ABSTRACT
The common lugworm exhibits two distinct types of spontaneous activity cycle. Each of them has been described in some detail, but the method of approach was rather different in the two cases, and their integration has not yet been discussed. The experiments to be described were made to throw light on their mutual relations. The work was done at the laboratory of the Marine Biological Association of the United Kingdom, at Plymouth, in March and April 1950.
The relevant features of the two cycles, as hitherto described, will first be summarized. For a more detailed review see Wells (1950).
THE OESOPHAGEAL CYCLE (FEEDING, OR f CYCLE)
This rhythm has been studied mainly on dissected preparations.
The ‘isolated extrovert’ preparation consists of the proboscis with a short length of oesophagus attached. It gives bursts of vigorous rhythmic contraction alternating with periods of relative rest. The time taken by a whole cycle is generally 6−7 min. Single preparations are very regular in their timing, and the variation is chiefly from preparation to preparation. The intermittent pattern arises in a diffuse structure, probably a nerve plexus, in the wall of the oesophagus, and is transmitted to the powerful muscles of the proboscis, to whose activity the movements of the writing lever are mainly due (Wells, 1937).
The pattern can be shown to spread further, by way of the nerve ring and cord, to the muscles of the body wall, but the type of effect which it there produces varies in the different regions of the body. The body wall of the first three segments exhibits vigorous rhythmic contractions during the outbursts of the oesophagus, and there is close integration between the individual contractions in the extrovert and body wall (Wells, 1937). The body wall of the branchiate segments behaves quite differently; it traces a continuous and often very regular wave pattern, and this is usually inhibited, by reduction of amplitude, at each outburst of the extrovert. Sometimes—but by no means invariably—the body wall of the branchiate segments shows a vigorous and rather prolonged contraction as the oesophageal outburst passes off (Wells, 1949b).
When one has acquired the trick of making the dissections, these main results can be got with great regularity, especially during the cooler months of the year. Their interpretation in terms of the activities of the whole worm is rather tentative, and rests on four pieces of evidence.
(a) If intact worms are watched in glass tubes, the majority show cycles of activity of the anterior end, whose vigour is somewhat variable. They may appear as outbursts of extrusion and withdrawal of the proboscis, or merely of swaying, stretching and gulping movements. But in either case their timing agrees with that of the f cycles of the oesophagus, whose outward expression they therefore seem to be. It was suggested, from these observations, that the oesophageal pacemaker determines intermittent feeding under natural conditions (Wells, 1937).
(b) The feeding of the closely related species A. cristaia Stimpson was described by MacGinitie & MacGinitie (1949, p. 202) in the following words: ‘..the process of everting and inverting the proboscis and then swallowing occurs about every 5 sec. Also, after a certain number of swallows, ranging from 8 to 15, the worm takes a rest period of a few minutes, then begins feeding again.’ This account agrees very well with the behaviour of the dissected preparations of A. marina.
(c) The difference in response between the anterior segments and the branchiate ones corresponds to a functional distinction. The first three segments co-operate with the proboscis itself in bringing about the acts of extrusion and withdrawal. The branchiate segments perform wave movements, which travel along that part of the body in creeping and in the driving of water through the burrow (Just, 1924; van Dam, 1938).
(d) The behaviour of intact A. marina, living normally in sand, can be studied by recording the water movements through the burrow. The tracings often show a periodicity whose timing agrees with that of the f cycle. As the outbursts of the oesophageal pacemaker cause inhibition of the body segments responsible for irrigation, we may reasonably attribute these oscillations of the water movement trace to the influence of this pacemaker, and accept them as confirmation that the f cycle is not merely a property of the dissected preparations, but a rhythm of real importance under natural conditions (Wells, 1949b).
THE IRRIGATION-DEFAECATION CYCLE (i-d CYCLE)
In this case, the observations have mostly been made on intact worms.
