a comparative stud of thye chilopod and diplopod cuticle...

A Comparative Study of the Chilopod and DiplopodCuticle

By GORDON BLOWER(From the Department of Zoology, Victoria University of Manchester)

SUMMARY

I . The histology of the cuticle and epidermis of certain chilopods and diplopodsis described. Two principal layers of the cuticle are recognized, an outer homogeneousand refractile exocuticle which is usually but not invariably pigmented and an innerendocuticle.

2. The endocuticle and the exocuticle both contain chitin. The exocuticle is con-sidered as a modification of the outer part of the chitinous matrix by an impregnatingsubstance.

3. Certain properties of the impregnating substance are described. It appears tobe a substance rich in phenolic groups, perhaps a protein, which has a stability andresistance to acids in its own right irrespective of the presence of pigment or aromaticcross-links. Pro-sclerotin is suggested as a name for this substance. Chemical testsshow that it is present in regions not optically definable as exocuticle.

4. The epidermis is virtually an epithelium of gland cells which appear to secretelipoid material. The lipoid passes on to the surface of the cuticle by means of ductspassing through the cuticle. Here it appears to form a superficial layer and to impreg-nate the sclerotin and pro-sclerotin.

5. There appears to be an intimate association of lipoid with the aromatic groupsof the pro-sclerotin and sclerotin. Destruction of the aromatic groups by means of anoxidizing agent appears to intensify the colouring of the lipoid by sudan.

6. The myriapod cuticle is shown to have many features in common with that ofother arthropods. The main difficulty in the way of extensive homology with otherarthropod types is the absence in myriapods of an outer non-chitinous and resistantlayer.

CONTENTSPAGE

I N T R O D U C T I O N . . . . . . . . . . . . . 142

M A T E R I A L A N D M E T H O D S . . . . . . . . . . . 142

T H E T W O P R I N C I P A L L A Y E R S O F T H E C U T I C L E A N D T H E V A R I A T I O N I N T H E I R F O R M A N D

E X T E N T . . . . . . . . . . . . . 143

S T A I N I N G R E A C T I O N S . . . . . . . . . . . 145

T H E P H E N O L I C S U B S T A N C E I M P R E G N A T I N G T H E C H I T I N O U S M A T R I X . . . . 1 4 9

A R G E N T A F F I N M A T E R I A L I N T H E C U T I C L E . . . . . . . . 1 5 3

T H E E P I D E R M A L G L A N D C E L L S A N D T H E O C C U R R E N C E O F L I P O I D I N T H E C U T I C L E . 154

P O R E C A N A L S . . . . . . . . . . . . . 1 5 6

R E S U M E A N D D I S C U S S I O N . . . . . . . . . . . 1 5 6

A C K N O W L E D G E M E N T . . . . . . . . . . . 1 6 1

R E F E R E N C E S 161

[ Q u a r t e r l y J o u r n a l of M i c r o s c o p i c a l S c i e n c e , V o l . 9 2 , p a r t 2 , J u n e 1 9 5 1 . ]

142 Blower—A Comparative Study of the

INTRODUCTION

DURING recent years there has been much interest in the integumentof certain arthropods. A large part of the work in this field has been

done on the insect cuticle and the present state of our knowledge of thissubject has been reviewed in a recent article by Wigglesworth (19486).

The integument in other classes of arthropods has not been neglected,however. Beginning with Yonge's (1932) suggestion that the two principallayers in Homarus are common to all arthropods, there has been much dis-cussion as to the probable homology of the described conditions in differentarthropods. Pryor (19406) pointed out the chemical similarity of the 'cuticle'of Homarus with the epicuticle in insects. Dennell (1946, 1947a and b) madean extensive comparison of insect and crustacean cuticles and saw a consider-able correspondence in detail between the fundamental layers of the cuticlein these two groups.

Apart from Browning's paper on the cuticle of Tegenaria (1942), a descrip-tion of the epicuticle in ticks (Lees, 1947) and a paper by Langner (1937) onthe cuticle of diplopods, arthropods other than insects and Crustacea havereceived little attention. It is the aim of the present paper to illustrate, byselecting a few clear-cut histochemical reactions, the general features whichhave emerged from an extensive study of the cuticle in various chilopods anddiplopods and which may contribute towards a clearer understanding of thefundamental features of the arthropod exoskeleton.

In this paper the word myriapod is used without classificatory significance,as a convenient term covering both chilopods and diplopods.

MATERIAL AND METHODS

The investigation has been carried out on British species of centipedes andmillipedes. Animals were kept alive in glass vessels in the laboratory. Thefollowing of the larger species have been used for detailed examination:Chilopoda—Lithobius forficatus (L.), Haplophilus subterraneus (Leach); Diplo-poda—Schizophyllum sabulosum (L.), Tachypodaiulus niger (Leach).

Most histochemical tests, unless otherwise stated, have been performed onfrozen sections, cut at 20 ft, of material fixed in 5 per cent, formaldehyde in 0-9per cent, saline. Fixed material was impregnated with 25 per cent, gelatine solu-tion and the blocks hardened in 5 per cent, formaldehyde (see Carleton, 1938).Paraffin sections have been used for a study of the histology of the epidermisand the staining reactions of the cuticle. For chilopods, Flemming withoutacetic in 0-9 per cent, saline proved the best fixative. Heidenhain's 'Susa'was also found useful. For diplopods, fixation by neutral formaldehyde (5 percent.) followed by decalcification with a 30 per cent, aqueous solution ofsodium hexametaphosphate (Wilks, 1938) provided the best pictures of theepidermis. The presence of calcium salts in the cuticle precluded the use ofan acid fixative such as Flemming, although fairly good results were obtainedwith Susa which also rendered subsequent decalcification unnecessary.

Chilopod and Dipbpod Cuticle 143

Material for paraffin sections was embedded by the dioxane technique(Carleton, 1938). Epidermal pigment in the diplopod integument was removedby immersing sections for 24 hours in ethylene chlorhydrin (see Lea, 1945).Mallory's triple stain and iron haematoxylin, both alone, and when combinedwith ponceau acid fuchsin and light green (Masson's trichrome) have beenthe most useful stains.

