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AMER. ZOOL., 29:5-18 (1989) Evolutionary Patterns of Axial Muscle Systems in Some Invertebrates and Fish1 Q. BONE The Marine Laboratory, Citadel Hill, Plymouth, U.K. SYNOPSIS. Axial muscles used for oscillatory swimming are found not only in fish and other vertebrates but also in some protochordates and invertebrates. Chaetognaths have unsegmented locomotor musculature with some unusual features, but larvacean tunicates and the tadpole larvae of ascidians show the simplest variant of the chordate segmented axial muscle arrangement for flexing a notochordal column, where all muscle cells along one side are electrically coupled. With amphioxus, the basic fish myotomal layout is established, with two main fibre types probably used for different patterns of swimming (as in fish). There are, however, several unique features, including the flattened fibre shape and the paramyosin system of the notochord. Agnatha have two fibre types in the myotomes, a third type perhaps being a developmental stage in the ontogeny of fast fibres. In lampreys, the central fibres of the characteristic fibre sandwiches in the myotomes are flattened (though less so than in amphioxus); they have a dual innervation of unknown function seen also in the fast fibre system of many Gnathostome fish groups. Hagfish fast fibres are not flattened nor do they have a dual innervation. Gnathostome fish axial muscles are strikingly uniform in design with two possible exceptions: (1) higher teleost fast fibres which, unlike those of other groups, are multiply-innervated and (2) tonic fibres in a few fish, which seem not to be involved in locomotion. INTRODUCTION As well as fish and some higher vertebrates, several deuterostome groups generate swimming thrust by using axial muscles to produce alternating lateral flexions of the body. In the tunicates which swim in this way, the axial musculature is segmented and operates to flex a notochord or vertebral column, just as in fish, so that the ascidian tadpole larva and the adult larvacean perhaps provide glimpses of an early stage in the evolution of the vertebrate axial musculature system. However, the rather uniform dual fibre pattern of the axial musculature in the myotomes of all fish groups, despite their distant taxonomic relationships, is first seen in amphioxus (Branchiostoma). There are good reasons to suppose that the fundamental duality of fibre types in the axial musculature arose in response to the stringent demands that the density and viscosity of an aquatic medium impose on fish-like forms swimming through it (Bone, 1978), so that it is less surprising than it might 1 From the Symposium on Axial Movement Systems: Biomechanics and Neural Control presented at the Annual Meeting of the American Society of Zoologists, 27-30 December 1986, at Nashville, Tennessee. seem at first sight that all fish groups have this arrangement. This review of axial muscle fibre types and their innervation deals briefly with the non-segmented chaetognath system, and the segmental electrically-coupled axial muscle cells of tunicates, before considering the myotomal axial system of amphioxus and fish in more detail. In these sections, it is argued that the functional division of the axial musculature into two differently specialised fibre types, each with a different innervation pattern, is an ancient one and homologous in all fish groups. The significance of other fibre types is considered and, paradoxically, it seems that true tonic fibres are found in some fish; however, they may play no part in locomotion. The innervation patterns of fish fast muscle fibres are considered, and it is concluded that the fast fibres of higher teleosts have been uniquely modified from an ancestral pattern seen today in all other fish and in urodele amphibia. Table 1 summarises the organisation of the axial muscle systems in the forms considered in this review. The group on the left of Table 1, the Chaetognatha, is very different from all the others; here there is an alternative solution to the design of an axial muscle system for providing thrust by TABLE 1. Comparisons between axial muscle fibres in different groups. Gnathostome fish Tunica la Chaelognathu Ascidian tadpole Segmented Non-segmented Many muscle fibres Gap junctions but no coupling Cholinergic Doubtful chordate relationships A different solution using axial muscle for oscillatory swimming Many muscle cells in tiers Agnatha Acrania Larvacean Few muscle cells All coupled Lampreys Hagfish Fibres flat and very thin Dual fibre types and intermediate Flattened Dual fibre types fast fibres and intermediate Coupled Fast coupled