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Ultrastructural study of the ovary of the sugarcane spittlebug Mahanarva fimbriolata (Hemiptera)
Micron 37 (2006) 633–639 www.elsevier.com/locate/micron
Ultrastructural study of the ovary of the sugarcane spittlebug Mahanarva fimbriolata (Hemiptera) De´bora Caperucci, Maria Izabel Camargo-Mathias * Departamento de Biologia, Instituto de Biocieˆncias, UNESP, Av 24 A, no. 1515. Cx. Postal 199, CEP: 13506-900 Rio Claro, SP, Brazil Received 13 October 2005; received in revised form 8 February 2006; accepted 9 February 2006
Abstract The present study describes the ultrastructure of meroistic telotrophic ovaries of the sugarcane spittlebug Mahanarva fimbriolata. In this type of ovary, nurse cells, oogonia, and prefollicular tissue are located at the terminal (distal) regions or tropharium of ovarioles. Oocytes in different developmental stages, classified from I to V, are observed in the vitellarium. Stage I oocytes do not exhibit intercellular spaces in the follicular epithelium, suggesting that synthesis and production of yolk during this stage occurs only through endogenous processes. Small yolk granules of different electron densities are present in the cytoplasm. Few lipid droplets are observed. Stage II oocytes exhibit small intercellular spaces in the follicular epithelium. More protein as well as lipid yolk granules are observed in the cytoplasm. In stage III oocytes, intercellular spaces in the follicular epithelium are larger than those observed in the previous stage. Electrondense protein granules of various sizes, larger than those observed in stage II oocytes predominate in the cytoplasm. Smaller lipid droplets are also present. In stage IV oocytes, the follicular epithelium exhibits large intercellular spaces. Our data clearly indicate that the opening of these spaces in the follicular epithelium of M. fimbriolata oocytes increases as the intake of exogenous proteins intensifies, that is, in stages IVand Voocytes. During these stages, granular yolk becomes viscous due to the lysis of granules. In stage Voocytes, viscous yolk predominates in the cytoplasm. This type of yolk, however, has not been described for other orders of insects. The chorion of M. fimbriolata oocytes consists of an external layer (exochorion) and an internal one (endochorion), which is in direct contact with the oocyte. Numerous small pores that probably facilitate oxygenation of the internal structures inside the eggs are observed in the exochorion. # 2006 Elsevier Ltd. All rights reserved. Keywords: Mahanarva fimbriolata; Ovary; Ultrastructure; Vitellogenesis
1. Introduction The reproductive system of insects usually performs several functions, among them: egg production, reception and sperm storage (occasionally for long periods), and coordination of events leading to fertilization and egg-laying (Gillot, 1995). The number of ovarioles in different orders varies greatly from one species to another; for example, viviparous aphids exhibit a functional ovariole in one ovary, while higher termite queens of the genus Eutermes may have more than 2000 ovarioles in each ovariole (Buning, 1994; Gillot, 1995; Chapman, 1998). Seven ovarioles per ovary were observed in M. fimbriolata (Caperucci and Camargo-Mathias, 2006a,b). Ovarioles of insects present three distinct regions: terminal filament located near the distal end and composed of connective
* Corresponding author. Tel.: +55 19 3526 4135; fax: +55 19 3534 0009. E-mail address: [email protected] (M.I. Camargo-Mathias). 0968-4328/$ – see front matter # 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.micron.2006.02.002
or conjunctive tissue; germarium or tropharium, the apical tip of the ovariole below the terminal filament, consisting of a mass of cells capable of differentiating into oocytes and nurse cells (when present); and vitellarium, which is the largest portion of the ovariole containing developing oocytes (undergoing vitellogenesis) surrounded by follicular cells. Studies on the female reproductive system of the sugarcane spittlebug are especially important to support basic research on insect reproduction, as well as applied research aiming at the development of mechanisms for the biological control of this emergent pest. Previous studies on this insect already described its ovaries as meroistic telotrophic, exhibiting follicular and nurse cells, as well as oocytes in the tropharium (Caperucci and Camargo-Mathias, 2006a,b). This study aimed to describe the ultrastructural analysis of the ovaries of the sugarcane spittlebug M. fimbriolata. Studies on this insect are especially relevant due to little morphological data available in the literature that could contribute for a better understanding of its biology. More specifically, studies on its
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reproductive system may provide additional information to control it. 2. Material and methods In this study, we utilized adult females of the sugarcane spittlebug M. fimbriolata collected at Copersucar, PiracicabaSa˜o Paulo State, Brazil, and kindly provided by Dr. Enrico de Beni Arrigone. We used the equipment available at the laboratories of the Laboratory/Biology Department of Electron Microscopy, UNESP, Rio Claro-SP. Spittlebugs were collected in the sugarcane plantation, anesthetized by thermal shock, dissected while immersed in Petri dish with physiological solution for insects to remove their ovaries. Ovaries were then fixed in 3% glutaraldehyde, postfixed in 1% OsO4, routinely processed, and embedded in Epon Araldite (Haddad et al., 1998). Ultrathin sections were stained with uranyl acetate and lead citrate. Grids containing ultrathin sections of the material were analyzed and photographed in a PHILLIPS 100 TEM transmission electron microscope. 3. Results In M. fimbriolata, tropharium cells range from elliptic to round, although most are irregular in shape, with homogeneous
and not very electrondense cytoplasm (Fig. 1A). Electrondense material is observed in intercellular spaces. The most frequently observed organelles are mitochondria (Fig. 1B), followed by ribosomes, and vesicular rough endoplasmic reticulum. The nucleus has an irregular in shape with little condensed chromatin, and nucleoli. The numerous pores perforate the nuclear envelope (Fig. 1B). Tropharium cells, especially those located in the periphery are supported by a thick basal lamina (Fig. 1B), and just below it, near the plasmatic membrane, very electrondense vacuoles containing membrane elements are frequently observed. As they reach the vitellarium, stage I oocytes are characterized by small yolk granules in the cytoplasm, most of them containing proteins, but yolk granules containing lipids are also observed (Fig. 1C). Intercellular spaces in the follicular epithelium are not observed, indicating that synthesis and production of yolk occur only by endogenous processes in these oocytes (Fig. 1D arrow). Stage II or pre-vitellogenic oocytes exhibit large quantities of lamellar endoplasmic reticulum, suggesting an intense endogenous synthesis of proteins (Fig. 1F). Also during this stage, intercellular spaces in the follicular epithelium are formed (Fig. 1E). Protein granules are more electrondense than lipid granules and increase in size as they join together and move from the periphery to the central region of the cytoplasm (Fig. 1E).
