Morphology and Function of Malpighian Tubules and

Associated Structures in the Cockroach,

Periplaneta americana
BETTY J. WALL, JAMES L. OSCHMAN A N D BARBARA A. SCHMIDT
D e p a r t m e n t of Bioloyicul Sciences, Northwestern University,
E u a n s t o n , Illinois 60201

ABSTRACT
This paper describes the different regions of the Malpighian
tubules and the associated structures (ampulla, midgut, ileum) in the cockroach, Periplaneta americana. There are about 150 tubules in each insect.
Each tubule consists of at least three parts. The short distal region is thinner
than the other parts and is highly contractile. The middle region comprises
most of the tubule length and is composed of primary and stellate cells. Primary cells contain numerous refractile mineral concretions, while stellate cells
have smaller nuclei, fewer organelles, simpler brush border, and numerous
multivesicular bodies. Symbiont protozoa are sometimes present within the
lumen of the middle region near where it opens into the proximal region of the
tubule. The latter is a short region that drains the tubular fluid into one of
the six ampullae. These are contractile diverticula of the intestine located at
the midgut-hindgut junction. The ampulla is highly contractile, and consists
of a layer of epithelial cells surrounding a cavity that opens into the gut via
a narrow slit lined by cells of unusual morphology. The proximal region of the
tubule and the ampulla resemble the midgut in that they have similar microvilli, basal infolds, and distribution of mitochondria. This suggests an endoderma1 origin and reabsorptive function for the proximal region of the tubule and
for the ampulla. A number of inclusions found within the tubule cells are
described, including peroxisomes and modified mitochondria. Current theories
of fluid transport are evaluated with regard to physiological and morphological
characteristics of Malpighian tubules. The possible role of long narrow channels such as those between microvilli and within basal folds is considered, as
is the mechanism by which these structures are formed and maintained. Also
discussed is the role of peroxisomes and symbionts in the excretory process.

Osmoregulation and excretion in insects

occur in two steps. First, a primary secretion or urine is produced by the Malpighian
tubules. This fluid, which resembles in
many ways a filtrate of the blood (Ramsay, '58; Farquharson, '74; Maddrell and
Gardiner, '74) flows into the gut and accumulates in the rectum, where the second, reabsorptive phase takes place (reviewed by Phillips, '70; Maddrell, '71 ; Wall
and Oschman, '75). Here substances that
are required for the metabolism of the
animal are reabsorbed into the blood while
wastes are retained in the rectal lumen,
concentrated, and excreted. Hence the
Malpighian tubule-rectum system corresponds functionally to the nephridia of
Annelida and Onychophora and to the kidney of vertebrates.
J. MORPH., 146: 265-306.

Current interest in Malpighian tubule

structure and function was stimulated by
the studies of Wigglesworth ('3la,b,c) and,
more recently, of Ramsay ('52, '53, '54,
'55a,b, '56, '58, '61) who devised methods
for isolating individual tubules in vitro
and collecting the secreted fluid. Berridge
('66) devised a defined medium in which
the tubules could secrete for very long
periods. Detailed information was then obtained on the composition of the secreted
fluid and the effects of bathing medium
composition on rate of formation and composition of the secreted fluid (Berridge,
'68, '69). These studies have contributed
to the development of our current theories
on the mechanism by which various epithelia form fluid secretions (reviewed by
Oschman and Berridge, '71; Berridge and

265

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B . J . WALL, J . L. OSCHMAN A N D B . A. SCHMIDT

icana were used in this study. Stock cultures were maintained in 12 hours light
with water and food (oatmeal or Lab Chow)
continuously available.
Physiology. To determine the concentration of the fluid secreted by cockroach
Malpighian tubules, animals were anesthetized with COz and then dissected open
under Ringer solution. The tubules were
dissected free and removed into a separate drop of Ringer solution under Paraffin oil (e.g. Berridge, '66). The Ringer
solution was the same as that used by
Treherne ('61). The secreted fluid as well
as a sample of the Ringer solution were
analyzed for freezing point depression
using the method of Ramsay and Brown
('55).
Morphology. Malpighian tubules and
associated structures (ampulla, midgut,
ileum) were fixed in 2.5% glutaraldehyde
with 0.05 M phosphate buffer and 5% sucrose, pH 7.2 to 7.4. Tissues were postfixed
in osmium in block in aqueous uranyl
acetate. They were then dehydrated in
ethanol, embedded in Araldite, and sectioned with the Huxley microtome. Sections were stained with lead citrate and
uranyl acetate. Micrographs were taken
with either the RCA-EMU-3F or the Hitachi HU-ll-E. To study regional changes
in morphology along the length of the
tubule, one tubule was divided into five
parts that were embedded and sectioned
separately.
Uranium-calcium staining. A method
for tracing intercellular spaces has been
developed in which fixation and uranium
in block staining are done in the presence
of calcium ions. The rationale was that
extracellular polymers might be present
that are cross-linked with calcium ions,
and that these substances might be retained in the tissue if calcium was present
in all of the solutions used to process the
specimens. Although the method worked
(fig. 22) further study will be required
to determine if the mechanism of staining
involves replacement of bound calcium
ions with uranium, or if calcium is acting
as a mordant for binding of colloidal uranium. The method consisted of fixing the
tissue in 2.5% glutaraldehyde buffered
in 0.1 M s-collidine with 0.005 M CaCh
and 0.005 M KCl added. The tissue was
MATERIALS A N D METHODS
then processed through wash and osmium
Animals. Adult male Periplaneta amer- solutions that were buffered in the same

Oschman, '72; Oschman et al., '74). It is

now thought that movement of the aqueous component of secretions may be the
consequence of an osmotic gradient established by pumping some solute into a confined space or infolding of the cell surface.
It was first proposed that intercellular
spaces might be the sites of the osmotic
gradients responsible for water absorption
in the gall bladder (Kaye et al., '66; Diamond and Tormey, '66a,b). Extension of
the model to foldings of the cell surface
such as microvilli and basal infoldings
was suggested by Diamond and Bossert
('68) and applied to Malpighian tubules
by Berridge and Oschman ('69). Although
this is not the only theory of fluid secretion (see DISCUSSION), it does achieve an
integration of physiological findings with
ultrastructural features of the transporting cells.
The morphology of Malpighian tubules
has been described for a number of insect species (Baccetti et al., '63; Beams
et al., '55; Berkaloff, '61; Berridge and
Oschman, '69; Bradfield, '53; Byers, '71;
Eichelberg and Wessing, '75; Fuller, '66;
Grinyer and Musgrave, '64; Jarial and
Scudder, '70; Kessel, '70; Mazzi and Baccetti, '63; Messier and Sandborn, '66;
Meyer, '57; Smith and Littau, '60; Sohal,
'74; Taylor, '71a,b, '73; Tsubo and Brandt,
'62; Wessing, '65; Wessing and Eichelberg, '69a,b; Wigglesworth and Salpeter,
'62). These studies have, however, provided little information on the region
where the tubules drain into the gut. Also,
with a few exceptions, there has been little
detailed information on tubules that are
differentiated into several regions. The
purpose of this paper is to describe in
detail the regional specializations along
the pathway of urine flow that may be
indicative of different functional capacities such as secretion, reabsorption, and
storage of metabolites. We also present
some measurements of the tubular fluid
composition and discuss the mechanism
of secretion. Finally, some of the information obtained from this study provides
clues about the embryological origin of
the Malpighian tubules as well as the manner in which foldings of the cell surface
are formed and maintained.

