vi. the physiology of excretion and the signifi- cance of ... · excretion in indian earthworms 347...

Studies on the Structure, Development, andPhysiology of the Nephridia of Qligochaeta.

VI. The Physiology of Excretion and the Signifi-cance of the Bnteronephric Type of NephridialSystem in Indian Earthworms.

By

Kara Narayan Bahl, D.Sc. (Panj.), D.PML, D.Sc. (Oxon),(Merton College, Oxford)

Professor of Zoology, University of Lucknow, India.

With 7 Text-figures and 11 Tables.

CONTENTS.

PAGE1. INTRODUCTION 344

2. T H E NATURE AND MASS OF EXCRETORY SUBSTANCES EVACUATED

B Y THE EARTHWORM ( P H E R E T I M A P O S T H U M A ) . . . 348

(a) ESTIMATION OF AMMONIA AND U R E A IN W A T E R CONTAIN-

ING EARTHWORMS . 348

(6) ANALYSIS OF THE U R I N E OF EARTHWORMS, AND ESTIMA-

TION OF AMMONIA AND U R E A THEREIN . . . 351

3. T H E EXCRETORY SUBSTANCES IN THE COELOMIC F L U I D . . 354

4. T H E EXCRETORY SUBSTANCES I N THE BLOOD . . . . 356

5. T H E INITIAL PLACES OF E X C R E T I O N — T H E BODY-WALL AND THE

INTESTINAL W A L L . . . . . . . . 358

6. T H E R O L E OF THE NEPHRIDIA I N EXCRETION AND OSMOTIC

REGULATION 361

(A) T H E OSMO-REGULATORY FUNCTION OF THE NEPHRIDIA 363

(i) VOLUME-REGULATION 363

(ii) T H E R A T E OF EXCRETION OF U R I N E . . . 3 6 9

(iii) T H E OSMOTIC PRESSURE OF BLOOD, COELOMIC

F L U I D , AND U R I N E 371

(iv) T H E PROTEIN CONTENTS OF BLOOD, COELOMIC


(v) T H E CHLORIDE CONTENTS OF BLOOD, COELOMIC


(vi) CONCLUSIONS . . . . . . . 376

344 K. N. BAHL

PAGE(B) T H E EXCRETORY INCLUSIONS OF T H E ' C I L I A T E D

M I D D L E T U B E ' (ATHEOPHAGOCYTIC SECTION) O F T H E

N E P H R I D I A 377

7. E X C E E T O E Y ORGANS OTHEE THAN N E P H R I D I A . . . . 385

8. SUMMARY 387

9. R E F E R E N C E S 388

I. INTRODUCTION.

OUE knowledge of the physiology of excretion in the Oligo-chaeta is still very incomplete. Stephenson (19) gave anexhaustive account of published work up to 1930, while Stolte(£§) has admirably summarized the latest accounts up to 1938.But, as remarked by Stolte, each one of the number of con-tributory processes involved in excretion has been investigatedwith varying results, so that it is not possible as yet to have acomplete picture of the whole process. Similarly, Heidermanns(14) states: 'The findings about the excretory material ofworms vary greatly even to-day. Apart from the non-unanimityof accounts about the material which appear in the innermetabolism and the materials of the purin-group which arestored in the cells of the chloragogen tissue, several authorsalso differ from one another in their published results withregard to the nature and mass of the excretory substanceseliminated as a result of nitrogen metabolism, as also withregard to ammonia and urea.' It seems hard to believe, butnevertheless it is a fact, that we do not yet completely under-stand the physiology of excretion in the common earthwormstudied by every elementary student of zoology.

Broadly speaking, 'the function of the excretory system isto keep constant the internal environment of the body, or inother words, the fluid content of the body as a whole. To thisend, the excretory organs eliminate or segregate unwantedsubstances, and retain or reabsorb constituents needful to theorganism' (Wigglesworth, 21). In an earthworm there are twocirculating fluids, i.e. the blood and the coelomic fluid, whichremain completely separated from each other, but togetherform the greater part of the internal environment of the body.

EXCRETION IN INDIAN EARTHWORMS 345

How does an earthworm keep these two fluids constant, andhow do its excretory organs or nephridia eliminate or segregateunwanted substances ?

The nephridia have, on the one hand, a fairly rich blood-supply1—Benham's diagram of the blood-supply of the nephri-dium of L u m b r i c u s (9) shows a copious blood-supply, andso does a diagram of the blood-supply of a septal nephridiumof P h e r e t i m a p o s t h u m a (Text-fig. 1)—and, on the other,they not only float within the coelomic fluid but are in opencommunication with it through their nephridiostomes. • Someworkers have, therefore, assumed that there is a division offunctions between these two fluids. Eogers (17 a), for example,says: 'The coelomic 'fluid receives from the gut the food-materials as they diffuse through the walls and conveys thesefood-materials to the various cells of the body. It also receivesfrom the active cells of the body the various metabolic wastesand conveys them to the nephridia, through which they areeliminated. The blood or haemolymph in the closed system oftubes, on the other hand, is a solution of haemoglobin andserves as the carrier of oxygen to the various cells and tissuesof the body.' This division of functions between the coelomicfluid and blood, in which blood has nothing to do with excre-tion, is an assumption which has never been tested and provedso far.

The problem of excretion in the earthworm may be resolvedunder the following heads: (1) Where do the metabolic wastesfirst originate and what is their chemical nature ? (2) Does thecoelomic fluid contain nitrogenous waste products in solutionand, if so, in what form ? (3) Does the blood take any part incarrying excretory products, or is it merely a carrier of oxygen ?(4) What exactly is the role of the nephridia ? Are they onlyexcretory or do they have any other function besides? Arethere any other excretory organs besides nephridia? (5) Areany excretory products stored within the body of the earth-worm ? If so, in what form and where ? (6) What are the

1 Willem and Minne's (22) statement that 'in many Oligochaeta the.nephridia have no blood-supply' does not hold for the nephridia of earth-worms. ,

NO. 340 A a

346 K. N. BAHL

characters of the excretory fluid as it is finally evacuated fromthe body? An attempt has been made in this memoir to dealwith all these aspects of the problem, and to present, as far aspossible, a complete picture of the whole process of excretion.

Almost all previous workers on the physiology of excretion

TEXT-ITC. 1.

A septal nephridium of Phe re t ima pos thuma showing thecourse of the blood-vessels and capillaries in it. affnv, afferentnephridial vessel; ce, connecting loop-capillaries between theafferent and efferent vessels; commv, commissural vessel; sb,septo-nephridial branch of the ventro-tegumentary vessel.(x dr. 120.)

in the Oligochaeta have studied the process in the Europeantype genus Lumbr icus ; little attention has been paid toother forms, particularly the tropical earthworms, which present


important differences in their nephridial system from that ofL u m b r i c u s and would thus be likely to throw considerablelight on the physiology of the different processes involved inexcretion. Another unfortunate defect in the publications ofprevious workers, with a few notable exceptions, is that theyseldom give a complete account of the biochemical methodsthey have employed in arriving at their results. This makesthe task of a subsequent worker difficult, as he cannot ade-quately check their results by using the same methods as theyemployed. I have, therefore, tried to give, as far as possible,full details of the methods employed by me in all my bio-chemical estimations.

I have studied the process of excretion mainly in the earth-worm P h e r e t i m a ( P e r i c h a e t a ) pos thuma (2), whichpossesses three types of nephridia: (1) open septal nephridia,80-100 in each segment, which discharge their excretory pro-ducts through an elaborate system of canals into each segmentof the i n t e s t i n e and not to the e x t e r i o r ; (2) closedintegumentary nephridia, about 200 in each segment, whichopen to the ex t e r i o r on the body-wall; and (3) closedpharyngeal tufted nephridia, which number several hundredsin each of the three segments (IV-VI) where they occur, andwhich open in to the buccal c a v i t y and pha ryngea llumen . All these three types of nephridia are extremelyminute and lack the terminal bladder of the nephridium ofL u m b r i c u s .

I am deeply indebted to my friend and colleague Dr. S. M.Sane of the Department of Chemistry who has readily helpedme in this work at all times. and without whose active co-operation this work would not have been possible. My bestthanks are due to Dr. M. L. Bhatia for his kind help in thepreparation of illustrations and to Mr. L. N. Johri for his pains-taking assistance in the greater part of this work. I am alsovery thankful to Dr. N. K. Panikkar of the University College,Trivandrum, for reading through my manuscript and makingseveral useful suggestions.

348 K. N. BAHL

2. THE NATURE AND MASS OF EXCRETORY SUBSTANCES

EVACUATED BY THE EARTHWORM (P 'HERETIMA POSTHUMA).

Before determining the place of origin and the course ofelimination of the excretory products, it is necessary to find outthe nature and mass of excretory substances as they are finallyevacuated by the earthworm, as that will provide the necessaryclue to the proceeding contributory processes of excretion. Thefirst step, therefore, was to analyse and estimate the nitro-genous excretory products as they are finally evacuated by theearthworm.

(a) E s t i m a t i o n of Ammonia and Urea inWate r c o n t a i n i n g E a r t h w o r m s .

Lesser and Delaunay (as quoted by Stolte, 20) investigatedthe nitrogenous contents of the excretory fluid voided by thenephridia of the earthworm L u m b r i c u s . Lesser found onlyammonia, while Delaunay found ammonia, urea, and alsonitrogen as amins, but no -uric acid. Lesser kept earthworms indistilled water for twenty-four hours, while Delaunay keptthem for eight days, since he found that evacuation of thecontents of the terminal bladder of a nephridium took placeonly once in three days. This observation of Delaunay does nothold in the case of P h e r e t i m a because its nephridia do notpossess a terminal bladder to retain the excretory fluid for anylength of time and must therefore go on discharging the urineall the time. I may state at once that although keeping earth-worms in distilled water is a convenient method of obtainingtheir nephridial fluid, the water is bound to contain in additionsubstances defaecated through the anus as well as those dis-charged through the mouth. Even an earthworm whose guthas been cleaned of its earthy contents by keeping it in waterfor several days, gives out watery drops from the mouth as wellas anus, after it is wiped dry with a piece of cloth. Lesser andDelaunay did not take worms with clean guts and, therefore,all their samples of water containing earthworms must havecontained not only the nephridial fluid but also faecal matterdischarged through the anus, as well as the watery discharge


given out through the mouth. They, therefore, tested not onlythe nephridial fluid but also the discharges from the two ends ofthe gut.

