The fully formed intermoult cuticle and associated ...jcs.biologists.org/content/joces/s3-104/66/253.full.pdf · 253 The fully formed intermoult cuticle and associated structures

253

The fully formed intermoult cuticle and associatedstructures of Podura aquatica (Collembola)

By J. NOBLE-NESBITT(From the Department of Zoology, University of Cambridge. Present

address: Department of Zoology, University of Toronto, Canada)

With 4 plates (figs. 6 to 9)

SummaryThe cuticle overlying most of the body consists of 2 major layers only, the lamellateendocuticle and the epicuticle, and is very thin (1 to z^i). Three major cuticularlayers are found in the slightly thicker cuticle of certain areas (2 to 3 fj. thick) and inthe thickest regions (8 to 10/x) a distinct sclerotized exocuticle is present. The epi-cuticle 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 isthrown into major tubercles of approximately 3 /x pitch, with minor tubercles super-imposed upon them. Modification of the cuticle at the bases of setae is described andrelated to the mechanical requirements of the setal insertion and suspension. Thenature of the muscle insertion is also considered. The epidermis is fully cellular andthe cells contain the granules of black pigment which impart the black coloration tothe insect. Specializations of the epidermis in the regions of ocelli, setae, muscleinsertions, and the vesicles of the ventral tube are described, along with modificationsof the overlying cuticle.

IntroductionEARLIER work on the cuticle of Collembola has shown that it has a structurewhich fits closely into the scheme for a generalized insect (or other arthropod)cuticle. Imms (1906) distinguished 2 layers in the cuticle of Anurida maritima(Guerin). The 2-layered nature of the cuticle was also reported for Tomo-cerus longicornes (Miiller) by Sommer (1885), and for Hypogastrura viatica(Tulbbergii) and Isotoma grisea Lubbock by Prowazek (1900). Koncek(1924) reported 3 layers in the cuticle of Tetrodonotophora gigas Reuter.Ogel (1958) and Lower (1958) also report 3 major divisions of the cuticle, inFolsomia Candida (Willem) and Smynthurus viridis L., respectively. Boelitz(1933) recognized lamellae in stomodaeal cuticle, and Ogel (1958) reportedlamellae in the endocuticle in her electron microscopical study. Lower(1958), however, concluded that lamellae were absent from the cuticle ofS. viridis L. Most of these earlier accounts indicated that the underlyingepidermis was syncitial, with few or no cell boundaries.

The cuticle of Collembola therefore appears, from these reports, to be a verythin, but otherwise typical, arthropod cuticle which exhibits a few peculiaritiesof 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 ofthe cuticle.[Quart. J. micr. Sci., Vol. 104, pt. 2, pp. 253-70, 1963.]

254 Noble-Nesbitt—Intermoult cuticle of Podura

Materials and methods

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

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

Specimens prepared for sectioning with the freezing microtome werefixed with Baker's formaldehyde/calcium fixative for 3 days, then stored inBaker's formaldehyde/calcium/cadmium storage solution. Prior to embedding,the specimens were washed for 1 h in tap-water, then left overnight in a25% gelatine solution, to which a little creosote was added to act as a pre-servative. This was followed by transferring the specimens to fresh gelatineand creosote solution and gelling in a refrigerator for 2 h. The gelatine blockwas then trimmed, and hardened for 4 days in Baker's formaldehyde/calcium/cadmium storage solution. Frozen sections were then cut on a freezingmicrotome, at 6/x, and transferred to slides coated with gelatine. The slideswere stored in Baker's formaldehyde/calcium/cadmium storage solution, andlater stained with Sudan black and Mayer's carmalum (see Pantin, 1948,p. 26).

Fixation of whole P. aquatica for the electron microscope was carried outin the cold, using 1% osmium tetroxide buffered to pH 7-2 after the methodof Palade, modified by Sjostrand (1956). The period of fixation was varied,but the usual period was about 24 h. Dehydration was carried out, also in thecold, by passing the tissues through a graded series of alcohols or acetones.After dehydration, the tissues were embedded usually in 'araldite' (Glauertand Glauert, 1958; Luft, 1961) polymerized for 72 h at 570 C, but also inpartially pre-polymerized 'methacrylate' (Borysko, 1956) polymerized for20 h at 6o° C. The embedding was carried out in gelatin capsules.

During the early stages of the investigation, sections were cut using aHodge thermal-advance ultra-microtome. The tissue block was orientatedon the microtome chuck, using sealing wax. Sections showing interferencecolours of pale straw, gold, silver, or grey were picked up on celloidin-covered

Noble-Nesbitt—Intermoult cuticle of Podura 255

grids which occasionally had the celloidin film stabilized with a very thinlayer of carbon. Later sectioning was carried out using a Huxley mechanical-advance ultra-microtome manufactured by the Cambridge InstrumentCompany. Orientation was achieved by remounting the small tissue blockusing 'araldite' adhesive on to a stem of 'araldite', which fitted the chuck ofthe microtome. Sections were cut at thicknesses of 20 to 80 m/A, and pickedup on grids as described above. Electron staining of the sections on the gridswas accomplished by using uranyl acetate in 50% alcohol (see Gibbonsand Grimstone, i960) or the lead hydroxide stain of Watson (1958) as modi-fied by Peachey (1959). Bulk staining with phospho-tungstic acid was alsoused.

