Salivary apyrase of Rhodnius prolixus. Kinetics and purification.

The salivary apyrase activity of the blood-sucking bug Rhodnius prolixus was found to reside in a true apyrase (ATP diphosphohydrolase, EC 3.6.1.5) enzyme. The crude saliva was devoid of 5'-nucleotidase, inorganic pyrophosphatase, phosphatase and adenylate kinase activities. ATP hydrolysis proceeded directly to AMP and Pi without significant accumulation of ADP. Km values for ATP and ADP hydrolysis were 229 and 291 microM respectively. Ki values for ATP and ADP inhibition of ADP and ATP hydrolysis were not different from the Km values, and these experiments indicated competitive inhibition. Activities were purified 126-fold by combined gel filtration and ion-exchange chromatography procedures with a yield of 63%. The purified enzyme displayed specific activities of 580 and 335 mumol of Pi released/min per mg of protein for ATP and ADP hydrolysis respectively. The action of the purified enzyme on several phosphate esters indicates that Rhodnius apyrase is a non-specific nucleosidetriphosphate diphosphohydrolase.     

Hormonal Integration of Egg Production in Rhodnius prolixus

SYNOPSIS. This paper reviews the evidence dealing with hormonalcontrols on egg production in Rhodnius prolixus by which theinputs of feeding and mating are integrated. Juvenile hormone,secreted by the corpus allatum, plays a central role in eggproduction, intervening at several sites to control the durationof egg production. The controls on the corpus allatum are complexand apparently highly redundant, and the necessity for thisredundancy is analysed in terms of the strategic advantage conferredon the insect by this network of controls.     

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.     

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.     

Lipophorin from Rhodnius prolixus: Purification and partial characterization

The lipophorin of adult females of Rhodnius prolixus was radioactively labelled with ³²P exclusively in the phospholipid moiety and purified on a KBr ultracentrifugation gradient. The density of purified [³²P]phospholipid labelled lipophorin on the fifth day after a blood meal was 1.1211 ± 0.0017 g/ml. By weight it contained 51.7% protein, 0.7% sugar and 47.6% lipid. The protein moiety was composed of three apoproteins of 226 ± 11, 86 ± 2 and 16 ± 1 kDa. Mannose and N-acetylglucosamine were the only sugars detected. Among the lipids, 66.3% were neutral lipids and 33.8% were phospholipids. Analysis by thin-layer chromatography showed that in the total phospholipids fraction ³²P was distributed as follows: phosphatidylethanolamine (54.4%), phosphatidylcholine (44.7%), cardiolipin (2.1%), phosphatidylserine (0.7%), phosphatidylinositol (0.4%), sphingomyelin (0.3%) and phosphatidic acid (0.2%). The total phosphate content was 0.53 ± 0.03 nmol/μg of protein.     

Transfer of phospholipids from fat body to lipophorin in Rhodnius prolixus

32P-Labeled fat bodies (32P-fat bodies) of Rhodnius prolixus females were incubated in the presence of non radioactive purified lipophorin and the release of radioactivity to the medium was analysed to answer the question of whether lipophorin is a reusable shuttle for phospholipids. The radioactivity found in the medium was associated with lipophorin phospholipids. When the 32P-fat bodies were incubated in the absence of lipophorin, only a small amount of radioactivity was released and it was not associated with lipophorin, indicating that there was no release of pre-labeled 32P-lipophorin by the tissue. Analysis of 32P-phospholipids transferred from fat bodies to the lipophorin particles by thin-layer chromatography revealed a predominance of phosphatidylethanolamine and phosphatidylcholine, with minor amounts of phosphatidylserine, phosphatidylinositol, and sphingomyelin. The transfer of phospholipids to lipophorin was linear with time up to 45 min and the process was inhibited at low temperature and by the metabolic inhibitors azide and fluoride. The transfer of phospholipids from the fat bodies to lipophorin was saturable with respect to the concentration of lipophorin, which was half-maximal at about 8 mg/ml. A directional movement of phospholipids from the fat body to lipophorin was observed. The net gain of phospholipids in 2 h of incubation with fat body was 8.54 nmol per insect, which corresponds to 6.69% of increase in the lipophorin phospholipid content. The rate of 32P-phospholipid transfer from fat body to lipophorin particles varied during the days after a blood meal increasing up to day 10 and then decreasing in parallel with the process of oogenesis.     

