J. Exp. Biol. (1965), 43, 385-395 3 8 5With 1 plate and 4 text-figures

Printed in Great Britain

THE SITE AND PERMEABILITY OF THE FILTRATIONLOCUS IN THE CRAYFISH ANTENNAL GLAND*

BY LEONARD B. KIRSCHNER AND STANLEY WAGNER

Department of Zoology, Washington State University, Pullman, Washington

(Received 19 February 1965)

INTRODUCTION

Study of the physiology of excretory organs in the invertebrates has lagged farbehind that in vertebrates, and questions for which answers have long existed for thelatter are still moot for most forms. Recent research on the crayfish antennal glandhas shown that this organ operates on the same general plan as the vertebrate kidney;a primary ultrafiltrate formed somewhere in the organ is modified by reabsorption,and possibly by secretion, to form a final urine (Riegel & Kirschner, i960). Thusinulin is excreted by the crayfish, the urine concentration being usually higher thanthat in blood. By analogy with the vertebrate kidney the concentration of this com-pound may be attributed to the reabsorption of water somewhere in the tubularlumina. Glucose is not normally excreted, but appears in the urine when bloodconcentrations exceed about 200 mg. per cent. Animals treated with phloridzin alsoexcrete glucose, and the maximum urine to blood concentration ratio (U/B) is aboutthe same as for inulin. It also appears that chloride reabsorption (Peters, 1935;Riegel, 1963) and dilution of the urine (Riegel, 1963) occur in the tubular portion ofthe antennal gland. Some evidence also points to the urinary bladder as a site ofdilution by solute reabsorption (Kamemoto, Keister & Spalding, 1962). Each of thesephenomena has also been described in the kidneys of fresh-water vertebrates (e.g.Smith, 1950), suggesting the analogy in function mentioned above.

Little is known, however, about the details of antennal-gland physiology. Forexample, the filtration locus has not been demonstrated, although the coelosomac issometimes cited because, like Bowman's capsule in the vertebrate nephron, it is themost proximal portion of the organ. And we have no data on the ' porosity' of thefiltration site; that is, on the molecular size compatible with free filtration. The sitesand mechanisms by which most of the filtered material is reabsorbed are unknown,and we are ignorant of what compounds may be added by secretion from blood totubular urine.

As a result of intensive studies on the vertebrate kidney during the past 30 years wepossess techniques for approaching many of these problems. The availability ofradioactive inulin may prove to be particularly useful. There exists a body of evidencethat the behaviour of this compound is similar in both the antennal gland and thevertebrate kidney; that is, that it is filtered into the tubular lumen, is excluded from

• This investigation was supported by funds for medical and biological research, State of WashingtonInitiative Measure 171, and by a grant (G12471) from the National Science Foundation.

25 Exp. Biol. 43, 2

386 LEONARD B. KIRSCHNER AND STANLEY WAGNER

the cells, and is neither secreted nor reabsorbed after filtration. Thus, in severalspecies of crayfish:

1. Inulin is excreted in the urine, and the U/B is usually 2-3 (Riegel & Kirschner,i960). The U/B for glucose in phloridzinized animals is also about 2-3, indicating thatthis value may be characteristic of a compound that is freely filtered but not reabsorbed.

2. The U/B is essentially constant when the inulin concentration of the blood isvaried over three orders of magnitude (Riegel & Kirschner, i960).

3. Micropuncture studies show that the inulin concentration of the tubular fluidin the coelomosac and labyrinth is nearly the same as that in blood. This situationmust obtain if free filtration occurs in one of these regions (Riegel, unpublishedexperiments).

Such observations indicate that inulin may be used as a reference compound instudies on the antennal gland as in the vertebrate kidney. The behaviour of othercompounds in various regions of the organ may be assessed by comparing theirconcentrations with the concentrations of inulin either in the tubular fluid taken bymicropuncture or in extracts of entire regions. Thus if a test-compound is injectedinto the blood the ratio of its blood concentration to that of inulin ought to remainunchanged in any portion of the antennal gland that treats both compounds in thesame fashion. If the ratio of the test-compound to inulin is not the same as in theblood the behaviour of the two compounds obviously differs in this region, and thenature of the difference may provide information about the organ's role in handling thetest-compound.

