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Arthropod Structure & Development 38 (2009) 31–44

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Arthropod Structure & Development

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Cytochemical characterization of microvillar and perimicrovillarmembranes in the posterior midgut epithelium of Rhodnius prolixus

Jose M. Albuquerque-Cunha a, Marcelo S. Gonzalez b,*, Eloi S. Garcia c, Cıcero B. Mello b,Patrıcia Azambuja c, Joao C.A. Almeida d, Wanderley de Souza e, Nadir F.S. Nogueira a

a Laboratorio de Biologia Celular e Tecidual, Centro de Biociencias e Biotecnologia, Universidade Estadual do Norte Fluminense,Avenida Alberto Lamego, 2000, Horto, Campos dos Goytacazes, Rio de Janeiro, CEP 28.015-620, Brazilb Laboratorio de Biologia de Insetos, Departamento de Biologia Geral, Instituto de Biologia, Universidade Federal Fluminense,Morro do Valonguinho S/N

�, Centro, Niteroi, Rio de Janeiro, CEP 24.001-970, Brazil

c Laboratorio de Fisiologia e Bioquımica de Insetos, Instituto Oswaldo Cruz, Fiocruz, Av. Brasil, 4365, Rio de Janeiro, CEP 21045-900, Brazild Laboratorio de Fisiologia e Bioquımica de Microrganismos, Centro de Biociencias e Biotecnologia, Universidade Estadual do Norte Fluminense,Avenida Alberto Lamego, 2000, Horto, Campos dos Goytacazes, Rio de Janeiro, CEP 28.015-620, Brazile Laboratorio de Ultraestrutura Celular Hertha Meyer, Instituto de Biofısica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro,Avenida Brigadeiro Trompowsky s/n. CCS, Bloco G-SS, Ilha do Fundao, Rio de Janeiro, CEP 21949-900, Brazil

a r t i c l e i n f o

Article history:Received 26 June 2007Accepted 1 June 2008

Keywords:Rhodnius prolixusCytochemistryPerimicrovillar membranes and peritrophicmatrixSurface anionic sitesCarbohydrate-binding molecules

* Corresponding author. Tel.: þ55 21 26292285; faxE-mail address: marcelo@ioc.fiocruz.br (M.S. Gonz

1467-8039/$ – see front matter � 2008 Elsevier Ltd.doi:10.1016/j.asd.2008.06.001

a b s t r a c t

Perimicrovillar membranes (PMM) are structures present on the surface of midgut epithelial cells of thehematophagous insect, Rhodnius prolixus. They cover the microvilli and are especially evident 10 daysafter blood meal, providing the compartmentalization of the enzymatic processes in the intestinalmicroenvironment. Using an enzyme cytochemical approach, Mg2þ-ATPase and ouabain-sensitiveNaþKþ-ATPase activities were observed in the plasma (or microvillar) membrane (MM) of midgut cellsand in the PMM. In contrast, alkaline phosphatase was only detected in MM. Using cationized ferritinand colloidal iron hydroxide particles, anionic sites were found only on the luminal surface of the PMM.Using fluorescein isothiocyanate (FITC)-labeled lectins, residues of a-D-galactose, mannose, N-acetyl-neuraminic acid, N-acetyl-D-galactosamine and N-acetyl-galactosamine-a-1,3-galactose were detectedon the apical surface of posterior midgut epithelial cells. On the other hand, using FITC-labeled neo-glycoproteins (NGP) it was possible to detect the presence of carbohydrate binding molecules (CBM)recognizing N-acetyl-D-galactosamine, a-D-mannose, a-L-fucose and a-D-glucose in the posterior midgutepithelium. The use of digitonin showed the presence of sterols in the MM and PMM. These resultshave led the authors to suggest that for some components the PMM resembles the MM liningthe midgut cells of R. prolixus, composing a system which covers the microvilli and stretches to theluminal space.

� 2008 Elsevier Ltd. All rights reserved.

1. Introduction

The intestines of many hemipterans have no true peritrophicmatrix (Peters, 1992; Jacobs-Lorena and Oo, 1996; Shao et al.,2001; Terra, 2001; Silva et al., 2004) but there is a system com-posed of extracellular membrane layers (Billingsley and Downe,1983) exerting a similar function, also termed perimicrovillarmembranes (PMM) by Terra (1988). On the apical surface of themidgut epithelium of hematophagous triatomines, the PMM coverthe microvillar membrane (MM) of the epithelial cells, connectingtheir microvilli and projecting themselves towards the intestinal

: þ55 21 2629 2376.alez).

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lumen (Lane and Harrison, 1979; Andries and Torpier, 1982; Bill-ingsley and Downe, 1986a). Previous studies have shown that10 days after a blood meal the PMM reach their maximum de-velopment in Triatoma infestans and Rhodnius prolixus (Burgos andGutierrez, 1976; Billingsley and Downe, 1983, 1986a,b; Nogueiraet al., 1997), two important species which are involved in thetransmission of Chagas disease (Garcia and Azambuja, 1991;Kollien and Schaub, 2000; Azambuja et al., 2005). Furthermore,Gutierrez and Burgos (1978) demonstrated that the PMM of T.infestans are composed of proteins and phospholipids and showa plexiform aspect. It has been suggested that the PMM areformed by lipoproteins that leave the endoplasmic reticulum ofthe epithelial cells and form double membrane vesicles seenbudding from some Golgi areas. These reach the midgut cell

J.M. Albuquerque-Cunha et al. / Arthropod Structure & Development 38 (2009) 31–4432

apices where they are secreted into the luminal space (Lane andHarrison, 1979; Andries and Torpier, 1982; Billingsley and Downe,1988; Silva et al., 1995).

