J. Exp. Biol. (1969). S1- 71-97
With 16 text-figures
Printed in Great Britain
71
SECRETION BY THE
MALPIGHIAN TUBULES OF RHODNIUS. THE
MOVEMENTS OF IONS AND WATER
BY S. H. P. MADDRELL
Department of Zoology, University of Massachusetts,
Amherst, Massachusetts, U.S.A., and Agricultural Research Council Unit at
Department of Zoology, Downing Street, Cambridge, England*
(Received 1 November 1968)
INTRODUCTION
The mechanism of operation of the Malpighian tubules of Rhodnius is of interest
for two main reasons. The first and more important one is that during diuresis they
can secrete at a very high rate of up to 3-3 /i\. min."1 cm."2. This can be compared with
the figure of 0-25 /A. min."1 cm."2 for the gall-bladder of the roach (Diamond, 1962a)
and I-I jul. min.- 1 cm."2 for the gall-bladder of the dog (Grim & Smith, 1957). The
rate of movement of ions is also impressive, e.g. chloride ions can move into the
lumen at a rate of about o-6o /i-equiv. min."1 cm."2 and sodium and potassium ions
each enter at about half this rate. The mechanism underlying this very high rate of
secretion is clearly of particular interest. Secondly, earlier work has shown that this
secretion is induced by the appearance in the insect's haemolymph of a very potent
diuretic hormone which speeds secretion by about one thousand times (Maddrell(
1962, 1963). Something is now known of the source of the hormone (Maddrell, 1963)
of the exact site of its release (Maddrell, 1966) and of the stimulus giving rise to
its release (Maddrell, 19646). Logically the next step is to consider how the diuretic
hormone has its effect.
It has long been pointed out that Malpighian tubules of insects are well suited for
the physiological investigation of secretory processes (Wigglesworth, 1948)—they
lie almost freely in the haemocoel of the insect and each consists practically entirely
of a single layer of cells arranged as a hollow tube as a single unravelled structure.
There is by now a considerable amount of information on secretion by Malpighian
tubules of insects, notably those of Carausius (Ramsay, 1953-6, 1958) and Calliphora
(Berridge, 1966a, b, 1967, 1968). Some work has already been done on Rhodnius
tubules (Ramsay, 1952; Maddrell, 1962, 1963, 1964a, b, 1966) but Ramsay moved on
to work with the tubules of Carausius to do his more detailed classic work with
in vitro tubules, while the author's work on Rhodnius tubules was directed towards
an investigation of their hormonal control, using them primarily as an assay method.
The particular advantage of working with Rhodnius tubules is that they secrete at
a rate about an order of magnitude higher than do those of other insects and to
anticipate the results to be presented in this and subsequent papers, the basis of
secretion by the tubules of Rhodnius is different in many respects from those of the
• Present address.
72
S. H. P. MADDRELL
other tubules so far examined. An additional advantage is that the interpretation of
results obtained with Rhodnius Malpighian tubules is likely to be facilitated by the
fact that the secretory part of the tubule consists of only one type of cell, whereas it
has been recently shown that the tubules in Calliphora consist of a heterogeneous
population of two cell types (Berridge & Oschman, 1969) and a similar interpretation
would appear to apply to a micrograph published in an earlier paper (Berridge, 1967).
L
Secreted
fluid
Glass rod
Glass rod
Fig. 1. The experimental arrangement used to investigate the secretory behaviour of lengths
of tubule isolated either (a) from the distal end or (6) from the more proximal portion of the
upper part of the Malpighian tubule.
MATERIALS AND METHODS
In these experiments 5th stage larvae of Rhodniusprolixus Stal taken from a laboratory culture were used.
There is good evidence that the Malpighian tubules of Rhodnius are divided physiologically and structurally into two regions (Wigglesworth, 19316, c; Ramsay, 1952).
The upper or distal lengths of tubules are thought to secrete fluid into the lumen
while the lower or proximal regions probably modify the fluid passing through them
possibly by reabsorption. The work described in this paper deals with the upper ends
of the tubules.
Isolated preparations of upper lengths of tubule were set up in drops of Ringer
solution under dense (s.g. = 0-865-0-890) liquid paraffin (mineral oil). Figure 1
shows the main features of the preparations. Pieces from the distal end discharging
from one cut end or pieces taken from farther down the tubule having two open ends
could be handled equally easily. Among the advantages of this preparation are the
Secretion by the Malpighian tubules of Rhodnius
73
ease of setting it up—it is fairly easy to run up to twenty at one time—and the fact
that one insect can supply enough lengths of tubule to make six or eight such preparations. The rate of secretion of such a preparation was followed at intervals by
measuring with an ocular micrometer the diameter of the drop of secretion produced
and calculating its volume, assuming it to be spherical. The solutions used to bathe
the tubules all contained glucose, for it has recently been shown that the Malpighian
tubules of Calliphora require an energy source in the medium (Berridge, 1966&).
Secretion by Rhodnius Malpighian tubules fails in about 10-30 min. without glucose,
whereas with it secretion persists for about 8-10 hr.* In most experiments the
solutions also had added to them diuretic hormone in the form of a part of a brei
of the mesothoracic ganglionic mass which contains large amounts of extractable
hormone (Maddrell, 1963). Secretion is about 1000 x faster after the addition of
hormone, so that the phenomenon becomes much more easily studied. It is possible
for example to measure the rate of secretion of a length of tubule as short as 0-4 mm.
which comprises fewer than twelve cells.
The compositions of the various Ringer's solutions used are summarized in Table 1.
Sodium and potassium concentrations in small samples of fluid were estimated
Table 1. The composition of the experimental solutions {concentrations in mMJl.)
1
NaCl
KC1
MgCl2
3
1290 1376 —
8-6 — 1376
85
85
85
CaCls
NaHCO 3
NaH2PO4
20
2-0
102
102
43
Glucose
KHCO 3
KH 2 PO 4
Na2SO4
K
2
34
—
—
—
43
34
—
—
—
—
—
_
—
6
7
8
9
—
—
—
—
—
_
_
_
_
_
_
—
—
—
—
—
—
—
—
—
—
—
550
—
102
102
—
102
5-0 120 10
34
—
—
810
QH
34
—
100
400
4-3
34
—
100
—
43
^ ^
~"
~~~
MgSO«
Sucrose
—
—
—
—
Na 2 HPO 4
NaNOa
KNO3
Mg(NO 3 ) 2
Ca(NO 3 ) 2
NaBr
KBr
MgBr2
—
—
_
—
—
—
_
_
i
2'O
—
—
2*O
~~~~
~~~
——
—
——
2*O
5 0 5 0 5 0
50
25
50
_
—
—
—
— '—
_
_
_
_
—
—
—
—
—
—
—
—
—
_
_
_
_
_
_
_
_
——
—
_
8-5
—
_
85
14
— —
_
_
8-5 —
2-0
20
20
—
—
—
—
—
—
—
—
—
34
—
—
—
34
—
—
—
34
—
—
—
^ ^
^ ^
^ ^
^ ^
^ ^
—
—
—
—
—
—
—
—
—
—
—
—
_
—
—
—
—
—
—
—
_
—
—
—
—
—
—
—
_
—
—
8-5
2-0
—
—
—
•"
™~
—
^ ^
—
—
—
—
—
—
_ 8
—
_
—
—
—
_
_
o
_
_
_
_
— 129 137-6 —
—
8-6 — 1376 —
—
8-5 8-5 8-5 —
—
2 0 2 0 2 0
—
—
—
—
— 129
_
_
_
8-6
_
_
_
8-5
—
—
• "
_
—
—
—
—
—
—
—
137*"
.
