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Follicular sheath (ovarian sheath) structure in virginoparae of the vetch aphid, Megoura viciae Buckton (Homoptera : Aphididae)
Int. J. lnsectMorphol. & Embryol., Vol. 18, No. 4, pp. 217-226, 1989 Printed in Great Britain
0020-7322/89 $3.00+ .00 ~) 1989 Pergamon Press plc
FOLLICULAR SHEATH (OVARIAN SHEATH) STRUCTURE IN VIRGINOPARAE OF THE VETCH APHID, M E G O U R A VICIAE BUCKTON (HOMOPTERA • APHIDIDAE)
C. N. BROUGH* a n d A. F. G. DIXON School of Biological Sciences, University of East Anglia, Norwich, NR4 7TJ, U.K. *Department of EnvironmentalBiology, University of Manchester, Manchester, M13 9PL, U.K.
(Accepted 19 April 1989)
Abstract--The anatomy of the follicular (ovarian) sheath surrounding the embryos in the virginoparae of the vetch aphid, Megoura viciae Buckton (Homoptera : Aphididae) is described. Enlarging oocytes released from the germaria are enveloped in a monolayer of cells derived from the prefollicular tissue, which is continuous with the sheath surrounding the germarium. Each embryo is compartmentalized. Initially, the sheath cells are cuboidal, but as division and elongation of the oocytes ensue, they become progressively stretched. Sheath cells surrounding large embryos show some variability in structure. In some areas, they are little more than a pair of membranes separated by a trace of cytoplasm. In other areas, they are thicker and have a structure that indicates a role in the uptake of materials. In these areas, the cells are often invaginated, with particulate and flocculent material present in the folds, and numerous organelles in the cytoplasm. Microvilli occur around some of the sheath cells and microtubule bundles are present in cells where the sheath is folded to form areas of contact between cells. A very thin acellular tunica propria is present to the exterior of the follicular sheath cells. Two acellular "membranes" present between the sheath cells and embryonic epidermal cells may have a role in the regulation of materials transfer. The structure of the epidermal cells of the embryos indicates role in the uptake of materials. Index descriptors (in addition to those in title): Embryos, epidermis, ovarioles,
parthenogenesis. INTRODUCTION APHIDS have t e l o t r o p h i c ovaries (Biining, 1985), whose f u n c t i o n a l a n a t o m y has b e e n the s u b j e c t of several studies. T h e recent investigations, which i n c l u d e u l t r a s t r u c t u r a l o b s e r v a t i o n s , c o n c e n t r a t e almost exclusively o n the structure of the p a r t h e n o g e n e t i c ovary a n d i g n o r e the process of e m b r y o g e n e s i s in aphids, in particular, the r e l a t i o n s h i p b e t w e e n the d e v e l o p i n g e m b r y o s a n d the s u r r o u n d i n g epithelial sheath cells. T h e only p r e v i o u s u l t r a s t r u c t u r a l study to e x a m i n e the follicular sheath is that of C o u c h m a n a n d King (1980). T h e r e has, h o w e v e r , b e e n c o n s i d e r a b l e work o n o t h e r groups of insects, i n c l u d i n g the H e m i p t e r a , o n the p r o d u c t i o n , growth, a n d m a t u r a t i o n of eggs (see, for e x a m p l e , review by Zissler a n d S a n d e r , 1982). T h e r e p r o d u c t i v e system of v i r g i n o p a r o u s aphids is p a r t i c u l a r l y i n t e r e s t i n g , as the ovarioles c o n t a i n o o g o n i a to fully f u n c t i o n a l lstinstar larvae. T h e process of r e p r o d u c t i o n is extensively modified w h e n c o m p a r e d with that o b s e r v e d in egg-producers. 217
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This p a p e r examines the ultrastructual anatomy of the follicular sheath that encloses the developing embryos in each ovariole, and describes the relationship between the developing embryos and the sheath cells.
MATERIALS AND METHODS Virginoparae of the vetch aphid, Megoura viciae were reared on seedlings of bean, Vicia faba (var. Aquadulce) in a glasshouse at approximately 20°C, and a 16-hr photoperiod. For transmission electron microscopy, aphids were taken at a moult, before cuticle sclerotization, and were fixed for 2 hr in 2.5% glutaraldehyde in 0.1M phosphate buffer containing 0.15M sucrose. To facilitate penetration of the fixative, the head, cauda and appendages were removed after partial fixation, and the aphids were split longitudinally. After washing in buffer, post-fixation was for 1.5 hr in 1% osmium tetroxide in 0.1M phosphate buffer. Dehydration was in a graded series of ethanol, and embedding was in London "White" medium-grade resin. Semi-thin sections were stained in toluidine blue. Ultra-thin sections, cut on an LKB III ultramicrotome, were stained in aqueous uranyl acetate followed by lead citrate (Reynolds, 1963). Sections were viewed with a JEOL 100CX transmission electron microscope. Material was prepared for scanning electron microscopy by fixing excised ovarioles in the fixatives given above. After dehydration in graded acetone, the material was critical-point dried, coated with gold-palladium and viewed with an ASID unit attached to a JEOL 100CX transmission electron microscope.