I have repeated the experiments of Lesser and Delaunaywith the earthworm P h e r e t i m a p o s t h u m a . Only wormswith clean guts were kept in distilled water for twenty-fourhours, and the water was tested for ammonia, urea, and uricacid. While ammonia and urea were present, there was notrace of uric acid. The criticism made above on the experimentsof Lesser and Delaunay holds equally in the case of my experi-ments, but there is an important difference. While L u m b r i -c u s possesses only funnelled nephridia opening directly to theexterior through comparatively large nephridiopores, the in-numerable integumentary nephridia of P h e r e t i m a (2) open-ing to the exterior on the integument are all closed nephridiawith no open communication with the coelomic fluid. Thenumerous funnelled septal nephridia of P h e r e t i m a (2) donot open to the exterior but into the intestine all along its length,so that their excretory fluid must necessarily pass out throughthe anus. Further, the closed pharyngeal nephridia of P h e r e -t i m a must discharge their fluid through the mouth. In thecase o f . P h e r e t i m a , therefore, one must include the dis-charges through the mouth and anus along with the nephridialfluid voided through integumentary nephridiopores, so as toestimate correctly the total quantity of the excretory sub-stances voided by the earthworm.

For quantitative estimations the water containing the earth-worms was collected every twenty-four hours; it was clear andcontained hardly any proteins. Ammonia and urea wereestimated and the results of the estimations are given inTable I.

It will be seen from this table that the quantity of ammoniaexcreted is two to four times that of urea. Delaunay (12 a)gives the percentage of ammonia-nitrogen and urea-nitrogenin L u m b r i c u s as 46 to 48 per cent, and 6 to 12 per cent,respectively; on converting these figures into ammonia andurea, I find that my results (proportions between ammonia andurea) are more or less in accord with those of Delaunay (12 a).

350 K. N. BAHL

TABLE I. Ammonia and Urea voided in Waterin Twenty-four Hours.

Serialnumber.

1.2.3.4.5.

average

Numberof

worms.

202020202020

Weightin

grams.

21-6828-4830-9827-8228-2027-43

N(urea andammonia) inmilligrams

(per 100 gm.of body-weight).

4-976-115-334-675-025-22

Ammonia inmilligrams

(per 100 gm.of body-weight).

5-246-544-874-305-525-29

Urea inmilligrams

(per 100 gm.of body-weight)

(by diff.).

1-391-652-822-290-881-80

These estimations, therefore, confirm Delaunay's conclusions:(1) that nitrogenous excretory substances in the earthworms areeliminated as ammonia and urea only, and that no uric acid isexcreted by the earthworm; (2) that there is more of ammoniathan of urea.

The procedure followed for these estimations was as follows:

Earthworms were kept in water for several days till their gutswere cleaned of all their earthy contents. Sets of twenty earthwormseach were kept in 50 c.c. of distilled water in several glass dishes fortwenty-four hours. At the end of twenty-four hours worms werewiped dry and weighed, and the water in each dish was divided intotwo equal parts. From one part ammonia was estimated directly byForeman's method of alcoholic distillation.1 The other part wastreated with urease2 to convert urea into ammonia and the totalammonia produced was estimated also by Foreman's method: thisammonia included both ammonia excreted as such, as well as am-monia from urea. Since free ammonia had been estimated separatelyfrom the same water, the difference between the two estimationsgave the ammonia obtained from urea, and from this figure thepercentage value of urea was calculated.

1 The estimations and their calculations were made according to themethod described for urine by S. W. Cole in his 'Practical PhysiologicalChemistry' (Heffer and Sons, Cambridge, 1942), pp. 334-5.

2 In. all estimations of urea tabloids of urease supplied by the BritishDrug Houses were used.


(&) A n a l y s i s of t h e Ur ine of E a r t h w o r m s , and t h eE s t i m a t i o n of Ammonia and Urea t h e r e i n .

Although keeping earthworms in distilled water is a con-venient method of collecting their excretory fluid, it is at bestan indirect method, giving urine only in a highly diluted condi-tion. As far as I have been able to find out from literature, noworker has so far succeeded in collecting a sufficient quantityof pure urine of an earthworm. Eustum Maluf (16) gives theosmotic pressure of the blood, but puts a query mark againstthat of the urine. Adolph (1) gives the osmotic pressure of thebody juice, but not that of the urine. Carter (10) says that in "the earthworm 'no analyses of the urine have yet been made'.The difficulty has, of course, been to collect a sufficient quantityof urine for either qualitative or quantitative work.

I have successfully made use of the following simple methodof collecting the urine of earthworms:

Freshly collected earthworms were brought into the laboratoryand kept in water for three or four days to remove most of the earthfrom their guts. A clean dry glass dish with high walls was dividedinto two parts by a closely fitting vertically placed glass plate. Theworms were taken out one by one, wiped dry with a piece of cloth,and kept in one half of the glass dish. On keeping the dish in aslanting position, worms collected against the vertical glass plate ina cluster; and droplets of colourless urine oozed out of the integu-ment, as also from the mouth and anus, and collected in the lowestpart of the other half of the dish. In about twenty minutes, 4 to5 drops of urine were excreted by forty earthworms. A fresh lot offorty worms was treated similarly and another 4 to 5 drops wereobtained. This process was repeated several times until about 8 c.c.of urine was collected in 4 to 5 hours.1

In order to prevent evaporation from the body surfaces of theworms and of the excreted urine itself in dry weather, the dish con-taining earthworms was placed in a larger dish containing water,and both dishes were covered with a large bell-jar so that the atmo-sphere for the worms was as humid as possible.

1 With practice and improvement of collecting technique I have beenable to collect 25 c.c. of urine in two and a half hours from 105 earthwormsin wet weather.

352 K. N. BAHL

A qualitative analysis of earthworm's urine revealed thepresence of basic radicles like sodium, potassium, calcium, andmagnesium, as well as acid radicles like chlorides and phos-phates, and the absence of sulphates. The reaction with litmus

bell-Jo. earfhwormi

wooden block

small ejlass-dish

larcje qlass-dish

water

TEXT-FIG. 2.

Apparatus used for collecting the urine of earthworms.

was markedly alkaline, the pH being 8-3. Xanthoproteic test(Cole, 1942, p. 80) showed the presence of a merest trace ofprotein.

Ammonia and urea were estimated quantitatively by Fore-man's alcoholic distillation method and the results obtained areas follows:

TABLE II. Ammonia and Urea in the Urine of Earthworms.

Serialnumber.

1.2.3.4.5.

Average

N(urea and ammonia)in milligrams per

100 c.c.

3-613-613-614-043-613-69

Ammonia inmilligramsper 100 c.c.

2-192-802-192-803-352-66

Urea in milligramsper 100 c.c.

{by diff.).

3-872-803-873-721-953-24

The test for uric acid was negative.

In this table it is noteworthy that the percentage of ammonia


and urea n i t r o g e n is almost constant in all the five estima-tions; only it is distributed in slightly different proportionsbetween ammonia and urea. Delaunay also found variation inthe distribution of nitrogen between ammonia and urea, andthought that it was possible that a part of excreted urea wastransformed rapidly into ammonia. That this is probable isseen by comparing the proportions of ammonia and urea inTables I and II. In Table I ammonia is, on an average, threetimes that of urea, since the urine was in water for twenty-fourhours, while in Table II the average percentage of ammoniais about 18 per cent, less than that of urea, since the urine waskept only for four to five hours and at a fairly low temperature.

The important conclusion is that in the earthworm, as inmost aquatic invertebrates, urea and ammonia form the mainbulk of the nitrogenous excretion, and that no uric acid isexcreted by the earthworm.

All previous workers collected urine in water and conse-quently it was a very dilute urine on which they made theirestimations. The percentages of ammonia and urea had to becalculated no t on the volume of urine, but either on theweight of earthworms excreting urine or on the total nitrogenexcreted, because the exact volume of urine was never known.This is how I have myself calculated the figures in Table I.But on obtaining urine as such, it has now become possible toestimate the percentage of urea and ammonia in relation to thevolume of urine excreted, as given in Table II, and thus to makea parallel comparison with similar percentages in the coelomicfluid and blood (Tables III and IV). But it must be realizedthat, as stated in chapter 6 a (i) (v ide i n f r a ) , the urinecollected is that of earthworms living like freshwater animals,and not as terrestrial animals, the urine of which would pre-sumably be more concentrated.

The method adopted for estimating urea and ammonia wasas follows:

6-5 c.c. of urine was diluted with 15 c.c. of distilled water and25 c.c. of absolute alcohol was added to precipitate the proteins,which were present in a fine colloidal state. The liquid was centri-fuged and filtered. The nitrate was divided into two equal parts:

354 K. N. BAHL

from one part ammonia was estimated directly by Foreman'salcoholic distillation method, while the other part was treated withurease to convert urea into ammonia, and distilled by Foreman'salcoholic method. The difference between the two estimations gavethe value for urea.

3. THE EXCRETORY SUBSTANCES IN THE COELOMIC FLUID.

Having ascertained that the chief nitrogenous excretory pro-ducts voided by the earthworm are urea and ammonia, the nextstep is to find out where these excretory products come from.All the three kinds of nephridia—septal, integumentary, andpharyngeal—float in the coelomic fluid; further, the septalnephridia are in open communication with the coelomic fluidthrough their nephridiostomes; at the same time all the threekinds are copiously supplied with blood. We must presume,therefore, that urea and ammonia in solution must come to thenephridia either from the coelomic fluid or from the blood orfrom both. Taking the coelomic fluid first, we know that it fillsthe entire coelomic cavity and keeps moving back and forth ina live earthworm. The eoelomic fluid of P h e r e t i m a is milk-white in colour and contains as many as five kinds of corpuscles(Kindred, 15), of which the most numerous are the phagocytes.What the respective functions of these five kinds of corpusclesare, is not yet known with certainty, but there is little doubtthat the phagocytes engulf bacteria, dead chloragogen cells,and other solid waste matters present in the coelomic fluid. Ithas been generally assumed that the coelomic fluid also containsmetabolic wastes in a dissolved state; for example, Stephenson(19) writes: 'A certain amount of coelomic fluid c o n t a i n i n ge x c r e t o r y s u b s t a n c e s in s o l u t i o n (the spaced wordsare mine) passes through the nephrostome and is propelleddown the nephridial tube by ciliary action.' But there is nomention in literature of any worker having tested and estimatedthe nitrogenous excretory substances in s o l u t i o n in thecoelomic fluid. The only statement I have come across is: that'the chief function of the coelomic fluid consists in the dis-tribution of fluid food-materials. Besides there is an excretoryfunction through the nephridia, about which up till now v e r y


l i t t l e is known ' 1 (Stolte, 80)/ There is no mention ofammonia, urea, uric acid, or any other nitrogenous excretorysubstance in solution having been found in the coelomic fluid.Even Heidermanns (14), who estimated the ammonia and ureacontents of the gut and the body-wall, did not think of estimat-ing ammonia and urea contents of the coelomic fluid or of blood.

