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Regulation of oogenesis in honey bee workers via programed cell death
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Regulation of oogenesis in honey bee workers via programed cell death
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Isobel Ronai ⇑, Deborah A. Barton, Benjamin P. Oldroyd, Vanina Vergoz
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School of Biological Sciences, Macleay Building A12, The University of Sydney, Sydney, NSW 2006, Australia
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Article history: Received 14 April 2015 Received in revised form 23 June 2015 Accepted 24 June 2015 Available online xxxx Keywords: Oogenesis Ovariole Programmed cell death Honey bee Worker sterility
a b s t r a c t Reproductive division of labour characterises eusociality. Currently little is known about the mechanisms that underlie the ‘sterility’ of the worker caste, but queen pheromone plays a major role in regulating the reproductive state. Here we investigate oogenesis in the young adult honey bee worker ovary in the presence of queen pheromone and in its absence. When queen pheromone is absent, workers can activate their ovaries and have well-developed follicles. When queen pheromone is present, even though workers have non-activated ovaries, they continually produce oocytes which are aborted at an early stage. Therefore, irrespective of the presence of the queen, the young adult worker ovary contains oocytes. By this means young workers retain reproductive plasticity. The degeneration of the germ cells in the ovarioles of workers in the presence of queen pheromone has the morphological hallmarks of programmed cell death. Therefore the mechanistic basis of ‘worker sterility’ relies in part on the regulation of oogenesis via programmed cell death. Our results suggest that honey bees have co-opted a highly conserved checkpoint at mid-oogenesis to regulate the fertility of the worker caste. Ó 2015 Published by Elsevier Ltd.
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1. Introduction
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In a social insect colony, the queen has reproductive hegemony over her sterile daughters. However there is a spectrum in the degree of ‘worker sterility’, even in bees. At one extreme there is the stingless bee genus Frieseomelitta where worker ovaries completely degenerate during development (Boleli et al., 1999). At the other extreme is the stingless bee genus Melipona where worker ovaries are well developed and workers contribute to male production (Tóth et al., 2004). The honey bee, Apis mellifera, is intermediate between these two extremes. The ovaries of honey bee workers are much reduced relative to those of queens, but remain functionally competent (Snodgrass, 1956). The honey bee worker has a high degree of reproductive plasticity. Queen mandibular pheromone suppresses ovary activation in workers (Hoover et al., 2003). In the absence of a queen the workers (which cannot mate) have the potential to activate their ovaries and lay haploid male-destined eggs. Conversely, if workers with activated ovaries are moved from a queenless colony to a queenright colony their ovaries regress (Malka et al., 2007). Workers also activate their ovaries on a seasonal basis, in particular during swarming season when colonies are temporarily queenless after the old queen has departed and the new queen has not begun to lay eggs (Holmes et al., 2013; Woyciechowski and Kuszewska,
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⇑ Corresponding author. E-mail address: [email protected] (I. Ronai).
2012). Another group of pheromones, emitted by the brood also contribute to suppressing ovary activation in workers (Alaux et al., 2010; Traynor et al., 2014). The ovarioles comprise the structural and functional units of the honey bee ovary. The major reproductive difference between the two female castes is that workers have 2–12 ovarioles per ovary whereas queens have 160–180 (Snodgrass, 1956). It is important to note that the term ‘ovariole’ is used to describe both the outer layer of cells of the ovariole (epithelial sheath, otherwise known as the tunica externa) and the inner, non-cellular membrane (tunica propria). Inside the tunica propria are both somatic cells and germ cells. The somatic cells encapsulate and support the germ cells. A germ cell gives rise not only to an oocyte but also to nutritive nurse cells, which remain directly connected to the oocyte to allow the transport of nutrients into the developing oocyte (Büning, 1994). The honey bee ovary is therefore classified as being polytrophic meroistic. The majority of studies on the adult honey bee ovary have focused on oogenesis in the queen (Berger and Abdalla, 2005; Patrício and Cruz-Landim, 2002; Paulcke, 1901; Snodgrass, 1956) with only one study examining early oogenesis in queenright and queenless workers (Tanaka and Hartfelder, 2004). Here, we systematically investigate oogenesis in the young adult worker ovary in the presence or absence of queen pheromone in a controlled environment without the confounding factor of brood pheromones. We were expecting no oocytes in the ovarioles of workers in the presence of queen pheromone and that oocytes would be
http://dx.doi.org/10.1016/j.jinsphys.2015.06.014 0022-1910/Ó 2015 Published by Elsevier Ltd.
Please cite this article in press as: Ronai, I., et al. Regulation of oogenesis in honey bee workers via programed cell death. Journal of Insect Physiology (2015), http://dx.doi.org/10.1016/j.jinsphys.2015.06.014
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present in the ovarioles of workers in the absence of queen pheromone. Instead we show that queen pheromone alters oocyte development in the worker, thereby leading to ‘worker sterility’.
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2. Materials and methods
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2.1. Biological material
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Age-matched adult workers of Australian commercial stock derived from A. m. ligustica were obtained by incubating overnight combs of emerging brood. The brood was from a single queenright colony which minimises the genetic and environmental heterogeneity among experimental treatments. The following day, the emerged workers were placed in four laboratory cages (n 150 per cage) each fitted with a 5 5 cm section of natural honey comb. Two of the cages contained a strip (0.5 queen equivalents) of queen mandibular pheromone (Phero Tech Inc., Canada) to simulate queen presence, whereas the other two cages contained no queen pheromone. The caged workers were provided with honey, ground pollen and water ad libitum. Workers were collected at 14 days of age and samples immediately frozen on dry ice.
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2.2. Tissue dissection
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We dissected a subset (n = 77) of the workers in cold PBS under a dissecting microscope (Olympus SZ40): workers exposed to queen pheromone (n = 41) and not exposed to queen pheromone (n = 36). The tergal surface was opened and the gut, fat body and first four tergites removed, leaving the abdominal cavity containing the paired ovaries (anchored to the last tergite) and the venom gland. During the dissection, the ovaries were scored as: non-activated (transparent, thread-like ovaries) or activated (opaque, swollen ovaries) (Vergoz et al., 2012). In cages containing queen pheromone, no workers had activated ovaries, whereas in cages with no queen pheromone 12.5% of workers had activated ovaries.
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2.3. Tissue preparation
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The ovaries were prepared as if for in situ hybridisation (Dallacqua and Bitondi, 2014; Zimmerman et al., 2013). Four abdominal cavities with their connected ovaries were placed in a vial containing heptane fixative (1 mL heptane, 80 lL HEPES/HEM buffer, 100 lL of 8% (w/v) paraformaldehyde and 20 lL DMSO) and shaken for 30 min. The samples were rinsed in PBS and then dehydrated with an increasing concentration of ethanol (25%, 50%, 75% and 100% v/v). These were stored overnight at 20 °C. The samples were rehydrated with a decreasing concentration of ethanol (75%, 50% and 25% v/v) and then PBS-Tween 20. Samples were fixed in triton fixative (89.01% PBS, 0.09% Triton X-100, 0.9% of 16% (w/v) paraformaldehyde and 10% DMSO v/v), shaken for 20 min and washed in PBS-Tween 20. The samples were incubated in proteinase K solution (approximately 20 lg/mL) for 15 min, then twice washed in glycine (1% w/v) and PBS-Tween 20. The samples were refixed in triton fixative and shaken for 20 min and washed in PBS-Tween 20. Nucleic acid was stained with SYTOX Blue Nucleic Acid Stain (Invitrogen) (0.002% v/v) for 20 min and washed in PBS-Tween 20. The paired ovaries were then dissected away from their abdominal cavity in PBS-Tween 20, immersed in glycerol (70% v/v) and mounted on individual slides.
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2.4. Imaging
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Paired ovaries were observed immediately under a laser scanning confocal microscope (Leica TCS SP5 II). SYTOX Blue was
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excited by a 458 nm laser and emitted light was collected between 465 and 484 nm. Red autofluorescence was excited by 561 nm and 633 nm lasers and emitted light was collected between 580 and 650 nm and 650–700 nm, respectively.
