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MORPHOLOGICAL CHARACTERIZATION OF CELL DEATH DURING THE OVARY DIFFERENTIATION IN WORKER HONEY BEE
Cell Biology International 2002, Vol. 26, No. 3, 243–251 doi:10.1006/cbir.2001.0839, available online at http://www.idealibrary.com on
MORPHOLOGICAL CHARACTERIZATION OF CELL DEATH DURING THE OVARY DIFFERENTIATION IN WORKER HONEY BEE REJANE DANIELE REGINATO and CARMINDA DA CRUZ-LANDIM Departamento de Biologia, Instituto de Biocieˆncias, Universidade Estadual Paulista (UNESP), 13506-900, Rio Claro, SP, Brasil Received 28 November 2000; accepted 27 September 2001
Cell death that occurs during ovary differentiation in the honeybee worker’s larval development accounts for ovariole reabsorption. From a morphological standpoint, three modes of death were detected. Germinative cells in the ovarioles die by an apoptotic-like process, whereas the somatic cells die by an autophagic process, type II cell death; and during pupation, stromatic and ovarian capsular cells die through cytoplasmic disintegration, releasing their components into the hemolymph. These modes of cell death are in part determined by the pattern of tissue 2002 Elsevier Science Ltd. All rights reserved. organization within which the cell occurs. K: honeybee; castes; ovary; differentiation cell death; tissue organization.
INTRODUCTION Marked caste dimorphism has evolved in the highly eusocial honey bee. The typical queen and worker characteristics are different from one another in many morphological, physiological and behavioural aspects, among which ovarian development is very prominent. The worker caste is distinguished by a very limited reproductive capacity and correspondingly has underdeveloped ovaries compared with the queen (Ribbands, 1953; Snodgrass, 1956; Free, 1981). Differences between queen and worker ovaries mainly concern ovariole number, which is extremely high in queens and very low in workers, and is due to an environmentally-induced ovariolar reabsorption during differentiation in the latter (Lotmar, 1915; Beetsma, 1979; Bueno, 1981). Progressive feeding of honey bee larvae leads to several assumptions, one of which is that the special ‘royal’ diet determines the queen type. The pioneering studies of Leuchart (1855) first showed that qualitative differences between royal jelly and worker food was responsible for queen differentiation. As with other social insects, however, development into a worker or queen is regulated by the corpora allata (Dixon and Shuel, 1963; Velthuis, 1970; Kerr et al., 1974; Rembold, 1987; Fax: 55-19-534-0009. E-mail: [email protected] 1065–6995/02/$-see front matter
Rachinsky et al. 1990), an endocrine organ producing juvenile hormone. This means that there is a caste-specific modulation of the general pattern of juvenile hormone synthesis based on the quality and amount of food ingested by the larvae, leading to the differences between castes (Rachinsky et al., 1990; Rachinsky and Hartfelder, 1990, 1991; Rachinsky and Engels, 1995), expressed in the worker ovaries as cell death, and resulting in diminished ovariole numbers. Cell death during development or the life of an organism is generally referred to as ‘programmed cell death’, an expression first used by Lockshin and Williams (1964) to describe the breakdown of intersegmental muscles of silkworms during metamorphosis. The original use was an adaptation of the ‘death clock’ concept of Saunders (1966) who suggested that cells contain instructions for their own destruction, without simultaneously being irreversibly damaged. The term distinguishes physiological (programmed) cell death (Wyllie et al., 1984a; Lockshin, 1985) from traumatic (pathological) cell death. Its structural manifestations are shrinkage of cell volume, loss of specialized plasma membrane regions, such as microvilli, conservation of most cytoplasmic organelles, progressive perinuclear chromatin condensation, and release of signals which cause engulfment by 2002 Elsevier Science Ltd. All rights reserved.
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adjacent phagocytes (Kerr, 1971; Wyllie et al., 1984a,b; Arends and Wyllie, 1991). Cell death with these characteristics was, in fact, named apoptosis in recognition of its significance to tissue and organism homeostasis (Kerr et al., 1972). Apoptotic cell death involves activators, effectors and negative regulators within a cascade of events (Barinaga, 1998), as is known to occur during insect metamorphosis, and specifically during ovary development in worker bees. However, the morphological and biochemical features characterizing cellular apoptosis are not always present in insects (Clarke, 1990; Lockshin et al., 1991; Lockshin and Zakeri, 1996), where other types of cell death have been described (Bowen et al., 1993, 1996; Gregorc and Bowen, 1997; Hartfelder and Steinbruck, 1997; Silva de Moraes and Bowen, 2000; Cruz-Landim and Silva de Moraes, 2000). Thus the present work is an attempt to characterize morphologically the types of cell death occurring during ovary differentiation in worker bees.
