J. Cell Set. 8, 413-425 (i970 413Printed in Great Britain

THE CONTRACTILE MECHANISM IN

OBLIQUELY STRIATED BODY WALL MUSCLE

OF THE EARTHWORM, LUMBRICUS

TERRESTRIS

M. F. KNAPPAND P. J. MILLDepartment of Zoology, University of Leeds, England

SUMMARY

Obliquely striated muscle fibres from the longitudinal and circular layers of the body wallof the earthworm were prepared in extended and contracted states for study in the electronmicroscope.

Contracted fibres differ from extended ones in the following respects: (i) the I-bands arenarrower, (ii) the A-bands are wider, and (iii) there are more rows of thick myofilaments in eachA-band.

The arrangement of the thick and thin myofilaments in interdigitating arrays and the occur-rence of cross-links between the 2 types of myofilament indicate a classical sliding-filamentmechanism of contraction as in cross-striated muscle, resulting in a reduction in the I-bandwidth.

The increase in the A-band width could be due to a moving apart of the myofilaments duringcontraction to preserve constant volume of the lattice.

The third change, the increase in the number of rows of thick myofilaments in the A-band,can be explained only by a shearing of these filaments past one another in such a way as to in-crease the amount of their overlap.

The role of the sliding-filament and shearing contraction mechanisms in bringing about thechanges observed in earthworm muscle fibres is considered and the possible correlation of thesemechanisms with certain physiological data is discussed. The function of the sarcoplasmicreticulum in the transmission of impulses to the interior of the fibre and/or in the control ofthe contraction mechanism is also discussed.

INTRODUCTION

A description of the arrangement of contractile elements in the obliquely striatedbody-wall muscle of the earthworm, Lumbricus terrestris, has been presented in aprevious paper (Mill & Knapp, 1970). Thick and thin myofilaments lie parallel to thelongitudinal axis of the fibre and interdigitate in much the same way as in cross-striated muscle, producing A-bands, I-bands and H-lines. The thin filaments areattached to transversely oriented rod-shaped structures called Z-rods. In cross-striated muscle the Z-line material tends to be in the form of a disk, thus giving a 3-dimensional array of thin and thick myofilaments; but in the earthworm muscle theZ-line is only a thin rod from which very few rows of thin filaments arise, giving asheet-like array. Furthermore, each array in the earthworm is displaced along thelongitudinal axis of the fibre with respect to the next one, so that the A- and I-bandsfollow a diagonal course and so give rise to oblique striations. These striations make an

414 M. F. Knapp and P. J. Mill

angle of 50 to the longitudinal axis in relaxed muscle, and this increases to 300 incontracted muscle (Hanson, 1957).

Ikemoto (1963), working on the earthworm Eisenia foetida, came to similar con-clusions to those outlined above on the structure of the body-wall muscles. He sug-gested that the thin filaments contract, by coiling, in the I-band region (F-region ofIkemoto) and that the resultant tension is applied to one point towards either end ofeach thick filament. This causes a change in the shape of the latter from a shallowS-shape to a more marked one. Consequently the thick filaments separate and shear,apparently without any sliding of the filaments past one another in the conventionalmanner of cross-striated muscle (Huxley & Hanson, 1954).

Straubesand & Kersting (1964), on the other hand, state that the thick and the thincontractile elements, which they named fibrils and filaments respectively, extend thefull length of the fibre; and argued that the fibrils are not homogeneous, but that theyare composed of filaments packed closely together. They noted that the A-bands(B-lamellae of Straubesand & Kersting) become more prominent in contracted muscleand suggested that this is the result of the aggregation of filaments into fibrils, and thatrelaxation is brought about by the reverse process.

In a paper on the translucent part of the adductor muscle of the oyster Crassostreaangulata, Hanson & Lowy (1961) concluded that the oblique striations are due to 2kinds of myofilaments arranged as in cross-striated muscle, but with the A- and I-bandsat an angle of io° to the fibre axis. They suggested that contraction is brought aboutby the filaments sliding past one another.

Other investigations on obliquely striated muscles have been carried out byRosenbluth (1965, 1967) on the nematode Ascaris and on the polychaete Glycera(Rosenbluth, 1968), and by Heumann & Zebe (1967) on Lumbricus terrestris. Rosen-bluth (1967) argued that contraction is by means of a sliding filament mechanism,but that in addition there is a second contraction mechanism which involves the'shearing' of thick filaments past one another with some degree of separation in theirmiddle region. Heumann & Zebe (1967) came to a similar conclusion for Lumbricus.

