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Elaborations of the basal surface of the cells of the Malpighian tubules of an insect
TISSUE 0
& CELL
1985 Longman
1985 17 (6) 865-881 Group
Ltd
M. J. O’DONNELL*,
S. H. P. MADDRELL, H. le B. SKAER and J. B. HARRISON
ELABORATIONS OF THE BASAL THE CELLS OF THE MALPIGHIAN AN INSECT Key words:
Insect.
Malpighian
tubule,
membrane
SURFACE OF TUBULES OF
architecture.
electrical
potentials.
mtrxellu-
lar regionalization ABSTRACT.
Electrical
mcasurcments
microelectrodcs
showed
that
have superficial
regions
different
membrane standard
to changes
chloride
by different
of the cells revealed with
was examined
similar
by marking
regions
from
was
by 30-40
concentration
many long cellular from
also differed projections
neighbouring
cells with
usmg mtraccllular
tubules body
of Rhodnru~
of the cell.
mV.
but
of the bathmg
to the drug. furosemide.
projections individual
the main
smaller
concentration
responses
and resistance
of the Malpighlan
properties
the potassium
in the external
and intcrdigitate
digitation
superficial
potential
parts
in electrxal
on changing
that was remforced
of the basal regions surface
m these
depolarization
response fmding
potential
of membrane
the fluid-secreting
alcian
medium.
in the two Electron
a
The
regions.
a
muoscopy
that run parallel cells. The
The
showed
to the cell
degree
blue or by horseradIsh
of mtcrperox-
ldase Injection. A survey similar
of the published
projectlons
on
their
micrographs basal
Introduction The cells of insect Malpighian tubules have apical microvilli projecting into the lumen (Beams et al., 1955; Meyer, 1957; Smith and Littau, 1960: Wigglesworth and Salpeter. 1962). On the basal, haemolymph-facing side, the cells also have elaborations of the plasma membrane. It has always been supposed that these were no more than simple pleating of the cell surface (Wigglesworth and Salpeter, 1962; Maddrell. 1980; Phillips, 1982). We now provide evidence to show that. in Rho&Gus, the cells have a complex array of long basal projections which run parallel to the ceil surface under the basement membrane. A survey of the literature shows that similar basal projections are found in the Malpighian tubules of many, though not all. other insects. Agricultural
and Food
Research
Neurophysiology
and
Zoology,
Street,
Downing
*Present University. Keceivcd
Cambridge
address:
Department
Downsvicw.
Ontario,
I8 June
Council
Pharmacology.
14X5.
Unit
of Insect
Department CB2 3EJ. of
Canada
Biology, M3J
of
England. York 1P3.
of insect
surfaces
and
Malpighian
not
the
tubules
simple
basal
hhows that infoldings
most
hd\c
prewou4)
These morphological findings provide an explanation for otherwise puzzling electrophysiological results. The basal membrane potential recorded by an intracellular microelectrode whose-tip was positioned close to the basal surface of the cell was as much as 40 mV less negative than the potential of about -65 mV recorded when the electrode tip was advanced into the main body of the cell cytoplasm. We suggest that such low basal membrane potentials are seen when the microelectrode tip is located in a long basal projection.
Materials and Methods Malpighian tubules were dissected from fifth instar Rhodnius prolixus Stal from a laboratory culture. Each tubule consists of a single layer of squamous epithelial cells forming a blind-ended tube approximately 90 Nm in diameter and 45 mm in length. We have worked only on the upper two-thirds of the tubule, which contains a single cell type, the fluidsecretingcells. When required. fast fluid
866
O’DONNELL,
secretion was stimulated by the inclusion of 10e6 M 5-hydroxytryptamine (5HT) in the bathing medium (Maddrell et al., 1969). Electrical measurements
Dissected tubules were placed in a perfusion apparatus which permitted rapid changes of the bathing fluid (Berridge and Prince, 1972; O’Donnell and Maddrell, 1984). The chamber consisted of a central compartment containing liquid paraffin (mineral oil) and two lateral compartments filled with saline, one of which could be perfused. The Malpighian tubule was arranged between the three compartments so that the blind end was in the perfusable compartment with the open end pulled through notches in the central compartment and secured with silicone grease to the wall of the non-perfused saline compartment. The tubule in this non-perfused compartment was nicked with ultrafine scissors so that the luminal contents were continuous with the saline. The basal cell membrane potential was measured from the potential difference between an intracellular microelectrode and a reference electrode in the perfused bath. The reference electrode was an agar bridge connected through a calomel half cell to ground. Thin-walled glass microelectrodes of electrical resistance of 8-10 MQ were mounted in a holder connected to an amplifier of high input impedance (lOn Q; W.P. Instruments, Inc.). The microelectrode holder and preamplifier probe were mounted on a Leitz micromanipulator. Initial contact of the microelectrode with the epithelium produced a visible dimpling of the cell surface; the microelectrode was then retracted and re-advanced more slowly towards the cell surface until there was a slight change in potential (<2 mV) and a coincident rise in electrode resistance; observation with a dissecting microscope (at 50 times) showed no apparent dimpling. Gentle tapping of the baseplate of the micromanipulator caused the electrode to ‘pop’ into the cell. Alternatively, the capacitance compensation control was momentarily turned to maximum. This caused the amplifier to oscillate and the oscillations acted as a microcautery (Purves, 1981) which facilitated microelectrode entry into the cell. Use of this technique (hereafter referred to as micorocautery) was identifi-
MADDRELL,
SKAER
AND HARRISON
able by vertical lines produced on recordings of the potential (Fig. la). The input resistance of the basal membrane was determined by injecting transepithelial current pulses and measuring the resulting deflection of the intracellular potential. The deflections decreased with distance from the interface between the paraffin-filled bath and the perfusion bath. Therefore, in these experiments, only cells adjacent to the interface were impaled. A stimulus generator was used to inject current pulses of 0.6 s at a frequency of 0.8 Hz into the lumen of the tubule through a 1 MQ resistor. The current intensity was continuously monitored by an operational amplifier which functioned as a current to voltage converter and also drove the bath to virtual ground. Minor changes in injected current were corrected by adjusting the output of the stimulus generator. Intracellular potential deflections were corrected for the resistance of the ground electrode by subtracting the extracellularly recorded voltage deflections from all results. The standard saline used to bathe the tubules had the following composition (mM): NaCl, 129; KCl, 8.6, CaCl,, 2; MgCl*, 8.5; NaHCO,, 10.2; NaH2P04, 4.3; glucose, 34. The pH of this saline was 6.8 and its osmotic concentration was 342 mosmol 1-i. A potassium-rich saline containing 137.6 mM K was made by replacing NaCl by KCl. A sodiumfree saline had 137.6 mM KC1 and the NaHC03 and NaH2P04 of the standard saline were replaced by the same concentrations of KHCOr and KH,PO,. A saline containing only 29.6 mM of chloride was made by replacing the NaCl in standard saline by sodium isethionate. A chloride-free saline had the following composition (mM): Na isethionate, 129; K2S04, 4.3; CaSO,, 2; MgS04, 8.5; NaHCO,, 10.2; NaH2P04, 4-3; glucose 34. The effects of furosemide (1O-4 M) in standard saline were also studied. Electron microscopy 1. Conventionalfixation.
Lengths taken from the fluid-secreting region of Malpighian tubules of fifth instar Rhodnius larvae were fixed for 1 hr at room temperature in a solution of 2.5% glutaraldehyde in 0.05 M cacodylate buffer at pH 7.2 with 6% sucrose added. After washing in buffer with 6% sucrose, specimens were post-fixed in 1% osmium tetroxide with 6% sucrose for 1 hr.
hlALI’IGHIAN
TUBULE
REGIONALIZATION
Following further buffer rinses, the tissue was stained en bloc with 2% aqueous uranyl acetate for 1 hr before dehydration through an ascending series of alcohols and embedding in Araldite resin. Sections, cut on an LKB Ultrotome III or Reichert FC4, were stained with lead citrate and uranyl acetate and were examined with a Philips EM300 at 60 kV. 2. Alcian blue-lanthanum staining. This method for enhancing lanthanum staining of the cell surface by addition of Alcian blue to the primary fixative was devised by Shea (1971). Specimens were fixed for 2 hr at room temperature in 2% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.4, with 3% sucrose and 0.5% Alcian blue (filtered using a millipore filter of 0.22 pm pore size). After three rinses in 0.1 M cacodylate buffer, pH 7.4, with 6% sucrose, specimens were post-fixed for 1 hr in a solution of osmium tetroxide containing 1% lanthanum nitrate and brought to pH 8.05. Tissues were then stained en bloc with uranyl acetate, dehydrated, embedded and sectioned as described above. Control preparations were incubated for 1 hr in the standard saline described above. They were then fixed and processed as described in section 1, but using 0.1 M cacodylate buffer throughout rather than 0.05 M. 3. Injection with peroxidase.
