Emergency queens in Tetragonula carbonaria (Smith, 1854) (Hymenoptera:Apidae: Meliponini)

Túlio M Nunes,1* Tim A Heard,2 Giorgio C Venturieri2,3 and Benjamin P Oldroyd1

1Behaviour and Genetics of Social Insects Laboratory, School of Biological Sciences A12, University of Sydney,Sydney, NSW 2006, Australia.2CSIRO Ecosystem Sciences, EcoSciences Precinct, Dutton Park, Brisbane, Qld 4001, Australia.3Empresa Brasileira de Pesquisa Agropecuária – Embrapa Amazônia Oriental, 66095-100 Belém, Pará, Brazil.

Abstract There is increasing interest in the management of stingless bees for crop pollination, honey production andrecreational beekeeping. Colony propagation is based on the division of one colony into two smaller colonies.The process generates a queenless colony and requires that the new colony is successful in the production ofa new queen. Three different mechanisms by which female larvae develop as queens have been described forstingless bees: (1) genetic determinism with no differentiation in the cells used for rearing queens and workers;(2) queen development is determined by large queen cells; and (3) queen cells are formed by the fusion of twoworker-sized brood cells. Colonies of species that utilise specially constructed queen cells cannot be dividedartificially unless there are queen cells present at the time of splitting. Here, we show that queenless coloniesof the Australian stingless bee Tetragonula carbonaria (Smith, 1854) can construct queen cells by the fusionof two worker-sized cells. This phenomenon is unknown from any other stingless bee species with a spiralbrood comb. It has the practical benefit that beekeepers can divide hives even when there are no queen cellspresent. Finally, it is shown that T. carbonaria workers remain completely sterile in queenless colonies.

Key words queenless, sterile worker, stingless bee.

INTRODUCTION

The keeping of stingless bees (meliponiculture) is a rapidlyexpanding activity in tropical and subtropical parts of theworld. Stingless bees have cultural significance (Eardley &Kwapong 2013) and are increasingly important for use in croppollination, honey production and recreational beekeeping(Heard 1999; Heard & Dollin 2000; Cortopassi-Laurino et al.2006). In Australia, the number of people involved in stinglessbeekeeping has increased 12% annually over the last decade(Halcroft et al. 2013). Most propagation techniques depend onthe artificial division of a single colony into two daughtercolonies. Most stingless bee species are monogynous and soon division of a colony, one of the daughter colonies will bequeenless. Nonetheless, most species have a constant supplyof immature virgin queens that are reared as an insuranceagainst the possibility that the reigning queen is lost(Sakagami 1982). Thus, it is thought that the most commonmechanism by which queenlessness is corrected in the dividedcolony is for one of the immature gynes to mature, take amating flight and assume the egg-laying role (Imperatriz-Fonseca & Zucchi 1995).

In honey bees (Apis spp.), workers produce queens byfeeding young female larvae with an excess of royal jelly (deWilde & Beetsma 1982). In addition, workers radically recon-

struct the brood cell of a queen-destined larva, increasing itssize and altering its shape so that it becomes a queen cell. Inhoney bees, the brood cell remains open throughout the larvalphase. Thus, honey bee workers can convert a worker larvainto a queen larva up until the larvae are about 3 days old(Hatch et al. 1999). In contrast, stingless bees produce theirbrood via a mass provisioning process and enclose the broodcell immediately after the egg is laid (Michener 1974). Thus,in most stingless bee species, the development of the larva ispredestined soon after the egg is laid.

In stingless bees, three different methods of queen produc-tion are known (Hartfelder et al. 2006). In the genus Melipona,queens are reared in brood cells of similar in size and shape tothose used for rearing workers. In this genus, the developmen-tal trajectory of a larva into a worker or a queen is determinedboth by genetic propensity and by the quality of the larval food(Jarau et al. 2010; Schwander et al. 2010; Fig. 1a). This modeof queen determination results in an excess of queens in thenest (e.g. 25% of all female larvae in Melipona marginataLepeletier, 1836, Kerr 1950). Many new queens are killedby workers as they emerge (Koedam et al. 1995), but themajority leave the nest, mate and attempt to infiltrate a foreignnest, becoming the reproductive queen of the host colony(Sommeijer et al. 2003; Wenseleers et al. 2011). In othergenera and more commonly, queens are reared in speciallyconstructed queen cells that are larger than worker cells andcontain up to eight times the volume of brood food (Darchen &Delage-Darchen 1971; Menezes et al. 2013; Fig. 1b). The*tulionunes@usp.br