I, therefore, estimated the ammonia and urea contents of thecoelomic fluid and the results obtained are given in the followingtable:

TABLE III. Ammonia and Urea in Coelomic Fluid.

Serialnumber.

Foreman'smethod.

1.2.3.4.5.

Average

JM essierizationmethod.

6.7.

N(urea and ammonia)in milligrams(per 100 ex.).

4-013-985015-474-50

4-79

—

Ammoniain milligrams(per 100 ex.).

3-384-04-244-943-9

4-13

Urea in milligrams(per 100 ex.)

(bydiff.).

2-291-673-023 02-61

2-52

— Ammonia+Urea —— 3-5 mgm. —— 4-5

The test for uric acid was negative.

It will be seen from this table that, on an average, every100 c.c. of coelomic fluid contain 4-13 mgm. of ammonia and2-52 mgm. of urea. By comparing these figures with those inTable II, it will be seen that while the percentage of ammoniavoided by the earthworm in its urine is, on an average, slightlylower than that contained in the coelomic fluid, the percentageof urea voided is slightly higher than that contained in thecoelomic fluid. It is likely, therefore, that the nephridia derivetheir urea Lorn some other source as well, and that other sourceto be tested is obviously blood.

1 The spaced words are mine.

356 K. N. BAHL

The method followed for estimating ammonia and urea in thecoelomic fluid was as follows:

Forty to fifty live earthworms were cut open and yielded about10 c.c. of coelomic fluid.1 The coelomic fluid obtained was verynearly pure; only a very small quantity of blood came with it. Thefluid was immediately centrifuged, and the corpuscle-free fluid wasdecanted off and measured, and then diluted with an equal amountof distilled water. Treatment with eight times its volume of absolutealcohol precipitated all proteins.2 After filtration the clear colourlessfluid obtained was measured and divided into two equal parts. Fromone part ammonia was estimated directly by Foreman's alcoholicdistillation method, while from the other part total ammonia (freeammonia+urea ammonia) was estimated by the same method aftertreatment with urease. The difference between the two estimationsgave the value for urea.

Urea in the coelomic fluid was also estimated by the Urease-Nesslerization method. The fluid was incubated with urease at37° C. for half an hour to convert urea into ammonium carbonate.Proteins of the fluid were precipitated by NaOH and zinc sulphatesolutions. Part of the supernatant fluid was treated with Nessler'sreagent. Standard solutions of an ammonium salt were also treatedwith Nessler's reagent, and compared with the Nesslerized coelomicfluid colorimetrically by the Klett-Bio Colorimeter and the amountof urea in 100 c.c. of the fluid was calculated. This is the routinemethod followed for estimation of urea in human blood in thePathological Department of the Lucknow University Hospital andI am indebted to Dr. V. S. Mangalik of the Pathology Departmentfor kindly making colorimetric estimations of coelomic fluid andblood at my request.

4. THE EXCRETORY SUBSTANCES IN THE BLOOD.

On looking through the literature I found that althoughsome authors like de Bock and Freudweiler had ascribed an

1 Care was taken not to let the intestinal contents get mixed with thecoelomic fluid; any worms in which the intestine got punctured werepromptly rejected.

2 Various reagents, like trichlor-acetie acid, sodium tiingstate andsulphuric acid, and absolute alcohol were tried for precipitating proteinsin the coelomic fluid before estimating the amount of urea. Of theseabsolute alcohol proved the most convenient and most efficient.


excretory function to the amoebocytes of the blood, no workerhad so far made a blood-urea estimation of earthworm'sblood, although it is so commonly done of human blood. In viewof the assumption commonly made and expressed by Eogers(17 a) it is clearly important to know whether blood takes anypart in the excretion of ammonia and urea. I, therefore,estimated the amounts of these two substances in the earth-worm's blood, and my results are given in the following table:

TABLE IV. Ammonia and Urea in Blood.

Serialnumber.

1.2.3.4.

Average

5.

N(urea-\-ammonia) inmilligrams

(per 100 c.c).

3-08 )2-984-012-98 .

3-26

Foreman'smethod.

Nesslerization 1method. / ""*"

Ammonia, inmilligrams

(per 100 c.c).

2-492-493-061-81

2-71

Urea in milli-grams

(per 100 c.c.)(by diff.)-

2-2071-973-193-18

2-638

Ammonia-)-Urea5-8 mgm.

By comparing the figures in this table with those in Table III(for coelomic fluid), it is readily seen that while the averagepercentage of urea in the blood is about the same as that in thecoelomic fluid, the average percentage of ammonia is distinctlylower. Since the nephridia are copiously supplied with blood,the conclusion is irresistible that the nephridia eliminateammonia and urea from the blood as well as from the coelomicfluid. The assumption of Eogers (17 a) that coelomic fluid isconcerned with the distribution of food-materials and excretion,,and that blood is a mere carrier of oxygen is, therefore, clearlyuntenable. We must conclude that the blood collects themetabolic wastes from the tissues of the body just in the samewpy as the coleomic fluid.

Circulating human blood has an ammonia value of zero orbelow analytical level. But after shedding, ammonia appearsalmost immediately, which in the rabbit is said to amount to

358 K. N. BAHL

1 mg. per 100 c.c. My estimations were carried out on sheddedblood of earthworms, and it is quite possible that part of theammonia is a post-mortem product and that the actual per-centage is much lower in the circulating blood.

At first I thought it would be difficult to collect enough blood tocarry out estimations of ammonia and urea in the blood; in fact, myattempts at taking out blood from the hearts and dorsal vessel bymeans of an injecting needle and syringe were not successful. Butwhile collecting the coelomic fluid, I found that a quantity of bloodoozed out of the cut blood-vessels when dissected worms were left ina glass dish. In order to obtain a sufficient quantity of blood in asclean and pure a condition as possible, earthworms were dissectedand as much of coelomic fluid removed as possible; the wall of theintestine was cut through to remove its contents, and then thehearts were cut open and such cut worms were left in a petri dish.The dish was kept in an inclined position in order to let the oozingblood collect at the lowest part of the dish. In this way about 5 c.c.of blood could be obtained in about two hours by cutting openthirty-five to forty earthworms.

At first a few crystals of potassium oxalate were added to preventcoagulation, but it was soon found that the blood of an earthwormdoes not coagulate,1 so that after the first collection, no oxalate wasadded for any of the subsequent estimations. Although all care wastaken to remove as much of the coelomic fluid as possible, the bloodobtained did contain a very small quantity of coelomic fluid.

For estimations 3 c.c. of blood was diluted with 21 c.c. of distilledwater, and then 40 c.c. of absolute alcohol were added to precipitatethe proteins. The fluid was then centrifuged and the supernatantfluid filtered. As in the case of the coelomic fluid, ammonia wasestimated directly by Foreman's alcoholic distillation method, whileammonia and urea together were estimated by the same method aftertreatment of the fluid with urease. The difference between the twoestimations gave the value for urea.

5. THE INITIAL PLACES OF EXCRETION : THE BODY-WALL

AND THE INTESTINAL WALL.

Having found that both the coelomic fluid and blood containammonia and urea, the next question was to find out as towhere these excretory products came to the coelomic fluid and

1 It indicates the absence of fibrinogen.


blood from. Bearing in mind the fact that the body of an earth-worm is made up essentially of two tubes, the body-wall and thealimentary canal, the body-wall being concerned primarily withlocomotion and the alimentary canal with assimilation of food,the natural presumption is that these would be the two mainseats of metabolism in the earthworm, and that excretoryproducts would be first formed at these two places. Withregard to the gut-wall, we may also bear in mind that theintestine is thickly covered all over with chloragogen cells whichhave been credited by Schneider (18) and others with hepaticstructure and function. Both the body-wall and the alimentarycanal are richly supplied with blood-vessels, and both of themare also in immediate contact with the coelomic fluid; theexcretory products of metabolism formed in the gut-wall andthe body-wall would, therefore, be discharged either into theblood or into the coelomic fluid or into both—it must be intoboth, since ammonia and urea are constantly present in boththe fluids. It does not preclude the possibility that smallamounts of urea and ammonia may be formed as metabolicwastes within the coelomic fluid and blood themselves.

Ammonia and urea were, therefore, estimated in both theintestine and the body-wall, and the results obtained are givenin the following table:

TABLE V. Comparative Amount of Ammonia and Ureain the Intestine and Body-wall.

Intestine. Body-wall.

1.2.\4.5.

Average

•§• ft.

fe-S3-03-03-03-03-0

3-0

2-493-604-766-124-76

4-34

i,4-014-414-396-004-39

4-64

fe-S30303-0303030

3-603-602-494-763-60

3-61

2012-011-992-402-01

2-08

360 K. N. BAHL

It will be seen from this table that while the percentageamount of ammonia excreted by the intestine is only slightlyhigher than that excreted by the body-wall, urea excreted bythe intestine is more than twice that excreted by the body-wall.

Heidermanns (14) carried out estimations of ammonia andurea in the intestine and body-wall of L u m b r i c u s and hisresults are given below (Table VI) for comparison with myresults in Table V.

TABLE VI. Comparative Amount of Ammonia and Urea in theIntestine and Body-wall (After Heidermanns).

Intestine.

Ammoniamilligramsper cent.

5-29-87-19-27-5

Ureamilligramsper cent.

13-2 TT28-8 U l e a S e

11-6 m e a -9-8/

16-5 ] Xanthydrol12-0 / urea.

Body-wall.

Ammoniamilligramsper cent.

4-62-1

Ureamilligramsper cent.

0-8 lUrease1-9 /urea.1-4 \ Xanthydrol2-3 /urea.

It should be noted at once that Heidermanns's figures forammonia and urea in the intestine are very high indeed ascompared with mine.

