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J. Cell Set. 19, 459-485 (i975) 459Printed in Great Britain

INCORPORATION OF LIPID INTO THE

EPICUTICLE OF RHODNIUS (HEMIPTERA)

V. B. WIGGLESWORTHDepartment of Zoology, University of Cambridge, Dotiming Street,Cambridge, England

SUMMARYThe incorporation of lipid into both the outer and inner epicuticle during deposition is de-

scribed. Waterproofing of the epicuticle by secretion of the wax layer, and sclerotization with orwithout melanization, are controlled from a distance by the epidermal cells by way of the porecanals.

The pore canals gradually narrow as they approach the epicuticle. On reaching the innerepicuticle the canal ends in a conical projection from the apex of which a permeable lipophilicchannel about 20-25 ran in diameter runs vertically to the surface.

Shortly before ecdysis, silver-binding material (perhaps protein rich in tyrosine, or other pre-cursors concerned in sclerotization) spreads radially from a point in the cuticular channels justbelow the outer epicuticle, and gradually impregnates the outer two thirds or more of the innerepicuticle. The precise pattern varies in different cuticular structures. Argentaffin materials(polyphenols) first appear in these same sites at the time of ecdysis and increase rapidly duringthe next 24 h.

Lipid appears in the lumen of the distal parts of the pore canals (with a patchy distribution)shortly before ecdysis. When digestion and absorption of the old endocuticle are almost com-plete, minute lipid droplets appear on the surface of the epicuticle, apparently exuded from theepicuticular channels, and spread to make a uniform layer. When first formed this layer stainsreadily with Sudan B, but the lipid becomes incorporated in a delicate non-lipid silver-bindingmembrane (also exuded from the epicuticular channels) and hardens just before ecdysis, to formthe so-called 'wax layer' which then no longer stains with Sudan B. Within half an hour afterecdysis the aJcian blue-staining cement layer is poured out by the dermal glands, and forms acontinuous but somewhat irregular covering over the 'wax layer'.

Changes in the epicuticle that accompany the repair of abrasions are described.

INTRODUCTION

An earlier paper (Wigglesworth, 1973) described the formation of the epicuticle inthe abdominal integument of Rhodnius prolixus, with special reference to the mech-anisms by which the underlying epidermal cells control the patterns of folding in thesurface. The present paper deals primarily with the incorporation of lipids; butthe work has included observations on the hardening and darkening of the epicuticleat the time of ecdysis and the relation between these changes and the waterproofingof the cuticle.

METHODS AND MATERIAL

Improved methods of lipid staining for the light microscope have consisted in the use ofSudan B, 0-25%, in 50% pyridine, applied too$-i-fim sections cut in agar and ester wax(Wigglesworth, 1959) after fixation in osmium tetroxide; and with graded exposure to sodium

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460 V. B. Wigglestoorth

hypochlorite to unmask the lipid (Wigglesworth, 1971). Osmium fixation followed by 'pro-gallin A' (ethyl gallate) (Wigglesworth, 1957) has also been used.

For the demonstration of lipid in the electron microscope the method recently described(Wigglesworth, 19750) has been used. This consists in fixation in osmium tetroxide followed byglutaraldehyde, or the mixture of these two fixatives according to Hinde (1971), partition in70% ethanol saturated with myrcene (0-5-1%) containing o-i% ethyl gallate, followed by re-newed osmium treatment and embedding in the medium of Spurr (1969).

All observations have been made on the abdominal cuticle of the 4th-stage larva of Miodniusand the newly moulted 5th instar.

RESULTS

Initial deposition of the epicuticle

The deposition of the epicuticle as seen in the electron microscope has been de-scribed in a number of insects, notably Calpodes (Locke, 1961, 1966, 1969), Tenebrio(Delachambre, 1970, 1971) and Hyalophera (Greenstein, 1972). The stages seem to bethe same in all insects and have been confirmed on the abdomen of the Rhodnius larva.The stages are set out briefly below with notes on the role of lipids in the process.

(i) Prominent slender microvilli form over the surface of the epidermal cells soonafter apolysis, first along the intercellular boundaries (as noted by Delachambre(1970) in Tenebrio), later everywhere (Fig. 2).

(ii) A continuous membrane, resembling a plasma membrane, appears at the levelof the tips of the microvilli (Figs. 3, 4).

(iii) A diffuse, lipid-rich zone appears over the apex of each microvillus (Fig. 4).(iv) In standard electron micrographs a clear central zone appears in this dark

deposit, with darkly staining walls above and below, to form a characteristic curvedplaque over each microvillus (see Wigglesworth, 1973, fig. 14). But after the myrcene-osmium procedure the plaques remain uniformly black throughout.

(v) These plaques extend laterally and fuse to form a continuous epicuticle some17 nm thick, which again appears uniformly black in the myrcene-osmium prepara-tions (Fig. 5). This is termed the outer epicuticle ('cuticulin' layer of Locke (1966)).

During stages (iv) and (v) there is a prodigious increase in the extent of the plasmamembrane, to cover the microvilli and to allow for expansion of the intercellularmembranes. Small vesicles with thicklipid-rich walls, presumably derived from the Golgibodies, move up to the apex of the cell and fuse with the plasma membranes (Fig. 6).

(vi) The microvilli then proceed to add the inner epicuticle (the ' protein epicuticle'or 'dense layer' of Locke, 1966). They commonly show lipid-rich tips often connectedby a continuous membrane. The inner epicuticle finally reaches a thickness of about0-3-0-4/im; the epidermal cells then turn over to the formation of fibrillar, lamellatecuticle (Wigglesworth, 19756).

During the period of epicuticle deposition numerous lipid droplets move up towardthe apex of the cell, and are incorporated into the cuticle by way of the microvilli(Figs. 7, 8) (cf. Locke, 1966). At this time great numbers of microtubules run towardthe epicuticle (cf. Wigglesworth, 1973), and are presumably engaged in the transport ofmaterial to the microvilli (Locke, 1969). But microtubules seem to be lipid free and arealmost invisible in the myrcene-osmium preparations.

Lipid in the epicuticle of Rhodnius 461

In the sockets at the base of the setae, the inner epicuticle is greatly thickened. Thewall of the seta is also to be regarded as a thickened epicuticle - although it does con-tain some chitin in its inner parts (Wigglesworth, 1933). Consequently the tormogenand trichogen cells, which lay down respectively the socket and the seta, show thesame cytological changes as the other epidermal cells show during epicuticle forma-tion, but in greatly exaggerated form (cf. Locke, 1969). In both tormogen and tricho-gen cells, microtubules are exceedingly abundant and rows of lipid droplets move upbetween them on their way from the Golgi bodies (and perhaps from the breakdown ofmitochondria) to the developing epicuticle. The tormogen and trichogen cells are con-spicuous in light-microscope sections by reason of their high lipid content (Figs. 11,12). Fig. 13 shows the appearance of their lipid-rich cytoplasm in the electron micro-scope. Lipid is also conspicuous in the fibrous rods which are a familiar feature at theperiphery of developing scales and setae (Paweletz & Schlote, 1964; Overton, 1967)and are later incorporated into the setal wall (Figs. 9, 10).

