A Comparative Study of the Chilopod and Diplopod Cuticle 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 diplopods is described. Two principal layers of the cuticle are recognized, an outer homogeneous and refractile exocuticle which is usually but not invariably pigmented and an inner endocuticle. 2. The endocuticle and the exocuticle both contain chitin. The exocuticle is considered as a modification of the outer part of the chitinous matrix by an impregnating substance. 3. Certain properties of the impregnating substance are described. It appears to be a substance rich in phenolic groups, perhaps a protein, which has a stability and resistance to acids in its own right irrespective of the presence of pigment or aromatic cross-links. Pro-sclerotin is suggested as a name for this substance. Chemical tests show that it is present in regions not optically definable as exocuticle. 4. The epidermis is virtually an epithelium of gland cells which appear to secrete lipoid material. The lipoid passes on to the surface of the cuticle by means of ducts passing through the cuticle. Here it appears to form a superficial layer and to impregnate the sclerotin and pro-sclerotin. 5. There appears to be an intimate association of lipoid with the aromatic groups of the pro-sclerotin and sclerotin. Destruction of the aromatic groups by means of an oxidizing 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 of other arthropods. The main difficulty in the way of extensive homology with other arthropod types is the absence in myriapods of an outer non-chitinous and resistant layer. CONTENTS PAGE INTRODUCTION............. 142 MATERIAL AND METHODS.. 142......... THE TWO PRINCIPAL LAYERS OF THE CUTICLE AND THE VARIATION IN THEIR FORM AND EXTENT............. 143 STAINING REACTIONS. 145.......... THE PHENOLIC SUBSTANCE IMPREGNATING THE CHITINOUS MATRIX....149 ARGENTAFFIN MATERIAL IN THE CUTICLE........153 THE EPIDERMAL GLAND CELLS AND THE OCCURRENCE OF LIPOID THE CUTICLE 154 IN. PORE CANALS.............156 RESUME AND DISCUSSION...........156 ACKNOWLEDGEMENT...........161 REFERENCES 161 [Quarterly Journal of Microscopical Science, Vol. 92, part 2, June 1951.]
142 Blower A Comparative Study of the INTRODUCTION DURING recent years there has been much interest in the integument of 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 this subject 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 principal layers in Homarus are common to all arthropods, there has been much discussion as to the probable homology of the described conditions in different arthropods. Pryor (19406) pointed out the chemical similarity of the 'cuticle' of Homarus with the epicuticle in insects. Dennell (1946, 1947a and b) made an extensive comparison of insect and crustacean cuticles and saw a considerable correspondence in detail between the fundamental layers of the cuticle in these two groups. Apart from Browning's paper on the cuticle of Tegenaria (1942), a description of the epicuticle in ticks (Lees, 1947) and a paper by Langner (1937) on the cuticle of diplopods, arthropods other than insects and Crustacea have received little attention. It is the aim of the present paper to illustrate, by selecting a few clear-cut histochemical reactions, the general features which have emerged from an extensive study of the cuticle in various chilopods and diplopods and which may contribute towards a clearer understanding of the fundamental 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 and millipedes. Animals were kept alive in glass vessels in the laboratory. The following of the larger species have been used for detailed examination: Chilopoda Lithobius forficatus (L.), Haplophilus subterraneus (Leach); Diplopoda Schizophyllum sabulosum (L.), Tachypodaiulus niger (Leach). Most histochemical tests, unless otherwise stated, have been performed on frozen sections, cut at 20 ft, of material fixed in 5 per cent, formaldehyde in 0-9 per cent, saline. Fixed material was impregnated with 25 per cent, gelatine solution 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 epidermis and the staining reactions of the cuticle. For chilopods, Flemming without acetic in 0-9 per cent, saline proved the best fixative. Heidenhain's 'Susa' was also found useful. For diplopods, fixation by neutral formaldehyde (5 per cent.) followed by decalcification with a 30 per cent, aqueous solution of sodium hexametaphosphate (Wilks, 1938) provided the best pictures of the epidermis. The presence of calcium salts in the cuticle precluded the use of an acid fixative such as Flemming, although fairly good results were obtained with 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 removed by immersing sections for 24 hours in ethylene chlorhydrin (see Lea, 1945). Mallory's triple stain and iron haematoxylin, both alone, and when combined with ponceau acid fuchsin and light green (Masson's trichrome) have been the 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 are optically discernible. The inner layer is colourless and is horizontally striated. The outer layer is homogeneous, that is to say, not obviously horizontally striated, is highly refractile, and is usually pigmented in part or in the whole of its thickness. In this work the terms endocuticle and exocuticle have been applied to the optically distinguishable inner and outer layers respectively. In a chilopod the exocuticle varies in its form and thickness in different regions of the body (fig. 1, A and B). The exocuticle of the sclerites is thicker than 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 thickness of the cuticle whereas the arthrodial membrane exocuticle is very thin and lobulated. The sclerite of Haplophilus (fig. IB) is in marked contrast to that of Lithobius; the exocuticle is here produced inwards in the form of cones. These cones appear to occupy the field of activity of individual epidermal cells as