Grids were viewed principally in Siemen's 'Elmiskop' electron microscopesat the Cavendish Laboratory and the Department of Anatomy. Later workwas carried out using the Philips E.M. 200 electron microscope at the Depart-ment of Zoology.

Observations with the light microscopeAs seen with the light microscope, most of the body is covered by a soft,

thin, transparent cuticle 0-5 to i/x thick (see fig. 1). It stains blue with

major tubercleminor tubercles

FIG. 1. Diagram showing the surface sculpturing in the cuticle ofP. aquatica.

Mallory's triple stain, except for an amber outer region, which is very thinand refractile. Another layer which stains red in Mallory occurs betweenthese layers in certain areas where the cuticle is slightly thicker. It alsooccurs occasionally in the thin cuticle, where it is exceedingly thin and notreadily distinguishable from the outer refractile layer. Applying the nomen-clature generally used for arthropod cuticles, the blue inner layer is theendocuticle, the outer refractile amber layer is the epicuticle, and wherepresent the red layer is an exocuticular layer equivalent to the 'mesocuticle'of Schatz (1952) and Lower (1956, 1958). A true, fully sclerotized exocuticleis absent from this thin cuticle.

The cuticle is thrown into small tubercles approximately 3 JJ, in diameterat their bases, approximately 3 fj. high, and with a peak to peak distance of


approximately 3JU. The tuberculate nature of the cuticle in this speciesis well known (Maynard, 1951). Tubercles are absent only in specialareas, 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 cuti-cular surface is smooth. These tubercles will be referred to as the 'majortubercles'.

FIG. 2. Diagram of a Mallory-stained section through the cuticlewhich occurs over most of the body in P. aquatica, showing a setal in-sertion. The cuticle is slightly lifted away from the epidermis, whichcontains pigment granules. The blue endocuticle is shown by hori-zontal lines, and the area at the setal insertion which stains red inMallory is shown by vertical lines. The epicuticle is amber in colour.

Superimposed on the major tubercles is a system of more minute tubercles,giving in surface views a granular, sculptured appearance to the generalsurface 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 beendescribed in other species by Goto (1956) and Ogel (1958).

Areas of the limbs, antennae, furcula, retinaculum, and head capsule, wheregreater mechanical strength may be required, have a thicker cuticle (1*5 to2/A thick) showing three distinct layers in Mallory-stained sections, an innerblue endocuticle, a middle red 'mesocuticle', and a superficial refractileamber epicuticle (see fig. 3). The tubercles referred to above are presentalso in these areas, but they involve only the outer layers of the cuticle. It isof interest to note that this type of cuticle occurs on the outside of the antennaewhen these are pointed forward. This is the area of impact when a surface ishit after a spring, and greater mechanical strength would be expected in suchan area. It is possible that the 'mesocuticle' provides this strength.


Over a limited area in the clypeal region of the head capsule, extending foronly about 40 jx. X 40 /x, the cuticle attains a thickness of 10 to 12 fj, and itsstructure 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 theoutermost epicuticular layer only. This layer is refractile and amber coloured.Beneath this is a layer about 5 ju. thick which stains red-orange in Mallory.It tapers away at the edges of the area. There is evidence of amber colorationwithin this layer, occurring as blotches, or as a layer bounded inside and outby regions which stain red. This indicates that tanning occurs in this layer,which, therefore, may be referred to as a true exocuticle. Underlying the

FIG. 3. Diagram of a Mallory-stained section through a thickenedregion of the cuticle of P. aquatica. Horizontal lines indicate a bluecoloration, and vertical lines indicate a red coloration. The super-

ficial epicuticle is amber in colour.

exocuticle is the endocuticle, which stains blue with Mallory. It is about4 /A thick and in some sections it is seen as a double layer, the innermost partstaining 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 theendocuticle and exocuticle being absent, and cell membranes pass up to thebristles. This is especially evident where fixation has caused the retractionof the cells from the cuticle (see fig. 4). The arrangement is similar to thatof bristle insertions in pterygote insects (Wigglesworth, 1933, and his report ofHsu in Wigglesworth, 1953).

