Lutzomyia longipalpis peritrophic matrix: formation, structure, and chemical composition.

Abstract

Sandflies are vectors of several pathogens, constituting serious health problems. Lutzomyia longipalpis (Lutz & Neiva, 1912) is the main vector of Leishmania chagasi, agent of visceral leishmaniasis. They synthesize a thick bag-like structure that surrounds the bloodmeal, named peritrophic matrix (PM). One of the major roles of PM in blood-fed insects includes protection against ingested pathogens by providing a defensive barrier to their development. We used traditional and modern morphological methods as well as biochemical and immunolabeling tools to define details of the PM structure of the Lu. longipalpis sandfly, including composition, synthesis, and degradation. The kinetics of PM formation and degradation was found to be related to the ingestion and time of digestion of the bloodmeal. The midgut changes its size and morphology after the blood ingestion and during the course of digestion. A striking morphological modification takes place in the midgut epithelium after the stretching caused by the bloodmeal, revealing a population of cells that was not observed in the unfed midgut. The transmission and scanning electron microscopies were used to reveal several morphological aspects of PM formation. The PM looks thicker and well formed 24 h after the bloodmeal. Presence of chitin in the PM was demonstrated by immunolabeling with an alpha-chitin monoclonal antibody. SDS-polyacrylamide gel electrophoresis showed at least five protein bands with molecular masses of 38.7-135 kDa, induced by the protein-free diet. Mouse polyclonal antiserum was produced against PMs induced by protein-free meal and used in Western blotting, which revealed at least three associated proteins.

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MORPHOLOGY, SYSTEMATICS, EVOLUTION
Lutzomyia longipalpis Peritrophic Matrix: Formation, Structure, and
Chemical Composition
N.F.C. SECUNDINO,1I. EGER-MANGRICH,1, 2, 3E. M. BRAGA,4M. M. SANTORO,3
AND P.F.P. PIMENTA1
J. Med. Entomol. 42(6): 928Ð938 (2005)
Sandßies are vectors of several pathogens, constituting serious health problems. Lut-
zomyia longipalpis (Lutz & Neiva, 1912) is the main vector of Leishmania chagasi, agent of visceral
leishmaniasis. They synthesize a thick bag-like structure that surrounds the bloodmeal, named
peritrophicmatrix(PM).OneofthemajorrolesofPMinblood-fedinsectsincludesprotectionagainst
ingested pathogens by providing a defensive barrier to their development. We used traditional and
modern morphological methods as well as biochemical and immunolabeling tools to deÞne details of
thePMstructureoftheLu.longipalpissandßy,includingcomposition,synthesis,anddegradation.The
kineticsofPMformationanddegradationwasfoundtoberelatedtotheingestionandtimeofdigestion
of the bloodmeal. The midgut changes its size and morphology after the blood ingestion and during
the course of digestion. A striking morphological modiÞcation takes place in the midgut epithelium
after the stretching caused by the bloodmeal, revealing a population of cells that was not observed
intheunfedmidgut.Thetransmissionandscanningelectronmicroscopieswereusedtorevealseveral
morphological aspects of PM formation. The PM looks thicker and well formed 24 h after the
bloodmeal. Presence of chitin in the PM was demonstrated by immunolabeling with an ?-chitin
monoclonalantibody.SDS-polyacrylamidegelelectrophoresisshowedatleastÞveproteinbandswith
molecular masses of 38.7-135 kDa, induced by the protein-free diet. Mouse polyclonal antiserum was
produced against PMs induced by protein-free meal and used in Western blotting, which revealed at
least three associated proteins.
ABSTRACT
KEY WORDS
peritrophic matrix, sandßy, chitin, morphology
SANDFLIES ARE INSECT VECTORS of pathogens, causative
agents of arbovirosis, bartonellosis, and leishmaniasis
in humans, constituting serious health problems in
tropical countries. Lutzomyia longipalpis (Lutz &
Neiva, 1912) is the most important vector of Leishma-
nia chagasi, the etiological agent of a life-threatening
form of the disease, American visceral leishmaniasis.
This New World sandßy is distributed throughout
southernMexicotonorthernArgentinaandParaguay.
The parasite life cycle starts in the adult sandßy with
the ingestion of an infective bloodmeal.
Severalhematophagous(blood-feeding)insects,in-
cluding adult female sandßies, synthesize a thick bag-
likestructurethatsurroundsthebloodmeal,calledthe
peritrophic matrix (PM) (Ramos et al. 1994). PMs are
present in most insects and are classiÞed into type I
and type II. Usually, PM II occurs in the larval stage
and some adult Diptera but not in the adult female
sandßy. PM I is a chitinous structure that envelops
the bloodmeal along the entire midgut, separating the
ingested food from the midgut epithelium. In general,
thename“PM”iscommonlyusedasareferenceforthe
type I in adult hematophagous insects.
