Ultrastructural and cytochemical aspects of Schistosoma mansoni cercaria

Micron 40 (2009) 394–400

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Ultrastructural and cytochemical aspects of Schistosoma mansoni cercaria M.G.S. Cavalcanti a,b,d, H.R.C. Arau´jo a,g, M.H.S. Paiva e, G.M. Silva a,c, C.C.G.S. Barbosa a, L.F. Silva a,f, F.A. Brayner a,c, L.C. Alves a,c,f,* a

Departamento de Parasitologia, Centro de Pesquisas Aggeu Magalha˜es (CPqAM/FIOCRUZ), Recife – PE, Brazil Departamento de Fisiologia e Patologia, Universidade Federal da Paraı´ba (UFPB), Joa˜o Pessoa, Brazil c Laborato´rio de Imunopatologia Keizo Asami (LIKA), Universidade Federal de Pernambuco, Recife – PE, Brazil d Departamento de Medicina Tropical, Universidade de Federal de Pernambuco, Recife – PE, Brazil e Associac¸a˜o Caruaruense de Ensino Superior, Caruaru – PE, Brazil f Universidade de Pernambuco (UPE), Recife – PE, Brazil g Departamento de Entomologia Me´dica, Instituto Rene´ Rachou, Belo Horizonte – BH, Brazil b

A R T I C L E I N F O

A B S T R A C T

Article history: Received 28 March 2008 Received in revised form 18 September 2008 Accepted 19 September 2008

An alternative to identify the critical processes necessary to the parasite establishment of the host is to focus on the evolutionary stage responsible for the primary invasion, i.e. the infection structure. The objective of this study was to ultrastructurally characterize Schistosoma mansoni cercariae, using cytochemical techniques. In order to identify basic proteins, techniques such as ethanolic phosphotungstic acid (EPTA) and ammoniacal silver staining were used. Calcium sites location was achieved using the Hepler technique and to evidence anionic groups, we used cationic ferritin particles and enzyme treatment with trypsin Vibrio cholerae, chondroitinase and neuraminidase. The EPTA technique highlighted the presence of basic tegument proteins, nucleus and nucleolus from subtegumental cells, inclusion bodies and preacetabular glands. After using ammoniacal silver, we observed a strong staining in all infective larvae, particularly in the nuclei of muscle cells, circular muscle tissue and preacetabular glands. Calcium site locations were shown to be uniform, thereby limiting the inner spaces of the larvae, especially muscle cells. Samples treated with cationized ferritin particles presented strong staining at the cuticular level. Neuraminidase treatment did not alter the stained shape of such particles on the trematode surface. However, trypsin or chondroitinase treatment resulted in absence of staining on the larval surface. This information on the biochemical composition of the infecting S. mansoni larvae provides data for a better understanding of the biology of this parasite and background on the intriguing parasite–host relationship. ß 2008 Elsevier Ltd. All rights reserved.

Keywords: Schistosoma mansoni cercaria Ultrastructural cytochemical

1. Introduction Schistosomiasis is one of the most serious and complex public health problems, mainly due to the lack of any vaccine, failures in attempts to eradicate the vector mollusk and the development of resistance to anti-schistosomiasis drugs (Kalife et al., 2000). Despite a century of discovery and great number of studies, schistosomiasis is still present in 74 countries, affecting millions of people in Latin American and African countries (World Health Organization, 1998).

