Structure of ovaries and oogenesis in entognathans (Apterygota)

Int. J. Insect Morpho,'. & Embryol.,

Printed in Great Britain

Vol, 22. Nos 2-4, pp. 255-269. 1993

STRUCTURE

OF OVARIES

0020-7322/93$6.00+.00 (~ 1993PergamonPress Ltd

AND

OOGENESIS

IN

ENTOGNATHANS (APTERYGOTA) SZCZEPAN M . BILIIqSKI Department of Systematic Zoology, Institute of Zoology, Jagiellonian University, 30-060 Krakdw, Poland

A b s t r a c t - Two categories of female gonads can be discerned among entognathans. In Protura, Collembola and Campodeina (Apterygota), the paired ovaries are sac-shaped, not divided into discrete ovarioles. In contrast, the ovaries of Japygina are composed of 7 metamerically arranged ovarioles. The ovaries (ovarioles) of proturans and japygids are panoistic, whereas those of collembolans and campodeids are polytrophic-meroistic. Germ-cell clusters of collembolans and campodeids are always chain-like (nonbranched). The oocyte develops from the cell placed centrally within the chain. Other cells become presumptive nurse cells. Differentiated nurse cells synthesize rRNA that is later transferred to the developing oocytes. In the panoistic ovaries (proturans and japygids), oocyte nuclei (germinal vesicles) are large and contain huge, active nucleoli. Three types of reserve materials are deposited in entognathan oocytes: lipid droplets, yolk spheres, and characteristic dense granules. Vitellogenesis is of a mixed type. Egg envelopes are secreted by the follicular cells and/or by the oocyte. The possible evolution (anagenesis) of entognathan ovaries is discussed.

Index ,descriptors (in addition to those in title): Oocyte, nurse cells, germ-cell cluster formation, follicular cells, vitellogenesis, formation of egg envelopes.

INTRODUCTION IN MOSTmodern classifications, entognathous insects are regarded as a sister group of the Insecta (s. str.), and subdivided into 3 orders: Protura, Collembola, and Diplura (Hennig, 1969; Kristensen, 1981). Recently, the analysis and reinterpretation of several morphological characters of recent and extinct diplurans (Testajapygidae; KukalovaPeck, 1987), have led to the suggestion that this order represents a paraphyletic taxon (Stys and Bilifiski, 1990). Consequently, it has been proposed that Diplura should be split into 2 equally ranked groups: Campodeina and Japygina. This genealogical hypothesis is strongly supported by the distribution of the ovary types among entognathans (see also section on Evolution of entognathan ovaries and Fig. 18). The structure of the female reproductive system, as well as the course of oogenesis, have been analysed, in the representatives of all 4 (or 3) entognathan groups (see Table 1). These studies have clearly shown that along with general similarities between the ovaries of the Entognatha and Insecta (s. str.) some fundamental differences occur. The aim of thJis article is to summarize the present knowledge on entognathan oogenesis with special reference to the germ-cell cluster formation, yolk accumulation, and deposition of egg envelopes. More detailed and comprehensive description of the female reproductive system of entognathans will be presented in the book, The Insect Ovary: 255

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S. M. BILUqSKI TABLE1. RECENTDESCRIPTIONSOFOOQENESISINENTOGNATHANTAXA Group

Species

References

Protura

Acerentomon dispar Acerentornon gallicum

Jura, 1975 Bilifiski, 1977; Biliriski and Klag, 1977; Klag, 1978; Klag and Bilifiski, 1984

Tetrodontophora bielansis Tomocerus minutus Folsomia candida lsotomurus palustris Orchesella flavescens Allacma fusca

Krzysztofowicz,1971, 1975; Bilifiski, 1976 Matsuzaki, 1973 Pal6vody, 1971, 1973, 1976 Pal6vody, 1971, 1976 This study This study

Campodeina

Campodea remyi Lepidocampa weberi Campodea sp.

