2O

THE MECHANISM OF URINE FORMATIONIN INVERTEBRATES

II. THE EXCRETORY MECHANISM IN CERTAIN MOLLUSCA

B Y L. E. R. P I C K E N

Zoological Laboratory, Cambridge, and the Freshwater Laboratory,Wray Castle, Westmorland

(Received 10 February 1936)

(With One Text-figure)

INTRODUCTION

ALTHOUGH some knowledge of the chemical nature of molluscan excretory productsand the process of excretion has been available for many years, it is not known towhat extent the excretory organs are concerned in the regulation of the salt andwater balance of these animals. The only published contributions to the subject ofosmotic conditions in freshwater molluscs are measurements of the freezing-pointof the blood in Anodonta and Limnaea made by Fredericq (1898), Botazzi (1908),and Koch (1917).

This paper is in outlook a continuation of the first of the series (Picken, 1936 a).Measurements have been made of (1) the vapour pressure of body fluids and ex-cretory fluid (urine), (2) the hydrostatic pressure of the blood, and (3) the refractiveindex and colloid osmotic pressure of body fluids and urine. In the case of thelamellibranch Anodonta, it has been possible to measure nitration rates, and tomake an estimate of the water flux in this animal. The methods used are describedin the appendices to Picken (1936 a).

THE EXCRETORY ORGANS OF THE MOLLUSCA

The reduction of the coelom in Mollusca recalls the reduction in Crustacea,but the molluscan condition differs from the crustacean in the intimate associationof the heart with that portion of the coelom drained by the nephridium. Thefollowing account of the excretory organs is based on Strohl (1924). The coelomicspace surrounding the heart is known as the pericardial cavity, and it is into thisthat the ciliated nephrostome of the nephridium opens. In certain groups, such asthe Cephalopoda and Gastropoda, the pericardium may develop glandular areas—the pericardial glands. The cilia beat in such a manner that liquid is withdrawn fromthe pericardial cavity and driven into the nephridium. Further movement of theliquid and the emptying of the nephridium are accomplished differently in differentgroups. In the lamellibranchs and heteropods the nephridium is contractile.

The Mechanism of Urine Formation in Invertebrates 21

Among the cephalopods, the respiratory contractions of the mantle probablyfacilitate discharge, while in the pulmonates, general contractions of the wholebody appear to be responsible for the movement of the liquid in the nephridium.The wall of the nephridium generally shows excretory cells, often with solid in-clusions. Frequently the nephridium has the form of a sac, and the wall may bethin, or sponge-like and of considerable thickness. In Chiton, nudibranchs andheteropods, the sac is branched, and diverticula extend throughout the body.There is always an abundant supply of blood to the nephridium.

THE EXCRETORY ORGANS OF THE LAMELLIBRANCHIATA

The paired kidneys of lamellibranchs have the form of a tube bent upon itself,and opening at one end into the pericardium, and at the other on the externalsurface of the body. The proximal portions of these coelomoducts are glandular,the distal portions thin walled and ciliated, forming reservoirs for the excretoryproduct. These bladder-like regions of the two nephridia communicate with eachother anteriorly. A large gland—Keber's Organ—discharges into the pericardium.

ANODONTA CYGNEA

The vapour pressures of blood, pericardial fluid and urine

In Table I are collected the values obtained for the concentrations of blood,pericardial fluid and urine. The animals I - I O were from hard Cambridge tap

Table I. Concentrations of body fluids in Anodonta cygnea

]

DlUUU

I2

3456789

1 0I I12

131 41516

o-o6O-OQ

O-II0-090-09I-I40 1 30 1 30-08O - I I0-070-05O-II0-05O-I20 0 8

Pericardialfluid

0-030-09o-io—

o-io—

O-I20 1 3O-O7

—O i l0-05o-io0-050-140-08

Urine

Right

0-04O'OD

—0-060 0 8

o-io—

O-II0-04

—O-O2O'O5o-ioO-O3o-io0-04

Left

—0-040-06

—

o-o6o-io——

0-08—————

o-oi

Concentrations expressed in terms of solutions of sodium chloride of equivalent vapour pressures,and accurate to ± o-oi per cent sodium chloride.

water, 11-16 were from soft Westmorland water. The low concentration of theblood is very striking, and agrees with Koch's value of A = o-O9°C. The peri-cardial fluid is in most cases approximately isotonic with the blood, but the urine is

2 2 L. E. R. PlCKEN

markedly hypotonic to the body fluid. The fluids from the two nephridia maydiffer in concentration. It is apparent that the blood and urine concentrations ofthe animals from hard water are on the whole higher than in those from soft water.

