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The peritrophic membrane of Spodoptera frugiperdaSecretion of peritrophins and role in immobilization and recycling digestive enzymes.

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62
Bolognesi et al.
Archives of Insect Biochemistry and Physiology 47:62–75 (2001)
The Peritrophic Membrane of Spodoptera frugiperda:
Secretion of Peritrophins and Role in Immobilization and
Recycling Digestive Enzymes
Renata Bolognesi,1 Alberto F. Ribeiro,2 Walter R. Terra,1 and Clélia Ferreira1*
1
Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, São Paulo, Brazil
Departamento de Biologia, Instituto de Biociências, Universidade de São Paulo, São Paulo, Brazil
2
A peritrophin from the Spodoptera frugiperda peritrophic membrane (PM) and microvillar proteins from S. frugiperda anterior midgut cells were isolated and used to raise antibodies in
a rabbit. These antibodies, as well as a Tenebrio molitor amylase antibody that cross-reacts with S. frugiperda amylases, and
wheat-germ aglutinin were used in immunolocalization experiments performed with the aid of confocal fluorescence and
immunogold techniques. The results showed that the peritrophin was secreted by anterior midgut columnar cells in
vesicles pinched-off the microvilli (microapocrine secretion).
The resulting double membrane vesicles become single membrane vesicles by membrane fusion, releasing peritrophin and
part of the amylase and trypsin. The remaining membranes
still containing microvillar proteins and membrane-bound amylase and trypsin are incorporated into a jelly-like material associated with PM. Calcofluor-treated larvae lacking a PM were
shown to lose the decreasing gradient of trypsin and chymotrypsin observed along the midgut of control larvae. This gradient is thought to be formed by a countercurrent flux of fluid
(in the space between PM and midgut cells) that powers enzyme recycling. Arch. Insect Biochem. Physiol. 47:62–75,
2001. © 2001 Wiley-Liss, Inc.
Key words: peritrophic membrane; enzyme immobilization; enzyme recycling; peritrophin secretion; microapocrine secretion; immunocytolocalization; endo-ectoperitrophic circulation
INTRODUCTION
The peritrophic membrane (PM) is a film
that surrounds the food bolus in most insects. It
is composed of a network of chitin and proteins
to which enzymes and other components associate (Peters, 1992). PM shares with the ancestral
gastrointestinal mucus the functions of protection
against food abrasion and microorganisms but has
also specific functions (Terra, 2001). These functions depend on the fact that PM compartmen© 2001 Wiley-Liss, Inc.
Contract grant sponsor: FAPESP; Contract grant sponsor:
CNPq (PRONEX program).
*Correspondence to: Clélia Ferreira, Departamento de
Bioquímica, Instituto de Química, Universidade de São
Paulo, C.P. 26077, 05513-970, São Paulo, Brazil.
E-mail: clfterra@iq.usp.br
URL:
http://www.iq.usp.br/wwwchem/bioquimica/laba1ing.html
Received 6 November 2000; Accepted in revised form 9 February 2001
S. frugiperda Peritrophin Secretory Mechanism
talizes the midgut lumen into an endoperitrophic
and an ectoperitrophic space and includes: (1) prevention of non-specific food binding onto cell surface; (2) prevention of enzyme excretion by allowing
enzyme recycling; and (3) restriction of oligomer
hydrolases to ectoperitrophic space in Lepidoptera
and Diptera (Terra, 2001). Although there is strong
indirect evidence for the proposed PM functions,
few have been experimentally tested.
PM is divided into two types according to its
site of synthesis. Type I PM is formed by either
the whole midgut epithelium, or by only part of it
(anterior or posterior regions) (Terra, 2001). This
type of PM is studied mainly in hematophagous
adult mosquitoes and Coleoptera and Lepidoptera
larvae. There are descriptions at the microscopic
level of the structure and formation of PM in mosquitoes and more recently some of their constituent peritrophins were isolated and characterized
(Jacobs-Lorena and Oo, 1996). Coleoptera larval
PM structure and formation were studied only at
the microscopic level (Ryerse et al., 1994).
Lepidopteran PM has been studied with immunocytochemical methods that revealed that PM
is usually formed in anterior midgut and peritrophins may be released before chitin fibers are
assembled (Ryerse et al., 1992; Harper and
Hopkins, 1997; Harper and Granados, 1999). A
peritrophin (intestinal insect mucin) has been isolated and sequenced from Trichoplusia ni (Wang
and Granados, 1997) and a model of the supramolecular structure of its PM proposed (Wang and
Granados, 2001).
Type II PM is formed by a few cells at the
entrance of the midgut (cardia) and is studied
mainly with mosquito larvae (Peters, 1992) and
fly larvae (Tellam et al., 1999). A model of the
supramolecular structure of Lucilia cuprina larval PM was recently proposed based on the sequence of several of its constituent peritrophins
(Schorderet et al., 1998).
In spite of the reviewed studies on PM structure and formation, there are no data on the
mechanisms of peritrophin secretion. This is particular interesting among Lepidoptera larvae, as
PM is usually formed in their anterior midgut,
where microapocrine secretion is observed.
