Structural features of muscle fibres in the cockroach leg

J. Insect Physwi.,

1969,Vol.

15, pp. 2255 to 2262. Perganm

Press. Printed in Great Britain

STRUCTURAL FEATURES OF MUSCLE IN THE COCKROACH LEG S. S. JAHROMI*

FIBRES

and H. L. ATWOOD

Department of Zoology, University of Toronto, Toronto, Canada (Received 12 June 1969) Abstract-Studies of muscle fibres in several leg muscles of the cockroach (Peripheta americana) were made with the electron microscope. The pink muscles of the coxa were found to have a thin : thick filament ratio of 3 : 1 compared with a ratio of 6 : 1 in most of the other leg muscles. Sarcomere lengths were uniform in some muscles and variable in others, Fibres with reduced sarcoplasmic reticulum and an unusual T-system were found in coxal muscle 13 5b. The significance of the findings is discussed in relation to contractile properties of arthropod muscle fibres. INTRODUCTION

DIFFERENTleg muscles of the cockroach (particularly

those in the coxa) are known be biochemically and physiologically diverse. In a recent paper, SMIT et al. (1967) reviewed earlier work on these muscles, and showed that the coxal muscles 135a and 135~ (which serve both as extensors of the trochanter and as flight muscles of the forewing) are more resistant to fatigue, and have a higher mitochondrial content, than the coxal muscles 136 and 137 (which function as extensors of the trochanter). Speed of contraction and peak twitch tension (referred to unit area of muscle cross-section) were found to be similar in the two groups by USHERWOOD (1962). In the present paper, attention is given to some of the structural features of these muscles which have not been analysed in previous work. In particular, the sarcomere organization and the myofilament arrangements are described. An attempt is made here to extract possible correlations between ultrastructural and physiological properties of arthropod muscle fibres, by comparison of the data for the cockroach coxal muscles with those available for certain crustacean muscles.

to

MATERIALS

AND METHODS

The muscles used in this study were the mesothoracic coxal muscles 135a, 135b, 135c, 136 and 137 (CARBONELL,1947) and the tibial extensor muscle of the mesothoracic leg, from the cockroach, Periplaneta americana L. For light microscopy, muscles were fixed, while stretched slightly past rest length, in Bouin’s fluid, embedded in paraffin, and sectioned at 5 ~1. Sarcomere * Present address: Department of Biology, Pahlavi University, Shiraz, Iran. 2255

2256

S. S. JAHROMIand H. L. ATWOOD

lengths for individual fibres were obtained by measuring the total length of a group of sarcomeres with an eyepiece scale, and dividing by the number of sarcomeres in the group (usually 5-10). In some cases, individual sarcomeres were measured. Certain muscles were also prepared for electron microscopy. They were first fixed in 4% glutaraldehyde in Millonig phosphate buffer (1 hr at room temperature). Subsequently, they were rinsed in buffer alone (2 hr) and post-fixed in 1 y0 buffered osmium tetroxide (1 hr). Sections approximately 500 A thick were cut and stained in ethanolic uranyl acetate and then by the lead citrate method of REYNOLDS(1963). RESULTS Sarcomere and A-band length Sarcomeres of fibres from the muscles under investigation were measured in teased-fibre preparations, in slides of muscles sectioned longitudinally, and from electron micrographs. Representative data for muscles of one male cockroach are presented in Table 1. Comparable results were obtained in muscles of other individuals. The main branches (a, c) of muscle 135 contain fibres of rather uniform sarcomere length, mostly 3-5 p. Sarcomeres of fibres from muscle 137 (and also 136) fall mostly within the same lengths. There is a small but statistically significant difference in mean sarcomere length for the two groups, provided that comparisons are made between muscles fixed in situ in the same leg. The finding was confirmed by measurements made from electron micrographs (Table 2). Fibres of muscle 135b (classified as ‘slow’ by BECHT and DRESDEN, 1956) showed a much wider range of sarcomere lengths. The calculated mean value of 7.31 p (Table 1) was considerably higher than for the other coxal muscles. Furthermore, the frequency distribution of sarcomere lengths was far from that expected for a sample from a normally distributed population. The sarcomeres of the extensor tibiae muscle fibres could not be directly compared with those of coxal muscle fibres, since a different segment of the leg was involved, and differences in the amount of stretch applied during fixation of the two muscles could have occurred. However, samples taken from the central part of the muscle (Table l), which is innervated mainly by the ‘fast’ motor axon (Smyth, quoted in HOYLE, 1965), showed fairly uniform sarcomeres, whereas samples from the proximal part of the muscle, which is innervated by both ‘fast’ and ‘slow’ axons, showed a wide range of sarcomeres and a higher mean value, as in muscle 135b. The fibres from the central part of the extensor tibiae appeared generally similar to those in muscles 136 and 137 when viewed with the electron microscope. We did not examine the long-sarcomere fibres of the proximal part of the extensor tibiae muscle with the electron microscope. A-zone lengths, measured from electron micrographs of fibres taken from the central part of the extensor tibiae muscle and from coxal muscles 135~ and 137, are presented in Table 2. The fibres of muscle 135c, which have the shortest sarcomeres, also have the shortest mean A-zone. The tibia1 extensor muscle fibres and the fibres of muscle 137 were similar in sarcomere length and in mean A-zone