Cholinergic Single Dual innervation innervation No myotomes Cholinergic Unusual central endplates Notochordal paramyosin First chordate-like arrangement, but only one muscle fibre type Some special features, but basic fish plan None coupled Elasmobranchs Dipnoi Chondroslei Lower teleosts Higher teleosts Dual fibre types and some apparently tonic fibres in a few species No coupling in adults Cholinergic Cholinergic Dual fast Single, fast innervation innervation In most, dual Multiple innervation of innervation of fast fibres fast fibres Onwards and The "typical" upwards to vertebrate the Amphibia pattern (since higher teleosts are the most abundant vertebrates) Lampreys closer to basic fish plan? AXIAL MUSCLE: FISH AND INVERTEBRATES 06 FIG. 1. Scheme of arrangement of axial musculature in the chaetognath Sagitta. The motor innervation of the two muscle fibre types (B fibres dark stipple) lies on the outer side of the basement membrane (BM) under the multi-layered outer epithelium (OE). Dark blocks indicate numerous gap junctions between muscle fibres. At lower right, an extracellular record of spontaneous swimming activity (from a suction electrode enclosing the tail tip) shows that Sagitta can vary its movements. Time marker: seconds. found, one much less abundant than the other. The A fibres make up some 85% of the total, and are larger than the B fibres that CHAETOGNATHA lie in small clumps between them. The two Chaetognaths or arrow worms are a small fibre types differ in myofibrillar array, and marine group, almost all planktonic, whose the B fibres contain relatively more SR and 50 or so species are of rather uniform mitochondria (Dress and Duvert, 1983). design so that although only a couple of The A fibres are some 300-400 pm long, species in the genus Sagitta have been 1-2 nm wide, and around 8 fim deep, and examined in detail, it is probably safe to unlike the smaller B fibres, span the whole suppose that what follows is typical of all. depth of the muscle layer. Unfortunately, The axial musculature is non-seg- nothing is known of possible physiological mented, and forms a tube around the fluid- differences between these two fibre types, filled body cavity. External to the muscle but it seems clear that the much more layer is a thick basement membrane con- abundant A fibres are fast fibres, used for taining helically wound collagen layers, the rapid darts the animal makes to catch which forms the outer limit of the hydro- its prey and for the brief swimming bursts static skeleton. Figure 1 illustrates the sys- made every 20 sec or so which drive it tem. Sagitta swims by oscillating its body in upwards in the water column (Feigenbaum the dorso-ventral plane (like a cetacean), and Reeve, 1977). Recent physiological and does so in rapid darts; the animals are studies (Duvert and Savineau, 1986) have formidable predators on copepods, which shown that acetylcholine is probably the they seize with the spines on the side of the neuromuscular transmitter. Curiously, head. Ultrastructural studies by Duvert and although the muscle fibres are coupled by his colleagues (Duvert and Salat, 1979; numerous gap junctions, they do not appear Duvert et al., 1980) have shown that two to be electrically coupled, and in line with types of cross-striated muscle fibre are this, each fibre is probably separately body oscillations that is interesting because it presents some rather mysterious features. 8 Q. BONE innervated (Bone et ai, 1987), by nerve terminals lying outside the basement membrane, and hence not in direct contact with the muscle fibre membrane (Duvert and Barets, 1983). Here then is a very unusual axial muscle system that evidently has little to do with the chordate arrangement. Chaetognath affinities are entirely obscure, and even though some zoologists have allied them with chordates, others have felt that molluscs, coelenterates, or annelids are their closest allies. Thus their possible chordate affinities cannot be taken too seriously! TUNICATES lower tier is also innervated (Fig. 2a). All muscle cells are coupled dorso-ventrally and antero-posteriorly by gap junctions, and in this way, all the muscle cells along one side of the body are electrically coupled (Fig. 2b, c). Electrophysiological investigations have been limited to Dendrodoa (Mackie and Bone, 1976 and unpublished observations) and to Ciona, and in Dendrodoa at least, three types of electrical event can be recorded from the muscle cells of intact larvae (Fig. 2d). The largest, rapid nonovershooting spikes are seen as the tail rapidly flexes unilaterally. Longer bursts of smaller spikes are seen during the short bursts of symmetrical swimming movements; and