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.     

Carbohydrate accumulation and utilization by oocytes of Rhodnius prolixus

The processes of accumulation and mobilization of carbohydrate stores in eggs of Rhodnius prolixus were analyzed. During oogenesis, the total amounts of glycogen, glucose, and trehalose increased with an accumulation of proteins, especially when oocytes grew from 1.0 to 1.5 mm in length. At 2.0 mm length, when oocytes were ready for oviposition, nutrient reserves did not increase appreciably and trehalose content decreased. Mating did not affect the final content of carbohydrates or proteins in oocytes of mated and virgin females. A trehalase activity was detected in follicles containing vitellogenic oocytes, 1.0 and 1.5 mm length, in both mated and virgin females. This activity was extremely low in chorionated, 2.0-mm oocytes. After oviposition, glycogen content decreased in fertilized eggs, but not in unfertilized ones, and some was present in newly hatched nymphs. Glucose content remained constant in unfertilized eggs, but increased in fertilized ones, while total protein amount was constant in both groups after egg laying.     

Extracellular glutathione peroxidase from the blood‐sucking bug,Rhodnius prolixus

Glutathione peroxidase (GPX) activity was measured in several tissues of the blood‐sucking bug, Rhodnius prolixus. In contrast to the pattern found in vertebrates, where GPX is predominantly intracellular, the highest levels of this enzyme in Rhodnius were found in the hemolymph. The hemolymph glutathione‐dependent peroxidase accepted both H₂O₂ and t‐butyl hydroperoxide as substrates. This fact, together with the absolute glutathione dependence, inhibition by mercaptosuccinate, insensitivity to cyanide, and a molecular mass (100.7 kDa) similar to vertebrate GPXs, led us to attribute this peroxidatic activity to a Se‐dependent enzyme. Hemolymph GPX specific activity increases during development and a twofold stimulation was observed after an oxidative challenge with hemin, suggesting that enzyme synthesis is under regulatory control. A role for extracellular GPX as an antioxidant protection against oxidative damage produced by heme derived from digestion of blood hemoglobin is discussed. Arch. Insect Biochem. Physiol. 41:171–177, 1999. © 1999 Wiley‐Liss, Inc.     

Rhodnius prolixus LIPOPHORIN: LIPID COMPOSITION AND EFFECT OF HIGH TEMPERATURE ON PHYSIOLOGICAL ROLE

Lipophorin is a major lipoprotein that transports lipids in insects. In Rhodnius prolixus, it transports lipids from midgut and fat body to the oocytes. Analysis by thin‐layer chromatography and densitometry identified the major lipid classes present in the lipoprotein as diacylglycerol, hydrocarbons, cholesterol, and phospholipids (PLs), mainly phosphatidylethanolamine and phosphatidylcholine. The effect of preincubation at elevated temperatures on lipophorin capacity to deliver or receive lipids was studied. Transfer of PLs to the ovaries was only inhibited after preincubation of lipophorin at temperatures higher than 55°C. When it was pretreated at 75°C, maximal inhibition of phospholipid transfer was observed after 3‐min heating and no difference was observed after longer times, up to 60 min. The same activity was also obtained when lipophorin was heated for 20 min at 75°C at protein concentrations from 0.2 to 10 mg/ml. After preincubation at 55°C, the same rate of lipophorin loading with PLs at the fat body was still present, and 30% of the activity was observed at 75°C. The effect of temperature on lipophorin was also analyzed by turbidity and intrinsic fluorescence determinations. Turbidity of a lipophorin solution started to increase after preincubations at temperatures higher than 65°C. Emission fluorescence spectra were obtained for lipophorin, and the spectral area decreased after preincubations at 85°C or above. These data indicated no difference in the spectral center of mass at any tested temperature. Altogether, these results demonstrate that lipophorin from R. prolixus is very resistant to high temperatures.     