_
13
34
—
—
—
34
102
43
—
pi
\sl
12
34
—
—
—
34
—
—
—
4-3
11
—
/ u
vSoUi
—
34
—
—
—
*rr\
guV/4
10
120
—
34
102
43
—
5
—
2-0
—
4
——
TOTA
——
1j ^ U
NH4C1
_
_
NH 4 HCO 3
—
—
—
_
—
_
—
_
—
_
—
_
—
_
—
_
—
_
—
_
—
I 3 7 -6
NH 4 H 2 PO 4 —
NH 4 NO 3
—
Tris:HCI
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
_
—
—
_
—
—
_
—
—
—
15
—
—
15
102
—
102
43 43
— i 37 -6
— —
* In haemolymph alone secretion also persists for about 8-10 hr., but if glucose is added to the
haemolymph, secretion may go on for as long as 24 hr. Clearly there is some as yet unidentified ingredient
of haemolymph which allows secretion to persist longer than in its absence. This may be similar to
the factor required for maintained secretion by tubules of Carausius (Ramsay, 1953). The requirement
for this material is not as marked with Rhodnius tubules as it is with Carausius tubules.
74
S. H. P. MADDRELL
using a Unicam SP 600 Flame Spectrophotometer. This instrument is quite sensitive
so that it is feasible to measure both Na and K on a single sample of 100-200 nl.
However with such small samples of fluid the errors are fairly considerable—up to
10%—so that where possible samples of around 1 fi\. were taken. Concentrations
of ammonium ion were measured using basically the colorimetric method of Brown,
Duda, Korkes & Handler (1957) in conjunction with a Unicam Spectrophotometer.
Chloride concentrations were estimated either by a tracer method using 36C1 supplied
as Na36Cl or by potentiometric titration with silver nitrate solution (Ramsay, Brown &
Croghan, 1955).
Radioactive samples were counted on a Nuclear Chicago Mark 1 Scintillation
counter or by using a thin window GM tube and a Labgear Fast Dekatron Counter.
Errors were less than about 4%.
Osmotic concentrations were measured using the cryoscopic method of Ramsay &
Brown (1955). This method enables very small samples (< 1 nl.) to be used and
yet gives very accurate results (errors less than 1 %).
Secretion by Malpighian tubules is affected by temperature changes (Maddrell,
1964 a). All the present experiments were carried out at room temperature which
was usually near 240 C. in the period that the experiments covered. All experiments
which involved comparisons in rates were carried out at exactly the same temperature.
RESULTS
The first objective was to show that the properties of the upper regions of the
Malpighian tubule are uniform along its length so that any part of it could be used in
subsequent experiments. To this end the rates of secretion and ionic composition
of the fluid produced were measured for a series of tubule lengths taken from various
points along the whole upper length of the tubule. It was found that there were no
detectable differences either in rate or in the composition of the secreted fluid between
different lengths of the same tubule. However, it was noticeable that while the rate
of secretion not surprisingly was higher for longer lengths the relation was not a
linear one. The results are plotted in Fig. 2 to illustrate this point. Possible reasons
for such a relation are brought up in the discussion. The fluid secreted when the
tubules were bathed in standard Ringer's solution containing 8-6 mM/1. K, 143 HIM/
1. Na and 155 mM/1. Cl (as well as CaCl2, MgCl2, NaHCO3, Na2HPO4 and glucosesee Table 1) was found to contain about 100 mM/1. Na, 85 mM/1. K and 180 mM/1. Cl.
Cations
Some preliminary experiments carried out in 1963 had suggested that the Malpighian tubules of Rhodnius might be able to secrete slowly in a fluid that contained
no potassium ions. Accordingly, lengths of tubules were set up in potassium-free
solutions. Rather surprisingly it was found that not only could the tubules secrete
but that they secreted at the same rate as do tubules bathed in standard Ringer's
solution or in haemolymph (Table 2). Clearly Rhodnius Malpighian tubules are
very different from the tubules of Carausius and Calliphora which secrete only very
slowly in solutions containing only small amounts of potassium (Ramsay, 1955;
Berridge, 1967). Also, while the tubules of both Calliphora and Carausius secrete
Secretion by the Malpighian tubules of Rhodnius
75
faster when bathed in solutions containing elevated concentrations of potassium,
Rhodnius tubules secrete at a rate which is independent of the potassium concentration
over the range 0-150 mM/1. (the sodium concentration being reduced concomitantly).
These results are based on experiments with 100 lengths of tubule.
In contrast to its independence of the presence of potassium ions, secretion is
70 r
60
so
• | 40
c
o
•
I 30
**•
4)
I 20
?:•
10
0
5
10
15
20
25
30
Length of tubule (mm.)
Fig. 2. The rates of secretion of different lengths of tubule (at 24° C ) .
Table 2. The secretory performance of isolated tubules in solutions
of various compositions
Solutions
(taken from
Table 1)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
Short description
of solution
Control Ringer's solution
K-free
Na-free
SO4 solution
Cl-free, high bicarbonate
Cl-free, high phosphate
Nitrate solution
K-free nitrate solution
Na-free nitrate solution
Bromide solution
Choline solution
Tetraethylammonium solution
Ammonium solution
Ammonium nitrate solution
Rate of
secretion as %
of control
100
Number of
tubules
250
90-100
75
40
03
54
5
24
5°
1
12
45
5
45
15
100
12
O'l
o-i
40
35
12
8
6
6
12
8
76
S. H. P. MADDRELL
slowed considerably in solutions containing no sodium ions. Secretion by 50 lengths
of tubule slowed on average to 40 % of the rate that would be expected in standard
Ringer's solution (Table 2). Strikingly, the addition of as little as 1-1-5 mM/l. of
sodium ions caused a rapid acceleration of secretion to about the normal level (Fig. 3).
In choline chloride or tetra-ethyl ammonium chloride solutions, containing no
sodium nor potassium, secretion stops (Table 2). Clearly, neither of these large
monovalent cations nor calcium nor magnesium can substitute for sodium or potassium.
1-2
1-1
+ 1-5 mM/l. Na
10
0-9
0-8
07
13
06
OS
I 0-4
o
0-3
>
0-2
0-1
10
20
30
40
SO
Time (min.)
Fig. 3. The effect, on the rates of secretion of tubules bathed in sodium-free solution,
of adding a small amount of sodium containing solution.
Analyses of the secreted fluid were performed for tubules bathed in solutions
whose sodium and potassium concentrations were altered so that the sum of their
concentrations was kept constant at 150 mM/l. The results are plotted in Fig. 4.
Several points emerge from these results. The first is that the sum of the concentrations
of sodium and potassium in the secreted fluid is constant over the whole range of
concentrations. This can be explained by the facts that the osmolarity of the fluid
produced is always very near to that of the bathing fluid (see p. 83 and Fig. 11)
and that sodium and potassium are virtually the only cations secreted from these
solutions.
