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Ultrastructure of the worker ovarioles in Formica ants (Lymenoptera : Formicidae)
Int. J. InsectMorphol. & EmbryoL, Vol. 14, No. I, pp. 21 to 32, 1985. Printed in Great Britain.
0020- 7322/85$3.00 + 00 .t; 1985PergamonPressLtd.
U L T R A S T R U C T U R E OF THE WORKER OVARIOLES IN FORMICA A N T S ( H Y M E N O P T E R A • FORMICIDAE) JOHAN BILLEN* Limburgs Universitair Centrum, Department SBM, B-3610 Diepenbeek, Belgium and Laboratorium voor Systematiek en Ecologie, K.U.Leuven, Naamsestraat 59, B-3000 Leuven, Belgium (Accepted 17 July 1984) Abstract--The ultrastructure of the ovarioles during oogenesis is examined in 4 species of Formica ants (Hymenoptera : Formicidae). During the early stages of development, yolk particles start to accumulate in the oocyte cytoplasm. Peripherally, an elaborate microvillar contact area forms where it borders the surrounding follicle cells. The nurse cells remain interconnected with one another and with the oocyte via cytoplasmic bridges, allowing intercellular transport. The nurse cell cytoplasm contains m a n y small mitochondria and large numbers of ribosomes. The fully grown oocyte contains numerous protein granules and lipid droplets as well as mitochondria and ribosomes. During oocyte maturation, the columnar follicle cells gradually become cuboidal, and finally flattened with large intercellular spaces. An a m o r p h o u s vitelline m e m b r a n e is deposited between these flattened cells and the fully grown oocyte. The yellow bodies that appear in degenerating ovarioles ar e characterized by numerous multilamellar bodies and probably represent the remnants of the nurse cells. Index descriptors (in addition to those in title): Oocytes, follicular epithelium, nurse cells, oogenesis, degeneration, Formica cunicularia, Formica fusca, Formica pratensis, Formica sanguinea.
INTRODUCTION
ALTHOUGH insect gametogenesis has been the subject of a number of ultrastructural investigations, ants have hitherto been neglected. To date only males (Caetano, 1980) and queens (Cruz-Landim, 1978 ; Cruz-Landim and Caetano, 1981) of the New World leafcutting ants have been studied. Ant workers, however, form an interesting subject for ovariole research. Influenced by the presence of the queen, the worker, in a relatively short period of time, displays a complete cycle of ovariole development. This cycle consists of the stages of growth, full development, regression, and final degeneration. During the last steps of this cycle, the ovarioles are characterized by the appearance of yellow bodies, whose origin is rather poorly understood. In a number of Formica species, this individual developmental cycle has been found to be age-dependent (Otto, 1958 ; Hohorst, 1972; Billen, 1982). Worker eggs are generally resorbed, or can produce males. Worker eggs producing workers are rarely found, even in normal colonies (Ehrhardt, 1962). The various stages in ovariole development are distinguished by the differing external appearance of the ovariole: the number of protuberant chambers, the general appearance of the terminal oocyte, and the occurrence of yellow bodies. These external characteristics are a manifestation of the oogenic processes, which are reflected by morphological changes in the oocyte and in its accompanying nurse and follicle cells. *Research assistant of the Belgian National Fund for Scientific Research. 21
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In this paper, we describe the ovariole ultrastructure in 4 F o r m i c a species with respect to this individual developmental cycle. The appearance of the oocyte, its nurse and follicle cells have been investigated, and a possible origin of the yellow bodies is discussed. MATERIAL AND METHODS Several nests including their queens were collected near Roermond in the Netherlands and were transferred to artificial nests at the University of Louvain. In this way a readily available stock of workers of Formica cunicularia Latr., F. fusca L., F. pratensis Retz. and F. sanguinea Latr. of varying ages and physiological conditions was maintained for investigations. After fixation in cold 2~/0glutaraldehyde in a buffer of 0.05 M sodium cacodylateand 0.15 M saccharose at pH 7.3, the ovaries were postfixed in 2°/o osmium tetroxide in the same buffer. Tissues were bloc-stained in 2°?0 uranyl acetate during the acetone dehydration process before being embedded in Araldite. Sectioning was performed with a Reichert Ultracut microtome and double-stained grids were viewed in a Philips EM 400 electron microscope. RESULTS The ant ovary consists of meroistic polytrophic ovarioles, their number in F o r m i c a species varying from 1 to 6 in workers, up to 30 in the queens. Each ovariole is composed of a chain of successive follicles, consisting of an oocyte and its accompanying nurse and follicle cells. An ovariole sheath, which mainly comprises muscle fibres and tracheoles, forms the external lining of each ovariole tube. In the apical part of the germarium, cystocytes, rich in free ribosomes and mitochondria, differentiate into oocytes and nurse cells (trophocytes). As a result of incomplete cytokinesis, clusters of cells with intercellular bridges appear (Figs. 1; 2). The cytoplasmic gap thus created between cystocyte daughter cells reaches a constant 1.2 ~tm diameter. The bridge walls, which appear densely stained, surround a cytoplasmic zone with vesicular material (Fig. 2). On leaving the germarium, one cell from each cluster of interconnected cells will develop as the future oocyte, its sibling cells becoming nurse cells. In addition to the oocyte and nurse cells that originate from the cystocyte germ cell line, another group of mesodermal cells gives rise to follicle cells. These appear as prefollicle cells in the apical part of the germarium and show a distinct structural relationship to the cystocytes, which they surround with numerous cytoplasmic branches (Fig. 1). After a few days of imaginal life, the ant ovariole starts to display the characteristic alternate succession of egg and trophic chambers (Fig. 3). During their descent through the vitellarium, the oocyte and its nurse and follicle cells will become involved in oogenesis. Soon after leaving the germarium, the oocyte occupies a central position in the midst of a single layer of surrounding follicle cells (Fig. 3). In very young workers, the oocyte still contains a distinct nucleus (Fig. 4), which after a few days completely vanishes. At the same time, a number of accessory nuclei of up to 10 Ixm in diameter begin to appear in the ooplasm. They exhibit a double membrane envelope that lines the clear matrix, which contains one or 2 electron-dense pseudonucleoli (Fig. 3). Many lipid droplets and numerous very small mitochondria are found along with large amounts of free ribosomes. Granular endoplasmic reticulum occurs as local accumulations of concentric whorls or linearly arranged rows (Fig. 5). As vitellogenesis proceeds, the number of yolk particles in the ooplasm increases, with many protein granules and lipid droplets filling the mature oocyte (Fig. 16). During its early growth, the oocyte still retains a cytoplasmic bridge with the accompanying nurse cell chamber (Fig. 4). Similar to the intercellular cystocyte bridges,
Ultrastructure of the Worker Ovarioles in Formica Ants (Hymenoptera : Formicidae)
FIG. 1 .Section through most apical part of germarium in a one-week-old F. f us c a worker. As a result of incomplete cytokinesis, clusters of interconnected cystocytes (CC) are formed. Note slender cytoplasmic strands from prefollicular cells (pFC) penetrating between cystocytes, os = ovariole sheath, x 6,150. F~G. 2. Detail of cystocytes showing interconnecting cytoplasmic bridges in a F. cunicularia queen, x 6,000.
