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Comparative analysis of carbohydrate residues in the midgut of phlebotomines (Diptera: Psychodidae) from colony and field populations from Amazon, Brazil
Accepted Manuscript Comparative analysis of carbohydrate residues in the midgut of phlebotomines (Diptera: Psychodidae) from colony and field populations from Amazon, Brazil Davi Marcos Souza de Oliveira, Bruno José Martins da Silva, Chubert Bernardo Castro De Sena, José Aprígio Nunes Lima, Thiago Vasconcelos dos Santos, Fernando Tobias Silveira, Edilene Oliveira Silva PII:
S0014-4894(16)30114-X
DOI:
10.1016/j.exppara.2016.06.002
Reference:
YEXPR 7257
To appear in:
Experimental Parasitology
Received Date: 1 March 2016 Revised Date:
30 May 2016
Accepted Date: 1 June 2016
Please cite this article as: de Oliveira, D.M.S., Martins da Silva, B.J., De Sena, C.B.C., Lima, J.A.N., Vasconcelos dos Santos, T., Silveira, F.T., Silva, E.O., Comparative analysis of carbohydrate residues in the midgut of phlebotomines (Diptera: Psychodidae) from colony and field populations from Amazon, Brazil, Experimental Parasitology (2016), doi: 10.1016/j.exppara.2016.06.002. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Comparative Analysis of Carbohydrate Residues in the Midgut of Phlebotomines
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(Diptera: Psychodidae) from Colony and Field Populations from Amazon, Brazil.
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Davi Marcos Souza de Oliveira1,2,3, Bruno José Martins da Silva1,2,3, Chubert Bernardo Castro De Sena2,
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José Aprígio Nunes Lima4, Thiago Vasconcelos dos Santos4, Fernando Tobias Silveira ,4,5, Edilene
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Oliveira Silva1,2,3*
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Brazil. Post code: 66075-900 2
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Laboratory of Parasitology Institute of Biological Sciences, Federal University of Pará; Belém, PA,
Laboratory of Structural Biology, Belém, Pará, Brazil. Post code: 66075-900
National Institute of Science and Technology in Structural Biology and Bioimaging, Rio de Janeiro, Rio
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de Janeiro, Brazil. Rio de Janeiro, Brazil. Post code: 21941-902; 4
Laboratory of Leishmaniasis ‘Prof Dr. Ralph Lainson’, Evandro Chagas Institute, Ministry of Health,
Ananindeua, Pará, Brazil. Post code: 67030-000 5
Tropical Medicine Nucleus, Federal University of Pará Bélem, Pará, Brazil. Post code: 66055-240
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* Author for correspondence: Dr. Edilene O. Silva-Federal University of Pará, Institute of Biological
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Sciences, Laboratory of Parasitology and Laboratory of Structural Biology, Augusto Corrêa Av., 01,
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Guamá, 660975-110, Belém, Pará, Brazil. Phone/Fax: 0559132017102, Email address: [email protected]
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Highlights
• Lutzomyia antunesi and Lutzomyia flaviscutellata, vectors of cutaneous leishmaniasis, present GalNAc on midgut epithelial.
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• Lutzomyia longipalpis s.l midgut presents residues of GalNAc, mannose, galactose and GlcNAc.
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• Carbohydrate characterization improve the knowledge of Phlebotomine-Leishmania interaction.
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Abstract
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Leishmaniasis are worldwide diseases that occur in 98 countries including Brazil, transmitted by the bite
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of female phlebotomines during blood feeding. In Brazil it is known that some species of sand flies as
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Lutzomyia longipalpis s.l. (vector of Leishmania infantum chagasi), Lutzomyia flaviscutellata (vector of
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Leishmania (Leishmania) amazonensis) and Lutzomyia antunesi (is the proven vector of Leishmania
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(Viannia) lindenbergi) are incriminated of transmitting the parasite Leishmania for the vertebrate host.
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The phlebotomine-parasite is mediated by the attachment of the promastigote lipophosphoglycan (LPG)
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to the midgut epithelium. However, another mechanism that is LPG-independent and mediated by N-
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acetyl-galactosamine (GalNAc). The aim of this study was to characterize the carbohydrate residues that,
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probably, play a role in parasite attachment to the midgut of phlebotomine from colony and field
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populations from the Brazilian Amazonian region. We observed the presence of GalNAc, Mannose,
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Galactose and GlcNAc in all phlebotomine species. A binding assay between L. (L.) amazonensis and L.
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infantum chagasi to the midguts of different species of phlebotomines was performed. The attachment of
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both Leishmania and vector species suggests the presence of GalNAc on the midgut surfaces. Thus, these
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results suggested that GalNAc is a possible binding sites of Leishmania in sand flies from the Brazilian
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Amazonian region.
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Keywords: Psychodidae, Leishmania, Midgut surface, Lectin, Host-parasite interaction, Glycoconjugates.
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1. Introduction
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Leishmaniasis are infectious diseases caused by protozoan parasites of the genus
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Leishmania (Trypanosomatidae: Kinetoplastidae) that are transmitted by the bite of female phlebotomines
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(Diptera: Psychodidae) during blood feeding (WHO, 2015). Phlebotomines become infected when they
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ingest mammalian blood that contains the amastigote forms inside of macrophages that release the
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parasites into the midgut. Inside the vector, amastigotes differentiate to an infective metacyclic stage
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promastigotes (Ramalho-Ortigao et al., 2010). However, some species of phlebotomines, classified as
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specific or restrictive, allow the development of only one Leishmania species, while others, which are
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classified as permissive, allow the development of more than one species of Leishmania (Myskova et al.,
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2007).
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The establishment of infection depends on a parasite’s capacity to overcome natural
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barriers such as digestive enzymes produced by epithelial cells (Pimenta et al., 1992). In the early stage of
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development, leishmanias are protected against hydrolytic activities by peritrophic matrix (Pimenta et al.,
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1997) However, parasites need to escape from the endoperitrophic space and attach to the midgut
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epithelium to avoid being excreted with the blood remnants (Pimenta et al., 1992). This adhesion depends
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on the interaction between molecules present on the surface of the parasite and the midgut epithelium.
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In a species-specific interaction, such as Phlebotomus papatasi and Leishmania major, the
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adhesion is mediated by lipophosphoglycan (LPG), a linear chain of phosphorylated disaccharide
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repeating units that are bound to cell membranes by a lipid anchor. This molecule is abundantly present
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on flagellar forms only and presents structure variations during the life cycle of the parasite ( Assis et al.,
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2012; Pimenta et al., 1994). During parasite-sand fly interactions, carbohydrate residues that are present
23
on LPG are recognized by lectins as binding sites on midgut epithelial cells (Pimenta et al., 1994). In the
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midgut of P. duboscqui and P. papatasi there is a lectin (PpGalec) that is specific for L. major LPG.
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PpGalec seems to be specific for L. major LPG since it was detected at low concentration in Lu.
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longipalpis s.l. and was absent in P. sergenti and P. argentipes (Kamhawi et al., 2004).
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Some studies showed the role of LPG in the Leishmania-parasite interaction. Butcher et al.
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(1996) and Jecna et al. (2013), showed that a strain of L. major LPG mutant was not able to adhere on
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epithelial surface of its natural vector P. papatasi. In addition, parasite-phlebotomine interactions seem to
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occur through a LPG-independent mechanism. Experimental infections showed that Phlebotomus
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perniciosus and Lu. longipalpis s.l. permitted the development of L. major wild types and LPG mutants in
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the same proportion, suggesting that the interaction between the parasite and these sand fly species was
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not mediated by LPG (Myskova et al., 2007; Svárovská et al., 2010).
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One of possible mechanism involved in the interaction of permissive phlebotomines and
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parasites probably depends on the carbohydrates that are present on the midgut epithelium which are
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recognized by lectins that are present on the surface of Leishmania (Azevedo-Pereira et al., 2007; Côrtes
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et al., 2012). Midgut biochemical analyses of the permissive phlebotomines Lu. longipalpis s.l.,
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Phlebotomus halepensis, Ph. perniciosus, Phlebotomus argentipes and Ph. arabicus showed that all of
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these species present N-acetyl-galactosamine (GalNAc) residues on the microvillar border whereas the
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midguts of specific vectors lack GalNAc (Myskova et al., 2007).
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Although several studies have established the structure and the role of LPG in Leishmania-
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sand fly interactions, there are few studies about the characterization of epithelial carbohydrate residues in
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the midgut of sand flies. Therefore, this is the first study of carbohydrate characterization of
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epidemiological important species from the Amazonian region using lectins to identify GalNAc,
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mannose, galactose and GlcNAc residues of the midgut epithelium of sand flies.
