Lutzomyia longipalpis Peritrophic Matrix: Formation, Structure, and ...

MORPHOLOGY, SYSTEMATICS, EVOLUTION

Lutzomyia longipalpis Peritrophic Matrix: Formation, Structure, and Chemical Composition N.F.C. SECUNDINO,1 I. EGER-MANGRICH,1, 2, 3 E. M. BRAGA,4 M. M. SANTORO,3 1 AND P.F.P. PIMENTA

J. Med. Entomol. 42(6): 928Ð938 (2005)

ABSTRACT Sandßies 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 deÞne details of the PM structure of the Lu. longipalpis sandßy, 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 modiÞcation 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 ␣-chitin monoclonal antibody. SDS-polyacrylamide gel electrophoresis showed at least Þve 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. 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 Leishmania chagasi, the etiological agent of a life-threatening form of the disease, American visceral leishmaniasis. This New World sandßy is distributed throughout southern Mexico to northern Argentina and Paraguay. The parasite life cycle starts in the adult sandßy with the ingestion of an infective bloodmeal. Several hematophagous (blood-feeding) insects, including adult female sandßies, synthesize a thick baglike structure that surrounds the bloodmeal, called the 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 1 Laboratory of Medical Entomology Centro de Pesquisas Rene ´ Rachou, Fundaç aõ Oswaldo Cruz, Minas Gerais, Brazil. 2 Centro de Cie ˆ ncias da Sau´ de, Universidade do Vale do Itajaõ´Ð Univali, Itajaõ´, Santa Catarina, Brazil. 3 Departament of Biochemistry and Immunology, Centro de Cie ˆ ncias Biolo´ gicas, Universidade Federal de Minas Gerais, Brazil. 4 Departament of Parasitology, Centro de Cie ˆ ncias Biolo´ gicas, Universidade Federal de Minas Gerais, Brazil.

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, the name “PM” is commonly used as a reference for the type I in adult hematophagous insects. The major roles of PM in blood-fed insects include 1) damage or clogging prevention of midgut microvilli by the luminal contents (Richards and Richards 1977, Rudin and Hecker 1982, Berner et al. 1983); 2) compartmentalization of digestive events by acting as a permeable barrier for digestive enzymes (Terra 1990); 3) protection against pathogens by providing a defensive barrier (Peters 1992, Miller and Lehane 1993); and 4) sequestration of heme produced by the digestion of the blood hemoglobin (Walters et al. 1995, Pascoa et al. 2002). Several physical and biochemical properties of the PM, such as thickness, structure, composition, synthesis, 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 novel role of the PM in protecting Leishmania from the hydrolytic activities of the sandßy midgut.

0022-2585/05/0928Ð0938$04.00/0 䉷 2005 Entomological Society of America

November 2005

SECUNDINO ET AL.: L. longipalpis PERITROPHIC MATRIX

PM synthesis starts immediately after accumulation of the bloodmeal in the midgut (Peters 1992). The kinetics of PM formation and degradation is totally related to the ingestion and the time of digestion of the 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-Coeˆ lho 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 modiÞed scanning electron microscopy, immunolabeling confocal laser microscopy, SDS-polyacrylamide gel electrophoresis (PAGE), and Western blot. The use of these traditional and morphological techniques, as well as biochemical and immunolabeling tools, enabled 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 closed laboratory colony established in 1995 and maintained in the Laboratory of Medical Entomology at Centro de Pesquisas Rene´ Rachou (Fiocruz-MG), Fundaç aõ Oswaldo Cruz in the city of Belo Horizonte, 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 insectary according to the conditions described by Killick-Kendrick et al. (1973). Induction of PM Formation by Bloodmeal and Protein-Free Diet. Groups of 3Ð 4-d-old adult females were fed directly on the skin of anesthetized hamsters, 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) latexbeads (LB-5, Sigma, St. Louis, MO) suspended in a phosphate-buffered saline (PBS) solution as described 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 sandßies (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. The entire midguts or isolated PMs were frozen for the biochemical assays or Þxed for morphological observations. 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 antichitin monoclonal antibody, the midguts were Þxed with a 4% formaldehyde solution in PBS at 4⬚C during