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MALPIGHIAN TUBULE STRUCTURE AND FUNCTION

way as the fixative, and with CaClz and

KC1 present. Finally, the tissue was stained
for two hours in 0.5% aqueous uranium
acetate plus 0.005 M CaC12 and KC1. The
tissues were then dehydrated and embedded in Spurr resin.
Lanthanum treatment. Additional information on the morphology of extracellular channels was obtained by fixing tubules in 2.5% glutaraldehyde in 0.1 M
s-collidine buffer containing 5 % sucrose
and 0.005 M each of CaCl2, LaCL, and
MgC12. These tissues were then processed
without osmium treatment and sections
were examined without further staining.
Stellate cells. A variety of staining
methods were used in attempts to locate
stellate cells in cockroach tubules. The
method that proved most reliable was to
dissect the tubules in Ringer solution,
place them on albumen-coated slides, fix
in Carnoys solution, wash in distilled water, and stain in 0.01% aqueous toluidine
blue for 20 minutes. The specimens were
then dehydrated in ethanol and mounted
in Permount.

xirnal

midgut

Ion
trachea

RESULTS

Fig. 1 General arrangement of t h e structures

described in this paper. T h e full length of only one
of the Malpighian tubules is shown. The middle
region i s the longest part. The short thin distal
region is highly contractile and the short proxim a l region inserts into the ampulla. Each of t h e
six ampullae drains about 24-32 tubules. The ampullae are contractile enlargements on the intestinal surface at the junction between midgut and
ileum. Tracheae branch over the midgut surface
a n d send processes into t h e ampullae. Fine tracbeoles also attach the tubules to other organs
such a s fat body.

General description
The general arrangement of the Malpighian tubules and associated structures
is illustrated in figure 1. There are 144192 tubules in this insect (Meyers and
Miller, 69; Crowder and Shankland, 72)
although figure 1 shows only one tubule
in its entirety. Each tubule is about 23 cm long and 4 0 4 0 p thick. The tubules
extend throughout the abdomen, and are
held in intimate contact with the fat body
and intestine by fine tracheae. The tubules
are highly contractile, owing to muscles
that extend in a spiral fashion along their
length. The contractility of the tubules
has been noted for some time (e.g. Leger
and Duboscq, 1899) and it appears that
true muscles are present in conjunction
with some but not all of the contractile
tubules (reviewed by Snodgrass, 35). The
structure of the musculature of the tubules has been described previously (Crowder and Shankland, 72) and will not be
elaborated upon here. The tubules contract vigorously in animals that have been
dissected open under Ringer solution, although there is much variation in the rate
of contraction.
We have identified three regions in these

tubules, distal, middle, and proximal. The

route of flow is from distal region to proximal region. The middle region is longest,
and further study could reveal that it is
divided into smaller sections. The clear
distal region of the tubule is much shorter
(about 0.8 mm or 3 % of the total length)
and is thinner (about 30 p ) than the
middle region of the tubule. Movements
of the distal region of the tubule are not
correlated with those of the middle region,
and are of a different sort. This region
exhibits rapid bending motions followed
by rapid return to its original position.
Contraction of the middle region results
in coiling of the tubules. The coiling can
be loose or tight, depending on the strength
of the contraction. The middle region is
variable in color. In some animals it is
completely yellow, while in others it is
white (also Meyers and Miller, 69). The
yellow color is probably the result of storage of some compound, possibly riboflavin
(Metcalf, 43) within the cells. Gersch
(42) divided the middle region into two
parts on the basis of the number of secretion vacuoles seen in living tubules,
although there was no sharp boundary

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B . J . W A L L , J . L. O S C H M A N AND B . A. SCHMIDT

between the two parts. He found many

more secretion vacuoles in cells of the distal part than in the proximal part of the
middle region. We have also observed a
gradual decrease in the density of secretion vacuoles toward the proximal part of
the middle region. The proximal region is
the short part (0.5 mm long) where the
tubule drains into the ampulla, and is
readily distinguished as a separate region
in sectioned material (fig. 27). The six
ampullae are muscular enlargements of
the surface of the gut. About 24-32 tubules
drain into each ampulla. The movement
of fluid from the tubules into the ampullae was followed in animals that were dissected open under Ringer solution containing Amaranth. Llke most other crystalline
or water soluble dyes (as compared to colloidal dyes such as sudan black) Amaranth
is secreted into the tubules (e.g. Wigglesworth, '31c; Palm, '52; Maddrell, '71). In
the tubule lumen adjacent to the ampulla
the red fluid can be seen to flow back and
forth as the tubules contract and relax.
We observed that repeated contractions
gradually force the contents of the tubules
into the ampullae, which then contract
to expel their contents into the gut lumen.
The six ampullae do not contract synchronously.
There is a n extensive tracheal supply
to the ampullae (fig. 1) and fine branching
tracheoles penetrate into the connective
tissue surrounding both ampullae and Malpig hi a n tubules.

TABLE 1

Osmolality (in m O s m ) of b a t h i n g m e d i u m and

u r i n e secreted b y isolated M a l p i g h i a n tubules

Urine

Bathing
medium

1
2
3
4
5
6
7
8
9
10
11
12
13

536
423
451
453
528
455
443
515
42 1
430
463
436
407

480
427
42 7
434
457
433
406
422
405
364
415
400
400

aver age

459

42 1

Differ eiic e
(urinemedium)

+
+
+
+
+
+
+
+
+
+
+

56
- 4
24
19
71
22
37
93
16
66
48
36

+ 7

+ 38

vitro without the ampullae. The fluid secreted was also hyperosmotic to the bathing medium by a n average of 38 mOsm
(table 1). Although these findings are preliminary, they suggest that the osmotic
pressure of the fluid secreted by the tubules does not change significantly as it
flows through the ampullae.

Distal region of the tubule

Figure 2 surveys the structure of the

distal region at low magnification. The
tubule lumen is narrow compared with
other regions of the tubule. In addition
to the basement membrane, there is a
connective tissue sheath around the tubule. Embedded in this is a single muscle
Measurements of urine composition
fiber which gives rise to the bending moA previous paper (Wall, '70) describes tion mentioned above. The basal plasma
a n in vivo method for collecting the fluid membrane is highly folded and adjacent
secreted into the gut from the ampullae. cells interdigitate extensively. The genThe portion of the gut into which the am- eral arrangement of the cells is similar
pullae drain was tied off and a cannula to that in the middle region, which will
inserted to collect the fluid. It was observed be illustrated and described below. How(Wall, '70) that: (1) The ratio of potassium ever, the cytoplasm of the distal region
to sodium was higher when the rate of of the tubule is free of the clear vacuoles
secretion was higher; (2) On a n average, that are so abundant in the middle rethe potassium concentration was only gion. It contains large numbers of mitoslightly higher than the sodium concen- chondria, some of which extend into the
tration; (3) The osmolality of the secreted microvilli (figs. 2, 4). The only inclusion
fluid was a n average of 32 mOsm (mil- that seems to be unique to this region is
liosmoles) higher than that of the hemo- a modified type of mitochondrion (fig. 3)
lymph.
that is very long and thin in sections,
During the present study, we have ex- apparently due to the presence of a solid
amined the concentration of the fluid se- core within the matrix. In appropriate seccreted by individual tubules isolated in tions this core has a crystalline appear-

MALPIGHIAN TUBULE STRUCTURE AND FUNCTION

269

Fig. 2 Survey view of the short distal region. There is a single strip of muscle ( m ) embed^
ded i n the connective tissue. T h e muscle is capable of short fast contractions. Distal region
differs from other portions in that cells lack vacuoles; lumen is narrow, and many of the long
microvilli contain extensions of filamentous mitochondria. x 4,600.

ance. We do not illustrate the crystalline similar in size to mitochondria but with
arrangement of the core as it has been a homogeneous granular interior (fig. 3 ) .
adequately described in other tissues (re- These may be microbodies. Other organviewed by Tandler and Hoppel, '72) in- elles include Golgi complexes, bits of
cluding the fat body of the American cock- smooth and rough endoplasmic reticulum,
roach (Gharagozlov, '69) and of the fruit and small vesicles. Two sorts of microvilli
fly (Takahashi et al., '70), and the mam- project into the lumen in the distal region,
malian kidney tubule (Suzuki and Mostolfi, those containing extensions of mitochon'67; Youson and McMillan, '70). There dria, and others containing extensions of
are also round membrane-bound organelles the smooth endoplasmic reticulum. The

2 70

B. J . WALL, J. L. OSCHMAN AND B . A . SCHMIDT

latter present circular profiles in transverse sections (fig. 4). Occasionally one
encounters a microvillus containing both a
mitochondrion and an extension of smooth
endoplasmic reticulum. The disc-shaped
mitochondria with the crystalline cores do
not penetrate into microvilli.
The lateral plasma membranes are only
slightly folded and adjacent cells are joined
by septate junctions along much of their
areas of contact. The remainder of the
interface between cells is a narrow inter-

Fig. 4 Transverse section of microvilli in distal tubule. Two sorts of microvilli are present:
those containing mitochondria and those containing extensions of smooth endoplasmic reticulum,
which appears a s a circle (arrow) in profile.
X 32,500.
Fig. 3 Atypical mitochondrion in distal portion of tubule. Mitochondria1 matrix contains a
solid core (c) that has a crystalline appearance in
some sections (see text). Another organelle (") consists of a n accumulation of fine granules bounded by a membrane. These may be microbodies.
X 46, 500.

