Oogenesis of stone flies. Development of the follicular epithelium and formation of the eggshell in ovaries of Perla marginata (Panzer) and Perla pallida Guérin (Plecoptera: Perlidae)

Pergamon

Im. J. Insecr Morphol.

& Embryoi., Vol. 24, No. 3, pp. 253-271, 1995 Copyright 0 1995 Elsevier Science Lfd Printed in Great Britain. All rights reserved OOZO-7322/95 $9.50 + 0.00

0020-7322(95)00003-S

OOGENESIS OF STONE FLIES. DEVELOPMENT OF THE FOLLICULAR EPITHELIUM AND FORMATION OF THE EGGSHELL IN OVARIES OF PERLA MARGINATA (PANZER) AND PERLA PALLIDA GUl?RIN (PLECOPTERA : PERLIDAE)

Elibieta Institute

of Zoology,

Jagiellonian

Rosciszewska

University,

(Accepted

Ingardena

3 January

6, 30-060

Krakow,

Poland

1995)

Abstract-Development of the follicular epithelium in panoistic ovaries of stone flies Perk marginata (Panzer) and Perlapallida (Plecoptera: Perlidae) has been described. The major function of the follicular epithelium in Perla sp. is the secretion of eggshells. In this contribution some other functions of the epithelium are postulated as well. The follicular epithelium passes through several distinct stages of oogenesis. Particularly, during the period of middle vitellogenesis, the follicular epithelium could serve as a cellular pathway of egzogenous yolk precursors into the oocyte. The intercellular spaces (patency) are engaged in the vitellogenin’s transport only during late vitellogenesis. The process of choriogenesis and a diversification of follicular cells into subpopulations responsible for formation of specialized elements (main body chorion, chorionic collar, attachment structure) of the chorion are also discussed.

Index descriptors (in addition structure,

oocyte

development,

to those in title): Vitelline envelope, transmission electron microscope.

chorion,

attachment

INTRODUCTION

In all the known types of insect ovaries, including those of entognathous insects (Margaritis, 1985; Kaulenas, 1992; Bilinski, 1993), the follicular cells (somatic), form a single-layered epithelium that envelops each oocyte. The function of insect follicle cells has been widely studied (Anderson, 1964; De Loof and Lagasse, 1970; Bassemir, 1977; Bilinski and Petryszak, 1978; Ksiazkiewicz-Ilijewa, 1978; Goltzene, 1979; Matsuzaki et al., 1979; Soldan, 1979; Bitsch, 1980; Brower et al., 1981; Telfer et al., 1982; Bilinski, 1983; Bilinski et al., 1985; Margaritis, 1985; Matsuzaki et al., 1985; Bilinski, 1987; Bilinski and Jankowska, 1987; Ogorzalek, 1987; Mtinz, 1988; Rosciszewska, 1989; Gaino and Mazzini, 1988, 1990; Kaulenas, 1992; Szklarzewicz, 1989, 1993). It is well established that the formation of the chorion is the primary function of follicular cells. The secretory activity is closely connected with the morphological changes the follicular cells undergo during late oogenesis. Several changes, however, occur in earlier stages, indicating other additional functions of follicular cells. The present paper presents the first light microscope and transmission electron microscope (TEM) study of the development of follicular epithelium in stone flies during oogenesis. This study is of interest because in stone flies, the complex egg capsule, equipped with the so-called attachment structure (Stark and Szczytko, 1988; Rosciszewska, 1991a) secreted by the follicular cells, is well adapted to both land and aquatic 253

254

E. Rosciszewska

habitats. During secretion, follicular cells undergo categorization (diversification) into subpopulations responsible for the formation of the different parts of the egg capsule. Very little is known about plecopteran oogenesis. The process of ovary development is of particular interest. Recent studies indicate that the stone fly ovary is of the secondary panoistic (neopanoistic) type (Gottanka and Btining, 1990), providing new insight into evolution of insect ovaries (Stys and Bilinski, 1990).

MATERIALS

AND

METHODS

The ovaries were dissected from adult females and from 3 larval stages (A, B and C) of Perla marginafa and Perlu pallida. The precise determination of the stage of development the individual larva was in, was difficult (larvae were not reared in a laboratory, therefore the number of moults was unknown). The classification into 3 stages of larval development was based on: body dimensions, pigmentation, and the stage of ovary development. Stage A is characterized by larval body length from 12 to 16 mm and yellow colour. The ovary is flattened and consists of the parallel array of panoistic ovarioles enveloped by a single epithelial layer. All the oocytes in vitellarium are pre-vitellogenic. Stage B-the larvae are from 20-22 mm long, yellowish brown, the (single) epithelial sheet is broken and the ovarioles are arranged around the oviduct. The growing oocytes are in previtellogenesis or early vitellogenesis. Stage C (subimago) is a yellowish brown, body length is about 27 mm and the epithelial sheet is no longer visible. The terminal oocytes are in the late vitellogenesis phase or in choriogenesis. Oviducts are filled with (fully chorionated) eggs. The ovaries of the stage C larvae are very similar to ovaries of the imago forms. The dissected ovaries were fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4 for 72 h, rinsed several times in phosphate buffer, pH 7.4, containing 5.8% sucrose and postfixed for 1.5 h with 1% 0~04 in 0.1 M phosphate buffer, pH 7.4. The specimens were rinsed several times in water, dehydrated in a graded series of alcohol and acetone and embedded in Epon 812. Blocks were cut using a Tesla BS 490 A ultramicrotome. Uftrathin sections contrasted with uranyl acetate and lead citrate were examined in a Tesla BS 500 transmission electron microscope (TEM). Semithin sections (1.5 pm) stained either using 1% methylene blue in 1% borax or using PAS method (according to Litwin, 1985) were examined under a Peraval Interphaco Zeiss light microscope.

