Comparative Physiology - Invertebrate Excretory Organs

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COMPARATIVE PHYSIOLOGY: INVERTEBRATE 
EXCRETORY ORGANS1,2 
By LEONARD B. KIRSCHNER 
Department of Zoology, Washington State University 
Pullman, Washington 
INTRODUCTION 
Until a decade ago research on invertebrate excretory organs was 
confined almost entirely to their roles in producing the adaptive responses 
required for a nutritional regimen, an activity pattern, and especially an 
osmotic milieu. But the processes by which urine was produced from blood 
received virtually no attention. Until 1940 analyses of renal processes ex­
isted in only a handful of papers: one on the structure and function of the 
crayfish antennal gland (79), one on filtration in mollusks (83), and four on 
tubular and rectal structure and function in insects (77, 134-136). Ten 
years later, exactly two more had been added: an analysis of tubular fluid 
composition in the earthworm nephridium (93), and an analysis of the 
"filtrate" formed by the clam (30). All have become classics, referred to in 
nearly every survey of invertebrate excretion, but the list is pitiful when 
compared with a single year's output on the mammalian kidney. One out­
come was that many questions could be formulated about renal mechanisms 
in the invertebrates, but few could be answered. Martin's review (69) coin­
cided with the end of this period. Intensive analytical work on the insect 
Malpighian tubule was being reported, and preliminary reports on kidney 
function in the octopus had already appeared. Only a few years later a 
significant amount of new material was integrated into a review on the 
production of hypotonic and hypertonic urines (117). Many useful surveys 
of invertebrate excretion have appeared since then. These include a mono­
graph on osmotic and ionic regulation (88) as well as more restricted re­
views of excretion in mollusks ( 115), crustaceans (75, 114), and earth­
worms (60). Excretion in insects has been especially well surveyed ( 121, 
126). 
Even the most recent reviews have emphasized the adaptive role of ex­
cretory organs, especially in osmotic and ionic regulation. Renal mecha­
nisms have received less attention, or none at all. In this review the order 
of stress is reversed; the adaptive significance of renal mechanisms will be 
noted where appropriate, but is a secondary theme. This reflects nothing 
1 The survey of literature for this review was concluded in June 1966. 
• Preparation of this review was aided by Grants G-12471 and GB 811 from 
the National Science Foundation, and by funds provided by Washington State 
Initiative 171. 
169 
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170 KIRSCHNER 
more than the reviewer's interests, but the emphasis has dictated the struc­
ture and some limitations of the survey. A phyletic organization was re­
jected in favor of topical treatment because the latter brings out general 
similarities that appear to exist among many invertebrate groups. Heavy 
emphasis has been placed on relatively few papers dealing with members of 
only two phyla, the Arthropoda and Mollusca. In contrast, the metanephri­
dium receives only a little attention, and protonephridial organs are almost 
disregarded. Such an uneven treatment accurately represents the present 
state of the art. No attempt has been made to cover the literature exhaus­
tively. Selection of papers was based on their relevance to the analysis of 
renal processes. It is hoped that no important ones were overlooked. 
FILTRATION-REABSORPTION ORGANS 
Evidence for filtration.-Less than a decade ago the question whether 
primary urine formation involved filtration or secretion could not be an­
swered with confidence in a single case. The following discussion will serve 
to describe and then briefly assess the evidence now available. The picture 
is drawn from five types of observation, including: (a) excretion of 
polymers in the urine, (b) excretion of glucose in the urine, (c) mor­
phological identification of a filtration site, (d) analyses of tubular fluid 
from this site, and (e) pressure sensitivity of tubular fluid formation. It 
should establish that filtration-type kidneys almost certainly exist among 
representatives of at least two invertebrate phyla. There are indications of 
an even broader distribution. 
A variety of invertebrates have now been shown to excrete inulin and 
other polymers. Among mollusks inulin excretion has been studied in fresh­
water forms such as theLameIlibranch Anodonta cygnaea (85) and a pro­
sobranch gastropod Viviparus viviparus (62), marine species including Oc­
topus dofteini (38), and the abalone Haliotis rufescens (37), as weII as the 
pulmonate land snail Achatina fulica (71). It also appears in the urine of 
crustaceans following injection into the hemoeoel. This has been reported 
for marine forms such as the fully aquatic lobster Homarus americanus 
(19, 32) and the semiterrestrial crabs Carcinus maenas and H emigrapsus 
nudus (110). Several species of crayfish also excrete inulin in the urine 
(68, 104, 109). Inulin was also shown to be eliminated from the blood of 
three marine crabs (29) as well as from the blood of two freshwater and 
four marine mollusks (70). Inulin clearances calculated from the data in 
these papers are in the same range as for the other animals. This makes it 
likely that excretion occurred in the urine, but a direct demonstra.tion 
would be desirable. Crayfish also excrete dextrans with molecular weights 
as high as 60,000 and human serum albumin, but not human serum globulin 
or measurable quantities of their own hemocyanin labeled with the dye 
Evans blue (SO). Excretion of vertebrate hemoglobin by the lobster has 
also been reported (19). Polymer excretion might occur in a secretory 
organ if secretion involved an indiscriminate process like pinocytosis. But 
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INVERTEBRATE EXCRETORY ORGANS 171 
the aglomerular vertebrate kidney does not excrete inulin (31 ) , and the in­
sect Malpighian tubule excretes it at such a low rate that the urine to blood 
(U/B) ratio is only 0.046 (103 ). In the examples cited above, the U/B ra­
tios for both inulin and the dextrans were nearly always in the range 1-30. 
In addition, U /B ratios for polymers decreased with increasing molecular 
weight in the crayfish (50 ) as in the mammalian kidney (133 ). This would 
be predicted if primary urine formation involved passage through a filter 
with "pore" sizes distributed through a range, but is difficult to rationalize 
by any secretory process. 
An even stronger argument is afforded by renal handling of glucose 
which appears in some invertebrates to resemble that in the glomerular 
vertebrate kidney. Normal urine from several animals has been shown to 
contain little or no glucose. However, when the blood concentration was 
elevated by injection, glucose was excreted by the snail (71 ) , the octopus 
(38 ), and the abalone (37 ) , as well as by the lobster (19, 32 ) and crayfish 
( l09) . The abalone appeared to show a tubular maximum (Tmax). More­
over, in each of these animals phlorizin induced a marked and prolonged 
glycosuria even at normal blood sugar levels. In the three mollusks and in 
the crayfish the glucose U/B approached inulin D/B in the presence of 
phlorizin. This compound isknown only to inhibit glucose transport across 
membranes. There is no report of stimulation of glucose movement, yet 
this would have to be its mode of action were it causing glycosuria in a 
secretory organ. 
The locus of primary urine formation is known for a few animals, but 
has not been demonstrated in most. In freshwater clams, filtration appar­
ently occurs directly through the wall of the heart into a pericardial cavity, 
because when the pericardial membrane is punctured fluid can be continual­
ly drained from the cavity (83 ). The pericardial fluid is conducted into the 
body of the kidney through a renopericardial canal. Pericardial fluid is also 
formed in the gastropod Viviparus viviparus, and when the pericardial 
membrane in the latter was punctured no urine could be collected from the 
distal end of the ureter (62 ) . Two marine mollusks also form fluid in a 
pericardial cavity. The abalone has a morphological plan essentially the 
same as those described above. In the octopus the location of primary urine 
formation is different. A pericardial cavity surrounds an appendage of each 
branchial heart instead of enclosing the auricles and ventricles of the main 
(systemic) heart (38) . Possibly this change is due to the importance of the 
systemic circulation in cephalopods. The site of fluid formation is unknown 
in terrestrial gastropods. No fluid could be obtained from the pericardial 
cavity in three species of snails (71, 130), which shows that the pericardial 
cavity was not involved. Otherwise, these kidneys behaved like those in other 
mollusks. In all but the terrestrial snails the location of the pericardial cav­
ity provides presumptive evidence for the dependence of fluid formation on 
blood pressure. The coelomosac was suggested as a filtration site in the 
crustacean antennal gland (75), but eyidence available at that time was not 
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172 KIRSCHNER 
very strong. Recent demonstration that the ultrastructure of the system of 
peritubular cells in the crayfish coelomosac is nearly identical with that in 
the vertebrate glomerulus must be regarded as significant (57, 58). In con­
trast, the ultrastructural characteristics of the labyrinth (2) and distal tu­
bule (7) resemble those of the proximal and distal tubules of the vertebrate 
nephron. The same system of peritubular cells with podocytes extending to 
the basement membrane bordering a blood sinus has been noted in the coe­
lomosac of the crab Uca mordach by B. Schmidt-Nielsen (personal com­
munication). Kiimmel has extended his descriptions of ultrastructure in in­
vertebrate excretory organs to protonephridial organs in invertebrates, in­
cluding the chordate Branchiostoma (17, 53-56, 59). It was concluded that 
each of the nephridia examined had a region whose structure and proximal 
location suggested that it was the site of a filtration process. However, 
physiological studies [summarized in (1 17)] cast little light on the mecha­
nism. 