The hydrolytic events take place in three distinct midgut com-partments of R. prolixus: the lumen, perimicrovillar space (limitedby PMM and MM) and lysosomes. It is possible that these spacesconstitute a physical or physiological barrier to enzymes, exertinga role similar to that played by true peritrophic matrix whichcompartmentalizes the digestive process in most insect orders(Billingsley and Downe, 1983, 1985; Terra, 1988; Ferreira et al.,1988; Silva et al., 1995; Terra et al., 2006). Furthermore, PMMplayed an essential role in heme crystallization and alsodetoxification in the midgut of R. prolixus (Oliveira et al., 2005; Silvaet al., 2006). It has been well established that the PMM are involvedin the process of interaction of Trypanosoma cruzi – a protozoanparasite responsible for Chagas disease (Chagas, 1909, 1911) – withthe intestinal epithelium of R. prolixus, a process fundamental for itsreplication (Isola et al., 1981, 1986; Burgos et al., 1989; Kollien et al.,1998; Gonzalez et al., 1999). Indeed, the disorganization of thePMM obtained by insect decapitation or azadirachtin treatmentblocked both the assembly of the PMM and the development of theprotozoan in R. prolixus (Garcia et al., 1989; Nogueira et al., 1997;Gonzalez et al., 1998, 1999).

Despite the biological importance of the PMM, little is knownabout its structure or composition (Ferreira et al., 1988; Silva et al.,1996; Terra et al., 2006). Therefore, the authors decided to usea cytochemical approach to localize sterol, membrane-associatedenzymes, carbohydrates and carbohydrate-binding molecules inthe MM and PMM of R. prolixus intestinal cells. The results obtainedare described in this paper.

2. Material and methods

2.1. Insects, feeding procedure and biological material

The specimens of Rhodnius prolixus (Hemiptera: Reduviidae)were reared and maintained at 28 � 2 �C and between 60 and 70%relative humidity in a BOD incubator. Six-month-old males wereseparated for the experiments just after the first blood meal postadult emergence. Ten days after human blood meal, using themembrane apparatus as previously described by Garcia et al.(1984), males were immobilized at 4 �C for 10 min and fixed ona polystyrene table with entomological pins. Fragments of the an-terior secretory segment of posterior midgut – located just after theanterior midgut or stomach (Billingsley and Downe, 1983; Schaub,1989) – were taken from the abdominal cavity of 10 males in bothcontrol and experimental groups, opened longitudinally to exposethe epithelium and microvilli and smoothly washed in a solution of0.9% NaCl. Approximately 100 thin sections from control and ex-perimental groups were stained and examined. Each thin sectionobserved contained 5–10 different cells depending on the magni-fication used. The results were only reported when more than 90%of the insects in each experimental group displayed the same ul-trastructural organization and/or labeling (Albuquerque-Cunhaet al., 2004).

2.2. Transmission electron microscopy

Tissue fragments of the posterior midgut obtained from controland experimental groups were fixed in 2.5% glutaraldehydein 0.1 M cacodylate buffer, pH 7.2, for 2 h at room temperature.After fixation, they were washed twice in 0.1 M cacodylate buffer,post-fixed in a solution of 1% osmium tetroxide in 0.1 M cacodylatebuffer, pH 7.2 for 1 h at room temperature in the dark. The tissuefragments were then dehydrated in acetone, infiltrated andembedded in Epoxy resin and polymerized at 60 �C for 3 days. Thin

sections were stained with uranyl acetate and lead citrate andobserved in a Zeiss 900 transmission electron microscope(Nogueira et al., 1997; Gonzalez et al., 1998).

2.3. Localization of sterol–digitonin complexes

Following previous observations made by Okros (1966, 1968),the posterior midgut fragments were washed with 0.9% NaCl andfixed for 2 h in a solution containing 4% paraformaldehyde, 1%glutaraldehyde and 0.16 mM of digitonin (Sigma Chemical Co.) in0.1 M sodium-cacodylate buffer, pH 7.2. Then, the tissue waswashed in cacodylate buffer, pH 7.2, post-fixed in 1% osmiumtetroxide containing 0.8% potassium ferricyanide and 2 mM CaCl2in 0.1 M cacodylate buffer, pH 7.2, for 2 h at room temperature.Digitonin was omitted from the control group. The tissue frag-ments were then washed in cacodylate buffer and processed forfreeze-fracture and transmission electron microscopy. In someexperiments, the tissue fragments were washed and slowlyinfiltrated with 40% glycerol solution in 0.1 M cacodylate bufferuntil reaching this final concentration. The midgut fragmentswere placed in a gold-device support and frozen in Freon 22�cooled by liquid nitrogen and then transferred to liquid nitrogen.Fracture was made in a Bal-Tec Freeze-Fracture device at �115 �Cand a vacuum of 2.0 � 10�7 bar. Replicas of platinum–carbonwere obtained, digested with 30% chromic acid and sodiumhypochlorite, washed several times in distilled water, collected on200 mesh copper grips and observed in a Zeiss 900 TEM (Okros,1966, 1968).

2.4. Localization of anionic groups

Following Houk et al. (1986), to localize anionic sites exposedat neutral pH, posterior midgut fragments were collected,washed in PBS, pH 7.2, and fixed with 2.5% glutaraldehyde in0.1 M sodium-cacodylate buffer, pH 7.2, for 2 h. After this thefragments were washed in PBS, and incubated for 30 min in thepresence of 50 mM ammonium chloride to block free aldehydegroups, then incubated for 15 min in a solution containing1.0 mg/mL of cationized ferritin in 0.1 M phosphate buffer, pH 7.2(Danon et al., 1972). After incubation, the material was washed inthe same buffer, post-fixed in 1% osmium tetroxide, dehydratedin acetone and embedded in Epoxy resin. To localize exposedanionic sites at low pH, the tissue fragments were washed inPBS, incubated in ammonium chloride and then left for 15 min ina solution containing colloidal iron hydroxide particles in PBSbuffer, pH 1.8 (Gasic et al., 1968). Subsequently, the fragmentswere washed twice in PBS, post-fixed with osmium tetroxide andprocessed for transmission electron microscopy as describedabove. In control experiments tissue fragments were previouslyincubated for 1 h at 37 �C in a PBS solution, pH 6.0, containing0.25 U/mL of neuraminidase from Clostridium perfringens (Danonet al., 1972).