When Heidenhain's haematoxylin is used, a less straightforward pictureof clypeal cuticle is given (see fig. 5). Beneath the refractile epicuticle is adarkly stained layer with areas of medium stain. This corresponds to themixed 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 bristleinsertions (see fig. 5). This area of medium stain is separated from the lightlystained innermost layer (equivalent to the blue layer of Mallory sections) bya darkly stained thin layer 1 to 2/x thick. These complex staining reactionsdo 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. Thedegree of staining seems to be greater where greater hardness would beexpected and less where the cuticle is soft (i.e. in the endocuticle). However,the refractile, and presumably fully tanned, epicuticle is not stained. It ispossible, therefore, that varying degrees, or types, of tanning cause thisdifferential staining with haematoxylin. Striations, which are somewhatrefractile and at right angles to the cuticle surface, pass from the inner sur-

epidermis cell membrane

FIG. 4. Diagram of clypeal cuticle stained with Mallory. Theoccurrence of amber coloration beneath the epicuticle is shownby diagonal lines. Horizontal lines indicate a blue coloration,and vertical lines indicate a red coloration. The epicuticle is

amber in colour.

face of the cuticle out to the darkly stained zone in Heidenhain-stainedsections. 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 muscleinsertions is to be expected. However, Weis-Fogh (i960) has recently shownthat an elastic endocuticle which stains red in Mallory occurs at certainmuscle insertions in pterygote insects. It is possible that a similar type ofcuticle may also be present over muscle insertions in P. aquatica.

At the insertion of each bristle over the general body area, the thin cuticleshows a distinct red ring when stained in Mallory (see fig. 1). This is probablyassociated with a need for greater mechanical strength, or rigidity, at thebase of the seta. The red layer overlies the blue layer and underlies the re-fractile amber epicuticle, which is only imperfectly visible. The red layerappears to taper off sharply, as indicated in fig. 1, and may come to an endcompletely at the edge of this confined region, leaving only the blue and amberlayers of the general body cuticle. It is, therefore, probably produced underthe influence of the tormogen cell only (cf. Wigglesworth, 1933). It is tobe noted also that the setae are light amber in colour, and it is suggestedthat complete tanning may occur in them. Further evidence accrued frominvestigations using the electron microscope (see below).


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

The cuticle is smooth also over each ocellus, there being no evidence ofthe tubercles which occur over the surrounding cuticle. This modificationdoubtless allows uninterrupted passage of the light rays through to the under-lying tissues. Tubercles would be expected to scatter the rays. Beneath the

exocuticle

'epidermis

FIG. 5. Diagram of clypeal cuticle stained with Heiden-hain's haematoxylin.

cuticle lies a lens, which is highly refractile and amber coloured. It is notshed at the moult. The epidermis with its refractile pigment granules sur-rounds the lens and obscures the sensory cells, but nerve-tracts from themcan be traced passing to the brain. The pigment-containing epidermal cellspenetrate deeper into the head at this point, associated with the sensorynerve-tracts.

Light microscope preparations of Orchesella villosa stained in Malloryshow that the cuticle in this larger collembolan is thicker than that ofP. aquatica, the cuticle of the general body surface being 2 to 3 /u, thick. The'mesocuticle' occurs over the whole of the general body-surface. The cuticlesurface is tuberculate, as in P. aquatica. In the thicker clypeal cuticle, thevertical lines of the pore canals are more in evidence than in P. aquatica.Pigment granules again occur in the epidermis. In general, the results fromthis collembolan agree with those from P. aquatica, and confirm the basicstructure of the collembolan cuticle.

Observations using the light microscope thus indicate that the basic struc-ture of the cuticle and the underlying epidermis of P. aquatica and O. villosaagrees closely with that reported for other species of Collembola and for thepterygote insects. In general, 3 cuticular layers are present, though only in


localized areas does the exocuticle appear to be fully tanned. Elsewhere, it iseither more akin to the 'mesocuticle' of Lower (1956, 1958) or it is absent.The surface is tuberculate, except over specialized areas. Evidence of porecanals can be seen in the thick regions of the cuticle. Cytoplasmic filamentsappear to pass to, or perhaps even into, the setae. The epidermis containsthe pigment which imparts the body-colour, and obscures much of thecellular detail. It is therefore not possible to comment on the presence orabsence of cell boundaries, or to determine whether the epidermis is a syncy-tium as reported for Folsomia Candida (Willem) (Ogel, 1958) and Smynthurusviridis L. (Lower, 1958). A basement membrane is apparently absent. Thisagrees 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 incompletebecause of the extreme thinness of the cuticle and the highly refractile natureof many of the epidermal and cuticular components, which makes resolutiondifficult. A more detailed investigation has therefore been carried out usingthe electron microscope. The results of this investigation are set out below.

