morphology and function of malpighian tubules and associated structures in the cockroach,...
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Morphology and Function of Malpighian Tubules and
Associated Structures in the Cockroach,
Periplaneta americana
BETTY
J.
WALL, JAMES L. OSCHMAN AND BARBARA A. SCHMIDT
Depar tment
of
Bi o l oy i c u l Sciences, Northwestern University,
Eua nst on, Illinois 60201
A B S T R A C T
This p aper describes the different regions of the Malpighian
tubules and the associated structures (ampulla, midgut, ileum) in the cock-
roach, Periplaneta americana. There are about 150 tubules in each insect.
Each tubule consists of a t least three parts. T he sho rt distal region
is
thinner
than the other parts and is highly contractile. The middle region comprises
most of t he tubule len gth and is composed of primary an d stellate cells. Pri-
mary cells contain numerous refractile mineral concretions, while stellate cells
have smaller nuclei, fewer organelles, simpler brush border, and numerous
multivesicular bodies. Symbiont protozoa are sometim es prese nt within the
lume n of t he middle region nea r where it opens into the proximal region of th e
tubule. The latter is a short region that drains the tubular fluid into one of
the six ampullae. These are contractile diverticula of the intes tine located a t
the midgut-hindgut junction. The ampulla
is
highly contractile, and consists
of a layer of epith elial cells surroun ding a cavity that opens in to 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 micro-
villi, basal infolds, and distribution of mitochondria. This suggests an endoder-
ma1 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 transp ort ar e evaluated with re gard to physiological and morphological
cha rac ter ist ics of Malp ighian tubules. The possible role of long narrow chan -
nels such a s those between microvilli and within basal folds is considered, as
is th e mechanism by which these structures are formed and maintained. Also
discussed i s the role of peroxisomes and sy mbionts in the excretory process.
Osmoregulation and excretion in insects
occur in two steps. First, a primary secre-
tion or urine is produced by the Malpighian
tubules. This fluid, which resembles in
many ways a filtrate
of
the blood (Ram-
say, '58; Farquharson, '74; Maddrell and
Gardiner, '74) flows into the gut and ac-
cumulates in the rectum, where the sec-
ond, reabsorptive phase takes place (re-
viewed 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 corre-
sponds functionally to the nephridia of
Annelida and Onychophora and to the kid-
ney 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 ob-
tained on the composition of the secreted
fluid and the effects of bathing medium
composition on rate of formation and com-
position of the secreted fluid (Berridge,
'68, '69). These studies have contributed
to the development of our current theories
on the mechanism by which various epi-
thelia form fluid secretions (reviewed by
Oschman and Berridge, '71; Berridge and
265
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266
B .
J . W A L L , J . L . O S C H M A N A N D
B .
A . S C H M I D T
Oschman, '72; Oschman et al., '74). It is
now thought that movement of the aque-
ous component of secretions may be the
consequence of an osmotic gradient estab-
lished by pumping some solute into a con-
fined 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; Dia-
mond 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 secre-
tion (see
DISCUSSION ,
i t
does achieve an
integration of physiological findings with
ultrastructural features of the transport-
ing cells.
The morphology of Malpighian tubules
has been described for a number of in-
sect 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 Bac-
cetti, '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 Eichel-
berg, '69a,b; Wigglesworth and Salpeter,
'62). These studies have, however, pro-
vided 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 capaci-
ties 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 informa-
tion obtained from this study provides
clues about the embryological origin of
the Malpighian tubules as well as the man-
ner in which foldings of the cell surface
are formed and maintained.
M A T E R I A L S A N D METHODS
Animals. Adult male Periplaneta amer-
icana were used in this study. Stock cul-
tures were maintained in 12 hours light
with water and food (oatmeal or Lab Chow)
continuously available.
Physiology. To determine the concen-
tration of the fluid secreted by cockroach
Malpighian tubules, animals were anes-
thetized with COz and then dissected open
under Ringer solution. The tubules were
dissected free and removed into a sepa-
rate drop of Ringer solution under Paraf-
fin 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
Morphology. Malpighian tubules and
associated structures (ampulla, midgut,
ileum) were fixed in 2.5% glutaraldehyde
with
0.05 M
phosphate buffer and
5%
su-
crose, 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 sec-
tioned with the Huxley microtome. Sec-
tions were stained with lead citrate and
uranyl acetate. Micrographs were taken
with either the RCA-EMU-3F or the
Hi-
tachi HU -l l-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 re-
tained 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 ura-
nium. 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
then processed through wash and osmium
solutions that were buffered in the same
('55).
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MALPIGHIAN TUBULE STRUCTURE AND FUNCTION
267
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 embed-
ded in Spurr resin.
L a n t h a n u m t r e a t m e n t .
Additional in-
formation on the morphology of extracel-
lular channels was obtained by fixing tu-
bules 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 wa-
ter, and stain in 0.01% aqueous toluidine
blue for 20 minutes. The specimens
were
then dehydrated
in
ethanol and mounted
in
Permount.
RESULTS
General descr ip t ion
The general arrangement of the Mal-
pighian tubules and associated structures
is illustrated in figure 1. There are 144-
192 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 2-
3
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 tu-
bules has been described previously (Crow-
der and Shankland, 72) and will not be
elaborated upon here. The tubules con-
tract vigorously
in
animals that have been
dissected open under Ringer solution, al-
though there is much variation in the rate
of contraction.
We have identified three regions in these
xirnal
Ion
idgut
trachea
Fig.