MATERIALS AND METHODS Ovaries excised from 2–5th instar larvae and prepupa were fixed in Karnowsky fluid for transmission electron microscopy, and postfixed in 1% osmium tetraoxide in 0.1 sodium cacodylate buffer, dehydrated in serial concentrations of acetone and embedded in Epon-Araldite. Thin sections (800–1000 nm) were stained with uranyl acetate and lead citrate before being examined in a Philips electron microscope. Thick sections were stained with 1% toluidine blue for light microscopic examination. Ovaries were also prepared for cytochemical investigation of acid phosphatase in the dying cells, using p-nitrophenylphosphate as substrate (Ryder and Bowen, 1975).
RESULTS The ovary is comprised of somatic and germinative cells, the former creating the framework of the gonad, and the germinative cells being reproductive. In the first instar newly-hatched larvae, a mass of cells of different sizes remain indistinguishable as somatic or germinative, enveloped by a sheath comprised of several layers of flat cells, the capsula (Fig. 1A). As the larval phase progresses, the ovary differentiates by creating ovarioles within the primitive cell mass. The individualization of
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ovariole structures begins in the 2nd instar larvae by inward proliferation of the flat capsula cells. The germinative cells become located in the ovarioles as these become elongated structures limited by layers of flat cells as part of the peritoneal membrane (Fig. 1B). The ovarioles are separated from one another by interovariolar cells of the larval ovarian stroma (Fig. 1B,C). The germinative cells proliferate and form clusters which later undergo oogenesis inside the ovarioles, supported by intraovariolar somatic cells. Therefore, the somatic cells in the ovary present different forms, with variable location and function, such as capsule cells, stromal cells, peritoneal membrane and intraovariolar somatic cells (Fig. 1B,C). The germinative cells appear early as big round cells, and later form clusters of interconnected elements (Fig. 1A,B). During ovary differentiation in the worker bee, cell death in germinative and somatic cells, both in the inner and outer parts of the ovariole, present different characteristics according their lineage and location. Cell death was first detected within ovarioles of 3rd instar larvae (Fig. 2i). Cell death inside the ovarioles continues at increasing rates during the 4th and 5th instar stages, but with the death of germinative and somatic cells within the ovarioles showing different characteristics. Two types of morphology were observed in ovariolar cell death, one of almost classic apoptotic nature, with the dying cell first acquiring an almost spherical shape, with dense cytoplasm and condensed nuclear chromatin. Later fragmentation and formation of apoptotic bodies could be seen, as well as their engulfment by neighbouring cells (Fig. 2A–E). The cells showing this kind of death are almost certainly germinative. At the same time and within the same ovariole, cells generally located more peripherally and containing numerous vacuoles (some including organelle remnants, multivesicular bodies and myelin figures) occur. In spite of the presence of these degenerative structures in their cytoplasm, their nuclear chromatin remains dispersed and well-structured nucleoli are present. The cytoplasm and its organelles also appear normal (Fig. 3A–C). These cells, which appear to be having problems with autophagic reabsorption or processing, are mostly somatic in type. These two kinds of cell death were observed during the 3rd instar larvae or when the nutritional differences between worker and queen became established.; both lead to a decrease in ovariole number in the worker bees. The ovariole where
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Fig. 1. Light micrographs of the larval ovary. A. Ovary (ov) of a 1st instar larvae showing the capsula (c) and its content of large (l) and small (s) cells. B. Longitudinal section and C. Cross section of a portion of an ovary of a 4th instar larva showing its organization. g=clusters of germinative cells; se=intra-ovariolar somatic cells; tf=terminal filament of the ovariole; pm=peritoneal membrane; st=stromatic cells.
these deaths occur collapses and its outline becomes irregular with disrupted contours. The somatic cells that remain filled with glycogen and sometimes also lipid droplets, becoming part of the stromal tissues around the remaining ovarioles (Fig. 4A,B,C). In the adult, the ovary capsule and stroma of ovarioles are not present. The ovarioles are elongated free structures, sometimes referred to as ‘tubules of eggs’, delimited by the peritoneal sheath. The capsular and stromal cells disappear during metamorphosis. In the prepupa, the start of ovarian modulation is already seen, and the cells generally contain large amounts of glycogen (Fig.
