The primary body-wall musculature in the arrow-worm Sagitta setosa (Chaetognatha): An ultrastructural study

0040-8 166/80/00540723

TISSUE & CELL 1980 12 (4) 7233738 ,Q 1980 Longman Group Ltd

MICHEL

DUVERT

and CHRISTlANE

SO200

SALAT

THE PRIMARY BODY-WALL MUSCULATURE IN THE ARROW-WORM SAG/VA SETOSA (CHAETOGNATHA): AN ULTRASTRUCTURAL STUDY ABSTRACT. The primary musculature of Sqirta is mainly made up of two kinds of alternating fibers, A and 9. These fibers differ markedly in their localization in the muscular tissue, by the development of their SR and their mitochondria, and the shape of their myofibrils. Their contractile apparatus is similar and possesses myofibrils of regular thickness with very short I bands, flanked by invaginations which are large compartments communicating with the extracellular space. This fiber diversity appears and is maintained in the presence of an apparent common innervation. Nerve endinglike structures are scattered in the epidermis against the basement membrane and there are no nerves beneath this. The presence of at least two kinds of fibers in the primary musculature and the presence of the secondary musculature would suggest that the displacements of sagitra may be more complex than is generally admitted. The specializations of the trunk musculature underline the degree of specialization in the Chaetognatha phylum.

B fibers; we called them by the generic term of A fibers. In the present work we shall describe in some detail the primary musculature and particularly the two kinds of fibers (A and B). Conventions used to describe these cells are illustrated in Figs. I and 2.

Introduction THE trunk

musculature of Sag&a contains two kinds of muscles, the primary musculature comprising two large longitudinal muscles (one dorsal, the other ventral), and the secondary musculature which is located in the medial and ventro-lateral parts of the trunk (Perrier, 1897; Duvert and Salat, 1979). The primary musculature is made up of four quadrants increasing in size from the lateral fields towards the mesenteries (Hyman, 1959). Each quadrant contains a myofibrillogenesis zone against the lateral fields, then a group of C fibers and an alternation of A and B fiber groups (Duvert and Salat, 1979). C fibers are young A fibers (Duvert, 1975) these two groups forming a homogeneous population which differs strikingly from the

Materials and Methods Specimens of Sugitta setosu were collected in the plankton from the Bassin d’Arcachon. Because of the great difficulty in achieving a good and homogeneous preservation of the tissues of this animal, particularly rich in water, various fixatives were tried. The best results were obtained with animals fixed in an aldehyde mixture (Karnovsky, 1965), or glutaraldehyde 3 % in sea water. After being washed in sea water they were post-osmicated in 0~04 I o/oin 0.2 M Na-cacodylate buffer pH 7.5 and impregnated in an uranyl acetate solution (Silva et al., 1971). Some specimens were impregnated in an 1y. Na-phosphotungstate solution, pH 5, after glutaraldehyde

Department of Laboratoire de Cytologie, Universite de Bordeaux II, Avenue des Fact&es, 33405 Talence Cedex, France. Received 27 July 1980. Revised 17 August 1980. 46

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A AF BF BL BM C co D E FF GC H I IN LT M MF MI N NS PM SR TLGC

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I

AF

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AF

A band A fiber B fiber basal lamina basement membrane cisternae coat lining the general cavity dyad epidermis forming face general cavity H band I band invagination longitudinal tubule primary musculature maturing face mitochondria nucleus nervous system plasma membrane sarcoplasmic reticulum tissue lining the general cavity

Fig. 1. Scheme of a transversal section in the primary musculature showing the alternation of A and B fiber groups. For clarity only the contractile apparatus is included.

BF

AF

Fig. 2. Diagram showing some characteristics of A fibers (left) and B fibers (right), in transverse section. The invaginations are indicated by a thick line; gap and columnar junctions are indicated. The myofibrils are cut at the A band level (dots) and 1 band level (dots and hatched areas). Between arrows: the fiber width: 1, the fiber thickness; 2, myofibrillar thickness.

