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The aberrant spermatozoa of hydropsychidae caddisflies (trichoptera): An electron microscope analysis on spermiogenesis
JOURNAL OF ULTRASTRUCTURE RESEARCH
78, 84-94 (1982)
The Aberrant Spermatozoa of Hydropsychidae Caddisflies (Trichoptera): An Electron Microscope Analysis on Spermiogenesis MICHAEL FRIEDL,~NDER AND JOHN C. MORSE
Department of Biology, Ben Gurion University of the Negev, Beer Sheva, Israel and Department of Entomology, Fisheries and Wildlife, Clemson University, Clemson, South Carolina 29631 Received March 23, 1981, and in revised form July 15, 1981 The spermatozoon of caddisflies in the family Hydropsychidae lacks acrosome and centriole adjunct. The basal body and nucleus are separated. The axoneme is cone shaped and its microtubular doublets diverge, forming separate filaments which trail backward from the cell surface. The mitochondria do not fuse, conserve their cristae, form no derivatives, and appear within the lumen of the aberrant axoneme. Some of those may be secondarily derived characteristics since they are not present in early spermatids. Spermatozoa isolated into a Ringer solution from ejaculatory ducts or inseminated females show no autonomous movements. In the female, the spermatozoa may be moved by peristaltic contractions of the genital duct. The direction of their movement may be determined by tubular projections of the duct wall which engage the trailing microtubular doublets, thus impeding backward movement of the spermatozoa. No apyrene (anucleated) spermatozoa, similar to those characterizing Lepidoptera, could be found.
choptera stems from the difficulties encountered in obtaining suitable material for such studies. First, spermatid differentiation appears to occur in this order only close to, or after, pupation (Lankhorst, 1970; Le Lannic, 1975; Kiuta and Lankhorst, 1969). In the larvae, spermatogenesis does not advance further than meiotic prophase, while the adults contain only mature spermatozoa. Trichoptera pupae, however, are aquatic and relatively difficult both to collect in the field and to rear in the laboratory. Second, few entomologists are specialized in this small order and are able to determine with certainty the species of the larval instars and the pupae. We report here the first ultrastructural analysis of spermatid differentiation in Trichoptera. Spermatozoa in the inseminated female were studied to determine whether they undergo further transformation after ejaculation.
" T h e numerous works that have appeared in recent years on spermatogenesis of insects have covered practically every order . . . . There still remain, however, a small number of small orders which have not been investigated . . . . The Trichoptera is one of these small orders" (Lutman, 1910). Since this statement was made, we are aware of only one light microscope study on T r i c h o p t e r a spermiogenesis (Gresson, 1935). The other light microscope data after 1910 on spermatogenesis refer mainly to the behavior and number of chromosomes during meiosis (Pchakadze, 1928, 1930; Klingstedt, 1931; Kiuta and Kiuta, 1979). The only published ultrastructural data on Trichoptera spermiogenesis refer to the mature spermatozoa (Baccetti et al.. 1970; Phillips, 1969, 1974). That is a remarkable situation since there is a vast literature on ultrastructural studies of spermiogenesis in numerous other systematic groups of insects (e.g., Baccetti, 1972; Baccetti and Afzelius, 1976). Most probably, this lack of information on spermatogenesis of Tri-
MATERIALS AND METHODS
Diplectrona modesta, Hydropsyche sparna, and Cheumatopsyche spp. (Hydropsychinae, Hydropsychidae) from the suborder Annulipalpia in caddis84
0022-5320/82/010084-11 $02.00/0 Copyright © 1982 by Academic Press, inc. All rights of reproduction in any form reserved.
SPERM OF HYDROPSYCHIDAE CADDISFLIES flies (Trichoptera) were collected in the Piedmont region of South Carolina. Testes of larvae, pupae, and imagoes, ejaculatory ducts of imagoes, and genital ducts of inseminated females were fixed in 3% glutaraldehyde in 0.2 M cacodylate buffer, pH 7.3. Subsequently, the tissues were postfixed in 1% OsO4, dehydrated, and embedded in Epon. For electron microscopy, ultrathin sections were contrasted with uranyl acetate and lead citrate. For light microscopy, thick sections were stained in 1% toluidine blue. Living cells of the same tissues were dispersed in a Ringer solution and studied with phase optics. For comparison, living testicular cells of Locusta migratoria (Orthoptera) and Laspeyresia pomonella (Lepidoptera) were also dispersed in the same Ringer solution and studied with phase optics. RESULTS
The results were similar in all the species studied and the micrographs are from cells of Diplectrona modesta. Primary spermatocytes (Figs. 2 and 3) contain two pairs of short centrioles which remain throughout meiosis about half-way between the nucleus and the cell membrane. These centrioles neither develop axonemes, nor do they attach to mitochondria or other membranous structures. At the end of the telophase (Fig. 4), the forming nucleus contains highly condensed (elec-
85
Fro. 1. Schematic drawing of a mature spermatozoon cut through the median plane of symmetry. The trailing microtubular doublets are represented by one solid line, surrounded by cell membrane.
