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Germ cell cluster formation in insect ovaries
Int. J. Insect Morphol. & Ernb~vol., Vol. 22. Nos 2-4, pp. 237-253, 1993
0020-7322/93 $6.00+.00 © 1993PergamonPress Ltd
Printed in Great Britain
GERM
(;ELL CLUSTER
FORMATION
IN INSECT
OVARIES
Jr0RGEN BrONING Institute ot Zoology I, University of Erlangen-Ntirnberg, Staudstr. 5, D8520 Erlangen, Germany
Abstract--Three different ovariole types exist in insects: panoistic, polytrophic- and telotrophic-meroistic. Their ontogenetic development is comparable to all insect orders. Each ovariole is composed of somatic tissues and germ cells. Panoistic ovarioles can be developed: (1) by totally blocking germ cell cluster division (e.g. in "primitive" insect orders; and (2) after germ cell cluster formation by final cleavage of cystocytes, which develop as oocytes (e.g. in stoneflies or thrips). Polytrophic-meroistic ovaries, showing a set of identical characters, are found among hemimetabolous and holometabolous insects, indicating a "basic type" of common origin. One characteristic feature is the differentiation of only one oocyte, which is derived from one central cell of the cluster, whereas all other siblings are transformed into nurse cells. Telotrophic ovaries differ from polytrophic ovaries by retention of all nurse cells in the anterior trophic chamber. In addition, oocyte-nurse cell determination can be shifted towards more oocytes in a cluster, and clusters or subclusters can fuse by cell membrane reduction among nurse cells. This type of ovary developed independently 3 times from polytrophic ancestors and once in mayflies directly from panoistic ancestors. Index descriptors (in addition to those in title): Oogenesis, ultrastructure, phylogeny.
INTRODUCTION INSECTS are the most successful group of animals in terms of number of species. Their success is the result of many evolutionary changes, including those in their organs of reproduction, i.e. testes and ovaries. This review will show the fundamental evolutionary events that have occurred during their evolution in Paleozoic eras, some 300-400 million years ago. It will be shown that only some fundamental characters can be used to establish the iinsect gonads. The different types of testes and ovaries can emerge by only a few shifts in the cascade of events, concerning their time, duration and place. Furthermore, it will be shown that only very few new changes have actually occurred to produce the different types of ovarioles found in special groups of insects.
THE INFLUENCE OF SEX TO GONAD DEVELOPMENT I n m o s t m e t a z o a , g e r m cells s e p a r a t e f r o m s o m a t i c cells e a r l y , d u r i n g c l e a v a g e . T h i s h a p p e n s also in m o s t i n s e c t s . H o w e v e r , this e a r l y d i f f e r e n t i a t i o n is o n l y s e e n in t h o s e insects which have a pronounced oosome formation during oogenesis. This ooplasmic r e g i o n is s i t u a t e d n e a r t h e p o s t e r i o r p o l e o f t h e o o c y t e a n d h o u s e s s p e c i a l g e n e p r o d u c t s , RNPs and proteins, necessary for early pattern formation and separation of the germ line. I n D r o s o p h i l a , o n e o f t h e s e p r o d u c t s c o m e s f r o m t h e g e n e v a s a a n d c a n b e u s e d as a s p e c i a l m a r k e r o f g e r m line cells, as p r o d u c t i o n o f v a s a p r o t e i n is n e c e s s a r y t o m a i n t a i n 237
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J. BONING
a AC SC Cb
S
°i
mCc
Mi
!
b
1
I
l Me
FIG. 1. Diagram of Drosophila melanogaster male gonad (b) and early events in cluster formation (a). Some cysts are outlined in testicular tube, indicating growth (G), meiosis (Me), and histogenesis (H). x 85. Enlargement (a) shows apical hub [apical cells (AC)]. Stem cell (SC) still connected to cystoblast cell (SC). Cystocytes(Cc) of a cyst remain connected via intercellular bridges. Polyfusome is stippled. Each cyst is enclosed by 2 somatic cells. During phase of synchronized mitosis (Mi) a rosette is formed, x 300.
the germ line character (Lasko and Ashburner, 1990). Thus, germ line cells can be directly shown by the presence of the vasa product. During germ band formation, germ cells are shifted from the most posterior position as pole cells, into the region between the developing m e s e n t o d e r m and the syncytial yolk masses. Along a pass, mediated by the developing m e s o d e r m , germ cells are transported, or transport themselves, to more anterior positions, and are finally situated in the abdominal segments 5-7. During this "walk", the germ cell mass splits into 2 lateral packages, which will be enclosed by mesodermal sheaths, forming the 2 gonadanlagen. U p to this point, male and female gonad-anlagen are identical. In Drosophila males, each gonad develops into a long, curved and coiled tube, at whose anterior end a hub of somatic cells is found, to which germ cells are anchored (Hardy et al., 1979; Lindsley and Tokuyashu, 1980; GSnczy et al., 1992). These germ cells behave as stem cells, i.e. they produce by differential mitosis, a cell, which maintains the character of a stem cell and a sister cell (cystoblast), which has a definitive program of mitosis (Fig. 1). In Drosophila, the cystoblast will divide, in 4 synchronized
Germ Cell Cluster Formationin Insect Ovaries
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cycles, into a clone of 16 cells, the cystocytes. The cystoblast and its following siblings will only be enclosed by 2 somatic cells and the whole assembly (the cyst) will be anchored to the lateral sheath of somatic cells. Each cyst runs down the tube during differentiation. Thus, a developmental gradient will be established in the testis, at whose apical end the stem cells are found, whereas at the basal or posterior end the ripe sperm will be housed. Germ cell siblings remain in direct contact by incomplete cytokinesis via intercellular bridges or ring,, canals. Each bridge has a special ultrastructural appearance, to which actin filaments belong (Warn et al., 1985). During the interphases between the mitotic cycles, the forrner spindle remnants transform or become replaced by fusomal material, which fills the bridges. Fusomes of different mitotic cycles fuse to form a large polyfusome, which stretches through all intercellular bridges. All bridges are kept tightly together in a central position. This assembly is also called the rosette formation. Following the mitotic cycles, all cells go into the prophase of meiosis. At this stage, the germ cell siblings enter a growth phase during which the volume increases 25-fold. During this time, meiosis goes on, and at the end of this period, 64 interconnected siblings will undergo histogenesis, during which a spherical germ cell sibling will become a small and slender sperm. The fate of germ cells in female gonads of Drosophila has also been known for decades, especially due to the works of King and coworkers (reviewed by King, 1970). Apical somatic cells of the female gonad-anlage split the mass of primordial germ cells into small groups from which the stem cells of each ovariole arise. The fate of the stem cells is similar to that of the male line. The same events create clones of interconnected cells, which run down the germarial region of each ovariole. However, more than 2 somatic cells encompass each cluster, and these cells are the progenitors of the follicular cells. By still unknown processes, one of the 2 oldest siblings, also called pro-oocytes, develops as an oocyte; all others become nurse cells. This happens until the end of the synchronous divisions and at the beginning of growth, which starts in the prophase of meiosis. Further events of oogenesis will not be reported here; only early events will be focussed on. As shown, in both sexes, the same events occur in the germ line, and we have seen that the first steps in gonad differentiation are visible in the behavior of somatic cells. What happens in other insect systems? In the male and female gonads of grasshoppers (e.g. Locusta migratoria) an elongated gonad-anlage also differentiates into long and slender tubes: follicles in males and ovarioles in females. In male follicles, germ cells become enclosed by somatic cells as seen in Drosophila. At the posterior end of each follicle, a somatic apical cell differentiates and germ cells adhere (Fig. 2). These germ cells are the stem cells, which undergo the same events as in Drosophila, with at least one remarkable exception: stem cells build clusters from which single cells finally detach, and behave as cystoblasts. Each cystoblast will undergo some synchronized mitotic cycles, in which all those characters that exist in both sexes of Drosophila, are found. This has recently been shown for another grasshopper, Chortophaga viridifasciata (Carlson and Handel, 1988). In the female gonads of grasshoppers, some germ cells will be enclosed by somatic cells, but no cystoblasts will be generated. Each germ cell develops directly into the definitive oocyte, as seen in Isophya leonorae (Brining, unpublished). In this species, a germarial zone is completely absent, even in young nymphs, and a constant
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AC
C-CI
~
c-CI
FiG. 2. Apical cells (AC) and germ cell clusters (SC-CI) of Locusta migratoria testicular follicle. Cystocytecluster (Cc-C1). Zone of cystocytemitosis (Mi). Fusomal material is stippled, x 280.
number of about 8 germ cells can grow into 8 oocytes in each ovariole. Some oocytes degenerate during previtellogenesis; thus, about 6 oocytes are found at the time of vitellogenesis. In other species (e.g. Locusta migratoria) some apical germ cells can behave as stem cells; however, their capacity to generate cystoblasts is limited to only a few divisions. In adults, about 2 weeks after the final molt, a germarium is completely absent (Fig. 3). Each cystoblast develops directly into an oocyte. Thus, here too, the number of oocytes is limited. It is commonly accepted that insects arose from only one stem (Hennig, 1981; Kristensen, 1981; Stys and Bilifiski, 1990). What has been pointed out above for Drosophila and Locusta, is also found in many other systems. Thus, the following characteristics of germ cell cluster formation can be elaborated on and these characteristics must be considered very ancient, and are therefore symplesiomorphic (Fig. 4). Up to the end of the division cycles, both female and male cluster formation follows the same pattern. Differences between sexes become apparent during the following stages. In males, cluster formation is obligate. In females, cluster formation can be stopped from the very beginning, as shown for grasshoppers. Such ovaries are of the panoistic type; when clusters are formed insect ovaries can be meroistic, in which oocyte-nurse cell determination occurs.
