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The trichurid egg-shell: Evidence in support of the Bouligand hypothesis of helicoidal architecture
TISSUE
& CELL
Puhliskd
by I.on~man
1978 10 (4) 647-658 Group Ltd.
Printed
in Great Britain
D. A. WHARTON
THE TRICHURID EGG-SHELL: EVIDENCE IN SUPPORT OF THE BOULIGAND HYPOTHES I S OF HELICOIDAL ARCHITECTURE ABSTRACT. Electron microscopy of thin sections and freeze etch replicas of the em of the nematodes Trichwis suis and T. muris is used to provide evidence in support of the Bouligand hypothesis of helicoidal architecture. The evidence presented is as follows : 1. The specific objections to the Bouligand model raised by Dennell (1974) and Dalingwater (1975b) are answered by reference to a pyramid of helicoidal tissue in which the corners are blunt. 2. Sections cut normal to the plane of the laminae do not show parabolic patterning. Parabolae appear if the section is tilted-their direction depending upon the direction of tilting. 3. Freeze etching allows the direct visualization of helicoidal architecture. Fibres are parallel within any one lamina but the fibre direction rotates by an angle of 9 in successive laminae. Parabolic arcs are made up of short lengths of straight fibres-curved fibres were not observed. Planes of sectioning producing single and double spiral artifacts are described and the formation of these artifacts discussed. The sense of rotation of the helicoid is shown to be asymmetrical about any mid-plane through the egg.
terms are used as defined by G ubb (1975) and Wharton and Jenkins (1978, Fig. 14). Since it was first proposed by Bouligand (1965), a number of workers have criticized the helicoidal model (Dennell, 1973, 1974, 1976; Dalingwater, 1975a, b; Mutvei, 1974; Ejike, 1973; Rudall, 1969). These authors have preferred the model proposed by Drach (1953) or modifications thereof (for diagrams illustrating the various versions of the Drach model see Bouligand, 1972, Fig. 32). They consider that the apparent lamellae are real structures and not artifacts as suggested by the Bouligand model. They have also suggested that arthropod cuticle contains curved microfibrils connecting the ‘lamellae’. In addition Dennell (1973, 1974, 1976), Ejike (1973) and Dalingwater (1975a, b) have reported the presence of sinuous ‘macrofibrils’. These have been shown by Gubb (1975) to be pore canal filaments, pulled out of the pore canals during sectioning. Gubb
Introduction BOULEAND (1965) has suggested that the parabolic patterning observed in a variety of biological structural materials (for lists see Bouligand, 1972; Neville, 1975; Neville et a/., 1976) is an artifact. Parabolae are formed by the oblique sectioning of a stack of fibres, in which the fibres within any lamina are oriented in parallel but the fibre direction changes by a fixed amount in successive laminae (Fig. I). This type of structure is known as helicoidal architecture. The use of the terms ‘lamina’, ‘lamella’ and ‘parabola’ has been confused. In the present work these
Department of Zoology, University of Bristol, Woodland Road, Bristol. Present address : Department of Zoology, The Unibersity College of Wales, Aberystwyth SY23 3DA. Received 16 November 1977. Revised 23 February 1978. 647
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WHARTON
(1975) has also dealt with various objections model raised by Rudall (1969). Evidence in support of the Bouligand hypothesis has been provided by Gubb (1975), Altner (1975) and Neville et al. (1976). In this paper it is hoped to answer the specific objections to the Bouligand model raised by Dennell (1973, 1974, 1976) and Dalingwater (1975a, b) and to provide further evidence in support of the Bouligand hypothesis. The eggs of Trichurids are especially suitable for the study of helicoidal architecture. They are small, allowing the whole of a transverse section to be seen in thin sections. Sectioning or fracturing a pellet of eggs reveals a variety of features of the structure of the shell. to the Bouligand
Materials and Methods The preparation
of thin sections of the eggs of Trichuris suis and T. muris and freeze-etch replicas of the eggs of T. suis for observation under the electron microscope, has been described previously (Wharton and Jenkins, 1978). Results
The main evidence against the Bouligand model put forward by Dennell (1974) and Dalingwater (1975b) is that, according to the model the apparent parabolae should be out of register on the four faces of a pyramid of tissue (see Figs. 1, 2). Dennell (1974) and Dalingwater (1975b), using cellulose acetate strips and scanning electron microscopy respectively, found that lamellae were continuous around right-angled breaks. Similar observations made by Drach (1953) were considered by Bouligand (1972) to be due to optical effects. This could not, however, explain the observations of Dennell (1974) and Dalingwater (1975b). Their observations can be explained by the helicoidal model. If a pyramid of tissue is cut, the apparent parabolae will be out of register on the four faces of the pyramid (Figs. 1, 2). If a cone of tissue is cut, the apparent parabolae will follow the course of a double spiral (Figs. 3, 4). If a pyramid of tissue is cut, in which the corners of the pyramid are blunt, the parabolae will appear to be in register and the lamellae continuous around the corners
Fig. 1. Diagram of a pyramid of helicoidal tissue. Fibres are parallel in each lamina and the fibre orientation rotates in successive laminae. The parabolae generated on the four faces of the pyramid all face in the same direction (clockwise). After Bouligand (1972).
(Fig. 5). The parabolae follow the course of a double spiral (Fig. 6). As it is difficult to cut a perfectly sharp corner to a pyramid of crab cuticle, it seems IikeIy that this is the explanation of Dennell’s (1974) and Dalingwater’s (1975b) observations. The pictures published by them appear /
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Fig. 2. Apparent parabolae on the four faces of a pyramid of helicoidal tissue. Taken from a tracing paper overlay of Fig. 1. The parabolae are out of register on the four faces of the pyramid.
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Fig. 3. Projections on to the cutting plane of fibrillar orientation in concentric laminae formed by sectioning a cone of helicoidal tissue. After Bouligand (1972).
to show that the corners of their pyramids were rather blunt. It is to be expected that if the course of the apparent lamellae of a pyramid of crab cuticle is followed, they would be found to form a double spiral. Tilting experimenter As has been pointed out by Neville (1973, convincing evidence in support of the Bouligand hypothesis can be obtained by reference to Neville and Luke’s (1969) perspex model of helicoidal insect cuticle. If
Fig. 4. Apparent parabolae of a cone of helicoidal tissue. Taken from a tracing paper overlay of Fig. 3. The apparent parabolae follow the course of a double spiral.
