The localization of a peroxidase associated with hard cuticle formation in an insect, Calpodes ethlius stoll, lepidoptera, hesperiidae

TISSUE 8 CELL 1969 1 (3) 555-574 Published by Oliver Et Boyd Ltd, Edinburgh. Printed in Great Britain

MICHAEL LOCKE*

THE L O C A L I Z A T I O N OF A P E R O X I D A S E A S S O C I A T E D W I T H HARD CUTICLE F O R M A T I O N IN AN INSECT, CALPODES ETHLIUS STOLL, LEPIDOPTERA, HESPERIIDAE ABSTRACT. The distribution of a peroxidase associated with the formation of hard cuticle has been studied in developing larvae of Calpodes ethfius. It occurs in granules in several cell types but is most easily observed in the cells making the proleg spines at the 4th to 5th molt. Light microscopy shows peroxidase in numerous granules about 0.5/z in diameter at the time the cuticle of the spine shaft is being deposited. Electron microscopy shows these granules to be multivesicular bodies with peroxidase in the matrix. Peroxidase is also found in cisternae of the rough ER near Golgi complexes, in vesicles of Golgi complexes and in the secretory vesicles which discharge to make cuticle at the apical surface. The cuticle above the plasma membrane where peroxidase is being deposited reacts with DAB in the absence of hydrogen peroxide. Presumably this cuticle has been "peroxidized" as a first stage in stabilization by cross-linking. Some of the peroxidase secreted at the apical surface is pinocytosed and transported to the multivesicular bodies, suggesting that there may be a precise control of the cuticular environment through the turnover of its soluble components.

It also occurs less intensely in other epidermal cells engaged in the formation of hard IN the course of using plant peroxidase to cuticle at molting. The electron microscope trace the uptake of proteins from the blood showed the granules to be multivesicular into multivesicular bodies and storage gran- bodies with the endogenous peroxidase in ules in several insect tissues (Locke and the matrix, exactly where tracer peroxidase Collins, 1966, 1967, 1968; Locke, 1969a, from the haemocoel is sequestered in the ex1969b), it was found that some tissues con- periments cited above. Since multivesicular tained an endogenous peroxidase at certain bodies are usually regarded as organeUes for stages of their development (Locke, 1968). heterolysis (DeDuve and Wattiaux, 1966), The endogenous peroxidase is most notice- this location for the endogenous enzyme was able in epidermal cells during the formation difficult to reconcile with a functional relaof hard brown cuticle. It occurs in granules tion to hard cuticle formation. To resolve the in Calliphora larval epidermis before pu- paradox, the localization of the endogenous parium formation, and at molting in the cells peroxidase was studied during the genesis of forming the proleg spines in Calpodes larvae. the proleg spines. These proved particularly favorable material since they are adjacent to other epidermal cells engaged in a develop* Case Western Reserve University, Department mental sequence similar except for the abof Biology, Cleveland, Ohio 44106. sence of peroxidase and hard cuticle. Received 15 August 1968. 555 Introduction

LOCKE

556 Materials and Methods

The development of the proleg spines has been studied at the 4th to 5th molt in Calpodes etklius. The notation M + or M refers to the time before or after ecdysis at the 4th to 5th molt. Larvae were inflated with fixative and allowed to fix for a few seconds before the prolegs were halved with a transverse cut. Other procedures for tissue preparation and the timing of epidermal events related to cuticle formation in Calpodes have already been described (Locke, 1966, 1969a). The procedure for visualizing the endogenous enzyme involved the oxidation of diaminobenzidine (DAB) in the presence of H202 as in the procedure for plant peroxidase used as a tracer (Locke and Collins, 1968). All identifications of peroxidase in particular organelles have been confirmed on sections unstained except for the DAB reaction and osmication in material PrePared for electron microscopy. Control tissues were incubated without hydrogen peroxide or after heat killing (100'~C for 10 minutes). Conclusions are based on comparisons between stained and unstained material although only one or the other may be figured in the text. The spines on different prolegs develop synchronously, so that several experimental procedures can be performed on similar material from one larva. Duplicate series of prolegs were prepared, one for whole mounts for light microscopy, the other fixed in osmium tetroxide for electron microscopy.

A. PRELIMINARYOBSERVATIONSON THE PROLEG SPINES

1. The structure o.1"the spines There are prolegs on abdominal segments 3, 4, 5, 6 and 9, making five pairs in all. Each proleg is oval in cross-section measuring about 1.8 • 0.9 mm (Fig. I). The spines arise in a single row on the outer rim of the flattened foot. They are of two lengths (200F and 60F), causing the tips to be arranged in two rows (Figs. 2 and 3). Each spine has a hooked tip pointing away from the body, so

that during walking it pierces the leaf centrifugally as the leg expands, and is withdrawn when the plantar region is depressed. Only the hooked tip of hard amber-colored cuticle extends beyond the flexible surface cuticle secreted by the supporting cells. Most of the spine forms a cylindrical root of hard but colorless cuticle below the surface (Fig. 4). The projecting hook of the spine has an epicuticle, and below it a layer of fibrous cuticle with the fibers for the most part axially oriented rather than in a lamellate pattern. The root is composed only of fibrous cuticle.

