Insulin effects on honeybee appetitive behaviour

24 Pages·2016·0.87 MB·English

©2016.PublishedbyTheCompanyofBiologistsLtd|JournalofExperimentalBiology(2016)219,3003-3008doi:10.1242/jeb.143511
RESEARCHARTICLE
Insulin effects on honeybee appetitive behaviour
CarolinaMengoniGoñalons1,‡,MarieGuiraud2,*,‡,Marıá GabrieladeBritoSanchez2andWalterM.Farina1,§
ABSTRACT Seeley, 1982). Nutrition, age and reproductive status interact to
regulate worker behaviour (Amdam et al., 2004). This temporal
Worker honeybees (Apis mellifera) carry out multiple tasks
phenotypicprogressionis,inpart,aconsequenceofphysiological
throughout their adult lifespan. It has been suggested that the
changesthattheadultworkerundergoes.
insulin/insulin-like signalling pathway participates in regulating
To explore these traits in honeybee workers, a hypothesis has
behavioural maturation in eusocial insects. Insulin signalling
beenproposed.Thereproductivegroundplanhypothesis(Amdam
increases as the honeybee worker transitions from nurse to food
etal.,2004;Huntetal.,2007)suggeststhattemporallabourdivision
processortoforager.Asbehaviouralshiftsrequiredifferentialusage
is controlled by ovarian development (Turillazzi and West-
of sensory modalities, our aim was to assess insulin effects on
Eberhard, 1996): nurses present rudimentary ovary development
olfactory and gustatory responsiveness as well as on olfactory
whereas foragers lose this feature (Amdam et al., 2004). These
learning in preforaging honeybee workers of different ages. Adults
resultsleadtoanintegrativepathwaywherejuvenilehormone(JH),
wererearedinthelaboratoryorinthehive.Immediatelyafterbeing
vitellogenin (Vg) and insulin have pleiotropic effects on caste
injected with insulin or vehicle (control), and focusing on the
differentiation and worker labour division (Amdam et al., 2004;
proboscis extension response, bees were tested for their
Amentetal.,2008;LattorffandMoritz,2013).JH,Vgandinsulin
spontaneous response to odours, sucrose responsiveness and
levelsdifferbetweenfemalecastes(queenandworkers),andvary
ability to discriminate odours through olfactory conditioning. Bees
throughouttheadultbee’slifespan.JHtitersshowaslightincrease
injected with insulin have higher spontaneous odour responses.
from2to4daysoftheadultageandthenremainextremelylowuntil
Sucrose responsiveness and odour discrimination are differentially
peaklevelsarereachedwhenworkersstopperformingtaskswithin
affected by treatment according to age: whereas insulin increases
the nest and start foraging. By contrast, maximum Vg levels are
gustatory responsiveness and diminishes learning abilities of
found in young bees and then decrease until they become
younger workers, it has the opposite effect on older bees. In
undetectable in foragers (Amdam et al., 2004; Hartfelder
summary, insulin can improve chemosensory responsiveness in
and Engels, 1998). Simultaneously, insulin signalling gene
young workers, but also worsens their learning abilities to
expression, including insulin-like peptides and their receptors, is
discriminateodours.Theinsulinsignallingpathwayisresponsivein
higherinforagersthaninnurses(Amentetal.,2008;Coronaetal.,
youngworkers,althoughtheyarenotyetinitiatingoutdooractivities.
2007).
Our results show strong age-dependent effects of insulin on
Nutrition has an important role in honeybee age polyethism
appetitivebehaviour,whichuncoverdifferencesininsulinsignalling
regulationthroughoutthehoneybeeworker’sadulthood. (Amdametal.,2007;Robinson,1992).Theonsetageofforagingis
affectedbythecolony’snutritionalstatus(Amdametal.,2007)and
KEYWORDS:Apismellifera,Sucrose,Insulin,Chemosensory correlates with changes in the expression of genes implicated in
perception,Associativelearning,Odourdiscrimination feeding behaviour (Ament et al., 2008). Changes in nutritional
status, which are regulated through the insulin signalling system,
INTRODUCTION playaroleindictatingbehaviouralshiftsasworkersage,whichare
ThehoneybeeApismelliferaLinnaeus1758isasocialinsectthat likelytobemediatedthroughsocialcues(Amentetal.,2008;Toth
presents caste polyphenism (Wilson, 1971). Drones and queens etal.,2005).Unlikevertebrates,expressionofinsulin-relatedgenes
constitute the reproductive castes whereas workers are sexually in honeybees is negatively correlated with nutrient store: insulin
immature female individuals that perform collective tasks to levelsincreasewhereaslipidstoresdecreasewithworkerage(Toth
maintain the nest’s welfare. This caste exhibits age polyethism, etal.,2005).Fromthis,itcanbepostulatedthatinsulinsignalling
y
whichresultsinyoungadultsperformingdutiesinsidethenestsuch relates to the forager’s sensitivity to nutritional changes. The g
o
as nursing, comb building, food processing, guarding or fanning. insulin-like peptide is mainly expressed in the brain and is
