepsfungithatexploitantsarealsounusualinthatthemajorgrowth

phase and all parasite reproduction occurs long after host death. The fruiting

body of the parasite has a latency period of at least two weeks before it can

reproduce(shootspores)forthefirsttime,andthefungussecuresthedeadhost‐

antbodysoefficientlythatitcancontinuewithsuccessiveboutsofreproduction

without succumbing to decay (Andersen et al. 2009). This implies that

Ophiocordycepshavelife‐historytraitsreminiscentofperennialplants,including

traits such as age at first reproduction and allocation to current versus future

reproduction thathavebeenshapedbyselectionandare likely tobe linked to

ratesofageingandinvestmentinsomaticrepair(Harper1977).

Inaclassicpaper,CharnovandSchaffer(1973)showedthatiteroparous

life cycleswith continuing investment in somatic tissue can only evolvewhen

juvenile mortality is high relative to adult mortality. To our knowledge, the

applicability of this logic has never been explicitly tested in fungi (where

iteroparous fruiting bodies are rare with the exception of some saprotrophic

fungi; Moore et al. 2008), but available natural history data suggest that

Ophiocordyceps may well have the appropriate combination of traits for this

conceptualframeworktoapply.Somaticinvestmenttosecurecontinuedgrowth

of the fruitingbody issubstantial intheonlyspeciesstudied indetailso far,O.

unilateralis s.l. (Andersen etal.2009),andOphiocordyceps sporesare fragile in

103

general, and easily killed by UV light and desiccation (Evans et al. 2011).

However,theCharnovandSchaffermodelwouldnotbesupportedif,inspiteof

investments insomaticmaintenance,onlysporesproducedshortlyaftersexual

maturityof fruitingbodieswouldpassongenes to futureparasitegenerations.

This seems a distinct possibility because a variety of fungal hyperparasites

colonizethedevelopingstalksandfruitingbodiesandpotentiallycauseeffective

castration (Evans1982a). It is thereforeessential toknowtherelative ratesat

whichfruitingbodiesbecomereproductivelydysfunctionalintheirearlyphases

ofdevelopment.

Applying iteroparity life‐history theory to a specialized host‐parasite

interaction such asOphiocordyceps has interesting additional complications, as

within colony transmission success may, paradoxically, limit between‐colony

reproductive success of parasites, no matter whether spores are produced

directlyaftersexualmaturityofafruitingbodyorlongafterthat.Withincolony

transmission needs a minimum number of dead ants per unit of time and a

particularrateofinfectivitytomaintainalocalpopulationofparasites,whereas

host colonies need to be large enough to sustain the ensuing level of worker

mortality without going extinct. When mortality happens in ‘graveyards’, this

wouldrequirethatthesegraveyardshaveagrowthrate(i.e.netinflowor‘birth

rate’ofdeadinfectedants)aboveoneor, incaseof long‐termequilibriumwith

thepopulationofhostants,agrowthrateequaltoone.

Inthepresentstudy,weuseddatafrompreviousstudiesonO.unilateralis

inThailand(Andersenetal.2009;Pontoppidanetal.2009)andanewdataset

fromO.camponotirufipedis(=O.unilateralis s.l.) fromBrazil toparameterizea

developmental‐stage‐structured model describing the interaction dynamics

between Ophiocordyceps and its host ants. By measuring the distribution of

parasitelifestagesandtheoccurrenceofhyperparasitismwithinantgraveyards

we estimated the realized parasite pressure on the ants. We show that most

parasite fruiting bodies are incapable of transmitting infectious propagules

becauseofhyperparasitism,butthatiteroparousreproductionappearsessential

formaintainingmarginallypositivegrowthrates inantgraveyards.Ourresults

suggest thatslowdevelopmentof fruitingbodiesand iteroparousreproduction

are likely to be adaptations that achieve long‐term persistence with host‐ant

104

colonies.Thisisbecauseinfectionsuccessofsporesislikelytobelowwhennew

hostantsaredifficulttotargetinbothtimeandspace,sothatprolongedsurvival

of fruiting bodies increases parasite reproductive success in spite of relatively

highcostsofhyperparasitism.

Materials and Methods

Fieldwork

The common ant Camponotus rufipes is host to the specialized parasite

OphiocordycepscamponotirufipedisintheAtlanticrainforestsofBrazil(Evanset

al. 2011). The ants form large, long‐lived colonies headed by a single queen

(monogyny)thathavebeenobservedtosurviveatthesamesiteformorethan

10 years (R.F. de Souza & S. Robeiro, personal communication). The ants

constructnestsof leaves,twigsandsoil, typicallyatthebaseoftreesandoften

connectedtosmallersatellitenests,andforageatnightalongtemporallystable

trails.TheoveralldistributionofO. camponotirufipedis hasbeen studied since

2006 and provided the stimulus for the present focal study, undertaken in

February2011inMatadoParaíso,a400haAtlanticrainforestnaturereservein

Minas Gerais, Brazil. The forest harbours a high density and diversity of

Ophiocordyceps that infect ants, of which O. camponotirufipedis is one of the

mostcommon(Evansetal.2011).

We identifiedants infectedwithO. camponotirufipedis by searching the

underside of leaves along a ca. 460 m stretch of forest path and found five

graveyards(sensuPontoppidanetal.2009)withahighdensityofdeadC.rufipes,

eachofthemsituatedaroundasinglehostantcolony.Wemarkedareascovering

approximately theentiregraveyard (graveyards1,2,3)or largepartsof them

(graveyards 4, 5), and tagged all dead infected ants ‐ found typically on the

undersideofleavesandontwigs(n=432)‐withpinktapearoundtheleafstem.

After death, Ophiocordyceps parasitized ants progress through several

developmentalstages.Foreachcadaverwethereforecharacterizedthestateof

parasitedevelopmentasbeing:1.afreshlykilledant,2.acadaverwithaparasite

stroma (stalk‐like structure that is meant to develop into a mature fruiting

105

body), 3. a cadaverwith amature parasite sexual fruiting body (ascoma), 4. a

cadaver at stage 2 or 3, but hyperparasitized by other fungi, or 5. a cadaver

whosestatuscouldnotbeidentifiedasithadbeendamagedbyunknowncauses

and lacked obvious fungal growth (see below for more detailed category

descriptions).ThecoordinatesofeachgraveyardwereobtainedwithaGarmin

etrexGPSandmappedinGoogleEarth.

Measuringfungalreproduction

Toestimatetheinfectivityofmatureparasitefruitingbodies,15deadantswith

developedsexualreproductivebodies(ascomata)werecollectedwiththeleaves

theywereattachedtoandsuspendedonawoodenplatformaboveamicroscope

slideintheforestcloseto,butoutside,agraveyardofdeadinfectedants.Spore

discharge takes place during the night and microscope slides were therefore

checkedonthethreefollowingmorningsfordepositionofthehighlydistinctive

sporeclouds,whicharevisibletothenakedeye(Evansetal.2011).

After thiscollectionperiod, the15parasitizedantswithmature fruiting

bodieswerebroughttothelab,inadditionto16newlycollecteddeadantswith

mature fruitingbodies.All31antswere individually attachedwithVaseline to

thelidofaPetridishwithamicroscopeslideoragaratthebottomandplacedin

a dew chamber with 100% relative humidity for 18 hours (18.00 to 12.00).

These lab‐generatedmicroscopeslidesandagarplateswerechecked forspore

depositseverymorningfor4‐6days.

