A M . ZOOLOGIST 10:413-436 (1970)
Apparent Transport of Water by Insect Excretory Systems
JOHN E. PHILLIPS
Department of Zoology, University of British Columbia, Vancouver, B. C.
SYNOPSIS. Insects are capable of producing strongly hyperosmotic urine but most species
do not possess the anatomical equivalent of the mammalian kidney's couiitercurrent
system. Concentration of the excreta occurs in the rectum where water is absorbed
against increasing osmotic gradients without strict dependence on simultaneous absorption of solute. Properties of this process are reviewed. It is currently postulated that
this apparent transport of water is driven by local transport and recycling of solute
within the lateral intercellular spaces of the epithelium of the rectal pad.
The most concentrated excreta so far reported are those of the mealworm, Tenebrio
molitor. This species possesses a cryptonephridial complex in which the posterior end
of the malpighian tubules is closely applied to the rectum and both are enclosed within
a complex membranous sheath. Active transport of potassium chloride by the malpighian tubules into the complex creates a local high osmotic pressure within the
complex which is responsible, in part if not completely, for removal of water from the
rectal lumen. This system bears some resemblance to the countercurrenl system of the
mammalian kidney.
The success of insects in the terrestrial
environment can be attributed in large
measure to the evolution of efficient
mechanisms for conservation of water.
These mechanisms include an impermeable cuticle (Beament, 1964), direct absorption of water vapor from air by the integument in some species (reviewed by
Beament, 1965), and production of excreta
which are hyperosmotic to the hemolymph
(first reported by Wigglesworth, 1931). Besides insects and other terrestrial arthropods, only mammals and some birds are
capable of producing hyperosmotic urine.
As Table 1 indicates, the maximum concentrations of urine produced by mammalian and insect excretory systems are similar.
Since insects do not possess the anatomical equivalent of the vertebrate kidney's
countercurrent system, the mechanism
whereby they produce hyperosmotic urine
is of some interest. The purpose of this
paper is to summarize present knowledge
about this process. Consideration will first
be given to the more general and primitive
type of excretory system found in insects
such as the desert locust (Schistocerca gregaria) and cockroach [Periplancta americana) which can produce urine up to 2-4X
blood concentration. The mealworm
(Tenebrio molitor) produces much more
concentrated excreta (excreta:hemolymph
ratio of 10) comparable to that of the best
mammalian kidney and possesses a more
complex type of system (the cryptonephridial condition). The latter is reminiscent of the vertebrate countercurrent arrangement. This will be considered in the
final part of the paper.
A typical insect excetory system (Fig.
3) consists of a series of blind-ended malpighian tubules, a single-cell layer thick,
lying free in the blood space (heinocoel)
and emptying their content into the gut.
Here the fluid, mixed with material from
the midgut in feeding animals, moves back
through the hindgut into the enlarged
terminal section, the rectum, from which
excretion occurs through the anus.
The basic nature o£ the excretory process was worked out by Ramsay (19501961) and has been reviewed recently by Stobbart and Shaw (1964). Basically
the malpighian tubules produce a primary
excretory fluid which is formed by active
secretion of potassium and to a lesser extent sodium and phosphate (Berridge,
1968, 1969; Maddrell, 1969) into the
lumen. As a consequence of the passive
inflow of water which accompanies this
413
JOHN E. PHILLIPS
TABLE 1. Maximum concentration of urine or excreta produced by some mammals, birds, and
insects
Maximum osmotie gradient
in osmoles/liter
(urine—blood)
Reference
MAMMALS AND BIRDS
1.4
2.0
5.0
0.5
Human
Rat (Laboratory)
(Kangaroo)
Chicken
1
\
|
j
Prosser and Brown
(19G1)
TERRESTRIAL INSECTS
Vixippus morosus
Calliphora crylhrocephala
Schistocerca gregaria
Tenebrio molitor (Cryptone phridial complex)
0.5
0.5
1.1
5.1
Ramsay
Phillip's
Phillips
Ramsay
(1955c/)
(1901)
(1904(i)
(1964)
0.7
2.0
2.9
Ramsay (1950)
Surcliffe (1960)
Sutcliffe (1960)
SALT-WATER INSECTS
Aedes detritus
Epkydra riparia
Coelopa frigida
transport, electrochemical gradients are set
up for other solutes (ions, amino acids,
sugars, urea) which thereby diffuse passively into the lumen at varying rates according to the permeability characteristics
of the tubule's wall. The concentration of
any solute in the tubular fluid thus tends
to be directly proportional to that of the
hemolymph. However, a peculiarity of the
system is that the relative concentration of
ions is quite unlike that of the hemolymph;
for example, potassium is much higher
and sodium lower in tubular fluid compared to hemolymph. Ramsay concluded
that the tubules alone are not responsible
for ionic and osmotic regulation in insects;
indeed tubular secretion alone would radically upset the composition of the hemolymph were it not for selective reabsorption
in the rectum. Basically then, the process
of excretion in many insects is similar to
that of other animals in that it involves
formation of a primary excretory fluid containing all constituents of the hemolymph,
followed by selective reabsorption of essential ions and metabolites in required
amounts in the rectum. In this respect, the
tubules serve primarily to vary the load
imposed upon the selective reabsortivc
mechanisms of the rectum.
The regulatory ability of the rectum and
its role in forming hyperosmotic urine
became apparent when the composition of
various body fluids was analyzed (reviewed
by Stobbart and Shaw, 1964). Examples
are shown in Figure 1. Hindgut fluid derived largely from the malpighian tubules
tends to be isosmotic to the hemolymph so
that hyperosmosity of the excreta must be
achieved during passage through the rectum. One group of insects, represented by
the desert locust which lives under extremely dry conditions and normally never
faces the problem of excess water, produces hyperosmotic urine under all conditions. Other terrestrial insects such as the
blowfly and cockroach are capable of producing either hypo- or hyperosmotic urine
depending on the availability of water. In
this respect they are like the majority of
salt-water insect larvae which have been
studied, represented by the mosquito,
Aedes detritus (Fig. 1). Only one case of a
terrestrial insect which cannot produce
hyperosmotic urine, the cotton stainer
(Berridge, 1965), has been reported to
date; this species is similar to the freshwater larvae (reviewed by Stobbart and
Shaw, 1964). Regardless of the range of
urine osmolalities observed in various insects, the regulatory role of the rectum is
evident in that small increases in blood
(and hence hindgut fluid) concentrations
lead to very large increases in osmolalities
415
EXCRETION IN INSECTS
2.0
Desert Locust
(Phi Hips."64) —
Stick Insect
(Ramsay.'55) •
•
DEHYDRATED
O
HYDRATED
Blowfly(PhiUips.'61)
Cockroach (Wall &
Oschman ,1969)
Rhodnius
•
_(Ramsay.'52>
1.0
B
o
<
I—
2
LU
o
2
O
Euryhaline mosquito
larvae (Ramsay,1950)
o
LU
I—
ID
Cotton Stainer
(Berridge.1965)
2.0
_J
o
<
o
D
1.0
O-
O
i
I
•o
1
Blood Hindgut Rectum
BODY
Blood Hindgut Rectum
FLUID
FIG. 1. Osmotic pressure of body fluids in some insects under hydraled and dehydrated conditions.
o[ excreta (Fig. 1).