The major roles of PM in blood-fed insects include
1)damageorcloggingpreventionofmidgutmicrovilli
by the luminal contents (Richards and Richards 1977,
Rudin and Hecker 1982, Berner et al. 1983); 2) com-
partmentalization of digestive events by acting as a
permeablebarrierfordigestiveenzymes(Terra1990);
3) protection against pathogens by providing a defen-
sive barrier (Peters 1992, Miller and Lehane 1993);
and 4) sequestration of heme produced by the diges-
tion of the blood hemoglobin (Walters et al. 1995,
Pascoa et al. 2002).
Several physical and biochemical properties of the
PM,suchasthickness,structure,composition,synthe-
sis, and degradation time, can be related to the ability
of distinct parasite species to survive within insect
vectors (Huber et al. 1991, Shahabuddin et al. 1993),
including Leishmania in sandßy vectors (Feng 1951,
Walters et al. 1992). Pimenta et al. (1997) described a
novelroleofthePMinprotectingLeishmaniafromthe
hydrolytic activities of the sandßy midgut.
1Laboratory of Medical Entomology Centro de Pesquisas Rene ´
Rachou, Fundac ¸a ˜o Oswaldo Cruz, Minas Gerais, Brazil.
2Centro de Cie ˆncias da Sau ´de, Universidade do Vale do Itajaõ ´Ð
Univali, Itajaõ ´, Santa Catarina, Brazil.
3Departament of Biochemistry and Immunology, Centro de Cie ˆn-
cias Biolo ´gicas, Universidade Federal de Minas Gerais, Brazil.
4DepartamentofParasitology,CentrodeCie ˆnciasBiolo ´gicas,Uni-
versidade Federal de Minas Gerais, Brazil.
0022-2585/05/0928Ð0938$04.00/0 ? 2005 Entomological Society of America

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PMsynthesisstartsimmediatelyafteraccumulation
of the bloodmeal in the midgut (Peters 1992). The
kinetics of PM formation and degradation is totally
relatedtotheingestionandthetimeofdigestionofthe
bloodmeal, which varies according to distinct insect
species (Gemetchu 1974). In several sandßy species,
the PM is formed between 12 and 24 h after the
bloodmeal, and it is degraded between 36 and 72 h,
when the digestion is Þnished (Walters et al. 1993,
1995; Andrade-Coe ˆlho et al. 2001).
We present some straightforward methods to study
the PM of Lu. longipalpis, such as its histology, thin
section of transmission electron microscopy, a modi-
Þed scanning electron microscopy, immunolabeling
confocal laser microscopy, SDS-polyacrylamide gel
electrophoresis(PAGE),andWesternblot.Theuseof
these traditional and morphological techniques, as
well as biochemical and immunolabeling tools, en-
abled us to unveil new details of the PM structure,
composition, degradation, and synthesis kinetics.
Materials and Methods
Sandfly Rearing. Lu. Longipalpis were reared in a
closedlaboratorycolonyestablishedin1995andmain-
tained in the Laboratory of Medical Entomology at
Centro de Pesquisas Rene ´ Rachou (Fiocruz-MG),
Fundac ¸a ˜oOswaldoCruzinthecityofBeloHorizonte,
Minas Gerais State, Brazil. This colony was started
from ßies collected in Lapinha cave situated 35 km
from Belo Horizonte and then maintained in the in-
sectary according to the conditions described by
Killick-Kendrick et al. (1973).
InductionofPMFormationbyBloodmealandPro-
tein-Free Diet. Groups of 3Ð4-d-old adult females
werefeddirectlyontheskinofanesthetizedhamsters,
Mesocricetus auratus, for blood feeding. A similar
group of sandßies was allowed to take protein-free
diets through a chick-skin membrane in a glass feeder
apparatus heated by circulating water at 37?C. The
protein-free meal consisted of 10% (vol:vol) latex-
beads (LB-5, Sigma, St. Louis, MO) suspended in a
phosphate-buffered saline (PBS) solution as de-
scribed by Billingsley and Rudin (1992). The fully
engorged sandßies were separated and maintained on
50% sucrose ad libitum at 25?C and 95% of humidity
until the time of dissection.
Midgut Dissection. Midguts from blood-fed sand-
ßies (1Ð96 h after bloodmeal) and from protein-free
fed sandßies (12 h after the meal) were dissected in
PBS, pH 7.2, and were collected under a stereoscope.