The hydrostatic pressure of the blood

In Table II the values for the hydrostatic pressures are recorded. They rangefrom 3 to 8 cm. of water. The reality of these pressure measurements was confirmed

Table II

Blood

Refractiveincrement

due tonon-mineral

solutes

0-000230-00019o-oooi80-000460-000290-000260-000250-000330-000370-000340-00033

%ofproteincalcu-latedfrom

nD 1 %protein

= 0-00172

0 1 3O-IIo-io0-370-17o i s0-150-19O-22O-2O0-19

M a x i m u mcolloidosmoticpressurein mm.of water

3-o2-42-3S-93-73-33-34-24-74'44-2

Pericardial fluid

Refractiveincrement

due tonon-mineral

solutes

0-000190-000150-000200-000460-000280-000220-000230-000250-000330-000320-00026

%ofprotein(calcu-lated)

O-II0-09O-I20-270-160-130-130-150-190-19o-i6

Maximumcolloidosmoticpressurein mm.of water

2-41-92-65'93-63-03 03-24-24-23"4

Differencein mm. of

water

o-60-50-3

o-i0-30-3i-o°'5O-2o-8

Hydro-static

pressurein cm.

of water

05488

by an accident; in opening the pericardium of a mussel, a small hole was made inthe heart, and for several seconds a jet of blood rose to a height of about 5 cm.

The refractive index measurements on blood, pericardial fluid and urine

Table II shows the refractive increment due to non-mineral solutes of the blood,the pericardial fluid and the urine from eleven animals. The refractive index isrecorded, since the actual osmotic pressures were too small to be measured with anyaccuracy by the direct method in use at the time (see Picken, 1936 a). It is clearthat the pericardial fluid contains less non-mineral substance than the blood; thisfact supports the view that the pericardial fluid is a nitrate derived from the blood.If the non-mineral substance is assumed to be protein, maximum values for possibleosmotic pressures can be calculated from the refractive increment for 1 per centalbumin (0-00172) and the graph for colloid osmotic pressure per per cent proteinin human serum, given by Krogh & Nakazawa (1927) (see Picken, 1936 a, Appen-dix 3). In all probability, the true osmotic pressures are less than these calculatedvalues (loc. cit.). Calculated colloid osmotic pressures of the blood range from 2to 6 mm. of water. The difference between blood and pericardial fluid varies fromo-i to i-o mm. of water. Sometimes the urine has a higher non-mineral refractive

The Mechanism of Urine Formation in Invertebrates 23

increment, or, as may be seen from Table II, the increment may be considerablyless than that of the pericardial fluid or blood. It is evident that the hydrostaticpressures are much greater than the maximum possible values for the colloidosmotic pressure of the blood, and are always considerably greater than the maximumdifference between blood and pericardial fluid.

(1) Material.The specimens were kept in aquaria in water from their environment.

(2) Technique.

It was impossible to dissect the animal out of its shell without loss of blood. A holewas drilled through the hinge with a |-in. bit, and the dorsal surface of the pericardiumwas exposed.

(a) The hydrostatic pressure was measured with the apparatus described elsewhere(Picken, 1936 a, Appendix 2). The wall of the pericardium was pierced by the hypodermicneedle.

(b) The pericardium was opened and the pericardial fluid removed with a pipette.(c) The heart was wiped with cotton-wool to remove surface liquid, the wall was

pierced with a hypodermic needle mounted on a glass tube, and the blood was with-drawn.

(d) The animal was removed from the shell by cutting through the anterior andposterior adductor muscles, the gills were reflexed from the foot, and the excretoryaperture was exposed. After wiping the surface with cotton-wool, and arranging swabsto prevent creeping of liquid, the excretory aperture was opened with the tip of a glasspipette, and the urine was collected. Occasionally the urine was obtained by breakingaway the edge of the shell until the mantle and gills could be reflected and the excretoryopening uncovered. Blood and pericardial fluid were collected subsequently. The resultsobtained from animals treated in this way did not differ from those from animals treatedin the first way.

(e) The refractive index of the various fluids was measured as described in Appendix 3of a previous paper (Picken, 1936 a).

(/) Vapour pressures of blood, pericardial fluid and urine were determined by theHill thermal method (Appendix 1, loc. cit.).