manifested by the hexagonal areas which are seen in surface views of the cuticle. This prismatic arrangement is also manifest at the external face of the cuticle by shallow convexities. Although the hexagonal areas can be seen 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 Haplophilus are intucked and the exocuticle from the two contiguous sclerites gradually decreases in thickness towards the membrane, where it is not developed at all. The region of transition from sclerite to arthrodial membrane has a characteristic exocuticle and is here termed the 'intermediate sclerite' condition. The inner face of the exocuticle in this region is produced inwards as minute conical papillae (see fig. 1, A and B 'intermediate sclerite'). The pleural regions of Lithobius and Haplophilus are quite different. In Lithobius there is very little sclerotization, the majority of the pleuron cuticle being in the condition of arthrodial membrane whereas in Haplophilus there is a series of pleurites developed in the pleuron, each separated from the next by an intucked arthrodial membrane. This difference and the difference in the form of the cuticle as a whole is characteristic of the orders to which Lithobius and Haplophilus belong. The intersegmental membranes of both animals are usually in the condition of 'intermediate sclerite'. In section the outer surface of the sclerites of Lithobius and Haplophilus
144 Blower A Comparative Study of the appears to be bounded by a thin colourless membrane. This is in all probability a diffraction effect since a similar appearance characterizes the inner edge of the endocuticle when this is separated from the epidermis during tergibe scleribe arbhrodial membrane inbermediabe scleribe' pleuribes pleuro-tergal arch of mebazombe stemibe arbhrodial membrane sssssso scleribe k -inbersegmental membrane pleuro-berqal s 'arch of prozonite sbernibe FIG. I. Transverse sections of the cuticle, diagrammatized from free-hand drawings. A, Lithobius forficatus. B, Haplophilus subterraneus. c, Schizophyllum sabidosum. Exocuticle black, endocuticle shaded. sectioning. Verhoeff (1925) and Fuhrmann (1921) describe three cuticular layers in a chilopod. The outermost layer in each case undoubtedly refers to that which has just been described as a diffraction effect. The histological validity of this outer layer will be discussed later. In the meantime a twolayered cuticle will be considered as a working hypothesis and the following synonyms may be tentatively tabulated:
Chilopod and Diplopod Cuticle 145 Verhoeff Oberflachenschicht Farbschicht Lammellenschicht Fuhrmann Grenzhautchen Aussenlage Innenlage This work Regarded as a diffraction effect Exocuticle Endocuticle The exocuticle of the diplopods Schizophyllum and Tachypodoiulus does not differ perceptibly in different regions of the body (fig. ic), for here it is the presence of a calcined outer endocuticle which distinguishes a sclerite from an arthrodial membrane, where the endocuticle is very thin or absent altogether. The inner surface of the exocuticle is produced inwards as numerous minute papillae as in the intermediate sclerite of a chilopod. The sclerite endocuticle can be divided into an inner part with conspicuous horizontal striae and an outer part where the striae are not so obvious (fig. ic). Langner (1937), besides two layers of endocuticle, distinguishes two outermost layers, an exocuticle and an overlying epicuticle in several species of diplopod. Cloudsley-Thompson (1950) also describes an epicuticle as distinct from an exocuticle in certain centipedes and millipedes. Even so, a twolayered cuticle will be taken as a basis for description and the presence or absence of an epicuticle overlying the exocuticle will be discussed later. STAINING REACTIONS In the unhardened fore-gut cuticle of Homarus (Yonge, 1932) and that of the larva of Sarcophaga (Dennell, 1946) there is a close correspondence of staining reaction, and Dennell suggests, as did Yonge, that the red- and bluestaining layers differentiated by Mallory's triple stain are probably fundamental to all arthropods. As the cuticle of the last larval instar of Sarcophaga is converted into the puparium, the inward extension of the hardening process modifies the staining reaction (Dennell, 1947a). The outer red-staining region loses its affinity for the stain and develops an amber-brown colour as also does the outer part of the endocuticle. The puparium cuticle, however, stains red at the junction of the 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 layer stained with acid fuchsin and an inner layer stained with aniline blue. The red-staining layer has been 'pushed inwards', however, to various depths, according to the extent to which the outer layers have been involved in the hardening process. In the sclerite cuticle of Schizophyllum and Tachypodoiulus the exocuticle stains red except for a very thin outermost layer and the whole of the endocuticle stains blue the inner endocuticle staining much more deeply than the outer. In Haplophilus the exocuticle stains red except for the outermost region of the sclerite. The cones do not stain so deeply as the continuous part of the exocuticle. In Lithobius none of the sclerite exocuticle stains red but practically the whole of the sclerite endocuticle takes the acid
146 A Blower A Comparative Study of the exocubicle basiphil zone beneath exocuticle gland ducb endocubicle gland cell nucleus of epidermal cell basemen b membrane ouber non-staining parb of exocuticle papillabe part of exocuticle gland 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, 'Intermediate sclerite'. c, Arthrodial membrane. Camera lucida drawings. Flemming without acetic, iron haematoxylin. fuchsin. In the arthrodial membranes however, the exocuticle stains red and the endocuticle blue. With Masson's trichrome stain the details are similar, the acid fuchsinophil zones of Mallory preparations taking iron haematoxylin in their outer parts
