The cuticle overlying most of the body consists of 2 major layers only, the lamellate endocuticle and the epicuticle, and is very thin (1 to μ). Three major cuticular layers are found in the slightly thicker cuticle of certain areas (2 to 3 μ thick) and in the thickest regions (8 to 10μ)a distinct sclerotized exocuticle is present. The epicuticle consists of 1 to 4 laminae, the ‘inner’, ‘cuticulin’, ‘wax’, and ‘cement’ layers, and the distribution of these laminae is described. The outer surface of the cuticle is thrown into major tubercles of approximately 3 μ pitch, with minor tubercles super-imposed upon them. Modification of the cuticle at the bases of setae is described and related to the mechanical requirements of the setal insertion and suspension. The nature of the muscle insertion is also considered. The epidermis is fully cellular and the cells contain the granules of black pigment which impart the black coloration to the insect. Specializations of the epidermis in the regions of ocelli, setae, muscle insertions, and the vesicles of the ventral tube are described, along with modifications of the overlying cuticle.

Earlier work on the cuticle of Collembola has shown that it has a structure which fits closely into the scheme for a generalized insect (or other arthropod) cuticle. Imms (1906) distinguished 2 layers in the cuticle of Anurida marítima (Guérin). The 2-layered nature of the cuticle was also reported for Tomo-cerus longicornes (Müller) by Sommer (1885), and for Hypogastrura viatica (Tulbbergii) and Isotoma grísea Lubbock by Prowazek (1900). Koncek (1924) reported 3 layers in the cuticle of Tetrodonotophora gigas Reuter. Ögel (1958) and Lower (1958) also report 3 major divisions of the cuticle, in Folsomia candida (Willem) and Smynthurus viridis L., respectively. Boelitz (1933) recognized lamellae in stomodaeal cuticle, and Ögel (1958) reported lamellae in the endocuticle in her electron microscopical study. Lower (1958), however, concluded that lamellae were absent from the cuticle of S. viridis L. Most of these earlier accounts indicated that the underlying epidermis was syncitial, with few or no cell boundaries.

The cuticle of Collembola therefore appears, from these reports, to be a very thin, but otherwise typical, arthropod cuticle which exhibits a few peculiarities of its own. The present study on Podura aquatica L. (Collembola, Isotomidae) has been made to determine how far this generalization applies to this insect, and also to provide the structural basis for a consideration of the functions of the cuticle.

Podura aquatica was collected from the surfaces of ponds and ditches in the Cambridgeshire area and kept in small aquaria in the laboratory. Either ditch-water or tap-water was used in the aquaria, and a plentiful supply of duckweed was maintained on the water surface to provide food for the insects.

Specimens prepared for the light microscope were usually fixed for 2 h in alcoholic Bouin’s fixative (Duboscq-Brasil), but Carnoy’s fixative was also used. Following the usual dehydration and impregnation procedures, the specimens were embedded in 54° C paraffin wax and sections cut at 6 μ.. Mallory’s triple stain and Heidenhain’s haematoxylin stain were used as general stains. The methods employed followed those given in Pantin (1948). The black colour of P. aquatica proved useful in preparing specimens for the microtome, rendering the insect more readily visible. Embedding of P. aquatica was carried out using an orientation technique based on that described by Overgaarde (1948). Useful information was obtained from specimens prepared using the osmium I ethyl gallate method of Wigglesworth (1957, 1959). These also provided a reliable check on the fixative used in preparing specimens for the electron microscope.

Specimens prepared for sectioning with the freezing microtome were fixed with Baker’s formaldehyde/calcium fixative for 3 days, then stored in Baker’s formaldehyde/calcium/cadmium storage solution. Prior to embedding, the specimens were washed for 1 h in tap-water, then left overnight in a 25% gelatine solution, to which a little creosote was added to act as a preservative. This was followed by transferring the specimens to fresh gelatine and creosote solution and gelling in a refrigerator for 2 h. The gelatine block was then trimmed, and hardened for 4 days in Baker’s formaldehyde/calcium/cadmium storage solution. Frozen sections were then cut on a freezing microtome, at 6/1, and transferred to slides coated with gelatine. The slides were stored in Baker’s formaldehyde/calcium/cadmium storage solution, and later stained with Sudan black and Mayer’s carmalum (see Pantin, 1948, P26).

Fixation of whole P. aquatica for the electron microscope was carried out in the cold, using 1% osmium tetroxide buffered to pH 7·2 after the method of Palade, modified by Sjöstrand (1956). The period of fixation was varied, but the usual period was about 24 h. Dehydration was carried out, also in the cold, by passing the tissues through a graded series of alcohols or acetones. After dehydration, the tissues were embedded usually in ‘araldite’ (Glauert and Glauert, 1958; Luft, 1961) polymerized for 72 h at 57° C, but also in partially pre-polymerized ‘methacrylate’ (Borysko, 1956) polymerized for 20 h at 6o° C. The embedding was carried out in gelatin capsules.

During the early stages of the investigation, sections were cut using a Hodge thermal-advance ultra-microtome. The tissue block was orientated on the microtome chuck, using sealing wax. Sections showing interference colours of pale straw, gold, silver, or grey were picked up on celloidin-covered grids which occasionally had the celloidin film stabilized with a very thin layer of carbon. Later sectioning was carried out using a Huxley mechanical-advance ultra-microtome manufactured by the Cambridge Instrument Company. Orientation was achieved by remounting the small tissue block using ‘araldite’ adhesive on to a stem of ‘araldite’, which fitted the chuck of the microtome. Sections were cut at thicknesses of 20 to 80 mμ, and picked up on grids as described above. Electron staining of the sections on the grids was accomplished by using uranyl acetate in 50% alcohol (see Gibbons and Grimstone, 1960) or the lead hydroxide stain of Watson (1958) as modified by Peachey (1959). Bulk staining with phospho-tungstic acid was also used.

Grids were viewed principally in Siemen’s ‘Elmiskop’ electron microscopes at the Cavendish Laboratory and the Department of Anatomy. Later work was carried out using the Philips E.M. 200 electron microscope at the Department of Zoology.

