279
THE FUNCTION OF THE CALCIFEROUS GLANDS
OF EARTHWORMS
BY JAMES D. ROBERTSON, B.Sc.
Robert Donaldson Scholar, University of Glasgow
(From the Zoology Department, Glasgow, and the Sub-Department of
Experimental Zoology, Cambridge)
(Received November 15, 1935)
(With Two Plates and Three Text-figures)
I. INTRODUCTION
THE calciferou9 glands, Kalkdriisen, or glandes de Morren, are various terms applied to certain specialisations of the oesophageal epithelium which occur in most
terrestrial oligochaetes. They are well developed in the family Lumbricidae.
In Lumbricus terrestris (Linn.) the calciferous glands are present in segments
X-XIV, and consist of a pair of oesophageal pouches in segment X and a pair of
oesophageal glands in each of segments XI and XII. In segment X the oesophageal
epithelium is raised up into long narrow longitudinal folds projecting into the lumen.
The tips of these folds fuse, enclosing a series of longitudinal tunnels elongated
radially in transverse section. The tunnels extend backwards from the posterior part
of segment X to segment XIII where they become smaller, and they end blindly in
segment XIV.
The cells of the lamellae separating the tunnels are the secretory cells, and in the
oesophageal glands they form a milky calcareous secretion, which passes forwards in
the tunnels of the oesophageal wall to the pouches. There the individual particles
coalesce and crystallise out as concretions of calcium carbonate, which escape into
the oesophagus, pass down the gut, and finally reach the soil.
Since Julius Leo (1820) first described the glands many theories have been advanced as to their function.
Morren (1829) thought they might be connected with either the reproductive or
the digestive system, while Lankester (1864) suggested that the oesophageal pouches
"may be connected with the formation of the egg capsule" which at that time was
considered to be calcareous. The milky secretion of the hinder glands he thought
might be concerned in the process of digestion.
Claparede (1869) concluded that the function of the glands was the production
of concretions which would help to triturate food in the gizzard. Both Perrier (1874)
JAMES D. ROBERTSON
280
and Darwin (1881) agreed that this function must be a very subsidiary one, since
small stones are already present in the earth which worms ingest. Darwin maintained that the calciferous glands "served primarily as organs of excretion", but
that the calcium carbonate would incidentally neutralise any humic acids arising by
the decomposition of half-decayed leaves in the intestine.
Robinet (1883) and Harrington (1899) supported the neutralisation theory, but
Harrington found no support for the excretionary view from his experiments. Combault (1909) regarded the glands as respiratory organs, absorbing oxygen and excreting carbon dioxide "fixed" as carbonate. M'Dowall (1926) also regards the
fixation of carbon dioxide as the chief function of the glands.
oes.
XIII
XIV
Text-fig. 1. The calciferous glands of Lumbricus terrestris: con. concretion; oes. oesophagus;
oes. gl. oesophageal glands; oes.p. oesophageal pouch.
Michaelsen (1895, 1928), however, believes the primary function of the calciferous glands, or " Chylustaschen " as he calls them, is the absorption of food products. Gieschen (1930) gives the glands a respiratory significance. He believes they
remove calcium bicarbonate which has accumulated in the blood as a result of the
binding of carbon dioxide.
Voigt (1933) considers that the importance of the glands lies in the fact that
when the carbon dioxide content of the environment is high the chalk can be used
to eliminate that gas. That the buffering action of the secretion is important in the
maintenance of reaction under such unfavourable conditions as a high carbon dioxide concentration in the surrounding medium or the presence of acid food is
supported experimentally by Dotterweich (1933).
Since the experimental evidence on which the older hypotheses of Darwin and
Michaelsen are based is both slight and contradictory, it seemed desirable to reexamine them and also the view that the glands have a respiratory significance.
The Function of the Calciferous Glands of Earthworms
281
II. CHEMICAL AND PHYSICAL NATURE OF SECRETION
The nature of the secretion of the calciferous glands in worms other than the
Lumbricidae seems to be little known. In that family it consists of calcium carbonate. As Kelly (1901) pointed out, the carbonate in the two posterior oesophageal
glands is in the amorphous state, while it is present in the anterior oesophageal
pouches as crystalline concretions of calcite.
The calcareous spherules formed in the oesophageal glands have a diameter of
°'75~5/x- Most of the concretions are 0-5-1 mm. in diameter, but larger ones up to
2 mm. are not uncommon. Microchemical tests for calcium, magnesium, barium,
strontium, ammonium salts, nitrates, phosphates and oxalates were applied to the
amorphous secretion of specimens of Lumbricus terrestris, Allolobophora caliginosa,
and Eisenia foetida. The secretion in all cases consisted of calcium carbonate, the
tests for other radicles being negative except that a slight phosphate reaction was
obtained in each case. No trace of ammonium salts was found with Nessler's reagent even in specimens of Eisenia freshly collected from manure, this being contrary to Combault'8 identification of ammonium carbonate in this worm. His test
consisted in the recognition and distinction of crystals of calcium and ammonium
chloroplatinates and must be regarded as of doubtful validity.
The crystalline concretions formed by Lumbricus terrestris consist wholly of
calcium carbonate and a little organic matter. In section, viewed under a petrological microscope the crystals were recognised as calcite by the optical properties
of high double refraction and twinkling, and distinguished from aragonite by the
presence of the black cross under polarised light indicating a uniaxial crystal. The
crystals differ from the concentric calcospherites described by Keilin (1921) in insect larvae by their irregular concretionary form, and in the presence of organic
matter acting as a nucleus and scattered throughout the crystal. A few of the
crystals obtained were, however, almost perfect rhombohedra.
Several analyses of the percentage of carbonate in the concretions were made by
estimating their carbon dioxide content gravimetrically. It was found to be 94-9—
96-7 per cent.
