Physiological and genetic mechanisms underlying caste ... · PDF filePhysiological and genetic mechanisms underlying caste ... us to re-exa mine the question of genetic caste determination

144Apidologie 37 (2006) 144–163© INRA/DIB-AGIB/ EDP Sciences, 2006DOI: 10.1051/apido:2006013

Review article

Physiological and genetic mechanisms underlying caste development, reproduction and division of labor

in stingless bees

Klaus HARTFELDERa*, Gustavo R. MAKERTb, Carla C. JUDICEc, Gonçalo A.G. PEREIRAc, Weyder C. SANTANAd, Rodrigo DALLACQUAd,

Márcia M.G. BITONDId

a Departamento de Biologia Celular e Molecular e Bioagentes Patogênicos, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Brazil

b Departamento de Genética, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Brazilc Departamento de Genética e Evolução, Instituto de Biologia, Universidade Estadual de Campinas, Brazil

d Departamento de Biologia, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, Brazil

Received 26 September 2005 – revised 29 November 2005 – accepted 23 December 2005

Abstract – Investigations on physiological and molecular mechanisms underlying developmental andreproductive differentiation in social bees center on the question of how different patterns of larval nutritioncan affect hormonal dynamics and how these drive differential gene expression. Differential expressionanalyses and the generation of AFLP markers now enable us to re-examine the question of genetic castedetermination in the genus Melipona. The comparison of vitellogenin expression in three species ofstingless bees suggests divergence in regulatory mechanisms that directly relate to the mode of workerreproduction. As in honey bees, this indicates alternative functions for vitellogenin in the life cycle of adultworkers. The diversity in life histories and their associated specific physiologies make the stingless bees arich resource for information on evolutionary trajectories that have generated phenotypic plasticity in socialHymenoptera.

stingless bee / caste development / juvenile hormone / vitellogenin / worker reproduction / Apidae /Meliponini

1. INTRODUCTION

The caste syndrome in highly social insectsis a life history trait that is based upon devel-opmental pathways capable of generating alter-native phenotypes. In most cases, alternativecaste phenotypes and their differential repro-ductive potential result from an environmentalstimulus that triggers a response in the endo-crine system. This, in turn, drives preimaginaldifferentiation processes into alternative path-ways, primarily during metamorphosis. Thus,not surprisingly, the hormones involved incaste development and reproductive physiol-

ogy are juvenile hormone (JH) and ecdyster-oids, the major morphogenetic and reproductivehormones of insects (Wheeler, 1986; Hart-felder, 2000; Hartfelder and Emlen, 2005). Assuch, the caste syndrome is built upon a basicgroundplan of insect development and repro-duction (West-Eberhard, 1996; Hunt andAmdam, 2005). Having set the general frame-work for the caste syndrome we next have toask how life histories of different groups ofsocial insects are linked to these basic physio-logical modules, and whether and how thesecan generate and explain the variability indevelopmental and reproductive traits.

* Corresponding author: [email protected] to Dr. Warwick E. Kerr

Article published by EDP Sciences and available at http://www.edpsciences.org/apido or http://dx.doi.org/10.1051/apido:2006013

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http://dx.doi.org/10.1051/apido:2006013

Caste development in stingless bees 145

Why are stingless bees an interesting groupto look for such traits? Their large number ofspecies and genera, as well as the variability inecological and behavioral traits make stinglessbees a microcosm to look for correlative asso-ciations between life history traits on the onehand and physiological and genetic mecha-nisms underlying caste development, differen-tial reproduction and division of labor on theother. In this review many of these observationson stingless bees will be contrasted with corre-sponding information for the honey bee.

2. ROYAL CELLS, ROYAL FOOD AND ROYAL GENES – THE INITIAL TRIGGERS OF CASTE DEVELOPMENT

All stingless bees follow a mass-provision-ing strategy in caring for their brood, that is,brood cells built out of a wax and resin mixtureare filled with well defined amounts of larvalfood regurgitated from the crops of nurse-stageworkers. In several species, nurse-stage work-ers lay trophic eggs at defined positions withinnewly provisioned brood cells (Sakagami et al.,1963; Velthuis and Velthuis, 1998, and refer-ences therein; Silva-Matos et al., 2000). Theseeggs are eaten by the queen, or occasionally byanother worker. Subsequently, the queen laysa reproductive egg on top of the liquid foodmass, and the cell is immediately sealed. Sincethis sequence of events marks a critical aspectof colony reproduction (reproductive integra-tion and competition), the exact sequence ofbehavioral events, that is, the temporal dynamicsof cell construction, provisioning, egg-layingby workers (trophic and reproductive eggs) andqueens, and even of cell sealing, is highly spe-cies-specific (Sakagami et al., 1973; Sakagamiand Zucchi, 1974). This sequence of events inthe cell Provisioning and Oviposition Process(POP) has been split into discrete units and hasbeen intensely studied and classified in termsof possible phylogenetic implications (Zucchiet al., 1999; see also references in other reviewswithin this Special Issue).

2.1. Trophogenic control of caste development

With respect to caste development it isimportant to know whether the larval food thatstingless bee workers regurgitate into brood

cells contains specific compounds that mightfavor queen development, just like royal jellydoes in the honey bees, or whether it is simplya matter of food quantity. This question alsoleads directly to the major dividing linebetween the trigonine group of stingless beesand the genus Melipona in terms of the role oflarval food in the triggering of queen/workerdevelopmental pathways (Michener, 1974;Sakagami, 1982). In this respect, all trigoninegenera (we use here the colloquial terminologywhere “trigonines” is taken to mean “all sting-less bee genera, except Melipona”) capitalizeon quantitative differences in larval food that isbeing made available to the growing larvae(Figs. 1A, 1B), but they have chosen differentstrategies. In genera with a clustered brood cellarrangement (Leurotrigona, Frieseomelitta), allcells are of similar size (Fig. 1A), so they wouldnormally give rise to workers or males. Yet,when queens are to be reared, a second cell isconstructed closely apposed over a previouslybuilt one. Once the larva developing in thelower cell has consumed its portion of larvalfood it then penetrates into the upper cell andconsumes an additional portion of larval food(Faustino et al., 2002). The question of how alarvae in a closed cell may come to know thatthere is a second food portion waiting in aneighboring cell may actually be a matter ofinformation transfer from workers through anovel structural element connecting the twocells. Faustino et al. (2002) showed that workersconstruct a special feeding connection from theoriginal to the auxiliary cell. Since this modeof queen production has so far only been care-fully recorded under conditions of emergencyqueen rearing, it is still an open question whetherthe same mechanism of queen determination isalso used for queen production under queen-right conditions, for colony reproduction.

The more frequent situation encountered inmost trigonine genera is a brood nest with anarchitecture of horizontal combs separated bypillars (Michener, 1974). At the margins ofthese combs, the workers occasionally buildroyal cells of much larger size, and these cellsare provisioned with royal quantities of larvalfood, usually double or triple the amountdeposited in normal cells (Fig. 1B). A larva ina queen cell can thus feed much longer than aworker larva and stops growing and enters met-amorphosis at a much larger size (Hartfelder

146 K. Hartfelder et al.

and Engels, 1992). As a consequence of theconsumption of larger amounts of larval foodby queen larvae in all trigonines, the onset ofmetamorphosis is much delayed and totaldevelopmental time of queens is longer whencompared to workers (Camargo, 1972a).

