Trypanosoma cruzi TcSMUG L-surface Mucins Promote Development and Infectivity in the Triatomine Vector Rhodnius prolixus

Background

TcSMUG L products were recently identified as novel mucin-type glycoconjugates restricted to the surface of insect-dwelling epimastigote forms of Trypanosoma cruzi, the etiological agent of Chagas disease. The remarkable conservation of their predicted mature N-terminal region, which is exposed to the extracellular milieu, suggests that TcSMUG L products may be involved in structural and/or functional aspects of the interaction with the insect vector.

Methodology and Principal Findings

Here, we investigated the putative roles of TcSMUG L mucins in both in vivo development and ex vivo attachment of epimastigotes to the luminal surface of the digestive tract of Rhodnius prolixus. Our results indicate that the exogenous addition of TcSMUG L N-terminal peptide, but not control T. cruzi mucin peptides, to the infected bloodmeal inhibited the development of parasites in R. prolixus in a dose-dependent manner. Pre-incubation of insect midguts with the TcSMUG L peptide impaired the ex vivo attachment of epimastigotes to the luminal surface epithelium, likely by competing out TcSMUG L binding sites on the luminal surface of the posterior midgut, as revealed by fluorescence microscopy.

Conclusion and Significance

Together, these observations indicate that TcSMUG L mucins are a determinant of both adhesion of T. cruzi epimastigotes to the posterior midgut epithelial cells of the triatomine, and the infection of the insect vector, R. prolixus.     

Lipid metabolism in Rhodnius prolixus (Hemiptera: Reduviidae): role of a midgut triacylglycerol-lipase

The utilization of dietary lipids was studied in adult females of Rhodnius prolixus with the use of radiolabeled triacylglycerol (TAG). It was shown that (3)H-triolein, when added to blood meal, was hydrolyzed to free fatty acids in the posterior midgut lumen. Subsequently, free fatty acids were absorbed by posterior midgut epithelium and used in the synthesis of phospholipids, diacylglycerol (DAG) and TAG. Phospholipids, DAG and free fatty acids were then found in hemolymph, from where they were rapidly cleared, and label was found in the fat body, mainly associated with TAG. Radioactive lipids, especially TAG and phospholipids, also accumulated in the ovaries. The TAG-lipase activities of posterior midgut luminal content and tissue were characterized by incubation of these samples with (3)H-triolein in the presence of the detergent Triton X-100 and determination of the amounts of released radioactive free fatty acids. Under the conditions employed here, the release of free fatty acids was proportional to the incubation time and to the amount of sample obtained from insect midgut (enzyme source) that was added. TAG-lipase activities were affected by pH and posterior midgut tissue showed optimum activity around pH 7.0-7.5, but the luminal content had the highest activities as pH decreased. Differences in activities were observed according to calcium concentration in the medium. TAG-lipase activities were also affected by the concentration of NaCl and were activated in the presence of increasing salt concentrations. These activities were inhibited by phenylmethylsulphonyl fluoride (PMSF). On the second day after blood meal, when digestion is very intense, TAG-lipase activities were maximal and then gradually decreased.     

Lipophorin density variation during oogenesis in Rhodnius prolixus

The density of lipophorin was determined in adult females of Rhodnius prolixus on different days after a meal. Several populations of lipophorins, differing in density but always in the range of HDL, were found in the hemolymph. The density of the major population was analyzed and a complex profile of density variation was found associated with the principal metabolic events in these insects digestion and oogenesis. During the initial three days after the blood meal, with the onset of the digestive process, the density of lipophorin decreased from 1.1185 g/l to 1.1095 g/l, associated with the transfer of lipids from midgut to the lipophorin particles. During the period of intense vitellogenesis and lipid uptake by the ovary, the lipophorin density started to increase and reached the value, 1.1322 g/l, and remained stable up to the end of oogenesis. As soon as the requirement of lipids to build up the oocytes ceased, the density of lipophorin decreased to its initial value associated with the transfer of lipids from fat body to lipophorin. Soon after the blood meal the midgut was the main source of lipids capable of replenishing the lipophorin particles, while the fat body assumed this function during the succeeding days and reached its maximum capacity around day 10, as estimated by the rate of lipid transfer. The principal lipids transferred were phospholipids and diacylglycerols. Except in the protein/lipid ratio no major changes were observed among different lipids isolated from lipophoin of different densities. Arch. Insect Biochem. Physiol. 35:301-313, 1997.© 1997 Wiley-Liss, Inc.     

