- Email: [email protected]
Ultrastructural and polarized light microscope studies on spermatophores of Dicyrtoma ornata (Insecta, Collembola)
JOURNAL
OF ULTRASTRUCTURE
RESEARCH
50, 355-361
(1975)
Ultrastructural and Polarized Light Microscope Studies on Spermatophores of Dicyrtoma ornata (Insecta, Collembola} ROMANO DALLAI Institute of Zoology of the University of Siena, Via Mattioli 4, Siena, Italy Received August 21, 1974 The author describes the ultrastructure of the spermatophore in Dicyrtoma ornata (Collembola). It presents a long stem and an apical spermatic droplet inside which there are many incysted sperms. No external membrane surrounds the apical droplet. dehyde at pH 7.2. After .5 1 hr, the spermatophores were washed in Hoyle buffer, fixed in 1% osmium tetroxide for 30 min, dehydrated in ethanol and embedded in Epon. Thin sections obtained with LKB microtome were examined with a Siemens Elmiskop I A electron microscope after staining in uranyl acetate and lead citrate. Enzyme digestion with 1.5% pepsin was carried out by Behnke and Forer's method (1). For polarization microscopy a Leitz Ortholux microscope with a mercury lamp was used. The retardation values were calculated with an elliptic compensator; the spermatophores were previously treated with a series of fluids having different refraction values: water, ethanol (absolute, 70°-90°), chloroform monobromonaphthaline and methylene-iodide, either pure or mixed at different percentages. The refractive values was determined with a Zeiss refractometer. PAS reaction and enzyme digestions were carried out with different concentrations of pepsin, trypsin, and chitinase. Topochemical tests were made with phenol, picric acid, and clove oil. The material for scanning electron microscopy was fixed for 5-15 rain in Hoyle buffered glutaraldehyde, washed in distilled water and mounted on little disks, metal shadowed with gold and studied with a JEOL electron microscope.
I n C o l l e m b o l a , like in m a n y other terrest r i a l A r t h r o p o d a (10), n o m a t i n g occurs; f e r t i l i z a t i o n is c a r r i e d o u t b y m e a n s of particular structures called spermatophores produced by the male. Such struct u r e s are u s u a l l y d e p o s i t e d after long q u a i n t c o u r t i n g s (2) t h o u g h t h e r e are m a n y i n s t a n c e s of s p e r m a t o p h o r e s p r o d u c e d b y t h e m a l e alone, w i t h o u t t h e p r e s e n c e of t h e f e m a l e (6). T h e r e are m a n y l i g h t m i c r o s c o p i c a l d a t a r e l a t i n g to t h e m o r p h o l o g y of C o l l e m b o l a s p e r m a t o p h o r e s . T h e first f i n d i n g s des c r i b e d b y S c h a l l e r (9) were followed b y t h o s e of M a y e r (8), H a l e (6), S c h l i w a (11), a n d r e c e n t l y b y t h e c o n t r i b u t i o n s of Cass a g n a u (3, 4) a n d M a s s o u d (7). T h e p u r p o s e of t h i s work is twofold: to specify t h e fine s t r u c t u r e a n d c h e m i c a l n a t u r e of C o l l e m b o l a s p e r m a t o p h o r e s , a n d to d e t e c t e v e n t u a l u l t r a s t r u c t u r a l differences between spermatozoa in spermatop h o r e s a n d i n t h e m a l e g e n i t a l d u c t s . As far as I a m a w a r e t h i s k i n d of a r t h r o p o d sperm a t o p h o r e s has n o t b e e n p r e v i o u s l y examined by electron microscopy.