Ever since the early description of brood nestarchitecture in the genus Melipona (Ihering,1903), this stingless bee genus has been con-sidered a curiosity in the context of caste devel-opment because no specific queen cells couldbe found on the brood combs (Fig. 1C). Instead,queens were found to develop in cells of thesame size as those giving rise to workers andmales. But it is not only in this respect thatMelipona species differ from the trigonine gen-era, they also produce queens at much higherfrequencies (up to 20–25%) (Kerr, 1946, 1948,1950a, b) than the trigonine species (usually

only 1–2% of queens in female brood), andMelipona queens develop faster than workers(Kerr, 1948; Kerr et al., 1966).

This variability in queen cell structure andlarval food portions in stingless bees and theirphylogenetic proximity to the honey bees hasprompted questions such as, is quantity of lar-val food more important than quality, and whois in control of gyne production, the workers,the queen, or even the larva itself? The fre-quency of queen rearing could be increased tonearly 100% in trigonine species by provision-ing larvae reared in artificial cells with doubleto triple amounts of larval food that was col-lected and mixed together from other newlyprovisioned cells (Camargo, 1972a; Buschiniand Campos, 1995). This apparent prevalenceof food quantity over quality in most stinglessbees was also corroborated when the specific

Figure 1. Modes of queen rearing instingless bees. (A) In the brood nestof the trigonine genera Leurotrigonaand Frieseomelitta, brood cells arearranged in a cluster where indivi-dual cells are separated from eachother by pillars. Brood cells for wor-kers and males are of the same size(left). For queen rearing (right), asecond brood cell is built over analready finished one containing afemale larva. After this larva hasconsumed the food portion in cell 1it perforates the wall to cell 2 andconsumes its contents as well. (B)The brood nest of most trigoninegenera is laid out in horizontalcombs. Brood cells for workers andmales are of the same size (left). Forqueen rearing (right), large cells arebuilt at the margin of a comb, andthese are filled with a large food por-tion before the queen lays an egg andthe cell is sealed. (C) In the genusMelipona, brood cells of workers,males and queens are all of the samesize and contain the same amount oflarval food. Queens (right) mayemerge from up to 25% of the broodcells containing female offspring.For references see text.


composition of larval diets was biochemicallyanalyzed (Hartfelder and Engels, 1989). Thenutritional signal for caste development instingless bees is, thus, clearly different fromthat in honey bees which rely on a nutritionalswitch from royal jelly to worker jelly in thelate larval instars of worker brood (for reviewsee, Rembold et al., 1974; Hartfelder, 1990). Inessence, this means that honey bee nursesswitch fourth to fifth instar worker larvae froma high quality glandular food to a pollen andnectar supplemented diet. The result is thatqueens grow faster during these last instars andenter and complete metamorphosis morequickly than workers do. In contrast, the trigo-nine genera of stingless bees do not rely on anutritional switch, but on a larger food supplyfor queen larvae, resulting in an extended feed-ing period and, consequently longer duration ofpreimaginal development for queens.

Once again, Melipona seems to run contraryto the rule because a minor increment in theamount of food available to larvae couldincrease the frequency of queens, but it couldnot be driven beyond the 25% threshold(Camargo et al., 1976). And when investigatingthe strong seasonal variation observed in thepercentage of queens in female brood, Kerret al. (1966) noted that the frequency of queenproduction in Melipona is inversely propor-tional to the number of workers that arerequired to provision a brood cell. Since it isusually under favorable conditions of colonyprovisioning that less workers are needed to filleach brood cell, one cannot exclude that minordifferences in food quality may actually tip thebalance to favor queen development in a con-siderable portion of female brood. So far, how-ever, there is no data on (seasonal) qualitativevariation in larval food of Melipona species,mainly because we have no clues what to lookfor in the food.

In terms of who is in control of queen pro-duction, in the trigonine genera it is the workersthat hold most of the direct power over the castefate of the brood since they build the cells eitherclosely together or of different size (Sakagami,1982). In the genus Melipona it seems, at firstsight, that caste fate is a characteristic of thelarva itself, but then, larval food still has a mod-ulatory effect on this decision. These aspectsare undoubtedly interesting in terms of socio-biological questions and for modeling social

organization (Ratnieks, 2001). Less obviousmay be that they also have important implica-tions when it comes to underlying developmentalmechanisms and how they may have evolved.

2.2. Melipona, a special case?

The genus Melipona has gained singular sta-tus ever since Kerr (1946, 1948, 1950b) firstproposed that the exceptionally high and near25% frequency of queens emerging from cellsof female brood may be explained by a geneticpredisposition to caste fate. He proposed a twoloci model, each with two alleles, where onlydouble heterozygote females should becomequeens. Since the mother queen would alwaysbe double heterozygous at these loci, this pre-diction would be fulfilled in exactly 25% of allfemales (Kerr, 1948, 1950a, b). In strong col-onies and during certain times of the year onemay actually observe such a proportion ofemerging queens, yet, most of the time, queensemerge at much lower queen-to-worker ratios(Velthuis, 1976). Interestingly, however, thequeen frequency in brood is much less variableduring the seasons than is the male/female ratio(Velthuis et al., 2005). The lower than predictedproportion of queens produced in colonies undernatural conditions has been attributed to the larvalfood effect mentioned above, where a suboptimalfeeding regime would redirect the developmentof genetic queens into a worker phenotype(Kerr and Nielsen, 1966; Kerr, 1969, 1974,Camargo et al., 1976; Maciel-Silva and Kerr,1991).

So far, two different approaches have beenused to find evidence for or against a geneticpredisposition to caste. The first approach wasto look for morphological markers that woulddistinguish true workers from genetic queensdisguised as workers. The first morphologicalmarker for genetic queens was proposed byKerr and Nielsen (1966) who noted that fusionof the abdominal ganglia during metamorpho-sis follows different dynamics in queens andworkers. But this is a transient developmentalmarker, because in the adult stage the numberof fused abdominal ganglia is the same for thetwo castes. The caste difference in the temporalpattern of ganglia fusion has now found anexplanation in the endocrine signatures of thetwo castes, with ecdysteroids acting as the mainplayers (Pinto et al., 2003; and see below,


Sect. 2.3). The other morphological marker pro-posed to detect genetic queens was the numberof tergal glands (Cruz-Landim et al., 1980).

A completely different approach to test fora genetic predisposition of caste made use ofthe finding that queen development in stinglessbees in general (Melipona and trigonine gen-era) can be induced in almost 100% of thefemale brood when larvae are treated with JHor JH analogs during the spinning stage of thelast instar (Campos et al., 1975, 1983; Velthuisand Velthuis-Kluppell, 1975; Campos, 1978,1979, 1983; Buschini and Campos, 1995;Bonetti et al., 1995). Since only 25% of theseJH-induced queens should actually have thequeen genotype, the frequency of gynes emerg-ing in colonies headed by JH-induced queensshould vary from 0% (if the parental pair hadidentical alleles at both loci) to 25% (if thequeen and her mating partner had different alle-les at both caste loci). This seemed like anexperimentally feasible approach (Velthuis,1976) since queens can be mated to singlemales in mating boxes (Camargo, 1972b). Upto now, however, none of the many studiesinvestigating JH action in stingless bees reallygave a conclusive answer to this question,because JH-induced queens apparently haveproblems in establishing colonies. Based onrecent evidence we consider the lack in queen-like behavior in JH-induced queens as a phys-iological phenomenon because of the surpris-ingly high divergence in gene expressionpatterns for JH-induced queens when com-pared to newly emerged natural queens (Judiceet al., 2006; and see below Sect. 2.4).

