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An electron microscopic study of flight muscle breakdown in an aphid Megoura viciae
0040-8160/80j00390529502.00
TISSUE & CELL (1980) 12 (3) 529-539 ( 1980 Longman Group Ltd
BRUCE JOHNSON
AN ELECTRON MICROSCOPIC FLIGHT MUSCLE BREAKDOWN APHID MEGOURA VlClAE
STUDY OF IN AN
ABSTRACT. Within two days of settling to feed on a host plant degenerative changes can be detected in the flight muscles of aphids at the ultrastructural level. The thick myosin filaments and the M Lines and later the Z bands disappear leaving the thin actin filaments in the cytoplasm. The mitochondria change their configuration and become enveloped in cytosegresomes. As mitochondria become reduced in number the numbers of lysosomes and residual bodies in the cells build up. The cells then appear to become dispersed as finger-like processes protrude from them and are pinched off.
in aphids is coincident with hypertrophy of the fat body and the resumption of development of embryos in the ovaries which is interrupted during the brief dispersal phase of the aphid’s life. The present study is concerned with the breakdown of the indirect flight muscles of the vetch aphid Megouva viciae Buck.
Introduction MUSCLE breakdown is a regular occurrence in insects during metamorphosis. It is less common in adult insects although breakdown of the flight muscles has now been recorded in over 20 families belonging to eight different orders : Coleoptera, Diptera, Dermaptera, Heteroptera, Homoptera, Hymenoptera, Isoptera and Orthoptera (Johnson, 1976). In some species of Coleoptera, the muscles become reduced but later redevelop; in the other species in which flight muscle breakdown has been recorded it appears to be an irrevocable one way process. There have been several electron microscope studies of the histolysis of skeletal muscles in insects. The subject was reviewed by Finlayson (1975). Most of the studies have been on muscles other than flight muscles, and the only studies on flight muscles have been on beetles in which the muscles become reduced but later redevelop. The flight muscles of several species of aphids were shown by Johnson (1957) to begin to break down within 2-3 days of the aphids settling down to feed on a new host plant. Muscle breakdown which is irreversible Department
of Zoology,
Received 29 October Revised 21 February
University
Materials and Methods The aphids used were Megoura viciae Buck. from a clone maintained by Professor A. D. Lees at Silwood Park Field Station in England. The colonies from which the slates were obtained were kept in controlled environment cabinets at 10°C with a 16 hr photoperiod. The young alates were collected from the tops of the glass cages containing the plants and all aphids were checked to ensure that they could fly before they were used in experiments. The aphids were released on young broadbean seedlings and kept at 15 or 20°C with a 16 hr photoperiod. They were removed and fixed after they had been on the plants for 1, 2, 3, 5 and 15 days. Aphids to be fixed were dipped very briefly in 90% alcohol to wet the cuticle and then immersed in glutaraldehyde in phosphate buffer with 0.1 M sucrose at 5°C. The muscles
of Tasmania.
1979. 1980. 529
530
JOHNSON
of the aphids were exposed in the fixative by cutting the head and abdomen from the thorax and then bisecting the thorax dorsoventrally along the medial line by cutting the dorsal and ventral tergites separately with iridectomy scissors to avoid distorting the muscles. The tissue was then post-fixed in 0~04 after which the muscles were removed from the cuticle and separately embedded in Epon. Both dorso-ventral and longitudinal indirect muscles were studied. Sections were stained with uranyl acetate and lead citrate and examined under Philips 200 and Hitachi 300 electron microscopes. Results
The fine structure of intact flight muscles of young alates of M. viciae was described by Smith (1965). The muscles are very similar in structure to the asynchronous flight muscles of other species of insects. One small difference is that the Z bands of the myofibrils are a little more complex than usual consisting of a thick dense band with a light zone and a narrower dense line on either side of it (Fig. 1). Apart from this the myofilaments, the mitochondria and the other cytoplasmic inclusions resemble in general those in other insects (Smith, 1965). No changes were seen in the muscles of aphids fixed 1 day after settling. At 2 days,
Fig. 1. Longitudinal
changes were apparent in the contractile elements and in the mitochondria. These changes became more apparent with successive days and in aphids 15 days old from the time they settled very little trace remained of what were originally voluminous muscles entirely filling the thorax. The uneven progression of muscle breakdown seen with the light microscope(Johnson, 1959) is further revealed by electron microscopy. In the early stages of muscle breakdown individual sarcomeres were frequently seen in an advanced stage of degeneration while little sign of breakdown was seen in adjacent sarcomeres within the same myofibril or adjacent sarcomeres in neighbouring myofibrils (Fig. 2). Similarly when muscle degeneration was well advanced isolated sarcomeres were sometimes found to be nearly intact while around them all other sarcomeres showed advanced degeneration (Fig. 3). Within each sarcomere breakdown begins with the dissolution of the outermost thick myosin filaments. This often leads to the periphery of the fibril assuming a more angular profile (Fig. 4) than the more rounded perimeter of intact fibrils. Following the dissolution of thick filaments on the periphery it sometimes but not invariably happens that erosion channels develop into the interior of the myofibril as rows of
section of intact indirect
flight muscle of M. viciae. x 17,000.