Parameterizinganagestructuredmodelforcadaverturnoveringraveyards

From fieldwork in Thailand on a similar system ofO. unilateralis s.l. infecting

Camponotus leonardi (Andersen et al. 2009) and from observations of newly

infected ants in Brazil we estimated the duration of the different parasite life

stages. The first freshly killed stage, from death to the first signs of a stroma,

takesca.4days,forparasitesinthestromalstagewearrivedatawiderangeof

stage‐duration times (7‐30days), andwe inferred that parasiteswith sexually

maturefruitingbodieswereatleast20daysold.Theserangesarelikelytovary

across the season, as growth conditions for fungi areprobablyproportional to

rainfall.Toremainconservativewhenparameterizingourmodel(seebelow),we

106

therefore used a relatively crude timescale assuming that development from

hostdeathtostromaappearancetakesoneweek,anddevelopmentfromstroma

tosexualmaturitytakesfourweeks.

Adopting the approximate approachdescribed abovehas the advantage

that estimates apply to bothOphiocordycepsunilateralis infections in Thailand

and O. camponotirufipedis infections in Brazil, allowing us to supplement

missingdatainourpresentstudy.DatafromThailandobtainedbyfollowing17

individuals for10monthswith intervalsof twomonthsproducedapproximate

figuresfortheriskofO.unilateraliss.l.becominghyperparasitizedatagivenage.

Wefoundthat30/31O.unilateraliss.lmonitoredfor18monthsbecameinfected

byhyperparasiticfungi.Basedonourfieldobservationweestimatedthatmature

parasites persist for at least 4 weeks before being hyperparasitized (DPH,

unpublished data). Finally, we assumed that the accumulation of freshly dead

cadavers was proportional to the number of spore‐producing parasites with

maturefruitingbodies,asourmeasurementswouldshowthatneitherimmature

stromatanormaturebuthyperparasitizedfruitingbodiesproducedanyspores.

We formalized the "life‐cycle" of parasitized cadavers as illustrated in

Figure2,wherearrows represent transitionsbetween life stages.Weassumed

thattherateofnewinfections,b,isdeterminedbytheavailabilityofliveantsand

is constant over time (assuming no colony growth or decline). We further

assumedthatcadaversinthestromalstagewouldbehyperparasitizedatarate

PsandcadaversinthematurestagewouldbehyperparasitizedatratePm;these

wouldthenabortthenormalcompletionofthesestagesandtransferthemtothe

effectivelysterilehyperparasitizedstage(seedatabelow).Writingthenumberof

cadaversineachclassasavectorN=(Fresh,Stroma,Mature,Hyperparasitized)',

we then summarized the cadaver "life cycle" as a population transitionmatrix

givenbyequation1:

€

A =

e−1 0 b 01− e−1 e−(0.25+Ps ) 0 00 0.25

0.25+Ps1− e−(0.25+Ps )( ) e−(0.25+Pm ) 0

0 Ps0.25+Ps

1− e−(0.25+Ps )( ) Pm0.25+Pm

1− e−(0.25+Pm )( ) e−0.019

107

In this matrix (Equation 1) time steps proceed in multiples of one week and

developmentratesaresuchthatthemeantimeineachclassisoneweekforthe

freshlykilledcadaverclass,andfourweeksforboththestromalandthesexually

maturefruitingbodystage.

IfweassumethattheratesoftransitionbetweenclassesinAareconstant

throughtimethen the long‐termstablestagedistributionof thematrixAgives

the expected distribution of cadavers observed in each class. To estimate the

unknownparametersofA(b,Ps,andPm),wefoundthevaluesthatminimizedthe

sumofsquareddifferencesbetweentheexpectedstablestagedistributionofA

and the stage distribution of cadavers observed in the field (Table 2). This

allowedustosubsequentlyestimatethegraveyardgrowthrate,λ,accountingfor

theunhyperparasitizedpartofthegraveyard,asthedominanteigenvalueofthe

transitionmatrix(Leslie1945;Lefkovitch1965).

Addingvariationinoverallgrowthrates

Thegrowthrateofgraveyards (λ)and thedevelopmental stagedistribution in

graveyards depends on the assumed fungal development rate, which in turn

dependsontemperatureandhumidity(e.g.Arthurs&Thomas2001;Arthurset

al. 2001; Hatzipapas et al. 2002). We first explored the sensitivity of the

estimatedparametersb,Ps,Pmandλtoeffectsofvariationintheassumedfungal

developmental rate. This was done by varying Ophiocordyceps development

rates relative to the fitted model by ± 50% from the original fitted value, by

incorporatingtheparameterθ(rangingfrom0.5to1.5,ratherthanbeingfixedat

1) to Equation 1, under the assumption that all parasite life stages would be

affectedequally.WethensetupfouralternativeversionsofEquation1tofurther

explore the effects of seasonal variation in the fungal developmental rate on

graveyardgrowthrateand theproportionof thegraveyardcadavers thathave

escapedhyperparasitism in relation to faster (θ > 1) or slower (θ < 1) overall

fungaldevelopment.Wehypothesizedthatseasonalvariationcouldaffectfungal

lifehistory inthreedifferentwaysby:1)onlyaffectingparasitedevelopmental

rate, 2) affecting parasite and hyperparasite developmental rates similarly so

they become positively correlated and 3) affecting the inflow rate of new

cadaversbbyapositivecorrelationbetweentimespentinthematurelifestage

108

and new ants infected. The four alternative scenarios capture the different

combinationsof thesepotentialeffectsofvariation in fungaldevelopmentrate.

In scenario 1A and 1B the inflow rate of new cadavers is assumed to be

uncorrelatedwith the parasite development rate, while cadaver inflow rate is

assumedtobepositivelycorrelatedwithparasitedevelopmentrateinscenario

2A and 2B. In scenario 1A and 2A hyperparasite development is uncorrelated

withparasitedevelopmenttime,whilethevariablesarepositivelycorrelatedin

scenario1Band2B(Table1).

Results

Lifestagedistributionofdeadinfectedantsingraveyards

In the fivegraveyardswe founda totalof432dead infectedants;12.5%were

fresh,12.9%carriedastroma,6.5%weremature,55.4%werehyperparasitized,

andtheremaining12.7%weredamagedwithnoobviousfungalgrowth(Fig.1,

Table2).

None of themature parasite fruiting bodies (0/15) dispersed spores at

ambient temperature and humidity at the time of our spore collection in the

forest. However, after exposure to higher humidity simulating nights of heavy

rainfall in the forest, 42%(13/31)of thematureparasite fruitingbodieswere

shootingsporesinthelab.

Modelfitting

We fitted the stage‐structured graveyard growth model to the observed

distributions of parasite life stages and performed simulations assuming that

fungaldevelopmentalratesrangedfrom50%to150%ofourestimatedmeansto

evaluate the sensitivity of parameter estimates to the assumed developmental

rates. The estimated cadaver inflow ratebwas 1.42new cadavers permature

cadaver,varyingfrom0.85to1.75acrossthetotalrangeof0.5to1.5timesthe

mean developmental rate (Fig. 3A). The estimated probability of

hyperparasitismamongparasitesinthestromalstagePswas0.55,varyingfrom

0.31 to0.75across the total rangeof0.5 to1.5 times themeandevelopmental

109

rate(Fig.3B).Theestimatedprobabilityofhyperparasitismamongparasitesin

the mature stage Pm was 0.057, and relatively invariant across the range of

developmentalrates.Thissuggeststhattheprobabilityofnewhyperparasitism

of mature parasites is very limited for the four weeks that the parasite is

assumedtospendinthislifestage(Fig.3C).Thegraveyardgrowthrateλforthe

modelshowingthebestoverallfitwithallparametervalueswas1.07andhada

verysmallrangeofvariation(1.035‐1.100)acrosstherangeof0.5to1.5times

the mean developmental rate, indicating that the observed stage distribution

wasconsistentwitharelativelyslowgraveyardgrowthrate(Fig.3D).

