the role of the epidermal cells in moulding the …j.cellsci. 13, 683-705 (1973 68) 3 printed in...

J.CellSci. 13, 683-705 (1973) 683

Printed in Great Britain

THE ROLE OF THE EPIDERMAL CELLS IN

MOULDING THE SURFACE PATTERN OF

THE CUTICLE IN RHODNIUS (HEMIPTERA)

V. B. WIGGLESWORTH

Department of Zoology, University of Cambridge,Downing Street, Cambridge, England

SUMMARY

The patterns of folding in the epicuticle of RJwdmus result from mechanical forces directedby the epidermal cells and not from, the action of physical forces arising spontaneously in thesecreted substance. This conclusion is supported by observations on the cuticular folds overthe abdomen of the larva and adult; on the formation of the plaques and the tactile setae arisingfrom them; and on the taenidial folds in the tracheae.

At the time of formation of the outer epicuticle the boundaries of the definitive epidermalcells are clearly defined by adhesion zones. The total surface area of the cuticle is thus the sumof contributions determined by the individual cells.

The area of outer epicuticle deposited is greater than that of the apical plane of the cell. Thecell surface is thrown into folds over which the epicuticle is laid down. But before the innerlayers are deposited the cuticle is moulded by mechanical forces controlled by the epidermalcells.

Microtubules and fibre bundles appear to play an active part, notably in the outgrowth ofsetae, and perhaps in the raising of the initial folds in the abdominal surface and in the tracheae.The taenidial folds in the tracheae arise mainly at the sites of the folds in the preceding instar.

Cellular movements or changes in shape lead to aggregation of epidermal cells to form the'stellate pattern' of the larval abdomen and the transverse 'ripple pattern' of the adult.

Distension of intercellular and intracellular vacuoles controls the moulding of the definitivestars in the larval cuticle, the transverse ridges in the adult, the smoothing of the cuticle overthe plaques, the raising of the plaques in dome-like form above the rest of the cuticle, theexpansion of the setae and the shaping of the taenidial folds in the tracheae.

The basement membrane provides a resistant substrate for these mechanical forces.

INTRODUCTION

An earlier paper (Wigglesworth, 1970) confirmed the widespread distribution ofstructural or bound lipid in the insect cuticle. Work in progress is concerned with thehistological details of the incorporation of this lipid during the deposition of the cuticle.The present paper deals with a single aspect of this study: the means by which thesurface epicuticle is moulded into its characteristic pattern before the deeper layersare laid down.

The selected elements in this pattern are (i) the stellate folding of the epicuticle inthe abdomen of the larva, and the transverse 'ripple' pattern in the abdomen of theadult; (ii) the dome-like plaques of the larval abdomen; (iii) the sockets and the bristlesarising from them; and (iv) the taenidial folds of the tracheal cuticle.

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684 v- B- Wigglesworth

MATERIALS AND METHODS

4th-stage larvae of RJwdnins were examined throughout the 14 days from feeding to ecdysis;43 specimens were studied during the 48 h (7-9 days after feeding, at 26 °C) when the epi-cuticle of the 5th-stage larva is formed. The integument of the abdomen was fixed in osmiumtetroxide 1 % for 2 h, followed by picric acid for 1 day to dissolve out the granules of pteridinepigment. The integument was (i) mounted whole after treatment with dilute sodium hypo-chlorite (' 10 per cent' solution, i.e. 10 g of available chlorine per 100 ml, diluted 1:1000 withwater) for 30 min and staining with Sudan black B (Wigglesworth, 1971); (ii) embedded inagar (2-5 %) and esterwax, cut both vertical and tangential to the surface, at 1 /im, treated withhypochlorite of graded strength (chiefly 1:1000 for 1-2 min), stained with Sudan black B andmounted in Farrants' medium. A smaller series of 5th-stage larvae (transforming to adult) wasstudied over the corresponding period, 11-13 days after feeding. Preparations made as de-scribed show the distribution of free triglyceride and bound lipid (Wigglesworth, 1971).

Blocks showing key stages were re-embedded in Araldite and examined in thin section withthe electron microscope. This procedure does not give electron micrographs of high quality;but the results are fully adequate for relating the light-microscope preparations showing boundlipids, with the established changes in the fine structure of the cuticle as described by Locke(1966, 1967) in Calpodes (Lep.), Filshie & Waterhouse (1969) in Nezara (Hemipt.) and Dela-chambre (1970) in Tenebrio (Col.). A shorter series was fixed in combined glutaraldehyde andosmium tetroxide (Hinde, 1971) before embedding in agar and Araldite.

RESULTS

Deposition of the epicuticle in the larval abdomen as seen with the light microscope

During the 48 h from 7 to 9 days after feeding of the 4th-stage larva, 14 stages(A-O) have been recognized for purposes of description.