Loading of lipophorin particles with phospholipids at the midgut of Rhodnius prolixus

³²P-Labelled midguts (³²P-midguts) of Rhodnius prolixus females were incubated in the presence of nonradioactive purified lipophorin and the release of radioactivity to the medium was analysed. The radioactivity found in the medium was associated with lipophorin phospholipids. When the ³²P-midguts were incubated in the absence of lipophorin, no ³²P-phospholipids were found in the medium. Comparative analysis by thin-layer chromatography of ³²P-phospholipids derived from metabolically labelled ³²P-midgut or lipophorin particles after incubation with ³²P-midgut showed some differences, revealing a possible selectivity in the process of phospholipids transfer. The transfer of phospholipids to lipophorin was linear with time up to 45 min, was saturable with respect to the concentration of lipophorin, and was half-maximal at about 5 mg/ml. The binding of ³²P-lipophorin to the midgut at O°C reached the equilibrium at about 1 h of incubation. The binding of ³²P-lipophorin was inhibited by an excess of nonradioactive lipophorin, which suggests a specific receptor for lipophorin. The capacity of midguts and fat bodies to transfer phospholipids to lipophorin varied during the days following the meal. When lipophorin enzymatically depleted of phospholipids by treatment with phospholipase A² was incubated with ³²P-midguts, the same amount of phospholipids was transferred, indicating a net gain of phospholipids by the particle. © 1995 Wiley-Liss, Inc.     

Biochemical and cellular characterization of lipophorin–midgut interaction in the hematophagous Panstrongylus megistus (Hemiptera: Reduviidae)

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 (¹²⁵I-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 μg/ml. However, membranes obtained after a blood meal exhibited higher binding activity. Saturation kinetics results using ¹²⁵I-Lp suggested a single binding site with high affinity for Lp in the midgut membranes (K_d = 5.1 ± 3.6 × 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.     

OVARIAN NUTRITIONAL RESOURCES DURING THE REPRODUCTIVE CYCLE OF THE HEMATOPHAGOUS DIPETALOGASTER MAXIMA (HEMIPTERA: REDUVIIDAE): FOCUS ON LIPID METABOLISM

In this study, we have analyzed the changes of the ovarian nutritional resources in Dipetalogaster maxima at representative days of the reproductive cycle: previtellogenesis, vitellogenesis, as well as fasting‐induced early and late atresia. As expected, the amounts of ovarian lipids, proteins, and glycogen increased significantly from previtellogenesis to vitellogenesis and then, diminished during atresia. However, lipids and protein stores found at the atretic stages were higher in comparison to those registered at previtellogenesis. Specific lipid staining of ovarian tissue sections evidenced remarkable changes in the shape, size, and distribution of lipid droplets throughout the reproductive cycle. The role of lipophorin (Lp) as a yolk protein precursor was analyzed by co‐injecting Lp‐OG (where OG is Oregon Green) and Lp‐DiI (where DiI is 1,10‐dioctadecyl‐3,3,30,30‐tetramethylindocarbocyanine) to follow the entire particle, demonstrating that both probes colocalized mainly in the yolk bodies of vitellogenic oocytes. Immunofluorescence assays also showed that Lp was associated to yolk bodies, supporting its endocytic pathway during vitellogenesis. The involvement of Lp in lipid delivery to oocytes was investigated in vivo by co‐injecting fluorescent probes to follow the fate of the entire particle (Lp‐DiI) and its lipid cargo (Lp‐Bodipy‐FA). Lp‐DiI was readily incorporated by vitellogenic oocytes and no lipoprotein uptake was observed in terminal follicles of ovaries at atretic stages. Bodipy‐FA was promptly transferred to vitellogenic oocytes and, to a much lesser extent, to previtellogenic follicles and to oocytes of ovarian tissue at atretic stages. Colocalization of Lp‐DiI and Lp‐Bodipy‐FA inside yolk bodies indicated the relevance of Lp in the buildup of lipid and protein oocyte stores during vitellogenesis.     