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2. Materials and Methods
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2.1 Sand flies
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2.1.1 Sand flies from colony: Lutzomyia longipalpis s.l. specimens were obtained from a long-term closed colony (F38)
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of Instituto Evandro Chagas (IEC) primarily captured in the municipality of Cametá. The insects were
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reared and maintained in a closed colony at 25 ºC, 80% relative humidity on a 50% sucrose diet.
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2.1.2 Sand flies from field:
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Phlebotomines were captured in Barcarena (01º30’24’’ S and 48º37’12’’ W) and Cametá
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(02º14'40" S and 49º29'45" W) municipalities of Pará State. The capture of phlebotomines was carried out
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from 2013 to 2015 for five consecutive days per year using CDC light traps (Center for Disease Control
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and Prevention) that were installed at 5:00 pm, and the sand flies were collected at 6:00 am of the
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following day. The traps were installed at 1.5 meters above ground level in the peridomiciliary and
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forested environments. Captured sand flies were transported to the laboratory and identified according to
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Young and Duncan (Young and Ducan, 1994).
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2.2 Detection of glycoconjugates on the sand fly midgut by FITC labeled lectins. After four days of sugar feeding, female sand flies from both the colony and field
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populations were dissected in drops of 1% paraformaldehyde. Three midguts from different species were
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used for each lectin. The midguts were fixed in 4% paraformaldehyde at 4 °C for 20 minutes and opened
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longitudinally by a 1 mL needle syringe under a Zeiss stereoscopic microscope. Non-specific sites were
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blocked by 1% Bovine Serum Albumin (BSA) for 30 minutes. After that, the midguts were incubated for
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1 hour in a dark, humid chamber, separately, with FITC- conjugated lectins Helix pomatia agglutinin
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(HPA), concanavalin-A (CON-A), peanut agglutinin (PNA) and wheat germ agglutinin (WGA) (1:100),
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that bind specifically to GalNAc, mannose, galactose and GlcNac, respectively (Sharon and Lis, 2004).
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Subsequently, the midguts were washed three times in PBS pH 8.0 for 10 minutes and incubated with
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4',6-diamidino-2-phenylindole (DAPI) DNA marker and actin/myosin marker (phalloidin) for 40 minutes.
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In the negative control, HPA lectin was incubated first with 250 mM of GalNAc for 1 h. To perform the
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negative control of mannose, galactose and GlcNAc, the midguts were first incubated with non-
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fluorescent lectins. After this time, samples were analyzed using an Axio Ziess fluorescent microscope at
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100x magnification. The experiments were repeated two times for each sample group.
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The Mean Fluorescence Intensity (MFI) was determined according to Tinel et al. (2014)
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using Image J software (National Institutes of Health USA). Differences between MFI were tested by a
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means ANOVA.
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2.3 Detection of glycoconjugates on sand fly midgut lysates by western blotting.
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Midguts were dissected in Grace Insect Medium (GIM). Malpighian tubes were removed
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and midguts were macerated in 25 µL of protease inhibitor cocktail (AEBSF, 2 mM, Aprotinin, 0.3 µM,
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Bestatin 130 µM, Ethylenediamine tetraacetic acid (EDTA) 1 mM, Epoxysuccinyl-64 (E-64) 14 µM,
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Leupeptin, 1 µM) and cell lysis buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM Na2 EDTA, 1 mM
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Ethylene glycol tetraacetic acid (EGTA), 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM
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glycerophosphate, 1 mM Na3VO4, 1 µg/mL leupeptin) at 4 °C, subsequently heated at 100 °C for 5
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minutes, and then frozen in liquid nitrogen. The samples were centrifuged at 10,000 RPM at 4 °C for 10
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minutes. The midgut proteins from supernatant were separated by SDS PAGE on 10% polyacrylamide gel
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using a Mini-Protean Tetra chamber (Bio-Rad), and then transferred from gel to nitrocellulose membrane
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followed and cut into strips. The strips were blocked with Tris-BSA-NaCl pH 7,6 overnight and then
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incubated with peroxidase conjugated lectins diluted 1:100 in BSA for 1 hour. Subsequently, the strips
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were washed three time with Tris-0,05% Tween-NaCl pH 7.6 followed the incubation with 3,3’-
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Diaminobenzidine (DAB) (10 mg per tablet) for five minutes. The protein migration was analyzed by
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densitometry, using the ImageJ software.
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2.4 Midgut-binding assay Midguts of Lu. longipalpis s.l., Lu. flaviscutellata and Lu. antunesi from the field were dissected in GIM medium as described in item 2.2. Promastigotes of L. (L.) amazonensis (MHOM/BR/26361) and L. infantum chagasi
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(MHOM/BR/27850) were obtained from the Evandro Chagas Institute and were maintained in RPMI
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1640 medium supplemented with Fetal Bovine Serum (FBS) at 27 ºC. Fourth day cultured promastigotes
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(log phase) were centrifuged (1500 RPM, 10 minutes) and washed with phosphate-buffered saline (PBS)
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pH 7.2. Parasites were labeled for 20 minutes at 26 ºC with 2.5 µL of VybrantTM DIO, washed four times,
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then re-suspended in RPMI medium with 10% FBS at a concentration of 108 cells/mL.
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The midguts were incubated with 108 cells/mL of each species of Leishmania labeled with
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VybrantTM DIO in a dark, humid chamber for 40 minutes. After that, the midguts were washed three times
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by transferring them to fresh drops of PBS pH 8.0 for 10 minutes, and then glasses were mounted in
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Prolong Gold antifade reagent. The samples were analyzed using an Axio Zeiss fluorescent microscope at
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100x magnification. In the control assays, 50 µL of medium containing Leishmania were pre-incubated
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with 20 µL of GalNAc (250 mM) for 40 minutes at room temperature.
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3. RESULTS
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3.1 Capture of Phlebotomines A total of 432 female phlebotomines, belonging to 14 species, were captured from April
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2013 to May 2015. A total of 180 (61,3.%) specimens of Lu. longipalpis s.l. were captured, representing
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the most abundant species, followed by 76 specimens of Lu. antunesi and 64 specimens of Lu.
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flaviscutellata, representing 13.2% and 7.3%, respectively. The other important epidemiological species,
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Psychodopygus complexa (6.6%) and Lu. migonei (1.1%), were captured in Cametá and Barcarena
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municipalities, respectively (Table 1).
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Species
Males (n)
Females (n)
Total
440
180
620
61.3
57
76
133
13.2
10
64
74
7.3
45
22
67
6.6
06
23
29
2.9
04
12
16
1.6
03
11
14
1.4
04
10
14
1.4
Lu. furcata
02
09
11
1.1
Lu. migonei
03
08
11
1.1
Lu. trinidadensis
05
06
11
1.1
Lu. davisi
0
06
06
0.6
Lu. pinotti
0
04
04
0.4
Lu. rorotaensis
0
1
01
0.1
579
432
1011
Lu. longipalpis s.l. Lu. antunesi Lu. flaviscutellata
Lu. sordellii Lu. pusilla Lu. gomezi
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Ps. complexa
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Lu. tuberculata
Total 10 11 12
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Table 1: Total number of phlebotomine species captured from April 2013 to May 2015 in the municipality of Barcarena and Cametá, state of Pará %
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3.2 Presence of lectins-binding epitopes on Phlebotomine midguts
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3.2.1 Lu. longipalpis s.l from colony and field populations
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The midguts of Lu. longipalpis s.l. from the colony presented positive fluorescence for GalNAc, mannose, galactose and GlcNAc carbohydrates.
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HPA, a lectin that is specific for GalNAc, presented the most fluorescent reactivity,
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followed by PNA on the midguts of the colony sand flies. The samples of Lu. longipalpis s.l. from the
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field were also analyzed and showed a different profile of fluorescence to the tested lectins since CON-A
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represented the second most fluorescence. Furthermore, samples from the colony presented a slightly greater intensity of fluorescence for all lectins compared to the field samples (Fig. 1).
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3.2.2 Lu. flaviscutellata and Lu. antunesi
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Midguts of two other species, Lu. flaviscutellata and Lu. antunesi from the field were
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investigated to evaluate the presence of carbohydrates. The midguts from all of the species reacted
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positively for all tested lectins, but showed different intensity of CON-A, PNA and WGA among the
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analyzed species.