THE TWO PRINCIPAL LAYERS OF THE CUTICLE AND THE VARIATION IN THEIR

FORM AND EXTENT '

In sections of the cuticle of any chilopod or diplopod two layers areoptically discernible. The inner layer is colourless and is horizontallystriated. The outer layer is homogeneous, that is to say, not obviouslyhorizontally striated, is highly refractile, and is usually pigmented in partor in the whole of its thickness. In this work the terms endocuticle andexocuticle have been applied to the optically distinguishable inner and outerlayers respectively.

In a chilopod the exocuticle varies in its form and thickness in differentregions of the body (fig. 1, A and B). The exocuticle of the sclerites is thickerthan that of the arthrodial membranes. In the sclerites of Lithobius (fig. IA)there is an exocuticle occupying from a quarter to a fifth of the total thicknessof the cuticle whereas the arthrodial membrane exocuticle is very thin andlobulated. The sclerite of Haplophilus (fig. IB) is in marked contrast to thatof Lithobius; the exocuticle is here produced inwards in the form of cones.These cones appear to occupy the field of activity of individual epidermalcells as manifested by the hexagonal areas which are seen in surface views ofthe cuticle. This prismatic arrangement is also manifest at the external faceof the cuticle by shallow convexities. Although the hexagonal areas can beseen in surface views of the cuticle of Lithobius, no evidence of the individual'cell-prisms' is to be seen in section. The arthrodial membranes of Haplo-philus are intucked and the exocuticle from the two contiguous scleritesgradually decreases in thickness towards the membrane, where it is notdeveloped at all. The region of transition from sclerite to arthrodial membranehas a characteristic exocuticle and is here termed the 'intermediate sclerite'condition. The inner face of the exocuticle in this region is produced inwardsas minute conical papillae (see fig. 1, A and B—'intermediate sclerite').

The pleural regions of Lithobius and Haplophilus are quite different. InLithobius there is very little sclerotization, the majority of the pleuron cuticlebeing in the condition of arthrodial membrane whereas in Haplophilus thereis a series of pleurites developed in the pleuron, each separated from the nextby an intucked arthrodial membrane. This difference and the difference inthe form of the cuticle as a whole is characteristic of the orders to whichLithobius and Haplophilus belong. The intersegmental membranes of bothanimals are usually in the condition of 'intermediate sclerite'.

In section the outer surface of the sclerites of Lithobius and Haplophilus


appears to be bounded by a thin colourless membrane. This is in all proba-bility a diffraction effect since a similar appearance characterizes the inneredge of the endocuticle when this is separated from the epidermis during

tergibe

scleribe

arbhrodialmembrane

inbermediabescleribe'

pleuribes

stemibe

pleuro-tergalarch of arbhrodial membrane

mebazombe s s s s s s o —

scleribe k

-inbersegmentalmembrane

pleuro-berqal'arch of s

prozonite

sbernibe

FIG. I . Transverse sections of the cuticle, diagrammatized from free-hand drawings. A, Litho-bius forficatus. B, Haplophilus subterraneus. c, Schizophyllum sabidosum. Exocuticle black,

endocuticle shaded.

sectioning. Verhoeff (1925) and Fuhrmann (1921) describe three cuticularlayers in a chilopod. The outermost layer in each case undoubtedly refers tothat which has just been described as a diffraction effect. The histologicalvalidity of this outer layer will be discussed later. In the meantime a two-layered cuticle will be considered as a working hypothesis and the followingsynonyms may be tentatively tabulated:

Chilopod and Diplopod Cuticle 145

Verhoeff

Oberflachenschicht

FarbschichtLammellenschicht

Fuhrmann

Grenzhautchen

AussenlageInnenlage

This work

Regarded as a diffrac-tion effect

ExocuticleEndocuticle

The exocuticle of the diplopods Schizophyllum and Tachypodoiulus doesnot differ perceptibly in different regions of the body (fig. ic), for here it isthe presence of a calcined outer endocuticle which distinguishes a scleritefrom an arthrodial membrane, where the endocuticle is very thin or absentaltogether. The inner surface of the exocuticle is produced inwards asnumerous minute papillae as in the intermediate sclerite of a chilopod. Thesclerite endocuticle can be divided into an inner part with conspicuous hori-zontal striae and an outer part where the striae are not so obvious (fig. ic).Langner (1937), besides two layers of endocuticle, distinguishes two outer-most layers, an exocuticle and an overlying epicuticle in several species ofdiplopod. Cloudsley-Thompson (1950) also describes an epicuticle as distinctfrom an exocuticle in certain centipedes and millipedes. Even so, a two-layered cuticle will be taken as a basis for description and the presence orabsence of an epicuticle overlying the exocuticle will be discussed later.

STAINING REACTIONS

In the unhardened fore-gut cuticle of Homarus (Yonge, 1932) and that ofthe larva of Sarcophaga (Dennell, 1946) there is a close correspondence ofstaining reaction, and Dennell suggests, as did Yonge, that the red- and blue-staining layers differentiated by Mallory's triple stain are probably funda-mental to all arthropods.

As the cuticle of the last larval instar of Sarcophaga is converted into thepuparium, the inward extension of the hardening process modifies the stainingreaction (Dennell, 1947a). The outer red-staining region loses its affinityfor the stain and develops an amber-brown colour as also does the outer partof the endocuticle. The puparium cuticle, however, stains red at the junction ofthe tanned and untanned regions. The red-staining zone is pushed inwards,as it were, by the tanning of the outer layers.

Sections of the myriapod cuticle stained with Mallory show an outer layerstained with acid fuchsin and an inner layer stained with aniline blue. Thered-staining layer has been 'pushed inwards', however, to various depths,according to the extent to which the outer layers have been involved in thehardening process. In the sclerite cuticle of Schizophyllum and Tachypodoiulusthe exocuticle stains red except for a very thin outermost layer and the wholeof the endocuticle stains blue—the inner endocuticle staining much moredeeply than the outer. In Haplophilus the exocuticle stains red except for theoutermost region of the sclerite. The cones do not stain so deeply as thecontinuous part of the exocuticle. In Lithobius none of the sclerite exocuticlestains red but practically the whole of the sclerite endocuticle takes the acid

146

A

Blower—A Comparative Study of the

exocubiclebasiphil zonebeneath exocuticlegland ducb

endocubicle

gland cell

nucleus ofepidermal cell

basemen bmembraneouber non-stainingparb of exocuticlepapillabe partof exocuticlegland ducb

endocubicle

gland cell

epidermal cell

exocuticle(viewed obliquely)

gland ducb

endocuticle

gland ceil

epidermal cell

FIG. 2. Transverse sections of the cuticle of Lithobius forficatus. A, Sclerite. B, 'Intermediatesclerite'. c, Arthrodial membrane. Camera lucida drawings. Flemming without acetic, iron

haematoxylin.

fuchsin. In the arthrodial membranes however, the exocuticle stains red andthe endocuticle blue.