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F]~. 3. Egg chamber during early ovariole development (F. fusca, 8-day-old). Oocyte (OC) is surrounded by a single layer of columnar follicle cells (FC) and starts formation of a microvillar border in contact area with follicle cells, an = accessory nuclei; NC = nurse cells; os = ovariole sheath. × 3,350.
the diameter of the oocyte to nurse cell passage also measures 1.2 ~tm a n d thus allows considerable cytoplasmic t r a n s p o r t of lipid droplets (Fig. 7). In the contact area between the oocyte a n d the s u r r o u n d i n g follicle cells, a very elaborate microvillar region develops, in which nearly all of the microvilli are part of the oocyte (Fig. 6). The d e v e l o p m e n t of this specialized intercellular contact area gradually increases with oocyte m a t u r a t i o n . The initially u n m o d i f i e d o o l e m m a (Fig. 4: o n e - d a y - o l d
Ultrastructure of the Worker Ovarioles in Formica Ants (Hymenoptera : Formicidae)
Figs. 4 - 9. Captions on p. 26.
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worker) soon starts to develop microvillar processes (Fig. 3: early ovariole development, 8-day old) which become fully formed in nearly mature oocytes (Fig. 11:1 V2-yr old). During early oogenesis, the follicle cells surrounding the oocyte appear as columnar cells with distinct and rather elongated nuclei (Fig. 8). With age, the follicle cells become more cuboidal, their nuclei being rather ovoid or rounded (Fig. 10). Near the end of oogenesis, large intercellular spaces begin to occur, with a distinct electron-dense granular layer appearing between the oocyte and the follicle cells (Fig. 11). The mature oocyte is finally surrounded by a layer of nearly squamous follicle cells with very flattened nuclei (Fig. 12). Throughout their development, the follicle cells contain numerous mitochondria and free ribosomes, together with a well-developed Golgi system (Figs. 8; 13). In the early stages, the granular endoplasmic reticulum is restricted to the columnar and cuboidal follicle cells. At later stages, it becomes more widespread. Neighbouring follicle cells also display intercellular bridges. These bridges, occurring only in the apical part of the cells, are of constant diameter (0.3 ~tm, Figs. 9; 13). The bridge walls are very densely stained and characterized by a granular endoplasmic reticulum-like strand close to the inner lining of the bridge (Figs. 9; 13). In contrast, the follicle cells surrounding the nurse cell group have an extremely flattened appearance with very long and slender cytoplasmic branches, penetrating between the nurse cells (Fig. 14). Their cytoplasm also contains numerous mitochondria and free ribosomes; intercellular bridges were not observed between these flattened follicle cells. The nurse cells themselves, after leaving the germarium, grow considerably to become polygonal cells, reaching a diameter of up to 100 txm. A large rounded nucleus is located in the center of the cell. The nurse cell cytoplasm contains large numbers of free ribosomes and small mitochondria; a few lipid droplets may occur, although endoplasmic reticulum and Golgi system are very scarce (Fig. 14)..Intercellular bridges with a diameter of approximately 1.2 ~tm are often found and have the same appearance as those among cystocytes (Fig. 15). Unlike the oocyte and follicle cells, the nurse cells do not show obvious changes during oogenesis. During the final stages of their developmental cycle, the ovarioles are characterized by the appearance, in their basal region, of deeply-yellow ovoid inclusions. These can attain a diameter of over 100 lam and are usually found adjacent to an oocyte (Fig. 16). Histological sections of these so-called yellow bodies ("GelbkOrpern") reveal a very FIG. 4. Oocyte with distinct nucleus at beginning of differentiation in a 2-day-old F. cunicularia worker. No special membrane modifications have occured in contact region with follicle cells; a cytoplasmic bridge with nurse cell compartment is shown in upper part of figure (white circle). × 2,250. FiG. 5. Detail of ooplasm showing accumulation of linearly arranged rows of rough endoplasmic reticulum (rer), F. cunicularia, x 6,450. FIG. 6. Detail of microvillate appearance of oolemma in contact area with follicle cells in a F. pratensis worker, x 12,400. FIG. 7. Contact area between a nurse cell and oocyte with intercellular transport of a lipid droplet (F. sanguinea). × 12,700. FIG. 8. Follicle cell epithelium in young ovarioles containing columnar cells with elongate nuclei, many mitochondria and a well-developed Golgi apparatus (*) in a one-week-old F. fusca worker. × 4,450. Fl6. 9. Detail of apical part of follicle cells in F. cunicularia showing an intercellular cytoplasmic bridge. Note large amounts of mitochondria and free ribosomes, x 13,000. FC = follicle cells; ld = lipid droplet; M = mitochondria; mv = microvilli; N = nucleus; NC = nurse cells; OC = oocyte; rer = rough endoplasmic reticulum.
Ultrastructure of the Worker Ovarioles in Formica Ants (Hymenoptera : Formicidae)
Figs. 1 0 - 15. Captions on p. 28.
