Division of labor during brood production in stingless bees with … Trigona species. In Melipona species, all bees are born from identical cells, while in most other genera, queen

Apidologie 37 (2006) 175–190 175c© INRA/DIB-AGIB/ EDP Sciences, 2006DOI: 10.1051/apido:2006018

Review article

Division of labor during brood production in stingless beeswith special reference to individual participation

Olga Inés C

Columbia Basin College, Science and Math Department, Pasco, WA 99301, USA

Received 31 October 2005 – revised 26 January 2006 – accepted 15 February 2006

Abstract – The focus of this paper is the process for brood production known as the Provisioning andOviposition Process (POP), and particularly the individual behavior observed in the facultatively polygy-nous stingless bee Melipona bicolor. Following individually marked bees revealed that ovarian developmentis correlated with individual behavior differences. While most of the eggs laid by workers are consumedby the queen (trophic eggs), workers contribute significantly in male production with reproductive eggs,illustrating the reproductive conflict at the individual level. From an evolutionary outlook, “benefactor”behaviors may evolve if workers conserve the “hope” of reproduction. This indicates that an importantfunction of trophic eggs is to keep the ovaries active. It is also possible that ovary development representsan internal factor promoting division of labor: reproductive workers are specialized or elite bees with lowresponse thresholds and high activity levels that restrain the participation of other workers.

stingless bees / division of labor / POP /Melipona bicolor / reproductive competition / Apidae

1. DIVISION OF LABORIN STINGLESS BEES

It has long been acknowledged that divi-sion of labor among hymenopteran workerspresents age-correlated patterns of task per-formance (temporal polyethism) that spatiallyfollow a centrifugal sequence (Wilson, 1985).In stingless bees, very young bees producewax and work in the brood nest where theywere born. They move further away from itas they age until they leave the nest to be-come foragers. Although the age ranges dur-ing which particular tasks are executed varyacross species, bees generally pass throughfour stages: callow, nurse bee, housekeeperand forager. The tasks associated with thesestages are: (1) incubation and repairs of thebrood chamber; (2) construction and provi-sioning of cells, cleaning of the nest, and feed-ing young adults and the queen; (3) furthercleaning of the nest, reconstruction of the in-volucrum, reception and ripening of nectar,

Corresponding author: [email protected]

and guard duty at the entrance of the nest; (4)foraging for pollen, nectar, propolis and othermaterials (Wille, 1983). Meliponine workersexhibit considerable flexibility in task allo-cation: tasks are not rigidly established butdepend on the conditions of the colony. For ex-ample it is possible to force a colony consist-ing only of old workers to rear brood by par-tially re-activating the hypopharyngeal glands(Sakagami, 1982).

2. DIVERSE APPROACHES TO THESTUDY OF DIVISION OF LABOR

Classic works representing purely etho-logical descriptions on division of labor inmeliponines date back to 1955. Since then, amultitude of studies have addressed generalaspects of division of labor, the provisioningand oviposition process (POP), conflict be-tween colony members, evolution of behav-ior, taxonomical comparisons, and systemat-ics versus behavior (Online supplementary list

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176 O.I. Cepeda

of references1). However, few focused on spe-cialization and individual behavior.

Although in many of the studies bees wereindividually marked, results were presentedfor age groups providing a general patternand thus lacking information on individualdifferences in behavior. Individual differenti-ation has been studied in other Hymenoptera,e.g. in ants (Gordon, 1999), wasps (O’Donnelland Jeanne, 1990) and in Apis bees (Visscherand Camazine, 1999). In stingless bees, in-tranidal individual behavior has been studiedin Melipona favosa (Sommeijer et al., 1982;Kolmes and Sommeijer, 1992), M. bicolor(Bego, 1983), Trigona (Tetragonula) minangk-abau (Inoue et al., 1996), and M. subnitida(Koedam et al., 1999). As for extranidal tasks,some works that addressed individual differen-tiation related to food and resin collection hasbeen described in M. beecheii (Biesmeijer andTóth, 1998), and concerning task partitioningin nectar collection, there is information com-piled for five species of stingless bees (Hartand Ratnieks, 2002).

Recent models of colony organization areinterested in how mechanisms at the individ-ual level generate organization and behaviorat the colonial level. It is now recognizedthat the genotype of a colony is distributedamong hundreds or thousand of genetically di-verse individuals. In computer simulations, di-vision of labor emerges by introducing min-imal variances in the responses of individualsto a task; such results may indicate that organi-zation in a colony emerges as a self-organizedsystem derived from the independent actionsof workers (Ronacher and Wehner, 1999). Inorder to clarify the mechanisms that gener-ate colonial organization and behavior, mod-els based on proximal mechanisms that incor-porate both internal and external factors havebeen developed (Beshers and Fewell, 2001).The response threshold model refers to inter-nal individual limits that regulate individualresponses to diverse tasks. Such a model as-sumes that response thresholds are specific foreach task and that each individual can havea different threshold for each task (Besherset al., 1999; Bonabeau and Theraulaz, 1999).