POLARIZION MICROSCOPE OBSERVATIONS; CHARACTERS OF THE BIREFRINGENCE
MATERIALS AND METHODS Male and female specimens of Dicyrtorna ornata found under stones in the Botanical garden of the University of Siena were placed in small containers with polytene lids partially filled with a mixture of plaster of Paris and clay at 8-10°C. The spermatophores were removed with a needle and fixed either in Hoyle buffered 5% glutaraldehyde with the addition of 5% sucrose or in phosphate buffered 4% paraformal-
S p e r m a t o p h o r e s are s e e n as c l e a r l y biref r a c t i v e s t r u c t u r e s w h e n e x a m i n e d in dist i l l e d water. T h e r e t a r d a t i o n v a l u e of t h e b i r e f r i n g e n c e is a b o u t 45. T h e axis of b i r e f r i n g e n c e is m a i n l y l o n g i t u d i n a l . T h e s p e r m a t o p h o r e s were t r e a t e d w i t h f l u i d s of p r o g r e s s i v e l y i n c r e a s i n g r e f r a c t i o n v a l u e s , from d i s t i l l e d w a t e r ( r e f r a c t i o n i n d e x 1.33) u p to m e t h y l e n e iodide (1.74).
355 Copyright© 1975by AcademicPress, Inc. Allrights of reproductionin any formreserved.
356
ROMANO
DALLAI
COLLEMBOLA SPERMATOPHORES The birefringence of these structures appears to be partially related to the refraction indices; the r e t a r d a t i o n values decrease as the refraction index increase up to 1.54 where it reaches a m i n i m u m . Thereafter the values increase again so as to reach values which are similar to those in water. On the basis of these d a t a the curve of the r e t a r d a t i o n values has been drawn (Fig. 9). The total birefringence has a double nature: one is an intrinsic birefringence which does not change with the refraction index, the other is textural, depending on the submicroscopic organization of the materials which build up a complex body according to Wiener. The structural birefringence of spermatophores treated with alcoholic phenol solution or with clove oil remains unaltered but when they are treated with picric acid, the birefringence appears inverted. Even after prolonged e n z y m e t r e a t m e n t with pepsin or trypsin a t concentrations ranging from 0.5% to 1.5%, and periodical renewal of the enzyme, the morphology of the s p e r m a t o p h o r e s appears unaltered; birefringence does not change. Also, treatm e n t with 1% chitinase does not seem to alter the structural material of the spermatophores. SCANNING ELECTRON MICROSCOPE OBSERVATIONS A s p e r m a t o p h o r e consists of a stem bearing a spermatic droplet (Fig. 2). The stem is about 500 t~m long with a diameter ranging from 8.5 tLm at the apex. The droplet m a y occasionally be almost spherical with a diameter of 37-45 p m but as a
357
m~ 656O555045
O
30-
25~ 20 15105-
"D
FIa. 9. Curve of retardation values. Abscissa: refractive index of the imbibition liquid. Ordinate: retardation in A units. rule it is elliptical with a diameter of 35 × 25 ttm. Under normal conditions h u m i d i t y is condensed on the spermatic droplet m a k i n g it appear spherical and larger t h a n it actually is (Fig. 1). The stem is m a d e of fibrous material oriented along its major axis but not strictly orthogonal at the basis (Fig. 6). The base of the stem is wide, made to adhere firmly to the s u b s t r a t u m (Fig. 7). M a n y times in a midportion the stem has a swelling (Fig. 2). At the apex a shieldlike swelling is constantly present (Fig. 3). This is where the spermatic droplet resides (the longer side of the shield measures a b o u t 15 ttm, the shorter 10-12 ttm). The surface of the droplet is very uneven showing tiny depressions and protrusions (Figs. 3, 8). A
FIc. 1. Dicyrtoma ornata male near two spermatophores (arrows). × 20. Fro. 2. Spermatophore as seen with the Scanning electron microscope. The stem shows a swelling. At the tip of the stem there is the spermatic droplet. × 300. FIG. 3. A shield-like swelling bears the spermatic droplet. Scanning electron microscope. × 2 000. FIo. 4. Stem of the spermatophore attached to a little piece of wood.Polysaccharides are present only at the level of the wood. PAS reaction. × 150. FIG. 5. Apical part of the stem as seen with the interference contrast microscope. × I 000. Fia. 6. Middle portion of the stem at the Scanning electron microscope, x 3 000. FIa. 7. Base of the stem adhering to the substratum. Scanning electron microscope, x 3 000. FIG. 8. The apical droplet showing a pointed structure at its tip. Scanning electron microscope. × 1 000.
