Hormonal control of morphogenetic cell death of the wing hypodermis in Lucilia cuprina

Hormonal control of morphogenetic cell death of the wing hypodermis in Lucilia cuprina

TISSUE & CELL 1975 7 (2) 281-296 Pohlishd by Longmat~ Group Ltd. Printed in Grent Britain I. M. SELIGMAN,” HORMONAL CONTROL CELL DEATH OF THE LUCILI...

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TISSUE & CELL 1975 7 (2) 281-296 Pohlishd by Longmat~ Group Ltd. Printed in Grent Britain

I. M. SELIGMAN,”

HORMONAL CONTROL CELL DEATH OF THE LUCILIA CUPRINA

B. K. FILSHIE, F. A. D0Y-t and A. C. CROSSLEYS

OF MORPHOGENETIC WING HYPODERMIS

IN

ABSTRACT. 1. Cytoplasmic fragments in the haemolymph of newly emerged flies derive from the degenerating wing hypodermis. 2. At the time of eclosion, dorsal and ventral cell layers of the wing are connected by processes containing bundles of microtubules and microfilaments. Cytoplasmic fragments contain similar bundles of microtubules but few microfilaments. 3. Extensive vacuolation marks the onset of hormonally initiated fragmentation of the wing hypodermal cells. Haemocytes containing lysosomes are present in the wing at this time, but do not invade the fragmenting hypodermis.

Introduction PREVIOUSwork has shown that in the haemolymph of the newly emerged adult blowfly, Luciliu cuprina, there is a transient change in the number of filamentous cellular fragments during the first hours after eclosion (Seligman and Doy, 1973). Despite much investigation of the hormonal regulation of fragment dispersal, the source of the fragments had not been determined. Fragments become detectable approximately 1 hr after the fly emerges from its puparium and reach a maximum concentration of 1.3 x IO7 fragments/ml 75 min after eclosion. During the following hour, the fragment concentration drops to about 3”/;, of the maximal titre. A ligature tied between head and thorax of newly emerged flies inhibits release of fragments from their aggregation site. An injection of haemolymph taken from older flies reverses this inhibition. Appreciable Division of Entomology, Commonwealth Scientific and Industrial Research Organization, Canberra, and fSchool of Biological Sciences, University of Sydney, Sydney, Australia. * Present address: Department of Physiology, University of Illinois, Urbana, Illinois, U.S.A. i Present address: Department of Biochemistry, Australian National University, Canberra, Australia. Received 22 October 1974. Revised 7 January 1975. 281

‘fragment dispersing hormonal activity’ (FDH) was detected in the haemolymph 5 min after eclosion. FDH and the tanning hormone, bursicon (Fraenkel and Hsiao, 1965; Cottrell, 1962a), could not be distinguished from each other experimentally and might be one and the same chemical entity (Seligman and Doy, 1973). A low concentration of cyclic AMP (5 x IO-” mol/fly) mimicked FDH activity. Stimulation of DOPA synthesis was demonstrated at this dosage of cyclic AMP (Seligman and Doy, 1972); a distinct tanning response was observed in Sarcophuga bullata (unpublished observations) and in Calliphora erythrocephalu only with a ten-fold greater dose of cyclic AMP (von Knorre et al., 1972). Van den Berg and Mills (1974) have shown that both tanning and l*C-tyrosine uptake into the cuticle of isolated thoraces of Periplanetu americana is stimulated by dibutyryl-cyclic AMP. Jones (1956) noted that anucleate filamentous fragments could be seen in the haemolymph of Sarcophuga bulluta during all stages of development, but he was unable to associate a change in concentration of these fragments with any one phase in the life of the insect. Jones postulated that the fragments were degradation products of plasmatocytes and/or podocytes, but did not