The observations described in this paper are concerned exclusively with questionsconcerning the nitration site. They suggest that the primary ultrafiltrate is, in fact,formed in the coelomosac, and provide an estimate of the permeability of the filtrationlocus.

METHODS

Most of the animals were specimens of Pacifastacus sp. collected locally; a fewexperiments were conducted on Orconectes virilus obtained from a commercialsupplier. They were stored at 150 C. in aerated tap-water and were fed twice weekly.During the period of storage the animals lived in a large porcelain holding tank andwere viable for at least 5 months under these conditions. Individuals isolated forexperiments were placed in small plastic boxes containing about 500 ml. tap-water.Test-compounds were injected into the sternal sinus, and blood and urine sampleswere taken as described in Riegel & Kirschner (i960).

In many of the experiments described the antennal glands were dissected to providepieces of coelomosac, labyrinth and tubule. The animals were packed in chipped iceto immobilize them, the antennal glands were quickly removed and samples of tissuewere taken from the three regions. Both glands could be dissected under a binocularmicroscope in 5-7 min. Contrast between coelomosac and tubule (which are physio-logically separated by the labyrinth, but morphologically contiguous) was enhancedby prior injection of Evans's blue (ioo/jg./gm. animal) since the dye becomes con-centrated in the coelomosac (cf. Results). The labyrinth was easily distinguished byits natural green colour. Most tissues were homogenized in and deproteinized bySomogyi's reagent (Somogyi, 1930), but those prepared for experiments with serumalbumin were treated differently, as described below.

Filtration locus in crayfish antemtal gland 387

Radiochemical procedures

One group of experiments involved a study of the pattern of excretion of polymersof different sizes and polarity. The compounds used were inulin-carboxy-^C, twodextrans labelled with MC (one with a molecular weight range of 15,000-20,000, theother 60,000-90,000), and human serum albumin labelled with 126I. In these experi-ments blood and urine samples were taken, and aliquots of known volume wereplated on aluminium planchets with no further treatment. After drying they werecounted with a gas flow GM tube through an ultra-thin window. The very low energyX-ray emitted by m I was found to be detected more efficiently by the GM tube thanby a scintillation crystal.

Some experiments involved the use of pairs of isotopes. For those in which 14C and3H were used aliquots of the deproteinized extracts were pipetted into Bray's solution(Bray, i960) and counted in a two-channel, liquid scintillation counter. Standardswere prepared in the same way for every experiment, hence there was no need toapply quenching corrections. Tissues containing mI-labelled protein and 14C-labelledcarbohydrate were treated as follows. One aliquot of an aqueous extract of tissue(blood, antennal gland or urine) was plated on an aluminium planchet, dried, and thetotal isotope (12SI + 14C) was counted with the thin-window GM tube. A second samplewas deproteinized as described above and centrifuged to remove the protein. Analiquot of the supernatant fluid was plated, dried, and counted with the GM tube.Nearly all of the remaining counts were due to 14C dextran, but a small correction wasapplied for m I remaining in the supernatant. This quantity was determined in aseparate experiment by dissolving the albumin and measuring the fraction remaining insolution after precipitation. In ten trials the range was 0-7-2-6% of the total. The meanvalue (1 -4 ± 0-5 %) was used as the correction factor. It was appreciable only in extractsof the coelomosac.

