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Sperm maturation in the male and female genital tracts of Anagasta kühniella (Lepidoptera: Pyralididae)
Int. J. Insect Morphol. & EmbryoL 1 (1): 11-19. 1971. P e r g a m o n Press. Printed in Great Britain.
SPERM MATURATION IN THE MALE A N D FEMALE GENITAL TRACTS OF ANAGASTA KUHNIELLA
(LEPIDOPTERA: PYRALIDIDAE)* JOHN G. RIEMANN a n d BARBARA J. THORSON Metabolism and Radiation Research Laboratory, Agricultural Research Service, U.S. Dept. of Agriculture, Fargo, North Dakota 58102, U.S.A.
(Accepted 7 May 1971) Abstract--In the testes of the Mediterranean flour moth, Anagasta (-- Ephestia) kiihniella (Zeller), a thick sheath forms around the plasma membrane, replacing a series of laminate proiections, the radial mantle; but another projection, the satellite body, is retained as an appendage of the sheath. This sheath quickly differentiates into an outer part made up o f a series of rings 150 A_wide, covered by a thin sleeve, and interrupted on one side by a narrow, dense plate to which the satellite body is attached, and an inner part made up of a mass of lamellae lacking definite orientation. Once the sperm are transferred to the female tract in spermatophores, marked changes occur in the sheath; the satellite body disappears, dense material appears between the inner margins of the radial discs, and the inner sheath differentiates into two distinct portions, both of which have a paracrystalline structure. Once the females deposit their eggs, most of the remaining sperm degenerate and leave empty sheaths. Evidence was found of a normal separation of sheaths and core components within the spermathecal duct, perhaps in preparation of the sperm for fertilization. During this separation, the sheath splits along the line of the dense plate. The sheath of the apyrene sperm was similar to the outer sheath of the eupyrene sperm. Index descriptors (in addition to those in title): Anagasta (=Ephestia) kiihniella, sperm ultrastructure, eupyrene apyrene sheath maturation.
INTRODUCTION TESTICULAR sperm of various lepidopterans have been studied at the ultrastructural level. A n d r 6 (1959, 1961, 1962) gave detailed descriptions o f the axial filament a n d mitochondrial derivatives of Macroglossum a n d Pieris respectively. He also described for the first time the series of appendages a r o u n d the plasma m e m b r a n e that have come to be recognized as characteristic of lepidopteran sperm n e a r i n g maturity in the testes. Y a s u z u m i a n d O u r a (1964, 1964b, 1965a, 1965b) made a detailed study of spermiogenesis in Bombyx mori L. with particular emphasis o n development of structures they called the t u b u l a r structure a n d the clear b a n d . Early stages of spermiogenesis in Bombyx were also studied by F r i e d l a n d e r a n d W a h r m a n (1970). Observations o n testicular sperm of various other lepidopterans were made by Phillips (1970), R i e m a n n (1970), Smith (1968), a n d Zylberberg (1963). Only a few observations have been made of lepidopteran sperm that have been released from the testes. Phillips (1970) showed that sperm in the ejaculatory duct of Desmiafuneralis *Supported in part by U.S. Atomic Energy Commission Contract AT(49-7)3028. 11
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JOHN G. RIEMANN and BARBARA J. THORSON
(Hfibner) were enclosed by a rather thick fibrous sleeve (sheath). Riemann (1970) found that eupyrene (nucleate) sperm of Trichoplusia ni (Hfibner) went through a rather elaborate metamorphosis during passage through the male and female tracts. The changes were most evident in the sheath, which formed at about the time the sperm left the testes. Anagasta eupyrene sperm go through a metamorphosis basically similar to those of Trichoplusia ni (Riemann, 1970), but they have a considerably more complex structure and more details have been established about their metamorphosis. In this paper, we briefly describe the appearance and metamorphosis of Anagasta sperm as they occur in the testes and post-testicular organs. A more detailed description (with emphasis on the sheath) is given of the eupyrene sperm in the female tract with some observations on the apyrene (anucleate) sperm which represent a considerable part of the total sperm mass received by the female during copulation. We also describe some changes that occurred in sperm after prolonged storage in the spermathecae. A recent article by Phillips (J. Ultrastruct. Res. 34: 547-85, 1971) on lepidopteran sperm was not seen until the present paper was sent to the publisher. M A T E R I A L S AND METHODS The parts of the reproductive systems of both sexes were dissected out into saline. They were then placed as quickly as possible into cold 2 °//o glutaraldehyde in 0"lu phosphate buffer adjusted to p H 7.4, fixed for 5-6 hr, left overnight in buffer, postfixed for 1-2 hr in 1o~, OsO4, and washed again in buffer for 1-2 hr. The tissue was then dehydrated in ethanol and propylene oxide and embedded in Araldite 502 or Spurr Low-Viscosity embedding medium. Silver or light gold sections were cut with a diamond knife, stained with uranyl acetate and lead citrate (Venable and Coggeshall, 1965), and examined with an RCA EM U-3H electron microscope at 50 kV. Magnifications were based on periodic calibration of the microscope with diffraction gratings and are approximate. OBSERVATIONS
In the male reproductive system Those parts of the male reproductive system involved in sperm storage are sequentially the testes, the seminal vesicles, and the paired (duplex) portion of the ejaculatory duct. We examined sperm from all three organs. In Anagasta, as in other lepidopterans (Virkki, 1963), the meiotic divisions leading to formation of nucleate sperm occur only in the larval stage. However, spermiogenesis is not synchronous and sperm in various stages of development can be found in both pupal and adult testes. A fairly late stage of spermiogenesis is shown in Fig. 1. In this transverse section through the tail of a eupyrene cell, the axial filament and mitochondrial derivatives are enclosed by a plasma membrane while outside the membrane there are a series of spokelike appendages. The plasma membrane seen around sperm of this stage seems to be derived from an envelope of smooth endoplasmic reticulum, which surrounds the central components of the sperm before a layer of peripheral cytoplasm is sloughed off. Following Riemann (1970), we have used satellite body as a convenient and descriptive term for the single nonlaminate appendage of Anagasta sperm. This name is apparently equivalent to the clear band (Yasuzumi and Oura, 1964a, 1964b, 1965a, 1965b). Sections of the satellite body cut at an appropriate angle showed the main mass attached to the plasma membrane by a series of septa with a diameter of about 50 ,~. Little else could be determined except that the main part of the body contained numerous fine lamellae often
Sperm Maturation in the Male and Female Genital Tracts of Anagasta kiihniella
13