358
ROMANO DALLAI
FIG. 10. Cross section of the apical droplet of Dicyrtoma ornata. S, section of the stem inside the droplet three sperms are also to be seen (arrows). x 21 000. FIG. 11. Section of the shieldlike swelling at the tip of the stem. x 30 000. FIG. 12. Cross-section through the middle of the stem. x 13 000.
Fro. 13. Cross-section of t h e apical droplet. M a n y s p e r m s (arrows) are to be seen. E a c h of t h e m is in a cyst a n d p r e s e n t s a n u c l e u s (n) a n d a flagellum (f) w o u n d u p r o u n d a bulky m a s s (b). G r a n u l a r m a t e r i a l is interposed b e t w e e n t h e s p e r m s . × 18 000. FIG. 14. Cross- a n d longitudinal-sections of t h e p e d u n c l e which in the m a l e genital a p p a r a t u s c o n n e c t e d t h e s p e r m s to t h e deferent wall. × 38 000. FIG. 15. T h e very t h i n dense layer p r e s e n t at the periphery of t h e droplet (arrows). × 30 000. 359
360
ROMANO DALLAI
by a formation 2 #m in diameter the position and structure of which indicate it to be the direct continuation of the stem (Figs. 5, 10); both the axial formation and the stem, in fact, consists of a compact bundle of fibres ranging in diameter from 500 /~ to 2500 /~ (Figs. 10-12). The constituting material is moderately electron dense and very likely has a proteinic nature; sections treated with 1.5% pepsin, in fact, present the material only partially digested. At the tip of the stem a certain amount of thicker and denser material than that of the stem itself forms a layer about 0.5-1.2 tLm thick. This layer is folded upon itself to form the shield-shaped body mentioned above (Fig. 11). Each spermatozoon in a cyst has a diameter of 4.5 #m. The acrosome, nucleus and flagellum are curiously wound up round a bulky mass (Figs. 10, 13L The nucleus forms three proximal helical turns; the flagellum another four or five turns (Fig. FIo. 16. Schematicdrawing of a spermatophore of 13). Longitudinal and transverse sections Dicyrtoma ornata. of the stem connecting the spermatozoon to the wall of the male genital ducts are pointed structure is located at the tip of the frequently present also in the material droplet and on the axis of the stem (Fig. 8). interposed between the spermatozoa (Fig. TRANSMISSION ELECTRONMICROSCOPE OB- 14). SERVATIONS The structure of the sperms in the apical Each spermatic droplet contains about droplet does not differ appreciably from 600 spermatozoa within a finely granular the structure of those in the male genital material (assuming a spherical droplet size ducts (5); both types of sperms are wound of 45 #m and a spherical spermatozoon size up round a dense central mass. Both have of 4.5 tLm). Calculations were carried out the same long anterior peduncle which on the basis of the electron microscope connected them to the deferent wall. observations concerning the number of CONCLUSIONS spermatozoa found in several cross sections The morphology of D i c y r t o m a ornata of the same spermatic droplet. A thick layer of the same granular material (7 urn) spermatophores is similar to that of D. is present also at the base and apex of the m i n u t a described by Hale (6). Our studies droplet. No external membrane seems to enable us to confirm Hale's observations exist; the spermatic droplet is externally with the light microscope regarding the limited by a very thin (600-700 A) layer of existence of an axis inside the spermatic material of the same kind as that found in droplet and extend his data by showing between the spermatozoa only more elec- that the droplet rests on a shieldlike suptron dense (Fig. 15). port. The spermatic droplet is axially crossed Polarization microscopic and electron
