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Development and glycoprotein composition of the perimicrovillar membrane in Triatoma (Meccus) pallidipennis (Hemiptera: Reduviidae)
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Development and glycoprotein composition of the perimicrovillar membrane in Triatoma (Meccus) pallidipennis (Hemiptera: Reduviidae) Q7
rrez-Cabrera a, Ricardo Alejandre-Aguilar b, Salvador Herna ndez-Martínez c, Ana E. Gutie Bertha Espinoza a, * noma de M Departamento de Inmunología, Instituto de Investigaciones Biom edicas, Universidad Nacional Auto exico, Ciudad Universitaria, 04510 Mexico City, D.F., Mexico b gicas, Instituto Polit Departamento de Parasitología, Escuela Nacional de Ciencias Biolo ecnico Nacional, Mexico c n Sobre Enfermedades Infecciosas, Instituto Nacional de Salud Pública, Av. Universidad 655, Col. Sta. María Ahuacatitlan, CP 62508 Centro de Investigacio Cuernavaca, Morelos, Mexico a
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a r t i c l e i n f o
a b s t r a c t
Article history: Received 14 April 2014 Received in revised form 25 June 2014 Accepted 1 July 2014 Available online xxx
Hemipterans and thysanopterans (Paneoptera: Condylognatha) differ from other insects by having an intestinal perimicrovillar membrane (PMM) which extends from the base of the microvilli to the intestinal lumen. The development and composition of the PMM in hematophagous Reduviidae depend on factors related to diet. The PMM may also allow the human parasite Trypanosoma cruzi, the etiological agent of human Chagas Disease, to establish and develop in this insect vector. We studied the PMM development in the Mexican vector of Chagas Disease, Triatoma (Meccus) pallidipennis. We describe changes in the midgut epithelial cells of insects in response to starvation, and at different times (10, 15 and 20 days) after bloodfeeding. In starved insects, the midguts showed epithelial cells closely connected to each other but apparently free of PMM with some regions being periodic acideSchiff (PASeSchiff) positive. In contrast, the PMM was evident and fully developed in the midgut region of insects 15 days after feeding. After this time, the PMM completely covered the microvilli and reached the midgut lumen. At 15 days following feeding the labeled PASeSchiff increased in the epithelial apex, suggesting an increase in carbohydrates. Lectins as histochemical reagents show the presence of a variety of glycoconjugates including mannose, glucose, galactosamine, N-acetyl-galactosamine. Also present were Nacetyl-glucosamine and sialic acid which contribute to the successful establishment and replication or T. cruzi in its insect vectors. By means of scanning electron microscopy (SEM) and transmission electron microscopy (TEM), the formation and structure of the PMM is confirmed at 15 days post feeding. Our results confirmed the importance of the feeding processes in the formation of the PMM and showed the nature of the biochemical composition of the vectors' intestine in this important Mexican vector of Chagas disease. © 2014 Published by Elsevier Ltd.
Keywords: Perimicrovilla membrane Triatoma (Meccus) pallidipennis Trypanosoma cruzi Glycoproteins
1. Introduction Insect members of the order Hemiptera and Thysanoptera (Paraneoptera: Condylognatha) have specific mouth adaptations that allow them to suck sap or liquefy food from different preys or hosts (Terra, 1988). This feeding mode has been associated with modifications of the alimentary canal to digest and absorb nutrients, which are present at extremely low concentration in the putative hemipteran ancestor's food source (Silva et al., 1995; Terra,
* Corresponding author. Tel.: þ52 555 622 8943/44. E-mail address: [email protected] (B. Espinoza).
1990). Nutrient absorption is dependent on the presence of an intestinal membrane with unique characteristics called the perimicrovillar membrane (PMM; Lane and Harrison, 1979) or the extracellular membrane layer. The PMM, comprises lipoproteins, glycoconjugates and carbohydrate-binding molecules (Albuquerque-Cunha et al., 2009), and is secreted from the endoplasmic reticulum of the epithelial cells, in double-membrane et al., vesicles that bud from the Golgi apparatus (Damasceno-Sa 2007). These vesicles reach the midgut cell apices where they are released into the luminal space (Silva et al., 1995; Terra et al., 1996). The PMM surrounds the microvilli of the midgut epithelial cells, which extends from the base of the microvilli to the lumen (Silva et al., 1995). The function of the PMM is similar to that of the
http://dx.doi.org/10.1016/j.asd.2014.07.001 1467-8039/© 2014 Published by Elsevier Ltd.
rrez-Cabrera, A.E., et al., Development and glycoprotein composition of the perimicrovillar membrane in Please cite this article in press as: Gutie Triatoma (Meccus) pallidipennis (Hemiptera: Reduviidae), Arthropod Structure & Development (2014), http://dx.doi.org/10.1016/ j.asd.2014.07.001
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Fig. 1. Histological sections of T. pallidipennis midgut with different feeding conditions showing single columnar epithelial cells with three distinct regions (A, apical; M, medial; B, basal) and the basal membrane (BM). The apical region shows brush border (BB) and an acidophil layer (AL) evidents 15 and 20 days after blood meal (c and d respectively), this layer surrounds the brush border and reaches the midgut lumen (L). The medial region shows round nucleus (N). The muscle layers (ML) was also evident. a, starved group; b, 10 days after blood meal; c, 15 days after blood meal; d, 20 days after blood meal. (100).
peritrophic membrane in other insect orders: it acts as a physical and physiological barrier for enzymes involved in the digestion and absorption processes (Shao et al., 2001). Unlike the peritrophic membrane, however, the PMM is not a chitinous extracellular membrane layer, and has more specific functions such as selective permeability and it is optimized for nutrient absorption (Terra et al., 1996). Despite the different feeding types of insects Hemiptera (phytophagous, zoophytophagous and hematophagous), the PMM composition is similar (Terra, 1990; Albuquerque-Cunha et al., 2004, 2009). However, various studies have shown that PMM production could be explained by the different feeding behaviors et al., 2007; Fialho et al., 2009; Azevedo et al., (Damasceno-Sa 2009). On the apical surface of the midgut epithelium of blood-sucking insects the PMM covers the microvillar membrane of epithelial cells, fills the space between microvilli, and extends into the intestinal lumen (Albuquerque-Cunha et al., 2009). In Rhodnius prolixus the PMM becomes more evident 10 days after a blood meal (Nogueira et al., 1997). More specifically, Gonzalez et al. (1998) and Albuquerque-Cunha et al. (2004), using different experimental protocols, showed that the PMM development in R. prolixus
depends on the insect abdominal distension, ingestion of blood diet components, humoral and hormonal factors. The glycoconjugates and carbohydrate binding molecules that are associated with the PMM of triatomine gut cells, also are involved in the digestive and nutrient absorption processes (Shao et al., 2001), and several investigations have linked glycoconjugates with the interaction with pathogens (Pereira et al., 1981; Dinglasan and Jacobs-Lorena, 2005). In this case, the PMM acts as an adhesion site for Trypanosoma cruzi epimastigotes, an essential process for parasite establishment in the insect vector (Garcia and Azambuja, 2004). Alves et al. (2007) showed that GalNAc, ManNAc, GlcNAc, D-Gal, D-Man and NANA are sites for recognition of PMM by T. cruzi. This explains why a PMM disruption has been correlated with blocking T. cruzi development in triatomine vectors (Cortez et al., 2002, 2012). Given this information, it seems clear that unraveling how the PMM develops is essential to understand how T. cruzi becomes established in the insect vector. However, our knowledge of the triatomine midgut morphology comes mainly from R. prolixus or Triatoma infestans, due to their medical relevance as vectors of Chagas disease in South America. Other species of triatomines may show similar or unique biological characteristics. Triatoma (Meccus) pallidipennis is considered the most important
rrez-Cabrera, A.E., et al., Development and glycoprotein composition of the perimicrovillar membrane in Please cite this article in press as: Gutie Triatoma (Meccus) pallidipennis (Hemiptera: Reduviidae), Arthropod Structure & Development (2014), http://dx.doi.org/10.1016/ j.asd.2014.07.001
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Fig. 2. Scanning electron microscopy of T. pallidipennis midgut in starved condition (a, 5000; b, 15,000) and 15 days after blood meal (c, 5000; d, 15,000). The surface view of midgut from starved insects shows cells with irregular apical surface (MM). 15 days post feeding the PMM (PMM) was arranged as a mesh on and between cells.
vector of T. cruzi in central Mexico due to its large abundance, high prevalence of infection with T. cruzi and extensive contact with human populations (Martínez et al., 2006; Ramsey et al., 2012). In this paper our main aim is to document how the PMM is produced and formed over time by glycoprotein component analysis and histological and ultrastructural studies in T. pallidipennis midgut after starvation and feeding, given that food source is key for PMM development.
2. Materials and methods 2.1. Insect experimental protocol T. pallidipennis were reared and maintained under controlled laboratory conditions in an insectary established at the Escuela gicas, Instituto Polite cnico Nacional, Nacional de Ciencias Biolo Mexico City, Mexico. Fifth-instar nymphs were starved for 30 ± 5 days and after their last molt two groups were formed: one maintained without feeding (starved group), other that was allowed to feed (fed group). Insects from the fed group were dissected 10, 15 and 20 days after feeding. These times were chosen from a pilot study that covered 5e55 days after feeding and which demonstrated major differences in PMM development after 10, 15 and 20 days. Sample sizes for each group (for the case
of the starved group) and subgroups (for the case of the fed group) comprised three animals and there were three independent experiments.