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20 min. After the Þxation procedures, the samples were washed and stored in PBS at 4⬚C until use. Morphometry of Blood-Fed Midguts. Glutaraldehyde-Þxed midguts from blood-fed sandßies were dissected and mounted into concave slides. The samples were observed and photographed under a light microscope. The measurements were performed under an optical microscope by using a microscopic scale. SEM and TEM. Glutaraldehyde-Þxed midguts were postÞxed with a 1% osmium tetroxide solution containing 0.8% potassium ferrocyanide (Pimenta and De Souza 1983). They were dehydrated in ascendant concentration of acetone and embedded in Epon for TEM or dried using a critical point apparatus for SEM. Ultrathin sections were obtained with an ultramicrotome, 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, they were covered with a layer of 20 nm of gold particles and observed in the scanning electron microscope (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) solution for 10 min, washed, and mounted on glass slides to be observed under optical microscopy (Pimenta et al. 1997). Production of Anti-PM Polyclonal Antibody (␣-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 induced PMs were isolated from midguts of latex-beadfed 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 obtained from mice inoculated with PBS plus adjuvant in the absence of PM extract. Immunolabeling with Anti-Chitin Monoclonal Antibody (␣-Chitin mAb). Midguts from sandßies fed on protein-free diets were dissected, washed, and incubated in 1% PBS/bovine serum albumin (Sigma) for 20 min. Then, the samples were incubated with ␣-chitin mAb [1:100 (vol:vol)] during 1 h. After washing, the samples were incubated with ßuorescent secondary antibody (ßuorescein isothiocyanate-IgG; Sigma). Finally, the midguts were mounted on a glass slide with the anti-fading Vectashield (Vector Laboratories, Burlingame, CA), observed, and photo-

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Table 1. Dimension of Lu. longipalpis midguts dissected at sequential times after bloodmeal 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

a b

Compared with control. Unfed midgut.

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 Western blotting. Unfed midguts and samples of blood drawn from mice or isolated latex-bead PMs were used as controls. Protein concentration was determined, and 10 ␮g was applied in each lane in 12% acryl amide gel under denaturant conditions (Laemmli 1970). After 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, conjugated with alkaline phosphatase (Sigma). Sera from normal mice were used as a control. Results and Discussion Structure of Midgut and Changes after Bloodmeal. Dimension and aspect of midguts dissected at sequential 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 cytoplasm (Fig. 2). The cytoplasm is Þlled with endoplasmic reticulum and mitochondria (Figs. 2 and 3). There is also an extensive basal labyrinth formed by invaginations 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-Coeˆ lho et al. 2001). This epi-

Fig. 1. Midguts dissected and photographed at distinct 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 magniÞcation. MagniÞcation, 40⫻.

thelium is in addition responsible for secretion of digestive enzymes and synthesis of PM (Walters et al. 1993, Pimenta et al. 1997). Histological sections of midguts dissected 24 h after bloodmeal revealed the PM as a thick and well built structure separating the epithelium from the bloodmeal (Fig. 6). A basophilic epithelium (blue color) was distinguished from the acidophilic bloodmeal (yellow color), probably due to the digestive enzymes. The PM showed intermediate staining afÞnity (green). A striking morphological modiÞcation takes place within the midgut epithelium after the bloodmeal ingestion. 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 have an electron-dense cytoplasm, few microvilli, and many mitochondria close to the apical membrane. There is also the presence of a very extensive basal labyrinth, compared with the electron-lucent principal cells. In a meticulous study, Leite and Evangelista (2001) showed two types of endocrine cells in Lu. longipalpis after a bloodmeal. Endocrine cells have rarely been observed in Nematocera. Such cells also were noted only after the bloodmeal, in small number and dispersed within the epithelium. These endocrine cells are completely distinct from the dark cells observed in this study. Therefore, this research suggests that both cell populations, in the early hours of the digestion are involved in the synthesis and secretion of the PM components.

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Figs. 2–8. (2Ð3) Aspects of Principal cells of the unfed midgut epithelium. The cell surfaces are covered by microvilli (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 throughout the cytoplasm. The dark cell (5) has an extensive and swollen basal labyrinth (*), some lucent vesicles, and several mitochondria close to its surface. N, nucleus; mv, microvilli. MagniÞcation, 9,000⫻. (6) Histological section of 24-h blood-fed midgut. There is a thick PM (arrows) beneath the epithelium (e) separating the bloodmeal (bl). MagniÞcation, 250⫻. (7Ð8) Histological sections of 12-h latex-fed midgut. In Fig. 1, there is a PM inside the midgut Þlled with latex-beads. Tangential section is showing the Þbrillar structure of the latex-bead PM. E, epithelium. MagniÞcation, 160⫻ (7) and 480⫻ (8).

