THE FUNCTION OF THE CALCIFEROUS GLANDS OF EARTHWORMS BY ...jeb.biologists.org/content/jexbio/13/3/279.full.pdf · THE FUNCTION OF THE CALCIFEROUS GLANDS OF EARTHWORMS BY JAMES D

279

THE FUNCTION OF THE CALCIFEROUS GLANDSOF EARTHWORMS

BY JAMES D. ROBERTSON, B.Sc.Robert Donaldson Scholar, University of Glasgow

(From the Zoology Department, Glasgow, and the Sub-Department ofExperimental 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 ap-plied to certain specialisations of the oesophageal epithelium which occur in mostterrestrial oligochaetes. They are well developed in the family Lumbricidae.

In Lumbricus terrestris (Linn.) the calciferous glands are present in segmentsX-XIV, and consist of a pair of oesophageal pouches in segment X and a pair ofoesophageal glands in each of segments XI and XII. In segment X the oesophagealepithelium is raised up into long narrow longitudinal folds projecting into the lumen.The tips of these folds fuse, enclosing a series of longitudinal tunnels elongatedradially in transverse section. The tunnels extend backwards from the posterior partof segment X to segment XIII where they become smaller, and they end blindly insegment XIV.

The cells of the lamellae separating the tunnels are the secretory cells, and in theoesophageal glands they form a milky calcareous secretion, which passes forwards inthe tunnels of the oesophageal wall to the pouches. There the individual particlescoalesce and crystallise out as concretions of calcium carbonate, which escape intothe oesophagus, pass down the gut, and finally reach the soil.

Since Julius Leo (1820) first described the glands many theories have been ad-vanced as to their function.

Morren (1829) thought they might be connected with either the reproductive orthe digestive system, while Lankester (1864) suggested that the oesophageal pouches"may be connected with the formation of the egg capsule" which at that time wasconsidered to be calcareous. The milky secretion of the hinder glands he thoughtmight be concerned in the process of digestion.

Claparede (1869) concluded that the function of the glands was the productionof concretions which would help to triturate food in the gizzard. Both Perrier (1874)

280 JAMES D. ROBERTSON

and Darwin (1881) agreed that this function must be a very subsidiary one, sincesmall stones are already present in the earth which worms ingest. Darwin main-tained that the calciferous glands "served primarily as organs of excretion", butthat the calcium carbonate would incidentally neutralise any humic acids arising bythe decomposition of half-decayed leaves in the intestine.

Robinet (1883) and Harrington (1899) supported the neutralisation theory, butHarrington found no support for the excretionary view from his experiments. Com-bault (1909) regarded the glands as respiratory organs, absorbing oxygen and ex-creting carbon dioxide "fixed" as carbonate. M'Dowall (1926) also regards thefixation 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 calci-ferous glands, or " Chylustaschen " as he calls them, is the absorption of food pro-ducts. Gieschen (1930) gives the glands a respiratory significance. He believes theyremove calcium bicarbonate which has accumulated in the blood as a result of thebinding of carbon dioxide.

Voigt (1933) considers that the importance of the glands lies in the fact thatwhen the carbon dioxide content of the environment is high the chalk can be usedto eliminate that gas. That the buffering action of the secretion is important in themaintenance of reaction under such unfavourable conditions as a high carbon di-oxide concentration in the surrounding medium or the presence of acid food issupported experimentally by Dotterweich (1933).

Since the experimental evidence on which the older hypotheses of Darwin andMichaelsen are based is both slight and contradictory, it seemed desirable to re-examine 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 SECRETIONThe 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 car-bonate. As Kelly (1901) pointed out, the carbonate in the two posterior oesophagealglands is in the amorphous state, while it is present in the anterior oesophagealpouches 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 to2 mm. are not uncommon. Microchemical tests for calcium, magnesium, barium,strontium, ammonium salts, nitrates, phosphates and oxalates were applied to theamorphous secretion of specimens of Lumbricus terrestris, Allolobophora caliginosa,and Eisenia foetida. The secretion in all cases consisted of calcium carbonate, thetests for other radicles being negative except that a slight phosphate reaction wasobtained in each case. No trace of ammonium salts was found with Nessler's re-agent even in specimens of Eisenia freshly collected from manure, this being con-trary to Combault'8 identification of ammonium carbonate in this worm. His testconsisted in the recognition and distinction of crystals of calcium and ammoniumchloroplatinates and must be regarded as of doubtful validity.