C L U S T E R F O R M A T I O N IN F E M A L E S O F " P R I M I T I V E " O R D E R S Most of the so-called primitive insect orders have panoistic ovarioles, as in grasshoppers (Fig. 5). It is easy to stop any cluster formation in these ovarioles, and this will result in panoistic ovarioles. We could not find any sign of cluster formation in female nymphs of dragonflies (Odonata), web spinners (Embioptera), grasshoppers (Orthoptera), stick insects (Phasmida), cockroaches (Dictyoptera) and termites (Isoptera), but it was found in stoneflies (Plecoptera) (Gottanka and Brining, 1990) and
G e r m Cell Cluster Formation in Insect Ovaries
241
FIG. 3. Ovariole of Locusta migratoria 2 weeks after final molt. Note that all oocytes are growing. A germarium is missing from that time on. Bar = 10/~m.
i) G e r m cells i n t e r a c t i n g w i t h somatic apical cells (A)become determined as stem cells (S).
2) Stem cells create c y s t o b l a s t s differential mitosis.
(C) by
3) Stem cell and c y s t o b l a s t s r e m a i n c o n n e c t e d f o r m i n g a p r e l i m i n a r y s t e m cell cluster.
4) C y s t o b l a s t s d e t a c h f i n a l l y and u n d e r g o a species- and sex-specific p r o g r a m of m i t o t i c c y c l e s (n). 5l C l u s t e r s of 2a c y s t o c y t e s synchronous divisions.
(Cc)are built by
6) The s y n c y t i a l c y s t o c y t e c l u s t e r shows
maximal branching.
//
7) F u s o m a l m a t e r i a l fills i n t e r c e l l u l a r b r i d g e s (B) and c o n n e c t s to a p o l y f u s o m e 8) B r i d g e s move c e n t r a l l y f o r m i n g a r o s e t t e of cystocytes. 9) A f t e r m i t o t i c cycles all cystocytes enter p r o p h a s e of meiosis.
FIG. 4. Di~tgram of early events during cluster formation of germ cells in males and females.
(P).
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J. BOninG
F~G. 5. Hypothetical dendrogram of insect ovariole types, based mainly on interpretation of insect phylogeny by Hennig (1981) and Kristensen (1981). (a) At the base of insects, cluster formation in female gonads was repressed, resulting in panoistic ovary. (b) In mayflies (Ephemeroptera), linear clusters are built and a telotrophic meroistic ovary emerges (Gottanka, 1992; this report). (c) In stoneflies (Plecoptera), clusters are built but all cystocytes remain oocytes, which detach later on (Gottanka and Biining, 1990). (d, d') Rise of basic polytrophic meroistic ovary (Brining and Sohst, 1990; this report). (e) Earwig (Dermaptera) pathway, which develop a polytrophic meroistic ovary with stem cell clusters and regular split of cystocyte clusters (Yamauchi and Yoshitake, 1983). (f) Assumed reduction to the panoistic status in earth lice (Zoraptera). (g) Telotrophic meroistic ovary of bugs (Hemiptera) (King and Brining, 1985; this report). (h) Reduction to the panoistic status in thrips (Thysanoptera) (Pritsch and Brining, 1989). (i) Reduction of somatic tissues in the polytrophic meroistic ovary of Strepsiptera (Brining, unpublished). (j) Evolution of telotrophic meroistic ovary in polyphage beetles (Coleoptera : Polyphaga) (King and Brining, 1985; this report). (k) Evolution of telotrophic meroistic ovary at the roots of Raphidioptera and Megaloptera : Sialidae (King and Brining, 1985). (1) Reduction to panoistic status in Megaloptera : Corydalidae (Matsuzaki et at., 1985). Reduction to panoistic status in snowfleas (Boreidae) (m) and fleas (Siphonaptera) (n) and rise to secondary polytrophic meroistic ovary in Hystrichopsylla (o) (Brining and Sohst, 1988). Orders in which extrachromosomal rDNA amplification occurs, are shown by a symbol of a ring showing 3 active genes. Amount of species of orders are shown by black sectors.
mayflies (Ephemeroptera) (Gottanka and Brining, submitted). In Plecoptera, cystoblasts undergo a program of synchronized mitoses. The cystocytes remain connected by intercellular bridges and polyfusomes are found. Cystocytes partly form rosettes, and all cystocytes enter the prophase of meiosis. However, transdetermination to nurse cells does not occur and clusters split into single cells at the basal region of the germarium. Each detached cell develops as an oocyte. In this ancient order, all elements of cluster formation are used in the female line, but all cells remain true oocytes.
Germ Cell Cluster Formation in Insect Ovaries k
243 C
© FIG. 6. Diagrams after ultrathin serial sections through mayfly terminal chambers. Positions of cells is shown correctly, but number of siblings is reduced. (a) Ovariole of larval Cloeon sp., several months before imaginal bolt. Only one, linear cluster is found. Bar = 6 Ixm. (b) Terminal chamber of ovariole of Siphlonurus armatus, last larval stage. Five clusters with one oocyte each are found, but only 2 are shown here. Bar = 25 ixm. (c) Terminal chamber qf ovariole of Ephemerella ignita, subimago. Degenerating germ cells are shown with stripes. Bar = 25 ixm.
T h e second example is that of mayflies. This o r d e r has b e e n considered to have panoistic ovarioles (Soldan, 1980). H o w e v e r , Gross (1903) has m e n t i o n e d that mayflies m a y be telotrophic, because they have an extremely large germarial region, which could be a terminal trophic c h a m b e r . Ultrathin serial sectioning of germarial regions o f 3 different d e v e l o p m e n t a l stages derived f r o m 3 different species, has shown that mayflies indeed have a telotrophic-meroistic ovary, which is unique a m o n g insects and whose a u t a p o m o r p h i c characteristics can be used well for phylogenetic interpretations ( G o t t a n k a and Brining, submitted) (Fig. 6). These characteristics are the following: (1) only one germ cell is successful in the germarial region and creates the whole population of cystocytes, i.e. there is no stem cell activity in ovarioles; (2) the cystoblast divides s y n c h r o n o u s l y into one u n b r a n c h e d chain o f siblings by several mitotic cycles. H o w e v e r , during later cycles, not all cells u n d e r g o an S-phase; thus, the whole chain has
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FIG. 7. Polyfusome a m o n g linearly ordered germ cell siblings of Siphlonurus armatus ovarioles. Left margin is stippled. Stars indicate intercellular bridges. Bar = 2 I~m.
different numbers of cells, which do not strictly follow the 2 n rule; (3) siblings remain interconnected by intercellular bridges throughout previtellogenesis; (4) fusomes connect to each other; thus, one huge and stretched polyfusome is found (Fig. 7); (5) the chain of unbranched siblings fills the whole terminal chamber, turning up and down randomly; (6) siblings, which become situated most basally, can develop as oocytes. All others serve as nurse cells; (7) after mitosis is finished and final position of siblings is apparent, the chain breaks into pieces of more or less equal lengths. Each part houses only one oocyte, whose position is random for each part of the chain; (8) during growth of the oocyte, the 2 intercellular bridges connecting the nurse cells to the oocyte, keep their tight positions in relation to each other, and the upper part of the oocyte develops as a nutritive cord, connecting the main body of the oocyte to the chain of nurse cells; (9) at the end of oogenesis, which in mayflies is coincidental with the molt of the subimago, the chain of nurse cells splits into smaller parts, which, finally degenerate. The mayflies are the 4th group of insects in which teletrophic ovaries are found. The other 3 groups are the Hemiptera, the polyphagous Coleoptera, and the Raphidoptera/ Megaloptera (Sialidae). In all 3 cases, telotrophy seems to have developed from polytrophic ancestors, because their relatives have polytrophic meroistic ovaries (see below). Mayflies may not have originated from polytrophic ancestors, because all groups
Germ Cell Cluster Formationin Insect Ovaries
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next to mayflies have panoistic ovaries. Thus, the telotrophic ovary has evolved directly from panoistic ancestors. The suggested key events are: (1) no stem cell activity occurs in ovarioles; (2) the rise of one linear cluster; (3) an unknown determination process, which enables the basal siblings to continue as oocytes, most probably by interaction with prefollicular cells, which are in intimate contact with the germ cell siblings; (4) by unknown processes, which enable a sufficient number of siblings to serve as nurse cells for each oocyte, and which subdivide the original cluster into subclusters. Siblings, which serve as nurse cells, show no increase in volume of their nuclei; thus, they apparently do not polyploidize their genomes. Special transport mechanisms, which are discussed for other types of telotrophic and polytrophic ovaries, do not seem to exist. There are no bundles of microtubules or other prominent cytoskeletal elements, which may serve for 1Eransport. There is also no sign of membrane reduction, as is found in all other telotrophic ovaries, and which is thought of as an advantage for nurse cells to nourish all oocytes. Thus, this ovary has by far the most "primitive" status of telotrophy found among insects.