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disorderly and degenerated structure. Initially, they exhibit a number of polygonal and membrane-lined areas at their center with a diameter of 10 - 15 lxm. In the peripheral region, there are long and slender strands penetrating between the polygonal fields. Some of these contain a nucleus, which may suggest a multicellular origin (Fig. 17). In the later stages of development, the yellow body becomes almost completely filled with extensive myeloid inclusions that consist of concentric membraneous layers, reaching a total diameter of a few micrometres (Fig. 18). In completely degenerated ovarioles, 2 or 3 such yellow bodies are the only contents of the ovariole sheath. Nothing remains of the formerly occurring cell types at this stage. Whether the mature oocyte gave rise to an egg or was resorbed by the haemolymph could not be established; no morphological evidence for either of these possibilities was found. DISCUSSION As a result of division of labor, which is one of the fundamental characteristics of the social insect community, the task of egg-laying is almost completely restricted to the queen. The worker caste is composed of females whose ovarioles display the same characteristic cellular differentiation required for oogenesis as in the queen. However, due to radical queen inhibition the development of their reproductive organs is suppressed, rendering them sterile. Owing to the meroistic nature of insect ovarioles, the sister germ ceils remain connected by cytoplasmic canals during their early development. Interconnections between cystocytes persist between their descendant oocyte and nurse cells. The diameter of these intercellular bridges is a constant 1.2 Ixm, similar to the corresponding measurements in the ant queen (Cruz-Landim, 1978). The diameter of the intertrophocytic ring canals are reported to be 3.5 p.m in the honeybee queen (Ramamurty and Engels, 1977) and in the range 0 . 3 - 1.3 ~tm in the braconid wasp, Habrobracon juglandis (Cassidy and King, 1972). In these examples, with bridge diameters exceeding 1 Ixm, considerable cytoplasmic transport between neighbouring cells may be expected. This has been shown to be the case for mitochondria (Mandelbaum, 1980), endoplasmic reticulum and lipid droplets (Cassidy and King, 1972) and large amounts of ribosomes (Engels, 1973; Cruz-Landim and Caetano, 1981). Attempts have recently been made to clarify the cellular regulation mechanisms underlying the unidirectional transport in the insect ovary (Woodruff and Telfer, 1980; Telfer et al., 1981; De Loof, 1983). In these studies, a substantial electrical potential gradient across the oocyte and nurse cell compartment was found to have an important influence on the distribution of cytoplasmic constituents. As a result of these FIG. 10. Follicle cell epithelium at a later stage of development in F. cunicularia, showing slightly cuboidal cells with an ovoid to rounded nucleus and extensive amounts of mitochondria. × 6,400. FIG. 11. Follicle cell epithelium near the end of oogenesis in a 1 ~2-year old F. sanguinea worker, showing appearance of large intercellular spaces (*). × 2,450. FIG. 12. Flattened follicle cell epithelium at end of oogenesis (F. cunicularia), x 5,500. Flo. 13. Detail of follicle cell cytoplasm showing 2 intercellular bridges, numerous free ribosomes and Golgi apparatus in F. sanguinea. × 17,800. FIG. 14. Nurse cells in a callow F. pratensis worker, surrounded by flattened follicle cells deeply penetrating (arrows) between nurse cells. × 1,650. FIG. 15. Intercellular bridges between neighbouring nurse cells in a one-week-old F. fusca worker. × 4,900. FC = follicle cells; ga = Golgi apparatus; M - mitochondria; mv = microvilli; NC = nurse cells; OC = oocyte; os = ovariole sheath.
Ultrastructure of the Worker Ovarioles in Formica Ants (Hymenoptera : Formicidae)
Fic,. 16. Semi-thin section showing yellow body (YB) adjacent to a nearly fully-grown oocyte (OC) in F. pratensis, x 275. FJc;. 17. Yellow body in empty ovariole tube, showing several nuclei and membrane-lined areas ¢F. sanguinea), x 1,650. Fro. 18. Detail of multilamellar bodies (MLB) in a "fully-grown" yellow body of a F. cunicularia worker, x 16,150.
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bioelectric properties, negatively charged ribosomes flow from the nurse cells into the oocyte since its potential is a few millivolts more positive. In addition to transport of cytoplasmic organelles, such as mitochondria and lipid droplets, the other important functions of the nurse cells include the synthesis and subsequent transfer of RNA into the ooplasm (Cassidy and King, 1972). In agreement with this, are the many free ribosomes that together with mitochondria occupy most of the nurse cell cytoplasm. The low levels of other organelles, such as endoplasmic reticulum and Golgi apparatus support the conclusion by Engels (1973) that nurse cells are not actively involved in yolk synthesis. The cytoplasm of the follicle cells is apparently a highly active tissue. Throughout their development, the follicle cells contain a well-developed Golgi complex and many mitochondria. With subsequent oocyte maturation, more granular endoplasmic reticulum is formed together with numerous intercellular spaces. The follicular epithelium acts as a selective barrier through which molecules pass for incorporation into the maturing oocyte. The development of the oolemma specialization of pinocytotic pits and vesicles along with the microvillar border hereby facilitates uptake of metabolic constituents. Until now, the most probable pathway of vitellogenic proteins is believed to be through the interfollicular spaces (De Wilde and De Loof, 1973; Engels, 1973; Koeppe et al., 1980; Bitsch and Bitsch, 1982; Telfer et al., 1982). In addition to their contribution to the passage of vitellogenic constituents, the follicle cells are known to produce ecdysone. This is transmitted to the oocyte at the end of vitellogenesis (Lagueux et al., 1977) and may influence the follicular bioelectric properties (De Loof, 1983). At the same time, the follicle cells begin to synthesize vitelline membrane precursors as a product of the Golgi apparatus (De Wilde and De Loof, 1973; Bitsch and Bitsch, 1982). In addition, the follicle cells show intercellular bridges having a diameter of about a quarter of that observed in the oocytes and nurse cells. Similar cytoplasmic channels also extend between follicle cells in the ovary of the honeybee queen (Ramamurty and Engels, 1977), and are a result of the delay in the completion of cytokinesis. The occurrence of long and slender cytoplasmic strands from the flattened follicle cells that deeply penetrate between the nurse cells is also observed in the prefollicular cells surrounding the cystocytes. By the end of vitellogenesis, the mature oocyte contains a considerable amount of yolk material of mainly extracellular origin. Following the deposition of the vitelline membrane and chorion, the oocyte becomes an independent entity that will eventually be either resorbed, or allowed to leave the oviduct as a full-grown egg. The now flattened follicle cells, being no longer functional, break down and disappear (Cruz-Landim, 1978). The appearance of yellow bodies in the final stages of ovariole development is a wellknown phenomenon (Weyer, 1928), but so far no report has dealt with their origin. From their close proximity to oocytes and histological characteristics, we may tentatively assume that they originate from degenerating nurse cells. Supporting evidence for this is provided by their multinuclear appearance, which definitely rejects any oocyte origin. Their general size, reaching over 100 I~m, also excludes any relationship with the follicle cells, taking into account their considerable reduction in volume near the end of oogenesis. On the basis of this evidence, the nurse cells seem to be the most likely origin for the yellow bodies. The polygonal areas in the latter, may conveniently represent the former nurse cells, with the narrow peripheral strands corresponding to the remainder of the flattened follicle cells, which initially surrounded the nurse cell compartment during earlier stages.