Fig. 2. Cross-section of muscle at an early stage of degeneration. The myofibrils show various degrees of breakdown. In the fibril in the upper centre of the picture (arrow) most of the thick filaments have disappeared, in other fibrils fewer filaments have gone. The fibril at the upper left has a large ‘erosion channel’ from which the thick filaments have dissolved. x 15,000. Fig. 3. Longitudinal section of muscle from which the thick filaments have gone except in one sarcomere where some are still intact. The Z bands are still conspicuous. x 26,000. Fig. 4. Cross-section of myofibril showing a halo of actin filaments where the outermost thick filaments have disappeared and also a hole (arrow) containing thin filaments in disarray and from which ten thick filaments have been dissolved. x 26,000. Fig. 5. Longitudinal section of a sarcomere showing thick filaments in the process of breaking down (arrow). Breakdown appears to begin in the region of the M line and proceed outwards towards the Z bands. x 30,000.
JOHNSON
532
adjacent thick filaments break down (Fig. 2). Sometimes also, but more rarely, isolated thick filaments deep within fibrils are dissolved; the dissolution process appears to begin with single filaments and then adjacent filaments are affected so that in crosssections of the fibrils at this stage holes develop where the dense myosin filaments have disappeared and the thin actin filaments remain in disarray (Fig. 4). Both the development of channels from the periphery inwards and the development of holes from isolated filaments breaking down within fibrils suggest that dissolving filaments in some way influence adjacent filaments causing them in turn to break down. The thick filaments appear to show the first sign of dissolution in the region of the M line (Fig. 5), breakdown then spreads along the filaments towards the Z bands. Here it stops, and there is no continuity of breakdown between the thick filaments in one sarcomere through the Z band with thick filaments in adjacent sarcomeres. Thick filaments in the process of dissolution can be seen in Fig. 6. Some filaments on the periphery of the fibril where breakdown is taking place can be seen to be much more diffuse than the normal intact filaments such as those deeper within the fibril. As the thick filaments disappear from the sarcomeres so does the M line, so that by the time all of the thick filaments have gone the M line has totally disappeared. In the intact muscle the thin filaments are positioned around the thick filaments
in a very regular arrangement with the thin filaments always equidistant between adjacent thick filaments. At the perimeter of the fibril the thick filaments always have six thin filaments regularly spaced around them. When the thick filaments break down the thin filaments immediately lose their uniform spacing and become more scattered (Fig. 6). When the thick filaments have completely disappeared the thin filaments remain anchored to the Z bands which remain intact for some time after all the thick filaments have gone. But because the thin filaments extend within each sarcomere only to the M line, the effect of removing the thick filaments is to separate the fibril into a series of short sections consisting of bundles of thin filaments extending outward from each side of the Z band. Mostly these sections remain orientated in the same way with respect to one another and remain in the positions they held in the intact muscle (Fig. 7). But some sections break adrift from the rest and become disorientated (Fig. 8). These sections, which I refer to as ‘butterfly-bodies’, often come to lie at right angles to the line of orientation of the original muscle. In them the thin filaments appear to become rather more splayed out at the edges than in sections of muscle which remain in their original positions. Such ‘butterfly-bodies’ bear a striking resemblance to isolated I segments of muscle artificially produced by the extraction of myosin (Huxley, 1963). The Z bands continue to hold the thin
Fig. 6. Cross-section of myofibril in the A zone. Few thick filaments remain and they are surrounded by the surviving thin filaments. Some of the outermost thick filaments (arrow) are in the process of breaking down. x 77,000. Fig. 7. Longitudinal lines have disappeared
section of muscle from which all of the thick filaments and M leaving the thin filaments and Z bands intact. x 20.000.
Fig. 8. Thin filaments held together at the Z band and forming a ‘butterfly-body’ I segment. In this segment a few thick filaments remain (arrow). x 30,000. Fig. 9. Granular vesicle possibly a lysosome young adult aphid before flight. x 30,000. Fig. 10. Lysosomes and residual breakdown (5 days). x 30,000.
bodies
in the cytoplasm
or
of a muscle cell of a
in a muscle cell in an advanced
stage of
JOHNSON
534
filaments together for a while after the disappearance of the thick filaments, but later they too disappear leaving the thin filaments lying in the cytoplasm. In 15 day old insects fragments of muscle cells have still been found to contain thin filaments and no evidence was found in this study of their breaking down. The mitochondria in intact flight muscles are abundant (Fig. 1) and are arranged in rows between myofibrils. They vary greatly in size and some of the larger ones are as large in cross-section as the diameter of the fibrils. They are closely packed with large numbers of cristae and have a dense matrix. As the thick filaments begin to undergo autolysis changes begin to become evident in the mitochondria. The cristae become fewer and more widely spaced, and the matrix becomes more flocculent (Fig. 11). The mitochondria also tend to become more uniformly circular in profile whereas in young intact muscle, although circular profiles tend to predominate, some more elongated profiles are found. As muscle breakdown proceeds the number of mitochondria becomes progressively reduced until by the time all of the myosin filaments have gone only scattered mitochondria remain. The reduction in the numbers of mitochondria appears to be due to their digestion in vacuoles. In muscle which is breaking down mitochondria are frequently found to be enveloped by vacuolar membranes (Fig. 11). The membranes are generally
closely apposed to the mitochondria but occasionally mitochondria are found in vacuoles with large spaces between them and the membrane. The membranes of these vacuoles or cytosegresomes are thicker and denser staining than other membranes and they tend to have an irregular profile as distinct from the smooth profile of the outer mitochondrial membranes (Fig. 11). In young intact muscles a few vesicles containing granular material can be found in the cytoplasm but they are not plentiful (Fig. 9). As the muscles break down a variety of lysosome-like bodies appear in the cells. Some of these contain granular material, some contain dense amorphous material, others contain parallel layers of membrane (Fig. 10). As muscle breakdown proceeds residual bodies begin to appear and these gradually increase in number. They consist of membrane limited vacuoles containing patches of densely staining amorphous material and numerous discrete bundles of multilamellate material. By the time the thick filaments, the Z bands and most of the mitochondria have disappeared, the muscle cells have become greatly reduced in size. This appears to come about by the cells budding off fragments which are lost into the surrounding medium. In Fig. 12 a section of a muscle cell at 15 days is shown. The cell profile contains a number of nuclei packed close together while in the cytoplasm there are numerous thin filaments together with
Fig. 11. Profiles of mitochondria which appear to be in the process of becoming enveloped in cytosegresomes. The mitochondria at the upper right is completely enveloped while the one at the upper left is only partially enveloped. No cytoplasmic membranes are associated with the mitochondrion at the lower left. Note that layers of cytoplasmic membrane adjacent to the enveloped mitochondria and the remains of thecontractile elements of the muscle. A, thin filaments; M, thick filaments; G, glycogen x 40,000. Fig. 12. Profile of the remains of a muscle cell after 15 days. The cell contains a number of nuclei packed tightly together as well as some mitochondria and residual bodies. Finger-like processes extend from its periphery and a number of profiles of processes are present which may or may not be still attached to the cell. The figure also contains a number of tracheae and tracheoles. A small broken segment of basement lamella can be seen at the lower right, the remainder is outside the frame of the picture. x 10,000.