2. The formation o f the spi~es The formation of the epicuticle and the timing of epidermal events at the 4th to 5th molt have already been described (cuticulin, Locke, 1966; protein epicuticle, Locke, 1969a). The timing of events in the spine precedes that in the surface cuticle. The shape of the spines is determined from about M - 2 6 to M - 2 0 hours when the plasma membrane of the projecting part of the spine cell is covered with cuticulin. By M - 1 8 hours the protein epicuticle has been deposited and the formation of fibrous cuticle has begun. The fibrous cuticle of the root is secreted by the spine cell, appearing at the tips of the microvilli between it and the supporting ceils, and is complete by about six hours before ecdysis. Most of the fibrous cuticle of the spine is not laid down in lamellae with laminae of parallel fibers rotating in their orientation as described by Bouligand (1966). The fibers are predominantly axially oriented at first and the 'microvilli' are really longitudinal ridges. The fibrous cuticle below the surface epicuticle, on the other hand, is lamellate and arises at the tips of more symmetrical microvilli. Fibers with the lamellate pattern are not laid down in the spines until shortly before ecdysis. The spine cuticle is at first colorless, but the exposed tip darkens progressively from M - 7, reaching the final amber color by ecdysis. The timing of the important TISSUE ~- CELL 1969 1 (3)

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Fig. 1. The prolegs bearing spines in a 5th instar Calpodes larva. :< 18. Fig. 2. Enlargement of the spines of one side on a living proleg. Only the hooked tip is dark and projects above the surface. • Fig. 3. The proleg spines seen in a whole mount of a proleg. Unstained. • Fig. 4. Enlargement of Fig. 3 showing the long and short spines causing the hooked tips to be arranged in t w o rows. • Fig. 5. Optical section in a w h o l e mount of spines during their development about 12 hours before ecdysis, showing the peroxidase containing granules below the tip. DAB reaction for peroxidase, :,: 500. TISSUE ~ CELL 1969 1 (3)

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Fig. 6. The sequence of events in the formation of the proleg spines. The coloration of the spine cuticle by diaminobenzidine in the absence of H20= begins at the tip and extends d o w n the hooked side of the spine and finally colors all the root. It persists to a small degree for up to 48 hours after ecdysis. The granules containing peroxidase are at first located where the tip joins the surface cuticle. Later, they appear more diffusely along the length of the cell b e l o w the cuticle colored by DAB. Top row of numbers : time from ecdysis at the 4th to 5th molt. Lower row : time from ecdysis at the 3rd to 4th molt.

ABBREVIATIONS USED h i basal infolds bro basement membrane c v coated vesicle D A B Diaminobenzidine e epicuticle fc cuticle of spine shaft with fibers axially oriented g Golgi complex h haemocoel Ic lamellate cuticle above the supporting cell

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time in hours before or after ecdysis at the 4th to 5th molt multivesicular bodies nucleus plasma membrane vesicles containing peroxidase spine cell supporting cell peroxidase in ER cisternae peroxidase between plasma membrane folds, presumably having just been discharged from a secretory vesicle

TISSUE ~ CELL 1969 1 (3)

Fig. 7. The structure of a developing spine. L.S. through the base of a spine during its formation about 13 hours before ecdysis to the 5th instar. The spine cell contains numerous multivesicular bodies. The cuticular shaft of the spine projects basally as a root between the spine cell and the supporting cells. The spine cell contains numerous axially oriented microtubules. Stained in uranyl acetate and lead citrate, e, epicuticle; fc, cuticle of spine shaft with fibers axially oriented ; Ic, lamellate cuticle ; mvb, multivesicular bodies ; Su, supporting cell; Sp, spine cell. x 8000. TISSUE 8- CELL 1969 1 (3)

560 events in spine development is summarized in Fig. 6.