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claim to have resolved the question of the origin of these structures. He conceded that they might be released from some other source. Whitten (1964) suggested that in the haemolymph of newly emerged Sarcophugu bulluta adults cellular fragments derive from degenerating multinucleate haemocytes. In the larvae of Calliphora the clotting sequence certainly involves haemocyte fragmentation (Zachary and Hoffmann, 1973; Crossley, 1975). Kerr et al. (1972) observed that the changes in morphology of dying cells are the same in embryonic tissues, in senescent cells, as well as in cells treated with drugs that induce degeneration. They proposed that the process be called ‘apoptosis’. In this paper we will show, both by experimental manipulation and by comparing the ultrastructure of the fragments with the ultrastructure of the wing, that most, if not all, the fragments in the haemolymph of newly emerged adult Lucilia are apoptotic derivatives of wing hypodermal cells.

was carried out in 1‘A osmium tetroxide buffered at pH 7.2 with Verona1 acetate containing sucrose. Dehydration was accomplished in a graded ethanol series and propylene oxide. Some tissues were treated with 1% uranyl acetate in alcohol during dehydration to enhance membrane contrast. Dehydrated tissue was embedded in Araldite, and thin sections were obtained on Porter-Blum or Reichert microtomes equipped with diamond knives. Contrast in sections was enhanced by treatment with 5 % uranyl acetate in 20 % methanol, followed by treatment with lead citrate (Reynolds, 1963). Sections were examined in either a Siemens Elmiskop I or a Hitachi HUl 1E electron microscope. Acid phosphatase activity was visualized in the electron microscope by incubation of glutaraldehyde-fixed cells in a modified Gomori medium (Gomori, 1950; Barka, 1964).

Materials and Methods

The appearance of fragments in the haemolymph is not an immediate response to FDH, but is delayed for about threequarters of an hour after the FDH titre in the haemolymph peaks (Seligman and Doy, 1973). We exploited this lag to determine which tagma of the fly contains the fragment precursors. Flies were allowed to emerge from their puparia, and ligatures were tied as indicated in Table 1. The flies were bled 75 min later. The first line in Table 1

Fly rearing, synchronization of adult eclosion and experimental manipulations are described elsewhere (Seligman and Doy, 1972, 1973). Substances with putative hormonal activity were injected into the thorax of ‘neckligated’ flies. ‘Neck-ligated flies are those in which a tight knot is tied between head and thorax soon after ecdysis is initiated. This inhibits release of bursicon and FDH from the CNS. The assay for substances having or affecting these two hormonal activities is based on this tactic. Each estimate was based on a pooled haemolymph sample from 6-10 flies. The means of duplicate determinations are presented in the tables.

Results (1) Delineation of the morphological source of fragments

Table 1. Effect of ligatures between body parts of the adult on fragment dispersal. Flies were released from their puparia and ligated 15 min later at the junction between the body parts indicated in the table. The flies were bled 90 min after eclosion

Electron microscopy

Tissue dissection and centrifugation of blood samples for electron microscopy were carried out in fixative at room temperature. After 15 min fixation the fixative was replaced and the temperature reduced to 4°C. Minimum fixation time was 1 hr, and the fixative used was 2.5 % glutaraldehyde solution, buffered at pH 7.2 with 0.05 M HCl-cacodylate containing 0.15 M sucrose. After washing in buffered sucrose solution, post-fixation

Ligature

Position

Fragments per ml x lO-4

Tanning score (%I

Head-thorax

Head Body

15 878

0 100

Thorax-abdomen

Thorax Abdomen

792 4

100 100

indicates that no fragments were released from the head. The data in the rest of the

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table show that the fragments were released from a source anterior to a ligature tied between the abdomen and the thorax. Thus, the fragments derive from a thoracic structure. This simple technique of ligating various body parts was used to locate the organ in the thorax from which the fragments derive. Ligatures tied at the base of the wings prevented release of fragments into the haemolymph (Table 2). Fragment dispersal Table 2. Purtially emergedpies were neck ligated rmd/or pulled out of their puparia and wing li,qrated. The neck ligated fries were injected with 1 pl ‘active blood’ or 5 nmol cyclic AMP in 1 PI wuter. ‘The flies were bled 75 min after emergence (Nos. 1 unri 2) or 90 min after they were injected (,Vos. 3 ~6)

Treatment

Fragments per mlx IO-”

Wings ligated soon after eclosion and the flies were bled 75 min later Unligated control of I Wing and neck ligated flies injected with active blood Neck ligated flies injected with active blood