RESULTS

The oocytes developing in (panoistic) ovaries of these stones flies are arranged linearly (Fig. l.), and gradually shift towards oviducts. The first batch of eggs is fully formed Figs. 1-7. Perk mnrginatu. Development of the follicular epithelium during early stages of oogenesis (previtellogenesis-middle vitellogenesis). Note changes in numbers, volumes and shapes of the follicular cells. Fig. 1. Semithin longitudinal section of a fragment of a panoistic ovary. Stage A larva. Early previtellogenesis. Methylene blue stained. X500. 0 = oocyte; short arrows = fohicular cells. Fig. 2. Semithin longitudinal section of previtellogenic oocyte of the stage B larva, surrounded by the follicular epithelium. Methylene blue stained. x500. 0 = oocyte; short arrows = follicular cells. Fig. 3. Smithin longitudinal section of terminal, early vitellogenic oocyte of the stage B larva. Note cubic shapes of the follicular cells. Methylene blue stained. x500. 0 = oocyte; short arrows = follicular cells. Fig. 4. TEM, middle vitellogenesis. Follicular cell - oocyte interface. Electron-dense fibrillar material is deposited close to oocyte microvilli. Note also pinocytotic vesicles at the oocyte microvilli bases. x 17000. FC = follicular cell; 0 = oocyte; long arrows = vitelline envelope precursor. Fig. 5. Semithin longitudinal section of subterminal, middle vitellogenic oocyte from the mature ovary. Note columnar shapes of the follicular cells. Methylene blue stained. x500. 0 = oocyte; short arrows = follicular cells; arrowhead = nucleolus. Fig. 6. TEM, middle vitellogenesis. A fragment of follicular cell (apical part). ~16000. RER = rough endoplasmic reticulum. Fig. 7. TEM, middle vitellogenesis. RER whorl encasing a lipid droplet (basal part of the follicular cell). x18300. L = lipid droplet; RER = rough endoplasmic reticulum.

Oogenesisof Stone Flies

255

in subimago individuals. This batch is subsequently deposited into the oviduct and stored until fertilization and oviposition. The consecutive stages of follicular epithelium development were examined in vitellaria of larval and imaginal ovaries. The 5 distinct stages of the follicular epithelium development (corresponding to the oocyte development) are as follows: I. Previtellogenesis. In ovaries of the stage A larvae, all oocytes in the vitellarium are previtellogenic (Fig. 1); in ovaries of the stage B larvae, all oocytes, with the exception

E. Rosciszewska

256

of the terminal ones, are previtellogenic (Fig. 2). The squamous follicular cells (FC) envelop the oocyte, forming compact, unilayered epithelium. The FC are not numerous. The oocyte diameter averages from 0.03 mm (stage A larva) to 0.05 mm (stage B larva). The corresponding numbers of the FC on the cross-sections are 5-7 and 20-25. The FC are flattened with oval nuclei (Figs. 1, 2, 14 and 26A.a). The chromatin is concentrated at the nuclear periphery. The nuclei are surrounded with scarce amount of the cytoplasm. The apical surface of the follicular cells adhere to the slightly folded oolemma (Fig. 14). The basal surface faces the basal lamina (Figs. 1 and 14). With mitosis, the number of follicular cells gradually increases. ZZ. Early vitelfogenesis. This occurs in terminal oocytes of the stage B larvae (oocyte dimensions: 0.09 mm x 0.04 mm) and also in all oocytes of the subimago and imago (with the exception of the subterminal and terminal oocytes) (Figs. 3 and 15). The follicle cells enlarge and become cuboidal. The nuclei are larger and spheroidal. The amount of the heterochromatin increases and the nucleoli (l-2) appear (not shown; compare later stage: Figs. 5 and 26A.b, c). Ribosomes become more prevalent. Mitochondria and RER (rough endoplasmic reticulum) remain sparse during stage B, but are more numerous in those of subimago and imago. The contact zone between the oocyte and the follicular cells is not only more extensive but more complex. The oocytes grow microvilli (Fig. 15). In close proximity to the bases of microvilli, micropinocytotic vesicles were observed (Fig. 15). The follicular cells develop projections. Distinct concentrations of microvilli were observed in association with these projections. ZZZ.Middle viteffogenesis. This stage of development is present in subterminal oocytes of stage C larva (subimago) and of the imago (Fig. 5). The oocyte and the FC undergo further increase in volume (the oocyte dimensions: 0.12mm x 0.06 mm). In the ooplasm, numerous droplets of lipid and yolk globules appear (Figs. 4 and 5). The Figs. 8-13.

Perlu

marginata. Development of the follicular epithelium during late stages of oogenesis (late vitellogenesis - choriogenesis). Fig. 8. TEM, late vitellogenesis. A fragment of two neighbouring follicular cells (perinuclear regions). Note a space between the cells and a small lateral process. x17540. G = Golgi complex; arrowhead = lateral process of follicular cell. Fig. 9. TEM, late vitellogenesis. Longitudinal section through follicular cell - oocyte interface. Note perioocytic space and vitelline envelope material deposited between oocyte microvilli. x20000. Mv = oocyte microvilli; long arrow = vitelline envelope material; rosette = perioocytic space. Figs. 10, 11. Semithin longitudinal sections of terminal oocyte during late vitellogenesis. Subpopulations of follicular cells can be distinguished. Fig. lo-main body cells (lateral oocyte surface). Fig. II-posterior pole cells. Note lack of patency at the anterior (Fig. 10) and at the posterior (Fig. 11) poles. Note the enlargement of the follicular cells with respect to the former stages (compare Figs. 1-5) Methylene blue stained, x500. I = interfollicular cells; 0 = oocyte; short arrows = follicular cells; asterisk contour = intercellular space. Fig. 12. Semithin longitudinal section of a fragment of the posterior pole region of the oocyte. Late choriogenesis. Note an elongated cell between the chorionic collar and the attachment disc. Methylene blue stained. x860. Ch = chorion; D = attachment disc; short arrows = follicular cells; asterisk = follicular cell producing collar. Fig. 13. Semithin longitudinal section of a fragment of oocyte and follicular cells producing the main body chorion. Late choriogenesis. Note two distinct chorionic layers. The outer one is composed of segments. Methylene blue stained. x500. Ch = chorion; Mi = micropyle; short arrow = follicular cell.

