http://www.collembola.org/publicat/integum/epicut.htm Last updated on 2006.11.21 by Frans Janssens
Checklist of the Collembola: Some notes on the Ultrastructure of the Cuticula. Epicuticula

Frans Janssens, Department of Biology, University of Antwerp, Antwerp, B-2020, Belgium
Jean-Auguste Barra, Laboratoire de Zoologie, Université Louis Pasteur, Strasbourg, 67000, France
Luc De Bruyn, Department of Biology, University of Antwerp, Antwerp, B-2020, Belgium

Fig.1. Epicuticular surface structure of Tomocerus sp.
Ghiradella, H.T. © 2000
This page is under construction.

Abstract

The formation of the surface ultrastructure of the laminate cuticula of Collembola and more in particular the formation of the regular (mostly hexagonal) pattern on the surface of the epicuticula is based on an epidermal by exocytosis regulated receptor mediated deposition of lipocuticulin micellae.

Introduction

Fig.n. Epicuticular surface structure of dorsal metanotal bladder-like vesicle of male Sminthurides sp.
Palacios-Vargas, J. © 2000.
The ultrastructure of the surface of the integument of the Collembola is one of their most striking features (Hopkin, 1997:51). While it is possible to make the epicuticular surface structure visible in light microscopy (fig.1'), the details of the surface texture was revealed in detail by scanning and transmission electron microscope studies by Massoud in 1969 and by Paulus in 1971 (Ghiradella & Radigan, 1974:301; Eisenbeis & Wichard, 1985:218). The basic pattern is a regular hexagonal tesselation of microtubercles interconnected by ridges (Fig.1, Fig.n, Fig.x, Fig.y) (Ghiradella & Radigan, 1974:302; Massoud & Barra, 1980:251; Eisenbeis & Wichard, 1985:205(Tafel 92 fig.d)); Palacios-Vargas, 2000:103(Foto 3)). The hexagonal pattern occurs on all integumental surfaces, even on the surface of the ocelli (Ghiradella & Radigan, 1974:302). Barra, 1971:322-353 described four variants of the ocellar surface ultrastructure.

The hexagonal pattern does not occur
- on the dorsal face of the unguis of Folsomia candida,
- on the retractile vesicles of the ventral tubus of Pogonognathellus flavescens (Eisenbeis & Wichard, 1985:207(Tafel 93 fig.d)),
- on the rami of the retinaculum of Pogonognathellus flavescens (Eisenbeis & Wichard, 1985:207(Tafel 93 fig.d)),
- on the teeth of the mucro of Orchesella villosa (Eisenbeis & Wichard, 1985:209(Tafel 94 fig.d)),
- on the vesicles of the post antennal organ of Onychiurus spec. (Eisenbeis & Wichard, 1985:213(Tafel 96 fig.d)),
- on the pseudocelli of Onychiurus spec. (Eisenbeis & Wichard, 1985:213(Tafel 96 fig.f)),
- on the post antennal organ of Isotoma aff. saltans (Eisenbeis & Wichard, 1985:227(Tafel 103 fig.f & fig.g)),
- on the distal part of the labrum of Isotomurus palliceps (Eisenbeis & Wichard, 1985:228(Tafel 104 fig.e)).

Fig.x. Epicuticular surface structure of Tomocerus sp.
Plant, N. © 2000
In Pogonognathellus flavescens, the centre to centre distance of the hexagons is about 750 nanometer on the body and about 300 nm on the eye; [on the body] the ridges are about 150 nm wide and 100 nm high (Ghiradella & Radigan, 1974:302).
Not only hexagonal but square arrangements, such as in Anurida maritima, also occur.
In species at risk from desiccation, the microtubercles are closely apposed and the cuticle is thickened to lower its permeability and reduce transpiration of water.
Adaptations of the arrangement of the microtubercles to cold do not seem to occur.