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The comparative analyses of MFI in the midguts of phlebotomine from field showed a
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higher fluorescence of HPA in Lu. longipalpis s.l. and Lu. flaviscutellata. We also observed that MFI of
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WGA of Lu. flaviscutellata midgut were significantly higher than WGA from other species (Fig. 2).
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All of the control samples of Lu. longipalpis s.l., Lu. flaviscutellata, and Lu. antunesi
showed low mean fluorescence intensity (MFI < 0) for all investigated lectins.
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3.3 Glycoconjugates containing carbohydrate residues on midguts lysates
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3.3.1 Lu. longipalpis s.l. from colony and field populations
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Midguts of sand flies from both the colony and the field displayed glycoconjugates containing GalNAc,
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mannose, galactose and GlcNAc. Peptides displaying carbohydrates ranged from 40 to 75 kilodaltons
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(kDa) for the colony samples (Fig. 3A) and from 37 to 100 kDa for field samples (Fig 3B).
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3.3.2 Lu. flaviscutellata and Lu. antunesi
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Lutzomyia flaviscutellata midguts reacted positively to HPA, confirming the results
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observed in the fluorescence analyses. In addition to the presence of GalNAc, the midguts were positive
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for mannose, galactose and GlcNAc. Despite low reactivity to PNA and WGA lectins, the densitometry
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analyses confirmed the positive reaction for both lectins (Fig. 4A). Analyses of Lu. antunesi midguts
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revealed a positive reactivity to GalNAc and mannose ranging from 37 to 250 kDa. This species was also
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positive to PNA and WGA suggesting the presence of galactose and GlcNAc residues on midgut
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epithelial cells (Fig. 4B).
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3.4. Binding of L. (L.) amazonensis and L. infantum chagasi to phlebotomine midguts Labeled promastigotes bound to the midguts of Lu. longipalpis s.l., Lu. flaviscutellata and
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Lu. antunesi shows that these species permitted adhesion in vitro of both species of Leishmania to the
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midguts of phlebotomines (Fig. 5). Although Lu. flaviscutellata is the natural vector of L. (L.)
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amazonensis, this parasite attached to the midgut of Lu. longipalpis s.l. and Lu. antunesi (Fig. 5 a-c).
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When the binding assay was performed with L. infantum chagasi, the results showed the parasite bound
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not only to the midgut of Lu. longipalpis s.l., its natural vector, but also to Lu. flaviscutellata and Lu.
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antunesi midguts (Fig. 5 d-f). Control of binding assays showed that L. (L.) amazonensis and L. infantum
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chagasi blocked with GalNAc bound weakly to the midgut epithelium of three species compared to
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parasites that were not pre-incubated with GalNAc (data not shown).
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4. DISCUSSION The vectorial competence of phlebotomines in transmitting Leishmania is related to the
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parasite’s ability to overcome natural barriers in the phlebotomine. One of the principal barriers can be
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overcome by the attachment to surface molecules on the epithelial cells, thus preventing expulsion during
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blood meal excretion (Ramalho-Ortigao et al., 2010).
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In species-specific interactions, LPG is the main Leishmania molecule that is recognized
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by lectins of the midgut cells (Pimenta et al., 1994). However, other LPG-independent mechanism seems
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to occur in phlebotomines that are classified as permissive vectors (Dostálová and Volf, 2012). Surface
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carbohydrates are found composing the glycocalyx of the cells. The glycocalyx glycans have an important
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role in pathogen-host interactions because they permit the binding of the parasite to the cell surface (Liu
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et al., 2015). Some carbohydrates are monosaccharides, such as GalNAc, mannose, galactose and
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GlcNAc. These glycans are part of the glycoconjugate structure and are recognized by lectins on the
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parasite surface (Liu et al., 2015).
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Lutzomyia longipalpis s.l., the main vector of L. infantum chagasi, has a broad
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geographical distribution, occurring in all federative States of Brazil (Lainson and Rangel, 2005). This
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was the most abundant (61.3%) species captured in all of the five studied areas, which is in agreement
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with other studies about phlebotomine fauna in Brazil (Barata et al., 2005; Oliveira et al., 2011; 2006).
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Although Lu. longipalpis s.l. is considered the vector of L. infantum chagasi in vivo
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experiments showed that this phlebotomine allowed the development of L. major (Myskova et al., 2007).
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Furthermore, the results of the in vitro experiments displayed that Leishmania tropica bound as
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successfully as L. infantum chagasi to Lu. longipalpis s.l. midguts (Wilson et al., 2010). These results
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permitted the classification of Lu. longipalpis s.l. as a permissive vector. However, a variety of conditions
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must be considered to determine the vectorial competence, including the localization of different forms of
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Leishmania in specific parts of the sand fly midgut, the presence of metacyclic forms in the vector’s
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anterior midgut and the possibility of a phlebotomine being experimentally infected (Bates et al., 2015).
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Permissive vectors, such as Lu. longipalpis s.l. and Ph. arabicus, that were infected by L.
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major are biochemically characterized by the presence of GalNAc-containing glycoconjugates (Myskova
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et al., 2007). Thus, in this study, epithelial carbohydrates of Lu. longipalpis s.l. maintained in a colony
4
were compared to carbohydrates of Lu. longipalpis s.l. and two other species (Lu. flaviscutellata and Lu.
5
antunesi) that were captured in the field. Our results showed that the midgut of Lu. longipalpis s.l. reacted
6
positively to CON-A, PNA and WGA by FITC-lectins and western blotting analyses. These results are
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consistent with Myscova et al., (2007) who showed a high reaction to HPA and CON-A lectins. However,
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studies using less sensitive techniques reported negative reactivity to the same lectins (Evangelista and
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Leite, 2002).
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Analysis of the midgut of Lu. longipalpis s.l. from colony and field populations showed a
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high MFI to GalNAc, suggesting this carbohydrate is implicated as an adhesion molecule of Leishmania
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to midgut epithelium. In addition, prior studies showed that Lu. longipalpis s.l. lack PpGalec, a galectin of
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the specific vectors Ph. papatasi and Ph. duboscqi midgut that recognizes Leishmania surface LPG
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(Jochim et al., 2008).
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The second and third most abundant species captured in the studied areas were Lu.
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antunesi (13.2%) and Lu. flaviscutellata (7.3%), respectively. Considering the captures were performed in
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impacted areas, our results are in accordance with other studies where both species were captured in
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anthropic areas (Azevedo et al., 2011; Oliveira et al., 2011; Trujillo et al., 2013). The midgut analysis also
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showed that HPA was the most reactive lectin in Lu. antunesi and Lu. flaviscutellata, confirming the
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presence of glycoconjugates containing GalNAc. Interestingly, Lu. flaviscutellata is known as vector of L.
21
(L.) amazonensis. However, it should be emphasized that Lu. flaviscutellata captured in a forested area of
22
French Guiana was naturally infected by Leishmania (Viannia) guyanensis (Fouque et al., 2007).
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Furthermore, although Lu. antunesi is the proven vector of L. (V.) lindenbergi in the Amazonian region
24
(Lainson and Shaw, 2005), this species was found naturally infected by suprapylarian Leishmania,
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probably L. infantum chagasi, in Marajo Island (Ryan et al., 1984). Thus, our data suggest that these
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species can be permissive vectors.
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All of the field species showed high reactivity to HPA lectin, suggesting that GalNAc may
2
be a common carbohydrate residue present on midgut epithelial cells. Thus, the GalNAc is probably
3
related to Leishmania attachment in a non-species-specific interaction, as observed with Lutzomyia neivai
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and Lutzomyia migonei naturally infected by L. infantum chagasi (Carvalho et al., 2010; Dias et al., 2013;
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Salomón et al., 2010).
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The four lectins tested reacted positively to carbohydrates present on the midgut of four
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species from the field, but only the midgut analyses of Lu. longipalpis s.l. and Lu. flaviscutellata showed
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significant differences between HPA and other lectins.
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The mannose, galactose and GlcNAc carbohydrates were present in all species examined
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in this study. The second most reactive lectin was CON-A, suggesting mannose as binding sites by
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Leishmania in the studied species, as observed with Lu. migonei that presented a high concentration of
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mannose in its epithelial midgut (Tínel et al., 2014). These differences of the midgut glycoconjugates
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found in the phlebotomines from colony and field populations is probably related to the variety of food
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sources in the habitats of these specimens. This finding is in accordance with observations by Jacobson et
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al., (2007) who showed phlebotomines from desert and oasis environments presented different
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carbohydrate and glycosidase patterns.
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This study confirmed the presence of GalNAc residues by binding assays with L. (L.)
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amazonensis and L. infantum chagasi with the midguts of Lu. longipalpis s.l., Lu. flaviscutellata and Lu.