so arranged as to give the appearance of tubules with a diameter of about 50 A. The other projections from the plasma membrane are those of the radial mantle (appendices lacina6 of Andr6, 1959, 1961), each appendage having alternate dark and light bands with a periodicity of 125 A. In the more anterior part of the nuclear region (Fig. 2), the satellite body is a massive crescent-shaped structure no longer attached to the plasma membrane. Also, the spokes of the radial mantle here come closer together to form finally a series of concentric rings around the nucleus. The eupyrene sperm remain in bundles until after they are transferred to the females during mating. However, before the bundles pass from the testes, both the individual sperm cells and the bundles as a whole undergo considerable reorganization. In the sperm cells, these changes involve loss of the radial mantle and the appearance and partial differentiation of the sheath material. These modifications are accompanied by lysis of the trophic cells and the formation of a supporting network of 600 A thick strands, which lie around the sperm cells and over the bundle as a whole. Details of these changes are still obscure and are being investigated. It is presently known, however, that the rays of the mantle are not converted in situ into the sheath because they become detached from the cells before sheath formation. Whatever the origin of the sheath, it apparently forms very quickly and differentiates into inner and outer portions (Figs. 3-4). In Anagasta, these changes may occur only in the testes, but in Trichoplusia, some sperm with unformed sheaths were found in seminal vesicles (Riemann, loc. cit.). Thus far, we have found no morphological differences between the sperm in the seminal veiscles and in the duplex of Anagasta, but the walls of the seminal vesicles are secretory, and the vesicles may have a role in sperm maturation. They do serve to hold bundles for release into the duplex after mating. Although it becomes smaller, the satellite body is retained after formation of the sheath. It is then attached to an electron-dense plate that forms a small part of the outer sheath. Except for the dense plates, few details could be seen in transverse sections of the outer part of the sheath aside from a series of cross striations (Fig. 4). In longitudinal sections (Fig. 5), the major structural unit appears as an open ring with a width of 150 A and a height of 200 A that seems to be continuous with components of the inner sheath. The material of the inner sheath is similar to that of the satellite body, an array of fibers (or lamellae) that often have parallel arrangements suggesting minute tubules.
In the female reproductive system During copulation, as many as 100 eupyrene bundles, masses of apyrene sperm, and quantities of secretory material from different parts of the male reproductive system are transferred within spermatophores to the bursa copulatrix of the female. Unpublished observations by Harold Hofmann of this laboratory show that sperm transfer occurs 45-60 rain after initiation of copulation. Disruption of the eupyrene bundles as evident 1 hr later is completed after another hour. Then the sperm pass through the seminal duct and a portion of the common oviduct and appear in the spermatheca at about 5-6 hr after the start of copulation. Some sperm remain in the spermatophore for at least 24 hr. Contents of spermatophores were fixed at 1, 3, and 8-9 hr after the start of copulation. Also, in the 8-9-hr group, the spermathecae were fixed so that sperm could be studied from the 2 sites in the same individual.
14
JOHN G. RIEMANN a n d BARBARA J. THORSON
A portion of a bundle fixed at 1 hr after the start of copulation is shown in Fig. 6. The bundle strands are still intact, but the satellite bodies have apparently separated from the cells. Higher magnification (Fig. 7) showed that remnants of the satellite bodies remained attached to the dense plates and that larger portions were nearby. The inner part of the
~. . . .
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FIGS.
~
1-12.
Sperm Maturation in the Male and Female Genital Tracts of Anagasta kiihniella
15
sheath appears largely u n c h a n g e d from its c o n d i t i o n in the duplex, b u t the outer sheath is more sharply defined, partly due to deposition of electron-dense material between the bases of the 150-A rings noted earlier (Fig. 8). I n transverse section, this material appears as 60-A line a n d seems to be c o n t i n u o u s with the dense plate, b u t the distinctiveness of the outer sheath is also partly caused by development of more clearly defined connections between the 150-A rings a n d the material of the i n n e r sheath. These connections, which look like pegs in l o n g i t u d i n a l sections, are seen to be part of the inner sheath when the c o m p o n e n t s of the sheath separate in degenerating sperm. The outer sheath n o w has a thickness of a b o u t 270 A as seen i n transverse sections. I n spermatophores fixed at 3 hr after copulation, the eupyrene sperm were diassociated from bundles. The outer sheath of the individual sperm still looked like that of cells fixed at 1 hr, b u t the inner sheath (Fig. 9) had now differentiated into two m a j o r portions. One of these, adjacent to the larger m i t o c h o n d r i a l derivative, seemed to correspond to a smaller structure called the intrasheath rod in Trichoplusia sperm ( R i e m a n n , loc. cit.). Very little material corresponding to the second a n d larger part of the i n n e r sheath of Anagasta sperm was f o u n d in the Trichoplusia sperm. I n both m a j o r parts of the i n n e r sheath, the lamellae n o w had a m u c h more ordered appearance t h a n before. Also, a definite dense b o u n d a r y was n o w present between the internal sheath a n d the plasma m e m b r a n e . FIG. 1. Transverse section of sperm tail in testis bundle, showing radial mantle (RM) and satellite body outside plasma membrane (PM); latter encloses mitochondrial derivatives (MD) and axial filament (AF). FIG. 2. Transverse section of nucleus (N) within testis bundle, showing massive crescentshaped satellite body (SB) and partially joined spokes of radial mantle. FIG. 3. Transverse section of part of bundle within testis following sheath formation, showing bundle strands (BS) between cells. F1G. 4. Transverse section of ce!l from bundle within seminal vesicle, showing inner (IS) and outer sheath (OS); satellite body (SB) is attached to dense plate (DP). FIc. 5. Longitudinal section of cell within ejaculatory duct, showing axial filament (AF), inner sheath (IS), outer sheath (OS), and mitochondrial derivative (MD). FIG. 6. Transverse section of part of eupyrene bundle in spermatophore 1 hr after start of copulation; a few apyrene cells and secretory granules are to right of bundle. FIG. 7. Enlargement of cell within bundle of Fig. 6, showing small part of satellite body (SB) attached to dense plate and larger part nearby; a layer of dense material (arrows) has formed beneath outer sheath. FIG. 8. Longitudinal section of cell at same stage as that shown in Figs. 6-7, showing dense material (arrows) beneath outer sheath; axial filament (AF). FIG. 9. Transverse section of cell in spermatophore 3 hr after start of copulation, showing differentiation of inner sheath into two parts with ordered arrangement of lamellae becoming apparent, especially in intrasheath rod (IR). FIG. 10. Longitudinal section of tail region of cell in spermatophore 8-9 hr after start of mating, showing paracrystalline structure of inner sheath (IS); axial filament (AF). FIG. l 1. Transverse section of tail region of cell in spermatheca; dense plate (DP), intrasheath rod (IR). FIG. 12. Longitudinal section of tail region of cell in spermatheca; axial filament (AF), mitochondrial derivative (MD).