TheentiremidgutsorisolatedPMswerefrozenforthe
biochemical assays or Þxed for morphological obser-
vations. Midguts dissected from unfed sandßies were
used as control.
Midgut Fixation. The samples were Þxed at room
temperature for 2 h with 2.5% glutaraldehyde diluted
in 0.1 M caccodylate buffer for transmission (TEM)
and scanning electron microscopy (SEM) (Pimenta
and De Souza 1983). For immunolabeling with anti-
chitin monoclonal antibody, the midguts were Þxed
with a 4% formaldehyde solution in PBS at 4?C during
20 min. After the Þxation procedures, the samples
were washed and stored in PBS at 4?C until use.
Morphometry of Blood-Fed Midguts. Glutaralde-
hyde-Þxedmidgutsfromblood-fedsandßiesweredis-
sected and mounted into concave slides. The samples
were observed and photographed under a light mi-
croscope. The measurements were performed under
an optical microscope by using a microscopic scale.
SEMandTEM.Glutaraldehyde-Þxedmidgutswere
postÞxed with a 1% osmium tetroxide solution con-
taining0.8%potassiumferrocyanide(PimentaandDe
Souza1983).Theyweredehydratedinascendantcon-
centrationofacetoneandembeddedinEponforTEM
or dried using a critical point apparatus for SEM.
Ultrathin sections were obtained with an ultrami-
crotome, stained with uranyl acetate and lead citrate
to be observed with a TEM. For SEM observations,
the dissected blood-fed midguts were fractured to
have the PM structures inside the organ exposed.
Then,theywerecoveredwithalayerof20nmofgold
particles and observed in the scanning electron mi-
croscope (JEOL JSM5600).
Histology. Twenty-four blood-fed sandßies and
12-h protein-free fed sandßies were dissected and the
midguts were Þxed as described for SEM and TEM.
These samples were processed for Historesin (Leica,
Wetzlar, Germany) or Epon embedding. Histological
sections of 1 ?m thickness were obtained using an
ultramicrotome, stained with 1% toluidine blue
(Sigma)solutionfor10min,washed,andmountedon
glass slides to be observed under optical microscopy
(Pimenta et al. 1997).
ProductionofAnti-PMPolyclonalAntibody(?-PM
Ab). To visualize PM proteins by Western blotting
analysis, we produced a mouse polyclonal antiserum
against PM in BALB/c mice. Eighty protein-free in-
duced PMs were isolated from midguts of latex-bead-
fed sandßies. The PMs were macerated in 250 ?l of
PBS containing a protease inhibitor cocktail (1 mM
EDTA, 10 ?M N-tosyl-L-lysine chloromethyl ketone,
and 10 ?M aprotinin) and added to the same volume
of complete FreundÕs adjuvant. Approximately 100 ?l
of the PM extract (containing 16 PMs, equal to 20 ?g
of protein) was inoculated into the peritoneal cavity
of Þve mice in intervals of 15 d during 2 mo by using
incomplete FreundÕs adjuvant. Seven days after the
last inoculation, sera were collected and ?-PM Ab
titles were tested by ELISA by using the same PM
extract. Sera were stored at ?70?C. Control sera were
obtainedfrommiceinoculatedwithPBSplusadjuvant
in the absence of PM extract.
Immunolabeling with Anti-Chitin Monoclonal
Antibody(?-ChitinmAb).Midgutsfromsandßiesfed
on protein-free diets were dissected, washed, and in-
cubatedin1%PBS/bovineserumalbumin(Sigma)for
20 min. Then, the samples were incubated with
?-chitinmAb[1:100(vol:vol)]during1h.Afterwash-
ing, the samples were incubated with ßuorescent sec-
ondary antibody (ßuorescein isothiocyanate-IgG;
Sigma). Finally, the midguts were mounted on a glass
slide with the anti-fading Vectashield (Vector Labo-
ratories, Burlingame, CA), observed, and photo-
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graphed with a laser confocal microscope (LSM 510;
Carl Zeiss, Jena, Germany).
SDS-PAGE and Western Blotting Analyses. Total
extracts from Þve midguts dissected from sandßies at
different times after the bloodmeal or protein-free
diet were used for SDS-PAGE electrophoresis or
Westernblotting.Unfedmidgutsandsamplesofblood
drawnfrommiceorisolatedlatex-beadPMswereused
as controls. Protein concentration was determined,
and10?gwasappliedineachlanein12%acrylamide
gel under denaturant conditions (Laemmli 1970). Af-
ter electrophoresis, the gel was stained with silver
nitrate. In a similar experiment, the protein bands
were blotted onto a nitrocellulose membrane (GE
Osmonics, Inc., Minnetonka, MN). The PM proteins
were detected after incubation in ?-PM Ab diluted
1:100 and then revealed by anti-mouse IgG, conju-
gated with alkaline phosphatase (Sigma). Sera from
normal mice were used as a control.
