THE MECHANISM OF URINE FORMATION IN INVERTEBRATES

THE MECHANISM OF URINE FORMATION ININVERTEBRATES

I. THE EXCRETION MECHANISM IN CERTAIN ARTHROPODA

BY L. E. R. PICKEN(Zoological Laboratory and Physiological Laboratory, Cambridge,

and the Freshwater Laboratory, Wray Castle, Westmorland)

(Received November 12, 1935)

(With Five Text-figures)

CONTENTSPAOE

Introduction 309

Crustacea 311

Car emus mamas . . . . . . . . . . . . 3 1 2

Resul t s of e x p e r i m e n t s 312(a) T h e tonic i ty of b lood a n d u r i n e . . . . . . . . 3 1 2(A) T h e colloid osmot ic p r e s su re and hydros t a t i c p res su re of t h e b lood . 313(c) T h e p ro te in con ten t of the u r i n e . . . . . . . . 3 1 4

P o t a m o b i u s f h w i a t i l i i 3 1 6

R e s u l t s o f e x p e r i m e n t s . . . . . ' 3 1 6( a ) T h e t o n i c i t y o f b l o o d a n d u r i n e . . . . . . . . . 3 1 6(b) The colloid osmotic pressure and hydrostatic pressure of the blood . 317(c) The protein content of the urine 318

Onychophora . 318

Peripatopris s p p . 318

Resul t s of e x p e r i m e n t s . . . . . . . . . . . 3 1 9

. Discuss ion . 320

(1) T h e osmot ic w o r k of t h e excre to ry o rgans . . • . . . . . 320

(2) F i l t r a t ion . . . . . . 3 2 1

(3) Secre t ion and resorp t ion 322

A p p e n d i x I * 322

A p p e n d i x I I . . . ' . . . . . . . . . . 323

A p p e n d i x I I I . . . . . . . . . . . . . 325

S u m m a r y 327

References . . . . . . . . . . . . . 328

INTRODUCTION .

THE suggestion that the excretory organs of fresh-water organisms assist in osmoticregulation was first made by Overton (1904). If an animal living in fresh water hasa surface permeable to water and a body fluid hypertonic to the external medium,water will pass from the environment to the internal medium. Overton postulated

310 L. E. R. PICKEN

that the excretory organs eliminated water by secreting a product hypotonic to thebody fluid. In this way he foresaw the possibility of an increased water flux infresh-water organisms, and anticipated the concept of a dynamic water and saltbalance.

Since Overton's paper it has often been assumed that the excretory organs ofinvertebrates are concerned in the regulation of the salt and water balance of theorganism (e.g. Pantin, 1931), but there has been little evidence to support thisassumption. Schlieper (1929) concluded that the antennary glands of the shorecrab, Carcinus maenas, play no part in the maintenance of the hypertonicity of theblood, since they secrete a fluid isotonic with the blood when the crab is living indilute sea water. On the other hand, Schlieper and Herrmann (1930) and Herrmann(1931) showed that in the crayfish, Potamobius fluviatilis, the secretion of the an-tennary glands is hypotonic to the blood. Schwabe (1933), also working withCrustacea, has correlated the relative development of the antennary gland inamphipods with their habitat, suggesting that the relatively large size of the glandin fresh-water forms is associated with its increased importance in osmotic regulation.

There exists a considerable literature on the qualitative and quantitative com-position of the fluid discharged by the excretory organs from the point of view ofnitrogenous excretion, but little work has been done in order to estimate to whatextent these take part in osmotic adjustment. Moreover, the occurrence of filtration,secretion, and resorption in the excretory organs has never been demonstrated, andinvestigators have had to be content" zu lernen, was sich aus der einfachen Betrach-tung der Nephridialorgane ergibt.... Beinahe alles was wir wissen, beschranktsich auf die Deutung des mikroskopischen Bildes, die naturgemass sehr unsicherist" (von Buddenbrock, 1928). Until recently methods were not available for com-paring the concentration of body fluid or blood and the excretory product; determi-nation of total osmotic pressure was impossible, since the samples of fluids ob-tainable are often very small. It was not known whether osmotic work is done inthe formation of the excretory product, and it was supposed impossible that thefirst stage in excretion, in invertebrates as in vertebrates, should be the filtrationfrom the blood of a liquid free from proteins and corpuscles. Thus von Budden-brock, writing of excretion in the higher Crustacea, states: "Es ist bei diesen z.T.sehr kleinen Tieren kaum moglich, den sowieso recht geringen Blutdruck alsfiltrierende Kraft zu Hilfe zu nehmen und so bleibt nur iibrig, eine aktive Sekretionder Exkrete anzunehmen."

The object of this investigation has been, firstly, to obtain values for the osmoticpressures of the body fluid and excretory product in the animals studied, and so todecide whether the excretory organs perform osmotic work by producing a fluidhypotonic or hypertonic to the body fluid; secondly, to measure the hydrostaticpressure and the colloid osmotic pressure of the body fluid, and the colloid osmoticpressure of the secretion, in order to estimate the possibility of filtration in these forms.

Relative measures of osmotic pressures have been obtained from vapour-pressure determinations made by the Hill thermal method. For this and othermethods see Appendices I, II, III.

The Mechanism of Urine Formation in Invertebrates 311

CRUSTACEA

Until recent years the accounts of the excretory process in Crustacea have beenbased on inferences from the morphology and histology of the antennary and maxil-lary glands. It is therefore necessary to outline the structure of these organs beforeconsidering the events which are supposed to occur in them. The following accountof the morphology of the excretory organs is based on Burian and Muth (1924).

Both glands consist of a tube, one end of which opens to the exterior, the otherinto a mesodermal coelomic sac. The tube of the antennary gland undergoesmodifications in the decapods so that its surface is very much increased, and in thiscondition it is known as the labyrinth (see Fig. 1). Before opening to the exterior thelabyrinth expands to form a bladder in which the secretion is stored, and at timesliberated to the exterior through the nephropore, which generally has some sort of

•bl.

•g.c.

it.