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3. Results
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The ovariole of the adult honey bee worker shows successive developmental stages of oogenesis from the tip to the base. The ovariole can be divided into regions depending on cell type (somatic or germ) and stage of germ cell development. These regions are the terminal filament, the germarium, the vitellarium, and the pedicel (which is sometimes referred to as the basal stalk) (Fig. 1A). Workers not exposed to queen pheromone can have activated ovaries and their ovarioles have well-developed oocytes. The developing oocytes cause the epithelial sheath to bulge (Fig. 1A) producing the defining feature of the activated ovary phenotype. The basal end of the tunica propria of an activated ovary is 71 lm ± 6 lm (n = 5) in diameter. The terminal filament, a stack of aligned disc-shaped cells that are orientated transversely (Fig. 1B), is located at the apical end of the tunica propria. This filament contains cells that are mainly of somatic origin but germline stem cells are also present (Tanaka and Hartfelder, 2004). In the early-germarium stage, there are undifferentiated germ cells known as oogonia (Fig. 1C). In this region, individual cells are hard to differentiate. In the mid-germarium stage, the early oocyte is centrally located and is differentiated by size from its surrounding cells that develop into trophocyte nurse cells (approximately 39– 59 per oocyte (Büning, 1994)) (Fig. 1D). The nucleus of the oocyte, known as the germinal vesicle, is relatively large and contains a small black dot indicative of the chromatin beginning to condense. In the late-germarium stage, the oocytes are now located at the basal end of their associated nurse cells (Fig. 1E). The germinal vesicle by this point appears as an unstained dark structure due to the condensed chromatin inside, known as the karyosome. The vitellarium stage of development is where self-contained follicles are present (Büning, 1994). At the vitellarium stage the oocyte is enlarged and encapsulated by a monolayer of columnar follicle cells (Fig. 1F), which are somatic in origin. In the nurse chamber the nurse cell size follows a gradient; those cells closest to the oocyte are the largest (Fig. 1A and F). The nurse cells have very large nuclei relative to the cytoplasm as well as bright staining around their nuclei (Fig. 1F) which is indicative of high levels of transcription in these cells (Gutzeit et al., 1993). Each ovariole terminates in a structure called the pedicel (Fig. 1G), which is somatic in origin. The pedicel acts as a plug to the oviduct opening and degrades when an egg is ready to be laid (Büning, 1994). Late oogenesis is characterised by developmental programmed cell death of the nurse cells (Fig. 1H). The basal nurse cells have a rounded appearance, condensed chromatin and an absent cytoplasm (Fig. 1H) due to the ‘dumping’ of their cytoplasmic contents into the oocyte (Cavaliere et al., 1998). Sometimes the ovarioles of workers not exposed to queen pheromone show what we term a ‘transition phenotype’ between non-activated ovaries and activated ovaries (Fig. 1I). In these ovarioles the germarium region has both healthy and dead oocytes. The developing oocytes in the middle of the tunica propria are healthy, as indicated by chromatin condensing in the germinal vesicles (Fig. 1I). However, at the basal end of the tunica propria the oocytes are dead as evidenced by the absence of staining (Fig. 1I). The oocytes’ nurse cells are dying, as evidenced by the pycnotic nuclei (central and highly compact, the condensed chromatin stains heavily) (Fig. 1I).
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Please cite this article in press as: Ronai, I., et al. Regulation of oogenesis in honey bee workers via programed cell death. Journal of Insect Physiology (2015), http://dx.doi.org/10.1016/j.jinsphys.2015.06.014
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Fig. 1. The ovarioles of adult workers not exposed to queen pheromone showing activated ovaries with well-developed oocytes or a ‘transition phenotype’. (A) A montage of images digitally reconstructing an entire ovariole (above) and a schematic representation of the ovariole (below) delineating the regions: terminal filament; germarium; vitellarium; and pedicel. The ovariole has a non-cellular membrane, the tunica propria (TP) and an outer layer of cells, the epithelial sheath (ES). Particular cell types of the ovariole include the trophocyte nurse cells (nc), oocytes (oo) and follicle cells (fc). The nucleus of the oocyte is termed the germinal vesicle (gv). (B) The terminal filament. (C) The early-germarium. (D) The mid-germarium. (E) The late-germarium. (F) The vitellarium. The oocyte (oo) is surrounded by follicle cells (fc) and is accompanied by nurse cells (nc). The germinal vesicle (gv) of the oocyte contains condensed chromatin, termed the karyosome (arrowhead). (G) The pedicel. (H) The basal nurse cells (nc) undergo developmental programmed cell death, the nuclei condense (arrows). (I) A montage of images digitally reconstructing an ovariole of a ‘transition phenotype’. At the apical end of the lower ovariole, oocytes are healthy and at the basal end oocytes are dying. Scale bars represent: (A, I) 50 lm; (B–H) 20 lm.
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Workers exposed to queen pheromone have non-activated ovaries and their ovarioles show widespread cell death. The dying cells are disorganised and have irregular nuclear staining, compared to the healthy epithelial sheath cells (Fig. 2) and cells of healthy ovarioles (Fig. 1A). The tunica propria often lacks a cohesive structure and is irregularly shaped, a characteristic of the non-activated ovary phenotype (Fig. 2A and B). The basal end of the tunica propria of a non-activated ovary is 41 lm ± 6 lm (n = 5) in diameter. The terminal filament is present and the cells look healthy. However, the widespread cell death occurring in the tunica propria causes the terminal filament to be positioned more basally inside the tunica propria (Fig. 2A and B compared to 1A). The germarium region is noticeably shorter than in workers not exposed to queen pheromone (Fig. 2A and B compared to 1A). The early-germarium oogonia are the last remaining healthy germ cells (Fig. 2B), however, these are sometimes in the process of
dying (Fig. 2A). In the mid to late germarium all oocytes and nurse cells are dead or dying. The oocytes shrink and their cytoplasm is degrading and contains numerous vacuoles, as indicated by the absence of staining (Fig. 2C and D). The nurse cells shrink and some have pycnotic nuclei (Fig. 2C) whereas others are dead, appearing as empty voids (Fig. 2D). Possible autophagic vacuoles are often present in highly degenerated germarium regions, and in the final stages of degeneration the tunica propria contains empty spaces (Fig. 2A and B). Non-activated ovaries do not develop follicles and therefore never reach the vitellarium stage of development (Fig. 2). It is important to note that among the workers exposed to queen pheromone there is considerable variation in the stage of cell death observed in the ovaries, even when workers are of the same age (Fig. 2). Using fluorescence microscopy, we found evidence of a lipofuscin-like pigment, an undegradable subcellular aggregate of
Please cite this article in press as: Ronai, I., et al. Regulation of oogenesis in honey bee workers via programed cell death. Journal of Insect Physiology (2015), http://dx.doi.org/10.1016/j.jinsphys.2015.06.014
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Fig. 2. The ovarioles of adult workers exposed to queen pheromone showing non-activated ovaries with widespread cell death. The terminal filament (TF) is present; the germarium region has dead or dying oocytes (oo) and nurse cells (nc); and no vitellarium stage develops. (A and B) Montage of images digitally reconstructing entire ovarioles in the final stages of degeneration. (C) An ovariole in the initial stages of degeneration. (D) An ovariole midway through degeneration. Tracheole (T). Scale bars represent 50 lm.
