Acta Tropica 106 (2008) 137–142

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Ecto-phosphatase activity on the external surface of Rhodnius prolixus salivaryglands: Modulation by carbohydrates and Trypanosoma rangeli

Suzete A.O. Gomesa, Andre L. Fonseca de Souzab, Tina Kiffer-Moreirab,Claudia F. Dickb, Andre L.A. dos Santosb, Jose R. Meyer-Fernandesb,∗

a Setor de Morfologia, Ultraetrutura e Bioquımica de Artropodes e Parasitos, Laboratorio de Transmissores de Leishmaniose.Instituto Oswaldo Cruz/FIOCRUZ, Av. Brasil 4365, Manguinhos, Rio de Janeiro, RJ, 22045900, Brazil

b Laboratorio de Bioquımica Celular, Instituto de Bioquımica Medica (IBqM), Bloco H, Centro de Ciencias da Saude (CCS), Universidade Federal doRio de Janeiro (UFRJ), Av. Brigadeiro Trompowsky s/n, Ilha do Fundao, Rio de Janeiro, RJ 21941-590, Brazil

ct’s vet vecte invs arecto-eT. ranhenovaria

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a r t i c l e i n f o

Article history:Received 28 September 2007Received in revised form 26 February 2008Accepted 28 February 2008Available online 6 March 2008

This work is dedicated to Leopoldo De Meison his 70th birthday.

Keywords:Rhodnius prolixusSalivary glandsEcto-phosphataseTrypanosoma rangelid-Galactosed-Mannose

a b s t r a c t

The salivary glands of insenius prolixus an importanthe present study we havglands. Ecto-phosphataseWe characterized these eto investigate R. prolixus–(4.30 ± 0.35 nmol p-nitropity was not affected by pHapproximately 50% by inophosphatase activity detecsurface efficiently releaserate of phosphate releasewas inhibited by carbohyinhibited salivary glands

epimastigote forms induced a lactivity. Interestingly, boiled lonphosphatase activity. Taken togprolixus salivary glands–T. rang

1. Introduction

Triatomines of the Rhodnius genera present a pair of salivaryglands that contains two close and independent units: one largerand elongated (principal) and other one smaller and translucent(accessory). Both gland units are formed by a single layer of epithe-lial gland cells, surrounded by a thick basal lamina (Meirelles et al.,2003).

Rhodnius prolixus an important vector of Trypanosoma cruzi,the ethiologic agent of Chagas’ disease, is also the vector for Try-panosoma rangeli (Anez, 1983), which is apparently nonpathogenicto humans, but can be pathogenic to triatomines (Watkins, 1971).T. rangeli develops as short epimastigotes in the gut of its insectvector, crossing from the intestine lumen to the hemocoel and

∗ Corresponding author. Fax: +55 21 25626781.E-mail address: meyer@bioqmed.ufrj.br (J.R. Meyer-Fernandes).

0001-706X/$ – see front matter © 2008 Elsevier B.V. All rights reserved.doi:10.1016/j.actatropica.2008.02.008

ctors are target organs to study the vectors–pathogens interactions. Rhod-or of Trypanosoma cruzi can also transmit Trypanosoma rangeli by bite. Inestigated ecto-phosphatase activity on the surface of R. prolixus salivaryable to hydrolyze phosphorylated substrates in the extracellular medium.nzyme activities on the salivary glands external surface and employed itgeli interaction. Salivary glands present a low level of hydrolytic activityl (p-NP) × h−1 × gland pair−1). The salivary glands ecto-phosphatase activ-tion; and it was insensitive to alkaline inhibitor levamisole and inhibitedc phosphate (Pi). MgCl2, CaCl2 and SrCl2 enhanced significantly the ecto-n the surface of salivary glands. The ecto-phosphatase from salivary glandssphate groups from different phosphorylated amino acids, giving a higher

phospho-tyrosine is used as a substrate. This ecto-phosphatase activitys as d-galactose and d-mannose. Living short epimastigotes of T. rangeliphosphatase activity at 75%, while boiled parasites did not. Living longower, but significant inhibitory effect on the salivary glands phosphataseg epimastigote forms did not loose the ability to modulate salivary glandsether, these data suggest a possible role for ecto-phosphatase on the R.

eli interaction.

