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.     

Lipophorin interaction with the midgut of Rhodnius prolixus: characterization and changes in binding capacity

Several classes of lipids are transported in insect hemolymph by lipophorin, a major hemolymphatic lipoprotein. The binding of lipophorin to the midgut of the hematophagous insect Rhodnius prolixus was characterized in a midgut membrane preparation, using purified lipophorin radiolabelled in protein moiety ((125)I-HDLp). Lipophorin specific binding to membranes achieved equilibrium after 30-40 min, was sensitive to pH, and was maximal at pH 7.0. In the presence of increasing concentrations of membrane protein, corresponding increases in lipophorin binding were observed. The specific binding of lipophorin to the membrane preparation was a saturable process, with K(d)=0.9+/-0.06 x 10(-7) M and a maximal binding capacity of 70+/-11 ng lipophorin/microg of membrane protein. Lipophorin binding did not depend on calcium, but it was affected by ionic strength and was inhibited in the presence of increasing salt concentrations. Suramin interfered with lipophorin binding to the midgut receptor, and it was abolished in the presence of 2 mM suramin, but at concentrations between 0.05 and 0.2 mM it was slightly increased. Condroitin 4-sulfate also affected lipophorin binding, which was reduced to 56% of control. Pre-incubation of the midgut membrane preparation with trypsin or at high temperature inhibited binding. Midgut capacity to bind lipophorin varied at different days after blood meal. It was highest at second day after feeding, and then gradually decreased.     

Purification and characterization of a lipid transfer particle in Rhodnius prolixus: phospholipid transfer

In this study we report the purification and characterization of a lipid transfer particle (LTP) from Rhodnius prolixus hemolymph, and its participation in phospholipid and diacylglycerol transfer processes. (3)H-diacylglycerol labeled low density lipophorin from Manduca sexta ((3)H-LDLp) was incubated with R. prolixus lipophorin (Lp) in the presence of Rhodnius hemolymph. Following incubation and isolation, both lipoproteins showed equivalent amounts of (3)H-labeled lipids. Hemolymph was subjected to KBr gradient ultracentrifugation. SDS-PAGE analysis of gradient fractions showed the enrichment of bands with molecular masses similar to the M. sexta LTP standard. LTP containing fractions were assayed and lipid transfer activity was observed. Purification of LTP was accomplished by (i) KBr density gradient ultracentrifugation, (ii) size exclusion, (iii) Cu(++) affinity and (iv) ion exchange chromatographies. LTP molecular mass was estimated approximately 770 kDa, comprising three apoproteins, apoLTP-I (315 kDa), apoLTP-II (85 kDa) and apoLTP-III (58 kDa). Phospolipid content of (32)P-LTP was determined after two-dimensional TLC. (32)P-phospholipid-labeled and unlabeled lipophorins, purified from R. prolixus were incubated in the presence of LTP resulting in the time-dependent transfer of phospholipids. LTP-mediated phospholipid transfer was not a selective process.     

Evidence and partial characterization of an inducible antibacterial factor in the haemolymph of Rhodnius prolixus

Antibacterial activity induced in the haemolymph of adult Rhodnius prolixus was investigated. Little or no antibacterial activity appeared in the first hours after Streptococcus mutans inoculation, but the activity increased reaching a maximum 5–6 days later and then declined gradually. The bacteria were destroyed in the haemolymph when the level of the antibacterial activity attained maximal value. The antibacterial activity appears to be related to the defence mechanism of the insect against bacterial infection. The activity was semi-purified by one-step Bio-Gel P-10 Gel-filtration and the estimated apparent mol. wt was 7,000 Daltons. It was found that this activity was due to a protein of small molecular weight. The activity was heat-stable, dialyzable and inactivated by trypsin treatment. The addition of haemolymph, obtained after inoculation of S. mutans into insects, to label-DNA growing cells (1) caused a dramatic reduction in the viable cells, measured by colony-forming units, and (2) increased approx 4-fold the releasing of labelled-DNA into the supernatant of the bacterial culture media. The possibility of this activity being lysozyme was excluded. The possible mechanism of action of this antibacterial activity is discussed.     

Purification and properties of a storage protein from the hemolymph of Rhodnius prolixus

A non-arylphorin hexameric storage protein (SPR) has been isolated from the larval hemolymph of the bug, Rhodnius prolixus. The purified protein contained 0.69% lipid and 1.04% carbohydrate. SPR had a mol. wt of 470,000, with a subunit mol. wt of 78,000. In electron micrographs, SPR molecules appeared very similar to calliphorin. After the metamorphic molt, SPR persisted at lower concentrations in the hemolymph of adult insects, and it disappeared only in fasting bugs.     