2.2. Light microscopy T. pallidipennis midguts were obtained by dissection from both starved and fed groups, and were fixed for 2 h at 4 C in 4% paraformaldehyde in sodium phosphate buffer (PBS, 2.5 mM Na2HPO4 (BAKER, Xalostoc, Mex); 7.97 mM NaH2PO4 (BAKER, Xalostoc, Mex); 154 mM NaCl (Sigma, St Louis, MO)) pH 7.2. After fixation, samples were washed twice in PBS followed by alcoholic dehydration (50%, 70%, 90%, 96% and 100% ethanol), and xylene clarification. Finally, tissues were embedded in Paraplast X-Tra (Sigma, St Louis, MO) and sections (5 mm-thick) were obtained with a microtome (Leica Mod Jang RM2025). Sections were stained with hematoxylineeosin (Kiernan, 2008) and the periodic acideSchiff method (PASeSchiff) to identify tissues containing neutral hexose sugars and sialic acids (Kiernan, 2008). Twelve histological sections per insect were assessed, and each section was analyzed with an optic microscope in bright field (Optiphot-2 Nikon) covering at least five fields. Thus, 540 fields at 100 objective for each histological analysis of starved and fed insect groups were analyzed over three replicates.
rrez-Cabrera, A.E., et al., Development and glycoprotein composition of the perimicrovillar membrane in Please cite this article in press as: Gutie Triatoma (Meccus) pallidipennis (Hemiptera: Reduviidae), Arthropod Structure & Development (2014), http://dx.doi.org/10.1016/ j.asd.2014.07.001
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2.3. Scanning electron microscopy Scanning electron microscopy (SEM) was used to study the formation of PMM in the starved and the fed groups up to 15 days after feeding. Midgut tissues were fixed in 4% paraformaldehyde in PBS pH 7.2 for 2 h at RT, washed twice in PBS, and then dehydrated with a gradual ethanol concentration series (50%, 70%, 90%, 96% and 100%). Tissues were critical point-dried with CO2, mounted on stubs, coated with a 20-nm-thick gold film, and examined with a JEOL Scanning Electron Microscope (JSM6360LV). 2.4. Transmission electron microscopy Midgut tissues from starved and fed (15 days post-feeding) samples were processed for transmission electron microscopy (TEM). Samples were obtained as described before and immediately fixed in 4% glutaraldehyde (Ted-Pella, Redding, CA) in phosphate buffered saline (PBS, pH 7.4). After 24 h at RT, the specimens were washed with PBS and post-fixation was performed with 1% of osmium tetroxide (EMS, FT Washington, PA) in PBS during 2 h at RT. Dehydrated using progressive ethanol concentrations and embedded in DER-ERL resin (EMS, FT Washington, PA). Semi-thin sections (0.5 mm) were stained with toluidine blue (EMS, FT Washington, PA). Semi-thin sections were obtained using an RMC ultramicrotome (Model MTX, Tucson, AZ) to observe the midguts under light microscopy. At least two areas were selected in semithin sections, and were prepared for electron microscopy. Thin sections (80 nm) were contrasted with 5% uranyl acetate and lead citrate (lead 80 mM nitrate, 120 mM sodium citrate and 160 mM NaOH; all from EMS, FT Washington, PA) (Reynolds, 1963), and examined in a Jeol transmission electron microscope (JEM-1011) (Hern andez-Martínez et al., 2013). 2.5. Lectins as histochemical markers Midguts from the starved and the fed subgroups (15 days postfeeding) were fixed, dehydrated, clarified and embedded in paraffin
as described above. These samples were later re-hydrated and incubated for 1 h at RT in the presence of 25 mg/mL of biotinylated lectins (all from Sigma, Toluca, Mexico) prepared in chloride buffer pH 7.4. The biotinylated lectins were Canavalia ensiformis (ConA), Arachis hypogeal (PNA) and Triticum vulgaris (WGA). After incubations, samples were washed with PBS, and incubated with Streptavidinefluorescein conjugate (Amersham Biosciences RPN1232, Freiburg Germany) diluted 1:200 in bicarbonate buffer (NaHCO3 0.1 M and ClNa 0.1 M, pH 8.3). Samples were washed again with PBS and counterstained with DAPI (40 ,6-diamidino-2-phenylindole) (Molecular Probes, Oregon) diluted 1:1000 in PBS. Finally, samples were washed with PBS and mounted on a glass slide using fluoro-gel (EMS, Tlalpan, Mexico), and observed under an Olympus confocal microscope (Model BX51WI) coupled to a Disk Scanning Unit (DSU). Using ConA, it was possible to identify mannose and glucose residues. With PNA galactosamine and Nacetyl-galactosamine were detected, and using WGA it was possible to identify sialic acid and N-acetyl-glucosamine. 3. Results 3.1. General morphology T. pallidipennis midguts comprised a simple columnar epithelium, surrounded by a muscular layer that consists of an inner oblique longitudinal layer and an outer oblique transverse layer. The midgut epithelial cells are typically single columnar composed of three distinct regions: apical, medial and basal. The basal membrane was also clearly observed (Fig. 1). The apical region shows brush border and an acidophilic layer. In the medial region there are round nuclei and also staining granules as can be seen in the basal region. Using the time course analysis, it was possible to see that the arrangement mainly of the apical region, as well as of some of its subcellular structures, becomes modified over time after ingestion of a meal (Fig. 1aed). In the starved group (Fig. 1a), the midgut epithelial cells were closely connected by the medial and basal
Fig. 3. Transmission electron microscopy of T. pallidipennis midgut in starved condition (a, 25,000) and 15 days after blood meal (b, 15,000). The starved condition midgut shows the microvilli (MM) extending into the lumen (L); while the vector midgut 15 days after blood meal shows the PMM on and surrounding the microvilli extending toward the lumen (L).
rrez-Cabrera, A.E., et al., Development and glycoprotein composition of the perimicrovillar membrane in Please cite this article in press as: Gutie Triatoma (Meccus) pallidipennis (Hemiptera: Reduviidae), Arthropod Structure & Development (2014), http://dx.doi.org/10.1016/ j.asd.2014.07.001
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region, with brush border at the cell apex and free of projections at the luminal surface of the apical region (Fig. 1a). By 10 days postfeeding, a general view of the cell surface revealed noncontinuous projections at the luminal surface of the apical region suggesting the initial formation of PMM (Fig. 1b). There was an increase in cytoplasmic contents and a large number of vesicles in the cytoplasm (Fig. 1b). Midgut epithelial cells of insects that were fed 15 days previously had epithelium with folds in the apical portion and a brush border densely-packed that was homogeneously-distributed. In this post-feeding time, we observed a full development of an acidophil layer (Fig. 1c). This layer surrounds the brush border and reaches the midgut lumen, suggesting the presence of the PMM (from here on this layer will be named PMM). The lumen is irregular in shape owing to longitudinal folds (Fig. 1c). Twenty days after feeding the layer that surrounds the brush border was less evident and had surface disruptions (Fig. 1d). 3.2. Scanning and transmission electron microscopy observation of the midgut In order to follow the development of membrane structure, T. pallidipennis midgut was observed on SEM and TEM. Based on the SEM, the cell surfaces of starved midgut show an irregular apical
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surface (Fig. 2a and b). The absence of PMM was supported by transversal sections viewed with TEM observing the microvilli devoid of PMM (Fig. 3a). The time of full development of an acidophil layer observed with light microscopy was 15 days post feeding. Using the SEM, we could observe that this layer, is arranged as a mesh on and between cells extending into the lumen, in the same day (Fig. 2c and d). According to TEM, the layer mentioned is the PMM and is on the cell surface as extending toward the lumen like thin pipes but in turn is found surrounding the microvilli (Fig. 3b). The microvilli in both the starvation and 15 days post feeding, seen by TEM, looks very well organized, especially at 15 days post feeding, appreciating its double membrane (Fig. 3a and b). 3.3. Localization of carbohydrates 3.3.1. PASeSchiff Animals from the starved group showed midgut regions with glycoconjugates mainly on the brush border and basal membrane (Fig. 4a). However, in midgut regions and cytoplasmic granules of digestive cells of fed animals, there was an increase in PASeSchiff positive regions on the epithelial apex and on the basal membrane (Fig. 4bed). By 10 days after feeding, the cell cytoplasm was more highly colored (Fig. 4b); midgut cells at 15 days after feeding had an
Fig. 4. PASeSchiff positive regions of T. pallidipennis midgut with different feeding conditions. The midgut region with Pass-Schiff positive staining was the brush border (BB) and basal membrane (BM). Midgut cells at 15 and 20 days after feeding had an evident layer surrounding the brush border (PMM), extending into the lumen (L) that was also Pass-Schiff positive. a, starved group; b, 10 days after blood meal; c, 15 days after blood meal; d, 20 days after blood meal. N: nucleus. (100).
