Mastophorus muris (Nematoda: Spirurina): Ultrastructure of somatic muscle development

MURZS (NEMATODA : SHRURTNA) : ULTRASTRUCTURE OF SOMATIC MUSCLE DEVELOPMENT GEORGE S. HAMADA* and GUTA WERT~EI~ Laboratory

of Helminthology,

The Hebrew University-Hadassah

Medical School, Jerusalem, Israel

(Received I2 September 1977) G. s. and WERTHEIM G. 19%. k'asfuplrauu~muris (Nematoda: Spirurina): ultrastructure of somatic muscle development. ~~ternfftiu~u~3uurnal for Parcsitolugy 8: 405-414. The ultrast~cture of the somatic muscle cells of the adult and six developmental stages of ~~~fop~or~.~ were studied. In all stages the cells consisted of a contractile region containing myofibrils separated by dense bands and a noncontractile region with nuclei, mitochondria, glycogen, lipid droplets and vesicles. Two sizes of myofilaments were present. The dense band contained T tubules and sarcoplasmic reticulum, and, in more advanced stages, support filaments, glycogen and dense bodies. The contractile region of the adult muscle cell consisted of several hundred irregularly shaped myofibrils arranged in a random pattern. This pattern of myofibrils was defined as irregular-coelomyarian. The third stage larva had a shallow-coelomyarian myofibril configuration, which changed to coelomyarian in the late third stage through the addition of new myofibrils at the apical contractile border. In the fourth stage larvae, the subdivision of existing myofibrils changed the pattern to irregular-

AbSfrBCt-HAMADA

coelomyarian.

INDEX KEYWORDS: ultrastructure.

Maslophovu.7 n:trris: muscle development;

INTRODUCTION of the obliquely striated somatic musculature of nematodes has been described in several species (reviewed by Bird, 1971). Ultrastructural analysis has been used to clarify the contractile mechanism of Ascaris muscles (Rosenbluth, 1967) and to refine the classification system of nematode musculature (Hirumi, Raski & Jones, 1971). Incidental to an ultrastructural study of Musrophws rnuris (unpublished), a stomach parasite of rodents, it was observed that the structure of the adult somatic muscle differs from that of hitherto described nematode species. The current study was undertaken to analyze M. muris adult muscle and to survey the developmental sequence of the muscle cell from the third stage larva to the adult in an attempt to clarify its organization.

THE ORGANIZATION

MATERIALS

AND METHODS

hf. mm’s was routinely maintained in the laboratory in Blatella germmica and male rats of the outbred Hebrew University Sabra strain. The roaches were in~_.. ^_ ~___.___ *On Fellowship leave from LaGuardia Community College, City University of New York, Long Jsland City, NY 11101, U.S.A.

nematode;

somatic

muscle;

fected with embryonated eggs dissected from uteri of female worms recovered 3-4 months post infection. Eggs were mixed with wet powdered oatflakes and fed to roaches starved for 48 h. Ten to 15 gelatinous cysts, containing third stage larvae, were dissected from roaches 4-5 weeks p.i. and fed to rats, SO-100 g with a Pasteur pipette. Infective larvae, parasitic larvae recovered 5, 8, 13, 18 days p.i., young adult worms (27 days p.i.) and adults from patent infections (60 days p.i.) were processed for electron microscopy. Worms were rinsed in saline and cut into appropriately sized cylinders. Tissue was fixed for l-2 h in ice cold 2.5% glutaraldehyde in either phosphate or cacodylate buffer pH 7.4. Worms were then rinsed in three changes of cold buffer for 1 h. Postfixation was carried out for 1 h with ice cold 1% osmium tetroxide in one of the above mentioned buffers. The tissue was rinsed in distilled water and in 2% uranyl acetate in distilled water, dehydrated in an ethanol series, transferred to propylene oxide and left overnight in a I : I mixture of propylene oxide-Epon. Following 2 h in fresh Epon ir? VQCUO (Bird, 1971), the tissue was embedded in fresh Epon in flat silicone rubber molds, oriented and polymerized at 60°C for 48 h. Some larvae were embedd~ h Spurr’s low viscosity medium (Spufl, 19691 and oolvmerized at 70°C for 24 h. Blocks were sect&ned with-glass knives on a LKB Ultrotome. Thick (O&l*0 pm) sections were stained with 1% toluidine blue in 0.5 y/, sodium borate at 6O’C. Selected areas were thin sectioned and picked up on naked or formvar-carbon coated grids. Epon embedded material was stained with 405