Chilopod and Diphpod Cuticle epidermal cell basemenb membrane 'inbermediabe. scleribe' exocufcicle cone of exocubicle gland cell Inbermediabe scleribe eaocutrcfe epidermis endocubicle gland cell 50]X FIG. 3. Transverse section of an arthrodial membrane between the tergite and a pleurite of Haplophilus subterraneus. Below are surface views of the cuticle corresponding to the three regions indicated on the section. Camera lucida drawing. Flemming without acetic, iron haematoxylin. and ponceau acid fuchsin inwardly; the region staining blue with Mallory taking the light green of the Masson combination. In the sclerite of Haplophilus, for example, the continuous part of the exocuticle stains blue-black with the haematoxylin, save at its free edge which does not stain. The cones stain with the ponceau and acid fuchsin, which, together with a little of the retained haematoxylin, give a purple colour. Figs. 2, 3, 4, and 5 illustrate the
148 Blower A Comparative Study of the various regions of the cuticle stained with iron haematoxylin alone. It will be noticed that the sclerite endocuticle of Lithobius stains with haematoxylin only in its outer layers; the inner layers stain red when the haematoxylin is outer non-sbaining parb of eiocubide continuous parb of exocubicle pnsmabic cone of exocubicle gland duct endocuticle epidermal cell gland cell basement membrane FIG. 4. Transverse section of the sclerite cuticle of Haplophilus subterraneus. Camera lucida drawing. Flemming without acetic, iron haematoxylin. 6uber non-staining and inner basiphif exocubicle swelling^ in gland 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 lucida drawing. Neutral formaldehyde fixation followed by decalcification in sodium hexametaphosphate and removal of epidermal pigment by ethylene chlorhydrin. followed with the other stains of Masson's combination. The figures show clearly the passage inwards of the basiphil zone (equivalent to the outer parts of the red-staining zones with Mallory) associated with greater degrees of tanning of the outer layers. The whole of the arthrodial membrane exocuticle of Lithobius is basiphil. The exocuticle of Schizophyllum, which is of a more distinct amber colour than that of the arthrodial membrane of Lithobius, fails
Chilopod and Diplopod Cuticle 149 to stain in its outermost layer. This non-staining zone is extended in the sclerite exocuticle of Haplophilus and in the sclerite of Lithobius the entire exocuticle fails to stain, the basiphil region occupying a large part of the endocuticle. In all cases, treatment with diaphanol (chlorine dioxide in glacial acetic acid) leads to the entire thickness of the exocuticle staining with either the acid fuchsin of Mallory or the iron haematoxylin used in Masson's combination the endocuticle staining with aniline blue or light green respectively. In Lithobius the intensity of blue or green staining of the endocuticle is greater in the sclerite than in the arthrodial membrane, and similarly in Schizophyllum the inner endocuticle still has a greater affinity for these stains after diaphanol treatment. Dennell (1946) has described the effect of diaphanol on the subsequent staining reaction of the cuticle of Sarcophaga puparia. The larval condition is restored as far as staining reaction is concerned and there appears to be a reversal of the effects of tanning. Since diaphanol treatment in this case restores the fundamental two-layered nature of the cuticle it might be assumed that the endocuticle and exocuticle of the described myriapods are the homologous fundamental layers. The two layers of Sarcophaga and Homarus are, however, further differentiated by the presence or absence of chitin, whereas no such distinction can be drawn in the case of the myriapod cuticle. It might be added here that the division of the thin outer layer of a diplopod into nonstaining and basiphil zones appears to be the basis which Langner (1937) adopts in distinguishing an epicuticle and an exocuticle. Since both regions of the outer layer contain chitin (see next section) and diaphanol treatment removes the distinction, it is doubtful whether this particular staining reaction is a suitable criterion to adopt. THE PHENOLIC SUBSTANCE IMPREGNATING THE CHITINOUS MATRIX The optical appearance of the exocuticle and the endocuticle is, in fact, the only criterion which constantly differentiates these two layers. Although certain chemical properties are always associated with the exocuticle, the same properties are sometimes shown by the endocuticle. Even such a characteristic feature as pigmentation is not shown by all regions of the exocuticle. A feature of both layers is the presence of chitin. This fact is made very clear in the case of Lithobius where the exocuticle often separates from the endocuticle during the potash treatment, but is nevertheless just as intensely positive to the chitosan test (see Campbell, 1929) as the endocuticle. The exocuticle is therefore to be regarded as the modification of the outer layers of a chitinous matrix by the addition of some impregnating substance or substances. The exocuticle of all forms always gives intensely positive xanthoproteic and Millon reactions, from which it can be assumed that phenolic groupings enter largely into the composition of the impregnating substance. The