THE EXCRETORY ORGANS OF PULMONATES

In the pulmonates a single nephridium only is found. Considerable differencesin the diameter of the nephro-pericardial canal exist in the Basommatophora ascompared with the Stylommatophora, i.e. between aquatic and terrestrial forms.In the former the diameter is noticeably greater than in the latter (Buchner, 1891;Rolle, 1908).

Among the pulmonates the nephridial sac is characterized by the presence ofconnective tissue lamellae and trabeculae in which are blood vessels. The secretorysurface of the nephridium is thus much enlarged. There are also muscle fibres inthe lamellae so that movements of the contents may be assisted by contraction ofthe nephridium. Occasionally the whole lumen may be filled by this sponge-likedevelopment, but generally the basal portion of the sac is free from this elaboration.The blood supply of the pulmonate nephridium is extremely well developed.

24 L. E. R. PICKEN

In the several suborders the nephridium varies considerably in general form.Thus in Arion it forms a ring round the pericardium, while in Planorbis and Limnaeait is straight and narrow. The land pulmonates have a long duct from the sac, whichruns along the rectum to the exterior.

It has been assumed that the fluid separated by the pericardial glands and dis-charged into the pericardium is mostly "water" and serves to wash out excretoryproducts discharged into the nephridial lumen. Rolle (loc. cit.) has argued from thewidth of the nephrostome in aquatic pulmonates that a considerable amount of wateris eliminated by the nephridium. As his experiments represent the only attemptto estimate the amount of water eliminated by a molluscan nephridium, they areworth quoting, although, as Strohl remarks, they are "keineswegs iiberzeugend".

Rolle removed specimens of L. stagnalis from water, dried them with blottingpaper, weighed them and left them in damp air for 18-24 hours. On reweighinghe recorded a loss of 4—6 per cent in weight. This loss he interpreted as representingwater eliminated by the nephridium in that time—assuming that evaporation wasprevented by the presence of a moist atmosphere.

In the terrestrial pulmonates the contents of the nephridium are discharged atintervals of a fortnight or three weeks. The snails, e.g. Helix, produce considerablequantities of excreta in the form of particles of uric acid, 4-6 mg. in weight.Hibernating snails do not eliminate, but the cells of the nephridium become filledwith uric acid concretions.

LIMNAEA PEREGRA

In Table III are collected the values obtained from Limnaea peregra for vapourpressures of blood, pericardial fluid and urine. Botazzi (1908) gives values for thedepression of freezing-point of the liquid obtained by squashing L. stagnalis of

Table III. Limnaea peregra

Hydrostaticpressure in

cm. of water

2'OO'O3 0S'O§•04-04-0vo8-o

2 5 016-019-0——

Concentration of bloodin terms of a solution

of sodium chlorideof the game vapour

pressure. G. ofsodium chloride in

100 ex . solution

0-49o-6i0-480-410-400-41o-6i0-350-280 3 20-320 3 90-520-270-41

Concentrationof pericardial

fluid

———

O'430-440-320-390-290-250 3 2O-2O0 3 3

O-23°-54

l ^ \ T\ f* ̂ n ̂ r o in ft TI

of urine

0-420-440-36o-330 2 30-29o-3S0-230-200-29O-2OO-280-430-290-42

Concentrations accurate to ± 0-02 per cent sodium chloride.

The Mechanism of Urine Formation in Invertebrates 25

ti = 0-22-0-23° C.; these are approximately equivalent to a solution of 0-35 per centsodium chloride. The average of the values for blood in Table III is 0-43 per centsodium chloride, which is not far removed from Botazzi's figure. From the tableit is clear that the pericardial fluid is generally isotonic with the blood, while theurine is hypotonic to the blood. Table III also includes the values for the hydro-static pressure in L. peregra. The considerable range of values measured is probablydue to the great differences in the tonus of the body wall in different animals at themoment of measuring the pressure. The xanthoproteic test gave positive resultson blood, pericardial fluid and urine.

LIMNAEA STAGNALIS

The results of these experiments are shown in Table IV. The calculations ofcolloid osmotic pressure were made as in the case of Anodonta. There is obviously

Table IV. Limnaea stagnalis

Colloid osmotic pressure incm. of water (calculated)

Blood

2-I2-73-13-23'ia-o1 "42-1

Pericardialfluid

0-3o-o03i-o07I-I1-20-7

Difference

i-82-71-82-22-409O'21-4

Hydrostaticpressure in

cm. of water

11

853

Colloid osmoticpressure in cm.

of water.Urine

1-2

7-4

2-72-2

a considerable difference between the protein content of the pericardial fluid andthat of the blood. The hydrostatic pressure may be considerably greater than thisdifference.