60 1400

29 1540

Wing and neck ligated flies injected with 5 nmol cyclic AMP Neck ligated flies injected with 5 nmol cyclic AMP

13 1840

induced by injected or endogenously produced hormone, or by cyclic AMP was inhibited when the wings were isolated from the rest of the body. The small fragment dispersal response observed in wing-ligated flies probably resulted from our inability to tie ligatures precisely at the junction between the wing and thoracic sclerites of each fly. These results indicate that the wings are the most important, possibly the only organ from which fragments are derived. (2) The ultrastructure of haemolymph ,fragmants and haemocytes The fragments present in the circulating haemolymph shortly after eclosion are

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heterogeneous. They range from 200 nm to 50 pm in maximum dimension, and are spherical, elongated or spindle shaped. All are bound by a limiting unit membrane, but lack any nucleus or nuclear envelope. Figs. 1 and 2 show a sample of fragment types. Many fragments contain granular subunits with the dimensions and staining properties of ribosomes. These are often arranged on the surface of membranous compartments (Fig. 1) presumably derived from rough endoplasmic reticulum. Other organelles recognizable in fragments include mitochondria, vesicles bounded by single unit membranes, and a variety of microtubules and microfilaments. Microtubules 25 nm in diameter are sometimes extraordinarily numerous and tightly packed within fragments in strictly parallel orientation (Fig. 2). Occasionally, bundles of microtubules up to 30 nm in diameter appear in fragments. Electron-lucent core material can be detected in transverse sections of most fragment microtubules, but some filaments appear as uniformly electron dense profiles 20-30 nm in diameter (Fig. 2, insert). Other fragments contain smaller microfilaments with diameters in the range 5-7 nm. The circulating haemolymph also contains many nucleated haemocytes, and most of these are apparently actively engaged in phagocytosis of fragments (Fig. 3). Similar phagocytic blood cells have already been described in Calliphora (Crossley, 1964, 1968, 1975). The plasma membrane of haemocytes engaged in phagocytosis is pleomorphic, and extends arms or sheets containing cytoplasm to surround fragments. By completely engulfing fragments, the haemocyte forms membrane-limited inclusion bodies, or phagosomes. The cytoplasm of such cells is seen to contain enormous numbers of phagosomes (Fig. 3) and, as in Calliphora, many were demonstrated to contain acid-phosphatase (see section (4). below). (3) The ultrastructure of the wing at eclosion The untanned, unexpanded wing of the newly emerged fly is composed of dorsal and ventral sheets of cuticle, each secreted by a single layer of epidermal cells (Fig. 4). The dorsal cuticle has a total thickness of 4-5 pm and is composed of the following layers (approximate thicknesses in paren-

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theses); (a) epicuticle (0.1 pm), (b) presumptive exocuticle (1.5 pm), (c) procuticle (2.4 pm> and (d) subcuticle (0.2 pm). The total thickness of the ventral cuticle is about 2 pm and is composed of (a) epicuticle (0.1 pm), (b) procuticle (1.5 pm) and (c) subcuticle (0.2 pm). No presumptive exocuticle appears to be present within the ventral cuticle. The basal extremities of epidermal cells from both dorsal and ventral integuments are extended into a complicated series of thin cytoplasmic processes which reach into the interior compartment of the wing, at this time filled with haemolymph (Fig. 4). Frequently, the nuclei of epidermal cells are contained within the cytoplasmic processes. The cells contain numerous fibrillar

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bundles containing microtubules 27-32 nm in diameter interspersed with microfilaments 4.5-7 nm in diameter (Figs. 6, 7, 8). Some of the microtubules possess internal electrondense material, which forms either a central filament or completely fills the central cavity (i.e., the microtubule becomes a large microfilament) (Fig. 8). In accurate transverse sections both the microtubules and the microfilaments are seen to possess external, irregular ‘halos’ of electron-lucent material, in each case 5-10 nm in thickness. An estimate of the relative numbers of microtubules and microfilaments is difficult to obtain, but in areas where both can be seen together in transverse section there appear to be about lo-15 microfilaments surrounding each microtubule.

