The histochemistry of the cuticle in different stages of the moult cycle of Carcinus maenas is described.
The newly formed cuticle of a crab exposed at moulting shows evidence of phenolic tanning in the epicuticle.
The subsequent tanning of the pigment layer of the endocuticle is confined to a brief period immediately after moulting.
The polyphenol oxidase of the epicuticle involved in tanning disappears soon after moulting, but some time later an oxidase is again indicated, this time in the pigment layer. It appears to be secreted by the tegumental glands.
From the concurrent appearance of a polyphenol oxidase and tyrosine and the subsequent formation of dihydroxyphenols and melanin-like substances in the cuticle, it is suggested that the oxidation products of tyrosine are responsible for the pigmentation of the cuticle.
Pigmentation of the cuticle is discussed in relation to phenolic tanning in Crustacea and Insecta.
Recent work has emphasized the fundamental similarity of the crustacean cuticle to that of insects (Drach, 1939; Pryor, 1940; Dennell, 1947 b). The last-mentioned author in addition to confirming the occurrence of phenolic tanning in Crustacea has suggested that its mechanism may be essentially similar to that in insects. Notwithstanding the homology of the cuticle in the two groups mentioned above it is seen that in Crustacea tanning of the cuticle is much abbreviated, the prime cause of hardening being calcification (Dennell, 1947 b). In insects it has been shown (Pryor, 1940; Fraenkel and Rudall, 1940, 1947; Dennell, 1946, 1947 a) that the hardening of the cuticle is due to the passage into it of a polyphenol, the oxidation product of which forms cross-linkages within the protein phase of the cuticle. The other concomitant changes are the reorientation of the chitin crystallites, a loss of solubility of proteins and a darkening effect. It has been pointed out (Pryor, 1940; Pryor et al., 1947) that hardening and darkening of the cuticle of insects are the result of tanning, and it therefore appeared of interest to discover the relation existing in Crustacea between tanning and the formation of melanin-like pigments in the cuticle.
The material used in this work was the merus of the walking legs of Carcinus maenas, obtained from Plymouth. Paraffin, frozen, and hand sections were prepared for study of the cuticle. Dehydration was carried out with dioxane to avoid the undue hardening that may result from treatment with higher grades of ethyl alcohol. The stains used were Mallory’s triple stain and Masson’s trichrome stain. The oxidases of the cuticle were studied using the nadi reagent (a mixture of dimethyl-para-phenylenediamine and a-naphthol (Lison, 1936) on frozen and hand sections. Melanin-like substances were detected by the solvent action of ethylene chlorhydrin. In addition, a number of other histochemical reagents were used for the detection of fats, phenols, and amino-acids, and are mentioned in appropriate places in the text.
Phenolic Tanning and Darkening
Dennell (1947 b) described the cuticle of Crustacea in the light of recent work on that of insects and brought forward evidence of the occurrence of tanning in the epicuticle and the outer layers of the endocuticle. The quinones responsible for tanning have been shown to be formed by the oxidative activity of a polyphenol oxidase which is located in the epicuticle in the very early stages after moulting. From the disappearance of the oxidase soon after moulting it appears that the tanning of the cuticle is a brief process confined to a period immediately following the formation of the new cuticle. With a view to throwing more light on this aspect of the problem, the cuticle was studied from its appearance at the time of moulting.
