275
DIGESTION IN ASCIDIANS AND THE INFLUENCE
OF TEMPERATURE
BY N. J. BERRILL,
Department of Zoology, McGill University, and Department of
Physiology, University of Leeds.
(Studies from the Atlantic Biological Station, St Andrews, N.B.)
{Received 30th December 1928.)
(With Eleven Text-figures.)
CONTENTS.
1.
3.
3.
4.
5.
6.
7.
8.
9.
10.
Introduction
Feeding mechanism
Morphology and histology
Liver pigment
Secretion of enzymes
pH. range for activity of amylase and protease
Relative strength of amylase and protease
Influence of temperature
Summary
Appendix
PAGE
275
376
376
378
379
380
383
383
389
291
1. INTRODUCTION.
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,
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
276
N. J. BERRILL
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.
2. FEEDING MECHANISM.
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 endostyle 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
oes
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 nesonliamis as Fig> *' I n t e s t i n e a n d U v e r of Tethyum pyriforme
ticies is carnea aown me oesopnagus as americanum. bSy branchial sac; kg, hind-gut;
a single Strand until it reaches the region /, liver; /', accessory lobes of liver; mg, mid-gut;
of the canal opposite the duct openings oe5' o^P* 13 ^ 8 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 arid 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.
3. MORPHOLOGY AND HISTOLOGY.
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 midgut 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
Digestion in Ascidians and the Influence of Temperature
277
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.
tig.
8 th
c.d
Fig. 2. Section through oesophagus, liver, and hind-gut: c, mucus food cord at base of oesophagus;
c', same coiled in hind-gut; c.d, ciliated ducts; d, d', main and secondary ducts of liver; hg, hind-gut;
lb, lobe of liver; s.tb, secreting tubules.
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
278
N. J. BERRILL
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.
Fig. 3. Three secreting tubules showing blood supply: bl.v, blood vessels; Im, lumen; s.ep, secretory
epithelium; t.r, testicular acini.
Except where otherwise stated, the results recorded in the following pages are
all obtained from experiments made with Tethyum pyriforme atnericanum. A somewhat 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.
4. LIVER PIGMENT.
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
Digestion in Ascidians and the Influence of Temperature
279
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.
Bile pigment. 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.
Bile salts. Pettenkoffer's test gave negative results.
Cholesterol. The Liebermann-Burchard and Salkowski tests also gave negative
results.
Fig. 4. Secretory, cilated, and absorptive epithelium from liver: a, secretory cells from distal
tubules; b, ciliated cells of middle region of liver; c, absorptive cells from proximal region of
liver and of intestine.
5. SECRETION OF ENZYMES.
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.
1 gm. liver extracted for 24 hours at 1800C. in 50 c.c. toluol water.
o-6 gm. gut extracted for 24 hours at 18 C. in 30 c.c. toluol water.
Amylase. 1 per cent, starch solution buffered to pH 7-2;
0-4 c.c. enzyme extract with 1 c.c. sea-water
and 10 c.c. substrate incubated for 4 hours at 280 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
N. J. BERRILL
280
Protease. 10 per cent, gelatin buffered to pU 7-2; 0-4 c.c. extract—1 c.c. sea-water to 5 c.c substrate;
incubated at 280 C. for 20 hours. Liver enzyme mixture fluid at 90 C.; controls solid below
220 C ; gut enzyme mixture solid at 21 ° C.
Lipase. (1) Emulsions of purified and neutralised olive oil stained red with Nile blue sulphate, in seawater of pH 7-3, underwent no colour change after incubation with gut and liver extracts at
280 C. for 4 days.
(2) 2 ! c.c. boiled milk; \ c c 2% Na2CO3; \ c.c. liver extract, adjusted to/>H 7-5; control with
boiled extract.
With phenol red: colour, at first red; after 15 hours, orange; after 25 hours, yellow Gust).
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; i c.c. extract per 10 c.c. substrate; incubated
at 280 C. for 6 hours.
