The fine structure and mechanism of breakdown of larval intersegmental muscles in the blowfly Calliphora erythrocephala

J. InsectPhyriol.,1968,Voi. 14,pp. 1389to 1407. Perpnon

Press. P&ted

in Great Btitain

THE FINE STRUCTURE AND MECHANISM OF BREAKDOWN OF LARVAL INTERSEGMENTAL MUSCLES IN THE BLOWFLY CALLIPHORA ERYTHROCEPHALA A. C. CROSSLEY* CSIRO, Division of Entomology, Canberra, A.C.T.,

Australia

(Received 17 May 1968) Abstract-The intersegmental muscles of Calliphora are composed of an array of thick and thin myofilaments, in which an average of ten thin myofilaments surrounds each thick myofilament. The Z-disks are perforated, and both thick and thin myofilaments pass through the perforations when the muscle is fully contracted. During puparium formation the intersegmental muscles contract only partially, so as to bring the ends of the thick myofilaments into contact with, but not to penetrate, the Z-disk. It is shown that invasion of muscles by phagocytes is under humoral control, and can be prematurely or artificially induced by injection of crustecdysone. A change in the haemocyte population induced by crustecdysone is the first morphological indication of the onset of metamorphosis. Breakdown of intersegmental muscles in Culliphma is not mediated by nervous stimuli. The signal inviting invasion of haemocytes into individual muscles is unknown, but morphological changes in the doomed muscles precede invasion. Attenuated folds of haemocyte plasma membrane penetrate muscles which possess normal contractile elements. Adjacent folds unite, isolating muscle fragments within the haemocyte cytoplasm. Soluble or colloidal elements are taken into the haemocyte by coated vesicles, which line the pockets of haemolymph between haemocyte and muscle. Coated vesicles in circulating haemocytes are capable of isolating non-proteinaceous substances such as thorium dioxide, and transporting them into the cell, where they accumulate in vacuoles. Within the phagocytic haemocyte muscle fragments are degraded in a welldeveloped vacuolar apparatus. Acid phosphatase activity is demonstrated in many vacuoles. The catabolic process within the vacuolar apparatus leads to the accumulation of lipid and glycogen reserves. Protein and protein+RNA vacuoles also accumulate, but the extent of degradation of macromolecules is unknown. Rupture of the haemocyte plasma membrane leads to the eventual release of all modified muscle components into the haemolymph. INTRODUCTION

OVERTHIRTY years ago FMENKEL (1934,193s) showed, by means of ligature experiments, that the hardening and darkening of the Calliphora larval cuticle to form the * Queen Elizabeth II Research Fellow. 1389

1390

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CROSSLEY

puparium is controlled by a hormone produced in the ring gland at the anterior of the larva. The development of the ligature procedure into the ‘Calhphora test’ for assay of moulting hormone by BECKERand PLAGGE(1939) made possible the eventual crystallization of an active moulting hormone, ‘ecdysone’, by BUTENANDT and KARLSON(1954). Ecdysone has now been characterized (KARLSONet al., 1963 ; EARLSON,1965) and a number of analogues prepared. It has recently been shown that crustacean and insect moulting hormones are very similar or identical and that the analogue crustecdysone (18ecdysone) although less active than 01ecdysone is much more abundant in both Antheraea pupae and crayfish, and may be the hormone chiefly responsible for moulting (HAMPSHIRE and HORN, 1966; HORN et al., 1966). The mode of action of ecdysone and its analogues is still imperfectly understood (review by NOVAK,1966). It is not clear how much influence ecdysone analogues have on the processes of differentiation that precede and follow puparium formation. Thus alterations in the haemocyte population and changes in the muscles appear to be under the control of external, and probably humoral, factors (CROSSLEY,1964,1965), but it is not known whether such changes are mediated by ecdysone. The present fine-structural study of muscle breakdown by phagocytic haemocytes provides an opportunity to relate the changes in the haemocyte-muscle complex with the titre of an ecdysone analogue. The cytology of degeneration in the intersegmental muscles of the adult silk moth Antheraea pernyi has been reported by LOCKSHINand WILLIAMS (1965a). In this insect alteration of the ecdysone/juvenile hormone balance potentiates the breakdown of these muscles but the final triggering signal that initiates breakdown is the cessation of the nervous input to the doomed muscle. This signal is thought to bring about rupture of lysosomes in apparently viable muscle, with the result that organelles are broken down within what might be called an ‘autophagic’ vacuolar apparatus (terminology of DE Duv~ and WATTIAUX,1966). Phagocytic haemocytes are not involved until the terminal stages of the process (LOCKSHIN and WILLIAMS, 1964, 1965a-d). The failure of juvenile hormone extracts from Lepidoptera, and of synthetic lepidopterous juvenile hormone analogues, to produce juvenilizing effects in Diptera suggests that some differences in the control of pupation exist in the two orders. Furthermore, preliminary results have suggested that the nervous system may not be responsible for triggering muscle breakdown immediately after puparium formation in Calliphora (CROSSLEY,1965). Both observations indicate that the present analysis of muscle breakdown in Diptera should be compared with the published analysis of muscle degeneration in an adult Lepidoptera (LOCKSHIN and WILLIAMS, 1964, 1965a-d). The numerous light microscope studies of muscle breakdown in Diptera have recently been reviewed (CROSSLEY,1965), intersegmental muscles from the reduviid bug Rhodnius (TOSELLI, 1965; AUBER-THOMAY,1967), the cockroach Periplaneta (SMITH, 1966), and the blowfly Phormia (OSBORNE, 1967) have been examined with the electron microscope. Studies on the intersegmental muscles of Phormia during larval life indicate

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1391

that these muscles can shorten by 76 per cent, and a hypothetical scheme of myofilament interaction has been suggested to account for this ‘supercontraction’ (OSBORNE,1967). MATERIALS