l
o
The oldest worker bees are foragers, who gather resources for the upregulated in forager brains compared with those of nurses i
B
whole colony and transport it back to the hive (Lindauer, 1952; (Ament et al., 2008). This, in turn, would make foragers more
l
sensitive to appetitive cues, contributing to their exaggerated a
t
1LaboratoriodeInsectosSociales,DepartamentodeBiodiversidadyBiologıá responsestonutritionalstimuli. en
Experimental,IFIBYNE-CONICET,FacultaddeCienciasExactasyNaturales, Honeybeesexhibitaninnatereflextowardsantennalstimulation m
UniversidaddeBuenosAires,PabellónII,CiudadUniversitaria,(C1428EHA) withsucrosesolution,theproboscisextensionresponse(PER).By i
lBauCeongosniAtioirnesA,nAimrgaelnet,inCaN.R2USn,iFv-e3r1si0te6́2d,eTToouulolouusseeC,UedPeSx,C9,eFnrtarendcee.Recherchessur assessingthePERtowardswaterandsucrosesolutionsvaryingin per
*Presentaddress:DepartmentofBiologicalandExperimentalPsychology,Queen concentration, it is possible to estimate the sucrose response x
MaryUniversityofLondon,London,UK. E
‡Theseauthorscontributedequallytothiswork threshold (SRT) of individuals, a measure of gustatory f
responsiveness (Page et al., 1998). This assay can give o
§Authorforcorrespondence([email protected]) information about the bees’ nutritional status (Martinez and al
Farina, 2008; Pankiw et al., 2004). Foragers often show high n
W.M.F.,0000-0001-6411-489X r
sucroseresponsiveness,whereastheoppositeisfoundinyoungand u
o
Received20May2016;Accepted15July2016 middle-aged bees (Scheineret al., 2004). Regarding the effect on J
3003
RESEARCHARTICLE JournalofExperimentalBiology(2016)219,3003-3008doi:10.1242/jeb.143511
libitum,andwerecheckedeveryotherday.Foodwasreplacedevery
Listofabbreviations 2daysanddeadbeeswereremovedwheneverneeded(aspreviously
AIC Akaike’sinformationcriterion describedinMengoniGoñalonsandFarina,2015).Forhiverearing,
CR conditionedresponse emergingbeesweremarkedwithacrylicpaint (ALBA-Argentina)
CS− unrewardedconditionedstimulus on their thorax. A specific colour was used for each day of
CS+ rewardedconditionedstimulus
emergencesoastodeterminetheirageatalaterstage.Markedbees
DI discriminationindex
were introduced into an observational hive that consisted of two
GLM generalisedlinearmodel
broodframes,amatedqueenandapproximately4000workers,and
GLMM generalisedlinearmixedmodel
GRS gustatoryresponsescore werereadilyacceptedbytherestofthecolony(Breedetal.,2004).
JH juvenilehormone Thehivewascontainedbetweenacrylicwallsthathada40×25cm
PER proboscisextensionresponse windowcoveredbyahingeddoorthatallowedaccesstothecolony.
QMP queenmandibularpheromone On the day of the experiment, marked bees were captured
SOR spontaneousodourresponse
individuallyinplastictubesandtakentothelaboratory.
SRT sugarresponsethreshold
The experiments comply with the ‘Principles of animal care’,
Vg vitellogenin
publication no. 86-23, revised 1985, of the National Institutes of
Health,aswellaswiththecurrentlawsofthecountryinwhichthe
experimentswereperformed.
cognitive abilities, evidence suggests that a JH analogue affects
short-term olfactory memory in recently emerged honeybees Experimentalseriesandinjections
(Maleszka and Helliwell, 2001). JH positively influences the Withthepurposeofassessingdifferentialinsulineffectsaccording
insulin-like signalling pathway in insects (Amdam and Seehuus, to adult age, four groups of pre-foraging bees were contemplated.
2006;Coronaetal.,2007;Huntetal.,2007;Tuetal.,2005).Thus, Therefore, young beeworkers were assessed when they were 2/3,
weexpectthatinsulin,withastrongagedependency,willnotonly 5/6,9/10or14/15daysold.
affect sucrose and olfactory responsiveness but will also have an Experimentalbeeswereanaesthetisedwithiceandharnessedin
effectonlearningabilitiestodiscriminateodoursinhoneybees. carvedpipette tips, whichrestrainedbodymovement,but allowed
Apis mellifera has been considered a reference model within them to freely move their mouthparts and antennae. Before they
invertebrates to study behavioural and neural plasticity (Brown regainedactivity,theywereinjectedwithamicrosyringe(NanoFil,
etal.,2004;GiurfaandSandoz,2012;MassonandArnold,1987; WorldPrecisionInstruments)throughthefourthandfifthsegments
Menzel,1999;Siggetal.,1997;Winningtonetal.,1996).Infact,at oftheabdomen.Treatmentbeeswereinjectedwith1µlofinsulin
earlyagesoftheadultstage,thecentralnervoussystemofhoneybee (4mgml−1, Human recombinant Zinc, Gibco, Grand Island, NY,
workerscompletesitsmaturation(MassonandArnold,1987),and USA) and control bees received 1µl of Hepes buffer solution
experiences undergone during this period can shape later (25mmoll−1) (Ament et al., 2011). Solutions were used
physiology and behaviour (Arenas and Farina, 2008; Arenas immediately after melting and kept in ice during the procedure.