T H E B A S I C T Y P E OF M E R O I S T I C O V A R I E S The evolution of the polytrophic meroistic ovary has been thought to be a unique event at the base of Holometabola and Paraneoptera (Brining and Sohst, 1990). The polytrophic meroistic ovary of earwigs (Dermaptera) has been considered as a parallel development, :since this ovary was the only one in which the stem cells built preliminary clusters (Fig. 20 in Yamauchi and Yoshitake, 1983) and in which the original cystocyte cluster of 8 cells split into 4 subclusters of 2 cells each, one oocyte and one nurse cell. Meanwhile, inlterconnected siblings adherent to apical cells have been found in the testis of Locusta (see Fig. 2), which can be interpreted as stem cell clusters. Furthermore, in some midges (Diptera, Nematocera, Chironomidae and Sciaridae), there is evidence from older literature and from our own investigations (Sachtleben, 1918; Brining, unpublished) that clusters may split into subclusters. In Lepidoptera, Miya (1975) found permanent intercellular bridges between germ cells in very young gonad-anlagen during embryogenesis. In Hymenoptera, single stem cells have not been found as yet in ovarioles. Even in very young Apis queens, germ cells seem to be interconnected to large clusters. However, most apical clusters contain fusomal areas not connected to a huge polyfusome, indicating that subclusters may detach later on. These data come from whole-mounts, labelled with F-actin-specific rhodaminyl phalloidin (Bianing, unpublished). These clues, of course, need further analysis by ultrathin serial sectioning and by investigation o:f other ontogenetic stages. If all these clues can be substantiated, we must accept that stem cells or even all germ cells have the potential to build clusters. Consequently, the point at which the polytrophic meroistic ovary has emerged from panoistic ancestors must include the earwigs. Despite this unresolved problem, the following characteristics are common to all polytrophic meroistic ovaries: (1) each polytrophic meroistic follicle emerges basically from a cystocyte cluster, containing 2n cells; (2) the cluster has all those characteristics mentioned above for general germ cell clusters: synchronized divisions, maximal branching, polyfusome, rosette formation; (3) all siblings enter the prophase of meiosis; (4) one of the 2 oldest siblings continues meiosis and develops as an oocyte, while all others will stop meiosis and become
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PSYLLINA
ALEURODINA
COCCINA
APHIDINA
AUCHENO-/ HETEROPTERRRHYNCHA OIDEA
FIG. 8. Diagrams of terminal chambers of subgroups of Hemiptera and possible relationships. N u m b e r s represent n u m b e r of siblings; N = nurse cells; O = oocytes; V = variable; C = consant. Short bars symbolize masses of microtubules. P e r m a n e n t polyfusome during cytocyte divisions exists only in the Psyllina/Aleurodina line. The hypothetical 4-cell stage is surrounded by a broken line,
since in Coccina cluster formation occurs in the ovary-anlage and not in the ovariole, as occurs in all other groups. Final terminal chambers are drawn by enlargement of about 200. For further details see text. transdetermined to develop as nurse cells; (5) nurse cells undergo additional S-phases by which transcriptional activity will be enhanced. These S-phases are not followed by cytokinesis, thus, nurse cell nuclei become polyploid; (6) the program of transcriptional activity of nurse cells remains the same as that of oocytes; (7) transport from nurse cells to oocytes follows the osmotic-electric gradient, which is generated by a high synthesis of nurse cells, which demands high pools of metabolites.
RISE OF TELOTROPHIC
OVARIES ANCESTORS
FROM
POLYTROPHIC
Hemiptera A m o n g hemimetabolous insects, there is only one group in which telotrophic ovaries have developed, whereas their next relatives have polytrophic meroistic ovaries: these are the H e m i p t e r a (true bugs, lantern flies, cicades, aphids, coccids, white flies and leaf hoppers). The telotrophic ovary of true bugs is by far the most well known telotrophic ovary. Only some characters, which can be used by phylogenetists for infraorder phylogeny will be concentrated on. In other words: can the following well-known characteristics of bugs, typical for the whole group, be used to indicate the basis for the stemline of H e m i p t e r a , or are they advanced and c o m m o n only to one or some subgroups (Fig. 8)?
Germ Cell Cluster Formation in Insect Ovaries
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The first characteristic in question concerns the dividing capacity of apical nurse cells. In most bugs apical nurse cells can divide mitotically throughout the growth phases of oocytes. However, in mirids and bedbugs, a constant number of nurse cells is found, as well as in aphids, coccids, white flies and leafhoppers. Cicades are not well investigated. There are some reports, which describe apical mitoses, while others could not find them. Since most "primitive" families of Heteroptera have not been investigated until now, a clear answer about the point at which the above characteristic has emerged, cannot be given. Most probably, the characteristic of continuous mitotic activity is an autapomorphic character at the base of the line that splits later on into Cicadiformes/ Fulgoriformes and Heteroptera/Coleorrhyncha. Such a position would include a regression to the status of fixed number of nurse cells at least twice among Heteroptera and perhaps once in Cicadiformes. The other possibility is that the characteristic of perpetuated n:titotic activity of upper nurse cells has evolved independently among Heteroptera and Cicadiformes/Fulgoriformes. The second characteristic is that of polyploidization of nurse cells. In most meroistic ovaries, nurse cells undergo polyploidization of their genomes. Among hemipterans, 2 groups apparently do not polyploidize their genomes: the Aleurodina (white flies) and the Psyllina (leafhoppers). This characteristic is weighted as synapomorphic. Another synapomorphic characteristic of these sister groups is the reduction of microtubules in the trophic core: in white flies, only the trophic core is free of tightly bundled microtubules, whereas the small and slender nutritive tubes, connecting arrested oocytes to the trophic core, are still filled with parallel bundles of microtubules (Nowak and B~ning, in preparation). In the sister group of leafhoppers (Psyllina), even the nutritive tubes do not have bundles of microtubules (Beyrich and Brining, in preparation). Synapomorphic for both groups is also the appearance of polyfusomes, which are missed in all other hemipteran groups studied so far. Autapomorphic for leafhoppers is the total reduction of rmrse cell membranes after mitotic cycles. In all 4 groups (Aphidina] Coccina; Aleurodina/Psyllina), only one cluster develops in each ovariole; i.e. the numbers of nurse cells and oocytes are constant. The basic event for all groups mentioned above, is the formation of only one cluster of germ cells, following the 2 n rule. In coccids, the cluster houses 4 (= 22) or 8 ( = 23) cells, of which one cell develops as the definitive oocyte, whereas all others become nurse cells. In aphids, the cluster contains 32 (= 25) cells in most cases, whereby half of these cells or a smaller compartment continue the oocyte-line. A similar situation is found in white flies: here, the cluster contains 64 ( = 26) cells, of which 8 cells continue as oocytes. In psyllids, the tendency to enhance the number of cells is more obvious and leads to irregular but constant numbers of nurse cells and oocytes: the cluster houses about 280 (near to 28) cells, of which 30-40 continue first as arrested oocytes. All others share in the syncytium of nurse cells, which become devoid of membranes. The above discussion regarding cluster formation in Hemiptera leads to the hypothesis that at the beginning of Hemipteran development, only one cluster has been developed in an ovariole and that the key event was a change in oocyte determination from only one oocyte to several. Such a situation, in which one syncytial cluster exists, housing nurse cells and several oocytes simultaneously, can only be resolved by the construction of a telotrophic ovary. Thus the key events can be listed as follows: (1) no stem cell activity in ovarioles occurs; (2) reduction to only one cluster per ovariole, which follows the 2 n rule; (3) maintenance of oocyte characteristics to more than one sibling; (4) development of a
248
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TRC
YTIUM BRIDGE CELL IA
LL ZONE OF PREVITELLO( GROWTH
FIG. 9, Ovary of Rhapidioptera/Megaloptera : Sialidae. From King and Brining (1985), modified.
Germ Cell Cluster Formationin Insect Ovaries
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trophic core region out of the central part of the former rosette; (5) parallel ordered microtubules in the trophic core region and in nutritive cords. At least the :following questions remain open: (1) Of what characteristic is the founder cell of this singular cluster: Is it a so-called cytoblast, or is it a stem cell? (2) How many potential founder cells assemble in each ovariole and by which processes will only one cell survive? Answers to both questions can only be given by comparative investigation of very early events during gonad formation in embryonic stages of Hemiptera.
