J. Cell Sci. 12, 197-215 (i973) 197Printed in Great Britain

FUNCTIONAL DEVELOPMENT OF THE

RETICULAR SYSTEM IN AN INSECT MUSCLE

WITH SYNCHRONOUSLY DIFFERENTIATING

CELLS

N. M. TYRERDepartment of Neurobiology, Research School of Biological Sciences,Australian National University, Box 475, Canberra, Australia

SUMMARY

In contrast to many developing muscles which contain a spectrum of cells at different stagesof differentiation, cell development in the locust abdominal intersegmental muscle tends to besynchronous, so that at any stage of development the cells are at the same stage of differentia-tion. This makes it feasible to relate the properties of the developing tissue as a whole to changesoccurring within the cells.

Muscle contraction in the abdominal dorsal muscles of locust embryos and young hoppershas been related to ultrastructural changes within the muscle. Early in development both con-traction and relaxation rates are very slow and the muscle shortens very little. The maximumregistered tension is achieved relatively early while the rates of contraction and relaxation remainslow. Contraction and relaxation become more rapid as the embryo develops.

These changes can be related to the development of the reticular system, various componentsof which mature at different rates. When the muscle is first able to develop tension the myofibrescontain scattered clusters of myofilaments while the transverse tubule system (T-system), thecisternae and the sarcoplasmic reticulum (SR) are not well developed. The myofibres reachtheir final size and the filaments are fully formed while the T-system is still irregular and theSR is sparse. The T-system and the cisternae become well developed before the SR. Duringthe time that the relaxation time decreases the SR becomes increasingly prominent.

INTRODUCTION

Physiological and biochemical features of developing muscle have been studiedextensively in vertebrates (e.g. Buller, Eccles & Eccles, i960; Close, 1964; Trayer& Perry, 1966; Klicka & Kasper, 1970; Dow & Stracher, 1971; Margreth, Salviati &Catani, 1971). Ultrastructural changes in developing vertebrate muscle have alsobeen described (Allen & Pepe, 1965; Ezerman & Ishikawa, 1967; Shimada, Fischman& Moscona, 1967; Fischman, 1967; Shiaffino & Margreth, 1969; Edge, 1970) and insome cases it has been possible to correlate these changes with the changing propertiesof the muscle (Aloisi & Margreth, 1967; Luff & Atwood, 1971).

In vertebrates 2 problems are encountered when making detailed comparisonbetween the changing ultrastructure and the changing properties of developing muscle.First, many adult muscles contain a heterogeneous population of muscle fibre types,usually described as red, white and intermediate (Edgerton & Simpson, 1969). Thedifferent developmental histories of these fibre types may confuse attempts to relate

198 N. M. Tyrer

cellular changes with the changing properties of the whole muscle. Secondly, manydeveloping muscles contain a spectrum of cells at different stages of differentiation(Allen & Pepe, 1965; Kelly, 1971) which precludes direct correlation between celldevelopment and the physiological or biochemical features of the muscle as a whole.

In this paper an insect muscle is described in which the differentiation, of the cellsis synchronous. All cells at any particular stage of development are at the same stageof differentiation. This offers a unique possibility for the study of development ofphysiologically important components of muscle cells. The development of the timecourse of contraction and relaxation of this muscle has been investigated, which isthe subject of a preliminary report (Tyrer, 1969). It is found that development oftension, contraction rate and relaxation rate all occur separately at easily identifiablestages, which can be related to developments in the structure of the muscle.

MATERIALS AND METHODSEggs and hoppers were obtained from a culture of the locust Schistocerca gregaria maintained

in the Department of Zoology, Cambridge, England, under conditions similar to those used bythe Anti-Locust Research Centre (now Centre for Overseas Pest Research) (Hunter-Jones,1961).

Definition and identification of developmental stages

The eggs of S. gregaria are laid in pods of 30 to 90. The development time of different podsof embryos is variable, so that the age of an embryo is not a precise way of describing the stageof development. Fortunately, all the eggs in any one pod tend to develop at the same rate(Tyrer, 1970) so it is possible to define the stage of development of an embryo as a percentageof the incubation period of the egg pod from which it came. Using this method, a newlyhatched hopper is described at 100 %, while an embryo which is three-quarters developed isa 75 % embryo. Two methods, which are described elsewhere, were used to stage embryos(Tyrer, 1970). Either the stage was calculated from accurate measurements of the times whenthe egg pod was laid and when the first hopper hatched (= timed), or the percentage develop-ment was estimated from external morphological criteria (= estimated).