Abbreviations used in figures AF = axial filament BS = bundle strand DP = dense plate
IS = inner sheath N ~ nucleus IR = intrasheath rod OS = outer sheath MD = mitochondrial derivative PM = plasma membrane RM = radial mantle SB = satellite body
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JOHN G. RIEMANNand BARBARAJ. THORSON
During bundle breakdown, the sheaths of eupyrene sperm in Trichoplusia rotate about 120° from their former position in relation to the axial filament and the mitochondria. A lesser rotation of about 30 ° (as marked by the position of the dense plate) occurs in the sheath of Anagasta sperm. The significance of this rotation is unknown. At 8-9 hr after mating, structural differentiation was apparently complete, both in those eupyrene sperm still in the spermatophore and in those that had reached the spermatheca (Figs. 10-13). An ordered paracrystalline structure was evident in the components of the inner sheath. The larger and less dense part appeared to be composed of lamellae with a diameter of 35 A and a center-to-center spacing of 85 A. Either 2 or 4 lamellae were attached to the projections leading to the ! 50 A units of the outer sheath to give the appearance of either simple parallel lines or a lattice. A similar organization occurred in the intrasheath rod, but there the center-to-center spacing of the lamellae was only 50 A. In sections of spermathecae, various numbers of apyrene sperm were found among the eupyrene cells. They were easily recognized (Fig. 14) by their small mitochondrial derivatives, their simple sheaths, and the absence of dense cores in the accessory fibers of the axial filament. Formation of the apyrene sheath occurs in the testes, and apparently replaces or incorporates the plasma membrane. The sheath apparently remains unchanged in its passage through the reproductive system. The sheath of apyrene sperm is similar to the outer sheath of eupyrene cells. In longitudinal section (Fig. 15), it contains 150-A thick rings covered with a thin layer of electrondense material. Cross striations within the rings can be seen in transverse sections (Fig. 12), but similar striations within the outer rings of the eupyrene cells are obscured after the bundle disrupts. Also, beneath the 150-A rings of the apyrene cells, there are at least 2 sleeves of material that contain rings of denser material. Possibly these sleeves are homologous with the electron-dense material that develops between the 150-A discs of the eupyrene sperm, although there the material is interrupted by connections to the inner sheath. In the apyrene cells, the rings of the outer sleeve are positioned beneath the spaces between the 150-A open rings, and the rings of succeeding sleeves are positioned alternately with those of the adjacent sleeve. In the posterior part of the apyrene cells, beyond the axial filament and mitochondrial derivatives, the various sleeves appear in transverse section as a series of concentric rings. In longitudinal sections of 2 or more adjacent apyrene sperm within the spermathecae, we noticed an exact pairing (in register) of the 150-A open rings of the cells. A similar point-to-point pairing was not noticed in eupyrene cells or in apyrene cells in the male reproductive system. The status of the sheath of the eupyrene sperm as a part 'added on' after formation of the usual sperm components was emphasized by observations of the contents of the spermathecae of old females. Female Anagasta lay few eggs until after mating, but they then FIG. 13. Transverse section through nucleus of cell in spermatheca; intrasheath rod (IR). FIG. 14. Transverse section of apyrene cell in spermatheca, showing striations within the sheath similar to those seen in outer sheath of eupyrene cells within male tract; mitochondrial derivative (MD). F16. 15. Longitudinal section of apyrene cell in spermatheca, showing repeating sheath units; axial filament (AF), mitochondrial derivative (MD). FIG. 16. Area in spermatheca 9 days after mating, showing many empty sheaths. FIG. 17. Degenerating eupyrene and apyrene cells in spermatheca 5 days after mating. FIG. 18. Empty sheath and naked eupyrene cells in spermathecal duct fixed 3 days after mating.
Sperm Maturation in the Male and Female Genital Tracts of Anagasta kiihniella
17
deposit all their eggs within 4-5 days. Thereafter, sperm could still be found in saline squashes of spermathecae examined with a phase light microscope, but they exhibited little motility. Examination of the ultrastructure of spermathecae 7-9 days after mating showed that a large part of the contents were empty sheaths (Fig. 16). These empty sheaths
FtGS. 13-18.