Individual cells of the upper Malpighian tubule were injected with horseradish peroxidase (HRP) made up as a 3.2% solution, with 4 mg in 125 ~1 of 0.2 M Tris with 0.5 M KC1 at pH 7.5. The solution was injected iontophoretically from electrodes with tip resistances of approximately 30-40 MQ, by applying a current of 10 nA for 612 min. The transmembrane potential was recorded during injection and for 5-6 min after current injection. Tubules were not processed further if the post-injection membrane potential differed from the pre-injection value by more than 10-15 mV. For ultrastructural localization of the tracer, sections of tubule containing the injected cell were fixed for 30 min in a solution of 2.5% glutaraldehyde in 0.05 M phosphate buffer at pH 7.4 with 6% sucrose. Following rinses in 0.05 M phosphate buffer with 6% sucrose and subsequently in 0.05 M Tris buffer with 6% sucrose, the tissue was incubated for 10 min in 0.5% cobalt chloride
made up in 0.05 M Tris buffer (Adams, 1977). After two washes in Tris buffer with 6% sucrose, the peroxidase activity was developed enzymatically by the action of glucose oxidase on P-D glucose (Lundquist and Josefsson. 1971: Itoh et ul., 1979). Following an overnight wash in phosphate buffer with 6% sucrose, the tissue was stained en bloc with uranyl acetate, dehydrated. embedded and sectioned as described above. Controls were prepared by treating noninjected tubules to exactly the same procedures of fixation and incubation. 4. Freeze-fracture. Lengths of Malpighian tubule were incubated, unfixed, for 10 min in 25% glycerol in standard saline before being mounted in gold rivets. They were frozen in Freon 22 cooled with liquid nitrogen. Freezefracture was carried out in a Balzers 360M apparatus under a vacuum of 0.5 x 10 + Torr The specimens were and at -100°C. shadowed with a mixture of tungsten and tantallum and backed with carbon. The replicas were cleaned in dilute solutions of sodium hypochlorite, mounted on copper grids and examined using a Philips EM300. The micrograph (Fig. 16) is mounted so that the direction of shadow is from the bottom. Results A. Electrical measurements
Several examples of potentials recorded across the basal membrane of tubules bathed in standard saline are shown in Figs 1 and 2. If the cell surface was visibly dimpled just prior to impalement, the potential difference recorded was typically 60-70 mV, cell interior negative (O’Donnell and Maddrell, 1984). Repeated further small advancements did not significantly change the potential. However, if the microelectrode touched the cell surface only very lightly prior to impalement, so that little or no dimpling occurred, then a stable smaller potential of -30 to -50 mV was often, though not always, observed after impalement. In other cases, the full potential was recorded. When low potentials were observed, a discrete change to a more negative value sometimes occurred if the microelectrode was advanced a few micrometres (Fig. 2a), or if the baseplate of the micromanipulator was lightly tapped (Fig. 2b). In other cases, application of
O’DONNELL,
+ b -
,
t +
FCS
I
cs
Fig. 1. Intracellular recordings of basal membrane potentials in two tubules (a, b). Tubules were initially bathed in control saline (CS); changes to chloride-free saline (0 Cl), potassium-rich saline (KR) or control saline containing 10e4 M furosemide (FCS) are indicated by the labelled solid arrows. The effects of changes in bathing saline composition on membrane potential are discussed in the text. Scales: vertical, 10 mV; horizontal, 2 min. (a) The microelectrode contacted the cell surface at the far left arrow and the cell was successfully impaled at A. Microcautery (MC) produced a discrete shift to a less negative potential. After the final return to CS the potential shifted spontaneously to a more negative value (B). The electrode was then retracted into the bathing saline (C). (b) In this cell the potential declined after impalement, and then shifted abruptly to a stable value. Exposure to FCS produced a large depolarization. On return to control saline, the potential shifted to a more negative value (open arrow), and subsequent exposures to FCS produced smaller depolarizations.
MADDRELL,
SKAER
AND HARRISON
microcautery was followed by a change in potential (Figs la, 2b), or the potential changed spontaneously (Figs lb, 2a), probably as a result of slight movements of the tubule during perfusion. In some cases, as many as three or four discrete potentials were recorded during advancement of the microelectrode. In addition, if the initial potential recorded was more negative than about -60 mV, then a discrete change to a less negative potential was sometimes observed if the microelectrode was very slowly retracted a few micrometres (Fig. 2a). One possible explanation for these results would be the existence of two intracellular regions of differing electrical potential, situated at different depths beneath the basal cell surface. Indeed, a first region was found very near to the cell surface and was characterized by low electrical potentials. In different cells, or in separate impalements of the same cell, these low potentials were found to be variable, from about -20 to -50 mV. In a second, deeper’ compartment, the potentials were more negative and much less variable, rangingfrom -50 to -70 mV. Characteristically, these more negative potentials were maxima; further advancements of the microelectrode did not produce further increases in potential. An obvious objection to the above interpretation is that the low potentials might simply have been caused by current leakage from the cell through an impalement shunt. Such shunting might result from ineffective sealing of the cell membrane to the glass wall of the microelectrode. However, gross membrane damage was unlikely because the low potentials were often stable for more than 10 min. Furthermore, the time course of potential change after impalement, microcautery, or movement of the electrode tip was similar, irrespective of the stable value of the potential difference (Figs lb, 2a, b); differences in time course would suggest differences in the speed, and therefore efficiency, of membrane sealing. More direct evidence against leakage artefacts was provided by measurements of the input resistance of the basal membranes in tubules which had not been stimulated with 5-HT. An impalement shunt would produce a conductance in parallel with the membrane conductance, and would be detectable by a decrease in basal membrane input resistance.
MALPIGHIAN
TUBULE
h
REGIONALIZATION
+
Fig. 2. Recordings of basal membrane potential during transepithelial current injection m two tubules (a, b). The basal membrane input resistance is proportional to the height of the potential deflection produced by current injection. Scales: vertical, 10 mV; horizontal, 1 min. (a) The potential recorded after the first impalement (A) was unstable. The second impalement (B) produced stable values of input resistance and potential. Subsequent small advancements (upward arrows) and retractions (downwards arrows) of the electrode usually produced shifts in potential. The open arrow indicates a spontaneous change in potential. (b) The initial impalement (A) was unsuccessful, resistance and potential declined to near zero in less than 1 min. The potential recorded after the second impalement (B) was stable. Microcautery (MC) or gentle tapping of the micromanipulator baseplate (T) produced changes in potential. In the latter instance, the potential was unstable. The microelectrode was withdrawn into the bathing saline at the arrow labelled R.
In measurements where a rapid decline in potential suggested an unsuccessful impalement, there was indeed a corresponding drop in input resistance, suggesting the presence of an impalement shunt (Figs Za, b). However, in impalements producing a low but stable potential, the input resistance was not significantly lower than that found in impalements giving potentials in the range -50 to -70 mV. We analysed data for paired experiments on unstimulated tubules in
which two or more potentials were measured at one site in one tubule. In 28 such experiments, the mean low potential was -28&2 mV, and the mean change in potential produced by a 200 nA current pulse was 11.5+2 mV. The mean high potential at these same sites was -6Okl mV, and a 200 nA current pulse produced a mean change in potential of 8.3+ 1.2 mV. The corresponding mean input resistances were 57.5+10 k& and 41.5+6 kQ for the low and
870
O’DONNELL,
high potential sites, respectively; the differences are not statistically significant. Low potential sites were not, therefore, associated with ineffective sealing of the cell membrane around the microelectrode. Changing the potassium concentration in the bathing saline provided another means of testing for leakage artefacts. Previous work has shown that the basal membrane of Rhodnius Malpighian tubules is selectively permeable to potassium; a change in [K] from 8.6 to 137.6 mM causes a basal membrane depolarization of 65 mV (O’Donnell and Maddrell, 1984). If the low potentials were caused by leakage around the microelectrode, one would expect to record a noticeably smaller change in potential in
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response to an increase in the potassium concentration of the bathing medium. The results of 36 experiments on stimulated tubules to test this are summarized in Fig. 3. They show no significant correlation between the potential recorded in control saline and the potential change on exposure to K-rich saline. Similarly, when tubules were bathed in Na-free saline containing 156.2 mM K, there was no correlation between the potential in the control saline and the size of the depolarization produced by the K-rich Na-free saline (Fig. 4). The low basal potentials could also be artefactual if the electrode tip had crossed both the basal and apical cell membranes and
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Potential8nconuol ~allne(mV) Fig. 3. Depolarization in potassium-rich saline as a function of the potential in control saline. N=22 tubules from 17 insects. In this figure and in Figs 4 and 6-8, each point represents a recording from a separate location in a tubule. All salines contained 5-HT (at 10-e M) unless noted otherwise. .
-20 -
, 0
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-40 -60 Potenttal in control saline (mV)
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Fig. 4. Depolarization in potassium-rich sodium-free saline as a function of the basal membrane potential in control saline. N=24 tubules from 18 animals.
