ABSTRACT
Triatomines are hematophagous arthropod vectors of Trypanosoma cruzi, the causative agent of Chagas Disease. Panstrongylus lignarius, also known as Panstrongylus herreri, is considered one of the most versatile triatomines because it can parasitize different hosts, it is found in different habitats and countries, it has sylvatic, peridomestic and domestic behavior and it is a very important vector of Chagas disease, especially in Peru. Molecules produced and secreted by salivary glands and fat body are considered of important adaptational value for triatomines because, among other functions, they subvert the host haemostatic, inflammatory and immune systems and detoxify or protect them against environmental aggressors. In this context, the elucidation of the molecules produced by these tissues is highly valuable to understanding the ability of this species to adapt and transmit pathogens. Here, we use high-throughput sequencing techniques to assemble and describe the coding sequences resulting from the transcriptome of the fat body and salivary glands of P. lignarius. The final assembly of both transcriptomes together resulted in a total of 11,507 coding sequences (CDS), which were mapped from a total of 164,676,091 reads. The CDS were subdivided according to their 10 folds overexpression on salivary glands (513 CDS) or fat body (2073 CDS). Among the families of proteins found in the salivary glands, lipocalins were the most abundant. Other ubiquitous families of proteins present in other sialomes were also present in P. lignarius, including serine protease inhibitors, apyrase and antigen-5. The unique transcriptome of fat body showed proteins related to the metabolic function of this organ. Remarkably, nearly 20% of all reads mapped to transcripts coded by Triatoma virus. The data presented in this study improve the understanding on triatomines' salivary glands and fat body function and reveal important molecules used in the interplay between vectors and vertebrate hosts.
Subject(s)
Fat Body/metabolism , Panstrongylus/genetics , Salivary Glands/metabolism , Transcriptome , Animals , Chagas Disease/transmission , Gene Expression Profiling , High-Throughput Nucleotide Sequencing , Insect Proteins/genetics , Insect Vectors/genetics , Insect Vectors/metabolism , Lipocalins/genetics , Panstrongylus/anatomy & histology , Panstrongylus/metabolism , Peru , Proteomics , Salivary Proteins and Peptides/genetics , Salivary Proteins and Peptides/metabolism , Serine Proteinase Inhibitors/genetics , Serine Proteinase Inhibitors/metabolismABSTRACT
Lipophorin is the main lipoprotein in the hemolymph of insects. During vitellogenesis, lipophorin delivers its hydrophobic cargo to developing oocytes by its binding to non-endocytic receptors at the plasma membrane of the cells. In some species however, lipophorin may also be internalized to some extent, thus maximizing the storage of lipid resources in growing oocytes. The ectopic ß chain of ATP synthase (ß-ATPase) was recently described as a putative non-endocytic lipophorin receptor in the anterior midgut of the hematophagous insect Panstrongylus megistus. In the present work, females of this species at the vitellogenic stage of the reproductive cycle were employed to investigate the role of ß-ATPase in the transfer of lipids to the ovarian tissue. Subcellular fractionation and western blot revealed the presence of ß-ATPase in the microsomal membranes of the ovarian tissue, suggesting its localization in the plasma membrane. Immunofluorescence assays showed partial co-localization of ß-ATPase and lipophorin in the membrane of oocytes as well as in the basal domain of the follicular epithelial cells. Ligand blotting and co-immunoprecipitation approaches confirmed the interaction between lipophorin and ß-ATPase. In vivo experiments with an anti-ß-ATPase antibody injected to block such an interaction demonstrated that the antibody significantly impaired the transfer of fatty acids from lipophorin to the oocyte. However, the endocytic pathway of lipophorin was not affected. On the other hand, partial inhibition of ATP synthase activity did not modify the transfer of lipids from lipophorin to oocytes. When the assays were performed at 4°C to diminish endocytosis, the results showed that the antibody interfered with lipophorin binding to the oocyte plasma membrane as well as with the transfer of fatty acids from the lipoprotein to the oocyte. The findings strongly support that ß-ATPase plays a role as a docking lipophorin receptor at the ovary of P. megistus, similarly to its function in the midgut of such a vector. In addition, the role of ß-ATPase as a docking receptor seems to be independent of the enzymatic ATP synthase activity.