150 Blower A Comparative Study of the exocuticle is not, however, the only part of the cuticle positive to these two tests. The whole of the sclerite endocuticle of Lithobius is likewise intensely positive. When frozen sections of the cuticle are treated with concentrated mineral acids the exocuticle survives the treatment, as also do the ducts of the epidermal gland cells which bridge the gap vacated by the endocuticle. The sclerite endocuticle of Lithobius dissolves much more slowly than that of the arthrodial membranes. In the sclerite of Haplophilus the inner part of the exocuticle, that is, the part divided into inwardly projecting cones, dissolves in several hours at room temperature. The outer, continuous part of the exocuticle is a typical amber qplour as is the whole exocuticle of Lithobius and, like that of Lithobius, survives acid treatment for days. The inner discontinuous part of the exocuticle of Haplophilus is not pigmented. The foregoing observations may be summarized thus: the typical amber-coloured exocuticle is very resistant to concentrated mineral acid. The colourless inner region of the exocuticle in Haplophilus is not quite so resistant to acid, and finally the sclerite endocuticle of Lithobius is even less resistant but nevertheless shows quite a degree of resistance when compared with the arthrodial membrane endocuticle, which dissolves almost immediately. All regions showing any degree of resistance are alike in giving positive xanthoproteic and Millon reactions. It appears that the chemical difference between the conical part of the exocuticle of Haplophilus and the sclerite endocuticle of Lithobius is one of degree and not kind the cones of Haplophilus being included as part of the exocuticle by virtue of their homogeneity and refractility. It appears from these facts that the process of sclerotization consists in the impregnation of a chitinous matrix by a substance rich in phenolic groups and a subsequent.process which renders this material resistant to acids. The development of refractility and, later, of an amber colour seems to be correlated with the development of this resistance, although resistance is developed by the phenolic substance present in the inner, discontinuous, part of the exocuticle of Haplophilus without the assumption of an amber colour and in the sclerite endocuticle of Lithobius without even the assumption of refractility. As suggested in a preliminary communication (Blower, 1950) it seems possible that this phenolic impregnating substance is a protein in contrast to the alcohol-soluble phenol which is one of the precursors of sclerotin in the cockroach ootheca (Pryor, 1940a). Evidence in favour of this view arises from a comparison of the arthrodial membrane endocuticle of Lithobius, on the one hand, with the sclerite endocuticle of this same animal where the phenolic substance appears to be present in a simple form, since here the substance has conferred a degree of resistance on the endocuticle but has not rendered it homogeneous or refractile. In the first place it will be remembered that the sclerite endocuticle gives positive Millon and xanthoproteic reactions whereas the arthrodial membrane endocuticle does not. This, and the following 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 performed on a transverse section where both regions under consideration are present together. The iso-electric points of the two regions have been determined by the method employed by Yonge (1932). The value for the sclerite endocuticle is between ph 5-4 and ph 5-6 whereas that for the arthrodial membrane endocuticle was between ph 2-8 and ph 3-0. It follows from this that the substance responsible for the difference between the two regions is probably amphoteric such as an amino-acid or protein. This difference in iso-electric points is reflected in the staining reactions of the two regions. With Mallory the sclerite endocuticle stains red whereas the arthrodial membrane endocuticle stains a light blue. The substance differentiating the sclerite endocuticle from the arthrodial membrane endocuticle is still present after treatment with water, alcohol, xylene, &c, and remains demonstrable by its staining reaction. It seems on this account that a simple amino-acid is not responsible and that a protein is probably involved. The fact that the additional substance in the sclerite endocuticle of Lithobius confers a degree of resistance to acids would also suggest that the substance concerned is something other than a free phenol or amino-acid. The resistant nature of the substance impregnating the sclerite endocuticle is manifest during the application of the chitosan test. In sections of potash-treated material the sclerites are still noticeably different from the arthrodial membranes. The two layers of the sclerite are quite obvious and the exocuticle is still homogeneous. The arthrodial membranes appear to have separated into individual laminae. At the first application of the iodine and sulphuric acid only the arthrodial membranes assume the violet colour. The sclerite endocuticle assumes a much lighter and more delicate tinge of violet, the exocuticle remains colourless. A few moments after flooding the slide the sclerite endocuticle and exocuticle split up into individual laminae, expand to the extent of the arthrodial membranes and assume the deep-violet colour first shown by these membranes. If the temperature of the potash during treatment has fallen below that specified by Campbell (1929), i.e. 160 0 C, or if the solution has not been fully saturated, the initial reluctance of the layers of the sclerite to assume a deep-violet colour is prolonged. Pieces of cuticle in this condition immersed in 3 per cent, acetic acid did not completely dissolve. The residue still gives a strongly positive Millon test. Lafon (1943), on treating the cuticle of a scorpion with 10 per cent, potash at ioo C, found that two layers survived the treatment a colourless layer composed of chitin and an outer very thin amber-coloured layer. Lafon suggests that this resistant outer layer is comparable with the cuticulin of insects. This layer bears similarity to the exocuticle of Lithobius, which remains as a discrete layer after potash treatment. This layer in Lithobius survives even more brutal treatment than that which Lafon used, still remaining homogeneous after 2 hours at 155 0 C. in concentrated potash. It may be