Earlier work on the composition of pericardial fluid in mollusks (30, 
83) and on tubular fluid from the coelomosac of the crayfish (79) was con­
sistent with ultrafiltration, but not very extensive. Recent studies increase 
both the number of animals surveyed and the variables examined. In the 
freshwater gastropod V. viviparus, the pericardial fluid had the same 
freezing point depression and pH as blood, and the same concentrations of 
sodium and chloride. The calcium concentration was slightly lower, as 
would be expected if some is bound to blood protein. Inulin appeared in the 
pericardial fluid at the same concentration as in blood after being injected 
into the latter (62). Both inulin and glucose injected into the anterior aorta 
in the abalone appeared in the pericardial cavity within minutes at the 
same concentration as in blood. Inulin, p-aminohippuric acid (PAR), 
Evans blue, and glucose were all excreted in the urine after being intro­
duced directly into the pericardial cavity. This shows that the renopericar­
dial canals in the abalone conduct pericardial fluid into the proximal region 
of the kidneys (37). Octopus pericardial fluid was isosmotic with blood and 
had an inorganic composition resembling that of an ultrafiltrate, although 
potassium concentration was slightly low (89) and its pH was somewhat 
higher (87). When the renopericardial canal was blocked, transfer of fluid 
from the pericardial cavity to the urinary sac did not occur (38). Analyses 
of fluid from the crayfish coelomosac (105, 106) showed that its inorganic 
composition is that of a slightly modified ultrafiltrate of blood. After inulin 
was injected into the hemocoel its concentration in the tubular fluid was 
about equal to that in blood. Since the filtration site in terrestrial snails has 
not been located, composition· of the original ultrafiltrate is not known. In 
many species of annelids the nephridia communicate with the coelom by an 
open ciliated nephrostome or funnel. The coelomic fluid has some character­
istics of an ultrafiltrate (45), containing a very low protein concentration 
(3). However, analytical equivalence for a series of substances found in 
blood and coelomic fluid (3, 4) was. not very convincing, and the codomic 
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INVERTEBRATE EXCRETORY ORGANS 173 
fluid was found to be slightly hypertonic to blood (93). Fluid taken from 
the nephrostomal end of the nephridium was isosmotic with the coelomic 
fluid and had the same chloride concentration. Thus coelomic fluid may be 
an ultrafiltrate of blood slightly modified by reabsorption or secretion even 
before entering the nephridia. 
The effect of pressure on tubular fluid formation has been investigated 
only in a few mollusks, but in each case pressure sensitivity was indicated. 
Picken originally estimated the rate of filtration in Anodonta by opening 
the pericardial membrane and withdrawing fluid as it was formed (83). 
His results indicated that the filtration rate was about 20 per cent of the 
body weight per hour, which would be exceedingly high even for a very 
active animal. When inulin clearances were used, the rates were an order 
of magnitude lower (70, 85). Since the high value was obtained in animals 
with pericardial membranes open, normal animals may generate a back 
pressure within the pericardial cavity as a result of limited drainage by the 
renopericardial canals. The efficacy of such a back pressure was demon­
strated on the snail Achatina futica in which retrograde pressure of 12 cm 
of water sufficed to stop urine flow, primarily because of a sharp decrease 
in filtration (71). In the snail Viviparus both pericardial fluid formation 
and urine flow varied directly with blood pressure (62). This was also true 
in the octopus (89). These observations suggest that arterial blood pres­
sure, colloid osmotic pressure, and intrarenal hydrostatic pressure play the 
same roles in urine formation in these animals as in the vertebrates. 
When one attempts to assess this picture two weaknesses are apparent 
in making a general case for filtration. Some of the evidence presented, no­
tably the material on sites of filtration, composition of the fluid formed, 
and pressure dependence, is consistent with ultrafiltration,but does not rule 
out secretion. The morphological observations can be dismissed as coinci­
dence, and it is possible to devise a pinocytotic model which is capable of 
producing an ultrafiltrate of blood and which is pressure sensitive. On the 
other hand, renal treatment of polymers and glucose is fairly compelling, 
especially the latter. The facts that glucose is normally not excreted unless 
the blood concentration is elevated, and that phlorizin causes it to be ex­
creted with U/B ratios about equal to inulin, argue strongly against a se­
cretion mechanism. The second weakness in generalizing from extant data 
is that only a few animals have been examined. On the other hand, these 
organisms are drawn from two phyla and from a spectrum o f habitats in­
cluding marine, freshwater, and semiterrestriaI. The observations do not 
appear biased by choice of animals. It is noteworthy that no observations 
have been reported that support secretion as opposed to filtration as the 
means for forming the primary urine. Ultrafiltration appears to be the 
main mechanism for forming a primary urine in the complex renal organs 
of mollusks and crustaceans. More work is needed to show whether this is 
also true of the open nephridia in annelids and of protonephridia. 
Rates of filtration.-Until recently, estimates of urine formation were 
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174 IcrRSCHNER 
obtained by blocking the excretory openings and measuring weight change 
in animals, or by collecting urine as it was excreted. The only attempt to 
measure the filtration rate was in the freshwater clam (83). As noted, the 
method used gave a very high value, probably because pericardial back 
pressure was abolished in collecting the filtrate. Essentially the same proce­
dure was used on the snail Helix pomatia with the same results. Formation 
o f tubular fluid amounted to about 28 per cent of the body weight per hour 
(130). Utilization of inulin has led to more reasonable estimates of filtra­
tion rates in a number of invertebrates. These are presented in the second 
column of Table I for a group of freshwater animals including, for com­
parison, the vertebrates Ambystoma tigrinum and Salmo gairdnerii. Filtra­
tion rates for some marine animals are also shown. 
Although many more invertebrate species must be investigated before it 
is safe to make generalizations, this group is notable for two reasons. It 
appears that the filtration rates in most animals lie in the range 1-10 per 
cent of the body weight per hour, and there is a great deal of overlap be-
TABLE I 
. FILTRATION RATES· IN FRESHWATER AND MARINE INVERTEBRATES 
Freshwater Marine 
Species CF Ref. Species CF Ref. ml kg-1 hr-1 ml kg-1 hr-1 
Achatina fulicab 50 (71) Octopus dolfleim" 3 (38) 
Anodonta 19 (85) Haliotis 12 (37) 
c'Ygneab rufescensb 
Margaritana 85 (70) Aplysia 13 (70) 
margaritifemb californicusb 
Viviparus 55 (62) Cryptochitan 35 (70) 
viviparusb stelter�"b 
Arion aterb 60 (70) My titus 23 (70) 
californicusb 
Procambarus 6 (104) Carcinus 28 (110) 
clarki· maenus· 
Orconectes 3 (104) Geocareinus 3* (29) 
virilis· latemUs" 
Ambystoma 25 (1) Ocypode 11* (29) 
tigrinumd albicans· 
Salmo 7.6 (39) Goniopsus 5* (29) 
gairdneriid cruentatis" 
a Filtration rate (CF) was measured by inulin clearance. Values marked (*) were 
obtained indirectly by measuring disappearance from the blood. 
b Mollusks. 