In this paper, S. frugiperda peritrophin
secretory mechanism is described based on immunocytochemical data and the role of PM in en-
63
zyme immobilization and enzyme recycling is experimentally evaluated.
MATERIALS AND METHODS
Animals
Spodoptera frugiperda (Lepidoptera: Noctuidae) were laboratory reared according to Parra
(1986). The larvae were individually contained in
glass vials with a diet based on kidney beans
(Phaseolus vulgaris), wheat germ, yeast, and agar,
and were maintained under a natural photoregime at 25°C. Adults were fed a 10% honey solution. Fifth (last)-instar larvae of both sexes were
used in the determinations.
Alternatively, larvae were fed during 8 h on
the same diet containing 1% calcofluor, before being dissected.
Preparation of Samples
Larvae were immobilized by placing them on
ice, after which they were rinsed in water, blotted
with filter paper, and their guts were dissected in
cold 125 mM NaCl. PM with its contents and the
midgut tissue were then separated. The first third
of midgut tissue was thoroughly rinsed with saline and used for microvillar membrane isolation.
When the larvae were fed on diet with calcofluor, PM plus contents isolated as described
above were divided into three parts of equal length,
homogenized in double distilled water with the aid
of a Potter-Elvehjem homogenizer, and centrifuged
at 10,000g for 10 min at 4°C. The resulting supernatants were stored at –20°C until use.
For the determination of enzymes in PM
fractions, PMs were isolated from their contents
and homogenized in 125 mM NaCl with the aid
of a Potter-Elvehjem homogenizer. The pellet of
centrifugation at 600g, 10 min, 4°C was collected
and named PM matrix, whereas the supernatant
was centrifuged (4°C) at 25,000g for 30 min. The
new pellet was labeled jelly (particles) and the
new supernatant, jelly (soluble). The preparations
were maintained at –20°C until use.
The PM matrix, obtained after thoroughly
washing the PM, was used for peritrophin solubilization.
Isolation of Microvillar Membranes From
Anterior Midgut
Microvillar membranes were prepared with
a procedure similar to that described by Capella
64
Bolognesi et al.
et al. (1997) in 50 mM mannitol, 2 mM Tris-HCl,
pH 7.5.
Solubilization of Proteins From Spodoptera
frugiperda PM
Proteins were extracted from PM matrix by
sequentially homogenizing (Potter-Elvehjem homogenizer), centrifuging (25,000g, 30 min, 4°C)
and exposing the PM to different treatments. The
following media were sequentially used for solubilization: 125 mM NaCl; 125 mM NaCl, 40 mM
Tris-HCl, pH 7.0, 65 mM CHAPS (3-[(3-Cholamidopropyl)dimethylammonio]-1-propane-sulfonate); 40 mM Tris-HCl pH 7.0, 12 M urea; and
6% SDS, 66 mM β-mercaptoethanol.
Protein Determination and Hydrolase Assays
Protein was determined according to Bradford (1976), using Coomassie Blue G with ovalbumin as a standard.
Aminopeptidase and trypsin were assayed in
100 mM Tris-HCl buffer (pH 7.5) using as substrate 1 mM L-leucine-p-nitroanilide and α-Nbenzoyl-DL- arginine p-nitroanilide, respectively,
and following the release of p-nitroaniline according to Erlanger et al. (1961).
Amylase activity was measured by determining the appearance of reducing groups (Noelting
and Bernfeld, 1948) from 0.5% soluble starch in
50 mM glycine-NaOH buffer (pH 9.5).
β-N-Acetylglucosaminidase was determined
by following the increase of nitrophenolate (according to Terra et al., 1979) produced from 1.25
mM p-nitrophenyl-N-acetyl-β-D-glucosaminide in
50 mM citrate sodium phosphate buffer pH 5.0.
Chymotrypsin was assayed with 1 µM N-succinyl-Ala-Ala-Phe 7-amido-4 methylcoumarin in
100 mM Tris-HCl buffer (pH 8.5). The substrate
is dissolved in dimethyl sulfoxide and then diluted 100 times with buffer. The reaction is stopped
with 5% acetic acid and the fluorescence was detected in a F2000 Hitachi fluorimeter, with excitation at 380 nm and detection at 460 nm.
Prior to enzymatic assays, the pellets resulting from centrifugation were resuspended by homogenization with the aid of a Potter-Elvehjem
homogenizer. If this procedure is not used, the
recovery of enzymes in pellets is lower.
Incubations were carried out at 30°C for at
least four different periods of time, and initial
rates of hydrolysis were calculated. All assays
were performed under conditions that the enzyme
product was proportional to enzyme concentration and to incubation time. Controls without enzyme and others without substrate were included.
One unit of enzyme (U) is defined as the amount
that hydrolyses 1 µmol of substrate (or bond) per
minute. Enzyme activities were expressed in milli
units (mU).
