J. Cell Sci. i, 49-57 (1966)

Printed in Great Britain

THE ORGANIZATION AND

MYOFILAMENT ARRAY OF INSECT

VISCERAL MUSCLES

D. S. SMITH, B. L. GUPTA AND UNA SMITHDepartment of Biology, University of Virginia, Charlottesville, Virginia, U.S.A.

SUMMARY

The cytological organization of three insect visceral muscles has been examined in theelectron microscope. In each instance, the fibres were found to be striated, and the striationpattern has been shown to reflect the distribution along the sarcomere of two sets of myo-filaments. In transverse sections of the fibre at the level of the A band, these muscles have beenfound to exhibit an unusual myofilament array in which each thick (myosin) filament is sur-rounded by twelve thin (actin) filaments rather than six, as in insect flight muscle and vertebrateskeletal muscle. The distribution of T-system tubules and cisternae of the sarcoplasmic reti-culum in these visceral fibres is described, and compared with the corresponding membranesystems in other striated muscles.

INTRODUCTION

In anatomical considerations of the insect body, the muscular systems are commonlydivided into two general categories: 'skeletal' and 'visceral' fibres. The former actupon the articulated exoskeleton, while the latter invest the various regions of theintestinal tract and other internal organs within the body cavity. Whereas many of theskeletal muscles, notably those concerned with flight and locomotion, may contractrapidly and often at high frequency, the visceral fibres, like their analogues in thevertebrate body, generally exhibit a slower peristaltic or irregular activity.

Hitherto, investigations on the cytological organization of insect muscles havemainly been focused upon the fibres involved in the flight mechanism, and thesestudies have revealed a consistent deviation in the arrangement of the myofilamentsof the contractile system from the pattern occurring in vertebrate skeletal muscles.In the latter (Huxley, 1957; Huxley & Hanson, i960) the thick and thin filaments ofthe sarcomere, respectively containing myosin and actin, are arranged in a doublehexagonal array in the A bands, with the actin components situated in the trigonalposition with respect to the myosin filaments. In the indirect flight muscles of theblowfly Calliphora, on the other hand, it was found (Huxley & Hanson, 1957) thatthe thin filaments are relatively more numerous and are situated opposite to, andmidway between, adjoining myosin filaments in the hexagonal array. In the formerinstance, each thin filament is 'shared' by three thick filaments and, in the latter, bytwo thick filaments.

The configuration of myofilaments reported in the asynchronous flight muscle of4 Cell Sci. 1

50 D. S. Smith, B. L. Gupta and U. Smith

Calliphora also occurs in the flight muscle fibres of Odonata (Anisoptera and Zygop-tera) (D. S. Smith, unpublished), which have synchronous contraction properties,and may be characteristic of insect flight muscles in general. An identical dispositionoccurs in the tymbal muscle of the cicada Tibicen (D. S. Smith, unpublished), but,in the absence of detailed studies on other insect skeletal muscle fibres, it is not possibleto generalize further on the organization of the myofilament array in this category ofmuscle cells.

This report is concerned with the structure of visceral muscle from three locationsin the insect body. It has been found that in each instance the contractile material isrepresented by a double array of thick and thin filaments, occurring in a configurationstrikingly different from that of insect flight muscle fibres. In the muscular sheathinvesting the midgut of Ephestia larvae (Lepidoptera), in that of the spermatheca ofPeriplaneta (Orthoptera) and of the seminal vesicle of Carausius (Orthoptera), it wasfound that, while the thick filaments within the A band maintain an hexagonal array,each of these filaments is surrounded by a ring of twelve thin filaments. These visceralmuscle fibres were found to be similar not only with respect to their fibrillar organiza-tion, but also in their general structure, and, while this account concerns primarily thelast of the above examples, the cytological features described are for the most partcommon to each of these visceral fibres.