MALPIGHIAN
TUBULE
x71
REGIONALIZATION
entered the tubule lumen. The electrode would then record the transepithelial potential (TEP), i.e. about -8 mV, lumen negative, in unstimulated tubules, or about -28 mV in stimulated tubules (O’Donnell and Maddrell, 1984). However, several types of evidence indicated that the low basal potentials were not TEPs. (1) Advancement of the electrode after an initial recording of a low potential usually resulted in a more negative potential up to about -60 mV. Subsequent retraction produced lower potentials. One would expect to encounter first a high basal potential and then a lower TEP if the electrode tip entered the lumen after further advancement. (2) In a related series of experiments, in which we deliberately tried to advance the electrode tip into the tubule lumen, we found this very difficult, possibly because the tip would have to cross the closepacked membranes of the numerous apical microvilli. (3) Our measurements of input resistance do not show the substantially higher values at the lower potential sites that one would expect if the electrode tip had crossed a second cell membrane. (4) The high conductivity of the secreted fluid means that the lumen of the tubule will be very nearly equipotential. The widely varying potentials within one cell and within different cells of the same tubule are, therefore, inconsistent with a luminal position of the electrode tip. Furthermore, if some of the potentials were TEPs. one would expect a bimodal distribution of the recorded potentials, with peaks at
-8 and -65 mV in unstimulated tubules and -28 and -65 mV in stimulated tubules. The lower peak would correspond to a luminal location of the microelectrode tip, and therefore represents a transepithelial potential (O’Donnell and Maddrell, 1984). The peak at -65 mV would correspond to the basal membrane potential. However, Fig. 5 shows that the potentials have a broad unimodal distribution. (5) The final argument against a luminal position of the electrode tip during measurement of low potentials is based upon the potential changes produced by chloride-free saline, furosemide and 5HT. Effects of chloride-reduced and chloride-free saline. In experiments with SHT-stimulated
tubules, the potential change produced across the basal membrane by a reduction in the chloride concentration of the bathing saline was inversely correlated with the potential recorded in control saline (Fig. 6). Low potential sites depolarized by about 5-15 mV when the bathing medium chloride concentration was reduced from 158.6 to 29.6 mM. In contrast, there was a slight hyperpolarization in chloride-reduced saline if the potential in control saline was greater than about 60 mV, inside negative. These effects were inconsistent with a significant chloride conductance of the basal membrane; chloride movements through conductive channels would produce, in accordance with the Nernst equation. a depolarization of
-20 Potential
dlfferencc
40
-60
-X(1
-l(K)
(mV)
Fig. 5. Frequency histogram of basal membrane potent]& warded from tubules m control saline or control saline with 5-HT. All the data arc based on experiments in which at least three impalements were made on one tubule. For experiments in i-HT-containmg salme. N=107 recordings from 21 tubules from 16 animals. For those in saline alone. N=200 recordings from seven tubules from five animals.
O’DONNELL,
872
MADDRELL,
SKAER
AND HARRISON
Fig. 6. Potential change in chloride-reduced saline (29.6 mM) as a function of the potential control saline. N=seven tubules from five animals.
in
explained by chloride movements through conductive channels. Because chloride is thought to enter the cells only through co-transport with sodium and potassium (O’Donnell and Maddrell, 1984), the effects of furosemide, a drug which inhibits such co-transport systems (Frizzell et al., 1979), were examined. Exposure of stimulated tubules to control saline containing 10m4M furosemide resulted in large depolarizations at the low potential sites and reduced or negligible depolarization at the
about 42 mV under the conditions described. of stimulated tubules to Exposure chloride-free saline produced much larger changes in potential across the basal membrane. At low potential sites the potential changes were as much as 100 mV (Fig. la) with the inside of the cell becoming positive relative to the bathing solution, whereas the high potential sites hyperpolarized by 3-5 mV, and, in some cases, by more than 10 mV. The data are summarized in Fig. 7. In this case as well, the results could not be
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Fig. 7. Potential change in chloride-free saline as a function of the basal membrane potential in control saline. N= 13 tubules from ten animals. In three tubules. indicated by the symbols +, A and n , impalements were made at four or more sites. These data indicate that the potential in control saline and the response to chloride-free saline varies widely at separate sites close to the surface of the same tubule.
MALPIGHIAN
TUBULE
X73
REGIONALIZATION
Fig. 8. Potential changes produced by furosemide (10 4 M in control saline) as a functmn of the basal membrane potential in control saline. N=ll tubules from ten animals. In two tubules, indicated by the symbols A and W, impalements were made at seven or more sites.
high potential sites (Figs lb, 8). High potential sites did not hyperpolarize in response to furosemide. in contrast to the effects of chloride-free solutions. The potential changes produced at the low potential sites by furosemide or chloride-free saline were qualitatively similar to the transepithelial changes previously described (O’Donnell and Maddrell, 1984). As noted above, this situation might have arisen if the low potential sites represented a luminal location of the microelectrode tip, i.e. that the low potentials were, in fact, transepithelial potentials. We have, therefore, compared the magnitudes of the potential changes produced by furosemide at low potential sites with the changes in TEP. If the low potentials were TEPs, the changes would be the same. However. the response of the basal potential to chloride-free saline and furosemide was highly variable (Figs 7, 8). whereas the TEP became about 80 mV more positive during either treatment (O’Donnell and Maddrell, 1984). Furthermore, in 19 tubules in which basal potentials and TEPs were measured simultaneously before and during exposure to furosemide, the change across the basal membrane at low potential sites (-30 to -50 mV) was within 5 mV of the transepithelial change in only seven tubules. In five tubules the difference was between 5 and 20 mV. and in the other seven
tubules. the changes in potential differed by more than 20 mV. In similar experiments in which ten tubules were exposed to chloridefree saline, the change in basal potential was within 5 mV of the change in TEP in only one tubule. In six tubules the potential changes differed by5-20 mV, and in three tubules the difference was more than 20 mV. Effects of .5-hydroxytryptamine. The differences in response of stimulated and untubules suggested a restimulated examination of the effects of SHT. Previously, we found only slight changes at the basal membrane in response to 5HTstimulation (O’Donnell and Maddrell. 1984). However. these experiments examined only high potential sites. We have now recorded potential changes at low potential sites in response to exposure to saline containing lo-’ M. A typical record is shown in Fig. 9 and a summary of five such experiments is given in Table 1, together with the changes observed transepithelially in the earlier investigation. There was a triphasic change in potential at the low potential sites. Although these were similar in some respects to transepithelial potential changes recorded previously (O’Donnell and Maddrell. 1984). there were significant differences prior to stimulation and during the first phase of the response.
x74
O’DONNELL,
MADDRELL,
SKAER
AND
HARRISON
Fig. 9. Effects of 5-HT on basal membrane potential at a low potential site. Scales: vertical. 10 mV, horizontal, 1 min. At the arrow, the bathing saline was changed from control saline to control saline containing 5-HT (lo-” M). The short horizontal bar at the left of the record indicates the potential measured by the microelectrode in the bathing saline. Note that the response to 5-HT has three distinct phases (labelled).
Table 1. Effects of 5HT upon transepithelial and ‘low’ basal membrane potentials
Potential difference (mV) Low
potentials
TEP*
Phase of Response:
(n=5)
(n=22)
Pre-stimulation Phase 1 Phase 2 Phase 3
-26+3 -41+3 12+s -36+1
-8+3 -23+3 18+3 -28+3
*Data from O’Donnell and Maddrell. 1984.
All the electrical measurements described above suggest that Malpighian tubules contain two distinct regions of differing electrical potential. Such a difference would require a significant electrical resistance between the two intracellular regions. This resistance could be produced, for example, by a long and/or narrow cytoplasmic connection between the two regions. We therefore carried out an ultrastructural examination of the basal side of the Malpighian tubules.
B. Ultrastructure of the basal surface of Malpighian tubules
Conventional electron microscopy showed that the folding of the basal membrane is far from simple. The cellular projections that touch the basement membrane (and are held there by hemidesmosomes; Fig. 10) seldom connect directly with the main body of cytoplasm of the cell. This is particularly clear if the extracellular spaces surrounding the cytoplasmic fingers are revealed either by filling them with lanthanum (Fig. 11) or by finding cases were the cytoplasm is in a slightly shrunken state (Fig. 10). It is clear that several layers of these extensions overlie one another at the basal surface of the cell. In grazing sections of the surface it is evident that the projections run parallel with each other for considerable distances just under the basement membrane (Fig. 12). Evidence from these sections suggests that the basal surface is thrown into long, narrow fingers or folds which run parallel with each other along the surface of the tubule and are frequently nestled one around another (as in Fig. 11).
Fig. 10. Section of a Malpighian tubule cell that has been inJected with horsxadlah pcroxldahc and then treated for the localization of the tracer. This procedure shrinks the cells slightly and gives clear definition of the basal projections. The complex organization of thcsc projections into several overlapping layers can be made out. Hcmidesmosomes mark the sites of attachment of the cell extensions to the basal lamina (BL) (arrows). P, proJections; TC. tracheal cell. x 16,000. Fig. 11. Section of the basal region of a cell from material stained with Alclan bloc-lanthanum. The cxtracellular clefts arc densely stamcd. delineating the basal projections. ~30.(NxI.
MALPIGHIAN
TUBULE
REGIONALIZATION
It is possible to assess the degree to which the cellular projections arising from adjacent cells interdigitate. By marking individual cells with horseradish peroxidase (Figs 13, 14) or alcian blue (Fig. 15) extensive interdigitation is revealed, in some cases projections can be seen halfway across a neighbouring cell (Fig. 13, arrow). In freeze-fracture preparations, where the fracture has passed through the membranes touching the basement membrane, one can see the ‘footprints’ of the basal projections. It is clear that they extend for distances up to 80x (15 pm) their width (cu. 180 nm) (Fig. 16).
looked at the published electron micrographs of insect Malpighian tubules. It appears that the tubules of many insects have long basal projections and not, as previously supposed. simple basal infoldings. The Malpighian tubule cells of the Orthopteran grasshoppers. Dissosteira (Tsubo and Brandt, 1962) and Melanoplus (Beams et al., 1955) clearly have numbers of definite basal projections. as do both cell types in the stick insect, Carausk (Taylor, 1971). In at least some of the regions of the tubules of the Homopteran. /Macrosteles, which has longitudinally differentiated tubules, there are numerous long thin basal projections (Smith and Littau, 1960). in Oncopeltus, a heteropteran Hemipteran. the basal regions of the tubule cells have narrow. extremely numerous basal projections (Smith, 1968). As is often the case. the Diptera are different: in the adult tubules of Calliphora and MUSCU,the basal regions do not have discrete projections but instead there is a network of anastomosing narrow channels which run deeply into the cell (Smith. 1968: Sohal. 1974). Similar canahculi are found in the basal regions of the tubule\ of the Lepidopteran. Calpodes (Ryerse. 1979). In the mosquito, Aedes tarr~rorhynchus (Diptera). the primary tubule cell\
Discussion The electrical measurements of the properties of the basal region of the upper Malpighian tubules of Rhodnius suggested that the cells might have regions of cytoplasm separated by relatively high resistances. Ultrastructural observations revealed that the basal surface of the cell, immediately under the basement membrane, is thrown into long (some are at least 15 pm) projections running parallel with the cell surface. With this observation in mind, we have
Fig.