Epicuticular surface structure of Megalothorax sp.
Walter, D.E. © 1999
In this paper, it will be shown that, while there are species-specific differences in the surface pattern, the formation of the ultrastructure is merely controled by the electro-chemical and physical characteristics of the film formation process of the by the epidermal cells secreted epicuticular material. This might indicate that the ultrastructure should not be used for taxonomic purposes, as already has been suggested (Hopkin, 1997:55).
Exceptions do exists: the epicuticular ultramorphology of the species of the Coenaletidae (see Fig.5) is considered as characteristic to the family (Palacios-Vargas, 2000:103(Foto.2),104). This family-specific ultrastructure might be an adaptation to the marine littoral and commensal life with Coenobita clypeata and might function as a means to form a distributed plastron on the body of suddenly in water submerged animals.
To be clarified:
In Tetradontophora? Eisenbeis found an ultramorphological structure similar to Coenaletidae (Eisenbeis:199Y:p).

Epicuticular surface structure observation in light microscopy

Fig.lm. Epicuticular surface structure of Tomocerus sp.
Janssens, F. © 2004
First of all, especially when observing the cuticular surface structures in light microscopy, the pattern of epicuticular microtubercles should not be confused with the pattern of exocuticular tubercles. The pattern of epicuticular microtubercles is superimposed upon the pattern of exocuticular tubercles.

The epicuticular ultrastructure can be recognised using light microscopy techniques. However, the details of the ultrastructure cannot be made visible due to the limited capabilities of the visible light to resolve the tiny substructures (the wavelength of the light is equal or larger than the size of the details of the ultrastructure). Using a magnification of 1000 times (ocular lens 10x, object lens 100x) (the conventional Abbe limit), a thin section of a specimen of Tomocerus sp., embeded in paraplast, and treated with a histological hemaluin-eosin colouring technique, was observed applying an immersion oil phase-contrast illumination system. A network of interconnected microtubercles in a hexagonal pattern is clearly revealed (fig.1m).

Structure of the epicuticula

Fig.y. Epicuticular surface structure of Entomobrya sp.
Plant, N. © 2000
The nonchitinous epicuticula is composed of substances that, it has been shown by Wigglesworth (1933b), are also constituents of the exocuticula. According to Kuhnelt (1928, 1928a), this surface film (Grenzlamelle) of the exocuticula is highly resistant to acids, but when heated in caustics it is saponified and can be shown to contain fatty acids and cholesterin (Snodgrass, 1935).
The epicuticula differentiates into four layers (Massoud & Barra, 1980:251): a cement layer, a wax layer, an external or outer epicuticular layer (of polyphenolic resins) and an internal or inner epicuticular layer (of cuticulin = a mixture of tanned lipoproteins and an enzyme phenoloxidase, that produces extra tanning in case of damage of the epicuticula - self-repairing).

Summary of the nomenclature used by several authors with respect to the laminate structure of the epicuticula (Edney, 1977:48-50):
the inner epicuticula, 300 to 1000 nm thick in Rhodnius, rich in lipids impregnating a highly tanned protein component. It is the "cuticulin layer" of Wigglesworth (1947), the dense layer or protein epicuticle of Locke (1961), the inner epicuticle of Weis-Fogh (1970);
the outer epicuticula, 17 nm thick, highly resistant and lipid rich. It is the resistant layer of Wiggleworth (1947), the paraffin layer of Dennell & Malek (1955), the cuticulin layer of Locke (1961) and Filshie (1970), the outer epicuticle of Weis-Fogh (1970);
the wax layer, 10 nm thick, lipid, hard, and hydrophobic. It is the surface monolayer or oriented lipid layer of Locke (1966), the outer epicuticle or superficial layer of Filshie (1970);
the cement layer, 30 to 100 nm thick, a stabilised mucopolysaccharide impregnated with wax. It is the tectocuticle of Richards (1953); it is a multilayer of which the inner most layer is the monomolecular lipid layer of Locke (1966) and Gluud (1968), the wax layer of Wigglesworth (1975).