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antunesi. Both Leishmania species bound to their natural vector’s midguts and to the midguts of other
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species. Leishmania (L.) amazonensis attached to the midgut of Lu. longipalpis s.l. and Lu. antunesi while
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L. infantum chagasi bound to the midgut of Lu. flaviscutellata and Lu. antunesi.
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Interestingly, Lu. antunesi presented a higher number of attached parasites than Lu.
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longipalpis s.l. and even Lu. flaviscutellata, the natural vector of L. (L.) amazonensis. Similar results was
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noted when L. infantum chagasi was used. The higher number of parasites was observed on L. antunesi
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midguts compared to Lu. longipalpis s.l. or to Lu. flaviscutellata midguts. Our study is consistent with
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that of Wilson et al., (2010), who showed that L. major and L. tropica bound more efficiently to the Lu.
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longipalpis s.l. midgut than that of L. infantum chagasi. Probably, these phlebotomine specimens
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presented different amounts of GalNAc residues at the moment of interaction. These results suggest that
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there is a possible participation of GalNAc as binding site in non-specific interaction Leishmania-
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phlebotomine. There are few studies about glycoconjugates of phlebotomine midguts and its relationship
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with Leishmania attachment (Myskova et al., 2007; Tínel et al., 2014). However, some studies have
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characterized the carbohydrates and their role in mosquito tissues. Studies of Anopheles tessellates
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midguts showed that this species presents mannose and GlcNAc as binding sites of Plasmodium
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(Ramasamy et al., 1997). Although mosquitoes and phlebotomines are insects from different Families,
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Culicidae and Psychodidae, respectively, these results are in accordance with the present study because
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the authors identified the same carbohydrates residues.
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This is the first study that provides the carbohydrate characterization of the midgut
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epithelium, including GalNAc, in phlebotomines from the Amazonian region. In addition, all of the
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phlebotomine species investigated are of epidemiological importance in that area, and the experiments
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compared the carbohydrate profile using specimens from both a colony and the field. Thus, these results
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demonstrate the importance of monitoring natural infection because the presence of GalNAc indicates a
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possible change in vectorial competence driven by evolution.
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In conclusion, our data showed that three important epidemiological phlebotomine species
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from the Amazonian region present midgut residues of GalNAc, mannose, galactose and GlcNAc that are
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probably used by the parasites as binding sites. These findings are important informations that increase
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the understanding of the Leishmania and phlebotomine interaction.
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Acknowledgments
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We are grateful to the technicians Iorlando Barata, Edna Leão, Fábio Silva, Luciene Santos, Roberto
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Brandão, and Sueli Pinheiro from the IEC “Ralph Lainson” Leishmaniasis Laboratory for their valuable
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contribution in the field and laboratory work. We are also grateful to PROPESP-UFPA. This work was
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supported by National Institute of Science and Technology in Structural Biology and Bioimaging-INBEB
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(CNPq, grant number 573767/2008-4).
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Assis, R.R., Ibraim, I.C., Nogueira, P.M., Soares, R. P., Turco, S.J., 2012. Glycoconjugates in New World species of Leishmania: Polymorphisms in lipophosphoglycan and glycoinositolphospholipids and interaction with hosts. Biochim. Biophys. Acta - Gen. Subj. 1820 (9), 1354–1365. doi.org/10.1016/j.bbagen.2011.11.001 1820. Azevedo, P.C.B., Lopes G.N., Fontes, R.S., Vasconcelos, G.C., Moraes, J.L.P., Rebêlo, J.M.M., 2011. The effect of fragmentation on phlebotomine communities (Diptera: Psychodidae) in areas of ombrophilous forest in São Luis, State of Maranhão, Brazil. Neotrop Entomol. 40: 271-277. doi.org/10.1590/S1519-566X2011000200018 Azevedo-Pereira, R.L., Pereira, M.C.S., Oliveria-Junior, F.O.R., Brazil, Rp Côrtes, L.M.C., Madeira, M.F., Santos, A.L.S., Toma, L., Alves, C.R., 2007. Heparin binding proteins from Leishmania (Viannia) braziliensis promastigotes. Vet. Parasitol. v.145 p. 234–239. doi:10.1016/j.vetpar.2006.12.019. Barata, R.A,, Silva, J.C.F., Mayrink, W., Silva, J.C., Prata, A., Lorosa, E.S,, Fiúza, J.A,, Gonçalves, C.M., Paula, K.M., Dias, E.D., 2005. Aspectos da ecologia e do comportamento de flebotomíneos em áreas endêmicas de leishmaniose visceral, Minas Gerais Rev. Soc. Bras. Med. Trop. 38(5), 421– 425. doi.org/10.1590/S0037-86822005000500012. Bates, P.A., Depaquit, J., Galati, E.A.B., Kamhaw, S., Maroli, M., Mcdowell, M.A., Picado, A., Ready, P.D., Salomón, O.D., Shaw, J.J., Traub-Csekö, Y.M., Warburget, A., 2015. Recent advances in phlebotomine sand fly research related to leishmaniasis control. Parasit. Vectors. 8, 131. doi.org/10.1186/s13071-015-0712-x Butcher, B.A., Turco, S.J., Hilty, B.A., Pimenta, P.F., Panunzio, M., Sacks, D., 1996. Deficiency in beta1,3-galactosyltransferase of a Leishmania major lipophosphoglycan mutant adversely influences the Leishmania-sand fly interaction. The Journal of Biological Chemistry v.271 (34) pp. 2057320579. doi:10.1074/jbc.271.34.20573. Carvalho, M.R., Valença, H.F., Silva, F.J., Pita-Pereira, D., Araújo, P.T., Britto, C., Brazil, R.P., Filho, S.P.B., 2010. Natural Leishmania infantum infection in Migonemyia migonei (França, 1920) (Diptera:Psychodidae:Phlebotominae) the putative vector of visceral leishmaniasis in Pernambuco State, Brazil. Acta Trop. 116: 108-110. doi:10.1016/j.actatropica.2010.03.009. Côrtes, L.M.C., Pereira, M.C.S., Silva, F.S., Pereira, B.A.S., Junior, F.O.O., Soares, R.O.A., Brazil, R.P., Toma, L., Vicente, C.M., Nader, H.B., Madeira, M.F., Bello, F., Alves, C.R., 2012. Participation of heparin binding proteins from the surface of Leishmania (Viannia) braziliensis promastigotes in the adhesion of parasites to Lutzomyia longipalpis cells (Lulo) in vitro. Parasit. Vectors v. 5:142. doi:10.1186/1756-3305-5-142 Dias, E.S., Michalsky, E.M., Nascimento, J.C., Ferreira, E.C., Lopes, J.V., Fortes-Dias, C.L., 2013. Detection of Leishmania infantum, the etiological agent of visceral leishmaniasis, in Lutzomyia neivai, a putative vector of cutaneous leishmaniasis. Journal of Vector Ecol. 38(1), 193–196. doi.org/10.1111/j.1948-7134.2013.12028.x Dostálová, A., Volf, P., 2012. Leishmania development in sand flies: parasite-vector interactions overview. Parasites Vectors. 5: 276. doi:10.1186/1756-3305-5-276. Evangelista, L.G., Leite, A.C., 2002. Histochemical localization of N-acetyl-galactosamine in the midgut Lutzomyia longipalpis (Diptera: Psychodidae). J Med. Entomol. 39: 432-439. Fouque, F., Gaborit, P., Issaly, J., Carinci, R., Gantier, J., Ravel, C., Dedet. J., 2007. Phlebotomine sand flies (Diptera: Psychodidae) associated with changing patterns in the transmission of the human cutaneous leishmaniasis in French Guiana. Mem. Inst. Oswaldo Cruz. 102(1), 35–40. doi.org/10.1590/S0074-02762007000100005. Jacobson, R.L., Studentsky, L., Schlein, Y., 2007.Glycolytic and chitinolytic activities of Phlebotomus papatasi (Diptera: Psychodidae) from diverse ecological habitats. Folia Parasitol. 54: 301–309. Jochim, R,C., Teixeira, C.R., Laughinghouse, A., Mu, J., Oliveira, F., Gomes, R.B., R.B., Elnaiem, D.E., Valenzuela, J.G., 2008. The midgut transcriptome of Lutzomyia longipalpis: comparative analysis of
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Figure 1: Analysis of lectins Mean Fluorescence Intensity (MFI) of Lu. longipalpis s.l. from colony and field populations. Helix pomatia agglutinin lectin was significantly more reactive than the three other lectins. ANOVA (p< 0.05). HPA – Helix pomatia agglutinin, CON-A – Concanavalin A, PNA – Peanut agglutinin, WGA – Weat germ agglutinin.
