Reproductive traits and mandibular gland pheromone of anarchistic honey bee workers Apis ...

13 Pages·2017·0.99 MB·English

Reproductive traits and mandibular gland pheromone of
anarchistic honey bee workers Apis mellifera occurring
in China
De-Fang Niu, Christian W. W. Pirk, Huo-Qing Zheng, Shun Ping, Jin-Hu Shi,
Lian-Fei Cao, Fu-Liang Hu
To cite this version:
De-Fang Niu, Christian W. W. Pirk, Huo-Qing Zheng, Shun Ping, Jin-Hu Shi, et al.. Reproductive
traits and mandibular gland pheromone of anarchistic honey bee workers Apis mellifera occurring in
China. Apidologie, 2016, 47 (4), pp.515-526. ￿10.1007/s13592-015-0396-4￿. ￿hal-01532305￿
HAL Id: hal-01532305
https://hal.science/hal-01532305
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Apidologie(2016)47:515–526 Original article
*INRA,DIBandSpringer-VerlagFrance,2015
DOI:10.1007/s13592-015-0396-4
Reproductive traits and mandibular gland pheromone
of anarchistic honey bee workers Apis mellifera occurring
in China
De-FangNIU1,2,ChristianW.W.PIRK3,Huo-QingZHENG1,ShunPING1,Jin-HuSHI4,
Lian-FeiCAO5,Fu-LiangHU1
1CollegeofAnimalSciences,ZhejiangUniversity,No.866YuhangtangRoad,Hangzhou310058,China
2JiangsuAgri-AnimalHusbandryVocationalCollege,Taizhou225300,China
3SocialInsectResearchGroup,DepartmentofZoologyandEntomology,UniversityofPretoria,PrivateBagX20,
Hatfield,Pretoria0028,SouthAfrica
4ZhejiangAnimalHusbandryTechnologyPromotionTerminus,Hangzhou310021,China
5TheAnimalHusbandryandVeterinaryResearchInstitute,ZhejiangAcademyofAgriculturalSciences,Hangzhou
310021,China
Received2March2015–Revised22August2015–Accepted23September2015
Abstract– Inhoneybeecolonies,workers,inparticularof“anarchistic”lineages,canactivatetheirovariesandlay
eggs,eveninthepresenceofthequeen.Weidentifiedthreequeenrightcoloniesshowingtypicalsignsofworker
reproduction. To characterize this new lineage, we extracted the mandibular gland and analyzed it using gas
chromatography. The total amounts of the five main components of the mandibular gland, namely methyl p-
hydroxyben-zoate (HOB), 9-oxo-2(E)-decenoicacid(9-ODA), (S)-9-hydroxy-(E)-2-decenoicacid(9-HDA), 10-
HDA, and 10-hydroxyde-canoic acid (10-HDAA) were significantly higher in the mandibular gland profiles of
workerswithactivatedovaries(AWs,8.88±1.71μg)comparedtoworkerswithinactivatedovaries(IAWs,4.00±
2.09μg).Furthermore,thechemicalprofilesofIAWsweredominatedbythe“workersubstances”10-HDA(34.64±
8.19%)anditsprecursor10-HDAA(22.88±4.95%),whilethechemicalprofilesinAWsweredominatedbythe
precursorofthequeensubstance9-HDA(40.04±7.55%).Theratiosoftwoprecursorsubstances10-HDAA/9-
HDAofIAWsweremoreworkerlike(>1.0)whereasAWsweremorequeenlike(≤1.0).Theseresultssuggestthat
themandibularpheromonesofanarchisticworkersresembleamorequeen-likereproductiveactiveprofileandthat
theseworkersmayrepresentareversiontoamorebasalreproductivephenotype.