Function of the epicuticular ornamentation

Epicuticular surface ornamentation of Tomocerus? sp.
Ghiradella, H.T. 2000
Respiration under water. The plastron respiration hypothesis of Imms (1906) or Noble-Nesbitt (1963).
When the littoral Anurida maritima and Anuridella marina are submerged in water, a layer of air is retained in the troughs between the microtubercles. The very thin firmly held layer of air is termed a 'plastron'. It is held in position by surface tension forces and its volume remains constant (Wigglesworth, 1965:343) as long as the gas exchange between the submerged animal and the plastron is in balance with the gas exchange between the plastron and the surrounding water; the animal can continue to respire when submerged, if the water is well aerated.

Collembola are terrestrial animals that do not normally enter water. Many species are soil inhabiting. In the soil they may be subjected by flooding due to rain. Specimens survive flooding through an epicuticular plastron. The plastron is a respiratory structure where the epicuticle is covered by protuberances that retain a constant volume of air covering the cuticle when the animal is submerged into water. This thin sheet of air works like a gill: oxigen depletion in the plastron air leads to the diffusion of more oxygen from the surrounding water into the plastron. The rigid epicuticular structures ensure that the air volume remains constant and that the gas is not dissolved in the surrounding water.

Sminthurides aquaticus may 'slip' into the water and stay completely submerged for about 4 days (Falkenhan, 1932 cited from Thibaud, 1970:181). Cave Collembola may traverse over the bottom of water pools (Franciscolo, 1951 cited from Thibaud, 1970:181). Adult Arrhopalites may live 17 days under water (Delamare Deboutteville 1952, cited from Thibaud, 1970:181). When Ceratophysella armata exits from the water and returns back into the water, its body is covered with a thin layer of air (Pritt, 1951 cited from Thibaud, 1970:181). Onychiurus, Tomocerus, and Orchesella breath under water through the thin layer of air that surrounds them, that functions like a "physical lung": the oxygen of the layer of air that is consumed due to the respiration of the animal is renewed by the oxygen of the water (Ruppel, 1953 cited from Thibaud, 1970:181). Submerged specimens of Typhlogastrura balzuci postpone moulting for 36 days; those that attempt to moult do not survive it (Thibaud, 1970:181-182). Possibly, Hypogastruridae may complete their life cycle sub-aquatic (Thibaud, 1970:182).
Cuticular plastron structures are also found in whip spiders (Amblypygi); e.g. in the under stones on beaches of Florida occuring Phrynus marginemaculatus, surrounding the book lung stigmata (Weygoldt, 2000:61).
The ability to stay alive under water for several hours may be an adaptation to living close to water.

Nomenclature of the epicuticular ornamentation

Epicuticular surface ornamentation
Sepsenwol, S. © 2004
A comprehensive nomenclature concerning the epicuticular ornamentation is suggested ( modified after Massoud & Barra, 1980:252-2591; and compiled from Dallai & Malatesta, 1973:1367; Ghiradella & Radigan, 1974:3022; Hale & Smith, 19665; Hopkin, 1997:51-553; Eisenbeis & Wichard, 1985:204,2184; Massoud, 19696; Paulus, 1971:38,397 ):

Interpreting cross-sections of the epicuticular ornamentation

Transmission Electron Micrographs (TEM's) of cross-sections of the epicuticular ornamentation are not always that easy to interprete. Depending on the angle relative to the cuticular surface the section was made, the image may look quite different and it is not always obvious to reconstruct from the image the three dimensional structure of the ornamentation. To be able to interprete the images correctly, it is required first to learn to recognise from the image at what angle the section was made.