Lipid in the epicuticle

When the outer epicuticle is newly formed its lipid content is readily demonstrated.Sections cut in esterwax, given mild treatment with sodium hypochlorite (1:20 dilu-tion of the stock 10% solution for 1 min) results in intense black Sudan B staining

(Fig- H)-Later, when the inner epicuticle has formed, the same treatment again leads to

intense staining of the outer epicuticle, but the inner epicuticle is hardly stained. How-ever, if exposed to longer treatment by sodium hypochlorite (1:2O dilution for 4 min)the outer epicuticle may be completely dispersed, the outer portions of the innerepicuticle are again unstained, but the inner parts are intensely stained. Indeed, as thehypochlorite treatment is prolonged the epicuticle is seen to be rich in lipid through-out its entire thickness (Fig. 15). These observations confirm conclusions reached byobserving the breakdown of the epicuticle in chlorated nitric acid with the liberation oflipid droplets, so that the inner epicuticle consists of tyrosine-rich protein intimatelyassociated with lipid (Wigglesworth, 1933). Lipid is plentiful also immediately belowthe inner epicuticle.

The epicuticular pores

The first evidence of the existence of pores in the epicuticle was obtained when asmall insect, such as a flea or a louse, was immersed in liquid paraffin or olive oil andobserved under the microscope in the fresh state. Minute droplets of water exudedfrom the surface of the cuticle; most rapidly over the soft cuticle at the joints of thelimbs, etc., but also over hard cuticle. It was inferred that invisible water-containingpores must come very close to the surface and that the crowding of polar lipid mole-cules, present at the surface of the cuticle, into the interface between oil and water,provided the energy for the formation of the water droplets (Wigglesworth, 1942).

The first visual evidence of such epicuticular pores was obtained by stripping awaythe remains of the old cuticle in Rhodnius a day before ecdysis and immersing the in-sect in ammoniacal silver oxide (Wigglesworth, 1947). Fig. 16 shows a vertical section

462 V. B. Wigglesworth

through the new cuticle so treated. Numerous silver-stained channels run through theepicuticle to end in a mass of reduced silver deposit below. These silver deposits, ofcourse, greatly exaggerate the apparent width of the channels.

The pores are readily seen in sections of cuticle prepared to visualize lipids in theelectron microscope. As described in the paper on the lamellate cuticle (Wiggles-worth, 19756) the pore canals in the Rhodnius larva are cylindrical structures with aclear lumen and with lipid-rich walls. They contain an axial filament (as described byLocke (1961)) but this does not seem to be particularly rich in lipid.

The pore canals extend up to the base of the epicuticle. Here they narrow, theaxial filament disappears and the lipid walls become approximated. Sections in thelight microscope stained with Sudan black B after sodium hypochlorite treatment showa conical termination of the pore canal as it comes to the epicuticle; and a faint verticalstriation of the epicuticle suggests the presence of fine channels.

This appearance is confirmed in electron-microscope sections prepared by themyrcene partition technique (Figs. 17-19). The channels appear as darkly stainingthreads about 20-25 nm in diameter. As seen in tangential section (Figs. 20, 21), theelectron-dense centre is surrounded by an electron-lucent zone which separates itfrom the moderate lipid staining of the inner epicuticle. The appearance of the innerepicuticle in Fig. 18 recalls the 'feltwork' of fine lipid filaments figured by Locke(1961) in Tenebrio; but I have been inclined to regard this appearance as an artifact ofcutting this hard cuticle.

At no stage do the channels in the epicuticle, in the myrcene—osmium preparations,have a clear lumen; they are always filled with osmiophilic material. But the rapid entryof ammoniacal silver shows that their contents are readily permeable. These channelspresumably furnish the connexion between the epidermal cells, the epicuticle and themoulting fluid; and thus provide for the discharge of protease and chitinase for thedigestion of the inner layers of the old cuticle (Wigglesworth, 1933), for the absorptionof the products of digestion, for the supply to the epicuticle of the substrates and/orenzymes necessary for sclerotization and melanin formation, and for the exudation ofthe lipid and other components of the wax layer.

Although the epicuticular channels always appear black after myrcene and osmiumtreatment, if the cuticle is fixed in glutaraldehyde alone, followed by dilute brominewater, to eliminate olefinic double bonds reacting with osmium tetroxide, and thesections stained with lead citrate, the channels appear colourless with darkly stainingwalls (Fig. 22), sometimes with a fine axial filament (cf. Locke, 1969, fig. 2).

Silver reduction as a guide to wax secretion

It is generally believed that the waterproofing of the insect is due to a layer of waxsecreted on to the surface of the epicuticle (Wigglesworth, 1945; Beament, 1945). Itwas noted that synthetic sapphire dust (crystalline alumina) induced a great increase intranspiration through the cuticle - but, in the case of Rhodnius, only if the insectmoved in contact with it. No microscopic evidence of abrasion of the cuticle could beseen. But since the insect cuticle had long been known to be rich in polyphenolicmaterial (Schmalfuss, Heider & Winkelmann, 1933) the insect, after walking on


dusted filter paper, was immersed in silver oxide solution. The crests of the folds andthe domes of the plaques, where the moving surface had come into contact with thedust, showed a deep brown localized stain (Wigglesworth, 1945) (Fig. 26).

The same procedure was adopted to study the development of impermeability inthe epicuticle at the time of ecdysis. Exposure of the new cuticle by stripping away theold cuticle at 2 days before ecdysis, and immersion in silver oxide, revealed finesilver-staining channels in the epicuticle with blackened material exuding from theirendings at the surface (Fig. 27). During the next 24 h silver-reducing material spreadradially from the distal extremities of these channels to form minute brown spots,each with a black centre (Fig. 23). Ultimately the spots arising from adjacent channelsfused to form a more or less continuous layer (Fig. 24). Later still, the non-reducingareas reappeared and gradually spread until, shortly before ecdysis, the silver-bindingmaterial was almost wholly covered (Fig. 25).

Finally, as the last remnants of the old endocuticle were digested, and absorbedthrough the new cuticle along with the residue of the moulting fluid, the space betweenthe old and new cuticle became dry and filled with air. Ecdysis then occurred and re-vealed the new cuticle surface as completely dry and waxy, wholly unwettable bywater, and giving no reaction when immersed in silver oxide solution. On the otherhand brief immersion in chloroform removed the wax, and the silver-reducing pro-perties throughout the epicuticle were again revealed.

In the original account of these observations the silver-reducing substance wasloosely referred to as probably 'protein material, very rich in dihydroxyphenols,which is tanned when the phenols are oxidized in part to quinones' (Wigglesworth,1947). But if the test for polyphenols is critically applied (immersion in silver oxide inthe dark for 30 min only, and washing out with sodium thiosulphate) the reaction doesnot become weakly positive until just before ecdysis and does not become stronglypositive until 24 h later (Figs. 28, 29).

In the original account the silver-binding material was believed to be spreading overthe surface of the epicuticle. As we shall see later, that is indeed correct; but sectionsexamined in the electron microscope show that when the silver staining spreads radiallyfrom each epicuticular channel, so as to form in surface view an amber brown spot witha black centre (the central channel) the greater part of the deposit is found to benot on the surface of the cuticle but within the substance of the inner epicuticle(Figs. 30-33).