Subject(s)
Lipid Metabolism , Lipoproteins/metabolism , Mitochondrial Proton-Translocating ATPases/metabolism , Oocytes/metabolism , Panstrongylus/metabolism , Animals , Endocytosis , Female , Fluorescent Antibody Technique , Immunoprecipitation , Ligands , Ovary/metabolismABSTRACT
Lipophorin, the main lipoprotein in the circulation of the insects, cycles among peripheral tissues to exchange its lipid cargo at the plasma membrane of target cells, without synthesis or degradation of its apolipoprotein matrix. Currently, there are few characterized candidates supporting the functioning of the docking mechanism of lipophorin-mediated lipid transfer. In this work we combined ligand blotting assays and tandem mass spectrometry to characterize proteins with the property to bind lipophorin at the midgut membrane of Panstrongylus megistus, a vector of Chagas' disease. We further evaluated the role of lipophorin binding proteins in the transfer of lipids between the midgut and lipophorin. The ß subunit of the ATP synthase complex (ß-ATPase) was identified as a lipophorin binding protein. ß-ATPase was detected in enriched midgut membrane preparations free of mitochondria. It was shown that ß-ATPase partially co-localizes with lipophorin at the plasma membrane of isolated enterocytes and in the sub-epithelial region of the midgut tissue. The interaction of endogenous lipophorin and ß-ATPase was also demonstrated by co-immunoprecipitation assays. Blocking of ß-ATPase significantly diminished the binding of lipophorin to the isolated enterocytes and to the midgut tissue. In vivo assays injecting the ß-ATPase antibody significantly reduced the transfer of [(3)H]-diacylglycerol from the midgut to the hemolymph in insects fed with [9,10-(3)H]-oleic acid, supporting the involvement of lipophorin-ß-ATPase association in the transfer of lipids. In addition, the ß-ATPase antibody partially impaired the transfer of fatty acids from lipophorin to the midgut, a less important route of lipid delivery to this tissue. Taken together, the findings strongly suggest that ß-ATPase plays a role as a docking lipophorin receptor at the midgut of P. megistus.
Subject(s)
ATP Synthetase Complexes/metabolism , Cell Membrane/metabolism , Digestive System/metabolism , Lipoproteins/metabolism , Panstrongylus/metabolism , Protein Binding , Animals , Biological Transport , Carrier Proteins , Lipid MetabolismABSTRACT
Studies were made on the ribosomal DNA intergenic region, comprising complete internal transcribed spacer (ITS)-1, 5.8S, and ITS-2 sequences, of populations of the triatomine Panstrongylus megistus, the most important vector of Chagas' disease in Brazil since Triatoma infestans eradication. Specimens were from 26 localities of Rio Grande do Sul, Santa Catarina, Paraná, São Paulo, Minas Gerais, Bahia, and Sergipe states. In total, 21 ITS-1 and 12 ITS-2 haplotypes were found. Nucleotide differences were higher in ITS-1 (3.00%) than in ITS-2 (1.33%). The intergenic region was 1,513-1,522-bp-long (mean 1,516.9 bp), providing 26 combined haplotypes. The combination of microsatellites found in both ITSs may be of applied usefulness, to assess interpopulation specimen exchange and potential recolonizations after vector elimination by control implementation. Network results suggest that São Paulo may be considered one of the spreading centers of this species. Molecular clock datation suggests that P. megistus populations are diversifying at least since 4.54 million years ago, with diversification still ongoing today by geographical isolation of populations. Evidence is provided about the relationship of genetic diversity with geographical spread that characterizes a major vector and explains its ability to colonize distant areas and different ecotopes, including human habitats, and consequently its importance in Chagas' disease epidemiology.
