In the Dogfish (Scyliorhinus Canicula)

Home , Branchial arch, Gill slit, Spiracle (vertebrates)

J. Exp. Biol. (1965), 43, 363-383 363 With 12 text-figures Printed in Great Britain THE MUSCULAR BASIS OF THE RESPIRATORY PUMPS IN THE DOGFISH (SCYLIORHINUS CANICULA)

BY G. M. HUGHES* AND C. M. BALLINTIJNf Marine Biological Laboratory, Plymouth, and Department of Zoology, Cambridge

{Received 17 March 1965)

The mechanism of gill ventilation in the dogfish has been shown to be fundamentally the same as that found in teleost fishes (Hughes, 19606; Hughes & Shelton, 1962). Water enters the respiratory system through both the mouth and the spiracle during expansion of the oro-branchial cavity (Woskoboinikoff, 1932) and after passing across the gills it enters the parabranchial cavities before being ejected to the outside through the five pairs of gill slits. The flow across the gills is maintained partly as a result of the increased pressure in front of the gill resistances but also because of the suction pump action of the parabranchial cavities. The muscular activities producing the changes in volume of these two cavities and hence the required pressure gradient across the gills have not been established and descriptions of the relationships of muscles and skeleton are not always clear in detail. Woskoboinikoff (1932) and others were of the opinion that the coraco-mandibularis muscle was of importance during the phase of the cycle when the mouth opens and the oro-branchial cavity expands, but this was categorically denied by Balabai (1938) in a footnote to his paper. From observations on dogfish, anaesthetized so that they no longer pumped water across their gills, it was suggested (Hughes, 1960 a) that the main muscular action during the cycle was due to the constrictor muscles. These compress the branchial region and on their relaxation the elastic properties of the skeleton and ligaments are sufficient to account for expansion of the parabranchial cavities. A dogfish in this condition can be made to pump water through its respiratory system by compressing the branchial region by hand. It was also clear in Scyliorhinus that, as had also been observed by Satchell (1959) in Squalus, the respiratory cycle begins with a compression and is followed by relaxation and a relatively long respiratory pause. As with the study of all muscular activities it is extremely difficult to describe the function of a given system from the morphology alone and for respiration it has been shown both in mammals (Campbell, 1958) and fishes (Hughes, 1961; Hughes & Shelton, 1962) that electromyography is an ideal technique. This technique has recently been applied to teleost fishes in greater detail (Ballintijn & Hughes, 1965). The purpose of the present work therefore was to study the action of the main muscles of the head and branchial region of the dogfish, Scyliorhinus canicula, in order to establish the phase of the respiratory cycle at which they are active and to ascertain their role in the action of the oro-branchial pressure pump and parabranchial • Present address: Department of Zoology, University of Bristol. f On leave from the Zoological Laboratory of the University, Groningen, The N< therlands. Sup- ported by a grant of the Netherlands Organization for the Advancement of Pure Research (Z.W.O.)- 364 G. M. HUGHES AND C. M. BALLINTIJN suction pumps. The results have shown that most muscles are active during the compression phase of the respiratory cycle and that relatively few are active during the phase of expansion of either the oro-branchial or parabranchial cavities. It appears, therefore, that the hypobranchial musculature plays a relatively minor role in normal respiration although it is important during hyperventilation and at other times when the volume of the pharynx is increased as, for example, during feeding.

MATERIAL AND METHODS The methods used in the present study were fundamentally the same as those described in Hughes (i960) and Ballintijn & Hughes (1965). The dogfish was anaesthetized in MS222 (Sandoz), initially at a concentration of 1 g. in 10,000 c.c, and after being fixed in the holder it was placed in a tank containing 25 1. of sea water and about 1 g. MS 222. Individual variations in susceptibility to the anaesthetic were often found. For this reason the exact concentration was varied either by diluting the bath containing the preparation or by pipetting small volumes of concentrated anaesthetic (1 g. in 250 c.c.) into the mouth. For the placement of the paired stainless steel electrodes it was necessary to make a small cut in the thick skin above the muscle to be investigated. Larger pins were used than in studies of the trout and it was also necessary to use thicker wire in the input leads to the amplifier. The animal was maintained in this condition with four pairs of electrodes in different muscles. In some cases the output of the preamplifiers was passed through an integrator circuit and gave a convenient record of the discharge pattern in a given muscle. Pressures in the oro-branchial and one of the parabranchial cavities were recorded using Hansen manometers (Hughes & Shelton, 1958). Movements of the lower jaw and branchial region just dorsal to a given slit were recorded using RCA 5734 mechano-transducer valves. All recordings were made while the fish breathed continuously and was under light anaesthesia. Each animal was used in two experiments, involving the study of muscles first on one side and later on the other side. For the latter it was found advantageous to keep the animal lighdy anaesthetized between experiments. Following the recording of electromyograms, pressures and movements, the positions of the electrode tips were determined by passing a small current through them and so depositing ferric ions. The fish was then killed and the electrode position visualized by immersing the head in a solution containing 4 % potassium ferrocyanide. Dissection revealed a spot of Prussian blue at the point from which the recordings had been made.