AND METHODS

CuZZiphora erythrocephuZu (Meig.) ( = vacina R.D.) larvae were reared at 25°C

on excess quantities of liver, and allowed to pupate on sand. Pupae were maintained at 25°C and 60 per cent r.h. Under these conditions the stages within the puparium occupied 200 to 220 hr. The time that had elapsed since the larva rounded up to form the puparium is abbreviated to ‘hr Pp’ throughout the text. Ventral oblique muscles (HEWITT, 1914), numbered 15, 16, and 17 in an earlier anatomical study (CROSSLEY,1965) were obtained by dissection. Fixatives used included 2.5% glutaraldehyde solution, buffered at pH 7.2 with 0.05 M HClcacodylate containing 0.015 M sucrose, and 1% osmium tetroxide solution buffered at pH 7.2 with veronal acetate with added sucrose. After thorough washing, and in the case of glutaraldehyde fixation after osmium post-fixation, dehydration was accomplished in a graded ethanol series. Dehydrated muscle was embedded in Araldite, and thin sections were obtained on a Porter-Blum MT-2 microtome equipped with a diamond knife. Contrast in electron micrographs was enhanced by treatment of sections with 5% aqueous uranyl acetate, followed by treatment with lead citrate (REYNOLDS, 1963). Sections were examined in a Siemens Elmiskop I, operated with an anti-contamination cold-stage, and 30 p ultra-thinmetal objective apertures (C. W. French, Weston, Massachusetts). The magnification of the instrument was calibrated using a germanium-shadowed carbon replica of a diffraction grating of spacing 463 rnp (Ladd Research Industries, Burlington, Vermont). A simple saline iso-osmotic with Culliphoru haemolymph, was used as a carrier for substances injected into larvae, and had the following composition; NaCl 8.86 g/l; KC1 2.86 g/l; CaCl, 1.13 g/l; glucose 9.72 g/l. Purified crystalline crustecdysone, extracted from crayfish (HAMPSHIREand HORN, 1966), was kindly supplied by Dr. D. H. A. Horn, CSIRO Division of Organic Chemistry, Melbourne, Australia. Ferritin (Horse spleen ) 2 x crystalline (Nutritional Biochemicals, Cleveland, Ohio) was washed in three changes of CaZZ$&ora saline. Two microlitres of 0.01% ferritin in saline were injected into larvae of average weight O-1 g. Larger doses of this ferritin preparation caused muscle paralysis. Colloidal thorium dioxide (Thorotrast), stabilized with 2.5% dextrin (aqueous), (Fellows Testagal, Detroit, Michigan), diluted 10 x , could be injected in 2 ~1 amounts without inducing obvious side effects, or delaying metamorphosis. The fine structure of the larval abdominal ventral oblique intersegmental muscles

These muscles are composed of fibres of polygonal cross-section. Myofibrils, composed of an array of thick and thin filaments, are surrounded by a sheath of sarcoplasm containing membranous organelles, microtubules, and nuclei. Each

1392

A. C. CROSSLHY

myofibril is traversed regularly by Z-disks which subdivide the fibril into sarcomeres and contribute to its striated appearance. The length of each sarcomere in fixed material varies according to the degree of stimulation to which the muscle was subjected during the fixation process. PORTER(1961) has pointed out that it is probably impossible to tix a muscle without stimulating it. In the present work muscles were fixed under various conditions and the widths of the A-band, I-band, and sarcomere were measured. A-band widths were derived from measurements of individual thick myofilaments in micrographs. As soon as the cuticle of the larva is punctured nearly all of the intersegmental muscles contract. This contraction is presumably brought about by the sudden drop in haemolymph hydrostatic pressure resulting from haemolymph leakage. It can be deduced that the contraction is under the control of nerve centres rather than a direct effect on each individual muscle, since contraction can be prevented by severing the nerve trunks leading to the intersegmental muscles. A method of severing the nerve trunks without puncturing the cuticle is considered in a later section. General muscle contraction is also prevented by ether anaesthesia. Carbon dioxide is less effective. In spite of the removal of stimuli by the above procedures some contraction still occurs when each muscle contacts fixative. Contraction of muscle can be opposed by injection of fixative into the intact larva, thus increasing haemolymph hydrostatic pressure and physically preventing contraction of the muscle. Relaxing solutions, such as those employed for glycerinated muscle (JEWELLet al., 1964) are not effective in preventing contraction in intact muscles from living larvae. Table 1 presents the values for sarcomere width, A-band width, and I-band width in muscles prepared in various ways. Dimensions thought to pertain to relaxed muscle were obtained by measurement of muscles from larvae subjected to deep but reversible ether anaesthesia, or by measurement of paralysed muscles. A method of muscle paralysis involving injection of 2 ~1 O-1y0 ferritin was accidentally discovered during the course of the work. Although the mechanism of the indicate that ferritin paralysis is unknown, sarcomere length measurements paralysis leaves the muscle in a relaxed state. The constancy of the value of 3.3 p obtained for the length of the thick myofilaments which make up the A-band in muscles fixed in partially contracted and relaxed states, suggests that the thick filaments do not shorten during contraction. However, variable and slightly greater A-band widths were recorded in stretched muscles taken from larvae subjected to abnormally high haemolymph .hydrostatic pressures by injection of fixative. Individual thick filaments measured more than 3.3 p in length, and were apparently slightly stretched, rather than displaced with respect to their neighbours. The thick myofilaments are hexagonally packed, with a centre to centre separation of 430 A in muscle from a white puparium. Each thick myofilament is surrounded by an orbital of nine to twelve thin myofilaments. The average orbital contains ten myofilaments (Figs. 2, 4). In relaxed muscle the thin filaments lose their orbital arrangement in the I-band. Cross-bridges interconnect thick and thin myofilaments in the regions of overlap (Figs. 3, 5). The Z-disk is made up

BFtBAKnOwN

OF LABVAL

INTERSEGMENTAL

1393

MUSCLES IN THE BLOWFLY

of an irregular sheet of electron-opaque finely granular material, pierced by numerous openings (Figs. 2-4). Thin myo&unents paas into the Z-disk material and each thin myofilament appears to be coated with electron-dense material in the region adjacent to the Z-disk (Fig. 5). TABLE1 Developmental stage of insect Larva Larva

Larva

White puparium

Sarcomere width Procedure Muscle from etherized insect t Injection of fixative to oppose muscle contraction Cuticle punctured, muscle fully contracted Muscle in state of contraction normal after rounding up to form puparium

0 5.5

A-band width*

I-band width

7.0

3.3

1.1-1.9

94%10.5

343

2.6-3.3

1.75-3.3

:

3*3- 3.6

3.3

0

O-O*15

* Based on measurements of ten or more individual thick myofilaments. t Similar values obtained for paralysed muscle, see text. $ Could not be determined accurately as individual thick myofilaments could not be obtained in longitudinal section. It appears that many thick myofilaments are non-linear in supercontracting muscle. Granules of electron-dense material 200 to 300 A in dia. are interposed between thin myofilaments in the I-band (Figs. 3, 7). Aflinity for lead salts but not for uranyl acetate suggests that the granules should be interpreted as glycogen. The occurrence of deposits of glycogen between the contractile elements of muscles from a number of sources, including insect viscera (SMITH et al., 1966), has been reported. In muscles taken from white puparia the sarcomere width is seldom less than 3 p, close to the normal width of the A-band. Shortening apparently stops when the thick myofilaments reach the level of the Z-disk. In mechanically stimulated muscles taken from larvae after puncture of the cuticle however, the sarcomere width was frequently found to be less than the normal width of the A-band (Fig. 7). This observation can be accounted for by a theory of supercontraction (MCALEAR and HOYLE, 1963; HOYLE et al., 1965; OSBORNE,1967). The supercontraction phenomenon can be substantiated by examination of micrographs of muscles sectioned in the region of the Z-disk. In transverse sections the apertures of the Z-disk are greatly expanded, and both thick and thin myofilaments pass through each aperture (Fig. 10). In longitudinal sections overlap of thick myofilaments from neighbouring sarcomeres is apparent (Fig. 7).