2.5. Ultrastructural enzyme cytochemistry

For cytochemical localization based on the enzyme activity oftypical plasma membrane markers (Trams and Lauter, 1974), thetissue fragments, as well as sections of mouse intestines, (ap-proximately 1 mm3), used as positive controls, were fixed for30 min at 4 �C in a solution containing 1% glutaraldehyde and 5%sucrose in 0.1 M cacodylate buffer, pH 7.2. After that, the sampleswere washed in cacodylate buffer containing 7% sucrose and in-cubated in cytochemical media designed for the localization ofmembrane-associated enzymes. For localization of alkaline phos-phatase (EC 3.1.3.1), the samples were washed in 0.1 M Tris–mal-eate buffer, pH 8.0, containing 7% sucrose, incubated in a medium

J.M. Albuquerque-Cunha et al. / Arthropod Structure & Development 38 (2009) 31–44 33

containing 2 mM cerium chloride, 2 mM b-glycerophosphate, 5%sucrose and 0.01% Triton X-100 in 0.1 M Tris–maleate buffer, pH9.0, washed in the same buffer and fixed again for 60 min in 2.5%glutaraldehyde in cacodylate buffer (Robinson, 1985). In negativecontrol samples, either the substrate (b-glycerophosphate) wasomitted or 5 mM levamisole, a general inhibitor of the enzyme,was added to the incubation medium. Culture of mouse peritonealmacrophages was used as positive controls (Robinson and Kar-novsky, 1983a,b).

For localization of NaþKþ-ATPase the tissue fragments werefixed for only 10 min at 4 �C, followed by washing and incubationfor 1 h at 37 �C in a reaction medium containing 20 mM para-nitrophenylphosphate (pNPP), 20 mM magnesium chloride, 30 mMpotassium chloride, 10 mM strontium chloride, and 5% sucrose in0.05 M Tris–HCl buffer, pH 9.0 (Ernst, 1972; Mayahara et al., 1980,1982). Thereafter, the samples were washed three times in thesame buffer and then incubated for 10 min in a solution containing2% lead nitrate, 10% sucrose in Tris–HCl buffer, pH 9.0. In controlexperiments, ouabain (10 mM) was added to the reaction mediumas described by Peacock (1981) and MacVicker et al. (1993) todistinguish ouabain-sensitive and insensitive NaþKþ-ATPasesactivities from each other.

For localization of Mg2þ-ATPase after fixation the tissue wasincubated for 1 h at 37 �C in a medium containing 5 mM adenosinetriphosphate, 10 mM magnesium sulfate, 3 mM cerium chloride, 5%sucrose in 0.05 M Tris–maleate buffer, pH 7.2. For the control thesubstrate (ATP) was omitted from the incubation medium. Aspositive control, mouse macrophages were used (Wachstein andMeisel, 1957).

For localization of 50-nucleotidase, the incubation mediumcontained 3 mM cerium chloride, 2 mM magnesium chloride,1 mM ATP and 5% sucrose in 0.1 M Tris–maleate buffer, pH 7.4(Robinson and Karnovsky, 1983a, b). ATP was omitted in thecontrol samples.

2.6. Localization of carbohydrates andcarbohydrate-binding molecules (CBM)

Midgut fragments were washed in a 0.015 M NaCl solutionand fixed for 1 h at room temperature in 4% paraformaldehyde in0.1 M cacodylate buffer, pH 7.2, followed by another wash incacodylate buffer and PBS containing 1% of BSA and incubationfor 1 h in 50 mM ammonium chloride solution. The fragmentswere then washed in PBS-BSA and incubated for 1 h at roomtemperature in the presence of either fluorescein isothiocyanate(FITC)-labeled neoglycoproteins (NGP) (E-Y Laboratories, USA),fluorescein isothiocyanate (FITC)-labeled lectins (Sigma ChemicalCo) or carbohydrates (Sigma Chemical Co) at a concentration of100 mg/ml. Subsequently, the samples were washed with PBS andmounted, with 0.2% n-propyl-gallate, on a glass slide and ob-served in a Zeiss confocal laser-scanning microscope using an

Fig. 1. (A) Transmission electron microscopy of posterior midgut epithelial cells (MC)of male Rhodnius prolixus. The apical regions of these cells show a large number of longand densely packed homogeneously distributed microvilli (thick arrow) covered byperimicrovillar membranes (black asterisk). Microvillar membrane (white asterisk).The apical cytoplasm presents a large number of mitochondria (M) and hemoxisomes(thin arrow). Bar: 0.5 mm. (B) Freeze-fracture microscopy of posterior midgut epithelialcells (MC) of male Rhodnius prolixus. Long and densely packed microvilli with severalparticles (thick arrow) in the cytoplasm were observed. Some intramembranousparticles in line are observed going through longitudinally for the whole microvilliextension or joining neighbor microvilli (arrow). These particles can also be detectedon the lumen forming continuous strings (thin arrow). P face of perimicrovillarmembranes (Ppmm). E face of microvillar membrane (Em). P face of microvilli (Pm).Bar: 0.25 mm.

Fig. 2. (A) Transmission electron microscopy of posterior midgut epithelial cells (MC) ofdigitonin-treated male Rhodnius prolixus. Microvillar membrane disruption (black aster-isk). Perimicrovillar membranes displayed some isolated tubular (thick arrow) or cylin-droid (thin arrow) structures. Bar: 0.25 mm. (B) Freeze-fracture microscopy of posteriormidgut epithelial cells (MC) of digitonin-treated male Rhodnius prolixus. Peripheral hemi-tubules were seen on the P face of perimicrovillar membranes (Ppmm), above the mi-crovilli (thin arrow). Bar: 0.4 mm.

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emission filter of 488 nm and a barrier filter of 575/640 nm(Etzler, 1979; Kataoka and Tavassoli, 1984). In all cases imagesfrom the following regions were analyzed: (a) apical, comprisingthe PMM and microvilli; (b) central region, where the nucleus of theepithelial cell is located; (c) basal region of the epithelial cell. Incontrol experiments the tissue fragments were incubated for 1 hat room temperature in the presence of 200 mM of the specificmonosaccharides recognized by the lectins or of the neo-glycoproteins tested.