COCKROACH LEG MUSCLEFIBRFS TABLE I-SARCOMERB LENGTHSIN COXALMUSCLE135b,

2257

135c,137,AND IN EXTENSORTIBIAE

Muscle Extensor tibiae 135b

135c

137

Proximal

Central

(%)

(%)

(%)

(%)

(%)

2 68

44

3

56

37 26 13 7 7 7

87 13

0.24 0.04

Sarcomere length (p) 2-3 3-4 4-5 5-6 6-7 7-8 8-9 9-10 10-l 1

Mean & S.D. k S.E. No. of fibres

13 14 23 10 17 16

28 2

7

(4

64

3.82* 0.51 0.07

4*01* 0.28 0.04

1.57 0.29

100

50

50

30

* Means differ significantly (t-test,

TABLE ~-LENGTHS

Muscle Tibia1 extensor

(137) Coxal extensor (135c)

30

P < 0.05).

OF SARCOMERES,A-ZONES, AND H-ZONF~ OF IWOFIBRILS IN FIBRBS FIXEDFOR ELECTRONMICROSCOPY Sarcomere length

A-zone length

H-zone length

(tL)

(p)

0

4-31(No.= 50) f0.57 S.D. f0.08

Coxal extensor

(4

7.31 1.76 0.18

O-49(No.= 15) +O-16 S.D.

3.51**(No.= 50) 50.25 S.D. + O-24S.E.

0.33(No.= 18) k 0.09S.D.

S.E.

4.30*(No.= 50) ~0.22 S.D. f0.03

3.34(No.= 50) kO.49 SD. + 0.07S.E.

S.E.

3.71*(No.= 50) f0.38 SD.

2.53**(No.= 50) kO.23 S.D.

kO.05 S.E.

** ** Means differ significantly (t-test,

f0.03

S.E.

P< O-001).

kO.04 S.E.

20.02

S.E.

