Salivary gland homogenates of Lutzomyia longipalpis and its vasodilatory peptide maxadilan cause plasma leakage via PAC1 receptor activation.

OBJECTIVES: Experiments were designed to determine if salivary gland homogenates (SGH) of the sand fly Lutzomyia longipalpis, the vasodilatory peptides maxadilan and pituitary adenylate cyclase-activating peptide (PACAP-38) may cause plasma leakage and to what extent these effects could be due to PAC1 receptor stimulation. METHODS: Using FITC-dextran as a plasma marker, intravital microscopy of the hamster cheek pouch (HCP) and a digital camera were used to assess arteriolar diameter and fluorescence of a selected area (5 mm(2)) representative of the HCP microcirculation. RESULTS: Cheek pouches prepared for intravital microscopy and exposed to topical application of SGH, maxadilan or PACAP-38 developed maximal dilation of arterioles in the range of 20-60 mum within 10 min, and this effect lasted for 30-90 min. The increase in fluorescence intensity induced by each of these compounds was due to plasma leakage from postcapillary venules. The mutant peptide of maxadilan (M-65), a PAC1 receptor antagonist, inhibited both dilation and plasma leakage induced by SGH or maxadilan. Plasma leakage induced by SGH was modestly inhibited by the bradykinin B(2) receptor antagonist HOE-140, but not by the antihistamine mepyramine or the nitric oxide synthase inhibitor L-NA. CONCLUSIONS: SGH of L. longipalpis and its vasodilatory peptide maxadilan caused long-lasting arteriolar dilation and plasma leakage in the cheek pouch via PAC1 receptor activation.

Study on the bacterial midgut microbiota associated to different Brazilian populations of Lutzomyia longipalpis (Lutz & Neiva) (Diptera: Psychodidae).

The bacterial community associated with the midgut of three Brazilian Lutzomyia longipalpis (Lutz & Neiva) populations, two from endemic areas for visceral leishmaniasis (Jacobina, Bahia State and São Luís, Maranhão State) and one from a non-endemic area (Lapinha Cave, Minas Gerais State), was identified. Five groups, 35 females each, from each population were separated; a total of 175 females per collecting area were analyzed. The species identification was based on molecular and traditional bacteriological methods. All bacteria were either affiliated to non-Enterobacteriaceae, such as Acinetobacter, Burkholderia, Flavimonas, Pseudomonas and Stenotrophomonas, or and to Enterobacteriaceae, such as Citrobacter, Enterobacter, Escherichia, Klebsiella, Serratia, Pantoea, Morganella and Weeksella. Stenotrophomonas was found to be associated with all three populations studied. In addition, Serratia spp., which are well documented as laboratory contaminant of insects, were detected only in the Jacobina population. We also discuss the impact of the colonization of insect gut by bacteria on the development and transmission of pathogens.

Study on the bacterial midgut microbiota associated to different Brazilian populations of Lutzomyia longipalpis (Lutz & Neiva) (Diptera: Psychodidae) / Estudo sobre as bactérias da microbiota intestinal de diferentes populações brasileiras de Lutzomyia Longipalpis (Lutz & Neiva) (Diptera: Psychodidae)

A flora bacteriana encontrada no intestino de populações brasileiras de Lutzomyia longipalpis (Lutz & Neiva) provenientes de duas áreas endêmicas para leishmaniose visceral (Jacobina, BA e São Luís, MA) e de uma área não endêmica (Gruta da Lapinha, MG) é descrita. Cinco grupos de 35 fêmeas de cada população foram separados, no total de 175 fêmeas analisadas por área de coleta. A identificação das espécies foi baseada em métodos moleculares e em métodos bacteriológicos tradicionais. As bactérias encontradas foram classificadas como pertencentes ao grupo das não-Enterobacteriaceae, como Acinetobacter, Burkholderia, Flavimonas, Pseudomonas, ou ao grupo das Enterobacteriaceae, como Stenotrophomonas, Citrobacter, Enterobacter, Escherichia, Klebsiella, Serratia, Pantoea, Morganella e Weeksella. Stenotrophomonas esteve presente nas três populações analisadas. Além disso, Serratia spp., grupo bem documentado como contaminante laboratorial de insetos, foi detectado apenas na população de Jacobina. O impacto da colonização do intestino de insetos por bactérias no desenvolvimento e transmissão de patógenos é discutido.