MALPIGHIAN TUBULE STRUCTURE AND FUNCTION

Fig. 5 Survey micrograph of middle region. Lumen is larger than in distal region and
cells contain numerous clear vacuoles and mineralized concretions. Large dense structures
(1) resemble lysosomes observed in other insect tissues (e.g. Locke and Collins, '65). Penetration of mitochondria into microvilli is variable, i.e. compare brush border at top and bottom
of picture with that on either side. Connective tissue (ct) is thick and muscle (m) is embedded
in it. Mitochondria present throughout cytoplasm. Portions of blood cells shown at upper left.
X 4,000.

271

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B . J. WALL, J. L. OSCHMAN AND B. A. SCHMIDT

Fig. 6 High magnification of basal portion. middle region of tubule. Basement membrane
consists of inner granular layer (gl) and fibrillar layer (0. Connective tissue is comprised of
collagen fibrils (col) and outer granular layer. Profiles of microtubules are observed in some
of t h e interdigitating cell processes (arrows). X 80,000.

cellular space. Thus the arrangement of

lateral membranes is similar to that of
many other tubular epithelia in invertebrates.

Middle region
Figure 5 provides a survey view of the
middle region of the tubule, which comprises most of the tubule length. As mentioned above, this region is either completely or partly yellow in freshly dissected
specimens. The lumen is larger than that
of the distal region, and the secretory cells
are larger, apparently because they are
swollen with various sorts of vacuoles.
These will be described in more detail below. The cells interdigitate with each other
and are joined by septate junctions as in

the distal region of the tubule. The middle region is comprised of two cell types,
the primary or secretory cells, and stellate
cells. The primary cells are more abundant and are described first. The following
description proceeds from basal to apical
surface.
Fig. 7 Basal portion, middle region of tubule.
Cells interdigitate extensively. Basement membrane
is laminated. Note profiles of microtubules (arows) and mitochondria (m) in cytoplasmic interdigitations. Profiles of rough a n d smooth endoplasmic reticulum (rer and ser) are present within
t h e interdigitations. X 40,000.
Fig. 8 Basal portion of tubule cell, middle region, fixed in glutaraldehyde-lanthanum.Unstained
section. L a n t h a n u m is deposited within basement
membrane (bm) and extracellular channels between interdigitations. X 52,000.

MALPIGHIAN TUBULE STRUCTURE A N D FUNCTION

2 73

274

B . J. WALL. J . L. OSCHMAN AND B . A . SCHMIDT

Basal surface
Figure 6 reveals details of the tubulehemolymph interface. The outermost layer
consists of fine granules adhering to the
collagen fibers. This connective tissue
sheath becomes much thicker toward the
proximal region of the tubule. The collagen fibers appear to be embedded in a
clear matrix. The underlying tubule basement membrane is comprised of a n outer
layer of fine filaments and a n inner granular zone. Connective tissues and basement membranes of insects are frequently
multi-layered (e.g. Ashhurst, '68; Locke
and Huie, '72; Oschman and Berridge,
'70) but the functional significance of the
various components is poorly understood.
We suspect that the collagen fibers are
necessary to provide a n elastic protective
sheath around the highly contractile tubules, and that the basement membrane
may be comprised of polyelectrolytes that
can act both as a mechanical filter and
as a charged sieve to restrict the movement of certain molecules into or out of
the basal infoldings (e.g. Oschman and
Berridge, '7 1).
The basal infoldings form deep channels extending perpendicular to the basement membrane. These channels probably
correspond to the fine cytoplasmic striations in the same region observed by light
microscopists (Snodgrass, '35, p. 418). The
infoldings are long channels of narrow
but uniform width that extend deep into
the basal cytoplasm of the cells. Although
the channels appear empty in conventionally prepared specimens (fig. 7), when
specimens are fixed in glutaraldehyde-lanthanum or in glutaraldehyde-calcium followed by in block treatment with uraniumcalcium (fig. 8) both the channels and the
basement membrane are dense. There
are two interpretations of this finding:
some of the lanthanum or uranium is in
a colloidal form which simply becomes
trapped in the channels and thus acts a s
an extracellular marker much like colloidal lanthanum; alternatively, fixing and
processing with lanthanum or calciumcontaining solutions may precipitate and
retain some negatively charged extracellular substance. Further study is needed
to clarify this finding.
Figures 7 and 9 reveal that the basal channels are not formed from simple

folding of the basal plasma membrane

but instead are developed from interdigitating finger-like extensions of neighboring cells. A similar arrangement occurs in vertebrate kidney and salivary
gland striated duct and is well illustrated
in 3-dimensional reconstructions published
by Rhodin ('58), Tandler ('62), and Bulger
('65). That these are indeed processes from
adjacent cells is documented in figure 9,
which is a tangential section through the
basal surface in a region where one of
the cells is more darkly stained than the
other. Comparison with figure 7 shows
that the infolds actually arise because of
irregular arborizing extensions from adjacent cells. Toward the basal surface
these extensions often contain microtubules, which are sectioned transversely in
figure 7 and longitudinally in figure 9.
The microtubules are often within 1 of
the basement membrane and parallel to
the direction of the extensions. The microtubules may have a role in the genesis
and maintenance of the basal labyrinth,
as will be discussed below. No specialized
structures such as slit diaphragms have
been observed at the openings of the basal
infolds.
In addition to the microtubules mentioned above, the cytoplasm within the
interdigitating processes from adjacent
cells contains mitochondria, ribosomes, and
segments of smooth and rough endoplasmic reticulum (fig. 7). In some regions
one encounters chains of smooth vesicles
and tubules (fig. lo). In surface view (e.g.
top of fig. 25) these structures form a n
extensive reticular network. A similar arrangement occurs in the ciliary epitheliu m of the vertebrate eye (Tormey, '64) in
which the reticular structure is known to
be an artifact of fixation caused by the
disruption and vesiculation of a flattened
smooth surface cisternal system. Because
of the similarity of this structure in the
Malpighian tubule with that of ciliary epithelium, we are unsure of its exact structure and think that the chains of vesicles
Fig. 9 Tangential section near surface, middle
region. One cell is lighter than its neighbor, emphasizing interdigitating processes. Microtubules
shown in transverse section in figures 6 and 7
occur here in longitudinal profile (arrows). oriented
parallel to t h e sides of the interdigitating processes. In upper left basement membrane components are observed with collagen and other fibrils
oriented parallel to surface as in figure 6 . x 40,000.

MALPIGHIAN TUBULE STRUCTURE AND FUNCTION

2 75

276

B . J . WALL. J. L. OSCHMAN AND B . A. SCHMIDT

Fig. 10 Association of mitochondria with membranes of interdigitating processes from

adjacent cells. Apparent c h a i n s of vesicles within the interdigitations may be sheets of smooth
endoplasmic reticulum that have vesiculated during fixation. X 40,500.

could be artifacts caused by distortion of

a smooth surfaced cisternal system.
Cytoplasmic inclusions
The conspicuous and distinguishing feature of the middle region is the presence
of large vacuoles. While many of these
appear to be empty in sectioned material,
others contain either a network of fine

filaments, fine granules, or large concretions apparently comprised of concentric

shells of densely staining material (figs.
5, 1 1 , 25). Although the latter are usually
within the cytoplasm (fig. ll), one was
found within a nucleus (fig. 12). These
structures are common in Malpighian tubules as well as in insect intestinal cells
(Gouranton, '68), insect utricles (Ballan-

M A L P I G H I A N T U B U L E STRUCTURE A N D FUNCTION

277

Fig. 11 Membranebound vacuole containing mineral concretion found within cytoplasm

of middle region. X 40,500.