Structure of Midguts after Protein-Free Diet. The histological sections of the midguts dissected 12 h after 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 latexbeads inside (Fig. 25). Small projections similar to the 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-

sitioned in the external area of the blood-induced PM, 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-free PM is shown as a satisfactory model in the understanding of which elements are related with the PM formation without the inßuences of blood proteins. PM Formation and Structure. The midguts dissected 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 midgut epithelium was even more stretched. The principal cells were visible, presenting vesicles and mitochondria distributed in the cytoplasm (Fig. 9). The dark cells were completely fulÞlled with vesicles, and

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Figs. 9–14. (9Ð12) Epithelial cell modiÞcations after the bloodmeal followed by PM formation. Figures 9 and 10 show 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). Figures 11 and 12 show distinct areas of a 6-h blood-fed midgut. Cells still have swelling basal labyrinth (*), 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).

the majority of the mitochondria were close to the apical plasma membrane (Fig. 10). They also showed extended endoplasmic reticulum cisterns. Outside the epithelium, it was interesting to note several smallsecreted granules between the epithelial cells and the

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 isolating the blood cells from the epithelial cells (Figs. 11 and 12). In this area, it was possible to observe se-

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Figs. 15–19. (15Ð22) SEM from fractured midguts. (15) General view of a midgut fractured in the middle showing internal 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 an early PM (PM) positioned between the epithelium (ep) and the bloodmeal (bl). mw, muscle network. MagniÞcation, 720⫻. (18Ð19). Twenty-four-hour fractured midgut exposing external (18) and internal faces (19) of the PMs. The external face has a wavy aspect (18, arrows) and the internal face presents marks with the format of the muscle network (19, arrows). mw, muscle network; bl, bloodmeal; asterisk, red blood cells. MagniÞcation, 1,600⫻ (18) and 2,500⫻ (19).

creted material in front of the two types of epithelial cells (Figs. 11 and 12). This study demonstrates that the PM synthesis components start in the Lu. longipalpis as soon as 1 h after the bloodmeal. This is very similar to Plebotomus perniciosus (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 becomes compact and distant from the midgut epithelium. In the 12-h midguts, nearly formed PM separating the epithelium from the bloodmeal was observed (Fig. 13). Finally, in the 24-h midguts, a thick wellformed PM presenting an enlarged aspect was seen separating the bloodmeal completely from the epithelium (Fig. 14). This PM showed two distinct regions: a thin Þbrillar layer, close to the epithelium, and a thicker granular layer in contact with the bloodmeal. Similar aspects of the kinetics of PM formation were observed in mosquitoes and other sandßies (Blackburn 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 pigments associated with the granular layer of the PM (Figs. 13 and 14). Probably, there are heme components originated from the digested hemoglobin of the blood cells. Recently, a new role of the PM in sequestrating 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 aspects visualized by TEM, showing blood cells close to the epithelium (Fig. 16). Six hours later, midguts showed empty areas, isolating the blood cells from the epithelium (Fig. 17). There already was a structured PM similar to a tiny lamina between the epithelium and the blood cells, which could not be well visualized

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Figs. 20–22. (20) Details of the PM of a 24-h blood-fed midgut. The PM is very well formed showing the Þbrillar layer (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).

by TEM. This early formed PM was very attached to the epithelium. The PMs became thicker in the 12and 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 PM were easily visualized. The Lu. longipalpis PM is an 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). These layers look morphologically separated as two linked, but distinct, structures. The Þbrillar layer is a laminar and thin structure close to the microvilli, which contrasts with the thick granular layer composed by amorphous elements in contact with the bloodmeal. Studies based on TEM observations, carried 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).

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Figs. 23–25. (23) Anti-chitin immunolabeling of a 12-h PM induced by feeding on protein-free diet. There is a strong reaction of ␣-chitin mAb showing a ßuorescent PM inside the lumen (arrows). MagniÞcation, Fig. 40⫻ (23). (24) General SEM view of an isolated PM induced by feeding on protein-free meal and dissected 12 h after the ingestion. Note the presence 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⫻.

The PM showed a progressive shrinking in the 36and 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 bloodmeal. The SEM revealed an outstanding muscle network covering the entire midgut surface (Figs. 20 Ð 22). SDS-PAGE and Western Blot Analysis of PM Proteins. Knowledge of PM protein contents and composition is not known in sandßies. This study aimed to understand the protein composition of the Lu. longipalpis 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 “disappearance” of two bands during the course of blood digestion, demonstrating the effects of digestive enzymes. One band had similar molecular mass to that of albumin (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 molecular masses ranging from 135 to 38.7 kDa. The ␣-PM Ab reacted with three protein bands from the latexbead PM with molecular masses of 55, 30, and 16 kDa.