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This concept generates a simple but powerfulfeedback system that distributes the appropri-ate number of workers for each task, in a self-organized manner. For social insects there ex-ist empirical evidences supporting such mod-els (Robinson, 1992; Bonabeau and Theraulaz,1999; Beshers and Fewell, 2001). Thresholdscan be fixed when they do not change with age,may be reinforced by repetition, or may be“forgotten” if not performed (Bonabeau et al.,1996). Literature also presents empirical evi-dence in favor of the existence of reinforce-ment thresholds in social insects (Theraulazet al., 1991; O’Donnell, 1998; Robson andTraniello, 1999). In stingless bees, indirect ev-idence for response thresholds in M. bicolor isfound in the pioneering work of Bego (1983).She perceived that each individual carries outdifferent percentages of the tasks and neverthe total repertoire of the colony. She verifiedthat, on average, an individual carries out 50%to 70% of the total number of tasks. This re-search demonstrates worker heterogeneity instingless bees.

Among a group of workers available forthe execution of a task "x," some individualsmay present a high preference for such taskand dedicate themselves more frequently whencompared with the average task performancefrequency of other workers of the same age.These workers are called specialists or elite(Robson and Traniello, 1999). There is em-pirical evidence in favor of the existence ofspecialization in Hymenoptera (Rissing, 1981;Calderone and Page, 1988; O’Donnell andJeanne, 1990). Kolmes and Sommeijer (1992),working with the stingless bee M. favosa,found that the construction of brood cells isa task performed by an elite group of work-ers. Inoue et al. (1996) presented evidenceon individual variation between workers forthe bee Trigona (Tetragonula) minangkabau.They verified that the population of a nest canbe divided into two main groups: nurse beesthat can spend all their lives taking care of thebrood, and foragers that hardly or never workon the comb. Here we see evidence for strongand very limited response thresholds leadingto extreme specializations.

Finally, individual behavior must be inte-grated with colonial organization. The model

Division of labor in stingless bees 177

of social inhibition explains that temporarypolyethism is the result of the interaction ofan intrinsic process of behavioral developmentwith an inhibiting effect produced by otherworkers (Huang and Robinson, 1999). Thismodel combines the physiological state in re-lation to the task together with the external in-fluence produced by the interactions betweenworkers (Beshers and Fewell, 2001).

3. THE PROVISIONINGAND OVIPOSITION PROCESSIN STINGLESS BEES

Brood production resembles that of soli-tary bees and follows a general pattern in allspecies of stingless bees. Workers constructand mass provision the cells; the queen thenlays her eggs on top of this liquid provision,and, finally, the workers seal the cells. Thissequence of behaviors has been termed theprovisioning and oviposition process (POP).Within each cell, the new individual devel-ops without any further interference from itskin. The first POP in meliponine was observedby Drory in 1872 in M. scutellaris and sincePOP has been recognized as a highly elabo-rate process with complex social interactions.The most outstanding fact commonly observedin this process is the laying of two types ofeggs by the workers: trophic eggs (TEs) thatare consumed by the queen, and reproductiveeggs (REs) that develop into males (Sakagami,1982). During the period when bees are mostengaged in brood care, it is noticeable thattheir wax production is at its peak, and dissec-tion reveals developed ovaries, while the cropcontains large amounts of pollen. Such factorsare not found when bees end their participa-tion in the POP (Sakagami et al., 1963; Bego,1990; Simoes and Bego, 1991).

The POP has been studied in at least 50species of stingless bees. Classic works onPOP observed characteristics such as age ofthe bees involved, number of provisions percell, time interval between ovipositions, dailyoviposition rate, duration of pre-provisioning,provisioning and post-provisioning phases andgeneral behavioral interactions between thequeen and the discharging workers. The pro-cess was divided in phases and definitions

for the diverse repertories were established(Sakagami and Zucchi, 1966). Comparisonsof the POP in different species have beenused to establish relations between systemat-ics and behavior (Sakagami and Zucchi, 1966;Drummond et al., 2000) and phylogenetic re-lationships (Zucchi et al., 1999).

For all meliponine, Sakagami (1982) rec-ognizes four patterns of cell construction andprovisioning:

- Successive: provisioning of cells occur oneby one followed by the oviposition of thequeen as happens in M. bicolor. In thisspecies, cells are at different stages of con-struction at any given time so there are sev-eral provision and oviposition cycles perday.

- Synchronous: several cells receive provi-sions at about the same time. The queencan oviposite all at once, or in batches.

- Semisynchronous: successive cell con-struction is followed by synchronous pro-visioning.

- Composite: in a given comb there isprimarily synchronous provisioning, butsome cells are provisioned in succession.

In meliponine bees, cells can be constructedin clusters or in horizontally or spirally or-ganized combs. Only in Dactylurina are thecombs vertical and double layered, whichis a remarkable parallel to the cell arrange-ment used by Apis. Cells can be spherical- orcolumnar-like; they are made of pure wax orcerumen (Wille, 1983; Nogueira-Neto, 1997).Each cell is built by several young wax-producing workers. Complete cells have a rimof wax, called a collar, which is used for seal-ing it after oviposition. The number of cellsproduced per day varies from about 10 inMelipona colonies up to several hundred inTrigona species. In Melipona species, all beesare born from identical cells, while in mostother genera, queen cells are larger than thosethat produce workers and males (Sakagami,1982). Sommeijer et al. (1982) followed in-dividuals of the stingless bee M. favosa anddemonstrated that groups of workers build lar-val cells and subsequently provision them asteams. However, inactivity is often recordedas one of the most common behavioral states

178 O.I. Cepeda

of workers in the nest. Later this same author(Sommeijer et al., 1984) states that the courtof workers that surround the queen during herresting periods, termed the extra-ovipositionperiods, consists of the same individuals thatwere mostly engaged in cell construction. Theauthors therefore conclude that the function ofsuch extra-oviposition courts is principally toconvey information to the queen and encour-age her to set out towards the completed cellsto lay.