RESULTS A. The skeletal and muscular systems (Fig. 1) The skeleton of the head and branchial region of the dogfish are so well known (see Daniel, 1934; Marinelli & Strenger, 1959) that a complete description here is not necessary; but certain important aspects of the morphology will be mentioned. The branchial arch skeleton is made up of a series of cartilages which are inclined forwards from the dorsal to the ventral elements. In transverse section the main curvature lies between the epi- and cerato-branchial cartilages and it is across this joint that the adductor branchialis muscle operates. From the lateral surfaces of the Respiratory muscles of the dogfish 365 hyoid and branchial arches there spread out a series of gill rays which support the septa separating the gill pouches from one another (Fig. 2). The gill rays attached to the hyoid arch are particularly long and bridge the wide space between the spiracle and the first gill slit. In addition to the gill rays each of the inter-branchial septa is supported by a large extra-branchial cartilage lying external to the gill rays. This cartilage is important in maintaining the shape of the parabranchial cavities. The pharyngo-branchials lie just beneath the vertebral column and are functionally anchored to it by means of a series of muscles (Fig. 1). The anterior muscle, the subspinalis, has its origin between the ventral part of the skull and the vertebral column and is inserted on the first pharyngo-branchial cartilage. The latter is connected by an interarcual muscle to the second pharyngo-branchial, which is similarly

Interarcualis dors alls Lev. palatoquadrati Lev- hyomandibulae Subspinalis

Arcuills Coraco-mandlbularls Coraco- Coraco-branchlales communls hyoldeus Fig. 1. Diagram of the skeleton and main muscles of the dogfish head seen from the left aide. The superficial constrictor sheets are not shown. The direction in which movement occurs when a given muscle contracts is shown by the arrow. Add. br., adductor branchialis; Add. md., adductor mandibulae; Pal.-pt. Qu., palato-pterygoid. connected to the third and so on. This system of dorsal interarcualis muscles appears to function by pulling forwards the whole of the dorsal part of the branchial arch skeletons. At the ventral ends of the branchial cartilages are inserted the coraco-branchial muscles. Contraction of these muscles would pull the ventral part of the skeleton downwards and backwards and so expand the oro-branchial and parabranchial cavities. These hypo-branchial muscles are some of the few clearly denned muscles of the head region, which is covered by a whole sheet of superficial constrictor muscles. Parts of the latter become concentrated to form more specific muscles as, for example, the levator hyomandibulae. The constrictor sheet is formed of several overlapping sheets, each associated with a given arch. Posteriorly the superficial constrictor of the hyoid segment extends to form the valve of the first slit. The superficial constrictor of the first branchial segment is overlain slightly by this and in its turn covers the second branchial constrictor and so on (Lighttoller, 1939). Most of the fibres in these sheets run in a dorso-ventral direction around the branchial region. In this work we have made an artificial distinction between dorsal, lateral and ventral regions. The former 366 G. M. HUGHES AND C. M. BALLINTIJN lies above the upper end of the gill slit and the ventral constrictors begin at the ventral end of the gill slits, while the lateral portions are in the region over which the slit extends on the side of the body. Another and most important part of each constrictor sheet, however, is not external but lies within the gill septum. In these septal constrictors the fibres run dorso-ventrally (Fig. 2) and in the same plane as the gill rays across which the fibres can be seen to pass, although in other elasmobranchs it is reported that they insert on individual rays. Dorsally this sheet is continuous

Dorsal superficial constrictor

Interarcualis dorsalis Pharyngo-. branchial Septal constrictor Levitor

Epl-branchlal

Adductor

Cerato- branchlal

Hypo- branchial Extrabranchlal cartilage

Ventral superficial constrictor Fig. 2. Diagram to show the relationships between the constrictors and the other muscles moving a single branchial arch in a dogfish. The plane of the section is the same as the inter- branchial septum and is supported by the branchial cartilages, its rays, and the extrabranchial cartilage (after Romer, 194.9).