Oogenesis

of Stone Flies

2.51

closely arranged FC become columnar with ovoid nuclei (containing 1 or 2 large nucleoli) (Fig. 5). The cytoplasm is rich in ribosomes (Figs. 6 and 7). In the central part of individual cells, numerous mitochondria and some Golgi complexes were observed (not shown). In basal parts, RER whorls are present. These surround large characteristic lipid droplets (Fig. 7). In apical part, organelles are abundant. In particular, the concentric RER whorls are present (just as in the basal parts of the cells)

258

E. Rosciszewska

(Fig. 6). In some cases, these whorls surround lipid droplets. Sometimes, within such a whorl, an inclusion is present. (Not shown. It might be a protein because it stains blue, using the methylene blue method.) However, most frequently neither lipid droplet nor any other inclusion is present. Besides RER whorls in the apical parts of the FC, a few vesicles filled with electron-dense fibrillar material were observed. During the middle vitellogenesis, the perioocytic space appears, which is penetrated with oocyte microvilli interdigitated with follicular cell processes (Figs. 4 and 26A.c; compare Figs. 14 and 15). Close to microvilli bases, pinocytotic vesicles were observed. An electron-dense fibrillar material accumulates between microvilli (Figs. 4 and 26A.c). The lateral surfaces of the FC are still in close contact with one another. The septate junctions are present between the cells (compare later stage, Fig. 18). During mid-vitellogenesis, the FC placed on posterior pole of the oocyte, gradually become higher than the cells elsewhere (Fig. 5). IV. Late vitellogenesis. This stage can be observed in terminal oocytes in ovaries of subimago and imago (Figs. 10, 11 and 26A.d). The steady increase in the oocyte volume continues. The oocyte becomes packed with yolk and lipid globules. The FC are being stretched on the oocyte and are slightly flattened (Figs. 10 and 26A.d). Septate junctions are disassembled. Intercellular spaces between follicle cells (patency) are formed (Figs. 10 and 26A.d). These are filled with the flocculent material similar to that found in perioocytic space (Figs. 8 and 9). The intercellular spaces are absent only at the oocyte poles (Figs. 10 and 11). On the basis of these morphological differences, the FC can be classified as belonging to the main body subpopulation and the subpopulation of the posterior pole of the oocyte (Figs. 10 and 11). The cells belonging to main body subpopulation are located at the lateral surfaces of the oocyte. They are flattened, with intercellular spaces between. In some cases, there are small projections on lateral cell surfaces (Fig. 8). The cells belonging to the anterior pole are arranged in a compact way (no intercellular spaces between them), but due to the lack of clear morphological differences, can be classified as belonging to the main body subpopulation. The FC on the posterior pole are arranged just like the ones at the anterior pole. However, they can be classified as belonging to a different subpopulation-those at the posterior pole. They are prismatic and taller than the main body FC. The cell nuclei are elongated along the long cell axes (Fig. 11). The perioocytic space on the posterior

Figs. 14-16. TEM, development of follicular cells during different stages of oogenesis. Fig. 14. Perla marginata. Stage A larva. Follicular cells on a previtellogenic oocyte adhere to a slightly folded oolemma. x 14000. B = basal lamina, FC = follicular cell; 0 = oocyte. Fig. 15. Perla marginata. Stage B larva. Longitudinal section through follicular cells on early vitellogenic oocyte. Note numerous ribosomes in the follicular cells cytoplasm and oocyte microvilli. x14100. FC = follicular cell; 0 = oocyte; Mv = oocyte microvilli. Fig. 16. Perla pallida. A fragment of main body follicular cell (mature ovary, early choriogenesis). Note secretory vesicles (V) and secretory grains (arrow contour) in the apical part of the follicular cell. In expanding perioocytic space (rosette) an electron-dense layer of chorion precursor (double arrowhead) is visible. Vitelline envelope (long arrow) adheres to the oocyte microvilli (Mv) x25100.

Oogenesis

of Stone Flies

pole is somewhat larger than the perioocytic space elsewhere. More distinct features, which distinguish the FC subpopulations, become visible during choriogenesis. Parallel with the external morphological changes and the emergence of the subpopulations, there are ultrastructural changes in all FCs. The large, flattened, lobate nuclei contain large nucleoli (one or two). The nuclear envelope contains numerous

E. Rosciszewska

260

pores (not shown). The cytoplasm is very rich in ribosomes. The other organelles such as: RER elements, Golgi complexes, and mitochondria, are concentrated mainly around the nucleus (Figs. 8 and 26A.d). In the basal parts of the cells, the RER whorls encase lipid droplets, now larger than before (not shown, see the earlier stage Fig. 7). In the apical parts, the vesicles of different sizes, filled with the electron-dense material, are visible (Fig. 26A.d). During late vitellogenesis, the perioocytic space expands (Fig. 9, compare with earlier stages, Figs. 14, 15 and 4). It is filled with flocculent material (precursor of vitelline envelope?) (Fig. 9). In comparison with middle vitellogenesis (Fig. 4), one can observe the emergence of condensations of fibrillar electron-dense material surrounding the microvilli (the already forming layer of vitelline envelope?) (Fig. 9). The apical membrane of the FC is more elaborate, compared with that of the former stages (Figs. 14, 15, 4, 9 and 26.A). The cell projections are branched and longer (Fig. 9). Numerous pits and vesicles at the base of the oocyte microvilli testify to the micropinocytotic activity of the oocyte (Figs. 9 and 26A.d). V. Choriogenesis. After the vitellogenesis is concluded and the electron-dense fibrillar material has been deposited around microvilli, forming an infant form of vitelline envelope (VE) (Figs. 16 and 26A.e), formation of the chorion begins. As choriogenesis progresses, the fibrillar material of the VE gradually compacts. In the perioocytic space, penetrated with FC processes, a thin layer of electron-dense material, a precursor of the chorion, appears (Fig. 16). The cells of follicular epithelium come close together again. Laterally, the neighbouring cells are joined by septate junctions and also by belt desmosomes (in apical parts of the cells) (Figs. 18 and 26A.f). Numerous vesicles of different sizes can be seen in the apical follicle cell cytoplasm (Fig. 16). The largest vesicles contain electron-dense material immersed in an electron-lucent matrix. Electron-dense grains can also be observed (Fig. 16). Choriogenesis can be divided into 2 clearly distinct stages: stage l-formation of the inner chorionic layer; stage 2-formation of the outer chorionic layer. By the end of the first stage, the formation of the attachment structure at the posterior pole of the oocyte begins (Fig. 19; compare Figs. 23 and 24).