Bareth, 1972 Asabo and Ando, 1978 Bilifiski, 1979, 1983a, b

Japygina

Catajapyx aquilonaris

Biliriski and Szklarzewicz,1992

Collembola Poduromorpha Entomobryomorpha

Symphypleona

Ultrastructure,

Previtellogenic

Growth

and

Evolution,

edited by J.

BOning (in

preparation).

ARCHITECTURE OF OVARIES Two well-characterized categories of female gonads can be discerned among the Entognatha. In Protura, Collembola and Campodeina the paired ovaries are sac (tube)shaped and not composed of discrete ovarioles (Pal6vody, 1971; Bareth, 1972; Jura, 1975; Krzysztofowicz, 1977; Asaba and Ando, 1978; Bilifiski, 1979). In some collembolans, however (e.g. Orchesella), ovarial sacs appear to be secondarily divided into 2 irregular lobes (Fig. 1) (Bilifiski, unpublished). The terminal filaments, as well as oviducts in the representatives of the above mentioned groups, are poorly understood, and therefore will not be discussed in this article. In investigated species, ovarial tubes are enclosed with basement lamina only (Figs 13, 17). The external epithelial sheath (equivalent to tunica externa of insects) has never been reported, even in larval or premature individuals (Bilifiski, 1983a; Klag and Witalis, 1990). Two regions: the germarium and vitellarium can be distinguished within the sacs. The germaria are localized laterally (collembolans, Fig. 6) or apically (proturans and campodeids). In the vitellaria, oocytes occur solitarily ( = panoistic ovaries; proturans) or are associated with nurse cells (= polytrophic meroistic ovaries; collembolans, campodeids). For further characterization and classification of the ovary types, see the section on Evolution of entognathan ovaries. An entirely different category of the female gonad, has been described in the representatives of Japygina (Grassi, 1887; Silvestri, 1901; Bilifiski and Szklarzewicz, 1992). Here, each of the 2 ovaries consists of 7 segmentally (metamerically) arranged

Ovaries and Oogenesis in E n t o g n a t h a n s

¢

FJ~. 1. Orchesellaflavescens. Scanning electron micrograph of the ovary. × 60. Fie. 2. Cataiapyx aquilonaris. Light micrograph of an ovariole. F = fat body; O = lateral oviduct. x i00. FIG. 3. Allacma fusca. Chain of nurse cells. × 800. F~G. 4. Carnpodea sp. Intercellular bridge connecting 2 prospective nurse cells. Arrows indicate rims of bridge, x 17,000. FIG. 5. Allacma fusca. Intermediate cell (I) lying between vitellogenic oocyte and "proper" nurse cells (N), Note n u m e r o u s lipid droplets in cytoplasm of intermediate cell. Yolk spheres = asterisks. × 800. FIG. 6. Allacmafusca. A g e r m a r i u m containing n u m e r o u s germ cells. × 800.

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panoistic ovarioles. The 2 lateral oviducts bearing the ovarioles extend throughout the abdomen and fuse posteriorly, forming a short common oviduct. Each ovariole is differentiated into an apical terminal filament, a germarium comprising gonial cells and a vitellarium, which contains 3-4 linearly arranged oocytes (Fig. 2). The terminal filaments are very short, and connect individual ovarioles to the closest lobes of the fat body (Bilifiski and Szklarzewicz, 1992). The ovarioles are covered with a thin layer of basement lamina (Fig. 12); the peritoneal envelope (epithelial sheath) is absent.