Sodium Dodecyl Sulfate (SDS)-Polyacrylamide
Gel Electrophoresis (PAGE)
SDS-PAGE of Spodoptera frugiperda samples
was carried out in 7–12 or 12% (w/v) polyacrylamide gels containing 0.1% (w/v) SDS, on a discontinuous pH system (Laemmli, 1970), using
Bio-Rad Mini-Protein II equipment. Samples were
mixed with sample buffer (2:1) containing 260
mM Tris-HCl, pH 6.8, 2.5% (w/v) SDS, 0.36 mM
2-mercaptoethanol, 10% glycerol, and 0.05% (w/
v) bromophenol blue and heated for 4 min at 95°C
in a water bath before being loaded onto the gels.
Electrophoresis was carried out at 200 V until the
front marker (bromophenol blue) reached the bottom of the gel. Proteins were silver-stained according to Blum et al. (1987).
Mr values were calculated according to Shapiro et al. (1967) using the following Mr standards:
aprotinin (Mr 6,500), lysozyme (Mr 18,500), soybean
trypsin inhibitor (Mr 21,500), carbonic anhydrase
(Mr 31,000), glyceraldehyde 3-phosphate dehydrogenase (Mr 36,000), ovoalbumin (Mr 45,000), bovine serum albumin (Mr 66,000), phosphorylase b
(Mr 97,400), β-galactosidase (Mr 116,250), and myosin (Mr 200,000).
Preparation of Peritrophin, Amylase, and
Microvillar Membrane Proteins Antisera
The peritrophin used to produce rabbit antiserum was prepared as follows. After solubilization of Spodoptera frugiperda PM proteins with
CHAPS and urea, 230 µg of the soluble proteins
were submitted to SDS-PAGE in a 7.5 to 12%
polyacrylamide gradient. The gel portion where
the Mr 33k protein band was found was cut apart
and homogenized to a final volume of 4 ml in
Freund’s complete adjuvant diluted twice. The
emulsion was injected subcutaneously into five
locations in the back of a rabbit. After 65 days, a
similar injection was administrated in the rab-
S. frugiperda Peritrophin Secretory Mechanism
bit, but with incomplete adjuvant, and 8 days
later the rabbit was bled. The blood was left
standing 30 min at 37°C and overnight at 4°C,
before being centrifuged at 3,000g for 10 min at
4°C. The supernatant was added to a suitable solution to become 50% saturated in ammonium sulfate, pH 6.8. After standing overnight at room
temperature (25°C), the resulting suspension was
centrifuged at 5,000g for 15 min at 4°C. This step
was repeated again and the final pellet was resuspended in 0.1 M NaCl, dialyzed for 20 h
against 1,000 volumes of 0.1M NaCl with one
change of saline. The dialysate was centrifuged
at 10,000g for 10 min at 4°C and the resulting
supernatant distributed into small aliquots and
kept at –20°C until used.
To produce antiserum against Spodoptera
frugiperda microvillar membranes, the pellet containing anterior midgut microvillar membranes
was suspended in Freund’s complete adjuvant diluted twice. The amount of protein applied in each
set of injection was 250 µg. Other procedures were
identical to those described for peritrophin antiserum preparation.
Tenebrio molitor amylase was purified and
the antiserum prepared as described by Cristofoletti et al. (2001).
Western Blots of Proteins
Western blots were made after SDS-PAGE
in 7.5–12% gels. The proteins and molecular
weight standards (Bio-Rad, Hercules, CA) were
electrophoretically transferred onto a nitrocellulose membrane filter (pore size 0.45 µm) (Towbin
et al., 1979). The transfer efficiency was estimated
by observing the pre-stained molecular weight
standards on the filter. After a blocking step, the
filters were reacted with the antiserum diluted
in TBS-T (Tris- buffered saline-Tween: 50 mM
Tris-HCl buffer, pH 7.5, containing 0.15 M NaCl
and 0.05% Tween) for 2 h at room temperature.
The filters, after being extensively washed with
TBS-T, were reacted with goat anti-rabbit IgG antibody coupled with peroxidase (Sigma, St. Louis,
MO), diluted 1:1,000 in TBS-T at pH 7.5, for 2 h
at room temperature. After extensive washing
with TBS-T and TBS (both at pH 7.5) the strips
were incubated with 20 mg of 4-chloro-1-naphtol
in 4 ml of methanol added to 20 ml of TBS at 37°C
and 15 µl of H2O2, for up to 15 min. Alternatively,
65
the strips were treated with ECL Western blotting kit (Amersham, UK) according to the manufacturer’s instructions, and then exposed to a
high-performance luminescence detection film
(Hyperfilm-ECL Amersham, UK). The following
proteins were used as pre-stained molecular weight
standards: phosphorylase b (107 kDa), bovine serum albumin (76 kDa), ovoalbumin (52 kDa), carbonic anhydrase (36.8 kDa), soybean trypsin
inhibitor (27.2 kDa), and lysozyme (19 kDa).
Pre-immune serum was used in control experiments to show that antisera were specific.