Fig. 1. (A and B) Details of the ultrastructure of tropharium cells (tro) of Mahanarva fimbriolata oocytes, n = irregular nucleus, np = nuclear pores, cl = cell limit, nu = nucleolus, mb = myelin bodies. Scale bars: A = 2 mm and B = 0.5 mm. (C) General view of stage I oocyte (ov I) and partial view of follicular epithelium (fe), with large amounts of protein granules (py), lipid granules (ly), mitochondria (m), interface follicular epithelium/oocyte (ie). Scale bar: 2 mm. (D) Cells of the follicular epithelium of stage I oocytes (ov I) with partially irregular nuclei (n), intercellular spaces (ie) with electrondense material (arrow), mitochondria (m), lamellar rough endoplasmic reticulum (rerl). Scale bar: 2 mm. (E) Detail of stage II oocyte (ov II) and follicular epithelium (fe) with intercellular spaces (ie), nuclei of follicular cells, lipid granules (ly) and protein granules (py), interface follicular epithelium/oocyte (ie) with microvilli (mic). Scale bar: 5 mm. (F) Cytoplasm of follicular cells of stage II oocytes II (ov II) with lamellar rough endoplasmic reticulum (rerl), nucleus (n) with several pores (arrow). Scale bar: 0.5 mm.
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Vitellogenesis intensifies in stage III oocytes. Electrondense protein granules of different sizes and smaller lipid granules predominate in the cytoplasm (Fig. 2A). Lamellar rough endoplasmic reticulum in the follicular cells is observed (Fig. 2B). During this stage oocytes exhibit follicular epithelium with larger intercellular spaces when compared to those of the previous stage (Fig. 2B). In stage IV oocytes, granular yolk becomes viscous due to breakdown of compounds followed by granule lysis (Fig. 2C). Yolk is present as protein granules and viscous yolk, the latter seems to be the result of granules that have undergone lysis, becoming unorganized structures (Fig. 2C and E). Large amounts of vesicular and lamellar rough endoplasmic reticulum are present in the cytoplasm of follicular cells, indicating that during this phase the follicular epithelium might still be involved in the synthesis of elements that will later form the chorion (Fig. 3C and D). Much larger intercellular spaces than in previous stages are also observed, clearly indicating that the opening of these spaces in the follicular epithelium of M. fimbriolata oocytes increases as the intake of exogenous proteins intensifies (Fig. 3A and B). Viscous yolk is more frequently observed in intermediate stages IV and V oocytes (Fig. 3E and F).
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In stage V oocytes, viscous yolk completely fills the cytoplasm, while granular yolk is no longer observed (Fig. 4B). Follicular cells in stage V oocytes are in the process of degeneration with several vacuoles in the cytoplasm (Fig. 4C). The chorion in M. fimbriolata oocytes consists of exochorion, more external and less electrondense layer in direct contact with follicular cells; and endochorion, the innermost and more electrondense layer (Fig. 4A and D). The exochorion is perforated by numerous pores (Fig. 4A arrow). 4. Discussion Studies on the spittlebug M. fimbriolata describe it as a pest to agriculture that attacks sugarcane. Therefore, studies on its reproductive system provide important information about its biology. The ovaries of M. fimbriolata are meroistic telotrophic, that is, follicular cells, as well as nurse cells, and oocytes are located in the tropharium. In most insects, ovarioles are functional units covered by two morphologically distinct sheaths, the outermost cellular sheath, also known as peritoneal sheath, and the innermost acellular one consisted of thin fibrils, called tunica propria. According to some authors, their role is to provide elasticity to
Fig. 2. (A) Cytoplasm of stage III oocytes (ov III) with protein granules (py) and mitochondria (m). Scale bar: 1 mm. (B) Cytoplasm of stage III oocytes (ov III) with intercellular spaces (ie), irregular nucleus (n) with pores (arrow), mitochondria (m) and lamellar rough endoplasmic reticulum (rerl). Scale bar: 1 mm. (C) Cytoplasm of stage IVoocytes (ov IV), containing protein granules (py), with more electrondense periphery and less electron dense interior, mitochondria (m). Viscous yolk (vy) is first observed. Scale bar: 2 mm. (D) Intercellular spaces (ie) between the follicular epithelium (fe) and the oocyte with electrondense material, protein granules (py) in the periphery of the oocyte. Scale bar: 1 mm. (E) Detail of the cytoplasm of stage IV oocytes (ov IV) with viscous yolk (vy) containing proteins, and mitochondria (m). Scale bar: 1 mm.
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Fig. 3. (A) Detail of the spaces between follicular cells (ie) of stage IV oocytes (ov IV) with homogeneous material. Scale bar: 1 mm. (B) Intercellular spaces (ie) in the follicular epithelium of stage IV oocytes (ov IV) and vesicular rough endoplasmic reticulum (rerv) and lamellar (rerl). Scale bar: 1 mm. (C and D) Cytoplasm of follicular cells of stage IVoocytes (ov IV), with lamellar rough endoplasmic reticulum (rerv) and lamellar (rerl), and mitochondria (m). Scale bars: 1 mm. (E) General view of the cytoplasm of intermediate stages IV and V oocytes showing the beginning of production of viscous yolk (vy) inside the granule, protein granules (py). Scale bar: 1 mm. (F) Cytoplasm of intermediate stages IV and V oocytes with viscous yolk (vy), and protein granules (py). Space bar: 5 mm.