3. The peroxidase distribution #1 the spine cells The distribution of the granules containing peroxidase was studied by light microscopy in a series of whole mounts prepared from M - 3 0 hours through ecdysis into the 5th stadium. The granules first appear in a cluster at the base of the projecting tip of the spine where its cuticle becomes confluent with that of the surface, at about M - 2 1 hours. By M - 1 6 hours them are more granules, some of them distributed along the length of the spine cell (Fig. 5). They disappear by about M - 7 hours. There are no obvious granules containing peroxidase visible in the epithelial cells adjacent to the spine cells, Diaminobenzidine (DAB) does not color the granules in heat-killed material or in the absence of H202 substrate. The peroxidase in the granules resists several hours fixation and is not affected by preincubation in 0"0Y';; H~O2. Neither substrate concentration nor pH is very critical for the adequate demonstration of the enzyme. The granules are still colored using ~ or x 100 the usual hydrogen peroxide concentration. They color slightly when the reaction is run at pH 4. The color is almost maximal at pH 5, and no difference could be detected between material incubated at pHs 6, 7, 8 and 9. Autophagic vacuoles in the fat body and oenocytes are occasionally colored by DAB. To test whether the coloration of the proleg spine granules could be due to a nonspecific reaction between an oxidized lipid and DAB, prolegs were incubated in 0-033,oH20~ before reaction with DAB in the absence of H20~. This procedure did not color the spine granules. The brown coloration appeared only when DAB and H202 were present concurrently. The peroxidase reaction in the spine granules is not inhibited by phenylthiourea (PTU) under conditions which block phenolase activity demonstrated with dopamine as a substrate in the cuticle and

LOCKE granules of developing bristles. We may conclude that the granules seen in proleg spines contain a peroxidase. in a control series of preparations without H202 substrate it was found that DAB colors some cuticles during their formation. The reaction is probably enzymatic since there is no coloration after heat killing (100"C for 10 minutes). The reaction is not due to a phenolase since it is not inhibited by PTU under conditions which stop the cuticle being colored by dopamine. Incubation in dopamine without an inhibitor gives a color only in the spine tip, anticipating the distribution of the natural amber coloration which appears shortly before ecdysis. The color due to DAB, on the other hand, begins at the tip ( M - 2 5 hours), spreads to the hooked side of the spine root by M - 14, and the whole root by M - 8 (Fig. 6). The color then fades but is still present at ecdysis and very slightly for the first 1-2 days of the 5th stadium. There may be a peroxidase in the spine cuticle in addition to, or as part of, this oxidizing system not needing H~O~, but the cuticle is not clearly darker after reactions with H202 present. The timing of events characterized by reactions with DAB is summarized in Fig. 6. 4. PeroxMase gramdes in other epMermal cells In addition to the proleg spine cells, several other epidermal cell types have peroxidase granules at a time when they are depositing cuticle, e.g. the socket cells of bristles, the cells forming 'lenticels' (the characteristic sense organs of Hesperiid and Lycaenid larvae), in the ducts of dermal glands and occasionally in muscle insertions. The cells have in common the secretion of a more or less rigid cuticle. We may therefore correlate the peroxidase with one sort of cuticle hardening. These observations made it important to determine the nature of the granules containing peroxidase and their relation to the formation of hard cuticle and the cuticular system oxidizing DAB. TISSUE 8- CELL 1969 1 (3)

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| Fig. 8. The structure of a developing spine. T.S. spine during its formation about 16 hours before ecdysis. There are numerous axially oriented microtubules and several multivesicular bodies. The plasma membrane appears microvillate but is actually folded in longitudinal pleats. Stained in uranyl acetate and lead citrate, e, epicuticle; fc, cuticle of spine shaft with fibers axially oriented; mvb, multivesicular bodies. • 10,000. TISSUE 8- CELL 1969 1 (3)

LOCKE

562 B. THE ULTRASTRUCTUREOF THE SPINE CELLS DURING THE DEPOSITION OF THE SPINE CUTICLE Figure 7 shows the appearance of a spine during its development. The fibrous cuticle of the shaft projects as a root between the spine cell and the supporting cells. The continuity between the root and the shaft shows that the root is deposited mainly by the spine cell. The cytoplasm of the spine cell contains many axially oriented microtubules, small dense secretory vesicles, late stages of autophagic vacuoles and numerous multivesicular bodies. The multivesicular bodies (Figs. 8 and 9) have about the size and distribution of the granules containing peroxidase seen by light microscopy.

1. The localization oJ'peroxMase in Ml/Bs and small vesicles Thick electron microscope sections (gold interference colors) of prolegs reacted for peroxidase, but otherwise unstained, show the enzyme localized in the matrix of the MVBs (Fig. 11). This osmiophilia is much more intense than that in unreacted material (Fig, 13). There is also peroxidase in the small

dense secretory vesicles. Peroxidase in both locations is confined to the spine cell (Fig. 12), but there is some extra osmiophilia in the fibrous cuticle, presumably the result of the reaction with DAB not requiring H~O~ substrate (Fig. 10).