finally, much smaller events can be recorded which do not correspond to obvious tail movements. At present, these results are rather hard to interpret, since it is difficult to see (for example) what the function of the dorsal and ventral innervation of the most anterior tiers of muscle cells may be, or indeed why each dorsal muscle cell should be innervated if the whole system is closely coupled. Perhaps the largest potentials accompanying unilateral flexions result from simultaneous activation of the dorsal and ventral innervation of the anterior cells, whilst symmetrical swimming only involves the dorsal innervation along the tail; but these are only speculations. What does seem clear, though, is that the result of this rather complicated arrangement is that different kinds of movement can be produced from a single type of axial muscle cell. Since Kowalewsky's demonstration (Kowalewsky, 1867, 1871) that the ascidian tadpole larva was chordate-like, it has been clear that the tunicates are related in some way to vertebrates, although their exact status has been disputed. In any event, ascidian tadpole larvae, adult larvacean tunicates, and some doliolid larvae all have a finned tail supported by a notochord, and flexed by segmented axial muscle cells. The myogenic tail movements of some doliolid larvae (others only have vestigial tails [Braconnot, 1970]) are desultory and can hardly be of much significance in distributing this short-lived larva of a planktonic adult, but those of the ascidian tadpole and the larvacean adult are quite different. Adult ascidians are sessile, and larval locomotion plays an essential role in distribution and site selection, whilst in larvaceans, tail movements are used not only for swimming, but also for producing the feeding current within the filtering house, and indeed, for expanding the house itself. Larvaceans In some respects, larvaceans are simpler Ascidian tadpole larvae than ascidian tadpole larvae, for there are The segmented caudal muscle cells have only ten caudal muscle cells, and they are been examined in several species, and are arranged in a single row rather than in tiers rather similar in all, apart from differences in each segment. Each is coupled electriin the sarcotubular systems probably cally to those adjacent (Fig. 3a) but in conresulting from size differences between trast to the ascidian tadpole, where there species (Burighel et ai, 1977). Three or are apparently no motoneuron cell bodies sometimes four tiers of muscle cells are along the dorsal nerve cord in the tail, in found in each segment; only the dorsal row larvaceans the muscle cells are innervated is innervated from axons in the dorsal nerve by motoneurons whose cell bodies are segcord, except at the anterior end, where the mentally arranged in the tail (Fig. 3c). Each AXIAL MUSCLE: FISH AND INVERTEBRATES FIG. 2. Dendrodoa. (a) Schematic diagram of tadpole larva. There are three tiers of muscle cells in each segment; the upper (U) is innervated by a few motor axons in the dorsal nerve cord above the notochord (N). A second motor innervation (VM) supplies the most anterior cells of the ventral row (L). The middle tier (M) and the caudal lower tier are not innervated, but are coupled to neighbouring cells. All motoneuron somata lie in the brain (B). (b) Current steps injected into a muscle cell of the upper tier (above) are seen in a cell of the ventral tier (below), (c) Simultaneous intracellular records from ipsilateral muscle cells (anterior below) during a burst of symmetrical swimming, showing close coupling, (d) Intracellular record from caudal muscle cell showing two series of unilateral flexions (u) followed by a burst of symmetrical swimming (s). Low amplitude electrical activity following the first series of unilateral flexions is not correlated with obvious tail movements. Vertical scale as (c). muscle cell receives two kinds of motor innervation. One type forms large terminal rosettes, the other elongate finer beaded terminals; under the former alone is acetylcholinesterase found (Flood, 1973). Here then is the same paradox as in the ascidian tadpole, viz. individual innervation of electrically coupled axial muscle cells, though in larvaceans the situation is complicated by the dual innervation of all muscle cells. Once again it has to be admitted that the operation of the system is not well understood, but as in ascidian tadpoles, it permits different types of movement from a single type of muscle cell. In the case of larvaceans, however, some clues to the variety of contractile responses from this single fibre type are provided by recordings of escape swimming responses (Bone, 1984). In