MATERIALS AND METHODS

An adult male specimen of the stick-insect Carausius morosus was found in a labora-tory culture of this parthenogenetic species, and was selected for a study of theorganization of the seminal vesicle. This paper is concerned mainly with the muscularsheath investing this region of the reproductive tract. The spermatheca was fixed for2 h in ice-cold 2-5 % glutaraldehyde, maintained at pH 7-4 in 0-05 M cacodylate buffercontaining o-i5M sucrose. After overnight washing in cold cacodylate-buffered0-3 M sucrose, the material was treated with veronal-acetate-buffered 1% osmiumtetroxide at the same pH, for 1 h, dehydrated in an ethanol series, and embedded inAraldite. Sections were cut on a Huxley microtome and examined in a PhilipsEM 200 electron microscope. Contrast in the sections was enhanced by double'staining', initially with saturated uranyl acetate in 50% ethanol (30 mm) and sub-sequently with lead citrate (Reynolds, 1963) for 3-5 min.

Visceral muscle fibres associated with the midgut of larvae of Ephestia kiihniella,and with the spermatheca of adult Periplaneta americana were also prepared accordingto the above schedule.

RESULTS

General organization of the muscle fibres

As is frequently the case in organs within the insect body cavity, and also in thebody wall of certain invertebrates which lack an exoskeleton (e.g. platyhelminths;annelids) the muscle fibres surrounding the seminal vesicle of Carausius are disposed

Insect visceral muscles 51

in both longitudinal and circular fashion, perpendicular to one another, over thesurface of the organ. This double investment is, however, incomplete, and manyfields include fibres oriented in only one direction.

Each of these muscle fibres is elongated in transverse section, varying betweenabout 1 and 2-5 fi in width (Figs. 2, 3 and 8), and the contractile material, except in thevicinity of the nucleus, almost fills the cell. The fibrillar system is not divided intolamellar or cylindrical fibrils as in insect skeletal fibres (Tiegs, 1955; Pringle, 1957;Smith, 1962). The nuclei are generally situated laterally in the muscle cell, and, whileskeletal fibres are typically multinucleate, these visceral fibres appear to contain onlya single nucleus. The sarcoplasm extending from the poles of the nucleus is extensiveand free from myofilaments, a circumstance similar to that occurring in vertebratesmooth muscle cells. This region of sarcoplasm contains mitochondria and (Fig. 9)ribosomes, sparse cisternae of the rough-surfaced endoplasmic reticulum and well-defined Golgi complexes. The fibres are linked at frequent intervals by adhesionplates or desmosomes (Fig. 6), exhibiting a layer of dense extracellular material. Inskeletal fibres of insects such desmosomes do not occur, and it is interesting to notethat in visceral fibres these structures differ from the ' septate desmosomes' generallyinterpolated in regions of close contact between insect cells (Locke, 1965).

Each fibre is surrounded externally by a basement membrane or sarcolemma(Fig. 2) in which, as in other insect muscles (Smith, 1961a), no collagen-like fibrilshave been resolved.

The cell membrane

Each fibre is limited by a typical plasma membrane, underlying the sarcolemma andbounding the contractile material. At intervals, this membrane is inflected or in-vaginated into the fibre to form blindly ending tubes, generally about 100-200 A indiameter (Figs. 7, 8) but sometimes (Fig. 11) dilating to form larger cavities, up to700 A in diameter. In the Carausius and Ephestia material, these invaginations areirregularly disposed, but in the muscle investing the spermatheca of Periplaneta, inwhich the fibres are larger (up to 15/i in diameter), these derivatives of the cellmembrane extend across the radius of the fibre, and are arranged in a more preciselyradial pattern. These tubules appear to correspond to the transverse tubular system(T-system) elements which form an array precisely oriented with respect to the myo-fibrillar striations in insect flight and leg muscle (Smith, 1961 a, b, 1962) and vertebrateskeletal muscle (Porter & Palade, 1957; Andersson-Cedergren, 1959; Fawcett & Revel,1961; Revel, 1962; Franzini-Armstrong & Porter, 1964; Peachey, 1965), where theyare believed to play an important part in contraction, by providing the pathway,electrically coupled with the surface cell membrane, along which excitation may bedistributed throughout the fibre. In the visceral muscles so far examined, the sarco-plasmic reticulum cisternae are very reduced in extent, and are apparently representedby flattened vesicles, closely adjoining the invaginated T-system tubules. The closejuxtaposition of these two membrane components, in the dyad configuration (cor-responding to the 'triads' of vertebrate muscle, see references above), is illustrated inFig. 10.