In view of the varied interpretations of the contractile mechanism the presentinvestigation was undertaken to attempt to resolve this problem in the body-wallmuscles of the earthworm, Lumbricus terrestris.

MATERIAL AND METHODSExtended preparations of the body-wall muscles of Lumbricus terrestris were obtained as

described in a previous paper (Mill & Knapp, 1970). To prepare body-wall muscles in a con-tracted state the animal was dropped directly into the fixative (25 % glutaraldehyde bufferedwith sodium cacodylate). This was sufficient to cause immediate contraction of the muscles.Pieces of body wall were removed and placed in fresh fixative for 1 h. Both extended and con-tracted muscle preparations were washed in cacodylate buffer. This was followed by post-fixation in osmium tetroxide (buffered with veronal-acetate buffer) for 1 h. They were thenwashed in veronal-acetate buffer, dehydrated in an ethanol series and embedded in Epon.Sections were cut on a Huxley microtome, mounted on coated grids and examined in anAEI EM6B electron microscope. Contrast was improved by staining the sections in uranylacetate and lead citrate.

Contractile mechanism of earthworm muscle 415

RESULTS

It is clear from an earlier paper in which the fine structure of earthworm musclefibres has been described (Mill & Knapp, 1970) that, in practice, it is not possible touse longitudinal sections for comparative studies of obliquely striated muscle fibres.The arrangement of the myofilament arrays, in thin, plate-like sarcomeres, is such thatuseful information can be obtained only from truly longitudinal sections which areexactly in the xz or yz planes. These are very difficult to obtain and, furthermore, thereare no constant reference points from which it can be established whether or notdifferent sections are in the same plane. Thus, most of the information on the differ-ences between extended and contracted muscle fibres has been obtained from trans-verse sections.

No differences have been observed between contracted and extended muscles ineither the diameter of the thick and thin myofilaments or in the dimensions of theZ-rods. However, transverse sections of muscle fibres in these 2 states do differstrikingly from one another, in both the number of rows of thick myofilaments in theA-bands and in the relative widths of the A- and I-bands. In extended muscle theA-band is composed of 3-4 rows of thick myofilaments (Fig. 2), compared with 5-9rows in contracted muscle (Fig. 3). In the former the thick myofilaments in the A-band are 20-60 nm apart. In contracted fibres a greater separation is often found,particularly in the centre of the A-band where the myofilaments may be separatedby some 60-100 nm, although near the edges of the bands the distance between myo-filaments is usually similar to that found in extended fibres. Consequently, in con-tracted fibres the middle of the A-band often appears less dense than the rest of theband, giving rise to an impression of an H-line. Both in extended and contractedmuscle, thin myofilaments may either be present or absent in the mid-region of theA-band (compare Fig. 3 A and B) and, therefore, the occurrence of an H-line does notindicate the state of contraction of the muscle fibre, as in cross-striated muscles.Rosenbluth (1968) likewise noted the variable occurrence of the H-line in the poly-chaete Glycera,

In extended muscle fibres the A- and I-bands are distinct and approximately equalin width (Fig. 2), whereas in the contracted state the A-bands are the more prominentand may extend from one Z-rod to the next, so that the I-band is almost entirelyeliminated (Fig. 3). There is considerable variation in the distance between adjacentZ-rods (Z-Z) and consequently in the widths of the A- and I-bands, both in extendedand in contracted fibres. Thus, in order to carry out a comparative study of the fibresin the 2 states, the ratios of the band widths to the Z-Z distances (A/Z-Z and I/Z— Z)were calculated, and also the I-band/A-band ratio (I/A).

The Z-Z distance, A-band width and I-band width (i.e. the total width of the 2 halfI-bands enclosed between 2 adjacent Z-rods) were measured and the ratios (I/A,A/Z-Z and I/Z-Z) calculated for both longitudinal and circular muscle fibres. Sincethere may be a difference between the 2 muscle layers (either intrinsic or introducedby the methods utilized to obtain extended and contracted fibres) a statistical analysiswas initially carried out on the data from these 2 layers. In extended fibres no

416 M. F. Knapp and P. J. Mill

differences in Z— Z distance or in A- and I-band widths were found between thelongitudinal and circular muscle layers, but the ratios I/A, A/Z-Z and I/Z—Z differedsignificantly (Table i). There were also significant differences between contractedlongitudinal and circular muscles in Z-Z distance, and A- and I-band widths, althoughin this case the ratios I/A, A/Z-Z and I/Z-Z did not differ (Table i).