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JOHNSON
many residual bodies and a few scattered mitochondria. Around the edge of the cell the plasma membrane is extended into long finger-like processes containing cytoplasm and thin filaments and sometimes mitochondria. The origin of some of these processes can be seen but sections of many more processes are found in the surrounding areas suggesting that after their development the processes become detached from the cell. Their further fate is unknown but at a later stage no trace of the cells or of the processes could be found and all that could be detected were layers of the basement lamella. Enclosed within it were profiles of numerous tracheae and tracheoles. Discussion Degeneration of the flight muscles of aphids as of other insects (Finlayson, 1975) is probably under the dual control of neural and endocrine factors. One way or another the muscles respond to some influence which causes them to begin a process complete disinteculminating in their gration. From the foregoing account it is apparent that muscle breakdown is not a haphazard process but is a highly programmed one in which specific events follow one another in a definite sequence. First the thick myosin filaments are dissolved together with the M line, later the Z band is removed leaving the thin actin filaments which persist. Meanwhile the mitochondria change their configuration and then become greatly reduced in number. Concurrently granular lysosomes and residual bodies increase in number. Finally the cells appear to undergo a process of budding off portions of the cytoplasm, later they completely disappear and the only traces left of the muscles are the basement layer and the tracheoles. In the breakdown of some muscles in insects phagocytic haemocytes are involved. This does not appear to be the case with flight muscle breakdown either in aphids or in other insects which have been studied. Neither in this study nor in studies of muscle breakdown in other animals (Bhakthan et al., 1970; Crossley, 1972; Lockshin and Beaulaton, 1974; Schiaffino and Hanzlikova, 1972) has any evidence been found of autophagic lysosomes being involved in the breakdown of the filaments. The thick
myosin filaments, the M line and later the Z band all appear to be dissolved in the cytoplasm. It is possible that they are dissolved as a result of the action of a series of specific enzymes. The pattern of dissolution of thick filaments within sarcomeres with the development of erosion channels and of holes is compatible with the hypothesis that an enzyme is involved in this step. How holes can begin to develop deep within sarcomeres is more difficult to account for. The fact that the thick filaments begin to dissolve in the region of the M line would suggest that if an enzyme is involved the enzyme molecules may only gain access to them there where they are not surrounded by thin filaments. Once they begin to break down in this region, breakdown would then proceed along the filament in both directions eliminating that filament but not influencing adjacent filaments except at the M line. The disappearance of the M line as the thick filaments disappear suggests that it may not require a separate enzyme for its dissolution. Its integrity may well depend upon the presence of intact thick filaments. It is likely that in the first instance the breakdown of the thick filaments would involve the separation of the myosin molecules from one another rather than their reduction to smaller fractions. Their further reduction would involve the presence in the cytoplasm of proteolytic enzymes and it is unlikely that these would be released into the cytoplasm while the cell is still functional albeit no longer as a contractile muscle cell. The fact that the Z lines remain for some time after the thick filaments have disappeared suggests that their reduction is brought about by some separate mechanism. Their breakdown could also be the result of enzyme activity but if so different enzymes would appear to be involved. The suggestion that more than one enzyme is involved in muscle breakdown is supported by the finding by Lockshin and Beaulaton (1974) that breakdown of the intersegmental muscles of Antherea can be influenced by actinomycin D and puromycin. Actinomycin D prevented all breakdown whereas puromycin only inhibited digestion of the thick filaments but not of the Z line material nor of the thin filaments. The persistence of the thin filaments in
MUSCLE
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BREAKDOWN
aphid muscle is interesting in that in some other accounts of muscle breakdown both thick and thin filaments seem to disappear concurrently. In fact Miledi and Slater (1969) in a study of the breakdown of denervated diaphragm muscle of the rat were unable to determine whether thick or thin filaments disappeared first. Several authors have commented on the persistence of the Z line and thin filaments after the dissolution of thick filaments ( Auber-Thomay, 1967; Bhakthan etal., 1970; Crossley, 1972; Lockshin and Beaulaton, 1974). In studies on muscular dystrophy in vertebrates there is accumulating evidence that mitochondria become sequestered into cytosegresomes originating from smooth surfaced endoplasmic reticulum where they are digested either by intrinsic enzymes or by later fusion with primary lysosomes (Topping and Travis, 1974; Christie and Stoward, 1977). The finding in this study that mitochondria become isolated in vacuoles suggests that the process leading to the breakdown of mitochondria in aphid muscles may be similar to that in vertebrate muscular