Biochemical properties of the major proteins from Rhodnius prolixus eggshell

Two proteins from the eggshell of Rhodnius prolixus were isolated, characterized and named Rp30 and Rp45 according to their molecular masses. Purified proteins were used to obtain specific antiserum which was later used for immunolocalization. The antiserum against Rp30 and Rp45 detected their presence inside the follicle cells, their secretion and their association with oocyte microvilli. Both proteins are expressed during the final stage of vitellogenesis, preserved during embryogenesis and discarded together with the eggshell. The amino terminals were sequenced and both proteins were further cloned using degenerated primers. The amino acid sequences appear to have a tripartite arrangement with a highly conserved central domain which presents a repetitive motif of valine-proline-valine (VPV) at intervals of 15 amino acid residues. Their amino acid sequence showed no similarity to any known eggshell protein. The expression of these proteins was also investigated; the results demonstrated that this occurred strictly in choriogenic follicles. Antifungal activity against Aspergillus niger was found to be associated with Rp45 but not with Rp30. A. niger exposed to Rp45 protein induced growth inhibition and several morphological changes such as large vacuoles, swollen mitochondria, multi-lamellar structures and a disorganized cell wall as demonstrated by electron microscopy analysis.     

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.     

Thermosensation and the TRPV channel in Rhodnius prolixus

The thermal sense of triatomine bugs, vectors of Chagas disease, is unique among insects. Not only do these bugs exhibit the highest sensitivity to heat known in any animal up to date, but they can also perceive the infrared radiation emitted by the body of their warm-blooded hosts. The sensory basis of this capacity has just started to be unravelled. To shed additional light on our understanding of thermosensation, we initiated an analysis of the genetic basis of the thermal sense in Rhodnius prolixus. We tested the hypothesis that a TRPV (transient receptor potential vanilloid) channel receptor is involved in the evaluation of heat in this species. Two different approaches were adopted. Initially, we analysed the expression of a TRPV candidate for this function, i.e., RproIav, in different tissues. Subsequently, we tested the effects of capsaicin and capsazepine, two molecules known to interact with mammal TRPV1, using three different behavioural protocols for evaluating thermal responses: (1) proboscis extension response (PER), (2) thermopreference in a temperature gradient and (3) spatial learning in an operant conditioning context. Bioinformatic analyses confirmed that the characteristic features typical of the TRPV channel subfamily are found in the RproIav protein sequence. Molecular analysis showed that RproIav is expressed in R. prolixus, not only in the antennae, but also in other body structures bearing sensory organs. Behavioural experiments consistently revealed that capsaicin treated insects are less responsive to heat stimuli and prefer lower temperatures than non-treated insects, and that they fail to orient in space. Conversely, capsazepine induces the opposite behaviours. The latter data suggest that triatomine thermoreception is based on the activation of a TRP channel, with a similar mechanism to that described for mammal TRPV1. The expression of RproIav in diverse sensory structures suggests that this receptor channel is potentially involved in bug thermoreception. This constitutes solid evidence that thermosensation could be based on the activation of TRP receptors that are expressed in different tissues in R. prolixus. Whether RproIav channel is a potential target for the compounds tested and whether it mediates the observed effects on behaviour still deserves to be confirmed by further research.     

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.     

The biological activity of diuretic factors in Rhodnius prolixus

The rapid post-feeding diuresis of Rhodnius prolixus is under neurohormonal control and involves the integrated activity of the crop, Malpighian tubules and hindgut. One of the factors which is involved in this rapid diuresis is serotonin, however a peptide(s) is also considered to be involved. In other insects, corticotropin releasing factor (CRF)-like and kinin-like, calcitonin-like peptides and CAP(2b) have been demonstrated to be diuretic factors/hormones.In the present study, serotonin and CRF-like peptides increased secretion rate and cAMP content of Rhodnius Malpighian tubules, while the kinin-like peptides tested did not increase secretion rate or cAMP content of the tubules. Extracts of the CNS were processed and several HPLC fractions revealed kinin-like immunoreactivity but these fractions did not increase secretion rate when tested on Malpighian tubules. However, these same fractions did possess activity when tested on the hindgut contraction assay. In addition, material eluting at higher acetonitrile concentrations from the HPLC increased secretion and cAMP content of Rhodnius Malpighian tubules. This material eluted at concentrations of acetonitrile consistent with the elution time of CRF-like peptide standards.Synergism was demonstrated using the pharmacological agent forskolin and serotonin, tested on the rate of secretion of Rhodnius Malpighian tubules, in agreement with data of Maddrell et al. As well, synergism could be demonstrated using mesothoracic ganglionic mass (MTGM) homogenates and serotonin at some concentrations of serotonin. However, combinations of CRF-like material and serotonin increased secretion additively, not synergistically. Kinin-like peptides, tested along with CRF-like material and serotonin, at low concentrations, did not increase secretion above that of those factors tested alone.     