Since morphological markers did not pro-vide the desired resolution, and offspring anal-ysis of JH-induced queens appears to behampered by the queens’ reproductive biology,we recently invested in a large-scale screeningof genetic markers. We established a protocolto generate large numbers of Amplified Frag-ment Length Polymorphism (AFLP) markers(Makert et al., 2006) and have started to lookfor markers that segregate with caste. One ofthese putative markers is shown in Figure 2. Inthis set of bees, which is a single age cohort ofa single colony, we detected this marker inthree of the four virgin queens (75%), in 13 ofthe 31 workers (42%), and in 5 of the 19 males(26%). We detected at least six more markerswith similar segregation ratios. While thesedata are still preliminary they already providesupport to the genetic predisposition hypothe-sis for caste determination in Melipona,although the final model derived from AFLPmarkers may actually look quite different fromthe originally proposed two loci/two alleles model.

These approaches were all designed toexplicitly test for a genetic predisposition ofcaste under the Kerr hypothesis. As this is aclassic Mendelian segregation model, it has itsfocus on the individual genotype, and conse-quently, considerations on individual versuscolony fitness acting in this system did not playa major role in explanations on how this systemmay have evolved (Kerr, 1969, 1974, 1987). Amore recent approach focuses exactly on thesefitness aspects to explain the high numberof queens produced in Melipona colonies(Ratnieks, 2001; Wenseleers et al., 2003).These authors suggest that caste fate in Melipona

Figure 2. Amplified Fragment Length Polymorphism (AFLP) markers visualized by electrophoresis in asequencing gel. DNA was extracted from bees emerging from a single brood comb of a Melipona quadri-fasciata colony. After digestion by restriction enzymes, adapter primers were ligated to the fragments per-mitting selective amplification. The section of a gel in this figure shows a band (arrow) that is present in42% of the workers, in 75% of the queens, and in 26% of the males. This AFLP marker is one of the candidatesfor a genetic mechanism acting in caste determination in the genus Melipona.


females can be decided by the larvae them-selves. Their inclusive fitness model predictsan optimal queen emergence frequency of 20%in female brood of colonies headed by a single-mated queen that produces not only the femalebut also all the male offspring. In contrast, if themale offspring were all worker-produced, theoptimal queen emergence frequency in such acolony would be 14%. This hypothesis findssupport in the fact that there are only minorquantitative differences in larval food availableto female larvae, so control by workers throughcell provisioning is only very limited. Eventhough plausible and attractive on theoreticalgrounds, the “self determination” hypothesisfor caste fate has a drawback as it does not pro-vide any lead on what might be the underlyingdevelopmental mechanism(s) for the queen/worker decision.

2.3. Hormonal regulation of caste development

Independent of what is the nature of the ini-tial trigger of caste determination, this informa-tion has to be transmitted to all tissues and cellsin a coordinated manner to synchronize andintegrate the development of the segmentalmorphological structures and internal organsduring metamorphosis. This integrative func-tion is played out by the endocrine systemthrough the release of JH and ecdysteroids bythe corpora allata and the prothoracic gland,respectively. The role of JH in caste develop-ment of stingless bees has been extensivelyexplored by the application of JH and JH ana-logs. These experiments unambiguously dem-onstrated the existence of a JH-sensitive periodfor the queen/worker decision in the spinningphase of the last larval instar (Campos et al.,1975, 1983; Velthuis and Velthuis-Kluppell,1975; Campos, 1978, 1979; Bonetti et al.,1994, 1995; Buschini and Campos, 1995).Endogenous JH levels during larval developmenthave, so far, only been measured for a singlespecies, Scaptotrigona postica (Scaptotrigonaaff. depilis) (Hartfelder and Rembold, 1991),and the observation of strikingly higher JH lev-els in queens have fully confirmed the assertionof a JH-critical period in the spinning phase ofthe last larval instar.

For S. postica, we also determined the larvaland pupal ecdysteroid titer profile and observed

major caste differences in the prepupal andpupal stages. During these stages, the ecdyster-oid titer was much higher in queens than inworkers (Hartfelder and Rembold, 1991).Ecdysteroid function has been investigated inM. quadrifasciata with respect to the caste-spe-cific program of abdominal ganglia fusion(Pinto et al., 2003). This study revealed thatventral nerve cord preparations of M. quadri-fasciata larvae exposed in vitro to a physiolog-ical concentration of 20-hydroxyecdysoneundergo rapid shortening of connectives andshow programmed cell death in response to thishormone. In vivo, these events would representthe first steps in the fusion process of theabdominal ganglia described by Kerr and Nielsen(1966).

The ecdysteroid titer profiles for pupae ofhighly eusocial bees have now been investi-gated for the honey bee (A. mellifera) and twostingless bees (S. postica and M. quadrifasciata).With data obtained for the latter species (Pintoet al., 2002) it became possible to relate the dif-ferent parameters in the modes of caste deter-mination (trophogenic versus genetic, earlyversus late nutritional signal, and fast versusslow developing queens) with their character-istic endocrine signatures. In the fast developers(Apis and Melipona queens), the pupal ecdys-teroid titer has its major peak earlier in thepupal phase than in the slow developers (Scap-totrigona queens), when compared to theirrespective workers. The phylogenetic differ-ence (honey bees versus stingless bees) isreflected in the peak levels that the ecdysteroidtiter attains in the early pupal phase and in theway the titer gradually declines in the late pupalphase. In honey bees, queens and workers dif-fer only in the timing of the pupal peak but notin peak height, whereas in the stingless bees,the two castes differ with respect to both timingand levels of the ecdysteroid titer peak, whichis always higher in the queen caste. Withrespect to the late pupal ecdysteroid titer, thisis higher in honey bee queens than in workers,whereas in the two stingless bees it is the work-ers that have higher titers than the queens.These comparative analyses show to what extentthe endocrine system can respond differently tocaste-determining stimuli, and which aspectsin this response may represent phylogeneticconstraints. Since the pupal ecdysteroid titer isa major regulating factor for the pigmentation


program of the adult cuticle of bees (Zufelatoet al., 2000), and probably also for other mor-phogenetic programs executed in the pupalperiod of metamorphosis, these titer differ-ences should have prime importance for caste-and group-specific characteristics apparent inthe adult life stages of the highly eusocial bees.

2.4. From caste to BLAST: differential gene expression in Melipona queens and workers

In stingless bees, the results of caste differ-entiation processes and their hormonal controlhave so far been evaluated primarily throughtheir effects on morphological characterexpression and effects on allometric change(Velthuis and Velthuis-Kluppell, 1975; Campos,1978; Hartfelder and Engels, 1992), yet theseeffects are far removed from the direct targetsof hormonal regulation. Since both JH andecdysteroids exert their effects through nuclearhormone receptors (Barchuk et al., 2004) thatimpact directly on gene expression, theprogress on differential gene expression anal-ysis in honey bee caste development (Coronaet al., 1999; Evans and Wheeler, 1999, 2000;Hepperle and Hartfelder, 2001; Guidugli et al.,2004) and in bumble bees (Pereboom et al.,2005) has prompted us to investigate differen-tial gene expression in caste development ofstingless bees. These studies were performedon the genus Melipona due to its distinct modeof caste determination. Yet, instead of lookingat differential gene expression during the latelarval stages, we focused on newly emergedadult bees. At this stage, queens and workersare morphologically distinct, but they are notyet influenced by social interactions that mayimpact gene expression soon after the bees par-ticipate in colony life.

