Irreversible Sterility of Workers and High
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© 2020. Published by The Company of Biologists Ltd | Journal of Experimental Biology (2020) 223, jeb230599. doi:10.1242/jeb.230599 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.