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oxidised proteins and lipids (Höhn and Grune, 2013), in the ovaries (Fig. 3). Under all fluorescent light channels lipofuscin has strong autofluorescence and this is used to detect its presence in tissues
(Terman and Brunk, 2004). The lipofuscin-like pigment is associated with the degenerating germarium region in non-activated ovaries (Fig. 3A), but is absent from this region in activated ovaries
Fig. 3. Lipofuscin-like pigment in the ovaries of adult workers. Ovaries stained with SYTOX Blue nucleic acid stain (blue) with autofluorescent (magenta) lipofuscin-like pigment and tracheoles (T). (A) Lipofuscin (arrowheads) in degenerating germarium regions of non-activated ovaries. (B) Lipofuscin is absent from the germarium region of an activated ovary. (C) Lipofuscin at the tip of the terminal filament (arrowhead). Scale bars represent: (A and C) 50 lm; (B) 20 lm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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(Fig. 3B). The lipofuscin-like pigment is also found at the tip of the terminal filament, regardless of ovary state (Fig. 3C).
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4. Discussion
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Our study reveals the cytological basis of queen pheromone ‘control’ over worker reproduction (de Groot and Voogd, 1954; Hoover et al., 2003; Malka et al., 2007; Winston and Slessor, 1992): queen pheromone causes the abortion of developing oocytes. Some workers exposed to queen pheromone have a tunica propria with such a highly degenerated germarium region that it seems unlikely ovarioles of this kind could ever recover. Tanaka et al. (2006) stated that in the ovaries of older workers engaged in foraging some of the ovarioles have only the epithelial sheath remaining. However, it is important to note that we always found the terminal filament (which contains germline stem cells (Tanaka and Hartfelder, 2004)) intact, regardless of queen presence. This finding leads us to hypothesise that oogenesis might be able to resume if the environmental conditions becomes favourable. We also suggest that exposure to queen pheromone as larvae might cause ‘scarring’ in the adult ovary. Workers that are not exposed to queen pheromone as adults but have been reared (as larvae and pupae) with a queen, sometimes have degenerating oocytes in the basal region of the tunica propria. These oocytes likely developed when the workers were larvae (Hodin, 2009) and reared with the queen. In contrast, a recent study showed that larvae reared in queenless colonies have high rates of ovary activation as adults (Woyciechowski and Kuszewska, 2012). The presence of the queen is the initial and most critical environmental signal for the reproductive fate of the worker. We observed that the degeneration of the ovariole of adult workers exposed to queen pheromone follows a characteristic sequence, which includes the morphological hallmarks of programmed cell death. First, we found that the oocyte dies. The same phenomena is observed in the queen honey bee, the death of the oocyte is thought to initiate the degeneration of the germarium region (Patrício and Cruz-Landim, 2008) and is also observed in rat ovaries (Sánchez et al., 2012). Second, we found that the nurse cells seem to show some characteristics of apoptosis (chromatin disorganisation, pycnotic nuclei and DNA fragmentation) followed by autophagy (formation of vacuoles and structural disorganisation). Studies in other organisms have showed that both apoptosis and autophagy occur during oogenesis and results in cells being rapidly removed. These two types of programmed cell death are complementary; autophagy efficiently removes the debris once apoptosis has completed (Mpakou et al., 2006; Sánchez et al., 2012; Velentzas et al., 2007a,b). Lastly, we showed that the degeneration culminates in dead cells and empty spaces inside the tunica propria. The empty spaces are most likely due to phagocytosis of the cellular debris after programmed cell death occurs (Mpakou et al., 2006; Nezis et al., 2006, 2001; Nezis et al., 2002), which allows the recycling of these resources. Interestingly, the ovariole degeneration in the honey bee is analogous to follicular atresia that occurs during vertebrate oogenesis (Tilly, 2001). Further molecular work is required to determine how programmed cell death regulates oogenesis in the adult worker. The presence of lipofuscin in the ovary is interesting for two reasons. First, lipofuscin is characteristic of degenerating ovarian follicles in primates (Hayama et al., 1992; Koering, 1969) and is associated with poor fertilisation rates and lower developmental competence in human oocytes (Otsuki et al., 2007). Second, lipofuscin is associated with programmed cell death (Höhn and Grune, 2013). In particular, lipofuscin is implicated in the process of autophagy (Mizushima et al., 2004; Terman and Brunk, 2005). As lipofuscin is present in non-activated ovaries when late stage
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degeneration is occurring, we propose that it could be used as a marker of this degeneration. The dimorphism between the worker and queen castes in the honey bee embodies the reproductive division of labour. As adults these genotypically identical castes have strikingly similar ovariole structure (though workers have an order of magnitude fewer ovarioles) and the key difference between the non-reproductive and reproductive castes is the progression through the stages of oogenesis. Queens progress through all the stages of oogenesis and lay eggs (Snodgrass, 1956), whereas we found that workers exposed to queen pheromone continually produce oocytes, but these oocytes never reach the vitellarium stage of oogenesis. In adult workers the progression from the germarium stage of oogenesis to the vitellarium stage means passing a critical checkpoint at mid-oogenesis. We found that queen pheromone represses follicle development and this has also been identified in queenright workers (Tanaka and Hartfelder, 2004). This checkpoint is employed by other organisms in response to a variety of adverse environmental conditions and utilises programmed cell death to arrest oogenesis (McCall, 2004). For example, when Drosophila do not receive sufficient food this triggers the degeneration of the oocytes at this checkpoint (Drummond-Barbosa and Spradling, 2001; Pritchett and McCall, 2012). Our study suggests that honey bees, and perhaps eusocial insects more generally have co-opted this evolutionary conserved oogenesis checkpoint to allow the evolution of ‘worker sterility’. Why has the mid-oogenesis checkpoint been selected to regulate oogenesis in the worker honey bee? Most likely utilising this checkpoint is an evolutionary trade-off that maximises the fitness of the worker. In a queenright environment there is little chance of a worker’s eggs surviving (Ratnieks and Visscher, 1989; Wenseleers et al., 2004) so the oocytes that are continually produced are aborted. The maturation of the oocyte is nutritionally demanding, so stopping oogenesis at this checkpoint reduces waste of resources. Conversely, the continual production of oocytes means that a young worker can rapidly switch to producing eggs if the environmental conditions become favourable for personal reproduction, for example, if the queen disappears. The use of this checkpoint allows the fine tuning of worker oogenesis to an environmental cue and thereby provides adaptive reproductive plasticity of the adult worker. On reflection the widespread use of the term ‘worker sterility’ in the honey bee literature needs to be reassessed. A workers’ ovary is thought to be in a state of ‘rest’ (Velthuis, 1970) as there are no visible oocytes and only under suitable environmental conditions do workers ‘activate’ their ovaries. Our systematic examination of worker oogenesis shows that oocytes are continually produced by young workers, irrespective of queen presence. Workers are therefore poised to exploit any reproductive opportunity to maximise their fitness. The term ‘worker sterility’ does not reflect the reproductive plasticity of the young adult honey bee worker.
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Acknowledgements
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We acknowledge the facilities, and the scientific and technical assistance, of the Australian Microscopy & Microanalysis Research Facility at the ACMM, the University of Sydney. The work was funded by Australian Research Council grants DP120101915 and DP1093491.
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References
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Alaux, C., Maisonnasse, A., Le Conte, Y., 2010. Chapter seventeen-pheromones in a superorganism: from gene to social regulation. Vitam. Horm. 83, 401–423. Berger, B., Abdalla, F.C., 2005. Programmed cell death during ovarian differentiation in queens of Apis mellifera Linné, 1758 (Hymenoptera, Apini), Brazilian. J. Morphol. Sci. 22, 1–4.