B

Fig. 1. The excretory organs of the decapods (diagrammatic): (A) Carcinus, (B) Potamobius;bl. bladder; c.t. coelomic sac; g.c. green canal; i.e. intermediary canal; /. labyrinth; t.c. transparentcanal; to.c. white canal. (After Marchal.)

closing device. The discharge of urine appears to be due to the secretion pressurebehind, since there are no muscle fibres in the bladder or in any other part of theorgan. From histological evidence it appears that all parts of the excretory organproduce both an indifferent, transporting fluid, and more obviously excretorysubstances. That is to say there are no histological grounds for division into regionsreserved for filtration, secretion and resorption. The coelomic sac consists ofseveral layers of cells, and those which line the cavity have inclusions which appearto be discharged into the liquid. This portion secretes carcinuric acid which isrelated to the pyridine carboxylic acids (Burian and Muth, 1924). The openingbetween the sac and the labyrinth—the nephrostome—is closed by a sphinctermuscle, and any passage of fluid from the labyrinth into the coelomic sac appears tobe prevented by a valve-like arrangement of cells. The labyrinth is composed of asingle layer of cells, which show basal striations recalling the appearance of cells inthe distal convoluted tubules and spiral tubules of the mammalian kidney. In the

312 L. E. R. PICKEN

bladder the lining cells are vacuolated and discharge droplets of fluid, so that thisportion is not a simple reservoir. This is evident in Homarus where the liquid in thebladder is not derived from the rest of the organ, since there is here no open con-nection with the labyrinth. Both labyrinth and bladder exhibit considerable varia-tion in complexity in the various groups of the decapods.

Carcinus maenas

Among the decapod Crustacea, the Brachyura, Anomura and certain Macrurasuch as Palinurus, Scyllarus and Gebia, possess a branched coelomic sac, the pro-cesses of which extend into depressions in the labyrinth wall, so that the labyrinthhas a sponge-like appearance in section. The only communication between coelomicsac and labyrinth is through the nephrostome. Blood reaches the gland by thenephridial artery, which supplies a system of blood lacunae completely surroundingthe coelomic sac and all its branches, and separating the sac from the labyrinth. Thelabyrinth is further complicated by the presence of trabeculae carrying blood vessels.This is the condition in Carcinus. The bladder is here lobed instead of being a simplerounded vesicle.

RESULTS OF EXPERIMENTS

(a) The tonicity of blood and urine

Schlieper (1929) and Schwabe (1933) record a normal hypertonicity of the bloodto the surrounding medium in specimens of Carcinus maenas from the North Seaand from the Mediterranean. According to these workers there is a difference infreezing-point depression of blood and sea water of 0-05° C. Schwabe finds thatthe tonicity varies with the position of the crab in the moult cycle, newly moultedindividuals being isotonic with or slightly hypotonic to the sea water.

Measurements of the vapour pressures of blood and sea water did not confirmthe normal hypertonicity of the blood in Carcinus. Table I contains the values ofblood, urine (= excretory fluid) and sea-water concentrations, expressed in terms ofsodium chloride solutions of equivalent strength. Of the fourteen crabs, six hadblood hypertonic, three isotonic and five hypotonic to the sea water. All thesecrabs were encrusted with Balanus, and therefore had not moulted recently. Otherindividuals (not included in Table I because their antennary glands contained nourine) gave similar values scattered above and below the concentration of sea water.

Schlieper and Herrmann (1930), using the cryoscopic method of Burian andDrucker (1910), compared the concentration of blood and urine. This method is theone which both Schwabe (1933) and Herrmann (1931) employed. With very carefultemperature control the accuracy obtainable is approximately the same as that of thevapour-pressure thermopiles. The latter have the advantage of requiring very muchless than the 1-5 c.c. of liquid necessary for the method of Burian and Drucker. Thedepressions of freezing-point given by Schlieper and Herrmann are measurementsmade on mixtures of urine from two or three individuals. Hence their resultscannot show small differences between the products of the glands of a singleindividual, or between the blood and urine of the same animal.

The Mechanism of Urine Formation in Invertebrates

Table I. Carcinus maenas

3*3

Concentration expressed asgm. NaCl in ioo c.c. solution

Sea water

3 5 23-523-583 6 03 4O3 5 13'473'593-633 6 23 6 23-5i3-5i3-5i

Blood

3-573-563 6 13 6 33 - 3 i3-383'493593-693-623-523-363H83 5 1

Urine

3'573-623-663'743 2 23-293-453-5i3-653 7 13-533'393'423 40

Hydrostaticpressure in

cm. of water

513252 0——182613121212

71 0

Colloid osmotic pressure incm. of water (calculated from

refractive index measurements)

Blood

————

13-515-2I5'96 17'5

14-8IS'O8'9

I3'26-3

Urine

————i ' ii - 8

2 '72 7i ' 52 23 - 0

3 60 90 7

Difference

————

1 2 4

1 3 4I3'23 46-o

12-6IZ'O5'3

12-35'6

Concentrations of solutions accurate to ± 002 per cent, sodium chloride.

Table I includes the values found for the tonicity of the urine. It appears thatthe urine may be hypertonic, isotonic or hypotonic to the blood.

(b) The colloid osmotic pressure and hydrostatic pressure of the blood

The hydrostatic pressure (see Appendix II) was measured in forty-one crabs,and an average value of 13 cm. of water pressure was obtained. Measurements ofthe refractive increment due to non-mineral constituents of the blood were made inthe same crabs, and the colloid osmotic pressure was calculated (see AppendixIII (b)). Direct measurements of colloid osmotic pressure were made on the bloodand urine of seven crabs (see Appendix III (a)). In Fig. 2 the refractive incrementdue to non-mineral solutes is plotted against the observed values for colloid osmoticpressure. It was found that the calculated values were rather high, but were of thesame order as the direct measurements. From Fig. 2 it is possible to read off valuesfor the colloid osmotic pressure of crabs' blood, knowing the refractive increment.The values for colloid osmotic pressure in Table I were determined in this way. Ofthe forty-one crabs, twenty-seven had a value for hydrostatic pressure greater thanthe colloid osmotic pressure of the blood.

It is possible to predict from the condition of the crab whether the hydrostaticpressure will be normal, or abnormally low. Thus of nine crabs showing poor reflexresponses, seven had a hydrostatic pressure less than 7-5 cm. of water. In no casewas an active crab found with a low hydrostatic pressure.

