Parasitol Res (2006) 98: 525533 DOI 10.

1007/s00436-005-0092-9

ORIGINA L PA PER

A. H. A. de Moraes Neto . G. S. P. Cunha . T. F. Ferreira . S. N. de Carvalho . E. V. Guimares . W. de Souza

Fine structure and cytochemical analysis of the intestinal wall along the body of adult female of Litomosoides chagasfilhoi (Nematoda: Filarioidea)
Received: 9 September 2005 / Accepted: 11 November 2005 / Published online: 17 January 2006 # Springer-Verlag 2006

Abstract Litomosoides chagasfilhoi is a filariid nematode parasite of the abdominal cavity of the wild rodent Akodon cursor (Winge, 1887), that has been described and used in Brazil as a new model for human filariasis. The fine structure of the intestine of this nematode was analyzed based on observations made by light and transmission electron microscopies of serial sections along the body. Cytochemical analysis was carried out to investigate the composition of the intestinal wall. This structure consisted of a basal lamina and an epithelium of variable thickness, composed of cells that have an irregular shape. The cytoplasm of intestinal cells contains few organelles: vacuoles, lysosomal bodies, spheroid bodies, endoplasmic reticulum, and many large lipid droplets. In the anterior portion of the intestine,

the lysosomal bodies, spheroid bodies, and vacuoles presented positive reaction for acid phosphatase, and carbohydrates were detected in lysosomal bodies. The midbody and posterior regions presented less organelles and lipid droplets, and nuclei were more abundant. Residues of L-fucose were detected by Ulex europaeus lectin binding in the midbody sections. Basic proteins were associated to lipid droplets, in the posterior region. In the whole extension of the intestine, carbohydrates were detected on tight junctions. These results indicate that the metabolized material in the epithelium can contribute to the microfilariae development and also probably can be involved with the excretory/secretory mechanism of these nematodes.

Introduction
A. H. A. de Moraes Neto . G. S. P. Cunha . T. F. Ferreira . S. N. de Carvalho Laboratrio de Biologia Celular e Tecidual, Centro de Biocincias e Biotecnologia, Universidade Estadual do Norte Fluminense (UENF), 28013-620, Campos dos Goytacazes, Rio de Janeiro, Brazil A. H. A. de Moraes Neto (*) Ncleo de Biologia e Controle de Endo e Ectoparasitos de Interesse Mdico e Veterinrio, Departamento de Biologia, Instituto Oswaldo Cruz, Fundao Oswaldo Cruz, 21045-900 Rio de Janeiro, Brazil e-mail: moraesnt@uenf.br Fax: +55-22-27251530 E. V. Guimares Laboratrio de Biologia Estrutural, Departamento de Ultraestrutura e Biologia Celular, Instituto Oswaldo Cruz, Fundao Oswaldo Cruz, Rio de Janeiro, Brazil W. de Souza Laboratrio de Ultraestrutura Celular Hertha Meyer, Programa de Biologia Celular e Parasitologia, IBCCF, UFRJ, 21949-900 Rio de Janeiro, Brazil

Considerable variation in the morphology of the body wall and the intestine between species of adult filariids have been described by electron microscope studies (Moraes Neto et al. 2001, 2002, 2003; Ogbogu and Storey 1996; Bird and Bird 1991; Franz et al. 1984). Most of these studies have focused on the analysis of the body wall of nematodes, because the cuticle is the portion that establishes a direct contact with the immune system of the host and is also physiologically important as a site of nutrient acquisition (Peixoto et al. 1997; Martinez and De Souza 1995, 1997; Johnstone 1994; Lee et al. 1986). However, there are few contradictory information about the nutrition mode and its relation with the excretory/secretory mechanism to escape from the attack of the host immunological system (Franz and Andrews 1986a,b; Blaxter et al. 1992). The filariids use transcuticular uptake of glucose, amino acids, and adenosine (Howels and Chen 1981). They can digest and absorb molecules that are metabolized and secreted by the intestinal wall; as such, small peptides that regurgited from the worm gut are secondarily absorbed via the transcuticular route (Peixoto et al. 1999). Immunolocalization studies have detected some proteins and enzymes in the intestine of filariids that can interact with the immune system of the host and may also be essential for nematodes