Observations with the electron microscopeFig. 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 epidermalcells, which is thrown into numerous small folds. It consists of lamellaelying 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 isthrown into the major tubercles seen in the light microscope. The epidermalsurface is thrown into corresponding folds. The peak-to-peak distance ofthese tubercles is approximately 3/x and they rise approximately 2ju, abovethe general level of the cuticle, which is slightly less than light microscopepreparations indicate. Superimposed on this gross sculpturing is a finer,epicuticular sculpturing. The epicuticle is thrown into minor tubercleswhich are approximately 0-2/x in diameter, 0-2/A high and 0-4 to o-6/n apart.The presence of these was indicated by the light microscope. They have beenreported by Ogel (1958) for F. Candida (Willem). The arrangement of thetubercles in P. aquatica is shown in fig. 6, B, which is an oblique section throughthe 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 ofthe epicuticular tubercles (Way, 1950). Strands of material pass up throughthe pore canals, and these strands are fine projections of the epidermal cells(see fig. 6, D). These pore canals with their cytoplasmic contents are there-fore typical of pore canals in general as described for insect cuticles (cf. Wiggles-worth, 1933; Richards and Anderson, 1942; Dennell, 1943, 1946; Tower,1906; Locke, i960) and for other arthropod cuticles (Richards, 1951).

The thicker cuticle which occurs in some areas of the body shows a few


modifications in the electron microscope. In general, the major tuberclesare confined to the outermost lamellae of the endocuticle (which follow asinuous course), the inner lamellae being very much straighter. This effectis accentuated as the cuticle becomes thicker (see fig. 6, E). The pore canalsare much more distinct and pass up to the major tubercles, which often havevesicles underlying them (see fig. 6, F). Within the sinuous region of thecuticle, the pore canals tend to be arborescent (see fig. 6, G). NO real differen-tiation of the cuticle below the epicuticle can be seen, apart from the differencein the course followed by the lamellae, as noted above, even though in Mallory-stained sections viewed under the light microscope differential staining wasseen in these areas. Nevertheless, the demarcation between sinuous andstraight lamellae in the electron micrographs is as sharp as the demarcationbetween the red and blue regions in the Mallory-stained sections. Further-more, in the sinuous region the pore canals are arborescent, whereas in theregion 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 influencewhich is not shown up in the electron micrographs by an obvious structuralchange within the lamellae but which causes the differential staining inMallory. As the evidence points to these areas having greater mechanicalstrength than the cuticle of the general body-area, it may be that the redcoloration signifies a slight hardening which binds the lamellae closer chemicallywithout disrupting the structural relationships. Certainly, a red colorationwith Mallory is an intermediate stage in the colour sequence undergone whenexocuticle formation occurs (see Dennell and Malek, 1955 a, b). The per-sistence of this red coloration in the fully formed cuticle indicates that it is notmerely a transitory phase during a series of chemical reactions, but moreprobably represents a distinct chemical bonding. This chemical state may bethe same as that passed through briefly during normal hardening of the exo-cuticle, but on the basis of this evidence it is not necessarily so. Red colorationwith Mallory is also given in the mesocuticle of Lower (1956, 1958), whichhe considers as an arrested intermediate stage in normal hardening, and inthe elastic cuticle of Weis-Fogh (i960), which is considered to be modifiedendocuticle which has undergone none of the processes associated withnormal exocuticle formation. Clearly, unless supported by further evidence,a red coloration with Mallory only shows that the endocuticle has undergonesome sort of modification, without it being possible to conclude anythingfurther, except perhaps that the modified endocuticle will be 'tougher'. Thisis in agreement with the distribution of the modified endocuticle over thebody. 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 3distinct layers, the endocuticle, the exocuticle, and the epicuticle, as forinsect cuticles in general and corresponding to the 3 layers seen in sectionsunder the light microscope (see fig. 6, H). The thickness of the cuticle

1p

FIG. 6

J. NOBLE-NESBITT

FIG. 7

J. NOBLE-NESBITT


increases from the 1 to z\i of the bulk of the cuticle, to 8 to 10 fi. The endo-cuticle extends about half-way through the cuticle and is 4 to 5 /x thick. Itis 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 infigs. 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 majortubercles, and sensory extensions through the cuticle, interrupt this layer.In this fully formed exocuticle we see a completely different structure fromthat of the cuticle of other regions and layers. This layer corresponds to theamber-coloured layer of light microscopy and therefore presumably representsa sclerotized region. Sclerotization thus involves a major reorganization ofthe basic cuticular fine structure, probably at the intermolecular level (Pryor,1940). Bounding the cuticle on its outermost side is the much-tuberculateepicuticle. The major tubercles also involve only the outer regions of theexocuticle. The minor tubercles are epicuticular only. We therefore see onthe basis of this structural evidence from the clypeal cuticle that this aptery-gote insect is capable of producing, even if in a confined region only, a 3-layered cuticle characteristic of pterygote insects, confirming the conclusionsreached in the light-microscope study.

The epicuticleThe seemingly ubiquitous layer of the epicuticle is the electron-dense

layer corresponding to the cuticulin layer (see Wigglesworth, 1933; Locke,1957, 1958, i960, 1961). This layer can be seen in fig. 7, c. It may be con-sidered as the basic epicuticular layer. It is bounded both externally and in-ternally by layers which differ over different parts of the cuticle. This is thefirst-formed layer of the cuticle (see Noble-Nesbitt, 1963a; Wigglesworth,1933, 1947, 1948). It is very thin, being only 20 to 30 m/x thick.