• Crustacea. 
d Vertebrates. 
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INVERTEBRATE EXCRETORY ORGANS 175 
tween the marine and the freshwater-terrestrial groups. The last observa­
tion is not really surprising. The sharp decrease in glomerular filtration in 
marine vertebrates is an adaptation to hypotonic regulation. Hypotonic reg­
ulation occurs in relatively few invertebrates, and in none does the degree 
of hypotonicity achieve vertebrate levels. Therefore the osmotic stress 
which makes a filtration kidney a liability is absent or much reduced. 
Among freshwater invertebrates inulin clearances for five mollusks and 
two species of crayfish are clearly comparable with values for aquatic ver­
tebrates. This set of values should dispel any illusion that a glomerular 
kidney is a special adaptation for excretion of water. Instead, it appears to 
be only one member of the class of filtration-type kidneys, and is quantita­
tively no more than equivalent to invertebrate types. The result of a rela­
tively impermeable body surface is illustrated by the two crayfish which 
have the lowest filtration rates among the group of freshwater inverte­
brates considered. The same thing can be seen in comparing filtration val­
ues for the salmon with those for the larval salamander which is much 
more permeable to water. The adaptive significance of decreased water 
permeability at the body surface has been recognized for a long time, 
and was discussed recently in relation to the ability to invade freshwater 
(120). 
The question whether filtration rates in invertebrates can be controlled 
is rarely considered. In freshwater most of the work done by the kidney 
involves operating on the filtered load of solute (see below), and this is an 
expensive process. It would be clearly adaptive to adjust the filtration rate 
to the minimum compatible with a variety of excretory requirements. 
There are a few indications that controls exist. The ability of lobsters to 
remain anuric for weeks has been described (19). This appeared to cor­
relate with a low blood protein concentration, and was corrected by inject­
ing blood from "normal" animals. In the crab Ocypode disappearance of 
inulin from the blood was dependent on environmental salinity (29). As 
mentioned above, when fluid is drained as rapidly as formed from the kid­
ney chamber in the snail or from the pericardial cavity of the clam, filtra­
tion rates are much faster than when determined by inulin clearance. The 
same phenomenon was noted in the aquatic snail (62). This indicates that 
the filtration potential is much higher than normally realized in the animal. 
One limiting factor may be the narrow renopericardial canal, another the 
arterial pressure. Control of arterial pressure and tubular back pressure at 
the filtration site by nervous or humoral mechanisms might be investigated 
profitably. 
Reabsorption of filtered solute: nonelectrolytes.-It is apparent that 
filtration rates vary widely, even among the relatively few animals exam­
ined. We can expect that as more species are investigated, and as filtration 
rates are correlated with environmental parameters and with nutritional 
and behavioral factors, the range will be extended. However, the one fac­
tor that stands out as common to the operation of all filtration-type organs 
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176 ICIRSCHNER 
is their potential for loss of useful solutes. A body of evidence is ac­
cumulating that reabsorptive processes modify the final fluid excreted. 
The only nonelectrolyteshown to be rea!bsorbed is glucose. The urine 
of invertebrates appears to contain little or no glucose. Since it appears in 
most animals' blood, its reabsorption from a filtrate is indicated. Two types 
of experiment provide conclusive evidence for this in several animals. Gly­
cosuria folIows elevation of blood glucose concentration in the lobster (19, 
32) and crayfish (109), which suggests that glucose is a threshold substance 
whose reabsorption rate is limited by a saturable transport mechanism. 
Thresholds may be about 100 mg per 100 ml in the lobster and 200 mg per 
100 ml in the crayfish, but these are rough approximations because they 
were obtained following injection, and blood glucose may decrease very 
rapidly during the periods of measurement. For the same reason T maX 
values still have not been estimated in these animals. More detailed data 
have been obtained in several mollusks. The snail Achatina excreted little 
glucose if blood levels were lower than 50 mg per 100 m!. At h igher blood 
concentrations U/B ratios increased in most animals. Reabsorption was 
blocked by phlorizin, and glucose clearances became equal to inulin clear­
ances (71). The T max was 42 mg kg-1 hr-1. In the octopus (38) some of 
the filtered glucose was reabsorbed immediately from the pericardial fluid, 
but most of the sugar was reabsorbed from the renal sac itself. Reabsorp­
tion at both loci was inhibited by phlorizin. Animals with blood concentra­
tions -below 60 mg per 100 ml excreted little glucose, but when glucose was 
infused glycosuria ensued. The U IB approached 1 as blood levels neared 
150 mg per 100 m!. Similar results were reported (37) in the abalone. Glu­
cose reabsorption appeared to be much more efficient from the left kidney 
than from the right. Since filtration rates were similar in both, the right 
kidney appears to lose glucose. The situation needs further investigation. 
Reabsorption of filtered solute.' electrolytes.-Where filtration occurs in 
freshwater animals, massive reabsorption of NaCl must follow unless 
water can be transported actively into the urine, because nearly all fresh­
water forms produce urine hypotonic to blood. Inulin data ( described 
below) show that water may be reabsorbed from the tubular system, but in 
no case do they suggest secretion. It is possible to make a rough estimate 
of a large part of the cost of operating filtration-type kidneys in freshwater, 
partly because more measurements of filtration rates and urine flows have 
been published. But it is also important that we now have a fair idea of the 
actual input energy required in transporting sodium which, with chloride, 
presents the largest load to the tubular reabsorbing mechanism. One esti­
mate of the cost of operating such a kidney was based on thermodynamic 
considerations (86). Calculations based on such an approach can provide 
only an absolute minimum value, and this need not be close to the actual 
energy expended. A more realistic estimate can be based on the observation 
that 16-18 sodium ions are transported per molecule of oxygen used in the 
frog's skin (141). This stoichiometric relationship has been demonstrated 
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INVERTEBRATE EXCRETORY ORGANS 177 
TABLE II 
THE ENERGY REQUIREMENT OF �ODIUM REABSORPTION IN FRESHWATER ANIMALS 
Specie� tF Uv [Nalb [Nalu tN. RN. 02N• Ref. 
Lumbricus terrestris 25 41 10 1025 825 0.9 (44, 140) 
Achatina fulica 50 50 60 35 3000 1250 1.6 (71) 
Anodonta cygnea 19 19 15 10 285 95 0.1 (85) 
Viviparus viviparus 55 15 34 13 1870 1675 2.1 (62) 
Procambarus clarki 6 2.4 200 10 1200 1175 1.4 (104) 
Orconectes virilis 3 1.3 200 10 600 590 0.7 (104) 
Palaemonetes antennarius 20 400 360 8000 800 1.0 (74) 
Gammarus duebeni 23 250 83 5750 3840 4.8 (63) 
Gammarus pulex 15 150 27 2250 1850 2.3 (63) 
Vertebrates 
A mbystoma tigrinum 25 10 100 5 2500 2450 3.0 (1) 
Salmo gardnerii 7.6 4.5 130 10 990 945 1.2 (39) 
Explanation of symbols heading columns: CF=filtration rate (ml kg-1 hr-I); 
Uv=urine flow (ml kg-I hr-I) ; [Nalb=blood sodium concentration (mmole I-I); 
[Nalu= urine sodium concentration (mmole I-I); CN.=filtered sodium load (I'rnole 
kg-I hr-I); RN.=rate of reabsorption (I'm ole kg-I hr-I); 02No=estimated oxygen 
consumption required for sodium reabsorption (ml kg-I hr-1). 
in many vertebrate systems, but there are reasons, discussed below, for be­
lieving that it may also obtain in invertebrates. 
An estimate of the filtered sodium load can be made from filtration rates 
and blood sodium concentrations. The rate of sodium loss can be obtained 
from urine flow rate and sodium concentration. Reabsorption is the 
difference between filtered load and quantity lost. With the relationship Na 
/02 = 16, the cost of sodium reabsorption can be expressed in terms of oxy­
gen consumption. The data are summarized in Table II. The most useful 
are those for which inulin clearances as well as urine flows are available. 