The crystalline concretions formed by Lumbricus terrestris consist wholly ofcalcium carbonate and a little organic matter. In section, viewed under a petro-logical microscope the crystals were recognised as calcite by the optical propertiesof high double refraction and twinkling, and distinguished from aragonite by thepresence of the black cross under polarised light indicating a uniaxial crystal. Thecrystals differ from the concentric calcospherites described by Keilin (1921) in in-sect larvae by their irregular concretionary form, and in the presence of organicmatter acting as a nucleus and scattered throughout the crystal. A few of thecrystals obtained were, however, almost perfect rhombohedra.

Several analyses of the percentage of carbonate in the concretions were made byestimating 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 intothe oesophagus as well as the concretions, and in two cases the amount was de-termined. Several earthworms were fed on moistened calcium sulphate which theyingested as they would earth. At the end of a fortnight the concretions greater than0-33 mm. were sieved from the casts and analysed for carbonate. The carbonate stillpresent 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. ofworms

32

Carbonate content of casts (mg.)

Concretions

86-839-i

Remainder

9"500

Total

96-339-i

% of total asconcretions

90-1100

JEB-Xiliiii


III. FEEDING EXPERIMENTS

The purpose of the following experiments was to ascertain (i) whether thecalciferous glands of earthworms fed on various calcium salts continued to secretecalcium carbonate, and (2) whether the glands secreted carbonate when leaves werethe sole source of calcium.

In the first experiments individual worms which had been kept 3 days inmoistened 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 leafwere given to the worms as food. The carbonate concretions were collected, usuallyat the end of successive 5-day periods, by putting the casts through a sieve whichretained particles of 0-5 mm. in diameter and over. After a considerable time thecalciferous glands of the worms were fixed and sectioned, and the concretions presentin the intestine counted.

Table II. Feeding experiments

Substances

CaCO,

Ca^PO,),

CaC,O4

Pear leaves

Filter paper

Moorland peat

No. of concretionsand period

5 days 5 days 5 days0 8 4

5 days 9 days5 13

5 days 9 days4 5

7 days 7 days 25 days4 15 4°

7 days 7 days3 0 _

4 days

State of oesophagealglands

Glands active; numerous cal-careous spherules present


Very few calcareous spherulespresent


Calcareous spherules still pre-sent

Very few spherules present

Concretions

Oeso-phagealpouches

2

3

0

0

0

0

Gizzardand/or

intestine

1

2

1

3

0

0

The results given in Table II show that the glands continue secreting whenworms are kept feeding on the carbonate or the phosphate of calcium, and also onpear leaves, since concretions continued to be formed and passed out. The worm infilter paper ceased forming concretions within a week, but at the end of 14 dayscalcareous 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 beaffected by the poisonous nature of the salt. The acid peat seems to have acteddirectly on the glands of the worm kept in it, since few calcareous spherules werepresent and fragments of protoplasm were found in the lumen of the glands, ap-parently owing to the disruption of the gland cells.

1 Pear leaves were readily eaten by worms and were convenient to obtain.


The concretions- in each case consisted of calcium carbonate. The numbers ofconcretions 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 theexperiments was already present in the worms.

In a second series of experiments an approximate measure of the carbonateexcreted was obtained by weighing the concretions formed by two worms in periodsof a week. Tap water was used to moisten the salts, but the amount of calcium pre-sent in it could not account for more than about 1 mg. of carbonate weekly.

Table III. Feeding experiments

Salt'

CaCO, (1)(2)

CaSO, (1)(2)

Ca.CPOJ, (1)(2)

Av. wt. ofworms (gm.)

7-S10-25

6-o10-75

909-5

Wt. of carbonate concretions (mg.) per week

1

16-5

I2'0

6-614-1

2

200149

4-62I-O

9-46-i

3

29-834-O

7-410-27-04-0

4

io-6

60

5

232

7-0

Av.

20-9218

6-2514-4

7-78-i

Experiment CaSO4 (1) was carried on for 8 weeks, the worms remaining healthyand 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 theglands. In leaves both inorganic and organic calcium compounds are present, butwhether 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 PHOTOGRAPHYVoigt (1933) has used X-rays for studying the calciferous glands. The following

observations and experiments were almost completed before the writer's notice wasdrawn to his paper.

The physiological state of the glands in earthworms kept in various media canbe determined at any time by X-rays. When in a state of secretion the small cal-careous particles present in the glands cause them to stand out when the worm isX-rayed. The more calcium present in the glands and the more compact it is, themore opaque are they to the rays.