10 c.c. Benedict required 12 c.c. mixture
Maltose solution
>25 c.c. control
10 c.c.
12 c.c. mixture
Lactose solution
10 c.c.
>25
c.c. control
10 c.c.
12 c.c. mixture
Sucrose solution
10 c.c.
>25 c.c. control
10 c.c.
>25 c.c. both control
Filter-paper pulp
10 c.c.
and mixture
solution
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.
6. £H RANGE FOR ACTIVITY OF AMYLASE AND PROTEASE.
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.
Amylase.
(1 per cent, starch solution in 50 per cent, distilled water,
50 per cent, buffer solution, \ c.c. enzyme extract per 10 c.c substrate
incubated for 18 hours at 350 C.)
pU
c.c. required to titrate
10 c.c. Benedict's sol.
3-0
4-0
>n-o
>n-o
5-o
6-o
6-5
>II*O
4'5
3'S
pH
c.c. required to titrate
10 c.c. Benedict's sol.
7*0
2*5
i*7 (Control > n )
8-5-
4'S
5
T
8-o
9-0
2'2
>II'O
There is an activity range for the amylase of pH 6-o topH 8-5, with an optimum
in the region of pH 7-5.
Digestion in Ascidians and the Influence of Temperature
281
Protease.
(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 350 C.)
Condition on being
cooled to 120 C.
3 -o
Solid
Solid
Solid
Solid
Solid
Semi-fluid
Fluid
Fluid
Fluid (control solid)
Fluid
Fluid
Fluid (control solid)
Fluid
4-0
4-5
5'O
5'5
6-o
6-5
7-0
7-5
8-o
8-S
9*0
IO'O
Condition on being
cooled to 3 0 C.
_
—
—
—
_
Semi-fluid
—
Fluid
—
Fluid
Semi-fluid
The protease is active irompH 6-o to apH value above io-o and has an optimum
probably near^H 8-o or 8-5.
No experiments other than those already described were carried out on lipase,
but extracts were made of the liver of Molgula dtrina and of the stomach walls of
Asddia prunum in order to determine whether there existed in these two forms a
protease similar to that of Tethyum and Boltenia.
Molgula dtrina.
(5 c.c. 20 per cent, gelatin in buffer solution, 5 c.c sea-water,
\ c.c. enzyme, incubated for 18 hours at 350 C.)
pU
S'O
6-o
7-0
8-o
9*o
IO'O
Condition on being cooled to 120 C.
Solid
Semi-fluid
Fluid
Fluid (control solid)
Fluid
Fluid
Asddia prunum.
(Same enzyme substrate mixture, conditions as in Molgula)
pH
5"°
6-o
7-0
8-o
9-0
IO'O
Condition on being cooled to 120 C.
Solid
Solid
Fluid
Fluid (control solid)
Fluid
Fluid
Therefore in Tethyum and Boltenia among the Pyuridae, in Molgula, and in Asddia
there is a protease, active only in alkaline and neutral media.
282
N.
J.
BERRILL
7. RELATIVE STRENGTHS OF THE AMYLASE AND PROTEASE
OF TETHYUM AND BOLTENIA.
In the case of Tethyum 3 gm. liver were extracted for 18 hours in 50 c.c. toluol
water at 160 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. z\ per cent, casein solution in \ per cent. Na2CO2
adjusted to^>H 7-5, for the protease.
Tethyum: protease. Formol titration.
Milligrams amino-acid Nitrogen per litre
Temperature
5°C.
15° C.
35° C.
After 3 hours
After 26 hours
42
70
217
448
35O
720
Tethyum-. amylase. Benedict's quantitative method.
Grams glucose per litre
Temperature
5°C.
15° C.
35° C
After 2 hours
After 22 hours
I'O
3'6
6-5
2-5
7-0
13-0
Boltenia: protease.
Milligrams amino-acid Nitrogen per litre
Temperature
15° C.
35° C.