Fig. 5. Diagram of a pyramid of helicoldal tissue. in which the corners of the pyramid are blunt. The basal lamina is emphasized to show the shape of the pyramid. The apparent pardbolae generated by the rotation of planes of parallel fibres, appear to be continuous around the corners of the pyramid. See
also Figs. 1, 3. viewed from the side, equivalent to sections cut precisely normal to the plane of the laminae, no parabolae are seen. If the model is tilted parabolae appear, their direction depending upon the direction of tilting (Fig. 7, inset). This experiment has recently been successfully performed using the cell wall of Chara vulgaris oospores (Neville c? u/.,
Fig. 6. The apparent parabolae generated by a pyramid of helicoidal tissue, in which the corners of the pyramid are blunt. Taken from a tracing paper overlay of Fig. 5. The apparent parabolae follow the course of a double spiral. See also Figs. 2. 4.
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1976), and with crab cuticle (Bouligand pers. comm). The eggs of Trichuris muris are suitable for this experiment as it is relatively easy to obtain a section normal to the surface of the shell. The experiment also requires that the specimen is tilted in a plane normal to the section and at right-angles to the shell (Fig. 8). Potassium permanganate-stained thin sections of the eggs of T. muris, in which the shell was not yet fully formed, were used. Parabolae were clearly visible (Fig. 9). The specimen was placed on the tilting goniometer stage of a Philips EM 300 electron microscope. A suitable magnification was chosen and the direction of tilt determined by reference to a fold in the section. A portion of egg-shell was selected which did not show parabolae when the section was horizontal (0 tilt) and was at right angles to the direction of tilt. The specimen was tilted about the X-axis (Fig. 8) and pictures were taken at various tilting angles. The specimen was centred each time by reference to a piece of dirt on the section. The results of the tilting experiment are shown in Fig. 7. At 0” tilt no parabolae are visible. When the specimen is tilted parabolae appear, their direction depending upon the direction of tilting. If the section is tilted in the same plane as the surface of the shell (about the Y-axis in Fig. 8), reversal of parabolae cannot be obtained. If the pattern consisted of actual parabolic fibres, the pattern could only be reversed by rotation about the Y-axis (Neville et cd., 1976). It is therefore concluded that the helicoidal model holds true for the parabolic patterning of the eggs of Trichuris n7zrri.s.
Y Fig. 8. Orientation of a section of egg-shell during the tilting experiment. When tilted about the X-axis parabolae appear, their direction depending upon the direction of tilting. When tilted about the Y-axis there is no change in appearance. The lamellae of the chitinous layer are represented by fine lines.
Direct visualization of helicoidal architecture using the ,freeze-etch technique
In oblique and tangential fractures of freezeetch preparations of the eggs of T. suis fibrils can be observed. These can be seen to be responsible for the formation of the parabolae of the chitinous layer (Fig. 10). Fig. 11 shows a tangential fracture which reveals fibrils within three laminae of the chitinous layer. Laminae can be seen to consist of straight parallel fibres, the fibril orientation changes by an angle of 9 in successive laminae. One complete rotation of the helicoid (360”-generating two lamellae) therefore consists of 40 laminae. In Xenopsyllu there is a change of 11 25’ in successive laminae (Neville, 1975). In Hub Cynthia test the angle is 12’ (Cubb, 1975); in cockroach maxillary palp cuticle, IO~~lZ (Altner, 1975) and in the cell wall of Pclwriu embryos, 35” (Peng and Jaffe, 1976). Fig. 12 shows an oblique fracture, reveaiing the parabolae of the chitinous layer. The
Fig. 7. Transverse section through the developing egg-shell of T. muris. The section was orientated and tilted as in Fig. 8 by of-20”. x 107,300. The insets show the results predicted by similarly tilting Neville and Luke’s (1969) model of helicoidal insect cuticle. The model consists of plastic laminae on which are ruled parallel lines. The orientation of the lines changes by a fixed angle in successive laminae. No parabolae are seen when the model is viewed end on (0”) but appear if the model is tilted ( +20”, -20”). The twisted ribbons represent the orientation of cuticular pore canals.
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parabolae can be seen to consist of straight lengths of fibrils. Parabolae are not visible in transverse fractures of the shell (Wharton and Jenkins, 1978, Fig. 9). In Fig. 13 it can be seen that the fracture goes from transverse in the left of the picture to oblique in the right. Parabolae are visible where the shell has fractured obliquely. The width of the lamellae depends on the angle of fracturing of the shell (this can also be seen in Fig. 17). This provides further evidence for the Bouligand hypothesis, as the lamellar periodicity should increase with the angle of obliquity of the section or fracture (Neville, 1975), theoretically becoming infinity for sections parallel to the plane of the laminae. Dennell (1973, 1974, 1976) considers that arthropod cuticle consists of areas of horizontal fibres (‘laminae’) and areas of vertically curved fibres (‘inter-laminae’). According to Dennell, the appearance of parabolae is due to the presence of these curved fibres. Mutvei (1974) and Dalingwater (1975a) have claimed to have observed curved fibres using
scanning electron microscopy. Gubb (1975), however, has shown that the apparent parabolae of the cuticle of Carcinus maenas and the test of Halocynthia papilIosa consist of short lengths of straight fibres. The macrofibrils of crustacean cuticle and tunicate test are large enough to be observed by scanning electron microscopy. In order to observe the fibrils of the chitinous layer of trichurid eggs, it was necessary to use the freeze-etch technique. This has been used recently by Peng and Jaffe (1976) to reveal the helicoidal arrangement of microfibrils in the cell wall of fucoid eggs and by Altner (1975) to reveal helicoidally arranged fibrils in the cockroach maxillary palp cuticle. Curved fibres were not observed in the eggshell of Trichuris suis. Observations on freeze-etch preparations thus do not support the interpretation of Dennell (1973, 1974, 1976). Fibrils are parallel within each lamina but rotate by a fixed angle (9”) in successive laminae, thus forming the parabolae seen in oblique sections. This corresponds to the interpretation of Bouligand (1965).