Where only the latter appear, the assumption has been made that filtration 
rate equals urine flow, i.e., that no water was reabsorbed from the tubular 
system. Values shown for sodium reabsorption in these animals are mini­
mum estimates. Two recent measurements on aquatic vertebrates are in­
cluded for purposes of comparison. It is clear that the filtered solute load is 
large in every case. This amounts to about 2 per cent of the total extracel­
lular sodium per hour in the crayfish, about 5 per cent in the clam, and 
about 12 per cent in the giant snail. As a corollary, the metabolic expendi­
ture for sodium reabsorption must be considerable in these animals. The 
rate at which sodium is reabsorbed is shown in column 7 and the calculated 
oxygen requirement for this process in column 8. The latter is a respect­
able proportion of the total oxygen consumed by the animal in some cases. 
It amounts to about 1-2 per cent of the total metabolism of the earthworm, 
and the crayfish, and 6 per cent in the clam. Even these values are mislead-
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178 KIRSCHNER 
ing, for if the animal is to remain in a sodium steady state the entire 
filtered load must be reabsorbed, if not in the kidney then from the envi­
ronment ( i.e. across gills or gut). If the same active transport mecha.nisms 
operate at the body surface as in the kidney (there is evidence for this in 
vertebrates, but not in invertebrates), the total cost of sodium regulation to 
an animal like Anodonta is on the order of 20 per cent of its total metabolism. 
The important point is that a filtration kidney is expensive to operate 
in freshwater whether the price is paid in the kidney itself or elsewhere. 
This has been overlooked in the past in assessing the relative importance 
of the body surface and kidney in ionic regulation. In most studies lack of 
information about renal function made it necessary to restrict considera­
tion to input-output relationships for the whole animal. In freshwater, "in­
put" is active uptake at the 'body surface; "output" can be divided into 
renal and extrarenal losses to the environment. On this basis the role of 
the kidneys may appear negligible. For example, urinary sodium loss in 
the crayfish is only about 20 (J.mole kg-I hr-1 while diffusion across the 
bodysurface accounts for 380 (J.mole kg-I hrl (18). But Table II shows 
that active transport within the kidney exceeds that across the gills by a 
factor of 2-3, and energetically is the costliest part of ionic regulation. It 
was recognized long ago that reduced water permea.bility at the body sur­
face and a decrease in osmotic pressure of the body fluids are both signifi­
cant adaptations to life in freshwater. Operation of a filtration kidney is 
obviously an important reason for this. 
Data summarized in Table I for some marine invertebrates do not show 
the clear tendency toward reduced filtration rates characteristic of verte­
brates. This is not surprising for two reasons. Few marine invertebrates 
are hyperosmotic regulators, and hence water loss through the kidney poses 
no special osmotic problem. In addition, since the bulk of the filtered solute 
load comprises easily replaced electrolytes, high filtration rates put little en­
ergetic'strain on tubular reabsorbing systems. Nevertheless, evidence for 
tubular reabsorptive mechanisms exists, especially in those organisms eapa­
ble of some degree of ionic regulation. It was noted (36 ) that the D/B for 
sodium in two species of fiddler crab was only about 0.8 when the animals 
were in seawater, and dropped to 0.6 in concentrated seawater. No evi­
dence was found for net anion reabsorption, so electrostatic neutrality must 
have been maintained by excretion of another cation. Sodium reabsorption 
was also seen in the crab Pachygrapsus with the U/B dropping to 0.4 in 
concentrated seawater. In addtiion, all three species produced slightly hy­
potonic urines, probably indicating some net solute reabsorption (23). 
Others do not appear to reabsorb sodium to the extent that U IB is appre­
ciably different from 1 (24). Sodium reabsorption was also indicated by a 
low U IB in the cephalopod Sepia. Earlier observations indicate that some 
crustaceans produce urine with low calcium and magnesium concentrations 
in freshwater or diluted seawater, indicating that these ions may be reab­
sorbed from the tubules [summarized in (91)]. 
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INVERTEBRATE EXCRETORY ORGANS 179 
The inulin data in Table II show that water is reabsorbed from the an­
tennal gland of crayfish (106). At low rates of urine flow it is also reab­
sorbed by the snail Achatina (71). This phenomenon is not advantageous 
for a hyperosmotic regulator, but is known to occur in aquatic vertebrates 
(cf. the examples in Table I). In some freshwater animals, water reab­
sorption may be an unavoidable concomitant of active solute transport 
from the tubular lumen. The clam Anodonta may not reabsorb filtered 
water, nor does the snail at moderate to high rates of urine flow. The crabs 
Hemigrapsus nudus and Carcinus maenas reabsorbed significant amounts 
of water from the filtrate when the animals were in air (110). Hemigrap­
sus reabsorbed water from the filtrate even when in normal seawater. The 
urine in these animals apparently cannot be made hypertonic to the hemo­
lymph, but any water conserved in the kidney under these conditions (sea­
water or air) minimizes the necessary osmotic work done elsewhere. 
Tubular secretion.-It has been known for a long time that the com­
position of normal urines, as well as the renal excretion of dyes, provided 
evidence for transport systems capable of moving solutes from blood into 
tubular fluid. Studies on organic secretory systems in three mollusks have 
recently been published. When phenolsulfonphthalein (PSP) was intro­
duced into the octopus the urine concentration rose rapidly, and within a 
few minutes exceeded that of the blood. After several hours U/B ratios 
greater than 100 were noted (38). Since the inulin U/B was 1, this concen­
tration cannot be explained on the basis of filtration. The PSP U IB was 
very dependent on blood concentration, decreasing from 100 at blood con­
centrations of 0.1 mg per 100 ml to approach unity asymptotically at concen­
trations in excess of 3 mg per 100 m!. This behavior is characteristic of a 
compound that can be excreted by a transport system as well as a filtration 
mechanism; transport predominates at low blood concentrations, filtration 
at high blood concentrations. The mechanism was shown to be energy re­
quiring by the infusion of dinitrophenol which caused a rapid decrease in 
urine PSP concentration. The same phenomena were observed for p-ami­
nohippuric acid (PAR). Since neither PSP nor PAR was concentrated in 
the pericardial fluid, this was obviously not the site of secretion. Secretion 
of PSP and PAR were also demonstrated in the abalone (37), and in the 
snail Achatina (71). The U/E was much lower in the abalone than in the 
octopus or snail and was similar to that in the lobster (19). 
The ability of marine invertebrates (like marine vertebrates) to con­
centrate magnesium in urine is often adaptative in this environment. The 
range of abilities to concentrate magnesium may be defined by M aia squi­
nado with a U IE of 1.1 (111), and the shrimp Palaemon serratus with a 
U/B of 6.7 (73), or the crab Pachygrapsus with a U/B of 6.1 (90). In 
grapsoid crabs there appears to be a general tendency for the U/E to in­
crease in salinities greater than that of seawater (24, 36, 90), and in 
Carcinus maenas kept in moist air (110). In Carcinus some, but not all, of 
the increase in urine concentration may have resulted from water reab-
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180 KIRSCHNER 
sorption in the antennal gland. When marine animals are in media more 
dilute than seawater the magnesium U/B may decrease and net reabsorp­
tion occur. Thus UjB ratios of less than 1 have been reported for Eriocheir 
sinensis in freshwater (119) and for the lobster in dilute seawater (19). 
The ability to turn a transport vector through 1800 is obviously useful for 
an animal like Eriocheir, able to live in both fresh- and seawater; how 
the animal is able to do this has apparently not been explored. Less is 
known about magnesium excretion in marine groups outside crustacea, al­
though it has been shown (111, 112) that magnesium is reabsorbed rather 
than secreted in the cephalopod mollusks Sepia and Eledone. 
Sulfate, like magnesium, is present in high concentration in seawater 
and is often concentrated by tubular secretion in marine animals (91). The 
secretory mechanisms appear to be independent, as shown by the lack of 
correspondence in U /B ratios for the two ions and by the fact that sulfate 
is secreted in Sepia and Eledone, both of which reabsorb magnesium. 