Most early studies of rectal function
were restricted to comparison of the composition of fluid entering and leaving the
rectum, while changes in volume were ignored; consequently, it was not possible to
make definite conclusions concerning the
mechanisms responsible for regulation in
this organ. Wigglesworth (1932) observed
many years ago that the contents of the gut
in a large number of insects from various
orders appeared to become drier as they
passed through the gut, and the change
was particularly marked in the rectum. He
therefoi-e suggested that the function of
the rectum was reabsorption of water,
416
JOHN E. PHILLIPS
Microscope
stage
Three-way
stopcock
Locust
Micromanipulator
FIG. 2. Method for injection and removal of fluid
from the rinsed ligated rectum of the desert locust.
Light stippling indicates aqueous solutions and
dark stippling indicates mercury. The micropipette containing a 25/xl aliquot of experimental
solution is sealed into the anus with a mixture of
Likewise, in his studies on the stick insect,
Ramsay (1955) estimated the rate of production of fluid and ionic secretion by
malpighian tubules is 5 to 10 times the
rate of excretion, so that most of the ions
and water must be reabsorbed in the rectum. These two observations suggested a
decrease in volume of fluid during passage
through the rectum, thus indicating that
hyperosmotic urine was probably formed
by reabsorption of water without a proportional amount of solute rather than by
secretion of solutes into the rectal lumen.
No rigorous evidence was available concerning mechanisms of reabsorption of ions
in the rectum of terrestrial insects from
these earlier studies.
DIRECT MEASUREMENT OF RECTAL REABSORrTION
To elucidate the nature of the reabsorptive processes in the rectum, the author
(Phillips, 1961, 1964a-c) developed a
method for measuring reabsorption directly from the isolated rectum of the blowfly
and locust in situ (Fig. 2). This consisted
Micrometer burette
beeswax and resin. The micrometer burette is used
to drive this fluid into the rectum and to
withdraw it. Samples of rectal fluid can be
removed periodically by inserting a long capillary
through the slopcock into the micro-injection
pipette.
of performing an operation to ligate the
hindgut and then rinsing out the rectal
contents by injecting and withdrawing
fluid through the anus. Small aliquots (25
ju.1) of experimental solutions were introduced (as shown in Fig. 2) and sampled at
various times for analysis. Changes in volume of fluid were followed using radioiodinated human serum albumin. The dye,
amaranth, was also included in injected
solutions to detect damage to and leakage
from the rectum.
Some of the basic observations made by
this method are summarized in Figure 3,
which describes the situation in hydrated
animals. Since a close coupling of movements of solute and water across biological
membranes has been widely observed (reviewed by Schultz and Curran, 1968; Diamond, 1965, 1968) a brief consideration of
electrolyte movement is in order. Sodium,
potassium, and chloride are absorbed from
the rectum under both hydrated and dehydrated conditions. This absorption can
lead to the development of 10 to 100-fold
concentration gradients, both under natu-
•117
EXCRETION IN INSECTS
MALPIGHIAN
TUBULES
passive
active
movement
t ran spo r t
SOLUTES
H2O
secret ion
DYES
K
t—t
EXCRETA
RECTUM
ACTIVE
ANUS
REABSORPTION
Na
K
Cl
I mM/1.
Q2 n
5
ii
<QI nl/ht.
1.52
A°C
4.7
HEMOLYMPH
IO8 mM/l.
Na
I I 11
K
115 11
Cl
A°C O.76
7.1
pH
H2O
935
Cl"
Na'
K*
H
O.4
0.2
5.O 0.4
>iM/hr.
initial
ra t e s
FIG. 3. Diagramalic summary of the various secretory and reabsorptive processes occurring in the
excretory system of the desert locust. The rates and
concentrations of body fluids are for hydra led
animals (Phillips, 196'l«-c). The reabsorptive rates
for individual ions are those observed in the isolated rectum when the initial concentration of
injected rectal fluid is the same as the normal fluid
in the hindgut.
ral conditions (see composition of excreta,
Fig. 3) and under experimental conditions
when absorption of water is prevented or
reversed (i.e., no drag effect) by including
sugars in the injected solutions. The average electropotential difference (20 mV
lumen positive) measured across the rectal
wall is too small to account for absorption
of cations against such large gradients
by passive mechanisms; therefore, it is
necessary to postulate active transport of
all three ions. Potassium is usually much
more rapidly absorbed than sodium, which
serves to compensate for the greater secretion of potassium relative to sodium by the
malpighian tubules. The transport rate of
chloride depends on the relative amount
of potassium and sodium present (the value in Fig. 3 is for a sodium chloride solution). This is due, at least partially, to a
change in electropotential gradient. The
content of the lumen is actively acidified
either by hydrogen ions secreted into the
lumen or by bicarbonate absorbed from
the lumen (Phillips, 1961). It should be
emphasized that absorption of ions represents the net difference between transport from the lumen and back-diffusion
into the lumen, i.e., the typical pump ami
leak system of biological membranes. In
the absence of a difference in concentration across the rectal wall, the efflux to
influx ratios in hydrated locusts were
found (using Cl'!(i and Na22) to be 2:1 for
chloride and 10:1 for sodium.
Ionic regulation in the desert locust does
not simply involve saturation of the ion
reabsorptive mechanisms by increased load
delivered to the rectum when blood levels
rise. In dehydrated animals with elevated
418
JOHN E. PHILLIPS
ionic concentrations in the blood, there is
also a reduction in rate of ionic reabsorption when concentrations in the rectal
lumen arc high. (Only dehydrated animals
show saturation kinetics).
Water is absorbed in the locust's rectum
from isosmotic or hyperosmotic solutions
regardless of their ionic composition.
Moreover, maximum water reabsorption
occurs in dehydrated locusts when active
reabsorption of ions in the rectum is reduced. These observations suggest a certain
degree of independence of water absorption against osmotic gradients and ionic
transport across the rectal wall as a whole.
Tndeed a degree of independence would
seem necessary in this system; for (unlike
the vertebrate nephron) in an insect system, such as that found in the locust, all
the reabsorptive activities concerned with
regulation of individual ions, osmotic pressure, bulk reabsorption of most of the primary excretory fluid, and concentration of
waste products, occur simultaneously in
one segment.
The clearest demonstration of this independence is that water is absorbed from
pure sugar solutions injected into recta of
the desert locust (Phillips, 1964r/) although the rectal wall is impermeable to
disaccharides (Phillips, 1964^, 1968; Wall,
1967). Figure 4 shows that the rate of
absorption of water from pure sugar solutions, or Ringer's with added sujrar, is inversely proportional to the osmotic gradient across the rectal wall. When there is no
osmotic gradient, water is absorbed at a
rate of 17 ^1/hr from the locust's rectum.
Absorption of water occurs against osmotic
gradients at higher luminal concentrations, reaching an equilibrium point (i.e.,
no net water-movement) when the concentration in the lumen exceeded that of the
blood by 500 milli-osmolar. This equilibrium point may exceed 1 osmolar in dehydrated locusts. (Factors which may normally control reabsorption of water in the
insect's rectum have been reported by
Wall, 1967: Highnam, el nl., 1965; and
A ford ue, 1969). At concentrations beyond
this point water moves into the rectum.