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survival, such as (a) gp29, a glycoprotein that is present in the cuticle and hypodermal cell layer of the Brugia malayi adult and is also found on the basement of the basal lamina of the intestine (Selkirk et al. 1990); (b) CuZn superoxide dismutase (Wildenburg and Henkle-Duhrsen 1999) and a cathepsin D-like lysosomal aspartic protease (Jolodar et al. 2004) that appears to function in intestinal digestion and tissue degradation of Onchocerca volvulus and is recognized in the sera of onchocerciasis patients; and (c) a phosphoglycerate mutase (Zhang et al. 2004) from the glycolytic and gluconeogenic pathways for all nematodes. With regard to the fine structure, the intestines of adults of O. volvulus (Franz and Bttner 1983), B. malayi (Vincent et al. 1975), Loa loa (Franz et al. 1984), and Litomosoides carinii (Franz and Andrews, 1986a,b) are composed of a basal lamina and flat to cubic epithelial cells bearing microvilli on the luminal surface, which are filled with materials. These cells are linked by desmosomes. The cytoplasm contains mitochondria, numerous vacuoles, some small lysosome dense bodies, and lipid droplets. The nuclei are surrounded by rough endoplasmic reticulum. In thirdstage larvae of Wuchereria bancrofti (Weber 1985), the digestive tract is fully differentiated, presenting lipid inclusions and microvilli. Wolbachia bacteria are present in the cytoplasm and in the luminal surface in W. bancrofti and in other organs of majority of filariids (Peixoto et al. 2001; Taylor et al. 2000). The intestinal epithelium of third-stage larvae of L. carinii is one-cell thick. A lumen is already identified, but no microvilli are present. The cytoplasm of the cells contains the same organelles found in adults, but its alimentary tract is nonfunctional (Ogbogu and Storey 1996). To obtain more information on the organization and composition of the intestinal wall and its relation with the excretory/secretory mechanism of the filariids, we extended our studies by examining the adult female of Litomosoides chagasfilhoi, a nematode that has been described in Brazil (Moraes Neto et al. 1997) by light and transmission electron microscopies (TEM) and cytochemical techniques.

male worms (87.095.0 mm long) were used in this study. Each one was cut in 32 portions, dehydrated in acetone series, and embedded in Spurrs resin. Thin sections were collected on copper grids, counterstained with uranyl acetate and lead citrate, and observed in a Zeiss 900 TEM. Light microscopy The semithin sections obtained from TEM preparations were stained in toluidine blue, mounted with entellan, and photographed under a Zeiss Axioplan microscope using a 20 objective. The measurements were made with the aid of an ocular with a scale in millimeters. Cytochemical labeling Lipids detection Fixed filariids were washed twice in 0.1 M cacodylate buffer and three times in 0.1 M imidazole buffer, pH 7.5, and postfixed in 2% osmium tetroxide in a 0.1 M imidazole buffer (Angermller and Fahimi 1982). They were washed again in the last buffer, dehydrated in an acetone series, and embedded in Spurr. Thin sections were collected on copper grids, counterstained with lead citrate, and observed by TEM. Basic protein detection Fixed filariids were washed three times in 0.1 M cacodylate buffer, pH 7.2, dehydrated in ethanol series, and incubated in 2% phosphotungstic acid (PTA) dissolved in ethanol for 24 h and embedded in Spurrs resin (Gordon and Bensch, 1968). Thin sections were collected on copper grids and observed by TEM with no counterstain. Control was incubated in pirydin for 90 min, at 37C after the fixation, and washed with 0.1 M cacodylate buffer. Acid phosphatase Fixed filariids were washed twice in 0.1 M cacodylate buffer and three times in 0.1 M Tris maleate buffer, pH 7.5 (Robinson and Karnovsky 1983; Barka and Anderson 1962). They were incubated in a medium containing 10 mM -glicerophosphate, 0.1 M Tris maleate buffer, pH 5.0, and 2 mM cerium chloride (Briggs et al. 1975; Hulstaert et al. 1983) for 1 h at room temperature, washed in the same buffer, postfixed in 1% osmium tetroxide, 5 mM calcium chloride in a 0.1 M cacodylate buffer. The filariids were dehydrated in acetone series and embedded in Spurr. Thin sections were collected on copper grids and observed by TEM with no counterstain. Control was incubated in the absence of the substrate. Detection of polysaccharides and glicoproteins Fixed filariids were prepared as described for TEM. Thin sections were collected on 300 mesh gold grids and incubated in a 1% periodic acid solution for 30 min at room temperature. The grids were rinsed four times in distilled water (twice under shaking) for 10 min, incubated in 1% thiosemicarbazide solution in 10% acetic acid for 72 h, rinsed in (10, 5, and 2%) acetic acid series and distilled water, and floated in a 1% silver proteinate for 30 min at room temperature in the darkness (Thiery 1967). They were rinsed again in distilled water and observed by TEM with no counterstain.