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Boleli, I.C., Paulino-Simões, Z.L., Gentile Bitondi, M.M., 1999. Cell death in ovarioles causes permanent sterility in Frieseomelitta varia worker bees. J. Morphol. 242, 271–282. Büning, J., 1994. The insect Ovary: Ultrastructure, Previtellogenic Growth and Evolution. Chapman and Hall, London, UK. Cavaliere, V., Taddei, C., Gargiulo, G., 1998. Apoptosis of nurse cells at the late stages of oogenesis of Drosophila melanogaster. Dev. Genes. Evol. 208, 106–112. Dallacqua, R.P., Bitondi, M.M.G., 2014. Dimorphic ovary differentiation in honeybee (Apis mellifera) larvae involves caste-specific expression of homologs of Ark and Buffy cell death genes. PLoS ONE 9, e98088. de Groot, A.P., Voogd, S., 1954. On the ovary development in queenless worker bees (Apis mellifica L.). Experientia 10, 384–385. Drummond-Barbosa, D., Spradling, A.C., 2001. Stem cells and their progeny respond to nutritional changes during Drosophila oogenesis. Dev. Biol. 231, 265–278. Gutzeit, H., Zissler, D., Fleig, R., 1993. Oogenesis in the honeybee Apis mellifera: cytological observations on the formation and differentiation of previtellogenic ovarian follicles. Roux’s Arch. Dev. Biol. 202, 181–191. Hayama, S.-I., Kamiya, S., Yamazaki, A., Daigo, M., Nigi, H., 1992. Lipofuscin in the corpus luteum of macaque ovaries. Primates 33, 133–137. Hodin, J., 2009. She shapes events as they come: plasticity in female insect reproduction. In: Whitman, D.W., Ananthakrishnan, T.N. (Eds.), Phenotypic Plasticity of Insects: Mechanisms and Consequences. Science publishers, U.S.A.. Höhn, A., Grune, T., 2013. Lipofuscin: formation, effects and role of macroautophagy. Redox Biol. 1, 140–144. Holmes, M.J., Oldroyd, B.P., Duncan, M., Allsopp, M.H., Beekman, M., 2013. Cheaters sometimes prosper: targeted worker reproduction in honeybee (Apis mellifera) colonies during swarming. Mol. Ecol. 22, 4298–4306. Hoover, S.E.R., Keeling, C.I., Winston, M.L., Slessor, K.N., 2003. The effect of queen pheromones on worker honey bee ovary development. Naturwissenschaften 90, 477–480. Koering, M.J., 1969. Cyclic changes in ovarian morphology during the menstrual cycle in Macaca mulatta. Am. J. Anat. 126, 73–101. Malka, O., Shnieor, S., Hefetz, A., Katzav-Gozansky, T., 2007. Reversible royalty in worker honeybees (Apis mellifera) under the queen influence. Behav. Ecol. Sociobiol. 61, 465–473. McCall, K., 2004. Eggs over easy: cell death in the Drosophila ovary. Developmental biology 274, 3–14. Mizushima, N., Yamamoto, A., Matsui, M., Yoshimori, T., Ohsumi, Y., 2004. In vivo analysis of autophagy in response to nutrient starvation using transgenic mice expressing a fluorescent autophagosome marker. Mol. Biol. Cell 15, 1101–1111. Mpakou, V.E., Nezis, I.P., Stravopodis, D.J., Margaritis, L.H., Papassideri, I.S., 2006. Programmed cell death of the ovarian nurse cells during oogenesis of the silkmoth Bombyx mori. Dev. Growth Differ. 48, 419–428. Nezis, I.P., Stravopodis, D.J., Papassideri, I., Margaritis, L.H., 2001. Actin cytoskeleton reorganization of the apoptotic nurse cells during the late developmental stages of oogenesis in Dacus oleae. Cell Motil. Cytoskelet. 48, 224–233. Nezis, I.P., Stravopodis, D.J., Papassideri, I., Robert-Nicoud, M., Margaritis, L.H., 2002. Dynamics of apoptosis in the ovarian follicle cells during the late stages of Drosophila oogenesis. Cell Tissue Res. 307, 401–409. Nezis, I.P., Stravopodis, D.J., Margaritis, L.H., Papassideri, I.S., 2006. Programmed cell death of follicular epithelium during the late developmental stages of oogenesis in the fruit flies Bactrocera oleae and Ceratitis capitata (Diptera, Tephritidae) is mediated by autophagy. Dev. Growth Differ. 48, 189–198. Otsuki, J., Nagai, Y., Chiba, K., 2007. Lipofuscin bodies in human oocytes as an indicator of oocyte quality. J. Assist. Reprod. Genet. 24, 263–270.
Patrício, K., Cruz-Landim, C., 2002. Mating influence in the ovary differentiation in adult queens of Apis mellifera L. (Hymenoptera, Apidae), Brazilian. J. Biol. 62, 641–649. Patrício, K., Cruz-Landim, C.D., 2008. Morphological aspects of cell reabsorption in laying queens and workers of Apis mellifera (Hymenoptera, Apidae), Iheringia. Série Zoologia 98, 421–424. Paulcke, W., 1901. Ueber die Differenzirung der Zellelemente im Ovarium der Bienenkönigin (Apis mellifica female). Zoologische Jahrbücher. Abteilung für Anatomie und Ontogenie der Tiere 14, 177–202. Pritchett, T., McCall, K., 2012. Role of the insulin/Tor signaling network in starvation-induced programmed cell death in Drosophila oogenesis. Cell Death Differ. 19, 1069–1079. Ratnieks, F.L., Visscher, P.K., 1989. Worker policing in the honeybee. Nature 342, 796–797. Sánchez, M.E., Martínez, O.E., Vázquez-Nin, G., 2012. Immunohistochemical and ultrastructural visualization of different routes of oocyte elimination in adult rats. Eur. J. Histochem. 56. Snodgrass, R.E., 1956. Anatomy of the honey bee. Cornell University Press, London, UK. Tanaka, E.D., Hartfelder, K., 2004. The initial stages of oogenesis and their relation to differential fertility in the honey bee (Apis mellifera) castes. Arthropod Struct. Dev. 33, 431–442. Tanaka, E., Schmidt-Capella, I., Hartfelder, K., 2006. Cell death in the germlinemechanisms and consequences for reproductive plasticity in social bees, Brazilian. J. Morphol. Sci. 23, 15–26. Terman, A., Brunk, U.T., 2004. Lipofuscin. Int. J. Biochem. Cell Biol. 36, 1400–1404. Terman, A., Brunk, U.T., 2005. Autophagy in cardiac myocyte homeostasis, aging, and pathology. Cardiovasc. Res. 68, 355–365. Tilly, J.L., 2001. Commuting the death sentence: how oocytes strive to survive. Nat. Rev. Mol. Cell Biol. 2, 838–848. Tóth, E., Queller, D.C., Dollin, A., Strassmann, J.E., 2004. Conflict over male parentage in stingless bees. Insectes Soc. 51, 1–11. Traynor, K.S., Le Conte, Y., Page Jr, R.E., 2014. Queen and young larval pheromones impact nursing and reproductive physiology of honey bee (Apis mellifera) workers. Behav. Ecol. Sociobiol. 68, 2059–2073. Velentzas, A.D., Nezis, I.P., Stravopodis, D.J., Papassideri, I.S., Margaritis, L.H., 2007a. Mechanisms of programmed cell death during oogenesis in Drosophila virilis. Cell Tissue Res. 327, 399–414. Velentzas, A.D., Nezis, I.P., Stravopodis, D.J., Papassideri, I.S., Margaritis, L.H., 2007b. Stage-specific regulation of programmed cell death during oogenesis of the medfly Ceratitis capitata (Diptera, Tephritidae). Int. J. Dev. Biol. 51, 57. Velthuis, H.H.W., 1970. Ovarian development in Apis mellifera worker bees. Entomol. Exp. Appl. 13, 377–394. Vergoz, V., Lim, J., Oldroyd, B.P., 2012. Biogenic amine receptor gene expression in the ovarian tissue of the honey bee Apis mellifera. Insect Mol. Biol. 21, 21–29. Wenseleers, T., Hart, A.G., Ratnieks, F.L.W., 2004. When resistance is useless: policing and the evolution of reproductive acquiescence in insect societies. Am. Nat. 164, E154–167. Winston, M.L., Slessor, K.N., 1992. The essence of royalty: honey bee queen pheromone. Am. Sci. 80, 374–385. Woyciechowski, M., Kuszewska, K., 2012. Swarming generates rebel workers in honeybees. Curr. Biol. 22, 707–711. Zimmerman, S.G., Peters, N.C., Altaras, A.E., Berg, C.A., 2013. Optimized RNA ISH, RNA FISH and protein-RNA double labeling (IF/FISH) in Drosophila ovaries. Nat. Protoc. 8, 2158–2179.