2258

S. S. JAHROMI and H. L. ATWOOD

length. The difference between the mean A-zone lengths, although small, was statistically significant (t-test, P< O-05). H-zones were measured in tibia1 extensor and white coxal(l37) muscle fibres (Fig. 1, Table Z), but were indistinct or lacking in many of the pink coxal(135a, c) muscle fibres (Fig. 2). A check was made to ensure that variation of sarcomere lengths and A-zone lengths within single muscle fibres was not great enough to invalidate comparisons between fibres. Ten fibres were selected at random from muscles 137, 135c, and 135b. For each fibre, seven individual sarcomeres were measured. A table for simple analysis of variance was then constructed; the resulting F ratios, which ranged from 7-O (for muscle 137) to 100 (for muscle 135b) all indicated significance at the 1 per cent level (9 and 60 degrees of freedom). Thus, although some variation in sarcomere length occurs within fibres, it is small compared with the differences between different fibres. The same type of analysis was performed for measurements of A-zones obtained from electron micrographs, with similar results. Mitochondria As reported by previous workers (SMIT et al., 1967), a high mitochondrial content was observed in the pink coxal muscles (135a, c). The mitochondria sometimes extended along the muscle fibre for several sarcomeres, as in other insect flight muscles (Fig. 2). In some cases, mitochondria appeared to be joined end-toend. Up to 35 per cent of the cross-sectional area of fibres from these muscles was composed of mitochondria (Fig. 3). By contrast, the mitochondria in fibres of the central part of the tibia1 extensor muscle and in the white coxal muscles (136, 137) were smaller in size and fewer in number. They occupied usually less than 10 per cent of the cross-sectional area of transverse sections of these fibres (Fig. 3). Myo$brils All of the fibres from muscles 135 (a, c), 136, 137, and the tibia1 extensor exhibited strap-shaped myofibrils, as in other cockroach leg muscles (HAGOPIAN, 1966). Sarcoplasmic reticulum was prominent between adjacent myofibrils. Diadic and triadic contacts were observed in the region of the A-zone, usually near its ends (Figs. l-3). Within each sarcomere, two to four such contacts usually were present between adjacent myofibrils. T-tubules were observed to invaginate from the surface near the ends of the A-zones (c$. HAGOPIANand SPIRO, 1967). The fibres of muscle 135b included in our sample, presented an entirely different

picture. More than one type of fibre appeared in the sample. In one type (Fig. 4), the sarcoplasmic reticulum and diads were relatively reduced (cf. USHERWOOD, 1967; COCHRANEet al., 1969). Numerous tubules, invaginating from the surface near the Z-line, together with basement membrane material, formed transversely T-tubules arising from these invaginations were seen oriented invaginations.

Fro. 1. Longitudinal sections of a fibre in coxal muscle 136 (inset), showing I, A, and H zones; and of a fibre from the central part of the extensor tibiae muscle at higher magnification, to show sarcoplasmic reticulum (SR) between the myofilaments, and diads (D) within the A zones. Z lines, and I, A, and H zones, are indicated. Scale mark, 1/~.

Fro. 2. Longitudinal sections through two muscle fibres of coxal muscle 135c. In A, large mitochondria can be seen between the myofibrils. Diads near the ends of the A zones are marked by arrows. SR, sarcoplasmic reticulum; T, triad; TR, tracheole. Scale mark, 1/z.

FIG. 3. Transverse sections through fibres of muscles 137 (A) and 135c (B), showing similarity in shape of the myofibrils, and differences in mitochondrial (M) content and in myofilament arrangement (insets). Diads (D) commonly occur in both muscles near the ends of the A zones of the myofibrils. Scale mark, 1/z. Insets are enlargements of approximately 4 x and 6 x (A) and 4 x and 6 × (B) to show myofilament arrays.

FIc. 4. Transverse sections through two muscle fibres in 135b. A, B: Fibre with very sparse sarcoplasmic reticulum and few diads (small arrows), with extensive sarcolemmal invaginations (large arrows), especially near the Z lines (Z). C ; Fibre with n~wnerous small but incompletely separated myofibrils (M) and frequent diads (small arrows). Note the lack of strap-shaped myofibrils in these muscles. Scale mark, 1/z,