Fig. 9. Transverse section of the developing egg-shell of T. muris, taken at the base of the opercular plug (P). The parabolae of the chitinous layer can be clearly seen. The parabolae point in the same direction (clockwise) on either side of the egg (boxes). x 13,650. Fig. 10. Freeze-etch replica revealing the microfibrils that make up the parabolae the chitinous layer. x 38,750. Circled arrow indicates the direction of shadowing. Fig. 11. Freeze-etch replica of laminae are shown. Fibrils are tibre orientation changes by an arrow indicates the direction of
of
a tangential fracture through the chitinous layer. Three straight and parallel within each lamina (lines). The angle of 9” in successive laminae. x 131,000. Circled shadowing.
Fig. 12. Freeze-etch replica of an oblique fracture through the chitinous fibres form the parabolae of the chitinous layer. x 58,500. Circled arrow direction of shadowing.
layer. The shows the
Fig. 13. Freeze-etch replica: the angle of fracture goes from transverse on the lefthand side of the picture to oblique on the right-hand side. The width of the parabolae increases as the angle of obliquity of the fracture increases. x 20,950. Circled arrow shows the direction of shadowing. Fig. 14. Tangential section of the egg-shell. of a single spiral. x 14,500.
The chitinous
layer has the appearance
Fig. 17. Freeze-etch replica of a tangential fracture of the chitinous layer. The apparent lamellae form a double spiral (see Fig. 18). The picture is complicated by the fracture jumping from one lamella to another (arrow). x 12,100. Circled arrow shows the direction of shadowing.
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WHARTON
Fig. 15. Diagram showing that the apparent lamellae of the shell follow the course of a double spiral. Taken from a tracing paper overlay of Wharton and Jenkins (1978. Fig. 7). One arm of the double spiral is emphasized.
Fig. 16. Planes of sectioning of trichurid eggs which produce the double spiral (A-B) and the single spiral (D-E) artifacts. Single spirals are produced by tangential sections. Double spirals are produced by transverse or longitudinal sections.
Helicoidal asymmetry
Optically active regions of beetle cuticle reflecting circularly polarized light extinguish simultaneously on both sides of the insect when viewed with a right circular analyser (Neville and Caveney, 1969). The sense of rotation of the helicoid is therefore the same on either side of the mid-line, i.e. it is asymmetrical (Neville, 1975). This has been demonstrated directly by determining the sense of rotation of the helicoid on either side of the mid-line (Neville, 1970). Trichurid eggs are small enough to allow complete sections to be observed with the electron microscope, In tranverse sections of developing egg-shells of T. murk it can be seen that the apparent direction of the parabolae is the same (clockwise) on either side of the egg (Fig. 9). The sense of rotation of the helicoid can be related to the direction of the parabolae (Neville and Luke, 1971). The sense of rotation is therefore asymmetrical about any mid-plane through the egg. The single and double spiral artifacts
In thin sections cut tangentially to the surface of the shell an optical density artifact is produced that has the appearance of a single spiral (Fig. 14). In transverse sections of the egg the apparent lamellae of the chitinous
layer can be seen to form a double spiral (Fig. 15 and Wharton and Jenkins, 1978, Fig. 7). The planes of sectioning of the egg which produce the single and double spiral artifacts are shown in Fig. 16. Bouligand (1972) has discussed the formation of the double spiral artifact (Fig. 3). He also gave two explanations of the single spiral. Both these explanations are considered to be inadequate by Gordon and Winfree (1978) and by Gubb (pers. comm.). The first explanation is that the single spiral is caused by the projection on to the cutting plane of fibrils of laminae arranged as a set of homofocal parabolae (Bouligand, 1972, Figs. 20, 21, 22). This would, however, give a single spiral as an alternative to a double spiralnot superimposed upon it, as is seen in sections of crab body wall tubercles (Bouligand, 1972). The second explanation is that the single spiral is caused by a microtomy artifact. The knife either cuts and turns up fibres or passes between them. As the direction of sectioning is constant and the orientation of fibres to the knife changes in successive laminae and according to its position in the spiral; a single spiral is superimposed upon the double spiral (see Bouligand, 1972, Figs. 28, 31). This predicts, however, that
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the spiral will be asymmetrical.
This is not
the case (Fig. 14 and Bouligand, 1972, Fig. 9). Gordon and Winfree (1978) suggest that to produce the single spiral artefact there must be some component that rotates only 180” for every 360” rotation of fibre direction. This condition can be satisfied by adding a set of parallel vectors individually to a set of radial vectors. The radial vectors are provided by the projection on to the cutting plane of the orientation of fibres in concentric conical laminae (as in Fig. 3). The parallel vectors are provided by a distortion of the fibres during sectioning. Gubb (1977) considers that it is not necessary to postulate a sectioning artifact, the parallel vector may be introduced by an optical effect, The formation of the single spiral is discussed more fully by Gubb (1977) and by Gordon and Winfree (1978). Whether a single or a double spiral was seen in sections of trichurid eggs depended on whether sections were cut tangentially or normally/obliquely to the plane of the laminae (Fig, 16). If the parallel vector is due to the distortion of the fibres, this is likely to be greater when sectioning in the plane of the laminae than when sectioning normally to the plane of the lam&e. Freeze etching also reveals tangential sections of the shell (Fig. 17). In this case the parabolae can be seen not to form a single spiral, but appear to form the double spiral artifact (Fig. IS). During freeze-fracture the shell brittle-fractures, so the microfibril pattern would not be subject to distortion.
Fig. 18. Course of the apparent lamellae revealed by a tangentiai fracture in a freeze-etch replica of the egg of T. suis. Taken from a tracing paper overlay of Fig. 17. The lamellae appear to form a double spiral.