© 2008 Elsevier B.V. All rights reserved.

from this compartment to the salivary gland lumen, a criticalstep of the life cycle of T. rangeli in the invertebrate host (Heckeret al., 1990). Meirelles et al. (2005) demonstrated a high num-ber of parasites, mostly long epimastigotes, adhered to the outersurface (basal lamina) of the salivary glands, suggesting that a pos-sible pore-forming protein (PFP) could be used by the parasites toreach the salivary gland lumen. Once in the lumen the parasitesdifferentiate into metacyclic trypomastigote forms, which are capa-ble of infecting the vertebrate host during the blood meal (Anez,1983).

Several studies indicated the presence of specific moleculeson the basal lamina of insects’ salivary glands (Rosenberg, 1985;Billingsley and Sinden, 1997; Arrighi and Hurd, 2002). Salivaryglands of R. prolixus are highly glycosylated and many lectinsbound to these glands indicate the presence of surface-associatedsugars. The surface of most of parasites presented lectins or lectins-like molecules that recognize specific carbohydrates and mediatethe adhesion or invasion to or into the host cells (Basseri et

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al., 2002). Invasion of salivary glands by parasites seems to bemediated by specific receptor–ligand interactions (Basseri et al.,2002).

Ecto-phosphatases may exert their function by dephosphory-lating surface proteins, whose phosphorylation can be mediatedby ecto-protein kinases, a phenomenon that has been widelydescribed in many organisms (Goding, 2000). Previous data of ourgroup showed that ecto-phosphatases may regulate the interac-tion of fungal with host cells, being considered an essential step ininfection process and adhesion to mammalian cells (Collopy-Junioret al., 2006). Recently, an ecto-phosphatase was localized on theexternal surface of Malpighian tubules basal membranes (Ferraroet al., 2004).

In the present work, we have characterized an ecto-phosphataseactivity on the external surface of salivary glands of R. prolixus anddemonstrated its modulation by carbohydrates and epimastigoteforms of T. rangeli.

2. Material and methods

2.1. Insects

All the insects used in this experiment were adult males of R.prolixus originating from a colony raised and maintained at 28 ◦C,70% relative humidity and fed on rabbit blood.

2.2. Isolation of salivary glands of R. prolixus

Intact salivary glands were collected carefully by cutting offthe head of the insects and immediately immersed in a cold solu-tion containing (100 mM NaCl; 8.6 mM KCl; 2.0 mM CaCl2; 8.5 mMMgCl2; 34 mM glucose; 2.4 mM alanine; 20 mM TRIS–HEPES pH 7.0)modified from Maddrell’s buffer (Maddrell, 1969). Cellular integritywas assessed in all conditions by measuring the lactate dehydroge-nase activity (Ferraro et al., 2004).

2.3. Parasites

T. rangeli H14 strain (CT-IOC 038, supplied by Dr. M.A. Sousa,Colecao de Tripanossomatıdeos, Fiocruz, Brazil) was grown at 28 ◦Cin liver infusion tryptose (LIT) medium (DIFCO) supplemented with20% heat-inactivated fetal bovine serum. The short epimastigotes(99% purity) were obtained from log-growth phase of the parasites(until 7 days of culture) and long epimastigotes of T. rangeli (>98%

purity) originated from later stationary growth phase after 12 daysof culture (Gomes et al., 2006).

2.4. Phosphatase activity on salivary glands surface

Intact salivary glands were incubated for 1 h at 25 ◦C ina reaction medium (final volume: 0.2 mL) containing 50 mMTris–HCl buffer, pH 7.2, 20 mM KCl, 100 mM sucrose and 5 mMp-nitrophenylphosphate (p-NPP) as substrate unless otherwisestated in the legends. The phosphatase activity was determinedmeasuring the rate of p-nitrophenol (p-NP) production from thep-nitrophenylphosphate used as substrate. After incubation, thereaction was interrupted by the addition of 0.4 mL NaOH 1N andthe concentration of p-NP released by the phosphatase action wasverified by the comparison with a p-NP standard curve measuredat wavelength of 405 nm using an ELISA spectrophotometer (DeAlmeida-Amaral et al., 2006). We also tested phosphorylated aminoacids, P-serine, P-threonine and P-tyrosine, as phosphatase sub-strates. In this case, after incubation time, the reaction was addedto 0.4 mL 0.1 N HCl. The tubes were centrifuged at 1500 × g for10 min and 0.2 mL aliquots of supernatant were used to measure

ica 106 (2008) 137–142

the released inorganic phosphate (Pi) from these substrates by Fiskeand Subbarow (1925) method and Pi concentration was verified bythe comparison with a Pi standard curve. The phosphatase activ-ity was calculated by subtracting the nonspecific p-NPP hydrolysismeasured in the absence of the glands. Ecto-phosphatase activ-ities were expressed as nmol p-NP × h−1 × gland pair−1 or nmolPi × h−1 × gland pair−1, for the phosphoaminoacids assays.