Raphidioptera/Megaloptera : Sialidae The second .group in which telotrophic ovaries have evolved from a basic polytrophic meroistic ovary are the common ancestors of snakeflies (Raphidioptera) and alderflies (Megaloptera : Sialidae). In both groups, the same type of telotrophic ovary occurs, and there is no doubt that this type has emerged only once (Fig. 9). Ontogenetic development of these groups was published some years ago (Biining, 1979 a-c; 1980; King and BiJning, 1985), and only some points, which can be considered as synapomorphic will be covered. Nurse cell nuclei do not polyploidize their genomes, and all prospective oocytes do not differ from nurse cells in their ultrastructure, except for one fact: nurse cells totally lose their cell membranes, whereas prospective oocytes remain in their original status, which they acquired at the end of cluster divisions, including the intercellular bridges by which prospective oocytes remain connected to the nurse cell syncytium. Autapomorphic for snakeflies are numerous microtubules found in the nutritive cords. Compared with the microtubules of nutritive cords in Hemiptera, those in snakeflies are not ordered in bundles parallel to the long axis of the cords, but show no preferential orientation at all. This telotrophic ovary is not as effective as the other 2 found in Hemiptera and polyphagous Coleoptera. This is shown by autoradiographic data on Sialis flavilatera ovaries. In this species, the synthesis of R N A is very slow and oocyte nuclei synthesize as much as a single nurse cell nucleus. R N A sythesized by the nurse cell syncytium is transported 10 times slower than in Hemiptera or polyphagous Coleoptera (Brining, 1972; in preparation; Mays, 1972). Coleoptera : Polyphaga All polyphagous beetles have a telotrophic meroistic ovary, which differs from all other types. Autapomorphic is the 3-dimensional network of interstitial cells (Fig. 10), by which nurse; cell nuclei are kept in place, when nurse cell-nurse cell membranes are reduced (Btini:ng, 1972; 1979a, b; King and Brining, 1985). Models of cluster formation have been published, but some questions remain open (Kloc and Matuszewski, 1977; Kozhanova and Pasichnik, 1979; King and Biining, 1985; Matuszewski et al., 1985). Common in all models published, is that nurse cells are derived from linear clusters, oriented more or less parallel to the long axis of the tropharium. Oocytes develop at the base of the tropharium, primarily connected to nurse cells by an intercellular bridge. However, the last 2 publications concerning this matter (King and BOning, 1985; Matuszewski et al., 1985) point out that ramifications of linear clusters occur, even between oocytes. Recent and ongoing investigations on 2 coleopteran species (Asclera coerulea, Propylea quatuordecimpunctata) have shown that cluster formation is more complex than had been previously assumed. One or several clusters are housed in each tropharium. The cluster seems to start with a ramified cluster as seen in adephagous
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FILAMENT
TROPHARIL
ITIAL CELL CELL NUCLEUS
ICULAR TISSUE 'E CORD
PROPRIA
CELL ZONE OF PREVITELLOGI GROWTH
)SOME
'2 501Jm
...
,k'.' FIG. 10. Ovary of Coleoptera : Polyphaga. From King and BOning (1985), modified.
Germ Cell Cluster Formation in Insect Ovaries
251
Fie. 11. Model of cluster formation in Coleoptera : Polyphaga. This model proposes normal cluster formation in beginning. Later on, additional intercalary divisions occur among apical siblings. One subcluster is emphasizedby stippling. For further details see text.
Coleoptera, which have polytrophic ovaries. This implies that in the beginning of cluster formation, the 2 n rule may be followed. Those siblings, which are connected tightly to the prefollicular tissue, can develop further as oocytes. All others, which are only embedded to interstitial cells, become transdetermined to develop as nurse cells. Potential nurse cells will divide preferentially by intercalary divisions, producing long and sometimes ramified chains of nurse cells, oriented parallel to the long axis of the tropharium (Fig. 11). Thus the key events for the evolution of the telotrophic meroistic ovary of polyphage Coleoptera may be: (1) no stem cell activity in ovarioles occurs; (2) reduction to one or few clusters; (3) during cluster formation, the 2 n rule is only followed in the beginning. Later on, intercalary divisions of prospective nurse cells follow; (4) somatic interstiitial cells form a 3-dimensional meshwork in the trophic chamber. This investigation is still ongoing and how many clusters are housed in a tropharium at the beginning is not known. During later developmental phases, clusters and/or subclusters can fuse, which makes an analysis very difficult and time-consuming (Brining, 1979a).
CONCLUSIONS Elucidation of germ cell clusters in combination with ultrathin serial sectioning has given new information about general construction of insect ovaries, and has opened this field of oogenesis to phylogenetic analysis. Further investigation of the evolution of 3dimensional structures of ovarioles, on germariai regions and on follicles by ultrathin serial sectioning, in combination with immunological methods and computer-enhanced
252
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3 - d i m e n s i o n a l r e c o n s t r u c t i o n t e c h n i q u e s , will g i v e n e w i n s i g h t s i n t o g e r m c e l l - s o m a t i c cell i n t e r a c t i o n s , as o c c u r s d u r i n g o o c y t e d e t e r m i n a t i o n p r o c e s s e s . T h e s e c o m p a r a t i v e investigations should be considered with the fundamental results of genetic and m o l e c u l a r a n a l y s e s o n o o g e n e s i s o f D r o s o p h i l a . O n l y s u c h a n i n t e g r a t i v e a p p r o a c h will show the main pathways of this organ during anagenesis and phylogeny of organisms.
Acknowledgements--I am greatly indebted to all my students, whose works have contributed to the understanding of the phylogeny of insect ovaries, as did the helpful discussions with many colleagues, among whom I will mention above all Dr R. C. King, Western Reserve University, Evanston, U.S.A., and Dr S. Bilifiski, Jagiellonian University, Krakow, Poland. This work is supported by the Deutsche Forschungsgemeinschaft.
REFERENCES BONING, J. 1972. Untersuchungen am Ovar von Bruchidius obtectus Say (Coleoptera-Polyphaga) zur Kl/arung des Oocytenwachstums in der Pr~ivitellogenese. Z. Zellforsch. 128: 241-82. BONING, J. 1979a. The trophic tissue of telotrophic ovarioles in polyphage Coleoptera. Zoomorphologie 93: 33-50. BONING, J. 1979b. The telotrophic nature of ovarioles of polyphage Coleoptera. Zoomorphologie 93" 51-7. BONtNG, J. 1979C. The telotrophic-meroistic ovary of Megaloptera. I. The ontogenetic development. J. Morphol. 162: 37-66. BONING, J. 1980. The ovary of Rhaphidia is telotrophic and of the Sialis type (Insecta : Rhaphidiopera). Zoomorphologie 95: 127-31. BON1NG, J. and S. SOHST. 1988. The flea ovary: ultrastructure and analysis of cell clusters. Tissue Cell 20: 783-95. BONING, J. and S. SOHST. 1990. Ultrastructure and cluster formation in ovaries of bark lice, Peripsocus phaeopterus (Stephens) and Stenopsocus stigmaticus (Imhof and Labram) (Insecta : Psocoptera). Int. J. Insect Morphol. Embryol. 19: 227-41. CARLSON, J. G.and M. A. HANDEL. 1988. Intercellular bridges and factors determining their patterns in the grasshopper testis. J. Morphol. 196: 173-85. GONCZY, P., S. VISWANATHANand S. DINARDO. 1992. Probing spermatogenesis in Drosophila with P-element enhancer detectors. Development 114: 89-98. GOTTANKA, J. 1992. Vergleichende Untersuchungen zur Ultrastruktur der Germarien urspriinglicher Insekten. Dr. Thesis, University of Erlangen-Ntirnberg, Germany. GOTrANKA,J. and J. BONING. 1990. Oocytes develop from interconnected cystocytes in the panoistic ovary of Nemoura sp. (Pictet) (Plecoptera : Nemouridae). Int. J. Insect Morphol. Embryol. 19: 219-25. GRoss, J. 1903. 121berdie Histologie des Insectenovariums. Zool. Jahrb. Abt. Anat. Ontog. 18: 71-186. HARDY, R. W., K. T. TOKUYASU, D. L. LINDSLEY and M. GAaAVITO. 1979. The germinal proliferation center in the testis of Drosophila melanogaster. J. Ultrastruct. Res. 69: 180-90. HENNIG, W. 1981. Insect Phylogeny. Translated and edited by A. C. PONT (with revisionary notes by D. Schlee). Wiley & Sons, New York. KINC, R. C. 1970. Ovarian Development in Drosophila melanogaster. Academic Press, New York. KtN(~, R. C. and J. BONING. 1985. The origin and functioning of insect oocytes and nurse cells, pp. 37-82. In G. A. KERKUT and L. I. GILBERT (eds) Comprehensive Insect Physiology, Biochemistry and Pharmacology, Vol. 1. Pergamon Press, Oxford. KLoc, M. and B. MATUSZEWSKI. 1977. Extrachromosomal DNA and the origin of oocytes in the telotrophic meroistic ovary of Creophilus maxillosus. (L.) (Staphylinidae, Coleoptera-Polyphaga). Wilhelm Roux's Arch. 183: 35l~58. KOZHANOVA, N. I. and M. I. PASlCHN*K. 1979. Differentiation of oocytes and nurse cells in telotrophic ovarioles of the beetle Coccinella septempunctata. Cytologia 21: 114549. KRISTENSEN, N. P. 1981. Phylogeny of insect orders. Annu. Rev. Entomol. 26: 144. LASKO, P. F. and M. ASHBURNER. 1990. Posterior localization of vasa protein correlated with, but is not sufficient for, pole cell development. Gen. Dev. 4: 905-21. LINDSLEY, D. L. and K. T. TOKUVASU. 1980. Spermatogenesis, pp. 225-94. In M. ASHBURNER and T. R. F. WEIGHT (eds) The Genetics and Biology of Drosophila, Vol. 2d. Academic Press, London. MATSUZAK~, M., A. SASAKIand M. KOMURO. 1985. Panoistic ovarioles of the dobson fly Protohermes grandis (Megaloptera : Corydalidae), pp. 13-23. In H. ANDO and K. MIYA (eds) Recent Advances in Insect Embryology in Japan. ISEBU, Tsukuba.