The lugworm lives in a burrow of characteristic structure (Wells, 1945). If the water movements through a burrow are recorded, the tracing commonly shows long stretches, lasting for many hours, of very regular cyclic activity. Besides the oscillations mentioned above, there are conspicuous diphasic peaks, occurring at intervals of about 40 min. Each excursion is due to a backward movement of the worm to the surface of the sand, for defaecation, followed by a spell of vigorous headward irrigation, during which it creeps back to the bottom of its burrow. This type of tracing is not always obtained, but it occurs whenever the worm is feeding in the normal manner, at the base of a gradually subsiding funnel, and building up a pile of faeces. At other times, there maybe other types of record, such as continuous, confused activity or complete rest; but when these appear, the worm is neither feeding nor defaecating (Wells, 1949b).
Identical cycles are often traced by worms in glass tubes, and it can be shown, by appropriate experimentation, that the excursions are not reflexly produced, either by the presence of faecal matter in the rectum, or by oxygen exhaustion or carbon dioxide accumulation. They are due to the activity of a second spontaneous pacemaker system (Wells, 1949 a, b).
Very little has been done with dissected preparations, to locate the pacemaker for the i-d cycle, or to trace the spread of its pattern through the neuromuscular system. Records of the spontaneous behaviour of longitudinal body-wall strips from the branchiate part of the body suggest that the ventral nerve cord is the seat, or a necessary part, of the pacemaker (Wells, 1949 a).
THE INTEGRATION OF THE TWO CYCLES
When considering the functional meaning of the f- and i-d cycles, the problem of their integration at once arises. One would suppose it hardly profitable, or likely, for the worm to indulge in an outburst of feeding while moving backwards to the surface for defaecation. Is there, then, an inhibition of either pacemaker by the other, by which such disharmonies can be avoided?
To explore this question, we set up a series of eighteen dissections, differing slightly in detail, but all on the general plan illustrated in Fig. 1. The worm is pinned ventral side downwards, and care is taken throughout the dissection to avoid injury to the nerve ring or cord. The extrovert is prepared as previously described (Wells, 1937). The oesophagus is cut across, and its oral end is connected to a light isotonic lever. A strip of circular muscle is taken from the more anterior segments and operates the second lever. The movements of the branchiate region are recorded by immobilising a couple of segments with pins passing through the neuropodia, and connecting a third lever to a fine hook passed through the dorsal body wall. Finally, the basal region of the tail is similarly recorded, though in this case, owing to the slenderness of the tail, the pinning is such that the longitudinal musculature takes a large part in determining the movements of the lever. The nerve cord was divided in the course of several of the experiments.
We used slowly moving drums (at 40 − 80 mm./hr.), because we were looking for signs of the long-period i-d cycle, and frontal writing points, to facilitate the analysis of the tracings. The pull on the preparation was 0 · 4 g. for each lever (see Wells & Ledingham (1942) for a note on the effect of lever tension).
The preparations were mounted in sea water, at temperatures ranging from 13 to 16 · 7 ° C. The salt balance of sea water is practically identical with that of Aremcola body fluid (Robertson, 1949). The isolated extrovert shows f cycles either in sea water or in body fluid (Wells, 1937).
The chief results can be seen in the extract of Fig. 2, which was traced by a preparation set up exactly as in Fig. 1. The following points are clear.
(a) The f cycle appears with great vigour and regularity on the extrovert trace. It is transmitted to the body wall and each outburst can be seen as a group of contractions in the anterior body-wall strip, and usually as a decrease in amplitude in the branchiate region. Line A marks a typical f cycle burst, showing these effects. On many occasions (e.g. at B) the decline of an oesophageal burst is accompanied by a vigorous contraction of the branchiate segments. Contractions of exactly similar appearance are often shown by the branchiate segments after division of the nerve cord between it and the extrovert, so they cannot be directly due to impulses arriving from the extrovert. Their correlation with the f cycle, in undivided preparations, is perhaps due to a ‘rebound’ period of raised excitability, as the inhibition caused by an oesophageal outburst passes off.
Records of the various effects of the f cycle, taken on faster drums, were published by Wells (1937, fig. 21 ; 1949 b, figs. 6, 7). They have not previously been recorded simultaneously from a single preparation.