bs_bs_banner

Austral Entomology (2014) ••, ••–••

© 2014 Australian Entomological Society doi:10.1111/aen.12104

extra food triggers the development of the larva as a queen.Some species rear large queens in queen cells and miniaturequeens in worker-sized cells. The presence of both types ofqueen rearing suggests an intermediate stage between the twomodes of queen production (Ribeiro et al. 2006). A thirdmethod of queen rearing is achieved by the fusion of broodcells. In some species with clustered brood comb such as theFrieseomelitta genus and Leurotrigona muelleri Friese 1900, alarva invades a neighbouring cell and feeds on its food con-tents (Fig. 1c). The overfed worker larva develops into a newqueen (Terada 1974; Faustino et al. 2002). In species thatproduce queens by the fusion of brood cells, the absence of thequeen leads to the production of auxiliary cells by workers.These cells are provisioned exclusively with food (i.e. no eggis laid in them) and are built on the top of an existing cellcontaining a female larva. The larva tunnels into the accessoryfood cell, resulting in the production of an emergency queen(Faustino et al. 2002; Fig. 1d).

The aim of this study is to investigate the mechanism ofqueen production in queenless Tetragonula carbonaria colo-nies. T. carbonaria is the most commonly kept species inAustralia, and their colonies are frequently divided by bee-keepers to increase the number of colonies (Halcroft et al.2013). The mechanism by which T. carbonaria rears a newqueen when the old queen is lost is unknown. We further assessthe degree of ovary activation of workers in queenright andqueenless colonies.

MATERIALS AND METHODS

The experiment was conducted using five colonies ofT. carbonaria in Brisbane Australia during November andDecember of 2013. The colonies were housed in woodenboxes (internal dimensions of 23 × 15 × 11 cm, volume 3.8 L)covered with a plastic lid that allowed observation of in-nestbehaviour. In order to observe the process of brood-combconstruction, the area of young brood (i.e. the advancing front)was positioned at the top of the brood in every nest (Brito et al.2012). The queen was removed from each colony at the begin-ning of the experiment and the behaviour of the coloniesobserved during the following 4 weeks.

At the third week of the experiment, all the queen cells werecollected and dissected under 10× magnification. The newcomb produced by the workers was also sampled in order todetermine if the queenless workers had laid eggs.

New virgin queens emerged in all the colonies during thefirst 2 weeks of observations. These queens were assumed tobe present in queen cells prior to our removing the motherqueens. To maintain the queenless condition of the colonies,these virgin queens were removed. After 4 weeks ofqueenlessness, 10 workers were collected from the broodcomb together with 10 workers from each of five controlqueenright colonies. We dissected the workers under 20× mag-nification and scored the ovaries for their degree of develop-ment, size and the presence of oocytes (Gloag et al. 2007;Cruz-Landim 2009).

In order to determine the potential for producing queensartificially, we constructed five microcolonies using 100-mmdiameter Petri dishes (Fig. 2). Each microcolony received40–60 newly emerged workers, a small amount of maturebrood (approximately 2 cm2) containing 90–110 brood cellsand a small piece of a freshly constructed brood comb con-taining from 20 to 40 cells. The top of the Petri dishes weredrilled with 6.5-mm diameter holes in order to accommodate

Fig. 1. Methods of queen production in stingless bees. (a)Genetic determinism. The size of the cell has no influence on castefate. Genetic determinism may be modified by the amount andquality of food in the cell (Jarau et al. 2010; Schwander et al.2010). (b) Caste fate is determined by the size of the brood celland the amount of food it contains. Queens arise as the result offusion of cells in (c) queenright colonies and in (d) queenlesscolonies (d).

Fig. 2. Artificial queen production in Tetragonula carbonariamicro-colonies. (a) Mature brood, (b) young brood, (c) construc-tion of auxiliary cell and (d) feeders.

2 T M Nunes et al.

© 2014 Australian Entomological Society

food supply and allow the maintenance of the microcolony.Sucrose syrup (34%, v/v) was offered ad libitum in 5-mLcentrifuge tubes with an aperture on the bottom to allow theworkers to access the syrup. Pollen from the original colonywas mixed with sucrose syrup (34%) and offered in the caps ofsimilar tubes. The microcolonies were kept in complete dark-ness in incubators with constant humidity at 80% and tempera-ture at 30°C. The microcolonies were inspected every 3 daysfor a period of 30 days.