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MATUSZEWSKI,B., K. CIECHOMSKI,J. NURKOWSKAand M. KLOC. 1985. The linear clusters of oogonial cells in the development of telotrophic ovarioles in polyphage Coleoptera. Roux's Archiv. Dev. Biol. 194: 462-9. MAYS, U. 1972. Autoradiographische Untersuchungen zum Stofftransport von den Nfihrzellen zur Oocyte der Feuel~vanze Pyrrhocoris apterus L. (Heteroptera). Z. Zellforsch. 123: 395-410. MIYA, K. 1975. Ultrastructural changes of embryonic cells during organogenesis in the silkworm, Bombyx mori. I. Tlae gonad. J. Fac. Agric. Iwate University 12: 329-64. PRITSCH, M. and J. BONING. 1989. Germ cell cluster in the panoistic ovary of Thysanoptera (Insecta). Zoomorphology 108: 309-13. SACHTLEBEN, H. 1918. l~lber die Entwicklung der Geschlechtsorgane von Chironomus mit besonderer Berticksichtigung der keimbahnbegleitenden Substanzen. Dr. Thesis, University of Miinchen, Germany. SOLDAN,Z. 1981. Comparative Anatomy of the Internal Reproductive Organs of Mayflies (Ephemeroptera). Academia, Praha. STYS, P. and S. BILIIqSKI. 1990. Ovariole types and the phylogeny of hexapods. Biol. Rev. 65: 401-29. WARN, R. M., H. O. GUTZEIT, L. SMITH and A. WARN. 1985. F-actin rings are associated with the ring canals of the Drosophila egg chamber. Exp. Cell Res. 157: 355-63. YAMAUCHI, H. and N. YOSHITAKE. 1983. Origin and differentiation of the oocyte-nurse cell complex in the germarium of the earwig, Anisolabis maritima (Dermaptera : Labiduridae). Int. J. Insect Morphol. Embryol. 11: 293-306.
0020-7322/93 $6.00+.00 © 1993PergamonPress Ltd
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GERM
(;ELL CLUSTER
FORMATION
IN INSECT
OVARIES
Jr0RGEN BrONING Institute ot Zoology I, University of Erlangen-Ntirnberg, Staudstr. 5, D8520 Erlangen, Germany
Abstract--Three different ovariole types exist in insects: panoistic, polytrophic- and telotrophic-meroistic. Their ontogenetic development is comparable to all insect orders. Each ovariole is composed of somatic tissues and germ cells. Panoistic ovarioles can be developed: (1) by totally blocking germ cell cluster division (e.g. in "primitive" insect orders; and (2) after germ cell cluster formation by final cleavage of cystocytes, which develop as oocytes (e.g. in stoneflies or thrips). Polytrophic-meroistic ovaries, showing a set of identical characters, are found among hemimetabolous and holometabolous insects, indicating a "basic type" of common origin. One characteristic feature is the differentiation of only one oocyte, which is derived from one central cell of the cluster, whereas all other siblings are transformed into nurse cells. Telotrophic ovaries differ from polytrophic ovaries by retention of all nurse cells in the anterior trophic chamber. In addition, oocyte-nurse cell determination can be shifted towards more oocytes in a cluster, and clusters or subclusters can fuse by cell membrane reduction among nurse cells. This type of ovary developed independently 3 times from polytrophic ancestors and once in mayflies directly from panoistic ancestors. Index descriptors (in addition to those in title): Oogenesis, ultrastructure, phylogeny.
INTRODUCTION INSECTS are the most successful group of animals in terms of number of species. Their success is the result of many evolutionary changes, including those in their organs of reproduction, i.e. testes and ovaries. This review will show the fundamental evolutionary events that have occurred during their evolution in Paleozoic eras, some 300-400 million years ago. It will be shown that only some fundamental characters can be used to establish the iinsect gonads. The different types of testes and ovaries can emerge by only a few shifts in the cascade of events, concerning their time, duration and place. Furthermore, it will be shown that only very few new changes have actually occurred to produce the different types of ovarioles found in special groups of insects.
THE INFLUENCE OF SEX TO GONAD DEVELOPMENT I n m o s t m e t a z o a , g e r m cells s e p a r a t e f r o m s o m a t i c cells e a r l y , d u r i n g c l e a v a g e . T h i s h a p p e n s also in m o s t i n s e c t s . H o w e v e r , this e a r l y d i f f e r e n t i a t i o n is o n l y s e e n in t h o s e insects which have a pronounced oosome formation during oogenesis. This ooplasmic r e g i o n is s i t u a t e d n e a r t h e p o s t e r i o r p o l e o f t h e o o c y t e a n d h o u s e s s p e c i a l g e n e p r o d u c t s , RNPs and proteins, necessary for early pattern formation and separation of the germ line. I n D r o s o p h i l a , o n e o f t h e s e p r o d u c t s c o m e s f r o m t h e g e n e v a s a a n d c a n b e u s e d as a s p e c i a l m a r k e r o f g e r m line cells, as p r o d u c t i o n o f v a s a p r o t e i n is n e c e s s a r y t o m a i n t a i n 237
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a AC SC Cb
S
°i
mCc
Mi
!
b
1
I
l Me
FIG. 1. Diagram of Drosophila melanogaster male gonad (b) and early events in cluster formation (a). Some cysts are outlined in testicular tube, indicating growth (G), meiosis (Me), and histogenesis (H). x 85. Enlargement (a) shows apical hub [apical cells (AC)]. Stem cell (SC) still connected to cystoblast cell (SC). Cystocytes(Cc) of a cyst remain connected via intercellular bridges. Polyfusome is stippled. Each cyst is enclosed by 2 somatic cells. During phase of synchronized mitosis (Mi) a rosette is formed, x 300.
the germ line character (Lasko and Ashburner, 1990). Thus, germ line cells can be directly shown by the presence of the vasa product. During germ band formation, germ cells are shifted from the most posterior position as pole cells, into the region between the developing m e s e n t o d e r m and the syncytial yolk masses. Along a pass, mediated by the developing m e s o d e r m , germ cells are transported, or transport themselves, to more anterior positions, and are finally situated in the abdominal segments 5-7. During this "walk", the germ cell mass splits into 2 lateral packages, which will be enclosed by mesodermal sheaths, forming the 2 gonadanlagen. U p to this point, male and female gonad-anlagen are identical. In Drosophila males, each gonad develops into a long, curved and coiled tube, at whose anterior end a hub of somatic cells is found, to which germ cells are anchored (Hardy et al., 1979; Lindsley and Tokuyashu, 1980; GSnczy et al., 1992). These germ cells behave as stem cells, i.e. they produce by differential mitosis, a cell, which maintains the character of a stem cell and a sister cell (cystoblast), which has a definitive program of mitosis (Fig. 1). In Drosophila, the cystoblast will divide, in 4 synchronized
Germ Cell Cluster Formationin Insect Ovaries
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cycles, into a clone of 16 cells, the cystocytes. The cystoblast and its following siblings will only be enclosed by 2 somatic cells and the whole assembly (the cyst) will be anchored to the lateral sheath of somatic cells. Each cyst runs down the tube during differentiation. Thus, a developmental gradient will be established in the testis, at whose apical end the stem cells are found, whereas at the basal or posterior end the ripe sperm will be housed. Germ cell siblings remain in direct contact by incomplete cytokinesis via intercellular bridges or ring,, canals. Each bridge has a special ultrastructural appearance, to which actin filaments belong (Warn et al., 1985). During the interphases between the mitotic cycles, the forrner spindle remnants transform or become replaced by fusomal material, which fills the bridges. Fusomes of different mitotic cycles fuse to form a large polyfusome, which stretches through all intercellular bridges. All bridges are kept tightly together in a central position. This assembly is also called the rosette formation. Following the mitotic cycles, all cells go into the prophase of meiosis. At this stage, the germ cell siblings enter a growth phase during which the volume increases 25-fold. During this time, meiosis goes on, and at the end of this period, 64 interconnected siblings will undergo histogenesis, during which a spherical germ cell sibling will become a small and slender sperm. The fate of germ cells in female gonads of Drosophila has also been known for decades, especially due to the works of King and coworkers (reviewed by King, 1970). Apical somatic cells of the female gonad-anlage split the mass of primordial germ cells into small groups from which the stem cells of each ovariole arise. The fate of the stem cells is similar to that of the male line. The same events create clones of interconnected cells, which run down the germarial region of each ovariole. However, more than 2 somatic cells encompass each cluster, and these cells are the progenitors of the follicular cells. By still unknown processes, one of the 2 oldest siblings, also called pro-oocytes, develops as an oocyte; all others become nurse cells. This happens until the end of the synchronous divisions and at the beginning of growth, which starts in the prophase of meiosis. Further events of oogenesis will not be reported here; only early events will be focussed on. As shown, in both sexes, the same events occur in the germ line, and we have seen that the first steps in gonad differentiation are visible in the behavior of somatic cells. What happens in other insect systems? In the male and female gonads of grasshoppers (e.g. Locusta migratoria) an elongated gonad-anlage also differentiates into long and slender tubes: follicles in males and ovarioles in females. In male follicles, germ cells become enclosed by somatic cells as seen in Drosophila. At the posterior end of each follicle, a somatic apical cell differentiates and germ cells adhere (Fig. 2). These germ cells are the stem cells, which undergo the same events as in Drosophila, with at least one remarkable exception: stem cells build clusters from which single cells finally detach, and behave as cystoblasts. Each cystoblast will undergo some synchronized mitotic cycles, in which all those characters that exist in both sexes of Drosophila, are found. This has recently been shown for another grasshopper, Chortophaga viridifasciata (Carlson and Handel, 1988). In the female gonads of grasshoppers, some germ cells will be enclosed by somatic cells, but no cystoblasts will be generated. Each germ cell develops directly into the definitive oocyte, as seen in Isophya leonorae (Brining, unpublished). In this species, a germarial zone is completely absent, even in young nymphs, and a constant
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AC
C-CI
~
c-CI
FiG. 2. Apical cells (AC) and germ cell clusters (SC-CI) of Locusta migratoria testicular follicle. Cystocytecluster (Cc-C1). Zone of cystocytemitosis (Mi). Fusomal material is stippled, x 280.