Other recent reports concern secretion of ammonia. In the crab Uca, 
blood ammonia is 20 mM. When the animals are in normal seawater, the 
concentration of ammonia in the urine is about 75 mM, and in concentrated 
seawater it rises to 116 mM (36). Urine concentration in the sephalopod 
Sepia was reported to be 146 mM, nearly 25 per cent of the total urine 
cation concentration (112). As with most secretory systems, the mecha­
nisms involved have not been investigated. However, it is striking that in 
both animals sodium reabsorption occurs from the urine and the sodium 
U/B is much less than 1. A more complete analysis has been made in the 
octopus (87). Urine usually contains appreciable NH4+ (10-30mM) and 
has a low pH, whereas blood and pericardial filtrate have much lower NH4+ 
concentrations and are less acid. Urinary ammonia was decreased by per­
fusing the renal sac with an alkaline buffer. Additional data throwing some 
light on underlying mechanisms are discussed below. 
Sites of tubular transport.-Studies on the site of nonelectrolyte reab­
sorption are also restricted to a few papers showing that glucose reabsorp­
tion almost certainly occurs from the proximal region of the kidney in both 
mollusks and crustaceans. This has been demonstrated directly for the octo­
pus and abalone in the work described above, since these animals have no 
distal tubular segments. Proximal reabsorption of glucose occurs in the lob­
ster for the same reason. On the basis of homology with the lobster, glu­
cose should be reabsorbed from the crayfish labyrinth, but no direct obser­
vations have been made. Similarly, organic compounds (PAH and PSP) 
are probably secreted in the proximal region of the invertebrate kidney 
since their transport has been demonstrated in kidneys with no distal seg­
ment (octopus, abalone, and lobster). Secretion of magnesium and ammo­
nium ion in marine invertebrates occurs in the labyrinth of the crustacean 
antennal gland or the (proximal) renal sac in marine mollusks since these 
animals have no distal tubular segment. Sodium reabsorption, possibly 
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INVERTEBRATE EXCRETORY ORGANS 181 
linked to ammonia excretion in grapsoid crabs (see above), must occur 
proximally in the crustacean antennal gland for the same reason. 
Localization of tubular function has been most thoroughly studied in 
the crayfish antenna! gland. This work is worth describing in detail because 
it provides, if not a prototype, at least a point of departure for work on 
other organs. It was established (79) that tubular fluid in the coelomosac 
and labyrinth were approximately isosmotic with hemolymph and that chlo­
ride concentrations were approximately the same. In the distal tubule the 
osmotic pressure and chloride concentration both decreased sharply. The 
picture has been expanded in a recent series of papers. Although tubular 
fluid in the coelomosac resembled an ultrafiltrate, sodium and chloride con­
centrations were about 15 per cent lower than in blood, and inulin compa­
rably higher (105, 106). This may indicate that reabsorption of some of the 
filtered electrolyte and of water already occurred in this region. Coelomo­
sac fluid has a high potassium concentration (DjB about 2). This could be 
caused by tubular secretion or by the existence of an electrical potential 
across the epithelium. Knowledge of transtubular potentials in different re­
gions of this organ is badly needed! When labeled protein and inulin were 
injected simultaneously, protein concentration in the coelomosac was about 
20 times as high as inulin (50). With a fluorescein-labeled globulin it was 
shown that the protein was intracellular and confined to the peritubular 
cells. Carbohydrate polymers, even dextrans with molecular weights as 
high as the proteins, did not become concentrated, hence the phenomenon 
seems to be concerned specifically with protein. Similar observations have 
been made on the concentration of protein by cells in the vertebrate glo­
merular epithelium (27). Protein is known to stimulate pinocytosis, result­
ing in the uptake by cells of molecules too large to traverse the membrane 
(20). Such a pinocytotic mechanism may serve to minimize loss of protein 
passing through the filter. 
Tubular fluid sodium, potassium, and chloride were unchanged in the 
labyrinth. However, inulin was more concentrated here, the DjB rising to 
about 1.3, and osmotic pressure was about 10 per cent lower than in the 
coelomosac. These data suggest that as much as 20 or 25 per cent of the 
filtered electrolyte load and some water are reabsorbed in this region. The 
dilution that occurs in the labyrinth is worth noting, even though it is 
small, for the following reasons. Kidneys capable of diluting urine usually 
have a distal tubular segment intercalated between a proximal region, such 
as the labyrinth in crustaceans, and the urinary bladder or excretory pore 
(117). However, cases are known in which animals without a distal tubule 
produce a slightly hypotonic urine, as for example the prawn Palaemon 
(d. Table I). The proximal kidney chamber in the snail also produces a 
slightly hypotonic fluid, although this is further diluted in the distal ureter 
(128). The same thing is true in the earthworm (15). If a fraction of the 
filtered solute load is reabsorbed in the proximal part of the kidney, pro-
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182 KIRSCHNER 
duction of a slightly hypotonic urine need involve nothing more than some 
depression of tubular permeability to water. Thus, this observation pro­
vides a. rationale for the occasional description of hypotonic urine produced 
proximally, without destroying the generalization that to produce a urine 
1-2 orders of magnitude hypotonic to blood requires a distal tubular seg­
ment. 
In the crayfish the concentration of chloride and the osmotic pressure in 
the distal tubular region dropped to half (105) or less (79) of the blood 
values. The direction of water movement was settled when it was shown 
that inulin became about twice as concentrated in the distal tubular fluid as 
in blood (106). This indicates that about half the filtered water is reab­
sorbed, and the combined inulin and osmotic pressure data indicate that at 
least three quarters of the filtered solute load has been reabsorbed by the 
time presumptive urine passes through the distal tubule. An effective distal 
electrolyte transport system is consistent with the high rate of oxygen con­
sumption reported for this region (46, 79). But the tubule is most notable 
in having a low water permeability. This, together with active solute reab­
sorption, is responsible for producing a dilute urine. The observation that 
distal tubular sodium concentration is as high as in blood (106) does not 
agree with chloride data pr osmotic pressure. It is either an experimental 
artifact or much of the tubular sodium must have been sequestered in an 
osmotically inactive form. Small vesicles have been seen free in the tubular 
lumena (67, 79). These have now been isolated and appear histochemically 
to have a high lipid content and to have proteolytic activity (107, 108). If 
these vesicles (which are especially numerous in the distal tubule) contain 
a large quantity of sodium, the lack of agreement between sodium values 
and osmotic pressure (or chloride) can be reconciled. 
Distal tubular fluid samples have never been as dilute as bladder urine, 
nor has the chloride concentration been as low (79, 105). The urinary 
bladder may be the site of final dilution of the urine, reabsorbing perhaps 
as much as 25 per cent of the solute filtered. Independent evidence for this 
has been reported. Isotopic sodium introduced directly into the urinary 
bladder appears in the hemolymph within minutes (43) . Tracer movement 
seemed to involve more than simple diffusion because it was inhibited by 
injection of eserine into the animal. This compound is an inhibitor of the 
enzymecholinesterase which was found in high concentration in the uri­
nary bladder. In contrast, urine in the distal tubular system of the c�arth­
worm nephridium is nearly as dilute as bladder urine. The main site of di­
lution in this animal appears to be the distal tubular segment (15, 9.3) . It 
may well be that the site of final dilution varies in different animals. 
Mechanisms of tubular transport.-Only a little work has been done 
on transport mechanisms concerned with solute movement. The system re­
sponsible for glucose· reabsorption resembles the one in the vertebrate ne­
phron in every regard tested. Thus, it appears to: (a) show saturation 
kinetics, ( b) be energy requiring and blocked by metabolic inhibitors, (c) 
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INVERTEBRATE EXCRETORY ORGANS 183 
be inhibited by phlorizin, (d) be located in cells with a brush border on 
which there is a high level of alkaline phosphatase. Whether there is any 
relation between glucose translocation in epithelia and perilumenal alkaline 
phosphatase is unknown; it has been questioned on experimental grounds 
(84). Yet the correlation found in vertebrate systems is still provocative 
, ( 16). Now the same distribution has been found in many invertebrate kid-
neys. A heavy concentration of peritubular alkaline phosphatase has been 
demonstrated in the proximal tubular regions of the crayfish (52) and the 
crab Cancer borealis (8 ) ; in protonephridia of a nemertine and a planarian 
(21); in annelid nephridia (25, 34, 35); and in the primitive mollusk 
Acanthochites fascicularis (33). Some insects have no alkaline phosphatase 
in the Malpighian tubules although a peritubular concentration has been 
described for others (9, 22). 