Similar results have been observed in the
blowfly, Calliphora erythrocepluda (Phillips, 1961, 1969) in vivo and the cockroach
(in vitro) by Wall (1967), who also
found that the rectal wall was impermeable to disaccharides. Stobbart (1968) has
confirmed the observations on absorption
from pure sugar solutions in the desert
locust in vivo.
The possibility that gradients of hydrostatic pressure, created by contraction
of the rectal musculature, might cause
filtration of water from the lumen can be
excluded on several grounds (Phillips,
1964rt) but perhaps the most convincing is
that absorption of water against osmotic
gradients also occurs across an everted rectal sac in vitro when a small difference in
hydrostatic pressure (about 2 cm H;.O)
actually opposes the net water movement
(Fig. 4).
The relationship between rate of movement of water and osmotic gradient suggests a water-permeable membrane (with
osmotic permeability given by the slope of
the line) containing a transport mechanism responsible for movement of water
against osmotic gradients. The decrease in
slope at high concentrations (Fig. 4)
might indicate a change in membrane permeability (6 juJ/hr, rectum at equilibrium)
or rectification of water movement as predicted by Patlak, el nl. (1963) for doublemenibraned systems.
It should be stressed that during absorption from hyperosmotic sugar solutions (or
indeed various ionic solutions) the osmotic gradient actually increases (Fig. 5) since
the rectal wall is impermeable to the sugar molecules in the lumen. Gradients established in such experiments are maintained for many hours and only slowly decrease as the preparation generally deteriorates. This absorption from sugar solutions
does not appear to be a transient event
since continuous transport of water is required to balance the passive diffusion or
osmotic flow previously suggested.
Considerable independence of water ab-
419
EXCRETION IN INSECTS
WATER ABSORPTION RATE
IN INSECT RECTA
(28°C)
40
LOCUST in_yivojpartial dehydration
and pure sugar in lumen.
(Phillips, 1964)
20
E a,
3 E
LOCUST in vitro(everted):
/ dehydrated-, Ringer-sucrose
/
in lumen. (Goh&Phillips. 1969)
COCKROACH '
in v i t r o , with
sucrose in i
0 Jumen.(Wall,1967
i
<D -^
-10
-500
0
500
1000
1500
Solute Concentration Difference
(C
' mosM/kg.)
lumen
OSMOSIS
TRANSPORT
A
Hemocoel
*
•• •
48-6
jJl/cm2hr.AosM
17
hr
Rectal
Wall
Lumen
Locust
FIG. 4. The upper graph shows the relationship
between the osmotic gradient and volume flow o£
water across the rectal wall of three insect preparations. As shown in the lower diagram, this relationship can be explained in terms of a relatively
constant transport process in series with a passive
leak (by diffusion or osmotic flow), the size and
direction of which is proportional to the osmotic
gradient.
420
JOHN E. PHILLIPS
40
30
oI
<3
O
.
O
Or
,-/ / / o20
1-0
Hemolymph
1
1
i
2
4
6
1
i
10
8
Time (hr.)
,
I
1
I
18
20
22
FIG. 5. The change in osmotic pressure of rectal
fluid with time following injection of pure trehalose solutions into ligated recta of two hydra ted
(solid circles) and two dehyraled (open circles)
locusts (Phillips, I964«).
sorption and net ionic absorption across
the rectal wall is further suggested by a
comparison of maximum osmotic gradients
developed following injection of pure
sugar solutions and Ringer's solution containing sugar (Table 2). These maximum
gradients are not significantly different although the rate of ionic transfer across the
rectal wall and the concentration of ions
in the lumen vary by two orders of magnitude. (The maintainance of low concentrations of ions in the lumen during absorption from pure sugar solutions is due,
as previously indicated, to active absorption, so that recycling of ions across the
rectal wall does occur during such experiments). Finally, a comparison of rates of
absorption from isosmolar sugar and
Ringer's solutions was carried out by Wall
(1967) using an in vitro preparation of
the cockroach. Over the first hour the rates
were identical. After three hours, the rate
of absorption from pure sugar was still 50%
of that for Ringer's solution.
As expected for an active process, the
transport of water across the rectal wall is
abolished or reduced by various respiratory
inhibitors (Table 3). On the other hand,
the specific inhibitor of sodium and potassium transport, ouabain, has no positive
effect at acceptable concentrations; however, ouabain does not inhibit transport of
potassium across several other insect epithelia (e.g., malpighian tubules, Berridge,
1966; midgut, Haskell, et a]., 1965; and
labial glands, Kafatos, 1968).
It has been concluded on the basis of
these observations that:
(1) Net movement of water from the
rectal lumen of such insects as the cockroach and desert locust occurs against
increasing osmotic gradients; i.e., the absorbate is hyposmotic to the contents of the
2. Comparison of oxmolic gradients developed following injection of hyperosmotic sugar
and Ringer's solutions in1o ligaird recia of locusts (Phillips, 1964a)
Solution injected
3) Maximum osmotic gradient developed
(osmoles/liter)
water-fed
saline-fed
2) Total concentration of monovalent ions in
rectal fluid (meq/liter)
water-fed—initial
—final
saline-fed—initial
—final
3) Net transfer of monovalent ions (^cq/hr/cm-)
water-fed
Pure treliaiose
0.30
0.96
5
(5
8
12
<0.03
100% Eingcr
-4- trelialose
0.43
1.07
710
368
700
—
1.36
421
EXCRETION IN INSECTS
TABLE 3. Inhibition of rectal absorption of water from isosmotic and hyperosmotic solutions
%
Inhibitor (molarity)
2,4-Dinitrophenol (10"*)
Cyanide (10~2)
2,4-Dinitrophenol (10"3)
2
)
Todoacetate (10~
Ouabain (10~2)
3
"
(10- )
Malonate (10"2)
J nsect
Cockroach
Stick insect
Desert locust
"
"
JJ
M
lumen. Secretion of solutes into the lumen
is not an important factor in the formation of hyperosmotic excreta.
(2) This movement of water against a
gradient is an energy-requiring process,
but does not require simultaneous net
transfer of solute across the rectal wall as a
whole, although recycling of ions occurs.
POSSIBLE MECHANISMS OF WATER TRANSPORT
At this point it might be useful to consider mechanisms which have from time to
time been proposed for moving water
across biological membranes (Table 4),
and to speculate on the mechanism for
absorbing fluid against a gradient in the
insect's rectum. The first two processes,
simple osmosis and filtration, have already
been excluded by demonstration of net
movement of water against gradients of
osmotic and hydrostatic pressure.
Pinocytosis is involved in the production
of hyposmotic fluid by contractile vacuoles of Protozoa (Schmidt-Nielsen and
Schranger, 1963; Riddick, 1968). A model
for rectal absorption involving recycling of
ions and reverse pinocytosis was previously suggested (Phillips, 1964a) as one possiTABLE 4. Possible mechanisms of net movement of
•water
1.
2.
3.
4.