Results fixation, then dehydrated and embedded in Araldite. Some animals were fixed in Mn04K 1% in sea water. Visualization of RNA was made according to Mentre (1972) and Palekar and Bernhard (1968). Alkaline phosphatase activity was carried out according to Mayahara et al. (Pearse, 1972). ‘pH signature’ and critical electrolyte concentration (CEC) methods were made according to Scott and Dorling (1965); glycan visualization with the PAS method and according to Thiery (1967). For electron microscopy, thin sections were ‘stained’ with uranyl acetate and lead citrate except for cytochemical preparations.

Features common fo A and Bfibers The fibers are composed of two kinds of sarcoplasm. One is electron dense and homogeneous after en bloc impregnation in uranyl acetate (Fig. 3); it contains very few organelles and occasionally SR of irregular shape. The other contains a great density of small granules (Figs. 3, 7, 8) which give a strong positive reaction with the methods of Palekar and Bernhard (1968) and of Mentre (1972) (Fig. 9). The ribosomal nature of these granules is also indicated by two data: the sarcoplasm is highly basophil with alcian blue or toluidin blue pH > 3, or pyronin, and for a CEC 6 0.25 M (alcian blue-MgCle). No

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glycan can be detected with the PAS method or with the PATAg method of Thiery (1967), whereas glycogen is found in other tissues, like mesenteries, with these methods. Fibers are probably mononucleated, the nucleus lies in the pole near the general cavity (Fig. 9). Their Golgi apparatus is well developed (Figs. 5, 6) and regularly present near the nucleus. The forming face of the dictyosomes is in relation with the outer nuclear membrane. The saccules contain an alkaline phosphatase activity (Fig. 6). Mitochondria are numerous, sometimes ramified (Figs. 14, 15). They can reach 10 pm in length. Their mean diameter does not exceed l-2 pm and is more important in B than in A fibers (Fig. 3). Their inner membrane bears tubular cristae regularly disposed in rows (Fig. 7); the matrix contains composite granules (Fig. 7, inset) about 22 nm in width, somewhat difficult to preserve. The plasma membrane bears a characteristic junctional complex (Duvert et al., 1980) and is not flanked by vesicles. It is coated by a basal lamina only at the basement membrane level (Fig. 4). The plasma membrane is in regular continuity, at the H band level, with large invaginations (Fig. 10). These invaginations are very flat and they are extended on each side of the myofibrils, essentially at the A band level (Figs. 3, 10, 14-20); they have a maximum extension at the H band level and can be scarce or even absent at the I band level (Figs. 18-20). Successive invaginations can be continuous over many sarcomeres, especially in A fibers (Fig. 18). They may have fenestrations (Fig. 17). Extracellular tracers (peroxidase and lanthanum) always fill these compartments which are very narrow and slightly widened at their margins. The SR constitutes a polymorphic network which is continuous with the nuclear envelope (Fig. 5). Three aspects can be distinguished: (a) Longitudinal tubules, about 60 nm in width, essentially localized against the plasma membrane or the invaginations, at the A band level (Figs. 14-20). (b) Transversal cisternae of irregular shape, at the I band level, between the myofibrils (Figs. 14-20). They bear fenestrations (Fig. 17). (c) Irregular sections, without contact with the contractile apparatus. Some-