2. The origin q/' the peroxidase in Golgi complexes The peroxidase reaction in the vesicles near the apical face of the cell where cuticle is secreted is more intense than in the MVBs nearby. The vesicles can be traced back to their presumed origin as secretory vesicles from Golgi complexes situated for the most part in the central and basal regions of the cell (Figs. 14 and 15). Peroxidase can be made out near the Golgi complexes in small dense masses within the cisternae of the rough ER. These are presumably the antecedents of transition vesicles transporting the peroxidase to the complexes and secretory vesicles. The complexes have an inconspicuous saccular component and are made up mainly of vesicles, some containing peroxidase (1200 1500 & in diameter), others (about 800 ~ diameter) having a coated sur-

Fig. 9. The multivesicular bodies of the spine. L.S. spine cell during the formation of the shaft cuticle about 13 hours before ecdysis. The longitudinal ridges of the plasma membrane are cut obliquely. Stained in uranyl acetate and lead citrate, fc, cuticle of spine shaft; mvb, multivesicular bodies. • 39,000.

Figs. 10 8 11. The distribution of peroxidase in the developing spine. Fig. 10. The DAB reaction in the cuticle of the spine shaft. L.S. spine at its junction with the supporting cell. Larva aged M - 1 3 8 9 Fig. 11, T.S. spine showing peroxidase in multivesicular bodies and smaller vesicles. Larva aged M - 1 7 . Peroxidase reactions, otherwise unstained, e, epicuticle ; fc, fibrous cuticle of the spine shaft reacting with D A B ; mvb, multivesicu[ar bodies containing peroxidase in the matrix; pro, plasma membrane; pv, vesicles containing peroxidase; Sp, spine cell; Su, supporting cell. • 30,000.

Figs. 1 2 8" 13. The distribution of peroxidase in the spine cell and supporting cells. Fig. 12. Thick L.S. through a spine cell and supporting cells, unstained except for the peroxidase reaction. Peroxidase is almost confined to the multivesicular bodies and secretory vesicles of the spine cell. Fig. 13. T.S. spine cell, control tissue unstained and unreacted. The multivesicular bodies and secretory vesicles have some osmiophilia but much less than the tissue reacted for peroxidase. Larvae aged M 17. fc, fibrous cuticle of the spine shaft; Ic, lamellate cuticle above the supporting cells ; rnvb, multivesicular bodies containing peroxidase in the matrix: Sp, spine ceil; Su, supporting cell, ;-; 10,000. TISSUE 8" CELL 1969 1 (3)

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accumulation of irregularly shaped vacuoles with electron transparent contents (Fig. 4). The cupular material surrounds circular or oval spaces, about 2 to 5/z in cross-sectional diameter. These spaces contain hair bundles (Fig. 13) and they presumably correspond to the canals seen with the light microscope. No connections between the bundles and the borders of the canals were seen. (iii) The receptor cells All the receptor ceils are pear-shaped and have basal nuclei. They contain several Golgi zones, granular membranes and ribosomes, and also large numbers of vesicles of various sizes. A few short agranular cisternae of the endoplasmic reticulum are also found, and these often lie in the apical half of the cell, beneath the lateral surface membrane. In many cells there is a wide variety of inclusion bodies, varying in diameter from about 0.2 to 2/x: these organelles may contain either smaller vesicles, membranes, or aggregates of densely staining material with a regularly repeating substructure. Each receptor cell hears, on its apical surface, an array of between 70 and 90 stereocilia and one kinocilium situated asymmetrically on one side of the ciliary group (Fig. 4). These cilia are arranged in a hexagonal pattern. The kinocilium contains the typical arrangement of '9 + 2' fibrils and arises from a basal body, bearing a basal foot (Fig. 5). This foot points away from the group of stereocilia in a direction approximately at

right angles to a line joining the two central fibres of the kinocilium. The stereocilia arise from a 'cuticular plate' lying in the apical cytoplasm of the receptor cell (Figs. 4 and 6). They vary in height from one side of the hair bundle to the other, the longest (about 4/~) lying near the kinocilium, and the shortest (about 1.5/~) on the opposite side of the cell. Throughout most of their length they are of relatively uniform diameter (about 0-25 k~to 0.4/+), but they taper at the base to about 0.08 tz. The flat surfaces at their distal ends lie at an angle to the surface of the macula but are roughly parallel to a line joining tile free ends of all the cilia in the group (Fig. 6). Internally, the cilia are composed of a number of longitudinally oriented fibres, each about 25 to 50 A in diameter. For the most part these fibres lie separately, but at the base of the cilium they are condensed into a rodlet (about 250 A in diameter) which extends into the underlying cuticular plate (Fig. 7). The point of termination of the rodlets has not been established; they may possibly connect with similar rodlets found at the lateral borders of the cuticular plate (Fig. 8). The arrangement of the cilia on the sensory cells shows an interesting pattern across the whole sensory macula. This pattern is revealed by comparing the orientation of a number of hair bundles in terms of their morphological axes. Each axis is defined as the line passing through the kinocilium and that row of stereocilia most nearly bisecting