oikopleurid larvaceans the outer epithelium is excitable, and mechanical or electrical stimulation of the epithelial cells evokes rapid escape swimming bursts. Intracellular records from the muscle cells during normal and escape swimming show that not only does the frequency of swim- 10 Q. BONE Fie. 3. Oikopleura. (a) Current steps injected into one caudal muscle cell (upper) are recorded in the next but one cell along the tail, (b) Intracellular record of muscle potentials during normal swimming (vertical and horizontal scales as in (d)). (c) Schematic diagram showing dual innervation of caudal muscle cells from motoneurons in cord driven by caudal ganglion (CG), and connexion of the outer excitable epithelia (stippled) with the caudal ganglion via the paired receptors whose bristles touch the walls of the house (H). The anterior epithelium (not stippled) is not excitable, (d) Intracellular record of muscle potentials during escape swimming burst (to same scales as (b)). ming movements increase during escape swimming (see also Bone and Mackie, 1975) but the amplitude of the spike-like potentials much increases (Fig. 3b, d). It is possible therefore that the second motor innervation of the caudal muscle cells modulates the responses evoked by the cholinergic endings. The transmitter employed at the second motor ending is unknown (unfortunately neuropeptide immunocytochemistry has yet to be applied successfully to larvaceans). Evidently, ascidian tadpole larvae and oikopleurid larvaceans (perhaps indirectly derived from ascidian tadpoles by neoteny) show two rather different variations on an axial muscle theme where only a single fibre type is found. These are almost all very small animals in which the tail is under 5 mm long (there are a few giant larvaceans whose tails may be 30 mm or longer, but these have not been examined in detail), and it is perhaps the small scale of their design that has prevented the "invention" of the added complication of two muscle fibre types specialised for different types of swimming such as are found in all fish. The obverse of this coin, however, is that to enable them to obtain a variety of patterns of tail movement from the single type of muscle fibre, they are obliged to use a more complex system of control than is found in fish, involving electrical coupling between muscle cells as well as dual innervation. ACRANIA Although there are several unique features, the axial musculature of amphioxus AXIAL MUSCLE: FISH AND INVERTEBRATES 11 FIG. 4. The amphioxus axial system (partly after Bone 1984). The notochord (N) contains paramyosin fibres, innervated by motor fibres (NM) in the ventral part of the cord. Two types of muscle fibre (SUP: superficial) and (D: deep) send tails to different regions of the central motor end-plate, where (inset above) their motor terminals contain vesicles of different sizes and where the muscle cell tails seem to be linked by gap junctions. Intermediate fibres (INT) send their tails with the deep fibres. (Re-drawn from Flood, 1968, 1969.) is constructed essentially on the fish plan, with myosepta dividing myotomes that contain two histologically distinct muscle fibre types (Flood, 1968), which probably (though not certainly) are used for different modes of swimming, as are the two main fibre types in the fish myotome. In amphioxus, the two types (superficial and deep) differ in mitochondrial and glycogen content, volume of sarcotubular system, and in position just as in fish. As in most fish, the mitochondria-rich fibres lie superficially in the myotome (Fig. 4), and form a much smaller part of the myotome than the deeper mitochondria-poor fibres, making up only around 1% of the total. An intermediate fibre type also occurs, but the significance of this fibre type is unclear; perhaps it represents a developmental stage in the formation of the deep mitochondriapoor fibres. Amphioxus muscle fibres are very different from those offish in their shape and mode of innervation (Fig. 4), for they are flattened, very thin plates with curious thin tails which approach the sheath of the central nervous system to reach the neuromuscular junction just outside the sheath (Flood, 1966). Here, the tails of the mitochondria-rich superficial fibres form a separate bundle above the much larger bundle formed by the tails of the deeper fibres. The nerve terminals associated with each type of muscle tail contain vesicles of different diameter: those in nerve terminals associated with the tails of the superficial fibres are electron-lucent and around 70 nm diameter, whereas those associated with the deeper fibres are also electron-lucent but around 117 nm in diameter. Several points