After 3 hours
After 24 hours
28
63
315
497
Boltenia: amylase.
Grams glucose per litre
Temperature
15° C.
35° C.
After 2 hours
2-0
5-5
After 22 hours
8-o
ii-5
The amylase, therefore, is many times more powerful than the protease.
8. INFLUENCE OF TEMPERATURE.
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.
32
28
24
20
u
§ 16
Tib
O
12
8
3hrs.
0
10
20
30
40
50
60°C
Temperature
Fig. 5. Graph to show effect of temperature on Tethyum liver amylase after various durations.
(Appendix, Series i.)
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.
SERIES 1. (See Appendix.)
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 mixturepH 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
284
N. J. BERRILL
15, and io° 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 0 C , after 3 hours, 300 C ; after 9 hours, 260 C ; after 20 hours, 23 0 C ; after
33 hours, 170 C ; and after 57 hours it is 130 C.
5°C
10
20
60
30
40
50
70 hours
Duration of experiment
Fig. 6. Graph showing results of Series 1 (Appendix) plotted to show approximate durations of
amylase activity at various temperatures.
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 150 C.
Normal digestion periods.
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 150 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 io° C. the time taken was
50 to 55 hours, while at 50 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
Digestion in Ascidians and the Influence of Temperature
285
and was passed at the rate of one to two inches per hour at 150 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 150 C. and io° C , and it is seen that at 150 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 io° C. the enzyme is about three-fourths destroyed after 62 hours, while the digestion period at that temperature is 55 hours.
20
16
" 70hrs.
S 12
c
0)
1
I
3
30
40
50
60 °C
Temperature
Fig. 7. Graph to show effect of temperature on Teihyum amylase. (Appendix, Series a.)
0
10
20
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 400 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
0
15 to 160 C , the temperature optimum is 170 C.
(c) In experiments lasting 57 hours, i.e. about the normal digestion period at
9° to io° C., the temperature optimum is 130 C.
Fig. 6. (d) At 150 C. the enzyme is about three-fourths destroyed during the
digestion period, at that temperature, of 35 hours.
(e) At io° 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 150 C. only 25 per
N. J. BERRILL
286
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.
840
10
20
30
40
50
60 °C
Temperature
Fig. 8. Graph to show the effect of temperature on Tethywn protease. (Appendix, Series 2.)
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.
SERIES 2. AMYLASE AND PROTEASE.
3 gm. liver were extracted in 50 c.c. toluol water for 18 hours at 180 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
Digestion in Ascidians and the Influence of Temperature
287
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 200 C. The optimum in the
case of the amylase drops from 480 C. after 2 hours to 280 C. after 47 hours and to
50
I
40
g 30
Tr
^-.«7^r
20
'
—Za.
-~u
•4rr>;.-
10
10
0
20
SO
40
50
60
Duration of experiment
Fig. 9. Graph showing the temperature optima obtained in Appendix, Series 1, 2 and 3, plotted
against duration of the experiment: 1, amylase of Series 1; za, amylase of Series 2; 26, protease
of Series 253, amylase of Series 3.
10
8
& 6
CO
8
*5b
10
20
30
50
40
Temperature
Fig. 10. Graph showing the effect of concentration of the enzyme (amylase) on the temperature
optimum and on the amount of substrate transformed.
26y C. after 70 hours, i.e. it falls 200 C. in 45 hours. In the case of the protease the
optimum after 2 hours is 490 C. (extrapolated), after 47 hours it is 270 C , ix, it
falls 220 C. in 45 hours. In the first series with amylase the optimum after 2 hours
was 350 C. (extrapolated), after 47 hours 150 C , Le. it fell 200 C. in 45 hours.
19-2
288
N. J. BERRILL
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 170 C. The
substrate was the 3 per cent, starch solution used in the last series of experiments.
(a) 5 c.c. undiluted extract were added to 50 c.c. starch solution.
(b) 5 c.c. extract diluted to ^ added to 50 c.c. starch solution.