12. Section
cut tangentially
long basal projections Fig.
13. This
horseradish project
degree Fig.
15. The
unstained the stained Fig.
ahgnment
region
of the field
relationship with
cell extend ccl1
T.
away,
between
shown with
the basal projections
heen
injected
wth tracer.
and in the area
of
x lS.l)(lO.
HRP
filled
the non-filled
basal prolcctlons
cell.
illustraring
the
x32,01JC of two adlaccnt
as shown
(BL)
hcrc.
cell‘. IS revealed
Baul
projections
in an area also occuplcd
when
from
by projection>
the from
~6l.W)
preparation
of a tuhulc,
the extracellular are striking
leaflets
(arrowheads).
m which
in the pattern
if this is the case, some projections x20,50(1.
almost
the cntlrc
of the hasal surface.
The
and the way in whrch they branch
can be made out. A discontinuity two cells and,
across this border
The
x26.001).
electron-denrc
ccl1 (arrows)
13. The
from
ha\
with
the two cells
in Fig.
those
blue-lanthanum.
of the basal projections
and ioterdigitate
from
between
only
filled
convcntionallq.
haaal lamina.
a cell that
of this cell.
cells.
tracheole.
BL.
containing
adjacent
Alcian
of a cell fixed
of the adjacent
along the basal lamina
leaving
region
each other
of the projections
detail
between
with
of a tubule,
the basal
16. Freeze-fracture
fractured
horder
into
power
IS stained
the basal
basal extensions
cell can be seen rnterdigitating
of overlap
one cell
part
The
IF interdigitation
14. Higher one
shows (*).
distance
there
Fig. from
section
peroxidase
some
overlap.
through
of the cell run parallel
(large
arrow)
extend
cell
has been
elongation (small
may represent
a conaidcrahlc
and
arrows) the
distance
880
O’DONNELL,
have basal regions similar to Calliphora and Musca, but the smaller stellate cells have short basal projections (Bradley et al., 1982). Finally, micrographs of the lower tubules of Rhodnius show that they too have basal projections, which in this case often have mitochondria in them (Wigglesworth and Salpeter, 1962). Cell membrane elaborations, such as the projections that we now describe, may be specialized for the rapid fluid secretion that these tubules can achieve. Recent work (Maddrell, 1980; O’Donnell etal., 1982) suggests that water may move into the lumen by a transcellular route in response to osmotic gradients of a few milliosmole per litre (but see McElwain, 1984). Bradley (1983) has suggested that the clumped microvilli on the apical (luminal) surface of Rhodniw upper Malpighian tubules provide narrow extracellular spaces which would help maximize osmotic solute-water coupling as fluid moves from the cells into the tubule lumen. The basal projections may also provide increased surface area and long narrow spaces to promote solute-water coupling, in this case in the movement of fluid from the haemolymph into the cell. The existence of long basal projections provides a reasonable explanation for the low electrical potentials which we observe. A variety of experimental tests show that the
MADDRELL,
SKAER
AND
HARRISON
low potentials are not TEPs or artefacts of impalement damage. Rather, we suggest that a low potential is recorded when the microelectrode tip enters a basal projection. At present we have no good explanation for the dramatically different effects of chloride-free saline on high and low potential sites. During reduced chloride entry across the basal membrane, it is likely that dramatic changes in the intracellular ion activities may occur, since the cation pumps on the apical membrane will continue to function. These activity changes would have correspondingly large effects upen. the movement of ions across the basal membrane and the resultant membrane potential. Our data also suggest that the potassium activity in the basal projections is less than in the main cell cytoplasm. The low potential sites depolarize by about 55 mV in K-rich saline, suggesting that the cell membrane there has a high permeability to potassium ions. However, if the basal membrane potential of the cellular projections is largely a potassium diffusion potential, then it follows that the potassium activity within these basal structures will be considerably lower than in the main body of the cell cytoplasm. Future experiments will examine possible mechanisms which might maintain reduced levels of potassium activity within the basal projections.
Adams, J. C. 1977. Technical considerations in the use of horseradish peroxidase as a neuronal marker. Neuroscience, 2, 141-14s. Beams, H. W., Tahmisian, T. N. and Devine, R. L. 1955. Electron microscope studies on the cells of the Malpighian tubules of the grasshopper (Orthoptera, Acrididae). 1. biophys. biochem. Cytol., 1, 197-202. Berridge, M. J. and Prince. W. T. 1972. Transepithelial potential changes during stimulation of isolated salivary glands with 5-hydroxytryptamine and cyclic AMP. J. exp. Biol., 56, 139-153. Bradley, T. J. 1983. Functional design of microvilli in the Malpighian tubules of the insect. Rhodnius prolixus. 1. Cell Sci., 60, 117-135. Bradley, T. J., Stuart, A. M. and Satir, P. 1982. The ultrastructure of the larval Malpighian tubules of a saline-water mosquito. Tissue & Cell, 14, 759-713. Friuell, R. A.. Field, M. and Schultz, S. G. 1979. Sodium-coupled chloride transport by epithelial tissues. Am. J. Physiol., 236, Fl-F8. Itoh, K., Konishi, A., Nomura, S., Mizuno, N., Nakamura, Y. and Sugimoto, T. 1979. Application of a coupled oxidation reaction to the electron microscopic demonstration of horseradish peroxidase: glucose oxidase method. Brain Res., 175, 341-346. Lundquist, I. and Josefsson, J-O. 1971. Sensitive method for determination of peroxidase activity in tissue by means of a coupled oxidation reaction. Analyr. Biochem., 41, 567-577. McElwain, D. L. S. 1984. A theoretical investigation of fluid transport in an insect, Rhodniwprolims Stal. Proc. R. Sot. Lond. B, 222, 36S372.
MALPIGHIAN Maddrell,
TUBULE
X81
REGIONALIZATION
S. H. P. 1980. Characteristics of eptthehal transport und Transport (eds F. Bonner and A. Kleinzeller).
Membranes
in insect Malpighian 14, 427-463.
tubules.
Current
Toprcs
rn
Maddrell. S. H. P., Pilcher. D. E. M. and Gardiner, B. 0. C. 1969. Stimulatory effect of 5-hydroxytryptamine (serotonin) on secretion by Malpighian tubules of insects. Nature, Lond.. 222, 784-785. Meyer, G. F. 1957. Elektronenmikroskopische Untersuchungen an den Malpighlgeflssen verxhiedener Insekten. 2. Zellforsch., 47, 1128. O’Donnell, M. J., Aldis, G. K. and Maddrell, S. H. P. 1982. Measurements of osmotic permeability in the Malplghlan tubules of an insect, Rhodniw prolixus Stal. Proc. R. Sot. Lond. B. 216, 267-277. O’Donnell, M. J. and Maddrell, S. H. P. 1984. Secretion by the Malpighian tubules of Rhodniusprolixus Stal: elcctrvzal events. J. exp. Biol., 110,275-290. Phillips, J. E. 1982. Comparative physiology of insect renal function. Am. J. Physiol.. 241, R241-R257. Pwves, R. D. 1981. Microelectrode Methods for Intracellular Recording and lontophoresis. Academic Press, London. Ryerse, J. S. 1979. Development changes in Malpighian tubule structure. Trrsue & Cell, 11, 533-551. Shea, S. M. 1971. Lanthanum staining of the surface coat of cells. Its enhancement by the use of fixatives containmg Alcian Blue or cetylpyridinium chloride. J. Cell Biol.. 51, 61 l-620. Smith, D. S. 1968. Insect CelLF. Their Structure and Function. Oliver & Boyd, Edinburgh. Smith, D. S. and Littau, V. C. 1960. Cellular specialization in the excretory epithelial of an insect, Macrostelesfasctfrons Stal (Homoptera). J. biophys. and biochem. Cyto/.. 8, 103-133. Sohal. R. S. 1974. Fine structure of the Malpighian tubules in the housefly, Musca domestrca. Trssue & Cell. 6,71%728. Taylor, H. H. 1971. Thefilm structure of type 2 cells in the Malpighian tubules of the stick insect. Carawus ~O~OSNS. 2. Zeliforsch.
Tsubo,
mikrosk.
Dissosteira
Wigglesworth, J
Anat.,
I. and Brandt,
Insect
Carolina.
J. Ultrastruct.
V. B. and Salpeter. Physiol.,
122, 41 l-424.
P. W. 1962. An electron
8, 299-307.
Res..
microscopic
study of the Malpighian
tubules
of the grasshopper.
6, 2x35.
M. M. 1962. Histology
of the Malplghian
tubules In Rhodniwprolixlrs
(Hermptcra).
& CELL
1985 Longman
1985 17 (6) 865-881 Group
Ltd
M. J. O’DONNELL*,
S. H. P. MADDRELL, H. le B. SKAER and J. B. HARRISON
ELABORATIONS OF THE BASAL THE CELLS OF THE MALPIGHIAN AN INSECT Key words:
Insect.