The glands of specimens of Lumbricus terrestris collected from garden soil, grasslawns, 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 keepingearthworms in such acid media as filter paper moistened with lemon juice, starch,acid potassium phosphate, and peat. Keeping them in acid peat was the mostsuccessful, as after .about 10 days the glands were practically free from calcium andno longer stood out in the X-ray plate. At this stage the earthworms were X-rayed,

19-2


and then put into various calcium salts moistened with distilled water. Controlswere kept feeding on filter paper, starch, and peat. Plates were taken at intervals of2-5 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 freefrom calcium after 11 or 13 days in peat. Two days afterwards, the glands of theworms kept in the carbonate, sulphate, phosphate, and chloride of calcium weremarkedly denser, showing that secretion had taken place, while in those worms keptas controls in filter paper, starch, and peat the density of the glands had only slightlyincreased. Concretions had already been formed in the glands of the worm fed oncalcium 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 contra-dictory to that obtained in the feeding experiments. It seems that the result ob-tained there must be disregarded, since the section through the glands was madeafter the worm had been 14 days in the salt and was apparently affected by itspoisonous nature.

The experiments as a whole indicate that some absorption of the ingested saltstakes place, resulting in an increased concentration of calcium in the blood. Thisresults in an active excretion of the excess calcium as carbonate by the calciferousglands.

V. HYDROGEN-ION CONCENTRATION OF GUT, SOIL, AND CASTINGS

Since there seems to be no precise information about the reaction of the wall ofthe alimentary canal, the pH of the gut and of the coelomic fluid was determined bya 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 chloroformvapour and dissected under liquid paraffin. An indicator was drawn into a capillarytube of about i-5-mm. bore. At a particular region of the coelom or gut it was ex-pelled and drawn up again several times, this operation being conducted under theparaffin. The colour was finally compared with standards made up in similar tubeswith buffer solution of known pH. The buffer solutions of Clark and Lubs wereused, 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 dif-ferent regions of the gut, most of the values lying between pH 6-3 and 6-6. A con-spicuous exception is seen in the calciferous glands or rather the two hind oeso-phageal glands, where the pH of the isolated secretion is usually about 9-4. Thereaction of the coelomic fluid is generally more alkaline than the gut wall. The tablegiven is for specimens of Lumbricus terrestris, but results with Allolobophora cali-ginosa and Eisenia foetida were closely similar.

The Function of the Calciferous Glands of Earthworms

Table IV. pH of coelomic fluid and gut

2 8 5

Region ofearthworm

Coelomic fluid:In front of cropAnterior intestineMid-intestinePosterior intestineRectum

Gut:PharynxOesophagusCalciferous glandsCropGizzardAnterior intestineMid-intestinePosterior intestineRectum

Duration in filter paper

1 day

pH

6-76-76-7676-7

6-S6-S9'36-66-66-46-66-56-S

pa

6-86-76-66-66-6

6-66-6946-76-76-S6-6

64

2 days

£H

6-96-96-9696-9

6-S6-59'46-66-66-46-66-464

PH

6-76-86-76-76-7

6-66-69'46-66-66-46-4646-s

4 days

pa

6-66-66-66-66-6

6-2629-26-26-2626-26263

pa

6-s6-5

6-S

6-36-39-26-46-46-26-26-2

Salisbury (1924) has shown that worm casts are generally less acid than the soilfrom which they are derived. This holds only for acid soils, since he found a reduc-tion in the pH of casts from worms living in alkaline soils. The percentage of carbo-nate in the casts was approximately double that of the soil, and he suggested that thecalcareous particles from the calciferous glands neutralise the soil acids as Darwinhad believed.

Salisbury's data on the ̂ >H of worm casts were confirmed by keeping specimensof 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 aqueousextract of soil and water in the proportion 1:5, adding indicator, and centrifugingthe extract. The colour was then compared with that of standard solutions.

Earthpa

6 26-26-i6-i6-i6-i

Av. 6-13

Table V

Worm castpa

6-46-56-46-36-46-3

Av. 6-38+ 0-25

. pH of earth and worm casts

Earthpa

6-56-66-66-66-66-6

Av. 6-58

Worm castpa

6 36-36-46-46-36-4

Av. 6-35-023

Earthpa

7-77-77-87'77-87-7

Av. 7-73

Worm castpa

7-47-57-67-47-57-4

Av>7-47— 0-26

The difference in pH of the soil and cast refer to the moist soil and cast. Whenthese were air-dried before the determinations, the difference was usually reducedin the case of the acid soil, and it disappeared entirely in the chalky alkaline soil.