(c) 5 c.c. extract diluted to j ^ added to 50 c.c. starch solution.
Each concentration mixture was incubated for twelve hours at temperatures
between 150 C. and 500 C , and the results are expressed in Fig. 10.
81 hrs.
I
12
Li
s
8
8
a
"3b
Zfhrs
7hrs.
0
10
20
30
40 °C
Temperature
Fig. 11. Graph to show the effect of temperature on Tethyum amylase. (Appendix, Series 3.)
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
Digestion in Ascidians and the Influence of Temperature
289
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 Tethyutn, 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.
9. SUMMARY.
1. The mechanism of feeding and digestion in the Pyurid Ascidians Tethyum
pyriforme americanum and Boltenia ovifera is described.
2. The structure and histology of the " liver " is described and it is shown that it
is primarily an organ of secretion.
3. It is found that the only digestive enzymes are those poured into the gut by
the liver, and consist of a powerful amylase, a protease, a very weak lipase, and also
an invertase, a maltase, and a lactase.
4. The brownish pigment of the liver gives reactions with acids somewhat like
those of bile pigment. There is no trace of bile salts, however, nor of cholesterol.
5. The amylase has an activity range from^>H 6*o topiH 8-5 with an optimum
near/>H 7-5. The protease is active from/>H 6-0 to above pH io-o. A similar protease is secreted by Molgula citrina and Ascidia prunum.
6. The relative strengths of the amylase and protease are compared, the
amylase being very much the stronger.
7. While experiments of brief duration indicate an optimum temperature for
enzyme activity above 400 C , the more prolonged the experiments the lower does
the optimum become. Whatever the optimum may be after an experiment of
2 hours' duration, it falls about 200 C. during the next 45 hours, if the experiments
be so prolonged.
8. At 150 C. and at io° C. the food takes about 35 and 55 hours respectively to
pass through the alimentary canal, and at 5 0 C. somewhere between 70 and 90 hours.
These temperatures approximately cover the normal range in temperature of the
environment, and therefore of the animal itself.
9. From experiments lasting 33 hours the optimum temperature for enzyme
activity was found to be about 170 C.; that is, within one or two degrees of the body
temperature. From experiments lasting 57 hours the optimum temperature was
found to be about 130 C ; that is, within three degrees of the body temperature.
10. These temperature optima not only represent the relative amounts of
substrate converted at different temperatures, but also represent the absolute
amounts converted and convertible.
290
N. J. BERRILL
11. The enzymes, amylase and protease, are two-thirds to three-quarters destroyed during their period of activity within the alimentary canal of the animal,
and in order to utilise the remainder the digestion mixture would have to be retained within the canal for twice as long a time.
12. Therefore it seems probable that the organism in making such a compromise between a high activity of the enzyme and its economical use is working to a
maximum efficiency; and it is possible that a permanent increase in the stability of
the digestive enzymes would be turned to advantage through a more prolonged
retention of the food within the gut.
Digestion in Asddians and the Influence of Temperature
291
APPENDIX.
SERIES 1.
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.
. 0*02 x i o o o n 100
1000 c.c. contain
=4 x
gm.
6
X
Therefore
IOO
x
S
X
represents the number of grams glucose per 5 litres digestion mixture.
For details of extract see text.
Duration in hours of experiments
Tem.
(°C.)
1
5O
14
7-i
40
12
8-3
i-i
c.c. required
5
2-5
40
1-9
53
59
25
1-4
25
23
4
37
5
i-6
62-5
4
10
c.c. required
4*2
23
25
14
4*2
23
i-6
60
20
27
7
I'2
——
ioo/ae
1
ioo/a?
c.c. required
100/a:
c.c. required
o-8
ioo/a;
125
0*65
154
c.c. required
125
I'O
100
07
c.c. required
148
o-8
2'O
c.c. required
105
"7*5
5O
24
100/a:
o*95
0-85
i-4
7i
4-1
—
I'O
100
I'O
100
35
9
9i
95
2'8
9
i-i
I'O5
1
60
ii'i
1*65
82
175
5
20
2*47
—
—
—
—
57
6
16-6
4
5-6
33
10
10
20
18
32
20
9
3
IOOJX
ioo/ac
SERIES 2.