3. Results

3.1. General morphology

Midgut epithelial cells are typically columnar and show on theapical surface homogenously distributed microvilli surrounded bythe plexiform PMM that connect the microvilli to each other andproject towards the midgut lumen. The apical cytoplasm displaysmitochondria and hemoxisomes (Fig. 1A). Long and densely packedmicrovilli were observed in freeze-fracture samples. The P face ofthe microvillar membrane showed numerous linearly arrangedintramembranous particles following the main axis of the micro-villi. These particles also appear as strings on the E fracture face,where only few and randomly arranged intramembranous particleswere seen (Fig. 1B).

3.2. Localization of sterol–digitonin complexes byfreeze-fracture and transmission electron microscopy

After digitonin treatment, the PMM displayed some isolatedtubular or cylindroids structures. Longitudinally running leaf-likestructures with a width of 50–60 nm, but with variable length,were observed among microvilli, where the cylindroids were notfound. Disruptions of MM were observed in some microvilli.Moreover, PMM were observed forming stick-like structures orhemi-tubules near the epithelium apex cells (Fig. 2A). Freeze-fracture replicas showed peripheral hemi-tubules on the P face ofthe PMM (Fig. 2B).

3.3. Detection of anionic groups

The presence of anionic sites was detected only on the luminalsurface of PMM when the midguts were incubated in the presenceof cationized ferritin particles at pH 7.2 (Fig. 3A), or colloidal ironhydroxide particles at pH 1.8 (Fig. 3B). Labeling was reduced whenthe tissues were previously incubated with neuraminidase fromClostridium perfringens (Fig. 3C).

3.4. Localization of enzymes

Diffuse reaction products indicative of Mg2þ-ATPase wereobserved both in PMM and MM (Fig. 4A). Labeling was not observedin control preparations (Fig. 4B). Intense deposition of reactionproduct, indicative of enzymatic activity, was observed only in theMM when the tissues were incubated in the presence of a mediumdesigned for the localization of alkaline phosphatase (Fig. 4C).Either the addition of levamisole or the omission of b-glycer-ophosphate from the incubation medium inhibited the depositionof reaction product (Fig. 4D,E). In the case of NaþKþ-ATPase, la-beling was observed on both the luminal side of PMM – but not inthe lumen – and mainly in MM among microvilli. Some de-marcation was detected at the basis of microvilli. No labeling ofbasolateral plasma membrane was seen (Fig. 5A–C). The addition ofouabain to the reaction medium inhibited the labeling on bothPMM and MM (Fig. 5D,E).

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No reaction was observed either in MM or PMM in tissueincubated in a cytochemical medium designed for the localizationof 50-nucleotidase (not shown).

3.5. Localization of carbohydrates andcarbohydrate-binding molecules (CBM)

Using concanavalin A, which recognizes mannose and glucoseresidues, intense labeling of the apical surface of the posteriormidgut epithelium of R. prolixus was observed. Cytoplasmic gran-ules were also stained (Fig. 6A). Labeling was inhibited by the ad-dition of mannose to the incubation medium (not shown).

Using WGA, which recognizes N-acetyl-D-glucosamine andN-acetyl-neuraminic acid (sialic acid), all regions of the epitheliumwere intensely labeled (Fig. 6B). Labeling was partially inhibited bythe addition of neuraminic acid to the incubation medium (Fig. 6C).

BSI-B4 from Bandeirae simplicifolia which recognize a-Dþ-

galactose and PNA from Arachis hypogea which recognize N-acetyl-galactosamine and N-acetyl-galactosamine, intensely labeled theepithelial surface, including the PMM, as well as granulesdistributed throughout the cytoplasm of the posterior midgutepithelial cell (Fig. 7A,B). Labeling was partially abolished byaddition of a-D

þ-galactose (Fig. 7C) but total inhibition wasobtained by addition of a-D

þ-galactose together with N-acetyl-galactosamine to the incubation medium (Fig. 7D).

Using Helix pomatia agglutinin, which recognizes N-acetyl-galactosamine, a slight labeling of the cell surface was observed.However, cytoplasmic granules were intensely labeled (Fig. 7E). Theaddition of N-acetyl-galactosamine to the incubation mediuminhibited the labeling (not shown).

Using the FITC-labeled a-D-galactopyranosylphenyl-albumin(Fig. 8A), a-D-mannopyranosylphenyl-albumin (Fig. 8B), a-L-fuco-pyranosylphenyl-albumin (Fig. 8C) and a-D-glucopyranosylphenyl-albumin (Fig. 8D) NGPs, which bind sugar residues of N-acetyl-D-galactosamine, a-D-mannose, a-L-fucose and a-D-glucose,respectively, labeling of the apical surface of the midgut epithelialcells was observed. Labeling was inhibited when the respectivecarbohydrates were added to the incubation medium (not shown).

4. Discussion

Since the hydrolytic events are primarily determined by theaction of digestive enzymes in the intestinal tract, PMM areconsidered important structures for the compartmentalization ofthe digestive process of triatomines (Billingsley and Downe, 1983,1986a,b, 1988; Ferreira et al., 1988; Terra and Ferreira, 1994). It hasbeen considered that the PMM development is directly related to T.cruzi growth and differentiation in the insect vectors (Isola et al.,1986; Burgos et al., 1989; Garcia et al., 1989; Nogueira et al., 1997;Gonzalez et al., 1998, 1999). Therefore, a better understanding ofthe PMM composition and function can clarify both fundamentalsteps of the process of triatomine digestion and the T. cruzi lifecycle.

Cholesterol is the major free sterol and a plasma membranecomponent present in most eukaryotic cells (Trauble and Sackman,1972). Digitonin combines with non-sterified 3-b-hydroxysterolmolecules, like cholesterol, producing relevant alterations in lipid

Fig. 3. Transmission electron microscopy of posterior midgut epithelial cells (MC) ofmale Rhodnius prolixus. (A) Incubated with cationized ferritin. Particles were onlydetected in the luminal surface of perimicrovillar membranes (arrow). Bar: 0.6 mm. (B)Incubated with colloidal iron. Particles were only detected in the luminal surface ofperimicrovillar membranes (arrow). Bar: 0.4 mm. (C) Incubated with colloidal iron afterprevious incubation with neuraminidase from Clostridium perfringens. Only reducedlabeling in the perimicrovillar membranes was seen (arrow). Bar: 0.4 mm.