GEORCZ S. HAMADA and GUTA WERTHCIM

406

1 % uranyl acetate in 50% ethanol for 30 min followed by lead citrate. Spurr’s embedded material was stained in 4% uranyl acetate in absolute methanol for 10 min followed by lead citrate. All thin sections were examined with a Philips 300 E.M. at 60 kV. RESULTS The development of M. nluris from infective larva to adult in the rat lasts approx 26-27 days. The first two moults occur in the intermediate host. The third moult occurs on days 8-9 after infection of the final host and the fourth on days 25-26. The infection is patent at 60 days p.i. In all stages the muscle cell is elongate, spindle shaped and oriented along the longitudinal axis of the worm. Each cell consists of a peripheral contractile region, a non-contractile region, a non-contractile region projecting into the pseudocoelom and an arm extending to the hypodermal cord. All stages are polymyarian. The various developmental stages differ in cell size as well as in size, shape and composition of the myofibrils and dense bands (Table 1). Ultrastructure

of the muscle cell of adult Mastophorus

The organization of the muscle cells of the adult male and female recovered from a patent 60 day old infection is similar, with the female muscle somewhat larger in size. In cross section the cell is 170260pm thick measured radially from the hypodermal border to the apical tip of the cell. The contractile region is 75-150 Mm thick and 75-90 llrn wide, measured perpendicular to the radial axis. Light microscopic observations show that the contractile elements extend from the base of the cell to the lateral periphery of the central part of the cell where it partia!ly flanks the noncontractile sarcoplasm. The TABLE

Stage

No. cells quadrant

Cell thickness (l(m)

I-Mastophoru,

I.J.P. VOL. x. 1978

contractile region is subdivided by randomly arranged dark lines, into irregular circular to elongate profiles 4-6 pm wide to 16 pm long (Fig. 1). Observed with the electron microscope these dark lines prove to be dense bands separating adjacent myofibrils (Fig. 2). They contain T tubules, flattened cisternae of sarcoplasmic reticulum, glycogen and dense fibrillar bodies (Fig. 3). Membrane bound electron dense bodies, possibly mitochondria, are also present, but cristae are not observed (Fig. 4). The myofibril contains myofilaments of two sizes: thick filaments 24 nm in diameter and thin filaments IO nm in diameter (Fig. 4). Large vacuoles are present at the apical contractile border. The noncontractile sarcoplasm contains glycogen, lipid droplets, Golgi bodies, rough endoplasmic reticulum, lysosomes, free ribosomes, small filament bundles and a peripheral band containing dense mitochondria with short cristae. Nucleci are present in the sarcoplasm that projects into the contractile region and up to three nuclei have been seen in a single cell. The sarcolemma is externally bounded by a basal lamina. Developmental

seyuetrce of’ Mastophorus

musc!e cells of the six stages, selected to describe the pattern of development, have a number of characteristics in common. In all stages the muscle cell has a peripheral contractile and a pseudocoelomic noncontractile region. The contractile region is divided into myofibrils by dense bands (Fig. 5). The myofibrils contain thick (range 21-26 nm) and thin (range 7-9 nm) myofilaments, with I I-12 thin filaments surrounding a single thick filament. Groups of thick filaments with few or no thin filaments can occasionally be seen but a linearly organized H band is not present. The dense bands contain T tubules

muris

SOMATICMUSCLE

Contractile Contractile region region thickness (pm) width (Mm)

Number of myofibrils

Width of myofibrils (pm)

J-3 9

10

2.7

2

D5

12

13.6

6.2

2.9

8- 10

I.5

DU

II

24

3.5-7.0

5.5

9-12

I .4

D13

12

21-40

6-10

7- 8

I6

1.8

D18

12

49-67

15-25

II~IX

20

2.0

II

70- 130

42

42

50

23

I I-12

260

150

90

200

446

12

170

75

75

200

6

infective

L4

adult D27 Female Male *Measurements

based on largest cells in sections

mrrsc~lecc1i.s

The somatic

0.9

6

Contractile region configuration Shallow coelomyarian Shallow coelomyarian, coelomyarian Coelomyarian