Microscopic observations

Proteins conjugated with dyes were observed microscopically in order to delimittheir distribution in the antennal gland. Animals were injected with Evans's blue(which becomes bound to blood protein), or with goat globulin conjugated withfluorescein. Both antennal glands were excised between 2 and 24 hr. later andimmediately plunged into isopentane cooled to — 160° C. with liquid nitrogen. In theearly experiments, including all those with Evans's blue, the organs were embeddedin paraffin in vacuo, and the paraffin sections were mounted conventionally, i.e. byfloating on warm water. This resulted in loss of most of the dye (see section onResults). Dry mounting (Branton & Jacobson, 1962) produced too much tissuedistortion in our hands, but the following technique was found to give excellent results.The frozen organs were either dried as above or were transferred to vials of anhydrousacetone at — 500 C. and subjected to desiccation by freeze-substitution (Pearse, 1961)for 6-9 days. The tissues were then fixed by transferring them, still in acetone, to4° C. overnight. They were then embedded in paraffin (m.p. 52-55° C). Sectionswere mounted on slides by the method suggested by J. F. Danielli (Harris, Sloane &King, 1950). After sectioning, ribbons were placed on warm (40° C.) mercury untilthey flattened. An albumin-coated slide was then placed over the sections and permittedto float there for about a minute. The slide was then lifted from the mercury with

25-2

388 LEONARD B. KIRSCHNER AND STANLEY WAGNER

flattened sections adhering. After deparaffining, a drop of fluorospar was added beforeplacing the coverslip on the slide. Sections were studied with a Leitz Ortholuxmicroscope fitted for either phase-contrast or ultraviolet optics.

RESULTS

Excretion of polymers

To attempt to estimate the permeability of the filtration site a series of experimentswas run with a group of polymers of increasing size. Text-fig, i shows the time-coursefor excretion of inulin (M.W. 5000) as a function of time. The behaviour of dextranof low molecular weight (15,000-20,000) (LMWD), shown in Text-fig. 2, is very

5s§c

50

40

30

20

10

-

-

X -

-

X—

-

48

24

l

-

-

O

I

2

~ ^

I

i

4

~ ~~x

x

1

o1

6

i

12 24 36 48

Hours

60 72 84

Text-fig. 1. Concentration of inulin in blood (— x —) and urine (— x —) following injectioninto the ventral haemocoel at o hr. The inset shows the time-course during the first 6 hr. TheU/B varied between z-i and 2'5 during the period 24—72 hr.

similar. In a series of ten determinations on three animals the mean U/B for thisdextran was 3-0+1-5 (s.D.). In a similar experiment using inulin the mean U/B fortwenty-three experimental periods in ten animals was 2-2 ± 1-2 (s.D.). The differencebetween mean values for LMWD and inulin is not significant (o-i > P > 0-05), andhence the compounds appear to behave similarly.

Text-fig. 3 shows the excretion pattern for a larger dextran (HMWD) with amolecular weight range of 60,000-90,000. This compound too is excreted, and as withthe LMWD the concentration in the urine exceeds that in the blood. Although thegeneral pattern of excretion resembles that of the other two, the U/B appears to besomewhat lower. In a series of sixteen determinations made on three animals themean U/B was 1-35 + 0-70 (s.D.). The difference between this value and the mean U/B

Filtration locus in crayfish antennal gland 389

for LMWD or inulin is statistically significant (P < o-oi), although the ranges ofvalues overlap.

These data suggest that filtration of compounds as large as HMWD may berestricted, but the use of small groups of animals made it desirable to examine thequestion by another method. Four animals were injected with a mixture of HMWD-14Cand inulin-3H. The animals were run in pairs at different times; two were O. virilus,the other two Pacifastacus sp. Blood and urine samples were removed as nearlysimultaneously as possible and assayed for the isotopes. Another three animals (twoO. virilus and one Pacifastacus sp.) were injected with LMWD-14C and inulin-3H. The

eo8

&

125

100

75

50

-

I " 11

I

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— • — ,

I 1 1 1 I

48 72

Hours

Text-fig. 2. Concentration of low molecular weight dextran in blood (— •—) and urine(— • — ) following injection of 052 mg. into the ventral haemocoel. The U/B varied between2-8 and 6-o during the period 24-72 hr.

results are shown in Table 1. For each group the mean ratio of 14C/3H (i.e. dextran/inulin) in blood is shown in column 4. If both compounds are treated identically thisratio should be unchanged in the urine. The data in columns 5 and 6 show that this isnearly true for LMWD-injected animals, but not for the others which excreted theHMWD only about 69% as fast as inulin.