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52 D. S. Smith, B. L. Gupta and U. Smith

The contractile system

As has been described above, the contractile material of each visceral muscle fibrecorresponds anatomically to a single fibril. This situation has been described in otherinvertebrate muscles; for example, in the striated fibres of an ostracod (Fahrenbach,1964) and a coelenterate (Chapman, Pantin & Robson, 1962). The striated nature ofthis visceral muscle is readily seen in longitudinal sections of the fibre (Figs. 2,4 and 5).Well-defined Z bands traverse the contractile system, following an irregular course,the sarcomere length defined by adjacent Z bands being about 7 to 8 ft. Each Z bandis flanked by similarly irregular I bands, about 0-7 to 1 -o fi in width. It should be notedthat, in these longitudinal sections, the light H band, occurring in the central regionof the sarcomere in insect and vertebrate skeletal muscles, is not clearly defined. Thelongitudinal sections (Figs. 4, 5) indicate that both thick (myosin) and thin (actin)filaments occur in the A band, while the former are excluded from the I bands, thesituation that obtains in vertebrate skeletal muscle (Huxley & Hanson, i960; Huxley,i960). Further details of the disposition of these myofilaments in visceral fibres arerevealed in transverse sections.

In transverse profiles of the A band of Carausius visceral muscle (Fig. 7) the doublearray of thick and thin myofilaments is distinct. At this relatively low magnification,the latter are resolved as rings, surrounding the myosin filaments; the unusual natureof the geometrical relationship between these two sets of filaments becomes apparentat higher magnification. The familiar pattern of six thin filaments surrounding each

W (b) W

Fig. 1. Diagrammat ic representation of the distr ibution of thick (myosin) and thin(actin) filaments in the A-band region of: (a) vertebrate skeletal muscle (Huxley, 1957;Huxley, i960; Huxley & Hanson, i960); (6) insect flight muscle fibres (Huxley &Hanson, 1957; Hanson & Lowy, i960; Smith, 1962); (c) insect visceral muscles,described in this paper (compare Fig. 12).

thick filament, as in the insect and vertebrate muscles so far described, does not occur;instead, the thin filaments form twelve-membered 'orbitals' around the thick fila-ments. Very slight distortion in the spacing of this double array is sufficient to obscurethe detailed geometry of the double lattice, and the diagram of this configurationshown in Fig. 1 c (and compared with other striated muscles, Fig. 1 a, b) accords withregions of transverse sections of visceral muscle fibres where distortion of the latticeappears to be minimal (Fig. 12).

In insect flight muscle and vertebrate skeletal muscle each myosin filament is sur-

Insect visceral muscles 53

rounded by six actin filaments but, as Huxley & Hanson (1957) and Smith (1962)noted, the trigonal situation of the actin filaments in vertebrate fibres reduces theratio of actin to myosin filaments, compared with the flight muscle fibres, in whicheach actin filament is shared by only two myosin filaments (Fig. 1 a, b). In insectvisceral muscles, each thin filament is shared, as in flight muscle, by two thick fila-ments, but the number of thin filaments is exactly doubled: in flight muscle a myosinfilament and its six neighbours are associated with thirty actin filaments (Fig. 1 b), whilein visceral muscle (Fig. 1 c) sixty similar myofilaments occur within the same area.