Heidermanns's estimations show that urea formed in theintestine is, on an average, t e n times as much as that formedin the body-wall, while in my estimations the proportion is2-2 : 1 . This discrepancy may be partly due to the fact thatP h e r e t i m a is a much more active worm than L u m b r i c u sso far as body movements are concerned and hence the meta-bolism in the body-wall would be greater in P h e r e t i m a thanin L u m b r i c u s . L u m b r i c u s is a comparatively sluggishworm, while P h e r e t i m a keeps moving about restlessly allthe time. This may account for the higher percentage ofammoma and urea in the body-wall of P h e r e t i m a as com-pared with that of L u m b r i c u s , but it is difficult to explainwhy the percentage of ammonia and urea is higher in the


intestine of L u m b r i c u s than in that of P h e r e t i m a .Either Heidermanns did not precipitate the proteins com-pletely or he allowed autolysis to take place before he made hisestimations. On looking through his figures for ammonia andurea in the intestine, one cannot fail to notice that while hisfigures for ammonia are more or less constant, those for ureashow a very wide variation indeed.

Heidermanns, on the basis of his estimations, held that ' thechloragogen tissue is the c e n t r a l o rgan of urea m e t a -b o l i s m ' . According to my estimations also, the percentageof urea is highest in the intestine (chloragogen tissue) as com-pared with that in the body-wall, blood, coelomic fluid, andurine. There is no doubt, therefore, that the chloragogen tissueis an important place, if not the central organ, of urea meta-bolism.

For estimation of ammonia and urea in the intestine and body-wall,twenty-five to thirty fresh earthworms were dissected and the body-wall and intestine separated and washed. 3 gm. of each was weighedand pounded with quartz sand with pestle and mortar. The pastewas mixed with a sufficient quantity of distilled Water (21 c.c), andthen 40 c.c. of absolute alcohol were added to precipitate all proteins.The fluid was then centrifuged and filtered. Ammonia and urea wereestimated by Foreman's alcoholic distillation method as before.

6. THE EOLE OF THE NEPHRIDIA IN EXCRETION AND

OSMOTIC EEGULATION.

Having come to the conclusion that ammonia and urea arefirst formed in the intestinal wall and the body-wall, that theypass therefrom into the coelomic fluid and blood, and arethence eliminated to the exterior, we shall now consider the partplayed by the nephridia in eliminating these excretory sub-stances from the coelomic fluid and blood, and in regulatingthe osmotic relations of these internal fluids.

As already stated, P h e r e t i m a has two kinds of nephridia:the closed integumentary and pharyngeal nephridia, andthe open septal nephridia. Both kinds possess a long windingintracellular canal which runs throughout the body of eachnephridium. In a septal nephridium (Text-fig. 3), the two

NO. 340 B b

362 K. N. BAHL

limbs—the straight and the twisted—are 225 //. and 480 \Krespectively in length, while the intracellular canal is as longas 4-45 mm., i.e. more than six times the length of the twolimbs put together. Further, the canal has four ciliated tracts

b.c.t.e.

a.'

TEXT-FIG. 3.

A septal nephridium of Phere t ima pos thuma showing thecourse of the intracellular canal and its ciliated tracts, a-a', thefirst ciliated tract; 6-6', the second; c-c', the third; aaAd-d', thefourth ciliated tract; bet, the brown ciliated tube (phagocyticsection); /, funnel; si, straight lobe; tl, twisted loop with itstwo limbs. (x dr. 120.)

in its course, and one can easily see in a live nephridium underthe low power of the microscope that liquid is driven down thetube by the beating of the cilia of the nephridiostome and thefour ciliated tracts. In the closed nephridia there are only twociliated tracts (&-&' and G-C') and no nephridiostome, but the

BXCEBTION IN INDIAN EARTHWORMS 363

cilia in these ciliated tracts keep beating, as in an open nephri-dium, and drive the fluid down the tube. There seems littledoubt that the nephridia derive the fluid in their intracellularcanals from the coelomic fluid and blood. In the closed inte-gumentary and pharyngeal nephridia the movement of thecilia in the ciliated tracts probably sets up a slight pressurewhich is enough to draw liquid by a process of nitration fromthe blood and coelomic fluid, through the exceedingly thin wallsof the nephridium into the lumen of the intracellular canal. Inthe open septal nephridia, however, the coelomic fluid plasmapasses directly through the nephridiostome into the intra-cellular canal of the nephridium, but the blood-plasma can beextracted by filtration alone even by the septal nephridia.

In vertebrates and even in some of the higher invertebratesthe mechanism of renal secretion has been analysed into pro-cesses of filtration, reabsorption, tubular excretion, and chemicaltransformation (23). We have to find out how far these pro-cesses can be detected and demonstrated in the nephridialsecretion of an earthworm.

We have referred above to the extremely long and muchcoiled intracellular canal in the nephridium of an earthworm:the glandular cells of the nephridium may remove somethingfrom the blood and coelomic fluid, and add it to the liquidcontained within the lumen of the intracellular canal, or asKogers (17 a) has pointed out, the long nephridial canal ' mayserve as a means of conserving water which might otherwisebe lost to the organism'.

(A) The Osmo- regu l a to ry F u n c t i o n of theN e p h r i d i a .

In order to regulate the osmotic relations of the coelomicfluid and blood, the excretory organs of the earthworm must(1) keep the volume of these internal fluids more or lessconstant, and (2) eliminate most of the basic and acidr id ic les formed in the body (23).

(i) V o l u m e - E e g u l a t i o n . Although an earthworm is aterrestrial animal, it is still aquatic in its respiratory habit, aswe know that a certain amount of moisture is always necessary

864 K. N. BAHL

to keep its skin moist, or else the skin becomes desiccated andthe animal dies of asphyxia. Besides it must require an adequateamount of water for its metabolic needs, considering that ithas two circulating fluids—blood and coelomic fluid—in itsbody. An earthworm does not drink water through its mouth,but it absorbs all the water it needs through its skin. It is wellknown that earthworms can remain in water for months withoutany harmful effects. Darwin quotes Perrier who kept largeworms alive for nearly four months completely submerged.I myself kept seven sets, each of fifteen earthworms (P h e r e -t i m a p o s t h u m a ) , submerged and starved in tap-water fortwenty-two days with only twelve casualties in all. EustumMaluf (16) also kept earthworms in water for several days, andconcluded from his experiments that earthworms are generallycapable of living indefinitely in fresh water. That water entersthe body of the earthworm by osmosis through its integumenthas been conclusively proved by experiment. Adolph (1) foundthat a group of large worms which were dug from wet groundon a very warm day gained 15 per cent, in weight in 100minutes after immersion in tap-water at 28° C. Eustum Maluf(16) repeated Adolph's experiment and confirmed his observa-tion. I have also repeated Adolph's experiment and found thatearthworms gained 7 to 16 per cent, in weight in tap-water inseven hours, as shown in the graph on p. 365. Actually the gainin weight must be more, since the earthworms defaecated a cer-tain amount of earth during these seven hours which was nottaken into account in my weighings. In a second lot of five setsof earthworms, with twelve worms in each set, I found that thegain in weight was as much as 11-12 to 26 per cent, on keepingthem in tap-water for five hours. The defaecated earth wasagain not taken into account.

In order to keep the volume of the internal fluids more or lessconstant, these large quantities of absorbed water must beeliminated, or else the internal fluids would become highlydiluted and the earthworm would burst through continualabsorption of water. Such a fatal result, however, seldomoccurs; in fact, the volume of the internal fluids as indicatedby the weight of the worms is kept constant, as is shown by the

Xpoq jo

TEXT-FIG. 4.

A graph showing changes in body-weight following immersion ofearthworms in tap-water. The initial weight of worms is taken as100. During the first six or seven hours there is an increase ofweight by 7 to 16 per cent. I t is worth noting that after thefirst five or six days the worms keep a more or less constantweight, varying only by 1*7 to 2-8 per cent, around the mean oraverage weight.

366 K. N. BAHL

fact that when worms have been in tap-water for four to fivedays and all the earth has been defaecated, they keep up moreor less a constant weight for the succeeding eight or nine days,the weight varying only by 1-7 to 2-8 per cent, around the meanor average weight, thus setting up, so to speak, a new equili-brium in tap-water (Text-fig. 4), wherein the water absorbedand the water excreted balance each other. The question arisesas to how the large quantity of absorbed water is eliminated bythe earthworm. When an earthworm ( P h e r e t i m a ) , whichhas been in tap-water for several days and whose gut has beenthoroughly cleaned, is mopped with a dry towel and examinedunder a binocular dissecting microscope, it is seen that the skinsoon becomes wet on account of the secretion of urine throughthe innumerable nephridiopores of the integumentary nephridia,a watery drop is ejected through the anus and next a drop fromthe mouth, and that water is ejected more frequently and there-fore more copiously through the anus than through the mouth.Adolph (1) could not see the fluid ejected through the nephri-diopores of L u m b r i c u s but could see the watery dischargethrough the anus; Eustum Maluf (1@) could see clear colourlessliquid spurting and oozing out of the nephridiopores andflowing into intersegmental furrows, and inferred the dischargeof water through the anus and mouth from his weighing experi-ments only; but I have been able to see water being dischargedthrough all these three openings. By Iigating the earthwormsat one or both ends and finding an increase as well as a decreasein weight, Eustum Maluf concluded that 'when a worm is firstintroduced into tap-water, its gut is of paramount importancein osmo- and volume-regulation, but that there is a definitevolume-regulative tendency on the part of the kidneys (nephri-dia) also, which, in t h e n o r m a l worm, 1 is however, com-pletely masked by such a function on the part of the alimentarytract'.

I have repeated Eustum Maluf's experiment of Iigating bothends of the worms and have confirmed bis observation thatthere is a distinct decrease in weight, but my conclusion fromthis observation is different. It must be remembered that

1 The spaced words are mine.


immersion in water is not the normal environment for an earth-worm. Adolph rightly concluded that 'in their usual environ-ment, moist ground, earthworms are partially desiccated'.Bustum Maluf did not observe the exudation of fluids in earth-worms freshly taken from the soil. If an earthworm (P h e r e -t i m a p o s t h u m a ) is taken direct from the soil, washed andmopped with a dry towel, and then observed under a binocularmicroscope, it is seen that although the skin becomes moist onaccount of exudation from the numerous nephridiopores, andthere is a small watery discharge from the mouth as the wormprotrudes its buccal chamber, there is no w a t e r y d i s c h a r g eat all from the anus—only more or less solid faecal pellets beingdefaecated at intervals. The discharge on the skin is apparentlythrough the integumentary nephridia, and that from the mouththrough the pharyngeal nephridia and the 'salivary glands',but the copious discharge through the anus is completelyabsent. Thus it is clear that in an earthworm living in the soilthe gut does not excrete water and so takes practically no partin osmo- and volume-regulation. My conclusion, therefore, isthat in its usual environment, the nephridia of the earthwormfunction adequately as volume- and osmo-regulatory organs asthey have to deal only with a small quantity of metabolic waterand water normally absorbed by the skin for respiratory andgeneral metabolic needs of the body. But when an earthwormis placed in water for several hours, the skin absorbs largequantities of water which cannot be eliminated by the nephridiaalone, and then the gut comes to their rescue, so to speak, andtakes a prominent part in osmo- and volume-regulation byeliminating through the anus the large quantity of waterabsorbed through the skin.