There is the possibility that some of the amorphous secretion may escape into
the oesophagus as well as the concretions, and in two cases the amount was determined. Several earthworms were fed on moistened calcium sulphate which they
ingested as they would earth. At the end of a fortnight the concretions greater than
0-33 mm. were sieved from the casts and analysed for carbonate. The carbonate still
present in the casts was also determined.
In these two cases the amount of secretion other than the concretions is small.
Table I. The physical nature of the excreted carbonate
No. of
worms
JEB-Xiliiii
Carbonate content of casts (mg.)
Concretions Remainder
Total
3
86-8
9"5
96-3
2
39-i
00
39-i
% of total as
concretions
90-1
100
JAMES D. ROBERTSON
282
III. FEEDING EXPERIMENTS
The purpose of the following experiments was to ascertain (i) whether the
calciferous glands of earthworms fed on various calcium salts continued to secrete
calcium carbonate, and (2) whether the glands secreted carbonate when leaves were
the sole source of calcium.
In the first experiments individual worms which had been kept 3 days in
moistened filter paper were fed on moistened calcium salts, decaying pear leaves,1
moorland peat (pH 4-2), and filter paper. At intervals equal portions of pear leaf
were given to the worms as food. The carbonate concretions were collected, usually
at the end of successive 5-day periods, by putting the casts through a sieve which
retained particles of 0-5 mm. in diameter and over. After a considerable time the
calciferous glands of the worms werefixedand sectioned, and the concretions present
in the intestine counted.
Table II. Feeding experiments
Concretions
Substances
No. of concretions
and period
CaCO,
5 days 5 days 5 days
0
8
4
Ca^PO,),
5 days 9 days
5
13
CaC,O4
5 days 9 days
4
5
Pear leaves
Filter paper
Moorland peat
State of oesophageal
glands
Oesophageal
pouches
Gizzard
and/or
intestine
Glands active; numerous calcareous spherules present
2
1
Glands active; numerous calcareous spherules present
3
2
Very few calcareous spherules
present
0
1
0
3
0
0
0
0
7 days 7 days 25 days
Glands active; numerous cal4
15
4°
careous spherules present
7 days 7 days
Calcareous
spherules still pre3
0
_
sent
4 days
Very few spherules present
The results given in Table II show that the glands continue secreting when
worms are kept feeding on the carbonate or the phosphate of calcium, and also on
pear leaves, since concretions continued to be formed and passed out. The worm in
filter paper ceased forming concretions within a week, but at the end of 14 days
calcareous spherules were still present in a section through the oesophageal glands.
The worm kept in calcium oxalate ingested very little of it and seemed to be
affected by the poisonous nature of the salt. The acid peat seems to have acted
directly on the glands of the worm kept in it, since few calcareous spherules were
present and fragments of protoplasm were found in the lumen of the glands, apparently owing to the disruption of the gland cells.
1
Pear leaves were readily eaten by worms and were convenient to obtain.
The Function of the Calciferous Glands of Earthworms
283
The concretions- in each case consisted of calcium carbonate. The numbers of
concretions found have no special significance, since the concretions varied in size.
It is probable that the calcium carbonate secreted during the first few days of the
experiments was already present in the worms.
In a second series of experiments an approximate measure of the carbonate
excreted was obtained by weighing the concretions formed by two worms in periods
of a week. Tap water was used to moisten the salts, but the amount of calcium present in it could not account for more than about 1 mg. of carbonate weekly.
Table III. Feeding experiments
Av. wt. of
worms (gm.)
Salt'
CaCO,
(1)
(2)
CaSO,
7-S
10-25
(1)
6-o
(2)
10-75
90
Ca.CPOJ, (1)
(2)
9-5
Wt. of carbonate concretions (mg.) per week
1
2
200
16-5
149
4-6
I2'0
6-6
14-1
2I-O
9-4
6-i
3
4
5
Av.
29-8
34-O
io-6
232
20-9
218
7-4
10-2
7-0
4-0
60
7-0
6-25
14-4
7-7
8-i
Experiment CaSO4 (1) was carried on for 8 weeks, the worms remaining healthy
and continuing to form concretions.
It seems clear from the preceding experiments that the inorganic carbonate,
sulphate, and phosphate are available as a source of calcium for the secretion of the
glands. In leaves both inorganic and organic calcium compounds are present, but
whether only the former or both are available to worms has not been shown.
Feeding experiments with several organic calcium compounds were unsuccessful.
IV. X-RAY PHOTOGRAPHY
Voigt (1933) has used X-rays for studying the calciferous glands. The following
observations and experiments were almost completed before the writer's notice was
drawn to his paper.
The physiological state of the glands in earthworms kept in various media can
be determined at any time by X-rays. When in a state of secretion the small calcareous particles present in the glands cause them to stand out when the worm is
X-rayed. The more calcium present in the glands and the more compact it is, the
more opaque are they to the rays.
The glands of specimens of Lumbricus terrestris collected from garden soil, grass
lawns, and from pasture over chalk were all in a state of active secretion.
Attempts were made to eliminate most of the calcium from the glands by keeping
earthworms in such acid media as filter paper moistened with lemon juice, starch,
acid potassium phosphate, and peat. Keeping them in acid peat was the most
successful, as after .about 10 days the glands were practically free from calcium and
no longer stood out in the X-ray plate. At this stage the earthworms were X-rayed,
19-2
284
JAMES D. ROBERTSON
and then put into various calcium salts moistened with distilled water. Controls
were kept feeding on filter paper, starch, and peat. Plates were taken at intervals of
2-5 days.
The X-ray technique was as follows: 30 kV., 140 mA., ^ sec. at 12 in. distance,
using a fine-focus Coolidge Tube supplied by a four-valve transformer unit.