Limnaea spp.

(1) Material.In the first experiments specimens of L. peregra were used. These were collected from

a pond in Westmorland and kept in pond water. Later experiments were made on L.stagnalis in Cambridge.

(2) Technique.

The shell was broken away with forceps, care being taken that the mantle was nottorn in so doing and the heart and pericardium thus injured.

(a) The measurement of hydrostatic pressure. The snail was placed on a microscopeslide and allowed to expand and creep. When the head was extended the needle wasinserted into the haemocoel behind the buccal mass.

26 L. E. R. PICKEN

(b) The collection of blood, pericardial fluid and urine. A cut was made through the lineof junction of the anterior mantle edge with the rest of the body. The mantle was thenreflected over the visceral mass and pinned down, exposing on the uppermost side thepericardium, heart and nephridium. The whole of this surface was carefully wiped withabsorbent wool to remove mucus and any water which might contaminate the fluids asthey were collected.

For collecting the fluids, fine pipettes were used drawn from i f m m . internal boreglass tubing and washed with distilled water and acetone. The heart could be seen beatingwithin the pericardium which always appeared distended with fluid. A pipette wasinserted into the pericardium and as much as possible of the pericardial fluid was removed.

The pericardium was opened with fine scissors and the wall laid back from the heartso that the remaining pericardial fluid could be wiped away with cotton-wool. A secondpipette was used to puncture the heart and withdraw the blood, which was usually notmore than o-i mm.3 in volume. In L. stagnalis blood was also collected from the haemocoel.

From the heart the yellow nephridium runs down the mantle edge to open at thepneumopore. An incision was made in the portion distal to the heart and the nephridialfluid collected in a third pipette.

The three samples of fluid were kept in a small test-tube plugged with cotton-wool.Since the fluid was contained in the capillary portion of the pipette, the ratio of surfaceexposed to volume was very small and hence the risk of evaporation changing the con-centration was reduced.

(c) The measurement of colloid osmotic pressures. The colloid osmotic pressures werecalculated from the refractive increment and the total concentration of the sample (seedescription for Anodonta).

(d) Vapour pressure measurements were made with strips of cigarette paper (seeAnodonta).

THE RATE OF FILTRATION IN ANODONTA

Since the maximum estimated values for the colloid osmotic pressure of theblood are so much lower than the hydrostatic pressures set up in the organism, itwas suspected that the rate of filtration of liquid into the pericardium is very rapid.Attempts were made to measure the rate of filtration. In the first experiments thepericardium was opened by a median dorsal longitudinal incision, and the twoflaps of the dorsal wall were turned back exposing the heart. The liquid in thepericardium was drained away and the cavity was rapidly wiped with cotton-wool.As the heart continued to beat, liquid collected in the pericardium and was removedin a pipette. The chief difficulty in this method was due to the creeping of liquidover the surface and in particular the creeping of blood from the cut edges of thepericardial wall. Alternatively a glass cannula was inserted into the pericardialcavity, but again there was the risk of bleeding from the edges of the incision (thefragility of the tissues makes it impossible to ligature round the cannula) or loss ofliquid through the imperfect fitting of the cannula into the incision. Finally acoarse hypodermic needle (about 1-5 mm. bore) was mounted in such a way thatit could be passed into the pericardium anterior to the auricles and tilted so thatthere was a difference in level of about 0-5 cm. between each end of the needle. Alength of about 2 cm. of glass tubing was sealed on to the needle. The arrangementof the experiment is shown in Fig. 1. Since there was no danger from the surfacecreeping of liquid, the animal could be partly submerged in water, the temperature

The Mechanism of Urine Formation in Invertebrates 27

of which was maintained at approximately the same level as that of the aquariumtanks. A piece of cotton-wool soaked in water covered the opening in the shell.Since the experiments were done in winter there was no need for more elaboratetemperature control; the temperature of the room was never more than a degreehigher than that of the bath. When the needle was slipped into the pericardium,fluid began to drip from the end of the glass tube. This was collected continuouslyfor periods of 5 min. at 10-min. intervals in a pipette discharging into a small flask(ca. 25 c.c.) connected to a filter pump. Table V shows the average volume ofliquid collected during 5 min. at 10-min. intervals over a period of at least 1 hour.