This supports the hypothesis that the single spiral artifact may be due to a distortion of the fibres during sectioning. It is difficult to explain the formation of double and single spirals if the Bouligand model is rejected. Summary The following evidence is now available support of the Bouligand hypothesis helicoidal architecture:
in of
experiments (present work; 1. Tilting Neville ef al., 1976). 2. Direct visualization of helicoidal architecture by scanning electron microscopy (Gubb, 1975) and freeze etching (present work; Altner, 1975; Peng and Jaffe, 1976). of double and single 3. The formation spiral artifacts (present work; Bouligand, 1972; Gubb, 1977). 4. The ability to relate pore canal orientation to the helicoidal structure of arthropod cuticle (Neville et ul., 1969). increases with 5. The width of parabolae the angle of obliquity of the section or fracture (present work; Neville, 1975: Gubb, 1975). in the 6. There is a sinusoidal variation intensity of birefringence observed under the polarizing microscope, when passing from one lamella to the next (Neville and Caveney, 1969). A similar sinusoidal variation in electron density is seen under the electron microscope (Neville and Luke, 1971). Helicoidal cuticle of suitable pitch reflects circularly polarized light (Neville and Caveney, 1969; Caveney, 1971). The analogy between helicoidal systems and cholesteric liquid crystals (Neville and Luke, 1971). The specific objections to the Bouligand model raised by Dennell (1974) and Dalingwater (1975b) are answered by reference to a pyramid of helicoidal tissue in which the corners are blunt (present work). Acknowledgements I would like to thank my Ph.D. supervisor, Dr C. J. Mapes, for his help and encourage-
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ment; the late Professor H. E. Hinton and Dr A. E. Dorey for many helpful conversations; Dr A. C. Neville, Dr S. E. Reynolds and Dr D. C. Gubb for introducing me to the wonders of helicoidal architecture; Mr J. A.
Clements and Mr A. Britton for technical assistance; Dr D. A. Erasmus and Dr A. C. Neville for reading the manuscript and the Science Research Council for financial support.
References ALTNER, H. 1975. The microfibril texture in a specialized plastic cuticle area within a sensillum field on the cockroach maxillary palp as revealed by freeze fracturing. Cell Tissue. Ref., 165, 79-88. BOULIGAND, Y. 1965. Sur une architecture torsadbe rt+pondue dans de nombreuses cuticles d’arthropods. C. r. hebd. Se!anc. Acad. Sci., Paris, 261, 3665-3668. BOULIGAND, Y. 1972. Twisted fibrous arrangements in biological materials and cholesteric mesophases. Tissue & CM, 4, 189-217. CAVENEY,S. 1971. Cuticle reflectivity and optical activity in scarab beetles, the role of uric acid. Proc. R. Sot. (B), 178,205-225. DALINGWATER, J. E. 1975a. SEM observations on the cuticles of some decapod crustaceans. Zool. J. Linn. Sm., 56, 327-330. DALINGWATER, J. E. 1975b. The reality of arthropod cuticular laminae. Cell. Tiss. Res., 163, 41 l-413. DENNELL, R. 1973. The structure of the cuticle of the shore crab Car&us maenas (L). 2001. J. Linn. Sm., 52, 159-163. DENNELL, R. 1974. The cuticle of the crabs Cancer pagurus (L) and Carcinus mamas (LX Zool. J. Linn. Sot., 54,241-245. DENNELL, R. 1976. The structure and lamination of some arthropod cuticles. 2001. J. Linn. Sot., 58, 159-164. DRACH, P. 1953. Structures des lamelles cuticulaires chez les crustaces. C. r. hebd. Siam. Acad. Sci., Paris, 237, 159-163. EJIKE, C. 1973. Macrofibrils in the cuticle of the crab Calinectes gladiator (Benedict). Zool. J. Linn. Sot., 53, 253-255. GORDON, H. and WINFREE, A. T. 1978. A single spiral artifact in arthropod cuticle. Tissue & Cell, 10, 39-50. GUBB, D. C. 1975. A direct visualization of helicoidal architecture in Car&us maenas and Halocynthia papillosa by scanning electron microscopy. Tissue & Cell, 7, 19-32. GUBB, D. C. 1977. Helicoidal architecture and the control of morphogenesis in extracellular matrices. Ph.D. thesis, Bristol University. MUTVEI, H. 1974. SEM studies on arthropod exoskeletons. Bull. Geol. Inst. Univ. Uppsala, NS, 4, 73-80. NEVILLE, A. C. 1970. Cuticle ultrastructure in relation to the whole insect. In Insect Ultrastructure (ed. A. C. Neville), Symp. R. Ent. Sot., 5, 17-39. Blackwell, London. NEVILLE, A. C. 1975. Biology of the Arthropod Cuticle. Springer-Verlag, Berlin. NEVILLE, A. C. and CAVENEY,S. 1969. Scarabaeid beetle exocuticle as an optical analogue of cholesteric liquid crystals. Biol. Rev., 44, 531-562. NEVILLE, A. C., GUBB, D. C. and CRAWFORD, R. M. 1976. A new model for cellulose architecture in some plant cell walls. Protoplasma, 90, 307-317. NEVILLE, A. C. and LUKE, B. M. 1969. A two-system model for chitin-protein complexes in insect cuticles. Tissue & Cell, 1, 689-707. NEVILLE, A. C. and LUKE, B. M. 1971. A biological system producing a self-assembling cholesteric protein liquid crystal. J. Cell. Sci., 8, 93-109. NEVILLE, A. C., THOMAS, M. G. and ZELAZNY, B. 1969. Pore canal shape related to molecular architecture of arthropod cuticle. Tissue & CeN, 1, 183-200. PENG, B. H. and JAFFE, C. F. 1976. Cell wall formation in Pelvetia embryos. A freeze fracture study. Planta, 133,57-71. RUDALL, K. M. 1969. Chitin and its organization with other molecules. J. Polymer Sri., 28, 83-102. WHARTON, D. A. and JENKINS, T. 1978. Structure and chemistry of the egg-shell of a nematode (Trichurissuis). Tissue & Cell, 10, 427-440.