tron-opaque) chromatin, is located close to the cell surface, and has a bowl-shaped outline with the concavity toward the former spindle pole. The centriole is attached by one end to the concave face of the nucleus, the other end is free and directed to the cell surface. This centriole is about twice as long as those of the early spermatocytes (compare with Fig. 3). The mitochondria are similar to those of the spermatocytes and are dispersed within the cytoplasm facing the convex side of the nucleus. At the beginning of spermatid elongation (Figs. 5 and 6) the nucleus is irregular, ovoid, and located at the anterior end of the
Fro. 2. A primary spermatocyte showing a transverse section of one of the four centrioles, about half-way between the nucleus and the cell surface, x 28 500. FIG. 3. A primary spermatocyte as in Fig. 2 showing a longitudinal section of one centriole which is about half as long as that at telophase (compare with Fig. 4) and is disconnected from the mitochondria or other membranous structures, x 28 500. FIG. 4. Meiotic telophase. The nucleus has a bowl-shaped outline. The centriole is about twice as long as those found at prophase (compare with Fig. 3) and is attached by one end of the concave face of the nucleus. The mitochondria (M) are similar to those of the spermatocytes (compare with Fig. 2) and are dispersed within the cytoplasm. × 28 500. FIG. 5. An early spermatid showing a transverse section of the basal body (centriole) which has an electronopaque tubular envelope (compare with Fig. 6). × 36 000. Fro. 6. An early spermatid as in Fig. 5. The basal body microtubular doublets (arrows) diverge backward from the basal body in a funnel-like pattern. The mitochondria (M) are longer than at telophase (compare with Fig. 4) and are located within the lumen of the "axoneme." x 28 500. FIG. 7. A spermatid later than in Fig. 6. An electron-opaque material forms a cylinder enclosing the basal body and protruding at the anterior tip of the cell. The chromatin forms a spongy net at the periphery of the nucleus. M, mitochondria, x 28 500. Fro. 8. A spermatid later than in Fig. 7. The electron-opaque material concealing the basal body forms a mushroom cap-like structure which is supported by a stalk containing the diverging microtubular doublets (arrows). The chromatin forms an entanglement of wavy threads. M, mitochondria. × 28 500. Fro. 9. A spermatid later than in Fig. 8. Flat vesicles appear at the internal face of the mushroom cap and at the portion of the nucleus close to the cap (compare with Figs. 10-12). The chromatin forms threads running in the direction of the long axis of the nucleus, x 28 500.
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cell. The chromatin forms electron-opaque, interconnected lumps which are dispersed within an electron-lucid background and, therefore, display a less compact appearance than at telophase. The centriole (basal body) has an electron-opaque tubular envelope and is no longer connected to the nucleus (Fig. 5) but is very close and tangential to it (Fig. 6). An axoneme-like structure develops from the posterior end of the basal body. The microtubular doublets of this structure, however, diverge posteriorly in a funnel or conical pattern (Figs. 10-17) rather than run in parallel as they do in axonemes of regular cilia and flagella. Consequently, in slices cut perpendicular to the long axis of the spermatid, these microtubules appear circular only when sectioned close to their origin. However, in more posterior transverse slices, each of the microtubules appears oval because of the angle it forms with the plane of the section. The scattered mitochondria fuse into several longer mitochondria which appear arranged in tandem within the lumen of the funnellike "axoneme." These mitochondria conserve their cristae and form neither a Nebenkern, nor mitochondrial derivatives. Afterward (Fig. 7), the chromatin forms a spongy net evenly distributed at the pe-
riphery of the nucleus, leaving a central electron-lucid core. The basal body, surrounded by the cylinder of electron-opaque material, is protruding at the anterior tip of the spermatid, tightly wrapped by the cell membrane. Later on (Fig. 8), the chromatin forms an entanglement of wavy threads that are arranged roughly following the longitudinal axis of the nucleus. Simultaneously, the electron-opaque cylinder concealing the basal body transforms into a mushroom cup-like structure which is supported by a stalk containing the funnel-like "axoneme." The anterior part of the nucleus remains close, but apparently not attached, to the mushroom cap. Subsequently (Fig. 9), a row of flat vesicles begins to appear, first at the internal face of the mushroom cap and then, at the internal face of the nucleus as well. These vesicles begin to fuse from the anterior cap region to the posterior end of the nucleus until they merge with the intercellular space, as their membranes unite with the cell membrane (Figs. 10-15). At the end of this process, the cap and the nucleus appear connected to the "axoneme" stalk only at the central lower area of the cap containing the basal body (Figs. 18-23).
FIGS. 10-12. Spermatids somewhat later than in Fig. 9. Transverse sections. Fro. 10. The lower spermatid is sectioned through the basal body and the mushroom cap, while the upper one is sectioned through the diverging microtubular doublets and mitochondria (M). x 36 500. FIG. 11. A section between the two shown in Fig. 10; flat vesicles are found between the mushroom cap and the stalk containing the diverging microtubular doublets. × 36 500. FIG. 12. Two spermatids at a further posterior position than that of the one at the upper portion of Fig. 10 showing the 10 additional microtubular doublets (but not the 10 doublets developing from the basal body which have not reached this level at this stage of development). × 36 000. FIGs. 13-15. Spermatids later than in Figs. 10-12. Transverse sections. FIG. 13. A section through the anterior portion of the nucleus and the posterior rim of the cap which is partially separated from the stalk of the cell. × 36 500. FIG. 14. A section through the nucleus and the stalk which contains more microtubular doublets than the more anterior section in Fig. 13. × 36 500. FIG. 15. Sections posterior to that in Fig. 14 showing incomplete separation between nucleus and stalk. x 36 500. FIG. 16. Transverse sections of almost mature spermatozoa showing the anterior portions of the microtubular doublets which are close to the internal face of the cell surface or within finger-like projections of the cell membrane (arrows). x 36 500. FIG. 17. Transverse sections of spermatids as in Fig. 16. The trailing portions of the microtubular doublets are circled by cell membrane and appear separated from the main body of the stalk of the cell (arrows), x 36 500.