RESULTS The virginoparae of M. viciae have between 12 and 22 ovarioles. Each consists of a spherical to pear-shaped germarium attached to the body wall by a terminal filament. F r o m each g e r m a r i u m a vitellarium extends posteriorly. In adult aphids, this contains a linear series of up to 8 follicles, with one embryo in each, increasing in developmental status towards the oviduct. The germaria and ovarioles are divided into 2 groups (i.e. ovaries), which are bilaterally symmetrical. The g e r m a r i u m of each ovariole is surrounded by a single layer of epithelial sheath cells that are continuous with the block of prefollicular cells at the base of each g e r m a r i u m (Fig. 1). A . Follicular sheath The follicular sheath of each ovariole consists of a continuous monolayer of epithelial cells extending from the prefollicular region to the oviduct. As an enlarging oocyte is released from a germarium, it is surrounded by a single layer of cells derived from the prefollicular tissue (Figs 1; 6). These remain with the oocyte throughout subsequent development, until embryogenesis is complete. A small n u m b e r of cells follows each oocyte, such that each follicle is complete, and oocytes are compartmentalized (Figs 2; 3). The epithelial plug between successive oocytes persists throughout subsequent development, separating adjacent embryos within an ovariole. This junction may be elongated or twisted between large embryos (Figs 4; 5). The ultrastructure of sheath cells surrounding newly extruded oocytes, oocytes during cleavage, blastulae, and embryos during early development showed no ultrastructural features indicative of nutrient transfer (Fig. 6). Vitellogenesis is absent. During symbiont invasion, sheath cells are absent in the region where the dorsal body wall of the e m b r y o is open, and so there is direct access of maternal h a e m o l y m p h to the e m b r y o throughout this developmental period. The sheath cells that surround oocytes during early cleavage (Fig. 6) b e c o m e progressively stretched and thinner as embryonic development within the follicle proceeds (Fig. 7). A very thin acellular basement m e m b r a n e (tunica propria)
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FIG. 1. Semi-thin section through a germarium (G), which has released 2 oocytes (O). F = cells of the prefollicular tissue; S = follicular sheath cells. Scale bar = 20 i~m. FIG. 2. Semi-thin section of 2 embryos (E) in an ovariole of an adult. P = epithelial plug; S = follicular sheath. Scale bar = 50 p,m. FIG. 3. Scanning electron micrograph (SEM) of an internal view of junction between 2 embryos in an ovariole. P = epithelial plug. Scale bar = 25 Ixm. FIGS 4; 5. SEM of epithelial plug (P) formed from follicular sheath between 2 embryos (E). Scale bars = 40 ~m.
b o u n d s the e x t e r n a l surface of the sheath cells (Fig. 8). This b a s e m e n t m e m b r a n e also s u r r o u n d s the g e r m a r i u m , a n d is c o n t i n u o u s along the whole length of the ovariole. T h e sheath s u r r o u n d i n g large e m b r y o s is in places very thin, a p p e a r i n g stretched (Figs 7; 8), both along the sides of the e m b r y o s and in the interfollicular regions b e t w e e n n e i g h b o u r i n g e m b r y o s . T h e sheath exists in some areas as only a cytoplasmic trace b e t w e e n two cell m e m b r a n e s , which may be less t h a n the d i a m e t e r of o n e m i t o c h o n d r i o n (Figs 8; 9). Pores or gaps were not seen in the sheath cells, and its r u p t u r e in living p r e p a r a t i o n s causes a r e d u c t i o n in the size of the follicle, often with expulsion of the
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FIGS 6--8. Transmission electron micrographs. FIG. 6. Follicular sheath (S) surrounding a dividing oocyte (O). Scale bar = 15 lam. Fxc. 7. Follicular sheath cells (S) surrounding an embryo that is ovulating. Ep = embryonic epidermis; G = sheath cells surrounding a germarium of embryo. Scale bar = 15 lam. FIc. 8. Follicular sheath (S) surrounding embryos (E) in 2 adjacent ovarioles. D = dense particulate and flocculent material; M = mitochondrion; Tp = tunica propria; 1 and 2 = " m e m b r a n e s " between sheath cells and embryonic surface. Scale bar = 5 I~m.
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embryo, indicating the high tension in the sheath caused by stretching as embryos grow. There is thickening of sheath cells in some areas (Figs 7; 8). It was not clear if thickening is a result of differential cytological properties of these cells making them more resistant to stretching, or if they are simply not as stretched as cells in other areas. Thickening often occurs in the region of sheath cell nuclei, suggesting that this part of a cell does not stretch as readily as the cytoplasm. Areas of cellular thickening do not appear to correspond to any particular area of an embryo, although they are most common in the mid-lateral regions where they often reach 1.2-1.5 Ixm in thickness. In such areas, the sheath commonly forms a series of folds, which vary in abundance and complexity (Figs 7; 8). The larger cells that form the folds have a relatively high content of rough endoplasmic reticulum (RER), often with distended cisternae and numerous vacuoles (Fig. 12). Microtubule-rich areas occur in the apical region of some sheath cells, especially in areas where the sheath is folded, such that the tunica propria is folded back on itself or touches that of another cell (Fig. 10). Small clusters of microtubules are also found in other sheath cells, often in the cell apices (Fig. 11), although these are few in number and not widespread. The invaginations in the sheath formed by folding frequently contain dense, particulate and flocculent material. These are presumably of haemolymph origin and possibly have a histotrophic function (Fig. 12). This may represent engulfing and/or uptake of haemolymph materials by the sheath cells. Particulate materials are also present in the apices of some sheath cells adjacent to the folds (Fig. 12). Some small aggregations of microvilli occur in localized areas at the inner and outer surfaces of the sheath cells (Fig. 13), occasionally in small lacunae within the cells (Fig. 14). These do not correspond to any particular region of the embryos but may serve to increase the surface area of the sheath cells. The space between the follicular sheath and embryo surface contains 2 acellular "membranes". These appear to regulate the transfer of materials, in that a greater amount of particulate and flocculent material occurs at their outer surfaces (Figs 8; 13). It is unclear whether they are secreted by the embryonic epidermal cells or by the sheath cells. They appear shortly after symbiont invasion, and persist throughout subsequent embryonic development until the formation of the cuticle. The sequestration of nutrients by the embryos involves transfer across 3 acellular (tunica propria, and 2 inner "membranes") and 2 cellular (sheath cells and embryonic epidermis) barriers. The follicular sheath cells may have specialized regions for the modification and/or gross uptake of materials, and transfer of some solutes may occur by diffusion.