2.5. T. rangeli interaction assays

Intact salivary glands were incubated in the presence or absenceof living or disrupted by heating short or long T. rangeli epi-mastigotes (1 × 107 cells), in eppendorff tubes at 25 ◦C for 60 min.Then the tubes were washed three times with buffer solution(50 mM Tris–HCl, pH 7.2, 20 mM KCl and 100 mM sucrose) sali-vary glands free parasites –were incubated in a reaction medium asdescribed before. Salivary glands’ viability was assessed after wash-ing by measurement of lactate dehydrogenase activity as describedbefore.

2.6. Reagents

Phosphatase inhibitors were purchased from Merck (Darm-staldt, Germany); p-NPP and all of the carbohydrates were obtainedfrom Sigma Chemical Co. (Saint Louis, MO, USA). All other reagentswere analytical grade.

2.7. Statistical analysis

All experiments were performed in triplicate, with similarresults obtained from at least three separate cell suspensions. Sta-tistical significance was determined by Student’s t-test. Significancewas considered as P < 0.05. Data from pH curve were analyzed bymeans of ANOVA One Way followed by the Tukey test using thePrism computer software (Graphpad Software Inc., San Diego, CA,USA).

3. Results

3.1. Salivary glands ecto-phosphatase activity characterization

Salivary glands of R. prolixus, whose integrity was assessedbefore and after the reactions by measure lactate dehydrogenaseactivity on the supernatant (data not shown) present an ecto-

phosphatase activity at a rate of 4.3 ± 0.35 nmol p-NP × h−1 × glandpair−1. The time course of phosphatase activity measured on thesurface of glands was linear for at least 60 min (r2 = 0.9692). Simi-larly, in assays to determine the influence of number of glands pairs,the phosphatase activity measured over 60 min was linear over anearly six-fold of number of glands pairs (r2 = 0.9596) (data notshown). Phosphatase activity was measured in the pH range from6.0 to 8.5. The phosphatase activity was not affected by pH variation(Fig. 1). In order to check the possibility that the p-NPP hydrolysiswas the result of a cytosolic contamination from disrupted cells,we prepared a reaction mixture with salivary glands that wereincubated in the absence of p-NPP. Subsequently, the suspensionwas centrifuged to remove salivary glands and the supernatant waschecked for phosphatase activity. This supernatant failed to showp-NPP hydrolysis (data not shown). These results also rule out thepossibility that the phosphatase activity here described could befrom disrupted salivary glands.

The phosphatase from salivary glands surface showed a moresignificant hydrolysis of P-tyrosine at a rate of 10.32 ± 0.46 nmolPi × h−1 × gland pair−1 in comparison with P-serine and P-threonine, 5.28 ± 0.37 and 5.21 ± 0.33 nmol Pi × h−1 × gland pair−1,

S.A.O. Gomes et al. / Acta Tropica 106 (2008) 137–142 139

Fig. 2. Substrate specificity of Rhodnius prolixus salivary glands ecto-phosphataseactivity. Three pairs of salivary glands were incubated at 25 ◦C for 1 h in a reactionmixture (0.2 mL) containing 100 mM sucrose, 20 mM KCl, 50 mM Tris–HCl, pH 7.2and 5 mM of each phosphoamino acids substrate: P-tyrosine (P-tyr) or P-serine (P-ser) or P-threonine (P-thr). The values represent the mean ± standard errors of threeindependent experiments expressed by one pair of glands.

Fig. 1. Effect of pH on the ecto-phosphatase activity of the salivary glands of R.prolixus. Three pairs of salivary glands were incubated at 25 ◦C in a reaction mix-ture (0.2 mL) containing 20 mM KCl, 100 mM sucrose, 5 mM p-NPP as substrate and100 mM MES–HEPES–citrate buffer adjusted to pH values between 6.0 and 8.5 for1 h. The salivary glands cells were intact during all the course of the experimentsin all conditions used. The values represent the mean ± standard errors of threeindependent experiments, which were performed in triplicate and expressed byone pair of glands. There was no statistically significant difference in phosphatase

activity between pH 6.0 and 8.5.