FIG. 6 (plate). A, section through epidermis and soft cuticle.B, oblique section through epidermis and soft cuticle, showing the major tubercles in cross-

section.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. Theblack granules in the epidermis are pigment granules.

br. p.c, branching pore canal; cutic. I., cuticulin layer; cyt. fil., 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.


In soft cuticle, which consists of endocuticle and epicuticle only, theendocuticle adjoins the 'cuticulin' layer, except where the minor tuberclesoccur. Here, an 'inner layer', probably equivalent to the layer described byLocke for Rhodnius prolixus as a tanned, homogeneous layer beneath theelectron-opaque cuticulin layer (see Locke, 1957, 1958), is found. Lockeconsidered that this layer was tanned chitin / protein. Certainly the 'innerlayer' of Podura is homogeneous and appears to be structurally similar to thetanned exocuticle (see above and compare figs. 7, c; 6, H). The occurrence ofthe 'inner layer' only under the minor tubercles suggests that it acts as aseries of structural pillars holding up the minor tubercles, which probablyplay an important role in the surface properties of the cuticle (see Noble-Nesbitt, 19636). Some support for the minute tubercles is to be expected.In the hard cuticle of the clypeus, the 'inner layer' is sometimes con-tinuous over the cuticle, and is not confined only to the minor tubercles (seefig. 7, D). It is possible that this is a reflection of the tanning of the underlyingexocuticle, which is also continuous in this region. However, it should benoted that in Rhodnius abdominal cuticle as described by Locke, the 'innerlayer' is underlain only by endocuticle. Outside the cuticulin layer, a thin,less-dense layer occurs, bounded externally by another thin and very denselayer. These layers, which are only about 5 m /x thick, are always most distinctnear the minor tubercles (see fig. 7, c). They are possibly wax and cementlayers, respectively (cf. Locke, 1957, 1961). Over the minor tuberclesthemselves, these layers are greatly modified. The wax layer is thicker andthe cement layer balloons out (see fig. 7, E), possibly because of being loosenedduring the histological treatment. Often the cement layer takes on a lenticularshape (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 ispossible that they are subject to a fair amount of damage. They are of great

FIG. 7 (plate), A, section through clypeal cuticle, showing pore canals traversing thelamellate endocuticle and the homogeneous exocuticle.

B, section through clypeal cuticle, showing the pore canals and their cytoplasmic contentsin 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; cent. /., cement layer; cutic. I., cuticulin layer; cyt. fil.,

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 L, outer wax layer; p.c, pore canal; sens,, sensory processes; wax L, wax layer.


importance in the surface properties of the cuticle (see Noble-Nesbitt, 19636).No evidence of pore canals penetrating the continuous layers of the epicuticleof 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 thecuticulin layer of the epicuticle. These seem to be distinct infoldings of theepicuticular layer, since they show evidence of being hollow structures.They are approximately 40 m/x in diameter, the core being approximately20 m/x in diameter, and the walls approximately 10 mfi thick. At the endproximal to the muscle, each tonofibrilla forms a Y-shaped cone which re-ceives a group of myofilaments.

Setal insertions

P. aquatica is sparsely clothed with setae. Those that have been seen in theelectron microscope all have a similar type of insertion. Fig. 7, H shows thestructure of the cuticle at the setal insertion. The seta inserts into the socketwhich is differentiated from the rest of the cuticle. The socket consists of a rimsurrounding an inner pad overlain by an epicuticular 'articular membrane'.The rim of the socket consists of structurally hard cuticle, which resemblesexocuticle (see fig. 7, H). It has a tapering connexion to the base of the hollowcylinder of the seta which encloses the strand of nerve-tissue, and this appearsto give the solid foundations upon which the seta is erected. The pad con-sists of lamellated soft cuticle, the lamellae being strung from the socket rim tothe 'epicuticular' inner cylinder of the seta and to the base of the setal wall. Itprobably forms an elastic suspension to hold the seta in place, whilst allowingmovement, and to return the seta to its resting position after such a move-ment. Figs. 7, H and 8, A distinctly show a 'guy-rope' type of suspension. It willbe noticed that the lamellae here exhibit a different pattern to those of otherregions of endocuticle. They appear to be distinctly interconnected givinga feathery appearance. This structural difference could well be associatedwith different mechanical properties such as elasticity (see Locke, i960).As we have seen, the cuticle of this area stains red in Mallory, suggesting achemical difference too. It is perhaps significant that 'Resilin' (Weis-Fogh,i960), which forms an elastic cuticle, is also associated with similar differen-tial staining properties. The rim is further connected to the seta by means ofthe epicuticle, which presumably is a tough, inelastic membrane (Wiggles-worth, 1933, 1947), giving a further suspension at this level between the setaand socket rim. This corresponds to the articular membrane recognized inlight microscope studies (see Hsu in Wigglesworth, 1953). The walls of theseta are structurally exocuticular (i.e. sclerotized) down to just below thelevel of the articular membrane only. Below that only the inner epicular cylin-der penetrates the pad of soft cuticle of the socket. The base of the setatherefore appears to be freely movable, as in mechanoreceptors (see Hsu in