Materials and methods

Nematodes Adult females of L. chagasfilhoi were collected during necropsies from the abdominal cavity of naturally infected Akodon cursor (Rodentia: Muridae) trapped in the locality of Catimbau Grande, Rio Bonito, Rio de Janeiro, Brazil (Moraes Neto et al. 1997), and from experimentally infected gerbils Meriones unguiculatus, by exposing to infected hematophagous mites Ornythonyssus bacoti under controlled conditions of temperature (25C) and humidity (8085%) (Bertram et al. 1946). Filariids were fixed for 2 h at room temperature or overnight at 4C in 2.5% glutaraldehyde, 4% freshly prepared paraformaldehyde, 5 mM calcium chloride in 0.1 M cacodylate buffer, pH 7.2. Transmission electron microscopy Fixed filariids were washed and postfixed in a solution containing 1% osmium tetroxide, 5 mM calcium chloride, and 0.8% potassium ferrocyanide in 0.1 M cacodylate buffer, pH 7.2. Ten fe-

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Control was processed as described above without previous incubation in periodic acid. Lectin-binding sites Adult females of L. chagasfilhoi were washed three times in phosphate buffered saline (PBS), pH 7.2, and fixed for 2 h at 4C in 0.1% glutaraldehyde, 4% freshly prepared paraformaldehyde, and 2% picric acid in 0.1 M cacodylate buffer (Bendayan 1984; Bendayan et al. 1987, 1990; Berryman and Rodewald, 1990). After fixation, they were washed in PBS and dehydrated at progressively lower temperatures in 3090% methanol. The infiltration was done in Unycril at 20C for 2 weeks, and polymerization was performed under ultraviolet light at 20C for 1 week. Ultrathin sections were collected on 300 mesh nickel grids, washed in distilled water for 10 min, and incubated for 20 min at room temperature in 50 mM ammonium chloride, pH 8.3. They were then washed for
Fig. 1 Light and transmission electron microscopies of the intestine of adult females of L. chagasfilhoi. a Light microscopy (LM) of semithin section at 4 mm behind the anterior end. Cuticle (C), uterus (U), and intestine (I). Bar=40 m. b TEM view of a longitudinal thin section of the intestine wall at the same distance in a, showing the cytoplasm of epithelial cells that contains few organelles: vacuoles (V), endoplasmic reticulum (ER), and many lysosomal bodies (L). Tight junctions (TJ) occur between the intestinal cells. The microvilli (MV) are distributed irregularly over the luminal surface (LU). Bar=0.6 m. c LM of semithin section at 7 mm behind the anterior end. The intestinal epithelium is thicker than in a. Cuticle (C), uterus (U), and intestine (I). Bar=32 m. d TEM view of a longitudinal thin section of the intestinal wall at 7 mm behind the anterior end, showing lipid droplets (LD), many lysosomal bodies (L), and few vacuoles (V) in the cytoplasm of epithelial cells. Intestinal wall (IW), uterine wall (UW), luminal surface (LU). Bar=0.6 m. e LM of semithin section at 10 mm behind the anterior end. The thickness of epithelium was close to c. Cuticle (C), uterus (U), and intestine (I). Bar=32 m. f TEM view of a longitudinal thin section of the intestinal wall at 10 mm behind the anterior end, showing a lot of lysosomal bodies (L) and large vacuoles (V) in the cytoplasm of epithelial cells. Luminal surface (LU). Bar=0.6 m