1394

A. c.

CROSSLRY

Termination of the thin myofilaments in the central region of each sarcomere does not produce a well defined H-band (Fig. 6) and it is likely that the ends of the thin filaments do not lie in register, masking the cross-banding pattern, as suggested by OSBORNE(1967). Mitochondria are disposed on each side of the Z-disk between adjacent myofibrils. The sarcoplasmic reticulum is well developed and anastomosing cisternae pass between myofibrils. Associations between cistemae of the sarcoplasmic reticulum and elements of the T-system take the form of dyads, usually at the level of the I-band. Each muscle fibre is enclosed by a thick basement lamella composed of finely granular material of moderate electron-density. Zones of finely granular or homogenous material of much greater electron-density occur at intervals beneath the basement lamella, on the cytoplasmic face of the plasma membrane (Figs. 3, 6). The control of phagocytosis of intersegmental muscle

Accumulations of haemocytes in the dorsal region of posterior segments of second instar Musca larvae were noted by ARVY (1953). It has since been shown that in Calliphora similar accumulations survive until shortly before puparium formation, and are important haemocytopoietic centres (CROSSLEY,1964). An increase in the proportion of phagocytic (Type F) haemocytes circulating at the time of puparium formation can be traced to a change in the differentiation pathway of stem cells in the haemocytopoietic centres, followed by a dissolution of the centres with the release of many phagocytic haemocytes. It has been suggested that these haemocyte alterations may be under humoral control, and represent an early indication of the onset of puparium formation (CROSSLEY,1965). In order to test this hypothesis experimentally, the titre of moulting hormone has been altered by the injection of a solution of crystalline crustecdysone (/3 ecdysone). Three types of test preparation were employed. In the first type, late third instar Calliphora larvae were tightly ligatured behind Weismann’s ring, before sufficient moulting hormone had been released to induce puparium formation. This tight ligature not only interrupted blood circulation, thus preventing pupation of the posterior region of the larva, but also severed the nerve trunks supplying the posterior intersegmental muscles, inhibiting crawling movements. Twenty-four hours after the anterior region had formed a partial puparium, moulting hormone was injected into the larval posterior region. The hormone dose was 2 Calliphora units ( = 2 CU = 0.004 mg) of crystalline crustecdysone (,9 ecdysone) dissolved in 2 ~1 Calliphora saline. Control preparations were injected with saline without hormone. Blood samples were analysed, and the condition of certain intersegmental muscles determined, at intervals after the injection. Analysis of the haemocyte picture included a phase contrast examination and a histochemical determination of acid phosphatase activity. In order to clarify the results of the experiments the seven types of haemocyte described for CaZZiphora larvae (CROSSLEY,1964) have been grouped into three classes. (i) Phagocytic haemocytes (types A and F);

B-OWN

OF LARVAL INTBRSBGMENTAL

MUSCLES IN THE BLOWFLY

09 Engorged phagocytic haemocytes, the

K6rdmkugeln

1395

of WEISMANN

(1864);

(iii) All other types of haemocyte, including the stem types that differentiate into phagocytic haemocytes. Relative numbers of haemocytes are shown in Fig. 1. Fig. 1 (A) represents the haemocyte counts in each class in tightly ligatured larval posterior regions injected

FIG. 1. Histogram in which changes in the haemocyte population are related to the titre of crustecdysone. (A), (C), (E) are saline-injected control preparations; (B), (D), (F) are crustecdysone-injected preparations. Solid line, class 1 (phagocytic haemoctyes) ; cross-hatched line, class 2 (engorged phagocytic haemocytes) ; open line, class 3 (all other types of haemocyte). Please refer to the text for discussion.

with saline 24 hr earlier (Control). Fig. 1 (B) s h ows the experimental picture 24 hr after injection of crustecdysone. Engorged phagocytic haemocytes make up 3 per cent of the population in control preparations, but this proportion increases to 29 per cent after crustecdysone injection. In order to maintain the proportion of phagocytic haemocytes at the level of control preparations, numbers of previously undifferentiated cells must transform into the phagocytic class. A whole mount was made from each experimental preparation for the demonstration of surviving or partially degraded muscles in the light microscope (Fig. 8). Muscles were also dissected, under chilled fixative, from experimentally induced pupae and prepared for examination in the electron microscope (see Fig. 17). In preparations examined 24 hr after injection, the breakdown of muscles that are normally destroyed in the prepupal histolysis phase was found to be complete, but no muscles that normally survive this phase had been destroyed. Elevation of the injected dose of crustecdysone to 10 Calliphora units increased mortality, but did not induce the breakdown of additional muscles.

1396

A. C. C~oss~gy

In the second type of test preparation a ligature was also employed, but this was not drawn tight, with the result that nerve trunks supplying intersegmental muscles remained intact. Nevertheless, blood circulation was interrupted at the ligature, and puparium formation by the posterior segments was prevented. Injections of crustecdysone in saline, or saline alone, were subsequently performed as in the first type of preparation. Injection of crustecdysone into ligatured larvae with intact nerve trunks did not produce results significantly different from those obtained after injection into ligatured larvae with severed nerve trunks. It was confirmed that muscles that normally survive the prepupal histolysis phase are preserved whether or not they are innervated, as noted in an earlier communication (CROSSLEY,1965). Although normal development, as evidenced by the phagocytosis of selected abdominal muscles, is initiated by crustecdysone, differentiation does not proceed beyond the stage reached at about 24 hr Pp in a normal puparium and development of new adult structures is not initiated. When the initial injection of 2 CU crustecdysone was followed by a second 2 CU booster dose, administered to the pupa 18 hr after the first dose, it was noted that differentiation was still arrested at about the 24 hr Pp stage. Furthermore, heart-beat ceased and degeneration of tissues ensued from 48 to 72 hr after the initial injection. In the third type of test preparation larvae were injected with crustecdysone prematurely. Larvae that could be predicted to pupate normally in 36 hr or in 72 hr could be induced to pupate in only 5 hr. Predictions of remaining larval life-span were made by recording the cessation of feeding and the subsequent shrinkage of the crop. Samples in which all individuals pupated within 9 hr were readily obtained. Feeding third instar larvae did not pupate naturally sooner than 72 hr after removal from the food supply. Such larvae were injected with 2 CU crustecdysone in saline. When the haemocyte picture was analysed 24 hr later, it was found that the number of phagocytic haemocytes was significantly greater than in control preparations injected with saline (cf. Fig. lC, D). When crustecdysone was injected into wandering larvae (larval life-expectancy 36 hr) and the haemocyte picture analysed 24 hr later, a similar result was obtained, but a greater number of engorged haemocytes was recorded (cf. Fig. lE, F). LOCKSHIN and WILLIAMS (1965~) have shown that injection of the parasympatheticomimetic drug pilocarpine into silkmoth larvae delays muscle breakdown, by preventing cessation of transmission of nervous impulses that are essential for maintenance of these muscles. Two microlitres of 0.05 M neutralized pilocarpine were injected into wandering Calliphora larvae with a larval life expectancy of 24 hr. A 50 per cent mortality was encountered, but in surviving insects puparium formation ensued within 48 hr. No preservation of muscles that are normally destroyed was detected. Higher concentrations of pilocarpine gave 100 per cent mortality. The structure of phagocytic haemocytes Identification of phagocytic haemocytes in preparations for the electron microscope is greatly facilitated by the injection of electron-dense substances into