Subject(s)
Genetic Variation , Insect Vectors/genetics , Panstrongylus/genetics , Animals , Brazil , Chagas Disease/parasitology , Chagas Disease/transmission , DNA, Ribosomal Spacer/genetics , DNA, Ribosomal Spacer/metabolism , Insect Vectors/metabolism , Molecular Sequence Data , Panstrongylus/metabolism , Phylogeography , Polymerase Chain Reaction , RNA, Ribosomal, 5.8S/genetics , RNA, Ribosomal, 5.8S/metabolism , Sequence Analysis, DNAABSTRACT
Chagas disease is a trypanosomiasis whose causative agent is the protozoan parasite Trypanosoma cruzi, which is transmitted to humans by hematophagous insects known as triatomines and affects a large proportion of South America. The digestive tract of the insect vectors in which T. cruzi develops constitutes a dynamic environment that affects the development of the parasite. Thus, we set out to investigate the chemical composition of the triatomine intestinal tract through a metabolomics approach. We performed Direct Infusion Fourier Transform Ion Cyclotron Resonance Mass Spectrometry on fecal samples of three triatomine species (Rhodnius prolixus, Triatoma infestans, Panstrongylus megistus) fed with rabbit blood. We then identified groups of metabolites whose frequencies were either uniform in all species or enriched in each of them. By querying the Human Metabolome Database, we obtained putative identities of the metabolites of interest. We found that a core group of metabolites with uniform frequencies in all species represented approximately 80% of the molecules detected, whereas the other 20% varied among triatomine species. The uniform core was composed of metabolites of various categories, including fatty acids, steroids, glycerolipids, nucleotides, sugars, and others. Nevertheless, the metabolic fingerprint of triatomine feces differs depending on the species considered. The variable core was mainly composed of prenol lipids, amino acids, glycerolipids, steroids, phenols, fatty acids and derivatives, benzoic acid and derivatives, flavonoids, glycerophospholipids, benzopyrans, and quinolines. Triatomine feces constitute a rich and varied chemical medium whose constituents are likely to affect T. cruzi development and infectivity. The complexity of the fecal metabolome of triatomines suggests that it may affect triatomine vector competence for specific T. cruzi strains. Knowledge of the chemical environment of T. cruzi in its invertebrate host is likely to generate new ways to understand the factors influencing parasite proliferation as well as methods to control Chagas disease.
Subject(s)
Insect Vectors/metabolism , Metabolome , Panstrongylus/metabolism , Rhodnius/metabolism , Triatoma/metabolism , Trypanosoma cruzi/metabolism , Animals , Cyclotrons , Feces/chemistry , Feces/parasitology , Gastrointestinal Tract/metabolism , Gastrointestinal Tract/parasitology , Host Specificity , Host-Parasite Interactions , Humans , Insect Vectors/parasitology , Mass Spectrometry/methods , Panstrongylus/parasitology , Rabbits , Rhodnius/parasitology , Triatoma/parasitology , Trypanosomiasis/parasitologyABSTRACT
In this work, we have analyzed the pathways by which lipophorin (Lp) delivers its lipid cargo to developing oocytes of Panstrongylus megistus, a hematophagous vector of Chagas' disease. Lp, vitellin, total lipids and proteins were measured in ovarian tissues at different stages of the reproductive cycle. Localization of Lp in developing oocytes, mainly at their cortical area, was demonstrated by immunofluorescence assays using an anti-Lp antibody labeled with FITC. In vivo approaches injecting fluorescently labeled Lp to follow the course of the entire particle (Lp-DiI or Lp-Oregon Green) or its lipid cargo (Lp-Bodipy-FA) were monitored by laser scanning confocal microscopy. Significant increases in the amounts of lipids, proteins and vitellin were observed in ovarian tissue with the progress of vitellogenesis. Unexpectedly, an increase in the amount of Lp was also observed. The experiments in vivo demonstrated that the uptake of fluorescent Lp labeled on its protein or lipid moiety by developing oocytes occurred very fast, being impaired at low temperatures. The co-injection of fluorescent Lp and vitellogenin (Vg) showed that both particles co-localized inside yolk bodies, confirming the endocytic pathway for Lp. When the fate of lipids transferred to oocytes was evaluated in vitellogenic females by co-injecting Lp-Bodipy-FA and Lp-DiI, the signal for Bodipy-FA was found in both lipid droplets and yolk bodies. In contrast, in injected females kept at 4°C the fluorescence was reduced, being observed exclusively in lipid droplets, implying that lipid transfer to the oocyte was diminished but not abolished. Taken together, the results demonstrate that in the hematophagous P. megistus, the storage of lipid resources by developing oocytes occurs by the convergence of different pathways by which Lp maximizes the delivery of its lipid cargo. In addition, it was also shown that, to some extent, lipids stored in the oocyte lipid droplets can also originate from endocytosed Vg. The relevance of these events in the context of the physiology of reproduction in P. megistus is discussed.