A. Mitosis in the epidermis is almost complete but the cells are still in part adherentto the cuticle.

B. Detachment from the old cuticle (apolysis) is probably complete; mitosis is over;the definitive epidermis is uniform, with closely packed nuclei all lying in one plane(Fig. 1B). Most of the bound lipid of the cells is proximal to the nucleus. Bristlerudiments are visible but only just emerging from the surface.

C. Bristles up to one quarter of their final length, with the emergent portion showingintense lipid staining. Epidermal cells with dark lipid staining proximal to the nuclei.

D. Bristles up to one half of the final length, showing intense blue-black staining.Cell boundaries at the apex of the epidermal cells difficult to see. Lipid-stainingcytoplasm moving around the nucleus (usually on one preferred aspect) to accumulatein a distal cap (Fig. 1D). In the early stages of this process a lipid-staining strandapplied to the nucleus often runs from its proximal to its distal aspect.

E. Bristles from one half to full final length; still showing deep lipid staining afterhypochlorite treatment. Apical boundaries of the epidermal cells seen as fine blacklines in surface view. In section the intercellular membrane ends distally above aterminal bar and sometimes projects as a small ridge at the surface (Fig. 1E). Thelipid-rich cytoplasm is now accumulating distal to the nucleus.

F. Bristles mostly at their definitive size. Apical boundaries of the epidermal cellsseen as conspicuous dark lines in surface view. In vertical section they form up-standing ridges (Fig. 1 F, to the left); in horizontal sections these ridges give a honey-

Cellular control of cuticle pattern 685

comb appearance (Fig. 3). In a slightly more advanced stage in the same insect theremay be a finely crinkled membrane all over the surface of the cell (Fig. 1F, to theright). This appearance, which may lead to the formation of folded stars over the apexof each cell, is seen in horizontal section in Fig. 4.

G. Intercellular boundaries no longer visible. The lipid-rich cytoplasm forms astar-shaped area overlying each nucleus, and the surface is thrown into folds whichtend to radiate from the centre (Fig. 5). In section the area of the deeply staining starprojects above each nucleus with the surface thrown into innumerable minute folds(Fig. i G ; Fig. 5).

In this description 2 different structures are referred to as 'stars', (i) As the outerepicuticle (p. 686) forms it is thrown into folds which tend to radiate from thecentre of the cell in a stellate fashion, (ii) Below these cuticular stars is the lipid-staining ergastoplasm (p. 686). At this level in the cell there is commonly a ringof intercellular vacuoles which indent the periphery of the ergastoplasm so as to giveit a star-like appearance in surface view (see Fig. iG ; Fig. 2H').

H. An intermediate stage; epidermal cells mostly with individual stars over eachnucleus, as in G, but many stars in process of fusion as in J.

J. Bristles show dark lipid staining only towards the base. The individual lipid-rich stars are fusing in groups of 2, 3, 4 or 5 to form large composite stars (Fig. 1 J:Fig. 6). At the point where the individual stars come together there is a narrow un-stained gap with clear radiating lines which mark the intercellular boundaries (Fig. 6).Occasional individual stars persist; they are indeed general at the intersegmentalmembranes.

K. A large vacuole appears below the centre of each composite star, produced bythe massive enlargement of the intercellular vacuoles at these sites; subsidiary vacuolesappear in the branches of the stars; and the cytoplasm of the distal portions of theepidermal cells becomes vacuolated (Fig. iK; Fig. 7). As a consequence of thisorganized distension of the epidermal cells the minute folds in the surface are smoothedout to give the large definitive stellate folds of the larval cuticle, each with its centreraised to a peak by the central vacuole.

L. The vacuolated state of the epidermis persists as at stage K, but a lipid-rich'inner epicuticle' is just beginning to form (Fig. iL ; Fig. 8).

M. Inner epicuticle thickening. The epidermal cytoplasm tends to become detached,leaving a narrow clear zone below the epicuticle. (This may be an artifact.) Muchbound lipid at the apex of the cells. Vacuolation less conspicuous.

N. Vacuoles in the epidermis have disappeared, except below the plaques (seebelow). Inner epicuticle almost fully formed and is thicker over the plaques (seebelow) (Fig. 9).

O. Epicuticle complete; laminated cuticle beginning to appear.The foregoing stages differ in duration. As judged by the number of examples found

in the series examined, stages F and G are very brief (lasting perhaps less than 1 h)and are liable to be overlooked. Stage K likewise is probably no more than 1 h induration.