Results and Discussion
Structure of Midgut and Changes after Bloodmeal.
Dimensionandaspectofmidgutsdissectedatsequen-
tial times after bloodmeal are shown in Table 1 and
Fig. 1, respectively. There are modiÞcations in the
midgut size related with the blood ingestion and the
digestion time. Thirty minutes after blood ingestion,
the midgut reaches its maximum volume (Þve times
larger than an unfed midgut). After 48 h, the midgut
volume is reduced to half its size compared with the
beginning (30 min). At the end of the digestion, the
size of the midgut is practically equal to an unfed
midgut.
The midgut epithelium from unfed Lu. longipalpis
showed a main population of typical columnar cells
(Figs. 2 and 3). This population of columnar cells is
predominately “principal cells” (Rudin and Hecker
1982). They are microvillar cells containing rounded
nucleus with loose chromatin and translucent cyto-
plasm (Fig. 2). The cytoplasm is Þlled with endoplas-
micreticulumandmitochondria(Figs.2and3).There
is also an extensive basal labyrinth formed by invagi-
nations of the basal cell membranes (Figs. 4 and 5).
Similar morphological aspects also were observed in
other sandßy midguts (Gemetchu 1974, Rudin and
Hecker 1982, Andrade-Coe ˆlho et al. 2001). This epi-
thelium is in addition responsible for secretion of
digestive enzymes and synthesis of PM (Walters et al.
1993, Pimenta et al. 1997).
Histologicalsectionsofmidgutsdissected24hafter
bloodmeal revealed the PM as a thick and well built
structure separating the epithelium from the blood-
meal (Fig. 6). A basophilic epithelium (blue color)
was distinguished from the acidophilic bloodmeal
(yellow color), probably due to the digestive en-
zymes. The PM showed intermediate staining afÞnity
(green).
A striking morphological modiÞcation takes place
within the midgut epithelium after the bloodmeal in-
gestion. The midgut is stretched and the epithelium
becomes a squamous-like structure. The principal
cells were stretched (Fig. 4). A small population of
cells, distinct from the principal cells, became visible
(Fig. 5). They are denominated “dark cells,” which
haveanelectron-densecytoplasm,fewmicrovilli,and
many mitochondria close to the apical membrane.
There is also the presence of a very extensive basal
labyrinth, compared with the electron-lucent princi-
pal cells. In a meticulous study, Leite and Evangelista
(2001) showed two types of endocrine cells in
Lu.longipalpisafterabloodmeal.Endocrinecellshave
rarely been observed in Nematocera. Such cells also
werenotedonlyafterthebloodmeal,insmallnumber
anddispersedwithintheepithelium.Theseendocrine
cells are completely distinct from the dark cells ob-
served in this study. Therefore, this research suggests
that both cell populations, in the early hours of the
digestionareinvolvedinthesynthesisandsecretionof
the PM components.
Table 1.
sequential times after bloodmeal
Dimension of Lu. longipalpis midguts dissected at
Time after
bloodmeal (h)
Diam of
midgut (mm)
Proportion
of increasea
Controlb
0.3
1
3
12
24
48
72
0.06
0.3
0.25
0.22
0.19
0.19
0.15
0.09
0
5
4.2
3.7
3.2
3.2
2.5
1.5
aCompared with control.
bUnfed midgut.
Fig. 1.
times after the bloodmeal. (a) Control (unfed midgut).
(b) 0.5 h. (c) 1 h. (d) 3 h. (e) 12 h. (f) 24 h. (g) 48 h. (h)
72 h. All midguts were photographed under the same mag-
niÞcation. MagniÞcation, 40?.
Midguts dissected and photographed at distinct
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Structure of Midguts after Protein-Free Diet. The
histologicalsectionsofthemidgutsdissected12hafter
the protein-free diet revealed a PM inside the organ
lumen (Fig. 7). This PM was composed by very dense
Þbrillar material surrounding the latex-beads, easily
observed in tangential section (Fig. 8). Isolated PMs
from midguts fed on protein-free diet showed a thick
and rigid surface (Figs. 24 and 25) when observed by
SEM. The PM surface was punctured, showing latex-
beadsinside(Fig.25).Smallprojectionssimilartothe
latex-beads were seen on the PM surface.
This study demonstrates for the Þrst time that a
protein-free diet is able to induce the sandßy PM
formation in response to the stretching of the midgut.