Classical osmosis
Filtration
Pinocytosis
Secondary transport coupled to transfer of
solute:
(a) electro-osmosis
(b) double-membrane effect
(c) local osmosis
(d) co-diffusion (drag effect)
5. Primary transport of water
0. Beament's model (cuticular valve)
Inhibition
50%
100%
100%
100%
100%
not significant
50%
Reference
Wall, 1967
Vietinghoff, 1965
Irvine, 1966
Goh and Phillips, unpublished
bility. However, ultrastructural studies (to
be discussed subsequently) on several species of insects indicate that micropinocytotic vesicles, while observed, are not numerous or pronounced in the rectal epithelium.
Therefore, this process is considered unlikely to make a major contribution to
water transfer.
Another membrane, the body surface of
some insects, is capable of absorbing water
from unsaturated air (as low as 50% R.H.)
against enormous activity gradients (reviewed by Beament, 1964, 1965). Obviously net transport of solutes does not accompany this water movement. This absorption of water depends on the integrity of a
continuous superficial layer of wax molecules. The latter causes the cuticle to act as
a rectifier, or valve, which favors entry of
water but restricts loss from the underlying
chitinous layers. Beament (1965) suggests
that the epidermal cells in some unknown
manner (e.g., by varying the isoelectric
point of proteins) alter the water activity
of the underlying layers of cuticle in a
cyclical manner, thereby providing a downhill gradient for passive entry of water from
the atmosphere during favorable periods
i n the cycle.
Since the insect's rectum is morphologically an inpushing of the body wall and is
lined with a chitinous cuticle, Beament
(1965) has suggested a common mechanism for apparent transport of water across
these two membranes. It is difficult to see
how this mechanism could be applied to
the rectal wall since a number of properties of integumentary cuticle which have
been clearly associated with a continuous
monolayer of wax molecules (Beament,
1961, 1964, 1965) are not observed for rec-
JOHN E. PHILLIPS
422
TABLE 5. A comparison
of integumentary and rectal cuticles of insects
Type of cuticle
Property"
1.
2.
3.
4.
Beatification of net movement of water
Permeability to electrolytes
Permeability of solute proportional to solubility of lipid molecules
Large electrostatic potential in absence of ionic concentration
gradients
Integument"
-+—
+
-j-
Rectum0
—
-f—
—
* These properties of integumentary cuticle are associated with a continuous monolayer of
wax molecules.
"c Beament, 1965.
Phillips, 1968. Observations on rectification (property 1) are unpublished.
tal cuticle of the desert locust (Phillips, ports water molecules". Rather the term
1968). The properties of the two cuticles was clearly defined (Phillips 1964rt) in
are compared in Table 5. These differ- terms of classical thermodynamics as origiences suggest the absence of a continuous nally applied by Rosenburg (1954) to
wax layer which might act as a valve in transport of solute. That is, considering
rectal cuticle. (Rather the rectal cuticle the rectal epithelium as a black box, enerbehaves as a molecular sieve with water- gy must be utilized to move water against
filled pores, having radii of 6.5 A; Phillips, osmotic and hydrostatic gradients without
1968). This general conclusion has been proportional movement of solute. As far as
confirmed recently by directly measuring the insect is concerned, faced with the
the osmotic flow of water across the iso- problem of reabsorbing water without a
lated rectal cuticle of the desert locust. proportional amount of solute, it has
While the osmotic permeability of this evolved a water pump. While the mammembrane decreases with increasing total malian kidney does this also, several epconcentration of solute in the bathing so- ithelial membranes in series are involved.
lutions, the value is independent of direc- The principal intention at the time was to
tion of flow when osmotic gradients are distinguish (Table 6) the rectal wall from
reversed.
those epithelia such as the mammalian
In discussing the remaining two mecha- ileum and gall bladder in which water can
nisms (4 and 5 of Table 4) it is move against osmotic gradients but in
necessary to clear up some misunderstand- which the absorbate is isomotic or hyperosing. In the original paper (Phillips, motic. Obviously the latter membranes
1964a), the author used the term "active cannot develop hyperosmotic solutions
transport of water" to describe absorption from isosmotic solutions as can the insect"s
of water in the locust, while pointing out rectum.
that the nature of the process was still a
Over the past 10 years the definition
matter for speculation and further exper- and usage of the term, active transport, has
imentation. The use of this term was taken changed with the advent of irreversible
by some workers as suggesting an energy- thermodynamics in the field of membrane
utilizing carrier molecule which directly physiology (discussed by Curran and
picks up and transports water molecules Schultz, 1968). To those concerned with
(i.e., a primary water-transporter). This is molecular events, the more recent definiin spite of the fact that care was subse- tion of primary transport as a flow (net
quently taken to clarify this misunderstand- movement) coupled directly to an energying (Phillips, 1965), to the effect that yielding chemical reaction is more mean"the use of the term active transport of ingful (e.g., Kedem, 1965). A net movewater is not intended to imply a carrier ment of water against its activity gradient
that specifically combines with and trans- which is coupled to (i.e., dependent on)
423
EXCRETION IN INSECTS
TABLE 6. Comparison of some properties of movement of water across terieorate epithelia and
rectal wall of insects
Property
1.
2.
3.
•A.
Absorption against osmotic gradients
Increase in osmotic gradient during (1)
Absorbate hyposmotie (direct analysis)
Dependence of (1) on net solute absorption:
(a) water movement with pure isosmotic sugar solution on
lumen side
(b) direct correlation between rate of transport of solute
and volume flow
Vertebrate
epithelia*
Bectal wall
+
—
—
+
—
+
-f—
+
+
—(?)
* Ileum (reviewed by Schultz and Curran, 1968) and gall bladder (reviewed by Diamond,
1968).
transport of solutes is now referred to as
secondary transport (Kedem, 1965). On
the basis of these definitions, it is not possible with the available evidence to make a
rigorous distinction between primary and
secondary transport in the case of the rectum of locust or cockroach. To explain,
two types of evidence (e.g., Schultz and
Curran, 1968) indicate that movement of
water against activity gradients is coupled
to movement of solutes in such membranes
as the mammalian ileum and gall bladder
(Table 6): (1) An obligatory dependence of movement of water on the
presence of net transport of solute, and
(2) the existence of a stoichiometric relationship between movement of solute and
water such that the absorbate is isosmotic
or hyperosmotic to the contents of the
lumen over a wide range of experimental
conditions. Since experimental evidence of
this type has not been obtained for the
insect's rectum, there is no rigorous evidence which excludes a primary water
pump, hence the use of the term, "apparent transport of water," in the title of this
paper.
While primary transport of water would
be energetically wasteful, the energy output of the locust's rectum (Phillips, 1964<v)
is six times the theoretical minimal energy requirement for such a mechanism
using a value of 6 //.1/hr/AOsM for permeability of the rectal wall to water at
equilibrium (Fig. 4). Such a mechanism
would permit the desired independence
between solute and osmotic regulation discussed earlier.
However, there has been no clear demonstration of primary water transport
(Robinson, 1965); moreover, double-membrane models have been proposed which
can account for hyposmotie fluid transport
in terms of transport of solute (Patlak, et
al., 1963; House, 1964). It would seem more
reasonable, therefore, to assume, as a first hypothesis, some type of coupling of water
movement to transport of solute. While
movement of water occurs in the locust's
rectum without net movement of solute,
the author (Phillips, 1965) previously
pointed out that "it is possible to envisage
water movements in terms of active solute
transport and back diffusion across individual membranes so that net flux of solute
across the rectal wall as a whole is not
involved." Such a cycling does occur in the
locust's rectum at all times. The difference then between vertebrate epithelia,
such as the ileum, and the insect's rectum
might be a system of recycling ions or other solutes within or across the rectal pad. A
few examples are suggested in Figure 6.