rrez-Cabrera, A.E., et al., Development and glycoprotein composition of the perimicrovillar membrane in Please cite this article in press as: Gutie Triatoma (Meccus) pallidipennis (Hemiptera: Reduviidae), Arthropod Structure & Development (2014), http://dx.doi.org/10.1016/ j.asd.2014.07.001
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evident layer surrounding the brush border (possibly, the PMM) which was PASeSchiff positive (Fig. 4c), and 20 days after feeding this layer was evident, however the coloration was discontinuous (Fig. 4d). 3.3.2. Glycan identification using lectins In the starved group ConA labeled the apical surface, membrane basal and cytoplasm of midgut cells, especially under the cellular apex (Fig. 5a). In insects 15 days after feeding, all regions of the epithelium were intensely labeled, and there was an increase in the cytoplasm labeling compared with the starved group, especially under the cellular apex (Fig. 5b). PNA was only detected in starved insects on the apical surface and the basal membrane (Fig. 5c). In the midguts of insects 15 days post feeding, there was an increase in the labeling of the epithelial surface, including the layer that surrounds the brush border (the PMM), as well as in granules distributed throughout the cytoplasm and especially under cellular apex (Fig. 5d). In starved groups, WGA was detected lightly on the apical surface of midgut cells and in the basal membrane (Fig. 5e). In contrast, in the insects 15 days after feeding, WGA was detected more strongly in the apical surface comprising the PMM and the cytoplasm (Fig. 5f). 4. Discussion In starved animals of T. pallidipennis, the microvilli were practically deprived of a PMM covering. At 10 days post-feeding the midgut cells had already developed a layer surrounding the brush border suggesting the formation of PMM, which was more evident and developed 15 days post-feeding. Twenty days post-feeding the covering membrane was reduced and PMM disruption was seen. The SEM and TEM of midguts of starved insects indicated that the PMM was still evident on the margins of individual cells and regions between the cells, also the apical microvilli not exhibit uniform organization in whole cell. Furthermore, the midgut of insects within 15 days after feeding, the PMM was located above and between microvilli and extended toward the lumen, apparently form a continuous domain from one cell to another. The PMM has a narrow tubular arrangement from the cellular surface to the lumen; however, Silva et al. (1995) mentioned that depending on the preparation, the perimicrovillar tubes may appear folded or almost perpendicular to the cell surface. Similar data have been described in other species of triatomines such as R. prolixus, T. infestans and Triatoma vitticeps; by 7 days after a blood meal the PMM layer was still incomplete although it was thick and had a smooth surface, hindering the visualization of individual cells (Billingsley and Downe, 1986; Rocha et al., 2010). Furthermore, at 10 days after a blood meal, the PMM reached its maximum development (Burgos and Gutierrez, 1976; Billingsley and Downe, 1983; Kollien and Schaub, 2000). Moreover, the development of PMM can be understood at a higher taxonomic level. In other hemipterans such as the hematophagous Cimex hemipterus (Hemiptera: Comocidae), the phytophagous Dysdercus peruvianus (Hemiptera: Pyrrhocoridae) and the zoophytophagous Brontocoris tabidus (Heteroptera: Pentatomidae), the PMM synthesis varies according to their feeding mode. In C. hemipterus, the PMM is evident 20 days post feeding (Azevedo et al., 2009); in D. peruvianus, PMM recovered all the cells 30 h post-feeding et al., 2007); while in B. tabidus, the PMM is (Damasceno-Sa
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evident in starved and fed condition (Fialho et al., 2009). Development of PMM in hemipterans varies according to how frequently the animal has access to food: in phytophagous and zoophytophagous species access to food seems frequent (Damasceno et al., 2007; Fialho et al., 2009), while in blood-suckers such Sa access is less frequent as hosts are harder to find (Billingsley, 1990; Nogueira et al., 1997; Azevedo et al., 2009). Thus, in comparative terms, it seems that digestion in T. pallidipennis takes place at a slower than the species mentioned. We observed more PASeSchiff positive regions in the midguts of fed animals than in starved animals. The PASeSchiff positive reaction suggests the presence of glycoconjugates. This means that starved midgut have fewer glycoconjugates in the epithelial apex, basal membrane and some cytoplasmic granules; contrary to what was found in midgut with 15 days after feeding, because the labeled PAS increased in the epithelial apex, above all in the extracellular membrane layer, and this corresponds to the PMM. Different papers have reported that glycoconjugates are associated with the plasma membrane of insect cells and are usually involved in the interaction mechanisms with pathogens (Pereira et al., 1981; Rudin and Hecker, 1988; Jacobson and Doyle, 1996; Dinglasan and JacobsLorena, 2005). In this paper we found that the PMM contained a variety of glycoconjugates that showed differences in the abundance of sugar residues in the midgut of starved insects or 15 days post feeding, which could impact the development of T. cruzi in T. pallidipennis. In this paper we used three lectins: Con A, PNA and WGA. According to previous descriptions, these lectins can detect important sugars involved for adhesion of parasites (Alves et al., 2007). Labeling of tissues from the starved group was less intense than in tissues 15 days after feeding with different lectins. Con A for example, leads to an intense labeling of the midgut of starved animals but increased in the midgut of animals within 15 days post feeding, indicating mannose and/or glucose as a major sugar residue in cross sections of midguts under conditions of starvation and feeding. However, there was an increase in labeling 15 days after the feeding, in all cross sections and PMM. Other sugars binding to molecules were N-acetyl-galactosamine and galactosamine, recognized by the lectin PNA. Actually, the labeling was more evident 15 days after feeding specially on the cellular apex. Nacetyl-D-glucosamine and sialic acid, recognized by the lectin WGA, was more strongly detected on PMM and cytoplasmic contents. It was possible to label intracellular glycoconjugates as we used paraformaldehyde as a tissue fixative to permeabilize the plasma membrane to facilitate the entrance of lectineFITC complex. Intracellular and extracellular labeling was expected as glycoconjugates are produced by the cells and exported to the apical surface where they are incorporated by both MM and PMM. This suggests the existence of some similarities between the midgut cells of starved and fed groups: an increase in the sugar residues, especially mannose, glucose, N-acetyl-D-glucosamine and sialic acid. According to Albuquerque-Cunha et al. (2009), the main differences between MM and PMM may reflect both changes in function of the prolonged contact with the intestinal microenvironment and enzymatic dispersion along the great extension of the system composed by the plasma membranes of the insect midgut. It is possible that the major expression of sialic acid (NANA) and mannose residues found in midgut cuts of T. pallidipennis within 15 days after feeding are related to the time of parasite multiplication.
Fig. 5. Confocal laser-scanning microscopy of the cross sections of midgut epithelial cells of T. pallidipennis. The starved group labeled with ConA (a), PNA (c) and WGA (e) showed less fluorescence compared with the group with15 days after feeding using the same lectins: ConA (b), PNA (d) and WGA (f). This last group showed intense staining in the brush border (BB), the layer surrounded it (PMM) and the cytoplasm of the apical region (double arrow). A higher staining in the cytoplasm was observed with WGA that with the other lectins (f). ML: muscle layer; N: nucleus; L: lumen. (60).
rrez-Cabrera, A.E., et al., Development and glycoprotein composition of the perimicrovillar membrane in Please cite this article in press as: Gutie Triatoma (Meccus) pallidipennis (Hemiptera: Reduviidae), Arthropod Structure & Development (2014), http://dx.doi.org/10.1016/ j.asd.2014.07.001
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Alves et al. (2007) demonstrated that T. cruzi epimastigote hydrophobic proteins and carbohydrate residues of R. prolixus PMM glycoproteins (mainly NANA and man) with 10 days after feeding are important factors for the parasite/vector interaction. Based on the above data, the main difference between T. pallidipennis and R. prolixus as vectors could be the time taken to form the PMM and produce the appropriate conditions for T. cruzi to replicate and continue its life cycle within the vector. This idea is supported by the observations showing that the development of the parasite is directly related to the PMM development (Gonzalez et al., 1998; Garcia et al., 1999, 2007; Cortez et al., 2002; Garcia and Azambuja, 2004; Alves et al., 2007). If this is true, then parasite development is constrained by the biochemical conditions of the vectors' intestine. Therefore, is necessary to continue a detail mapping of the proteins and carbohydrates, especially of the PMM, which will give us basis for understanding the processes of digestion but mostly to understand the relationships between triatomines and T. cruzi. Q6
Uncited reference García et al., 1989. Acknowledgments
We thank L.A. Morales Arreola, Ma. L. Martínez Ramírez and L. Romero Romero from Departamento de Patología, Facultad de noma Medicina Veterinaria y Zootecnia, Universidad Nacional Auto xico, Mexico City, Mexico (UNAM) for processing of samples; de Me Y. Hornelas from the Instituto de Ciencias de Mar y Limnología, UNAM, for providing access to SEM facilities; M. Tapia Rodríguez for microscopy assistance; W. Espinoza and I. Martínez for logistic rdoba-Aguilar and C. Lowenberger provided comsupport. A. Co ments to the manuscript. This paper is part of the doctoral thesis of dicas of A. Gutierrez-Cabrera in the Doctorado de Ciencias Biome noma de Me xico (UNAM). A.E. the Universidad Nacional Auto rrez-Cabrera acknowledges the scholarship and financial Gutie Q2 support provided by the National Council of Science and Technology (CONACyT), and Program in Biomedical Sciences of the UNAM. Q3 This project was partially supported for a grant to Bertha Espinoza n Miguel Alema n (2013) and DGAPA-UNAM Q4,5 from Fundacio (IN206512). References Albuquerque-Cunha, J.M., Mello, C.B., Garcia, E.S., Azambuja, P., de Souza, W., lez, M.S., Nogueira, N.F., 2004. Effect of blood components, abdominal Gonza distension, and ecdysone therapy on the ultrastructural organization of posterior midgut epithelial cells and perimicrovillar membranes in Rhodnius prolixus. Mem. Inst. Oswaldo Cruz 99, 815e822. Albuquerque-Cunha, J.M., Gonz alez, M.S., Garcia, E.S., Mello, C.B., Azambuja, P., Almeida, J.C., de Souza, W., Nogueira, N.F., 2009. Cytochemical characterization of microvillar and perimicrovillar membranes in the posterior midgut epithelium of Rhodnius prilixus. Arthropod Struct. Dev. 38, 31e44. Alves, C.R., Albuquerque-Cunja, J.M., Mello, C.B., Garcia, E.S., Nogueira, N.F., Bourguingnon, S.C., de Souza, W., Azambuja, P., Gonzalez, M.S., 2007. Trypanosoma cruzi: attachment to perimicrovillar membrane glycoproteins of Rhodnius prolixus. Exp. Parasitol. 116, 44e52. Azevedo, D.O., Neves, C.A., Santos-Mallet, J.R., Goncalves, T.C.M., Zanuncio, J.C., Serrao, J.E., 2009. Notes on midgut ultrastructure of Cimex hemipterus (Hemiptera: Cimicidae). J. Med. Entomol. 46, 435e441. Billingsley, P.F., Downe, A.E.R., 1983. Ultrastructural changes in posterior midgut cells associated with blood feeding in adult female Rhodnius prolixus Stal (Hemiptera: Reduviidae). Can. J. Zool. 61, 2574e2586. Billingsley, P.F., Downe, A.E., 1986. The surface morphology of the midgut cells of Rhodnius prolixus Stal (Hemiptera Reduviidae) during blood digestion. Acta Trop. 43, 355e366. Billingsley, P.F., 1990. The midgut ultrastructure of hematophagous insects. Annu. Rev. Entomol. 35, 219e248.