Figs. 17-20.

Perlupallida.

TEM. The formation of the inner layer of chorion. Longitudinal sections of follicular cells. Fig. 17. Early stage of formation of the inner layer of chorion. Note Golgi complexes (G), secretory grains in the folli&ar cell cytoplasm (arrow contour) and electron-dense material at follicular ceil microvilli tins The chorion (Ch) is pierced bv irregularly placed channels (arrowheads). . (Mv). \ ~‘13000. FC = folhcujar cell; long arrows= vi&me envelope. Fig. 18. Early stage of formation of the inner layer of chorion. A fragment of adjacent follicular cells. Septate junction (SJ) and belt desmosome (D) are visible. x43000. Fig. 19. A fragment (perinuclear region) of folhcular cell belonging to posterior pole subpopulation. The early stage of formation of the attachment structure. Note expanded RER containing fine granular, electron-dense material. x 12500. N = nucleus; RER = rough endoplasmic reticulum; FC = follicular cell; asterisk = electron-dense granular material stored in RER. Fig. 20. Longitudinal section of a fragment of main body follicular cells. Late stage of formation of the inner layer of chorion. Note bundles of channels (arrowhead) piercing the layer. x9000. Ch = chorion; FC = follicular cell; G = Golgi complex; Mv = follicular cell microvilli; arrow contour = secretory grains; double asterisk contour = fibrous material of a medium electrondensity.

Oogenesis

of Stone Flies

261

Stage 1 The formation of the inner chorionic layer does not proceed synchronously over the different oocyte regions. At the anterior pole, the forming chorion layer is thinner and the process of its deposition seems to go slowly, whereas at the posterior pole, it is rapid (Figs. 17 and 20). The end result, however, is that the inner layer is of uniform thickness over the whole oocyte.

262

E. Rosciszewska

At the beginning of its formation, the inner chorion layer is composed of electron-lucent matrix pierced by numerous irregularly arranged channels (Fig. 17). At the end of stage 1, the inner chorion layer is organized as a fine granular, medium electron-density matrix, penetrated by regularly spaced bundles of channels (Fig. 20). The channels are perpendicular to the oocyte surface, filled with electron-dense material, and each individual bundle of channels is cupped on the outer surface of the inner layer by an extra fibrous layer of medium electron-density (Fig. 20). The inner layer at the posterior pole forms a shallow pit surrounded by a collar. The bottom of the pit is the place where the stem of the attachment structure is anchored (Fig. 26B; compare Rosciszewska, 1991a, Figs. 1, 2 and 6). The FCs during the first stage of choriogenesis are packed with RER, mitochondria and Golgi complexes. The electron-dense grains are present in their apices (Figs. 17 and 20). The apical membrane of the FCs forms microvilli. The electron-dense material adheres to the microvilli tips (Figs. 17 and 20). The ultrastructure of posterior pole FC subpopulation differs from that of main body FC subpopulation. The posterior pole FC have expanded RER cisternae filled with fine granular material (Fig. 19). The FC cells belonging to main body subpopulation, which are located in close proximity to the posterior pole, differentiate into collar sub-subpopulation (Figs. 12, 21 and 26B). They are markedly elongated. Their apical parts squeeze in between the above-mentioned prismatic follicular cells and the collar (Figs. 12 and 26B). The diagrammatic representation of the FC categorization (diversification) into subpopulations is illustrated in Table 1. Stage 2 During this stage of choriogenesis, cytoplasm of main body FCs is densely packed with organelles (Figs. 21, 22 and 26A.g-i). The large nucleus is shifted towards the cell base. RER cisternae are organized in parallel rows (Fig. 21). Electron-dense grains (usually associated with Golgi complexes) can be observed (Fig. 21). As in stage 1, a granular electron-dense material is visible attthe microvilli tips (Fig. 22). This material also appears in close proximity to the forming outer chorionic layer (Fig. 22). The latter consists of segments (Fig. 13), which are composed of smaller blocks of granular material (Fig. 22). During stage 2, the inner chorionic layer also undergoes further modifications. The matrix substance becomes more electron-dense than before (compare Figs. 17, 20, 22 and 25). At the posterior oocyte pole, the secretion of the attachment structure proceeds. TEM studies reveal that at the beginning of formation of the attachment disc

Figs. 21-22. Perla marginata. TEM. The formation of the outer layer of chorion. Fig. 21. Longitudinal section of posterior pole follicular cells (compare with Fig. 26B) showing a border between cells belonging to 2 subpopulations. Note different RER organization in the cells. ~8300. FCAt = follicular cell which produces the attachment disc; FCCo = follicular cell which produces the chorionic collar; G = Golgi complex; N = nucleus; RER = rough endoplasmic reticulum; arrow contour = secretory grains; asterisk = electron-dense granular material stored in RER of FCAt. Fig. 22. Longitudinal section of fragments of follicular cell (FC) and chorion (Ch) during formation of the outer chorionic layer (OL). Note secretory grains (arrow contour), electron-dense material at microvilli tips (arrows) and identical-looking electron-dense material close to the forming chorion. Inner layer of chorion is capped with the material of medium electron-density (double asterisk contour). ~17000. Arrowhead = a channel in the inner layer of chorion.

Oogenesis

2 different groups belonging 23 and 24). In one of them, than the similar material formation of the stem and existence of the 2 groups of

of Stone Flies

263

to subpopulation of prismatic FC can be distinguished (Figs. the material stored in RER is much more electron-dense of the second group. This feature disappears when the the central part of the attachment disc is concluded. (The prismatic FCs can probably be connected with the different

E. Rosciszewska

264 Table

1. Categorization

of the follicular

epithelial

cells during

mid-

oogenesis

columnar

of Perk

follicular

marginata (Plecoptera).