Ultrastructure of the Worker Ovarioles in Formica Ants (Hymenoptera : Formicidae)
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T h e very disorderly u l t r a s t r u c t u r a l a p p e a r a n c e o f the yellow b o d y c o n t e n t s clearly suggests that a process o f d e g e n e r a t i o n is in progress. The latter hypothesis is s u p p o r t e d by the occurrence of extensive a m o u n t s o f m u l t i l a m e l l a r inclusions. T h e y p r o b a b l y form part o f the lysosome complex (Noirot a n d Q u e n n e d e y , 1974) a n d c o r r e s p o n d histochemically to glycoproteins ( Q u e n n e d e y a n d Brossut, 1975). G e n e r a l ovariole d e g e n e r a t i o n with oocyte r e s o r p t i o n has been f o u n d to be a n orderly process regulating the recycling o f n u t r i e n t s ( H o p k i n s a n d King, 1964). I n the presence o f the queen, eggs of a n t workers are thus generally r e a b s o r b e d soon after reaching their m a x i m a l growth. Their n u t r i t i o u s c o m p o n e n t s o n being c o n s u m e d by the p o s t p h a r y n g e a l glands b e c o m e available for larval trophallaxis (G0rtz, 1973; Schmidt, 1974). A similar nutritive goal is achieved in other species, such as M y r m i c a , t h r o u g h the p r o d u c t i o n o f trophic eggs (Brian a n d Rigby, 1978). I n the genus F o r m i c a , workers as a rule are n o t supposed to be egg-laying. I n special c o n d i t i o n s , however, where the q u e e n i n h i b i t i o n is suppressed or removed, they will readily p r o d u c e male b r o o d (Billen, 1984). In the wood ant, F. p o l y c t e n a , male p r o d u c t i o n from worker eggs has also been established in n o r m a l c o n d i t i o n s in the presence of the q u e e n ( E h r h a r d t , 1962). Other species are even k n o w n to be able to rear worker individuals from worker eggs; this was observed for L a s i u s n i g e r (Reichenbach, 1902) a n d O e c o p h y l l a l o n g i n o d a (Ledoux, 1950). T h u s , the existance of a cycle o f ovariole d e v e l o p m e n t in worker ants can be related to their ability to p r o d u c e viable eggs in queenless or even n o r m a l c o n d i t i o n s . Therefore, should the need arise, provided the workers are in the necessary physiological c o n d i t i o n , they m a y c o n t r i b u t e either actively in egg p r o d u c t i o n or passively by regulating larval trophallaxis t h r o u g h the nutrients r e a b s o r b e d from their ovarioles. Acknowledgements--I am very grateful to Els Plaum for technical assistance and to Marc Withofs for the
photographs. I am also indebted to Professor J. van Boven and Professor E. Schockaert for the valuable advice and discussion during the preparation of the manuscript, and thank Dr R. P. Evershed for revising the English. REFERENCES Bil[ fiN, J. 1982. Ovariole development in workers of Formica sanguinea Latr. (Hymenoptera, Formicidae). lnsectes Sociaux 29:86 - 94. BH I try, J. 1984. Stratification in the nest of the slave-making ant Formica sanguinea Latreille (Hymenoptera, Formicidae). Ann. Soc. R. Zool. Belg. ll4:231 -41. BJlSCH, J. and C. B]TSCH. 1982. Les htapes de la vitellogenesechez Thermobia domestica (Packard) (Thysanura : Lepismatida). Int. J. Insect Morphol. Embryol. I I: 197 - 212. BRIAN, M. V. and C. RIGt3¥. 1978. The trophic eggs of Myrmica rubra L. ]nsectes Sociaux 25: 89- 110. CA~-TANO, F. H. 1980. Ultra-estrutura dos EspermatOzoides de Atta capiguara e Atta sexdens rubropilosa (Formicidae). Naturalia (Sffo Paulo) 5: 105- 11. CASSID¥, J. D. and R. C. KING. 1972. Ovarian development in Habrobracon juglandis (Ashmead) (Hymenoptera : Braconidae). 1. The origin and differentiation of the oocyte - nurse cell complex. Biol. Bull. 143:483 - 505. CRUZ-LANDIM, C. 1978. Structural dynamics of oogenesis in Atta sexdens rubropilosa (Hymenoptera, Formicidae). Rev. BrasiL Biol. 38: 363- 81. CRUZ-LANDIM,C. and F. H. CAETANO.1981. The histochemistry and fine structure of the vitellarium in Atta (Formicidae, Myrmicinae). Rev. Brasil. Biol. 41:363-70. DE LOOE,A. 1983. The meroistic insect ovary as a miniature electrophoresis chamber. Comp. Biochem. Physiol. 74A 3 - 9. DE WILDE, J. and A. DE LOOF. 1973. Reproduction, pp. l l - 9 5 . In. M. ROCKSTEIN(ed.) The Physiology o f lnsecta, Vol. l, Academic Press, New York, London. EHRHARDT, H.-J. 1962. Ablage tibergroBer Eier durch Arbeiterinnen von Formica polyctena FOrster (Ins., Hym.) in Gegenwart yon K0niginnen. Naturwissenschaften 22:524 - 25 ENGELS, W. 1973. Das zeitliche und ratimliche Muster der Dottereinlagerung in die Oocyte yon Apis mellifica. Z. Zellforsch. 142: 409- 30.