1
General arrangemen t of th e structures
described in th is paper. T he full length of only one
of the Malpighian tubules is shown. The mid dle
region is th e longest part. T he short thin dis tal
region is highly contractile and the short proxi-
ma l region inserts into the am pulla. Each of th e
six ampullae drains about
24-32
tubules. The am-
pullae are contractile enlargements on the intes-
tinal surface at the juncti on between midgut and
ileum. Trach eae branch over the midg ut surface
an d send processes into the am pullae. Fine tra-
cbeoles also attach the tubules to other organs
such as fat body.
tubules, distal, middle, and proximal. The
route of flow is from distal region to proxi-
mal 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 stor-
age 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 se-
cretion vacuoles seen in living tubules,
although there was no sharp boundary
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MALPIGHIAN TUBULE STRUCT URE AND FUNCTION
269
Fig.
2
Survey view of the short distal region. There is a single strip of muscle (m)
embed^
ded in the connective tissue. Th e muscle is capable of shor t fast contractions. Distal region
differs from other portions in th at cells lack vacuoles; lume n i s narro w, a nd m any of the long
microvilli contain extensions of filam entou s mitochondria.
x 4,600.
ance. We
do
not illustrate the crystalline
arrangement of the core as it has been
adequately described in other tissues (re-
viewed by Tandler and Hoppel, '72)
in-
cluding the fat body of the American cock-
roach (Gharagozlov,
'69)
and of the fruit
fly
(Takahashi et al.,
70),
and the mam-
malian kidney tubule (Suzuki and Mostolfi,
'67;
Youson and McMillan, '70). There
are also round membrane-bound organelles
similar in size to mitochondria but with
a homogeneous granular interior (fig.
3) .
These may be microbodies. Other organ-
elles include Golgi complexes, bits of
smooth and rough endoplasmic reticulum,
and small vesicles. Two sorts
of
microvilli
project into the lumen in the distal region,
those containing extensions of mitochon-
dria, and others containing extensions of
the smooth endoplasmic reticulum. The
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MALPIGHIAN TUBULE STRUCTURE AND FUNCTION
Fig.
5
Survey micrograph
of
middle region. Lumen i s larger tha n in distal region and
cells contain nu me rou s clear vacuoles an d mineralized concretions. Large dense structures
1)
resemble lysosomes observed in other insect tissues (e.g. Locke and Collins,
'65).
Penetra-
tion of mitochondria into microvilli i s variable, i.e. compar e bru sh border a t top and bottom
of picture w ith tha t on either side. Connective tissue (ct) is thick an d mu scle (m ) is embedded
in it. Mitochondria present throughout cytoplasm. Portions of blood cells show n at u pper left.
X 4,000.
271
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272
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
th 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
inverte-
brates.
M i d d l e region
Figure
5
provides a survey view
of
the
middle region of the tubule, which com-
prises most of the tubule length.
A s
men-
tioned above, this region is either com-
pletely 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 be-
low. The cells interdigitate with each other
and are joined by septate junctions as
in
the distal region of the tubule. The mid-
dle region is comprised of two cell types,
the primary or secretory cells, and stellate
cells. The primary cells are more abun-
dant and are described first. The following
description proceeds from basal to apical
surface.
Fig.
7
Basal portion, m iddle region
of
tubule.
Cells interd igitat e extensively. Basement m emb rane
is laminated. Note profiles of microtubules
a-
rows) and mitochondria (m) in cytoplasmic inter-
digitations. Profiles
of
rough and smooth endo-
plasmic reticulum (rer and ser) are present within
th e interdigitations.
X 40,000.
Fig.
8
Basal portion
of
tubule cell, middle re-
gion, fixed i n
glutaraldehyde-lanthanum.
Unstained
section. Lanthanum is deposited within basement
mem brane (bm) an d extracellular ch anne ls be-
tween interdigitations. X 52,000.
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M A L P IG H I AN T U B U L E S T R U C TU R E A N D F U N C T I O N
2
73
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274 B . J.
WALL.
J .
L. OSCHMAN AND
B . A .
SCHMIDT
Basal surface
Figure
6
reveals details of the tubule-
hemolymph 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 colla-
gen fibers appear to be embedded in a
clear matrix. The underlying tubule base-
ment membrane
is
comprised of an outer
layer of fine filaments and an inner gran-
ular zone. Connective tissues and base-
ment 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.
W e
suspect that the collagen fibers are
necessary to provide a n elastic protective
sheath around the highly contractile tu-
bules, 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 move-
ment of certain molecules into or out of
the basal infoldings (e.g. Oschman and
Berridge, '7
1 .
The basal infoldings form deep chan-
nels extending perpendicular to the base-
ment membrane. These chan nels probably
correspond to the fine cytoplasmic str ia-
tions 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 convention-
ally prepared specimens (fig. 7), when
specimens are fixed in glutaraldehyde-lan-
thanum or in glutaraldehyde-calcium fol-
lowed by
in
block
treatment with uranium-
calcium (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 as
an extracellular marker much like col-
loidal lan tha num; alternatively, fixing and
processing with lanthanum or calcium-
containing solutions may precipitate and
retain some negatively charged extracel-
lular substance. Further study is needed
to clarify this finding.
Figures 7 and
9
reveal that the ba-
sal channels are not formed from simple
folding of the basal plasma membrane
but instead ar e developed from interdig-
itating finger-like extensions of neigh-
boring cells. A similar arrangement oc-
curs 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). Th at these a re indeed processes from
adjacent cells is documented in figure 9,
which
is
a tangential section through the
basal surfa ce 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 ad-
jacent cells. Toward the basal surface
these extensions often contain microtu-
bules, 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 mi-
crotubules 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 a t the openings of the basal
infolds.