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Figure 2: Midgut Mean Fluorescence Intensity (MFI) analyses of Lu. longipalpis s.l., Lu. flaviscutellata, and Lu. antunesi from the field with positive fluorescence to FITC-Lectins HPA, CON-A, PNA and WGA. Although all species presented different MFI to four lectins, only in Lu. longipalpis s.l. and Lu. flaviscutellata MFI was significantly different ANOVA (p< 0.05).
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Figure 3. Western blotting analysis of midgut glycoproteins of Lu. longipalpis s.l. from colony and field. Glycoproteins of Lu. longipalpis s.l. midgut lysates from colony (A) and filed (B) were identified with HPA, CON-A, PNA and WGA lectins. The densitometry analysis showed that HPA was the highest reactive lectin and CON-A was the lowest one. Each labeled protein of the molecular weight (MW) marker is on the bottom of densitometry boxes.
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Figure 4. Western blotting analysis of midgut glycoproteins of Lu. flaviscutellata and Lu. antunesi from field. Analyses of Lu. flaviscutellata midguts showed that all lectins were positive for glycoconjugates containing peptides ranging from 37 kDa to 100 kDa. Despite low reactivity for PNA and WGA, these lectins were positive according to the densitometry analyses (A). Lu. antunesi midgut lysates were positive for glycoconjugates containing peptides ranging from 37 kDa to 150 kDa. This species revealed strong reactivity for HPA and CON-A lectins (B). Each labeled protein of the molecular weight (MW) marker is on the bottom of densitometry boxes.
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Figure 5. Binding assay of phlebotomine midguts with parasite. Midguts of Lu. longipalpis s.l., Lu. flaviscutellata and Lu. antunesi were incubated with 50 µL of medium containing 108 cell/mL. Leishmania (L.) amazonensis bound to midguts of Lu. longipalpis s.l. (a), Lu. flaviscutellata (b), and Lu. antunesi (c). L. infantum chagasi bound to the midguts of its natural vector Lu. longipalpis s.l. (d), Lu. flaviscutellata (e), and Lu. antunesi (f). Interestingly, the attachment of L. (L.) amazonensis and L. infantum chagasi to Lu. antunesi (c and f) midguts was higher than observed in Lu. flaviscutellata and Lu. longipalpis s.l., respectively.
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Highlights •
Lutzomyia antunesi and Lutzomyia flaviscutellata, vectors of cutaneous leishmaniasis, present GalNAc on midgut epithelial. Lutzomyia longipalpis s.l midgut presents residues of GalNAc, mannose,
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galactose and GlcNAc. •
Carbohydrate characterization improve the knowledge of Phlebotomine-
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Leishmania interaction.
S0014-4894(16)30114-X
DOI:
10.1016/j.exppara.2016.06.002
Reference:
YEXPR 7257
To appear in:
Experimental Parasitology
Received Date: 1 March 2016 Revised Date:
30 May 2016
Accepted Date: 1 June 2016
Please cite this article as: de Oliveira, D.M.S., Martins da Silva, B.J., De Sena, C.B.C., Lima, J.A.N., Vasconcelos dos Santos, T., Silveira, F.T., Silva, E.O., Comparative analysis of carbohydrate residues in the midgut of phlebotomines (Diptera: Psychodidae) from colony and field populations from Amazon, Brazil, Experimental Parasitology (2016), doi: 10.1016/j.exppara.2016.06.002. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Comparative Analysis of Carbohydrate Residues in the Midgut of Phlebotomines
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(Diptera: Psychodidae) from Colony and Field Populations from Amazon, Brazil.
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Davi Marcos Souza de Oliveira1,2,3, Bruno José Martins da Silva1,2,3, Chubert Bernardo Castro De Sena2,
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José Aprígio Nunes Lima4, Thiago Vasconcelos dos Santos4, Fernando Tobias Silveira ,4,5, Edilene
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Oliveira Silva1,2,3*
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Brazil. Post code: 66075-900 2
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Laboratory of Parasitology Institute of Biological Sciences, Federal University of Pará; Belém, PA,
Laboratory of Structural Biology, Belém, Pará, Brazil. Post code: 66075-900
National Institute of Science and Technology in Structural Biology and Bioimaging, Rio de Janeiro, Rio
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de Janeiro, Brazil. Rio de Janeiro, Brazil. Post code: 21941-902; 4
Laboratory of Leishmaniasis ‘Prof Dr. Ralph Lainson’, Evandro Chagas Institute, Ministry of Health,
Ananindeua, Pará, Brazil. Post code: 67030-000 5
Tropical Medicine Nucleus, Federal University of Pará Bélem, Pará, Brazil. Post code: 66055-240
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* Author for correspondence: Dr. Edilene O. Silva-Federal University of Pará, Institute of Biological
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Sciences, Laboratory of Parasitology and Laboratory of Structural Biology, Augusto Corrêa Av., 01,
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Guamá, 660975-110, Belém, Pará, Brazil. Phone/Fax: 0559132017102, Email address: [email protected]
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Highlights
• Lutzomyia antunesi and Lutzomyia flaviscutellata, vectors of cutaneous leishmaniasis, present GalNAc on midgut epithelial.
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• Lutzomyia longipalpis s.l midgut presents residues of GalNAc, mannose, galactose and GlcNAc.
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• Carbohydrate characterization improve the knowledge of Phlebotomine-Leishmania interaction.
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Abstract
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Leishmaniasis are worldwide diseases that occur in 98 countries including Brazil, transmitted by the bite
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of female phlebotomines during blood feeding. In Brazil it is known that some species of sand flies as
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Lutzomyia longipalpis s.l. (vector of Leishmania infantum chagasi), Lutzomyia flaviscutellata (vector of
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Leishmania (Leishmania) amazonensis) and Lutzomyia antunesi (is the proven vector of Leishmania
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(Viannia) lindenbergi) are incriminated of transmitting the parasite Leishmania for the vertebrate host.
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The phlebotomine-parasite is mediated by the attachment of the promastigote lipophosphoglycan (LPG)
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to the midgut epithelium. However, another mechanism that is LPG-independent and mediated by N-
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acetyl-galactosamine (GalNAc). The aim of this study was to characterize the carbohydrate residues that,
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probably, play a role in parasite attachment to the midgut of phlebotomine from colony and field
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populations from the Brazilian Amazonian region. We observed the presence of GalNAc, Mannose,
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Galactose and GlcNAc in all phlebotomine species. A binding assay between L. (L.) amazonensis and L.
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infantum chagasi to the midguts of different species of phlebotomines was performed. The attachment of
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both Leishmania and vector species suggests the presence of GalNAc on the midgut surfaces. Thus, these
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results suggested that GalNAc is a possible binding sites of Leishmania in sand flies from the Brazilian
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Amazonian region.
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Keywords: Psychodidae, Leishmania, Midgut surface, Lectin, Host-parasite interaction, Glycoconjugates.
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1. Introduction
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Leishmaniasis are infectious diseases caused by protozoan parasites of the genus
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Leishmania (Trypanosomatidae: Kinetoplastidae) that are transmitted by the bite of female phlebotomines
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(Diptera: Psychodidae) during blood feeding (WHO, 2015). Phlebotomines become infected when they
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ingest mammalian blood that contains the amastigote forms inside of macrophages that release the
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parasites into the midgut. Inside the vector, amastigotes differentiate to an infective metacyclic stage
8
promastigotes (Ramalho-Ortigao et al., 2010). However, some species of phlebotomines, classified as
9
specific or restrictive, allow the development of only one Leishmania species, while others, which are
10
classified as permissive, allow the development of more than one species of Leishmania (Myskova et al.,
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2007).
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The establishment of infection depends on a parasite’s capacity to overcome natural
13
barriers such as digestive enzymes produced by epithelial cells (Pimenta et al., 1992). In the early stage of
14
development, leishmanias are protected against hydrolytic activities by peritrophic matrix (Pimenta et al.,
15
1997) However, parasites need to escape from the endoperitrophic space and attach to the midgut
16
epithelium to avoid being excreted with the blood remnants (Pimenta et al., 1992). This adhesion depends
17
on the interaction between molecules present on the surface of the parasite and the midgut epithelium.