686 V. B. Wigglesworth

Changes in fine structure during formation of the stellate cuticle

Transfer of'ergastoplasm' to form the 'stars'. The darkly staining substance of thecytoplasm is shown up (a) by haematoxylin after Carnoy fixation (protein and nucleicacid); (b) by gallocyanin at pH 2-o after Carnoy fixation (nucleic acid); this staining isabsent after treatment with ribonuclease; (c) by Sudan black after hypochlorite (boundlipid). Electron-microscope sections confirm that it is rich in rough endoplasmicreticulum (ER). At stages A-C when it lies proximal to the nucleus it is largely inthe form of laminated stacks of membranes. As these move around the nucleus toform the distal cap or 'star' they break up into small vesicles of rough ER. Associatedwith the movement of the ER are numerous mitochondria which contribute to thelipid staining of the star (Fig. 12). There may well be other lipid-rich componentsnot visible in the electron microscope.

Formation of the outer epicuticle. The 'outer epicuticle' (the 'cuticulin' of Locke(1966)) is a trilaminate structure about 17 nm thick, which first appears in the form ofminute curved plaques each laid down at the apex of a single microvillus of the epi-dermal cells, so that when they first appear, during stage E, each is separated from itsneighbours (Fig. 14). At stage F these minute plaques are still largely independent,but they are increasing in number and so crowded that the surface is thrown intoirregular folds (Fig. 15). It is this process of expansion which leads to the formationof the individual stars centred over each nucleus.

By the time stage G (Fig. 1G; Fig. 5) is reached the epicuticular plaques have fusedto form a continuous membrane which will become the definitive outer epicuticle ofthe developing instar (Fig. 16). At this stage bundles of microtubules are often con-spicuous in the vertical axis of the projecting folds (Figs. 16, 17) (cf. Filshie & Water-house, 1969).

Intercellular boundaries. The cell boundaries faintly visible at the apex of the epi-dermal cells at stage E are of the standard type described, for example, in the developingwings of cecropia (Greenstein, 1972). A conspicuous adhesion zone, or intermediatejunction, lies just below the apical boundary (Figs. 12, 13). Proximal to this the celljunctions show ordinary apposed plasma membranes and occasional zones with septatedesmosomes. MicroviUi are conspicuous at the apex of the cell (Fig. 12). They are oftenenlarged and elongated at the intercellular boundary and here they may lead to theformation of a projecting ridge of outer epicuticle (Fig. 1 E, F) (cf. Delachambre, 1970).

Small vacuoles appear in the intercellular boundary, some distance below the inter-mediate junction; they usually arise between the 2 plasma membranes (Figs. 24, 25).It is these small vacuoles which sometimes enlarge and indent the darkly stainingergastoplasm below the cell apex to give it a star-like appearance in surface view.

Fusion of individual 'stars' in groups; moulding of the stellate pattern of the epkuticle.The nature of the force which impels the aggregation of the dark ergastoplasm ofadjacent cells to form the composite stars (Fig. 6) is unknown. The metabolicmachinery of the cell, which constitutes the ergastoplasm, piles up at the points ofconvergence, becoming thinned out over the remote aspect of the cell. Electron-microscope sections show that at this stage the outer epicuticle is complete. Figs. 18—21


show vertical sections through the meeting point of the cells at stage K when eachcomposite star is being raised by the expanding vacuole below. In similar sections seenwith the light microscope the large vacuoles often appear to communicate with theexterior through a pore at the apex of the star, but the electron-microscope sectionsshow that the gap is always bridged by the continuous epicuticle, often with a minimalamount of cytoplasm below it (Fig. 21).

In the earliest stage of its formation the vacuole appears to arise between the2 plasma membranes just proximal to the intermediate junction. As it expands it mayremain intercellular, with widely separated plasma membranes (Fig. 25); but quiteoften a double plasma membrane is seen crossing a large vacuole (Fig. 19). The accu-mulated fluid may thus be largely intracellular. Many vacuoles are crossed bytrabeculae visible in the light microscope.

Fig. 20 shows the outlines of the 4 cells investing the vacuole shown in Fig. 18. Thesection has just missed the apex of the star; it is possible that cell 4 has been missedand the tip of the cell so labelled belongs to cell 2.

Finally, smaller intracellular vacuoles appear within the radiating ridges of thestars. These appear to arise from the expansion of vacuoles of the endoplasmicreticulum (Fig. 22). By this time (stage L) the inner epicuticle is forming and all thefiner folds in the surface have been smoothed out to be replaced by the larger foldsradiating from the definitive stars centred over the major vacuoles (Fig. 8).

Moulding of the adult epicuticle

In the 5th-stage larva at 13 days after feeding, when the new epidermis of theabdominal tergites is fully organized and is detachable as a uniform smooth sheet ofcells, as in the 4th-stage larva at 7 days (stage E), the intercellular boundaries arefaintly visible in the Sudan-stained whole mounts, by virtue of the terminal bars attheir distal extremities. These boundaries have a roughly hexagonal form as in the4th-stage larva (Fig. 29).

At the next stage, corresponding to stage F (Fig. 1F) of the 4th-stage larva, whenthe boundaries are very conspicuous and fine folds are appearing over the freesurface, each cell has become elongated in the transverse direction (Fig. 30).