COCKROACH LEGMUSCLE PIBRES

2259

running longitudinally between myofibrils. Myofibrils were irregular in shape, rather than strap-shaped. In another type of fibre, myofibrils were small and numerous, and both sarcoplasmic reticulum and diads well represented (Fig. 4). Clearly, muscle 135b is quite heterogeneous in its fibre composition, as suggested by the sarcomere measurements (Table 1). Myojilaments An interesting difference in myoiilament arrangement was observed in comparisons of muscle 135 (a, c) with muscles 136,137, and the tibia1 extensor. In the last three muscles, the array of thin filaments around thick ones in the A-zone (Fig. 3A) is similar to that observed in other cockroach leg muscles (HAGOPIAN, 1966) and in insect visceral muscles (SMITH et al., 1966). Each thick filament is surrounded by twelve thin ones, the ratio of thin : thick filaments being 6 : 1. In muscle 135 (a, c), the myofilament arrangement (Fig. 3B) resembles that of other insect flight muscles (HUXLEYand HANSON, 1957) and of crustacean ‘fast’ abdominal muscles (JAHROMI and ATWOOD, 1967). Each thick filament is surrounded by six thin filaments in the A-zone, and each thin filament is equidistant from two adjacent thick filaments. The ratio of thin : thick filaments is 3 : 1. In muscle 135b, the myofilament arrangement in the fibres we sampled was similar to that in muscles 136 and 137, though in some cases the thin : thick filament ratio appeared to be slightly greater for the 135b fibres. DISCUSSION The results of the present investigation are of particular interest when taken in conjunction with the measurements of USHERWOOD (1962) on tension output of the cockroach coxal muscles. The observations of JAHROMIand ATWOOD(1967, 1969) on structural and contractile properties of crayfish and lobster abdominal extensor muscles are also relevant. A summary of these studies is provided in Table 3. The cockroach muscles 135 (a, c), 136, and 137 have a similar average sarcomere length and a similar overlap of thin and thick filaments within the sarcomere. (Statistically, the values are significantly smaller for the pink muscles, but the amount of the difference is slight.) The ratio of thin to thick myofilaments in the A-zone in fibres of muscle 136 and 137 is twice that in muscles 135a and 135~. The number of thick filaments per $ cross-section is slightly greater in the latter muscles. The speed of contraction and the peak twitch tension are roughly the same in these muscles. The agreement is even closer if the latter values are adjusted for the differences in mitochondrial content by increasing the value for muscle 135 by 30 per cent and that for muscle 137 (and 136) by 5 per cent. The preliminary conclusion to be drawn from these data is that the higher ratio of thin filaments in muscles 136 and 137 does not confer a different speed of contraction nor the ability to develop significantly more tension. In terms of the sliding filament hypothesis, (HUXLEY and NIEDERGERKE,1954), it is possible that the

* From t From

(1962).

ATWOOD

and

USHBRWOOD

9

JAHROMI

Lobster : Superficial (tonic) abdominal extensors t

4

3.7

Cockroach: Coxal muscle 13%

Lobster : Deep (phasic) abdominal extensors t

4.2

0

6

3

2.5

3.2

OL)

Average overlap of thick and thin filaments within sarcomeres near rest length

OF STRUCTURAL

(1969).

Average sarcomere length

3--COMPARISON

Cockroach : Coxal muscle 137

Muscle

TABLE

CONTRACTILE

2

2-4

2-4

2-4

Diads/ sarcomere between adjacent myofibrils

AND

6:l

3:l

3:l

6:l

Ratio of thin to thick myofilaments

FEATURES

IN

350400

450-600

650

600

Thick filaments/ pa crosssection

MUSCLES

AND

0.85 *

Peak tension

5-14 kg/cm8 (TEA spike)

0.8-l *4 kg/cm2 (TEA spike)

0.70 * kg/cm2 (twitch)

kg/cm (twitch)

LOBSTER

Slow (peak twitch time for TEA spike, 400 msec)

Fast (Peak twitch time 50 msec)

Fast * (Peak twitch time 10 msec)

Fast * (Peak twitch time 13 msec)

Contraction speed

OF COCKROACH

COCKROACH LEG MUSCLEF1RRF.S

2261

number of cross-bridges per unit length of the thick filaments is the same for both types of muscles. This would require that each thin filament form (on the average) only half the number of cross-connexions per unit length in muscles 136 and 137. This preliminary conclusion requires further testing through more accurate tension measurements (preferably on single muscle fibres). In the crustacean abdominal extensor muscle, the myofilament arrangements in deep and superficial fibres resemble those of the cockroach muscles, 135~ and 137, respectively. In addition, the general organization of the sarcomere is similar for all four muscles: in particular, two to four diadic contacts are usually evident between myofibrils in the region of the A-zone in longitudinal sections. However, in the crustacean superficial extensor muscle fibres, the sarcomeres and the zone of overlap between thick and thin filaments both average about twice that of the other muscles. The physiological performance of these muscle fibres is strikingly different from the others as well: they contract much more slowly, and develop a greater peak tension. In the experiments quoted in Table 3, total tension was measured during the occurrence of a prolonged spike set up in tetraethylammonium chloride (TEA) solution. The conclusion from this qualitative survey is that, in arthropod skeletal muscle fibres, the length of the sarcomere and the extent of overlap of thin and thick myofilaments are more important in determining contractile properties, than is the ratio of thin : thick myofilaments, or the absolute number of thick filaments per unit area of cross-section (cf. R~~EGG,1968). The relationship between sarcomere length, contraction speed, and total tension is in qualitative agreement with that proposed by HUXLEY and NIEDERGERKE (1954). A further point of interest arising from the present investigation concerns the long-sarcomere fibres in coxal muscle 135b and in the proximal part of the tibia1 extensor muscle. Both muscles receive innervation from ‘slow’ motor axons, whereas muscles 135a, 135c, 136, and 137 do not (USHERWOOD, 1962). By analogy with certain crustacean long-sarcomere are preferentially work is needed