BREAKDOWN OFLARVAL INTERsEGMENTAL MUSCLES IN THEBLOWFLY

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the living larva or pupa. Ferritin and thorotrast, for example, are both rapidly engulfed by phagocytic haemocytes but the latter is less toxic. Within 1 hr from the introduction of thorotrast into the haemolymph, electron-dense granules can be obsenred in cup-shaped depressions of the plasma-membrane of certain haemocytes (Fig. 13 inset). These depressions are coated on their cytoplasmic surface by alveolariform thickenings, and are interpreted as coated vesicles. Coated vesicles containing thorotrast separate from the plasma membrane and are to be seen in the interior of the cell, where they presumably contribute to the large vacuoles containing engulfed material (Fig. 13). Large vacuoles containing ferritin or thorotrast often also contain recognizable cell organelles such as mitochondria and are assumed to be cytolysomes. Acid-phosphatase activity has been recorded in phagocytic haemocytes in preparations for the light microscope (CROSSLN, 1964). The phagocytic haemocytes have now been re-investigated with the electron microscope to localize the sites of activity. After incubation in Gomori-type medium, lead deposits are confined to cytolysomes and membrane-limited vacuoles containing granular material of moderate electron density. No deposits are seen in the nucleus or free in the cytoplasm (Fig. 14). Phagocytosis of muscle by haemocytes The ventral oblique muscles in the anterior segments of the puparium are invaded by phagocytic haemocytes about 10 hr after their involvement in the rounding-up of the puparium. The first indication of the impending invasion is the appearance in doomed muscles, at about 5 hr Pp, of three structures not found in surviving muscles at this time. The first structure is a large vacuole containing flocculent material of low electron density. The second structure is a whorl of regularly periodic concentric lipoprotein membranes, resembling a myeloid body, similar to a structure described in mouse kidney (MILLER, 1960, Fig. 6). The third structure is made up of paired concentric membrances, probably smooth surfaced endoplasmic reticulum, also similar to structures seen in mouse kidney (MILLER, 1960, Fig. 28). Shortly before the haemocyte invasion the subdivisions of the fenestrated sarcoplasmic reticulum partly break down, with the creation of abnormal elongated vacuoles between myofibrils. All the changes that precede haemocyte invasion are confined to the sarcoplasm, and the myofilaments are eventually engulfed without disruption of hexagonal packing or loss of striation (Figs. 15, 17). It might be expected that dissolution of the sarcolemma would precede haemocyte invasion. Haemocytes were, however, recorded attacking muscles enclosed in a full thickness of basement lamella, and rolls of finely granular lamella material could be distinguished in vacuoles within the haemocytes. The plasma membrane of phagocytic haemocytes is extended into pseudopodia and flattened folds. Ground-plasm containing ribosomes and occasionally endoplasmic reticulum extends into each fold (Figs. 11, 13, 17). When the haemocyte is engaged in phagocytosis of muscle fragments the extensions become 89

1398

A. C. CRORXEY

attenuated, and infiltrate between organelles of the muscle sarcoplasm. Basement lamellae, nuclei, and mitochondria are engulfed first, but the engulfment of fibrils follows when haemocyte extensions pass between myofibrils in areas occupied by sarcoplasmic reticulum, or into tubules of the T-system. Fracture of the myofibril sarcomere occurs at the level of the I-band, and fragments made up of FIG. 2. Transverse section of intersegmental muscle from a white puparium. Electron-dense material adheres to thin filaments (arrow) in the region of the perforated Z-disks (2). S, Sarcoplasmic reticulum. x 60,000. FIG. 3. Longitudinal section of intersegmental muscle from a white puparium, showing part of a contracted sarcomere. The thick filaments reach the Z-disks (2). S, Sarcoplasmic reticulum; P (d) electron-dense deposit beneath the plasma membrane, thought to be associated with the deposition of the thick basement lamella. x 50,000. FIG. 4. Transverse section of intersegmental muscle from white puparium. An average of ten thin myofilaments surrounds each thick myofilament in the A-band region (A). Absence of thin myofilaments is taken to indicate that the section passes through the H-band (H). Note tbe perforated Z-disk (Z) and the electrondense coating on thin myofilaments in the vicinity of the Z-disk (arrow). M, mitochondrion. x 100,000. FIG. 5. Longitudinal section of a sarcomere in the vicinity of the Z-disk (Z) showing continuation of thin filaments within the electron-dense deposit in the vicinity of the Z-disk (arrow). Cross-bridges are to be seen in the region of myofilament overlap. FIG. 6. Longitudinal section of relaxed but not stretched muscle from a wandering third instar larva. The fenestrated nature of the sarcoplasmic reticulum is shown, and a T-canal can be seen at the dyad (dy). I, I-band; Z, Z-disk; d, electron-dense deposit beneath plasma membrane adjacent to basement lamella. x 23,000. FIG. 7. Longitudinal section of a fully contracted, mechanically stimulated, muscle from a wandering third instar larva. The passage of thick myofilaments through the Z-disk (Z) and the resulting overlap of myofilaments from neighbouring sarcomeres can be seen (arrow). Granular deposits near the Z-disks are interpreted as glycogen (g). x 23,000. FIG. 8, Light micrograph of dissected pupa 24 hr after puparium formation to show the muscles that survive the histolysis phase induced by the injection of crustecdysone. D, dorsal; V, ventral. FIG. 9. Transverse section of intersegmental muscle from a white puparium to show the perforated nature of the Z-disks (Z). x 36,000. FIG. 10. Transverse section of fully contracted intersegmental muscle from a wandering larva. Note the expansion of the perforations in the Z-disk (Z), and the passage of both thick and thin myofilaments through the perforations (arrow). x 75,000. FIG. 11. Phagocytic haemocyte (ph) engulfing fragments of muscle composed of intact myofilaments (mf) and mitochondria (m). Note the infiltration of extended folds of haemocpe plasma membrane (e) between myofibrils, and the pockets of haemolymph (h) between haemocyte and muscle. v, Vacuole within haemocyte; li, lipid within haemocyte. x 22,000. FIG. 12. Longitudinal section of a muscle stretched in glutaraldehyde. Individual thick myofilaments in this preparation measure 3.8 /.Lin length. x 12,300.

!B

..........