Table i. A comparison of transverse sections of longitudinal and circular musclefibres in (a) the extended and (b) the contracted state

Z-Z distance

Extended

A-band width

-v- (fim)

I-band width

.v (/an)

Longitudinal 0-41 ± 0-022 > 0 7Circular 0-42 ±0-015

I/A

O-I9 ± O'OII > O-2O-2I ±OOO9

A/Z-Z

Ratio P Ratio PLongitudinal 1-2010-045 < 002 046 ±0011 < 005Circular I-OI ±0-055 0-5010013

O-22±OOI2 > 0-6

o-2i ±0009

I/Z-Z( -̂

Ratio P053 ±0010 < 0050-49 ±0014

Contracted

Z-Z distance

x (/(m)

A-band width

x (fim)

I-band width

.v (/tm)

LongitudinalCircular

LongitudinalCircular

043 ± 0 0 2 0 < O-Oi 0-32 + 0016 < 0010-57 ±0-026 044 ±0026

I/A A/Z-Z

Ratio P Ratio P035 ±0022 > 09 075 ±0014 > 0-7033 ±0043 0-7610023

o n ±0006 < ooi0-1610014

I/Z-Z( • \

Ratio P025 ±0012 > 0 70-24 ±0-023

All distances were measured in transverse sections and values are stated ± s.E.Significant P values are in bold type.

In view of these differences the 2 layers are treated separately for the purpose ofcomparing extended with contracted muscle. The results are set out in Table 2. Incontracted fibres the A-band is wider than in extended ones (P = <̂ o -ooi) and the I-band narrower (longitudinal, P = < o-ooi; circular, P = < o-oi). The A- and I-bands are approximately equal in width in extended muscle (I/A ratio for longitudinal,1-20; and for circular, I-OI), although the ratio does show considerable variation. Incontrast, in the contracted state, the I-band is only about one third the width of theA-band (I/A ratio for longitudinal, 0-35; for circular, 0-33). Since, in the longitudinalmuscle layer, the Z-Z distance is approximately the same in extended and contractedfibres, the changes in the A- and I-band widths are reflected in the A/Z-Z and I/Z-Zratios, with the former increasing in value in contracted muscle (P = <̂ o-ooi) andthe latter decreasing (longitudinal, P = <^ o-ooi; circular, P = 4. o-ooi). In circularmuscle, despite a significant increase in Z—Z distance in contracted muscle, the in-crease in the width of the A-band is sufficient for the A/Z-Z ratio to be significantly

Contractile mechanism of earthworm muscle 417

larger in contracted than extended muscle fibres (P = -̂ o-ooi). It is these differ-ences that produce a significant difference in the I/A ratio (P = <̂ o-ooi).

From longitudinal sections it was seen that thick and thin myofilaments are approxi-mately parallel to the longitudinal axis of the fibres in both extended and contractedfibres, and are generally more or less straight. It should also be noted that the ends ofthe thick myofilaments have not been observed to extend beyond the Z-material incontracted fibres, and so do not change their actin partners as may be the case insmooth muscle (Riiegg, 1968).

Table 2. A comparison of transverse sections of extended and contracted muscle fibresfrom (a) the longitudinal and (b) the circular muscle layer

Longitudinal

ExtendedContracted

ExtendedContracted

Circular

ExtendedContracted

ExtendedContracted

Z-Z distance

041 ±0022043 ± 0020

I/A

Ratioi-2o± 0045O-35±OO22

P

> 0-5

P<£ OOOI

Z-Z distance

.v (/tm)

0-42 ±0-015o-57±o-026

I/AA

Ratio

I-OI ±0055o-33±o-O43

p

< OOOI

p<£ OOOI

A-band width

x(fim)

o-i9±o-on <032 ±0016

A/Z-Z

Ratio

046 ±0011 <0-75 ±0-014

P

: 0001

Pt OOOI

A-band width

x(fim) p

0-21 ± 0 0 0 9 <g OOOI

o-44± 0-026

A/Z-ZA

Ratio

0-50 ±0-013 <076 ±0-023

pi OOOI

I-band widtht

x (fim)

0 2 2 ± O O I 2

on ±0006

I/Z-Z

Ratio

0 5 3 ± 0 0 1 00 2 5 ± 0 0 1 2

P

< OOOI

p<( OOOI

I-band widthA

x (/tm)

O-2I ±0009

O i 6 ± o - o i 4

I/Z-ZA

Ratio

0-49 ± 0-0140-24 + 0-023

p

< 001

p<3 OOOI

All distances were measured in transverse sections and values are stated ± S.E.Significant P values are in bold type.