Hypodermic needlepassing into pericardium

•Water level

Exposed body surface

Glass tube

Fig. 1

In some cases liquid could be collected after the preparation had been filtering foras long as 4 hours, but generally the rate of nitration fell off considerably after thefirst half-hour. This is not generally due to failure of the heart beat, but is probablydue to the reduced rate of water uptake into the animal through the body surface.The liquid collected from the pericardium would normally pass through the ne-phridia and (since the urine is hypotonic to the blood) salt would probably beresorbed into the blood. There is therefore a great loss of salt when the pericardialfluid is collected. This will result in a fall in the osmotic pressure of the blood and aslowing down of the rate of water uptake. In support of this interpretation of thedecline in filtration rate is the fact that after perhaps an hour's continuous collectionfrom the pericardium, the volume of the heart is very much reduced. This maybe due to a reduction in the total blood volume, which is to be expected if the rateof water uptake diminishes more rapidly than the decline in mechanical efficiencyof the heart.

28 L. E. R. PlCKEN

Since there is a possibility of a tonic contraction of the pericardium squeezingliquid into the needle and so augmenting the apparent rate of filtration, plasticenemodels of the pericardium were made from several individuals; the volume of themodels was determined by displacement. The average volume was about 3 c.c. andthe values ranged from 2-5 to 3-5 c.c. These models were made to the externaldimensions of the pericardium and therefore included the volume of the heart.Hence the maximum volume of pericardial fluid at the moment of inserting thehypodermic needle must be less than 3 c.c. It is obvious that the figures for thevolume of liquid filtered over long periods can be only slightly influenced by anyslow tonic contraction of the pericardium. From Table V it is seen that the averagerate of filtration at a temperature of approximately 170 C. is about 1 c.c. in 5 min.

Table V

Technique

(1) Opened pericardium andcollected liquid accumulatingin pericardium

(2) Collected through glasscannula

(3) Collected through hypo-dermic needle. Animal partlysubmerged in water

Averagevolumeof liquidcollectedin 5 min.

c.c.

0-41

0-04o-o80-04C 1 42-561-030-750-310-97——

Totalvolume

collectedc.c.

—0-310-3

2 1

631

22 21 2

Periodin whichcollected

hours

—o-6i -o—

o-7so-S0-30-150-153"3i -o

Volumefiltered

in24 hours

(calculated)c.c.

118

—124

72

670288216144288160288

TIT i

Wetweight

8-

45

3459

33

4964495363

—

»r-«

1 em-perature

Of

—

—

18-0—

i7'617-8l8"2i6-51 6 518-117-8

This means that the animal will lose 288 c.c. of urine in 24 hours. Of the liquidfiltered, a small volume will of necessity be resorbed as water of hydration with thesalt resorbed, but it is improbable that there will be considerable resorption ofwater, since this would only increase the waterlogged condition of the tissues. Theaverage wet weight of the animal (without its shell) is about 50 g. It is thereforepossible that at a temperature of 180 C. Anodonta cygnea can excrete approximatelysix times its own weight of water in 1 day.

THE SALT UPTAKE IN ANODONTA

If the urine has an average concentration of 0-06 per cent sodium chloride andthe average daily output of the excretory organs is about 250 c.c. of fluid, the totalloss of osmotically active substances will be equivalent to ca. 0-15 g. of sodiumchloride. It has been estimated that the volume of water passing through thesiphons of a mussel in 24 hours is ca. 40 gallons, i.e. approximately 214 litres. From

The Mechanism of Urine Formation in Invertebrates 29

Ruttner's figures (1913-14) for the number of unicellular algae in 10 c.c. of lake waterit is possible to make an approximate calculation of the amount of salt obtainablefrom the food ingested in 1 day (assuming (1) that the animal removes all algae fromthe water passing throughiits siphons, (2) that the phytoplankton is the chief con-stituent of the food, and (3) that the valves are open for 24 hours). In making thecalculatiori it is supposed that the concentration of the cell sap of unicellular algaeis the same as that of fresh-water Protozoa, i.e. ca. 025 per cent sodium chloride(mean of estimates by Gelfan (1928), Kamada (1935), and Picken (1936 b)). Thevolume of the algal cells has been estimated approximately by treating them ascylinders having length and diameter equal to the maximum length and diameterof the cell. Having determined the volume of unicellular algae in 10 c.c. of lakewater, the volume in 214 litres was found, and without making any correction forthe volume of the cell wall, the volume of plant cells in 214 litres was consideredas a certain volume of 0-25 per cent sodium chloride solution, and the weight ofsodium chloride absorbed as food in 24 hours was calculated. The details of thecalculation appear in Table VI. The dimensions of the algae are mean values taken