respectively (Fig. 2). In the Table 1, is shown that the ammo-nium molybdate an acid phosphatase inhibitor and levamisolean alkaline phosphatase inhibitor, failed to inhibit phosphataseactivity. Moreover, sodium vanadate, a phosphotyrosine phos-phatase inhibitor was able to inhibit the phosphatase activity in23% and inorganic phosphate, which is the natural product ofreactions catalyzed by phosphoprotein phosphatases, inhibitedapproximately 50% the phosphatase activity. Sodium tartrate, aninhibitor of secreted phosphatases was not capable to influenceecto-phosphatase activity. Several divalent cations and metal chela-tors were tested and the results are shown in Table 2. MgCl2,CaCl2 and SrCl2 enhanced significantly the ecto-phosphatase activ-ity detected on surface of salivary glands. On the other hand, EDTA,ZnCl2 and CuCl2 had no significant effects on the salivary glandsecto-phosphatase activity (Table 2). We also investigated the effectsof carbohydrates on the salivary glands ecto-phosphatase activity.The results showed that glands phosphatase activity was inhib-ited by galactose and mannose (46.3%) and no effect was observedin the presence of glucose and fructose (Fig. 3, panel A). The

Table 1Influence of different compounds on the phosphatase activity of intact salvary glandscells of Rhodnius prolixus

Addition Relative activity (%)

None 100 ± 8.2Levamisole (1 mM) 108.8 ± 13.7Sodium orthovanadate (1 mM) 77.0 ± 7.7*

Sodium fluoride (10 mM) 93.7 ± 12.0Ammonium molybdate (1 mM) 118.8 ± 11.9Sodium tartrate (10 mM) 107.4 ± 7.4Pi (10 mM) 44.2 ± 4.3*

The ecto-phosphatase activity was measured in the standard assay described underSection 2. Phosphatase activity is expressed as a percentage of that measuredunder control conditions, i.e., without other additions. The phosphatase activity ofthe salivary glands of Rhodnius prolixus (4.3 ± 0.35 nmol Pi × h−1 × gland pair−1 forphosphatase) was taken as 100%. The standard errors were calculated from abso-lute values of three experiments, performed in triplicate, expressed by one pair ofglands and converted to percentage of the control value. Asterisks denote signif-icant differences (p < 0.05) after comparison with the enzyme activity of control(none addition).

Fig. 3. (A) Effect of carbohydrates on the ecto-phosphatase activity of salivary glands of R. prolixus. Three pairs of salivary glands were incubated with different carbohydratesat 25 ◦C for 1 h in a reaction mixture (0.2 mL) containing 20 mM KCl, 100 mM sucrose, 50 mM Tris–HCl buffer pH 7.2, and 5 mM p-NPP. (B) Effects of increasing concentrationsof d-galactose on ecto-phosphatase of salivary glands of R. prolixus. Three pairs of salivary glands were incubated with different concentrations of d-galactose (5–25 mM)at 25 ◦C for 1 h in a reaction mixture (0.2 mL) containing 20 mM KCl, 100 mM sucrose, 50 mM Tris–HCl buffer pH 7.2, and 5 mM p-NPP. The glands were intact and viableduring all the course of the experiments in all conditions used. The values represent the mean ± standard errors of three independent experiments, which were performedin triplicate. Asterisks denote significant differences (p < 0.05) after comparison with the enzyme activity of control (none addition).

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Table 2Influence of different divalent cations on the phosphatase activity of intact salvaryglands cells of Rhodnius prolixus

Addition Relative activity (%)

None 100 ± 8.2EDTA (1 mM) 126.1 ± 18.8MgCl2 (5 mM) 159.8 ± 10.2*

CaCl2 (5 mM) 147.2 ± 9.3*

SrCl2 (5 mM) 156.5 ± 12.2*

ZnCl2 (5 mM) 135.6 ± 16.1CuCl2 (1 mM) 130.7 ± 18.1

The ecto-phosphatase activity was measured in the standard assay described underMaterials and methods. Phosphatase activity is expressed as a percentage of that

measured under control conditions. The phosphatase activity of the salivary glandsof Rhodnius prolixus (4.3 ± 0.35 nmol Pi × h−1 × gland pair−1 for phosphatase) wastaken as 100%. The standard errors were calculated from absolute values of threeexperiments, performed in triplicate, expressed by one pair of glands and were con-verted to a percentage of the control value. Asterisks denote significant differences(p < 0.05) after comparison with the enzyme activity of control (none addition).

inhibition of ecto-phosphatase activity by galactose occurred in adose-dependent manner (Fig. 3 panel B).