This transverse elongation of the epidermal cells and of the lipid-rich stars ofergastoplasm below their surface, is the basis of the transverse ripple pattern of theepicuticle in the adult; for the elongated 'stars' tend to fuse into transverse chainsacross the abdomen (Fig. 31), as opposed to the multiple radial or stellate groups ofthe larva. As the epicuticle is laid down abundant vacuoles appear in the epidermalcells and the finely folded epicuticle on the surface is smoothed out (Figs. 32, 33).Meanwhile the epidermal cells aggregate in the form of transverse ridges; usually3 or 4 rows of cells to a ridge with valleys between. The smoothed-out epicuticle thusadopts the form of transverse ridges with irregular lateral folds running down into thevalleys and occasionally crossing them to unite with folds from the neighbouring ridge

(F'g- 33)-There is, of course, much local variation in the details of this pattern; in some areas

the epicuticle may assume a stellate pattern resembling that of the larva.


Moulding of the plaques and bristles of the larva

Plaques. At stage E in the 4th-stage larva, apart from the presence of the developingsensory bristle, the future plaques are almost indistinguishable from the interveningregions of the epidermis. The lipid-rich ergastoplasm is condensed below the apicalsurface, the terminal bars of the intercellular boundaries are evident in surface viewand project upwards as minute ridges as seen in section (Fig. 2E) but the epidermisin the areas of the plaques is no higher than elsewhere.

At stages F and H (Fig. 2F, H) small intercellular vacuoles appear in the region ofthe terminal bar giving a star-like appearance to the lipid-rich cytoplasm seen insurface view (cf. Fig. 11). The outer epicuticle is forming and is thrown into minutefolds which later form a stellate arrangement over each cell; but this folding is lessconspicuous than over the general surface of the abdominal cuticle. Small intracellularvacuoles are just beginning to appear in the cytoplasm proximal to the nuclei andthese serve to extend the cells and raise the plaques slightly above the surroundingepidermis. The apical cytoplasm stains more deeply with Sudan B than in the inter-vening epidermis.

By stage K, when the vacuolation of the general epidermis is moulding the stellateepicuticle, large intracellular vacuoles distend the basal parts of the plaques, raisingthem into a dome-like form (Fig. 2K; Fig. 10); the intercellular vacuoles at the apexof the cells also enlarge (cf. Fig. 11), with the result that the newly formed epicuticleis smoothed out. In section the apex of each cell has a mushroom-like appearance, butthe rim of each 'mushroom' is connected to the next by continuous epicuticle whichbridges over the enlarged intercellular vacuoles (Fig. 2K). This moulding process isa modification of that operating in the general stellate cuticle (Fig. 1).

Bristles. The formation of bristles and their sockets in Rhodnius was describedearlier and the term ' tormogen' was introduced for the socket-forming cell (Wiggles-worth, 1933). The course of events in relation to cuticle formation is outlined above(p. 684). The changes in fine structure during the process have been described forthe scales and setae of other insects by a number of authors (Paweletz & Schlote, 1964;Overton, 1967; Lawrence, 1966; Locke, 1969; Greenstein, 1972). The present findingsin Rhodnius agree with these earlier reports: the formation of an outgrowth rich inparallel microtubules, apparently stiffened by peripheral bundles of fibrous material.One observation only will be recorded.

Fig. 26 shows a part of a bristle at stage F in longitudinal section, with the micro-tubules, about 15 nm in diameter, packed at distances apart ranging from 12-5 to25 nm. The outer epicuticle is beginning to form, but the constituent 'plaques' arestill separated. When the epicuticle is completed it forms a loose sleeve around thebristle. At stage L, when the epidermis is highly vacuolated and the cuticular folds arebeing smoothed out, there are corresponding changes in the bristles.

Fig. 27 shows a section through the base of a bristle emerging from the socket inprocess of formation by the tormogen cell. The cytoplasm of the tormogen is notunduly vacuolated, but the outgrowth of the trichogen, forming the base of the bristle,is diffusely vacuolated. The bristles themselves are becoming distended to fill the


overlying epicuticle and they often contain visible vacuoles. Fig. 28 shows an obliquesection through such a bristle with the microtubules more widely dispersed than inFig. 26. Their diameter is unchanged at about 15 nm but they are separated by intervalsranging from 37-5 to 50 nm. Similar observations were made by Lawrence (1966) inOncopeltus where 'the second phase in the growth of the hair appears to involveinflation of the cytoplasm of the trichogen cell. As the hair becomes turgid it loses itsragged outline.'

