The physical properties of the isolated mesogloea of Calliactis and Metridium are described and the behaviour of the tissue on loading recorded. It can be compared with a spring and dashpot model.
It is shown that the viscous-elastic properties of the body-wall, which have previously been ascribed to the muscles, are the attributes of the mesogloea.
On being heated in water under a small load, isolated mesogloea of Calliactis contracts, at temperatures which are only a little higher than those at which vertebrate collagen contracts. This is regarded as additional evidence for the collagenous nature of the mesogloea protein.
It is shown that the physical behaviour of the material is consonant with the crossed fibrillar collagenous nature of the mesogloea described elsewhere.
In another paper some account is given of the histology and chemical properties of the mesogloea of certain coelenterates, among which are Calliactis parasitica and Metridium senile. It is shown there that the mesogloea of these animals contains fibres arranged in a crossed spiral pattern which is determined largely by the mechanical forces acting on the tissue (Chapman, 1953). This tissue is tough and cartilage-like to handle when it is cut out of the animal, and yet it appears to be soft and flexible in the animal during life. It would seem that the physical properties of the mesogloea might be of considerable importance in the life of the animal, yet nowhere was it possible to obtain any precise information about the isolated mesogloea; indeed, in one recent paper on the physical properties of Metridium body-wall (Kipp, 1940) the properties of the mesogloea are ignored. It was therefore thought to be worth while to attempt to examine the physical properties of isolated actinian mesogloea in order to see if they are consonant with the histological structure, and to see how far the physical properties ascribed by Kipp and others to the muscle may properly be ascribed to the mesogloea.
The work which is described in the present paper consists of an examination, from several aspects, of the properties of the isolated mesogloea of the two sea anemones, Calliactis and Metridium, which belong to adjacent families of the subtribe Acontiaria, so that, although they differ in their construction and habits to a certain extent, the differences are of degree rather than of kind. Metridium is relatively thin-walled and changes its volume enormously in an apparently spontaneous fashion, while Calliactis has a much thicker body-wall (calleo—to be thick-skinned), is exposed in life to rougher treatment and changes its volume but little. In its normal movements it has been shown (Chapman, 1949) to act as a closed fluid-muscle system.
While it was not found easy to make good ring preparations from the body-wall of Metridium, Calliactis has so thick a mesogloea that the body-wall does not corrugate much, even when the disk has been sliced off, making it possible to prepare a fairly cleanly cut ring of tissues from the unanaesthetized animal. It is possible to scrape off with a scalpel the layers inside the mesogloea, and thus to prepare a ring of mesogloea with only the ectoderm adhering; with Metridium it is not so easy to make a satisfactory mesogloea preparation on account of the corrugations of the body-wall and the thinness of the mesogloea. Calliactis was therefore used in the main, but the results obtained with this animal have been compared with the results of experiments made on Metridium in most instances. Of the viability of such body-wall preparations there is no doubt, for specimens in which the muscle layer was left intact and which had been kept in sea water at 10–15° C. for 42 hr. underwent spontaneous contractions at the end of that time.
Three main sets of experiments were carried out, the experimental details and the results for each being described separately in the following paragraphs.
In the first set of experiments, ring preparations of Calliactis and Metridium were kept in sea water at room temperature (usually about 12–15° C) and were connected to a light, counterbalanced lever system writing on a smoked drum. The lever gave a fivefold increase in the length changes of the preparation. The speed of the drum was 0·01 cm./sec. throughout the experiments. A clamp was arranged so that the length of the preparation could be kept constant without its being under tension. The experimental arrangement is shown in Fig. 1.
Experiments were made on the body-wall ring preparations of Calliactis cut from the middle of the column. They were prepared, as a rule, in the afternoon and were left overnight in sea water until they were used on the following day.
A series of experiments was made comparing the behaviour of preparations in which the muscle layer was left untouched, with that of preparations from which the muscle layer had been scraped with a scalpel. That there was no doubt of the complete removal of the muscle layer by scraping was shown by two methods.
