Irreversible Sterility of Workers and High

SHORT COMMUNICATION Irreversible sterility of workers and high-volume egg production by queens in the stingless bee Tetragonula carbonaria Francisco Garcia Bulle Bueno1,*, Rosalyn Gloag1, Tanya Latty2 and Isobel Ronai1

ABSTRACT et al., 2016). First, the presence of the queen, through aggressive Social insects are characterised by a reproductive division of labour interactions or pheromones, triggers programmed cell death in the between queens and workers. However, in the majority of social ovaries of the workers (Luna-Lucena et al., 2019; Ronai et al., insect species, the workers are only facultatively sterile. The 2015); workers may therefore develop activated ovaries only in the ’ Australian stingless bee Tetragonula carbonaria is noteworthy as queen s absence. Second, ovary development is trophogenic workers never lay eggs. Here, we describe the reproductive anatomy (Lisboa et al., 2005), as the quantity or quality of nutrients has a of T. carbonaria workers, virgin queens and mated queens. We then strong effect on ovary development (Boleli et al., 2000, 1999; conduct the first experimental test of absolute worker sterility in the Corona et al., 2016; Cruz-Landim, 2000; Jarau et al., 2010; Luna- social insects. Using a controlled microcolony environment, we Lucena et al., 2019; Schwander et al., 2010). For example, in the investigate whether the reproductive capacity of adult workers can be honey bee Apis mellifera, females fed highly nutritious royal jelly as rescued by manipulating the workers’ social environment and diet. adults have activated ovaries even in the presence of queen The ovaries of T. carbonaria workers that are queenless and fed pheromone (Kamakura, 2011; Lin and Winston, 1998; Pirk et al., unrestricted, highly nutritious royal jelly remain non-functional, 2010). indicating they are irreversibly sterile and that ovary degeneration is Stingless bees (Meliponini) are a species-diverse pantropical fixed prior to adulthood. We suggest that T. carbonaria might have clade of social insects, comprising Neotropical, Afrotropical and – evolved absolute worker sterility because colonies are unlikely to ever Indo-Malayan Australasian subclades that diverged 50 60 MYA be queenless. (Michener, 1974; Rasmussen and Cameron, 2010; Roubik, 1995). The reproductive habits of stingless bee workers have been best KEY WORDS: Activated ovaries, Ovarioles, Spermatheca, studied in the Neotropical subclade, where workers have been Programmed cell death, Reproductive system, Eusociality variously shown to produce males under queenright conditions (Beig et al., 1985; Boleli et al., 2000; Contel and Kerr, 1976; INTRODUCTION Vollet-Neto et al., 2018), produce males only when queenless In social insects, the reproductive labour is divided between queens (Cruz-Landim, 2000; Tóth et al., 2004), lay trophic eggs, which and workers (Hammond and Keller, 2004; Naug and Wenzel, serve as nourishment for the queen (Wille, 1983), or never lay eggs 2006). The queen is typically the sole reproductive female and her (Boleli et al., 1999; Staurengo da Cunha, 1979). However, the specialised function in the colony is to lay eggs. In contrast, the non- reproductive habits of workers in the Indo-Malayan Australasian reproductive female workers forage for food, defend the colony subclade are relatively unknown. against predators and care for the queen’s brood (Michener, 1974). In this study, we describe the reproductive anatomy of mated In the majority of social insect species, workers are only queens, virgin queens and female workers of the Australian facultatively sterile and have the ability to lay unfertilised haploid stingless bee Tetragonula carbonaria (Smith 1854). A colony of eggs that develop as males (Ebie et al., 2015; Khila and Abouheif, this Indo-Malayan Australasian subclade species has one mated 2010; Wenseleers et al., 2004). The contribution of workers to the queen, thousands of non-reproductive female workers, some males colony’s male output is highly variable across social insect species, and a few virgin queens (Gloag et al., 2007; Heard, 2016; Nunes and is determined by kinship-mediated cooperation and within- et al., 2015). Notably, when the queen is removed from the colony, colony conflict over male production (Alves et al., 2009; Herbers, T. carbonaria female workers do not activate their ovaries (Nunes 1984; Mehdiabadi et al., 2003; Tóth et al., 2002). Facultative worker et al., 2015), suggesting that they may be irreversibly sterile. To sterility means that if the queen is lost and the colony cannot confirm this, we tested whether manipulating the nutritional and requeen, then workers can secure some reproductive success by social environment of age-matched adult female workers in a producing males (Bourke, 1988). controlled microcolony environment can rescue their reproductive The key determinants of the reproductive capacity of social insect capacity (i.e. stimulate oogenesis). We hypothesised that if adult workers are their nutritional status and social environment (Ronai T. carbonaria workers are irreversibly sterile, then those fed an unrestricted, highly nutritious diet in the absence of a queen would