Coelomyarian Irregular coelomyarian Irregular coelomyarian Irregular coelomyarian Irregular coelomyarian

I.J.P. VOL. 8. 1978

Masfophorus:

ultrastructure

of somatic muscle

Figure legends on p. 408

407

GEORGE S. HAMADAand GUTA WERTHEIM

408

(invaginated sarcolemma) and profiles of sarcoplasmic reticulum and, beginning with day 8 p.i., groups of thin filaments, glycogen and small bundles of support filaments. The noncontractile sarcoplasm contains a nucleus, glycogen, mitochondria, lipid droplets, ribosomes, rough endoplasmic reticulum, microtubules, an occasional Golgi apparatus, small (40 nm) and large (0.25 pm) vesicles. A basal lamina is present external to the sarcolemma. Third stage larva (represented by the infective larva and by larvae recovered 5 and 8 days p.i.). The muscle cells of the infective larva, recovered from the insect haemocoel, are IO pm thick. The contractile region is 2.7 pm thick and 2 pm wide. The contractile region contains 6 rectangular myofibrils divided into two lateral rows by a dense band passing along the midline of the cell (Fig. 6). The width of the myofibril (distance between adjacent dense bands) is 0.9 pm. The myofilaments in many cells are indistinct. Short invaginations of sarcolemma and dilated cisternae of sarcoplasmic reticulum are sometimes visible at the lateral basal border of the noncontractile region. Growth and development of the muscle cell proceeds directly after infection. By the 5th day p.i. the thickness of the cell increased to 13.6 pm; thickness of the contractile region has reached 6.2 pm and its width 2.9 pm. The number of myofibrils is now 8-10, and the width of each myofibril I.5 km (Fig. 7). The large 0.25 pm vesicles in the noncontractile region often enclose whorls of membranes. New, smaller myofibrils develop at the apical border of the contractile region, around a central core of sarcoplasm. The sarcolemma at the level of the basal noncontractile border shows numerous invaginations (Fig. 8). In the larva, recovered 8 days p.i., just prior to moulting, the muscle cell begins to show expansion in width. The cell reaches 24 pm in thickness; the contractile region is between 3.5 pm to 7.0 em thick and is 5.5 Hrn wide. The number of myofibrils along the basal border may reach 5, each about I .4 pm and becoming smaller apically (Fig. 9). The number of myofibrils in the cell is 9-12. lnvaginations of sarcolemma are seen only rarely at the basal noncontractile border. Four/h stage larvae (represented by worms recovered I3 and 18 days p.i.). (Legends Line marker

1.J.P. VOL.8. 1978

In larvae recovered I3 days p.i., the lateral cords are large enough to affect the size of the muscle cells. The noncontractile region, in cells close to the lateral cords, is smaller than in cells further removed, but the contractile regions in cells of both sizes remain approximately the same. The cells are 21-40 pm thick and the contractile region is 6-10 pm thick and 7-8 pm wide. The myofibrils, which now number up to 16, are I.8 pm across in the basal contractile region. The basal border of the contractile region is subdivided by dense bands perpendicular to the cuticle, into 4-5 myofibrils (Fig. IO). The dense bands bend and curve outlining myofibrils in slightly irregular shapes. No invaginations of sarcolemma are observed at the basal level of the noncontractile border. In the I8 day larva, the muscle cells are 49-67 pm thick, and the contractile region is 15-25 Hrn thick and I l-18 brn wide. Approximately 20 myofibrils are arranged linearly along the lateral margins, up to 8 at the basal border of the cell and a few begin to take shape in the center of the contractile region (Fig. I I ). Basally, U-shaped myofibrils are commonly seen, formed by short dense bands apparently dividing existing myofiblils (Fig. 12). Whorls of membranes are present at the basal sarcolemma and in the interior of the contractile region (Fig. 13). Myofibril width is now 2.0 pm. In addition to the other elements, the dense bands contain whorls of membrane and microtubules. The region of sarcolemma invaginations is very well developed (Fig. 14). Young adult. In the worms examined 27 days p.i.. immediately following the fourth moult, the muscle cells are 70-l 30 pm thick, and the contractile region is 42 pm thick and 42 pm wide. The dense bands appear increasingly irregular causing the myofibrils to lose their rectangular shape. Irregular oval profiles of myofibrils, up to 3 pm in diameter, fill the contractile region (Fig. 15). The myofibrils, now over 50 in number, have completely lost the perpendicular, regular arrangement of the previous stageb. It is often difficult to define myofibril boundariks since there is a continuity of myofilaments between otherwise divided myofibrils. Some sarcolemma invaginations are present at the level of the basal noncontractile border.