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In a species-specific interaction, such as Phlebotomus papatasi and Leishmania major, the
19
adhesion is mediated by lipophosphoglycan (LPG), a linear chain of phosphorylated disaccharide
20
repeating units that are bound to cell membranes by a lipid anchor. This molecule is abundantly present
21
on flagellar forms only and presents structure variations during the life cycle of the parasite ( Assis et al.,
22
2012; Pimenta et al., 1994). During parasite-sand fly interactions, carbohydrate residues that are present
23
on LPG are recognized by lectins as binding sites on midgut epithelial cells (Pimenta et al., 1994). In the
24
midgut of P. duboscqui and P. papatasi there is a lectin (PpGalec) that is specific for L. major LPG.
25
PpGalec seems to be specific for L. major LPG since it was detected at low concentration in Lu.
26
longipalpis s.l. and was absent in P. sergenti and P. argentipes (Kamhawi et al., 2004).
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Some studies showed the role of LPG in the Leishmania-parasite interaction. Butcher et al.
2
(1996) and Jecna et al. (2013), showed that a strain of L. major LPG mutant was not able to adhere on
3
epithelial surface of its natural vector P. papatasi. In addition, parasite-phlebotomine interactions seem to
4
occur through a LPG-independent mechanism. Experimental infections showed that Phlebotomus
5
perniciosus and Lu. longipalpis s.l. permitted the development of L. major wild types and LPG mutants in
6
the same proportion, suggesting that the interaction between the parasite and these sand fly species was
7
not mediated by LPG (Myskova et al., 2007; Svárovská et al., 2010).
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One of possible mechanism involved in the interaction of permissive phlebotomines and
9
parasites probably depends on the carbohydrates that are present on the midgut epithelium which are
10
recognized by lectins that are present on the surface of Leishmania (Azevedo-Pereira et al., 2007; Côrtes
11
et al., 2012). Midgut biochemical analyses of the permissive phlebotomines Lu. longipalpis s.l.,
12
Phlebotomus halepensis, Ph. perniciosus, Phlebotomus argentipes and Ph. arabicus showed that all of
13
these species present N-acetyl-galactosamine (GalNAc) residues on the microvillar border whereas the
14
midguts of specific vectors lack GalNAc (Myskova et al., 2007).
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Although several studies have established the structure and the role of LPG in Leishmania-
16
sand fly interactions, there are few studies about the characterization of epithelial carbohydrate residues in
17
the midgut of sand flies. Therefore, this is the first study of carbohydrate characterization of
18
epidemiological important species from the Amazonian region using lectins to identify GalNAc,
19
mannose, galactose and GlcNAc residues of the midgut epithelium of sand flies.
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2. Materials and Methods
2
2.1 Sand flies
3
2.1.1 Sand flies from colony: Lutzomyia longipalpis s.l. specimens were obtained from a long-term closed colony (F38)
5
of Instituto Evandro Chagas (IEC) primarily captured in the municipality of Cametá. The insects were
6
reared and maintained in a closed colony at 25 ºC, 80% relative humidity on a 50% sucrose diet.
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2.1.2 Sand flies from field:
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Phlebotomines were captured in Barcarena (01º30’24’’ S and 48º37’12’’ W) and Cametá
10
(02º14'40" S and 49º29'45" W) municipalities of Pará State. The capture of phlebotomines was carried out
11
from 2013 to 2015 for five consecutive days per year using CDC light traps (Center for Disease Control
12
and Prevention) that were installed at 5:00 pm, and the sand flies were collected at 6:00 am of the
13
following day. The traps were installed at 1.5 meters above ground level in the peridomiciliary and
14
forested environments. Captured sand flies were transported to the laboratory and identified according to
15
Young and Duncan (Young and Ducan, 1994).
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2.2 Detection of glycoconjugates on the sand fly midgut by FITC labeled lectins. After four days of sugar feeding, female sand flies from both the colony and field
19
populations were dissected in drops of 1% paraformaldehyde. Three midguts from different species were
20
used for each lectin. The midguts were fixed in 4% paraformaldehyde at 4 °C for 20 minutes and opened
21
longitudinally by a 1 mL needle syringe under a Zeiss stereoscopic microscope. Non-specific sites were
22
blocked by 1% Bovine Serum Albumin (BSA) for 30 minutes. After that, the midguts were incubated for
23
1 hour in a dark, humid chamber, separately, with FITC- conjugated lectins Helix pomatia agglutinin
24
(HPA), concanavalin-A (CON-A), peanut agglutinin (PNA) and wheat germ agglutinin (WGA) (1:100),
25
that bind specifically to GalNAc, mannose, galactose and GlcNac, respectively (Sharon and Lis, 2004).
26
Subsequently, the midguts were washed three times in PBS pH 8.0 for 10 minutes and incubated with
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4',6-diamidino-2-phenylindole (DAPI) DNA marker and actin/myosin marker (phalloidin) for 40 minutes.
2
In the negative control, HPA lectin was incubated first with 250 mM of GalNAc for 1 h. To perform the
3
negative control of mannose, galactose and GlcNAc, the midguts were first incubated with non-
4
fluorescent lectins. After this time, samples were analyzed using an Axio Ziess fluorescent microscope at
5
100x magnification. The experiments were repeated two times for each sample group.
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The Mean Fluorescence Intensity (MFI) was determined according to Tinel et al. (2014)
7
using Image J software (National Institutes of Health USA). Differences between MFI were tested by a
8
means ANOVA.
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2.3 Detection of glycoconjugates on sand fly midgut lysates by western blotting.
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Midguts were dissected in Grace Insect Medium (GIM). Malpighian tubes were removed
12
and midguts were macerated in 25 µL of protease inhibitor cocktail (AEBSF, 2 mM, Aprotinin, 0.3 µM,
13
Bestatin 130 µM, Ethylenediamine tetraacetic acid (EDTA) 1 mM, Epoxysuccinyl-64 (E-64) 14 µM,
14
Leupeptin, 1 µM) and cell lysis buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM Na2 EDTA, 1 mM
15
Ethylene glycol tetraacetic acid (EGTA), 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM
16
glycerophosphate, 1 mM Na3VO4, 1 µg/mL leupeptin) at 4 °C, subsequently heated at 100 °C for 5
17
minutes, and then frozen in liquid nitrogen. The samples were centrifuged at 10,000 RPM at 4 °C for 10
18
minutes. The midgut proteins from supernatant were separated by SDS PAGE on 10% polyacrylamide gel
19
using a Mini-Protean Tetra chamber (Bio-Rad), and then transferred from gel to nitrocellulose membrane
20
followed and cut into strips. The strips were blocked with Tris-BSA-NaCl pH 7,6 overnight and then
21
incubated with peroxidase conjugated lectins diluted 1:100 in BSA for 1 hour. Subsequently, the strips
22
were washed three time with Tris-0,05% Tween-NaCl pH 7.6 followed the incubation with 3,3’-
23
Diaminobenzidine (DAB) (10 mg per tablet) for five minutes. The protein migration was analyzed by
24
densitometry, using the ImageJ software.
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2.4 Midgut-binding assay Midguts of Lu. longipalpis s.l., Lu. flaviscutellata and Lu. antunesi from the field were dissected in GIM medium as described in item 2.2. Promastigotes of L. (L.) amazonensis (MHOM/BR/26361) and L. infantum chagasi
5
(MHOM/BR/27850) were obtained from the Evandro Chagas Institute and were maintained in RPMI
6
1640 medium supplemented with Fetal Bovine Serum (FBS) at 27 ºC. Fourth day cultured promastigotes
7
(log phase) were centrifuged (1500 RPM, 10 minutes) and washed with phosphate-buffered saline (PBS)
8
pH 7.2. Parasites were labeled for 20 minutes at 26 ºC with 2.5 µL of VybrantTM DIO, washed four times,
9
then re-suspended in RPMI medium with 10% FBS at a concentration of 108 cells/mL.
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The midguts were incubated with 108 cells/mL of each species of Leishmania labeled with
11
VybrantTM DIO in a dark, humid chamber for 40 minutes. After that, the midguts were washed three times
12
by transferring them to fresh drops of PBS pH 8.0 for 10 minutes, and then glasses were mounted in
13
Prolong Gold antifade reagent. The samples were analyzed using an Axio Zeiss fluorescent microscope at
14
100x magnification. In the control assays, 50 µL of medium containing Leishmania were pre-incubated
15
with 20 µL of GalNAc (250 mM) for 40 minutes at room temperature.
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3. RESULTS
2
3.1 Capture of Phlebotomines A total of 432 female phlebotomines, belonging to 14 species, were captured from April
4
2013 to May 2015. A total of 180 (61,3.%) specimens of Lu. longipalpis s.l. were captured, representing
5
the most abundant species, followed by 76 specimens of Lu. antunesi and 64 specimens of Lu.