cells

vitellogenesis

choriogenesis

produce

main

produce

chorionic

produce

attachment

~~~~~

chemical composition of the attachment structure: the stem and the central part of the disc are strongly PAS positive, and do not stain with bromophenol blue, whereas the other parts of the disc are PAS negative and are rich in proteins; compare Rosciszewska, 1991a). During the late choriogenesis, both the inner and the outer chorionic layers are electron-dense. The outer chorionic layer is composed of segments, while the inner layer is more homogeneous (Figs. 13,25,26A. and 26B). Each segment is pierced by 2 coaxial central channels (compare: Rosciszewska, 1991a, Fig. 29). In the later phase of the eggshell formation, a so-called gelatinous sheet (decorated with mushroom-like structures) is deposited on the external surface of the outer chorionic layer (Fig. 25). At the same time, there are first indications of degeneration of FC (mitochondria filled with an electron-opaque material, lamellar bodies, Fig. 25).

Perla marginafa. Longitudinal section of the posterior pole follicular cell subpopulation during formation of the attachment structure. Fig. 23. Semithin section, PAS method. Note strongly PAS positive stem of the forming attachment structure. Arrows indicate cells which in TEM are seen as electron-dense (compare Fig. 24). x640. At = a stem of the attachment structure. Fig. 24. TEM. As in Fig. 23, enlarged fragment with the adjacent follicular cells. Note different electron-density of the cells. ~10000. N = nucleus; RER = rough endoplasmic reticulum; asterisk = granular material stored in RER. Fig. 25. Perla pallida. TEM. A fragment of longitudinal section of the follicular cells during terminal period of the main body chorion formation. Chorion (Ch) composed of 2 layers, extrachorion composed of gelatinous sheet (asterisk contour) and mushroom-like structure (arrowhead) are visible. Note degenerating follicular cells (M = abnormal mitochondria). X8ooo. FC = follicular cell; Mv = follicular cell microvilli; N = nucleus; arrow contour = secretory grains. Figs. 23-24.

Oogenesis

of Stone Flies

265

266

E. Rosciszewska

Fig. 26A. oogenesis. cell shapes; space (d-i) logenesis; BL = basal

A diagrammatic representation of follicular cell development in consecutive stages of Note an increase in cell volumes and numbers of organelles; characteristic changes of intercellular spaces during late vitellogenesis (d); and gradually expanding perioocytic in which the chorionic material is deposited. a = previtellogenesis; b, c, d = vitele, f, g, h, i = choriogenesis; arrow = chorion precursor; arrowhead = extrachorion; lamina; FC = follicular cell; IL = inner layer of chorion; OL = outer layer of chorion; 0 = oocyte; VE = vitelline envelope. Fig. 26B. Posterior pole region of the oocyte. A categorization of follicular cells into 3 subpopulations (FC, FCAt, FCCo) during the chorion and the attachment structure formation period (left) and after the process is finished (right). Asterisk = attachment structure; FC = follicular cells producing main body chorion; FCAt = follicular cells producing attachment structure; FCCo = follicular cells producing collar.

Oogenesis

of Stone

Flies

267

DISCUSSION The follicle cells of the plecopterans Perk marginatu and Perk pallida undergo a sequence of characteristic morphological changes in coordination with the consecutive stages of the oocyte development. Similar changes were reported in FC of other insect species (Ksigikiewicz-Ilijewa, 1978; Goltzene, 1979; Matsuzaki et al., 1979; Bitsch, 1980; Kaulenas, 1992; Szklarzewicz, 1993). The coordination of the development process in both the oocyte and the follicular epithelium is possible thanks to heterocellular gap junctions present between the oocyte microvilli and the FC processes (compare Wollberg et al., 1976; Woodruff, 1979; Huebner and Injeyan, 1981; Bilinski and Klag, 1982; 1987; Munz, 1988; Woodruff and Anderson, 1984; Bilinski et al., 1985; Bilinski, Kaulenas, 1992). The adjacent FC in some insects can also communicate via gap junctions (Wollberg et al., 1976) or via cellular bridges (in Apis mellifica and in some dipterans, Kaulenas, 1992). Both types of interconnections can serve to synchronize the process of the development of follicular epithelium. In the investigated Perk sp. there is no indication of cellular bridges; however, the FC are coupled laterally via small processes. Most probably gap junctions are present in these places. Developmental changes occurring in FCs are associated with the successive tasks the FCs perform during different stages of oogenesis. Previtellogenesis in these stone flies is unusually long; it persists for 2 years. During this time, the oocyte growth proceeds slowly together with a slow gradual increase of the number of FCs enveloping the oocyte. During the previtellogenesis and early vitellogenesis, the FC’s changes are similar to those in many other insect species. They are tightly apposed and form a barrier, which prevents vitellogenic proteins (present in the insect haemolymph) from prematurely entering the oocyte (Lauverjat et al., 1984). The follicular epithelium may also mediate hormonal information regulating oogenesis (Kessel and Ganion, 1979; Hagedorn, 1985; Koeppe et al., 1985; Bitsch and Bitsch, 1988; Belles et al., 1993). During active viteflogenesis in FCs of several insect species, the whorls of RER encasing lipid droplets were reported (Kessel and Ganion, 1979; Bitsch, 1980; were observed in Perk marginatu during Matsuzaki et al., 1985). Similar structures vitellogenesis (also at the beginning of choriogenesis). These droplets may store ovarian hormone stimulating the synthesis of vitellogenins in fat body (Kessel and Ganion, 1979; Bitsch, 1980). On the other hand, the droplets may store energy providing raw materials much needed later during energy demanding choriogenesis. During vitellogenesis, insect