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Fie. 18. Mature spermatozoa. The mitochondria are smaller than in early developing spermatids (compare with Fig. 6) and are dispersed within the stalk of the cell. x 4700. Close to the b a s a l b o d y the " a x o n e m e " has in t r a n s v e r s e s e c t i o n s 10 m i c r o t u b u l a r doublets, 1 o f e a c h pair with e l e c t r o n - o p a q u e , the o t h e r with e l e c t r o n - t r a n s l u c e n t , l u m e n (Figs. 10 a n d 11). A n a d d i t i o n a l 11 d o u b l e t s
of i d e n t i c a l a p p e a r a n c e d e v e l o p at m o r e p o s t e r i o r p o s i t i o n s (Fig. 12). D u r i n g develo p m e n t , t h e s e t w o r o w s of d o u b l e t s are inc o r p o r a t e d into a single " a x o n e m e " (Figs. 13-15). B u t t r a n s v e r s e s e c t i o n s close to the
FIGS. 19-23. Spermatozoa in the female genital duct. FIG. 19. Tubular projections of the chitinous internal layer of the duct crisscross the trailing microtubular doublets of the spermatozoa (arrows). The spermatozoa contain small, isolated mitochondria (M). x 7880. FIG. 20. The cell membrane at the cap of the spermatozoa evaginates into pores of the chitinous internal layer of the duct. x 25 300. FIGS. 21-23. Transverse sections of the spermatozoa through the basal body (Fig. 21), the posterior tip of the nucleus and the anterior portion of the stalk (Fig. 22), and the posterior portion of the stalk (Fig. 23). x 25 300.
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SPERM OF HYDROPSYCHIDAE CADDISFLIES
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mushroom cap show fewer doublets than do those at more posterior locations (Figs. 13 and 14). At later stages of development (Figs. 1317): (1) The nucleus continues to elongate and the chromatin threads became gradually parallel and fuse into thicker structures until the nucleus is completely filled with highly electron-opaque chromatin. (2) The angle of divergence of the microtubules increases and the microtubular doublets " p u s h " the cell membrane and protrude from the cell surface wrapped by sleeves of cell membrane. The spermatid then resembles a maypole with trailing doublets suspended from the central "axoneme" pole. In more anterior transverse sections (Figs. 16 and 17), the nonprotruding portion of each doublet appears either close to the cell membrane of the "axoneme" stalk or within finger-like projections of this membrane. In more posterior transverse sections, the trailing portions of the doublets give the illusion of being isolated from the spermatid, within ovals of cell membrane. (3) The electron-opaque material of the mushroom cap, which surrounds the basal body at earlier stages, disappears and the basal body separates from the nucleus, which now appears posterior to the basal body (Fig. 18). (4) The long mitochondria divide into smaller isolated mitochondria, which appear to be distributed randomly within the ~'axoneme" stalk of the spermatozoon (Fig. 18). Testes of imagoes contain exclusively the mature spermatozoa, which develop as described here and which are shown schematically in Fig. 1. No other kind of spermatozoa could be detected. This is also the only kind of spermatozoa found in the diverse parts of the genital duct of inseminated females (Figs. 19-23). Their chromatin, however, is more fibrillar and less condensed than that of the intratesticular spermatozoa. They are oriented with the nuclei in the same direction within the duct. The internal wall of the duct is covered by a thin layer of chitin which has both numerous small pores and cylindrical pro-
cesses projecting into the lumen. The cell membrane evaginates from the anterior portion of the spermatozoa into these pores (Fig. 20), while the trailing microtubular doublets are in contact with the chitinous processes of the duct wall (Fig. 19). Under the light phase microscope, the spermatozoa isolated into a Ringer solution from either the ejaculatory duct or the inseminated female show no autonomous movements. We found, however, that spermatozoa having the typical kind of flagella, for example those of Locusta migratoria (Orthoptera) and Laspeyresia pomonella (Lepidoptera), do have strong autonomous movements when isolated under similar conditions. DISCUSSION
Polymorphism of Trichoptera spermatozoa. The spermatozoa we describe here belong to three different genera of the family H y d r o s p y c h i d a e (Annulipalpia). They share, however, the following characteristics which are not found in the representative model of insect spermatozoa (Baccetti, 1972): (a) lack of a connection between the nucleus and basal body; (b) lack of a centriole adjunct; (c) lack of an acrosome; (d) an aberrant axoneme with numerous doublets of microtubules which trail from the basal body and protrude as individual filamentous structures from the body of the cell; and (e) isolated mitochondria which conserve their original cristae, form no mitochondrial derivatives, and are located within the lumen of the aberrant axoneme. These characteristics, however, may have been secondarily derived during phylogenesis since at early stages of spermiogenesis the spermatid shows: (a) close relation between the nucleus and the basal body and (b) an electron-opaque material which surrounds the basal body and resembles a centriole adjunct. Phillips (1969) mentioned that in one Hydropsyche sp. the mature spermatozoon has an axoneme with the typical nine peripheral doublets of microtubules, but with,