Moulding of the taenidial folds in the tracheae

In each segment of the ventral abdomen there is a transverse trachea which anasto-moses with its opposite number in the midventral line. Tangential sections of the bodywall give longitudinal sections of these tracheae for the light and electron microscope.As had been noted (Wigglesworth, 1954), there is a progressive development along thecourse of the tracheae. It has now been found that at stage N, when the outer epicuticleof the abdominal surface is complete, the inner epicuticle is thickening, and thevacuoles below the stellate folds have disappeared, separation of the trachea is be-ginning in the immediate neighbourhood of the spiracle but does not extend very farinwards. In such specimens it is possible to follow all the early stages in the mouldingof the cuticle. For purposes of description 6 stages in tracheal cuticle formation havebeen recognized.

(i) A smooth membrane, rich in lipid, appears between the existing tracheal cuticleand the epidermis. The surface of the cytoplasmic layer is seen as a thin lipid-stainingline in the light microscope. Electron-microscope sections show closely packedmicrovilli.

(ii) The sub-tracheal membrane is thrown into transverse folds and is becomingseparated from the taenidia and the old tracheal cuticle (Fig. 38). It is seen in theelectron microscope to be a fibrous 'ecdysial membrane' in process of digestion (seeLocke, 1958). The lipid-rich surface of the cytoplasm shows in the electron microscopeas a more or less level sheet of outer epicuticle made up of the independent curvedplaques laid down by individual microvilli, giving the surface a scalloped appearancein longitudinal section (Figs. 38, 39). There is an electron-dense layer of cytoplasmbelow the outer epicuticle.

(iii) Taenidial folds now appear below the sites of the existing taenidia (Fig. 40).They are covered with independent plaques of outer epicuticle irregularly disposed togive a highly crinkled surface.

(iv) The plaques of outer epicuticle have now fused to form a continuous membraneover the taenidial evaginations, but the surface of these is still highly convoluted(Fig. 41). The substance enclosed by the epicuticle stains intensely with Sudan blackafter hypochlorite (Fig. 23).

(v) The taenidial folds form upstanding ridges with their inner extremity dilated(Fig. 42). Filaments resembling microtubules often run vertically into them. They arefilled with cytoplasm which forms conspicuous microvilli in the inflated rim, withvacuolated spaces between.

(vi) The microvilli proceed to lay down the glassy contents which gradually fill the


inflated rim to form the new taenidia. Fig. 43 shows this process about half completed.Fig. 44 is an oblique section of a taenidial ring from the same specimen as Fig. 43showing the microvilli and the vacuolated spaces between.

This process is a further modification of the mechanism described in the surfacecuticle: the production of folds covered by the growing epicuticle, moulded perhapsby the deposition of microtubules and certainly by the hydrostatic pressure of intra-cellular vacuoles. The question arises as to how the spacing of the spiral folds isregulated. The vast majority of the folds appear at the same sites within the epidermalcells as were responsible for the folds in the preceding instar. That can be seen in theglancing section of the trachea shown in Fig. 23. It can be seen at the right-hand endof the trachea shown in Fig. 37; and this same relation occurs likewise in small tracheae3 /tm in diameter.

It is not to be expected that there will be an identical number of taenidial folds inthe new and the old cuticle. The taenidia form discontinuous spirals and these spiralsmay well be longer in the new cuticle and thus the number of folds visible in longi-tudinal section will increase. In the trachea shown in Fig. 23 the number of foldsbetween 2 points where the old and the new taenidia were obviously superimposed was48 in the old cuticle and 49 in the new cuticle. In the trachea shown in Fig. 34 thefigures were 85 in the old cuticle and 93 in the new.

DISCUSSION

Major changes in the form of the cuticle in Rhodnius, such as occur in the genitalia,the intersegmental boundaries, the lateral pleats of the abdomen, the wing lobes andwings, etc., are controlled by local differences in the intensity of mitosis. In theRhodnius abdomen the area of the epidermal layer over the central regions of thetergites and sternites is determined by the extent of the old cuticle when it has con-tracted down after the large meal of blood (Wigglesworth, 1964; Bennet-Clark, 1971).The intensity of mitosis in these areas is adapted to this demand and by 6—7 days afterfeeding the epidermal cells form a regular level sheet with nuclei at a standarddistance apart. The mechanisms which regulate subsequent changes in the form ofthe epicuticular surface are the subject of this paper.

Stellate pattern of the larval abdomen

The stellate folding of the cuticle surface was earlier believed to result from thespontaneous expansion of the epicuticle as it was formed (Wigglesworth, 1933). It hasnow been shown that the process is more complex. The total area of the epicuticle isdetermined by the amount of outer epicuticle formed within the boundaries of eachepidermal cell. The outer epicuticle is laid down in the form of discrete patches overeach microvillus of the cell surface; these patches grow at their margins and ultimatelyfuse to give a continuous membrane (Locke, 1966, 1967; Filshie & Waterhouse, 1969;Delachambre, 1970). In the larval abdomen this membrane assumes a highly crinkledstate, commonly with the folds radiating in stellate fashion over the apex of each cell.But the final stellate pattern is on a larger scale; the apical ergastoplasm (p. 686) of


2, 3, 4 or even 5 neighbouring cells converges upon a central point to form a compositestar; the cell boundaries at the surface disappear; and a large vacuole develops belowthe point of convergence which thus forms the centre and apex of the composite star.The minute folds of the epicuticle are smoothed out by pressure from the vacuolesbelow and are replaced by larger folds, often themselves containing small vacuoles,which radiate from the centre of each star. As soon as the moulding process is completeand the inner epicuticle is formed the vacuoles disappear.

