Larval helpers and age polyethism in ambrosia beetles

15 Pages·2011·1.13 MB·English

Larval helpers and age polyethism in ambrosia beetles
PeterH.W.Biedermann1andMichaelTaborsky
DepartmentofBehavioralEcology,InstituteofEcologyandEvolution,UniversityofBern,CH-3012Bern,Switzerland
EditedbyBertHölldobler,ArizonaStateUniversity,Tempe,AZ,andapprovedSeptember1,2011(receivedforreviewMay14,2011)
Division of labor among the workers of insect societies is a the role of division of labor is unknown. Ambrosia beetles live
conspicuous feature of their biology. Social tasks are commonly inside trees, which is a habitat extraordinarily favoring social
sharedamongagegroupsbutnotbetweenlarvaeandadultswith evolution (12), apparently having fostered at least seven in-
completely different morphologies, as in bees, wasps, ants, and dependent origins of fungiculture in beetles (13). Hence, they
beetles(i.e.,Holometabola).Auniqueyethardlystudiedholome- represent a unique model system to study the evolution of so-
tabolous group of insects is the ambrosia beetles. Along with cialityinrelationtofungiculture.Interestingly,ambrosiabeetles
one tribe of ants and one subfamily of termites, wood-dwelling varyintheirmatingsystem(inbreedingvs.outbreedingspecies)
ambrosiabeetlesaretheonlyinsectlineageculturingfungi,atrait and ploidy level (haplodiploid vs. diploid species), which are
predictedtofavorcooperationanddivisionoflabor.Theirsociality factorsthathavebeenassumedtocontributetosocialevolution,
hasnotbeenfullydemonstrated,becausebehavioralobservations although their respective roles in social evolution are contro-
havebeenmissing.Herewepresentbehavioraldataandexperi- versial (1). The ambrosia beetle subtribe Xyleborini is charac-
mentsfromwithinnestsofanambrosiabeetle,Xyleborinussax- terizedbyregularinbreeding,haplodiploidy,andfungiculture(8,
esenii.Larvalandadultoffspringofasinglefoundresscooperate 14).Highrelatednessandhaplodiploidyincombinationwithan
inbroodcare,gallerymaintenance,andfungusgardening,show- extremelyfemale-biasedsexratioarefactorspredisposingthem
ingacleardivisionoflaborbetweenlarvalandadultcolonymem- to advanced sociality (1). Additionally, cooperation in fungi-
bers.Larvaeenlargethegalleryandparticipateinbroodcareand culture is likely because a single individual can hardly maintain
gallery hygiene. The cooperative effort of adult females in the afungusgardenonitsown(10).Indeed,itwasshownthatadult ON
colony and the timing of their dispersal depend on the number female offspring delay dispersal from their natal gallery, which UTI
L
of sibling recipients (larvae and pupae), on the presence of the results in an overlap of eggs, larvae, pupae, and at least two O
V
mother,andonthenumberofadultworkers.Thissuggeststhat generationsofadultoffspringwithinacolony(8).Ahelpereffect E
altruistic help is triggered by demands of brood dependent on of philopatric females has been indicated by the fact that the
care. Thus, ambrosia beetles are not only highly social but also number of staying females that do not reproduce correlates
show a special form of division of labor that is unique among positively with gallery productivity in Xyleborinus saxesenii Rat-
holometabolousinsects. zeburg (8). Behavioral observations of ambrosia beetles within
their galleries have been missing so far, however, because it is
| | | |
altruism cooperativefungiculture insectagriculture larvalworkers virtually impossible to observe them in nature inside the wood.
mutualism The only report on eusociality in ambrosia beetles is not based
onbehavioraldata,butreproductiveroleshavebeeninferredby
Divisionoflaborenhancesworkefficiencyandisfundamental destructivesamplingofactivenestsofAustroplatypusincompertus
to the biological evolution of social complexity (1). This (9).Tofacilitateobservationsofbeetlebehaviorsinsidegalleries,
we developed artificial observation tubes to contain entire col-
conspicuous feature of social insects largely explains their eco-
oniesofreproducingbeetles(15,16).Hereweusethisbreeding
logical success (2). Task specialization in insects usually occurs
techniqueofX.saxeseniitoask(i)whetheroffspringproducedin
between different age groups. Individuals either pass through
a gallery engage in alloparental brood care and fungus mainte-
consecutive molts during development (Hemimetabola; e.g.,
nance, (ii) whether different types of individuals specialize in
termites, aphids), with immatures resembling small adults and
divergent tasks, and (iii) whether decisions to help and to dis-
divisionoflaboroccurringtypicallybetweensuchlarvalnymphs
that may show morphological specializations [e.g., first-instar perse relate to the number of potential beneficiaries and the
numberofpotentialworkerspresentinthecolony.Furthermore,
soldieraphids(3)];orindividualsmetamorphosebydramatically
weevaluateexperimentally(iv)whetherfemaledispersaldepends
reorganizing their morphology during the pupal stage (Hol-
onthepresenceofanegg-layingfoundress,becauseherremoval
ometabola; e.g., bees, wasps, ants, beetles), which predestines
should affect the need for alloparental care. We compare our
larvaeandadultstospecializeindifferenttasksbecauseoftheir
results with the patterns of sociality known from other major
morphological and physiological differentiation. Indeed, larvae
insecttaxa.
of the eusocial Hymenoptera, for example, may produce nest-
building silk [weaver ants (4)] and may supply adults with extra Results
enzymesandnutrients[trophallaxisinseveralwaspsandants(2, Age-andSex-SpecificBehavior.Gallerymaintenanceandbroodcare
5, 6)]. However, all known cooperative actions of holometabo- were allocated differently between different age classes within
lous larvae are apparently completely under adult control (2), anest(Fig.1andTableS1).Thegallerywasextendedmainlyby
anddespite the potential for the evolution of highlyspecialized larvae (digging), which also reduces the spread of mold (SI Text
immature helper morphs, larvae of these taxa are largely im- andFig.S1),whereasfunguscare(cropping)andbroodprotection
mobileandhelplessandinneedofbeingmoved,fed,andcared (blocking)wereexclusivelyconductedbyadults.Blockingwasonly
for by adults (7). In ambrosia beetles, however, in which co-
operative breeding (8) and eusociality (9) also have been as-
sumed, larvae can move and forage independently in the nest,
Authorcontributions:P.H.W.B.andM.T.designedresearch;P.H.W.B.performedresearch;
which provides great potential for division of labor between P.H.W.B.analyzeddata;andP.H.W.B.andM.T.wrotethepaper.
larvaeandadults. Theauthorsdeclarenoconflictofinterest.