dystrophy. Although no primary lysosomes were seen fused with the vacuoles many were seen in the cytoplasm of the cells. The many large residual bodies which were found in the muscles of old insects contained dense lamellate material which probably originated from mitochondria. No intact or recognizable mitochondria were found in these bodies suggesting that mitochondrial breakdown takes place individually in cytosegresomes and that these later fuse to produce the large residual bodies. Well before the mitochondria were enveloped in vacuoles they did show definite changes in configuration with a reduction in numbers of cristae and a change in matrix density. It is very likely that these changes were brought about in response to the changed internal environment of the cells coincident with the beginning of the dissolution of the fibrils and that they were unrelated to their eventual destruction in vacuoles. It is interesting that although there was a rapid initial reduction in mitochondria some mitochondria were still
present in the cells in the final stages when the cells were very much reduced in size. The reduction in the volume of the muscle cells following dissolution of the contractile apparatus appears to result from cytoplasmic processes being budded off from the cells. There does not appear to be an extensive expulsion of material by the process of exocytosis. Randall (1970) also found that in denervated proleg muscles in Galleria cytoplasmic projections were budded off the muscle cells, and Miledi and Slater (1969) described a similar budding off of cell processes in degenerating muscle cells in the rat. The latter authors also found that the basement lamella was thrown into extensive folds which again is very similar to the situation in aphids where folded layers of lamella are a conspicuous feature when the muscles are in an advanced stage of degeneration, suggesting that there is no reduction in the size of the basement layer and as the muscle volume shrinks it inevitably becomes folded. If the cells continue to bud off small portions of themselves the question arises of what is the fate of the fragments. As the fragments bud off they are still enclosed within the basement lamella. They must then either pass through this in an intact form, or undergo autolysis and then the products of their dissolution pass through it. A small number of profiles of cytoplasmic fragments were in fact seen outside the basement lamella suggesting that the former method may take place but it would be premature to suggest that this is the normal method of dispersal of the remains of the muscles. One way or another however the breakdown products of the degenerating flight muscles must enter the haemocoel and from there they no doubt eventually find their way into other cells to become involved in further anabolic processes.
Acknowledgements I wish to thank Professor A. D. Lees for supplying the insects and for providing laboratory facilities while this study was being carried out. 1 am also grateful to Dr J. Holmes for his help in many ways.
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JOHNSON
References AUBER-THOMAY, M. 1967. Modifications ultrastructurales au tours de la d&g&n&escence et de la croissance de fibres musculaires chez un insecte. J. Microscopic, 6, 627-638. BHAKTHAN, N. M. G., BORDEN, J. H. and NAIR, K. K. 1970. Fine structure of degenerating and regenerating muscles in a bark beetle, Zps confiws. I. Degeneration. J. Cell Sci., 6, 807-811. CHRISTIE, K. N. and STOWARD, P. J. 1977. A cytochemical study of acid phosphatase in dystrophic hamster muscle. J. Ultrastruct. Res., 58, 219-234. CROSSLEY,A. C. S. 1972. Ultrastructural changes during transition of larval to adult intersegmental muscle at metamorphosis in the blowfly Calliphora erythrocephala. 1. Dedifferentiation and myoblast fusion. J. Embryol. enp. Morph., 21, 43-74. FINLAYSON,L. H. 1975. Development and degeneration. In InsectMuscle (ed. P. N. R. Usherwood), Chap. 2, pp. 75-149. Academic Press, London. HUXLEY, H. E. 1963. Electron microscope studies on the structure of natural and synthetic protein filaments from striated muscle. J. Mol. Biol., 7, 281-308. JOHNSON, B. 1957. Studies on the degeneration of the flight muscles of alate aphids. I. A comparative study of the occurrence of muscle breakdown in relation to reproduction in several species. J. Insect Physiol., 1, 248-256. JOHNSON,B. 1959. Studies on the degeneration of the flight muscles of alate aphids. II. Histology and control of muscle breakdown. J. Insect Physiol., 3, 367-377. JOHNSON,C. G. 1976. Lability of the flight system: a context for functional adaption. In Insect Flight (ed. R. C. Raincy), Symp. R. Ent. Sot. Lond. No. 7. pp. 217-234. LOCKSHIN R. A. and BEAULATON, J. 1974. Programmed cell death: cytochemical evidence for lysosomes during breakdown of the intersegmental muscles. J. Ultrastruct. Res., 46,43-62. MILEDI, R. and SLATER,C. R. 1969. Electron microscope study of denervated skeletal muscle. Proc. R. Sot. Land. B, 174, 253-269. RANDALL, W. 1970. Ultrastructural changes in the proleg retractor muscles of Galleria melloneZla after denervation. J. Insect Physiol., 16, 1927-1943. SCHIAFFINO,S. and HANZLIKOVA, U. 1972. Studies on the effect of denervation in developing muscles. II. The lysosomal system. J. Ukrastruct. Ref., 39, 1-14. SMITH, D. S. 1965. The organization of flight muscles in an aphid Megoura viciae (Homoptera). J. Cell Biol., 27, 379-393. TOPPING, T. M. and TRAVIS, D. F. 1974. An electron cytochemical study of mechanisms of lysosomal activity in the rat left ventricular mural myocardium. J. Ultrastruct. Res., 46, l-22.