With Masson's trichrome stain the details are similar, the acid fuchsinophilzones of Mallory preparations taking iron haematoxylin in their outer parts

Chilopod and Diphpod Cuticle

epidermal cell

basemenbmembrane

'inbermediabe .scleribe' exocufcicle

cone of exocubicle

gland cell

Inbermediabe „scleribe eaocutrcfe

epidermis

endocubicle

gland cell

50]XFIG. 3. Transverse section of an arthrodial membrane between the tergite and a pleurite ofHaplophilus subterraneus. Below are surface views of the cuticle corresponding to the threeregions indicated on the section. Camera lucida drawing. Flemming without acetic, iron

haematoxylin.

and ponceau acid fuchsin inwardly; the region staining blue with Mallorytaking the light green of the Masson combination. In the sclerite of Haplo-philus, for example, the continuous part of the exocuticle stains blue-blackwith the haematoxylin, save at its free edge which does not stain. The conesstain with the ponceau and acid fuchsin, which, together with a little of theretained haematoxylin, give a purple colour. Figs. 2, 3, 4, and 5 illustrate the


various regions of the cuticle stained with iron haematoxylin alone. It willbe noticed that the sclerite endocuticle of Lithobius stains with haematoxylinonly in its outer layers; the inner layers stain red when the haematoxylin is

outer non-sbainingparb of eiocubidecontinuous parbof exocubiclepnsmabic coneof exocubicle

gland duct

endocuticle

epidermal cell

gland cell

basementmembrane

FIG. 4. Transverse section of the sclerite cuticle of Haplophilus subterraneus. Camera lucidadrawing. Flemming without acetic, iron haematoxylin.

6uber non-stainingand inner basiphifexocubicleswelling^ ingland aucb

outer endocuticle

gland duct

inner endocuticle

gland cell

j^i_—epidermal cell

FIG. 5. Transverse section of the sclerite cuticle of Schizophyllum sabulosum. Camera lucidadrawing. Neutral formaldehyde fixation followed by decalcification in sodium hexameta-

phosphate and removal of epidermal pigment by ethylene chlorhydrin.

followed with the other stains of Masson's combination. The figures showclearly the passage inwards of the basiphil zone (equivalent to the outer partsof the red-staining zones with Mallory) associated with greater degrees oftanning of the outer layers. The whole of the arthrodial membrane exocuticleof Lithobius is basiphil. The exocuticle of Schizophyllum, which is of a moredistinct amber colour than that of the arthrodial membrane of Lithobius, fails


to stain in its outermost layer. This non-staining zone is extended in thesclerite exocuticle of Haplophilus and in the sclerite of Lithobius the entireexocuticle fails to stain, the basiphil region occupying a large part of theendocuticle.

In all cases, treatment with diaphanol (chlorine dioxide in glacial aceticacid) leads to the entire thickness of the exocuticle staining with either theacid fuchsin of Mallory or the iron haematoxylin used in Masson's combina-tion—the endocuticle staining with aniline blue or light green respectively.In Lithobius the intensity of blue or green staining of the endocuticle isgreater in the sclerite than in the arthrodial membrane, and similarly inSchizophyllum the inner endocuticle still has a greater affinity for these stainsafter diaphanol treatment.

Dennell (1946) has described the effect of diaphanol on the subsequentstaining reaction of the cuticle of Sarcophaga puparia. The larval conditionis restored as far as staining reaction is concerned and there appears to bea reversal of the effects of tanning. Since diaphanol treatment in this caserestores the fundamental two-layered nature of the cuticle it might be assumedthat the endocuticle and exocuticle of the described myriapods are the homo-logous fundamental layers. The two layers of Sarcophaga and Homarus are,however, further differentiated by the presence or absence of chitin, whereasno such distinction can be drawn in the case of the myriapod cuticle. It mightbe added here that the division of the thin outer layer of a diplopod into non-staining and basiphil zones appears to be the basis which Langner (1937)adopts in distinguishing an epicuticle and an exocuticle. Since both regionsof the outer layer contain chitin (see next section) and diaphanol treatmentremoves the distinction, it is doubtful whether this particular staining reactionis a suitable criterion to adopt.

THE PHENOLIC SUBSTANCE IMPREGNATING THE CHITINOUS MATRIX

The optical appearance of the exocuticle and the endocuticle is, in fact, theonly criterion which constantly differentiates these two layers. Althoughcertain chemical properties are always associated with the exocuticle, thesame properties are sometimes shown by the endocuticle. Even such acharacteristic feature as pigmentation is not shown by all regions of the exo-cuticle.

A feature of both layers is the presence of chitin. This fact is made veryclear in the case of Lithobius where the exocuticle often separates from theendocuticle during the potash treatment, but is nevertheless just as intenselypositive to the chitosan test (see Campbell, 1929) as the endocuticle. Theexocuticle is therefore to be regarded as the modification of the outer layersof a chitinous matrix by the addition of some impregnating substance orsubstances.

The exocuticle of all forms always gives intensely positive xanthoproteic andMillon reactions, from which it can be assumed that phenolic groupingsenter largely into the composition of the impregnating substance. The


exocuticle is not, however, the only part of the cuticle positive to these twotests. The whole of the sclerite endocuticle of Lithobius is likewise intenselypositive.