Observations with the light microscope

As seen with the light microscope, most of the body is covered by a soft, thin, transparent cuticle 0·5 to 1μ thick (see fig. 1). It stains blue with Mallory’s triple stain, except for an amber outer region, which is very thin and refractile. Another layer which stains red in Mallory occurs between these layers in certain areas where the cuticle is slightly thicker. It also occurs occasionally in the thin cuticle, where it is exceedingly thin and not readily distinguishable from the outer refractile layer. Applying the nomenclature generally used for arthropod cuticles, the blue inner layer is the endocuticle, the outer refractile amber layer is the epicuticle, and where present the red layer is an exocuticular layer equivalent to the ‘mesocuticle’ of Schatz (1952) and Lower (1956, 1958). A true, fully sclerotized exocuticle is absent from this thin cuticle.

FIG. 1

Diagram showing the surface sculpturing in the cuticle of P. aquatica.

FIG. 1

Diagram showing the surface sculpturing in the cuticle of P. aquatica.

The cuticle is thrown into small tubercles approximately 3 μ. in diameter at their bases, approximately 3μ high, and with a peak to peak distance of approximately 3 μ. The tuberculate nature of the cuticle in this species is well known (Maynard, 1951). Tubercles are absent only in special areas, such as over the eyes (lens area only), on some areas of the limbs (unguis), and over the vesicles of the ventral tube; in these cases the cuticular surface is smooth. These tubercles will be referred to as the ‘major tubercles’.

Superimposed on the major tubercles is a system of more minute tubercles, giving in surface views a granular, sculptured appearance to the general surface of the cuticle, which is absent over the smooth areas (see fig. 2). These smaller tubercles, which will be referred to as the ‘minor tubercles’, are too small to be resolved fully in the light microscope. They have been described in other species by Goto (1956) and Ogel (1958).

FIG. 2.

Diagram of a Mallory-stained section through the cuticle which occurs over most of the body in P, aquatica, showing a setal insertion. The cuticle is slightly lifted away from the epidermis, which contains pigment granules. The blue endocuticle is shown by horizontal lines, and the area at the setal insertion which stains red in Mallory is shown by vertical lines. The epicuticle is amber in colour.

FIG. 2.

Diagram of a Mallory-stained section through the cuticle which occurs over most of the body in P, aquatica, showing a setal insertion. The cuticle is slightly lifted away from the epidermis, which contains pigment granules. The blue endocuticle is shown by horizontal lines, and the area at the setal insertion which stains red in Mallory is shown by vertical lines. The epicuticle is amber in colour.

Areas of the limbs, antennae, furcula, retinaculum, and head capsule, where greater mechanical strength may be required, have a thicker cuticle (1·5 to 2 μ thick) showing three distinct layers in Mallory-stained sections, an inner blue endocuticle, a middle red ‘mesocuticle’, and a superficial refractile amber epicuticle (see fig. 3). The tubercles referred to above are present also in these areas, but they involve only the outer layers of the cuticle. It is of interest to note that this type of cuticle occurs on the outside of the antennae when these are pointed forward. This is the area of impact when a surface is hit after a spring, and greater mechanical strength would be expected in such an area. It is possible that the ‘mesocuticle’ provides this strength.

FIG. 3.

Diagram of a Mallory-stained section through a thickened region of the cuticle of P. aquatica. Horizontal lines indicate a blue coloration, and vertical lines indicate a red coloration. The superficial epicuticle is amber in colour.

FIG. 3.

Diagram of a Mallory-stained section through a thickened region of the cuticle of P. aquatica. Horizontal lines indicate a blue coloration, and vertical lines indicate a red coloration. The superficial epicuticle is amber in colour.

Over a limited area in the clypeal region of the head capsule, extending for only about 40 μ × 40 μ, the cuticle attains a thickness of 10 to 12 p and its structure is more readily discerned in the light microscope (see figs. 4 and 5). Surface tubercles are again present. They seem to be due to folding in the outermost epicuticular layer only. This layer is refractile and amber coloured. Beneath this is a layer about 5 μ thick which stains red-orange in Mallory. It tapers away at the edges of the area. There is evidence of amber coloration within this layer, occurring as blotches, or as a layer bounded inside and out by regions which stain red. This indicates that tanning occurs in this layer, which, therefore, may be referred to as a true exocuticle. Underlying the exocuticle is the endocuticle, which stains blue with Mallory. It is about 4 μthick and in some sections it is seen as a double layer, the innermost part staining a lighter blue (cf. Dennell, 1946). At the edges of the clypeal region, the blue layer tapers off into the thinner blue layer of the thin body cuticle. At bristle insertions, the clypeal cuticle is very much thinner, most of the endocuticle and exocuticle being absent, and cell membranes pass up to the bristles. This is especially evident where fixation has caused the retraction of the cells from the cuticle (see fig. 4). The arrangement is similar to that of bristle insertions in pterygote insects (Wigglesworth, 1933, and his report of Hsü in Wigglesworth, 1953).

FIG. 4.

Diagram of clypeal cuticle stained with Mallory. The occurrence of amber coloration beneath the epicuticle is shown by diagonal lines. Horizontal lines indicate a blue coloration, and vertical lines indicate a red coloration. The epicuticlc is amber in colour.

FIG. 4.

Diagram of clypeal cuticle stained with Mallory. The occurrence of amber coloration beneath the epicuticle is shown by diagonal lines. Horizontal lines indicate a blue coloration, and vertical lines indicate a red coloration. The epicuticlc is amber in colour.

FIG. 5.

Diagram of clypeal cuticle stained with Heidenhain’s haematoxylin.

FIG. 5.

Diagram of clypeal cuticle stained with Heidenhain’s haematoxylin.

When Heidenhain’s haematoxylin is used, a less straightforward picture of clypeal cuticle is given (see fig. 5). Beneath the refractile epicuticle is a darkly stained layer with areas of medium stain. This corresponds to the mixed red-orange and amber exocuticle zone seen with Mallory’s stain. Underlying this mixed zone is a distinct zone of medium stain, which is, however, almost wholly replaced by the darkly stained layer near bristle insertions (see fig. 5). This area of medium stain is separated from the lightly stained innermost layer (equivalent to the blue layer of Mallory sections) by a darkly stained thin layer 1 to 2 μ thick. These complex staining reactions do not readily fit in with Dennell and Malek’s scheme (Dennell and Malek, 1955 a, b) for the state of tanning as shown by affinity for haematoxylin. The degree of staining seems to be greater where greater hardness would be expected and less where the cuticle is soft (i.e. in the endocuticle). However, the refractile, and presumably fully tanned, epicuticle is not stained. It is possible, therefore, that varying degrees, or types, of tanning cause this differential staining with haematoxylin. Striations, which are somewhat refractile and at right angles to the cuticle surface, pass from the inner surface of the cuticle out to the darkly stained zone in Heidenhain-stained sections. These are thought to represent pore canals.