ovarioles, assisting oocytes to move down towards the exterior (Chapman, 1998). The origin of the tunica propria in the ovarioles is debated. Some authors believe that it is synthesized in the terminal filament area (Chapman, 1998), while others hypothesize that it is produced by phagocytes, which have been frequently found in the ovaries of several insects (Schreiner, 1977; Secco, 1990). Evidences on its origins in M. fimbriolata were not found. The tropharium is the next region after the terminal filament of ovarioles. In this region oocytes are formed from germinative cells through successive mitotic divisions and will be covered with a single epithelium of follicular cells from mesoderm (Bonhag, 1955; Rockstein, 1964; Buning, 1979; Wigglesworth, 1979). Ultrastructural results on the ovaries of the spittlebug M. fimbriolata revealed that the morphology of tropharium cells is irregular, corroborating the observed by Buning (1994) on Dysdercus intermedius, an insect of the same order. The most frequently cytoplasmic organelles found in the tropharium cells of M. fimbriolata were mitochondria, suggesting that large amounts of energy might be needed, mainly for cell division. The cellular material found in spaces between tropharium cells indicates an intense cell activity, as well as material exchange between cells. The basal lamina or tunica propria in the tropharium region of M. fimbriolata was thick and electrondense. According to Silva (1999), the fibrous region of the basal lamina surrounding
germinative cells of lobsters of the genus Panulirus may be able to adapt to volume changes undergone by oocytes throughout the differentiation process, in addition to facilitate the release of mature oocytes from the vitellarium, which will be ovulated and later fertilized. In addition to cell support, the basal lamina might also select elements entering the ovariole from the hemolymph and vice-versa in M. fimbriolata. These data corroborate those found by Reddy and Locke (1990), suggesting that the basal lamina serves as the primary barrier between hemolymph elements and the interior of oocyte, monitoring the traffic of macromolecules. The following region in the ovarioles after the tropharium is the vitellarium, where oocytes accumulate yolk through vitellogenesis, consequently increasing in size many times until ready to be fertilized. Synthesis (endogenous production) and deposition (exogenous incorporation) of yolk in oocytes occur in this region with participation of oocyte, as well as follicular cells. This was also observed in M. fimbriolata ovarioles. The oocytes of telotrophic ovaries of M. fimbriolata develop in the vitellarium in close association with follicular cells, which surround them as an epithelium. These cells originate from mitosis of pre-follicular cells that underwent a series of morphological and physiological changes during oogenesis and vitellogenesis. To date, the presence of cytoplasmic bridges between adjacent follicular cells in telotrophic ovaries has not been observed, as described for polytrophic ovaries. This was also not observed for M. fimbriolata.
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The large morphological diversity found in the oocytes, as well as follicular cells present in M. fimbriolata ovarioles is due to the developmental stage in which the oocytes are observed. This suggests a simultaneous, although asynchronous, development of oocytes in different ovarioles, where oocytes in early developmental stages and mature oocytes can be found throughout the ovarioles. Therefore, our data have shown that vitellogenesis occurs in a distal to proximal sequence, with less developed oocytes, also
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called primary, located in the distal region and more developed or vitellogenic oocytes located in the proximal region. An ultrastructural analysis of oocytes in the vitellarium of M. fimbriolata ovarioles revealed that during stage I, small yolk granules of different electron densities are present in the cytoplasm, most of them containing proteins, but yolk granules containing lipids are also observed. These oocytes are in early stages of vitellogenesis and produce yolk granules through endogenous synthesis.
Fig. 4. (A) Intermediate stages IVand Voocytes showing the beginning of chorion deposition (ch), apical portion of follicular cells (fe). Periphery of cytoplasm with a modified cell membrane (arrow), protein granules (py) and mitochondria (m), viscous yolk (vy). Scale bar: 2 mm. (B) Stage Voocytes with homogeneous and electron dense viscous yolk (vy). Scale bar: 1 mm. (C) Stage V oocytes with follicular cells in the process of degeneration (fed), where organelles with unorganized structure and intercellular spaces (ie) are observed. Scale bar: 1 mm. (D) Stage V oocytes: ultrastructural aspect of chorion with exochorion (exo) with pores (arrow) and endochorion (end). Scale bar: 2 mm.
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We did not observe intercellular spaces between follicular cells in the follicular epithelium of stage I oocytes of M. fimbriolata, confirming that synthesis and production of yolk might occur only by endogenous processes. Camargo-Mathias and Caetano (1998) also did not observe intercellular spaces in the follicular epithelium of stage I oocytes of the ant Pachycondyla (Neoponera) villosa. Openings of intercellular spaces of the follicular epithelium have been usually described as a process that requires the presence of cytoskeleton elements (Huebner and Injeyan, 1980). Some authors have pointed out that opening and closing processes of these spaces are strongly associated with these elements, corroborating data found for Rhodnius prolixus (King and Akai, 1984). In M. fimbriolata oocytes, cytoskeleton elements are not observed in the follicular cells surrounding them. Mahanarua fimbriolata oocytes in developmental stage II, also known as pre-vitellogenic, unlike stage I oocytes, exhibited many small vesicles in the cytoplasm. The ones containing proteins and lipids were in the periphery of the oocyte while larger granules were found in the center, probably due to the fusion of smaller vesicles, as observed for D. intermedius (Buning, 1994). Large amounts of rough endoplasmic reticulum found in stage II oocytes of M. fimbriolata suggest that during this stage intense endogenous protein synthesis still occurs, providing protein elements that will be used in the development of the oocyte. During the first two developmental stages of M. fimbriolata oocytes, we observed the traffic of material from the germinal vesicle to the cytoplasm through nuclear pores. This suggests that endogenous synthesis of elements that will probably be part of the final yolk composition is being carried out inside the oocyte. The traffic of material through nuclear pores was also observed in pre-vitellogenic oocytes in Oncopeltus fasciatus and R. prolixus (Hubner, 1981). The author described this material as probably RNAr. The ultrastructural data obtained in this study revealed that incorporation of extra-ovarian proteins from the hemolymph begins in stage II oocytes. We observed morphological changes in the cells of the follicular epithelium, especially regarding the opening of intercellular spaces. During this stage, spaces begin to be formed, which were not observed in the previous stage. Large quantities of mitochondria observed in the cytoplasm of follicular cells near lateral membranes indicate the need of energy to open these spaces through which extra-ovarian material will flow. Our data suggest that vitellogenesis in oocytes of the spittlebug M. fimbriolata probably occurs through four processes: (a) endogenous synthesis of elements carried out by the oocyte; (b) synthesis of elements by follicular cells followed by transport into the oocyte by endocytosis; (c) selective intake of extra-ovarian material by follicular cells followed by transport to the oocyte; (d) transport of extraovarian material into the oocyte through intercellular spaces of the follicular epithelium. Anderson and Telfer (1970) have demonstrated in previous studies that proteins transported into the oocyte might also be
selected at the intercellular spaces of the follicular epithelium. These authors also suggested that follicular cells might produce elements involved in the removal of proteins from the hemolymph to be incorporated by the oocyte. Ultrastructural data obtained for stage III oocytes of M. fimbriolata indicate that vitellogenesis intensifies during this stage. Lipid droplets and protein granules enclosed by a membrane were also observed in larger amounts than in stage II oocytes. The presence of rough endoplasmic reticulum in the cytoplasm of stage III oocytes of M. fimbriolata indicates that endogenous synthesis of yolk still occurs during this stage. In addition to rough endoplasmic reticulum, some authors have pointed out the participation of the Golgi complex during vitellogenesis. This organelle, however, was not observed in M. fimbriolata oocytes, indicating little or no participation in the production of elements. Norrevang (1968), reviewing the ultrastructural characteristics of oogenesis in several animal groups, suggested that in most oocytes, the Golgi complex does not seem to be an important element and little has been described about its role in the dynamics of vitellogenesis in insects. In follicular cells of M. fimbriolata oocytes, lamellar rough endoplasmic reticulum was more abundant than vesicular one. According to Han and Bordereau (1982), the presence of two kinds of rough endoplasmic reticulum in the same cells could indicate different functions. The vesicular rough endoplasmic reticulum would be more active while the lamellar form, the less active between these organelles. On the other hand, it is known that ribosomes only bind to rough endoplasmic reticulum membranes when these are active and involved in protein synthesis. Therefore, we believe that both kinds are active and producing proteins in the follicular cells of stage III oocytes. In stage IVoocytes of M. fimbriolata, two types of yolk were observed: granular and viscous. The latter seems to be the result of lysis of the yolk granule, which becomes unorganized. Studies conducted by Gilbert and Kerkut (1985) and Buning (1994) revealed that granular yolk is usually found in oocytes of insects. Viscous yolk, however, is still under debate and data on it are not available in the literature. Granular yolk becomes viscous in M. fimbriolata oocytes due to the breakdown of granule content followed by lysis. Also, intercellular spaces in the follicular epithelium in stage IV oocytes were larger than those observed in previous stages with fine and electrondense granules. This material then enters the oocyte through the cell membrane, and is later incorporated into yolk. This process was also observed in oocytes of the ant P. (N.) villosa by Camargo-Mathias and Caetano (1998). Therefore, it is clear that the opening of intercellular spaces in the follicular epithelium of M. fimbriolata oocytes increases as the incorporation of exogenous proteins intensifies. Viscous yolk was more frequently observed in intermediary stages IV and V oocytes of M. fimbriolata. This form of yolk, however, has not been described in the literature for other insects. In stage V oocytes viscous yolk completely fills the cytoplasm. Granular yolk is no longer observed.
