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Ultrastructural properties of the Theromyzon (Annelida: Hirudinae) cocoon membrane
Micron 35 (2004) 281–285 www.elsevier.com/locate/micron
Ultrastructural properties of the Theromyzon (Annelida: Hirudinae) cocoon membrane Corneliu Dimitriu, Daniel H. Shain* Department of Biology, Rutgers, The State University of New Jersey, 315 Penn St, Camden, NJ 08102, USA Received 9 September 2003; accepted 2 October 2003
Abstract The cocoon of the leech Theromyzon trizonare consists of fibrils packed into an arrangement that produces both C- and S-like patterns of bow-shaped lines in sections oblique through the membrane. Sections normal to the cocoon membrane show layers containing cross-sections of fibrils (approximately 16 nm dia.) that are separated by a center-to-center distance of approximately 23 nm. In cross-section, each fibril presents a central hole approximately 5 nm in diameter. A structureless layer covers most of the exterior surface of the cocoon membrane, and short protuberances are apparent in some zones. q 2004 Elsevier Ltd. All rights reserved. Keywords: Leech; Theromyzon; Cocoon; Ultrastructure; Transmission electron microscopy; Membrane; Fibril
Cocoons provide microenvironments suitable for embryonic development in a limited number of metazoans. Cocoon production in aquatic annelids is unique because they are secreted and assembled under water, in contrast with arthropod cocoons that are typically spun in a terrestrial environment (e.g. Bombyx mori; Rui, 1998). Little is known about the cocoon structure of clitellate annelids and the specificity of transmission electron microscopy (TEM) techniques that best process cocoons of various species. The TEM study performed by Knight and Hunt (1974) on the cocoon of the leech Erpobdella octoculata revealed that its hardened shell, difficult to section and stain, contained arrays of fibrils that produced patterns of bow-shaped lines in oblique sections. Similar fibrillar networks have been reported in the secretory glands of other clitellate annelids (Hess and Vena, 1974; Richards, 1977; Morris, 1983), and patterns resembling bowshaped lines were observed within the cocoon wall of the leech Branchiobdella pentodonta (Farnesi and Tei, 1975). The current study was prompted by the observation that the flexible, membranous cocoons secreted by the aquatic leech Theromyzon trizonare (formerly referred * Corresponding author. Tel.: þ 1-856-225-6144; fax: þ1-856-225-6312. E-mail address: [email protected] (D.H. Shain). 0968-4328/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.micron.2003.10.049
to as Theromyzon rude; Davies and Oosthuizen, 1993) are exceptionally resistant to heat and denaturing chemicals (e.g. autoclaving in guanidinium isothiocyanate; T.A. Mason and D.H. Shain, personal communication). In an effort to understand the structural basis of this resiliency, we sought to determine the underlying architecture of the Theromyzon cocoon membrane by electron microscopy. T. trizonare leeches were collected in the ponds of Golden Gate Park, San Francisco. In contrast with egg laying behavior observed in most leeches, which pass the cocoon sheath over their head following its synthesis around clitellar segments (Sawyer, 1986), T. trizonare appears to secrete its cocoon ventrally, through a process similar to that described for Glassiphonia lata (Nagao, 1958). After removing eggs from the capsule, cocoon halves were fixed in 2.5% glutaraldehyde and 2% paraformaldehyde in 0.1 M sodium cacodylate buffer (pH 7.2) for 24 h at þ 4 8C, then post-fixed with 2% OsO4 for 90 min at room temperature. After dehydration with graded ethanol concentrations, followed by graded acetone – ethanol concentrations, the specimens were embedded in EMbed 812 and polymerized at 74 8C. Thin sections were cut with a LKB ultramicrotome equipped with a diamond
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Fig. 1. Approximate shape and dimensions of a mature Theromyzon cocoon. Zone Z of the membrane was sectioned in planes normal (1) and parallel (2) to the median plane (AA) of the cocoon. Sections included portions from both the convex and flat side of the cocoon membrane.
