Ultrastructure of the ovariole sheath in Diatraea saccharalis (Lepidoptera: Pyralidae)

The ultrastructure of the ovariole sheath along the Diatraea saccharalis ovariole was studied by scanning and transmission electron microscopy. Each ovariole is surrounded by an epithelial sheath, a tunica propria and scattered lumen cells. These three components of the ovariole sheath show different ultrastructural features along the ovariole, in the germarium or in the vitellarium; these differences are more evident in the epithelial sheath cells. The epithelial sheath is composed by two layers of cells, the external one running longitudinally and the internal one running circularly in the ovariole. These cells, in vitellarium, present cytoplasmic bundles of myofilaments that are arranged parallel to the long axis of the cells; these myofilaments are apparently related to the contraction movements of the follicles within the ovariole. The acellular tunica propria, composed of finely f ilamentous material, is attached to the adjacent follicle cells by adhesive dense plates. Between the epithelial sheath and the tunica propria there is a population of lumen cells, with morphological features of secretory activity. BIOCELL 2002, 26(2): 229-235 ISSN 0327 9545 PRINTED IN ARGENTINA


Introduction
The great majority of insects has each of their two ovaries encompassed by an ovarian capsule, in the beginning of the female reproductive system development (Machida, 1926;Miya et al., 1970b;Büning, 1994).
During the growth of the ovaries, this ovarian capsule tears at its attachment points to the oviduct and the ovarioles emerge gradually by elongation into the abdominal cavity of the insect (Machida, 1926;Snodgrass, 1935;Koch et al., 1967;King and Aggarwal, 1965;Miya et al., 1970a, b;Büning, 1994).
Each ovariole is enclosed separately by an ovariole sheath, which is composed by: the cellular epithelial sheath on the outside, the amorphous tunica propria on the inside and a layer of isolated lumen cells between the epithelial sheath and tunica propria. (Bonhag and Arnold, 1961;King and Koch, 1963;King and Aggarwal, 1965;Koch and King, 1966;Miya et al., 1970a, b;Cruickshank, 1973;Gregório et al., 1990;Büning, 1994). In this paper, we describe the ultrastructural organization of the ovariole sheath along the germarium and vitellarium of Diatraea saccharalis ovarioles. There are no ultrastructural studies on ovarian sheath organization in Diatraea saccharalis, although this insect has long been considered the most destructive pest attacking sugarcane in several Latin American countries.

Material and Methods
The larvae of Diatraea saccharalis were reared on an artificial diet (Hensley and Hammond, 1968), and the pupae were maintained in recipient, without the artificial diet, until the emergence as adults. Both larvae and pupae were maintained under controlled temperature (25-27°C) and humidity (70%).
Ovaries removed from insects of the last larval instar and from pupae (5-7 days) were fixed for 24h in 2% glutaraldehyde -4% paraformaldehyde solution buffered in 0.1M buffer phosphate (pH 7.3).
For the transmission electron microscopy (TEM) observations, the ovaries were post-fixed in 1% osmium tetroxide for 2h, dehydrated through a graded series of acetone and embedded in Araldite. Ultra-thin sections were double stained with uranyl acetate and lead citrate.
For the scanning electron microscopy (SEM) observations, the pre-fixed ovarioles were immersed in 10% NaOH solution for 17h, post-fixed in 1% osmium tetroxide for 2h, dehydrated through a graded series of acetone, critical point dried with liquid CO 2 and sputtered gold-coated (10nm thick).

Results
Each ovariole of Diatraea saccharalis is surrounded by a continuous sheath composed of the epithelial sheath, the lumen cells and the tunica propria. These three components of the ovariole sheath show different ultrastructural features along the ovariole.
In the germarium, the epithelial sheath cells are flat and could be visualized as juxtaposed "disks", not easily distinguishable from the cells of the adjacent connective tissue (Figs. 1,3,4). Their cytoplasms present elongated mitochondria, few rough endoplasmic reticu-lum, glycogen deposits and some dense vesicles (Fig.  3). It is not possible to distinguish a basal membrane separating the epithelial sheath cells from the adjacent connective tissue (Fig. 4). The tunica propria is very thin and is composed of a homogeneous layer of finely filamentous material (Figs. 1-5). There is a discreet but progressive increase in the thickness of the tunica propria along the germarium. The fluid-filled lumen which extends between the tunica propria and epithelial sheath contains small populations of ameboid cells, the lumen cells. These cells possess dense mitochondria, few cisterns of rough endoplasmic reticulum, heterogeneous vacuoles and vesicles, Golgi complexes, in addition to free ribosomes (Figs. 1-3). Their nuclei are ovoid in shape, exhibiting uncondensed chromatin and evident nucleoli. There are no adhesive junctions between lumen cells, except interdigitations (Figs. 1 and 5); finely flocculated material is observed in the intercellular spaces, when the interdigitations are enlarged (Figs. 1 and 5).
In the vitellarium, the cells of the epithelial sheath are disposed in two layers (external and internal cellular layer) (Figs. 6,7,8). SEM of the NaOH digestion preparation shows that in the external layer the cells are arranged parallel to the long axis of the ovariole and in the internal layer they run circularly around the ovariole . TEM shows that these cells in both layers present large amount of cytoplasmatic myofilaments parallel to the longest axis of the cells; there is an increase in the amount of myofilaments along the vitellarium (Figs. 6 and 7). The epithelial sheath is surrounded by both inner and outer continuous basal membranes (Figs. 6 and 7). There are interdigitations and desmosomes attaching the epithelial sheath cells of the same layer as well as between the cells of the two adjacent layers (Figs. 6,7,9). Besides myofilaments, the cytoplasm shows mitochondria, rough endoplasmic reticulum, small vesicles with flocculent material and free ribosomes (Fig. 7). The tunica propria is thicker     than that in the germarium and exhibits bundles of tubular structures in addition to the filamentous material (Fig. 10). There are electron-dense adhesive plates attaching the tunica propria to the adjacent follicle cells, with adjacent microtubules (Fig. 10). The lumen cells show the same ultrastructural morphology as those described in the germarium (Fig. 8).