Histological examination of the fixed test piece, stained in ‘Azan’ stain, showed no trace of the endoderm and its musculature. Secondly, a scraped test piece did not show any sign of those spontaneous contractions characteristic of intact body-wall rings which begin very soon after excision from the living animal. In addition, it was shown that the same type of loading curve was given by test pieces, both scraped and intact, immersed in pure sea-water and immersed in magnesium sea-water which completely anaesthetizes the muscle (Fig. 2). Tracings were also made with an unloaded ring of intact body-wall, prepared up to 42 hr. before use, to show the spontaneous activity of the muscle.
Comparison of the behaviour, under simple loading, of intact and scraped rings, that is, of the mesogloea plus muscle and of the mesogloea alone, showed that there were no differences between the specimens except such as could be attributed to differences in cross-sectional area of the test piece or to differences brought about by the compacting of the mesogloea which occurs on scraping. In both the scraped and intact rings on loading there was an immediate elastic extension followed by a slow elongation, rapid at first but slowing down to a nearly constant rate of elongation. On removal of the load there was an immediate elastic shortening followed by a slow shortening, which, however, was not as great as the slow extension so that the ring did not return to its original length. Examples of simple loading tests with intact and scraped body-wall preparations are given in Fig. 3.
By loading the mesogloea alone with different weights it was shown that the immediate elastic extension was nearly proportional to the load for all but small loads. It was also seen that, on unloading, the mesogloea shortened by very nearly, but not quite, the same amount as that by which it had elongated immediately on loading. These experiments are illustrated in Figs. 4 and 5, and in Table 1 in which the percentage extension of the test piece of mesogloea is shown when it is loaded with different weights.
In another type of experiment the body-wall ring was stretched by a load for 2 or 3 min. Its attachment was then clamped so that it could not shorten but it was no longer subject to load. After many minutes at constant length without load (see Fig. 6) the clamp was removed and the body-wall allowed to adopt its own length without constraint. On releasing the clamp it immediately underwent an elastic shortening, a similar behaviour being shown by rings of mesogloea from which the muscle had been scraped and by intact rings.
In a third set of experiments a load was applied, stretching the tissues, the lever was returned to its position at the beginning of the experiment and then clamped. The ring was, therefore, under no tension nor was it kept at a fixed length. It was free to shorten. After a brief interval the clamp was released and the length of the preparation again registered. As will be seen from Fig. 7, both the intact body-wall ring and the scraped, muscle-free specimen show quite clearly that the ring recovers to a certain extent, but does not completely recover, its original length. This is, of course, only what would be expected from the simple loading tests and provides little more information about the behaviour of the preparation than they do.
Finally, spontaneous contractions of the muscle were demonstrated in specimens of body-wall prepared as long as 42 hr. before use. In another experiment spontaneous activity was shown during a loading test 4 hr. after cutting out from the animal. Such activity of the muscle may lead to confusion in the loading tests, and examples are given in Fig. 8 in which spontaneous contractions appear at the end of a loading test. Spontaneous activity would not appear to be a significant feature of the experiments, however, because the loads employed appear to be greater than those which the muscle can exert, and because the time scale for plastic recovery of the mesogloea being much slower than that for muscular contractions the two can be distinguished.
Another set of experiments was made in which the mesogloea was loaded to destruction in a relatively short time, usually in about 6–7 min. These experiments were made to gain an idea of the strength of the tissue, although during the experiments the tissue was under, admittedly, artificial conditions. In addition, it was hoped that some information might be gained about the strength of the material in various directions with respect to the crossed fibrillar orientation of its fibres. Pieces of tissue were prepared as for the kymograph recordings, and their width and thickness were measured with a micrometer. Their length was measured when they were set up in a simple form of extensimeter which is illustrated in Fig. 9. An upper fixed and a lower movable clamp held the test piece tightly between pieces of moistened filter-paper. The clamps had to be screwed up tightly, otherwise the material slipped, and although the test pieces were crushed by this means they did not appear to lose their physical strength as they rarely broke at the clamps. Usually they fractured in the middle. This method of holding the specimen was therefore considered to be satisfactory because, although it caused deformity, it did not produce weakness. It should be mentioned that the dimensions of the specimen are not those which it possessed in the living animal because the fibres became compacted during preparation. The tests occupied various times, but a typical experiment took 6–7 min. from the beginning of loading to the rupture of the test piece. Strips of body-wall were cut in a longitudinal, diagonal and horizontal manner in order to test whether greater extension was possible along the length of the geodetic fibres or diagonal to them and whether those fibres were stronger along their length than they were at their interfibre junctions. The results of a number of experiments are given in Table 2.