Secondly, the tubules' preferential handling of potassium is clearly seen—potassium
appears at a higher concentration in the secreted fluid than does sodium at all concentrations of potassium in the bathing fluid higher than about 15 mM/l.; with the
sodium and potassium concentrations in the bathing fluid equal, potassium is about
30% more concentrated in the secreted fluid than is sodium; and finally, potassium
Secretion by the Malpighian tubules of Rhodnius
77
is more concentrated in the secreted fluid than in the bathing fluid over the range
0-110 mM/1. while this is only true for sodium in the range 0-85 mM/1. Nevertheless,
it is also clear that sodium moves through the tubule wall relatively little slower
than does potassium. Furthermore, it will be recalled that the rate of secretion is not
lowered in solutions containing no potassium but it is lowered in sodium-free solutions.
10 20 30 40 50 60 70 80 90 100110120130140150
150140.130120110100 90 80 70 60 50 40 30 20 10 0
[Na + ]
Concentration in bathing fluid (min/l.)
Fig. 4. The dependence of the concentrations of sodium and potassium in the fluid secreted
by tubules on the concentrations of sodium and potassium in the bathing solution.
The third point is that when sodium is at quite low concentrations in the bathing
fluid it appears at a substantially elevated concentration in the secreted fluid (up to
30 times as concentrated as in the bathing fluid when this is near 1 mM/1., Fig. 4). The
same is true for potassium when it is at low concentration. This ability to concentrate
one ion from a solution containing a very large excess of the other leaves one with
the decided impression that there must be separate mechanisms for the handling of
sodium and for the handling of potassium.
It is also worth pointing out that at least over the range K = o, Na = 150 to
K = 149, Na = 1 mM/1., the rate of fluid secretion is constant. It follows that at
78
S. H. P. MADDRELL
low concentrations of potassium (below about 6-7 mM/1.) the rate of secretion of
sodium increases rapidly as the potassium concentration is reduced to nothing.
Similarly, the rate of secretion of potassium rapidly increases as the sodium concentration decreases below about 6-7 mM/1. (however, below about 1 mM/1. of sodium,
the rate of secretion of potassium decreases rapidly again). These points are brought
out in Fig. 5. Again, this is explicable if one supposes there to be separate mechanisms
10 r
10 20 30 40 50 60 70 80 90 100 110120130140150
Concentration in bathing fluid (cationic balance made up in
each case by the other ion) (mM/1.)
Fig. 5. Dependence of the rates of secretion of sodium and potassium on
their concentrations in the bathing fluid.
for the handling of sodium and potassium. At low concentrations of potassium
either the mechanism for potassium ions switches to handling sodium ions or the
sodium system is very much stimulated. A similar argument applies at low sodium
concentrations where presumably either the potassium system is stimulated or the
sodium machinery switches to moving potassium. At concentrations of sodium of
less than about 1 mM/1., potassium movement is abruptly slowed which suggests
that either the sodium system ceases to be able to move potassium or, perhaps less
likely, that the potassium system far from being stimulated by a further drop in
sodium concentration is partially inhibited in its action. A third possible explanation
that receives support from an examination of the electrical events involved (Maddrell,
Secretion by the Malpighian tubules of Rhodnius
79
1969), is that chloride movements are slowed at these very low concentrations of
sodium.
A further series of experiments was carried out in which the rate of secretion
was followed in potassium-free solutions containing varying amounts of sodium and
also in sodium-free solutions containing varying amounts of potassium, the balance
12s r
150
Concentration of sodium in the bathing fluid (mM/1.)
Fig. 6. The rates of fluid secretion of tubules bathed in (a) potassium-free solutions and (6)
solutions containing 10 mM/1. of potassium, at different concentrations of sodium (the
cationic balance being made up with choline ions). The points and the attached vertical
lines represent mean values and the extent of twice the standard error respectively.
100
_ + 4 6 m M / l . Na +
75
50
s
CO
pin
c
CO
Na+-free
+ 3 mM/1. Na'
da:
ion i
•4
Rin
+ 2 7 mM/l. Na +
25
25
50
75
100
125
150
Concentration of potassium in the bathing fluid (mM/1.)
Fig. 7. The rates of fluid secretion of tubules bathed (a) in sodium-free solutions at different
concentrations of potassium, and (6) in solutions containing 10 mM/1. of potassium but
with various amounts of sodium added. The points and the attached vertical lines represent
mean values and the extent of twice the standard error respectively.
8o
S. H. P. MADDRELL
of cationic concentration in each case being made up with choline ions. The results
are plotted in Figs. 6 and 7. In each case the rate of secretion is more or less proportional
to the concentration of the cation present. Also plotted in Figs. 6 and 7 are the results
of adding small amounts of the other cation. Such treatment results in a disproportionately large acceleration of the rate of secretion. Analyses of the fluid produced
by tubules secreting slowly in a fluid containing 13 mM/l. of sodium but no potassium
•200
r
180
160
3
•a
140
S 120 100 .3
*t'
80 60
40
c
20 h
o
O
150
25
50
75
100
125
125
100
75
50
25
150 [NH 4 + ]
0 [Na+]
Concentration in the bathing fluid (mM/l.)
Fig. 8. Concentrations of ammonium ions in fluid secreted by tubules bathed in solutions
containing sodium and ammonium ions. The discontinuous line is taken from Fig. 4 and gives
for comparison the values obtained for potassium in similar experiments.
were compared with analyses of the fluid produced by the same tubules after their
rate of secretion had been stimulated by the addition of 10 mM/l. of potassium.
These analyses taken together with the rates of secretion showed that the addition
of this small amount of potassium stimulates the inward movements not only of
potassium ions but also of sodium ions.
Rather surprisingly, the tubules would secrete at substantial rates (Table 2) in an
ammonium-based solution (Table 1) containing no sodium or potassium ions. Indeed
in a solution containing 148 mM/l. of ammonium ions and 3 mM/l. of sodium ions
the tubules secreted for up to an hour at the same rate that they would be expected
to achieve in standard Ringer's solution (eight tubules). The tubules seem to be
behaving as if potassium and ammonium ions were indistinguishable. The one
Secretion by the Malpighian tubules of Rhodnius
81
difference appears to be that secretion slows and stops sooner in ammonium-based
solutions than it does in conventional potassium-containing solutions. Probably the
lack of potassium ions has an effect on secretion by an indirect route. The analysis
of the fluid secreted by tubules bathed in ammonium-based solutions also supports
the suggestion that the Malpighian tubules of Rhodnius, at least in the short term,
behave as if they were unable to distinguish between ammonium and potassium
ions (Fig. 8).
The handling of unions
The dependence of the tubules on the presence of chloride ions is shown by the
fact that the rate of secretion was reduced practically to nothing (Table 2 and Fig. 9)
when the tubules were bathed in solutions where sulphate ions replaced chloride
ions (solution in Table 1). It may be argued that this is not surprising since the
solution now only contains as anions (other than sulphate ions, which are well known
to penetrate cell membranes only very slowly) 10-2 mM/1. of bicarbonate ions and
4-3 mM/1. of phosphate ions (Table 1). But even in solutions containing large concentrations of bicarbonate ions or of phosphate ions (solutions 5 and 6 in Table 1)
secretion was still slowed to about 5 % of its normal rate in high bicarbonate solutions
and was still slower in high phosphate solutions (Table 2). The lack of effect of
phosphate ions is not surprising in view of the very large size of the hydrated ion
(Eccles, 1964), but it can be contrasted with the situation found with the tubules
of Calliphora (Berridge, 1967) where phosphate ions readily support secretion and
indeed promote a faster flow than do chloride ions. Similarly, hydrated bicarbonate
ions although smaller than hydrated sulphate ions are still a good deal larger than
chloride ions. The apparent dependence of the tubules on the presence of chloride
ions may therefore reflect only the relatively small size of the hydrated chloride ion
compared with the other anions so far mentioned; chloride ions may be the only
ones capable of entering the cells fast enough.