SPERM OF HYDROPSYCH1DAE CADDISFLIES atypically, seven instead of the two central microtubules generally found in most axonemes. In another related species of Hydropsychidae (Macronema sp.), Phillips (1974) reported the typical 9+2 kind of axoneme. Either the spermatozoa of the Hydropsyche sp. and the Macronema sp. studied by Phillips differ greatly from those of the hydropsychid species we examined, or his species were misidentified. Phillips (1969) found the 9+7 type of axoneme also in the spermatozoa of Polycentropus sp. which belongs to another family, Polycentropodidae, of the same suborder, Annulipalpia. In the other suborder, Integripalpia, species of the families Leptoceridae (Baccetti et al., 1970) and Phryganeidae (Phillips, 1970) have spermatozoa with the regular 9+2 type of axoneme. Phillips (1969) mentioned that other species of Trichoptera have spermatozoa with the typical 9+2 axoneme. However, he did not indicate the species or families to which they belong. The scarce available data, which we review here, suggest that Annulipalpia species develop aberrant spermatozoa while the Integripalpia species maintain the basic model of insect spermatozoa. Phylogenetic dichotomy of the structure of the spermatozoa is well documented in other orders of insects (Dallai, 1979). In Trichoptera, however, further studies will be necessary to corroborate our hypothesis and to determine w h e t h e r any other e v o l u t i o n a r y changes may be inferred. Functioning of Hydropsychidae spermatozoa. It appears that Hydropsychidae spermatozoa have no autonomous movements since: (a) they lack real flagella and display extremely aberrant distribution of organelles which are generally related to sperm motility and (b) they do not move after being isolated in a Ringer solution from the male or female genital ducts, although the flagellate spermatozoa of other Trichoptera families do move under similar conditions (Khalifa, 1949). Therefore, another mechanism of spermatozoa move-
93
ment can be inferred to occur during fertilization in Hydropsychidae. In such a mechanism, movement of the spermatozoa could be caused by the peristaltic contractions of the female genital duct. The direction of movement, however, may depend on the presence of the tubular projections of the female genital wall duct into its lumen and the fact that the spermatozoa are all pointing in the same direction. The spermatozoa can move forward throughout the projections, but could be prevented from going backward as their trailing microtubular doublets engage the projections. Are apyrene spermatozoa present in Trichoptera? Trichoptera and Lepidoptera gametogenesis have in common several cytogenetic features such as female heterogamety, achiasmatic oogenesis, and holocentric chromosomes (Kiuta, 1971). In addition, light microscope studies (Klingstead, 1931) indicate that Trichoptera spermatogenesis is dichotomous, producing eupyrene (nucleated) and apyrene (anucleated) spermatozoa. Such a system characterizes lepidopteran spermatogenesis (Meves, 1903; Friedliinder et al., 1981). In Hydropsychidae, however, we found only one type of spermatozoa in both males and inseminated females. Also, the previously quoted ultrastructural studies on Trichoptera spermatozoa do not indicate the presence of any anucleated spermatozoa. These studies, however, were not intended to deal with the problem of the two kinds of spermatozoa. It is possible, therefore, that even if apyrene spermatozoa were present in the species they considered, such spermatozoa could have remained either unreported or unseen. Thus, the question of whether Hydropsychidae spermatogenesis is exceptional in Trichoptera by producing only nucleated spermatozoa or whether apyrene spermatozoa are generally absent from the order merits further attention. We are gratefulto Mr. CamilliusLay for his enthusiastic assistance in obtaininga regular, fresh supply of study specimensfor this research. The research was funded by a ClemsonUniversityAlumniVisitingPro-
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fessorship to M.F. This is the Technical Contribution 1902 of the South Carolina Agricultural Experiment Station, Clemson University. REFERENCES BACCETT1, B. (1972) Advan. Insect Physiol. 9, 315397. BACCETTL B., AND AFZELIUS, B. A. (1976) Monogr. Develop. Biol. 10, 1-254. BACCETT1, B., DALLAI, R., AND ROSATI, F. (1970) J. Ultrastruct. Res. 31, 212-228. DALLAI, R. (1979) in FAWCETT, D. W., AND BEDFORD, J. M. (Eds.), The Spermatozoon, pp. 253265, Urban & Schwarzenberg, Baltimore/Munich. FRIEDLA.NDER, M., JANS, P., AND BENZ, G. (1981) J. Insect Physiol. 27, 267-269. GRESSON, R. A. R. (1935) Quart. J. Microsc. Sci. 78, 311-327. KHALIFA, A. (1949) Trans. R. Entomol. Soc. London 100, 449471. KIUTA, B. (1971) Ergeb. Wiss. Unters. Schweiz. Natn. Park 9, 174-185.
KIUTA, B., AND KIUTA, M. A. J. E. (1979) Genetica (The Hague) 50, 119-126. K1UTA, B., AND LANKHORST,L. (1969) Genetica (The Hague) 40, 1-6. KLINGSTEDT, }-I. (1931) Acta Zool. Fenn. 10, 1-69. LANKHORST, L, (1970) Genen Phaenen 14, 9-14. LE LANN~C, J. (1975) Bull. Soc. Zool. Ft. 100, 539550. LtJTMAN, B. F. (1910) Biol. Bull. 19, 55-72. MEVES, F. (1903) Arch. Mikrosk. Anat. Entwicklungsmech. 61, 1-82. PCHAKAI~ZE, G. M. (1928) Arch. Russ. Anat. Histol. Embryol. 7, 297-303. PCHAKADZE, G. M. (1930) Arch. Russ. Anat. Histol. Embryol. 9, 311-321. PHILLIPS, D. M. (1969) J. Cell Biol. 40, 28--43. PHILLIPS, D. M. (1970) in BACCETTI, B. (Ed.), Comparative Spermatology, pp. 263-273, Academic Press, New York. PHILLIPS, D. M. (1974) Spermiogenesis, Academic Press, New York.
