Studies on the fine structure of cercarial tail muscle of Schistosoma sp. (trematoda)

JOURNAL

OF ULTRASTRUCTURE R E S E A R C H

57, 77-86 (1976)

Studies on the Fine Structure of Cercarial Tail Muscle of Schistosoma sp. (Trematoda) ~ J A M E S F . REGER

Department of Anatomy University of Tennessee Memphis, Tennessee 38163 Received March 16, 1976 Three muscle cell types occur in the forked tail of Schistosoma sp. (Trematoda) cercarial larvae. These include a fast, cross-striated muscle functioning in rapid tail movements; a slow, smooth muscle at the body-tail juncture functioning in body and tail shape changes; and a slow, smooth muscle in the tail forks functioning to maintain fork ridgidity during backward swimming movements. The cross-straited muscle observed in this study is unique to platyhelminthes. The striations are the result of transversely aligned Z densities interspersed with branched elements of subsurface sarcoplasmic reticulum cisternae. The myofibrils of striated muscle cells are divided into sarcomeres with typical A and I bands reflecting overlapping thick and thin myofilaments. The smooth muscle cells found at the body-tail juncture are typical for platyhelminthes. They contain irregularly arranged Z densities, overlapping thick and thin myofilaments, and small amounts of subsurface sarcoplasmic reticulum cisternae. The smooth muscle cells found in the two caudal branched forks of the tail are unique in that their thick filaments exhibit a repeating cross-period striation of 350 to 400 A. The morphology of these three muscle cell types is a direct reflection of the three functions known to occur during backward and forward swimming movements in forked tail cercarial larvae.

Fine structure studies on trematode muscle in cercarial tail muscle have been made on Schistosoma mansoni (10), Tetrapapillatrema concavocorpa (10), Himasthla quissetensis (4), Heterobilharzia americana (12), Parorchis acanthus (15), and Cryptocotyle lingua (5). Lumsden and Foor (12) presented excellent evidence for differences between the body wall smooth muscle and the tail cross-striated muscle, particularly with respect to the underlying significance of the cross-striated nature of the longitudinal tail muscle which subserves rapid tail movements in these animals. The fact that the longitudinal tail muscle of cercarial larvae was crossstriated, and probably functioned in rapid tail movements, had been known since the early light microscope studies of Cable (3) and Vickers (21). Cercaria larvae of the forked tail species (7) undergo some rather unusual shape changes and high speed tail movements (20-24 tail oscillations per second) (7) dur-

ing swimming. When cercaria swim backward, the two forked branches of the tail are spread stiffly laterally and perpendicularly to the long axis of the tail, while in forward swimming they are extended caudally and parallel to the longitudinal axis of the tail. The shape and poise of the body and the tail, and the position of the two caudal forks of the tail determine whether cercaria swim backward or forward. It is the purpose of the following study to demonstrate some of the unique morphological features of the three muscle cell types observed in the tail which underly tail and fork movements during swimming in these animals. MATERIAL AND METHODS Cercerial larval stages of Schistosoma sp. (Trematoda) were washed from the pond snail, Cerithidea sp., intestinal tract in saline and immediately immersed in a cold (0-5°C) solution of glutaraldehyde fixative (pH 7.6; 0.1 M phosphate buffer). The tail portion was then dissected into small pieces and allowed to fix for 1 to 2 hr. The tissue was then washed for an hour in cold (0-4°C) phosphate buffer (pH 7.6, 0.1 M), and postfixed for an hour in cold (0-

Supported by USPHS Grant No. 156612 (GRS). 77 Copyright © 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.

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4°C) 2% OsO4 (pH 7.6, 0.1 M phosphate buffer). Subsequently the tissue was dehydrated in 5-rain changes of successive grades of methanol (beginning with 50%), and embedded in Epon 812. Sections, 1-2 t~m thick, were made of the Epon-embedded tissue and stained with Mallory Azure II-Methylene blue for purposes of orientation of tissue prior to thin sectioning. Thin sections were cut with a diamond knife fitted to an LKB Ultratome II, floated on distilled water, mounted on carbon-coated grids, stained with uranyl acetate and lead citrate, and examined with a Hitachi HU l l A electron microscope. Micrographs were taken at initial magnifications of 750040 000x at exposures of 3-5 sec on Cronar, Ortholitho, Type A sheet film. Negatives were enlarged up to 10 times with a Durst S-45 enlarger equipped with a 250-W mercury vapor point source. OBSERVATIONS