times they are associated with the ribosome-like particles, as the outer surface of the nuclear envelope (Fig. 8). Tubules and cisternae are continuous and form units extending along successive sarcomeres (Figs. 16, 17, 19,20); the other aspects of SR are not disposed according to a sarcomere pattern. Dyads are essentially differentiations of the longitudinal tubules which are against the plasma membrane or the invaginations. They are rarely found between cisternae and the margins of the invaginations. They lack ‘junctional granules’ in our preparations (Figs. 8, 10, 11, 14). The junctional processes are contrasted with phosphotungstate (Fig. 12); inversely one can see only the SR membranes after Mn04K fixation (Fig. 13). The SR membranes do not show any indentation at the junctional process level. These junctional processes are disposed in two parallel rows along the tubules; because of the very high frequency of these processes in crosssections, it is possible that dyads constitute the main part of the tubules. Fig. 21 shows the mean dimensions of these dyads. Diflkrences between A and B/ibers I. Myofibrilar shape. A and B fibers are clearly distinguished in transversal sections by the shape of their myofibrils (Figs. l-3), but the fine structure of their contractile apparatus is the same (Duvert, 1969). A fibers: they have a characteristic pattern constituted by the succession of groups of fibers extending from the basement membrane towards the general cavity (Fig. 1). The width of individual fibers increases more than its thickness; the largest ones are near the mesenteries, they reach about 15 pm width for a mean thickness of about 2.5 pm. The number of their myofibrils increases but their thickness remains constant (Figs. l-3), about 450 nm. These myofibrils fill about all the spindle-shaped section of the fibers in transversal section; they are rectangular except at the two extremities where they have a trapezoidal shape (Figs. 2, 3). Bfibers: Groups of B fibers alternate with A fiber groups. They have a triangular shape in transversal section and they do not reach the general cavity (Figs. I, 3). The fibers are irregular and not clearly individualized in transversal section (Fig. 3). They increase in

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dimension towards the mesenteries where their mean thickness is about 2.5 pm. The number of myofibril increases but their shape is very irregular (Fig. l), although their mean thickness remains constant, about 550 nm. The mitochondria are large and numerous they are sandwiched between the branched myofibrils (Fig. 3). II. Other constituents. The amount and relative distribution of different organelles (essentially: SR, invaginations, mitochondria) varies markedly according to the fiber kind. A jibers: the invaginations are between the myofibrils (Figs. 2, 3, 10, IQ, they form partition-like structures. The SR cisternae at the I band level connect each set of tubules running mainly under the plasma membrane and rarely under the invaginations (Figs. 3, 10, 18). The mitochondria are not intermingled with the myofibrils, they are generally in two rows at each side of the contractile apparatus (Figs. 2, 3, 10). B.fibers: the myofibrils of irregular shape

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are largely bordered by invaginations (Fig. 3). The SR cisternae at the I band level connect sets of tubules running under the plasma membrane and the invaginations (Figs. 14-17, 19, 20). Consequently B fibers are provided with peripheral and interior couplings, since dyads are mainly localized at the tubule level. The mitochondria are large and they fill the spaces between the contractile apparatus and the plasma membrane (Figs. 2, 3). Discussion This work is the first account of the fine structure of the primary musculature in Sag&a. The great difficulty with Chaetognaths is that we have, at present, very little information about their physiology, and particularly about that of the muscular tissue. In this discussion we shall attempt to correlate our morphological results and physiological data (Hyman, 1959). We also intend to note peculiarities of the Chaetognath phylum.

Fig. 3. Transversal section; a group of four B fibers is surrounded by two groups of A fibers. Note the abundance of the SR and the large mitochondria in B fibers. The asterisk shows the homogeneous sarcoplasm in one fiber. Arrow-heads point out two myotendinous junctions. Above the basement membrane lies a pluristratified epidermis with the nervous system and nervous ending-like structures. x 17,000. Fig. 4. Nervous ending-like structure lying against the basement membrane, above the primary musculature. One can see two kinds of vesicles: clear vesicles and densecored vesicles. x 70,000. Fig. 5. The outer nuclear envelope contributes membranes to the forming face of dictyosomes. These vesicles fuse into saccules. The arrow-head shows the continuity between SR and the perinuclear space. x 44,000. Fig. 6. Alkaline

phosphatase

activity in dictyosome.

x 46,000.

Fig. 7. Tangential section in the inner mitochondrial membrane showing the regular disposition of the tubular cristae, x 15,000. Inserr: matrix granules. x 42,000. Fig. 8. Portion

of granular

SR. x 68,000.