The newly formed cuticle of a crab at the time of shedding the old shell (fig. 1) consists of a thin epicuticle and a homogeneous endocuticle staining red and blue respectively with Mallory’s triple stain. Overlying the red epicuticle is a very thin blue-staining membrane, the outer epicuticle, whose integrity as a discrete layer is borne out by its separation from the inner epicuticle due sometimes to mechanical factors and noticed in fixed preparations of the cuticle. The inner epicuticle even before the old shell is cast shows evidence of tanning. At this stage in frozen and hand sections the untreated epicuticle shows an amber coloration but the endocuticle is colourless. That the amber coloration of the epicuticle is due to tanning is indicated by the bleaching of the amber-coloured zone by diaphanol, which has a selective action on tanned regions. The occurrence of tanning in the epicuticle is further confirmed by the results obtained with the Millon and argentaffin reactions, both of which are positive, indicating the presence of aromatic substances. The endocuticle is unaffected by such treatment. Staining of sections with Sudan black B. indicates that both the inner and outer epicuticles contain lipoid substances. Ferric chloride gives a slight green coloration in the inner epicuticle and in the outer layers of the endocuticle, indicating the presence of a dihydroxyphenol. This is consistent with the observation (Dennell, 1947 a) that tanning spreads inwards as a result of a wave of quinone formation induced by the polyphenol oxidase activity in the epicuticle. The presence of polyphenols in the outer layers of the endocuticle as indicated by the ferric chloride test suggests impending tanning of this region.
Sections of the cuticle of a soft crab some time after moulting (fig. 2) show that both the epicuticle and the outer layers of the endocuticle are tanned. That the tanning of the cuticle is completed by this stage in the moult cycle is suggested by the lack of a reaction with ferric chloride, indicating the absence of a dihydroxyphenol. In the absence of an oxidase and extension of the tanned zone at this stage, dihydroxyphenols involved in tanning may not now occur in the cuticle. Fig. 3 shows a section of the hardened cuticle of Carcinus in the middle intermoult stage. It corresponds to that given by Dennell (1947 b) of the merus of the first walking leg of Astacus. A comparison of a section of the hardened cuticle with the exoskeleton of a soft crab shows that the epicuticle and the tanned zone of the endocuticle are similar and changes are observed only in the rest of the endocuticle, which is considerably extended and differentiated into a calcified zone and a narrow inner non-calcified layer. At the junction of the calcified and pigmented layer is a narrow zone such stains intensely blue after Mallory stain. The ducts of the tegumental glands passing through the cuticle to open on its surface form a prominent feature of the sections. The contents of the ducts stain red with Mallory and at the openings of the ducts red-staining globules are often seen, and may represent the secretion of the glands. Their appearance recalls that observed by Wigglesworth (1947) in regard to the dermal glands of Rhodnius.
The nature of the changes undergone by the cuticle during the intermoult stage after the initial occurrence of tanning are shown in Table I, which gives the results of histochemical tests on the cuticle at an early stage, middle stage, and a late stage in the intermoult period.
The positive Millon and argentaffin reaction in the epicuticle and pigment layer is consistent with the observation that these regions are hardened by phenolic tanning at a very early stage in the moult cycle. The negative reaction with ferric chloride in the earlier stages of the intermoult period points to the absence of free dihydroxyphenols at this stage, though they reappear later. The presence of dihydroxyphenols at a comparatively late stage in the moult cycle appeared inexplicable in view of the observation that tanning is confined only to a very early stage after moulting. An interesting result was obtained by treating hand sections of the cuticle with the nadi reagent which gave an intense blue colour in the pigment layer of the endocuticle. The presence of an oxidase appears to be indicated by the inhibition of this reaction by potassium cyanide and by heating. Treatment of hand sections of the cuticle with aromatic substances showed rapid coloration with catechol, protocatechuic acid, and hydroquinone. Dennell (1947 b) observed that in Astacus and other decapods the epicuticle soon after moulting shows the presence of a polyphenol oxidase but later the nadi reaction is obtained even in the presence of cyanide. This later reaction is apparently due, however, to the presence of osquinones, which are capable of oxidizing the nadi reagent, and is distinct from the true oxidase reaction mentioned above. The absence of a positive reaction with nadi some time after tanning has taken place, as has been observed in Carcinus, may be taken to indicate not only the absence of an oxidase, but also that the quinones which reacted at an earlier stage to give a pseudo-phenoloxidase reaction have now lost their reactive properties, possibly due to polymerization or to changes consequent on their condensation with the proteins of the cuticle. Later still, however, phenol oxidase reappears, now in the pigment layer, and represents either a fresh secretion on the part of the structures elaborating it, or secretion by some other structure. From an analogy with the condition obtaining in insects where the polyphenol oxidase of the cuticle has been shown to be derived from the epidermis (Dennell, 1947 a) it might be expected that this layer in Carcinus would be the site of elaboration of the oxidase. Treatment with nadi reagent did not give a positive reaction in the epidermis at any stage of the moult cycle, but the tegumental glands which.during this stage are found in an active secretory state gave with nadi a blue coloration which was thermolabile and cyanide-sensitive. The ducts of the glands penetrating the cuticle also gave a positive reaction apparently indicating the transport of the oxidase to the cuticle. Hand sections treated with catechol rapidly gave a dark brown coloration in the tegumental glands and their ducts, so confirming the presence here of a polyphenol oxidase.