4B,C). This content increases in the prepupae and lipid droplets also appear mainly in the stromal cells. Large vacuoles are formed beside the nucleus, and the cell membrane eventually bursts, liberating its contents (Fig. 4C). The stromal cells become more dispersed and the intercellular spaces fill up with fluid similar in nature to that of the vacuolar content (Fig. 4C). Although haemocytes could be found among the stroma, they were never observed to be phagocytic. It seems, therefore, that the disappearance of the capsule and stroma is due to cellular disintegration, apparently without any observable changes in the organelles. The only changes observed seem to
Fig. 2. TEM micrographs of cell death in germinative cells. i=insert of light micrograph of a cross section of an ovariole of a 3rd instar larva ovary showing cell death (asteriscs). A. Apoptotic—like cell, with fragmented nucleus (n), condensed chromatin (cr) and little cytoplasmic. B. Dying cell showing membrane delimitation (stars) of cytoplasmic regions. Note that this cell is apparently inside a vacuole of other cell. C. Phagocitic vacuole containing a dying cell (stars). D. Autophagic vacuoles (va) in apoptotic-like cells (stars) and in apparently normal cells. nu=nucleolus; n=nucleus.
Fig. 3. TEM micrographs of dying intra-ovariolar somatic cells. A, B, C. Features of the autophagic structures (va) and lipid (l) accumulation in intra-ovariolar somatic cells. Note the normal aspect of mitochondria (m) in A and B and the apparently swollen mitochondria in a more damaged cell (c).
Fig. 4. TEM micrographs of cellular death in the stromatic cells. A. Disrupting ovariole (ol) with cells filled with glycogen (gl) being liberated in the stroma. B. Stromatic cell with ‘bags’ of glycogen (gl) lateral to the nucleus (n). C. Disintegrating stromatic cells from a pre-pupa. pm=peritoneal membrane.
Fig. 5. TEM micrographs of ovary cells treated to show acid phosphatase location. A. Positive reaction in autophagic vacuoles (asteriks) of intra-ovariolar somatic cells. B. Lead precipitation in the nuclear chromatin (asteriks) of ovariolar cells, in a 4th instar larva. Note the negative nucleoli (arrows). C. Nucleus of a stromatic cell showing positive chromatin (arrows) and negative nucleolus. D. Positive nucleolus (nu) and negative chromatin in another stromatic cell.
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be in the arrangement of glycogen deposits and vacuole size. No acid phosphatase activity was found in the apoptotic ovariolar cells or in stromal cells, while in the intra-ovariolar somatic cells showing autophagic degeneration some activity was present in the vacuoles (Fig. 5A). However, lead precipitation was observed in nuclei and nucleoli of apparently intact cells in the ovarioles and the stroma (Fig. 5B,C,D). The lead precipitate never occurred at the same time in the chromatin and the nucleolus, and seemed to be more frequent and strong in stromatic cells of the 4th stage ovarioles. DISCUSSION Although cell death in the honey bee worker is environmentally induced in the sense that it is determined by diet, it might be considered a programmed cell death because alterations in the juvenile hormone levels produced by differential feeding triggers the suicide programme of certain ovary cells. The resulting cell death, therefore, involves the action of activators, effectors, and negative regulators (Barinaga, 1998), although it does not always have the morphological characteristics of apoptosis. Several authors (Clarke, 1990; Lockshin et al., 1991; Lockshin and Zakeri, 1996) have considered that in insects the morphological and physiological phenomena taking place during cell death are not so well structured and organized as in vertebrates, mainly in mammals. But even in those where some discrepancy has been observed from the classical process, it seems that the pattern of tissue organization may have some function (Roach and Clarke, 1999) in the type of reabsortive process of the dead cell. When phagocytes, or neighbouring cells with phagocytic capacity, are present, heterophagic elimination of the cellular debris, as seen in Figure 2A–D, can occur. The cells where acid phosphatase activity was found possess numerous autophagic and possibly heterophagic vacuoles, the latter resulting from the engulfment of cells that have died by ‘apoptosis’. Some of these cells will die by a process classified by Lockshin et al. (1991) as type II cell death, or autophagic cell death, which is common in insects. Besides the three types of cell death reported here in insects, another type was observed in the midgut. In this case, the dead cells seemed to be eliminated into the lumen and were digested there (Cavalcante, 1998; Cruz-Landim and Silva de Moraes, 2000). Therefore, it seems that when cell