remain to be clarified about this remarkable arrangement. First, Flood (1966) observed apparent gap junctions not only between the tails of the muscle fibres (including between tails of deep and superficial fibres), but also between the tails and sheath cell processes, and suggested that the apparent gap junctions might be artefactual. However, Hagiwara and Kidokoro (1971) have demonstrated electrical coupling between amphioxus muscle cells, so that it is unclear whether the two types of muscle fibre can be activated independently. Secondly, Flood (1974) has shown acetylcholinesterase at both superficial and deep muscle fibre cen- 12 Q.BONE tral endings, despite their different vesicle morphology, so that both fibre types seem to have a cholinergic innervation. The other striking feature of the axial system, peculiar to amphioxus, is that the notochordal lamellae are contractile and contain a thick and thin fibre array very like those of the slow adductor or "catch" muscles of lamellibranch molluscs. The thick filaments are up to 1,500 A in diameter and show a regular 145 A periodicity; they contain paramyosin (tropomyosin A) (Flood, 1967), as do the molluscan muscles. Contraction of the notochordal cells via cholinergic synapses at the base of the spinal cord modifies the stiffness of the notochord (Flood, 1969; Flood et al., 1969; Guthrie and Banks, 1970). Guthrie and Banks have shown that electrical events in the notochordal cells follow spikes in the axons of the giant Rhode cell system of the spinal cord which is also involved in myotomal muscle contraction, and suggest that the notochordal system is chiefly important during fast swimming. During bursts of rapid swimming, amphioxus contracts its myotomes at frequencies up to 20 Hz (Guthrie and Banks, 1970), and at these frequencies the notochordal muscles respond tonically. In fast swimming the angles of flexion of the body are small, so Guthrie and Banks suggest that the tonic notochordal response effectively doubles the stiffness of the notochord and hence the elastic recoil of the system. During slow swimming at contraction frequencies of 12 Hz, notochordal responses are phasic and do not impede initial body flexures. Webb (1973) suggests in addition, that control of notochordal stiffness along the body permits amphioxus to swim and burrow in the sand equally well in either direction (as indicated by its common name). From the evolutionary point of view it is remarkable that this complex notochordal system is unique to amphioxus, and considering that it is alone amongst the chordates in being able to burrow very rapidly head or tail first into the sediment, it seems most likely that Webb's (1973) opinion is correct, and that the notochordal system arose in conjunction with the burrowing habit. It would be worth examining the notochord of amphioxus larvae, in particular, those of the apparently neotenous pelagic "amphioxides" larvae (Wickstead, 1964). AGNATHA In lampreys and hagfish, two muscle fibre types are arrayed in characteristic sandwiches in the myotomes; in hagfish both types are circular, but in lampreys the central fast fibres in each sandwich are flattened plates (Fig. 5). In both groups the slow parietal fibres are multiply-innervated, whilst the fast fibres are focallyinnervated2; the two fibre types differ also in mitochondrial content, myofilament pattern and so forth, as they do in other fish. As in amphioxus, there are some central fibres in the muscle sandwiches of both lampreys and hagfish, which lie next to the parietal fibres and are in some respects intermediate between the two main fibre types (Flood and Storm-Mathisen, 1962; Dahl and Nicolaysen, 1971 for hagfish; Lie, 1974 for lampreys). In lampreys these intermediate fibres are electrically-coupled to the other central fast fibres (Teravainen, 1971) so it does not seem that they may be separately activated. This problem has been pointed out previously, in considering tunicates and amphioxus (pp. 8 and 11), and it may perhaps be resolved by supposing that the degree of gap junction permeability and hence electrotonic coupling can be regulated in vivo, to permit separate activation of coupled muscle fibre types. Whilst there is no direct evidence for this suggestion, gap junction permeability can be altered experimentally, e.g., by changes in intracellular pH (Turin and Warner, 1980). In hagfish, where the fibres are not electrically coupled, Andersen,?