In its normal environment the amount of water absorbedby an earthworm is small: the worm is never fully hydrated oras Adolph puts it, it is partially desiccated; in a form likeL u m b r i c u s , with open exonephric nephridia, the water of thecoelomic fluid plasma passing freely into the nephridial canaland that of the blood filtering into it are probably reabsorbedby the glandular cells of the nephridium and also-by the wallof its terminal bladder. Delaunay's observation that urine is

868 K. N. BAHL

excreted from the terminal bladder only once in three dayslends support to my conclusion. But in a form like P h e r e -t i m a with enteronephric nephridia the loss of water is com-pletely reduced, as the greater part of the nephridial fluid is notdischarged to the outside but passes into the gut which effec-tively absorbs a large part of the water. The part of thenephridial fluid which is discharged to the outside directly isexcreted by the integumentary nephridia which have noterminal bladders. By estimating the percentage of moisturein the fresh 'castings' (faeces) of P h e r e t i m a and E u t y -p h o e u s and always finding the percentage higher in E u t y -p h o e u s than in P h e r e t i m a , I have already (4) shown thatthe gut of P h e r e t i m a is much more efficient in absorbingwater than the gut o f E u t y p h o e u s which possesses exonephricnephridia like those o f L u m b r i c u s .

We have already seen that ammonia and urea form the mainbulk of nitrogenous excretion of the earthworm and that theseare excreted in low concentration (Table II). A certain mini-mum amount of water must be excreted to eKminate even theselow amounts of excretory products. In a form like L u m -br i cus or E u t y p h o e u s , therefore, in its normal environ-ment, the necessary amount of water is excreted and the restconserved by the nephridia, and the gut takes practically nopart in water-conservation, as is shown by the fact that itsvermicelli-like loosely semi-solid castings contain a large per-centage of water. But in a form like P h e r e t i m a , thenephridia of which lack a terminal bladder, the task of water-conservation is taken over largely by the gut into which thenephridial fluid is discharged and which gives off solid pellet-like castings, with a comparatively small percentage of water.

It seems, therefore, that an earthworm, when submerged inwater, can live like a freshwater animal, like its freshwaterallies, absorbing water through its skin and eliminating itlargely through its gut and partly through its-nephridia. Asthere is abundance of water, there is no need of conservation ofwater. But in its normal environment, moist earth, an earth-,worm is partially desiccated, and conservation of water is ofgreat importance to it as it is to a terrestrial animal; the water


is conserved by the nephridia and their bladders in Lum-b r i c u s , and by the nephridia and the gut together inP h e r e t i m a .

(ii) The E a t e of E x c r e t i o n of Urine.—Consideringthat the rate of excretion of urine would throw light on volume-regulation, I measured the rate at which urine is excreted byearthworms, as is shown in Table VII.

It will be seen from this table that I could collect 3-7 c.c. to7-2 c.c. of urine in three and a half hours. When it was rainingand the humidity in the atmosphere was high, the quantity ofurine was very much more than that obtained on a dry day,because there was little evaporation from the body-surface ofearthworms and the urine itself. In the first ten minutes, thequantity of urine excreted is at its maximum, but it goes ondecreasing in successive ten minutes. In experiment 3, thevolume of urine collected was 6-5 c.c, while the weight lost byearthworms was 10-16 gm., which is accounted for by theevacuation of faeces and the evaporation of moisture from thebody-surface of earthworms during the period of collection ofthe urine. The volume of urine c a l c u l a t e d for twenty-fourhours comes to 49-2 c.c, i.e., about 45 per cent, of the weight ofthe body. In the experiment the excretion of urine slows downconsiderably after an hour and a half, and earthworms have tobe kept in water again for some time before we can get urineout of them a second time. But there is little doubt that whenearthworms are continually kept in water, the intake and out-flow of water in twenty-four hours must be much more than45 per cent, of the body weight. It is clear that earthworms inwater excrete urine very largely from the water absorbed bythem through their skin, and that the quantity of metabolicwater is very small indeed. From experiment 8, one cancalculate that a fully hydrated earthworm will excrete at least0-82 c.c. of urine in twenty-four hours, the average weight ofan earthworm being 1-6-1-8 gm.

Earthworms for collection of urine had been kept in water forthree to four days to get rid of most of the earth from the gut. Freshearthworms from the soil excrete very little urine and even that isdifficult to collect as it gets mixed with the faeces. It would be

TA

BL

E V

II.

Bat

e of

Exc

reti

on o

f U

rine

.

OS

O

Num

ber

ofE

xpt.

1. 2. 3. 4. 5. 6. 7. 8. 9.

Num

ber

ofw

orm

s.

72 70 72 70 65 60 65 60 75

Vol

ume

ofur

ine

ex-

cret

ed in

10 m

inut

esin

c.c

.

— — 1-7

0-8

0-55

0-5

one

drop

"Will

IN 1

1.

0-3

0-2

Nil

.N

il.

— — — — —

Tot

alvo

lum

eco

llec

ted

in c

.c.

4-3

4-0

6-5

4-8

4-1

5-1

3-7

7-2

6-9

Per

iod

inw

hich

col

lect

edin

hou

rs.

Hou

rs

min

utes

3 35

2 50

3 10

3 30

3 30

4 30

3 30

3 30

3 45

Exc

rete

dvo

lum

e ca

lcu-

late

d fo

r 24

hour

s in

c.c

.

28-8

33-8

49-2

32-9

26-6

27-2

25-3

49-3

744

-16

Wei

ght o

f ear

th-

wor

ms

befo

re a

ndaf

ter

coll

ecti

onin

gm

.

108-

26-9

7-06

106-

86-9

8-99

109-

36-9

9-20

104-

71-9

6-49

100-

18-8

9-87

93-2

7-82

-97

98-9

9-89

-01

Not

rec

orde

d.N

ot r

ecor

ded.

Tem

pera

ture

in°C

.

27-2

926

-5-2

8-5

25-5

-28

27-5

-29

28-3

028

-8-3

228

-5-3

1-0

Rem

arks

.

Mor

ning

cool

and

hum

id d

ueto

a l

ittl

esh

ower

.

Coo

l ra

iny

day.


interesting if one could collect a sufficient quantity of urine fromworms fresh from the soil and compare this quantity with thatcollected from worms progressively subjected to exposure anddesiccation.

(iii) The Osmot i c P r e s s u r e of Blood , CoelomicF lu id , and Urine.—In order to form an idea of the role ofthe nephridia in regulating the osmotic relations of the internal

Z2S&. C O ^ : ^ ^ BLoT^SSEL .ux»*.O^-O*fc,

MVPOTONIC URINE(A»O-O5ff-OOS8*C)

TEXT-ETG. 5.

A diagrammatic representation of the body of Phere t imaposthuma showing the depression of the freezing-point ofthe different fluids of its body (plan adapted from Bustum Maluf).

fluids, I determined the osmotic pressure of blood, coelomicfluid, and urine by measuring the depression of the freezing-point of each by Beckmann's method, and my results are asfollows:1

TABLE VIII. Depression of the Freezing-point of theFluids of Pheretima posthuma.

1. Blood-plasma A = 0-40° 0.-0-50° C.2. Coelomic fluid-plasma A = 0-285° C.-O-310 0.3. Urine . . . A = 0-050° 0.-0-065° C.

These figures are diagrammatically represented in Text-fig. 5.The depression of the freezing-point (A) is a measure of themolecular concentration, and therefore of the osmotic pressureof a solution. Bustum Maluf (16) gives 0-45° C. as the figure fortne depression of freezing-point of the blood of L u m b r i c u s ,

1 I am indebted to Mr. M. Raman Nayar of the Chemistry Departmentfor determining the depression of the freezing-point of these fluids for me.

372 K. N. BAHL

while Adolph (1) gives 0-31° 0. as the figure for the depressionof freezing-point of the body juice o f L u m b r i c u s . My figuresare in agreement with theirs. The water content of the body ofP h e r e t i m a i s a variable factor and this is apparently reflectedin the slight variation in the osmotic pressures of the threefluids. As far as I know, no worker has so far estimated thedepression of the freezing-point of the urine of an earthworm, asno one was able to obtain it in sufficient quantity. From thefigures of the depression of the freezing-point given above, itwill be seen that the coelomic fluid is h y p o t o n i c to the blood,and that the urine is markedly h y p o t o n i c to both thecoelomic fluid as well as the blood.

The difference in the depression of the freezing-point betweenblood and coelomic fluid is very striking and forms an interest-ing osmotic problem by itself. How is the blood maintainedhypertonic to the coelomic fluid, and what are the factorsresponsible for it? As we shall see presently (vide infra),the blood has a higher protein content but a lower chloridecontent than the coelomic fluid, so that these two factorscannot account for the whole story. It seems that a detailedchemical analysis of blood as well as coelomic fluid is called forto solve the question of the difference in osmotic pressurebetween these two fluids.

Samples of blood and coelomic fluid for the determination of thedepression of freezing-point were obtained in as pure a condition aspossible. Samples of urine were taken from worms which had beenin water for some days and had been, so to speak, living like fresh-water animals, eliminating water in large quantities through the gutas well as through the nephridia.

(iv) The P r o t e i n C o n t e n t s of B lood , CoelomicFlu id , and Urine .— It has been proved conclusively that inthe amphibian kidney the fluid passing from the glomerularcapillaries into the Bowman's capsule is a protein*free filtratepractically identical with the blood-plasma except for itscolloids (i.e. proteins and fats), and that it passes out of thecapillaries as a result of the purely physical process of filtration.It was, therefore, considered advisable to estimate the protein

BXCBBTION IN INDIAN EARTHWORMS 373

contents of the blood, coelomic fluid, and urine of the earthwormto find out if its urine was really a protein-free nitrate. Myestimations of proteins are as follows:

TABLE IX. Protein Contents of the Fluids of Pheretima.

Blood-plasma.

gm. per 100 c.c.

1. 3-4572. 3-9493. 3-7574. 3-376

Average 3-643

Coelomic fluid-plasma.

gm. per 100 c.c.

0-5500-4790-4580-429

0-479

Urine.

gm. per 100 c.c.