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bilayers once the digitonin–cholesterol complexes are formed(Szabo et al., 1971; Elias et al., 1978, 1979). Digitonin is also a po-tential detergent that can solubilize membrane constituents andpromote the dissolution of these components before (Williamson,1969) or after (Okros, 1966, 1968; Scallen and Dietert, 1969)fixation. Our observations showed that digitonin readily interactedwith the epithelium and PMM promoting a visible disruption, asshown by the rupture of MM and compaction of the PMM whichlost its characteristic plexiform array over the microvilli. Theseobservations indicated that in addition to the MM, sterols also existin PMM. The extensive effects of digitonin in the structuralorganization of MM and PMM suggest that these membranes arerich in sterols. Several data obtained using model systems showthat cholesterol in condensing the membrane bilayer regulatesmembrane fluidity and permeability (Shinitzky and Henkart, 1980;Benveniste, 2002). In insects, the lipid/protein ratio of microvillarmembranes seems to be related to the amount of microvillardigestive enzymes and carrier proteins (Jordao et al., 1994; Capellaet al., 1997). Higher Diptera have microvillar membranes with highdensities, poor in cholesterol and rich in proteins and the initial andintermediate stages of digestion occur in the midgut lumen withmost terminal digestion carried out by microvillar enzymes (Jordaoet al., 1994). In R. prolixus, oligomers formed in the luminalcompartment through partial digestion of blood by cathepsins arehydrolyzed down to monomers by enzymes trapped between MMor on the surface of posterior midgut cells. Both MM and PMM havelow apparent buoyant density but, PMM display a lower densitythan MM. Also, the PMM is poor and MM is rich in integral proteins(Ferreira et al., 1988). Previous authors investigated the cholesterolmetabolism in blood sucking triatomines (McGuire et al., 1980;Soulages and Brenner, 1991; Canavoso et al., 1998) and in themidgut membranes of phytophagous species (Shen et al., 1997). InR. prolixus, immobilized digestive enzymes are usually integralmembrane proteins mostly entrapped in the midgut cell glycocalyx(Ferreira at al. 1988). This is a particularly interesting observationsince we observed the presence of tubular and cylindroid structuresin MM and PMM of digitonin-treated posterior midguts as well asthe stick-like structures or hemi-tubules near the epithelium apexcells, point to a disorganization of membranes directly ensheathingthe microvilli which are normally set in position by columnsobliquely disposed between MM and PMM (Lane and Harrison,1979). Thus, it is suggested that in the case of MM and PMM of R.prolixus, the cholesterol molecules guarantee the necessary fluidityto maintain the spatial architecture without losing eitherpermeability or the flow of inserted components, providing bettercompartmentalization between lumen and perimicrovillar spacecompartments since MM and PMM maintain a constant distancefrom each other (Lane and Harrison, 1979; Andries and Torpier,1982; Billingsley and Downe, 1983). This is probably the bestsolution to maintain a suitable compartmentalization for theenzymatic digestive activities (Terra, 1990; Bonfanti et al., 1992;Terra and Ferreira, 1994; Silva et al., 1995, 1996; Terra, 2001; Silvaet al., 2004) allowing the control of the active transport of ions andcompounds from the lumen and perimicrovillar space intoposterior midgut cells (see below).

Studies with mammalian cells showed that the net negativesurface charge of cells is mainly due to carboxylic groups of gly-coproteins and/or glycolipids, especially sialic acid, sulfated

Fig. 4. Transmission electron microscopy of posterior midgut epithelial cells (MC) of male RhATPase. (A) Diffuse reaction products were observed in microvillar and perimicrovillar memincubation medium inhibited the deposition of reaction product in microvillar and perimicroof posterior midgut epithelial cells (MC) of male Rhodnius prolixus incubated in a cytochemobserved only in the microvillar membrane (arrow). Bar: 0.4 mm. Either addition of levamisothe deposition of reaction product in microvillar membrane. Bars: 0.25 mm and 0.4 mm, res

polysaccharides and phosphate groups (Cook et al., 1961; Grinnelet al., 1975). Sialic acid, or N-acetyl-neuraminic acid, has a negativecharge in the function of its dissociation characteristics and it playsa role in several biological phenomena (Fenderson et al., 1990;Feize, 1991). Present experiments using colloidal iron hydroxideparticles showed the presence of anionic groups detected at low pH(about 1.8), indicating the presence of anionic groups with ionizedcarboxylic groups at low pH only on the luminal surface of thePMM. Similar results were obtained using cationized ferritin. Alsothe authors’ observations show that previous incubation of thetissue fragments with Clostridium perfringens neuraminidaseabolishes the binding of the cationic particles to the membranes.These observations point to sialic acids as the main moleculesresponsible for the negative charge on the epithelial cell surface ofdigestive tract of R. prolixus.