DISCUSSION

In obliquely striated muscles thick and thin myofilaments overlap one another as incross-striated muscle, and cross-links have been seen between the 2 types of filament(Mill & Knapp, 1970). In addition, the I-band is much reduced in contracted fibres,so that it seems likely that this muscle contracts by means of the sliding-filamentmechanism. Fig. 1 is a schematic diagram of the muscle in the xz plane, showing thethick myofilaments and the Z-rods; the relative positions of these elements in therelaxed state are represented in A, and after sliding-filament contraction in B. It isclear that this mechanism of contraction would result in a reduction in the width ofthe I-band in both transverse and longitudinal planes.

27 CEL 8

4 i8 M. F. Knapp and P. J. Mill

By investigations using X-ray diffraction techniques it has been demonstrated thatin cross-striated muscles the filament lattice maintains a constant volume duringchanges between the relaxed and contracted states and that as a consequence themyofilaments are further apart in contracted than in relaxed muscle (Elliott, Lowy &Worthington, 1963). There is no reason to believe that similar changes do not occurin obliquely striated muscles; indeed, in the present investigation, increased separa-tion of thick myofilaments has been observed in the centre of the A-band in con-tracted muscle fibres. Such changes would be accompanied both by an increase in theangle of the A- and I-bands to the longitudinal fibre axis and, in transverse sections,

Fig. 1. This diagram demonstrates the changes which occur in the xz plane as aresult of the contraction of an obliquely striated muscle fibre, A, relaxed fibre; B,fibre contracted by the sliding filament mechanism; c, fibre contracted by the shearingmechanism. The thick vertical lines represent thick myofilaments and the dotsrepresent sections through Z-rods. There is the same number of myofilaments ineach of the 3 figures and the middle myofilament in the central row is in the equi-valent position in each case (marked by the horizontal line). The angle of the stria-tions is exaggerated for clarity. The inset shows the angle of the striations, which isthe same in A and B but is increased with respect to the longitudinal fibre axis in c.Changes in the position of the myofilaments due to the maintenance of constantvolume of the filament lattice have not been represented in this diagram (see text).

Contractile mechanism of earthworm muscle 419

by increases in A- and I-band widths (although the increase in I-band width wouldprobably be more than compensated for by the reduction in the width of this band dueto sliding-filament contraction).

There is one change observed in contracted muscles which is not accounted foreither by the sliding-filament mechanism of contraction or by the changes due tomaintenance of constant volume, namely, the greater number of rows of thick myo-filaments in each A-band. This must be caused by the thick myofilaments shearingpast one another and so increasing their overlap (Heumann & Zebe, 1967; Rosenbluth,1967). The changes in the number of rows is sufficiently consistent to suggest thatshearing always occurs during contraction of obliquely striated muscles (in additionto sliding of the thick and thin filaments with respect to one another); and that ashearing mechanism is an integral part of the contraction process. In order to empha-size the differences between this and the sliding-filament mechanism, the afore-mentioned changes in the separation of myofilaments due to maintenance of constantvolume have not been represented in Fig. 1. Fig. i c shows the arrangement of thethick myofilaments after shearing, as compared with the relaxed state (Fig. 1 A), anddemonstrates that some of the changes which result from shearing would reinforce someof those which must occur if constant volume is maintained; namely, the increase inthe angle of the A- and I-bands to the longitudinal fibre axis, and the increase inA- and I-band widths in the transverse plane. It seems unlikely that the increasein the angle of striation from 50 in extended muscle to 30° in contracted (Hanson,1957) could be explained entirely by the sliding-filament/constant-volume process.From our figures it looks as though there is an increase in the separation of thethick myofilaments of at most about 100%, whereas calculations indicate an increaseconsiderably greater than this even for a change of angle from 10 to 250. In con-tracted muscle, a reduction in transverse distance between Z-rods would be expectedif contraction were by means of the sliding-filament mechanism alone, but bothmaintenance of constant volume and shearing would result in an increase in thisdistance. The Z-Z distance remained more or less constant in longitudinal fibres, buta significant increase was found in contracted circular fibres.