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Figs. 26–27. (26) SDS-PAGE analysis of the midguts dissected 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. Migration of protein size markers (in kilodaltons) is indicated on the left. (27) Western blotting analysis of midguts processed as described for Fig. 26 by using mouse polyclonal antiserum against latex-bead PM proteins. Unfed, control midguts that never received bloodmeal; PMl, peritrophic matrix dissected from a 12-h latex-bead fed midgut; B, control (hamster blood); MW, molecular weights. Immunolabeled protein sizes (in kilodaltons) are indicated on the left.

Such bands were found in the 1- and 12-h blood-fed midguts. In addition, the 55-kDa band also was observed in the unfed midguts and another band, 91-kDa, was exclusively seen in the Þrst digestion times (1Ð 12-h blood-fed midgut). There was no antibody reaction with the blood sample used as a control (Fig. 27). Our results showed the existence of at least Þve protein bands in the sandßy PM induced by the protein-free diet. The molecular masses ranged from 135 to 38.7 kDa. In contrast, the analysis of Ae. aegypti and Anopheles gambiae (Giles 1902) revealed a more complex PM composition with 40 proteins, from which 15 presented similar proÞles in both mosquitoes (JacobsLorena and Oo 1996). These authors also analyzed the protein-free induced PMs from these two mosquitoes fed on latex beads. In this case, 15Ð20 pro-

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teins showed similarity to the blood induced PM. The PM from Simulium vitatum (Zetterstedt 1838) revealed 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 Þndings with the Lu. Longipalpis, which presented few PM proteins. In our experiments, we observed that mouse polyclonal 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 molecules were present in Lu. longipalpis midgut immediately 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 exclusively from the PM or digestive enzymes, required during the blood digestion. The proteins revealed by Western blotting seem to be involved in the PM structures. Detection of Chitin in PMs. The monoclonal antibody 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-free midguts were dissected and processed for chitin immunolabeling, showing a very speciÞc and strong reaction observed under confocal laser microscope. 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 mongolensis (Osborn 1923), and Plebotomus squamirostris (Feng 1951), the hardness of PMs was attributed to the presence of chitin (Feng 1951), which was later demonstrated 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. papatasi with chitinase mixed with the bloodmeal and demonstrated the absence of PM synthesis in the insect midgut. In contrast, they also fed the sandßies with a chitinase inhibitor (allosamidin), and the PM that formed was disorganized. Chitinase involvement with the modulation of physical properties of PMs also has been suggested in An. gambiae (Shen and JacobsLorena 1997). Ramalho-Ortigaõ and Traub-Cesko¨ (2003) found two chitinase genes expressed in bloodfed Lu. longipalpis midgut. Besides the experimental evidence on chitin presence in the PM, few studies have visualized indirectly its localization with ßuorescent wheat germ agglutinin

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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, we report for the Þrst time the localization of the PM chitin by using a speciÞc monoclonal antibody. We detected the chitin in Lu. longipalpis PM induced 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 organized free of blood proteins and synthesized by simple distention of the midgut cells. Certainly, due to the absence of blood proteins, PM formation and degradation were quicker than those processes in blood-fed sandßies, similar to previous observations in mosquitoes (Jacobs-Lorena and Oo 1996). The sandßy PM is recognized as an important structure inßuencing the vector competence for Leishmania. Pimenta et al. (1997) described a novel role for the sandßy PM in promoting parasite survival, because the 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 infection when they are speciÞcally vulnerable to proteolyses. In the later stages of infection, Schlein et al. (1991) demonstrated that Leishmania produces chitinase to destroy part of the PM and escape from the excretion of the digested bloodmeal. This study unveiled details of the Lu. longipalpis PM that should be considered in further studies related to the interaction of the sandßy vector with pathogens. Acknowledgments We Denise N. Pimenta and Paola R. S. Eiras for reading and comments on this article. This research was supported by Conselho Nacional de Desenvolvimento Cientõ´Þco e Tecnolo´ gico, Fundaç aõ de Amparo a Pesquisa de Minas Gerais, and Fundaç aõ Oswaldo Cruz.

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Received 30 August 2004; accepted 9 March 2005.