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GORTZ,H.-D. 1973. Herkunft und Weg der Dotterproteine bei Formicapolyctena Foerst. Proc. 7th Int. Congr. I.U.S.S.I., London, 1 4 0 - 4 3 . HOHORST, B. 1972. Entwicklung und Ausbildung der Ovarien bei Arbeiterinnen yon Formica (ServiJormica) rufibarbis Fabricius (Hymenoptera : Formicidae). Insectes Sociaux 19: 3 8 9 - 402. HOPKINS, C. R. and P. E. KING. 1964. Egg resorption in Nasonia vitripennis (Walker) (Hymenoptera : Pteromalidae). Proc. R. Entomol. Soc. Lond. (,4) 39: lOl - 19. KOEPPE, J. K., K. HOBSON and S. E. WELLMAN. 1980 Juvenile hormone regulation of structural changes and D N A synthesis in the follicular epithelium of Leucophaea maderae. J. Insect Physiok 26: 2 2 9 - 40. LAGUEUX, M., M. HIRN and J. A. HOFFMANN. 1977. Ecdysone during ovarian development in Locusta migratoria. J. Insect Physiol. 23: 1 0 9 - 19. LEDOUX, A. 1950. Recherches sur la biologie de la fourmi fileuse (Oecophylla longinoda LatE). Ann. Sci. Nat. Zool. 12: 3 1 2 - 461 MANDELBAUM, I. 1980. Intercellular bridges and the fusome in the germ cells of the cecropia moth. J. Morphol. 166:37 - 50. NOIROT, C. and A. QUENNEDEY. 1974 Fine structure of insect epidermal glands. Annu. Rev. Entomol. 19: 61 - 80. OTTO, D. 1958. Ueber die Arbeitsteilung im Staate yon Formica rufa rufo-pratensis Gt~ssw. und ihre verhaltensphysiologischen Grundlagen. Wiss. Abh. 50:1 - 166. QUENNEDEY, A.and R. BROSSUT. 1975. Les glandes mandibulaires de Blaberus craniifer Burm. (Dictyoptera, Blaberidae). d(~veloppement, structure et fonctionnement. Tissue Cell 7: 5 0 3 - 17. RAMAMURTY, P. S. and W. ENGELS. 1977. Occurrence of intercellular bridges between follicle epithelial cells in the ovary of Apis mellifica queens. J. Cell Sci. 24: 1 9 5 - 202. REICHENBACH, H. 1902. Uber Parthenogenese bei Ameisen und andere Beobachtungen an Ameisenkolonien in kilnstlichen Nestern. Biok Zentralbk 22:461 - 6 5 . SCHMIDT, G. H. 1974. Steuerung der Kastenbildung und Geschlechtstierregulation im Waldameisenstaat, pp. 4 0 4 - 5 1 2 . In. G. H. Schmidt (ed.) Sozialpolymorphismus bei Insekten, Wiss. Verl. Ges. m b H , Stuttgart. TELFER, W. H., R. I. WOODRUFF and E. HUEBNER. 1981. Electrical polarity and cellular differentiation in meroistic ovaries. Amer. Zool. 21:675 - 86. TELFER, W. H., E. HUEBNER and D. S. SMITH. 1982. The cell biology of vitellogenic follicles in Hyalophora and Rhodnius, pp. 1 1 8 - 49. In R. C. KJ~; and H. AKAI (eds.) Insect Ultrastructure, Vol. I, Plenum Press, New York, London. WEVER, F. 1982. Untersuchungen iiber die Keimdrt~sen bei Hymenopterenarbeiterinnen. Z. Wiss. Zool. 131: 3 4 5 - 501. WOODRUFF, R. I. and W. H. TELFER. 1980. Electrophoresis of proteins in intercellular bridges. Nature (Lond.) 286:84 - 6.
0020- 7322/85$3.00 + 00 .t; 1985PergamonPressLtd.
U L T R A S T R U C T U R E OF THE WORKER OVARIOLES IN FORMICA A N T S ( H Y M E N O P T E R A • FORMICIDAE) JOHAN BILLEN* Limburgs Universitair Centrum, Department SBM, B-3610 Diepenbeek, Belgium and Laboratorium voor Systematiek en Ecologie, K.U.Leuven, Naamsestraat 59, B-3000 Leuven, Belgium (Accepted 17 July 1984) Abstract--The ultrastructure of the ovarioles during oogenesis is examined in 4 species of Formica ants (Hymenoptera : Formicidae). During the early stages of development, yolk particles start to accumulate in the oocyte cytoplasm. Peripherally, an elaborate microvillar contact area forms where it borders the surrounding follicle cells. The nurse cells remain interconnected with one another and with the oocyte via cytoplasmic bridges, allowing intercellular transport. The nurse cell cytoplasm contains m a n y small mitochondria and large numbers of ribosomes. The fully grown oocyte contains numerous protein granules and lipid droplets as well as mitochondria and ribosomes. During oocyte maturation, the columnar follicle cells gradually become cuboidal, and finally flattened with large intercellular spaces. An a m o r p h o u s vitelline m e m b r a n e is deposited between these flattened cells and the fully grown oocyte. The yellow bodies that appear in degenerating ovarioles ar e characterized by numerous multilamellar bodies and probably represent the remnants of the nurse cells. Index descriptors (in addition to those in title): Oocytes, follicular epithelium, nurse cells, oogenesis, degeneration, Formica cunicularia, Formica fusca, Formica pratensis, Formica sanguinea.
INTRODUCTION
ALTHOUGH insect gametogenesis has been the subject of a number of ultrastructural investigations, ants have hitherto been neglected. To date only males (Caetano, 1980) and queens (Cruz-Landim, 1978 ; Cruz-Landim and Caetano, 1981) of the New World leafcutting ants have been studied. Ant workers, however, form an interesting subject for ovariole research. Influenced by the presence of the queen, the worker, in a relatively short period of time, displays a complete cycle of ovariole development. This cycle consists of the stages of growth, full development, regression, and final degeneration. During the last steps of this cycle, the ovarioles are characterized by the appearance of yellow bodies, whose origin is rather poorly understood. In a number of Formica species, this individual developmental cycle has been found to be age-dependent (Otto, 1958 ; Hohorst, 1972; Billen, 1982). Worker eggs are generally resorbed, or can produce males. Worker eggs producing workers are rarely found, even in normal colonies (Ehrhardt, 1962). The various stages in ovariole development are distinguished by the differing external appearance of the ovariole: the number of protuberant chambers, the general appearance of the terminal oocyte, and the occurrence of yellow bodies. These external characteristics are a manifestation of the oogenic processes, which are reflected by morphological changes in the oocyte and in its accompanying nurse and follicle cells. *Research assistant of the Belgian National Fund for Scientific Research. 21
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In this paper, we describe the ovariole ultrastructure in 4 F o r m i c a species with respect to this individual developmental cycle. The appearance of the oocyte, its nurse and follicle cells have been investigated, and a possible origin of the yellow bodies is discussed. MATERIAL AND METHODS Several nests including their queens were collected near Roermond in the Netherlands and were transferred to artificial nests at the University of Louvain. In this way a readily available stock of workers of Formica cunicularia Latr., F. fusca L., F. pratensis Retz. and F. sanguinea Latr. of varying ages and physiological conditions was maintained for investigations. After fixation in cold 2~/0glutaraldehyde in a buffer of 0.05 M sodium cacodylateand 0.15 M saccharose at pH 7.3, the ovaries were postfixed in 2°/o osmium tetroxide in the same buffer. Tissues were bloc-stained in 2°?0 uranyl acetate during the acetone dehydration process before being embedded in Araldite. Sectioning was performed with a Reichert Ultracut microtome and double-stained grids were viewed in a Philips EM 400 electron microscope. RESULTS The ant ovary consists of meroistic polytrophic ovarioles, their number in F o r m i c a species varying from 1 to 6 in workers, up to 30 in the queens. Each ovariole is composed of a chain of successive follicles, consisting of an oocyte and its accompanying nurse and follicle cells. An ovariole sheath, which mainly comprises muscle fibres and tracheoles, forms the external lining of each ovariole tube. In the apical part of the germarium, cystocytes, rich in free ribosomes and mitochondria, differentiate into oocytes and nurse cells (trophocytes). As a