knife. Confined to the central area of the cocoon, the sectioning was performed in various planes whose orientations varied relative to the median plane of the cocoon (Fig. 1). Special attention was given to sectioning through planes perpendicular and parallel to the median plane of the cocoon (Fig. 1); in the latter case, the direction of cutting stroke was normal to the capsule surface. Sections were picked up on 50, 100 or 200 mesh grids and stained with uranyl acetate and lead citrate. Micrographs were taken with a Zeiss EM 902 electron microscope at 80 kV electron gun voltage. Each section contained a maximum of half the total length of the cocoon contour. Representative micrographs were taken along each contour. All aspects shown in Fig. 2 were found in sections throughout the membrane. During processing for TEM, the cocoon membrane became wavy; consequently, the apparent membrane thickness varied along the cocoon contour. The magnitude of the apparent membrane thickness suggests a normal section in Fig. 2a, and an oblique section in Fig. 2c; a less oblique section is shown in Fig. 2b. Alternate layers of cross-sections through the fibrils are seen in normal sections, whereas alternate layers of bow-shaped lines predominate in oblique sections. The bow-shaped lines present both C-like patterns (Fig. 2a –c) and S-like patterns (Fig. 2b). An 11 nm axial banding pattern appears to be superimposed over the fibrils in some regions of the cocoon membrane (Fig. 2b). A structureless layer covers most of the exterior side of the cocoon membrane (Fig. 3a); this layer was detached from the membrane in some zones of the cocoon (e.g. Fig. 3b). Polygon-shaped spaces were observed in all
sections throughout the cocoon membrane (Figs. 2b, 3a). In some zones of the cocoon, the exterior side of the membrane displayed small protuberances, the tips of which appear to be formed from fibrils that intersect with each other (Fig. 4). Similar protuberances were observed on the exterior side of the cocoon of the leech B. pentodonta (Farnesi and Tei, 1975); in that study, it was proposed that protuberances might play a role in protecting the cocoon’s integrity. Ordered fibrous elements, seen in conjunction with an amorphous mass (Fig. 5), may represent transitions of the fibril protein between liquid and solid phases. Following nucleation, for instance, the fibrils may crystallize from a supersaturated solution of noncrystalline fibril protein, as described previously (Feher and Kam, 1985). Thus, this finding may depict an intermediate stage related to the process of cocoon membrane formation. Cross-sections of fibrils are shown in Fig. 6. A centerto-center distance (d; Fig. 6a) of approximately 23 nm separates the fibrils. Each fibril (approximately 16 nm dia.) has a central hole (h; Fig. 6b) of approximately 5 nm in diameter. Fibrillar dimensions are approximately the same in Erpobdella (Knight and Hunt, 1974), but the Erpobdella cocoon wall is , 10 £ thicker than that of Theromyzon which may contribute to overt differences in the consistency of these cocoons (i.e. hard-shelled vs. membranous, respectively). By comparison, silk fibers spun by arthropods are much larger (, 1 mm in diameter; Vollrath, 1992). The present study was performed in a limited zone of the cocoon. Consequently, there are no data to formulate a model for the entire cocoon structure. However,
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Fig. 2. (a) Section perpendicular to the Theromyzon cocoon membrane. Layers of cross-sectioned fibrils (FC) alternate with layers of fibrils sectioned longitudinally (FL). A limited zone shows a C-like pattern of bow-shaped lines (C). (b) Section oblique through the cocoon membrane. Layers of bow-shaped lines, in C-like patterns (C) and S-like patterns (S), alternate with layers of fibrils sectioned longitudinally (FL). An 11 nm banding pattern (lines) is superimposed over the fibrils. The arrowhead shows a space having a polygonal shape. (c) Section more oblique than the section shown in Fig. 2b. The C-like patterns (C) of the bow-shaped lines predominate. Cj represents the conjunction between two C-like patterns. FL represents fibrils sectioned longitudinally. Scale bars 200 nm.
Fig. 3. (a) Normal section through the cocoon membrane. Both sides of the membrane, interior (I) and exterior (E), are visible. A structureless layer (asterisk) covers the exterior side (E). Layers of cross-sectioned fibrils (FC) alternate with layers of fibrils sectioned longitudinally (FL); some layers may contain only one row of fibrils (arrows). Polygon-shaped spaces of various dimensions (arrowheads) are found throughout the cocoon membrane. (b) Portions of a structureless layer (asterisk) that appear detached from underlying layers. Fibrils (arrow) underline the structureless layer. The two fibrillar masses (p) that are intermediary between the structureless layer and the cocoon membrane may represent intermediate phases during cocoon membrane formation (see text). Scale bars 400 nm.