G E R M - C E L L C L U S T E R S AND THE F U N C T I O N I N G OF N U R S E C E L L S IN C O L L E M B O I _ A N S AND C A M P O D E I D S Germaria of collembolans and campodeids are relatively large and contain dividing and/or differentiating germ-cell clusters (Fig. 6). As in other insects, here each clone also arises as the result of several consecutive mitotic divisions of an initial cystoblast (Pal6vody, 1976; Bilifiski, 1983a). In both groups, germ-cell clusters are always unbranched and chain-like. The sibling cells (= cystocytes), comprised of an individual chain, are morphologically identical and remain connected via simple intercellular bridges. Obviously, each cystocyte, excluding the first and the last one, possesses 2 bridges. The number of cystocytes per cluster is variable and varies from 8 to 9 (collembolan Folsomia; Pal6vody, 1976) to more than 30 (Campodea; Bilifiski, 1983a). In entognathans, all cystocytes within the clone enter meiotic prophase and form synaptonemal complexes during pachytene (Pal6vody, 1973; Bilifiski, unpublished). The distinction between the oocyte and presumptive nurse cells is therefore possible as late as in the postpachytene stages. Ultrastructural analysis of germ-cell clones has revealed that the oocyte always differentiates from the cell placed more or less centrally within the chain (Pal6vody, 1976; Bilifiski, 1983a). Other cells become the presumptive nurse cells. At least in some species (collembolans Tetrodontophora bielanensis, Allacma fusca and the campodeid Campodea), the presumptive nurse cells also diversify into 2 categories (Krzysztofowicz, 1971, 1975; Bilifiski, 1983a; Bilifiski and Tylek, 1987). The 2 cells adjoining the oocyte and connected with it via intercellular bridges, develop into specific "intermediate cells", while the others become the nurse cells proper (Fig. 5). Differentiated germ-cell clones are invested with the prefollicular cells and migrate to the vitellarium. A functional unit composed of the descendants of one germ-cell cluster and surrounded by the somatic follicular cells constitutes an egg chamber. Usually, all nurse cells of the chamber form single compact group attached to the anterior pole of the oocyte. An interesting exception has recently been found in the collembolan Allacma fusca (Bilifiski, unpublished). Here, each oocyte is equipped with 2 long, irregularly arranged, chains of nurse cells (Fig. 3). The chains are attached separately, via intermediate cells, to the same oocyte pole. Differentiated nurse cells of entognathans, similar to those of insects, contain large spherical or slightly irregular polyploidal nuclei that possess well-developed, active nucleoli. This is in line with histochemical and autoradiographical investigations showing that intensive syntheses of RNA (presumably rRNA) take place in these nuclei (Krzysztofowicz, 1971). The cytoplasm of the nurse cells contains large accumulations of nuage material, mitochondria and an abundance of free ribosomes (Figs 7, 8). The intermediate cells are morphologically and functionally distinct from typical nurse cells (Fig. 5). Histochemical and autoradiographic studies have demonstrated that they

Ovaries and Oogenesis in Entognathans

Flo. 7. Allacma fusca. Two neighbouring nurse cells. Note close association of nuage material (asterisks) with small rod-like mitochondria (arrow). x 16,400. FIG. 8. Campodea sp. Accumulations of nuage material (N) in perinuclear cytoplasm of nurse cell. × 13,500. FIG. 9. Acerentomon gallicum. An apical chamber. Note accumulations of nuage material (N) in close contact with mitochondria, x 11,200.