Excretion of protein was even more restricted. Text-fig. 4 shows the urine and bloodconcentrations of human serum albumin-126! following its injection into the haemocoel.It can be seen that the concentration in the urine was always well below that in blood.Three animals were injected with labelled albumin, and the mean U/B in sevenmeasurements was 0-50 ± 0-18 (s.D.). In no case was the concentration in the urine asgreat as that in blood. Goat serum globulin (MW about 170,000) labelled withfluorescein was injected into four animals and no fluorescence appeared in the urine.In addition, Evans's blue was injected into a series of animals but was not excreted

39° LEONARD B. KIRSCHNER AND STANLEY WAGNER

in the urine even when the blood concentration was so high that a dilution of 20-foldwas necessary to obtain a spectrophotometric reading. This dye is known to be boundto plasma protein in vertebrates, and appears to be bound in crayfish blood sincedeproteinization left none in the supernatant fluid (unpublished observations).

1200 -

1000 -

Text-fig. 3. Concentration of high molecular weight dextran in blood (—O—) and urine(—O—) following injection of 3-0 mg. into the ventral haemocoel. The U/B varied between0-7 and 1-7 during this experiment. The first urine sample was taken early and the low con-centration may have been the result of dilution by bladder urine formed before the dextran wasinjected.

Table 1. Blood and urine concentration of dextran and inulin

(See text for description of the experimental protocol. The U/B ratio (column 6) is a ratio ofdextran/inulin values, i.e. column 5/column 4.)

Dextran

HMWDLMWD

No. ofanimals

43

No. ofsampleperiods

167

Dextran : Inulin ratios

Blood Urine

2 0 4

U / B ± S . D .

o-6o±o-i90-92 ±0-24

Concentration of polymers in the coelomosac

Although Evans's blue was not excreted in the urine it appeared in the antennalgland as shown in PI. 1, fig. 1. In this experiment the gland on the left was taken froman untreated animal. The coelomosac shows as a darker region near the centre of thedorsal surface. It is light brown, and stands out from the surrounding tubule, whichis white. The outer rim of the organ, shown in the photograph as a very light border,

Filtration locus in crayfish antennal gland 391

comprises the labyrinth, which is distinctly green. The gland on the right was takenfrom another animal which had been injected with 35 fi\. of a 0-5% aqueous solutionof Evans's blue. The organ was removed 24 hr. after injection and immediatelyimmersed in a physiological saline. The dye was perceptible over the entire surface,but was obviously very concentrated in the coelomosac. This suggested either that thecoelomosac is more vascular than other regions, or that the dye concentration was

100 -

.5 50 -

.a<

Fext-fig. 4. Concentration of albumin in blood (—O—) and urine ( O—) followinginjection of 0-024 m g - into t n e ventral haemocoel. The U/B was 052 at 24 hr. It was o-6iafter 98 hr. when the blood concentration had fallen to 184 /ig./ml.

Table 2. Albumin-inuKn ratios in blood and antennal gland

(See text for experimental protocol. Values for the ratios have been normalized to a bloodvalue of i-oo in order to facilitate comparison of the two animals.)

AnimalTime

hr.