18
JOHN G. RIEMANNand BARBARAJ. THORSON
probably resulted mostly from degeneration of the core components since various stages of disassociation of the mitochondrial derivatives and axial filaments were found 5-9 days after mating (Fig. 17). Another possible source of empty sheaths was suggested by examination of sections of spermathecal ducts. Two ducts examined 3 days after mating contained empty sheaths and what seemed to be intact inner portions of eupyrene sperm (Fig. 18). These observations suggested that the sheath of eupyrene sperm is discarded before fertilization of eggs. All the empty sperm sheaths seen in spermathecae or in spermathecal ducts seemed to be intact except for longitudinal breaks along one side of the dense plate of the outer sheath. Thus, the plate may be a seam or suture used specifically to permit separation of the sheath and other sperm components. DISCUSSION Lepidopteran testicular sperm have been examined in a number of species (Phillips, 1970). In all the species the eupyrene sperm have basically the same type of appendages as those occurring around Anagasta sperm. From species to species, however, some variation does occur in the form of the appendages. Thus, in Ancyloxpha the lamellae of the radial mantle have a periodicity of 90 A (Phillips, 1970), while in Anagasta the spacings are about 125 A. Also, the satellite body of Anagasta sperm does not contain vesicles like those found in the same structure of silkworm sperm (Yasuzumi and Oura, 1965b). According to these latter authors, all appendages around silkworm sperm have a common origin. In their analogy, the satellite body (clear band) and the spokes of the radial mantle are compared to the trunk and branches of a tree. No definite conclusions have been reached as to functions of the sperm appendages or the origin of the sheath, and only a few statements concerning these points seem justified at this time. It would seem plausible that the radial mantle is converted into the sheath, but if this occurs, it is only indirectly. As stated earlier, the mantle rays become detached from the plasma prior to formation of the sheath. In some of our micrographs, it appears that the detached rays are being transformed into the bundle strands that bind the sperm cells together after lysis of the surrounding nurse cells. In any case, there is a stage, apparently of brief duration, in which the sperm are bare except for the satellite bodies. Then masses of amorphous material appear over the plasma membrane to eventually form the sheath. A marked enlargement of the satellite body occurs during at least part of the period during which the radial mantle is being replaced by the sheath. The significance of the enlargement is unknown. Eupyrene sperm in Anagasta and Trichoplusia (Riemann, loc. cit.) differ considerably in complexity and organization, but the manner of formation (as far as this is known), basic components, and differentiation of the sheath seem similar. Also, we have found a similar type of sheath in four other species of moths belonging to three families. Thus, formation of eupyrene sperm with complex sheaths outside the plasma membrane is probably characteristic of Lepidoptera. The distinctiveness of the sheath from components within the plasma membrane is emphasized in Anagasta by the degeneration of the internal components, which leaves intact sheaths, and by an apparently natural separation of the sheath, perhaps in preparation for fertilization. Changes in the structure of sperm after they enter the female genital system have been
Sperm Maturation in the Male and Female Genital Tracts of Anagasta kiihniella
19
r e p o r t e d for several insect species. The subject is briefly reviewed by Hughes a n d D a v e y (1969) in a p a p e r in which they describe changes a r o u n d the a c r o s o m e o f Periplaneta sperm after entry into the spermathecae. The changes t h a t have been described are various and Hughes and D a v e y e m p h a s i z e d that viewing t h e m as types o f capacitation, as this term is used for m a m m a l i a n sperm, m a y n o t be justified. It also seems advisable at present to a v o i d the use o f the term c a p a c i t a t i o n in describing the changes in A n a g a s t a sperm after they reach the female tract. The function o f the sheath o f the eupyrene sperm in A n a g a s t a is presently entirely speculative. A m e t a b o l i c role for the sheath seems indicated b y the g r a d u a l d e v e l o p m e n t o f a highly ordered structure within the s p e r m a t o p h o r e . This d e v e l o p m e n t is at least roughly c o r r e l a t e d with the beginning o f motility o f eupyrene sperm several hours after bundle disruption. W h e t h e r the two p h e n o m e n a are actually related, and if so, how, remains to be determined. P r o t e i n a c e o u s paracrystalline structures have been f o u n d in the m i t o c h o n d r i a l derivatives o f m a n y insect sperm, a n d Phillips (1970) suggested that suitable p r e p a r a t i o n s will reveal p e r i o d i c structure in the m i t o c h o n d r i a l derivatives o f other insect sperm r e p o r t e d to have none, The function o f the paracrystalline m a t e r i a l is n o t known, a n d we were unable to find a n y crystalline-like structure in the rather simple m i t o c h o n d r i a l derivatives o f Anagasta. Thus, the size a n d p e r i o d i c o r g a n i z a t i o n o f the inner sheath suggested to us that the sheath h a d acquired some o f the functions o f the m i t o c h o n d r i a l derivatives o f other insect sperm. A s s a y o f the sheath for possible enzymatic activity is planned. REFERENCES
ANDRe, J. 1959. Etude au microscope 61ectronique de l'6volution du chondriome pendant la spermatog(~nese du Papillon du chous, Pieris brassicae. Ann. Sci. Natur. 1: 283-305. ANDRe, J. 1961. Sur quelques d6tails nouvellement connus de l'ultrastructure des organites vibratiles. J. Ultrastruct. Res. 5: 86-108. ANDRe, J. 1962. Contribution fi la connaissance du chondriome, 6tude de ses modifications ultrastructurales pendant la spermatog6n(~se. J. Ultrastruct. Res. (Suppl.) 3: 1-185. Fa~EDLANDER,M. and J. WAHRMAN.1970. The spindle as a basal body distributor: A study in the meiosis of the male silkworm moth, Bombyx mori. J. Cell Sci. 7: 65-89. HUGHES, M. and K. G. DAVEY. 1969. The activity of spermatozoa of Periplaneta. J. Insect Physiol. 15: 1607-16. PmLLIPS, D. M. 1970. Insect sperm: Their structure and morphogenesis. J. Cell Biol. 44: 243-77. RIEMANN, J. G. 1970. Metamorphosis of sperm of the cabbage looper, Trichoplusia ni, during passage from the testes to the female spermathecae, pp. 321-331, In: Baccio Baccetti ted.) Comparative Spermatologv, Accademia Nazionale Dei Lincei, Quaderono N. 137. SMITH, D. S. 1968. Insect cells, their structure and function. Oliver & Boyd, Edinburgh. VENABLE,J. H. and R. COGGESHALL.1965. A simplified lead citrate strain for use in electron microscopy. J. Cell Biol. 25: 407-8. VmKKL N. 1963. Gametogenesis in the sugarcane borer moth, Diatrae sacchaarllis (F.) Crambidae. J. Agr. Univ. Puerto Rico 47: 102-37. YASUZUMI, G. and C. OURA. 1964a. Differential analysis by various staining techniques of structures present in developing spermatids of the silkworm. Nature (London) 204 (5964): 1197-98. YASUZUML G. and C. (SURA. 1964b. Spermatogenesis in animals as revealed by electron microscopy. XIII. Formation of a tubular structure and two bands in the developing spermatid of the silkworm, Bombyx mori Linne. Z. ZellIbrsch. Mikroskop. Anat. 64: 210-26. YASUZUMI, G. and C. OUaA. 1965a. Spermatogenesis in animals as revealed by electron microscopy. X1V. The fine structure of the clear band tubular structure in late stages of development of spermatids of the silkworm, Bombyx mori Linne. Z. Zellforsch. Mikroskop. Anat. 66:182-96. YASUZUMI, G. and C. OURA. 1965b. Spermatogenesis in animals as revealed by electron microscopy. XV. The fine structure of the middle piece in developing spermatid of the silkworm, Bombyx mori Linne. Z. Zellforsch. Mikroskop. Anat. 67: 502-20. ZYLBERBERG, L. 1963. Remarques sur l'ultrastructure des flagelles des spermatozoides typiques et atypiquest de Pieris brassicae L. C. R. Acad. Sci. (Paris) 245: 2702-03.