* Corresponding author at: Department of Parasitology, Centro de Pesquisas Aggeu Magalha˜es/FIOCRUZ, Av. Moraes Rego s/n, Campus da UFPE, CEP 50670-420, Recife, Pernambuco, Brazil. Tel.: +55 81 2101 2643; fax: +55 81 2101 2516. E-mail address: lcalves@cpqam.fiocruz.br (L.C. Alves). 0968-4328/$ – see front matter ß 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.micron.2008.09.006

Helminth disease control needs to be considered from two perspectives: morbidity and transmission. For morbidity control, which aims to reduce the numbers of cases of severe forms of the disease, diagnosis and treatment are sufficient, although the success of chemotherapy for treating schistosomiasis in highprevalence areas has not been long-term, culminating in fast reinfection. Regarding transmission control, simply treating the infected population is not enough. It also becomes necessary to interrupt the parasite’s life cycle in the intermediate host (Katz, 1980). Even with strong efforts towards studying possible controls, from a variety of researchers and research institutions around the world, this parasitosis continues to spread. In spite of various studies relating to the parasitic stage responsible for the infection (the cercariae), none of these studies have placed emphasis on cytochemical characterization at the ultrastructural level.

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Most studies emphasize the morphological characterization of Schistosoma mansoni cercariae, but only a few of them associate morphology with chemical composition. By using the cytochemical characterization, the biochemical composition of a certain biological system can be identified. Associating this technique with ultrastructure enables not only ‘‘in loco’’ identification of proteins, carbohydrates, lipids and other compounds, but also their location and quantification (De Souza, 1998). The present study focuses on finding the locations of basic protein, calcium and anionic sites in S. mansoni cercariae, through the use of cytochemistry techniques applied to ultrastructure. Understanding the relationships between these biochemical components and their locations may facilitate identification of the probable functions of these constituents.

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3.3. Calcium The cercariae obtained were washed and fixed in a 2.5% glutaraldehyde solution in 0.1 M cacodylate buffer (pH 7.2) with 5 mM CaCl2, overnight at room temperature. After fixing, the samples were washed in cacodylate buffer (pH 7.2) containing 10 mM calcium chloride for 10 min (twice). Post-fixing was performed using 1% osmium in 0.1 M cacodylate buffer (pH 7.2), containing 10 mM calcium chloride and 0.8% potassium ferricyanide for 2 h in a darkened room. Two washes were performed with 0.1 M cacodylate buffer (pH 7.2), containing 10 mM calcium chloride for 10 min. Following this, contrasting was performed, with 2% aqueous uranyl acetate for 2 h. Dehydration and inclusion were performed in accordance with the routine method. 3.4. Anionic site location

2. Materials and methods 2.1. Parasites S. mansoni cercariae were obtained from Biomphalaria glabrata snails. These mollusks were infected with S. mansoni, Belo Horizonte strain, and kept in the Schistosomiasis Laboratory of the Aggeu Magalha˜es Research Center, Recife, Brazil. 2.2. Ultrastructure Parasites were fixed for 2 h at 4 8C in a solution containing 4% paraformaldehyde, 2.5% glutaraldehyde in 0.1 M cacodylate buffer at pH 7.2. After washing them in the same solution, they were post-fixed for 1 h in a solution containing 1% OsO4 in 0.1 M cacodylate buffer, dehydrated in acetone and embedded in Epon. Ultrathin sections were picked up on uncoated 200 mesh copper grids, double-stained with uranyl acetate and lead citrate, and observed using a Jeol JEM 100CX or a Zeiss 900 electron microscope.

To detect anionic sites, infective larvae were fixed in a 2.5% glutaraldehyde solution in cacodylate buffer (pH 7.2). The larvae were then incubated for 1 h at room temperature in a solution containing 1 mg/ml of cationized ferritin particles at pH 7.2 (Danon et al., 1972). The samples remained under agitation for 1 h. They were rinsed in PBS, washed in cacodylate buffer, dehydrated in acetone and embedded in Epon 812 (Electron Microscopy Sciences). Thin sections were counterstained with uranyl acetate and lead citrate and examined. 3.5. Enzyme treatment Cercariae were incubated for 5 min in a solution containing 500 mg/ml trypsin (Sigma, St. Louis, MO) at pH 7.2, or for 18 h in