Growthandlongevitytradeoff

Exploringthefourdifferentscenariosforimplementationofvariationinoverall

fungal development rate (the x‐axis in Fig. 4) showed that, as expected, slow

development increases the time spent in themature, infectious stage and the

likelihood of a susceptible ant coming in contact with spores, while fast

development increases the likelihood of reaching the mature stage prior to

hyperparasitism.Inscenario1Aand1B,wheretheinflowrateofnewcadaversis

uncorrelated with the Ophiocordyceps developmental rate, the overall growth

rateofthegraveyardremains>1forthetotalrangeoffungaldevelopmentrates

(dashedcurve,Fig.4A;scenario1Aand1Bgivethesameresult,asdo2Aand2B,

because they only differ in whether the probability of hyperparasitized

individualsremaininginthegraveyardiscorrelatedtotheparasitedevelopment

rate or not, which in the model does not affect the graveyard growth rate).

However, if faster fungal development also results in lower infectivity due to

reduced likelihoodofantencounters (scenario2Aand2B), then thegraveyard

growthpeaksat even slower ratesof fungaldevelopmentand rapidlydeclines

whendevelopmentalratesincrease(solidcurve,Fig.4A).

Thefourscenariosdifferintheproportionofcadaversthatremainfreeof

hyperparasites (Fig. 4B). If parasite and hyperparasite developmental rates

affect only the transitions among cadaver categories (1A andB; black and red

curves),thenfasterdevelopmentratesresultinfewerhyperparasitizedcadavers

(Figure 4B). Faster developmental rates in the hyperparasite lead to faster

senescence of hyperparasitized cadavers and thus a greater proportion of

110

unhyperparasitized cadavers (i.e. the difference between scenario 1A and 1B,

Figure4B).Ifthecadaverinflowrateispositivelycorrelatedwiththetimespent

inthematurestage(scenario2Aand2B,greenandbluecurves)theproportion

ofunhyperparasitizedcadaversismaximizedatslowdevelopmentalratesasfast

developmentleadstorelativelylowreplenishmentoffresh,unhyperparasitized

cadavers.

Discussion

Low density and limited interaction efficiency between infective parasites and

susceptiblehosts

We found thatonlyca. 6.5%of theO. camponotirufipedis fruitingbodieswere

effectivelyproducingspores,asmostdeadantsweresterilebecausetheywere

immature (25.5%), damaged (12.7%) or hyperparasitized (55.4%) by other

fungi that arenot pathogensof ants. Field and lab trials further indicated that

only 42% (13 out of 31 tested) of the apparent fertile fruiting bodies were

shooting spores at a particular time interval, illustrating that detailed

environmental conditions matter as well. Finally, upon dissection some

apparentlyhealthyO.camponotirufipediscadaverswerefoundtobeinvadedby

larvaeof smallunidentifiedarthropods(SBAandDPH,unpublisheddata).This

may also have reduced the probability of the parasite reaching maturity and

wouldhavemoved a number of them to the sterile hyperparasitized category.

This demonstrates that most cadavers are not infectious to foraging ants and

implies that disease pressure at the colony‐level ismuch lower than the high

numbersofdeadgraveyardantssuggest.

In addition to the low number of infective parasites, only a small

percentage of the ant colony members are actually available as targets for

Ophiocordyceps spores, as all brood andmost workers remain inside the safe

nestboundaries,sothatonlyforagersfacetheriskofbeinginfected(Mirenda&

Vinson 1981, Porter & Jorgensen 1981, MacKay 1985). The local interaction‐

interface between parasite and host is therefore limited, so that colony‐level

infections canonlybe stablewhengraveyards continue to growuntil a steady

111

statethatmaintainshostandparasiteindividualsatrelativelyconstantdensities

of chronic colony infection. The finding of a graveyard growth rate just above

onesupportssuchascenario.

Infecting not just workers of the same colony but also those of other

colonies, would require that the founding rate of graveyards exceeds their

extinctionrate, i.e. thathostcoloniesandthegraveyardsaroundthemproduce

enough dead ants over a sufficient number of years to replace themselves by

founding ‘offspring’ graveyardsarounduninfectedhost colonies.The scale and

durationofourstudywereinsufficienttoobtainharddataonsuchpopulation‐

levelequilibria,butsheds interesting lightonhowparasite iteroparityhelpsto

maintain the stability of infections within graveyards. We will therefore first

evaluate our present understanding of within‐colony transmission, and then

briefly address what kind of studies would be needed to comprehend

transmissionacrosscolonies.

ThelogicofiteroparousreproductioninOphiocordyceps

It seems likely that the absence of persistent spores or alternative non‐host

reservoirs is crucial for understanding iteroparity in Ophiocordyceps. Many

generalistentomopathogenicfungisuchasthewell‐studiedgeneraMetarhizium

and Beauvaria, which are asexual anamorphs of Cordyceps‐like teleomorphs,

combine rapid semelparous asexual reproduction with the production of

persistentspores.Theyarealsoincreasinglysuspectedtohave‘hiddenlives’as

endophytes of leaves and in the rhizosphere, with the possibility to produce

spores outside the bodies of insect hosts (St. Leger 2008; Vega 2008). This

suggests that sexual reproduction in the Ophiocordyceps sexual morphs of

hypocrealean fungi has somehow necessitated a life‐history focusing on

producingshort‐livedsporesforalongtimeratherthanlongerlivedsporesfora

short time as asexual forms do. Another specialist ant pathogen, the

entomophtoraleanfungusPandora infectingFormicawoodants inEurope,also

hasasemelparousstrategyofrapidasexualreproduction(Marikovsky1962).As

withOphiocordyceps, thePandora hosts aremanipulated intobitingvegetation

close to ant trails just before dying, but spores develop quickly from conidial

matsontheantbodysurface.Thesenormallyonlysurviveforafewdays,butthe

112

sporesproducedtowardstheendoftheantforagingseasontendtobedurable

(J. Malagocka & A.B. Jensen, personal communication) and may thus play a

decisiveroleinmaintainingcolony‐levelinfectionsyearafteryear.

While it appears logical that tropical rainforesthabitatdoesnot require

dormantrestingspores,thisdoesnotnecessarilyimplythatsporesshouldbeso

short‐livedasthoseofOphiocordyceps.Protectionofsporesbye.g.pigmentation

hasevolvedrepeatedlyinfungi(Butler&Day1998;Belletal.2009)soitseems

likely thatOphiocordyceps spores that would have remained viable for weeks

rather thandayscouldhaveevolved.However,selection for increasedviability

wouldbeunlikelytoariseinhabitatswherefrequenttorrentialrainswouldtend

towashsporesawayfromtheterritoriesoftheforagingants.Ifthatisso,thekey

question remaining is how parasite iteroparity secures a sufficiently high

infectionratewithephemeralspores.