IO figipurrs on p. 407)

on the light micrograph represents SO Mm and line markers on all electron micrographs represent 1 pm. Figs. l-4 Somatic muscle cells of adult M. nwris. FIG. I. Light micrograph of the body wall of M. murk: hypodermis (h), contractile region of somatic muscle cell Cc), noncontractile region (n), oval profiles in the contractile region (arrows). FIG. 2. Low power electron micrograph of the contractile region containing numerous irregular myofibrils (m) bordered by dense bands (d) which contain deeply invaginated T tubules (arrows). Fig. 3. A portion of a peripheral myofibril (m) showing invagination of sarcolemma forming the T tubule (t), cisternae of sarcoplasmic reticulum (arrow) and fibrillar dense bodies (f). FIG. 4. Membrane bound dense body (db) (perhaps a mitochondrion. but no cristae are visible): thick (mT) and thin (arrow<) myofilaments.

Figure legends on p. 410

GEORGE S. HAMADA and GUTA WERTHEIM

410 DISCUSSION

The organization of the muscle cell of adult M. rn14ris is basically similar to that of other nematodes (Bird, 1971). The cell is longitudinally oriented, elongate, spindle shaped, and contains a basal contractile region, an apical pseudocoelomic noncontractile region and an arm leading to the hypodermal cord. Ultrastructural similarities include components of the noncontractile region (nuclei, glycogen, mitochondria, lipid, vesicles), and of the contractile region (thick and thin myofilaments, T tubules, sarcoplasmic reticulum, support filaments). However, the organization of the components within the contractile region of the adult differs considerably from other nematode muscles. In the previously described coelomyarian nematodes : Ascaris lumbricoides Reger, 1964 ; Rosenbluth, 1965a; 1965b, 1967; Watson, 1965), Brugia malayi (Vincent, Ash & Frommes, 1975), Capillaria hepatica (Wright, 1964), Contracaecum multipapillatum third stage larva (Larsh. Huizinga, Race and Martin, 1968), Deontostoma calijhwica (Hope, 1969), Diroj’/aria immitis (Lee & Miller, 1967), and Parascaris quorum (Auber-Thomay, 1964; Hinz, 1959, 1963) the myofibrils are rectangular and arranged in regular rows perpendicular to the cuticle. The myofibrils of adult M. muris muscle are irregularly oval in profile and several hundred of them fill the contractile region in a randomly (Leged

I.J.P. VOL. 8. 1978

oriented pattern. The term irregular-coelomyarian is suggested for this type of organization. The developmental sequence of the somatic muscle ceil of M. muris is illustrated in Fig. 16. The irregularcoelomyarian muscle cell develops from the shallowcoelomyarian type muscle cell of the infective larva first described in Longidorus elongatus by Hirumi. Raski & Jones (1971), through a continuous process of cellular growth, the addition of new myofibrils and of elements of the dense bands, and changes in the pattern of the myofibrils within the contractile region. Growth involves an increase in cell thickness from IO urn in the infective larva to 170-260 urn in the adult; an increase in width of the contractile region from 2 urn to 75-90 urn and in thickness from 2.7 urn to 65-80 urn. During the course of development from infective larvae to adult the number of myofibrils increases from 6 to over 200 and their width from 0.9 urn to 6 urn. The increase in the number of myofibrils is the result of two processes: (I) the formation of new, small myofibrils at the apical border of the contractile region, thereby adding myofibrils radially; (2) the subdivision of existing .myofibrils at the basal border and in the interior of the contractile region, responsible for growth in width. Two observations are suggestive of the formation of new apical myofibrils. The constant presence of

to figures on pp. 409, 41 I and 412)

FIG. 5. Myofibril of a young adult worm (27 days p.i.) showing ultrastructural elements typical of developmental stages. The myofibril contains two sizes of myofilaments. Some areas contain thick filaments with few thin filaments (arrows). The dense bands contain T tubules (t), sarcoplasmic reticulum (sr), bundles of thin filaments (mt), glycogen (g) and bundles of support filaments (sf). FIG. 6. Muscle cell of an infective larva (L,). Six myofibrils (m) separated by dense bands(d) containing dilated cisternae of sarcoplasmic reticulum (sr). Mitochondria in the noncontractile region. FIG.