We noticed that this PM material is similar to the
Þbrillar layer of the blood-induced PM. This result
suggested that the Þbrilar components, which are po-
sitionedintheexternalareaoftheblood-inducedPM,
are mainly synthesized by the sandßy midgut without
any association with or induction of blood proteins.
There is a possibility that the chitin is present only in
the Þbrillar layer of the blood-induced PM. Thus, the
protein-freePMisshownasasatisfactorymodelinthe
understanding of which elements are related with the
PM formation without the inßuences of blood pro-
teins.
PM Formation and Structure. The midguts dis-
sected 1 h after the bloodmeal showed a stretched
epithelium with blood cells close to it (Fig. 9) and a
widespread basal labyrinth. Three hours later, the
midgutepitheliumwasevenmorestretched.Theprin-
cipal cells were visible, presenting vesicles and mito-
chondria distributed in the cytoplasm (Fig. 9). The
dark cells were completely fulÞlled with vesicles, and
Figs. 2–8.
(arrows). They have rounded nucleus (N) and lucent cytoplasm containing endoplasmic reticulum (er) and several
mitochondria (m) close to their surfaces. MagniÞcation, 1,300? (2) and 1,100? (3). (4Ð5) Stretched epithelial cells of a 24-h
blood-fed midgut. The principal cell (4) has an extensive basal labyrinth (*) and several lucent vesicles (v) distributed
throughoutthecytoplasm.Thedarkcell(5)hasanextensiveandswollenbasallabyrinth(*),somelucentvesicles,andseveral
mitochondriaclosetoitssurface.N,nucleus;mv,microvilli.MagniÞcation,9,000?.(6)Histologicalsectionof24-hblood-fed
midgut. There is a thick PM (arrows) beneath the epithelium (e) separating the bloodmeal (bl). MagniÞcation, 250?.
(7Ð8)Histologicalsectionsof12-hlatex-fedmidgut.InFig.1,thereisaPMinsidethemidgutÞlledwithlatex-beads.Tangential
section is showing the Þbrillar structure of the latex-bead PM. E, epithelium. MagniÞcation, 160? (7) and 480? (8).
(2Ð3) Aspects of Principal cells of the unfed midgut epithelium. The cell surfaces are covered by microvilli
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the majority of the mitochondria were close to the
apical plasma membrane (Fig. 10). They also showed
extendedendoplasmicreticulumcisterns.Outsidethe
epithelium, it was interesting to note several small-
secretedgranulesbetweentheepithelialcellsandthe
bloodmeal. There were granules close to the surfaces
of the principal cells and of the dark cells (Figs. 9 and
10). The 6-h blood-fed midguts showed an area iso-
latingthebloodcellsfromtheepithelialcells(Figs.11
and 12). In this area, it was possible to observe se-
Figs. 9–14.
principal and dark cells, respectively, from 1- and 3-h blood-fed midgut. They are stretched cells presenting a swelled basal
labyrinth (*) and vesicles. Outside the cells, it is possible to see small granules close to the principal cell and to the dark cell
(arrows).Figures11and12showdistinctareasofa6-hblood-fedmidgut.Cellsstillhaveswellingbasallabyrinth(*),several
vesicle with contents (v), secretory products (arrows) close to their surfaces, and the bloodmeal (bl) distant from the
epithelium. MagniÞcation, 10,600?; 11,500? (10); 9,600? (11); and 12,000? (12). (13Ð14) Twelve- and 24-h blood-fed
midguts showing progressive formation of the PM. The 12-h midgut still has an extensive basal labyrinth (13, asterisks). The
PM became thicker and separated from the epithelial cells (14, double arrow). It is possible to observe a laminar layer of the
PM close to the epithelial cell and an amorphous region close to the bloodmeal (13 and 14, asterisks). Arrows in both Þgures
are showing heme-like pigments. MagniÞcation, 12,000? (13) and 13,000? (14).
(9Ð12) Epithelial cell modiÞcations after the bloodmeal followed by PM formation. Figures 9 and 10 show
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creted material in front of the two types of epithelial
cells (Figs. 11 and 12).
ThisstudydemonstratesthatthePMsynthesiscom-
ponents start in the Lu. longipalpis as soon as 1 h after
the bloodmeal. This is very similar to Plebotomus per-
niciosus (Rondani 1843) (Walters et al. 1993) and
distinct from Phlebotomus papatasi (Scopoli 1786),
which start the production of the PM 4 h after the
bloodmeal (Blackburn et al. 1988).