Wigglesworth, 1953). The 'elastic' cuticle and suspension, of course, maymerely serve to keep the hair erect, whilst allowing a certain amount offlexibility to guard against breakage and ensuring that the hair returns to itsnormal erect position after being displaced. In support of this interpretation,a similar insertion is seen in the chemoreceptors shown in their electronmicrographs by Slifer, Prestage, and Beams (1957, plate 5). As in thosechemoreceptors, 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 asthose described by Slifer, Prestage, and Beams, the nerve-strands appear topass 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 bySlifer, 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 roughsurface on an otherwise smooth area of cuticle. This is of importance insurface properties (see Noble-Nesbitt, 19636), and may keep this part of theseta hydrofuge. It may be noted that the ridges occur at the general level ofthe surrounding cuticle.

The tormogen cell, which forms the socket, surrounds the hair-formingtrichogen cell (cf. Wigglesworth, 1933, 1953). The trichogen cell thereforeappears to pierce the tormogen cell, but close examination reveals that it liesin a deep fold in the tormogen cell (see fig. 8, F). Thus, the tormogen cellsends out two arms, which invest the trichogen cell and meet on the oppositeside of it (cf. Lees and Picken, 1945). The cytoplasms of the 2 cells areseparated by the plasma membranes of both cells. Sections parallel to theepidermal surface and transverse to the cells show that they effectively havea circular cross-section, although the actual cross-section is C-shaped. Thisarrangement is reflected in the circular socket and seta. The tormogen cellbears numerous microvillii at its cuticular surface (see fig. 8, G, H). Thesignificance of these is not clear, but they possibly are concerned with thesecretion of the specialized cuticle of the socket. The tormogen cell furtherhas lateral projections a little way beneath the epidermal surface, forming aflange 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 theseare absent from the trichogen cell. The trichogen cell, surrounded by thetormogen cell, likewise surrounds the distal processes of the sensory nerves.Accordingly, it is probable that the investing of the dendrites and of thetrichogen cell occurs at the same time and in the same manner. Thus, by arolling of these 2 cells, both the distal process and the trichogen cell are sur-rounded, and the circular hair and socket pattern produced. The boundariesof the cells clearly define the limits of the differentiation of the cuticle of thehair and socket (see fig. 8, G, H). Intercellular cytoplasmic connexions (plas-modesmata 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. Eachone 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,i960; Slifer and Sekhon, i960). Each neurofilament is circular in transversesection and hollow. Fig. 8, B to D show the distal processes passing up thesensillum.

The ventral tube vesicles

The cuticle is very thin (0-5 /x thick) and the epicuticle modified over thisarea. The endocuticle appears to consist of 2 or 3 dense lamellae, and isbounded 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 thatthese laminae represent the wax and cement layers, with the latter splittingto form further apparent layers externally. This suggests that this outermostlayer is a composite one, with its constituents normally intimately boundtogether, but which is prone to having an outer membrane peel off (see fig.9, c). These layers are very thin (approximately 10 m/x thick). Their presenceis important in considering the surface properties of the cuticle overlying thevesicles (see Noble-Nesbitt, 1963&). At the edges of the area, the cuticle ismodified to form cuticular flaps (see fig. 9, D, E).

The epidermis is glandular (see fig. 9, F) and reminiscent of active excretoryand resorptive epithelia as found in the Malpighian tubules and the rectum ofinsects (Smith and Littau, i960). The epidermal cell surface is deeply folded,forming a honeycomb border beneath the thin cuticle. Elongated mito-chondria occur within the cellular projections, and large mitochondria areseen in a layer at the base of these projections. Between the cellular pro-jections, extracellular spaces appear to be pinched off at the bases of thecellular projections, indicating that pinocytosis may occur (see fig. 9, E).Certainly, smooth-walled vesicles appear beneath the intuckings. This may

FIG. 8 (plate), A, oblique section through the base of a seta and its cuticular socket, show-ing 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 pigmentgranules, some of which have been partially pulled away during sectioning.

G, section through the epidermis, cuticle, and a seta, showing the arrangement of the tor-mogen and trichogen cells in the epidermis and with respect to the overlying cuticular struc-tures.

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 tormogencell (as indicated by the arrows).

artic. m., articular membrane; cut., cuticle; epid., epidermis;/?., flange of tormogen cell;lin., epicuticular lining of setal lumen; him., setal lumen; microv., microvilli of tormogencell; mit., mitochondrion; neurof., neurofilament; sens., sensory processes; torm., tormogencell; trich., trichogen cell.