The biochemical effects of potassium chloride on the silkworm, （ Bombyx mori L.）

The supplementation with 50, 100 and 150μg/mL potassium chloride to the fifth instar larvae of the silkworm Bombyx mori on fat body glycogen, protein, total lipids and haemolymph protein and trehalose were analyzed. The fat body glycogen and protein and haemolymph protein were increased significantly in all the treated groups; whereas fat body total lipids increased only in 100 and 150μg/mL and haemolymph trehalose increased only in 150μg/mL potassium chloride-treated groups when compared with those of the corresponding parameters of the carrier controls.     

Pretarsal structures in Reduviidae (Heteroptera, Ins ecta)

A survey of pretarsal structures in Reduviidae of 22 higher‐level taxa and several outgroup representatives is conducted using scanning electron microscopy (SEM) and light microscopy. Based on histology and SEM, pretarsal structures are described in detail for Rhodnius prolixus. Structures of the distal tarsomer, which appear to be functionally correlated with the pretarsus, are documented for the first time in Heteroptera. These comprise lateral oval sclerites, two campaniform sensilla and two marginal setae, observed in Reduviidae and other heteropteran taxa. The presence of a peg‐like dorsomedian sensillum medially between the claws in Reduviidae and Nabidae is demonstrated. As a result of its structure and position, homology of this sensillum with the ‘dorsal arolium’ in other heteropteran groups is proposed. Within Reduviidae, the transformation of the peg‐like dorsomedian sensillum to a campaniform sensillum, as seen in most representatives of the harpactoroid complex, is hypothesized. The first record of parempodia other than setiform within Reduviidae is noted in nymphs of Harpactorinae, which may possess lamellate parempodia. Several characters that are possibly useful for clarifying relationships among the harpactoroid groups are described and discussed. The pretarsal structures, including the unguitractor plate and the tarsal marginal setae among reduviid groups are discussed in a phylogenetic context.     

Carbohydrate metabolism during the pupal molt of the tobacco hornworm,Manduca sexta

During the pupal molt of the tobacco hornworm, Manduca sexta, the percentage of active fat body glycogen phosphorylase increased from 5–10 to 20%, but only for a period of 5 h prior to the molt. From the time of the appearance of two sclerotized dorsal bars to the time of the molt, the concentration of total hemolymph carbohydrates doubled to 100 mM trehalose. Initially, the glucose level was high (16 mM) when compared with feeding larvae (approximately 1 mM) but decreased to zero just prior to the molt. The amount of cuticular chitosan decreased from approximately 100 mg to 10 mg at pupation; the exuvia contained approximately 7 mg. While the levels of total lipids in hemolymph were not affected, the lipid content of the fat body decreased significantly prior to the molt but increased sharply thereafter. Fat body glycogen phosphorylase in pharate pupae and pupae of M. sexta was substantially activated by the Manduca adipokinetic peptide hormone, which in pharate pupae, produced the same response at 2 and 20 pmol per insect as in ligated larval abdomens. In pupae the response was clearly reduced. Using chilling to stimulate glycogen phosphorylase, it was found that the enzyme in pharate pupae and pupae responded both in vivo and in vitro as in ligated abdomens of larvae. Thus, a transition to the adult response seems to occur during the pupal and pharate adult development. © 1995 Wiley-Liss, Inc.     

Third instar nymphs of Rhodnius prolixus exposed to alpha-cyanopyrethroids: from hyperactivity to death

The hyperactivity, incoordination, recovery, and mortality produced by four alpha-cyanopyrethroids usually used for Chagas disease vector control (beta-cypermethrin, beta-cyfluthrin, lambda-cyhalothrin, and deltamethrin) were evaluated on third instar nymphs of Rhodnius prolixus. All pyrethroids modified the locomotor activity of the nymphs, which increased linearly as a function of the log of insecticide concentration. lambda-Cyhalothrin showed the lowest values of Effective Concentration 50%, Lethal Concentration 50%, Effective Time 50%, and Lethal Time 50% when insecticides were applied by contact with treated filter papers. Recovery from incoordination was observed after topical application of the insecticides. The recovery was inhibited by the simultaneous application of piperonyl butoxide, suggesting that biotransformation by mixed-function microsomal oxidases is involved in the process of recovery.     

Identification of yolk platelet-associated hydrolases in the oocytes of Rhodnius prolixus.