1Behaviour, Ecology and Evolution Laboratory, School of Life and Environmental show no change in reproductive capacity. Sciences A12, University of Sydney, Sydney, NSW 2006, Australia. 2Insect Behaviour and Ecology Laboratory, School of Life and Environmental Sciences MATERIALS AND METHODS A12, University of Sydney, Sydney, NSW 2006, Australia. Biological material *Author for correspondence ([email protected]) Our study was carried out at the University of Sydney (Sydney, Australia) in December–January 2017 and December–February F.G.B.B., 0000-0003-1521-7109; R.G., 0000-0002-2037-4267; T.L., 0000-0002- 7469-8590; I.R., 0000-0002-1658-857X 2019. As the source colonies, we used seven T. carbonaria colonies (Colony 1–7) housed in wooden Original Australian Tetragonula

Received 5 June 2020; Accepted 27 July 2020 Hives (Heard, 2016). These colonies were queenright, weighed Journal of Experimental Biology

1 SHORT COMMUNICATION Journal of Experimental Biology (2020) 223, jeb230599. doi:10.1242/jeb.230599 above average (8–9 kg; Heard, 2016) and had high forager activity Xin et al., 2016). Stingless bees are not known to use royal jelly, but at the entrance of the colony (∼100 bees exiting per minute). a high protein diet increases fertility in stingless bee workers (Costa To obtain age-matched workers, we extracted two discs of late and Venturieri, 2009). stage worker brood comb (approximately 200 bees per disk) from a The microcolonies were checked daily. Food, pollen and water source colony and placed the discs in a plastic container. The were replenished when needed. Any dead workers were removed containers were then placed in an incubator (34.5°C) for and recorded (see Table S1). We collected two workers from each approximately 5 days. Tetragonula carbonaria worker brood comb microcolony at 0 days of age and all remaining workers at 14 days of contains both male and female brood in identical cells. Any males that age. All collected workers were immediately placed into a −80°C emerged were removed from the container. freezer.