Malpighian
tubule,
membrane
SURFACE OF TUBULES OF
architecture.
electrical
potentials.
mtrxellu-
lar regionalization ABSTRACT.
Electrical
mcasurcments
microelectrodcs
showed
that
have superficial
regions
different
membrane standard
to changes
chloride
by different
of the cells revealed with
was examined
similar
by marking
regions
from
was
by 30-40
concentration
many long cellular from
also differed projections
neighbouring
cells with
usmg mtraccllular
tubules body
of Rhodnru~
of the cell.
mV.
but
of the bathmg
to the drug. furosemide.
projections individual
the main
smaller
concentration
responses
and resistance
of the Malpighlan
properties
the potassium
in the external
and intcrdigitate
digitation
superficial
potential
parts
in electrxal
on changing
that was remforced
of the basal regions surface
m these
depolarization
response fmding
potential
of membrane
the fluid-secreting
alcian
medium.
in the two Electron
a
The
regions.
a
muoscopy
that run parallel cells. The
The
showed
to the cell
degree
blue or by horseradIsh
of mtcrperox-
ldase Injection. A survey similar
of the published
projectlons
on
their
micrographs basal
Introduction The cells of insect Malpighian tubules have apical microvilli projecting into the lumen (Beams et al., 1955; Meyer, 1957; Smith and Littau, 1960: Wigglesworth and Salpeter. 1962). On the basal, haemolymph-facing side, the cells also have elaborations of the plasma membrane. It has always been supposed that these were no more than simple pleating of the cell surface (Wigglesworth and Salpeter, 1962; Maddrell. 1980; Phillips, 1982). We now provide evidence to show that. in Rho&Gus, the cells have a complex array of long basal projections which run parallel to the ceil surface under the basement membrane. A survey of the literature shows that similar basal projections are found in the Malpighian tubules of many, though not all. other insects. Agricultural
and Food
Research
Neurophysiology
and
Zoology,
Street,
Downing
*Present University. Keceivcd
Cambridge
address:
Department
Downsvicw.
Ontario,
I8 June
Council
Pharmacology.
14X5.
Unit
of Insect
Department CB2 3EJ. of
Canada
Biology, M3J
of
England. York 1P3.
of insect
surfaces
and
Malpighian
not
the
tubules
simple
basal
hhows that infoldings
most
hd\c
prewou4)
These morphological findings provide an explanation for otherwise puzzling electrophysiological results. The basal membrane potential recorded by an intracellular microelectrode whose-tip was positioned close to the basal surface of the cell was as much as 40 mV less negative than the potential of about -65 mV recorded when the electrode tip was advanced into the main body of the cell cytoplasm. We suggest that such low basal membrane potentials are seen when the microelectrode tip is located in a long basal projection.
Materials and Methods Malpighian tubules were dissected from fifth instar Rhodnius prolixus Stal from a laboratory culture. Each tubule consists of a single layer of squamous epithelial cells forming a blind-ended tube approximately 90 Nm in diameter and 45 mm in length. We have worked only on the upper two-thirds of the tubule, which contains a single cell type, the fluidsecretingcells. When required. fast fluid
866
O’DONNELL,
secretion was stimulated by the inclusion of 10e6 M 5-hydroxytryptamine (5HT) in the bathing medium (Maddrell et al., 1969). Electrical measurements
Dissected tubules were placed in a perfusion apparatus which permitted rapid changes of the bathing fluid (Berridge and Prince, 1972; O’Donnell and Maddrell, 1984). The chamber consisted of a central compartment containing liquid paraffin (mineral oil) and two lateral compartments filled with saline, one of which could be perfused. The Malpighian tubule was arranged between the three compartments so that the blind end was in the perfusable compartment with the open end pulled through notches in the central compartment and secured with silicone grease to the wall of the non-perfused saline compartment. The tubule in this non-perfused compartment was nicked with ultrafine scissors so that the luminal contents were continuous with the saline. The basal cell membrane potential was measured from the potential difference between an intracellular microelectrode and a reference electrode in the perfused bath. The reference electrode was an agar bridge connected through a calomel half cell to ground. Thin-walled glass microelectrodes of electrical resistance of 8-10 MQ were mounted in a holder connected to an amplifier of high input impedance (lOn Q; W.P. Instruments, Inc.). The microelectrode holder and preamplifier probe were mounted on a Leitz micromanipulator. Initial contact of the microelectrode with the epithelium produced a visible dimpling of the cell surface; the microelectrode was then retracted and re-advanced more slowly towards the cell surface until there was a slight change in potential (<2 mV) and a coincident rise in electrode resistance; observation with a dissecting microscope (at 50 times) showed no apparent dimpling. Gentle tapping of the baseplate of the micromanipulator caused the electrode to ‘pop’ into the cell. Alternatively, the capacitance compensation control was momentarily turned to maximum. This caused the amplifier to oscillate and the oscillations acted as a microcautery (Purves, 1981) which facilitated microelectrode entry into the cell. Use of this technique (hereafter referred to as micorocautery) was identifi-
MADDRELL,
SKAER
AND HARRISON
able by vertical lines produced on recordings of the potential (Fig. la). The input resistance of the basal membrane was determined by injecting transepithelial current pulses and measuring the resulting deflection of the intracellular potential. The deflections decreased with distance from the interface between the paraffin-filled bath and the perfusion bath. Therefore, in these experiments, only cells adjacent to the interface were impaled. A stimulus generator was used to inject current pulses of 0.6 s at a frequency of 0.8 Hz into the lumen of the tubule through a 1 MQ resistor. The current intensity was continuously monitored by an operational amplifier which functioned as a current to voltage converter and also drove the bath to virtual ground. Minor changes in injected current were corrected by adjusting the output of the stimulus generator. Intracellular potential deflections were corrected for the resistance of the ground electrode by subtracting the extracellularly recorded voltage deflections from all results. The standard saline used to bathe the tubules had the following composition (mM): NaCl, 129; KCl, 8.6, CaCl,, 2; MgCl*, 8.5; NaHCO,, 10.2; NaH2P04, 4.3; glucose, 34. The pH of this saline was 6.8 and its osmotic concentration was 342 mosmol 1-i. A potassium-rich saline containing 137.6 mM K was made by replacing NaCl by KCl. A sodiumfree saline had 137.6 mM KC1 and the NaHC03 and NaH2P04 of the standard saline were replaced by the same concentrations of KHCOr and KH,PO,. A saline containing only 29.6 mM of chloride was made by replacing the NaCl in standard saline by sodium isethionate. A chloride-free saline had the following composition (mM): Na isethionate, 129; K2S04, 4.3; CaSO,, 2; MgS04, 8.5; NaHCO,, 10.2; NaH2P04, 4-3; glucose 34. The effects of furosemide (1O-4 M) in standard saline were also studied. Electron microscopy 1. Conventionalfixation.
Lengths taken from the fluid-secreting region of Malpighian tubules of fifth instar Rhodnius larvae were fixed for 1 hr at room temperature in a solution of 2.5% glutaraldehyde in 0.05 M cacodylate buffer at pH 7.2 with 6% sucrose added. After washing in buffer with 6% sucrose, specimens were post-fixed in 1% osmium tetroxide with 6% sucrose for 1 hr.
hlALI’IGHIAN
TUBULE
REGIONALIZATION
Following further buffer rinses, the tissue was stained en bloc with 2% aqueous uranyl acetate for 1 hr before dehydration through an ascending series of alcohols and embedding in Araldite resin. Sections, cut on an LKB Ultrotome III or Reichert FC4, were stained with lead citrate and uranyl acetate and were examined with a Philips EM300 at 60 kV. 2. Alcian blue-lanthanum staining. This method for enhancing lanthanum staining of the cell surface by addition of Alcian blue to the primary fixative was devised by Shea (1971). Specimens were fixed for 2 hr at room temperature in 2% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.4, with 3% sucrose and 0.5% Alcian blue (filtered using a millipore filter of 0.22 pm pore size). After three rinses in 0.1 M cacodylate buffer, pH 7.4, with 6% sucrose, specimens were post-fixed for 1 hr in a solution of osmium tetroxide containing 1% lanthanum nitrate and brought to pH 8.05. Tissues were then stained en bloc with uranyl acetate, dehydrated, embedded and sectioned as described above. Control preparations were incubated for 1 hr in the standard saline described above. They were then fixed and processed as described in section 1, but using 0.1 M cacodylate buffer throughout rather than 0.05 M. 3. Injection with peroxidase.