Fig. 1. Cellular fragments from the haemolymph 60 min after adult eclosion. The large fragment at upper left contains ribosomes and rough endoplasmic reticulum. The large fragment at the lower right contains a bundle of oriented microtubules. Numerous smaller fragments are present in the field. x 28,000. Fig. 2. Fragments at higher magnification than Fig. I showing transverse sections of oriented microtubules. Most of the microtubules have electron-lucent centres but others appear as solid rods (insert). x 25,000 (insert x 120,000.) Fig. 3. A phagocytic haemocyte Sample is taken from the circulating

engulfing numerous small cellular fragments. blood of a fly 60 min after eclosion. x 13,500.

Fig. 4. Transverse section of the distal part of the wing of a newly emerged adult. Numerous hypodermal processes containing bundles of microtubules (arrows) traverse the haemolymph-filled space between upper and lower wing integuments. Epicuticle, epi; exocuticle, 1~x0; endocuticle, endo; subcuticle, sub. x 4500. Fig. 5. Transverse section through the wing of a newly emerged adult. A hypodermal process from the ventral surface stretches across the central cavity of the wing and joins via a desmosomal attachment (shown at higher magnification in the upper insert) to the dorsal hypodermal layer. The lower insert shows higher magnification of an oriented bundle of microtubules present within the hypodermal process. Nucleus, n; cuticle, cu. x 11,000 (top insert, x 60,000; bottom insert, x 50,000). Fig. 6. Transverse section of the wing of a newly emerged adult showing the cuticlehypodermal junction of the upper surface. Fibrillar bundles (f) attach to hemidesmosomes on invaginations of the apical plasma membrane (arrows). Adjacent gap and septatejunctions(shownat higher magnification in the left and right inserts respectively) are present between neighbouring hypodermal cells. Subcuticle, sub; endocuticle, endo. x 30,000 (inserts, x 100,000). Fig. 7. Section through a wing hypodermal cell showing five separate fibrillar bundles (f) containing microtubules and microfilaments. Subcuticle, sub; endocuticle, endo; epicuticle, epi. x 10,000. Fig. 8. High magnification of cross sections of microtubules (mt) and microfilaments (mf) in a fibrillar bundle. Note that some microtubules appear hollow whilst others contain a central electron-dense core which may completely fill the centre of the microtubule. x 100,000.

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These bundles of microtubules and microfilaments stretch from basal to apical borders of the cells and follow the orientation of the cytoplasmic extensions (Figs. 4, 5). At both borders, bundles appear to be embedded in layers of electron-dense material approximately 300 nm in thickness on the interior face of the cell membrane (Fig. 5, insert, and Fig. 6). At the cuticular border, the cell membrane is indented to form fingerlike pits in the regions where bundles make contact with the cuticle via hemidesmosomes (Fig. 6). At the basal borders of the cells the membrane is usually not indented, but frequently cells from dorsal and ventral integuments join in these regions with the addition of a three-layered intercellular cementing material to the external surfaces of the cells (Fig. 5, insert). Intercellular material exhibits transverse striation with a periodicity of approximately 17 nm. Where junctions are formed between adjacent cells of either the dorsal or ventral integuments, these are of the septate or gap types (Fig. 6, insert). The septate junctions are invariably located towards the cuticular border and the gap junctions towards the interior compartment of the wing. The rest of the cytoplasm of epidermal cells contains large numbers of free ribosomes and rough endoplasmic reticulum, mitochondria and a few phagosomes (Figs. 5. 6). Other types of cells are found in the haemolymph compartment of the wing at this namely haemocytes, tracheae and stage, pigment cells. An example of a pigment cell is illustrated in Fig. 9. The cell lies closely adpressed to the basal lamina of the epidermis and is identified as a pigment cell by the numerous, densely stained granules up to 0.5 km in diameter located mainly in the peripheral cytoplasm. Neither function nor fate of these cells at later stages of development is known.