The bacterial community associated with the midgut of three Brazilian Lutzomyia longipalpis (Lutz & Neiva) populations, two from endemic areas for visceral leishmaniasis (Jacobina, Bahia State and São Luís, Maranhão State) and one from a non-endemic area (Lapinha Cave, Minas Gerais State), was identified. Five groups, 35 females each, from each population were separated; a total of 175 females per collecting area were analyzed. The species identification was based on molecular and traditional bacteriological methods. All bacteria were either affiliated to non-Enterobacteriaceae, such as Acinetobacter, Burkholderia, Flavimonas, Pseudomonas and Stenotrophomonas, or and to Enterobacteriaceae, such as Citrobacter, Enterobacter, Escherichia, Klebsiella, Serratia, Pantoea, Morganella and Weeksella. Stenotrophomonas was found to be associated with all three populations studied. In addition, Serratia spp., which are well documented as laboratory contaminant of insects, were detected only in the Jacobina population. We also discuss the impact of the colonization of insect gut by bacteria on the development and transmission of pathogens.

Single and concomitant experimental infectionsby Endotrypanum spp. and Leishmania (Viannia) guyanensis (Kinetoplastida: Trypanosomatidae) in the Neotropical sand fly Lutzomyia longipalpis (Diptera: Psychodidae)

Lutzomyia longipalpis females received single and mixed infections with Endotrypanum and Leishmania. Two biological parameters were analyzed: the percentage of infected females and the distribution of flagellates in the gut of the females. The principal comparisons were performed between (1) two strains of Endotrypanum, (2) cloned versus primary sample of one strain of Endotrypanum, (3) Endotrypanum versus Leishmania guyanensis, and (4) the pattern of flagellates behaviour by optical microscopy in females with single or mixed infection versus the identification of parasites isolated from digestive tracts by isoenzyme electrophoresis. Flagellates of Endotrypanum showed distinct patterns of infection suggesting that there is variation between and within strains. The distribution of Endotrypanum and L. guyanensis differed significantly in relation to the colonization of the stomodeal valve. In co-infection with L. guyanensis, a large number of flagellates were seen to be plentifully infecting the stomodeal valve in significantly more specimens than in females infected by Endotrypanum only. However, the electrophoretic profiles of isoenzymes of parasites recovered from all co-infected specimens corresponded to Endotrypanum. This suggests that the mere correlation sand fly infection-biochemical analysis of isolates may induce parasitological incorrect consideration.

Single and concomitant experimental infections by Endotrypanum spp. and Leishmania (Viannia) guyanensis (Kinetoplastida: Trypanosomatidae) in the neotropical sand fly Lutzomyia longipalpis (Diptera: Psychodidae).

Lutzomyia longipalpis females received single and mixed infections with Endotrypanum and Leishmania. Two biological parameters were analyzed: the percentage of infected females and the distribution of flagellates in the gut of the females. The principal comparisons were performed between (1) two strains of Endotrypanum, (2) cloned versus primary sample of one strain of Endotrypanum, (3) Endotrypanum versus Leishmania guyanensis, and (4) the pattern of flagellates behaviour by optical microscopy in females with single or mixed infection versus the identification of parasites isolated from digestive tracts by isoenzyme electrophoresis. Flagellates of Endotrypanum showed distinct patterns of infection suggesting that there is variation between and within strains. The distribution of Endotrypanum and L. guyanensis differed significantly in relation to the colonization of the stomodeal valve. In co-infection with L. guyanensis, a large number of flagellates were seen to be plentifully infecting the stomodeal valve in significantly more specimens than in females infected by Endotrypanum only. However, the electrophoretic profiles of isoenzymes of parasites recovered from all co-infected specimens corresponded to Endotrypanum. This suggests that the mere correlation sand fly infection-biochemical analysis of isolates may induce parasitological incorrect consideration.

Structure and distribution of sensilla on maxillary palps and labella of Lutzomyia longipalpis (Diptera: Psychodidae) sand flies.