Individual cells of the upper Malpighian tubule were injected with horseradish peroxidase (HRP) made up as a 3.2% solution, with 4 mg in 125 ~1 of 0.2 M Tris with 0.5 M KC1 at pH 7.5. The solution was injected iontophoretically from electrodes with tip resistances of approximately 30-40 MQ, by applying a current of 10 nA for 612 min. The transmembrane potential was recorded during injection and for 5-6 min after current injection. Tubules were not processed further if the post-injection membrane potential differed from the pre-injection value by more than 10-15 mV. For ultrastructural localization of the tracer, sections of tubule containing the injected cell were fixed for 30 min in a solution of 2.5% glutaraldehyde in 0.05 M phosphate buffer at pH 7.4 with 6% sucrose. Following rinses in 0.05 M phosphate buffer with 6% sucrose and subsequently in 0.05 M Tris buffer with 6% sucrose, the tissue was incubated for 10 min in 0.5% cobalt chloride
made up in 0.05 M Tris buffer (Adams, 1977). After two washes in Tris buffer with 6% sucrose, the peroxidase activity was developed enzymatically by the action of glucose oxidase on P-D glucose (Lundquist and Josefsson. 1971: Itoh et ul., 1979). Following an overnight wash in phosphate buffer with 6% sucrose, the tissue was stained en bloc with uranyl acetate, dehydrated. embedded and sectioned as described above. Controls were prepared by treating noninjected tubules to exactly the same procedures of fixation and incubation. 4. Freeze-fracture. Lengths of Malpighian tubule were incubated, unfixed, for 10 min in 25% glycerol in standard saline before being mounted in gold rivets. They were frozen in Freon 22 cooled with liquid nitrogen. Freezefracture was carried out in a Balzers 360M apparatus under a vacuum of 0.5 x 10 + Torr The specimens were and at -100°C. shadowed with a mixture of tungsten and tantallum and backed with carbon. The replicas were cleaned in dilute solutions of sodium hypochlorite, mounted on copper grids and examined using a Philips EM300. The micrograph (Fig. 16) is mounted so that the direction of shadow is from the bottom. Results A. Electrical measurements
Several examples of potentials recorded across the basal membrane of tubules bathed in standard saline are shown in Figs 1 and 2. If the cell surface was visibly dimpled just prior to impalement, the potential difference recorded was typically 60-70 mV, cell interior negative (O’Donnell and Maddrell, 1984). Repeated further small advancements did not significantly change the potential. However, if the microelectrode touched the cell surface only very lightly prior to impalement, so that little or no dimpling occurred, then a stable smaller potential of -30 to -50 mV was often, though not always, observed after impalement. In other cases, the full potential was recorded. When low potentials were observed, a discrete change to a more negative value sometimes occurred if the microelectrode was advanced a few micrometres (Fig. 2a), or if the baseplate of the micromanipulator was lightly tapped (Fig. 2b). In other cases, application of
O’DONNELL,
+ b -
,
t +
FCS
I
cs
Fig. 1. Intracellular recordings of basal membrane potentials in two tubules (a, b). Tubules were initially bathed in control saline (CS); changes to chloride-free saline (0 Cl), potassium-rich saline (KR) or control saline containing 10e4 M furosemide (FCS) are indicated by the labelled solid arrows. The effects of changes in bathing saline composition on membrane potential are discussed in the text. Scales: vertical, 10 mV; horizontal, 2 min. (a) The microelectrode contacted the cell surface at the far left arrow and the cell was successfully impaled at A. Microcautery (MC) produced a discrete shift to a less negative potential. After the final return to CS the potential shifted spontaneously to a more negative value (B). The electrode was then retracted into the bathing saline (C). (b) In this cell the potential declined after impalement, and then shifted abruptly to a stable value. Exposure to FCS produced a large depolarization. On return to control saline, the potential shifted to a more negative value (open arrow), and subsequent exposures to FCS produced smaller depolarizations.
MADDRELL,
SKAER
AND HARRISON
microcautery was followed by a change in potential (Figs la, 2b), or the potential changed spontaneously (Figs lb, 2a), probably as a result of slight movements of the tubule during perfusion. In some cases, as many as three or four discrete potentials were recorded during advancement of the microelectrode. In addition, if the initial potential recorded was more negative than about -60 mV, then a discrete change to a less negative potential was sometimes observed if the microelectrode was very slowly retracted a few micrometres (Fig. 2a). One possible explanation for these results would be the existence of two intracellular regions of differing electrical potential, situated at different depths beneath the basal cell surface. Indeed, a first region was found very near to the cell surface and was characterized by low electrical potentials. In different cells, or in separate impalements of the same cell, these low potentials were found to be variable, from about -20 to -50 mV. In a second, deeper’ compartment, the potentials were more negative and much less variable, rangingfrom -50 to -70 mV. Characteristically, these more negative potentials were maxima; further advancements of the microelectrode did not produce further increases in potential. An obvious objection to the above interpretation is that the low potentials might simply have been caused by current leakage from the cell through an impalement shunt. Such shunting might result from ineffective sealing of the cell membrane to the glass wall of the microelectrode. However, gross membrane damage was unlikely because the low potentials were often stable for more than 10 min. Furthermore, the time course of potential change after impalement, microcautery, or movement of the electrode tip was similar, irrespective of the stable value of the potential difference (Figs lb, 2a, b); differences in time course would suggest differences in the speed, and therefore efficiency, of membrane sealing. More direct evidence against leakage artefacts was provided by measurements of the input resistance of the basal membranes in tubules which had not been stimulated with 5-HT. An impalement shunt would produce a conductance in parallel with the membrane conductance, and would be detectable by a decrease in basal membrane input resistance.
MALPIGHIAN
TUBULE
h
REGIONALIZATION
+
Fig. 2. Recordings of basal membrane potential during transepithelial current injection m two tubules (a, b). The basal membrane input resistance is proportional to the height of the potential deflection produced by current injection. Scales: vertical, 10 mV; horizontal, 1 min. (a) The potential recorded after the first impalement (A) was unstable. The second impalement (B) produced stable values of input resistance and potential. Subsequent small advancements (upward arrows) and retractions (downwards arrows) of the electrode usually produced shifts in potential. The open arrow indicates a spontaneous change in potential. (b) The initial impalement (A) was unsuccessful, resistance and potential declined to near zero in less than 1 min. The potential recorded after the second impalement (B) was stable. Microcautery (MC) or gentle tapping of the micromanipulator baseplate (T) produced changes in potential. In the latter instance, the potential was unstable. The microelectrode was withdrawn into the bathing saline at the arrow labelled R.
In measurements where a rapid decline in potential suggested an unsuccessful impalement, there was indeed a corresponding drop in input resistance, suggesting the presence of an impalement shunt (Figs Za, b). However, in impalements producing a low but stable potential, the input resistance was not significantly lower than that found in impalements giving potentials in the range -50 to -70 mV. We analysed data for paired experiments on unstimulated tubules in
which two or more potentials were measured at one site in one tubule. In 28 such experiments, the mean low potential was -28&2 mV, and the mean change in potential produced by a 200 nA current pulse was 11.5+2 mV. The mean high potential at these same sites was -6Okl mV, and a 200 nA current pulse produced a mean change in potential of 8.3+ 1.2 mV. The corresponding mean input resistances were 57.5+10 k& and 41.5+6 kQ for the low and
870
O’DONNELL,
high potential sites, respectively; the differences are not statistically significant. Low potential sites were not, therefore, associated with ineffective sealing of the cell membrane around the microelectrode. Changing the potassium concentration in the bathing saline provided another means of testing for leakage artefacts. Previous work has shown that the basal membrane of Rhodnius Malpighian tubules is selectively permeable to potassium; a change in [K] from 8.6 to 137.6 mM causes a basal membrane depolarization of 65 mV (O’Donnell and Maddrell, 1984). If the low potentials were caused by leakage around the microelectrode, one would expect to record a noticeably smaller change in potential in
MADDRELL,
. .
$
3
. le
:
‘gyj-
.*
.
.
. . .
.
.*
e
B
AND HARRISON
.
.
. - .
.a:.
l
SKAER
response to an increase in the potassium concentration of the bathing medium. The results of 36 experiments on stimulated tubules to test this are summarized in Fig. 3. They show no significant correlation between the potential recorded in control saline and the potential change on exposure to K-rich saline. Similarly, when tubules were bathed in Na-free saline containing 156.2 mM K, there was no correlation between the potential in the control saline and the size of the depolarization produced by the K-rich Na-free saline (Fig. 4). The low basal potentials could also be artefactual if the electrode tip had crossed both the basal and apical cell membranes and
l
*()_
2
5 jj
o-
E
E
-20 ,
11
I -20
I - 40
I
I -Ml
I
I -x0
I
I -100
Potential8nconuol ~allne(mV) Fig. 3. Depolarization in potassium-rich saline as a function of the potential in control saline. N=22 tubules from 17 insects. In this figure and in Figs 4 and 6-8, each point represents a recording from a separate location in a tubule. All salines contained 5-HT (at 10-e M) unless noted otherwise. .
-20 -
, 0
I
I -20
I
,
1
-40 -60 Potenttal in control saline (mV)
I
-Ro
I
I
I
I -100
Fig. 4. Depolarization in potassium-rich sodium-free saline as a function of the basal membrane potential in control saline. N=24 tubules from 18 animals.