Divisionoflaborsetsthestagefortheoriginoffungiculturein
ThisarticleisaPNASDirectSubmission.
fungus-growing ants and termites (10). Ambrosia beetles origi- 1Towhomcorrespondenceshouldbeaddressed.E-mail:[email protected].
nate from solitary or colonial ancestors, and their fungus agri-
Thisarticlecontainssupportinginformationonlineatwww.pnas.org/lookup/suppl/doi:10.
culture may have coevolved with sociality (8, 10, 11). However, 1073/pnas.1107758108/-/DCSupplemental.
www.pnas.org/cgi/doi/10.1073/pnas.1107758108 PNASEarlyEdition | 1of6
Fig.1. DivisionoflaborandagepolyethismbetweenageandsexclassesinX.saxesenii.Barsshowthemean(±SE)proportionsoftimelarvae,teneral
females,maturefemales,andmalesperformeddifferentcooperativetasks.Statisticallysignificantdifferencesbetweentheclassesaredenotedbydifferent
letters(P<0.05;GEE,detailsinTableS1).NotescaledifferencesbetweenA–CandD–F.
performed by foundresses and mature females, and in 33 of 35 fungal layer on each other’s bodies by allogrooming. In males,
casesonlyoneindividualblockedatatime.Inthe25cases(in19 allogroomingwasfrequentlyfollowedbyamatingattempt;itmay
of 93 galleries) when we did not observe a blocking female, the thusservealsotoobtaininformationaboutfemalematingstatus.
followingbehavioralscanrevealedthatlarvaehadcrawledoutof
thegallery.Atleast71larvae((cid:1)x=3.7larvaepergallery)diedin Adult Female Behaviors Depending on Gallery Composition. The
thismanner,whichsuggeststhatanimportantfunctionofblocking proportion of time adult females exhibited cropping and allog-
istopreventlarvaefromgettingaccidentallylost. rooming and the occurrence of blocking were higher during gal-
Larvaeandadultstookdifferentsharesinhygienicbehaviors. lery stages when brood dependent on care was present in the
Only larvae compressed dispersed waste into compact balls colony(preadultandlarval-adultgallerystages)thanduringother
(balling;MovieS1).Frass-balls,piecesofwood,orpartsofdead times (postlarval gallery stage; Table S2). Shuffling was shown
siblings were moved within the gallery and pushed out of the equally often before, during, and after larval presence within
entrance (shuffling; Movie S2) mainly by mature females but to a gallery, whereas adult female digging tended to increase per
someextentalsobylarvaeandteneralfemales(Fig.1andTable capitaafterpupationofthelastlarva.Cannibalismwasnotshown
S1). The ultimate waste disposer was always the mature female before the hatching of adult daughters, and it occurred more
thatwasblockingtheentrance.Cannibalismwasdirectedtoward often when dependent offspring were still present. Dispersal of
adultbeetles(1case),pupae(3cases),andlarvae(37cases)that adultfemalesseemedtobecontingentonbroodcaredemands:it
were already dead (in most cases) or that did not respond to significantly increased in the 10 d after a major proportion of
beinggroomed.Insuchcasesthegroomer(larvaoradult)would larvaehadcompletedtheirdevelopmentrelativetothe10dbe-
useitsmandiblestoopenthebodyofthegroomedsiblingwithin fore (Wilcoxon: z = −3, P < 0.001, n = 23; Fig. S3). Because
seconds. Allogrooming was shown by all stages and both sexes, broodnumberslikelycorrelatewithfungusproductivity,dispersal
and it seemed to be crucial for individual survival: in an exper- couldbetriggeredbyalternativefactors,however,likethequality
imentwith10pupaethatweresinglykepteitherwithoneorsix of thewood/medium or fungus.Nevertheless,in summarythere
larvae,thepupaesurvivedinfiveoffivecaseswithsixlarvae,but arehintsthatsomebehavioraltasksofadultfemalesfunctionally
they survived in only one of five cases with one larva (Fisher relatetothedemandsofcare-dependentbrood.
exacttest:P=0.048,n=10;detailsinSITextandFig.S2).In In a second analysis we tested for the relationship between
the other four cases with one larva, the pupae were overgrown adult female behaviors and the numbers of adult females and
and killed by fungi. In addition, the body surface of single brood (pupae and larvae) present in the galleries during the
foundressesthathadnotyetsuccessfullyestablishedabroodwas larval-adult gallery stage only (Table S2). Analyzed per capita,
overgrown by a fungal layer (mainly Paecilomyces variotii and adult female digging, shuffling, cannibalism, and blocking were
Fusarium merismoides) within a few weeks, which caused the all independent of the numbers of adult females and younger
deathofatleast7of29solitaryfemales.Wheneverindividualsof nestmates.However,percapitaallogroomingandfunguscropping
differentstagesencounteredeachother,theyremovedthevisible activities significantly increased with increasing numbers of pu-
2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1107758108 BiedermannandTaborsky
pae and larvae, whereas with increasing adult female numbers cialize in different tasks than the colony foundress, and they
allogrooming significantly decreased. Adult female dispersal seemtoadjusttheirworkeffortflexiblytothevaryingsizeofthe
correlated negatively with the number of dependent offspring brood produced by the latter. Dispersal propensity of adult
[generalizedestimationequation(GEE):P=0.003;Fig.2Aand females(i)correlatespositivelywithlownumbersofdependent
Table S2] and positively with adult female numbers (GEE: P < youngandhighnumbersofadultworkers,and(ii)isincreasedby
0.001;TableS2). experimental removal of the foundress. This suggests that phil-
In 34 of 43 galleries with mature offspring, females delayed opatry may be related to indirect fitness benefits, because the
their dispersal from the natal nest after maturation. This phil- group memberssharing agallery areall very highlyrelated (for
opatric period (i.e., the latency from the first appearance of anotherxyleborinebeetleseeref.14).Thereisnomorphological
a mature female within a gallery until the first dispersal event) differentiation among adult females, and they are all fully ca-
correlatedpositivelywiththeaveragenumberofdependentbrood pable of breeding and establishing their own gallery. However,
(eggs,larvae,pupae)peradultfemalepresentduringthisperiod dissections of all females in a number of field galleries showed
(Spearmanrankcorrelation:R =0.379,P=0.027,n=34). thatdaughterscobreedintheirnatalgalleryinonlyaquarterof
S
colonies (17). In summary, the social and reproductive patterns
EffectofFoundressRemoval.Eggs are produced primarily by the of X. saxesenii conform to primitive eusociality, defined by
foundress,andin4of16galleriesdissectedinthefieldeggswere overlapping generations, cooperative brood care, and some re-
producedalso byatleastonedaughter (onaveragereproduced productivedivisionoflabor,despitetotipotencyinreproduction
23.9%ofallfemalesinthese4galleries:range,2–4egg-layerson ofallindividuals.