et al., 2009a,b, 2012). Recently emerged and middle-aged Twenty-fourbeesofthesameagewereusedperinjectionsession,
workers show reliable learning performances in an olfactory PER which lasted approximately 45min overall. In order to reduce
conditioning (Mengoni Goñalons and Farina, 2015), similar to effectsresultingfromdifferencesinthetimebetweeninjectionand
those of foragers. However, gustatory responsiveness and its evaluation,treatmentassignmentwasdonebyblocks,inwhich12
sensitivity to environmental changes vary with age (Mengoni beeswereinjectedwithinsulinandtheother12wereinjectedwith
GoñalonsandFarina,2015;Ramírezetal.,2010). buffersolution.Astwoinjectionsessionswereperformedeachday,
Previous reports showed that endocrine secretions can tune the order in which the treatments were given in the morning was
chemosensorysystem in Drosophila (Ko et al., 2015; Root et al., switched in the afternoon. The following day, the entiretreatment
2011). However, these responses could differ strongly in social assignmentwasalternated.Allfouragegroupswererepresentedina
insects. Here, we assess the effect of insulin on appetitive pairofdays.Theorderinwhichagegroupsweretestedwithineach
behaviours such as chemosensory responsiveness and learning pair was randomised. Behavioural assays were performed 5min
abilities in preforaging bees, especially in the case of young hive afterinjectionofthelastbee.Theorderinwhichbeeswereinjected
bees. waskeptinthefollowingprocedure.Timebetweeninjectionandthe
y
startoftheassaywas25minforallbees. g
o
MATERIALSANDMETHODS l
o
Studysiteandanimals Spontaneousodourresponse i
B
The study was carried out during the summer–autumn season of The harnessed bee was placed between a device that produced a
l
a
2014intheexperimentalfieldoftheFacultaddeCienciasExactasy constantairflowandanairextractorthatremovedreleasedodours.The
t
NaturalesoftheUniversidaddeBuenosAires,Argentina(34°32′S, airstream(2.5mls−1)wasdeliveredtotheheadofthebee2cmaway n
e
58°26′W).NewlyemergedEuropeanhoneybees(A.mellifera)were fromit.Anodourisconsideredaneutralstimulusandusuallyelicits m
obtained from sealed brood frames taken from the experimental no PER. Nevertheless, a naïve bee can still show a spontaneous ri
e
apiary and placed in an incubator at 36°C and 55% relative response towards a certain odour. For this procedure, pure odours
p
humidity. After emergence, workers were collected and reared in (Raguso and Pichersky, 1999), linalool and phenylacetaldehyde x
E
different environments: in the laboratory under controlled (Sigma-Aldrich, Steinheim, Germany), were used. Each was
f
conditions or in an observational hive. For the former, emerging delivered for 6s when, by means of an electric valve, the airflow o
beeswerecollectedingroupsofupto150individualsandconfined wasredirectedtopassthroughasyringecontaining4µlofthepure al
n
inwoodenboxes(10×10×10cm)withametallicmeshononeside odourimpregnatedona30×3mmfilterpaper.Aspontaneousodour
r
andaplasticdooronother.Cageswerekeptinanotherincubatorat response (SOR) was considered when the bee fully extended its u
o
31°C. They offered 16% w/w sucrose solution and pollen ad proboscis during odour delivery. Bees that responded to the J
3004
RESEARCHARTICLE JournalofExperimentalBiology(2016)219,3003-3008doi:10.1242/jeb.143511
mechanicalairstimulus(16scleanairflowbeforeodourpresentation) first,butnotthelatter.TheglobalDIwascalculatedasthenumber
were discarded, as well as bees that did not respond to 50% w/w oftrialpairsthebeesucceededindiscriminating betweenthetwo
sucrose solution after the gustatory response assay (see following odours(valuesinclude0through4).
section).Odourpresentationswere10minapart,andtheorderwas Aperiodof20minelapsedbetweenthelasttrialandthetesting
alternated frombeetobee(partsofthisprocedurewerepreviously phase.Thelatterconsistedofnon-rewardedpresentationsoftheCS+
describedinMengoniGoñalonsandFarina,2015). andtheCS−,alternatingtheirorderfrombeetobee.Afterthetesting
phase, the response to 50% w/w sucrose solution was verified and
Gustatoryresponsiveness only responding bees were taken into account (a similar procedure
ImmediatelyafterSORevaluation,beeswerestimulatedwithsucrose waspreviouslydescribedinMengoniGoñalonsandFarina,2015).
solutionsofincreasingconcentrations(0.1,0.3,1,3,10,30and50%
w/w) by touching their antennae (Page et al., 1998). The lowest Statisticalanalysis
sucroseconcentrationatwhichanindividualrespondedbyextending The effects of factors on all variables were assessed by means of
itsprobosciswasinterpretedasitsSRT.Beeswerelinedupandtested generalised linear models (GLM) or generalised linear mixed
sequentiallyforeachconcentration,i.e.allbeeswerepresentedwith models (GLMM). Models were fitted in R (R Foundation for
0.1%solutionfirst,thenwith0.3%solutionandsoon.Beforeeach StatisticalComputing,Vienna,Austria)usingtheglmfunctionfor
sucrosesolutionpresentation,allbeesweretestedfortheirresponseto theformercaseandtheglmerfunctionofthelme4package(Bates
water (0%). This controlled potential effects of repeated sucrose et al., 2015) for the latter. Alternative models were assessed and
stimulationthatcouldleadtoincreasedsensitizationorhabituation,as compared,andonewaschosendependingonitsparsimonyandits
wellasensuringthatextensionoftheprobosciswasnotduetothirst. Akaike’sinformationcriterion(AIC)value.Posthoccomparisons
Theinter-stimulusintervalbetweenwaterandsucrosesolutionwas wereperformedwiththeglhtfunctionoftheRpackagemultcomp
4minlong.Attheendoftheexperiment,agustatoryresponsescore (Hothornetal.,2008).