In glutaraldehyde-fixed visceral muscle of Carausius, the diameter of the thick andthin myofilaments is respectively about 160-180 A, and about 40-50 A. In osmium-fixed vertebrate muscles, the corresponding values have been measured as n o and50 A (Huxley & Hanson, i960). An increase in the apparent diameter of the myosinfilaments after glutaraldehyde fixation, to about 150 A, has been described by Franzini-Armstrong & Porter (1964) in fish skeletal muscle. The centre-to-centre spacingof the thick filaments in the A-band array of Carausius visceral muscle is about440-460 A; somewhat greater, that is, than the 370-A spacing encountered in osmium-fixed asynchronous insect flight muscle (Smith, 1962).

In transverse sections, as in the longitudinal plane, the region of junction betweenthe A and I bands of visceral muscle is clearly demarcated. Fig. 11 illustrates a regionof junction between these sarcomere regions in Carausius visceral muscle. An A-bandprofile, containing both myofilament populations, adjoins a well-defined thoughirregularly disposed area containing only thin I-band filaments. In the I band, how-ever, the orbital arrangement of the I filaments is lost, and these filaments appear tobe closely and irregularly packed. The irregularity of the margin between these bandsin precisely transverse sections conforms with their zigzag disposition, viewed inlongitudinal section. In longitudinal sections of Carausius seminal vesicle muscle, theZ bands appear as well-defined regions of increased density, in the middle of the spanof I filaments, paralleling the contours of the A/I junction. In transverse sections ofthis muscle (Fig. n ) the Z bands are less well defined; however, in the midgut muscleof Ephestia, the I-filament region is divided by an area containing dense structures,perhaps representing aggregates of Z-band material (Fig. 8).

The apparent absence of an H zone is a striking feature of longitudinal sections ofCarausius visceral muscle. In vertebrate skeletal muscle (Huxley & Hanson, i960)this region of the sarcomere in a relaxed fibre is defined- by the inner extremities ofthe actin filaments, and is traversed by the mid-region of the myosin filaments. Althoughlongitudinal sections of insect visceral muscle appear to lack H bands, this region ofthe sarcomere is more clearly defined in transverse profiles of the fibre, as is illustratedin Fig. 13. At the margin of this band, the thin filaments terminate more or lessabruptly, and, as is seen in the case of the A/I junction of these fibres (Figs. 2, 4), theA/H junction appears to be similarly irregular.

In their detailed construction, then, these visceral muscles exhibit several points ofresemblance with other striated fibres of insects and vertebrates; though, as regardstheir sarcomere organization, the striation repeat in visceral muscles has been achievedby a hitherto undescribed myofilament configuration.

54 D. S. Smith, B. L. Gupta and U. Smith

DISCUSSION

The sliding-filament model of striated muscle contraction and relaxation, as pro-posed by Huxley & Hanson (i960), has been most amply substantiated in the skeletalfibres of vertebrates. However, as Hanson & Lowy (i960) point out, a similar mech-anism may operate not only in invertebrate striated fibres, but perhaps also in certain'smooth' muscles in which two sets of cross-linked myofilaments are present, thoughnot aligned in the sarcomere register characteristic of striated fibres.

In vertebrate skeletal muscle fibres it is clear (Huxley & Hanson, i960; Page &Huxley, 1963) that the length of the A and I filaments (the myosin and actin components)remains constant during the activity cycle; furthermore, the length of these filaments ismore or less constant in different vertebrate skeletal muscles. In invertebrates, on theother hand, the relaxed sarcomere length, and hence presumably the lengths of theconstituent myofilaments, may vary considerably from one muscle to another. Thequestion remains whether invertebrate muscles operate by the same sliding-filamentmechanism as occurs in vertebrate fibres, or whether change in filament length is analternative or additional method of changing sarcomere length. There is no evidencethat the length of the A band changes during contraction in insect flight muscle fibres(Hanson, 1956), but such change has been reported elsewhere in invertebrates, as inLimulus skeletal muscle (de Villafranca, 1961) and in femoral muscle of Locusta(Gilmour & Robinson, 1964). While no observations have as yet been made on thechanges in band pattern in insect visceral muscle fibres, an important preliminary tosuch studies is the recognition of the dimensions and distribution of their constituentmyofilaments.