Burgos, M.H., Gutierrez, L.S., 1976. The intestine of Triatoma infestans I. Cytology of the midgut. J. Ultrastruct. Res. 57, 1e9. Cortez, M.G.R., Gonzalez, M.S., Cabral, M.M.O., Garcia, E.S., Azambuja, P., 2002. Dynamic development of Trypanosoma cruzi in Rhodnius prolixus: role of decapitation and ecdysone therapy. Parasitol. Res. 88, 697e703. Cortez, M.R., Procencano, A., Silva, C.E., Mello, C.B., Zimmermann, L.T., Schaub, G.A., Garcia, E.S., Azambuja, P., Gonzalez, M.S., 2012. Trypanosoma cruzi: effects of azadirachtin and ecdysone on the dynamic development in Rhodnius prolixus larvae. Exp. Parasitol. 131, 363e371. , J., Carneiro, N.B., DaMatta, R.A., Samuels, R.I., Terra, W.R., Silva, C.P., Damasceno-Sa 2007. Biphasic perimicrovillar membrane production following feeding by previously starved Dysdecus peruvians (Hemiptera: Pyrrhocoridae). J. Ins. Physiol. 53, 592e600. Dinglasan, R.R., Jacobs-Lorena, M., 2005. Insight into a conserved lifestyle: proteincarbohydrate adhesion strategies of vector-borne pathogens. Infect. Immun. 73, 7797e7807. Fialho, M.D.C., Zanuncio, J.C., Neves, C.A., Ramalho, F.S., Serrao, J.E., 2009. Ultrastructure of the digestive cells in the midgut of the predator Brontocoris tabidus (Heteroptera: Pentatomidae) after different feeding periods on prey and plants. Ann. Entomol. Soc. Am. 102, 119e127. García, E.S., Gonzalez, M.S., Azambuja, P., Rembold, H., 1989. Chagas' disease and its insect vector. Effect of azadirachtina A on the interaction of a triatomine host (Rhodnius prolixus) and its parasite (Trypanosoma cruzi). J. Biosci. 44, 317e322. Garcia, E.S., Gonzalez, M.S., Azambuja, P., 1999. Biological factors involving Trypanosoma cruzi life cycle in the invertebrate vector, Rhodnius prolixus. Mem. Inst. Oswaldo Cruz 94, 213e216. Garcia, E.S., Azambuja, P., 2004. Lignoids in insects: chemical probes for the study of ecdysis, excretion and Trypanosoma cruzidtriatomine interactions. Toxicon 44, 431e440. Garcia, E.S., Ratcliffe, N.A., Whitten, M.M., Gonzalez, M.S., Azambuja, P., 2007. Exploring the role of insect host factors in the dynamics of Trypanosoma cruzieRhodnius prolixus interactions. J. Ins. Physiol. 53, 11e21. Gonzalez, M.S., Nogueira, N.F., Feder, D., de Souza, W., Azambuja, P., Garcia, E.S., 1998. Role of the head in the ultrastructural midgut organization in Rhodnius prolixus larvae: evidence from head transplantation experiments and ecdysone therapy. J. Ins. Physiol. 44, 553e560. ndez-Martínez, S., Lanz-Mendoza, H., Martínez-Barnetche, J., Rodríguez, M.H., Herna 2013. Antimicrobial properties of Anopheles albimanus pericardial cells. Cell Tiss. Res. 351, 127e137. Jacobson, R.L., Doyle, R.J., 1996. Lectin-parasite interactions. Parasitol. Today 12, 55e61. Kiernan, J.A., 2008. Histological and Histochemical Methods: Theory and Practice, fourth ed. Scion Publishing Ltd, Kent, UK, p. 587. Kollien, A.H., Schaub, G.A., 2000. The development of Trypanosoma cruzi in Triatominae. Parasitol. Today 16, 381e387. Lane, N.J., Harrison, J.B., 1979. An unusual cell surface modification: a double plasma membrane. J. Cell Sci. 39, 355e372. Martínez, F.H., Villalobos, G., Cevallos, A.M., de la Torre, P., Laclette, J.P., AlejandreAguilar, R., Espinoza, B., 2006. Taxonomic study of the Phyllosoma complex and other triatomine (Insecta: Hemiptera: Reduviidae) species of epidemiological importance in the transmission of Chagas disease: using ITS-2 and mtCytB sequences. Mol. Phylogenet. Evol. 41, 279e287. Nogueira, N.F., Gonzalez, M.S., Garcia, E.S., de Souza, W., 1997. Effect of Azadirachtin A on the fine structure of the midgut of Rhodnius prolixus. J. Invertebr. Pathol. 69, 58e63. Pereira, M.E., Andrade, A.F., Ribeiro, J.M., 1981. Lectins of distinct specificity in Rhodnius prolixus interact selectively with Trypanosoma cruzi. Science 211, 597e600. rrez-Cabrera, A.E., Salgado-Ramírez, L., Peterson, A.T., S Ramsey, J.M., Gutie anchez~ a, C.N., 2012. Ecological connectivity of Trypanosoma Cordero, V., Ibarra-Cerden cruzi reservoirs and Triatoma pallidipennis hosts in an anthropogenic landscape with endemic Chagas disease. PLoS ONE 7, e46013. Reynolds, E.S., 1963. The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J. Cell. Biol. 17, 208e212. ~o, J.E., 2010. Digestive cells in the Rocha, L.L.V., Neves, C.A., Zanuncio, J.C., Serra midgut of Triatoma vitticeps (Stal, 1859) in different starvation periods. C.R. Biol. 333, 405e415. Rudin, W., Hecker, H., 1988. Lectin-binding sites in the midgut of the mosquitoes, Anopheles stephensi Liston and Aedes aegypti L. (Diptera: Culicidae). Parasitol. Res. 75, 268e279. Shao, L., Devenport, M., Jacobs-Lorena, M., 2001. The peritrophic matrix of hematophagous insects. Arch. Ins. Biochem. 47, 119e125. Silva, C.P., Ribeiro, F.A., Gulbenkian, S., Terra, W.R., 1995. Organization, origin and function of the outer microvillar (perimicrovillar) membranes of Dysdercus peruvianus (Hemiptera) midgut cells. J. Ins. Physiol. 41, 1093e1103. Terra, W.R., 1988. Physiology and biochemistry of insect digestion: an evolutionary perspective. Braz. J. Med. Biol. Res. 21, 675e734. Terra, W.R., 1990. Evolution of digestive systems of insects. Annu. Rev. Entomol. 35, 181e200. Terra, W.R., Ferreira, C., Baker, J.E., 1996. Compartmentalization of digestion. In: Lehane, M.J., Billingsley, P.F. (Eds.), Biology of the Insect Midgut. Chapman & Hall, London, pp. 207e234.