Apismellifera /anarchisticcolony/ovaryactivation/eggproduction/workerpolicing/workermandibular
glandpheromone
1. INTRODUCTION workers (Michener 1974). Under normal condi-
tions, the queen isusually the sole femalerepro-
Likemanyeusocialinsects,thehoneybeeApis ductive active individual in a colony, while
mellifera L.ischaracterizedbyextremereproduc- workers refrain from reproducing despite having
tive division of labor between the queen and functional ovaries and in general, can lay only
haploidmaleeggs(arrhenotoky) (Winston 1987;
Hepburn and Radloff 1998; Hoover et al. 2003;
ElectronicsupplementarymaterialTheonlineversionof
thisarticle(doi:10.1007/s13592-015-0396-4)contains Slessoretal.2005).Inwild-typecoloniesofEu-
supplementarymaterial,whichisavailabletoauthorized ropean subspecies, approximately 0.01 % of
users. workers lay7% ofallmale eggs, ofwhichonly
Correspondingauthor:F.Hu,[email protected] one egg in 1000 is reared and the other are re-
Manuscripteditor:DavidTarpy moved by other workers; the so-called worker
516 D.-F.Niuetal.
policing(Ratnieks1993;Visscher1996).Howev- (Ratnieks and Visscher 1989; Oldroyd and
er,effectiveworkerpolicingisnotpresentinarare Ratnieks2000)bylayingawayfromherandthe
strain of A. mellifera “anarchistic” colonies, in central brood nest (Neumann et al. 2003). How-
which a large number of worker-laid eggs are ever,thethirdcharacteristichasnotbeenreported
reared (Oldroyd et al. 1994; Montague and in anarchistic honey bees and which is often as-
Oldroyd1998;Châlineetal.2002).Inanarchistic sociated with reproductive active workers in the
colonies, approximately 1 % of workers show Capehoneybee(Pettey1922;Moritzetal.1999,
highlyactivatedovariesandfrequentlylayunfer- 2001, 2008; Martin et al. 2002a, b; Pirk et al.
tilized eggs. That proportion of workers can be 2002, 2012; Zheng et al. 2010)—a queen-like
increasedupto9%inselectivelybredcoloniesin pheromonalbouquet(Hooveretal.2005b).
the presence of a laying queen (Montague and The queen mandibular gland pheromone
Oldroyd 1998; Oldroyd and Osborne 1999; (QMP) is a complex mixture of 9-oxo-2(E)-
Oldroyd et al. 1999). This results in the unusual decenoic acid (9-ODA), the two enantiomers of
situation that majority of males are offspring of 9-ODA’s biosynthetic precursor, (R)- and (S)-9-
workers rather than the queen in these colonies hydroxy-(E)-2-decenoic acid (9-HDA), and two
(Oldroyd et al. 1994; Montague and Oldroyd aromaticcompoundsmethylp-hydroxyben-zoate
1998;Barronetal.2001),whichissimilartothe (HOB) and 4-hydroxy-3-methoxyphenylethanol
naturalsituationintheCapehoneybeewherethe (HVA) (Slessor et al. 1988), which is central to
layingworkerssignificantlycontributetothepop- the regulation of queen-worker and worker-
ulation(Moritzetal.1998).IntheCapehoneybee, worker conflict resolution (Plettner et al. 1993;
Apis mellifera capensis from South Africa, Moritzet al.2000,2004).QMPisdominatedby
workers are able to produce diploid offspring via the compound of 9-ODA, the so-called queen
thelytokousparthenogenesis(Onions1914;Lundie substance,whichisessentialfortheinhibitionof
1954;Anderson1963;VermaandRuttner1983).It ovary activation and egg-laying behavior in
has been suggested that workers discriminate be- workers (Hoover et al. 2003; Dietemann et al.