6
flaviscutellata, representing 13.2% and 7.3%, respectively. The other important epidemiological species,
7
Psychodopygus complexa (6.6%) and Lu. migonei (1.1%), were captured in Cametá and Barcarena
8
municipalities, respectively (Table 1).
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Species
Males (n)
Females (n)
Total
440
180
620
61.3
57
76
133
13.2
10
64
74
7.3
45
22
67
6.6
06
23
29
2.9
04
12
16
1.6
03
11
14
1.4
04
10
14
1.4
Lu. furcata
02
09
11
1.1
Lu. migonei
03
08
11
1.1
Lu. trinidadensis
05
06
11
1.1
Lu. davisi
0
06
06
0.6
Lu. pinotti
0
04
04
0.4
Lu. rorotaensis
0
1
01
0.1
579
432
1011
Lu. longipalpis s.l. Lu. antunesi Lu. flaviscutellata
Lu. sordellii Lu. pusilla Lu. gomezi
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Ps. complexa
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Lu. tuberculata
Total 10 11 12
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3.2 Presence of lectins-binding epitopes on Phlebotomine midguts
2
3.2.1 Lu. longipalpis s.l from colony and field populations
3
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The midguts of Lu. longipalpis s.l. from the colony presented positive fluorescence for GalNAc, mannose, galactose and GlcNAc carbohydrates.
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HPA, a lectin that is specific for GalNAc, presented the most fluorescent reactivity,
7
followed by PNA on the midguts of the colony sand flies. The samples of Lu. longipalpis s.l. from the
8
field were also analyzed and showed a different profile of fluorescence to the tested lectins since CON-A
9
represented the second most fluorescence. Furthermore, samples from the colony presented a slightly greater intensity of fluorescence for all lectins compared to the field samples (Fig. 1).
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3.2.2 Lu. flaviscutellata and Lu. antunesi
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Midguts of two other species, Lu. flaviscutellata and Lu. antunesi from the field were
14
investigated to evaluate the presence of carbohydrates. The midguts from all of the species reacted
15
positively for all tested lectins, but showed different intensity of CON-A, PNA and WGA among the
16
analyzed species.
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The comparative analyses of MFI in the midguts of phlebotomine from field showed a
18
higher fluorescence of HPA in Lu. longipalpis s.l. and Lu. flaviscutellata. We also observed that MFI of
19
WGA of Lu. flaviscutellata midgut were significantly higher than WGA from other species (Fig. 2).
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All of the control samples of Lu. longipalpis s.l., Lu. flaviscutellata, and Lu. antunesi
showed low mean fluorescence intensity (MFI < 0) for all investigated lectins.
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3.3 Glycoconjugates containing carbohydrate residues on midguts lysates
2
3.3.1 Lu. longipalpis s.l. from colony and field populations
3
Midguts of sand flies from both the colony and the field displayed glycoconjugates containing GalNAc,
4
mannose, galactose and GlcNAc. Peptides displaying carbohydrates ranged from 40 to 75 kilodaltons
5
(kDa) for the colony samples (Fig. 3A) and from 37 to 100 kDa for field samples (Fig 3B).
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3.3.2 Lu. flaviscutellata and Lu. antunesi
2
Lutzomyia flaviscutellata midguts reacted positively to HPA, confirming the results
4
observed in the fluorescence analyses. In addition to the presence of GalNAc, the midguts were positive
5
for mannose, galactose and GlcNAc. Despite low reactivity to PNA and WGA lectins, the densitometry
6
analyses confirmed the positive reaction for both lectins (Fig. 4A). Analyses of Lu. antunesi midguts
7
revealed a positive reactivity to GalNAc and mannose ranging from 37 to 250 kDa. This species was also
8
positive to PNA and WGA suggesting the presence of galactose and GlcNAc residues on midgut
9
epithelial cells (Fig. 4B).
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3.4. Binding of L. (L.) amazonensis and L. infantum chagasi to phlebotomine midguts Labeled promastigotes bound to the midguts of Lu. longipalpis s.l., Lu. flaviscutellata and
13
Lu. antunesi shows that these species permitted adhesion in vitro of both species of Leishmania to the
14
midguts of phlebotomines (Fig. 5). Although Lu. flaviscutellata is the natural vector of L. (L.)
15
amazonensis, this parasite attached to the midgut of Lu. longipalpis s.l. and Lu. antunesi (Fig. 5 a-c).
16
When the binding assay was performed with L. infantum chagasi, the results showed the parasite bound
17
not only to the midgut of Lu. longipalpis s.l., its natural vector, but also to Lu. flaviscutellata and Lu.
18
antunesi midguts (Fig. 5 d-f). Control of binding assays showed that L. (L.) amazonensis and L. infantum
19
chagasi blocked with GalNAc bound weakly to the midgut epithelium of three species compared to
20
parasites that were not pre-incubated with GalNAc (data not shown).
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4. DISCUSSION The vectorial competence of phlebotomines in transmitting Leishmania is related to the
4
parasite’s ability to overcome natural barriers in the phlebotomine. One of the principal barriers can be
5
overcome by the attachment to surface molecules on the epithelial cells, thus preventing expulsion during
6
blood meal excretion (Ramalho-Ortigao et al., 2010).
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In species-specific interactions, LPG is the main Leishmania molecule that is recognized
8
by lectins of the midgut cells (Pimenta et al., 1994). However, other LPG-independent mechanism seems
9
to occur in phlebotomines that are classified as permissive vectors (Dostálová and Volf, 2012). Surface
10
carbohydrates are found composing the glycocalyx of the cells. The glycocalyx glycans have an important
11
role in pathogen-host interactions because they permit the binding of the parasite to the cell surface (Liu
12
et al., 2015). Some carbohydrates are monosaccharides, such as GalNAc, mannose, galactose and
13
GlcNAc. These glycans are part of the glycoconjugate structure and are recognized by lectins on the
14
parasite surface (Liu et al., 2015).
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Lutzomyia longipalpis s.l., the main vector of L. infantum chagasi, has a broad
16
geographical distribution, occurring in all federative States of Brazil (Lainson and Rangel, 2005). This
17
was the most abundant (61.3%) species captured in all of the five studied areas, which is in agreement
18
with other studies about phlebotomine fauna in Brazil (Barata et al., 2005; Oliveira et al., 2011; 2006).
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Although Lu. longipalpis s.l. is considered the vector of L. infantum chagasi in vivo
20
experiments showed that this phlebotomine allowed the development of L. major (Myskova et al., 2007).
21
Furthermore, the results of the in vitro experiments displayed that Leishmania tropica bound as
22
successfully as L. infantum chagasi to Lu. longipalpis s.l. midguts (Wilson et al., 2010). These results
23
permitted the classification of Lu. longipalpis s.l. as a permissive vector. However, a variety of conditions
24
must be considered to determine the vectorial competence, including the localization of different forms of
25
Leishmania in specific parts of the sand fly midgut, the presence of metacyclic forms in the vector’s
26
anterior midgut and the possibility of a phlebotomine being experimentally infected (Bates et al., 2015).
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Permissive vectors, such as Lu. longipalpis s.l. and Ph. arabicus, that were infected by L.
2
major are biochemically characterized by the presence of GalNAc-containing glycoconjugates (Myskova
3
et al., 2007). Thus, in this study, epithelial carbohydrates of Lu. longipalpis s.l. maintained in a colony
4
were compared to carbohydrates of Lu. longipalpis s.l. and two other species (Lu. flaviscutellata and Lu.
5
antunesi) that were captured in the field. Our results showed that the midgut of Lu. longipalpis s.l. reacted
6
positively to CON-A, PNA and WGA by FITC-lectins and western blotting analyses. These results are
7
consistent with Myscova et al., (2007) who showed a high reaction to HPA and CON-A lectins. However,
8
studies using less sensitive techniques reported negative reactivity to the same lectins (Evangelista and
9
Leite, 2002).
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Analysis of the midgut of Lu. longipalpis s.l. from colony and field populations showed a
11
high MFI to GalNAc, suggesting this carbohydrate is implicated as an adhesion molecule of Leishmania
12
to midgut epithelium. In addition, prior studies showed that Lu. longipalpis s.l. lack PpGalec, a galectin of
13
the specific vectors Ph. papatasi and Ph. duboscqi midgut that recognizes Leishmania surface LPG
14
(Jochim et al., 2008).
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The second and third most abundant species captured in the studied areas were Lu.