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Regulation of oogenesis in honey bee workers via programed cell death
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Isobel Ronai ⇑, Deborah A. Barton, Benjamin P. Oldroyd, Vanina Vergoz
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School of Biological Sciences, Macleay Building A12, The University of Sydney, Sydney, NSW 2006, Australia
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a r t i c l e
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Article history: Received 14 April 2015 Received in revised form 23 June 2015 Accepted 24 June 2015 Available online xxxx Keywords: Oogenesis Ovariole Programmed cell death Honey bee Worker sterility
a b s t r a c t Reproductive division of labour characterises eusociality. Currently little is known about the mechanisms that underlie the ‘sterility’ of the worker caste, but queen pheromone plays a major role in regulating the reproductive state. Here we investigate oogenesis in the young adult honey bee worker ovary in the presence of queen pheromone and in its absence. When queen pheromone is absent, workers can activate their ovaries and have well-developed follicles. When queen pheromone is present, even though workers have non-activated ovaries, they continually produce oocytes which are aborted at an early stage. Therefore, irrespective of the presence of the queen, the young adult worker ovary contains oocytes. By this means young workers retain reproductive plasticity. The degeneration of the germ cells in the ovarioles of workers in the presence of queen pheromone has the morphological hallmarks of programmed cell death. Therefore the mechanistic basis of ‘worker sterility’ relies in part on the regulation of oogenesis via programmed cell death. Our results suggest that honey bees have co-opted a highly conserved checkpoint at mid-oogenesis to regulate the fertility of the worker caste. Ó 2015 Published by Elsevier Ltd.
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1. Introduction
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In a social insect colony, the queen has reproductive hegemony over her sterile daughters. However there is a spectrum in the degree of ‘worker sterility’, even in bees. At one extreme there is the stingless bee genus Frieseomelitta where worker ovaries completely degenerate during development (Boleli et al., 1999). At the other extreme is the stingless bee genus Melipona where worker ovaries are well developed and workers contribute to male production (Tóth et al., 2004). The honey bee, Apis mellifera, is intermediate between these two extremes. The ovaries of honey bee workers are much reduced relative to those of queens, but remain functionally competent (Snodgrass, 1956). The honey bee worker has a high degree of reproductive plasticity. Queen mandibular pheromone suppresses ovary activation in workers (Hoover et al., 2003). In the absence of a queen the workers (which cannot mate) have the potential to activate their ovaries and lay haploid male-destined eggs. Conversely, if workers with activated ovaries are moved from a queenless colony to a queenright colony their ovaries regress (Malka et al., 2007). Workers also activate their ovaries on a seasonal basis, in particular during swarming season when colonies are temporarily queenless after the old queen has departed and the new queen has not begun to lay eggs (Holmes et al., 2013; Woyciechowski and Kuszewska,
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⇑ Corresponding author. E-mail address: [email protected] (I. Ronai).
2012). Another group of pheromones, emitted by the brood also contribute to suppressing ovary activation in workers (Alaux et al., 2010; Traynor et al., 2014). The ovarioles comprise the structural and functional units of the honey bee ovary. The major reproductive difference between the two female castes is that workers have 2–12 ovarioles per ovary whereas queens have 160–180 (Snodgrass, 1956). It is important to note that the term ‘ovariole’ is used to describe both the outer layer of cells of the ovariole (epithelial sheath, otherwise known as the tunica externa) and the inner, non-cellular membrane (tunica propria). Inside the tunica propria are both somatic cells and germ cells. The somatic cells encapsulate and support the germ cells. A germ cell gives rise not only to an oocyte but also to nutritive nurse cells, which remain directly connected to the oocyte to allow the transport of nutrients into the developing oocyte (Büning, 1994). The honey bee ovary is therefore classified as being polytrophic meroistic. The majority of studies on the adult honey bee ovary have focused on oogenesis in the queen (Berger and Abdalla, 2005; Patrício and Cruz-Landim, 2002; Paulcke, 1901; Snodgrass, 1956) with only one study examining early oogenesis in queenright and queenless workers (Tanaka and Hartfelder, 2004). Here, we systematically investigate oogenesis in the young adult worker ovary in the presence or absence of queen pheromone in a controlled environment without the confounding factor of brood pheromones. We were expecting no oocytes in the ovarioles of workers in the presence of queen pheromone and that oocytes would be
http://dx.doi.org/10.1016/j.jinsphys.2015.06.014 0022-1910/Ó 2015 Published by Elsevier Ltd.
Please cite this article in press as: Ronai, I., et al. Regulation of oogenesis in honey bee workers via programed cell death. Journal of Insect Physiology (2015), http://dx.doi.org/10.1016/j.jinsphys.2015.06.014
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present in the ovarioles of workers in the absence of queen pheromone. Instead we show that queen pheromone alters oocyte development in the worker, thereby leading to ‘worker sterility’.
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2. Materials and methods
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2.1. Biological material
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Age-matched adult workers of Australian commercial stock derived from A. m. ligustica were obtained by incubating overnight combs of emerging brood. The brood was from a single queenright colony which minimises the genetic and environmental heterogeneity among experimental treatments. The following day, the emerged workers were placed in four laboratory cages (n 150 per cage) each fitted with a 5 5 cm section of natural honey comb. Two of the cages contained a strip (0.5 queen equivalents) of queen mandibular pheromone (Phero Tech Inc., Canada) to simulate queen presence, whereas the other two cages contained no queen pheromone. The caged workers were provided with honey, ground pollen and water ad libitum. Workers were collected at 14 days of age and samples immediately frozen on dry ice.
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2.2. Tissue dissection
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We dissected a subset (n = 77) of the workers in cold PBS under a dissecting microscope (Olympus SZ40): workers exposed to queen pheromone (n = 41) and not exposed to queen pheromone (n = 36). The tergal surface was opened and the gut, fat body and first four tergites removed, leaving the abdominal cavity containing the paired ovaries (anchored to the last tergite) and the venom gland. During the dissection, the ovaries were scored as: non-activated (transparent, thread-like ovaries) or activated (opaque, swollen ovaries) (Vergoz et al., 2012). In cages containing queen pheromone, no workers had activated ovaries, whereas in cages with no queen pheromone 12.5% of workers had activated ovaries.
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2.3. Tissue preparation
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The ovaries were prepared as if for in situ hybridisation (Dallacqua and Bitondi, 2014; Zimmerman et al., 2013). Four abdominal cavities with their connected ovaries were placed in a vial containing heptane fixative (1 mL heptane, 80 lL HEPES/HEM buffer, 100 lL of 8% (w/v) paraformaldehyde and 20 lL DMSO) and shaken for 30 min. The samples were rinsed in PBS and then dehydrated with an increasing concentration of ethanol (25%, 50%, 75% and 100% v/v). These were stored overnight at 20 °C. The samples were rehydrated with a decreasing concentration of ethanol (75%, 50% and 25% v/v) and then PBS-Tween 20. Samples were fixed in triton fixative (89.01% PBS, 0.09% Triton X-100, 0.9% of 16% (w/v) paraformaldehyde and 10% DMSO v/v), shaken for 20 min and washed in PBS-Tween 20. The samples were incubated in proteinase K solution (approximately 20 lg/mL) for 15 min, then twice washed in glycine (1% w/v) and PBS-Tween 20. The samples were refixed in triton fixative and shaken for 20 min and washed in PBS-Tween 20. Nucleic acid was stained with SYTOX Blue Nucleic Acid Stain (Invitrogen) (0.002% v/v) for 20 min and washed in PBS-Tween 20. The paired ovaries were then dissected away from their abdominal cavity in PBS-Tween 20, immersed in glycerol (70% v/v) and mounted on individual slides.