Discussion
The ovarioles in Diatraea saccharalis are individually covered by an epithelial sheath, a population of lumen cells and a tunica propria. These three elements of the ovariole coating were also observed in many others insects (Bonhag, 1958;King and Aggarwal, 1965;Koch and King, 1966;Miya et al., 1970b;Cruickshank, 1973), although the morphologic variations along the ovariole coating were not described in a systematized way.
In the germarium of the Diatraea saccharalis ovariole, the epithelial sheath cells are not distinguishable from those of the adjacent connective tissue; we did not detect basal membrane separating the two tissues, as observed by Miya et al. (1970b) in Bombyx mori. The epithelial sheath cells present a flat shape, with oval nuclei and reduced cytoplasm, with small elongated mitochondria, few cisterns of rough endoplasmic reticulum, small glycogen deposits and some dense vesicles; these ultrastructural features composing the cytoplasm are very similar to those found in Bombyx mori (Miya et al., 1970b).
In the vitellarium of the Diatraea saccharalis ovariole, the long epithelial sheath cells are disposed in two layers: an external cellular layer and an internal cellular layer, both attached by desmosomes. Adjacent to previtellogenic follicles, these epithelial sheath cells show characteristics of muscles cells, exhibiting bunches of myofilaments, distributed in the following way: longitudinally (external cellular layer) and transversally (internal cellular layer) to the long axis of the ovariole. There is an increase in the amount of myofilaments along the vitellarium. These characteristics of the epithelial sheath cells observed along the vitellarium of Diatraea saccharalis seem to be a common feature in the great majority of the insects (Machida, 1926;Bonhag, 1958;King and Koch, 1963;King and Aggarwal, 1965;Koch and King, 1966;Koch et al., 1967;Miya et al., 1970a;Mahowald, 1972;Cruickshank, 1973;Mandelbaum, 1980;Cook and Peterson, 1989;Carcupino et al., 1992;Büning, 1994). The authors relate the presence of these myofilaments to the capacity of the contraction of the ovarioles for the deposition of eggs. Pulsation and synchronized contractions of muscle fibers of the epithelial sheath were recorded in complete cycles of oscillations ranging from 200mm to 4s (Büning, 1994).
The lumen cells observed between epithelial sheath and tunica propria do not show morphologic variations along the germarium or the vitellarium of the Diatraea saccharalis ovarioles. Lumen cells are cuboid in shape, with various interdigitations; the nucleus is oval and its cytoplasm exhibits mitochondria, rough endoplasmic reticulum, some vacuoles and small vesicles, small glycogen deposits and Golgi complexes. In the extracellular spaces between adjacent lumen cells, we observed a considerable amount of finely flocculated material. The lumen cells described above are similar to those found in other insects. The same holds true for the flocculent material found here and there between lumen cells of Hyalophora cecropia (King and Aggarwal, 1965), Drosophila melanogaster (Koch and King, 1966), Bombyx mori (Miya et al., 1970a) and Anagasta kuhniella (Cruickshank, 1973). Some of these authors described these cells as blood cells (macrophages) (Koch    and King, 1966;Cruickshank, 1973), capable of secreting material to repair the tunica propria at its torn points due to ovariole growth and to the moment of eggs deposition; Cruickshank (1973) confirms this function for the lumen cells, suggesting that there seems to be a correlation between its secretory activity and the increase in thickness of the tunica propria during development. Although we did not observe significant modifications in the ultrastructural organization of the lumen cells, their morphological features indicate its contribution to the tunica propria thickness, that occurs along the Diatraea saccharalis ovariole.
In the germarium, the tunica propria is extremely thin, composed of finely filamentous material. There is an increase in the tunica propria thickness from the germarium to the vitellarium, where some dense structures were visualized among its filamentous material. The ultrastructural aspect of the tunica propria is very similar in many insects (Machida, 1926;Bonhag, 1958;King and Koch, 1963;King and Aggarwal, 1965;Koch and King, 1966;Koch et al., 1967;Miya et al., 1970a;Mahowald, 1972;Cruickshank, 1973;Gregório et al., 1990;Büning, 1994). According to these authors, the nature of the tunica propria material is still uncertain. It is known that this acellular layer presents proteins and carbohydrate complexes (Bonhag and Arnold, 1961) and that these proteins are rich in S-S groups (Cruickshank, 1973). Büning (1994) suggests that collagen type I, laminine and proteoglycans compose the tunica propria of insects. This author describes that, adjacent to the vitelogenic follicles, the tunica propria exhibits structures in stick shape, oriented parallel to the long axis of the ovariole, as we saw in Diatraea saccharalis at the end of previtellogenesis. However, Büning (1994) does not suggest a function for these structures. The basal membranes (tunica propria) of the insect ovarioles present three functions, as proposed by Büning (1994): (1) they serve as a mechanical structure to keep the elongated structure of the ovarioles stable; (2) they serve as physical sieves against the haemolymph; (3) they have some chemical selectivity for macromolecules which play a role in hormone access during previtellogenesis. The presence of adhesive plates attaching the tunica propria to the follicle cells, and their relation to the cytoplasmic microtubules, suggest that the tunica propria becomes stretched during the oogenesis.