In the late stages of digestion, the bloodmeal be-
comes compact and distant from the midgut epithe-
lium. In the 12-h midguts, nearly formed PM separat-
ing the epithelium from the bloodmeal was observed
(Fig. 13). Finally, in the 24-h midguts, a thick well-
formed PM presenting an enlarged aspect was seen
separating the bloodmeal completely from the epi-
thelium (Fig. 14). This PM showed two distinct re-
gions:athinÞbrillarlayer,closetotheepithelium,and
athickergranularlayerincontactwiththebloodmeal.
Similar aspects of the kinetics of PM formation were
observed in mosquitoes and other sandßies (Black-
burn et al. 1988; Walters et al. 1992, 1995; Jacobs-
Lorena and Oo 1996; Pimenta et al. 1997). This aspect
seems to be related with the digestion process of the
bloodmeal and differs according to temperature and
species studied (Lawyer et al. 1990).
It was also interesting to note several heme-like
pigmentsassociatedwiththegranularlayerofthePM
(Figs. 13 and 14). Probably, there are heme compo-
nents originated from the digested hemoglobin of the
blood cells. Recently, a new role of the PM in seques-
trating heme has been proposed by Pascoa et al.
(2002) in studying the mosquito Aedes aegypti L.
The SEM exposed outstanding structural aspects
unveiling the microanatomy of the PM structure. The
modiÞed SEM technique by fracturing the blood-fed
midguts exposed their internal structures (Fig. 15).
One-hour and 3-h blood-fed midguts conÞrmed as-
pects visualized by TEM, showing blood cells close to
the epithelium (Fig. 16). Six hours later, midguts
showedemptyareas,isolatingthebloodcellsfromthe
epithelium (Fig. 17). There already was a structured
PM similar to a tiny lamina between the epithelium
andthebloodcells,whichcouldnotbewellvisualized
Figs.15–19.
structures. mw, midgut wall; bl, bloodmeal. MagniÞcation, 280? (15). (16) One-hour blood-fed midguts showing bloodmeal
inside the organ lumen. Large magniÞcation is showing red cells separated from each other (asterisks). ep, epithelium; bl,
bloodmeal. MagniÞcation, 4,300?. (17) Six-hour blood-fed midgut showing a fracture, which exposes a tiny lamina forming
anearlyPM(PM)positionedbetweentheepithelium(ep)andthebloodmeal(bl).mw,musclenetwork.MagniÞcation,720?.
(18Ð19). Twenty-four-hour fractured midgut exposing external (18) and internal faces (19) of the PMs. The external face
hasawavyaspect(18,arrows)andtheinternalfacepresentsmarkswiththeformatofthemusclenetwork(19,arrows).mw,
muscle network; bl, bloodmeal; asterisk, red blood cells. MagniÞcation, 1,600? (18) and 2,500? (19).
(15Ð22)SEMfromfracturedmidguts.(15)Generalviewofamidgutfracturedinthemiddleshowinginternal
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by TEM. This early formed PM was very attached to
the epithelium. The PMs became thicker in the 12-
and 24-h midguts, easily separating themselves from
the epithelium and exposing large areas (Figs. 18 and
19).
In the 24-h midgut, Þne details of the well formed
PMwereeasilyvisualized.TheLu.longipalpisPMisan
envelope that consists of one thin Þbrillar layer and
another thick granular layer (Fig. 20). The Þbrillar
region facing the epithelium presented wavy aspects
(Fig.18).Theselayerslookmorphologicallyseparated
as two linked, but distinct, structures. The Þbrillar
layer is a laminar and thin structure close to the mi-
crovilli, which contrasts with the thick granular layer
composedbyamorphouselementsincontactwiththe
bloodmeal. Studies based on TEM observations, car-
ried out by other investigators, suggest that the PMs
from Lu. spinacrassa and P. papatasi are formed of
microÞbrilis and granules (Blackburn et al. 1988,
Walters et al. 1995).
Figs. 20–22.
(Fla) close to the epithelium (ep) and the granular layer (Gla) close to the bloodmeal (bl). Double arrows indicate the
thicknesses of the epithelium (ep) and of the PM. mw, muscle network. MagniÞcation, 10,000? (20). (21Ð22) Thirty-six and
48-h blood fed midguts, respectively, showing progressive shrinking aspects of the PM. At 36 h, the PM is contracting with
several clefts (Fig. 21, arrows) and at 48 h it is completely corrugated with sinusoidal aspect (22, white line). mw, muscle
network; ep, epithelium; bl, bloodmeal. MagniÞcation, 4,000? (21) and 1,600? (22).