Darwin had found that the intestinal wall of worms was acid to litmus, and alsofresh casts except those from chalky soils. Casts i day old were usually no longeracid, and he attributed this to the breakdown of humic acids formed in the gut bythe 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 tothe amorphous secretion (pH 9-4) of the calciferous glands. Concretions from theglands 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 tendencytowards neutralisation of the worm cast is due, mainly at least, to the secretions ofthe gut wall, the secretion of the calciferous glands having no appreciable effectowing to the form in which most of the calcium carbonate is passed out of theglands. In connection with the last point, it seems that the earthworm has no con-trol over the form in which the carbonate is passed into the gut, since concretions ofno value in neutralisation are formed whether it is kept in alkaline soil, acid soil, orfeeding on decaying leaves. Powdered calcium carbonate immediately neutraliseshumus as Robinet observed, but concretions kept in acid peat (pH about 4-2) didnot alter its acidity and seemed to be quite unaffected by the humic acid9 even aftera week.

VI. OPTIMUM HYDROGEN-ION CONCENTRATION OF ENZYMES

Darwin, Robinet, and Harrington have suggested that the secretion of the cal-ciferous glands will neutralise any acids present in the soil ingested or formed bythe decomposition of organic matter in the intestine, and thus bring the contents ofthe gut to a more favourable reaction for digestion. The evidence that neutralisationis 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 alkalinemedium, while Lesser and Taschenberg (1908) found the proteolytic enzyme to beactive in both weak acid and weak alkaline media.

It was thought of interest to investigate the optimum pH's of the main intestinalenzymes. A crude distilled water extract of the intestine was used in the followingexperiments. Its pH was about 6-2-6-4.

The sugars produced in amylase digestion were estimated by the Hagedorn andJensen method as modified by Boyland (1928). Fatty acids were directly titratedwith sodium hydroxide in a carbon-dioxide-free atmosphere with phenolphthaleinmethylene blue as indicator. When the buffer was suitable the acidimetric titrationof Glick (1934) to apH of 6-6 with brom thymol blue was used. Amino-N was de-termined by the formol titration, the formaldehyde being added after neutralisationto phenolphthalein.

The duration of the amylase experiment was 23 hours, the average time takenfor 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 percent., 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; figures for experiments subtracted from average of controls.

The Function of the Calciferous Glands of Earthworms

Table VI. Optimum pH of amylase

287

4-65'45-86-o6-26 46-66-87-07-27"47-67-88-o

Controlc.c. N/100

thiosulphate

1 6 7i6-7516-7516-81 6 8167516-81 6 81 6 81 6 716-81 6 716-816-75

Experimentc.c. N/100

thiosulphate

9-27'97-56-55-65'25 04'2S4-35'256-o7-29-059 1

Difference

7-68-99 3

10-311-2n - 6"•75125512-5" 5 51 0 8

9-67'7S7'7

Mg. reducingsugar per c.c.

2-362-762-883-i93-473-603 643-893-883-583'352-982-402-39

4-0 -

3-5

oc3

3-000

2-5

2-0

Text-fig. 2. The optimum pH of amylase.

Under the conditions of the experiment, the optimum reaction for amylasedigestion lies between pH 6-8 and 7-0.

Since the calcium carbonate secreted by the glands may increase the activity ofthe amylase, the following experiment was performed.

Substrate, enzyme extract and buffer solution as before; CaCO3, powdered (p) or asconcretions (c); 23 hours at 17-180 C.;̂ >H 6-8.

Calcium carbonate seems to have no specific effect on amylase digestion either inpowdered form or as concretions. Initially when the powdered carbonate wasadded, 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 concretionsdid not affect the pK.


Table VII. Calcium carbonate and amylase digestion

Substrate

Starch alone0-05 gm. CaCOj (p)o-O2 gm. CaCO3 (p)0-05 gm. CaCO3 (c)0-02 gm. CaCO, (c)

Controlc.c. N/100

thiosulphate

168167

Experimentc.c. N/100

thiosulphate

4-854-84-84-8475

Difference

11-911-95" 9 5u-95120

Mg. reducingsugar per c.c.

3-693-7037037o3-72

Lipase digestion. 10 c.c. Clark's phosphate-NaOH buffer; 1 c.c. 2 per cent, enzymeextract; 0-5 c.c. methyl butyrate; 9 hours at 180 C ; enzyme extract added to controls im-mediately before titration with phenolphthalein methylene blue as indicator.