The storage of nutritional resources during vitellogenesis of Panstrongylus megistus (Hemiptera: Reduviidae): the pathways of lipophorin in lipid delivery to developing oocytes

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.     

Phenol oxidases from Rhodnius prolixus: Temporal and tissue expression pattern and regulation by ecdysone

Rhodnius prolixus 5th instar nymphs have significant PO enzymatic activity in the anterior midgut, fat body and hemolymph. The tissue with the major amount of PO activity is the anterior midgut while those with higher specific activities are the fat body and hemolymph. In this work the temporal pattern of PO enzymatic activity in different tissues was investigated. In fat body, PO peaks occur at 7, 12 and 16 days after a blood meal. In hemolymph, PO diminishes until day 7, and then recovers by day 14. In the anterior midgut tissue, PO peaks on day 9 and just before ecdysis; a similar pattern was observed in the anterior midgut contents. Some of these activities are dependent on the release of ecdysone, as feeding blood meal containing azadirachtin suppresses them and ecdysone treatment counteracts this effect. These results suggest that during the development of the 5th instar, the insect has natural regulating cycles of basal PO expression and activation, which could be related to the occurrence of natural infections. The differences in temporal patterns of activity and the effects of azadirachtin and ecdysone in each organ suggest that, at least in R. prolixus, different tissues are expressing different PO genes.     

Effects of the season and growth stage on the contents of lipids and photosynthetic pigments in brown alga <Emphasis Type="Italic">Undaria pinnatifida</Emphasis>

Seasonal changes in the contents of lipids and photosynthetic pigments (PSP) in the brown alga Undaria pinnatifida (Harvey) Suringar (Phaeophyceae, Alariaceae) on different stages of its growth were studied. Lipids of all plant growth group comprised glyceroglycolipids (GL), phospholipids, and neutral lipids (NL). The ratio between these lipid groups and the content of particular lipids depended on the season and algal growth stage: NL predominated in seedlings; juvenile algae comprised approximately similar amounts of NL and GL; and in adult algae, GL predominated. In winter and spring, algal tissues contained relatively more free sterols than in summer. Total lipid content in seedlings and juvenile algae was higher then in adult plants. Lipid fatty acid (FA) composition was similar on all growth stages, but the content of major components differed; this is mainly related to 18:4 n-3, 20:4 n-6, and 20:5 n-3 acids. The predominant FAs in seedling lipids were saturated FAs, whereas in the lipids of juvenile and adult algae, polyunsaturated FAs predominated.     

Phosphorus supply drives rapid turnover of membrane phospholipids in the diatom Thalassiosira pseudonana

In low-phosphorus (P) marine systems, phytoplankton replace membrane phospholipids with non-phosphorus lipids, but it is not known how rapidly this substitution occurs. Here, when cells of the model diatom Thalassiosira pseudonana were transferred from P-replete medium to P-free medium, the phospholipid content of the cells rapidly declined within 48 h from 45±0.9 to 21±4.5% of the total membrane lipids; the difference was made up by non-phosphorus lipids. Conversely, when P-limited T. pseudonana were resupplied with P, cells reduced the percentage of their total membrane lipids contributed by a non-phosphorus lipid from 43±1.5 to 7.3±0.9% within 24 h, whereas the contribution by phospholipids rose from 2.2±0.1 to 44±3%. This dynamic phospholipid reservoir contained sufficient P to synthesize multiple haploid genomes, suggesting that phospholipid turnover could be an important P source for cells. Field observations of phytoplankton lipid content may thus reflect short-term changes in P supply and cellular physiology, rather than simply long-term adjustment to the environment.     