Sections can be classified according to several criteria related to the plane the section was made:

Fig.c1. Section of the epicuticula of Pseudosinella subduodecima
Barra, J.-A. © 1973 (unpublished)

An example: Fig. c1 is a section of the epicuticular ornamentation of a specimen of Pseudosinella subduodecima (Barra, 1973), one day after moulting. In the left part of the image, the section is at a surface oblique tangential angle. The 'air-bubble'-like objects are the oblique tangential projections of the sectioned troughs of the crowns.
Note that these 'air-bubble' like artefacts have misled Paulus (1971) in his interpretation of the three dimensional epicuticular structure.
In the centre of the image, the section becomes more surface oblique perpendicular, indicating a slightly downwards curved local topography of the cuticular surface (note also the flattened cuticular horizon). And at the right it is again more surface oblique tangential. Given the relative wide ridges (in left and right part of image), the section is crown saggital oriented. Given the almost perfect symmetry of the tesselation, the section is perpendicular to opposite ridges of a crown. The asymmetry at the right is an indication of an anomaly in the hexagonal tesselation. Applying this interpretation to the section results in a projection of the ornamental tesselation as represented in the schematic diagram of Fig. 2c.

Fig.c2. Schematic projection of the ornamental tesselation deduced from the section of the epicuticula of Pseudosinella subduodecima

Three dimensional Model of the epicuticular Ultrastructure

Fig.p. Three dimensional reconstruction
of the exocuticula and epicuticula
Paulus, 1971.

Paulus (1971:37-44) proposed a three dimensional model (fig. p) of the epicuticular structure based on an interpretation of TEM images of a tangential section through the cornea of Neanura sp. and a cross-section through the cuticula of Entomobrya muscorum. Paulus, apparently not aware that the cuticular cross-section was quite oblique, misinterpreted the TEM image and concluded that the epicuticula rests on short pillars. See chapter "Interpreting cross-sections of the epicuticular ornamentation" for a more elaborate discussion.
Paulus' model does support the plastron respiration hypothesis of Imms (1906) or Noble-Nesbitt (1963).

Tuberculate Cuticula

Fig.8. Tuberculate cuticula
of Sminthurides sp.
Palacios-Vargas, J. © 2000.
Fig.7. Tuberculate dentes
of Sminthurides sp.
Palacios-Vargas, J. © 2000.
The hexagonal primary granular ultrastructure of the surface of the epicuticle is a primary characteristic of the cuticula of all Collembola. While the primary granules (minor tubercles or 'tubercoli minori' of Dallai, 1973) are epicuticular structures, the tubercles (major tubercles or 'tubercoli maggiori' of Dallai, 1973) are exocuticular structures. In the latero-ventral view of the mid part of the dens of Sminturides sp. (Fig.7 after Palacios-Vargas, 2000:103(Foto 4)) each tubercle is covered by the epicuticula with its primary granular ultrastructure. Note also the lateral exocuticular folds. Due to the curvature of the tubercular surface, the regular hexagonal primary granular pattern is strongly deformed. The largest deformation occurs at the apex of the tubercle. Each tubercle is covered by a helicoidal texture of hexagons. The helicoidal hexagon winding can be explained by a mechanism purely based on geometrical considerations. The most important geometrical constraint in this model is: the hexagon formation process can be viewed as analogous to winding strings around a cone in such a way that its surface is fully covered. Therefore, the number of strings being wound simultaneously per cone circumference must be dependant on the angle they make with a plane perpendicular to the cone axis. A terminal pentagon closes the helicoidal winding at the apex of the tubercle (Fig.7). The tuberculation (Fig.8) can be seen as a secondary characteristic of the cuticula. Tuberculation originates from exocuticular folding. Periodically spaced repeated linear folding results in crenulation. Tuberculation can be described as the result of two orthogonal crenulations. Tuberculation of the cuticula increases the surface of the cuticula dramatically. Assuming that tuberculation is a derived character, the phylogenetic position of the Poduromorpha can be questioned.
Tubercles of Hypogastrura tullbergi
Fig.9a. SEM of tubercle
of Hypogastrura tullbergi
After Cassagnau, 1977.
The cuticula of Hypogastrura shows one of the most basic primary tubercles of all Collembola. While the relative long tubiform tubercles of Sminthurides are characterised by an epicuticular helicoidal winding of hexagonal crowns, the relative stout tubercles of Hypogastrura tullbergi with a more tetrahedral architecture have three epicuticular hexagonal crowns that are joined at the top vertex of the tetrahedron (the apex of the tubercle) (see Fig.9a after Cassagnau, 1977; planche II, fig. a; x 11,000: a tubercle of the tergite of the fourth abdominal segment). The hexagonal primary arrangement in Hypogastrura tullbergi is constituted of triangular headed primary granules. The tubercles are more elevated than the triangular primary granules, and have a superimposed hexagonal arrangement of triangular primary granules. The triangular head at the apex of the tubercle is more raised than any other primary granule and its size is much larger.