7. Muscle

cells of a 5-day p.i. larva (L,). Smaller newly developed apical border of the nontractile region.

myofibrils

(arrows)

at the

FIG. 8. Muscle cells of a 5 day p.i. larva (L:,). Border between contractile and noncontractile r-egions. The noncontractile region contains mitochondria with a few short cristae, small vesicles and glycogen (g). lnvaginations of the sarcolemma are present in this area (arrows). FIG. 9. Muscle

FIG.

cell of larva (L,) recovered 8 days p.i. showing the beginning of growth basal contractile region in this cell contains 5 myofibrils (m).

IO. Muscle

in width.

The

cell of a I.3 day old larva (L,) showing additional myofibrils in the apical contractile region. The myofibrils retain much of their rectangular shape. Figs. I l-14.

Muscle cells of larva (L,)

recovered

I8 days p.i.

Fm.

I I.

FIG.

12. U-shaped

FIG.

13. Basal myofibril (m) containing whorls of membranes (arrows) at the sarcolemma adjacent hypodermis (h) and within the dense band (d). These whorls may represent deposits of membranes to be used for membraneous components of the developing dense bands.

Additional

myofibrils are forming in width and at the apical contractile are forming in the interior of the contractile region (arrows). basal myofibril

(m) appears as a dense band (d) develops into two.

border.

Myofibrils

to divide the myofibril

to the FIG.

14. Two

adjacent

muscle cells. The

area of sarcolemma horder (arrow).

invagination

at basal noncontractile

I.J.P. \‘oL. 8. 1978

Ma.sfophor~s: ultrastructure

of somatic muscle

Figure legend7 OHprevious page smaller myofibrils at the apical border of the contractile region and the numerous invaginations of sarcolemma which occur in this area. These invaginations are interpreted as the site of formation of new membrane components of the dense bands (T tubules and sarcoplasmic reticulum). Larvae recovered 5 and 18 days p.i. contain abundant invaginations which indicate that growth in this direction occurs early in the L, larvae and in the mid to late LS larvae. Their relative absence in larvae recovered on days 8 and I3 suggest that the process of apical myofibril formation slows down considerably during the L,-L4 moult. The appearance of U-shaped myofibrils in the basal contractile region (Fig. 12) in worms from

days 13 and 18 p.i., the division of the myofibril into two equal parts by the newly formed dense band and the presence of membrane whorls at the cell periphery and in its interior suggest that the existing myofibrils are being subdivided. The whorls of membranes may represent sites of membrane elaboration for the formation of new T tubules and new sarcoplasmic reticulum during the process of myofibril subdivision. The increase in the number of myofibrils in the apical contractile region changes the muscle cell organization from shallow-coelomyarian to coelomyarian by mid to late third stage larva (days 5 and 8). Myofibril subdivision at the basal contractile region (days 13 and 18) and in the interior (day 18)

(Legends fbr figure on p. 414) FIG. 16. Diagrammatic representation of the developmental sequence of M. ncwis scmatic muscles from the infective third stage larva to the adult. Line marker represents IO Wm. (A) Infective larva (L,). Transverse section showing organization of muscle cells in the body quadrants. Shallow coelomyarian. (B) Muscle cell of larva Sdays p.i. (LJ. Developing into coelomyarian configuration. (C) Larva I-days p.i. (L,). Coelomyarian. (D) Larva 13-days p.i. (L,). Coelomyarian. (E) Larva 18-days p.i. (L,). Irregular myofibrils begin to form in interior of contractile region. Irregular coelomyarian. (F) Young adult 27-days p.i. Irregular coelomyarian. (G) Adult male 60-days p.i. Irregular coelomyarian. Actual thickness 170 Wm. Ahbreviations: c-contractile region; d-dense band; db-membrane bound dense body: f-fibrillar dense bodies; g-glycogen; h-hypodermis; m-myofibril; mT--thick myofilaments; mt--thin myofilaments; n-noncontractile region; sf-support filaments; sr-sarcoplasma reticulum; tT tubule.

I.J.P.

VOL. 8.

1978

Mf&o&rus:

ultrastructure

of somatic muscle

413

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,fiir Zel(forschung

49: 339-343.