When frozen sections of the cuticle are treated with concentrated mineralacids the exocuticle survives the treatment, as also do the ducts of the epi-dermal gland cells which bridge the gap vacated by the endocuticle. Thesclerite endocuticle of Lithobius dissolves much more slowly than that ofthe arthrodial membranes. In the sclerite of Haplophilus the inner part of theexocuticle, that is, the part divided into inwardly projecting cones, dissolvesin several hours at room temperature. The outer, continuous part of theexocuticle is a typical amber qplour as is the whole exocuticle of Lithobius and,like that of Lithobius, survives acid treatment for days. The inner discontinu-ous part of the exocuticle of Haplophilus is not pigmented. The foregoing obser-vations may be summarized thus: the typical amber-coloured exocuticle isvery resistant to concentrated mineral acid. The colourless inner region of theexocuticle in Haplophilus is not quite so resistant to acid, and finally thesclerite endocuticle of Lithobius is even less resistant—but nevertheless showsquite a degree of resistance when compared with the arthrodial membraneendocuticle, which dissolves almost immediately. All regions showing anydegree of resistance are alike in giving positive xanthoproteic and Millonreactions. It appears that the chemical difference between the conical part ofthe exocuticle of Haplophilus and the sclerite endocuticle of Lithobius is oneof degree and not kind—the cones of Haplophilus being included as part ofthe exocuticle by virtue of their homogeneity and refractility.

It appears from these facts that the process of sclerotization consists in theimpregnation of a chitinous matrix by a substance rich in phenolic groupsand a subsequent.process which renders this material resistant to acids. Thedevelopment of refractility and, later, of an amber colour seems to be cor-related with the development of this resistance, although resistance is deve-loped by the phenolic substance present in the inner, discontinuous, partof the exocuticle of Haplophilus without the assumption of an amber colourand in the sclerite endocuticle of Lithobius without even the assumption ofrefractility.

As suggested in a preliminary communication (Blower, 1950) it seemspossible that this phenolic impregnating substance is a protein in contrast tothe alcohol-soluble phenol which is one of the precursors of sclerotin in thecockroach ootheca (Pryor, 1940a). Evidence in favour of this view arises froma comparison of the arthrodial membrane endocuticle of Lithobius, on theone hand, with the sclerite endocuticle of this same animal where the phenolicsubstance appears to be present in a simple form, since here the substancehas conferred a degree of resistance on the endocuticle but has not renderedit homogeneous or refractile. In the first place it will be remembered thatthe sclerite endocuticle gives positive Millon and xanthoproteic reactionswhereas the arthrodial membrane endocuticle does not. This, and the follow-ing histochemical details, even if their absolute meaning is not clear, give a

Chilopod and Diphpod Cuticle 151

comparison under closely controlled conditions since each test has been per-formed on a transverse section where both regions under consideration arepresent together.

The iso-electric points of the two regions have been determined by themethod employed by Yonge (1932). The value for the sclerite endocuticle isbetween pH 5-4 and pH 5-6 whereas that for the arthrodial membrane endo-cuticle was between pH 2-8 and pH 3-0. It follows from this that the sub-stance responsible for the difference between the two regions is probablyamphoteric such as an amino-acid or protein. This difference in iso-electricpoints is reflected in the staining reactions of the two regions. With Mallorythe sclerite endocuticle stains red whereas the arthrodial membrane endo-cuticle stains a light blue. The substance differentiating the sclerite endocuticlefrom the arthrodial membrane endocuticle is still present after treatmentwith water, alcohol, xylene, &c, and remains demonstrable by its stainingreaction. It seems on this account that a simple amino-acid is not responsibleand that a protein is probably involved. The fact that the additional substancein the sclerite endocuticle of Lithobius confers a degree of resistance to acidswould also suggest that the substance concerned is something other than afree phenol or amino-acid.

The resistant nature of the substance impregnating the sclerite endocuticleis manifest during the application of the chitosan test. In sections ofpotash-treated material the sclerites are still noticeably different from thearthrodial membranes. The two layers of the sclerite are quite obvious andthe exocuticle is still homogeneous. The arthrodial membranes appear tohave separated into individual laminae. At the first application of the iodineand sulphuric acid only the arthrodial membranes assume the violet colour.The sclerite endocuticle assumes a much lighter and more delicate tinge ofviolet, the exocuticle remains colourless. A few moments after flooding theslide the sclerite endocuticle and exocuticle split up into individual laminae,expand to the extent of the arthrodial membranes and assume the deep-violetcolour first shown by these membranes.

If the temperature of the potash during treatment has fallen below thatspecified by Campbell (1929), i.e. 1600 C , or if the solution has not beenfully saturated, the initial reluctance of the layers of the sclerite to assume adeep-violet colour is prolonged. Pieces of cuticle in this condition immersedin 3 per cent, acetic acid did not completely dissolve. The residue still gives astrongly positive Millon test.

Lafon (1943), on treating the cuticle of a scorpion with 10 per cent, potashat ioo° C , found that two layers survived the treatment—a colourless layercomposed of chitin and an outer very thin amber-coloured layer. Lafonsuggests that this resistant outer layer is comparable with the cuticulin ofinsects. This layer bears similarity to the exocuticle of Lithobius, whichremains as a discrete layer after potash treatment. This layer in Lithobiussurvives even more brutal treatment than that which Lafon used, still remain-ing homogeneous after 2 hours at 1550 C. in concentrated potash. It may be


that in the scorpion this outer layer contains chitin but shows a reluctance todisplay itself similar to that of the exocuticle of Lithobius, and the unsuspectedpresence of chitin would account for the anomalous figures obtained fromanalysis of this layer. Lafon subjected Lithobius to the same treatment, butin view of the fact that he was dealing with whole pieces of cuticle, the smallamount obtainable from Lithobius may have led to his overlooking a survivingouter layer in this case.

As has been suggested (Blower, 1950) the substance impregnating thesclerite endocuticle of Lithobius and the exocuticle of all forms may be aprotein with a high tyrosine content, since it is very rich in phenolic groupsand has an iso-electric point similar to that of tyrosine. If this were so it seemspossible that there is a development of cross-links by the oxidation of theside chains of this tyrosine-rich protein. Brown (1950) has suggested thatthis method of tanning may take place in certain regions of Mytilus and in theegg-capsule of Fasciola. Whatever the mechanism of tanning, it seems pos-sible from the foregoing facts that the substance differentiating the scleriteendocuticle of Lithobius from the arthrodial membrane is a protein rich inphenolic groups and probably represents the precursor of typical sclerotin.This substance will accordingly be termed pro-sclerotin. The prismatic conesof the exocuticle of Haplophilus may then be regarded also as consisting ofpro-sclerotin.