Subject(s)
Insect Proteins/metabolism , Lipid Metabolism , Lipoproteins/metabolism , Oocytes/metabolism , Panstrongylus/metabolism , Vitellogenesis , Animals , Female , Oocytes/growth & development , Panstrongylus/growth & development , Vitellogenins/metabolismABSTRACT
In order to better understand the metabolism of dietary lipids in hematophagous insects, we have performed a biochemical and cellular characterization of lipophorin (Lp)-midgut interaction in Panstrongylus megistus, a vector of Chagas' disease. The study was accomplished by solid-phase binding assays or with iodinated Lp ((125)I-Lp), using midgut membranes from fifth instar nymphs after ecdysis and after insects received a blood meal. Results obtained from both physiological conditions indicated that Lp interacted specifically with the midgut, implying the participation of receptors. Binding capacity of lipophorin to membranes was dependent on the amount of membranes added in the system, reaching saturation at 0.1 microg/ml. However, membranes obtained after a blood meal exhibited higher binding activity. Saturation kinetics results using (125)I-Lp suggested a single binding site with high affinity for Lp in the midgut membranes (K(d) = 5.1 +/- 3.6 x 10(-8) M). The unrelated lipoprotein, human LDL, did not compete with Lp for its binding site in the midgut. The binding was dependent on pH and the treatment of membranes with trypsin or heat causes a significant inhibition of the binding. Midgut-Lp interaction was affected by changes in ionic strength and by suramin, but showed no requirement of calcium. Ligand blotting assays revealed two membrane proteins that specifically bound Lp (61 and 45 kDa). At cellular level, Lp binding sites were located mainly at the basal plasma membrane of isolated enterocytes. Labeled Lp with fluorescent probes directed to its proteins or its phospholipids fraction co-localized mainly at the basement membrane of the midgut. In addition, no intracellular Lp was observed at any condition. The lack of an endocytic pathway for Lp in the midgut of P. megistus is analyzed in the context of insect physiology.
Subject(s)
Insect Proteins/metabolism , Lipoproteins/metabolism , Panstrongylus/metabolism , Animals , Cell Membrane/metabolism , Digestive System/metabolism , Protein BindingABSTRACT
The metabolism of lipids and carbohydrates related to flight activity in Panstrongylus megistus was investigated. Insects were subjected to different times of flight under laboratory conditions and changes in total lipids, lipophorin density and carbohydrates were followed in the hemolymph. Lipids and glycogen were also assayed in fat body and flight muscle. In resting insects, hemolymph lipids averaged 3.4 mg/ml and significantly increased after 45 min of flight (8.8 mg/ml, P < 0.001). High-density lipophorin was the sole lipoprotein observed in resting animals. A second fraction with lower density corresponding to low-density lipophorin appeared in insects subjected to flight. Particles from both fractions showed significant differences in diacylglycerol content and size. In resting insects, carbohydrate levels averaged 0.52 mg/ml. They sharply declined more than twofold after 15 min of flight, being undetectable in hemolymph of insects flown for 45 min. Lipid and glycogen from fat body and flight muscle decreased significantly after 45 min of flight. Taken together, the results indicate that P. megistus uses carbohydrates during the initiation of the flight after which, switching fuel for flight from carbohydrates to lipids.