0-0250-0290-036

0-030

In man the blood-plasma protein concentration is 6-5-8-5 gm.per 100 c.c, i.e. about double that of the earthworm.

It will be seen from this table that the protein content of theblood-plasma is seven to eight times that of the coelomic fluid-plasma and that the urine is not protein-free, since it containsmeasurable traces of colloidal proteins. Picken (17) has found asimilar condition in the urine of Arthropods studied by him andsays: ' An examination of the urine has shown almost certainlyin Carc inus and possibly in P o t a m o b i u s and Pe r i -p a t o p s i s , that it contains a little protein.' He traces theseproteins in the urine of Ca rc inus either to proteins derivedfrom blood, or to cell breakdown in the kidney, or to mucus.In the earthworm the mucus secreted by the skin, and themucin and the proteolytic enzyme of the saliva dischargedthrough the mouth may account for the traces of proteins foundin the urine. If this supposition be correct, then we can holdthat the urine is really a protein-free nitrate, but that traces ofprotein find their way into it from these sources during theprocess of collection of the urine.

In an earthworm the nephridia must derive their urinaryf lid both from the blood and the coelomic fluid. On an analogywith what has been proved in the case of the amphibian kidneywe may presume that the part of the urine derived from theblood is really a protein-free filtrate, filtered from the blood-

374 K. N. BAHL

capillaries of the nephridia, as through a semi-permeable mem-brane, but this cannot be true of the part of the urine derived fromthe coelomic fluid. As the coelomic fluid-plasma passes directlyand freely into the septal nephridia through their open funnels, itmust contain colloidal proteins in the concentration of about 480mg. per 100 c.c, and since proteins are too valuable to the earth-worm to be allowed to be lost with the urine, we must presumethat the cells of the nephridia keep on reabsorbing the proteinsof the coelomic fluid-plasma as it passes through the nephridiaas urine.

In a form like P h e r e t i m a in which the urine of the septalnephridia is discharged into the intestine, it is probable that anyproteins still left over in the urine after their reabsorption bythe nephridia would be reabsorbed by the intestine, but in aform like L u m b r i c u s in which the urine passes out directlythrough the nephridia, the nephridia alone must be reabsorbingefficiently all the proteins passing into them in the coelomicfluid-plasma. The protein estimations were made on fluidscollected from earthworms which had been in water for severaldays and were living like freshwater animals, eliminating asmuch water as they were absorbing. The fact that proteinconcentration of the coelomic fluid-plasma is about 16 t i m e sthat of the urine strongly supports the conclusion that proteinsare reabsorbed by the nephridia.

The average percentage proportion of the protein contents ofthe three fluids works out as—blood plasma 100: coelomic fluid-plasma 13-1: urine 0-82. The difference between the proteincontents of the blood-plasma and coelomic fluid-plasma is con-siderable, but there is no doubt that there is a large amount ofprotein matter contained in the corpuscles of the coelomic fluid,while the blood has very few corpuscles in it, and all its proteinsare suspended in a colloidal state. The differences in the osmoticpressures of the three fluids cannot be due to their proteincontents, as the protein osmotic pressure in any case must bevery small indeed. In man it is 25 mm. Hg; in the earthworm itwill be only about 12 mm. Hg.

Each fluid (blood, coelomic fluid, or urine) was centrifuged and10 c.c. of the supernatant fluid was treated with 70 c.c. of strong


alcohol to precipitate all the proteins. The liquid was filtered througha previously weighed filter-paper and the precipitate was washedseveral times with warm distilled water. The precipitate on thefilter-paper was then dried and weighed several times till the weightwas constant. This weight minus the original weight of the filter-paper gave the weight of the proteins in 10 c.c. of each fluid, fromwhich the weight per cent, was calculated.

(v) The Chlo r ide C o n t e n t s of Blood , CoelomicF lu id , and Urine.—Since a qualitative analysis had shownthe presence of chlorides in all the three fluids, it was thoughtthat a quantitative estimation of the chloride contents of theblood, coelomic fluid, and urine would throw light on the differ-ences in the osmotic pressures of the three fluids. Further, acomparison of the chloride contents of the three fluids wouldgive us an idea of the reabsorption of chlorides, if it occurs,within the nephridia. In order to form as accurate an idea aspossible of the reabsorption of chlorides, worms were kept inoxygenated distilled water for six days, so that all the earth inthe gut had been defaecated and the worms were living likefreshwater animals, eliminating as much water as they wereabsorbing. In such worms there was no question of conservationof water by its reabsorption by the nephridia and the gut, andtherefore a comparison of the concentration of chlorides in thethree fluids would indicate directly the amount of reabsorptionof chlorides. The results of chloride estimations are as follows:

TABLE X. Chloride Contents of the Fluids of Pheretima.

Blood-plasma.

(mgm. per 100 c.c.)

1. 45-802. 50-823. 51-42

Average 49-35

Coelomic fluid-plasma.

(mgm. per 100 c.c.)

79-2677-2381-30

79-26

Urine.

(mgm. per 100 c.c.)

3-5563-862

3-7

In terms of NaCl the proportions work out roughly to blood82 : coelomic fluid 132 : urine 6. From these figures two impor-tant conclusions can be readily drawn: (1) that there is areabsorption of chlorides on a large scale by the nephridia, and

376 K. N. BAHL

(2) that the chloride content cannot account for the higherosmotic pressure of the blood, since the chloride content ofblood is decidedly lower than that of coelomic fluid. It is evidentthat some other factor or factors are involved which needfurther investigation.

The method followed for chloride estimations is that recommendedby Cole, 1942 (p. 378). 10 c.c. of each fluid was diluted with 70 c.c.of distilled water, and treated with 10 c.c. of 10 per cent, sodiumtungstate and then with 10 c.c. of 2/3 normal sulphuric acid toprecipitate the proteins. The filtrate (coloured yellow in the case ofblood, pale yellow in the case of coelomic fluid, and colourless in thecase of urine) was titrated with acid silver nitrate (N/50) and am-monium thiocyanate (N/50) as directed by Cole. The results obtainedby this method were confirmed by the ignition method as describedby Skinner.1 10 c.c. of each fluid was treated with 20 c.c. of 5 per cent,sodium carbonate and evaporated to dryness in a platinum dish andthen ignited at dull red heat. The product was extracted with hotwater and filtered through ashless filter-paper and the residue igniteda second time. The ignition product was extracted with dilutenitric acid and added to the main nitrate. The combined filtratewas titrated with acid silver nitrate and thiocyanate as before.

I am deeply indebted to Mr. M. Eaman Nayar of the Chemis-try Department who has taken great pains in making theseestimations for me.

(vi) Conclusions.—Taking into account the observationsand their interpretations as recorded under the preceding fivesub-headings, the conclusions arrived at may now be summarizedas follows: (1) That the part of urine which is excreted from theblood is probably a protein-free filtrate, but that the coelomic fluid-plasma entering the nephridia through funnels must containproteins suspended in a colloidal form and these proteins are re-absorbed by the nephridia. (2) That minute traces of proteinpresent in the urine as finally collected probably come from themucus secreted by the skin, and the proteolytic enzyme secretedby the salivary gland. (3) That the urine as finally excreted andcollected is h y p o t o n i c to both the blood and the coelomicfluid. (4) That there is a reabsorption of the chlorides on a large

1 Skinner and others—' Official and Tentative Methods of Analysis of theAssociation of Official Agricultural Chemists' (Washington, 1935).


scale from the initial nephridial filtrate during its passagethrough the nephridia. (5) That in the normal environment, ina form like E u t y p h o e u s or L u m b r i c u s , the nephridiaare concerned with nitrogenous excretion, water-conservation,and protein and salt reabsorption, but when the worm is keptin water the gut takes a prominent part in the regulation ofwater exchange by excreting water through the anus and themouth. In enteronephric forms, like P h e r e t i m a , however,the nephridia and the gut together are normally concerned withwater-conservation. (6) That the osmotic relations of thecoelomic fluid and blood are regulated (a) by keeping the volumeof these fluids more or less constant through the nephridia whenworms are in soil, and through the nephridia and the gut whenthe worms are in water, and (b) by eliminating acid radicleslike chlorides and phosphates, and basic radicles like sodium,calcium, potassium, and magnesium through the nephridialfluid. (7) That the higher osmotic pressure of the blood ascompared with that of the coelomic fluid cannot be accountedfor by the chloride content alone, since it is actually lower inthe blood than in the coelomic fluid. Further investigation onthis point is called for.

(B) The E x c r e t o r y Inc lus ions of the ' C i l i a t e dMiddle T u b e ' (A th rophagocy t i c Sect ion) of

the N e p h r i d i a .

It is well known that in the nephridium of Lumbr i cus theso-called 'ciliated middle tube' and the adjoining ampulla havea brownish semi-opaque appearance due to the presence ofbrownish granules within the nephridial cells surrounding theciliated tract. Even in preserved specimens of Lumbr icus ,which alone have been available to me, one can easily see theopaque brown colour of the ciliated middle tube. Schneider(18), on the basis of bis injection experiments, called this areathe p h a g o c y t i c sec t ion of the nephridium. Cuenot (IE)confirmed Schneider's observation and found that the middletube alone was stained by physiological injections. Schneiderrecognized this phagocytic section in the nephridia of all theOligochaeta investigated by him except in P h e r e t i m a , and

NO. 340 c c

378 K. N. BAHL

'Phagocytare Abschnitte in den Nephridien fehlen beiP e r i c h a e t a ( P h e r e t i m a ) ' . Unfortunately Schneidermade a mistake here, as the phagocytic section is as clearlypresent in the septal nephridia of P h e r e t i m a (Text-fig. 6)as in those of other Oligochaetes. I have found that the phago-

A

TEXT-ITG. 6.

A. A microphotograpb. of a few septal nephridia of Phere t imapos thuma (X cir. 50). The elongated black areas mark theheavy deposits of brownish yellow granules in the 'ciliatedmiddle tube' (athrophagocytic section). B. A section passingthrough two nephridia, showing deposits of brownish yellowgranules in the ciliated middle tubes ( x cir. 275). nph, nephri-dium; peg, deposits of pigmented excretory granules.

cytic ciliated tract is present also in the septal nephridia ofE u t y p h o e u s (6), L a m p i t o (3), H o p l o c h a e t e l l a (6),and Tonosco lex (5), and is yellowish-brown or even blackishin colour. The granules are stored within the cytoplasm of thecells of the nephridia surrounding the ciliated canal and areapparently harmless storage excretory products; they can beclearly seen in whole mounts (Text-fig. 6 A) as well as in sec-tions of septal nephridia (Text-fig. 6 B). It is noteworthy that


the deposits in P h e r e t i m a and Tonoscolex are muchheavier than in L u m b r i c u s . In P h e r e t i m a the depositis heaviest at the beginning of the ciliated canal and becomesprogressively lighter towards the distal end of the canal, thusforming a gradient of thickness of the deposit. Further, thedeposit appears in the form of a linear series of cylindrical rings,appearing like the nodes' and internodes of a bamboo stem.The determination of the chemical nature of these excretorygranules has been a subject of great difficulty and I have spenta considerable amount of time and labour on it. I am statingmy conclusions as follows:

(1) That the P igmented E x c r e t o r y Granules arenot Guanine .