The distended fluid-filled spaces between the plasma membranes of adjacent epi-thelial cells are commonly regarded as representing fluid transfer across the epi-thelium by the intercellular route (DiBona, 1972). That aspect of the matter has notbeen considered in this paper; but it is clear that the accumulation of fluid in thesespaces, as well as in the intracellular vacuoles, is used to exert mechanical pressure inmoulding the surface pattern both in the stellate larval cuticle and in the dome-likeplaques.

A striking feature is the rapidity with which the vacuoles arise, induce changes inform, and then disappear again. The precise length of time involved is difficult toassess, but the fact that different stages in the process are often visible in a singlepreparation, and the infrequency of preparations at the peak of vacuole formation,suggest that it is quite brief.

Ripple pattern of the adult abdomen

A similar process takes place in the adult, but instead of the ergastoplasm of theepidermal cells aggregating in small radial groups before moulding of the epicuticlebegins, the apices of the cells become elongated in the transverse axis of the abdomenand the ergastoplasm associates in transverse chains. Three or four such chains areaggregated together to form a ridge. Under the pressure of epidermal vacuoles theepicuticle of these ridges is smoothed out with folds running from the ridges into andacross the valleys between. There is much local variation in the details of this pattern.

Dome-like plaques of the larva

It was earlier recognized that the dome-like plaques of the larval cuticle are raisedabove the level of the intervening stellate cuticle by the pressure of expanding vacuolesat the base of the epidermal cells (Wigglesworth, 1933, text-fig. 7B, p. 286). The epi-dermal cells differ from those of the stellate cuticle in laying down an epicuticle that isonly slightly folded in star-shaped form over each nucleus. Under pressure from theintracellular vacuoles below, the epicuticle is expanded to form a smooth surface fromone cell to the next, with a firm exocuticle below.

In this process the ring of expanding vacuoles around the neck of each cell may actas a constriction which will apply pressure to the apical pole. These vacuoles areusually quite small in the general epidermal cells (Fig. 1 G); but in the plaques theybecome greatly enlarged, and reach their peak just at the time when the apical cuticleis being smoothed out (Fig. 2K).

The moulding of the cuticle by the pressure of localized vacuoles in this way canbe effective only if there is a firm foundation for resistance below. This resistant


background is supplied by the basement membrane which is fully thickened, by thesecretory activity of the haemocytes, just before deposition of the epicuticle begins(Wigglesworth, 1973).

Sockets and bristles

Fibre bundles and microtubules appear to provide the skeletal support for thesocket and the out-growing seta, as described by a number of authors. In the finalstages in the moulding of the epicuticle of the seta the vacuolation of the trichogen andits outgrowth plays an important part, as it does in Oncopeltus (Lawrence, 1966).

Tracheal taenidia

In an earlier paper (Wigglesworth, 1954) I wrote as follows: 'When the new trachealmembrane is first laid down at moulting it is smooth and is little larger than thecuticular membrane of the preceding instar. The new trachea then increases in dia-meter and at the same time the membrane is thrown into folds which graduallydeepen. There can be little doubt that these spiral and annular folds result from anexpansion in area of the newly formed cuticle - such as is seen in the epicuticle of thebody surface (Wigglesworth, 1933). The formation of the so-called "taenidium" orspiral thread is doubtless a secondary phenomenon resulting from the deposition ofcuticular material within these folds.' This interpretation was elaborated in detail byLocke (1958). It has now been shown to be based on misleading appearances.

As Locke (1958, 1967) has shown, the first component of the new trachea to be laiddown is the epicuticle. But the raising of this layer into spiral or annular folds is nota spontaneous physical change but an active process of growth.

When the outer epicuticle first appears it is seen in longitudinal section as the levelscalloped boundary of the epidermal surface; the tiny unit plaques over each micro-villus are still separated. It is at this stage that the inward folding takes place. Insection the folds, as they form, have an irregular and highly crinkled surface with theepicuticular plaques still separated. As the definitive area of the outer epicuticle isattained the individual plaques unite to form a continuous outer epicuticle. The innerepicuticle is laid down over the tips of the elongated microvilli and the expansion ofvacuolar spaces between the microvilli distends the cavity of the inner border of thefold which will contain the taenidium. The substance of the taenidium is then laiddown by the microvilli.