The hydrostatic pressure measured represents only the value found in thesternal sinus; the actual pressure in the vessels supplying the antennary gland iscertainly greater. It is probable that the blood pressure is mainly determined by thetone of the body muscles, the heart superposing on this pulsations due to itsactivity. If the hypodermic needle (see Appendix II) is driven into the pericardial

J E D - X I I I i l l 2 1

L. E. R. PlCKEN

space the observed pressure increases, and pulsations appear which may havea maximum value of 40 cm. of water.

(c) The protein content of the urine

The lowest values for colloid osmotic pressure in Fig. 2 were measurementsmade on urine samples. Since they fell within the limit of accuracy of the osmo-meter (see Appendix III (a)) it was necessary to have confirmation of the presencein the urine of some substance with large molecules. Refractive index measurements

201-

3i 10sa

o

'3

0

0

G

0 500 1000 1500Refractive increment of non-mineral solutes (blood and urine)

Fig. 2.

showed that there was a considerable non-mineral refractive increment, from whicha colloid osmotic pressure of the same order as the value directly determined couldbe calculated (see Appendix HI (b)), assuming the increment to be due to protein.The xanthoproteic test was applied to the urine and gave a positive result on each ofsix samples (see Appendix III (b)). This indicated the presence of amino acids suchas tryptophane, tyrosine and phenyl alanine. A drop of acetic acid added to theurine gave a white precipitate, most of which dissolved in excess of the acid; butthere remained a little stringy precipitate. This was entirely soluble in hydrochloricacid or in a dilute solution of sodium carbonate. Samples of urine from which the


precipitate had been removed by filtration had a lower nitrogen content than un-filtered samples. These tests indicated the presence in the urine of a nitrogenouscompound precipitated by dilute acetic acid, and in part insoluble in excess of thisacid. Such reactions would be given by protein, part of which was mucin. It wasconcluded that the urine contains proteins derived from the blood, or from cellbreakdown in the gland, or from mucus, and possibly from all three sources.

(1) Material.The crabs were kept in aerated sea water at a temperature of 160 C.

(2) Technique.(a) Measurement of hydrostatic pressure (see Appendix II). The zero of the spoon gauge

was determined, and after opening the reservoir tap the crab was forced on to the tip ofthe needle, which penetrated the soft arthrodial membrane at the base of the last leg.

(b) Collection of blood. The propodite of one chela was cut through and the bloodcollected in a chemically clean, dry ignition tube, cooled on ice. Cooling facilitatedaggregation of the leucocytes and left the blood clear. The blood was kept on ice in order toreduce the effects of bacterial action.

(c) Collection of the urine. Pipettes with small bulbs were used for collecting and storingthe urine. They had the advantage of holding the urine so that spilling was impossible, andsince the liquid was exposed to a very limited volume of air, evaporation was not likely toproduce concentration changes in the time between collecting the sample and measuringthe vapour pressure. The operculum closing the antennary gland was lifted with a cataractknife, the pipette was inserted and the urine withdrawn. A separate pipette was used tocollect from the other gland so that the fluid from the two glands could be compared.

(d) The measurement of colloid osmotic pressure (see Appendix III). The blood wasshaken and the clot of leucocytes removed. Some of the remaining liquid was used in theosmometer. Sea water was put on one side of the membrane. Before use the membranewas soaked in sea water for hah0 an hour. Table II is a protocol of one experiment. Since itwas possible that the clotting of the leucocytes would remove protein from solution and solead to low values for the colloid osmotic pressure of the blood, estimations of total nitrogenwere made on samples of blood centrifuged immediately after collection, and on sampleswhich were cooled on ice, and from which the leucocyte clot had been removed. In nocase was the total nitrogen of the second sample lower than that of the first; it was con-cluded that determinations of colloid osmotic pressure made on blood from which theleucocyte clot had been removed gave a true picture of the values for normal blood.

Table II. Crab (5). June 5, 1934.

Timep.m.

4-15S-oo5-356.007-058.059-os

Manometerreading

15-019-519920-321721-721-7

Height ofmeniscus

9-59-8999'9999-999

Colloid osmoticpressure, blood

cm. H,0

5-S97

io-o1C4II-8n-8n-8

(e) The xanthroproteic test for proteins in the urine. This was performed as directed byCole (1928).

(/) The measurement of vapour pressures of blood and urine. The procedure adopted wasto compare the blood with the urine from each gland, separately, then the blood with sea

316 L. E. R. PICKEN

water collected from the aquarium when the crab was removed, and then sea water with astandard solution of sodium chloride. Finally the thermopiles were calibrated with astandard solution of sodium chloride. Both blood and urine were kept on ice until thedeterminations were made. Solutions were applied to the papers on the thermopiles bymeans of small pipettes holding by capillarity enough liquid to wet the paper thoroughly.Before applying the urine a drop was expelled from the pipette, and the tip wiped withabsorbent wool so that any concentrated liquid was removed.

Potamobius fluviatilis

The antennary glands of the Caridea and Astacidea differ from those of theremaining decapods in that the coelomic sac is unbranched, but its surface is in-creased by the development of septa, giving it a chambered appearance in section.The sac is embedded in the spongy mass of the labyrinth. In contrast to the con-dition found in the Brachyura, the coelomic sac and labyrinth have separate bloodsupplies.

Among the crayfishes, the nephridial canal is more highly developed than inother decapods. The labyrinth is divided into three portions (see Fig. i B), (i) the" green canal"—a flat, chambered sac, connected at one end to the coelomic sac, andat the other opening through an "intermediary canal" into (2) a simple tube—the"transparent canal"—which in turn opens into (3) the "white canal".


(a) Tfie tonicity of blood and urine

The existence of a hypertonic body fluid in fresh-water invertebrates was knownfrom the work of Fre'dericq (1898) and Botazzi (1908), but until Herrmann (1931)demonstrated the hypotonicity of the urine in Potamobius there was no evidence thatthe excretory organs were concerned in the maintenance of this hypertonicity of thebody fluid.

Before Herrmann's experiments almost the only evidence of the performance ofosmotic work by invertebrate excretory organs was that obtained by Botazzi (1906),who showed that in Octopus vulgaris the urine is hypotonic to the blood (A blood =2-296° C, A urine = 2-24° C.). The average value for A crayfish blood given byHerrmann (and also by Botazzi),is o-8o° C, which is approximately equivalent to thedepression for a 1-26 per cent, solution of sodium chloride.

