Digestion in Ascidians and the Influence of Temperature

The physiology of digestion in Ascidians is but poorly known, and most of the existing knowledge is due to the work of Yonge (1925) on Ciona intestinalis. This is a genus, however, that is by no means typical of the class as a whole in that the stomach is relatively simple and has no well developed folds or liver.

In most of the Phlebobranchiata, to which Ciona belongs, the wall of the stomach is thrown into prominent ridges or folds that secrete a yellow-brown fluid. Among the Stolidobranchiata this development is taken further ; in the Styelidae folds alone are present; in the Botryllidae the folds are extended to form simple tubules ; while in the Pyuridae and Molgulidae a definite organ, commonly called the liver, is formed by an evagination of the stomach wall that divides into a mass of innumerable small tubules. The species the digestion system of which is described in this paper belong to the Pyuridae, and the structure of the liver and its relation to the alimentary tract as a whole are shown in Figs. 1 to 4. The form used primarily was Tethyum pyriforme americanum, and to a lesser extent Boltenia ovifera, while a few experiments were made with Molgula citrina and Ascidia prunum (see Huntsman, 1912).

The purpose of this paper is twofold, to compare the digestive system of a more typical Ascidian with that of Ciona, and to determine the connection, if any, between the optimum temperatures for enzyme activity and the body temperature of the animal. In the course of a conversation with Mr C. F. A. Pantin the suggestion arose that the temperature optimum of an enzyme would approximate more and more to the body temperature of the animal as the duration of the experiments approached the normal digestion period, and that the animal would retain the food within the gut until 70 per cent, to 90 per cent, of the enzyme had become inactivated. It is this problem of adaptation that the following account most concerns.

The experimental work was done at the Atlantic Biological Station at St Andrews, New Brunswick, while on the staff of the Physiological Department, University of Leeds, from which department most of the reagents used had been obtained. Thanks are due to the Biological Board of Canada for its hospitality at St Andrews during this period.

The feeding mechanism in Tethyum and Boltenia is similar to that described by Orton (1913) and Roule (1884) for other forms. The mucus secreted by the endo-style is carried by the beating cilia of the inner lining of the branchial sac across to the dorsal lamina. In so doing, the food particles drawn in through the inhalent or branchial siphon are swept against the field of mucus and are eventually found along the dorsal lamina welded by the mucus into a string that passes down and forwards to the oesophagus.

This cord of mucus and attached particles is carried down the oesophagus as a single strand until it reaches the region of the canal opposite the duct openings of the liver. Here the brownish secretion of that gland is poured on to the cord, which then, probably due to the resistance of the food mass preceding it, becomes coiled and doubled upon itself, until a short distance beyond the liver a cross-section through the gut would cut the cord in ten or twelve places. Thus a Tethyum or Boltenia individual possessing a gut three inches in length and about one-third of an inch internal diameter could contain a cord four or five feet long. The food mass as a whole is carried through the alimentary canal by the cilia of the lining epithelium.

In Ciona the epithelium of the stomach consists of a large number of absorption cells with a smaller number of secreting cells scattered among them. In the mid-gut the secretory cells are absent and are replaced by glycogen and mucus cells. All the cells of the alimentary tract are ciliated.

In Tethyum, however, the secretory cells seem to be confined to the distal parts of the liver tubules, while the proximal regions consist mainly of absorption cells like those typical of the gut as a whole, and of specialised ciliated cells.

The liver is a relatively large organ divided up externally into a limited number of lobes, each of which contains up to about five hundred small tubules. These tubules are formed by the folding of the epithelium so that the lumina and folds are of about the same diameter. Each fold has its interior distended by one or more blood vessels, while the organ as a whole is very greatly vascularised.