number of about 8 germ cells can grow into 8 oocytes in each ovariole. Some oocytes degenerate during previtellogenesis; thus, about 6 oocytes are found at the time of vitellogenesis. In other species (e.g. Locusta migratoria) some apical germ cells can behave as stem cells; however, their capacity to generate cystoblasts is limited to only a few divisions. In adults, about 2 weeks after the final molt, a germarium is completely absent (Fig. 3). Each cystoblast develops directly into an oocyte. Thus, here too, the number of oocytes is limited. It is commonly accepted that insects arose from only one stem (Hennig, 1981; Kristensen, 1981; Stys and Bilifiski, 1990). What has been pointed out above for Drosophila and Locusta, is also found in many other systems. Thus, the following characteristics of germ cell cluster formation can be elaborated on and these characteristics must be considered very ancient, and are therefore symplesiomorphic (Fig. 4). Up to the end of the division cycles, both female and male cluster formation follows the same pattern. Differences between sexes become apparent during the following stages. In males, cluster formation is obligate. In females, cluster formation can be stopped from the very beginning, as shown for grasshoppers. Such ovaries are of the panoistic type; when clusters are formed insect ovaries can be meroistic, in which oocyte-nurse cell determination occurs.
C L U S T E R F O R M A T I O N IN F E M A L E S O F " P R I M I T I V E " O R D E R S Most of the so-called primitive insect orders have panoistic ovarioles, as in grasshoppers (Fig. 5). It is easy to stop any cluster formation in these ovarioles, and this will result in panoistic ovarioles. We could not find any sign of cluster formation in female nymphs of dragonflies (Odonata), web spinners (Embioptera), grasshoppers (Orthoptera), stick insects (Phasmida), cockroaches (Dictyoptera) and termites (Isoptera), but it was found in stoneflies (Plecoptera) (Gottanka and Brining, 1990) and
G e r m Cell Cluster Formation in Insect Ovaries
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FIG. 3. Ovariole of Locusta migratoria 2 weeks after final molt. Note that all oocytes are growing. A germarium is missing from that time on. Bar = 10/~m.
i) G e r m cells i n t e r a c t i n g w i t h somatic apical cells (A)become determined as stem cells (S).
2) Stem cells create c y s t o b l a s t s differential mitosis.
(C) by
3) Stem cell and c y s t o b l a s t s r e m a i n c o n n e c t e d f o r m i n g a p r e l i m i n a r y s t e m cell cluster.
4) C y s t o b l a s t s d e t a c h f i n a l l y and u n d e r g o a species- and sex-specific p r o g r a m of m i t o t i c c y c l e s (n). 5l C l u s t e r s of 2a c y s t o c y t e s synchronous divisions.
(Cc)are built by
6) The s y n c y t i a l c y s t o c y t e c l u s t e r shows
maximal branching.
//
7) F u s o m a l m a t e r i a l fills i n t e r c e l l u l a r b r i d g e s (B) and c o n n e c t s to a p o l y f u s o m e 8) B r i d g e s move c e n t r a l l y f o r m i n g a r o s e t t e of cystocytes. 9) A f t e r m i t o t i c cycles all cystocytes enter p r o p h a s e of meiosis.
FIG. 4. Di~tgram of early events during cluster formation of germ cells in males and females.
(P).
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F~G. 5. Hypothetical dendrogram of insect ovariole types, based mainly on interpretation of insect phylogeny by Hennig (1981) and Kristensen (1981). (a) At the base of insects, cluster formation in female gonads was repressed, resulting in panoistic ovary. (b) In mayflies (Ephemeroptera), linear clusters are built and a telotrophic meroistic ovary emerges (Gottanka, 1992; this report). (c) In stoneflies (Plecoptera), clusters are built but all cystocytes remain oocytes, which detach later on (Gottanka and Biining, 1990). (d, d') Rise of basic polytrophic meroistic ovary (Brining and Sohst, 1990; this report). (e) Earwig (Dermaptera) pathway, which develop a polytrophic meroistic ovary with stem cell clusters and regular split of cystocyte clusters (Yamauchi and Yoshitake, 1983). (f) Assumed reduction to the panoistic status in earth lice (Zoraptera). (g) Telotrophic meroistic ovary of bugs (Hemiptera) (King and Brining, 1985; this report). (h) Reduction to the panoistic status in thrips (Thysanoptera) (Pritsch and Brining, 1989). (i) Reduction of somatic tissues in the polytrophic meroistic ovary of Strepsiptera (Brining, unpublished). (j) Evolution of telotrophic meroistic ovary in polyphage beetles (Coleoptera : Polyphaga) (King and Brining, 1985; this report). (k) Evolution of telotrophic meroistic ovary at the roots of Raphidioptera and Megaloptera : Sialidae (King and Brining, 1985). (1) Reduction to panoistic status in Megaloptera : Corydalidae (Matsuzaki et at., 1985). Reduction to panoistic status in snowfleas (Boreidae) (m) and fleas (Siphonaptera) (n) and rise to secondary polytrophic meroistic ovary in Hystrichopsylla (o) (Brining and Sohst, 1988). Orders in which extrachromosomal rDNA amplification occurs, are shown by a symbol of a ring showing 3 active genes. Amount of species of orders are shown by black sectors.
mayflies (Ephemeroptera) (Gottanka and Brining, submitted). In Plecoptera, cystoblasts undergo a program of synchronized mitoses. The cystocytes remain connected by intercellular bridges and polyfusomes are found. Cystocytes partly form rosettes, and all cystocytes enter the prophase of meiosis. However, transdetermination to nurse cells does not occur and clusters split into single cells at the basal region of the germarium. Each detached cell develops as an oocyte. In this ancient order, all elements of cluster formation are used in the female line, but all cells remain true oocytes.
Germ Cell Cluster Formation in Insect Ovaries k
243 C
© FIG. 6. Diagrams after ultrathin serial sections through mayfly terminal chambers. Positions of cells is shown correctly, but number of siblings is reduced. (a) Ovariole of larval Cloeon sp., several months before imaginal bolt. Only one, linear cluster is found. Bar = 6 Ixm. (b) Terminal chamber of ovariole of Siphlonurus armatus, last larval stage. Five clusters with one oocyte each are found, but only 2 are shown here. Bar = 25 ixm. (c) Terminal chamber qf ovariole of Ephemerella ignita, subimago. Degenerating germ cells are shown with stripes. Bar = 25 ixm.
T h e second example is that of mayflies. This o r d e r has b e e n considered to have panoistic ovarioles (Soldan, 1980). H o w e v e r , Gross (1903) has m e n t i o n e d that mayflies m a y be telotrophic, because they have an extremely large germarial region, which could be a terminal trophic c h a m b e r . Ultrathin serial sectioning of germarial regions o f 3 different d e v e l o p m e n t a l stages derived f r o m 3 different species, has shown that mayflies indeed have a telotrophic-meroistic ovary, which is unique a m o n g insects and whose a u t a p o m o r p h i c characteristics can be used well for phylogenetic interpretations ( G o t t a n k a and Brining, submitted) (Fig. 6). These characteristics are the following: (1) only one germ cell is successful in the germarial region and creates the whole population of cystocytes, i.e. there is no stem cell activity in ovarioles; (2) the cystoblast divides s y n c h r o n o u s l y into one u n b r a n c h e d chain o f siblings by several mitotic cycles. H o w e v e r , during later cycles, not all cells u n d e r g o an S-phase; thus, the whole chain has
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FIG. 7. Polyfusome a m o n g linearly ordered germ cell siblings of Siphlonurus armatus ovarioles. Left margin is stippled. Stars indicate intercellular bridges. Bar = 2 I~m.