78, 84-94 (1982)
The Aberrant Spermatozoa of Hydropsychidae Caddisflies (Trichoptera): An Electron Microscope Analysis on Spermiogenesis MICHAEL FRIEDL,~NDER AND JOHN C. MORSE
Department of Biology, Ben Gurion University of the Negev, Beer Sheva, Israel and Department of Entomology, Fisheries and Wildlife, Clemson University, Clemson, South Carolina 29631 Received March 23, 1981, and in revised form July 15, 1981 The spermatozoon of caddisflies in the family Hydropsychidae lacks acrosome and centriole adjunct. The basal body and nucleus are separated. The axoneme is cone shaped and its microtubular doublets diverge, forming separate filaments which trail backward from the cell surface. The mitochondria do not fuse, conserve their cristae, form no derivatives, and appear within the lumen of the aberrant axoneme. Some of those may be secondarily derived characteristics since they are not present in early spermatids. Spermatozoa isolated into a Ringer solution from ejaculatory ducts or inseminated females show no autonomous movements. In the female, the spermatozoa may be moved by peristaltic contractions of the genital duct. The direction of their movement may be determined by tubular projections of the duct wall which engage the trailing microtubular doublets, thus impeding backward movement of the spermatozoa. No apyrene (anucleated) spermatozoa, similar to those characterizing Lepidoptera, could be found.
choptera stems from the difficulties encountered in obtaining suitable material for such studies. First, spermatid differentiation appears to occur in this order only close to, or after, pupation (Lankhorst, 1970; Le Lannic, 1975; Kiuta and Lankhorst, 1969). In the larvae, spermatogenesis does not advance further than meiotic prophase, while the adults contain only mature spermatozoa. Trichoptera pupae, however, are aquatic and relatively difficult both to collect in the field and to rear in the laboratory. Second, few entomologists are specialized in this small order and are able to determine with certainty the species of the larval instars and the pupae. We report here the first ultrastructural analysis of spermatid differentiation in Trichoptera. Spermatozoa in the inseminated female were studied to determine whether they undergo further transformation after ejaculation.
" T h e numerous works that have appeared in recent years on spermatogenesis of insects have covered practically every order . . . . There still remain, however, a small number of small orders which have not been investigated . . . . The Trichoptera is one of these small orders" (Lutman, 1910). Since this statement was made, we are aware of only one light microscope study on T r i c h o p t e r a spermiogenesis (Gresson, 1935). The other light microscope data after 1910 on spermatogenesis refer mainly to the behavior and number of chromosomes during meiosis (Pchakadze, 1928, 1930; Klingstedt, 1931; Kiuta and Kiuta, 1979). The only published ultrastructural data on Trichoptera spermiogenesis refer to the mature spermatozoa (Baccetti et al.. 1970; Phillips, 1969, 1974). That is a remarkable situation since there is a vast literature on ultrastructural studies of spermiogenesis in numerous other systematic groups of insects (e.g., Baccetti, 1972; Baccetti and Afzelius, 1976). Most probably, this lack of information on spermatogenesis of Tri-
MATERIALS AND METHODS
Diplectrona modesta, Hydropsyche sparna, and Cheumatopsyche spp. (Hydropsychinae, Hydropsychidae) from the suborder Annulipalpia in caddis84
0022-5320/82/010084-11 $02.00/0 Copyright © 1982 by Academic Press, inc. All rights of reproduction in any form reserved.
SPERM OF HYDROPSYCHIDAE CADDISFLIES flies (Trichoptera) were collected in the Piedmont region of South Carolina. Testes of larvae, pupae, and imagoes, ejaculatory ducts of imagoes, and genital ducts of inseminated females were fixed in 3% glutaraldehyde in 0.2 M cacodylate buffer, pH 7.3. Subsequently, the tissues were postfixed in 1% OsO4, dehydrated, and embedded in Epon. For electron microscopy, ultrathin sections were contrasted with uranyl acetate and lead citrate. For light microscopy, thick sections were stained in 1% toluidine blue. Living cells of the same tissues were dispersed in a Ringer solution and studied with phase optics. For comparison, living testicular cells of Locusta migratoria (Orthoptera) and Laspeyresia pomonella (Lepidoptera) were also dispersed in the same Ringer solution and studied with phase optics. RESULTS
The results were similar in all the species studied and the micrographs are from cells of Diplectrona modesta. Primary spermatocytes (Figs. 2 and 3) contain two pairs of short centrioles which remain throughout meiosis about half-way between the nucleus and the cell membrane. These centrioles neither develop axonemes, nor do they attach to mitochondria or other membranous structures. At the end of the telophase (Fig. 4), the forming nucleus contains highly condensed (elec-
85
Fro. 1. Schematic drawing of a mature spermatozoon cut through the median plane of symmetry. The trailing microtubular doublets are represented by one solid line, surrounded by cell membrane.