3.2. R. prolixus salivary glands –T. rangeli interaction assays

In vitro assays were carried out in order to investigate thesalivary glands phosphatase activity on the interaction betweenthe surface of R. prolixus salivary glands and short or long epi-mastigote forms of T. rangeli. The capacity of living and disruptedheating of short or long epimastigotes of T. rangeli to modulatethe salivary gland ecto-phosphatase activity was studied. Livingshort epimastigote forms induced an inhibitory effect (75%) in sali-vary glands ecto-phosphatase activity. However, short epimastigoteforms die by heating thereby loosing the ability to modulate thisecto-phosphatase activity (Fig. 4). On the other hand, living long

Fig. 4. Influence of Trypanosoma rangeli on ecto-phosphatase activity of R. prolixussalivary glands. Three pairs of salivary glands were incubated with live or heatedshort or long T. rangeli epimastigotes (1 × 107 cells), at 25 ◦C for 1 h in the inter-action medium containing 50 mM Tris–HCl buffer, pH 7.2, 20 mM KCl and 100 mMsucrose. After this time the salivary glands were removed (free of parasites) and incu-bated at 25 ◦C for 1 h in a reaction mixture (0.2 mL) containing 20 mM KCl, 100 mMsucrose, 50 mM Tris–HCl buffer pH 7.2, and 5 mM p-NPP. Black bar: salivary glandscontrol; gray bar: living short epimastigotes plus salivary glands; hatched gray bar:disrupted by heating short epimastigotes plus salivary glands; white bar: living longepimastigotes plus salivary glands; hatched white bar: disrupted by heating longepimastigotes plus salivary glands. Data are means ± standard errors of three inde-pendent experiments, which were performed in triplicate and expressed by one pairof glands. Asterisks denote significant differences (p < 0.05) after comparison withthe enzyme activity of control (none parasite interaction).

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epimastigote forms induced a significant lower inhibitory effect onthis phosphatase activity. Interestingly, long epimastigote formsdisrupted by heating did not loose the ability to modulate thisecto-phosphatase activity (Fig. 4).

4. Discussion

Phosphatases are fundamental components in cellular eventsregulated by phosphorylation–dephosphorylation systems, whichare coordinately controlled by the concerted action of proteinkinases and phosphoprotein phosphatases (Hunter, 1995). In spiteof the above, ecto-phosphatases have been characterized in severalunicellular organisms, such as the parasitic protozoan Leishma-nia donovani (Gottlieb and Dwyer, 1981), Trypanosoma brucei(Fernandes et al., 1997), T. cruzi (Furuya et al., 1998), T. rangeli(Gomes et al., 2006), Herpetomonas muscarum muscarum (Dutraet al., 1998), Phytomonas spp. (Dutra et al., 2000), Herpetomonassamuelpessoai (Santos et al., 2002), Crithidia deanei (Lemos et al.,2002), Entamoeba histolytica (De Sa Pinheiro et al., 2007), Tri-chomonas vaginalis (De Jesus et al., 2002a) and Tritrichomonasfoetus (De Jesus et al., 2006) only recently this class of membraneproteins in insects has become a subject of interest (Ferraro etal., 2004). In insects, phosphatases were thought to be restrictedto the internal cells or inside vacuoles and the presence of anecto-phosphatase has never been described. Recently, this kind ofecto-enzyme was described on the surface of Malpighian tubulesof R. prolixus (Ferraro et al., 2004).

In fungi cells, as Fonsecaea pedrosoi was demonstrated that cellsexpressing high ecto-phosphatase activity were significantly morecapable to adhere to epithelial cells than fungi that expressedlow levels of ecto-phosphatase activity (Kneipp et al., 2004). Theecto-phosphatase activity also mediates the adhesion of fungi,Cryptococcus neoformans to animal epithelial cells (Collopy-Junioret al., 2006). In T. rangeli life cycle, a critical step is the adhe-sion/invasion of long epimastigote forms to the outer surface ofR. prolixus salivary glands. In this context, we characterized theecto-phosphatase profile on the surface of salivary glands from R.prolixus and investigated the possible role of this enzyme in thevector–parasite interaction.