Measurements of vapour pressures were made with the more reliable thermalmethod, and confirmed the hypertonicity of the blood to the external medium, andthe hypotonicity of the urine to the blood. Table III shows the values obtained forcrayfish from a Westmorland stream. The measurements on blood agree withthose made by the earlier workers, but Herrmann's figure of A urine = o-i6° C,i.e. 0-25 per cent, sodium chloride, is higher than the values shown here in all butone of the specimens examined. It is apparent that here, as in Cardnus, the con-centration of the secretion produced by the two glands differs in the same animal,and that the difference is of the same order as in Cardnus. Experiments showed thatthe salt concentration of the stream water from which the animals came was less thancould be measured with the thermopiles.

The Mechanism of Urine Formation in Invertebrates

Table III. Potamobius fluviatilis

317

Sex

Female

Male

Hydrostaticpressurecm. H,0

7-51 o-o180

Colloidosmotic

pressure, bloodcm. H,O

2-O6-5

I I - O

Concentrationblood

% NaCl

•27[•22[•29[•33f29[•40

Concentration urine% NaCl

Right

0-080060-39

^•08"0-140-08

Left

OilC140-20O-O7O-O5O'i6

Concentrations accurate to ±002 per cent, sodium chloride.

Table IV. Potamobius fluviatilis

Sex

Female

MaleFemale

>»

Colloidosmotic

pressure,blood

cm. H , 0

18-017-0II-OIO-O20-5

Colloidosmotic

pressure,urine

cm. HtO

Right

3-S4-00-63"8o-i

Left

0-8i"5o-sS'3o-i

Meandifference

15-71421045'4

20-4

Hydro-static

pressurecm. H,O

I4-S2216-52022

Con-centration

blood% NaCl

1-25I'2O1-191-261-25

Concentrationurine

% NaCl

Right

0-480-320-300-480-29

Left

0280-250-25068O'3O

Concentrations accurate to ±o-oi per cent, sodium chloride.

In Table IV are collected the results of measurements made on crayfish fromCambridge tap water. It is seen that the concentration of the urine is higher inthese animals than in the Westmorland forms (with one exception), and it would beinteresting to know whether this difference is correlated with the higher salt contentof Cambridge tap water.

(b) The colloid osmotic pressure and hydrostatic pressure of the blood

With the exception of the values given in Table III no direct determinations ofcolloid osmotic pressure have been made; but since there is such a close agreementbetween non-mineral refractive increment and colloid osmotic pressure in fish andmammalian sera, it was assumed that the graph obtained for Carcinus could be usedfor calculating colloid osmotic pressure in Potamobius. Accordingly the values inTable V are obtained from the refractive index, making an allowance for salts. Theaverage value for the calculated colloid osmotic pressure of the blood was 15 cm. ofwater.

Of the eleven individuals in Tables IV and V, ten have a mean value for thehydrostatic pressure greater than the colloid osmotic pressure of the blood. Theaverage value for the hydrostatic pressure is about 20 cm. of water.

L. E. R. PlCKEN

Table V. Potamobius fluviatilis

Sex

FemaleMale

»>FemaleMaleFemale

Colloidosmotic

pressure,blood

cm. H,O

'5'4II-O •i3"S14-014-7198

Colloidosmoticpressure,

urinecm. H,0

Right

o-i

o-s0-4o-oo-i

Left

o-ii'4o-i0-3o-i3-8

Meandifference

iS-39-6

13-213614-216-0

Hydro-static

pressurecm. H,0

211922-52O25-S25

Xanthoproteic test

Right

+ + +

+ + ++ + ++ +

Left

+ ++ + + +

++ + +

++ + + + +

Note: a — 9ign shows that the test was not made: in no case was the xanthoproteic test performedon the urine with a negative result.

(c) The protein content of the urine

It wa9 found that the urine contained a considerable quantity of non-mineralsubstances contributing to the refractive increment, and that the fluids from the twoglands differed in the concentrations of these substances. Table V contains values forcolloid osmotic pressure and hydrostatic pressure in six crayfish, together with theresults of xanthoproteic tests on the urine from each gland. The number of + signsindicates roughly the relative intensity of the colour produced, and hence theamount of " protein " in the urine. The tests were made before the refractive indexmeasurements, so that there was no danger of the subjective intensity estimate beinginfluenced by a knowledge of the refractometer reading.

(1) Material.The crayfish were collected from a stream in Westmorland, and kept in a depth of

about 2 in. of their stream water, the water being constantly aerated. They were rathersmall specimens, never more than 4 in. in length and often smaller. The experiments wererepeated with larger specimens from Surrey, kept in Cambridge tap water.

(2) Technique.Those points only will be mentioned which differed from the procedure in the case of

Carcinus.(a) Measurement of hydrostatic pressure. The needle was inserted into the sternal sinus

from the posterior end of the thorax, through the intersegmental membrane.(b) Collection of urine. The opening of the antennary gland in Potamobius is protected

by a membrane with a very small slit-like aperture. The membrane was pierced and theurine collected by fine hypodermic needles, mounted on glass tubes with de Khotinskycement.

ONYCHOPHORA

Per ipa tops is spp.

In the Onychophora the excretory organs are segmentally arranged. They arestructurally similar to the excretory organs of the Crustacea; each has a blind end-sac (a coelomic rudiment) communicating with a coiled duct, which opens into abladder. The excretory product is discharged from the bladder through a fine tube


opening to the exterior at the base of the leg. In these experiments several species ofPeripatopsis were used.


The values for the various measurements are collected in Table VI. In sevenout of nine cases in which the concentrations of blood and urine were determinedfor the same animal, it was found that the urine was hypotonic to the blood. Thehydrostatic pressure was large compared with the difference in colloid osmoticpressure of blood and urine (a value of 3 cm. of water was obtained from a specimen1 day after birth). As in the case of Potamobius and Carcinus, the urine gave apositive xanthoproteic test.

Table VI. Peripatopsis spp.