~ii~!~i~I~

~i~~i~ ~

~~

~

i

~~

~

~i~.,

BRJ3AKDOWN OF LARVAL INTERSWMENTAL MUSCLESIN ‘IIiE BLOWFLY

1399

several sarcomeres can be contained within a vacuole in the cytoplasm of a single haemocyte. The irregular configuration of the haemocyte plasma membrane adjacent to disintegrating muscle leads to the formation of pockets of haemolymph between the two systems (Figs, 11, 17). Fragments of flocculent material of low electron density are present in the pockets. The haemocyte plasma membrane bordering on the pockets bears numerous cup-shaped identations which are interpreted as coated vesicles (Fig. 16). Muscle preparations were incubated in a Gomori-type acid phosphatase reagent. Lead deposits were not detected in muscle fibres, nor in any vacuoles within the muscle, although haemocytes nearby showed massive deposits and thus

FIG. 13. Phagocytic haemocyte from the haemolymph of a larva injected with thorotrast 8 hr earlier. Numerous coated vesicles are visible (arrows) and in the enhugement (inset), thorotrast particles can be seen within coated vesicles at the cell surface, and within the cell. Thorotrast accumulates in vacuoles (v) within the haemocyte. e, Extension of the plasma membrane into folds. x 25,000. Inset x 58,000. FIG. 14. Haemocyte from preparation incubated in Gomori-type acid-phosphatase medium. Lead deposits indicating sites of activity of this enzyme are confined to certain vacuoles (a), and to cytolysomes. n, nucleus; e, extension of plasma membrane. x 18,500.

FIG. 15. Vacuoles within a haemocyte containing muscle contractile elements. Thick myofilaments alone are visible in this preparation (m). One vacuole contains the residue that remains after the destruction of the myofilaments (arrow). x 28,000. FIG. 16. The interaction of phagocytic haemocyte (ph) and muscle. Pockets of haemolymph (h) separate the two systems, and are partially filled with debris (db). Coated vesicles (cv) communicate with the pockets of haemolymph. v, vacuole within haemocyte, probably containing acid-phosphatase. m, muscle mitochondrion; mf, obliquely sectioned myofilaments. x 72,000. FIG. 17. Premature, crustecdysone induced, engulfment of myofilaments (mf) by phagocyte (ph). 2, Z-disk; h, haemolymph; e, attenuated extension of haemocyte plasma membrane; cy, cytolysome within haemocyte. x 23,000. FIGS. 18-20. Vacuoles within phagocytic haemocytes from puparia 24 hr Pp. FIG. 18. A cytolysome (cy) containing elements of rough endoplasmic reticulum and a central core of lipid droplets (li). Lipid droplets are also present free in the cytoplasm. x 25,000. FIG. 19. Vacuoles (v) derived from muscle contractile elements. li, saturated lipid;

g, glycogen.

x 35,000.

FIG. 20. Protein + RNA granule containing condensed sheets of ribosomes (r) and electron lucid droplets possibly unsaturated lipid. x 15,500. FIG. 21. Contents of haemocyte 36 hr Pp. Myofilaments czn still be detected in one elongated vacuole (mf), although others contain ribosomes (r) and lipid (Ii). Lipid can be seen partially extruded from one cytolysome (arrow). x 9000. FIG. 22. Phagocytic haemocyte from puparium 96 hr Pp. The cytoplasm is filled with lipid (li) and electron dense droplets. Lipid droplets are also numerous in the haemolymph (h). x 16,000.

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A. C. CROSSLEY

served as a control on the technique. Lead deposits were not detected in pockets of haemolymph enclosed between haemocyte and muscle. These observations are consistent with the view that acid-phosphatases (and probably other lysosomal enzymes) are confined within the vacuolar apparatus of the haemocyte. Breakdown of muscle fragments within the vacuolar apparatus of the haemocyte The circulation in cyclorrhaphous pupae during histolysis phases is sluggish, and engorged haemocytes tend to adhere to each other in the positions formerly occupied by newly engulfed tissues (WHITTEN, 1964). It is possible to dissect out groups of haemocytes containing material derived almost exclusively from the ventral oblique muscles. When such haemocytes are examined in the electron microscope it is found that newly engulfed contractile elements retain their hexagonally packed filamentous structure in vacuoles within the cell. However, the thin myofilaments soon disappear, leaving regularly spaced thick filaments which survive for many hours (Fig. 15). Eventually the thick filaments also disappear leaving an electron dense residue within elongated vacuoles. Many non-contractile elements of muscle sarcoplasm can be recognized in vacuoles within the phagocytic haemocyte. Muscle sarcolemma gives rise to large spherical vacuoles filled with flocculent material of low electron density. Muscle mitochondria, ribosomes, and concentric membrane whorls find a place in cytolysomes of varied structure (Figs. 18, 21). Many ribosomes and ribosome studded membranes are sequestered in special vacuoles. In these vacuoles condensation and breakdown of the membranous material gives rise to sheets of closely spaced ribosome residues in a hexagonal array (Fig. 20). These vacuoles are very similar to the protein+ RNA vacuoles recorded in insect fat body (LOCKE and COLLINS, 1965). As development within the pupa advances numerous droplets composed of homogeneous material of low electron-density appear in cytolysomes (Figs. 18, 21,22). The droplets are not enclosed within unit membranes, and are interpreted as lipid. This interpretation is supported by histochemical studies with the light Glycogen is also observed in preparations for the light and electron microscope. microscopes (Fig. 19). A dynamic concept of lipid transfer can be developed from a study of images in which lipid droplets appear partially extruded from cytolysomes (Fig. 21), free in the cytoplasm of haemocytes (Fig. 22), protruding as blebs from the plasma membrane of haemocytes, and free in the haemolymph. Such a concept is limited by the usual difficulty of directional interpretation of static images, but is supported by the observation of a gradual increase in the number of droplets of lipid in the haemolymph within the puparium. The population of lipid droplets and miscellaneous granules present in the haemolymph is greatly augmented between 100 and 150 hr after formation of the Protein vacuoles and protein+ RNA vacuoles appear in the haemopuparium. lymph. Evidently many engorged phagocytic haemocytes rupture, releasing the A preparation of haemocytes mounted between contents of the vacuolar apparatus. slide and coverslip can normally be compressed considerably before rupture of the

BBEAKDOWN OF LARVALINTERSEGMENTAL MUSCLES

IN THE BLOWFLY

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cells occurs. However, haemocytes taken from insects 100 or more hours after formation of the puparium are excessively fragile, and rupture with the release of vacuoles when compressed. DISCUSSION