Fatty acid incorporation by Rhodnius prolixus midgut

[(14)C]Oleic acid injected into the hemocoel of Rhodnius prolixus females was shown to rapidly associate with lipophorin particles. Half of the lipophorin-associated [(14)C]oleic acid was transferred in about 5 min to different organs, but the midgut was the main organ to take it up on day 10 after a blood meal. The rate of [(14)C]oleic acid incorporation by the midgut was high up to 15 min after injection and then declined. The [(14)C]oleic acid incorporated by the midgut was found in phospholipids (58.6%) and neutral lipids (37.4%). The midgut capacity to incorporate [(14)C]oleic acid varied on different days after a meal: it increased up to day 10 and then decreased. The fate of the [(14)C]lipids synthesized by the midgut was followed and it was observed that 10 days after feeding diacylglycerol was the main lipid released to hemolymph and that most of phospholipids and triacylglycerols remained associated with the midgut. The metabolism of free fatty acids in Rhodnius prolixus females is discussed in the context of major biological events that follow a blood meal such as digestion and oogenesis.     

Composition of DNA-bound lipids in the regenerating rat liver

Using thin-layer chromatography, the qualitative and quantitative composition of specific DNA-bound neutral lipids (NL) and phospholipids (PL) of regenerating rat liver 22 hours (S-phase) and 28 hours (G2-phase) after hepatectomy was studied. These lipids are represented by light and tightly bound components. The intact liver DNA contains minor amounts of NL and PL (15.02 micrograms and 5.82 micrograms per mg of DNA, respectively). The composition of DNA-bound lipids in rat liver differs markedly from that of nuclear membrane and chromatin total lipids. The former are strongly deficient in free cholesterol (FC), but are rich in cholesterol esters (CE), very rich in cardiolipin (CL) and deficient in phosphatidylcholine. The basic parameters of DNA-bound lipids of rat liver (NL/PL, CE/FC and cholesterol/PL) are more than unity and depend on the cell cycle. It was shown that in the S-phase the content of DNA-bound NL and PL increases 1.5-fold, in the G2-phase the NL content shows a still greater increase--2.3-fold, while that of DNA-bound PL decreases to normal values. The basic changes of the DNA-bound lipids in regenerating rat liver are due to FC, CE and CL, which determine the tissue specificity of these lipids.     

Activity cycles and the control of four digestive proteinases in the posterior midgut of Rhodnius prolixus Stål (Hemiptera: Reduviidae)

Proteinase levels in the posterior midgut of fifth-instar and adult Rhodnius prolixus follow a cyclic pattern after ingestion of the bloodmeal. In the fifth instar, cathepsin B showed two peaks: the first occurred 6 days after ingestion and the second at the time of ecdysis. Cathepsin D, cathepsin B and lysosomal carboxypeptidase B reached maximal levels 6 days after ingestion. At this time the highest levels of these proteinases were found in mated females, the lowest in males and intermediate levels in virgin females. Maximal levels of aminopeptidase occurred later than catheptic enzymes, and the decline, after maximal levels were achieved, was much more gradual.Catheptic-proteinase levels within the posterior midgut in fifth-instar larvae and adults correlated positively with the amount of protein contained in this gut region. This indicates that production of these proteinases is controlled by a secretagogue mechanism. Aminopeptidase levels were controlled in a different manner. The mated state or sex of adults altered the proteinase levels by changing the amount of protein that was passed into the digestive midgut from the crop.     

Control of protease secretion in the intestine of fifth instar larvae of Rhodnius prolixus.

Groups of four Rhodnius prolixus larvae in the 5th-instar received no food or were given once only through a special feeder, similar weights of food solutions. These were either protein solutions (whole human blood, its plasma or erythrocytes, egg albumin, bovine serum albumin, haemoglobin) or non-protein solutions (saline, casein hydrolysate, dextran, maltose). Following feeding, the protein content and caseinolytic activity of the larval midgut homogenates, determined between the 3rd and 14th days, were practically the same in starved larvae and in larvae receiving non-protein solutions. In the protein-fed larvae the protein content and the specific protease activity increased. These findings exclude neurosecretory control and indicate that ingested protein stimulate the proteolytic activity of R. prolixus midgut.     