follicular epithelium may take part in providing the oocyte with yolk precursors. In a few insect species, only the FCs take an active part in vitellogenin synthesis as reported in Diptera (Chia and Morrison, 1972; Huebner et al., 1975; Brennan et al., 1982; Shepherd et al., 1985), in Lepidoptera (Zhu et al., 1986) and Coleoptera (Ullmann, 1973; Chaminade and Laverdure, 1980; Zhai et al., 1984). More frequently, the epithelium undergoes a transformation from a tightly packed arrangement to one with large intercellular spaces (patency), which provide a pathway for yolk precursors from the fat body via haemolymph into the oocyte (Huebner and Anderson, 1972; Huebner and Injeyan, 1980; Kunkel and Nordin, 1985; Kaulenas, 1992). This is also the case with Perk sp. but only during late vitellogenesis. In early and mid-vitellogenesis, the FCs in Perk sp. are closely apposed. Similar observations were reported in only a few insects (De Loof and Lagasse, 1970; Chia and Morrison, 1972; Huebner et al., 1975; Bitsch, 1980; Bilinski and Szklarzewicz, 1987; Szklarzewicz,

E. RoSciszewska

268

1989). In these insects, the follicular epithelium serves as a cellular pathway of exogenous yolk precursors into oocyte. In middle-vitellogenesis, the FCs in Perlu sp. prepare for secretion of the eggshell. The appearance of organelles such as RER elements, mitochondria, Golgi complexes (all connected with synthesis processes), the presence of large nucleoli and the accumulation of electron-dense fibrillar material close to the oocyte microvilli, testify to the beginning of the secretion. In lute vitellogenesis, an important event takes place. This is the development of large intercellular spaces (patency), which enable rapid vitellogenin transport to the oocyte. The patency is typical in insects; for a review see Kaulenas, 1992. Consequently, in a very short time the oocyte is considerably enlarged and packed with yolk and lipid droplets. During the same time, the FC organelles again greatly increase. Morphogenesis

of the eggshell

The structure, chemical composition, and functions of the eggshell (egg capsule) of stone flies were the subjects of earlier investigations (Rosciszewska, 1991a, 1991b; Rosciszewska and Jankowska, 1993). The eggshell consists of the VE, chorion (the inner and the outer layer), and extrachorion (gelatinous sheet decorated with mushroom-like structures). On the posterior pole, there is the attachment structure. The present results reveal that all the elements of the egg capsule are secreted by the FCs. This is not so in the majority of insects. It was established that the follicular epithelium is responsible for VE and chorion formation (Cummings, 1972; Regier et al., 1982; Margaritis, 1985, 1986; Bilinski and Jankowska, 1987; Mazzini and Gaino, 1988; Gaino and Mazzini, 1990; Kaulenas, 1992). However, the extrachorion is usually produced by accessory reproductive glands (Kaulenas, 1992). In Plecoptera and in some Ephemeroptera, the accessory glands are lacking (Matsuda, 1976; Mazzini and Gaino, 1988); therefore their function is transferred to the FC. VE formation starts as early as middle-vitellogenesis and proceeds until the stage of advanced choriogenesis. During this time, the FCs show pronounced synthetic activity. It is noteworthy that for the majority of insects the VE formation starts much later, close to the conclusion of vitellogenesis. Then, choriogenesis starts after the VE is already constituted (Mazzini and Gaino, 1988; Mouzaki and Margaritis, 1991; Kaulenas, 1992). In this respect, the Perlu sp. are different. Not only does VE formation start much earlier but, in addition, the process overlaps with choriogenesis. This very long period of VE secretion corresponds with the unusually long larval development in stone flies (morphogenesis of the eggshell has already occurred in the oldest larvae). Chorion formation occurs in subimago just before emergence on to land and the fertile period. The process is so rapid that attempts to collect specimens at all the consecutive stages of choriogenesis were not successful; some important stages were missed. This is why examples of exocytosis by the FCs were not presented. Nevertheless, the presented microphotographs with electron-dense material close to the FC microvilli tips, clearly suggest that chorion precursors are released from the FC. Similar observations were reported in other insects (Cummings, 1972). The characteristic changes in follicular epithelium morphology take place during choriogenesis. Patency is lacking, the FCs again draw close together and the septate junctions are rearranged between the adjacent cells. Belt desmosomes at the apical parts of FC ensure reliable mechanical connections between the cells. Such connections, i.e. are characteristic of the insect follicular septate junctions and belt desmosomes,

Oogenesis

of Stone

Flies

269

epithelium (Huebner and Injeyan, 1981; Telfer et al., 1982; Lauverjat et al., 1984; Szklarzewicz, 1993). There is a great increase in the number of organelles, and electron-dense (secretory) grains appear, pointing out to the secretory activity of the follicular epithelium. The precursors of the chorion are being gradually deposited in (extensive) perioocytic space. The material deposited in the perioocytic space is very similar to that stored in the FC cytoplasm. It could be suggested that this is the same material. The choriogenesis in insects usually follows 1 of the 2 typical pathways. In the first, the chorion is assembled by apposition of newly secreted material, as in Drosophila. In the second, the additional chorion deposition involves intercalation phenomenon as in Lepidoptera (Margaritis, 1985). In Perla sp., the chorion layers are being deposited consecutively, but in addition the electron-density of the layers significantly increases towards the end of choriogenesis. It appears both pathways are used. This possibility merits further investigation. In summary, choriogenesis involves formation of the main body chorion, the attachment structure and the collar, and it is the different subpopulations of FCs that are responsible for the formation of these various elements. Acknowledgements--I am grateful to Professor Cz. Jura for critical reading of the manuscript, to Professors B. Weglarska and J. Klag for many discussions and helpful suggestions. Special thanks are due to Msc. S. Kuzyk and Msc. W. Jankowska for their technical assistance. The present investigations were supported by Polish Committee of Scientific Research - Grant PB 0507/P2/92/02.