Fig. 2. Section through the amphibian papilla in the transverse plane of the animal, and in the plane of the long axis of the papilla. The receptor cells in the sensory macula (sin) are inclined at varying angles to the macular surface, which is not appreciably curved in this plane. Remains of the cupula (c) can be seen. The non-cellular material across the utriculo-saccular canal (usc) and across the opening of the amphibian papilla (o) is an artefact probably produced by irregular preservation of gelatinous material from the utriculus or saceulus, ct, Connective tissue ; m, membrane separating 1umen of papilla from lumen of perilymph duct; n, branch of auditory nerve supplying papilla; pc/, perilymph d u c t ; s , mesial wall ofsacculus; v, ventral wall of utriculus. • Fig. 3. The macular surface of an isolated cupula, showing the arrangement of the "canals" which, in the living animal, contain the hair bundles. Phase contrast. - 500. TISSUE 8- CELL 1969 1 (3)

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P E R O X I D A S E AND C U T I C L E F O R M A T I O N face (Fig. 16). These cells are like other epidermal cells in that their Golgi complexes never have numerous saccules and vesicles of the sort found in vertebrate cells, even though they are at the height of cuticle deposition and are presumed to be active in protein synthesis. Both tlme small coated vesicles and the vesicles containing peroxidase can be traced along routes to the apical surface outlined by tracks of microtubules. The microtubules extend from the dense folds of RER :in time basal region along the length of the spine (Locke, 1969a). The vesicles containing peroxidase discharge through the apical plasma membrane at the base of 'microvilli~ (Fig. 17). The contents presumably disperse and reaggregate around other more peripheral cuticular components in the way that epicuticle is laid down (Locke, 1969a). We may conclude from these observations that the peroxidase originates in Golgi complexes and is discharged apically as one of the components involved in the formation of new cuticle. How, then, do the MVBs come to contain endogenous peroxidase, since the MVBs of the epidermis have been shown to be organelles involved in the sequestration of exogenous protein taken up from the haemocoel (Locke and Collins, 1967)?

3. The source o f the peroxidase in the M VBs

All of the apical plasma membrane of the spine ceil, except that apposing the cuticle of the root, has large coated vesicles (about 1200 A in diameter) inserted at the base of 'microvilli'. The surface next to the root is the only one where the cuticle abuts on another cell rather than molting fluid. This has given rise to the hypothesis that the large

coated vesicles on the apical face may be concerned with uptake of some components of the molting fluid, and inevitably of some free components of the new cuticle (Locke, 1969a). Sorne of the smaller MVBs have a coated surface and appear to arise, at least in part, from tile fusion of the large coated vesicles. If some of the peroxidase discharged into the subcuticular environment from the secretory vesicles :fails to find a permanent site in the cuticle, we might expect that it would appear in nearby coated vesicles and MVBs. In agreement with this, the supporting cells have a few MVBs containing peroxidase in the cytoplasm immediately adjacent to the spine cuticle where peroxidase is being deposited. The supporting cells themselves have Golgi complexes and secretory vesicles completely free of peroxidase (Figs. 18 and 19). After this was found by electron microscopy, careful focusing on the whole mounts prepared for light microscopy revealed it as a general phenomenon. There are always a few MVBs and coated vesicles containing peroxidase in the supporting cells where they are close to the spine cuticle. The region of the cuticle into which secretory vesicles disperse must be very localized, as the MVBs containing peroxidase are never far removed from cuticle reacting with DAB. The observations also suggest that the MVBs may be rather static in position within the cell, and not part of the streaming columns presumed to distribute the secretory vesicles. We may conclude that the peroxidase in the MVBs probably arrives there by pinocytosis from the apical face. This could be part of a mechanism for controlling the composition of the subcuticular environment or an inevitable consequence of a rather nonspecific uptake of molting fluid.