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death occurs in organs that are isolated from the body-fluids (haemolymph, in the case of insects), the debris of the dead cells must be eliminated by phagocytic digestion by neighbouring cells, or dispersal into the organ lumen. When the organ does not have an envelope, or is in direct contact with the body-fluid, dying cells may be eliminated by simple disruption that liberates the cell contents into the haemolymph. In the ovarioles, the germinative cells are engulfed by the somatic neighbor cells, while the somatic cells are liberated by disruption of the whole ovariole into the stroma. Later these cells disrupt and liberate their contents into the haemolymph. The cells that are in direct contact with the body fluids, such as those of the larval salivary gland or Malpighian tubules (Cruz-Landim, 2000; Cruz-Landim and Silva de Moraes, 2000), or in the present case the stromal cells, are eliminated by disintegration. This cell disintegration, however, does not correspond to necrosis in the sense that it is a programmed event in ovary development, and does not cause any inflammation or adverse reaction. Since this generally occurs during metamorphosis, it may be seen as part of insect body reorganization during development. In the case of stromal and capsule cells it may be considered that they are first integrated into the fat body (which also disintegrates during metamorphosis), later liberating their contents into the haemolymph, by disruption. The event called ‘cell paralysis’ seen in cartilage by Roach and Clarke (1999) may in some ways be considered similar to that occurring in the ovarioles, where the peritoneal sheath impairs liberation of the cellular residues of germinative cells—which die early on in the ovary differentiation—into the body fluids. In spite of the morphological and biochemical differences that might be involved in these types of cell death, all of them are programmed and do not cause damage to the organism, thereby contributing to their correct development and function. If this is the case, then they might all be called apoptosis in the strict sense of the Greek word.
REFERENCES A MJ, W AH, 1991. Apoptosis mechanisms and roles in pathology. Int Rev Exp Pathol 32: 223–254. B M, 1998. Death by Dozens of Cuts. Science 280: 32–34. B J, 1979. The process of queen-worker differentiation in the honeybee. Bee Wld 60(1): 24–39.
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B ID, M SM, M K, 1993. Cell death in salivary glands of metamorphosing Caliphora vomitoria. Cell Biol Internat 17(1): 13–33. B ID, M K, M SM, 1996. Programmed cell death in the salivary glands of blow fly Calliphora vomitoria. Microsc Res Tech 34: 202–207. B OC, 1981. Diferenciac¸ao˜ dos ova´rios e determinac¸a˜o do nu´mero de ovarı´olos em Apis mellifera L. (Hymenoptera, Apidae). Tese de Doutorado, Universidade de Sa˜o Paulo, SP, 59p. C VM, 1998. Reorganizac¸a˜o do intestino me´dio de Apis mellifera (Hymenoptera, Apidae) durante a metamorfose: estudos ultra-estruturais e citoquı´micos. Dissertac¸a˜o de Mestrado, Instituto de Biocieˆncias, Universidade Estadual Paulista, Rio Claro, SP, 134p. C PGH, 1990. Developmental cell death: morphological diversity and multiple mechanisms. Anat Embryol 181: 195– 206. C-L C, S M RLM, 2000. Morte celular programada em abelhas como uma forma de redirecionar a morfologia e a fisiologia adaptativa. Ed. Tipografia Costa Ltda. Rio Claro, SP. 47p. C-L C, 2000. Replacement of larval by adult Malpighian tubules during metamorphosis of Melipona quadrifasciata anthidioides Lep. (Hymenoptera, Apidae, Meliponinae): Morphological studies. Cieˆncia e Cultura Journal of the Brazilian Association for Advancement of Science 52(1): 59–63. D SE, S RW, 1963. Studies in the mode of action of royal jelly in honeybee development. III. The effect of experimental variation in diet on growth and metabolism of honeybee larvae. Can J Zool 41: 733–739. F JB, 1981. A organizac¸a˜o social das abelhas (Apis). Universidade de Sa˜o Paulo, p. 79. G A, B ID, 1997. Programmed cell death in the honeybee (Apis mellifera L.) larvae midgut. Cell Biol Internat 21: 151–158. H K, S¨ G, 1997. Germ cell cluster formation and cell death are alternatives in caste-specific differentiation of the larval honey bee ovary. Invert Reprod Develop 31(1–3): 237–250. K JFR, H B, S J, 1974. An electron microscope study of cell delection in the anuran tadpole tail during spontaneous metamorphosis with special reference to apoptosis of striated muscle fibers. J Cell Sci 14: 571–585. K JFR, W AH, C AR, 1972. Apoptosis: a basic biological phenomenon with wide ranging implications in tissue kinetics. Br J Cancer 26: 239–257 K JFR, 1971. Shrinkage necrosis: a distinct mode of cellular death. J Pathol 105: 8–20. L R, 1885. Seebacher studien. Erchstadt Bienenz 11: 199–211. L RA, 1985. Programmed cell death. In: Kerkut GA, Gilbert LI, eds. Comprehensive insect physiology, biochemistry and pharmacology, Vol. 2. Oxford, Pergamon Press. 301–317. L RA, Z Z, 1996. The biology of cell death and its relationship to aging in Cellular Aging and Cell Death.