Provisioning and oviposition begin once thequeen appears and positions herself near anempty cell. The queen must be near a cell toencourage regurgitation. A court of bees formsaround the queen and the finished cell(s), andthe workers sequentially insert their head intothe cells and regurgitate food until the cell isabout two thirds full. The first regurgitationmay or may not be stimulated by the queen,depending on the species (Sakagami, 1982). InM. favosa, the workers engaged in provision-ing seek food from different workers appar-ently specialized in carrying provisions fromthe storage pots to bees participating in thePOP (Sommeijer et al., 1985).

Once the appropriate food level is reached,workers may lay TEs. The place where theTE is laid varies among species: it could beplaced on top of the larval food (as in M. bi-color), at the rim of the cell collar, or just ontop of the comb. In Cephalotrigona, TE-layerspresent their egg directly to the queen by flip-ping their abdomen, while in Lestrimelitta,the worker eats her own egg in the pres-ence of the queen (Michener, 1974; Sakagami,1982). Trigonisca, Duckeola, Frieseomelittaand Tetragonula are the only meliponine thatdo not produce trophic eggs (Sakagami andZucchi, 1968, 1974; Terada, 1970; Sakagamiand Inoue, 1990). The behavior of these in-fertile workers during the POP is consideredsimple and is characterized by the absenceof the typical excitement and the elaboratequeen-worker interactions found in all otherspecies where workers do lay eggs (Sakagamiand Zucchi, 1968, 1974; Sakagami and Inoue,1990). The simplicity of the POP in these ster-ile bees suggests that the ritualized interactionsbetween workers and queens are associatedwith the conflict over the production of males;

thus, ritualization partially resolves the con-flicts and produces cooperation (Crespi, 1992).TEs are considered specialized eggs for queenconsumption (Sakagami, 1982; Koedam et al.,2001; Velthuis et al., 2003a) because of theirhigh protein content (Velthuis et al., 2001,2003b). However, it could very well be thatTEs serve another very important function, aswill be discussed.

To finalize the POP, a worker seals the cellby folding the rim collar inward. This is doneusing their mandibles and working around thecell or most commonly by inserting the tip ofthe abdomen and folding the collar in a rotat-ing motion (as in M. bicolor). The seal is com-pleted with final mandibular work (Sakagami,1982). Occasionally, a sealing worker maystop rotating and lay a reproductive egg (RE);it could also happen that, after a cell is sealed,a “sneak” worker reopens the cell and lays.REs are very different from TEs because REsdevelop into males. The reproductive workermay or may not consume the egg laid by thequeen; so, in some cases, there can be two eggsper cell (Beig, 1972; Contel and Kerr, 1976;Machado et al., 1984; Bego, 1990; Koedamet al., 1999; Sommeijer et al., 1999; Koedamet al., 2001). Beig (1972) states that, in Scap-totrigona, worker REs develop faster so thatthese larvae hatch and consume the fertilizedeggs laid by the queen; however, in M. bi-color, 50% of cells that contain eggs fromboth queen and reproductive worker producea m6027SP female, demonstrating that thereis also the possibility for competition amongthe larvae (Koedam et al., unpublished data).The production of eggs in queenright condi-tions represents one great distinction betweenmost meliponine and Apis bees: in stinglessbees, many workers present ovarian develop-ment (Sakagami et al., 1963), providing evi-dence for reproductive competition. In severalmeliponine species, workers contribute sub-stantially in the production of males (Beig,1972; Contel and Kerr, 1976; Koedam et al.,1999; Sommeijer et al., 1999; Tóth et al.,2002). Sneak RE-laying workers present ex-tremely different behaviors when compared toTE-laying workers and to non-laying workers.During the POP, workers laying REs act ag-itated and when they succeed in laying, they


engage in an extremely prolonged sealing ac-tivity. This behavior has been considered to bea mechanism to avoid the consumption of theireggs by nest-mates (Koedam et al., 2001).

There are two hypotheses about the natureof TEs. Koedam et al. (2001) protected TEsfrom being consumed by the queen; they re-port that some TEs in M. bicolor can produceweak larvae when compared to other larvae ofthe same age. Such information indicates thatTEs could be degenerate reproductive eggs. Ina recent article, Velthuis et al. (2003b) assertthat, due to their high content in proteins, TEsare produced specifically to feed the queen.However, some of the proteins found in TEscould actually be enzymes in the process ofdenaturing the egg. Besides the lack of a typ-ically patterned chorion, TEs are also biggerand rounder than REs (Koedam et al., 1996;Koedam et al., 2001); workers may perceivethis morphological difference in size, or maytime the egg from the moment it is ready tobe laid and thus, modify their laying behavior.The nature of TEs and REs would be an in-teresting factor to clarify. If TEs are proved tobe degenerating REs, such information wouldgive more support to the hypothesis on the hi-erarchies of participation that workers presentin the POP where reproductive workers areclosest to the ancestral type while the non-laying or sterile workers are the modified type.The present study lends support to the ideathat the order of involvement in POP should begreatest for the reproductive workers that con-tribute more to POP than for TE-laying work-ers and least for bees that never lay any type ofegg.