tweenqueen-laidandworker-laideggsbythepres- 2007; Strauss et al. 2008). Anarchistic workers
ence or absence of an egg-marking pheromone are less inhibited by the queen pheromones than
(Ratnieks 1995), and the Dufour gland has been wild-typeworkers,andithasbeensuggestedthat
suggested as a potential origin (Katzav-Gozansky thisisdue toa reduced sensitivity toqueen- and
et al.1997) sincelayingworkers mimicthecom- larvae-produced signals (Oldroyd et al. 2001;
position of the queen Dufour’s gland secretion Hoover et al. 2005b). The worker mandibular
(Sole et al. 2002). Thus, eggs laid by anarchistic gland secretion under queenright conditions is
workers are more acceptable presumably due to dominated by the “worker substances” 10-hy-
theirproducedqueen-likesecretions,includinges- droxy-2(E)-decenoic acid (10-HDA) and 10-
ters, from their Dufour’s glands (Oldroyd and hydroxyde-canoic acid (10-HDAA) (Crewe
Ratnieks 2000; Martin et al.2004). However, the 1982; Plettner et al. 1993). However, workers
exact mechanism or pheromone involved in dis- are able to shift the synthesis towards the queen
criminationofworker-andqueen-laideggshasnot pathway and queens can also produce worker
beendetermined. substances (Yusuf et al. 2015). Workers mostly
For the reproductive phenotype to arise, three changethepheromonalbouquetunderqueen-less
predispositions facilitating worker reproduction conditions,inparticularworkersofA.m.capensis
have to be present. First, reproductive active quickly develop into “pseudoqueens” which lay
workers must be less affected by the queen and eggsandproducequeen-likeglandsecretiondom-
brood pheromones that normally suppress their inated by the queen substance (9-ODA) in their
ovary activation and eggs-laying behavior mandibular glands (Crewe and Velthuis 1980;
(Barron and Oldroyd 2001), and second they Moritz et al. 2000; Simon et al. 2001).
should increase the survival of their eggs either Pseudoqueen-produced pheromones play an im-
bylayingmoreacceptableeggs,whichavoidbe- portant role in establishing reproductive domi-
ing removed by other workers (worker policing) nance hierarchies among queen-less workers,
ReproductivetraitsandpheromoneprofilesofApismellifera 517
because9-ODAthatisproducedinregularcolo- colonies.Furthermore,threecoloniesnotshowingany
nies by a functional queen has been shown to featuresofanarchisticactivitieswereusedasdiscrimi-
inhibitovaryactivationandsuppresstheproduc- natorcolonies.
tion of a queen-like pheromone signal in other
workers (Crewe 1988; Moritz et al. 2000, 2004, 2.1. Experiment1.Ovaryactivationanalysis
2008;Dietemannetal.2007).Thus,theratiosof
9-ODA/(9-ODA + 9-HDA + 10-HDA + 10- Honey bee abdomens were dissected according to
HDAA) and 10-HDAA/9-HDA have been used Dade(1977)andassignedasbeinginactivated(ovaries
as strong indicators of individual fecundity thread-likeandlackingdefinedova,stages1–3)orfully
(Plettner et al. 1993; Moritz et al. 2004; Hoover activated(clearlydefinedova, stage4, or at least one
etal.2005a;Schäferetal.2006). eggpresentinovarioles,stage5)(Hess1942;Pirketal.
Anarchists, as a special case of worker repro- 2010).Sixtyworkersperanarchisticcolony(n=3)were
duction,provideanexperimentalresourceforin- obtainedfromdronecombsabovethequeenexcluder,
vestigatingtheproximatemechanismsunderlying andtheprocedurewasreplicatedin3-dayintervalsfor
the worker reproduction in wild-type colonies. fourtimesinJune2012,resultingin720workersbeing
However,anarchyisacomplexsyndrome,which dissectedandscored.
is influenced by pheromones, worker policing,
queen and worker genetic component, and other 2.2. Experiment 2. Egg-laying behavior
factors.Themajorityofresearchoftheanarchistic analysis
traits is based on the selectively bred line main-
tainedbytheUniversityofSydney,whichreliably Each anarchistic colony (n=3) was split into two
show higher levels of anarchistic phenotype halves(queenrighthalf:theareabelowqueenexcluder,
(Oldroydetal.1999;OldroydandOsborne1999). andqueen-excludedhalf:theareaabovequeenexclud-
In 2012, we first saw drone eggs and larvae er)usingaqueenexcluder,whichallowspassagefreely
appeared above the queen excluder in several ofworkersbutnotofthequeen.Inordertomeasurethe
queenright A. mellifera L. colonies, in Shanxi reproductiveoutputofthequeenandworkersinterms
province, China. In order to confirm their anar- of drones eggs produced within colonies, one drone
chistic traits, we investigated the following: (1) combwasputineachhalf.After8h,dronecombswere
thepercentageofworkerwithstage4–5activated removed from the colonies and the eggs in each half
ovaries (Hess 1942; Pirk et al. 2010) (2) and if werecounted.