Fig. 9. Method of Mentre; strong reaction in the coat lining the general cavity and in granules in nucleus, mitochondria and sarcoplasm. x 56,000. Fig. 10. Transversal sections in A fibers; arrows point out continuity membrane and invaginations at the H band level. x 38,000.

between plasma

Figs. 1 l-1 3. Dyads respectively seen with conventional procedures; after glutaraldehyde fixation and sodium phosphotungstate impregnation; after potassium permanganate fixation. Fig. I 1, x 175,000; Fig. 12, x 104,000; Fig. 13, x 66,000.

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The main result in this work is the description, in a Chaetognath muscle (the primary musculature), of two ultrastructurally different kinds of muscle fibers, segregated in clusters. These results are corroborated by stereological studies on the development of the primary musculature (Duvert, 1975). Fiber diversity is well documented in several vertebrates and invertebrate phyla where different kinds of fibers can be segregated in distinct muscles (Millman, 1967; Wissocq, 1970; Smith, 1972; Smith et al., 1973; Franzini-Armstrong, 1973 ; Hoyle, 1975 ; Huddart, 1975; Miller, 1975); this diversity is also documented in Aschelminthes (Lanzavecchia, 1977; Clement, 1977) a somewhat artificial group sometimes related to Chaetognaths (Hyman, 1959). Fiber diversity in a single muscle is also well known (Hoyle, 1967, 1975; Brooke and Kaiser, 1970, 1974; Franzini-Armstrong, 1973 ; Huddart, 1975 ; Elder, 1975). In Sag&a there are two kinds of muscle fibers segregated in two muscles in the trunk, the primary and the secondary musculature (Perrier, 1897; Duvert and Salat, 1979); the primary musculature is a mixed population of two kinds of fibers, A and B.

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Frequently, in Vertebrates and Invertebrates, well-determined kinds of muscles or fibers give different responses to stimulation and are innervated by appropriate axons, but there are exceptions to this scheme (Dorai Raj, 1964; Franzini-Armstrong, 1973; Huddart, 1975; Hoyle, 1975). In Sagittu this point remains obscure. The primary musculature fibers are not individually innervated (Duvert and Salat, 1979) and the nervous system, in the trunk wall, seems exclusively confined to the epidermis (Burfield, 1927; Hyman, 1959). We have never seen nerves crossing the basement membrane towards the fibers, even at the ventral ganglion level, contrary to the hypothesis of Bullock (1965). In fact the assertion of Burfield (1927) seems correct when he says about the peripheral intraepidermic nervous system that ‘it would appear that the fibers of this system innervate both the muscles of the trunk and the tail’. In this line we have very frequently seen abundant nerve ending-like structures (as reported in Figs. 3 and 4) scattered against the basement membrane (Duvert and Salat, 1979); in spite of a careful search we have never encountered in the trunk end plates as those reported in the head musculature and

Figs. 14, 15. Serial sections in B fibers; at the I band level there are no imagination but cisternae (arrows) continuous with longitudinal tubules on each side of the invaginations, at the A band level. Mitochondria (asterisk) ‘can be branched as invaginations (arrow-head). The two kinds of sarcoplasm are clearly seen. Note the great development of SR and mitochondria. x 32,000. Fig. 16. Longitudinal section tangential to a myofibril in a B fiber; two sets of cisternae at each I band level are connected at the Z band level; these cisternae are continuous with longitudinal tubules along the A bands. Note the very large mitochondria. x 28,000. Fig. 17. Longitudinal section, tangential to a myofibril in a B fiber. Grey areas are invaginations continuous with the plasma membrane (asterisk). They seem to have fenestrations (arrow-heads). Above these invaginations are the sets of cisternae, with fenestrations (arrows), connected at the Z band level where the cytoskeleton is well developed (Duvert ef al., 1980). Cisternae are continuous with longitudinal tubules extending along the A band level. x 65,000. Fig. 18. Longitudinal section in an A fiber: the invaginations are devoid of surrounding SR. Cisternae are present at the I band level. Many invaginations are continuous along several sarcomeres (arrow-heads). Ribosomes are scattered in the contractile apparatus, mainly at the H band level. x 25,000. Figs. 19, 20. Longitudinal sections in B fibers; the invaginations side by longitudinal tubules continuous with the cisternae of the x 37,000; Fig. 20, x 65,000.

are flanked at each

1 band level. Fig. 19.