30 min in PBS containing 4% bovine serum albumin (BSA). Sections were then treated for 2 h with the following 10 nm gold-labeled lectins (1:10 dilutions): Arachis hypogaea (PNA), Triticum vulgaris (WGA), Ulex europaeus (UEA-I) in PBS containing 4% BSA. After incubation, the grids were rinsed three times in PBS and twice in distilled water, counterstained with uranyl acetate and lead citrate, and examined in a Zeiss 900 transmission electron microscope. Controls consisted of addition of 200 and 500 mM of D-galactose, N-acetyl-D-glucosamine, or L-fucose to the incubation.

Results
In general, the size of the intestine was rather small (Figs. 1a, c,e, 2c,e, and 3c,e), occupying from 1/2 to 1/6 the body width (diameter of intestine/width of the body) (Table 1).

A
V U C

ER

TJ

I LU MV L

C UW L LD IW

D
V

I U LU

C L

V LU

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A
C

L MV IW LU C I N ER

PS

L TJ F I LD MV C U LU V

Fig. 2 Light and transmission electron microscopies of the intestine of adult females of L. chagasfilhoi. a TEM view of a longitudinal thin section of the intestinal wall at 10 mm behind the anterior end, showing a lot of lysosomal bodies (L) in the cytoplasm of epithelial cells. Microvilli (MV), luminal surface (LU). Bar=0.3 m. b TEM view of a longitudinal thin section of the intestinal wall (IW) and transversal section of the cuticle (C), at 10 mm behind the anterior end, showing a vesicle (arrow) at the pseudocelom region (PS), suggesting transport of macromolecules between the intestinal epithelium and the cuticle. Bar=1.1 m. c Light microscopy (LM) of semithin section at 34 mm behind the anterior end, close to the midbody region. The intestine reaches the maximum diameter (125 m), and the epithelium is thinner (2.5 m). Cuticle (C), uterus

(U), and intestine (I). Bar=32 m. d TEM view of a longitudinal thin section of the intestinal wall at 34 mm behind the anterior end, showing a lot of lysosomal bodies (L) surrounding the nuclei (N) and endoplasmic reticulum (ER) in the cytoplasm of epithelial cells. Bar=1.1 m. e LM of semithin section at 50 mm behind the anterior end, at the midbody region. The diameter of the intestine is reduced (50 m), but the thickness remains constant (2.5 m). Cuticle (C), uterus (U), and intestine (I). Bar=32 m. f TEM view of a longitudinal thin section of the intestinal wall at 50 mm behind the anterior end, showing many lipid droplets (LD) and vacuoles (V) in the cytoplasm of the epithelial cells. Tight junctions (TJ), microvilli (MV), and luminal surface (LU). Bar=1.1 m