In the diplopod Schizophyllwm there is a thin exocuticle, only the outerpart of which is amber coloured. The endocuticle is optically divisible intotwo distinct layers. The outer layer is impregnated with calcium salts—a factascertained by the application of the alkaline pyrogallol test (Lison, 1936).The inner layer is much more conspicuously laminated and appears to beimpregnated with pro-sclerotin. It gives positive xanthoproteic and Millontests and survives acid treatment for much longer than the outer endocuticle,but is not.so resistant as the exocuticle. As would be expected, the innerendocuticle has a higher iso-electric point than the outer (pH 36 comparedwith pH 2-8).

The behaviour of the cuticle towards diaphanol casts a little more light onthe nature of sclerotin and pro-sclerotin. The effect of this reagent on a tannedcuticle as described by Dennell (1946) has been mentioned. The fully tannedpuparial case of Sarcophaga is bleached by diaphanol and the condition ofthe cuticle in the last larval instar appears to be restored as is evidenced bythe staining reaction of the diaphanol-treated cuticle. Its effect on the chilo-pod and diplopod cuticle is to remove all traces of the amber colour residentin the exocuticle, but it has little effect on the resistance of this layer to acids.Theoretically it would be expected to remove the cross-links of sclerotin bydestroying the aromatic nuclei by oxidation. That it does in fact destroy therings is evidenced by the fact that a diaphanol-treated cuticle gives no traceof a positive xanthoproteic or Millon reaction in any region. The fact that itdoes not, however, destroy the resistance of the exocuticle to acids, pointsagain to the fact that pro-sclerotin itself is a substance with a degree of


resistance and stability which does not depend on the presence of aromaticcross-links. It will be recalled that the sclerite and arthrodial membrane endo-cuticles of Lithobius still stain differently after diaphanol treatment—withMallory the sclerite endocuticle stains a deep purplish-blue in contrast to thearthrodial membrane which stains a very pale blue.

ARGENTAFFIN MATERIAL IN THE CUTICLE

It is stated (Lison, 1936) that a positive argentaffin reaction (reduction ofan ammoniacal silver nitrate solution) indicates the presence of polyphenols,amino-phenols, or polyamines. This reaction has often been employed todemonstrate the distribution of phenolic substances in the cuticle. Hackman,Pryor, and Todd (1948) describe phenolic substances in the epicuticle (herereferred to as 'exocuticle') of Tachypodoiulus on the evidence furnished bythis test.

When sections of a chilopod or a diplopod are treated with a 5 per cent,solution of ammoniacal silver nitrate there is a browning of the exocuticle.The sclerite endocuticle of Lithobius eventually reduces the silver solution,but only after a much longer period in the reagent. The arthrodial membranenever reduces the silver. Ammoniacal silver has also been used in order todetermine whether a polyphenol layer covered by a wax layer lies external tothe exocuticle as described in Rhodnius and Tenebrio (Wigglesworth, 1947,1948a). A specimen of Lithobius was allowed to crawl in carborundum powderfor several hours and then plunged into silver solution for 24 hours. Thecuticle was then cleaned and washed well, and a portion embedded in wax andsectioned. Another portion of the cuticle was mounted whole. The wholemount revealed numerous criss-crossing brown lines on the surface of thesclerite representing the scratching of its surface by the carborundum par-ticles. In sections of the sclerite these brown scratch lines were representedby lens-shaped areas at the surface of the cuticle which had reduced thesilver more strongly than elsewhere. Beneath the exocuticle in the outerthird of the endocuticle numerous vertically disposed filaments were stained—much more intensely than the exocuticle itself. Furthermore, the contents ofthe epidermal glands and their ducts had reduced the silver nitrate solutionand appeared almost black.

It will be remembered that the whole of the sclerite endocuticle of Lithobiusis intensely positive to Millon's reagent, and the whole has a higher iso-electric point than the arthrodial membrane endocuticle. If the argentaffinreaction indicates the presence of phenolic substances one would expect thewhole of the sclerite endocuticle of Lithobius to darken evenly. This it doesnot do. As will be seen later the epidermal glands appear to secrete a lipoidmaterial, particularly evident in Haplophilus; and since these and the contentsof their ducts reduce the silver solution in Lithobius and in Haplophilus, itseems possible that the lipoid itself may be argentaffin.

Although Lison (1936) does not include lip'oids amongst argentaffin sub-stances, it seems theoretically possible that unsaturated fats at least would be


capable of reducing ammoniacal silver nitrate. To ascertain whether there isa possibility that cuticular lipoid is argentaffin, animals were cut into twopieces, the cuticle freed from underlying tissue and one piece boiled for2 hours in chloroform. The untreated piece of cuticle was retained as acontrol. Both pieces were embedded in paraffin wax and sectioned. Tworibbons, one from each piece, were mounted on a slide, freed from wax andthe slide immersed in 5 per cent, ammoniacal silver nitrate for 24 hours. Thesections were then fixed in a 1 per cent, solution of sodium thiosulphate (Lee,1937). The intensity of the silver staining of the exocuticle was definitely lessin the chloroform-treated cuticle. Furthermore, the extent of the argentaffinzone beneath the sclerite exocuticle was considerably reduced. The chloro-form used for the extraction was allowed to evaporate in a watch-glass. Anorange-coloured material remained which stained readily with sudan black.As will be made clear in the next section the distribution of lipoid in thecuticle appears to follow the distribution of sclerotin and pro-sclerotin. It ispossible that the results of an argentaffin test may indicate the presence ofother material besides phenolic substances.

THE EPIDERMAL GLAND CELLS AND THE OCCURRENCE OF LIPOID IN THE

CUTICLE

The cuticle of all the myriapods examined is pierced by numerous ductsarising from glandular cells in the epidermis. The various types of gland andduct are shown in figs. 2, 3, 4, and 5. In general there are more glandsbeneath the sclerites than elsewhere, or, in other words, those regions of theanimal most likely to come into contact with its solid environment are wellsupplied with glands. The sclerite epidermis is in fact an epithelium of thesegland cells. Their ducts pass through the cuticle and open to the exteriorbetween the cuticular prisms. The staining reaction of the glands in theepidermis leads to a supposition that they show an asynchronous activity—some glands having dense basiphil inclusions whilst others have not.

In frozen sections of Haplophilns stained with sudan black the contents ofthe glands and their ducts are coloured blue. In Lithobius the whole of theepidermis is weakly sudanophil, but sometimes inclusions of a strongly posi-tive material are to be found within the gland ducts. In Schizophyllum andTachypodoiulus one can only say that the epidermis generally is stronglysudanophil—the epidermal pigment granules making the precise location ofthe lipoid material difficult to see.