From the table of results it will be seen that there is not a very great difference in the breaking stresses of strips of tissue cut in a horizontal, diagonal and longitudinal manner, though in Calliactis the extensibility of the diagonal strips is perhaps greater than that of the others.
If all the fibres were in a 45° crossed fibrillar arrangement then diagonal strips should be relatively inextensible and vertical and horizontal strips relatively extensible. That this is not so is additional evidence that the fibres are arranged, not simply in a manner parallel to the surface, but in such a way that they can be pulled straight in a diagonal strip of tissue. This is in agreement with the arrangement of the fibres as loose sheets of warp and weft with fibrous connexions between the sheets.
The similarity of stress at the breaking point shown by strips cut in the three directions may perhaps be accounted for by the fact that when any fibrous material is stretched the fibres become orientated along the line of stretch and approach more closely to parallel orientation the more the material is stretched so that at the end of the stretching test the test piece, no matter from which orientation it originally came, consists of fibres of nearly parallel orientation. Histological examination of stretched mesogloea supports the view that the fibres become more nearly parallel in their orientation.
Similar experiments were performed on Metridium, the mean results of which are also given in Table 2. Despite its appearance in life it will be noted that the tensile strength of material from this animal is somewhat greater than that from Calliactis and that the diagonal strips are stronger and less extensible than the longitudinal ones, and of about the same strength and extensibility as the horizontal ones. This may perhaps be accounted for by the fact that, in the expanded animal, the mesogloea of Metridium is thinner than that of Calliactis so that the fibres are arranged more nearly as a two-than as a three-dimensional lattice, although the fact that the diagonal strips do show some extensibility points to the existence of a three-dimensional lattice in the moderately extended animal. This can indeed be seen in section and resembles the structure seen in Calliactis (Chapman, 1953).
On being heated in water collagen contracts in length and eventually, at higher temperatures, loses its strength and becomes gelatinous. It was considered that if similar behaviour could be demonstrated for Calliactis mesogloea it would provide some additional evidence for the collagenous nature of the connective tissue. An attempt to investigate the thermo-elastic properties of Calliactis mesogloea was therefore made with specimens prepared in the same way as those which were used for strength tests. The test strips were placed in the extensimeter previously described and were extended only by the weight of the extensimeter clamp (about 10 g.). The whole apparatus was immersed in a large beaker of tap water or of sea-water, and the change in length of the specimen observed at different temperatures. The length at room temperature was noted as the initial length, the temperature of the bath quickly raised to a new value and the length noted after 2, 5 and 10 min. During the 10 min. at which measurements at each temperature were made the temperature of the bath was kept approximately constant.
At 60 and 70° C., in both tap and sea-water, there is increase in length of up to 12% of the original length, but at 80 and 90° C. contraction occurs, the maximum amount being 25 % at the higher temperature. This behaviour is not quite the same as that of collagen from rat tail tendons whose contraction may begin at 55–60° C. in distilled water (Partridge, 1948). There is, nevertheless, a general likeness in the response of the two materials which points to their similarities rather than to their differences. They are dissimilar in the degree of orientation of their fibres, the rat tail tendon consisting of very nearly parallel fibres, so that it might well be expected that contraction, along the length of the fibre, would be more readily observable in the tendon than in the mesogloea in which the fibres are not predominantly parallel to the length of the test piece.