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In addition to protein yolk, lipid yolk was also observed in M. fimbriolata oocytes. As oocytes develop (stages IV and V) and vitellogenesis is completed, the number and size of interdigitations of the cell membrane of M. fimbriolata oocytes decrease until they completely disappear. Therefore, this specialization of the cell membrane might be closely related to incorporation of exogenous materials used to produce yolk. After completing its development, the oocyte is enclosed by two protective membranes: vitelline membrane and chorion, which is thicker and more complex regarding its components. The chorion of eggs of insects usually consists of proteins, lipids and carbohydrates (King and Koch, 1963; Paul et al., 1972; Legay, 1979), and it is synthesized by follicular cells (King, 1960; Hinton, 1969). Several authors hypothesized that the chorion is produced by the oocyte, from vesicles of the rough endoplasmic reticulum and dictyosomes. Vesicles merge with the cell membrane of more developed oocytes, discharging their content in the extracellular space, where it polymerizes. Ultrastructural observations revealed that the chorion of M. fimbriolata oocytes preserves the eggs consequently preserving the species (King, 1960); protects against mechanical shock; desiccation and predation (Wigglesworth, 1974; Hinton, 1969); and specially promote gas exchange providing oxygen for the embryo (Hinton, 1982a,b). Chorion is secreted by follicular cells and it consists of exochorion, the outermost and less electron dense sheath in direct contact with follicular cells, and endochorion, the innermost and more electron dense sheath enclosing and facing the vitelline membrane, as observed in most insects. The ultra-morphological data obtained in this study revealed several pores perforating the exochorion in M. fimbriolata, that might be involved in the aeration of the internal structures of the future egg. Acknowledgments FAPESP (Fundac¸a˜o de Amparo a` Pesquisa do Estado de Sa˜o Paulo) Grant no. 03/13472-6 for the financial support. Enrico de Beni Arrigone at Copersucar, Piracicaba-Sa˜o Paulo State, Brazil, for the spittlebug supply. Cristiane Marcia Mile´o, Ge´rson Mello Souza, Moˆnika Iamonte and Antonio Yabuki for the technical support. References Anderson, L.M., Telfer, W.H., 1970. Extracellular concentrating of proteins in Cecropia moth follicle. J. Cell Physiol. 76, 37–54. Bonhag, P.F., 1955. Histochemical studies of the ovarian nurse tissue and oocytes of the milkweed bug Oncopeltus fasciatus. I. Cytology, nucleic acids, and carbohydrates. J. Morphol. (Dallas).
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Buning, J., 1979. The trophic tissue of telotrophic ovarioles in polyphaga, Coleo´ptera. Zoomorphologie 93, 33–50. Buning, J., 1994. The Insect Ovary. Ultrastructure, Previtellogenic Growth and Evolution. Chapman & Hall. Camargo-Mathias, M.I., Caetano, F.H., 1998. Ultrastrucutural study of the follicular epithelium in oocytes of Pachycondyla (Neoponera) villosa ants. Hymenoptera: Ponerinae. Biocell 22, 53–60. Caperucci, D., Camargo-Mathias, M.I., 2006a. Lipids in oocytes of ants Neoponera villosa (Hymenoptera: Ponerinae). Sociobiology 47 (2) in press. Caperucci, D., Camargo-Mathias, M.I., 2006b. Female reproductive system of the sugarcane spittlebug Mahanarva fimbriolata (Auchenorrhyncha). Vitellogenesis dynamics and protein quantification, Micron, in press. Chapman, R.F., 1998. The Insects Structure and Function. Cambridge University Press/Hodder and Stoughton Educacional, New York. Gilbert, L.I., Kerkut, G.A., 1985. Embryogenesys and Reproduction. Pergamon Press, USA, pp. 391–419. Gillot, C., 1995. Entomology. Plenum Press, New York, p. 798. Haddad, A., et al., 1998. Te´cnicas Ba´sicas de Microscopia Eletroˆnica Aplicadas a`s Cieˆncias Biolo´gicas. Wanderley de Souza, pp. 66–73. Han, S.I., Bordereau, C., 1982. Origin and formation of the royal fat body of higher termite queens. J. Morphol. Lisse 173, 17–28. Hinton, H.E., 1969. Respiratory systems of insect egg shells. Annu. Rev. Entomol. 14, 343–368. Hinton, H.E., 1982a. Insect egg shells. Sci. Am. 223, 84–91. Hinton, H.E., 1982b. Biology of Insect Egg Shells. Pergamon Press, Oxford. Hubner, E., 1981. Nurse cell–oocyte interaction in the telotrophic ovaries of an insect, Rhodnius prolixus. Tissue Cell 13, 105–125. Huebner, E., Injeyan, H.S., 1980. Potency of the follicular epithelium in Rhodnius prolixus. A reexamination of the hormone response and technic refinement. Can. J. Zool. 58, 1617–1625. King, R.C., Koch, E.A., 1963. Studies on the ovarian follicle cells of Drosophila. Q. J. Microscop. Sci. 104, 297–320. King, R.C., 1960. Oogenesis in adult Drosophila melanogaster. IX. Studies on the cytochemistry and ultrastructure of developing oocytes. Growth 24, 265–323. King, R.C., Akai, H., 1984. Insect Ultrastucture, vol. 2. Plenum Press, pp. 3–40. Legay, J.M., 1979. Oocyte growth. Biochimie 61, 137–145. Norrevang, A., 1968. Electron microscopic morphology of oogenesis. Int. Rev. Cytol. 23, 114–186. Paul, M., Goldsmith, M.R., Hunsley, J.R., Kafatos, F.C., 1972. Specific protein synthesis in cellular differentiation. Production of eggshell proteins by silkmoth follicular. J. Cell Biol. 55, 653–680. Reddy, J.T., Locke, M., 1990. The size limited penetration on gold particles through insect basal laminae. J. Physiol. 36 (6), 397. Rockstein, M., 1964. The Physiology of Insecta, vol. I. Academic Press, New York (Chapter III), 640 p. Schreiner, B., 1977. Vitellogenesis in the milk weed bug Oncopeltus fasciatus: a light and electron microscopic investigation. J. Morphol. 35–80. Secco, V.N.D.P., 1990. Ultra-estrutura do desenvolvimento do ova´rio de Dermatobia hominis (Dı´ptera: Cuterebridae). Dissertac¸a˜o (Mestrado). UNESP, Botucatu, IB, 71 p. Silva, J.R.F., 1999 Estudo morfolo´gico em ova´rios de lagostas do geˆnero Panulirus. White 1847 (Crusta´cea: Decapoda: Palinuridae). Thesis (Doutor em Zoologia). Instituto de Biocieˆncias da Universidade de Sa˜o Paulo, p. 187. Wigglesworth, V.B., 1974. Insect Physiology. Chapman and Hall, London, 434 p. Wigglesworth, V.B., 1979. The Principles of Insect Physiology. Chapman and Hall, London, 277 p.