DUVERT

II 14nm

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19

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24nm

Fig. 2 I. Mean dimensions

of a dyad.

which are reminiscent of Nematodes (Duvert and Barets, 1972). It is somewhat difficult to conceive that a neurotransmitter has to cross the collagen network of the thick (about 0.25 pm) basement membrane, to induce depolarization. Furthermore invertebrate muscular synapses have generally a very different structure (Mill and Knapp, 1970; Osborne, 1975) but there is the notable exception of the myoepithelial cells of the proventriculus of a syllid (Smith et al., 1973). This latter organ has structural analogies with Sagittu trunk: nerves are separated from muscular tissue by connective tissue and nerve endings lie against a collagen network I .54.3 pm thick (vs. 0.25 pm in Sugitta). Another well-documented and analogous situation is reported in vertebrate sympathic nervous system where the effecters (smooth muscles) can be located several hundred microns from the liberation sites (Burnstock, 1970). We can conclude that the nervous ending-like structures (Fig. 4) can be interpreted as elements playing a role in the innervation control of the primary musculature. The different kinds of fibers are joined by a characteristic junctional complex with numerous gap junctions. The apparent absence of individual innervation for A and B fibers fits well with the possible electrical coupling of cells via gap junctions (Duvert and Salat, 1979), a situation closely related to that already mentioned in the proventriculus of SyIIid worms (Smith et al., 1973), and to the heart and smooth muscle of vertebrates for instance. Because of all these particularities, two main consequences arise: (1) in spite of the apparent common innervation a diversity of fiber kind is maintained, (2) the myofibrillogenesis zone adds new clusters of fibers

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allowing the enlargement of the trunk. The clusters of A and B fibers are primarily different (Duvert, 1975) and it seems that the differentiation and the regular disposition of A and B fiber groups are not under nervous control but are ‘myogenic’ properties. This apparent common innervation fits well with the syncytium-like functioning of the primary musculature (Hyman, 1959); but from our histological data (Duvert and Salat, 1979) one can conceive that, at least, one quadrant may have an autonomous functioning (and obviously the secondary musculature). Work is now in progress to elucidate the swimming behaviour of Sugitta for better understanding of this muscular diversity. In Vertebrates and Invertebrates the fiber kinds can differ largely on morphological grounds: structure and amount of SR relative development of T-system, density of couplings, myofibrillar characteristics as M and Z lines structure for instance and, particularly in invertebrates, sarcomere length and actin/myosin ratio (Auber, 1967; Hoyle, 1967; Baskin and Deamer, 1969; Hess, 1970; Smith, 1972; Huddart, 1975). Differences are also obvious at the physiological, biochemical and cytochemical levels (Barany, 1967; Peachey, 1968; Brooke and Kaiser, 1970), and different classifications have appeared. If the primary and secondary musculature differ markedly by their contractile apparatus (Duvert, 1975) this is not the case for A and B fibers in which the myofibrillar structure and the actin/myosin ratio are the same (Duvert, 1969). A and B fibers differ essentially at the membranous level (relative amount of SR and mitochondria, density of dyads . . .). This does not mean that there are no functional differences within each fiber kind. We know of, for instance, physiological or cytochemical differences between fibers which otherwise have identical contractile apparatus at the ultrastructural level. Furthermore, the primary musculature is a gradient of fibers, the youngest near the myofibrillar zone, the oldest near the mesenteries. We cannot say at present what is an ‘adult’ or a functional group of A or B fibers, Consequently the two fiber kinds we describe in this work are only indicative. If we compare two contiguous A and B fibers (i.e. of the same age) we note three striking differences (Figs. 2, 3). In B fibers:

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Dyads, which are mainly on longitudinal tubules, are more numerous, uniformly distributed, between the contractile apparatus and the plasma membrane and invaginations. (b) The SR is more abundant. (c) Mitochondria are more developed.