Concentration expressedas gm. NaCl in100 c.c. solution

Blood

0-46063062068o-6o0 5 0O-730900G0060

0-62080o-6o

Urine

0440-58o-540-64ogo0500̂ 460600-47

Hydrostaticpressurecm. H,O

1039

11-20

Colloid osmoticpressure cm. H»O

(calculated)

Blood

39

7-04'44'4

6-3303'4

2-75-8

Urine

20

2'I

3'3

Concentrations accurate to ±002 per cent, sodium chloride.

(1) Material.1

The animals were kept in damp fragments of rotten bark away from the light and werefed on small arthropods and sheep's liver. The specimens examined were mostly Peri-patopsis sedgwicki, occasionally P. moseleyi and rarely P. balfouri.

(2) Technique.(a) Measurement of hydrostatic pressure. The specimen was allowed to creep on a

microscope slide and was then pierced with the hypodermic needle.(b) Collection of urine. This was done in two ways. If the animals are killed with

chloroform vapour the body wall contracts violently and drops are discharged from thenephridial apertures. The second method is to collect drops of liquid spontaneously dis-charged. Animals are transferred to small clean glass vessels with a covering Petri dishlined with wet cotton-wool. The animals usually excrete drops simultaneously from all thenephridial apertures within 2 hours of the transfer, and the drops are collected in capillarypipettes and kept until required in ignition tubes with wet cotton-wool at the bottom toreduce evaporation.

1 I am indebted to Dr S, M. Manton for this material and for making the collections of blood andurine.

320 L. E. R. PICKEN

(c) Collection of blood. The animals were opened and the blood collected in a capillarypipette and kept until use in the manner described for the urine. When opening the bodycavity care was taken to avoid puncturing the slime glands.

(if) The measurement of colloid osmotic pressure. This was done indirectly, the colloidosmotic pressure being calculated from the refractive increment of blood and urine respec-tively. A Zeiss stop attachment made it possible to use very small drops of liquid in the re-fractometer. For calculating the colloid osmotic pressure, the graph for Carcinus (Fig. i)was used, and a mean salt correction of o-6o per cent, sodium chloride was applied in thosecases in which the total concentration of the sample had not been determined by a vapour-pressure measurement.

(e) The measurement of vapour pressures of blood and urine. These measurements weremade using strips of cigarette paper (see Appendix I) and the medium-sized thermopileswhen possible. It was often necessary to use microthermopiles (Appendix I) owing to thesmall size of the samples available.

DISCUSSION

(i) THE OSMOTIC WORK OF THE EXCRETORY ORGANS

The relationships between the concentrations of body fluid and urine, andbetween colloid osmotic pressure and hydrostatic pressure of the body fluid, havenow been described for the shore crab, the crayfish and Peripatopsis. In each ofthese animals vapour-pressure measurements have shown that there is a difference,large or small, between the concentration of the body fluid and that of the urine.The excretory product may be hypotonic or hypertonic to the blood, and it isdifficult to escape the conclusion that the osmotic difference is due to secretory workin the excretory organs.

In the case of Carcinus, the use of the vapour-pressure thermopiles has made itpossible to detect in the same organism small but significant differences between thesecretion and the blood. This means that the excretory organs in this animal may beof more importance in osmotic adjustment than Schlieper supposed. There is alsoevidence for osmotic work in respect of individual ions. Nagel (1934) has shownthat iodide is removed from the blood by the antennary gland and concentrated inthe urine, while the work of Biafaszewicz (1930) suggests that the excretory mech-anism may be important in maintaining the normal concentration of S0*4 and Mg-in the blood. Recently Bethe, von Hoist and Huf (1935) have shown from chlorideanalyses that the urine of crabs in dilute sea water is hypotonic to the blood—contrary to the results of Schlieper's freezing-point measurements, but supportingthe view that the excretory organs are active in osmotic regulation.

In the crayfish it is obvious to suggest that the removal of salt from the urine isassociated with the development of the labyrinth. Schlieper (1933) has measuredthe freezing-point of the urine in Homarus vulgaris, and finds approximate iso-tonicity between urine and blood. Homarus (a macruran like Potamobius) has nowhite canal, and Schlieper concludes that the appearance of this structure in theAstacidea is correlated with the ability of the antennary gland to resorb salt. Thework of Gicklhorn (1931) on the maxillary gland of Daphnia also supports the viewthat resorption takes place in the nephridial canal.


Scholles (1933) finds that in Potamobius, as in Eriocheir and Carcinus, theexcretory organs are concerned in the adjustment of the ionic balance of the bodyfluid. The antennary glands of the crayfish seem to differ chiefly from those of theshore crab in the increased importance of salt resorption. Even in Carcinus, how-ever, vapour-pressure measurements have shown that the urine may be hypotonic tothe blood; the salt-resorbing mechanism of the fresh-water Macrura is perhaps onlya specialisation of a mechanism already slightly developed in marine forms.

The observation that the urine of Peripatopsis is hypotonic to the blood is atfirst sight rather surprising, since Peripatopsis is a terrestrial animal and might beexpected to conserve water rather than salt. On the other hand, Peripatopsis emptiesthe nephridia comparatively infrequently (about once a fortnight), so that as com-pared with an aquatic organism the amount of water lost in this way is much re-duced (Potamobius excretes 4 per cent, of its weight per day (Herrmann, 1931)).Dr S. M. Manton finds that on an average 0-06 mg. of uric acid are eliminateddaily from the gut in a solid condition. This suggests that the bulk of the nitrogenousexcretion does not take place through the nephridia, and hence the urine may not beimportant in removing nitrogenous waste. The hypotonic urine might perhaps beinterpreted as a functional vestige surviving from a time when excretion took placethrough the nephridia, and when, moreover, the retention of salt was an importantfactor in the control of the internal medium.