Two different methodological approacheswere employed to investigate differential geneexpression in Melipona queens and workers.The first one was a Differential DisplayReverse Transcription (DDRT) PCR strategy(Judice et al., 2004) which permitted cloningand sequencing of 14 Expressed SequenceTags (ESTs). These represent 12 genes that areoverexpressed in newly emerged workers andtwo genes overexpressed in queens. Ten ofthese Melipona ESTs are also represented inthe EST library generated from honey bee brain

mRNA (Whitfield et al., 2002), and nine of theMelipona ESTs showed significant blastxmatches to sequences deposited in the non-redundant (nr) database (GenBank). Thesematching ESTs could be grouped into fourGene Ontology categories: transcriptional reg-ulators, cell signaling, structural genes, andmetabolic enzymes. Interestingly, the two tran-scriptional regulators overexpressed in work-ers are both putatively involved in repressionand silencing of gene expression, and the struc-tural genes encode muscle-specific proteins,indicating structural differences in queen andworker flight muscle. The metabolic enzymeoverexpressed in workers represents a memberof the cytochrome-P450 family, and thus,reflects a general pattern apparent in castedevelopment of social bees, namely, the prom-inent role of energy metabolism in the castesyndrome (Corona et al., 1999; Guidugli et al.,2004; Pereboom et al., 2005).

The second strategy towards identifyingcaste-specific gene expression in M. quadrifas-ciata was a high throughput approach that hasseveral advantages over DDRT-PCR. The Rep-resentational Difference Analysis (RDA) is aversion of a suppression subtractive hybridiza-tion strategy, and it was used to generate twolibraries enriched for queen and for workertranscripts, respectively. This approach gener-ated 1278 high quality ESTs, representing 337unique sequences (Judice et al., 2006). As in theDDRT-PCR study on caste-specific gene expres-sion in Melipona, this approach also revealed amuch broader functional spectrum of transcriptsin the worker library (22 unique sequences withexclusive occurrence in workers, versus 5exclusive to queens). At first view this wouldindicate that workers are transcriptionally morecomplex than queens, but this must be takenwith a caveat, as it may actually reflect an anno-tation problem. We recently performed a re-annotation of honey bee ESTs that were iso-lated in differential gene expression screens forcastes (Evans and Wheeler, 1999, 2000;Corona et al., 1999; Hepperle and Hartfelder,2001). Prior to the release of the honey beegenome sequence, many of these ESTs werelisted as “no matches” that fell through the anal-ysis because they mapped to little conserved 3'-UTR regions. In this re-annotation it nowbecame apparent that queens are transcription-ally quite complex and, even more importantly,


seem to express considerably more “novel”genes than workers, that is, genes which do nothave functionally annotated homologs in anyother genome.

To validate the RDA results for Meliponaqueens and workers, we tested the differentialexpression of 5 worker unique sequences byquantitative (realtime) RT-PCR. In this quan-titative expression analysis we also includedmRNA samples of newly emerged queens pro-duced by application of the JH analogpyriproxifen (PPN) to last instar larvae. Con-trary to expectation, the hormone inducedqueens did not show the queen-typical expres-sion pattern for these genes (Judice et al.,2006). In four of these, the JH-induced queenseven outmatched the workers (Fig. 3). Thisquite astonishing result apparently indicates aseparation between hormonal effects on adultphenotype and corresponding gene expressionpattern. It also represents a potentially interest-ing facet of the genetic predisposition to casteproposed for the genus Melipona. It may actu-ally explain why attempts to test the geneticcaste determination hypothesis with JH-induced queens have failed, because morpho-logical phenotype and gene expression patternsapparently do not match up in JH-inducedqueens.

A second source of hormonal regulation,acting possibly in addition to the JH and ecdys-teroid cascades, also became apparent throughthe differential gene expression analyses. Thisstrategy led to the cloning of a 320 bp fragmentwith significant blastx similarity (6e–12) to theDrosophila gene lilliputian (Judice, unpub-lished). In Drosophila, lilliputian acts not onlyas a regulator of cell size, where it integrateswith PTEN in the insulin signaling pathway(Wittwer et al., 2001), but it is also involved inaxis formation through the decapentaplegic(Dpp) pathway (Su et al., 2001). The detectionof a putative lilliputian homolog in caste devel-opment of a stingless bee may, thus, representa glimpse at a gene expression network thatintegrates the growth-regulating insulin signal-ing pathway with the morphogenetic regula-tors, juvenile hormone and ecdysteroids.

The entire list of differentially expressedgenes in Melipona queens and workers has nowbeen made available and searchable at http://www.lge.ibi.unicamp.br/abelha, after user reg-istration. This site also provides information onelectronic Northerns that represent transcriptabundance in the respective subtractive librar-ies. Current efforts are made to generate micro-array chips for M. quadrifasciata genes as atool for high-throughput gene expression anal-ysis in this stingless bee.

Figure 3. Comparative gene expression (realtime RT-PCR) in newly emerged queens and workers, and inqueens induced by application of the juvenile hormone analog pyriproxifen (PPN). The studied genes wereobtained from an RDA library where they were initially characterized as overexpressed in workers (Judiceet al., 2006). Queens induced by PPN application in the last larval instar showed an expression pattern thatwas clearly distinct from that observed in naturally reared queens. Either they showed an intermediate expres-sion level between natural queens and workers (DAG gene), or they displayed an expression level that evenexceeded that of workers. Means and standard errors of relative expression differences are shown. Statisticaldifferences for means are indicated by different letters over bars (modified after Judice et al., 2006).


3. PHYSIOLOGY OF REPRODUCTION AND DIVISION OF LABOR

Similar efforts in functional genomics per-formed on the honey bee are already dramati-cally changing how we view the castesyndrome and division of labor in eusocial bees(Evans and Wheeler, 2001; Kucharski andMaleszka, 2002; Cash et al., 2005). Yet, this bigpicture is mainly derived and based on studiesand experiments performed to a great extent onthe honey bee only. There is very little informa-tion available on the genomic and physiologicalregulatory architecture underlying reproduc-tion and division of labor in the stingless bees.Yet, even though still preliminary in manyaspects, results obtained for a select set of sting-less bees already indicate that surprises areawaiting with respect to the endocrine regula-tion of fertility and division of labor.

One of our research projects investigates theJH and ecdysteroid titer profiles in M. quadri-fasciata queens and workers. With respect toecdysteroid titers, both castes showed a minorpeak during the first days after emergence, andsubsequently their hemolymph titers remainedat basal levels throughout the adult life cycle(Hartfelder et al., 2002). This finding comparedfavorably well with that obtained for honeybees (Robinson et al., 1991; Hartfelder et al.,2002), and it sets the highly eusocial honeybees and stingless bees clearly apart from theprimitively eusocial bumble bees. In the latter,ovarian ecdysteroid levels and the ecdysteroidtiter correlate with reproductive activity andsocial dominance (Bloch et al., 2002; Gevaet al., 2005).