fibres

muscles (which

innervated to verify

(ATWOOD, presumably by

the

‘slow’

1965),

one might

contract motor

more axons.

expect

slowly

to find that the

than

Further

the others)

physiological

this proposal.

Acknowledgements-This study was supported by grants from The National Research Council of Canada and The Muscular Dystrophy Association of Canada.

REFERENCES ATWOODH. L. (1965) Excitation and inhibition in crab muscle fibres. Comp. Biochem. Physiol. 16, 409-426. BECHT G. and DRIZSDEN D. (1956) Physiology of the locomotory muscles in the cockroach. Nature, Lond. 177, 836. CARRONELLC. S. (1947) The thoracic muscles of the cockroach Periplaneta americana L. Smithson. misc. Coll. 107, l-23. COCHRANED. G., ELDER H. Y., and USHERWOODP. N. R. (1969) Electrical, mechanical and ultrastructural properties of tonic and phasic muscle fibres in the locust (Schistocerca gregaria). J. Physiol. 200, 68P-69P.

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S. S. JAHROMIand H. L. ATWOOD

HAGOPIANM. (1966) The myofilament arrangement in the femoral muscle of the cockroach, Leucophaea moderae Fabricius. r. Cell Biol. 28, 545-562. HAGOPIANM. and SPIRO D. (1967) The sarcoplasmic reticulum and its association with the T system in an insect. r. Cell Biol. 32, 535-545. HOYLE G. (1965) Neural control of skeletal muscle. In Physiology of Insecta, (Ed. by ROCKSTEIN,M.) 2, 203-232. Academic Press, New York. HUXLEY A. F. and NIEDERGERKE R. (1954) Interference microscopy of living muscle fibres. Nature, Land. 173, 971-973. HUXLEY H. E. and HANSON J. (1957) Preliminary observations on the structure of an insect flight muscle. In Electron Microscopy, Proceedings of the Stockholm Conference of 1956, pp. 202-203. Ahnqvist Sz Wiksell, Stockholm. JAHROMIS. S. and ATWOODH. L. (1967) Ultrastructural features of crayfish phasic and tonic muscle fibres. Can. J. Zool. 45, 601-606. JAHROMIS. S. and ATWOODH. L. (1969) Correlation of structure, speed of contraction, and total tension in fast and slow abdominal muscle fibres of the lobster (Homarusamericanus). J. exp. Zool. In press. &WNOLDS E. S. (1963) The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J. Cell Biol. 17, 208-212. R~~EGGJ. C. (1968) Contractile mechanisms of smooth muscle. Symp. Sot. exp. Biol. 22, 45-66. SMITH D. S., GUPTA B. L., and SMITH U. (1966) The organization and myofilament array of insect visceral muscles. J, Cell Sci. 1, 49-57. SMIT W. A., BECHT G., and BEENAKKERS A. M. T. (1967) Structure, fatigue, and enzyme activities in ‘fast’ insect muscles. J. Insect Physiol. 13, 1857-1868. USHERWOODP. N. R. (1962) The nature of ‘slow ’ and ‘fast’ contractions in the coxal muscles of the cockroach. J. Insect Physiol. 8, 31-52. USHERWOOD P. N. R. (1967) Insect neuromuscular mechanisms. Am. Zool. 7, 553-582.