(2) FILTRATION

It has been shown (pp. 313, 317, 319) that the hydrostatic pressure is of the sameorder as the colloid osmotic pressure of the blood, and in the case of the crayfishand Peripatopsis is almost always greater than the blood colloid osmotic pressure.This means that, contrary to the opinions of earlier writers, filtration is a possiblestage in the formation of the urine in invertebrates. An examination of the urinehas shown almost certainly in Carcinus, and possibly in Potamobius and Peripatopsis,that it contains a little protein. Some of this protein may be derived from mucusproduced in the gland; the remainder is perhaps the result of the disintegration ofgland cells, or the wall of the antennary gland may be slightly permeable to protein.In the last case the pressure necessary for filtration is less than the colloid osmoticpressure measured against a membrane impermeable to proteins, and is perhapsbetter represented by the difference between the colloid osmotic pressures of bloodand urine. In thirty-four crabs the hydrostatic pressure and the refractive incre-ments for blood and urine were determined, and the colloid osmotic pressurescalculated: twenty-six individuals had a hydrostatic pressure greater than thedifference between the colloid osmotic pressures of blood and urine. Of the eightcases in which this was not so, five had been noted as in poor condition—as judgedby their reflex responses. The condition of the remaining three was not recorded.The differences between the colloid osmotic pressure of blood and urine are shownin Tables I, IV and V.

Bethe, von Hoist and Huf (1935) have shown, by connecting a sea-water mano-meter to the crab, that increasing the hydrostatic pressure increases the rate of urine

322 L. E. R. PlCKEN

formation in the antennary gland of Carcinus. They find that the crab will maintaina constant pressure head of c. 3 cm. of sea water, and suggest that this represents thenormal hydrostatic pressure. It must be remembered that this is not an isometricmeasurement: this value represents rather the pressure at which filtration ceases.The fact that this value is lower than the average value for the colloid osmoticpressure of the blood may be due to the considerable dilution of the blood by seawater. These results confirm the possibility of filtration as a first stage in urineformation.

(3) SECRETION AND RESORPTION

The occurrence of secretion in the antennary gland is indicated by Nagel's (1934)work on iodide excretion in Carcinus. In both Carcinus and Potamobius the secretionof nitrogenous waste has been shown by previous workers (see Burian and Muth,1924). There is as yet no direct evidence of the occurrence of salt resorption in thearthropodan nephridium. It is, however, extremely probable that the hypotonicityof the urine in Potamobius and Peripatopsis, and perhaps in Carcinus in dilute seawater, is due to the conservation of salt in this way.

The results of this examination of arthropodan excretory organs suggest thatthese structures may be of the same importance to the animal as their analogues inthe vertebrates. It has been shown in each case that the excretory organs are able toproduce urine differing considerably in osmotic pressure from the blood, and it ispossible that a salt-resorbing mechanism is responsible for the difference in totalconcentration between blood and urine in forms such as the crayfish and Peripatopsis.The opinion of earlier writers, that only secretion could account for the formation ofthe urine, has been shown to be extremely improbable, since the hydrostatic pressureof the blood is generally greater than its colloid osmotic pressure, and is thereforesufficient to account for a preliminary filtration into the nephridium. It seemsprobable that the excretory organs not only assist in the regulation of the saltand water balance, as they do in the vertebrates, but that the mechanism by whichthis is accomplished is similar in both groups, and involves in arthropods, as invertebrates, filtration, resorption and secretion.

APPENDIX I

THE HILL THERMAL METHOD FOR COMPARING VAPOUR PRESSURES

Descriptions of the apparatus, its use, and the accuracy obtainable, are given byHill (1928, 1929, 1931) and Margaria (1930). It was found that evaporation is animportant factor in the use of the thermopiles; in order to increase the significanceof single measurements of vapour pressure made on samples too small for duplicatedeterminations, all manipulations were made in a small moist chamber.

Thorough wetting of the thermopile face was obtained by using cigarette papersto hold the solutions in contact with the junctions. With a paper 1 cm. square about5 x io~4 c.c. were required for thorough wetting. The volume could be reduced byusing strips of paper, 1 x 0-5 sq. cm., which required only 3 x io"4 c.c. of solution.

c.c.


It was found that the growth of stray E.M.F. in the thermopiles could be re-strained by keeping them shorted when not in use.

Owing to the uncertainty of vapour-pressure measurements on 1 x io~4

samples, an attempt was made to construct microthermopilessuitable for use with these small samples. The volume requiredfor a determination with these is a few hundredths of a cubicmillimetre.

The appearance of the thermopile is shown in Fig. 3. It issimilar to the .Hill-Downing type and suffers in the same way fromheat loss by conduction along the junctions. A coil of about eightturns of constantan wire (42 s.w.G.) was wound on a thin-walledglass tube. The coil was cleaned with zinc chloride solution, dis-tilled water and absolute alcohol, dried and painted over half itsarea with molten paraffin wax. It was then electroplated with silver,the wax was dissolved in boiling alcohol and the coil was givenseveral coats of " Elo>n varnish and slowly dried in an oven.

The moist cups for the thermopiles were made from ignitiontubes drawn off to a convenient length. One is shown in position inFig. 3. Cigarette papers were used for lining the cups. A moistchamber was made in which these microthermopiles could be setup. The procedure adopted consisted in depositing a minute dropon one line of junctions, and then placing over this a piece of Fl8- 3-cigarette paper about 2 mm. square. This was repeated with the other set ofjunctions.

These thermopiles were about one-third as sensitive as the medium-sizedthermopiles made by Mr A. C. Downing for this work, but they had only one-sixth the number of junctions. The increased sensitivity per junction may be due tothe much reduced gap between the thermopile face and the chamber wall, whichsteepens the diffusion gradient, and also to the cylindrical form, which gives a radiallysymmetrical gradient.

APPENDIX II

MEASUREMENT OF THE HYDROSTATIC PRESSURE OF THE BODY FLUID

The measurement of hydrostatic pressures in invertebrates is a matter of somedifficulty, as their small size makes manipulation difficult, and the small volume ofbody fluid makes it impossible to use any simple type of manometer for the pressuremeasurement, since the pressure within the system falls to zero when a very smallvolume change has taken place.