In vertical sections the silver deposit can be seen to extend down the central channel.It appears to enter the inner epicuticle only at a point just below the outer epicuticle,thus leaving a narrow clear zone, investing the central channel which is not invaded bythe silver-staining material (Figs. 1, 35). This clear zone corresponds with the electron-lucent zone which is seen around the epicuticular channels in tangential sections of theepicuticle (Figs. 20, 21). It is of interest to note the slight dilatation of the epicuticularchannels just below the outer epicuticle after myrcene-osmium staining, as seen inFig. 18. This material spreads radially, but as it approaches the material spreadingfrom adjacent channels there is commonly a narrow clear space partially separating theterritories (Figs. 33, 35). It gradually enters the deeper part of the inner epicuticle, but

464 V. B. Wigglestoorth

does not usually occupy more than two thirds of the total thickness. It does not invadethe outer epicuticle. Some of the material is exuded on to the surface of the epicuticle(Fig. 30). Sometimes the silver oxide will spread down the epicuticular channels to theinner limit of the epicuticle and here it may form more extensive deposits of reducedsilver in the endings of the pore canals (Figs. 32, 33). Sometimes it will spread far intothe pore canals (Fig. 38).

,1

N,

b

a —

Fig. 1. Idealized drawing of epicuticle showing the entry and distribution of silver-binding material from the pore canal to the inner epicuticle and to the cuticle surface.a, lamellate endocuticle; b, inner epicuticle; c, outer epicuticle with no silver im-pregnation ; d, exposed layer of silver-binding material; e, exudation of the same fromthe epicuticular pore; / , epicuticular channel surrounded by clear unimpregnatedzone: g, silver-binding material spreading through substance of inner epicuticle;h, pore canal.

The foregoing description applies to the ordinary stellate cuticle of the abdomen.Over the smooth cuticle of the plaques the epicuticle is thicker, the thickness of thesilver-binding substance is greater and the pattern of reduced silver somewhat different(Fig. 36). Over the future black pigment spots, the material does not extend deeplyinto the epicuticle, but it forms a dense continuous layer below the outer epicuticle(Fig. 34). At the sites of insertion of the dorso-ventral muscles the material extends farmore deeply, in a tongue-like manner, and may even spread some distance into thelamellate endocuticle (Figs. 37, 56).

The chemical significance of these changes, as in all histological effects of silver, willnot be easy to interpret precisely. But they serve to illustrate the route by whichsecretory products of the epidermal cells can reach the epicuticle; and for practicalpurposes, they furnish a means of determining the presence or absence of the water-proofing wax layer.


The 'wax layer'

Although the so-called wax layer was believed to have a substantial thickness of some250 nm, as calculated from the amount of wax extractable from a known area ofcuticle (Beament, 1945) this layer cannot normally be seen in histological preparations;its location is commonly inferred from the occurrence of a space between the epicuticleand the cement layer (Locke, 1966; Gluud, 1968). The existence of a wax layer wasoriginally inferred from the elimination of waterproofing by raising the temperatureabove a critical level, or by very mild abrasion (Wigglesworth, 1945); from the agree-ment between the critical temperature in the waterproofing of artificial membranes bythe isolated waxes and that observed in the insects from whose cuticle they had beenextracted (Beament, 1945; Wigglesworth, 1945); and from the fact that the initialwaterproofing of the new cuticle, which develops immediately before ecdysis, co-incides with the formation of a waxy impermeable surface on the new cuticle, that iseliminated by brief extraction with chloroform (Wigglesworth, 1947). The wax layerwas pictured as a persistent lamina covered after ecdysis by the cement layer.

In studying the epicuticle of the cockroach Periplaneta, it became evident that thecement layer served as a sponge in which the soft waterproofing grease is held (Kramer& Wigglesworth, 1950). It was suggested that the idea of an epicuticle made up ofsuccessive discrete laminae was over-simplified and that in other insects also, theoutermost layers of the cuticle might be permeated by wax. It had been observedthat although the surface of the newly moulted Rhodnius larva becomes relativelyhydrophil when the cement layer is poured out, it later becomes more hydrophobe again(Wigglesworth, 1947).

There are certain difficulties about investigating the secretion of the wax layer.During the final 6-12 h before ecdysis many active processes are going forward in thecuticle, simultaneously or in rapid succession. The inner layers of the lamellate cuticleare still being added. The changes that prelude the hardening and darkening of thecuticle entail the introduction into the epicuticle of substrates for the sclerotizingenzymes. (It is generally supposed that phenoloxidases and other enzymes needed arealready in position in the epicuticle.) The last remnants of the old endocuticle are beingdigested, so that the moulting fluid contains partially digested protein and chitin withsome lipid droplets in suspension.

By this time, perhaps, the discharge of protease and chitinase has ended; but ab-sorption of the moulting fluid and the products of digestion must be exceedinglyactive. It is unlikely that these processes of secretion and absorption take place throughthe substance of the epicuticle; they must surely be occurring by way of the permeablechannels connected with the pore canals. It is these same channels which must conveyto the surface the lipids and other materials that compose the wax layer whose forma-tion is the final act before ecdysis. It is indeed common for the contents of the porecanals, particularly in their distal parts, to stain darkly after the osmium-myrceneprocedure during the period immediately before and after ecdysis (Figs. 43, 44). It isto be noted in Fig. 44 that in the pore canals seen in cross-section, the axial filamentappears pale in contrast with the deeper staining of the lipid contents.


We have seen that both the outer and inner epicuticle are rich in lipid from theearliest stages of their formation, and although more osmium-binding material seems toappear at about the time of ecdysis, no good evidence of the timing of the secretoryprocess has emerged from electron-microscopic studies. Well before ecdysis occurs theouter epicuticle appears to have undergone some hardening process which tends toprevent adhesion between its surface and the embedding plastic. It is now so highlyrefractile that, in the absence of any staining, it appears as a dense black line in opticalsection in the light microscope when, for example, the stellate cuticle is observed insurface view (Fig. 45). It is important to distinguish this appearance from genuinelipid staining.

Lipid secretion

During the hours immediately preceding ecdysis, the old cuticle becomes ex-ceedingly thin, the moulting fluid is absorbed, and finally the space below the oldcuticle becomes dry and filled with air. The old cuticle has been stripped away duringthese late stages; the intact insect has been fixed briefly in 5% glutaraldehyde, stainedin Sudan B and the new cuticle separated and mounted whole in surface view. Lipidsecretion leading to the formation of the wax layer is a rapid process; a large number ofpreparations have been needed in order to demonstrate a range of intermediate stages.

At the earliest stage there is no sign of lipid staining of the surface. At the peak level,the epicuticle is covered by a blue-grey film which is continuous over the entire sur-face (Fig. 52) including the surface of the setae. Finally, just before ecdysis the hydro-phobe surface no longer stains with Sudan B (or stains very faintly indeed).

Figs. 46, 47 show an early stage (at 2 levels of focus) in which minute droplets oflipid are exuding from the epicuticular channels in the stellate cuticle. In Fig. 48 thedroplets are enlarging. In Fig. 50 they are seen fusing over the crests of the stars.Fig. 49 shows similar stages over the points of insertion of the dorso-ventral muscles.The insertion of each muscle fibre forms a truncated cylinder (cf. Fig. 56). To the leftof Fig. 49 droplets of lipid are spreading over the upper surface of the cylinders; to theright and above, at a slightly lower focus, the spreading droplets can be seen in opticalsection at points on the sides of the cylinders. In Fig. 51 there is extensive spreadingof lipid over the stellate cuticle, sometimes forming thicker deposits in the narrowfolds. Fig. 52 shows continuous diffuse lipid staining all over the surface.

The presence of such a lipid-containing membrane can be confirmed in sections ofthe cuticle, a day or so before ecdysis, stained with Sudan B after dilute hypochlorite,or by the myrcene-osmium procedure: it takes the form of a detached membraneforming a boundary to the residue of the moulting fluid, and showing definite lipidstaining (Fig. 53).