Subject(s)
Carbohydrate Metabolism , Flight, Animal/physiology , Hemolymph/metabolism , Lipid Metabolism , Panstrongylus/physiology , Animals , Carbohydrates/analysis , Fat Body/metabolism , Female , Hemolymph/chemistry , Lipids/analysis , Male , Panstrongylus/metabolism , RestABSTRACT
Modifications in content and lipid composition induced by fasting were examined in fat bodies from adults of Triatominae, Dipetalogaster maximus, Triatoma infestans and Panstrongylus megistus. With fasting, total lipid stores dropped approximately 50% for T. infestans and more than 70% for P. megistus. Total lipids analyzed by thin layer chromatography and fractionated by column chromatography on Unisil showed triacylglycerols as the main component in the three species, although P. megistus showed high levels of diacylglycerols (31-46%). Cholesterol amounted to 8-15%. In diacylglycerol fractions, C16:0, C18:1 and C18:0 fatty acids were detected; their ratio varied with species but it was not dependent on nutritional status. In triacylglycerol fractions C18:1 fatty acid was the major component at different times (48-68%); the ratio of monounsaturated to saturated in this fraction was 1.3, 2.6 and 1.2 for D. maximus, T. infestans and P. megistus respectively. The remarkable drop in lipid stores without noticeable changes in their relative composition would suggest that all types of lipid are used at similar rates. The higher content of diacylglycerols in P. megistus may be associated with the better flight performance of this species.
Subject(s)
Fat Body/metabolism , Lipid Metabolism , Triatominae/metabolism , Animals , Cholesterol/analysis , Diglycerides/analysis , Fasting/metabolism , Fatty Acids/analysis , Insect Vectors/metabolism , Lipids/analysis , Panstrongylus/metabolism , Triatoma/metabolismABSTRACT
A hemolinfa de Panstrongylus megistus mostrou uma atividade lectínica natural para eritrócitos de vários vertebrados e näo mostrou especificidade para os diversos tipos de eritrócitos testados (humano ABO, pato, coelho,c amundongo,carneiro, galinha e boi). Com relaçäo aos eritrócitos humanos a atividade lectínica foi similar nos tipos A+, B+ e AB+ enquanto a atividade mais alta foi observada no tipo O+. O título de aglutinaçäo entre eritrócitos animais näo mostrou diferença apreciável, excluindo eritrócitos de boi, que apresentaram o título mmais baixo. A determinaçäo da concentraçäo mínima de inibiçäo foi realizada com eritrócitos humanos O+. A aglutinaçäo foi inibida por vários carboidratos (ramnose, D-dalactose, rafinose, D-lactose e D-fucose). A ramnose foi o inibidor mais potente (0,78 mM). Os resultados sugerem a presença de mais de uma lectina na hemolinfa de P. megistus
Subject(s)
Erythrocytes/metabolism , Hemolymph/metabolism , Lectins/blood , Panstrongylus/metabolism , Agglutination TestsABSTRACT
The haemolymph of Panstrongylus megistus showed a natural lectin activity for a wide range of vertebrate erythrocytes. Agglutination was observed against all vertebrate erythrocytes tested (human ABO, duck, rabbit, mouse, sheep, chicken and cow). Cow erythrocytes showed the lowest titre. Concerning human erythrocytes, the lectin activity was similar in the types A+, B+ and AB+ while the highest activity was observed in the type O+. Determination of minimal inhibitory concentrations was carried out with human erythrocytes type O+. Agglutination was inhibited by several carbohydrates (rhamnose, D-galactose, raffinose, D-lactose and D-fucose). Rhamnose was reported as the strongest inhibitor (0.78 mM). The results suggest the presence of more than one lectin in the haemolymph of P. megistus.