152 Blower A Comparative Study of the that in the scorpion this outer layer contains chitin but shows a reluctance to display itself similar to that of the exocuticle of Lithobius, and the unsuspected presence of chitin would account for the anomalous figures obtained from analysis of this layer. Lafon subjected Lithobius to the same treatment, but in view of the fact that he was dealing with whole pieces of cuticle, the small amount obtainable from Lithobius may have led to his overlooking a surviving outer layer in this case. As has been suggested (Blower, 1950) the substance impregnating the sclerite endocuticle of Lithobius and the exocuticle of all forms may be a protein with a high tyrosine content, since it is very rich in phenolic groups and has an iso-electric point similar to that of tyrosine. If this were so it seems possible that there is a development of cross-links by the oxidation of the side chains of this tyrosine-rich protein. Brown (1950) has suggested that this method of tanning may take place in certain regions of Mytilus and in the egg-capsule of Fasciola. Whatever the mechanism of tanning, it seems possible from the foregoing facts that the substance differentiating the sclerite endocuticle of Lithobius from the arthrodial membrane is a protein rich in phenolic groups and probably represents the precursor of typical sclerotin. This substance will accordingly be termed pro-sclerotin. The prismatic cones of the exocuticle of Haplophilus may then be regarded also as consisting of pro-sclerotin. In the diplopod Schizophyllwm there is a thin exocuticle, only the outer part of which is amber coloured. The endocuticle is optically divisible into two distinct layers. The outer layer is impregnated with calcium salts a fact ascertained by the application of the alkaline pyrogallol test (Lison, 1936). The inner layer is much more conspicuously laminated and appears to be impregnated with pro-sclerotin. It gives positive xanthoproteic and Millon tests and survives acid treatment for much longer than the outer endocuticle, but is not.so resistant as the exocuticle. As would be expected, the inner endocuticle has a higher iso-electric point than the outer (ph 36 compared with ph 2-8). The behaviour of the cuticle towards diaphanol casts a little more light on the nature of sclerotin and pro-sclerotin. The effect of this reagent on a tanned cuticle as described by Dennell (1946) has been mentioned. The fully tanned puparial case of Sarcophaga is bleached by diaphanol and the condition of the cuticle in the last larval instar appears to be restored as is evidenced by the staining reaction of the diaphanol-treated cuticle. Its effect on the chilopod and diplopod cuticle is to remove all traces of the amber colour resident in 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 by destroying the aromatic nuclei by oxidation. That it does in fact destroy the rings is evidenced by the fact that a diaphanol-treated cuticle gives no trace of a positive xanthoproteic or Millon reaction in any region. The fact that it does not, however, destroy the resistance of the exocuticle to acids, points again to the fact that pro-sclerotin itself is a substance with a degree of
Chilopod and Diplopod Cuticle 153 resistance and stability which does not depend on the presence of aromatic cross-links. It will be recalled that the sclerite and arthrodial membrane endocuticles of Lithobius still stain differently after diaphanol treatment with Mallory the sclerite endocuticle stains a deep purplish-blue in contrast to the arthrodial membrane which stains a very pale blue. ARGENTAFFIN MATERIAL IN THE CUTICLE It is stated (Lison, 1936) that a positive argentaffin reaction (reduction of an ammoniacal silver nitrate solution) indicates the presence of polyphenols, amino-phenols, or polyamines. This reaction has often been employed to demonstrate the distribution of phenolic substances in the cuticle. Hackman, Pryor, and Todd (1948) describe phenolic substances in the epicuticle (here referred to as 'exocuticle') of Tachypodoiulus on the evidence furnished by this 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 membrane never reduces the silver. Ammoniacal silver has also been used in order to determine whether a polyphenol layer covered by a wax layer lies external to the exocuticle as described in Rhodnius and Tenebrio (Wigglesworth, 1947, 1948a). A specimen of Lithobius was allowed to crawl in carborundum powder for several hours and then plunged into silver solution for 24 hours. The cuticle was then cleaned and washed well, and a portion embedded in wax and sectioned. Another portion of the cuticle was mounted whole. The whole mount revealed numerous criss-crossing brown lines on the surface of the sclerite representing the scratching of its surface by the carborundum particles. In sections of the sclerite these brown scratch lines were represented by lens-shaped areas at the surface of the cuticle which had reduced the silver more strongly than elsewhere. Beneath the exocuticle in the outer third of the endocuticle numerous vertically disposed filaments were stained much more intensely than the exocuticle itself. Furthermore, the contents of the epidermal glands and their ducts had reduced the silver nitrate solution and appeared almost black. It will be remembered that the whole of the sclerite endocuticle of Lithobius is intensely positive to Millon's reagent, and the whole has a higher isoelectric point than the arthrodial membrane endocuticle. If the argentaffin reaction indicates the presence of phenolic substances one would expect the whole of the sclerite endocuticle of Lithobius to darken evenly. This it does not do. As will be seen later the epidermal glands appear to secrete a lipoid material, particularly evident in Haplophilus; and since these and the contents of their ducts reduce the silver solution in Lithobius and in Haplophilus, it seems possible that the lipoid itself may be argentaffin. Although Lison (1936) does not include lip'oids amongst argentaffin substances, it seems theoretically possible that unsaturated fats at least would be