The lumen was filled with material in all preparations (Figs. 1a c,e, 2ac,e,f, 3af, 4a, and 5b). The microvilli were irregularly distributed along the luminal surface (Figs. 2a,f and 3d,f). Its wall had variable thickness (Table 1) and was composed of epithelial cells with an irregular shape. Tight junctions occurred between these cells (Figs. 1b, 2f, 4d, and 5c,d). At 4 mm behind the anterior end (Fig. 1a), the cytoplasm of the cells contained vacuoles, spheroid bodies (not shown), endoplasmic reticulum, and many lysosomal bodies (Fig. 1b). Just below, at 7 mm (Fig. 1c), the diameter of

intestine was slightly bigger, and the epithelium was thicker (Table 1); many lipid droplets, lysosomal bodies, and few vacuoles were observed (Fig. 1d). At 10 mm (Fig. 1e), the diameter of the intestine and the thickness of the epithelium were close to the anterior segment (7 mm) (Fig. 1c; Table 1). The cytoplasm of the epithelium had larger amounts of lysosomal bodies and large vacuoles (Figs. 1f and 2a). At this region, some vesicles were visualized at the pseudocelom region (Fig. 2b), suggesting transport of macromolecules between the intestinal epithelium and the cuticle. At 34 mm

529 Fig. 3 Light and transmission electron microscopies of the intestine of adult females of L. chagasfilhoi. a, b TEM views of longitudinal thin sections of the intestinal wall (IW) and of the uterine membrane (UW), at 50 mm behind the anterior end, showing vesicles (arrows) at the pseudocelom region (PS), suggesting transport of macromolecules between the intestinal epithelium and the uterus. Note lysosomal bodies (L) at uterine membrane. Nuclei (N), luminal surface (LU). Bars=1.1 m. c Light microscopy (LM) of semithin section at 70 mm behind the anterior end, the diameter of intestine was the same of the middle portion, but the epithelium was thicker (17.5 m). Cuticle (C), uterus (U), and intestine (I). Bar=30 m. d TEM view of a longitudinal thin section of the intestinal wall at 70 mm behind the anterior end, showing large vacuoles (V). Microvilli (MV) and luminal surface (LU). Bar=0.6 m. e LM of semithin section at 78 mm behind the anterior end. The intestine had a small diameter (37.5 m), and the thickness of the epithelium was reduced (5 m). Bar=30 m. f TEM view of a longitudinal thin section of the intestinal epithelium at 78 mm behind the anterior end, showing lipid droplets (LD), endoplasmic reticulum (ER), and nuclei (N). Microvilli (MV) and luminal surface (LU). Bar=1.1 m

UW L

UW

PS

IW

IW N

LU

C
C V

I U LU MV

E
U

C ER LD N

MV I LU

behind the anterior end (Fig. 2c), close to the midbody region, the intestine reached the maximum diameter, and the epithelium was thinner (Table 1). Some lipid droplets (not shown), many lysosomal bodies surrounding nuclei, and endoplasmic reticulum were observed (Fig. 2d). At the midbody region (4450 mm) (Fig. 2e), the diameter of the

intestine was reduced, but the thickness remained constant (Table 1). The cytoplasm of the cells contained few organelles, and lipid droplets were more abundant; vacuoles and nuclei were present (Figs. 2f and 3b). At this region, close to the posterior portion of intestine, some vesicles in the pseudocelom and lysosomal bodies in the uterine membrane

Table 1 Measurements of ten females of L. chagasfilhoi intestine in serial sections along the body

Region of Distance from intestine anterior end (mm) Anterior 4.0 7.0 10.0 34.0 44.050.0 70.0 78.0

Width of the Diameter of Ratio of diameter of body (m) intestine (m) intestine/width of the body 240.0250.0 77.5102.5 250.0 77.5110.0 260.0 72.5112.5 260.0 107.0125.0 210.0260.0 42.550.0 200.0230.0 40.055.0 220.0240.0 37.5 1/3 1/3 1/3 1/2 1/5 1/4 1/6