COLLEMBOLA SPERMATOPHORES
microscopic observations give evidence for the presence of a mixed filamentous body which probably is proteinic, although it is resistant to pepsin and trypsin. The proteinic nature of the material is evidenced by its PAS negativity (Fig. 4). This is in contrast with Schliwa's findings (11) which instead indicated glycogen as one of the components of the stem. Chitin seems to be absent in the spermatophore since enzyme digestion with chitinase does not affect its structure. A mixed filamentous body is found also by electron microscopy where the stem presents bundles of structures having the same orientation as the major axis of the spermatophore. The fact that the topochemical data differ from those obtained with the structures containing collagen is most singular and excludes the possibility that the components of the spermatophore belong to this class of substances. Many authors who have studied the
361
morphology of the apical spermatic droplet conclude that it is limited by an external membrane. It does not seem proper to speak of a real membrane around the spermatic droplet, however, but rather of a condensation of the fine granular material surrounding the spermatozoon. REFERENCES 1. BEHNKE, O., AND FORER, A., J. Cell Sci. 2, 169 (1967). 2. BRETFELD,G., Sond. Z. fl zool. Syst. Evol. 8, 259 (1970). 3. CASSAGNAU,P., Bull. Soc. Hist. natur. Toulouse 107, 279 (1971). 4. CASSAGNAU,P., Rev. Ecol. Biol. Sol 8, 609 (1971). 5. DALLAI,R., in BACCETTI, B. (Ed.), Comparative Spermatology, p. 275. Academic Press, New York, 1970. 6. HALE, W. G., Pedobiologia 5, 146 (1965). 7. MASSOUD,Z., in PESSON, P. (Ed.), La vie dans les sols, p. 337. Gauthier-Villars, Paris, 1971. 8. MAYER,H., Zool. Jb. Syst. 85, 501 (1957). 9. SCHALLER,f., Naturwissenscha#en 39, 48 (1952). 10. SCHALLER,F., Ann. Rev. Entomol. 16, 407 (1971). 11. SCHLIWA,W., Zool. Jb. Ana[. 82,445 (1965).
OF ULTRASTRUCTURE
RESEARCH
50, 355-361
(1975)
Ultrastructural and Polarized Light Microscope Studies on Spermatophores of Dicyrtoma ornata (Insecta, Collembola} ROMANO DALLAI Institute of Zoology of the University of Siena, Via Mattioli 4, Siena, Italy Received August 21, 1974 The author describes the ultrastructure of the spermatophore in Dicyrtoma ornata (Collembola). It presents a long stem and an apical spermatic droplet inside which there are many incysted sperms. No external membrane surrounds the apical droplet. dehyde at pH 7.2. After .5 1 hr, the spermatophores were washed in Hoyle buffer, fixed in 1% osmium tetroxide for 30 min, dehydrated in ethanol and embedded in Epon. Thin sections obtained with LKB microtome were examined with a Siemens Elmiskop I A electron microscope after staining in uranyl acetate and lead citrate. Enzyme digestion with 1.5% pepsin was carried out by Behnke and Forer's method (1). For polarization microscopy a Leitz Ortholux microscope with a mercury lamp was used. The retardation values were calculated with an elliptic compensator; the spermatophores were previously treated with a series of fluids having different refraction values: water, ethanol (absolute, 70°-90°), chloroform monobromonaphthaline and methylene-iodide, either pure or mixed at different percentages. The refractive values was determined with a Zeiss refractometer. PAS reaction and enzyme digestions were carried out with different concentrations of pepsin, trypsin, and chitinase. Topochemical tests were made with phenol, picric acid, and clove oil. The material for scanning electron microscopy was fixed for 5-15 rain in Hoyle buffered glutaraldehyde, washed in distilled water and mounted on little disks, metal shadowed with gold and studied with a JEOL electron microscope.