Some insight into the mechanism of ammonia excretion is afforded by a 
recent study on the octopus (87). Urinary ammonia concentration was very 
pH dependent, decreasing in more alkaline urines. It was proposed that the 
mechanism involved hydrogen ion transport with NHa diffusing into the 
renal sac passively to be trapped as ammonium ion. The source of urinary 
ammonia appeared to be glutamine and taurine, especially the latter, since 
both were extracted from blood passing through the kidney. Glutaminase 
was present in the kidney. 
There is some indication that sodium reabsorption may also be based on 
the same mechanisms as in vertebrate epithelia. A membrane-bound ATPase 
has been implicated in sodium transport in a variety of preparations both 
cellular and epithelial. Since most of the research on this enzyme has uti­
lized vertebrate material it is worth noting that it was first isolated and 
characterized in an invertebrate tissue, axons of Carcinus maenas (122). It 
has been shown (14) that the enzyme activity in both vertebrate cells and 
epithelia and in invertebrate cells varies with the intensity of sodium trans­
port, and that the ratio of Na transported to ATP hydrolyzed was always 
2-3, indicating an identity in mechanism. The enzyme has also been isolated 
from invertebrate gills known to transport sodium (92, 118). However, no 
measurements have been made on invertebrate excretory organs. 
Evidence of a different sort has been obtained in studies on the crayfish 
antennal gland. Inhibitors of the enzyme cholinesterase have been shown to 
stop active sodium transport in both vertebrate (28, 48) and invertebrate 
(51) epithelia. Injection of one of these compounds (eserine) into crayfish 
was followed by a rise in urine sodium concentration to near blood levels 
(41), indicating that tubular reabsorption was blocked. Urine flow was not 
measured, but the naturesis was appreciable because blood sodium de� 
creased by about 10 per cent in 24 hr following the injections. Analyses of 
different parts of the excretory system showed that cholinesterase was low 
in the coelomosac and labyrinth, but high in the distal tubule and even 
higher in the urinary bladder. Eserine inhibited the cholinesterase activity 
of antennal gland homogenates. It also blocked the absorption of isotopic 
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184 KIRSCHNER 
sodium introduced directly into the bladder (43), which seems to be one 
site of sodium reabsorption ( see above ) . While these scattered observations 
are hardly overwhelming, there is no reason to suppose that the molecular 
basis for sodium and potassium transport should differ much among ani­
mals. Evidence is accumulating that active transport of alkali metals may 
have evolved to maintain a high cytoplasmic potassium concentration. I f so, 
the requirement, and perhaps the mechanism involved, is primitive. 
Modification of the mucosal membrane in an epithelial cell appears to per­
mit translocation of sodium without altering the molecular mechanism on 
the serosal or nutrient membrane. This proposition has ample support in 
studies on vertebrate kidney, but needs confirmation in invertebrate excre­
tory systems. Measurement of oxidative enzyme activity in the crayfish an­
tenna! gland is consistent with a high level of energy expenditure. It has 
recently been shown to have the highest endogenous oxygen consumption 
among a group of organs studied (47) . 
In most marine invertebrates there is little evidence for active chloride 
reabsorption. In freshwater animals producing a dilute urine, chloride is 
clearly absorbed. Unfortunately, measurement of transtubular potential 
differences are needed before net absorption data can be characterized as 
active transport or diffusive. Recent development of both free-flow, and 
stopped-flow tubular perfusion in the earthworm shows that such data can 
be obtained in the invertebrates ( 15 ) . Chloride reabsorption was diffusive 
in the earthworm proximal segment, while sodium was transported. The 
techniques used in this elegant study merit close attention. In the snail kid­
ney the pH of tubular urine became alkaline during passage through the 
distal tubular segment, which had a carbonic anhydrase concentration 
about an order of magnitude greater than in any other part of the organ 
( 130) . Apparently bicarbonate is secreted into the tubular urine distally, 
and a chloride-bicarbonate exchange mechanism may exist here. The entire 
question of acid-base balance must be as important for invertebrates as for 
vertebrates, and it deserves more attention than it has received. 
SECRETORY KIDNEYS 
While the question whether a primary urine is formed by filtration in 
the organs just described has been vexing, there is another group for 
which the answer is clearly negative. This is the system of Malpighian tu­
bules and posterior alimentary canal which together comprise the exeretory 
system in most insects. Some differences in morphology in different insects 
havc been described ( 137) , and we will have occasion later to consider one 
of these special structures. At the outset it suffices to consider a general­
ized system consisting of a set of Malpighian tubules opening at one (prox­
imal ) end into the alimentary canal at the junction of the midgut and hind� 
gut, but 'Closed at the other (distal) end which lies in the hemocoeI. Be­
cause the distal end is closed, fluid formed in the tubule will flow proximal­
ly and enter the intestine. After flowing through the hindgutand rectum, 
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INVERTEBRATE EXCRETORY ORGANS 185 
the final urine is voided through the anus. The development of tracheolar 
respiration in insects has been accompanied by a striking reduction in the 
importance of the circulatory system. As a corollary the blind ends of the 
Malpighian tubules usually lie in a general hemocoel, and do not receive a 
direct arterial blood supply. Blood pressure clearly plays little role in fluid 
formation in these tubules. It was shown that the rate of fluid secretion 
was uninfluenced by hydrostatic pressures up to 4 cm H20 (77) . This sys­
tem therefore must secrete the tubular fluid. 
The insects are an enormously varied group having adapted to virtually 
every ecological niche. Since the excretory system is critical in homeostasis 
it is too much to suppose that uniformity will be found in its functions. In­
deed, many differences have already been described. For example, most in­
sects appear to be uricotelic, with the Malpighian tubules secreting the uric 
acid. In Rhodnius, secretion seems to be most intense in the distal region 
of the tubule, and involves the formation of an alkaline urine containing 
soluble urates. The urine is acidified in the proximal region of the tubule 
and uric acid precipitated even before it enters the intestine. But in the 
stick insect Dixippus, the rate of secretion is highest in the middle segment 
of the tubule, and the urine remains alkaline until it passes through the in­
testine. It is acidified and uric acid is precipitated in the rectum (98). In 
the cockroach Periplanata americana, uric acid is secreted directly into the 
hindgut and the Malpighian tubules apparently play no role in its elimina­
tion ( 124) . 
Even the generalization that uric acid i s the major nitrogenous excreto­
ry product has exceptions. Allantoin, but practically no uric acid, is excret­
ed by the cotton stainer Dysderctts ( 13 ) , and the urine of Corrix dentipes 
contains a high concentration of ammonium carbonate and little else ( 125 ) . 
Patterns o f nitrogen excretion in a number o f insects were surveyed re­
cently (72 ) . From an osmoregulatory point of view, the final urine may be 
dilute when water is abundant and the animal is hyperosmotic to its medi­
um. In salt-loaded animals urine may be isotonic or even hypertonic. At 
least one organism stores urine formed during an early larval stage, and it 
reabsorbs the stored fluid during a later larval state when water is in short 
supply ( 11 ) . 
However, some features of Malpighian tubular function appear to be 
common to all insects. Modern work really began with studies on Rhodnius 
prdlixus ( 134-136) . On feeding, this animal ingests a large quantity of 
mammalian blood and quickly becomes diuretic. Most of the water and 
potassium taken in is excreted within a few hours. Fluid excretion then di­
minishes, but the excretory system continues to operate at a high rate for 
many days as nutrients are metabolized. Uric acid excretion is elevated 
even though urine volume is reduced during this period. Fluid secretion 
was initiated in the distal portion of the tubule ( i.e. the blind end) . Soluble 
urates secreted distally at a pH of 7.8 were precipitated in the proximal re­
gion where the urine was acidified by a still uncharacterized mechanism. 