Table VI

Number of unicellularalgae in io c.c. of lake

water (Ruttner)

Chrysomonads 600Mallomonas 12Gymnodinium 30Cyclotella 1320Staurastrum 240Oocystis 18

Dimensionsof cell in fi

6-5. n-514. 4528, 36-520, 695. 12511, 20

Volumeof cell in /i3

3826,93O

22,5001,885

889,0001,900

Volumein 10 c.c. p3

2-3 x 10s

08 x io5

6-8 x i o '25-0 x 10'

21300 x io1

0-3 x io5

Total volume in 10 c.c. = 2-17 x io9 /J'.Volume in 214 litres = 4-65 cm.*

Daily salt intake in food = 4-65 c.c. of a solution of 0-25 per cent sodium chloride= 0012 g. of sodium chloride.

from Fritch & West's British Fresh-water Algae (1927). The total volume in 214litres is approximately 5 c.c. If this is considered as 5 c.c. of a 0-25 per cent solutionof sodium chloride the weight of salt ingested as algae in 1 day is ca. 1-25 x io~2 g.

THE WATER CONTENT OF THE MUSCLES IN ANODONTA

In view of the extraordinarily small amount of protein in the blood and theprobable correlation of this with the state of hydration of the tissue proteins, thewet and dry weights of the posterior and anterior adductor muscles were determinedfor each of three mussels. The muscles were dissected out, scraped free of othertissue, freed of superficial liquid by squeezing between filter papers until no moreliquid could be removed, and dried to constant weight in an electric oven at 95 ° C.For comparison with these, three pairs of gastrocnemii from three frogs were alsoweighed and dried. From these determinations an average value for the percentage

30 L. E. R. PICKEN

dry weight of Anodonta muscle of 17-06 was obtained. The value for frog's gastroc-nemius was 20-38. If the dry weight of a given wet weight of frog's muscle is 100 g.the equivalent dry weight of the same wet weight of Anodonta muscle would beonly 86 g. It is evident that Anodonta muscle contains considerably less solid thanfrog's muscle, and since the proteins are the most important solid constituent theconcentration of protein in Anodonta muscle must be less than the concentration infrog's muscle.

DISCUSSION

Salt and water balance in the Mollusca

The experiments on Anodonta and Limnaea show that the pericardial fluid isgenerally isotonic with the blood but contains less non-mineral substance con-tributing to the refractive index; moreover, the blood has a considerable hydro-static pressure. It is reasonable to suppose that the pericardial fluid is a filtratefrom the blood through the wall of the heart. In Anodonta and Limnaea peregra ithas been shown that the urine is hypotonic to the blood and to the pericardial fluid;that is to say, the pericardial fluid having passed through the nephrostome into thenephridium loses salt. (It is possible, but improbable, that water is resorbed fromthe liquid as it passes along the nephridium.) The hypotonicity of the fluid may bedue either to the resorption of salt or to the secretion from the wall of the nephridiumof a solution hypotonic to the blood—there is perhaps only a verbal distinctionbetween these two processes. Whatever may be the mechanism by which hypo-tonicity arises, the excretory organs are certainly doing osmotic work in producinga relatively dilute urine.

Until something is known of the rates of water movement through the excretoryorgans it is impossible to assess their importance in the regulation of salt and waterbalance. Furthermore, without an estimate of the contribution of the food to thesalt store of the body it is impossible to decide how much of the total salt exchangeis to be ascribed to this source and how much to osmotic work at the surface betweenorganism and environment. In Anodonta it has been possible to make roughestimates both of the rate of movement of water through the animal and of the gainin salt from food ingested. The daily salt loss through the excretory organs has beenestimated (see p. 28) at 0-15 g. of sodium chloride. In Table VI it is shown thatthe daily salt intake from food ingested is probably about 5 c.c. of a solution of0-25 per cent sodium chloride, that is ca. 0-0125 £• °f sodium chloride. It is obviousthat more salt is lost in the urine than can be replaced by the fobd ingested in agiven period; that is to say, that if the animal is to maintain the hypertonicity of itsblood to the external medium, salt must be taken into the system at some point asyet unknown. In other words, osmotic work must be done at a surface other thanthat of the excretory organs.