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Fig. 4. A protuberance (P) on the exterior side of the cocoon. Fibrils (short arrows) intersect with each other forming the tip (T) of the protuberance. A structureless layer (asterisk) covers the cocoon membrane. FC represents fibrils sectioned transversally. Scale bar 250 nm.
the presence of the bow-shaped lines (C; Fig. 2) in oblique sections and of circular shapes (FC, Fig. 2) alternating with fibrillar shapes (FL, Fig. 2) in normal sections, suggest that the twisted model of fibrous arrangements (Bouligand, 1965, 1972) could explain most aspects of the membrane ultrastructure within the central zone. Still, regions displaying axial banding and protuberances (e.g. Figs. 2b and 4, respectively) are difficult to explain by Bouligand’s model and may represent a different fibrillar arrangement. Similar axial banding patterns were reported by Knight and Hunt (1974) within the structure of the E. octoculata cocoon,
and the presence of a collagen-type protein was proposed. Thus, the possibility that axial banding results from a multi-protein network, or from a different arrangement of a common fibril cannot be deciphered at this juncture. Nonetheless, the dominant feature in both Theromyzon and Erpobdella cocoons is a twisted arrangement of adjacent, fibrous layers. In Theromyzon, some 60– 70 layers are stacked upon each other at various angles to form the cocoon wall. It seems likely that this dimensional, lattice-like configuration contributes, at least in part, to the unusual thermal and chemical resilience of the Theromyzon cocoon, although other factors may also
Fig. 5. Fibrous elements (arrows) in conjunction with zones of amorphous material (A). I represents the interior side of the cocoon membrane. Scale bar 300 nm.
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Fig. 6. (a) Section through the cocoon membrane showing fibrils sectioned transversally (FC) and longitudinally (FL). The average distance ðdÞ between the centers of the neighboring fibrils is 23 nm. Scale bar 100 nm. (b) Higher magnification of fibrils sectioned transversally. The fibril has a central hole (h) of approximately 5 nm in diameter. The fibril has an outside diameter ðDÞ of approximately 16 nm. Scale bar 20 nm.
play important roles (e.g. molecular composition, protein structure).
References Bouligand, Y., 1965. Sur une architecture torsade´e re´pandue dans de nombreuses cuticules d’Arthropodes. C.r. hebd. Se´anc. Acad. Sci., Paris 261, 3665–3668. Bouligand, Y., 1972. Twisted fibrous arrangements in biological materials and cholesteric mesophases. Tissue Cell 4 (2), 189–217. Davies, R.W., Oosthuizen, J.H., 1993. A new species of duck leech from North America formerly confused with Theromyzon rude (Rynchobdellida: Glossiphoniidae). Can. J. Zool. 71, 770 –775. Farnesi, R.M., Tei, S., 1975. Histochemical and ultrastructural studies of the cocoon of Branchiobdella pentodonta with (Annelida, Oligochaeta). Bollettino della Societa` italiana di biologia sperimentale 51 (18), 1184–1189.
Feher, G., Kam, Z., 1985. Nucleation and growth of protein crystals: general principles and assays. Meth. Enzymol. 114, 77–111. Hess, R.T., Vena, J.A., 1974. Fine structure of the clitellum of the annelid Enchytraeus fragmentosus. Tissue Cell 6, 503– 514. Knight, D.P., Hunt, S., 1974. Molecular and ultrastructural characterization of the egg capsule of the leech Erpobdella octulata L. Comp. Biochem. Physiol. A 47, 871 –880. Morris, G.M., 1983. The cocoon-producing cells of Eisenia foetida (Annelida, Oligochaeta): a histochemical and ultrastructural study. J. Morphol. 177, 41–50. Nagao, Z., 1958. Some observations on the breeding habits of a freshwater leech Glassiphonia lata Oka. Jpn. J. Zool. 12, 219– 223. Richards, K.S., 1977. The histochemistry and ultrastructure of the clitellum of the enchytraeid Lumbricillus rivalis (Oligochaeta: Annelida). J. Zool., Lond. 183, 161–176. Rui, H.G., 1998. Silk Reeling: (Cocoon Silk Study). Science Publishers. Sawyer, R.T., 1986. Leech Biology and Behavior. Oxford University Press, Oxford. Vollrath, F., 1992. Spider webs and silks. Sci. Am. 166, 70– 76.