The location of the lipoid material in the cuticle is not made very clear bysudan staining. In Lithobius only the arthrodial membrane and the inter-mediate sclerite exocuticle shows signs of the blue colour. There is, however,a very thin sudanophil layer external to the exocuticle. In Haplophilus thecones of the exocuticle are slightly sudanophil and again there is a very thinsudanophil layer external to the exocuticle. In the intermediate sclerites ofthis animal the exocuticle is strongly positive, and over the intucked arthrodial

Chibpod and Diplopod Cuticle 155

membranes there is always quite an accumulation of sudanophil material.In Schizophyllum the outer part of the exocuticle is sudan positive over theentire animal.

Treatment with diaphanol has an interesting effect on the subsequentstaining with sudan black, in so far as it definitely intensifies it. In a frozensection of diaphanol-bleached material of Schizophyllum or Tachypodoiulusthe exocuticle is coloured almost black with sudan black and this colouringextends inwards for about a quarter the thickness of the outer endocuticleas numerous very fine filamentous processes. There appears to be a penetra-tion of lipoid material down the pore canals (see next section).

The mechanism by which treatment with diaphanol leads to the lipoidmaterial becoming sudanophil is perhaps connected with the fact that itdestroys the aromatic links. Possibly the lipoid is in some way intimatelyattached to these groups and on their destruction is more readily availableto the sudan stain. This intensification of sudan staining is also evident inHaplophilus. The effect of diaphanol on the subsequent staining with haema-toxylin may possibly be explained on similar lines. It will be rememberedthat in the sclerite of Lithobius after diaphanol treatment the exocuticle stainswith haematoxylin, whereas in the untreated cuticle the exocuticle does notstain at all but the outer third of the endocuticle is stained with haematoxylin(see fig. 2A). Here it appears that the diaphanol, on destroying the aromaticgroups in the exocuticle, has made available some basiphil substance origi-nally firmly attached to these groups. By analogy with the above facts con-cerning the intensification of sudan staining by diaphanol and the fact thatthe contents of the gland ducts and glands are basiphil, it might be suggestedthat the substance made available to the haematoxylin is in fact a lipoid. Thehaematoxylin staining substance present beneath the exocuticle of Lithobius,however, does not remain after diaphanol treatment.

Pieces of cuticle warmed gently in a saturated solution of potassium chlo-rate in concentrated nitric acid show first a dissolution of the endocuticleand then a breaking down of the exocuticle into an oily material which stainsreadily with sudan black. This appears to be an oxidative process, similar tothat produced by diaphanol, but more vigorous, and presumably it leads inthe same way to a liberation of the lipoid by a destruction of the aromaticgroups with which it seems to be associated. Nitric acid alone can effect thisprocess. If whole pieces of animals are immersed in cold concentrated nitricacid the process can be studied more critically since the oxidation proceedsmuch more slowly. Pieces of Haplophilus thus treated show first a dissolutionof the endocuticle. After an hour nothing remains but the exocuticle. Beforethe amber colour has disappeared oily droplets float to the surface. Thesedroplets, smeared on to a slide, colour with sudan black. The outer amber-coloured part of the exocuticle is still intact at this stage and therefore thedroplets have probably come from the dissolution of the cones of the exo-cuticle. If the amber-coloured portion is left overnight it is bleached and onwashing and immersing in sudan black it takes up the colour rather unevenly.


This latter observation applies also to Lithobius, although no oily dropletsfree themselves from the cuticle as in Haplophilus. In Lithobius, during treat-ment with cold nitric acid, the intermediate sclerites and the arthrodialmembranes dissolve completely before the sclerites have lost their ambercolour. In both cases several days' treatment with acid results in the completesolution of all but the exocuticle of the sclerites.

As was pointed out in the preceding section it seems possible that thecuticular lipoid is capable of reducing silver nitrate. The vertically-disposedargentafEn filaments beneath the exocuticle of Lithobius are thus possiblylipoid in nature. Here it seems possible that the lipoid material penetratesdown the pore canals, as is indicated by sudan colouring in Schizophyllumand Tachypodoiulus. It is not clear, however, if this be the case, why sudancolouring in Lithobius does not give the same picture.

PORE CANALS

When a section of Tachypodoiulus or Schizophyllum is immersed in concen-trated mineral acid the outer endocuticle dissolves rapidly. Between the innerendocuticle and the exocuticle, besides the persistent ducts of the epidermalgland cells, there are to be seen numerous fine filamentous processes emergingfrom the inner endocuticle and continuous distally with the inwardly project-ing papillae from the underside of the exocuticle. By analogy with otherarthropods these structures appear to represent the solid contents of the porecanals. No such clear demonstration of pore canal filaments has been seen ina chilopod. It will be remembered, however, that the under surface of theintermediate sclerite exocuticle in Lithobius and Haplophilus is producedinwards as minute papillae, and these may represent the distal ends of originalpore canals into which exocuticular material has penetrated. Furthermore,treatment with silver has revealed filaments beneath the sclerite exocuticle ofLithobius. Unfortunately no developmental history of what appear to be thepore canals is available. Plotnikow (1904) figures pore canals in the form ofconical tufts with their apices arising from the epidermis in the developinglarval cuticle of Tenebrio. The* fully formed cuticle of this larva has the innerpart of its exocuticle in the form of cones as has Haplophilus. If the surfaceof the cuticle of Haplophilus is examined over the region of transition fromintermediate sclerite to arthrodial membrane (fig. 3, A, B, c), the impression isobtained that each cone of the exocuticle is formed by the merging togetherof exocuticular material in the pore canals. Possibly the original pore canalsof Haplophilus are tufted and give rise to the conical arrangement of theinner region of the exocuticle.

RisuMi AND DISCUSSION

A two-layered cuticle has been taken throughout as a working hypothesis.It has been seen, however, that the chemical reactions of the two layers donot neatly arrange themselves on each side of the optical dividing line between

Chilopod and Dipbpod Cuticle 157

endocuticle and exocuticle. This division is one of convenience only. Byconsidering different regions of the same animal, and different animals, thepossible changes attendant to the formation of a typical amber-coloured andhighly refractile exocuticle may be inferred. In the animals studied eachregion of the cuticle can be placed in an ascending scale of conditions approach-ing a typical exocuticle (see Table I).