The yolk platelets from Rhodnius prolixus, a blood-sucking bug, are composed mostly of vitellin and here are shown to contain at least two hydrolytic enzymes, a phosphatase and a cathepsin D-like proteinase. Both the proteinase and the phosphatase have an acid pH optimum. No hydrolytic activity was observed under alkaline or neutral conditions. Among several proteinase inhibitors tested, only pepstatin could abolish vitellin breakdown in vitro. The proteinase appears to be bound to the yolk platelet membranes. The phosphatase activity, using p-nitrophenyl phosphate as substrate, was enhanced after disruption of the platelet membrane by Triton X-100. This activity could be inhibited by tartrate but not by p-cloromercuribenzoate.     

Female metasternal gland odours mediate male aggregation in Rhodnius prolixus, a triatomid bug

We investigated whether metasternal gland (MG) products mediate male aggregation during mating in Rhodnius prolixus (Heteroptera: Reduviidae) Stål, 1859, through selective occlusion of female MGs. Pairs of insects presenting intact MGs mated and other males aggregated around them confirming conclusions from previous studies. However, when female MGs were occluded, males did not aggregate. Conversely, the occlusion of male MGs did not affect aggregation of males. The present experiments demonstrated that the products emitted by the MGs of a mating R. prolixus female induce other males to aggregate around the mating pair. Aggregation behaviour of males probably results from the eventual release of volatiles from the female's MGs. We reinforce the hypothesis suggesting that the compounds produced by the MGs of R. prolixus act as sexual communication signals for this species.     

Uptake of Rhodnius heme-binding protein (RHBP) by the ovary of Rhodnius prolixus

The uptake of RHBP (Rhodnius heme-binding protein) by the ovaries of Rhodnius prolixus was characterized. RHBP purified from oocyte was labeled with ¹²⁵I and used to study the process of uptake by the ovary in vivo and in vitro. After injection, the [¹²⁵I]RHBP was readily removed from the hemolymph and accumulated especially in the ovary. The capacity of the ovary to take up [¹²⁵I]RHBP from the hemolymph varied during the days following blood meal. It increased up to day 2, remained stable until day 5, and then decreased up to the end of oogenesis. In vitro, the uptake of [¹²⁵I]RHBP was linear at least up to 60 min. The uptake was dependent on [¹²⁵I]RHBP concentration and showed to be a saturable process. The addition of a molar excess of non-related proteins such as Vitellin (Vt), Lipophorin (Lp), and Bovine Serum Albumin (BSA) did not reduce [¹²⁵I]RHBP uptake. Using immunogold technique the RHBP was localized at the microvilli, coated pits, and yolk granules. The main yolk protein, Vt, did not compete with RHBP for the uptake. Thus, it is discussed here that they bind to independent binding sites of the oocytes, and are directed later on to the same compartment. The need of both proteins for the completion of mature oocyte was verified in vivo. The reduction of heme-RHBP in the hemolymph, by changing the diet, decreased the number of eggs laid. Increasing the concentration of heme-RHBP in the hemolymph, the number of eggs produced increased in a dose dependent manner. In vitro, both apo-RHBP and heme-RHBP can be taken up by the oocyte. Since the mature oocyte contains only heme-saturated RHBP, the possible fate of apo-RHBP is also discussed. Arch. Insect Biochem. Physiol. 39:133–143, 1998. © 1998 Wiley-Liss, Inc.     

东方粘虫飞行初期糖类的动用和消耗

本文研究了静态和飞翔开始阶段东方粘虫Mythimna separta(Walker)飞翔肌、脂肪体及肠道内糖元和海藻糖以及血淋巴内海藻糖含量的动态变化。飞行1h后脂肪体内消耗糖元最多,约占总消耗糖类的80%-90%;其次是肠道组织的糖元消耗,飞翔肌内糖类含量是有限的,仅够飞翔最初时期(3min)使用。在此时期,飞翔肌海藻糖含量上升然后下降,脂肪体和肠道海藻糖经历下降和继而上升的变化。然而,血淋巴内海藻糖含量一直下降到流入和流出量持平。雄蛾飞翔肌糖类代谢水平高于雌娥飞翔肌。     