(GRS)wasobtainedforeachbee.Thisscorewasbasedonthenumber An SOR was defined asthe extension of the proboscistowards
ofsucroseconcentrationstowhichthebeesresponded.Theresponse any of the two odours. Effect of insulin injection on SOR was
wasarbitrarilyquantifiedwithscoresfrom1to7,where1represented assessed by means of a GLM with binomial error structure. The
abeethatonlyrespondedtothehighestsucroseconcentration,whilea initial model included rearing environment, age and treatment as
score of 7 represented an individual that responded to all fixed factors. In addition, to test for any odour bias, spontaneous
concentrations tested. If a bee failed to respond to sucrose responsestoeachodourwereconsideredinaseparateanalysis.In
concentration in the middle of a response series (e.g. responded to thiscase,aGLMMwasusedandtheinitialmodelincludedrearing
0.3,3and10%,butdidnotrespondto1%),this‘failed’responsewas environment,age,treatmentandodourasfixedfactorsandsubject
consideredtobeanerrorandthebeewasdeemedtohaverespondedto bee as a random factor. Gustatory responsiveness was estimated
thatconcentrationaswell.Abeethatdidnotrespondtoanyofthe throughtheGRS,whichisasumoftheunconditionedresponsesto
sucrose concentrations (score of 0) was excluded from further the sugar solutions presented in the procedure. Values include 1
analyses. In addition, those bees that responded to all sucrose through7.TheeffectofinsulininjectiononGRSwasassessedby
concentrations and all presentations of water were excluded from means of a GLM with binomial error structure. The initial model
analyses as they appeared not to be able to discriminate between includedrearingenvironment,ageandtreatmentasfixedfactors.
sucrose solution and water (as previously described in Mengoni Abeethatextendeditsproboscistowardsodoursinthefirsttrial
GoñalonsandFarina,2015). pair was considered to showa spontaneous response and was not
takenintoaccountforconditioninganalysis.Theeffectofinsulinon
OdourdiscriminationinclassicalPERconditioning olfactory discrimination was assessed by means of a GLM with
For this procedure, only 5/6- and 14/15-day-old laboratory-reared binomial error structure. The initial model included treatment and
bees were used. These naïve bees were presented with the same age as fixed factors. In the testing phase, no bee extended its
odoursusedintheSORassay,butinthiscaselinaloolwaspaired proboscis towards the CS−. Therefore, effect of insulin injection
with 50% w/w (rewarded conditioned stimulus, CS+; wasonlystudiedonCRtowardstheCS+andassessedbymeansofa
unconditioned stimulus) and phenylacetaldehyde was not GLMthatincludedtreatmentandageasfixedfactors.
(unrewarded conditioned stimulus, CS−). The harnessed bee was
placedinthesamecontextasdescribedintheSpontaneousodour RESULTS
y
responsesection,above.Duringconditioning,odourwasdelivered Spontaneousodourresponse g
o
for 6s and, in the case of the CS+ presentation, the reward was Bees were presented with two odour stimuli (linalool and l
o
presentedduringthelast3softhisperiodbytouchingtheantennae phenylacetaldehyde). We defined a spontaneous response as the i
B
with 50% w/w sucrose solution and then feeding the bee. A extensionoftheproboscistowardsanyofthetwoodours.Insulin-
l
a
conditionedresponse(CR)wasconsideredtohaveoccurredwhen injected bees, independently of age or rearing environment, had
t
the bee fully extended its proboscis during the first 3s of odour higher SOR than control bees. As the minimum model did not n
e
delivery.Onetriallastedfor39sandwascomposedof16sofclean includethesefactors,rearingenvironmentoragehadnoeffecton m
airflow,6sofodourand17sofcleanairflow.Trainingconsistedof SOR(SOR∼treatment,AIC=625.13,Z=2.585,P=0.0097;Fig.1; ri
fiveCS+andfiveCS−trialsarrangedinapseudo-randomisedorder TableS1).Inaddition,therewasnobiastowardsacertainstimuli e
p
(CS−, CS+, CS+, CS−, CS−, CS+, CS−, CS+, CS+, CS−). The (SOR ∼ odour+bee, Z=0.00, P=1; Table S2). Therefore, we can x
E
inter-trial interval lasted approximately 15min. To estimate the statethattherewasnoodourpreference.
f
ability to discriminate between the two odours in the differential o
conditioning,wedefinedaglobaldiscriminationindex(globalDI) Gustatoryresponsiveness al
n
for each bee. In each trial pair, a bee was considered to Immediately after SOR evaluation, bees were presented with
r
have discriminated between the CS+ (linalool) and the CS− increasing concentrations of sucrose solutions. A GRS was u
o
(phenylacetaldehyde)onlyifitextended itsproboscistowardsthe definedasthesumofpositiveresponsesthroughouttheprocedure J
3005
RESEARCHARTICLE JournalofExperimentalBiology(2016)219,3003-3008doi:10.1242/jeb.143511
A B global DI (5/6days old: Z=−3.493, P<0.001; 14/15days old:
100 100 Z=4.919,P<0.001;Fig.3A).Inaddition,contrololderbeesshowed
lower global DI than control younger bees (Z=4.428, P<0.001;
80 80
Fig.3A,whitebars).Inthetestingphase,injectionofinsulinhada
%) 60 60 similareffectonCRtotheCS+(CR∼age×treatment,AIC=206.66;
SOR ( 40 * 40 n.s. Finigo.l3dBer;Tbeaebsle(5S/56).dIanyssuloinldr:eZd=uc−e2d.3C4R9,inPy=o0u.0n3g8e3r;be1e4s/,1b5udtarayisseodldit:
Z=2.399, P=0.0341; Fig. 3B). In addition, control older bees
340
20 333 20 352 321 showed lower conditioned responses than control younger bees
(Z=2.829,P=0.0103;Fig.3B,whitebars).