RESULTS

Workers of T. carbonaria continue to build brood cells underqueenless conditions. The shape and position of the new cellsdiffers from those found in queenright colonies. In queenlesscolonies, the spiral shape of the brood comb (Fig. 3a) is lostand the overall morphology of the brood comb resembles thatfound in the close related species T. hockingsi Cockerell 1929(see figures in Franck et al. 2004; Brito et al. 2012). The cellsbuilt in queenless colonies became irregular in shape and posi-

tion relative to neighbouring cells. One week after the queenremoval, the workers construct a great number of queen-sizedcells that are full of food but lack eggs (Fig. 3b). A weeklysampling showed that on average, 4.6 ± 2.5 (mean ± SD) cellswere constructed per colony per day during the 3 followingweeks. Half of the newly constructed cells were queen-sized(2.4 ± 1.2, n = 33) and the remainder normal cell size(2.2 ± 1.7, n = 37). Both cell sizes are provisioned with foodand capped despite the absence of an egg.

Dissection of the queen cells constructed in the first weeksuggests that T. carbonaria rears emergency queens fromexisting female larvae. All queen cells sampled during thethird week of the experiment were attached to an empty broodcell that did not contain larvae or food (Fig. 4a).

We dissected 20 worker brood cells from each colonyduring the third week of the experiment, and none of themcontained worker-laid eggs (Fig. 5). Dissection of the workersshowed no differences between the degree of ovary activationbetween queenright and queenless colonies. In both cases,workers’ ovaries consisted of a pair of small ovarioles without

(a)(a)(a) (b)

Fig. 3. (a) Brood comb of Tetragonula carbonaria in a queenright colony. Note the spiral shape of the brood comb. (b) Brood combof T. carbonaria 1 week after queen removal showing royal-sized cells (‘a’) constructed by workers, provisioned with food and cappedwithout egg laying.

(a) (b)

Fig. 4. (a) Mature brood comb of Tetragonula carbonaria from a queenless colony showing an emergency queen cell attached to anempty auxiliary cell on the top. (b) Queen larva of T. carbonaria adjacent to an empty brood cell.

Emergency queens in Tetragonula carbonaria 3

© 2014 Australian Entomological Society

oocytes (10 workers per colony, 5 queenless colonies and 5queenright colonies).

Queen-sized brood cells were constructed in allmicrocolonies. In three microcolonies, one queen cell wasobserved, and two were recorded in the remaining twomicrocolonies. Queen cell construction commenced 3–6 daysfollowing the establishment of the microcolonies and were builtadjacent to the young brood provided. After 30 days, a queenpupa was found in each queen cell constructed by themicrocolonies.

DISCUSSION

T. carbonaria builds emergency queen cells when queenlessand workers never lay eggs, even after a period of protractedqueenlessness. Emergency queen rearing is previously unre-ported in the Tetragonula. The discovery of emergency queensin T. carbonaria means that after the artificial reproduction ofcolonies through nest division, workers can correct thequeenless condition of the nest through the production ofqueen cells from ordinary worker cells that contain eggs oryoung larvae. Thus, we have shown that common beekeeperpractice of confirming that queen cells are present in thecolony to be split is not required.

The microcolony technique described here successfully pro-duced queen pupae. Potentially, this technique could be scaledup so beekeepers could sell queens or attempt geneticimprovement as is practised with honey bees. At a smallerscale, the technique would allow the increase of nest numbersnot just by production of two small colonies from one largecolony, but several mini-colonies from a single hive. Furtheranalysis should address the minimum number of workers andbrood material necessary to produce a queen, and whetherthese queens can be mated or introduced to other colonies.

A few species of neotropical stingless bees also producequeens by the use of auxiliary cells (Frieseomelitta variaLepeletier 1836: Faustino et al. 2002; Plebeia lucii Moure

2004: Teixeira 2007; Leurotrigona muelleri: Teixeira 2012).These neotropical species are all characterised by brood combsof open structure. T. carbonaria appears to be the first instanceof a species that builds a spiral brood comb (Brito et al. 2012) inwhich colonization of an adjacent cell by a larva leads to anemergency queen. It is interesting that the normally spiral broodcomb of T. carbonaria is built as an open structure similar tothat observed in T. hockingsi in queenless colonies. This differ-ence in brood comb morphology may arise as an epiphenom-enon of the bees’ need to use auxillary cells when constructingqueen cells.

According to Rasmussen and Cameron (2010), theneotropical stingless bees separated from the paleotropicalgroup about 80 million years ago. Around 75 million years ago,a second dichotomy separated the Indo-Malay/Australasiangroup from the Afrotropical group. The production of queensthrough the fusion of brood cells and overfeeding larvae is acharacteristic shared by species in the three groups of stinglessbees: the basal neotropical species typified by L. muelleri, theIndo-malay/Australasian bees typified by T. carbonariadescribed in this study and Austroplebeia australis, originallyfrom the Afrotropical group that has apparently dispersed intoAustralia via the Malay region (Rasmussen & Cameron 2010;Teixeira 2012). Assuming homology of the queen cell buildingbehaviour, the shared elements in these disparate groupssuggest that the ancestral state of queen production in stinglessbees was by the fusion of worker brood cells and not by thespecial construction of a queen cell or by genetic determinism.