worker-laideggsareabletoescapeworkerpolic- Tocomparethenumberofworker-laideggsbetween
ing.Inordertofurtherunderstandtheroleofthe thetwohalvesandtoevaluatethepotentialcontribution
workermandibularglandsecretionsintheacqui- ofworkerstotheeggslaidinthe queenrighthalf,we
sitionofreproductivestatusandintheproximate placedonedronecombeachinaplasticqueenexcluder
mechanismsunderlyingtheanarchisticsyndrome, boxesaboveandbelowthequeenexcluder.Again,the
(3)weinvestigatedthecompositionofmandibular numberofworker-laideggs wascounted ineach half
glandprofilesofworkerswithinactivatedovaries after8h,andthiswasreplicatedonthreeorfourtimes
(IAWs)andworkerswithactivatedovaries(AWs) within1week.Analysisforstatisticaldifferencesofthe
sampledinthreecolonieswithtypicalanarchistic egg production between queenright half and queen-
traitsand determined ifAWs producequeen-like excluded half was performed using Student’s t test.
substancesintheirmandibularglandsecretions. Dataarepresentedasmeansandstandarddeviations.
2. MATERIALSANDMETHODS 2.3. Experiment3.Workerpolicinganalysis
Eggsandlarvaewerefoundabovethequeenexclud- 2.3.1. Sourcesofeggs
er in ten queenright A. mellifera L. colonies, in the
apiaryofShanxiprovince,China,ofwhichthreewere Three wild-type queenright colonies (WT1, WT2,
transported to Hangzhou and the University’s apiary. WT3) are the sources of wild-type queen-laid eggs
All of them were suspected of being “anarchistic” (WTQE),threewild-typequeen-lesscolonies(WTL1,
518 D.-F.Niuetal.
WTL2,WTL3)asthesourcesofwild-typeworker-laid this analysis, eggs removed at 1, 2, 4, or 6 h were
eggs (WTWE), three anarchistic queenright colonies completedatapointswhereasremainingeggsafter6h
(AC1,AC2,AC3)asthesourcesofanarchisticqueen- were treated as censored data. Egg source, day, and
laid eggs (ACQE), and anarchistic worker-laid eggs discriminatorwereusedasvariablesintheCoxregres-
(ACWE).Thesecolonies(n=9)weredividedintothree sion survival model, and we used it to calculate the
groups;thefirstgroupWT1,WTL1,AC1;thesecond likelihood ratio for the survival rate of the different
group WT2, WTL2, AC2; and the third group WT3, eggsourcesafteraddingthefactorsofdayanddiscrim-
WTL3,AC3.Eachgroupprovidedfourdifferenttypes inator. The survival function was modeled without
of eggs, including WTQE, WTWE, ACQE, and (“Null”) and then with (overall) source of eggs, day,
ACWE. and discriminator colony (overall) as factors, and the
improvementoftheabilityofthemodeltodescribethe
2.3.2. Discriminatorcolonies data tested with a χ2 test. The model then tested the
effectofaddingsourceofeggs,day,ordiscriminatoras
To assay the removal rates of eggs from various factors. Lastly, we presented a pairwise comparison
sources, we used three wild-type colonies (WT1, between the survival eggs of four different sources.
WT2, WT3) and three anarchistic colonies (AC1, Wepresentedthedatagraphicallyasthemeanpropor-
AC2, AC3) as discriminator colonies. Each hive was tion (±standard deviations) of eggs remaining at each
separatedintotwopartsbyaqueenexcluder,inwhich timeperiod.
thequeenwasconfinedtothebottom,yieldingqueen-
excluded areas above the queen excluder and 2.4. Experiment 4. Workers’ mandibular
queenrightareasbelow. pheromoneanalysis
2.3.3. Eggremovalbioassays Pheromonecompositionsanalysiswasconductedat
Pretoria University, South Africa. Ten heads of IAWs
Toperformtheassay,wetransferred20eggsfrom andAWseachcolony(n=3)wereremovedandstored
eachsourceofeggs;thefourdifferentsources(WTQE, in200 μl dichloromethane foratleast24h toextract
WTWE, ACQE and ACWE) into drone cells of the compounds of their mandibular glands. Half of the
same test comb that has been left overnight in their extractswereevaporatedtodrynessunderastreamof
correspondingdiscriminatorcolonies.Allofthesetest nitrogenandanalyzedbygaschromatography(Zheng
eggswerecollectedfromtheir originaldrone cellsby et al. 2010). Chromatograms were recorded using an
usingsterilizedtoothpick.Anewtoothpickwasusedfor Agilent 6890GCandpeakareasquantifiedusingHP
eachrow.Aftertransferring,thetestcombwith80eggs ChemStation software. HOB, 9-ODA, HVA, 9-HDA,
wassandwichedbetweenbroodcombsthatpreviously 10-HDA, and 10-HDAAwereidentified basedon the
placed in the center of the upper chamber of each retentiontimesofsyntheticcompoundsandtheirreten-
discriminatorcolony(Pirketal.2002).Eggswerenot tiontimerelativetotheinternalstandards(Simonetal.