TISSUE & CELL (1980) 12 (3) 529-539 ( 1980 Longman Group Ltd
BRUCE JOHNSON
AN ELECTRON MICROSCOPIC FLIGHT MUSCLE BREAKDOWN APHID MEGOURA VlClAE
STUDY OF IN AN
ABSTRACT. Within two days of settling to feed on a host plant degenerative changes can be detected in the flight muscles of aphids at the ultrastructural level. The thick myosin filaments and the M Lines and later the Z bands disappear leaving the thin actin filaments in the cytoplasm. The mitochondria change their configuration and become enveloped in cytosegresomes. As mitochondria become reduced in number the numbers of lysosomes and residual bodies in the cells build up. The cells then appear to become dispersed as finger-like processes protrude from them and are pinched off.
in aphids is coincident with hypertrophy of the fat body and the resumption of development of embryos in the ovaries which is interrupted during the brief dispersal phase of the aphid’s life. The present study is concerned with the breakdown of the indirect flight muscles of the vetch aphid Megouva viciae Buck.
Introduction MUSCLE breakdown is a regular occurrence in insects during metamorphosis. It is less common in adult insects although breakdown of the flight muscles has now been recorded in over 20 families belonging to eight different orders : Coleoptera, Diptera, Dermaptera, Heteroptera, Homoptera, Hymenoptera, Isoptera and Orthoptera (Johnson, 1976). In some species of Coleoptera, the muscles become reduced but later redevelop; in the other species in which flight muscle breakdown has been recorded it appears to be an irrevocable one way process. There have been several electron microscope studies of the histolysis of skeletal muscles in insects. The subject was reviewed by Finlayson (1975). Most of the studies have been on muscles other than flight muscles, and the only studies on flight muscles have been on beetles in which the muscles become reduced but later redevelop. The flight muscles of several species of aphids were shown by Johnson (1957) to begin to break down within 2-3 days of the aphids settling down to feed on a new host plant. Muscle breakdown which is irreversible Department
of Zoology,
Received 29 October Revised 21 February
University
Materials and Methods The aphids used were Megoura viciae Buck. from a clone maintained by Professor A. D. Lees at Silwood Park Field Station in England. The colonies from which the slates were obtained were kept in controlled environment cabinets at 10°C with a 16 hr photoperiod. The young alates were collected from the tops of the glass cages containing the plants and all aphids were checked to ensure that they could fly before they were used in experiments. The aphids were released on young broadbean seedlings and kept at 15 or 20°C with a 16 hr photoperiod. They were removed and fixed after they had been on the plants for 1, 2, 3, 5 and 15 days. Aphids to be fixed were dipped very briefly in 90% alcohol to wet the cuticle and then immersed in glutaraldehyde in phosphate buffer with 0.1 M sucrose at 5°C. The muscles
of Tasmania.
1979. 1980. 529
530
JOHNSON
of the aphids were exposed in the fixative by cutting the head and abdomen from the thorax and then bisecting the thorax dorsoventrally along the medial line by cutting the dorsal and ventral tergites separately with iridectomy scissors to avoid distorting the muscles. The tissue was then post-fixed in 0~04 after which the muscles were removed from the cuticle and separately embedded in Epon. Both dorso-ventral and longitudinal indirect muscles were studied. Sections were stained with uranyl acetate and lead citrate and examined under Philips 200 and Hitachi 300 electron microscopes. Results
The fine structure of intact flight muscles of young alates of M. viciae was described by Smith (1965). The muscles are very similar in structure to the asynchronous flight muscles of other species of insects. One small difference is that the Z bands of the myofibrils are a little more complex than usual consisting of a thick dense band with a light zone and a narrower dense line on either side of it (Fig. 1). Apart from this the myofilaments, the mitochondria and the other cytoplasmic inclusions resemble in general those in other insects (Smith, 1965). No changes were seen in the muscles of aphids fixed 1 day after settling. At 2 days,
Fig. 1. Longitudinal
changes were apparent in the contractile elements and in the mitochondria. These changes became more apparent with successive days and in aphids 15 days old from the time they settled very little trace remained of what were originally voluminous muscles entirely filling the thorax. The uneven progression of muscle breakdown seen with the light microscope(Johnson, 1959) is further revealed by electron microscopy. In the early stages of muscle breakdown individual sarcomeres were frequently seen in an advanced stage of degeneration while little sign of breakdown was seen in adjacent sarcomeres within the same myofibril or adjacent sarcomeres in neighbouring myofibrils (Fig. 2). Similarly when muscle degeneration was well advanced isolated sarcomeres were sometimes found to be nearly intact while around them all other sarcomeres showed advanced degeneration (Fig. 3). Within each sarcomere breakdown begins with the dissolution of the outermost thick myosin filaments. This often leads to the periphery of the fibril assuming a more angular profile (Fig. 4) than the more rounded perimeter of intact fibrils. Following the dissolution of thick filaments on the periphery it sometimes but not invariably happens that erosion channels develop into the interior of the myofibril as rows of
section of intact indirect
flight muscle of M. viciae. x 17,000.