Confocal Fluorescence Microscopy and
Electron Microscopy
For immunofluorescent visualization of
peritrophin, α-amylase, and microvillar membrane proteins, tissue samples were fixed in
Zamboni’s fixative (Stefanini et al., 1967) for 16
h at 4°C. The samples were dehydrated in graded
ethanol, embedded in paraffin wax, and cut at
10 µm. The tissue sections were collected on
glass slides and the paraffin removed with xylene. After hydration, the sections were washed
in PBS (20 mM phosphate buffer pH 7.4, containing 0.15 M NaCl), followed by immersion in
PBS containing 0.2% Triton X-100, for 2 h at
room temperature. The preparations were then
rinsed in PBS and incubated with the primary
antibody (peritrophin, α-amylase, or microvillar
membrane proteins) diluted 1:1,000 in PBS plus
0.1% bovine serum albumin for 16 h at 4°C. As
controls, sections were incubated with non-immune serum in the same conditions. The samples
were then rinsed in PBS at room temperature
and incubated with the secondary antibody
(FITC- Amersham Little Chalfont, UK) diluted
1:1,000 in PBS plus 0.1% bovine serum albumin,
for 1 h at room temperature. After rinsing in PBS
at room temperature, the sections were mounted
in Vectashield (Vector Labs, Inc., Burlingame,
CA) mounting medium and examined in a Zeiss
LSM 410 confocal microscope.
For fluorescent visualization of chitin, sections prepared as described above were incubated
with FITC-WGA (fluorescein isothiocyanatewheat germ agglutinin) in the presence of 0.2 M
N-acetylglucosamine. The binding of WGA with
chitin is specific in the presence of excess Nacetylglucosamine (Peters and Latka, 1986).
66
Bolognesi et al.
For immunolabeling of peritrophin or amylase at the ultrastructural level, midgut pieces
were fixed (paraformaldehyde-glutaraldehyde),
embedded in L.R. White acrylic resin, incubated
with the primary and secondary (goat anti-rabbit IgG coupled to 15 nm gold) antibodies and examined in a Zeiss EM 900 electron microscope as
detailed elsewhere (Silva et al., 1995). As controls,
sections were incubated with non-immune serum
in the same conditions.
RESULTS
Isolation of a Peritrophin From S. frugiperda PM
The S. frugiperda PM is sufficiently strong
to be picked up with forceps, but, as observed under the dissecting microscope, it is composed of a
resistant solid structure (PM matrix) to which a
jelly-like material (PM jelly) is associated. The
volume of jelly is larger than the solid material
and it is stepwise solubilized during the washings
with the dissecting saline.
Microvillar membranes from S. frugiperda
anterior midgut cells were isolated as previously
described (Capella et al., 1997). The isolated
membranes presented an aminopeptidase specific activity 5-fold higher than the initial tissue
homogenate (not shown), which is comparable
to the 5.6-fold ratio described by Capella et al.
(1997). Some of the microvillar membrane proteins are apparently also present in the ectoperitrophic vesicle membranes and in the PM jelly
(Fig. 1A). There are other proteins occurring only
in vesicle membrane and PM jelly (Fig. 1A).
After washing out the jelly material, the remaining PM matrix was sequentially extracted
with 65 mM CHAPS, 12M urea, 6% SDS containing 66 mM β-mercaptoethanol and, then, the final sediment was discarded and the extracts
submitted to SDS-PAGE. Figure 1B shows that
urea extracts more proteins from the PM matrix
than CHAPS, but in both extracts the major proteins with Mr values close to other peritrophins
are those with Mr values of 27k and 33k. Taking
into account that most of these proteins are isolated after urea treatment of PM, they must be
PM integral proteins or peritrophins (Tellam et
al., 1999).
Peritrophin-33k was isolated by SDS-PAGE
from CHAPS and urea extracts in sufficient
amounts to raise antibodies in a rabbit. The resulting antiserum recognized peritrophin-33k in
Fig. 1. Silver-stained SDS-PAGE of proteins from anterior
midgut cell microvillar membranes, ectoperitrophic vesicles,
and solubilized from PM of S. frugiperda. A: 12% gel. Asterisks correspond to proteins occurring in vesicles and PM jelly,
but not in the microvillar membranes. Lane 1, anterior midgut microvillar membranes; lane 2, ectoperitrophic vesicles;
lane 3, PM jelly. B: 7–12% gel. Asterisk indicates the
peritrophin-33k. Lane 1, PM matrix solubilized in CHAPS;
lane 2, PM matrix solubilized in urea; lane 3, PM matrix
solubilized in SDS containing β-mercaptoethanol. Samples
were prepared as described in the legend of Table 1.
Western blots of the CHAPS and urea extracts (not
shown). Anti-peritrophin-33k serum recognizes PM
in microscopical preparations (Fig. 2A,B).
It was not possible to transfer peritrophin-27k
and peritrophin-33k to polyvinylidene difluoride
membranes, which hindered N-terminal sequencing of these proteins. When PM urea extracts containing more than 10 µg of protein were submitted
to SDS-PAGE, only peptides larger than 50 kDa
are observed. This suggests that in some conditions
peritrophin-33k forms insoluble aggregates.