Previous work on tegumental glands records a wide range of functions performed by them. Yonge (1932) has given a critical and comprehensive account of the work of earlier authors and has suggested that the tegumental glands are primarily concerned with the secretion and preservation of the epicuticle. The intimate association of the glands with the cuticle led Dennell (1947 b) to point out that ‘it is difficult to avoid the view that in Crustacea the activity of the tegumental glands is closely connected with the structure of the cuticle’. He suspected that if they are not concerned directly with the secretion of the cuticle, they may yet be related to the subsequent hardening by the elaboration of the oxidase involved in tanning. It is seen from the foregoing observation in Carcinus maenas that the tegumental glands secrete a polyphenol oxidase some time after tanning and are concerned with an aspect of cuticular development which, it will be shown in this paper, is related to pigmentation.
Tyrosine and Melanin Formation
In the search for a possible substrate for the oxidase occurring late in the development of the cuticle, it was of interest to note the positive reaction given with Mörner’s reagent in the pigment layer of the cuticle at this time. That the substance giving the green coloration with Mörner’s reagent is indeed tyrosine is confirmed by the use of α--nitroso-β-naphthol which gives a red coloration with tyrosine in the presence of nitric acid (Feigl, 1947). The occurrence of tyrosine is in itself not surprising as Trim (1941) had already recorded its presence in the cuticle of the lobster. The condition observed in Carcinus recalls that found in Sarcophaga falculata (Dennell, 1947 a), in which tyrosine accumulates in the outer endocuticle preparatory to the tanning of this region to form the exocuticle of the puparium. But in Carcinus the presence of tyrosine in the pigment layer, which has already undergone tanning, may. not have the same significance as in Sarcophaga.
In the absence of any further extension of the tanned zone of the cuticle during the stages when the oxidase and substrate are present, it would appear that at this time they are not involved in further tanning of the cuticle. The assumption that a later process of tanning, if it occurs, may result only in an intensification of tanning that has taken place, seems inconsistent with the positive reaction obtained with ferric chloride in the endocuticle, showing the presence of free dihydroxyphenols presumably formed as a result of the oxidation of tyrosine by the phenolase. Tyrosine, as indicated by a feeble reaction with Mörner’s reagent, gradually disappears, although free dihydroxyphenol persists for some time.
Additional support for this view is afforded by examination of sections of hard and thickened cuticle after prolonged treatment with diaphanol. Such sections show in the pigment zone and the calcified layer a black coloration, revealed by the disappearance of colour due to tanning. When such sections are treated with ethylene chlorhydrin, which is a solvent for melanins, it is observed that the black substance of the cuticle is rapidly dissolved. That melanin-like substances may be formed from dihydroxyphenols present in the cuticle is suggested by the observation that hand sections of the cuticle when treated with dihydroxyphenylalanine (DOPA) show within a few minutes a black coloration in the endocuticle. When such sections are treated with ethylene chlorhydrin, they are decolorized in a manner similar to that occurring in naturally blackened cuticles under such conditions. From the concurrent appearance of a phenol oxidase and tyrosine in the pigment layer and the subsequent formation of dihydroxyphenols and pigmented products, it may be justifiable to assume that tyrosine is oxidized in the cuticle to form dihydroxyphenols, giving rise to pigmented products of the nature of melanins.