Drs J. M. Gillis and J. Van Den Bosshe (personal communication) have found, usinginterference microscopy, that in glycerinated muscle fibres from the body wall ofLumbricus contraction involves 2 consecutive phases. In the first the I-band disappearsand there is no change in the angle of striation; in the second, striations reappearand both the width of the bands and their angle with respect to the longitudinal axis ofthe fibre steadily increase. This implies that contraction involves a sliding mechanismat first, followed by shearing of the thick myofilaments.

In response to direct electrical stimulation with pulse durations of up to at least25 ms, earthworm muscle contracts phasically. With a stimulus duration of 50 ms ormore the muscle relaxes with a similar time course, but when the tension has fallen toa value of 20-30 % of its peak value the remaining tension is maintained for severalseconds to give a sustained contraction (Hidaka, Kuriyama & Yamamoto, 1969).Contractions with similar phasic and tonic components were obtained by Budington(1902) in response to stimulation with an induction coil. In this case greater stimulus

27-2

420 M. F. Knapp and P. J. Mill

strength caused a relative increase in the tonic component.The contraction mechanismsdiscussed above could provide an explanation of the phasic and tonic components ofcontraction if, initially, stimulation activates the sliding-filament mechanism, untilmaximal contraction by this mechanism has been attained, while after more pro-longed stimulation, the shearing mechanism is brought into play, thus agreeing withthe observations of Gillis. This differs from Rosenbluth's idea that the early activephase of contraction involves both sliding-filament and shearing mechanisms.Rosenbluth also suggested that externally applied stress might then remove the thinfilaments, so that cross-links could form between adjacent thick filaments and themuscle be reset at the new length (Rosenbluth, 1967).

Ikemoto's observation that in contracted muscle fibres of Eisenia the thinmyofilaments coil in the I-band region (Ikemoto, 1963) has not been confirmed inLumbricus. Ikemoto used different techniques from those used in this paper for thepreparation of extended and contracted muscle. In addition his extended musclefibres were glycerinated before fixation in osmium tetroxide, whereas his contractedones were not. This may be the reason for the differences observed by him in theappearance of the thin filaments in the 2 states (Heumann & Zebe, 1967).

In a muscle fibre in which shearing occurs there need be no relation between thedegree of overlap between thick and thin filaments and the degree of stagger of adja-cent thick filaments (Rosenbluth, 1967). Consequently length and tension could bedissociated from one another in these fibres. However, if shearing is accompaniedby some form of linkage (or some other mechanism for increasing tension) this couldbe expected to contribute to the total tension developed by the muscle and there wouldbe a length/tension relationship, although this may well be different from that obtainedin a muscle which contracts by the sliding-filament mechanism alone. Experiments onthe mechanical properties of a muscle of this type might help to elucidate this point.

Since shearing provides an additional mechanism for shortening, obliquely striatedmuscle is capable of a greater range of length change than cross-striated muscle(Rosenbluth, 1967). It is of interest to note that locomotory muscles which functionby acting on a rigid skeleton (as in vertebrates and arthropods) are cross-striated,whereas obliquely striated muscles seem to be restricted to animals without a rigidskeleton and in which locomotion is brought about by the action of muscles on ahydroskeleton (e.g. nematodes and annelids) or on a contractile chamber (cephalopodmantle), and consequently considerable length changes are incurred.

It has previously been suggested that tension developed in individual muscle fibresmaybe transmitted byway of peripheral fibrillar bundles to the surrounding connec-tive tissue (Rosenbluth, 1967; Mill & Knapp, 1970). If this is so it is presumablyimportant that the latter should remain reasonably taut. However, large variationsoccur in both the diameter and length of the earthworm. Mill & Knapp (1970)described single muscle fibres which are situated in the connective tissue of the bodywall. The function of these could be to take up the slack in the connective tissue. Itmay well be significant that the orientation of these fibres is perpendicular to the longaxis of the fibres in the muscle layer in which they are found. Possibly the transverselyoriented fibres in the longitudinal muscle layer contract when the diameter of the

Contractile mechanism of earthworm muscle 421

worm decreases during relaxation of the longitudinal muscles (and contraction of thecircular ones) and conversely, the longitudinally oriented fibres in the circular musclelayer contract when the circular muscles relax. It must be emphasized that this ispurely speculative as it is not known whether the contraction of these single musclefibres is in or out of phase with the fibres of the muscle layer in which they are found.