Hardening of the lipid

But it is not lipid alone which is being exuded upon the surface (Fig. 30). Prepara-tions made by stripping away the old cuticle at the height of production of the silver-binding material, exposing for 1 h in 5%ammoniacal silver and fixing in Carnoy (cf.Fig. 24) and cutting in vertical section, show silver-binding material forming a dense


layer adherent to the epicuticle. (This is the material formerly called the 'polyphenollayer'.) In some preparations the layer is detached from the surface and appears as acolourless membrane packed with silver granules (Fig. 54).

We saw that by the time the cuticle reaches the 'dry' stage its reactivity to directsurface exposure in ammoniacal silver is limited to scattered spots (see Fig. 25).Sections of the cuticle at this stage show that each of these brown silver-staining spotsis coated by a thin detachable membrane blackened by a granular silver precipitate(Figs. 39, 40). Beyond the limits of each silver-stained spot of epicuticle this mem-brane is now colourless and invisible.

In the electron microscope (Figs. 41, 42) it can again be seen that the outer epicuticleforms a silver-free barrier between the silver deposit in the inner epicuticle and a densecontinuous layer of silver on the outer surface of the cuticle. (The irregular silvergranules lying above this layer probably just represent diffusion of reactive materialinto the ammoniacal silver solution.)

It is clearly the newly acquired impermeability of the silver-binding membranewhich prevents the reaction of the epicuticle with ammoniacal silver by blocking theaccess of the silver solution to the epicuticular channels. Fig. 41^ indicates this im-permeable layer, almost free of silver, which has become separated from the surface.This gives a truer picture of the tenuous nature of this impermeable membrane.

Is the impermeability of this membrane the result of sclerotization of a protein con-stituent? Or is it due to the hardening of the lipid to form a solid wax? The fact thatpermeability is at once restored by brief contact with chloroform (Fig. 38) stronglysuggests that it is the change in the lipid component that is responsible.

The nature of the material responsible for the silver precipitate in this membrane isan open question. It could be a protein rich in carboxylic or phenolic groups, or anacid mucopolysaccharide; fatty acids or acid phosphatides would presumably be ex-tractable by chloroform. Chloride is not responsible: these silver precipitates do notform with silver nitrate in 1 N nitric acid. The silver-binding granules visible in theepidermal cells in some preparations (Fig. 38) consist of uric acid and pteridines.

It is this lipid-impregnated membrane, responsible for the hydrophobe surface ofthe insect at the moment of ecdysis, its wax extractable at this time by brief exposure tochloroform at room temperature, which has been called the 'wax layer'. When thelipid is first exuded on the surface of the epicuticle it stains readily with Sudan B; butwhen hardened and incorporated in the non-lipid membrane, it no longer stains inthis way.

The dermal glands and the cement layer

At the moment of ecdysis the dermal glands of type B* are tensely distended withsecretion. It was claimed (Wigglesworth, 1947) that this secretion gave rise to a'cement layer' which forms a protective cover for the wax. The evidence was asfollows. The dermal glands are still distended with secretion immediately after ecdysis;

• There are two types of dermal glands in the Rhodnius larva (Wigglesworth, 1933)- By farthe most numerous (type B) has a large cuticle-lined vesicle which becomes distended withsecretion. Type A has vacuoles distributed throughout the glandular cell.


the vesicles are being emptied within half an hour, and almost all have collapsed with-in an hour after moulting. At the moment of ecdysis the surface of the cuticle is com-pletely non-wettable. When the dermal glands discharge their contents, droplets ofwater readily adhere to the surface.

After publishing a paper on the connective tissues of Rhodnius (Wigglesworth,1956) I stained some sections with alcian blue (unpublished observations) and notedthat this was the only stain I had used which was actively taken up by the contents ofthe dermal glands. Alcian blue also stained the cement layer (Figs. 57, 58), the base-ment membrane and (less strongly) the inner layers of the old cuticle in process ofdigestion, including that of the tracheae.

If the larva, an hour or so after moulting, is immersed intact for 5 min in alcian blue(o-i% in 1% acetic acid) the dye being stabilized in 60% ethanol made alkaline withammonia, and the cuticle is then dissected off, mounted whole and observed in sur-face view, the recently discharged blue-green secretion is most conspicuous around theopenings of the ducts of the dermal glands; but it spreads out from these points toform a delicate coating over the entire cuticle (Fig. 59). These observations confirm theorigin of the cement layer from the dermal glands.

The cement layer, as a whole, is not soluble in lipid solvents; nor is it digested bypepsin-HCl or by trypsin (Wigglesworth, 1947). It gives a positive argentaffine reac-tion (Fig. 28). Baldwin & Salthouse (1959) who described the alcian blue staining ofthe secretory product in the vesicles of the dermal glands in Rhodnius, regarded thissecretion as being a mucus which serves for lubrication during ecdysis; they showedthat it was digested by hyaluronidase. Hyaluronic acid, or something like it, seems tobe widespread in insect secretions (Estes & Faust, 1964).

The cement layer is poured out over the 'wax layer', which is itself a compound oflipid and non-lipid components. The question arises whether the non-lipid elementsin the wax layer have been contributed by the dermal glands before ecdysis. Thiscannot be altogether excluded (it has been claimed to be the case in Tenebrio (Wiggles-worth, 1948)) but in Rhodnius it seems unlikely because the 'wax layer', unlike thecement layer, stains only very faintly with alcian blue and when first secreted it reactsdirectly with ammoniacal silver which the cement layer does not.

The cement layer does not extend over the surface of the setae. But, as we haveseen, the 'wax layer' does; and if the intact 5th-stage larva, some days after ecdysis, isimmersed briefly in sodium hypochlorite (1:20 dilution, 2 min) before staining withSudan B, a delicate membrane, staining with the lipid dye, or breaking down toliberate minute black droplets, can be seen extending over the whole surface of thecuticle, including the setae (Fig. 60).

It was shown in earlier work (Wigglesworth, 1945) that the wax is very readilyremoved from the surface of the newly moulted Rhodnius larva by brief immersion inchloroform at room temperature; and this is true even several hours after ecdysiswhen the cement layer is already formed. As the cuticle hardens and darkens so thecement layer becomes more resistant to extraction of the wax; this is effective only at50-60 °C.

That would imply that the hardening of the cement is similar to the sclerotization of


the cuticle. But later experiments (Wigglesworth, 1947) showed that the two pro-cesses were independent: for exposure to coal gas for 4-6 h causes a permanent arrestof the discharge (or of the hardening) of the dermal gland secretion, and the silver-binding structures are exposed by brief extraction with chloroform at room tempera-ture; whereas cuticle hardening and darkening go forward normally on restoration0 the air. On the other hand, if the insect is allowed three quarters of an hour after

ecdysis for discharge of the cement layer and is then exposed to hydrogen cyanide for24 h, darkening of the cuticle is arrested, but the dermal gland secretion has hardenedas usual, and chloroform at room temperature for 5 min leads to hardly any exposure ofthe silver-reducing epicuticle. These results would suggest that the hardening of thecement is a different process from sclerotization of the cuticle.

In electron-microscope sections of the cuticle after the myrcene-osmium procedurethe combined 'wax layer' and 'cement layer' appears as a weakly staining membranewith an irregular surface; there is a very thin inner boundary which shows intensestaining and sometimes another thin stained layer on the outer surface (Figs. 61, 62).This appearance agrees closely with that seen in sections of the integument of variousHemiptera as described by Gluud (1968). (Gluud follows Locke (1966) in regardingthe inner boundary as a 'monomolecular lipid layer'.)