Ultrastructural properties of the Theromyzon (Annelida: Hirudinae) cocoon membrane Corneliu Dimitriu, Daniel H. Shain* Department of Biology, Rutgers, The State University of New Jersey, 315 Penn St, Camden, NJ 08102, USA Received 9 September 2003; accepted 2 October 2003
Abstract The cocoon of the leech Theromyzon trizonare consists of fibrils packed into an arrangement that produces both C- and S-like patterns of bow-shaped lines in sections oblique through the membrane. Sections normal to the cocoon membrane show layers containing cross-sections of fibrils (approximately 16 nm dia.) that are separated by a center-to-center distance of approximately 23 nm. In cross-section, each fibril presents a central hole approximately 5 nm in diameter. A structureless layer covers most of the exterior surface of the cocoon membrane, and short protuberances are apparent in some zones. q 2004 Elsevier Ltd. All rights reserved. Keywords: Leech; Theromyzon; Cocoon; Ultrastructure; Transmission electron microscopy; Membrane; Fibril
Cocoons provide microenvironments suitable for embryonic development in a limited number of metazoans. Cocoon production in aquatic annelids is unique because they are secreted and assembled under water, in contrast with arthropod cocoons that are typically spun in a terrestrial environment (e.g. Bombyx mori; Rui, 1998). Little is known about the cocoon structure of clitellate annelids and the specificity of transmission electron microscopy (TEM) techniques that best process cocoons of various species. The TEM study performed by Knight and Hunt (1974) on the cocoon of the leech Erpobdella octoculata revealed that its hardened shell, difficult to section and stain, contained arrays of fibrils that produced patterns of bow-shaped lines in oblique sections. Similar fibrillar networks have been reported in the secretory glands of other clitellate annelids (Hess and Vena, 1974; Richards, 1977; Morris, 1983), and patterns resembling bowshaped lines were observed within the cocoon wall of the leech Branchiobdella pentodonta (Farnesi and Tei, 1975). The current study was prompted by the observation that the flexible, membranous cocoons secreted by the aquatic leech Theromyzon trizonare (formerly referred * Corresponding author. Tel.: þ 1-856-225-6144; fax: þ1-856-225-6312. E-mail address: [email protected] (D.H. Shain). 0968-4328/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.micron.2003.10.049
to as Theromyzon rude; Davies and Oosthuizen, 1993) are exceptionally resistant to heat and denaturing chemicals (e.g. autoclaving in guanidinium isothiocyanate; T.A. Mason and D.H. Shain, personal communication). In an effort to understand the structural basis of this resiliency, we sought to determine the underlying architecture of the Theromyzon cocoon membrane by electron microscopy. T. trizonare leeches were collected in the ponds of Golden Gate Park, San Francisco. In contrast with egg laying behavior observed in most leeches, which pass the cocoon sheath over their head following its synthesis around clitellar segments (Sawyer, 1986), T. trizonare appears to secrete its cocoon ventrally, through a process similar to that described for Glassiphonia lata (Nagao, 1958). After removing eggs from the capsule, cocoon halves were fixed in 2.5% glutaraldehyde and 2% paraformaldehyde in 0.1 M sodium cacodylate buffer (pH 7.2) for 24 h at þ 4 8C, then post-fixed with 2% OsO4 for 90 min at room temperature. After dehydration with graded ethanol concentrations, followed by graded acetone – ethanol concentrations, the specimens were embedded in EMbed 812 and polymerized at 74 8C. Thin sections were cut with a LKB ultramicrotome equipped with a diamond
282
C. Dimitriu, D.H. Shain / Micron 35 (2004) 281–285
Fig. 1. Approximate shape and dimensions of a mature Theromyzon cocoon. Zone Z of the membrane was sectioned in planes normal (1) and parallel (2) to the median plane (AA) of the cocoon. Sections included portions from both the convex and flat side of the cocoon membrane.