The typology, number, size, and distribution pattern of labellar and palpal sensilla of male and female Lutzomyia longipalpis (Lutz and Neiva, 1912) sand flies were analyzed by scanning electron and light microscopy, contributing to the construction of a gustatory and olfactory map in this important vector species. About 35 trichoid sensilla, sorted into three different classes according to their lengths, are present on the two labellar lobes. Five distinct structures are observed in the maxillary palps, which could be classified as noninnervated (microtrichia and scales) or the following innervated structures: 1) campaniform sensilla, found near the distal end of palpal segments II and III; 2) multiporous capitate peg sensilla, found in reduced number (1-6) on segment II, but located in a group of 15-25 on the distal third part of segment III; and 3) trichoid sensilla, distributed linearly on segment IV and spirally on segment V. Light microscopy analysis of silver-stained specimens indicated the presence of pores in the trichoid sensilla and their chemosensory function is discussed, as well as the possible function of the other sensilla.

The 3A1-La monoclonal antibody reveals key features of Leishmania (L) amazonensis metacyclic promastigotes and inhibits procyclics attachment to the sand fly midgut.

In this work, we characterise metacyclic promastigotes of Leishmania amazonensis, the causative agent of cutaneous and diffuse cutaneous leishmaniasis in the New World. To purify metacyclics from stationary culture by negative selection, we used the monoclonal antibody 3A1-La produced against procyclic promastigotes. The purified forms named 3A1-La(-) promastigotes, present key metacyclic characteristics: slender cell body and long flagella, ultrastructural features, resistance to complement lysis, high infectivity for macrophages and mice and reduced capacity for binding to the sand fly midgut. Moreover, the epitope recognised by 3A1-La is important for the promastigote attachment to the insect vector midgut epithelium. These results further characterise 3A1-La(-) promastigotes as metacyclic forms of L. amazonensis.

Interaction of insect trypanosomatids with mosquitoes, sand fly and the respective insect cell lines.

Interaction experiments between hematophagous insects and monoxenous trypanosomatids have become relevant, once cases of human infection involving these protozoa have been reported. Moreover, investigations related to the interaction of insects with trypanosomatids that harbour an endosymbiotic bacterium and thereby lack the paraflagellar rod structure are important to elucidate the role of this structure in the adhesion process. In this work, we compared the interaction of endosymbiont-bearing trypanosomatids and their aposymbiotic counterpart strains (without endosymbionts) with cell lines of Anopheles gambiae, Aedes albopictus and Lutzomyia longipalpis and with explanted guts of the respective insects. Endosymbiont-bearing strains interacted better with insect cells and guts when compared with aposymbiotic strains. In vitro binding assays revealed that the trypanosomatids interacted with the gut epithelial cells via flagellum and cell body. Flagella attached to the insect gut were enlarged, containing electrondense filaments between the axoneme and flagellar membrane at the point of adhesion. Interactions involving the flagellum lacking paraflagellar rod structure were mainly observed close to tight junctions, between epithelial cells. Endosymbiont-bearing trypanosomatids were able to colonise Aedes aegypti guts after protozoa feeding.

Leishmania (Viannia) braziliensis metacyclic promastigotes purified using Bauhinia purpurea lectin are complement resistant and highly infective for macrophages in vitro and hamsters in vivo.

In this study we characterised metacyclogenesis in axenic culture of Leishmania (Viannia) braziliensis, the causative agent of mucocutaneous leishmaniasis in the New World. Metacyclogenesis of other species of Leishmania has been shown by morphological changes as well as molecular modifications in the lipophosphoglycan, the major cell surface glycoconjugate of the promastigotes. In order to obtain metacyclic forms of L. braziliensis we tested a panel of different lectins. Our results showed that Bauhinia purpurea lectin facilitated the purification of metacyclic promastigotes from stationary-phase culture by negative selection. The B. purpurea non-agglutinated promastigotes had a slender short cell body and long flagella, typical of metacyclic morphology. The ultrastructural analysis showed that B. purpurea non-agglutinated promastigotes have a dense and thicker glycocalyx. They are resistant to complement lysis, and highly infective for macrophage in vitro and hamsters in vivo. Contrary to procyclic promastigotes, B. purpurea non-agglutinated forms were poorly recognised by sand fly gut epithelial cells. These results suggest that the B. purpurea non-agglutinated promastigotes are the metacyclic forms of L. braziliensis.