ABSTRACT
In insects, lipid transfer to the tissues is mediated by lipophorin, the major circulating lipoprotein, mainly through a nonendocytic pathway involving docking receptors. Currently, the role of such receptors in lipid metabolism remains poorly understood. In this work, we performed a histological characterization of the fat body of the Chagas' disease vector, Panstrongylus megistus (Burmeister), subjected to different nutritional conditions. In addition, we addressed the role of the ß-chain of ATP synthase (ß-ATPase) in the process of lipid transfer from lipophorin to the fat body. Fifth-instar nymphs in either fasting or fed condition were employed in the assays. Histological examination revealed that the fat body was composed by diverse trophocyte phenotypes. In the fasting condition, the cells were smaller and presented a homogeneous cytoplasmic content. The fat body of fed insects increased in size mainly due to the enlargement of lipid stores. In this condition, trophocytes contained abundant lipid droplets, and the rough endoplasmic reticulum was highly developed and mitochondria appeared elongated. Immunofluorescence assays showed that the ß-ATPase, a putative lipophorin receptor, was located on the surface of fat body cells colocalizing partially with lipophorin, which suggests their interaction. No changes in ß-ATPase expression were found in fasting and fed insects. Blocking the lipophorin-ß-ATPase interaction impaired the lipophorin-mediated lipid transfer to the fat body. The results showed that the nutritional status of the insect influenced the morphohistological features of the tissue. Besides, these findings suggest that ß-ATPase functions as a lipophorin docking receptor in the fat body.
Subject(s)
ATP Synthetase Complexes/metabolism , Fat Body/cytology , Insect Proteins/metabolism , Lipid Metabolism , Lipoproteins/metabolism , Panstrongylus/cytology , Animals , Fat Body/enzymology , Nymph/cytology , Nymph/enzymology , Panstrongylus/enzymology , Panstrongylus/growth & developmentABSTRACT
A cDNA encoding a trypsin-like protease from the salivary glands of the haematophagous reduviid Panstrongylus megistus was cloned and sequenced. The deduced protein sequence showed similarities to serine proteases of other hemipterans but with substitutions in the catalytic triad and the substrate binding site. The expression of the gene increased more than sixfold after feeding. Saliva showed the highest proteolytic activity at neutral to slightly basic pH. Substrate and inhibitor profiles and zymography indicated the presence of a trypsin-like protease with preference for Arg and Lys at P1. Using chromatography, a fibrinolytic enzyme was purified whose sequence was identified by tandem mass spectrometry as that encoded by the cDNA.
Subject(s)
Feeding Behavior , Panstrongylus/enzymology , Protein Processing, Post-Translational , Salivary Glands/enzymology , Serine Proteases/genetics , Amino Acid Sequence , Animals , Base Sequence , DNA, Complementary/genetics , Electrophoresis, Polyacrylamide Gel , Feeding Behavior/drug effects , Fibrinolysis/drug effects , Gene Expression Profiling , Gene Expression Regulation/drug effects , Hydrolysis/drug effects , Larva/drug effects , Larva/enzymology , Molecular Sequence Data , Molecular Weight , Panstrongylus/drug effects , Panstrongylus/genetics , Protein Processing, Post-Translational/drug effects , Salivary Glands/drug effects , Sequence Alignment , Serine Proteases/chemistry , Serine Proteases/isolation & purification , Serine Proteases/metabolism , Serine Proteinase Inhibitors/pharmacology , Substrate Specificity/drug effects , Time FactorsABSTRACT
The metabolism of dietary lipids in the anterior midgut of Panstrongylus megistus during blood digestion was studied. Fifth instar nymphs were fed a blood meal containing 7.1 +/- 0.4 mg of lipids, consisting mainly of triacylglycerol (TAG), and completed the overall process of digestion in about 20 days. Lipolysis of TAG and pathways for diacylglycerol (DAG) biosynthesis in the midgut were investigated by feeding the insects with [9,10-3H]-oleic acid-labeled triolein. Lumenal [3H]-triacylglycerol was hydrolyzed, generating mainly fatty acids (FA) and glycerol and to lesser extent, DAG. Almost no radioactivity associated with monoacylglycerol was found at any time. In midgut tissue, labeled fatty acids were incorporated into phosphatidic acid, DAG and TAG, whereas no significantly labeled monoacylglycerol was observed. In addition, the activities of enzymes related to DAG metabolism were assayed in non-blood fed midgut homogenates and at different times after feeding on a blood meal. Significant changes in the activities of phosphatidate phosphohydrolase (EC 3.1.3.4) and triacylglycerol lipase (EC 3.1.1.3) were observed during blood digestion, suggesting that these enzymes are important in regulating intracellular DAG synthesis and mobilization in midgut cells. Finally, the histological changes of lipid stores observed in anterior midgut confirmed the active process of uptake and trafficking of lipids performed by the enterocytes during blood digestion.
Subject(s)
Dietary Fats/blood , Dietary Fats/metabolism , Digestive System/metabolism , Panstrongylus/metabolism , Animals , Dietary Fats/pharmacokinetics , Dietary Fats/pharmacology , Digestive System/enzymology , Diglycerides/biosynthesis , Lipase/metabolism , Lipid Metabolism , Lipids/administration & dosage , Lipids/classification , Lipids/pharmacokinetics , Nymph/enzymology , Nymph/metabolism , Panstrongylus/enzymology , Panstrongylus/growth & development , Phosphatidate Phosphatase/metabolismABSTRACT
The alpha-glycerophosphate dehydrogenase (alpha-GPDH) activity in flight muscles of Panstrongylus megistus and Triatoma sordida, vectors of Chagas disease in Brazil, was studied. Both species showed higher enzymatic activities in fliers than in non-fliers insects. T. sordida exhibited a higher proportion of flier insects than P. megistus. A possible role of alpha-GPDH on triatomines flight is discussed.
Subject(s)
Animals , Flight, Animal/physiology , Glycerolphosphate Dehydrogenase/metabolism , Insect Vectors/enzymology , Muscles/enzymology , Triatominae/enzymology , Glycerolphosphate Dehydrogenase/physiology , Panstrongylus/enzymology , Triatoma/enzymologyABSTRACT
The alpha-glycerophosphate dehydrogenase (alpha-GPDH) activity in flight muscles of Panstrongylus megistus and Triatoma sordida, vectors of Chagas disease in Brazil, was studied. Both species showed higher enzymatic activities in fliers than in non-fliers insects. T. sordida exhibited a higher proportion of flier insects than P. megistus. A possible role of alpha-GPDH on triatomines flight is discussed.