REFERENCES Anderson, E. 1964. Oocyte differentiation and vitellogenesis in the roach Periplaneta americana. J. Cell Biol. 20: 131-55. Bassemir, U. 1977. Ultrastructural differentiations in the developing follicle cortex of Locusta migrutoria with special reference to vitelline membrane formation. Cell Tissue Res. 185: 247-62. Belles, X., P. Cassier, X. Cerda, et. al. 1993. Induction of choriogenesis by 20-hydroxyecdysone in the German cockroach. Tissue Cell 25(2): 195-204. Bilit’tski, S. M. 1983. Oogenesis in Campodea sp. (Insecta, Diplura): chorion formation and the ultrastructure of follicle cells. Cell Tissue Res. 228: 167-70. Bilinski, S. M. 1987. Heterocellular gap junctions in vitellogenic follicles of Cumpodea sp. (Diplura), pp. 19-22. In H. Ando and C. Jura (eds.) Recent Advances in Insect Embryology in Japan and Poland. Arthropod. Embryol. Sot. Jap.,‘ISSEBU Co. Ltd., Tsukuba. . -. . Bilinski. S. M. 1993. Structure of ovaries and ooeenesis in Entognathans (Antervzota). Int. J. Insect Morohol. Embryol. 22: 255-69. Bilinski, S. M. and W. Jankowska. 1987. Oogenesis in the Bird Louse Eomenacanthus strumineus (Insecta, Mallophaga). General description and structure of the egg capsule. Zool. Jahrb. Anat. 116: 1-12. Bilinski, S. M. and J. Klag. 1982. Gap junctions between oocyte and follicle cells in Aceremtomon sp. (Insecta, Protura). Int. J. Invertebr. Reprod. 5: 331-35. Bilidski, S. M. and B. Petryszak. 1978. The ultrastructure and the function of follicle cells in Foucartia squamulutu (Herbst) -(Curculionidae). Cell Tissue Res. 189: 347-53. Bilinski. S. M. and T. Szklarzewicz. 1987. Ultrastructural modifications of the follicular eoithelium accompanying the onset of vitellogenesis in the whirligig beetle, Gyrinus natator. Cell Tissue’Res. 249: 209-14. Bilinski, S. M., W. J. Hage and J. G. Bluemink. 1985. Gap junctions between the follicle cells and the oocyte during oogenesis in an insect Tribolium destructor (Coleoptera). Wilhelm Roux’s Arch. Dev. Biol. 194: 296300. Bitsch, J. 1980. Ultrastructure de I’epithtlium folliculaire et des enveloppes de I’ovocyte chez Lepismachilis turgionii (Grassi) (Thysanura : Machilidae). Int. J. Insect. Morphol. Embryol. 9: 2540. Bitsch, V. and J. Bitsch. 1988.20-hydroxyecdysone and ovarian maturation in the firebat Thermobia domestica (Thysanura: Lepismatidae). Arch. Znsect Biochem. Physiol. 7: 281-93. Brennan, M. D., A. J. Weiner, T. J. Goralski and A. P. Mahowald. 1982. The follicle cells are a major site of vitellogenin synthesis in Drosophila melanogaster. Dev. Biol. 89: 225-36.

270

E. Rosciszewska

D. L., R. J. Smith and M. Wilcox. 1981. Differentiation within the gonads of Drosophila revealed by immunofluorescence. J. Embryol. Exp. Morphol. 63: 23342. Chaminade, M. and A. Laverdure. 1980. La vitellogenese endogene chez Tenebrio molitor (Coleoptere). Bull. sot. Zool. 105: 43744. Chia, W. K. and P. E. Morrison. 1972. Autoradiographic and ultrastructural studies on the origin of yolk protein in housefly, Musca domestica L. Can. J. Zool. 50: 1569-76. Cummings, M. R. 1972. Formation of vitelline membrane and chorion in developing oocytes of Ephestiu kiihniella. Z. Zellforsch. 127: 175-88. De Loof, A. and A. Lagasse. 1970. The ultrastructure of the follicle cells of the ovary of the Colorado beetle in relation to yolk formation. J. Insect Physiol. 16: 211-20. Gaino, E. and M. Mazzini. 1988. Oogenesis of the Mayfly Hubrophlebia eldae: Synthesis of vitelline and chorionic envelopes. Gamete Res. 21: 439-50. Gaino, E. and M. Mazzini. 1990. Follicle cell activitv in the ovarioles of Habrophlebia eldae (Eohemeroptera: . . Leptophlebiidae). Trans. Amer. Microsc. Sot. 109: 300-10. Goltzene, F. 1979. Etude ultrastructurale de developpement normal des ovocytes et des cellules folliculaires chez l’orthoptere Locusta migratoria (L.) Arch. Anat. Hist. Embryol. Norm. Exp. 62: 55-72; Gottanka, J. and Jr Butting. 1990. Gocytes develop from interconnected-cystocytes in the panoistic ovary of Nemouru sp. (Pictet) (Plecoptera : Nemouridae). ht. J. Insect Morphol. Embryol. 19: 219-25. Hagedorn, H. H. 1985. Role of ecdysteroids in reproduction, pp. 20561. In G. A. Kerkut and L. I. Gilbert (eds.) Comprehensive Insect Physiology Biochemistry and Pharmacology, Vol. 8. Pergamon, Oxford. Huebner, E. and E. Anderson. 1972. A cytological study of the ovary of Rhodnius prolixus. I. The ontogeny of the follicular epithelium. J. Morphol. 136: 45994. Huebner, E. and H. S. Injeyan. 1980. Patency of the follicular epithelium in Rhodnius prolixus. A re-examination of the hormone response and technique refinement. Can. J. Zool. 58: 1617-25. Huebner, E. and H. S. Injeyan. 1981. Follicular modulation during oocyte development in an insect: formation and modification of septate and gap junctions. Dev. Biol. 83: 101-13. Huebner, E., S. S. Tobe and K. G. Davey. 1975. Structural and functional dynamics of oogenesis in Glossina austeni: vitellogenesis with special reference to the follicular epithelium. Tissue Cell 3: 535-58. Kaulenas, M. S. 1992: Insect accessory reproductive structures, Zoophysiology 31: 38-7.5. Kessel, R. G. and L. R. Ganion. 1979. Localization of horseradish peroxidase in the panoistic dragonfly ovary. J. Submicrosc. Cytol. 11: 313-24. Koeppe, J. K., M. Fuchs, T. T. Chen, L. M. Hunt, G. E. Kovalick and T. Briers. 