* al. (1963) have not observed any physiological dif- 2 The patterns of innervation of axial muscle fibers distinguished are: Focal: the fiber is innervated at a single site only. Multiple: the fiber is innervated at several sites along its length; if by several different axons, this is polyneuronal multiple innervation. Terminal: the innervation site is at one or both ends of the fibre. Dual: two different axons contribute to closely adjacent innervation sites on a fibre with terminal innervation. 13 AXIAL MUSCLE: FISH AND INVERTEBRATES Lampetra Myxine FIG. 5. Myotomes of lamprey and hagfish seen from inner aspect. Note lack of innervation of some central and intermediate fibres in lamprey. There is no coupling between muscle fibres in hagfish. 2 axons (1 & 2) supply lamprey central fibres. (Modified from Bone, 1978.) ference between intermediate and fast fibres. Perhaps, as in amphioxus, these intermediate fibres may be developmental stages in the formation of the central fast fibres (they are lacking in ammocoete larvae of lampreys). An interesting and puzzling feature of the innervation of lamprey fast central fibres is that their motor end-plates are supplied by two apparently separate axons, whose terminals contain vesicles of different diameters (Kashapova and Sakharov, 1976). In hagfish, only a single axon supplies each fast muscle fibre (Bone, 1963). The dual innervation of lamprey fast fibres is found also in elasmobranchs and some other fish groups (see below) and deserves further investigation. In this difference in innervation pattern, as in muscle fibre shape and in many other features, hagfish differ from lampreys, and lampreys are closer to the cephalochordate arrangement. GNATHOSTOME FISH In all gnathostome fish, from elasmobranchs to dipnoi, there are two main axial muscle fibre types, very similar in all; where investigations have been made, each type performs a different function (references in Bone, 1978). Slow red fibres usually lie superficially, forming up to 15% of the total myotomal cross-sectional area, and beneath them is the main mass of white fast fibres. It is this zoning of the fibre types within the fish myotome that makes the study of the structure, biochemistry and function of the different fish fibre types much simpler than in higher vertebrates. Histological, ultrastructural, histochemical, electrophysiological, and biochemical studies all support the view that the slowfibresare designed for sustained aerobic operation during cruise swimming, whilst the fast fibres are specialised for anaerobic glycolysis during short bursts of rapid swimming. Technical difficulties have so far prevented in amphioxus the same kind of direct evidence for this generalisation provided by electromyographic studies in gnathostome fish, but there seems no reason to doubt that the duality of function in gnathostome axial muscle arose early in chordate phylogeny. No biologist will be surprised that there are complications which have to be introduced into this simple picture of the gnathostome fish axial musculature; probably the most interesting are the questions raised by other fibre types, and by the patterns of innervation found in different groups. Whilst recognising the fundamental division of myotomal fibres into slow red 14 Q. BONE W2 different manner, and the slow fibres do not propagate action potentials. The distinction between them is one of kind, rather than degree, as it is in mammals. It is therefore inappropriate to consider fish fast and slow fibres as part of a continuum (as in mammals), although within fast and slow categories it seems sensible to adopt this approach rather than distinguishing a multiplicity of sub-types. FIG. 6. Outer part of Scyliorkinus myolome in trans- Slow fibres verse section, showing superficial (tonic?) fibres (S), The slow fibres are invariably multiplyand the inner and outer red (Rl & R2), and white (Wl & W2) fibres. A coiled indirect proprioceptive innervated by two or more axons, bearing ending is seen between skin and outer myotomal sur- many cholinergic end-formations along face. their length, and despite Stanfield's finding that, in dogfish, some possessed sufficiently large inward Na+ currents in voltage clamp fibres and fast white fibres, several workers experiments as to suggest that they could have sub-divided these categories, or dis- propagate action potentials (Stanfield, tinguished other fibre types that do not fit 1972), action potentials have never been into them. For example, dogfish slow and observed from slow fibres in any fish fast fibres may each be sub-divided on the species. Slow red fibres specialised for susbasis of enzymatic activity and position tained low speed swimming are rather uniwithin the myotome (Bone and Chubb, form in design in all fish, but in a few fish 1978). In dogfish and some higher teleosts, small numbers of a different type of slow a small category of