The results presented here confirm the previous work of Fer-reira et al. (1988) who found alkaline phosphatase in the posteriormidgut cells of R. prolixus associated with the MM but not withPMM, as a plasma membrane-bound enzyme. However, labelingfor alkaline phosphatase inside the cells or in the luminal contentwas not observed here as was detected by Terra et al. (1988) usinghydrolases assays. It is possible that soluble alkaline phosphataseis synthesized as an inactive form (Terra et al., 1988) only turningactive when reaching the MM. On the other hand, Mg2þ-ATPasewas located in both MM and PMM. Furthermore, an ouabain-sensitive NaþKþ-ATPase, the most reliable enzymatic marker ofthe plasma membrane (Ernst, 1972; Trams and Lauter, 1974), wasobserved mainly in the MM among microvilli and also a reactionwas detected in PMM. This is an unexpected observation since thesodium/potassium pump was traditionally described as anapparently universal feature restricted to the basolateral plasmamembrane of secretory epithelia, thus allowing directed fluid andion transport to occur (MacVicker et al., 1993). Despite the factthat the essential function of the NaþKþ pump in animal epitheliahas been contradicted over the years, the NaþKþP-type ATPase orsodium pump is recognized as an intrinsic plasma membraneenzyme that hydrolyses ATP to maintain transmembrane gradi-ents of Naþ and Kþ and is inhibited specially by cardiac glycosidessuch as ouabain which bind to sites in the catalytic a-subunit(Mercer, 1993; Lingrel and Kuntzweiller, 1994; Huang et al., 1997).Thus, ouabain is the glycoside of practical choice for specific in-hibition of NaþKþP-type ATPase activity (Joubert, 1990). Althoughthe specific mechanisms of ouabain inhibition are not preciselyunderstood once the binding sites for ATP, Naþ, Kþ and cardiacglycosides all reside within the a-subunit whereas the b-subunitis essential for activity of the NaþKþ-ATPase (Horowitz et al.,1990), the inhibition of NaþKþ-ATPase activity in MM and PMM inthe reaction medium containing the glycoside is not surprisingand confirms our demarcation since ouabain apparently onlybinds to the extracellular surface of NaþKþ-ATPase enzyme(Hootman and Ernst, 1988). It has long been known the crucialrole of NaþKþ-ATPase on the flux of water, ions and organiccompounds from midgut lumen – where it drives water absorp-tion – to hemolymph throughout the basolateral plasma mem-brane of the insect midgut epithelial cells by its eletrogenicactivity of exporting Naþ and importing Kþ (Wigglesworth, 1933;Treherne, 1959; Berridge, 1983; MacVicker et al., 1993; Levenson,1994; Emery et al., 1998). Besides, the tissue-specific expression of

odnius prolixus incubated in a cytochemical medium designed for localization of Mg2þ-branes (white asterisk). Bar: 0.15 mm. (B) The omission of the substrate (ATP) from thevillar membranes (white asterisk). Bar: 0.25 mm. (C) Transmission electron microscopyical medium designed for localization of alkaline phosphatase. Reaction product wasle (D) to or omission of b-glycerophosphate (E) from the incubation medium inhibited

pectively.

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J.M. Albuquerque-Cunha et al. / Arthropod Structure & Development 38 (2009) 31–44 39

NaþKþ-ATPase in insects depends on the evolutionary history andecology of that particular species (Emery et al., 1998). In this way,the localization of ionic drivers for homeostasis seems to be verydifferent in Hemiptera when compared to the other insect orderswhere the NaþKþ pump has been detected (Ferreira et al., 1988;Maddrell, 2004; Gutierrez et al., 2004; Terra and Ferreira, 2006).For example, the absorption of water mainly occurs in anteriormidgut of R. prolixus and requires dietary sodium ions (but notpotassium), which are supposed to be actively absorbed providinga driving force for the movement of water (Farmer et al., 1981;Barret, 1982). Moreover, an unusual NaþKþ-ATPase activity in theapical membrane surface was previously described in peripheralcells of Periplaneta americana salivary glands (Just and Walz,1994), Malpighian tubules cells of Ischnura elegans (Khodabandeh,2006), anterior midgut of Aedes aegypti (Patrick et al., 2006)anterior and central midgut of Anopheles gambiae (Okech et al.,2008) and follicle cells from the vitellogenic follicles of R. prolixus(Ilenchuck and Davey, 1982; Sevala and Davey, 1993).

Magnesium is an important cofactor for many biologicalprocesses, such as protein synthesis and nucleic acid stability but,the chemistry of Mg2þ is unique amongst biological cations and theproperties of Mg2þ transport systems are not well known (Konradet al., 2004; de Graaf et al., 2007). The Mg2þ transporters belong tothe P-type ATPase super family and mediate Mg2þ influx down itselectrochemical gradient (Maguire, 2006). The extracellularmagnesium concentration is tightly regulated by the extent ofintestinal absorption and most physiological studies favor a so-dium-dependent exchange mechanism of transport related to theg-subunit of renal NaþKþ-ATPase as a critical component fortranscellular magnesium reabsorption (Konrad et al., 2004;Schlingmann and Gudermann, 2005). In addition, the Mg2þ-ATPasecomplex is considered the true enzyme substrate for the ouabain-sensitive P-Type NaþKþ-ATPase (Hexum et al., 1970; Robinson,1983). In insects, high concentrations of Mg2þ have been reportedin Malpighian tubule fluid of Aedes campestrs (Phillips andMaddrell, 1974) and Teleogrillus oceanicus (Xu and Marshall, 1999).

Since PMM acts as a mechanical barrier for immobilizingmolecules (Ferreira et al., 1988; Terra et al., 1988; Silva et al., 2004), itis tempting to speculate from our results that both Mg2þ-ATPase andNaþKþ-ATPase studding MM and PMM could be involved with theactive transport of ions from the lumen and perimicrovillar space intoposterior midgut cells. This transport is assumed to be related to theabsorption of organic substances, like amino acids, from the lumen tothe perimicrovillar space by appropriate protein carriers present inPMM. As pointed out by Ferreira et al. (1988), if MM actively trans-ports potassium ions from the perimicrovillar space into midgut cellsthere will be a Kþ gradient between the midgut lumen and the per-imicrovillar space. Once in the perimicrovillar space, amino acids maydiffuse into midgut cells by specific transporters – as potassium ion-amino acids co-transporters – on the microvillar surface. In this way,the inward Kþ gradient and outward Naþ gradient could also con-stitute a cationic pathway for Naþ-conserving perimicrovillar spacealkalinization (or de-acidification) as previously observed in Aedesaegypti (Patrick et al., 2006) and An. gambiae (Okech et al., 2008) sincethe deduced co-localization of HþV-ATPase and NaþKþP-ATPase inthese vectors alternates between apical and basolateral regions alongthe alimentary channel conserving the necessary midgut lumen al-kalinization and de-alkalinization. On the other hand, former obser-vations from Miranda and Ribeiro (1980) and Terra et al. (1988) point