Figs. 1 4 8- 1 5. The distribution of peroxidase in the rough ER, Golgi complexes and secretory vesicles of the spine cells. Larvae aged M - 1 7 8 9 Peroxidase reaction but cont[ast slightly enhanced by in block staining in u[anyl acetate. The peroxidase can be made out in packets in the ER cistemae and in secretory vesicles. ,i, peroxidase in ER cisternae; g, Golgi complex; n, n u c l e u s ; p v , secretory vesicle containing peroxidase, Fig. 14, >: 16,000; Fig. 15, :.:30,000. TISSUE & CELL 1969 1 (3)

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4. The basal smface of tile spine cells Although many of the peroxidase containing secretory vesicles from the basal Golgi complexes appear to stream toward the apical face and be concerned in cuticle formation, others may discharge basally to the haemocoel (Figs. 20 and 21). Some of the basal infoldings surround cytoplasm with clusters of vesicles containing peroxidase which appear to arise in nearby Golgi complexes. The basal aggregation of vesicles seen in Figs. 20 and 21 could be caused by a block to basal discharge, with vesicles accumulating locally until they

get caught in a stream of vesicles directed apically. Alternatively, they couId be vesicles discharging to the haemocoe[. The contents of a vesicle discharging basally would disperse so quickly that we should not expect to find local concentrations of peroxidase in the blood near the plasma membrane. Although fixed blood gives tao histocbemical reaction for peroxidase, peroxidase is weakly detectable colorimetrically in tl~e blood plasma during the formation of spines and only very faintly in midinstar larvae. Some haemocytes react strongly for peroxidase at all times and the possibility cannot be excluded that the

Fig. 16. A centrally located Golgi complex in a spine cell from a larva 1789 hours before ecdysis. Peroxidase reaction. Stained in block with uranyl acetate. Sections stained with uranyl acetate and lead citrate. Packets of secretory material can be made out in the ER cisternae ( ~ ). There are also coated vesicles associated with the Golgi complexes. J , secretion in ER cisternae; cv, coated vesicle ; v, secretory vesicle. - 60,000. Fig. 17. The localization of peroxidase in secretory vesicles and their discharge at the apical surface. Larva aged M - 1 7 . Thick section reacted for peroxidase, otherwise unstained. ~ , peroxidase between plasma membrane folds, presumably having just been discharged from a secretory vesicle; e, epicuticle; fc, cuticle of spine shaft; mvb, multivesicular bodies containing peroxidase ; pv, secretory vesicles containing peroxidase. 9 27,000.

Fig. 18. The multivesicular bodies containing peroxidase in the supporting cells exposed to spine cuticle. Larva aged M - 1 7 . L.S. through parts of a supporting cell between t w o spine cells. The supporting cell has a few multivesicular bodies containing peroxidase in the apical region near the cuticle of the spine cell. This peroxidase is presumed to be derived from the secretory vesicles containing peroxidase seen abundantly in the spine cell. Thick section unstained except for the peroxidase reaction, e, epicuticle; fc, fibrous cuticle secreted above spine cell, part of which has reacted with DAB ; /c, lamellate cuticle above supporting cell; mvb, multivesicular bodies in the supporting cells containing peroxidase; pv, secretory vesicles containing peroxidase; Sp, spine cell ; Su, supporting cell. :- 16,000. Fig. 19. The apical cuticle secreting surface of a spine cell where it abuts on a supporting cell. The spine cell has many secretory vesicles containing peroxidase. The supporting cell only has peroxidase in this region in what are believed to be coated vesicles, Larva aged M - 1 7 hours. Thick section unstained except for the peroxidase reaction, x 28,000,

Figs. 20 8- 21. The basal surface of spine cells next to the haemocoel. Near the basal surface there are numerous vesicles containing peroxidase which may be discharging to the haemocoel. Fig. 20, larva aged M - 1 7 { hours. Thick sections, peroxidase reaction. Contrast slightly enhanced by in block staining in uranyl acetate. Fig. 21, larva aged M - 1389 hours. Thick sections unstained except for the peroxidase reaction, hi, basal infolds; bin, basement membrane; g, Golgi complex; h, haemocoel; n, nucleus. -,32,000. TISSUE 8 CELL 1969 1 (3)

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T H E A M P H I B I A N P A P I L L A OF T R I T U R U S the whole bundle. In all cases where the basal foot or the two central fibres of the kinocilium could be seen, this axis was approximately parallel to the direction of the basal foot and at right angles to a line joining the two central fibres. On this basis, it appears that the axes of all the hair bundles are roughly parallel to the long axis of the amphibian papilla (that running from the mesial end to the opening into the sacculus); the variation in orientation was not seen to exceed about 20 ~ on either side of the papillary axis. Moreover, the hair bundles in the saccular half of the macula have their kinocilium on the side nearest to the sacculus, whereas all those in the other half have their kinocilium on the opposite side. About halfway along the macula, where the junction between the two populations occurs, there is a zone where adjacent bundles may have their kinocilia on opposite sides. No orientations intermediate between the two were seen. Flock and Wers/ill (1962) describe two slightly different arrangements of the stereocilia of the receptor cells in the lateral-line neuromasts. In the amphibian papilla, the existence of two distinct arrangements is not apparent, although there is some variation between different cells in the number of stereocilia in the bundle and :in the number of rows in which they are arranged. The receptor cells are innervated by two different types of nerve terminal, corresponding to the granulated and non-granulated endings that have been described in many other labyrinthine organs (see, for example,