The double-membrane model first proposed by Curran (1960) and formally described by Patlak, et al., (1963) involves
two membranes or diffusion barriers in
series, one of which has restricted permeability to solutes relative to water (i.e. high
reflection coefficient, e.g., a = 1) while the
second is less selective to passage of water
and solutes (low reflection coefficient, e.g.,
JOHN E. PHILLIPS
424
LUMEN
(IOO0 mOsm)
diffusion H 2 0
t
Barrier I
6 -- I
I
> 1000 mOsm
A high
hydrostatic
pressure
*
I
back
diffusion
of cation
diffusion H 2 0
I
solute
transport
I
I
back
diffusion
or
solute
transport
I
I
> 1000 mOsm
high
hydrostatic
pressure
laminar
flow
'"i
H 2 0 by
electroosmosis
Barrier II
a a 0
laminar flow of
solute and H 2 0
transport
of cation
Intracellular
( 4 0 0 mOsrrO
solute transport
and
back diffusion
(400 mOsm)
HEMOLYMPH
ELECTRO-OSMOSIS
LOCAL OSMOSIS
DOUBLE MEMBRANE
and SOLUTE RETURN
EFFECT
FIG. 6. Three hypothetical mechanisms whereby
rectal wall against an osmotic gradient (explanaactive transport and recycling of solute might cause
tion in the text),
a hyposmotic absoibate to move across the
tr = 0 if the membrane is completely nonselective). Active transport of solute into
the intermediate compartment across the
first membrane will create an osmotic gradient which in turn will cause water to
flow into the intermediate compartment
between the membranes. Since the osmotic
gradient across the non-selective membrane is less effective (or completely
ineffective if a = 0), this docs not lead to
entry of water from the blood side. The
hydrostatic pressure arising from entry of
fluid into the intermediate compartment
causes water to flow in the direction of
least resistance across the non-selective and
more permeable second membrane. A modification of this model placing the solute
pump on the non-selective membrane or any
membrane other than lumen-facing membrane (a hypothetical situation described
by Patlak, 1963) could cause movement of
water against an osmotic gradient driven
by local recycling across the non-selective
membrane (Fig. 6). House (1964) has described a model for frog's skin which bears
some resemblance to this hypothesis but
which involves differences in permeability
to specific ions for the two membranes.
Diamond's (1965, 1968) hypothesis of
local osmosis may be considered a special
case of the double-membrane hypothesis.
According to this model, ionic transport
into long, narrow, restricted channels
creates a local osmotic gradient, causing
water to follow by osmosis. The hydrostatic
pressure which develops due to entry of
water into the restricted spaces causes fluid
to flow down the channels which are in
direct continuity with the second compartment (blood side). If the dimensions of
the channel are sufficiently long and narrow to permit attainment of osmotic
equilibrium as fluid flows down the channel, the fluid entering the second compartment will be isosmotic under all conditions. Again this model could be modified
EXCRETION IN INSECTS
to explain the situation in the locust's rectum if ions were subsequently reabsorbed
across a water-impermeable membrane
(Fig. 6). This model then requires two
membranes with different permeability
and transport properties, one responsible
for secretion and the other for reabsorption. The lateral intercellular channels
have been identified as the site of local
osmosis in various vertebrate epithelia by
demonstration of a correlation between the
distension of these spaces and the rate of
movement of fluid (Kaye, et ah, 1966;
Tormey and Diamond, 1967; Diamond
and Bossert, 1967; Schmidt-Nielsen and
Davis, 1968). The lateral channels are
closed when transport is stopped by cooling, metabolic inhibitors (ouabain), and
adverse gradients (Diamond and Bossert,
1967). The basal infoldings and microvilli
of epithelial cells have also been suggested
as possible sites (Diamond and Bossert,
1968; Berridge and Oschman, 1969).
A third possible mechanism of coupling
water movement to active recycling of ions
is electro-osmosis (Fig- 6). Preliminary
studies (Phillips, 1961) suggest that this
mechanism is not important since absorption of water against osmotic gradients in
the locust's rectum is relatively unaffected
by reversal of an electric current applied
across the rectal wall.
Recent ultrastructural studies, notably
by Gupta and Berridge (1966r/) and Berridge and Gupta (1967) on the blowfly
and Oschman and Wall (1969) on the
cockroach, suggest a possible structural basis for applying these models to the insect's
rectum. Studies have also been carried out
by Hopkins (1966), Noirot and NoirotThimothee (1960, 1966), Wessing (1966),
Baccetti (1962), and Baccetti, Mazzi, and
Massimello (1963). Irvine (1966), in the
author's laboratory, has carried out a preliminary study of the locust. Basic similarities in ultrastructure of these various rectal
epithelia are apparent. Fig. 7 shows diagrammatically the major features of the
epithelium of the blowfly's rectal papillae
(left) and on the right is illustrated the
epithelium found in the rectal pad of
425
such orthopterans as the cockroach and
desert locust.
In all cases the rectal lumen is lined
with a chitinous cuticle. The apical plasma
membrane is highly infolded with associated mitochondria (few in dipterans). On
the cytoplasmic surface of this membrane
there is a coat of subunits (125 A wide with
gaps of 40 A) first described by Gupta and
Berridge (1966£>). The basal plasma membrane lacks infolding (except Drosophila;
Wessing, 1966).
The lateral plasma membranes are remarkable for the complexity of their infoldings. At both apical and basal borders,
junctional complexes consisting of septate
desmosomes and tight junctions are observed. These are thought to reduce exchange between the lateral intercellular
spaces and the rectal lumen or subepithelial sinus, respectively (Loewenstein and
Kanno, 1964; Loewenstein, et ah, 1965).
Extensive finger-like interdigitations of adjacent cell membranes, each containing a
mitochondrion, are the most striking
feature in orthopteran cells. The two
membranes in such regions are maintained
at a constant spacing of 200 A by very fine
20 A fibers in the intercellular space. The
equivalent structure in the dipteran cell
consists of stacks of evenly spaced membranes surrounded by mitochondria. Berridge and Gupta (1967) estimate that the
lateral surface is thereby increased 100 to
1000 times over that of the plane surface
in the blowfly's papillae. At intervals the
lateral membranes form dilations in continuity with the narrow channels. These
dilations and the narrow channels ultimately connect with larger intercellular
sinuses characterized by the presence of a
basement membrane and containing tracheal branches to the rectal cells. Exit of
fluid from this system of spaces is limited
to a few points of tracheal penetration.