knife. Confined to the central area of the cocoon, the sectioning was performed in various planes whose orientations varied relative to the median plane of the cocoon (Fig. 1). Special attention was given to sectioning through planes perpendicular and parallel to the median plane of the cocoon (Fig. 1); in the latter case, the direction of cutting stroke was normal to the capsule surface. Sections were picked up on 50, 100 or 200 mesh grids and stained with uranyl acetate and lead citrate. Micrographs were taken with a Zeiss EM 902 electron microscope at 80 kV electron gun voltage. Each section contained a maximum of half the total length of the cocoon contour. Representative micrographs were taken along each contour. All aspects shown in Fig. 2 were found in sections throughout the membrane. During processing for TEM, the cocoon membrane became wavy; consequently, the apparent membrane thickness varied along the cocoon contour. The magnitude of the apparent membrane thickness suggests a normal section in Fig. 2a, and an oblique section in Fig. 2c; a less oblique section is shown in Fig. 2b. Alternate layers of cross-sections through the fibrils are seen in normal sections, whereas alternate layers of bow-shaped lines predominate in oblique sections. The bow-shaped lines present both C-like patterns (Fig. 2a –c) and S-like patterns (Fig. 2b). An 11 nm axial banding pattern appears to be superimposed over the fibrils in some regions of the cocoon membrane (Fig. 2b). A structureless layer covers most of the exterior side of the cocoon membrane (Fig. 3a); this layer was detached from the membrane in some zones of the cocoon (e.g. Fig. 3b). Polygon-shaped spaces were observed in all
sections throughout the cocoon membrane (Figs. 2b, 3a). In some zones of the cocoon, the exterior side of the membrane displayed small protuberances, the tips of which appear to be formed from fibrils that intersect with each other (Fig. 4). Similar protuberances were observed on the exterior side of the cocoon of the leech B. pentodonta (Farnesi and Tei, 1975); in that study, it was proposed that protuberances might play a role in protecting the cocoon’s integrity. Ordered fibrous elements, seen in conjunction with an amorphous mass (Fig. 5), may represent transitions of the fibril protein between liquid and solid phases. Following nucleation, for instance, the fibrils may crystallize from a supersaturated solution of noncrystalline fibril protein, as described previously (Feher and Kam, 1985). Thus, this finding may depict an intermediate stage related to the process of cocoon membrane formation. Cross-sections of fibrils are shown in Fig. 6. A centerto-center distance (d; Fig. 6a) of approximately 23 nm separates the fibrils. Each fibril (approximately 16 nm dia.) has a central hole (h; Fig. 6b) of approximately 5 nm in diameter. Fibrillar dimensions are approximately the same in Erpobdella (Knight and Hunt, 1974), but the Erpobdella cocoon wall is , 10 £ thicker than that of Theromyzon which may contribute to overt differences in the consistency of these cocoons (i.e. hard-shelled vs. membranous, respectively). By comparison, silk fibers spun by arthropods are much larger (, 1 mm in diameter; Vollrath, 1992). The present study was performed in a limited zone of the cocoon. Consequently, there are no data to formulate a model for the entire cocoon structure. However,
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283
Fig. 2. (a) Section perpendicular to the Theromyzon cocoon membrane. Layers of cross-sectioned fibrils (FC) alternate with layers of fibrils sectioned longitudinally (FL). A limited zone shows a C-like pattern of bow-shaped lines (C). (b) Section oblique through the cocoon membrane. Layers of bow-shaped lines, in C-like patterns (C) and S-like patterns (S), alternate with layers of fibrils sectioned longitudinally (FL). An 11 nm banding pattern (lines) is superimposed over the fibrils. The arrowhead shows a space having a polygonal shape. (c) Section more oblique than the section shown in Fig. 2b. The C-like patterns (C) of the bow-shaped lines predominate. Cj represents the conjunction between two C-like patterns. FL represents fibrils sectioned longitudinally. Scale bars 200 nm.
Fig. 3. (a) Normal section through the cocoon membrane. Both sides of the membrane, interior (I) and exterior (E), are visible. A structureless layer (asterisk) covers the exterior side (E). Layers of cross-sectioned fibrils (FC) alternate with layers of fibrils sectioned longitudinally (FL); some layers may contain only one row of fibrils (arrows). Polygon-shaped spaces of various dimensions (arrowheads) are found throughout the cocoon membrane. (b) Portions of a structureless layer (asterisk) that appear detached from underlying layers. Fibrils (arrow) underline the structureless layer. The two fibrillar masses (p) that are intermediary between the structureless layer and the cocoon membrane may represent intermediate phases during cocoon membrane formation (see text). Scale bars 400 nm.