Fig.10a. 2D scheme of tubercle
of Hypogastrura tullbergi
Janssens, F., © 2001.
Based on the SEM of a tubercle (fig.9a) a simplified two dimensional scheme of the epicuticular arrangement of the primary granules on the tubercle can be represented as a kind of exploded, flat view (fig.10a: top view that can be mapped to the SEM of fig.9a). Three regular hexagonal crowns are joined at the center. This center is the apex of the tubercle.

The three dimensional epicuticular arrangement of the primary granules is modelled as a ball and stick wire-frame. The sticks represent the ridges interconnecting the primary granules. The balls represent the 3D position of the primary granules. The heads of the primary granules are modeled as intersected tori that are placed at the ends of the sticks. In this way, by putting the sticks together in the proper geometrical configurations, the polygonal heads of the primary granules are modelled implicitly. Not only the shape but also the size of the heads are properly modelled: concave joined sticks result in smaller heads; convex joined sticks result in larger heads.

The model allows us to formulate a relation between the size of the primary granular head and the angle between the longitudinal axes of the ridges connected to the granule:
s = r*(1+2*sin(30+(alpha/2)))
- alpha is the angle between the longitudinal axes of the sticks;
- s is the size of the primary granular head measured from the center of the head to the edge orthogonally.
This formula is based on the follwing simplified assumptions on the dimensioning of the ridges:
- radius of the sticks, minor radius of the tori and radius of the balls are all equal (r);

We can also derive the the type of cuticular surface curvature from the variability of the size of the primary granular heads. Given a regular primary arrangement of primary granules, heads that are smaller than those of the regular primary arrangement indicate a concave cuticular surface while larger heads indicate a convex cuticular surface.

Fig.11b. 3D model of tubercle
of Hypogastrura tullbergi
lateral view
Janssens, F., © 2001.
Fig.11a. 3D model of tubercle
of Hypogastrura tullbergi
top view
Janssens, F., © 2001.
The tubercle is modelled as a flat topped tetrahedron. At the top of the tetrahedron three hexagons of primary granules intersect in such a way that a small platform is made that serves as a carrier for the large triangular head of the tubercle (fig.11a: top view that can be mapped to the SEM of fig.9a; fig.11b: lateral view). Note that this apical triangular head is that large due to the fact that it is the combination of three convex tilted primary granules. Note also that the three triangular heads at the corners of the platform are the combination of three convex tilted primary granules. Due to the fact that they are relatively less tilted, they are smaller then the apical head, but still larger than the regular heads. The bases of the hexagons line-up with the hexagonal primary arrangement of the triangular primary granules of the non-tubercular epicuticula.