Immunocytolocalization of Peritrophin in
S. frugiperda Midgut Cells
Peritrophin-33k is localized in PM in both
anterior and posterior midguts and in anterior
midgut tissue (Fig. 2A,B). Controls with pre-immune serum were negative.
The extracellular peritrophin-33k-labeled
structure was confirmed to be PM by labeling
with WGA (Fig. 2C,D). WGA is a lectin that binds
specifically to chitin in the presence of excess Nacetylglucosamine (Peters and Latka, 1986). WGA
S. frugiperda Peritrophin Secretory Mechanism
67
Fig. 2. Immunofluorescence visualization of peritrophin-33k
(A, B) and chitin (C, D) in S. frugiperda midguts. A: Anterior midgut showing highly stained PM and midgut cells
with fluorescence in vesicles inside columnar cells. Note nonstained goblet cells (arrows). B: Posterior midgut with fluo-
rescence restricted to PM. C: Anterior midgut with labeling
in PM and in tracheas underlying epithelium. D: Posterior
midgut with labeling in PM and in tracheas underlying epithelium. Bars = 50 µm.
labeling seen at the base of the midgut epithelial
cells (Fig. 2C,D) should correspond to the chitin
rings of tracheas.
In anterior midgut cells, peritrophin-33k is
found in secretory vesicles within columnar cells
(Fig. 2A), whereas the long-neck bottle-shaped
goblet cells are not labeled (Fig. 2A, shadow indicated by arrows).
Immunogold labeling shows the occurrence
of peritrophin-33k in PM along the whole midgut
and in glycocalyx and secretory vesicles in anterior midgut cells and its virtual absence from pos-
68
Bolognesi et al.
terior midgut cells (Fig. 3A,B). In the rare fields
that peritrophin-33k is localized in posterior midgut cells, the protein is always observed inside
the smaller and more dense vesicles (like those
in anterior midgut) (Fig. 3C), whereas no labeling is found in the larger vesicles, typical of posterior midgut cells (Fig. 3C).
Immunolocalization of Amylase and Microvillar
Membrane Proteins in S. frugiperda Midgut Cells
Antibodies raised in rabbits against purified
T. molitor amylase (Cristofoletti et al., 2001) recognize S. frugiperda amylases in Western blots (not
shown). Immunolocalization was performed with
this heterologous antiserum because attempts to
purify S. frugiperda amylase were not successful.
Amylase labeling follows peritrophin labeling
both with confocal immunofluorescence microscopy
(Fig. 4C,D) and immunogold ultrastructural localization (Fig. 3D–F). Nevertheless, amylase labeling of midgut cell microvilli is more intense than
peritrophin labeling (compare Figs. 3A and D) and
it is occasionally found associated with vesicle
membranes in the apical cytoplasm and inside the
microvilli (Fig. 3E, arrows).
A suspension of microvillar membranes prepared as described above was injected into a rabbit. The resulting antiserum recognized in a
Western blot, after SDS-PAGE of the anterior
midgut microvillar proteins, proteins with the
following Mr values: 21.6k, 27.3k, 30k, 41.4k,
54.7k, 79.2k, 93.1k, 112.2k, 120k (not shown).
The microvillar proteins with Mr 120k and
112.2k were also recognized by the anti-amylase
serum, whereas that with 93.1k reacted with the
anti-trypsin serum (not shown). Those microvillar proteins are supposed to correspond to amylase and trypsin bound to microvillar proteins
(Jordão et al., 1999). The anti-microvillar proteins serum reacts with proteins 21.6k, 27.3k,
and 30k of the ectoperitrophic vesicle membranes
(not shown). This confirms that at least part of
the proteins of the ectoperitrophic fluid vesicles
derive from the anterior midgut microvillar
membranes.
Microvillar protein labeling is more evident
in anterior midgut cell apexes, than in posterior
midgut ones, whereas PM labelling seems evenly
distributed along the whole midgut (Fig. 4A,B).
Association of Digestive Enzymes With PM and
Midgut Contents of S. frugiperda
More digestive enzymes are incorporated
into the jelly part of the PM than in the matrix
(Table 1). Actual differences are even higher than
observed, if one takes into account that the matrix is necessarily contaminated with non-dispersed food. Non-dispersed food binds large
amounts of digestive enzymes (see pellet corresponding to PM contents, Table 1), mainly amylase and trypsin. The association of the enzymes
with PM is not a result of simple adsorption phenomenon, as specific activity ratios differ between
the ectoperitrophic fluid and jelly (Table 1). The
enzymes more restricted to PM jelly are those
found membrane-bound to vesicles in ectoperitrophic fluid. Some of these enzymes remain membrane bound in jelly. The specific activities of
amylase and trypsin increase from the ectoperitrophic vesicles to PM jelly particles, the contrary
being true for aminopeptidase. This suggests that
aminopeptidase-carrying vesicles are more easily
solubilized in the ectoperitrophic fluid before incorporation than those carrying amylase and
trypsin. The significant amounts of aminopeptidase
recovered inside PM may correspond to a minor
aminopeptidase distinct from the major microvillar aminopeptidase (Ferreira et al. 1994a).