4–22 females in total; details in ref. 17). If fewer eggs are pro-
duced, the need for alloparental care declines. Therefore, we Division of Labor Between Larvae and Adults. Task sharing in X.
predicted that the number of dispersing daughters will increase saxeseniiisunequalbetweenthesexesandageclassesformostof
if the foundress is experimentally removed from the gallery. the social behaviors described (Fig. 1 and SI Text). Regarding
Our experimental interference raised the dispersal activity of gallery hygiene, for example, larvae form balls from dispersed
daughters (relative to the same galleries before the manipula- frass,whereasthetransportandremovaloftheseballsismainly
tion;Fig.2B)inthetreatment(Wilcoxon:z=−2.637,P=0.008, performed by adult females. Gallery extension is almost exclu- ON
n = 10) and in the control groups (Wilcoxon: z = −1.82, P = sively accomplished by larvae. A social role of larvae has never UTI
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0.034, n = 10). Removal of the foundress, however, raised the beenreportedinambrosiabeetles,andgallerymaintenanceand O
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dispersal of daughters much more strongly than the control sit- fungiculturehavebeenattributedsolelytothefoundress(11,15, E
uation (i.e., removal of the medium without the foundress; 18). Larvae might serve a cooperative function not only in
Utest:z=−3.708,P<0.001,n= 10+10). Xyleborini but also in other beetles, as anecdotal observations
andspeculationssuggestfromtheScolytinae[e.g.,Dendroctonus
Discussion sp.(19); the Xyloterini (20)], Platypodinae [e.g., Trachyostus
Here we report on division of labor and age polyethism in Co- ghanaensis (21); Doliopygus conradti (22)], and Passalidae [Pas-
leoptera,whichapparentlyrelatetothefungicultureinambrosia salus cornatus (23)]. The ultimate cause for the larval speciali-
beetles. It confirms the predicted high degree of sociality of zation in digging may relate to their repeated molting: as the
ambrosia beetles (9, 11). In X. saxesenii, all group members mandibles gradually wear during excavation (for wood-dwelling
contribute to the divergent tasks of gallery maintenance and termites see ref. 24), adult females showing extensive digging
brood care, and there is correlative evidence that female off- behavior would suffer from substantial long-term costs. In con-
springdelay their dispersaldepending onthe number of poten- trast,larvalmandiblesregenerate ateachmolt.
tialbeneficiariespresentintheirnatalgallery.Tasksaretypically Teneralandmaturefemalestakeoverfunguscare.Blockingof
shared differentially between larval and adult colony members, the gallery entrance is done exclusively by mature females and
whichresemblesthedivisionoflaborreportedfromtermitesand almostonlybythefoundress(seeref.14forsimilarobservations
otherhighlysocialinsecttaxa(Table1).Amongholometabolous in Xylosandrus germanus). Direct brood care by allogrooming is
insects,however,X.saxeseniiistheonlyspeciestodateknownto performedbyallageclasses.Malesgroomfemalesathighrates,
exhibit an active behavioral task specialization between larvae whichmayprimarilyservecourtshipbecausematingattemptsare
and adults. Furthermore, adult daughters in this species spe- alwaysinitiatedbyallogrooming(cf.20).Malesdonottakepartin
other social behaviors except for low levels of digging and crop-
ping.Asymmetriesinrelatednesscausedbyhaplodiploidyshould
favor females to become helpers (25, 26). Inbreeding can nev-
ertheless reduce relatedness asymmetries and thus favor also
males to help (27). Male soldiers and brood-caring males have
been documented in haplodiploid and sib-mating thrips and
Cardiocondyla ants, respectively (27). In Xyleborini, very few
malesareproduced[approximately5–12%ofoffspring(28)],and
malesdonotseemtocontributesignificantlytogalleryfunction,
hygiene, and fungus growth. Instead, they seem to specialize in
their reproductive role by continually attempting to locate and
fertilize their sisters. Nevertheless, both male larvae and adults
arefullycapableofperformingallofthebehaviorsexhibitedby
females, as demonstrated in galleries that contain only males
[approximately 2% of X. saxesenii galleries are founded by
unfertilizedfemalesthatproducesolelymalebroods(16,28)].