Only preliminary results are, so far, avail-able on the JH titer in adult stingless bees. SuchRIA measurements are currently being per-formed on M. quadrifasciata queens and work-ers. For queens, the only results available arefor newly emerged gynes, revealing very hightiters (around 300 pg JH-III equivalents per µLhemolymph) at this early age. For workers, JHtiters were measured in a pilot study where theywere kept for up to 30 days in Petri dishes insmall social units. Under these conditions, theJH titer of workers started out at around 25 pg(JH-III equivalents per µL hemolymph) atemergence. It gradually increased and reacheda maximum of around 75 pg between days 7and 8 and then rapidly declined again to basal

levels, fluctuating around 25 pg until the end ofthe observation period. Provided that this resultcan be confirmed by RIA analysis of JH titers inworkers kept under normal conditions and per-forming activities corresponding to their respec-tive age (Waldschmidt and Campos, 1997),stingless bees would differ quite drasticallyfrom honey bees in the physiological parametersunderlying division of labor in workers and alsowith respect to the association of JH and repro-ductive activity in queens. If confirmed in a moreextensive study, these findings clearly will havean impact on how we perceive the role of JH inreproduction and behavioral development ineusocial Hymenoptera (Robinson and Vargo,1997; Sullivan et al., 2000; Bloch et al., 2002).

Even though the older Melipona workerswere clearly different from honey bee workerswith respect to their hormone titers, the twospecies showed traces of similarity in the youngbees. Like in the honey bee, where a small JHpeak has been detected in 2–4 day-old honeybee workers (Elekonich et al., 2003), M. quad-rifasciata workers also showed a peak in theirJH titers in the first days after emergence. Sucha minor peak at an early adult age was alsoapparent for the ecdysteroid titer of M. quadri-fasciata workers and for honey bee workers(Hartfelder et al., 2002). The physiological sig-nificance of these early JH and ecdysteroidpeaks in young workers of social bees is still amystery, but it coincides with a transition froma still immature behavioral state to that of aworker bee participating in activities relevantto colony performance. This early adult hormo-nal signature may also be relevant to programsinvolved in determining adult life span, asrecently shown for winter bees infested withVarroa mites. These bees had an altered ecdys-teroid profile in the first days after emergenceand showed immunological characteristics relatedto reduced life span (Amdam et al., 2004).

The main parameter of female fertility ininsects, the vitellogenin titer in hemolymph hasbeen profoundly investigated in the honey bee,but only preliminarily so in stingless bees. Thestudy by Engels and Engels (1977) on S. pos-tica and M. quadrifasciata showed that queens,as expected, had a much higher vitellogenintiter than workers. Less clear and evident was,however, the relationship between the vitello-genin titer and the program of division of laborin adult workers in these stingless bees.


In honey bees, the vitellogenin titer in workersincreases when they are approximately 7 daysold, and it reaches a maximum while they areperforming nursing activities (Hartfelder andEngels, 1998). At the transition from nursing toforaging behavior, the vitellogenin titer declinesand stays at low levels for the remainder of theadult life cycle. The vitellogenin titer of honeybee workers, thus, shows exactly the oppositeprofile to the JH titer (Robinson et al., 1991).In accordance with this, experimental evidencehad already previously suggested an inhibitoryrole of JH on vitellogenin expression (Rutz et al.,1976) and its relation to the behavioral transitionfrom nursing to foraging behavior (Jaycox, 1976).It is only in the late pupal stages that JH actuallyacts as an activator of vitellogenin synthesis inhoney bees queens and workers (Barchuk et al.,2002), just as it does in the adult life cycle of manyother insects (Raikhel et al., 2005). This dynamicbehavior of the vitellogenin titer has been con-firmed not only at the level of protein titers inhemolymph, but also by the temporal expres-sion pattern of the recently sequenced honeybee vitellogenin gene (Piulachs et al., 2003).

The honey bee vitellogenin sequence hasnow been used to clone and partially sequencethe vitellogenin gene of M. quadrifasciata(Andrade and Simões, unpublished). Due to itshigh conservation, the honey bee vitellogeninprimers could also be employed to study thetemporal expression profiles in a set of sting-less bee species that show interesting differ-ences with respect to worker reproduction.These first results already show a completelydifferent picture from that observed in thehoney bee. First of all, vitellogenin geneexpression could be detected by semiquantita-tive RT-PCR throughout the entire pupal stagein all three stingless bee species (Fig. 4), quitedifferent from the honey bee, where vitello-genin expression appears to be strongly sup-pressed by the high ecdysteroid titer (Guidugliet al., 2005). This already could reflect a majorchange in the regulatory architecture underlyingvitellogenin gene expression. Second, vitello-genin transcript and protein levels are ratherdisparate in stingless bees, indicating additionalcontrol at the translational level or in the secretionprocess. Third, the three stingless bee speciesdiffer considerably in how they modulate vitel-logenin gene expression and its correspondinghemolymph protein level.

Figure 4. Developmental patterns of vitellogeningene expression (determined by RT-PCR) andvitellogenin titer (determined by Western blot ana-lysis) in hemolymph of workers representing threestingless bee genera. (A) In Melipona scutellaris,the level of vitellogenin transcription graduallyincreases during pupal development, as the ecdys-teroid titer diminishes. Transcription reaches a peakin nurse stage workers, when vitellogenin becomesclearly detectable in hemolymph. (B) In Frieseo-melitta varia, vitellogenin gene expression appearsto be constitutive in pupae and in adult workers, andalso, the vitellogenin titer shows no age-relatedmodulation. Together with the fact that in F. variaworkers the ovaries degenerate completely duringpupal development, this finding strongly suggestsalternative functions for this protein. (C) In Scapo-totrigona postica, vitellogenin gene expression alsoappears to be constitutive during pupal develop-ment and in the adult stages, but the presence ofvitellogenin protein in the hemolymph could onlybe evidenced in the nurse bee stage. Pw, Pb, Pbl,Pbm, Pbd are pupal stages characterized by progres-sive eye and body pigmentation. Re, N, F are newlyemerged workers, nurses, and foragers, respecti-vely. The pupal ecdysteroid titer profile is a com-posite representation for M. quadrifasciata (Pinto et al.,2002), and S. postica (Hartfelder and Rembold,1991).


In the genus Melipona, nurse-stage workersproduce not only trophic eggs, but also repro-ductive eggs and, thus, they contribute signifi-cantly to male production (Tab. I). Accordingly,vitellogenin protein is detected in hemolymphprimarily during the nursing stage (Fig. 4A). Inaddition, vitellogenin transcription in the pupalstage most closely approximates to the patternshown in honey bees. In Frieseomelitta variaworkers, the ovary anlagen degenerate com-pletely during metamorphosis (Boleli et al.,1999), thus disabling adult workers from pro-ducing any type of egg. In this case, vitello-genin appears to be expressed constitutively(Fig. 4B). This is quite a surprising finding inview of the fact that these workers invest heav-ily in producing a protein for which they appar-ently have no use. This puzzle may be resolvedonce we consider that vitellogenin may actuallybe involved in processes other than reproduc-tion. Such additional roles have recently beendemonstrated for honey bee vitellogenin whichwas found to be associated with immune sys-tem integrity and, consequently, longevity ofworker bees (Amdam et al., 2004). Such find-ings can provide a lead as to where to look forvitellogenin functions, even in the sterile Frie-seomelitta workers. Stingless bee workersappear to live about 20 days longer than honeybees (Bego, 1982).

Scaptotrigona postica workers present anintermediate picture when compared to the pre-vious two stingless bees. Vitellogenin transcriptlevels are not (negatively) correlated withecdysteroids during the pupal stage, but theprotein makes its appearance in hemolymphprimarily during the nurse bee stage (Fig. 4C).These results confirm a previous study thatshowed a strong correlation between vitello-genin titer and ovary size in relation to workerage (Engels and Engels, 1977).