SPERM MATURATION IN THE MALE A N D FEMALE GENITAL TRACTS OF ANAGASTA KUHNIELLA
(LEPIDOPTERA: PYRALIDIDAE)* JOHN G. RIEMANN a n d BARBARA J. THORSON Metabolism and Radiation Research Laboratory, Agricultural Research Service, U.S. Dept. of Agriculture, Fargo, North Dakota 58102, U.S.A.
(Accepted 7 May 1971) Abstract--In the testes of the Mediterranean flour moth, Anagasta (-- Ephestia) kiihniella (Zeller), a thick sheath forms around the plasma membrane, replacing a series of laminate proiections, the radial mantle; but another projection, the satellite body, is retained as an appendage of the sheath. This sheath quickly differentiates into an outer part made up o f a series of rings 150 A_wide, covered by a thin sleeve, and interrupted on one side by a narrow, dense plate to which the satellite body is attached, and an inner part made up of a mass of lamellae lacking definite orientation. Once the sperm are transferred to the female tract in spermatophores, marked changes occur in the sheath; the satellite body disappears, dense material appears between the inner margins of the radial discs, and the inner sheath differentiates into two distinct portions, both of which have a paracrystalline structure. Once the females deposit their eggs, most of the remaining sperm degenerate and leave empty sheaths. Evidence was found of a normal separation of sheaths and core components within the spermathecal duct, perhaps in preparation of the sperm for fertilization. During this separation, the sheath splits along the line of the dense plate. The sheath of the apyrene sperm was similar to the outer sheath of the eupyrene sperm. Index descriptors (in addition to those in title): Anagasta (=Ephestia) kiihniella, sperm ultrastructure, eupyrene apyrene sheath maturation.
INTRODUCTION TESTICULAR sperm of various lepidopterans have been studied at the ultrastructural level. A n d r 6 (1959, 1961, 1962) gave detailed descriptions o f the axial filament a n d mitochondrial derivatives of Macroglossum a n d Pieris respectively. He also described for the first time the series of appendages a r o u n d the plasma m e m b r a n e that have come to be recognized as characteristic of lepidopteran sperm n e a r i n g maturity in the testes. Y a s u z u m i a n d O u r a (1964, 1964b, 1965a, 1965b) made a detailed study of spermiogenesis in Bombyx mori L. with particular emphasis o n development of structures they called the t u b u l a r structure a n d the clear b a n d . Early stages of spermiogenesis in Bombyx were also studied by F r i e d l a n d e r a n d W a h r m a n (1970). Observations o n testicular sperm of various other lepidopterans were made by Phillips (1970), R i e m a n n (1970), Smith (1968), a n d Zylberberg (1963). Only a few observations have been made of lepidopteran sperm that have been released from the testes. Phillips (1970) showed that sperm in the ejaculatory duct of Desmiafuneralis *Supported in part by U.S. Atomic Energy Commission Contract AT(49-7)3028. 11
12
JOHN G. RIEMANN and BARBARA J. THORSON
(Hfibner) were enclosed by a rather thick fibrous sleeve (sheath). Riemann (1970) found that eupyrene (nucleate) sperm of Trichoplusia ni (Hfibner) went through a rather elaborate metamorphosis during passage through the male and female tracts. The changes were most evident in the sheath, which formed at about the time the sperm left the testes. Anagasta eupyrene sperm go through a metamorphosis basically similar to those of Trichoplusia ni (Riemann, 1970), but they have a considerably more complex structure and more details have been established about their metamorphosis. In this paper, we briefly describe the appearance and metamorphosis of Anagasta sperm as they occur in the testes and post-testicular organs. A more detailed description (with emphasis on the sheath) is given of the eupyrene sperm in the female tract with some observations on the apyrene (anucleate) sperm which represent a considerable part of the total sperm mass received by the female during copulation. We also describe some changes that occurred in sperm after prolonged storage in the spermathecae. A recent article by Phillips (J. Ultrastruct. Res. 34: 547-85, 1971) on lepidopteran sperm was not seen until the present paper was sent to the publisher. M A T E R I A L S AND METHODS The parts of the reproductive systems of both sexes were dissected out into saline. They were then placed as quickly as possible into cold 2 °//o glutaraldehyde in 0"lu phosphate buffer adjusted to p H 7.4, fixed for 5-6 hr, left overnight in buffer, postfixed for 1-2 hr in 1o~, OsO4, and washed again in buffer for 1-2 hr. The tissue was then dehydrated in ethanol and propylene oxide and embedded in Araldite 502 or Spurr Low-Viscosity embedding medium. Silver or light gold sections were cut with a diamond knife, stained with uranyl acetate and lead citrate (Venable and Coggeshall, 1965), and examined with an RCA EM U-3H electron microscope at 50 kV. Magnifications were based on periodic calibration of the microscope with diffraction gratings and are approximate. OBSERVATIONS
In the male reproductive system Those parts of the male reproductive system involved in sperm storage are sequentially the testes, the seminal vesicles, and the paired (duplex) portion of the ejaculatory duct. We examined sperm from all three organs. In Anagasta, as in other lepidopterans (Virkki, 1963), the meiotic divisions leading to formation of nucleate sperm occur only in the larval stage. However, spermiogenesis is not synchronous and sperm in various stages of development can be found in both pupal and adult testes. A fairly late stage of spermiogenesis is shown in Fig. 1. In this transverse section through the tail of a eupyrene cell, the axial filament and mitochondrial derivatives are enclosed by a plasma membrane while outside the membrane there are a series of spokelike appendages. The plasma membrane seen around sperm of this stage seems to be derived from an envelope of smooth endoplasmic reticulum, which surrounds the central components of the sperm before a layer of peripheral cytoplasm is sloughed off. Following Riemann (1970), we have used satellite body as a convenient and descriptive term for the single nonlaminate appendage of Anagasta sperm. This name is apparently equivalent to the clear band (Yasuzumi and Oura, 1964a, 1964b, 1965a, 1965b). Sections of the satellite body cut at an appropriate angle showed the main mass attached to the plasma membrane by a series of septa with a diameter of about 50 ,~. Little else could be determined except that the main part of the body contained numerous fine lamellae often
Sperm Maturation in the Male and Female Genital Tracts of Anagasta kiihniella
13