I n C o l l e m b o l a , like in m a n y other terrest r i a l A r t h r o p o d a (10), n o m a t i n g occurs; f e r t i l i z a t i o n is c a r r i e d o u t b y m e a n s of particular structures called spermatophores produced by the male. Such struct u r e s are u s u a l l y d e p o s i t e d after long q u a i n t c o u r t i n g s (2) t h o u g h t h e r e are m a n y i n s t a n c e s of s p e r m a t o p h o r e s p r o d u c e d b y t h e m a l e alone, w i t h o u t t h e p r e s e n c e of t h e f e m a l e (6). T h e r e are m a n y l i g h t m i c r o s c o p i c a l d a t a r e l a t i n g to t h e m o r p h o l o g y of C o l l e m b o l a s p e r m a t o p h o r e s . T h e first f i n d i n g s des c r i b e d b y S c h a l l e r (9) were followed b y t h o s e of M a y e r (8), H a l e (6), S c h l i w a (11), a n d r e c e n t l y b y t h e c o n t r i b u t i o n s of Cass a g n a u (3, 4) a n d M a s s o u d (7). T h e p u r p o s e of t h i s work is twofold: to specify t h e fine s t r u c t u r e a n d c h e m i c a l n a t u r e of C o l l e m b o l a s p e r m a t o p h o r e s , a n d to d e t e c t e v e n t u a l u l t r a s t r u c t u r a l differences between spermatozoa in spermatop h o r e s a n d i n t h e m a l e g e n i t a l d u c t s . As far as I a m a w a r e t h i s k i n d of a r t h r o p o d sperm a t o p h o r e s has n o t b e e n p r e v i o u s l y examined by electron microscopy.
POLARIZION MICROSCOPE OBSERVATIONS; CHARACTERS OF THE BIREFRINGENCE
MATERIALS AND METHODS Male and female specimens of Dicyrtorna ornata found under stones in the Botanical garden of the University of Siena were placed in small containers with polytene lids partially filled with a mixture of plaster of Paris and clay at 8-10°C. The spermatophores were removed with a needle and fixed either in Hoyle buffered 5% glutaraldehyde with the addition of 5% sucrose or in phosphate buffered 4% paraformal-
S p e r m a t o p h o r e s are s e e n as c l e a r l y biref r a c t i v e s t r u c t u r e s w h e n e x a m i n e d in dist i l l e d water. T h e r e t a r d a t i o n v a l u e of t h e b i r e f r i n g e n c e is a b o u t 45. T h e axis of b i r e f r i n g e n c e is m a i n l y l o n g i t u d i n a l . T h e s p e r m a t o p h o r e s were t r e a t e d w i t h f l u i d s of p r o g r e s s i v e l y i n c r e a s i n g r e f r a c t i o n v a l u e s , from d i s t i l l e d w a t e r ( r e f r a c t i o n i n d e x 1.33) u p to m e t h y l e n e iodide (1.74).