Fig.2. (A)Adultfemaledispersalcorrelatesnegativelywiththenumberof Thelife-historytrajectoryofX.saxeseniiismostsimilartothat
cared brood in the gallery (larvae and pupae; GEE: P = 0.003; statistical
of social aphids and some termite families, where individuals
detailsinTableS2).(B)Effectsofremovaloftheblockingfoundress.The
serve as helpers (e.g., workers and soldiers) in their natal colo-
numbersofdispersingX.saxeseniiadultfemales(mediansandquartiles)are
nies before maturation (Table 1). Either sex may help in these
shown4dbeforeand4daftertheexperimentaltreatments.Wilcoxontests:
*P<0.05,**P<0.01;Mann-WhitneyUtest:***P<0.001;samplesizeswere taxa, and their flexible developmental period allows individuals
10controlgalleriesand10galleriesfromwhichthefoundresswasremoved. to adjust the length of their immature stage to maximize their
BiedermannandTaborsky PNASEarlyEdition | 3of6
Table1. Formsofdivisionoflaborinexemplaryspeciesofthemostprominentsocialinsecttaxa
Behavioraltask-specialization
Betweenimmatures*and Amongadult Cooperationby
Socialinsectspecies adultstages females males Modeofnestingandnourishment Reference
Hemimetabola
Cryptotermescavifrons + − − Wooddweller,gutsymbionts 45
(Kalotermitidae)
Hodotermopsisjaponica + − + Wooddweller,gutsymbionts 46
(Termopsidae)
Reticulitermesfukienensis + − ? Subterranean,gutsymbionts 47
(Rhinotermidae)
Macrotermessubhyalinus + + + Subterraneanandmoundbuilding, 48
(Termitidae) funguscultivation
Pemphigusspyrothecae + − − Plantgalls,sapsucking 49
(Aphidae)
Kladothripsintermedius − + + Plantgalls,sapsucking 50
(Thysanoptera)
Holometabola
Lassioglossumzephyrum − + − Groundnesting,pollenandnectar 51
(Halictinae)
Exoneurabicolor(Allodapinae) − + − Stalk-nesting,pollenandnectar 51
Apismellifera(Apidae) − + − Cavitynesting,pollenandnectar 52
Liostenogasterflavolineata − + − Freenesting,arthropodsandnectar 53
(Stenogastrinae)
Vespulagermanica(Vespinae) − + − Freenesting,arthropodsandnectar 7
Attatexana(Formicidae) − + − Subterranean,funguscultivation 2
Oecophyllalonginoda −† + − Treenesting,arthropodsandhoneydew 4
(Formicidae)
Austroplatypusincompertus − + + Woodboring,funguscultivation 9
(Platypotinae)
Xyleborinussaxesenii + + + Woodboring,funguscultivation Thisstudy
(Scolytinae)
+,present;−,absent;?,unknown.
*Nymphalandlarvalstages.
†Larvaeofweaverantsproducenest-building-silk,buttheweavingactioniscontrolledbytheworkerants(2,4).
inclusivefitness.Theymayeitherremaininanimmaturehelper In Xyleborini, ideal conditions for the evolution of both mu-
phase at the nest or develop into adults that can disperse or tualism and altruism seem to prevail: (i) they are well pre-
reproduce in their natal colony (3, 29–32). Likewise, the de- adapted to brood care because they originate from a beetle
velopmentalperiod ofsecond-andthird-instarlarvae ofX.sax- lineage (Scolytinae) with parental care (38); (ii) they breed in
esenii can vary between 4 and 17 d (16). In addition, there is isolated galleries that are founded by one reproductive that
astrikingsimilarityofthesetaxainecologyandmatingpatterns. dominates offspring production [i.e., the foundress (17)]; (iii)
Theyallinhabitdefensiblenests,oftenwithinwood,whichfavors they are haplodiploid and mate predominantly among full sib-
local mating and inbreeding—conditions claimed as strongly lings [e.g., inbreeding coefficients of approximately 0.9 in Xylo-
favoring social evolution (12). In many of these cases helping sandrusgermanusReiter(8)],whichincreasesrelatednesswithin
tasksdonotseemtocurtailahelper’sfuturereproduction(i.e., natal colonies; and (iv) they disperse solitarily after maturation
becausehelpingisriskfreeanddoesnotreduceahelper’senergy toreproduceelsewhere.Inaddition,(v)colonymembersbenefit
stores), which may weaken the tradeoff between helping and greatly from cooperation due to their dependence on fungi-
futurereproduction(33,34). culture; and (vi) the wood used as a resource for shelter and
substratum for fungi is virtually nondepreciable, which renders
RoleofIndividualSelectionandKinSelection.Wedefinedabehav- resource competition negligible. Larval digging, for example,
ior as cooperative if social partners potentially benefit from its whichmightberegardedasaby-productofselfishlarvalfeeding,
performance,independentlyofwhetherthisbehaviorentailsnet reduces competition because it generates a common good (i.e.,
costs to the actor (35). This definition includes (i) mutualistic space and substratum for fungiculture). Similarly, other group
behaviorsthatareregardedasselfishactsgeneratingbenefitsto members benefit from gallery hygiene resulting as a by-product
otherindividuals(commongoods)asaby-product(36),and(ii) from the seemingly selfish adult feeding activities cropping and
altruisticbehaviorsthatbringaboutnetcoststotheactor,which cannibalism.Bycontrast,balling,shuffling,allogrooming,blocking,
are compensated through indirect fitness benefits via kin-selec- and adult digging may rather be altruistic. These behaviors ap-
tion;theywillthusonlyevolveingroupsofrelatives(25).Inthe parentlybenefitothergroupmembersattheexpenseoftimeand
course of social evolution and task specialization shaped by kin energy costs to the actors, without immediate benefits to the
selection, mutualistic behaviors may lose their original function latter. Particularly dangerous are allogrooming and blocking,
andchangeintotrulyaltruisticbehaviors(36).Highrelatedness becausetheyexposetheperformerstopathogens,parasites,and
andspatialseparationbetweengroupsareveryfavorabletothe predators.