100
Amylase. — represents the number of grams glucose per 5 litres digestion mixture.
X
Duration in hours of (experiments
Temp.
(° C.)
61
2
20
5
51
9
11
41
10
10
34
11
29
6-4
5'2
19
16
3*4
2"2
30
2-8
45
36
i'5
9
34-5
14
3-2
3i
i-4
70
29
4-6
21
5
22
66
i'45
68
7
1
7*
—
—
—
22
8
12-5
14-2
5
—
7
2*2
47
5'i
70
c.c. required per
i9'5
—
2-O
—
—
c.c. required
5O
i-4
70
—
—
ex. required
I-I5
c.c. required
I "2
83
I-O5
95
10 c.c. Benedict
100/a:
87
1-05
IOO
1-2
100/a:
100/a:
100/a;
c.c. required
ioo/a?
c.c. required
46
i'4
70
83
3'i
32
i'75
56
i'4
70
c.c. required
2'5
40
17
c.c. required
58
5-5
18
100/ac
ioo/»
ioo/ac
N. J. BERRILL
292
SERIES 2.
N
Protease, i c.c. — NaOH=o-i4 milligram amino-acid nitrogen,
ioo
Duration in hours of experiments
Temp.
62
3
10
26
50
i-6
2-4
3-2
3'5
51
3-8
5-o
6-2
6-6
41
4-0
7-2
IO-I
34
3 -o
7-0
9-5
10-4
29
2-3
6-o
9-8
12-5
24
—
4'75
8-4
12-7
21
19
4'3
7-6
12-2
15
i-o
2-8
II-O
5
o-6
2-1
6-4
5-o
I2-O
8-5
c.c. N/100 NaOH required
to titrate 2 c.c. digestion
mixture
c.c.
c.c.
c.c.
c.c.
c.c.
c.c.
c.c.
c.c.
required
required
required
required
required
required
required
required
SERIES 3.
Amylase.
Temp.
(°C.)
41
34
28
100
represents the number of grams glucose per 5 litres digestion mixture.
Duration in hours of experiments
7
15
6-6
12-5
8
12-5
8
25
15*4
6-5
21
20
11
21
6-6
15
5-6
18
4-5
22
4-2
24
4
5
25
(40)
2'5
22
4'5
45
5
20
82
22
c.c. required per 10 c.c.
ioo/ae
c.c. required per 10 c.c.
24
100 fx
4*5
3-4
2-4
42
i-95
2-05
49
56 ? 5
i-6
62-5
7i' 4
1-85
54
74
c.c. required per 10 c.c.
100fx
c.c. required per 10 c.c.
ioo/ac
c.c. required per 10 c.c.
c.c. required per 10 c.c.
100/x
10. REFERENCES.
HUNTSMAN, A. G. (1912). " Ascidians from the coasts of Canada." Trans. Canad. Inst. 9, i n .
ORTON, J. H. (1913). " The ciliary mechanisms on the gill and the mode of feeding in Amphioxus,
Ascidians, and Solenomya togata." Journ. Mar. Biol. Assoc. (N.S.), 10, 19.
L. (1884). " Recherches sur les Ascidies simples
" Ann. du. Mus. d'Hist. Nat. Marseille
Zool. 2, 1.
WAGNER, N. (1885). "Die Wirbellosen des Weissen Meeres." Zool. Forschungen an der Kiiste des
Solowetskischen Meerbusens in den Sommermonaten der Jahre 1877, 79, 8 2 , 1 , 121. Leipzig.
YONGE, C. M. (1925). " Studies on the comparative physiology of digestion. 3. Ciona intestinalis."
Brit. Journ. Exp. Biol. 2, 373.
ROULE,