introducedintheircolonyoforigintoavoidanyinter- 2001). Their relative mass ratios (RMRs) were mea-
ferences of nestmate recognition (Pirk et al. 2007). suredrelativetotetradecane.Thestandardsolutioncon-
Drone combs with four types of eggs were placed in tainingallofsixcompoundswasrundailytoensurethat
thediscriminatorcoloniesensuringthatcombsandeggs RMRswerewithinthelimitofthevariabilityfoundin
wereoriginatingfromothercoloniesthanthediscrimi- theseriesofstandardruns.
nator colonies. The number of remaining eggs was We calculated the absolute amounts (micrograms)
counted1,2,4,and6hafterintroduction,anditwas andrelativeamounts(theirpercentagecomposition)of
repeatedfourtimesonseparatedays. HOB,9-ODA,HVA,9-HDA,10-HDA,and10-HDAA
inIAWsandAWs.Theratioof9-ODA/(9-ODA+9-
2.3.4. Statisticalanalysis HDA + 10-HDA + 10-HDAA) (Moritz et al. 2004;
Hoover et al. 2005a; Schäfer et al. 2006) and 10-
Survival data of (WTQE, WTWE, ACQE, and HDAA/9-HDA(Plettneretal.1993)wereusedasindi-
ACWE)wereanalyzedusingaCoxregressionsurvival cators of individual fecundity. Analysis for statistical
analysis (Collett 1994) as implemented in SPSS. For differences between IAWs and AWs were performed
ReproductivetraitsandpheromoneprofilesofApismellifera 519
using Mann-Whitney U test. Data are presented as From that, we can calculate that the contri-
meansandstandarddeviations. bution of the queen was on average 209 eggs
or 94 % of the eggs laid in the queenright
3. RESULTS section of the hive.
3.1. Experiment1.Ovaryactivationanalysis 3.3. Experiment3.Workerpolicing
Atotalof720workerswererandomlycollect- A Cox regression analysis showed that there
edfromeachcolonyforovaryactivationanalysis. were significant differences in the removal rates
ThemeanpercentageofAWsinthreeanarchistic forthefourdifferentsourcesofeggs(“Sourceof
colonies was 6.25–49.58 %, and the mean per- eggs” P<0.001). The experimental day did not
centage of workers with at least one mature egg significantly affected the removal rates (“Day of
appeared in their ovarioles (stage 5, Pirk et al. trial” P>0.05) nor did discriminator colony
2010)was3.00–21.25%(TableI). (“Discriminator”P>0.05)(TableII).
In anarchistic discriminator colonies, WTWE
3.2. Experiment 2. Egg-laying behavior were removed significantly faster than ACWE
analysis (P<0.001); however, ACWE were removed at a
similar rate as WTQE (P=0.182) and ACQE
According to analysis of variance, the mean (P=0.290)(Figure3a,TableII).
rate of egg production of queenright half In wild-type discriminator colonies, the
(222.83±85.91), to which potentially the queen pairwisecomparisonsshowedthatthefoursource
andworkerscontributed,wassignificantlyhigher eggs were treated significantly different: ACQE
than that of the queen-excluded half (11.42± were removed significant less than WTQE
21.38) within 8 h in anarchistic colonies (t= (P<0.05) and WTWE were removed fastest
8.273, df=12.358, P<0.001) (Figure 1). The whereas ACQE were removed significantly
mean rate of egg production of workers in slower than WTQE (P<0.001) (Figure 3b,
queenright half (13.00±20.72) was lower TableII).
than that of queen-excluded half (25.67± Inbothanarchisticandwild-typediscriminator
36.88) in anarchistic colonies (Figure 2). colonies,WTWEwereremovedfasterthanother
TableI.Thepercentageofworkerswithactivatedovaries(AWs)andatleastonematureeggappearedintheir
ovariolesinthreeanarchisticcolonies.