Effect of Calcofluor in the Distribution of
Proteinases Along the Midgut Contents of
S. frugiperda Larvae
Calcofluor inhibits the formation of chitincontaining microfibrils by binding the polysaccharide (Maeda and Ishida, 1967). In agreement with
a previous report with Trichoplusia ni (Wang and
Granados, 2000), S. frugiperda larvae fed for 8 h
with artificial diet containing 1% calcofluor lose
their PM (not shown). These larvae, in contrast
Fig. 3. Immunocytochemical localization of peritrophin-33k
(A–C) and amylase (D–F) in S. frugiperda midgut cells. A:
Anterior midgut. Peritrophin labeling in PM and microvilli.
B: Anterior midgut. Peritrophin labeling in secretory vesicles.
C: Posterior midgut. Note that peritrophin labeling is restricted to the smaller and more dense secretory vesicles.
In most observed fields there is no labeling. D,E: Anterior
midgut, amylase labeling. Comments for amylase are the
same as for peritrophin, except that amylase labeling is occasionally seen associated with membranes of vesicles (arrows). F: Posterior midgut, amylase labeling. MV, microvilli.
Bars = 0.5 µm.
S. frugiperda Peritrophin Secretory Mechanism
Figure 3.
69
70
Bolognesi et al.
Fig. 4. Immunofluorescence visualization of microvillar proteins (A,B) and amylase (C,D) in S. frugiperda midguts. A:
Anterior midguts showing highly stained PM and brush borders. B: Posterior midgut with PM heavily labeled and with
brush borders less labeled than in anterior midgut. C: An-
terior midgut with labeling in brush borders and in vesicles
inside columnar cells. Note non-stained goblet cells (arrows).
D: Posterior midgut with fluorescence restricted to PM. Bars
= 25 µm (A–C), 50 µm (D).
TABLE 1. S. frugiperda Digestive Enzymes in Microvillar Membranes, Ectoperitrophic Fluid Vesicles, Washed PM, and PM Contents*
Enzyme
Acetylglucosaminidase
Aminopeptidase
Amylase
Trypsin
Microvillar
membranesa
n.d.
3,100
430
7
Washed PMc
Ectoperitrophic contents
PM contentsd
Vesicles
Fluid
Jelly (s)
Jelly (p)
Matrix
Soluble
Pellet
0.42
1,680
2,160
86
10
50
1,700
75
1.1 (3.4)
65 (6.0)
1,900 (5.5)
160 (12.1)
0.001 (0.01)
870 (3.8)
4,800 (0.9)
350 (2.0)
0.7 (0.47)
120 (2.9)
8,300 (6.6)
190 (3.7)
0.66 (0.5)
19 (18.2)
730 (32.1)
190 (61.9)
0.26 (0.1)
34 (8.6)
5,120 (55)
174 (20.2)
S. frugiperda Peritrophin Secretory Mechanism
*Results are specific activities (mU/mg protein) and relative activities (in parentheses) displayed as percentage of the sum of units of activity in midgut
lumen (ectoperitrophic fluid + PM contents). Data are expressed as means calculated from four assays performed in each of three different preparations
obtained from five animals. SEM were found to be 20–35% of the means. n.d., no data.
a
Data correspond to anterior midgut cells (Capella et al., 1997).
b
Ectoperitrophic contents were collected by rinsing the luminal surface of the midgut tissue with saline. The rinsing saline was then centrifuged at 600g
for 10 min. The resulting supernatant was centrifuged at 25,000g for 30 min. The new pellet corresponds to the vesicles and the new supernatant to the
fluid. Calculated from Ferreira et al. (1994a).
c
PM cleaned from contents was homogenized in dissecting saline. The pellet of a centrifugation at 600g for 10 min was collected as PM matrix, whereas
the supernatant was centrifuged at 25,000g for 30 min. The new pellet was labeled jelly (particles) and the new supernatant, jelly (soluble).
d
PM contents were centrifuged at 600g for 10 min and the supernatant and pellets were collected.
71
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Bolognesi et al.
TABLE 2. Distribution of Proteinases (%) Along the
Midgut Contents in Control and Calcofluor-Treated
S. frugiperda Larvae*
Trypsin
Chymotrypsin
Midgut
contents
Control
Calcofluor
Control
Calcofluor
Anterior
Middle
Posterior
51 ± 5
38 ± 5
11 ± 2
24 ± 2
43 ± 2
33 ± 2
58 ± 1
34 ± 1
9±2
35 ± 5
42 ± 2
23 ± 4
*Figures are means and SEM based on determinations of
units of activity performed in 4 different preparations obtained from 4 larvae each. Experimental larvae were fed
with 1% calcofluor in the artificial diet for 8 h before being
dissected. Midgut contents after being divided into 3 portions of similar length were assayed for trypsin and chymotrypsin.
to control larvae, do not have a decreasing gradient of trypsin and chymotrypsin along their midguts (Table 2).