154 Blower A Comparative Study of the capable of reducing ammoniacal silver nitrate. To ascertain whether there is a possibility that cuticular lipoid is argentaffin, animals were cut into two pieces, the cuticle freed from underlying tissue and one piece boiled for 2 hours in chloroform. The untreated piece of cuticle was retained as a control. Both pieces were embedded in paraffin wax and sectioned. Two ribbons, one from each piece, were mounted on a slide, freed from wax and the slide immersed in 5 per cent, ammoniacal silver nitrate for 24 hours. The sections 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 less in the chloroform-treated cuticle. Furthermore, the extent of the argentaffin zone beneath the sclerite exocuticle was considerably reduced. The chloroform used for the extraction was allowed to evaporate in a watch-glass. An orange-coloured material remained which stained readily with sudan black. As will be made clear in the next section the distribution of lipoid in the cuticle appears to follow the distribution of sclerotin and pro-sclerotin. It is possible that the results of an argentaffin test may indicate the presence of other 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 ducts arising from glandular cells in the epidermis. The various types of gland and duct are shown in figs. 2, 3, 4, and 5. In general there are more glands beneath the sclerites than elsewhere, or, in other words, those regions of the animal most likely to come into contact with its solid environment are well supplied with glands. The sclerite epidermis is in fact an epithelium of these gland cells. Their ducts pass through the cuticle and open to the exterior between the cuticular prisms. The staining reaction of the glands in the epidermis 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 of the glands and their ducts are coloured blue. In Lithobius the whole of the epidermis is weakly sudanophil, but sometimes inclusions of a strongly positive material are to be found within the gland ducts. In Schizophyllum and Tachypodoiulus one can only say that the epidermis generally is strongly sudanophil the epidermal pigment granules making the precise location of the lipoid material difficult to see. The location of the lipoid material in the cuticle is not made very clear by sudan staining. In Lithobius only the arthrodial membrane and the intermediate sclerite exocuticle shows signs of the blue colour. There is, however, a very thin sudanophil layer external to the exocuticle. In Haplophilus the cones of the exocuticle are slightly sudanophil and again there is a very thin sudanophil layer external to the exocuticle. In the intermediate sclerites of this 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 the entire animal. Treatment with diaphanol has an interesting effect on the subsequent staining with sudan black, in so far as it definitely intensifies it. In a frozen section of diaphanol-bleached material of Schizophyllum or Tachypodoiulus the exocuticle is coloured almost black with sudan black and this colouring extends inwards for about a quarter the thickness of the outer endocuticle as numerous very fine filamentous processes. There appears to be a penetration of lipoid material down the pore canals (see next section). The mechanism by which treatment with diaphanol leads to the lipoid material becoming sudanophil is perhaps connected with the fact that it destroys the aromatic links. Possibly the lipoid is in some way intimately attached to these groups and on their destruction is more readily available to the sudan stain. This intensification of sudan staining is also evident in Haplophilus. The effect of diaphanol on the subsequent staining with haematoxylin may possibly be explained on similar lines. It will be remembered that in the sclerite of Lithobius after diaphanol treatment the exocuticle stains with haematoxylin, whereas in the untreated cuticle the exocuticle does not stain 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 aromatic groups in the exocuticle, has made available some basiphil substance originally firmly attached to these groups. By analogy with the above facts concerning the intensification of sudan staining by diaphanol and the fact that the contents of the gland ducts and glands are basiphil, it might be suggested that the substance made available to the haematoxylin is in fact a lipoid. The haematoxylin 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 chlorate in concentrated nitric acid show first a dissolution of the endocuticle and then a breaking down of the exocuticle into an oily material which stains readily with sudan black. This appears to be an oxidative process, similar to that produced by diaphanol, but more vigorous, and presumably it leads in the same way to a liberation of the lipoid by a destruction of the aromatic groups with which it seems to be associated. Nitric acid alone can effect this process. If whole pieces of animals are immersed in cold concentrated nitric acid the process can be studied more critically since the oxidation proceeds much more slowly. Pieces of Haplophilus thus treated show first a dissolution of the endocuticle. After an hour nothing remains but the exocuticle. Before the amber colour has disappeared oily droplets float to the surface. These droplets, smeared on to a slide, colour with sudan black. The outer ambercoloured part of the exocuticle is still intact at this stage and therefore the droplets have probably come from the dissolution of the cones of the exocuticle. If the amber-coloured portion is left overnight it is bleached and on washing and immersing in sudan black it takes up the colour rather unevenly.