Measurement of the mechanical response of the muscles

The dorsal longitudinal intersegmental muscles from the fourth abdominal segment wereused (Fig. 1). Details of the methods used to record the mechanical response of these musclesin the embryo are described elsewhere (Tyrer, 1969). The dorsal part of one side of the fourthabdominal segment was isolated mechanically from the rest of the abdomen and hooked to therecording lever system. The response of the muscles to stimulation of their motor nerve witha burst of stimuli for 0-7 s at a frequency of 50/s was recorded (Fig. 2). Responses of themuscles were recorded from 40 preparations. Sixteen of these were made from animalswhose age was calculated as a percentage of the total development time. These preparationswere from the period of development 67-109 %. Twenty-four preparations were from animalswhose age was estimated on morphological criteria. Four of these were at the 70% stage, 6 at80, 7 at 93 and 7 at the 100 % stage. The relaxation time from peak tension to half the peaktension, T£R, was measured during 12 maximal responses for each preparation. It is notpossible to compare quantitatively the contraction times and degree of tension developed atdifferent stages of development since the stimulus used may have imposed false minima onthese values in the later stages of development.

Development of locust SR 199

Dorsalmuscles

70%

80%

92%

100%

109%1 mm

40 A

1 s

Fig. 1 Fig. 2

Fig. 1. Diagram of a newly hatched hopper dissected to show the position of the dorsalintersegmental muscles. Those in the fourth segment are shown in black. The develop-ment of the dorsal muscles of other segments parallels that of the fourth.

Fig. 2. Examples of the response of the dorsal muscles to stimulation of their motornerve at a frequency of 50/s for 0-7 s at different stages of development. (Base linesand stimulus artifacts are retouched.)

Electron microscopy

The dorsal muscles from the fourth abdominal segment (Fig. 1) of several embryonic stages,of the 100 % stage and the 5-day-old first instar hopper, were prepared for electron microscopy.In an initial investigation material was obtained from accurately timed embryos and was cal-culated to be from the 68, 77 and 92 % stages of development. Subsequently, material wasobtained from embryos which were staged according to morphological criteria. Stages estimatedto be 70, 80 and 92 % were used (Tyrer, 1970). The 5-day-hopper material was from animalsin which the development time of the embryos was unknown.

Embryos and hoppers were decapitated and a mid-dorsal incision made along the length ofthe body. The body wall was pinned out, well extended, on plasticine in a dish and fixed for2 h in ice-cold glutaraldehyde fixative. This comprised 2-5 % glutaraldehyde maintained atpH 7-0 in 0-05 M sodium cacodylate buffer containing 0-17 M sucrose. After overnight washingin cold sodium cacodylate-buffered 0-34 M sucrose, the material was treated with sodiumcacodylate-buffered 1 % osmium tetroxide at pH 7-0 for 1 h, dehydrated in an ethanol seriesand embedded in Araldite. For examination with the light microscope i-/«n thick sections werecut with glass knives, dried down on to microscope slides, and stained either with 1 % methy-lene blue in 1 % borax or with 1 % toluidine blue in 1 % borax. For electron microscopy thin

2 0 0 N. M. Tyrer

40 r

30

\ 20

1 0

70 80 90

% development

100 110

Fig. 3. The time of relaxation from peak to half peak tension of the dorsal muscles atdifferent stages of development. The mean of 12 responses is shown for each prepara-tion. The responses were obtained by maximal stimulation of the motor nerve ata frequency of 50/s for 0-7 s (see Fig. 2). The stages defined according to morphologicalcriteria are shown as open symbols and the timed stages as closed symbols. Thelimits indicated are 2 standard deviations from the mean.

sections were cut using glass knives and either a Huxley or a Reichert ultramicrotome. Contrastwas enhanced by double staining, initially with saturated uranyl acetate in 50 % ethanol(1-5 h) and subsequently with Reynold's lead citrate (0-5 h). Longitudinal sections proved tobe particularly difficult to stain. Some success was achieved by pretreating the sections with amylacetate vapour for 0-5 h and then staining for 3 h in uranyl acetate and 1 h in lead citrate. Evenso, satisfactory contrast was obtained only in relatively thick sections. Sections were examinedeither with a Philips EM 200 or a Hitachi HU11E electron microscope.