The Z bands of Carausius visceral muscle, although not precisely aligned trans-versely to the long axis of the fibre, define a sarcomere length of about 7-8 ju,, that isbetween three and four times that of insect flight muscle or vertebrate skeletal muscle.In the present material, although no assessment has been made of the degree of con-traction or relaxation of the fibres examined, the I-band width is in the range, of0-7-1-o p. Considerably greater sarcomere lengths have been recorded in invertebratestriated muscles; Haswell (1889) described fibres in the pharynx of syllids (Annelida)with a sarcomere length of 33 /i.

The apparent absence of an H band in longitudinal sections of visceral muscle fibresdeserves further mention. In the relaxed sarcomere of vertebrate muscle, this regionis traversed by the medial portions of the myosin filaments, and is delimited by theinner ends of the actin filaments. It has recently been reported (Franzini-Armstrong &Porter, 1964) that in glutaraldehyde-fixed vertebrate muscle the H band may also betraversed by narrow connecting strands linking the inner extremities of the actinfilaments. H bands are clearly demarcated in transverse sections of insect visceralfibres, and some indication of similar linking strands traversing this region is evident,and it is possible that this feature, together with the irregularity of this narrow region,serves to obscure this portion of the sarcomere in longitudinal sections.

In summary, this preliminary study suggests that the slowly contracting visceralmuscles of insects contain contractile material constructed on essentially the same

Insect visceral muscles 55

plan as that of vertebrate fibres in that it consists of two morphologically distinct setsof myofilaments which, though differing from those of vertebrate striated muscle intheir geometrical arrangement about the long axis, are nevertheless distributed in acomparable fashion along the sarcomere bands.

The electron microscope, coupled with biochemical and physiological studies, hascontributed much to a more complete understanding of the role of the membranesystems of striated muscle fibres; notably, the function of membranes within thefibre in controlling the phases of the activity cycle. In insect flight muscles, it is knownthat the plasma membrane at the surface of the fibre is continuous with open tubularinvaginations passing radially into the fibre at regular intervals, at a level either mid-way between the Z and H bands (synchronous fibres) or at other levels (asynchronousfibres) (Smith, 1961a, b, 1962). These tubules correspond to the T-system invagina-tions that occur in vertebrate striated muscle fibres (H. E. Huxley, 1964; Page, 1964;Franzini-Armstrong & Porter, 1964). Micro-depolarization experiments (A. F. Huxley,1959, 1964; Huxley & Peachey, 1964) on vertebrate and crab muscle have suggestedthat these invaginations may be responsible for the triggering of myofibrillar activationby acting as the pathway along which surface excitation is passively conducted into thefibre. In rapidly contracting synchronous muscles of insects and vertebrates, theseT-system tubules are flanked by, and in close association with, cisternae of the sarco-plasmic reticulum in triad or dyad configurations (Smith, 1961a, b, 1962; Porter &Palade, 1957; Andersson-Cedergren, 1959; Fawcett & Revel, 1961; Revel, 1962;Franzini-Armstrong & Porter, 1964; Peachey, 1965). Peachey & Porter (1959) com-pared the disposition of internal membrane systems in striated and smooth musclefibres, in the context of the speed of contraction of these muscles, and pointed out that,if some derivative of the cell membrane (now identified as the T-system tubules) isresponsible for internal conduction of excitation in striated muscle fibres, then it ispossible that the absence of such invaginations in smooth muscle fibres of vertebratesmay be correlated with the slowness of their contractile response.