Ourmodelofferssuggestions for theselection forces thatareultimately

responsible fortheoriginandmaintenanceof iteroparity inOphiocordyceps.As

outlinedintheIntroduction, iteroparousreproductiontendstobeevolutionary

stable when externally imposed juvenile mortality is high relative to adult

mortality(Charnov&Schaffer1973). Inadditiontoshort livedspores,alsothe

immature fruiting body stages appear to be highly vulnerable, but here the

external factors are biotic rather than abiotic, because hyperparasitism risk is

highinthestromalparasitelifestage(ca.55%;Fig.3B),butexpectedtobevery

lowinthematurelife‐stage(ca.5.5%;Fig.3C).Thisisundertheassumptionthat

thematurelifestagehasadurationofonemonth;inthefieldinThailandmature

fruiting bodies were observed to accumulate hyperparasites after this time

periodbutwespeculatethatsporeproductionmayhaveceasedatthisstage.A

low risk of hyperparasitism may well be related to mature fruiting bodies

expressing a much more efficient immune defence than the rapidly growing

stromata, because it takes time for the growing parasite mycelium to

compartmentalize the dead host body into specific fungal tissues with

complementary roles in protecting the elaborate fruiting body structures that

producethespores(Andersenetal.2009).Fungalimmunedefencesarepoorly

understood (but see e.g. Soanes & Talbot 2010), butOphiocordyceps fungi are

known to produce a range of secondarymetabolites thatmay be relevant for

113

maintaining cadavers (Isaka et al. 2005). Thus, the likelihood for spores to

survive,infectandproduceastomaisverylow,butafruitingbodythathasmade

ittowardsmaturityisworthmaintainingforalongtime.

Though not known in any detail, it is probable based onmorphological

structuressuchasstalksandsporeproducingbodiesthatiteroparityalsooccurs

in a range of the other Clavicipitaceaeous fungi, such as those infecting

lepidoteran and coleopteran larvae and spiders (Sung et al. 2007). While our

knowledgeof these groups is cursory it is likely that the life‐historiesof these

parasitesarealsocharacterizedbyahighdegreeofhostspecificityandlimited

contactbetweenhostsandinfectivespores,andalsothesefungiappeartohave

evolved and be most diverse in (sub)tropical regions. This suggests that the

evolution andmaintenance of iteroparity in these obligate insect pathogens is

primarilyrelatedtoclimateandhostspecificity,andthatanthostshavemerely

required the additional evolutionof thewell‐knownextendedphenotypes that

manipulateinfectedworkerstoleavetheirnests.

Ourmodelalsoexploredwhetherandhowitmattersthatthetimespent

in the mature parasite stage is positively correlated with cadaver inflow rate

(scenario2Aand2B)ornot(scenario1Aand1B).Suchcorrelationsarelikelyto

exist because the number of new infected ants should be somemonotonously

increasing function of the number of spores produced in graveyards,which in

turn should be positively correlatedwith the reproductive life span ofmature

fruitingbodies.Aslifespanwouldlikelytrade‐offagainstdevelopmentrate,this

suiteoftraitswouldthenalsobeaccompaniedbyslowdevelopment.Ourmodel

confirmsthatpositivecorrelationsbetweenfruitingbodylifespanandcadaver

inflow rate maximize graveyard growth and the proportion of cadavers that

escapebeinghyperparasitized,provideddevelopmentisslow(Fig.4).However,

when fruitingbody life spanand cadaver inflow rate arenot correlated, faster

ratesofdevelopmentappear tobeoptimal forparasite reproduction (Scenario

1A and 1B).We believe that correlations between parasite and hyperparasite

developmental rates are likely to occur as environmental fluctuations in

temperature and humidity may affect different fungal species similarly.

However, such correlationsmay also vary considerably because hyperparasite

114

growthisgenerallyfasterthanOphiocordycepsgrowth,duetothemuchsimpler

morphologyofthehyperparasites.

Graveyardgrowthanddiseasespreadacrossspatialscales

Ourestimatesofgraveyardgrowthratesjustabove1areconsistentwithcolony‐

specificaggregationsofdeadantsbeingsustainable,witheachmatureparasite

onaverageproducingslightlymorethanonenewmatureparasite.Toappreciate

the significanceof this result it is important to realize that the colony, not the

individual ant, is thehost forparasites suchasOphiocordyceps (Sherman et al.

1988;Schmid‐Hempel1998:p.204). If antworkersdie close to theirnestand

endupinfectingtheiryoungersiblings,thisisequivalenttoverticaltransmission

(Boomsmaetal.2005;Cremeretal.2007;Hughesetal.2008).Bycontrast,true

horizontal transmission would then be restricted to spores produced by

parasitesofonecolonyinfectingworkersofanothercolony.Thiscouldeitherbe

achievedby rare infectedworkersdyingmuch further away from their colony

thanthe ‘resident’graveyard,ortosporesproducedingraveyardsoccasionally

dispersingovermuchlongerdistances.

Long distance spore dispersal seems unlikely asOphiocordyceps spores

areheavyandnoteasilydispersedbywind. Itcouldbepossible thatvectoring

occurs but no evidence is known. However, if horizontal transmission would

primarilydependonthemovementof the infectedants themselves, thiswould

suggest the intriguing possibility of disruptive selection for both short and

(occasionally)long‐distancedispersalofparasiteextendedphenotypes.Infected

antsshouldtheneitherdieverycloseorrelativelyfarfromtheircolonybecause

ant territories are geographicalmosaicswithmost if not all interactionsbeing

restricted to nearest neighbor colonies (Leston 1973;Majer 1993; Blüthgen&

Stork2007).Futurestudiesmayaddressthisbyestimatingthegeneflowwithin

andbetweengraveyardsandbylookingatthegeneticdiversityofthedeadhosts

andtheirparasites.

115

Acknowledgments We thank Roberto Barreto at the Federal University of Viçosa, Minas Gerais,Brazil for kind hospitality.We are grateful to Anna Mosegaard Schmidt andRaquel Loreto for discussion, Jørgen Eilenberg for comments on an earlierversionofthemanuscriptandGöstaNachmanforsuggestionsforimprovementofthemodel.SBAwasfundedbyaPhD.ScholarshipfromtheScienceFacultyoftheUniversity of Copenhagen, and JJB and SBAwere supported by theDanishNational Research Foundation. HCE and SLE were funded by the Braziliansciencefoundation(CNPq).DPHwasfundedbyanOutgoingInternationalMarieCurieFellowship.

116

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Figure Legends

Figure 1. Aerial photo (from GoogleEarth) of the sampling areawith the five

graveyards marked and the distribution of parasite life stages plotted as pie

charts.Atotalof432deadinfectedantswereencountered,distributedwith41,

35,44,149&163individuals,respectively,ingraveyard1‐5.

Figure2. Idealizedparasitelife‐cycle. Boxesindicatelifestages(fresh,stroma,

mature and hyperparasitized) and arrows indicate transitions between stages.

Newcadaversenterthesystemwithbirthratebandremaininthe‘fresh’stage

foraweekonaverage.Theythenmovetothe‘stroma’lifestageandstaythere

for an average of four weeks, during which a proportion is lost to the

‘hyperparasitized’lifestageatratePs.Thoseindividualsthatmovetothemature

stagespendonaveragefourweeksthere,duringwhichaproportionislosttothe

‘hyperparasitized’lifestageatratePm.Individualsinthehyperparasitizedstage

remainonhereforanaverageof52weeksbeforebeinglost.

Figure3. Sensitivityanalysisoftheestimatedlife‐historyparametersbasedon

thestagestructuredgraveyardgrowthmodelthatfittedtheempiricaldatabest.

PanelA:thenewcadaverinflowrateb,panelB:thehyperparasitismrateinthe

stromallifestatePs,panelC:thehyperparasitismrateinthematurelifestagePm,

PanelD:thegraveyardgrowthrateλ.Thevariationinfungaldevelopmentalrate

from 50% to 150% is plotted along the x‐axes, relative to the average fungal

development rate thatwasestimated from the fielddata (here representedby

the relativevalueof1).Dashed lines connect theexpectedmean for they‐axis

estimatewiththisoverallmeandevelopmentrate.