In insect intersegmental and visceral muscles nine to twelve thin myofilaments surround each thick myofilament (TOSELLI, 1965; SMITH, 1966; SMITH et al., 1966; ANDERSONand ELLIS, 1967; AUBER-THOMAY, 1967; HEHN, 1967; SANDBORN et al., 1967; SCHAEFER et al., 1967). Examination of published micrographs reveals that in these muscles an average thin myofilament orbital contains about ten elements, and the same average value is obtained for Calhphora intersegmental muscle. As SMITH (1966) has pointed out, it is not clear whether a fibre is intrinsically variable and undetermined in its ratio of thin to thick myofilaments or whether the mode of preparation induces some disorder. In the femoral muscle of the cockroach Leucophaea, ten to twelve thin myofilaments encircle each thick myofilament (HAGOPIAN, 1966), but HOYLE (1967) reports that a 4 : 1 actin/ myosin ratio (providing orbitals of ten thin myofilaments) is normal in the locust extensor tibiae. Hoyle notes that, as in many muscles with a high actin/myosin ratio, the orbitals are not regular, and concludes that a neat array is not an essential feature of the molecular architecture of a striated muscle fibre. This conclusion is supported by the present work. From a comparative study of insect flight muscles AUBER (1966, 1967) concludes that a high thin/thick myofilament ratio can be correlated with a slow work rhythm. Blowfly intersegmental muscles have a slow work rhythm and conform to Auber’s hypothesis. Measurements of thick myofilament length in Calliphora intersegmental muscles indicate that there is no measurable length change during contraction. This constancy is in agreement with precise measurements made on vertebrate muscle with X-ray techniques (ELLIOT et al., 1965 ; HUXLEY et al., 1965). A measurable increase in thick myofilament length was, however, recorded in muscles stretched during fixation Stretching of thick myofilaments could partially account for the in glutaraldehyde. relatively greater (4.1 p) A-band width in intersegmental muscles of Phormia, reported by OSBORNE(1967). Although the morphological terms ‘thick myofilament’ and ‘thin myofilament’ are used in this report, it is generally accepted that the filaments are mainly composed of myosin and actin molecules respectively. The presence of myosin and actin in insect muscles has been established (GILMOUR and CALABY, 1953 ; MARUYAMA, 1959; KOMINZ et al., 1962), but the location of the proteins in the fibril is less certain. The accepted location is queried by GILMOUR and ROBINSON (1964). The Z-disks of Calliphora abdominal intersegmental muscles are perforated by numerous holes. When the muscle contracts strongly after the cuticle has been punctured, both thick and thin myofilaments pass through these holes, causing the holes to enlarge. This appearance has been recorded in Phormia intersegmental muscles, and is one manifestation of the phenomenon of ‘supercontraction’

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(OSBORNE, 1967). The present study indicates that supercontraction does not occur when intersegmental muscles contract strongly at the time of puparium formation. Perhaps maintenance of normal haemolymph hydrostatic pressure reduces the incidence of supercontraction. The highly disorganized nature of the supercontracting myofibril seems to argue against a rapidly reversible recovery, and physiological data would be of considerable interest. In Calliphora intersegmental muscles the configuration of elements of the sarcoplasmic reticulum, T-tubule system, and chondriome is very similar to that reported for Periplaneta (SMITH, 1966) and Phomia (OSBORNE, 1967). Deposits of electron-dense granular material at intervals beneath folds in the plasma membrane are of unknown function, but it is suggested that they may be associated with the secretion or attachment of the thick basement lamella component of the sarcolemma. Similar deposits are found in insect pericardial cells, which also bear a thick basement lamella. The injection of a solution of crystalline crustecdysone initiates a series of changes in which specific intersegmental muscles are engulfed by haemocytes. The changes can be induced in ligatured abdomens, isolated before the critical period for the moulting hormone; these abdomens would otherwise remain in the larval state indefinitely. Similar changes can be initiated 48 or more hours before the anticipated normal time of puparium formation by the premature injection of crustecdysone. Identical changes ensue whether or not a nervous connexion to the intersegmental muscle is present. Furthermore the injection of the parasympatheticomimetic drug pilocarpine does not delay breakdown in normally innervated muscles. It must be concluded that, in CaZZiphoralarvae, breakdown of certain intersegmental muscles is induced, perhaps indirectly, by an increase in the titre of crustecdysone in the haemolymph, in the presence of nervous stimuli. This finding contrasts with the situation reported in the adult silkmoth by LOCKSHIN and WILLIAMS (1964, 1965a-d), where the correct humoral environment is only the prerequisite for nervous control of muscle degeneration. The situation in Calliphora may be more comparable with that reported in the Colorado beetle Leptinotarsa, where the degeneration of the flight muscle is under direct control of hormones derived from the post-cerebral complex of endocrine glands (STEGWEE et al., 1963). Crustecdysone injection sets in motion a programme of destruction that is essentially normal, and does not induce breakdown of any muscles that are normally preserved. Individual muscles must differ in their reaction to crustecdysone, since some muscles resist phagocytosis although surrounded by phagocytic haemocytes engaged in the destruction of adjacent muscles. Whilst it is known that invasion of muscles by haemocytes is preceded by cytological changes in the doomed muscle, the nature of the specific signal that invites haemocyte invasion into a particular muscle remains unknown. Dissolution of the muscle basement lamella is not a prerequisite for haemocyte invasion. In the larval intersegmental muscles of CaZZiphora there are no signs of degeneration of the contractile elements at the time of haemocyte invasion. This situation was noted by PEREZ(1910) but is apparently not universal in cyclorrhaphous Diptera, since extensive fragmentation

BREAKDOWNOF

LARVAL INTBRBEOMBNTALMUSCLESINTHEBLOWFLY

with loss of fibre striation is reported

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to precede invasion of phagocytes into the muscles of Surcophugu and &oqMu (WHITTEN, 1964). The appearance of the vacuoles and concentric membrane structures is probably a morphological indication of the train of events that leads to the invasion of haemocytes, although no evidence has been obtained to suggest that the vacuoles themselves are released onto the surface of the muscle. The actual signal, which is most probably chemical, is unlikely to be visible in preparations for the electron microscope. The development induced by crustecdysone proceeds only to the stage reached by normal larvae at about 24 hr Pp. Repeated or abnormally large injections of crustecdysone do not extend the period of development, and death normally ensues about 48 hr after the initial injection. The factors necessary for further development may be hormonal, but it is perhaps more likely that the ligature procedure so disrupts the developmental process that complete differentiation would not be possible in a normal hormonal environment. Phagocytic haemocytes are shown to possess a well developed ‘vacuolar apparatus’ as defined by DE Duv~ and WATTIAUX(1965). Acid-phosphatase activity reported in phagocytic haemocytes (CROSSLEX,1964) has been localized in certain types of vacuole, which presumably also contain other hydrolases of the lysosome complement. Coated vesicles are demonstrated in insect haemocytes for the first time. Smooth-surfaced pinocytotic vesicles (FAWCETT, 1965) were not found. Coated vesicles have been reported in a number of insect tissues including pericardial cells (BOWERS,19M; PORTERet al., 1967; CROSSLEY,1968a, b), oacytes (ROTH and PORTER, 1964; DROLLER and ROTH, 1966), rectal epithelium (GUPTA and BERRIDGE,1966), and epidermal cells secreting cuticle (LOCKE, 1966; FILSHIE and WATERHOUSE,1968). Coated vesicles are considered to play an important part in the uptake of proteins by the cell (ROSENBLUTHand WISSIG, 1964; FAWCETT,1965). The coated vesicles of CuZZipho~ahaemocytes can, however, take up molecules of non-proteinaceous nature such as thorium dioxide, and transport them into the cell. The influences of the dextrins used to stabilize the colloidal thorium particles on the cell membranes are unknown, but it is clear that coated vesicles may be involved in the transport of non-proteinaceous substances in these cells. The recent work of FRIENDand FARQUHAR (1967) suggests that coated vesicles may function not only as heterophagosomes for transport of absorbed protein to lysosomes, but also as transport vesicles to convey enzymes or surface coat material made within the cell to the cell surface. The latter function might be ascribed to coated vesicles observed at the surface of insect epidermal and tracheal cells at the time of cuticle deposition (LOCKE, 1966). It could also be argued that the coated vesicles bordering on pockets of haemolymph trapped between haemocyte and disintegrating muscle in CuZZiphoru may function to transport hydrolytic enzymes to the muscle, although it appears from histochemical data that an active acidphosphatase is not released. Perhaps the coated vesicles in this situation are only concerned with the absorption of components released from the disintegrating muscle.