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play only a minor role in RNA synthesis (Krzysztofowicz, 1975; Bilifiski and Tylek, 1987). On the other hand, the intermediate cells maintain some features in common with the oocyte and, most significantly, participate in the formation of lipid droplets and dense granules (cf. section on Accumulation of reserve materials). The occurrence of such "transitional" cells within egg chambers of campodeids and collembolans, led to the suggestion that in entognathans the factor responsible for the differentiation of the oocyte has a gradient character and that ultimate fate of a given cell is correlated with the received concentration of a hypothetical determinant (Bilifiski, 1983a; and in preparation). As mentioned above, sibling germ cells are connected by intercellular bridges. Ultrastructural investigations have revealed that intercellular bridges of collembolans (Pal6vody, 1976; Krzysztofowicz, 1975) and campodeids (Bilifiski, 1983a) are morphologically identical. Their rims are slightly dilated and covered with filamentous material (Fig. 4) so that tangential sections show a regular striation. Since within bridges, mitochondria, free ribosomes, lipid droplets and dense granules are observed, it has been suggested that these organelles are transferred from the nurse (and intermediate) cells to the oocyte (Krzysztofowicz, 1975; Bilifiski, 1983a). All morphological and embryological characters, as well as phylogenetical argumentation (Stys and Bilifiski, 1990) indicate that the polytrophic meroistic ovaries of entognathans and those of insects must have evolved independently. Basic characteristics of germ-cell clusters that are specific for collembolans and campodeids are as follows: (1) clones of sibling cells are always linear and chain like; (2) all siblings enter mitotic prophase (this character is shared with some "true" insects, see Brining this issue); (3) oocytes differentiate from the central cell; (4) presumptive nurse cells diversify into intermediate cells and nurse cells proper; (5) intercellular bridges are devoid of fusomes: consequently, polyfusomes are never formed (the origin and function of these structures are discussed by King et al., 1982; King and Brining, 1985). It should be added here, that linear clusters of germ cells have recently been described in mayflies (Ephemeroptera) (Gottanka and Brining, in press; Brining, this issue), however, in this case fusomes and long unbranched polyfusomes occur in the bridges.

G E R M A R I A OF " P A N O I S T I C O V A R I E S " The germaria of proturans consist of 2 clearly defined regions. In the apical one, a large cytoplasmic area, termed the apical chamber, occurs; the basal one contains meiotic (pachytene) oocytes. The apical chamber is equipped with one large highly branched nucleus which is associated with numerous, conspicuous aggregations of nuage material (Fig. 9). The cytoplasm of the chamber, containing free ribosomes and mitochondria, resembles that of nurse cells (trophocytes). The whole chamber is firmly enveloped by somatic cells (or their extensions) and never contacts the basement lamina. All these data led to the conclusion that the chamber is of germ-line origin and represents "retained trace of original meroism" (Klag and Bilifiski, 1984; Stys and Bilifiski, 1990). This idea agrees well with the current genealogical hypotheses on entognathan phylogeny (cf. section on Evolution of entognathan ovaries). In contrast, the germaria of the japygid, Catajapyx aquilonaris are small and comprise only a limited number of germ cells (Bilifiski and Szklarzewicz, 1992). These cells are not connected by the intercellular bridges and therefore never form clusters. In light of this it

Ovaries and Oogenesisin Entognathans

261

has been proposed that ovaries of japygids are primarily panoistic (Bilifiski and Szklarzewicz.. 1992).

A C T I V I T Y OF THE O O C Y T E N U C L E U S The structure and function of the oocyte nucleus (germinal vesicle) are entirely different in the panoistic (proturans and japygids) and polytrophic meroistic (collembolans and campodeids) ovaries. In the latter, the oocyte nuclei are generally small, transparent and inactive in RNA synthesis (Krzysztofowicz, 1975; Bilifiski, 1983a). Usually, they are localized close to intercellular bridges connecting the oocyte with the nurse (or intermediate) cells. In contrast, germinal vesicles of proturans and japygids are large and centrally located. They contain huge, dense nucleoli (Fig. 10). Ultrastructural investigations have shown that the nucleoli of the oocytes of Acerentomon (Bilifiski, 1977) and Catajapyx (Bilifiski and Szklarzewicz, 1992) are very active. The resulting RNA is transported, via nuclear pores, to the ooplasm, which in consequence is densely and uniformly filled with free ribosomes (Fig. 10).