The epithelium in the distal portions of the liver tubules is definitely secretory, and secretory cells can be identified forming about 30 per cent, of the total ; whether the cells that stain less deeply with haematoxylin are inactive secretory cells or not is unknown. The cells of the epithelium form a single layer in direct contact with the wall of a blood vessel, as may be seen in Fig. 3. Where tubules fuse proximally to form first a number of large canals and finally two or three very large ducts entering the stomach region, the nature of the epithelium changes and it has the same appearance as that lining the alimentary tract generally. This epithelium is apparently almost wholly absorptive and mucus producing. Where sharp bends occur in the tubules, and along the side of the larger canals, the cilia of the epithelial cells are greatly developed, while the cell bodies are about half the usual length. All the cells lining the alimentary tract are ciliated, those of the secreting cells having the shortest, the cells immediately proximal to them having the longest, cilia.

It seems then that the secretion formed at the blind ends of the tubules is passed actively through the middle region of the liver to pour through the large ducts opening at the base of the oesophagus, where it mixes with the slowly moving column of food.

The problems of absorption and transportation of the products of digestion and in particular the interesting question of carbohydrate storage (Wagner, 1885) have been left for a future occasion.

Except where otherwise stated, the results recorded in the following pages are all obtained from experiments made with Tethyum pyriforme americanum. A some-what less complete series of experiments was made with the closely related Boltenia ovifera, with regard to pigment, pH range for the activity of the amylase and protease, and temperature effects, but as the results, beyond indicating an essential similarity between the two forms, failed to throw further light on the problems involved, they have been omitted.

The liver is situated in approximately the same relative position as the liver of the vertebrates, and it was thought that the nature of the secretion might determine whether the organs are homologous or not, but the results of experiments are decisive. The yellow-brown colour of the secretion suggested that true bile pigment might be present, though the absence of haemoglobin in the blood made it unlikely. The following tests were made for bile pigment, bile salts, and cholesterol.

Gmelin’s nitric acid test was made on a solution of the pigment n distilled water. The solution, at first yellow, changed to a vivid green on addition of the acid, and the same change could be produced by either sulphuric acid or acetic acid. In no case, however, was there any sign of the intermediate colours, red, violet, and blue that would be expected in the case of bile pigment proper.

Pettenkoffer’s test gave negative results.

The Liebermann-Burchard and Salkowski tests also gave negative results.

Before investigating the nature of the gut and liver enzymes, the hydrogen ion concentration of the alimentary tract was first determined. Freshly caught individuals were used and their guts quickly isolated, out of water. Drops of liquid were immediately obtained from various parts of the lumen by means of a pipette and when tested with dilute Brom-thymol-blue on a white plate invariably gave a greenish-blue reaction. The same indicator added directly on to the epithelial lining and contents of guts that had been slit open also showed a like colour. Thus it was found, with the aid of colour standards, that the pH of the gut lumen was approximately the same from the oesophagus to the hind-gut and had a value between 6.8 and 7.4, usually above 7.0.

Extracts were then made of the mid-gut and hind-gut together, and of the liver, and it was found, as the following experiments show, that enzymes are apparently secreted by the liver alone.

I gm. liver extracted for 24 hours at 18° C. in 50 c.c. toluol water.

0·6 gm. gut extracted for 24 hours at 18° C. in 30 c.c. toluol water.

Amylase. 1 per cent, starch solution buffered to 7·2 ; 0·4 c.c. enzyme extract with 1 c.c. sea-water and 10 c.c. substrate incubated for 4 hours at 28° C., and titrated into 10 c.c. Benedict’s quantitative solution (or aliquot part).

10 c.c. Benedict required 5·5 c.c. liver enzyme mixture.

10 c.c. Benedict required 78·0 c.c. gut enzyme mixture.

10 c.c. Benedict required 80 to 85 c.c. in the case of the controls with boiled extract

Protease, 10 per cent, gelatin buffered to pH 7·2 ; 0·4 c.c. extract—1 c.c. sea-water to 5 c.c substrate ; incubated at 28° C. for 20 hours. Liver enzyme mixture fluid at 9° C. ; controls solid below 22° C.; gut enzyme mixture solid at 21° C.