Fig. 5. Transmission electron microscopy of posterior midgut epithelial cells (MC) of male RhATPase. (A,B) Labeling was observed in microvillar and perimicrovillar membranes (thickproducts were observed not only in the luminal side of perimicrovillar membranes (thick arreaction are also detected at the basis of microvilli (arrowhead). Mitochondria (M). Bar: 0.4reaction product in microvillar and perimicrovillar membranes (white thick arrow). Mitoch

out that the acid pH (5.5) in the midgut lumen of R. prolixusprovide a suitable environmental for the activity of cathepsin B andD-like enzymes that digest hemoglobin resulting in oligopeptidesfurther transported to the perimicrovillar space to fuel amino-peptidase and alkaline phosphatase, which products were digestedby dipeptidases and then absorbed. Unfortunately, there is noavailable information concerning the pH of the perimicrovillarspace specifically but, perhaps the atypical location of NaþKþ-ATPase in the MM could be related to the ejection of Naþ intoperimicrovillar membrane space increasing pH to supply the ap-propriate alkaline environment for the activity of both aminopep-tidase (optimal pH 8.0) and alkaline phosphatase (optimal pH 10.4)(Terra et al., 1988). The Naþ gradient could also aid in the ab-sorption of amino acids by providing the proximal energy, in theform of favorable electrochemical gradients, to move ions andorganic solutes across MM and PMM. In the absence of relevantperimicrovillar space pH measurements it is not really possible todiscuss the physiological effects of pH on the enzyme activity.Therefore, further evaluation of the possible roles of NaþKþ-ATPase,transporters, exchangers and ions channels in MM and PMM willrequire measurements of pH, membrane potential and theintracellular activities of the ions involved so that electrochemicalpotentials can be calculated for ionic species. So, the location ofNaþKþ-ATPase at the apical cell membrane remains unclear but ourcytochemical observations – supported by conventional electronmicroscopy – are consistent with an ouabain-sensitive NaþKþ-ATPase and Mg2þ-ATPase acting together to guarantee the inwardflow of Kþ ions and outward flow of Naþ ions necessary to the fluxof water, ions and nutrients inside the posterior midgut cells andalso a suitable pH in the perimicrovillar space for the activity ofdigestive enzymes. Thus, the authors’ results support thehypothesis previously proposed by Ferreira et al. (1988), thatabsorption of compounds in Hemiptera, Homoptera and theirancestors depends on transfer of water from midgut lumen tomidgut cells generating a concentration gradient used as a drivingforce for trans-membrane transport of compounds and ionsthrough appropriate symporters in MM and PMM (Ferreira et al.,1988; Terra, 1988, 1990; Terra and Ferreira, 1994; Silva et al., 1995,1996; Terra et al., 2006). Also, detailed studies on the presence andactivity of Mg2þ-ATPase and NaþKþ-ATPase in MM and PMM arenecessary to elucidate their functions at the posterior midgut ofR. prolixus.

It is known that glycoconjugates and carbohydrate-bindingmolecules are associated with the plasma membrane of insectcells and are usually involved in the interaction mechanisms withpathogens (Rudin and Hecker, 1988; Jacobson and Doyle, 1996;Dinglasan and Jacobs-Lorena, 2005). This paper shows that thePMM contain a variety of glycoconjugates in agreement with theearly description about the presence of high-mannose glycans inthe digestive tract of reduviid insects (Bonay et al., 2001). Theintense labeling observed after midgut incubation with Con Alectin indicates mannose as a major sugar residue in PMM. Similarresults were obtained with BSI-BSA4 which recognizes a-D-ga-lactose and N-acetyl-galactosamine residues. D-Galactose, man-nose and N-acetyl-galactosamine are also detected inside thecells. The use of paraformaldehyde induced the appearance ofpores in the plasma membrane facilitating the entrance of lectin–FITC complex and consequently the labeling of intracellular gly-coconjugates. Once both intra- and extracellular demarcations

odnius prolixus incubated in a cytochemical medium designed for localization of NaþKþ-arrow). Mitochondria (m) and hemoxisomes (arrowhead). Bar: 0.5 mm. (C) Reactionrow) but mainly in microvillar membrane among microvilli (thin arrow). Few areas ofmm. (D,E) Addition of ouabain to the incubation medium inhibited the deposition of

ondria (m) and hemoxisomes (arrowhead). Bars: 0.5 mm and 0.2 mm, respectively.

Fig. 6. Confocal laser-scanning microscopy of the posterior midgut epithelial cells(MC) of male Rhodnius prolixus. (A) Incubated with FITC-labeled concanavalin A. In-tense labeling of sugar residues (mannose and/or glucose) was observed both in thecytoplasm (arrow) and among cells (thick arrow). (B) Incubated with FITC-labeledWGA. Intense labeling of sugar residues (N-acetyl-D-glucosamine and/or N-acetyl-neuraminic) was observed on the apical surface of the epithelium (arrow). (C) Labelingof WGA residues on the apical surface of the epithelium (arrow) was partially inhibitedby addition of sialic acid to the incubation medium. Bars: 25 mm.

J.M. Albuquerque-Cunha et al. / Arthropod Structure & Development 38 (2009) 31–4440

were obtained, it is reasonable to suppose that the glyco-conjugates are produced by the cells and later on exported to theapical surface where they are incorporated by both MM and PMM.On the other hand, Helix pomatia only localized N-acetyl-galac-tosamine-a1,3-galactose residues inside the epithelial cells. Incontrast, extracellular exclusive labeling was obtained only withWGA which detected residues of sialic acid in the PMM. Thisobservation reinforces that PMM can use sialic acid to acquirea negative charge.