DufranCais, '70), protozoa (Andre and

Faure-Fremiet, '67), and many other invertebrates. The nature of these concretions has been a matter of controversy.
Earlier studies (Berkaloff, '58, '59; Srivastava, '62; Fuller, '66) suggested that
these structures contained urates. Wigglesworth and Salpeter ('62) noted that
the intracellular refractile concretions
were not the same as the excretory granules in the lumen, as only the latter gave
a positive urate test with ammoniacal silver nitrate. Gouranton ('68) was unable to
detect uric acid or guanine by chromatography of intestinal concretions. Instead,
he detected calcium, magnesium, iron,
phosphate, carbonate, protein, and acid
mucopolysaccharide. Similarly, Mello and

Bozzo ('69) tentatively recognized lipoprotein or phospholipid in excretory globules

in Malpighian tubules of larval bees. Stadhouders and Jacobs ('61) did histochemical studies on the structures in cockroaches and found they contained calcium and
phosphorus. We thus suspect that the concentric concretions of Periplaneta tubules
are sites of calcium phosphate storage.
They are probably not uric acid since:
(1) uric acid is not a major excretory
product in cockroaches (Mullins and Cochran, '72); (2) urate crystals observed in
electron micrographs of cornea of gout
patients (Slansky and Kuwabara, '68) show
a cuboidal crystalline structure that differs considerably from the concentric concretions in the Malpighian tubules; and

278

B. J. W A L L , J. L. O S C H M A N AND B . A. S C H M I D T

ble role of these organelles in nitrogen

excretion will be considered in the DISCUSSION.

Fig. 12 Mineral concretion within a nucleus,

middle region. X 9.000.

( 3 ) the structures in Malpighian tubules

resemble closely the calcified concretions
(lithosomes) in protozoa (Andre and
Faure-Fremiet, 67) and in experimentally
induced calcification of mammalian kidney tubules (Giacomelli et al., 64). The
concretions are occasionally found in the
tips of microvilli (fig. 20), and may enter
the lumen in this way.
In addition to mitochondria, ribosomes,
and endoplasmic reticulum, one occasionally encounters structures resembling annulate lamellae (fig. 13). This figure also
illustrates a dense body that is similar to
that shown in figure 3. The functional
significance of these inclusions is unclear.
However, we suspect that they may be
analogous to the microbodies or peroxisomes of vertebrate tissues. Microbodies
typically have a crystalline core, as do at
least some of the dense granules in Malpighian tubules (fig. 14). In many species
microbodies participate in the metabolism
of ammonia and uric acid, and the possi-

In addition to the spherical vacuoles

described above, some sections of the tubules contain clear vacuoles with a more
prismatic shape (fig. 15). These structures
could contain either a substance with low
intrinsic electron opacity, or their content
may be extracted at some stage of processing. If urates are stored in these Malpighian tubules, it is more likely that they
would be within prismatic granules such
as these than in the larger refractile concretions. We suggest this because the
urate-containing crystals in insects usually have needle, lens, or lozenge shapes
(Noel and Tahir, 29) similar to many of
the inclusions in figure 15. Further, Wigglesworth (53) reported that the uratic
granules in Rhodnius tubules are soluble
in aqueous fixative and are rapidly dissolved by osmium. Figure 15 also illustrates an autolysosome, Golgi complex, and
multivesicular body.
The mitochondria in the middle region
lack the crystalline inclusions encountered in the distal tubule. However, in
one specimen we have observed a region
in which most of the mitochondria were
irregular or doughnut shaped, containing central clear regions of density and
texture similar to the cytoplasm (figs. 16,
17). In some cases (fig. 17) the mitochondrial cristae are organized in a spoke-like
fashion around the central core. We have
only observed these mitochondria in one
specimen, and are unable to determine
their signficance.
Other inclusions are arrays of tubular
structures (fig. 18) that are sometimes
associated with granular material (fig. 19)
that is not bounded by a membrane. The
nature of these structures is unknown,
but we have observed similar inclusions
in nearby tracheal cells, indicating that
they may be due to a non-specific pathological condition such as a virus.
Apical surface
The apical cell surface is folded into
microvilli of the same sort as those found
in the distal region. However, the degree
to which mitochondria penetrate into the
microvilli seems to be less than in the distal region (compare figs. 2 and 5 ) and in

MALPIGHIAN TUBULE STRUCTURE AND FUNCTION

2 79

Fig. 1 3 Annulate lamellae (al) and vacuoles containing dense granular material (d). T h e
latter may be precursors of the large concentric concretions such a s illustrated in figures 1 1
and 12. or they may be microbodies or peroxisomes, as shown in figure 1 4 . x 80.000.
Fig. 1 4 Dense body with crystalline core (arrow) similar to that found in vertebrate
peroxisomes. X 49,000.

some areas, no mitochondria are found

within microvilli (figs. 16, 20, 21). There
is variation in the extent of penetration
of mitochondria into microvilli within the

same cells. Note that in figure 5 the microvilli in the cell shown in profile at the
bottom and the cell shown at the top have
areas where no mitochondria are within

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B . J . WALL, J. L. OSCHMAN AND B. A. S C H M I D T

Fig. 15 Second type of inclusion (::') observed within cells of middle region. Low density
of these prismatic structures suggests that their crystalline contents were dissolved during

fixation. Autolysosome (A), Golgi complex (G) and multivesicular body (MVB). X 22,000.

microvilli and other areas that have many. extensions of the network of smooth enIn the areas where mitochondria are ab- doplasmic reticulum that is abundant in
sent, there are still two sorts of microvilli, the apical zone of cytoplasm interior to
those penetrated by smooth ER and small- the microvilli (fig. 20). We are uncertain
er ones, lacking inclusions (fig. 21). The if this smooth endoplasmic reticulum is
tubules within the microvilli are clearly continuous with that extending into the

MALPIGHIAN TUBULE STRUCTURE AND FUNCTION

281

Fig. 16 Atypical mitochondria, middle region of tubule. These mitochondria contain o n e

or more pockets of cytoplasm ("). Their functional significance is unknown. Also included is
a profile of Golgi complex (G). X 21,000.

interdigitating processes at the basal cell

surface (figs. 6, 7, 10).

Cell junctions
At the interface between adjacent cells
we have noted two sorts of specialized junctions. The most extensive in terms of area

of contact is the septate junction, which

is characterized by a series of thin diaphragms or septa extending perpendicular
to the plasma membrane (fig. 23). Uranium-calcium treatment (fig. 22) renders
the spaces between septa opaque to electrons. so that the 3-dimensional structure

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B . J. WALL, 3. L. OSCHMAN AND B. A . SCHMIDT

Fig. 17 Higher magnification of atypical mitochondria containing pockets of cytoplasm. Note

spoke-like configuration of mitochondrion at top,
and multivesicular body (mvb). X 75,000.

Fig. 18 Cells of middle region sometimes contain early stages of autophagy (structures bounded
by isolation membranes, i m ) and later stages (lysosome, 1). Included is a t.s. of a crystalline array of
tubules (t) of unknown significance. Golgi complex (G) and endoplasmic reticulum a r e also included. X 29,000.