4. MELIPONA BICOLOR:A FACULTATIVE POLYGYNOUSBEE

In stingless bees, several species have beenreported to have provisional polygyny whenthere is queen replacement (Silva et al., 1972);but this is a small and specific time-periodwithin the colony cycle far from real polyg-yny. In Melipona, a nest of M. subnitida mayhave 5 queens, and M. marginata presentedtwo queens in laboratory conditions (Bego,

1989; Imperatriz-Fonseca, unpublished data).In contrast, only M. bicolor presents a trulyfacultative polygyny (Kerr, 1949; NogueiraNeto, 1997; Velthuis et al., 2001) and has beenobject of intense investigation (Velthuis, 2006,this issue).

In polygynous M. bicolor colonies, con-flicts potentially arise among queens overwhether and how to divide reproduction, sincecell provisioning occurs cell-by-cell. Indeedconsiderable reproductive skew has been ob-served among queens (Velthuis et al., 2001;Velthuis, 2006, this issue). However, encoun-ters among queens at a given cell are placidand interactions are ritualized (Bego, 1989).At the end of this paper, I will describe andcomment on an interesting behavioral differ-ence that may arise among individual queensin polygynous conditions.

5. VARIOUS IMPLICATIONSOF OVARIAN DEVELOPMENTAND ITS RELATION TO WORKERACTIVITY LEVELS: A CASESTUDY IN MELIPONA BICOLOR

In M. bicolor, individual weight and extentof ovarian development was correlated withthe levels of activity presented by individualworkers during POP. These results that willbe further presented, may indicate that ovar-ian development is necessary for workers toeffectively assist in brood production, explain-ing the so called idiosyncratic inclination to at-tend the brood (Oster and Wilson, 1978). Be-havioral differences in M. bicolor divide work-ers into three groups: non-layers, layers of TEsand layers of REs. Egg-layers, regardless ofthe type of egg, participate most in POPs bothin terms of presence (constancy) and their sig-nificant contributions during each POP (as-siduity). The correlations may be indicatingthat ovarian development plays an importantrole in task partition in the colony by influenc-ing the degree of involvement of each worker.

To obtain insight into individual variationin activity during POP, the behavior of in-dividual bees was tracked (495 continuousfilming hours per colony) in one polygynous(Col1) and one monogynous (Col2) colony of

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Figure 1. Coefficient of variation for theovarian area and behavioral assiduity be-tween age groups in M. bicolor. (Noovarian measurements were made forcolony 1. Lack of CV means there wasdata of only one or no specimens).

M. bicolor. Research methods include stan-dard procedures for colony maintenance andworker marking (Sakagami, 1966; Koedamet al., 1999; for details see Cepeda-Aponte,2003). The occurrence of the behaviors ofbody insertion (partial or total), larval fooddischarge, and egg laying (trophic or reproduc-tive) were noted for each worker of known ageparticipating in a POP. For each individual Icalculated:

Assiduity: total frequency of each basicbehavior monitored;Constancy: total number of POP in whichan individual participated.

At the end of the monitoring period, allmarked workers in Col2 were sacrificed, in-dividually weighted, and dissected. Ovarieswere fixed and photographed; ovarian areawas measured (for details see Cepeda-Aponte,2003).

In Figure 1 the coefficient of variation cal-culated for each age group revealed that in-dividual workers of the same age group werevery different morphologically (ovary size) aswell as behaviorally. The coefficient of varia-tion of the ovary area was found to have a widerange of values, varying from 0.98 to 65.4. Thecoefficient of variation for the total participa-tion or assiduity per age group also presentedgreat variations that fluctuated between 29.2and 172.6. Spearman correlation of these co-efficients presented no significance when thedata was treated as a whole. Correlations ap-peared only once the sample was separated

into types of workers: non egg-layers versusegg-layers.

5.1. Behavioral differences between egglayers and non egg layers

Only Col1 presented eleven reproductiveindividuals that laid from one to three REs.Figures 2 and 3 depict their participation (as-siduity and constancy respectively) in all be-haviors, demonstrating their extraordinary per-formance: reproductive workers were involved7 times more often in POPs (Fig. 3) than TE-layers, and were extremely active (Fig. 2).When the performance of the TE-layers wasanalyzed in Col1 and Col2, TE-layers of Col2were significantly more active than TE-layersof Col1 that presented RE-layers. Behavioraldata of the non-laying bees presented no statis-tical differences between the colonies. All TE-layers differed significantly from non-layers.(Kruskall-Wallis and U test post hoc P <0.0125). TE-layers presented a two-fold higherPOP activity than did non-laying bees (Figs. 2,3). So, it appears that the hierarchy of presenceand participation in POP is RE-layers > TE-layers > non layers.

Another way to perceive the great contribu-tion of egg layers in the POP is by calculat-ing an average participation per bee as shownin Table I. In Col1 the extraordinary effortsof only eleven reproductive workers (3% ofa total of 353 bees) are evident as they par-ticipated in almost 1/4 of the total number of


Figure 2. Comparison of behavioral as-siduity for the three types of workers par-ticipating in POP in M. bicolor. (Signif-icant difference (>0.0125) between andamong all groups).

Figure 3. Comparison of behavioral con-stancy of the three types of workers par-ticipating in POP in M. bicolor. (Signif-icant difference (>0.0125) between andamong all groups).

Table I. Average participation in POP per individual in M. bicolor.