Wigglesworth (1948a) as a result of his observations on Tenebrio suggested that the tyrosine of the cuticle may give rise to melanins, for the regions of the cuticle which later become darkened show the presence of tyrosine. In Carcinus tyrosine is not found in the cuticle in the early stages of the moult cycle. A similar condition to that in Carcinus has been observed in Sarcophaga where no free tyrosine is found in the larval cuticle until just before puparium formation (Dennell, 1946). It may be inferred that the tyrosine of the cuticle is derived from the blood. Pinhey (1930) found that the blood of Maia squinado and Cancer pagurus contains about 0·004 per cent, of tyrosine, at all stages of the intermoult period. Tyrosine estimation of the blood of Carcinus shows that it is similarly more or less constant at about 0-003 per cent. If, as has been shown by Dennell (1947 a) and Fraenkel and Rudall (1947), the blood tyrosine is the source of the phenol involved in tanning of the cuticle, the more or less constant percentage of the blood tyrosine in Carcinus maenas would support the view that tyrosine of the cuticle is derived from the blood. This is probable because if tyrosine is oxidized by tyrosinase in the blood to form dihydroxyphenols in the early stages of the moult cycle as a preliminary to tanning, in the later stages when there is no evidence of tanning, tyrosine as such may be transported to the cuticle to serve as a substrate for the formation of pigmented products. Such a condition might explain why the blood tyrosine remains always more or less constant although tanning is confined to a period just about the time of moulting. This is in contrast to what has been observed in Sarcophaga where the tyrosine content of the blood varies, being almost absent in the young feeding larva, then gradually increasing in the maturing larva, and again decreasing, presumably as a result of its consumption during the hardening and darkening of the cuticle. This feature is probably related to the fact that both hardening and darkening of the cuticle takes place simultaneously, whereas in Carcinus tanning is followed by the development of pigment.
Trim (1941) pointed out that a proportion of the tyrosine derivatives of the cuticle may be in the form of dihydroxyphenols. The occurrence of free dihydroxyphenols has been noted in the cuticles of various insects (Pryor, 1940). Possibly such dihydroxyphenols are derivatives of tyrosine formed in excess of the requirements for tanning, and therefore not linked up with the protein constituents of the cuticle. In Carcinus histochemical tests show that dihydroxyphenols are transformed into melanin-like substances formed probably as a result of further oxidation. Support for this view is found in the observation of Verne (1923) that the chromogen which gives rise to the black pigmentation of the eyes, legs, and carapace of crabs is an amino-acid.
It may be seen from the work of Pryor (1940), Fraenkel and Rudall (1947), and Dennell (1947 a) that in insects the polyphenol formed in the blood is probably deaminated before reaching the cuticle where it is oxidized to quinones. From the close correspondence of the process of tanning in Crustacea, a similar deamination of the blood phenol may take place there also. If so, the melanins formed in the cuticle are not to be regarded as arising from the deaminated blood phenol, as melanins contain the NH group. Keilin and Hartree (1936) observed that if tyrosine is deaminated and the products oxidized, the reaction never gives rise to melanins. Therefore it may be reasonable to conclude that where darkening is brought about by the formation of melanin-like substances, tyrosine derivatives are formed in the cuticle possibly by the transport of tyrosine as such to the cuticle where further oxidation results in the formation of pigmented products and the excess of tyrosine derivatives may occur as free dihydroxyphenols. In insects, since darkening and hardening of the cuticle occur together, both the oxidation of deaminated products of the blood phenol and the oxidation of the tyrosine of the cuticle taking place coincidentally, it may not appear obvious that the two processes give rise to different end-products. But in Carcinus it is found that the early and slight tanning of the cuticle is separated by an interval of time from the process in which tyrosine appearing in the cuticle is oxidized by the phenolase. The sequence of appearance of the tyrosine and oxidase, the dihydroxyphenols, and lastly the melanins make it possible to infer that pigmentation of the cuticle is a separate process though chemically not unrelated to the earlier tanning.