If the ability to perform osmotic work is a fundamental property of the wholeof the surface, then the excretory tubes, in resorbing salt from the liquid bathingthe morphologically external surface, are only doing something which takes place

The Mechanism of Urine Formation in Invertebrates 31

over the whole of the external surface. Under such circumstances the liquid takenup by the blood from the surrounding medium will not be isotonic with the latter,but hypertonic to the environment. In the case of the excretory organs, whosesurfaces do work to maintain a concentration difference between that face of theepithelium in contact with the excretory fluid and that in contact with the blood,the "external" medium is the pericardial fluid and the work done is sufficientto produce a urine the concentration of which is equivalent to 0-06 per cent sodiumchloride and a blood with a concentration of o-io per cent sodium chloride. Sup-pose that the osmotic work done at the external body surface is of the same order asthat at the surface of the excretory organs, then the work at the excretory organswill be proportional to

, (concentration of internal medium) . o-io° (concentration of excretory fluid) ~ ^ 0-06'

while at the external surface, since the external medium has a concentrationapproximately equivalent to 0-02 per cent sodium chloride, the concentration ofthe solution absorbed is given by

. o-io . xlog —,- = log ;

6 0-06 6 0-02

that is x = ca. 0-03 per cent sodium chloride.If 250 c.c. of fluid are excreted daily, the same volume must be absorbed, and

if we suppose that this solution is concentrated during absorption, it follows that250 c.c. of a solution of 0-03 per cent sodium chloride are taken in daily. This willlead to a gain of 0-075 £• °f sodium chloride. We now have a balance sheet for theexchange of osmotically active substance expressed in equivalent concentrations ofsodium chloride.

Loss in excretion Gain015 g. °'°75 through surface

coi2 in food0087

Deficit = 0063 g.It is clear that either the salt contribution of the food has been underestimated

or that the surface is doing considerably more work than the excretory organs.The estimate of the food ingested was based on figures for lake water; it is probablethat the phytoplankton of ponds is much more abundant than that of lakes. Toaccount for the deficit, the volume of phytoplankton ingested would have to beincreased from 5 c.c. in 214 litres to about 25 c.c. It may be argued that the hypo-thesis of what might be called (by analogy with the marine teleosts) extrarenal saltresorption, is unnecessary and that the whole of the salt lost may be replaced fromthe food. But it is certainly impossible that the food is the only source of salt sincestarved animals will live for months in aquaria supplied with running tap water(and therefore containing very little in the way of micro-organisms) and will main-tain the blood hypertonic to the external medium. On the other hand there issome evidence for the dependence of the concentration of the blood on that of the

32 L. E. R. PICKEN

external medium. From Table I it can be seen that the average concentration ofthe blood is o-o8 per cent sodium chloride in specimens 11-16 from soft water, ascompared with o-io per cent sodium chloride in the specimens 1-10 from Cam-bridge tap water (the concentration of which is equivalent to ca. 0-02 per centsodium chloride). The urine in the Westmorland forms has an average concentration

of 0-05 per cent sodium chloride. Log has then the value of 1-2241, while in

the case of the specimens from Cambridge tap water, log —7 = 1-2218. The ratioO'OO

is approximately constant, but the osmotic pressure of the blood is lower in thespecimens from soft water.

The state of hydration of the muscle proteins

The recognition of the fact that in spite of the extremely low concentration ofprotein in the blood, Anodonta is able to preserve its tissues from oedema, leads tothe conclusion that the state of hydration of the tissue proteins is not entirelycontrolled by the colloid osmotic pressure and hydrostatic pressure of the bodyfluid. It has hitherto been tacitly supposed that the tissue proteins are in equilibriumwith the body fluids so that an increase in hydrostatic pressure or a fall in colloidosmotic pressure of the body fluid will lead to oedema of the tissue. If, however,the percentage of protein in the muscles is determined, this is found to be verymuch larger than would be possible if the muscle cell were simply a solution ofproteins bounded by a membrane impermeable to proteins but permeable to waterand other solutes. In the case of Anodonta the body fluid contains approximately0-2 per cent of protein; if equilibrium were established between the tissues andthe blood, the concentration of protein in the tissues would fall to this level and it isdifficult to imagine any organization persisting at such a low protein concentration.In particular it is difficult to suppose that a condensed protein structure, such as ispresent in a muscle fibre, could be developed in so dilute a solution of protein.Since the concentration of protein in the muscle appears to be much higher thanthat in the blood, there is reason to suppose that the distribution of water betweenthe cell proteins and the body fluid is controlled by factors other than the hydro-static pressure of the body fluid and the concentration of protein in cell and bodyfluid. There is perhaps an active control of the state of hydration of the tissueproteins.