In addition to the microtubules men-
tioned above, the cytoplasm within the
interdigitating processes from adjacent
cells contains mitochondria , ribosomes, and
segments of smooth and rough endoplas-
mic reticulum (fig. 7). In some regions
one encounters chain s 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 ar-
rangement occurs in the ciliary epitheli-
um 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 struc ture in the
Malpighian tubule with that of ciliary epi-
thelium, we a re unsure of its exact struc-
ture and think that the chains of vesicles
Fig. 9
Tangential section n ear surface, middle
region. One cell is lighter t han its neighbor, em-
phasizing int erdig itatin g processes. Microtubules
shown in
transverse section
in
figures 6 and
7
occur he re in lo ngitu dinal profile (arrows). oriented
parallel to the sides
of
the interdigitating proc-
esses. In upper
left
basement membrane compo-
nen ts are observed with collagen and other fibrils
oriented parallel to surface as in figure 6 . x 40,000.
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M A L P I G H IA N T U B U L E S T R U C T U R E A N D F U N C T I O N 275
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276
B . J . WALL. J .
L.
O S C H M A N AND B . A. SCHMIDT
Fig.
10
Association
of
mitochondria with membranes
of
interdigitating processes
from
adjacent
cells.
Apparent ch ains
of
vesicles within t he 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 fea-
ture
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 concre-
tions 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 tu-
bules as well as in insect intestinal cells
(Gouranton, 68), insect utricles (Ballan-
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MALPIGHIAN TUBULE STRUCTURE AND 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 in-
vertebrates. The nature of these concre-
tions has been a matter of controversy.
Earlier studies (Berkaloff, '58, '59; Sri-
vastava, '62; Fuller, '66) suggested that
these structures contained urates. Wig-
glesworth and Salpeter ('62) noted that
the intracellular refractile concretions
were not the same as the excretory gran-
ules
in
the lumen, as only the latter gave
a positive urate test with ammoniacal sil-
ver nitrate. Gouranton ('68) was unable to
detect uric acid or guanine by chromatog-
raphy
of
intestinal concretions. Instead,
he detected calcium, magnesium, iron,
phosphate, carbonate, protein, and acid
mucopolysaccharide. Similarly, Mello and
Bozzo ('69) tentatively recognized lipopro-
tein or phospholipid in excretory globules
in Malpighian tubules of larval bees. Stad-
houders and Jacobs ('61) did histochemi-
cal studies on the structures in cockroach-
es and found they contained calcium and
phosphorus. We thus suspect that the con-
centric 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 Coch-
ran, '72); (2) urate crystals observed in
electron micrographs of cornea of gout
patients (Slansky and Kuwabara, '68) show
a cuboidal crystalline structure that dif-
fers considerably from the concentric con-
cretions in the Malpighian tubules; and
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278
B . J . W A L L , J .
L .
O S C H M A N A N D
B.
A . S C H M I D T
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 kid-
ney tubules (Giacomelli et al., 64). The
concretions are occasionally found in the
tips of microvilli (fig. 2 0 , and may enter
the lumen in this way.
In addition to mitochondria, ribosomes,
and endoplasmic reticulum, one occasion-
ally encounters structures resembling an-
nulate 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 peroxi-
somes of vertebrate tissues. Microbodies
typically have a crystalline core, as do at
least some of the dense granules in Mal-
pighian tubules (fig. 14). In many species
microbodies participate in the metabolism
of ammonia and uric acid, and the possi-
ble role of these organelles in nitrogen
excretion will be considered in the D I S -
In addition to the spherical vacuoles
described above, some sections of the tu-
bules 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 proc-
essing. If urates are stored in these Mal-
pighian tubules, it is more likely that they
would be within prismatic granules such
as these than in the larger refractile con-
cretions. We suggest this because the
urate-containing crystals in insects usu-
ally have needle, lens, or lozenge shapes
(Noel and Tahir,
29)
similar
to
many of
the inclusions
in
figure 15. Further, Wig-
glesworth
(53)
reported that the uratic
granules in
Rhodnius
tubules are soluble
in aqueous fixative and are rapidly dis-
solved by osmium. Figure 15 also illus-
trates an autolysosome, Golgi complex, and
multivesicular body.
The mitochondria in the middle region
lack the crystalline inclusions encoun-
tered in the distal tubule. However, in
one specimen we have observed a region
in which most of the mitochondria were
irregular or doughnut shaped, contain-
ing central clear regions of density and
texture similar to the cytoplasm (figs.
16,
17). In some cases (fig. 17) the mitochon-
drial 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 patho-
logical condition such as a virus.
Apical
surface
The apical cell surface is folded into
microvilli of the same sort a s those found
in
the distal region. However, the degree
to which mitochondria penetrate into the
microvilli seems to be less than in the dis-
tal region (compare figs. 2 and
5 )
and in
CUSSION.
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MALPIGHIAN TUBULE STRUCTURE AND FUNCTION
2
79
Fig.
1 3
Annulate lam ellae (al) and vacuoles containing dense granular m aterial (d). Th e
latter may be precursors of the large concentric concretions such a s illustrated in figures 1 1
and 12. or they ma y be microbodies or peroxisomes, as shown in figure 1 4 .
x
80.000.
Fig. 1 4 Dense body w ith crystalline core (arro w) similar to that found in vertebrate
peroxisomes. X
49,000.
some areas , no mitochondria are found same cells. Note tha t in figure
5
the micro-
within microvilli (figs.
16,
20,
21).
There villi
in
the cell shown in profile at the
is
variation
in
the exten t of penetration bottom and the cell shown at the top have
of mitochondria into microvilli within the areas where no mitochondria are within
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280 B . J. WALL,
J.