Table VIII. Optimum pH of lipase

pH

Initial

5-76-26-46-66-87-07-27-67'77-9

Final

566-o6-2636-66-8707'47'57-7

Controlc.c. JV/20 NaOH

8-427-67-05-984-863-982-861-56I-2Oo-75

Experimentc.c. N/20 NaOH

8628-028168646-544723-622-16i-88i-57

Difference

0200-421162 6 6i-680-740-76o-6oo-68082

3-Or

2-5

2-0

Oa

1-0

0-5

5-5 6-0 7-56-5 7-0

paText-fig. 3. The optimum pH of lipase.

8-0


The above is one of several experiments in which the optimum pH in phosphatebuffer varied from pH 6-4 to 6-6 with different extracts. When a borate buffer wasused, an increase in the liberation of fatty acids up to the alkaline limit of the bufferwas observed. With ethyl butyrate as substrate, optima exactly similar to those ob-tained with methyl butyrate were found, but with the neutral fat tributyrin a morealkaline optimum was found in the phosphate buffer, pH 7-3-77.

Proteinase digestion. Unfortunately the proteinase even in a 20 per cent, extractof the intestine was very weak, and sufficient digestion at 180 C. in 24 hours couldnot be obtained in order to determine any optima. However, when the digestion ofcasein 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 differentconditions. Nevertheless, 50 per cent, of the activity of the enzymes has taken placebetween pH 6-o and 7-5 in the case of the amylase, and between pH 6-4 and 6-9 inthe case of the esterase. Only in the case of the weak proteolytic enzyme is an alka-line medium apparently specially favourable to digestion.

The reaction at which digestion occurs in the earthworm will largely depend onthe amount of soil present in the gut and its acidity or alkalinity. Decomposingorganic matter in the gut and the products of digestion may also be expected to in-fluence the reaction in some cases. Salisbury has found earthworms in soils rangingfrom pH. 5-1 to 7-5, while Allee and co-workers (1930) have found them in soilsvarying from pH 56 to 8-3. They are most abundant in soils of approximatelyneutral reaction. That the reaction of the soil can be slightly altered in the gut isshown by the tendency towards neutralisation shown by worm casts, but this wouldappear to be of only small benefit in digestion. But the secretion of the calciferousglands certainly plays no dominant part in the neutralisation, and the suggestion ofDarwin 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 in-gested various calcium salts secrete calcium carbonate, which passes into the gut ascalcite concretions. Since in all cases, except the carbonate, the salt of calcium seemsto be changed in its intermediary metabolism to carbonate, it is inferred that thecarbonate radicle has come ultimately from the carbonic acid of the blood. Theamount of carbon dioxide bound in this way as carbonate can be measured byestimating the carbonate present in the worm casts, and can be compared with therespiratory carbon dioxide.

In each of these experiments two worms were used as a unit. They were kept infilter paper moistened with 5 c.c. of a dilute solution of calcium nitrate or chlorideand 2 c.c. of a nutrient solution.1 Others were kept in the sulphate and carbonatemoistened with distilled water and 2 c.c. of the nutrient solution, or feeding on pearleaves. 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.


measurements were made. The experiments were conducted in a constant tempera-ture 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 wasmeasured in a Haldane type of respiration apparatus. Carbon-dioxide-free airsaturated with moisture was passed over the vessel containing the worms, dried incalcium chloride U-tubes, and the respired carbon dioxide absorbed in soda-limeU-tubes. Blank experiments were carried out between the respiratory measure-ments, the correction being 0-2 mg. or less. At the end of the week the casts wereanalysed for carbonate in the same apparatus, by adding hydrochloric acid and ex-pelling the carbon dioxide by gentle heating while carbon-dioxide-free air waspassed through. The apparatus was again checked by analysing small amounts ofpure calcium carbonate of similar order to that found in the casts, the results beingcorrect within 2 per cent. In most cases the whole experiment was repeated withthe same worms. In the casts of the worms in calcium carbonate the concretionsonly could be analysed.

Since the factor of movement was uncontrolled there was naturally considerablevariation in the amounts of carbon dioxide respired, the mean deviations from thefigures given in the table ranging from o-8 to io-o per cent, with an average of5-1 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.

8-5i4'510-510-5

12-512-5'

13-013-0

noI I - O

CO,, average per day (24 hours) in mg.

Respiratory

13-80020-633

I5-93315367

1836716-900

15-96714967

2066717967

In carbonate

1-0290-486

0-9571 000

1-8290-914

0-771081406790-886

Total

14-8292 1 - 1 1 9

16-89016-367

2019617-814

16-73815-781

21-34618-853

% of totalCO, bound

as car-bonate

692-3

5-76-i

9 15-i4-65-2

3-24-7

As might have been expected the results vary considerably even in the case ofduplicate experiments with the same worms. But in no case has the amount ofcarbon dioxide excreted as carbonate exceeded 10 per cent, of the total, the averageamount being 5-3 per cent.