myotomal fibres are fibre have been found. unlike either slow or fast fibres and appear to be true tonic fibres (Kilarski and Tonic fibres Kozlowska, 1983; Bone etal., 1986). Again, In several freshwater teleosts such as in many higher teleosts, there are distinct Gasterosteus (Kilarski and Kozlowska, 1983) pink or intermediate fibres lying between "tonic" fibres have been described, which the outer slow red fibres and the deeper differ markedly from typical slow fibres. white fibres, which are not only interme- They stain weakly for succinic dehydrodiate in mitochondrial and enzyme content genase, have relatively sparse mitochonand so on, but also appear to be recruited dria and at the ultrastructural level show at sustained swimming speeds intermediate long sarcomere lengths, thick Z-lines, and between slow cruise and maximum burst lack M-lines. In these respects they resemspeed (Johnston et ai, 1977). ble amphibian tonic fibres, but so far the In mammals, where all fibre types are mechanical properties of this kind of fibre focally-innervated and propagate action have only been examined in dogfish (Bone potentials, their inherent plasticity is man- et al., 1986). In each myotome of Scylioifested by the way in which their ultrastruc- rhinus an interrupted layer of 80-90 superture, biochemistry and mechanical prop- ficial fibres lies outside the slow fibre zone, erties can be altered experimentally by just under the connective tissue sheets of appropriate chronic electrical stimulation, the sub-dermis (Fig. 6). They are entirely so that different fibre types represent stages distinct from the underlying slow red fibres within a dynamic equilibrium, as Pette for they are of much larger diameter, con(1985) remarks. Although there is no rea- tain2+few mitochondria, and stain weakly for son to suppose that fish muscle fibres are CA -activated ATPase (Table 2). They any less plastic, they differ importantly from make up less than 0.5% of total myotomal mammalian fibres, because the slow and fibre number (0.6% of total cross-sectional fast fibre types are innervated in a quite area at the post-anal level, compared with 15 AXIAL MUSCLE: FISH AND INVERTEBRATES TABLE 2. Comparison of some structural and mechanical properties of muscle fibre types in the myotomes o/Scylio- rhinus.* Fibre type Innervation % mitochondria! volume Maximum Ca2+-activated force developed (kN m~2) Maximum Ca2+-activated force developed corrected for myofibril density (kN m"2) Unloaded contraction velocity V,bcl(L0 sec"1) V ^ U sec-) Superficial Multiple 2.39 ± 0.49 49 ± 4 Slow red Multiple 21.55 + 3.39 70 ± 8 Fast white Focal terminal 0.99 ± 0.16 180 ± 5 65 113 231 0.47 ± 0.03 0.58 1.44 ± 0.01 1.53 4.4 ± 0.3 4.5 * From Bone ti al, 1986. 24.4% for slow fibres and 75% for fast importance) has not yet been carefully fibres). Single skinned superficial fibres examined and, so far as I am aware, elecproduce around half the maximum force tromyographic and mechanical studies of per unit cross-sectional area produced by the axial musculature in terrestrial slow fibres and around a quarter of that amphibia have yet to be performed to see produced by fast fibres, and their contrac- if some of the fibre types there can be tion velocity is correspondingly lower than regarded as tonic and equivalent, for that of the other fibre types (Table 2). example, to the tonic type 5 fibres of XenAs can be seen from Table 2, the mul- opus limb muscles (Lannergren, 1978). tiply-innervated Scyliorhinus superficial fibres fulfil many tonic fibre criteria, dif- Fast fibres fering from those of amphibia mainly in In all fish groups apart from the higher their large diameter. It is difficult to know teleosts, the fast fibres are focally-innerwhat role they play, for they have only been vated and almost always terminally-innerfound in Scyliorhinus canicula and S. stellaris, vated (Bone, 1970; Bone and Ono, 1982). and they are absent from all other sharks In contrast to the slow fibres, they propaexamined as well as from batoid axial mus- gate muscle action potentials. In sharks, cles. In view of their number and the low fast fibres are innervated only at one end, forces they can exert, it seems very improb- but in some teleosts, such as catfish (Barets, able that they can play any part in loco- 1961) they are innervated at each end. An motion. interesting feature of the terminal innerIn some cases at least, the large end-for- vation in several groups (including urodele mations along the superficial fibres are sup- amphibia) is that the terminal endplate is plied by axons which branch also to inner- derived from the endings of two separate vate the outermost slow fibres, but it is