MALPIGHIAN
TUBULE
x71
REGIONALIZATION
entered the tubule lumen. The electrode would then record the transepithelial potential (TEP), i.e. about -8 mV, lumen negative, in unstimulated tubules, or about -28 mV in stimulated tubules (O’Donnell and Maddrell, 1984). However, several types of evidence indicated that the low basal potentials were not TEPs. (1) Advancement of the electrode after an initial recording of a low potential usually resulted in a more negative potential up to about -60 mV. Subsequent retraction produced lower potentials. One would expect to encounter first a high basal potential and then a lower TEP if the electrode tip entered the lumen after further advancement. (2) In a related series of experiments, in which we deliberately tried to advance the electrode tip into the tubule lumen, we found this very difficult, possibly because the tip would have to cross the closepacked membranes of the numerous apical microvilli. (3) Our measurements of input resistance do not show the substantially higher values at the lower potential sites that one would expect if the electrode tip had crossed a second cell membrane. (4) The high conductivity of the secreted fluid means that the lumen of the tubule will be very nearly equipotential. The widely varying potentials within one cell and within different cells of the same tubule are, therefore, inconsistent with a luminal position of the electrode tip. Furthermore, if some of the potentials were TEPs. one would expect a bimodal distribution of the recorded potentials, with peaks at
-8 and -65 mV in unstimulated tubules and -28 and -65 mV in stimulated tubules. The lower peak would correspond to a luminal location of the microelectrode tip, and therefore represents a transepithelial potential (O’Donnell and Maddrell, 1984). The peak at -65 mV would correspond to the basal membrane potential. However, Fig. 5 shows that the potentials have a broad unimodal distribution. (5) The final argument against a luminal position of the electrode tip during measurement of low potentials is based upon the potential changes produced by chloride-free saline, furosemide and 5HT. Effects of chloride-reduced and chloride-free saline. In experiments with SHT-stimulated
tubules, the potential change produced across the basal membrane by a reduction in the chloride concentration of the bathing saline was inversely correlated with the potential recorded in control saline (Fig. 6). Low potential sites depolarized by about 5-15 mV when the bathing medium chloride concentration was reduced from 158.6 to 29.6 mM. In contrast, there was a slight hyperpolarization in chloride-reduced saline if the potential in control saline was greater than about 60 mV, inside negative. These effects were inconsistent with a significant chloride conductance of the basal membrane; chloride movements through conductive channels would produce, in accordance with the Nernst equation. a depolarization of
-20 Potential
dlfferencc
40
-60
-X(1
-l(K)
(mV)
Fig. 5. Frequency histogram of basal membrane potent]& warded from tubules m control saline or control saline with 5-HT. All the data arc based on experiments in which at least three impalements were made on one tubule. For experiments in i-HT-containmg salme. N=107 recordings from 21 tubules from 16 animals. For those in saline alone. N=200 recordings from seven tubules from five animals.
O’DONNELL,
872
MADDRELL,
SKAER
AND HARRISON
Fig. 6. Potential change in chloride-reduced saline (29.6 mM) as a function of the potential control saline. N=seven tubules from five animals.
in
explained by chloride movements through conductive channels. Because chloride is thought to enter the cells only through co-transport with sodium and potassium (O’Donnell and Maddrell, 1984), the effects of furosemide, a drug which inhibits such co-transport systems (Frizzell et al., 1979), were examined. Exposure of stimulated tubules to control saline containing 10m4M furosemide resulted in large depolarizations at the low potential sites and reduced or negligible depolarization at the
about 42 mV under the conditions described. of stimulated tubules to Exposure chloride-free saline produced much larger changes in potential across the basal membrane. At low potential sites the potential changes were as much as 100 mV (Fig. la) with the inside of the cell becoming positive relative to the bathing solution, whereas the high potential sites hyperpolarized by 3-5 mV, and, in some cases, by more than 10 mV. The data are summarized in Fig. 7. In this case as well, the results could not be
. .
.
.
.
. l
.
I
0
t
I
- 20
t
t
t
- 40 Potential
.
. .*: .
. t
t
-60 in control
l
.
.
saline
.
t
- 80
I
I
I
-l(X)
(mV)
Fig. 7. Potential change in chloride-free saline as a function of the basal membrane potential in control saline. N= 13 tubules from ten animals. In three tubules. indicated by the symbols +, A and n , impalements were made at four or more sites. These data indicate that the potential in control saline and the response to chloride-free saline varies widely at separate sites close to the surface of the same tubule.
MALPIGHIAN
TUBULE
X73
REGIONALIZATION
Fig. 8. Potential changes produced by furosemide (10 4 M in control saline) as a functmn of the basal membrane potential in control saline. N=ll tubules from ten animals. In two tubules, indicated by the symbols A and W, impalements were made at seven or more sites.
high potential sites (Figs lb, 8). High potential sites did not hyperpolarize in response to furosemide. in contrast to the effects of chloride-free solutions. The potential changes produced at the low potential sites by furosemide or chloride-free saline were qualitatively similar to the transepithelial changes previously described (O’Donnell and Maddrell, 1984). As noted above, this situation might have arisen if the low potential sites represented a luminal location of the microelectrode tip, i.e. that the low potentials were, in fact, transepithelial potentials. We have, therefore, compared the magnitudes of the potential changes produced by furosemide at low potential sites with the changes in TEP. If the low potentials were TEPs, the changes would be the same. However. the response of the basal potential to chloride-free saline and furosemide was highly variable (Figs 7, 8). whereas the TEP became about 80 mV more positive during either treatment (O’Donnell and Maddrell, 1984). Furthermore, in 19 tubules in which basal potentials and TEPs were measured simultaneously before and during exposure to furosemide, the change across the basal membrane at low potential sites (-30 to -50 mV) was within 5 mV of the transepithelial change in only seven tubules. In five tubules the difference was between 5 and 20 mV. and in the other seven
tubules. the changes in potential differed by more than 20 mV. In similar experiments in which ten tubules were exposed to chloridefree saline, the change in basal potential was within 5 mV of the change in TEP in only one tubule. In six tubules the potential changes differed by5-20 mV, and in three tubules the difference was more than 20 mV. Effects of .5-hydroxytryptamine. The differences in response of stimulated and untubules suggested a restimulated examination of the effects of SHT. Previously, we found only slight changes at the basal membrane in response to 5HTstimulation (O’Donnell and Maddrell. 1984). However. these experiments examined only high potential sites. We have now recorded potential changes at low potential sites in response to exposure to saline containing lo-’ M. A typical record is shown in Fig. 9 and a summary of five such experiments is given in Table 1, together with the changes observed transepithelially in the earlier investigation. There was a triphasic change in potential at the low potential sites. Although these were similar in some respects to transepithelial potential changes recorded previously (O’Donnell and Maddrell. 1984). there were significant differences prior to stimulation and during the first phase of the response.
x74
O’DONNELL,
MADDRELL,
SKAER
AND
HARRISON
Fig. 9. Effects of 5-HT on basal membrane potential at a low potential site. Scales: vertical. 10 mV, horizontal, 1 min. At the arrow, the bathing saline was changed from control saline to control saline containing 5-HT (lo-” M). The short horizontal bar at the left of the record indicates the potential measured by the microelectrode in the bathing saline. Note that the response to 5-HT has three distinct phases (labelled).
Table 1. Effects of 5HT upon transepithelial and ‘low’ basal membrane potentials
Potential difference (mV) Low
potentials
TEP*
Phase of Response:
(n=5)
(n=22)
Pre-stimulation Phase 1 Phase 2 Phase 3
-26+3 -41+3 12+s -36+1
-8+3 -23+3 18+3 -28+3
*Data from O’Donnell and Maddrell. 1984.
All the electrical measurements described above suggest that Malpighian tubules contain two distinct regions of differing electrical potential. Such a difference would require a significant electrical resistance between the two intracellular regions. This resistance could be produced, for example, by a long and/or narrow cytoplasmic connection between the two regions. We therefore carried out an ultrastructural examination of the basal side of the Malpighian tubules.
B. Ultrastructure of the basal surface of Malpighian tubules
Conventional electron microscopy showed that the folding of the basal membrane is far from simple. The cellular projections that touch the basement membrane (and are held there by hemidesmosomes; Fig. 10) seldom connect directly with the main body of cytoplasm of the cell. This is particularly clear if the extracellular spaces surrounding the cytoplasmic fingers are revealed either by filling them with lanthanum (Fig. 11) or by finding cases were the cytoplasm is in a slightly shrunken state (Fig. 10). It is clear that several layers of these extensions overlie one another at the basal surface of the cell. In grazing sections of the surface it is evident that the projections run parallel with each other for considerable distances just under the basement membrane (Fig. 12). Evidence from these sections suggests that the basal surface is thrown into long, narrow fingers or folds which run parallel with each other along the surface of the tubule and are frequently nestled one around another (as in Fig. 11).
Fig. 10. Section of a Malpighian tubule cell that has been inJected with horsxadlah pcroxldahc and then treated for the localization of the tracer. This procedure shrinks the cells slightly and gives clear definition of the basal projections. The complex organization of thcsc projections into several overlapping layers can be made out. Hcmidesmosomes mark the sites of attachment of the cell extensions to the basal lamina (BL) (arrows). P, proJections; TC. tracheal cell. x 16,000. Fig. 11. Section of the basal region of a cell from material stained with Alclan bloc-lanthanum. The cxtracellular clefts arc densely stamcd. delineating the basal projections. ~30.(NxI.
MALPIGHIAN
TUBULE
REGIONALIZATION
It is possible to assess the degree to which the cellular projections arising from adjacent cells interdigitate. By marking individual cells with horseradish peroxidase (Figs 13, 14) or alcian blue (Fig. 15) extensive interdigitation is revealed, in some cases projections can be seen halfway across a neighbouring cell (Fig. 13, arrow). In freeze-fracture preparations, where the fracture has passed through the membranes touching the basement membrane, one can see the ‘footprints’ of the basal projections. It is clear that they extend for distances up to 80x (15 pm) their width (cu. 180 nm) (Fig. 16).
looked at the published electron micrographs of insect Malpighian tubules. It appears that the tubules of many insects have long basal projections and not, as previously supposed. simple basal infoldings. The Malpighian tubule cells of the Orthopteran grasshoppers. Dissosteira (Tsubo and Brandt, 1962) and Melanoplus (Beams et al., 1955) clearly have numbers of definite basal projections. as do both cell types in the stick insect, Carausk (Taylor, 1971). In at least some of the regions of the tubules of the Homopteran. /Macrosteles, which has longitudinally differentiated tubules, there are numerous long thin basal projections (Smith and Littau, 1960). in Oncopeltus, a heteropteran Hemipteran. the basal regions of the tubule cells have narrow. extremely numerous basal projections (Smith, 1968). As is often the case. the Diptera are different: in the adult tubules of Calliphora and MUSCU,the basal regions do not have discrete projections but instead there is a network of anastomosing narrow channels which run deeply into the cell (Smith. 1968: Sohal. 1974). Similar canahculi are found in the basal regions of the tubule\ of the Lepidopteran. Calpodes (Ryerse. 1979). In the mosquito, Aedes tarr~rorhynchus (Diptera). the primary tubule cell\
Discussion The electrical measurements of the properties of the basal region of the upper Malpighian tubules of Rhodnius suggested that the cells might have regions of cytoplasm separated by relatively high resistances. Ultrastructural observations revealed that the basal surface of the cell, immediately under the basement membrane, is thrown into long (some are at least 15 pm) projections running parallel with the cell surface. With this observation in mind, we have
Fig.