For making isometric pressure measurements a "spoon" gauge was employed.The gauge is made by blowing a bubble in a length of glass tubing. While being

blown, the two ends of the tube are drawn apart so that the bubble is elliptical. Ifnow a yellow-tipped flame is allowed to play on one side until the glass collapses a

1 Kindly supplied by Birkbys, Ltd., Liversedge, Yorkshire.

324 L. E. R. PlCKEN

spoon-shaped bubble results. One end of the tube is drawn out to make a long thinlever, sufficiently rigid to have a short period of vibration. When the pressure on theair within the bubble is raised, the curvature of the spoon decreases and the tip isdeflected. By varying the thickness of the bulb, gauges of different sensitivity canbe made. Since it was desirable that the system should be isometric, the change involume of the gauge with pressure was measured. A length of capillary tubing wasfused on to the gauge, and the gauge was filled with water so that the meniscus roseinto the capillary tubing. The displacement of this meniscus could be observed asthe pressure was raised. Small and relatively insensitive gauges were made in whichthe volume change for 20 or 30 cm. of water was imperceptible. The movements ofthe gauge were observed under the high power of a microscope. A fibre of darkblue glass was sealed to the tip of the gauge, and this was seen projected on the

Fig. 4-

ground-glass screen of a periscope, with an eyepiece scale superimposed. Pressurescould be read to within +0-5 cm. of water.

Such a gauge was filled with cold boiled saline (to avoid the formation of gasbubbles) and connected to a reservoir of saline. The reservoir enabled the gauge tobe filled with saline under pressure. The gauge was connected by a three-way tap toa water manometer and a hypodermic needle mounted with de Khotinsky cementon one limb of the tap. Fig. 4 shows the appearance of the apparatus. The tap aenables the reservoir to be isolated from the gauge. Since the tube from the reservoiris not included when the tap a is closed (as it would be were b closed), the drop ofliquid which emerges before the pressure falls to zero is extremely small—a fractionof a cubic millimetre. By means of the manometer known pressures can be appliedand the resulting deflection observed. Over the range of pressures measured, i.e.0—30 cm. of water, the deflection varies linearly with the pressure applied.

Determinations of hydrostatic pressure are made with the spoon gauge under apressure somewhat greater than the pressure to be measured. With tap c turned sothat the needle is connected to the gauge, taps a and b are opened, and the glass


fibre is displaced to a position which depends on the height of liquid in the reservoir,and the resistance offered by the needle to the escape of fluid. If a is closed thepressure falls instantly to zero. It is necessary to observe this zero with the tip of theneedle under water, since evaporation from the liquid at the tip leads to a change incurvature of the meniscus, and hence the pressure in the gauge will change withevaporation. The zero return is tested three or four times before making a determina-tion. In general, successive positions for the zero differ from each other by less thanhalf a scale division. Tap a is now opened until saline drips slowly from the needle.The organism is forced on to the tip, so that the puncture is made as rapidly aspossible (to avoid clogging the tip with mucus), and tap a is closed. If the operationis successful the fibre immediately begins to fall, and comes to rest at a positionconsiderably removed from the zero (in most cases). On stimulating the animalto movement the pressure rises and subsequently falls again. The values of body-fluid pressures recorded are minimal values obtained when the animal is quiescent.Occasionally the glass fibre does not return towards zero when tap a is closed. Inthese cases the needle is clogged. Readings are only recorded when this return iscomplete.

It is necessary to have the gauge under a pressure greater than the final pressuremeasured since the tip is blocked if the animal is pierced when the pressure in thegauge is atmospheric.

Since the amount of liquid injected into the animal is infinitesimal (if the heightof the reservoir is suitably adjusted), it is scarcely necessary to change the saline forevery organism, as in most animals the injection of a cubic millimetre of distilledwater would not produce a detectable change in the concentration of the body fluid.At most it is sufficient to wash through with one reservoir full of the new saline.

APPENDIX III

DETERMINATION OF THE COLLOID OSMOTIC PRESSURE OF BIOLOGICAL FLUIDS

(a) The apparatus first used for the measurement of colloid osmotic pressureswas a modification of that described by Krogh and Nakazawa (1927). The changeswere directed to facilitating construction and reducing the volume of the samplenecessary for a determination. A serious difficulty was encountered when thevolumes of the samples were only a few cubic millimetres, since the determinationof the rise of fluid in the osmometer due to capillarity cannot be made withouta free liquid surface. To avoid this difficulty the apparatus was redesigned, so thatthe movement of the saline was observed and not that of the sample.

The apparatus is shown in Fig. 5. The flanges a and b had to be plane and circular.From the reservoir c the capillary and tube d can be flooded with saline. With thesaline just level with flange b, the height of the meniscus in the capillary is marked,and all measurements of the position of the meniscus are made with this mark aszero. A small perforated silver disc 0-9 cm. in diameter is placed on b, and over it arubber washer reaching almost to the edge of the flange and bored to take the silverdisc. A suitable membrane, about 1-5 cm. in diameter, is laid over the disc and over

326 L. E. R. PlCKEN

this is placed a second washer, bored with a smaller tool, so that only the portion ofthe membrane over the perforations is uncovered. Any saline on the membrane isremoved with filter paper and the manometer e with its attached flange is lowereduntil the flange covers the washer, etc., on flange b. The two flanges are then clampedtogether by a brass screw clamp which grips them evenly. Through the hole / thedrop of saline, squeezed from between the washer and the membrane, can be re-moved with a capillary pipette. To prevent evaporation from the sample a fragmentof cotton-wool soaked in isotonic saline is introduced through the hole/, and pusheda few millimetres up the tube towards the manometer. With another pipette thesample of fluid is run on to the membrane and the hole is then closed with a plug ofplasticine. In tightening the screw clamp thesaline meniscus rises above the capillary zero,and the height of the manometer is now ad-justed until the difference between the heightof the saline meniscus and the manometerreading is somewhat greater .than the expectedcolloid osmotic pressure. Since the sample isvery small the transfer of saline resulting indilution must be prevented. The meniscus iswatched through a vernier microscope and themanometer is adjusted every half hour untilthe meniscus neither rises nor falls. At theend of 5 hours, when the meniscus is steady,the difference between the manometer readingand the height of the meniscus above the capil-lary zero gives the colloid osmotic pressure ofthe liquid on the membrane.

Throughout the determination the osmo-meter is kept in a stirred water bath. Care has to be taken that tap g does notleak and that the membrane is tightly clamped. This inverted osmomeler avoidsthe difficulties due to the clotting of blood samples which is apt to occur incapillary tubes.

The membranes used were made by Elford's method (1931). They are collodionmembranes prepared for virus filtration and are quite flat and uniform in thickness.The pore size is c. 0-05/x. They can be stored in chloroform water and cut to sizewith cork borers.