B. Epidermal cells of embryos Examination of post-katatrepsis embryos revealed that their epidermal cells (including those of the appendages) are involved in the uptake and transfer of materials from the space between the embryo and the external acellular "membranes". The epidermal cells form a layer 1-3 I~m thick, which in many areas has an extensive development of microvilli present on both the internal and external surfaces (Fig. 15). Microvilli are numerous in some areas, and occur in small clusters in others. Cells internal to the epidermis commonly have extensive microvillus areas, which infill intercellular spaces between these cells and the inner surface of the epidermis (Fig. 16). Microvilli presumably facilitate the sequestration of materials by the embryos. The embryonic epidermal cells have abundant organelles throughout embryogenesis
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FIGS 9-14. Transmission electron micrographs. FIG. 9. The follicular sheaths (S) surrounding 2 adjacent embryos. Ep = embryonic epidermis; M = mitochondrion; T = tracheole. Scale bar = 2 Ixm. FIG. 10. Clusters of microtubules (Mt) in apical region of follicular sheath cells that form a fold such that tunica propria (Tp) of adjacent cells meet. Scale bar = 0.2 v,m. FIG. 11. Microtubules (Mt) present in apical region of a follicular sheath cell (S). Tp = tunica propria. Scale bar = 0.5 wm. FIG. 12. A n area where follicular sheath cells are folded. D = dense particulate and flocculent material present in invagination; P = particulate material in the cell apices; Tp = tunica propria; V = vesicles. Scale bar = 1 wm. FIG. 13. Microvilli (Mv) present at inner and outer surfaces of follicular sheath cells (S). D = dense particulate and flocculent materials; Ep = embryonic epidermis; Tp = tunica propria. Scale bar = 2 p,m. Fro. 14. Microvilli (Mv) adjacent to a lacuna (L) in a follicular sheath cell. Ep = embryonic epidermis. Scale bar = 2 i~m.
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(Figs 15-18), including RER, Golgi complexes, and some small vacuoles. During the late stages of embryogenesis, the embryonic epidermal cells secrete the cuticle (Fig. 19). During this period, the abundance and distribution of microvilli decline, especially on the outer surfaces of the epidermal cells (Figs 16; 19), although the cells retain an extensive organelle content (Figs 16-18). Mature embryos have a complete covering of cuticle. The secretion of the cuticle is presumably under hormonal control, and it may terminate the sequestration of maternally derived nutrients, in a similar way to the termination of vitellogenesis during chorionogenesis in oviparous insects. After this, the final stages of development must be accomplished by the utilization of embryonic reserves, unless the unsclerotized cuticle allows diffusion of nutrients. Each ovariole terminates at a lateral oviduct. The 2 lateral oviducts are circular in cross-section and they lead into a median oviduct and vagina, which forms a dorsoventrally compressed slit when an embryo is not present. DISCUSSION Oocytes are surrounded by a monolayer of cells produced by the prefollicular tissue at the base of germaria. These investing cells form the follicular sheath. In virginoparae of aphids, the oocytes are invested by far fewer cells than the oocytes of the oviparous morph, or those of other oviparous insects. The stretching of the cells of the follicular sheath and relatively small size of the oocyte when released from the germarium requires few follicle cells per embryo, and consequently a reduced prefollicular tissue zone. During "previtellogenic" growth and early cleavage of oocytes prior to blastulation, no nutritive role could be attributed to the sheath cells on the basis of their ultrastructure. This is not surprising, as vitellogenesis is absent, and the nutritive cord is connected and presumably functional until blastulation, shortly before symbiont invasion (Brough and Dixon, in preparation). During symbiont invasion, the follicular sheath is disrupted, which presumably enables direct access of maternal haemolymph to embryos. These early developmental features negate any functional role of the follicular sheath cells relating to the uptake and transfer of nutrients to embryos. Consequently, the organelle content of the prefollicular tissue remains low throughout the morphological transition from columnar to cuboidal to squamous epithelium, which results from stretching during early development. Couchman and King (1980) found that the follicular (ovarian) sheath had little or no trophic role before the loss of the trophic cord in Brevicoryne brassicae, but found protein-like materials between the sheath and blastoderm. However, they described the sheath as lying exterior to the prefollicular tissue (Couchman and King, 1979). In Megoura, the prefollicular tissue differentiated to produce the sheath, which is bordered externally only by a very thin tunica propria and the sheath around early embryos was not seen to engulf materials. The follicular sheath of aphids (= ovarian and ovariole sheaths) is very different from that described for other groups of insects having telotrophic ovaries where it consists of an external and internal sheath, the former containing muscles and tracheoles (Bonhag and Arnold, 1961; Huebner, 1984). In contrast, in aphids, it is made up of a thin, unicellular layer, bordered by a very thin tunica propria, which constitutes the basement membrane. The tunica propria is continuous, surrounding the germarium and the ovariole. It appears that even though the follicular sheath cells surrounding each embryo are stretched, the tunica propria is of similar thickness throughout. During embryonic differentiation and development, the protein demands of embryos
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FIGs 15-19. Transmission electron micrographs. FIG. 15. Microvilli (Mv) at the inner (I) and outer (O) surfaces of the embryonic epidermal cells. ER = swollen tubules of the endoplasmic reticulum; N = nucleus; M = mitochondria. Scale bar = 1.5 p,m. Fro. 16. Embryonic epidermal cells during the initial stages of cuticle (C) formation. Mv = microvilli internal to the epidermal cells; S = follicular sheath. Scale bar = 2 ~tm. FIG. 17. An embryonic epidermal cell, containing a stack of endoplasmic reticulum, (ER). N = nucleus. Scale bar = 1 i~m. FIG. 18. Embryonic epidermal cell during cuticle formation. ER = swollen tubules of endoplasmic reticulum; N = nucleus; V = vesicles. Scale bar = 0.5 p~m. FIG. 19. Outer surface of the embryonic epidermis (Ep) after cuticle (C) formation, with absence of microvilli. Scale bar = 0.5 ~,m. g r e a t l y i n c r e a s e . T h e y r e a c h t h e 1st i n s t a r b e f o r e b i r t h , a n d also b e g i n r e p r o d u c t i o n shortly after katatrepsis. Hence large, rapidly developing embryos must have substantial nutrient requirements. Ultrastructural observations presented provide evidence of how t h e m a t e r i a l s a r e s u p p l i e d . T h e s h e a t h s u r r o u n d i n g t h e l a r g e e m b r y o s is v a r i a b l e in s t r u c t u r e . I n a r e a s w h e r e it is v e r y t h i n , c o n s i s t i n g o f a c y t o p l a s m i c t r a c e b e t w e e n a p a i r of membranes, with very few organelles, there was no structural evidence of atrophic r o l e . It is p o s s i b l e t h a t t h e r e is d i f f u s i o n a c r o s s t h e s h e a t h a n d a s s o c i a t e d m e m b r a n e s in