Sarcoplasmic reticulum

In cross-striated muscle it is generally thought that the T-system is concerned withthe conduction of electrical excitation into the centre of the fibre. The sarcoplasmicreticulum (SR) on the other hand, is thought to be intimately involved in the controlof the contraction process and is capable of concentrating and releasing calcium(Ebashi, 1961) and of releasing a relaxing factor (Nagai, Makinose &Hasselbach, i960;Muscatello, Andersson-Cedergren, Azzone & von der Decken, 1961). The T-systemis absent in annelid obliquely striated muscles and the SR is in the form of a systemof transverse tubules and peripheral vesicles (Heumann & Zebe, 1967; Mill & Knapp,1970). The role of the SR in these muscles is not clear. Rosenbluth (1968) put for-ward the idea that in Glycera the limiting membranes of the SR may be electricallycoupled to the sarcolemma and could act as capacitors capable of conveying rapidchanges in potential from the cell surface into the sarcotubules. Pucci & Afzelius(1962) and Rohlich (1962), working on leech, likewise suggest that the function of thetubules is electrical conduction and, in view of the absence of a T-system in thesemuscles, this view cannot be completely excluded at the present. Thus, although, inother muscles which do not have a T-system (obliquely striated oyster adductormuscle and vertebrate 'slow' muscle) the development of tension is slow, the obliquelystriated muscles of annelids are capable of quite rapid contractions. Furthermore, thearrangement of the tubules in Lumbricus, Glycera and Hirudo is such that no myofila-ment is more than 0-5 /«m from a tubule. However, the theory is dependent on themembranes concerned being electrically coupled at the dyad, which is situated betweenthe peripheral vesicles and the sarcolemma (Heumann & Zebe, 1967; Mill & Knapp,1970), and upon the ability of the unilaminar membrane of the tubular system to pro-pagate electrical excitation. In muscles with both a T-system and SR there is no evidenceto suggest that action potentials are transferred across the dyads or triads from theextracellular T-system membranes to the intracellular, unilaminar membranes of theSR. Present evidence is insufficient to establish whether or not the annelid SR isconcerned with the conduction of electrical excitation into the interior of the musclefibre.

Other functions attributed to the SR in annelids are concerned with the transfer ofchemicals across membranes. Ikemoto (1963) considers that exchange occurs betweenthe peripheral vesicles and the surface protoplasm, material being taken into thevesicles and waste products being discharged from them. Heumann & Zebe (1967)have demonstrated that the SR is capable of actively absorbing calcium in the presenceof ATP, as can the SR of vertebrate striated muscle (Ebashi, 1961). Hidaka, Ito,Kuriyama & Tashiro (1969) postulated that muscle action potentials are generated bymovement of calcium ions into the fibres. If contraction in annelid muscles at the

422 M. F. Knapp and P. J. Mill

molecular level is in any way similar to that in cross-striated muscles, as seems likely,then it is possible that the SR in the 2 types of muscle functions in the same way.However, further physiological and biochemical evidence is required before specificfunctions can be attributed to the sarcoplasmic reticulum in annelid muscle.

We would like to thank Professor G. F. Elliott for his valuable criticism and suggestionsregarding this work; also Drs J. M. Gillis and J. Van Den Bosshe for sending us informationon their unpublished observations.

REFERENCES

BUDINGTON, R. A. (1902). Some physiological characteristics of annelid muscle. Am. J. Physiol.7, I55-I79-

EBASHI, S. (1961). Calcium binding activity of vesicular relaxing factor. J. Bioctiem., TokyoSO, 236-244.

ELLIOTT, G. F., LOWY, J. & WORTHINGTON, C. R. (1963). An X-ray and light-diffraction studyof the filament lattice of striated muscle in the living state and in rigor. J. molec. Biol. 6,295-3O5-

HANSON, J. (1957). The structure of the smooth muscle fibres in the body wall of the earth-worm. J. biophys. biochem. Cytol. 3, m-121 .