Ultrastructural study of the ovary of the sugarcane spittlebug Mahanarva fimbriolata (Hemiptera) De´bora Caperucci, Maria Izabel Camargo-Mathias * Departamento de Biologia, Instituto de Biocieˆncias, UNESP, Av 24 A, no. 1515. Cx. Postal 199, CEP: 13506-900 Rio Claro, SP, Brazil Received 13 October 2005; received in revised form 8 February 2006; accepted 9 February 2006
Abstract The present study describes the ultrastructure of meroistic telotrophic ovaries of the sugarcane spittlebug Mahanarva fimbriolata. In this type of ovary, nurse cells, oogonia, and prefollicular tissue are located at the terminal (distal) regions or tropharium of ovarioles. Oocytes in different developmental stages, classified from I to V, are observed in the vitellarium. Stage I oocytes do not exhibit intercellular spaces in the follicular epithelium, suggesting that synthesis and production of yolk during this stage occurs only through endogenous processes. Small yolk granules of different electron densities are present in the cytoplasm. Few lipid droplets are observed. Stage II oocytes exhibit small intercellular spaces in the follicular epithelium. More protein as well as lipid yolk granules are observed in the cytoplasm. In stage III oocytes, intercellular spaces in the follicular epithelium are larger than those observed in the previous stage. Electrondense protein granules of various sizes, larger than those observed in stage II oocytes predominate in the cytoplasm. Smaller lipid droplets are also present. In stage IV oocytes, the follicular epithelium exhibits large intercellular spaces. Our data clearly indicate that the opening of these spaces in the follicular epithelium of M. fimbriolata oocytes increases as the intake of exogenous proteins intensifies, that is, in stages IVand Voocytes. During these stages, granular yolk becomes viscous due to the lysis of granules. In stage Voocytes, viscous yolk predominates in the cytoplasm. This type of yolk, however, has not been described for other orders of insects. The chorion of M. fimbriolata oocytes consists of an external layer (exochorion) and an internal one (endochorion), which is in direct contact with the oocyte. Numerous small pores that probably facilitate oxygenation of the internal structures inside the eggs are observed in the exochorion. # 2006 Elsevier Ltd. All rights reserved. Keywords: Mahanarva fimbriolata; Ovary; Ultrastructure; Vitellogenesis
1. Introduction The reproductive system of insects usually performs several functions, among them: egg production, reception and sperm storage (occasionally for long periods), and coordination of events leading to fertilization and egg-laying (Gillot, 1995). The number of ovarioles in different orders varies greatly from one species to another; for example, viviparous aphids exhibit a functional ovariole in one ovary, while higher termite queens of the genus Eutermes may have more than 2000 ovarioles in each ovariole (Buning, 1994; Gillot, 1995; Chapman, 1998). Seven ovarioles per ovary were observed in M. fimbriolata (Caperucci and Camargo-Mathias, 2006a,b). Ovarioles of insects present three distinct regions: terminal filament located near the distal end and composed of connective
* Corresponding author. Tel.: +55 19 3526 4135; fax: +55 19 3534 0009. E-mail address: [email protected] (M.I. Camargo-Mathias). 0968-4328/$ – see front matter # 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.micron.2006.02.002
or conjunctive tissue; germarium or tropharium, the apical tip of the ovariole below the terminal filament, consisting of a mass of cells capable of differentiating into oocytes and nurse cells (when present); and vitellarium, which is the largest portion of the ovariole containing developing oocytes (undergoing vitellogenesis) surrounded by follicular cells. Studies on the female reproductive system of the sugarcane spittlebug are especially important to support basic research on insect reproduction, as well as applied research aiming at the development of mechanisms for the biological control of this emergent pest. Previous studies on this insect already described its ovaries as meroistic telotrophic, exhibiting follicular and nurse cells, as well as oocytes in the tropharium (Caperucci and Camargo-Mathias, 2006a,b). This study aimed to describe the ultrastructural analysis of the ovaries of the sugarcane spittlebug M. fimbriolata. Studies on this insect are especially relevant due to little morphological data available in the literature that could contribute for a better understanding of its biology. More specifically, studies on its
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reproductive system may provide additional information to control it. 2. Material and methods In this study, we utilized adult females of the sugarcane spittlebug M. fimbriolata collected at Copersucar, PiracicabaSa˜o Paulo State, Brazil, and kindly provided by Dr. Enrico de Beni Arrigone. We used the equipment available at the laboratories of the Laboratory/Biology Department of Electron Microscopy, UNESP, Rio Claro-SP. Spittlebugs were collected in the sugarcane plantation, anesthetized by thermal shock, dissected while immersed in Petri dish with physiological solution for insects to remove their ovaries. Ovaries were then fixed in 3% glutaraldehyde, postfixed in 1% OsO4, routinely processed, and embedded in Epon Araldite (Haddad et al., 1998). Ultrathin sections were stained with uranyl acetate and lead citrate. Grids containing ultrathin sections of the material were analyzed and photographed in a PHILLIPS 100 TEM transmission electron microscope. 3. Results In M. fimbriolata, tropharium cells range from elliptic to round, although most are irregular in shape, with homogeneous