L. O S C H M A N A N D 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 en-
In 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
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MALPIGHIAN TUBULE STRUCTURE AND FUNCTION
281
Fig. 16 Atypical mitoch ondr ia, mid dle region of tubule. These mitochondria conta in on e
or more pockets
of
cytoplasm
( ) .
Their functional significance is unknown. Also included i s
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 junc-
tions. The most extensive in terms of area
of contact is the septate junction, which
is characterized
by
a series of thin dia-
phragms or septa extending perpendicular
to the plasma membrane (fig. 23). Ura-
nium-calcium treatment (fig. 22) renders
the spaces between septa opaque to elec-
trons.
so
that the 3-dimensional structure
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282
B.
J .
WALL,
3. L.
OSCHMAN AND B.
A .
SCHMIDT
Fig. 18 Cells of middle region sometimes con-
tain early stages of autophagy (st ructu res bounded
by isolation m emb ranes , im ) and later stages (lyso-
Fig.
17
Higher magnification
of
atypical mi- some,
1).
Included
is
a t.s.
of
a crystalline array
of
tochondria containing pockets
of
cytoplasm. Note tubules (t) of unknown significance. Golgi com-
spoke-like configuration
of
mitochondrion at top, plex (G) and endoplasmic reticulum ar e also in-
and m ultivesicular body (mvb). X
75,000.
cluded. X 29,000.
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M A L P I G H I A N TUBULE S T R U C T U R E A N D F U N C T I O N
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 gra nular inclusions (*), on e of which
is surrounded by tub ular struc tures similar to those illustrated in ficure 18. X 41 000.
of the septa is seen
in
relief as parallel ignated as comb desmosomes by Danilova
folded sheets of intermembranous mate- et al.
('69).
Images similar to those
in
rial. Junctions
of
this sort were first de- figure
22
are obtained with lanthanum
scribed by Locke
('65)
and have been des- staining, and have been integrated with
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284
B .
J .
WALL, J . L.
OSCHMAN
AND B . A . SCHMIDT
Fig. 21 Transverse section of microvilli, mid-
dle region. Profiles of smooth endoplasmic reticu-
lum ar e circular in tran sverse sections. Smaller
microvilli lack mitochondria or smooth endoplas-
mic reticulum. X 66,000.
freeze fracture data (e.g. Flower, '70) to
provide a structural model for this junc-
tion (Gilula et al.,
'70).
Gap junctions are
also encountered occasionally, and, again,
they have a characteristic
appearance
n
face views of material treated with ura-
nium-calcium
in
block. This type
of
image
has been obtained previously
n
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).
Symbiotic pro tozoa
In some of the specimens we have ob-
served symbiont protozoa within the proxi-
mal portion of the middle region. Figure
Fig.
20
Apical surface, middle region. Micro-
28
illustrates the ultrastructural appear-
ance of these symbionts, while their
illi contain tubular structures that ar e continu-
ous with smooth endoplasmic reticulum (arrow).
Note dense concretion
hat
appears to
be pinch. tion within the tubule is illustrated in
ing
off
into
lumen
(L). X
40,000.
figure 27. These symbionts are similar
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MALPIGHIAN TUBULE STRUCTURE AND FUNCTION
285
Fig.
22
Face view of sept ate junc tion after glu-
taraldehyde-calcium fixation and uranium acetate-
calcium
in block
stain ing. Urani um fills extracellu-
lar space delineating septa as pleated sheets.
X 18,000.
Fig.
23
Septate junct ion, middle region.
X
65,000.
in structure to the haplosporidians de-
scribed by Woolever ('66) in the cockroach,
Leucophaea .
These protozoa attach to the
tubule cells by means of microvilli tha t
insert between the microvilli of the tubule
cells. These sporozoa produce spores wi th
a thick cuticle that is difficult to section.
Fig.
24
Stellate cells. Note in figure b that
A
detailed description
of
the protozoa
is
stellate cell is comp arabl e in size to nucle us (n p)
not
appropriate in
this paper, although we
o f
primary cell. Photomicrographs
of
middle
re-
gion fixed in Carnoy's fixative and stained with
will discuss below their possible involve- aqueous toluidine blue. Stellate cells have acquired
ment in excretion. a blue color. x 1,250.
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286
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
Fig.
25
Basal portion, ste llate cell. Note ab sence
of
vacuoles and presence
of
numerous
multivesicular
bodies.
Stellate cell nucleus (N).
X 21,000.
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M A L P I G H I A N T U B U L E S T R U C T U R E A N D FUNCTION
87
Fig. 26
Stellate cell in region whe re it exten ds across whole tubule. Organelles are less
abundant than
in
prim ary cells. and m icrovilli lack inclu sion s such a s mitochondria or smooth
endoplasmic reticu lum . Basal interdigitations ar e sim ilar to those of primary cells.
X
11,500.
Stellate
cells
Whole-mounts stained with toluidine
blue show that the tubules contain a sec-
ond smaller sort
of
cell that occurs at
regular intervals along the length of the
tubule (fig. 24). A comparable second cell
type has been noted in a number of pre-
vious studies (reviewed by Taylor, '71b),
but their function remains obscure. In the
cockroach the stellate cells have smaller
nuclei than primary cells, and have nar-
row processes that extend between adja-
cent primary cells. Because of their small-
er size they present narrow profiles in
transverse sections and are encountered
infrequently in electron micrographs. The
stellate cells are characterized by the ab-
sence of vacuoles or concretions
so
abun-
dant in primary cells (figs.