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In: Holbrook NJ, Lockshin RA, eds. Cellular aging and cell death. New York, Willey-Liss. 167–180. L RA, A A, K N, Z ZF, 1991. Programmed cell death and apoptosis: Early DNA degeneration does not appear to be prominent in either embryonic cell death or metamorphosis of insects. J Cell Biol Abstr 115: 41. L RA, W CM, 1964. Programmed cell death. II. Endocrine potentiation of the breakdown of the intersegmental muscles of silkmoths. J Insect Physiol 10: 643–649. L R, 1915. Die Metamorphosis des Bienendarms (Apis mellifera). Beihefte Schweiz Bienen-Zeitung 1: 443–506. R A, E W, 1995. Caste development in honeybees (Apis mellifera): juvenile hormone turns on ecdysteroids. Naturwiss 82: 378–379. R A, H K, 1990. Corpora allata, a prime regulating element for caste-specific juvenile hormone titre in honey bee larvae (Apis mellifera carnica). J Insect Physiol 36: 189–194. R A, H K, 1991. Differential production of juvenile hormone and its desoxy precursor by corpora allata of honey bees during a critical caste development. Naturwiss 78: 270–272. R A, S C, S A, H K, 1990. Caste and metamorphosis: hemolymph titers of juvenile hormone and ecdysteroids in last instar honeybee larvae. Gen Comp Endocr 00: 31–38. R H, 1987. Caste differentiation of the honeybee, fourteen years of biochemical research at Martinsried. In: Eder J, Rembold H, eds. Chemistry and Biology of Social Insects. Mu¨nchen, Verlag Peperny. 3–13. R CR, 1953. The behaviour and social life of honeybees. London. Bee Research Ass. 352pp. R IH, C NMP, 1999. ‘Cell Paralysis’ as an intermediate stage in the programmed cell death of epiphysical chondrocytes during development. Journal of Bone and Mineral Research 14: 1367–1378. R TA, B ID, 1975. A method for the fine structural localization of acid phosphatase activity using p-nitrophenyl phosphate as substrate. J Histochem Cytochem 23: 235–237. S JW, 1966. Death in embryonic systems. Science 154: 604–612. S M RLM, B ID, 2000. Modes of Cell Death in the Hypopharyngeal gland of the Honey Bee (Apis mellifera) L. Cell Biology International 10: 737–743. S RE, 1956. Anatomy of the honeybee. New York, Comstock Publishing Ass.. 334. V HHW, 1970. Ovarian development in Apis mellifera worker bees. Ent Exp Appl 13: 377–394. W AH, D E, B JJ, 1984a. Intracelular mechanisms in cell death in normal and pathological tissues. In: Davies I, Sigee DC, eds. Cell Aging and Cell Death. Cambridge, Cambridge Univ. Press. 269–284. W AH, M RG, S AL, D D, 1984b. Chromatin cleavage in apoptosis: association with condensed morphology and dependence in macromolecular synthesis. J Pathol 142: 67–77.
MORPHOLOGICAL CHARACTERIZATION OF CELL DEATH DURING THE OVARY DIFFERENTIATION IN WORKER HONEY BEE REJANE DANIELE REGINATO and CARMINDA DA CRUZ-LANDIM Departamento de Biologia, Instituto de Biocieˆncias, Universidade Estadual Paulista (UNESP), 13506-900, Rio Claro, SP, Brasil Received 28 November 2000; accepted 27 September 2001
Cell death that occurs during ovary differentiation in the honeybee worker’s larval development accounts for ovariole reabsorption. From a morphological standpoint, three modes of death were detected. Germinative cells in the ovarioles die by an apoptotic-like process, whereas the somatic cells die by an autophagic process, type II cell death; and during pupation, stromatic and ovarian capsular cells die through cytoplasmic disintegration, releasing their components into the hemolymph. These modes of cell death are in part determined by the pattern of tissue 2002 Elsevier Science Ltd. All rights reserved. organization within which the cell occurs. K: honeybee; castes; ovary; differentiation cell death; tissue organization.