Worker and queen reproductive capacity Ovary dissections To assess the differences in reproductive anatomy between castes We extracted the paired ovaries from each bee collected from both under natural conditions, we collected newly emerged workers natural colonies and microcolonies. The tiny ovaries of the workers (n=200, per colony), recognised by their pale colour, from four of need to be carefully dissected as they are fragile and likely to break. the source colonies (Colony 1–4), colour-marked their thorax using We placed the ovaries in a drop of distilled water on a microscope POSCA pens and returned them to their source colony. After slide and covered them with a cover slip. The ovaries were imaged 14 days, the majority of marked workers had lost their mark or died, under a dissecting microscope (Leica IC80 HD) at 0.67× (mated but we collected 10 marked workers from each source colony. queens and virgin queens) and 4× (workers) magnification. We We collected the mated queen (unknown age) and one virgin queen analysed the images with ImageJ (https://imagej.nih.gov/ij/) while (unknown age) from each of these four source colonies (colonies 1– blind to treatment. 4). To extract the queens, we set up observation hives with a We evaluated the reproductive capacity and anatomy of transparent plastic lid so that we could observe the brood comb. Once T. carbonaria females following the protocols used in our the mated queen was collected, we waited 24 h for a virgin queen to previous studies of honey bee ovaries (Ronai et al., 2017, 2015). emerge on the brood comb and then extracted a virgin queen. These For each bee, we assessed the ovary activation state (deactivated, virgin queens were walking on top of the brood comb flapping their semi-activated or activated) and counted the number of ovarioles wings and were being fed via trophallaxis by the workers. Note that (filaments) in an ovary. We then quantified the length of the ovary we observed male aggregations outside nearby colonies. All collected as a proxy for the amount of ovary development or degeneration. workers and queens were immediately placed into a −80°C freezer. For workers, we measured the length of both ovaries (from the tip of the longest ovariole of each ovary to where the two oviducts join; Manipulation of workers in microcolonies Fig. S2A). For mated queens and virgin queens, it was impossible to To manipulate the social environment and diet of adult workers, we measure the length of the ovarioles as they are tightly curled, so we designed laboratory-based microcolonies (see Fig. S1) using 500 ml measured the elliptical area of the ovary (area=a×b×π, where a is the plastic containers. Each microcolony was provided with a 1.5–2cm major diameter divided by 2 and b is the minor diameter divided by (diameter) pellet of propolis (a mix of wax and plant resins that 2; Fig. S2B). If still attached after dissection, we noted the presence stingless bees use as nest building material; Heard, 2016) and a and status (semen present or absent) of the spherical spermatheca, 250 mg pellet of pollen was taken from a pot-pollen (pollen is stored the specialised organ used to store the sperm, and measured its in pots made of propolis, and is the main protein source for stingless diameter if present (Fig. S2). bees; Heard, 2016), both obtained from a nearby colony. Into each For all workers, we investigated whether the length of the right microcolony we placed newly emerged workers (n=50 per and left ovary was correlated using a Pearson correlation so we could microcolony, 8 microcolonies) from one of three source colonies then compare the length of the longest ovary in our experimental (colonies 5–7). The eight microcolonies contained no queen and treatments. To compare the length of the longest ovary of workers fed also no brood. a control diet versus royal jelly diet, we conducted a Student’s t-test Four of the microcolonies were randomly assigned a control diet (all assumptions were met) using R Studio (version 1.3.959), https:// consisting of stingless bee honey and the other four paired (from the www.rstudio.com/), with the function t.test in the R package stats same source colony) microcolonies were assigned a royal jelly diet v4.0.0 (https://www.r-project.org/). consisting of 50% stingless bee honey and 50% frozen royal jelly (Royal Jelly, Australia). A volume of 0.2 ml of each diet was placed RESULTS AND DISCUSSION onto plastic dishes (1 cm diameter) using a 3 ml plastic pipette. We Reproductive morphology of workers in queenright colonies established that the workers were ingesting the food by observing Queenright 14-day-old T. carbonaria workers had deactivated the bees feeding and that the food containers were emptied. In ovaries as no developing oocytes were present inside the ovarioles addition, when dissecting the workers, we observed that their crop (Fig. 1). The number of ovarioles in the ovary of workers varied was full of food. from 4 to none. The mean (±s.d.) length of the longest ovary was Royal jelly is high in protein, contains important fatty acids 0.65±0.15 mm (n=40; Table S2). The length of the right and left (Altaye et al., 2010) and is fed to honey bee (Apis sp.) queens ovary was asymmetric (P=0.098; Table S3). Only 20% of workers throughout their lifespan (Howe et al., 1985; Melampy and Jones, (n=8) had an identifiable, but vestigial, spermatheca with a mean 1939; Viuda-Martos et al., 2008). Notably, feeding royal jelly to diameter of 0.05±0.01 mm (Fig. 2A, Table S2). adult honey bee workers causes them to activate their ovaries (Cardoso-Júnior et al., 2020 preprint; Lin and Winston, 1998; Wang Reproductive morphology of virgin queens and mated et al., 2014; Yang et al., 2017), and feeding it to female fruit flies, queens crickets and silkworms increases their fecundity (Hodin, 2009; Tetragonula carbonaria queens had four ovarioles per ovary Hodin and Riddiford, 2000; Kamakura, 2011; Kayashima et al., (Fig. 2). We observed that the ovaries of T. carbonaria are of the

2012; Kunugi and Mohammed Ali, 2019; Miyashita et al., 2016; meroistic polytrophic type (Büning, 1994; Martins and Serrão, Journal of Experimental Biology

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Worker Virgin queen Mated queen

Fig. 1. The ovaries of Tetragonula carbonaria females. Queenright 14-day-old workers have deactivated ovaries (no developing oocytes present) with zero to four ovarioles. Virgin queens have semi-activated ovaries (oocytes not mature) with four ovarioles. Mated queens have activated ovaries (mature oocytes present) with four ovarioles. The ovaries of mated and virgin queens are covered by an extensive tracheal network. Image background has been removed. Scale bar: 1 mm.