MALPIGHIAN TUBULE STRUCTURE AND FUNCTION

283

Fig. 19 Basal surface, middle region. There is some evidence of pinocytosis (arrow) but
this is not frequently observed in this tissue. Cells contain granular inclusions (*), o n e of which
is surrounded by tubular structures similar to those illustrated in ficure 18. X 41,000.

of the septa is seen in relief as parallel

folded sheets of intermembranous material. Junctions of this sort were first described by Locke ('65) and have been des-

ignated as comb desmosomes by Danilova

et al. ('69). Images similar to those in
figure 22 are obtained with lanthanum
staining, and have been integrated with

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B . J. WALL, J . L . OSCHMAN AND B . A. SCHMIDT

Fig. 21 Transverse section of microvilli, middle region. Profiles of smooth endoplasmic reticulum are circular in transverse sections. Smaller
microvilli lack mitochondria or smooth endoplasmic reticulum. X 66,000.

freeze fracture data (e.g. Flower, '70) to

provide a structural model for this junction (Gilula et al., '70). Gap junctions are
also encountered occasionally, and, again,
they have a characteristic appearance in
face views of material treated with uranium-calcium in block. This type of image
has been obtained previously in vertebrate
tissues with lanthanum infiltration (Revel
and Karnovsky, '67). Again, freeze etch
evidence has been utilized to ascertain a
possible 3-dimensional reconstruction of
the junctional structure (McNutt and
Weinstein, '70).

Fig. 20 Apical surface, middle region. Microvilli contain tubular structures that are continuous with smooth endoplasmic reticulum (arrow).
Note dense concretionthat appears to be pinch.
ing off into lumen (L). X 40,000.

Symbiotic protozoa
In some of the specimens we have observed symbiont protozoa within the proximal portion of the middle region. Figure
28 illustrates the ultrastructural appearance of these symbionts, while their
tion within the tubule is illustrated in
figure 27. These symbionts are similar

MALPIGHIAN TUBULE STRUCTURE A N D FUNCTION

285

Fig. 22 Face view of septate junction after glutaraldehyde-calcium fixation and uranium acetatecalcium in block staining. Uranium fills extracellular space delineating septa a s pleated sheets.
X 18,000.
Fig. 23 Septate junction,
middle region.
X 65,000.

in structure to the haplosporidians described by Woolever ('66) in the cockroach,

Leucophaea. These protozoa attach to the
tubule cells by means of microvilli that
insert between the microvilli of the tubule
cells. These sporozoa produce spores with
a thick cuticle that is difficult to section.
A detailed description of the protozoa is
not appropriate in this paper, although we
will discuss below their possible involvement in excretion.

Fig. 24 Stellate cells. Note i n figure b that

stellate cell is comparable in size to nucleus ( n p )
o f primary cell. Photomicrographs of middle region fixed in Carnoy's fixative and stained with
aqueous toluidine blue. Stellate cells have acquired
a blue color. x 1,250.

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B . J. WALL. J. L. OSCHMAN AND B. A. S C H M I D T

Fig. 25 Basal portion, stellate cell. Note absence of vacuoles and presence of numerous
multivesicular bodies. Stellate cell nucleus (N). X 21,000.

MALPIGHIAN T U B U L E S T R U C T U R E A N D FUNCTION

287

Fig. 26 Stellate cell in a region where it extends across whole tubule. Organelles are less
abundant than in primary cells. and microvilli lack inclusions such a s mitochondria or smooth
endoplasmic reticulum. Basal interdigitations a r e similar to those of primary cells. X 11,500.

Stellate cells
nuclei than primary cells, and have narWhole-mounts stained with toluidine row processes that extend between adjablue show that the tubules contain a sec- cent primary cells. Because of their smallond smaller sort of cell that occurs at er size they present narrow profiles in
regular intervals along the length of the transverse sections and are encountered
tubule (fig. 24). A comparable second cell infrequently in electron micrographs. The
type has been noted in a number of pre- stellate cells are characterized by the abvious studies (reviewed by Taylor, '71b), sence of vacuoles or concretions so abunbut their function remains obscure. In the dant in primary cells (figs. 25, 26). The
cockroach the stellate cells have smaller basal surface of stellate cells is elaborated

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B. J. WALL, J. L. OSCHMAN AND B. A. SCHMIDT

into long processes that interdigitate with

those of the primary cells (fig. 25). The
cytoplasm often appears less dense than
that of primary cells, apparently because
of fewer free ribosomes (fig. 9 probably
shows the interdigitation between a primary cell and a lightly stained stellate
cell). There are numerous multivesicular
bodies, lysosomes, segments of rough endoplasmic reticulum, and Golgi complexes
with associated small vesicles (figs. 25,
26). The mitochondria are of similar structure to those in primary cells, although
they seem to be less abundant (fig. 26).
The apical surface is folded into microvilli, but these differ from those in primary cells in that they do not contain
extensions of smooth tubular endoplasmic
reticulum or mitochondria (fig. 26).

Proximal region
We have designated a s the proximal
region the short portion of the tubule
(about 0.5 mm long) that drains into the
ampulla. The transition in morphology of
the tubule cells at the junction between
middle and proximal regions is apparent
in light micrographs of methylene bluestained thick sections (fig. 27). The proximal region does not contain symbionts
and there is a n abrupt change in the form
of the brush border. Electron micrographs
(figs. 29, 30) reveal that cells of the proximal region have microvilli that are thinner and farther apart than those of middle
and distal regions. It will be seen below
that microvilli in the proximal region
closely resemble those within the ampulla
and midgut. For purposes of comparison
the reader is referred to other studies that
illustrate aspects of insect midgut structure (Oschman et al., '74; Berridge, '70;
Oschman and Wall, '72; Smith et al., '69).
Another feature in common with midgut
is that the basal channels are somewhat
distended to form a compartment of relatively large volume that opens to the hemocoel in relatively few places (fig. 30; see
Berridge, '70). Finally there is a n inclusion in these cells that is also found in
the ampulla. This is a dense membranebound accumulation of membrane-like
leaflets with a wavy appearance in sections. This inclusion is illustrated in the
proximal tubule (figs. 29, 30) and in the
ampulla (fig. 32).

Fig. 27 Photomicrograph of 1 p thick methylene blue-stained section of region where tubule

drains into ampulla (A). Symbionts (S) are present in middle region. Note abrupt transition in
cell density and in brush border at junction between middle and proximal regions (arrow). X 820.

The connective tissue and musculature

investing the tubules is most fully developed in the proximal region (figs. 29, 30).
The cells lack the vacuoles and concretions abundant in the middle region. However, the proximal portion of the middle
region also lacks vacuoles (e.g. fig. 28).
In general, the mitochondria within the
proximal tubule cells seem more concentrated toward the apical or luminal surface, rather than scattered throughout the
cells as in middle and distal regions of the
Fig. 28 Section of middle region tubule near
proximal end. Protozoan symbionts (s) (haplosporidia) are in lumen. Thin processes from the protozoan cells mingle with microvilli of Malpighian
tubule. Muscle (m) is well developed in this region. x 12,200.

MALPIGHIAN T U B U L E S T R U C T U R E AND FUNCTION

289

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B . J. WALL, J. L. OSCHMAN AND B. A. S CH M I D T

Fig. 29 Survey view, proximal region of tubule. Lumen is triangular in shape, basal surface is heavily invested with connective tissue and musculature (m). Microvilli are further
apart than in other regions. Basal infolds are distended. Dense bodies (arrows) have lamellar
interiors similar to those in ampulla cells (figs. 32, 34). X 4,100.

tubules (figs. 2, 5). The cytoplasm also

contains lysosomes, rough endoplasmic reticulum, Golgi complexes, and numerous
multivesicular bodies. There is generally
a region at the cell apex that is free of
organelles except for small vesicles and
filaments (fig. 30). Neither mitochondria

nor smooth tubules extend into the microvilli. Instead, each microvillus has a core
of fine filaments similar to those within
midgut microvilli (e.g. Smith et al., '69).
Cells of the proximal region interdigitate
extensively along both basal and lateral
borders.

MALPIGHIAN TUBULE STRUCTURE AND FUNCTION

291

Fig. 30 Proximal region a t higher magnification. Connective tissue (ct) is thicker t h a n

basement membrane (bm) and h a s muscles ( m ) embedded in it. Cells interdigitate, but basal
channels (bc) between adjacent cell processes a r e wider than in middle region (compare with
figs. 7, 19). Microvilli a r e not closely packed and resemble those of ampullar cells (figs. 32, 33):
Dense lamellate inclusions (arrow) a r e similar to those found in ampullae (fig. 3 2 ) . X 14.000.