The layer which stains red in Mallory also occurs over muscle insertions. If this represents a mechanically stronger cuticle then its occurrence at muscle insertions is to be expected. However, Weis-Fogh (1960) has recently shown that an elastic endocuticle which stains red in Mallory occurs at certain muscle insertions in pterygote insects. It is possible that a similar type of cuticle may also be present over muscle insertions in P. aquatica.

At the insertion of each bristle over the general body area, the thin cuticle shows a distinct red ring when stained in Mallory (see fig. 1). This is probably associated with a need for greater mechanical strength, or rigidity, at the base of the seta. The red layer overlies the blue layer and underlies the refractile amber epicuticle, which is only imperfectly visible. The red layer appears to taper off sharply, as indicated in fig. 1, and may come to an end completely at the edge of this confined region, leaving only the blue and amber layers of the general body cuticle. It is, therefore, probably produced under the influence of the tormogen cell only (cf. Wigglesworth, 1933). It is to be noted also that the setae are light amber in colour, and it is suggested that complete tanning may occur in them. Further evidence accrued from investigations using the electron microscope (see below).

A smooth, very thin cuticle (about 0 · 5 μ thick) covers the vesicles of the ventral tube, an eversile organ on the first abdominal segment. It stains blue with Mallory, and sometimes seems to have a refractile outermost layer. It overlies a glandular epithelium, which may be involved in exchange of materials over the thin cuticle (see Noble-Nesbitt, 1963c). Further details were made apparent in the electron microscope (see below).

The cuticle is smooth also over each ocellus, there being no evidence of the tubercles which occur over the surrounding cuticle. This modification doubtless allows uninterrupted passage of the light rays through to the underlying tissues. Tubercles would be expected to scatter the rays. Beneath the cuticle lies a lens, which is highly refractile and amber coloured. It is not shed at the moult. The epidermis with its refractile pigment granules surrounds the lens and obscures the sensory cells, but nerve-tracts from them can be traced passing to the brain. The pigment-containing epidermal cells penetrate deeper into the head at this point, associated with the sensory nerve-tracts.

Light microscope preparations of Orchesella villosa stained in Mallory show that the cuticle in this larger collembolan is thicker than that of P. aquatica, the cuticle of the general body surface being 2 to 3 μ thick. The ‘mesocuticle’ occurs over the whole of the general body-surface. The cuticle surface is tuberculate, as in P. aquatica. In the thicker clypeal cuticle, the vertical lines of the pore canals are more in evidence than in P. aquatica. Pigment granules again occur in the epidermis. In general, the results from this collembolan agree with those from P. aquatica, and confirm the basic structure of the collembolan cuticle.

Observations using the light microscope thus indicate that the basic structure of the cuticle and the underlying epidermis of P. aquatica and O. villosa agrees closely with that reported for other species of Collembola and for the pterygote insects. In general, 3 cuticular layers are present, though only in localized areas does the exocuticle appear to be fully tanned. Elsewhere, it is either more akin to the ‘mesocuticle’ of Lower (1956, 1958) or it is absent. The surface is tuberculate, except over specialized areas. Evidence of pore canals can be seen in the thick regions of the cuticle. Cytoplasmic filaments appear to pass to, or perhaps even into, the setae. The epidermis contains the pigment which imparts the body-colour, and obscures much of the cellular detail. It is therefore not possible to comment on the presence or absence of cell boundaries, or to determine whether the epidermis is a syncytium as reported for Folsomia candida (Willem) (Ogel, 1958) and Smynthurus viridis L. (Lower, 1958). A basement membrane is apparently absent. This agrees with results reported for some Collembola (cf. Lower, 1958; Ogel, 1958) but differs from the situation in pterygote insects (cf. Wigglesworth, 1953) and other Collembola (cf. Sommer, 1885; Imms, 1906).

Much of the information obtained using the light microscope is incomplete because of the extreme thinness of the cuticle and the highly refractile nature of many of the epidermal and cuticular components, which makes resolution difficult. A more detailed investigation has therefore been carried out using the electron microscope. The results of this investigation are set out below.

Observations with the electron microscope

Fig. 6, A is an electron micrograph of the cuticle which covers most of the body in P. aquatica. The endocuticle overlies the surface of the epidermal cells, which is thrown into numerous small folds. It consists of lamellae lying one above the other and is bounded on its outer side by the very thin, electron-dense epicuticle. The whole of the cuticle in these thin areas is thrown into the major tubercles seen in the light microscope. The epidermal surface is thrown into corresponding folds. The peak-to-peak distance of these tubercles is approximately 3 μ and they rise approximately 2 μ, above the general level of the cuticle, which is slightly less than light microscope preparations indicate. Superimposed on this gross sculpturing is a finer, epicuticular sculpturing. The epicuticle is thrown into minor tubercles which are approximately 0 · 2 μ in diameter, 0 · 2 μ high and 0 · 4 to 0 · 6 μ apart. The presence of these was indicated by the light microscope. They have been reported by Ogel (1958) for F. candida (Willem). The arrangement of the tubercles in P. aquatica is shown in fig. 6, B, which is an oblique section through the cuticle, glancing the surface. Pore canals pass up through this cuticle, especially to the major tubercles, but also to the minor tubercles (see fig. 6, c). In Diataraxia, pore canals similarly pass up through the cuticle to the tips of the epicuticular tubercles (Way, 1950). Strands of material pass up through the pore canals, and these strands are fine projections of the epidermal cells (see fig. 6, D). These pore canals with their cytoplasmic contents are therefore typical of pore canals in general as described for insect cuticles (cf. Wigglesworth, 1933; Richards and Anderson, 1942; Dennell, 1943, 1946; Tower, 1906; Locke, 1960) and for other arthropod cuticles (Richards, 1951).

FIG. 6 (plate).

A, section through epidermis and soft cuticle.

B. oblique section through epidermis and soft cuticle, showing the major tubercles in crosssection.

c, section through epidermis and soft cuticle, showing large pore canals beneath the major tubercles and finer pore canals between the major tubercles.

D.section through epidermis and cuticle of the clypeus, showing cytoplasmic strands in the fine pore canals.