As shown in Plates I and II, the glands of the worms used were practically free
from calcium after 11 or 13 days in peat. Two days afterwards, the glands of the
worms kept in the carbonate, sulphate, phosphate, and chloride of calcium were
markedly denser, showing that secretion had taken place, while in those worms kept
as controls in filter paper, starch, and peat the density of the glands had only slightly
increased. Concretions had already been formed in the glands of the worm fed on
calcium carbonate. After 4 or 5 days concretions were present in the oesophageal'
pouches of the worms fed on calcium chloride and on calcium sulphate.
The worm in calcium oxalate also showed secretion, this result being contradictory to that obtained in the feeding experiments. It seems that the result obtained there must be disregarded, since the section through the glands was made
after the worm had been 14 days in the salt and was apparently affected by its
poisonous nature.
The experiments as a whole indicate that some absorption of the ingested salts
takes place, resulting in an increased concentration of calcium in the blood. This
results in an active excretion of the excess calcium as carbonate by the calciferous
glands.
V. HYDROGEN-ION CONCENTRATION OF GUT, SOIL, AND CASTINGS
Since there seems to be no precise information about the reaction of the wall of
the alimentary canal, the pH of the gut and of the coelomic fluid was determined by
a colorimetric method.
Earthworms were kept in moistened filter paper to free them from ingested earth.
After periods varying from 1 to 4 days, specimens were anaesthetised in chloroform
vapour and dissected under liquid paraffin. An indicator was drawn into a capillary
tube of about i-5-mm. bore. At a particular region of the coelom or gut it was expelled and drawn up again several times, this operation being conducted under the
paraffin. The colour was finally compared with standards made up in similar tubes
with buffer solution of known pH. The buffer solutions of Clark and Lubs were
used, and also their indicators brom thymol blue, brom cresol purple, phenol red,
and thymol blue.
From Table IV it is seen that there is not much variation in the pH of the different regions of the gut, most of the values lying between pH 6-3 and 6-6. A conspicuous exception is seen in the calciferous glands or rather the two hind oesophageal glands, where the pH of the isolated secretion is usually about 9-4. The
reaction of the coelomic fluid is generally more alkaline than the gut wall. The table
given is for specimens of Lumbricus terrestris, but results with Allolobophora caliginosa and Eisenia foetida were closely similar.
The Function of the Calciferous Glands of Earthworms
285
Table IV. pH of coelomicfluidand gut
Duration in filter paper
Region of
earthworm
2 days
1 day
4 days
pH
pa
£H
PH
pa
pa
Coelomic fluid:
In front of crop
Anterior intestine
Mid-intestine
Posterior intestine
Rectum
6-7
6-7
6-7
67
6-7
6-8
6-7
6-6
6-6
6-6
6-9
6-9
6-9
69
6-9
6-7
6-8
6-7
6-7
6-7
6-6
6-6
6-6
6-6
6-6
6-s
6-5
Gut:
Pharynx
Oesophagus
Calciferous glands
Crop
Gizzard
Anterior intestine
Mid-intestine
Posterior intestine
Rectum
6-S
6-S
9'3
6-6
6-6
6-4
6-6
6-5
6-S
6-6
6-6
94
6-7
6-7
6-S
6-6
6-S
6-5
9'4
6-6
6-6
6-4
6-6
6-4
64
6-6
6-6
9'4
6-6
6-6
6-4
6-4
64
6-2
62
9-2
6-2
6-2
62
6-2
62
63
6-3
6-3
9-2
6-4
6-4
6-2
6-2
6-2
64
6-s
6-S
Salisbury (1924) has shown that worm casts are generally less acid than the soil
from which they are derived. This holds only for acid soils, since he found a reduction in the pH of casts from worms living in alkaline soils. The percentage of carbonate in the casts was approximately double that of the soil, and he suggested that the
calcareous particles from the calciferous glands neutralise the soil acids as Darwin
had believed.
Salisbury's data on the ^>H of worm casts were confirmed by keeping specimens
of Lumbricus terrestris in well-mixed and sifted natural soils of different reaction.
The pH of the casts and the soil was estimated each day by making an aqueous
extract of soil and water in the proportion 1:5, adding indicator, and centrifuging
the extract. The colour was then compared with that of standard solutions.
Table V . pH of earth and worm casts
Earth
Worm cast
Earth
Worm cast
Earth
Worm cast
pa
pa
pa
pa
pa
pa
62
6-2
6-i
6-i
6-i
6-i
Av. 6-13
6-4
6-5
6-4
6-3
6-4
6-3
Av. 6-38
+ 0-25
6-5
6-6
6-6
6-6
6-6
6-6
Av. 6-58
63
6-3
6-4
6-4
6-3
6-4
Av. 6-35
-023
7-7
7-7
7-8
7'7
7-8
7-7
Av. 7-73
7-4
7-5
7-6
7-4
7-5
7-4
Av>7-47
— 0-26
The difference in pH of the soil and cast refer to the moist soil and cast. When
these were air-dried before the determinations, the difference was usually reduced
in the case of the acid soil, and it disappeared entirely in the chalky alkaline soil.
286
JAMES D. ROBERTSON
Darwin had found that the intestinal wall of worms was acid to litmus, and also
fresh casts except those from chalky soils. Casts i day old were usually no longer
acid, and he attributed this to the breakdown of humic acids formed in the gut by
the decomposition of organic material.
It is clear that the fall in the reaction of the soils of pH 7-7 and 6-6 is not due to
the amorphous secretion (pH 9-4) of the calciferous glands. Concretions from the
glands were present in the casts, but they do not affect the reaction in the slightest.
From the data obtained on the reaction of the gut, it is suggested that the tendency
towards neutralisation of the worm cast is due, mainly at least, to the secretions of
the gut wall, the secretion of the calciferous glands having no appreciable effect
owing to the form in which most of the calcium carbonate is passed out of the
glands. In connection with the last point, it seems that the earthworm has no control over the form in which the carbonate is passed into the gut, since concretions of
no value in neutralisation are formed whether it is kept in alkaline soil, acid soil, or
feeding on decaying leaves. Powdered calcium carbonate immediately neutralises
humus as Robinet observed, but concretions kept in acid peat (pH about 4-2) did
not alter its acidity and seemed to be quite unaffected by the humic acid9 even after
a week.