Lipase. (1) Emulsions of purified and neutralised olive oil stained red with Nile blue sulphate, in sea-water of pH 7·3, underwent no colour change after incubation with gut and liver extracts at 28° C. for 4 days.

(2) boiled milk; 2% Na₂CO₃; liver extract, adjusted to pH 7·5 ; control with bofled extract.

With phenol red: colour, at first red; after 15 hours, orange; after 25 hours, yellow (just). Colour of control red throughout.

In addition to testing the liver extract for amylase, tests were made for invertase, maltase, lactase, and cellulase.

Extract as before; mixtures buffered to pH 7.3; extract per 10 c.c. substrate; incubated at 28° C. for 6 hours.

Thus the liver secretes a powerful amylase with associated invertase, maltase, and lactase, a moderately strong protease, and a very weak lipase. No cellulase is present. While the enzymes produced by the gut proper, if produced at all, are too weak to be detected.

In Ciona somewhat different conditions hold. There is a powerful amylase, an invertase though no maltase or lactase, a lipase, but a protease too weak to be investigated.

The following experiments were made to determine the range of the hydrog ion concentration over which the various enzymes are active, and at the same time the optimum concentration.

(1 per cent, starch solution in 50 per cent, distilled water, 50 per cent, buffer solution, enzyme extract per 10 c.c substrate incubated for 18 hours at 35° C.)

There is an activity range for the amylase of pH 6·0 to pH 8·5, with an optimum in the region of pH 7·5.

(10 c.c. 20 per cent, gelatin in buffer solution, 5 c.c. sea-water, 1 c.c. enzyme extract, incubated for 18 hours at 35° C.)

The protease is active from pH 6·0 to a pH value above 10·0 and has an optimum probably near pH 8·0 or 8·5.

No experiments other than those already described were carried out on lipase, but extracts were made of the liver of Molgula citrina and of the stomach walls of Ascidia prunum in order to determine whether there existed in these two forms a protease similar to that of Tethyum and Boltenia.

(5 c.c. 20 per cent, gelatin in buffer solution, 5 c.c sea-water, enzyme, incubated for 18 hours at 35° C.)

(Same enzyme substrate mixture, conditions as in Molgula)

Therefore in Tethyum and Boltenia among the Pyuridae, in Molgula, and in Ascidia there is a protease, active only in alkaline and neutral media.

In the case of Tethyum 3 gm. liver were extracted for 18 hours in 50 c.c. toluol water at 16° C.; in the case of Boltenia 2 gm. were extracted in 50 c.c. under the same conditions.

10 c.c. extract were added to 100 c.c. 3 per cent, starch solution in buffer mixture pH 7·5 50 per cent, and sea-water 50 per cent., for the amylase ; 20 c.c extract were added to 100 c.c. per cent, casein solution in per cent. Na₂CO₂ adjusted to pH 7·5, for the protease.

The amylase, therefore, is many times more powerful than the protease.

Experiments were made to determine the influence of temperature on the relative amounts of substrate hydrolysed after digestion periods varying from two to eighty hours, so that not only was the relative degree of hydrolysis determined for various temperatures after a given length of time, but also the absolute amounts Digestion in Ascidians and the Influence of Temperature 283 converted after varying periods for any one temperature. In this way some indication was obtained of the true optimum temperature of a given enzyme.

Experiments were also devised to determine the time taken for any food particle to pass through the alimentary canal, at several temperatures, in order to discover the degree of economy and efficiency obtaining in the production of digestive enzymes by the organism.

The experimental results are expressed in the form of graphs, the data from which they were constructed being contained in tables to be found at the end of this paper.

Tethyum liver. 5 gm. extracted in 50 c.c. toluol water for 15 hours at 18⁰ C.