with the dorsal superficial constrictors but ventrally it ends in a connective tissue strand lying dorsal to the coraco-hyoideus and appears to join its fellow from the opposite side. Some fibres at the ventral end of the sheet are inserted upon the cerato-branchial cartilage. Contraction of the septal constrictors would clearly decrease the volume of the parabranchial cavities, not only by a compression of the cavities next to one another but also in a dorso-ventral direction. Some of the muscles which are more clearly defined include the adductor mandibulae, which is extremely well developed and has fibres which run right round the angle of the jaw. On to a medial connective tissue sheet of the adductor mandibulae is inserted Respiratory muscles of the dogfish 367 the tendon of thepre-orbital muscle. Anterior to the spiracle the levator palatoquadrati runs between the otic capsule and the palato-pterygo-quadrate. In Scyliorhinus the separation of a spiracular muscle from the levator palato-quadrati was not always clear. Table 1 arranges the various muscles into four groups according to their function. The levator series have their origin on the axial skeleton or musculature and insertion on the dorsal ends of a visceral arch. The second group are muscles of the constrictor series with their division into dorsal, lateral, ventral and septal elements. These muscles form a sheet around the whole branchial region, which is compressed by contraction of their dorsoventrally running fibres. The third group operate between individual cartilages of either the same or different arches of the visceral skeleton. The final group have their origin on the coracoid and insertion at the ventral ends of the visceral arches. The action of this group of muscles is to lower and pull backwards the jaw, hyoid and branchial arches. Table 1 Muscles and their position Mandibular arch Hyoid arch Branchial arches Levator series (axial Levator palatoquadrati Levator hyomandi- Levatores branchiales; trapezius skeleton or musculature bulae to visceral arch) Superficial constrictor (Dorsal Dorsal \ stries Intermandibularis Constrictor ] Lateral Lateral [ External hyoideus (Ventral {Ventral/ Septal Between cartilages of T (Dorsal 1 (Daniel) visceral arches Preorbitalis Inter- ..t=Sub- 81X113168 (Lateral) spinalis (upper jaw Adductores branchiales Adducto... r . -Meep mandibulae ( iowerjaw Hypobranchial muscles Coraco-mandibularis Coraco-hyoideus Coraco-branchisdes (pectoral girdle to Arcualis communis visceral arch) B. The respiratory cycle (1) Muscle activity in relation to movements The electromyograms included potentials of varying sizes and were evidently due to activity in many units. In general they begin with a series of small potentials which build up to larger ones of higher frequency towards the end of the burst of activity. In a few cases recordings from single units were obtained. There are some variations in the relationship between electrical activity and movement found in different animals and to some extent between individual respiratory cycles. The following account is based upon the relationship found in the majority of cases but there were certainly some exceptions. It is possible, for example, that local differences within a given muscle would lead to differences in phase relative to the activity in other muscles and consequently relative to the movements in superimposed records. Where no electrical activity was apparent such negative results were not considered seriously unless it could be demonstrated that electrical activity could be recorded with the same electrode placement by encouraging the fish to make other types of movement. This is particularly important because one of the major findings of this work has been the relatively small amount of activity in the hypobranchial muscles during a normal respiratory cycle. However, by stimulating the dogfish to expand 368 G. M. HUGHES AND C. M. BALLINTIJN the branchial regions widely, or by getting it to bite a wooden stick placed inside the mouth, it was found that electrical activity could be recorded and hence the electrodes were definitely placed in the muscle. In many cases it was found that a clearer picture of the pattern of a muscle activity was given by the integrator recordings (Fig. 5). The movements recorded were raising and lowering of the lower jaw and lateral movements in the neighbourhood of the gill slits. The recording levers were usually placed slightly dorsal to the flaps of the gill slits. Movements of the lower jaw indicate changes in the volume of the oro-branchial cavity except in so far as they take place in the anterior region. Some of the major changes in volume of the cavity occur more

1 1 1 1 + 10 cm.

Oro-branchial pressure 0 ci Lower jaw \ movement \ \ 0 ^-^T c cm + Hl Parabranchlal \ I / \ \ (3rd) ° \ pressure _ \ CI Glll-sllt (3rd) movement 1 / f 0 r Add. md. Lev. pq. Lev. hmd. Con. hy. Con. br. Interarc. dor. Coraco-hy. —*MM Coraco-br. _ Fig. 3. Diagram showing the pressures in the oro-branchial cavity and parabranchial cavities in relation to movements of the lower jaw and gill region. Superimposed on these is shown the main phase of activity of most of the muscles from which recordings have been made in the present work. No indication is given of the intensity of activity in a given muscle except that of the adductor mandibulae, which is more active in the part of the cycle where the line is thicker. The respiratory cycle is subdivided into phases in relation to the movement recordings. posteriorly where there is no suitable place to record from. For this reason relationships between the movements recorded from the lower jaw, pressures in the oro- branchial cavity and electrical activity in the muscles must be interpreted with caution. There is, however, a general relationship between activity in the adductor mandibulae, levator palatoquadrati, levator hyomandibulae and closing of the jaws. Most activity in these muscles occurs during the closing phase of the cycle, but the adductor mandibulae is also active as the jaw is lowered. When subdividing the respiratory cycle it is advisable to do so in relation to movements of the lower jaw because those of the gill region are complicated by some being passive and others active. The movements recorded in the branchial region can be Respiratory muscles of the dogfish 369 divided into three phases: (1) an initial small expansion; (2) rapid adduction; (3) abduction, which is followed by a respiratory pause before phase (1) begins the cycle once again. The relative size of the initial abduction increases when recordings are made in identical positions towards the posterior slits (Hughes, i960). It is probable that the first phase in this cycle is produced entirely passively by the movement of water into the parabranchial cavities due to contraction of the oro-branchial cavity. In Fig. 3 is summarized the main phase of activity for most of the muscles. It is clear that, with the exception of the adductor mandibulae, most muscles from which recordings were made are only active during the relatively brief period at the end of the respiratory pause and during phases 1 and 2 of the parabranchial movements. There are some other muscles from which electrical activity has been recorded during other phases of the cycle but not during normal quiet respiration. It must be emphasized, however, that by no means all muscles have been recorded from in the present work and the possibility remains that some of them are active at other phases of the normal cycle. The precise timing of the individual muscle activities depends upon the muscle concerned. Generally there is a peristaltic relationship in which the anterior parts contract before those more posteriorly placed in the system.

(2) The activity of specific muscles in the respiratory cycle (a) Adductor mandibulae (Add. md.). Recordings were made from this muscle in all specimens investigated. It was always possible to divide the activity recorded into