0 0
Hepes Insulin Hive Cage
DISCUSSION
Treatment Rearing environment
Our study reveals that insulin improves spontaneous response to
Fig.1.Insulininjectionincreasedtheprobabilityofspontaneousodour odours independent of rearing environment and age. In addition,
response,independentlyofageorrearingenvironment,inthehoneybee whereas it improves sucrose responsiveness and reduces odour
Apismellifera.Percentageofbeesthatextendedtheirproboscistowards
discrimination in younger honeybees, it has the opposite effect in
eitherofthetwoodours,linaloolandphenylacethaldehyde(spontaneous
older ones. We expected insulin to have an effect on gustatory
odourresponse,SOR).Responsesofbeesofallages(A)afterbeinginjected
withHepesbuffer(white)orinsulin(black)and(B)obtainedfroman responsiveness in younger workers and little or no effect in older
experimentalhive(darkgrey)orfromcageskeptinthelaboratory(lightgrey). ones.Theresultspartiallyverifyourprediction,as2/3-day-oldbees
Minimaladequatemodel:SOR∼treatment.Asteriskindicatesasignificant were affected by insulin injection, but 5/6- and 9/10-day-old bees
difference(P<0.05);n.s.,notsignificant.Valuesabovebarsindicatethe werenot.Giventhatinsulinlevelsincreasewithworkerage(Corona
numberofbeestested.
etal.,2007),theseresultsindicatethatexogenousinsulinartificially
induces the youngest workers to exhibit higher responsiveness to
(Pageetal.,1998).Hive-rearedbeeshadlowerGRSthanlaboratory- sucrose, a trait associated with foragers (Scheiner et al., 2004). In
reared bees (GRS ∼ rearing environment+age×treatment, addition, the youngest bees also increased their probability to
AIC=2083.1, Z=5.497, P<0.001; Fig. 2A; Table S3). This effect respondspontaneouslytoodoursaftertreatmentadministration.To
was independent of age and treatment, as models containing sum up, insulin injection improves chemosensory responsiveness.
interactionsbetweenrearingenvironmentandthesetwofactorshad Thisseemstobeanadaptivefunctionintermsofdivisionoflabour,
higher AIC values than the one chosen. Effect of insulin on as nurse bees would be able to modify their chemosensory
gustatoryresponsivenessdependedonageofinjection.Treated2/3- thresholds, which is required when social structure undergoes a
day-old bees presented higher GRS than control bees (Z=4.685, changeasaresultofaselectivepressure,suchasthesuddendeathofa
P<0.001), which means their SRT was lower. On the contrary, portionoftheforagers.Inthiscase,nursesandfoodprocessorsare
insulin injectionof 14/15-day-old bees had adepressing effect on forcedtoperformoutsidetasksatayoungeragethanexpected.These
GRS,raisingtheirSRT(Z=−4.375,P<0.001).Bees5to10daysold precociousbeesexhibitadecreaseinVglevelsandlipidreservesin
werenotaffectedbyinsulin(5/6daysold:Z=0.890,P=0.8080;9/ the hemolymph and an increase in JH and insulin levels (Corona
10daysold:Z=−1.521,P=0.3770;Fig.2B). et al., 2007; Hartfelder and Engels, 1998), and they show a poor
foraging performance (Chang et al., 2015; Perry et al., 2015).
OdourdiscriminationinclassicalPERconditioning Similarly,pollenforagers,whobeginoutsidetasksearlierthannectar
For this procedure, 5/6- and 14/15-day-old laboratory-reared bees foragers,showhighgustatoryresponsiveness,aqualitythatindicates
wereused.AglobalDIwasdefinedforeachbeeasthenumberof apoornectarforagingability(Amdametal.,2006).Thissuggests
trial pairs the bee succeeded in discriminating between the two thatyounghivebeesthatareartificiallyinducedtobecomingforagers
odours (if it extended its proboscis towards the CS+, but not the throughariseininsulinlevelswouldbepoornectargatherers.
CS−).Aneffectofinsulinwasobservedinbeesofbothages(global Contrarytowhatwasexpected,ourresultsindicatethat14/15-day-
DI∼ age×treatment, AIC=576.84; Table S4).Treated 5/6-day-old oldtreatedbeeshadlowerGRSvaluesthancontrolbees.Thus,the
beespresentedlowerglobalDIthancontrolbees.Onthecontrary, biologicaleffectfoundwastheinverseofthatfoundintheyoungest
insulininjectionof14/15-day-oldbeeshadanincreasingeffecton bees.Ontopofthehighinsulinlevelsthatwouldbefoundinthese
y
g
o
A * B * * Fig.2.Effectofinsulininjectionongustatory ol
responsivenesswasagedependentin i
7 7 A.mellifera.Gustatoryresponsescores(GRS)of B
l
(A)beesobtainedfromanobservationalhive a
6 6 Hepes (darkgrey)orfromcageskeptinthelaboratory nt
Insulin (lightgrey)and(B)2/3-,5/6-,9/10-and14/15-day- e
m
5 5 oldbeesafterbeinginjectedwithHepesbuffer
i
(white)orinsulin(black).Minimaladequatemodel: r
S e
R 4 4 GRS∼rearingenvironment+age×treatment. p
G 66 Thethickline,boxandwhiskersrepresentthe x
71 E
3 3 median,interquartileintervalanddatarange,
f
respectively.Asterisksindicateforstatistical o
260 286 66 71 78 68 63
2 2 differencesbetweeninjectiontreatmentswithin l
a
anagegroup(P<0.05).Valuesinbarsindicate n
1 1 63 thenumberofbeestested. ur
Hive Cage 2/3 5/6 9/10 14/15
o
Rearing environment Age (days) J
3006
RESEARCHARTICLE JournalofExperimentalBiology(2016)219,3003-3008doi:10.1242/jeb.143511
A B Fig.3.Effectofinsulininjectiononodour
*
discriminationinPERconditioningof
* * Hepes A.melliferawasagedependent.(A)Global
4 100 Insulin discriminationindices(globalDI)duringacquisition
and(B)percentageofbeesthatextendedtheir
80 * proboscistowardstherewardedconditioned
3 stimulus(conditionedresponse,CR)duringthe
Global DI 2 CR (%) 6400 * *43 taiwnefhstseuetirnlnibngae(psibnshlgaeacssinksej)ee.pdcBetaeerfndeodsrwmrwieetheadrrHee2de05p/um6ensiadnnbeadurfftl1faee4brr/oa1(rcw5aqhtduoiatiresyyi)stiooornld,
44 conditions.Minimaladequatemodels:globalDI∼
1 age×treatment,CR∼age×treatment.InA,thethick
44 43
20 line,boxandwhiskersrepresentthemedian,inter-
quartileintervalanddatarange,respectively.