INTRODUCTION Marked caste dimorphism has evolved in the highly eusocial honey bee. The typical queen and worker characteristics are different from one another in many morphological, physiological and behavioural aspects, among which ovarian development is very prominent. The worker caste is distinguished by a very limited reproductive capacity and correspondingly has underdeveloped ovaries compared with the queen (Ribbands, 1953; Snodgrass, 1956; Free, 1981). Differences between queen and worker ovaries mainly concern ovariole number, which is extremely high in queens and very low in workers, and is due to an environmentally-induced ovariolar reabsorption during differentiation in the latter (Lotmar, 1915; Beetsma, 1979; Bueno, 1981). Progressive feeding of honey bee larvae leads to several assumptions, one of which is that the special ‘royal’ diet determines the queen type. The pioneering studies of Leuchart (1855) first showed that qualitative differences between royal jelly and worker food was responsible for queen differentiation. As with other social insects, however, development into a worker or queen is regulated by the corpora allata (Dixon and Shuel, 1963; Velthuis, 1970; Kerr et al., 1974; Rembold, 1987; Fax: 55-19-534-0009. E-mail: [email protected] 1065–6995/02/$-see front matter
Rachinsky et al. 1990), an endocrine organ producing juvenile hormone. This means that there is a caste-specific modulation of the general pattern of juvenile hormone synthesis based on the quality and amount of food ingested by the larvae, leading to the differences between castes (Rachinsky et al., 1990; Rachinsky and Hartfelder, 1990, 1991; Rachinsky and Engels, 1995), expressed in the worker ovaries as cell death, and resulting in diminished ovariole numbers. Cell death during development or the life of an organism is generally referred to as ‘programmed cell death’, an expression first used by Lockshin and Williams (1964) to describe the breakdown of intersegmental muscles of silkworms during metamorphosis. The original use was an adaptation of the ‘death clock’ concept of Saunders (1966) who suggested that cells contain instructions for their own destruction, without simultaneously being irreversibly damaged. The term distinguishes physiological (programmed) cell death (Wyllie et al., 1984a; Lockshin, 1985) from traumatic (pathological) cell death. Its structural manifestations are shrinkage of cell volume, loss of specialized plasma membrane regions, such as microvilli, conservation of most cytoplasmic organelles, progressive perinuclear chromatin condensation, and release of signals which cause engulfment by 2002 Elsevier Science Ltd. All rights reserved.
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adjacent phagocytes (Kerr, 1971; Wyllie et al., 1984a,b; Arends and Wyllie, 1991). Cell death with these characteristics was, in fact, named apoptosis in recognition of its significance to tissue and organism homeostasis (Kerr et al., 1972). Apoptotic cell death involves activators, effectors and negative regulators within a cascade of events (Barinaga, 1998), as is known to occur during insect metamorphosis, and specifically during ovary development in worker bees. However, the morphological and biochemical features characterizing cellular apoptosis are not always present in insects (Clarke, 1990; Lockshin et al., 1991; Lockshin and Zakeri, 1996), where other types of cell death have been described (Bowen et al., 1993, 1996; Gregorc and Bowen, 1997; Hartfelder and Steinbruck, 1997; Silva de Moraes and Bowen, 2000; Cruz-Landim and Silva de Moraes, 2000). Thus the present work is an attempt to characterize morphologically the types of cell death occurring during ovary differentiation in worker bees.
MATERIALS AND METHODS Ovaries excised from 2–5th instar larvae and prepupa were fixed in Karnowsky fluid for transmission electron microscopy, and postfixed in 1% osmium tetraoxide in 0.1 sodium cacodylate buffer, dehydrated in serial concentrations of acetone and embedded in Epon-Araldite. Thin sections (800–1000 nm) were stained with uranyl acetate and lead citrate before being examined in a Philips electron microscope. Thick sections were stained with 1% toluidine blue for light microscopic examination. Ovaries were also prepared for cytochemical investigation of acid phosphatase in the dying cells, using p-nitrophenylphosphate as substrate (Ryder and Bowen, 1975).