Thickness of intestinal wall (m) 5.0 7.510.0 5.010.0 2.5 2.5 5.017.5 2.55.0

Middle Posterior

530 Fig. 4 Cytochemical labeling of longitudinal thin sections of the intestine of adult females of L. chagasfilhoi. ac Detection of acid phosphatase at the anterior portion. The lysosomal bodies (L) and vacuoles (V) presented positive reaction. Lipid droplets (LD) and luminal surface (LU). Bar=1.7 m (a), Bar=0.6 m (b, c). d Detection of carbohydrates at the anterior portion, in the lysosomal bodies (L) and at tight junctions (TJ). Bar=0.4 m. e Detection of UEA-I binding sites (arrowheads) at the midbody region. Lipid droplets (LD) and luminal surface (LU). Bar=1 m. f Detection of acid phosphatase at the posterior portion. The vacuoles (V) presented positive reaction. Lipid droplets (LD). Bar=1 m

LU

C
V

LD V

TJ

E
V

LD

LD LU

were observed, suggesting transport of molecules among the intestine epithelium and the uterine wall (Fig. 3a,b). In the posterior region (70 mm behind the cephalic end) (Fig. 3c), the diameter of the intestine was the same as observed in the midbody region, but the epithelium was thicker (Table 1). Many lipid droplets, few lysosomal bodies, and many large vacuoles were present in this region (Fig. 3d). Just below, at 78 mm from anterior end (Fig. 3e), the intestine was very thin, and the thickness of the epithelium was reduced (Table 1). However, the lipid droplets became more abundant; endoplasmic reticulum and various nuclei were observed in the cytoplasm of intestinal cells (Fig. 3f); lysosomal bodies were not observed. At the anterior portion of the intestine, the lysosomal, spheroid bodies, and vacuoles presented positive reaction for acid phosphatase (Fig. 4ac); carbohydrates were detected in the lysosomal bodies (Fig. 4d). At the midbody region, the epithelial cells presented residues of L-fucose that were recognized by UEA-I lectin (Fig. 4e). However,

the other two lectins tested, WGA and PNA, did not label the intestinal epithelium. In the posterior portion of the intestine, vacuoles presented positive reaction for acid phosphatase (Fig. 4f), and basic proteins were associated to lipids (Fig. 5a). In the whole extension of the epithelium of L. chagasfilhoi intestine, lipid droplets were labeled by the osmiumimidazole technique (Fig. 5b), and carbohydrates were detected at tight junctions (Figs. 4d and 5c,d).

Discussion
Serial sections along the intestine showed that the fine structure of the intestinal epithelium of adult female L. chagasfilhoi is extremely variable in thickness and abundance of organelles. Although, in others filariids such as L. loa (Franz et al. 1984), B. malayi (Vincent et al. 1975), and O. volvulus (Franz and Bttner 1983), the epithelium is a layer of constant thickness; our observations of L.

531 Fig. 5 Cytochemical labeling of longitudinal thin sections of the intestine of adult females of L. chagasfilhoi. a Detection of basic proteins associated to lipids (LD), at the posterior portion. Bar=1 m. b Detection of lipids (LD), at the posterior portion. Luminal surface (LU). Bar=1 m. cd Detection of carbohydrates (polysaccharides) on the tight junctions (TJ) at the anterior and posterior portions. Luminal surface (LU). Bar= 0.15 m (c), Bar=1 m (d)

LD LD LU

TJ TJ LU

chagasfilhoi confirm those of Franz and Andrews (1986a,b) for L. carinii, showing that the epithelium displays a variable thickness. In the cytoplasm of the anterior portion of the intestinal epithelium of L. chagasfilhoi, many lipid droplets, lysosomal bodies, and large vacuoles were observed and confirmed by cytochemical techniques for lipid detection and for acid phosphatase activity, respectively. The abundance of lysosomal bodies, also previously described in the third-stage larvae of L. carinii (Ogbogu and Storey 1996) and in adult of O. volvulus (Franz et al. 1984), can be related with the digestion of hemoglobin from the host, which takes place at this region (Bonner et al. 1971). Vacuoles and lipid droplets are common in the epithelium of all filariids, but lysosomal bodies associated or surrounding the droplets have not been described before. These droplets and vacuoles are extremely large compared to the epithelial diameter. In the midbody and posterior regions, the intestinal epithelium presented a decrescent amount of lysosomal bodies and an increment of lipid droplets and vacuoles, confirming previous observations made in L. carinii by Franz and Andrews (1986a,b). It is known that in the anterior portion of the intestine of helminths, secretion and digestion of macromolecules occur, while in the middle and posterior regions, secretion, absorption, and storage of nutrients take place (Bird and Bird 1991). Thus, the distribution of various organelles along the intestine of filariids can be due to the functional role played by each portion. On the other hand, the presence of lipid droplets in the intestine of adult helminths has also been attributed to adverse effects of the hosts immune response (Wright et al. 1985). Transcuticular uptake of glucose, amino acids, and adenosine occurs in filariids (Howels and Chen 1981).