RESULTS

The time course of the muscle response

The youngest preparation from which a mechanical response was obtained onstimulation of the dorsal nerve was a 67% embryo. Shortening was slight and thecontraction and relaxation times were long and variable (Figs. 2, 3). In older pre-parations, shortening increased, until by the 80% stage the muscle shortened to thesame extent and developed the same tension as in the newly hatched animal (Fig. 2).

In the 80% embryo, however, both contraction and relaxation times of the re-sponses were still longer and more variable than those of the newly hatched animal(Figs. 2, 3). Not only was there variation from preparation to preparation, butdifferent records from the same preparation gave variable results. In older stagescontraction and relaxation times became progressively shorter and variation wasconsiderably reduced. While the relaxation rate continued to increase throughout

Development of locust SR 201

the period of development investigated, there appeared to be little change in thecontraction rate after the 92% stage (Fig. 2).

The ultrastructure of the muscle

There are 2 surprising features in the developing intersegmental muscle. First,the differentiation of the myofibres in any one muscle is synchronous. Virtually nodifferences could be detected in the organization of the individual myofibres in anygiven muscle. Secondly, except in the earliest embryos, the myofibre is the only celltype present in the muscle (excluding nerves and their sheath cells (Figs. 8, 12, 16,20)).

In each of the 4 dorsal muscles there are between 9 and 20 myofibres usuallyarranged in 2 rows (Figs. 4, 8, 12, 16, 20). In all stages later than 80% these aregenerally between 10 and 15 /tm in diameter and about 0-5 mm long. Earlier than the80% stage the myofibres are smaller in cross-section - between 5 and 8/tm in dia-meter in the 70% stage (Fig. 4), but they are the same length as in later stages. Inthese early stages, myoblasts are present although all the myofibres are at the samestage of differentiation. The number of myoblasts decreases as the myofibres increasein size until by the 80% stage they are very rare. Presumably the fusion of themyoblasts with the myofibres results in the increase in their diameter as occurs inother insect muscles during development (Crossley, 1972).

The organization of the myofibre

In mature locust intersegmental muscle, as in many insect skeletal muscles (seeMill & Lowe, 1971), the sarcomere is long, 7-8 /tm, the Z-bands are irregular andimprecisely aligned in adjacent fibrils, and there are 9-12 thin filaments surroundingeach thick filament. The myofilaments occur in groups, myofibrils, which arebetween 0-5 and i-o/tm in diameter. Surrounding each myofibril in the A-bandregion is a network of tubules, the sarcoplasmic reticulum (SR), communicating withcisternae which occur close to the junction between the A-band and the I-band.In this region also the transverse tubules (T-tubules) descend from the externalmembrane and are closely apposed to the cisternae in such a way that, in section,2 profiles are seen, one of the T-tubule and the other of the cisternal element. Thisis the dyad, which is analagous to the triad found in vertebrate reticular system.Mitochondria occupy the space between the myofibrils in the I-band region butthey are sparsely distributed and so are not seen between every myofibril in any onesection.

The 70% stage (Figs. 4-7)

Already by this stage the myofilaments are organized into sarcomeres of the samedimensions as in the mature muscle (Fig. 6). The myofilaments are grouped intomyofibrils which may be up to 1 /tm in diameter, although they are usually rathersmaller than this (Figs. 5, 7). The myofibrils are separated by large areas of sarco-plasm which contain a few membrane-bound profiles, some irregularly orientedfilaments, ribosomes and an occasional microtubule (Figs. 6, 7). Mitochondria, as yet

202 N. M. Tyrer

small and containing few cristae, are arranged irregularly on either side of the Z-band(Fig. 6).

The reticular system at this stage is poorly developed. The outer cell membraneis invaginated in a few places to form T-system elements about 40 nm in diameter,but there are few of these and they usually extend only a short distance into the cells(Figs. 5, 7). Between the myofibrils a few thin-walled profiles, without contents, areseen in the A-band region, and these appear to be the beginning of the SR (Fig. 6).Irregularly shaped, thin-walled elements are associated with the T-system elementsin the A-I band region. Sometimes these elements have weakly electron-dense con-tents and have electron-dense material between them and the T-tubule: this suggeststhat differentiation of cisternal elements has begun (Figs. 6, 7).