axons, which may contain different types unclear whether this means that the super- of synaptic vesicles (Bone, 1972). Ono ficial fibres are activated during slow swim- (1983) has surveyed the occurrence of such ming driven by the slow fibres. It seems dual innervation of fast fibres, adding one most probable that they are not and, par- clupeid and holocephali to the forms preadoxical as it may seem, the superficial viously reported, which include sturgeons fibres are not involved in locomotion; per- and axolotls, as well as elasmobranchs (Best haps they have a postural role in keeping and Bone, 1973; Sakharovand Kashapova, head and tail slightly raised when the fish 1979). There seems good reason to suprests on the bottom. If this is correct, the pose that dual innervation is the primitive superficial fibres would be equivalent in innervation pattern of vertebrate fast axial function (as well as in their structure and muscle fibres, but there is as yet no evimechanical properties) to the tonic fibres dence for (a) different transmitters in the two axons, nor (b) any difference in their of amphibian limb muscles. function if both are cholinergic. It is an The origin of tonic fibres in terrestrial obvious possibility that one axon might forms subject to gravity from the ancestral release a "modulator" neuropeptide to aquatic condition (where gravity is of little 16 Q. BONE alter the contractile response resulting from cholinergic release by the other, but it is not clear why the fish should need to grade the fast muscle system in this way if it is only employed during burst swimming. In a very few teleost fish, fast muscle fibres appear to have both terminal and some degree of multiple innervation. One of the groups of the deep sea Stomiiformes, the Photoichthya (Weitzman, 1974) has a few axons passing through the fast zone of the myotome, and innervating fibres in a distributed manner, but also has fibres which are innervated terminally (Bone and Ono, 1982). It would be interesting to examine larval stomiiforms to see if the beginnings of multiple-innervation, as seem to be shown by the adults, appears only at metamorphosis. In higher teleosts, fast fibres are multiply-innervated and may have relatively more mitochondria and a more extensive vascular bed than the apparently entirely anaerobic fast fibres in those groups where they are focally innervated. Electromyographic investigations (e.g., Bone et al., 1978) of fish with focal and multiple fast fibre innervation swimming in a water treadmill showed that the herring with focal innervation only used their fast fibres at high speeds and were unable to drive the fast system for other than short bursts, whereas in carp, where the fast fibres are multiply-innervated, there was low-amplitude electrical activity from the fast fibres at intermediate swimming speeds; only at higher swimming speeds were muscle action potentials recorded from the fast fibres. The multiple innervation of fast muscle fibres in higher teleosts is polyneuronal, ranging from only a few separate axons innervating each muscle fibre in zebra fish (Westerfield, et al, 1986), up to 22 in the marine teleost Cottus (Hudson, 1969). In their interesting study of the zebra fish, Westerfield et al. found that motoneuron EPPS generate action potentials in the majority of muscle fibres, but it is not yet clear whether this is always the case for those species where the degree of polyneuronal innervation is much greater, and where fast fibres are known from electromyography to be active in sustained locomotion. CONCLUDING REMARKS This brief survey of axial muscle patterns in protochordates and fish suggests that originally, axial muscle fibres were of a single type, and rather than being organised into serial myotomes, electrically "separate" from each other, formed a series of electrically-coupled muscle cells along the length of the body. With size increase, perhaps related to a more efficient technique of filter-feeding, serial activation of the axial muscle fibres along the body was required (and myosepta may have been needed for mechanical reasons) so that the system became transformed into separate myoseptal units, in which two major fibre types were specialised in different directions. The universal distinction in fish between slow red aerobic fibres employed during slow sustained cruising, and the fast white fibres for burst swimming shows not only how successful this solution has been to the need for a wide range of power output from the axial musculature over even modest speed ranges, but also that it must have early appeared in chordate evolution. 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