Fig. 2. Cross-section of muscle at an early stage of degeneration. The myofibrils show various degrees of breakdown. In the fibril in the upper centre of the picture (arrow) most of the thick filaments have disappeared, in other fibrils fewer filaments have gone. The fibril at the upper left has a large ‘erosion channel’ from which the thick filaments have dissolved. x 15,000. Fig. 3. Longitudinal section of muscle from which the thick filaments have gone except in one sarcomere where some are still intact. The Z bands are still conspicuous. x 26,000. Fig. 4. Cross-section of myofibril showing a halo of actin filaments where the outermost thick filaments have disappeared and also a hole (arrow) containing thin filaments in disarray and from which ten thick filaments have been dissolved. x 26,000. Fig. 5. Longitudinal section of a sarcomere showing thick filaments in the process of breaking down (arrow). Breakdown appears to begin in the region of the M line and proceed outwards towards the Z bands. x 30,000.
JOHNSON
532
adjacent thick filaments break down (Fig. 2). Sometimes also, but more rarely, isolated thick filaments deep within fibrils are dissolved; the dissolution process appears to begin with single filaments and then adjacent filaments are affected so that in crosssections of the fibrils at this stage holes develop where the dense myosin filaments have disappeared and the thin actin filaments remain in disarray (Fig. 4). Both the development of channels from the periphery inwards and the development of holes from isolated filaments breaking down within fibrils suggest that dissolving filaments in some way influence adjacent filaments causing them in turn to break down. The thick filaments appear to show the first sign of dissolution in the region of the M line (Fig. 5), breakdown then spreads along the filaments towards the Z bands. Here it stops, and there is no continuity of breakdown between the thick filaments in one sarcomere through the Z band with thick filaments in adjacent sarcomeres. Thick filaments in the process of dissolution can be seen in Fig. 6. Some filaments on the periphery of the fibril where breakdown is taking place can be seen to be much more diffuse than the normal intact filaments such as those deeper within the fibril. As the thick filaments disappear from the sarcomeres so does the M line, so that by the time all of the thick filaments have gone the M line has totally disappeared. In the intact muscle the thin filaments are positioned around the thick filaments
in a very regular arrangement with the thin filaments always equidistant between adjacent thick filaments. At the perimeter of the fibril the thick filaments always have six thin filaments regularly spaced around them. When the thick filaments break down the thin filaments immediately lose their uniform spacing and become more scattered (Fig. 6). When the thick filaments have completely disappeared the thin filaments remain anchored to the Z bands which remain intact for some time after all the thick filaments have gone. But because the thin filaments extend within each sarcomere only to the M line, the effect of removing the thick filaments is to separate the fibril into a series of short sections consisting of bundles of thin filaments extending outward from each side of the Z band. Mostly these sections remain orientated in the same way with respect to one another and remain in the positions they held in the intact muscle (Fig. 7). But some sections break adrift from the rest and become disorientated (Fig. 8). These sections, which I refer to as ‘butterfly-bodies’, often come to lie at right angles to the line of orientation of the original muscle. In them the thin filaments appear to become rather more splayed out at the edges than in sections of muscle which remain in their original positions. Such ‘butterfly-bodies’ bear a striking resemblance to isolated I segments of muscle artificially produced by the extraction of myosin (Huxley, 1963). The Z bands continue to hold the thin
Fig. 6. Cross-section of myofibril in the A zone. Few thick filaments remain and they are surrounded by the surviving thin filaments. Some of the outermost thick filaments (arrow) are in the process of breaking down. x 77,000. Fig. 7. Longitudinal lines have disappeared
section of muscle from which all of the thick filaments and M leaving the thin filaments and Z bands intact. x 20.000.
Fig. 8. Thin filaments held together at the Z band and forming a ‘butterfly-body’ I segment. In this segment a few thick filaments remain (arrow). x 30,000. Fig. 9. Granular vesicle possibly a lysosome young adult aphid before flight. x 30,000. Fig. 10. Lysosomes and residual breakdown (5 days). x 30,000.
bodies
in the cytoplasm
or
of a muscle cell of a
in a muscle cell in an advanced
stage of
JOHNSON
534
filaments together for a while after the disappearance of the thick filaments, but later they too disappear leaving the thin filaments lying in the cytoplasm. In 15 day old insects fragments of muscle cells have still been found to contain thin filaments and no evidence was found in this study of their breaking down. The mitochondria in intact flight muscles are abundant (Fig. 1) and are arranged in rows between myofibrils. They vary greatly in size and some of the larger ones are as large in cross-section as the diameter of the fibrils. They are closely packed with large numbers of cristae and have a dense matrix. As the thick filaments begin to undergo autolysis changes begin to become evident in the mitochondria. The cristae become fewer and more widely spaced, and the matrix becomes more flocculent (Fig. 11). The mitochondria also tend to become more uniformly circular in profile whereas in young intact muscle, although circular profiles tend to predominate, some more elongated profiles are found. As muscle breakdown proceeds the number of mitochondria becomes progressively reduced until by the time all of the myosin filaments have gone only scattered mitochondria remain. The reduction in the numbers of mitochondria appears to be due to their digestion in vacuoles. In muscle which is breaking down mitochondria are frequently found to be enveloped by vacuolar membranes (Fig. 11). The membranes are generally
closely apposed to the mitochondria but occasionally mitochondria are found in vacuoles with large spaces between them and the membrane. The membranes of these vacuoles or cytosegresomes are thicker and denser staining than other membranes and they tend to have an irregular profile as distinct from the smooth profile of the outer mitochondrial membranes (Fig. 11). In young intact muscles a few vesicles containing granular material can be found in the cytoplasm but they are not plentiful (Fig. 9). As the muscles break down a variety of lysosome-like bodies appear in the cells. Some of these contain granular material, some contain dense amorphous material, others contain parallel layers of membrane (Fig. 10). As muscle breakdown proceeds residual bodies begin to appear and these gradually increase in number. They consist of membrane limited vacuoles containing patches of densely staining amorphous material and numerous discrete bundles of multilamellate material. By the time the thick filaments, the Z bands and most of the mitochondria have disappeared, the muscle cells have become greatly reduced in size. This appears to come about by the cells budding off fragments which are lost into the surrounding medium. In Fig. 12 a section of a muscle cell at 15 days is shown. The cell profile contains a number of nuclei packed close together while in the cytoplasm there are numerous thin filaments together with
Fig. 11. Profiles of mitochondria which appear to be in the process of becoming enveloped in cytosegresomes. The mitochondria at the upper right is completely enveloped while the one at the upper left is only partially enveloped. No cytoplasmic membranes are associated with the mitochondrion at the lower left. Note that layers of cytoplasmic membrane adjacent to the enveloped mitochondria and the remains of thecontractile elements of the muscle. A, thin filaments; M, thick filaments; G, glycogen x 40,000. Fig. 12. Profile of the remains of a muscle cell after 15 days. The cell contains a number of nuclei packed tightly together as well as some mitochondria and residual bodies. Finger-like processes extend from its periphery and a number of profiles of processes are present which may or may not be still attached to the cell. The figure also contains a number of tracheae and tracheoles. A small broken segment of basement lamella can be seen at the lower right, the remainder is outside the frame of the picture. x 10,000.