and not very electrondense cytoplasm (Fig. 1A). Electrondense material is observed in intercellular spaces. The most frequently observed organelles are mitochondria (Fig. 1B), followed by ribosomes, and vesicular rough endoplasmic reticulum. The nucleus has an irregular in shape with little condensed chromatin, and nucleoli. The numerous pores perforate the nuclear envelope (Fig. 1B). Tropharium cells, especially those located in the periphery are supported by a thick basal lamina (Fig. 1B), and just below it, near the plasmatic membrane, very electrondense vacuoles containing membrane elements are frequently observed. As they reach the vitellarium, stage I oocytes are characterized by small yolk granules in the cytoplasm, most of them containing proteins, but yolk granules containing lipids are also observed (Fig. 1C). Intercellular spaces in the follicular epithelium are not observed, indicating that synthesis and production of yolk occur only by endogenous processes in these oocytes (Fig. 1D arrow). Stage II or pre-vitellogenic oocytes exhibit large quantities of lamellar endoplasmic reticulum, suggesting an intense endogenous synthesis of proteins (Fig. 1F). Also during this stage, intercellular spaces in the follicular epithelium are formed (Fig. 1E). Protein granules are more electrondense than lipid granules and increase in size as they join together and move from the periphery to the central region of the cytoplasm (Fig. 1E).
Fig. 1. (A and B) Details of the ultrastructure of tropharium cells (tro) of Mahanarva fimbriolata oocytes, n = irregular nucleus, np = nuclear pores, cl = cell limit, nu = nucleolus, mb = myelin bodies. Scale bars: A = 2 mm and B = 0.5 mm. (C) General view of stage I oocyte (ov I) and partial view of follicular epithelium (fe), with large amounts of protein granules (py), lipid granules (ly), mitochondria (m), interface follicular epithelium/oocyte (ie). Scale bar: 2 mm. (D) Cells of the follicular epithelium of stage I oocytes (ov I) with partially irregular nuclei (n), intercellular spaces (ie) with electrondense material (arrow), mitochondria (m), lamellar rough endoplasmic reticulum (rerl). Scale bar: 2 mm. (E) Detail of stage II oocyte (ov II) and follicular epithelium (fe) with intercellular spaces (ie), nuclei of follicular cells, lipid granules (ly) and protein granules (py), interface follicular epithelium/oocyte (ie) with microvilli (mic). Scale bar: 5 mm. (F) Cytoplasm of follicular cells of stage II oocytes II (ov II) with lamellar rough endoplasmic reticulum (rerl), nucleus (n) with several pores (arrow). Scale bar: 0.5 mm.
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Vitellogenesis intensifies in stage III oocytes. Electrondense protein granules of different sizes and smaller lipid granules predominate in the cytoplasm (Fig. 2A). Lamellar rough endoplasmic reticulum in the follicular cells is observed (Fig. 2B). During this stage oocytes exhibit follicular epithelium with larger intercellular spaces when compared to those of the previous stage (Fig. 2B). In stage IV oocytes, granular yolk becomes viscous due to breakdown of compounds followed by granule lysis (Fig. 2C). Yolk is present as protein granules and viscous yolk, the latter seems to be the result of granules that have undergone lysis, becoming unorganized structures (Fig. 2C and E). Large amounts of vesicular and lamellar rough endoplasmic reticulum are present in the cytoplasm of follicular cells, indicating that during this phase the follicular epithelium might still be involved in the synthesis of elements that will later form the chorion (Fig. 3C and D). Much larger intercellular spaces than in previous stages are also observed, clearly indicating that the opening of these spaces in the follicular epithelium of M. fimbriolata oocytes increases as the intake of exogenous proteins intensifies (Fig. 3A and B). Viscous yolk is more frequently observed in intermediate stages IV and V oocytes (Fig. 3E and F).
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In stage V oocytes, viscous yolk completely fills the cytoplasm, while granular yolk is no longer observed (Fig. 4B). Follicular cells in stage V oocytes are in the process of degeneration with several vacuoles in the cytoplasm (Fig. 4C). The chorion in M. fimbriolata oocytes consists of exochorion, more external and less electrondense layer in direct contact with follicular cells; and endochorion, the innermost and more electrondense layer (Fig. 4A and D). The exochorion is perforated by numerous pores (Fig. 4A arrow). 4. Discussion Studies on the spittlebug M. fimbriolata describe it as a pest to agriculture that attacks sugarcane. Therefore, studies on its reproductive system provide important information about its biology. The ovaries of M. fimbriolata are meroistic telotrophic, that is, follicular cells, as well as nurse cells, and oocytes are located in the tropharium. In most insects, ovarioles are functional units covered by two morphologically distinct sheaths, the outermost cellular sheath, also known as peritoneal sheath, and the innermost acellular one consisted of thin fibrils, called tunica propria. According to some authors, their role is to provide elasticity to
Fig. 2. (A) Cytoplasm of stage III oocytes (ov III) with protein granules (py) and mitochondria (m). Scale bar: 1 mm. (B) Cytoplasm of stage III oocytes (ov III) with intercellular spaces (ie), irregular nucleus (n) with pores (arrow), mitochondria (m) and lamellar rough endoplasmic reticulum (rerl). Scale bar: 1 mm. (C) Cytoplasm of stage IVoocytes (ov IV), containing protein granules (py), with more electrondense periphery and less electron dense interior, mitochondria (m). Viscous yolk (vy) is first observed. Scale bar: 2 mm. (D) Intercellular spaces (ie) between the follicular epithelium (fe) and the oocyte with electrondense material, protein granules (py) in the periphery of the oocyte. Scale bar: 1 mm. (E) Detail of the cytoplasm of stage IV oocytes (ov IV) with viscous yolk (vy) containing proteins, and mitochondria (m). Scale bar: 1 mm.