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The ingestion of large fragments of muscle is accomplished by a distinct process of phagocytosis, involving extensions of the haemocyte plasma membrane. Similar extensions are observed on vertebrate cells engaged in phagocytosis, and have been termed ‘marginal folds’ by FAWCETT(1965). In Cdliphora haemocyte vacuoles containing muscle fragments become acid-phosphatase positive, indicating that a lysosomal vacuolar apparatus is involved in the breakdown of muscle fragments. NOVIKOFF(1960) notes that most tissues contain acid phosphatases, with the exception of striated muscle. Lysosomes containing acid phosphatase were reported in lepidopterous muscle by LOCK~HINand WILLIAMS (1965a), but this enzyme has not been detected in Calliphma muscle by the histochemical method used in the present work. Visible breakdown of muscle occurs within the vacuolar apparatus of the haemocyte, and lipid and glycogen reserves accumulate. Some of the reserve material appears to be passed into the haemolymph from intact haemocytes, but eventually rupture of the haemocyte plasma membrane releases all reserves directly into the haemolymph. As SAUNDERS (1966) has pointed out, cell death should bring about the release of a spectrum of cellular components ranging from low molecular weight building blocks to macromolecules, but it is not known whether macromolecules are utilized as such by developing tissues. Uptake of specific RNA’s appears to result in specific morphogenetic effects, or in synthesis of tissue specific proteins (references in SAUNDERS,1966). It has recently been found (CROSSLEY,1968a) that developing adult myotubes in Calliphora are provided with numerous coated vesicles, structures associated with the cellular uptake of proteins and other large molecules. Relatively large molecules derived from larval muscles could be stored by haemocytes and transferred to developing adult muscles through the haemolymph. The degree of degradation of different tissue components within phagocytes remains to be explored. The cyclorrhaphous pupa should provide favourable Fat body and pericardial cells also have the material for such an exploration. property of absorbing and modifying proteins from the haemolymph (LOCKE and COLLINS, 1965, 1966; CROSSLEY,1968b) and may also play an important part in the modification of histolysed larval macromolecules prior to their incorporation into developing adult tissues. REFERENCES ANDERSON W. A. and ELLIS R. A. (1967) A comparative electron microscope study of visceral muscle fibres in Cambarus, Drosophila and Lumbricus. Z. Zellforsch. mikr. Anat. 79, 581591. ARVY L. (1953) Contribution a l’etude de la leucopoiese chez quelques Dipteres. Bull. SOC. zool. Fr. 78, 158. AUBER J. (1966) Distribution des deux types de myofilaments dans diverse muscles de Dip&es. J. Microscopic 5, 28a. AUBER J. (1967) Distribution of the two kinds of myofilaments in insect muscles. Am. Zool. 7, 451456. AUBER-THOMAY M. (1967) Modifications ultrastructurales au tours de la degenerescence et de la croissance de fibres musculaires chez un insecte. J. Microscopic 6, 627-638.

BREAKDOWN OF LARVALINTBRSEOMENTAL MUSCLESIN THE BLOWFLY BBCE. Fliegen. Bowws B. (Sulz.)).

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and PLAGGEE. (1939) Ueber die Puparium-Bildungauslbsende Hormon der Biol. Zbl. 59, 326-341. (1964) Coated vesicles in the pericardial cells of the aphid (Myztis perticue Protoplasmu 59, 351-367. BUTENANDT A. and KARLSON P. (1954) Uber die Isolierung eines Metamorphose-Hormons der Insekten in kristallisierter Form. Z. Naturf 96, 389-391. CROSSLEYA. C. (1964) An experimental analysis of the origins and physiology of haemocytes in the blue blowfly Culliphoru erythrocephalu (Meig.). g. exp. Zool. 157, 375-397. CROSSLEYA. C. (1965) Transformations in the abdominal muscles of the blue blowfly Culliphorn erythrocephaba (Meig.). 3. Embryol. exp. Morph. 14, 89-110. CROSSLEYA. C. (1968a) Degeneration and redifferentiation of intersegmental muscle during metamorphosis of a blowfly. In preparation. CROSSLEYA.C. (1968b) The fine-structure of pericardial cells in the blowfly CaZZi@ora erythrocephah. In preparation. DE DUVE R. and WATTIAUXR. (1966) Functions of lysosomes. A. Rev. Physiol. 28,435-492. DROLLER M. J. and ROTH T. F. (1966) An electron microscope study of yolk formation during oogenesis in Lebistes reticulatus Guppy. J. Cell Biol. 28, 209-232. ELLIOT G. F., LOWY J., and MILLMANB. M. (1965) X-Ray diffraction from living striated muscle during contraction. Nature, Lond. 206, 1357-1358. FAWCET~D. W. (1965) Surface specializations of absorbing cells. J. Histochem. Cytochem. 13, 75-91. FILSH~ B. K. and WATERHOUSE D. F. (1968) The structure and development of a surface pattern on the cuticle of the green vegetable bug Nezura oiridula. In preparation. FRAWK~L G. (1934) Pupation of flies initiated by a hormone. Nature, Lond. 133, 834. FRAHNKELG. (1935) A hormone causing pupation in the blowfly Culliphora erythrocephahz. Proc. R. Sot. (B) 118, l-12. FRIEND D. S. and FARQUHARM. G. (1967) Functions of coated vesicles during protein absorption in the rat vas deferens. J. Cell Biol. 35, 357-376. GILMOURD. and CALABYJ. H. (1953) Physical and enzymic properties of actomyosins from the femoral and thoracic muscles of an insect. Enzymologia 16, 23-33. GILMOURD. and ROBINSON P. M. (1964) Contraction in glycerinated myofibrils of an insect (Orthoptera, Acrididae). r. Cell Biol. 21, 385-396. GUPTA B. L. and BERRIDGEM. J. (1966) Fine structural organization of the rectum in the blowfly, Culliphoru erythrocephalu (Meig.) with special reference to connective tissue, tracheae and neurosecretory innervation in the rectal papillae. g. Morph. 120, 23-82. HAGOPIANM. (1966) The myofilament arrangement in the femoral muscle of the cockroach Leucophaea maderae F. J. Cell Biol. 28, 545-562. HAMPSHIREF. and HORND. H. A. (1966) Structure of crustecdysone, a Crustacean moulting hormone. Chem. Comm. 2, 37-38. HENN G. (1967) Die Muskulatur des Eileiters von Curuusius morosus-I. Histologische Untersuchungen. Z. ZeEforsch. m&r. Anat. 78, 511-545. HEWITT C. G. (1914) The House FZy. Cambridge University Press, London. HORND. H. S., MIDDLETONE. J., WUNDERLICHJ. A., and HAMPSHIREF. (1966) Identity of the moulting hormones of insects and crustaceans. Chem. Comm. 11, 339-341. HOYLE G. (1967) Diversity of striated muscle. Am. Zool. 7, 43.549. HOYLE G., MCALEARJ. H., and SELVERSTON A. (1965) Mechanism of supercontraction in a striated muscle. J. Cell Biol. 26, 621-640. HUXLEY H. E., BRO~VNW., and HOLMESK. C. (1965) Constancy of axial spacings in frog sartorius muscle during contraction. Nature, Lond. 206, 1358. JEWELL B. R., F'RINGLE J. W. S., and R~?EGGJ. C. (1964) Oscillatory contraction of insect fibrillar muscle after glycerol extraction. g. Physiol., Land. 173, 6-8. KARLSONP. (1965) Biochemical studies of ecdyson control of chromosomal activity. J. cell. camp Physiol. (Supp. 1) 66, 69-75.