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these areas, possibly facilitated by small areas of microvilli. However, in areas where cellular thickening occurs, the greater abundance of organeUes (e.g. RER) indicates a trophic role. The occurrence of large invaginations, often where cellular thickening and folds occur, may reflect engulfing of haemolymph materials. Couchman and King (1980) similarly reported engulfing of materials by sheath cells in B. brassicae. In Megoura, the invaginations are filled with particulate and flocculent material, similar to that occurring between the sheath and embryos in other areas. If engulfing does occur, it could deliver the large quantities of materials a rapidly developing embryo requires. There was no ultrastructural evidence of large amounts of active internalization by the sheath cells, or of substantial release at their internal surfaces. The materials seen in the sheath folds possibly have a histotrophic function. The microtubular bundles present in sheath cell apices may provide additional rigidity to the cytoskeleton, for the formation and/or maintenance of folds in the sheath. The convolutions that occur in areas where the sheath cells are cytologically specialized may be a means of increasing the cell surface area available for nutrient uptake. Accumulation of materials to the exterior (on the maternal side) of the 2 acellular "membranes", between the sheath and embryos, suggests that they may have a function in the regulation of uptake. The presence of 4 distinct layers between embryo and maternal haemolymph presents a considerable barrier to nutrient flow, and consequently there must be an efficient transport mechanism. Uptake by the embryonic epidermal cells prior to cuticle formation is indicated by the presence of microvilli on their external surfaces and numerous organelles within the cytoplasm. The demands of rapidly developing embryos must be great, and they will require an array of materials, which could be taken up in a readily assimilable form (e.g. monosaccharides, amino acids, and fatty acids). These materials could be selectively sequestered and concentrated by sheath or epidermal cells. It is possible that different methods of uptake exist for various materials. Active uptake of materials by the epidermal cells of embryos indicates how such demands may be met, and also means that materials have only a short distance to be transferred within an embryo. However, the biochemical form of the sequestered materials, and their pathways to and within embryos need to be studied. This paper gives an appreciation of how uptake mechanisms may operate. At the onset of cuticle secretion, the epidermal cells appear to lose their ability for nutrient uptake, and the presence of the cuticle prevents further uptake unless low molecular weight metabolites can diffuse across it. Hence, during the final stages of embryogenesis it is likely that the embryo utilizes its stored reserves for tissue maintenance, respiration, the completion of development, and reproduction. The absence of muscles associated with the follicular sheath discounts the possibility of ovariole contraction moving embryos distally. Embryos are compartmentalized within individual follicles, and growth and development of the mother are accompanied by elongation of the ovariole, production of oocytes, and continued development of existing oocytes and embryos. Degeneration of sheath cells around the mature, terminal embryos was not seen. Some early workers suggested that these sheath cells are expelled with the embryo (as a "pseudochorion"), or discarded in the posterior end of the genital duct at birth (see Hagan, 1951). Hille Ris Lambers (1950) also noted that larvae from several aphid species were born in sacs, from which they soon hatched. In Megoura, the sheath cells surrounding terminal embryos are extremely thin, and embryos in the oviducts are not surrounded by sheath cells. The cells of each ovariole are continuous with those of a
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lateral oviduct, a n d are n o t a distinct tissue, simply enclosing the e m b r y o s . S o m e diversity m a y t h e r e f o r e exist b e t w e e n aphids in the fate of the sheath cells. T h e folds in the s h e a t h a r o u n d large, m a t u r i n g e m b r y o s m a y indicate the m e c h a n i s m by which e m b r y o s are t r a n s f e r r e d to the oviducts, b e i n g the cause or effect of e m b r y o s b e i n g s q u e e z e d i n t o the oviducts. T h e passage of a fully d e v e l o p e d e m b r y o into the lateral oviduct, a n d t h e n into the m e d i a n o v i d u c t , followed by its b i r t h will e n a b l e the next e m b r y o in the ovariole to m o v e to the lateral oviduct. H o w e v e r , for a fuller u n d e r s t a n d i n g , the o v a r i o l e / o v i d u c t j u n c t i o n requires further examination. Acknowledgement--This work was supported by a S.E.R.C. Studentship (C.N.B.).
REFERENCES
BONHAG,P. F. and W. J. ARNOLD. 1961. Histology, histochemistry and tracheation of the ovariole sheaths in the American cockroach Periplaneta americana (L.). J. Morphol. 108: 107-29. BONI~t;, J. 1985. Morphology, ultrastructure and germ cell cluster formation in ovarioles of aphids. J. Morphol. 186: 209-21. COOCrtMAN, J. R. and P. E. KINt;. 1979. Germarial structure and oogenesis in Brevicoryne brassicae (L.) (Hemiptera : Aphididae). Int. J. Insect Morphol. Embryol. g: 1-10. COUC,MAN,J. R. and P. E. KIN6. 1980. Ovariole sheath structure and its relationship with developing embryos in a parthenogenetic viviparous aphid. Acta Zool. (Stockh.) 61: 147-55. HAt;AN,H. R. (editor) 1951. Embryology of the Viviparous Insects. Ronaid Press, New York. HILLE RIS LAMBERS,D. 1950. An apparently unrecorded mode of reproduction in aphididae. Proc. 8th Int. Congr. Entomol. 235. HUEBNER,E. 1984. The ultrastructure and development of the telotrophic ovary, pp. 3-48. In R. C. KINCand H. AKAI(eds) Insect Ultrastructure, Vol. 2. Plenum Press, New York. REYNOLDS,E. S. 1963. The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J. Cell Biol. 17: 208-12. ZISSLER,O. and K. SANDER. 1982. The cytoplasmic architecture of the insect egg cell, pp. 189-221. In R. C. KINGand H. AKAI.(eds) Insect Ultrastructure, Vol. 1. Plenum Press, New York.