Fig. 4. Oscilloscope records of electromyograms from four muscles and a movement recording (a) activity in the levator palatoquadrati, constrictor hyoideus lateralis, adductor mandibulae deep portion, and portion in the lower jaw (the movement record is of the lower jaw, closing being upwards); (6) activity in the adductor mandibulae, constrictor hyoideus, coraco- branchialis and levator hyomandibulae. The mechanical recording is from the region of third gill slit, adduction being upwards. Notice the absence of activity (except movement artifact) in the coraco-branchialis, which becomes active when the mouth is closed. There is a corresponding decrease in activity of the adductor mandibulae. two parts; (i) the tonic phase; (ii) the rapid burst of larger action potentials recorded as the mouth closed and passive expansion of the branchial region began (Figs. 3 and 4). Attempts were made to gain more detailed information concerning activity in different regions of this muscle. Electrodes were inserted into portions of the muscle in the upper jaw, the lower jaw or into a region between the two where the muscle fibres are 24 Exp. Blol. 43, 2 37° G. M. HUGHES AND C. M. BALLINTIJN quite red in colour. This deep portion tended to have less tonic activity in many preparations but showed a particularly marked increase in this phase when the mouth was held open, as did the burst component. The other two parts of the muscle usually showed a tonic phase plus the burst activity in which larger spikes of higher frequency became prominent. In some preparations it was found that electrodes placed in the lower-jaw part of the muscle did not show any activity in normal respiration but only became active during a coughing action, whereas electrodes placed in other regions Add. md. ^^^T^^\A^

Sec Add. md.

Coraco-br. k .1 i A .1 1 A.

Fig. 5. Integrator recordings of electrical activity in the muscles together with movement recordings from the lower jaw (L. j.) and region of the second gill slit (G. s.). (a) Adductor mandibulae and constrictor hyoideus. Notice the phasic activity in the constrictor hyoideus. (6) Adductor mandibulae and coraco-branchialis. Coraco-branchialis active in this preparation; its activity occurs when the adductor mandibulae is inactive, (c) Adductor mandibulae and coraco-branchialis with mouth held in closed position. Same preparation as (6). Respiratory muscles of the dogfish 371 of this muscle showed a reduction in their activity. It is clear that there is some func- tional division within the adductor muscle which is probably related to differences in the detailed arrangement of the fibres as shown in the drawings of Marinelli & Strenger (1959). Following the burst activity there is usually a silent period during which the oro-branchial cavity is expanding. This period lengthens if the mouth is held closed and the tonic activity becomes markedly reduced in intensity during such inter- ference (Fig. 46). The function of this muscle is to close the mouth and therefore to prevent the reflux of water from the oro-branchial cavity. The significance of the tonic phase of activity, however, is probably concerned with the slow expansion of the branchial region. Because of the mechanical coupling between the jaws and the rest of the visceral arches, the elastic forces responsible for expansion of the parabranchial cavities also exert a traction on the jaws. If the jaws are partly closed, they serve as an anchor against which the parabranchial cavities are rotated forwards and thus expansion is facilitated (Fig. 8 a). When the animal is swimming it is clear that variations in the activity of this muscle will control the volume of water entering the mouth as a result of the animal's movement through the water. The reflex responses to opening or closing of the mouth are very clearly defined and it would be of interest to know the receptor mechanisms involved. (b) Levator palatoquadrati {Lev. pq.) and levator hyomandibulae {Lev. htnd.). Both muscles are active as the jaws become completely closed and the oro-branchial cavity decreases in volume. Their activity coincides with, or is a little later than, the burst phase of the adductor mandibulae. Usually the lev. pq. is active a little before the lev. hmd. but this is not invariable. The lev. pq. is situated in front of the spiracle and the phase of its action coincides with the closing of this aperture. It is of interest that these muscles form part of the levator series and might have been expected to be concerned with expansion of the branchial region but are clearly concerned with reduction of the oro-branchial cavity. Because of their relationship to the spiracle the action of these muscles is important in some of the coughing activities during which the spiracle stays open longer than normally to allow 'spouting' to take place. In some preparations a tonic phase of activity has been recorded in the levator palatoquadrati. (c) Constrictor hyoideus {Con. hy.). This broad sheet of muscle lies in the space between the first gill slit and the jaws and is the easiest part of the constrictor sheet from which to record action potentials. The activity of this muscle produces a reduction in volume of the oro-branchial cavity and also has a marked effect on the movements of the first and other branchial arches because of the mechanical couplings between them. Many recordings were made from the different parts of the muscle, but in all cases the timing was the same in the dorsal, lateral or ventral parts (Fig. 7) and over- lapped that of the lev. pq. and the lev. hmd. It usually coincided with the burst activity of the adductor mandibulae and the beginning of the abductor movement of the gill region. {d) Constrictor branckiales {Con. br.). These parts of the superficial constrictor sheet are not so easy to record from as the constrictor hyoideus because of their smaller extent and the danger of interfering with the action of the gill flap. A convenient place to record from them was usually just above and anterior to a slit. These muscles 24-2 372 G. M. HUGHES AND C. M. BALLINTIJN become active slightly later than the constrictor hyoideus but not markedly so. All regions of a given constrictor are active at the same time, including the septal constrictor. In some cases these muscles are not very active and constriction of the branchial region is produced as a result of activity in the constrictor hyoideus because of the mechanical connexions between the hyoid and first branchial arch. This link was emphasized by the result obtained by Balabai (1938) when he cut the connexion between the 1st branchial arch and the hyoid arch, which produced a reduction in volume of the parabranchial cavities. A similar effect follows when the hyoid segment is constricted.

Fig. 6. Superimposed tracings from electromyogramB, pressure and movement recordings. The muscles recorded from are the adductor mandibulae, levator hyoideus, levator palatoquadrati and interarcualis dorsalis. The difference in rhythm before and after the ' cough' is not always present.