The massive extension of the outer epicuticle at the site of the taenidial folds isclearly an active process of growth, the extent of which is controlled by the epidermalcells in conformity with the instructions latent in the pattern. The great majority ofthe taenidial flanges occupy the same sites as in the preceding instar. They are alreadydetermined by a growth pattern which continues without interruption from one cellto the next. As in the cuticle of the abdominal surface, no cell boundaries are to beseen in the tracheal cuticle. It is not uncommon to find a cell junction immediatelybelow a taenidial fold.

I have not observed the initial appearance of taenidial folds in the newly developing1-2/tm tracheae and in tracheoles. Beaulaton (1968), in his plate jb, shows a longi-


tudinal section of a tracheole in Antheraea, in which the outer epicuticle is almostcompletely fused; it is thrown into highly complex small folds, but the regular annulartaenidial folds are not yet formed.

This work has been supported by a grant from the Agricultural Research Council. I thankProfessor T. Weis-Fogh for research facilities, Dr J. E. Treherne and-Dr Nancy J. Lane forthe use of the electron microscope, Dr B. L. Gupta for advice and Miss Y. R. Carter forinvaluable technical assistance.

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pattern on the cuticle of the green vegetable bug Nezara viridula. Tissue & Cell 1, 367-385.GREENSTEIN, M. E. (1972). The ultrastructure of developing wings in the giant silkmoth,

Hyaloplwra cecropia. II. Scale-forming and socket-forming cells. J. Morph. 136, 23-52.HINDE, R. (1971). The fine structure of the mycetome symbiotes of the aphids Brevicoryne

brassicae, Myzus persicae, and Macrosiphum rosae. J. Insect Physiol. 17, 2035-2050.LAWRENCE, P. A. (1966). Development and determination of hairs and bristles in the milkweed

bug, Oncopeltus fasciatus (Lygaeidae, Hemiptera). J. Cell Sci. 1, 475-498.LOCKE, M. (1958). The formation of tracheae and tracheoles in PJiodttius prolixus. Q.Jlmicrosc.

Sci. 99, 29-46.LOCKE, M. (1966). The structure and formation of the cuticulin layer in the epicuticle of an

insect, Calpodes ethlius (Lepidoptera, Hesperidae). J. Morph. 118, 461-494.LOCKE, M. (1967). The development of the patterns in the integument of insects. Adv. Morpho-

gen. 6, 33-88.LOCKE, M. (1969). The structure of an epidermal cell during the development of the protein

epicuticle and the uptake of molting fluid in an insect. J. Morph. 127, 7-40.OVERTON, J. (1967). The fine structure of developing bristles in wild type and mutant Droso-

pliila melanogaster. J. Morph. 122, 367-380.PAWELETZ, N. & SCHLOTE, F. W. (1964). Die Entwicklung der Schmetterlingsschuppe bei

Ephestia kUhniella Zeller. Z. Zellforsch. mikrosk. Anat. 63, 840-870.WIGGLESWORTH, V. B. (1933). The physiology of the cuticle and of ecdysis in Rliodnius prolixus

(Triatomidae, Hemiptera) with special reference to the function of the oenocytes and of thedermal glands. Q.Jlmicrosc. Sci. 76, 269-318.

WIGCLESWORTH, V. B. (1954). Growth and regeneration in the tracheal system of an insect,Rliodnius prolixus (Hemiptera). Q. Jlmicrosc. Sci. 95, 115-137.

WIGGLESWORTH, V. B. (1964). Homeostasis in insect growth. Symp. Soc. exp. Biol. 18, 265-281.WIGGLESWORTH, V. B. (1970). Structural lipids in the insect cuticle and the function of the

oenocytes. Tissue & Cell 2, 155-179.WIGCLESWORTH, V. B. (1971). Bound lipid in the tissues of mammal and insect: a new histo-

chemical method. J. Cell Sci. 8, 709-725.WIGGLESWORTH, V. B. (1973). Haemocytes and basement membrane formation in Rhodnius.

J. Insect Physiol. 19, 831-844.

(Received 18 September 1972)


WillmmmiMm(Wimm

B

Fig. i. Semi-diagramatic sections of epidermis of 4th-stage larva during formation andmoulding of the epicuticle. The letters represent the stages as described in the text.Staining shows distribution of lipid (hypochJorite - Sudan black after osmium fixa-tion) but cytological details are omitted. The arrows in J and K indicate the directionof movement of the ergastoplasm.


H K

Fig. 2. Above, the raising of the dome-like plaque by the pressure from enlargedvacuoles in the cells beneath (from Wigglesworth, 1933). Below, semi-diagramaticsections of epidermis below the plaque at 4 stages in epicuticle formation. The lettersindicate the stages as described in the text. Staining as in Fig. 1.