Under natural conditions it is probable that this percentage will vary widely atdifferent times. The carbonate excretion will depend on the presence of suitablecalcium compounds in the earth and organic matter ingested, and the various factorsinfluencing their absorption and excretion, while the amount of carbon dioxiderespired will vary with the muscular activity of the worms, with changes in the tem-perature and humidity of the environment, and also with the degree of starvation.


It would seem improbable, however, that the excretion of carbon dioxide boundas carbonate could ever account for more than a fraction of the total metaboliccarbon dioxide.

VIII. THE SUPPOSED ABSORPTIVE FUNCTION

Michaelsen has repeatedly stated that the primary function of the calciferousglands 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 alsowell known that some external digestion can take place in earthworms, leaves beingoften digested in this manner until only the network of veins remains.

Liebmann (1927) in investigating fat absorption found that only isolated cells inthe calciferous glands absorbed the fat, absorption taking place chiefly in the in-testine but also in the crop.

Attempts were made to demonstrate absorption by the iron saccharate techniqueused by Yonge (1926) and others, but they were not very satisfactory. A dilutesolution 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 werecoloured blue by the Prussian blue reaction, but only a few scattered cells in thelamellae of the calciferous glands.

The great vascularity of the glands and the large surface afforded by the epi-thelial folding are advantageous to secretion and do not necessarily imply an ab-sorptive function.

Stephenson (1930) remarks on the well-marked tendency in the elaboration ofthe 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 be-ing obviously unsuitable for an absorptive function.

Michaelsen's view can be upheld neither from the experimental evidence norfrom physiological probabilities.

IX. DISCUSSION

In Darwin's important work—Vegetable Mould and Earthzvorms—he argued thatworms which consume fallen leaves containing usually a large percentage of limewould be liable to become charged with too much calcium were it not for its excre-tion as the carbonate by the calciferous glands. He observed that the glands ofworms from mould over chalk'' contained as many free calciferous cells (amorphoussecretion), and fully as many and large concretions as did the glands of worms whichlived where there was little or no lime; and this indicates that the lime is an excretionand 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 2-5days. Only a few particles were forming in the glands and no concretions were


found in the intestine. He concluded that his experiments did not support the ex-cretionary view, but no great weight can be attached to them because of theirshort duration.

Combault states that he confirmed Harrington's experiment with the carbonatebut he gives no details.

Voigt found some earthworms in which the glands did not stand out whenX-rayed, and he kept them in the carbonate, sulphate, and oxalate of calcium for8-10 days. The glands did not appear even then in an X-ray plate, and he concludedthat worms could not make use of these salts and that perhaps only organiccalcium compounds in their food were available as a source of calcium for thesecretion.

Voigt's results are inconsistent with the experiments of the writer in which byvarious methods it has been shown that excretion of calcium carbonate is continuedfor considerable periods (from 14 days to 8 weeks) in the carbonate, sulphate, phos-phate (tribasic), oxalate, chloride, and nitrate of calcium, thus affording evidence ofthe 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 carbondioxide is being excreted bound as carbonate, and they maintain that it is this par-ticular function that is important. Combault found an increased carbonate contentof worm casts as compared with a sample of the original soil, and this has been fullyconfirmed by Salisbury. Combault further states that ammonium carbonate isfound in the glands of Eisenta foetida of manure heaps (which the writer has failedto confirm), and that therefore the carbon dioxide is fixed by the most abundant basein the environment—lime in the soil and ammonia in manure. He believed that thecarbon dioxide thus fixed would otherwise be liable to asphyxiate the worms, but itis highly improbable that the relatively small amount which seems to be bound ascarbonate has any importance in this connection. He also believed that the calci-ferous glands were the actual site of this fixation and that they constituted "unappareil respiratoire" absorbing oxygen as well. His special fixing and stainingmethod to demonstrate oxygenation in the blood of the efferent vessels of the calci-ferous glands is questioned by Winterstein (1921). The assertion that water is drawninto a diverticulum of the oesophagus in front of the glands, and passed backwardsbetween the lamellae to re-enter the oesophagus by a second opening in segmentXIV is inaccurate. This opening has never been found by subsequent workers in-cluding the writer, and there is never sufficient water present in the oesophagus tojustify even the nominal correctness of the term "branchies internes" which healso gives to the calciferous glands.