evolution of cooperative behaviors (12), as long as local com- Adult X. saxesenii females showed a strong incentive to stay
petitionbetweenrelativesdoes notoppose thisforce(37). and cooperate in a productive natal nest. Partly this may be
4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1107758108 BiedermannandTaborsky
selfish,becauseuptoonequarteroffemalesmaygetthechance Adultfemalebehaviorsdependingongallerycomposition.Tocheckforpotential
to reproduce (17), and females might build up reserves during effectsofgalleryageonbehavior,wediscriminatedbetweenthefollowing
their philopatric period for subsequent dispersal and nest successivestagesofgallerydevelopment:(i)preadultgallerystage:founder
foundation. An experimental study in the ambrosia beetle femaleanddependentoffspring(larvaeandpupae)withorwithouteggs
Xyleborusaffinissuggests,however,thatfemalesdonotbuildup present(n=2galleries);(ii)larval-adultgallerystage:allageclasses(mature/
teneral beetles,larvae,and pupae)withor without eggs present (n = 31
reservesduring their philopatricperiod but rathersuffer direct
galleries);and(iii)postlarvalgallerystage:onlyadultswithouteggs,larvae,
fitness costs(39). Alternatively, indirect fitnessbenefits maybe
orpupaepresent(n=16galleries).Variationinsamplesizeofgallerieswas
involved in helping to raise siblings (8). Three results suggest causedbythefactthatnotallageclasseswerevisibleineverygallery.We
that adult female cooperation is triggered by the demands of usedafirstseriesofGEEstoidentifytheeffectoftheparticularstageof
brood dependent on care: (i) helping effort of adult females gallerydevelopmentontheproportionoftimeadultfemales(=teneraland
rose with a greater number of brood dependent on care, (ii) maturefemales)spentwithacertainbehavior.InasecondGEEseriesper-
adult females dispersed at a higher rate with an increasing formed only with data of the larval-adult gallery stage, we analyzed
number of workers in the colony, and (iii) dispersal rate of whetherandhowtaskperformanceofadultfemalesrelatedtothenumber
females increased in response to experimental removal of the ofadultfemalespresentandtothenumberofpupaeandlarvaepresentin
thegallery(TableS2).
foundress, which indicates that delayed dispersal of females is
Femaledispersal.Foreachgallerywemeasuredtheretentionperiodbetween
not primarily motivated by the potential to breed in the natal thefirstappearanceofamaturedaughter(fullysclerotized,readytodisperse)
gallery (see also ref. 8). withinthegalleryandthefirstfemaledispersalevent(i.e.,thefemalehadleft
Inconclusion,thehighdegreeofsocialityinambrosiabeetles throughthegalleryentranceandsatontopofthemedium,whereittriedto
seems to result from a combination of four major factors: (i) escapethroughthecap).UsingaSpearmanrankcorrelationanalysiswetested
parentalcareasapreadaptationfortheevolutionofsocialityin whetherthedispersaldelayintervalrelatedtothemeannumberofoffspring
the ancestors of modern ambrosia beetles (38); (ii) very high attended,dividedbythemeannumberofadultfemalespresentatthattime.
relatednesswithinfamiliesduetohaplodiploidyandinbreeding; Influenceoffoundressonoffspringdispersal.Intheentrancetunnelfoundresses
(iii) a proliferating, monopolizable resource providing ample eitherblock(sitstillandfullyclosethetunnel)ormovebackandforthwhen
food for many individuals, which needs to be tended and pro- shufflingmaterialtothedumps.Todeterminetheinfluenceofthefoun-
tbeecettelde;seaendref(.iv4)0)hdiguhetcoostthseodfiffidcisuplteiressaolf(fifonrdianngoathseuritasbcolelyhtionset dwreeses’xppreersimenecnetaolnlythreembeohveadviotrhaenfidrdstispceernstaimlpertoepreonfsittyhoefemnatrtaunrecefetmuanlnees,l UTION
whenafemalewaspresentinthere(n=11galleries),atastagewheneggs, L
tree,ofnestfoundation,andasuccessfulstartoffungiculture(11, larvae,andadultdaughterswerepresenttogetherwiththefoundress.We VO
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16), which render predispersal cooperation particularly worth- determinedthereproductivestatusoftheremovedfemalebydissectionto
while.X.saxeseniilarvaearepredisposedtoassumecertaintasks checkwhetherwehadsuccessfullyremovedthefoundress.Weexcludedone
likeballingoffrassandgalleryenlargement(digging)becauseof gallery from the treatment group, where we found an immature female
theirbodymorphologyandthefrequentrenewalofmandiblesby blockinginsteadofthefoundress.Inthecontrolgroup(n=10galleries)we
molting. Thus, behavioral tasks are shared between larval and removedthefirstcentimeterofmediumwhennofemalewaspresentinthis
adult stages. This has not been shown for beetles, and the de- partoftheentrancetunnel.Experimentalgallerieswererandomlyassigned
scribed division of labor between immature and adult stages totreatmentandcontrolgroups.Dispersalofthedaughterswasmeasured
inbothgroupsfor4dbeforeand4daftertheinterventionbycollectingthe
seemstobeuniqueamongholometaboloussocialinsectsatlarge.
females on top of the medium that had left the gallery throughthe en-
tranceandtriedtodispersethroughthecapofthetube(Fig.2B).
MaterialsandMethods
StudySystem.X.saxeseniigalleriesarefoundedbyindividualfemalesthat
Statistics.WeusedaseriesofGEEs[lmerinR(43)],whichareanextensionof
transmitsporesofthespecies-specificambrosiafungusAmbrosiellasulfurea
generalizedlinearmodelswithanexchangeablecorrelationstructureofthe
Batraintheirgutfromthenataltothenewgallery(41,42).Thisfungus
responsevariablewithinacluster(=galleryidentity),toanalyzeeffectsof
formsayellowlayeroffruitingcellsonthesurfaceofgallerywalls(Fig.S4A).
dependentvariablesoncorrelatedbinaryresponsevariables(proportional
Afterlandingonatreetrunk,thefoundressexcavatesastraighttunnelinto
dataweretransformedtobinarydata)andtoidentifyfactorsaffectingthe
thexylemwithasmalleggnicheatitsend.Assoonasfungusbedsemerge,
relativebehavioralfrequenciesperclass(larvae,adultfemales,andmales)
shefeedsonthemandstartsegg-laying(16).Duringoffspringdevelopment
(44).First,wetestedwhetherthelarvae,teneralfemales,maturefemales,
theeggniche isenlarged toasingle flatbroodchamberof uptoa few
andmalesshowdifferenttendenciestoexpressthecooperativebehaviors
square centimeters in size and ≈1-mm height. There, most of the fungus
(Fig. 1 and Table S1). Second, we compared these frequencies between
gardengrows, andindividualsofallageclasses liveinclosecontactwith
foundressesandtheirmaturedaughters(SITextandTableS4).Inathird
eachother(Fig.S4C).InthisstudywebredX.saxeseniiinglasstubesfilled seriesofGEEswedeterminedtheinfluenceofaparticulardevelopmental
with artificial medium that mainly contained sawdust (for details on this
stageofthegallery(preadult,larval-adult,andpostlarvalgallerystages)on
methodseeSITextandref.16).