channels penetrating between these cells (Fig. 9). The vacuoles and channels appear to become confluent, with the result that the hypodermis becomes dissected into isolated fragments. Detachment of cells from the cuticle (Fig. 10) and breakdown of the basal lamina follow, and general tissue disintegration ensues. Blood spaces adjacent to the disintegrating hypodermis are occupied by haemocytes, and these contain numerous lysosomes, as evidenced by acid-phosphatase histochemistry (Fig. I I ). Although the haemocytes are apparently engaged in phagocytosis, disintegration of the hypodermis occurs in areas free of haemocytes, and on morphological grounds it appears that haemocytes play a secondary role in apoptosis. This is further evidenced by the detection of acid phosphatase-reactive lysosomal vacuoles in the wing hypodermis itself (Fig. 12). Reaction product is confined to membrane limited vacuoles, and does not appear in the groundplasm of cells, or between haemocytes and adjacent tissues. Fragments of hypodermis entering circulation often contain acidphosphatase activity, again localized in vacuoles (Fig. IO). In recently emerged adults the distribution of acid phosphatase activity is variable, as evidenced by the modified Gomori technique. However. vacuoles within nerve and tracheal cells are often reactive in preparations that are otherwise negative. It may be that changes in the innervation and oxygen supply resulting from death of nerve and tracheal cells trigger the breakdown of the entire wing hypodermis. Cells adjacent to the basement lamella containing numerous granules with affinity for heavy metals give no indication of acid phosphatase content in Gomori-type preparations lacking uranium and lead section stain, strengthening the supposition that they are pigment cells (Fig. 9).

(4) Ulfrust~rrctrrrc~ qf’ cell dmth

Table I shows that the fragments derive from somewhere in the thorax, because no fragments were found in isolated heads or abdomens. In our previous communications (Seligman and Doy, 1972, 1973) we showed that FDH activity appears in the haemolymph soon after eclosion. Fragment dispersal starts about I hr after eclosion.

it1 wing

n1orphogetwsi.s

The wing hypodermis begins to degenerate at eclosion, and fragmentation (apoptosis) is completed about 2 hr later. The first morphological indication of degeneration is the appearance of large vacuoles within hypodermal cells, and of extracellular

Discussion

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Injecting FDH containing haemolymph (‘active blood’) or cyclic AMP into neckligated flies also initiates fragment dispersal. The response to these hormonal stimuli are substantially reduced when the wings are separated from the body of the fly with ligatures tied at the base of each wing (Table 2). These results indicate that the fragments, or their immediate precursors, should be associated with a component of the wing. Ultrastructure emerged flies

oj’ the wings of newly

A distinctive feature of wings in newly emerged flies is the complex array of cytoplasmic processes that traverse the wings and join the opposite hypodermal layers (Figs. 4, 5). The processes contain microtubules and microfilaments and although both have been detected frequently in insect cells of ectodermal origin, our present results are the first to indicate that microtubules and microfilaments may be intimately associated with one another. For instance, during morphogenesis of scale cells in Ephestia kuhniella, separate bundles of 6 nm filaments and 25 nm microtubules are found (Overton, 1966; Paweletz and Schlote, 1964) in the cells prior to deposition of cuticle. The distribution and orientation of these bundles is related to the final cuticular pattern of the scale. Similarly, in forming bristles of Calpodes ethlius (Locke, 1966,

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and see review by Locke, 1967), bundles of 6 nm filaments are found just beneath the surface plasma membrane prior to cuticulin deposition, in addition to axially oriented microtubules found towards the centre of the cell. In bristles and scale cells it seems that the microfilament-microtubule complex is associated with the direction of cuticle deposition and, ultimately, with the determination of the surface pattern. In the present study microfilaments and microtubules are intermingled and, although there is no semblance of regular packing of either class of fibrils, the overall arrangement bears some similarities to the ordering of thick and thin filaments of muscle; i.e. each microtubule (= thick filament) is surrounded by IO-15 microfilaments (= thin filament). On the other hand, apart from the presence of microfilaments, the other structural components of the processes of Lucilia wing hypodermis are identical to those present in epidermal cells associated with muscle insertions in insects (Auber, 1963; Hagopian, 1970; Lai Fook, 1967; Moulins, 1968; White and Gregory, 1972) namely, microtubular bundles traversing the cells and connecting to the apical and basal plasma membranes by hemidesmosomes. Microfilaments and microtubules in the wing hypodermis are not associated with the direction of cuticle deposition as they are in bristles and scales. They probably