A secondary layer of relatively undifferentiated medullary cells is observed in the
blowfly, while this layer is completely absent in the cockroach. The small secondary
cells of locusts, however, are characterized
by extensive irregular infolding of the ap-
JOHN E. PHILLIPS
~=~ Cuticle
Apical
Infoldings
Intercellular
dilations
Intercellular
channels
(200A)
Intercellular
dilat ions
Junctional complex
Infundibular or
Subepithelial space Trachea
Basal Plasma
Membrane
Secondary Cell
Muscle
I'IG. 7. Diagrammatic summary of basic ultrastruclural features o£ the epithelium of the recial pad
(or papillae) in some adult dipteran and orthop-
teran species. See text for further description and
references.
ical membrane with associated mitochondria, suggesting a second transporting layer. Exit of fluid from the infundibular
spaces o£ the blowfly's papillae is controlled by a valve allowing- only exit of
fluids. In the cockroach, fluid exists only
where large tracheae penetrate the circular
muscle. The latter acts as a simple valve
around the trachea. In summary, the rectal
epithelium possesses a series of spaces similar to, if more elaborate than, those observed in various vertebrate epithelial
membranes.
The basic ultrastructural organization
(Fig. 7) suggests several possibilities for
applying the modified double-membrane
427
EXCRETION IN INSECTS
Rectal Pad
Lumen
Cuticle
Subintimal
space
Primary cells
of pad
Reduced
epithelium
between pads
Intercellular
spaces
Secondary
cells ot pad
Hemocoel
I
^> solute transport
> solute diffusion
FIG. 8. A diagram of the major membrane-bound
compartments in the rectal pad's epithelium in an
insect such as the desert locust. Large arrows indicate possible sites and direction of transport of
solutes based on ultrastrucunal observations summarized in Fig. 7. Broken lines with small arrows
indicate possible direction and routes of solute
recycling by active or passive mechanisms. Any one
or more of these solute cycles might cause movement of water (not shown on this diagram) in the
lumen-to-heniocoel direction as a result of mechanisms proposed in Figure 6. Solute cycles 1, 3, and 5
are applications of the hypothesis o£ local osmosis
with return of solute. Cycles 2, 4, and 6 are
applications of the double-membrane hypothesis
with direction of solute pump opposite to the
direction of water flow. Cycle 4 does not represent
a single event but a continuous process which
recurs throughout the full length of the lateral
spaces; e.g., potassium which might be transported
into the cell with a subsequent back-diffusion into
the lateral spaces at the apical end might he
repeatedly re-transported into the cell as fluid
moves down the lateral spaces to the hemocoel.
and local-osmosis hypotheses presented in
Figure 6. Some of these possibilities are
shown schematically (Fig. 8). Cycles 1, 3,
and 5 involve local osmosis and return of
solute across the lateral (1, 3) and apical
(5) plasma membranes, respectively. Cycles 1 and 3 differ in the route of return of
solute, cycle 1 involving passive return to
the lumen via the reduced epithelium between the rectal pads, while in cycle 3 solute is reabsorbed within the more proximal region of the lateral intercellular
spaces. The even-numbered cycles (2, 4, 6)
involve the modified double-membrane
hypothesis with solute transported in the
opposite direction to the flow of water.
428
JOHN E. PHILLIPS
These models make a number of predictions which suggest an experimental basis
Cor distinguishing between them. Only a few
examples will be mentioned. Cycles 2, 3,
and 4 require that the intracellular osmotic pressure of the primary epithelial cells
must be greater or at least equal to the
lumen content (and thus two to three times
the hemolymph value) in order to permit simple osmotic flow of water across the
apical membrane and cuticle. Cycles 3, 4,
and 5 and a primary water pump all predict that the absorbate emerging from the
rectal pad should be strongly hyposmotic
to the contents of the lumen under dehydrated conditions. Only cycle 1 does not
involve local recycling of solute within the
rectal pad. This cycle predicts a hypcrosmotic or isosmotic absorbate leaving the
rectal pad. Cycle 6, while obviously not required for apparent transport of water
since the secondary cells are absent in the
cockroach, might represent a supplementary mechanism responsible for the larger
osmotic gradients developed in the rectum
of the desert locust.
Berridge and Gupta (1967) demonstrated a correlation between the distension of
the lateral intercellular dilations and the
relative rate of absorption of fluid previously reported (Phillips, 1961). These observations indicate that the lateral channels are the route taken by water through
the rectal epithelium. Oschman and Wall
(1969), however, were unable to find a
clear correlation between distension of the
intercellular spaces and physiological state
in the cockroach. On this basis, Berridge
and Gupta (1967) have proposed that
water moves by local osmosis into the narrow intercellular spaces caused by active
secretion of potassium chloride. In support
of this, they demonstrated histochemically
the localization of a Mg-activated ATPase
specifically on the stacks of lateral membrane (Berridge and Gupta, 1968). However, both this histochemically visualized
ATPase and one isolated in biochemical
studies are not stimulated by sodium and
potassium so that it is not clear whether
this enzyme is comparable to the Na- and
K-activated ATPase of other biological
membranes. In the absence of ions from
the lumen, Berridge and Gupta (1967)
suggest that solute might be recycled via
the reduced epithelium between the rectal
pad or from the blood or infundibular
space. In essence they propose a simple
local osmosis of the vertebrate type (involving an isosmotic or hyperosmotic absorbate) through the lateral spaces of the
epithelium of the rectal papillae (cycle 1
of Fig. 8) and return of solute by another
route.
Recent experimental observations indicate that the absorbate from the rectal
pads is hyposmotic to the lumen's content.
This is not compatible with simple local
osmosis across the epithelium of the pad,
which would lead to isosmotic or hyperosmotic absorbate. This is concluded from
comparison of total back-diffusion of ions
into the rectal lumen with the passive permeability of the rectal wall to water as
previously estimated (Phillips, 1964a).
Thus, to balance the passive leakage of
water, the ligated rectum of the locust
must absorb minimally at least 6 pi water
per hour (Fig. 4) just to maintain a 1000
mOsM gradient (which the locust does for
long periods). According to local osmosis,
this absorbate must be at least isosmotic
to the contents of the lumen (i.e., its concentration must be 1000 mOsM). Thus, according to local osmosis, at least 6
/xOsM/hr/cm2 of solute would be required
to diffuse into the lumen to maintain such
a gradient, assuming solute returns via the
lumen. The measured back-diffusion averages 0.5 ^OsM/hr. This value is high compared to the estimate of Stobbart (1968)
for the exchange rate of Na22 and K42
between hemolymph and rectal epithelium. These preliminary calculations suggest
that recruiting of ions from the lumen via
reduced epithelium or directly from the
hemolymph is probably too low to account
for observed movement of water assuming
local osmosis across the rectal pads. This
has been more clearly demonstrated by
Wall and Oschman (unpublished) by a
direct analysis of absorbate collected from
429
EXCRETION IN INSECTS
the sub-epithelial space of the cockroach's
rectal pad by micropuncture. Under conditions of water-deprivation, the absorbate is
considerably hyposmotic to the lumen's
content. It would appear then that any
recycling of ions, if it occurs, is likely to be
located within the rectal pads, so that absorbate is hyposmotic when it enters the
hemocoel.