A C C U M U L A T I O N OF R E S E R V E M A T E R I A L S Three types of reserve materials are deposited in the oocytes of entognathans: lipid droplets, yolk spheres, and specific dense granules (Fig. ll). Lipid droplets and dense granules arise in the ooplasm (and in the cytoplasm of intermediate cells) in the final phase of previtellogenesis (Bilifiski, 1976). The latter structures are formed within elements of rough endoplasmic reticulum (vesicles or short cisternae) (Fig. 11), without the participation of Golgi complexes (Bilifiski, 1976, 1979). Histochemical tests have revealed that they are basophilic, in contrast to acidophilic yolk spheres (Bareth, 1972; Asaba and Ando, 1978). Moreover, in some species the dense granules are predominantly localized in the peripheral ooplasm. It is especially evident in the oocytes of Campodea (Bareth, 1972; Bilifiski, 1983b) and Lepidocampa (Asabo and Ando, 1978), where they form a distinct cortical layer. All the above data led to the assumption that the dense granules do not represent reserve material, but participate in the formation and/or reinforcement of egg envelopes (Bilifiski and Tylek, 1987). This opinion has been supported by electron microscopical studies of the oocytes of the collembolan Orchesella, showing that in this species the dense granules are extruded from the ooplasm, fuse and build a simple envelope (Figs 16, 17) (Bilifiski and Kisiel, unpublished). Proteinaceous yolk spheres (granules) undoubtedly represent the main reserve material of entognathans. In some species, especially in those characterized by long embryonic development (e.g. collembolan Tetrodontophora bielanensis), they are accumulated in extremely large quantities. Yolk spheres may be up to 20 p.m in diameter and .often contain paracrystalline structures immersed in a homogeneous matrix (Matsuzaki, 1973; Bilifiski, 1976). Vitellogenesis (= yolk formation) is of a mixed type in entognathans i.e. some yolk proteins are incorporated into the ooplasm via pinocytosis (Fig. 13) and ,;ome are synthesized by the oocyte (= autosynthesis) (Bilifiski, 1976, 1979; Pal6vody, 1976; Bitsch and Pal6vody, 1980). In the latter process, numerous elements of rough endoplasmic reticulum and elaborated Golgi complexes take part (Fig. 11).

262

S.M. BILINSK!

FIG. 10. Acerentomon gallicum. Previtellogenicoocyte. M = mitochondria; NU large amounts of ribosomes in cytoplasm, x 11,000.

FOLLICULAR

=

nucleolus. Note

EPITHELIUM AND THE FORMATION OF EGG ENVELOPES According to the fundamental light microscopical investigations, the ovaries of entognathans are devoid of the follicular epithelium (Matsuzaki, 1973; Pal6vody, 1976; Bareth, 1978), but instead, possess single "parietal cells". Recently, however, electron microscopical studies have shown that, at least in some developmental stages, the oocytes of entognathans are surrounded by a continuous layer of flat somatic cells (Klag, 1978; Bilifiski, 1983b). Their location beneath the basement lamina (Figs 12, 13), as well as their participation in the formation of egg coverings, indicate that they constitute simple follicular epithelium. This opinion has also been supported by the discovery of heterocellular gap junctions between the oocyte and enveloping follicular cells in the ovaries of proturans (Acerentomon) and campodeids (Campodea) (Bilifiski and Klag, 1982; Bilifiski, 1987). It should be added that analogous junctions are characteristic of the oocyte-follicular cells interface in insects proper (Huebner, 1981; Bilifiski et al., 1985).

Ovaries and Oogenesisin Entognathans

263

FIG. 11. Tetrodontophora bielanensis. ActiveGolgicomplexin vitellogenicoocyte.L = lipiddroplet; Y = yolkspheres. Note dense granuleswithinelementsof rough endoplasmicreticulum(asterisks). x 45,000.