Some attempts were therefore made to find smaller anions which will substitute
for chloride ions. Bromide ions which are very similar in size are perhaps not surprisingly an admirable substitute, and tubules secrete at normal rates when bathed
in a solution in which all the chloride ions are replaced by bromide ions (Table 2).
Nitrate ions, however, which although they are only slightly larger than chloride
ions (by 7% (Coombs, Eccles & Fatt, 1955)), support only a much reduced rate of
secretion by the Malpighian tubules of Rhodnius (Table 2).
So although sodium ions, whose hydrated radius is 43 % larger than that of hydrated
nitrate ions (Coombs et al. 1955), will support secretion at the normal rate, secretion
is considerably slowed in a nitrate-based solution. This does argue for a special role
for the chloride ion rather than for one based solely on its small size. By contrast, for
example, the inhibitory post-synaptic potential of the cat motoneurone where it is
thought that the ability of an ion to carry the current flowing through the membrane
is dependent solely on ion size, is supported equally well by chloride and nitrate
ions but not by the larger sodium ion (Coombs et al. 1955).
The rate of secretion by the tubules depends markedly on the concentration of
chloride ions in the bathing solution, the rate increasing rapidly as the concentration
increased from zero and reaching its maximum at about 30 mM/1. (Fig. 9). This
6
Exp. Biol. 51, 1
82
S. H. P. MADDRELL
may be contrasted with the performance of tubules bathed in sodium-free and
potassium-free solutions where the maximum rate is not reached until the concentrations of sodium and potassium reach 150 mivi/1. and about 130 ITIM/1. respectively
(Figs. 6, 7). In solutions containing both sodium and potassium, however, the
maximum rate is attained when the sum of the sodium and potassium concentrations
reaches about 60 mM/1. (see Fig. 6, for example).
The concentration of chloride ions in the secreted fluid is more or less constant
at chloride concentrations in the bathing fluid of 8 mM/1. or more. At lower concentrations the concentration in the secreted fluid is reduced (Fig. 10) but is still
125
a
x
<u ^
.100
C3 O
*
•
%
"So
75
50
o w
w C
T3 2
Pas
o
1 1
5 10
J
20
L
30
JL
50
J_
100
155
Concentration of chloride ions in bathing fluid (mM/1.)
Fig. 9. Rates of fluid secretion of tubules bathed in solutions containing different concentrations
of chloride ions. The points and the attached vertical lines represent mean values and the
extent of twice the standard error respectively.
many times higher than in the bathing fluid. Presumably at these very low concentrations, when secretion is much slowed, bicarbonate and/or phosphate ions make
up a large proportion of the anionic balance. Since Wigglesworth (1931a) has shown
that the urine of adult Rhodnius during diuresis contains the equivalent of 5-10 mM/1.
of bicarbonate ions and because bicarbonate solutions support secretion better than
do phosphate solutions (Table 2), it seems more likely that it is bicarbonate ions
which make up the anionic balance under these conditions.
At low chloride concentrations the rate of secretion becomes sensitive to the
potassium concentration so that at a concentration of 10 mM/1. of chloride ions,
secretion with 8-6 mM/1. of potassium ions is about half as fast as it is when the
potassium concentration is 25 mM/1. A somewhat similar result comes from experiments
with a potassium-free nitrate solution where secretion was much slower than in
a potassium-containing nitrate solution (Table 2). The omission of sodium from
a nitrate-based solution has no effect on the rate of secretion of tubules bathed in it.
These results with nitrate solutions stand in contrast to those with standard Ringer's
Secretion by the Malpighian tubules of Rhodnius
83
(solutions where it will be recalled that potassium-free solutions supported a normal
rate of secretion but that the rate of secretion in sodium-free solutions is reduced by
60%. All these cases serve to show how the effect of the presence or absence of one
ion may have an effect which is dependent on the concentration of another ion.
0
10 20 30 40 50 60 70 80 90 100110120130:140150
Concentration of chloride ions in the bathing fluid (mni/1.)
Fig. io. The dependence of the chloride concentration in the secreted fluid
on the concentration of chloride ions in the bathing fluid.
Water movements
In these experiments the osmotic concentration of the secreted fluid was measured
using tubules bathed in solutions of a wide range of osmolarities. Solutions of lower
osmolarity were produced by diluting standard Ringer's solution with distilled water.
More concentrated solutions were made up either by adding appropriate amounts of
sucrose to the standard solution, or by making up a solution twice as concentrated
as the standard and diluting it with distilled water. The results are presented in
Fig. 11, from which it is clear that over a wide range the osmotic concentration of
the secreted fluid is always almost iso-osmotic with the bathing fluid. In that the
majority of the points lie very slightly above the iso-osmotic line, it may be said that
the secreted fluid is marginally hyper-osmotic to the bathing fluid (by about o-oioo-ois0 C).
In other experiments the rates of secretion were measured, and these are plotted
6-2
84
S. H. P. MADDRELL
against the osmotic concentration of the bathing medium in Fig. 12. Clearly there isj
an almost inverse relation between rate of secretion and osmolarity of the bathing
medium. Taken with the fact that the secreted fluid is practically iso-osmotic with
the bathing fluid, it follows that the rate of secretion of solute varies relatively little
in solutions of differing osmolarities but that the rate of water movement is inversely
proportional to the osmolarity of the bathing solution. It is worth mentioning at
this point that the osmotic concentration of the secreted fluid does not depend on
the length of the tubule portion taken. Different lengths varying from 1 to 26 mm.
all produce fluids of the same osmotic concentration practically iso-osmotic with the
bathing solution (Table 3).
0
0-1 0-2 0-3 0-4 0-5 0-6 07 0-8 0-9 10 1-1 1-2 1-3 1-4 1-5.
Osmotic pressure of the bathing fluid (A° C.)
Fig. 11. Osmotic pressure of the fluid secreted by tubules bathed in solutions of different
osmotic pressures. The line is the iso-osmotic line.
Table 3. The osmotic concentrations of fluids secreted by
tubules of various lengths
Serial
I
2
3
4
Length of tubule
(mm.)
26-0
22-0
2-01
1-08
"secretion
^bathing solution
PC)
PC.)
c-6551
0657 I
06541
0-6677
0-647
Effects of drugs and hormones {other than the insect diuretic hormone)
A series of experiments investigated the effects of drugs on the secretory performance of the tubules. Not surprisingly it was found that metabolic poisons such
as cyanide, iodoacetate, azide and 2,4-dinitrophenol all stopped secretion quickly
Secretion by the Malpighian tubules of Rhodnius
85
when added to the bathing medium; Table 4 records their effectiveness in slowing
secretion. More interestingly, ouabain even at concentrations as high as io" 3 M/1.
had no effect on the rate of secretion (25 tubules) nor on the sodium and potassium
concentrations in the secreted fluid (10 tubules) even when the tubules were bathed
in ouabain-containing solutions for 1 hr. before fast secretion was started by adding
the diuretic hormone. Neither did such pretreatment with ouabain affect the
100
90
80
? 70
60
••\
*o 50
•\
§
40
o
2 30
20
"
-
*
•
-
.