Abrasion and repair of the wax layer

Gentle abrasion of the cuticle in Rhodnius by rubbing with synthetic sapphire dustleads to a great increase in transpiration, such that in a dry atmosphere the recently fed5th-stage larva is completely dried up and dead in less than 24 h. It was noted that ifthe insect was kept in a humid atmosphere, about 80% of the extra transpiration waseliminated within 24 h. Repair must therefore be a rapid process (Wigglesworth,1945). Larvae abraded in this way with Linde B powder at one day after feeding havenow been studied by the use of ammoniacal silver (without thiosulphate extraction)combined with alcian blue and Sudan B staining.

Abrasion removes the alcian blue-staining 'cement layer' over the domes of theplaques and the crests of the stellate folds of the cuticle, and this layer is not replaced.The silver-binding material in the epicuticle is exposed over the same areas; but with-in 24 h silver staining had almost disappeared. Where the abrasion had injured thesurface of the epicuticle, there was an amber-brown coloration of the cuticle withoutexposure to silver oxide. This darkening of gross abrasions serves to restore the im-permeability of the cuticle to methylene blue (Lai-Fook, 1966).

Within 3 days after abrasion a waxy bloom appears on the abraded area. Sudan Bstaining shows deposits of solid wax which is virtually unstained but which containsnumerous minute droplets that stain quite strongly. Although waterproofing has forthe most part been restored, lipid secretion continues. After abrading the cuticle it iscustomary to wash away most of the adhering dust in a jet of water; but it was noted(Wigglesworth, 1945) that if the dust is left in contact with the cuticle after abrasion,the restoration of waterproofing is long delayed. This was ascribed to the newlysecreted lipid being adsorbed by the dust.

Fig. 63 shows a section of the normal unabraded cuticle of a 4th-stage larva, fixed

47© V. B. Wigglesworth

with glutaraldehyde, followed by myrcene partition and osmium treatment, showingthe combined wax and cement layer detached from the surface. Fig. 64 shows a sectionof cuticle from another area of the same insect which had been abraded 3 days before,similarly treated. The wax forms an irregular deposit in the folds of a detached mem-brane. This membrane is quite without interruption; whereas the original cementlayer had been destroyed over all the prominent folds. It is therefore a new structure.

Figs. 65 and 66 show sections of this membrane and the over-lying wax in theelectron microscope. The membrane is thicker than the normal cement layer (cf.Figs. 61, 62). After fixation in osmium tetroxide followed by myrcene partition andrenewed osmium, it is for the most part electron lucent, but on its lower surface thereis always a lipid-staining layer. This membrane is presumably the new waterproofinglayer; it does not stain with Sudan B; it may be supposed to consist of wax incorporatedinto a carrier protein, and to correspond with the compound ' wax layer' formed beforeecdysis. The reason for the continued accumulation of free wax is not known.

DISCUSSION

A brief account of the initial deposition of the epicuticle, as observed with theelectron microscope after the myrcene partition procedure for the visualization oflipids, has confirmed the incorporation of lipid into both the outer and inner epicuticle.And the breakdown of the newly deposited epicuticle by exposure to sodium hypo-chlorite has likewise confirmed the presence of bound lipid in both epicuticular layers.

This paper has been mainly concerned with the functional connexion between theepidermal cells and the remote epicuticle at the time of ecdysis.

Epicuticular channels as the means of access

The fine extensions from the pore canals, running vertically through the innerepicuticle and piercing the outer epicuticle, have been re-investigated. These channelswere first detected in Rhodnius by the entry along them of silver oxide applied to thesurface of the new cuticle exposed before ecdysis, and the penetration of the silver intothe pore canals (Wigglesworth, 1947). Their presence was confirmed by the samemethods in Tenebrio (Wigglesworth, 1948), in Periplaneta (Kramer & Wigglesworth,1950) and in caterpillars (Way, 1950; Takahashi, 1956).

The epicuticular channels have now been revealed in the electron microscope by themyrcene-osmium procedure, after which the contents show up as solid black threads(20-25 nm diameter) each surrounded in cross-section by a halo of non-stainingcuticle, giving an overall diameter of about 50 nm. After glutaraldehyde fixation andexposure to bromine water, followed by lead citrate staining, they appear as clearchannels with darkly staining walls, and sometimes with a fine axial filament.

The epicuticular channels are presumably concerned in the discharge and re-absorption of the moulting fluid and of the products of digestion of the old endocuticle.But they are involved also in the transfer of diphenols to the epicuticle. Argentaffincomponents in the epicuticle are barely detectable at ecdysis. They increase greatly,particularly in the melanized regions of the epicuticle, during the next 24 h. But it

Lipid in the epicuticle of Rhodnius

seems probable that the argentafRn diphenols are derivatives of tyrosine and otherprecursors introduced into the epicuticle in the last day or two before ecdysis.

It had been shown in the past (Wigglesworth, 1947) that silver-binding materialwas transferred to the epicuticle shortly before ecdysis; such material, wrongly be-lieved to contain diphenols, was described as spreading from the epicuticular channelsover the surface of the epicuticle. By the use of the electron microscope it has now beenshown that most of the silver-binding precursors spread radially from a point in thecuticular channels just below the outer epicuticle, not over the surface, but impreg-nating the substance of the inner epicuticle; a very thin layer only is deposited on thesurface. This interpretation was in fact suggested by Dennell & Malek (1955) inPeriplaneta and by Takahashi (1956) in Bombyx morihrva; Way (1950) had been unableto observe any discharge of silver-reducing material on the surface of the cuticle inDiataraxia (Lep.).

The course and pattern of spreading, as determined by the application of silveroxide to the exposed surface of the cuticle, varies in the different types of epicuticle(plaques, stellate cuticle, pigment spots, etc.). In the case of the dorso-ventral muscleinsertions it may extend into the outer layers of the fibrous endocuticle. The close re-lation between this distribution and the subsequent hardening and blackening of theinner epicuticle suggests that the silver-binding material is concerned in the sclerotiza-tion process. Towards the time of ecdysis there is commonly a massive transfer oftyrosine and its derivatives into the integument; for example in Drosophila (Mitchell,Weber-Tracy & Schaar, 1971) and Periplaneta (Wirtz & Hopkins, 1974).

In more recent years epicuticular channels have been shown to be widely distributedin insects. They were described by Locke (1961, 1964) under the name of 'wax fila-ments' or 'lipid-water liquid crystals' in Calpodes, Galleria, Tenebrio larva and Apis.Similar 'epicuticular filaments' were found by Noirot & Noirot-Timothe'e (1969) inthe rectal and peripheral cuticle of Isoptera, Dictyoptera, Phasmoptera, Neuroptera,Siphonaptera, Trichoptera, Hymenoptera and Coleoptera. And under the name of'pore tubules' they have been studied in wireworms, larvae of Elateridae (Col.), byZacharuk (1972).

It has long been known that the phenoloxidase enzymes are established in the integu-ment early in development, the substrates much later (Gortner, 1911). Likewise in thesclerotized cuticle of Schistocerca it is the supply of substrate and not the abundance ofenzyme which controls hardness (Andersen, 1974ft). ^n ^ne l a r v a of Calpodes, Locke &Krishnan (1971) showed that the phenoloxidases are laid down early in the outerepicuticle ('cuticulin layer'), in the inner epicuticle and in those parts of the fibrouscuticle that will be sclerotized or melanized. Polyphenols appear shortly beforeecdysis and are localized in and below the inner epicuticle and along the epicuticularfilaments - as described in the present paper for the silver-binding precursors inRhodnius (cf. Locke & Krishnan (1971), fig. 30, and Fig. 1 of this paper).