result of incomplete cytokinesis, clusters of cells with intercellular bridges appear (Figs. 1; 2). The cytoplasmic gap thus created between cystocyte daughter cells reaches a constant 1.2 ~tm diameter. The bridge walls, which appear densely stained, surround a cytoplasmic zone with vesicular material (Fig. 2). On leaving the germarium, one cell from each cluster of interconnected cells will develop as the future oocyte, its sibling cells becoming nurse cells. In addition to the oocyte and nurse cells that originate from the cystocyte germ cell line, another group of mesodermal cells gives rise to follicle cells. These appear as prefollicle cells in the apical part of the germarium and show a distinct structural relationship to the cystocytes, which they surround with numerous cytoplasmic branches (Fig. 1). After a few days of imaginal life, the ant ovariole starts to display the characteristic alternate succession of egg and trophic chambers (Fig. 3). During their descent through the vitellarium, the oocyte and its nurse and follicle cells will become involved in oogenesis. Soon after leaving the germarium, the oocyte occupies a central position in the midst of a single layer of surrounding follicle cells (Fig. 3). In very young workers, the oocyte still contains a distinct nucleus (Fig. 4), which after a few days completely vanishes. At the same time, a number of accessory nuclei of up to 10 Ixm in diameter begin to appear in the ooplasm. They exhibit a double membrane envelope that lines the clear matrix, which contains one or 2 electron-dense pseudonucleoli (Fig. 3). Many lipid droplets and numerous very small mitochondria are found along with large amounts of free ribosomes. Granular endoplasmic reticulum occurs as local accumulations of concentric whorls or linearly arranged rows (Fig. 5). As vitellogenesis proceeds, the number of yolk particles in the ooplasm increases, with many protein granules and lipid droplets filling the mature oocyte (Fig. 16). During its early growth, the oocyte still retains a cytoplasmic bridge with the accompanying nurse cell chamber (Fig. 4). Similar to the intercellular cystocyte bridges,
Ultrastructure of the Worker Ovarioles in Formica Ants (Hymenoptera : Formicidae)
FIG. 1 .Section through most apical part of germarium in a one-week-old F. f us c a worker. As a result of incomplete cytokinesis, clusters of interconnected cystocytes (CC) are formed. Note slender cytoplasmic strands from prefollicular cells (pFC) penetrating between cystocytes, os = ovariole sheath, x 6,150. F~G. 2. Detail of cystocytes showing interconnecting cytoplasmic bridges in a F. cunicularia queen, x 6,000.
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F]~. 3. Egg chamber during early ovariole development (F. fusca, 8-day-old). Oocyte (OC) is surrounded by a single layer of columnar follicle cells (FC) and starts formation of a microvillar border in contact area with follicle cells, an = accessory nuclei; NC = nurse cells; os = ovariole sheath. × 3,350.
the diameter of the oocyte to nurse cell passage also measures 1.2 ~tm a n d thus allows considerable cytoplasmic t r a n s p o r t of lipid droplets (Fig. 7). In the contact area between the oocyte a n d the s u r r o u n d i n g follicle cells, a very elaborate microvillar region develops, in which nearly all of the microvilli are part of the oocyte (Fig. 6). The d e v e l o p m e n t of this specialized intercellular contact area gradually increases with oocyte m a t u r a t i o n . The initially u n m o d i f i e d o o l e m m a (Fig. 4: o n e - d a y - o l d
Ultrastructure of the Worker Ovarioles in Formica Ants (Hymenoptera : Formicidae)
Figs. 4 - 9. Captions on p. 26.
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worker) soon starts to develop microvillar processes (Fig. 3: early ovariole development, 8-day old) which become fully formed in nearly mature oocytes (Fig. 11:1 V2-yr old). During early oogenesis, the follicle cells surrounding the oocyte appear as columnar cells with distinct and rather elongated nuclei (Fig. 8). With age, the follicle cells become more cuboidal, their nuclei being rather ovoid or rounded (Fig. 10). Near the end of oogenesis, large intercellular spaces begin to occur, with a distinct electron-dense granular layer appearing between the oocyte and the follicle cells (Fig. 11). The mature oocyte is finally surrounded by a layer of nearly squamous follicle cells with very flattened nuclei (Fig. 12). Throughout their development, the follicle cells contain numerous mitochondria and free ribosomes, together with a well-developed Golgi system (Figs. 8; 13). In the early stages, the granular endoplasmic reticulum is restricted to the columnar and cuboidal follicle cells. At later stages, it becomes more widespread. Neighbouring follicle cells also display intercellular bridges. These bridges, occurring only in the apical part of the cells, are of constant diameter (0.3 ~tm, Figs. 9; 13). The bridge walls are very densely stained and characterized by a granular endoplasmic reticulum-like strand close to the inner lining of the bridge (Figs. 9; 13). In contrast, the follicle cells surrounding the nurse cell group have an extremely flattened appearance with very long and slender cytoplasmic branches, penetrating between the nurse cells (Fig. 14). Their cytoplasm also contains numerous mitochondria and free ribosomes; intercellular bridges were not observed between these flattened follicle cells. The nurse cells themselves, after leaving the germarium, grow considerably to become polygonal cells, reaching a diameter of up to 100 txm. A large rounded nucleus is located in the center of the cell. The nurse cell cytoplasm contains large numbers of free ribosomes and small mitochondria; a few lipid droplets may occur, although endoplasmic reticulum and Golgi system are very scarce (Fig. 14)..Intercellular bridges with a diameter of approximately 1.2 ~tm are often found and have the same appearance as those among cystocytes (Fig. 15). Unlike the oocyte and follicle cells, the nurse cells do not show obvious changes during oogenesis. During the final stages of their developmental cycle, the ovarioles are characterized by the appearance, in their basal region, of deeply-yellow ovoid inclusions. These can attain a diameter of over 100 lam and are usually found adjacent to an oocyte (Fig. 16). Histological sections of these so-called yellow bodies ("GelbkOrpern") reveal a very FIG. 4. Oocyte with distinct nucleus at beginning of differentiation in a 2-day-old F. cunicularia worker. No special membrane modifications have occured in contact region with follicle cells; a cytoplasmic bridge with nurse cell compartment is shown in upper part of figure (white circle). × 2,250. FiG. 5. Detail of ooplasm showing accumulation of linearly arranged rows of rough endoplasmic reticulum (rer), F. cunicularia, x 6,450. FIG. 6. Detail of microvillate appearance of oolemma in contact area with follicle cells in a F. pratensis worker, x 12,400. FIG. 7. Contact area between a nurse cell and oocyte with intercellular transport of a lipid droplet (F. sanguinea). × 12,700. FIG. 8. Follicle cell epithelium in young ovarioles containing columnar cells with elongate nuclei, many mitochondria and a well-developed Golgi apparatus (*) in a one-week-old F. fusca worker. × 4,450. Fl6. 9. Detail of apical part of follicle cells in F. cunicularia showing an intercellular cytoplasmic bridge. Note large amounts of mitochondria and free ribosomes, x 13,000. FC = follicle cells; ld = lipid droplet; M = mitochondria; mv = microvilli; N = nucleus; NC = nurse cells; OC = oocyte; rer = rough endoplasmic reticulum.