Protein 2DE reference map of the anterior midgut of the blood‐sucking bug Rhodnius prolixus

Rhodnius prolixus is an important vector of Trypanosoma cruzi, the causative agent of Chagas’ disease, an illness that affects 20% of Latin America population. The obligatory course of the parasite in the vector digestive tract has made it an important target for investigation in order to control the parasite transmission and thus interrupt its biological cycle in the insect vector. Therefore, an insight into the vector midgut physiology is valuable for insect control as well as to provide potential novel targets for drugs and vaccines development and thus disease treatment. In this study, the first 2DE map of R. prolixus anterior midgut is described. Proteins were separated by 2DE and analyzed by nano‐LC MS/MS. The results yielded 489 proteins from 475 spots. These proteins were classified into 28 functional groups and their physiological roles in the insect midgut are discussed. All MS data have been deposited in the ProteomeXchange with identifiers PXD001488 and PXD001489 ( http://proteomecentral.proteomexchange.org/dataset/PXD001488 , http://proteomecentral.proteomexchange.org/dataset/PXD001489 ).     

Decrease in DEET repellency caused by nitric oxide in Rhodnius prolixus

N,N-diethyl-3-methylbenzamide (DEET) is widely used as an insect repellent; however, little is known about its mode of action. On the other hand, nitric oxide (NO) participates in the olfaction transduction pathway of insects. In this work, nitroso-acetyl-cysteine (SNAC), a nitric oxide donor, or dibutyril-cyclic-GMP (db-cGMP), the cyclic nucleotide analog, were applied on fifth instar nymphs of Rhodnius prolixus before exposing them to DEET, to obtain information about the possible role of NO/cGMP system in the olfaction process. In the first place, we exposed the nymphs to several DEET concentrations (70, 700, 1,750, and 3,500 microg/cm2). All these concentrations produced a repellent effect. A decrease in repellency during the course of the experiment was observed when the nymphs were exposed to high concentrations of DEET (700 and 1,750 microg/cm2), suggesting an adaptation phenomenon. The pre-treatment of the insects with 15 microg /insect of SNAC or 2 microg/insect of db-cGMP produced a reduction of the repellency. An increase in locomotor activity was observed in insects exposed to 350 or 700 microg/cm2 DEET. Although exposure to 70 microg/cm2 DEET produced a high repellency response, it did not modify the insects' locomotor activity. Insects treated with two doses of SNAC before being exposed to 350 microg/cm2 of DEET showed no differences in locomotor activity compared to controls.     

Characterization of an ecto-phosphatase activity in malpighian tubules of hematophagous bug Rhodnius prolixus

We have characterized a phosphatase activity present on the external surface of intact Malpighian tubules in Rhodnius prolixus. This phosphatase hydrolyses the substrate p-nitrophenyl phosphate at a rate of 3.38 +/- 0.07 nmol Pi x mg(-1) x min(-1). Phosphatase activity decreased with the increase of the pH from 6.4 to 7.6 pH, a range in which tubules cellular integrity was maintained for at least 1 h. Classical inhibitors of acid phosphatase, such as ammonium molybdate, fluoride, vanadate, mpV-PIC, and bpV-PHEN, caused different patters of inhibition. The ecto-phosphatase present an apparent Km of 1.67 +/- 0.34 mM and Vmax of 5.71 +/- 0.37 nmol Pi x mg(-1) x min(-1) for p-NPP. Zinc chloride inhibited 78.2% of ecto-phosphatase activity, with Ki of 0.35 mM. Such inhibition was reversed by incubation with cysteine and GSH, but not DTT, serine, and GSSG, showing that cysteine residues are important for enzymatic activity. Phosphatase activity increased 141% three days after blood meal, and returned to basal levels 2 days later. These results suggest that ecto-phosphatase activity could be involved in a diuretic mechanism, essential in the initial days after a blood meal for the control of Rhodnius homeostasis.     