HINZ E. 1963. Electronmikroskopische Untetscchungen an Puruscuris eqnorum (Integument, Isolationsgewete, Muskulatur und Nerven). Prorop/asn.a 56: 202-241. HIRUMI H., RASKI D. J. & JOTYESN. 0. 1571. Primitive muscle cells of nematodes: morphological aspects of platymyarianand shallow coelomyarian muscles in two plant parasitic nematodes, Triehodorus christiei and Longidorns elongatus. search 34: 517-543.

Journal

of

Ultrcstr rrcinre

Re-

HOPE W. D. 1969. Fine structure of the somatic muscles of the free-living marine nematode Deontostoma culi./ornirum Steiner and Albin. 1933 (Leptosomatidae). Proceedings oJ‘ rhe Helminthologiral ington 36: 10-29.

Society

of Wush-

LARSHJ. E., HUIZIKGAH. W., RACEG. J. & MARTINJ. H. 1968. A study of the body wall of the third-stage larva of Contracaecum n;ultipapillutum by electron microscopy. The Journal of the Elisha Mitchell Scientif;c Society

84: 285-292.

LEE C. C. & MILLER J. H. 1967. Fir.e structure of Diro$/aria immiti,e body wall musculature. Experimental FIG. 15. Somatic muscle cell of young adult worm (27 day p.i.). The dense bands (d) are randomly branched and irregularly shaped. The myofibrils (m) have varying shapes and sizes.

Purasitolo.gy

Ultrastructure

of the contractile region shifts the organization in the L4 larva to the irregular-coelomyarian of the adult (Fig. 16). In the evolutionary sequence of somatic muscle cells proposed by Hirumi et nl. (1971) the irregularcoelomyarian musculature of M. muris would represent an additional stage in complexity beyond that of the coelomyarian. The coelomyarian nematode Deontostoma californicum, in which a few irregularly shaped myofibrils were observed (Hope 1969). may represent a transitory stage between coelomyarian and irregular-coelomyarian. We speculate that in the coelomyarian nematodes the number of myofibrils increases only through addition of new apical myofibrils and not through a subdivision of existing myofibrils, as observed in A4. muris. wish to express our gratitude to Dr. B. M. Zuckerman, Laboratory of Experimental Biology, University of Massachusetts, U.S.A., for reviewing the manuscript. This work was supported, in part, by Biomedical Sciences Support Grants PHS 5 SO 5 RRO7132-05 and PHS 5 SO 7 RR07132-07 to P. L. Krupa, The City College of the City University of New York.

Acknowledgements-We

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ROSENBLUTHJ. 1965a. Ultrastructural organization of obliquely striated mcscle fibers in Asrnris /umbricoide.r. Journal of Cell Biolopy 25

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ROSE~~LUTHJ. 1965b. Ultrastructure of somatic muscle cells in Ascarfs lumbricoides. II. Intermuscular junc-

tions, neuromuscular

junctions,

Journal of Cell Biology 26

and glycogen stores.

: 579-59 I .

ROSENBLUTHJ. 1967. Obliquely striated muscle. III. Contraction mechanism of Ascari.s body muscle. Jonrnul of Ceil Biology 34: 15-33. SPURR A. R. 1969. A low-viscosity epoxy resin embedding medium for electron microscopy. Jorrrna/ of Ultrustrncfnre Research 26: 3 I-43. VINCENT A. L., A?H L. R. Rc FROMMESS. P. 1975. The ultrastructure of adult Brngiu malayi (Brug, 1927) (Nematoda: Filarioidea). ./ourncl of Purasitokogy 61 : 499-5 12. WATSONB. D. 1965. The fine structure

of the body-wall and the growth of the cuticle in the adult nematode

Ascaris Imrbricoides. Quarterly .scopical Science 106: 83-91.

Jonrnul

of

Micro-

WRIGHT K. A. 1964. The fine structure of the somatic muscle cells of the nematode Capilluriu heputicu (Bancroft, 1893). Canadian Journal of Zoology 42: 483-490.

414

GEORGE

S. HAMANDA

and GUTA WERTHEIM

Hypodermis

\ Contractile .ti,_l

reqon

Non,- contractile regto” C

B

FE. Figure

16.

fegend on p. 41

I

1.1.1”. VOL. 8. 1978