Although the secretion would seem to represent a certain amount of bound car-bon dioxide, the calciferous glands themselves are in no true sense respiratory organsat all.

In a recent attempt to demonstrate a connection of the calciferous glands withthe respiratory function Gieschen measured by means of a Krogh respiration mano-meter the respiratory quotients of worms kept under different conditions.


When worms were fed on chalk the R.Q. was lowered. Gieschen interprets this asindicating that chalk absorbed into the blood has combined with carbon dioxideaccording to the reaction

CaCO8 + CO2+H2O =but the possibility that the reaction has taken place not in the blood but in the gutbetween respired carbon dioxide and ingested chalk must not be disregarded.

Gieschen also found that the R.Q. was lowered when worms were kept in atmo-spheres of high carbon dioxide content, and that in experiments of 3 days' durationthe 'R.Q. after falling became stationary when the percentage of carbon dioxidereached 20 per cent, or higher. Since the R.Q. in similar experiments of worms inwhich the glands had been removed increased to nearly unity, due apparently to asinking of oxygen consumption, he concluded that the glands made oxygen absorp-tion possible in high carbon dioxide tensions, and prevented the tension of the lattergas in the blood from exceeding a certain limit. The glands are supposed to achievethis 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 glandsexcrete the carbonate and not the bicarbonate of calcium. The apparatus he used isso sensitive that very small changes in the experimental conditions give rise to con-siderable differences in the measurements. It is also probable that at the high carbondioxide tensions Gieschen used some of the respiratory carbon dioxide was retainedin combination within the tissues of the earthworms, thus making difficult the in-terpretation of his respiratory quotients.

Voigt's experiments on the availability of the carbonate, sulphate, and oxalate asa source of the calcium of the secretion have already been mentioned. By X-rayphotography he confirmed the mode of excretion, the concretions in the anterioroesophageal pouches being formed from the amorphous carbonate in the oeso-phageal glands, and then passed into the oesophagus. He also found that the densityof the oesophageal glands was much reduced when worms had been kept in anatmosphere containing 25 per cent, carbon dioxide for 3 days, or in one of 14 percent, for 5 days, indicating that the carbonate had gone into solution in the bloodas the bicarbonate. He asserts, however, that this bicarbonate escapes through theskin, a statement unsupported by any conclusive evidence.

[n Voigt's view, the biological importance of the glands rests on the fact thatunder unfavourable carbon dioxide conditions (over 14 per cent.) the chalk formedin the glands can be utilised in the elimination of that gas.

Dottenveich has brought forward considerable evidence to support his viewthat 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 removedis lower than that of normal worms after being 3 days in an atmosphere containing5 per cent, carbon dioxide. The reaction of the coelomic fluid of normal worms fellonly slightly after 16 hours in 25 per cent, carbon dioxide. Analyses of the calciumcontent of worms, excluding gut and glands which were dissected out before eachanalysis, showed an increased amount per unit weight after they had been kept in


high carbon dioxide percentages. This seems to indicate that the calcium in theglands has been distributed throughout the body.

Dotterweich concludes that under unfavourable conditions such as a high per-centage 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 thefunction of the glands is the storage and excretion of superfluous calcium salts.

The validity of the conclusions of these two authors depends on whether earth-worms are ever exposed for long periods to such high percentages of carbon dioxideas were used in their experiments. The percentage of carbon dioxide in the'soilatmosphere depends largely on the activities of micro-organisms, and has an averagevalue at the depth of 6 in. of 0-25 per cent. (Russel and Appleyard, 1915). Duringwet conditions, these authors found the high value of 9-1 per cent, in grasslandunder Festuca ovina. To this will be added the amount of carbon dioxide re-spired by the worm, but the aeration afforded by the burrow itself would in thewriter's opinion prevent any considerable increase of long duration in the carbondioxide content of the burrow from that source.

The possibility that the calcium carbonate present in the calciferous glandsmight act as a buffer reserve under exceptional conditions must, however, be ad-mitted.

The evidence adduced to support the neutralisation hypothesis is as follows:

(1) Histological appearances of "over-secretion" were found by Harringtonafter 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 soilfrom 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 theglands of worms kept in acid moorland peat, but the writer agrees with Combaultin 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 theacids by increasing the secretion.