Membrane structures similar to those associated with the T-tubules frequentlyapproach the plasma membrane and run parallel to it (Fig. 7). These resemble theperipheral couplings described in developing rat intercostal muscle by Kelly (1971)-

The 80% stage {Figs. 8-11)

As can be seen in transverse sections, the sarcoplasm is now packed with myofila-ments (Fig. 9). The myofibrils are, as in the previous stage, still usually 0-5-1 -o/tmacross, but a greater proportion are closer to 1 jum across than in the 70% embryo.Some of the peripheral myofibrils may be a little larger. No change in the myofibrilsize occurs in subsequent stages. Between the myofibrils profiles of SR are sparse, buta small number of cisternae are easily identified associated with T-tubules in theA-I band region (Fig. 9). They form flattened sacs about 60 nm across, usually haveweakly electron-dense contents and frequently have electron-dense material inter-posed between them and the T-tube. Peripheral couplings of SR elements and thesurface are rare at this stage.

Some of the tubes of the T-system descending from the plasma membrane extendmuch deeper into the cell than before while others are still ill-developed. Mitochon-dria are still fairly small and have few cristae.

The 9 2 % stage (Figs. 12—15)

The reticular elements are now relatively well differentiated. The plasma membraneis invaginated at regular intervals around the fibre and the T-tubules descend uni-formly into the fibre (Fig. 13). Dyads at the A-I band region are well formed andnumerous and the contents of the cisternae are more electron-dense than in earlierstages (Fig. 13). The dyads now appear to be fully developed (Figs. 14, 15). Nochange in their structure or number occurs after this stage. In the A-band region,however, the SR is still sparse. Mitochondria are larger and contain more cristae(Fig- 13)-

The 100% stage (Figs. 16-19)

The most important difference between the 100 and 92% stage is the increaseddevelopment of the SR. The profiles of the tubules between the myofibrils in theA-band region are better defined and rather more numerous (Fig. 17). The organiza-

Development of locust SR 203

tion of the T-system and dyads, however, is little changed. Mitochondria have morecristae than in earlier stages (Figs. 17, 18) and the variability of their shape in trans-verse section (Fig. 17) suggests that they now have a more complicated structurethan the simple cylindrical condition in the earlier embryonic stages. They may bearprocesses like those described in cockroach femoral muscle (Hagopian, 1966).

The 5-day stage (Figs. 20-23)

A striking difference between this stage and the earlier ones is the abundance ofthe SR. Whereas the region between myofibrils previously contained SR which wasone or two profiles wide, in this stage there are tiers of 4 and even 5 profiles. Otherfeatures of the muscle are little changed.

DISCUSSION

Structurally the myofilaments appear to have attained their full development bythe 80% embryonic stage even though the T-system and SR are still poorly developed.By the 92 % stage the T-system and dyads are as highly organized as in later stages,while the SR is still sparse. In subsequent stages the SR continues to develop and bythe 5-day stage has become quite elaborate.

Concepts of the roles of the various components of the reticular system in insects(Smith, 1966) have been based largely on cytological similarities with vertebrates(see Peachey, 1968). Thus, it is assumed that in insects, as in vertebrates, the T-systemconveys a signal into the cell following surface excitation, which, at the dyad, causescalcium to be released into the sarcoplasm. from the cisternae, so causing the myofila-ments to contract. Relaxation is supposed to be achieved, as in vertebrates, by rapiduptake of calcium into the SR which then returns it to the cisternae.

The experimental observations on the developmental changes in relaxation rate inthis muscle correlate well with the structural changes in the SR. The mean valuesfor the relaxation rate, T^R, (Fig. 3) decrease progressively at the same time that theSR elaborates. This supports the supposition that the process of relaxation in thisinsect is similar to that in vertebrates.

There is also a progressive increase in the rate of contraction as development pro-ceeds, particularly in the early stages. It appears, under these experimental condi-tions, that the maximal contraction rate is attained before the maximal relaxation rate(Fig. 2). It may be significant that the T-system and dyads appear to be fully formedbefore the SR is fully developed, although of course, SR development should alsoaffect the contraction time since the rate at which the SR accumulates calcium isa factor determining when contraction ends.

A possibility which must be considered is that the time course of the membranepotential produced by stimulating the motor nerve may be slower in the earlierstages of development. Microelectrode recordings from the muscle during stimulationof the nerve showed no change in the time course of the membrane response betweenthe 92 and 100% stages (Tyrer, 1968, 1969). Records were not obtained, however,from earlier embryos so this remains a possible explanation for all or part of theslower time course of the mechanical response in the earlier stages.