E. section through a thickened region of the cuticle, showing the outer sinuous and inner straight lamellae.

F.section through a thickened region of the cuticle, showing cuticular vesicles beneath the major tubercles and also the outer sinuous and inner straight lamellae.

G.section through soft cuticle at a major tubercle to show the branching pore canal.

H, section through epidermis and cuticle of the clypeus, showing the three major cuticular layers, the endocuticular lamellae, the homogeneous exocuticle, and the pore canals. The black granules in the epidermis are pigment granules.

br. p.c., branching pore canal; cutie. I., cuticulin layer; cyt. fit., cytoplasmic filament; endo., endocuticle; epi., epicuticle; epid,, epidermis; epid. pr., epidermal projections; exo., exocuticle; inner I,, inner layer; maj. tub., major tubercle; min. tub., minor tubercle; nucl., nucleus; p.c., pore canal; p.c. ves., pore canal vesicle; pig. gr., pigment granule; sin. lam., sinuous lamellae; str. lam., straight lamellae.

FIG. 6 (plate).

A, section through epidermis and soft cuticle.

B. oblique section through epidermis and soft cuticle, showing the major tubercles in crosssection.

c, section through epidermis and soft cuticle, showing large pore canals beneath the major tubercles and finer pore canals between the major tubercles.

D.section through epidermis and cuticle of the clypeus, showing cytoplasmic strands in the fine pore canals.

E. section through a thickened region of the cuticle, showing the outer sinuous and inner straight lamellae.

F.section through a thickened region of the cuticle, showing cuticular vesicles beneath the major tubercles and also the outer sinuous and inner straight lamellae.

G.section through soft cuticle at a major tubercle to show the branching pore canal.

H, section through epidermis and cuticle of the clypeus, showing the three major cuticular layers, the endocuticular lamellae, the homogeneous exocuticle, and the pore canals. The black granules in the epidermis are pigment granules.

br. p.c., branching pore canal; cutie. I., cuticulin layer; cyt. fit., cytoplasmic filament; endo., endocuticle; epi., epicuticle; epid,, epidermis; epid. pr., epidermal projections; exo., exocuticle; inner I,, inner layer; maj. tub., major tubercle; min. tub., minor tubercle; nucl., nucleus; p.c., pore canal; p.c. ves., pore canal vesicle; pig. gr., pigment granule; sin. lam., sinuous lamellae; str. lam., straight lamellae.

The thicker cuticle which occurs in some areas of the body shows a few modifications in the electron microscope. In general, the major tubercles are confined to the outermost lamellae of the endocuticle (which follow a sinuous course), the inner lamellae being very much straighter. This effect is accentuated as the cuticle becomes thicker (see fig. 6, E). The pore canals are much more distinct and pass up to the major tubercles, which often have vesicles underlying them (see fig. 6, F). Within the sinuous region of the cuticle, the pore canals tend to be arborescent (see fig. 6, G). NO real differentiation of the cuticle below the epicuticle can be seen, apart from the difference in the course followed by the lamellae, as noted above, even though in Mallorystained sections viewed under the light microscope differential staining was seen in these areas. Nevertheless, the demarcation between sinuous and straight lamellae in the electron micrographs is as sharp as the demarcation between the red and blue regions in the Mallory-stained sections. Furthermore, in the sinuous region the pore canals are arborescent, whereas in the region with straight lamellae the pore canals are confined to vertical trunks. It is possible that, associated with their arborescence in the sinuous region, the cytoplasmic filaments of the pore canals exert some chemical influence which is not shown up in the electron micrographs by an obvious structural change within the lamellae but which causes the differential staining in Mallory. As the evidence points to these areas having greater mechanical strength than the cuticle of the general body-area, it may be that the red coloration signifies a slight hardening which binds the lamellae closer chemically without disrupting the structural relationships. Certainly, a red coloration with Mallory is an intermediate stage in the colour sequence undergone when exocuticle formation occurs (see Dennell and Malek, 1955 a, b). The persistence of this red coloration in the fully formed cuticle indicates that it is not merely a transitory phase during a series of chemical reactions, but more probably represents a distinct chemical bonding. This chemical state may be the same as that passed through briefly during normal hardening of the exocuticle, but on the basis of this evidence it is not necessarily so. Red coloration with Mallory is also given in the mesocuticle of Lower (1956, 1958), which he considers as an arrested intermediate stage in normal hardening, and in the elastic cuticle of Weis-Fogh (1960), which is considered to be modified endocuticle which has undergone none of the processes associated with normal exocuticle formation. Clearly, unless supported by further evidence, a red coloration with Mallory only shows that the endocuticle has undergone some sort of modification, without it being possible to conclude anything further, except perhaps that the modified endocuticle will be ‘tougher’. This is in agreement with the distribution of the modified endocuticle over the body. Structural differentiation also occurs in some red-staining regions, such as at the insertion of setae (see below).

Electron micrographs of the thick cuticle of the clypeal region show 3 distinct layers, the endocuticle, the exocuticle, and the epicuticle, as for insect cuticles in general and corresponding to the 3 layers seen in sections under the light microscope (see fig. 6, H). The thickness of the cuticle increases from the 1 to 2 μ of the bulk of the cuticle, to 8 to 10 μ. The endo-cuticle extends about half-way through the cuticle and is 4 to 5 μ thick. It is distinctly lamellated and has pore canals passing through it (see fig, 7, A). The cytoplasmic strands passing up through the pore canals can be seen in figs. 6, D and 7, B. Outside the endocuticle, the exocuticle is delimited sharply. This layer is homogeneous in appearance, with no evidence of lamellae. Only the pore canals, which branch but mainly pass to the tips of the major tubercles, and sensory extensions through the cuticle, interrupt this layer. In this fully formed exocuticle we see a completely different structure from that of the cuticle of other regions and layers. This layer corresponds to the amber-coloured layer of light microscopy and therefore presumably represents a sclerotized region. Sclerotization thus involves a major reorganization of the basic cuticular fine structure, probably at the intermolecular level (Pryor, 1940). Bounding the cuticle on its outermost side is the much-tuberculate epicuticle. The major tubercles also involve only the outer regions of the exocuticle. The minor tubercles are epicuticular only. We therefore see on the basis of this structural evidence from the clypeal cuticle that this apterygote insect is capable of producing, even if in a confined region only, a 3layered cuticle characteristic of pterygote insects, confirming the conclusions reached in the light-microscope study.