2004) as is typical for Hymenoptera. As an oocyte moves 0.59±0.11 mm for those fed a royal jelly diet (n=40, Table S2). The from the tip of the ovariole to the base, it progresses through mean length of the right and left ovary was significantly correlated stages of development (stem cell, germarium and vitellarium) for both control diet and royal diet workers (P=0.001 and 0.007, (Ronai et al., 2015). respectively; Table S3). There was no significant difference in the Virgin queens had semi-activated ovaries with oogenesis mean length of the longest ovary between the control diet and royal underway (Fig. 1), and the mean area of the largest ovary was diet treatments after 14 days (t=−0.68194, d.f.=75.203, P=0.4974; 1.02±0.12 mm2 (n=4; Table S4). The spermatheca had a mean Fig. 3). diameter of 0.55±0.03 mm (n=3; Table S4). For two of the virgin queens, the spermatheca reservoir was transparent, indicating it was Conclusions empty (Fig. 2C); however, the third virgin queen had a filled An unrestricted high quality diet and the absence of the queen failed spermatheca reservoir, indicating that she mated within the 24 h that to rescue the reproductive capacity of T. carbonaria adult workers, there was no mated queen in the colony (Fig. 2D). indicating that they are irreversibly sterile. This absolute sterility is Mated queens had activated ovaries full of mature oocytes consistent with observations from natural nests in which workers (Fig. 1). Some of the activated ovaries had yellow bodies present, a never lay eggs, even if queenless (Gloag et al., 2007; Nunes et al., consequence of previous oviposition (Fénéron and Billen, 1996; 2014). Absolute sterility of workers is relatively rare among social Gobin et al., 1998). The mean area of the largest ovary was 13.41± insect species. Only 13 genera have so far been classified as having 2.99 mm2 (n=4; Table S4), approximately 13 times larger than that truly sterile workers based on observations under natural conditions of the virgin queens. In order to fit inside the abdomen (Ronai et al., 2016); whether sterility is irreversible in these species (approximately 6.5 mm in length) of the queen, the ovaries are has not yet been tested experimentally. curled tightly in a spiral and occupy the majority of the abdominal Why have a few genera of social insects evolved absolute worker cavity, which causes the mated queen to become highly sterility and forgone the obvious benefits of facultative sterility? It is physogastric. The spermatheca had a mean diameter of 1.05± likely that the life history of some species predisposes them to the 0.81 mm (n=2; Table S4) and for both mated queens, the reservoir evolution of absolute worker sterility. Tetragonula carbonaria have was filled with a milky white substance, indicating the presence of a high and constant rate of queen production (Heard, 2016) semen (Fig. 2B). compared with other stingless bee species with facultative worker sterility (Grosso et al., 2000; Ribeiro et al., 2003; Van Veen and Reproductive morphology of queenless workers fed royal Sommeijer, 2000) and also honey bees that have facultative worker jelly diet and control diet sterility (Jay, 1968; Kropácová̌ and Haslbachová, 1969). In our Both royal jelly diet and control diet queenless workers had study, we found a mated ‘virgin’ queen within 24 h of the original degenerated and deactivated ovaries. The mean length of the longest mated queen being removed. In addition, T. carbonaria workers are ovary of 0-day-old workers was 0.56±0.1 mm (n=16; Table S2). able to create emergency queen cells from worker cells (Nunes et al., The mean length of the longest ovary of 14-day-old workers was 2015). A T. carbonaria colony is therefore likely to always have a

0.57±0.14 mm for those fed a control diet (n=40; Table S2) and queen present and there would never be a need for workers to lay Journal of Experimental Biology