3. Cytochemical staining 3.1. Ethanolic phosphotungstic acid (EPTA) To detect of basic proteins, the parasites were fixed as described above for the ultrastructure analysis, dehydrated in ethanol and incubated in a solution containing 2% phosphotungstic acid in absolute ethanol (EPTA) for 2 h at room temperature (Bloom and Aghajanian, 1968). The specimens were then washed in ethanol, incubated for 10 min in propylene oxide and embedded in Epon to be examined without counterstaining. 3.2. Ammoniacal silver The concentrates of cercariae were centrifuged at low speeds and the pellets were fixed for 1 h at room temperature in 2.5% glutaraldehyde in 0.1 M cacodylate buffer at pH 7.2. After fixing, the specimens were recentrifuged and rinsed with distilled water. The cercariae were then suspended in ammoniacal silver solution (AS) for 5 min and then alternately washed with distilled water and centrifuged several times. Next, the cercariae were resuspended for several minutes in 3% formalin. After centrifugation and washing, the specimens were dehydrated by means of increasing concentrations of ethanol and embedded in Epon 812. Thin sections were cut and stained for contrast using uranyl acetate followed by lead citrate, or were left unstained, before viewing.

Fig. 1. S. mansoni cercariae with routine processing. Note the presence of spines (sp) and cercarial granules (gc) in the tegument (t). Below the tegument, the basal lamina (bl), circular-fiber muscles (cm), longitudinal-fiber muscles (lm), inclusion bodies (ib) and nuclei (n) of subtegumental cells can be seen. Bar = 1 mm.

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the presence of 0.1 U/ml of chondroitinase AB (Sigma) at pH 8.0. All of these treatments were carried out at 37 8C with agitation (De Souza et al., 1989). Subsequently, the larvae were processed for detection of anionic sites. 4. Results 4.1. General morphology The S. mansoni cercariae were analyzed by means of a routine procedure for transmission electron microscopy (Fig. 1). It was shown that the cercariae presented thorns that were easily observed on the body as well as on the tail. On the cercaria tegument, the basal lamina (bl) and a group of circular-fiber muscles (cm) and longitudinal-fiber muscles (lm) were found consecutively. Electrondense structures called cercarial bodies could also be seen on the tegument (Fig. 1). The inclusion bodies could present spherical, elongated or discoid shape and generally displayed a central electrondense region and a translucent peripheral region (Fig. 1).

4.2. Location of basic proteins (EPTA and ammoniacal silver) Basic proteins were detected by using the EPTA technique and ammoniacal silver. The material was processed in accordance with the EPTA and was incubated for a 24–72 h period in 2% PTA in pure ethanol. The ethanolic phosphotungstic acid technique enabled identification of basic proteins present on the tegument (Fig. 2A), nuclei and subtegumental cell nucleoli (Fig. 2B and C), inclusion bodies (not shown) and preacetabular glands (Fig. 2D). We identified strong staining on all infective larvae, after using the ammoniacal silver technique (Fig. 3A and B). We also demonstrated staining in the nuclei of muscle cells (not shown), circular muscle tissue (Fig. 3C) and preacetabular glands (Fig. 3D). 4.3. Calcium site location By using the Hepler technique (1980), which uses CaCl2 in glutaraldehyde, post-fixed in osmium tetroxide with potassium ferricyanide, we identified the presence of cellular sites containing calcium. Staining was shown inside all of the cercariae. The distribution of the staining showed the presence of calcium

Fig. 2. (A–D) Transversal sections through S. mansoni cercariae subjected to EPTA, to detect basic proteins. (A) EPTA staining over whole tegument. (B and C) Stained nuclei (N) and nucleoli of subtegumental cells. (D) Stained basic protein (arrows) from preacetabular glands. Bars in (A) and (C) = 0.5 mm, bars in (B) and (D) = 1 mm.