204 N. M. Tyrer

Other factors which have not been considered here may affect the time course ofthe mechanical response during development. Changes may occur in the contractileproteins themselves, or in the metabolism of the muscle. These questions can onlybe answered by further analysis of the properties of the muscle. Such detailed analysesof the biophysical, biochemical and physiological changes during development arefeasible in these muscles because of the synchronized differentiation of the musclecells.

The initial investigation for this study was done as part of a Ph.D. dissertation in the Depart-ment of Zoology in the University of Cambridge, while I held a Research Studentship from theAgricultural Research Council. I am indebted to Dr J. E. Treherne for his advice and encourage-ment during this period and for allowing me the facilities of the A.R.C. unit in the Departmentof Zoology, Cambridge, in 1971 to obtain the material to finish the work in Australia. I am grate-ful to Miss A. B. Poyser and Mr R. Whitty for their skilled technical assistance. I thank mywife Dr J. Altman for continually pressing me to publish the work and for reading themanuscript.

REFERENCES

ALLEN, E. R. & PEPE, F. A. (1965). Ultrastructure of developing muscle cells in the chickembryo. Am. J. Anat. 116, 115-148.

ALOISI, M. & MARGRETH, A. (1967). In Exploratory Concepts in Muscular Dystrophy and RelatedDisorders (ed. A. T. Milhorat), pp. 305-317. Amsterdam: Exerpta Medica Foundation,International Congress Series 147, 305-317.

BULLER, A. J., ECCLES, J. C. & ECCLES, R. M. (i960). Differentiation of fast and slow musclesin the cat hind-limb. J. Physiol., Lond. 150, 399-416.

CLOSE, R. (1964). Dynamic properties of fast and slow skeletal muscles of the rat duringdevelopment. J. Physiol., Lond. 173, 74-95.

CROSSLEY, A. C. (1972). Ultrastructural changes during transition of larval to adult inter-segmental muscle at metamorphosis in the blow-fly, Calliphora erythrocephala. I. Dedif-ferentiation and myoblast fusion. J. Embryol. exp. Morph. 27, 43-74.

Dow, J. & STRACHER, A. (1971). Changes in the properties of myosin associated with muscledevelopment. Biochemistry, N.Y. 10, 1316-1321.

EDGE, M. B. (1970). Development of apposed sarcoplasmic reticulum at the T system andsarcolemma and the change in orientation of triads in rat skeletal muscle. Devi Biol. 23,634-650.

EDGERTON, V. R. & SIMPSON, D. R. (1969). The intermediate muscle fibre of rats and guineapigs. J. Histochem. Cytochem. 17, 828-838.

EZERMAN, E. B. & ISHIKAWA, H. (1967). Differentiation of the sarcoplasmic reticulum andT-system in developing chick skeletal muscle in vitro. J. Cell Biol. 35, 405—420.

FISCHMAN, D. A. (1967). An electron microscope study of myofibril formation in embryonicchick skeletal muscle. J. Cell Biol. 32, 557-575.

HAGOPIAN, M. (1966). The myofilament arrangement in the femoral muscles of the cockroachLeucophaea maderae Fabricius. J. Cell Biol. 28, 545-562.

HUNTER-JONES, P. (1961). Rearing and Breeding Locusts in the Laboratory, pp. 1-12. London:Anti-Locust Research Centre.

KELLY, A. M. (1971). Sarcoplasmic reticulum and T tubules in differentiating rat skeletalmuscle. J. Cell Biol. 49, 335-344.

KLICKA, J. & KASPA, J. L. (1970). Changes in enzyme activities of the hatching muscle of thechick (Gallus domesticus) during development. Comp. Biochem. Physiol. 36, 803-809.

LUFF, A. R. & ATWOOD, H. L. (1971). Changes in the sarcoplasmic reticulum and transversetubular system of fast and slow skeletal muscles of the mouse during postnatal development.J. Cell Biol. 51, 369-383.

Development of locust SR 205

MARGRETH, A., SALVIATI, G. & CATANI, C. (1971). Electron transport in sarcoplasmic reticulumof fast and slow muscles. Archs Biochem. Biophys. 144, 768-772.

MILL, P. J. & LOWE, D. A. (1971). Ultrastructure of the respiratory and non-respiratory dorso-ventral muscles of the larva of a dragonfly. J. Insect Physiol. 17, 1947-1960.