& CELL
Puhliskd
by I.on~man
1978 10 (4) 647-658 Group Ltd.
Printed
in Great Britain
D. A. WHARTON
THE TRICHURID EGG-SHELL: EVIDENCE IN SUPPORT OF THE BOULIGAND HYPOTHES I S OF HELICOIDAL ARCHITECTURE ABSTRACT. Electron microscopy of thin sections and freeze etch replicas of the em of the nematodes Trichwis suis and T. muris is used to provide evidence in support of the Bouligand hypothesis of helicoidal architecture. The evidence presented is as follows : 1. The specific objections to the Bouligand model raised by Dennell (1974) and Dalingwater (1975b) are answered by reference to a pyramid of helicoidal tissue in which the corners are blunt. 2. Sections cut normal to the plane of the laminae do not show parabolic patterning. Parabolae appear if the section is tilted-their direction depending upon the direction of tilting. 3. Freeze etching allows the direct visualization of helicoidal architecture. Fibres are parallel within any one lamina but the fibre direction rotates by an angle of 9 in successive laminae. Parabolic arcs are made up of short lengths of straight fibres-curved fibres were not observed. Planes of sectioning producing single and double spiral artifacts are described and the formation of these artifacts discussed. The sense of rotation of the helicoid is shown to be asymmetrical about any mid-plane through the egg.
terms are used as defined by G ubb (1975) and Wharton and Jenkins (1978, Fig. 14). Since it was first proposed by Bouligand (1965), a number of workers have criticized the helicoidal model (Dennell, 1973, 1974, 1976; Dalingwater, 1975a, b; Mutvei, 1974; Ejike, 1973; Rudall, 1969). These authors have preferred the model proposed by Drach (1953) or modifications thereof (for diagrams illustrating the various versions of the Drach model see Bouligand, 1972, Fig. 32). They consider that the apparent lamellae are real structures and not artifacts as suggested by the Bouligand model. They have also suggested that arthropod cuticle contains curved microfibrils connecting the ‘lamellae’. In addition Dennell (1973, 1974, 1976), Ejike (1973) and Dalingwater (1975a, b) have reported the presence of sinuous ‘macrofibrils’. These have been shown by Gubb (1975) to be pore canal filaments, pulled out of the pore canals during sectioning. Gubb
Introduction BOULEAND (1965) has suggested that the parabolic patterning observed in a variety of biological structural materials (for lists see Bouligand, 1972; Neville, 1975; Neville et a/., 1976) is an artifact. Parabolae are formed by the oblique sectioning of a stack of fibres, in which the fibres within any lamina are oriented in parallel but the fibre direction changes by a fixed amount in successive laminae (Fig. I). This type of structure is known as helicoidal architecture. The use of the terms ‘lamina’, ‘lamella’ and ‘parabola’ has been confused. In the present work these
Department of Zoology, University of Bristol, Woodland Road, Bristol. Present address : Department of Zoology, The Unibersity College of Wales, Aberystwyth SY23 3DA. Received 16 November 1977. Revised 23 February 1978. 647
648
WHARTON
(1975) has also dealt with various objections model raised by Rudall (1969). Evidence in support of the Bouligand hypothesis has been provided by Gubb (1975), Altner (1975) and Neville et al. (1976). In this paper it is hoped to answer the specific objections to the Bouligand model raised by Dennell (1973, 1974, 1976) and Dalingwater (1975a, b) and to provide further evidence in support of the Bouligand hypothesis. The eggs of Trichurids are especially suitable for the study of helicoidal architecture. They are small, allowing the whole of a transverse section to be seen in thin sections. Sectioning or fracturing a pellet of eggs reveals a variety of features of the structure of the shell. to the Bouligand
Materials and Methods The preparation
of thin sections of the eggs of Trichuris suis and T. muris and freeze-etch replicas of the eggs of T. suis for observation under the electron microscope, has been described previously (Wharton and Jenkins, 1978). Results
The main evidence against the Bouligand model put forward by Dennell (1974) and Dalingwater (1975b) is that, according to the model the apparent parabolae should be out of register on the four faces of a pyramid of tissue (see Figs. 1, 2). Dennell (1974) and Dalingwater (1975b), using cellulose acetate strips and scanning electron microscopy respectively, found that lamellae were continuous around right-angled breaks. Similar observations made by Drach (1953) were considered by Bouligand (1972) to be due to optical effects. This could not, however, explain the observations of Dennell (1974) and Dalingwater (1975b). Their observations can be explained by the helicoidal model. If a pyramid of tissue is cut, the apparent parabolae will be out of register on the four faces of the pyramid (Figs. 1, 2). If a cone of tissue is cut, the apparent parabolae will follow the course of a double spiral (Figs. 3, 4). If a pyramid of tissue is cut, in which the corners of the pyramid are blunt, the parabolae will appear to be in register and the lamellae continuous around the corners
Fig. 1. Diagram of a pyramid of helicoidal tissue. Fibres are parallel in each lamina and the fibre orientation rotates in successive laminae. The parabolae generated on the four faces of the pyramid all face in the same direction (clockwise). After Bouligand (1972).
(Fig. 5). The parabolae follow the course of a double spiral (Fig. 6). As it is difficult to cut a perfectly sharp corner to a pyramid of crab cuticle, it seems IikeIy that this is the explanation of Dennell’s (1974) and Dalingwater’s (1975b) observations. The pictures published by them appear /
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Fig. 2. Apparent parabolae on the four faces of a pyramid of helicoidal tissue. Taken from a tracing paper overlay of Fig. 1. The parabolae are out of register on the four faces of the pyramid.
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Fig. 3. Projections on to the cutting plane of fibrillar orientation in concentric laminae formed by sectioning a cone of helicoidal tissue. After Bouligand (1972).
to show that the corners of their pyramids were rather blunt. It is to be expected that if the course of the apparent lamellae of a pyramid of crab cuticle is followed, they would be found to form a double spiral. Tilting experimenter As has been pointed out by Neville (1973, convincing evidence in support of the Bouligand hypothesis can be obtained by reference to Neville and Luke’s (1969) perspex model of helicoidal insect cuticle. If
Fig. 4. Apparent parabolae of a cone of helicoidal tissue. Taken from a tracing paper overlay of Fig. 3. The apparent parabolae follow the course of a double spiral.