To make sense of these disparate patterns ofvitellogenin expression in workers of highlyeusocial bees we will have to consider them inrelation to life tables and task performance. Atleast in the honey bee, vitellogenin expression,together with the hemolymph JH titer, consti-tutes the primary regulatory machinery forbehavioral development (Amdam and Omholt,2002). Studies on behavioral development,unfortunately, may still be called the blind spotin stingless bee biology. Much ethologicalwork has been dedicated to the complex behav-ioral interactions between the queen and workersin the POP context (Sakagami, 1982; Zucchiet al., 1999) in normal colonies, and in a par-ticularly fine example, also in artificial mixed-species colonies (Silva, 1977). Yet, the “whodoes what and when” in the colony has littlebeen explored. Fortunately, two of the focal

Table I. Patterns of worker reproduction under queenright conditions. The listing of stingless bee specieswithin each reproductive type is far from complete and is only intended to provide examples; for furtherreferences see Sakagami (1982), Engels and Imperatriz-Fonseca (1990) and Toth et al. (2004).

Type of worker reproduction Species References

Ovaries active in presence of queen, workers produce reproductive and trophic eggs

Melipona subnitidaMelipona favosa

Paratrigona subnuda Scaptotrigona postica

Koedam et al., 1999Chinh et al., 2003Toth et al., 2002

Sakagami and Zucchi, 1963; Beig, 1972; Paxton et al., 2003

Ovaries active in presence of queen, but workers produce primarily or only trophic eggs

Tetragonisca angustulaTrigona carbonaria

Geotrigona mombuca

Grosso et al., 2000Green and Oldroyd, 2002Silva-Matos et al., 2000

Ovaries mostly inactive, no trophic eggs but occasional laying of reproductive eggs

Friesella schrottkyi Imperatriz-Fonseca and Kleinert, 1998

Ovaries inactive in the presence of the queen

Leurotrigona muelleri Sakagami, 1982

Ovaries degenerate during pupal development

Frieseomelitta varia Boleli et al., 1999


species of this review, S. postica and M. quad-rifasciata, have been reasonably well studied interms of worker life tables (Bego, 1982;Waldschmidt and Campos, 1997). These stud-ies have revealed that stingless bees are muchmore plastic in their age-related tables of taskperformance, reflecting either a higher level ofidiosyncrasy (workers remain on the respectivetask longer) or a higher degree of flexibility inswitching tasks, possibly as a consequence ofsmaller colony size and the necessity for “fill-ing in when there is need”. Colony size variesconsiderably within the stingless bees (Nogueira-Neto, 1997), from honey bee-sized colonies inTrigona and Scaptotrigona species to colonysizes comprising only a few hundred workers(Plebeia, Melipona, and many other genera),so, their worker life tables and associated taskperformance may actually look quite different.A closer look at vitellogenin and JH titers inworkers of known age and task may, in thefuture, provide insight into the regulatoryarchitecture underlying division of labor instingless bee workers. The above mentionedresults on vitellogenin expression patterns instingless bees only opened the trail and cer-tainly leave this question far from beinganswered, because, in this study we controlledonly for task (nurse of forager) but not forworker age. In fact, future studies may profitconsiderably when comparing stingless beesnot only to honey bees, but also to bumble bees.Similarities between stingless bees and bumblebees emerge not only in the reproductive biol-ogy of workers (egg-laying in the presence ofthe queen) but also in an apparently more flex-ible and JH-independent system of division oflabor (van Doorn, 1987; Cameron and Robinson,1990; O’Donnell et al., 2000).

4. DEVELOPMENT OF THE WORKER REPRODUCTIVE SYSTEM AND WORKER REPRODUCTION IN STINGLESS BEES – “EVERYTHING GOES”?

The results presented above on vitellogeninprotein titers in hemolymph and vitellogenintranscript abundance in fat body of stinglessbee workers are difficult to reconcile in astraightforward manner with the predictionsfrom kin selection theory on worker reproduc-

tion. With few exceptions, stingless bee coloniesare headed by a single queen that has matedwith a single male (Paxton et al., 1999; Peterset al., 1999), and this sociogenetic constellationwould favor male production by workers (Crozierand Pamilo, 1996). Quite amazingly though,there is hardly anything more variable than thepattern of reproductive activity in workers ofstingless bees, which ranges from a strongworker contribution to male production to com-plete worker sterility (Silva-Matos et al., 2000).A main complicating factor in our comprehen-sion of worker fertility in stingless bees is theproduction of trophic eggs that workers ofmany species produce in addition to or insteadof reproductive eggs. These trophic eggs arelaid during the POP process and are generallyconsumed by the queen (Akahira et al., 1970),thus, helping her to maintain her own optimumreproductive activity (for review see, Sakagami,1982; Engels and Imperatriz-Fonseca, 1990).Whereas oophagy is a common feature in socialinsects, used by dominant females to monopo-lize colony reproduction, stingless bees haveclearly gone one step further, so as to make theproduction of trophic eggs a constitutive fea-ture of colony integration in many genera. Thisevolutionary trend is evident in the fact thattrophic eggs differ from reproductive eggs inmany aspects, they are not only bigger and havea modified chorion structure, they also appearto be cytochemically immature and possiblyhave even lost the oocyte nucleus (Akahiraet al., 1970; Cruz-Landim and Cruz-Höfling,1971; Koedam et al., 1996).

The reproductive potential and activity ofworkers in the normal colony context, i.e. in thepresence of the queen, can roughly be dividedinto five classes: (I) workers are totally incapa-ble of producing eggs of any type, (II) workersproduce eggs but do not lay them, (III) workersproduce/lay primarily trophic eggs, (IV) work-ers produce/lay primarily reproductive eggs,and (V) workers produce and lay both types ofeggs. The first class is best represented by thegenus Frieseomelitta, where ovaries have beenshown to start developing normally during thelarval stages, but then they degenerate com-pletely during pupal development (Boleli et al.,1999, 2000). As a consequence, no germ cellsare left and, in fact, the adult gonads show thecytological characteristics of a storage organ.Examples for the other classes are shown in


Table I. Obviously, these different modes ofegg production by workers have consequenceson individual and colony fitness, since it is onlythe workers of categories IV and V that can andwill contribute significantly to the productionof males (Beig, 1972; Contel and Kerr, 1976;Toth et al., 2004; Velthuis et al., 2005).

In physiological and cell biological terms,the way that trophic eggs are produced is stilla little understood phenomenon. Their role,however, is fairly clear, namely to provide thequeen with a protein- and lipid-rich diet so thatshe can sustain the high levels of vitellogeninsynthesis in her fat body. In this respect, thestingless bees differ from the honey bees whichfeed the queen trophallactically with glandularsecretions. Yet, the detection of vitellogeninreceptor-like proteins on the hypopharyngealgland of honey bee workers suggests that thisprotein may actually be taken up from thehemolymph and converted by the gland intoroyal jelly proteins (Amdam et al., 2003). In thehoney bee, the vitellogenin transfer from nurse-stage workers to the queen would, thus, passthrough the hypopharyngeal gland secretions,while in the stingless bees it is passed ondirectly through the trophic eggs. This may bepartly a consequence of, or at least coherentwith the restriction imposed on reproductiveactivity of workers in honey bees, where thepolyandrous mating system apparently hasresulted in a suppression of ovarian activitythrough queen pheromones on the one hand andworker policing on the other.