1985. The role of juvenile hormone in reproduction, pp. 165-203. In G. A. Kerkut and L. I. Gilbert (eds.) Comprehensive Insect Physiology, Biochemistry and Pharmacology, Vol. 8 (II). Pergamon, Oxford. Ksiaikiewicz-Ilijewa, M. 1978. Ultrastructural changes in follicle cells during the oocyte growth in Gromphadorhina bruuneri (Shelf.) (Blattodea). Acta Biol. Crucoviensiu Ser. Zool. 21: 13742. Kunkel, J. G. and J. H. Nordin. 1985. Yolk proteins, pp. 83-111. In G. A. Kerkut and L. I. Gilbert (eds.) Comprehensive Insect Physiology, Biochemistry and Pharmacology, Vol. 1. Pergamon, Oxford. Lauverjat, S., A. Szollosi and C. Marcaillou. 1984. Permeability of the ovarian follicle during oogenesis in Locustu migratoria L. (Insecta, Orthoptera). J. Ultrastruct. Res. 87: 197-211. Litwin, J. A. 1985. Light microscopy histochemistry on plastic sections. Progr. Histochem. Chytochem. 16: l-84. Margaritis, L. H. 1985. Structure and physiology of egg shell, pp. 153-226. In G. A. Kerkut and L. I. Gilbert (eds.) Comprehensive Insect Physiology, Biochemistry and Pharmacology, Vol. I. Embryogenesis and Reproduction. Pergamon, Oxford. Margaritis, L. H. 1986. The eggshell of Drosophila melunogaster. II. New staging characteristics and fine structural analysis of choriogenesis. Can. J. Zool. 64: 2152-57. Matsuda, R. 1976. Morphology and Evolution of the Insect Abdomen. Pergamon, Oxford. Matsuzaki, M., H. Ando and S. N. Visscher. 1979. Fine structure of oocyte and follicular cells during oogenesis in Galloisiana nipponensis (Caudell and King) (Grylloblattodea : Grylloblattidae). Int. J. Insect Morphol. Embryol. 8: 257-63. Matsuzaki. M., A. Sasaki and M. Komuro. 1985. Panoistic ovarioles of the dobsonfly Protohermes grandis (Megaloptera, Corydalidae), pp. 13-23. In H. Ando and K. Miya (eds.) Recent Advances in Insect Embryology in Japan, Isebu, Tsukuba. Mazzini, M. and E. Gaino. 1988. Oogenesis of the mayfly Habrophlebia eldae: Synthesis of vitelline and chorionic envelopes. Gamete Res. 21: 439-50. Mouzaki, D. G. and L.-H. Margaritis. 1991. Choriogenesis in the medfly Ceratitis capitutu (Wiedermann) (D&era : Tephritidae). Int. J. Insect Morphol. Embrvol. 20: 5148. Miinz, A. ‘1988. Heterologus gap junctions between oocytes and follicle cells of an insect, Dysdercus intermedius, and their potential role as ion current pathways. Cell Tissue Res. 252: 147-55. Ogorzalek, A. 1987. Inductive effect of oocyte nucleus on ovarian follicle morphogenesis in water bugs Brower,

Oogenesis

of Stone

Flies

271

Heteroptera, pp. 5167. In H. Ando and C. Jura (eds.) Recent Advances in Insect Embryology in Japan and Poland, Arthropod. Embryol. Sot. Jap., ISSEBU, Tsukuba. Regier, J. C., G. D. Mazur, F. C. Kafatos and M. Paul. 1982. Morphogenesis of silkworm chorion: initial framework formation and its relation to synthesis of specific proteins. Dev. Biol. 92: 159-74. Rosciszewska, E. 1989. Oogenesis in the stone-fly, Perla sp. (Insecta, Plecoptera). I. Morphology of adult female gonad. 2001. Jahrb. Anat. 118: 335-64. Rosciszewska, E. 1991a. Ultrastructural and histochemical studies of the egg capsules of Perla marginata (Panzer, 1799) and Dinocras cephalotes (Curtis, 1827) (Plecoptera: Perlidae). Znt.J. Insect Morphol. Embryol. 20: 189-203. Rosciszewska, E. 1991b. Morphological changes developing after oviposition on egg surface of Zsoperla rivulorum (Plecoptera : Perlodidae). Zool. Jahrb. Anat. 121: 30@11. Rosciszewska, E. and W. Jankowska. 1993. Morphological studies of the egg capsule during eclosion and the first instar nymph of stone fly Perla pallida (Plecoptera, Perlidae). Zool. Jahrb. Anat. 123: 347-52. Shepherd, B., M. J. Garabedian, M. C. Hung and P. C. Wensink. 1985. Developmental control of Drosophila yolk protein 1 gene by cis-acting DNA elements. Cold Sprmg Harbor Symp. @ant. Biol. 50: 521-26. Soldan, T. 1979. The structure and development of the female internal reproductive system in six European species of Ephemeroptera. Acta Entomol. Bohemoslov. 76: 353-65. Stark. B. P. and S. W. Szczytko. 1988. Egg morphology and phylogeny in Arcynopterygini (Plecoptera : Perlodidae). J. Kansas Entomol. Sot. 61: 14360. Stys, P. and S. M. Bilinski. 1990. Ovariole types and the phylogeny of hexapods. Biol. Rev. 65: 401-29. Szklarzewicz, T. 1989. Ultrastructural studies on the oogenesis of Nicoletia phytophila (Zygentoma, Nicoletiidae), pp. 237-44. In R. Dallai (ed.) 3rd International Seminar on Apterygota, University of Siena, Siena, Italy. Szklarzewicz, T. 1993. Ultrastructural changes in the follicular cells during the oocyte growth in Nicoletia phytophila (Insecta : Zygentoma). Zool. Polon. 38: 85-95. Telfer, W. H., E. Huebner and D. S. Smith. 1982. The cell biology of vitellogenic follicles in Hyalophora and Rhodnius, pp. 118-46. In R. C. King and H. Akai (eds.) Insect Ultrastructure. The Ultrastructure of Gametes. Vol. 1. Plenum, New York. Ullmann, S. L. 1973. Oogenesis in Tenebrio molitor: histological and autoradiographical observations on pupal and adult ovaries. J. Embryol. Exp. Morphol. 30: 179-217. Wollberg, Z., E. Cohen and M. Kalina. 1976. Electrical properties of the developing oocytes of the migratory locust, Locusta migratoria. J. Insect Physiol. 88: 145-58. Woodruff, R. I. 1979. Electrotonic junctions in Cecropia moth ovaries. Dev. Biol. 69: 281-95. Woodruff, R. I. and K. L. Anderson. 1984. Nutritive cord connection and dye-coupling of the follicular epithehum to the growing oocytes in the telotrophic ovarioles in Oncopeltus fusciatus, the milkweed bug. Wilhelm Roux’s Arch. Dev. Biol. 193: 15863. Zhai. Q. H., J. H. Postlethwait and J. W. Bodley. 1984. Vitellogenin synthesis in the lady beetle Coccinellu septempunctata. Insect Biochem. 14: 299-305. Zhu, J., L. S. Indrasith and 0. Yamashita. 1986. Characterization of vitellin, egg specific protein and 30 kD a protein from Bombyx eggs and their fates during oogenesis and embryogenesis. Biochim. Biophys. Acta 882: 427-36.