therelativebehavioralfrequencyperclass(TableS2).Finally,wemodeled
whetherlarvaeandadultfemalenumbersaffectedtherelativefrequencies
BehavioralRecordingsandAnalyses.Intotal,93ofroughly500gallerieswere
ofcooperative behaviorsinadultfemales(TableS2).Fortheremovalex-
foundedsuccessfullyinthelaboratory(i.e.,eggswerelaid),andin43ofthese
periment we compared behavioral frequencies and dispersal activity be-
galleriesindividualsreachedadulthood.Thesegallerieswereusedforbe-
tween the groups using Mann-Whitney U tests, and within groups using
havioralanalyses.Wedistinguished11behaviors(TableS3),whichcomprised
Wilcoxonmatched-pairssigned-rankstests(Fig.2B).Allstatisticalanalyses
seven cooperative behaviors that apparently raise the fitness of colony
wereperformedwithSPSSversion15.0andRversion2.8.1(43).
members.Everysecondtothirddayweperformedscanobservationsofall
individualsvisiblewithinagallery(detailsinSIText).
Age- and sex-specific behavior. We used GEEs (details below) with an ex- ACKNOWLEDGMENTS.ThepreviousworkofK.Peerprovidedthebasisof
thisstudy.WethankC.ArnoldandS.Knechtforhelpinapilotstudy,and
changeablecorrelationstructureoftheresponsevariablewithinacluster(=
U.G. Mueller, T. Turrini, R. Bergmueller, K. Peer, and two anonymous
galleryidentity)toidentifyeffectsofthefourclassesofindividuals(larvae, reviewersforhelpfulcommentsonthemanuscript.P.H.W.B.waspartially
teneralfemales,maturefemales,males)ontheproportionoftimespentwith supported by the Roche Research Foundation and a DOC grant from the
acertainbehavior,usingbinomialerrordistributions(Fig.1andTableS1). AustrianAcademyofScience.
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6of6 | www.pnas.org/cgi/doi/10.1073/pnas.1107758108 BiedermannandTaborsky
Supporting Information
Biedermann and Taborsky 10.1073/pnas.1107758108
SIText affectsurvivalofpupae,andthat(ii)sixdigginglarvaeshouldbe
Activity of Individuals Dependent on Light and Gravity. All behav- more successful than one digging larva to hinder the spread of
ioralobservationsofXyleborinussaxeseniiinthisstudyweredone moldonthe chamberwalls.
underamicroscopewitha6-Wartificiallightsource.Forstorage, Survival of pupae was significantly affected by the number of
however,tubeswerewrappedinpapertokeepthegallerydarkas coinhabitinglarvae(Fisherexacttest:P=0.048,n=10).Allfive
if in wood. Therefore, we tested whether the changing of light pupaesurvivedinthechamberswithsixlarvae,whereasonlyone
conditionsandoftheaxisofgravitywouldaffectbeetleactivity. of the five pupae survived in the chamber with one larva (Fig.
Fivegalleriesduringthelarval-adultgallerystagewereusedto S2).Pupae diedbecausethey were overgrownby fungi.
obtainfiveactivitymeasuresafterthreedifferenttreatments:the Thepercentageofchamberwallareacoveredwithmoldafter
numbers of active and inactive individuals (larvae and adults 14dwassignificantlylowerwhenthechamberwasinhabitedbysix
combined) in eachgallery were counted (i) rightafter uncover- larvae(median24.1%)thanbyonelarva[median90.5%;gener-
ingthetubes,(ii)after30minofexposuretoa6-Wlightsource, alizedestimationequation(GEE):coefficient±SE=−3.072±
and(iii)after 60minoflightexposure,rightafterchangingthe 0.163,z=18.86,P<0.001,n=26;Fig.S1].Moldfungicovered
axisofgravity by 90°. 93%ofthechamberareaintwochambersthatwereleftempty
Separate pairwise comparisons (Wilcoxon matched-pairs for 14d.
signed-rankstest)ofthefiveobservationswithineachgallerywere In summary, this experiment revealed the importance of (i)
combined in a metaanalysis according to Sokal and Rohlf (1): larval allogrooming for the survival of pupae and of (ii) larval
χ2(2×N) =−2 ×Σln(π). digging tohinder the spreadof moldfungi within the gallery.
This revealed no significant influence of the light [χ2(10) <
18.31;P>0.05]andgravitytreatments(P>0.05)ontheactivity Behaviors of Foundresses Vs. Mature Daughters. Usually the foun-
ofbothlarvae and adults. dresscannotbedistinguishedfromhermaturedaughtersoncethe
BehaviorsofthebarkbeetleIpspinialsodonotdifferbetween latterarefullysclerotized.Insixgalleries,however,thefoundress
individuals kept in the dark or exposed to light, nor between could be distinguished from her mature daughters because they
individualsrearedinchambersinaverticalpositionorinahor- remained lighter than their mothers for an extended period. In
izontalposition (2). thesegallerieswemade10focalobservationsoffoundressesand
14ofmaturedaughters(10mineach).GEEmodelsshowedthat
TendencyofX.SaxeseniiLarvaetoAggregate.Forthisexperiment foundressesandtheirmaturedaughtersdifferedsignificantlyinthe
we removed all individuals and tunnels from seven galleries frequency of cooperative behaviors, except for allogrooming
during the larval-adult gallery stage, leaving only some centi- (Table S4) and cannibalism, which were never observed by
meters of fungus-infested medium within the tubes. In these foundresses.The individual blockingthegalleryentrancewas al-
seven tubes we created three artificial chambers of ≈20 mm2 ways the foundress during the focal animal observations. In the
(and ≈1-mm height) within the medium, extending next to the removalexperiment,however,1of11dissectionsoftheblocking
glassfromthetopofthemediumalongsidethetubewall.Sixto femalerevealedimmatureovaries,indicatingthatdaughtersmay
twelve second/third-instar larvae were placed ontop ofthe me- also block the gallery entrance occasionally. Per capita, foun-
diumineachofthetubesandallowedtomovefreely.After24h dresses showed more fungus cropping and shuffling behavior but
werecordedthe location of alllarvae. less gallery extension behavior (digging) than their mature
Insixreplicates,alllarvaehadmovedinsideoneandthesame daughters. In summary, foundresses spent more time with co-
ofthethreechambers.In onlyonereplicate hadlarvaesplitup operativebehaviorsthantherematuredaughters.