Figure 4. Graveyard growth rate λ (panel A) and proportion of cadavers un‐

hyperparasitized (panelB) as a functionofdevelopmental rate (x‐axis) for the

four different modelled scenarios. The variation in fungal developmental rate

from .25% to 400% is plotted along the x‐axes, relative to the average fungal

development rate thatwasestimated from the fielddata (here representedby

therelativevalueof1).A.Thegraveyardgrowthrate,whichonlyaccounts for

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theunhyperparasitizedindividuals,isidenticalinscenario1Aand1B,andis>1

acrossalldevelopmentalratesbutpeaksat fast to intermediatedevelopmental

rate. Scenario 2A and B are also identical, with negative growth rates at fast

development rates but peaks with growth rates >1 at intermediate to slow

developmental rates. B. The four scenarios differ in the proportion of un‐

hyperparasitizedcadaversacrossthedevelopmentalrange.Scenario1Aand1B

have high rates of hyperparasitism at slow developmental rates. Note that as

development rates increase, a greater proportion of cadavers escape hyper‐

parasitism in scenario1Bdue to the faster senescenceof thehyperparasitized

cadavers.Scenario2Aand2Bshowhaveincreasingratesofhyperparasitismas

the developmental rate increases due to the relative decrease in the inflow of

newcadavers.

121

Figures

Figure1

Figure2

122

Figure3

123

Figure4

124

Tables

Table1

Fouralternativescenariosoftheimpactofvariationinfungaldevelopmentrate

forOphiocordycepsandhyperparasitic fungi. Inscenario1AandBthe infection

rate (b) is uncorrelated with parasite developmental rate while the rates are

correlatedinscenario2AandB,meaningthatthetimetheparasitespendsinthe

mature life stage, as determined by development rate, is positively correlated

with the rateatwhichnew infected individuals appear. In scenario1Aand2A

thehyperparasitedevelopmentalrateisuncorrelatedwithparasitedevelopment

rate while the rates are correlated in scenario 1B and 2B, implying that

environmental factors determining fungal growth affect the parasite and

hyperparasitesinthesameway.

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Table2

Parasite life‐stage distribution across the five graveyards with numbers of

sampledcadaversineachofthefourcategories.

Graveyard Fresh Stroma Mature Hyperparasitized Other Total

1 26 13 5 93 12 149

2 7 6 1 15 6 35

3 4 10 8 17 5 44

4 8 4 2 24 3 41

5 9 23 12 90 29 163

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CHAPTER 4

HOST SPECIFICITY OF PARASITE MANIPULATION

–ZOMBIE ANT DEATH LOCATION IN THAILAND

VS. BRAZIL

129

Host specificity of parasite manipulation –zombie ant death location in Thailand vs. Brazil

SandraB.Andersen1&DavidP.Hughes2

1CentreforSocialEvolution,DepartmentofBiology,UniversityofCopenhagen,

Universitetsparken15,2100Copenhagen,Denmark2DepartmentofEntomologyandDepartmentofBiology,PennStateUniversity,

PA16802,USA

InvitedAddendumto:

AndersenSB,GerritsmaS,YusahKM,MayntzD,Hywel‐JonesNL,BillenJ,

BoomsmaJJ,HughesDP(2009)Thelifeofadeadant:theexpressionofan

adaptiveextendedphenotype.TheAmericanNaturalist174:424‐433

Keywords:Parasitemanipulation,hostspecificity,zombieants

Correspondenceto:

SandraBreumAndersen,Email:[email protected]

andDavidP.Hughes,Email:[email protected]

INPROOF,non‐peerreviewedarticle

130

Abstract

Recently we presented how Camponotus ants in Thailand infected with the

fungusOphiocordyceps unilateralis are behaviorallymanipulated into dyingwhere

the conditions are optimal for fungal development. Death incurred in a very

narrow zone of space and here we compare this highly specific manipulation

witharelatedsysteminBrazil.Weshowthatthebehavioralmanipulationisless

fine‐tuned and discuss the potential explanations for this by examining

differencesinanthostandenvironmentalcharacteristics.

Text

Parasite manipulation of host behavior is an intriguing example of parasite

adaptation. The change of host behavior is considered to be an extended

phenotypeoftheparasite,asitcanbeexplainedasanexpressionofparasitegenes

in the host phenotype to increase parasite fitness.1‐3 One of the most dramatic

examplesofaparasiteextendedphenotypeisthemanipulationofantbehaviorby

thefungusOphiocordycepsunilateralissl.4,5

In a recent study we showed that the manipulation of the host ants by O.

unilateralisslishighlyspecificandbeneficialtoparasitefitness,therebyfulfilling

the criteria of an extended phenotype 6. InfectedCamponotus leonardi worker

ants leave their nest in the canopy and seekout theundersideof a leaf in the

undergrowth, bite into a leaf vein anddie. Theparasite thenquickly colonizes

the ant and grows for > 2 weeks before achieving reproduction. The death

locationof theantswas found tobe far fromrandom:dead infectedantswere

located 25.20 ± 2.46 SE cm above the ground, where the humidity and

temperaturewereoptimal for fungal growth, andon thenorth‐northwest sideof

theplantbitingontoaveinofthe leaf.Parasites inthedeadantsrelocatedfrom

this ‘manipulative zone’ did not grow, confirming the adaptive value of the

behavioral change. O. unilateralis was until recently believed to be a globally

distributedspecies,butmorphologicalstudiesinBrazilrevealedaspeciescomplex

with high host specificity.7 Here we wanted to explore the data on adaptive

131

manipulationfurtherandinfertheroleofhostcharacteristics,bycomparisonof

the data on the death position from Thailand with that of ants in the related

systemofOphiocordycepscamponotirufipedis(=unilateraliss.l.) infectingtheant

CamponotusrufipesinBrazil.Inaddition,ashyperparasitismbymycoparasitesis

extensiveandlikelyverycostlyfortheparasite,wewishedtoelucidatewhether

thepositionwhere theantwasmanipulated todiehadaneffecton theriskof

hyperparasitism.

Fieldwork took place in February 2011 inMata do Paraíso, a 400 ha Atlantic

rainforestnature reserve inMinasGerais,Brazil.The locationandconditionof

132 dead C. rufipes ants infected with O. camponotirufipedis was registered

along a 460 m stretch of forest path. For each dead ant we noted the height

above ground, the orientationof theant (whichcompassdirection theheadwas

pointing)andtheparasitelifestageasoneofthefollowingfourcategories:1.a

freshlykilledant(n=26),2.acadaverwithastroma(stem‐likestructurethat

is the precursor to the production of a mature fruiting body; n = 28), 3. a

cadaverwithamaturesexualfruitingbody(ascoma;n=19),4.acadaveratstage2

or3,buthyperparasitizedbyotherfungi(n=59).ThedatawereanalyzedinJMP

9.0.2 for Mac and PAST 1.80 (available as free download at

http://folk.uio.no/ohammer/past/) and comparedwith thedataonheight and

orientationofdeadC. leonardi ants infectedwithO.unilateralis s.l. obtained in

ThailandasreportedinAndersenetal.6

At theBraziliansitewe foundnodifference in theheightofdead infectedants

betweentheparasitelifestagesorinthevariationaroundthemean.Wesuggest

thatthereisnorelationshipbetweentheheightatwhichantsaremanipulatedto

die and theprobability of the fungus reachingmaturity or subsequently it self

becomingahosttohyperparasites(Fig.1A,One‐WayANOVABrazil:F3,128:0.329,

p=0.804,Levene’stestofunequalvariance:F3,128:2.390p=0.0718).Thedead

infectedantswerefoundhigherupinBrazilthaninThailandandwithagreater

variance around the mean (Fig.1B, t‐test assuming unequal variances t174.5: ‐

15.506,p≤0.0001,Levene’stestofunequalvariance:F1,181:89.555p≤0.0001).