25,
26). The
basal surface
of
stellate cells
is
elaborated
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288 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 interdigita tion between a pri-
mary cell and a lightly stained stellate
cell). There are numerous multivesicular
bodies, lysosomes, segments of rough en-
doplasmic reticulum, and Golgi complexes
with associated small vesicles (figs. 25,
26). The mitochondria are of similar struc-
ture to those in primary cells, although
they seem to be less abundant (fig. 26).
The apical surface is folded into micro-
villi, but these differ from those in pri-
mary 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 . Th e transition in morphology of
the tubule cells at the junction between
middle and proximal regions is apparent
in light micrographs of methylene blue-
stained thick sections (fig. 27). The proxi-
mal region does not contain symbionts
and there
is
an abrupt change in the form
of the brush border. Electron micrographs
(figs. 29, 30) reveal that cells of the prox-
imal region have microvilli that are thin-
ner 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 struc-
ture (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 compar tment of rela-
tively large volume that opens to the hemo-
coel in relatively few places (fig. 30; see
Berridge, '70). Finally there
is
an inclu-
sion in these cells that
is
also found in
the ampulla. This is a dense membrane-
bound accumulation of membrane-like
leaflets with a wavy appearance in sec-
tions. This inclusion
is
illustrated in the
proximal tubule (figs. 29, 30) and in the
ampulla (fig. 32).
Fig. 27
Photomicrog raph of
1
p thick methyl-
ene blue-stained section of region where tubule
drains into ampulla A) . Symbionts (S) are pres-
ent in middle region. Note abrupt transition in
cell density an d in b rush border at junctio n be-
tween middle and proximal regions (arrow). X 820.
The connective tissue and musculature
investing the tubules is most fully devel-
oped in the proximal region (figs. 29,
30).
The cells lack the vacuoles and concre-
tions a bundant in the middle region. How-
ever, 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 concen-
trated toward the apical or luminal sur-
face, rather than scattered throughout the
cells a s in middle and dist al regions of the
Fig. 28 Section
of
middle region tubule near
proximal en d. Protozoan sym bionts (s) (haplospo-
ridia) a re in lum en. Thin processes from th e pro-
tozoan cells mingle with microvilli
of
Malpighian
tubule. M uscle ( m ) is well developed in th is re-
gion.
x
12,200.
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MALPIGHIAN
TUBULE STRUCTURE AND FUNCTION
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290
B . J . WALL J. L . OSCHMAN AND B.
A .
SCHMIDT
Fig. 29
Survey view, proximal region of tubule. Lumen
is
triangular in shape, basal sur-
face is heavily invested w ith connective tissue an d mu scula ture
(m).
Microvilli ar e fur the r
apart than in other regions. Basal infolds are distend ed. Dense bodies (arro ws) have lam ellar
interiors similar to those in am pul la cells (figs. 32, 34). X 4,100.
tubules (figs. 2,
5).
The cytoplasm also nor smooth tubules extend into the micro-
contains lysosomes, rough endoplasmic
re-
villi. Instead, each microvillus has a core
ticulum, Golgi complexes, and numerous of fine filaments similar to those within
multivesicular bodies. There is generally midgut microvilli (e.g. Smith et al.,
'69).
a region at the cell apex that is free of Cells
of
the proximal region interdigitate
organelles except for small vesicles and extensively along both basa l and latera l
filaments (fig.
30).
Neither mitochondria borders.
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MALPIGHIAN TUBULE STRUCTURE AND FUNCTION
291
Fig. 30 Proximal region a t higher m agnification. Connective tissue (ct) is thicker th an
basemen t mem brane (bm) a nd ha s muscles (m ) embedded in it . Cells interdigitate, but basal
cha nne ls (bc) between a djace nt cell processes ar e wider tha n in middle region (compar e with
figs. 7,
19).
Microvilli ar e no t closely pack ed and r esemb le thos e of am pu lla r cells (figs.
32,
33):
Dense lamellate inclusions (arrow) ar e similar to those found in amp ullae
(fig. 3 2 ) . X 14.000.
Ampulla
interdigitations similar to those of the prox-
Figure
31
summarizes the organization imal region (figs. 29, 30). As mentioned
of
the ampulla. The lumen of the
proxi-
above, the ampulla contains large mem-
ma1 region narrows at the region where brane-enclosed structures (about 2-3
p
in
it
drains into the ampulla
(figs.
27, 31). diameter) with a laminated interior (figs.
The epithelium lining the ampulla is col- 32, 34). Although we do not know the
umnar and has thin microvilli
and
basal function
of
these structures, they resem-
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292
midgut
B . J . W A L L , J.
L.
OSCHMAN AND B .
A .
SCHMIDT
-Middle
leum
Fig. 31 Sum mary diag ram of ampu lla a nd associated structures. Muscles are not in-
cluded. Proximal part of middle region contains protozoa (fig.
27).
Proximal region of tubu le
is composed of cells similar to ampulla. Tubule lum en narrows at region where it d rains into
ampu lla. Cavity of am pulla d ra in s via nar row slit into gut. Apical surfaces of tubules, am -
pullae, an d midgut ar e 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 re-
ticulum 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 pro-
jecting inward and containing a small
amount of membranous and granular ma-
terial. These structures can be observed
in methylene blue-stained thick sections.
By tracing these structures in serial sec-
tions, we have found that they are deeply
penetrating channels that approach close-
ly 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 sec-
tions, and shows a region where one cana-
liculus 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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MALPIGHIAN TUBULE
STRUCTURE
AND FUNCTION
293
Fig.
33
Transverse section
of
microvilli of am-
pulla. Th ese microvilli a re irregular i n sh ap e and
are not closely packed as in tubules (compa re 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 some-
thing resembling them but on a smaller
scale in cockroach midgut (unpublished).