Prothoracicotropic hormone of Rhodnius prolixus: partial characterization and rhythmic release of neuropeptides related to Bombyx PTTH and bombyxin

Summary

The brain-retrocerebral complex (br-complex) of Rhodnius prolixus was found both to contain and release neuropeptides related to Bombyx mori PTTH and bombyxin. A > 10 kDa peptide fraction obtained from extracts and incubation media of br-complex exhibited high steroidogenic activity on Rhodnius prothoracic glands and reacted with a Bombyx PTTH antibody on dot blots. The release from the Br-complex of this immunoreactive peptide fraction showed a daily rhythm: high release during the night and little on no release during the day. On Western blots, a single 68 kDa peptide in the > 10 kDa peptide fraction was recognized by the Bombyx PTTH antibody and was also released rhythmically during a day. This peptide was reduced to a doublet of about 17 kDa that retained immunoreactivity. Double immunoprecipitation of the > 10 kDa peptide fraction from brain media using the Bombyx PTTH antibody and agarose-bound secondary antibody removed the steroidogenic activity in this fraction; it also removed the 68 kDa peptide on Western blots. A bombyxin antiserum recognized a 3–5 kDa peptide in a <10 kDa peptide fraction; this peptide fraction was also released with a daily rhythm but possessed weak steroidogenic activity. The natural PTTH of Rhodnius, therefore, appears to be a 68 kDa peptide, possibly composed of several 17 kDa subunits, that is related to Bombyx PTTH.     

Sulfated glycosaminoglycans from ovary of Rhodnius prolixus

We have characterized sulfated glycosaminoglycans from ovaries of the blood-sucking insect Rhodnius prolixus, and determined parameters of their synthesis and distribution within this organ by biochemical and histochemical procedures. The major sulfated glycosaminoglycan is heparan sulfate while chondroitin 4-sulfate is a minor component. These glycosaminoglycans are concentrated in the ovarian tissue and are not found inside the oocytes. Besides this, we detected the presence of a sulfated compound distinguished from sulfated glycosaminoglycans and possibly derived from sulfated proteins. Conversely to the compartmental location of sulfated glycosaminoglycans, the unidentified sulfated compound is located in the ovarian tissue as well as inside the oocytes. Based on these and other findings, the possible roles of ovarian sulfated glycosaminoglycans on the process of oogenesis in these insects are discussed.     

Lipophorin lipase from the yolk of Manduca sexta eggs: identification and partial characterization.

In the hawkmoth Manduca sexta high density lipophorin from adult insects (HDLp-A) delivers lipids to developing oocytes. During this lipid delivery HDLp-A is taken up by the oocyte and converted to a very high density lipophorin (VHDLp), which is stored in protein storage granules (yolk bodies). A membrane-free lysate of isolated M. sexta yolk bodies was demonstrated to contain lipoprotein lipase activity that hydrolyses the diacylglycerol of HDLp-A. With HDLp-A as a substrate yolk body lipophorin lipase (YBLpL) activity was shown to be maximal between pH 9 and pH 9.5. NaCl concentration was optimal between 0.7 M and 1 M. YBLpL activity required neither bovine serum albumin nor calcium ions but appeared to be stimulated by 5 mM EDTA. Diisopropyl fluorophosphate effectively inhibited YBLpL activity, indicating the presence of a serine in the active site of the enzyme. The identified lipase activity co-eluted with lipophorins and vitellins from the yolk in the void volume of a Sephadex G-75 gel filtration column. This observation suggests that the lipase has a Mr of more than 80,000, or that the enzyme is associated with the lipoproteins. Incubation of HDLp-A with yolk body lysate converted HDLp-A to two classes of higher density lipophorins. The highest density lipophorins produced during this incubation approached the density of VHDLp as it is isolated from mature eggs. The possible role of YBLpL activity in the delivery of lipids to developing oocytes is discussed.     

Isolation and purification of the diuretic hormone from Rhodnius prolixus

Extracts of Rhodnius prolixus mesothoracic ganglionic masses were subjected to column chromatographic separation procedures. The eluates were assayed biologically for diuretic hormone activity.Gel filtration through columns of polyacrylamide gel was found to separate soluble diuretic activity into two distinct zones. Poor recovery was obtained with respect to the activity of the crude extract. No diuretic activity was found to co-chromatograph with a sample of 5-hydroxytryptamine. When samples of haemolymph from freshly fed Rhodnius were chromatographed under similar conditions only the second zone (low molecular weight) was found to have diuretic activity. It is this low molecular weight zone which would more reasonably qualify as the physiologically active diuretic hormone. Likewise, the diuretic activity released from isolated mesothoracic ganglionic masses by K⁺-rich Ringer's solution showed only low molecular weight activity after chromatography.     