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186 KIRSCHNER 
Uric acid crystals were then washed into the gut by the fluid flowing proxi­
mally. 
Tubular mechanisms.-The tubular fluid in Rhodnius had a high po­
tassium concentration, the KINa ratio being 10-20 in the distal end (95). 
The ratio decreased proximally but was always greater than 1. Tubular 
fluid in the mosquito larva also had a high potassium concentration. The 
ratio KINa could be varied -by appropriate pretreatment of the animal, but 
it was always greater than 1 (96). This prompted a broad investigation of 
the base composition of tubular fluid in eight species drawn from five or­
ders of insects. The members of this group varied greatly in a number of 
characteristics, one of the most notable of which was the base composition 
of the hemolymph. Values for both the sum K + Na, and the ratio KINa 
varied over more than an order of magnitude. Yet in every case the tubular 
fluid secreted was approximately isotonic with hemolymph and had a high 
KINa (97). This list has recently been extended to include the locust 
Schistocerca gregaria (81), and larval Dysdercus fasciatus (12). No ex­
ceptions have been reported. The V IB for potassium is much greater than 
1 in all species examined. The tubUles in five species showed potential dif­
ferences of 15-30 mV with the tubular lumen positive to hemolymph (97). 
_thus potassium must be actively transported. Two species developed po­
tential differences with the lumen negative, but the electrical asymmetry 
was too small to account for the potassium distribution found. It was con­
cluded that active transport occurred in these cases also. 
A more detailed examination was undertaken on the stick insect Dixip­
pus morosus from which a functional Malpighian tubule preparation could 
be obtained for in vitro studies. It was established that isolated prepara­
tions secreted continuously for more than 30 hr, and produced tubular 
urine similar in composition to that obtained in vivo with a KINa of more 
than 20 (98, 99). Active transport of potassium from hemolymph to lumen 
was established ; weaker transport of sodium also occurred. The two ions 
appeared not to compete for a common mechanism. The rate of secretion 
was strongly dependent on potassium concentration in the bathing medium, 
while sodium affected the secretion rate only slightly. It was also shown 
that the tubular fluid was always more alkaline than the bathing solution. 
The rate of secretion was little affected by the pH of the medium over the 
range 5.5-7.2. Outside this range it dropped sharply ( 100). Concentrations 
of chloride and phosphate in the urine were always substantial and depen­
dent on concentration in the medium. Calcium and magnesium concentra­
tions in the urine were very low. Later work showed that tubular secretion 
contained amino acids when these were added to the bath and that glucose, 
fructose, sucrose, and urea also appeared in the tubular urine ( 101) . No ev­
idence was found for saturation kinetics, nor was there competition among 
related compounds. The VIB ratios were also less than 1, and it was con­
cluded that these compounds entered the tubule by diffusion. Inulin was 
also secreted ( 1 03), but its concentration was an order of magnitude lower 
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INVERTEBRATE EXCRETORY ORGANS 187 
than that of the other organic compounds tested. Three of the observations 
are particularly significant in regard to the mechanism of fluid secretion : 
(a ) the tubular fluid is approximately isotonic to hemolymph ; ( b ) in all 
cases examined the secretionhas a high potassium concentration ; and (c ) 
the rate of secretion is critically dependent o n potassium concentration in 
the bathing solution. It was proposed that secretion is induced by active 
potassium transport into the tubular lumen, with water and other solutes 
diffusing passively. Sodium is weakly transported into the tubule, and 
transport mechanisms for uric acid and both acidic and basic dyes must 
exist to account for their high concentrations. 
The electron microscope (6) adds significant detail to this model mech· 
anism. When secretion was induced in the tubules of Rhodnius many small 
vesicles were seen in the micro filaments of the distal brush border. Vesicles 
appeared to discharge from filament tips into the lumen, and such vesicles 
have now been isolated intact from tubules of Dixippus, and from the tubu­
lar system of the frog and crayfish kidney as well (107, 108, 138). The 
suggestion that secretion in excretory systems generally may be associated 
with the formation of such vesicles merits further investigation. Some of 
the data on the DiziPpus tubule could be rationalized if secretion involved 
the formation and extrusion of vesicles containing a fluid . resembling de­
proteinized cytoplasm. For example, the high KINa is characteristic of all 
nucleated animal cells. Measurable, but low concentrations of alkaline earth 
metals and the otherwise puzzling appearance of sugar and amino acids in 
the urine could be explained if their concentrations in the peritubular cells 
were of the same order. Even the appearance of inulin in very low concen­
tration might only reflect a slight permeability of the nutrient membrane to 
this compound. Within the framework of such a hypothesis the role of 
potassium concentration in the hemolymph would be to control the rate of 
vesicle formation. It should be possible to test this by comparing electron­
photomicrographs taken in low potassium medium with those in high potas­
sium, as has been done for water diuresis ( 10 ) . 
The role of the alimentary canal.-Evidently the fluid secreted by the 
Malpighian tubules is fairly uniform in composition among insects differing 
enormously in regard to the homeostatic demands placed on the excretory 
system. It was also shown (96 ) that this was true of a single animal accli­
mated to different ionic environments. Larval Aedes aegypti survived in 
distilled water and near-isotonic solutions of sodium chloride or potassium 
chloride, maintaining the hemolymph reasonably constant in all three 
media. The tubular fluid formed was always isotonic to hemolymph and in­
variably had a high KINa. Results such as those show that the tubules 
themselves cannot be the main site of ionic regulation. The final urine of 
Aedes was examined after it passed through the rectum, and in distilled­
water animals the concentration of both ions was very low. The fluid ex­
creted was hypotonic to hemolymph. It was nearly isotonic in animals 
adapted to either saline solution, but with a high Na/K ratio in animals 
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188 KIRSCHNER 
immersed in NaC ! and the reverse in animals that had been in KCl . Fluid 
passing through the hindgut was altered little in composition although there 
was evidence of some reabsorption. Obviously the rectum contained trans­
port mechanisms responsible for determining the composition of the final 
urine, and hence for regulating hemolymph composition in the face of en­
vironmental variation. 
Probably the most thorough study of the insect rectum has appeared in 
a series of papers on the desert locust Schistocerca gregaria (80-82). 'Nhen 
a solution of hemolymph was injected into the rectum, sodium, potassium, 
chloride, and water were all absorbed. A potential difference of 20-30 mV 
was found across the rectal wall, lumen positive to hemolymph. This was 
of the wrong sign to account for chloride movement, and too small for the 
equilibrium concentrations found for sodium and potassium. Thus the rec­
tal epithelium contains active transport mechanisms for all three ions ( 81 ) . 
The net flux of these ions depended on their rectal fluid concentration. In 
normal hydrated animals saturation kinetics for ion absorption could not be 
demonstrated, but in salt-loaded animals flux maxima occurred at rates far 
below those observed in controls. In both groups potassiUm absorption was 
about an order of magnitude faster than sodium or chloride. The behavior 
of these transport mechanisms showed how a normally hydrated, starved 
animal could maintain ionic homeostasis even though it produced an iso­
tonic, high-K fluid in the Malpighian tubules. In addition, the depression of 
all fluxes in salt-loaded animals obviously enables the animal to excrete 
ions under these conditions. 
Starved, normally hydrated locusts produced Malpighian tubular fluid 
at the rate of about 8 tJ.I per hr ( about 5 ml kg-l hr-1) ; but little fluid could 
be collected from the rectum, which indicates that water as well as ions were 
reabsorbed there (82). Animals given hypertonic saline to drink were able to 
regulate the ionic concentration of their hemolymph at levels a little above 
normal, which indicates that they were in water balance. Since these animals 
extracted osmotically active water from the ingested saline, rectal water 
reabsorption is indicated. This requires that the rectum be able to produce 
a final urine hypertonic to the hemolymph. When rectal fluid concentration 
was measured it was found to be markedly hypertonic in such salt-loaded 
animals, the total osmolar U IE averaging more than 3. The importance of 
hypertonic urine production for a terrestrial animal or one regulating its 
body fluid hypotonic in an aquatic environment is obvious. Several welI­
documented cases have now been reported for rectal fluid hypertonicity 
(82, 94, 95, 98, 102, 127). 