Ampulla
Figure 31 summarizes the organization
of the ampulla. The lumen of the proxima1 region narrows at the region where
it drains into the ampulla (figs. 27, 31).
The epithelium lining the ampulla is columnar and has thin microvilli and basal

interdigitations similar to those of the proximal region (figs. 29, 30). As mentioned
above, the ampulla contains large membrane-enclosed structures (about 2-3 p in
diameter) with a laminated interior (figs.
32, 34). Although we do not know the
function of these structures, they resem-

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B. J . WALL, J. L. OSCHMAN AND B. A . SCHMIDT

-Middle

midgut

leum

Fig. 31 Summary diagram of ampulla and associated structures. Muscles are not included. Proximal part of middle region contains protozoa (fig. 27). Proximal region of tubule
is composed of cells similar to ampulla. Tubule lumen narrows at region where it drains into
ampulla. Cavity of ampulla drains via narrow slit into gut. Apical surfaces of tubules, ampullae, and midgut are folded to form microvilli. Ileum is lined with cuticle.

ble the osmiophilic lamellate bodies found

within atrial and parabronchial cells of
vertebrate lung (Lambson and Cohn, '68;
Hatasa and Nakamura, '65; Smith and
Ryan, '73). The dense bodies in the lung
are formed in the rough endoplasmic reticulum and, when released to the cell
surface, contribute to the surface active
agent (surfactant) that coats the alveolar
membrane. Although we have not studied
the fate of the dense bodies in the insect
ampulla, they also may be released at the
cell surface, possibly contributing to the
surface coating of the drainage canals
(see below) or hindgut.
Another striking feature of the ampulla
is the presence of a deeply penetrating
canalicular system. Sections through these
canaliculi (e.g. fig. 34) give the appear-

ance of clear vacuoles lined with a surface

that has some rudimentary microvilli projecting inward and containing a small
amount of membranous and granular material. These structures can be observed
in methylene blue-stained thick sections.
By tracing these structures in serial sections, we have found that they are deeply
penetrating channels that approach closely the apical cell surfaces. The canaliculi
do not seem to open into the ampullar
lumen, but we are not entirely certain of
this point. Fig. 27 is one of a series of sections, and shows a region where one canaliculus approaches the ampullar lumen
near the region where a proximal tubule
drains. We have no information on the
function of this system, but suspect that
it could provide a stationary or unstirred

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293

Fig. 33 Transverse section of microvilli of ampulla. These microvilli a r e irregular i n s h a p e and

are not closely packed a s in tubules (compare with
figs. 4, 21). x 66,000.

fluid compartment from which solutes

could be reabsorbed by the ampullar cells.
We are not aware that such structures
have been described in other transporting
epithelia, although we have observed something resembling them but on a smaller
scale in cockroach midgut (unpublished).
Clearly, this part of the excretory system
should be studied further.

Drainage canal
Figure 31 illustrates the region where
fluid from the ampulla drains into the gut.
We have not been able to work out the
precise structure of this region, which appears to consist of a complex labyrinth
of channels. The individual cells lining
the channels (fig. 35) are thin and highly
Fig. 32 Survey view of ampulla. Only a portion of thick connective tissue is included. Cells
contain peculiar lamellated inclusions (arrows). Mitochondria are particularly abundant near apical
surface. X 5,000.

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B . J . WALL, J. L. OSCHMAN AND B . A . SCHMIDT

Fig. 34 Cavities within ampullar cells. These

clear spaces seem to b e bounded by apical membrane, since short microvilli protrude into them.
They do not appear to be permanently open to
the ampullar lumen. They contain membranous
fragments (arrows) and particulate material that
renders their contents somewhat more dense than
the lumen (compare with fig. 32). X 5,000.

folded on their apical but not their basal

surfaces. Canaliculi are present. The microvilli resemble those of the midgut even
more closely than do those of the ampulla.
The cells contain lipid droplets, small mitochondria, numerous ribosomes, and occasional bits of rough endoplasmic reticulum. The basal surface faces the gut and
appears to have little if any basement
membrane. The apical surface faces the
drainage canal and is coated with a loose
layer of material that looks like a disorganized jumble of membranes resembling a peritrophic membrane. Although
direct evidence is lacking, we suspect that
this layer could be formed by release of
the contents of the dense lamellated bodies
in the ampulla.
IZeum
We have mentioned the morphological
resemblance between cells of the ampulla
and those of the midgut, and referred for
comparison to the numerous published descriptions of midgut structure. To emphasize this point, we include a brief description of the beginning of the hindgut,' the
ileum. The ileum of Periplaneta has not
been described elsewhere although BallanDufrancais ('72) has described Blattella
ileum. The ileum is lined by a thin cuticular intima secreted by columnar cells.
The basal surface is irregular (fig. 36)
and there is a layer of connective tissue.
Mitochondria are abundant only toward
the apical pole of the cells. At low magnifications (fig. 36) one observes dense
bands of material lying adjacent to the
lateral plasma membranes. Higher magnification (fig. 37) reveals that these are
bundles of microtubules. The apical plasm a membrane is elaborated into closely
packed tubular infolds (figs. 36, 37). The
cuticle consists of a dense epicuticle and
a pale endocuticle. In some areas the endocuticle contains clear oval or lens-shaped
inclusions that either arise from or give
rise to clear prismatic crystals in the apical cytoplasm of the cells (fig. 37). Very
little information is available on the physiology of the ileum, and further anatomical description would be pointless at
present.
DISCUSSION

Mechanism of secretion
The striking anatomical feature of Mal-

MALPIGHlAN TUBULE STRUCTURE AND FUNCTION

295

Fig. 35 Cells bordering drainage canal from ampulla. Canal lumen at bottom, midgut
lumen at top. Cells are thin and have microvilli resembling those of midgut. Canaliculi ( x c )
are abundant. Cells contain rough endoplasmic reticulum (rer), mitochondria (m), and lipid
droplets (L). Loose surface coat (sc) resembles a peritrophic membrane. x 30,000.

pighian tubules revealed by this and previous ultrastructural studies is the presence of highly folded cell surfaces. This
feature is characteristic of many other secretory tissues such as pancreas, sweat
and salivary glands, choroid plexus, aglo-

merular kidney, proximal and distal kidney tubules, etc. (Pease, '56; Fawcett, '62;
Berridge and Oschman, '72). All of these
epithelia secrete a fluid that is about isosmotic with blood and that is nearly protein-free (except where protein secretion

296

B . J . WALL, J . L. OSCHMAN A N D B . A . SCHMIDT

r i g . au
JUIVCY
UI I I C U I I L . uJiurnriar cells nave irregular Dasai surrace racing tnlCK layer
of connective tissue (ct). Mitochondria a r e abundant near apical cell surface, which is lined
with cuticle (cut). Dense bands in cytoplasm (b) have proven to be bundles of mictotubules

(fig.37). X 7,000.

is part of the gland function, as in the sorptive epithelia such as gall bladder and
pancreas). The secreted fluids exert a pos- intestine have characteristics similar to
itive hydrostatic pressure, and active ion those of secretory epithelia. Specifically,
transport is required for water flow. Ab- water flow is directly proportional to ion

MALPIGHIAN TUBULE STRUCTURE AND FUNCTION

Fig. 37 Higher magnification, apical portion of ileum. Cytoplasm contains bundles of

microtubules (mt). Lumen is a t bottom. Apical membrane is highly folded. Clear crystalline
inclusions () with rectangular profiles occur within cytoplasm a n d cuticle. Those n e a r outermost surface of the cuticle a r e more rounded i n profile. x 30,000.