VI. OPTIMUM HYDROGEN-ION CONCENTRATION OF ENZYMES
Darwin, Robinet, and Harrington have suggested that the secretion of the calciferous glands will neutralise any acids present in the soil ingested or formed by
the decomposition of organic matter in the intestine, and thus bring the contents of
the gut to a more favourable reaction for digestion. The evidence that neutralisation
is advantageous to digestion rests on the observations of Willem and Minne (1899),
who found an extract of the intestine to digest fibrin most actively in an alkaline
medium, while Lesser and Taschenberg (1908) found the proteolytic enzyme to be
active in both weak acid and weak alkaline media.
It was thought of interest to investigate the optimum pH's of the main intestinal
enzymes. A crude distilled water extract of the intestine was used in the following
experiments. Its pH was about 6-2-6-4.
The sugars produced in amylase digestion were estimated by the Hagedorn and
Jensen method as modified by Boyland (1928). Fatty acids were directly titrated
with sodium hydroxide in a carbon-dioxide-free atmosphere with phenolphthalein
methylene blue as indicator. When the buffer was suitable the acidimetric titration
of Glick (1934) to apH of 6-6 with brom thymol blue was used. Amino-N was determined by the formol titration, the formaldehyde being added after neutralisation
to phenolphthalein.
The duration of the amylase experiment was 23 hours, the average time taken
for filter paper to pass through the gut in a number of worms at room temperature.
Amylase digestion. Substrate, 2 per cent, starch solution, 5 c.c.; enzyme extract, 2 per
cent., 5 c.c; buffer solution, McIlvaine's, 5 c.c; toluene; 23 hours at 17-5° C ; controls,
boiled extract ;^>H's measured at beginning and end of experiment withB.D.H. Capillator,
no change after incubation;figuresfor experiments subtracted from average of controls.
The Function of the Calciferous Glands of Earthworms
287
Table VI. Optimum pH of amylase
Control
c.c. N/100
thiosulphate
4-6
5'4
5-8
6-o
6-2
64
6-6
6-8
7-0
7-2
7"4
7-6
7-8
8-o
Experiment
c.c. N/100
thiosulphate
Difference
167
9-2
7-6
i6-75
16-75
16-8
7'9
7-5
8-9
93
168
6-5
5-6
10-3
11-2
n-6
"•75
1255
12-5
"55
1675
16-8
5'2
168
168
167
4'2S
50
4-3
16-8
5'25
6-o
167
7-2
9-6
16-8
16-75
9-05
7'7S
91
7'7
108
Mg. reducing
sugar per c.c.
2-36
2-76
2-88
3-i9
3-47
3-60
3 64
3-89
3-88
3-58
3'35
2-98
2-40
2-39
4-0 -
3-5
oc
3
3-0
00
2-5
2-0
Text-fig. 2. The optimum pH of amylase.
Under the conditions of the experiment, the optimum reaction for amylase
digestion lies between pH 6-8 and 7-0.
Since the calcium carbonate secreted by the glands may increase the activity of
the amylase, the following experiment was performed.
Substrate, enzyme extract and buffer solution as before; CaCO3, powdered (p) or as
concretions (c); 23 hours at 17-180 C.;^>H 6-8.
Calcium carbonate seems to have no specific effect on amylase digestion either in
powdered form or as concretions. Initially when the powdered carbonate was
added, it caused a change in the pH of the buffer solution owing to its alkalinity.
Thus 5 c.c. of buffer solution of approximately pH 6-6 were added to the substrate,
and the addition of the powdered carbonate brought thepH to 6-8. The concretions
did not affect the pK.
288
JAMES D.
ROBERTSON
Table VII. Calcium carbonate and amylase digestion
Experiment
Control
Substrate
c.c. N/100
c.c. N/100
thiosulphate
thiosulphate
168
167
4-85
4-8
4-8
4-8
475
Starch alone
0-05 gm. CaCOj (p)
o-O2 gm. CaCO3 (p)
0-05 gm. CaCO3 (c)
0-02 gm. CaCO, (c)
Difference
Mg. reducing
sugar per c.c.
3-69
3-70
370
37o
3-72
11-9
11-95
"95
u-95
120
Lipase digestion. 10 c.c. Clark's phosphate-NaOH buffer; 1 c.c. 2 per cent, enzyme
extract; 0-5 c.c. methyl butyrate; 9 hours at 180 C ; enzyme extract added to controls immediately before titration with phenolphthalein methylene blue as indicator.
Table VIII. Optimum pH of lipase
pH
Initial
Final
5-7
56
6-o
6-2
63
6-6
6-8
6-2
6-4
6-6
6-8
7-0
7-2
Experiment
Control
c.c. JV/20 NaOH c.c. N/20 NaOH
70
7-6
7'7
7-9
7'4
7'5
7-7
8-42
862
7-6
7-0
5-98
4-86
3-98
2-86
1-56
8-02
816
I-2O
i-88
o-75
i-57
Difference
020
0-42
116
266
i-68
0-74
0-76
o-6o
o-68
082
864
6-54
472
3-62
2-16
3-Or
2-5
2-0
O
a
1-0
0-5
5-5
6-0
6-5
7-0
7-5
pa
Text-fig. 3. The optimum pH of lipase.
8-0
The Function of the Calciferous Glands of Earthworms
289
The above is one of several experiments in which the optimum pH in phosphate
buffer varied from pH 6-4 to 6-6 with different extracts. When a borate buffer was
used, an increase in the liberation of fatty acids up to the alkaline limit of the buffer
was observed. With ethyl butyrate as substrate, optima exactly similar to those obtained with methyl butyrate were found, but with the neutral fat tributyrin a more
alkaline optimum was found in the phosphate buffer, pH 7-3-77.