.
10
0 0-1 0-2 0-3 0-4 OS 0-6 07 0-8 0-9 .10 M 1-2 1-3
Osmotic pressure of the bathing fluid (A° C.)
Fig. iz. Rates of fluid secretion in various solutions of differing osmotic pressures. The
discontinuous line is that of an exactly inverse relationship between rate of secretion and
osmotic concentration of the bathing fluid.
Table 4. The effectiveness of various inhibitors in slowing secretion
by isolated Malpighian tubules
Concentration giving
50 % reduction in rate
Inhibitor
Cyanide
Iodoacetate
Azide
2,4-dinitrophenol
Copper ions
Acetazolamide
Ouabain
of secretion (M/1.)
3X10- 4
3 x io-»
10- 3
10-6
35 x i o - 6
No effect at io~2
No effect at io~3
86
S. H. P. MADDRELL
acceleration produced by adding 1-2 mM/1. of sodium ions to tubules secreting in
a sodium-free solution (six tubules).
As reported elsewhere (Maddrell, Pilcher & Gardiner, 1969) 5-hydroxytryptamine
(serotonin) is a very potent stimulator of secretion by the Malpighian tubules, the
threshold concentration being about 5 x io~8 M/1. (55 tubules).
In ten cases the reaction of the tubules to mammalian ADH was tested. This
hormone had no effect on the rate of secretion nor on the composition of the secreted
fluid even at concentrations as high as 10 pressor units/ml.
Cupric ions, as in other systems, have an inhibitory effect, in this case that of
slowing secretion markedly at concentrations higher than 2 x io~5 M/1. (Fig. 13).
10
0-9
08
.•+5x10-5M/l. Cu2+
Untreated
controls
.07
06
0-5
0-4
0-3
0-2
0-1
J
10
1
I
20
I
'
30
'
•
40
0
10
Time (min.)
J
20
30
40
I
50
Fig. 13. The effect of copper ions on secretion.
Nervous supply to the tubules
During a preliminary examination of the tubules with the electron microscope
there were found very occasional apparent neurosecretory axon terminals swollen
with packed elementary neurosecretory granules and lying just beneath the basement
membrane of the tubule. The axons were traced a short way back from the tubules
and were found to arrive running alongside tracheae. Such a nervous supply to
Malpighian tubules of insects has not been reported before—in Rhodnius probably
because there are only rather few small axons and they run to the tubule together
with the much larger and more obvious elements of the tracheal supply. The possibility
that these axon endings might contain the diuretic hormone in appreciable amounts
was tested by making extracts of whole tubules and adding them to isolated tubules
to see if they would induce a fast flow of secretion. In only two cases of ten did such
an extract elicit an increase in rate of flow and the increase was small and short-lived.
The amount of diuresis-inducing substance in these two cases was calculated to be
less than o-oi % of the hormone extractable from one mesothoracic ganglionic mass.
It may be concluded that these neurosecretory axons do not provide the main channel
whereby the diuretic hormone reaches the Malpighian tubules. This is as expected,
because earlier work has shown that in vivo more than enough hormone to induce the
Secretion by the Malpigkian tubules of Rhodnius
87
maximum rate of secretion by the tubules is released into the insects' haemolymph
(Maddrell, 1964a) from the surface of the proximal regions of the abdominal nerves
(Maddrell, 1966). It is conceivable that the neurosecretory axons might play some
role in the extremely rapid onset of diuresis in vivo (Maddrell, 1964a) or of course
that they are involved in some other way in affecting the Malpighian tubules.
DISCUSSION
The Malpighian tubules of Rhodnius may appear to be rather catholic in their
ionic dependences. They will secrete in solutions containing either no sodium or no
potassium or if there are ammonium ions present they will secrete in the absence of
both sodium and potassium. Similarly, they can secrete at the normal rate in a solution
in which the chloride ions are replaced by bromide ions, and at a reduced but still
considerable rate in a nitrate-based solution, though they cannot secrete in a solution
in which chloride ions are replaced by bicarbonate or phosphate ions. It is even
possible to elicit about 35% of the normal rate of secretion in a solution based on
ammonium nitrate and containing no sodium, potassium or chloride ions (Table 2).
Nonetheless, under normal conditions the tubules preferentially handle sodium,
potassium and chloride ions, any of which are highly concentrated by the tubules if
they are present at low concentration in the bathing solution. The unusual ions that
they can handle are in fact rather similar in size to these preferred ions, bromide
and nitrate ions being respectively 2 % smaller and 7 % larger than the chloride ion
in aqueous solution and ammonium ions being 1 % smaller than potassium ions.
The tubules cannot handle choline or tetraethylammonium ions, nor calcium, magnesium, sulphate or phosphate ions and can move bicarbonate ions only very slowly
(Table 2).
Even without the electrical evidence to be presented in the next paper (Maddrell,
1969) it is arguable that there are separate pumps which handle sodium and potassium.
In particular this study has shown that each of these ions can be concentrated from
solutions containing large excesses of the other (Fig. 4). It is difficult to see how this
could be the case were there not separate mechanisms for handling these two ions.
While this argument has a good deal of force, one has to reconcile it with the observation that the rate of secretion of fluid in the presence of both ions is independent
of the concentration of one of them when the cationic balance is made up by the
other (Fig. 4) but dependent on the concentration of one when the other is replaced
by choline (Figs. 6, 7). In addition, in the absence of sodium, potassium secretion
is slower than is sodium secretion in the absence of potassium. There is a somewhat
similar situation in Calliphora where secretion by the tubules is much slower at
low concentrations of potassium with the cationic balance made up with choline
ions than it is if the other monovalent cation is sodium (Berridge, 1967, 1968).
Berridge has suggested as a working hypothesis in this case that the movement of
the ions across the tubule wall might involve two steps, the first being across the
blood-side cell membrane which carries a sodium-potassium exchange pump. This
pump controls access to the luminal-side membrane on which is sited an electrogenic
potassium pump and through which the ions move the second step. This neatly
explains the accelerating effect of adding sodium ions to tubules secreting in a low-
88
S. H. P. MADDRELL
potassium/high-choline solution; the sodium ions can be involved in the sodium,
potassium exchange pump (though they would first have to enter the cells) and
potassium thus enters the cells faster and becomes available in higher concentration
to the electrogenic pump, which is the rate-defining step. Secretion by Calliphora
tubules is not sensitive to ouabain (Berridge, 1968), even in a solution containing
84 ITIM/1. sodium and 56 mM/1. potassium, so that it follows that the blood-side
sodium/potassium exchange pump is not sensitive to ouabain.