Smith and Sj6strand, 1961 a and b; Engstr6m and Ades, 1965). There is no evidence in these organs of the third type of ending described by Lowenstein et al. (1964) in the sensory epithelia of the labyrinth of Raia. The non-granulated endings contain large numbers of mitochondria and usually also a few vesicles (about 300 to 1000 ~ in diameter), with electron transparent contents; the supposed synaptic regions are associated with densely staining masses (about 0 2 to 0-6 t9 in diarneter) surrounded by small vesicles (about 300 to 500 /~ in diameter). The granulated endings are found less commonly than the other type. They are associated with membrane pairs in the base of the receptor cell (Fig. 10), and contain relatively few mitochondria but large numbers of small vesicles about 500 to 800 ~ in diameter. A few densely cored vesicles (of total diameter about 700 to 800 A, Fig. 10) are also found in the granulated endings; these vesicles resemble structures which in other nervous tissues are thought to contain amines (see, for example, Wood, 1966; Aghajanian and Bloom, 1967). Densely cored vesicles have occasionally also been seen in the nongranulated endings. (iv) The accessory cells" Each accessory cell bears one short 'cilium' and a large number of microvilli on the apical surface (Figs. 4, 5 and 14). The microvilli are much shorter (under 1 /~) and of smaller diameter than the stereocilia of the receptor cells. Moreover, they are arranged

Fig. 4. Oblique section passing through part of the macula and lumen of the amphibian papilla. In the upper region of the micrograph the section passes through several hair bundles. Each bundle is composed of many stereocilia and one kinocilium ( k ) ; all the kinocilia lie in similar positions relative to their associated stereocilia. The section also passes through many irregularly shaped vesicles which lie between the cupuia and the surface of the macula. The accessory cells (ac) bear microvilli (m) and 'cilia" (c), Dense material lines the cellular junctions near the lumenal border of the epithelium, b, basa~ b o d y ; cp, cuticular plate ; rc, receptor cell. ~-,:6200. Fig. 5. Oblique section through a hair bundle of a receptor cell. The foot (f) on the basal body points away from the stereoci[ia (s). ac, accessory cell; m, microvillus of accessory cell. ;,: 17,000. TISSUE & CELL 1969 1 (3)

409

i84 ii~,iii~ili~ !84184184 ~ ~i~i~,~

572

LOCKE

Cuticle reacting with DA

Epicuticle

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Supporting Cerl

i Supporting Cell 9: \ 7\

Spine Cell

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Fig. 22. An interpretation of the distribution of peroxidase during the formation of the hard cuticle of the shaft of a proleg spine. One component of the secretory vesicles arising in the Golgi complexes of the spine cells is a peroxidase. The vesicles move to the apical face of the cell and are discharged during the deposition of the cuticle of the shaft. The peroxidase is presumed to be involved in cross-linking the structural proteins of the cuticle. This 'peroxidized" cuticle reacts with DAB in the absence of hydrogen peroxide and may be a halfway stage in stabilization. Some of the peroxidase secreted at the surface is taken back up by coated vesicles and transported to multivesicular bodies in both spine cedis and supporting ceils. TISSUE 8 CELL 1969 1 (3)

P E R O X I D A S E AND C U T I C L E F O R M A T I O N plasma is contaminated by ruptured cells. However, the results suggest that there may be some basal discharge of vesicles containing peroxidase. Discussion and Conclusions THE PEROX1DASE IN THE P R O L E G SPINE CFLLS

An interpretation of the observations on tile distribution of peroxidase in the spine cells and supporting cells is presented in Fig. 22. Only the spine cell makes peroxidase, which it secretes at the apical face where it plays some role in tile formation of the hard fibrous cuticle of the shaft and hook. The apical plasma membranes of both epidermal cell types are active in pinocytosis at this time, and peroxidase is taken up from any surface near cuticle which contains the newly secreted peroxidase. This hypothesis answers the question with which the work began-how can the peroxidase have a functional role in cuticle formation when it is located in multivesicular bodies, organelles for lysis. PEROXIDASE AND C U T I C L E STABILIZATION

Two methods of stabilizing insect cuticular proteins have been studied extensively, the cross-linking of resilin by dityrosine and trityrosine (Andersen, 1966) and sclerotization, the cross-linking of proteins by the reaction of their free amino groups with quinones (see, for example, the reviews by Brunet, 1965; Cottrell, 1964). The two processes differ in that the former involves tyrosyl residues and the latter free amino groups, but they are alike in that the crosslinking depends upon small reactive molecules in solution. In either event the problem facing the cell would be how to control such reactive molecules as quinones or peroxides (presumably, but not necessarily, hydrogen peroxide). In the cross-linking of resiiin, peroxidases are probably involved, and similar principles may apply in the stabilization of other sorts of cuticle. Andersen (1966) has described a