(e) InterarcuaUs dorsalis (Int. d.). The muscles of this series seem to have their main activity shortly after the burst in the adductor mandibulae and therefore very close to that of the levator palatoquadrati and levator hyomandibulae. There may be some slight differences in timing between the muscles of different branchial arches but insufficient evidence was obtained to be certain of this. The action of these muscles is to reduce the distance between the dorsal elements of adjacent arches and to help to constrict both the oro-branchial and parabranchial cavities. The most anterior muscle of this series forms the subspinalis, and recordings shown in Fig. 6 indicate Respiratory muscles of the dogfish 373 that they are active a little before the levator hyomandibulae. This figure also shows that, during a spontaneous cough, the levator palatoquadrati becomes more active during the expansion phase, which is exaggerated during these manoeuvres. It is possible that the subspinalis, by drawing the pharyngo-branchial cartilage forward, might function by producing a ventralwards movement of the branchial arch. If this were the case these muscles might function during the expansion phase of the cycle. How- ever, the normal recordings support the view that they are more concerned with reduction in the volume of the cavities.

Levator hyomandibulae

Constrictor hyoideus <

Oro-branchlal pressure

Lower law

Parabranchlal + pressure

3rd gill silt

Sec. Fig. 7. Superimposed tracings from electromyograms, pressure and movement recordings. The muscles recorded from are levator hyomandibulae, and the dorsal, lateral, and ventral portions of the constrictor hyoideus. The third cycle is a ' cough'. (/) Hypobranchial muscles (Cor. md., Cor. hy., Cor. br.). Electrodes were inserted into this large group of muscles many times but little activity was recorded from them during the normal respiratory cycle. That the electrodes were so inserted in the muscles that a myogram could be led off was confirmed by the appearance of potentials when the mouth was forcibly closed (Figs. 46, 5 c). In the upper beam the coraco-hyoideus muscle comes into action as the adductor mandibulae becomes less active. The reverse experiment of opening the mouth increases activity in the adductor 374 G. M. HUGHES AND C. M. BALLINTIJN and there is none in the coraco-hyoideus. The activity of muscles in this series initiated by forcible closing of the mouth sometimes continued when the lower jaw was released. In some preparations when the fish ventilated very actively, a discharge was recorded from the muscles during the expansion phase of the cycle (Fig. 5). The alternation

Parabranchial Oro-branchlil cavity cavity

Fig. 8. Diagram to illustrate the changes in volume of a parabranchial cavity produced as a result of (a) the backwards movement of the branchial arches, and (6) their flexion producing a decrease in volume in the transverse plane. w Con. hy. lat. Con. br. 3 dors. Add. md. Coraco-md.

Con. hy. lat. Con. br.3 dors. Add. md. Coraco-md.

0-5 sec Fig. 9. Scyliorhimu camcula. Electromyograms during a 'bite'. Notice the marked activity in the coraco-mandibularis which is absent from the normal respiratory cycles. between activity of the hypobranchial muscles and the adductor mandibulae was found several times during such hyperventilation and fits in with the generally suggested function of these muscles based upon their morphological position. Their relationship in moving the lower jaw is emphasized by forcibly moving the jaw. Evidently there are reflex mechanisms which control the balance of activity between these two groups of muscles, and perhaps the hypobranchial muscles are normally inactive because the elasticity of the skeleton is sufficient to produce a sufficiently Respiratory muscles of the dogfish 375 rapid expansion of the branchial region. When this expansion is reduced by closing the mouth then the hypobranchial muscles come into action. Activity was recorded several times in the arcualis communis (arc. com.) during heavy breathing as the branchial region expanded. In one preparation activity of the arcualis communis alternated with that of the adductor mandibulae but sometimes it was only active during every other pause in the activity of the adductor mandibulae. Simultaneous recordings from the coraco-hyoideus showed that it often came into action during these alternative cycles. At other times, however, the coraco-hyoideus and arcualis communis were active synchronously during the same cycle. Besides their being active during hyperventilation, a fairly certain way of evoking contraction of the hypobranchial muscles was found by touching the inside of the upper jaw, which usually resulted in the dogfish biting. During such responses the mouth and oro-branchial cavity firsto f all opened wide and then were rapidly shut. The electromyograms clearly showed (Fig. 9) a burst of activity in the hypobranchial muscles succeeded immediately by bursts of action potentials in the adductor mandibulae. During the expansion phase which precedes the bite the levator hyomandibulae was also very active.

(3) Muscle activity in relation to the pressure recordings As the causal sequence in the production of pressure changes in the respiratory cavities is: muscle activity ->• movements -*• volume changes in respiratory cavity -*• pressure changes, it might be expected that the correlation between electrical activity in the muscles and movements would be a more direct one than that between muscle activity and pressures. In general this has been shown to be true but is complicated because of the difficulty of accurately recording the relevant movements. Thus, because of the nature of the system being investigated, it has not yet been possible to record the movements of individual skeletal elements. Some inaccuracies are due to differences in the positioning of the recording levers and there are also variations between individual fishes. The analysis is further complicated because some of the movements recorded are produced passively by other parts of the system; those actively produced are more clearly related to the electrical activity. In contrast the pressure recordings provide a summated picture of the total muscular action on the respiratory cavities and the correlation of the electromyograms with them is often clearer than with the movements. The increase in pressure which occurs in the oro-branchial and parabranchial cavities usually occurs during the phase of the cycle when most of the respiratory muscles are active (Fig. 3). The termination of the bursts in these muscles tends to coincide with the maximal positive pressure recorded in the cavities. This is particularly clear for the constrictor muscles and also the adductor mandibulae, which has its phasic activity during the period when the mouth is rapidly closing and the pressure in the oro-branchial cavity is increasing. Nevertheless it is difficult to make an exact correlation between electrical activity and the pressure produced. Certainly there is not necessarily a direct correlation between pressure and the amount of electrical activity recorded from a given muscle. This is well illustrated in experiments when the respiratory system is interfered with by forcibly closing the mouth or holding it -wide open. When the mouth is closed the positive pressures in the oro-branchial cavity 376 G. M. HUGHES AND C. M. BALLINTIJN are markedly increased but not those in the parabranchial cavities, and usually there is a slowing of the respiratory rhythm. However, there is no corresponding increase in the activity in the constrictor musculature. Sometimes when the mouth is closed and the pressures are particularly large there may be an increase in activity. This disparity between changes in electrical activity and pressures is well shown in recordings just before the mouth is released and shortly after it has returned to the normal respiratory rhythm (Fig. 10b). It can be seen that the electrical activity increases in these muscles although the positive pressure in the oro-branchial cavity