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KARLSONP., HOFFMEISTERH., HOPPE W., and HUBERR. (1963) Zur Chemie des Ecdysons. Jushu Liebigs Annln Chem. 662, l-20. KOMINZD.R., MAR~YAMAK., LEVHNBOOK L., and LEWIS M. (1962) Tropomyosin, myosin and actin from the blowfly, Phormia regina. Biochim. biophys. Acta 63, 106-116. LOCKE M. (1966) The structure and formation of the cuticulin layer in the epicuticle of an insect, Calpodes ethlius (Lepidoptera, Hesperiidae). J. Morph. 118, 461-494. LOCKEM. and COLLINSJ. V. (1965) The structure and formation of protein granules in the fat-body of an insect. J. Cell Biol. 26, 857-884. LOCKE M. and COLLINSJ. V. (1966) Sequestration of protein by the fat-body of an insect.

Nature, Lond. 210, 552-553. LOCK~HINR. A. and WILLIAMSC. M. (1964) Programmed cell death-II. Endocrine potentiation of the breakdown of intersegmental muscles of silkmoths. g. Insect Physiol. 10,

643-649. L~CK~HIN R. A. and WILLIAMS C. M. (1965a) Programmed cell death-I. Cytology of degeneration in the intersegmental muscles of the pemyi silkmoth. g. Insect Physiol. 11, 123-133. LOCKSHINR. A. and WILLIAMSC. M. (196Sb) Programmed cell death-III. Neural control of the breakdown of the intersegmental muscles of silkmoths. J. Insect Physiol. 11,601-610. LOCK~HINR. A. and WILLIAMSC. M. (1965~) Programmed cell death-IV. The influence of drugs on the breakdown of the intersegmental muscles of silkmoths. r. Insect Physiol. 11, 803-809. LOCK~HINR. A. and WILLIAMS C. M. (1965d) Programmed cell death-V. Cytolytic enzymes in relation to the breakdown of the intersegmental muscles of silkmoths. J. Insect Physiol. 11, 831-844. MARUYAMAK. (1959) Insect actomyosin, a flow birefringence study. J. Insect Physiol. 3,

271-292. MCALEAR J. H. and HOYLE G. (1963) The mechanism of supercontraction in a striated muscle fibre of the barnacle Balamrs nubilus. J. Cell Biol. 19, 49A. MILLER F. (1960) Hemoglobin absorption by cells of the proximal convuluted tubule in mouse kidney. J. biophys. biochem. Cytol. 8, 689-718. NOVAKV. J. A. (1966) Insect Hormones. Methuen, London. NOVIKOFFA. B. (1960) Biochemical and staining reactions of cytoplasmic constituents. In Developing Cell-systems and Their Control. (Ed. by RUDNICKD.) pp. 167-203, Ronald Press, New York. OSBORNEM. P. (1967) Supercontraction in the muscles of the blowfly larva: An ultrastructural study. r. Insect Physiol. 13, 1471-1482. PEREZ C. (1910) Recherches histologiques sur la metamorphose des muscides, Calliphora erythrocephala (Meig.). Arch. 2001. exp. g&n. (5) 4, l-274. PORTERK. R. (1961) The sarcoplasmic reticulum: Its recent history and present status. J. biophys. biochem. Cytol. 10, 219-226. PORTERK. R., KENYON K., and BADBNHAUSEN S. (1967) Specializations of the unit membrane. Protoplasma 63, 262-274. REYNOLDSE. S. (1963) The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J. Cell Biol. 17, 208-212. RO~ENBLUTH J. and W~ssrcr S. L. (1964) The distribution of exogenous ferritin in toad spinal ganglia and the mechanism of its uptake by neurons. J. Cell Biol. 23,307-325. ROTH T. F. and PORTERK. R. (1964) Yolk protein uptake in the oocyte of the mosquito Aedes aegypti L. g. Cell Biol. 20,313-332. SUBORN E. B., DUCLOSS., Mgssr~~ P. E., and ROBERGEJ. J. (1967) Atypical intestinal striated muscle in Drosophila melanogaster. J. Ultrasttwt. Res. 18, 695-702. SAUNDERS J. W. (1966) Death in embryonic systems. Science, N.Y. 154,604-612. SCHAEFBRC. W., VANLXRBERG J. P., and RHODINJ. (1967) The fine structure of mosquito midgut muscle. r. Cell Biol. 34, 905-911.

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D. S. (1966) The structure of intersegmental muscle fibres in an insect, Periplatteta americana L. g. Cell Biol. 29,449+59. SMITH D. S., GUPTAB. L., and SMITH U. (1966) The organization and myof?lsment array of insect visceral muscles. g. Cell Sci. 1, 49-57.

SMITH

STEGWBED., KIMMEL E. C., DE Bonn J. A., and HEN~TRAS. (1963) Hormonal control of reversible degeneration of flight muscles in the colorado potato beetle Leptinotmsa decimlineata Say (Coleoptera). J. Cell Biol. 19, 519-527. TOSBLLIP. A. (1965) Fine structure of the fully developed intersegmental abdominal muscles of Rhodmk.r prolixus. Anat. Rec. 151,427. WEISMANNA. (1864) Die nachembryonale Entwicldung der Musciden nach Beobachtungen an Musca vomitmia und Sarcophaga cantaria. Z. wiss. Zool. 14, 187. WHIITEN J. M. (1964) Haemocytes and the metamorphosing tissues in Sarcophaga bullata, Drosophila melanogaster and other cyclorrhaphous diptera. J. Insect Physiol. 10,447-+69.