The situation in Rhodnius is different in that the addition of a solution containing
a low concentration of either cation to a solution containing a low concentration of
the other cation produces an acceleration of secretion of both cations (p. 80), so that
an explanation based on an exchange pump will not fit the facts. Such an exchange
pump would, however, offer an explanation for the remarkable accelerating effect
of adding 1 mM/1. of sodium to tubules secreting in high-potassium/sodium-free
solution. Here again, as in Calliphora, ouabain has no effect, so that such a pump, if it
exists, is ouabain-insensitive. As far as cations are concerned one can say that the
presence of one cation accelerates the transfer of the other across the tubule wall
from a low concentration, but decelerates its transfer from a high concentration; at
a low concentration of the other cation it acts synergistically but at a high concentration it is in competition with the other. As an explanation of this one could
imagine that transfer across the tubule wall might occur in two steps in series. The
first, which controls access to the second, would allow sodium and potassium through
more quickly when both are present than if one were absent, in which case entry
would merely be by diffusion. This would explain the synergistic action of the
cations at low concentrations. The second step might involve pumps capable of
moving the ions actively. The pump for one cation would also be able to move the
other but only when its own cation was at a very low concentration and when the
other pump was saturated. This would explain why an increase in a very low ambient
concentration of, say, sodium would slow movement of potassium from a high ambient
concentration; the extra sodium ions would compete successfully with the potassium
ions present for the attention of the sodium pump. When only one cation was present,
there would be no facilitated passage through the first step and secretion would be
proportional to the movement of ions by simple diffusion through the first step;
the rate of secretion would increase in a more or less linear fashion with increase in
concentration of the cation, as indeed is observed (Figs. 5 and 6).
This model as far as it goes is little more than a summary of the facts. Further, it
does not explain the accelerating effect of adding a solution containing sodium ions
to a bathing solution containing no sodium and a high concentration of potassium.
Nor does it explain why the secretion rate should be dependent on the potassium
concentration at low chloride concentration or in chloride-free nitrate-based solutions.
It is clear that the model is too simple and that the level of chloride concentration
is in some way involved in the movements of the cations. Further speculation is
left until the electrical evidence to be presented in the next paper (Maddrell, 1969)
has been considered.
The manner in which the tubules handle anions leaves one with the impression
that chloride ions are especially important. Nitrate ions though rather similar in
size to chloride ions, nonetheless, are a rather inferior substitute for chloride ions.
Secretion by the Malpighian tubules of Rhodnius
89
Of the other anions tested only bromide ions, which are rather similar in size and
chemical properties to chloride ions, could replace chloride ions. The evidence suggests
at least the possibility of a specific transporting mechanism, perhaps a pump for
chloride ions.
The movement of water through the tubule cells is probably consequent upon the
movement of ions. Berridge (1967, 1968; Berridge & Oschman, 1969) has fully
discussed the possible mechanisms whereby this might occur, and has pointed out
that the basal infoldings and luminal microvilli of the cells of Malpighian tubules
are well suited to a coupling of solute and water transport in isotonic proportions
by means of standing osmotic gradients (Diamond & Tormey, 1966; Diamond &
Bossert, 1967) within the infoldings and in the spaces between the microvilli. The
observations that in Rhodnius the tubules secrete a fluid which is practically isotonic
to the bathing fluid (over the range A = o-i3°-i>3° C , Fig. 11), and that the rate
of fluid secretion is inversely proportional to the osmotic pressure (Fig. 12) are entirely
consistent with the suggestion that water movement follows, and is osmotically
linked with, the movement of ions. The observation that the osmotic concentration
of the fluid secreted by extremely short lengths of tubule is the same as that produced
by longer lengths (p. 84) argues that the fluid emerging into the lumen from one
cell is already isotonic and shows that the standing osmotic gradient that couples
ion and water movement is not, as might have been thought possible, along the
lumen of the tubule with the distal contents more concentrated than the fluid further
down the tubule.
From the standpoint of comparative physiology the most striking thing to emerge
from this work is that the basic mechanism underlying the secretion is so different
from that of other insects' Malpighian tubules. In other insects the central role of
potassium ions is well established (Ramsay, 1953; Berridge, 1967) and this has led
to the suggestion that potassium secretion is the prime mover in generating the
flow of urine (Ramsay, 1956). The rate of secretion in these tubules is very dependent
on the potassium content of the bathing solution and it falls to a very low level in
the absence of potassium ions. By contrast the rate of secretion by Rhodnius tubules
is unaffected by changes in the potassium concentration and proceeds at a normal
rate in the absence of potassium ions from the bathing solution. Again, the tubules
of Calliphora secrete at their maximum rate in a sodium-free solution containing
potassium as the sole monovalent cation (Berridge, 1967, 1968), whereas the tubules
of Rhodnius are very sensitive to lowered concentrations of sodium, the rate of secretion
being reduced to 40 % of normal by the exclusion of sodium ions from the bathing
medium (Fig. 5). Figures 14 and 15 compare the ways in which Rhodnius and Calliphora
handle sodium and potassium ions and they clearly show that the basic difference is
the ability of Rhodnius tubules to transport sodium ions at speed, so fast in fact that
fluid secretion is not slowed in the absence of potassium. Secretion by the tubules
of Calliphora persists in solutions containing a wide variety of anions (Berridge, 1967);
in Rhodnius secretion requires the presence of chloride (or bromide) ions and falls
to very low levels in solutions containing bicarbonate and/or phosphate ions. Nitrate
ions can substitute for chloride ions to a limited extent but these ions are nearly the
same size as chloride ions. Copper ions stop secretion by the tubules of Rhodnius
(Fig. 13), whereas these ions are said not to affect the transfer of phosphate or
9o
S. H. P. MADDRELL
potassium ions by the tubules of Calliphora (Berridge, 1967) although they appear
(perhaps at a higher concentration?) to check net solute transport so that fluid secretion
stops (Berridge, 1968). In their ability to handle ammonium ions apparently as
100 125 150 [K+]
75
125
100
75
0 20 40 60 80 100 120 140 [K+]
50 25
0 [Na+]
140 120 100 80
Concentration in the bathing fluid (mM/l.)
60 40
20
0 [Na+]
Fig. 14 A comparison of the ways in which the Malpighian tubules of Rhodnius and Calliphora
handle sodium and potassium ions. Figures for tubules of Calliphora taken from Berridge
(1968).
0
25
50
75
100
125 150
20 40
60
80 100 120 140
Concentration in the bathing fluid (cationic balance made up in each case by the other ion) (mln/1.)
Fig. 15. A comparison of the rates at which the Malpighian tubules of Rhodnius and Calliphora
secrete potassium and sodium at different concentrations of these ions in the bathing fluids.
Figures for Calliphora are calculated from data of Berridge (1968).
Secretion by the Malpighian tubules of Rhodnius
91
indistinguishable from potassium ions (at least for periods of up to an hour) the
Malpighian tubules of Rhodnius again differ sharply from those of Calliphora (Berridge, 1967) and Carausius* which cannot, and indeed they differ from the midgut
of Hyalophora cecropia where again ammonium ions will not substitute for potassium
(Nedergaard & Harvey, 1968), although in this case the pH of the solution used was
fairly high, so that there may have been present a small amount of ammonia which
being toxic may have masked any substitution (Harvey, personal communication).
To some extent these differences from other Malpighian tubules might be expected
from the differences in function. Rhodnius tubules are called upon to secrete at very
high rates only for a matter of hours and this is followed by a period as long as months
during which secretion is very slow indeed. The function of the tubules of most other
insects is in general to provide a much more constant flow of secretion at a rate an
order of magnitude slower than is the case of the fast secretion in Rhodnius. This can
be correlated with the much greater effect the diuretic hormone of Rhodnius has on
secretion (an acceleration of about 1000 times) than has that of Dysdercus, for example
(an acceleration about 6 times) (Berridge, 1966a). Perhaps potassium secretion is
a feature of tubules which secrete at ordinary rates, but secretion at very elevated
rates involves the transport of two or more ions in parallel. In this connexion it will
be recalled that the very fast fluid movement in the gall-bladder of vertebrates is
generated by a simultaneous (and linked) transport of sodium and chloride ions in
the same direction (Diamond, 19626). It appears from the published micrographs of
the Malpighian tubules of various insects that the microvilli on the luminal surface
of the tubule cells of Rhodnius are exceptionally long (Wigglesworth & Salpeter, 1962)
so that the surface area across which it is speculated that active pumping occurs
(Berridge & Oschman, 1969) may be higher than in other species; this would also
help to explain the very high rates of secretion by Rhodnius tubules.