Times(n=12) Colony(n=3) AWs(%) X (cid:1)SD Workerswithatleast X (cid:1)SD
onematureegg(%)
1 AC1 10.00 16.25±7.50 6.67 7.25±4.72
2 26.67 23.33
3 11.67 6.67
4 16.67 11.67
1 AC2 3.33 49.58±14.23 3.33 21.25±7.59
2 8.33 8.33
3 11.67 8.33
4 1.67 0
1 AC3 58.33 6.25±4.59 33.33 3.00±2.45
2 61.67 45.00
3 30.00 18.33
4 48.33 45.00
520 D.-F.Niuetal.
Figure1.Therateofeggproductionofqueenrighthalfandqueen-excludedhalfinthreeanarchisticcolonies(AC).
Thetwopartswereseparatedbyaqueenexcluder,sothequeenwastheonlyindividualconfinedtoonartofthehive.
Meanandstandarddeviationsareshown.
egg sources, while the survival rates of ACWE whereastheremainingfourcompoundswerenot
were intermediate between those of queen-laid significantly different between the two groups
eggs (WTQE, ACQE) and WTWE. In addition, (lowestP value=0.565)(Figure4,TableSI).
the anarchistic colonies were less discriminatory Therelativeamountsof10-HDAAand9-HDA
than wild-typecolonies towards the four sources were significantly different between IAWs and
ofeggs(Figure3). AWs (P<0.001) with 10-HDA (34.64±8.19 %)
and 10-HDAA (22.88±4.95 %) being the domi-
3.4. Experiment 4. Workers’ mandibular nant components in the profile of IAWs, while
pheromoneanalysis AWs was dominated by 9-HDA (40.04±
7.55 %), the queen substance, 9-ODA was
Thetotalamountsofallidentifiedcompounds a relativelyminorcomponentinbothIAWs(5.14
weresignificantlyhigherinAWs(8.88±1.71μg, ±3.24 %) and AWs(2.09±1.14 %),andwas not
n=30)comparedtoIAWs(4.00±2.09μg,n=28) significantly different between the two groups,
(P<0.05, Table SI). Furthermore, the absolute similar to the remaining three components
amounts of HOB and 9-HDAwere significantly (lowest P value =0.063) (Figures 4 and 5,
higher in AWs compared to IAWs (P<0.05), Table SI). The ratio of 9-ODA/(9-ODA + 9-
Figure 2.The rate of egg production of workers in queenright and queen-excluded halves of three anarchistic
colonies(AC),whenthequeenwascagedontoonedronecomb.Meanandstandarddeviationsareshown.
ReproductivetraitsandpheromoneprofilesofApismellifera 521
TableII.Likelihoodratiocomparingthesurvivalofeggslaidbywild-typequeens(WTQE),anarchisticqueens
(ACQE),wild-typeworkers(WTWE),andanarchisticworkers(ACWE)inanarchisticcolonies(n=180foreach
eggsource)andwild-typecolonies(n=240foreacheggsource)workerpolicingbioassays.