so arranged as to give the appearance of tubules with a diameter of about 50 A. The other projections from the plasma membrane are those of the radial mantle (appendices lacina6 of Andr6, 1959, 1961), each appendage having alternate dark and light bands with a periodicity of 125 A. In the more anterior part of the nuclear region (Fig. 2), the satellite body is a massive crescent-shaped structure no longer attached to the plasma membrane. Also, the spokes of the radial mantle here come closer together to form finally a series of concentric rings around the nucleus. The eupyrene sperm remain in bundles until after they are transferred to the females during mating. However, before the bundles pass from the testes, both the individual sperm cells and the bundles as a whole undergo considerable reorganization. In the sperm cells, these changes involve loss of the radial mantle and the appearance and partial differentiation of the sheath material. These modifications are accompanied by lysis of the trophic cells and the formation of a supporting network of 600 A thick strands, which lie around the sperm cells and over the bundle as a whole. Details of these changes are still obscure and are being investigated. It is presently known, however, that the rays of the mantle are not converted in situ into the sheath because they become detached from the cells before sheath formation. Whatever the origin of the sheath, it apparently forms very quickly and differentiates into inner and outer portions (Figs. 3-4). In Anagasta, these changes may occur only in the testes, but in Trichoplusia, some sperm with unformed sheaths were found in seminal vesicles (Riemann, loc. cit.). Thus far, we have found no morphological differences between the sperm in the seminal veiscles and in the duplex of Anagasta, but the walls of the seminal vesicles are secretory, and the vesicles may have a role in sperm maturation. They do serve to hold bundles for release into the duplex after mating. Although it becomes smaller, the satellite body is retained after formation of the sheath. It is then attached to an electron-dense plate that forms a small part of the outer sheath. Except for the dense plates, few details could be seen in transverse sections of the outer part of the sheath aside from a series of cross striations (Fig. 4). In longitudinal sections (Fig. 5), the major structural unit appears as an open ring with a width of 150 A and a height of 200 A that seems to be continuous with components of the inner sheath. The material of the inner sheath is similar to that of the satellite body, an array of fibers (or lamellae) that often have parallel arrangements suggesting minute tubules.
In the female reproductive system During copulation, as many as 100 eupyrene bundles, masses of apyrene sperm, and quantities of secretory material from different parts of the male reproductive system are transferred within spermatophores to the bursa copulatrix of the female. Unpublished observations by Harold Hofmann of this laboratory show that sperm transfer occurs 45-60 rain after initiation of copulation. Disruption of the eupyrene bundles as evident 1 hr later is completed after another hour. Then the sperm pass through the seminal duct and a portion of the common oviduct and appear in the spermatheca at about 5-6 hr after the start of copulation. Some sperm remain in the spermatophore for at least 24 hr. Contents of spermatophores were fixed at 1, 3, and 8-9 hr after the start of copulation. Also, in the 8-9-hr group, the spermathecae were fixed so that sperm could be studied from the 2 sites in the same individual.
14
JOHN G. RIEMANN a n d BARBARA J. THORSON
A portion of a bundle fixed at 1 hr after the start of copulation is shown in Fig. 6. The bundle strands are still intact, but the satellite bodies have apparently separated from the cells. Higher magnification (Fig. 7) showed that remnants of the satellite bodies remained attached to the dense plates and that larger portions were nearby. The inner part of the
~. . . .
~ ~,
~ , .........~ i
FIGS.
~
1-12.
Sperm Maturation in the Male and Female Genital Tracts of Anagasta kiihniella
15
sheath appears largely u n c h a n g e d from its c o n d i t i o n in the duplex, b u t the outer sheath is more sharply defined, partly due to deposition of electron-dense material between the bases of the 150-A rings noted earlier (Fig. 8). I n transverse section, this material appears as 60-A line a n d seems to be c o n t i n u o u s with the dense plate, b u t the distinctiveness of the outer sheath is also partly caused by development of more clearly defined connections between the 150-A rings a n d the material of the i n n e r sheath. These connections, which look like pegs in l o n g i t u d i n a l sections, are seen to be part of the inner sheath when the c o m p o n e n t s of the sheath separate in degenerating sperm. The outer sheath n o w has a thickness of a b o u t 270 A as seen i n transverse sections. I n spermatophores fixed at 3 hr after copulation, the eupyrene sperm were diassociated from bundles. The outer sheath of the individual sperm still looked like that of cells fixed at 1 hr, b u t the inner sheath (Fig. 9) had now differentiated into two m a j o r portions. One of these, adjacent to the larger m i t o c h o n d r i a l derivative, seemed to correspond to a smaller structure called the intrasheath rod in Trichoplusia sperm ( R i e m a n n , loc. cit.). Very little material corresponding to the second a n d larger part of the i n n e r sheath of Anagasta sperm was f o u n d in the Trichoplusia sperm. I n both m a j o r parts of the i n n e r sheath, the lamellae n o w had a m u c h more ordered appearance t h a n before. Also, a definite dense b o u n d a r y was n o w present between the internal sheath a n d the plasma m e m b r a n e . FIG. 1. Transverse section of sperm tail in testis bundle, showing radial mantle (RM) and satellite body outside plasma membrane (PM); latter encloses mitochondrial derivatives (MD) and axial filament (AF). FIG. 2. Transverse section of nucleus (N) within testis bundle, showing massive crescentshaped satellite body (SB) and partially joined spokes of radial mantle. FIG. 3. Transverse section of part of bundle within testis following sheath formation, showing bundle strands (BS) between cells. F1G. 4. Transverse section of ce!l from bundle within seminal vesicle, showing inner (IS) and outer sheath (OS); satellite body (SB) is attached to dense plate (DP). FIc. 5. Longitudinal section of cell within ejaculatory duct, showing axial filament (AF), inner sheath (IS), outer sheath (OS), and mitochondrial derivative (MD). FIG. 6. Transverse section of part of eupyrene bundle in spermatophore 1 hr after start of copulation; a few apyrene cells and secretory granules are to right of bundle. FIG. 7. Enlargement of cell within bundle of Fig. 6, showing small part of satellite body (SB) attached to dense plate and larger part nearby; a layer of dense material (arrows) has formed beneath outer sheath. FIG. 8. Longitudinal section of cell at same stage as that shown in Figs. 6-7, showing dense material (arrows) beneath outer sheath; axial filament (AF). FIG. 9. Transverse section of cell in spermatophore 3 hr after start of copulation, showing differentiation of inner sheath into two parts with ordered arrangement of lamellae becoming apparent, especially in intrasheath rod (IR). FIG. 10. Longitudinal section of tail region of cell in spermatophore 8-9 hr after start of mating, showing paracrystalline structure of inner sheath (IS); axial filament (AF). FIG. l 1. Transverse section of tail region of cell in spermatheca; dense plate (DP), intrasheath rod (IR). FIG. 12. Longitudinal section of tail region of cell in spermatheca; axial filament (AF), mitochondrial derivative (MD).