rrez-Cabrera, A.E., et al., Development and glycoprotein composition of the perimicrovillar membrane in Please cite this article in press as: Gutie Triatoma (Meccus) pallidipennis (Hemiptera: Reduviidae), Arthropod Structure & Development (2014), http://dx.doi.org/10.1016/ j.asd.2014.07.001
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Development and glycoprotein composition of the perimicrovillar membrane in Triatoma (Meccus) pallidipennis (Hemiptera: Reduviidae) Q7
rrez-Cabrera a, Ricardo Alejandre-Aguilar b, Salvador Herna ndez-Martínez c, Ana E. Gutie Bertha Espinoza a, * noma de M Departamento de Inmunología, Instituto de Investigaciones Biom edicas, Universidad Nacional Auto exico, Ciudad Universitaria, 04510 Mexico City, D.F., Mexico b gicas, Instituto Polit Departamento de Parasitología, Escuela Nacional de Ciencias Biolo ecnico Nacional, Mexico c n Sobre Enfermedades Infecciosas, Instituto Nacional de Salud Pública, Av. Universidad 655, Col. Sta. María Ahuacatitlan, CP 62508 Centro de Investigacio Cuernavaca, Morelos, Mexico a
Q1
a r t i c l e i n f o
a b s t r a c t
Article history: Received 14 April 2014 Received in revised form 25 June 2014 Accepted 1 July 2014 Available online xxx
Hemipterans and thysanopterans (Paneoptera: Condylognatha) differ from other insects by having an intestinal perimicrovillar membrane (PMM) which extends from the base of the microvilli to the intestinal lumen. The development and composition of the PMM in hematophagous Reduviidae depend on factors related to diet. The PMM may also allow the human parasite Trypanosoma cruzi, the etiological agent of human Chagas Disease, to establish and develop in this insect vector. We studied the PMM development in the Mexican vector of Chagas Disease, Triatoma (Meccus) pallidipennis. We describe changes in the midgut epithelial cells of insects in response to starvation, and at different times (10, 15 and 20 days) after bloodfeeding. In starved insects, the midguts showed epithelial cells closely connected to each other but apparently free of PMM with some regions being periodic acideSchiff (PASeSchiff) positive. In contrast, the PMM was evident and fully developed in the midgut region of insects 15 days after feeding. After this time, the PMM completely covered the microvilli and reached the midgut lumen. At 15 days following feeding the labeled PASeSchiff increased in the epithelial apex, suggesting an increase in carbohydrates. Lectins as histochemical reagents show the presence of a variety of glycoconjugates including mannose, glucose, galactosamine, N-acetyl-galactosamine. Also present were Nacetyl-glucosamine and sialic acid which contribute to the successful establishment and replication or T. cruzi in its insect vectors. By means of scanning electron microscopy (SEM) and transmission electron microscopy (TEM), the formation and structure of the PMM is confirmed at 15 days post feeding. Our results confirmed the importance of the feeding processes in the formation of the PMM and showed the nature of the biochemical composition of the vectors' intestine in this important Mexican vector of Chagas disease. © 2014 Published by Elsevier Ltd.
Keywords: Perimicrovilla membrane Triatoma (Meccus) pallidipennis Trypanosoma cruzi Glycoproteins
1. Introduction Insect members of the order Hemiptera and Thysanoptera (Paraneoptera: Condylognatha) have specific mouth adaptations that allow them to suck sap or liquefy food from different preys or hosts (Terra, 1988). This feeding mode has been associated with modifications of the alimentary canal to digest and absorb nutrients, which are present at extremely low concentration in the putative hemipteran ancestor's food source (Silva et al., 1995; Terra,
* Corresponding author. Tel.: þ52 555 622 8943/44. E-mail address: [email protected] (B. Espinoza).
1990). Nutrient absorption is dependent on the presence of an intestinal membrane with unique characteristics called the perimicrovillar membrane (PMM; Lane and Harrison, 1979) or the extracellular membrane layer. The PMM, comprises lipoproteins, glycoconjugates and carbohydrate-binding molecules (Albuquerque-Cunha et al., 2009), and is secreted from the endoplasmic reticulum of the epithelial cells, in double-membrane et al., vesicles that bud from the Golgi apparatus (Damasceno-Sa 2007). These vesicles reach the midgut cell apices where they are released into the luminal space (Silva et al., 1995; Terra et al., 1996). The PMM surrounds the microvilli of the midgut epithelial cells, which extends from the base of the microvilli to the lumen (Silva et al., 1995). The function of the PMM is similar to that of the
http://dx.doi.org/10.1016/j.asd.2014.07.001 1467-8039/© 2014 Published by Elsevier Ltd.
rrez-Cabrera, A.E., et al., Development and glycoprotein composition of the perimicrovillar membrane in Please cite this article in press as: Gutie Triatoma (Meccus) pallidipennis (Hemiptera: Reduviidae), Arthropod Structure & Development (2014), http://dx.doi.org/10.1016/ j.asd.2014.07.001
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Fig. 1. Histological sections of T. pallidipennis midgut with different feeding conditions showing single columnar epithelial cells with three distinct regions (A, apical; M, medial; B, basal) and the basal membrane (BM). The apical region shows brush border (BB) and an acidophil layer (AL) evidents 15 and 20 days after blood meal (c and d respectively), this layer surrounds the brush border and reaches the midgut lumen (L). The medial region shows round nucleus (N). The muscle layers (ML) was also evident. a, starved group; b, 10 days after blood meal; c, 15 days after blood meal; d, 20 days after blood meal. (100).
peritrophic membrane in other insect orders: it acts as a physical and physiological barrier for enzymes involved in the digestion and absorption processes (Shao et al., 2001). Unlike the peritrophic membrane, however, the PMM is not a chitinous extracellular membrane layer, and has more specific functions such as selective permeability and it is optimized for nutrient absorption (Terra et al., 1996). Despite the different feeding types of insects Hemiptera (phytophagous, zoophytophagous and hematophagous), the PMM composition is similar (Terra, 1990; Albuquerque-Cunha et al., 2004, 2009). However, various studies have shown that PMM production could be explained by the different feeding behaviors et al., 2007; Fialho et al., 2009; Azevedo et al., (Damasceno-Sa 2009). On the apical surface of the midgut epithelium of blood-sucking insects the PMM covers the microvillar membrane of epithelial cells, fills the space between microvilli, and extends into the intestinal lumen (Albuquerque-Cunha et al., 2009). In Rhodnius prolixus the PMM becomes more evident 10 days after a blood meal (Nogueira et al., 1997). More specifically, Gonzalez et al. (1998) and Albuquerque-Cunha et al. (2004), using different experimental protocols, showed that the PMM development in R. prolixus
depends on the insect abdominal distension, ingestion of blood diet components, humoral and hormonal factors. The glycoconjugates and carbohydrate binding molecules that are associated with the PMM of triatomine gut cells, also are involved in the digestive and nutrient absorption processes (Shao et al., 2001), and several investigations have linked glycoconjugates with the interaction with pathogens (Pereira et al., 1981; Dinglasan and Jacobs-Lorena, 2005). In this case, the PMM acts as an adhesion site for Trypanosoma cruzi epimastigotes, an essential process for parasite establishment in the insect vector (Garcia and Azambuja, 2004). Alves et al. (2007) showed that GalNAc, ManNAc, GlcNAc, D-Gal, D-Man and NANA are sites for recognition of PMM by T. cruzi. This explains why a PMM disruption has been correlated with blocking T. cruzi development in triatomine vectors (Cortez et al., 2002, 2012). Given this information, it seems clear that unraveling how the PMM develops is essential to understand how T. cruzi becomes established in the insect vector. However, our knowledge of the triatomine midgut morphology comes mainly from R. prolixus or Triatoma infestans, due to their medical relevance as vectors of Chagas disease in South America. Other species of triatomines may show similar or unique biological characteristics. Triatoma (Meccus) pallidipennis is considered the most important
rrez-Cabrera, A.E., et al., Development and glycoprotein composition of the perimicrovillar membrane in Please cite this article in press as: Gutie Triatoma (Meccus) pallidipennis (Hemiptera: Reduviidae), Arthropod Structure & Development (2014), http://dx.doi.org/10.1016/ j.asd.2014.07.001
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Fig. 2. Scanning electron microscopy of T. pallidipennis midgut in starved condition (a, 5000; b, 15,000) and 15 days after blood meal (c, 5000; d, 15,000). The surface view of midgut from starved insects shows cells with irregular apical surface (MM). 15 days post feeding the PMM (PMM) was arranged as a mesh on and between cells.
vector of T. cruzi in central Mexico due to its large abundance, high prevalence of infection with T. cruzi and extensive contact with human populations (Martínez et al., 2006; Ramsey et al., 2012). In this paper our main aim is to document how the PMM is produced and formed over time by glycoprotein component analysis and histological and ultrastructural studies in T. pallidipennis midgut after starvation and feeding, given that food source is key for PMM development.
2. Materials and methods 2.1. Insect experimental protocol T. pallidipennis were reared and maintained under controlled laboratory conditions in an insectary established at the Escuela gicas, Instituto Polite cnico Nacional, Nacional de Ciencias Biolo Mexico City, Mexico. Fifth-instar nymphs were starved for 30 ± 5 days and after their last molt two groups were formed: one maintained without feeding (starved group), other that was allowed to feed (fed group). Insects from the fed group were dissected 10, 15 and 20 days after feeding. These times were chosen from a pilot study that covered 5e55 days after feeding and which demonstrated major differences in PMM development after 10, 15 and 20 days. Sample sizes for each group (for the case
of the starved group) and subgroups (for the case of the fed group) comprised three animals and there were three independent experiments.