Willem and Minne (22) who tested these brownish granulesin the nephridia of Lumbr icus came to the conclusion thatthey were granules of guan ine . This statement has neverbeen questioned but has been implicitly accepted by subsequentworkers, and has been incorporated as such both by Stephenson(19) and Stolte (20). Willem and Minne relied exclusively onsolubility tests, and say that 'the granules resist the action ofalcohol, ether, chloroform, and ammonia, but are soluble inpotash and hydrochloric acid—these chemical charac-t e r s cor respond to g u a n i n e ' . (The spaced words aremine.) I have repeated these solubility tests of Willem andMinne, and find that my results are greatly at variance withtheirs. For these solubility tests I have used the nephridia ofP h e r e t i m a as well as those of Lumbr i cus , and am givingmy results in Table XL

Taking Willem and Minne's second statement first, that thegranules are soluble in potash and hydrochloric acid, I find thatalthough the granules are soluble in 2 per cent, caustic soda orpotash, they are abso lu te ly inso luble in hydroch lor icac id . I have tried all strengths of this acid, even concen-trated, in cold and also boiling 5 per cent, and 2 per cent,hydrochloric acid—still the granules will not dissolve. If thegranules were of guanine, they would dissolve in 5 per cent,boiling hydrochloric acid forming guanine-hydrochloride. I

TA

BL

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XI.

Incl

usio

nsof

the

'Cil

iate

dM

iddl

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ube.

'

Ph

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bric

us.

Solu

bili

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n

1

Alc

ohol

(Rec

tifi

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irit

s).

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in12

day

s.

Sol.

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day

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2

Eth

er.

Inso

l,*

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l. *

3

Chl

oro-

form

.So

l. in

32 d

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in32

day

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4

Am

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day

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5K

OH 2

per

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.

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(with

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.

Sol.

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.

6H

OI

5pe

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nt.

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l.

Inso

l.

7G

laci

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ceti

cA

cid.

Sol.

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days

.

Sol.

in5

days

.

8

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tone

.

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l.al

thou

ghth

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o.

9G

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rine

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r ce

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l.

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l.

10

Pyr

idin

e.

Sol.

(with

in24

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tried pure guanine as a control and found that it readily dis-solved in 5 per cent, boiling hydrochloric acid. Coming now toWillem and Minne's first statement that the granules resist theaction of alcohol, ether, chloroform, and ammonia, I am afraidI find that even this statement of theirs is not quite correct.Although the pigmented granules are insoluble in ether, theydissolve, though very slowly, in ammonia, alcohol, and chloro-form as shown in the table on p. 380. Further, they alsodissolve slowly in glacial acetic acid. If the granules were ofguanine, they would be precipitated by both ammonia andacetic acid instead of being dissolved in them.

As a further test I took a very large number of the nephridiaof P h e r e t i m a and kept them in 5 per cent, hydrochloric acidat 65° C. for twenty-four hours. The pigmented granules, ofcourse, showed no sign of solution, but in order to make sure ifthere was even a trace of guanine dissolved in the hydrochloricacid, I filtered the material and allowed the filtrate to evaporate.If there had been any guanine in the filtrate (hydrochloricacid) I should have got needle-like crystals of guanine hydro-chloride. But no such crystals were formed, showing that therewas no guanine in the pigmented granules which could dissolvein hydrochloric acid.

These reactions, therefore, with hydrochloric acid, ammonia,and acetic acid prove that the pigmented granules in the middletube of the nephridia of L u m b r i c u s and P h e r e t i m a arenot of g u a n i n e .

(2) Tha t the P i g m e n t e d E x c r e t o r y Granules arenot Uric Acid or U r a t e s .

The fact that these excretory inclusions form an apparentlyamorphous pigmented deposit, soluble in alkalies but insolublein hydrochloric acid, led me to suspect that they may be de-posits of uric acid or urates. In fact, the deposit closelyresembles the deposit of amorphous urates as figured in booksof clinical medicine (13). I therefore tried again and again themurexide test for uric acid or urates not only on the nephridiaon the slide, but also on the caustic soda and sodium carbonatesolutions of the excretory material. In none of these trials

882 K. N. BAHL

could I get a positive murexide test, showing that the depositsare n o t of uric acid or urates.

For a time I thought that possibly the quantities are toosmall for giving the murexide test. I, therefore, tried with alarge quantity of nephridial material with the same negativeresult. Bearing in mind the fact that a small crystal of uricacid is enough to give the murexide test on a cavity slide, I haveno doubt that, had there been any uric acid in the nephridialgranules, I should have got the murexide test with the largequantities of material I used time after time for this test.

(3) Tha t t he P i g m e n t e d E x c r e t o r y Granules areof B lood-p igmen t (haemochromogen) .

Besides alkalies like caustic soda and sodium carbonate,pyridine1 quickly dissolves the excretory granules. Pyridine isa colourless liquid solvent, and I found that when nephridia ofP h e r e t i m a with heavy deposits of pigmented granules werekept in it, the deposits were dissolved within twenty-four hours,giving pyridine a brownish red colour. Large quantities2 ofnephridial material were used to get a satisfactorily intensecolour of pyridine. The coloured pyridine was then subjectedto spectroscopic examination. Two clear bands could be seen.The band near the D line (the a-band) is narrow with its centreabout 5579 A.U., i.e., about mid-way between the D and Elines. The /2-band is broad and has its centre about 5267 A.U.The actual figures are:

First Absorption Band: AA 5659-5500; centre 5579 A.U.Second Absorption Band: AA 5405-5130; centre 5267 A.U.3

These figures are identical with those of the two bands ofhaemochromogen . A photograph of the absorption spec-trum of the pyridine solution of the granules is shown in Text-fig. 7. These spectroscopic bands, therefore, conclusively prove

1 I am indebted to Dr. S. B. Dutt of the University of Allahabad forsuggesting this solvent for a spectroscopic examination of the material.

2 I am thankful to Messrs. V. G. Jhingran and S..D. Misra for dissectingout and collecting a large quantity of septal nephridia for making a pyridinesolution.

3 I am indebted to my friends Dr. D. B. Deodhar and Dr. P. N. Sharmafor making these spectroscopic examinations for me.

EXCRETION IN INDIAN EAETHWOHMS

that the excretory granules consist of the blood-pigment, astheir pyridine solution gives the two bands characteristic ofh a e m o c h r o m o g e n .

The pyridine solution was made from septal nephridia takenfrom earthworms ( P h e r e t i m a ) preserved in formalin. Itwas at once suspected that pyridine may have dissolved theblood-pigment from the minute capillaries and blood-vessels

TEXT-FIG. 7.

A photograph of the absorption spectrum of a pyridine solution ofthe pigment granules of the septal nephridia of Phere t imapos thuma, showing the two typical absorption bands of hae-mochromogen. The spectrum of iron arc accompanies that ofpigment granules in order to locate the exact position of the bands.

that necessarily accompanied the nephridia, when they werekept in pyridine. But examination under the microscopeclearly revealed that while the pigmented excretory granuleshad been completely and quickly dissolved, the blood-vesselsand capillaries retained their full red colour and had apparentlysuffered no change. But in order to make the assurance doublysure, I kept pieces of blood-vessels only from the same materialfor a m o n t h in pyridine and examined this pyridine solutionspectroscopically. Although a very faint band (the a-band)could be seen, nothing of the /3-band was visible. The septalnephridia had never been kept in pyridine for more thantwenty-four hours. As soon as the pigment granules got dis-solved, this lot of septal nephridia was taken out and a fresh lotput in, and so on, until the pyridine had an orange-red colour.I have no doubt in my mind, therefore, that the haemochro-mogen in the pyridine solution of septal nephridia came from

384 K. N. BAHL

the pigmented excretory granules and no t from the blood-vessels.

The question naturally arises whether these pigmented granulesare only of haemochromogen or whether there is some othersubstance accompanying them. There seems no doubt thathaemochromogen must ultimately come from blood, and it leadsone to the conclusion that the glandular cells surrounding theciliated tract abstract haemoglobin from the blood, transform itto haemochromogen, and store it there. There is one importantconsideration which makes it possible that there is some othersubstance along with haemochromogen in the pigmented excre-tory granules, a substance which probably comes from thecoelomic fluid. It has been noticed by me time after time thatthe pigmented granules are completely absent in the closedintegumentary and pharyngeal nephridia, and are presentonly in the open septal nephridia, into which alone the plasmaof the coelomic fluid enters directly through their nephridio-stomes. The possibility is that as this plasma passes throughthe nephridiostome into the intracellular canal of the nephri-dium, it carries with it extremely minute solid granules whichmay be very finely divided broken bits of corpuscles or yellowcells—in fact, any particles which can go through the very fineand efficient sieve of the cilia of the nephridiostome; theseparticles are taken up by the ciliated cells of the middle tubeand stored there along with haemochromogen extracted fromthe blood-capillaries. In this connexion it is pertinent to recallthe injection experiments of Cordier (11 a) who injected colloidalfluids of varying degrees of dispersion into the coelomic fluidand found that they collected in the walls of the ciliated middletubes of the nephridium. Cordier believed that, like his artificialcolloidal fluids, natural colloidal solutions and solid particlesare absorbed by the ciliated tract and are deposited in the formof brown granulations, and he, therefore, recognized thefunction of the cells of the ciliated middle tube as a t h r o -p h a g o c y t o s i s (Gk. a t h r o i s , to collect). Cordier says thatparticles taken up by the athrophagocytic cells are retained fortwo months and even longer; I have no doubt that once theyare taken in, they are retained throughout life. Cordier pro-

EXCRETION IN INDIAN EABTHWORMS 385

bably did not continue his observations longer than two months.The fact that blood-pigment granules are present only in theseptal nephridia and not in the integumentary and pharyngealones can also be explained by the possibility that the ' ciliatedtract' is athrophagocytic only in the septai nephridia and not inthe integumentary and pharyngeal ones.