It is worth examining some of the consequences of this very high rate of secretion.
It can be calculated that at a temperature of 370 C. in vivo fluid enters the tubules
at a rate which corresponds to the removal in each minute of a layer of fluid surrounding
the tubule to an average thickness of 26 /i. This point is illustrated in Fig. 16, which
also shows how dilated the tubule is during fast secretion. It also follows that the
contents of the lumen are replaced on average every 30 sec. and that fluid leaves the
upper end of the tubule flowing at nearly 1 mm./sec. If this fluid passes through the
cells it means that a volume of fluid equivalent to the volume of the cells goes through
them every 16 sec. Even in an in vitro preparation at 24° C. with a 5 mm. length
of tubule a layer of fluid 11 fi deep is taken into the tubule every minute, replacing
the contents of the lumen every 90 sec. This centripetal flow of fluid, while it is not
fast enough to have more than a negligible effect on the rate of movement of ions to
the surface of the tubule, would be fast enough to have an appreciable effect on the
supply of oxygen. This may help to explain why it is not necessary to oxygenate or
stir the drop of solution bathing the tubule; there is a continuous flow of oxygencontaining solution towards the tubule without it.
These very high rates of passage of chloride, potassium and sodium ions and of
water raises the problem of how the cells maintain their integrity in the face of such
intense traffic through them of ions and water whose intracellular balance is thought
• Miss D. E. M. Pilcher, personal communication.
92
S. H. P. MADDRELL
to be vital to the cell. One can speculate that either the organelles and cytoplasm are
very tolerant or possibly they are insulated or compartmentalized in some way from
regions of the cell where the traffic flow occurs. There is also the possibility that
some of the movement might occur along a route not involving passage through the
cell interior. A model for this type of movement is presented in an intriguing paper
by Cereijido & Rotunno (1968) who suggest that ions may cross epithelia carried in
the cell membrane. However, the cells of the Malpighian tubules of Rhodnius appear
structurally modified to admit material at one face and discharge it from the other;
they have pronounced basal infoldings and very many close-packed microvilli on
Fig. 16. Tracings of cross-sections of upper parts of Malpighian tubules of Rhodnius. Both
tubules were taken from the same insect and treated identically except that the tubule at the
left was bathed in Ringer's solution to which had been added the diuretic hormone, while the
tubule on the right was bathed in a control drop of Ringer's solution which contained no
diuretic hormone. Immediately prior to its fixation the tubule on the left was secreting at
a rate of 53 nl./min. The line drawn round the profile of this tubule indicates the extent of
the amount of fluid outside the tubule which would enter it in one minute during fast secretion
in vivo: x 1000.
the apical surface, nearly all of them containing a long mitochondrion (Wigglesworth &
Salpeter, 1962). There is already evidence that water flow in the gall-bladder is
routed through the epithelial cells (Tormey & Diamond, 1967), so that it does seem
probable that the water goes through the cells of the Malpighian tubules. Indeed, in
view of the massive volume passing per unit time it is difficult to see how it could
follow any route with a much smaller cross-sectional area than that of the route
through the cells. Nonetheless, the ions might conceivably follow an extracytoplasmic
route, perhaps in the cell membrane, to emerge from the surface of the microvilli and
thus produce an osmotically coupled flow of water through the cell in the manner
discussed earlier. It might be possible to test this idea by allowing the tubules to
secrete in a solution containing a tracer ion such as 36C1 until the specific activity of
the secreted fluid reached 100%, when the specific activity of the chloride in the
cells could be tested. If this were substantially below 100%, it would show that the-
Secretion by the Malpighian tubules of Rhodnius
93
transport did not involve all the cell volume, suggesting either that transport is
compartmentalized within (or excluded from) the cytoplasm or, perhaps less likely,
that not all the cells are involved in transport.
Long lengths of tubule do not, in vitro, secrete proportionately faster than do short
lengths (p. 74). This might be due to the pressure developed in the lumen which would
be slightly greater the faster the flow of fluid. However, just such a non-linearity is
also seen with lengths of tubule bathed in sodium-free solutions where the rates of
secretion are only 40 % of the rates expected in standard solutions. This argues that
the non-linearity is not due to higher pressures in longer lengths of tubule but to
some other factor. This might be, for example, a reflexion of the greater oxygen
needs of a long length of tubule in that diffusion and the centripetal flow of fluid
might not supply oxygen fast enough for long tubules in a small drop of fluid. In any
case it will be interesting to see if secretion is affected by experimentally applied low
oxygen tensions. Other transport systems in insects such as the midgut of larvae of
Hyalophora cecropia are extremely sensitive to lowered oxygen tensions (Haskell,
Clemons & Harvey, 1965).
Table 5. Comparison of the composition of the fluid secreted by the upper part
of the Malpighian tubule with the fluid excreted by the insect
Fluid leaving the
Fluid leaving the
upper tubule*
insecff(concentrations in mM/1.) (concentrations in miu/l.)
Sodium
Potassium
Chloride
Osmotic pressure
(as mM/1. NaCl)
IOO
135
85
180
185
144
149
* Figures from the present work.
t
Figures from Maddrell (1964a) and Ramsay (1952).
Estimated.
By comparing the composition of the fluid produced by the secretory upper part
of the Rhodnius Malpighian tubule with the composition of the urine as finally
voided from the insect, one can estimate the contribution of the other parts of the
excretory system, the lower part of the tubule and the rectum. As has been argued
before (Maddrell, 1963), the contribution of the rectum is probably a relatively
minor one because of its low surface area/volume ratio and the speed with which the
urine flows through it. It seems probable that most of the differences can be
attributed to the activity of the lower end of the tubule with perhaps some lesser
contribution from the ampullae (Wigglesworth, 193ib) through which the fluid
flows to enter the rectum. The differences in composition of the fluids leaving the
upper end of the tubule and leaving the insect are summarized in Table 5. What one
does not know is whether or not water is added to or removed from the fluid
as it passes through the rest of the excretory system. However, none of twenty
isolated preparations of the lower ends of tubules secreted any fluid at all, so that
it is unlikely that any fluid is added. And from the high rates of fluid excretion
observed in intact insects it seems likely that fluid is not absorbed at a high rate from
the fluid delivered by the upper lengths of tubule. From the figures in Table 5 one
94
S. H. P. MADDRELL
can estimate that the lower parts of the tubules remove potassium and chloride ions
from the fluid passing through them, and depending on how fast water follows this
movement sodium ions may or may not be added to the fluid in the lumen. For
example, if the lower tubules removed 75 fiM of potassium chloride from every
millilitre of fluid entering them and as a result 260 /A. of water followed this movement,
this would leave behind in the lumen a fluid like that actually excreted. At the other
extreme, if no water moved out of the tubule the actually excreted fluid could be
achieved by the removal of 71 mM/1. of potassium and the addition of 35 ITIM/I. of
sodium (with of course the net removal of 36 mM/1. of chloride ions to maintain
electrical neutrality). If the former interpretation is correct, then the figures for the
secretory ability of the upper part of the tubule given on p. 71 are in error and in
fact the upper tubule would have to secrete fluid at a rate of up to 4-4 [i\. min."1 cm."2
with chloride ions entering at up to o-8o /i-equiv. min."1 cm.~2 to give the same final
rate of excretion. In either case one can calculate that at 240 C. potassium ions are
removed from the lumen of the lower part of the tubule at a rate equivalent to about
0-25 /i-equiv. min."1 cm.~2 which is slower than, say, chloride ions are secreted at
the upper end (about 0-37 /i-equiv. min."1 cm."2 at 24° C). This might reflect the
fact that the microvilli on the luminal side of the cells of the lower tubule are more
widely separated and so present a smaller surface area to the lumen per unit length
of tubule than do the microvilli of the upper part of the tubule (Wigglesworth &
Salpeter, 1962). It is clear that a study of the activity of the lower part of the tubule
would be most rewarding—it moves ions very actively, consists of a single layer
of cells of one type only and presumably is stimulated in its action by the diuretic
hormone in the same dramatic way that the upper tubule is.