DISCUSSION
Secretory Mechanisms of Trypsin and Amylase
in S. frugiperda Anterior Midgut
Ultrastructural immunolocalization shows
that peritrophin-33k occurs inside secretory
vesicles identical to those reported to contain
trypsin (Jordão et al., 1999) and amylase (this
paper). These vesicles were shown to be involved
in microapocrine secretory mechanisms. Therefore, before discussing peritrophin secretion, it is
necessary to review the known secretory mechanisms of trypsin and amylase, including new data
presented in this paper.
According to the model proposed by Jordão
et al. (1999), after being synthesized trypsin is
maintained bound to membranes through a hydrophobic peptide anchor. In this form it is processed in the Golgi complex and transported in
secretory vesicles. These vesicles migrate through
the cell microvilli and, before or after being fused
with the microvillar membrane at the tips of the
microvilli, are discharged into the lumen by a
pinching-off process, resulting in double- and
single-membrane vesicles, respectively. Doublemembrane vesicles become single-membrane
vesicles by membrane-fusing processes. Part of
the trypsin on the luminal surface of single-membrane vesicles becomes soluble by limited proteolysis or by partial dissolution of the vesicles
in the highly alkaline midgut lumen. The remain-
ing vesicle membrane, with part of the trypsin
molecules still bound, is finally incorporated into
the forming external layer of PM.
The above-mentioned model is supported by
the following observations: (1) Immunolabelling
data showed that trypsin is associated with the
membrane of vesicles that migrate through the
microvilli and that are ultimately pinched-off from
the microvilli (Jordão et al., 1999); (2) Solubilization of membrane-bound trypsin by several agents
suggested that a hydrophobic peptide is the trypsin
anchor; (3) The specific activity of trypsin in
trypsin-carrying membranes from S. frugiperda
ectoperitrophic contents is much higher than in
microvillar membranes, whereas the specific activity of the microvillar aminopeptidase is approximately the same in both membranes (Ferreira et
al., 1994b; Table 1, this paper). This suggested that
ectoperitrophic trypsin-carrying membranes are
produced from a specific region of the columnar
cell microvillar membrane (to account for the differences found in specific activities); (4) About 18%
of the midgut luminal activity of trypsin was found
incorporated in PM (Ferreira et al. 1994b; Table
1, this paper).
This paper lends support to the proposed
trypsin secretory model in several instances. (1)
Western blots revealed the presence of trypsin in
microvillar membranes and the occurrence of microvillar proteins in ectoperitrophic vesicle membranes; (2) some vesicle membrane proteins are
seen in PM jelly but not among microvillar proteins (Fig. 1A), supporting the assertion that
ectoperitrophic vesicles pinch-off from specific regions in anterior microvilli and become partly incorporated into PM jelly; (3) membrane-bound
trypsin is found in PM together with microvillar
aminopeptidase (Table 1); and (4) immunocytochemical data confirmed the presence of other
microvillar proteins in PM (Fig. 4A,B).
Amylase secretion in S. frugiperda larval
midgut was considered to follow a route similar
to that described for trypsin in anterior midgut,
except that secretory vesicles contain a fraction
of soluble molecules derived from membranebound molecules by a putative pH increase inside vesicles (Jordão et al., 1999) or by limited
proteolysis or another kind of processing. In this
paper, this model is supported by Western blots
revealing the presence of amylase in microvillar
S. frugiperda Peritrophin Secretory Mechanism
membranes, immunocytochemical data (Fig. 3)
showing the occurrence of soluble and membranebound forms of amylase, and by results confirming the occurrence of membrane-bound amylase
associated with PM (Table 1).
Secretory Mechanisms and the Formation of PM
in S. frugiperda and Other Lepidopteran Larvae
S. frugiperda PM is composed of a solid matrix to which a jelly-like material (PM jelly) is
associated. Urea extracts of the PM matrix have
two major proteins (27k and 33k) that display the
same solubility properties and Mr values as other
peritrophins (for review see Tellam et al., 1999).
Peritrophin-33k was isolated and an antiserum raised in rabbits showed that this protein is
secreted in major amounts by columnar cells only
in the anterior two thirds of S. frugiperda midguts. Similar results were obtained with Trichoplusia ni, in spite of apparent contrasting
reports. T. ni peritrophin secretion was at first
assigned to goblet cells (Wang and Granados,
1997), but a later study showed that secretion was
actually carried out by columnar cells (Harper and
Granados, 1999). Although these authors concluded that peritrophin is secreted along the
whole T. ni midgut (Harper and Granados, 1999),
a close inspection at the published immunocytolocalizations reveals that labeling in posterior
midgut is found only outside the cells. This means
that peritrophin is not secreted by posterior midgut cells and suggests that labeling may correspond to excess peritrophin molecules remaining
after PM formation, as described for Lucilia
cuprina peritrophin-95 (Tellam et al., 2000). The
formation of PM in anterior midgut was also described in the larvae of the Lepidoptera Ostrinia
nubilalis (Harper and Hopkins, 1997), based on
WGA data. Contrary to the classic view that lepidopterans have type I PM, Heliothis virescens PM
seems to be of type II, based on immunocytolocalization data with an anti-PM antibody
(Ryerse et al., 1992). Perhaps these experiments
should be repeated with antibodies prepared
against a purified peritrophin, instead of using
an antiserum raised against whole washed PM.