TABLE I

1. Arthrodial membrane endocuticle of Haplo-philus and Lithobius and outer endocuticle ofSchizophyllum.

Sclerite endocuticle of Lithobius and the innerendocuticle of Schizophyllum.

3. Prismatic cones of Haplophilus. Inner layers ofexocuticle of Schizophyllum.

4. Stainable portion of the continuous exocuticleof Haplophilus. Arthrodial membrane exocut-icle of Lithobius. Inner portion of the exocuticleof intermediate sclerite in Lithobius and Hap-lophilus.

5. Whole of sclerite exocuticle of Lithobius andthe non-staining outermost regions of the exo-cuticle of Haplophilus and Schizophyllum.

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The process of exocuticle formation seems to be heralded by the appearanceof a substance, possibly a protein, rich in phenolic groups, in the chitinousmatrix of the cuticle. This substance has been given the name 'pro-sclerotin'.It is resistant to acids even before the more characteristic features of anexocuticle are manifested. The substance eventually renders the impregnatedregion homogeneous and refractile, by which time the region may be calledexocuticle on the definition here adopted. Later there is a development ofeven greater resistance, the development of an amber colour and finally theloss of staining reaction.


The process of sclerotization as described in detail by Pryor (1940 a and b)in the cockroach ootheca and in insects, and by Dennell (1947a) in the pupa-rial case of Sarcophaga, is regarded as a tanning of cuticular protein attendedby the development of an amber-brown colour. Pryor, however, states thata water-soluble protein is passed into the cuticle to form the basis of thesclerotin whereas Dennell considers that the pre-existent cuticular proteinmay be involved in the tanning process.

It does not seem that the whole of the process of exocuticle formation assuggested in this work is comparable with the tanning process as describedby Pryor and Dennell. Neither in Sarcophaga nor in the cockroach ootheca isthere a substance described,similar to pro-sclerotin. In the cockroach oothecaneither of the precursors of sclerotin appears to have any degree of resistancebefore they are linked together as sclerotin.

Browning (1942) describes a colourless exocuticle in certain regions of thespider Tegenaria, and Brown (1950) records the fact that the precursor ofsclerotin in certain structures of Mytilus and in the egg-capsule of Fasciolais not water- or alcohol-soluble but is probably a protein. Brown also suggeststhat the deamination of the phenol destined to tan the protein as described byPryor (1940 a and b) is possibly a derived condition. Both the cockroachootheca and the puparium of Sarcophaga appear to be special cases. In Sarco-phaga the sclerotin of the puparium is formed at the end of the instar, and itmay be that there has been a modification of the precursors of sclerotin todiffusible substances since they have to pass to a region of the cuticle distalto the epidermis.

A feature invariably associated with the exocuticle of myriapods is thepresence of lipoid material. This lipoid appears to be secreted by the glandcells of the epidermis through ducts which open on to the surface of thecuticle. At the surface it appears to form a thin film. It appears also that theexocuticle is impregnated with lipoid where there appears to be an intimateassociation of the lipoid and sclerotin or pro-sclerotin. It may be that thelipoid in the exocuticle is responsible for this layer (and adjacent regions ofpro-sclerotin) assuming an avidity for iron haematoxylin.

Pryor (19406) suggested that the sclerotin in insects is impregnated withlipoid. He pointed out that sclerotin and any material rich in aromatic groupsis very lipophil. In myriapods this observation is consistent with the fact thatboth sclerotin and pro-sclerotin appear to be impregnated with lipoid. InHaphphilus, it will be recalled, there is a dense accumulation of sudanophilmaterial over the arthrodial membranes. This region of the cuticle is unmodi-fied and no demonstrable exocuticle is formed at all. This may account forthe whole of the lipoid being stainable at the surface, since in this regionthere is no lipophil layer to absorb it.

It is interesting to consider the possible explanation of the scratch-patterneffect obtainable by treating the cuticle with an abrasive dust followed byimmersion in ammoniacal silver nitrate. It may be that the outer surface ofthe lipoid film covering the exoeuticle undergoes a process similar to the

Chihpod and Diplopod Cuticle 159

drying of a coat of oil-bound paint. Perhaps the abrasive agent removes thisinert layer and reveals the reactive lipoid beneath which may reduce thesilver solution. Wigglesworth (1947, 1948, &c.) explains scratch patterning ininsects on the basis of removal of a wax layer and exposure of a polyphenollayer which lies beneath the wax layer. In view of the fact that there appearsto be evidence, in myriapods, that the lipoid itself will reduce the silversolution and that no developmental details are available for myriapods, it isunwise to speculate further on this point.

In the myriapods studied there appears to be no cuticular layer correspond-ing to the cuticulin layer described by Wigglesworth in Rhodnius and Tenebrio(1947, 1948a). Cloudsley-Thompson (1950) on the basis of treatment withchlorated nitric acid suggests that a similar layer does exist in certain speciesof myriapods which he has examined. From this evidence,. however, thereseems nothing to differentiate between a layer of sclerotin impregnated withlipoid and a cuticulin layer. Just as Langner (1937) speaks of an epicuticleand an exocuticle in the outer part of the cuticle of a diplopod on the basisof an outer non-staining and an inner basiphil zone, the differential solubilityof the outer and innermost parts of the sclerotin of the cuticle is apparentlytaken by Cloudsley-Thompson as evidence for the presence of an epicuticle.

Wigglesworth (1947) has been careful to distinguish between cuticulin asa lipo-protein subsequently tanned with quinones, and Pryor's sclerotinsecondarily impregnated with lipoid. Only developmental evidence can settlethis point, but in view of the chemical similarity between sclerotized cuticulinand lipoid-impregnated sclerotin the difference in time relation may not beof fundamental importance, for pro-sclerotin may be impregnated withlipoid before tanning as generally understood takes place. In this case therewould seem to be nothing to distinguish between lipoid-impregnated pro-sclerotin (e.g. the exocuticular cones in Haplophilus) and cuticulin.

If the sequence of events during deposition of the cuticle is the same inmyriapods as in Rhodnius and there is no essential difference between sclero-tized cuticulin and lipoid impregnated sclerotin or pro-sclerotin, then thewhole of the myriapod exocuticle and parts of the endocuticle (sclerite endo-cuticle of Lithobius) can be considered as being impregnated with cuticulin.Even so, these regions still cannot be homologized with a cuticulin layer,since they contain chitin.