Fig. 10. Superimposed electromyograms, pressure and movement recordings showing the effect of (a) forcibly closing the mouth, (6) its subsequent release. is markedly reduced. The adductor mandibulae illustrates this admirably for it shows a marked decrease in activity when the mouth is closed. The converse is true when the mouth is held wide open and the muscles are more active but the pressure changes are less than during the normal cycle. These observations emphasize the importance of the integrity of the whole respiratory system if it is to function in an efficient and economic way. When damaged or interfered with experimentally the different parts of the system are no longer integrated with one another and despite increased muscular activity there can only be less efficient ventilation of the gills. In these systems passive valves are often important but they are not well developed in the mouth of Scyliorhinus. In this connexion the action of the lower jaw in closing the mouth is important, for in specimens where the mouth Respiratory muscles of the dogfish 377 is not closing completely the oro-branchial pressure changes will be reduced. Thus for two individuals making equivalent muscular efforts there will be differences in the ventilation of the gills. In cases where the mouth is not closing completely it is sometimes difficult to correlate the phasing of the movement with the muscular activity producing it and the mouth may appear to be fully closed just as the positive pressure in the oro-branchial cavity begins to increase. The correlation of electromyograms with pressure changes is of particular value in the analysis of the changes which occur during coughs and other modifications of the normal respiratory rhythm. A number of different types of 'cough' have been observed which occur 'spon- taneously ' during the normal respiratory rhythm and others only when the animal is interfered with by opening or closing its mouth. In some of the latter cases the pattern of movements, pressures and electromyograms seems to correspond with that of a normal' cough'. A complete investigation and analysis of these patterns has not been undertaken as yet, but one or two general principles are included here. As in teleost fish the most common modification is where there is an increase in the parabranchial pressure which precedes that in the oro-branchial cavity and therefore produces a reversal in the flow of water through the gills. Sometimes this increased pressure in the parabranchial cavities is associated with an inhibition of the adductor mandibulae, but in other cases the large parabranchial pressure is followed by a rapid contraction and closing of the mouth associated with an increase in pressure within the oro- branchial cavity. In some instances there is an extra burst of activity in the adductor mandibulae during this phase. The timing of the adductor mandibulae and other muscles relative to one another often changes during coughs and other modified respiratory manoeuvres. For example, the constrictor hyoideus may contract before the levator hyomandibulae, whereas normally the reverse is true. In other cases, as mentioned above, the adductor mandibulae may be less active; particularly, its tonic phase during the increased contraction of the branchial region and its burst activity comes later than normally—for example after the levator palatoquadrati and not before it. The analysis of such records makes it clear that these increases in pressure are produced by particular muscles and are active contractions of the parabranchial region before the oro-branchial region. They are, of course, associated with differences in the movement recordings. C. The mechanism of ventilation It is possible to divide the respiratory cycle into the following main phases: (1) A phase during which most of the respiratory muscles are active. This produces a decrease in volume of the oro-branchial cavity, but despite activity in the constrictor branchialis muscles the parabranchial cavities first passively increase in volume because of the passage of water into them through the gills; after that they contract. (2) A passive expansion of the oro-branchial cavity during which there is no activity in the adductor mandibulae. (3) A phase with a more rapid expansion of the oro-branchial cavity. When the hypobranchial muscles are active they fit into this phase, which may therefore be an active one. During this expansion phase of the oro-branchial cavity there is also an expansion of the parabranchial cavities, which, after a respiratory pause of varying duration is followed by phase (1). During phase (1) there is burst activity in the adductor mandibulae and most 378 G. M. HUGHES AND C. M. BALLINTIJN muscles are active. The oro-branchial cavity contracts, and through the positive pressure produced by it the parabranchial system initially expands, although its constrictor muscles contract. After the initial expansion, the flow of water pressed in by the oro-branchial cavity ceases, and as the constrictor muscles are still active the parabranchial system starts to contract. The parabranchial cavities continue to contract although there is no more activity in the constrictor muscles. This can be ex- plained as follows: (1) The movement record is from gill slit 3 and most constrictor records are from more anterior segments. Hence the peristaltic sequence will reduce the difference. (2) The constrictor muscles compress a tube-like structure, filled with fluid. Near the gill slits, where the movements are recorded, the walls of the tube and the constrictor sheath are thinner and weaker than in other places. At these places the wall will bulge during contraction, under the pressure of the fluid, and thus contract less than in the other regions. When at last the muscles stop contracting, the water accumulated in the bulge has to flow out before complete adduction is achieved. After adduction both the oro- and parabranchial system expand (the parabranchial more slowly, because water has to flow in through the gill resistances). This expansion is passive because the resting position is expanded. Therefore during the contraction phase elastic forces are accumulated. These elastic forces tend to expand the branchial system by rotating forward the branchial arches as in Fig. 8 a and to pull the jaw wide open. This is counteracted by the tonic phase of the adductor mandibulae, which keeps the mouth half-closed and so serves as an anchor against which the elastic forces expand the branchial system. It must be remembered that in the present experiments it has not been possible to record from the adductor branchialis muscles and this is the main group that might well be active when the parabranchial cavities actively decrease in volume. If, however, it should prove that their activity is also coincident with that of the adductor mandibulae, etc., then the delay in the contraction of the parabranchial cavities must be due to the release of water from them during the active contractile phase.