The follicular epithelium of entognathans develops from somatic prefollicular cells that are usually scattered throughout the germaria, between dividing and/or developing germ cells. The oocytes (or oocyte-nurse cell complexes) are invested with the prefollicular cells, while migrating to the vitellarium. Here, follicular cells do not form an epithelium, but lie on the surface of the germ cells singly or in small groups. In this stage, the follicular cells are markedly flattened and equipped with cytoplasmic projections by which individual cells may communicate. During vitellogenesis, the follicular cells multiply and enlarge forming, in the final phase of this stage, continuous epithelium, Simultaneously, in the cytoplasm of these cells numerous elements of rough endoplasmic reticulum and Golgi complexes are accumulated (Fig. 13). These organelles are subsequently involved in the secretory processes that led to the formation of egg envelopes (Figs 14, 15). The eggs of entognathans are roughly spherical and covered with 2 simple envelopes (Bernard, 1979; Bilifiski and Larink, 1989; Larink and Bilifiski, 1989). Their surface is devoid of any regional specializations e.g. micropyles or respiratory appendages. Because

264

S . M . BILIg~SK1

FIG. 12.

Catajapyx aquilonaris.

Follicular cells during previtellogenesis. B = basement lamina; O = oocyte, x 17,500. FlG. 13. Campodea sp. Follicular cells during vitellogenesis. B = basement lamina; G = Golgi complex; O = oocyte; V = secretory vacuoles. Arrows indicate pinocytotic vesicles, x 20,000. FIG. 14. Acerentomon gallicum. Follicular cells during formation of egg envelopes. O = oocyte; V = secretory vacuoles. Note transparent envelope (E) on oocyte surface, x 13,000.

Ovaries and Oogenesis in Entognathans

265

)

F~G. 15. Allacrna fusca. Secretory vacuoles (asterisks) in cytoplasm of follicular cell. ER = rough endoplasmic reticulum. × 15,600. FxGs 16, 17. Orchesellaflavescens. Peripheral ooplasm during formation of first egg envelope. Note numerous dense granules (white asterisks) and accumulations of already extruded material (black asterisks). B = basement lamina, x 30,000.

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S.M. BILIr~SKI

homologies of entognathan egg coverings with those of insects are not clear, the widely used (but specific) terms; vitelline envelope and chorion, will be replaced here by more general ones, the outer and inner envelope. Detailed ultrastructural investigations on the formation of egg coverings of entognathans are rare; it is evident however, that this process follows various strategies in different species. In the collembolan Tetrodontophora bielanensis, the first (and definitively outer) envelope is formed by the follicular epithelium, whereas the inner one is synthesized by the deposited egg cell (Krzysztofowicz and Kisiel, 1989). Similarly, the first envelope of Campodea and proturan Acerentomon is secreted by the follicular epithelium (Bilifiski and Klag, 1977; Bilifiski, 1983b). Finally, in some collembolans (Tomocerus, Orchesella) the formation of the inner envelope depends on the secretory activity of the oocyte (Matsuzaki, 1973). Ultrastructural analysis of the oocytes of Orchesella suggests that dense granules (see above) participate in this process.

E V O L U T I O N OF E N T O G N A T H A N O V A R I E S Two basic types of ovaries (ovarioles) are traditionally distinguished among hexapods: panoistic and meroistic. In panoistic ovaries (1) all germ cells (oogonia) are transformed into oocytes. In meroistic ovaries (2) mitotic divisions of the oogonia generate clusters (clones) of interconnected cells; in each syncytial clone usually one (or rarely several) cell(s) develop(s) into the oocytes, whereas the others become the nurse cells (-trophocytes). The cells within clusters remain joined by specialized structures, termed intercellular bridges. The distribution of ovary types among various insect orders suggests that panoistic ovaries represent the plesiomorphic grade inherited from the hexapod ancestor (King and BOning, 1985; Stys and Bilifiski, 1990). It is also believed that meroistic ovaries constitute the next anagenetic grade that evolved at least twice among hexapodans (Stys and Bilifiski, 1990) (see also section on germ-cell clusters and the functioning of nurse cells). Furthermore, it has recently been proposed that secondary panoistic ovaries developed (by a reductional process) from the meroistic ones (Pritsch and Brining, 1987; Brining and Sohst, 1988; Stys and Bilifiski, 1990). Such ovaries are devoid (secondarily) of nurse cells, but usually retain traces of original meroism (e.g. germ-cell clusters) (for further discussion see Brining and Sohst, 1988; Stys and Bilifiski, 1990). Among entognathans, 2 groups (Collembola and Campodeina) possess advanced meroistic ovaries. Ultrastructural analysis has indicated that the process of oogenesis in these groups also retains important similarities, including germ-cell cluster formation and yolk accumulation. In light of this, convergent origin of meroism (polytrophy) in Collembola and Campodeina is rather improbable. Consequently, it has been postulated (Stys and Bilifiski, 1990) that among entognathans, meroistic ovaries evolved once -- in the common ancestor of Campodeina and Parainsecta (= Collembola + Protura) (cf. Fig. 18). In all modern genealogical hypotheses (Hennig, 1969; Kristensen, 1981; KukalovaPeck, 1987; Stys and Bilifiski, 1990), the third entognathan order, proturans, is classified as a sister-group of Collembola. The acceptance of such hypothesis implies that the ovaries of proturans had evolved from the meroistic ones (cf. Fig. 18), and therefore should be denoted as secondary panoistic (neopanoistic) (Stys and Bilifiski, 1990). In light