0020-7322/89 $3.00+ .00 ~) 1989 Pergamon Press plc
FOLLICULAR SHEATH (OVARIAN SHEATH) STRUCTURE IN VIRGINOPARAE OF THE VETCH APHID, M E G O U R A VICIAE BUCKTON (HOMOPTERA • APHIDIDAE)
C. N. BROUGH* a n d A. F. G. DIXON School of Biological Sciences, University of East Anglia, Norwich, NR4 7TJ, U.K. *Department of EnvironmentalBiology, University of Manchester, Manchester, M13 9PL, U.K.
(Accepted 19 April 1989)
Abstract--The anatomy of the follicular (ovarian) sheath surrounding the embryos in the virginoparae of the vetch aphid, Megoura viciae Buckton (Homoptera : Aphididae) is described. Enlarging oocytes released from the germaria are enveloped in a monolayer of cells derived from the prefollicular tissue, which is continuous with the sheath surrounding the germarium. Each embryo is compartmentalized. Initially, the sheath cells are cuboidal, but as division and elongation of the oocytes ensue, they become progressively stretched. Sheath cells surrounding large embryos show some variability in structure. In some areas, they are little more than a pair of membranes separated by a trace of cytoplasm. In other areas, they are thicker and have a structure that indicates a role in the uptake of materials. In these areas, the cells are often invaginated, with particulate and flocculent material present in the folds, and numerous organelles in the cytoplasm. Microvilli occur around some of the sheath cells and microtubule bundles are present in cells where the sheath is folded to form areas of contact between cells. A very thin acellular tunica propria is present to the exterior of the follicular sheath cells. Two acellular "membranes" present between the sheath cells and embryonic epidermal cells may have a role in the regulation of materials transfer. The structure of the epidermal cells of the embryos indicates role in the uptake of materials. Index descriptors (in addition to those in title): Embryos, epidermis, ovarioles,
parthenogenesis. INTRODUCTION APHIDS have t e l o t r o p h i c ovaries (Biining, 1985), whose f u n c t i o n a l a n a t o m y has b e e n the s u b j e c t of several studies. T h e recent investigations, which i n c l u d e u l t r a s t r u c t u r a l o b s e r v a t i o n s , c o n c e n t r a t e almost exclusively o n the structure of the p a r t h e n o g e n e t i c ovary a n d i g n o r e the process of e m b r y o g e n e s i s in aphids, in particular, the r e l a t i o n s h i p b e t w e e n the d e v e l o p i n g e m b r y o s a n d the s u r r o u n d i n g epithelial sheath cells. T h e only p r e v i o u s u l t r a s t r u c t u r a l study to e x a m i n e the follicular sheath is that of C o u c h m a n a n d King (1980). T h e r e has, h o w e v e r , b e e n c o n s i d e r a b l e work o n o t h e r groups of insects, i n c l u d i n g the H e m i p t e r a , o n the p r o d u c t i o n , growth, a n d m a t u r a t i o n of eggs (see, for e x a m p l e , review by Zissler a n d S a n d e r , 1982). T h e r e p r o d u c t i v e system of v i r g i n o p a r o u s aphids is p a r t i c u l a r l y i n t e r e s t i n g , as the ovarioles c o n t a i n o o g o n i a to fully f u n c t i o n a l lstinstar larvae. T h e process of r e p r o d u c t i o n is extensively modified w h e n c o m p a r e d with that o b s e r v e d in egg-producers. 217
218
C.N. BROUGHand A. F. G. DIxoN
This p a p e r examines the ultrastructual anatomy of the follicular sheath that encloses the developing embryos in each ovariole, and describes the relationship between the developing embryos and the sheath cells.
MATERIALS AND METHODS Virginoparae of the vetch aphid, Megoura viciae were reared on seedlings of bean, Vicia faba (var. Aquadulce) in a glasshouse at approximately 20°C, and a 16-hr photoperiod. For transmission electron microscopy, aphids were taken at a moult, before cuticle sclerotization, and were fixed for 2 hr in 2.5% glutaraldehyde in 0.1M phosphate buffer containing 0.15M sucrose. To facilitate penetration of the fixative, the head, cauda and appendages were removed after partial fixation, and the aphids were split longitudinally. After washing in buffer, post-fixation was for 1.5 hr in 1% osmium tetroxide in 0.1M phosphate buffer. Dehydration was in a graded series of ethanol, and embedding was in London "White" medium-grade resin. Semi-thin sections were stained in toluidine blue. Ultra-thin sections, cut on an LKB III ultramicrotome, were stained in aqueous uranyl acetate followed by lead citrate (Reynolds, 1963). Sections were viewed with a JEOL 100CX transmission electron microscope. Material was prepared for scanning electron microscopy by fixing excised ovarioles in the fixatives given above. After dehydration in graded acetone, the material was critical-point dried, coated with gold-palladium and viewed with an ASID unit attached to a JEOL 100CX transmission electron microscope.