The three basic types of muscle cells examined in cercarial larvae of S c h i s t o s o m a sp. include the longitudinally directed, fast, cross-striated muscle cells in the tail (Figs. 1-5); the slow, smooth muscle cells at the body-tail juncture (Figs. 68); and the slow, smooth muscle cells of the forked portions of the tail (Figs. 9 and 10). The longitudinally directed, fast, tail muscle is situated beneath a single layer of circular smooth muscle cells (Sm, Figs. 1 and 2). Each striated muscle cell is divisible into a nonmyofibrillar and a myofibrillar region (Figs. 1 and 2). The nonmyofibrillar region (bottom, Fig. 2) is directed toward the center of the tail axis. This region of the muscle cell contains mitochondria (M, Fig. 2) and its surface is innervated (A, Fig. 2). Each muscle cell contains a single, irregularly shaped myofibril (Top, Fig. 2) situated in the outer surface of the tail. Subsurface, sarcoplasmic reticulum cisternae (SSR, Fig. 2) are confined to the myofibrillar region of the muscle cell. Myofibrillae of the muscle cells are divisible into typical sarcomeres composed of A and I bands resulting from overlaping thin and thick myofilaments. Thin filaments (Ti, Figs. 3 and 4) extend from repeating, transversely aligned Z densities (Z, Figs. 3 and 5). Thick filaments (Tk, Figs. 3 and 4) are approximately 250-300/~

t h i c k and t a p e r t o w a r d the I bands. Branched elements of the sarcoplasmic reticulum (BSR, Figs. 3 and 5) extend from the subsurface cisternae (SSR, Fig. 4) and come to lie in a regularly arranged pattern among the Z densities (BSR, Z, Figs. 3 and 5). Cross sections of muscle cells at the level of the Z densities (Fig. 5) demonstrate this alternating repeat pattern of SR and Z densities. The smooth muscle cells found at the body-tail juncture (Figs. 6-8) are composed of two sets of cells, each perpendicularly arranged with respect to the other. These cells are morphologically similar to other platyhelminthes smooth muscle cells in that they contain two sets of myofilaments, subsurface sarcoplasmic reticulum cisternae and scattered Z densities into which the thin filaments insert (Figs. 7 and 8). Relative to the longitudinal axis of the body-tail juncture the longitudinally oriented muscle cells (L, Figs. 6-8) have a highly scalloped surface at their origin in the more caudal regions of the body-tail juncture (Fig. 7). The thin filaments here insert into s u b p l a s m a l e m m a l densities (Ti, Fig. 7) on the deeply infolded scalloped surface. At the more rostral level of the body-tail juncture (Fig. 8) the scalloping disappears and the cytoplasm contains typically interdigitating thin and thick myofilaments. The transversely oriented set of smooth muscle cells (T, Fig. 6) tends to be short, and they extend from the outer surface of the animal toward a central, nephridial canal (N, Fig. 6). More caudally the transversely oriented smooth muscle cells become circularly arranged (arrows, Fig. 6) and extend into the tail as the outer circular smooth muscle cell layer overlying the longitudinal cross-striated muscle cells. The smooth muscle cells of the two forked terminal portions of the tail (Figs. 9 and 10) are also composed of two sets of muscle cells, perpendicularly arranged to each other (Fig. 9). The cells are relatively smaller in cross-section compared to either

FIo. 1. Longitudinal view of cross-striated tail muscle cells. Note t h a t the cross-striation is due to regularly repeating, transversely aligned Z densities (Z) and interspersed elements of branched sarcoplasmic reticulum (BSR). The cells are divided into myofibrillar (left) and nonmyofibrillar (right) regions. The latter region contains mitochondria. (Sm) Circularly arranged smooth muscle cells, x 16 000. FIo. 2. Cross-sectional view of portions of three cross-striated tail muscle cells. The innervation (A) of the muscle cells occurs along the nonmyofibrillar surface of the cell. The subsurface, sarcoplasmic reticulum cisternae (SSR) are confined to the myofibrillar region only (see arrows). (Sm) Circularly arranged smooth muscle cells; (M) mitochondria, x 32 000. 79

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the cross-striated muscle cells or the smooth muscle cells of the body-tail juncture. The smooth muscle cells in the fork contain very small amounts of subsurface sarcoplasmic reticulum cisternae (SSR, Fig. 9) and an interdigitating set of thick and thin myofilaments (Figs. 9 and 10). The thick myofilaments range in crosssectional diameter from 300-700/~ and exhibit a unique cross-period repeat pattern of 350 to 400 A (arrows, Fig. 10).