Juvenoid effects on Rhodnius prolixus embryogenesis

Protein electrophoretic techniques demonstrate that the normal patterns of protein synthesis accompanying embryogenesis of Rhodnius prolixus are altered by the juvenoid compound Ro 13.5223 (fenoxycarb). Electrophoretic analysis reveals that embryonic perturbation by fenoxycarb is due in part to alterations in the normal molecular events accompanying development. Using high-resolution 2-D gel electrophoresis, major differences in protein patterns were observed. Three proteins normally absent, were expressed in fenoxycarb perturbed embryos (two on day 3 and one on day 7).     

The neuropeptidome of Rhodnius prolixus brain

We show a sensitive and straightforward off‐line nano‐LC‐MALDI‐MS/MS workflow that allowed the first comprehensive neuropeptidomic analysis of an insect disease vector. This approach was applied to identify neuropeptides in the brain of Rhodnius prolixus, a vector of Chagas disease. This work will contribute to the annotation of genes in the ongoing R. prolixus genome sequence project. Peptides were identified by de novo sequencing and comparisons to known neuropeptides from different organisms by database search. By these means, we were able to identify 42 novel neuropeptides from R. prolixus. The peptides were classified as extended FMRF‐amide‐related peptides, sulfakinins, myosuppressins, short neuropeptide F, long neuropeptide F, SIF‐amide‐related peptides, tachykinins, orcokinins, allatostatins, allatotropins, calcitonin‐like diuretic hormones, corazonin, and pyrokinin. Some of them were detected in multiple isoforms and/or truncated fragments. Interestingly, some of the R. prolixus peptides, as myosuppressin and sulfakinins, are unique in their characteristic C‐terminal domain among insect neuropeptides identified so far.     

Structural and morphological characterization of hemozoin produced by Schistosoma mansoni and Rhodnius prolixus

Hemozoin (Hz) is a heme crystal produced upon the digestion of hemoglobin (Hb) by blood-feeding organisms as a main mechanism of heme disposal. The structure of Hz consists of heme dimers bound by reciprocal iron-carboxylate interactions and stabilized by hydrogen bonds. We have recently described heme crystals in the blood fluke, Schistosoma mansoni, and in the kissing bug, Rhodnius prolixus. Here, we characterized the structures and morphologies of the heme crystals from those two organisms and compared them to synthetic β-hematin (βH). Synchrotron radiation X-ray powder diffraction showed that all heme crystals share the same unit cell and structure. The heme crystals isolated from S. mansoni and R. prolixus consisted of very regular units assembled in multicrystalline spherical structures exhibiting remarkably distinct surface morphologies compared to βH. In both organisms, Hz formation occurs inside lipid droplet-like particles or in close association to phospholipid membranes. These results show, for the first time, the structural and morphological characterization of natural Hz samples obtained from these two blood-feeding organisms. Moreover, Hz formation occurring in close association to a hydrophobic environment seems to be a common trend for these organisms and may be crucial to produce very regular shaped phases, allowing the formation of multicrystalline assemblies in the guts of S. mansoni and R. prolixus.     

Metalloproteases in Trypanosoma rangeli-infected Rhodnius prolixus

Protease activities in the haemolymph and fat body in a bloodsucking insect, Rhodnius prolixus, infected with Trypanosoma rangeli, were investigated. After SDS-polyacrylamide gel electrophoresis containing gelatin as substrate, analysis of zymograms performed on samples of different tissues of controls and insects inoculated or orally infected with short or long epimastigotes of T. rangeli, demonstrated distinct patterns of protease activities: (i) proteases were detected in the haemolymph of insects which were fed on, or inoculated with, short epimastigotes of T. rangeli (39 kDa and 33 kDa, respectively), but they were not observed in the fat body taken from these insects; (ii) protease was also presented in the fat bodies derived from naive insects or controls inoculated with sterile phosphate-saline buffer (49 kDa), but it was not detected in the haemolymph of these insects; (iii) no protease activity was observed in both haemolymph and fat bodies taken from insects inoculated with, or fed on, long epimastigotes of T. rangeli. Furthermore, in short epimastigotes of T. rangeli extracts, three bands of the protease activities with apparent molecular weights of 297, 198 and 95 kDa were detected while long epimastigotes preparation presented only two bands of protease activities with molecular weights of 297 and 198 kDa. The proteases from the insect infected with T. rangeli and controls belong to the class of either metalloproteases or metal-activated enzymes since they are inhibited by 1,10-phenanthroline. The significance of these proteases in the insects infected with short epimastigotes of T. rangeli is discussed in relation to the success of the establishment of infection of these parasites in its vector, R. prolixus.