The authors also showed the presence of carbohydrate bindingmolecules on the apical surface of R. prolixus posterior midgutepithelial cells using FITC-labeled neoglycoproteins (NGP) able torecognize surface exposed carbohydrate residues (Etzler, 1979;Kataoka and Tavassoli, 1984). The observations revealed thepresence of CBM to N-acetyl-D-galactosamine, a-D-mannose, a-D-glucose and a-L-fucose. These observations confirm previousstudies showing the presence of lectins in the midgut of R. pro-lixus (Ribeiro and Pereira, 1984; Mello et al., 1996). Indeed, it ispossible that these sugar-binding molecules are involved in thebinding of T. cruzi epimastigotes to the midgut epithelial cellsurface (Pereira et al., 1980, 1981; Zimmermann et al., 1987;Doyle, 1994; Schottelius and Aisien, 1994; Bonay and Fresno, 1995;Bonay et al., 2001), a process where PMM may play some role(Kollien et al., 1998; Gonzalez et al., 1999; Alves et al., 2007;Nogueira et al., 2007).

Little is known about the biogenesis and composition of thePMM and there are controversies about their origin. Some authorssuggested that they are formed by lipoproteins leaving from theendoplasmic reticulum towards coalition of membranous vesicles,which reach the apical surface of intestinal epithelial cells (Laneand Harrison, 1979; Andries and Torpier, 1982).

The fact that the same specific markers are localized both inthe PMM and MM suggests the existence of some similaritiesbetween them. This idea is supported by the cytochemical local-ization of several of the same membrane-associated enzymes,sterol, carbohydrate residues and CBM. In fact, former observa-tions obtained by Ferreira et al. (1988) already suggested that MMare still associated with fragments of PMM. Thus, as pointed outby Silva et al. (1995) the complex of membranes constitutedof MM and PMM are probably a system of tubular structureswhich ensheath the microvilli and extend toward the luminalsurface making the perimicrovillar membrane space a closedcompartment.

Our results provide additional evidence for the model –previously proposed by Silva et al. (1995) and Terra et al. (2006) –which points out that MM and the PMM of hemipterans posteriormidgut cells are from the same origin, actually composing a systemof plasma membranes of insect midgut cells, formed by thecontinuous flux of vesicles seen budding from some Golgi areas tothe apical portion of the insect midgut cells. The differencesbetween the molecular components found in the MM and PMMmay reflect both changes in function of the prolonged contact withthe intestinal microenvironment and the enzymatic dispersionalong the great extension of the system composed by the plasmamembranes of insect midgut. Cholesterol molecules seem toguarantee the necessary fluidity to maintain the spatial architec-ture essential to the compartmentalization of enzymatic digestiveactivities (Ferreira et al., 1988) without losing the flow of compo-nents inserted. Finally, the authors believe that their mapping ofproteins and carbohydrates present in the R. prolixus’ PMM supplya basis not only for the study of triatomine digestion but may helpin the understanding of the relationship established betweentriatomine midgut and T. cruzi development in this vector. This ideais supported by the observations showing that the development ofthe parasite is directly related to the development of the PMM(Burgos et al., 1989; Nogueira et al., 1997; Gonzalez et al., 1998,

Fig. 7. Confocal laser-scanning microscopy of the posterior midgut epithelial cells (MC) of male Rhodnius prolixus. (A) Incubated with FITC-labeled BSI-B4. Intense labeling of a-Dþ-

galactose and/or N-acetyl-galactosamine residues was observed in the cytoplasm (thick arrow), apical surface of the cells (thin arrow) and perimicrovillar membranes (whiteasterisk). Bar: 25 mm. (B) Incubated with FITC-labeled PNA. Intense labeling of N-acetyl-galactosamine residues was observed both in cytoplasmic granules (thick arrow) and theapical surface of the cells (thin arrow). Tracheole (T). Bar: 50 mm. (C) Incubated with a-D

þ-galactose and then FITC-labeled BSI-B4. Only a light labeling was detected (arrow). Bar:50 mm. (D) Incubated with a-D

þ-galactose together with N-acetyl-galactosamine and then FITC-labeled BSI-B4. No labeling was observed. Bar: 50 mm. (E) Incubated with FITC-labeled HPA/HPE. Residues of N-acetyl-galactosamine were detected in cytoplasmic granules (arrow). Bar: 25 mm.

Fig. 8. Confocal laser-scanning microscopy of the posterior midgut epithelial cells (MC) of male Rhodnius prolixus. (A) Incubated with FITC-labeled a-D-galactopyranosylphenyl-albumin NGP. Labeling for N-acetyl-D-galactosamine was detected on the apical surface of the epithelium (arrow). (B) Incubated with FITC-labeled a-D-mannopyranosylphenyl-albumin NGP. Labeling for a-D-mannose was detected on the apical surface of the epithelium (black asterisk). (C) Incubated with FITC-labeled a-L-fucopyranosylphenyl-albuminNGP. Labeling for a-L-fucose was detected on the apical surface of the epithelium (white asterisk). Tracheole (T). (D) Incubated with FITC-labeled a-D-glucopyranosylphenyl-albuminNGP. Labeling for a-D-glucose was detected on the apical surface of the epithelium (white arrow). Bars: 25Dmm.

J.M. Albuquerque-Cunha et al. / Arthropod Structure & Development 38 (2009) 31–4442

1999; Garcia et al., 1999, 2007; Cortez et al., 2002, Alves et al., 2007;Nogueira et al., 2007). Furthermore, an antiserum against R. prolixusPMM and midgut tissue interfered with the midgut structuralorganization and reduced the development of Trypanosoma cruzi inthe insect vector (Gonzalez et al., 2006).

Acknowledgments

We thank Dr Renato da Matta and Dr Carlos Peres Silva fromLBCT and Laboratorio de Quımica e Funçao de Proteınas e Peptıdeos(LQFPP) of the Centro de Biociencias e Biotecnologia of theUniversidade Estadual do Norte Fluminense (UENF) for the criticalrevision. We also thank Rodrigo Mexas, Bruno Eschenazi andGenilton Jose Vieira (Laboratorio de Analise e Produçao de Imagens/FIOCRUZ) as well as Marcia Adriana Dutra and Arthur Rodriguez(LBCT/UENF) for the photography treatment and Professor ShaunWalker for the English revision. This work was supported by

Conselho Nacional de Desenvolvimento Cientıfico e Tecnologico(CNPq), Programa de Nucleos de Excelencia (Pronex) and FundaçaoEstadual do Norte Fluminense (FENORTE).

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