RESULTS The ovary is comprised of somatic and germinative cells, the former creating the framework of the gonad, and the germinative cells being reproductive. In the first instar newly-hatched larvae, a mass of cells of different sizes remain indistinguishable as somatic or germinative, enveloped by a sheath comprised of several layers of flat cells, the capsula (Fig. 1A). As the larval phase progresses, the ovary differentiates by creating ovarioles within the primitive cell mass. The individualization of
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ovariole structures begins in the 2nd instar larvae by inward proliferation of the flat capsula cells. The germinative cells become located in the ovarioles as these become elongated structures limited by layers of flat cells as part of the peritoneal membrane (Fig. 1B). The ovarioles are separated from one another by interovariolar cells of the larval ovarian stroma (Fig. 1B,C). The germinative cells proliferate and form clusters which later undergo oogenesis inside the ovarioles, supported by intraovariolar somatic cells. Therefore, the somatic cells in the ovary present different forms, with variable location and function, such as capsule cells, stromal cells, peritoneal membrane and intraovariolar somatic cells (Fig. 1B,C). The germinative cells appear early as big round cells, and later form clusters of interconnected elements (Fig. 1A,B). During ovary differentiation in the worker bee, cell death in germinative and somatic cells, both in the inner and outer parts of the ovariole, present different characteristics according their lineage and location. Cell death was first detected within ovarioles of 3rd instar larvae (Fig. 2i). Cell death inside the ovarioles continues at increasing rates during the 4th and 5th instar stages, but with the death of germinative and somatic cells within the ovarioles showing different characteristics. Two types of morphology were observed in ovariolar cell death, one of almost classic apoptotic nature, with the dying cell first acquiring an almost spherical shape, with dense cytoplasm and condensed nuclear chromatin. Later fragmentation and formation of apoptotic bodies could be seen, as well as their engulfment by neighbouring cells (Fig. 2A–E). The cells showing this kind of death are almost certainly germinative. At the same time and within the same ovariole, cells generally located more peripherally and containing numerous vacuoles (some including organelle remnants, multivesicular bodies and myelin figures) occur. In spite of the presence of these degenerative structures in their cytoplasm, their nuclear chromatin remains dispersed and well-structured nucleoli are present. The cytoplasm and its organelles also appear normal (Fig. 3A–C). These cells, which appear to be having problems with autophagic reabsorption or processing, are mostly somatic in type. These two kinds of cell death were observed during the 3rd instar larvae or when the nutritional differences between worker and queen became established.; both lead to a decrease in ovariole number in the worker bees. The ovariole where
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Fig. 1. Light micrographs of the larval ovary. A. Ovary (ov) of a 1st instar larvae showing the capsula (c) and its content of large (l) and small (s) cells. B. Longitudinal section and C. Cross section of a portion of an ovary of a 4th instar larva showing its organization. g=clusters of germinative cells; se=intra-ovariolar somatic cells; tf=terminal filament of the ovariole; pm=peritoneal membrane; st=stromatic cells.
these deaths occur collapses and its outline becomes irregular with disrupted contours. The somatic cells that remain filled with glycogen and sometimes also lipid droplets, becoming part of the stromal tissues around the remaining ovarioles (Fig. 4A,B,C). In the adult, the ovary capsule and stroma of ovarioles are not present. The ovarioles are elongated free structures, sometimes referred to as ‘tubules of eggs’, delimited by the peritoneal sheath. The capsular and stromal cells disappear during metamorphosis. In the prepupa, the start of ovarian modulation is already seen, and the cells generally contain large amounts of glycogen (Fig.
4B,C). This content increases in the prepupae and lipid droplets also appear mainly in the stromal cells. Large vacuoles are formed beside the nucleus, and the cell membrane eventually bursts, liberating its contents (Fig. 4C). The stromal cells become more dispersed and the intercellular spaces fill up with fluid similar in nature to that of the vacuolar content (Fig. 4C). Although haemocytes could be found among the stroma, they were never observed to be phagocytic. It seems, therefore, that the disappearance of the capsule and stroma is due to cellular disintegration, apparently without any observable changes in the organelles. The only changes observed seem to
Fig. 2. TEM micrographs of cell death in germinative cells. i=insert of light micrograph of a cross section of an ovariole of a 3rd instar larva ovary showing cell death (asteriscs). A. Apoptotic—like cell, with fragmented nucleus (n), condensed chromatin (cr) and little cytoplasmic. B. Dying cell showing membrane delimitation (stars) of cytoplasmic regions. Note that this cell is apparently inside a vacuole of other cell. C. Phagocitic vacuole containing a dying cell (stars). D. Autophagic vacuoles (va) in apoptotic-like cells (stars) and in apparently normal cells. nu=nucleolus; n=nucleus.
Fig. 3. TEM micrographs of dying intra-ovariolar somatic cells. A, B, C. Features of the autophagic structures (va) and lipid (l) accumulation in intra-ovariolar somatic cells. Note the normal aspect of mitochondria (m) in A and B and the apparently swollen mitochondria in a more damaged cell (c).