'TISSUE 8 CELL 1s

1 (3)

573

model system which may throw light on the way resilin is stabilized. He incubated a gel of silk fibroin with horseradish peroxidase and hydrogen peroxide and found that it became more rigid and insoluble. He was able to extract dityrosine and trityrosine residues from the stabilized gel. Thus protein + peroxidase + hydrogen peroxide can form a complete stabilizing system. Coles (1966) found peroxidase in extracts from several locust tissues including the epidermis secreting resilin. He presumed that this peroxidase could be involved in tile formation of tile dityrosine and trityrosine cross-links as Andersen had suggested. A similar sort of peroxidation of tyrosine residues could be taking place in the hardening of Calpodes spine cuticle in the presence of a natural peroxidase. A biologically harmless low level of hydrogen peroxide might be maintained in the cuticular environment. The spine cell could then determine the cuticle to be stabilized through the local secretion of peroxidase. The spatial and temporal precision of the process would be further enhanced by the constant turnover of the peroxidase, secretion being followed by pinocytosis. The spine cuticle colored by DAB has presumably been partially oxidized but is not yet cross-linked. The observations on Calpodes spine peroxidase could be interpreted as a morphological confirmation of Andersen's hypothesis. Calliphora larvae stabilize their late larval cuticle to form the puparium by tanning with an o-quinone derived from N-acetyl dopamine (Karlson and Sekeris, 1962; Sekeris and Karlson, 1962). The relation between this process and the peroxidase observed in the epidermis is unknown, but deserves to be explored. Acknowledgements I am grateful to J. T. McMahon for technical assistance. The work was generously supported by a grant from the National Institutes of Health G M 09960.

574

LOCKE

References ANDERSEN, SVV.ND OLAV. 1966. Covalent cross-links in a structural protein, resilin. Acta physiol. scand., 66, 1-81. BOULIGAND, M. YVES. 1965. Sur une architecture torsadde rdpandue dans de nombreuses cuticles d'Arthropodes. C.r. hebd. Sdanc. Acad. Sci., Palls, 261, 3665. BRUNET, P. C. J. 1965. The metabolism of aromatic compounds. Aspects of Insect Biochemistly (T. W. Goodwin, editor). Biochemical Society Symposium 25, pp. 49-77, Academic Press, New York. COLES, G. C. 1966. Studies on resilin biosynthesis. J. Insect Physiol., 12, 679. COTTRELL, C. B. 1964. Insect ecdysis with particular emphasis on cuticular hardening and darkening. Advances in Insect Physiology (J. W. L. Beament, J. E. Treherne and V. B. Wigglesworth, editors), Vol. 2. Academic Press, New York. DEDuvE, C. and WATTIAUX, R. 1966. Functions of lysosomes, A. Rev. Physiol., 28, 435. KARLSON, P. and SEKERIS, C. E. 1962. Zum Tyrosinstoffwechsel der Insekten. IX. Kontrolle des Tyrosinstoffwechsels dutch gcdyson. Biochim. biophys. Acta, 63, 489. LOCKE, M. 1966. The structure and formation of the cuticulin layer in the epicuticle of an insect, Colpodes ethlius (Lepidoptera, Hesperiidae). J. Morph., 118, 461. LOCKE, M. 1968. The localization of a peroxidase associated with the formation of hard cuticle in insects. 26th Annual Proceedings E M S A (C. J. Arceneaux, editor). LOCKE, M. 1969a. The structure of an epidermal cell during the formation of the protein epicuticle and the uptake of molting fluid in an insect. J. Morph., 127, 7-40. LOCKe, M. 1969b. The ultrastructure of the oenocytes in the molt/intermolt cycle of an insect. Tissue & Cell, 1, 103. LOCKE, M. and COLLINS, J. V. 1966. The sequestration of protein by the fat body of an insect. Nature, Lond., 210, 552. LOCKE, M. and COLLINS, J. V. 1967. Protein uptake in multivesicular bodies in the molt/intermolt cycle of an insect. Science, N.Y., 155, 467. LOCKE, M. and COLLINS, J. V. 1968. Protein uptake into multivesicular bodies and storage granules in the fat body of an insect. J. Cell Biol., 36, 453. S~KEmS, C. E. and KARLSON, P. 1962. Zum Tyrosinstoffwechscl der Insekten. VII. Der katabolische Abbau des Tyrosins und die Biogenese der Sklerotisierungsubstanz, N-Acetyl-dopamin. Biochim. biophys. Acta, 62, /03.

TISSUE 8- CELL 1969 1 (3)