PEACHEY, L. D. (1968). Muscle. A. Rev. Physiol. 30, 401-440.SHIAFFINO, S. & MARGRETH, A. (1969). Co-ordinated development of the sarcoplasmic reticu-

lum and T system during postnatal differentiation of rat skeletal muscle. J. Cell Biol. 41,

SHIMADA, Y., FISCHMAN, D. A. & MOSCONA, A. A. (1967). The fine structure of embryonicchick skeletal muscle cells differentiated in vitro. J. Cell Biol. 35, 445-453.

SMITH, D. S. (1966). The organisation and function of the sarcoplasmic reticulum and T-systemof muscle cells. Prog. Biophys. molec. Biol. 16, 107-142.

TRAYER, I. P. & PERRY, S. V. (1966). The myosin of developing skeletal muscle. Biochem. Z.345, 87-100.

TYRER, N. M. (1968). Some Aspects of Functional Development in the Locust Embryo. Ph.D.Thesis University of Cambridge.

TYRER, N. M. (1969). Time course of contraction and relaxation in embryonic locust muscle.Nature, Lond. 224, 815-817.

TYRER, N. M. (1970). Quantitative estimation of the stage of embryonic development in thelocust, Schistocerca gregaria. J. Embryol. exp. Morph. 23, 705-718.

{Received 25 May 1972)

ABBREVIATIONSINS

AcepifImmbmf

ON PLATESA-bandcisternal elementcuticle and epidermisfat bodyI-bandmitochondrionmyoblastmyofibre

mtnnvPsrtZ

microtubulenucleusmotor nervemyoblast processsarcoplasmic reticulumT-tubuleZ-band

2o6 N. M. Tyrer

Figs. 4-7. The 68 % (timed) stage.Fig. 4. Light micrograph of a i-/Mn transverse section of 1 of the 4 dorsal

muscles. Scale 10 fim.Fig. 5. Low-magnification electron micrograph of the muscle cut in TS in the

A-band region close to the I-band. A myofibre, containing clusters of myofilaments0-5-1-0 /Jm across is seen in the centre of the field. Myofibre nuclei (n) are seen middleleft and lower right and a portion of a myoblast (mb) is seen lower left. T-tubules (i)usually descend only a short distance into the fibre, but one deeply descending oneis seen in the centre of the field. A few dyads (arrowed) are present. The mitochondria(m) are small, x 14000.

Fig. 6. Longitudinal section of the muscle showing myofibrils 0-5 /m\ acrossseparated by regions of sarcoplasm containing a few profiles of sarcoplasmic reticulum(sr) and some poorly oriented filaments. Mitochondria (m) occur in the I-band region.A possible dyadic association with a T-tubule is arrowed, x 19000.

Fig. 7. A transverse section of the myofibre in the A-band region close to theI-band. The myofibrils are separated by sarcoplasm containing irregular, apparentlyswollen, elements (sr). These are interpreted as elements of sarcoplasmic reticulumwhich are poorly fixed. Some elements similar to these are loosely associated withthe plasma membrane (cf. Kelly, 1971). A dyadic association with a short T-systemelement is arrowed. Note that the cisternal element (c) contains little material. Theslightly corrugated appearance of the T-tubule suggests an origin similar to that ofthe chick (Ezerman & Ishikawa, 1967). Note the large number of thin filamentssurrounding each thick filament, x 57500.

Development of locust SR

5

207

J

208 N. M. Tyrer

Figs. 8-11. The 80% stage.Fig. 8. Light micrograph of a 1 -/tm transverse section of 1 of the 4 dorsal muscles

from a 77 % (timed) embryo. Note that the size of the myofibres (mf) is comparablewith that of later stages (cf. Figs. 12, 16 and 20). n, myofibre nucleus. Scale 10 /an.

Fig. 9. Low-magnification electron micrograph of a myofibre from the 80 %(estimated) muscle cut in TS. The myofibre is now packed with filaments. SomeT-system elements (t) descend deeply into the fibre and some dyads (arrowed) areseen. Between the myofibrils are seen profiles of a sparse SR. x 14000.

Fig. 10. Longitudinal section of a muscle at the 80% (estimated) stage showingsome incompletely divided myofibrils between O'5 and i'O/tm in diameter. Note thelong sarcomeres and irregular Z-bands. Between the myofibrils are a few profiles ofsarcoplasmic reticulum (sr). Mitochondria (m) are present in the I-band region,x 19000.