Clearly, this part
of
the excretory system
should be studied further.
D rainage c ana l
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 ap-
pears 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 por-
tion of thick connective tiss ue is inc lud ed. Cells
conta in peculiar lamellated inclu sion s (arrows). Mi-
tochondria are particularly abu nda nt near apical
surface.
X 5,000.
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294
B . J .
WALL,
J.
L. OSCHMAN AND B . A . SCHMIDT
Fig. 34 Cavities within ampullar cells. These
clear spaces seem to be bounded by apical me m-
brane, since short microvilli protrude i nto 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 no t their basal
surfaces. Canaliculi are present. The mi-
crovilli resemble those of the midgut even
more closely than do those
of
the ampulla.
The cells contain lipid droplets, small mi-
tochondria, numerous ribosomes, and oc-
casional bits of rough endoplasmic reticu-
lum. 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 dis-
organized jumble of membranes resem-
bling 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 num erous published de-
scriptions of midgut structure. To empha-
size this point, we include a brief descrip-
tion
of
the beginning of the hindgut,' the
ileum. The ileum of
Periplane ta
has not
been described elsewhere although Ballan-
Dufrancais ('72) has described Blatte l la
ileum. The ileum
is
lined by a thin cutic-
ular 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 mag-
nifications (fig. 36) one observes dense
bands of material lying adjacent to the
lateral plasma membranes. Higher mag-
nification (fig. 37) reveals that these are
bundles of microtubules. The apical plas-
ma 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 en-
docuticle contains clear oval or lens-shaped
inclusions that either arise from or give
rise to clear prismatic crystals
in
the api-
cal cytoplasm of the cells (fig. 37). Very
little information is available on the phys-
iology of the ileum, and further ana-
tomical description would be pointless at
present.
DISCUSSION
M e c h a n i s m
of
secre t ion
The striking anatomical feature of Mal-
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MALPIGHlAN
T U B U L E
S T R U C T U R E
AND F U N C T I O N
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 pre- merular kidney, proximal and distal kid-
vious ultrastructural studies is the pres- ney tubules, etc. (Pease, '56; Fawcett, '62;
ence
of
highly folded cell surfaces.
This
Berridge and Oschman, '72). All
of
these
feature is characteristic of many other se-
epithelia secrete a fluid that is about is-
cretory tissues such as pancreas, sweat osmotic with blood and that is nearly pro-
and salivary glands, choroid plexus, aglo- tein-free (except where protein secretion
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AND
FUNCTION
297
Fig. 37 Higher magnification, apical portion of ileum. Cytoplasm con tain s bundle s of
microtubules (mt). Lumen is a t bottom. Apical mem bra ne is highly folded. Clear crystalli ne
inclusions ( ) with rectang ular profiles occur wi thin cytoplasm an d cuticle. Those ne ar outer-
most surface of the cuticle a re more rounded in profile. x 30,000.
-
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298 B .
J.
W A L L ,
J.
L . O S C H M A N 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 con-
centration of the transported ion, and os
molality of the transported fluid is not
dependent on rate of fluid flow (Oschman
and Berridge, 71). A s mentioned in the
beginning of the article, a local osmotic
gradient model has been proposed to ac-
count 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 estab-
lishing the gradients in Malpighian tu-
bules of many insect species, since the
secreted fluid usually contains a high con-
centration of potassium (Ramsay,
53;
Ber-
ridge, 68).
Figure
8
compares the transporting
Malpighian
tubule
38
Fig. 38 Comparison of gallbladder and Malpighian tubule ultrastructure, approximately
to the scale indicat ed. Gallbladder produces isosmotic fluid reabsorption and h as long narr ow
intercellular spaces that are folded into thin leaflets. Malpighian tubule secretes a nearly
isosmotic fluid secretion, an d has num erou s closely packed microvilli. Gallbladder structur e
based on micrographs of Kaye et al.
(66)
and Tormey and Diamond (67). Malpighian
tubule dimensions based on present study.
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MALPIGHIAN TUBULE STRUCTURE AND FUNCTION
299
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 propor-
tions, it is implied that the thousands of
short microvillar and basal channels of
the Malpighian tubule cell may be func-
tionally equivalent to a single intercellular
space of gall bladder. While the contribu-
tion of each microvillar channel is prob-
ably quite small (Taylor, 71a) the effect
of many such small gradients, when
summed over the entire cell surface, may
be comparable to that of the intercellular
spaces of gall bladder.
Taylor (71a) has presented an alternate
simple osmotic model in which the cells
are hyperosmotic to the hemolymph and
the lumen hyperosmotic to the cytoplasm.
This model might seem to apply well to
Periplaneta
Malpighian tubules, since the
secreted fluid is hyperosmotic to the bath-
ing medium (table 1). However, the tubu-
lar fluid in many insects is isosmotic or
even hypoosmotic to the hemolymph (Ram-
say, 54; Maddrell, 71). Indeed, i t was the
failure of such simple osmotic theories
to account for the general phenomenon
of isosmotic fluid secretion that led to the
development of the local osmosis concept
(e.g. Auricchio and Biirany, 59).
Another explanation of fluid secretion
is the formed body hypothesis of Riegel
(70), summarized in figure 39. Formed
bodies are lysosome-like spherical vesicles
20
p or more in diameter that are ob-
served in micropuncture samples obtained
from various transporting tissues (reviewed
by Riegel, 70). The formed bodies are
thought
to
contain protein and proteases.