Fig. 9. An epidermal cell process in the central wing cavity 90 min after eclosion. Large vacuoles (v) and extracellular channels (ch) are indicative of impending cellular fragmentation. Nerve cell, nc; pigment cell, pi; tracheole, fu; nucleus, n. x 11,000. Fig. 10. An epidermal cell process 90 min after eclosion in a region of advanced fragmentation. A small fragment (fr) containing an acid-phosphatase reactive vacuole (ap) maintains tenuous contact with the cuticle (cu) through a single cell process. Another nearby epidermal cell (ep) is relatively firmly attached by numerous cell processes (modified Gomori acid-phosphatase technique). x 39,000. Fig. II. A haemocyte (h) adjacent to an epidermal cell (ep) in the wing of a larva 30 min after eclosion (modified Gomori acid-phosphatase technique). Acid phosphatase activity is indicated by lead deposits, leading to interpretation of vacuoles as lysosomes (1~). Cytolysome, cy. x 43,000. Fig. 12. Wing epidermis 30 min after eclosion. Modified Gomori acid-phosphatase technique. Many large vacuoles in epidermal cells contain lead deposits, indicating the presence of acid phosphatase (ap). In nerve cells, acid phosphatase reaction in mitochondria (m), and abnormal vacuolation. x 33,000.

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serve a reinforcing function similar to their role in muscle insertions. The presence of microfilaments is unexplained. Perhaps the microfilaments are similar to the thin (actin?) filaments of muscle and add contractile properties to the hypodermal processes? Further studies of haemolymph flow in Luciliu wings during the period just prior to adult eclosion may be able to determine whether or not the hypodermal processes possess contractile properties. The elongated junctions between the hypodermal cells may limit access to the wing by holding the two hypodermal layers close to each other. Mayer (1896) described similar structures in the wings of some lepidopterous species and proposed that these cytoplasmic bridges ‘serve to hold the two walls of the wing membrane close together during the great expansion of the wing which occurs upon emergence from the chrysalis’. Similar structural elements have been described in the wings of the newly emerged adult aphid, Brevicoryne hrcrssicue (White and Gregory, 1972). Large areas of the wing of newly emerged flies did not expand when the haemolymph pressure was increased artificially (Cottrell, 1962c). The elasticity of the wing undergoes drastic change after release of the fragments into the haemocoele. If pressure is applied to the abdomen, the wings distend and are converted into haemolymph-filled bladders. Cottrell (1962b) observed that when internal pressure was increased by pumping saline into the fly, ‘the upper and lower lamellae became separated from each other, allowing large quantities of blood and saline to flow between them converting the wings into fluid filled sacs’. Cottrell suggested that a transient plastic phase precedes tanning of the cuticle and he also invokes a hypothetical ‘change in the mechanical properties of the components responsible for maintaining the spatial relationships of the upper and lower lamellae of the wing’. Degeneration of the contiguous hypodermal layers in the wing explains these observations, and an increase in the intrinsic plasticity of the wing cuticles becomes a superfluous postulate. Four hours after eclosion the two cuticular layers of the wing are pressed against each other (unpublished observation) and the degraded hypodermal cells have been ingested by phagocytes. Snodgrass (1935)

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observed that when wing development is completed, the epidermis has largely disappeared and the mature wing is almost entirely a cuticular structure. However, Bland and Nutting (1969) stated that this generalization can only be applied to the Holometabola. Morphology