Proteinase digestion. Unfortunately the proteinase even in a 20 per cent, extract
of the intestine was very weak, and sufficient digestion at 180 C. in 24 hours could
not be obtained in order to determine any optima. However, when the digestion of
casein or gelatin in Clark's phosphate buffer was prolonged at 25 ° C. for 48 hours,
there was more digestion at a/>H of about 8-o than at either pH 6-o or 7-0.
It is realised that the optimum pH's of these enzymes may vary under different
conditions. Nevertheless, 50 per cent, of the activity of the enzymes has taken place
between pH 6-o and 7-5 in the case of the amylase, and between pH 6-4 and 6-9 in
the case of the esterase. Only in the case of the weak proteolytic enzyme is an alkaline medium apparently specially favourable to digestion.
The reaction at which digestion occurs in the earthworm will largely depend on
the amount of soil present in the gut and its acidity or alkalinity. Decomposing
organic matter in the gut and the products of digestion may also be expected to influence the reaction in some cases. Salisbury has found earthworms in soils ranging
from pH. 5-1 to 7-5, while Allee and co-workers (1930) have found them in soils
varying from pH 56 to 8-3. They are most abundant in soils of approximately
neutral reaction. That the reaction of the soil can be slightly altered in the gut is
shown by the tendency towards neutralisation shown by worm casts, but this would
appear to be of only small benefit in digestion. But the secretion of the calciferous
glands certainly plays no dominant part in the neutralisation, and the suggestion of
Darwin and Harrington concerning this cannot be substantiated.
VII. AMOUNT OF CARBON DIOXIDE BOUND AS CARBONATE
It has been shown that the calciferous glands of earthworms which have ingested various calcium salts secrete calcium carbonate, which passes into the gut as
calcite concretions. Since in all cases, except the carbonate, the salt of calcium seems
to be changed in its intermediary metabolism to carbonate, it is inferred that the
carbonate radicle has come ultimately from the carbonic acid of the blood. The
amount of carbon dioxide bound in this way as carbonate can be measured by
estimating the carbonate present in the worm casts, and can be compared with the
respiratory carbon dioxide.
In each of these experiments two worms were used as a unit. They were kept in
filter paper moistened with 5 c.c. of a dilute solution of calcium nitrate or chloride
and 2 c.c. of a nutrient solution.1 Others were kept in the sulphate and carbonate
moistened with distilled water and 2 c.c. of the nutrient solution, or feeding on pear
leaves. The worms were kept for over a week in their respective salts before any
1
This was quite empirical and consisted of peptone ops per cent., glucose o-i per cent.
JAMES D. ROBERTSON
290
measurements were made. The experiments were conducted in a constant temperature room at 180 C , the worms being in darkness the whole time.
On three alternate days the respiratory carbon dioxide of each pair of worms was
measured in a Haldane type of respiration apparatus. Carbon-dioxide-free air
saturated with moisture was passed over the vessel containing the worms, dried in
calcium chloride U-tubes, and the respired carbon dioxide absorbed in soda-lime
U-tubes. Blank experiments were carried out between the respiratory measurements, the correction being 0-2 mg. or less. At the end of the week the casts were
analysed for carbonate in the same apparatus, by adding hydrochloric acid and expelling the carbon dioxide by gentle heating while carbon-dioxide-free air was
passed through. The apparatus was again checked by analysing small amounts of
pure calcium carbonate of similar order to that found in the casts, the results being
correct within 2 per cent. In most cases the whole experiment was repeated with
the same worms. In the casts of the worms in calcium carbonate the concretions
only could be analysed.
Since the factor of movement was uncontrolled there was naturally considerable
variation in the amounts of carbon dioxide respired, the mean deviations from the
figures given in the table ranging from o-8 to io-o per cent, with an average of
5-1 per cent.
Table IX. Amount of carbon dioxide bound as carbonate
Salt, etc.
CaCl.,1-5%:
(1)
(2)
Ca(NO3),, 1 %:(:)
(2)
CaSO«:
(1)
(2)
CaCO,:
(1)
(2)
Pear leaves:
(i)
(2)
Average wt.
of worms
in gm.
8-5
i4'5
10-5
10-5
12-5
12-5'
13-0
13-0
no
II-O
CO,, average per day (24 hours) in mg.
Respiratory
In carbonate
13-800
20-633
1-029
0-486
I5-933
15367
18367
16-900
15-967
14967
20667
17967
0-957
1 000
1-829
0-914
0-771
0814
0679
0-886
Total
14-829
21-119
16-890
16-367
20196
17-814
16-738
15-781
21-346
18-853
% of total
CO, bound
as carbonate
69
2-3
5-7
6-i
91
5-i
4-6
5-2
3-2
4-7
As might have been expected the results vary considerably even in the case of
duplicate experiments with the same worms. But in no case has the amount of
carbon dioxide excreted as carbonate exceeded 10 per cent, of the total, the average
amount being 5-3 per cent.
Under natural conditions it is probable that this percentage will vary widely at
different times. The carbonate excretion will depend on the presence of suitable
calcium compounds in the earth and organic matter ingested, and the various factors
influencing their absorption and excretion, while the amount of carbon dioxide
respired will vary with the muscular activity of the worms, with changes in the temperature and humidity of the environment, and also with the degree of starvation.
The Function of the Calciferous Glands of Earthworms
291
It would seem improbable, however, that the excretion of carbon dioxide bound
as carbonate could ever account for more than a fraction of the total metabolic
carbon dioxide.
VIII. THE SUPPOSED ABSORPTIVE FUNCTION
Michaelsen has repeatedly stated that the primary function of the calciferous
glands or " Chylustaschen " is probably the absorption of food products.