Ecto-nucleotidase activities in the fat body of Rhodnius prolixus

In this study, we describe the ability of intact fat body of an insect, Rhodnius prolixus, to hydrolyze extracellular ATP. In these fat bodies, the ATP hydrolysis was low in the absence of any divalent metal, and was stimulated by MgCl(2). Both activities (in the absence or presence of MgCl(2)) were linear with time for at least 30 min. In order to confirm the observed nucleotidase activities as ecto-nucleotidases, we used an impermeant inhibitor, DIDS (4, 4'-diisothiocyanostylbene 2'-2'-disulfonic acid). This reagent inhibited both nucleotidase activities and its inhibitory effect was suppressed by ATP. Both ecto-nucleotidase activities were insensitive to inhibitors of other ATPase and phosphatase activities, such as oligomycin, sodium azide, bafilomycin, ouabain, vanadate, molybdate, sodium fluoride, levamizole, tartrate, p-NPP, sodium phosphate, and suramin. Concanavalin A, activator of some ecto-ATPases, was able to stimulate the Mg(2+)-independent nucleotidase activity, but not the Mg(2+)-dependent one. The Mg(2+)-independent nucleotidase activity was enhanced with increases in the pH in the range between 6.4-8.0, but the Mg(2+)-dependent nucleotidase activity was not affected. Besides MgCl(2) , the ecto-ATPase activity was also stimulated by CaCl(2),() MnCl(2), and SrCl(2), but not by ZnCl(2). ATP, ADP, and AMP were the best substrates for the Mg(2+)-dependent ecto-nucleotidase activity, and CTP, GTP, and UTP produced very low reaction rates. However, the Mg(2+)-independent nucleotidase activity recognized all these nucleotides producing similar reaction rates, but GTP was a less efficient substrate. The possible role of the two ecto-nucleotidase activities present on the cell surface of fat body of Rhodnius prolixus, which are distinguished by their substrate specificity and their response to Mg(2+), is discussed.     

Hormonal Control of Vitellogenin Uptake in Rhodnius prolixus Stal

SYNOPSIS. In Rhodnius, vitellogenesis is characterized by theappearance of large spaces between the cells of the follicularepithelium, a condition which is called patency. Patency isa response to juvenile hormone (JH) secreted by the corpus allatum.JH brings about an increase in patency by causing the folliclecells to shrink, an effect which may be mediated by (Na⁺-K⁺)ATPase. Follicle cells which have differentiated in the absenceof JH fail to respond to JH with an increase in patency. Thefollicle cells are also affected by an andgonadotropin fromneurosecretory cells in the abdomen. This anti-JH antagonizesthe patency-inducing action of JH.     

Terpenoids from Rhizomes of Alpinia japonica Inhibiting Nitric Oxide Production

A new sesquiterpenoid, 1 , and three new diterpenoids, 3 – 5 , along with five known compounds, 2 and 6 – 9 , were isolated from rhizomes of Alpinia japonica. The structures of the new compounds were determined as (1R,4R,6S,7S,9S)‐4α‐hydroxy‐1,9‐peroxybisabola‐2,10‐diene ( 1 ), methyl (12E)‐16‐oxolabda‐8(17),12‐dien‐15‐oate ( 3 ), (12R)‐15‐ethoxy‐12‐hydroxylabda‐8(17),13(14)‐dien‐16,15‐olide ( 4 ), and methyl (11E)‐14,15,16‐trinorlabda‐8(17),11‐dien‐13‐oate ( 5 ) by means of spectroscopic data. The absolute configurations at C(4) in 1 and C(12) in 4 were deduced from the circular dichroism (CD) data of the in situ‐formed [Rh₂(CF₃COO)₄] complexes. Inhibitory effects of the isolates on NO production in lipopolysaccharide‐induced RAW264.7 macrophages were evaluated, and 2 – 4, 6 , and 7 were found to exhibit inhibitory activities with IC₅₀ values between 14.6 and 34.3 μM .