Insect visceral muscle fibres appear to represent an intermediate condition betweensmooth muscle and striated muscles with a highly developed system of transversetubules (T-system tubules) and associated sarcoplasmic reticulum cisternae. Thefibres have a diameter below that of most vertebrate smooth muscle fibres; shortT-system invaginations are present, associated in dyad configurations with cisternaeof the sarcoplasmic reticulum, which are reduced to small flattened vesicles. Thedistance across which diffusion of an ' activating substance' must take place to triggercontraction of the fibrillar material in these visceral muscles is of the order of 1 {i;that is, the distance between the cell membrane and tubular derivatives, and the centreof the contractile apparatus. This distance is similar to that separating the centre ofthe sarcomere from the T-system tubules in the fast-acting fibres of insects andvertebrates, and the speed of contraction of visceral fibres falls well within the limitsimposed by the length of the excitation-contraction coupling pathway (comparePeachey & Porter, 1959). Furthermore, if the activating substance in insect visceralmuscles represents an influx of calcium ions to the contractile system (Porter, 1961;Ebashi, 1961; Hasselbach, 1964) then it is possible that the slow relaxation of these

56 D. S. Smith, B. L. Gupta and U. Smith

fibres is mediated by active sequestration of calcium ions within the reduced cisternaeof the sarcoplasmic reticulum, perhaps augmented by a calcium-pump mechanismat the cell membrane.

The most unusual feature of these visceral muscles is the disposition of the myo-filaments within the sarcomere. It should be pointed out that, while comparison hasbeen drawn between the filament array in vertebrate fibres and insect visceral fibres,there is at present no evidence available on the chemical composition of the thick andthin filaments in the latter, and any analogy must therefore be tentative. Nevertheless,the doubling of the number of thin filaments associated with the thick filamentsin the A-band region of these muscles divides them sharply from other insect fibreshitherto examined. The extent of occurrence of this pattern of myofilament distri-bution in insects and other animals, and its physiological significance, remain to bedetermined.

One of us (D. S. S.) gratefully acknowledges support from the National Science Foundation(Grant number GB-1291).

REFERENCES

ANDERSSON-CEDERGREN, E. (1959). Infrastructure of motor end plate and sarcoplasmic com-ponents of mouse skeletal muscle fiber. J. Ultrastruct. Res. (Suppl. 1), 1-191.

CHAPMAN, D. M., PANTIN, C. F. A. & ROBSON, E. A. (1962). Muscle in coelenterates. Revuecan. Biol. 21, 267-278.

EBASHI, S. (1961). The role of 'relaxing factor' in contraction-relaxation cycle of muscle.Prog, theor. Phys., Osaka (Suppl. 17), 35-40.

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FAWCETT, D. W. & REVEL, J. P. (1961). The sarcoplasmic reticulum of a fast-acting fish muscle.J. biophys. biochem. Cytol. 10 (Suppl.), 89-110.

FRANZINI-ARMSTRONG, C. & PORTER, K. R. (1964). Sarcolemmal invaginations constituting theT system in fish muscle fibers. J. Cell Biol. 22, 675-696.

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HANSON, J. (1956). Studies on the cross-striation of the indirect flight myofibrils of the blowflyCalliphora. J. biophys. biochem. Cytol. 2, 691-710.

Fig. 2. A low-power electron micrograph of the muscle investing the seminal vesiclein a male Carausius morosus. The fibre is associated with a layer of extracellularbasement-membrane material (bm) constituting the sarcolemma. Note the obliquelysituated Z bands {Z) flanked by I bands (/) and the apparent absence, in this plane ofsection, of a mid-sarcomere H band. The contractile material in this muscle consistsof a single fibril (fi), bordered on one side by a layer of sarcoplasm (sp). Note thesmall mitochondria (m). x 25000.Fig. 3. A low-power transverse section of Carausius seminal vesicle muscle. Note thatin this section the contractile material virtually fills the muscle cell, and that theplasma membrane is invaginated into the fibre at irregular intervals, to form T-systemtubules (T). x 45000.