Oschman and Wall (1969) and Phillips
(1969) point out the necessity for modifying the original hypothesis of Berridge and
Gupta (1967) to allow for reabsorption of
solute in the intercellular sinus (i.e., cycle
3 of Fig. 8). That is, the intercellular spaces
within a single cell-layer form a tubular
system containing secretory and reabsorptive areas. Wall and Oschman suggest that
recycling of ions involves sodium rather
than potassium. In support of this, Phillips
(1965) concluded on the basis of electropotential profiles through the rectal pad of
locusts (also Vietinghoff, et al., 1969) that
the electropotential gradient would permit passive entry of sodium into the epithelium of the rectal pad, but sodium
must be removed actively at the hemocoelfacing membrane or, on present ultrastructural evidence, the membranes bounding
the lateral intercellular spaces which are in
continuity with the hemocoel.
There is one problem concerning this
hypothesis. It requires an osmotic pressure
of the cell's interior slightly in excess of
that in the lumen in order to draw water
from the latter space into the rectal pad's
epithelium by osmosis during absorption
from hyperosmotic sugar solutions. Since
the concentration of monovalent ions in
the rectal tissue of the locust is lower than
that of the blood (Phillips, 1964b; Stobbart, 1968) this hypothesis requires postulation of a very high concentration of other solutes in epithelial cells, or some other
means of lowering activity of water within
the cell (e.g., organization of water at the
surface of abundant microtubules).
In summary, while the hypothesis that
water moves by local osmosis with subsequent reabsorption of solute within the lat-
eral spaces of the rectal pad's epithelium is
appealing, critical physiological observations which might permit a distinction to
be made between this model and others,
have not yet been obtained (See Phillips,
1969, for an assessment of evidence for the
hypothesis of local osmosis). The required
evidence includes (I) measurement of intracellular osmotic pressure to determine
the role of the apical membrane, (2) a
demonstration of the dependence of net
water transport on transport of solutes and
recycling, and (3) analysis of intercellular
fluid from the lateral spaces possibly by
micropuncture to demonstrate hyperosmosity of a primary secretory fluid prior to reabsorption of solutes.
CRYPTONEPHRIDIAL SYSTEM OF
TeiiebriO
molitor
The diversity of insect excretory systems
is strikingly illustrated by the cryptonephridial system of the mealworm, Tenebrio molitor. According to Ramsay (1964)
and Grimstone, Mullinger, and Ramsay
(1968) who have carried out an intensive
study of this system over the last 10 years,
the fecal material is so dry in dehydrated
mealworms that water must be removed in
the posterior rectum as water vapor. In
order to estimate their maximum osmotic
pressure, Ramsay determined the relative
humidity with which the fecal pellets were
in equilibrium. The vapor pressure in the
posterior rectum of dehydrated mealworms averaged 90% R.H. (maximum 75%
R.H.) which corresponds to a freezingpoint depression of 10.5°C. and an excretato-blood osmolarity ratio of nearly 10:1.
The mealworm, in common with other
Coleoptera and some larval Lepidoptera,
has an unusual anatomical arrangement of
the excretory system (referred to as cryptonephridial), whereby the distal ends of
the malpighian tubules, which normally lie
free in the hemocoel, are closely applied
to the rectum (Fig. 9). Both the rectum
and the distal ends of the tubules are completely enclosed by a complex multilaminar membrane, the perinephric membrane, the inner part of which resembles
430
JOHN E. PHILLIPS
BlistQr
x " " " * ~ ^ - Leptophragma
Trache,
Perinephric
Membrane
Circular
muscle
Rectal
epithelium
TRANSVERSE
SECTION OF THE WALL OF THE RECTAL
COMPLEX
KIG. i). Diagramalic cross-section of the mealworm's rectal complex to illustrate the series of
membranous baniers and compartments associated
with the cryptonephridial arrangement
Grimstone, Mullinger, and Ramsay, 1968).
(after
somewhat a rayelin sheath. The perinephric space so enclosed between the rectal
epithelium and the malpighian tubules,
here renamed perirectal tubules, is almost isolated from the hemocoel. At intervals the perinephric membrane is reduced to thin, circular, window-like structures, the leptophragmata, separating the
lumen of the perirectal tubules from the
hemocoel. A small specialized cell, the leptophragmata cell, forms an extremely thin
diaphragm across the window. The leptophragmata have long been suspected as
sites of ionic movement (Lison, 1937) because they selectively turn black on exposure to silver nitrate and light.
What is the significance of the cryptonephridial arrangement? All insect mal-
pighian tubules so far studied (Ramsay,
1953; Phillips, I946«; Berridge, 1968, 1969;
Aiaddrell, 1969) transport potassium to the
lumen side. The perirectal tubules of the
mealworm might likewise transport potassium chloride from the blood into the rectal complex without accompanying movement of water. The result would be to
create a local high concentration of solute
within the perirectal tubules and perinephric space, thereby reducing the osmotic
gradient against which the rectal epithelium must move water. Alternatively, this
might permit simple passive reabsorption
of water from the rectal lumen down an
osmotic gradient. (Jn essence the transporting capacity of the malpighian tubules
might be added to that of the rectal ep-
EXCRETION IN INSECTS
431
ithelium.) Reabsorbed water and ions rectal lumen. These predictions were
might then leave the rectal complex by tested by injecting solutions of extreme osway of the common malpighian trunk. molarity into various compartments and
Ramsay (1964) and Grimstone, Mullinger, observing the change in rate of flow and
and Ramsay (1968) have conducted an composition of fluid leaving the rectal
impressive series o£ experiments and ul- complex through the malpighian tubules.
trastructural observations which provide This fluid was collected by allowing the
•cut end of the common malpighian trunk
strong support for this hypothesis.
To study movement of ions between the to empty into a small vasoline cup built up
hemolymph and rectal complex, an in vi- around it. Extreme changes in osmolarity
tro preparation of this organ was used of the external medium produced by add(Fig. 10). The isolated preparation was ing 3 M sucrose or distilled water failed
placed under oxygenated mineral oil. to cause changes in turgor pressure of periMovement of substances into and out of tubular cells or in the rate of flow from the
the rectal complex could be estimated by common malpighian trunk. This confirms
following' changes in composition of small the relative impermeability of the perinephaliquots (0.3 ^1) of physiological salines ric membrane to water. When distilled
applied to the outside. After adding a water was injected into the rectal lumen or
chloride-free Ringer, the external concen- perinephric space, tubular flow increased
tration of potassium fell rapidly indicating three-to-four-fold, and osmolarity of the
absorption (Fig. 10). That this is due to collected fluid dropped. Similar results
net movement of potassium into the rectal were obtained after injecting 3 M sucrose
complex rather than outward movement of into the rectal lumen. These results
water is suggested by the lack of signifi- demonstrate the relatively high permeabilcant change in external osmotic pressure ity of the rectal epithelium, muscular layand concentration of sodium. In dehy- er, and tubule epithelium to water. They
drated mealworms the lumen of the also indicate that water which was reperirectal tubules is on average 49 mV moved from the rectal lumen without a
positive to the hemocoel and the concen- proportional amount of solute (the experitration of potassium at least ten times ex- ment with sucrose) does leave the rectal
ternal concentrations; therefore, the move- complex via the common malpighian trunk.
Finally the model for cryptonephridial
ment of potassium is an active process. Tn
support of this, inward movement of potas- function in the mealworm postulates high
sium is abolished by 10 mM sodium cyan- concentrations of solute within the perinephric space and perirectal tubules. Using
ide (Fig-10).