Thismaysuggesta less fine tunedhostmanipulation inBrazil,potentiallyasa

132

consequence of a wider height range of appropriate growth conditions. A

temperatureandhumidityprofilebyheightfromthegroundwasnotmeasured

in Brazil, butmay likely differ from Thailand, as the forestwas denser.While

bothlocationsexperienceawetandadryseason,thedryseasonintheAtlantic

Rainforests of Minas Gerais, Brazil is correlatedwith lower temperatures and

highhumidity8,whichmaylowertheriskofparasitedesiccation.Inaddition,no

pattern in theorientationof theantswas found inBrazil, incontrast to thatof

the Thai ants (Fig. 2). Note that the direction of the ant head is depicted, in

contrasttolocationaroundtheplantasinAndersenetal.6,sothisisoppositeto

whatwepreviouslyreportedforThailandasthemajorityofantswerefacingthe

plantinThailand.Wedonotknowwhetheritisthelocationaroundtheplantor

thedirectionoftheheadthatisrelevanttotheantdeathlocationinThailand,but

neither appeared tomatter in Brazil.We suggest that this is because infected

ants in Brazil likely bite during the night, when the ants are most active, in

contrast to at noon inThailand 9. Thiswould eliminate the potential for using

solarcuesfororientation,e.g.byshade‐seekingbehavior.TheantC.rufipesisthe

dominantantspeciesatthefieldsiteinBrazil,wheretheynestonthegroundin

contrasttothecanopydwellingC.leonardi,whichwerecordedfromover20m

up in the canopy.How this affects theparasite strategy isunknown,but itmay

makethehostmoreaccessibletotheparasite inBrazil,as thedead infectedants

arepositionedincloseproximitytotheforagingtrailsofthehost(R.Loretoetal.in

preparation).However,thesocialimmunityoftheanthost10islikelystillthemain

challengefortheparasite,selectingforpersistenceintheenvironmenttoensure

transmission(S.B.Andersenetal. inpreparation). Inall, thecomparisonof the

two host‐parasite systems suggests that both parasites are highly adapted to

their hosts but that environmental and host differences confer different

strengthsofselectivepressureonthespecificityofhostmanipulation.

133

Acknowledgements

WethankRobertoBarretoandSimonElliotattheFederalUniversityofViçosa,

MinasGerais,Brazil,andHarryC.EvansattheFederalUniversityofViçosaand

CAB International, Surrey, UK for their kind hospitality. We are grateful to

JacobusJ.BoomsmaandRaquelLoretofordiscussion.SBAwasfundedbyaPhD.

Scholarship fromtheScienceFacultyof theUniversityofCopenhagenandDPH

wasfundedbyanOutgoingInternationalMarieCurieFellowship.

134

References

1. PoulinR.Manipulationofhostbehaviourbyparasites:aweakening

paradigm?ProcRSocLond(Biol)2000;267:787‐92.2. MooreJ.Parasitesandthebehaviorofanimals.OxfordUniversityPress,

2002.3. DawkinsR.Theextendedphenotype.OxfordUniversityPress,1982.4. EvansHC,SamsonRA.Cordycepsspeciesandtheiranamorphspathogenicon

ants(Formicidae)intropicalforestecosystemsII.TheCamponotus(Formicinae)complex.TransactionsoftheBritishMycologicalSociety1984;82:127‐50.

5. PontoppidanM‐B,HimamanW,Hywel‐JonesNL,BoomsmaJJ,HughesDP.Graveyardsonthemove:thespatio‐temporaldistributionofdeadOphiocordyceps‐infectedants.PLoSONE2009;4:e4835.

6. AndersenSB,GerritsmaS,YusahKM,MayntzD,Hywel‐JonesNL,BillenJ,BoomsmaJJ,HughesDP.Thelifeofadeadant:theexpressionofanadaptiveextendedphenotype.AmerNat2009;174:424‐33.

7. EvansHC,ElliotSL,HughesDP.Hiddendiversitybehindthezombie‐antfungusOphiocordycepsunilateralis:fournewspeciesdescribedfromcarpenterantsinMinasGerais,Brazil.PLoSONE2011;6:e17024.

8. CarmoPintoSd,VenâncioMartinsS,BarrosNFd,TeixeiraDiasHC.ProduçãodeserapilheiraemdoisestádiossucessionaisdeflorestaestacionalsemidecidualnaReservaMatadoParaíso,emViçosa,MG.RevistaÁrvore2008;32:545‐56.

9. HughesDP,AndersenSB,Hywel‐JonesNL,HimamanW,BillenJ,BoomsmaJJ.Behavioralmechanismsandmorphologicalsymptomsofzombieantsdyingfromfungalinfection.BMCEcol2011;11:13.

10.CremerS,ArmitageSAO,Schmid‐HempelP.Socialimmunity.CurrBiol2007;17:R693‐R702.

135

Figure Legends Figure1

Theheightabovegroundofdeadinfectedants.PanelAshowsthattherewereno

significant differences between the height of dead ants in four different life

stages inBrazil (fresh,stroma,matureandhyperparasitized(HP);meanheight

cm ± SD). The death height therefore does not affect which parasites reach

maturity and which succumb to hyperparasitic fungi. Panel B shows the

differencebetweentheheightatwhichdeadinfectedantsarefoundinBraziland

Thailand,wheretheantsdieatagreaterheightinBrazilwithagreatervariance

aroundthemean(meanheightcm±SD).

Figure2

ThedirectionoftheheadsofdeadinfectedantsinBrazilandThailand.InBrazil

therewasnopatterninthedirectionwhilethedeadantspointedtowardsSouth

inThailand.Theblueslicesshowthenumberofdeadants inagivendirection

while the red lines indicate themean head direction and the 95% confidence

interval.

136

Figures

Figure1

Figure2

137

CONCLUSIONS AND

PERSPECTIVES

139

Conclusions and perspectives The chapters of this thesis cover different aspects of heterogeneity in the

symbioticinteractionsbetweenantsandmicrobes.Whilethesystemsdealtwith

are quite different there are some overall conclusions to be drawn and new

questionstobeaddressedgeneratedbythefindings.

Genetic diversity of symbionts Thegeneticdiversityofbacterial symbiontswasaddressed in chapter1and2

andinbothsystemsarangeofgenotypeswasfoundinmosthosts.Theeffectson

thehostsofthisdiversitywerehowevernotcompletelyclearfromtheobtained

results. In the case of the Wolbachia bacteria, the overall consequences of

infectionarestillnotunderstood,yet thestudypresentedheresuggests that it

may be more in the mutualistic range of the symbiosis spectrum than the

parasitic, which has been the prevalent view. The unexpected location of the

bacteria extracellularly in parts of the ant gut suggests that theymay have an

undiscovered role in the ant digestion, an inference that holds promise for

furtherstudies.Theapparentinteractionbetweenthestrainsatcertainhostlife

stages, and the potential for the strains to have different phenotypic effects,

suggeststhatthediversitylikelywillbeofimportancetothehost.Thepresence

ofWolbachiabacteriainthefecaldropletsopensthepossibilityfortransmission

between workers and not just between queen and offspring. The partial

segregationofthetwostrainsfoundisinadditionrelevanttoscreeningstudies

of various insect species, where perhaps only a leg is used for the DNA

extraction. If some strains only are located in e.g. gut, they would likely be

missedbysuchascreeningapproach.