Tubercles of Podura aquatica
Fig.9. SEM of tubercle
of Podura aquatica
After Dallai & Malatesta, 1973.
The tubercles of Podura aquatica are quite larger than those of Hypogastrura: three epicuticular hexagonal crowns are joined at the top vertex of the tetrahedral tubercle (the apex of the tubercle). The hexagons are extended with two pentagonal crowns at the base of the faces of the tetrahedron (see Fig.9 after Dallai & Malatesta, 1973; x 10,000). The tetragonal primary arrangement in Podura aquatica is constituted of square headed primary granules (Dallai & Malatesta, 1973:136). The tubercles (major tubercles cf. definition of Hale & Smith, 1966, or granules secondaires cf. Massoud, 1969, or tubercoli maggiori cf. Dallai & Malatesta, 1973) are more elevated than the tetragonal primary granules, and have a superimposed hexagonal/pentagonal arrangement of triangular primary granules (Dallai & Malatesta, 1973:136). The triangular head at the apex of the tubercle is more raised than any other primary granule and its size is up to 5 times larger, up to 400 nm long (Dallai & Malatesta, 1973:136). Effectively, the cuticula of Podura and in particular the formation of the tubercles is realised according to a scheme that is analog to that observed in Hypogastruridae (Dallai & Malatesta, 1973:137).

Fig.10. 2D scheme of tubercle
of Podura aquatica
Janssens, F., © 2001.
Based on the SEM of a tubercle (fig.9) a simplified two dimensional scheme of the epicuticular arrangement of the primary granules on the tubercle can be represented as a kind of exploded, flat view (fig.10: top view that can be mapped to the SEM of fig.9). Three regular hexagonal crowns are joined at the center. This center is the apex of the tubercle. The hexagons are surrounded by irregular pentagons. In this simplified scheme, all pentagons are identical and are derived from the regular hexagons. The bases of the pentagons line-up with the tetragonal primary arrangement of the primary granules of the epicuticula.
The intrinsic triangular nature of the tubercle is clearly demonstrated.

Fig.11b. 3D model of tubercle
of Podura aquatica
lateral view
Janssens, F., © 2001.
Fig.11a. 3D model of tubercle
of Podura aquatica
top view
Janssens, F., © 2001.
The three dimensional epicuticular arrangement of the primary granules on the tubercle are modelled as a ball and stick wire-frame (fig.11a: top view that can be mapped to the SEM of fig.9; fig.11b: lateral view). At the top of the tetrahedron, three regular hexagons of primary granules intersect in such a way that a small platform is made that serves as a carrier for the large triangular head of the tubercle. Note that this apical triangular head is that large due to the fact that it is the combination of three convex tilted primary granules. Note also that the three triangular heads at the corners of the platform are the combination of three convex tilted primary granules. Due to the fact that they are relatively less tilted, they are smaller then the apical head, but still larger than the regular heads. This architecture is similar to that of Hypogastrura. However, the podural tubercle is more than twice as high as the hypogastrural tubercle. It is also more steep. As a result, each hexagon is at its basis extented into the face of the tetrahedron with two pentagons that form the basis of each face of the tetrahedral tubercle. At the flanks of the tetrahedron, the faces are joined through strongly deformed pentagons. The base pentagons line-up with the tetragonal primary arrangement of the tetragonal primary granules of the non-tubercular epicuticula.

Tubercles of Tetrodontophora bielanensis
Cf. Dallai (1973):
steep conic shape of tubercle: Tav. II fig 1
tetragonal arrangement of primary granules: Tav. II fig 2 and 3
primary granules with tetragonal head: Tav. II fig 2 and 3
resembles tubercles of Podura aquatica
but more elevated
base of tubercle = pentagonal

less elevated tubercles on ventral side of furca: Tav. VI fig 2
with tetragonal base
and 4 uptilted pentagons joined at the apex of the tubercle
apical granular head = tetragonal and much larger than the other tetragonal heads of the granules of the primary arrangement

To be completed.

Conclusion

To be completed.

Acknowlegdements

We would like to thank Dr Nico Büsscher for preparing the in paraplast sectionned specimens.

Fig.x. Animation to show that the hexagonal pattern of epicuticular ridges improves the tangential rigidity of the cuticle.