Our behavioural observations, combined with the dissectionsof the brood cells and examination of worker ovaries, suggestcomplete absence of worker reproduction during the 4 weeks ofqueenlessness. Worker sterility has been reported previously inT. carbonaria, T. clypearis and T. mellipes in queenright colo-nies (Drumond et al. 2000; Gloag et al. 2007) and is somewhatsurprising given that worker egg laying is widespread inneotropical stingless bees (Tóth et al. 2004) and inAustroplebia (Drumond et al. 2000; Palmer et al. 2002). In themajority of neotropical species, workers are able to activatetheir ovaries and lay eggs even in the queens’ presence(Sakagami & Zucchi 1963; Sakagami 1982; Zucchi 1993;Palmer et al. 2002). Workers lay both trophic eggs that are eatenby the queen or reproductive eggs that develop into males(Zucchi 1993). The only other group of stingless bees withcompletely sterile workers is the genus Frieseomelitta. In thisgenus, development of workers’ ovaries stops at the pupal stage,and the lateral oviducts regress via apoptosis (Boleli et al.1999). As a result, workers are completely sterile even underqueenless conditions (Boleli et al. 1999). Based on the diversityof the genus, the study of more species could confirm if thesterility is a synapomorphy for the group as it is forFrieseomelitta.

ACKNOWLEDGEMENT

This work was supported by Fundação de Amparo a Pesquisado Estado de São Paulo (FAPESP) post-doctoral grant to TMN(BEPE Proc. 2013/09263-4).

Fig. 5. Brood comb of Tetragonula carbonaria 2 weeks afterqueen removal. (a) Brood produced in queens’ presence and (b)brood produced in queenless state, provisioned with food and notcontaining eggs.

4 T M Nunes et al.

© 2014 Australian Entomological Society

REFERENCES

Boleli IC, Simões ZLP & Bitondi MMG. 1999. Cell death in ovariolescauses permanent sterility in Frieseomelitta varia workers bees.Journal of Morphology 242, 271–282.

Brito RM, Schaerf TM, Myerscough MR, Heard TA & Oldroyd BP. 2012.Brood comb construction by the stingless bees Tetragonula hockingsiand Tetragonula carbonaria. Swarm Intelligence 6, 151–176.

Cortopassi-Laurino M, Impertriz-Fonseca VL, Roubik DW et al. 2006.Global meliponiculture: challenges and opportunities. Apidologie 37,275–292.

Cruz-Landim C. 2009. Abelhas: Morfologia e função de sistemas. EditoraUNESP, Rio Claro, Brazil.

Darchen R & Delage-Darchen B. 1971. Le déterminisme des castes chezles Trigones (Hyménoptères, Apidés). Insectes Sociaux 18, 121–134.

Drumond PM, Oldroyd BP & Osborne K. 2000. Worker reproduction inAustroplebeia australis Friese (Hymenoptera, Apidae, Meliponini).Insectes Sociaux 47, 333–336.

Eardley C & Kwapong P. 2013. Taxonomy as a tool for conservation ofAfrican stingless bees and their honey. In: Pot-Honey: A Legacy ofStingless Bees (eds P Vit, S Pedro & DW Roubik), pp. 261–268.Springer, New York, USA.

Faustino CD, Silva-Matos EV, Mateus S & Zucchi R. 2002. First record ofemergency queen rearing in stingless bees (Hymenoptera, Apidae,Meliponini). Insectes Sociaux 49, 111–113.

Franck P, Cameron E, Good G, Rasplus J-Y & Oldroyd BP. 2004. Nestarchitecture and genetic differentiation in a species complex of Aus-tralian stingless bees. Molecular Ecology 13, 2317–2331.

Gloag RS, Beekman M, Heard TA & Oldroyd BP. 2007. No workerreproduction in the Australian stingless bee Trigona carbonaria Smith(Hymenoptera, Apidae). Insectes Sociaux 54, 412–417.

Halcroft M, Spooner-Hart R, Haigh A, Heard TA & Dollin A. 2013. TheAustralian stingless bee industry: a follow-up survey, one decade on.Journal of Apicultural Research 52 (2), 1–7.

Hartfelder K, Makert GR, Judice CC et al. 2006. Physiological andgenetic mechanisms underlying caste development, reproduction anddivision of labor in stingless bees. Apidologie 37, 144–163.