Colony 1workers n = 353

Total % Colony 2Workers n = 214

Total %

Total number of events in480 consecutive hours

13 689 15 380

Non egg layers Each of 217 workers par-ticipated in average in 24events

38 Each of 154 workers par-ticipated in average in 50events

50

Trophic egg layers Each of 125 workers par-ticipated in average in 44events

40 Each of 60 workers par-ticipated in average in128 events

50

Reproductive egg layers Each of 11 workers par-ticipated in average in 271events

22 No reproductive workers

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recorded events. In this same Col1, TE-layers,represented about 1/3 of the workers (n = 125)and participated in 40% of the total events. InCol2, where there were no reproductive work-ers, a small number of TE-layers (n = 60)worked 2.5 times more than non-laying work-ers (n = 154) and participated in 50% of theevents. As was mentioned earlier, there was asignificant difference in activity between theTE-layers of both colonies, and such a differ-ence is also perceptible here. From this analy-sis it may be inferred that, even though therewere only eleven reproductive workers, theirhigh levels of performance competitively re-duced the participation of other workers ofCol1 (social inhibition model).

Workers in both colonies presented an evi-dent variability in individual behavior withineach age group. By separating workers intogroups characterized by the type of egg laidor its lack, significant differences appeared be-tween these three types of workers for all vari-ables measured. These analyses illustrate thegreat “interest” laying workers appear to havein the POP. Sommeijer (1984) reports that inM. favosa, laying workers are not the princi-pal provisioners of the cell they lay in. Thisdoes not necessarily mean that these egg lay-ers do not participate in POPs: they may havebeen active provisioners of other cells. That is,laying bees are more involved in POP and par-ticipate frequently, but they do not necessarilysecure or monopolize a specific cell.

5.2. Morphological differences betweenegg layers and non egg layers

According to the present results, layingworkers are behaviorally very different fromnon-laying ones because laying workers (bothTE- or RE-layers) are more constant andmore assiduous in the POP. Now let us com-pare these behavioral characteristics againstmorphological factors (ovarian area and bodyweight) and maximum age reached. Weightwas considered to be an indirect indicator ofnutritional influence. It was found that beeshave a tendency to loose weight as they age.There is great variability of weight within eachage group but two clusters may be seen: a first

Figure 4. Weight changes per age group of workersparticipating in POP in M. bicolor.

heavier group of young bees up to 14 days old,and a second, lighter group (Fig. 4).

When analyzing the TE-layers, there wasno correlation between ovarian area and be-havioral variables (Tab. II). This result was dueto age differences within the complete groupof egg layers: when sacrificed, bees were be-tween 13 and 25 days old, which means that, inyoung bees, ovarian development should havebeen at its peak while ovaries had degeneratedin older bees. However, in the same group theheaviest laying workers (n = 48) were moreconstant and assiduous in the POP, inspectingthe cells, and laying their eggs; these same be-haviors were found to be correlated with age inTE-layers. As for the non-egg-laying group, itcould contain bees that were either too youngor about to lay, plus old bees that effectivelyhad never laid. So, they were divided and an-alyzed in two subgroups: bees between 4 and12 days of age, the prime time and maximumage for egg production (Cepeda-Aponte, un-published data), and older bees between 13and 23 day old. For the young non-layers, itwas seen that, as they age, grow heavier andtheir ovaries mature, their apparent involve-ment in the POP increases, and they becomemore engaged in inspecting cells and discharg-ing larval food. However, since correlation co-efficients were never higher than 0.49, an in-tricate feedback relation between nutritionalinput, ovarian development, hormonal levels,social interactions and even learning capac-ity might exist, besides the effect introducedby those young bees that would never lay.For the old non-layers, there was a negative


Table II. Significant correlation coefficients (Spearman) between physical and behavioral variables in M.bicolor.TE = trophic eggs

correlation between age and ovarian area, asanticipated. However, the expected negativecorrelations of the level of POP participationwith ovarian area or weight were not found,and instead, behavior was positively correlatedwith age. These last results demonstrate thedifficulty for the analysis due to the variabilitywithin each group plus the already mentionedinterweaving of internal and external factors.Larger sample size taken from more colonieswould be necessary to test the validity of thesepreliminary but interesting observations.

5.3. Egg laying: proximate and ultimatemechanisms

Egg layers in M. bicolor are so involved inthe POP that they could be called elite, excep-tional or specialized individuals (Robson andTraniello, 1999; Velthuis, unpublished data).In this study, egg layers represented between28% and 39% of the total marked work-ers. This seems to indicate that differencesin ovarian development might influence re-sponse thresholds leading to the diverse levels

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of participation in the POP. Individuals drawnto the POP could improve the execution of atask through repetition or auto-reinforcement.When non-layers with high response thresh-olds, and therefore low competitiveness, ap-proach a POP, the high competitiveness ofegg-layers would drive them away and thelack of “practice” would make them “forget”such tasks and cause them to forage for workelsewhere. The behavior observed in M. bi-color nicely fits models on response thresh-old, auto-reinforcement and social inhibition(Beshers and Fewell, 2001). It will be neces-sary to pay more attention to egg-layers sincethey may represent key individuals in the POPand, hence, in colony growth (Robson andTraniello, 1999).