2.2. Light microscopy T. pallidipennis midguts were obtained by dissection from both starved and fed groups, and were fixed for 2 h at 4 C in 4% paraformaldehyde in sodium phosphate buffer (PBS, 2.5 mM Na2HPO4 (BAKER, Xalostoc, Mex); 7.97 mM NaH2PO4 (BAKER, Xalostoc, Mex); 154 mM NaCl (Sigma, St Louis, MO)) pH 7.2. After fixation, samples were washed twice in PBS followed by alcoholic dehydration (50%, 70%, 90%, 96% and 100% ethanol), and xylene clarification. Finally, tissues were embedded in Paraplast X-Tra (Sigma, St Louis, MO) and sections (5 mm-thick) were obtained with a microtome (Leica Mod Jang RM2025). Sections were stained with hematoxylineeosin (Kiernan, 2008) and the periodic acideSchiff method (PASeSchiff) to identify tissues containing neutral hexose sugars and sialic acids (Kiernan, 2008). Twelve histological sections per insect were assessed, and each section was analyzed with an optic microscope in bright field (Optiphot-2 Nikon) covering at least five fields. Thus, 540 fields at 100 objective for each histological analysis of starved and fed insect groups were analyzed over three replicates.
rrez-Cabrera, A.E., et al., Development and glycoprotein composition of the perimicrovillar membrane in Please cite this article in press as: Gutie Triatoma (Meccus) pallidipennis (Hemiptera: Reduviidae), Arthropod Structure & Development (2014), http://dx.doi.org/10.1016/ j.asd.2014.07.001
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2.3. Scanning electron microscopy Scanning electron microscopy (SEM) was used to study the formation of PMM in the starved and the fed groups up to 15 days after feeding. Midgut tissues were fixed in 4% paraformaldehyde in PBS pH 7.2 for 2 h at RT, washed twice in PBS, and then dehydrated with a gradual ethanol concentration series (50%, 70%, 90%, 96% and 100%). Tissues were critical point-dried with CO2, mounted on stubs, coated with a 20-nm-thick gold film, and examined with a JEOL Scanning Electron Microscope (JSM6360LV). 2.4. Transmission electron microscopy Midgut tissues from starved and fed (15 days post-feeding) samples were processed for transmission electron microscopy (TEM). Samples were obtained as described before and immediately fixed in 4% glutaraldehyde (Ted-Pella, Redding, CA) in phosphate buffered saline (PBS, pH 7.4). After 24 h at RT, the specimens were washed with PBS and post-fixation was performed with 1% of osmium tetroxide (EMS, FT Washington, PA) in PBS during 2 h at RT. Dehydrated using progressive ethanol concentrations and embedded in DER-ERL resin (EMS, FT Washington, PA). Semi-thin sections (0.5 mm) were stained with toluidine blue (EMS, FT Washington, PA). Semi-thin sections were obtained using an RMC ultramicrotome (Model MTX, Tucson, AZ) to observe the midguts under light microscopy. At least two areas were selected in semithin sections, and were prepared for electron microscopy. Thin sections (80 nm) were contrasted with 5% uranyl acetate and lead citrate (lead 80 mM nitrate, 120 mM sodium citrate and 160 mM NaOH; all from EMS, FT Washington, PA) (Reynolds, 1963), and examined in a Jeol transmission electron microscope (JEM-1011) (Hern andez-Martínez et al., 2013). 2.5. Lectins as histochemical markers Midguts from the starved and the fed subgroups (15 days postfeeding) were fixed, dehydrated, clarified and embedded in paraffin
as described above. These samples were later re-hydrated and incubated for 1 h at RT in the presence of 25 mg/mL of biotinylated lectins (all from Sigma, Toluca, Mexico) prepared in chloride buffer pH 7.4. The biotinylated lectins were Canavalia ensiformis (ConA), Arachis hypogeal (PNA) and Triticum vulgaris (WGA). After incubations, samples were washed with PBS, and incubated with Streptavidinefluorescein conjugate (Amersham Biosciences RPN1232, Freiburg Germany) diluted 1:200 in bicarbonate buffer (NaHCO3 0.1 M and ClNa 0.1 M, pH 8.3). Samples were washed again with PBS and counterstained with DAPI (40 ,6-diamidino-2-phenylindole) (Molecular Probes, Oregon) diluted 1:1000 in PBS. Finally, samples were washed with PBS and mounted on a glass slide using fluoro-gel (EMS, Tlalpan, Mexico), and observed under an Olympus confocal microscope (Model BX51WI) coupled to a Disk Scanning Unit (DSU). Using ConA, it was possible to identify mannose and glucose residues. With PNA galactosamine and Nacetyl-galactosamine were detected, and using WGA it was possible to identify sialic acid and N-acetyl-glucosamine. 3. Results 3.1. General morphology T. pallidipennis midguts comprised a simple columnar epithelium, surrounded by a muscular layer that consists of an inner oblique longitudinal layer and an outer oblique transverse layer. The midgut epithelial cells are typically single columnar composed of three distinct regions: apical, medial and basal. The basal membrane was also clearly observed (Fig. 1). The apical region shows brush border and an acidophilic layer. In the medial region there are round nuclei and also staining granules as can be seen in the basal region. Using the time course analysis, it was possible to see that the arrangement mainly of the apical region, as well as of some of its subcellular structures, becomes modified over time after ingestion of a meal (Fig. 1aed). In the starved group (Fig. 1a), the midgut epithelial cells were closely connected by the medial and basal
Fig. 3. Transmission electron microscopy of T. pallidipennis midgut in starved condition (a, 25,000) and 15 days after blood meal (b, 15,000). The starved condition midgut shows the microvilli (MM) extending into the lumen (L); while the vector midgut 15 days after blood meal shows the PMM on and surrounding the microvilli extending toward the lumen (L).
rrez-Cabrera, A.E., et al., Development and glycoprotein composition of the perimicrovillar membrane in Please cite this article in press as: Gutie Triatoma (Meccus) pallidipennis (Hemiptera: Reduviidae), Arthropod Structure & Development (2014), http://dx.doi.org/10.1016/ j.asd.2014.07.001
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region, with brush border at the cell apex and free of projections at the luminal surface of the apical region (Fig. 1a). By 10 days postfeeding, a general view of the cell surface revealed noncontinuous projections at the luminal surface of the apical region suggesting the initial formation of PMM (Fig. 1b). There was an increase in cytoplasmic contents and a large number of vesicles in the cytoplasm (Fig. 1b). Midgut epithelial cells of insects that were fed 15 days previously had epithelium with folds in the apical portion and a brush border densely-packed that was homogeneously-distributed. In this post-feeding time, we observed a full development of an acidophil layer (Fig. 1c). This layer surrounds the brush border and reaches the midgut lumen, suggesting the presence of the PMM (from here on this layer will be named PMM). The lumen is irregular in shape owing to longitudinal folds (Fig. 1c). Twenty days after feeding the layer that surrounds the brush border was less evident and had surface disruptions (Fig. 1d). 3.2. Scanning and transmission electron microscopy observation of the midgut In order to follow the development of membrane structure, T. pallidipennis midgut was observed on SEM and TEM. Based on the SEM, the cell surfaces of starved midgut show an irregular apical
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surface (Fig. 2a and b). The absence of PMM was supported by transversal sections viewed with TEM observing the microvilli devoid of PMM (Fig. 3a). The time of full development of an acidophil layer observed with light microscopy was 15 days post feeding. Using the SEM, we could observe that this layer, is arranged as a mesh on and between cells extending into the lumen, in the same day (Fig. 2c and d). According to TEM, the layer mentioned is the PMM and is on the cell surface as extending toward the lumen like thin pipes but in turn is found surrounding the microvilli (Fig. 3b). The microvilli in both the starvation and 15 days post feeding, seen by TEM, looks very well organized, especially at 15 days post feeding, appreciating its double membrane (Fig. 3a and b). 3.3. Localization of carbohydrates 3.3.1. PASeSchiff Animals from the starved group showed midgut regions with glycoconjugates mainly on the brush border and basal membrane (Fig. 4a). However, in midgut regions and cytoplasmic granules of digestive cells of fed animals, there was an increase in PASeSchiff positive regions on the epithelial apex and on the basal membrane (Fig. 4bed). By 10 days after feeding, the cell cytoplasm was more highly colored (Fig. 4b); midgut cells at 15 days after feeding had an
Fig. 4. PASeSchiff positive regions of T. pallidipennis midgut with different feeding conditions. The midgut region with Pass-Schiff positive staining was the brush border (BB) and basal membrane (BM). Midgut cells at 15 and 20 days after feeding had an evident layer surrounding the brush border (PMM), extending into the lumen (L) that was also Pass-Schiff positive. a, starved group; b, 10 days after blood meal; c, 15 days after blood meal; d, 20 days after blood meal. N: nucleus. (100).