A partial neutralisation of acid soil does take place in the worm, but it wouldseem to be effected chiefly by the secretions of the gut other than that of the calci-ferous glands. The reduction in the pH of worm casts in alkaline and slightly acidsoil demonstrating that the amorphous secretion is not being passed into the oeso-phagus, the analyses of the amount of carbonate excreted as concretions of no valuein neutralisation, and the fact that concretions are still passed out when worms arekept in acid soil, peat, and decaying leaves, all point to the ineffectiveness of thesecretion as an agent for neutralisation.


Only the weak proteolytic enzyme seems to be more efficient in an alkalinemedium. The optimum hydrogen-ion concentrations of the stronger and more im-portant amylase and lipase give no definite support to the hypothesis.

The position of the calciferous glands in the oesophagus suggests that the secre-tion may be of importance in intestinal digestion, and if the organs are solely forexcretion it seems peculiar that their position is not in a more posterior part of thealimentary tract. However, the position is advantageous in that a considerableamount of blood from the dorsal vessel which has collected absorbed food productsand salts from the dorso-intestinal vessels, courses directly through the glands.

The formation of carbonate concretions has the physiological advantage that thecalcium is now in a form which is not reabsorbed by the intestine but passes un-changed to the exterior. It also means that no neutralisation can be effected, sincethe compact crystals are chemically inactive in such acidities as are likely to be ex-perienced by earthworms.

The theory that the glands absorb food products has already been discussed, andit 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 saltsto the needs of the earthworm as a whole, and when ordinary requirements for thatelement have been met, the excess is concentrated as carbonate in the calciferousglands and excreted in a relatively inactive form.

The formation of calcium carbonate is more or less continuous and takes placeunder acid, neutral, and alkaline conditions provided that calcium compounds areavailable in the soil and food ingested.

The carbonate radicle may often represent fixed metabolic carbon dioxide, andits elimination in conjunction with calcium by the calciferous glands points to theimportance of these organs in connection with the acid-base balance of the blood.

Under conditions of a high, sustained carbon dioxide content in the environ-ment, the carbonate in the glands will go into solution as the bicarbonate, thusacting as a buffer reserve in the maintenance of reaction.

There is no evidence that neutralisation of acid substances in the gut is a vitalfunction or even an incidental process, and the theory of food absorption is quiteimprobable.

XI. SUMMARY

1. In the Lumbricidae the secretion of the calciferous glands consists mainly ofcalcium carbonate, the percentage of carbonate in the calcite concretions being95-97 per cent.

2. Feeding experiments indicate that the calcium of the secretion can be derivedfrom the common inorganic salts such as the carbonate, sulphate, phosphate, oxa-late, chloride, and nitrate, and also from pear leaves.


3. Measurements of the hydrogen-ion concentration of the gut, soil, and castingsof specimens of Lumbricus terrestris show that the tendency of the cast to be moreneutral than the soil is due to the secretions of the gut as a whole, and not to thesecretion of the calciferous glands.

4. The optimum ^>H's of two of the main intestinal enzymes have been mea-sured. Amylase has an optimum at pH 6-8-yo, and lipase at pH 6-4-6-6 and 7-3-7-7 depending on the substrate.

5. The amount of carbon dioxide bound as carbonate by the glands was mea-sured 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, ingroups 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 carbonatebeing passed into the gut as crystals of calcite which are chemically inactive in thegut.

I wish to express my sincere thanks to Prof. Graham Kerr for suggesting thiswork and for his continued interest in it, and especially to Dr James Gray for adviceand many helpful suggestions. My thanks are also due to Drs A. E. Barclay andD. W. Lindsay of the Radiology Department, University of Cambridge, for theirco-operation and assistance in taking the X-ray plates.

REFERENCES

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.KELLY, A. (1901). Jena Z. Naturw. 36, 453.LANKESTEH, E. R. (1864). Quart. J. nticr. Sci., N.S., 4, 258.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.ROBINET, C. (1883). C.R. Acad. Sci., Paris, 97, 192.RUSSEL, E. J. and APPLEYARD, A. (1915). J. agric. Sci. 7, 1.

JOURNAL OF EXPERIMENTAL BIOLOGY, XIII, 3. PLATE I.

1t 12

ROBERTSON—THE FUNCTION OF THE CALCIFEROUS GLANDSOF EARTHWORMS (pp. 279—397)

The Function of the Calciferous Glands of Earthworms 297SALISBURY, 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.

Jber, 1899.)WrNTKRSTErN, H. (1931). Handbuch der vergleichenden Pkysiologie, 1, 2, 70.

YONGE, C. M. (1926). J. Mar. biol. Ass., N.S., 14, 342.

EXPLANATION OF PLATES

PLATE IFig. 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 ar'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 IIFig. 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).

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