The figures in Table VII are taken from Tabulae Biologicae; in the first columnare given the percentages of water in various muscles. The second column containsthe percentage of protein in the muscles. In the third column the values of the

percentage of water , „ ., c . , ,ratio — -—7 •- are shown. From these figures it appears that the

percentage of protein ° r r

percentage of water in the muscles falls in the higher vertebrates as compared withthe lower, and in the vertebrates as compared with the invertebrates. This de-hydration of the muscle proteins is shown clearly in the third column. It appearsthat not only is the distribution of water in the tissue proteins controlled by some

The Mechanism of Urine Formation in Invertebrates 33

factor other than hydrostatic pressure and the colloid osmotic pressure of the bodyfluid, but that this control increases and is in some way linked with what is called"evolution".

The scheme of salt and water balance outlined above explains many of theproperties of molluscan bodies; the impression of fragility, the ease with which thetissues disintegrate, the excessive " wateriness" of the tissues, and the rapidity with

Table VII (from Tabulae Biologicae)

Muscle

Ox (lean)Ox (heart)HenSalmonHerringPlaicePerchSkateCodLobsterOyster

Percentageof water

76-476634722267-0175 °978-3579-48801381-5076618038

Percentageof protein

20-5619-3521-331973I6-II187118-531800169318-31I3-3I

Percentage of waterPercentage of protein

3-73-43-43-44-74-24-34-54-94-26-o

which the body fluid is squeezed out of the animal, are all in keeping with the lowcolloid osmotic pressure and with the fact that the hydrostatic pressure may reachvalues very much greater than the colloid osmotic pressure of the blood. The grossmechanical properties of the animal—the molluscan "facies"—are the consequencesof the relative magnitudes of hydrostatic pressure and colloid osmotic pressure,and the state of hydration of its tissue proteins. In measuring these we measurean imponderable—that quality which stamps a mollusc almost as certainly as anymorphological criterion.

SUMMARY

1. In Anodonta cygnea:(a) The blood has a vapour pressure equivalent to that of a solution of ca.

o-io per cent sodium chloride.(b) The pericardial fluid is isotonic with the blood.(c) The urine has a vapour pressure equivalent to that of a solution of ca.

0-06 per cent sodium chloride.(d) The hydrostatic pressure of the blood is ca. 6 cm. of water.(e) The calculated colloid osmotic pressure is ca. 3-8 mm. of water.(/) The average rate of filtration of fluid into the pericardium is ca. 250 c.c. in

24 hours.(g) The salt uptake from ingested phytoplankton is estimated as equivalent to

0-012 g. sodium chloride in 24 hours.(h) The loss of osmotically active substance in the urine is estimated as equivalent

to 0-15 g. sodium chloride in 24 hours.JEB-XJVi 3

34 L. E. R. PICKEN

2. In Limnaea peregra the vapour pressure of the blood is equivalent to thatof a solution of ca. 0-43 per cent sodium chloride. The pericardial fluid is isotonicwith the blood, and the urine has a concentration equivalent to ca. 0-30 per centsodium chloride.

3. In Limnaea stagnalis the hydrostatic pressure of the blood is ca. 8 cm. ofwater. The colloid osmotic pressure of the blood is ca. 2-5 cm. of water (calculated);that of the pericardial fluid is ca. 0-7 cm. of water.

REFERENCES

BOTAZZI, F. (1908). Ergebn. Pkytiol. 7, 161-402.BUCHNER, O. (1891). Jh. Ver. vaterl. Naturk. Wiirttemb. 47.FREDERICQ, L. (1898). Bull. Acad. Belg. Cl. Sci. HI ser. 35, 831-3.GELFAN, S. (1928). Protoplama, 4, 192-200.KAMADA, T. (1935). J. Fac. Sci. Tokyo Univ. (4) Zoo!. 4, 49.KOCH, W. (1917). Pfltig. Arch. get. Physiol. 166, 281.KROGH, A. & NAKAZAWA, F. (1927). Biochem. Z. 188, 241-58.PICKEN, L. E. R. (1936a). J. exp. Biol. 13, 309-28.

(19366) J. exp. Biol. 13, 387.ROLLE, G. (1908). Z. Natunu. 43, N.F. 36.RUTTNKR, F. (1913-14). Int. Rev. Hydrobiol. 6, 518-27).STROHL, J. (1924). Winterstein's Handb. vergl. Pkytiol. 2, 2. H&lfte, 443.Tabulae Biologicae, Berlin (1926), 3, 438.WEST, G. S. & FRITSCH, F. E. (1927). A Treatise on the Freshwater Algae. Cambridge.