rrez-Cabrera, A.E., et al., Development and glycoprotein composition of the perimicrovillar membrane in Please cite this article in press as: Gutie Triatoma (Meccus) pallidipennis (Hemiptera: Reduviidae), Arthropod Structure & Development (2014), http://dx.doi.org/10.1016/ j.asd.2014.07.001
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evident layer surrounding the brush border (possibly, the PMM) which was PASeSchiff positive (Fig. 4c), and 20 days after feeding this layer was evident, however the coloration was discontinuous (Fig. 4d). 3.3.2. Glycan identification using lectins In the starved group ConA labeled the apical surface, membrane basal and cytoplasm of midgut cells, especially under the cellular apex (Fig. 5a). In insects 15 days after feeding, all regions of the epithelium were intensely labeled, and there was an increase in the cytoplasm labeling compared with the starved group, especially under the cellular apex (Fig. 5b). PNA was only detected in starved insects on the apical surface and the basal membrane (Fig. 5c). In the midguts of insects 15 days post feeding, there was an increase in the labeling of the epithelial surface, including the layer that surrounds the brush border (the PMM), as well as in granules distributed throughout the cytoplasm and especially under cellular apex (Fig. 5d). In starved groups, WGA was detected lightly on the apical surface of midgut cells and in the basal membrane (Fig. 5e). In contrast, in the insects 15 days after feeding, WGA was detected more strongly in the apical surface comprising the PMM and the cytoplasm (Fig. 5f). 4. Discussion In starved animals of T. pallidipennis, the microvilli were practically deprived of a PMM covering. At 10 days post-feeding the midgut cells had already developed a layer surrounding the brush border suggesting the formation of PMM, which was more evident and developed 15 days post-feeding. Twenty days post-feeding the covering membrane was reduced and PMM disruption was seen. The SEM and TEM of midguts of starved insects indicated that the PMM was still evident on the margins of individual cells and regions between the cells, also the apical microvilli not exhibit uniform organization in whole cell. Furthermore, the midgut of insects within 15 days after feeding, the PMM was located above and between microvilli and extended toward the lumen, apparently form a continuous domain from one cell to another. The PMM has a narrow tubular arrangement from the cellular surface to the lumen; however, Silva et al. (1995) mentioned that depending on the preparation, the perimicrovillar tubes may appear folded or almost perpendicular to the cell surface. Similar data have been described in other species of triatomines such as R. prolixus, T. infestans and Triatoma vitticeps; by 7 days after a blood meal the PMM layer was still incomplete although it was thick and had a smooth surface, hindering the visualization of individual cells (Billingsley and Downe, 1986; Rocha et al., 2010). Furthermore, at 10 days after a blood meal, the PMM reached its maximum development (Burgos and Gutierrez, 1976; Billingsley and Downe, 1983; Kollien and Schaub, 2000). Moreover, the development of PMM can be understood at a higher taxonomic level. In other hemipterans such as the hematophagous Cimex hemipterus (Hemiptera: Comocidae), the phytophagous Dysdercus peruvianus (Hemiptera: Pyrrhocoridae) and the zoophytophagous Brontocoris tabidus (Heteroptera: Pentatomidae), the PMM synthesis varies according to their feeding mode. In C. hemipterus, the PMM is evident 20 days post feeding (Azevedo et al., 2009); in D. peruvianus, PMM recovered all the cells 30 h post-feeding et al., 2007); while in B. tabidus, the PMM is (Damasceno-Sa
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evident in starved and fed condition (Fialho et al., 2009). Development of PMM in hemipterans varies according to how frequently the animal has access to food: in phytophagous and zoophytophagous species access to food seems frequent (Damasceno et al., 2007; Fialho et al., 2009), while in blood-suckers such Sa access is less frequent as hosts are harder to find (Billingsley, 1990; Nogueira et al., 1997; Azevedo et al., 2009). Thus, in comparative terms, it seems that digestion in T. pallidipennis takes place at a slower than the species mentioned. We observed more PASeSchiff positive regions in the midguts of fed animals than in starved animals. The PASeSchiff positive reaction suggests the presence of glycoconjugates. This means that starved midgut have fewer glycoconjugates in the epithelial apex, basal membrane and some cytoplasmic granules; contrary to what was found in midgut with 15 days after feeding, because the labeled PAS increased in the epithelial apex, above all in the extracellular membrane layer, and this corresponds to the PMM. Different papers have reported that glycoconjugates are associated with the plasma membrane of insect cells and are usually involved in the interaction mechanisms with pathogens (Pereira et al., 1981; Rudin and Hecker, 1988; Jacobson and Doyle, 1996; Dinglasan and JacobsLorena, 2005). In this paper we found that the PMM contained a variety of glycoconjugates that showed differences in the abundance of sugar residues in the midgut of starved insects or 15 days post feeding, which could impact the development of T. cruzi in T. pallidipennis. In this paper we used three lectins: Con A, PNA and WGA. According to previous descriptions, these lectins can detect important sugars involved for adhesion of parasites (Alves et al., 2007). Labeling of tissues from the starved group was less intense than in tissues 15 days after feeding with different lectins. Con A for example, leads to an intense labeling of the midgut of starved animals but increased in the midgut of animals within 15 days post feeding, indicating mannose and/or glucose as a major sugar residue in cross sections of midguts under conditions of starvation and feeding. However, there was an increase in labeling 15 days after the feeding, in all cross sections and PMM. Other sugars binding to molecules were N-acetyl-galactosamine and galactosamine, recognized by the lectin PNA. Actually, the labeling was more evident 15 days after feeding specially on the cellular apex. Nacetyl-D-glucosamine and sialic acid, recognized by the lectin WGA, was more strongly detected on PMM and cytoplasmic contents. It was possible to label intracellular glycoconjugates as we used paraformaldehyde as a tissue fixative to permeabilize the plasma membrane to facilitate the entrance of lectineFITC complex. Intracellular and extracellular labeling was expected as glycoconjugates are produced by the cells and exported to the apical surface where they are incorporated by both MM and PMM. This suggests the existence of some similarities between the midgut cells of starved and fed groups: an increase in the sugar residues, especially mannose, glucose, N-acetyl-D-glucosamine and sialic acid. According to Albuquerque-Cunha et al. (2009), the main differences between MM and PMM may reflect both changes in function of the prolonged contact with the intestinal microenvironment and enzymatic dispersion along the great extension of the system composed by the plasma membranes of the insect midgut. It is possible that the major expression of sialic acid (NANA) and mannose residues found in midgut cuts of T. pallidipennis within 15 days after feeding are related to the time of parasite multiplication.
Fig. 5. Confocal laser-scanning microscopy of the cross sections of midgut epithelial cells of T. pallidipennis. The starved group labeled with ConA (a), PNA (c) and WGA (e) showed less fluorescence compared with the group with15 days after feeding using the same lectins: ConA (b), PNA (d) and WGA (f). This last group showed intense staining in the brush border (BB), the layer surrounded it (PMM) and the cytoplasm of the apical region (double arrow). A higher staining in the cytoplasm was observed with WGA that with the other lectins (f). ML: muscle layer; N: nucleus; L: lumen. (60).
rrez-Cabrera, A.E., et al., Development and glycoprotein composition of the perimicrovillar membrane in Please cite this article in press as: Gutie Triatoma (Meccus) pallidipennis (Hemiptera: Reduviidae), Arthropod Structure & Development (2014), http://dx.doi.org/10.1016/ j.asd.2014.07.001
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Alves et al. (2007) demonstrated that T. cruzi epimastigote hydrophobic proteins and carbohydrate residues of R. prolixus PMM glycoproteins (mainly NANA and man) with 10 days after feeding are important factors for the parasite/vector interaction. Based on the above data, the main difference between T. pallidipennis and R. prolixus as vectors could be the time taken to form the PMM and produce the appropriate conditions for T. cruzi to replicate and continue its life cycle within the vector. This idea is supported by the observations showing that the development of the parasite is directly related to the PMM development (Gonzalez et al., 1998; Garcia et al., 1999, 2007; Cortez et al., 2002; Garcia and Azambuja, 2004; Alves et al., 2007). If this is true, then parasite development is constrained by the biochemical conditions of the vectors' intestine. Therefore, is necessary to continue a detail mapping of the proteins and carbohydrates, especially of the PMM, which will give us basis for understanding the processes of digestion but mostly to understand the relationships between triatomines and T. cruzi. Q6
Uncited reference García et al., 1989. Acknowledgments
We thank L.A. Morales Arreola, Ma. L. Martínez Ramírez and L. Romero Romero from Departamento de Patología, Facultad de noma Medicina Veterinaria y Zootecnia, Universidad Nacional Auto xico, Mexico City, Mexico (UNAM) for processing of samples; de Me Y. Hornelas from the Instituto de Ciencias de Mar y Limnología, UNAM, for providing access to SEM facilities; M. Tapia Rodríguez for microscopy assistance; W. Espinoza and I. Martínez for logistic rdoba-Aguilar and C. Lowenberger provided comsupport. A. Co ments to the manuscript. This paper is part of the doctoral thesis of dicas of A. Gutierrez-Cabrera in the Doctorado de Ciencias Biome noma de Me xico (UNAM). A.E. the Universidad Nacional Auto rrez-Cabrera acknowledges the scholarship and financial Gutie Q2 support provided by the National Council of Science and Technology (CONACyT), and Program in Biomedical Sciences of the UNAM. Q3 This project was partially supported for a grant to Bertha Espinoza n Miguel Alema n (2013) and DGAPA-UNAM Q4,5 from Fundacio (IN206512). References Albuquerque-Cunha, J.M., Mello, C.B., Garcia, E.S., Azambuja, P., de Souza, W., lez, M.S., Nogueira, N.F., 2004. Effect of blood components, abdominal Gonza distension, and ecdysone therapy on the ultrastructural organization of posterior midgut epithelial cells and perimicrovillar membranes in Rhodnius prolixus. Mem. Inst. Oswaldo Cruz 99, 815e822. Albuquerque-Cunha, J.M., Gonz alez, M.S., Garcia, E.S., Mello, C.B., Azambuja, P., Almeida, J.C., de Souza, W., Nogueira, N.F., 2009. Cytochemical characterization of microvillar and perimicrovillar membranes in the posterior midgut epithelium of Rhodnius prilixus. Arthropod Struct. Dev. 38, 31e44. Alves, C.R., Albuquerque-Cunja, J.M., Mello, C.B., Garcia, E.S., Nogueira, N.F., Bourguingnon, S.C., de Souza, W., Azambuja, P., Gonzalez, M.S., 2007. Trypanosoma cruzi: attachment to perimicrovillar membrane glycoproteins of Rhodnius prolixus. Exp. Parasitol. 116, 44e52. Azevedo, D.O., Neves, C.A., Santos-Mallet, J.R., Goncalves, T.C.M., Zanuncio, J.C., Serrao, J.E., 2009. Notes on midgut ultrastructure of Cimex hemipterus (Hemiptera: Cimicidae). J. Med. Entomol. 46, 435e441. Billingsley, P.F., Downe, A.E.R., 1983. Ultrastructural changes in posterior midgut cells associated with blood feeding in adult female Rhodnius prolixus Stal (Hemiptera: Reduviidae). Can. J. Zool. 61, 2574e2586. Billingsley, P.F., Downe, A.E., 1986. The surface morphology of the midgut cells of Rhodnius prolixus Stal (Hemiptera Reduviidae) during blood digestion. Acta Trop. 43, 355e366. Billingsley, P.F., 1990. The midgut ultrastructure of hematophagous insects. Annu. Rev. Entomol. 35, 219e248.