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Fig. 3. (A–D) Transversal sections through S. mansoni cercariae processed using the ammoniacal silver technique. Staining all over infective larvae (arrows) (A and B), presence of basic proteins (arrows) in circular muscle tissue (mc) (C) and in preacetabular glands (arrows) (D). Bar in (A) = 0.5 mm and bars (B–D) = 2 mm.

between larval compartments and indicated uniform distribution among the inner spaces of infective larvae (Fig. 4A and B).

treatment with chondroitinase (Fig. 5C) or trypsin (Fig. 5D) resulted in absence of staining on the infective larval surface.

4.4. Enzyme treatment

5. Discussion

Samples treated with cationized ferritin showed good preservation of internal structures and strong staining at the cuticular level and presented uniform staining on all surface (Fig. 5A). Sample treatment with neuraminidase followed by incubation with cationized ferritin particles did not alter the staining pattern of these particles on the trematode surface (Fig. 5B). However,

The present study used two cytochemical techniques (EPTA and ammoniacal silver) to detect basic proteins at the ultrastructural level. Although both methods detect basic proteins, they have different specificities: EPTA reveals proteins rich in histidines, while ammoniacal silver reveals arginine and lysine (De Souza, 1998).

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Fig. 4. (A and B) Transversal sections through S. mansoni cercariae processed using the Hepler technique. Staining (arrows) was evident through the interior of the cercariae. Distribution shows the presence of calcium between compartments of the infective larvae. Bar = 2 mm, TEM.

Fig. 5. (A) S. mansoni cercariae processed in order to detect anionic sites. Uniform staining is observed on the surface (arrows). (B) Neuraminidase treatment allowing bonding with cationic particles (arrows). (C) Chondroitinase AB and (D) trypsin, completely abolishing the bonding with cationic ferritin particles. Bar = 1 mm.

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Using the EPTA technique, we identified the presence of basic proteins in the tegument, nuclei and nucleoli of subtegumental cells, inclusion bodies and acetabular glands. Although EPTA detects proteins rich in histidines, these are compromised through a series of cellular functions such as cell differentiation and gene function regulation in eukaryotic cells. Thus, locating such proteins in the nuclei and nucleoli of subtegumental cells can only confirm the reaction specificity. According to Dorsey et al. (2002), the inclusion bodies present in the cytoplasm of cercarial tegument originate from the Golgi complex and are responsible for forming the external heptalaminar membrane. Since this membrane presents proteins in its constitution, it is possible that many of them originate from inclusion bodies. Studies conducted by Stirewalt (1959) showed that the preacetabular glands present secretions that are important in relation to the penetration of cercariae into the host. These secretions have proteolytic action. According to our results, the EPTA staining in these glands indicates the presence of basic proteins rich in histidines that may be associated with the host penetration process. We identified strong staining after using the ammoniacal silver method, all over the infecting larvae. We also demonstrated staining in muscle cell nuclei, in circular muscle tissue and, just as seen with EPTA, in preacetabular glands. Calcium ions play an important role in many cellular activities and are involved in muscle contraction, depolarization, secretion, activation of microtubules and microfilaments, endocytosis and exocytosis (De Souza, 1998). Despite the existence of some studies on the presence of calcium in cercaria, none of these studies attempted an ultrastructural approach. Elucidating the calcium storage locations in infective larvae at the ultrastructural level would be of extreme importance, because this would show in detail the ion distribution. From this, correlations between ‘‘in loco’’ locations and function will become possible. In our study, the presence of calcium in all internal regions of the cercariae was evident. According to the staining, the ions are distributed in regular shapes, and seem to limit the inner spaces of the infective larvae. Calcium was also shown in the preacetabular glands (data not shown). Several studies using histochemical techniques have managed to detect calcium in preacetabular glands (Gordon and Griffith, 1951; Stirewalt, 1959). According to Dresden and Asch (1977), calcium found in these glands is combined forming calcium carbonate. This finding is based on gland secretion analysis and on physicochemical analysis of the cercaria. It has been indicated that the enzyme secretion complex of S. mansoni cercariae is responsible for histological and histochemical changes in cornea extracts and in acellular connections from the host tissue barriers during the passage through the skin (Gordon and Griffith, 1951; Stirewalt, 1959). There is much evidence that some of these enzymes are present in the preacetabular glands and may have proteolytic action (Stirewalt, 1959). Other studies have brought the possibility that calcium ions may act as enzyme activators in this process. The selective staining of preacetabular glands by alizarin red S (ARS) suggests that calcium ions are present in this gland. Cytochemical detection of calcium ions using the Schiff base glyoxal-bis (2-hydroxyanil) (GBHA) has shown the presence of these ions in preacetabular glands and in S. mansoni ducts. This latter method is even more specific for calcium ion detection than ARS is (Lewert et al., 1966). Examination of the material released from S. mansoni cercariae following stimulation confirms that there are physical and histochemical differences in composition between pre and