47 44 46 47
0 0 Asterisksindicateforstatisticaldifferencesbetween
5/6 14/15 5/6 14/15 injectiontreatmentswithinanagegroup(P<0.05).
Age (days) Valuesinorabovebarsindicatethenumberofbees
tested.
middle-aged bees, an additional insulin shock did not increase endocrine systems – including the insulin system – ready for the
sucroseresponsiveness,butdecreasedit. Sucroseresponsivenessis transition from young hive beesto foragers is valuable but it also
closely related to nutritional status and, thus, feeding motivation introducespenaltiesintermsofforagingefficiency.Inaddition,we
(MartinezandFarina,2008;Pankiwetal.,2004).Ashighlevelsof foundcontrastingeffectsofinsulinonbotholfactorylearningand
insulinleadtoforagingactivities(Amentetal.,2008),itseemsclear gustatoryresponsivenessbetweenyoungandmiddle-agedworkers.
thattheforagingtaskisdirectlytunedbyinsulinlevelspresentinthe Ultimately, the same results were observed in both acquisition
bee hemolymph. Despite the fact that we did not include actual andtestingphasesoftheolfactoryconditioning,whichimpliesthat
foragersinourstudy,weclaimthatthe14/15-day-oldbeesusedinour insulin modifies the bee’s chemosensory responsiveness, and this
experimentswereinacorrespondingphysiologicalstatetoforagers, modificationlastsatleast20minafterconditioning,suggestingan
astheircoetaneoussisters–emergedasadultsonthesameday–were effectonmedium-termmemory.
seen flying to an artificial feeder 2days later. A honeybee forager Vertebrate insulin (bovine: Mott and Breed, 2012; human:
doesnotseekfoodforindividualsustenance,butinsteadcontributes present study) has been shown to be bioreactive in honeybees. In
tocolonyreserves.Nonetheless,itstillneedsmotivationtoinitiatea our case, the mode of administration was systemic and general,
foraging trip. In other words, insulin would be comparatively low and we assessed variables in a short-term period. Therefore, it is
whentheforagerisinthehive,allowingittoeat.Oncetheforageris not possible to elucidate the hormone’s targets and the source of
satiated, insulin levels rise until reaching a threshold that would its behavioural effects. We infer that the insulin pathway is a key
trigger a foraging flight (Ament et al., 2008). This fine insulin to understanding how the different metabolic pathways act in
regulation would then explain why insulin-injected 14/15-day-old concert to synchronise the development of chemosensory and
beesshowlowerGRScomparedwithcontrolbees. physiological processes. Many questions remain unanswered
Laboratory-reared bees had higher gustatory responsiveness about what other pathways are involved after an artificial rise in
than hive bees. These results are not surprising as bees reared in insulinlevelinthehemolymphofhoneybees.However,thisstudy
the hive had been exposed to the queen mandibular pheromone reveals one of the main roles of insulin in adult honeybee
(QMP),whichisreleasedbythequeen,andQMPreducessucrose developmentandprovidestoolsforresearchonhowinsulinaffects
responsiveness (Pankiw and Page, 2003). In addition, laboratory labour division in bees as well as a worker’s individual
bees exposed to QMP, which simulates queen presence, has no physiological state.
effect on the expression genes involved in the insulin signalling
peptide (Fischer and Grozinger, 2008). This last result Acknowledgements
corroborates our own findings that the effect of insulin did not WealsothankK.Lukowiakandtwoanonymousrefereesfortheirvaluable
y
depend on whether workers were reared in the hive or queen commentsandsuggestionsonanearlierversionofthismanuscript. g
o
deprived in the laboratory. Competinginterests ol
Insulinalsoaffectedolfactorylearningperformance.Inyounger Theauthorsdeclarenocompetingorfinancialinterests. Bi
workers, insulin improved chemosensory responsiveness but
l
a
worsened olfactory discrimination. Therefore, contrary to our Authorcontributions t
expectations,highergustatoryresponsivenessasaresultofinsulin C.M.G.,M.G.,M.G.d.B.S.andW.M.F.conceivedanddesignedtheexperiments. n
e
injectiondidnotresultinbettercognitiveabilitiesintermsofodour C.M.G.andM.G.performedtheexperiments.C.M.G.performeddataanalysis. m
discrimination(MengoniGoñalonsandFarina,2015;Ramírezetal., C.M.G.,M.G.,M.G.d.B.S.andW.M.F.draftedthemanuscript.Allauthorsrevised ri
andcommentedonthemanuscript. e
2010;Scheineretal.,2004).Infact,thecorrelationwastheopposite.