RESULTS The virginoparae of M. viciae have between 12 and 22 ovarioles. Each consists of a spherical to pear-shaped germarium attached to the body wall by a terminal filament. F r o m each g e r m a r i u m a vitellarium extends posteriorly. In adult aphids, this contains a linear series of up to 8 follicles, with one embryo in each, increasing in developmental status towards the oviduct. The germaria and ovarioles are divided into 2 groups (i.e. ovaries), which are bilaterally symmetrical. The g e r m a r i u m of each ovariole is surrounded by a single layer of epithelial sheath cells that are continuous with the block of prefollicular cells at the base of each g e r m a r i u m (Fig. 1). A . Follicular sheath The follicular sheath of each ovariole consists of a continuous monolayer of epithelial cells extending from the prefollicular region to the oviduct. As an enlarging oocyte is released from a germarium, it is surrounded by a single layer of cells derived from the prefollicular tissue (Figs 1; 6). These remain with the oocyte throughout subsequent development, until embryogenesis is complete. A small n u m b e r of cells follows each oocyte, such that each follicle is complete, and oocytes are compartmentalized (Figs 2; 3). The epithelial plug between successive oocytes persists throughout subsequent development, separating adjacent embryos within an ovariole. This junction may be elongated or twisted between large embryos (Figs 4; 5). The ultrastructure of sheath cells surrounding newly extruded oocytes, oocytes during cleavage, blastulae, and embryos during early development showed no ultrastructural features indicative of nutrient transfer (Fig. 6). Vitellogenesis is absent. During symbiont invasion, sheath cells are absent in the region where the dorsal body wall of the e m b r y o is open, and so there is direct access of maternal h a e m o l y m p h to the e m b r y o throughout this developmental period. The sheath cells that surround oocytes during early cleavage (Fig. 6) b e c o m e progressively stretched and thinner as embryonic development within the follicle proceeds (Fig. 7). A very thin acellular basement m e m b r a n e (tunica propria)
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FIG. 1. Semi-thin section through a germarium (G), which has released 2 oocytes (O). F = cells of the prefollicular tissue; S = follicular sheath cells. Scale bar = 20 i~m. FIG. 2. Semi-thin section of 2 embryos (E) in an ovariole of an adult. P = epithelial plug; S = follicular sheath. Scale bar = 50 p,m. FIG. 3. Scanning electron micrograph (SEM) of an internal view of junction between 2 embryos in an ovariole. P = epithelial plug. Scale bar = 25 Ixm. FIGS 4; 5. SEM of epithelial plug (P) formed from follicular sheath between 2 embryos (E). Scale bars = 40 ~m.
b o u n d s the e x t e r n a l surface of the sheath cells (Fig. 8). This b a s e m e n t m e m b r a n e also s u r r o u n d s the g e r m a r i u m , a n d is c o n t i n u o u s along the whole length of the ovariole. T h e sheath s u r r o u n d i n g large e m b r y o s is in places very thin, a p p e a r i n g stretched (Figs 7; 8), both along the sides of the e m b r y o s and in the interfollicular regions b e t w e e n n e i g h b o u r i n g e m b r y o s . T h e sheath exists in some areas as only a cytoplasmic trace b e t w e e n two cell m e m b r a n e s , which may be less t h a n the d i a m e t e r of o n e m i t o c h o n d r i o n (Figs 8; 9). Pores or gaps were not seen in the sheath cells, and its r u p t u r e in living p r e p a r a t i o n s causes a r e d u c t i o n in the size of the follicle, often with expulsion of the
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FIGS 6--8. Transmission electron micrographs. FIG. 6. Follicular sheath (S) surrounding a dividing oocyte (O). Scale bar = 15 lam. Fxc. 7. Follicular sheath cells (S) surrounding an embryo that is ovulating. Ep = embryonic epidermis; G = sheath cells surrounding a germarium of embryo. Scale bar = 15 lam. FIc. 8. Follicular sheath (S) surrounding embryos (E) in 2 adjacent ovarioles. D = dense particulate and flocculent material; M = mitochondrion; Tp = tunica propria; 1 and 2 = " m e m b r a n e s " between sheath cells and embryonic surface. Scale bar = 5 I~m.
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embryo, indicating the high tension in the sheath caused by stretching as embryos grow. There is thickening of sheath cells in some areas (Figs 7; 8). It was not clear if thickening is a result of differential cytological properties of these cells making them more resistant to stretching, or if they are simply not as stretched as cells in other areas. Thickening often occurs in the region of sheath cell nuclei, suggesting that this part of a cell does not stretch as readily as the cytoplasm. Areas of cellular thickening do not appear to correspond to any particular area of an embryo, although they are most common in the mid-lateral regions where they often reach 1.2-1.5 Ixm in thickness. In such areas, the sheath commonly forms a series of folds, which vary in abundance and complexity (Figs 7; 8). The larger cells that form the folds have a relatively high content of rough endoplasmic reticulum (RER), often with distended cisternae and numerous vacuoles (Fig. 12). Microtubule-rich areas occur in the apical region of some sheath cells, especially in areas where the sheath is folded, such that the tunica propria is folded back on itself or touches that of another cell (Fig. 10). Small clusters of microtubules are also found in other sheath cells, often in the cell apices (Fig. 11), although these are few in number and not widespread. The invaginations in the sheath formed by folding frequently contain dense, particulate and flocculent material. These are presumably of haemolymph origin and possibly have a histotrophic function (Fig. 12). This may represent engulfing and/or uptake of haemolymph materials by the sheath cells. Particulate materials are also present in the apices of some sheath cells adjacent to the folds (Fig. 12). Some small aggregations of microvilli occur in localized areas at the inner and outer surfaces of the sheath cells (Fig. 13), occasionally in small lacunae within the cells (Fig. 14). These do not correspond to any particular region of the embryos but may serve to increase the surface area of the sheath cells. The space between the follicular sheath and embryo surface contains 2 acellular "membranes". These appear to regulate the transfer of materials, in that a greater amount of particulate and flocculent material occurs at their outer surfaces (Figs 8; 13). It is unclear whether they are secreted by the embryonic epidermal cells or by the sheath cells. They appear shortly after symbiont invasion, and persist throughout subsequent embryonic development until the formation of the cuticle. The sequestration of nutrients by the embryos involves transfer across 3 acellular (tunica propria, and 2 inner "membranes") and 2 cellular (sheath cells and embryonic epidermis) barriers. The follicular sheath cells may have specialized regions for the modification and/or gross uptake of materials, and transfer of some solutes may occur by diffusion.