However, our results suggest the existence of an alternative route for nutrition of the intrauterine larvae and transport of molecules to the cuticle. The observation of vesicles located in the pseudocelom, and also seen in close association with the uterine wall and the cuticle, suggests a migration of these vesicles from the intestine to these regions. The observation of lysosomal bodies at the uterine wall, as observed by Franz et al. (1984) in adult female of O. volvulus, which could participate in the digestion of those vesicles, supports that idea. Thus, our observations are in agreement with those of Jolodar et al. (2004), Peixoto et al. (1999), Selkirk et al. (1990), and Zhang et al. (2004), which have demonstrated that filariids can absorb and digest molecules that are metabolized and secreted by the intestinal wall. Filarial gut-associated antigens that are not detected by normally infected hosts, but that are accessible to hosteffector molecules and cells, inducing an immune response that is partially protective and therefore could be a potential useful vaccine antigens, as shown by McGonigle et al. (2001), could probably be exported by this alternative route. Carbohydrates were detected in the lysosomal bodies at the anterior portion of the intestine and in tight junctions of the whole epithelium. Increasing evidence indicates that the tight junctions also play a role in membrane transport. Various signaling and trafficking molecules localize to the sites of cellcell junctions in epithelial cells, including Rab proteins, a family of small GTPases that regulate different steps of vesicular transport along the endocytic and exocytic pathways (Kohler and Zahraoui 2005). Thus, carbohydrates could be transported to other regions of the nematode via membrane transport, in addition to the pseudocelomatic fluid distribution.

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The lysosomal bodies at the anterior portion and vacuoles of the anterior and posterior portions of the intestine were positive for acid phosphatase. Basic proteins were associated to lipids at posterior portion of the intestine. Using gold-labeled lectins, UEA-I positive sites were only found at the midbody region, showing L-fucose residues. Nevertheless, in the cuticle of L. carinii, W. bancrofti, and B. malayi, residues of N-acetyl-D-glucosamine, D-galactose, and N-acetyl-D-galactosamine have been recognized by WGA and PNA lectins (Rao et al. 1987; Schraermeyer et al 1987a,b; Arajo et al. 1993), demonstrating heterogeneity in the constitution of the nematode body. The variations in the composition of the intestine can probably be related to the functional role of each portion and to the interspecific variations common to all nematodes, indicating that the epithelium is not a homogeneous structure.
Acknowledgements We are grateful to Ms. Beatriz Ferreira Ribeiro, Mrcia Adriana Dutra, and Giovana Alves de Moraes from the Laboratrio de Biologia Celular e Tecidual, CBB, UENF, and Izaias Aparecido Pimenta, from the Departamento de Biologia, IOC, FIOCRUZ, for technical assistance; to Dr Renato Augusto DaMatta and Dr Joo CA Almeida, for critical review of the manuscript; and to Ms. Maria de Ftima Leal Alencar for secretarial assistance. This work was supported by Conselho Nacional de Desenvolvimento Cientfico e Tecnolgico (CNPq) grant 150.115/2003-2, Fundao Carlos Chagas Filho de Amparo Pesquisa do Estado do Rio de Janeiro (FAPERJ), Universidade Estadual do Norte Fluminense Darcy Ribeiro, and Departamento de Biologia, Instituto Oswaldo Cruz, FIOCRUZ.

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