2-5

6o

Albumin : Inulin ratios

Gland

Right!Left JRight)Left J

Blood Coelomosac Labyrinth

(47-W'(17-I 12-

3o

73

o-45° 5 3o-6oo-57

Tubule

0-58o-57

1-29

higher in this compartment than elsewhere. Since Evans's blue is bound to proteinthe second alternative would indicate that concentration of protein had occurred. Totest this possibility two animals were injected with a mixture of uC-labelled inulinand mI-labelled human serum albumin. Each animal received o-io ml. of a solutioncontaining 2-2 fiC 14C and 13-1 fiC. m I . One animal was sacrificed z\ hr. later, theother 6 hr. In both cases blood samples were removed just before the antennal glandswere removed. The ratio of albumin to inulin was determined in the blood, and in thecoelomosac, labyrinth and tubules for each organ. Values for these ratios appear inTable 2, and it is obvious that the albumin was much more concentrated in thecoelomosac than in the blood or in other parts of the organ.

392 LEONARD B. KIRSCHNER AND STANLEY WAGNER

If the concentration of protein in the coelomosac is related to filtration, visualizationof the protein might provide evidence concerning the location of the filtration site.Cross-sections of organs taken from Evans's blue-injected animals showed that thedye was concentrated exclusively around the tubular lumina in the coelomosac.However, the colour was not sufficiently intense to warrant photographing, probablydue to dye loss when sections were floated on water (see Methods). Instead, goatserum globulin conjugated with fluorescein was injected into the haemocoel of aseries of animals and sections of antennal gland were studied with an ultravioletmicroscope. Plate i, figs. 2 and 3, shows a pair of photomicrographs from a singleexperimental animal. The latter had been injected with o-i ml. of globulin solutionand the glands were removed 2 hr. later. The labyrinth and tubular portion show onlypunctile spots of fluorescence, possibly indicating the location of blood vessels. Incontrast, fluorescence was intense in the coelomosac. The protein is obviously con-centrated around the tubular spaces and nowhere else. It is especially noteworthythat the tubular lumina are completely devoid of fluorescence. On the other hand, theperitubular cells appear to be completely saturated with the protein.

DISCUSSION

The data presented here indicate that the filtration site in the crayfish antennalgland is freely permeable to compounds with molecular weights less than 20,000,while the restriction on excretion of HMWD suggests that restraint on filtration beginsin the molecular weight range 50,000-100,000. Wallenius (1954) found that thelimiting molecular weight for detection in the urine of several mammalian speciesvaried between 37,800 and 62,700. Some restraint on filtration could be demonstratedat any molecular weight greater than 5000. Thus, evidence at both ends of the rangeof molecular sizes indicates that the permeability of the antennal gland exceeds thatof the vertebrate glomerulus. However, comparison of the excretion patterns forserum albumin and for HMWD shows that molecular size cannot be the sole para-meter governing polymer behaviour. The two compounds have approximately thesame molecular weight, yet the carbohydrate is excreted three times as rapidly asalbumin. If permeability of the filter is involved then factors such as molecularstructure or charge must account for this difference. However, it is also possible thatprotein is filtered, then completely reabsorbed at low blood concentrations as has beendemonstrated for haemoglobin in vertebrates (Bayliss, Kerridge & Russell, 1933;Lippman, Ureen & Oliver, 1951). Since we have never seen either Evans's blue orfluorescence in any tubules after injection into crayfish the first alternative seems themore likely. Whatever the mechanism underlying this selectivity its significance isobvious. The data suggest an approximate upper MW limit for excretion of protein of150,000. Like the dextran measurements this shows that the antennal gland isappreciably more permeable than the glomerulus. However, nearly all the protein incrayfish blood is haemocyanin with a reported MW of about 875,000 (Goodwin, i960),so these estimates of ' porosity' are commensurate with conservation of blood protein.

Our data also support suggestions that the coelomosac is the site of formation of aprimary ultrafiltrate. The observation that LMWD is handled similarly to inulin inthis region is in accord with this observation, as is Riegel's demonstration that the

Filtration locus in crayfish antennal gland 393

coelomosac tubular fluid contains inulin in virtually the same concentration as blood.Moreover, the fact that the protein circulating in the blood becomes highly concentratedin the coelomosac is compatible with filtration at this site. Indeed, it is difficult torationalize this observation on any other basis.