FIG. 8

J. NOBLE-NESBITT

FIG. 9

J. NOBLK-NESBITT


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 allow-ing 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) whichpresumably secrete it and the overlying smooth cuticle. Pigment granulesare absent from these cells, and the passage of the light to the lens is thereforeuninterrupted. The lens is not shed at the moult, and whilst appearing to becomposed of cuticular material, it may perhaps be better thought of as aspecial inclusion formed for sensory purposes. Beneath the lens, the sensorycells have a regular structure such as would be given by closely packedfibrils (see fig. 9, H). These are thought to be the light-sensitive terminations.The lens presumably focuses the incident light on them. They pass inwardstowards the brain, still surrounded by pigmented epidermal cells, and stillthen retain their regular substructure.

FIG. 9 (plate), A, section through the tormogen, trichogen, and glial cells just below theouter epidermal surface, showing the substructure of the sensory processes enclosed by thesecells. Note the pigment granules (some of which have been partially pulled away duringsectioning) in the tormogen cell, and the regular array of tubular neurofilaments in the sen-sory processes.

B, section through the two adpressed vesicles of the ventral tube, showing the epidermalprocesses beneath each cuticle. The diagonal strip separating the two cuticles is morphologic-ally 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 waxlayer outside the cuticulin layer, and the composite cement layer outside the wax layer. Thecement layer consists of two dense laminae with a less dense lamina between them (indicatedby 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,shovving 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 edgesof 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 epidermalprocesses beneath the cuticle, with elongated mitochondria within the processes, large mito-chondria at their bases, and enclosed vesicles.

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

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

cutic. 1., cuticulin layer; endo., endocuticle; epid,, epidermis; gl., glial cell; lent., lentigencells; mit., mitochondrion; neurof., neurofilament; nucl., nucleus; o. epi., outer epicuticle;ret., retinal elements; sens., sensory processes; torm., tormogen cell; trich., trichogen cell; ves.,vesicle.


Discussion

The structure of the cuticle in Collembola, as we have seen, is not markedlydifferent from the structure of the cuticle in pterygote insects or other arthro-pods. Lower (1958) suggested that apterygote cuticle was simpler in struc-ture than pterygote cuticle, and that a 2-layered epicuticle was the basicepicuticle from which other types could be derived. He considered that fromthe simple apterygote cuticle, the more complex cuticle of pterygote insectscould be derived. In this simple apterygote cuticle, he described no cuticularlamellae and no pore canals. Furthermore, he distinguished no true exo-cuticle, in which full sclerotization occurred, and no cement layer. Thepresent study indicates that in Collembola, at least some of these features arefound. Cuticular lamellae are clearly visible in electron micrographs ofendocuticle and mesocuticle, whilst fine pore canals penetrate the cuticle fromthe epidermis to the epicuticle, and contain cytoplasmic filaments. In limitedregions 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, distinctcell boundaries occur in the epidermis, which Lower regarded as a syncytium.In these respects, then, it must be concluded that the cuticle of apterygoteinsects does not differ from that of pterygotes. Any difference lies only inthe distribution of the cuticular layers over the body. In this aspect, theapterygote insects are more akin to the larval forms of the pterygote insectsthan to the imaginal forms. In soft-bodied pterygote larvae, it is usual tofind an almost complete absence of an exocuticle, except in mouthparts andassociated structures, where hardness is a prime necessity. Larval formsare growing stages, intent mainly on increasing body size and weight to theadult level. Metabolic effort wasted in producing exocuticle would be ex-pected to retard this process. Furthermore, at the moult, soft cuticle canbe almost completely recovered, but hard exocuticle cannot; further wastageof valuable materials would occur if larvae were covered with exocuticle.Once adult, of course, with no further moulting, exocuticle represents nospecial potential loss to the pterygote insect. But in apterygote insects, whichcontinue to moult after becoming adult, production of exocuticle continues torepresent a potential loss to the insect, only part of which subsequently maybe recovered by eating the exuviae. It is not then surprising to discoverthat, on the one hand, exocuticular production is minimal, and, on the otherhand, 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 havethe apterygote insects. It is therefore incorrect to claim that apterygoteinsects represent an earlier phase in phytogeny when exocuticle could notyet be formed. A similar situation exists in Crustacea. In the cuticles ofCrustacea (see Richard, 1951) hardness is obtained principally by use ofincrustations of crystals of inorganic salts (e.g. calcium carbonate). Even inthese 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 aresclerotized (Dennell, 1947). Again we can infer an economy of metaboliceffort. Crustacea moult continuously and produce little proteinaceous exo-cuticle. But this does not necessarily mean that they have great affinities withthe Apterygota. Rather it means that they have solved a similar problem in asimilar way. In the pterygotes, this problem is confined only to the larvalstage, and the solution is again similar. In the imaginal stage, made possibleby major changes in the organization of the body, the problem does not ariseand no solution is required. Thus, lack of moulting in the adult stadium maybe associated with extensive exocuticle because of the lack of a selectivepressure against its formation. Indeed, the mechanical requirements offlight provide a selective pressure favouring its formation.