In the analysis of the actinomycete community composition on A.

echinatiorwefoundalowdiversity,withoverall justtwosegregatingstrainsof

Pseudonocardiadominating.Theresultsthusconfirmedthefindingsoftheinitial

studiesontheissue,byculturingofbacteria.Ithoweveralsohighlightedsomeof

the challenges of the analysis of 454 pyrosequencing data, in both the sample

collectionstepandthedataanalysis.Whileonlyasmallfragmentofcuticlewas

collected,withacoverofthetargetbacteriavisibletothenakedeye,arangeof

other confirmed and suspected contaminants from the tissue, and potentially

140

elsewhere,was present in the sequencing output of some of the samples. It is

thusnottoosurprisingthatotherstudiessequencingthebacteriaofwholeants

or culturing washes of ants identify a higher bacterial and actinomycete

diversity. More relevant to the questions of cooperation and conflict, and

stability of mutualisms, is it that a low diversity was generally found on the

laterocervicalplates,theareaofthecuticlewheretheantsapparentlysupplythe

bacteriawithnutrients.Thissuggeststhateithertheantsorthebacteriaareable

to control the community composition. The data was analysed in a range of

differentwaysbyadjustingthefilteringparametersandOTUselectionsettings,

all within the ‘standard’ range with the employed method, yet very variable

results were obtained, especially for the number of Pseudonocardia OTUs

present.Inthiscase,onlythesubsequentmanualvalidationagainsthighquality

sequences revealed a lowerdiversity.While the exact number ofOTUs froma

givengenusmaynotmatterinsomestudies,itwascentraltothis.

GiventhefocusonsymbiontdiversityinthefirsttwochaptersIwasalso

very interested in the diversity of Ophiocordyceps infecting ants, within and

between the individual ants in the graveyard.Parasitediversitywithin the ant

could either be a welcome opportunity for recombination or result in

competition with suboptimal host exploitation as outcome. Also, with‐in host

diversitycouldopenupthepossibilityofcheaterstrains,gainingmorethantheir

fair share of the reproduction by preferentially locating in the reproductive

tissues. Parasitediversity in the graveyardwouldbe equivalent towithinhost

variation(seebelow),aset‐upoftenobservedtoincreaseparasitevirulenceby

competition for host resources. The autonomy of the individual infected ants

may however limit this effect. The dead infected ants, for which the death

locationdatawasusedinchapter4,wereoriginallycollectedtostudytheeffect

of genetic diversity on host manipulation. DNA was extracted from whole

individuals and the amplification of variable regions of fungalDNA (ITS 1&2

and elongation factor 1a) was attempted. While amplification was successful,

cloning of the obtained PCR product proved challenging for unknown reasons

and when accomplished, a lot of sequence diversity was found, suggesting

amplificationofarangeofotherfungigrowingonorattachedtothecadavers.As

many fungal sequences deposited in GenBank, especially of environmental

141

samples, are not accurately named, the identification of Ophiocordyceps was

difficultso incombinationwith limitedtimeitwasnotpossibletoaddressthis

question further. Hopefully sequencing of pure cultures ofOphiocodyceps will

allowforthedesignofspecies‐specificprimers,whichwilleasethischallenge.

Environmental impacts – field vs. lab colonies In chapter 1 and 2 the bacterial symbionts were compared between field‐

collected colonies and colonies kept in culture rooms at relatively constant

conditions in regards to temperature,humidityand food for twomonths to10

years.ForWolbachia, theoveralldensityofbacteriawas found tobehigher in

the samples from lab reared colonies compared to those from the field,which

was particularly obvious for the rarer strainWSinvictaA. It also worth noting

thatallcoloniessampledfromthelabweredoubleinfected,incontrastto63%in

thefield.Whilethesamplesizeisnotlargeenoughtoinferwhetherallcolonies

in the lab indeed are double infected it is interesting to speculate on the

implicationsofsuchascenario.Thiscouldoccurifonlydoubleinfectedcolonies

survived the transition from the field to the lab, because the rarerWolbachia

strain provided some essential service needed in the new environment.

Alternatively,itmightbetheresultofwhatwouldbeconsideredsingleinfected

colonies,ifsampledinthefield,becomingdoubleinfectedinthelab.Thiscould

eitherbebecauseWSinvictaA isactuallypresent inallcolonies inthe field,but

repressedinsometoimmeasurableamounts,whileinthelabitisabletoachieve

ahigherdensity.SuchamechanismcouldalsobethereasonthatWSinvictaAis

foundinmeasurableamountsinthefieldinsomecolonies.Anotherexplanation

may be that the rarerWolbachia strainmay be vectored between colonies by

some unknown mechanism. Unfortunately, the field‐sampled colonies for this

studywerenotbroughttothelabandfieldsamplesfromthelabcolonieswere

onlyavailableforthreecolonies.Thesehaveyettobetestedbutthesamplesize

eitherwayhastobeincreasedtoconcludeanythingonthematter.

The Pseudonocardia community of A. echinatior was found to be

incrediblystableunderlabconditions,consideringitsexternal locationandthe

high proximity of colonies with different strains and species of bacteria.

InteractionswithEscovopsis areexpected tobeamajor selective factor for the

142

associationbetweenattineantsandPseudonocardia in the field.Howeveronce

successfully established in the Copenhagen lab, colonies will rarely encounter

Escovopsis.Theeliminationofthisthreatinthelabmayremovetheadvantageof

carrying thebacteria. It could thusbeargued that in theabsenceofEscovopsis

thecost:benefitratioofharboringthebacteria,whicharesustainedbytheants

atasignificantcost,shouldfavourtheantslosingthementirelywhenkeptinthe

lab.Unless thismaintenance cost isnegatedby the stableenvironmentandad

libitumfeedingintheculturerooms.Thecontinuedassociationbetweentheants

and bacteria could suggest that the ants are not actively controlling the

interaction. This raises the interesting question of to what extent the host is

capableofcontrollingthebacterialgrowthandthemechanismsavailabletothe

host for doing so and thus formaintaining amutualistic interaction. I find the

questions of how the association is controlled especially worthy of future

studies. The ants apparently support the bacterial growth by nutritional

secretions but how is the growth controlled to either just the laterocervical

plates or up‐regulated to the whole ant in case of an Escovopsis attack?

Actinobacteriainotherconditionsarefoundtoproduceantibioticswhenstarved

(M. Hutchings, personal communication), suggesting that the inducement of

growth, to fully cover the ant, and increased antibiotic productionmaynot be

easilycorrelated.Whydonewlyenclosedantsbecomefullycoveredinbacteria

that then subsequently are limited to the laterocervical plates? Andwhat role

does competition between bacterial strains play in the specificity of the

association versus e.g. host control via secretions? The bacteriamay however

also have yet undiscovered functions in addition to their activity against

Escovopsis.Othertraitscouldthusbecomerelevanttothehostinthelaboratory

settingandtherebystabilizetheinteraction.

The ant colony as a host Awell‐recognizedconsequenceofthesocialstructureofanantcolonyisthatthe

entirecolony,andnottheindividualants,isthehostoftheassociatedsymbionts.

In the studies ofWolbachia and Pseudonocardia we found no within colony

variation insymbiontdiversity.TheantsareknowntoacquirePseudonocardia

bacteria from the fungal garden and other colony members, which would

143

effectively maintain the colony specific community. While Wolbachia are

transmitted vertically from the queen, the presence of bacteria in the fecal

droplets asmentioned could serve as another route of transmission, primarily

within the colony. More intriguing is the aspect of horizontal transmission of

Ophiocordyceps, which requires the establishment of a new graveyard around

another ant colony.When searching formature parasites in the fieldwe on a

numberofoccasions foundbeautifulsamples inareaswithvery lowdensityof

dead infected ants, that is, not in graveyards, while these specimens were

difficult to locatewithingraveyards.This leadus tohypothesize thatwhile the

majority of ants die within an established graveyard, a few travel a longer

distanceand in thisnon‐graveyardarea theyhaveahigher chanceof reaching

maturitybyavoidingthehyperparasitismplaguingthehigh‐densitygraveyards.