The osmotic concentration of the urine produced by fed Rhodnius depends on the
temperature and is higher the higher the temperature (Maddrell, 1964a). Since the
upper tubule produces an isotonic fluid at temperatures between 18 and 240 C , it
probably also does so at elevated temperatures. It follows then that the activity of
the lower part of the tubule has less effect at a higher temperature and this might be
a result of the higher rate of secretion by the upper tubule—the lower tubule might
not be able to keep pace. This could easily be tested in vitro by investigating the
effects of perfusing the lower tubule at various rates.
How do the present results compare with those of Ramsay (1952)? As far as the
upper part of the tubule is concerned Ramsay found that in vivo the fluid it contained
had a much higher concentration of potassium and a lower concentration of sodium
than did the haemolymph—this was for a range of concentrations in the haemolymph
of from 140 to 188 mM/1. of sodium and from 4 to 23 mM/1. of potassium. These
results compare well with those shown in Fig. 4. However, Ramsay also found that
in seven cases of nine (four cases out of four of those measured during diuresis) that
the osmotic concentration of the fluid in the upper tubule was significantly higher
than that of the haemolymph, whereas the present work has shown that in vitro
tubules produce afluidwhich is practically iso-osmotic with the bathing fluid (Fig. 11).
A possible reason for this discrepancy could be that Ramsay collected fluid from the
tubules by pushing a cannula through the wall of the tubule until it filled the lumen.
This treatment and the fact that the internal diameter of the cannula was much less
than that of the tubule must have caused the internal pressure in the tubule to rise.
Secretion by the Malpighian tubules of Rhodnius
95
There is electrical evidence that an increase in internal pressure alters the functioning
of the tubules (Maddrell, 1969). Also if one supposes that the tubules work by
pumping ions into the long spaces between the luminal microvilli to create a standing
osmotic gradient which could give rise to an isotonic flow of fluid (Diamond &
Bossert, 1967), then an increase in internal pressure might well lead to a greater
separation of the microvilli, which change in geometry would be predicted to lead to
the production of a hypertonic rather than isotonic secretion. The observation that
in vitro the tubules produce a fluid with a slight tendency towards hypertonicity
(Fig. 11) suggests that if the tubules do use a standing osmotic gradient in the manner
described then the osmotic concentration of the secreted fluid might well be sensitive
to increased pressure in the lumen of the tubule.
It is noticeable, for instance, that some of Ramsay's fluid samples (two cases of
five) from Rhodnius upper tubules at a time when diuresis must have slackened were
more or less iso-osmotic with the haemolymph; if the tubules were secreting at a much
lower rate than during diuresis, the increase in pressure due to secretion would be
relatively low. This can be contrasted with the tubules sampled during diuresis, all
four of which contained hypertonic fluid. These results are consistent with the
explanation advanced above but, of course, do not go very far towards proving it.
The suggestion that the osmotic concentration of the secreted fluid might be sensitive
to increased internal pressure could be tested by forcing tubules to secrete against
various pressures applied as a head of fluid through a cannula.
This work has its roots in some preliminary experiments done while the author
held a National Research Council of Canada Postdoctorate Research Fellowship at
the Department of Biology, Dalhousie University, Halifax, Nova Scotia, Canada.
It is a pleasure to record my thanks to Professor K. E. von Maltzahn for his hospitality.
Much of the work described in this paper was done at Professor W. R. Harvey's
laboratory in the Department of Zoology, University of Massachusetts, supported
by a generous grant from the U.S. Public Health Service (grant AI-04291-06 to
Professor Harvey). It was continued at the Department of Zoology, University of
Cambridge, while the author was a Research Fellow of Gonville and Caius College
and of the Agricultural Research Council. I thank these bodies for their generous
support. I am grateful also to Professor W. R. Harvey, Dr R. B. Moreton and J. L.
Wood who kindly read the manuscript.
SUMMARY
1. The Malpighian tubules of Rhodnius will secrete at a normal rate in solutions
containing no potassium ions and the rate is unaffected by changes in the potassium
concentration, when the balance of cationic concentration is made up by sodium
ions.
2. In the absence of sodium ions the rate of secretion is much reduced. The
addition of very small amounts of sodium-containing solution brings about an
abrupt recovery in the rate and thereafter the rate is unaffected by further increases
in the sodium concentration.
3. In solutions containing either sodium or potassium with choline making up
the balance of monovalent cations, the rate of fluid secretion depends linearly on
the concentration of either sodium or potassium.
96
S. H. P. MADDRELL
4. The tubules will concentrate either sodium or potassium when they are present
at low concentration in the bathing fluid, even in the face of a very much larger
concentration of the other cation. This suggests that there are separate mechanisms
for the handling of these two ions.
5. Secretion can be supported by a solution containing ammonium ions in place
of sodium and potassium. The tubules behave, at least in the short term, as if they
were unable to distinguish ammonium from potassium.
6. Chloride ions appear to play a special role in that only bromide ions and, to
a limited extent, nitrate ions will substitute for them. The rate of secretion depends
linearly on the chloride concentration.
7. The tubules secrete a fluid which is practically iso-osmotic to the bathing
fluid. The rate of secretion depends inversely on the osmolarity of the bathing fluid.
The rate of movement of solute is little affected by these changes of osmolarity.
It appears that water movements follow, and are closely linked with, solute movements.
8. Copper, cyanide, iodoacetate and azide ions and 2,4-dinitrophenol all stop
secretion when added to the bathing medium. Ouabain, acetazolamide and mammalian
ADH all have no effect on the rate of secretion and ouabain has no effect on the
composition of the secreted fluid. 5-Hydroxytryptamine (serotonin) will stimulate
a rapid flow of secretion.
9. Apparent neurosecretory axons have been found which supply the tubules.
They do not contain enough diuretic activity to do more than play a minor role in
diuresis. They may contribute to the rapid onset of diuresis or may affect the Malpighian tubules in some other way.
10. The evidence suggests that the tubules function by secreting chloride, potassium and sodium ions into the lumen (it is speculated that this may conceivably
involve the active transport of all three ions) and that water movements closely follow
these ion movements so that a rapid flow of iso-osmotic fluid is achieved.
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