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2.4. Imaging
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Paired ovaries were observed immediately under a laser scanning confocal microscope (Leica TCS SP5 II). SYTOX Blue was
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excited by a 458 nm laser and emitted light was collected between 465 and 484 nm. Red autofluorescence was excited by 561 nm and 633 nm lasers and emitted light was collected between 580 and 650 nm and 650–700 nm, respectively.
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3. Results
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The ovariole of the adult honey bee worker shows successive developmental stages of oogenesis from the tip to the base. The ovariole can be divided into regions depending on cell type (somatic or germ) and stage of germ cell development. These regions are the terminal filament, the germarium, the vitellarium, and the pedicel (which is sometimes referred to as the basal stalk) (Fig. 1A). Workers not exposed to queen pheromone can have activated ovaries and their ovarioles have well-developed oocytes. The developing oocytes cause the epithelial sheath to bulge (Fig. 1A) producing the defining feature of the activated ovary phenotype. The basal end of the tunica propria of an activated ovary is 71 lm ± 6 lm (n = 5) in diameter. The terminal filament, a stack of aligned disc-shaped cells that are orientated transversely (Fig. 1B), is located at the apical end of the tunica propria. This filament contains cells that are mainly of somatic origin but germline stem cells are also present (Tanaka and Hartfelder, 2004). In the early-germarium stage, there are undifferentiated germ cells known as oogonia (Fig. 1C). In this region, individual cells are hard to differentiate. In the mid-germarium stage, the early oocyte is centrally located and is differentiated by size from its surrounding cells that develop into trophocyte nurse cells (approximately 39– 59 per oocyte (Büning, 1994)) (Fig. 1D). The nucleus of the oocyte, known as the germinal vesicle, is relatively large and contains a small black dot indicative of the chromatin beginning to condense. In the late-germarium stage, the oocytes are now located at the basal end of their associated nurse cells (Fig. 1E). The germinal vesicle by this point appears as an unstained dark structure due to the condensed chromatin inside, known as the karyosome. The vitellarium stage of development is where self-contained follicles are present (Büning, 1994). At the vitellarium stage the oocyte is enlarged and encapsulated by a monolayer of columnar follicle cells (Fig. 1F), which are somatic in origin. In the nurse chamber the nurse cell size follows a gradient; those cells closest to the oocyte are the largest (Fig. 1A and F). The nurse cells have very large nuclei relative to the cytoplasm as well as bright staining around their nuclei (Fig. 1F) which is indicative of high levels of transcription in these cells (Gutzeit et al., 1993). Each ovariole terminates in a structure called the pedicel (Fig. 1G), which is somatic in origin. The pedicel acts as a plug to the oviduct opening and degrades when an egg is ready to be laid (Büning, 1994). Late oogenesis is characterised by developmental programmed cell death of the nurse cells (Fig. 1H). The basal nurse cells have a rounded appearance, condensed chromatin and an absent cytoplasm (Fig. 1H) due to the ‘dumping’ of their cytoplasmic contents into the oocyte (Cavaliere et al., 1998). Sometimes the ovarioles of workers not exposed to queen pheromone show what we term a ‘transition phenotype’ between non-activated ovaries and activated ovaries (Fig. 1I). In these ovarioles the germarium region has both healthy and dead oocytes. The developing oocytes in the middle of the tunica propria are healthy, as indicated by chromatin condensing in the germinal vesicles (Fig. 1I). However, at the basal end of the tunica propria the oocytes are dead as evidenced by the absence of staining (Fig. 1I). The oocytes’ nurse cells are dying, as evidenced by the pycnotic nuclei (central and highly compact, the condensed chromatin stains heavily) (Fig. 1I).
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Fig. 1. The ovarioles of adult workers not exposed to queen pheromone showing activated ovaries with well-developed oocytes or a ‘transition phenotype’. (A) A montage of images digitally reconstructing an entire ovariole (above) and a schematic representation of the ovariole (below) delineating the regions: terminal filament; germarium; vitellarium; and pedicel. The ovariole has a non-cellular membrane, the tunica propria (TP) and an outer layer of cells, the epithelial sheath (ES). Particular cell types of the ovariole include the trophocyte nurse cells (nc), oocytes (oo) and follicle cells (fc). The nucleus of the oocyte is termed the germinal vesicle (gv). (B) The terminal filament. (C) The early-germarium. (D) The mid-germarium. (E) The late-germarium. (F) The vitellarium. The oocyte (oo) is surrounded by follicle cells (fc) and is accompanied by nurse cells (nc). The germinal vesicle (gv) of the oocyte contains condensed chromatin, termed the karyosome (arrowhead). (G) The pedicel. (H) The basal nurse cells (nc) undergo developmental programmed cell death, the nuclei condense (arrows). (I) A montage of images digitally reconstructing an ovariole of a ‘transition phenotype’. At the apical end of the lower ovariole, oocytes are healthy and at the basal end oocytes are dying. Scale bars represent: (A, I) 50 lm; (B–H) 20 lm.
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Workers exposed to queen pheromone have non-activated ovaries and their ovarioles show widespread cell death. The dying cells are disorganised and have irregular nuclear staining, compared to the healthy epithelial sheath cells (Fig. 2) and cells of healthy ovarioles (Fig. 1A). The tunica propria often lacks a cohesive structure and is irregularly shaped, a characteristic of the non-activated ovary phenotype (Fig. 2A and B). The basal end of the tunica propria of a non-activated ovary is 41 lm ± 6 lm (n = 5) in diameter. The terminal filament is present and the cells look healthy. However, the widespread cell death occurring in the tunica propria causes the terminal filament to be positioned more basally inside the tunica propria (Fig. 2A and B compared to 1A). The germarium region is noticeably shorter than in workers not exposed to queen pheromone (Fig. 2A and B compared to 1A). The early-germarium oogonia are the last remaining healthy germ cells (Fig. 2B), however, these are sometimes in the process of
dying (Fig. 2A). In the mid to late germarium all oocytes and nurse cells are dead or dying. The oocytes shrink and their cytoplasm is degrading and contains numerous vacuoles, as indicated by the absence of staining (Fig. 2C and D). The nurse cells shrink and some have pycnotic nuclei (Fig. 2C) whereas others are dead, appearing as empty voids (Fig. 2D). Possible autophagic vacuoles are often present in highly degenerated germarium regions, and in the final stages of degeneration the tunica propria contains empty spaces (Fig. 2A and B). Non-activated ovaries do not develop follicles and therefore never reach the vitellarium stage of development (Fig. 2). It is important to note that among the workers exposed to queen pheromone there is considerable variation in the stage of cell death observed in the ovaries, even when workers are of the same age (Fig. 2). Using fluorescence microscopy, we found evidence of a lipofuscin-like pigment, an undegradable subcellular aggregate of
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Fig. 2. The ovarioles of adult workers exposed to queen pheromone showing non-activated ovaries with widespread cell death. The terminal filament (TF) is present; the germarium region has dead or dying oocytes (oo) and nurse cells (nc); and no vitellarium stage develops. (A and B) Montage of images digitally reconstructing entire ovarioles in the final stages of degeneration. (C) An ovariole in the initial stages of degeneration. (D) An ovariole midway through degeneration. Tracheole (T). Scale bars represent 50 lm.
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oxidised proteins and lipids (Höhn and Grune, 2013), in the ovaries (Fig. 3). Under all fluorescent light channels lipofuscin has strong autofluorescence and this is used to detect its presence in tissues
(Terman and Brunk, 2004). The lipofuscin-like pigment is associated with the degenerating germarium region in non-activated ovaries (Fig. 3A), but is absent from this region in activated ovaries
Fig. 3. Lipofuscin-like pigment in the ovaries of adult workers. Ovaries stained with SYTOX Blue nucleic acid stain (blue) with autofluorescent (magenta) lipofuscin-like pigment and tracheoles (T). (A) Lipofuscin (arrowheads) in degenerating germarium regions of non-activated ovaries. (B) Lipofuscin is absent from the germarium region of an activated ovary. (C) Lipofuscin at the tip of the terminal filament (arrowhead). Scale bars represent: (A and C) 50 lm; (B) 20 lm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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(Fig. 3B). The lipofuscin-like pigment is also found at the tip of the terminal filament, regardless of ovary state (Fig. 3C).