Darwin demonstrated the presence of an amylase in the pharyngeal secretion,
while Willem and Minne found a proteinase in an extract of the pharynx. It is also
well known that some external digestion can take place in earthworms, leaves being
often digested in this manner until only the network of veins remains.
Liebmann (1927) in investigating fat absorption found that only isolated cells in
the calciferous glands absorbed the fat, absorption taking place chiefly in the intestine but also in the crop.
Attempts were made to demonstrate absorption by the iron saccharate technique
used by Yonge (1926) and others, but they were not very satisfactory. A dilute
solution of the salt was injected into the alimentary canal by way of the mouth,
since filter paper moistened in it was not ingested at all.
When absorption did take place, groups of adjacent cells in the intestine were
coloured blue by the Prussian blue reaction, but only a few scattered cells in the
lamellae of the calciferous glands.
The great vascularity of the glands and the large surface afforded by the epithelial folding are advantageous to secretion and do not necessarily imply an absorptive function.
Stephenson (1930) remarks on the well-marked tendency in the elaboration of
the glands for segregation from the alimentary canal culminating in the Lumbricidae,
where connection is retained only by one end of a long tunnel, such a condition being obviously unsuitable for an absorptive function.
Michaelsen's view can be upheld neither from the experimental evidence nor
from physiological probabilities.
IX. DISCUSSION
In Darwin's important work—Vegetable Mould and Earthzvorms—he argued that
worms which consume fallen leaves containing usually a large percentage of lime
would be liable to become charged with too much calcium were it not for its excretion as the carbonate by the calciferous glands. He observed that the glands of
worms from mould over chalk'' contained as many free calciferous cells (amorphous
secretion), and fully as many and large concretions as did the glands of worms which
lived where there was little or no lime; and this indicates that the lime is an excretion
and not a secretion poured into the alimentary canal for some special purpose ".
Harrington kept worms in calcium carbonate crystallised in minute rhomboids
(to distinguish it from the amorphous secretion) and sectioned the glands after 2-5
days. Only a few particles were forming in the glands and no concretions were
292
JAMES D. ROBERTSON
found in the intestine. He concluded that his experiments did not support the excretionary view, but no great weight can be attached to them because of their
short duration.
Combault states that he confirmed Harrington's experiment with the carbonate
but he gives no details.
Voigt found some earthworms in which the glands did not stand out when
X-rayed, and he kept them in the carbonate, sulphate, and oxalate of calcium for
8-10 days. The glands did not appear even then in an X-ray plate, and he concluded
that worms could not make use of these salts and that perhaps only organic
calcium compounds in their food were available as a source of calcium for the
secretion.
Voigt's results are inconsistent with the experiments of the writer in which by
various methods it has been shown that excretion of calcium carbonate is continued
for considerable periods (from 14 days to 8 weeks) in the carbonate, sulphate, phosphate (tribasic), oxalate, chloride, and nitrate of calcium, thus affording evidence of
the availability of these inorganic salts as a source of the calcium of the secretion.
It is possible that organic compounds can be utilised also.
Emphasis has been laid by Combault and M'Dowall on the fact that carbon
dioxide is being excreted bound as carbonate, and they maintain that it is this particular function that is important. Combault found an increased carbonate content
of worm casts as compared with a sample of the original soil, and this has been fully
confirmed by Salisbury. Combault further states that ammonium carbonate is
found in the glands of Eisenta foetida of manure heaps (which the writer has failed
to confirm), and that therefore the carbon dioxide isfixedby the most abundant base
in the environment—lime in the soil and ammonia in manure. He believed that the
carbon dioxide thus fixed would otherwise be liable to asphyxiate the worms, but it
is highly improbable that the relatively small amount which seems to be bound as
carbonate has any importance in this connection. He also believed that the calciferous glands were the actual site of this fixation and that they constituted "un
appareil respiratoire" absorbing oxygen as well. His special fixing and staining
method to demonstrate oxygenation in the blood of the efferent vessels of the calciferous glands is questioned by Winterstein (1921). The assertion that water is drawn
into a diverticulum of the oesophagus in front of the glands, and passed backwards
between the lamellae to re-enter the oesophagus by a second opening in segment
XIV is inaccurate. This opening has never been found by subsequent workers including the writer, and there is never sufficient water present in the oesophagus to
justify even the nominal correctness of the term "branchies internes" which he
also gives to the calciferous glands.
Although the secretion would seem to represent a certain amount of bound carbon dioxide, the calciferous glands themselves are in no true sense respiratory organs
at all.
In a recent attempt to demonstrate a connection of the calciferous glands with
the respiratory function Gieschen measured by means of a Krogh respiration manometer the respiratory quotients of worms kept under different conditions.
The Function of the Calciferous Glands of Earthworms
293
When worms were fed on chalk the R.Q. was lowered. Gieschen interprets this as
indicating that chalk absorbed into the blood has combined with carbon dioxide
according to the reaction
CaCO8 + CO2+H2O =
but the possibility that the reaction has taken place not in the blood but in the gut
between respired carbon dioxide and ingested chalk must not be disregarded.
Gieschen also found that the R.Q. was lowered when worms were kept in atmospheres of high carbon dioxide content, and that in experiments of 3 days' duration
the 'R.Q. after falling became stationary when the percentage of carbon dioxide
reached 20 per cent, or higher. Since the R.Q. in similar experiments of worms in
which the glands had been removed increased to nearly unity, due apparently to a
sinking of oxygen consumption, he concluded that the glands made oxygen absorption possible in high carbon dioxide tensions, and prevented the tension of the latter
gas in the blood from exceeding a certain limit. The glands are supposed to achieve
this latter function by removing the bicarbonate which accumulates in the blood.