Deposition of the waterproofing wax

During the late stages of development before ecdysis the distal parts of the porecanals are often filled with lipid-rich material. When the old cuticle is almost fully


digested, the surface of the new cuticle becomes readily stainable with Sudan B:minute droplets of lipid are exuded and fuse with adjacent droplets to form a continu-ous lipid layer on the surface. As the space between the old and new cuticle becomesdry, the surface lipid becomes incorporated in the delicate non-lipid silver-bindingmembrane, likewise exuded at this time; it hardens and becomes non-stainablewith Sudan B; and the exposed surface becomes completely non-wettable. It is thislipid-impregnated membrane which is termed the 'wax layer'.

In Periplaneta, in which the waterproofing lipid is a soft grease, the free proto-catechuic -acid -and other 3,4-dihydricphenols in the cuticle, besides being precursorsof the o-quinone involved in tanning, serve also as antioxidants which normally pre-vent autoxidation and hardening of the unsaturated cuticle lipid (Atkinson, Brown &Gilby, 1973). It may be that the polyphenols in the pore system of Rhodnius are like-wise responsible for maintaining the fluidity of the lipid until it is freely exposed.

Cement layer

At the moment of ecdysis the dermal glands are tensely distended by their muco-polysaccharide secretion. Within half an hour or so the glands have emptied and theresultant membrane, staining intense blue-green with alcian blue, forms the continu-ous but somewhat uneven cement layer over the 'wax layer'. Subsequently the cementlayer becomes hardened and hydrophobe. Perhaps it too is impregnated with wax, likethe cement layer of Periplaneta (Kramer & Wigglesvvorth, 1950). All one can see is anirregular membrane, some 30—100 nm thick, bounded below by an intensely lipid-staining layer not more than 10 nm thick and often appearing much less (Figs. 61, 62).

Repair of abrasions

Abrasion of the surface of the cuticle with synthetic sapphire dust causes a greatincrease in transpiration. This defect is rapidly repaired and an amorphous deposit ofexcess wax sometimes appears on the surface. The alcian blue-staining cement layer isnot replaced. The new waterproofing membrane is a glassy structure, some ioo-i5onmthick, with a lipid-staining layer some 25-50 nm thick on its lower surface (Figs. 65,66).

At no time has it been possible to see a 'wax layer' of a thickness comparable withthe 0-25 /tm which has been calculated from wax extraction of the cast cuticle ofRhodnius (Beament, 1945). Presumably much of this wax must represent reserve waxextracted from the substance of the exuvia.

The nature of the non-lipid components of the 'wax layer' and of the thickenedglassy membrane that is formed during the repair of abrasions (which is perhaps of thesame nature) will require further study. As an initial suggestion, it might be con-sidered whether these structures represent colourless sclerotin of the type which iswidespread in insects (Andersen, 1974a). The positive argentaffin reaction of thecement layer (Fig. 28) is suggestive.


Extracuticular layers

There seems to be good agreement among authors on the layers which compose theinsect epicuticle: (i) an outer epicuticle (the 'resistant layer' of the epicuticle ofWigglesworth (1947), 'paraffin layer' of Dennell & Malek (1955), 'cuticulin' layer ofLocke (1961)) with a constant thickness around 10-12 nm, of unknown compositionbut rich in lipid. (ii) An inner epicuticle ('cuticulin layer' of Wigglesworth (1947),'dense layer' or 'protein epicuticle' of Locke (1961)), varying in thickness up toabout 1 /tm, consisting of highly tanned protein associated with abundant lipid. Bothepicuticular layers are traversed by the epicuticular channels.

On the surface of the epicuticle are deposits of secretion the homologies of which arestill uncertain: the proteinaceous 'polyphenol layer' and the 'wax layer' (Wiggles-worth, 1945, 1947); the 'surface monolayer' or 'oriented lipid layer' (Locke, 1966);the 'outer epicuticle' sensu novo (Filshie, 1970a); the 'superficial layer' (Filshie,19706); the 'cement layer' (Wigglesworth, 1947) = 'tectocuticle' (Richards, 1952).

In Rhodnius the silver-binding material exuded from the epicuticular channels(formerly referred to as the proteinaceous 'polyphenol layer') is shown in the presentpaper to become impregnated with lipid exuded from the pore canals. This compositelayer hardens to give a very thin hydrophobic structure referred to here as the 'waxlayer'. After ecdysis the wax layer is covered by the cement layer discharged from thedermal glands and consisting largely of mucopolysaccharide, later impregnated bymore wax. The resultant extracuticular covering can be resolved in the electron micro-scope into several layers, lipid staining being most evident along narrow lines aboveand below an electron-lucent zone. When dispersed by hypochlorite this compositesurface membrane releases free lipid.

This work has been supported by a grant from the Agricultural Research Council. I thankProfessor T. Weis-Fogh and Dr D. A. Parry for research facilities, Dr J. E. Treheme andDr Nancy J. Lane for the use of the electron microscope, and Miss L. S. Swales and Mr W. M.Lee for valued technical assistance.

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ATKINSON, P. W., BROWN, W. V. & GILBY, A. R. (1973). Autoxidation of insect cuticular lipids:stabilization of alkyl dienes by 3,4-dihydricphenols. Insect Biochem. 3, 103-112.

BALDWIN, W. F. & SALTHOUSE, T. N. (1959). Dermal glands and mucin in the moulting cycle ofRhodnius prolixus Stal. J. Insect Physiol. 3, 345-349.

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DENNELL, R. & MALEK, S. R. A. (1955). The cuticle of the cockroach Periplaneta. II . Theepicuticle. Proc. R. Soc. B 143, 239-257.

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ESTES, Z. E. & FAUST, R. M. (1964). Studies on the mucopolysaccharides of the greater waxmoth, Galleria mellonella (Linnaeus). Comp. Biochem. Physiol. 13, 443-452.

FILSHIE, B. K. (1970a). The resistance of epicuticular components of an insect to extractionwith lipid solvents. Tissue & Cell 2, 181-190.

FILSHIE, B. K. (19706). The fine structure and deposition of the larval cuticle of the sheepblowfly (Lucilia cuprina). Tissue & Cell 2, 479-498.

GLUUD, A. (1968). Zur Feinstruktur der Insectencuticula. Ein Beitrag zur Frage des Eigen-giftschutzes der Wanzencuticula. Zool.Jb. (Anat.) 85, 191-227.

GORTNER, R. A. (1911). Studies on melanin. IV. The origin of the pigment and the colorpattern in the elytra of the Colorado Potato Beetle (Leptinotarsa decemlineata Say). Am. Nat.45. 743-755-

GREENSTEIN, M. E. (1972). The ultrastructure of developing wings in the giant silkmoth,Hyalophora cecropia. I. Generalized epidermal cells. J. Morpli. 136, 1-22.

HINDE, R. (1971). The fine structure of the mycetome symbiotes of the aphids Brevicorynebrassicae, Myzus persicae, and Macrosiphum rosae. J. Insect Physiol. 17, 2035-2050.

KRAMER, S. & WIGGLESWORTH, V. B. (1950). The outer layers of the cuticle in the cockroachPeriplaneta americana and the function of the oenocytes. Q. Jl microsc. Set. 91, 63-72.