As has been shown, hardening and darkening of the insect cuticle are due to the tanning action of quinones resulting from the oxidation of dihydroxyphenols formed in the blood and transported to the cuticle. Pryor and others (1947) observed that a most likely mode of participation of such dihydroxyphenols in the hardening of the cuticle is by enzymic oxidation followed by condensation of the oxidized material with the proteins of the cuticle so that stable cross-linked structures, in which the nitrogen of the amino-groups becomes directly attached to the aromatic nuclei, are formed. It is also observed that proteins tanned by quinones are dark in colour (Pryor, 1948). From this circumstance it seems to have been inferred that the darkening of the cuticle is only incidental to that hardening process, being its natural consequence. Wigglesworth (1948 b), however, observed that the ‘coloration of the tanned cuticles may be due to the presence of chromatophore groups such as the quinonoid group in the molecule or it may be due to coloured by-products arising from the oxidation of phenols not attached to any protein chain’. Such a view is consistent with the findings recorded in this paper, for it contemplates the occurrence of two processes, one primarily if not solely leading to hardening and the other contributing to pigmentation. Since phenolic substances are the materials involved in both processes, they appear to be related though separated in point of time. Wigglesworth believes that some undeaminated tyrosine in the cuticle probably serves for melanin formation. It is interesting also to note that Fraenkel and Rudall (1947) in their study of the structure of insect cuticles observe that there may be a degree of protein tanning by deaminated tyrosine as well as formation of melanin from undeaminated tyrosine. That tyrosine as such may pass into the cuticle is supported by the observation that in Sarcophaga (Dennell, 1946) tyrosine is indicated in the presumptive exocuticle and is used up during puparium formation as seen by the fall of tyrosine content of the cuticle from 3·5 to 2·0 per cent. (Trim, 1941). This represents only a fraction of the total tyrosine consumption as indicated by the fall in tyrosine content of the whole organism from 1·7 to 0·87 per cent. It is possible that the cuticular tyrosine contributes directly to melanin formation and may not take part in tanning of the cuticle. The observations made in Carcinus maenas support this view.
The presence of melanins in insect cuticles has long been recognized. Gessard (1904) and Gortner (1911a, 1911 b) have shown that the colouring of the insect cuticle is a fermentative process involving tyrosine and tyrosinase of the blood, dihydroxyphenylalanine being an intermediary product in the formation of melanins. With the revival of interest in the subject of the hardening of the insect cuticle as a result of the work of Pryor (1940) it has been shown that hardening is an enzymatic process very similar to that noted by Gortner and others, and so hardening and darkening have been regarded by some as two aspects of the same physico-chemical phenomenon. From the observations in Carcinus recorded here, phenolic hardening and darkening appear to be separate processes although the mechanisms underlying the two processes are similar. In insects this separateness is obscured, as in Sarcophaga, where tyrosine, although it accumulates in the cuticle before hardening, was not recognized as a pigment precursor; but a suggestion that they may be independent processes is gleaned from the work of Dennell (1947 a), who observed that the ‘first sign of darkening of the cuticle is seen actually before pupal contraction begins’ and presumably before the hardening of the cuticle has commenced. He pointed out that the ‘occurrence of darkening before as well as after pupal contraction gives an indication that the colouring of the cuticle and the muscular contraction which shapes the puparium are independent events although both are presumably induced by the liberation of the pupal hormone’.
I have great pleasure in acknowledging my indebtedness to Professor R. Dennell for help and guidance given me in the course of this study. I am grateful also to Professor H. Graham Cannon, F.R.S., for his continued interest in the work. My thanks are due to the Government of Madras for the award of an overseas scholarship during the tenure of which this study was made.