16
antunesi (13.2%) and Lu. flaviscutellata (7.3%), respectively. Considering the captures were performed in
17
impacted areas, our results are in accordance with other studies where both species were captured in
18
anthropic areas (Azevedo et al., 2011; Oliveira et al., 2011; Trujillo et al., 2013). The midgut analysis also
19
showed that HPA was the most reactive lectin in Lu. antunesi and Lu. flaviscutellata, confirming the
20
presence of glycoconjugates containing GalNAc. Interestingly, Lu. flaviscutellata is known as vector of L.
21
(L.) amazonensis. However, it should be emphasized that Lu. flaviscutellata captured in a forested area of
22
French Guiana was naturally infected by Leishmania (Viannia) guyanensis (Fouque et al., 2007).
23
Furthermore, although Lu. antunesi is the proven vector of L. (V.) lindenbergi in the Amazonian region
24
(Lainson and Shaw, 2005), this species was found naturally infected by suprapylarian Leishmania,
25
probably L. infantum chagasi, in Marajo Island (Ryan et al., 1984). Thus, our data suggest that these
26
species can be permissive vectors.
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All of the field species showed high reactivity to HPA lectin, suggesting that GalNAc may
2
be a common carbohydrate residue present on midgut epithelial cells. Thus, the GalNAc is probably
3
related to Leishmania attachment in a non-species-specific interaction, as observed with Lutzomyia neivai
4
and Lutzomyia migonei naturally infected by L. infantum chagasi (Carvalho et al., 2010; Dias et al., 2013;
5
Salomón et al., 2010).
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The four lectins tested reacted positively to carbohydrates present on the midgut of four
7
species from the field, but only the midgut analyses of Lu. longipalpis s.l. and Lu. flaviscutellata showed
8
significant differences between HPA and other lectins.
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The mannose, galactose and GlcNAc carbohydrates were present in all species examined
10
in this study. The second most reactive lectin was CON-A, suggesting mannose as binding sites by
11
Leishmania in the studied species, as observed with Lu. migonei that presented a high concentration of
12
mannose in its epithelial midgut (Tínel et al., 2014). These differences of the midgut glycoconjugates
13
found in the phlebotomines from colony and field populations is probably related to the variety of food
14
sources in the habitats of these specimens. This finding is in accordance with observations by Jacobson et
15
al., (2007) who showed phlebotomines from desert and oasis environments presented different
16
carbohydrate and glycosidase patterns.
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This study confirmed the presence of GalNAc residues by binding assays with L. (L.)
18
amazonensis and L. infantum chagasi with the midguts of Lu. longipalpis s.l., Lu. flaviscutellata and Lu.
19
antunesi. Both Leishmania species bound to their natural vector’s midguts and to the midguts of other
20
species. Leishmania (L.) amazonensis attached to the midgut of Lu. longipalpis s.l. and Lu. antunesi while
21
L. infantum chagasi bound to the midgut of Lu. flaviscutellata and Lu. antunesi.
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Interestingly, Lu. antunesi presented a higher number of attached parasites than Lu.
23
longipalpis s.l. and even Lu. flaviscutellata, the natural vector of L. (L.) amazonensis. Similar results was
24
noted when L. infantum chagasi was used. The higher number of parasites was observed on L. antunesi
25
midguts compared to Lu. longipalpis s.l. or to Lu. flaviscutellata midguts. Our study is consistent with
26
that of Wilson et al., (2010), who showed that L. major and L. tropica bound more efficiently to the Lu.
ACCEPTED MANUSCRIPT 15 1
longipalpis s.l. midgut than that of L. infantum chagasi. Probably, these phlebotomine specimens
2
presented different amounts of GalNAc residues at the moment of interaction. These results suggest that
3
there is a possible participation of GalNAc as binding site in non-specific interaction Leishmania-
4
phlebotomine. There are few studies about glycoconjugates of phlebotomine midguts and its relationship
6
with Leishmania attachment (Myskova et al., 2007; Tínel et al., 2014). However, some studies have
7
characterized the carbohydrates and their role in mosquito tissues. Studies of Anopheles tessellates
8
midguts showed that this species presents mannose and GlcNAc as binding sites of Plasmodium
9
(Ramasamy et al., 1997). Although mosquitoes and phlebotomines are insects from different Families,
10
Culicidae and Psychodidae, respectively, these results are in accordance with the present study because
11
the authors identified the same carbohydrates residues.
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This is the first study that provides the carbohydrate characterization of the midgut
13
epithelium, including GalNAc, in phlebotomines from the Amazonian region. In addition, all of the
14
phlebotomine species investigated are of epidemiological importance in that area, and the experiments
15
compared the carbohydrate profile using specimens from both a colony and the field. Thus, these results
16
demonstrate the importance of monitoring natural infection because the presence of GalNAc indicates a
17
possible change in vectorial competence driven by evolution.
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In conclusion, our data showed that three important epidemiological phlebotomine species
19
from the Amazonian region present midgut residues of GalNAc, mannose, galactose and GlcNAc that are
20
probably used by the parasites as binding sites. These findings are important informations that increase
21
the understanding of the Leishmania and phlebotomine interaction.
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Acknowledgments
2
We are grateful to the technicians Iorlando Barata, Edna Leão, Fábio Silva, Luciene Santos, Roberto
3
Brandão, and Sueli Pinheiro from the IEC “Ralph Lainson” Leishmaniasis Laboratory for their valuable
4
contribution in the field and laboratory work. We are also grateful to PROPESP-UFPA. This work was
5
supported by National Institute of Science and Technology in Structural Biology and Bioimaging-INBEB
6
(CNPq, grant number 573767/2008-4).
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Figure 1: Analysis of lectins Mean Fluorescence Intensity (MFI) of Lu. longipalpis s.l. from colony and field populations. Helix pomatia agglutinin lectin was significantly more reactive than the three other lectins. ANOVA (p< 0.05). HPA – Helix pomatia agglutinin, CON-A – Concanavalin A, PNA – Peanut agglutinin, WGA – Weat germ agglutinin.
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Figure 2: Midgut Mean Fluorescence Intensity (MFI) analyses of Lu. longipalpis s.l., Lu. flaviscutellata, and Lu. antunesi from the field with positive fluorescence to FITC-Lectins HPA, CON-A, PNA and WGA. Although all species presented different MFI to four lectins, only in Lu. longipalpis s.l. and Lu. flaviscutellata MFI was significantly different ANOVA (p< 0.05).
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Figure 3. Western blotting analysis of midgut glycoproteins of Lu. longipalpis s.l. from colony and field. Glycoproteins of Lu. longipalpis s.l. midgut lysates from colony (A) and filed (B) were identified with HPA, CON-A, PNA and WGA lectins. The densitometry analysis showed that HPA was the highest reactive lectin and CON-A was the lowest one. Each labeled protein of the molecular weight (MW) marker is on the bottom of densitometry boxes.
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Figure 4. Western blotting analysis of midgut glycoproteins of Lu. flaviscutellata and Lu. antunesi from field. Analyses of Lu. flaviscutellata midguts showed that all lectins were positive for glycoconjugates containing peptides ranging from 37 kDa to 100 kDa. Despite low reactivity for PNA and WGA, these lectins were positive according to the densitometry analyses (A). Lu. antunesi midgut lysates were positive for glycoconjugates containing peptides ranging from 37 kDa to 150 kDa. This species revealed strong reactivity for HPA and CON-A lectins (B). Each labeled protein of the molecular weight (MW) marker is on the bottom of densitometry boxes.
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Figure 5. Binding assay of phlebotomine midguts with parasite. Midguts of Lu. longipalpis s.l., Lu. flaviscutellata and Lu. antunesi were incubated with 50 µL of medium containing 108 cell/mL. Leishmania (L.) amazonensis bound to midguts of Lu. longipalpis s.l. (a), Lu. flaviscutellata (b), and Lu. antunesi (c). L. infantum chagasi bound to the midguts of its natural vector Lu. longipalpis s.l. (d), Lu. flaviscutellata (e), and Lu. antunesi (f). Interestingly, the attachment of L. (L.) amazonensis and L. infantum chagasi to Lu. antunesi (c and f) midguts was higher than observed in Lu. flaviscutellata and Lu. longipalpis s.l., respectively.
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Highlights •
Lutzomyia antunesi and Lutzomyia flaviscutellata, vectors of cutaneous leishmaniasis, present GalNAc on midgut epithelial. Lutzomyia longipalpis s.l midgut presents residues of GalNAc, mannose,
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galactose and GlcNAc. •
Carbohydrate characterization improve the knowledge of Phlebotomine-
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Leishmania interaction.