Fig. 11. A TS of the 77 % (timed) muscle in the A-band region close to the I-band.A sparse sarcoplasmic reticulum (sr) occurs between the myofibrils. A dyadic struc-ture is arrowed, x 57500.

Development of locust SR

2io N.M. Tyrer

Figs. 12-15. The 92% stage.

Fig. 12. A light micrograph of a i-/im section of 1 of the 4 dorsal muscles from the92 % (timed) stage. This micrograph shows very clearly that all the myofibres (mf)are in identical states of development. Scale 10 /tm.

Fig. 13. Low-magnification electron micrograph of a myofibre in TS from a 92 %(estimated) muscle. The myofibrils are now well defined by reticular elements. T-sys-tem elements (t) descend at regular intervals from the plasma membrane. The sarco-plasmic reticulum (sr) is still sparse but dyads (arrowed) are well formed and numerous.Mitochondria (m) have more cristae than in earlier stages and some are less regular insection suggesting that they are no longer simple cylindrical structures, x 14000.

Fig. 14. Longitudinal section of the muscle at the 92 % (estimated) stage. Thesparse sarcoplasmic reticulum is seen between the myofibrils in the A-band regionand well denned dyads (arrowed) occur close to the A-I band junction, x 19000.

Fig. 15. A transverse section of the 92 % (timed) muscle in the A-band region closeto the I-band showing myofibrils well defined by profiles of the sarcoplasmic reticu-lum. A T-tubule (t) is seen descending from the plasma membrane and in associationwith a cisterna to form a dyad which is very similar in organization to those of laterstages (cf. Figs. 19, 23). Note the dense material between the T-tubule and the cis-terna and also the dense contents of the cisterna. x 57 500.

Development of locust SR

14-2

212 N.M. Tyrer

Figs. 16-19. The 100% stage.

Fig. 16. Light micrograph of a i-fim section of 1 of the 4 dorsal muscles. As in the9 2 % stage (Fig. 12) the myofibres (mf) are all at the same stage of differentiation.Their condensed appearance is due to stretching of the muscle. The motor nerve(nv) is seen descending into the muscle. Scale 10 /tm.

Fig. 17. Low-magnification electron micrograph of a myofibre in TS. The sarco-plasmic reticulum is better defined than in the previous stage but it is still sparse.Dyads (arrowed) are similar in structure and number to the previous stage (Fig. 13)and to the next stage (Fig. 21). x 14000.

Fig. 18. Longitudinal section showing the sparse sarcoplasmic reticulum betweenthe myofibrils in the A-band region and well denned dyads (one arrowed) close tothe A—I band junction. Mitochondria occur in the I-band region as in the otherstages, x 19000.

Fig. 19. A transverse section in the A-band region close to the I-band. Elements ofthe sarcoplasmic reticulum can be distinguished from the T-tubules by their thinnerwalls. Note the periodic nature of the electron-dense material between the T-tubuleand the cisterna in the dyad (arrowed). In favourable sections this periodicity isseen in the dyads in all stages from the 80 % onwards, x 57 500.

Development of locust SR 213

214 N.M. Tyrer

Figs. 20-23. The 5-day hopper.

Fig. 20. Light micrograph of a 1 /tm section of 1 of the 4 dorsal muscles. Themyofibres (»«/) are the same size as in the previous stages and all are at the samestage of differentiation. Scale 10 fira.

Fig. 21. A low-power electron micrograph of a transverse section showing thatthe SR is highly developed compared with previous stages. In many places tiers ofprofiles are seen between the myofibrils and beneath the plasma membrane. Dyads(arrowed), however, are similar in structure and number to the 92 and 100 % stages.Note the irregular profiles of the mitochondria suggesting that they branch (cf.Hagopian, 1966). x 14000.

Fig. 22. Longitudinal section. Note the development of the sarcoplasmic reticulumin the A-band region where several tiers of profiles occur between the myofibrils.The dyads (arrowed), however, are little different from the previous stages. Mito-chondria are seen in the I-band region, x 19000.

Fig. 23. A transverse section in the A-band close to the I-band. Note the welldeveloped sarcoplasmic reticulum and the unchanged appearance of the cisterna (c)and the T-tubule (t). There is some indication of periodicity in the electron-densematerial between the T-tubule and the cisterna. x 57000.

D/mt>JfrhmfiU.t nf Inrust SR

20