Abbreviations used in figures AF = axial filament BS = bundle strand DP = dense plate
IS = inner sheath N ~ nucleus IR = intrasheath rod OS = outer sheath MD = mitochondrial derivative PM = plasma membrane RM = radial mantle SB = satellite body
16
JOHN G. RIEMANNand BARBARAJ. THORSON
During bundle breakdown, the sheaths of eupyrene sperm in Trichoplusia rotate about 120° from their former position in relation to the axial filament and the mitochondria. A lesser rotation of about 30 ° (as marked by the position of the dense plate) occurs in the sheath of Anagasta sperm. The significance of this rotation is unknown. At 8-9 hr after mating, structural differentiation was apparently complete, both in those eupyrene sperm still in the spermatophore and in those that had reached the spermatheca (Figs. 10-13). An ordered paracrystalline structure was evident in the components of the inner sheath. The larger and less dense part appeared to be composed of lamellae with a diameter of 35 A and a center-to-center spacing of 85 A. Either 2 or 4 lamellae were attached to the projections leading to the ! 50 A units of the outer sheath to give the appearance of either simple parallel lines or a lattice. A similar organization occurred in the intrasheath rod, but there the center-to-center spacing of the lamellae was only 50 A. In sections of spermathecae, various numbers of apyrene sperm were found among the eupyrene cells. They were easily recognized (Fig. 14) by their small mitochondrial derivatives, their simple sheaths, and the absence of dense cores in the accessory fibers of the axial filament. Formation of the apyrene sheath occurs in the testes, and apparently replaces or incorporates the plasma membrane. The sheath apparently remains unchanged in its passage through the reproductive system. The sheath of apyrene sperm is similar to the outer sheath of eupyrene cells. In longitudinal section (Fig. 15), it contains 150-A thick rings covered with a thin layer of electrondense material. Cross striations within the rings can be seen in transverse sections (Fig. 12), but similar striations within the outer rings of the eupyrene cells are obscured after the bundle disrupts. Also, beneath the 150-A rings of the apyrene cells, there are at least 2 sleeves of material that contain rings of denser material. Possibly these sleeves are homologous with the electron-dense material that develops between the 150-A discs of the eupyrene sperm, although there the material is interrupted by connections to the inner sheath. In the apyrene cells, the rings of the outer sleeve are positioned beneath the spaces between the 150-A open rings, and the rings of succeeding sleeves are positioned alternately with those of the adjacent sleeve. In the posterior part of the apyrene cells, beyond the axial filament and mitochondrial derivatives, the various sleeves appear in transverse section as a series of concentric rings. In longitudinal sections of 2 or more adjacent apyrene sperm within the spermathecae, we noticed an exact pairing (in register) of the 150-A open rings of the cells. A similar point-to-point pairing was not noticed in eupyrene cells or in apyrene cells in the male reproductive system. The status of the sheath of the eupyrene sperm as a part 'added on' after formation of the usual sperm components was emphasized by observations of the contents of the spermathecae of old females. Female Anagasta lay few eggs until after mating, but they then FIG. 13. Transverse section through nucleus of cell in spermatheca; intrasheath rod (IR). FIG. 14. Transverse section of apyrene cell in spermatheca, showing striations within the sheath similar to those seen in outer sheath of eupyrene cells within male tract; mitochondrial derivative (MD). F16. 15. Longitudinal section of apyrene cell in spermatheca, showing repeating sheath units; axial filament (AF), mitochondrial derivative (MD). FIG. 16. Area in spermatheca 9 days after mating, showing many empty sheaths. FIG. 17. Degenerating eupyrene and apyrene cells in spermatheca 5 days after mating. FIG. 18. Empty sheath and naked eupyrene cells in spermathecal duct fixed 3 days after mating.
Sperm Maturation in the Male and Female Genital Tracts of Anagasta kiihniella
17
deposit all their eggs within 4-5 days. Thereafter, sperm could still be found in saline squashes of spermathecae examined with a phase light microscope, but they exhibited little motility. Examination of the ultrastructure of spermathecae 7-9 days after mating showed that a large part of the contents were empty sheaths (Fig. 16). These empty sheaths
FtGS. 13-18.