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Fig. 3. (A) Detail of the spaces between follicular cells (ie) of stage IV oocytes (ov IV) with homogeneous material. Scale bar: 1 mm. (B) Intercellular spaces (ie) in the follicular epithelium of stage IV oocytes (ov IV) and vesicular rough endoplasmic reticulum (rerv) and lamellar (rerl). Scale bar: 1 mm. (C and D) Cytoplasm of follicular cells of stage IVoocytes (ov IV), with lamellar rough endoplasmic reticulum (rerv) and lamellar (rerl), and mitochondria (m). Scale bars: 1 mm. (E) General view of the cytoplasm of intermediate stages IV and V oocytes showing the beginning of production of viscous yolk (vy) inside the granule, protein granules (py). Scale bar: 1 mm. (F) Cytoplasm of intermediate stages IV and V oocytes with viscous yolk (vy), and protein granules (py). Space bar: 5 mm.
ovarioles, assisting oocytes to move down towards the exterior (Chapman, 1998). The origin of the tunica propria in the ovarioles is debated. Some authors believe that it is synthesized in the terminal filament area (Chapman, 1998), while others hypothesize that it is produced by phagocytes, which have been frequently found in the ovaries of several insects (Schreiner, 1977; Secco, 1990). Evidences on its origins in M. fimbriolata were not found. The tropharium is the next region after the terminal filament of ovarioles. In this region oocytes are formed from germinative cells through successive mitotic divisions and will be covered with a single epithelium of follicular cells from mesoderm (Bonhag, 1955; Rockstein, 1964; Buning, 1979; Wigglesworth, 1979). Ultrastructural results on the ovaries of the spittlebug M. fimbriolata revealed that the morphology of tropharium cells is irregular, corroborating the observed by Buning (1994) on Dysdercus intermedius, an insect of the same order. The most frequently cytoplasmic organelles found in the tropharium cells of M. fimbriolata were mitochondria, suggesting that large amounts of energy might be needed, mainly for cell division. The cellular material found in spaces between tropharium cells indicates an intense cell activity, as well as material exchange between cells. The basal lamina or tunica propria in the tropharium region of M. fimbriolata was thick and electrondense. According to Silva (1999), the fibrous region of the basal lamina surrounding
germinative cells of lobsters of the genus Panulirus may be able to adapt to volume changes undergone by oocytes throughout the differentiation process, in addition to facilitate the release of mature oocytes from the vitellarium, which will be ovulated and later fertilized. In addition to cell support, the basal lamina might also select elements entering the ovariole from the hemolymph and vice-versa in M. fimbriolata. These data corroborate those found by Reddy and Locke (1990), suggesting that the basal lamina serves as the primary barrier between hemolymph elements and the interior of oocyte, monitoring the traffic of macromolecules. The following region in the ovarioles after the tropharium is the vitellarium, where oocytes accumulate yolk through vitellogenesis, consequently increasing in size many times until ready to be fertilized. Synthesis (endogenous production) and deposition (exogenous incorporation) of yolk in oocytes occur in this region with participation of oocyte, as well as follicular cells. This was also observed in M. fimbriolata ovarioles. The oocytes of telotrophic ovaries of M. fimbriolata develop in the vitellarium in close association with follicular cells, which surround them as an epithelium. These cells originate from mitosis of pre-follicular cells that underwent a series of morphological and physiological changes during oogenesis and vitellogenesis. To date, the presence of cytoplasmic bridges between adjacent follicular cells in telotrophic ovaries has not been observed, as described for polytrophic ovaries. This was also not observed for M. fimbriolata.
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The large morphological diversity found in the oocytes, as well as follicular cells present in M. fimbriolata ovarioles is due to the developmental stage in which the oocytes are observed. This suggests a simultaneous, although asynchronous, development of oocytes in different ovarioles, where oocytes in early developmental stages and mature oocytes can be found throughout the ovarioles. Therefore, our data have shown that vitellogenesis occurs in a distal to proximal sequence, with less developed oocytes, also
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called primary, located in the distal region and more developed or vitellogenic oocytes located in the proximal region. An ultrastructural analysis of oocytes in the vitellarium of M. fimbriolata ovarioles revealed that during stage I, small yolk granules of different electron densities are present in the cytoplasm, most of them containing proteins, but yolk granules containing lipids are also observed. These oocytes are in early stages of vitellogenesis and produce yolk granules through endogenous synthesis.
Fig. 4. (A) Intermediate stages IVand Voocytes showing the beginning of chorion deposition (ch), apical portion of follicular cells (fe). Periphery of cytoplasm with a modified cell membrane (arrow), protein granules (py) and mitochondria (m), viscous yolk (vy). Scale bar: 2 mm. (B) Stage Voocytes with homogeneous and electron dense viscous yolk (vy). Scale bar: 1 mm. (C) Stage V oocytes with follicular cells in the process of degeneration (fed), where organelles with unorganized structure and intercellular spaces (ie) are observed. Scale bar: 1 mm. (D) Stage V oocytes: ultrastructural aspect of chorion with exochorion (exo) with pores (arrow) and endochorion (end). Scale bar: 2 mm.