Since peritrophin-33k occurs in the same anterior midgut secretory vesicles as trypsin and
amylase, the following events should occur during
PM formation in S. frugiperda larvae. Secretory
73
vesicles with trypsin and amylase associated with
their limiting membranes and containing free
peritrophin molecules, migrate from Golgi complex
into the cell microvilli. During this path, a gradual
increase in vesicle pH (or limited proteolysis or an
unknown processing mechanism) solubilizes part
of the amylase. The vesicles pinch-off at the tips
of the microvilli, resulting in double membranes
that become single-membrane vesicles by membrane fusing. During this process, peritrophins and
most amylase are liberated, whereas trypsin is
partly solubilized by limited proteolysis or by pHdependent partial dissolution of vesicles. The remaining membrane, with microvillar and vesicular
domains, is incorporated into the PM jelly.
Peritrophins probably associate with chitin
microfibrils to form PM matrix, as reviewed by
Tellam et al. (1999). Microapocrine secretion of
peritrophins is probably an adaptation to enhance
the dispersion of secretory vesicle contents into
the midgut lumen of water-absorbing tissues, such
as the anterior midgut of Lepidoptera larvae
(Cristofoletti et al., 2001 and references therein).
Role of S. frugiperda PM in Immobilization and
Recycling Digestive Enzymes
The first report on the existence of enzymes
partly immobilized on the surface of PM was published in 1979 (Terra et al., 1979). Since then, several papers confirmed this finding in different
insects (for review see Terra, 2001), although to
date the only quantitative determination of digestive enzymes associated with washed PM was performed in S. frugiperda. The results showed that
PM may contain up to 13 and 18% of the midgut
luminal activity of amylase and trypsin, respectively (Ferreira et al., 1994b). In this paper, it was
shown that trypsin, amylase, and microvillar enzymes are incorporated into the PM jelly when the
enzymes, still bound to membranes, are released
from anterior midgut cells by a microapocrine process. In contrast, enzymes soluble in the ectoperitrophic fluid, such as acetylglucosaminidase, occur
in negligible amounts in PM.
Although enzyme immobilization on PM may
have a role in digestion, it seems to be a consequence of microapocrine secretion rather than an
adaptation to improve digestion efficiency. The
advantages of microapocrine secretion was discussed before. It is tempting to speculate that in-
74
Bolognesi et al.
sects control proteinase activity by rendering
them bound to membranes up to the moment they
are released into the midgut lumen, in spite of
proteinases are usually synthesized as inactive
zymogens. The same reasoning is probably also
valid for amylase, since S. frugiperda midgut cells
are rich in glycogen (Capella et al., 1997) and its
uncontrolled hydrolysis could result in a lethal
osmotic shock.
Based on the distribution of digestive enzymes among different midgut compartments and
on the study of midgut fluid fluxes with dyes, a
model was proposed for the organization of digestive events in most insects (Terra, 1990, 2001). According to the model, food flows inside PM from
the anterior to the posterior midgut, whereas outside PM water flows from the posterior to the anterior midgut. The enzymes involved in initial
digestion should penetrate and be retained within
the anterior endoperitrophic space, since the enzyme-substrate complexes may be too large to diffuse back across PM. Nevertheless, as the food
progresses along the endoperitrophic space, the
molecular size of the food particles decreases until they are able to pass through PM together with
the enzymes to which they are bound. The enzymes
and oligomeric molecules are then displaced towards the anterior midgut, where terminal digestion and absorption occur. The enzymes may
diffuse back to the anterior endoperitrophic space
and a new cycle starts. A consequence of enzyme
recycling is that the concentration of digestive enzymes decrease along the midgut and they are not
excreted with faeces.
Although there is strong indirect evidence
in favor of the model, before this work there was
only one direct source of evidence supporting the
model: the finding that an excessive increase in
dietary protein fed to larvae led to both a decrease
in the trypsin gradient along the midgut and to
an increase in the trypsin excretory rate (Terra
and Ferreira, 1981; Espinoza-Fuentes and Terra,
1987). The existence of the recycling mechanism
can also be tested by disrupting PM. As expected
from the model, calcofluor-treated S. frugiperda
larvae lacking a PM lose the decreasing gradient
of trypsin and chymotrypsin along the midgut
that is observed in control larvae.
The importance of PM in the compartmentalization of digestive events in normal conditions (no
experimental infection attempts) is probably greater
than its role in preventing microorganism infection.
This assertion is supported by the findings of Wang
and Granados (2000). They fed Trichoplusia ni larva
with calcofluor and noted high mortality in larvae
lacking a PM. Examination of dead larvae showed
no signs of microbial infection.
ACKNOWLEDGMENTS
We are indebted to Dr. J.E. Baker for his
critical review of this manuscript. We also thank
L.Y. Nakabayashi, W. Caldeira, and M.V. Cruz for
technical assistance. R.B. is a graduate fellow of
FAPESP and A.F.R., C.F., and W.R.T. are staff
members of their respective departments and fellows of CNPq.
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