3 SHORT COMMUNICATION Journal of Experimental Biology (2020) 223, jeb230599. doi:10.1242/jeb.230599

C D

S S

Fig. 2. The spermatheca of T. carbonaria females. (A) Queenright 14-day-old worker with a vestigial spermathecal (S). (B) Mated queen with a full spermatheca reservoir containing semen. (C) Virgin queen with an empty spermatheca reservoir. (D) Virgin queen with a full spermatheca reservoir containing semen and semi-activated ovaries, suggesting mating occurred recently. Image background has been removed. Scale bars: 0.5 mm. eggs (Engels and Imperatriz-Fonseca, 1990; Michener, 1974). Neotropical sublclade stingless bees, have both monogamous Species that have a constant reservoir of virgin queens from which queens and workers that lay eggs (Vollet-Neto et al., 2018). the workers select the most fecund one or rear new queens very We found that T. carbonaria workers emerge from pupation quickly may be more likely to evolve absolute worker sterility with non-functional ovaries that contain no germ cells. The (Sommeijer et al., 1994). Another possible explanation for the degeneration of the ovary in T. carbonaria workers must therefore evolution of absolute worker sterility is the high rate of colony occur prior to adulthood. To date, only one other genus of stingless takeovers in T. carbonaria (Cunningham et al., 2014; Gloag et al., bee in the Neotropical subclade has been proposed to have 2008). This behaviour reduces the chance that a colony would ever absolute worker sterility, Frieseomelitta (Boleli et al., 2000, 1999; have a prolonged period where it was queenless and that the workers Luna-Lucena et al., 2019; Ronai et al., 2016), and their ovaries would need to lay eggs. degenerate during the last pupal stage via apoptosis (Boleli et al., Alternatively, absolute worker sterility may resolve reproductive 1999). Further work is needed to establish whether the conflict within colonies. Tetragonula carbonaria is a monogamous degeneration of the ovaries in T. carbonaria workersisinitiated species (Green and Oldroyd, 2002). Under monogamous queens, during the embryonic, larval or pupal stage. In T. carbonaria,the workers in a colony are all full sisters and workers would be more ovary degeneration in workers is likely determined by their related to their own sons (r=0.5) than to the sons of the queen nutritional environment pre-emergence as worker-destined larvae (r=0.25). If workers have the ability to lay eggs, there would be are provisioned with a smaller quantity of food than queen- worker–queen conflict for male production in the colony (Trivers destined larvae (Nunes et al., 2015). and Hare, 1976). The evolution of absolute worker sterility in Even when the workers of social insects have activated ovaries, monogamous species eliminates the worker–queen conflict to the they may not have a storage organ for semen. The spermatheca is benefit of the queen. However, many social insects, including most thus normally absent from workers (Gobin et al., 2008; Hölldobler Journal of Experimental Biology

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Fig. 3. Ovary length of 14-day-old queenless T. carbonaria workers. The workers were fed a control diet (n=40, 10 per microcolony) or a royal jelly diet (n=40, 10 per microcolony). Image background has been removed. Scale bars: 0.25 mm. There was no significant difference between the treatment groups.