Ultrastructure of the Worker Ovarioles in Formica Ants (Hymenoptera : Formicidae)
Figs. 1 0 - 15. Captions on p. 28.
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disorderly and degenerated structure. Initially, they exhibit a number of polygonal and membrane-lined areas at their center with a diameter of 10 - 15 lxm. In the peripheral region, there are long and slender strands penetrating between the polygonal fields. Some of these contain a nucleus, which may suggest a multicellular origin (Fig. 17). In the later stages of development, the yellow body becomes almost completely filled with extensive myeloid inclusions that consist of concentric membraneous layers, reaching a total diameter of a few micrometres (Fig. 18). In completely degenerated ovarioles, 2 or 3 such yellow bodies are the only contents of the ovariole sheath. Nothing remains of the formerly occurring cell types at this stage. Whether the mature oocyte gave rise to an egg or was resorbed by the haemolymph could not be established; no morphological evidence for either of these possibilities was found. DISCUSSION As a result of division of labor, which is one of the fundamental characteristics of the social insect community, the task of egg-laying is almost completely restricted to the queen. The worker caste is composed of females whose ovarioles display the same characteristic cellular differentiation required for oogenesis as in the queen. However, due to radical queen inhibition the development of their reproductive organs is suppressed, rendering them sterile. Owing to the meroistic nature of insect ovarioles, the sister germ ceils remain connected by cytoplasmic canals during their early development. Interconnections between cystocytes persist between their descendant oocyte and nurse cells. The diameter of these intercellular bridges is a constant 1.2 Ixm, similar to the corresponding measurements in the ant queen (Cruz-Landim, 1978). The diameter of the intertrophocytic ring canals are reported to be 3.5 p.m in the honeybee queen (Ramamurty and Engels, 1977) and in the range 0 . 3 - 1.3 ~tm in the braconid wasp, Habrobracon juglandis (Cassidy and King, 1972). In these examples, with bridge diameters exceeding 1 Ixm, considerable cytoplasmic transport between neighbouring cells may be expected. This has been shown to be the case for mitochondria (Mandelbaum, 1980), endoplasmic reticulum and lipid droplets (Cassidy and King, 1972) and large amounts of ribosomes (Engels, 1973; Cruz-Landim and Caetano, 1981). Attempts have recently been made to clarify the cellular regulation mechanisms underlying the unidirectional transport in the insect ovary (Woodruff and Telfer, 1980; Telfer et al., 1981; De Loof, 1983). In these studies, a substantial electrical potential gradient across the oocyte and nurse cell compartment was found to have an important influence on the distribution of cytoplasmic constituents. As a result of these FIG. 10. Follicle cell epithelium at a later stage of development in F. cunicularia, showing slightly cuboidal cells with an ovoid to rounded nucleus and extensive amounts of mitochondria. × 6,400. FIG. 11. Follicle cell epithelium near the end of oogenesis in a 1 ~2-year old F. sanguinea worker, showing appearance of large intercellular spaces (*). × 2,450. FIG. 12. Flattened follicle cell epithelium at end of oogenesis (F. cunicularia), x 5,500. Flo. 13. Detail of follicle cell cytoplasm showing 2 intercellular bridges, numerous free ribosomes and Golgi apparatus in F. sanguinea. × 17,800. FIG. 14. Nurse cells in a callow F. pratensis worker, surrounded by flattened follicle cells deeply penetrating (arrows) between nurse cells. × 1,650. FIG. 15. Intercellular bridges between neighbouring nurse cells in a one-week-old F. fusca worker. × 4,900. FC = follicle cells; ga = Golgi apparatus; M - mitochondria; mv = microvilli; NC = nurse cells; OC = oocyte; os = ovariole sheath.
Ultrastructure of the Worker Ovarioles in Formica Ants (Hymenoptera : Formicidae)
Fic,. 16. Semi-thin section showing yellow body (YB) adjacent to a nearly fully-grown oocyte (OC) in F. pratensis, x 275. FJc;. 17. Yellow body in empty ovariole tube, showing several nuclei and membrane-lined areas ¢F. sanguinea), x 1,650. Fro. 18. Detail of multilamellar bodies (MLB) in a "fully-grown" yellow body of a F. cunicularia worker, x 16,150.
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bioelectric properties, negatively charged ribosomes flow from the nurse cells into the oocyte since its potential is a few millivolts more positive. In addition to transport of cytoplasmic organelles, such as mitochondria and lipid droplets, the other important functions of the nurse cells include the synthesis and subsequent transfer of RNA into the ooplasm (Cassidy and King, 1972). In agreement with this, are the many free ribosomes that together with mitochondria occupy most of the nurse cell cytoplasm. The low levels of other organelles, such as endoplasmic reticulum and Golgi apparatus support the conclusion by Engels (1973) that nurse cells are not actively involved in yolk synthesis. The cytoplasm of the follicle cells is apparently a highly active tissue. Throughout their development, the follicle cells contain a well-developed Golgi complex and many mitochondria. With subsequent oocyte maturation, more granular endoplasmic reticulum is formed together with numerous intercellular spaces. The follicular epithelium acts as a selective barrier through which molecules pass for incorporation into the maturing oocyte. The development of the oolemma specialization of pinocytotic pits and vesicles along with the microvillar border hereby facilitates uptake of metabolic constituents. Until now, the most probable pathway of vitellogenic proteins is believed to be through the interfollicular spaces (De Wilde and De Loof, 1973; Engels, 1973; Koeppe et al., 1980; Bitsch and Bitsch, 1982; Telfer et al., 1982). In addition to their contribution to the passage of vitellogenic constituents, the follicle cells are known to produce ecdysone. This is transmitted to the oocyte at the end of vitellogenesis (Lagueux et al., 1977) and may influence the follicular bioelectric properties (De Loof, 1983). At the same time, the follicle cells begin to synthesize vitelline membrane precursors as a product of the Golgi apparatus (De Wilde and De Loof, 1973; Bitsch and Bitsch, 1982). In addition, the follicle cells show intercellular bridges having a diameter of about a quarter of that observed in the oocytes and nurse cells. Similar cytoplasmic channels also extend between follicle cells in the ovary of the honeybee queen (Ramamurty and Engels, 1977), and are a result of the delay in the completion of cytokinesis. The occurrence of long and slender cytoplasmic strands from the flattened follicle cells that deeply penetrate between the nurse cells is also observed in the prefollicular cells surrounding the cystocytes. By the end of vitellogenesis, the mature oocyte contains a considerable amount of yolk material of mainly extracellular origin. Following the deposition of the vitelline membrane and chorion, the oocyte becomes an independent entity that will eventually be either resorbed, or allowed to leave the oviduct as a full-grown egg. The now flattened follicle cells, being no longer functional, break down and disappear (Cruz-Landim, 1978). The appearance of yellow bodies in the final stages of ovariole development is a wellknown phenomenon (Weyer, 1928), but so far no report has dealt with their origin. From their close proximity to oocytes and histological characteristics, we may tentatively assume that they originate from degenerating nurse cells. Supporting evidence for this is provided by their multinuclear appearance, which definitely rejects any oocyte origin. Their general size, reaching over 100 I~m, also excludes any relationship with the follicle cells, taking into account their considerable reduction in volume near the end of oogenesis. On the basis of this evidence, the nurse cells seem to be the most likely origin for the yellow bodies. The polygonal areas in the latter, may conveniently represent the former nurse cells, with the narrow peripheral strands corresponding to the remainder of the flattened follicle cells, which initially surrounded the nurse cell compartment during earlier stages.