(a)

Since the myofibrils have approximately the same thickness (about 500 nm), the diffusion distances between the invaginations and the center of the myofibrils are roughly the same. On the contrary, the distances between SR and mitochondria on the one hand, and the center of the myofibrils on the other, are considerably lowered in B fibers. Then it appears clearly that sites which can contain Ca2 1,the trigger of muscular contraction, are more developed in B fibers (Ebashi, 1976; Carafoli ef al., 1976). This means that Ca2+, (1) can be delivered in much larger quantity and more uniformly in B fibers and (2) can be sequestered more effectively in B fibers. All other characteristics being equal we can postulate that B fibers could be more rapid than A fibers. Many authors have noted a correlation between the speed of contraction and/or relaxation and the development of the stores able to sequester Ca2+, in Vertebrates (Jewett ef a/., 1971; Kilarski, 1973; Eisenberg et al., 1974, etc.) and Invertebrates (Rosenbluth, 1969; Elder, 1971; Cochrane and Elder, 1972, etc.). There is also an apparent correlation between the relative abundance of couplings and the speed of contraction in vertebrates (Page, 1965, 1969) and invertebrates (Huddart and Oates, 1970; Jahromi and Atwood, 1971; Atwood, 1973). Ail these observations fit well with our hypothesis (but there are notable exceptions as, for instance, the heart of Limulus (Forbes et al., 1972); in comparable muscles where the rate of length change is related to cyclic calcium fluxes, the relative importance and distribution of SR and couplings seem related to the speed of activation and de-activation of the contractile apparatus, and to the speed of the contraction-relaxation cycle. Sag&a movements are dependent on extracellular Ca*+; animals cannot swim in sea water without calcium (this phenomena is reversible) or containing lanthanum or manganese (Duvert, 1975). Devine et al. ( 1972) have noted a correlation, in smooth

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muscles, between the dependence on the extracellular Ca and the development of the SR. It is also possible that B fibers are less dependent on the extracellular Ca than A fibers because of the larger development of SR and mitochondria (and these animals are collected in the Bassin d’Archacon where the salinity is not uniform and constant, but we must point out that animals are particularly abundant in regions where physicochemical parameters of water are stable and comparable with those of the ocean; nevertheless they can bear a fall of 50% in salinity (Duvert, 1975). The relative development of mitochondria and their distribution in B fibers put these organelles in a central position for the regulation and distribution of metabolites (Ca, ATP .). They can also underline differences between A and B fibers in terms of fatigability for instance (Elder, 1975), particularly in these fibers without glycogen and lipid reserve cytochemically detectable. The movement of Sagitta in sea water is not well documented. As Hyman points out : ‘swimming consists of short swift forward darts, each covering a distance of around 5 cm, this is followed by gliding on the momentum of the darts and return to floating’; the forward dart is extremely rapid: ‘the movement is so rapid that it appears to the naked eye like a trembling’. This very fastness of contraction (Sagitta= arrow) can be correlated with some characteristics. The subdivision of the contractile apparatus into small ribbon-like myofibrils of about 500 nm thickness (among the lowest values reported in vertebrate and invertebrate myofibril mean thickness-Huddart, 1975) and the very short sarcomeres (1.5 pm length) with narrow I bands (Duvert, 1969, 1975) are two characteristics found in many fast acting invertebrate muscles (Smith, 1966; Elder, 1975). At least in B fibers mitochondria constitute 30% of the fiber volume (vs. 10% in A fibers) (Duvert, 1975), a volume related to that found in flight muscles (Elder, 1975) and which can indicate a high level of aerobic metabolism. This last property was noted by Reeve (1966) and Reeve et al. (1970), who observe a very high oxygen consumption in Sagitta. Two data can also account for this high level of consumption: (I ) the musculature is devoid of glycogen and lipid reserves, as cytochemical results indicate (and Sagitta