Egg-laying has been related to division oflabor. In several social hymenopteran species,behavioral changes of aging workers mirrorwith changes in the development of theirovaries: young individuals that work in thenest present developed ovaries, while olderones forage and present degenerate ovaries(Wilson, 1985). From an ultimate perspective,it is possible that the generation of ovaries ofdiverse sizes made division of labor possible,so, less fertile females gave up reproduction tohelp probably related and more fertile females(Bourke, 1988). Another interpretation couldbe that laying workers would prefer to remainin the nest, close to their reproductive interests,while not exposing themselves to predators(Franks and Scovell, 1983). A modified typeis presented by Inoue et al. (1996) who foundthat, in Trigona (Tetragonula) minangkabau,the population of a nest could be divided intoworkers dedicating all their lives to care for thebrood, and foragers that hardly remained withthe brood and yet, all these worker bees are to-tally sterile.

It is also feasible to discuss possible ul-timate mechanisms. Why have workers re-tained ovarian development? What is seen inM. bicolor is females trying to reproduce:in these eusocial bees the ancient conflictover individual reproduction appears to per-sist. Even in Apis bees, where queen and larvaestrongly inhibit ovarian development in work-ers, some may escape control and lay eggs(Bourke, 1988; Ratnieks and Visscher, 1989).

And when honeybee workers of the same agewere isolated, they presented individual differ-ences in ovarian development (Velthuis, 1970).Furthermore, in genera of stingless bees thatpresent totally sterile workers, their behaviorduring POP lacks the typical excited traits ob-served in species where workers do produceeggs (Sakagami and Zucchi, 1968, 1974). Linand Michener (1972) argue that a benefactorbehavior may develop even where no altru-ism is involved, provided there is a signifi-cant contribution to male production by work-ers. Whenever a female conserves the “hope”of reproduction, her participation in colonytasks may be considered as an investment inher future reproduction. In many meliponinespecies exists a percentage of workers that layreproductive eggs and contribute significantlyto male production. Furthermore, the sameindividual worker may produce both trophicand reproductive eggs (Koedam et al., 1999;Cepeda-Aponte, unpublished data). This evi-dence may indicate that there is selection tomaintain the production of TEs in an indi-vidual that can produce both types of eggs(Kukuk, 1992); it could also indicate that sonsof reproductive workers have a high reproduc-tive success. TEs may have the function of de-ceiving the queen, but West-Eberhard (1981)proposes a more interesting hypothesis. Thisauthor states that the function of TEs is tomaintain ovary activity. This would indicatethat their potential role as the principal sourceof nutrients for the queen (Sakagami, 1982)may be a byproduct for the cooperative inter-action (and competition) between worker andqueen.

6. THE BEHAVIORAL “DILEMMAS”OF A QUEEN

While the focus of my research was onworker behavior in M. bicolor, the time exten-sive observation also yielded interesting, novelinformation on queen behavior as well. Bystudying and comparing POPs in the polyg-ynous and in the monogynous colony, it waspossible to perceive the great pressure thatmultiple queens place on each other. Nutri-tion is a determinant factor in queen behavior


Figure 5. Comparing averageand confidence intervals on theduration of POPs dependingon number of queens, for themonogynous (Col1) and polyg-ynous (Col2) colonies of M. bi-color. Data grouped by ranksbased on the presence of TEs inprevious POP.

and her performance as an egg-layer (Wheeler,1996; Velthuis et al., 2001; Velthuis, 2006,this issue). Queens have three ways of feed-ing: by trophallaxis with workers; or duringPOP by ingesting larval food directly fromthe provisioned cell, and/or consuming trophiceggs (Sakagami, 1982). TEs are laid during thepost-provisioning phase: the full cell seems toserve as an incentive for the workers to lay,but, at the same time, the close presence of thequeen restrains the worker from doing so. Aspreviously stated, in the POP, the ovipositionsof both queen and workers involve stereotypedrituals in their behavior. Sakagami et al. (1973)explain that at the core of this process lies theconflict of the worker that fears the queen butfeels compelled to defend the finished cell tolay her own egg. In polygynous conditions,queens are constantly inspecting the cell and,with their proximity to the cell, intimidate theworkers. Workers consequently should reducethe number of TEs they lay (Velthuis et al.,2001; Velthuis, 2006, this issue).

In this case study, a total of 393 continu-ous POPs for the monogynous colony and atotal of 508 continuous POPs for the polygy-nous colony were filmed for the same amountof time. Information for this latter colony wasdivided into period I and period II; the firstwith 241 POPs with three queens laying, whilethe second period presented 267 POPs, buthad only two laying queens. In the polygy-nous colony, the three queens were similar invarious aspects. Egg laying and consumptionof trophic eggs presented no statistical differ-

ences or worker discrimination, confirming aprevious report (Velthuis et al., 2001), so eachqueen had an equal chance to lay her eggs andfeed. In addition, the queens were of similarage: when observations began, queen A was176 days old, queen B was 165 days whilequeen C was 222 days. These differences werenot considered significant since queens in lab-oratory conditions have been reported to liveup to two years (Imperatriz-Fonseca, unpub-lished data). Since M. bicolor is a species thatpresents no direct aggression (Bego, 1989), thefollowing results may reveal yet another wayin which competition is manifested in thesepolygynous bees.