This does not mean, of course that trophiceggs are the exclusive source of nutrition forqueens of stingless bees, especially since thereare species were egg production in workers israre to absent. Queens do not only feed ontrophic eggs but also have frequently beenobserved to insert their heads into newly pro-visioned brood cells and, thus, may consumesome of the larval food before laying an egg(Sakagami, 1982).

5. CONCLUDING REMARKS AND PERSPECTIVES

Their variability in natural history and thephylogenetic proximity to the honey bee, themodel organism for polyphenism and division

of labor in the social Hymenoptera, makesstingless bees excellent candidates for compar-ative studies. In addition, the genus Meliponapresents a long-time enigma for its mechanismof caste determination that is now being deci-phered with respect to underlying genetic andphysiological mechanisms. The comparativeapproaches in hormone and gene expressionanalyses also gradually reveal where and towhat extent the regulatory machinery of insectdevelopment is conserved in the eusocial bees,and at which point development can be suffi-ciently plastic to drive the differentiation ofpolyphenic traits.

A particularly interesting facet is the emer-gent parallelism between caste determinationand sex determination, both of which exhibittransitions from environmental to geneticmechanisms of determination. Whilst the tran-sition from environmental (ESD) to genetic sexdetermination (GSD) is an ancient and recur-rent phenomenon in different lines of meta-zoans (Bull, 1981; Sarre et al., 2004; Yao andCapel, 2005), the transition from environmen-tal to genetic caste determination (ECD toGCD) is, obviously, a much more recent eventassociated with socioevolution in insects, andthere are only few examples in bees (the genusMelipona) and in ants (Winter and Buschinger,1986; Fraser et al., 2000; Julian et al., 2002;Volny and Gordon, 2002; Helms Cahan andKeller, 2003). In this sense, the ECD-GCDtransition in social insect castes, as well as othertransitions from environmental to geneticdetermination of seasonal polyphenisms, forinstance in butterfly color patterns (for reviewsee, Hartfelder and Emlen, 2005) can serve asscenarios that illustrate how environmental sig-nals and genetic factors become interchange-able in terms of their impact on developmentalpathways (West-Eberhard, 2003). This inter-changeability depends on ganged switch mech-anisms triggered by environmental or geneticfactors. In caste development, such switchesare manifest in the caste-specific hormone titersthat drive character expression in morphoge-netic fields.

An intriguing consequence of the apparentECD-GCD transition in stingless bees is thehigh percentage of gynes produced in Meliponacolonies under favorable colony conditions,and the way workers eventually adjust queenproduction to the colony reproductive cycle.


Gynes are kept in reserve for some days andmost are then eventually killed (Engels andImperatriz-Fonseca, 1990). This is a ‘tragedyof the commons’ (Wenseleers and Ratnieks,2004) in a double sense, not only for each indi-vidual superfluous gyne, but also in terms ofcolony investment.

Another interesting facet in the castes ofstingless bees is the fact that stingless bee work-ers are morphologically more similar to malesthan to queens (Kerr, 1974, 1987, 1990a), quiteopposite to what is observed in honey bees.And this male/worker similarity in stinglessbees is not only one of phenotype, but there arealso reports on similarities in behavior. Malesof stingless bees have been reported to sporadi-cally perform activities related to colony main-tenance, such as nectar dehydration, waxmanipulation and others (Nogueira-Neto, 1997;Van Veen et al., 1997; Velthuis et al., 2005). Ifverified consistently, this not only has implica-tions on sociobiological interpretations of castesystems in Hymenoptera (Kerr, 1990b), but,more directly, it also raises the question on howtask performance is physiologically regulatedand integrated in stingless bees, and whetherthey are distinct from honey bees in thisrespect.

The physiological background to age-relatedbehavioral development in bees involves char-acteristic changes in the juvenile hormone titerand in the expression of the yolk protein pre-cursor vitellogenin. Even though still prelimi-nary, the emergent results on juvenile hormonetiters and vitellogenin expression in adult workersin stingless bees indicate that the highly euso-cial bees differ significantly in these primecomponents of the reproductive groundplan offemale insects. In honey bees, the vitellogeninand JH titer have been mathematically modeledas a double repressor constellation for divisionof labor (Amdam and Omholt, 2003) and thisconstellation has recently been experimentallyconfirmed through a vitellogenin gene silenc-ing approach by RNA interference (Guidugliet al., 2005). In contrast, stingless bee workersappear to constitutively transcribe the vitello-genin gene, to maintain a low JH titer throughoutadult life (see preliminary results presented above),and to show a much more flexible age-relatedpattern of task performance (Waldschmidt andCampos, 1997). The only clear similarity wecould note in hormone titers in adult workers

of meliponine and apine bees is the minor peakdetectable in very young workers, which can beinterpreted as a prerequisite to adult maturation.The currently emerging results on regulatorymechanisms underlying caste development andreproductive physiology in stingless bees, thus,clearly illustrate the potential of this large andvariable group of bees for comparative studies.And it is through such comparative studies thatwe can expect to gain insight into evolutionarytrends in developmental and reproductive plas-ticity in social insects.

ACKNOWLEDGEMENTS

We thank Paulo Nogueira Neto for kindly provi-ding some of the Melipona quadrifasciata coloniesused in our experiments cited above, and we ack-nowledge financial support by Fundação de Ampa-rao a Pesquisa do Estado de São Paulo (FAPESP),Conselho Nacional de Desenvolvimento Científicoe Tecnológico (CNPq), Coordenação de Aperfei-çoamento de Pessoal de Nível Superior (CAPES),and the Deutsche Akademische Austauschdienst(DAAD).

Résumé – Mécanismes physiologiques et généti-ques sous-tendant le développement des castes, lareproduction et la division du travail chez lesabeilles sans aiguillon. Le nombre d’espèces et lavariabilité des caractères écologiques et éthologi-ques rendent les abeilles sans aiguillon remarquableset en font un groupe intéressant pour rechercher descorrélations entre les caractères du cycle évolutifd’une part et les mécanismes physiologiques etgénétiques qui sous-tendent le développement descastes, la reproduction et la division du travaild’autre part. Dans cette synthèse nous mettons encontraste les nombreuses observations sur lesabeilles sans aiguillon avec celles de leur groupesœur, les abeilles domestiques.Toutes les abeilles sans aiguillon approvisionnent enmasse les cellules de leur couvain avant que la reinene ponde un œuf et ne l’opercule. Il y a néanmoinsdes différences remarquables dans la structure du nidà couvain et dans la manière dont les reines, lesouvrières et les mâles sont élevés (Fig. 1). Dans legroupe des trigones, les reines sont produites dans degrandes cellules à couvain ou dans des cellules cons-truites les unes sur les autres, ce qui permet aux larvesd’ingérer beaucoup plus de nourriture. Dans le genreMelipona au contraire, toutes les cellules de couvainsont de même taille et un nombre relativement élevéde reines émerge de ces cellules dans des conditionsoptimales de la colonie. Cette observation a conduità émettre l’hypothèse que la détermination des cas-tes chez Melipona a peut-être une base génétique.Bien qu’elle ait été proposée il y a plus de 50 ans,