p
It appears that the adaptive function of modifying gustatory x
Funding E
responsiveness suggested earlier does not apply to olfactory C.M.G.andW.M.F.thanktheConsejoNacionaldeInvestigacionesCientıf́icasy f
learning. In this case, young bees forced to collect resources Tecnológicas(CONICET)andtheUniversidaddeBuenosAires(UBA)forsupport. o
wouldbecomeforagers,butbadlearnersintermsofdiscriminating ThisstudywaspartlysupportedbygrantsfromAgenciaNacionaldePromoción al
Cientıf́icayTecnológica(PICT20131060),UBA(20020130100185BA)and n
floral odours. This olfactory discrimination is beneficial when
selectingforagingsites,butisnotessentialifthecolonyisfacinga CONICET(PIP112-201501-00633)toW.M.F.M.G.andM.G.d.B.S.thankthe ur
CentredeRecherchessurlaCognitionAnimaleandtheUniversityPaulSabatier o
drastic change in social structure. Therefore, having all the (grantAPIGENE)forsupport. J
3007
RESEARCHARTICLE JournalofExperimentalBiology(2016)219,3003-3008doi:10.1242/jeb.143511
Supplementaryinformation Ko, K. I., Root, C. M., Lindsay, S. A., Zaninovich, O. A., Shepherd, A. K.,
Supplementaryinformationavailableonlineat Wasserman,S.A.,Kim,S.M.andWang,J.W.(2015).Starvationpromotes
http://jeb.biologists.org/lookup/doi/10.1242/jeb.143511.supplemental concerted modulation of appetitive olfactory behavior via parallel
neuromodulatorycircuits.eLife4,e08298.
References Lattorff,H.M.G.andMoritz,R.F.(2013).Geneticunderpinningsofdivisionof
Amdam,G.V.andSeehuus,S.-C.(2006).Order,disorder,death:lessonsfroma laborinthehoneybee(Apismellifera).TrendsGenet.29,641-648.
superorganism.Adv.CancerRes.95,31-60. Lindauer,M.(1952).Einbeitragzurfragederarbeitsteilungimbienenstaat.Z.Vgl.
Amdam,G.V.,Norberg,K.,Fondrk,M.K.andPage,R.E.(2004).Reproductive Physiol.34,299-345.
groundplanmaymediatecolony-level selection effects onindividualforaging Maleszka,R.andHelliwell,P.(2001).Effectofjuvenilehormoneonshort-term
behaviorinhoneybees.Proc.Natl.Acad.Sci.USA101,11350-11355. olfactory memory in young honeybees (Apis mellifera). Horm. Behav. 40,
Amdam,G.V.,Norberg,K.,Page,R.E.,Jr.,Erber,J.andScheiner,R.(2006). 403-408.
Downregulation of vitellogenin gene activity increases the gustatory Martinez, A. and Farina, W. M. (2008). Honeybees modify gustatory
responsivenessofhoneybeeworkers(Apismellifera).Behav.BrainRes.169, responsiveness after receiving nectar from foragers within the hive. Behav.
201-205. Ecol.Sociobiol.62,529-535.
Amdam, G. V., Nilsen, K.-A., Norberg, K., Fondrk, M. K. and Hartfelder, K. Masson, C.andArnold, G. (1987). Organization and plasticityof theolfactory
(2007).Variationinendocrinesignalingunderliesvariationinsociallifehistory. system of the honeybee, Apis mellifera. In Neurobiology and Behavior of
Am.Nat.170,37-46. Honeybees(ed.R.MenzelandA.Mercer),pp.280-295.Berlin:SpringerVerlag.
Ament,S.A.,Corona,M.,Pollock,H.S.andRobinson,G.E.(2008).Insulin MengoniGoñalons,C.andFarina,W.M.(2015).Effectsofsublethaldosesof
signalingisinvolvedintheregulationofworkerdivisionoflaborinhoneybee imidaclopridonyoungadulthoneybeebehaviour.PLoSONE10,e0140814.
colonies.Proc.Natl.Acad.Sci.USA105,4226-4231. Menzel,R.(1999).Memorydynamicsinthehoneybee.J.Comp.Physiol.ASens.
Ament,S.A.,Velarde,R.A.,Kolodkin,M.H.,Moyse,D.andRobinson,G.E. NeuralBehav.Physiol.185,323-340.
(2011).NeuropeptideY-likesignallingandnutritionallymediatedgeneexpression Mott,C.M.andBreed,M.D.(2012).Insulinmodifieshoneybeeworkerbehavior.
andbehaviourinthehoneybee.InsectMol.Biol.20,335-345.
Insects3,1084-1092.
Arenas,A.andFarina,W.M.(2008).Ageandrearingenvironmentinteractinthe
Page,R.,Jr.,Erber,J.andFondrk,M.(1998).Theeffectofgenotypeonresponse
retentionofearlyolfactorymemoriesinhoneybees.J.Comp.Physiol.A194,
thresholdstosucroseandforagingbehaviorofhoneybees(ApismelliferaL.).
629-640.
Arenas,A.,Fernández,V.M.andFarina,W.M.(2009a).Associativelearning J.Comp.Physiol.ASens.NeuralBehav.Physiol.182,489-500.
Pankiw,T.andPage,R.Jr.(2003).Effectofpheromones,hormones,andhandling
duringearlyadulthood enhanceslatermemoryretentionin honeybees.PLoS
on sucrose response thresholds of honey bees (Apis mellifera L.). J. Comp.
ONE4,e8046.
Arenas,A.,Giurfa,M.,Farina,W.M.andSandoz,J.C.(2009b).Earlyolfactory Physiol.ASens.NeuralBehav.Physiol.189,675-684.
experiencemodifiesneuralactivityintheantennallobeofasocialinsectatthe Pankiw,T.,Nelson,M.,Page,R.E.,Jr.andFondrk,M.K.(2004).Thecommunal
adultstage.Eur.J.Neurosci.30,1498-1508. crop:modulationofsucroseresponsethresholdsofpre-foraginghoneybeeswith
Arenas,A.,Giurfa,M.,Sandoz,J.C.,Hourcade,B.,Devaud,J.M.andFarina, incomingnectarquality.Behav.Ecol.Sociobiol.55,286-292.