Ultrastructure of the Worker Ovarioles in Formica Ants (Hymenoptera : Formicidae)
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T h e very disorderly u l t r a s t r u c t u r a l a p p e a r a n c e o f the yellow b o d y c o n t e n t s clearly suggests that a process o f d e g e n e r a t i o n is in progress. The latter hypothesis is s u p p o r t e d by the occurrence of extensive a m o u n t s o f m u l t i l a m e l l a r inclusions. T h e y p r o b a b l y form part o f the lysosome complex (Noirot a n d Q u e n n e d e y , 1974) a n d c o r r e s p o n d histochemically to glycoproteins ( Q u e n n e d e y a n d Brossut, 1975). G e n e r a l ovariole d e g e n e r a t i o n with oocyte r e s o r p t i o n has been f o u n d to be a n orderly process regulating the recycling o f n u t r i e n t s ( H o p k i n s a n d King, 1964). I n the presence o f the queen, eggs of a n t workers are thus generally r e a b s o r b e d soon after reaching their m a x i m a l growth. Their n u t r i t i o u s c o m p o n e n t s o n being c o n s u m e d by the p o s t p h a r y n g e a l glands b e c o m e available for larval trophallaxis (G0rtz, 1973; Schmidt, 1974). A similar nutritive goal is achieved in other species, such as M y r m i c a , t h r o u g h the p r o d u c t i o n o f trophic eggs (Brian a n d Rigby, 1978). I n the genus F o r m i c a , workers as a rule are n o t supposed to be egg-laying. I n special c o n d i t i o n s , however, where the q u e e n i n h i b i t i o n is suppressed or removed, they will readily p r o d u c e male b r o o d (Billen, 1984). In the wood ant, F. p o l y c t e n a , male p r o d u c t i o n from worker eggs has also been established in n o r m a l c o n d i t i o n s in the presence of the q u e e n ( E h r h a r d t , 1962). Other species are even k n o w n to be able to rear worker individuals from worker eggs; this was observed for L a s i u s n i g e r (Reichenbach, 1902) a n d O e c o p h y l l a l o n g i n o d a (Ledoux, 1950). T h u s , the existance of a cycle o f ovariole d e v e l o p m e n t in worker ants can be related to their ability to p r o d u c e viable eggs in queenless or even n o r m a l c o n d i t i o n s . Therefore, should the need arise, provided the workers are in the necessary physiological c o n d i t i o n , they m a y c o n t r i b u t e either actively in egg p r o d u c t i o n or passively by regulating larval trophallaxis t h r o u g h the nutrients r e a b s o r b e d from their ovarioles. Acknowledgements--I am very grateful to Els Plaum for technical assistance and to Marc Withofs for the
photographs. I am also indebted to Professor J. van Boven and Professor E. Schockaert for the valuable advice and discussion during the preparation of the manuscript, and thank Dr R. P. Evershed for revising the English. REFERENCES Bil[ fiN, J. 1982. Ovariole development in workers of Formica sanguinea Latr. (Hymenoptera, Formicidae). lnsectes Sociaux 29:86 - 94. BH I try, J. 1984. Stratification in the nest of the slave-making ant Formica sanguinea Latreille (Hymenoptera, Formicidae). Ann. Soc. R. Zool. Belg. ll4:231 -41. BJlSCH, J. and C. B]TSCH. 1982. Les htapes de la vitellogenesechez Thermobia domestica (Packard) (Thysanura : Lepismatida). Int. J. Insect Morphol. Embryol. I I: 197 - 212. BRIAN, M. V. and C. RIGt3¥. 1978. The trophic eggs of Myrmica rubra L. ]nsectes Sociaux 25: 89- 110. CA~-TANO, F. H. 1980. Ultra-estrutura dos EspermatOzoides de Atta capiguara e Atta sexdens rubropilosa (Formicidae). Naturalia (Sffo Paulo) 5: 105- 11. CASSID¥, J. D. and R. C. KING. 1972. Ovarian development in Habrobracon juglandis (Ashmead) (Hymenoptera : Braconidae). 1. The origin and differentiation of the oocyte - nurse cell complex. Biol. Bull. 143:483 - 505. CRUZ-LANDIM, C. 1978. Structural dynamics of oogenesis in Atta sexdens rubropilosa (Hymenoptera, Formicidae). Rev. BrasiL Biol. 38: 363- 81. CRUZ-LANDIM,C. and F. H. CAETANO.1981. The histochemistry and fine structure of the vitellarium in Atta (Formicidae, Myrmicinae). Rev. Brasil. Biol. 41:363-70. DE LOOE,A. 1983. The meroistic insect ovary as a miniature electrophoresis chamber. Comp. Biochem. Physiol. 74A 3 - 9. DE WILDE, J. and A. DE LOOF. 1973. Reproduction, pp. l l - 9 5 . In. M. ROCKSTEIN(ed.) The Physiology o f lnsecta, Vol. l, Academic Press, New York, London. EHRHARDT, H.-J. 1962. Ablage tibergroBer Eier durch Arbeiterinnen von Formica polyctena FOrster (Ins., Hym.) in Gegenwart yon K0niginnen. Naturwissenschaften 22:524 - 25 ENGELS, W. 1973. Das zeitliche und ratimliche Muster der Dottereinlagerung in die Oocyte yon Apis mellifica. Z. Zellforsch. 142: 409- 30.
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