Hatch S, Tarpy DR & Fletcher DJC. 1999. Worker regulation of emer-gency queen rearing in honey bee colonies and the resultant variationin queen quality. Insectes Sociaux 46, 372–377.

Heard TA. 1999. The role of stingless bees in crop pollination. AnnualReview of Entomology 44, 183–206.

Heard TA & Dollin AE. 2000. Stingless beekeeping in Australia: snapshotof an infant industry. Bee World 81, 116–125.

Imperatriz-Fonseca VL & Zucchi R. 1995. Virgin queens in stingless bee(Apidae, Meliponinae) colonies: a review. Apidologie 26, 231–244.

Jarau S, van Veen JW, Twele R et al. 2010. Workers make the queens inMelipona bees: identification of geraniol as a caste determining com-pound from labial glands of nurse bees. Journal of Chemical Ecology36, 565–569.

Kerr WE. 1950. Genetic determination of castes in the genus Melipona.Genetics 35, 143–152.

Koedam D, Aguilar Monge I & Sommeijer MJ. 1995. Social interactionsof gynes and their longevity in queenright colonies of Meliponafavosa (Apidae: Meliponinae). Netherlands Journal of Zoology 45,480–494.

Menezes C, Vollet-Neto A & Imperatriz-Fonseca VL. 2013. An advance inthe in vitro rearing of stingless bee queens. Apidologie 44, 491–500.

Michener CD. 1974. The Social Behavior of Bees: A Comparative Study.Belknap Press, Harvard University Press, Cambridge, Massachusetts,USA.

Palmer KA, Oldroyd BP, Quezada-Euán JJG, Paxton RJ & May-Itza WJ.2002. Paternity frequency and maternity of males in some stinglessbee species. Molecular Ecology 11, 2107–2113.

Rasmussen C & Cameron SA. 2010. Global stingless bee phylogenysupports ancient divergence, vicariance, and long distance dispersal.Biological Journal of the Linnean Society 99, 206–232.

Ribeiro MF, Wenseleers T, Santos-Filho P & Alves DA. 2006. Miniaturequeens in stingless bees: basic facts and evolutionary hypotheses.Apidologie 37, 191–206.

Sakagami SF. 1982. Stingless bees. In: Social Insects (ed. HR Hermann),pp. 361–423. Academic Press, New York, USA.

Sakagami SF & Zucchi R. 1963. Oviposition process in a stingless beeTrigona (Scaptotrigona) postica Letr. (Hymenoptera). StudiaEntomologica 6, 497–510.

Schwander T, Lo N, Beekman M, Oldroyd BP & Keller L. 2010. Natureversus nurture in social insect caste differentiation. Trends in Ecology& Evolution 25, 275–282.

Sommeijer MJ, De Bruijn LLM, Meeuwsen FJAJ & Slaa EJ. 2003. Repro-ductive behaviour of stingless bees: nest departures of non-acceptedgynes and nuptial flights in Melipona favosa (Hymenoptera: Apidae:Meliponini). Entomologische Berichten 63 (1), 7–13.

Teixeira LV. 2007. Produção de rainhas em colônias de Plebeia lucii(Hymenoptera, Apidae, Meliponina). Masters Dissertation.Universidade Federal de Viçosa, Viçosa, MG, Brazil.

Teixeira LV. 2012. Produção de rainhas em espécies de abelhas semferrão com células de cria dispostas em cacho (Hymenoptera, Apidae,Meliponini). PhD Thesis. Universidade Federal de Viçosa, Viçosa,MG, Brazil.

Terada Y. 1974. Contribuição ao estudo da regulação social emLeurotrigona muelleri e Frieseomelitta varia (Hymenoptera, Apidae).Masters Dissertation, Universidade de São Paulo, São Paulo, Brazil.

Tóth E, Queller DC, Dollin A & Strassmann JE. 2004. Conflict over maleparentage in stingless bees. Insectes Sociaux 51, 1–11.

Wenseleers T, Alves DA, Francoy TM, Billen J & Imperatriz-Fonseca VL.2011. Intraspecific queen parasitism in a highly eusocial bee. BiologyLetters 7, 173–176.

de Wilde J & Beetsma J. 1982. The physiology of caste development insocial insects. Advances in Insect Physiology 16, 167–246.

Zucchi R. 1993. Ritualized dominance, evolution of queen-worker inter-actions and related aspects in stingless bees (Hym., Apidae). In:Evolution of Insect Societies (eds T Inoue & S Yamane), pp. 207–249.Tokyo, Japan.

Accepted for publication 24 April 2014.

Emergency queens in Tetragonula carbonaria 5

© 2014 Australian Entomological Society