18
JOHN G. RIEMANNand BARBARAJ. THORSON
probably resulted mostly from degeneration of the core components since various stages of disassociation of the mitochondrial derivatives and axial filaments were found 5-9 days after mating (Fig. 17). Another possible source of empty sheaths was suggested by examination of sections of spermathecal ducts. Two ducts examined 3 days after mating contained empty sheaths and what seemed to be intact inner portions of eupyrene sperm (Fig. 18). These observations suggested that the sheath of eupyrene sperm is discarded before fertilization of eggs. All the empty sperm sheaths seen in spermathecae or in spermathecal ducts seemed to be intact except for longitudinal breaks along one side of the dense plate of the outer sheath. Thus, the plate may be a seam or suture used specifically to permit separation of the sheath and other sperm components. DISCUSSION Lepidopteran testicular sperm have been examined in a number of species (Phillips, 1970). In all the species the eupyrene sperm have basically the same type of appendages as those occurring around Anagasta sperm. From species to species, however, some variation does occur in the form of the appendages. Thus, in Ancyloxpha the lamellae of the radial mantle have a periodicity of 90 A (Phillips, 1970), while in Anagasta the spacings are about 125 A. Also, the satellite body of Anagasta sperm does not contain vesicles like those found in the same structure of silkworm sperm (Yasuzumi and Oura, 1965b). According to these latter authors, all appendages around silkworm sperm have a common origin. In their analogy, the satellite body (clear band) and the spokes of the radial mantle are compared to the trunk and branches of a tree. No definite conclusions have been reached as to functions of the sperm appendages or the origin of the sheath, and only a few statements concerning these points seem justified at this time. It would seem plausible that the radial mantle is converted into the sheath, but if this occurs, it is only indirectly. As stated earlier, the mantle rays become detached from the plasma prior to formation of the sheath. In some of our micrographs, it appears that the detached rays are being transformed into the bundle strands that bind the sperm cells together after lysis of the surrounding nurse cells. In any case, there is a stage, apparently of brief duration, in which the sperm are bare except for the satellite bodies. Then masses of amorphous material appear over the plasma membrane to eventually form the sheath. A marked enlargement of the satellite body occurs during at least part of the period during which the radial mantle is being replaced by the sheath. The significance of the enlargement is unknown. Eupyrene sperm in Anagasta and Trichoplusia (Riemann, loc. cit.) differ considerably in complexity and organization, but the manner of formation (as far as this is known), basic components, and differentiation of the sheath seem similar. Also, we have found a similar type of sheath in four other species of moths belonging to three families. Thus, formation of eupyrene sperm with complex sheaths outside the plasma membrane is probably characteristic of Lepidoptera. The distinctiveness of the sheath from components within the plasma membrane is emphasized in Anagasta by the degeneration of the internal components, which leaves intact sheaths, and by an apparently natural separation of the sheath, perhaps in preparation for fertilization. Changes in the structure of sperm after they enter the female genital system have been
Sperm Maturation in the Male and Female Genital Tracts of Anagasta kiihniella
19
r e p o r t e d for several insect species. The subject is briefly reviewed by Hughes a n d D a v e y (1969) in a p a p e r in which they describe changes a r o u n d the a c r o s o m e o f Periplaneta sperm after entry into the spermathecae. The changes t h a t have been described are various and Hughes and D a v e y e m p h a s i z e d that viewing t h e m as types o f capacitation, as this term is used for m a m m a l i a n sperm, m a y n o t be justified. It also seems advisable at present to a v o i d the use o f the term c a p a c i t a t i o n in describing the changes in A n a g a s t a sperm after they reach the female tract. The function o f the sheath o f the eupyrene sperm in A n a g a s t a is presently entirely speculative. A m e t a b o l i c role for the sheath seems indicated b y the g r a d u a l d e v e l o p m e n t o f a highly ordered structure within the s p e r m a t o p h o r e . This d e v e l o p m e n t is at least roughly c o r r e l a t e d with the beginning o f motility o f eupyrene sperm several hours after bundle disruption. W h e t h e r the two p h e n o m e n a are actually related, and if so, how, remains to be determined. P r o t e i n a c e o u s paracrystalline structures have been f o u n d in the m i t o c h o n d r i a l derivatives o f m a n y insect sperm, a n d Phillips (1970) suggested that suitable p r e p a r a t i o n s will reveal p e r i o d i c structure in the m i t o c h o n d r i a l derivatives o f other insect sperm r e p o r t e d to have none, The function o f the paracrystalline m a t e r i a l is n o t known, a n d we were unable to find a n y crystalline-like structure in the rather simple m i t o c h o n d r i a l derivatives o f Anagasta. Thus, the size a n d p e r i o d i c o r g a n i z a t i o n o f the inner sheath suggested to us that the sheath h a d acquired some o f the functions o f the m i t o c h o n d r i a l derivatives o f other insect sperm. A s s a y o f the sheath for possible enzymatic activity is planned. REFERENCES
ANDRe, J. 1959. Etude au microscope 61ectronique de l'6volution du chondriome pendant la spermatog(~nese du Papillon du chous, Pieris brassicae. Ann. Sci. Natur. 1: 283-305. ANDRe, J. 1961. Sur quelques d6tails nouvellement connus de l'ultrastructure des organites vibratiles. J. Ultrastruct. Res. 5: 86-108. ANDRe, J. 1962. Contribution fi la connaissance du chondriome, 6tude de ses modifications ultrastructurales pendant la spermatog6n(~se. J. Ultrastruct. Res. (Suppl.) 3: 1-185. Fa~EDLANDER,M. and J. WAHRMAN.1970. The spindle as a basal body distributor: A study in the meiosis of the male silkworm moth, Bombyx mori. J. Cell Sci. 7: 65-89. HUGHES, M. and K. G. DAVEY. 1969. The activity of spermatozoa of Periplaneta. J. Insect Physiol. 15: 1607-16. PmLLIPS, D. M. 1970. Insect sperm: Their structure and morphogenesis. J. Cell Biol. 44: 243-77. RIEMANN, J. G. 1970. Metamorphosis of sperm of the cabbage looper, Trichoplusia ni, during passage from the testes to the female spermathecae, pp. 321-331, In: Baccio Baccetti ted.) Comparative Spermatologv, Accademia Nazionale Dei Lincei, Quaderono N. 137. SMITH, D. S. 1968. Insect cells, their structure and function. Oliver & Boyd, Edinburgh. VENABLE,J. H. and R. COGGESHALL.1965. A simplified lead citrate strain for use in electron microscopy. J. Cell Biol. 25: 407-8. VmKKL N. 1963. Gametogenesis in the sugarcane borer moth, Diatrae sacchaarllis (F.) Crambidae. J. Agr. Univ. Puerto Rico 47: 102-37. YASUZUMI, G. and C. OURA. 1964a. Differential analysis by various staining techniques of structures present in developing spermatids of the silkworm. Nature (London) 204 (5964): 1197-98. YASUZUML G. and C. (SURA. 1964b. Spermatogenesis in animals as revealed by electron microscopy. XIII. Formation of a tubular structure and two bands in the developing spermatid of the silkworm, Bombyx mori Linne. Z. ZellIbrsch. Mikroskop. Anat. 64: 210-26. YASUZUMI, G. and C. OUaA. 1965a. Spermatogenesis in animals as revealed by electron microscopy. X1V. The fine structure of the clear band tubular structure in late stages of development of spermatids of the silkworm, Bombyx mori Linne. Z. Zellforsch. Mikroskop. Anat. 66:182-96. YASUZUMI, G. and C. OURA. 1965b. Spermatogenesis in animals as revealed by electron microscopy. XV. The fine structure of the middle piece in developing spermatid of the silkworm, Bombyx mori Linne. Z. Zellforsch. Mikroskop. Anat. 67: 502-20. ZYLBERBERG, L. 1963. Remarques sur l'ultrastructure des flagelles des spermatozoides typiques et atypiquest de Pieris brassicae L. C. R. Acad. Sci. (Paris) 245: 2702-03.