Substrate. 5 per cent, starch solution in 75 per cent, buffer mixture pH 7.3, 25 per cent, sea-water ; 1 c:c. extract per 10 c.c. substrate.

Samples of the digestion mixture were kept at temper 15, and 10° C., and titrations with Benedict’s solution were made for each temperature after 1, 3, 9, 20, 33, and 57 hours.

In Fig. 5 the results are plotted to show the temperature curve obtained after each of the above periods, and to show the optimum temperature in each case. This optimum is seen to vary with the duration of the experiment: after 1 hour it is 45° C., after 3 hours, 30° C.; after 9 hours, 26° C.; after 20 hours, 23° C.; after 33 hours, 17° C.; and after 57 hours it is 13° C.

In Fig. 6 the results are plotted to show the titration curve obtained at each temperature, and the approximate length of time for which the enzyme remains active at those temperatures is shown. The vertical dotted lines show the point at which the enzyme is about 75 per cent, inactivated at io° C. and at 15° C.

The time taken for any given particle of food to pass through the alimentary canal was determined only approximately. Freshly caught individuals with full guts were placed in filtered sea-water of known and constant temperature and the time taken to evacuate the food column determined by opening individuals at various intervals. In this way it was found that at 15° C., the highest temperature at which the animals could be kept in a definitely healthy condition, the food took about 35 hours to pass from the branchial sac to the anus. At 10° C. the time taken was 5° to 55 hours, while at 5° C. it was between 70 and 90 hours.

In individuals kept in filtered sea-water after the food was finally eliminated, the cord of mucus remained as a thread passing slowly out from the atrial siphon and was passed at the rate of one to two inches per hour at 15° C., a rate which tends to confirm the time obtained for the complete passage of food through the gut, found by the first method. Vertical continuous lines have been drawn in Fig. 6, at 35 and 55 hours, to show the approximate degree of destruction of the enzyme at 15° C. and 10° C., and it is seen that at 15° C. the 75 per cent, destruction line almost coincides with the line representing the normal time of digestion at that temperature, i.e. 35 hours. At 10° C. the enzyme is about three-fourths destroyed after 62 hours, while the digestion period at that temperature is 55 hours.

Therefore, on the basis of the results expressed graphically in Figs. 5 and 6, the following conclusions can be made.

Fig. 5. (a) While experiments of brief duration indicate an optimum temperature for enzyme activity above 40° C., the more prolonged the experiments the lower does the optimum temperature become.

(b) In experiments lasting 33 hours, i.e. about the normal digestion period at 15° to 16° C., the temperature optimum is 17° C.

(c) In experiments lasting 57 hours, i.e. about the normal digestion period at 9° to 10° C., the temperature optimum is 13° C.

Fig. 6. (d) At 15° C. the enzyme is about three-fourths destroyed during the digestion period, at that temperature, of 35 hours.

(e) At 10° C. the enzyme is roughly two-thirds destroyed during the digestion period, at that temperature, of 55 hours.

From these conclusions, if valid, it follows that the organism makes almost as full use of the digestive enzyme as is possible, in that the greater part of the enzyme is destroyed before it passes out from the alimentary canal. At 15° C. only 25 per cent, of the enzyme secreted remains active at the time it ceases to be used, and in order to utilise this remainder the digestion time at this temperature would have approximately to be doubled. That is, the enzyme is discarded by the organism about at the point on the curve shown in Fig. 6 when the curvature or rate of decrease in activity is most pronounced. In other words, the enzyme ceases to be used as soon as its activity decreases at all markedly from that which it first possessed. At io° C. in Fig. 6 the curve is almost a straight line for the time during which digestion normally proceeds at that temperature, viz. 55 hours, but after that it begins to curve towards the horizontal.