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postacetabular glands. This difference in gland content reflects their difference in function, since postacetabular glands play adhesive, protective and enzyme-stimulating roles, in contrast to the lithic function of the contents released by preacetabular glands (Lewert et al., 1966). Dresden and Edlin (1975) confirmed from physicochemical methods that the cercariae present an average of 10–15 ng of calcium. According to Lewert and Lee (1954), calcium and magnesium present functions similar to those of coenzymes or lithic enzyme activators in relation to gland secretion. Moreover, there is an in vitro correlation between calcium, magnesium and zinc ion effects on the enzyme activity of S. mansoni cercariae, and an in vivo effect on the function of these ions during penetration. It was noticed in the present study that the success of cercariae in penetrating the host depends on the ion concentrations in water. This finding may partially explain the differences in infection rates found between different laboratories. Murrell et al. (1983) associated the presence of negatively charged groups on the surfaces of helminths with protection against dissection. On the other hand, it is possible that such groups activate complementary systems via the Hageman factor or VII coagulation factor, consequently triggering inflammatory reactions. Using the cationized ferritin at pH 7.2 technique, we observed that the anionic sites were associated with locations in the external layer of the larvae. This result is similar to what has been observed with regard to another three helminths: Wuchereria bancrofti L3 (Silva et al., 2006), and Strongyloides ratti and Trichinella spirallis (Murrell et al., 1983). Sample treatment with neuraminidase, followed by incubation with cationized ferritin, did not alter the staining pattern of these particles on the trematode surface. Our results using highspecificity neuraminidase indicate that carboxyl groups are not involved in surface charges in S. mansoni cercariae. These results are in agreement with the findings of Himmelhoch and Zuckerman (1978), who studied Caenorhabditis briggsae. Trypsin treatment resulted in absence of staining. This observation indicates that, differing from what is found in other biological systems, glycoproteins sensitive to trypsin are expected to contribute significantly towards negative surfaces in cercariae (De Souza et al., 1989). Our results also indicate that in vivo cercariae treatment with AB chondroitinase, which has glycosaminoglycan chondroitin-4sulfate (A) and dermatan sulfate (B) specificity, inhibits bonding with cationic ferritin particles. This observation suggests that glycosaminoglycans are present in the tegument of S. mansoni cercariae. However, biochemical studies in greater depth are needed to confirm this cytochemical observation. Based on the analysis of basic proteins, calcium and anionic groups from S. mansoni cercariae, we were able to conclude that the locations of these compounds are strongly related to large numbers of larval functions, going from movement to reach their hosts to the survival of cercariae within the environment. Acknowledgements This study was developed at the Aggeu Magalha˜es Research Center, Oswaldo Cruz Foundation (FIOCRUZ), National Council for Science and Technology (Conselho Nacional Cientı´fico e Tecnolo´gico, CNPq) and ‘‘Keizo Asami’’ Immunopathology Laboratory. References Bloom, F.E., Aghajanian, G.K., 1968. Fine structure and cytochemical analysis of the staining of synaptic junctions with phosphotungstic acid. J. Ultrastr. Res. 22, 361–375.

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