Fig. 4. TEM micrographs of cellular death in the stromatic cells. A. Disrupting ovariole (ol) with cells filled with glycogen (gl) being liberated in the stroma. B. Stromatic cell with ‘bags’ of glycogen (gl) lateral to the nucleus (n). C. Disintegrating stromatic cells from a pre-pupa. pm=peritoneal membrane.
Fig. 5. TEM micrographs of ovary cells treated to show acid phosphatase location. A. Positive reaction in autophagic vacuoles (asteriks) of intra-ovariolar somatic cells. B. Lead precipitation in the nuclear chromatin (asteriks) of ovariolar cells, in a 4th instar larva. Note the negative nucleoli (arrows). C. Nucleus of a stromatic cell showing positive chromatin (arrows) and negative nucleolus. D. Positive nucleolus (nu) and negative chromatin in another stromatic cell.
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be in the arrangement of glycogen deposits and vacuole size. No acid phosphatase activity was found in the apoptotic ovariolar cells or in stromal cells, while in the intra-ovariolar somatic cells showing autophagic degeneration some activity was present in the vacuoles (Fig. 5A). However, lead precipitation was observed in nuclei and nucleoli of apparently intact cells in the ovarioles and the stroma (Fig. 5B,C,D). The lead precipitate never occurred at the same time in the chromatin and the nucleolus, and seemed to be more frequent and strong in stromatic cells of the 4th stage ovarioles. DISCUSSION Although cell death in the honey bee worker is environmentally induced in the sense that it is determined by diet, it might be considered a programmed cell death because alterations in the juvenile hormone levels produced by differential feeding triggers the suicide programme of certain ovary cells. The resulting cell death, therefore, involves the action of activators, effectors, and negative regulators (Barinaga, 1998), although it does not always have the morphological characteristics of apoptosis. Several authors (Clarke, 1990; Lockshin et al., 1991; Lockshin and Zakeri, 1996) have considered that in insects the morphological and physiological phenomena taking place during cell death are not so well structured and organized as in vertebrates, mainly in mammals. But even in those where some discrepancy has been observed from the classical process, it seems that the pattern of tissue organization may have some function (Roach and Clarke, 1999) in the type of reabsortive process of the dead cell. When phagocytes, or neighbouring cells with phagocytic capacity, are present, heterophagic elimination of the cellular debris, as seen in Figure 2A–D, can occur. The cells where acid phosphatase activity was found possess numerous autophagic and possibly heterophagic vacuoles, the latter resulting from the engulfment of cells that have died by ‘apoptosis’. Some of these cells will die by a process classified by Lockshin et al. (1991) as type II cell death, or autophagic cell death, which is common in insects. Besides the three types of cell death reported here in insects, another type was observed in the midgut. In this case, the dead cells seemed to be eliminated into the lumen and were digested there (Cavalcante, 1998; Cruz-Landim and Silva de Moraes, 2000). Therefore, it seems that when cell
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death occurs in organs that are isolated from the body-fluids (haemolymph, in the case of insects), the debris of the dead cells must be eliminated by phagocytic digestion by neighbouring cells, or dispersal into the organ lumen. When the organ does not have an envelope, or is in direct contact with the body-fluid, dying cells may be eliminated by simple disruption that liberates the cell contents into the haemolymph. In the ovarioles, the germinative cells are engulfed by the somatic neighbor cells, while the somatic cells are liberated by disruption of the whole ovariole into the stroma. Later these cells disrupt and liberate their contents into the haemolymph. The cells that are in direct contact with the body fluids, such as those of the larval salivary gland or Malpighian tubules (Cruz-Landim, 2000; Cruz-Landim and Silva de Moraes, 2000), or in the present case the stromal cells, are eliminated by disintegration. This cell disintegration, however, does not correspond to necrosis in the sense that it is a programmed event in ovary development, and does not cause any inflammation or adverse reaction. Since this generally occurs during metamorphosis, it may be seen as part of insect body reorganization during development. In the case of stromal and capsule cells it may be considered that they are first integrated into the fat body (which also disintegrates during metamorphosis), later liberating their contents into the haemolymph, by disruption. The event called ‘cell paralysis’ seen in cartilage by Roach and Clarke (1999) may in some ways be considered similar to that occurring in the ovarioles, where the peritoneal sheath impairs liberation of the cellular residues of germinative cells—which die early on in the ovary differentiation—into the body fluids. In spite of the morphological and biochemical differences that might be involved in these types of cell death, all of them are programmed and do not cause damage to the organism, thereby contributing to their correct development and function. If this is the case, then they might all be called apoptosis in the strict sense of the Greek word.
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