cette hypothèse n’a jamais été validée de façon con-cluante. Selon les recherches en cours, les marqueursgénétiques générés par un protocole AFLP se sépa-rent en fonction du phénotype de la caste (Fig. 2).D’autres preuves proviennent d’une analyse del’expression génique différentielle sur des reinesrécemment écloses et des ouvrières de Meliponaquadrifasciata. Cette étude a été produite sur 1200ESTs (étiquettes de séquences exprimées) qui ont étécomparées aux profils d’expression génique spéci-fiques des castes chez Apis mellifera, soulignantl’importance de la régulation métabolique dans ledéveloppement des castes. Nous avons testé les pro-fils d’expression de cinq des gènes représentés parles ESTs dans une comparaison entre reines naturel-les et ouvrières et reines induites par un traitementhormonal des larves au stade de filage du cocon ;nous avons constaté que les adultes issus des larvestraitées par hormones sur-exprimaient la plupart deces gènes marqueurs, même s’ils présentaient le phé-notype de reine (Fig. 3). Ceci indique que le phéno-type morphologique et le profil d’expressiongénique ne concordent pas nécessairement.Il existe aussi chez les abeilles sans aiguillon uneforte variation en ce qui concerne la reproduction desouvrières, certaines espèces présentant une contri-bution significative des ouvrières à la production desmâles. Chez de nombreuses espèces, les ouvrièresproduisent des œufs trophiques qui sont consomméspar la reine peu de temps avant qu’elle ne ponde dansune cellule fraîchement approvisionnée. Chez cer-taines espèces, pourtant, la reproduction des ouvriè-res est complètement bloquée par la dégénérescencedes ovaires au cours du développement nymphal.Ces différences dans les stratégies de reproductiondes ouvrières se reflètent dans les différences del’expression génique de la vitellogénine et dans lesteneurs de l’hémolymphe en vitellogénine. (Fig. 4).Le résultat le plus frappant se rencontre chez Frie-seomelitta, où les ouvrières présentent une expres-sion constitutive de la vitellogénine même si ellessont complètement stériles, indiquant par là desfonctions alternatives pour ce précurseur de la pro-téine du vitellus. Des résultats préliminaires surl’hormone juvénile et les teneurs en ecdystéroïdechez les ouvrières adultes de M. quadrifasciata mon-trent aussi des différences considérables avec leparadigme du développement comportemental del’abeille domestique. Les résultats qui se dégagentactuellement concernant les mécanismes de régula-tion qui sous-tendent le développement des castes etla physiologie de la reproduction chez les abeillessans aiguillon illustrent clairement le potentiel de cegroupe d’abeilles, vaste et varié, pour des étudescomparatives.

abeille sans aiguillon / développement des castes /hormone juvénile / vitellogénine / reproductiondes ouvrières / Meliponini

Zusammenfassung – Physiologische und geneti-sche Mechanismen in der Kastenentwicklung,Reproduktion und Arbeitsteilung bei stachello-sen Bienen. Stachellose Bienen sind bemerkenswerthinsichtlich ihrer Artenvielfalt und ihrer Variabilitätin ökologischen und Verhaltensmerkmalen. Diesmacht sie zu einer interessanten Gruppe für Unter-suchungen zu korrelativen Verbindungen zwischenLife History Merkmalen auf der einen Seite und phy-siologischen und genetischen Mechanismen derKastenentwicklung, differentiellen Reproduktionund Arbeitsteilung auf der anderen. In diesem Über-sichtsartikel werden wir viele Beobachtungen an sta-chellosen Bienen mit denen an Honigbienengegenüberstellen. Alle stachellosen Bienen massenverproviantierenihre Brutzellen kurz bevor die Königin ein Ei ablegtund die Brutzelle danach verschlossen wird undbleibt. Trotz dieses gemeinsamen Merkmals zeigensie jedoch beachtliche Variabilität hinsichtlich derNeststruktur und in der Art und Weise wie Königinnen,Arbeiterinnen und Männchen aufgezogen werden(Abb. 1). Die gemeinhin unter dem Begriff Trigoninenzusammengefassten Genera produzieren Königinnenin vergrösserten Brutzellen oder in Brutzellen, dieeng übereinandergebaut wurden. Im Gegensatz hierzusind die Brutzellen des Genus Melipona alle vongleicher Grösse und es schlüpfen eine vergleichsweisegrosse Anzahl von Jungköniginnen aus deren Brut-waben. Diese Beobachtung hat zu der HypotheseAnlass gegeben, dass die Kastendetermination beiMelipona eine genetische Komponente beinhaltenkönnte. Obwohl diese Hypothese nun schon vor über50 Jahren formuliert wurde, konnte sie nie schlüssigbelegt werden. Laufende Studien basierend aufeinem AFLP-Protokoll lieferten nun erstmals gene-tische Marker, die mit den Kastenphänotypen segre-gieren (Abb. 2). Weitere Evidenzen für einegenetische Grundlage der Kastendeterminierungkamen nun auch aus Analysen zur differentiellenGenexpression an frischgeschlüpften Königinnenund Arbeiterinnen von Melipona quadrifasciata.Diese Studie produzierte über 1200 ESTs, die mitkastenspezifischen Genexpressionsmustern bei Apismellifera verglichen werden konnten. Dabei wurdedie Bedeutung der metabolischen Regulation in derKastenbildung klar herausgestellt. Als wir dann dieExpressionsmuster von fünf der durch ESTs reprä-sentierten Gene von Melipona Arbeiterinnen undKöniginnen auch an Königinnen testeten, die durchJuvenilhormon-Applikation in der Spinnma-denphase produziert worden waren, zeigten diemeisten dieser Gene ein Expressionsmuster, dasweit mehr mit dem von Arbeiterinnen übereinstim-mte als mit dem von natürlichen Königinnen(Abb. 3). Dies weist darauf hin, dass der morpholo-gische Phänotyp nicht notwendigerweise kongruentist mit den Genexpressionsmustern. Auch hinsichtlich der Arbeiterinnenreproduktionzeigen stachellose Biene eine ausgeprägte Variabi-lität, und bei manchen Arten produzieren Arbeite-rinnen einen Grossteil der Männchen. Bei vielen


Arten legen Arbeiterinnen sogenannte Nähreier, dievon der Königin kurz vor der eigenen Eiablage ineiner frischverpoviantierten Brutzelle verzehrt wer-den. Bei anderen Arten hingegen, ist die Arbeiterin-nenreproduktion bedingt durch die Degeneration derOvarien schon während der Puppenphase vollstän-dig blockiert. Diese Unterschiede in den Reproduk-tionsstrategien der Arbeiterinnen traten auch in derExpression des Vitellogenin-Gens und der Vitello-genin-Titer bei Arbeiterinnen zutage (Abb. 4). Miteiner der interessantesten Befunde war die konstitu-tive Expression des Vitellogenin-Gens und ein kon-stant hoher Vitellogenin-Titer bei Frieseomelittavaria Arbeiterinnen, obwohl diese vollständig sterilsind. Dies weist auf alternative Funktionen vonVitellogenin in der Biologie dieser Bienen hin. Vor-läufige Ergebnisse zu den Juvenilhormon- undEcdysteroid-Titern bei adulten Arbeiterinnen vonMelipona quadrifasciata weisen ebenfalls auf erheb-liche Abweichungen von dem für die Honigbieneerstellten Paradigma der Verhaltensentwicklunghin. Diese neuen Ergebnisse zu regulatorischenMechanismen in der Kastenentwicklung und Repro-duktionsphysiologie stachelloser Bienen illustrierendas hohe Potential dieser artentreichen und vielfäl-tigen Gruppe für vergleichende Studien über sozialeBienen.

Stachellose Bienen / Kastenentwicklung / Juvenil-hormon / Vitellogenin / Arbeiterinnenreproduk-tion

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