156 Blower A Comparative Study of the This latter observation applies also to Lithobius, although no oily droplets free themselves from the cuticle as in Haplophilus. In Lithobius, during treatment with cold nitric acid, the intermediate sclerites and the arthrodial membranes dissolve completely before the sclerites have lost their amber colour. In both cases several days' treatment with acid results in the complete solution of all but the exocuticle of the sclerites. As was pointed out in the preceding section it seems possible that the cuticular lipoid is capable of reducing silver nitrate. The vertically-disposed argentafen filaments beneath the exocuticle of Lithobius are thus possibly lipoid in nature. Here it seems possible that the lipoid material penetrates down the pore canals, as is indicated by sudan colouring in Schizophyllum and Tachypodoiulus. It is not clear, however, if this be the case, why sudan colouring in Lithobius does not give the same picture. PORE CANALS When a section of Tachypodoiulus or Schizophyllum is immersed in concentrated mineral acid the outer endocuticle dissolves rapidly. Between the inner endocuticle and the exocuticle, besides the persistent ducts of the epidermal gland cells, there are to be seen numerous fine filamentous processes emerging from the inner endocuticle and continuous distally with the inwardly projecting papillae from the underside of the exocuticle. By analogy with other arthropods these structures appear to represent the solid contents of the pore canals. No such clear demonstration of pore canal filaments has been seen in a chilopod. It will be remembered, however, that the under surface of the intermediate sclerite exocuticle in Lithobius and Haplophilus is produced inwards as minute papillae, and these may represent the distal ends of original pore canals into which exocuticular material has penetrated. Furthermore, treatment with silver has revealed filaments beneath the sclerite exocuticle of Lithobius. Unfortunately no developmental history of what appear to be the pore canals is available. Plotnikow (1904) figures pore canals in the form of conical tufts with their apices arising from the epidermis in the developing larval cuticle of Tenebrio. The* fully formed cuticle of this larva has the inner part of its exocuticle in the form of cones as has Haplophilus. If the surface of the cuticle of Haplophilus is examined over the region of transition from intermediate sclerite to arthrodial membrane (fig. 3, A, B, c), the impression is obtained that each cone of the exocuticle is formed by the merging together of exocuticular material in the pore canals. Possibly the original pore canals of Haplophilus are tufted and give rise to the conical arrangement of the inner 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 do not 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. By considering different regions of the same animal, and different animals, the possible changes attendant to the formation of a typical amber-coloured and highly refractile exocuticle may be inferred. In the animals studied each region of the cuticle can be placed in an ascending scale of conditions approaching a typical exocuticle (see Table I). TABLE I 1. Arthrodial membrane endocuticle of Haplophilus and Lithobius and outer endocuticle of Schizophyllum. Sclerite endocuticle of Lithobius and the inner endocuticle of Schizophyllum. 3. Prismatic cones of Haplophilus. Inner layers of exocuticle of Schizophyllum. 4. Stainable portion of the continuous exocuticle of Haplophilus. Arthrodial membrane exocuticle of Lithobius. Inner portion of the exocuticle of intermediate sclerite in Lithobius and Haplophilus. 5. Whole of sclerite exocuticle of Lithobius and the non-staining outermost regions of the exocuticle of Haplophilus and Schizophyllum. < co I" *4 T P ^ O -52 - W OS o «s o < The process of exocuticle formation seems to be heralded by the appearance of a substance, possibly a protein, rich in phenolic groups, in the chitinous matrix of the cuticle. This substance has been given the name 'pro-sclerotin'. It is resistant to acids even before the more characteristic features of an exocuticle are manifested. The substance eventually renders the impregnated region homogeneous and refractile, by which time the region may be called exocuticle on the definition here adopted. Later there is a development of even greater resistance, the development of an amber colour and finally the loss of staining reaction.
158 Blower A Comparative Study of the 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 puparial case of Sarcophaga, is regarded as a tanning of cuticular protein attended by the development of an amber-brown colour. Pryor, however, states that a water-soluble protein is passed into the cuticle to form the basis of the sclerotin whereas Dennell considers that the pre-existent cuticular protein may be involved in the tanning process. It does not seem that the whole of the process of exocuticle formation as suggested in this work is comparable with the tanning process as described by Pryor and Dennell. Neither in Sarcophaga nor in the cockroach ootheca is there a substance described,similar to pro-sclerotin. In the cockroach ootheca neither of the precursors of sclerotin appears to have any degree of resistance before they are linked together as sclerotin. Browning (1942) describes a colourless exocuticle in certain regions of the spider Tegenaria, and Brown (1950) records the fact that the precursor of sclerotin in certain structures of Mytilus and in the egg-capsule of Fasciola is not water- or alcohol-soluble but is probably a protein. Brown also suggests that the deamination of the phenol destined to tan the protein as described by Pryor (1940 a and b) is possibly a derived condition. Both the cockroach ootheca and the puparium of Sarcophaga appear to be special cases. In Sarcophaga the sclerotin of the puparium is formed at the end of the instar, and it may be that there has been a modification of the precursors of sclerotin to diffusible substances since they have to pass to a region of the cuticle distal to the epidermis. A feature invariably associated with the exocuticle of myriapods is the presence of lipoid material. This lipoid appears to be secreted by the gland cells of the epidermis through ducts which open on to the surface of the cuticle. At the surface it appears to form a thin film. It appears also that the exocuticle is impregnated with lipoid where there appears to be an intimate association of the lipoid and sclerotin or pro-sclerotin. It may be that the lipoid in the exocuticle is responsible for this layer (and adjacent regions of pro-sclerotin) assuming an avidity for iron haematoxylin. Pryor (19406) suggested that the sclerotin in insects is impregnated with lipoid. He pointed out that sclerotin and any material rich in aromatic groups is very lipophil. In myriapods this observation is