The exact site of filtration cannot be delimited from these studies. Concentrationof protein, as shown in PI. 1, fig. 3, clearly occurs only in the peritubular regions. Thefluorescent dye appears to be dispersed throughout the peritubular cells rather thanbeing confined to the vascular spaces bathing them. This would create some interestingproblems regarding the mechanism by which a filtrate is formed. If the filtrate is, infact, formed across the luminal border of the peritubular cells the mechanism mustdiffer considerably from that in the vertebrates. It is worth noting that such a site forfiltrate formation differs from that suggested by Kummel's electron microscopicobservations (1964), but it might provide a rationale for the appearance of apicalvacuoles in these cells during urine formation (Peters, 1935).

Concentration of protein in the coelomosac raises another question. Our albumin/inulin ratios appear to suggest that 90-95% of the water entering the coelomosac asblood must be filtered and pass into more distal regions of the organ.

If the colloid osmotic pressure of crayfish blood reported by Picken, (about10 mm. Hg) were all due to protein, filtration of this magnitude would be impossible.However, calculations based on published values show that blood protein cannotcontribute more than a fraction of this value. Thus a reasonable mean protein con-centration is about 4% (Florkin, i960), most of it haemocyanin with a MW of about875,000 (Goodwin, i960). On this basis the blood protein itself contributes less than1 mm. Hg. The much larger value reported by Picken may have been due to smallcounterions (probably Na+) associated with the protein. This means that an increaseof 10-20-fold in protein concentration would probably still leave a positive filtrationpressure, for Picken reported hydrostatic pressures of 15 mm. Hg in the haemocoel,and the value in a pathway as direct as that between the antennary artery* andcoelomosac may be even higher (as in the vertebrate glomerulus).

Concentration of protein by ultrafiltration should also generate a Donnan situationin the coelomosac with the result that the concentration of diffusible cations (primarilyNa+) in the tubule should be lower than in the blood. Riegel has recently shown(unpublished experiments) that the sodium concentration in the tubular fluid averagedabout 20% less than in blood taken from the ventral haemocoel. The discrepancymight have been even more pronounced had it been possible to sample blood from theperitubular vascular spaces in the coelomosac. Riegel's observation, which appearsto be incompatible with free filtration, is instead a predictable consequence of a largefiltration-fraction.

Thus the ability of the coelomosac to concentrate both endogenous and foreignproteins points to this region as the site of formation of the ultrafiltrate. The data onpolymer excretion shows that a MW of above 150,000 is incompatible with filtration,although molecular parameters other than size are undoubtedly involved. This is

* It has been reported (e.g. Peters, 1935 ; KUmmel, 1964) that the coelomosac is supplied by a branchof the sternal artery. However, dye-perfusion experiments in this laboratory show that only theposteromedial region of the green gland (comprising a portion of the labyrinth) is so supplied. Onlywhen a dye is perfused via the antennary artery does it appear in the coelomosac.

394 LEONARD B. KIRSCHNER AND STANLEY WAGNER

consistent with the blood-protein picture in decapods since the main constituent,haemocyanin, is much too large to be filtered. However, the data presented hereindicate that further study will be required before we understand the mechanics offiltrate formation in the antennal gland.

SUMMARY

1. Inulin (MW 5000) and two dextrans (MW 15,000-20,000 and 60,000-90,000)appear in the urine of crayfish after injection into the blood. All three compounds aremore concentrated in the urine than in blood, indicating that water is reabsorbed froma filtrate formed within the antennal gland.

2. Inulin and the low molecular weight dextran seem to be handled in the samefashion since their urine/blood concentration ratios are about the same. However, thehigh-molecular-weight dextran is excreted only about 70% as effectively as the otherpair. This suggests that filtration of polymers in the MW range 50,000-100,000 isrestrained.

3. Excretion of human serum albumin occurs but it is even more severely restrictedthan the large dextran. Mammalian globulin (MW about 180,000) and crayfish bloodprotein are not excreted in the urine.