In this work, we observed that the ecto-phosphatase activitywas not changed by the increase of pH (Fig. 1). This result wassupported by the absence of inhibition by levamisole, an alka-line phosphatase inhibitor (Van Belle, 1976) and by ammoniummolybdate, an acid phosphatase inhibitor (De Almeida-Amaral

et al., 2006). Sodium tartrate, a secreted phosphatases inhibitor(Meyer-Fernandes et al., 1999), did not influence salivary glandsecto-phosphatase activity (Table 1). In addition, the supernatantof glands was not able to hydrolyze p-NPP (data not shown).Taken together, these results suggest that phosphatase activityis located at membrane surface not secreted to the extracellu-lar medium. From all classical inhibitors of phosphatses, onlysodium orthovanadate and Pi (natural product of phosphatasereaction) caused phosphatase inhibition (23% and 50%, respec-tively) on salivary glands ecto-phosphatse activity (Table 1). Thephosphatase activity was stimulated by MgCl2, CaCl2 and SrCl2and was not influenced by EDTA (Table 2) suggesting that thisform may belong to protein-serine/threonine phosphatases fam-ily PP2B/PP2C (Mumby and Walter, 1993). However, experimentscarried out using phosphoamino acids as substrates demonstrateda higher phosphotyrosine phosphatase activity than phosphoser-ine or phosphothreonine hydrolysis (Fig. 2). These results andphosphatase inhibition by sodium orthovanadate together can berelated to the phosphotyrosine phosphatase (PTP) family. Membersof the PTP superfamily are subdivided by the presence of a signaturemotif, H–C–X–X–G–X–X–R; they can be further subdivided as clas-

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sical PTPs or dual-specificity phosphatases (DSPs), this later beingable to hydrolyze phosphorylated tyrosine, serine and threonineresidues (Tonks and Neel, 2001).

Short and long epimastigotes of T. rangeli were able to modulatethe salivary glands ecto-phosphatase. Gomes et al. (2006) describeddifferential expression of cell surface polypeptides of short andlong epimastigote forms of T. rangeli. The salivary glands ecto-phosphatase modulation by short forms could be due the presenceof some surface protein component once boiled short epimastig-otes forms loosed the ability to modulate glands phosphataseactivity (Fig. 4). Long forms showed a lower ability to modulatesalivary glands ecto-phosphatase activity (Fig. 4) probably due tothe difference between surface components. Interestingly, boiledlong epimastigote forms did not loose the ability to modulate sali-vary glands phosphatase activity, corroborating the data obtainedby Basseri et al. (2002), that showed a role for carbohydrates, d-galactose residue specially, associated to the adhesion events. Aslong epimastigote forms are the parasite stages that invade salivaryglands in vivo, we may suggest that modulation of salivary glandsecto-phosphatase activity by long epimastigote forms could involveinteraction sites in parasites and salivary glands with d-galactoseand specific lectin-receptors.d-Galactose exposed on the surface of host cells is involved

on several pathogens adhesions (De Jesus et al., 2002b; Jesuset al., 2002). Sugar residues were observed acting as recogni-tion sites for invasion of malarial parasites in mosquito midgut(Ramasamy et al., 1997). Carbohydrate-binding protein (CBP), lectinor lectin-like molecules, which apparently have a role in the attach-ment of the parasite to host cells, are found on T. cruzi andT. rangeli surface (Bonay and Fresno, 1995; Bonay et al., 2001).Basseri et al. (2002) reported diversity in carbohydrate residuesand lectins-like molecules present on R. prolixus salivary glandssurface. T. rangeli–R. prolixus salivary glands interaction involvesrecognition between d-galactose/N-acetyl-d-galactosamine and itsspecific lectins receptors (Basseri et al., 2002). Our results showed asignificant inhibitory effect (46.3%) on ecto-phosphatase of R. pro-lixus salivary glands by d-mannose and d-galactose (Fig. 3 panelA). d-Galactose inhibited this activity in a dose-dependent manner(Fig. 3, panel B). As mannose residues are probably not involved inT. rangeli–R. prolixus salivary glands interaction, the effects of thissugar on salivary glands ecto-phosphatase activity remain to beelucidate.

In conclusion, the detection and characterization of ecto-phosphatases activities present on the outer surface of cell

membranes of targets organs of insects, as salivary glands, arousea particular interest since they can also mediate vector–parasiteinteractions and/or reception and transduction of external stimuli,which include phosphorylation and dephosphorylation of proteins(Dutra et al., 2006). The role of ecto-phosphatase on the recognitionmechanism remains under investigation.

Acknowledgements

We thank Dr. Hatisaburo Masuda for kindly providing theinsects, R. prolixus and Fabiano Ferreira Esteves for excellent tech-nical assistance.

This study was supported by grants from Fundacao de Amparoa Pesquisa do Estado do Rio de Janeiro (FAPERJ) and ConselhoNacional de Desenvolvimento Cientıfico e Tecnologico (CNPq).

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