Ovaries and Oogenesis in Entognathans

267

0 o 0 ! ! I I ! !

Rp ............

~ /

!. . . .

! I

I L

. . . .

I. . . . .

-I

I I I

MP!

I

pl

FIG. 18. Cladogram of major hexapodan taxa suggested by Stys and Bilifiski (i990). Assumed evolution of ovaries is superimposed. M = meroism (polytrophy); P = panoism; SP = secondary panoism. of this, the apical c h a m b e r has b e e n interpreted as a retained trace of original m e r o i s m (see above). T h e 4th (and the last) e n t o g n a t h a n group, Japygina, is characterized by the p r i m a r y panoistic ovarioles. This is evidenced by the small n u m b e r of g e r m cells within the g e r m a r i u m , and especially by the absence of intercellular bridges b e t w e e n t h e m (see above). Distinct structure of ovaries in "dipluran" groups ( C a m p o d e i n a -- polytrophic meroistic; J a p y g i n a -- p r i m a r y panoistic) agrees and supports the genealogical hypothesis p r o p o s e d by Stys and Bilifiski (1990) (see also Introduction). A c c o r d i n g to this hypothesis. Diplura do not represent a m o n o p h y l e t i c unit (1) and Japygina are m o r e closely related to the Insecta (s. str.) than C a m p o d e i n a (2). T h e c l a d o g r a m based on these ideas is shown in Fig. 18. T h e p r o p o s e d evolution of e n t o g n a t h a n ovaries is s u p e r i m p o s e d on the cladogram. Acknowledgements-I am greatly indebted to Dr P. Stys (Charles University, Prague) for the helpful

discussions and to Mrs W. Jankowska and Miss E. Kisiel for technical assistance. I am also grateful to Professor A. Szeptycki (Polish Academy of Sciences, Krak6w), Dr J. Pomorski (University of Wroclaw) and Dr K. Hurka (Charles University, Prague) for the determination of the specimens. REFERENCES ASABA, A. and H. ANDO. 1978. Ovarian structures and oogenesis in Lepidocampa weberi Oudemans (Diplura : Campodeidae). Int. J. Insect Morphol. Embryol. 7: 405-14. BARETH, C. 1972. L'ovogen6se chez un Arthropode du sol: Campodea (C.) remyi (Diploures). Structure et evolution de l'ovaire. C. R. Acad. Sci. Paris 275: 1887-90. BERNARD, E. C. 1979. Egg types and hatching of Eosentomon Berlese (Protura : Eosentomidae). Trans. Amer. Mierosc. Soc. 98: 123-26. BILIrqSKl, S. 1976. Ultrastructural studies on the vitellogenesis of Tetrodontophora bielanensis (Waga) (Collembola). Cell Tissue Res. 168: 399410.

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