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Our study reveals the cytological basis of queen pheromone ‘control’ over worker reproduction (de Groot and Voogd, 1954; Hoover et al., 2003; Malka et al., 2007; Winston and Slessor, 1992): queen pheromone causes the abortion of developing oocytes. Some workers exposed to queen pheromone have a tunica propria with such a highly degenerated germarium region that it seems unlikely ovarioles of this kind could ever recover. Tanaka et al. (2006) stated that in the ovaries of older workers engaged in foraging some of the ovarioles have only the epithelial sheath remaining. However, it is important to note that we always found the terminal filament (which contains germline stem cells (Tanaka and Hartfelder, 2004)) intact, regardless of queen presence. This finding leads us to hypothesise that oogenesis might be able to resume if the environmental conditions becomes favourable. We also suggest that exposure to queen pheromone as larvae might cause ‘scarring’ in the adult ovary. Workers that are not exposed to queen pheromone as adults but have been reared (as larvae and pupae) with a queen, sometimes have degenerating oocytes in the basal region of the tunica propria. These oocytes likely developed when the workers were larvae (Hodin, 2009) and reared with the queen. In contrast, a recent study showed that larvae reared in queenless colonies have high rates of ovary activation as adults (Woyciechowski and Kuszewska, 2012). The presence of the queen is the initial and most critical environmental signal for the reproductive fate of the worker. We observed that the degeneration of the ovariole of adult workers exposed to queen pheromone follows a characteristic sequence, which includes the morphological hallmarks of programmed cell death. First, we found that the oocyte dies. The same phenomena is observed in the queen honey bee, the death of the oocyte is thought to initiate the degeneration of the germarium region (Patrício and Cruz-Landim, 2008) and is also observed in rat ovaries (Sánchez et al., 2012). Second, we found that the nurse cells seem to show some characteristics of apoptosis (chromatin disorganisation, pycnotic nuclei and DNA fragmentation) followed by autophagy (formation of vacuoles and structural disorganisation). Studies in other organisms have showed that both apoptosis and autophagy occur during oogenesis and results in cells being rapidly removed. These two types of programmed cell death are complementary; autophagy efficiently removes the debris once apoptosis has completed (Mpakou et al., 2006; Sánchez et al., 2012; Velentzas et al., 2007a,b). Lastly, we showed that the degeneration culminates in dead cells and empty spaces inside the tunica propria. The empty spaces are most likely due to phagocytosis of the cellular debris after programmed cell death occurs (Mpakou et al., 2006; Nezis et al., 2006, 2001; Nezis et al., 2002), which allows the recycling of these resources. Interestingly, the ovariole degeneration in the honey bee is analogous to follicular atresia that occurs during vertebrate oogenesis (Tilly, 2001). Further molecular work is required to determine how programmed cell death regulates oogenesis in the adult worker. The presence of lipofuscin in the ovary is interesting for two reasons. First, lipofuscin is characteristic of degenerating ovarian follicles in primates (Hayama et al., 1992; Koering, 1969) and is associated with poor fertilisation rates and lower developmental competence in human oocytes (Otsuki et al., 2007). Second, lipofuscin is associated with programmed cell death (Höhn and Grune, 2013). In particular, lipofuscin is implicated in the process of autophagy (Mizushima et al., 2004; Terman and Brunk, 2005). As lipofuscin is present in non-activated ovaries when late stage
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degeneration is occurring, we propose that it could be used as a marker of this degeneration. The dimorphism between the worker and queen castes in the honey bee embodies the reproductive division of labour. As adults these genotypically identical castes have strikingly similar ovariole structure (though workers have an order of magnitude fewer ovarioles) and the key difference between the non-reproductive and reproductive castes is the progression through the stages of oogenesis. Queens progress through all the stages of oogenesis and lay eggs (Snodgrass, 1956), whereas we found that workers exposed to queen pheromone continually produce oocytes, but these oocytes never reach the vitellarium stage of oogenesis. In adult workers the progression from the germarium stage of oogenesis to the vitellarium stage means passing a critical checkpoint at mid-oogenesis. We found that queen pheromone represses follicle development and this has also been identified in queenright workers (Tanaka and Hartfelder, 2004). This checkpoint is employed by other organisms in response to a variety of adverse environmental conditions and utilises programmed cell death to arrest oogenesis (McCall, 2004). For example, when Drosophila do not receive sufficient food this triggers the degeneration of the oocytes at this checkpoint (Drummond-Barbosa and Spradling, 2001; Pritchett and McCall, 2012). Our study suggests that honey bees, and perhaps eusocial insects more generally have co-opted this evolutionary conserved oogenesis checkpoint to allow the evolution of ‘worker sterility’. Why has the mid-oogenesis checkpoint been selected to regulate oogenesis in the worker honey bee? Most likely utilising this checkpoint is an evolutionary trade-off that maximises the fitness of the worker. In a queenright environment there is little chance of a worker’s eggs surviving (Ratnieks and Visscher, 1989; Wenseleers et al., 2004) so the oocytes that are continually produced are aborted. The maturation of the oocyte is nutritionally demanding, so stopping oogenesis at this checkpoint reduces waste of resources. Conversely, the continual production of oocytes means that a young worker can rapidly switch to producing eggs if the environmental conditions become favourable for personal reproduction, for example, if the queen disappears. The use of this checkpoint allows the fine tuning of worker oogenesis to an environmental cue and thereby provides adaptive reproductive plasticity of the adult worker. On reflection the widespread use of the term ‘worker sterility’ in the honey bee literature needs to be reassessed. A workers’ ovary is thought to be in a state of ‘rest’ (Velthuis, 1970) as there are no visible oocytes and only under suitable environmental conditions do workers ‘activate’ their ovaries. Our systematic examination of worker oogenesis shows that oocytes are continually produced by young workers, irrespective of queen presence. Workers are therefore poised to exploit any reproductive opportunity to maximise their fitness. The term ‘worker sterility’ does not reflect the reproductive plasticity of the young adult honey bee worker.
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Acknowledgements
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We acknowledge the facilities, and the scientific and technical assistance, of the Australian Microscopy & Microanalysis Research Facility at the ACMM, the University of Sydney. The work was funded by Australian Research Council grants DP120101915 and DP1093491.
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References
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Alaux, C., Maisonnasse, A., Le Conte, Y., 2010. Chapter seventeen-pheromones in a superorganism: from gene to social regulation. Vitam. Horm. 83, 401–423. Berger, B., Abdalla, F.C., 2005. Programmed cell death during ovarian differentiation in queens of Apis mellifera Linné, 1758 (Hymenoptera, Apini), Brazilian. J. Morphol. Sci. 22, 1–4.