B. Epidermal cells of embryos Examination of post-katatrepsis embryos revealed that their epidermal cells (including those of the appendages) are involved in the uptake and transfer of materials from the space between the embryo and the external acellular "membranes". The epidermal cells form a layer 1-3 I~m thick, which in many areas has an extensive development of microvilli present on both the internal and external surfaces (Fig. 15). Microvilli are numerous in some areas, and occur in small clusters in others. Cells internal to the epidermis commonly have extensive microvillus areas, which infill intercellular spaces between these cells and the inner surface of the epidermis (Fig. 16). Microvilli presumably facilitate the sequestration of materials by the embryos. The embryonic epidermal cells have abundant organelles throughout embryogenesis
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FIGS 9-14. Transmission electron micrographs. FIG. 9. The follicular sheaths (S) surrounding 2 adjacent embryos. Ep = embryonic epidermis; M = mitochondrion; T = tracheole. Scale bar = 2 Ixm. FIG. 10. Clusters of microtubules (Mt) in apical region of follicular sheath cells that form a fold such that tunica propria (Tp) of adjacent cells meet. Scale bar = 0.2 v,m. FIG. 11. Microtubules (Mt) present in apical region of a follicular sheath cell (S). Tp = tunica propria. Scale bar = 0.5 wm. FIG. 12. A n area where follicular sheath cells are folded. D = dense particulate and flocculent material present in invagination; P = particulate material in the cell apices; Tp = tunica propria; V = vesicles. Scale bar = 1 wm. FIG. 13. Microvilli (Mv) present at inner and outer surfaces of follicular sheath cells (S). D = dense particulate and flocculent materials; Ep = embryonic epidermis; Tp = tunica propria. Scale bar = 2 p,m. Fro. 14. Microvilli (Mv) adjacent to a lacuna (L) in a follicular sheath cell. Ep = embryonic epidermis. Scale bar = 2 i~m.
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(Figs 15-18), including RER, Golgi complexes, and some small vacuoles. During the late stages of embryogenesis, the embryonic epidermal cells secrete the cuticle (Fig. 19). During this period, the abundance and distribution of microvilli decline, especially on the outer surfaces of the epidermal cells (Figs 16; 19), although the cells retain an extensive organelle content (Figs 16-18). Mature embryos have a complete covering of cuticle. The secretion of the cuticle is presumably under hormonal control, and it may terminate the sequestration of maternally derived nutrients, in a similar way to the termination of vitellogenesis during chorionogenesis in oviparous insects. After this, the final stages of development must be accomplished by the utilization of embryonic reserves, unless the unsclerotized cuticle allows diffusion of nutrients. Each ovariole terminates at a lateral oviduct. The 2 lateral oviducts are circular in cross-section and they lead into a median oviduct and vagina, which forms a dorsoventrally compressed slit when an embryo is not present. DISCUSSION Oocytes are surrounded by a monolayer of cells produced by the prefollicular tissue at the base of germaria. These investing cells form the follicular sheath. In virginoparae of aphids, the oocytes are invested by far fewer cells than the oocytes of the oviparous morph, or those of other oviparous insects. The stretching of the cells of the follicular sheath and relatively small size of the oocyte when released from the germarium requires few follicle cells per embryo, and consequently a reduced prefollicular tissue zone. During "previtellogenic" growth and early cleavage of oocytes prior to blastulation, no nutritive role could be attributed to the sheath cells on the basis of their ultrastructure. This is not surprising, as vitellogenesis is absent, and the nutritive cord is connected and presumably functional until blastulation, shortly before symbiont invasion (Brough and Dixon, in preparation). During symbiont invasion, the follicular sheath is disrupted, which presumably enables direct access of maternal haemolymph to embryos. These early developmental features negate any functional role of the follicular sheath cells relating to the uptake and transfer of nutrients to embryos. Consequently, the organelle content of the prefollicular tissue remains low throughout the morphological transition from columnar to cuboidal to squamous epithelium, which results from stretching during early development. Couchman and King (1980) found that the follicular (ovarian) sheath had little or no trophic role before the loss of the trophic cord in Brevicoryne brassicae, but found protein-like materials between the sheath and blastoderm. However, they described the sheath as lying exterior to the prefollicular tissue (Couchman and King, 1979). In Megoura, the prefollicular tissue differentiated to produce the sheath, which is bordered externally only by a very thin tunica propria and the sheath around early embryos was not seen to engulf materials. The follicular sheath of aphids (= ovarian and ovariole sheaths) is very different from that described for other groups of insects having telotrophic ovaries where it consists of an external and internal sheath, the former containing muscles and tracheoles (Bonhag and Arnold, 1961; Huebner, 1984). In contrast, in aphids, it is made up of a thin, unicellular layer, bordered by a very thin tunica propria, which constitutes the basement membrane. The tunica propria is continuous, surrounding the germarium and the ovariole. It appears that even though the follicular sheath cells surrounding each embryo are stretched, the tunica propria is of similar thickness throughout. During embryonic differentiation and development, the protein demands of embryos
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FIGs 15-19. Transmission electron micrographs. FIG. 15. Microvilli (Mv) at the inner (I) and outer (O) surfaces of the embryonic epidermal cells. ER = swollen tubules of the endoplasmic reticulum; N = nucleus; M = mitochondria. Scale bar = 1.5 p,m. Fro. 16. Embryonic epidermal cells during the initial stages of cuticle (C) formation. Mv = microvilli internal to the epidermal cells; S = follicular sheath. Scale bar = 2 ~tm. FIG. 17. An embryonic epidermal cell, containing a stack of endoplasmic reticulum, (ER). N = nucleus. Scale bar = 1 i~m. FIG. 18. Embryonic epidermal cell during cuticle formation. ER = swollen tubules of endoplasmic reticulum; N = nucleus; V = vesicles. Scale bar = 0.5 p~m. FIG. 19. Outer surface of the embryonic epidermis (Ep) after cuticle (C) formation, with absence of microvilli. Scale bar = 0.5 ~,m. g r e a t l y i n c r e a s e . T h e y r e a c h t h e 1st i n s t a r b e f o r e b i r t h , a n d also b e g i n r e p r o d u c t i o n shortly after katatrepsis. Hence large, rapidly developing embryos must have substantial nutrient requirements. Ultrastructural observations presented provide evidence of how t h e m a t e r i a l s a r e s u p p l i e d . T h e s h e a t h s u r r o u n d i n g t h e l a r g e e m b r y o s is v a r i a b l e in s t r u c t u r e . I n a r e a s w h e r e it is v e r y t h i n , c o n s i s t i n g o f a c y t o p l a s m i c t r a c e b e t w e e n a p a i r of membranes, with very few organelles, there was no structural evidence of atrophic r o l e . It is p o s s i b l e t h a t t h e r e is d i f f u s i o n a c r o s s t h e s h e a t h a n d a s s o c i a t e d m e m b r a n e s in