different numbers of cells, which do not strictly follow the 2 n rule; (3) siblings remain interconnected by intercellular bridges throughout previtellogenesis; (4) fusomes connect to each other; thus, one huge and stretched polyfusome is found (Fig. 7); (5) the chain of unbranched siblings fills the whole terminal chamber, turning up and down randomly; (6) siblings, which become situated most basally, can develop as oocytes. All others serve as nurse cells; (7) after mitosis is finished and final position of siblings is apparent, the chain breaks into pieces of more or less equal lengths. Each part houses only one oocyte, whose position is random for each part of the chain; (8) during growth of the oocyte, the 2 intercellular bridges connecting the nurse cells to the oocyte, keep their tight positions in relation to each other, and the upper part of the oocyte develops as a nutritive cord, connecting the main body of the oocyte to the chain of nurse cells; (9) at the end of oogenesis, which in mayflies is coincidental with the molt of the subimago, the chain of nurse cells splits into smaller parts, which, finally degenerate. The mayflies are the 4th group of insects in which teletrophic ovaries are found. The other 3 groups are the Hemiptera, the polyphagous Coleoptera, and the Raphidoptera/ Megaloptera (Sialidae). In all 3 cases, telotrophy seems to have developed from polytrophic ancestors, because their relatives have polytrophic meroistic ovaries (see below). Mayflies may not have originated from polytrophic ancestors, because all groups
Germ Cell Cluster Formationin Insect Ovaries
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next to mayflies have panoistic ovaries. Thus, the telotrophic ovary has evolved directly from panoistic ancestors. The suggested key events are: (1) no stem cell activity occurs in ovarioles; (2) the rise of one linear cluster; (3) an unknown determination process, which enables the basal siblings to continue as oocytes, most probably by interaction with prefollicular cells, which are in intimate contact with the germ cell siblings; (4) by unknown processes, which enable a sufficient number of siblings to serve as nurse cells for each oocyte, and which subdivide the original cluster into subclusters. Siblings, which serve as nurse cells, show no increase in volume of their nuclei; thus, they apparently do not polyploidize their genomes. Special transport mechanisms, which are discussed for other types of telotrophic and polytrophic ovaries, do not seem to exist. There are no bundles of microtubules or other prominent cytoskeletal elements, which may serve for 1Eransport. There is also no sign of membrane reduction, as is found in all other telotrophic ovaries, and which is thought of as an advantage for nurse cells to nourish all oocytes. Thus, this ovary has by far the most "primitive" status of telotrophy found among insects.
T H E B A S I C T Y P E OF M E R O I S T I C O V A R I E S The evolution of the polytrophic meroistic ovary has been thought to be a unique event at the base of Holometabola and Paraneoptera (Brining and Sohst, 1990). The polytrophic meroistic ovary of earwigs (Dermaptera) has been considered as a parallel development, :since this ovary was the only one in which the stem cells built preliminary clusters (Fig. 20 in Yamauchi and Yoshitake, 1983) and in which the original cystocyte cluster of 8 cells split into 4 subclusters of 2 cells each, one oocyte and one nurse cell. Meanwhile, inlterconnected siblings adherent to apical cells have been found in the testis of Locusta (see Fig. 2), which can be interpreted as stem cell clusters. Furthermore, in some midges (Diptera, Nematocera, Chironomidae and Sciaridae), there is evidence from older literature and from our own investigations (Sachtleben, 1918; Brining, unpublished) that clusters may split into subclusters. In Lepidoptera, Miya (1975) found permanent intercellular bridges between germ cells in very young gonad-anlagen during embryogenesis. In Hymenoptera, single stem cells have not been found as yet in ovarioles. Even in very young Apis queens, germ cells seem to be interconnected to large clusters. However, most apical clusters contain fusomal areas not connected to a huge polyfusome, indicating that subclusters may detach later on. These data come from whole-mounts, labelled with F-actin-specific rhodaminyl phalloidin (Bianing, unpublished). These clues, of course, need further analysis by ultrathin serial sectioning and by investigation o:f other ontogenetic stages. If all these clues can be substantiated, we must accept that stem cells or even all germ cells have the potential to build clusters. Consequently, the point at which the polytrophic meroistic ovary has emerged from panoistic ancestors must include the earwigs. Despite this unresolved problem, the following characteristics are common to all polytrophic meroistic ovaries: (1) each polytrophic meroistic follicle emerges basically from a cystocyte cluster, containing 2n cells; (2) the cluster has all those characteristics mentioned above for general germ cell clusters: synchronized divisions, maximal branching, polyfusome, rosette formation; (3) all siblings enter the prophase of meiosis; (4) one of the 2 oldest siblings continues meiosis and develops as an oocyte, while all others will stop meiosis and become
246
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PSYLLINA
ALEURODINA
COCCINA
APHIDINA
AUCHENO-/ HETEROPTERRRHYNCHA OIDEA
FIG. 8. Diagrams of terminal chambers of subgroups of Hemiptera and possible relationships. N u m b e r s represent n u m b e r of siblings; N = nurse cells; O = oocytes; V = variable; C = consant. Short bars symbolize masses of microtubules. P e r m a n e n t polyfusome during cytocyte divisions exists only in the Psyllina/Aleurodina line. The hypothetical 4-cell stage is surrounded by a broken line,
since in Coccina cluster formation occurs in the ovary-anlage and not in the ovariole, as occurs in all other groups. Final terminal chambers are drawn by enlargement of about 200. For further details see text. transdetermined to develop as nurse cells; (5) nurse cells undergo additional S-phases by which transcriptional activity will be enhanced. These S-phases are not followed by cytokinesis, thus, nurse cell nuclei become polyploid; (6) the program of transcriptional activity of nurse cells remains the same as that of oocytes; (7) transport from nurse cells to oocytes follows the osmotic-electric gradient, which is generated by a high synthesis of nurse cells, which demands high pools of metabolites.
RISE OF TELOTROPHIC
OVARIES ANCESTORS
FROM
POLYTROPHIC
Hemiptera A m o n g hemimetabolous insects, there is only one group in which telotrophic ovaries have developed, whereas their next relatives have polytrophic meroistic ovaries: these are the H e m i p t e r a (true bugs, lantern flies, cicades, aphids, coccids, white flies and leaf hoppers). The telotrophic ovary of true bugs is by far the most well known telotrophic ovary. Only some characters, which can be used by phylogenetists for infraorder phylogeny will be concentrated on. In other words: can the following well-known characteristics of bugs, typical for the whole group, be used to indicate the basis for the stemline of H e m i p t e r a , or are they advanced and c o m m o n only to one or some subgroups (Fig. 8)?
Germ Cell Cluster Formation in Insect Ovaries
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The first characteristic in question concerns the dividing capacity of apical nurse cells. In most bugs apical nurse cells can divide mitotically throughout the growth phases of oocytes. However, in mirids and bedbugs, a constant number of nurse cells is found, as well as in aphids, coccids, white flies and leafhoppers. Cicades are not well investigated. There are some reports, which describe apical mitoses, while others could not find them. Since most "primitive" families of Heteroptera have not been investigated until now, a clear answer about the point at which the above characteristic has emerged, cannot be given. Most probably, the characteristic of continuous mitotic activity is an autapomorphic character at the base of the line that splits later on into Cicadiformes/ Fulgoriformes and Heteroptera/Coleorrhyncha. Such a position would include a regression to the status of fixed number of nurse cells at least twice among Heteroptera and perhaps once in Cicadiformes. The other possibility is that the characteristic of perpetuated n:titotic activity of upper nurse cells has evolved independently among Heteroptera and Cicadiformes/Fulgoriformes. The second characteristic is that of polyploidization of nurse cells. In most meroistic ovaries, nurse cells undergo polyploidization of their genomes. Among hemipterans, 2 groups apparently do not polyploidize their genomes: the Aleurodina (white flies) and the Psyllina (leafhoppers). This characteristic is weighted as synapomorphic. Another synapomorphic characteristic of these sister groups is the reduction of microtubules in the trophic core: in white flies, only the trophic core is free of tightly bundled microtubules, whereas the small and slender nutritive tubes, connecting arrested oocytes to the trophic core, are still filled with parallel bundles of microtubules (Nowak and B~ning, in preparation). In the sister group of leafhoppers (Psyllina), even the nutritive tubes do not have bundles of microtubules (Beyrich and Brining, in preparation). Synapomorphic for both groups is also the appearance of polyfusomes, which are missed in all other hemipteran groups studied so far. Autapomorphic for leafhoppers is the total reduction of rmrse cell membranes after mitotic cycles. In all 4 groups (Aphidina] Coccina; Aleurodina/Psyllina), only one cluster develops in each ovariole; i.e. the numbers of nurse cells and oocytes are constant. The basic event for all groups mentioned above, is the formation of only one cluster of germ cells, following the 2 n rule. In coccids, the cluster houses 4 (= 22) or 8 ( = 23) cells, of which one cell develops as the definitive oocyte, whereas all others become nurse cells. In aphids, the cluster contains 32 (= 25) cells in most cases, whereby half of these cells or a smaller compartment continue the oocyte-line. A similar situation is found in white flies: here, the cluster contains 64 ( = 26) cells, of which 8 cells continue as oocytes. In psyllids, the tendency to enhance the number of cells is more obvious and leads to irregular but constant numbers of nurse cells and oocytes: the cluster houses about 280 (near to 28) cells, of which 30-40 continue first as arrested oocytes. All others share in the syncytium of nurse cells, which become devoid of membranes. The above discussion regarding cluster formation in Hemiptera leads to the hypothesis that at the beginning of Hemipteran development, only one cluster has been developed in an ovariole and that the key event was a change in oocyte determination from only one oocyte to several. Such a situation, in which one syncytial cluster exists, housing nurse cells and several oocytes simultaneously, can only be resolved by the construction of a telotrophic ovary. Thus the key events can be listed as follows: (1) no stem cell activity in ovarioles occurs; (2) reduction to only one cluster per ovariole, which follows the 2 n rule; (3) maintenance of oocyte characteristics to more than one sibling; (4) development of a
248
J. BONING
TRC
YTIUM BRIDGE CELL IA
LL ZONE OF PREVITELLO( GROWTH
FIG. 9, Ovary of Rhapidioptera/Megaloptera : Sialidae. From King and Brining (1985), modified.