of’flagments

and haemocytes

Muscle insertion-like structures in the hypodermal cells of newly emerged flies are convenient, unique markers for tracing the fate of degenerating cells, as the bundles of microtubules retain their morphological form and are easily identified among the debris jettisoned from the expanded wings (compare Figs. 1 and 2 with Figs. 5, 6, 7 and 8). Hence, the hypodermal cells fragment, cellular debris accumulates in the haemolymph and the fragments are then engulfed by phagocytes (Fig. 3). Remnants of the degraded hypodermal cells can be seen between the two adjacent cuticular layers of the wings in flies 4 hr after eclosion (unpublished observation). Microfilaments have not been definitely identified in the fragments. This could be as a result of more rapid digestion of microfilaments than of microtubules. The cellular site of action of FDH and the localization of the cells that synthesize cyclic AMP in response to the presence of the hormonal agonist are unknown. Phagocytosis is undoubtedly important in the removal of the fragmented hypodermis from the haemolymph, but it is unknown whether haemocytes mediate the initiation of apoptosis in the wing hypodermis. Wigglesworth (1961) states that phagocytes are concerned only with removal of dead or dying cells, although such cells may show little histological change. Degeneration of larval dipteran salivary glands is triggered by ecdysone. Hendrickson and Clever (1972) have correlated increased cathepsin D-like proteolytic activity with regression of the gland in Chironotmrs tentuns. Histolysis occurs both by autophagy and phagocytic attack in the salivary glands of Drosophila psedoohscwrr (Harrod and Kastritsis, 1972). Degeneration of the wing hypodermis is reminiscent of the localized degenerating cells in the imaginal discs of Drosophila melanogaster having congenital structural defects (Fristrom, 1968, 1969: Sprey, 1971). A few morphological

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studies on wing development state that the wings contain hypodermal cells prior to eclosion, and that the wings of the mature fly are bilaminate, acellular cuticular sheets (Snodgrass, 1935; Waddington, 1940). Available evidence indicates that the only hypodermal cells in the fly degenerating at the time of adult eclosion are restricted to the wings. Hypodermal cells in the other appendages and in the abdomen of the newly emerged adult fly are unimpaired (Fogal and Fraenkel, 1970). Very little is known about the biochemical processes intervening between release of FDH from the CNS and dissolution of the wing hypodermal cells. Seligman and Doy (1972) showed that cyclic AMP mimicked FDH activity but were unable to demonstrate FDH-induced cyclic AMP synthesis in viva or in cell free extracts. These negative results could be attributed to a lack of experimental subtlety. Whole animals were used in that study, not selected hormonesensitive tissues. A mediating role for cyclic AMP has also been suggested for isopreneinduced cardiac necroses in rats (Martorana, 1971). Induction of cyclic AMP synthesis might be one of the early steps in apoptosis. The work of Weissman et al. (1971) suggests that wing hypodermal cells and phagocytes respond differently to cyclic AMP. Weissman and his colleagues showed that cyclic AMP stabilizes the lysosomal membranes in phagocytes. The results of this study indicate that membranous elements in the hypodermal cell are more labile in the presence of cyclic nucleotides Wigglesworth (1955) postulated that in

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a hormone is released from the CNS soon after adult eclosion that specifically initiated degeneration of the prothoracic gland, thus eliminating the source of the moulting hormone. It would be extremely interesting if both wing and prothoracic gland degeneration are initiated by the same hormonal stimulus. The cytolytic mechanism for degeneration of the intersegmental muscles in saturniid moths is fully developed at the time of adult Cessation of motor-nerve emergence. impulses after eclosion is the ultimate signal which initiates the histolytic reaction in these muscles (Lockshin, 1969). The ‘eclosion hormone’ inhibits electrical activity in the motor neurons supplying the intersegmental muscles; injection of hormonally active preparations into isolated pharate-adult abdomens resulted in normal breakdown of these muscles (Truman, 1970, 1973a). This hormone is demonstrably different from bursicon (Truman, 1973b). Breakdown of larval intersegmental muscles in Calliphora is not mediated by nervous stimuli. A change in the phagocytic haemocytes is the first morphological indication of hormonally induced metamorphosis in blowflies (Crossley, 1968). Two days after emergence of the adult fly all but two muscles associated with the ptilinal apparatus have disappeared (Laing, 1935) and the internal layer of body musculature in the abdomen is considerably reduced (Cottrell, 1962b). The respective roles of hormones and the efferent nervous system in the death of the specialized eclosion musculature and of the wing hypodermis in Diptera remains to be determined.

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