LAI-FOOK, J. (1966). The repair of wounds in the integument of insects. J. Insect Physiol. 12,195-226.

LOCKE, M. (1961). Pore canals and related structures in insect cuticle.^, biophys. biochem. Cytol.10, 589-618.

LOCKE, M. (1964). The structure and formation of the integument in insects. Physiology ofInsects, vol. 3 (ed. M. Rockstein), pp. 379—470. New York: Academic Press.

LOCKE, M. (1966). The structure and formation of the cuticulin layer in the epicuticle of aninsect, Calpodes ethlius (Lepidoptera, Hesperidae). J. Morpli. 118, 461-494.

LOCKE, M. (1969). The structure of an epidermal cell during the development of the proteinepicuticle and the uptake of molting fluid in an insect. J. Morpli. 127, 7-40.

LOCKE, M. & KRISHNAN, N. (1971). The distribution of phenoloxidases and polyphenolsduring cuticle formation. Tissue & Cell 3, 103-126.

MITCHELL, H. K., WEBER-TRACY, U. M. & SCHAAR, G. (1971). Aspects of cuticle formation inDrosophila melanogaster. J. exp. Zool. 176, 429-444.

NoiROT, C. & NOIROT-TIMOTHEE, C. (1969). La cuticule proctode'ale des insectes. I. Ultra-structure compared. Z. Zellforsch. mikrosk. Anat. 101, 477-509.

OVHRTON, J. (1967). The fine structure of developing bristles in wild type and mutant Droso-phila melanogaster. J. Morph. 122, 367-380.

PAWELETZ, N. & SCHLOTE, F. W. (1964). Die Entwicklung der Schmetterlingsschuppe beiEphestia kuhniella Zeller. Z. Zellforsch. mikrosk. Anat. 63, 840-870.

RICHARDS, A. G. (1952). The Integument of Arthropods. Minneapolis: University of MinnesotaPress. 411 pp.

SCHMALFUSS, H., HEIDER, A. & WINKELMANN, K. (1933). 3, 4-Dioxyphenylessigsaure, Farb-vorstufe der Flugeldecken des Mehlkafers, Tenebrio molitor L. Biochem. Z. 257, 188-193.

SPURR, A. R. (1969). A low viscosity epoxy resin embedding medium for electron microscopy.J. Ultrastruct. Res. 26, 31-43.

TAKAHASHI, Y. (1956). Studies on the cuticle of the silkworm, Bovibyx mori L. VIII . Sublayersof epicuticle. Annotnes zool. jap. 29, 187-195.

WAY, M. J. (1950). The structure and development of the larval cuticle of Diataraxia oleracea(Lepidoptera). Q. Jl microsc. Sci. 91, 145-182.

WIGGLESWORTH, V. B. (1933). The physiology of the cuticle and of ecdysis in Rliodnius pro-lixus (Triatomidae, Hemiptera); with special reference to the function of the oenocytes andof the dermal glands. Q. Jl microsc. Sci. 76, 269-318.

WIGGLESWORTH, V. B. (1942). Some notes on the integument of insects in relation to the entry ofcontact insecticides. Bidl. ent. Res. 33, 205-218.

WIGGLESWORTH, V. B. (1945). Transpiration through the cuticle of insects. J. exp. Biol. 21,97-114.

WIGGLESWORTH, V. B. (1947). The epicuticle in an insect, Rhodnius prolixus (Hemiptera).Proc. R. Soc. B 134, 163-181.

WIGGLESWORTH, V. B. (1948). The structure and deposition of the cuticle in the adult meal-worm, Tenebrio molitor L. (Coleoptera). Q. Jl microsc. Sci. 89, 197-217.


WIGCLESWORTH, V. B. (1956). The haemocytes and connective tissue formation in an insect,Rhodnius prolixus (Hemiptera). Q. jfl microsc. Sci. 97, 89-98.

WICGLESWORTH, V. B. (1957). The use of osmium in the fixation and staining of tissues. Proc.R. Soc. B 147, 185-199.

WIGCLESWORTH, V. B. (1959). A simple method for cutting sections in the 0 5 to 1 fi range, andfor sections of chitin. Q. Jl microsc. Sci. 100, 315-320.

WIGGLESWORTH, 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). The role of the epidermal cells in moulding the surface pattern ofthe cuticle in RJiodnius (Hemiptera). J. Cell Sci. 12, 683-705.

WIGCLESWORTH, V. B. (1975a). Lipid staining for the electron microscope: a new method.J. Cell Sci. 19, 425-437.

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(Received 29 May 1975)


Fig. 2. 4th-stage larva at 6 days after feeding; numerous slender microvilli at apex ofepidermal cells, x 26000.Fig. 3. At 7 days after feeding. Delicate membrane connects tips of microvilli.x 26000.

Fig. 4. At 7 days after feeding. Lipid deposit over apex of microvilli. x 26000.Fig. 5. At 8 days after feeding; a, outer epicuticle formed as continuous lipid-staininglayer, x 40000.

Fig. 6. 7 days after feeding; thick-walled vesicles becoming incorporated into plasmamembranes (arrows). Abundant mitochondria, x 26000.Fig.r7-r9 days after feeding. Numerous lipid or lipoprotein droplets being incorporatedinto the inner epicuticle, by way of the microvilli. x 20000.Fig. 8. As Fig. 7. x 20000.Fig. 9. Longitudinal section of surface of developing seta showing fibrous rods rich inlipid. x 26000.Fig. 10. Transverse section of developing seta with lipid-rich rods below epicuticle.x 26000.Fig. 11. Longitudinal section of developing seta at 7 days after feeding, a, oenocyteshowing intense lipid staining; b, tormogen cell; c, trichogen cell, x 750.Fig. 12. Seta at 9 days. Tormogen and trichogen cells show strong lipid staining.X75°-


Fig. 13. At g days, section through trichogen (a), tormogen (6), and adjacent epidermalcell (c). x 26000.

Fig. 14. Newly forming epicuticle showing Sudan B staining after NaOCl. x 720.

Fig. 15. Slightly later stage, inner epicuticle forming. Lipid staining after NaOClthroughout the epicuticle. Note the thickening cuticle of the plaque (a) and the socketof the seta (A), x 720.

Fig. 16. Cuticle just before moulting; epicuticular channels (arrows) revealed byapplication of ammoniacal silver to the surface, x 1200.

Figs. 17-21. Epicuticular channels stained by the myrcene—osmium procedure.

Fig. 17. At 9 days; formation of lamellate cuticle just beginning. Channels arisefrom conical endings of pore canals, x 20000.

Fig. 18. 5th-stage larva at 2 days after moulting. Note the slight dilatation (arrows)just below the outer epicuticle. x 30000

Fig. 19. 4th-stage larva at 9 days after feeding. Pore canals, at bottom right, end incones leading to the epicuticular channels, x 20000.

Fig. 20. Tangential section of inner epicuticle showing lipid-staining channels eachsurrounded by a pale ring (arrows), x 30000.

Fig. 2i . As Fig. 20, slightly oblique section, x 30000.

Fig. 22. Epicuticle at 12 days after feeding. Lead staining after glutaraldehyde andbromine water. Epicuticular channels (arrows) have a clear lumen and dark walls; thaton the left has a fine axial filament, x 60000.

Fig. 23. Early stage of deposition of silver-binding material in epicuticle: minuteamber spot round each epicuticular channel. Surface view of the stellate epicuticle.X720.