DISCUSSION From this study it has become apparent that there are many interactions between different parts of the dogfish respiratory system. Some are due to mechanical couplings between elements of the visceral arch skeleton, which include both ligamentous and skeletal linkages, and also linkages through the connective tissue and skin. Evidently a model of the ventilation mechanism based on a complete separation of two pumps, one on either side of the gills (Hughes, i960), is over-simplified. It is now clear that such interactions and the elasticity of different parts of the system must be taken into account. Thus electromyography has emphasized the importance of a relatively brief phase of activity in the superficial constrictor muscles, accompanied by the storage of energy in the elastic components, which later plays a part in expanding the respiratory cavities. Furthermore, contraction of these muscles affects not only the oro-branchial pump but also the parabranchial pumps. A diagram of the pumping system modified to incorporate some of these features is shown in Fig. 11. The oro-branchial pump is represented by a large piston and each of the para- Respiratory muscles of the dogfish 379 branchial pumps on one side by separate pistons. The couplings between the individual pumps are indicated by springs which are drawn to suggest a stronger coupling between neighbouring pumps. One coupling which is particularly important is that between the oro-branchial and the first parabranchial pump, as was emphasized by Balabai (1938). Each of the pumps has a wall with a certain elasticity. The elasticity of the more posteriorly placed cavities is relatively greater. Between the oro-branchial

Fig. 11. Diagram of a model to show the main couplings and elastic components of the oro- branchial and parabranchial pumps of a dogfish during (a) the contraction, (ft) expansion phases of the respiratory cycle. cavity and the parabranchial cavities are situated the gill resistances provided by the filaments of each gill slit. Entrance to the oro-branchial cavity is through the mouth, which is guarded in many dogfishes by passive valves, but water also enters through the spiracle, guarded by a muscular valve. Each parabranchial cavity communicates with the exterior through its gill flap, which is a passive valve in these fish. A system such as that shown would serve to explain the time course of pressure and movement recordings and fits in with the general properties of the contracting musculature. The pattern of muscle activity found electromyographically confirms several general observations which previously suggested a subdivision of the cycle into an active expiratory movement followed by a passive inspiration and then a respiratory pause before 380 G. M. HUGHES AND C. M. BALLINTIJN the next expiratory contraction takes place. The passive nature of inspiration confirmed here is contrary to what has often been taught in elementary courses about the importance of the ventral hypobranchial musculature. During normal quiet ventilation these muscles are scarcely active at all and only come into action during very active ventilation, following forcible closure of the mouth, or during expansion of the whole pharyngeal cavities before feeding. It is generally considered that these muscles only became connected with the branchial apparatus late in phylogeny, so that the conclusion that the superficial constrictor and other lateral plate muscles play the major role in respiration fits in with expectations based on their morphological origins. Also of interest from a phylogenetic point of view is the fact that evidence for an active expiration and passive inspiration was also obtained in the lamprey using electro- myographic techniques (Roberts, 1950). On the other hand, in teleosts, the derivatives of the myotome become increasingly important during respiration. It is clear that the resting position of the skeletal system is one in which the mouth is open and the branchial basket is fully expanded. In such a position there would automatically be ventilation of the system when the fish swims through the water. There are many observations which suggest that many sharks ventilate their gills by such a mechanism during normal swimming (Hughes, unpublished). Adjustment of the ventilation volume could be achieved by varying the contraction of the adductor mandibulae and this may be one of the functions of the tonic phase of activity found in this muscle. Not only does it maintain the mouth in a certain degree of closure but it also forms an anchor which enables the branchial basket to expand. The connexion between the hyoid and 1st branchial arch is a particularly important link in this mechanism. Comparison with teleosts The mechanism of ventilation in both cartilaginous and bony fishes seems to be essentially the same in that the respiratory cavities may be divided into two parts, one on either side of the gill resistance. Differences in the number of respiratory cavities following these resistances have already been mentioned. In both groups of fish there are mechanical couplings between the different pumping mechanisms (Hughes, 1964) which in elasmobranchs are mainly due to the elasticity of the branchial tube, ligamentous connexions between the visceral arches and the external connective tissues. In the teleosts, however, although these types of coupling are present there are also important couplings due to skeletal levers abutting against one another (Ballintijn & Hughes, 1965). Furthermore, in both groups many of the muscles operate on both pumps. Thus, in the dogfish when the superficial constrictor sheet contracts the volume will be altered not only of the parabranchial cavities but also to some extent of the oro-branchial cavity. And in the trout activity of the muscles which move the palatal complex (e.g. the levator hyomandibulae et arcus palatini) will affect not only the volume of the buccal cavity but also of the opercular cavity, because the operculum is attached to this complex. The interaction between the two cavities resulting from the flow of water appears to be greater in the dogfish. Thus there is an initial expansion phase of the parabranchial region because the water passes into these cavities when the oro-branchial cavity decreases in volume. In the teleost such an effect must be present to some extent but it does not form such a significant part of the movement records. The correlation between the pressures in the cavities Respiratory muscles of the dogfish 381 on either side of the gill resistance are of interest and show some variability. In some cases a marked increase in the parabranchial pressure is not accompanied by a significant change in the oro-branchial cavity, as for instance during certain coughs. At other times the effect is quite distinct as there is a large increase in pressure in the two cavities. When the mouth is held open (Fig. 12 a) the pressure changes in the