Germ Cell Cluster Formationin Insect Ovaries
249
trophic core region out of the central part of the former rosette; (5) parallel ordered microtubules in the trophic core region and in nutritive cords. At least the :following questions remain open: (1) Of what characteristic is the founder cell of this singular cluster: Is it a so-called cytoblast, or is it a stem cell? (2) How many potential founder cells assemble in each ovariole and by which processes will only one cell survive? Answers to both questions can only be given by comparative investigation of very early events during gonad formation in embryonic stages of Hemiptera.
Raphidioptera/Megaloptera : Sialidae The second .group in which telotrophic ovaries have evolved from a basic polytrophic meroistic ovary are the common ancestors of snakeflies (Raphidioptera) and alderflies (Megaloptera : Sialidae). In both groups, the same type of telotrophic ovary occurs, and there is no doubt that this type has emerged only once (Fig. 9). Ontogenetic development of these groups was published some years ago (Biining, 1979 a-c; 1980; King and BiJning, 1985), and only some points, which can be considered as synapomorphic will be covered. Nurse cell nuclei do not polyploidize their genomes, and all prospective oocytes do not differ from nurse cells in their ultrastructure, except for one fact: nurse cells totally lose their cell membranes, whereas prospective oocytes remain in their original status, which they acquired at the end of cluster divisions, including the intercellular bridges by which prospective oocytes remain connected to the nurse cell syncytium. Autapomorphic for snakeflies are numerous microtubules found in the nutritive cords. Compared with the microtubules of nutritive cords in Hemiptera, those in snakeflies are not ordered in bundles parallel to the long axis of the cords, but show no preferential orientation at all. This telotrophic ovary is not as effective as the other 2 found in Hemiptera and polyphagous Coleoptera. This is shown by autoradiographic data on Sialis flavilatera ovaries. In this species, the synthesis of R N A is very slow and oocyte nuclei synthesize as much as a single nurse cell nucleus. R N A sythesized by the nurse cell syncytium is transported 10 times slower than in Hemiptera or polyphagous Coleoptera (Brining, 1972; in preparation; Mays, 1972). Coleoptera : Polyphaga All polyphagous beetles have a telotrophic meroistic ovary, which differs from all other types. Autapomorphic is the 3-dimensional network of interstitial cells (Fig. 10), by which nurse; cell nuclei are kept in place, when nurse cell-nurse cell membranes are reduced (Btini:ng, 1972; 1979a, b; King and Brining, 1985). Models of cluster formation have been published, but some questions remain open (Kloc and Matuszewski, 1977; Kozhanova and Pasichnik, 1979; King and Biining, 1985; Matuszewski et al., 1985). Common in all models published, is that nurse cells are derived from linear clusters, oriented more or less parallel to the long axis of the tropharium. Oocytes develop at the base of the tropharium, primarily connected to nurse cells by an intercellular bridge. However, the last 2 publications concerning this matter (King and BOning, 1985; Matuszewski et al., 1985) point out that ramifications of linear clusters occur, even between oocytes. Recent and ongoing investigations on 2 coleopteran species (Asclera coerulea, Propylea quatuordecimpunctata) have shown that cluster formation is more complex than had been previously assumed. One or several clusters are housed in each tropharium. The cluster seems to start with a ramified cluster as seen in adephagous
250
J. Bt3r~ING
FILAMENT
TROPHARIL
ITIAL CELL CELL NUCLEUS
ICULAR TISSUE 'E CORD
PROPRIA
CELL ZONE OF PREVITELLOGI GROWTH
)SOME
'2 501Jm
...
,k'.' FIG. 10. Ovary of Coleoptera : Polyphaga. From King and BOning (1985), modified.
Germ Cell Cluster Formation in Insect Ovaries
251
Fie. 11. Model of cluster formation in Coleoptera : Polyphaga. This model proposes normal cluster formation in beginning. Later on, additional intercalary divisions occur among apical siblings. One subcluster is emphasizedby stippling. For further details see text.
Coleoptera, which have polytrophic ovaries. This implies that in the beginning of cluster formation, the 2 n rule may be followed. Those siblings, which are connected tightly to the prefollicular tissue, can develop further as oocytes. All others, which are only embedded to interstitial cells, become transdetermined to develop as nurse cells. Potential nurse cells will divide preferentially by intercalary divisions, producing long and sometimes ramified chains of nurse cells, oriented parallel to the long axis of the tropharium (Fig. 11). Thus the key events for the evolution of the telotrophic meroistic ovary of polyphage Coleoptera may be: (1) no stem cell activity in ovarioles occurs; (2) reduction to one or few clusters; (3) during cluster formation, the 2 n rule is only followed in the beginning. Later on, intercalary divisions of prospective nurse cells follow; (4) somatic interstiitial cells form a 3-dimensional meshwork in the trophic chamber. This investigation is still ongoing and how many clusters are housed in a tropharium at the beginning is not known. During later developmental phases, clusters and/or subclusters can fuse, which makes an analysis very difficult and time-consuming (Brining, 1979a).
CONCLUSIONS Elucidation of germ cell clusters in combination with ultrathin serial sectioning has given new information about general construction of insect ovaries, and has opened this field of oogenesis to phylogenetic analysis. Further investigation of the evolution of 3dimensional structures of ovarioles, on germariai regions and on follicles by ultrathin serial sectioning, in combination with immunological methods and computer-enhanced
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3 - d i m e n s i o n a l r e c o n s t r u c t i o n t e c h n i q u e s , will g i v e n e w i n s i g h t s i n t o g e r m c e l l - s o m a t i c cell i n t e r a c t i o n s , as o c c u r s d u r i n g o o c y t e d e t e r m i n a t i o n p r o c e s s e s . T h e s e c o m p a r a t i v e investigations should be considered with the fundamental results of genetic and m o l e c u l a r a n a l y s e s o n o o g e n e s i s o f D r o s o p h i l a . O n l y s u c h a n i n t e g r a t i v e a p p r o a c h will show the main pathways of this organ during anagenesis and phylogeny of organisms.
Acknowledgements--I am greatly indebted to all my students, whose works have contributed to the understanding of the phylogeny of insect ovaries, as did the helpful discussions with many colleagues, among whom I will mention above all Dr R. C. King, Western Reserve University, Evanston, U.S.A., and Dr S. Bilifiski, Jagiellonian University, Krakow, Poland. This work is supported by the Deutsche Forschungsgemeinschaft.
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MATUSZEWSKI,B., K. CIECHOMSKI,J. NURKOWSKAand M. KLOC. 1985. The linear clusters of oogonial cells in the development of telotrophic ovarioles in polyphage Coleoptera. Roux's Archiv. Dev. Biol. 194: 462-9. MAYS, U. 1972. Autoradiographische Untersuchungen zum Stofftransport von den Nfihrzellen zur Oocyte der Feuel~vanze Pyrrhocoris apterus L. (Heteroptera). Z. Zellforsch. 123: 395-410. MIYA, K. 1975. Ultrastructural changes of embryonic cells during organogenesis in the silkworm, Bombyx mori. I. Tlae gonad. J. Fac. Agric. Iwate University 12: 329-64. PRITSCH, M. and J. BONING. 1989. Germ cell cluster in the panoistic ovary of Thysanoptera (Insecta). Zoomorphology 108: 309-13. SACHTLEBEN, H. 1918. l~lber die Entwicklung der Geschlechtsorgane von Chironomus mit besonderer Berticksichtigung der keimbahnbegleitenden Substanzen. Dr. Thesis, University of Miinchen, Germany. SOLDAN,Z. 1981. Comparative Anatomy of the Internal Reproductive Organs of Mayflies (Ephemeroptera). Academia, Praha. STYS, P. and S. BILIIqSKI. 1990. Ovariole types and the phylogeny of hexapods. Biol. Rev. 65: 401-29. WARN, R. M., H. O. GUTZEIT, L. SMITH and A. WARN. 1985. F-actin rings are associated with the ring canals of the Drosophila egg chamber. Exp. Cell Res. 157: 355-63. YAMAUCHI, H. and N. YOSHITAKE. 1983. Origin and differentiation of the oocyte-nurse cell complex in the germarium of the earwig, Anisolabis maritima (Dermaptera : Labiduridae). Int. J. Insect Morphol. Embryol. 11: 293-306.