(b) Outbursts of another type appear very plainly, though at rather irregular intervals, in the lower three lines (e.g. at C). They are seen as vigorous spells of activity in the anterior body wall and tail, and as a marked acceleration in the branchiate segments. They have no effect at all on the extrovert trace; nor is there any reciprocal effect, for they may appear during either the outbursts or the pauses of the f cycle.
The second type of outburst has not previously been recorded. It was traced by twelve of the eighteen preparations in this series. We interpret it as the expression, by the dissected worm, of the i-d cycle. Our reasons are as follows.
(i) The intervals between the outbursts, though rather variable, are of the same order as in the case of the i-d cycle. The worm of Fig. 3, for example, gave bursts at intervals of 15 − 20 min. in the first part of the experiment, then the period lengthened out to about 50 − 55 min. (The appearance of the outbursts on the extrovert trace, in the second extract, is unusual, and is discussed below.) The interval is generally 20 − 60 min., but the range encountered in the whole series of experiments was 10 min. to 2 hr. The intact worm, living under favourable conditions in sand, makes defaecatory excursions about once every 40 min. It exhibits corresponding outbursts of activity in glass tubes, and they often occur very regularly at about the same interval. However, their frequency can vary; they are accelerated if oxygen is admitted after a period of oxygen lack; and they may also change their timing without discernible external cause. A rather extreme example of the latter type of variation is given in Fig. 4. The worm was put in a glass U-tube for 48 hr., and the water movements were recorded by the technique described elsewhere (Wells, 1949a). For 10 hr., on the afternoon of the first day, it gave a very regular series of bursts of pumping, at intervals of about 50 min. (upper extract). These, of course, are i-d bursts. There followed 5 hr. of continuous, but rather fluctuating, activity; then a period of i-d bursts at intervals of up to hr. (second extract). The third extract is the direct continuation of the second, and shows a period of continuous, fluctuating activity, followed by i-d bursts whose intervals are at first short (15 min.) and then lengthen out again. There is in fact a close resemblance between the intact worms in glass tubes and the dissected ones, not only in the most usual intervals between the bursts, but also in the kind of variations that appear, and in the extreme values of the range.
(ii) Whole worms in glass tubes are generally motionless, or nearly so, between the irrigation outbursts. During the latter, the waves travel along their bodies at frequencies of the order of 8 waves per min. (van Dam, 1938). In sand, they pump water gently between the bursts, and the water movement trace often shows a periodicity suggesting inhibition of pumping by the f cycle ; the i-d outbursts involve a great increase in the velocity of the stream (Wells, 1949 b, figs. 3, 5). The branchiate segments of the dissected preparations show waves at about 1−2 per min. between the bursts, with amplitude inhibition by the f cycle. This frequency is too slow for vigorous irrigation, but it might produce the gentle water movement seen with worms in sand between the i-d outbursts. The bursts in the dissected preparations involve acceleration of the rhythm of the branchiate segments, and this is consistent with the suggestion that they represent i-d outbursts.
(iii) We have seen nothing else in the dissected preparations that could correspond with the i-d cycle ; neither do we know of any other periodic activity in intact worms with which the bursts of the dissected ones could be equated.
The behaviour of the intact worm is of course variable. The i-d cycle is not always shown, and when it is, its frequency is modifiable, for example by previous oxygen lack. Similarly, the worm is capable of sustained spells of vigorous proboscis activity, for example when dug up and replaced on the surface of the sand. Nevertheless, it has a characteristic behaviour pattern, governed by the two cycles, into which it settles whenever conditions are favourable. According to our interpretation, the operated worms tend to settle into the same routine, showing an indifference to dissection which is indeed remarkable.
The i-d cycle is sometimes traced by the body wall after complete isolation from the extrovert (Fig. 5).
According to our results, neither of the two pacemakers has any direct influence on the rhythm of the other. It will however be noted that the oesophagus has been cut across, when the experiment is set up as shown in Fig. 1. There is no reason to assume that the nerve cord is the only conducting path between extrovert and body wall; the two pacemakers might conceivably modify each other’s activity by way of the gut. To test this possibility, we kept the oesophagus intact in a further series of experiments, and recorded the movements of the extrovert by means of a fine glass hook passed through one side of the oesophagus, at about the level of the septal pouches. The main result was the same as before. The f and i-d cycles showed no mutual reactions.