4. Blood protein becomes concentrated in the coelomosac. Localization of fluor-escein-labelled mammalian globulin shows that the peritubular cells in the coelomosacare the main sites of protein accumulation. Concentration of blood protein in thecoelomosac suggests that filtration occurs in this region, but the intracellular locationsuggests that the filtration mechanism differs from that in the vertebrate nephron.

REFERENCES

BAYLISS, L. E., KERRIDGE, P. M. T. & RUSSELL, D. S. (1933). Excretion of protein by the mammaliankidney. J. Phytiol. 77, 386-98.

BRANTON, D. & JACOBSON, L. (1962). Dry, high resolution autoradiography. Stain Tech. 37, 239-42.BRAY, G. A. (i960). A simple efficient scintillator for counting aqueous solutions in a liquid scintillation

counter. Analyt. Biochem. 1, 279-85.FLORKIN, M. (i960). Blood chemistry. In The Physiology of the Crustacea, ed. T. H. Waterman.

New York: Academic Press.GOODWIN, T. W. (i960). Biochemistry of pigments. In The Physiology of the Crustacea, ed. T. H.

Waterman. New York: Academic Press.HARRIS, J. E., SLOANE, J. F. & KING, D. T. (1950). New techniques in autoradiography. Nature,

Land., 166, 25.KAMEMOTO, F. I., KEISTER, S. M. & SPALDING, A. E. (1962). Cholinesterase activities and sodium

movement in the crayfish kidney. Comp. Biochem. Physiol. 7, 81-7.KOMMEL, G. (1964). Morphologischer Hiiiweis auf einen Filtrationsvorgang in der Antennendruse

von Cambarus affims. Naturtoissenschaften, 8, 200-1.LIPPMAN, R. W., UREEN, H. J. &OLIVER,J. (1951). Mechanism of proteinuria. J.Exp Meet. 93,325-36.PEARSE, A. G. E. (1961). Histochemistry. Boston: Little Brown and Co.PETERS, H. (1935). tJber den Einfluss des Saltsgehaltes im Aussenmedium auf den Bau und die

Funktion der Exkretionsorgane dckapoder Crustacean. Z. Morph. Okol. Tiere, 30, 355—81.RIEGEL, J. A. (1963). Micropuncture studies of chloride concentration and osmotic pressure in the

crayfish antennal gland. J. Exp. Biol. 40, 487—92.RIBGEL, J. A. & KIRSCHNER, L. B. (i960). The excretion of inulin and glucose by the crayfish antennal

gland. Biol. Bull. 118, 296-307.SMITH, H. W. (1951). The Kidney. Oxford University Press.SOMOOYI, M. (1930). A method for the preparation of blood filtrates for the determination of blood

sugar. J. Biol. Chem. 86, 655-63.WALUBNIUS, G. (1954). Renal clearance of dextran as a measure of glomerular permeability. Acta Soc.

Med. Upsala. 59, (Suppl. 4).

Journal of Experimental Biology, Vol. 43, No. 2 Plate 1

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LEONARD B. KIRSCHNER AND STANLEY WAGNER LFadng p. 395)

Filtration locus in crayfish antennal gland 395

EXPLANATION OF PLATE

Fig. 1. Evans's blue accumulation in the coelomosac. Experimental protocol is described in the text.Fig. 2. Section through the labyrinth and tubule of an antennal gland removed 4 hr. after injection offluorescein-labelled globulin. The labyrinth comprises the outer border (upper edge) of the section;the tubular tissue is more loosely arranged around larger lumina. The section is illuminated withultraviolet light. Magnification, x 100. The symbols delimit regions of the organ. Ib, labyrinth;tu, tubule.

Fig. 3. Section through the coelomosac of the same organ shown in fig. 2. The coelomosac—tubuleborder can be seen in the upper edge of the photomicrograph. Illumination and magnification as infig. 2. tu, Tubule; co, coelomosac; ptc, peritubular cells of coelomosac; lu, lumen of coelomosac.