The only layer of the myriapod cuticle which appears to be in any wayhomologous with the insect epicuticle as described by Wigglesworth is thethin layer of lipoid at the surface. This layer may be responsible for thediffraction effect at the surface of the cuticle which lead Verhoeff and Fuhr-mann (see page 144) to speak of an outermost colourless layer (Oberflachen-schicht, Grenzhautchen). As regards the possible homology of the layers ofthe myriapod cuticle with those of Homarus (Yonge, 1932) and Sarcophaga(Dennell, 1946, 1947a), there appears to be close correspondence of themyriapod exocuticle with the 'cuticle' of Homarus and the epicuticle ofSarcophaga larva. Here again the main difficulty of comparison arises from


the fact that the exocuticle of myriapods contains chitin whereas the similarlayers in Homarus and Sarcophaga are described as being free from chitin.As has been seen, extensive sclerotization of the sclerite of Lithobius has ledto the whole of the exocuticle being unstainable. The epicuticle of Sarcophagalarvae stains red with Mallory but loses its affinity for the stain on beingconverted into the sclerotin of the puparium. Diaphanol treatment restoresthe larval staining reaction. Similarly in Lithobius diaphanol treatment leadsto the exocuticle staining red with Mallory—the two cases seem quite com-parable. Then again, where sclerotization does not seem to be so advanced,as in the exocuticle of the arthrodial membrane of Lithobius, this layer stainsred without previous diaphanol treatment.

The outer layer of the epicuticle of Sarcophaga larvae (the outer epicuticle—see Dennell, 1946) is much more resistant than the bulk of the epicuticleand is lipoid in nature. Were both layers of the epicuticle not described asbeing free from chitin it would be natural to compare, the inner and outerepicuticle with a region of pro-sclerotin underlying a region of completedsclerotin such as obtains in the sclerite exocuticle of Haplophilus or the wholeof the sclerite cuticle of Lithobius.

The suggested condition in myriapods where the fat is believed to impreg-nate the whole of the lipophil outer layers and to originate from epidermalgland cells recalls the condition in Homarus in which the 'cuticle' containslipoid material. Thomas (1944) records that the 'cuticle' of Lepas containslipoid. This latter author also records a fat reaction in the tegumental glandsof Lepas. If the glands of Homarus secrete the lipoid of the 'cuticle' then it isnot surprising that the glands show a periodicity in relation to the layingdown of the new integument and that Yonge believed that these glands wereresponsible for the secretion of the whole of the 'cuticle'. Pryor (1940&)suggested that the lipoid which he believed to impregnate the sclerotin ofinsects might be secreted from epidermal gland cells. Lastly, on this samepoint, it may be mentioned that Langner (1937) records a fat reaction in theepidermal glands of the diplopods she studied.

It has been suggested (Blower, 1950) that the myriapod cuticle might beconsidered as a chitinous matrix impregnated to varying extents by pro-sclerotin which may or may not be tanned, that is to say, which may or maynot have the amber colour and inertness usually associated with completesclerotin. To this generalization may be added the fact that lipoid materialappears to be secreted on to the surface of the cuticle from glands in theepidermis and also to impregnate the regions of sclerotin and pro-sclerotin.Here, there appears to be an intimate association of the lipoid with the aroma-tic groups of the sclerotin (evidenced by the fact that destruction of thearomatic groups by diaphanol or nitric acid renders the lipoid available tosudan colouring agents).

The myriapod cuticle, then, is characterized by the absence of an outerlayer which is both resistant and non-chititious, by the presence of a materialallied to sclerotin but peculiar in being stable and resistant independent of

Chilopod and Diphpod Cuticle 161

tanning, as usually understood, and by the apparent presence of lipoid in allthe modified regions of the cuticle. These characteristics, in the absence ofdetailed information as to the mode of development of the cuticle, makeextensive comparison with other arthropod cuticles a matter of some difficulty.

ACKNOWLEDGEMENT

My sincere thanks are due to Professor R. Dennell for the original sugges-tion of this problem and for the help and encouragement that he has freelygiven to me at all times.

REFERENCESBLOWER, J. G., 1950. Nature, 165, 569.BROWN, C. H., 1950. Ibid., 275.BROWNING, H. C , 1942. Proc. Roy. Soc. B, 131, 65.CAMPBELL, F. L., 1929. Ann. Ent. Soc. Amer., 22, 401.CARLETON, H. M., 1938. Histological technique. Oxford.CLOUDSLEY-THOMPSON, J. L., 1950. Nature, 165, 692.DENNELL, R., 1946. Proc. Roy. Soc. B, 133, 348.

1947a. Ibid., 134, 79.19476- Ibid., 134, 485.

FUHRMANN, H., 1921. Z. W1SS. Zool., 119, I.HACKMAN, R. H., PRYOR, M. G. M., and TODD, A. R., 1948. Biochem. J., 43, 474.LAFON, M., 1943. Ann. Sci. nat. zool., 5, 113.LANGNER, E., 1937. Zool. Jb. (Abt. Anat.), 63, 483.LEA, A. J., 1945. Nature, 156, 478.LEE, A. B., 1937. The microtomist's vade-mecum. London (Churchill).LEES, A. D., 1947. J. exp. Biol., 23, 379.LISON, L., 1936. Histochimie animate. Paris (Gauthier-Villars).PLOTNIKOW, W., 1904. Z. wiss. Zool., 76, 333.PRYOR, M. G. M., 1940a. Proc. Roy. Soc. B, 128, 378.

19406. Ibid., 393.THOMAS, H. J., 1944. Quart. J. micr. Sci., 84, 257.VERHOEFF, K. W., 1925. 'Chilopoda' in Bronn's Klass. u. Ordn. Tierreichs, 5, Abt. 11.WIGGLESWORTH, V. B., 1947. Proc. Roy. Soc. B, 134, 163.

1948a. Quart. J. micr. Sci., 89, 197.19486. Biol. Rev., 23, 408.

WILKS, R. A. C, 1938. Nature, 142, 958.YONGE, C. M., 1932. Proc. Roy. Soc. B, 111, 298.

a comparative stud of thye chilopod and diplopod cuticle...

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