The activities evoked by the two pacemakers could be integrated, either by directly controlling the initiation of their respective outbursts, or by regulating the extent to which their influences spread through the neuromuscular system. Our results give no support to the first possibility, but there are the following indications that the second may occur.
(a) As already stated, the oesophageal rhythm may appear in the intact worm, either as vigorous proboscis action, or, with the same timing, as gentler acts (Wells, 1937). This presumably means that the number of muscle fibres reached by impulses from the oesophagus varies with conditions.
(b) The trace of the anterion body wall, in Fig. 2, clearly shows that it serves two masters. It follows the extrovert, in between the i-d outbursts, but when these appear, they take charge and dominate its behaviour. Similarly, the f cycles are commonly seen as periodic inhibition of the branchiate segments in the intervals between the i-d outbursts, but there is no evidence in our records that they have any inhibitory influence while the latter are in progress.
(c) In a few preparations, the i-d cycles could clearly be detected on the extrovert trace. An example is seen in the second half of Fig. 3. The extrovert of this preparation began quite vigorously, but, in the 8 hr. which elapsed before the beginning of the second extract, it gradually weakened and failed ; the f cycle disappeared, first from the anterior body wall trace, then from that of the extrovert itself. At the same time, a spread of the i-d cycle into the extrovert trace occurred. The muscles responsible may be those of the proboscis itself, or those of the body wall round the mouth; for it is impossible, when setting up preparations of this type, to prevent a certain amount of invagination of the body wall of the head, which thus adds itself to the extrovert proper. In any case, however, a comparison of Figs. 2 and 3 shows that the extent of spread of the i-d cycle is variable.
The results rather suggest a competition for territory between the two pacemakers. It is, perhaps, by the physiological control of this competition, that their integration is achieved. In particular, if both pacemakers happen to discharge their outbursts simultaneously, the i-d cycle prevails over most of the body, and the f cycle in the region of the extrovert only. There is apparently nothing to prevent the occurrence of an oesophageal outburst during a defaecatory exclusion; but as, at such a moment, practically the whole of the body wall would be under the domination of the i-d pacemaker, we may presume that the f outburst could only evoke extremely localized acts, such as internal movements of the proboscis and perhaps opening and closing of the mouth.
An incidental point was noticed during the experiments. In six of the ten experiments in which the nerve cord was divided, there was temporary inhibition of the hinder end of the worm immediately after the cut. In one experiment there was definitely no inhibition, while in three the point was doubtful. There was never any inhibition of the front end. These facts suggest that a condition may follow section of the cord, similar to the well-known spinal shock in vertebrates.
SUMMARY
Lugworms were dissected in such a way that the movements of the following parts could be simultaneously recorded: extrovert, body wall from the anterior three segments, body wall from the branchiate segments, tail. The preparations were set up in sea water and tracings were taken for many hours in each case. The preparations typically settled down to give cyclic behaviour patterns, remarkably similar to those which intact worms exhibit under favourable conditions, and in which two components were conspicuous.
The first, and most invariable, component is the feeding cycle (f cycle), of period 6 − 7 min. This rhythm originates in the oesophagus, and is transmitted to the muscles of the proboscis (where it causes outbursts of vigorous contraction) and body wall (where it causes correlated contractions in the first three segments, but periodic inhibition in the branchiate segments).
The second component was seen in two-thirds of the experiments. It consists of bursts of vigorous rhythmic activity in the body wall and tail, and can appear after their connexion with the extrovert has been severed. Under exceptional circumstances (exhaustion of the f cycle) it may spread to the extrovert trace. Its period is generally 20-60 min. It is apparently identical with the irrigation-defaecation cycle (i-d cycle) of intact worms.
Neither pacemaker directly affects the rhythm of the other. The integration of the activities which they determine probably depends on variation in the extent to which their influences spread through the neuromuscular system. They appear to compete for territory. If they happen to discharge outbursts simultaneously, the i-d pacemaker dominates over most of the body wall, and the/pacemaker over the proboscis and mouth region.