directed fork stiffening which occurs during backward swimming movements (7). As to the structural features of the fast, cross-striated muscle cells of the tail which produce the high speed tail movements, some of the observations in the present study confirm the results of Lumsden and Foor (12) on Heterobilharzia americana. However, some of the results are at variance with their study. First, Lumsden and Foor (12) stated that "the myofibrils of the axial musculature are branched." (By axDISCUSSION ial they m e a n the l o n g i t u d i n a l crossThe fine structure of muscle cells ob- striated muscle of the tail.) We did not served in the tail, the body-tail juncture, observe myofibrillar branching in the and the caudal forks of Schistosoma sp. present study. In fact, each muscle cell cercarial larvae have direct functional cor- contains a single, irregularly shaped myrelates with other invertebrate muscle ofibril, about 6-7 by 25-30 ~m in crosscells where fast, slow, or tonic type con- sectional dimensions. Second, Lumsden tractions occur. The structural aspects of and Foor (12) stated t h a t the myofilamuscle usually discussed relative to func- ments ~are not arranged to form distinct A tion include myofibrillar size, myofila- and I bands." In the present study distinct ment structure, Z line structure, numbers A and I bands occur. This is a reflection of ofmitochondria, types of striation, interre- the interdigitation of thin and thick myofilationships of the T-tubules and sarco- laments, as for other cross-striated musplasmic reticulum, etc. (9, 18, 20). Con- cle. Third, Lumsden and Foor (12) stated cerning the three muscle cell types ob- that the thin filaments extend from ~Tusiserved in the present study, the following form densities" which, except for their three main points may be made. The longi- transverse regularity, are similar to those tudinal, cross-striated tail muscle cell~ in the body wall smooth, muscle. In the function in the rapid tail oscillations of 20 present study it is seen that the Z densities to 24 per second. The two perpendicularly are, in fact, a network with regularly disarranged sets of smooth muscle cells in the posed openings within which the branched body-tail juncture function in the alter- elements of sarcoplasmic reticulum occur. nate constriction and relaxation in this One of the most significant results of region when the animal shifts from back- Lumsden and Foor (12), and of the present ward to f o r w a r d m o v e m e n t (7). The study, is the discovery t h a t the sarcosmooth m u s c l e cells of t h e c a u d a l l y plasmic reticulum penetrates within the branched forks function in the laterally structure of the myofibril to come to lie as Fla. 3. Longitudinal view of a single sarcomere and portions of two others to the right and left. Note t h a t the sarcomere is divided into typical A and I bands with interdigitating thin (Ti) and thick (Tk) filaments. (Z) Z densities; (BSR) branching portions of the SR. × 64 000. FIG. 4. Cross-sectional view through a myofibril to demonstrate the high degree of filament register and the fact t h a t thick (Tk) filaments (arrows) are hollow. (SSR) Subsurface sarcoplasmic reticular cisternae; (Ti) thin filaments. × 64 000. Fla. 5. Cross-sectional view at the level of the Z densities (Z) to demonstrate the regular repeating interdigitation of the Z densities (Z) with elements of the branched sarcoplasmic reticulum (BSR). Also notice the point of attachment of Z line densities with the plasma membrane (P), as well as the branched SR (BSR) originating from the subsurface cisternum. × 64 000.

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FIG. 6. Cross-sectional view at body-tail juncture. Note the two sets of smooth muscle cells oriented perpendicularly to each other. The longitudinally oriented muscle cells (L) extend rostrally into the body wall. The transversely oriented muscle cells (T) continue caudally as the circular smooth muscle cells overlying the surface of the cross-striated muscle cells of the tail (arrows). The smooth muscle cells of both sets contain interdigitating thick and t h i n myofilaments, small amounts of subsurface sarcoplasmic reticulum cisternae, and irregularly disposed Z densities. (N) Nephridial canal. × 16 000.

FIGS. 7 and 8. These two micrographs are at caudal (Fig. 7) and rostral (Fig. 8) levels of the body-tail juncture. Note t h a t the longitudinally oriented muscle cells (L) have a highly plicated t e r m i n a t i o n at the most caudal level (Fig. 7). Thin (Ti) filaments insert into the subplasmalemmal electron densities in this region. >< 64 000. 83

FIG. 9. Low power longitudinal view of a portion of the forked tail d e m o n s t r a t i n g the two sets of s m o o t h muscle cells found here which are oriented p e r p e n d i c u l a r to each other. Note the wide r a n g e in crosssectional d i a m e t e r exhibited by the thick filaments. The cells contain i n t e r d i g i t a t i n g thick a n d t h i n f i l a m e n t s and small a m o u n t s of subsurface sarcoplasmic r e t i c u l u m cisternae (SSR). x 64 000. FIG. 10. H i g h magnification view of a portion of a smooth muscle cell in the forked tail to d e m o n s t r a t e the 350 to 400 A, cross-period repeat of the thick f i l a m e n t s (arrows). (Ti) T h i n filaments, x 110 000. 84