and were distributed over two chambers. This suggests that X.
saxeseniilarvae show astrongtendency toaggregate. Details on Function of Different Cooperative Behaviors. Gallery
extension—digging.Animportantcommon good created bylarval
Effect of Larval Numbers on the Survival of Pupae and on Fungus digging is the enlargement of the gallery. Nevertheless, it is
Growth. For this experiment we removed all individuals from probably a mutually beneficial behavior with selfish benefits for
13galleriesduringthelarval-adultgallerystage.Ineachofthese thelarvaebecauseitisalsopartoftheirfeedingonfungalhyphae
galleries we created two flat chambers with a height of ≈ 1 mm penetratingthewood.Theconsumedwoodpassesthegutwithout
and an area between 22.86 mm2 and 91.89 mm2 next to one of any sign of digestion (3, 4), however, because the enzymes re-
the existing tunnels and next to the tube glass (to allow ob- quiredtodigestwoodarelikelymissing(5,6).Becausehyphens
servations). Thereafter, one chamber was filled with one larva ofseveralfungicooccurtogetherwiththeambrosiafungusinthe
and the other one with six larvae (chambers were randomly as- medium/wood (especially when the gallery gets older), larval
signed).In5ofthe13gallerieswealsoplacedonepupaeachin digging apparently also hindered the spread of mold (see above
both chambers, which contained one or six larvae, respectively. andFig.S1).Moldfungididnotcompletelyovergrowchambers
For the next 14 d we tracked the survival of the pupae (in 5 in the presence of larvae. They probably serve the larvae as ad-
galleries) and the appearance of mold on the walls of the ditionalfoodsource,despitethefactthatthesefungiareusually
chambers (in all 13 galleries). Several fungi coexist in the me- toxic to arthropods. Previous studies have shown that toxic sec-
dium at this gallery stage, and mold fungi are expected to ondary metabolites produced by mold fungi as a response to
overtakeiftheyarenotcontrolledbylarvae.Therefore,wetook feeding by arthropods can be overcome if the arthropods feed
pictures of the chambers on day 14after the treatment and an- gregariously on these fungi (7–9). X. saxesenii larvae showed
alyzed the chamber area covered and uncovered by mold ac- astrongtendencytoaggregatewithinthegallery(seeabove).
cordingtolarvalnumbers,byusingtheprogramImageJ(version The digging of adults is clearly different from feeding and
1.44p).Wepredictedthat(i)fungallayersgrowingonthebody solely servesgalleryextension.
surface of pupae should be removed more frequently by six Gallery enlargement by digging is a mutual benefit to colony
allogrooming larvae than by one allogrooming larva, which may members, because it increases the surface where the ambrosia
BiedermannandTaborskywww.pnas.org/cgi/content/short/1107758108 1of9
fungi can grow, in this way lowering within-family competition retain workers in the nest, because females dispersed at higher
for food and space (10). Gallery surface area positively affects ratesafter the removalof the blockingfoundress (Fig.2B).
fungusandthuscolonyproductivity(11–13).Thecontributionof Brood care and body hygiene—allogrooming. Allogrooming was fre-
adultsto galleryextension is negligible compared withlarvae. quently observed between individuals of all stages, and its im-
Fungus care—cropping. Adult females are constantly walking and portance was shown by (i) pupae being overgrown by fungi and
screeningthefungallayerswiththeirlarge,disk-shapedantennae dying more frequently in the presence of one allogrooming larva
(Fig.S4D). They stopfrequently tomove their hairy, comb-like compared with six allogrooming larvae (see above), and (ii) the
mouthpartsthroughthefungus(Fig.S4E),probablybrushingoff repeated observation of single foundresses (before a brood was
sproutingambrosiastructures(Fig.S4B).Thiscroppingbehavior successfully produced) dying due to a lack of getting groomed.
of the adults apparently serves both nutrient intake and fungus Four solitary foundresses died because they got adhered to the
care. It induces the characteristic ambrosial growth of the fun- gallery wall by a fungal layer on their elytra, or fungi grew un-
gus, that is, the formation of copious ambrosia cells (fruiting derneaththeelytraandcausedthemtoswellsothatmovingwas
bodies)andsporodochia[clustersoffungusspores(14–18)](Fig. prevented. Additionally, it has been reported from ambrosia
S4 A and B). The presence of beetles is thus crucial for the beetles that eggs do not hatch (41) and larvae die (21) in the
growth of consumable ambrosia fungus structures (15, 19). Ad- absenceofthegroomingfoundress.Thegregariouslifeofallage
ditionally,croppingislikelytopreventinvasionsoftheambrosia classes that constantly groom each other might also explain the
garden by foreign fungi and microbes (14, 20, 21). Oral secre- puzzlinglowfrequencyofparasitoidsandmitesfoundinnestsof
tions of X. saxesenii contain various bacteria (e.g., refs. 22 and ambrosiabeetlescomparedwiththoseoftheircloserelatives,the
23) that possibly support fungus growth either by providing nu- nongregarious, phloem-feeding bark beetles (35, 42, 43). Recent
trients or by controlling the spread of pathogens, as shown for studies of other taxa have shown that grooming can greatly de-
bacterial mutualists of fungus-culturing ants (24–26). creasethesuccessofparasitoids[e.g.,bygroomingtheireggs(44)],
Galleryhygiene—balling,shuffling,andcannibalism.Galleryhygieneis parasites[e.g.,phoreticmites(45,46)andentomopathogenicfungi
important for a flourishing fungus garden as well as a healthy (47,48)].Costsofgroomingincludethetimeandenergyspentand
colony.Thelarvalballingbehaviorhasnotbeendescribedbefore. theriskofparasitetransmissiontothegroomer(30,49,50).