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Fig. 4. A protuberance (P) on the exterior side of the cocoon. Fibrils (short arrows) intersect with each other forming the tip (T) of the protuberance. A structureless layer (asterisk) covers the cocoon membrane. FC represents fibrils sectioned transversally. Scale bar 250 nm.
the presence of the bow-shaped lines (C; Fig. 2) in oblique sections and of circular shapes (FC, Fig. 2) alternating with fibrillar shapes (FL, Fig. 2) in normal sections, suggest that the twisted model of fibrous arrangements (Bouligand, 1965, 1972) could explain most aspects of the membrane ultrastructure within the central zone. Still, regions displaying axial banding and protuberances (e.g. Figs. 2b and 4, respectively) are difficult to explain by Bouligand’s model and may represent a different fibrillar arrangement. Similar axial banding patterns were reported by Knight and Hunt (1974) within the structure of the E. octoculata cocoon,
and the presence of a collagen-type protein was proposed. Thus, the possibility that axial banding results from a multi-protein network, or from a different arrangement of a common fibril cannot be deciphered at this juncture. Nonetheless, the dominant feature in both Theromyzon and Erpobdella cocoons is a twisted arrangement of adjacent, fibrous layers. In Theromyzon, some 60– 70 layers are stacked upon each other at various angles to form the cocoon wall. It seems likely that this dimensional, lattice-like configuration contributes, at least in part, to the unusual thermal and chemical resilience of the Theromyzon cocoon, although other factors may also
Fig. 5. Fibrous elements (arrows) in conjunction with zones of amorphous material (A). I represents the interior side of the cocoon membrane. Scale bar 300 nm.
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285
Fig. 6. (a) Section through the cocoon membrane showing fibrils sectioned transversally (FC) and longitudinally (FL). The average distance ðdÞ between the centers of the neighboring fibrils is 23 nm. Scale bar 100 nm. (b) Higher magnification of fibrils sectioned transversally. The fibril has a central hole (h) of approximately 5 nm in diameter. The fibril has an outside diameter ðDÞ of approximately 16 nm. Scale bar 20 nm.
play important roles (e.g. molecular composition, protein structure).
References Bouligand, Y., 1965. Sur une architecture torsade´e re´pandue dans de nombreuses cuticules d’Arthropodes. C.r. hebd. Se´anc. Acad. Sci., Paris 261, 3665–3668. Bouligand, Y., 1972. Twisted fibrous arrangements in biological materials and cholesteric mesophases. Tissue Cell 4 (2), 189–217. Davies, R.W., Oosthuizen, J.H., 1993. A new species of duck leech from North America formerly confused with Theromyzon rude (Rynchobdellida: Glossiphoniidae). Can. J. Zool. 71, 770 –775. Farnesi, R.M., Tei, S., 1975. Histochemical and ultrastructural studies of the cocoon of Branchiobdella pentodonta with (Annelida, Oligochaeta). Bollettino della Societa` italiana di biologia sperimentale 51 (18), 1184–1189.
Feher, G., Kam, Z., 1985. Nucleation and growth of protein crystals: general principles and assays. Meth. Enzymol. 114, 77–111. Hess, R.T., Vena, J.A., 1974. Fine structure of the clitellum of the annelid Enchytraeus fragmentosus. Tissue Cell 6, 503– 514. Knight, D.P., Hunt, S., 1974. Molecular and ultrastructural characterization of the egg capsule of the leech Erpobdella octulata L. Comp. Biochem. Physiol. A 47, 871 –880. Morris, G.M., 1983. The cocoon-producing cells of Eisenia foetida (Annelida, Oligochaeta): a histochemical and ultrastructural study. J. Morphol. 177, 41–50. Nagao, Z., 1958. Some observations on the breeding habits of a freshwater leech Glassiphonia lata Oka. Jpn. J. Zool. 12, 219– 223. Richards, K.S., 1977. The histochemistry and ultrastructure of the clitellum of the enchytraeid Lumbricillus rivalis (Oligochaeta: Annelida). J. Zool., Lond. 183, 161–176. Rui, H.G., 1998. Silk Reeling: (Cocoon Silk Study). Science Publishers. Sawyer, R.T., 1986. Leech Biology and Behavior. Oxford University Press, Oxford. Vollrath, F., 1992. Spider webs and silks. Sci. Am. 166, 70– 76.