(20) Details of the PM of a 24-h blood-fed midgut. The PM is very well formed showing the Þbrillar layer
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The PM showed a progressive shrinking in the 36-
and 48-h midguts (Figs. 21 and 22, respectively). The
characteristic of the granular area in contact with the
bloodmeal also were revealed (Fig. 19). This region
showed several marks, probably from the tight blood-
meal. The SEM revealed an outstanding muscle net-
work covering the entire midgut surface (Figs. 20Ð
22).
SDS-PAGE and Western Blot Analysis of PM Pro-
teins. Knowledge of PM protein contents and com-
positionisnotknowninsandßies.Thisstudyaimedto
understand the protein composition of the Lu. longi-
palpis PM by analyzing and comparing gels from
blood-fed midguts and isolated latex-bead PMs
(Fig. 26). Several bands can be seen in the blood-fed
midguts, but it was possible to note the “disappear-
ance” of two bands during the course of blood diges-
tion, demonstrating the effects of digestive enzymes.
One band had similar molecular mass to that of albu-
min (66 kDa) and the other with 28 kDa. Therefore,
during digestion, four new bands (94, 49, 22, and
18 kDa) were visualized.
The latex-bead PM showed Þve bands with molec-
ular masses ranging from 135 to 38.7 kDa. The ?-PM
Ab reacted with three protein bands from the latex-
bead PM with molecular masses of 55, 30, and 16 kDa.
Figs. 23–25.
reaction of ?-chitin mAb showing a ßuorescent PM inside the lumen (arrows). MagniÞcation, Fig. 40? (23). (24) General
SEMviewofanisolatedPMinducedbyfeedingonprotein-freemealanddissected12haftertheingestion.Notethepresence
of punctured holes (arrows), which allowed us to observe internal aspects of the PM. MagniÞcation, 140? (24). (25) Large
magniÞcation of an isolated PM induced by feeding on protein-free diet showing latex-beads (Lb) inside the structure. Note
the thickness of the PM (arrows) The PM is revealing in its surface marks (asterisks) derived from the ingested latex-beads.
MagniÞcation, 9,000?.
(23) Anti-chitin immunolabeling of a 12-h PM induced by feeding on protein-free diet. There is a strong
November 2005SECUNDINO ET AL.: L. longipalpis PERITROPHIC MATRIX
935

Page 9

Such bands were found in the 1- and 12-h blood-fed
midguts. In addition, the 55-kDa band also was ob-
servedintheunfedmidgutsandanotherband,91-kDa,
was exclusively seen in the Þrst digestion times (1Ð
12-h blood-fed midgut). There was no antibody reac-
tionwiththebloodsampleusedasacontrol(Fig.27).
Our results showed the existence of at least Þve
protein bands in the sandßy PM induced by the pro-
tein-free diet. The molecular masses ranged from 135
to38.7kDa.Incontrast,theanalysisofAe.aegyptiand
Anophelesgambiae(Giles1902)revealedamorecom-
plex PM composition with 40 proteins, from which 15
presentedsimilarproÞlesinbothmosquitoes(Jacobs-
Lorena and Oo 1996). These authors also analyzed
the protein-free induced PMs from these two mos-
quitoes fed on latex beads. In this case, 15Ð20 pro-
teins showed similarity to the blood induced PM. The
PM from Simulium vitatum (Zetterstedt 1838) re-
vealed a proÞle with a composition relatively
simple, comprising two main proteins with molecular
masses of 61 and 66 kDa (Ramos et al. 1994). This
simple protein composition is very similar to our Þnd-
ingswiththeLu.Longipalpis,whichpresentedfewPM
proteins.
In our experiments, we observed that mouse poly-
clonal antiserum was produced against the latex-bead
PM recognized at least three bands (55, 30, and
16.5 kDa). This supports that the latex-bead ingestion
induces some midgut gene expressions related with
PM synthesis, as well as the bloodmeal. These mole-
cules were present in Lu. longipalpis midgut immedi-
ately after the blood ingestion until 12 h later. It was
interesting to note that one of these bands (55 kDa)
was revealed in the midgut even before the blood
feeding, suggesting that some PM components are
synthesized in the midgut before the meal. There is
also the possibility that they may be remaining from
the former PM II, usually present in the insect larval
stages. The absence of cross-breeding reactivity with
blood components suggested that the bands are ex-
clusively from the PM or digestive enzymes, required
during the blood digestion. The proteins revealed by
WesternblottingseemtobeinvolvedinthePMstruc-
tures.
Detection of Chitin in PMs. The monoclonal anti-
body produced in our laboratory showed to be very
efÞcient and speciÞc in binding to chitin structures
present in several organisms (Martins et al. 1998).
Protein-freemidgutsweredissectedandprocessedfor
chitin immunolabeling, showing a very speciÞc and
strong reaction observed under confocal laser micro-
scope. This strong reaction revealed a ßuorescent PM
structure inside the abdominal region of the sandßy
midgut (Fig. 23).