Fig. 5. Diagram of a pyramid of helicoldal tissue. in which the corners of the pyramid are blunt. The basal lamina is emphasized to show the shape of the pyramid. The apparent pardbolae generated by the rotation of planes of parallel fibres, appear to be continuous around the corners of the pyramid. See
also Figs. 1, 3. viewed from the side, equivalent to sections cut precisely normal to the plane of the laminae, no parabolae are seen. If the model is tilted parabolae appear, their direction depending upon the direction of tilting (Fig. 7, inset). This experiment has recently been successfully performed using the cell wall of Chara vulgaris oospores (Neville c? u/.,
Fig. 6. The apparent parabolae generated by a pyramid of helicoidal tissue, in which the corners of the pyramid are blunt. Taken from a tracing paper overlay of Fig. 5. The apparent parabolae follow the course of a double spiral. See also Figs. 2. 4.
TRlCHURID
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1976), and with crab cuticle (Bouligand pers. comm). The eggs of Trichuris muris are suitable for this experiment as it is relatively easy to obtain a section normal to the surface of the shell. The experiment also requires that the specimen is tilted in a plane normal to the section and at right-angles to the shell (Fig. 8). Potassium permanganate-stained thin sections of the eggs of T. muris, in which the shell was not yet fully formed, were used. Parabolae were clearly visible (Fig. 9). The specimen was placed on the tilting goniometer stage of a Philips EM 300 electron microscope. A suitable magnification was chosen and the direction of tilt determined by reference to a fold in the section. A portion of egg-shell was selected which did not show parabolae when the section was horizontal (0 tilt) and was at right angles to the direction of tilt. The specimen was tilted about the X-axis (Fig. 8) and pictures were taken at various tilting angles. The specimen was centred each time by reference to a piece of dirt on the section. The results of the tilting experiment are shown in Fig. 7. At 0” tilt no parabolae are visible. When the specimen is tilted parabolae appear, their direction depending upon the direction of tilting. If the section is tilted in the same plane as the surface of the shell (about the Y-axis in Fig. 8), reversal of parabolae cannot be obtained. If the pattern consisted of actual parabolic fibres, the pattern could only be reversed by rotation about the Y-axis (Neville et cd., 1976). It is therefore concluded that the helicoidal model holds true for the parabolic patterning of the eggs of Trichuris n7zrri.s.
Y Fig. 8. Orientation of a section of egg-shell during the tilting experiment. When tilted about the X-axis parabolae appear, their direction depending upon the direction of tilting. When tilted about the Y-axis there is no change in appearance. The lamellae of the chitinous layer are represented by fine lines.
Direct visualization of helicoidal architecture using the ,freeze-etch technique
In oblique and tangential fractures of freezeetch preparations of the eggs of T. suis fibrils can be observed. These can be seen to be responsible for the formation of the parabolae of the chitinous layer (Fig. 10). Fig. 11 shows a tangential fracture which reveals fibrils within three laminae of the chitinous layer. Laminae can be seen to consist of straight parallel fibres, the fibril orientation changes by an angle of 9 in successive laminae. One complete rotation of the helicoid (360”-generating two lamellae) therefore consists of 40 laminae. In Xenopsyllu there is a change of 11 25’ in successive laminae (Neville, 1975). In Hub Cynthia test the angle is 12’ (Cubb, 1975); in cockroach maxillary palp cuticle, IO~~lZ (Altner, 1975) and in the cell wall of Pclwriu embryos, 35” (Peng and Jaffe, 1976). Fig. 12 shows an oblique fracture, reveaiing the parabolae of the chitinous layer. The
Fig. 7. Transverse section through the developing egg-shell of T. muris. The section was orientated and tilted as in Fig. 8 by of-20”. x 107,300. The insets show the results predicted by similarly tilting Neville and Luke’s (1969) model of helicoidal insect cuticle. The model consists of plastic laminae on which are ruled parallel lines. The orientation of the lines changes by a fixed angle in successive laminae. No parabolae are seen when the model is viewed end on (0”) but appear if the model is tilted ( +20”, -20”). The twisted ribbons represent the orientation of cuticular pore canals.
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parabolae can be seen to consist of straight lengths of fibrils. Parabolae are not visible in transverse fractures of the shell (Wharton and Jenkins, 1978, Fig. 9). In Fig. 13 it can be seen that the fracture goes from transverse in the left of the picture to oblique in the right. Parabolae are visible where the shell has fractured obliquely. The width of the lamellae depends on the angle of fracturing of the shell (this can also be seen in Fig. 17). This provides further evidence for the Bouligand hypothesis, as the lamellar periodicity should increase with the angle of obliquity of the section or fracture (Neville, 1975), theoretically becoming infinity for sections parallel to the plane of the laminae. Dennell (1973, 1974, 1976) considers that arthropod cuticle consists of areas of horizontal fibres (‘laminae’) and areas of vertically curved fibres (‘inter-laminae’). According to Dennell, the appearance of parabolae is due to the presence of these curved fibres. Mutvei (1974) and Dalingwater (1975a) have claimed to have observed curved fibres using
scanning electron microscopy. Gubb (1975), however, has shown that the apparent parabolae of the cuticle of Carcinus maenas and the test of Halocynthia papilIosa consist of short lengths of straight fibres. The macrofibrils of crustacean cuticle and tunicate test are large enough to be observed by scanning electron microscopy. In order to observe the fibrils of the chitinous layer of trichurid eggs, it was necessary to use the freeze-etch technique. This has been used recently by Peng and Jaffe (1976) to reveal the helicoidal arrangement of microfibrils in the cell wall of fucoid eggs and by Altner (1975) to reveal helicoidally arranged fibrils in the cockroach maxillary palp cuticle. Curved fibres were not observed in the eggshell of Trichuris suis. Observations on freeze-etch preparations thus do not support the interpretation of Dennell (1973, 1974, 1976). Fibrils are parallel within each lamina but rotate by a fixed angle (9”) in successive laminae, thus forming the parabolae seen in oblique sections. This corresponds to the interpretation of Bouligand (1965).