1.0

0.8

0.6

0.4

0.2

Length of the longest ovary (mm) 0

Control diet Royal jelly diet and Wilson, 1990; Khila and Abouheif, 2010; Van Eeckhoven and Author contributions Duncan, 2020). However, T. carbonaria workers sometimes have a Conceptualization: F.G.B.B., I.R.; Methodology: F.G.B.B., I.R.; Validation: I.R.; Formal analysis: F.G.B.B., I.R.; Investigation: F.G.B.B.; Writing - original draft: vestigial spermatheca, the first reported case in the Indo-Malayan F.G.B.B., I.R.; Writing - review & editing: F.G.B.B., R.G., T.L., I.R.; Visualization: Australasian subclade of stingless bees. Vestigial spermatheca are F.G.B.B., I.R.; Funding acquisition: R.G., T.L. present in workers of the one other stingless bee genus with absolute worker sterility (Boleli et al., 2000), and also in honey bee species Funding with facultative worker sterility (Gobin et al., 2006; Gotoh et al., This research received no specific grant from any funding agency in the public, 2013). The presence of a vestigial spermatheca in species where the commercial or not-for-profit sectors. workers have degenerated ovaries provides evidence that spermatheca Supplementary information degeneration is a separate process to the degeneration of the ovaries. Supplementary information available online at Our study of T. carbonaria ovaries highlights that the https://jeb.biologists.org/lookup/doi/10.1242/jeb.230599.supplemental exceptionally high reproductive capacity of queens is achieved in different ways across the clades of eusocial bees. Stingless bees and References Altaye, S. Z., Pirk, C. W. W., Crewe, R. M. and Nicolson, S. W. (2010). honey bees (Apis spp.) each evolved highly eusocial life histories Convergence of carbohydrate-biased intake targets in caged worker honeybees independently, from an ancestral corbiculae apid with more simple fed different protein sources. J. Exp. Biol. 213, 3311-3318. doi:10.1242/jeb. sociality (Cardinal and Danforth, 2011). Tetragonula carbonaria 046953 queens lay approximately 300 eggs per day (Heard, 2016) but have Alves, D. A., Imperatriz-Fonseca, V. L., Francoy, T. M., Santos-Filho, P. S., Nogueira-Neto, P., Billen, J. and Wenseleers, T. (2009). The queen is dead—long only 8 ovarioles in total; therefore, each ovariole is elongated and live the workers: intraspecific parasitism by workers in the stingless bee Melipona produces around 37 eggs per day. In contrast, A. mellifera queens scutellaris. Mol. Ecol. 18, 4102-4111. doi:10.1111/j.1365-294X.2009.04323.x lay approximately 2000 eggs per day (Page and Erickson, 1988) but Beig, D., Bueno, O. C. and Muller, T. (1985). Caracteristicas dos alveolos de cria e postura de operarias em Melipona quadrifasciata anthidioides Lep. (Hym., have on average 320 ovarioles in total (Jackson et al., 2011); Apidae, Meliponinae). Naturalia (São Josédo Rio Preto) 10, 75-81. therefore, each ovariole is relatively short and produces only around Boleli, I. C., Paulino-Simões, Z. L. and Gentile Bitondi, M. M. (1999). Cell death in 6 eggs per day. That is, the lower number of ovarioles in stingless ovarioles causes permanent sterility in Frieseomelitta varia worker bees. bee queens when compared with honey bee queens is compensated J. Morphol. 242, 271-282. doi:10.1002/(SICI)1097-4687(199912)242:3<271:: AID-JMOR6>3.0.CO;2-7 by the high volume of egg production in each ovariole of the ovary. Boleli, I. C., Paulino-Simões, Z. L. and Bitondi, M. M. G. (2000). Regression of the In summary, our results provide strong evidence that T. carbonaria lateral oviducts during the larval-adult transformation of the reproductive system of workers are irreversibly sterile as adults. Conducting similar studies Melipona quadrifasciata and Frieseomelitta varia. J. Morphol. 243, 141-151. doi:10.1002/(SICI)1097-4687(200002)243:2<141::AID-JMOR3>3.0.CO;2-Y of workers in the other Tetragonula spp. would establish Bourke, A. F. G. (1988). Worker reproduction in the higher eusocial Hymenoptera. whether absolute sterility is a characteristic of this genus. Studies of Q Rev. Biol. 63, 291-311. doi:10.1086/415930 absolute worker sterility, and the factors that favour its evolution, are Büning, J. (1994). The Insect Ovary: Ultrastructure, Previtellogenic Growth and likely to provide a deeper understanding of the evolution of Evolution. Springer Science & Business Media. Cardinal, S. and Danforth, B. N. (2011). The antiquity and evolutionary history of eusociality. social behavior in bees. PLoS ONE 6, e21086. doi:10.1371/journal.pone. 0021086 Acknowledgements Cardoso-Júnior, C. A. M., Oldroyd, B. P. and Ronai, I. (2020). Vitellogenin We thank Liz Gibson and Peter Clarke for their support in lending colonies of expression in the ovaries of adult honeybee workers provides insights into the T. carbonaria. evolution of reproductive and social traits. bioRxiv, 547760. Contel, E. P. B. and Kerr, W. E. (1976). Origin of males in Melipona subnitida Competing interests estimated from data of an isozymic polymorphic system. Genetica 46, 271-277.

The authors declare no competing or financial interests. doi:10.1007/BF00055470 Journal of Experimental Biology

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