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;i l\ /! A P Mouth closed J\J\J\J\J +1 cm. H2O

k li L_h A_L_k JI_J\_J_JI_JI-JL_LJ\_ILJI_L Fig. ia. Movement and pressure recordings from the respiratory cavities of a dogfish during (a) forced opening, (b) forced closing, of the mouth. oro-branchial cavity fall, but those in the parabranchial cavities do not show any noticeable change in amplitude, suggesting that the gill resistance is fairly large under these conditions. At other times there are indications that it is different in the two directions. When the mouth is opened and the pressure falls in the oro-branchial cavity there is, however, a change in the shape of the curve recorded in the parabranchial cavity during the expansion phase. This is because water then enters the 382 G. M. HUGHES AND C. M. BALLINTIJN parabranchial cavities only via the oro-branchial cavity and the lowered resistance to flow resulting from the open mouth leads to a more rapid return of the pressure to the external level. Correspondingly when the mouth is closed the rate of the pressure increase is reduced and also the rate of expansion of the parabranchial cavities. Similar observations have been made on teleosts (Hughes & Ballintijn, unpublished). The presence of the spiracle in elasmobranchs provides an additional entrance for the water current but is also used during the expulsion of noxious fluids. It is not as large an opening in the dogfish as in rays, but there are occasions when the mouth is closed, the branchial region contracts and water is forced out through the spiracle in order to clear the aperture. In conclusion, then, gill ventilation of the dogfish is mainly brought about by the superficial constrictor sheet contracting against the elasticity of the visceral arch skeleton. This reduces the volume of both the oro-branchial and parabranchial cavities and forces water out of the system. The rise in pressure in both cavities is more or less synchronous and similar in amplitude. When these muscles relax the parabranchial cavities gradually expand because water can only enter through the gill filaments as the external flaps are closed and prevent the entry of water through the external gill slits. During this expansion phase the main muscle that is active is the adductor mandibulae, whose tonic phase serves to maintain the position of the mandible and hyoid arch so that the branchial arches are drawn forwards by the mechanical couplings of the skin and ligaments. The rate of entry of water into the whole system during this phase is regulated by the degree of opening of the mouth, which is adjusted by the tonic action of the adductor mandibulae. During swimming this may be the only muscular activity involved in the branchial region and water irrigates the whole system as the fish swims forwards. In general, the constrictor muscles contract in a peristaltic sequence and closing of the lower jaw takes place before the main compression phase increases the pressure in the oro-branchial and parabranchial cavities. In this way reflux of water through the mouth is reduced when the fish is stationary.

SUMMARY 1. A study has been made of the muscular basis of the respiratory pumps in the dogfish by recording electromyograms, pressures and movements simultaneously. 2. Electrical activity in the muscles takes place more or less synchronously during the phase when the oro-branchial and parabranchial cavities are decreasing in volume. Besides being active at this time the adductor mandibulae has a tonic phase of activity which accompanies expansion of the branchial region. 3. There is a slight delay between the activity of the muscles in the branchial region such that those more anteriorly placed precede the posterior ones. All muscles of a given constrictor sheet are active synchronously regardless of their dorsal, lateral or ventral position. 4. During normal resting ventilation no electrical activity was recorded in the hypobranchial musculature. Activity was recorded in these muscles, however, during hyperventilation and also when the fish was made to bite by stimulating the inside of its mouth. In the latter activity the pharyngeal cavity is expanded by contraction of the coraco-muscles and subsequently the adductor mandibulae rapidly closes the jaws. Respiratory muscles of the dogfish 383 5. In a fully anaesthetized dogfish the relaxed position of the skeleton is with the mouth open and branchial region expanded. It is concluded that ventilation is primarily achieved by contraction of the superficial constrictor muscles, which produces an increase in pressure in the oro-branchial and parabranchial cavities. Water is forced through the gills because the pressure in the oro-branchial cavity exceeds that in the parabranchial cavities during both this phase and the expansion phase which succeeds it. Expansion is produced when the constrictor muscles relax by the elastic recoil of the visceral skeleton. This action is aided by tonic activity in the adductor mandibulae. 6. During swimming of certain sharks the whole branchial region remains in a relaxed condition and water enters the mouth. The volume entering could be regulated by variations in the amount of tonic activity in the adductor mandibulae. 7. It is suggested that the importance of the lateral plate musculature during normal ventilation is because it is the phylogenetically older system. Only later in phylogeny did the myotomic component of the head musculature (hypobranchial muscles) become concerned with respiration.

We wish to thank Dr. F. S. Russell, Director of the Plymouth Laboratory, and his staff for providing such excellent facilities and their friendly help.

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