There appears to be no published work on the physical properties of the coelenterate mesogloea apart from references to it such as a ‘stiff jelly’ or ‘cartilage-like’ in consistency. Its properties are easier to investigate in actinians than in other coelenterates because it is tougher and is present in sufficient quantity to make possible its isolation for direct experiment. The consistency of an intact Calliactis can vary from soft when open, to hard and springy when closed. The anaesthetized animal (18 hr. menthol + 3 hr. MgCl2 and menthol) can be inflated by a pressure head of a few cm. of water, but when strips of the body-wall are cut out they contract and cannot easily be extended. They contract both along the long axis and at right angles to it, for, although there are only circular muscles in the column wall, the longitudinal parietals fie very close to it. A marked area on the animal measuring 2·0 × 1·5 cm. became 1·2×1·1 cm. on excision and 1·0 × 0·8 cm. on further squashing with the handle of a scalpel, a total reduction of area of 75 %. And after this treatment the mesogloea was of an almost cartilaginous consistency. It is difficult to avoid supposing that the fibres have become compacted together by the loss of interfibrillar fluid, and that they cannot by themselves regain their original separation. Indeed, sections of the excised and squashed mesogloea show the fibres as a much more compact mass than in the animal fixed in the expanded condition, although in animals closed by their own efforts there is no great increase in fibre density above that of the open specimen. These observations are made under highly artificial conditions for it cannot be imagined that the mesogloea is subject to very great stretching or compacting stresses during life. Nevertheless, an examination of the physical properties as such may throw some light on the part which the mesogloea plays in the life of the animal and vis-à-vis the muscle with which it is so intimately connected. In the latest and most detailed study of the physical properties of Metridium ‘muscle’ (Kipp, 1940) no account has been taken of the properties of the mesogloea, although in a ring of body-wall of Metridium, there is, bulk for bulk, very little muscle compared with mesogloea. The first part of Kipp’s paper describes the responses of a ring of Metridium body-wall to loading without electrical stimulation of the muscle. All of the effects recorded he ascribes to the muscle, but it has been shown here, by experiments similar to Kipp’s, that the viscous-elastic properties of the body-wall of Calliactis are all clearly shown by the mesogloea alone without the muscle at all. The behaviour of the tissue can be exactly described in terms of the familiar spring and dashpot combinations used to describe the mechanical behaviour of substances which show viscosity and elasticity together. In Fig. 10 is set out pictorially a number of possible combinations of springs and dashpots, together with their corresponding (extension) × (time) curves. It can be seen that the behaviour of the mesogloea agrees with the ‘elastic, viscous-elastic parallel, viscous’ combination.
The description of the behaviour of the mesogloea as ‘plastic’ (implying a definite ‘yield-point’) is probably best avoided since the tissue appears to be deformed under very small loads. While it is clear that this analogy throws some light on the structure of the mesogloea it throws none at all on that of the muscle. The analogy of the spring and dashpot is not applicable to the material of the fibres themselves, but rather to an interlacing system of fibres in a matrix.
It can be said, then, that the mesogloea can be deformed by small forces acting for a long time, that it shows a component of elasticity and that it has an element of viscosity or ‘plasticity’ and it can also be said that, if the unstimulated muscle has properties which are different from these they are so weak in comparison that they cannot be detected by ordinary mechanical tests but that they are, in all probability, very similar.
The tension which can be exerted by actual muscle fibre material of Metridium has been found by Batham & Pantin (1950) to reach 40 kg./sq.cm., and it has been shown here that the breaking strain of mesogloea material of Calliactis is of the same order. This would seem to imply that, in life, since the muscle fibres are present only as a single layer on the surface of the mesogloea, and since this latter is of much greater cross-sectional area than the muscle, the strength of the body-wall resides in the mesogloea rather than in the muscle.
It is a pleasure to acknowledge help from my colleagues at Queen Mary College, from the Director of the Marine Biological Laboratory, Plymouth, and especially from Dr C. F. A. Pantin, F.R.S.