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many residual bodies and a few scattered mitochondria. Around the edge of the cell the plasma membrane is extended into long finger-like processes containing cytoplasm and thin filaments and sometimes mitochondria. The origin of some of these processes can be seen but sections of many more processes are found in the surrounding areas suggesting that after their development the processes become detached from the cell. Their further fate is unknown but at a later stage no trace of the cells or of the processes could be found and all that could be detected were layers of the basement lamella. Enclosed within it were profiles of numerous tracheae and tracheoles. Discussion Degeneration of the flight muscles of aphids as of other insects (Finlayson, 1975) is probably under the dual control of neural and endocrine factors. One way or another the muscles respond to some influence which causes them to begin a process complete disinteculminating in their gration. From the foregoing account it is apparent that muscle breakdown is not a haphazard process but is a highly programmed one in which specific events follow one another in a definite sequence. First the thick myosin filaments are dissolved together with the M line, later the Z band is removed leaving the thin actin filaments which persist. Meanwhile the mitochondria change their configuration and then become greatly reduced in number. Concurrently granular lysosomes and residual bodies increase in number. Finally the cells appear to undergo a process of budding off portions of the cytoplasm, later they completely disappear and the only traces left of the muscles are the basement layer and the tracheoles. In the breakdown of some muscles in insects phagocytic haemocytes are involved. This does not appear to be the case with flight muscle breakdown either in aphids or in other insects which have been studied. Neither in this study nor in studies of muscle breakdown in other animals (Bhakthan et al., 1970; Crossley, 1972; Lockshin and Beaulaton, 1974; Schiaffino and Hanzlikova, 1972) has any evidence been found of autophagic lysosomes being involved in the breakdown of the filaments. The thick
myosin filaments, the M line and later the Z band all appear to be dissolved in the cytoplasm. It is possible that they are dissolved as a result of the action of a series of specific enzymes. The pattern of dissolution of thick filaments within sarcomeres with the development of erosion channels and of holes is compatible with the hypothesis that an enzyme is involved in this step. How holes can begin to develop deep within sarcomeres is more difficult to account for. The fact that the thick filaments begin to dissolve in the region of the M line would suggest that if an enzyme is involved the enzyme molecules may only gain access to them there where they are not surrounded by thin filaments. Once they begin to break down in this region, breakdown would then proceed along the filament in both directions eliminating that filament but not influencing adjacent filaments except at the M line. The disappearance of the M line as the thick filaments disappear suggests that it may not require a separate enzyme for its dissolution. Its integrity may well depend upon the presence of intact thick filaments. It is likely that in the first instance the breakdown of the thick filaments would involve the separation of the myosin molecules from one another rather than their reduction to smaller fractions. Their further reduction would involve the presence in the cytoplasm of proteolytic enzymes and it is unlikely that these would be released into the cytoplasm while the cell is still functional albeit no longer as a contractile muscle cell. The fact that the Z lines remain for some time after the thick filaments have disappeared suggests that their reduction is brought about by some separate mechanism. Their breakdown could also be the result of enzyme activity but if so different enzymes would appear to be involved. The suggestion that more than one enzyme is involved in muscle breakdown is supported by the finding by Lockshin and Beaulaton (1974) that breakdown of the intersegmental muscles of Antherea can be influenced by actinomycin D and puromycin. Actinomycin D prevented all breakdown whereas puromycin only inhibited digestion of the thick filaments but not of the Z line material nor of the thin filaments. The persistence of the thin filaments in
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aphid muscle is interesting in that in some other accounts of muscle breakdown both thick and thin filaments seem to disappear concurrently. In fact Miledi and Slater (1969) in a study of the breakdown of denervated diaphragm muscle of the rat were unable to determine whether thick or thin filaments disappeared first. Several authors have commented on the persistence of the Z line and thin filaments after the dissolution of thick filaments ( Auber-Thomay, 1967; Bhakthan etal., 1970; Crossley, 1972; Lockshin and Beaulaton, 1974). In studies on muscular dystrophy in vertebrates there is accumulating evidence that mitochondria become sequestered into cytosegresomes originating from smooth surfaced endoplasmic reticulum where they are digested either by intrinsic enzymes or by later fusion with primary lysosomes (Topping and Travis, 1974; Christie and Stoward, 1977). The finding in this study that mitochondria become isolated in vacuoles suggests that the process leading to the breakdown of mitochondria in aphid muscles may be similar to that in vertebrate muscular dystrophy. Although