12. Section
cut tangentially
long basal projections Fig.
13. This
horseradish project
degree Fig.
15. The
unstained the stained Fig.
ahgnment
region
of the field
relationship with
cell extend ccl1
T.
away,
between
shown with
the basal projections
heen
injected
wth tracer.
and in the area
of
x lS.l)(lO.
HRP
filled
the non-filled
basal prolcctlons
cell.
illustraring
the
x32,01JC of two adlaccnt
as shown
(BL)
hcrc.
cell‘. IS revealed
Baul
projections
in an area also occuplcd
when
from
by projection>
the from
~6l.W)
preparation
of a tuhulc,
the extracellular are striking
leaflets
(arrowheads).
m which
in the pattern
if this is the case, some projections x20,50(1.
almost
the cntlrc
of the hasal surface.
The
and the way in whrch they branch
can be made out. A discontinuity two cells and,
across this border
The
x26.001).
electron-denrc
ccl1 (arrows)
13. The
from
ha\
with
the two cells
in Fig.
those
blue-lanthanum.
of the basal projections
and ioterdigitate
from
between
only
filled
convcntionallq.
haaal lamina.
a cell that
of this cell.
cells.
tracheole.
BL.
containing
adjacent
Alcian
of a cell fixed
of the adjacent
along the basal lamina
leaving
region
each other
of the projections
detail
between
with
of a tubule,
the basal
16. Freeze-fracture
fractured
horder
into
power
IS stained
the basal
basal extensions
cell can be seen rnterdigitating
of overlap
one cell
part
The
IF interdigitation
14. Higher one
shows (*).
distance
there
Fig. from
section
peroxidase
some
overlap.
through
of the cell run parallel
(large
arrow)
extend
cell
has been
elongation (small
may represent
a conaidcrahlc
and
arrows) the
distance
880
O’DONNELL,
have basal regions similar to Calliphora and Musca, but the smaller stellate cells have short basal projections (Bradley et al., 1982). Finally, micrographs of the lower tubules of Rhodnius show that they too have basal projections, which in this case often have mitochondria in them (Wigglesworth and Salpeter, 1962). Cell membrane elaborations, such as the projections that we now describe, may be specialized for the rapid fluid secretion that these tubules can achieve. Recent work (Maddrell, 1980; O’Donnell etal., 1982) suggests that water may move into the lumen by a transcellular route in response to osmotic gradients of a few milliosmole per litre (but see McElwain, 1984). Bradley (1983) has suggested that the clumped microvilli on the apical (luminal) surface of Rhodniw upper Malpighian tubules provide narrow extracellular spaces which would help maximize osmotic solute-water coupling as fluid moves from the cells into the tubule lumen. The basal projections may also provide increased surface area and long narrow spaces to promote solute-water coupling, in this case in the movement of fluid from the haemolymph into the cell. The existence of long basal projections provides a reasonable explanation for the low electrical potentials which we observe. A variety of experimental tests show that the
MADDRELL,
SKAER
AND
HARRISON
low potentials are not TEPs or artefacts of impalement damage. Rather, we suggest that a low potential is recorded when the microelectrode tip enters a basal projection. At present we have no good explanation for the dramatically different effects of chloride-free saline on high and low potential sites. During reduced chloride entry across the basal membrane, it is likely that dramatic changes in the intracellular ion activities may occur, since the cation pumps on the apical membrane will continue to function. These activity changes would have correspondingly large effects upen. the movement of ions across the basal membrane and the resultant membrane potential. Our data also suggest that the potassium activity in the basal projections is less than in the main cell cytoplasm. The low potential sites depolarize by about 55 mV in K-rich saline, suggesting that the cell membrane there has a high permeability to potassium ions. However, if the basal membrane potential of the cellular projections is largely a potassium diffusion potential, then it follows that the potassium activity within these basal structures will be considerably lower than in the main body of the cell cytoplasm. Future experiments will examine possible mechanisms which might maintain reduced levels of potassium activity within the basal projections.
Adams, J. C. 1977. Technical considerations in the use of horseradish peroxidase as a neuronal marker. Neuroscience, 2, 141-14s. Beams, H. W., Tahmisian, T. N. and Devine, R. L. 1955. Electron microscope studies on the cells of the Malpighian tubules of the grasshopper (Orthoptera, Acrididae). 1. biophys. biochem. Cytol., 1, 197-202. Berridge, M. J. and Prince. W. T. 1972. Transepithelial potential changes during stimulation of isolated salivary glands with 5-hydroxytryptamine and cyclic AMP. J. exp. Biol., 56, 139-153. Bradley, T. J. 1983. Functional design of microvilli in the Malpighian tubules of the insect. Rhodnius prolixus. 1. Cell Sci., 60, 117-135. Bradley, T. J., Stuart, A. M. and Satir, P. 1982. The ultrastructure of the larval Malpighian tubules of a saline-water mosquito. Tissue & Cell, 14, 759-713. Friuell, R. A.. Field, M. and Schultz, S. G. 1979. Sodium-coupled chloride transport by epithelial tissues. Am. J. Physiol., 236, Fl-F8. Itoh, K., Konishi, A., Nomura, S., Mizuno, N., Nakamura, Y. and Sugimoto, T. 1979. Application of a coupled oxidation reaction to the electron microscopic demonstration of horseradish peroxidase: glucose oxidase method. Brain Res., 175, 341-346. Lundquist, I. and Josefsson, J-O. 1971. Sensitive method for determination of peroxidase activity in tissue by means of a coupled oxidation reaction. Analyr. Biochem., 41, 567-577. McElwain, D. L. S. 1984. A theoretical investigation of fluid transport in an insect, Rhodniwprolims Stal. Proc. R. Sot. Lond. B, 222, 36S372.
MALPIGHIAN Maddrell,
TUBULE
X81
REGIONALIZATION
S. H. P. 1980. Characteristics of eptthehal transport und Transport (eds F. Bonner and A. Kleinzeller).
Membranes
in insect Malpighian 14, 427-463.
tubules.
Current
Toprcs
rn
Maddrell. S. H. P., Pilcher. D. E. M. and Gardiner, B. 0. C. 1969. Stimulatory effect of 5-hydroxytryptamine (serotonin) on secretion by Malpighian tubules of insects. Nature, Lond.. 222, 784-785. Meyer, G. F. 1957. Elektronenmikroskopische Untersuchungen an den Malpighlgeflssen verxhiedener Insekten. 2. Zellforsch., 47, 1128. O’Donnell, M. J., Aldis, G. K. and Maddrell, S. H. P. 1982. Measurements of osmotic permeability in the Malplghlan tubules of an insect, Rhodniw prolixus Stal. Proc. R. Sot. Lond. B. 216, 267-277. O’Donnell, M. J. and Maddrell, S. H. P. 1984. Secretion by the Malpighian tubules of Rhodniusprolixus Stal: elcctrvzal events. J. exp. Biol., 110,275-290. Phillips, J. E. 1982. Comparative physiology of insect renal function. Am. J. Physiol.. 241, R241-R257. Pwves, R. D. 1981. Microelectrode Methods for Intracellular Recording and lontophoresis. Academic Press, London. Ryerse, J. S. 1979. Development changes in Malpighian tubule structure. Trrsue & Cell, 11, 533-551. Shea, S. M. 1971. Lanthanum staining of the surface coat of cells. Its enhancement by the use of fixatives containmg Alcian Blue or cetylpyridinium chloride. J. Cell Biol.. 51, 61 l-620. Smith, D. S. 1968. Insect CelLF. Their Structure and Function. Oliver & Boyd, Edinburgh. Smith, D. S. and Littau, V. C. 1960. Cellular specialization in the excretory epithelial of an insect, Macrostelesfasctfrons Stal (Homoptera). J. biophys. and biochem. Cyto/.. 8, 103-133. Sohal. R. S. 1974. Fine structure of the Malpighian tubules in the housefly, Musca domestrca. Trssue & Cell. 6,71%728. Taylor, H. H. 1971. Thefilm structure of type 2 cells in the Malpighian tubules of the stick insect. Carawus ~O~OSNS. 2. Zeliforsch.
Tsubo,
mikrosk.
Dissosteira
Wigglesworth, J
Anat.,
I. and Brandt,
Insect
Carolina.
J. Ultrastruct.
V. B. and Salpeter. Physiol.,
122, 41 l-424.
P. W. 1962. An electron
8, 299-307.
Res..
microscopic
study of the Malpighian
tubules
of the grasshopper.
6, 2x35.
M. M. 1962. Histology
of the Malplghian
tubules In Rhodniwprolixlrs
(Hermptcra).