HANSON, J. & LOWY, J. (1961). The structure of the muscle fibres in the translucent part of theadductor of the osyter, Crassostrea angulata. Proc. R. Soc. B 154, 173-196.

HEUMANN, H.-G. & ZEBE, E. (1967). t)ber Feinbau und Funktionweise der Fasern aus demHautmuskelschlauch des Regenwurms, Lumbricus terrestris L. Z. Zellforsch. mikrosk. Anat.78, 131-150.

HIDAKA, T., ITO, Y., KURIYAMA, H. & TASHIRO, N. (1969). Effects of various ions on theresting and active membrane of the somatic muscle of the earthworm. J. exp. Biol. 50,405-415.

HIDAKA, T., KURIYAMA, H. & YAMAMOTO, T. (1969). The mechanical properties of the longi-tudinal muscle in the earthworm. J. exp. Biol. 50, 431-443.

HUXLEY, H. E. & HANSON, J. (1954). Changes in the cross striations of muscle during con-traction and stretch and their structural interpretation. Nature, Lond. 173, 973.

IKEMOTO, N. (1963). Further studies in electronmicroscopic structures of the oblique-striatedmuscle of the earthworm Eiseniafoetida. Biol. J. Okayama Univ. 9, 81—126.

MILL, P. J. & KNAPP, M. F. (1970). The fine structure of obliquely striated body wall musclesin the earthworm Lumbricus terrestris Linn. J. Cell Sci. 7, 233-261.

MUSCATELLO, U., ANDERSSON-CEDERGREN, E., AZZONE, G. F. & DECKEN, A. VON DER (1961).The sarcotubular system of frog skeletal muscle: A morphological and biochemical study.J. biophys. biochem. Cytol. 10, Suppl., 201-208.

NAGAI, T., MAKINOSE, N. & HASSELBACH, W. (i960). Der physiologische Erschlaffungsfaktorund die Muskelgrana. Biochim. biophys. Acta 43, 223-238.

Pucci, I. & AFZELIUS, B. A. (1962). An electron microscope study of sarcotubules and relatedstructures in the leech muscle. J. Ultrastruct. Res. 7, 210-224.

RSHLICH, P. (1962). The fine structure of the muscle fibre of the leech, Hirudo medicinalis.J. Ultrastruct. Res. 7, 399-408.

ROSENBLUTH, J. (1965). Ultrastructural organisation of obliquely striated muscle fibres inAscaris lumbricoides. J. Cell Biol. 25, 495-515.

ROSENBLUTH, J. (1967). Obliquely striated muscle. III. Contraction mechanism of Ascarisbody muscle. J. Cell Biol. 34, 15-33.

ROSENBLUTH, J. (1968). Obliquely striated muscle. IV. Sarcoplasmic reticulum, contractileapparatus, and endomysium of the body muscle of a polychaete, Glycera, in relation to itsspeed, J. Cell Biol. 36, 245-259.

ROEGG, J. C. (1968). Contractile mechanisms of smooth muscle. Syvip. Soc. exp. Biol. 22,45-66.

STRAUBESAND, J. & KERSTING, K. H. (1964). Feinbau und Organisation des Muskelzellen desRegenwurms. Z. Zellforsch. mikrosk. Anat. 62, 416—442.

{Received 26 August 1970)

Contractile mechanism of earthworm muscle 423

ABBREVIATIONS ON PLATES

A A-band t tubule of sarcoplasmic reticulumH H-line v peripheral vesicle of sarcoplasmic reticulum/ I-band Z Z-rod

The scale on the micrographs is equivalent to 1 /tm.

424 M. F. Knapp and P. J. Mill

Fig. 2. A transverse section of an extended longitudinal muscle fibre. A- and I-bandsare distinct and approximately equal in width. There are 3-5 rows of thick myofila-ments in each A-band. x 27500.Fig. 3. Transverse sections of A a contracted longitudinal muscle fibre and B a con-tracted circular muscle fibre. The A-bands occupy most of the space between adjacentZ-rods, so that the I-bands are almost entirely eliminated. There are 6-9 rows of thickmyofilaments in each A-band. In A an H-line can be seen in the centre of the A-bandbut in B the H-line is absent. The presence or absence of an H-line is not a constantcharacteristic of fibres from either muscle layer, x 27 500.

Contractile mechanism of earthworm muscle 425

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