In both monogynous and polygynouscolonies, I ranked each POP depending on howmany preceding POP lacked TE presentation:that is, if the preceding POP presented TE,the rank of the following POP was “0”, rank“1” if only the anterior POP lacked TE, etc.An additive effect was considered when therewas more than 1 TE per cell. In the monogy-nous colony, the duration of the POP was re-lated to the amount and frequency of TEs pre-viously ingested: the queen was apparently ca-pable of modulating the duration of each POPby postponing her oviposition and waiting fora worker to lay a TE. As seen in Figure 5, theaverage duration of a POP increased as morePOPs without TE accumulated resulting in ex-tremely long POPs lasting more than 30 min;however most POPs (n = 242) lasted on aver-age 15 min. In the polygynous colony, whenthe colony had only two queens, a subtle but

186 O.I. Cepeda

similar pattern was still perceivable but thischanged when the colony had three queens:there was no such pattern and instead mostPOPs were rather short (n = 102, mean =12 min), the longest POP lasting not more than16 min. These POPs of short duration couldreflect the urge each queen had in laying herown egg in what seems to be a competitive sit-uation, where queens prefer to lay rather thanto wait and feed on TEs. From Figure 5 it isalso clear that in the monogynous colony therewere hardly more than three successive POPswithout TE (n = 10 of 393 POP), while in thepolygynous colony, up to 11 consecutive POPscan occur without release of TEs. Other stud-ies in polygynous colonies with few queens,revealed that each queen had a higher proba-bility of presiding alone over a POP (Velthuiset al., 2001), than in polygynous colonies withnumerous queens, where the probability of anencounter with other queens at the same cellincreased. This factor therefore interferes withthe possibility of modulating the duration ofthe POP and of waiting to receive a TE. In con-sequence the lack of such nutrition may lead tocessation of egg-laying for some queens and tothe development of a reproductive skew.

7. CONCLUSION

In M. bicolor, the best-fed, heavy workerskeep their ovaries activated probably in “hopeof reproduction”, and they participate effec-tively in as many POPs as possible. This ob-servation places trophic eggs into a new per-spective: is it a specialized nutritional packagefor the queen (Velthuis et al., 2003b), or is it abyproduct of worker ovarian activity, that is, isit an over-mature reproductive egg?

As for the queens, we see that when she isalone, a queen can modulate the duration ofthe post-provisioning phase in order to receivetrophic eggs. In a polygynous condition, thepriority of the queens is to place their eggs asfast as possible, and this could result in a dis-advantageous nutritional condition that couldexplain the establishment of reproductive hier-archies or skews (Velthuis et al., 2001) and tothe abandonment of reproductive functions.

ACKNOWLEDGEMENTS

The investigation on individual behavior of M.bicolor was part of the Project “Organização colo-nial e padrões de reprodução em abelhas indigenas”(Colony organization and reproductive patterns innative stingless bees) performed at the Bee Lab inthe University of Sao Paulo, S.P. and was sponsoredby FAPESP (process No. 99/00180-0), Brazil. Theauthor thanks institutions, colleagues and personnelinvolved in its accomplishment, and very especiallyDr. Vera Lucia Imperatriz Fonseca and Dr. JacobusBiesmeijer.

Résumé – Division du travail pendant la produc-tion de couvain chez les abeilles sans aiguillon,en particulier du point de vue de la participa-tion individuelle. La division du travail est unemarque de socialité. Des études récentes ont ré-vélé que des individus, qui avaient été considéréscomme des entités désintéressées, faisaient preuvede plasticité. Le but de cet article est de fournir uneinformation sur les approches adoptées pour étu-dier le comportement des insectes sociaux, en sefocalisant principalement sur les modèles récentsqui concernent plus le comportement individuel etmontrent comment celui-ci s’intègre dans la divi-sion du travail au sein de la colonie. L’article pré-sente un cas d’étude réalisé sur l’abeille sans ai-guillon Melipona bicolor qui, comme la plupartdes abeilles sans aiguillon, possède des ouvrièrescapables de pondre deux types d’œufs. Certains,les œufs reproducteurs, se développent en mâles etcontribuent à la production des mâles de la colonie.D’autres, les œufs trophiques, constituent la princi-pale source de nourriture des reines. Des ouvrièresont été marquées et filmées individuellement durant20 j consécutifs, de façon à suivre leur comporte-ment dans le processus de production du couvainappelé Processus d’Approvisionnement et de Ponte(POP). A la fin de la période d’observation, les indi-vidus ont été pesés et la surface de leurs ovaires me-surée. Le poids constituait une mesure indirecte dela consommation de nourriture. Les ouvrières d’unmême groupe d’âge ont présenté une grande va-riation dans leurs caractéristiques morphologiqueset comportementales (Fig. 1). L’analyse a en outremontré que les ouvrières qui pondaient des œufsétaient plus impliquées dans le processus de pro-duction de couvain que celles qui n’avaient ponduaucun type d’œuf (Figs. 2, 3 ; Tab. I). Les ouvrièresavaient aussi tendance à perdre du poids avec l’âge(Fig. 4), mais les ouvrières les plus lourdes qui pon-daient des œufs participaient plus au POP (Tab. II).Le groupe des non-pondeuses comprend les jeunesabeilles et il n’a pas été possible de dire si celles-ci auraient pu devenir ou non des pondeuses. On apourtant noté que la participation des jeunes abeillesau POP augmentait parallèlement à leur poids et au