tron-opaque) chromatin, is located close to the cell surface, and has a bowl-shaped outline with the concavity toward the former spindle pole. The centriole is attached by one end to the concave face of the nucleus, the other end is free and directed to the cell surface. This centriole is about twice as long as those of the early spermatocytes (compare with Fig. 3). The mitochondria are similar to those of the spermatocytes and are dispersed within the cytoplasm facing the convex side of the nucleus. At the beginning of spermatid elongation (Figs. 5 and 6) the nucleus is irregular, ovoid, and located at the anterior end of the
Fro. 2. A primary spermatocyte showing a transverse section of one of the four centrioles, about half-way between the nucleus and the cell surface, x 28 500. FIG. 3. A primary spermatocyte as in Fig. 2 showing a longitudinal section of one centriole which is about half as long as that at telophase (compare with Fig. 4) and is disconnected from the mitochondria or other membranous structures, x 28 500. FIG. 4. Meiotic telophase. The nucleus has a bowl-shaped outline. The centriole is about twice as long as those found at prophase (compare with Fig. 3) and is attached by one end of the concave face of the nucleus. The mitochondria (M) are similar to those of the spermatocytes (compare with Fig. 2) and are dispersed within the cytoplasm. × 28 500. FIG. 5. An early spermatid showing a transverse section of the basal body (centriole) which has an electronopaque tubular envelope (compare with Fig. 6). × 36 000. Fro. 6. An early spermatid as in Fig. 5. The basal body microtubular doublets (arrows) diverge backward from the basal body in a funnel-like pattern. The mitochondria (M) are longer than at telophase (compare with Fig. 4) and are located within the lumen of the "axoneme." x 28 500. FIG. 7. A spermatid later than in Fig. 6. An electron-opaque material forms a cylinder enclosing the basal body and protruding at the anterior tip of the cell. The chromatin forms a spongy net at the periphery of the nucleus. M, mitochondria, x 28 500. Fro. 8. A spermatid later than in Fig. 7. The electron-opaque material concealing the basal body forms a mushroom cap-like structure which is supported by a stalk containing the diverging microtubular doublets (arrows). The chromatin forms an entanglement of wavy threads. M, mitochondria. × 28 500. Fro. 9. A spermatid later than in Fig. 8. Flat vesicles appear at the internal face of the mushroom cap and at the portion of the nucleus close to the cap (compare with Figs. 10-12). The chromatin forms threads running in the direction of the long axis of the nucleus, x 28 500.
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cell. The chromatin forms electron-opaque, interconnected lumps which are dispersed within an electron-lucid background and, therefore, display a less compact appearance than at telophase. The centriole (basal body) has an electron-opaque tubular envelope and is no longer connected to the nucleus (Fig. 5) but is very close and tangential to it (Fig. 6). An axoneme-like structure develops from the posterior end of the basal body. The microtubular doublets of this structure, however, diverge posteriorly in a funnel or conical pattern (Figs. 10-17) rather than run in parallel as they do in axonemes of regular cilia and flagella. Consequently, in slices cut perpendicular to the long axis of the spermatid, these microtubules appear circular only when sectioned close to their origin. However, in more posterior transverse slices, each of the microtubules appears oval because of the angle it forms with the plane of the section. The scattered mitochondria fuse into several longer mitochondria which appear arranged in tandem within the lumen of the funnellike "axoneme." These mitochondria conserve their cristae and form neither a Nebenkern, nor mitochondrial derivatives. Afterward (Fig. 7), the chromatin forms a spongy net evenly distributed at the pe-
riphery of the nucleus, leaving a central electron-lucid core. The basal body, surrounded by the cylinder of electron-opaque material, is protruding at the anterior tip of the spermatid, tightly wrapped by the cell membrane. Later on (Fig. 8), the chromatin forms an entanglement of wavy threads that are arranged roughly following the longitudinal axis of the nucleus. Simultaneously, the electron-opaque cylinder concealing the basal body transforms into a mushroom cup-like structure which is supported by a stalk containing the funnel-like "axoneme." The anterior part of the nucleus remains close, but apparently not attached, to the mushroom cap. Subsequently (Fig. 9), a row of flat vesicles begins to appear, first at the internal face of the mushroom cap and then, at the internal face of the nucleus as well. These vesicles begin to fuse from the anterior cap region to the posterior end of the nucleus until they merge with the intercellular space, as their membranes unite with the cell membrane (Figs. 10-15). At the end of this process, the cap and the nucleus appear connected to the "axoneme" stalk only at the central lower area of the cap containing the basal body (Figs. 18-23).
FIGS. 10-12. Spermatids somewhat later than in Fig. 9. Transverse sections. Fro. 10. The lower spermatid is sectioned through the basal body and the mushroom cap, while the upper one is sectioned through the diverging microtubular doublets and mitochondria (M). x 36 500. FIG. 11. A section between the two shown in Fig. 10; flat vesicles are found between the mushroom cap and the stalk containing the diverging microtubular doublets. × 36 500. FIG. 12. Two spermatids at a further posterior position than that of the one at the upper portion of Fig. 10 showing the 10 additional microtubular doublets (but not the 10 doublets developing from the basal body which have not reached this level at this stage of development). × 36 000. FIGs. 13-15. Spermatids later than in Figs. 10-12. Transverse sections. FIG. 13. A section through the anterior portion of the nucleus and the posterior rim of the cap which is partially separated from the stalk of the cell. × 36 500. FIG. 14. A section through the nucleus and the stalk which contains more microtubular doublets than the more anterior section in Fig. 13. × 36 500. FIG. 15. Sections posterior to that in Fig. 14 showing incomplete separation between nucleus and stalk. x 36 500. FIG. 16. Transverse sections of almost mature spermatozoa showing the anterior portions of the microtubular doublets which are close to the internal face of the cell surface or within finger-like projections of the cell membrane (arrows). x 36 500. FIG. 17. Transverse sections of spermatids as in Fig. 16. The trailing portions of the microtubular doublets are circled by cell membrane and appear separated from the main body of the stalk of the cell (arrows), x 36 500.