Examination of input-output relationships in unperturbed systems 
leaves the mechanism of hypertonic urine production unapproachable. The 
appropriate perturbations in the locust produced evidence for active water 
transport. Thus, when hypertonic trehalose solutions were introduced into 
the rectum, water was reabsorbed but solute reabsorption was negligible 
(80). Salt-loaded animals were able to absorb more water and achieve a 
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INVERTEBRATE EXCRETORY ORGANS 189 
higher degree of hypertonicity than hydrated animals. It appeared to 
make little difference whether the solute used consisted of transportable 
ions (Na, K, Cl) or an impermeant nonelectrolyte, so the phenomenon 
seems not to be linked to solute movement. Water permeability of the rec­
tal wall was high, and maximum hypertonicity o f the urine depended on 
the osmotic gradient. 
The locust belongs ,to a group of insects capable of producing urines 
with maximum D/B osmolarities between 2 and 4. Another group produces an 
excretory pellet that is apparently dry. The latter group has a special ana­
tomical arrangement of the excretory system. The distal ends of the Mal­
pighian tubules, instead of lying free in the hemocoel, are applied closely 
to the wall of the rectum, and the entire system of distal tubules and rec­
tum is enclosed by a membrane called the perirectal membrane.The cham­
ber formed by the perirectal membrane is filled with fluid (perirectal fluid) 
which therefore bathes both tubular and rectal epithelium, but is separated 
from the general hemolymph by the perirectal membrane. This disposition, 
found in Coleoptera and some larval Lepidoptera, was termed cryptonephric 
(137) or cryptosolenic (61) . Its function has been investigated in the meal­
worm T enebrio mo'litor ( 102). This animal produces a solid fecal pellet, 
and water reabsorption must occur from a vapor filled rectum. The vapor 
pressure at the posterior end of the rectum averaged about 90 per cent rela­
tive humidity in dehydrated animals. This corresponds to a freezing point 
depression of more than 10° C or about 5 osmolar ; the D/B osmolarity was 
nearly 10. This concentration is much greater than in the urines described 
above, and in the range of the most concentrated fluids produced by the 
mammalian countercurrent system ( 1 16). In dehydrated animals, fluids 
taken from the perirectal tubule and from the perirectal chamber ( outside 
the tubule, but within the perirectal membrane) were isotonic, and hyper­
tonic to the hemolymph. Fluid from the anterior part o f the perirectal 
chamber had a freezing point depression of about 4° C, while fluid from 
the posterior region was even more hypertonic with a freezing point de­
pression greater than 8° C. Tubular fluid emerging from the perirectal com­
plex was essentially a solution of KCI in osmotic equilibrium with anterior 
perirectal fluid. It was also noted that by the time the tubular fluid entered 
the midgut it was approximately isotonic with hemolymph. 
The observations are consistent with the following : (a) tubular secre­
tion within the rectal complex involves active inward transport of potassium 
as in other Malpighian tubules ; ( b ) osmotic equilibrium is established be­
tween the tubular fluid and whatever peritubular fluid bathes it ; and (c) op­
eration of the rectal complex stratifies the osmotic concentration of the peri­
rectal fluid so that the posterior region is more than twice as concentrated 
as the anterior. Stratification of the perirectal fluid suggests that the r ectal 
complex may operate as a countercurrent multiplier ( 139) system, pro­
viding an increasing osmotic gradient for transferring water from the rec­
tum to the perirectal fluid. A key requirement for operating such a system 
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190 KIRSCHNER 
is that the rectal epithelium be permeable to water. This was established, 
and it was shown that water transfer from rectum to perirectal tubule 
could occur even when 3M sucrose was introduced into the former (hemo­
lymph was about 0.6 osmolar). Even though the mechanisms underlying op­
eration of this system remain to be worked out, the possibility that the rec­
tal complex functions as a countercurrent multiplier is provocative:. This 
paper deserves careful study both for the elegance in design and execution 
of the experimental work, and for the important implications of the data. 
ENDOCRINE REGULATION OF EXCRETION 
Some work is beginning to appear on the regulation of excretion by 
hormones. Removal of the brain in earthworms caused them to increase 
their water content. The concentration of sodium in body fluid decreased 
( 42) . The concentration of urine chloride increased by an order of magni­
tude (44) . These changes were prevented by injection of qrain homog­
enates. Some evidence was presented that neuroendocrine factors 
influence secretion by isolated Malpighian tubules from the cockroach ( 131 , 
132). The technique used was based on dye secretion (78), and gives little 
information about the normal processes controlled. Two humoral factors 
were isolated from brain homogenates of the stick insect. One was shown 
to increase dye and fluid secretion by the Malpighian tubule ; the other de­
creased both ( 128, 129). It was suggested that one was a diuretic, the other 
an antidiuretic hormone. 
Hormonal initiation of post feeding diuresis in Rhodnius has been dem­
onstrated unequivocally (64-66). The rate of fluid secretion by isolated 
Malpighian tubules increased greatly when exposed to hemolymph or ex­
tract� of mesothoracic ganglia from newly fed animals. The active factor 
was rendered inactive by exposing it to the tubules and also appeared to 
become inactive in the hemolymph with time. Release of this hormone from 
the mesothoracic ganglion was caused by afferent impulses from stretch re� 
ceptors in the abdomen. 
SOME EXTRARENAL IMPLICATIONS 
Filtration kidneys and vertebrate evolution.-It was proposed that the 
development and widespread distribution of the glomerular kidney p rovided 
evidence that the vertebrates evolved in freshwater (123). The argument 
needs only a brief summary here. Because the glomerular capillaries are 
located close to a main arterial truk, they provide a high-pressure system 
capable of generating large volumes of filtrate. The lower region of the ex­
cretory tubule reabsorbs solutes faster than water, and hence the nephron 
is adapted to efficient excretion of water loads that could be expected to de­
velop only in freshwater. Such a device becomes a liability in seawater, 
and glomerular activity is reduced in marine vertebrates. It was concluded 
that this type of kidney evolved in freshwater as a part of the adaptation 
to that environment; therefore, it provided evidence that early vertebrates 
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INVERTEBRATE EXCRETORY ORGANS 191 
were freshwater animals. The entire question of the habitat of primitive 
vertebrates has been surveyed, and a freshwater origin emphatically re­
jected (113). Nevertheless, the proposition regarding the evolution of the 
glomerular kidney is pertinent because it raises an interesting question. 
The basic premises o f its physiology are as sound today as when they were 
proposed : the organ is admirably suited for excreting water in a fresh­
water environment. The extrapolation that it evolved to aid in hyperosmo­
tic regulation was a reasonable hypothesis thirty years ago, but this is no 
longer the case. Filtration-type kidneys are neither uniquely vertebrate nor 
exclusively freshwater ; filtration has been demonstrated unequivocally in 
members of two large invertebrate phyla drawn from marine, freshwater, 
and terrestrial habitats. It would be hard to support an argument that the 
crustacean antennal gland evolved as an adaptation to freshwater. This 
would require that the progenators of modern marine decapods were fresh­
water animals, for it is clear that marine as well as freshwater species 
form ultrafiltrates. The same is true for mollusks. Inulin clearances appear 
to be in the same range for aquatic invertebrates as for vertebrates, so 
there is nothing even quantitatively unusual about the latter. 
Thus it appears that organs of this type are widely distributed. This dis­
tribution indicates that the basic mechanism has been an unqualified biolog­
ical success, and raises the question why this should be so. The reason may 
be that it is enormously flexible both in an immediate sense and in terms of 
evolutionary potential. The capacity of a secretory kidney for excreting un­
usual, possibly deleterious compounds