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Boleli, I.C., Paulino-Simões, Z.L., Gentile Bitondi, M.M., 1999. Cell death in ovarioles causes permanent sterility in Frieseomelitta varia worker bees. J. Morphol. 242, 271–282. Büning, J., 1994. The insect Ovary: Ultrastructure, Previtellogenic Growth and Evolution. Chapman and Hall, London, UK. Cavaliere, V., Taddei, C., Gargiulo, G., 1998. Apoptosis of nurse cells at the late stages of oogenesis of Drosophila melanogaster. Dev. Genes. Evol. 208, 106–112. Dallacqua, R.P., Bitondi, M.M.G., 2014. Dimorphic ovary differentiation in honeybee (Apis mellifera) larvae involves caste-specific expression of homologs of Ark and Buffy cell death genes. PLoS ONE 9, e98088. de Groot, A.P., Voogd, S., 1954. On the ovary development in queenless worker bees (Apis mellifica L.). Experientia 10, 384–385. Drummond-Barbosa, D., Spradling, A.C., 2001. Stem cells and their progeny respond to nutritional changes during Drosophila oogenesis. Dev. Biol. 231, 265–278. Gutzeit, H., Zissler, D., Fleig, R., 1993. Oogenesis in the honeybee Apis mellifera: cytological observations on the formation and differentiation of previtellogenic ovarian follicles. Roux’s Arch. Dev. Biol. 202, 181–191. Hayama, S.-I., Kamiya, S., Yamazaki, A., Daigo, M., Nigi, H., 1992. Lipofuscin in the corpus luteum of macaque ovaries. Primates 33, 133–137. Hodin, J., 2009. She shapes events as they come: plasticity in female insect reproduction. In: Whitman, D.W., Ananthakrishnan, T.N. (Eds.), Phenotypic Plasticity of Insects: Mechanisms and Consequences. Science publishers, U.S.A.. Höhn, A., Grune, T., 2013. Lipofuscin: formation, effects and role of macroautophagy. Redox Biol. 1, 140–144. Holmes, M.J., Oldroyd, B.P., Duncan, M., Allsopp, M.H., Beekman, M., 2013. Cheaters sometimes prosper: targeted worker reproduction in honeybee (Apis mellifera) colonies during swarming. Mol. Ecol. 22, 4298–4306. Hoover, S.E.R., Keeling, C.I., Winston, M.L., Slessor, K.N., 2003. The effect of queen pheromones on worker honey bee ovary development. Naturwissenschaften 90, 477–480. Koering, M.J., 1969. Cyclic changes in ovarian morphology during the menstrual cycle in Macaca mulatta. Am. J. Anat. 126, 73–101. Malka, O., Shnieor, S., Hefetz, A., Katzav-Gozansky, T., 2007. Reversible royalty in worker honeybees (Apis mellifera) under the queen influence. Behav. Ecol. Sociobiol. 61, 465–473. McCall, K., 2004. Eggs over easy: cell death in the Drosophila ovary. Developmental biology 274, 3–14. Mizushima, N., Yamamoto, A., Matsui, M., Yoshimori, T., Ohsumi, Y., 2004. In vivo analysis of autophagy in response to nutrient starvation using transgenic mice expressing a fluorescent autophagosome marker. Mol. Biol. Cell 15, 1101–1111. Mpakou, V.E., Nezis, I.P., Stravopodis, D.J., Margaritis, L.H., Papassideri, I.S., 2006. Programmed cell death of the ovarian nurse cells during oogenesis of the silkmoth Bombyx mori. Dev. Growth Differ. 48, 419–428. Nezis, I.P., Stravopodis, D.J., Papassideri, I., Margaritis, L.H., 2001. Actin cytoskeleton reorganization of the apoptotic nurse cells during the late developmental stages of oogenesis in Dacus oleae. Cell Motil. Cytoskelet. 48, 224–233. Nezis, I.P., Stravopodis, D.J., Papassideri, I., Robert-Nicoud, M., Margaritis, L.H., 2002. Dynamics of apoptosis in the ovarian follicle cells during the late stages of Drosophila oogenesis. Cell Tissue Res. 307, 401–409. Nezis, I.P., Stravopodis, D.J., Margaritis, L.H., Papassideri, I.S., 2006. Programmed cell death of follicular epithelium during the late developmental stages of oogenesis in the fruit flies Bactrocera oleae and Ceratitis capitata (Diptera, Tephritidae) is mediated by autophagy. Dev. Growth Differ. 48, 189–198. Otsuki, J., Nagai, Y., Chiba, K., 2007. Lipofuscin bodies in human oocytes as an indicator of oocyte quality. J. Assist. Reprod. Genet. 24, 263–270.
Patrício, K., Cruz-Landim, C., 2002. Mating influence in the ovary differentiation in adult queens of Apis mellifera L. (Hymenoptera, Apidae), Brazilian. J. Biol. 62, 641–649. Patrício, K., Cruz-Landim, C.D., 2008. Morphological aspects of cell reabsorption in laying queens and workers of Apis mellifera (Hymenoptera, Apidae), Iheringia. Série Zoologia 98, 421–424. Paulcke, W., 1901. Ueber die Differenzirung der Zellelemente im Ovarium der Bienenkönigin (Apis mellifica female). Zoologische Jahrbücher. Abteilung für Anatomie und Ontogenie der Tiere 14, 177–202. Pritchett, T., McCall, K., 2012. Role of the insulin/Tor signaling network in starvation-induced programmed cell death in Drosophila oogenesis. Cell Death Differ. 19, 1069–1079. Ratnieks, F.L., Visscher, P.K., 1989. Worker policing in the honeybee. Nature 342, 796–797. Sánchez, M.E., Martínez, O.E., Vázquez-Nin, G., 2012. Immunohistochemical and ultrastructural visualization of different routes of oocyte elimination in adult rats. Eur. J. Histochem. 56. Snodgrass, R.E., 1956. Anatomy of the honey bee. Cornell University Press, London, UK. Tanaka, E.D., Hartfelder, K., 2004. The initial stages of oogenesis and their relation to differential fertility in the honey bee (Apis mellifera) castes. Arthropod Struct. Dev. 33, 431–442. Tanaka, E., Schmidt-Capella, I., Hartfelder, K., 2006. Cell death in the germlinemechanisms and consequences for reproductive plasticity in social bees, Brazilian. J. Morphol. Sci. 23, 15–26. Terman, A., Brunk, U.T., 2004. Lipofuscin. Int. J. Biochem. Cell Biol. 36, 1400–1404. Terman, A., Brunk, U.T., 2005. Autophagy in cardiac myocyte homeostasis, aging, and pathology. Cardiovasc. Res. 68, 355–365. Tilly, J.L., 2001. Commuting the death sentence: how oocytes strive to survive. Nat. Rev. Mol. Cell Biol. 2, 838–848. Tóth, E., Queller, D.C., Dollin, A., Strassmann, J.E., 2004. Conflict over male parentage in stingless bees. Insectes Soc. 51, 1–11. Traynor, K.S., Le Conte, Y., Page Jr, R.E., 2014. Queen and young larval pheromones impact nursing and reproductive physiology of honey bee (Apis mellifera) workers. Behav. Ecol. Sociobiol. 68, 2059–2073. Velentzas, A.D., Nezis, I.P., Stravopodis, D.J., Papassideri, I.S., Margaritis, L.H., 2007a. Mechanisms of programmed cell death during oogenesis in Drosophila virilis. Cell Tissue Res. 327, 399–414. Velentzas, A.D., Nezis, I.P., Stravopodis, D.J., Papassideri, I.S., Margaritis, L.H., 2007b. Stage-specific regulation of programmed cell death during oogenesis of the medfly Ceratitis capitata (Diptera, Tephritidae). Int. J. Dev. Biol. 51, 57. Velthuis, H.H.W., 1970. Ovarian development in Apis mellifera worker bees. Entomol. Exp. Appl. 13, 377–394. Vergoz, V., Lim, J., Oldroyd, B.P., 2012. Biogenic amine receptor gene expression in the ovarian tissue of the honey bee Apis mellifera. Insect Mol. Biol. 21, 21–29. Wenseleers, T., Hart, A.G., Ratnieks, F.L.W., 2004. When resistance is useless: policing and the evolution of reproductive acquiescence in insect societies. Am. Nat. 164, E154–167. Winston, M.L., Slessor, K.N., 1992. The essence of royalty: honey bee queen pheromone. Am. Sci. 80, 374–385. Woyciechowski, M., Kuszewska, K., 2012. Swarming generates rebel workers in honeybees. Curr. Biol. 22, 707–711. Zimmerman, S.G., Peters, N.C., Altaras, A.E., Berg, C.A., 2013. Optimized RNA ISH, RNA FISH and protein-RNA double labeling (IF/FISH) in Drosophila ovaries. Nat. Protoc. 8, 2158–2179.
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Please cite this article in press as: Ronai, I., et al. Regulation of oogenesis in honey bee workers via programed cell death. Journal of Insect Physiology (2015), http://dx.doi.org/10.1016/j.jinsphys.2015.06.014