The rapid increase in external concen- micropuncture, Ramsay described the contration of chloride during these experi- centration gradients within the compartments indicates the permeability of the ments of the rectal complex. Tn each comleptophragmata to this anion. Ramsay con- partment (Fig. 11) the first figure is the
cluded that, normally, passive absorption freezing-point depression in hydrated
of chloride accompanies active transport of mealworms, and the second figure is the
potassium into the rectal complex since average maximum value under dehydrated
the observed electropotential difference of conditions. The following points should
49 mV is adequate to cause net diffusion be noted: (1) Under hydrated conditions
against the average three-fold gradient of the osmotic pressure of all compartments is
the same as the hemolymph and the aniconcentration observed for chloride.
mals
produce moist fecal pellets (osmotic
The hypothesis further requires that the
pressure
not measured). (2) As the osmotperinephric membrane be relatively imic
pressure
of the blood increases during
permeable to water while the rectal epwater-deprivation,
the concentration of solithelium, muscular layer, and perirectal tuute
in
all
compartments
of the rectal combule allow rapid passage of water from the
432
JOHX E. PHILLIPS
plex increases more rapidly and becomes
hyperosmotic to the hemolymph. (3) The
perirectal space is either isosmotic (anteri-
or) or hyposmotic (posterior) to the
lumen of the tubule. However, the solute
in tubular fluid consists almost completely
0.4pi.External Medium
Ligature
Rectal Complex
Malphigian
Fluid
Oxygenated
Mineral Oil
r-
_._,A
,/
O
o
o
K
Cl-Free
Ringer
Cl-Free Ringer
+ 10mM/l. KCN
20
0
20
TIME (minutes)
FIG. 10. The upper diagram illustrates the in vitro
preparation used to measure uptake of ions
from the hemolymph by the mealworm's rectal
complex. The lower graphs indicate the change in
ionic concentrations in the external medium with
time following the application of small aliquots of
chloride-free Ringer (with and without cyanide)
to the exterior of the isolated rectal complex (after
Grimstone, Mullinger, and Ramsay, 1968).
EXCRETION I N INSECTS
433
- A F . P . OF COMPARTMENTS - TENEBRIO-RECTAL COMPLEX
Perinephric
Membrane
0.7-7.3
(100% KCt)
Valve
0.7-2.5
Perinephric
0.7-4.8
(<40% K.Na.Cl)_ £ P f ^ _ ^ . «20% K.NajCl)
0.7-2.2
—.^.Direction of
flow
• Net HjO transfer
Rectal
Lumen
I - I• I»T
? — 10.5
Dry Feces
Anus
I* I
'Net KCI transfer
FIG. 11. Diagram of the mealworm's rectal complex in longitudinal section to illustrate the major
membranous barriers and compartments. The unbracketed figures in each compartment indicate
measured osmotic pressures expressed as freezingpoint depression (_,i°C). The first figure indicates the average value for hydra ted larvae, while
the figure after the arrows indicates the average
maximum values reached in dehydrated larvae.
Contributions of individual ions to the total osmotic pressure are shown in brackets in some cases.
Postulated sites and direction of potassium transport and volume flow o£ water across membranes
are indicated by large arrows. Movements of fluid
within compartments are indicated by broken lines
with small arrows. (Based on data from Ramsay,
1964, and Grimstone, Mullinger, and Ramsay,
19G8).
of potassium chloride (up to 2 M) whereas
inorganic monovalent ions account for
less than 40% of the osmotic pressure of
fluid in the perinephric space. The concentration of sodium is much higher in the
perinephric space than in the tubule's
lumen. These observations suggest that the
tubules stand in the same relationship to
perinephric space as free malpighian tubules of other insects do to the hemolymph.
That is, potassium is transported from the
perinephric space to the lumen of the
perirectal tubules, and water follows passively in isosmotic amounts. This in turn
leads to concentration of sodium and
larger organic molecules, which are thereby largely responsible for the high osmotic
pressure of the perinephric space. (4) In
all compartments (rectal lumen, perinephric space, lumen of perirectal tubule) of
the rectal complex, the osmotic pressure
increases dramatically in the anterior to
posterior direction. This stratification is
reminiscent of that observed in the medulla
and papillae of the mammalian kidney.
What enters the rectum from the hindgut
is a fluid isosmotic to the hemolymph.
This stratification facilitates the progressive removal of water from the fecal material as it moves posteriorly since it encoun-
434
JOHN E. PHILLIPS
ters increasingly greater tissue osmolality.
The origin and maintainance of this
stratification is possibly the following: Net
transport of potassium from the hcmocoel
into the rectal complex increases towards
the posterior end as indicated by the frequency of leptophragmata. Most of the
water is withdrawn from the fecal material
(which is isosmotic to the blood on entering the rectum) in the anterior part of the
rectum. This is facilitated by the high
osmolality of the tissues and by removal of
osinotically active solute due to active reabsorption of ions. [It seems reasonable to
postulate active ionic absorption aaoss the
anterior rectal epithelium since this has
been demonstrated in the locust (Phillips,
1964&); moreover, Patton and Craig
(1939) demonstrated the unidirectional
efflux of Na22 from rectal lumen to hemocoel in Tenebrio.] Water and ions are similarly drawn across the anterior wall of the
peri rectal tubule and follow the path of
least resistance out of the common trunk.
There is progressively less water available
for absorption in the rectal pellets as they
move posteriorly, whereas secretion o£
postassium chloride into the rectal complex is unchanged or increases. Hence, the
potassium chloride entering the rectal
complex is less diluted in the posterior
rectum.
Grimstone, Mullinger, and Ramsay
(1968) suggest that the gradient is further
maintained by a posteriorly-directed flow
of fluid in the perinephric space. This flow
would thus oppose the tendency of the
osmotic gradient within the perinephric
space to dissipate itself by diffusion. The
arrangement bears resemblance to the
countercurrent multiplier system of the
mammalian kidney, since the ability to
transport ions against a limited gradient
across the perirectal tubule's epithelium is
multiplied by the opposing direction of
fluid flow. This is likewise the case for
movement of water between rectal lumen
and perirectal tubules (Kirschner, 1967).
The role of the rectal epithelium remains to be considered. Both Saini (1962,
1964) and Ramsay (1964) observed that
the osmotic concentration of the perirectal
fluid is below that in the lumen of the
rectum. This iniitally suggested that the
rectal epithelium must make an active
contribution to reabsorption of water similar to that observed in other insects such as
the locust and cockroach. However, the
rectal epithelium of the mealworm lacks
many of the ultrastructural features typical
of other insect recta involved in transport
of ions and water (Grimstone, Mullinger,
and Ramsay, 1968). Nevertheless, according to these workers, "it seems unlikely
that the rectal epithelium is wholly passive toward movement of water through
it". They suggest that since absorption of
water in the posterior rectum takes place
from water vapor, and since the rectal epithelium is morphologically an invagination of the body surface, water transport, as
described by Beament (1965), may occur
in the posterior part of the rectum.
In conclusion, it is generally held at
present that the ability of the common
insect's rectum and the cyptonephridial
system of Tenebrio molitor to absorb
water from the lumen without the proportional absorption of solute is possibly due
to active recycling of ions within the rectal
epithelium and cryptonephridial complex,
respectively.
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