Anarchisticdiscriminatorcolonies Wild-typediscriminatorcolonies
Term −2log χ2 df P −2log χ2 df P
likelihood likelihood
Null 6436.147 10,897.387
Overall 6416.886 19.274 3 <0.001 10,859.700 37.590 3 <0.001
Sourceofeggs 19.195 1 <0.001 34.441 1 <0.001
Dayoftrial 0.057 1 0.811 1.277 1 0.259
Discriminator 0.075 1 0.784 2.272 1 0.132
Pairwisecomparisonofeggssource:
WTQEvsACQE 0.074 1 0.786 4.526 1 0.033
WTQEvsACWE 1.780 1 0.182 17.556 1 <0.001
ACQEvsACWE 1.118 1 0.290 38.669 1 <0.001
WTQEvsWTWE 30.350 1 <0.001 101.960 1 <0.001
ACQEvsWTWE 27.738 1 <0.001 132.645 1 <0.001
ACWEvsWTWE 19.443 1 <0.001 43.047 1 <0.001
Thelikelihoodratioscomparedthesurvivaloffourdifferentsourcesofeggs(WTQE,WTWE,ACQE,andACWE).Thesurvival
functionwasmodeledwithout(BNull^)andthenwith(Boverall^)sourceofeggs,day,anddiscriminatorcolony(overall)asfactors,
andtheimprovementoftheabilityofthemodeltodescribethedatatestedwithaχ2-test.Themodelthentestedtheeffectofadding
sourceofeggs,day,ordiscriminatorasfactors.Lastly,wepresentedapairwisecomparisonbetweenthesurvivaleggsoffour
differentsources
HDA + 10-HDA + 10-HDAA) showed no the queenright part, it still suggests that the ma-
significant differences between IAWs and jorityisqueenderived.Anarchisticworkerswere
AWs (ratio IAWs=0.057±0.074, AWs=0.024± pronetolayeggsinqueen-excludedhalfrevealed
0.037, P=0.421), whereas the ratio of 10- thatanarchistsreproductionstillinfluencedbythe
HDAA/9-HDAwassignificantlyhigherinIAWs queen pheromones inhibitory effects but need
than in AWs (ratio IAWs=1.490±1.319>1.0, higher levels, because they could avoid suppres-
AWs=0.501±0.700<1.0,P<0.001)(TableSI). sion increasing the spatial distance to the phero-
mone source. A behavior has also been seen in
4. DISCUSSION otherlayingworkers(Moritzetal.2001;Pirketal.
2002), which might be facilitated by spatial dif-
Inourcolonies,theratesofworkerswithfully ferences in worker policing behavior (Neumann
activatedovaries(3–21%)werehigherthanpre- etal.2003).
vious naturally occurring anarchistic colonies In general, the efficiency of egg removal be-
(around 1 %; Montague and Oldroyd 1998; haviorseemssufficientlyhighenoughtoremove
Oldroydetal.1999),whichwassimilartotypical the majority of the worker-laid eggs (Ratnieks
selectively bred colonies (3–9 %; Oldroyd and 1993;Visscher1996;Pirketal.2003).However,
Osborne 1999; Barron and Oldroyd 2001) and the eggs laid by workers from our anarchistic
queen-less colonies of Apis mellifera ligustica colonies seems to be more acceptable since in
(5–24%;MillerandRatnieks2001). both anarchistic and wild-type discriminator col-
The rate of egg production of queenright half onies,ACQEwereremovedlessthanotherthree
was significantly higher than that of queen- sourcesofeggs,whilethesurvivalratesofACWE
excludedhalfworkers,andevenwhensubtracting were intermediate between queen-laid eggs
thenumberofeggspotentiallylaidbyworkersin (WTQE, ACQE) and WTWE. All of the four
522 D.-F.Niuetal.
Figure3.Removalratesofeggslaidbywild-typequeens(WTQE)andworkers(WTWE)andanarchisticqueens
(ACQE)andworkers(ACWE)whenintroducedintothreeunrelatedanarchistic(a) andwild-type(b) queenright
discriminatorcolonies,respectively.Valueswerethemeansofall3or4daysforalldiscriminators(n=3).Thebars
representedthestandarderrorsofthemeans.
sourcesofeggs,especiallyWTWEwererejected producequeen-likesignaltoestablishtheirrepro-
morestronglyinwild-typediscriminatorcolonies ductivedominance.Whencomparingwithlitera-
than in anarchistic discriminator colonies. These ture data, the total amounts in both IAWs and
resultsstronglysupportpreviousstudiesshowing AWswerehigherthaninA.mellifera queenright
that anarchistic workers evade egg policing by workers (2.59±0.62 μg), while they were less
laying more acceptable eggs (Oldroyd and than A. mellifera queen-less workers (10.73±
Ratnieks 2000) and that worker policing seems 2.30 μg) (Tan et al. 2012). Since pheromonal
to be reduced in anarchistic colonies. However, dominance cantranslateintothrophallactic dom-
theproximatemechanismunderlyingthediscrim- inance and greater ovarian activation potential
ination of worker and queen-laid eggs in honey (Schäfer et al. 2006), it would suggest that our
beesand,inparticular,inanarchisticcolonieshas anarchistic workers have a higher reproductive
not been determined, besides a potential egg- potential than the “standard” A. mellifera
markingpheromone(Ratnieks1995),eggviabil- queenrightworkersandperhapseventhanlaying
itymightalsoplayarole(Pirketal.2004). workers of other anarchistic lines or other Euro-
Theanalysisofthemandibularglandproducts pean subspecies, but most likely not laying
of workers collected from these three colonies workers of African subspecies (Zheng et al.