In general, the PM is considered to contain chitin
associated to proteins and proteoglycans (Richards
and Richards 1977). In sandßy species such as
Plebotomus chinensis (Engler 1905), Plebotomus mon-
golensis (Osborn 1923), and Plebotomus squamirostris
(Feng1951),thehardnessofPMswasattributedtothe
presence of chitin (Feng 1951), which was later dem-
onstrated in P. papatasi (Blackburn et al. 1988). The
most conclusive experiments related to the presence
of constitutive chitin in the sandßy PM were carried
out by Pimenta et al. (1997). The authors fed P. pa-
patasi with chitinase mixed with the bloodmeal and
demonstrated the absence of PM synthesis in the in-
sect midgut. In contrast, they also fed the sandßies
with a chitinase inhibitor (allosamidin), and the PM
that formed was disorganized. Chitinase involvement
withthemodulationofphysicalpropertiesofPMsalso
has been suggested in An. gambiae (Shen and Jacobs-
Lorena 1997). Ramalho-Ortiga ˜o and Traub-Cesko ¨
(2003) found two chitinase genes expressed in blood-
fed Lu. longipalpis midgut.
Besides the experimental evidence on chitin pres-
ence in the PM, few studies have visualized indirectly
itslocalizationwithßuorescentwheatgermagglutinin
Figs. 26–27.
sected at different times after the bloodmeal (1Ð72 h) and
isolated PMs induced by feeding on protein-free diet (6 and
12 h) or on bloodmeal (PM). Unfed, control midguts that
never received bloodmeal; PM, peritrophic matrix dissected
from a 24-h blood-fed midgut; MW, molecular weights. Mi-
gration of protein size markers (in kilodaltons) is indicated
on the left. (27) Western blotting analysis of midguts pro-
cessed as described for Fig. 26 by using mouse polyclonal
antiserum against latex-bead PM proteins. Unfed, control
midguts that never received bloodmeal; PMl, peritrophic
matrixdissectedfroma12-hlatex-beadfedmidgut;B,control
(hamster blood); MW, molecular weights. Immunolabeled
protein sizes (in kilodaltons) are indicated on the left.
(26) SDS-PAGE analysis of the midguts dis-
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Page 10

lectin or ßuorescent stain Calcoßuor, both bound to
N-acetylglucosamine residues (Walters et al. 1992,
Shen and Jacobs-Lorena 1997, Evangelista and Leite
2002). Chitin is a structural polysaccharide composed
of ?-(134)-linked N-acetylglucosamine residues.
Here,wereportfortheÞrsttimethelocalizationofthe
PM chitin by using a speciÞc monoclonal antibody.
We detected the chitin in Lu. longipalpis PM in-
duced by protein-free diet by using latex-beads. This
technique was introduced by Billingsley and Rudin
(1992) and has been successfully used to study PM
composition of mosquitoes (Jacobs-Lorena and Oo
1996, Tellam et al. 1999). The sandßy PM was orga-
nizedfreeofbloodproteinsandsynthesizedbysimple
distention of the midgut cells. Certainly, due to the
absence of blood proteins, PM formation and degra-
dationwerequickerthanthoseprocessesinblood-fed
sandßies, similar to previous observations in mosqui-
toes (Jacobs-Lorena and Oo 1996).
ThesandßyPMisrecognizedasanimportantstruc-
ture inßuencing the vector competence for Leishma-
nia.Pimentaetal.(1997)describedanovelroleforthe
sandßyPMinpromotingparasitesurvival,becausethe
PM creates a barrier for rapid diffusion of digestive
enzymes. The sandßy PM also limits the exposure of
parasites to these enzymes during the early stages of
infectionwhentheyarespeciÞcallyvulnerabletopro-
teolyses. In the later stages of infection, Schlein et al.
(1991) demonstrated that Leishmania produces chiti-
nase to destroy part of the PM and escape from the
excretion of the digested bloodmeal. This study un-
veiled details of the Lu. longipalpis PM that should be
consideredinfurtherstudiesrelatedtotheinteraction
of the sandßy vector with pathogens.
Acknowledgments
WeDeniseN.PimentaandPaolaR.S.Eirasforreadingand
comments on this article. This research was supported by
Conselho Nacional de Desenvolvimento Cientõ ´Þco e Tecno-
lo ´gico,Fundac ¸a ˜odeAmparoaPesquisadeMinasGerais,and
Fundac ¸a ˜o Oswaldo Cruz.
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Received 30 August 2004; accepted 9 March 2005.
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