Riegel suggests that the formed bodies
are secreted into the lumen of the Mal-
pighian tubule or other transporting tissue
(fig. 39a). The proteases become activated,
resulting in the hydrolysis of the proteins
(fig. 39b). This increases the osmotic pres-
sure within the formed bodies, water en-
ters them by osmosis, and the formed bod-
ies swell (fig. 39c). Solutes within the
lumen that are unable to penetrate into
formed bodies are concentrated as water
is drawn into the formed bodies (fig. 39d).
The increased osmotic pressure of the lu-
minal fluid then draws water across the
39
Fig. 39 Formed body hypothesis for fluid se-
cretion (Riegel,
70):
(a ) Formed body is secreted
from cell into lume n. (b) Digestive enzy mes within
formed body hydrolyse proteins. ( c ) Increase in
solute concentration within formed body causes
HzO to enter by osmosis, and formed body swells.
(d) Lumen becomes filled with swollen formed
bodies. S olutes un ab le to enter formed body be-
come concentrated in lumen . (e) Water is draw n
into lumen by osmosis.
f)
Hydrostatic pressure
increases within lumen d ue to water entry, and
fluid flows through lum en.
epithelial cell layer by osmosis (fig. 39e).
While Riegel
(68,
70)
presents an assort-
ment of evidence supporting this hypoth-
esis, formed bodies fail to explain a num-
ber of the important characterist ics of fluid
secretion by Malpighian tubules and other
transporting epithelia. For example, if
formed bodies are responsible for fluid se-
cretion, why is there such a strict depen-
dence of fluid secretion on ion secretion
(e.g. Oschman and Berridge, 71)? Absence
of transportable solutes in the bathing
medium brings about immediate cessation
of transport in gall bladder, intestine, Mal-
pighian tubules, and other transporting
systems. Long term secretion by the formed
body mechanism would require a contin-
uous
synthesis of polypeptides. However,
Malpighian tubules isolated from Calli-
phora can secrete in a simple medium
containing a single substrate such as mal-
tose, pyruvate, or an individual amino acid
(Berridge, 66). This result, together with
the finding that inhibitors of oxidative
phosphorylation stop fluid secretion (Ber-
ridge, 66) indicate that fluid secretion
depends on ion transport driven by ATP
hydrolysis, rather than by the alternate
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300
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 histochem-
ical studies of Drosophila Malpighian tu-
bules. 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.
G e n e s i s a n d m a i n t e n a n c e of c h a n n e l
ge om e t ry .
An aspect of secretion that
ha s been neglected
is
the manner in which
highly folded cell surfaces are formed dur-
ing
development, and how they are main-
tained during secretion, when there are
local changes in hydrostatic pressure pro-
duced by water flow. In the Malpighian
tubules we find microtubules within the
basal inte rdigitations. 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 simi-
lar 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 mater ial that
is apparently stained with lanthanum and
uranium (figs. 7, 8) could be involved in
maintaining the channel in the optimal
sha pe for fluid absorption.
Role of t h e a m p u l l a . Observations on
freshly dissected cockroaches show that
the ampullae accumulate the fluid se-
creted 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 gu t 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 modi-
fied while in the ampulla. Ultrastructural
evidence supports
an
absorptive role for
the ampulla. since its cells resemble those
gan of nutrient absorption in the insect.
Particularly striking are similarities in
configuration of basal interdigitations,
brush border microvilli, and concentra-
tion of mitochondria toward the apical
surface. Although one cannot be certain,
these structural similarities are at least
suggestive of common functional charac-
teristics. The tubular fluid contains amino
acids, sugars, and other substances that
are essential and therefore must be re-
absorbed (Ramsay,
58;
Farquha rson, '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 Sarc ophuga larvae absorbs
bicarbonate ions in exchange for amm onia
(Prusch, '71). In the cockroach the colon
fluid i s usually hypoosmotic to the primary
urine and hemolymph (Wall, '70) suggest-
ing so lute 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 re-
garded anatomically as extensions of the
gut, and hence may be the first sites of sol-
ute reabsorption from the primary urine.
This could be of particular advantage dur-
ing antidiuresis, as it would allow more
complete reabsorption of essential metab-
olites 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 re-
absorption.
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
Calpode s
Malpighian
tubules reabsorbs ions (Irvine, '69) while
the comparable portion of
R h o d n i u s
tu-
bules reabsorbs potassium and water (Wig-
glesworth, 31c).
Finally, the ampullae of cockroaches are
not structurally analogous to the pylorus-
bladder (the first part of the hindgut) of
Core thra larvae (Schaller, '49) since the
pylorus-bladder is not a diverticulum but
a segment of the gut. However, the py-
lorus-bladder contracts rhythmically at a
rate that
is
regulated by certain neuro-
hormones (Gersch, '67) and the same may
of the hidgut, which
is
the principal or-
be true of the ampulla.'
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MALPIGHIAN TUBULE STRUCTURE AND FUNCTION
30 1
Em bry oge ne s i s of a m p u l l a e a n d
M a l p i g h ia n t u b u l e s
The close ultrastructural resemblance
between the cells of proximal tubule, am-
pulla, and midgut has a bearing on the
embryological origin of the Malpighian tu-
bules, a controversial topic in the past.