no primary lysosomes were seen fused with the vacuoles many were seen in the cytoplasm of the cells. The many large residual bodies which were found in the muscles of old insects contained dense lamellate material which probably originated from mitochondria. No intact or recognizable mitochondria were found in these bodies suggesting that mitochondrial breakdown takes place individually in cytosegresomes and that these later fuse to produce the large residual bodies. Well before the mitochondria were enveloped in vacuoles they did show definite changes in configuration with a reduction in numbers of cristae and a change in matrix density. It is very likely that these changes were brought about in response to the changed internal environment of the cells coincident with the beginning of the dissolution of the fibrils and that they were unrelated to their eventual destruction in vacuoles. It is interesting that although there was a rapid initial reduction in mitochondria some mitochondria were still
present in the cells in the final stages when the cells were very much reduced in size. The reduction in the volume of the muscle cells following dissolution of the contractile apparatus appears to result from cytoplasmic processes being budded off from the cells. There does not appear to be an extensive expulsion of material by the process of exocytosis. Randall (1970) also found that in denervated proleg muscles in Galleria cytoplasmic projections were budded off the muscle cells, and Miledi and Slater (1969) described a similar budding off of cell processes in degenerating muscle cells in the rat. The latter authors also found that the basement lamella was thrown into extensive folds which again is very similar to the situation in aphids where folded layers of lamella are a conspicuous feature when the muscles are in an advanced stage of degeneration, suggesting that there is no reduction in the size of the basement layer and as the muscle volume shrinks it inevitably becomes folded. If the cells continue to bud off small portions of themselves the question arises of what is the fate of the fragments. As the fragments bud off they are still enclosed within the basement lamella. They must then either pass through this in an intact form, or undergo autolysis and then the products of their dissolution pass through it. A small number of profiles of cytoplasmic fragments were in fact seen outside the basement lamella suggesting that the former method may take place but it would be premature to suggest that this is the normal method of dispersal of the remains of the muscles. One way or another however the breakdown products of the degenerating flight muscles must enter the haemocoel and from there they no doubt eventually find their way into other cells to become involved in further anabolic processes.
Acknowledgements I wish to thank Professor A. D. Lees for supplying the insects and for providing laboratory facilities while this study was being carried out. 1 am also grateful to Dr J. Holmes for his help in many ways.
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References AUBER-THOMAY, M. 1967. Modifications ultrastructurales au tours de la d&g&n&escence et de la croissance de fibres musculaires chez un insecte. J. Microscopic, 6, 627-638. BHAKTHAN, N. M. G., BORDEN, J. H. and NAIR, K. K. 1970. Fine structure of degenerating and regenerating muscles in a bark beetle, Zps confiws. I. Degeneration. J. Cell Sci., 6, 807-811. CHRISTIE, K. N. and STOWARD, P. J. 1977. A cytochemical study of acid phosphatase in dystrophic hamster muscle. J. Ultrastruct. Res., 58, 219-234. CROSSLEY,A. C. S. 1972. Ultrastructural changes during transition of larval to adult intersegmental muscle at metamorphosis in the blowfly Calliphora erythrocephala. 1. Dedifferentiation and myoblast fusion. J. Embryol. enp. Morph., 21, 43-74. FINLAYSON,L. H. 1975. Development and degeneration. In InsectMuscle (ed. P. N. R. Usherwood), Chap. 2, pp. 75-149. Academic Press, London. HUXLEY, H. E. 1963. Electron microscope studies on the structure of natural and synthetic protein filaments from striated muscle. J. Mol. Biol., 7, 281-308. JOHNSON, B. 1957. Studies on the degeneration of the flight muscles of alate aphids. I. A comparative study of the occurrence of muscle breakdown in relation to reproduction in several species. J. Insect Physiol., 1, 248-256. JOHNSON,B. 1959. Studies on the degeneration of the flight muscles of alate aphids. II. Histology and control of muscle breakdown. J. Insect Physiol., 3, 367-377. JOHNSON,C. G. 1976. Lability of the flight system: a context for functional adaption. In Insect Flight (ed. R. C. Raincy), Symp. R. Ent. Sot. Lond. No. 7. pp. 217-234. LOCKSHIN R. A. and BEAULATON, J. 1974. Programmed cell death: cytochemical evidence for lysosomes during breakdown of the intersegmental muscles. J. Ultrastruct. Res., 46,43-62. MILEDI, R. and SLATER,C. R. 1969. Electron microscope study of denervated skeletal muscle. Proc. R. Sot. Land. B, 174, 253-269. RANDALL, W. 1970. Ultrastructural changes in the proleg retractor muscles of Galleria melloneZla after denervation. J. Insect Physiol., 16, 1927-1943. SCHIAFFINO,S. and HANZLIKOVA, U. 1972. Studies on the effect of denervation in developing muscles. II. The lysosomal system. J. Ukrastruct. Ref., 39, 1-14. SMITH, D. S. 1965. The organization of flight muscles in an aphid Megoura viciae (Homoptera). J. Cell Biol., 27, 379-393. TOPPING, T. M. and TRAVIS, D. F. 1974. An electron cytochemical study of mechanisms of lysosomal activity in the rat left ventricular mural myocardium. J. Ultrastruct. Res., 46, l-22.