FIG. 7 (plate).

A, section through clypeal cuticle, showing pore canals traversing the lamellate endocuticle and the homogeneous exocuticle.

B, section through clypeal cuticle, showing the pore canals and their cytoplasmic contents in oblique transverse section.

c, section through soft cuticle showing the substructure of the epicuticle and endocuticle.

D.section through the exocuticle and epicuticle of clypeal cuticle showing the continuous inner layer of the epicuticle.

E.section through the epidermis and soft cuticle, showing the ballooning cement layer over the minor tubercles.

F.section through soft cuticle showing the cement layer widening into a lens-shaped mass over the minor tubercles.

G.section through clypeal cuticle showing the modification of the outer epicuticular laminae over the tubercles.

H.section through the base of a seta and its cuticular socket showing the suspending lamellae of the soft cuticle forming the pad, and the epicuticular lining of the lumen of the seta.

artic. m., articular membrane; cem. I., cement layer; cutic. I., cuticulin layer; cyt. fit., cytoplasmic filament; endo., endocuticle; epi., epicuticle; epid., epidermis; exo., exocuticle; inner I., inner layer; lin., epicuticular lining of setal lumen; min. tub., minor tubercle; o. wax I., outer wax layer; p.c., pore canal; sens., sensory processes; wax I., wax layer.

FIG. 7 (plate).

A, section through clypeal cuticle, showing pore canals traversing the lamellate endocuticle and the homogeneous exocuticle.

B, section through clypeal cuticle, showing the pore canals and their cytoplasmic contents in oblique transverse section.

c, section through soft cuticle showing the substructure of the epicuticle and endocuticle.

D.section through the exocuticle and epicuticle of clypeal cuticle showing the continuous inner layer of the epicuticle.

E.section through the epidermis and soft cuticle, showing the ballooning cement layer over the minor tubercles.

F.section through soft cuticle showing the cement layer widening into a lens-shaped mass over the minor tubercles.

G.section through clypeal cuticle showing the modification of the outer epicuticular laminae over the tubercles.

H.section through the base of a seta and its cuticular socket showing the suspending lamellae of the soft cuticle forming the pad, and the epicuticular lining of the lumen of the seta.

artic. m., articular membrane; cem. I., cement layer; cutic. I., cuticulin layer; cyt. fit., cytoplasmic filament; endo., endocuticle; epi., epicuticle; epid., epidermis; exo., exocuticle; inner I., inner layer; lin., epicuticular lining of setal lumen; min. tub., minor tubercle; o. wax I., outer wax layer; p.c., pore canal; sens., sensory processes; wax I., wax layer.

The epicuticle

The seemingly ubiquitous layer of the epicuticle is the electron-dense layer corresponding to the cuticulin layer (see Wigglesworth, 1933; Locke, 1957, 1958, 1960, 1961). This layer can be seen in fig. 7, c. It may be considered as the basic epicuticular layer. It is bounded both externally and internally by layers which differ over different parts of the cuticle. This is the first-formed layer of the cuticle (see Noble-Nesbitt, 1963a; Wigglesworth, 1933, 1947, 1948). It is very thin, being only 20 to 30 m μ. thick.

In soft cuticle, which consists of endocuticle and epicuticle only, the endocuticle adjoins the ‘cuticulin’ layer, except where the minor tubercles occur. Here, an ‘inner layer’, probably equivalent to the layer described by Locke for Rhodnius prolixus as a tanned, homogeneous layer beneath the electron-opaque cuticulin layer (see Locke, 1957, 1958), is found. Locke considered that this layer was tanned chitin /protein. Certainly the ‘inner layer’ of Podura is homogeneous and appears to be structurally similar to the tanned exocuticle (see above and compare figs. 7, c; 6, H). The occurrence of the ‘inner layer’ only under the minor tubercles suggests that it acts as a series of structural pillars holding up the minor tubercles, which probably play an important role in the surface properties of the cuticle (see NobleNesbitt, 19636). Some support for the minute tubercles is to be expected. In the hard cuticle of the clypeus, the ‘inner layer’ is sometimes continuous over the cuticle, and is not confined only to the minor tubercles (see fig. 7, D). It is possible that this is a reflection of the tanning of the underlying exocuticle, which is also continuous in this region. However, it should be noted that in Rhodnius abdominal cuticle as described by Locke, the ‘inner layer’ is underlain only by endocuticle. Outside the cuticulin layer, a thin, less-dense layer occurs, bounded externally by another thin and very dense layer. These layers, which are only about 5 m/z thick, are always most distinct near the minor tubercles (see fig. 7, c). They are possibly wax and cement layers, respectively (cf. Locke, 1957, 1961). Over the minor tubercles themselves, these layers are greatly modified. The wax layer is thicker and the cement layer balloons out (see fig. 7, E), possibly because of being loosened during the histological treatment. Often the cement layer takes on a lenticular shape (see fig. 7, F) and it may be bounded by a further filamentous ‘wax’ layer, which is not always retained in preparations for the electron microscope (but see fig. 7, G). These modifications are not always in evidence, and it is possible that they are subject to a fair amount of damage. They are of great importance in the surface properties of the cuticle (see Noble-Nesbitt, 19636). No evidence of pore canals penetrating the continuous layers of the epicuticle of Podura has been obtained.

Muscle insertions

Further modifications of the cuticle occur at areas of muscle attachment. The muscles insert on to what are apparently inward projections of the cuticulin layer of the epicuticle. These seem to be distinct infoldings of the epicuticular layer, since they show evidence of being hollow structures. They are approximately 40 m/z in diameter, the core being approximately 20 m/z in diameter, and the walls approximately 10 mp thick. At the end proximal to the muscle, each tonofibrilla forms a Y-shaped cone which receives a group of myofilaments.