Fig. 9. Transverse section of the developing egg-shell of T. muris, taken at the base of the opercular plug (P). The parabolae of the chitinous layer can be clearly seen. The parabolae point in the same direction (clockwise) on either side of the egg (boxes). x 13,650. Fig. 10. Freeze-etch replica revealing the microfibrils that make up the parabolae the chitinous layer. x 38,750. Circled arrow indicates the direction of shadowing. Fig. 11. Freeze-etch replica of laminae are shown. Fibrils are tibre orientation changes by an arrow indicates the direction of
of
a tangential fracture through the chitinous layer. Three straight and parallel within each lamina (lines). The angle of 9” in successive laminae. x 131,000. Circled shadowing.
Fig. 12. Freeze-etch replica of an oblique fracture through the chitinous fibres form the parabolae of the chitinous layer. x 58,500. Circled arrow direction of shadowing.
layer. The shows the
Fig. 13. Freeze-etch replica: the angle of fracture goes from transverse on the lefthand side of the picture to oblique on the right-hand side. The width of the parabolae increases as the angle of obliquity of the fracture increases. x 20,950. Circled arrow shows the direction of shadowing. Fig. 14. Tangential section of the egg-shell. of a single spiral. x 14,500.
The chitinous
layer has the appearance
Fig. 17. Freeze-etch replica of a tangential fracture of the chitinous layer. The apparent lamellae form a double spiral (see Fig. 18). The picture is complicated by the fracture jumping from one lamella to another (arrow). x 12,100. Circled arrow shows the direction of shadowing.
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Fig. 15. Diagram showing that the apparent lamellae of the shell follow the course of a double spiral. Taken from a tracing paper overlay of Wharton and Jenkins (1978. Fig. 7). One arm of the double spiral is emphasized.
Fig. 16. Planes of sectioning of trichurid eggs which produce the double spiral (A-B) and the single spiral (D-E) artifacts. Single spirals are produced by tangential sections. Double spirals are produced by transverse or longitudinal sections.
Helicoidal asymmetry
Optically active regions of beetle cuticle reflecting circularly polarized light extinguish simultaneously on both sides of the insect when viewed with a right circular analyser (Neville and Caveney, 1969). The sense of rotation of the helicoid is therefore the same on either side of the mid-line, i.e. it is asymmetrical (Neville, 1975). This has been demonstrated directly by determining the sense of rotation of the helicoid on either side of the mid-line (Neville, 1970). Trichurid eggs are small enough to allow complete sections to be observed with the electron microscope, In tranverse sections of developing egg-shells of T. murk it can be seen that the apparent direction of the parabolae is the same (clockwise) on either side of the egg (Fig. 9). The sense of rotation of the helicoid can be related to the direction of the parabolae (Neville and Luke, 1971). The sense of rotation is therefore asymmetrical about any mid-plane through the egg. The single and double spiral artifacts
In thin sections cut tangentially to the surface of the shell an optical density artifact is produced that has the appearance of a single spiral (Fig. 14). In transverse sections of the egg the apparent lamellae of the chitinous
layer can be seen to form a double spiral (Fig. 15 and Wharton and Jenkins, 1978, Fig. 7). The planes of sectioning of the egg which produce the single and double spiral artifacts are shown in Fig. 16. Bouligand (1972) has discussed the formation of the double spiral artifact (Fig. 3). He also gave two explanations of the single spiral. Both these explanations are considered to be inadequate by Gordon and Winfree (1978) and by Gubb (pers. comm.). The first explanation is that the single spiral is caused by the projection on to the cutting plane of fibrils of laminae arranged as a set of homofocal parabolae (Bouligand, 1972, Figs. 20, 21, 22). This would, however, give a single spiral as an alternative to a double spiralnot superimposed upon it, as is seen in sections of crab body wall tubercles (Bouligand, 1972). The second explanation is that the single spiral is caused by a microtomy artifact. The knife either cuts and turns up fibres or passes between them. As the direction of sectioning is constant and the orientation of fibres to the knife changes in successive laminae and according to its position in the spiral; a single spiral is superimposed upon the double spiral (see Bouligand, 1972, Figs. 28, 31). This predicts, however, that
TRLCHURID
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EGG-SHELL
the spiral will be asymmetrical.
This is not
the case (Fig. 14 and Bouligand, 1972, Fig. 9). Gordon and Winfree (1978) suggest that to produce the single spiral artefact there must be some component that rotates only 180” for every 360” rotation of fibre direction. This condition can be satisfied by adding a set of parallel vectors individually to a set of radial vectors. The radial vectors are provided by the projection on to the cutting plane of the orientation of fibres in concentric conical laminae (as in Fig. 3). The parallel vectors are provided by a distortion of the fibres during sectioning. Gubb (1977) considers that it is not necessary to postulate a sectioning artifact, the parallel vector may be introduced by an optical effect, The formation of the single spiral is discussed more fully by Gubb (1977) and by Gordon and Winfree (1978). Whether a single or a double spiral was seen in sections of trichurid eggs depended on whether sections were cut tangentially or normally/obliquely to the plane of the laminae (Fig, 16). If the parallel vector is due to the distortion of the fibres, this is likely to be greater when sectioning in the plane of the laminae than when sectioning normally to the plane of the lam&e. Freeze etching also reveals tangential sections of the shell (Fig. 17). In this case the parabolae can be seen not to form a single spiral, but appear to form the double spiral artifact (Fig. IS). During freeze-fracture the shell brittle-fractures, so the microfibril pattern would not be subject to distortion.
Fig. 18. Course of the apparent lamellae revealed by a tangentiai fracture in a freeze-etch replica of the egg of T. suis. Taken from a tracing paper overlay of Fig. 17. The lamellae appear to form a double spiral.