Figs. 3-11. Tangential sections cut at 1 fun through the epidermis of 4th-8tage larvaeshowing stages in the moulding of the epicuticle. Hypochlorite-Sudan black afterosmium tetroxide. x 750.

Fig. 3. Stage F (cf. left side of Fig. 1 F); raised folds over cell boundaries.Fig. 4. Stage F (cf. right side of Fig. 1 F); epicuticle forming star-shaped folds over

each cell.Fig. 5. Stage G (cf. Fig. iG); individual 'stars' over each cell.Fig. 6. Stage J (cf. Fig. 1J); fusion of individual stars to form composite stars.Fig. 7. Stage K (cf. Fig. 1 K); vacuolation of composite stars begins.Fig. 8. Stage L (cf. Fig. iL); peak of vacuolation in the composite stars.Fig. 9. Stage N; showing definitive stars after disappearance of vacuoles.Fig. 10. Stage L; section through plaque showing intracellular vacuoles.Fig. 11. Stage L; section through surface of plaque showing bristle. Note intercellular

vacuoles around the ergastoplasm in the cells surrounding the base of the bristle.

Cellular control of cuticle pattern

45'

698 V. B. Wiggiesworth

Fig. 12. Border of epidermis at stage E, showing microvilli and ergastoplasm withmassed mitochondria below, x 17000.

Fig. 13. Detail of sub-apical desmosome of zonula adhaerens type at stage E.x 33000.

Fig. 14. Independent plaques of outer epicuticle at stage E. x 60000.

Fig. 15. Outer epicuticle at stage F with independent plaques being laid down oversurface folds, x 60000.

Figs. 16, 17. Stage G with folds containing microtubules covered by a continuousouter epicuticle. x 35000.

Cellular control of cuticle p"

1 6 5


Fig. 18. Stage K; aggregation of 4 epidermal cells around large vacuole forming thecentre of composite star, x 8600.Fig. 19. Two cells at apex of star bridging intercellular vacuole. x 10000.Fig. 20. Outlines of the 4 cells represented in Fig. 18.Fig. 21. Detail of the epicuticle over the vacuole in Fig. 19. x 60000.Fig. 22. Stage L; epidermal cell containing numerous intracellular vacuoles ap-parently derived from endoplasmic reticulum. x 8000.Fig. 23. Glancing 2-fim section of trachea showing new taenidial folds arising at sitesof the old folds, x 750.


Fig. 24. Stage H; intercellular junction between epidermal cells of a plaque. Foldedepicuticle to left; to the right a large intercellular vacuole is forming between the2 plasma membranes, x 15000.Fig. 25. As Fig. 24. A very small vacuole is forming within the junctional complexclose to the apex of the cell, x 15 000.Fig. 26. Stage F; longitudinal section of developing bristle with packed microrubulesand widely separated epicuticular plaques, x 40000.Fig. 27. Stage L; base of a bristle as it emerges from its socket. The contents of thebristle are highly vacuolated. x 14000.Fig. 28. The same: oblique section of bristle with microtubules more widely separatedby vacuolated cytoplasm than in Fig. 26. x 40000.Fig. 29. 5th-stage larva at 13 days after feeding; whole mount of epidermis of develop-ing adult; cells of approximately hexagonal outline, x 750.Fig. 30. sth-stage larva showing surface view of epidermis as adult epicuticle is aboutto be formed. Cell boundaries show transverse elongation, x 750.Fig. 31. The same at a slightly later stage. Epicuticle forming crumpled stars over eachcell, elongated and tending to fuse in transverse rows, x 750.Fig. 32. Whole mount of integument during the formation of the ripple pattern show-ing vacuoles in the cells.Fig. 33. The same specimen photographed at the level of the new epicuticle.

Cellular control of cuticle pattern 7°3


Figs. 34-37. 1-2 /im sections of tracheae of 4th-stage larvae during formation of newtracheal cuticle, to show the relation between the old and new taenidial folds.Fig. 35 shows an early stage of development, x 750.Fig. 38. Stage (ii) in the formation of new trachea, with old taenidia above; then theconvoluted ecdysial membrane; then the epicuticular plaques of new epicuticle; andbelow this the nuclear membrane, x 18000.Fig. 39. Detail of the new epicuticle from the middle of Fig. 38. x 100000.Fig. 40. Stage (iii); taenidial folds with epicuticular plaques not yet fused, x 52500.Fig. 41. Stage (iv); epicuticle continuous but taenidial folds highly crumpled,x 14000.

Fig. 42. Stage (v); folds with dilated extremities, showing microvilli. x 14000.Fig. 43. Stage (vi); microvilli laying down substance of the taenidia. x 23300.Fig. 44. As Fig. 43; tangential section of taenidia] fold showing microvilli cut at alllevels, x 23 000.

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