Setal insertions

P. aquatica is sparsely clothed with setae. Those that have been seen in the electron microscope all have a similar type of insertion. Fig. 7, H shows the structure of the cuticle at the setal insertion. The seta inserts into the socket which is differentiated from the rest of the cuticle. The socket consists of a rim surrounding an inner pad overlain by an epicuticular ‘articular membrane’. The rim of the socket consists of structurally hard cuticle, which resembles exocuticle (see fig. 7, H). It has a tapering connexion to the base of the hollow cylinder of the seta which encloses the strand of nerve-tissue, and this appears to give the solid foundations upon which the seta is erected. The pad consists of lamellated soft cuticle, the lamellae being strung from the socket rim to the ‘epicuticular’ inner cylinder of the seta and to the base of the setal wall. It probably forms an elastic suspension to hold the seta in place, whilst allowing movement, and to return the seta to its resting position after such a movement. Figs. 7, H and 8, A distinctly show a ‘guy-rope’ type of suspension. It will be noticed that the lamellae here exhibit a different pattern to those of other regions of endocuticle. They appear to be distinctly interconnected giving a feathery appearance. This structural difference could well be associated with different mechanical properties such as elasticity (see Locke, 1960). As we have seen, the cuticle of this area stains red in Mallory, suggesting a chemical difference too. It is perhaps significant that ‘Resilin’ (Weis-Fogh, 1960), which forms an elastic cuticle, is also associated with similar differential staining properties. The rim is further connected to the seta by means of the epicuticle, which presumably is a tough, inelastic membrane (Wiggles-worth, 1933, 1947), giving a further suspension at this level between the seta and socket rim. This corresponds to the articular membrane recognized in light microscope studies (see Hsü in Wigglesworth, 1953). The walls of the seta are structurally exocuticular (i.e. sclerotized) down to just below the level of the articular membrane only. Below that only the inner epicular cylinder penetrates the pad of soft cuticle of the socket. The base of the seta therefore appears to be freely movable, as in mechanoreceptors (see Hsü in Wigglesworth, 1953). The ‘elastic’ cuticle and suspension, of course, may merely serve to keep the hair erect, whilst allowing a certain amount of flexibility to guard against breakage and ensuring that the hair returns to its normal erect position after being displaced. In support of this interpretation, a similar insertion is seen in the chemoreceptors shown in their electron micrographs by Slifer, Prestage, and Beams (1957, plate 5). As in those chemoreceptors, strands of nerve-tissue pass up the core of the seta (see fig. 8, B, c, D). Although the terminations have not been seen in such detail as those described by Slifer, Prestage, and Beams, the nerve-strands appear to pass out through the thick wall of hair towards the surface (see fig. 8, D), suggesting that we are dealing with structures similar to those described by Slifer, Prestage, and Beams. The annular ridges seen at the base of the seta (see fig. 8, E) seem to have no obvious function. However, they form a rough surface on an otherwise smooth area of cuticle. This is of importance in surface properties (see Noble-Nesbitt, 19636), and may keep this part of the seta hydrofuge. It may be noted that the ridges occur at the general level of the surrounding cuticle.

FIG. 8 (plate).

A, oblique section through the base of a seta and its cuticular socket, showing the lamellae of the pad.

B longitudinal section through a seta, showing also its cuticular socket.

C. almost transverse section through a seta and its cuticular socket.

D.oblique section through a seta near to its tip. The arrows indicate thin points in the wall where sensory processes, with neurofilaments, appear to pass towards the outer surface.

E.section through the epidermis, cuticle, and a seta, showing a surface view of the base of the seta with its annular ridges.

F, section just below the outer epidermal surface, showing the arrangement of the tormogen and trichogen cells round the sensory processes. The dense oval structures are pigment granules, some of which have been partially pulled away during sectioning.

G.section through the epidermis, cuticle, and a seta, showing the arrangement of the tormogen and trichogen cells in the epidermis and with respect to the overlying cuticular structures.

H.section near the periphery of a seta, passing through the microvilli of the tormogen cell, showing the demarcation of the limits of the socket cuticle by the periphery of the tormogen cell (as indicated by the arrows).

artic. m., articular membrane; cut., cuticle; epid., epidermis; fl., flange of tormogen cell; tin., epicuticular lining of setal lumen; lum., setal lumen; microv., microvilli of tormogen cell; mit., mitochondrion; neurof., neurofilament; sens., sensory processes; torm., tormogen cell; trich., trichogen cell.

FIG. 8 (plate).

A, oblique section through the base of a seta and its cuticular socket, showing the lamellae of the pad.

B longitudinal section through a seta, showing also its cuticular socket.

C. almost transverse section through a seta and its cuticular socket.

D.oblique section through a seta near to its tip. The arrows indicate thin points in the wall where sensory processes, with neurofilaments, appear to pass towards the outer surface.

E.section through the epidermis, cuticle, and a seta, showing a surface view of the base of the seta with its annular ridges.

F, section just below the outer epidermal surface, showing the arrangement of the tormogen and trichogen cells round the sensory processes. The dense oval structures are pigment granules, some of which have been partially pulled away during sectioning.

G.section through the epidermis, cuticle, and a seta, showing the arrangement of the tormogen and trichogen cells in the epidermis and with respect to the overlying cuticular structures.

H.section near the periphery of a seta, passing through the microvilli of the tormogen cell, showing the demarcation of the limits of the socket cuticle by the periphery of the tormogen cell (as indicated by the arrows).

artic. m., articular membrane; cut., cuticle; epid., epidermis; fl., flange of tormogen cell; tin., epicuticular lining of setal lumen; lum., setal lumen; microv., microvilli of tormogen cell; mit., mitochondrion; neurof., neurofilament; sens., sensory processes; torm., tormogen cell; trich., trichogen cell.

The tormogen cell, which forms the socket, surrounds the hair-forming trichogen cell (cf. Wigglesworth, 1933, 1953). The trichogen cell therefore appears to pierce the tormogen cell, but close examination reveals that it lies in a deep fold in the tormogen cell (see fig. 8, F). Thus, the tormogen cell sends out two arms, which invest the trichogen cell and meet on the opposite side of it (cf. Lees and Picken, 1945). The cytoplasms of the 2 cells are separated by the plasma membranes of both cells. Sections parallel to the epidermal surface and transverse to the cells show that they effectively have a circular cross-section, although the actual cross-section is C-shaped. This arrangement is reflected in the circular socket and seta. The tormogen cell bears numerous microvillii at its cuticular surface (see fig. 8, G, H). The significance of these is not clear, but they possibly are concerned with the secretion of the specialized cuticle of the socket. The tormogen cell further has lateral projections a little way beneath the epidermal surface, forming a flange which appears to anchor the cell in the epidermis (see fig. 8, G, H). In common with the epidermal cells, it contains pigment granules, but these are absent from the trichogen cell. The trichogen cell, surrounded by the tormogen cell, likewise surrounds the distal processes of the sensory nerves. Accordingly, it is probable that the investing of the dendrites and of the trichogen cell occurs at the same time and in the same manner. Thus, by a rolling of these 2 cells, both the distal process and the trichogen cell are surrounded, and the circular hair and socket pattern produced. The boundaries of the cells clearly define the limits of the differentiation of the cuticle of the hair and socket (see fig. 8, G, H). Intercellular cytoplasmic connexions (plasmodesmata or desmosomes) bridge the gaps between the cells (see figs. 8, F; 9, A).