The experiments recorded in Series 2 (see Appendix), however, do not confirm these conclusions as well as was hoped. What does appear is that the temperature has exactly the same influence on protease activity as it has on amylase. For while the amylase results in Series 1 and 2 differ, the amylase and protease in Series 2, i. e. from one and the same extract, are almost identical in their behaviour to differences of temperature.

3 gm. liver were extracted in 50 c.c. toluol water for 18 hours at 18° C. 10 c.c. extract were added to 100 c.c. 3 per cent, starch solution (buffer mixture pH 7.5 75 per cent., sea-water 25 per cent.).

Figs. 7 and 8 show the results obtained, and it can be seen that in both amylase and protease the enzyme is considerably more stable at any given temperature than is the case in Series 1, and that the temperature optima, while falling as the experiments are prolonged, never reaches as low a value as 20° C. The optimum in the case of the amylase drops from 48° C. after 2 hours to 28° C. after 47 hours and to 26° C. after 70 hours, i.e. it falls 20° C. in 45 hours. In the case of the protease the optimum after 2 hours is 49° C. (extrapolated), after 47 hours it is 27° C., i.e. it falls 22° C. in 45 hours. In the first series with amylase the optimum after 2 hours was 35° C. (extrapolated), after 47 hours 15° C., i.e. it fell 20° C. in 45 hours.

Fig. 9 shows the various temperature optima plotted against time. Thus the only difference of importance between the two series is that in the second the enzyme is relatively more stable at all temperatures. This may be due to the presence of a greater proportion of protective substances in the second extract, although in case the concentration of the enzymes, which is obviously different in the two series, may bear on the question, the following experiments were carried out.

5 gm. liver were extracted in 50 c.c. toluol water for 18 hours at 17° C. The substrate was the 3 per cent, starch solution used in the last series of experiments.

• 5 c.c. undiluted extract were added to 50 c.c. starch solution.

• 5 c.c. extract diluted to added to 50 c.c. starch solution.

• 5 c.c. extract diluted to added to 50 c.c. starch solution.

  Each concentration mixture was incubated for twelve hours at temperatures between 15° C. and 50° C., and the results are expressed in Fig. 10.

It will be seen that there is very little difference in the optimum temperature for concentrations 1 and ⁠, but that for concentration it is very high for such a prolonged experiment. But since in no case has an enzyme concentration been used approaching such a low value as that in the mixture containing extract that had been diluted to one-tenth the original strength, the concentration of the enzyme probably has nothing to do with the differences discussed above. Fig. 10 also shows that the amount of substrate converted is roughly proportional to the square root of the concentration of the enzyme.

Experiments recorded in Series 3 (see Appendix), the results of which are expressed in Fig. 11, were carried out to determine whether the results of Series 1 or of Series 2 are the most likely to represent the condition in nature, and it is seen that the lower values of the first series are obtained. While extremely desirable that experiments on a much more extensive scale should have been made, it was impossible in the time available to do so owing primarily to the difficulty encountered in obtaining material in the necessary quantity, and all that can be said at present is that the results of and therefore the conclusions from the first series of experiments on amylase are the more characteristic.

Therefore, in the case of the Ascidian Tethyum, it seems probable that the organism, by determining the time taken by the food to pass through the alimentary canal, has made possibly the best compromise obtainable between economy in the use of its digestive enzymes and rapidity of their action ; and that the true temperature optimum for activity of either amylase or protease is within a very few degrees of the temperature of the body.

Whether such a condition of maximum efficiency exists in animals generally can only be discovered through extensive investigations.

10 c.c. Benedict’s solution correspond to 0·02 gm. glucose. If x is the number c.c. digestion mixture required to titrate 10 c.c. Benedict, then x c.c. contain 0·02 gm. glucose.

Therefore represents the number of grams glucose per 5 litres digestion mixture.

For details of extract see text.

Amylase. represents the number of grams glucose per 5 litres digestion mixture.

Protease, milligram amino-acid nitrogen.

Amylase. represents the number of grams glucose per 5 litres digestion mixture.