The endocuticle in P. aquatica is typically lamellated, but the arrangementof the lamellae differs in different parts of the cuticle. On the whole, thelamellae appear to have no regular interconnexions. However, in certainareas (such as in the sockets of the setae), a regular arrangement of inter-connexions does occur, and it is suggested that this arrangement may beassociated with greater elasticity. It is interesting to note that Locke (i960)has described this arrangement as being typical in the endocuticle of the larvaof Calpodes ethlius Stoll, and he suggests that it may be associated withelasticity. It is possible that the arrangement of the cuticular micelles in andbetween the lamellae may vary according to the type of cuticle, but obviouslymuch more high-resolution electron microscopy of endocuticles is requiredbefore any generalizations can be made.

The documentation of the 2-layered nature of the epicuticle in insects hasbeen summarized by Locke (i960). Lower (1958) suggested that this was thebasic structure from which other epicuticles were derived. In the presentstudy, it has been established that only one basic layer of the epicuticle canbe recognized. This layer, which is assumed to be the cuticulin layer, corre-sponds to the very dense layer recognized by Locke (1957, 1958, i960), whichhe calls the cuticulin layer. It occurs in all regions of the cuticle. Under-lying it is an 'inner layer', which is often discontinuous, and which corre-sponds to the homogeneous, chitin/protein layer, described by Locke (1957,1958, i960). This inner layer, at least in some instances, apparently supportsthe raised parts of the cuticulin layer. Wax and cement layers may overlie thecuticulin layer, but their occurrence is closely linked with the properties ofthe cuticular surface (see Noble-Nesbitt, 19636).

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 amvery grateful also to Dr. V. E. Cosslett for the electron-microscope facilitiesprovided in his department of the Cavendish Laboratory, and to Mr. R. W.Home and Mr. I. M. Wardell for taking the electron micrographs. I greatlyappreciated the electron-microscope facilities provided for a limited period

27° Noble-Nesbitt—Intermoult cuticle of Podura

at short notice by Dr. J. D. Lever of the Department of Anatomy. This workwas carried out during the tenure of an Agricultural Research CouncilResearch Studentship.

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19556. Ibid., 143, 427.GIBBONS, I. R., and GRIMSTONE, A. V., i960. J. biophys. biochem. Cytol., 7, 697.GLAUERT, A. M., and GLAUERT, R. H., 1958. Ibid., 4, 191.GOTO, H. E., 1956. Scot. Nat., 69, 1.IMMS, A. D., 1906. L.M.B.C. memoirs XIII. Anurida.KONCEK, S. K., 1924. Zool. Anz., 61, 238.LEES, A. D., and PICKEN, L. E. R., 1945. Proc. roy. Soc. B, 132, 396.LOCKE, M., 1957. Quart. J. micr. Sci., 98, 487.

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OGEL, S., 1958. Thesis, Dept. of Zool. and Appl. Ent., Imp. Coll., Lond.OVERGAARDE, C , 1948. Quart. J. micr. Sci., 89, 437.PANTIN, C. F. A., 1948. Notes on microscopical techniques for zoologists. Cambridge.PEACHEY, L. D., 1959. J. biophys. biochem. Cytol., 5, 511.PROWAZEK, S., 1900. Arb. Zool. Inst. Wien., 12, 335.PRYOR, M. G. M., 1940. Proc. roy. Soc. B, 128, 393.RICHARDS, A. G., 1951. The integument of arthropods. Minnesota.

and ANDERSON, T. F., 1942. J. Morph., 71, 135.SCHATZ, L., 1952. Ann. Ent. Soc. Amer., 45, 678.SjOSTRAND, F. R., 1956. In Physical techniques in biological research. 3. Cells and tissues,

edited by Oster, G., and Pollister, A. W. New York (Academic Press).SLIFER, E. H., PRESTAGE, J. J., and BEAMS, H. W., 1957. J. Morph., 101, 359.

and SEKHON, S. S., i960. Exp. Cell Res., 19, 410.SMITH, D. S., and LITTAU, V. C, i960. J. biophys. biochem. Cytol., 8, 103.SOMMER, A., 1885. Z. wiss. Zool., 41, 683.TOWER, W. L., 1906. Biol. Bull. Wood's Hole, 10, 176.WATSON, M. L., 1958. J. biophys. biochem. Cytol., 4, 727.WAY, M., 1950. Quart. J. micr. Sci., 91, 145.WEIS-FOGH, T., i960. J. exp. Biol., 37, 889.WHITEAR, M., i960. Nature, Lond., 187, 522.WIGGLESWORTH, V. B., 1933. Quart. J. micr. Sci., 76, 269.

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