Usually not more than 5 distal processes pass up into the sensillum. Each one contains a variable number of tubular neurofilaments (approximately 7 to 12) arranged in a fairly regular array (see figs. 8, B to D; 9, A; and Whitear, 1960; Slifer and Sekhon, 1960). Each neurofilament is circular in transverse section and hollow. Fig. 8, B to D show the distal processes passing up the sensillum.

The ventral tube vesicles

The cuticle is very thin (0 · 5 μ thick) and the epicuticle modified over this area. The endocuticle appears to consist of 2 or 3 dense lamellae, and is bounded externally by the epicuticle which consists of a dense cuticulin layer, with up to 4 laminae visible external to it (see fig. 9, B, c). It is probable that these laminae represent the wax and cement layers, with the latter splitting to form further apparent layers externally. This suggests that this outermost layer is a composite one, with its constituents normally intimately bound together, but which is prone to having an outer membrane peel off (see fig. 9, c). These layers are very thin (approximately 10 m p thick). Their presence is important in considering the surface properties of the cuticle overlying the vesicles (see Noble-Nesbitt, 19636). At the edges of the area, the cuticle is modified to form cuticular flaps (see fig. 9, D, E).

FIG. 9 (plate)

A, section through the tormogen, trichogen, and glial cells just below the outer epidermal surface, showing the substructure of the sensory processes enclosed by these cells. Note the pigment granules (some of which have been partially pulled away during sectioning) in the tormogen cell, and the regular array of tubular neurofilaments in the sensory processes.

B, section through the two adpressed vesicles of the ventral tube, showing the epidermal processes beneath each cuticle. The diagonal strip separating the two cuticles is morphologically external to the insect. Note the outer epicuticle and the very thin endocuticle.

c, section through the two adpressed vesicles of the ventral tube, showing the very thin wax layer outside the cuticulin layer, and the composite cement layer outside the wax layer. The cement layer consists of two dense laminae with a less dense lamina between them (indicated by two arrows). The outer dense lamina tends to peel away, leaving only the inner one (indicated by a single arrow).

D.section through the inner boundaries of the two adpressed vesicles of the ventral tube, showing the cuticular flaps at the edges of the vesicles.

E.section through the outer boundaries of the two adpressed vesicles of the ventral tube, showing the cuticular flaps (one of which has been damaged during sectioning) at the edges of the vesicles, the tuberculate cuticle beyond the flaps, and the smooth cuticle of the vesicles. Note also the epidermal processes and the enclosed vesicles at their bases.

F.section through the closely adpressed vesicles of the ventral tube, showing the epidermal processes beneath the cuticle, with elongated mitochondria within the processes, large mitochondria at their bases, and enclosed vesicles.

G.section through an ocellus, showing the smooth cuticle over the lens area, passing into tuberculate cuticle to the left.

H, section through part of the lens and its associated retinal elements, showing the fine structure of the retinal elements.

cutic. I., cuticulin layer; endo., endocuticle; epid., epidermis; gl., glial cell; lent., lentigen cells; mit., mitochondrion; neurof., neurofilament; nucl., nucleus; o. epi., outer epicuticle; ret., retinal elements; sens., sensory processes; torm., tormogen cell; trich., trichogen cell; ves., vesicle.

FIG. 9 (plate)

A, section through the tormogen, trichogen, and glial cells just below the outer epidermal surface, showing the substructure of the sensory processes enclosed by these cells. Note the pigment granules (some of which have been partially pulled away during sectioning) in the tormogen cell, and the regular array of tubular neurofilaments in the sensory processes.

B, section through the two adpressed vesicles of the ventral tube, showing the epidermal processes beneath each cuticle. The diagonal strip separating the two cuticles is morphologically external to the insect. Note the outer epicuticle and the very thin endocuticle.

c, section through the two adpressed vesicles of the ventral tube, showing the very thin wax layer outside the cuticulin layer, and the composite cement layer outside the wax layer. The cement layer consists of two dense laminae with a less dense lamina between them (indicated by two arrows). The outer dense lamina tends to peel away, leaving only the inner one (indicated by a single arrow).

D.section through the inner boundaries of the two adpressed vesicles of the ventral tube, showing the cuticular flaps at the edges of the vesicles.

E.section through the outer boundaries of the two adpressed vesicles of the ventral tube, showing the cuticular flaps (one of which has been damaged during sectioning) at the edges of the vesicles, the tuberculate cuticle beyond the flaps, and the smooth cuticle of the vesicles. Note also the epidermal processes and the enclosed vesicles at their bases.

F.section through the closely adpressed vesicles of the ventral tube, showing the epidermal processes beneath the cuticle, with elongated mitochondria within the processes, large mitochondria at their bases, and enclosed vesicles.

G.section through an ocellus, showing the smooth cuticle over the lens area, passing into tuberculate cuticle to the left.

H, section through part of the lens and its associated retinal elements, showing the fine structure of the retinal elements.

cutic. I., cuticulin layer; endo., endocuticle; epid., epidermis; gl., glial cell; lent., lentigen cells; mit., mitochondrion; neurof., neurofilament; nucl., nucleus; o. epi., outer epicuticle; ret., retinal elements; sens., sensory processes; torm., tormogen cell; trich., trichogen cell; ves., vesicle.

The epidermis is glandular (see fig. 9, F) and reminiscent of active excretory and resorptive epithelia as found in the Malpighian tubules and the rectum of insects (Smith and Littau, 1960). The epidermal cell surface is deeply folded, forming a honeycomb border beneath the thin cuticle. Elongated mitochondria occur within the cellular projections, and large mitochondria are seen in a layer at the base of these projections. Between the cellular projections, extracellular spaces appear to be pinched off at the bases of the cellular projections, indicating that pinocytosis may occur (see fig. 9, E). Certainly, smooth-walled vesicles appear beneath the intuckings. This may be of importance in uptake of water and other substances from the medium (see Noble-Nesbitt, 1963c).