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We did not observe intercellular spaces between follicular cells in the follicular epithelium of stage I oocytes of M. fimbriolata, confirming that synthesis and production of yolk might occur only by endogenous processes. Camargo-Mathias and Caetano (1998) also did not observe intercellular spaces in the follicular epithelium of stage I oocytes of the ant Pachycondyla (Neoponera) villosa. Openings of intercellular spaces of the follicular epithelium have been usually described as a process that requires the presence of cytoskeleton elements (Huebner and Injeyan, 1980). Some authors have pointed out that opening and closing processes of these spaces are strongly associated with these elements, corroborating data found for Rhodnius prolixus (King and Akai, 1984). In M. fimbriolata oocytes, cytoskeleton elements are not observed in the follicular cells surrounding them. Mahanarua fimbriolata oocytes in developmental stage II, also known as pre-vitellogenic, unlike stage I oocytes, exhibited many small vesicles in the cytoplasm. The ones containing proteins and lipids were in the periphery of the oocyte while larger granules were found in the center, probably due to the fusion of smaller vesicles, as observed for D. intermedius (Buning, 1994). Large amounts of rough endoplasmic reticulum found in stage II oocytes of M. fimbriolata suggest that during this stage intense endogenous protein synthesis still occurs, providing protein elements that will be used in the development of the oocyte. During the first two developmental stages of M. fimbriolata oocytes, we observed the traffic of material from the germinal vesicle to the cytoplasm through nuclear pores. This suggests that endogenous synthesis of elements that will probably be part of the final yolk composition is being carried out inside the oocyte. The traffic of material through nuclear pores was also observed in pre-vitellogenic oocytes in Oncopeltus fasciatus and R. prolixus (Hubner, 1981). The author described this material as probably RNAr. The ultrastructural data obtained in this study revealed that incorporation of extra-ovarian proteins from the hemolymph begins in stage II oocytes. We observed morphological changes in the cells of the follicular epithelium, especially regarding the opening of intercellular spaces. During this stage, spaces begin to be formed, which were not observed in the previous stage. Large quantities of mitochondria observed in the cytoplasm of follicular cells near lateral membranes indicate the need of energy to open these spaces through which extra-ovarian material will flow. Our data suggest that vitellogenesis in oocytes of the spittlebug M. fimbriolata probably occurs through four processes: (a) endogenous synthesis of elements carried out by the oocyte; (b) synthesis of elements by follicular cells followed by transport into the oocyte by endocytosis; (c) selective intake of extra-ovarian material by follicular cells followed by transport to the oocyte; (d) transport of extraovarian material into the oocyte through intercellular spaces of the follicular epithelium. Anderson and Telfer (1970) have demonstrated in previous studies that proteins transported into the oocyte might also be
selected at the intercellular spaces of the follicular epithelium. These authors also suggested that follicular cells might produce elements involved in the removal of proteins from the hemolymph to be incorporated by the oocyte. Ultrastructural data obtained for stage III oocytes of M. fimbriolata indicate that vitellogenesis intensifies during this stage. Lipid droplets and protein granules enclosed by a membrane were also observed in larger amounts than in stage II oocytes. The presence of rough endoplasmic reticulum in the cytoplasm of stage III oocytes of M. fimbriolata indicates that endogenous synthesis of yolk still occurs during this stage. In addition to rough endoplasmic reticulum, some authors have pointed out the participation of the Golgi complex during vitellogenesis. This organelle, however, was not observed in M. fimbriolata oocytes, indicating little or no participation in the production of elements. Norrevang (1968), reviewing the ultrastructural characteristics of oogenesis in several animal groups, suggested that in most oocytes, the Golgi complex does not seem to be an important element and little has been described about its role in the dynamics of vitellogenesis in insects. In follicular cells of M. fimbriolata oocytes, lamellar rough endoplasmic reticulum was more abundant than vesicular one. According to Han and Bordereau (1982), the presence of two kinds of rough endoplasmic reticulum in the same cells could indicate different functions. The vesicular rough endoplasmic reticulum would be more active while the lamellar form, the less active between these organelles. On the other hand, it is known that ribosomes only bind to rough endoplasmic reticulum membranes when these are active and involved in protein synthesis. Therefore, we believe that both kinds are active and producing proteins in the follicular cells of stage III oocytes. In stage IVoocytes of M. fimbriolata, two types of yolk were observed: granular and viscous. The latter seems to be the result of lysis of the yolk granule, which becomes unorganized. Studies conducted by Gilbert and Kerkut (1985) and Buning (1994) revealed that granular yolk is usually found in oocytes of insects. Viscous yolk, however, is still under debate and data on it are not available in the literature. Granular yolk becomes viscous in M. fimbriolata oocytes due to the breakdown of granule content followed by lysis. Also, intercellular spaces in the follicular epithelium in stage IV oocytes were larger than those observed in previous stages with fine and electrondense granules. This material then enters the oocyte through the cell membrane, and is later incorporated into yolk. This process was also observed in oocytes of the ant P. (N.) villosa by Camargo-Mathias and Caetano (1998). Therefore, it is clear that the opening of intercellular spaces in the follicular epithelium of M. fimbriolata oocytes increases as the incorporation of exogenous proteins intensifies. Viscous yolk was more frequently observed in intermediary stages IV and V oocytes of M. fimbriolata. This form of yolk, however, has not been described in the literature for other insects. In stage V oocytes viscous yolk completely fills the cytoplasm. Granular yolk is no longer observed.
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In addition to protein yolk, lipid yolk was also observed in M. fimbriolata oocytes. As oocytes develop (stages IV and V) and vitellogenesis is completed, the number and size of interdigitations of the cell membrane of M. fimbriolata oocytes decrease until they completely disappear. Therefore, this specialization of the cell membrane might be closely related to incorporation of exogenous materials used to produce yolk. After completing its development, the oocyte is enclosed by two protective membranes: vitelline membrane and chorion, which is thicker and more complex regarding its components. The chorion of eggs of insects usually consists of proteins, lipids and carbohydrates (King and Koch, 1963; Paul et al., 1972; Legay, 1979), and it is synthesized by follicular cells (King, 1960; Hinton, 1969). Several authors hypothesized that the chorion is produced by the oocyte, from vesicles of the rough endoplasmic reticulum and dictyosomes. Vesicles merge with the cell membrane of more developed oocytes, discharging their content in the extracellular space, where it polymerizes. Ultrastructural observations revealed that the chorion of M. fimbriolata oocytes preserves the eggs consequently preserving the species (King, 1960); protects against mechanical shock; desiccation and predation (Wigglesworth, 1974; Hinton, 1969); and specially promote gas exchange providing oxygen for the embryo (Hinton, 1982a,b). Chorion is secreted by follicular cells and it consists of exochorion, the outermost and less electron dense sheath in direct contact with follicular cells, and endochorion, the innermost and more electron dense sheath enclosing and facing the vitelline membrane, as observed in most insects. The ultra-morphological data obtained in this study revealed several pores perforating the exochorion in M. fimbriolata, that might be involved in the aeration of the internal structures of the future egg. Acknowledgments FAPESP (Fundac¸a˜o de Amparo a` Pesquisa do Estado de Sa˜o Paulo) Grant no. 03/13472-6 for the financial support. Enrico de Beni Arrigone at Copersucar, Piracicaba-Sa˜o Paulo State, Brazil, for the spittlebug supply. Cristiane Marcia Mile´o, Ge´rson Mello Souza, Moˆnika Iamonte and Antonio Yabuki for the technical support. References Anderson, L.M., Telfer, W.H., 1970. Extracellular concentrating of proteins in Cecropia moth follicle. J. Cell Physiol. 76, 37–54. Bonhag, P.F., 1955. Histochemical studies of the ovarian nurse tissue and oocytes of the milkweed bug Oncopeltus fasciatus. I. Cytology, nucleic acids, and carbohydrates. J. Morphol. (Dallas).
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