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Fie. 18. Mature spermatozoa. The mitochondria are smaller than in early developing spermatids (compare with Fig. 6) and are dispersed within the stalk of the cell. x 4700. Close to the b a s a l b o d y the " a x o n e m e " has in t r a n s v e r s e s e c t i o n s 10 m i c r o t u b u l a r doublets, 1 o f e a c h pair with e l e c t r o n - o p a q u e , the o t h e r with e l e c t r o n - t r a n s l u c e n t , l u m e n (Figs. 10 a n d 11). A n a d d i t i o n a l 11 d o u b l e t s
of i d e n t i c a l a p p e a r a n c e d e v e l o p at m o r e p o s t e r i o r p o s i t i o n s (Fig. 12). D u r i n g develo p m e n t , t h e s e t w o r o w s of d o u b l e t s are inc o r p o r a t e d into a single " a x o n e m e " (Figs. 13-15). B u t t r a n s v e r s e s e c t i o n s close to the
FIGS. 19-23. Spermatozoa in the female genital duct. FIG. 19. Tubular projections of the chitinous internal layer of the duct crisscross the trailing microtubular doublets of the spermatozoa (arrows). The spermatozoa contain small, isolated mitochondria (M). x 7880. FIG. 20. The cell membrane at the cap of the spermatozoa evaginates into pores of the chitinous internal layer of the duct. x 25 300. FIGS. 21-23. Transverse sections of the spermatozoa through the basal body (Fig. 21), the posterior tip of the nucleus and the anterior portion of the stalk (Fig. 22), and the posterior portion of the stalk (Fig. 23). x 25 300.
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SPERM OF HYDROPSYCHIDAE CADDISFLIES
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mushroom cap show fewer doublets than do those at more posterior locations (Figs. 13 and 14). At later stages of development (Figs. 1317): (1) The nucleus continues to elongate and the chromatin threads became gradually parallel and fuse into thicker structures until the nucleus is completely filled with highly electron-opaque chromatin. (2) The angle of divergence of the microtubules increases and the microtubular doublets " p u s h " the cell membrane and protrude from the cell surface wrapped by sleeves of cell membrane. The spermatid then resembles a maypole with trailing doublets suspended from the central "axoneme" pole. In more anterior transverse sections (Figs. 16 and 17), the nonprotruding portion of each doublet appears either close to the cell membrane of the "axoneme" stalk or within finger-like projections of this membrane. In more posterior transverse sections, the trailing portions of the doublets give the illusion of being isolated from the spermatid, within ovals of cell membrane. (3) The electron-opaque material of the mushroom cap, which surrounds the basal body at earlier stages, disappears and the basal body separates from the nucleus, which now appears posterior to the basal body (Fig. 18). (4) The long mitochondria divide into smaller isolated mitochondria, which appear to be distributed randomly within the ~'axoneme" stalk of the spermatozoon (Fig. 18). Testes of imagoes contain exclusively the mature spermatozoa, which develop as described here and which are shown schematically in Fig. 1. No other kind of spermatozoa could be detected. This is also the only kind of spermatozoa found in the diverse parts of the genital duct of inseminated females (Figs. 19-23). Their chromatin, however, is more fibrillar and less condensed than that of the intratesticular spermatozoa. They are oriented with the nuclei in the same direction within the duct. The internal wall of the duct is covered by a thin layer of chitin which has both numerous small pores and cylindrical pro-
cesses projecting into the lumen. The cell membrane evaginates from the anterior portion of the spermatozoa into these pores (Fig. 20), while the trailing microtubular doublets are in contact with the chitinous processes of the duct wall (Fig. 19). Under the light phase microscope, the spermatozoa isolated into a Ringer solution from either the ejaculatory duct or the inseminated female show no autonomous movements. We found, however, that spermatozoa having the typical kind of flagella, for example those of Locusta migratoria (Orthoptera) and Laspeyresia pomonella (Lepidoptera), do have strong autonomous movements when isolated under similar conditions. DISCUSSION
Polymorphism of Trichoptera spermatozoa. The spermatozoa we describe here belong to three different genera of the family H y d r o s p y c h i d a e (Annulipalpia). They share, however, the following characteristics which are not found in the representative model of insect spermatozoa (Baccetti, 1972): (a) lack of a connection between the nucleus and basal body; (b) lack of a centriole adjunct; (c) lack of an acrosome; (d) an aberrant axoneme with numerous doublets of microtubules which trail from the basal body and protrude as individual filamentous structures from the body of the cell; and (e) isolated mitochondria which conserve their original cristae, form no mitochondrial derivatives, and are located within the lumen of the aberrant axoneme. These characteristics, however, may have been secondarily derived during phylogenesis since at early stages of spermiogenesis the spermatid shows: (a) close relation between the nucleus and the basal body and (b) an electron-opaque material which surrounds the basal body and resembles a centriole adjunct. Phillips (1969) mentioned that in one Hydropsyche sp. the mature spermatozoon has an axoneme with the typical nine peripheral doublets of microtubules, but with,