355 Copyright© 1975by AcademicPress, Inc. Allrights of reproductionin any formreserved.
356
ROMANO
DALLAI
COLLEMBOLA SPERMATOPHORES The birefringence of these structures appears to be partially related to the refraction indices; the r e t a r d a t i o n values decrease as the refraction index increase up to 1.54 where it reaches a m i n i m u m . Thereafter the values increase again so as to reach values which are similar to those in water. On the basis of these d a t a the curve of the r e t a r d a t i o n values has been drawn (Fig. 9). The total birefringence has a double nature: one is an intrinsic birefringence which does not change with the refraction index, the other is textural, depending on the submicroscopic organization of the materials which build up a complex body according to Wiener. The structural birefringence of spermatophores treated with alcoholic phenol solution or with clove oil remains unaltered but when they are treated with picric acid, the birefringence appears inverted. Even after prolonged e n z y m e t r e a t m e n t with pepsin or trypsin a t concentrations ranging from 0.5% to 1.5%, and periodical renewal of the enzyme, the morphology of the s p e r m a t o p h o r e s appears unaltered; birefringence does not change. Also, treatm e n t with 1% chitinase does not seem to alter the structural material of the spermatophores. SCANNING ELECTRON MICROSCOPE OBSERVATIONS A s p e r m a t o p h o r e consists of a stem bearing a spermatic droplet (Fig. 2). The stem is about 500 t~m long with a diameter ranging from 8.5 tLm at the apex. The droplet m a y occasionally be almost spherical with a diameter of 37-45 p m but as a
357
m~ 656O555045
O
30-
25~ 20 15105-
"D
FIa. 9. Curve of retardation values. Abscissa: refractive index of the imbibition liquid. Ordinate: retardation in A units. rule it is elliptical with a diameter of 35 × 25 ttm. Under normal conditions h u m i d i t y is condensed on the spermatic droplet m a k i n g it appear spherical and larger t h a n it actually is (Fig. 1). The stem is m a d e of fibrous material oriented along its major axis but not strictly orthogonal at the basis (Fig. 6). The base of the stem is wide, made to adhere firmly to the s u b s t r a t u m (Fig. 7). M a n y times in a midportion the stem has a swelling (Fig. 2). At the apex a shieldlike swelling is constantly present (Fig. 3). This is where the spermatic droplet resides (the longer side of the shield measures a b o u t 15 ttm, the shorter 10-12 ttm). The surface of the droplet is very uneven showing tiny depressions and protrusions (Figs. 3, 8). A
FIc. 1. Dicyrtoma ornata male near two spermatophores (arrows). × 20. Fro. 2. Spermatophore as seen with the Scanning electron microscope. The stem shows a swelling. At the tip of the stem there is the spermatic droplet. × 300. FIG. 3. A shield-like swelling bears the spermatic droplet. Scanning electron microscope. × 2 000. FIo. 4. Stem of the spermatophore attached to a little piece of wood.Polysaccharides are present only at the level of the wood. PAS reaction. × 150. FIG. 5. Apical part of the stem as seen with the interference contrast microscope. × I 000. Fia. 6. Middle portion of the stem at the Scanning electron microscope, x 3 000. FIa. 7. Base of the stem adhering to the substratum. Scanning electron microscope, x 3 000. FIG. 8. The apical droplet showing a pointed structure at its tip. Scanning electron microscope. × 1 000.
358
ROMANO DALLAI
FIG. 10. Cross section of the apical droplet of Dicyrtoma ornata. S, section of the stem inside the droplet three sperms are also to be seen (arrows). x 21 000. FIG. 11. Section of the shieldlike swelling at the tip of the stem. x 30 000. FIG. 12. Cross-section through the middle of the stem. x 13 000.
Fro. 13. Cross-section of t h e apical droplet. M a n y s p e r m s (arrows) are to be seen. E a c h of t h e m is in a cyst a n d p r e s e n t s a n u c l e u s (n) a n d a flagellum (f) w o u n d u p r o u n d a bulky m a s s (b). G r a n u l a r m a t e r i a l is interposed b e t w e e n t h e s p e r m s . × 18 000. FIG. 14. Cross- a n d longitudinal-sections of t h e p e d u n c l e which in the m a l e genital a p p a r a t u s c o n n e c t e d t h e s p e r m s to t h e deferent wall. × 38 000. FIG. 15. T h e very t h i n dense layer p r e s e n t at the periphery of t h e droplet (arrows). × 30 000. 359
360
ROMANO DALLAI