Burgos, M.H., Gutierrez, L.S., 1976. The intestine of Triatoma infestans I. Cytology of the midgut. J. Ultrastruct. Res. 57, 1e9. Cortez, M.G.R., Gonzalez, M.S., Cabral, M.M.O., Garcia, E.S., Azambuja, P., 2002. Dynamic development of Trypanosoma cruzi in Rhodnius prolixus: role of decapitation and ecdysone therapy. Parasitol. Res. 88, 697e703. Cortez, M.R., Procencano, A., Silva, C.E., Mello, C.B., Zimmermann, L.T., Schaub, G.A., Garcia, E.S., Azambuja, P., Gonzalez, M.S., 2012. Trypanosoma cruzi: effects of azadirachtin and ecdysone on the dynamic development in Rhodnius prolixus larvae. Exp. Parasitol. 131, 363e371. , J., Carneiro, N.B., DaMatta, R.A., Samuels, R.I., Terra, W.R., Silva, C.P., Damasceno-Sa 2007. Biphasic perimicrovillar membrane production following feeding by previously starved Dysdecus peruvians (Hemiptera: Pyrrhocoridae). J. Ins. Physiol. 53, 592e600. Dinglasan, R.R., Jacobs-Lorena, M., 2005. Insight into a conserved lifestyle: proteincarbohydrate adhesion strategies of vector-borne pathogens. Infect. Immun. 73, 7797e7807. Fialho, M.D.C., Zanuncio, J.C., Neves, C.A., Ramalho, F.S., Serrao, J.E., 2009. Ultrastructure of the digestive cells in the midgut of the predator Brontocoris tabidus (Heteroptera: Pentatomidae) after different feeding periods on prey and plants. Ann. Entomol. Soc. Am. 102, 119e127. García, E.S., Gonzalez, M.S., Azambuja, P., Rembold, H., 1989. Chagas' disease and its insect vector. Effect of azadirachtina A on the interaction of a triatomine host (Rhodnius prolixus) and its parasite (Trypanosoma cruzi). J. Biosci. 44, 317e322. Garcia, E.S., Gonzalez, M.S., Azambuja, P., 1999. Biological factors involving Trypanosoma cruzi life cycle in the invertebrate vector, Rhodnius prolixus. Mem. Inst. Oswaldo Cruz 94, 213e216. Garcia, E.S., Azambuja, P., 2004. Lignoids in insects: chemical probes for the study of ecdysis, excretion and Trypanosoma cruzidtriatomine interactions. Toxicon 44, 431e440. Garcia, E.S., Ratcliffe, N.A., Whitten, M.M., Gonzalez, M.S., Azambuja, P., 2007. Exploring the role of insect host factors in the dynamics of Trypanosoma cruzieRhodnius prolixus interactions. J. Ins. Physiol. 53, 11e21. Gonzalez, M.S., Nogueira, N.F., Feder, D., de Souza, W., Azambuja, P., Garcia, E.S., 1998. Role of the head in the ultrastructural midgut organization in Rhodnius prolixus larvae: evidence from head transplantation experiments and ecdysone therapy. J. Ins. Physiol. 44, 553e560. ndez-Martínez, S., Lanz-Mendoza, H., Martínez-Barnetche, J., Rodríguez, M.H., Herna 2013. Antimicrobial properties of Anopheles albimanus pericardial cells. Cell Tiss. Res. 351, 127e137. Jacobson, R.L., Doyle, R.J., 1996. Lectin-parasite interactions. Parasitol. Today 12, 55e61. Kiernan, J.A., 2008. Histological and Histochemical Methods: Theory and Practice, fourth ed. Scion Publishing Ltd, Kent, UK, p. 587. Kollien, A.H., Schaub, G.A., 2000. The development of Trypanosoma cruzi in Triatominae. Parasitol. Today 16, 381e387. Lane, N.J., Harrison, J.B., 1979. An unusual cell surface modification: a double plasma membrane. J. Cell Sci. 39, 355e372. Martínez, F.H., Villalobos, G., Cevallos, A.M., de la Torre, P., Laclette, J.P., AlejandreAguilar, R., Espinoza, B., 2006. Taxonomic study of the Phyllosoma complex and other triatomine (Insecta: Hemiptera: Reduviidae) species of epidemiological importance in the transmission of Chagas disease: using ITS-2 and mtCytB sequences. Mol. Phylogenet. Evol. 41, 279e287. Nogueira, N.F., Gonzalez, M.S., Garcia, E.S., de Souza, W., 1997. Effect of Azadirachtin A on the fine structure of the midgut of Rhodnius prolixus. J. Invertebr. Pathol. 69, 58e63. Pereira, M.E., Andrade, A.F., Ribeiro, J.M., 1981. Lectins of distinct specificity in Rhodnius prolixus interact selectively with Trypanosoma cruzi. Science 211, 597e600. rrez-Cabrera, A.E., Salgado-Ramírez, L., Peterson, A.T., S Ramsey, J.M., Gutie anchez~ a, C.N., 2012. Ecological connectivity of Trypanosoma Cordero, V., Ibarra-Cerden cruzi reservoirs and Triatoma pallidipennis hosts in an anthropogenic landscape with endemic Chagas disease. PLoS ONE 7, e46013. Reynolds, E.S., 1963. The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J. Cell. Biol. 17, 208e212. ~o, J.E., 2010. Digestive cells in the Rocha, L.L.V., Neves, C.A., Zanuncio, J.C., Serra midgut of Triatoma vitticeps (Stal, 1859) in different starvation periods. C.R. Biol. 333, 405e415. Rudin, W., Hecker, H., 1988. Lectin-binding sites in the midgut of the mosquitoes, Anopheles stephensi Liston and Aedes aegypti L. (Diptera: Culicidae). Parasitol. Res. 75, 268e279. Shao, L., Devenport, M., Jacobs-Lorena, M., 2001. The peritrophic matrix of hematophagous insects. Arch. Ins. Biochem. 47, 119e125. Silva, C.P., Ribeiro, F.A., Gulbenkian, S., Terra, W.R., 1995. Organization, origin and function of the outer microvillar (perimicrovillar) membranes of Dysdercus peruvianus (Hemiptera) midgut cells. J. Ins. Physiol. 41, 1093e1103. Terra, W.R., 1988. Physiology and biochemistry of insect digestion: an evolutionary perspective. Braz. J. Med. Biol. Res. 21, 675e734. Terra, W.R., 1990. Evolution of digestive systems of insects. Annu. Rev. Entomol. 35, 181e200. Terra, W.R., Ferreira, C., Baker, J.E., 1996. Compartmentalization of digestion. In: Lehane, M.J., Billingsley, P.F. (Eds.), Biology of the Insect Midgut. Chapman & Hall, London, pp. 207e234.
rrez-Cabrera, A.E., et al., Development and glycoprotein composition of the perimicrovillar membrane in Please cite this article in press as: Gutie Triatoma (Meccus) pallidipennis (Hemiptera: Reduviidae), Arthropod Structure & Development (2014), http://dx.doi.org/10.1016/ j.asd.2014.07.001
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