Bending their bodies ventrally, larvae form cordlike frass into
DetailedMaterialsandMethods.Studysystemandlaboratorybreeding.
ballsthatcanbeshiftedwithinthegalleryordumpedoutofthe
Of 350 galleries of X. saxesenii studied in the field, the majority
entrance more easily (Movie S1). Frass balls are shifted by
shuffling (Movie S2) them toward certain areas, where they are produced approximately 10–25 dispersing individuals, 3.6% pro-
duced more than 100 individuals, and one gallery exceeded 300
used either to close parts of tunnels, possibly to regulate hu-
midity (14); to isolate diseased areas [so-called “death cham- individuals (51). The beetles show a strong sexual dimorphism:
bers”(27,28)];ortoberecycledbythefungus.Weobservedthat theraremales[theaveragesexratioisapproximately1:8to1:20
male/female (52)] donot fully sclerotize, stay small, and are un-
the wood chippings in the larval frass are often integrated into abletofly.Mostofthemdieintheirnatalgalleryafterthefemales
the fungal beds (see also ref. 27), which suggests that the mas-
havedispersed(52).
tication of the wood may facilitate resource utilization by the We bredX.saxeseniiinglasstubesfilledwithartificialmedium
ambrosia fungi. This would explain why nitrogen from beetle thatmainlycontainedsawdust(“testmedium”describedinref.53).
excretions had been detected in growing fungi (29). Perhaps
We used one founder female per tube that had either been col-
mostimportantly,frassanddebrisaredisposed,becausespaceis lected in the field or originated from a brood raised in the labo-
required for fungi to grow and for beetles to move (3, 12). Oc-
ratory. Females excavated galleries and brood chambers largely
casionallyweobservedsiblingcannibalism,whichmayserveboth
along the transparent tube wall, which enabled observations of
nutrient recycling and the removal of dead and diseased speci-
activity and development (Fig. S4A). When a gallery had been
mens.Thelatterisprobablyessentialforhinderingthespreadof
successfullyestablished,thetubewaswrappedinpapertokeepit
diseases and parasites [including mold (30, 31)], which is par-
darkasifinwood,butlightcouldenterthroughtheentranceatthe
ticularly threateningfor highly inbred communities (32, 33).
top of the tube. The tube was kept in a constant light/dark cycle
Galleryprotection—blocking.Blocking of the entrance by a gallery (11 h light/13 h dark) at 28 °C/22 °C and 70% humidity, and the
member is ubiquitous in ambrosia beetles, although it exposes
paperwrapwasonlyremovedduringobservationsunderamicro-
theblockingindividualtopredation,forinstancebybirds(34,35) scope (×6.4 to ×16 magnification) with an artificial light source
orpredatorybeetles(36).Parasitoidsalsohavebeenreportedto (maximum6W).Behaviorofscolytinebeetlesisaffectedneither
layeggsonblockingambrosia beetles(37). bylight(keptinthedarkvs.exposedtolight)norwhenthegallery
Blocking provides a variety of essential services to the gallery isturnedby90°,changingtheaxisofgravity(seeaboveandref.2).
members (see ref. 38 for review), like regulating the microcli- Behavioral recordings and analyses. Every second to third day we
mate, preventing larvae from falling out of the gallery, and ex- performed scan observations of all individuals visible within
cluding parasites, parasitoids,predators, andforeignfungi from a gallery: we noted the gallery identity, the number of visible
the gallery, which are common threats in bark and ambrosia individuals, and the respective behaviors they showed at that
beetles.Additionally,itmayhinderotherambrosiabeetlesfrom moment,thenumberofdispersingindividualsfoundonthesurface
entering a proliferating gallery and foreign males from mating ofthemedium(wheretheytriedtoescapethroughthecap),and
with females in the gallery, which can detrimentally affect their wecountedthenumberofeggsandpupae.Ineachscanallgallery
future reproductive success due to an outbreeding depression partswerebrowsedonetimeforvisibleindividualsasdescribed.
(39). Our study cannot distinguish between these potential, not We did not discriminate between the three larval instars.Pupae
mutually exclusive functions, but the data suggest that blocking andadultbeetlesweresexedonthebasisofmorphologyandsize.
byadult females is essential for the safety of larvae. Larvae are Wediscernedteneraladultfemalesthathadrecentlyhatchedand
verymobile,andinadditiontoourownresults,theonlyprevious showedweaksclerotizationandbrownishelytra,andmatureadult
removalexperimentofablockingfemaleinscolytinebeetleswe femalesthatwerefullysclerotizedwithdarkbrowntoblackelytra.
know of also resulted in the loss of larvae [and eggs; species: From each scan observation we noted the proportion of
Coccotrypes dactyliperda Fabricius (40)]. In line with this, block- individuals per class (larvae, teneral females, mature females,
ing is most common during the presence of larvae and tends to males) performing arespective behavior. In total we conducted
decrease in frequency after the first female offspring has ma- 500scanobservationsof43galleriesperageclass((cid:1)x=11.9scans
tured(TableS2).Inaddition,wefoundthatblockingmightalso pergallery, range2–40).
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BiedermannandTaborskywww.pnas.org/cgi/content/short/1107758108 3of9
d ***
ol 1.0
m
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d 0.8
e
er
v
o
c
all 0.6
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er
all 0.4
g
of
n
o 0.2
orti
p
o
pr 0.0
Fig.S1. EffectofX.saxeseniilarvaeonthegrowthofmoldfungi.Thenumberoflarvaepresentwithinachambernegativelyaffectedthepercentageofwall
areacoveredwithmoldfungiofartificiallycreatedchambersinfungusinfestedmedium.Generalizedestimationequation:coeff.±SE=-3.072±0.163,z=
18.86,P<0.001,n=26chambersin13galleries(paireddesign).Box-whiskerplotswithmedians(bold),10th,25th,75thand90thpercentilesareshown.
6
dead and
5 overgrown
by fungi
pae 4 alive
u
p
of
er 3
b
m
nu 2
1
0
1 larva 6 larvae
number of grooming larvae in the same chamber
Fig.S2. SurvivalofexperimentallyisolatedpupaeofX.saxeseniiwithone(Left)orsix(Right)tendinglarvaepresent.Singlepupaekeptwithonlyonelarva
weresignificantlymoreoftenovergrownbyfungiandkilledwithin14dcomparedwithpupaekeptwithsixlarvae(Fisherexacttest:P=0.048,n=10
chambers).
BiedermannandTaborskywww.pnas.org/cgi/content/short/1107758108 4of9