showed that anarchistic workers had ability to 2010; Yusuf et al. 2015). The extracts of IAWs
Figure4.Comparisonoftheabsoluteamount(micrograms,mean±SD)betweenworkerswithinactivatedovary
(IAWs)andworkerswithactivatedovaries(AWs)sampledinthreeanarchistichoneybeecolonies.
ReproductivetraitsandpheromoneprofilesofApismellifera 523
Figure5.Comparisonoftherelativeamounts(mean±SD)ofthefivecompoundsofmandibularglandsbetween
workerswithinactivatedovaries(IAWs)andworkerswithactivatedovaries(AWs)sampledinthreeanarchistic
honeybeecolonies.
hadthetypicalworker-likeprofilesdominatedby missinginallpreviouscasesonanarchisticbees.
“worker substances” 10-HDA and 10-HDAA. The ratio of 10-HDAA to 9-HDA produced by
AWsshowedamorequeen-likemandibulargland honey bee worker mandibular glands could be
secretionprofile,whichisdominatedby9-HDA, consideredamechanismthatexplainsworker“re-
theprecursorofthemajorqueenpheromonecom- production” in queenright colonies in
pound (9-ODA). In this study, the mandibular A. mellifera. Understanding the mechanisms of
gland phenomenon signals of IAWs were more workerreproductionwillprovideaninsideinthe
worker-likewhereasthatofAWswerequeenlike evolutionofsocialityingeneral.
(Table SI). Our results are consistent with previ-
ous studies on reproductive active workers. We
ACKNOWLEDGMENTS
showthatAWsproducemoreof9-HDAsimilarto
A.m.capensis workers bypreferentiallyfollow-
ing the “queen-specific” synthetic pathway This work was supported by the National Natural
Science Foundation of China (No. 31101773), the
(Zheng et al. 2010), a physiological priming for
earmarkedfundsforModernAgro-industryTechnology
pheromoneproductionandreproduction(Plettner
Research System (No. CARS-45), the National Re-
et al. 1996, 1998). Therefore, AWs just have to
searchfoundation(NRF)ofSouthAfrica,andtheUni-
synthesize the final compound 9-ODA by versityofPretoria,SouthAfrica.
oxidization of 9-HDA, to shift from a worker-
likemandibularglandsignaltoaqueen-likesignal
(Hepburn and Allsopp 1994) dominated by 9-
ODA, which allows them to rapidly dominate Traits reproductifs et phéromone de la glande
mandibulaire des ouvrières 'anarchistes' de l'abeille
reproduction (Ruttner and Hesse 1981; Zheng
Apismellifera présenteenChine
etal.2010).
Our results confirmed anarchistic syndrome
colonie anarchiste/ activation de l'ovaire/ production
occurredinthreeA.mellifera queenrightcolonies d'oeufs/ouvrière/'policing'
ofEuropeansubspeciesoriginsampledinChina,
whichisobservedonlyinthefourothercountries Reproduktive Eigenschaften und
before,NewZealand,Australia,America,andthe Mandibeldrüsenpheromon von anarchistischen
ArbeiterinnenderHonigbiene Apismellifera inChina
UKundernaturalconditions(Oldroydetal.1994;
Ratnieks 1995; Montague and Oldroyd 1998;
Apismellifera/anarchistischesBienenvolk/Aktivierung
Châline et al. 2002).Moreover, our resultsshow
der Ovarien / Eiproduktion / „Policing“durch
for the first time that the pheromonal signal of
Arbeiterinnen / Mandibeldrüsenpheromon der
anarchistic workers is more queen-like, a feature Arbeiterinnen