by a formation 2 #m in diameter the position and structure of which indicate it to be the direct continuation of the stem (Figs. 5, 10); both the axial formation and the stem, in fact, consists of a compact bundle of fibres ranging in diameter from 500 /~ to 2500 /~ (Figs. 10-12). The constituting material is moderately electron dense and very likely has a proteinic nature; sections treated with 1.5% pepsin, in fact, present the material only partially digested. At the tip of the stem a certain amount of thicker and denser material than that of the stem itself forms a layer about 0.5-1.2 tLm thick. This layer is folded upon itself to form the shield-shaped body mentioned above (Fig. 11). Each spermatozoon in a cyst has a diameter of 4.5 #m. The acrosome, nucleus and flagellum are curiously wound up round a bulky mass (Figs. 10, 13L The nucleus forms three proximal helical turns; the flagellum another four or five turns (Fig. FIo. 16. Schematicdrawing of a spermatophore of 13). Longitudinal and transverse sections Dicyrtoma ornata. of the stem connecting the spermatozoon to the wall of the male genital ducts are pointed structure is located at the tip of the frequently present also in the material droplet and on the axis of the stem (Fig. 8). interposed between the spermatozoa (Fig. TRANSMISSION ELECTRONMICROSCOPE OB- 14). SERVATIONS The structure of the sperms in the apical Each spermatic droplet contains about droplet does not differ appreciably from 600 spermatozoa within a finely granular the structure of those in the male genital material (assuming a spherical droplet size ducts (5); both types of sperms are wound of 45 #m and a spherical spermatozoon size up round a dense central mass. Both have of 4.5 tLm). Calculations were carried out the same long anterior peduncle which on the basis of the electron microscope connected them to the deferent wall. observations concerning the number of CONCLUSIONS spermatozoa found in several cross sections The morphology of D i c y r t o m a ornata of the same spermatic droplet. A thick layer of the same granular material (7 urn) spermatophores is similar to that of D. is present also at the base and apex of the m i n u t a described by Hale (6). Our studies droplet. No external membrane seems to enable us to confirm Hale's observations exist; the spermatic droplet is externally with the light microscope regarding the limited by a very thin (600-700 A) layer of existence of an axis inside the spermatic material of the same kind as that found in droplet and extend his data by showing between the spermatozoa only more elec- that the droplet rests on a shieldlike suptron dense (Fig. 15). port. The spermatic droplet is axially crossed Polarization microscopic and electron
COLLEMBOLA SPERMATOPHORES
microscopic observations give evidence for the presence of a mixed filamentous body which probably is proteinic, although it is resistant to pepsin and trypsin. The proteinic nature of the material is evidenced by its PAS negativity (Fig. 4). This is in contrast with Schliwa's findings (11) which instead indicated glycogen as one of the components of the stem. Chitin seems to be absent in the spermatophore since enzyme digestion with chitinase does not affect its structure. A mixed filamentous body is found also by electron microscopy where the stem presents bundles of structures having the same orientation as the major axis of the spermatophore. The fact that the topochemical data differ from those obtained with the structures containing collagen is most singular and excludes the possibility that the components of the spermatophore belong to this class of substances. Many authors who have studied the
361
morphology of the apical spermatic droplet conclude that it is limited by an external membrane. It does not seem proper to speak of a real membrane around the spermatic droplet, however, but rather of a condensation of the fine granular material surrounding the spermatozoon. REFERENCES 1. BEHNKE, O., AND FORER, A., J. Cell Sci. 2, 169 (1967). 2. BRETFELD,G., Sond. Z. fl zool. Syst. Evol. 8, 259 (1970). 3. CASSAGNAU,P., Bull. Soc. Hist. natur. Toulouse 107, 279 (1971). 4. CASSAGNAU,P., Rev. Ecol. Biol. Sol 8, 609 (1971). 5. DALLAI,R., in BACCETTI, B. (Ed.), Comparative Spermatology, p. 275. Academic Press, New York, 1970. 6. HALE, W. G., Pedobiologia 5, 146 (1965). 7. MASSOUD,Z., in PESSON, P. (Ed.), La vie dans les sols, p. 337. Gauthier-Villars, Paris, 1971. 8. MAYER,H., Zool. Jb. Syst. 85, 501 (1957). 9. SCHALLER,f., Naturwissenscha#en 39, 48 (1952). 10. SCHALLER,F., Ann. Rev. Entomol. 16, 407 (1971). 11. SCHLIWA,W., Zool. Jb. Ana[. 82,445 (1965).