The ocelli

The electron microscope confirms that the cuticle over the ocelli is smooth. Not even minor tubercles are present. This, of course, is important in allowing light to pass uninterrupted to the sensitive areas beneath the cuticle. The cuticular lens lies amongst a group of specialized cells (see fig. 9, G) which presumably secrete it and the overlying smooth cuticle. Pigment granules are absent from these cells, and the passage of the light to the lens is therefore uninterrupted. The lens is not shed at the moult, and whilst appearing to be composed of cuticular material, it may perhaps be better thought of as a special inclusion formed for sensory purposes. Beneath the lens, the sensory cells have a regular structure such as would be given by closely packed fibrils (see fig. 9, H). These are thought to be the light-sensitive terminations. The lens presumably focuses the incident light on them. They pass inwards towards the brain, still surrounded by pigmented epidermal cells, and still then retain their regular substructure.

The structure of the cuticle in Collembola, as we have seen, is not markedly different from the structure of the cuticle in pterygote insects or other arthropods. Lower (1958) suggested that apterygote cuticle was simpler in structure than pterygote cuticle, and that a 2-layered epicuticle was the basic epicuticle from which other types could be derived. He considered that from the simple apterygote cuticle, the more complex cuticle of pterygote insects could be derived. In this simple apterygote cuticle, he described no cuticular lamellae and no pore canals. Furthermore, he distinguished no true exocuticle, in which full sclerotization occurred, and no cement layer. The present study indicates that in Collembola, at least some of these features are found. Cuticular lamellae are clearly visible in electron micrographs of endocuticle and mesocuticle, whilst fine pore canals penetrate the cuticle from the epidermis to the epicuticle, and contain cytoplasmic filaments. In limited regions of the body, an exocuticle is also found, with full sclerotization, which also occurs in the setae. A cement layer, which may be discontinuous, is present at least in the epicuticle of Collembola. Furthermore, distinct cell boundaries occur in the epidermis, which Lower regarded as a syncytium. In these respects, then, it must be concluded that the cuticle of apterygote insects does not differ from that of pterygotes. Any difference lies only in the distribution of the cuticular layers over the body. In this aspect, the apterygote insects are more akin to the larval forms of the pterygote insects than to the imaginai forms. In soft-bodied pterygote larvae, it is usual to find an almost complete absence of an exocuticle, except in mouthparts and associated structures, where hardness is a prime necessity. Larval forms are growing stages, intent mainly on increasing body size and weight to the adult level. Metabolic effort wasted in producing exocuticle would be expected to retard this process. Furthermore, at the moult, soft cuticle can be almost completely recovered, but hard exocuticle cannot; further wastage of valuable materials would occur if larvae were covered with exocuticle. Once adult, of course, with no further moulting, exocuticle represents no special potential loss to the pterygote insect. But in apterygote insects, which continue to moult after becoming adult, production of exocuticle continues to represent a potential loss to the insect, only part of which subsequently may be recovered by eating the exuviae. It is not then surprising to discover that, on the one hand, exocuticular production is minimal, and, on the other hand, that the exuviae are often eaten. It is to be emphasized, however, that, just as the pterygote larvae have the potential to produce exocuticle, so have the apterygote insects. It is therefore incorrect to claim that apterygote insects represent an earlier phase in phylogeny when exocuticle could not yet be formed. A similar situation exists in Crustacea. In the cuticles of Crustacea (see Richard, 1951) hardness is obtained principally by use of incrustations of crystals of inorganic salts (e.g. calcium carbonate). Even in these arthropods, however, the areas of the cuticle which require great hardness (such as in the limbs and mouthparts) are hardened and darkened, i.e. they are sclerotized (Dennell, 1947). Again we can infer an economy of metabolic effort. Crustacea moult continuously and produce little proteinaceous exocuticle. But this does not necessarily mean that they have great affinities with the Apterygota. Rather it means that they have solved a similar problem in a similar way. In the pterygotes, this problem is confined only to the larval stage, and the solution is again similar. In the imaginai stage, made possible by major changes in the organization of the body, the problem does not arise and no solution is required. Thus, lack of moulting in the adult stadium may be associated with extensive exocuticle because of the lack of a selective pressure against its formation. Indeed, the mechanical requirements of flight provide a selective pressure favouring its formation.

The endocuticle in P. aquatica is typically lamellated, but the arrangement of the lamellae differs in different parts of the cuticle. On the whole, the lamellae appear to have no regular interconnexions. However, in certain areas (such as in the sockets of the setae), a regular arrangement of inter-connexions does occur, and it is suggested that this arrangement may be associated with greater elasticity. It is interesting to note that Locke (1960) has described this arrangement as being typical in the endocuticle of the larva of Calpodes ethlius Stoll, and he suggests that it may be associated with elasticity. It is possible that the arrangement of the cuticular micelles in and between the lamellae may vary according to the type of cuticle, but obviously much more high-resolution electron microscopy of endocuticles is required before any generalizations can be made.

The documentation of the 2-layered nature of the epicuticle in insects has been summarized by Locke (1960). Lower (1958) suggested that this was the basic structure from which other epicuticles were derived. In the present study, it has been established that only one basic layer of the epicuticle can be recognized. This layer, which is assumed to be the cuticulin layer, corresponds to the very dense layer recognized by Locke (1957, 1958, 1960), which he calls the cuticulin layer. It occurs in all regions of the cuticle. Underlying it is an ‘inner layer’, which is often discontinuous, and which corresponds to the homogeneous, chitin/protein layer, described by Locke (1957, 1958, 1960). This inner layer, at least in some instances, apparently supports the raised parts of the cuticulin layer. Wax and cement layers may overlie the cuticulin layer, but their occurrence is closely linked with the properties of the cuticular surface (see Noble-Nesbitt, 1963b).

My thanks are due to Professor Sir James Gray, Professor C. F. A. Pantin, and Professor V. B. Wigglesworth for providing facilities, and to Dr. J. W. L. Beament, who supervised this work, for his constant encouragement. I am very grateful also to Dr. V. E. Cosslett for the electron-microscope facilities provided in his department of the Cavendish Laboratory, and to Mr. R. W. Horne and Mr. I. M. Wardell for taking the electron micrographs. I greatly appreciated the electron-microscope facilities provided for a limited period at short notice by Dr. J. D. Lever of the Department of Anatomy. This work was carried out during the tenure of an Agricultural Research Council Research Studentship.

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