Gieschen's work has been adversely criticised by both Voigt and Dottenveich,
who point out that the basis of his reasoning is false, since the calciferous glands
excrete the carbonate and not the bicarbonate of calcium. The apparatus he used is
so sensitive that very small changes in the experimental conditions give rise to considerable differences in the measurements. It is also probable that at the high carbon
dioxide tensions Gieschen used some of the respiratory carbon dioxide was retained
in combination within the tissues of the earthworms, thus making difficult the interpretation of his respiratory quotients.
Voigt's experiments on the availability of the carbonate, sulphate, and oxalate as
a source of the calcium of the secretion have already been mentioned. By X-ray
photography he confirmed the mode of excretion, the concretions in the anterior
oesophageal pouches being formed from the amorphous carbonate in the oesophageal glands, and then passed into the oesophagus. He also found that the density
of the oesophageal glands was much reduced when worms had been kept in an
atmosphere containing 25 per cent, carbon dioxide for 3 days, or in one of 14 per
cent, for 5 days, indicating that the carbonate had gone into solution in the blood
as the bicarbonate. He asserts, however, that this bicarbonate escapes through the
skin, a statement unsupported by any conclusive evidence.
[n Voigt's view, the biological importance of the glands rests on the fact that
under unfavourable carbon dioxide conditions (over 14 per cent.) the chalk formed
in the glands can be utilised in the elimination of that gas.
Dottenveich has brought forward considerable evidence to support his view
that the calciferous glands act as a buffer reserve in the maintenance of reaction.
Thus thepH of the coelomic fluid of worms in which the glands have been removed
is lower than that of normal worms after being 3 days in an atmosphere containing
5 per cent, carbon dioxide. The reaction of the coelomic fluid of normal worms fell
only slightly after 16 hours in 25 per cent, carbon dioxide. Analyses of the calcium
content of worms, excluding gut and glands which were dissected out before each
analysis, showed an increased amount per unit weight after they had been kept in
294
JAMES D. ROBERTSON
high carbon dioxide percentages. This seems to indicate that the calcium in the
glands has been distributed throughout the body.
Dotterweich concludes that under unfavourable conditions such as a high percentage of carbon dioxide in the atmosphere or the presence of acid food in the gut,
the buffering action of the glands comes into play, but under normal conditions the
function of the glands is the storage and excretion of superfluous calcium salts.
The validity of the conclusions of these two authors depends on whether earthworms are ever exposed for long periods to such high percentages of carbon dioxide
as were used in their experiments. The percentage of carbon dioxide in the'soil
atmosphere depends largely on the activities of micro-organisms, and has an average
value at the depth of 6 in. of 0-25 per cent. (Russel and Appleyard, 1915). During
wet conditions, these authors found the high value of 9-1 per cent, in grassland
under Festuca ovina. To this will be added the amount of carbon dioxide respired by the worm, but the aeration afforded by the burrow itself would in the
writer's opinion prevent any considerable increase of long duration in the carbon
dioxide content of the burrow from that source.
The possibility that the calcium carbonate present in the calciferous glands
might act as a buffer reserve under exceptional conditions must, however, be admitted.
The evidence adduced to support the neutralisation hypothesis is as follows:
(1) Histological appearances of "over-secretion" were found by Harrington
after sectioning the glands of worms kept in acid media such as lemon juice, starch,
sawdust, and calcium phosphate.
(2) Salisbury found that worm casts were generally more neutral than the soil
from which they were derived.
(3) The proteolytic enzyme seems to be most efficient in an alkaline medium.
(4) The position of the glands suggests that they may have some function in
digestion.
(5) Robinet pointed out that calcium carbonate neutralises humus.
"Over-secretion"—the cytoplasm of the gland lamellae reduced in thickness,
crystals and shreds of protoplasm present jn the gland cavities—is found also in the
glands of worms kept in acid moorland peat, but the writer agrees with Combault
in attributing this appearance to the direct action of the acids on the secretory cells,
disrupting them, and not to an attempt on the part of the worm to neutralise the
acids by increasing the secretion.
A partial neutralisation of acid soil does take place in the worm, but it would
seem to be effected chiefly by the secretions of the gut other than that of the calciferous glands. The reduction in the pH of worm casts in alkaline and slightly acid
soil demonstrating that the amorphous secretion is not being passed into the oesophagus, the analyses of the amount of carbonate excreted as concretions of no value
in neutralisation, and the fact that concretions are still passed out when worms are
kept in acid soil, peat, and decaying leaves, all point to the ineffectiveness of the
secretion as an agent for neutralisation.
The Function of the Calciferous Glands of Earthworms
295
Only the weak proteolytic enzyme seems to be more efficient in an alkaline
medium. The optimum hydrogen-ion concentrations of the stronger and more important amylase and lipase give no definite support to the hypothesis.
The position of the calciferous glands in the oesophagus suggests that the secretion may be of importance in intestinal digestion, and if the organs are solely for
excretion it seems peculiar that their position is not in a more posterior part of the
alimentary tract. However, the position is advantageous in that a considerable
amount of blood from the dorsal vessel which has collected absorbed food products
and salts from the dorso-intestinal vessels, courses directly through the glands.
The formation of carbonate concretions has the physiological advantage that the
calcium is now in a form which is not reabsorbed by the intestine but passes unchanged to the exterior. It also means that no neutralisation can be effected, since
the compact crystals are chemically inactive in such acidities as are likely to be experienced by earthworms.
The theory that the glands absorb food products has already been discussed, and
it would seem that such a function is improbable, in the Lumbricidae at least.
X. CONCLUSIONS
There seems to be no adjustment of the amount of absorption of calcium salts
to the needs of the earthworm as a whole, and when ordinary requirements for that
element have been met, the excess is concentrated as carbonate in the calciferous
glands and excreted in a relatively inactive form.
The formation of calcium carbonate is more or less continuous and takes place
under acid, neutral, and alkaline conditions provided that calcium compounds are
available in the soil and food ingested.