297

298

B. J. WALL, J. L. OSCHMAN A N D B. A . SCHMIDT

flux, the osmolality of the transported fluid

is proportional to the osmolality of the
bathing fluid, addition of non-permanent
solutes to the bathing medium brings
about a proportional increase in the concentration of the transported ion, and osmolality of the transported fluid is not
dependent on rate of fluid flow (Oschman
and Berridge, 71). As mentioned in the
beginning of the article, a local osmotic
gradient model has been proposed to account for fluid transport in these diverse
epithelia. Solute uptake from basal infolds
of Malpighian tubules is thought to gener-

ate the osmotic driving force that causes

water to be taken up from the hemolymph
into the tubule cells. Solute pumping into
spaces between microvilli may provide the
local or standing osmotic gradient that
draws water from the cell into the lumen
(Berridge and Oschman, 69). Apparently
potassium pumps are involved in establishing the gradients in Malpighian tubules of many insect species, since the
secreted fluid usually contains a high concentration of potassium (Ramsay, 5 3 ; Berridge, 68).
Figure 38 compares the transporting

Malpighian
tubule

38
Fig. 38 Comparison of gallbladder and Malpighian tubule ultrastructure, approximately
to the scale indicated. Gallbladder produces isosmotic fluid reabsorption and has long narrow
intercellular spaces that are folded into thin leaflets. Malpighian tubule secretes a nearly
isosmotic fluid secretion, and has numerous closely packed microvilli. Gallbladder structure
based on micrographs of Kaye et al. (66) and Tormey and Diamond (67). Malpighian
tubule dimensions based on present study.

MALPIGHIAN TUBULE STRUCTURE AND FUNCTION

2 99

cells of gall bladder and Malpighian tubule

at about the same scale. Each Malpighian
tubule cell has thousands of microvilli,
while a gall bladder cell has only a single
intercellular space. Since both of these
cells transport water in isosmotic proportions, it is implied that the thousands of
short microvillar and basal channels of
the Malpighian tubule cell may be functionally equivalent to a single intercellular
space of gall bladder. While the contribution of each microvillar channel is probably quite small (Taylor, 71a) the effect
of many such small gradients, when
summed over the entire cell surface, may
39
be comparable to that of the intercellular
spaces of gall bladder.
Fig. 39 Formed body hypothesis for fluid seTaylor (71a) has presented an alternate cretion (Riegel, 70): (a) Formed body is secreted
simple osmotic model in which the cells from cell into lumen. (b) Digestive enzymes within
are hyperosmotic to the hemolymph and formed body hydrolyse proteins. ( c ) Increase in
solute concentration within formed body causes
the lumen hyperosmotic to the cytoplasm. HzO
to enter by osmosis, and formed body swells.
This model might seem to apply well to (d) Lumen becomes filled with swollen formed
Periplaneta Malpighian tubules, since the bodies. Solutes unable to enter formed body besecreted fluid is hyperosmotic to the bath- come concentrated in lumen. (e) Water is drawn
into lumen by osmosis. (f) Hydrostatic pressure
ing medium (table 1). However, the tubu- increases
within lumen d u e to water entry, and
lar fluid in many insects is isosmotic or fluid flows through lumen.
even hypoosmotic to the hemolymph (Ramsay, 54; Maddrell, 71). Indeed, it was the
failure of such simple osmotic theories epithelial cell layer by osmosis (fig. 39e).
to account for the general phenomenon While Riegel (68, 70) presents an assortof isosmotic fluid secretion that led to the ment of evidence supporting this hypothdevelopment of the local osmosis concept esis, formed bodies fail to explain a number of the important characteristics of fluid
(e.g. Auricchio and Biirany, 59).
Another explanation of fluid secretion secretion by Malpighian tubules and other
is the formed body hypothesis of Riegel transporting epithelia. For example, if
(70), summarized in figure 39. Formed formed bodies are responsible for fluid sebodies are lysosome-like spherical vesicles cretion, why is there such a strict depen20 p or more in diameter that are ob- dence of fluid secretion on ion secretion
served in micropuncture samples obtained (e.g. Oschman and Berridge, 71)? Absence
from various transporting tissues (reviewed of transportable solutes in the bathing
by Riegel, 70). The formed bodies are medium brings about immediate cessation
thought to contain protein and proteases. of transport in gall bladder, intestine, MalRiegel suggests that the formed bodies pighian tubules, and other transporting
are secreted into the lumen of the Mal- systems. Long term secretion by the formed
pighian tubule or other transporting tissue body mechanism would require a contin(fig. 39a). The proteases become activated, uous synthesis of polypeptides. However,
resulting in the hydrolysis of the proteins Malpighian tubules isolated from Calli(fig. 39b). This increases the osmotic pres- phora can secrete in a simple medium
sure within the formed bodies, water en- containing a single substrate such as malters them by osmosis, and the formed bod- tose, pyruvate, or an individual amino acid
ies swell (fig. 39c). Solutes within the (Berridge, 66). This result, together with
lumen that are unable to penetrate into the finding that inhibitors of oxidative
formed bodies are concentrated as water phosphorylation stop fluid secretion (Beris drawn into the formed bodies (fig. 39d). ridge, 66) indicate that fluid secretion
The increased osmotic pressure of the lu- depends on ion transport driven by ATP
minal fluid then draws water across the hydrolysis, rather than by the alternate

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B . J. W A L L , J . L . O S C H M A N AND B. A . SCHMIDT

synthesis and breakdown of polypeptides,

However, the formed bodies should not be
ignored, and further study may reveal that
they have a role in the excretory process.
Finally, Wessing and Eichelberg ('75)
have arrived a t a n entirely different model
of secretion based on extensive histochemical studies of Drosophila Malpighian tubules. These workers have suggested that
ions are transported across the tubules in
association with mucosubstances, a model
similar to that envisioned by Philpott ('68)
for the chloride cells of teleost fish.
Genesis and maintenance of channel
geometry. An aspect of secretion that
has been neglected is the manner in which
highly folded cell surfaces are formed during development, and how they are maintained during secretion, when there are
local changes in hydrostatic pressure produced by water flow. In the Malpighian
tubules we find microtubules within the
basal interdigitations. Microtubules are
known to be involved in the formation
and maintenance of cell shape (de-The,
'64; Branson, '68; Byers and Porter, '64;
Gibbins et al., '69; Tilney and Gibbins,
'69), and their position within the basal
interdigitations is consistent with a similar role in the Malpighian tubule. If it is
correct that fluid is actively absorbed from
the basal channels, they would then tend
to collapse. The extracellular material that
is apparently stained with lanthanum and
uranium (figs. 7, 8) could be involved in
maintaining the channel i n the optimal
shape for fluid absorption.
Role of the ampulla. Observations on
freshly dissected cockroaches show that
the ampullae accumulate the fluid secreted by the tubules and then, when full,
contract vigorously to force that fluid into
the intestine. This arrangement has two
consequences. First, the tubular fluid is
forced through the labyrinthine drainage
canal, which may act as a one-way valve
preventing back-diffusion from the gut into
the ampulla. Secondly, the tubular fluid
remains in contact with the cells lining
the ampulla during the interval between
ampullar contractions. The possibility thus
arises that the tubular fluid may be modified while in the ampulla. Ultrastructural
evidence supports an absorptive role for
the ampulla. since its cells resemble those
of the h i d g u t , which is the principal or-

gan of nutrient absorption in the insect.