I have carefully observed that the pigmented granules arevery sparse in the nephridia of young earthworms, but thedeposit becomes heavier and heavier as the earthworm growsin age. In fact, one would be justified in saying that the septalnephridia of P h e r e t i m a and other earthworms function as'storage kidneys' which go on storing these brownish yellowgranules throughout life. Further, the fact that pigmentedgranules dissolve so quickly in pyridine while the blood takesa very long time indicates that the pigment in the granules ishaemoglobin which has already been alkalined and reduced, and,therefore, dissolves quickly in pyridine to give the absorptionbands of haemoehromogen. It is probable that pyridine dis-solves out only haemoehromogen and leaves the other substance,if there is any, behind in the nephridia.

We have already discussed the processes of nitration andreabsorption as they occur in the nephridial secretion ofthe earthworm (Chapter 6 A); the storage of blood-pigmentgranules with or without another substance in the walls of thenephridial tubules demonstrates the process of chemical trans-formation (p. 363), whereby the products of blood destructionare rendered innocuous and stored within the nephridial cells.

7. EXCRETORY ORGANS OTHER THAN NEPHRIDIA.

Just as the athrophagocytic section of the nephridium storesup the destruction products of blood, it is reasonable to expecta similar organ in the body of the earthworm to deal withsimilar products of coelomic fluid, which contains severalkinds of innumerable corpuscles. Schneider (18) found in allthe species of Oligochaeta investigated by him do r sa l phago-cytic organs with the function of cleansing the coelomic fluidof dead particles. P h e r e t i m a possesses these phagocyticorgans as white fluffy bodies situated on either side of the

386 K. N. BAHL

dorsal vessel, and attached to it, from the twenty-sixth segmentbackwards. I have recently (8) described paired v e n t r a lphagocytic organs in Megasco lex t e m p l e t o n i a n u s ;this earthworm has no funnelled nephridia at all in its body andits closed integumentary and pharyngeal nephridia do notpossess a phagocytic section in them. But it has very largeventral phagocytic organs which are richly supplied with blood;it is possible that these phagocytic organs or 'septal sacs' ofthis earthworm deal with the destruction products both ofblood and coelomic fluid.

The nephridia of P o n t o s c o l e x c o r e t h f u r u s (7) possessvery large funnels with the largest nephrostomial opening Ihave seen, through which the coelomic corpuscles can passeasily; immediately behind the funnel each nephridium pos-sesses a 'receptacle', which is filled with coelomic corpuscles inall stages of degeneration. Towards the distal end of thenephridium, immediately preceding the terminal bladder, thereis a thick-walled glandular duct which contains yellowish browngranules in its walls. It would seem, therefore, that the nephri-dium of P o n t o s c o l e x has two phagocytic sections, one fordealing with the destruction products of coelomic fluid andanother with those of blood.

In mammals phagocytic cells are scattered throughout thebody and are grouped together as the r e t i c u l o - e n d o t h e -l ia l system. It comprises mainly the endothelial cells of thespleen, certain branched cells in the bone marrow, the Kupffercells of the liver, and the reticulum cells in lymph glands. Ther e t i c u l o - e n d o t h e l i a l system is concerned with blooddestruction and carries the disintegration of haemoglobin as faras bilirubin (23). In the earthworm it would seem that it issimilarly scattered and comprises the dorsal and ventral phago-cytic organs, the phagocytic section or sections of the septalnephridia, and the aggregates of phagocytic cells within andabove the typhlosole. It is concerned with the disintegrationof both the coelomic fluid and blood, the disintegration ofhaemoglobin being carried as far as h a e m o c h r o m o g e n onlyin the septal nephridia.


8. SUMMARY.

1. In an earthworm, as in most aquatic invertebrates, ureaand ammonia form the main bulk of nitrogenous excretion andthere is no trace of uric acid. These excretory products are firstformed in the body-wall and gut-wall, pass therefrom into thecoelomic fluid and blood, and are thence eliminated to theexterior by the nephridia. In P h e r e t i m a urea and ammoniapass out from the nephridia to the exterior either directlythrough the skin or through the two ends of the gut.

2. Ammonia and urea have been estimated for the first timein the blood, coelomic fluid, and urine of the earthworm. It hasbeen shown that blood is not a mere carrier of oxygen, asEogers believed, but that it also takes part in carrying urea andammonia from the body-wall and gut-wall to the nephridia.The blood of the earthworm does not coagulate, indicatingabsence of fibrinogen.

3. The role of the nephridia in excretion and osmotic regula-tion has been determined. A comparison of the osmotic pres-sures of blood, coelomic fluid, and urine shows that the coelomicfluid is h y p o t o n i c to the blood, and that urine is markedlyh y p o t o n i c both to the blood and coelomic fluid. The proteinand chloride contents of the blood, coelomic fluid, and urinehave been determined with a view to elucidate the differencesin their osmotic pressures. It has been found that the urinecontains the merest trace of protein, but that the amount ofproteins in the blood is about eight times that contained in theplasma of the coelomic fluid. On the contrary, the chloridecontent of the coelomic fluid-plasma is about 60 per cent,higher than that of the blood-plasma.

4. The part of urine which is excreted from the blood isprobably a protein-free filtrate, but the nephridia reabsorb allthe proteins passing into them with the coelomic fluid-plasma.Similarly, there is a reabsorption of chlorides on a large scalefrom the initial nephridial filtrate during its passage throughthe nephridia.

5. A convenient method has been devised for collectingurine of the earthworm, which has made it possible to collect asmuch as 25 c.c. of urine in two and a half hours. The rate of

388 K. N. BAHI,

excretion of the urine has been determined and it has beenfound that in an earthworm living in water the outflow of urinein twenty-four hours must be more than 45 per cent, of itsbody-weight.

6. It seems that an earthworm, when submerged in water,can live like a freshwater animal, and its gut acts as an osmo-regulatory organ in addition to the nephridia, but in the soil itlives like a terrestrial animal and the osmo-regulatory functionis adequately discharged by the nephridia alone which reabsorbchlorides and proteins, and are also active in the conservationof water. In P h e r e t i m a and other earthworms with anenteronephric type of nephridial system, the gut takes a pro-minent part in reabsorbing the water of the nephridial fluid andconserving water to its maximum extent.

7. The phagocytic section (ciliated middle tube) believed bySchneider to be absent in the nephridia of P h e r e t i m a hasbeen shown to be distinctly present; it is also present in thenephridia of L a m p i t o , B u t y p h o e u s , and T o n o s c o l e x .The brownish yellow granules characteristic of this phagocyticsection form a heavy deposit in the septal nephridia of P h e r e -t i m a p o s t h u m a , heavier than that described in L u m b r i -c u s. The deposit increases with the age of the earthworm andforms a ' storage excretory product'.

8. Spectroscopic examination has revealed that these brown-ish yellow granules, so far believed to be of guanine, are reallyblood-pigment granules, since a pyridine solution of themshows the two characteristic bands of h a e m o c h r o m o g e n .With regard to the blood-pigment, the nephridia function as' storage kidneys'. •

9. The mechanism of nephridial excretion of the earthwormcan be analysed into processes of filtration, reabsorption, andchemical transformation.

10. It, is probable that the dorsal and ventral phagocyticorgans of earthworms are additional excretory organs.

9. REFERENCES.

1. Adolph, Edward F., 1927.—"Regulation of volume and concentrationsin the body fluids of earthworms ", 'Journ. Exptl. Zool.', 47.


2. Bahl, K. N., 1919.—"New Type of Nephridial System found in . . .Pheretima", 'Quart. Journ. Micr. Sci.', 64.

3. 1924.—"Enteronephric Type of Nephridial System in Lampito",ibid., 68.

4. 1934.—"Significance of Enteronephrie System in Indian Earth-worms", ibid, 76.

5. 1941.—"Enteronephric nephr. syst. in Tonoscolex", ibid., 82.6. 1942.—"Nephridia of the sub-family Octochaetinae", ibid., 83.7. 1942.—"Nephridia of Pontoscolex", ibid., 84.8. 1945.—"Nephridia of Megascolex etc.", ibid., 86.9. Benham, W. B.,- 1891.—"Nephridium of Lumbricus and its Blood-

Supply ; with Remarks on Nephridia in other Chaetopoda ", ibid., 32.10. Carter, G. S., 1940.—'General Zoology of the Invertebrates'. London.11. Cole, S. W., 1942.—'Practical Physiological Chemistry'. Cambridge.11 a. Cordier, R., 1933.—"Sur les phenomenes d'athrophagocytose dans

le segment cilie de la nephridie du Lombric", 'C. R. Soc. Biol.Paris', 113.

12. Cuenot, L., 1898.—"Etudes Physiol. sur les Oligochetes", 'Arch.Biol.', 15.

12 a. Delaunay, H., 1931.—"L'excretion azotee des invertebres", 'Biol.Rev.', 6.

13. Harrison, G. A., 1937.—'Chemical Methods in Clinical Medicine'.London.

14. Heidermanns, C, 1937-8.—"tlber die Harnstoffbildung beim Regen-wurm", 'Zool. Jahrbuch.', 58.

15. Kindred, J. E., 1929.—"Leucocytes and Leucocytopoietic Organs ofan Oligochaete", 'Journ. Morph.', 47.

16. Maluf, N. S. Rustum, 1939.—"Volume- and Osmo-regulative Func-tions of the Alimentary Tract of Lumbricus terrestris", 'Zool. Jahr-buch.', 59.

17. Picken, L. E. R., 1936.—"Excretory Mechanism in certain Arthro-poda", 'Brit. Journ. Exptl. Biol.', 13.

17 a. Rogers, C. G., 1938.—'Text-Book of Comparative Physiology'.New York and London.

18. Schneider, G., 1896.—"PhagocytSre Organe und chloragogenzellender Oligochaten", 'Z. wiss. zool.', 61.

19. Stephenson, J., 1930.—'Oligochaeta'. Oxford.20. Stolte, H. A., 1938.—"Oligochaeta" in Bronn's 'Tierreichs'.21. Wigglesworth, V. B., 1939.—'Principles of Insect Physiology".

London, 1939.22. Willem, V., and Minne, A.,—1900.—"Rech. sur l'excretion chez

quelques Annelides", 'Mem. Acad. Roy. Belg.', 58.23. Wright, Samson, 1942.—'Applied Physiology'. Oxford.

vi. the physiology of excretion and the signifi- cance of ... · excretion in indian earthworms 347...

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