The carbonate radicle may often represent fixed metabolic carbon dioxide, and
its elimination in conjunction with calcium by the calciferous glands points to the
importance of these organs in connection with the acid-base balance of the blood.
Under conditions of a high, sustained carbon dioxide content in the environment, the carbonate in the glands will go into solution as the bicarbonate, thus
acting as a buffer reserve in the maintenance of reaction.
There is no evidence that neutralisation of acid substances in the gut is a vital
function or even an incidental process, and the theory of food absorption is quite
improbable.
XI. SUMMARY
1. In the Lumbricidae the secretion of the calciferous glands consists mainly of
calcium carbonate, the percentage of carbonate in the calcite concretions being
95-97 per cent.
2. Feeding experiments indicate that the calcium of the secretion can be derived
from the common inorganic salts such as the carbonate, sulphate, phosphate, oxalate, chloride, and nitrate, and also from pear leaves.
296
JAMES D. ROBERTSON
3. Measurements of the hydrogen-ion concentration of the gut, soil, and castings
of specimens of Lumbricus terrestris show that the tendency of the cast to be more
neutral than the soil is due to the secretions of the gut as a whole, and not to the
secretion of the calciferous glands.
4. The optimum ^>H's of two of the main intestinal enzymes have been measured. Amylase has an optimum at pH 6-8-yo, and lipase at pH 6-4-6-6 and 7-37-7 depending on the substrate.
5. The amount of carbon dioxide bound as carbonate by the glands was measured in a series of experiments with earthworms kept in different calcium salts.
The percentage of carbon dioxide excreted as carbonate never exceeded 10 per cent,
of the total metabolic carbon dioxide.
6. Absorption of iron saccharate injected into worms took place occasionally, in
groups of adjacent cells in the intestine and in isolated cells in the calciferous glands.
7. The true function of the calciferous glands is excretion, calcium carbonate
being passed into the gut as crystals of calcite which are chemically inactive in the
gut.
I wish to express my sincere thanks to Prof. Graham Kerr for suggesting this
work and for his continued interest in it, and especially to Dr James Gray for advice
and many helpful suggestions. My thanks are also due to Drs A. E. Barclay and
D. W. Lindsay of the Radiology Department, University of Cambridge, for their
co-operation and assistance in taking the X-ray plates.
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ALLEE, W.' C , TORVIK, M. M. et al. (1930). Pkysiol. Z06I. 3, 164.
BOYLAND, E. (1928). Biochetn.J. 22, 238.
CLAPAREDE, E. (1869). Z. iciss. Zool. 19, 602.
COMBAITLT, A. (1909). J. Anat., Paris, 45, 358 and 474.
DARWIN, C. (1881). Vegetable Mould and Earthworms, ist ed.
DOTTERWEICH, H. (1933). Pflug. Arch. get. Physiol. 232, 263.
GIESCHEN, A. (1930). Zool.Jb. (Physiol.), 48, 121.
GLICK, D. (1934). C.R. Lab. Carlsberg, 20, 5.
HARRINGTON, N. R. (1899). J. Morph. 13, Suppl. 105.
KEILIN, D. (1921). Quart. J. Truer. Set., N.S., 66, 611.
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LEO, J. (1820). De ttructura Lumbrici terrestris. Dissertatio inauguralis, Regiomonti.
LESSER, E. J. and TASCHENBERG, E. W. (1908). Z. Biol. 50, 446.
LIEBMANN, E. (1927). Zool.Jb. (Physiol.), 44, 281.
M'DOWALL, J. (1926). Proc. phys.Soc. Edinb. 21, 65.
MICHAELSEN, W. (1895). Abhr'Natune. Hamburg, 13, 21.
(1928). " Oligochaeten." In Kiikenthal's Handbuch der Zoologie, Teil 8, 32.
MORREN, C. F. A. (1829). De Lumbrici Terrestris. Bruxelles.
PERKIER, E. (1874). Arch. Zool. exp. gen. 3, 417.
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JOURNAL OF EXPERIMENTAL BIOLOGY, XIII, 3.
PLATE I.
1t
ROBERTSON—THE FUNCTION OF THE CALCIFEROUS GLANDS
OF EARTHWORMS (pp. 279—397)
12
The Function of the Calciferous Glands of Earthworms
297
SALISBURY, E. J. (1924). J. lirm. Soc. (Bot.), 46, 415.
STKPHKNSON, J. (1930). The Oligochaeta. Oxford.
VOIGT, O. (1933). Zool.Jb. (Physiol.), 52, 677.
WILLEM, V. and MINNE, A. (1899). Livre jubilaire Ch. van Bambeke, Bruxelles. (Extract in Zool.
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WrNTKRSTErN, H . (1931). Handbuch der vergleichenden Pkysiologie, 1, 2, 70.
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EXPLANATION OF PLATES
PLATE I
Fig. 1. Worm after 11 days in peat.
Fig. 2. Same worm after 2 days in calcium carbonate.
Fig. 3. Same worm after 4 days in calcium carbonate.
Figs. 4—6. Same procedure in calcium phosphate.
Figs. 7—9. Control worm photographed after 11,13 a r 'd '5 days in peat.
Fig. 10. Worm after 13 days in peat.
Fig. I I . Same worm after 2 days in calcium sulphate.
Fig. 12. Same worm after 5 days in calcium sulphate.
Fig. 13. Worm after 13 days in peat.
Fig. 14. Same worm after 2 days in filter paper moistened with calcium chloride (1 per cent.).
PLATE II
Fig. 15. Same worm after 5 days in this filter paper.
Figs. 16—18. Same procedure in calcium ozalate.
Figs. 19—21. Same procedure in calcium lactate.
Figs. 22—24. Same procedure in starch (control).
Figs. 25—27. Same procedure in filter paper (control).
IBB-XIII 111