Fig. 24. Later stage with almost complete fusion of the silver-binding material. Overthe crests of the epicuticular folds fusion is not quite complete, x 720.

Fig. 25. Later, dry, stage; silver-binding material accessible only over small scatteredpatches which colour intensely, x 720.

Lipid in the epicutkle of Rhodnius 479


Fig. 26. Argentaffin reaction in cuticle of 5th-stage larva at 10 days after moulting.Ammoniacal silver, and sodium thiosulphate, applied to intact cuticle after gentleabrasion of surface. Reaction positive only over the crests, and the dome of the plaque(arrows). (The other dark areas due to melanin.) x 720.

Fig. 27. Section of cuticle at earliest stage of appearance of silver-binding material, 2days before ecdysis (cf. Fig. 23). x 720.

Fig. 28. Argentaffin reaction in section through the integument of 4th-stage larva 1-2days before ecdysis. a, the new epicuticle, is quite negative; b, the old epicuticle, ispositive throughout, particularly in the melanized area to the left; c, the detachedcement layer is also positive, x 720.

Fig. 29. Argentaffin reaction in section of cuticle of 5th-stage larva at 1 day after moult-ing. The reaction is now positive throughout the epicuticle; notably in the melanizedareas (a), in the thickened cuticle below the plaques (b), and at the base of the seta andits socket (c). x 720.

Figs. 30-37 show electron-microscope sections of the silver-binding material accumu-lating in the epicuticle during the 48 h before ecdysis.

Fig. 30. An early stage with some of the material exuding at the surface (arrow),x 14000.

Fig. 31. Early stage with the material from each epicuticular channel widelyseparated (cf. Fig. 23). x 14000.

Fig. 32. As Fig. 31 but a thick deposit of silver extends down the epicuticularchannels to the endings of the pore canals, x 14000.

Fig- 33- The material is spreading laterally and more deeply into the epicuticle.x 14000.

Fig. 34. Epicuticle of pigment spot, early stage, x 14000.

Fig. 35. Advanced stage in stellate cuticle (cf. Fig. 24); material from adjacentchannels fusing, but often a narrow clear zone persists. In the middle region below,the individual patches are seen in tangential section. These show the epicuticularchannel as a black centre with a silver-free ring round it. (cf. Figs. 20, 21). x 14000.

Fig. 36. Epicuticle of a plaque, x 14000.

Fig. 37. Epicuticle over 2 muscle insertions. Silver-binding material extends intolamellate cuticle, x 9500.

Fig. 38. Section of integument of newly moulted sth-stage larva, treated with am-moniacal silver after extraction in warm chloroform. Silver staining of detached 'waxlayer' (a) of epicuticular channels (6), pore canals (c), and granules of uric acid andpteridine pigment in the epidermal cells (d). x 720.

Lipid in the epicuticle of Rhodnitis

1•.*?*.

28

t* X >% IK%J

.3 t


Fig. 39. 4th-stage larva at the dry stage; see Fig. 25: silver-binding material accessiblein small scattered patches. Vertical section. To right: silver-staining area covered bysilver-staining membrane, just separating at left. To left: epicuticle broken away, onlysilver-staining membrane persists, x 1000.

Fig. 40. As Fig. 39; adjacent section. To right: silver-staining membrane detached,x 1000.

Fig. 41. The same material; electron micrograph: a, patch of silver-staining epi-cuticle; b, outer epicuticle, silver free; c, continuous silver-staining layer on surfaceof outer epicuticle over silver-staining inner epicuticle; d, membrane ('wax layer')almost silver free, detached from outer epicuticle in non-staining area, x 14000.

Fig. 42. As Fig. 41, the same lettering, x 14000.

Fig. 43. Cuticle at 11 days after feeding; distal parts of pore canals filled with lipid-richmaterial, x 20000.

Fig. 44. Cuticle at 1-2 days before moulting, horizontal section, immediately belowepicuticle. Pore canals filled with lipid-rich material. Axial filaments appear as palecentres (arrow), x 20000.

Figs. 45-52. Surface views of exposed epicuticle during the last few hours beforeecdysis; whole mounts, Sudan B after glutaraldehyde.

Fig. 45. Unstained: refractile epicuticle can give a black surface line which may bemistaken for lipid staining, x 720.

Fig. 46. Focused on crests of stellate cuticle: lipid droplets exuding from epicuticularchannels, x 720.

Fig. 47. The same field as Fig. 46, at lower focus: lipid droplets, x 720.

Fig. 48. Another example of lipid droplets exuding, x 720.

Fig. 49. Insertions of dorso-ventxal muscles. Lower left: fusion of lipid droplets insurface view; above and to the right; lipid droplets seen in optical section at the sides ofthe raised sites of insertion, x 720.

Fig. 50. Fusion of droplets over surface of stellate cuticle, x 720.

Fig. 51. Lipid accumulations visible in narrow folds of epicuticle. x 720.

Fig. 52. Lipid forming a uniform blue grey-staining layer over entire surface ofepicuticle. x 720.

Fig. 53. Larva at 1 day before moulting. Osmium tetroxide. Sudan B staining after verymild NaOCl treatment of section. 'Wax layer' detached as a delicate lipid-stainingmembrane (arrow), x 720.

Fig. 54. Larva shortly before moulting. Exposed in ammoniacal silver: silver stainingthroughout epicuticle. Also with silver-staining membrane (' wax layer') on the surface.To the left this membrane (arrows) is seen detached from the epicuticle which is outof focus, x 720.

Lipid in the epicuticle of Rhodnius

b a

±*M

54

484 V. B. Wiggksworth

Fig- 55' 5th-stage lar\'a at 1 day after moulting. Chloroform extraction for 15 min at50 °C; exposed to ammoniacal silver. Silver binding throughout epicuticle; also in thedetached membrane consisting of combined 'wax layer' and 'cement layer' (arrows).X720.

Fig. 56. Material and treatment as Fig. 55. a, silver staining of detached wax andcement layer; b, intense silver staining of the points of insertion of dorso-ventralmuscles, x 720.

Fig- 57- 5th-8tage larva at 1 day after moulting. i-/Jm section showing deep alcian bluestaining of cement layer (arrow) detached from the unstained cuticle, x 720.

Fig. 58. As Fig. 57. x 720.

Fig. 59- sth-stage larva; surface view of colourless cuticle around a plaque. Alcian bluestaining shows thin irregular deposit of cement, best seen to the left (arrow), x 720.

Fig. 60. 5th-stage larva; surface view of plaque with tactile seta arising from it. NaOCl1:2O dilution (2 min) followed by Sudan B. Lipid-staining film on surface of cuticle,breaking down to give lipid droplets, x 720.

Fig. 61. Electron micrograph of epicuticle of unfed 4th-stage larva showing cementlayer on surface, x 28000.

Fig. 62. As Fig. 61; cement layer partially detached.

Fig. 63. Cuticle of 4th-stage larva 4 days after feeding; control side; showing detachedcement layer (arrow), x 720.

Fig. 64. As Fig. 63: abraded side, 3 days after abrasion. Irregular wax deposits on de-tached surface membrane, x 720.

Fig. 65. As Fig. 64; electron micrograph, a, repair membrane detached from cuticle;b, deposit of free wax. x 20000.

Fig. 66. As Fig. 65; repair membrane detached and withdrawn from a fold in the cuticlesurface, x 20000.


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incorporation of lipid into the epicuticle of ...(polyphenols) first appea inr these same site ast...

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