Lipid accumulation and utilization by oocytes and eggs of Rhodnius prolixus

Insect eggs must contain the necessary nutrients for embryonic growth. In this article, we investigated the accumulation of triacylglycerol (TAG) in growing oocytes and its utilization during embryonic development. TAG makes up about 60% of the neutral lipids in oocytes and accumulates as oocytes grow, from 2.2 ± 0.1 µg in follicles containing 1.0 mm length oocytes to 10.2 ± 0.8 µg in 2.0 mm length oocytes. Lipophorin (Lp), the hemolymphatic lipoprotein, radioactively labeled in free fatty acid (FFA) or diacylglycerol (DAG), was used to follow the transport of these lipids to the ovary. Radioactivity from both lipid classes accumulated in the oocytes, which was abolished at 4°C. The capacity of the ovary to receive FFA or DAG from Lp varied according to time after a blood meal and reached a maximum around the second day. ³H‐DAG supplied by Lp to the ovaries was used in the synthesis of TAG as, 48 hr after injection, most of the radioactivity was found in TAG (85.7% of labeling in neutral lipids). During embryogenesis, lipid stores were mobilized, and the TAG content decreased from 16.4 ± 2.1 µg/egg on the first day to 10.0 ± 1.3 µg on day 15, just before hatching. Of these, 7.4 ± 0.9 µg were found in the newly emerged nymphs. In unfertilized eggs, the TAG content did not change. Although the TAG content decreased during embryogenesis, the relative lipid composition of the egg did not change. The amount of TAG in the nymph slowly decreased during the days after hatching. © 2011 Wiley Periodicals, Inc.     

Microclimatic factors influencing refugium suitability for Rhodnius prolixus

Abstract.  Rehydration in the triatomine bug Rhodnius prolixus (Stål) is dependent on the blood meal, and water balance regulation is crucial for survival of starving bugs. In an experimental arena with zones at different climatic conditions, starved R. prolixus nymphs prefer a cooler and more humid zone, stopping there more often and for longer periods. This is probably a behavioural adaptation to limit water loss and reduce metabolic rate. In the Venezuelan State of Portuguesa, temperature and humidity were monitored in three kinds of potential refugia during the dry season: (i) in a palm roof; (ii) in a crack of the wall of a house; and (iii) in a palm tree crown. Fluctuations in temperature and saturation deficit are not very different inside and outside the palm roof except during a few hours of the day when the sun is at its zenith. In the crack of the wall, the diurnal temperature range is reduced to 6.5 °C compared with 12.4 °C outside, and the saturation deficit varies by only 7.6 hPa compared with 28.6 hPa outside. In the palm tree crown, the daily temperature range is only 4.2 °C compared with 13.8 °C outside, and the saturation deficit is permanently < 5 hPa. The microclimatic conditions in the palm tree crown would appear to be ideal for starving R. prolixus , but this kind of refugium generally harbours low densities of bugs, probably related to a combination of predation, pathogens and lower mean temperature within the crown. Such biotic and abiotic constraints play a lesser role in the less ideal palm roof and wall crack refugia where bugs can proliferate as long as hosts are readily available nearby.     

Hydrogen peroxide detoxification in the midgut of the blood-sucking insect, Rhodnius prolixus

Here we investigated H2O2 production and detoxification in the hematophagous hemiptera, Rhodnius prolixus. Superoxide dismutase (SOD) catalyzes the dismutation of superoxide radical (O2-). This reaction produces hydrogen peroxide, which is scavenged by antioxidant enzymes such as catalase (CAT). SOD and CAT activities were found in all tissues studied, being highest in the midgut. CAT was dose-dependently inhibited in vivo by injections of 3-amino-1,2,4-triazole (AT). Insects treated with AT showed a twofold increase in H2O2 levels. Injection of DL-buthionine-[S, R]-sulfoximine (BSO), an inhibitor of glutathione synthesis, also resulted in a fourfold increase in H2O2, together with stimulation of CAT activity. Simultaneous administration of both AT and BSO had a synergistic effect on midgut H2O2 content. Taken all together, our results suggest that CAT and glutathione-dependent mechanisms cooperate to control H2O2 concentration in the midgut cell and prevent hydroxyl radical generation by Fenton reaction in this tissue.