SPERM OF HYDROPSYCH1DAE CADDISFLIES atypically, seven instead of the two central microtubules generally found in most axonemes. In another related species of Hydropsychidae (Macronema sp.), Phillips (1974) reported the typical 9+2 kind of axoneme. Either the spermatozoa of the Hydropsyche sp. and the Macronema sp. studied by Phillips differ greatly from those of the hydropsychid species we examined, or his species were misidentified. Phillips (1969) found the 9+7 type of axoneme also in the spermatozoa of Polycentropus sp. which belongs to another family, Polycentropodidae, of the same suborder, Annulipalpia. In the other suborder, Integripalpia, species of the families Leptoceridae (Baccetti et al., 1970) and Phryganeidae (Phillips, 1970) have spermatozoa with the regular 9+2 type of axoneme. Phillips (1969) mentioned that other species of Trichoptera have spermatozoa with the typical 9+2 axoneme. However, he did not indicate the species or families to which they belong. The scarce available data, which we review here, suggest that Annulipalpia species develop aberrant spermatozoa while the Integripalpia species maintain the basic model of insect spermatozoa. Phylogenetic dichotomy of the structure of the spermatozoa is well documented in other orders of insects (Dallai, 1979). In Trichoptera, however, further studies will be necessary to corroborate our hypothesis and to determine w h e t h e r any other e v o l u t i o n a r y changes may be inferred. Functioning of Hydropsychidae spermatozoa. It appears that Hydropsychidae spermatozoa have no autonomous movements since: (a) they lack real flagella and display extremely aberrant distribution of organelles which are generally related to sperm motility and (b) they do not move after being isolated in a Ringer solution from the male or female genital ducts, although the flagellate spermatozoa of other Trichoptera families do move under similar conditions (Khalifa, 1949). Therefore, another mechanism of spermatozoa move-
93
ment can be inferred to occur during fertilization in Hydropsychidae. In such a mechanism, movement of the spermatozoa could be caused by the peristaltic contractions of the female genital duct. The direction of movement, however, may depend on the presence of the tubular projections of the female genital wall duct into its lumen and the fact that the spermatozoa are all pointing in the same direction. The spermatozoa can move forward throughout the projections, but could be prevented from going backward as their trailing microtubular doublets engage the projections. Are apyrene spermatozoa present in Trichoptera? Trichoptera and Lepidoptera gametogenesis have in common several cytogenetic features such as female heterogamety, achiasmatic oogenesis, and holocentric chromosomes (Kiuta, 1971). In addition, light microscope studies (Klingstead, 1931) indicate that Trichoptera spermatogenesis is dichotomous, producing eupyrene (nucleated) and apyrene (anucleated) spermatozoa. Such a system characterizes lepidopteran spermatogenesis (Meves, 1903; Friedliinder et al., 1981). In Hydropsychidae, however, we found only one type of spermatozoa in both males and inseminated females. Also, the previously quoted ultrastructural studies on Trichoptera spermatozoa do not indicate the presence of any anucleated spermatozoa. These studies, however, were not intended to deal with the problem of the two kinds of spermatozoa. It is possible, therefore, that even if apyrene spermatozoa were present in the species they considered, such spermatozoa could have remained either unreported or unseen. Thus, the question of whether Hydropsychidae spermatogenesis is exceptional in Trichoptera by producing only nucleated spermatozoa or whether apyrene spermatozoa are generally absent from the order merits further attention. We are gratefulto Mr. CamilliusLay for his enthusiastic assistance in obtaininga regular, fresh supply of study specimensfor this research. The research was funded by a ClemsonUniversityAlumniVisitingPro-
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fessorship to M.F. This is the Technical Contribution 1902 of the South Carolina Agricultural Experiment Station, Clemson University. REFERENCES BACCETT1, B. (1972) Advan. Insect Physiol. 9, 315397. BACCETTL B., AND AFZELIUS, B. A. (1976) Monogr. Develop. Biol. 10, 1-254. BACCETT1, B., DALLAI, R., AND ROSATI, F. (1970) J. Ultrastruct. Res. 31, 212-228. DALLAI, R. (1979) in FAWCETT, D. W., AND BEDFORD, J. M. (Eds.), The Spermatozoon, pp. 253265, Urban & Schwarzenberg, Baltimore/Munich. FRIEDLA.NDER, M., JANS, P., AND BENZ, G. (1981) J. Insect Physiol. 27, 267-269. GRESSON, R. A. R. (1935) Quart. J. Microsc. Sci. 78, 311-327. KHALIFA, A. (1949) Trans. R. Entomol. Soc. London 100, 449471. KIUTA, B. (1971) Ergeb. Wiss. Unters. Schweiz. Natn. Park 9, 174-185.
KIUTA, B., AND KIUTA, M. A. J. E. (1979) Genetica (The Hague) 50, 119-126. K1UTA, B., AND LANKHORST,L. (1969) Genetica (The Hague) 40, 1-6. KLINGSTEDT, }-I. (1931) Acta Zool. Fenn. 10, 1-69. LANKHORST, L, (1970) Genen Phaenen 14, 9-14. LE LANN~C, J. (1975) Bull. Soc. Zool. Ft. 100, 539550. LtJTMAN, B. F. (1910) Biol. Bull. 19, 55-72. MEVES, F. (1903) Arch. Mikrosk. Anat. Entwicklungsmech. 61, 1-82. PCHAKAI~ZE, G. M. (1928) Arch. Russ. Anat. Histol. Embryol. 7, 297-303. PCHAKADZE, G. M. (1930) Arch. Russ. Anat. Histol. Embryol. 9, 311-321. PHILLIPS, D. M. (1969) J. Cell Biol. 40, 28--43. PHILLIPS, D. M. (1970) in BACCETTI, B. (Ed.), Comparative Spermatology, pp. 263-273, Academic Press, New York. PHILLIPS, D. M. (1974) Spermiogenesis, Academic Press, New York.