consistent with the fact that both sclerotin and pro-sclerotin appear to be impregnated with lipoid. In Haphphilus, it will be recalled, there is a dense accumulation of sudanophil material over the arthrodial membranes. This region of the cuticle is unmodified and no demonstrable exocuticle is formed at all. This may account for the whole of the lipoid being stainable at the surface, since in this region there is no lipophil layer to absorb it. It is interesting to consider the possible explanation of the scratch-pattern effect obtainable by treating the cuticle with an abrasive dust followed by immersion in ammoniacal silver nitrate. It may be that the outer surface of the 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 this inert layer and reveals the reactive lipoid beneath which may reduce the silver solution. Wigglesworth (1947, 1948, &c.) explains scratch patterning in insects on the basis of removal of a wax layer and exposure of a polyphenol layer which lies beneath the wax layer. In view of the fact that there appears to be evidence, in myriapods, that the lipoid itself will reduce the silver solution and that no developmental details are available for myriapods, it is unwise to speculate further on this point. In the myriapods studied there appears to be no cuticular layer corresponding to the cuticulin layer described by Wigglesworth in Rhodnius and Tenebrio (1947, 1948a). Cloudsley-Thompson (1950) on the basis of treatment with chlorated nitric acid suggests that a similar layer does exist in certain species of myriapods which he has examined. From this evidence,. however, there seems nothing to differentiate between a layer of sclerotin impregnated with lipoid and a cuticulin layer. Just as Langner (1937) speaks of an epicuticle and an exocuticle in the outer part of the cuticle of a diplopod on the basis of an outer non-staining and an inner basiphil zone, the differential solubility of the outer and innermost parts of the sclerotin of the cuticle is apparently taken by Cloudsley-Thompson as evidence for the presence of an epicuticle. Wigglesworth (1947) has been careful to distinguish between cuticulin as a lipo-protein subsequently tanned with quinones, and Pryor's sclerotin secondarily impregnated with lipoid. Only developmental evidence can settle this point, but in view of the chemical similarity between sclerotized cuticulin and lipoid-impregnated sclerotin the difference in time relation may not be of fundamental importance, for pro-sclerotin may be impregnated with lipoid before tanning as generally understood takes place. In this case there would seem to be nothing to distinguish between lipoid-impregnated prosclerotin (e.g. the exocuticular cones in Haplophilus) and cuticulin. If the sequence of events during deposition of the cuticle is the same in myriapods as in Rhodnius and there is no essential difference between sclerotized cuticulin and lipoid impregnated sclerotin or pro-sclerotin, then the whole of the myriapod exocuticle and parts of the endocuticle (sclerite endocuticle 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 way homologous with the insect epicuticle as described by Wigglesworth is the thin layer of lipoid at the surface. This layer may be responsible for the diffraction effect at the surface of the cuticle which lead Verhoeff and Fuhrmann (see page 144) to speak of an outermost colourless layer (Oberflachenschicht, Grenzhautchen). As regards the possible homology of the layers of the myriapod cuticle with those of Homarus (Yonge, 1932) and Sarcophaga (Dennell, 1946, 1947a), there appears to be close correspondence of the myriapod exocuticle with the 'cuticle' of Homarus and the epicuticle of Sarcophaga larva. Here again the main difficulty of comparison arises from
160 Blower A Comparative Study of the the fact that the exocuticle of myriapods contains chitin whereas the similar layers in Homarus and Sarcophaga are described as being free from chitin. As has been seen, extensive sclerotization of the sclerite of Lithobius has led to the whole of the exocuticle being unstainable. The epicuticle of Sarcophaga larvae stains red with Mallory but loses its affinity for the stain on being converted into the sclerotin of the puparium. Diaphanol treatment restores the larval staining reaction. Similarly in Lithobius diaphanol treatment leads to the exocuticle staining red with Mallory the two cases seem quite comparable. Then again, where sclerotization does not seem to be so advanced, as in the exocuticle of the arthrodial membrane of Lithobius, this layer stains red 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 epicuticle and is lipoid in nature. Were both layers of the epicuticle not described as being free from chitin it would be natural to compare, the inner and outer epicuticle with a region of pro-sclerotin underlying a region of completed sclerotin such as obtains in the sclerite exocuticle of Haplophilus or the whole of the sclerite cuticle of Lithobius. The suggested condition in myriapods where the fat is believed to impregnate the whole of the lipophil outer layers and to originate from epidermal gland cells recalls the condition in Homarus in which the 'cuticle' contains lipoid material. Thomas (1944) records that the 'cuticle' of Lepas contains lipoid. This latter author also records a fat reaction in the tegumental glands of Lepas. If the glands of Homarus secrete the lipoid of the 'cuticle' then it is not surprising that the glands show a periodicity in relation to the laying down of the new integument and that Yonge believed that these glands were responsible for the secretion of the whole of the 'cuticle'. Pryor (1940&) suggested that the lipoid which he believed to impregnate the sclerotin of insects might be secreted from epidermal gland cells. Lastly, on this same point, it may be mentioned that Langner (1937) records a fat reaction in the epidermal glands of the diplopods she studied. It has been suggested (Blower, 1950) that the myriapod cuticle might be considered as a chitinous matrix impregnated to varying extents by prosclerotin which may or may not be tanned, that is to say, which may or may not have the amber colour and inertness usually associated with complete sclerotin. To this generalization may be added the fact that lipoid material appears to be secreted on to the surface of the cuticle from glands in the epidermis and also to impregnate the regions of sclerotin and pro-sclerotin. Here, there appears to be an intimate association of the lipoid with the aromatic groups of the sclerotin (evidenced by the fact that destruction of the aromatic groups by diaphanol or nitric acid renders the lipoid available to sudan colouring agents). The myriapod cuticle, then, is characterized by the absence of an outer layer which is both resistant and non-chititious, by the presence of a material allied to sclerotin but peculiar in being stable and resistant independent of