Particularly striking are similarities in
configuration of basal interdigitations,
brush border microvilli, and concentration of mitochondria toward the apical
surface. Although one cannot be certain,
these structural similarities are a t least
suggestive of common functional characteristics. The tubular fluid contains amino
acids, sugars, and other substances that
are essential and therefore must be reabsorbed (Ramsay, '58; Farquharson, '74;
Maddrell and Gardiner, '74). Little is
known of the location or mechanism of
sugar and amino acid absorption, although
Balshin and Phillips ('71) reported active
reabsorption of amino acids by the rectum.
The hindgut of Sarcophuga larvae absorbs
bicarbonate ions in exchange for ammonia
(Prusch, '71). In the cockroach the colon
fluid is usually hypoosmotic to the primary
urine and hemolymph (Wall, '70) suggesting solute reabsorption in this portion of
the gut. The present study indicates that
the ampullae and possibly the proximal
part of the Malpighian tubules can be regarded anatomically as extensions of the
gut, and hence may be the first sites of solute reabsorption from the primary urine.
This could be of particular advantage during antidiuresis, as it would allow more
complete reabsorption of essential metabolites from the tubular fluid when the
rate of urine formation is probably slow.
The large vacuoles or canaliculi in the
ampulla (fig. 34) may be involved in reabsorption.
There have been other suggestions that
the proximal regions in some tubules are
reabsorptive (e.g. Patton and Craig, '39;
Srivastava, '62; Wigglesworth, '31c). The
proximal region of Calpodes Malpighian
tubules reabsorbs ions (Irvine, '69) while
the comparable portion of Rhodnius tubules reabsorbs potassium and water (Wigglesworth, '31c).
Finally, the ampullae of cockroaches are
not structurally analogous to the pylorusbladder (the first part of the hindgut) of
Corethra larvae (Schaller, '49) since the
pylorus-bladder is not a diverticulum but
a segment of the gut. However, the pylorus-bladder contracts rhythmically a t a
rate that is regulated by certain neurohormones (Gersch, '67) and the same may
be true of the ampulla.'

30 1

MALPIGHIAN TUBULE STRUCTURE AND FUNCTION

Embryogenesis of ampullae and

Malpighian tubules
The close ultrastructural resemblance
between the cells of proximal tubule, ampulla, and midgut has a bearing on the
embryological origin of the Malpighian tubules, a controversial topic in the past.
Opinion has differed on whether the tubules are derived from midgut or hindgut
(reviewed by Snodgrass, ' 3 5 ; Srivastava
and Khare, '66). The hindgut or proctodaeum is thought to be entirely ectodermal, although Henson ('32) suggested that
the anterior part of the proctodael invagination is endodermal in origin. The midgut or ventriculus, on the other hand,
arises by regeneration of the mesenteron
rudiments, cells from the endodermal cell
layer surrounding the archenteron. In most
insects the Malpighian tubules seem to
have a histological resemblance to the midgut, and their endodermal origin has been
supported by Tirelli ('29), Savage ('56,
'62), and Butt ('49). Srivastava and Khare
('62) disagree with this view, since the
Malpighian tubules in Philosamia (Lepidoptera) bud off from the blind end of
the proctodaeal invagination before the
mesenteron has formed.
In the present study we have found that
the histological resemblance between the
Malpighian tubules and midgut is even
more striking at the ultrastructural level.
The proximal tubules have similar microvilli and basal infoldings, and are anatomically continuous with the ampulla and
midgut. Srivastava and Khare ('62) point
out that histological similarity does not
prove common embryological origin. However, the ultrastructural and probable
functional similarities between the proximal region of the tubule, ampulla, and
midgut imply that these tissues are derived from endoderm. There is an abrupt
change in the morphology of the cells at
the junction of the proximal and middle
regions of the tubules, leaving us uncertain as to the origin of the middle and
distal regions of the tubule. Henson ('32)
has suggested that tubules originating
from the midgut can become secondarily
attached to the hindgut. It is conceivable
that the reverse could occur, i.e., the tubules could arise from the anterior end
of the proctodaeum and later become attached to the midgut. Such "migrations"

of developing tubules seem improbable

however, and clearly do not occur in Blatta
orientalis and Periplaneta, in which the
tubules grow out from the ampulla (Henson, '44; Schmidt, unpublished).

Excretion - the role of peroxisomes

It has been generally thought that insects are uricotelic. Ammonia is toxic and
therefore has been considered suitable as
an excretory product only in aquatic forms
and in a few terrestrial invertebrates (reviewed by Campbell, '73). Although many
insects do excrete uric acid, at least during a part of their life cycle, ammonia
has been found to be a major excretory
product in the cockroach, Periplaneta
americana (Mullins and Cochran, '72,
'73a,b). Ammonia accounted for up to 91 %
of the total excretory nitrogen, depending
on diet. Uric acid was not detected in fecal
extracts, even in animals fed high protein
diets. Little uric acid was excreted even
when it was fed to the animals. Instead,
much of the uric acid was absorbed and
stored in the fat body.
Cockroach Malpighian tubules contain
structures resembling mammalian microbodies or peroxisomes (fig. 14) and these
structures may be involved in nitrogen
excretion. Peroxisomes have common functional properties in organisms as diverse
as Tetrahymena, yeasts, beans, and man
(DeDuve, '69). Peroxisomes contain a number of enzymes related to nitrogen metabolism, including various L and D-amino
acid oxidases, uricase, or urate oxidase,
and catalase (reviewed by Hruban and
Rechcigl, '69). The amino acid oxidases
catalize the direct formation of ammonia
from amino acids. Likewise, peroxisomes
are involved in the metabolism of uric
acid in many species (Shnitka, '66) according to the scheme:
uric acid
2

uricase
+ 2 allantoin
H20
+ COn + 2 HzOz
catalase
2H20 +

H202

0 2

Hydrogen peroxide is formed and rapidly

decomposed by catalase, which apparently is universally present in peroxisomes.
These organelles have also been observed
in Calliphora Malpighian tubules (Berridge
and Oschman, '69) as well as in other
insect tissues (Locke, '69; Locke and McMahon, '71) but their precise role in ni-

302

B. J. WALL. J. L. OSCHMAN AND B. A. SCHMIDT

trogen metabolism has not been established in insects.

may be able to synthesize essential amino

acids that the cockroach is not able to
manufacture de nouo. A symbiotic relaExcretion - the role of syrnbionts
tionship of this sort has already been demThe symbionts observed within the mid- onstrated between Blattella and the bacdle region of the tubules of Periplaneta terial symbionts within the mycetocytes
resemble the haplosporidians found in of the fat body. Blattella that lack these
Blattella gennanica. Woolever ('66) has symbionts are smaller and less fecund than
made a remarkably thorough study of the infected roaches (Donnellan and Kilby,
latter. She found that the Blattella sym- '67), are unable to synthesize six amino
bionts enter the host orally, multiply with- acids that normal animals can (Henry and
in the Malpighian tubule cells, migrate Block, '60, '62), and have heavier deposits
into the tubule lumen where they under- of urates in their fat body (Brooks and
go schizogony and sporogony, and release Richards, '56; Pierre, '62). The bacterial
their spores into the gut. The close ultra- symbionts can be isolated from the host
structural resemblance between vegeta- and grown on a medium in which uric
tive and spore stages in Blattella and Peri- acid is the only carbon source and can
planeta indicates that the Periplaneta sym- degrade the uric acid to pyruvate or ammonia (Donnellan and Kilby, '67). Combiont could also be a haplosporidian.
Little is known about the metabolic in- parable studies done on Periplaneta have
teractions between the tubule symbionts indicated that the fat body symbionts are
and their hosts. The symbionts are located responsible for the synthesis of folic, ascorat the proximal end of the middle region bic, and pantothenic acids (Gallagher, '63).
of the tubule (figs. 27, 28). They are not Thus the symbionts within the Malpighian
present in the proximal region of the tu- tubules could have a n important role in
bule, possibly because the symbiont micro- metabolism in the insect.
villi are adapted for attachment only to
ACKNOWLEDGMENTS
the closely packed brush border of the
This study was begun in the laboratories
middle region. Since the primary urine
resembles a filtrate of the hemolymph, of Professors Michael Locke and Bodil
the symbionts are bathed in a constantly Schmidt-Nielsen at Case Western Reserve
renewed medium that is rich in nutrients. University, Cleveland, Ohio, with support
I n addition they can readily release their from National Institutes of Health Grants
spores into the digestive tract so they will AM09975 and GM09960. The research
was also supported by USPHS Fellowship
be extruded with the feces.
Dr. K. G. Purohit has pointed out to us GM24015 to B. J. Wall and AM 29555 to
that the symbionts might benefit their J. L. Oschman, a s well as National Instihosts in much the same manner as do tutes of Health grants FR-7028 and AM
those or ruminants (cows, sheep, etc.). 14993. We are indebted to our colleagues
The rumen symbionts synthesize proteins who have provided us with advice and
from amino acids and ammonia. These support.
proteins become available to the host when
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