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has not a sustained swimming), (2) the musculature has a large system of invaginations which increases the fiber surface in this tissue devoid of circulatory and respiratory apparatus. A and B fibers bear in common some characteristics unusual among muscle fibers : the two kinds of cytoplasm, as in hepatocyte for instance (Bloom and Fawcett, 1968); well-developed dictyosomes, the nuclear envelope contributing to the forming face as in the vertebrates, myocardium for instance ‘(Bloom and Fawcett, 1968); the SR polymorphism with the rough endoplasmic reticulum portions; the abundance of ribosome-like particles in the sarcoplasm and the contractile apparatus at the I and H bands level. Some of these characteristics are more generally associated with secretory cells than muscle fibers. As we have pointed out, Ggittu has no circulatory, respiratory or excretory apparatus, so many organelles may be implicated in various functions not necessarily exclusively related to Ca2+ regulation (Martonosi, 1972). Dyads are related to those described in a large variety of muscles (Rosenbluth, 1972; Franzini-Armstrong, 1973; Smith, 1966). They are periodically disposed along the A bands according to the myofibrillar striation. In spite of the absence of a true T-system (periodically and transversally developed), the SR is composed of units (Figs. 16, 17). Their periodicity is the myofibrillar periodicity. These two facts point out the importance of the contractile apparatus in the organization of the fibers. Our data show that muscular contractions

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can act through three important mechanical devices: (1) the basement membrane on which muscle fibers insert; it is a flexible skeleton; (2) the incompressible volume of the general cavity filled with a liquid (Burfield, 1927); it represents an hydrostatic skeleton; (3) an important cytoskeleton (Duvert et a/., 1980), it shears out the contraction forces in the musculature. In the present work and elsewhere (Duvert and Salat, 1979) we have presented data which seem to indicate that the movements of Sag&a trunk may be complex and modulated in spite of their well-known speed. We think that the functioning of the trunk musculature must be more sophisticated than previous works suggested (Burfield, 1927; Hyman, 1959). All the cytological differentiations we have described (and those supplying the absence of true apparatuses) underline the importance of the locomotive muscles and show up the high degree of specialization in this very particular phylum. The fine structure of the contractile apparatus of the primary and secondary musculature (Duvert, 1975) and the cytological characteristics of the A and B fibers separate the Chaetognaths from the animals with ‘obliquely striated muscles’ or ‘helical muscles’, primarily annelids, nematodes and molluscs. Acknowledgements We wish to thank Mr C. Cazaux, co-director of the Institute of Marine Biology from Arcachon, and the sailors who provide us with Chaetognaths.

References ATWOOD, H. L. 1973. Crustacean muscle. In The Structure and Function of Muscle (ed. G. H. Bourne), Vol. 1, pp. 421-489. Academic Press, New York and London. AUBER, J. 1967. Distribution of the two kinds of myofilaments in insect muscles. Am. Zoo/., 7, 451-456. BARANY, M. 1967. ATPase activity of myosin correlated with speed of muscle shortening. J. gen. Physiol., 50, 197-216. BASKIN, R. J. and DEAMER, D. W. 1969. Comparative ultrastructure and calcium transport in heart and skeletal muscle microsomes. J. Cell Biol., 43, 610-617. BLOOM, W. and FAWCETT, D. W. 1968. A Textbook qf Histology, 1033 pp. W. B. Saunders Company. BROOKE, M. H. and KAISER, K. K. 1970. Muscle fiber types: how many and what kind? Arch. Neural., 23, 369-379. BROOKE,M. H. and KAISER, K. K. 1974. The use and abuse of muscle histochemistry. 228, 121-144.

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BULLOCK, T. H. and HORRIDGE, G. A. 1965. Struc’turr and Function irr the Nervous Systems Vol. II, pp. 1561-1564. W. H. Freeman and Company. San Francisco and London.

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