(b) Owing to the difficulty of making direct determinations of the colloidosmotic pressure of samples as small as io~3 c.c, other methods for determining theprotein content were considered.

It was found that the xanthoproteic test could be made on a micro scale. Thecolour was lasting, and the intensity varied considerably with changes in concentra-tion. The possibility was considered of developing this reaction into a quantitativemethod for protein estimation. Since the proportion of tryptophane, phenyl alanineand tyrosine (to the presence of which the reaction is due) varies considerably in

Fig. 5-


those few proteins which have been analysed, the method was only used for com-paring the concentrations of two solutions of the same protein.

It is possible to measure the refractive index of samples of about 1 c.mm. witha Zeiss dipping refractometer, using the auxiliary prism and a stop diaphragmattachment. Making an allowance for the salts present, the percentage of proteinpresent can be calculated from refractive index measurements of vertebrate sera.The colloid osmotic pressure can be determined (considering the liquid as vertebrateserum) from the graph given by Krogh and Nakazawa (1927) for the colloid osmoticpressure per percentage of protein. Alternatively, the colloid osmotic pressure can beread off from the graph given by Keys and Hill (1934) for the relation between thecolloid osmotic pressure of fish sera and the refractive increment due to non-mineralsolutes. Values determined in this way by the two methods agree within a centi-metre or so, and they have been compared with direct determinations made with theosmometer. The calculated values are rather higher than those found by directmeasurement, so that it is necessary to determine the refractive increment for 1 percent, protein in various invertebrates before the measurement of refractive indexalone will suffice for a determination of colloid osmotic pressure. In all the calcula-tions of protein content from refractive index it has been assumed that sodiumchloride is the chief mineral constituent.

SUMMARY1. In Carcinus maenas:(a) The blood may be hypertonic, isotonic or hypotonic to the external medium.(b) The urine may be hypertonic, isotonic or hypotonic to the blood, and its

concentration may differ in the two antennary glands.(c) The hydrostatic pressure of the body fluid is c. 13 cm. of water.(d) The colloid osmotic pressure of the blood is c. 11 cm. of water.(e) The urine probably contains protein and has a colloid osmotic pressure of

c. 3 cm. of water.

2. In Potamobius fluviatUis:(a) The blood is hypertonic to the external medium.(b) The urine is hypotonic to the blood but hypertonic to the external medium

and its concentration may differ in the two antennary glands.(c) The hydrostatic pressure of the body fluid is c. 20 cm. of water.(d) The colloid osmotic pressure of the blood is c. 15 cm. of water.(e) The urine may contain protein and has a colloid osmotic pressure (calculated)

of c. 2 cm. of water.3. In Peripatopsis spp.:(a) The blood is hypertonic to the urine.(b) The hydrostatic pressure of the body fluid is c. 10 cm. of water.(c) The colloid osmotic pressure (calculated) of the blood is c. 5 cm. of water.(d) The urine may contain protein and has a colloid osmotic pressure (calculated)

of c. 2-5 cm. of water.

328 L. E. R. PICKEN

4. It is concluded that filtration is possible and that secretion and resorptionalmost certainly occur in the formation of the urine.

5. A microthermopile is described.6. Methods are described for measuring the hydrostatic pressure and the

colloid osmotic pressures of the body fluids in small animals.

I wish to express my indebtedness to Prof. J. Barcroft, F.R.S., for permission towork in the Physiological Laboratory; to Dr A. B. Keys for advice and encourage-ment during the most critical stages of this research; to Dr K. Smith for membranesand to Mr S. Smith for microanalyses. The work was made possible by a grant fromthe Department of Scientific and Industrial Research, and by grants for apparatusfrom the Government Grant Committee of the Royal Society.

REFERENCES

BETHE, A., VON HOLST, E. and HUF, E. (1935). Pfliig. Arch. get. Phytiol. 235, 330-4.BiAtASZEWicz, K. (1930). Acta Biol. exp., Varsovie, 5, 57-84.BOTAZZI, F. (1906). Arch. Fisiol. 3, 416-46.

(1908). Ergebn. Phytiol. 7, 161-402.VON BUDDENBROCK, W. (1928). Grundritt der vergleiclienden Phytiologie. Berlin.BURIAN, R. and DRUCKER, K. (1910). Zbl. Phytiol. 23, 772-7.

and MUTH, A. (1924). Winterstein's Handbiich der vergleichenden Physiologie, 2, 2. Hfilfte,pp. 633-95.

COLE, S. W. (1928). Practical Phytiological Chemistry. Cambridge: Heffer.ELFORD, W. J. (1931). J. Path. Bad. 34, 505-21.FREDERICQ, L. (1898).. Bull. Acad. Belg. Cl. Set., in s., 35, 831-3.GICKLHORN, J. (1931). Protoplasma, 13, 707-24.HERRMANN, F. (1931). Z. vergl. Phytiol. 14, 479-524.HILL, A. V. (1928). Proc. roy. Soc. B, 103, 117-37.

(1929). Proc. roy. Soc. A, 127, 9-19.(1931). Adventuret in Biophysics. Oxford.

KEYS, A. B. and HILL, R. M. (1934). J. exp. Biol. 11, 28-34.KROGH, A. and NAKAZAWA, F. (1927). Biochem. Z. 188, 241-58.MARGARLA, R. (1930). J. Physiol. 70, 417-33.NAGEL, H. (1934). Z. vergl. Physiol. 21, 468-91.OvERTON, E. (1904). Verh. phys.-med. Ges. Wiirzburg, 36, 277-95.PANTIN, C. F. A. (1931). Biol. Rev. 6, 459-82.SCHLIEPER, C. (1929). Z. vergl. Physiol. 9, 478-513.

(i933)- Z. vergl. Physiol. 20, 255-7.SCHLIEPER, F. and HERRMANN, F. (1930). Zool.Jb., Abt. 1, Anatomie und Ontogenie, 52, 624-30.SCHOLLES, W. (1933). Z. vergl. Physiol. 19, 533-53.SCHWABE, E. (1933). Z. vergl. Physiol. 19, 183-236.

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THE MECHANISM OF URINE FORMATION IN INVERTEBRATES