FINE STRUCTURE OF CERCARIAL MUSCLE a network among the Z densities of each sarcomere. A n o t h e r example o f sarcoplasmic reticulum penetration within myofibrillae occurs in high speed synchronous flight muscle of certain Lepidoptera (1, 16) where branches of the SR penetrate among thick filaments at the M line. Based on this and a variety of types of other direct and indirect evidence it appears t h a t the amounts and/or the spatial relationships of the SR with myofilaments are a direct reflection of its role in calcium release and/ or uptake during contraction-relaxation cycles. As may be determined from data on cercarial tail muscle oscillation speeds (7) and the wing beat frequencies of the Lepidoptera studied above (1, 16, 19), the contraction-relaxation cycles in these muscles would fall between 20- to 100-msec duration. For durations of this magnitude it can be calculated from Hill's diffusion constant for CaC12 (8) that the maximal allowable myofibrillar diameter would fall within the range of 2.4 to 5.4 ~m. In fact, the cross-sectional dimensions of the cercarial cross-striated muscle and of the Lepidopteran wing muscle noted above are larger than this; being 6-7 by 25-30 ~m in the former and about 9 by 10 t~m in the l a t t e r (1, 16). Thus it is conceivable, though hardly provable on the basis of this study, that the intramyofibrillar penetration of SR in the two animals reflects the limitations of calcium diffusion rates. Support for such a possibility may be derived from comparisons of myofibrillar sizes in other fast-acting muscle with similar contraction-relaxation cycle durations. For example, in the fast-acting gas bladder muscle of the Atlantic toadfish, Opsanus tau (6) and the fast-acting bat (Myotis lucifigus) cricothyroid muscle used in echo location (17) the myoflbrillar sizes fall well within the range to satisfy their 20- to 200-msec contraction-relaxation cycles as allowable by Hill's diffusion constant for CaC12 (8). In the former the maximum distance from the center of a myoflbril to the SR is only 1.5 tLm and in the latter it is

85

closer to 1 t~m. Unfortunately such comparisons become relatively useless since so many factors govern cell physiology which go far beyond our capacity to develop comparisons with physicochemical models such as CaC12 diffusion rates. Further one might ask what the functional significance is of the fact that the penetration of the SR occurs at the Z densities in cercarial tail muscle and at the M line in the Lepidopteran muscle noted above (1, 16). There is no obvious answer to this question unless it has something to do with sarcomere length, or some as yet unknown functional characteristic. If it is assumed in both cases that the penetrating SR is concerned with calcium movements and not an as yet unknown function, the significance of its repeat penetration at the Z line in one muscle and at the M line in another muscle remains unknown. The smooth muscle found at the bodytail juncture no doubt functions in the alternately slow constrictions and relaxations which occur at this region and which are partly responsible for body and tail shape changes as the animal shifts from backward to forward swimming movements (7). These muscle fibers are quite identical to those of several other platyhelminthes smooth muscle cells (2, 5, 11-14). Their only unique feature concerns the interesting thin filament insertion regions found in the caudal ends of the cells in the body-tail juncture. This morphology is likely a reflection of mechanical tension forces exerted in this region. The smooth muscle cells found in the two, caudally branched tail forks are unique by the fact that their thick filaments exhibit a periodicity not found in the other two muscle cell types. These smooth muscle cells are likely responsible for the stiffly lateral projection of the tail forks which occurs during backward swimming movements (7). The uniqueness of the thick filament structure, which resembles certain invertebrate catch muscle, may reflect this function.

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In conclusion, the morphological characteristics of the three muscle cell types described in this study certainly reflect the known functions which occur in the forked tail species of cercaria during backward and forward swimming movements and the data serve to emphasize the high degree of morphological specialization which occurs in muscle with different functions. REFERENCES 1. 2. 3. 4.

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8. HILL, A. V., Proc. Roy. Soc. London, Ser. B, 135, 446 (1948). 9. HOYLE, G.,Amer. Zool. 7, 435 (1967). 10. KRUIDENIER,F., AND VATTER,A., Proc. 4th. Int. Cong. Elect. Micro. 2, 332 (1958). 11. LUMSDEN, R., AND BRYAM, J., J. Parasitol. 53, 326 (1967). 12. LUMSDEN,R., AND FOOR, W. E., J. Parasitol. 54, 78O (1968). 13. MAcRAE, E. K., J. Cell Biol. 18, 651 (1963). 14. MORITA, M., J. Ultrastruct. Res. 13, 383 (1965). 15. REES, G., Parasitology 62, 489 (1971). 16. REGER, J. F., ANDCOOPER, D. P., J. Cell Biol. 33, 531 (1967). 17. REVEL, J. P., J. Cell Biol. 12, 571 (1962). 18. ROSENBLUTH,J., in BOURNE, G. H. (Ed.), The Structure and Function of Muscle, Vol. 1, p. 389. Academic Press, New York, 1972. 19. SOTAVALTA,O., Ann. Entomol. Fennicae. 4, 1 (1947). 20. USHE~WOOD,P. N. R., Insect Muscle. Academic Press, New York, 1975. 21. VmKERS, G., Quart. J. Microsc. Sci. 82, 311 (1940).