This supports the hypothesis that the single spiral artifact may be due to a distortion of the fibres during sectioning. It is difficult to explain the formation of double and single spirals if the Bouligand model is rejected. Summary The following evidence is now available support of the Bouligand hypothesis helicoidal architecture:
in of
experiments (present work; 1. Tilting Neville ef al., 1976). 2. Direct visualization of helicoidal architecture by scanning electron microscopy (Gubb, 1975) and freeze etching (present work; Altner, 1975; Peng and Jaffe, 1976). of double and single 3. The formation spiral artifacts (present work; Bouligand, 1972; Gubb, 1977). 4. The ability to relate pore canal orientation to the helicoidal structure of arthropod cuticle (Neville et ul., 1969). increases with 5. The width of parabolae the angle of obliquity of the section or fracture (present work; Neville, 1975: Gubb, 1975). in the 6. There is a sinusoidal variation intensity of birefringence observed under the polarizing microscope, when passing from one lamella to the next (Neville and Caveney, 1969). A similar sinusoidal variation in electron density is seen under the electron microscope (Neville and Luke, 1971). Helicoidal cuticle of suitable pitch reflects circularly polarized light (Neville and Caveney, 1969; Caveney, 1971). The analogy between helicoidal systems and cholesteric liquid crystals (Neville and Luke, 1971). The specific objections to the Bouligand model raised by Dennell (1974) and Dalingwater (1975b) are answered by reference to a pyramid of helicoidal tissue in which the corners are blunt (present work). Acknowledgements I would like to thank my Ph.D. supervisor, Dr C. J. Mapes, for his help and encourage-
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ment; the late Professor H. E. Hinton and Dr A. E. Dorey for many helpful conversations; Dr A. C. Neville, Dr S. E. Reynolds and Dr D. C. Gubb for introducing me to the wonders of helicoidal architecture; Mr J. A.
Clements and Mr A. Britton for technical assistance; Dr D. A. Erasmus and Dr A. C. Neville for reading the manuscript and the Science Research Council for financial support.
References ALTNER, H. 1975. The microfibril texture in a specialized plastic cuticle area within a sensillum field on the cockroach maxillary palp as revealed by freeze fracturing. Cell Tissue. Ref., 165, 79-88. BOULIGAND, Y. 1965. Sur une architecture torsadbe rt+pondue dans de nombreuses cuticles d’arthropods. C. r. hebd. Se!anc. Acad. Sci., Paris, 261, 3665-3668. BOULIGAND, Y. 1972. Twisted fibrous arrangements in biological materials and cholesteric mesophases. Tissue & CM, 4, 189-217. CAVENEY,S. 1971. Cuticle reflectivity and optical activity in scarab beetles, the role of uric acid. Proc. R. Sot. (B), 178,205-225. DALINGWATER, J. E. 1975a. SEM observations on the cuticles of some decapod crustaceans. Zool. J. Linn. Sm., 56, 327-330. DALINGWATER, J. E. 1975b. The reality of arthropod cuticular laminae. Cell. Tiss. Res., 163, 41 l-413. DENNELL, R. 1973. The structure of the cuticle of the shore crab Car&us maenas (L). 2001. J. Linn. Sm., 52, 159-163. DENNELL, R. 1974. The cuticle of the crabs Cancer pagurus (L) and Carcinus mamas (LX Zool. J. Linn. Sot., 54,241-245. DENNELL, R. 1976. The structure and lamination of some arthropod cuticles. 2001. J. Linn. Sot., 58, 159-164. DRACH, P. 1953. Structures des lamelles cuticulaires chez les crustaces. C. r. hebd. Siam. Acad. Sci., Paris, 237, 159-163. EJIKE, C. 1973. Macrofibrils in the cuticle of the crab Calinectes gladiator (Benedict). Zool. J. Linn. Sot., 53, 253-255. GORDON, H. and WINFREE, A. T. 1978. A single spiral artifact in arthropod cuticle. Tissue & Cell, 10, 39-50. GUBB, D. C. 1975. A direct visualization of helicoidal architecture in Car&us maenas and Halocynthia papillosa by scanning electron microscopy. Tissue & Cell, 7, 19-32. GUBB, D. C. 1977. Helicoidal architecture and the control of morphogenesis in extracellular matrices. Ph.D. thesis, Bristol University. MUTVEI, H. 1974. SEM studies on arthropod exoskeletons. Bull. Geol. Inst. Univ. Uppsala, NS, 4, 73-80. NEVILLE, A. C. 1970. Cuticle ultrastructure in relation to the whole insect. In Insect Ultrastructure (ed. A. C. Neville), Symp. R. Ent. Sot., 5, 17-39. Blackwell, London. NEVILLE, A. C. 1975. Biology of the Arthropod Cuticle. Springer-Verlag, Berlin. NEVILLE, A. C. and CAVENEY,S. 1969. Scarabaeid beetle exocuticle as an optical analogue of cholesteric liquid crystals. Biol. Rev., 44, 531-562. NEVILLE, A. C., GUBB, D. C. and CRAWFORD, R. M. 1976. A new model for cellulose architecture in some plant cell walls. Protoplasma, 90, 307-317. NEVILLE, A. C. and LUKE, B. M. 1969. A two-system model for chitin-protein complexes in insect cuticles. Tissue & Cell, 1, 689-707. NEVILLE, A. C. and LUKE, B. M. 1971. A biological system producing a self-assembling cholesteric protein liquid crystal. J. Cell. Sci., 8, 93-109. NEVILLE, A. C., THOMAS, M. G. and ZELAZNY, B. 1969. Pore canal shape related to molecular architecture of arthropod cuticle. Tissue & CeN, 1, 183-200. PENG, B. H. and JAFFE, C. F. 1976. Cell wall formation in Pelvetia embryos. A freeze fracture study. Planta, 133,57-71. RUDALL, K. M. 1969. Chitin and its organization with other molecules. J. Polymer Sri., 28, 83-102. WHARTON, D. A. and JENKINS, T. 1978. Structure and chemistry of the egg-shell of a nematode (Trichurissuis). Tissue & Cell, 10, 427-440.