développement de leurs ovaires (Tab. II). On en adéduit que les abeilles les mieux nourries et pos-sédant des ovaires actifs pouvaient représenter lacondition ancestrale de la compétition pour la re-production entre les femelles et que les œufs ponduspar ces ouvrières auraient la fonction principale demaintenir leurs ovaires actifs afin de contribuer aupool génétique par la production de mâles. M. bico-lor a aussi la particularité d’être la seule abeille eu-sociale pour laquelle la polygynie facultative a étéprouvée. En comparant la durée du POP dans unecolonie mono-reine et une colonie à trois reines, ona remarqué qu’une reine toute seule pouvait prolon-ger la durée du POP jusqu’à ce qu’elle obtienne desœufs trophiques pour s’en nourrir. Par contre dansune colonie à plusieurs reines, la compétition pourla ponte est représentée par des POPs plus courts aucours desquels les reines « préfèrent » pondre plu-tôt que se nourrir (Fig. 5). Ainsi, dans une colonie àplusieurs reines, la priorité des reines est de placerleurs œufs le plus vite possible ; cela peut débou-cher sur une mauvaise nutrition qui pourrait expli-quer la mise en place de hiérarchies de reproductionet l’abandon des fonctions de reproduction par desreines individuelles.

abeille sans aiguillon / division du travail / POP /compétition pour la reproduction / Melipona bi-color / Apidae

Zusammenfassung – Arbeitsteilung in der Brut-aufzucht bei Stachellosen Bienen, mit besonde-rer Berücksichtigung der individuellen Betei-ligung. Ziel dieser Arbeit ist es, über Untersu-chungsansätze zum Studium von Verhalten bei so-zialen Insekten zu informieren, wobei besondersneuere Modelle in Betracht gezogen werden, diesich auf das individuelle Verhalten und dessen In-tegration in die Arbeitsteilung innerhalb der Kolo-nie beziehen. In dieser Fallstudie an der Stachel-losen Biene Melipona bicolor geht es um die Ei-ablage von Arbeiterinnen, die wie bei den meistenStachellosen Biene zwei Typen von Eiern produ-zieren können. Zum einen sind dies reproduktiveEier, aus denen Männchen entstehen können unddie damit einen Anteil an der Männchenprodukti-on der Kolonie darstellen. Zum anderen produzie-ren sie Nähreier, die die Hauptnahrungsquelle fürdie Königin darstellen.Arbeiterinnen bekannten Alters wurden individu-ell markiert, und über einen Zeitraum von 20 Ta-gen wurde ihr Verhalten im Brutproduktionspro-zess kontinuierlich gefilmt. Dieser Prozess wirdals Verproviantierungs- und Eiablageprozess (Pro-visioning and Oviposition Process, POP) bezeich-net. Am Ende der Beobachtungsperiode wurdendie Bienen gewogen und eine Flächenmessung derOvarien vorgenommen. Das Gewicht stellt hier-bei ein indirektes Mass für die Nahrungsaufnahmedar. Arbeiterinnen der gleichen Altersklasse zeig-

ten grosse Variabilität hinsichtlich morphologischerMerkmale und im Verhalten (Abb. 1). Die weitereAnalyse zeigte, dass Arbeiterinnen, die Eier legten,sich stärker im Brutproduktionsprozess engagiertenals Arbeiterinnen, die niemals Eier legten (Abb. 2,3; Tab. I). Auch bei einem tendenziellen Gewichts-verlust mit zunehmendem Alter (Abb. 4) waren esstets die schwereren Arbeiterinnen, die Eier legtenund in den POP eingebunden waren (Tab. II). Inner-halb der nichtlegenden Arbeiterinnen befand sichauch eine Gruppe junger Arbeiterinnen, für die esnicht möglich war zu entscheiden, ob sie später Ei-er legen würden oder nicht. Jedoch auch bei diesenjungen Arbeiterinnen waren es die schwereren, diegrössere Ovarien hatten und in stärkerem Mass amPOP teilnahmen (Tab. II).Daraus kann geschlossen werden, dass die am be-sten gefütterten Bienen mit den aktiven Ovariendie anzestralen Bedingungen der Reproduktions-konkurrenz zwischen Weibchen repräsentieren unddass die Produktion von Nähreiern bei diesen Ar-beiterinnen vor allem dazu dienen könnte, die Ova-rien in einem aktiven Zustand zu halten, um so auchgegebenenfalls Männchen zu produzieren und da-mit zum Genpool beitragen zu können.M. bicolor ist zudem von speziellem Interesse, weiles die einzige eusoziale Bienenart ist, für die einefakultative Polygynie nachgewiesen ist. Beim Ver-gleich der POP-Dauer in einer Kolonie mit nur einerKönign mit einer Kolonie mit drei Königinnen zeig-te sich, dass eine Königin, solange sie alleine ist, diePOP-Dauer hinauszögern kann, bis sie ein Nähreierhält. In einer Kolonie mit mehreren Königinnenwaren die POPs wesentlich kürzer, was auf eineKonkurrenz zur Eiablage hindeutet, d.h. die Köni-ginnen ziehen es vor, zu legen statt auf ein Nähreizu warten (Abb. 5). Diese verstärkte Legetendenzin Kolonien mit mehreren Königinnen könnte beidiesen zu einer unvorteilhaften Ernährungssituationführen und damit die Etablierung von Reproduk-tionshierarchien fördern, bzw. bei einzelnen Köni-ginnen ganz zur Aufgabe der reproduktiven Funk-tionen führen.

Stachellose Bienen / Arbeitsteilung / POP /Meli-pona bicolor / Verhalten

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Division of labor during brood production in stingless bees with … Trigona species. In Melipona species, all bees are born from identical cells, while in most other genera, queen