The effect of adipokinetic hormone on midgut characteristics in Pyrrhocoris apterus L. (Heteroptera)

Digestive processes and the effect of adipokinetic hormone (Pyrap-AKH) on the amount of nutrients (lipids, proteins, and carbohydrates), and on the activity of digestive enzymes (lipases, peptidases, and carbohydrases) were studied in the midgut of the firebug, Pyrrhocoris apterus. The analyses were performed on samples of anterior (AM), middle (MM) and posterior (PM) midgut parts. The results revealed that the digestion of lipids, carbohydrates and proteins take place in the acidic milieu. The Pyrap-AKH treatment increased significantly the level of lipids and proteins in the midgut, and also the level of triacylglycerols (TGs) predominantly in the AM, and the level of diacylglycerols (DGs) in the MM. The increase was not uniform for all present TG and DG species - those containing the linoleic fatty acid were predominant. No hormonal effect on lipase activity was recorded, while peptidase and glucosidase activity was increased in the MM and PM. All these facts indicate that the Pyrap-AKH probably stimulates digestion by more intensive food ingestion or turnover, and perhaps by the stimulation of metabolite absorption; the activation of digestive enzymes seems to be secondary or controlled by other mechanisms.     

The digestive system of the “stick bug” Cladomorphus phyllinus (Phasmida,Phasmatidae): A morphological,physiological and biochemical analysis

This work presents a detailed morphofunctional study of the digestive system of a phasmid representative, Cladomorphus phyllinus. Cells from anterior midgut exhibit a merocrine secretion, whereas posterior midgut cells show a microapocrine secretion. A complex system of midgut tubules is observed in the posterior midgut which is probably related to the luminal alkalization of this region. Amaranth dye injection into the haemolymph and orally feeding insects with dye indicated that the anterior midgut is water-absorbing, whereas the Malpighian tubules are the main site of water secretion. Thus, a putative counter-current flux of fluid from posterior to anterior midgut may propel enzyme digestive recycling, confirmed by the low rate of enzyme excretion. The foregut and anterior midgut present an acidic pH (5.3 and 5.6, respectively), whereas the posterior midgut is highly alkaline (9.1) which may be related to the digestion of hemicelluloses. Most amylase, trypsin and chymotrypsin activities occur in the foregut and anterior midgut. Maltase is found along the midgut associated with the microvillar glycocalix, while aminopeptidase occurs in the middle and posterior midgut in membrane bound forms. Both amylase and trypsin are secreted mainly by the anterior midgut through an exocytic process as revealed by immunocytochemical data.     

Changes in lipoprotein(a), oxidized phospholipids, and LDL subclasses with a low-fat high-carbohydrate diet

Low-fat diets have been shown to increase plasma concentrations of lipoprotein(a) [Lp(a)], a preferential lipoprotein carrier of oxidized phospholipids (OxPLs) in plasma, as well as small dense LDL particles. We sought to determine whether increases in plasma Lp(a) induced by a low-fat high-carbohydrate (LFHC) diet are related to changes in OxPL and LDL subclasses. We studied 63 healthy subjects after 4 weeks of consuming, in random order, a high-fat low-carbohydrate (HFLC) diet and a LFHC diet. Plasma concentrations of Lp(a) (P < 0.01), OxPL/apolipoprotein (apo)B (P < 0.005), and OxPL-apo(a) (P < 0.05) were significantly higher on the LFHC diet compared with the HFLC diet whereas LDL peak particle size was significantly smaller (P < 0.0001). Diet-induced changes in Lp(a) were strongly correlated with changes in OxPL/apoB (P < 0.0001). The increases in plasma Lp(a) levels after the LFHC diet were also correlated with decreases in medium LDL particles (P < 0.01) and increases in very small LDL particles (P < 0.05). These results demonstrate that induction of increased levels of Lp(a) by an LFHC diet is associated with increases in OxPLs and with changes in LDL subclass distribution that may reflect altered metabolism of Lp(a) particles.