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these areas, possibly facilitated by small areas of microvilli. However, in areas where cellular thickening occurs, the greater abundance of organeUes (e.g. RER) indicates a trophic role. The occurrence of large invaginations, often where cellular thickening and folds occur, may reflect engulfing of haemolymph materials. Couchman and King (1980) similarly reported engulfing of materials by sheath cells in B. brassicae. In Megoura, the invaginations are filled with particulate and flocculent material, similar to that occurring between the sheath and embryos in other areas. If engulfing does occur, it could deliver the large quantities of materials a rapidly developing embryo requires. There was no ultrastructural evidence of large amounts of active internalization by the sheath cells, or of substantial release at their internal surfaces. The materials seen in the sheath folds possibly have a histotrophic function. The microtubular bundles present in sheath cell apices may provide additional rigidity to the cytoskeleton, for the formation and/or maintenance of folds in the sheath. The convolutions that occur in areas where the sheath cells are cytologically specialized may be a means of increasing the cell surface area available for nutrient uptake. Accumulation of materials to the exterior (on the maternal side) of the 2 acellular "membranes", between the sheath and embryos, suggests that they may have a function in the regulation of uptake. The presence of 4 distinct layers between embryo and maternal haemolymph presents a considerable barrier to nutrient flow, and consequently there must be an efficient transport mechanism. Uptake by the embryonic epidermal cells prior to cuticle formation is indicated by the presence of microvilli on their external surfaces and numerous organelles within the cytoplasm. The demands of rapidly developing embryos must be great, and they will require an array of materials, which could be taken up in a readily assimilable form (e.g. monosaccharides, amino acids, and fatty acids). These materials could be selectively sequestered and concentrated by sheath or epidermal cells. It is possible that different methods of uptake exist for various materials. Active uptake of materials by the epidermal cells of embryos indicates how such demands may be met, and also means that materials have only a short distance to be transferred within an embryo. However, the biochemical form of the sequestered materials, and their pathways to and within embryos need to be studied. This paper gives an appreciation of how uptake mechanisms may operate. At the onset of cuticle secretion, the epidermal cells appear to lose their ability for nutrient uptake, and the presence of the cuticle prevents further uptake unless low molecular weight metabolites can diffuse across it. Hence, during the final stages of embryogenesis it is likely that the embryo utilizes its stored reserves for tissue maintenance, respiration, the completion of development, and reproduction. The absence of muscles associated with the follicular sheath discounts the possibility of ovariole contraction moving embryos distally. Embryos are compartmentalized within individual follicles, and growth and development of the mother are accompanied by elongation of the ovariole, production of oocytes, and continued development of existing oocytes and embryos. Degeneration of sheath cells around the mature, terminal embryos was not seen. Some early workers suggested that these sheath cells are expelled with the embryo (as a "pseudochorion"), or discarded in the posterior end of the genital duct at birth (see Hagan, 1951). Hille Ris Lambers (1950) also noted that larvae from several aphid species were born in sacs, from which they soon hatched. In Megoura, the sheath cells surrounding terminal embryos are extremely thin, and embryos in the oviducts are not surrounded by sheath cells. The cells of each ovariole are continuous with those of a
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lateral oviduct, a n d are n o t a distinct tissue, simply enclosing the e m b r y o s . S o m e diversity m a y t h e r e f o r e exist b e t w e e n aphids in the fate of the sheath cells. T h e folds in the s h e a t h a r o u n d large, m a t u r i n g e m b r y o s m a y indicate the m e c h a n i s m by which e m b r y o s are t r a n s f e r r e d to the oviducts, b e i n g the cause or effect of e m b r y o s b e i n g s q u e e z e d i n t o the oviducts. T h e passage of a fully d e v e l o p e d e m b r y o into the lateral oviduct, a n d t h e n into the m e d i a n o v i d u c t , followed by its b i r t h will e n a b l e the next e m b r y o in the ovariole to m o v e to the lateral oviduct. H o w e v e r , for a fuller u n d e r s t a n d i n g , the o v a r i o l e / o v i d u c t j u n c t i o n requires further examination. Acknowledgement--This work was supported by a S.E.R.C. Studentship (C.N.B.).
REFERENCES
BONHAG,P. F. and W. J. ARNOLD. 1961. Histology, histochemistry and tracheation of the ovariole sheaths in the American cockroach Periplaneta americana (L.). J. Morphol. 108: 107-29. BONI~t;, J. 1985. Morphology, ultrastructure and germ cell cluster formation in ovarioles of aphids. J. Morphol. 186: 209-21. COOCrtMAN, J. R. and P. E. KINt;. 1979. Germarial structure and oogenesis in Brevicoryne brassicae (L.) (Hemiptera : Aphididae). Int. J. Insect Morphol. Embryol. g: 1-10. COUC,MAN,J. R. and P. E. KIN6. 1980. Ovariole sheath structure and its relationship with developing embryos in a parthenogenetic viviparous aphid. Acta Zool. (Stockh.) 61: 147-55. HAt;AN,H. R. (editor) 1951. Embryology of the Viviparous Insects. Ronaid Press, New York. HILLE RIS LAMBERS,D. 1950. An apparently unrecorded mode of reproduction in aphididae. Proc. 8th Int. Congr. Entomol. 235. HUEBNER,E. 1984. The ultrastructure and development of the telotrophic ovary, pp. 3-48. In R. C. KINCand H. AKAI(eds) Insect Ultrastructure, Vol. 2. Plenum Press, New York. REYNOLDS,E. S. 1963. The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J. Cell Biol. 17: 208-12. ZISSLER,O. and K. SANDER. 1982. The cytoplasmic architecture of the insect egg cell, pp. 189-221. In R. C. KINGand H. AKAI.(eds) Insect Ultrastructure, Vol. 1. Plenum Press, New York.