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wmmD. S. SMITH, B. L. GUPTA AND U. SMITH {Facing p. 56)

Fig. 4. Longitudinal section of Carausius visceral muscle, including a Z band and theadjoining regions of the sarcomere. Note the irregular disposition of this band (Z), andthe adjoining I bands containing only thin filaments (about 50 A in diameter) (/). Fromthe edge of the I bands extend the A bands of the sarcomere, in which thick and thinfilaments interdigitate (A). The dense particles present in the I-band region probablyrepresent glycogen deposits (g). x 75000.

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Fig. 5. Longitudinal section of Caraushis visceral muscle, including a Z band (Z)flanked by A regions (A). Note that the I region extending from the Z band containsonly thin filaments (2) but that the neighbouring A band contains both thick (1) andthin (2) myofilaments. x 140000.Fig. 6. The intercellular linkage between muscle fibres investing the seminal vesicleof Caraushis. In these regions of close apposition the cell membranes are separatedby an intercellular gap containing a layer of dense material (arrows), a situationresembling that occurring in the 'desmosomes' or adhesion plates between manyepithelial cells. Note that a layer of dense material occurs within the muscle cells inthe desmosome region, and that I filaments (/) appear to terminate beside the cellmembrane in this region. It is possible that the dense sarcoplasm bordering the desmo-some represents Z-band material, and that the Z bands traversing the fibre may some-times terminate at the cell surface, x 60000.

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Fig. 7. Transverse section of visceral muscle of Carausius, through an A-band region.Note the two sets of myofilaments, and the invaginated T-system tubules (T); alsothe microtubules (mf) situated near the surface of the fibre, x 80000.

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Fig. 8. Transverse section of visceral muscle investing the midgut of larval Ephestia.Note the A-band profiles (A) containing thick and thin myofilaments, the I bandscontaining only thin filaments (/) and the aggregations of dense material in the middleof the I bands, apparently representing Z-band material (Z). As in Carausius visceralmuscle, the plasma membrane of these fibres is invaginated into the cell at irregularintervals, to form T-system tubules (T). x 75000.

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Fig. 9. Longitudinal section of Carausius visceral muscle, including a portion of thenucleus («) and the adjoining sarcoplasm. The latter contains sparsely distributedcisternae of the rough-surfaced endoplasmic reticulum (er) and well-developedsmooth-membraned Golgi complexes (go), x 56000.Fig. 10. Longitudinal section of Carausius visceral muscle, illustrating the dispositionof membrane systems within the fibre. A profile of the plasma membrane is includedat left (pm). Within the fibre, a portion of a T-system tubule (T) invaginated from thesurface plasma membrane (compare Fig. 7) lies alongside the fibrillar material (fi),and is closely associated with a flattened vesicle or cisterna of the sarcoplasmicreticulum (sr) containing electron-dense material, x 105000.

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Fig. I I . The distribution of myofilaments at the junction of A and I bands in Caraitsiusvisceral muscle. In the A bands (.-?) the thick filaments are surrounded by orbitals ofthin filaments, while in the I bands (/) only thin filaments occur. Dense material, prob-ably representing the Z band (Z) is also included in this section. Note the invaginatedtubules of the T-system (T): the mouth of the upper tubule contains a layer of extra-cellular material (indicated by an asterisk), bordered by regions of increased density(arrows) within the sarcoplasm, possibly confluent with the Z bands (compare Fig. 6).x noooo.

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Fig. 12. The disposition of myofilaments in the A band of Caraiisius visceral muscle.The thick filaments form a more or less regular hexagonal array (particularly withinthe marked rectangle), and each thick filament is surrounded by a ring of twelve thinfilaments. The geometry of the disposition of these myofilaments is illustrated indiagrammatic form in Fig. i c. x 180000.Fig. 13. The disposition of myofilaments at the A/H junction in Caraiisius visceralmuscle. In the A band (.4) thick and thin filaments occur together, while in theH band (H) only the former are present. There is some evidence that fine strands tra-verse the H band (arrows). Note the small mitochondrion (m) and transverse profilesof intrafibrillar microtubules (mi), x 120000.

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(Received 23 May 1965)