If in proximity of another ant colony, a new graveyardmay be founded. This

wouldcreatemeta‐populationdynamicsof graveyardsaspatchesestablishing

and going extinct over periods of years. In addition, this would give the

expectationthatwithingraveyardparasitediversityislowasitwouldonlytake

oneorafewinfectedantstoinitiateandmaintainthegraveyard.

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Pictures FieldworkinBrazil.

1)Field‐stationinMatadoParaiso,MinasGerais,Brazil.Thelowpalmnexttothescooter continually attracted infected ants biting into the leaves, in spite of itslocation intheburningsun.2)A freshlydeadC.rufipesantwith fungalhyphaegrowingfromthejoints.3)Commonlymorethanonedeadantisfoundononeleafwithinagraveyard.Hereonehasbitontoanotherdeadinfectedant.4)Deadinfected ant biting onto pink tag used to mark another dead ant (photo H.C.Evans).

146

5)AtagraveyardwithAnnaMosegaard‐Schmidt,deadantsaremarkedwithpinktape,over40cadaverswerefoundonthistreealone(PhotoD.P.Hughes).6)Matureparasiteinantbitingontoatwig.7)Deadinfectedantcoveredinhyperparasites.

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ACKNOWLEDGEMENTS

Thecompletionofthisthesishadnotbeenpossiblewithoutthecontributionsofalonglistoffantasticpeople.

FirstandforemostthankyoutoKoosandDavid,forintotalalmostfiveyearsofencouraging, challenging and enthusiastic supervision with countless greatdiscussions,shapingmeasthescientistIknowstartfeelinglike.

Thanks to the CSE and CMEC communities and others in the building for anoutstanding work environment, providing great discussions on socialinteractions and even greater actual social interactions: Line Vej Ugelvig,Susanne den Boer, Henrik de Fine Licht, Matthias Fuerst, Lisi Fuerst, NanaHesler,Maj‐BrittPontoppidan,AnnaMosegaardSchmidt,JellevanZweden,DóraHuszar, Sämi Schär, Panos Sapountzis, Andreas Kelager, Anne Andersen, LukeHolman, Aniek Ivens, Birgitte Hollegaard, David Nash, Janni Larsen, Jes SøePedersen, Luigi Pontieri, Marlene Stürüp, Michael Poulsen, Morten Schiøtt,Patriziad´Ettorre,PepijnKooij,RachelleAdams,RasmusStenbakLarsen,SanneNygaard,SeanByars,SzeHueiYek,VolkerNehring,DaniMoore,NickBos,SylviaMathiasen, Charlotte Olsen, Bettina Markussen, Henning Bang Madsen, RuthBruus Jakobsen, Rikke Anker Jensen, Jonas Geldman, Irina Levinsky, MichaelBorregaard, David Nogues, Anna‐Sofie Steensgaard, Ben Holt, Katie Marske,ChristianHof,SusanneFritzandeveryoneIaccidentlyforgot…SpecialthankstoLineandHenrikforcommentingonthesynopsis.

Thanks to the more or less ‘external’ collaborators: Lars Hestbjerg and KarinVestbjergintheMicrobialMolecularBiologygroupatUniversityofCopenhagen;MetteBoye,JoannaAmenuvorandAnnieRavnPedersenattheVeterinarylabofthe Technical University of Copenhagen; Tom Gilbert from the Centre forGeoGenetics at University of Copenhagen; Matt Ferrari and Raquel Loreto atCentre for Infectious Diseases at Penn State University; Harry Evans at CABI,England; Sam Elliot from Federal University of Viçosa, Brazil; HermogenesFernandez‐Marín,STRI,Panama;LisbethHaugkrogh,AaseJespersenandJørgenLützen from Zoomorphology at University of Copenhagen and MichaelWilliamson then at the section for Cell and Neurobiology at University ofCopenhagen.

Thanks to Charissa de Bekker and Roel Fleuren for support and company inencounteringtheAmerican‘culture’ofStateCollegePA.

And last but not least many thanks to my family, friends and Patrick forproviding the necessary support and distractions to function outside theuniversityaswell!

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CURRICULUM VITAE Personaldata

SandraBreumAndersen 27.051983Slotsgade7,st.tv.2200KbhNTlf.nr.:[email protected]/[email protected]

Education

2008‐2012PhD,CentreforSocialEvolution,UniversityofCopenhagen2006‐2008 MScBiology,UniversityofCopenhagen2003‐2006 BScBiolog,UniversityofCopenhagen1998‐2001 Mathematicalhighschool,VirumGymnasium

Relevantworkingexperience

2011 PennStateUniversity,USA,2monthshostedbyDr.DavidHughes2011 FieldworkinViçosa,Brazil2008‐2011 Teachingassistantin’Populationbiology’atUniversityofCopenhagen.2008‐2011 NOVOscienceambassador.Disseminationofsciencetoschoolchildren2008 CommunicationcoursearrangedbySwift&GelindeandDNS.2008/2011 FieldworkinGamboa,PanamawithSTRI2007 FieldworkinKhao‐Chong,Thailand2006 BSc‐project at University of Queensland, Australien with Prof. Ove

Hoegh‐Guldberg, including 6 weeks of fieldwork at Heron IslandResearchStation.

2002‐2007 GuideatØresundsAquarium,UniversityofCopenhagen

Conferences

2011 ESEB,Tübingen,Germany,posterpresentation2010 IUSSI,Copenhagen,Denmark,posterpresentation2010 Evolutionarypotentialofwildpopulations,Sønderborg,Denmark2009 ESEB,Turin,Italy,posterpresentation2008 SocialInsectBiology,Oulanka,Finland,oralpresentation2008 BiologyofSocialInsects,Tartu,Estland,oralpresentation2007 PopulationandEvolutionaryBiologyofFungalSymbionts,Ascona,

Switzerland

150

Publications,peerreviewed

Hughes,D.P.,Andersen,S.B.,Hywel‐Jones,N.,Himaman,W.,Billen,J.,Boomsma,J.J.2011:Convulsionsandlock‐jaws:behavioralmechanismsandmorphologicalsymptomsofzombieantsdyingfromfungalinfection,BMCEvolutionaryBiology(11)1Andersen,S.B.,Vestergaard,M.L.,Ainsworth,T.D.,Hoegh‐Guldberg,O.,Kühl,M.2010:Acutetissuedeath(whitesyndrome)affectsthemicroenvironmentoftabularAcroporacorals,AquaticBiology(10)1Andersen,S.B.,Gerritsma,S.,Yusah,K.M.,Mayntz,D.,Hywel‐Jones,N.L.,Billen,J.,Boomsma,J.J.&DavidP.Hughes2009:Thelifeofadeadant–theexpressionofanadaptiveextendedphenotype,TheAmericanNaturalist(174)3

Publications,nonpeerreviewed

Andersen,S.B.,Hughes,D.P.2010:Zombiemyrerpåvejtilkirkegården.AktuelNaturvidenskab(4)26‐29(Danishpopularsciencejounal)

Funding

2008 NOVOScholarshipforMaster’sstudents,36.000DKKR2007 OticonfoundationgrantforfieldworkinThailand,2000DKKR2006 Oticonfoundation,Frimodt‐HeinekefoundationandCopenhagen

EducationgrantsforBScprojectinAustralia,intotal15.000DKKRPeerreviewingexperience

ReviewedforJournalofInsectPathology

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