Opinion has differed on whether the tu-
bules are derived from midgut or hindgut
(reviewed by Snodgrass, 35; Srivastava
and Khare, '66). The hindgut or procto-
daeum is thought to be entirely ectoder-
mal, although Henson ( 32) suggested that
the anterior part of the proctodael invag-
ination is endodermal in origin. The mid-
gut 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 mid-
gut, 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
Phi losamia
(Lepi-
doptera) 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 micro-
villi and basal infoldings, and are ana-
tomically continuous with the ampulla and
midgut. Srivastava and Khare ('62) point
out that histological similarity does not
prove common embryological origin. How-
ever, the ultrastructural and probable
functional similarities between the proxi-
mal region of the tubule, ampulla, and
midgut imply that these tissues are de-
rived 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 uncer-
tain 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 tu-
bules could arise from the anterior end
of the proctodaeum and later become at-
tached to the midgut. Such migrations
of developing tubules seem improbable
however, and clearly do not occur in
Blat ta
or iental i s and Periplane ta in which the
tubules grow out from the ampulla (Hen-
son, '44; Schmidt, unpublished).
Ex c re t ion he ro l e
of
pe rox i som e s
It has been generally thought that in-
sects 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 (re-
viewed by Campbell, 73). Although many
insects do excrete uric acid, at least dur-
ing a part of their life cycle, ammonia
has been found to be a major excretory
product in the cockroach, Periplane ta
am e r i c ana
(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 i t 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 micro-
bodies or peroxisomes (fig. 14) and these
structures may be involved in nitrogen
excretion. Peroxisomes have common func-
tional properties in organisms as diverse
as T e t r a h y m e n a yeasts, beans, and man
(DeDuve, '69). Peroxisomes contain a num-
ber of enzymes related to nitrogen metab-
olism, 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) ac-
cording to the scheme:
uricase
uric acid 2 H 2 0
llantoin COn 2 HzOz
catalase
2
H202
H20 2
Hydrogen peroxide is formed and rapidly
decomposed by catalase, which apparent-
ly is universally present in peroxisomes.
These organelles have also been observed
in
Call iphora
Malpighian tubules (Berridge
and Oschman, '69) as well as in other
insect tissues (Locke, '69; Locke and
Mc-
Mahon, '71) but their precise role in ni-
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302
B.
J. WALL. J. L. OSCHMAN AND B . A .
SCHMIDT
trogen metabolism has not been estab-
lished in insects.
Ex c re t ion he ro le of syrnbionts
The symbionts observed within the mid-
dle region of the tubules of
Periplane ta
resemble the haplosporidians found in
Bla t t e l l a ge nnan ic a .
Woolever ('66) has
made
a
remarkably thorough study of the
latter. She found that the
Blatte l la
sym-
bionts enter the host orally, multiply with-
in the Malpighian tubule cells, migrate
into the tubule lumen where they under-
go schizogony and sporogony, and release
their spores into the gut. The close ultra-
structural resemblance between vegeta-
tive and spore stages in
Blatte l la
and
Peri-
p lane ta
indicates that the
Periplane ta
sym-
biont could also be a haplosporidian.
Little is known about the metabolic
in-
teractions between the tubule symbionts
and their hosts. The symbionts ar e located
at the proximal end of the middle region
of the tubule (figs. 27, 28). They are not
present in the proximal region of the tu-
bule, possibly because the symbiont micro-
villi are adapted for attachment only to
the closely packed brush border of the
middle region. Since the primary urine
resembles a filtrate of the hemolymph,
the symbionts are bathed in a constantly
renewed medium that
is
rich in nutrients.
In addition they can readily release their
spores into the digestive tract so they will
be extruded with the feces.
Dr. K.
G .
Purohit has pointed out to us
that the symbionts might benefit their
hosts in m uch the same manner as do
those or ruminants (cows, sheep, etc.).
The rumen symbionts synthesize proteins
from amino acids and ammonia. These
proteins become available to the host when
the symbionts pass on into the intestine
and
are
digested. The symbionts also pro-
duce volatile fatty acids that are absorbed
directly across the rumen epithelium (Hun-
gate,
'68;
Dobson and Phillipson, '68).
The protozoan symbionts
in
the Malpighi-
an tubules produce spores which are not
attacked by digestive enzymes in the gut
(Woolever, '66). However, the vegetative
cell disintegrates when the spores are re-
leased. Fragments of the vegetative cells
thus pass into the gut and
are
digested
(Woolever, '66), possibly providing an ad-
ditional source of nutrients. The symbionts
may be able to synthesize essential amino
acids that the cockroach is not able to
manufacture d e
nouo. A
symbiotic rela-
tionship of this sor t has already been dem-
onstrated between
Blatte l la
and the bac-
terial symbionts within the mycetocytes
of the fat body.
Blat te l la
that lack these
symbionts a re smaller and less fecund than
infected roaches (Donnellan and Kilby,
'67), are unable to synthesize six amino
acids that normal animals can (Henry and
Block, '60, '62), and have heavier deposits
of urates in their fat body (Brooks and
Richards,
'56;
Pierre, '62). The bacterial
symbionts can be isolated from the host
and grown on a medium in which uric
acid is the only carbon source and can
degrade the uric acid to pyruvate or am-
monia (Donnellan and Kilby, '67). Com-
parable studies done on
Periplaneta
have
indicated that the fat body symbionts
are
responsible for the synthesis of folic, ascor-
bic, and pantothenic acids (Gallagher, '63).
Thus the symbionts within the Malpighian
tubules could have an important role in
metabolism in the insect.
ACKNOWLEDGMENTS
This study was begun in the laboratories
of Professors Michael Locke and Bodil
Schmidt-Nielsen at Case Western Reserve
University, Cleveland, Ohio, with support
from National Institutes of Health Grants
AM09975 and GM09960. The research
was also supported by USPHS Fellowship
GM24015 to
B.
J.
Wall and
AM
29555 to
J.
L. Oschman, as well as National Insti-
tutes of Health grants FR-7028 and
AM
14993. We are indebted to our colleagues
who have provided us with advice and
support.
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