W.M.(2012).Earlyolfactoryexperienceinducesstructuralchangesintheprimary Perry, C. J., Søvik, E., Myerscough, M. R. and Barron, A. B. (2015). Rapid
olfactorycenterofaninsectbrain.Eur.J.Neurosci.35,682-690. behavioralmaturationacceleratesfailureofstressedhoneybeecolonies.Proc.
Bates,D.,Maechler,M.,Bolker,B.,Walker,S.,Christensen,R.H.B.,Singmann, Natl.Acad.Sci.USA112,3427-3432.
H.,Dai,B.,Grothendieck,G.,Eigen,C.andRcpp,L.(2015).Package‘lme4’. Raguso,R.A.andPichersky,E.(1999).Newperspectivesinpollinationbiology:
Convergence12,1. floralfragrances.Adayinthelifeofalinaloolmolecule:chemicalcommunication
Breed, M. D., Diaz, P. H. and Lucero, K. D. (2004). Olfactory information inaplant-pollinatorsystem.Part1:Linaloolbiosynthesisinfloweringplants.Plant
processinginhoneybee,Apismellifera,nestmaterecognition.Anim.Behav.68, SpeciesBiol.14,95-120.
921-928. Ramıŕez,G.P.,Martıń ez,A.S.,Fernández,V.M.,Bielsa,G.C.andFarina,W.M.
Brown, S. M., Napper, R. M. and Mercer, A. R. (2004). Foraging experience, (2010).Theinfluenceofgustatoryandolfactoryexperiencesonresponsivenessto
glomerulusvolume,andsynapsenumber:astereologicalstudyofthehoneybee rewardinthehoneybee.PLoSONE5,e13498.
antennallobe.J.Neurobiol.60,40-50. Robinson,G.E.(1992).Regulationofdivisionoflaborininsectsocieties.Annu.
Chang,L.-H.,Barron,A.B.andCheng,K.(2015).Effectsofthejuvenilehormone Rev.Entomol.37,637-665.
analoguemethopreneonrateofbehaviouraldevelopment,foragingperformance Root,C.M.,Ko,K.I.,Jafari,A.andWang,J.W.(2011).Presynapticfacilitationby
andnavigationinhoneybees(Apismellifera).J.Exp.Biol.218,1715-1724. neuropeptidesignalingmediatesodor-drivenfoodsearch.Cell145,133-144.
Corona,M.,Velarde,R.A.,Remolina,S.,Moran-Lauter,A.,Wang,Y.,Hughes, Scheiner, R., Page, R. E. and Erber, J. (2004). Sucrose responsiveness and
K. A. and Robinson, G. E. (2007). Vitellogenin, juvenile hormone, insulin
behavioralplasticityinhoneybees(Apismellifera).Apidologie35,133-142.
signaling, and queen honey bee longevity. Proc. Natl. Acad. Sci. USA 104,
Seeley, T. D. (1982). Adaptive significance of the age polyethism schedule in
7128-7133.
honeybeecolonies.Behav.Ecol.Sociobiol.11,287-293.
Fischer, P. and Grozinger, C. M. (2008). Pheromonal regulation of starvation
Sigg,D.,Thompson,C.M.andMercer,A.R.(1997).Activity-dependentchanges
resistance in honey bee workers (Apis mellifera). Naturwissenschaften 95,
tothebrainandbehaviorofthehoneybee,Apismellifera(L.).J.Neurosci.17,
723-729.
7148-7156.
Giurfa,M.andSandoz,J.-C.(2012).Invertebratelearningandmemory:fiftyyears
Toth,A.L.,Kantarovich,S.,Meisel,A.F.andRobinson,G.E.(2005).Nutritional
of olfactory conditioning of the proboscis extension response in honeybees.
statusinfluencessociallyregulatedforagingontogenyinhoneybees.J.Exp.Biol.
Learn.Mem.19,54-66.
Hartfelder, K. and Engels, W. (1998). Social insect polymorphism: hormonal 208,4641-4649.
regulationofplasticityindevelopmentandreproductioninthehoneybee.Curr. Tu,M.-P.,Yin,C.-M.andTatar,M.(2005).Mutationsininsulinsignalingpathway y
Top.Dev.Biol.40,45-77. alter juvenile hormone synthesis in Drosophila melanogaster. Gen. Comp. g
Hothorn, T., Bretz, F., Westfall, P. and Heiberger, R. M. (2008). Multcomp: Endocrinol.142,347-356. o
SimultaneousInferenceinGeneralParametricModels.Rpackageversion1.0-0. Turillazzi,S.andWest-Eberhard,M.J.(ed.)(1996).Naturalhistoryandevolution ol
Vienna,Austria:RFoundationforStatisticalComputing. ofpaperwasps.Oxford:OxfordUniversityPress. Bi
Hunt,G.J.,Amdam,G.V.,Schlipalius,D.,Emore,C.,Sardesai,N.,Williams, Wilson,E.O.(1971).TheInsectSocieties.Cambridge,MA:BelknapPress.
l
C.E.,Rueppell,O.,Guzmán-Novoa,E.,Arechavaleta-Velasco,M.,Chandra, Winnington,A.P.,Napper,R.M.andMercer,A.R.(1996).Structuralplasticityof a
S.etal.(2007).Behavioralgenomicsofhoneybeeforagingandnestdefense. identifiedglomeruliintheantennallobesoftheadultworkerhoneybee.J.Comp. nt
Naturwissenschaften94,247-267. Neurol.365,479-490. e
m
i
r
e
p
x
E
f
o
l
a
n
r
u
o
J
3008